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" the empirical dietary index for hyperinsulinemia edih score is a validated foodbased dietary scorethat assesses the ability of wholefood diets to predict plasma cpeptide concentrations although the edih hasbeen extensively applied and found to be predictive of risk of developing major chronic diseases its influence oncancer survival has not been evaluated we applied the edih score in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patterns after diagnosis and determine its influence on survivaloutcomesmethods we calculated edih scores to assess the insulinemic potential of postdiagnosis dietary patterns andexamined survival outcomes in a sample of stage iiii colorectal cancer patients in the nurses health studyand health professionals followup study cohorts multivariableadjusted cox regression was applied to computehazard ratios hr and confidence intervals ci for colorectal cancerspecific mortality and allcause mortalitywe also examined the influence of change in diet from pre to postdiagnosis period on mortalityresults during a median followup of years there were deaths which included colorectal cancerspecific deaths in the multivariableadjusted analyses colorectal cancer patients in the highest compared tolowest edih quintile had a greater risk of dying from colorectal cancer hr ci and a greater risk of allcause death hr 95ci compared to patients who consumed low insulinemicdiets from pre to postdiagnosis period patients who persistently consumed hyperinsulinemic diets were at higherrisk of colorectal cancer death hr151 95ci and allcause death hr 95ci our findings suggest that a hyperinsulinemic dietary pattern after diagnosis of colorectal cancer isassociated with poorer survival interventions with dietary patterns to reduce insulinemic activity and impactsurvivorship are warrantedkeywords colorectal cancer survival insulinemic dietary patterns insulin cpeptide correspondence fredtabungosumcedu1division of medical oncology department of internal medicine the ohiostate university college of medicine west 12th avenue 302b wisemanhallccc columbus oh usa2the ohio state university comprehensive cancer center arthur g jamescancer hospital and richard j solove research institute columbus oh usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctabung bmc cancer page of canceristhefourth most colorectalcommonlydiagnosed cancer in the united states while there ishigh potential for dietary patterns as a modifiable riskfactor for colorectal cancer development [ ] verylimited evidence exists among colorectal cancer survivors for example in a recent review we identified s published up to that reported on theassociation between dietary patterns and colorectalcancer development but only about five s onthe association between dietary patterns and outcomesamong colorectal cancer survivors [] the evidenceshowed that the western dietary pattern often characterized by high intakes ofred andprocessed meats desserts and potatoesis associatedwith higher risk of allcause mortality but generally notwith colorectal cancerspecific mortality in patients withcolorectal cancer the prudent dietary pattern oftencharacterized by high intakes of fruits vegetables wholegrains and poultry showed similar results with inverseassociations for allcause mortality but no consistent association with colorectal cancerspecific mortality []higher adherence to other dietary patterns including themediterranean diet score dietary approaches to stophypertension meal plan american cancer society cancerprevention guidelines score healthy eating index scorewere generally associated with lower risk of allcausemortality but the associations were inconsistent acrossstudies [ ]refined grainschronic diseasesfurther research is therefore needed to clarify ifdietary patterns are importantfor colorectal cancerprognosis and if dietary changes can maximally impactoverall and cancerspecific survival biomarkerbaseddietary patterns may be helpfulin this regard forexample hyperinsulinemia and insulin resistance areconsidered important underlying mechanisms linkingpoor dietary patterns and lifestyle behaviors to the development of multipleincludingcolorectal cancer [] studies have shown positiveassociations between circulating cpeptide concentrations a marker of beta cell secretory activity and colorectal cancer risk and prognosis [] therefore adietary pattern associated with hyperinsulinemia may bemore predictive of outcomes following colorectal cancerdiagnosis than a dietary pattern not associated with thispathway we previously derived the empirical dietaryindex for hyperinsulinemia edih score to assess thepotential of dietary patterns to influence insulinemia which has been extensively applied in large cohortstudies and found to be predictive of nonfasting cpeptide concentrations [ ] longterm weight gain risk of developing colorectal cancer other digestive system cancers and other cancers however the influence of dietary insulinemic potentialon cancer survival outcomes has not yet been evaluatedthe objective of the current study is to apply the edihscore in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patternsafter diagnosis and determine its influence on survivaloutcomesmethodsstudy populationwe used data from the nurses health study nhs andthe health professionals followup study hpfs twoongoing cohorts in the united states hpfs was initiatedin and enrolled male health professionalsbetween the ages of and years nhs initiatedin enrolled registered female nurses aged to years fig data on medical lifestyleand other healthrelated factors was collected at baselineand have been updated every years thereafter ethicalapproval for our study was provided by the harvardth chan school of public health and those of participating registries as required and the institutional reviewboards of the brigham and womens hospital studyparticipants provided consent by completing and submitting study questionnaires participants were free toterminate participation in the study at any timeassessment of diet and the empirical dietary index forhyperinsulinemia edih scorein both cohorts diet was assessed using a validated selfadministered food frequency questionnaire ffq thatassessed how often on average participants consumed astandard portion size of various foods in the past yearin the nhs diet was assessed in andevery years thereafter whereas in the hpfs diet wasassessed in and every years thereafter theedih score developed to empirically measure the insulinemic potential of whole diets using food groups hasbeen described in detail briefly thirtynine foodgroups were entered into stepwise linear regressionmodels to identify a dietary pattern most predictive ofplasma cpeptide levels the edih score represents aweighted sum of food groups with higher scores indicating hyperinsulinemic diets hyperinsulinemia andlower scores indicating low insulinemic diets the foodgroups contributing to lower edih scores are winecoffee fullfat dairy products whole fruit and green leafyvegetables whereas the food groups contributing to highedih scores arelowfat dairy products french frieslowenergy beverages cream soups processed meat redmeat margarine poultry nondark fish high tomatoesenergy beverage and eggs in the current study we calculated edih scores foreach participant based on the selfadministered ffqspostdiagnosis edih score was calculated based on the 0ctabung bmc cancer page of fig flow chart describing the flow of participants from the full cohorts to the final analytic sample in the nurses health study nhs andhealth professionals followup study hpfsfirst ffq returned at least months but not more than years after colorectal cancer diagnosis thus avoidingdiet assessment during active cancertherapy themedian time from diagnosisto postdiagnosis dietassessment was years prediagnosis edih score wascalculated based on the cumulative average of edihscores up to the last diet assessment before colorectalcancer diagnosis the median time from prediagnosisdiet assessment to diagnosis was yearspatients with colorectal cancer and mortality assessmentwhen a colorectal cancer diagnosis was reported duringthe previous years on the followup biennial questionnaires we requested permission to obtain hospital records and pathology reports blinded study physiciansthen reviewed these records and recorded data on tumorcharacteristics for nonrespondents the national deathindex was used to identify deaths and ascertain anydiagnosis of colorectal cancer that contributed to deathafter years of followup for disease diagnoses in nhs and to in hpfs we identified patients with pathologically confirmed colorectalcancer we excluded participants who died before in nhs or in hpfs had reported any cancerexcept nonmelanoma skin cancer before colorectalcancer diagnosis who died at diagnosis who did nothave prediagnosis diet or postdiagnosis diet patientswho did not complete a diet assessment between months and years after diagnosis or had diet assessedoutside of this period who had diabetes at colorectalcancer diagnosis and patients with stage iv or unknownstage at diagnosis therefore the current analysis included patients with stage i ii or iii colorectal cancerincluding participants from hpfs and from nhs fig deaths were ascertained throughreporting by family for persistent nonresponders wequeried the national death index with their names up 0ctabung bmc cancer page of to june for nhs and january for hpfs cause of death was assigned by blinded physicianscovariate assessmentboth cohorts assessed covariate data eg medical historylifestyle and health factorsthrough selfadministeredquestionnaires every years these factors included physical activity smoking habits alcohol intake multivitaminuse endoscopy status regular use of aspirin and othernonsteroidal antiinflammatory drugs nsaids familyhistory of colorectal cancer weight height menopausalstatus and postmenopausal hormone use only forwomenin both cohorts as previously described dietassessment was conducted every years [ ]statistical analysiswe categorized the edih score into quintiles withcohortspecific cutoffs then pooled the data for analysispersontime of followup was calculated from the dateof postdiagnosis diet assessment to death or to lastfollowup date january in hpfs or june innhs whichever was first we used the kaplanmeiermethod to generate survival curves by quintiles of edihscore and tested group differences highest vs lowestquintile using the logrank test for this test the edihscore was adjusted for total energy intake and bmi usingthe residual methodcox proportional hazards regression was used to calculate hazard ratios hrs of colorectal cancerspecificdeath or allcause death in edih quintiles quintile cutpoints were created separately by sex and applied in thepooled sample given that participants must survivefrom diagnosis until postdiagnosis diet assessment weused time since diagnosis as the underlying time scale toaccount for left truncation due to staggered entry thecox models were tested for the assumption of proportionality using timecovariate interaction terms andstratified by age sex and stage we fitted two models tothe data as follows model minimally adjusted modelincluded bmi demographic factors sex age at diagnosis and tumor characteristics stage subsite within thecolon grade of tumor differentiation model fullyadjusted model included all the covariates in model and postdiagnosisfactors packyears ofsmoking physical activity regular aspirin use pre topostdiagnosis weight change total alcohol intake andprediagnosis dietary pattern edih score testforlinear trend of risk across edih quintiles was performedusing the median postdiagnosis edih score in eachedih quintile as a continuous variable in the cox regression models and interpreting the pvalue of thisvariable as the pvalue for linear trendlifestyleto determine how changes in the insulinemic potential of diet before and after diagnosis influence survivalwe dichotomized pre and postdiagnosis edih scores atthe median and used to create a change variable withlow indicating a score below the median and high ascore above the median lowlow consistently lowdietary insulinemic potential before and after diagnosisie both scores below the median lowhigh patientsconsuming low insulinemic diets before diagnosis andmore hyperinsulinemic diets after diagnosis highlowpatients consuming hyperinsulinemic diets before diagnosis and then changed towards low insulinemic dietsafter diagnosis and highhigh patients who consistentlyconsumed hyperinsulinemic dietary patterns before andafter diagnosis we then applied these dietary patternchanges in multivariableadjusted cox models to examine risk of death from colorectal cancer and from othercauseschange preand postdiagnosisfrom postdiagnosis weightwe conducted exploratory subgroup analysesincategories of the following potential effect modifiers sexweightand prediagnosis edih score we categorized prediagnosisedih at the median median and ¥ median weightchange was calculated by subtracting prediagnosisweightthe continuousweight change variable was categorized as follows thosewho gained more than kg had a stable weight kg to kg or lost more than kg we also conducted subgroup analyses by time since diagnosis years ¥ years and age at diagnosis years ¥years tests of interaction between postdiagnosis edihscore and the potential effect modifiers were assessed byentering in the modelthe cross product of postdiagnosis edih score and the stratification variable andevaluated by the wald test all analyses were performedusing sas for unix all pvalues were two sidedresultscharacteristics of patients women with colorectal cancer after diagnosis are shown in table meanage at diagnosis was years and mean postdiagnosisbmi was kgm2 with of patients classified asoverweight or obese regarding disease stage hadstage i or ii and had stage iii disease during amedian followup of years there were allcausedeaths which included colorectal cancerspecificdeaths median overall survival by cancer stage was years for those with stage i disease years for thosewith stage ii and years for those with stage iiidisease fortyone percent of patients maintained astable weight between kg and kg between theprediagnosis and postdiagnosis period while lostmore than kg body weight and gained kgbody weight in the same period colorectal cancer patients with the most hyperinsulinemic dietary patterns 0ctabung bmc cancer page of table postdiagnosis characteristics of colorectal cancer patients by quintiles of postdiagnosis empirical dietary index forhyperinsulinemia edih score n characteristictotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile quintile to to n n quintile to n quintile to n quintile to n female age at diagnosisdage at diagnosis by sexdfemalemalecurrent smoker packyears of smokingbody mass index kgm2overweight bmi ¥ obese bmi ¥ physical activity methweekcphysical activity methweekcd by sex femalemale nonalcohol drinkers regular aspirin use location of cancer in the colon proximal colondistal colonrectumunspecifiedstage at diagnosis stage istage iistage iiimedian survival time yearsmedian survival time by staged years stage istage iistage iiiweight change categoriesd stable weight to kggained more than kglost more than kg avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intakecmetabolic equivalents from recreational and leisuretime activitiesdvalue is not age adjustedafter diagnosis quintile tended to have higher bodyweight and lower physical activity for example theaverage bmi among those classified in quintile was kgm2 and the average physical activity was methourweek compared with kgm2 and methourweek among those in quintile alsopatients consuming the most hyperinsulinemic dietarypatterns were less likely to have stage i disease and theyexperienced shorter survival times table patients consuming low insulinemic dietary patternshad higher intakes of wholegrains nuts vegetables wholefruits and coffee and lower intakes of refined grainscream soup eggs french fries butter margarine sugarsweetened beverages red meat and processed meat in 0ctabung bmc cancer page of table median 5th 95th percentile food and nutrient intake profiles of colorectal cancer patients by quintiles of postdiagnosisempirical dietary index for hyperinsulinemia edih scoretotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile n quintile n quintile n quintile n quintile n foods servingsweekprocessed meatred meat highenergy sugary beverages lowenergy sugary beverages margarinebutterfrench friesnondark fisheggslowfat dairycream souprefined grainstomatopoultrydark fishfullfat dairycoffeeteawhole fruitfruit juicepotatoesgreenleafy vegetablesdarkyellow vegetablesother vegetablesnuts total alcohol intake drinksweek whole grainsnutrient profile total carbohydrates gdtotal protein gd branchedchain amino acids gd total fat gdtotal fiber gd avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intaketerms of the nutrient profile resulting from this postdiagnosis dietary pattern patients consuming a lowinsulinemic dietary pattern had higher intakes of totalcarbohydrates and total fiber and lower intakes of total fattotal protein and branchedchain amino acids table kaplanmeier curves by quintiles of edih score areshown in fig with patients consuming a lowinsulinemic diet quintile experiencing better survivalfor colorectal cancerspecific and overall mortalitycompared to those consuming hyperinsulinemic dietsquintile in the multivariableadjusted analyses wefound that a hyperinsulinemic postdiagnosis dietarypattern was associated with higher risk of colorectalcancerspecific mortality and allcause mortality table 0ctabung bmc cancer page of fig kaplanmeier curves of a colorectal cancerspecific and b overall survival among patients with colorectal cancer by quintile of postdiagnosis empirical dietary index for hyperinsulinemia edih score logrank pvalues were calculated to test group differences quintile vs and adjusted for postdiagnosis total energy intake and postdiagnosis body mass indexcomparing colorectal cancer patients classified in the highestedih quintile to those in the lowest quintile there was a higher risk of colorectal cancerspecific death hr 95ci ptrend and a higher risk of allcause death hr 95ci ptrend afteraccounting for prediagnosis dietary insulinemic potentialamong other confounding variables table in relation to changes in the insulinemic potential ofthe diet before and after diagnosis patients whoconsumed a more hyperinsulinemic dietary patternconsistently before and after diagnosis were at higherrisk of dying from colorectal cancer hr ci and from other causes hr ci compared to patients who consistentlytable hazard ratios ci for colorectal cancerspecific and allcause mortality among patients with colorectal cancer byquintile of postdiagnosis edih scorestatistical modelcolorectal cancerspecific mortalityquintiles of the empirical dietary index for hyperinsulinemia edih scorequintile quintile quintile quintile ptrendquintile deathspatients aliveminimallyadjusted model reference fully adjusted model reference allcause mortalitydeathspatients aliveminimallyadjusted model reference fully adjusted model reference the minimallyadjusted models was adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stagegrade of tumor differentiation and location of primary tumor within the colon the fullyadjusted model was additionally adjusted for postdiagnosis physicalactivity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake andprediagnosis edih score 0ctabung bmc cancer page of fig hazard ratios for the association of change in dietary insulinemic potential between prediagnosis diet and postdiagnosis diet and risk ofdying form colorectal cancer crcsurvival and from all causes combined overall survival edih scores were dichotomized at the median lowlow the reference category represents participants who persistently consumed low insulinemic diets below the median edih from pre to postdiagnosis period lowhigh are those who changed from low insulinemic diets towards more hyperinsulinemic diets highlow represents thosewho changed from consuming hyperinsulinemic diets prior to diagnosis towards consuming low insulinemic diets after diagnosis whereas highhigh represents those who persistently consumed hyperinsulinemic diets prior to diagnosis and after diagnosis the number of deaths patientsalive in the four categories were as follows crcsurvival lowlow lowhigh highlow highhigh overallsurvival lowlow lowhigh highlow highhigh models were adjusted for age at diagnosis postdiagnosisbody mass index total energy intake sex race year of diagnosis cancer stage grade of tumor differentiation location of primary tumor withinthe colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake and prediagnosis edih scoreconsumed a low insulinemic dietary pattern before andafter diagnosis fig in subgroups of potential effect modifiers risk of colorectal cancerspecific mortality was significantly elevatedamong women and among those who lost body weightthose who were consuming a hyperinsulinemic dietarypattern before diagnosis and those younger than years for these subgroup analysesinteractions werestatistically significant only for sex in allcause mortalitytable discussionin the current study we showed that habitual consumption of hyperinsulinemic dietary patterns after colorectalcancer diagnosis or consumption of a hyperinsulinemicdietary pattern consistently before and after diagnosiswas associated with higher risk of dying from colorectalcancer and from all causes combinedthe insulinemic potential of diet was first estimatedby the insulin index which is based on a conceptsimilar to the more widely used glycemic index thatcharacterizes carbohydratecontaining foods accordingto their ability to raise blood glucose concentrationspostprandially compared with a reference food glucoseor white bread though carbohydrate content isone important factor influencing insulin response foodscan also stimulate insulin secretion in a carbohydrateindependent manner the insulin index directly quantifies the postprandial insulinemic potential of a food andtakes into account foods with a low or no carbohydratecontent it is important to understand that theinsulin index which was used in most previous studiesof dietary insulinemic potential and colorectal cancersurvival is conceptually and technically different fromthe edih and essentially uncorrelated spearman r the principle ofthe insulin index is how aparticularfood item stimulated insulin secretionindependent of underlying insulin resistance whereasthe edih is primarily driven by insulin resistance forcolorectal cancer the only other paper using the edihwas on cancer incidence both the insulin index and glycemic index assess thepostprandial shortterm effects of the diet unlike theedih score which predicts integrated insulin exposureie both fasting and nonfasting based on habituallongterm dietary intake postdiagnosis insulinindex and insulin load have been linked to higher risk ofdying from colorectal cancer [ ] higher dietary insulin load and insulin index after diagnosis of colorectalcancer were associated with increased risk of colorectalcancerspecific and overall mortality the association of postdiagnosis glycemic indices with colorectal 0ctabung bmc cancer page of table subgroup analyses of the association between dietary insulinemic potential and colorectal cancerspecific and allcausemortalitysubgroupptrendpinteractionquartile quartile quartile deathspatientsaliveedih quintilesquartile colorectal cancerspecific mortalitysexmenwomenweight change post minus prediagnosis weightstable weight to kgweight gain kgweight loss kgprediagnosis edih score median¥ medianage group at diagnosis years¥ yearstime since diagnosis years¥ yearsallcause mortalitysexmenwomenweight change post minus prediagnosis weightstable weight to kgweight gain kgweight loss kgprediagnosis edih score median¥ medianage group at diagnosis years¥ yearstime since diagnosis years¥ years ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref models were adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stage grade of tumordifferentiation and location of primary tumor within the colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirinuse weight change pre to postdiagnosis and postdiagnosis total alcohol intake and prediagnosis edih scorecancer prognosis has been inconsistent whereas onestudy found higher risk of colorectal cancer recurrenceand death associated with higher glycemic load but nothigher glycemic index another found no associationbetween glycemic load or glycemic index and colorectalcancer survival glycemic scores are primarilyreflective ofthe postprandial glucose responses ofcarbohydratecontaining foods whereas the edih scoredirectly reflects insulin increases induced by componentsof the dietary pattern that may or may not be contributing to calories eg coffee current study findingstherefore suggest that the direct effect of the diet on 0ctabung bmc cancer page of insulin may be more important than the effect of diet onglucose for colorectal cancer prognosis though theglycemic index is a measure of the shortterm postprandial effect of the diet on glucose concentrations it ispossible that such a habitual dietary pattern could overtime lead to sustained hyperinsulinemia and insulin resistance which could then mediate colorectal cancerprognosis however a previous study in these cohortsdid not observe an association between an overall lowcarbohydrate diet score and colorectal cancer or overallmortality although those who consumed a plantrichlowcarbohydrate diet which emphasized plant sourcesof fat and protein with moderate consumption of animalproducts had lower risk of colorectal cancerspecificmortality insulin is a growth factor and major regulator of cellmetabolism and its effects in target cells are mediatedby the insulin receptor a transmembrane protein withenzymatic activity evidence suggest that insulinstimulates growth mainly via its own receptor and notthe igf1 receptor and that in many cancer cells theinsulin receptor is overexpressed and the a isoformwhich has a predominant mitogenic effectis morerepresented than the b isoform the metabolicpathway stimulated by the activated insulin receptor toregulate glucose protein and lipid metabolism involvesthe pi3kakt pathwaycharacteristicsprovide a selective growth advantage to cancer cellswhen exposed to insulin therefore all conditions ofhyperinsulinemia both endogenousdiabetes metabolic syndrome obesity and exogenouseg hyperinsulinemic diets which also influence someof the endogenous conditions [ ] willincreasecancer risk and mortality theseegtypefor most ofalthough interactionsthe subgroupanalyses were not statistically significant some of thefindings merit some discussion the associations werestronger among women than among men which may berelated to severalfactors the larger sample size andstatistical power in our evaluation of women potentialconfounding with age as women were younger on average than men and a true biological interaction basedupon endocrine and associated metabolic factors wealso observed that there were worse outcomes amongpatients who lost weight than among those who maintained a stable weightto postdiagnosisperiod which may be consistent with complications ofprogressing disease leading to poor diet intakefrom premajor strengths of our study include the use of afoodbased edih score that is correlated with circulating cpeptide concentrations [ ] we had access tocomprehensive pre and postdiagnosis data on diet andimportant covariates which reduces the potential for residual confounding and recall bias our findings alsoaccounted for potential bias from staggered entry due todifferences between participants in the time betweendiagnosis and postdiagnosis diet assessment limitations to be considered in interpreting our findings include potential measurement error in the selfreporteddietary and lifestyle data though prior studies in thehpfs and nhs that evaluated the relative validity offfq data have shown reasonably good correlations between ffq and diet records [ ] though we adjusted for several potential confounding variables ahyperinsulinemic dietary pattern may be associated withother factors not included in the current study therefore we cannot completely rule out confounding byunmeasured variables given that we did not have information on cancer treatment which could influence dietary choices of cancer patients or modify the diet andsurvival association we adjusted all analyses by cancerstage at diagnosis which is the principal determinant ofcolorectal cancer treatmentin this large prospective study a higher edih scorereflecting higher insulinemic potential of the diet wasassociated with higher risk of death from colorectalcancer and from all causes taken together our resultssuggest that this association may be mediated partlythrough mechanisms involving hyperinsulinemia interventions with dietary patterns to reduce insulinemia mayenhance survivorship among colorectal cancer patientsabbreviationsbmi body mass index ci confidence interval edih empirical dietary indexfor hyperinsulinemia score ffq food frequency questionnairehpfs health professionals followup study hr hazard ratio methourweek metabolic equivalent hours per week nhs nurses health study nsaids nonsteroidal antiinflammatory drugs pi3kakt phosphatidylinositol kinaseprotein kinase b sas® statistical analysis software®acknowledgementswe would like to thank the participants and staff of the nurses health studyand health professionals followup study for their valuable contributions asw
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"Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunityNatasha D Sheybani1 Alexandra R Witter2 Eric A Thim1 Hideo Yagita3 Timothy N J Bullock Richard J Price To cite Sheybani a0ND Witter a0AR Thim a0EA et a0al Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunity Journal for ImmunoTherapy of Cancer 20208e001008 101136jitc2020001008 º Additional material is published online only To view please visit the journal online http dx jitc NDS and ARW contributed equallyAccepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Biomedical Engineering University of Virginia Charlottesville Virginia USA2Pathology University of Virginia Charlottesville Virginia USA3Department of Immunology Juntendo University Graduate School of Medicine Bunkyo ku Tokyo Japan4Radiology Medical Imaging University of Virginia Charlottesville Virginia USACorrespondence toDr Richard J Price rprice virginia eduDr Timothy N J Bullock tb5v virginia eduBackground Triple negative breast cancer TNBC remains recalcitrant to most targeted therapy approaches However recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy We therefore tested a strategy for immune sensitization of murine TNBC 4T1 tumors through combination of focused ultrasound FUS thermal ablation and a chemotherapy gemcitabine GEM known to attenuate myeloid derived suppressor cells MDSCsMethods We applied a sparse scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity We also tested this combination in Rag1 mice or T cell depleted wild type mice to determine the essentiality of adaptive immunity Further we layered Programmed cell death protein PD1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalResults The immune modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1 consistent with the dominant MDSC driven immunosuppression evident in this model The combination of FUSGEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice Tumor control correlated with increased circulating antigen experienced T cells and was entirely dependent on T cell mediated immunity The ability of FUSGEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti PD1Conclusion Thermally ablative FUS in combination with GEM restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic TNBC model This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluationTrial registration numbers NCT03237572 and NCT04116320BACKGROUNDMetastatic breast cancer BrCa particularly the triple negative breast cancer TNBC phenotype is resistant to most chemical and molecularly targeted therapeutic approaches Interestingly TNBC is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 Early studies in the use of immunotherapies targeting the PD1Programmed death ligand PD L1 checkpoint inhibitory axis showed some efficacy2 in TNBC compared with other BrCa subtypes which are generally recalcitrant to checkpoint blockade Activity in the TNBC subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in TNBC Greater immunotherapy efficacy in TNBC has been recently observed with the use of antibodies targeting the PD1PD L1 checkpoint inhibitory axis in combination with Nab paclitaxel5 This outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment TME found in TNBCAmong the potential networks in TNBC that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets These have the capacity to impair adaptive immunity and promote tumor growth and metastasis Among these cell types myeloid derived suppressor cells MDSCs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both T cell activation and effector functions6 Increased levels of this cell type have been demonstrated in tumor tissues of patients with primary BrCa while those with metastatic disease bear the highest abundance of circulating MDSCs8 Studies have Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate MDSC can improve antitumor immunity9To this end the central premise put forth in this study is that focused ultrasound FUSa safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissuescan synergize with immunotherapy in a murine model of metastatic TNBC FUS is capable of rapidly heating tumors to thermally ablative temperatures Its extracorporeal application obviates the need for catheterization injection or implantation FUS can be targeted with millimeter precision under MRI or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues The bioeffects of FUS hold distinct implications for tumor antigenicity immune cell activation and trafficking13 Thermally active FUS regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17 colon20 kidney21 and BrCa23 Pertaining to the challenge of myeloid cell immunosuppression in TNBC thermally ablative FUS has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin IL12 interferonÎ IFNÎ and tumor necrosis factorα TNFα from a variety of cancer cell lines and after in vivo treatment of tumors26 Whether the ability of FUS to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of BrCa is currently under debate with some studies showing activation of antigen presenting cells and T cell recruitment in patients with BrCa treated with thermally ablative FUS28 while others show that additional innate stimuli are needed to support antitumor immunity23 Notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells DCs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31Based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation FUS regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine TNBC with extensive granulocytic MDSC involvement that is recalcitrant to anti PD1 While some activity is evident with the partial ablation approach significantly greater control was achieved by targeting MDSC inhibition in combination with thermally ablative FUS This control was completely dependent on the adaptive immune responseMoreover we demonstrate that layering anti PD1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction These data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative FUS Once immunosuppressive myeloid cells are accounted for FUS treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockadeMETHODSCell line maintenance4T1 and E0771 cell lines were maintained in RPMI L glut or Dulbeccos Modified Eagles Medium DMEM gL D glucose L glutamine respectively supplemented with Fetal Bovine Serum FBS at °C and CO2 Thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments Cells tested negative for mycoplasmaEight week old to week old female BALBc or C57Bl6 mice were obtained from NCI Charles River NCI CRL or The Jackson Laboratory Female BALBc Rag1 mice were obtained from The Jackson Laboratory 4T1 or E0771 cells à were subcutaneously implanted into the right flank of mice Mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum Tumor outgrowth was monitored via digital caliper measurements Tumor volume was calculated as follows volume lengthÃwidth22 Approximately days 4T1 or days E0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsIn vivo ultrasoundguided FUS partial thermal ablationMice were treated with FUS either days 4T1 cohorts or days E0771 postimplantation On treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg Zoetis and dexdomitor mgkg Pfizer in sterilized saline Mouse flanks were shaved and depilated following which ultrasound guided FUS thermal ablation was performed using one of the two systems System and treatment details are provided in online supplementary materials and methods Mice that did not receive FUS treatment consistently underwent anesthesia and depilation of the flank Additionally these mice underwent a sham treatment consisting of exposure to the °C degassed water bath exposure for min Following sham or FUS treatment all mice were moved to a heating pad and given Antisedan for anesthesia reversal and recoveryGemcitabine therapyGemcitabine GEM mgmouse in µL volume Mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of FUS treatment following which administration was repeated for an additional weeks Administration of GEM doses was based on existing literature demonstrating the use of GEM for inhibition of MDSCs in 4T112 The initial dose of GEM was administered immediately prior to sham or FUS treatment Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cMice that did not receive GEM received an intraperitoneal injection of vehicle treatment µL of sterile saline at the time points specifiedPD1 blockade therapyFor checkpoint inhibitor therapy the rat anti mouse PD1 antibody αPD1 RMP114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse Treatment was initiated on day early αPD1 or day delayed αPD1T cell depletionsT cell depletion antibodiesanti CD8 clone Bio X Cell and anti CD4 GK15 clone Bio X Cellwere diluted in sterilized saline and administered intraperitoneally every to days starting at day days post FUS for a total of seven doses µg of each antibody for a total µg per mouseImmunohistochemistryOn day sham or FUS exposed tumors were excised and fixed in neutral buffered formalin Sigma Fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin Digital images of stained slides were acquired using the Vectra Automated Quantitative Pathology Imaging System Akoya Biosciences Whole slide screening and image capture were subsequently performed using Phenochart Akoya BiosciencesFlow cytometryMice were bled at days and via tail vein and samples were RBC lysed Hybri Max Sigma and stained for flow cytometry analysis At days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment In order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse CD45 FITC clone F11 BD Biosciences min prior to euthanasia 4T1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor DLNs pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis Additional details are provided in online supplementary materials and methodsSamples were acquired on an Attune NxT flow cytometer ThermoFisher Scientific and data were analyzed with FlowJo TreeStar or FCS Express De Novo Software A representative gating strategy for granulocytic myeloid derived suppressor cell G MDSC and CD44 T cells is provided in online supplementary figure Statistical analysisAll statistical analyses were performed in GraphPad Prism GraphPad Software A detailed description of statistical methods for each experiment is provided in the corresponding figure legend accessAnimal study approvalAll animal work was performed under a protocol approved by the Animal Care and Use Committee at the University of Virginia and conformed to the National Institutes of Health guidelines for the use of animals in researchRESULTSPartial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsetsTo achieve partial thermal ablation of 4T1 tumors we used an ultrasound guided FUS system equipped with a single element therapeutic transducer driven at MHz figure 1A online supplementary figure A grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under B mode ultrasound guidance figure 1BC The exceptionally small focus of this system rendered a low ablation fraction of total tumor volume Immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1D One week following FUS partial thermal ablation tumors and secondary lymphoid ans were excised for immunological characterization by flow cytometry figure 1B FUS partial thermal ablation of 4T1 tumors conferred a significant increase fold in the absolute number of CD11c hi DCs within the axillary tumor draining lymph node aDLN of mice figure 1E While this was accompanied by a nearly threefold elevation in the absolute number of CD86 DCs within the aDLN figure 1F the percentage of DCs expressing CD86 did not change figure 1G Increased numbers of DCsand CD86 DCs in particularsuggest FUS is promoting the maturation or trafficking of these cells in the DLNs where they could encounter and activate T cells However this did not translate to tumor growth restriction data not shown We also did not observe significant differences in the absolute number of activated T cells in 4T1 tumors figure 1H or DLNs data not shown following FUS exposure suggesting limitations in the ability of FUS activated DC to further drive an antitumor T cell responseImmune profiling by flow cytometry revealed that irrespective of FUS exposure of the intratumoral CD45 immune cell population is comprised of CD11b myeloid cells figure 1I Similarly approximately of the circulating immune cell population in 4T1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure Notably Ly6G granulocytic myeloid derived suppressor cells G MDSCs significantly dominated the immune cell repertoire within 4T1 tumors relative to other myeloid including F480 macrophages Ly6C cell subsets monocytic myeloid derived suppressor cells M MDSCs and CD11c hi DCs figure 1J FUS partial thermal ablation did not significantly alter the absolute number per Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Partial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsets A Design overview of a custom ultrasound guided FUS system consisting of a MHz single element transducer orthogonally co registered to an MHz linear ultrasound imaging array The tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °C Sham mice were similarly positioned but did not undergo sonications B Schematic illustration of FUS partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4T1 tumor bearing mice A grid of sonications was applied in a raster pattern onto the B mode ultrasound visible tumor In total two planes of sonication spaced mm apart were applied to each tumor Grid points were spaced mm apart within a single plane One week following thermal ablation tumors and secondary lymphoid ans were excised for sham n6 or FUS treated n5 mice and processed for flow cytometry C Representative B mode ultrasound images of ectopic 4T1 tumors either before top or during bottom FUS exposure Sonication grid depicting targets red points is superimposed on B mode image during treatment Subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed D Representative HE staining of either sham 4T1 tumors or those resected immediately following FUS partial thermal ablation Zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively E Absolute number of CD11c hi DCs in the axillary tumor draining lymph node aDLN of 4T1 tumor bearing mice p00136 vs sham F Absolute number of CD86 CD11c hi DCs in the aDLN p00063 vs sham G Percentage of CD86 subset out of total CD11c hi DCs within aDLN H Absolute number of intratumoral CD44 CD8 and CD44 CD4 T cells and regulatory T cells Tregs per gram tumor I Percentage of CD11b myeloid cells out of total CD45 immune cells across tumor spleen aDLN inguinal DLN iDLN and nontumor draining axillary and inguinal LNs nDLNs p005 vs all other groups irrespective of FUS exposure specifically tumor vs spleen p00226 tumor spleen vs all other ans p00001 J Absolute number of intratumoral myeloid cells CD11c hi DCs F480 macrophages Ly6C monocytic myeloid derived suppressor cells M MDSCs Ly6G granulocytic myeloid derived suppressor cells G MDSCs per gram 4T1 tumor p00001 vs all other cell types irrespective of FUS exposure All data represented as mean±SEM Significance assessed by unpaired t test FH or two way analysis of variance followed by Tukey multiple comparison correction IK nsnot significant DCs dendritic cells FUS focused ultrasound HIFU high intensityfocused ultrasoundSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets These observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4T1 TME must be addressed in order to facilitate the T cell response to FUSFUS partial thermal ablation in combination with GEM constrains primary TNBC tumor outgrowth and extends overall survivalOur observation of the overwhelming MDSC burden following 4T1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier To this end we tested a combinatorial paradigm incorporating GEM a myelosuppressive chemotherapy demonstrated to inhibit MDSCs transiently in the 4T1 model without consequence to T cell phenotype or function12To evaluate the efficacy of FUS and GEM in combination we used a preclinical ultrasound guided FUS system to achieve partial thermal ablation of established 4T1 tumors 14d after tumor implantation average tumor volume of mm3 In combination with the single session of FUS thermal ablation we initiated GEM therapy mgmouse which was then readministered weekly for a total of three GEM doses figure 2A Combinatorial therapy synergized to produce significant constraint of 4T1 tumor outgrowth compared with sham and monotherapy groups figure 2BCBy termination of treatments at day 4T1 tumors exposed to FUSGEM combination saw nearly à and à reductions in average volume compared with sham or GEM exposed tumors respectively figure 2B Two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2DE In a fraction of mice treated with FUSGEM we observed complete regression of 4T1 tumors although transient figure 2C tumor outgrowth eventually rebounded after termination of treatments 4T1 tumor bearing mice receiving FUSGEM treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and GEM groups respectively HRs and for FUSGEM relative to sham and GEM groups respectively figure 2F We additionally observed that FUSGEM significantly constrained outgrowth in a separate C57Bl6 metastatic mammary carcinoma model E0771 online supplementary figure To further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided FUS system Theraclion Echopulse that is already CE marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging FUS thermal ablation in combination with cancer immunotherapy We observed that partial thermal ablation using the Theraclion visualization and treatment unit MHz in combination accesswith GEM controlled 4T1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure These findings lend credence to the notion that the impact of combining GEM with FUS may be conserved across partial thermal ablation regimens Moreover they demonstrate that the efficacy of FUS partial thermal ablation in combination with GEM can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallyCombination of FUS partial thermal ablation with GEM increases the levels of circulating T cellsLymphocytesin particular CD8 and CD4 T cellsplay an important role in responding to tumor antigen and generating a durable antitumor response Based on the extended protective effect observed in mice treated with FUSGEM flow cytometry analysis was performed to evaluate the contribution of T cells in generating systemic and local tumor control We sampled the circulating immune cell repertoire in 4T1 tumor bearing mice via serial tail bleeds days and prior to readministration of GEM and a terminal cardiac bleed at the time of spleen harvest day figure 3A Combinatorial therapy significantly elevated absolute number of CD8 and CD4 T cells in the circulation at days and figure 3BC and EF Moreover a trend threefold to fivefold increase in circulating T cells was noted in the FUS group relative to sham figure 3BC and EF From days to systemic CD44 expressing antigen experienced T cell populations both CD8 and CD4 saw a steady significant increase after combinatorial therapy figure 3D and G A similar modest trend was noted for the FUS monotherapy group relative to sham and GEM figure 3D and G These changes were concordant with a decrease in circulating myeloid CD11b cells in GEM recipient groups demonstrating the ability of GEM to partially alleviate circulating myeloid burden figure 3HSplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4T1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression We observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6AB Consistent with this observation immunological characterization of spleens revealed a significant decrease in CD11b myeloid cellsa reduction in FUSGEM spleens relative to sham or monotherapy figure 3I While there appeared to be a trend toward more CD11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute CD11b cell numbers within the spleen data not shown The decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following FUSGEM treatment Relative to these sham and GEM groups combination therapy elevated splenic CD8 T lymphocytes by fold and Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM constrains primary triple negative breast cancer outgrowth and extends overall survival A Overview of experimental design for evaluation combination of FUS with serial GEM treatment in murine mammary carcinoma B Average 4T1 tumor outgrowth in sham n7 FUS monotherapy n5 GEM monotherapy n10 and combinatorial FUSGEM therapy groups n10 Data are represented up to select time points corresponding with mouse dropout due to humane endpoints All data represented as mean±SEM Significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Tukey multiple comparison correction p005 vs all other groups specifically sham vs FUSGEM p00001 FUS vs FUSGEM p00001 shamGEM vs FUSGEM p00026 C 4T1 tumor outgrowth from individual mice in sham FUS shamGEM or FUSGEM groups Data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint D Representative images of 4T1 tumors excised at day Scale bar1 cm E Quantification of 2D tumor areas from images in previous panel F Kaplan Meier curve depicting overall survival of sham treatment n9 FUS monotherapy n6 GEM monotherapy n10 and combinatorial FUSGEM therapy n10 recipient mice Significance assessed by log rank Mantel Cox test p005 vs all other groups specifically sham vs FUS p02154 sham vs FUSGEM p00001 sham vs shamGEM p00050 FUS vs FUSGEM p00021 FUS vs shamGEM p00312 FUSGEM vs shamGEM p00041Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c accessFigure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM increases the levels of circulating T cells A Overview of experimental design to understand the impact of FUS andor GEM treatment on circulating immune cells BC Absolute number of circulating CD8 T cells at day B and day C D Percentage of circulating CD8 T cells expressing CD44 from days to EF Absolute number of circulating CD4 T cells at day E and day F G Percentage of circulating CD4 T cells expressing CD44 from days to H Percentage of CD11b myeloid cells out of total CD45 immune cell in circulation from days to IK Percentage of myeloid cells I CD8 T cells J and CD4 T cells K out of total CD452 immune cells All data represented as mean±SEM All data representative of sham n6 FUS monotherapy n4 GEM monotherapy n9 and combinatorial FUSGEM therapy n6 groups Significance assessed by analysis of variance followed by Tukey multiple comparison correction for B C E F or Fishers least significant difference LSD without multiple comparisons correction for IK Significance for D G and H assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Fishers LSD without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access fold figure 3J and CD4 T lymphocytes by fold and fold figure 3K These elevations were accompanied by a modest increase in percentage of Foxp3 regulatory T cells Tregs online supplementary figure 6E Additionally increases in percentage of NK and B cells were noted twofold to fivefold online supplementary figure 6CD These findings indicate that combinatorial therapy with FUSGEM promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasesCombinatorial FUSGEM therapy does not promote robust local antitumor T cell responsesGiven the robust systemic immune signatures within the blood and spleen following FUSGEM we assayed 4T1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral T cell response figure 4A Approximately hours prior to euthanasia mice received intravenous brefeldin A injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry Immune characterization of tumors at days postimplantationthat is days subsequent to final GEM administrationrevealed no significant changes in absolute number of antigen experienced CD44 CD8 or CD4 T lymphocytes figure 4BC Moreover the polyfunctionality of these T cells as denoted by IFNÎ and granzyme B expression was not significantly altered figure 4DE However intratumoral functional changes were noted in the myeloid compartment GEM monotherapy modestly increased IL 12p40 production by DCs fold but this was not conserved in the combinatorial therapy group figure 4F Moreover while FUS monotherapy generated a trend in elevated TNFα production by intratumoral G MDSCs GEM recipient groups saw a significant increase threefold relative to sham figure 4G These findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely Interestingly intratumoral T cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionProtection conferred by combination of FUS and GEM is dependent on adaptive immunitySince our findings revealed no obvious advantage or function of adaptive immunity in the local TME we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with FUSGEM To this end we utilized an Rag1 model that is deficient in T and B cells to address the hypothesis that mature T andor B cells play a role in the observed response Wild type WT or Rag1 mice bearing 4T1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes Mice were subsequently treated with either GEM monotherapy or the combination of FUSGEM The tumor growth inhibition offered by FUSGEM was entirely lost in Rag1 mice relative to their WT counterparts with average 4T1 tumor volume in Rag1 mice being over fivefold higher than that of WT mice on terminati
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Intervertebral disc degeneration IDD is the most common degenerative disease all over the word Our previous studyconï¬rmed that the downregulated circGRB10 directly interacts with miR3285p which modulate ERBB2 and leads tothe degeneration of intervertebral disc however the underpinning mechanism of circGRB10 dysregulation remainsunclear We identiï¬ed that FUS and demonstrated that circGBR10 biosynthesis in nucleus pulposus NP cells waspromoted by FUS whose expression was controlled by miR1413p In addition ERBB2 downregulation led todecreased Erk12 phosphorylation which enhanced miR1413p production in NP cells In vivo data indicated that circGRB10 inhibited IDD in rat model The present study revealed that miR1413p and FUS are key factors that regulatecircGRB10 synthesis in NP cells In addition circGBR10 participates in the molecular circuitry that controls human IDDdevelopment These ï¬ndings provide a basis for further functional diagnostic and therapeutic studies of circGRB10in IDDIntroductionThe Global Burden of Disease Study stated that lowback pain LBP represents an important cause of disability worldwide1 LBP is tightly associated with intervertebral disc degeneration IDD which involves ofall LBP cases causing significant economic and socialburdens worldwide23 According to previous reports of the world population have low back pain during theirlifetime with being chronically disabled4Currently IDD pathogenesis is largely unclear howeverit could be due to microenvironmental alterations in theintervertebral discs caused by various factors such asgenetic features aging sex a predisposing injury and theCorrespondence Wei Guo guow0319163com1Department of Orthopaedics Hebei Province Cangzhou Hospital ofIntegrated Traditional and Western Medicine Cangzhou No2 Hospital Huanghe Road Cangzhou Hebei Province P R China2Department of Breast Surgery Hebei Province Cangzhou Hospital ofIntegrated Traditional and Western Medicine Cangzhou No2 Hospital Huanghe Road Cangzhou Hebei Province P R ChinaFull list of author information is available at the end of the Edited by G Calinenvironment56 The main feature of IDD pathology iselevated biosynthesis of catabolic enzymes combined withreduced extracellular matrix ECM accumulation causedby imbalanced anabolism and catabolism7 Intervertebraldiscs comprise a central nucleus pulposus NP a peripheral annulus ï¬brosus AF and cartilaginous endplates which connect overlying capillary beds craniallyand caudally The NP maintains homeostasis by producing an ECM mostly comprising type II collagen andproteoglycans the main functional components of intervertebral discs which are indispensable to maintain thedisc height and absorb various mechanical loads8 It iswell known that loss of collagenII and aggrecan is anearly critical event in the degenerative cascade in Intervertebral disc tissue9 MMP13 is the most importantenzymes that hydrolyze collagens10 ADAMTS5 is classiï¬ed as the major aggrecanases due to their high efï¬ciency in cleaving aggrecan11 A large body of evidencesupporting the involvement of MMP13 and ADAMTS5in IDD pathogenesis12 During IDD the main histologicalalteration involves the centrally located NP cells which The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of after a phenotypic transformation are substituted bysmaller ï¬brochondrocytelike cells with reduced proteoglycan production and a global shift towards synthesizing ï¬brotic materials and compromising the structuralintegrity of discs1314 Therefore unveiling the mechanisms underpinning such imbalance is urgently needed forthe development of new therapeutic targets in IDDMounting evidence supports roles for circular RNAscircRNAs in IDD15 Previous research demonstratedthat circRNAs are closed RNAs produced by backsplicingof single premRNAs18 It is not completely known howcircRNAs are biosynthesized although complementaritybetween inverted sequences in ï¬anking introns and theactivity of RNAbinding proteins RBPs increase thecontiguity of splice sites contributing to backsplicing inmammalian cells19The RBP FUS affects splicing regulation23 with manysplicing factors termed FUS interactors24 FUS mutations could lead to protein mislocalization to the cytosolwith decreased nuclear FUS amounts and occurrence ofabnormal cytosolic aggregates2728 The FUS protein isinvolved in regulating intracellular RNA transport mRNAsynthesis alternative splicing and polyadenylation siteselection29 He demonstrated that FUS combinedwith circ_002136 and promoted the generation ofcirc_002136 in Glioma30 It was recently shown that FUScontrols the expression of circRNAs by binding tointrons that ï¬ank the splicing junction31 Moreover FUSwas reported to be regulated by many miRNAs includingmiR1413p3233 Studies revealed miR1413p is upregulated in NP tissue specimens from IDD cases anddemonstrated that miR1413p is associated with discdegeneration34 However the function and mechanism ofFUS as well as the interaction between FUS and miR1413p in IDD have not been reportedOur previous research conï¬rmed that circGRB10amounts are markedly reduced in NP cells from IDDpatients which accelerates IDD development by enhancing miR3285p mediated ERBB2 suppression in NPcells15 However the mechanism of circGRB10 downregulation in degenerative NP cells remains unclear Inthis study we demonstrated that the miR1413p whichis significantly increased in degenerative NP cells34 regulate expression of the FUS which is responsible for thegeneration of circGRB10 in NP cells Furthermore weshowed that ERBB2 downregulation led to decreasedErk12 phosphorylation and the decreased levels of Erk1 phosphorylation enhanced miR1413p biogenesis indegenerative NP cells promoting IDD developmentTaken togetherthese ï¬ndings suggested circGBR10contributes to the molecular circuitry controlling IDDdevelopment in humansOfï¬cial journal of the Cell Death Differentiation AssociationResultsCircGRB10 regulates NP cell functions through the ERBB2Erk signaling pathwayOur previous study revealed circGRB10 promotes NPcell survival by increasing ERBB2 amounts via suppression of miR3285p However the effect of circGRB10expression on NP cell anabolism or catabolism remainsobscure To further assess circGBR10s functions in IDDpathogenesis circGRB10 or circGRB10 small interferingRNA siRNA was transiently transfected into culturedprimary human NP cells As shown in Supplementary FigS1 overexpression and knockdown of circGRB10 haveno effect on linear GRB10 but only affect circular GRB10The immunoï¬uorescence results demonstrated that afteroverexpressing circGRB10 in NP cellssignificantlyupregulation of collagen II and aggrecan and decreasedamounts of MMP13 and ADAMTS5 were found Conversely circGRB10 knockdown resulted in oppositeeffects Fig 1a b These ï¬ndings were conï¬rmed by qRTPCR Fig 1csignificantlyincreased while MMP13Our previous research demonstrated circGRB10 inhibits IDD development by regulating ERBB2 expression inNP cells Increasing evidence supports an important rolefor the ERBB2 gene and Erk signaling pathways in theprogression of many human diseases35 Meanwhile theErk pathway is altered during IDD38 and plays a significant role in extracellular metabolism39 These resultsprompted us to assess the plausible association of circGRB10 with ERBB2 Erk signaling In this study primaryhuman NP cells underwent transfection with circGBR10circGRB10 siRNA and respective negative controlsrespectively As shown in Fig 1d e western blot assayshowed that pErk12 collagen II and aggrecan amountswereandAMADT5 levels were reduced in NP cells overexpressingandcircGRB10 Conversely pErk12aggrecan were downregulatedandAMADT5 amounts were increased in NP cells transfected with circGRB10 siRNA Fig 1d e FurthermoreERBB2 affected pErk12 in a similar way as circGRB10Fig 1d e suggesting cricGRB10 modulates IDD progression via ERBB2Erk signaling Therefore in order tofurther validated whether ERBB2 was the downstreammediator of circGRB10 in the NP cells We cotransfectedcircGRB10 and ERBB2 siRNA into NP cells andobserved that the positive effects of circGRB10 on NPcells functions were attenuated in the absence of ERBB2Fig 1f g Moreover upregulation of ERBB2 counteracted the inhibitory effect of circGRB10 knockdown onNP cells function Fig 1hi Collectively the aboveï¬ndings indicated that circGRB10 associated protectionin IDD may involve ERBB2Erk signalingcollagen IIand MMP13 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see ï¬gure on previous pageFig circGRB10 regulates NP cell functions through ERBB2Erk signal pathway a CollagenII aggrecan MMP13 ADAMT5 expression wereanalyzed in circGRB10 or circGRB10 siRNA transfected cultured primary human NP cells using Immunoï¬uorescence staining analysis b Thecorresponding bar graphs show quantitative analysis of the relative ï¬uorescent value of each group n replicates per group p p Scale bar μm c qRTPCR showing the expression levels of collagen II aggrecan MMP13 ADAMT in human NP cells after circGRB10overexpression or knockdown Three independent experiments are presented as mean ± SEM error bars P P d The expressionlevels of CollagenII aggrecan MMP13 ADAMT5 and pErk12 were detected by western blot Quantitative analysis was shown in e and threeindependent repeats were performed in each experiment p f NP cell were cotransfected with circGRB10 and ERBB2 siRNA Western blotassay showed that ERBB2 siRNA blocked the effect of circGRB10 on CollagenII aggrecan MMP13 and ADAMT5 expression Quantitative analysis wasshown in g and three independent repeats were performed in each experiment p h NP cell were cotransfected with circGRB10 siRNAand ERBB2 Western blot assay showed that ERBB2 attenuated the effect of circGRB10 siRNA on CollagenII aggrecan MMP13 and ADAMT5expression Quantitative analysis was shown in i and three independent repeats were performed in each experiment p Key factors regulating circGRB10 formationIn this study we found a highly reverse complementarysequence nt upstream the ² splice site in intron andone nt downstream the ² splice site in intron which were named 2RC reversecomplementarysequence in intron and 6RC reverse complementarysequence in intron respectively Then wildtypesequence spanning from intron to intron of theGRB10 gene and multiple deletion constructs for circGRB10 were introduced into pcDNA31respectively Fig 2a Upon transfection the wildtypevector unlike the 2RC andor 6RC deletion constructs overexpressed circGRB10 indicating 2RCand 6RC may contain the binding sites that regulate circGRB10 biogenesis Fig 2bcould beregulated by RBPs postAs circRNAs are derived from premRNAs and circRNAstranscriptionally18212240 we hypothesized that circGBR10 ismodulated by RBPs posttranscriptionally in IDD development To identify the RBPs which might regulateGRB10 premRNA splicing to generate circGRB10 weincubated biotin labeled sequences cloned from circGRB10 back splicing site nt upstream P1 or ntdownstream P2 with nuclear protein extracts fromnormal human NP cells Fig 2c Nuclear proteins boundto RNA underwent separation by SDSPAGE and silverstaining Fig 2dfollowed by mass spectrometry foridentiï¬cation A total of proteins SupplementaryTable S1 were retrieved and mapped to the STRINGdatabase screening significant interactions with scoresabove Fig 2e Enrichment analysis demonstratedthat these proteins were mainly involved in thepathways of gene expression processing of capped introncontaining premRNA mRNA splicing mRNA splicingmajor pathway mRNA processing and formation andmaturation of mRNA transcript related signaling pathways Fig 2f Among these proteins were involved inthe mRNA splicing and mRNA splicingmajor pathwaySupplementary Table S2 In addition the web tool CircInteractome predicted RBPs which can potentiallybind circGRB10 premRNA Fig 2g Notably FUS wasOfï¬cial journal of the Cell Death Differentiation Associationthe only RBP that was involved in mRNA splicing andcould potentially bind to circGRB10 premRNA suggesting circGRB10 generation may be associated withFUS expression in NP cellsFUS promotes the generation of circGRB10 in NP cellsRecently FUS was reported to have a role in regulatingcircRNA biosynthesis via binding of introns surroundingthe backsplicing junctions31 As shown in Fig 3A FUSamounts in IDD NP tissues were remarkably lower thanthose of controls In addition Western blot further conï¬rmed this result Figs 3b and S2 To assess whetherFUS contributes to circGRB10 production in NP cells weoverexpressed or suppressed FUS and determined circGRB10 amounts qRTPCR demonstrated that FUSoverexpression led to significantly increased circGRB10amounts in NP cells while FUS knockdown reduced theexpression of circGRB10 Fig 3c Moreover FUS had noeffects on linear GRB10 expression Fig 3c Overexpression of FUS resulted in increased collagenII andaggrecan amounts and decreased MMP13 and ADAMT levels in NP cells while the circGRB10 siRNA attenuated these changes Fig 3d FUS knockdown resultedin downregulated collagenII and aggrecan and upregulated MMP13 and ADAMT5 in NP cells while circGRB10 markedly counteracted the effects of FUS knockdown indicating that FUS exerted its functions throughcircGRB10 Fig 3eTo assess whether FUSbinding sequences are important in circGRB10 biosynthesis FUSbinding sequenceswere searched in circGRB10 and surrounding intronsand two putative FUSbinding sites were detected Fig3f Next two short circGRB10 minigenes were engineeredincluding circGRB10s and circGRB10sEmPrecisely circGRB10s comprises presumed FUSbingingsites on both ï¬anking introns preserved with the inverselyinserted ²intron in circGRB10 removed to preventcomplementary sequences from reacting Fig 3f circGRB10sEm resembles circGRB10s but with FUS sitesdeleted from the surrounding introns Fig 3f RIPrevealed an overt interaction of FUS with circGRB10s 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see ï¬gure on previous pageFig RBPs interact with GRB10 premRNA a A schematic drawing of four types of circGRB10 overexpressing vectors to The genomicregion for circGBR10 green bars with its wildtype ï¬anking introns black lines was inserted into the pcDNA31 expression vector 2RC and 6RCare indicated by red bars A series of deletions are indicated by black crosses to b qRTPCR showed the expression of circGBR10 aftertransfection with the four types of circGRB10 overexpressing vectors to Three independent repeats were performed in each experimentp c Schematic diagram of RNAs corresponding to different fragments of GRB10 premRNA P1 P2 produced by in vitro transcription inthe presence of biotin for RNA pulldown experiments d Silver stain acrylamide gel of total nuclear proteins before Input and after pulldown withbiotinlabeled RNA probe P1 P2 M molecular weight marker kDa e Proteins identiï¬ed from mass spectrometry were integrated to STRINGdatabase and constructed Proteinprotein interaction PPI network A densely connected module which contains proteins including FUS wasscreened from the PPI network and these proteins were participate in biological process of mRNA splicing via spliceosome f Pathways enrichmentanalysis of proteins in PPI network g RBPs which can potentially bind circGRB10 premRNAunlike circBsEm Fig 3g indicating FUS required theputative sites in surrounding introns for binding We nextexpressed circGBR10sin FUS overexpressing orknocked down NP cells and circGRB10s yielded elevated amounts of circGRB10 transcript after FUS overexpression and reduced amounts upon FUS knockdownconï¬rming FUS is important in circGRB10 biosynthesisin NP cellsFig 3h Next circGRB10sEm wasexpressed in NP cells and it yielded markedly reducedcircGRB10 amounts in comparison with circGRB10sFig 3h This indicated that the putative FUSbindingsequences in the surrounding introns were important incircGRB10 biosynthesis Taken togetherthe aboveï¬ndings demonstrated that FUS had a critical regulatoryfunction in circGRB10 biosynthesis in NP cells viabinding to recognition sites in the introns surrounding thecircGRB10forming exonsFUS in NP cells is regulated by miR1413pThe mechanism of FUS downregulation in NP cells ofIDD patients remains unclear Previous studies havedemonstrated that FUS is regulated by miRNAs in manydeseases3233 Therefore we hypothesized that FUS maybe regulated by miRNAs in NP cells Using the Targetscan Microt4 miRanda PITA and RNAhybird databasesall predicted miRNAs were retrieved and submitted toVenn analysis Fig 4a The results showed that FUS waspredicted to be regulated by miRNAs SupplementaryTable S3 including miR1413p Svetoni conï¬rmedthat miR1413p regulates FUS expression during neuraldifferentiation and Ji revealed miR1413p is associated with disc degeneration3334 Furthermore qRTPCRshowed that miR1413p was markedly upregulated in NPtissue samples from IDD cases in comparison with controlvalues Fig 4b Therefore we supposed that FUSexpression was regulated by miR1413p in NP cells Tofurther assess miR1413p interaction with FUS luciferasereporter assays were carried out Cotransfection of FUSWT wild type and miR1413p mimic in primary humanNP cells resulted in markedly decreased luciferase activityin comparison with the FUSmut mutantmiR1413pmimic cotransfection group Fig 4c d These ï¬ndingsOfï¬cial journal of the Cell Death Differentiation Associationwere further conï¬rmed at the gene and protein levels inhuman NP cells in vitro Fig 4e f pointing to FUS as amiR1413p target Then primary human NP cellsunderwent transfection with miR1413p mimic and miR1413p inhibitor and the corresponding negative controls respectively The results showed that circGBR10was significantly downregulated in cells overexpressingmiR1413p Fig 4g Conversely circGRB10 was upregulated in the miR1413p inhibitor group Fig 4gMoreover upregulation of FUS alleviated the suppressiveeffects of miR1413p on circGRB10 expression Fig 4hwhile FUS knockdown attenuated the effects of miR1413p inhibitor on circGRB10 upregulation Fig 4i Theabove results indicated that miR1413p regulates circGRB10 expression in NP cells primarily through targetingof FUSERBB2 regulates miR1413p in NP cells byphosphorylating Erk12Induced Erk12 signaling causes widespread miRNArepression via suppression of the main steps of miRNAbiogenesis4142 In this study we found decreased levels ofErk12 phosphorylation in circGRB10 or ERBB2 knockeddown NP cells Fig 1d e Previous studies demonstratedthat phosphorylated Erk12 can cause widespreadmiRNA repression through suppressing the major stepsof miRNA biogenesis41 As ERBB2 amounts werereduced in degenerative NP cells we hypothesized thatmiR1413p may be regulated by Erk12 phosphorylationin NP cells To explore this possibility we overexpressedor knocked down ERBB2 in NP cells qRTPCR resultsdemonstrated that overexpression of ERBB2 significantlydownregulated miR1413p while ERBB2 knockdownincreased miR1413p amounts Fig 5a In addition pretreatment of NP cells with the Erk12 phosphorylationinhibitor U0126 downregulated ERBB2 and attenuatedERBB2 induced phosphorylation of Erk12 decreasing theexpression of FUS Fig 5b Moreover blocking Erk12phosphorylation in NP cellssignificantly attenuatedERBB2s effects on miR1413p biogenesis Fig 5c anddecrease the expression of circGRB10 Fig 5d Collectivelythe above ï¬ndings demonstrated that ERBB2 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see ï¬gure on previous pageFig FUS regulates the generation of circGRB10 in NP cells a qRTPCR showing FUS mRNA levels in normal and IDD NP tissues Threeindependent repeats were performed in each experiment p b Western blot showing FUS protein amounts were decreased in IDD NPtissues c qRTPCR analysis of circGRB10 expression level after FUS overexpression or knockdown in NP cells FUS overexpression led to significantlyincreased circGRB10 amounts in NP cells while its knockdown reduced circGRB10 levels Moreover FUS had no linear effects on GRB10 expressionThree independent repeats were performed in each experiment p d e qRTPCR analysis of the expression of CollagenII aggrecan MMP and ADAMT5 in NP cells f Schematic illustrating the putative FUSbinding sites on the ï¬anking introns in the circGRB10s minigene The ²terminus of the circular exons of circGRB10 was deï¬ned as position Putative FUSbinding sites A and B are located in the intron at the ² terminusof the circGRB10 exon position to and on the intron at the ² terminus of the circGRB10 exon position g RIP analysis ofFUSbinding to circGRB10s and circGRB10sEm minigenes in NP cells Bound complexes were pulleddown using an antibody against FUS qRTPCRwas then used to measure circGRB10s binding to FUS Values were normalized to the level of background RIP as detected by an IgG isotype controlh qRTPCR analysis of the expression of circGRB10 relative to GAPDH in NP cells Cells were cotransfected with FUS or FUS siRNA and a circGRB10minigene circGRB10s or circGRB10 minigene containing deleted FUSbinding sites circGRB10Em Quantitative data from three independentexperiments is presented as mean ± SEM error bars P P regulated miR1413p expression in NP cells by phosphorylating Erk12Next we found that decreased ERBB2 amounts indegenerative NP cells could promote miR1413p generation which suppressed the expression of FUS resultingin circGRB10 downregulation our previousstudydemonstrated that circGRB10 downregulation leads toERBB2 reduction by enhancing miR3285p mediatedsuppression of ERBB2 in NP cells15 These ï¬ndings suggested circGBR10 contributed to the molecular circuitrycontrolling IDD development in humans Fig 5ephosphorylation collagenII and aggrecan upregulationand inhibited the expression of MMP13 ADAMT5 inthe rat model of IDD Fig 6g Moreover Immunoï¬uorescence staining also conï¬rmed that the increasedexpression of collagenII aggrecan and the downregulation of MMP13 ADAMT5 expression in the circGRB10 group compared with noninjection group at weeks Fig 6h Jointly the above ï¬ndings suggested atherapeutic role for circGRB10 in protecting the discsrevealing circGRB10 as a candidate therapeutic target inIDDIntradiscal injection of circGRB10 alleviates IDD in a ratmodelNeedle puncture has been one of the most commonmethods to establish animal models of IDD4445 We havesuccessfully established a rat model of IDD by needlepuncture according to the above methods in this studySupplementary Fig S3 At and days upon modelingadenoviral human circGRB10 was injected into thepunctured intervertebral discs with 33G needles In vivoRNA FISH indicated circGRB10 in the NP region at weeks after surgery Fig 6b CT scan at and weeks revealed progressive disc space narrowing in allpunctured animals and a significant increase in DHI wasnoted at and weeks post surgery in rats treated bycircGRB10 Fig 6c CT scan revealed that overexpression of circGRB10 markedly preserved the progressive disc space narrowing in rat IDD modelFig 6dAnd safranin O staining results demonstrated that overexpression of circGRB10 can inhibit the degradation ofextracellular matrix of NP cells Fig 6e These resultssuggesting circGRB10 exerted protective effects in surgically induced IDD After injection of adenoviral circGBR10 FUS and ERBB2 amounts in degenerative NPtissues were remarkably elevated Fig 6f while miR1413p amounts were decreased Fig 6f In addition injectionof adenoviral circGBR10 alleviated the degenerativealterations ofincluding enhanced Erk12the NPDiscussionNumerous circRNAs are found in the human transcriptome playing critical roles in the regulation of cellfunctions174647 Our previous study showed that circGRB10 downregulation is associated with human NP cellapoptosis15 Howeverthe mechanism of circGRB10dysregulation in IDD has not been previously describedHere we found that FUS regulated and promoted circGRB10 biosynthesis by interacting with its ï¬ankingintrons In addition FUS expression in NP cell wasregulated by miR1413p Our ï¬ndings suggest a regulatory network in NP cells FUS bound to GRB10 premRNA to regulate circGRB10 synthesis while circGRB10 acted as a molecular sponge for miR3285p withsuppressive effects on ERBB2 production and modulatedIDD development downregulation of ERBB2 decreasedErk12 phosphorylation and promoted the generation ofmiR1413p which bound to the ²UTR region of FUS toinhibit its expression constituting a positive feedbackloop promoting intervertebral disc degenerationThe differential expression of circGBR10 betweennormal and degenerative NP tissues indicates that circGRB10 biosynthesis is controlled differently in variouscells with NP cells possessing speciï¬c factors required forcircRNA biogenesis Indeed introns and of the GRB10premRNA had binding sites to regulate circGRB10biogenesis Furthermore multiple RBPs were highlyOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see ï¬gure on previous pageFig miR1413p inhibits FUS expression in NP cells a The Venn diagram indicates the predicted miRNAs regulate FUS expression miR1413p was intersected predicted by different databases b Expression of miR1413p in IDD NP tissues showing that miR1413p expression wassignificantly higher than that of controls Quantitative data from three independent experiments is presented as mean ± SEM error bars P c Sequence alignment of a putative miR1413pbinding site within the ²UTR of FUS mRNA Bottom mutations in the ²UTR of FUS mRNAsequence to create the mutant luciferase reporter constructs d Luciferase reporter assay in NP cells after transfected with scramble oligo or miR1413p mimics Renilla luciferase vector and the reporter constructs Both ï¬reï¬y and Renilla luciferase activities are measured in the same sample Fireï¬yluciferase signals were normalized with Renilla luciferase signals Quantitative data from three independent experiments is presented as mean ± SEMerror bars P e f FUS expression level was detected by qRTPCR western blot in primary human NP cells Three independent experimentsis presented as mean ± SEM error bars P g NP cells from control tissues were transfected with miR1413p mimic or miR1413p inhibitorqRTPCR was used to detect the relative expression level of circGRB10 compared with controls Three independent experiments is presented asmean ± SEM error bars P h NP cells from control tissues were transfected with miR1413p with or without FUS overexpress plasmidqRTPCR was used to detect the relative expression level of circGRB10 compared with controls i miR1413p inhibitor with or without FUS siRNA wastransfected into NP cells from control tissues and the expression level of FUS Three independent experiments is presented as mean ± SEM errorbars P Fig ERBB2 regulate miR1413p expression in NP cells a miR1413p expression level in NP cells with ERBB2 overexpression or ERBB2knockdown Three independent experiments is presented as mean ± SEM error bars P b NP cells overexpressing ERBB2 were treated withU0126 or not for one hour Western blot was used to detect the phosphorylated level of Erk12 c d NP cells overexpressing ERBB2 were treated withU0126 or not qRTPCR was used to detect the expression level of miR1413p and circGRB10 Three independent experiments are presented asmean ± SEM error bars P e Schematic representation of mechanisms by which circGRB10 mediates IDD development On the basis ofï¬ndings described in the manuscript miR1413p downregulates FUS level in NP cells leading to circGRB10 decreased This downregulated circGRB10 in turn enhanced miR3285p mediated suppression of ERBB2 in NP cells leads to decreased Erk12 phosphorylation level And the decreasedErk12 phosphorylation level enhances the generation of miR1413p in NP cellsOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of Fig cricGRB10 attenuates IDD development in vivo a Overview of the experimental setup with injections of circGRB10 or their negativecontrol at and days after surgery b Six weeks after surgery in vivo RNA FISH found circGRB10 located in the NP region Blue ï¬uorescence diamidino2phenylindole DAPI indicating cell nucleus Red ï¬uorescence indicating circGRB10 Scale bar μm c Changes in disc height indexDHI of the indicated groups after needle puncture The DHI was measured at and weeks A significant decrease of the DHI was observedin all puncture groups at week after surgery P A significant increase in DHI was noted at and weeks post surgery in rat treated withcircGBR10 P d CT scan of the indicated groups were obtained weeks after needle puncture e The intervertebral disc degenerationevaluated by Safranin O staining Scale bar µm f qRTPCR showed that the increased levels of FUS ERBB2 and the decreased level of miR1413p in the punctured discs treated with circGRB10 Three independent experiments are presented as mean ± SEM error bars P g Westernblot showing the expression levels of collagen II aggrecan pErk12 MMP13 ADAMT5 in rat NP tissues h Immunostaining for collagenII andaggrecan in IDD model treated by circGRB10 at and weeks Scale bar µmOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of enriched in circGRB10s ï¬anking introns and FUS contributed to circGRB10 biogenesis as shown aboveAlthough FUS induced circGRB10 biosynthesisitssilencing only decreased circGRB10 levels by asshown above It is known that two or more RBPs couldthe synthesis of circRNAs synergistically4048controlwhich might explain the above incomplete suppressionTherefore circGRB10 modulation in NP cells deservesfurther examinationAs shown above altered FUS expression might profoundly affect circGRB10 biogenesis Further deletion ofFUSbinding sequences in the introns ï¬anking of circGBR10 dramatically reduced circGRB10 amounts Takentogether the above ï¬ndings indicate FUS may directlycontrol backsplicing to upregulate circGRB10 in NPcells by interacting with putative binding sequences onboth ï¬anking introns of circGRB10Recently miR141 has been detected in NP tissuesamples from IDD cases and its levels correlate with discdegeneration Therefore miR141 NPs have been used inIDD treatment with commendable efï¬cacy34 As shownabove miR1413p which is a key regulator of IDDdirectly regulated FUS further revealing the FUScircGRB10 axis as an IDDrelated regulatory pathwayAccumulating evidence indicates that Erk signaling hasan important role in IDD394950 In the present study wefound that circGRB10 significantly upregulated collagenII and aggrecan in NP cells and mediated the protectiveeffects in IDD likely via ERBB2Erk signaling There mightalso be cellular pathways that compensate for ERBB2expression after its knockdown For example the longintergenic noncoding RNA lincRNA BCLIN25 upregulates ERBB2 by inducing promoter CpG methylation ofmiR125b resulting in miR125b repression44 A previousstudy indicated the Erk pathway regulates the miRNAgenerating complex43 In additi
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"pd1pdl1 blockade therapy is a promising cancer treatment strategy which has revolutionized the treatmentlandscape of malignancies over the last decade pd1pdl1 blockade therapy has been trialed in a broad range ofmalignancies and achieved clinical success despite the potentially curelike survival benefit only a minority ofpatients are estimated to experience a positive response to pd1pdl1 blockade therapy and the primary oracquired resistance might eventually lead to cancer progression in patients with clinical responses accordingly theresistance to pd1pdl1 blockade remains a significant challenge hindering its further application to overcomethe limitation in therapy resistance substantial effort has been made to improve or develop novel antipd1pdl1based immunotherapy strategies with better clinical response and reduced immunemediated toxicity in thisreview we provide an overview on the resistance to pd1pdl1 blockade and briefly introduce the mechanismsunderlying therapy resistance moreover we summarize potential predictive factors for the resistance to pd1pdl1blockade furthermore we give an insight into the possible solutions to improve efficacy and clinical response inthe following research combined efforts of basic researchers and clinicians are required to address the limitation oftherapy resistancekeywords pd1pdl1 blockade cancer immunotherapy resistance immunotherapy is a validated and significant cancertreatment strategy which eliminates tumors by normalizing the antitumor immune responses [ ] over thelast decade cancer immunotherapy has revolutionizedthe treatment landscape of malignancies and achievedclinical success especially in immune checkpoint inhibitors correspondence 189whueducn lschrjjs163com jinyu sun and dengke zhang are cofirst authors4department of general surgery the first affiliated hospital of nanjingmedical university nanjing china2key laboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui chinafull list of author information is available at the end of the signalsandprogrammed death1 pd1 is a class of receptorexpressed on the t cell surface which could downregulate the immune system by abrogating t cellreceptorinducedantigenmediated t cell activation the interaction betweenpd1 and its ligand programmed deathligand pdl1 plays an essential role in maintaining selftoleranceand avoiding autoimmune diseases however pd1pdl1 could also prevent the activation of t cells in thetumor and thus result in immune resistance preventingpd1pdl1 blockade is a breakthrough in cancerimmunotherapy and it has been trialed in a broadrange of malignancies in the preclinical or clinicalincluding melanoma hodgkins lymphomastage breast cancer [ ] nonsmall celllung cancer as well as hepatocellular carcinomansclc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun biomarker research page of [ ] despite the longterm potentially curelikeclinical benefits therapy resistance remains a significant challenge for the further application of pd1pdl1 blockade therapy only a minority of patientsin general are estimated to experience apositive response to pd1pdl1 blockade therapy[] and the primary or acquired resistance mighteventually lead to cancer progression in patients withclinical response [ ]in this review we provide an overview on the resistance to pd1pdl1 blockade and its underlying mechanisms moreover we summarize potential predictivefactors for the resistance to pd1pdl1 blockade furthermore we give an insight into the possible solutionsto improve efficacy and clinical response of pd1pdl1blockade therapyresistance to pd1pdl1 blockade therapycheckpoint inhibitors targeting pd1 or pdl1 coulddisturb the interaction between pd1 and pdl1 whichwould preserve antitumor properties of t cells withdraw immune escape and normalize their ability to induce tumor cell death currently pd1pdl1 blockadehas shown sustained survival benefits in multiple malignancies and is at the forefront of cancer immunotherapy howeverjust as tumor cells can avoid immuneevasion several cancers may evolve to resist pd1pdl1 blockade therapy clinical evidence indicated thateven for patients with tumors highly positive for pdl1more than of them might not respond to pd1pdl1 blockade due to tumor heterogeneity and manyother reasons clinical responses vary largely across different tumor entities the objective response rate was in melanoma in nsclc in head and neck carcinoma and in kidneycancer besides for most patients experiencing initial clinical response acquired resistance remains another problem which would lead to cancer progressionor relapse after a few years [ ]many studies have demonstrated that antipd1therapy can significantly improve survival outcomes forpatients with metastatic or unresectable melanoma however only a small number of patients approximately could achieve a complete response in arecent phase i trial of atezolizumab antipdl1 involving patients with metastatic melanoma the overall response rate was among efficacy evaluablepatients and the median response duration was months moreover in another study on the longtermoutcomes of melanoma patients receiving antipd1therapy complete responses were only observed in of patients after a median followup of months of patients were alive without additional melanoma therapy additionally in the retreatedpatients after disease progression the response was onlyobserved in retreated patients receiving singleagent pd1 blockade therapy and of patientsescalated to pd1 blockade plus ipilimumab therapy inthis cohort most complete responses were durable withthe treatment failure rate of at three years whilethe response to retreatment remained relatively infrequent with a response rate of for patients withsingleagent pd1 blockade therapy moreover in aphase ii study of pembrolizumab on patients withadvancedobjectiveresponse was observed in of patients with a diseasecontrol rate of after a median followup of months adrenocorticalcarcinomatheinterestingly the response rate of some malignanciesis relatively high in hematological malignancies for example for patients with relapsed or refractory classicalhodgkin lymphoma tislelizumab antipd1 achievedan objective response rate of and a completeresponse of in a phase ii singlearm multicenterstudy similarly the complete response rate of camrelizumab antipd1 was with a partial remission rate of mechanisms underlying the resistance to pd1pdl1 blockadesince therapy resistance remains a significant limitationof pd1pdl1 blockade in clinical practice interest isgrowing in understanding the mechanisms underlyingthe resistance the response to pd1pdl1 blockaderelies on a preexisting immune response and determinants of adaptive immunity currently multiple factorshave been discovered to be involved in the efficacy ofpd1pdl1 blockade therapy such as tumor immunogenicity t celltumormicroenvironment and so forthfunction pdl1 expressionthe lack of tumor antigensthe genetic alterations are centralin the oncogenicprocess which could lead to tumor immunogenicity andprovide an opportunity for cancer immunotherapy tumor immunogenicity is positively associated with theability of the t cell to recognize tumor cells which isessential for the antitumor effect of pd1pdl1 blockade however the lack of tumor antigen will significantlyimpede the recognition ability of t cells and eventuallyresult in the failure of immunotherapymicrosatellites are prone to dna replication errorswhich will usually be repaired in normal cells however in tumors with mismatch repair mmr deficiencythese errors will accumulate which eventually result in alarge number of mutations and lead to microsatellite instability msi importantly high msi positivelycontributes to increased neoantigen production greater 0csun biomarker research page of immunogenicity and a more robust immune response moreoverthe resultant high tumor mutationburden would contribute to tumor immunogenic andenhance the response to pd1pdl1 blockade therapy[ ]multiple studies have demonstrated that the tumormutation burden is positively correlated with neoantigenburden as well as response to immunotherapy [ ]for example in colorectal cancer with mmr deficiencywhich usually exhibits a high tumor mutation burdenantipd1 therapy showed a higher response rate andbetter survival outcome compared to other subtypeswith mmr proficiency [] yarchoan analyzed the objective response rates of pd1pdl1blockade therapy for the corresponding tumor mutationburden in various cancers and their results showed thatthe mutation burden was closely associated with the objective response rate moreover pancreatic cancer generally exhibits a lowermutation load compared with other solid tumors andtherefore pd1pdl1 blockade is usually ineffective forthose patients and fails to improve their survival outcomes nevertheless in pancreatic cancer patients harboring an mmr deficiency they appear to be responsiveto pd1pdl1 blockade therapy mmr deficiency significantly increases the somatic mutation rate whichcould be translated into neoantigens and recognized bythe immune system thus making these patients responsive to pd1pdl1 blockade therapy [ ] accordingly pembrolizumab has been approved for selectedcancer patients with mmr deficiencyt cell dysfunctioneffective pd1pdl1 blockade therapy relies on the tcell function and any disruption in the processes of tcell immune function will result in the failure of pd1pdl1 blockade therapy a recent review by ren has provided an indepth insight into the mechanisms underlying the t cell dysfunctionmediated resistance with a focus on t cell recognition activationdifferentiation infiltration depletion as well as chemotaxis identification byantigen presentation is a critical process for the tumorantigensinitial t cells beta2microglobulin b2m is a significant hla1 moleculewhose mutation will hinder tumor antigen presentationand result in therapy resistance [] zaretsky analyzed biopsy samples from patients with metastatic melanoma receiving pembrolizumab who exhibited disease progression after an initial tumor regressionand they found a truncating mutation in the b2m genein the following research gettinger identifiedacquired homozygous loss or downregulation of b2m inlung patients with resistance to pd1pdl1 blockadeto further explore the role of b2m in mediating resistance they knocked out the b2m gene in immunocompetent lung cancer mice by crispr technology and theloss of b2m resulted in the resistance to pd1pdl1blockade additionally b2m mutationinducedresistance primarily occurred in an environment ofactivated pd1 positive t cellinfiltration whichresistance to pd1pdl1 blockadesuggested thattherapy might be particularly common in patients withhigh pd1 positive t cell for example t cellmoreover t cell activation is another critical processfor pd1pdl1 blockade therapy after blocking pd1pdl1 tumor cells can still counteract the activity ofimmune checkpoints and activate additional inhibitorypathways via expression of other immune checkpointsand their ligands within the tumor immune microenvironmentimmunoglobulinmucin3 tim3 is another type of immune checkpointreceptor expressed on tumorinfiltrating lymphocytes inhuman head and neck squamous cell carcinoma tumorinfiltrating lymphocytes pd1 blockade was demonstrated to upregulate tim3 expression which inhibitedt cells activation and contributed to tim3mediatedescape from pd1 blockade in the tumor microenvironment via pi3kakt pathway pd1 or pdl1physiologicallyinteractions between pd1 and pdl1block t cell activation pathways related to the immuneresponse against specific antigens and the expression ofpd1 or pdl1 has gained importance as a significantplayerin regulating the response to pd1pdl1blockade therapy pd1 and pdl1 are upregulated inthe tumor immune microenvironment of various malignancies which is considered as a strategy to evadeimmunosurveillance and imposes a significant barrier ofthe antitumor immune response importantly pdl1 primarily exhibits two distinct expression patternson tumor cells or on tumorinfiltrating immune cellspdl1 expression on immune cells reflects the adaptiveregulation meditated by ifnγ which is accompanied byincreased effector t cells as well as tumorinfiltratinglymphocytes effector t cells differently the expressionof pdl1 on tumor cells is less prevalent and it indicates the epigenetically dysregulated pdl1 gene whichis correlated with reduced immune infiltration scleroticor desmoplastic stroma as well as mesenchymal molecular features multiple studies have revealed a significantly higherobjective response rate in tumor pdl1 positive patientsthan pdl1 negative subgroups together with an improved progressionfree and overall survival [ ]kowanetz observed that atezolizumab antipdl1 achieved an objective response rate of in 0csun biomarker research page of patients with high pdl1 levels on tumor cells alone andof in those with a high expression on immune cellsalone although these observations indicated that thefunctional importance of pdl1 expression in regulatingpd1pdl1 blockadeinduced t cellthemechanistic significance of pdl1 on tumor cells or immune cells remains vagueresponsenoncoding rnasa large amount of micrornas mirnas and some longnoncoding rnas lncrnas have emerged as players inregulating tumor immunity [] and resistance topd1pdl1 blockade therapy recently huber identified a panel of circulating mirnas mir146a mir155 mir125b mir let7e mir125a mir146b mir99b which wereassociated with phenotypic and functional features ofmyeloidderived suppressor cells mdscs in melanomapatients importantly mdscs are a subclass of immature myeloid cells pathologically associated with cancerand play an inhibitory role against antitumor t cell immunity the transcriptional analysis showed thatthese mirnas could facilitate the conversion of monocytes into mdscs by melanoma extracellular vesiclesand the expression level ofthese mirna was upregulated in circulating cd14 monocytes and tumorsamples which was associated with myeloid cell infiltration and could predict the resistance to pd1 blockadetherapy moreover hu revealed the role of oncogeniclncrna for kinase activation linka in losing antigenicity and evading immune checkpoints and demonstrated lncrnadependent antigenicity downregulationsuppression for patients withand intrinsic tumortriplenegative breast cancer and resistantto pd1blockade therapythey showed upregulated linkalevels and downregulated peptideloading complex components the analysis suggested that linka expressioncould attenuate protein kinase amediated phosphorylation of the e3 ubiquitinprotein ligase trim71 via facilitating the crosstalk between phosphatidylinositol []trisphosphate and inhibitory gproteincoupled receptor pathways consequently linka could contribute to the degradation of the antigen peptideloadingcomplex and upregulate intrinsic tumor suppressors gut microbiomethe gut microbiome is a complex system composed ofmore than trillion microanisms which has beendemonstrated to regulate the efficacy and toxicity ofcancer immunotherapy many studies have reported theinfluence of the gut microbiome on cancer immunotherapy and the therapeutic response of pd1pdl1blockade therapy can be improved or diminished via gutmicrobiome modulationin mice models with distinct microbiome a significantly different response to pd1pdl1 blockade therapy was observed for example melanoma mice with anincreased bifidobacterium species in the gut microbiomeexhibited an effective response to pd1 blockade therapy similarly antibiotic administration was reported toreduce the diversity and aggravate dysbiosis of the gutmicrobiome thus influencing the clinical response topd1pdl1 blockade in tumorbearing mice as well ascancer patients [] compared to patients withoutantibiotic treatment the oral antibiotic application couldsignificantly diminish the clinical benefit of pd1pdl1blockade therapy and decrease progressionfree survivaland overall survival therefore dysbiosis of the gut microbiome is considered as one of the putative mechanisms underlying poorresponse to pd1pdl1 blockade therapy and thedualdirectional modulation of the gut microbiome oncancer immunotherapy is increasingly revealed howeverit is still unclear how gut microbiome regulatestherapy response and whether a specific bacterial taxaor gut microbiome as a whole plays a primary role remains largely unclear further research is required toprovide a more indepth understanding of the underlying mechanismspredictive factors for pd1pdl1 blockadetherapydespite the clinical success achieved in pd1pdl1blockade across multiple cancers the knowledge concerning therapy selection criteria is relatively limitedconsidering the potential adverse events and high costof immune checkpoint inhibitor agents there is a substantial need to identify predictive factors to select patients likely to benefit from this therapy currently apartfrom the functional status of immune cells [] ortumor infiltrating lymphocytes multiple factorshave been identified to predict the response to pd1pdl1 blockade therapy such as pd1pdl1 expression antigen recognition gut microbiome and so forthtable pd1 or pdl1 expressioninhibiting the pd1 pathwaymediated immune suppression is the basis and premise of pd1pdl1 blockadetherapy accumulating research has suggested that pdl1 is a biomarker to predict therapeutic response to pd1pdl1 blockade across multiple tumor types forexample atezolizumab achieved overall survival benefitacross all pdl1 expression subgroups in nsclc patients while those with high pdl1 expression experienced a more substantial survival benefit currently 0csun biomarker research page of table predictive factors for pd1pdl1 blockade therapytumor typenonsmall cell lung canceragentatezolizumabmultiple cancerscolorectal cancerurothelial carcinomaurothelial carcinomaurothelial cancermelanomamelanomamelanomapembrolizumabnivolumabatezolizumabatezolizumabatezolizumabantipd1 therapyantipd1 therapyantipd1 therapymmr mismatch repair msi microsatellite instability tmb tumor mutation burdenpredictive factorpdl1pdl1mmr msitmbtmbtmbgut microbiomegut microbiomegut microbiomereference pdl1 testing is recommended as a predictive test fornsclc urothelial carcinoma or head andneck cancers and so forthott assessed the predictive value of pdl1expression in patients with advanced solid tumors receiving pembrolizumab and the analysis showed that tumors with higher pdl1 expression and tumor mutationburden were significantly associated with higher response rate and more prolonged progressionfree survival heat map analysis revealed a close correlationbetween pdl1 expression and a broader pattern ofcoregulated gene expression which involved cytokine recruitment of t cells t cell activation markers as well asantigen presentation also the regression metaanalysisdemonstrated that pdl1 expression level was positivelyassociated with objective response rate p aswell as progressionfree survival p moreover nct02853305 and nct02807636 evaluated the efficacy of pembrolizumab or atezolizumab asfirstline treatment and the current data showed reduced survival in patients with low expression of pdl1accordingly it is advised that pembrolizumab or atezolizumab should be used for adult patients with a relativelyhigh pdl1 expression pdl1 expression of ¥ foratezolizumab and a combined positive score of ¥ forpembrolizumab however the efficacy of pd1pdl1blockade therapy as firstline therapy for advancedurothelial carcinoma still remains unclear [ ]importantly pdl1 positive only is not a predictivefactor for the response to pd1pdl1 blockade sincemultiple factors are involved in the pd1pdl1 blockade therapy in a study on patients with metastaticmelanoma receiving pembrolizumab preexisting cd8t cells were demonstrated as a prerequisite for thetumor regression after pd1pdl1 blockade therapy besidesin advanced adrenocortical carcinomatumor pdl1 expression status was not associated withtherapy response additionally it was reported thatpdl1 expression on tumor cells was not associatedwith therapy response in resected head and necksquamous cell cancer additionalinvestigation isrequired to illustrate the mechanisms accounting for thedifferenceantigen recognitionantigen recognition plays a vital role in initiating theadaptive immune response while the lack of tumor antigens significantly impedes the response to pd1pdl1blockade therapycurrently the fda has approved pembrolizumab totreat unresectable solid tumors with high msi or mmrdeficiency in a study on recurrent or metastaticcolorectal cancer patients with mmr deficiency or highmsi nivolumab showed an objective response rate of and of the patients had a disease control rateof ¥ weeks which indicated that patients with highmmr deficiency or high msi might exhibit better responses to pd1pdl1 blockade therapy [ ] interestingly the responses of tumors with mmrdeficientare highly variable and approximately half are resistantto pd1pdl1 blockade therapy mandal revealed that msi and the resultant mutation load wereresponsible for the variable response to pd1 blockadetherapy in mmrdeficiency tumors and the responsedegree was significantly correlated with the degree ofinsertiondeletion mutation loadseveral studies have revealed the association betweentumor mutation burden and the response to pd1pdl1blockade therapy [ ] mariathasan examined samples from patients with metastatic urothelial cancer receiving atezolizumab treatment and identified highneoantigen and tumor mutation burden as major determinants of clinical outcome their results showed that thetumor mutation burden was closely correlated with the response in the excluded and inflamed phenotypes 0csun biomarker research page of gut microbiome compositionclinical experiments on the human gut microbiomehave identified several specific bacteria genres that playimportant roles in human immunity and can be used asprognostic biomarkers for clinical response to pd1pdl1 blockade therapy based on the gut microbiome analysis of melanomapatients receiving pd1 blockade gopalakrishnan found that patients with prolonged progressionfree survival showed a higher multiplicity of bacteriaand clostridiales ruminococcaceae and faecalibacterium were abundant in therapy responders moreovermatson evaluated the baseline stool samplesfrom patients with metastatic melanoma before pd1pdl1 blockade treatment and the results showed thatcommensal microbial composition was significantly associated with the clinical response bifidobacteriumlongum collinsella aerofaciensand enterococcusfaecium were more abundant in responders similarlyin patients with epithelial tumors routy revealed that akkermansiacea muciniphila and enterococcus hirae were significantly abundant in those withbetter clinical response progressionfree survival months all these results indicate that gut microbiomecomposition may be a potential determinant of therapyresponse and might be used as a predictive factor inthe following research more studies are needed to validate the predictive value of gut microbiome in largercohorts and explore their efficiency in the context ofvarious types of tumorsstrategies and it hasfuture perspectivesimmunotherapy is one of the most promising cancertreatmentrevolutionized thelandscape of cancer management over the last decadehowever together with the costly and timeconsumingtrialanderror approach the limited therapy responseremains a tricky problem which hinders the furtherapplication of pd1pdl1 blockade to overcome therapy resistance and potential adverse events substantialeffort has been made on developing novel antipd1pdl1 based immunotherapy strategies with better clinical response and limited immunemediated toxicityfigs tobetterclinicallikelyachievesystem issince the interaction between cancer and the immunecomplex and involves multiplefactors strategies in combination with multiple agentsareoutcomescompared with singleagent administration a largenumber ofcombinedtherapy is an effective therapeutic strategy againstcancers for example transforming growth factor βtgfβblocking agents concomitantly with combinedpd1pdl1 blockade combined provides a clinicallyrevealed thatstudies haveexperimentson mice withfeasible strategy to improve efficacy and reduce toxicity mariathasan revealed that metastaticurothelial cancer with upregulated tgfβ signalingbefore treatmentresponded poorly to pd1pdl1blockade therapy the tumors with dense collagenfibrils could trap t cells in the stromal compartmentthus preventing them from playing their functions inpreclinicalimmuneexcluded phenotype they demonstrated that the coadministration of pdl1 blockade and tgfβblockingagents could reduce tgfβ signaling facilitate t cellinfiltration and achieve active antitumor immunityand tumor regression similarly the combination ofpd1pdl1 blockade with tumor necrosisfactorinhibitor [ ] metformin antivegf agents or otherinhibitors egcxcr4 has been demonstrated as a clinicallyfeasible strategy with improved antitumor efficacyand reduced toxicityimmune checkpointinhibitor agentspd1pdl1 blockade usually acts on the whole hostimmune system instead ofsitespecifically targetingtumorspecific immune cells while nanomedicine technology provides a powerful tool to selectively deliverimmune checkpointto tumors orlymphoid ans using drugloaded nanops usually to nm in diameter recent studies suggest that the pd1pdl1 antibody could be conjugatedor modified on the surface of nanops which couldmaintain their stability enhance efficiency and minimizethe toxicity of pd1pdl1 blockade [ ] forexamplein gastric cancer cells the pdl1 blockadeconjugated nanops contributed to significantlyhigher cellular uptake and achieved more effective inhibition of pdl1 expression compared with the controlgroups moreover in patients with metastatic triplenegative breast cancer the coadministration of nabpaclitaxelatezolizumabprolonged progressionfree survival owing to thesuccess in previous research clinical trials on nanoimmunotherapysuch asnct03589339 and nct03684785 these clinical trialsshould provide substantial evidence for the combinationof nanomedicine and pd1pdl1 blockade in the nextfew yearscurrently underwayblockadepdl1plusarethe manipulation offurthermore accumulating evidence has demonstrated that gut microbiome significantly impacts theefficacy of cancer immunotherapy which in turn indithe gut microbiomecates thatcould latently affectthe response to pd1pdl1blockade therapy [] currently antibiotic applicationfecal microbiota transplantation fmt anddiet regulation are considered as practical approachesto manipulate gut microbiome for example fmtfrom patients with a positive response to germfree or 0csun biomarker research page of fig overview on the strategies to improve the resistance to pd1pdl1 blockade therapy multiple strategies have been proposed toimprove the resistance to pd1pdl1 blockade therapy including combined therapy nanoimmunotherapy gut microbiome manipulation andso forthin contrastantibiotictreated mice could improve tumor controlaugment t cell responses and ameliorate the antitumor effects of pd1 blockadethetransplantation from resistant patients did not resultin improvement similarly responses to pdl1blockade are distinctin mice with different commensal microbes and the positive response of micewith advantageous gut microbiome can be transplanted to mice with negative responses by fmt orcohousing conclusionsdespite the success across multiple types of cancersonly a minority of patients are estimated to exhibit apositive response to pd1pdl1 blockade therapy andthe primaryacquired resistance might eventually leadto progression in patients with clinical responses thelimitation in clinical response impairs the efficacy andhinders its further application since the understandingof the mechanisms underlying therapy resistance remains vague only a few therapeutic options areavailable for those patients currently illustrating thedeterminants of response or resistance is significant toaccelerate improving survival outcomes and developingimproved treatment options for cancer patients tobetter realize the therapeutic potential of pd1pdl1blockade therapyit is essential to identify predictivebiomarkers for therapy response develop novel therapeutic strategies and improve therapeutic strategies incombination with other agents in the following research combined efforts of basic researchers and clinicians are required to address the pd1pdl1 blockadetherapy resistanceabbreviationspd1 programmed death1 pdl1 programmed deathligand nsclc nonsmall cell lung cancer mmr mismatch repair msi microsatelliteinstability b2m beta2microglobulin tim3 t cell immunoglobulin mucin3mirnas micrornas lncrnas long noncoding rnas mdscs myeloidderived suppressor cells tgfβ transforming growth factor β fmt fecalmicrobiota transplantationacknowledgmentsnot applicableauthors contributionsjys dk z mx and xz wrote original draft preparation sq w jsj and xjprovided critical revision all authors read and approved the final manuscriptfundingthis study was supported by national key research and developmentprojects intergovernmental cooperation in science and technology of chinano 2018yfe0126900 to jiansong ji the key research and developmentproject of zhejiang province no 2018c03024 to jiansong ji the nationalnatural science foundation of china to xl 0csun biomarker research page of availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1the first college of clinical medicine the first affiliated hospital of nanjingmedical university nanjing medical university nanjing china 2keylaboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui china 3college of medicine lishui college lishui china 4department of general surgery the first affiliated hospitalof nanjing medical university nanjing china 5department of radiologyaffiliated lishui hospital of zhejiang university lishui chinareceived april accepted august referenceshellmann md pazares l bernabe caro r zurawski b kim sw carcerenycosta e nivolumab plus ipilimumab in advanced nonsmallcell lungcancer n engl j med niglio sa jia r ji j ruder s patel vg martini a programmed death1or programmed death ligand1 blockade in patients with platinumresistant metastatic urothelial cancer a systematic review and metaanalysis eur urol sun jy lu xj cancer immunotherapy current applications and challengescancer lett andrews lp yano h vignali daa inhibitory receptors and ligands beyondpd1 pdl1 and ctla4 breakthroughs or backups nat immunol prestipino a zeiser r clinical implications of tumorintrinsic mechanismsregulating pdl1 sci transl med betof warner a palmer js shoushtari an goldman da panageas ks hayessa et
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coronavirus disease COVID19 pandemic access to surgical care for patients with head and neck cancer HNC is limited and unpredictable Determining which patients should be prioritized is inherently subjective and difficult to assess The authors have proposed an algorithm to fairly and consistently triage patients and mitigate the risk of adverse outcomes METHODS Two separate expert panels a consensus panel participants and a validation panel participants were constructed among international HNC surgeons Using a modified Delphi process and RAND CorporationUniversity of California at Los Angeles methodology with consensus rounds and meetings groupings of highpriority intermediatepriority and lowpriority indications for surgery were established and subdivided A pointbased scoring algorithm was developed the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN Agreement was measured during consensus and for algorithm scoring using the Krippendorff alpha Rankings from the algorithm were compared with expert rankings of case vignettes using the Spearman rank correlation coefficient RESULTS A total of indications for surgical priority were rated Weights for each indication ranged from to scale range to The response rate for the validation exercise was The SPARTANHN demonstrated excellent agreement and correlation with expert rankings Krippendorff alpha [ CI ] and rho [ CI ] S The SPARTANHN surgical prioritization algorithm consistently stratifies patients requiring HNC surgical care in the COVID19 era Formal evaluation and implementation are required Cancer American Cancer Society LAY SUMMARY ¢ Many countries have enacted strict rules regarding the use of hospital resources during the coronavirus disease COVID19 pandemic Facing delays in surgery patients may experience worse functional outcomes stage migration and eventual inoperability¢ Treatment prioritization tools have shown benefit in helping to triage patients equitably with minimal provider cognitive burden¢ The current study sought to develop what to the authors knowledge is the first cancerspecific surgical prioritization tool for use in the COVID19 era the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN This algorithm consistently stratifies patients requiring head and neck cancer surgery in the COVID19 era and provides evidence for the initial uptake of the SPARTANHN KEYWORDS coronavirus disease COVID19 delivery of health care head and neck cancer health priorities patient selection surgical procedures waiting listsCorresponding Author John R de Almeida MD MSc Division of Surgical Oncology Department of OtolaryngologyHead and Neck Surgery 8NU883 Toronto General Hospital University Health Network Elizabeth St Toronto ON M5G 2C4 Canada Johndealmeidauhnca Division of Surgical Oncology Department of OtolaryngologyHead and Neck Surgery Princess Margaret Cancer Center University Health Network University of Toronto Toronto Ontario Canada Institute of Health Policy Management and Evaluation Dalla Lana School of Public Health University of Toronto Toronto Ontario Canada Division of OtolaryngologyHead and Neck Surgery Dalhousie University Halifax Nova Scotia Canada Division of OtolaryngologyHead and Neck Surgery McMaster University Hamilton Ontario Canada Department of OtolaryngologyHead and Neck Surgery Western University London Ontario Canada Department of OtolaryngologyHead and Neck Surgery Memorial Sloan Kettering Cancer Center New York New York Head and NeckEndocrine Oncology Moffitt Cancer Center Tampa Florida Department of OtolaryngologyHead and Neck Surgery Medical University of South Carolina Charleston South Carolina Department of OtolaryngologyHead and Neck Surgery Stanford University Palo Alto California Department of OtolaryngologyHead and Neck Surgery University of Michigan Ann Arbor Michigan Department of OtolaryngologyHead and Neck Surgery The University of Texas MD Anderson Cancer Center Houston Texas Head and Neck Unit The Royal Marsden Hospital London United Kingdom Department of OtolaryngologyHead and Neck Surgery Icahn School of Medicine at Mount Sinai New York New York Department of OtolaryngologyHead and Neck Surgery Sunnybrook Health Sciences Centre University of Toronto Toronto Ontario Canada Department of OtolaryngologyHead and Neck Surgery Sinai Health System University of Toronto Toronto Ontario CanadaThe first authors contributed equally to this Additional supporting information may be found in the online version of this 101002cncr33114 Received June Revised June Accepted June Published online Month in Wiley Online Library wileyonlinelibrarycomCancer Month 0cOriginal INTRODUCTIONOn March the World Health anization declared a global pandemic due to the novel coronavirus severe acute respiratory syndrome coronavirus SARSCoV2 and the resulting coronavirus disease COVID191 As a result in many jurisdictions operating room capacity has been limited to only emergent or urgent surgical procedures2 Several advisory bodies have issued recommendations to safeguard access to oncologic surgery while still acknowledging that treatment delays may be necessary The American College of Surgeons has recommended postponing elective surgery including for patients with lowrisk cancers while recommending that other urgent cancer surgeries proceed34 Cancer Care Ontario has issued similar guidance recommending that hospitals include cancer surgery in their care delivery plan5The time from the diagnosis of head and neck cancer HNC to surgery is a metric with prognostic importance with treatment delays portending poorer oncologic outcomes68 In a recent systematic review evaluating delays in time from diagnosis to treatment initiation of studies demonstrated a decrease in survival to be associated with treatment delays68 These data support the urgency of initiating treatment for patients with HNC but to our knowledge do not inform a stratification schema when operating room access is not available for all patientsAs a result of these new imposed constraints difficult decisions regarding prioritization for cancer surgery are obligatory and require the consideration of broader principles regarding scarce resource allocation9 Key among these is the need for consistency and transparency to achieve fairness and to avoid engendering disparities in both access and outcomes1011 Prioritization on a casebycase basis using expert clinical judgment can be logistically challenging carries a cognitive burden and is susceptible to the biases of practitionersSurgical prioritization tools or algorithms offer decisionmaking transparency and provide equitable and timesensitive access to care to the patients who need it most1213 Although tools for surgical prioritization in the era of COVID19 continue to emerge to our knowledge oncology patients have not been explicitly considered14 Herein we have presented the development and validation of a novel algorithm Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN for the prioritization of surgery for patients with HNCMATERIALS AND METHODSThe current study was granted a waiver from the research ethics board at the University Health NetworkParticipants and SettingFor instrument development a group of expert HNC surgeons JRD DPG RG JCI DBC DB AE DJE KMH EM and IJW from institutions University Health Network Sinai Health Systems and Sunnybrook Health Sciences Centre at the University of Toronto participated in the consensus process consensus panel At the time of the consensus process all institutions were operating under significant resource constraints with limited availability of operating room time For instrument validation a group of participants JRD CWN DF DPG and EM completed the scoring algorithm designed after the consensus process Fifteen external head and neck surgeons HZ ACN RJW MAC CM EMG VD AGS AJR CML EYH JM VP BM and EG from institutions across Canada institutions the United States institutions and the United Kingdom institution participated in a ranking exercise of clinical vignettes validation panelScopeThe scope of variables considered in the prioritization algorithm was established and vetted by the consensus panel see Supporting Information All indications for prioritization were presented to the consensus panel using an online survey platform Google Forms httpsdocsgooglecomforms With exceptions survey respondents were asked to consider each of the indications in isolation For wait times panel members were asked to also consider histologic grade Similarly for surgical site the panel was asked to simultaneously consider extent of surgery Related indications were presented sequentially to facilitate pairwise comparison eg stage I and II vs stage III and IV were presented in sequence AJCC 8th edition The list of indications was pilot tested by surgeons JRD DPG EM and RG for sensibility readability content validity language and comprehensibilityConsensus ProcessThe consensus panel participated in a Delphi consensus process with rounds of rating see Supporting Information The first rounds aimed to achieve consensus regarding the priority grouping high intermediate or low High priority was defined as an indication to Cancer Month 0cproceed to surgery within weeks The second rounds of rating involved ranking each indication less important neutral or more important within their respective priority grouping Two teleconference meetings were conducted between the first and second rounds and between the third and fourth rounds with anonymized results from the prior round presented for discussion and to address inconsistencies and misinterpretationsA modification of the RANDUniversity of California at Los Angeles UCLA method was used to achieve consensus15 This methodology typically is used to determine the appropriateness of an intervention but in this setting was used to determine surgical priority We used a scale ranging from to in rounds and to indicate the decision to not operate or low priority scores intermediate priority scores or high priority scores For rounds and we used a scale from to to rate each indication compared with other indications within each of the priority groupings as either less important neutral or more important Consensus was determined based on RANDUCLA criteria15 For the first rounds to determine surgical priority a hierarchical logic was adopted to determine consensus regarding whether surgery should be performed and to then determine the priority of surgery based on the given indication Agreement on the decision to not operate was defined as a minimum of of the panelists rating a given indication with a zero score If there was no agreement to avoid surgery agreement for surgical priority then was defined as ¤ panelists rating the indication outside the 3point range containing the median as per RANDUCLA guidelines15 For rounds and any indication that failed to achieve consensus was classified as being of intermediate priority and for rounds and any indication failing to achieve consensus was classified as neutral within the priority groupingDevelopment of the SPARTANHNThe algorithm uses a pointbased system to assign a total score based on the sum of the individual indication scores see Supporting Information with higher scores corresponding to higher priority Scoring weights were based on consensus from both sets of rounds such that highpriority indications were assigned scores ranging from to intermediatepriority indications were assigned scores ranging from to and lowpriority indications were assigned scores ranging from to Within each priority grouping 3point range the scores were assigned based on the consensus ratings from the third and fourth rounds For any patients with the same total score the SPARTANHNde Almeida et alpatient with the longer surgical wait time was assigned the higher priority rankClinical VignettesTwelve clinical vignettes were constructed see Supporting Information after the consensus rounds to validate the SPARTANHN The vignettes described a variety of clinical scenarios incorporating multiple prioritization indications and additional clinical information Experts were asked to consider only the patientlevel information provided to them and not their own unique clinical and community practice environments Twelve scenarios were selected for diversity of cases The number was considered appropriate while avoiding the excessive cognitive burden associated with ranking too many scenariosStatistical AnalysisAgreementAgreement between raters during the Delphi process was calculated at each round and within each priority grouping using the Krippendorff alpha Kalpha Because typical coefficients of reliability are not suitable for coded data agreement for the rank orders generated by coders JRD CWN DF EM and DPG applying the SPARTANHN algorithm to the clinical vignettes was assessed using Kalpha calculated with bootstrap samples16 The Kalpha allows for estimation of reliability for any number of raters and categories and may be used when there are missing data17Validity of the SPARTANHN AlgorithmConvergent validity of the median rankings from the coders of each of the vignettes using the SPARTANHN and the expert panel rankings were assessed using the Spearman rank correlation coefficient The strength of the correlation was considered weak if the rho was moderate if the rho was between and and strong if the rho was In addition to SPARTANHN a second algorithm using a decisionmaking flowchart was developed SPARTANHN2 The tool and associated performance characteristics are included in Supporting Information Sample Size ConsiderationsFor determination of an adequate sample size for the expert panel we assumed that for model validity there was a strong correlation between the model rank order and expert rank order ie rho ¥ an alpha of power of and a nonresponse rate of Therefore the calculated sample size requirement was participantsCancer Month 0cOriginal All analyses were 2sided and statistical significance was set at P\xa0¤\xa0 Analyses were conducted using SAS University Edition statistical software SAS Institute Inc Cary North CarolinaRESULTSEstablishing Consensus Priority Groupings First Consensus RoundsAfter the first rounds the panel failed to achieve consensus for any indications that would result in a decision to not operate More than respondents indicated that they would not operate for the following indications the availability of alternative nonsurgical treatment with a similar prognosis respondents poor performance status ie Eastern Cooperative Oncology Group [ECOG] performance status of respondents and very severe comorbidity as indicated by a noncancerspecific survival rate of at year respondents In the first round consensus was achieved for indications for surgical prioritization of which were considered high priority of which were considered intermediate priority and of which were considered low priority After review of firstround results consensus was achieved for an additional indications indications were rated as being of intermediate priority and indications were rated as low priority Table Establishing Ranking Within Each Priority Grouping Second Consensus RoundsOf the lowpriority indications consensus for the importance of factors was achieved for scenarios both of which were deemed less important Table Of the intermediatepriority indications consensus for the importance of factors was achieved for of scenarios Of highpriority factors consensus for the importance of factors was achieved for scenarios all of which were deemed to be more importantAgreement during consensus rounds was found to be weak to moderate for all rounds ranging from to The agreement was similar when measured as per priority grouping in which the Kalpha ranged from to Table SPARTANHN Surgical Prioritization Scoring SystemPriority weights for each indication ranged from to spanning a 9point range and translated from the rounds of priority groupings into categories Four indications were assigned a weight of based on consensus that these factors were both high priority and more important Supporting Information Table All other highpriority indications were assigned a weighted score because there was no consensus that they were either less or more important For intermediatepriority indications a weighted score of was assigned for of the indications deemed to be more important by consensus The other indication deemed to be more important thyroid cancer with tracheal invasion was assigned a score of because of the fact that this indication can be associated with lowgrade histology which is assigned a negative weighted score Three intermediatepriority indications that were rated as more important were resource use indications which generally are colinear As such the decision was made to assign a maximum score of for the presence of any or all of these indications One intermediatepriority indication was deemed to be less important by consensus and was assigned a score of All other intermediatepriority indications were assigned scores of For the lowpriority indications those deemed to be less important were assigned a weight of and all other indications were assigned a weight of The total scale score ranged from to Fig Reliability and Validity AssessmentAgreement between the coders for the SPARTANHN was excellent Kalpha Agreement between the expert raters was moderate Kalpha Convergent validity was demonstrated by a strong correlation between the rank orders generated by the SPARTANHN and external experts rho CI [P\xa0 a0 ] Agreement between expert rankings and SPARTANHN rankings for the vignettes is shown in Figure DISCUSSIONIn the setting of the COVID19 pandemic in which the availability of operating room time as well as hospital and intensive care unit beds is limited the prioritization of surgical oncology cases is imperative to mitigate downstream adverse outcomes1920 The current methodology was adopted based on expert consensus In the current study we have proposed the SPARTANHN with the objective of providing transparency and facilitating surgical prioritization for treatment providersCreating COVID19era allocation schemas that are ethically sound is both critical and challenging Emanuel et al have advocated ethical principles with which to Cancer Month 0cSPARTANHNde Almeida et ali ytidbrom lanoitcnufi tnacifings laitnetoP fi ytilibareponi rohtw romut fi tnemriapmi citem etaredom laitnetoPsoc ro lanoitcnuf htiw recnac doryhTiinosavni laehcarthtw romut inossergorp esaeisd citamotpmySENEtsil tiawno e lihwTR suoverPi dedeecxe emit tiaW dedeecxe emit tiaWihgh rof kw¥ yb liygootsh edargihgh rof kw yb liygootsh edarg igngami ro lacniilC hpmyl decnavdAegats gncnavdai ei inossergorpi cpocsorcam roN ge esaesd edoni esaesdV inoitceserI ot III egatSnoitceser enob htiw recnac ytivac larOnoitide ht CCJA yregrus fo htgneL latot ro latotraen gniriuqer recnac ytivac larO yats fo htgnel latipsoHh tinu erac evsnetni ioNtinu nwodpets rod yregrus larosnart htiw recnac laegnyrahporOymotcessogllymotoubdnam htiiw recnac laegnyrahporO ymotcegnyral latot htiw recnac laegnyrahpopyHymotcegnyral latot gniriuqer recnac laegnyraLi cpocsodne gniriuqer recnac laegnyrahposaNymotcegnyrahp laitrapdna odne gniriuqer recnac suns lasanarap roi lasaNymotolli xamgniriuqer recnac laegnyrahposaNnoitceser cpocsinoitceseri noitceser nks gniriuqer recnac nks decnavdAinoitcurtsnocer palf lanoger dnai edon hpmyl detimil htiw renac kcen dna daeH edon hpmyl on htiw recnac kcen dna daeHycnangilam enob laropmeTesaesdiII ot I egatSy egAy egAy egAesaesdi SPGOCE y ¥ egAycnangil amditorap edarghgHinoitcurtsnocer palfeerf gniriuqer recnac nks decnavdAi laitrapgniriuqeryregrus laegnyral recnac laegnyraL gniriuqer recnac laegnyrahpopyHnyrahpognyral latotymotcegriuqer recnac sunsi roiretna nepogn i lasanarap ro lasaN ¥i lacgrus lacafonarciili ygootsh edargwol rof dedeecxe emit tiaWkwnoitceser eussittfos htiw recnac ytivac larOi rof gnhcaorppa tubdedeecxe ton em it tiaW dedeecxe emit tiaWliygootsh edarghghiwoliygootsh edargl rof kw deecxe ton emit tiaWromoc ereves yreV SP GOCE ei sutats ecnamrofrep rooP ypareht evitanretlAlebaliava ni dehcaorppa tub edargwol rof kw ditorap edargwoLycnangilam edon hpmyl htiw recnac doryhTiesaesdilygootshi recnacnonge ytidb i ta i lavvrusy srotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLi gnknaR fo sdnuoR retfA serocSdna snoitacdn iI noitazitiroirP ELBATCancer Month 0cOriginal TRj tnavuda dna esaesddecnavda hti iw tneitaPderiuqer tinu nwodpets ro tinu erac evsnetniIderiuqer ebut ymotsoehcart oNderiuqer palf eerf oNd yats fo htgnel latipsoHh yregrus fo htgneLd yats fo htgnel latipsoHderiuqer palf eerFh yregrus fo htgneLnoitpo na si SPGOCEsrotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLdeunitnoC ELBATyparehtodar iTRi nosnetxe ladonartxe ENE sutats ecnamrofreppu ygoocnO evitarepooCnretsaE l SPGOCE snoitaverbbAiTABLE Agreement Between Experts During the Delphi ProcessRoundOrdinal ScaleaLCL UCLPer Priority GroupLCL UCLAbbreviations LCL lower confidence limit UCL upper confidence limitThere were raters and agreement was measured using the Krippendorff alphaaOrdinal scale refers to the scale of to used to rate priority of surgery and Per Priority Group refers to the lowpriority mediumpriority and highpriority groups related to the scoring scaleguide the allocation of scarce resources maximizing the benefits produced by scarce resources treating people equally promoting and rewarding instrumental value and giving priority to those patients who are worst off9 These have been contextualized for cancer care more broadly and are manifest in the SPARTANHN algorithm21 The highpriority indications implicitly embrace an underlying premise of saving the most lives andor preserving the most lifeyears Many procedures for patients with HNC are aerosolgenerating and increase the risk to health care workers and other hospitalized patients22 Our process accounted for these by giving consideration to these factors during the consensus process although indications associated with potential exposure to health care workers did not emerge as lowpriority ones Indications associated with lower resource use did achieve consensus for higher importance This may help to avoid the opportunity cost of treating fewer patients with longer surgeriesAnecdotal and institutionspecific prioritization schemas for patients with HNC and general otolaryngology have been suggested213 These parallel similar efforts for general surgery cardiac surgery and orthopedic surgery12132328 In many of these patients are prioritized by the scoring of several criteria and summing of the scores to achieve a total patient score Many of these systems have been validated against expert rankings of surgical priority2728We used a methodology for developing a pointbased prioritization system similar to those previously described29 To the best of our knowledge pointbased surgical prioritization systems have been very well studied to date Hansen et al previously proposed a methodology for developing a pointbased prioritization system using the following steps ranking patient case vignettes Cancer Month 0cSPARTANHNde Almeida et alFIGURE The Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN scoring system ECOG indicates Eastern Cooperative Oncology Group ENE extranodal extensionusing clinical judgment drafting the criteria and categories within each criteria pretesting the criteria and categories consulting with patient groups and other clinicians determining point values for criteria and categories checking the testretest reliability and face validity and revising the points system as new evidence emerges29 Our approach to the development of the SPARTANHN was similar However given the relatively expedited nature of the process we did not directly involve patientsOne method proposed for establishing the priority of all indications in a pointbased scoring system is known as Potentially All Pairwise Rankings of all Alternatives PAPRIKA30 In the current study we chose to use the RANDUCLA process instead of pairwise comparison to minimize computational burden We established Cancer Month 0cOriginal FIGURE External validation rank results A total of experts were asked to rate the scenarios provided shown on the xaxis and the results were compared with the rank generated by models and shown on the yaxis Green shading reflects high priority ranked yellow shading indicates medium priority ranked and red shading indicates low priority ranked Asterisk denotes ties from the algorithm SPARTANHN indicates Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancerindications for surgical prioritization that would create an enormous computational burden using pairwise comparison methodology One problem inherent in the PAPRIKA methodology is the assumption that all indications are not equal and can be ranked However clinically certain indications may be equivalent in priority Furthermore pairwise comparisons assume mutual exclusivity of each of the indications which is not always the case Use of the RANDUCLA consensus process avoids the need for multiple pairwise comparison and allows for consideration of each factor in isolation The goal of the consensus rounds was not to establish a rank order for all indications but mainly to understand which indications result in high intermediate or low priorityThe SPARTANHN algorithm has demonstrated preliminary reliability and validity We demonstrated good agreement between raters and the SPARTANHN algorithm suggesting minimal interpretive error Many of the highpriority indications accounted for some component of interpretation because raters were forced to consider imminent disease progression that may result in an adverse outcome Despite the subjective decisions that must be made as part of SPARTANHN agreement remained high In fact true interrater reliability was found to be higher because the Kalpha is a conservative measure of reliability Other measures of reliability such as the Kendall coefficient of concordance tend to overestimate reliability and cannot be applied to missing data31 Perhaps most important the SPARTANHN correlated highly with expert rankings With established validity this algorithm may be ready for preliminary clinical use although further testing against realworld data to validate it with other cancer outcomes such as survival is neededThe results of the current study must be interpreted within the context of the study design Although externally validated by other surgeons across North America and the United Kingdom the criteria for which consensus was achieved for prioritization were not vetted by patients advocacy groups or other stakeholders such as medical or radiation oncologists The latter groups represent essential providers in the multidisciplinary care of patients with HNC and may have important insight into the availability and effectiveness of nonsurgical treatments1920 Nonetheless the actual prioritization of surgical waitlists remains the sole responsibility of surgeons and their practice partners In addition the SPARTANHN algorithm is intended to assist in making difficult prioritization decisions and is not intended to make recommendations for the time frame in which patients should receive treatment Instead established guidelines should be adhered to for treatment targets Patient wait times as they relate to those targets should be considered when using the SPARTANHN algorithm The validation process in the current study used expert opinion as the gold standard of prioritization which is potentially biased and reflected the opinions of surgeons practicing in academic medical centers from resourcerich nations Subsequently use of the SPARTANHN algorithm in other geographic regions Cancer Month 0cand health care systems requires additional investigation because local treatment paradigms and risk factors may vary substantiallyThe current study has presented the development and validation of a novel algorithm for the prioritization of surgery for patients with HNC Further evaluation of its implementation in various practice settings will be obligatory However the results of the current study have provided data with which to inform realworld use as the current pandemic has obviated our ability to more rigorously study the instrument prior to making necessary and difficult realtime allocation decisionsFUNDING SUPPORTNo specific funding was disclosedCONFLICT OF INTEREST DISCLOSURESEvan M Graboyes has received grants from the National Cancer Institute and the Doris Duke Charitable Foundation for work performed outside of the current study Vinidh Paleri offers his services as a proctor for a transoral robotic surgery proctoring program run by Intuitive Surgical and has been remunerated for his time Antoine Eskander has received a research grant from Merck and acted as a paid consultant for BristolMyers Squibb for work performed outside of the current study Ian J Witterick has stock in Proteocyte Diagnostics Inc and has received honoraria from Sanofi Genzyme and Medtronic Canada for work performed outside of the current study The other authors made no disclosuresAUTHOR CONTRIBUTIONSJohn R de Almeida Study idea and design writing and editing Christopher W Noel Study design writing data analysis and editing David Forner Study design writing data analysis and editing Han Zhang Data acquisition and editing Anthony C Nichols Data acquisition and editing Marc A Cohen Data acquisition and editing Richard J Wong Data acquisition and editing Caitlin McMullen Data acquisition and editing Evan M Graboyes Data acquisition and editing Vasu Divi Data acquisition and editing Andrew G Shuman Writing data acquisition and editing Andrew J Rosko Data acquisition and editing Carol M Lewis Data acquisition and editing Ehab Y Hanna Data acquisition and editing Jeffrey Myers Data acquisition and editing Vinidh Paleri Data acquisition and editing Brett Miles Data acquisition and editing Eric Genden Data acquisition and editing Antoine Eskander Data acquisition and editing Danny J Enepekides Data acquisition and editing Kevin M Higgins Data acquisition and editing Dale Brown Data acquisition and editing Douglas B Chepeha Data acquisition and editing Ian J Witterick Data acquisition
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"objectives to describe the benefits and limitations of using individual and combinations of linked english electronic health data to identify incident cancersdesign and setting our descriptive study uses linked english clinical practice research datalink primary care cancer registration hospitalisation and death registration dataparticipants and measures we implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and we calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers we described completeness of grade and stage information in the cancer registration data setresults gold standard cancers were identified positive predictive values of all case definitions were ¥ and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity for case definitions that used cancer registration alone or in combination was ¥ for the four most common cancers and ¥ across all cancer sites except bladder cancer using cancer registration alone for case definitions using linked primary care hospitalisation and death registration data sensitivity was ¥ for the four most common cancers and ¥ for all cancer sites except kidney oral cavity and ovarian cancer when primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancerss ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority strengths and limitations of this study º this is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in england to identify cases of the most common incident cancers º using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions º we described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data º a key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataintroductionthe clinical practice research datalink cprd provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 in the field of cancer epidemiology cprd primary care data linked to hospital episode statistics admitted patient care strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access data hes apc office of national statistics ons mortality and national cancer registration and analysis service ncras cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for ncras data research teams therefore commonly choose not to use all available linked data3 cancer epidemiology studies can also be conducted using ncras and hes apc data provided by national health service nhs digital and public health england phe without linkage to cprd primary care data4 this provides national coverage at the expense of the detailed health data that are available in primary care recordsvalidation studies assessing concordance between cprd gold hes apc and ncras data have estimated high positive predictive values ppvs for cprd gold data and varying proportions of registered cancers that are not captured in cprd gold and hes apc5 the most up to date analysis by arhi et al included the five most common cancers and all papers focussed on concordance between cprd gold only and ncras existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs national data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and clinical commissioning groups10 we aim to describe and compare the benefits and limitations of using different combinations of linked cprd primary care data hes apc ons mortality and ncras cancer registration data for conducting cancer epidemiology studies our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets we have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets we also describe the availability of stage grade and treatment variables over time in the cancer registration data for the cprd linked cohort this reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesmethodsstudy design and settingwe completed a concordance study using linked2 english cprd gold hes apc ons mortality and ncras data cprd gold data were extracted from the january monthly release and the 13th update to cprds linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 malignant neoplasms of urinary organ unspecified was considered consistent with both bladder and kidney cancer for ncras we accessed coded records for the most common cancers we included cancers recorded in the clinical or referral file for cprd gold cancers recorded in any diagnosis field for hes apc and the underlying or strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure gold standard algorithm to identify incident site specific cancers using all data sources hes hospital episode statistics ncras national cancer registration and analysis service ons office of national statisticsmost immediate cancer cause of death in ons mortality datacancer case definitions based on individual sources and combinations of sourceswe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources ncras alone ncras and hes apc all sources cprd gold hes apc and ons mortality cprd gold alone hes apc alone multiple malignant cancers recorded on the index date in cprd gold or hes apc were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the ncras data if available and as multiple site cancer if not for each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upgold standard cancer case definitionwe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure cancers recorded in ncras alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in hes apc alone or gold alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in ncras and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in ncras if there were more data sources in total with records for cancer at that site than data sources with contradictory recordswe used ncras data to identify stage grade and treatment where available in the cancer registry only cohort binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisstatistical analysisfor each cancer site and each individual or combined data source we combined our applied study definitions strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordwe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex we calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourceswe used kaplan meier methods to describe mortality over time for cancers identified using each definition the ons mortality death date was used for these analyseswe used the ncras only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timepatient public involvementpatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsresultsof research quality patients in the cprd gold january build were eligible for linkage to hes ons mortality and ncras data in set were excluded due to unknown sex of the remainder and had at least year of follow up between january and december and were included in the study population using the gold standard algorithm incident cases of cancer were identified the number of patients identified with each cancer is presented in online supplementary appendix table half n82 of these patients were male aged to aged to and aged or olderfigure presents ppvs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm when using ncras data alone to of cancers were confirmed by the algorithm for out of cancer sites the ncras only case definition gave the highest ppv case definitions using data sources not including ncras generally had lower ppvs ranging from to for individual cancer sites for the four most common cancers breast lung colorectal and prostate ppvs were at least for all case definitions minimal differences in ppvs were observed between age groups years and sexes online supplementary appendix figures figure presents sensitivity values for each case definition sensitivity was generally higher for the case definitions that included ncras data ranging from to for individual cancer sites except bladder cancer identified using ncras data alone and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity was also generally high for definitions using a combination of cprd gold hes apc and ons mortality data ranging from to ¥ for the four most common cancers sensitivity was lower for case definitions that used cprd gold alone range to for individual cancer sites or hes apc alone range to sensitivity values for cprd gold alone and hes apc alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures post hoc analysis suggested that the low sensitivity of cprd gold only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary organ cancer codes n1108 in cprd gold rather than contradictory information about the first cancer record n625 these incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 bladder cancers that were not recorded in ncras data n3445 were commonly recorded in both hes apc and cprd gold n2228 or in hes apc only with a subsequent unspecified or bladder cancer record in hes apc within months n995 table describes the number of days median iqr and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used case definitions using ncras alone and combinations of ¥ data sources captured cancers close to the gold standard date median lag ¤ days for all cancer sites whereas median lags were generally longer for the case definitions using cprd gold alone and hes apc alonefigure describes mortality over time following incident cancer diagnoses ascertained from each case definition minimal differences in mortality were observed between cancers identified from different case definitions where variability was observed cancers identified using cprd gold only had the lowest mortality rates eg kidney cancer and cancers identified using hes apc only or ncras only had higher mortality rates eg prostate cancer and bladder cancer respectivelyfigure describes completeness of grade and stage for cancers identified using ncras only recording of grade was highly variable between cancers with gradual increases in completeness over time completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure online supplementary appendix figure describes recording of treatment modalities identified using ncras strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure positive predictive value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service onsoffice of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0celitnecrep hthtrqi inademelitnecreprqi inadem hthtelitnecrep htht inademrqielitnecrep hthtcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l ebatopen access recnac lanoitan sarcn atad erac tneitapdetti mda scitsitats edospei latipsoh cpaseh knil atadhcraeser ecitcarp lacniilc drpc metsys suovren lartnec sncl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iia setis recnac nommoc tsom ruofdcii nosrev sesaesdi fonoitacfissacliscitsitats lanoitan rof ecfifo snoi atadnoitartsgerrecnac ecvres ssyanadna liinoitartsgeri lanoitanretni eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaci snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmun ot ot ot ot ot cl amoeym epitluml ot ci ameakuel ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep hthtsarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure mortality following first ever record of cancer in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service nhl non hodgkin's lymphoma ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure completeness of grade and stage for cancers identified using ncras data only cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites grading information is not applicable to braincns sarcoma or haematological cancers and not required by in the national data standard cosd for prostate cancer core staging is not applicable to haematological and gynaecological cancers other types of staging are recommended by cosd cns central nervous system cosd cancer outcomes and services data set ncras national cancer registration and analysis service nhl non hodgkin's lymphomastrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access only missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordeddiscussionstatement of principal findingswe investigated the use of different sources of electronic health record data to identify incident cancers for all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers combining cprd gold hes apc and ons mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date finally while we observed minimal changes in ppvs and sensitivities between and completeness of ncras cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 completeness of cancer treatment recording was difficult to assess due to the absence of a missing categorystrengths and weaknesses of the studythe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from cprd to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies we have also assessed the value of using ncras cancer registration data to measure stage grade and cancer treatment modalitiesa limitation of the study is that our gold standard algorithm is not validated we feel that we were justified in pre weighting ncras data as more reliable that other data sources as ncras is a highly validated data set that matches merges and quality checks data from multiple sources4 we did not consider ncras to be the outright gold standard as it is plausible that ncras does not identify all tumours diagnosed and treated in primary and secondary care for most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in hes apc cprd gold or ons mortality data but not in ncras these tumours may have been diagnosed and coded as invasive in primary or secondary care but not by ncras been incorrectly coded in hes apc cprd gold or ons mortality data not have been notified to ncras eg tumours treated in private hospitals or be the result of linkage errors between the data sets the proportion of cancers identified in hes apc but not in ncras is particularly high for bladder cancer this is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in ncras which are greatest for bladder cancer4 this explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in ncras compared with other data sources to identify incident cancers we required months of research quality follow up in cprd gold prior to inclusion in the study previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 the identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as ncras data collection started in hes apc in and ons mortality data in and by the inclusion of all diagnostic codes in hes apc assuming that the first ever primary or secondary record identified incident cancer reassuringly ppvs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases requiring internal confirmation within months for cancers recorded in cprd gold alone in our gold standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up our data cut only included ncras data for the top cancers earlier cancers at other sites will have been missed in this studyit is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesstrengths and weaknesses in relation to other studies discussing important differences in resultsthe most up to date study describing concordance between linked cprd gold hes apc and ncras data sets demonstrated that to of the five most common cancers recorded in cprd gold are not confirmed in either hes apc or cancer registration data and to of registered cancers are not recorded in cprd gold8 for cancers recorded in both sources the diagnosis date was a median of to days later in cprd gold than in the cancer registration data using cprd gold alone to identify these strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis use of hes apc only identified a higher proportion of patients with the correct diagnosis date than cprd gold but over represented older patients and those diagnosed through the emergency route the majority of registered cancers were picked up using both cprd gold and hes apc ranging from for lung cancer to for breast cancer previous research demonstrated similar results with substantial differences between cancer types5 additionally a study using data from to found that using hes data in addition to ncras data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancerswe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards levels of data completeness of staging information in the cprd extract in were similar to those reported by the united kingdom and ireland association of cancer registries ukaicr9meaning of the study possible explanations and implications for clinicians and policymakersuse of ncras cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately case definitions based on a combination of cprd gold hes apc and ons mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersthese findings should be considered when deciding which data sources to include in research studies and which sources to use to define cancer exposures outcomes and covariatesunanswered questions and future researchfurther research is required to investigate the validity of cancer recorded in cprd gold and hes apc that are not recorded in the ncras data and to understand differences in cancer data recording with cprd gold and cprd aurum cprds recently launched primary care database based on records from practices that use emis software17 further investigation would be required to confidently identify subtypes of cancer either using codes available in each data set eg colon and rectal cancer or additional information available in hes apc or ncras data use of ncrass recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu
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Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly citedCutaneous metastases are unusual presenting symptoms of lung cancer erefore they are prone to be misdiagnosed and missede report describes a case of a fortynineyearold female with painful zosteriform rashes showing multiple vesiclelike papuleslocalized on the left breast for days e patient had been diagnosed as lung adenocarcinoma at the department of oncology oneyear ago Skin biopsy revealed blue nodular lesions in the dermis composed of clustered heterogeneous tumor cells with glandularformation Immunohistochemical stains conï¬rmed the diagnosis of metastatic lung adenocarcinoma IntroductionLung cancer can metastasize to almost all ans butmore often invades the hilar nodes liver adrenal glandsbones and brain [] e incidence of lung cancer withmetastases to the skin varies between [] A lungcancer metastasis is usually classiï¬ed only as adenocarcinoma squamous cell carcinoma SCC or undiï¬erentiated carcinoma Until the 1980s SCC was reported asthe most common type of lung cancer However adenocarcinoma has replaced SCC as the most common lungcancer subtype especially in women and in neversmokers Sun reported that the type of adenocarcinoma was times more frequent than that of SCC []Skin metastases can appear on any cutaneous surface andthe most common sites are the chest abdomen head andneck [ ] Cutaneous metastases have various manifestations such as single papulesnodules or multiplelesions on anywhere of the skin while other rare formsmay show plaquelike lesions erysipelaslike papuleszosteriform lesions and scars [ ] Case PresentationA fortynineyearold nonsmoker female was admitted toour department with multiple painful papules localizedon the left breast ey appeared eruptively for about days and initially diagnosed as herpes zoster in anotherhospital e patient had been diagnosed as lung adenocarcinoma at the department of oncology one year agoShe was given oxitinib mesylate a targeted drug for thetreatment of nonsmallcell lung cancer In addition thepatient exhibited symptom of pain signs of weight lossanorexia and fatiguePhysical examination showed zosteriform vesiclelikepapules measuring cm on the left breast elesions were pink or red ï¬rm and tender Figure Excisional biopsy was performed revealing blue nodularlesions ltrating in the dermis composed of clusteredheterogeneous tumor cells with glandular formationSome tumor cells were detected within vessels or lymphatic vessels Some cells were transparent Mitosis wassignificant Figures 2a2c In immunohistochemistumor cells were positive for cytokeratin CKtrycytokeratin7 CK7thyroid transcription factor1TTF1 and EMA and negative for cytokeratin20CK20 carcinoembryonic antigen CEA and grosscysticGCDFP15Figures 3a3c Proliferative index as measured byKi67 was approximately oftumor cellsAccording to the clinical and pathological features cutaneous metastatic lung adenocarcinoma was madeprotein15diseaseï¬uid 0cCase Reports in Dermatological MedicineFigure Zosteriform vesiclelike papules measuring cm on the left breast Pink or red ï¬rm and tenderabcFigure Skin biopsy revealed a blue nodular lesions ltrating in the dermis composed of clustered heterogeneous tumor cells withglandular formation HE magniï¬cation b some tumor cells were detected within vessels or lymphatic vessels HE magniï¬cation c some cells were blue and transparent and mitosis was significant HE magniï¬cation abcFigure Immunohistochemical stain highlighting the tumor cells showing a CK7 b EMA and c TTF1 positive DiscussionSkin metastases suggest the progression of primary cancerand portend a poor clinical prognosis Skin metastases fromlung cancer are rare e percentage of patients with lungcancer that develops cutaneous metastases ranges from to percent [] It is seen more often in men than in women[] It does not show any speciï¬c presentation It is oftenpainless and less likely to be noticed making it more diï¬cultto be diagnosed correctly which may delay treatment Although described cases show that metastatic nodules arepainless our patient showed severe pain e presence ofzosteriform painful vesiclelike lesions really mimics herpeszoster clinically in our casee mechanisms determining the metastasis of lungcancer in skin remain unknown Pathogenesis is suggested tobe by lymphovascular invasion with poor diï¬erentiationand upper lobe tumors increasing the risk [] Usually skinmetastasis develops after initial diagnosis of the primarymalignancy and late in the course of the disease Occasionally skin lesions that arise from lung cancer may develop before the primary tumor is recognized In our case 0cCase Reports in Dermatological Medicine[] R Koca Y Ustundag E Kargi G Numanoglu andH C Altinyazar A case with widespread cutaneous metastases of unknown primary origin grave prognostic ï¬ndingin cancer Dermatology Online Journal vol no p [] N A Babacan S Kilickap S Sene A case of multifocalskin metastases from lung cancer presenting with vasculitictype cutaneous nodule Indian Journal of Dermatologyvol p skin metastases occurred during the immunotherapy Histology shows most commonly adenocarcinoma and thensquamoussmallcell followed by largecell carcinoma []Immunohistochemical markers are useful for the identiï¬cation of the primary cancer or when a shorter diï¬erential isdesired AntiTTF is both sensitive and speciï¬c for primaryadenocarcinomas bronchioalveolar carcinomas and smallcell carcinomas when thyroid primary is excluded []CK7and CK20 are sensitive but not speciï¬c for primaryadenocarcinomas and bronchioalveolar carcinomas eCK7CK20tumors usually include the lung breast endometrium ovary thyroid salivary gland and mesothelioma [ ]Treatment of a single solitary skin lesion usually includessurgery alone or combined with chemotherapy andor radiation If lesions are more disseminated chemotherapy isthe primary option but may elicit an inadequate response[] Radiation can also be used alone andor in combinationwith chemotherapy andor surgery However despite thecombination of radiotherapy and chemotherapy patientswith lung cancer developing cutaneous metastases have apoor outcome Mean survival is short usually to monthsafter diagnosis of cutaneous metastasis []Conflicts of Intereste authors declare they have no conï¬icts of interestAcknowledgmentsis work was supported by a grant from the NationalNatural Science Foundation of China References[] T W Mollet C A Garcia and G Koester Skin metastasesfrom lung cancer Dermatology Online Journal vol no [] S Sun J H Schiller and A F Gazdar Lung cancer in neversmokersa diï¬erent disease Nature Reviews Cancer vol no pp [] S Dreizen H M Dhingra D F Chiuten T Umsawasdi andM Valdivieso Cutaneous and subcutaneous metastases oflung cancer Postgraduate Medicine vol no pp [] M Khaja D Mundt R A Dudekula Lung cancerpresenting as skin metastasis of the back and hand a caseseries and literature review Case Reports in Oncology vol no pp [] W T McSweeney and K Tan Cutaneous metastases as apresenting sign of metastatic NSCLC Journal of Surgical CaseReports vol no [] M H Brownstein and E B Helwig Metastatic tumors of theskin Cancer vol no pp [] R B McGrath S P Flood and R Casey Cutaneous metastases in nonsmall cell lung cancer BMJ Case Reportsvol [] V Jerome Marson J Mazieres O Groussard Expression of TTF1 and cytokeratins in primary and secondaryepithelial lung tumours correlation with histological type andgrade Histopathology vol no pp 0c'
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we report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus cryosurgical excisionwas performed tissue pathology conï¬rmed the diagnosis of metastatic embryonal carcinoma the patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin bep chemotherapy regimenkeywordscryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourcorrespondencewenchien cheng division of pulmonary and critical care medicine department of internal medicinechina medical university hospital no yuderoad north dis taichung city taiwanemail wcchengdrgmailcomreceived july revised july accepted july associate editor james horespirology case reports e00644 101002rcr2644introductionlung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases ebms from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [] primary lung cancer is the most common cause of endobronchialtumours extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [] although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma ebms from embryonal carcinomas are extremely rare in this report we present acase of ebm from a primary embryonal carcinomacase reporta 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days he reported no fever chills coldsweats weight loss or decreased appetite a chest radiograph at admission revealed complete collapse of the leftlung fig 1a computed tomography ct was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumbronchoscopytumourobstructing thefig 1b theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 ttf1negative sallike protein sall4positive and cluster of differentiation cd30positive these ï¬ndings were consistentwith a ï¬nal diagnosis of metastatic embryonal carcinomafig we checked the levels of tumour markers in thepatient including those of alphafetoprotein afp betahuman chorionic gonadotropin bhcg and lactate dehydrogenase ldh each tumour marker was found to be the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirologythis is an open access under the terms of the creative commons attributionnoncommercialnoderivs license which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modiï¬cations or adaptations are made vol iss e00644page 0cebm from embryonal carcinomack teng figure chestradiograph and bronchoscopic view of the endobronchial metastasesebm a complete collapse of the left lungon chest radiograph b bronchoscopic viewof the endobronchial tumour within the leftmain bronchusfigure tumour pathology of metastatic embryonal carcinoma a embryonal carcinoma with a complex glandular growth pattern the characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magniï¬cation b immunohistochemical staining with antithyroid transcription factor1 ttf1 highlighting cells in the alveolar space original magniï¬cation c immunohistochemical staining with antisallikeprotein sall4 revealed diffuse nuclear staining original magniï¬cation d immunohistochemical staining with anticluster of differentiation cd30 highlighting diffuse membranous staining original magniï¬cation presentin high levels afp ngml bhcg miuml and ldh iul the patientunderwent a right orchiectomy followed by a bepbleomycin etoposide cisplatin chemotherapy regimendiscussionwe report here the case of a young man with an ebmfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumourslikewise manykindslung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverebms from extrapulmonary malignancies are rare [] primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith ebm primarily breast colon and renal carcinomas[] ebms from testicular seminomas are also extremelyrare the majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0cck teng ebm from embryonal carcinomatable reports of previous cases of ebmslocationdiagnostic methodpathology¶zt¼rk moreirameyer case case the oriï¬ce of right upper loberight main bronchusleft main bronchus main carina andright main bronchus fibreoptic bronchoscopy mixed gctfibreoptic bronchoscopy mixed gctvideobronchoscopyembryonal carcinoma¶zsu the oriï¬ce of the right upper lobefibreoptic bronchoscopytesticular seminomaturan varkey our caseand right intermediary loberight intermediate bronchusleft main bronchusleft main bronchusebm endobronchial metastases gct germ cell tumourembryonic carcinomas or seminomas there are only a few published reports of primary testicularembryonic carcinomas resulting in ebms []the mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently howeversome patients are asymptomatic in our patient symptoms on presentation included haemoptysis and shortnessof breathresults from chest radiography in patients with ebmcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyhowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difï¬cult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these ï¬ndings alone toconï¬rm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue the ï¬exible bronchoscopy ï¬breoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy the former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure the patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the ï¬nal pathology reportconï¬rmed the diagnosis of metastatic embryonal carcinomawe had evaluated the presence of afp bhcg andldh tumour markers elevated afp levels can be secretedby germ cell tumours gcts including embryonal carcinoma yolk sac tumour or teratoma in gcts detectablerigid bronchoscopybronchoscopyfibreoptic bronchoscopyand cryosurgerysomatictype gctembryonal carcinomaembryonal carcinomabhcg elevation is observed in both seminomas and nonseminomas the serum level of ldh was directly correlated with tumour burden in nonseminomatous gctswhich is also useful for the surveillance of patients withadvanced seminoma the tumour markers in ourpatient showed elevated levels of afp bhcg and ldhthis was compatible with the diagnosis of embryonal carcinoma moreirameyer also evaluated the patienttumour markers and found elevated levels of afp ngml and bhcg miuml the elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma ¶zsu onlyevaluated the patients bhcg level which was found to beelevated miuml and the ï¬nal diagnosis wasmetastatic testicular seminoma on comparison with previous case reports table ours was the ï¬rst case in which the tissue was obtainedusing cryosurgery other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation apc to control bleedingcryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree sampleshaemorrhage was observed to be similar during both procedures further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesin conclusion ebms from primary gcts notably thoseassociated with total or partial collapse are extremely rarewe have presented this case to emphasize the importanceof distinguishing ebm from primary lung carcinoma andto report the ï¬rst case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0cebm from embryonal carcinomadisclosure statementappropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisreferences ¶zt¼rk a aktas¸ z and yılmaz a endobronchialmetastasis of mixed germ cell tumors two cases tuberktoraks lee sh jung jy kim dh endobronchialmetastases from extrathoracic malignancy yonsei med j ikemura k lin dm martyn cp endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing lung moreirameyer a bautistaherrera d hern¡ndezembryonal endobronchialgonz¡lez mck teng carcinomaj bronchologyinterv pulmonol ¶zsu s erol mm oztuna f endobronchial metastasis from testicular seminoma med princ pract tumoraltesticular germ cell turan d akif ¶zg¼l m kirkil gendobronchial metastasis ofeurasian j pulmonol et varkey b and heckman mg diagnosis of a case ofembryonal carcinoma by bronchial biopsy chest paradis tj dixon j and tieu bh the role of bronchoscopy in the diagnosis of airway disease j thorac dis aktas z gunay e hoca nt endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisann thorac med barlow lj badalato gm and mckiernan jm serumtumor markers in the evaluation of male germ cell tumorsnat rev urol the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0c'
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of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified dtpa complexes21 the original substance of the modified dtpa dtpamod was synthesized in tomsk polytechnic university preparation of colloid solution dtpamod was produced using the following method a sample of modified dtpa with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of nahco3 solution by heating to a0°c after that the volume was adjusted with the same solvent up to the mark in order to reduce the p size the container with suspension was heated in water to a0°c and treated with ultrasound for a0min 1tomsk polytechnic university lenina avenue tomsk russia 2tomsk national research medical center russian academy of sciences kooperativny street tomsk russia email sadkintpuruscientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorfigure a0 the general scheme for the synthesis of 99mtcdtpamodwhich reduced the average p radius up to a0nm the general scheme for the synthesis of 99mtcdtpamod is shown in fig a0the second type of colloids is iron nanops coated with a carbon shell of fec fig a03a these ps were obtained from the institute of metal physics urb ras ekaterinburg russia in order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates adt onto the surface of these ps has been developed an effective method for the synthesis of adt followed by their application onto the carbon surface of ps was developed at the tomsk polytechnic university22 the general scheme for the synthesis of fec ps and their subsequent interaction with 99mtc is shown in fig a03bin the third type of colloids technetium99m was adsorbed on aluminum oxide powder a powder of lowtemperature cubic modification of gammaoxide al2o3 prepared from aluminum hydroxide powder aloh3 by its calcination in a muffle furnace was used the substance was synthesized in tomsk polytechnic universitya reducing agenttin ii chloride dihydrate was used in order to obtain complexes of 99mtc with colloidsgelatin powdered ph eur uspnf pure pharma grade cas number was purchased from applichem gmbh darmstadt germanymethods method for preparation of 99mtc labeled nanocells the introduction of the radioactive label 99mtc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent sncl22h2o a0mgml in different ratios and then adding a a0ml of eluate of 99mtc a0mbqml to the mixtures the mixtures were incubated for a0min at a temperature of a0°c the preparation is ready for use after cooling at room temperature the reducing agent sncl22h2o was used as a hydrochloric acid solution the amount of a0g of tin chloride ii is added to the vial and a0ml of a0m hydrochloric acid hcl scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cfigure a0 a carbon encapsulated iron nanop b the general scheme for the synthesis of fec psis then added for its preparation after dissolution the volume is adjusted with distilled water to a0ml dissolution was carried out in an inert gas argon mediumdetermination of the size of 99mtc labeled colloidal ps the determination of the size of the labeled nanocolloids was carried out by spectroscopy on a nanophox p size analyzer sympatec gmbh germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity calculations of the yield of products with different p sizes were determined according to the formulas given belowc220 avc a1avc c100 a1 a2a1 c50 a2 a3a2where avc is the activity of the initial suspension prior to filtration a1 is the activity measured after filtration through a a0nm filter a2 is the activity after filtration through a0nm filter a3 is the activity measured after filtration through a0nm filterin parallel determination of the radiochemical purity rcp of preparations by thin layer chromatography method was carried outthinlayer chromatography tlc procedure to determine radiochemical purity of 99mtcnanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip sorbfil russia a0 cm to determine scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0camount of sncl22h2o mga[sn99mtc]a atcviia table change in relative activities of the complex [sn99mtc] and 99mtc viipertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min in this system pertechnetate migrated with the front of the mobile phase rf to determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min in this system the 99mtcnanocolloid migrated with the front of the mobile phase rf stability the stability of 99mtcnanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mtcnanocolloid following by incubation at a0°c for a0h at different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using tlc24determination of the functional suitability of preparations for scintigraphic detection of sln a study to assess the functional suitability of new nanocolloid rps was performed in series of experiments involving white wistar male rats weighing a0g injection of rp in a dose of a0mbq was performed between the first and second fingers of the rats hind paw the animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame a0s in a pixel matrix static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of with a set of pulses scintigraphy of animals was performed on an ecam signature gamma camera siemens germany the results of scintigraphic studies determined the percentage of accumulation of rp in the lymph node and the injection site the maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the european convention for the protection of vertebrates used for experiments or other scientific purposes strasbourg the experimental protocols were approved by cancer research institute biomedical ethics committee protocol number all invasive manipulations with animals were performed using inhalation or drug anesthesiastatistical analysis all mean values are expressed as idg ± sd data were analyzed statistically using methods of general statistics with a commercially available software package statistics for windows statsoft inc version results and discussionto carry out the labeling of colloids 99mtc extracted from a standard generator in the form of pertechnetate ions contained in a nacl solution was used it has a higher degree of oxidation vii in this chemical form and is not prone to complex formation a reducing agenttin ii chloride dihydrate widely used for the preparation of various compounds labeled with 99mtc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 as a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess sncl2·2h2o or the additional formation of a complex of reduced 99mtc with tin26 all this required preliminary experimental studies to establish the necessary and sufficient amount of sncl2·2h2o in the reaction mixtureduring the experiments it was found that the optimal concentration of sn ii in the composition of the reaction mixture when it interacts with 99mtc should be in the range of a0mgml table a0it was found that when the eluate with the preliminarily reduced 99mtc vii was added to the nanops the sn ii concentration introduced in the rp was csn a0mgml almost the entire amount of 99mtc has time to enter the composition of the largesize complex with tin even before its mixing with nanops this means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the sn ii solution into the reaction mixture in this connection the reduction of 99mtc with tin ii was carried out in the presence of the selected substance in this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex the technique of applying of the 99mtc label to the surface of nanosized ps is given in the previous sectionas a result of the studies reagent compositions and conditions for obtaining three nanocolloid rps were determined table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of a0nmproceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mtc vii in the obtained preparations is preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in fig a0scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0ccomposition of the preparation per a0mldtpamod a0mg 99mtc a0mbq sncl22h2o a0mg n fec a0mg 99mtc a0mbq sncl22h2o a0mg n al2o3 a0mg 99mtc a0mbq sncl22h2o a0mg n colloid yield a0nm rcp ± ± ± table composition of reagents for production of technocium99 a0m nanocolloidsfigure a0 distribution of the preparation in the rat when the preparation is administered [al2o3 99mtc sn ii] a immediately after the administration of the drug b a0min after the administration c a0min after the administrationcomposition of preparations per a0mlal2o3 a0mg 99mtc a0mbq sncl22h2o a0mg g a0mg n dtpamod a0mg 99mtc a0mbq sncl22h2o a0mg g a0mg n fec a0mg 99mtc a0mbq sncl22h2o a0mg g a0mg n yield of colloid a0nm rcp ± ± ± table indicators of rcp and the yield of a colloid with a fraction of a0nm after the introduction of gelatin in the reagentsscintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mtc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed gelatin g was introduced into the reaction mixture in this connection to increase the mobility of the labeled ps and increase the speed of their movement through the lymph system matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mtc the results of the experiments showed that the addition of gelatin a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of a0nm table a0in addition the size of these ps was determined on a nanophox p analyzer the obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in fig a0 a b c the average p size diameter is and a0nm respectivelystability test showed that complex 99mtcnanocolloid was stable in normal serum at least for a0h radiochemical purity of the tracer at the end of the experiment was ± a study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes fig a0 table a0 displays the al2o3 99mtc dtpamod 99mtc fec 99mtc biodistribution data at different time points postinjectionthe level of accumulation of preparations in the lymph nodes is of the total injected activityconclusionas a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid rps were determined an experimental dependence of the change in the content of 99mtc vii impurities on the concentration of tin ii was established and its minimum amount a0mgml was determined to reach a rhp greater than in this case the yield of the target colloid with p sizes of ± a0nm is preliminary tests of the developed preparations on experimental animals showed that accumulation of rp in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition a0mgml in addition there was an increase in the yield of the colloid scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 change in the density of the distribution of the number of ps from their size in radiopharmaceuticals a 99mtcal2o3 b 99mtcfec c 99mtcdtpamodwith p sizes of a0nm to with radiochemical purity of the preparations of repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cstomachtime h99mtcal2o399mtcdtpamod99mtcfec ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± figure a0 distribution of the preparation in the rat with injection of suspension [al2o3 99mtc sn ii gelatin] a immediately after the administration of the preparation b a0min after the administration c a0min after the administration d a0min after the administration the numbers indicate lymph node site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± bloodmlheart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± table biodistribution data up to a0h after injection of a0mbq of 99mtc in healthy male rats data represent the average value n accumulation in the sln thus the level of accumulation of rp 99mtcdtpamod and rp 99mtcfecadt in the sln is and respectively at the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityreceived march accepted july references jakobsen j k sentinel node biopsy in urooncology a history of the development of a promising concept urol oncol weixler b et al sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival a prospective singlecenter trial world j surg 101007s0026 beasley g m et al sentinel lymph node biopsy for recurrent elanoma a multicenter study ann surg oncol moser j et al sentinel node biopsy in melanoma a singlecentre experience with consecutive patients br j dermatol 101245s1043 buda a et al optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer comparison of realtime fluorescence with indocyanine green and methylene blue int j gynecol cancer scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0c sahbai s et al pericervical injection of 99mtcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in spectct clin nucl med hoogendam j p et al 99mtcnanocolloid spectmri fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer j nucl med stoffels i leyh j p¶ppel t schadendorf d klode j evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies prospective randomized clinical trial to compare icg99mtcnanocolloid hybrid tracer versus 99mtcnanocolloid eur j nucl med mol imaging beisani m et al initial experience in sentinel lymph node detection in pancreatic cancer rev esp med nucl imagen mol schubert t uphoff j henke r p wawroschek f winter a reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer verification in consideration of the european guidelines bmc urol jaukovic l et al lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions hell j nucl med subramanian s pandey u shah s rangarajan v samuel g an indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection nucl med commun ruizdomnguez j m ibarzservio l garcade manuel g calaf peris© o intraoperative injection of 99mtcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery actas urol schauer a j specific developments in sentinel node labling using 99mtccolloids in the sentinel lymph node concept springer berlin wang y et al gasphase chemistry of technetium carbonyl complexes chem phys oconnor m k et al comparison of tc99m maraciclatide and tc99m sestamibi molecular breast imaging in patients with wang j zhang r evaluation of 99mtcmibi in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer ejnmmi res br j radiol costa p et al scintigraphic imaging with technetium99mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats acta cir bras vera d r wallace a m hoh c k mattrey r f a synthetic macromolecule for sentinel node detection 99mtcdtpamannosyldextran j nucl med hoh c k wallace a m vera d r preclinical studies of [99mtc]dtpamannosyldextran nucl med biol skuridin v et al modified dtpa moleculebased nanocolloid radiopharmaceuticals j radioanal nucl chem filimonov v d et al unusually stable versatile and pure arenediazonium tosylates their preparation structures and synthetic applicability lett lukasz k thin layer chromatography in drug analysis crc press london skuridin v et al radiopharmaceutical drug based on aluminum oxide indian j sci technol 1017485 ijst2015v8i36 sazonova s i et al synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging iran j nucl med skuridin v s et al synthesis and biological characterization of 99mtclabeled ciprofloxacin pharm chem j acknowledgementsthis work was financially supported by ministry of education and science of the russian federation rfmefi57514x0034author contributionsvs conducting experimental research analysis and interpretation of the data final approval for manuscript publication vs development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication en development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication es development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication ar conducting experimental research analysis and interpretation of the data final approval for manuscript publication nv conducting experimental research analysis and interpretation of the data final approval for manuscript publication rz conducting tests of the functional suitability of drugs preparation of the section evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and figures final approval of the manuscript for publication of the manuscriptcompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to vsreprints and permissions information is available at wwwnaturecomreprintspublishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0copen access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate if changes were made the images or other third party material in this are included in the s creative commons license unless indicated otherwise in a credit line to the material if material is not included in the s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0c'
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structures assigned to the products were concordant with the microanalytical andspectral data Compounds 4e18 were screened for their ability to induce the antioxidant enzyme NADPH quinone oxidoreductase NQO1 in cells a classical target for transcription factor nuclear factorerythroid268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamide showed the most potent NQO1inducer activity in vitro Compound had low toxicity in mice LD50 ¼ mgkg It also reduced thedamaging effects of gamma radiation as assessed by the levels of Nrf2 NQO1 reactive oxygen speciesROS and malondialdehyde MDA in liver tissues In addition compound showed amelioration in thecomplete blood count of irradiated mice and enhanced survival over a period of days followingirradiation Molecular docking of inside the Nrf2binding site of Kelchlike ECH associated protein Keap1 the main negative regulator of Nrf2 showed the same binding interactions as that of the cocrystallized ligand considering the binding possibilities and energy scores These ï¬ndings suggest thatcompound could be considered as a promising antioxidant and radiomodulatory agent The Authors Published by Elsevier Masson SAS This is an access under the CC BYlicense httpcreativecommonslicensesby40 IntroductionThe extensive use of radiotherapy and the damage caused to thesurrounding normal ans have provoked researchers to ï¬nd newstrategies to protect normal tissues from radiation hazards []The risk of injury from radiation can diminish the value of radiotherapy and contribute to complications for longterm cancer survivors [] Ionizing radiation interrupts cell functions throughradiolysis of water and the production of reactive oxygen speciesROS or reactive nitrogen species RNS [] Excessive productionof ROS and RNS promotes oxidative stress which can affect allcellular components including single or double DNA strand breaks Corresponding authorEmail address mmsghorabyahoocom MM Ghorab[] This ROSmediated toxicity can lead to mutations and consequently cause cardiovascular neurological toxicities and sexualdysfunction as well as cancer [7e10] In order to reduce theseradiationinduced side effects radioprotective drugs are used []Also the use of multitarget antioxidants that act as radioprotectorscan help limit normal tissue damage caused by ionizing radiation[12e14]Nuclear factor erythroid 2related factor Nrf2 is a transcription factor that regulates the expression of various antioxidantproteins to protect against oxidative damage in the cell [] Theabundance of Nrf2 is negatively regulated by Kelchlike ECH associated protein Keap1 a substrate adaptor for a Cullin3Rbx1ubiquitin ligase that binds and continuously targets Nrf2 for ubiquitination and proteasomal degradation [16e18] Under conditionsof oxidative stress redoxsensitive cysteine sensors of Keap1 aremodiï¬ed leading to loss of its ability to target Nrf2 for degradation101016jejmech2020112467 The Authors Published by Elsevier Masson SAS This is an access under the CC BY license httpcreativecommonslicensesby40 0cAM Soliman European Journal of Medicinal Chemistry consequently Nrf2 transports into the nucleus where it initiates thetranscription of its downstream target genes such as NADPHquinone oxidoreductase1 NQO1 []Quinazolinone is a strategic scaffold that has a wide range ofpharmacological activities such as antioxidant antiammatoryand anticancer activities [20e23] Sulfonamides in addition totheir use as antibiotics [24e27] have many pharmacological activities and can be used as antiviral [] antiammatory []antioxidant [] and anticancer agents [32e35] These versatilepharmacological activities make the two chemical classes excellentcandidates for developing new multitarget agents through a slightalteration in the structure that might lead to diversity in the biological activity []In addition numerous studies haverevealed iodine to be a potent antioxidant with higher potency thanthat of ascorbic acid [] Iodine can act as an electron donor that 0fquenches ROS such as OHand H2O2 [] or decreases thedamaging effects of ROS thus increasing the total antioxidant status in human serum []In this context it seemed of interest to search for new compounds with the ability to scavenge ROS and protect cells A seriesof new 68diiodoquinazolin43Hone conjugated to benzenesulfonamide was synthesized by the introduction of the sulfonamide group at the N3 of quinazolinone with the incorporation ofvarying acetamide terminal aimed at exploring the potential antioxidant and radioprotective activity The antioxidant potential ofthe target compounds was ï¬rst measured using a quantitative androbust NQO1 inducer activity bioassay in cells Acute toxicity studyfor the most active compound was then performed in vivo A nontoxic dose was subsequently selected to investigate the potentialprotective effect against wholebody gamma irradiationinducedoxidative stress in experimental mice All groups were observed days after irradiation for survival and weight changes Additionally molecular docking was performed inside the Nrf2bindingsite of Keap1 to gain insights into the molecular interactions andpossible mode of action Results and discussion Chemistry wasreactionofpreparedfrom theScheme shows the synthesis of thioacetamide quinazolinonebenzenesulfonamide derivatives 5e18 The starting material 68diiodo2mercapto4 oxoquinazolin34Hyl benzenesulfonamideisothiocyanatobenzenesulfonamide [] and 2amino35diiodobenzoic acid The coupling of with the 2chloroNsubstituted acetamide in dry acetone and anhydrous K2CO3 yieldedthe corresponding 268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNsubstituted acetamide 5e18 IRspectra of 5e18 displayed additional NH CH2 aliphatic and CObands at their speciï¬ed regions 1H NMR spectra of 5e18 revealedthe acetamide group through the presence of two singlets one at417e431 ppm referring to the CH2and the other at966e1121 ppm attributed to the NH protons with the disappearance of SH singlet of at ppm 13C NMR of 5e18 exhibited twosignals peculiar to the CH2 and CO carbons 1H NMR spectra of 6e8displayed singlets at and ppm assigned to the CH3group at the ortho meta and parapositions of the phenyl group 13CNMR of 6e8 showed signals at and ppm for theCH3 group 1H NMR spectra of 9e11 revealed triplets at and ppm attributed to the CH3 ethyl and quartet at and ppm referring to the CH2 ethyl at the ortho meta and parapositions 13C NMR of 9e11 showed two signals at due to CH3 ethyl and due to the CH2 ethylgroups respectively 1H NMR spectra of revealed singlet at ppm attributed to the OCH3 protons while 13C NMR of showed a signal at ppm due to the OCH3 carbon 1H NMRspectra of revealed triplet at ppm and quartet at ppmdue to the ethoxy group 1H NMR spectra of revealed a singlet at ppm due to the 2OCH3 protons while revealed two singletsat and ppm due to the 3OCH3 protons IR of 16e18 showedNO2 bands Biological activityIn vitro screeningThe antioxidant activity of compounds 4e18 was screened usingthe NQO1 inducer activity assay The Concentration of the novelcompounds to Double the speciï¬c enzyme activity of NQO1 CDvalue was used as a measure of inducer potency and results obtained are presented in Fig Table Evaluation of the NQO1inducer activity showed that compounds and wereinactive whereas compounds and had activityhowever CD value was not reached Compounds CD ¼ mMand CD ¼ mM showed concentrationdependent induceractivity These diiodoquinazolinones represent a new chemicalclass of NQO1 inducers thus adding to the existing knowledge ofthe diversity of the many chemical scaffolds that have been reported to induce this antioxidant enzyme The classical NQO1 inducers are primarily oxidants and electrophiles or othercompounds that react or are metabolized to products that reactand chemically modify cysteine sensors of Keap1 [] A newgeneration of NQO1 inducers is also emerging that of noncovalentsmallmolecule modulators of the Keap1eNrf2 proteinproteininteraction [44e46] Because our diiodoquinazolinones havesome common features with the Keap1eNrf2 proteinproteininteraction inhibitors in this study we tested the potential abilityof these compounds to directly disrupt the binding of Keap1 to Nrf2by molecular modeling see section In vivo evaluation Determination of toxicity lethal dose ï¬fty LD50 of compound The most promising compound was investigatedin vivo for acute toxicity LD50 in albino mice and the value wasfound to be mgkg body weight ip Subsequently onetenthof this dose was selected as the therapeutic dose for further evaluation of the potential radioprotective effects of compound Evaluation of the radiomodulatory effect of compound inmice Four groups of mice were used the ï¬rst group served ascontrol the second group was irradiated at a dose of Gy as a singledose the third group was injected ip with compound only for consecutive days and the last group received compound thenexposed to Gy of gamma radiation After days from irradiationï¬ve mice were checked for liver and hematopoietic system toxicities The residual mice in all groups were monitored over daysto evaluate the survival rate and body weight changes The effect of compound on radiationinduced livertoxicity Gamma radiationinduced hepatic oxidative stress asshown by a signiï¬cant increase in hepatic levels of nuclear Nrf213fold NQO1 32fold ROS 15fold and the lipid peroxidation product malondialdehyde MDA 2fold as compared to nonirradiated control mice This was in agreement with other studies[]Ionizing radiation is believed to induce damage through thegeneration of ROS resulting in an imbalance in the oxidantantioxidant ratio in cells [] In the current experiment the presenceof ROSmediated damage was conï¬rmed by the increase in MDAlevels in irradiated liver in addition to the increase in the expression of the enzymatic antioxidant system Moreover these results 0cAM Soliman European Journal of Medicinal Chemistry Scheme The synthetic pathways for the development of the diiodoquinazolinone derivatives 4e18support the notion that Nrf2 is an initial regulator of cellular responses to radiation exposure [] Once Nrf2 translocates to thenucleus it induces expression of endogenous antioxidant enzymessuch as NQO1 [] a ï¬avoprotein involved in cellular protectionagainst oxidative stress []Treatment of nonirradiated mice with compound led to anincrease in NQO1 and ROS levels and a decrease in Nrf2 with nosigniï¬cant change in MDA level as compared to normal nonirradiated mice Fig A signiï¬cant increase in Nrf2 levels aswell decrease in the levels of NQO1 ROS and MDA was observed in irradiated mice livers treated with compound when compared to the group subjected to radiation aloneFig Moreover treatment with compound improved bothsurvival and body weight of the animals following irradiation 0cAM Soliman European Journal of Medicinal Chemistry Additionally it has been reported that Nrf2 modiï¬es ROS production partly by regulating NQO1 expression [] On the other handthe NQO1 levels were signiï¬cantly higher than the nonirradiatedcontrols in agreement with the cell culture results this studyNotably the increased levels of ROS in nonirradiated mice treatedwith compound are consistent with the increased levels of ROSfollowing genetic Nrf2 activation by Keap1 knockdown []Importantly however the increased ROS production that accompanies NQO1 induction does not lead to damage as evidenced bythe lack of increase in the levels of MDA this study The effect of compound on the hematopoietic systemTo examine the possible role of compound in protecting thehematopoietic system against irradiation we measured the peripheral blood cell counts of red blood cells RBCs white bloodcells WBCs hemoglobin HGB and platelets PLT The irradiatedmice exhibited a signiï¬cant decrease in RBCs WBCs HGB and PLTcompared with the control group Fig These results are mainlyattributed to the fact that irradiation causes the formation of freeradicals which initiate a chain of events leading to the decline in thelevels of hematological parameters [] Indeed it has been wellestablished that gamma irradiation induces RBC injury includingmorphological and quantitative changes of RBCs These alternations may be partly attributed to radiationinduced oxidative stressin RBCs Exposure to radiation results in the formation of reactiveoxygen species ROS and reactive nitrogen species RNS as well asDNA damage which can then lead to severe injury to the hematopoietic system [] This is in harmony with Wang []who stated that injury to the hematopoietic system is the mostcommon injury induced by irradiation This was attributed to theeffect of ionizing radiation on hematopoietic stem cells and hematopoietic progenitor cells which are principally responsible forhematopoietic recovery Treatment of irradiated mice with compound ameliorated the decrease in peripheral blood cellsparticularly RBCs HGB and PLT Hence the antioxidant propertiesof compound may contribute to the amelioration of RBC countsand HGB in irradiated mice This is consistent with other studies forantioxidants effects on the hematopoietic system [] Thismight be explained through the promotion effect of radioprotectorsto proliferate hematopoietic stem cells and they also could increasethe levels of leukocyte growth factors [] Besides severalpotent radioprotectors protect various membrane systems as wellas hematopoietic stem cells from peroxidative damages thatFig Concentration dependence of the NQO1 inducer activity of compounds 4e18Fig without affecting the liver weight Fig as compared toirradiated mice The present results indicate that compound hasan antioxidant capacity as the treatment of irradiated mice with prevents oxidative stress reducing the increase in lipid peroxidation markers and maintaining the expression of Nrf2 comparedwith the irradiated group suggesting improved hepatic antioxidantcapacity Hence compound validated its radiomodulatory andantioxidant effect through its main structure quinazolinone andsulfonamide that goes in line with Soliman [] Also thisï¬nding was reinforced by Cuadrado and his colleagues whoemphasized the importance of therapeutic targeting for Nrf2because of its resourceful cytoprotective roles against a plethora ofdiseases that are associated with oxidative stress []At the same time it was found that NQO1 expression levels ofirradiated mice treated with were signiï¬cantly lower ascompared to vehicletreated irradiated ones but still signiï¬cantlyhigher than normal levels Interestingly the levels of NQO1 in allexperimental groups correlate with the levels of ROS suggestingROS involvement in the NQO1 induction The lower levels of NQO1and ROS in the irradiated group that also received could be theresults of increased antioxidant capacity due to Nrf2 activation []Table NQO1 inducer activity and CD values of compounds 4e18Conc mMCompound noCDaNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNR means not recordeda CD values are the averages of three independent experiments each with eight replicate wells of cells and SD for each data point was within of the value 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A Nrf2 B NQO1 C ROS and D MDA levels in liver of nonirradiated control and irradiated mice after days of irradiation The results wereexpressed as mean ± SE Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test signiï¬cantly different from control group signiï¬cantly different from irradiated group at p n ¼ happened after irradiation so it could protect blood componentsagainst irradiation [] Taken all together these results demonstrate the protective effect of compound against gammaradiation Molecular dockingMolecular docking was performed to assess the ability ofcompound to block the Kelch domain of Keap1 Through its Kelchdomain Keap1 binds to Nrf2 promoting its degradation resultingin low cytoprotective gene levels [] The PDB ï¬le 4IQK was obtained from the Protein Data Bank The binding site of Kelch domainhas been reported to have ï¬ve subpockets P1 P2 P3 P4 and P5[] P1 and P2 are positively charged pockets that contain thearginine triad Arg Arg and Arg This triad is crucialfor the selectivity of the molecular recognition together with a 0cAM Soliman European Journal of Medicinal Chemistry Fig A Survival percent and B Body weight changes of control irradiated compound and compound þ irradiated mice through days after irradiation Theresults were expressed as mean ± SE n ¼ Statistical analysis was carried out byKaplanMeier method followed by the ManteleCox test for survival analysis Bodyweight changes between groups were analyzed by twoway ANOVA followed byBonferronis post test signiï¬cantly different from control group signiï¬cantlydifferent from irradiated group at p group of hydrophobic residues contributes to the stability of thecomplex P1 is formed by residues Arg Ile Gly Phe Arg and Ser P2 is formed by Ser Arg Asn andAsn P3 is a neutrally charged pocket composed of Gly Ser Ala Gly Ser and Gly P4 is formed by Tyr Gln and Tyr whereas P5 is formed by Tyr and Phe The main interactions observed by the cocrystallized ligand NN0naphthalene14diylbis4methoxybenzenesulfonamidearetwo cationpi interaction with Arg piepi interaction with Tyr and two hydrogen bonds with Ser and Ser with S ¼ kcalmol Fig Compound showed the same key interactions exhibited by the cocrystallized ligand Compound S ¼ kcalmol RMSD ¼ has adopted a conformationallowing the presence of two cationpi interaction between Arg and the aromatic rings in addition to a hydrogen bond with themethoxy group Fig three hydrogen bonds made by ser andArg towards the methoxy groups and another hydrogen bondbetween Leu and NH2 group of the sulfonamide Superimposition between compound and the cocrystallized ligand showedthat they adopt the same orientation inside the binding site Fig Finally compound possessing the highest NQO1 inducer activityCD ¼ mM in this series showed the same interactions and thesame orientation of the native ligand inside the receptor indicatinga possible correlation between those multiple interactions and thenoted higher potency Based on the abovementioned resultscompound could possibly bind to Keap1 and disrupt its interaction with Nrf2The results from this study complement previous reportsshowing that the classical electrophilic Nrf2 activator sulforaphaneprotects cells including human retinal pigment epithelial cellskeratinocytes and mouse leukemia cells against oxidative damageFig Effect of compound on relative liver weight in nonirradiated control andirradiated mice after days of irradiation The results were expressed as mean ± SEn ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test There were no signiï¬cant differences between groupscaused by oxidative stressors of four different types namelymenadione tertbutyl hydroperoxide 4hydroxynonenal and peroxynitrite as well as by exposure to ultraviolet radiation []Furthermore unlike the effects of most direct antioxidants theindirect antioxidant effect of sulforaphane which results from Nrf2activation persists for several days after sulforaphane is no longerpresent in the cell culture medium This is because direct antioxidants such as ascorbic acid tocopherols carotenoids and polyphenols which neutralize ROS and other chemical oxidants areconsumed in these reactions whereas Nrf2 activation results intranscriptional upregulation of antioxidant defences which aremediated by proteins with long halflives often several days Thenew compounds generated in the current study have an additionaladvantage in that they are nonelectrophilic and are therefore expected to have a broader therapeutic window compared to electrophilic Nrf2 activators This is supported by the very low toxicityof compound in mice Taken together these results demonstratethe powerful effect of Nrf2 activation and induction of NQO1 inprotecting cells and animals against high levels of ROS and preventing ROSmediated damage This is of particular relevance toprotecting the hematopoietic system which is highly sensitive toROS Conclusiontheacetamide268diiodo4oxo34sulfamoylphenyl3In summary a hybridization strategy was adopted using theiodinated quinazolinone scaffold and sulfonamide moiety to producedihydroquinazolin2ylthioNsubstitutedderivatives 5e18 Different substitutions were introduced to theacetamide group to study the structureactivity relationship All thecompounds were screened for their antioxidant potential using theNQO1 inducer activity assay The 345trimethoxyphenyl derivative showed the highestinducer activity in this seriesCD ¼ mM and had low toxicity LD50 ¼ mgkg Treatmentof gammairradiated mice with compound lowered oxidativestress as evidenced by the lower levels of MDA ROS and NQO1 inliver Furthermore compound ameliorated the complete bloodpicture of irradiated mice as well as enhanced the survival of mice 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A RBCs B WBCs C HGB concentration and D PLT counts in nonirradiated control and irradiated mice after days of irradiation The resultswere expressed as mean ± SE n ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test signiï¬cantly different fromcontrol group signiï¬cantly different from irradiated groupover a period of days postirradiation Molecular docking of inside the active site of Keap1 conï¬rmed that it binds in the samemanner as that of the cocrystallized ligands The inducer activity ofcompound in upregulating NQO1 strongly suggests that it couldbe used as a lead antioxidant and radiomodulatory agent for furtheroptimization of the quinazolinone scaffold Materials and methods ChemistryAll chemicals were purchased from SigmaAldrich and are of ARgrade Melting points were determined in capillary on aGallen Kamp melting point apparatus Sanyo Gallen Kamp UKThin layer chromatography using precoated silica gel plates Kieselgel mm F254 Merck Germany was performed with asolvent system of chloroformmethanol to detect the spots byIR spectra KBr disc were recorded using an FTIRUV lightspectrophotometer Perkin Elmer USA NMR spectra were scannedon NMR spectrophotometer Bruker AXS Inc Switzerland operating at MHz for 1H and MHz for 13C Mass spectra wererecorded on the ISQ LT Thermo Scientiï¬c GCMS model Massachusetts USA Chemical shifts are expressed in dvalues ppmrelative to TMS as an internal standard using DMSOd6 as a solventElemental analyses were done on a model CHNSO analyserPerkin Elmer USA All the values were within ± of thetheoretical values 8diiodo2mercapto4 oxoquinazolin34Hylbenzenesulfonamide A mixture of 2amino35diiodobenzoic acid g mol and isothiocyanatobenzenesulfonamide g mol in absolute ethanol mL containing drops of triethylamine was reï¬uxed for h The solid product formed wascollected by ï¬ltration and crystallized from ethanol to give 00 NH2 Yield mp 0eC IR KBr Ê cm 0cAM Soliman European Journal of Medicinal Chemistry the NN0naphthalene14diylbis4Fig 2D and 3D interaction poses ofmethoxybenzenesulfonamide showing cationp pp interaction and hydrogenbonds with the key amino acids inside the binding pocket arom CO CN SO2 1H NMRDMSOd6 d ppm s 1H d 2H J ¼ Hz AB d2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s2H 13C NMR DMSOd6 d ppm Anal Calcd for C14H9I2N3O3S2 C H N Found C H N 34Dihydroquinazolinsulfonamide derivatives General procedure A mixture of g mol and chloroNsubstituted acetamide derivatives mol in dryacetone mL and anhydrous K2CO3 g mol was stirredat room temperature for h ï¬ltered and the solid product formedwas crystallized from dioxane to give 5e18 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNphenylacetamide Yield 00 NH NH2 mp 0eC IR KBr Ê cmarom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 2H 703e730 m 3H760e783 m 4H s 2H d 2H J ¼ Hz AB d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6d ppm þ ] [Mþ1 MS mz [] [M] [] Anal Calcd for C22H16I2N4O4S2 C Fig 2D and 3D interaction pose of compound showing cationp pp interactionsinside the binding pocket of 4IQKH N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNotolylacetamide Yield 00 NH NH2 mp 0eC IR KBr Ê cmarom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H ddd 1H J ¼ Hz 730e755 m 3H d 2H J ¼ HzAB s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl34 Yielddihydroquinazolin2ylthioNmtolylacetamide 00 NH NH2 mp 0eC IR KBr Ê cm arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H 0cAM Soliman European Journal of Medicinal Chemistry Hz 721e748 m 2H 770e804 m 5H s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6 dppm Anal CalcdforC24H20I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethylphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz 780e805 m 4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm Anal Calcd forC24H20I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioN 4methoxyphenyl acetamide 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d2H J ¼ Hz AB d 2H J ¼ Hz s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6d ppm Anal Calcdfor C23H18I2N4O5S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethoxyphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz AB 803e810 m4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSOd6 d ppm Anal Calcdfor C24H20I2N4O5S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34acetamidedihydroquinazolin2ylthioN35dimethoxyphenyl 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s6H s 2H dd 1H J ¼ Hz dd 2H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB s2H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSOd6 d ppm Anal CalcdforC24H20I2N4O6S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamideFig Superimposition of compound magenta and the cocrystallized ligand redshowed that they adopt the same orientation inside the receptor For interpretation ofthe references to color in this ï¬gure legend the reader is referred to the Web version ofthis m 1H 731e756 m 3H d 2H J ¼ Hz AB d2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34Yielddihydroquinazolin2ylthioNptolylacetamide 00 NH NH2 mp 0eC IR KBr Ê cm arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H d 2H J ¼ Hz AB m 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm þ MS mz [] [M ] [] Anal Calcd forC23H18I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2ethylphenyl 00 NH Yield mp 0eC IR KBr Ê cmNH2 arom aliph 2CO1619 CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H dd 1H J ¼ Hz m 1H ddd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB m 2H d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm MS mz []þ ] [Mþ1 ] [] Anal Calcd for [MC24H20I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN3ethylphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H ddd 1H J ¼ acetamide 0c 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 6H s 3H s 2H d 2H J ¼ Hz d 2HJ ¼ Hz AB s 2H d 2H J ¼ Hz d 1HJ ¼ Hz AB d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm MS mz [] þ[M ] [] Anal Calcd for C25H22I2N4O7S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2methyl4nitrophenyl acetamide 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSOd6 dppm s 3H s 2H d 1H J ¼ Hz d 2HJ ¼ Hz AB 790e805 m 6H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H17I2N5O6S2 C H N Found C H N N2methyl6nitrophenyl 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioacet 00 amide Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMRDMSOd6 d ppm s 3H s 2H dd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz 801e810m 5H d 1H J ¼ Hz d 1H J ¼ Hz s 1H13C NMR DMSOd6 d ppm MS mz [] þ[M ] [] Anal Calcd for C23H17I2N5O6S2 C H N Found C H N 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthio N24dinitrophenyl acetamide 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSOd6 dppm s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz800e804 m 2H d 2H J ¼ Hz AB d 1H J ¼ Hz830e834 m 3H s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C22H14I2N6O8S2 C H N Found C H N Biological evaluation NQO1 in vitro inducer activityHepa1c1c7 murine hepatoma cells were grown in a humidiï¬edatmosphere at 0eC CO2 The cells were tested routinely toensure that they were mycoplasmafree The aminimum essentialmedium aMEM supplemented with vv heat andcharcoalinactivated g100 mL min at 0eC fetal bovineserum was used For evaluation of the potential NQO1 inducer activity cells 104well were grown in transparent ï¬atbottomplastic 96well plates for h after which the cell culture medium was replaced with fresh medium containing each inducerdissolved in DMSO and diluted in the medium and theAM Soliman European Journal of Medicinal Chemistry cells were grown for further h Three replicates of each treatment of eight serial dilutions of inducers were used The ï¬nal DMSOconcentration in the cell culture medium was maintained at vv in all wells Cell lysates were prepared in digitonin and thespeciï¬c activity of NQO1 was determined using menadione as asubstrate as described [] Brieï¬y the cell culture medium wasremoved from each well and the cells were washed three timeswith mL of phosphate buffered saline PBS and subsequentlylysed in mL of digitonin suspension in the presence of EDTA for min with shaking Of the cell lysate mL was transferred to t
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Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedObjective To investigate whether preventive administration of a proton pump inhibitor PPI can reduce the occurrence anddevelopment of traumatic granuloma TG following type IVVI cordectomy Methods We retrospectively analyzed the statusof postoperative granulomas in patients who underwent type IVVI cordectomy due to glottic cancer and determinedwhether postoperative administration of a PPI had any impact on granuloma formation and development Results Thepercentage and number of patients with granuloma in the PPI treatment group experimental group at the 1st 2nd 3rd and6th month following surgery were and respectively The percentageand number of patients with granuloma in the noPPI group control group were and respectively The granuloma percentage of the PPI treatment group was lower than that of thecontrol group at all postoperative time points assessed The diï¬erences were not statistically significant at the 1st monthp but were statistically significant at the 2nd and 3rd months after surgery p p ConclusionPreventive use of a PPI in patients after type IVVI cordectomy can shorten the TG recovery duration and may reduce theseverity of TG but it cannot prevent TG from occurring Our results should be conï¬rmed by prospective randomized controlledtrials with large sample sizes IntroductionLaryngeal squamous cell carcinoma LSCC is a commonhead and neck cancer It had an incidence of approximately in China between and [] and new cases were reported worldwide in [] Itis estimated that there will be new cases worldwidein [] Approximately twothirds of LSCCs originatein the glottic area The presence of hoarseness in patientswith earlystage glottic cancer GC prompts the patients toseek medical treatment Anatomically the larynx is surrounded by cartilage and has sparse lymphatic tissue As aresult patients with GC are mostly diagnosed at an earlystage which is clinically deï¬ned as T12N0M0 []In recent years surgery has been gradually abandoned in the treatment of earlystage GC and has beenreplaced with transoral microsurgery TM or radiotherapyTM has the advantages of being minimally invasive and having a high laryngeal preservation rate and high costeï¬ectiveness Transoral laser microsurgery TLM is the mostcommon surgical method used for GC although a few studies in the literature have adopted transoral coblation microsurgery TCM Although TM methods diï¬erthermalinstruments are often needed for the surgery []Traumatic granuloma TG is a common complication ofTM especially after type IVV cordectomy Mild TG mayonly manifest as hoarseness foreign body sensation and frequent throat clearing The severe granuloma may cause or becomplicated by perichondritis which leads to severe symptoms such as dyspnea Additionally this granulation isbelieved to be an important factor in the formation of glotticweb and larynx stenosis [] Reï¬ux is an important 0cBioMed Research Internationalfactor that aï¬ects granuloma formation Proton pump inhibitors PPI are often used empirically in the treatment ofpatients with postoperative granulomas [ ]We found in our previous clinical practice that patientswith a wide range of resections such as type IVVI cordectomies have a higher risk of postoperative granuloma Thiscan sometimes be very severe even requiring temporary tracheotomy to alleviate dyspnea which greatly and adverselyimpacts patients quality of life PPIs have been empiricallyused for the treatment of patients with postoperative granuloma and evidence of its eï¬ectiveness has been published[] However there has not been any research on the prevention of postoperative granuloma formation with PPIsTherefore we initiated PPI treatment for patients who hadundergone type IVVI cordectomies and compared theresults with those of patients who had also undergone thistype of surgery but were not treated with PPIs to evaluatethe eï¬ect of PPI treatment for preventing postoperative granuloma formation in this patient population Materials and Methods Patients This was a retrospective study Patient inclusioncriteria i patients with vocal cord cancer who agreed toundergo type IV V and VI cordectomies ii patients whodid not undergo preoperative and postoperative radiotherapy Exclusion criteria i patients who were complicatedwith diabetes and were not treated routinely ii patientswho used PPIs regularly iii patients who continued tosmoke and drink after surgeryA total of patients between January and December were recruited as the control group who did not use PPIsimmediately after surgery and did not take PPIs persistentlyA total of patients between January and June were recruited as the experimental group PPI treatment group who took PPIs immediately after the surgeryand routinely Since this was a retrospective study only thepatients in the experimental group underwent preoperativereï¬ux symptom index RSI and reï¬ux ï¬nding score RFSassessments Patients with an RSI score points andoran RFS ¥ points were considered to have laryngopharyngeal reï¬ux disease LPRD [ ]All patients signed an informed consent form prior to thesurgery All clinical experiments conformed to the guidelinesissued by the committee on clinical research of Peking UnionMedical College Hospital PUMCH Ethics Committeeapproval was obtained at PUMCH and all patients providedspeciï¬c written informed consent Surgical Procedure According to the European Laryngological Society classiï¬cation endoscopic cordectomies includethe following types type IV total cordectomy type Vaextended cordectomy encompassing the contralateral cordtype Vb extended cordectomy encompassing the arytenoidtype Vc extended cordectomy encompassing the ventricularband type Vd extended cordectomies encompassing thesubglottis and type VI extended cordectomies encompassing the anterior commissure [ ] During the surgerythe larynx was fully exposed with a selfretaining laryngo°scope Karl Storz Tuttlingen Germany and the tumor°was resected en bloc under direct visualization of a orlaryngoscope Karl Storz Tuttlingen Germany using amodel coblator ArthroCare Corp Sunnyvale CA witha coblation level of and a coagulation level of Postoperative Treatment Procedure and FollowUp Thepatients in the experimental group were treated with intravenous omeprazole mg per day before the recovery of oralfeeding cases recovered oral feeding on the ï¬rst day aftersurgery and cases recovered oral feeding on the third dayThen they were given mg oral omeprazole twice daily minutes before breakfast and dinner for consecutiveweeks The patients in the control group were not treatedwith PPIs but if severe granulomas formed during followup and required intervention they were also given PPIs routinely patients in the control group developedgranulomas But only patients developed severe granulomas on the 2nd 3rd and 3rd month after surgery respectively and began to be given PPIs for weeks the same asthe experimental group while the other cases in the control group were not given PPIs throughoutthe wholefollowup period All patients were treated with cefuroximeat mg twice daily for week to prevent infection Afterthe surgery the patients were required to quit smoking anddrinking and engage in reasonable vocal useThe followup procedure included regular checkups at and months after surgery If a granuloma was detectedduring the 3month checkup the patients were followedmonthly until the granuloma disappeared Postoperativegranuloma was deï¬ned as relatively smooth tissue in the surgery region that protruded from the surrounding area thatmay be attached to a pseudomembrane The granulomaand the surrounding area did not have obvious vascularhyperplasia The proportion of patients with postoperativegranuloma was used as an observation indicator Additionally the number of unscheduled visits and the rate of reoperationincluding tracheotomy and granulectomy wererecorded as indicators of granuloma severityDuring followup if the granuloma was found to severelyimpact vocalization andor breathing or if patients were suspected of having a recurrent tumor the granuloma was surgically resected and the specimen was sent for examination Statistical Analysis Data were statistically analyzed withSPSS software SPSS Chicago IL Nonnormally distributed quantitative data are represented as median and interquartile range and were subjected to the Wilcoxon ranksumtest Normally distributed measurement data are representedas mean ± standard deviation and were subjected to theindependent sample t test Count data were subjected to thechisquared test Diï¬erences with p were consideredstatistically significant Results Baseline Characteristics The baseline data of the patientsin the PPI treatment group and the control group are shownin Table Among the patients in the PPI treatment 0cBioMed Research InternationalTable General information DiscussionVariableSex MFAge yearsTumor staging T1aT1bT2Surgery type IVVVIPPIn ± Controln ± p valuegroup patients were male and patient was female withan average age of ± years patients were at theT1 stage and patients were at the T2 stage patients underwent type IV surgery patients underwent type V surgeryand patients underwent type VI surgery Among the patients in the control group patients were male and patient was female with an average age of ± years patients were at the T1 stage and patients were at theT2 stage patients underwent type IV surgery patientsunderwent type V surgery and patients underwent typeVI surgery The two groups of patients did not diï¬er significantly in the baseline conditions Table Postoperative Granulation The numbers and percentageof patients with granuloma in the PPI treatment group atthe 1st 2nd 3rd and 6thmonth followup were and respectively The numbers and percentage of patients with granuloma in the control group at those time points were and respectively Although the percentage of granuloma in the PPItreatment group was lower than that of the control group ateach stage only the diï¬erences at the 2nd and 3rd monthsafter surgery were statistically significant p and p respectively Only one patient in the PPItreatment group required a second surgery due to persistentgranulation patients in the control group underwent a second surgery among whom patients had granuloma complicated with chondronecrosis and required totracheotomy due to dyspnea Figure However the diï¬erence between the two groups was not statistically significantp In the PPI treatment group only patients hadtwo unscheduled visits In the control group patients had unscheduled visits among them one patient had visitsdue to dyspnea The diï¬erence in the number of unscheduledvisits was not statistically significant p Table Eï¬ects of PPI Treatment in Patients with LPRD in theExperimental Group Among the patients in the experimental group were diagnosed with LPRD according to preoperative RSI and RFS scores and patients did not have LPRD The numbers of LPRDpatients with granuloma at the 1st 2nd 3rd and 6th monthsafter surgery were and respectively and the numbers of LPRD patients with granuloma at these time pointswere and While the data showed that the percentageof granuloma in the LPRD patients was higher than that inthe LPRD patients only the diï¬erence at the 3rd month aftersurgery was statistically significant p Table Transoral microsurgery TM for earlystage glottic cancerGC can achieve oncological therapeutic eï¬ects similar tothose of radiotherapy TM has the advantages of being minimally invasive and having a high laryngeal preservation rateand a low tracheotomy rate its disadvantage includes postoperative complications such as bleeding infection airwayburns and granuloma formation Therefore currently thetreatment selected for patients with earlystage GC is determined by the disease conditions as well as patient needs[ ] Postoperative traumatic granuloma TG is a common complication during the healing process after TLM surgery Severe TG may cause or be complicated by chondritis orchondronecrosis leading to severe complications includingdyspnea With the increase in the range of cordectomy theincidence of TG is also significantly increased Therefore itis necessary to investigate ways to reduce the incidence andseverity of postoperative TG [ ] Like TLMthecoblationassisted endoscopic cordectomy or TCM used inour study is also based on thermal damage whose working°C much lower than lasers workingtemperature is °°temperature which is C1000C and as a result its healing process and the mechanism of TG formation are alsosimilar to those of TLM Current literature indicates thatreï¬ux may be an important factor in the occurrence of postoperative TG [ ] Therefore we aimed to investigatewhether antiacid therapy could reduce TG through an analysis of the eï¬ects of PPI treatment in patients who underwenttype IVVI cordectomy which has rarely been reported°C70Our study showed that the incidences of TG after transoral surgery were as high as and in the twogroups which were much higher than the incidences of reported by Nerurkar and Shah and reported by Wang The reason for this discrepancy may be that the patients in our study all underwent typeIV or higher surgeries Enlarged wounds and damage to theperichondrium or cartilage may lead to increased TG andchondronecrosis Nerurkar and Shah reported that the incidence of TG in patients who have undergone type IV TLMwas and the study by Wang showed that the incidences of TG in patients who have undergone type IV andtype V TLM were and respectively [ ]Meanwhile the incidence of chondronecrosis was not rare especially on whom the surgeon had to expose thethyroid cartilage during tumor resection in TLM [] Thesestudies suggest that type IV or higher surgeries lead to a highlikelihood of TG occurrenceOur study showed that PPI treatment did not suppressthe formation of TG at the 1st month after surgery but thepercentage of TG in the PPI treatment group graduallybecame lower than that of the control group and the diï¬erences were statistically significant at the 2nd and 3rd monthsafter surgery At the 6th month granulomas disappeared inboth groups Our ï¬ndings suggest that although PPI treatment cannot reduce the incidence of TG it can significantlyshorten the duration of granulomas a ï¬nding that is similarto the study by Wang [] Additionally we did notobserve any severe cases of TG that were complicated with 0cBioMed Research InternationalabcdefghiFigure af Shows a typical case in the control group who was a male patient for years old with glottic cancer T2N0M0 at the left sidefollowed by type V cordectomy a One month after surgery a granuloma was found in the left vocal cord under a ï¬brolaryngoscope b months after surgery the granuloma enlarged and the right vocal cord become edema obviously c months after surgery d monthsafter surgery e Computerized tomography CT scan taken before surgery f months after surgery chondronecrosis was found in the CTscan where the white arrow points out gi Shows a typical case in the experimental group who was a male patient for years old withglottic cancer T2N0M0 at the right side followed by type V cordectomy g h and i were taken under a ï¬brolaryngoscope in month months and months after surgery respectively A granuloma could be found in g but disappeared in h and idyspnea in the PPI treatment group however TG in patients in the control group caused or was complicated bychondritis or chondronecrosis which led to severe dyspneaThere were only unscheduled visits among the PPI treatment group compared to unscheduled visits in the controlgroup Unfortunately the diï¬erences in the two indicators ofTG severity between the two groups were not statistically significant although we believe PPI treatment did alleviate the 0cBioMed Research InternationalTable Percentage and severity of granuloma in the two groups1st month2nd month3rd month6th monthResurgeryNumber of unscheduled visits asmedian and interquartile rangePPI treatment groupControl groupp valueNote aindicates that the diï¬erence is statistically significant0005a0037a Table Percentage of granuloma in patients with or without LPRDin the experimental group1st month 2nd month 3rd month 6th monthLPRD n LPRD n p valueNote aindicates that the diï¬erence is statistically significant0029aseverity of TG In this study postoperative PPI was used for weeks referring to the antiacid duration weeks in amedical routine of vocal process granulation and laryngopharyngeal reï¬ux disease J R Lechiens report in andChinse experts consensus on diagnosis and treatment of laryngopharyngeal and reï¬ux disease in [] The resultsshowed that PPI could shorten the recovery time and potentially prevent severe complicationsOur study showed that the percentage of TG did not differ significantly between the LPRD and LPRD patientsp However it took longer for granulomas to disappear in LPRD patients than in LPRD patients and the difference in the number of patients with granulomas wasstatistically significant between the two groups at monthsafter surgery p The reason for this phenomenonmay be that during the early stage of recovery after vocalcord injury changes in the extracellular matrix are the mainmanifestation and injury impacts a lot while acid reï¬ux hasa little eï¬ect Acid reï¬ux may impact the repair process in themiddle and late stages [ ]This was a retrospective nonrandomized controlledstudy with a small sample size We only analyzed granulomaformation in the patients and did not assess their oncologicaloutcome We based the diagnosis of LPRD on RSI and RFSscores and did not perform dualprobe 24hour pH monitoring Therefore we were unable to determine whethernonacid reï¬ux had an impact on the formation and development of postoperative granuloma ConclusionThe preventive use of PPI in patients who have undergonetype IVVI cordectomy cannot reduce the incidence of TGwhile it can shorten the TG recovery duration and may alsoreduce the severity of TG Our ï¬ndings should be conï¬rmedby prospective randomized controlled studies with largersample sizesData AvailabilityAccess to these anonymized data will be made available bythe corresponding author Dr Jian Wang upon reasonablerequestConflicts of InterestThe authors declare that they have no conï¬icts of interestï¬nancial or nonï¬nancial to discloseAuthors ContributionsXiaofeng Jin and Yanyan Niu contributed equally to thisstudyAcknowledgmentsThis study was funded by the Nature Science Foundation ofBeijing China Grant No References[] L B Du W M Mao W Q Chen Incidence and mortality of larynx cancer in China during ZhonghuaLiu Xing Bing Xue Za Zhi vol no pp [] F Bray J Ferlay I Soerjomataram R L Siegel L A Torreand A Jemal Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA A Cancer Journal for Clinicians vol no pp [] B Gupta N W Johnson and N Kumar Global epidemiology of head and neck cancers a continuing challenge Oncology vol no pp [] M F Vaculik C A MacKay S M Taylor J R B Trites R DHart and M H Rigby Systematic review and metaanalysisof T1 glottic cancer outcomes comparing CO2 transoral lasermicrosurgery and radiotherapy Journal of Otolaryngology Head and Neck Surgery vol no p [] W Steiner Results of curative laser microsurgery of laryngealcarcinomas American Journal of Otolaryngology vol no pp [] M S Strong Laser excision of carcinoma of the larynxLaryngoscope vol no pp [] A S Carney M S Timms C N Marnane S KrishnanG Rees and S Mirza Radiofrequency coblation for the resection of head and neck malignancies Otolaryngology and Headand Neck Surgery vol no pp [] M Lee M A Buchanan F Riï¬at and C E Palme Complications after CO2 laser surgery for early glottic cancer an 0cBioMed Research Internationalinstitutional experience Head Neck vol no S1 pp E987E990 [] B Liu L Cheng H Ming and C Zhong Treatment of theearlystage glottic cancer using lowtemperature radiofrequency coblation Journal of Cancer Research and Therapeutics vol no pp [] Y Zhang B Wang G Sun G Zhang L Lu and G LiangCarbon dioxide laser microsurgery versus lowtemperatureplasma radiofrequency ablation for T1a glottic cancer asingleblind randomized clinical trial BioMed Research International vol Article ID pages [] M Remacle H E Eckel A Antonelli Endoscopic cordectomy A proposal for a classiï¬cation by the Working Committee European Laryngological Society European Archivesof OtoRhinoLaryngology vol no pp [] L Wang S Sun S Wang D Liang and W Ji Clinical observation of traumatic granuloma after CO laser cordectomy andlaryngopharyngeal reï¬ux Zhonghua Er Bi Yan Hou Tou JingWai Ke Za Zhi vol no pp [] M Canis F Ihler A Martin C Matthias and W SteinerTransoral laser microsurgery for T1a glottic cancer reviewof cases Head Neck vol no pp [] A Galli L Giordano D Sarandria D di Santo and M BussiOncological and complication assessment of CO2 laserassisted endoscopic surgery for T1T2 glottic tumours clinicalexperience Acta Otorhinolaryngologica Italica vol no pp [] N K Nerurkar and R Shah Factors responsible for the development of carbon granuloma post transoral laser cordectomy Lasers in Medical Science vol no pp [] P C Belafsky G N Postma and J A Koufman The validityand reliability of the reï¬ux ï¬nding score RFS Laryngoscopevol no pp [] P C Belafsky G N Postma and J A Koufman Validity andreliability of the reï¬ux symptom index RSI Journal of Voicevol no pp [] M Remacle C van Haverbeke H Eckel Proposal forrevision of the European Laryngological Society classiï¬cationof endoscopic cordectomies European Archives of OtoRhinoLaryngology vol no pp [] J Yoo C Lacchetti J A Hammond R W Gilbert and Headand Neck Cancer Disease Site Group Role of endolaryngealsurgery with or without laser versus radiotherapy in themanagement of early T1 glottic cancer a systematic reviewHead Neck vol no pp [] C M Chiesa Estomba F A Reinoso A O Velasquez J LFernandez J L Conde and C S Hidalgo Complications inCO2 laser transoral microsurgery for larynx carcinomas IntArch Otorhinolaryngol vol no pp [] J R Lechien F Mouawad M R Barillari Treatment oflaryngopharyngeal reï¬ux disease a systematic review WorldJournal of Clinical Cases vol no pp [] M K Wani and G E Woodson Laryngeal contact granuloma Laryngoscope vol no pp [] C Ling M Yamashita J Zhang D M Bless and N V Welham Reactive response of ï¬brocytes to vocal fold mucosalinjury in rat Wound Repair and Regeneration vol no pp 0c'
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"Dietary macronutrients may indirectly affect body weight through their interactions with the fat massand obesity associated FTO gene This study aimed to investigate the association between FTO gene rs9939609polymorphism with macronutrients intake in overweight adultsMethods This study was carried out on overweight adults of Shiraz Iran Dietary intake was assessed using avalidated 168item semiquantitative food frequency questionnaire FFQ The FTO gene was genotyped forrs9939609 polymorphism The association between dietary macronutrients and the FTO genotype were assessedusing linear regression after adjustments for sex age physical activity and the serum levels of triglycerides fastingblood sugar FBS and low density lipoprotein LDLResults The higher intake of carbohydrates P fat P and calorie P were significantlyassociated with rs9939609 AA genotype P Carriers of the AA genotype of rs9939609 had significantlyhigher calorie fat and carbohydrate intake than the carriers of the TT genotype after adjusting for age and sex P P and P respectively Further adjustments for physical activity TG LDL and FBS did notchange these resultsConclusion The amounts of dietary calorie carbohydrate and fat intake were associated with FTO genotypeFurther studies are warranted to confirm these associations and to identify the underlying mechanismsKeywords FTO Polymorphism Genotype Macronutrient Carbohydrate Protein Fat FiberIntroductionThe prevalence of obesity as a healthrelated problemhas been dramatically increased in both developed anddeveloping countries [ ] More than of adultspopulation of the United States are obese [] Obesity isassociated with other chronic diseases such as cancerhypertension dyslipidemia cardiovascular disease type diabetes and psychological disorders [] Obesity is a Correspondence sdoaeeyahoocom2Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran3Research Center of Health and Environment Guilan University of MedicalSciences Rasht IranFull list of author information is available at the end of the multifactorial disorder caused by genetics lifestyle andenvironmental factors [ ]The role of some genes in obesity has been reported inmany studies [] The fat mass and obesity associatedFTO gene is located on the chromosome region16q122 and was reported to be strongly associated withobesity [ ] The FTO gene is widely expressed in several tissues such as brain visceral fat liver and hypothalamus Several studies reported that FTO genotypehas a strong association with body mass index BMIand obesity [ ] FTO rs9939609 polymorphism is associated with the increased risk of obesity People withrs9939609 FTO variant alleles homozygous AA and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMehrdad Lipids in Health and Disease Page of heterozygous AT are predisposed to greater adipositythan are those with wildtype alleles TT The minorallele frequency of rs9939609 is much different based onethnicity ie it is about and inEuropean Chinese Japanese and African populationsrespectively []FTO gene has an important role in regulation of foodintake energy balance appetite and basal metabolic rateBMR [ ] Polymorphisms in the intron regions ofFTO gene may act as a regulator of other genes such asIroquois homeobox IRX3 and obesityassociated single nucleotide polymorphisms of FTO were associatedwith expression of IRX3 but not FTO in human brains[] On the other hand FTO genotypes may influencethe association of dietary macronutrients with BodyMass Index BMI body weight food intake energy balance appetite and hormone secretion [] Dietarymacronutrients including carbohydrate fat and proteinas the main sources of energy play key roles in regulation of body weight and BMI [ ] However the effects of polymorphisms in obesityrelated genes on theamount of macronutrients intake is not clear So thisstudy aimed to investigate the interactions between theamount of dietary carbohydrate protein and fat withthe FTO genotype in overweight adultsMethodologyThis study was carried out from September to October on randomly selected participants referred to the Shohadaye Valfajr health center ShirazIran Participants were overweight adults aged to years with BMI between to kgm2 The Inclusioncriteria was defined as healthy people with overweightwillingness to participation in the study not participating in a weight management programs during two pastmonths and no weight loss greater than over the last months Participants with alcohol or drugs addictionn smoking certain weightrelated diseases including specific psychological or neurological disorders insulin resistancerenalfailure and infectious diseases n and pregnant orlactating women n were excluded Thus the finalnumber of participants in this study was All participants signed a consent form before participation in thestudythyroid diseasesliver diseasesAnthropometric measuresThe height of the participants was measured with a calibrated tape line fastened to a wall and without shoeswith a precision of cm A bio impedance analysisBIA scale BC418 Tanita Cooperation Tokyo Japanwas then used to measure anthropometric indices suchas BMI skeletal muscle percentage SM body fatBF skeletal muscle SM and body fat percentageBF after entering their height age and genderGenotypingDNA was extracted from whole peripheral blood sampleusing the DNA extraction kitCinnagen CompanyTehran Iran and were stored at °C before genotyping The concentration of the extracted material wasassessed using spectrophotometer by the NanoDrop®ND1000 UVVis Spectrophotometer Nanodrop technologies Rockland USA FTO gene was genotyped forrs9939609 polymorphism via tetraprimer amplificationrefractory mutation systempolymerase chain reactionTetraARMS PCR The sequences of the primers arepresented in supplementary file Macronutrients intakeUsual Macronutrients intakes of the participants wereassessed using a validated 168item semiquantitativefood frequency questionnaires FFQ [] The FFQ wasconsisted of food items with standard portion sizescommonly consumed by Iranian people Facetoface interviews were conducted by a trained dietitianDietary intake was analyzed using the Nutritionist4software program which was modified for Iranian foods[] Daily intakes of calorie were measured for eachperson by using the US Department of Agriculture foodconsumption database which was modified for IranianfoodsPhysical activityA validated international physical activity questionnaireIPAQ was used to measure participants physical activity [] Results obtained from IPAQ were expressed asmetabolic equivalents MET per minuteLaboratory measurementThe levels of serum triglyceride TG total cholesterolTC high density lipoprotein HDL lowdensity lipoprotein cholesterol LDL and glucose were measuredafter h of an overnight fastingStatistical analysisThe ShapiroWilk normality normality test was used todetermine if the quantitative variables had a normal distribution ANOVA test was used to compare demographic anthropometric measurements macronutrientsintake and physical activity between different FTO genotypes The post hoc Tukeys test was then used to identify significant differences of calorie and macronutrientsintake between three genotypes Linear regression wasused to adjust the effects of confounders including agesex PA TG TC LDL and FBS Statistical analyses wereperformed using SPSS version IBM SPSS IBM 0cMehrdad Lipids in Health and Disease Page of Corp Chicago USA The results were considered statistically significant at P Ethics approval and consent to participateThis study has been approved by ethics review board of ShirazUniversity of Medical Sciences Code irsumsrec1395100ResultsAll data were normally distributed according to theShapiroWilk normality test Regarding FTO rs9939609genotype about half of the participants were heterozygote n about of them were TT wild type n and about of them were AA homozygote n The genotype distribution of the study populationwas in HardyWeinberg equilibrium Significant differences were found in BMI P fat mass P calorie intake P fat intake P and carbohydrate intake P status of three FTOgenotypes Table Carriers of the AA genotype ofFTO rs9939609 polymorphism had significantly highercalorie fat and carbohydrate intake than the carriers ofthe TT genotype P P and P respectively Table Linear association of FTO rs9939609 genotype withintake carbohydrate fatthe level of macronutrientsprotein and fiber was then assessed after adjustmentthe effects of confounders This association remainedsignificant for carbohydrate calorie and fat intake afteradjustment for age and sex P P and P respectively model Further adjustments forphysical activity TG LDL and FBS did not change theresults P P and P respectivelymodel Table DiscussionThe present study evaluated the associations betweenrs9939609 FTO gene polymorphism with caloriefatcarbohydrate protein and fiber intake The results identified that there was a significant association betweenFTO genotype with calorie carbohydrate and fat intakeThis association remained significant for calorie carbohydrate and fat intake after adjustments for sex agephysical activity LDL HDL and FBS In carriers of AAgenotype of rs9939609 polymorphism dietary carbohydrate fat and calorie intake were higher than TT carriers However the results of recent studies about theassociation between dietary macronutrients and FTOpolymorphism were inconsistent [] Oyeyemi et alin a casecontrol study on people with obesity estimated as BMI ¥ and controls identified kcalTable characteristics of the subjects categorized by FTO rs9939609 genotypes N VariablesMale sex NAT n TT n Age years mean SDWeight kgHeight m mean SDBMI kgm2 mean SDFat Mass kg mean SDFM mean SDFFM kg mean SDFFM mean SDIPAQ METminute mean SD±±±±±±±±±Calorie intake Kcal mean SD ±Fat gday mean SDProtein gday mean SDCarbohydrate gday mean SDFiber gday mean SDFBS mg dL mean SDLDLC mg dL mean SDHDLC mg dL mean SDT Chol mg dL mean SD±±±±±±±±±±±±±±±±±±±±±±±±±±AA n ±±±±±±±±± ±±±±±±±±±TG mgdL mean SDAbbreviations BMI body mass index HDL highdensity lipoprotein FFM fat free mass IPAQ International Physical Activity Questionnaire LDL lowdensitylipoprotein T Chol total cholesterol TG triglycerides FBS fasting blood sugar FAT fat intake carbohydrate carbohydrate intake Protein protein intake Fiberfiber intake±±±P value 0cMehrdad Lipids in Health and Disease Page of TTTable Tukey test for comparison the calorie andmacronutrient intake between three genotypesAAVariableCalorieATP valueCarbohydrateProteinFatFiberPvalueP valued more energy intake per risk A allele of rs9939609 []Timpson reported higher calorie and fat intakeamong rs9939609 AA genotype carriers They suggestthat FTO polymorphism may influence on appetite andfood intake [] Some other studies also reported thatcarriers of risk allele FTO received higher energy intake[] Consistent with the results of this study Daya et alreported that carriers of ATAA genotype had higher fatintake times and had higher risk of obesity compared with TT genotype [] The FTO variants were reported to be associated with intake of energydensefoods such as fatrich foods [] FTO gene variantsplayed important roles in appetite regulation food intake tendency to choose energydense food high fatand high carbohydrate diet [] The carriers of A alleleFTO rs9939609 had energydense food choices higherbody weight and overeating behaviors [] On theother hand Qi in a crosssectional study on whitepopulation n found a lower energy intake perA risk allele à kcald [] Another study foundno association between a highfat diet and a high carbohydrate diet with the FTO gene in rats [] Drabsch in a systematic review reported that there is noconsistent evidence that the FTO gene SNPs are associated with total energy carbohydrate and fat intakes[] The cause of this discrepancy between the studiesremained unclear However the relationship betweenFTO genotype and dietary intake seems to be very complex and many factors may have a role in this associationTable Association of FTO genotypes with macronutrientsintakevariablesCalorieFATProteinCarbohydrateFiberR2 Model Beta PModel Beta PR2Model adjusted for age and sexModel Further adjustments for physical activity the serum levels oftriglycerides fasting blood sugar FBS and low density lipoprotein LDLPvalue such as the obesity [] level of physical activity []serum leptin [] and other dietary components [] However only overweight subjects were includedbecause of the possible effect of BMI on the associationbetween FTO genotype and dietary intakeRegarding to dietary carbohydrate the AA genotypecarriers had higher carbohydrate intake than TT genotype carriers which was in line with the results of theprevious studies [] Sonest found that FTOgenetic variants are associated with the amounts ofcarbohydrate intake Some study reported that carbohydrate intake especially glucose intake increased FTOgene expression [ ] In homozygous people for therisk allele of FTO gene rs9930506 polymorphism higherdietary carbohydrate intake had a positive associationwith FTO gene expression []This study found no association between protein intake and FTO genotype While some studies indicatedthat protein intake was associated with FTO genotype[ ] However another study reported that leucineintake increased FTO gene expression [] Doaei et alfound that higher protein intake upregulated the FTOgene and also indicated that only in A allele carrier []The mechanism of the interactions between the FTOgenotype and dietary macronutrients is not fully understood The FTO gene polymorphisms may change theamounts of macronutrients intake On the other handthe association of FTO polymorphisms with obesity maybe influenced by dietary intake It was observed that theA risk allele of FTO rs9939609 polymorphism had nosignificant association with obesity in subjects whosedietary fat intake was below of total energy but increased central and total adipose tissues in subjects withfat intake higher than [] Another study reportedthat the risk allele carriers who received Mediterraneandiet for years had lower BMI compared with the others[] Dietary macronutrients may also change the levelof FTO gene expression NowackaWoszuk indicated that a highfat diet could increase FTO gene expression in white adipose cells in rats [] Ronkainen investigated the association between fat intake andthe FTO gene expression They found that a highfat dietcould suppress FTO expression []Some studies suggested that FTO play a crucial role inregulating energy homeostasis FTO gene is expressed inhypothalamus that controls feeding and energy expenditure [ ] Interestingly FTO expression level in hypothalamus is regulated by dietary intake It was reportedthat a highfat diet can downregulate FTO expression inshortterm and up regulate it in longterm [ ]On the other hand the FTO gene is related with guthormones such as orexigenic hormone acylghrelin satiety hormone peptide YY that regulate food intake andappetite [] FTO gene polymorphism AA genotype 0cMehrdad Lipids in Health and Disease Page of influence on circulating PYY3 and acylghrelin levelsthat lead to increased food intake especially energydense foods and reduced satiety [ ] In rs9939609AA carriers suppression of acylated ghrelin led to overeating and obesity [] So it is plausible that FTO genepolymorphisms could change appetite and food intakethat may lead to weight gain and obesityAuthors contributionsMM and MHE designed the study involved in the data collection analysisand drafting of the manuscript MM SD and MGh were involved in theanalysis of the data and writing the manuscript All authors read andapproved the final manuscriptFundingFunding for this study was provided by Shiraz University of Medical SciencesStudy strengths and limitationsThe main strength of this study was the relatively highsample size of overweight adults and adjustments forsugar and lipid profiles as the possible factors affectingdietary intake This study also included only overweightsubjects because of the possible effect of BMI on the association between FTO genotype and dietary intake Inaddition information on a wide range of potential confoundersmodifiers and their potential effects were takeninto account The present study also has several limitations to acknowledge First the study was limited bycrosssectional design Second dietary intake was determined according to a selfreported questionnaire thisparameter was not measured objectively although similarto many prior epidemiological studiesAvailability of data and materialsNot applicableEthics approval and consent to participateThis study has been approved by Local ethics review boards at ShirazUniversity of medical sciences irsumsrec1395100Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Clinical Nutrition School of Nutrition and food SciencesShiraz University of Medical Sciences Shiraz Iran 2Cancer Research CenterShahid Beheshti University of Medical Sciences Tehran Iran 3ResearchCenter of Health and Environment Guilan University of Medical SciencesRasht Iran 4Student research committee Cancer Research Center ShahidBeheshti University of Medical Sciences Tehran IranReceived January Accepted August forcalorieremainedsignificantConclusionThe genotype of FTO may influence the amount of dietary intake in overweight people FTO gene rs9939609polymorphism was associated with dietary intake Theintake of calorie carbohydrate and fat intake were associated with FTO gene polymorphisms and this associationandmacronutrients after adjustments for sex age physicalactivity LDL HDL and FBS In AA carriers dietarycarbohydrate fat calorie was higher than TT carriersGenetic profile can play a key role in future nutritionalrecommendations especially for weight management andalso for prevention of dietrelated chronic diseases Diettherapy in people with risk allele of FTO rs9939609polymorphism may require to consider their desire toeat more carbohydrate fat and calorie Further studiesare needed to increase understanding of the underlyingmechanisms of the association between FTO gene anddietary intakeSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1294402001372xAdditional file AcknowledgementsThis study was conducted at the Department of Public Health Nutrition ofthe Shiraz University of Medical Sciences Shiraz IranReferencesHaslam DW James WP Obesity Lancet Ogden CL Carroll MD Kit BK Flegal KM Prevalence of Childhood and AdultObesity in the United States JAMA Alwan A Global status report on noncommunicable diseases WorldHealth anization Fall T Ingelsson E Genomewide association studies of obesity andmetabolic syndrome Mol Cell Endocrinol Fruhbeck G GomezAmbrosi J Muruzabal FJ Burrell MA The adipocyte amodel for integration of endocrine and metabolic signaling in energymetabolism regulation Am J Physiol Endocrinol Metab Fredriksson R Hagglund M Olszewski PK Stephansson O Jacobsson JAOlszewska AM Levine AS Lindblom J Schiöth HB The obesity gene FTO isof ancient origin upregulated during food deprivation and expressed inneurons of feedingrelated nuclei of the brain Endocrinology May Doaei S Hajiesmaeil M Aminifard A MosaviJarrahi SA Akbari MEGholamalizadeh M Effects of gene polymorphisms of metabolic enzymeson the association between red and processed meat consumption and thedevelopment of colon cancer a literature review J Nutritional Sci Doaei S Kalantari N Mohammadi NK Izadi P Gholamalizadeh M EiniZinabH Salonurmi T Jarrahi AM Rafieifar S Janipoor R Sadeghypor M Upregulation of FTO gene expression was associated with increase in skeletalmuscle mass in overweight male adolescents Arch Med Sci Sep155Hakanen M Raitakari OT Lehtimäki T Peltonen N Pahkala K Sillanmäki LLagstrom H Viikari J Simell O Ronnemaa T FTO genotype is associatedwith body mass index after the age of seven years but not with energyintake or leisuretime physical activity J Clin Endocrinol Metab Apr Thorisson GA Smith AV Krishnan L Stein LD The international HapMapproject web site Genome Res Nov Smemo S Tena JJ Kim KH Gamazon ER Sakabe NJ GómezMarÃn C AneasI Credidio FL Sobreira DR Wasserman NF Lee JH Obesityassociatedvariants within FTO form longrange functional connections with IRX3Nature Mar5077492371 Antonio J Knafo S Kenyon M Ali A Carson C Ellerbroek A Weaver CRoberts J Peacock CA Tartar JL Assessment of the FTO gene 0cMehrdad Lipids in Health and Disease Page of Ronkainen J Huusko TJ Soininen R Mondini E Cinti F Mäkelä KAKovalainen M Herzig KH Järvelin MR Sebert S Savolainen MJ Fat massandobesityassociated gene Fto affects the dietary response in mouse whiteadipose tissue Sci Rep Mar Poritsanos NJ Lew PS Fischer J Mobbs CV Nagy JI Wong D Rüther UMizuno TM Impaired hypothalamic Fto expression in response to fastingand glucose in obese mice Nutr Diab 2011110e19 Doaei S Kalantari N Izadi P Salonurmi T Jarrahi AM Rafieifar S Azizi Tabesh GRahimzadeh G Gholamalizadeh M Goodarzi MO Interactions between macronutrients intake FTO and IRX3 gene expression and FTO genotype in obeseand overweight male adolescents Adipocyte Jan Olszewski PK Fredriksson R Olszewska AM Stephansson O Alsiö JRadomska KJ Levine AS Schiöth HB Hypothalamic FTO is associated withthe regulation of energy intake not feeding reward BMC Neurosci Dec1011 Razquin C Martinez JA MartinezGonzalez MA A 3year intervention with aMediterranean diet modified the association between the rs9939609 genevariant in FTO and body weight changes Int J of Obesity McTaggart JS Lee S Iberl M Church C Cox RD Ashcroft FM FTO isexpressed in neurones throughout the brain and its expression is unalteredby fasting PLoS One 2011611e27968 httpsdoi101371journalpone0027968Stratigopoulos G Padilla SL LeDuc C A Watson E Hattersley AT McCarthyMI Zeltser LM Chung WK Leibel RL Regulation of FtoFtm gene expressionin mice and humans Am J Physiol Integr Comp Physiol 2008294R1185 Wang P Yang FJ Du H Guan YF Xu TY Xu XW Su DF Miao CYInvolvement of leptin receptor long isoform LepRbSTAT3 signalingpathway in brain fat massand obesityassociated FTO downregulationduring energy restriction Mol Med May Batterham RL Cohen MA Ellis SM Le Roux CW Withers DJ Frost GS GhateiMA Bloom SR Inhibition of food intake in obese subjects by peptide YY3 N Engl J Med Sep Wardle J Carnell S Haworth CM Farooqi IS ORahilly S Plomin R Obesityassociated genetic variation in FTO is associated with diminished satiety JClin Endocrinol Metab httpsdoi101210jc2008 Velders FP De Wit JE Jansen PW Jaddoe VW Hofman A Verhulst FCTiemeier H FTO at rs9939609 food responsiveness emotional control andsymptoms of ADHD in preschool children PLoS One Nov e49131Karra E ODaly OG Choudhury AI Yousseif A Millership S Neary MT ScottWR Chandarana K Manning S Hess ME Iwakura H A link between FTOghrelin and impaired brain foodcue responsivity J Clin Invest Aug Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationspolymorphisms rs1421085 rs17817449 and rs9939609 in exercisetrainedmen and women the effects of a 4week hypocaloric diet J Int Soc SportsNutr Dec Blundell JE Lawton CL Cotton JR Macdiarmid JI Control of humanappetite implications for the intake of dietary fat Annu Rev Nutr Ludwig DS The glycemic index physiological mechanisms relating toobesity diabetes and cardiovascular disease Jama Sonestedt E Roos C Gullberg B Ericson U Wirfält E OrhoMelander M Fatand carbohydrate intake modify the association between genetic variationin the FTO genotype and obesity Am J Clin Nutr Sep Esfahani FH Asghari G Mirmiran P Azizi F Reproducibility and relativevalidity of food group intake in a food frequency questionnaire developedfor the Tehran lipid and glucose study J Epidemiol Azar M Sarkisian E Food composition table of Iran National Nutrition andfood research institute Tehran Shahid Beheshti University Press [Persian] VasheghaniFarahani A Tahmasbi M Asheri H Ashraf H Nedjat S Kordi RThe Persian last 7day long form of the international physical activityquestionnaire translation and validation study Asian journal of sportsmedicine Jun22106 Oyeyemi BF Ologunde CA Olaoye AB Alamukii NA FTO gene associatesand interacts with obesity risk physical activity energy intake and timespent sitting pilot study in a Nigerian population J Obes May212017 Villagran M Petermann R Mardones L Garrido MA Natalia MM Associationbetween the polymorphism rs9939609 of the FTO gene with energy intakemacronutrients and alcohol consumption in the Chilean populationMedium Chile Dhurandhar NV Schoeller D Brown AW Heymsfield SB Thomas DSørensen TI Speakman JR Jeansonne M Allison DB Energy balancemeasurement when something is not better than nothing Int J Obes Daya M Pujianto DA Witjaksono F Priliani L Susanto J Lukito W Malik SGObesity risk and preference for high dietary fat intake are determined byFTO rs9939609 gene polymorphism in selected Indonesian adults Asia PacJ Clin Nutr Mar281183Livingstone MB Robson PJ Black AE Coward WA Wallace JM McKinley MCStrain JJ McKenna PG An evaluation of the sensitivity and specificity ofenergy expenditure measured by heart rate and the Goldberg cutoff forenergy intake basal metabolic rate for identifying misreporting of energyintake by adults and children a retrospective analysis Eur J Clin Nutr Mar573455 Zheng Y Huang T Zhang X Rood J Bray GA Sacks FM Qi L Dietary fatmodifies the effects of FTO genotype on changes in insulin sensitivity JNutr May Hardy DS Racette SB Hoelscher DM Macronutrient intake as a mediatorwith FTO to increase body mass index J Am Coll Nutr 201433256e66 Qi L Kraft P Hunter DJ Hu FB The common obesity variant near MC4Rgene is associated with higher intakes of total energy and dietary fatweight change and diabetes risk in women Hum Mol Genet Nov Zhong T Duan XY Zhang H Li L Zhang HP Niu L Angelica sinensissuppresses body weight Gaiand alters expression of the FTO gene in highfatdiet induced obese mice BioMed Res Int Drabsch T Gatzemeier J Pfadenhauer L Hauner H Holzapfel C Associationsbetween single nucleotide polymorphisms and total energy carbohydrateand fat intakes a systematic review Adv Nutr Jul Dorling JL Clayton DJ Jones J Carter WG Thackray AE King JA Pucci ABatterham RL Stensel DJ A randomized crossover trial assessing the effectsof acute exercise on appetite circulating ghrelin concentrations andbutyrylcholinesterase activity in normalweight males with variants of theobesitylinked FTO rs9939609 polymorphism Am J Clin Nutr Nov Katus U Villa I Ringmets I Vaht M Mäestu E Mäestu J Veidebaum T HarroJ Association of FTO rs1421085 with obesity diet physical activity andsocioeconomic status a longitudinal birth cohort study Nutr MetabCardiovasc Dis NowackaWoszuk J PruszynskaOszmalek E Szydlowski M Szczerbal INutrition modulates Fto and Irx3 gene transcript levels but does not altertheir DNA methylation profiles in rat white adipose tissues Gene 0c"
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acute myeloid leukemia aml is the most common type ofacute leukemia in adults caused by the clonal expansion ofundiï¬erentiated myeloid progenitor cells although mostpatients with aml can achieve complete remission by inductionchemotherapy the recurrence rate remains high and thus is themain factor aï¬ecting the outcomes of aml patients relapseoften develops from minimal residual disease in the protectivebone marrow bm microenvironment a comprehensiveunderstanding of the bm microenvironment is conducive to thediagnosis and personalized treatment of aml leukemic cellscytogenetics and molecular aberrations are the main factorsfor risk stratiï¬cation in patients with aml in additionthe bm microenvironment also plays a very important role thein the homing and survival ofbm microenvironment contains various componentssuchas immune cells stromal cells endothelial progenitor cellsextracellular matrix growth factors and cytokines theinteraction between leukemic cells and bm microenvironmentaï¬ects resistance to chemotherapy in aml the modulationof bm microenvironment in aml is currently undergoingpreclinical research and early clinical trials molecular inhibitorssuch as cxcr4 inhibitors vla4 inhibitors and eselectininhibitors are currently undergoing clinical trials immunecells and stromal cells are important components of the bmmicroenvironment and are also the main uencing factorsof leukemia development the estimate program isa common method to explore the microenvironment of manytumors recently it has also been used to explore theprognostic genes in the microenvironment of aml patients most studies have focused on diï¬erentially expressedgenes degs however the interaction and relationship betweengenes are open to investigate moreover the coding genes wereextensively explored but regions that encoded lncrnas andmirnas were less wellstudiedweighted gene coexpression network analysis wgcnais an algorithm commonly used in systems biology toexplore the correlation between gene sets and the clinic functionalization is achieved by constructing a freescalecoexpression gene network wgcna can identify highlyrelated genes and aggregate them into the same genetic modulecurrently wgcna is used in multiple ï¬elds such as cancer andnervous system or to identify potential biomarker candidates ornew therapeutic targets from genetic data the competition endogenous rna cerna hypothesis was anew regulatory mechanism between noncoding rna ncrnaand messenger rna mrna proposed by salmena in in this theory crosstalk between cernas is achieved byabbreviations aml acute myeloid leukemia auc area under curve bmbone marrow cam cell adhesion molecules cerna competing endogenousrnas deg diï¬erentially expressed gene david the database for annotationvisualization and integrated discovery go gene ontology icam1 intercellularadhesion molecule il10ra interleukin receptor subunit alpha keggkyoto encyclopedia of genes and genomes ppi proteinprotein interactiontcga the cancer genome atlas tlr6 toll like receptor tlr8 toll likereceptor tom topological overlap matrix wgcna weighted correlationnetwork analysisimmunerelated cerna network of amlcompetitively combining shared mirnas in recent yearsthe cernas hypothesis has attracted widespread attention in thestudy of molecular and biological mechanisms of tumorigenesisand development for example previous studies have foundthat lncrnarelated cernas were involved in the biologicalprocesses of glioblastoma and breast cancer theresearch on cernas of leukemia was generally based on thediï¬erential genes screened by leukemia and normal controls but no known module based on cernas network related tomicroenvironment in leukemia has been set upinformation ofthe aml cohortin this study mrnas mirnas and lncrnas data andclinicalfrom the cancergenome atlas tcga database were used to calculate theimmune and stromal scores of these aml cases using theestimate algorithm diï¬erentially expressed mrnas andlncrnas were applied to wgcna to identify the modulesmost relevant to the aml immune microenvironment thenthe immunerelated lncrnamirnamrna cerna networkwas established to screen genes with clinical signiï¬cance theseï¬ndings will help to better understand the role oftumormicroenvironment in aml and shed light on the developmentand progression of amlmaterials and methodsdata acquisitionall data sets of aml patients were downloaded from tcgadatabase1 the data used in this study met the following criteria excluding samples combined with other malignancies samples with lncrnas and mirnas and mrnas detection datafinally all lncrnas mrnas and mirnas expression proï¬lesof aml specimens and the corresponding clinical followupdata were downloadedidentiï¬cation of differentially expressedgenesthe estimate algorithm2 was used to calculate the immunescores and stromal scores of aml samples diï¬erentiallyexpressed genes degs such as lncrnas mirnas and mrnaswere identiï¬ed between high and low score groups stratiï¬ed bythe median value of immune scores and stromal scores usinglimma package all q values use fdr to correct the statisticalsigniï¬cance of the multiple test lncrnas and mrnas withlog fc and fdr were regarded as diï¬erentiallyexpressed while mirnas with log fc and fdr were regarded as diï¬erentially expressed then all the degs wereentered into r version auckland nz united states forcluster analysis based on the expression value of each sample inits respective data set the results were expressed in a clustergrameach column represents a sample and each row represents theexpression level of a given gene1portalgdccancergov2sourcefenetprojectsestimateprojectfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amldavid3 wasgo and pathway enrichment analysesthe database for annotation visualization and integrateddiscoveryenrichedbiological themes of degs functions particularly go geneontology terms and kegg kyoto encyclopedia of genes andgenomes pathway enrichment p was set as thecutoï¬ criterionapplied to analyzeweighted gene coexpression networkanalysiswgcna is an algorithm for identiï¬cation of gene coexpressionnetworksthrough highthroughput expression proï¬les ofmrnas or lncrnas with diï¬erent characteristics pairwisepearson correlation analysis was used to evaluate the weightedcoexpression relationship between all datasettopics in theadjacency matrix in this study wgcna was used to analyzemrnas and lncrnas to obtain the mrnas or lncrnas mostrelevant to aml immune microenvironmentcerna network construction andanalysisaccording to the results of wgcna we selected all mrnasand lncrnas in the most relevant module turquoise anddiï¬erentially expressed mirnas to construct a cerna networkbrieï¬y the associated cerna network in aml was constructedfollowing three stages prediction of lncrnamirna inorder to make lncrnas and mirnas map into the interactionssuccessfully we used the miranda4 and pita5 to get targetedlncrnas that mirnas may regulate prediction of mirnamrna three online databases miranda4 targetscan6 andmirwalk7 were used simultaneously for target mrna prediction construction of lncrnamirnamrna cerna networkthe cerna network was constructed based on the negativelyregulating target relationships of mirnamrna and mirnalncrna correlation pairsppi network constructionthe retrieval of interacting genes string database8 wasutilized to construct a proteinprotein interaction ppi networkof the degs identiï¬ed in the cerna network the interactingpairs with a conï¬dence score greater than were considered assignificant and were retained the degree represents the numberof interaction partnerssurvival analysiskaplanmeier plots weretherelationship between the overall survival of aml patientsand the expression level of mrnas lncrnas and mirnasthe statistical signiï¬cance of the correlation was tested by theconstructed to illuminatelogrank test and p was considered significant theanalysis was conducted using the r package of survivalstatistical analysisgraphpad prismtm san diego ca united states or rversion auckland nz united states software was usedfor all data analyses diï¬erences across groups were comparedusing kruskalwallis test for continuous variables diï¬erenceswere considered significant when p resultsimmune and stromal scores areassociated with aml clinicalparameterswe obtained gene expression proï¬les and clinical informationof aml patients from tcga database supplementarymaterial among them were male and were female with a median age range years oldaccording to the fab classiï¬cation there were cases of m0 m1 cases m2 cases m3 cases m4 cases m5 cases m6 cases and m7 case the estimate algorithm was used tocalculate the immune scores and stromal scores of aml patientsthe median immune score was range from to and the median stromal score was range from to we analyzed the relationship between immune scores andstromal scores and clinical parameters of aml patients caseswith m4 subtype aml had the highest immune scores while caseswith m3 subtype had the lowest immune scores p figure 1a similarly m4 cases had the highest stromal scoreswhereas m0 subtypes had the lowest p figure 1baccording to cytogenetics aml patients were divided intothree groups favorable intermediatenormal and poor therewas an obvious correlation between the cytogenetic risk andthe immune scores p figure 1cfavorable vsintermediate p favorable vs poor p intermediate vs poor p but no significant correlationbetween the cytogenetic risk and the stromal scores was observedp figure 1dscoreand high immunestromalusing the median immune or stromal score as a thresholdaml patients were divided into two groups with lowimmunestromalscoresurvival analysis showed that the survival rate of aml patientswith low immune scores was significantly higher than that ofpatients with high immune scores p figure 1ehowever there was no significant diï¬erence in survival betweenpatients with low stromal scores and those with high stromalscores p figure 1f3httpdavidncifcrfgov4httpwwwmicrorna5genieweizmannacilpubsmir07mir07_exehtml6httpwwwtargetscan7http12920671508tringdbidentiï¬cation of differentially expressedgenes based on immune scoresand stromal scoressetting the cutoï¬ criteria as log fc and fdr we identiï¬ed mrnas figure 2a and lncrnasfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure immune and stromal scores are associated with aml clinical parameters ab distribution of immune scores a and stromal scores b for aml fabsubtypes cd the correlation between immune scores c and stromal scores d and aml cytogenetic risk ef kaplanmeier survival curve based on immunese and stromal scores f aml acute myeloid leukemiafigure heatmap of differentially expressed genes in the high and low immunestromal score groups a mrnas b lncrnas and e mirnas based onimmune scores c mrnas d lncrnas and f mirnas based on stromal scoresfigure 2b based on immune scores and mrnasfigure 2c and lncrnasfigure 2d based onstromal scores setting the cutoï¬ criteria as log fc and fdr we identiï¬ed and mirnas based onimmune scores figure 2e and stromal scores figure 2frespectively the degs oflow vs high immunethefrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top go terms in each of biological process were performed for functional enrichment clustering analysis a top25 significant go terms based onupregulated genes in immune scores b top25 significant go terms based on upregulated genes in stromal scores c top25 significant go terms based ondownregulated genes in immune scores d top25 significant go terms based on downregulated genes in stromal scores go gene ontologyscore or stromal score groups were illustrated in the heatmap figure aml and thus require further research to determine theirbiological contributionfunctional enrichment analysis of degsbased on the david the databasefor annotationvisualization and integrated discovery gene annotation tool weperformed go analyses of both upregulated and downregulateddegs the top go biological process indicated that theupregulated degs based on immune or stromal scores wereprimarily enriched in neutrophil degranulation regulationof immune response signal transduction and ammatoryresponse figures 3ab while the downregulated degs basedon immunestromal scores were primarily enriched in rrnaprocessing regulation of translation regulation of transcriptionand cell diï¬erentiation figures 3cd subsequently weperformed kegg kyoto encyclopedia of genes and genomespathway enrichment and interrelation analysis kegg analysisrevealed that the upregulated degs were mainly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingpathway hematopoietic cell lineage and cell adhesion moleculescams pathways figures 4ab while the downregulateddegs were mainly enriched in ribosome metabolism pi3kaktsignaling pathway transcriptional dysregulation in cancer andmirnas in cancer figures 4cd above analyses revealedthatthese degs play a vital role in the development ofconstruction of weighted correlationnetwork analysis and identiï¬cation ofkey modulesbased on the results of survival analysis degs based on immunescores were selected for subsequent analysis the best β valuein the lncrnamrna coexpression network was which wascalculated using the diï¬erential lncrnas diï¬erentialmrnas and their expression data in leukemia samples nextthe method of dynamic tree cutting was used to producecoexpression modules finally modules of lncrnamrnacoexpression networks were generated and the heat map plot oftopological overlap matrix tom was shown figure 5a eachmodule was calculated and plotted with its corresponding clinicalcharacteristics correlation analysis showed that turquoisemodule displayed the highest relationship with aml immunescores r which included mrnas and lncrnasfigure 5b these mrnas were further used to performthe gene enrichment analysis the genes were most relatedto neutrophil degranulation immune response ammatoryresponse signal transduction and tolllike receptor signalingpathway figure 5c in addition genes were highly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top kegg pathway analysis were performed for functional enrichment clustering analysis a top25 significant kegg pathways based onupregulated genes in immune scores b top25 significant kegg pathways based on upregulated genes in stromal scores c top25 significant kegg pathwaysbased on downregulated genes in immune scores d top25 significant kegg pathways based on downregulated genes in stromal scores kegg kyotoencyclopedia of genes and genomespathway metabolic pathways and hematopoietic cell lineage bykegg analysis figure 5dcerna network constructionsince the turquoise module showed the highest relationshipwith aml immune scores we selected lncrnas and mrnas inthe turquoise module and diï¬erentially expressed mirnasbased on immune scores to construct a cerna network firstlybased on the pita and mircode online database that matchespotential mirnas with lncrnas a total of lncrnamirnapairs contained lncrnas and mirnas then we searchedfor the mrnas targeted by the diï¬erentially expressed mirnasusing three target gene prediction websites miranda mirwalkand targetscan using these websites we detected and target mrnas respectively based on the venn intersectionanalysis target mrnas were selected subsequently wematched the predicted target genes with the mrnas in theturquoise module then we constructed the cerna networkby integrating the mirnalncrnamrna interactions at lasta ï¬nal lncrnamirnamrna cerna network was constructedwith lncrnas mirnas and mrnas figure 6aproteinprotein interaction ppinetwork analysisto further explore the interplay among the mrnas in cernawe constructed a ppi network based on the string theretrieval of interacting genes online database figure 6b inthe network tlr8 toll like receptor icam1 intercellularadhesion molecule tlr6 toll like receptor and il10rainterleukin receptor subunit alpha had higher degrees and respectively supplementary table the genes encoding these proteins have been conï¬rmed tobe associated with immune microenvironment and leukemiaprogression association between mrnas mirnasand lncrnas in cerna and overall amlsurvivalwe further analyzed the prognostic values of mrnas mirnasand lncrnas in the cerna network subjects were dividedinto highexpression and lowexpression cohorts accordingto the median value ofthese genes for overall survivalfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure wgcna was used to analyze genetic modules a cluster diagram of coexpression network modules based on topological overlap in mrna andlncrna b study the relationship between each module with their corresponding clinical characteristics c go analysis showed the gene symbols and geneinteractions in the turquoise module d kegg analysis was used to study the pathway enrichment in the turquoise module wgcna weighted correlation networkanalysis go gene ontology kegg kyoto encyclopedia of genes and genomesthrough the package survivalthe highexpression and lowexpression cohorts were splitfor the logrank testin rsoftware out of lncrnas ac0099485 cmahp cta331p31 fcgr2c grk6p1 linc00539 linc01272 mipepp3psmb8as1 rp11266l95 rp11320g101 rp11421f163rp11439e1910 rp11792a83 and stag3l2 out of mirnas hsamir125b5p and hsamir3383p and out of mrnas agpat3 ankrd27 cbx2 ccnd2cd300lb cyth1 erg gdf11 igf1r kiaa0513 kiaa0930larp1 lfng lpcat1 nrep nudt16 pou2f2 ppm1hptafr qsox2 rab3d ralgps2 siglec7 slc43a2 srsf6tnfaip2 tns3 trib1 zbtb5 znf70 and znrf1 wereassociated with overall survival according to the logrank testp representative genes are shown in figure age ¥ years vs years and risk group favorablevsintermediatenormal vs poor were also associated withoverall survival according to the logrank test p and respectively the above mentioned lncrnas mrnas and mirnas were brought into further multivariatecox proportional hazard regression analysis with age and riskgroup finally mrnas ccnd2 erg lpcat1 nudt16ralgps2 tnfaip2 and znf70 lncrnas cmahp fcgr2cpsmb8as1 rp11266l95 rp11320g101 rp11792a83 andstag3l2 and mirnas hsamir125b5p and hsamir3383p were independently associated with overall survival table discussionin recent years studies about the roles of gene mutationsand chromosomalin the occurrence anddevelopment of aml and their prognostic values have madesignificant progress however the bm microenvironmentwhich also plays an important role in the pathophysiologytranslocationsfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure cerna network construction and proteinprotein interaction network a a lncrnamirnamrna cerna network was constructed by lncrnas mirnas and mrnas b a ppi network was constructed based on the string database the rectangle represents micrornas the circle represents mrnasand the triangle represents lncrnas the red represents upregulation in ishigh and the green represents downregulation in ishigh the size of the dot representsthe regulatory capacity of the mrna and larger points indicate stronger regulatory capability cerna competing endogenous rnas ppi proteinprotein interactionprocess in aml are poorly understood therefore mosttreatments previously targeted only tumor cells butfewtargeted the tumor microenvironmentindepth researchon the microenvironment of leukemia will help to furtherunderstand the mechanism of leukemia development and mayï¬nd new targets for microenvironment treatment this studyscreened microenvironmentrelated genes based on the tcgadatabase and further established microenvironmentrelatedlncrnamirnamrna cerna networks through wgcnafirst we calculated the immune scores and stromal scores ofaml patients based on the estimate algorithm and found thatthese scores were related to the fab typing of aml in additionimmune scores were significantly correlated with cytogenetic riskand overall survival estimate is a new algorithm to inferthe level of stromal and immune cells in tumor tissues andtumor purity using gene expression data high immunescores in the bm samples from patients with poor prognosisindicated that more immune cells were recruited in their bmfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure correlation between mrnas mirnas and lncrnas in cerna and overall aml survival in tcga kaplanmeier survival curves with the logrank testwere performed for the representative mrnas mirnas and lncrnas cerna competing endogenous rnas aml acute myeloid leukemia tcga the cancergenome atlasmicroenvironment this may be due to that aml cells activelyshape the bm environment and immune cells to promote diseaseprogression through cellular structural and functional changes however there was no significant correlation betweenstromal scores and cytogenetic risk or survival of aml patientssuggesting that the proportion of stromal cells were comparablein diï¬erent group possible explanation may be that stromal cellsplay an important role in solid tumors while its role inleukemia is not as strong as in solid tumors then we identiï¬ed diï¬erentially expressed mrnas mirnasstromaland lncrnas based on the immune scores orscores functional enrichment analysis indicated thatthesedegs were mainly involved in immune and ammatoryresponses consistent with these results previous studies haveshown that the biology of the immune system is essentialfor the formation of a complex bm microenvironment in recent yearsthe immunologicalcharacteristics of aml has increased and the developmentof eï¬ective aml immunotherapy strategies has attractedwidespread attention the understanding ofin recent years important advances in cerna coexpressionnetwork research have developed rapidly the disruption offrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amltable multivariate cox proportional hazard regression analysis of lncrnas mrnas and mirnasgenesccnd2erglpcat1nudt16ralgps2tnfaip2znf70cmahpfcgr2cpsmb8as1rp11266l95rp11320g101rp11792a83stag3l2hsamir125b5phsamir3383phr 95ci pthe cerna network balance is a major cause for tumorigenesis therefore understanding the complex interactions betweendiï¬erent cerna networks will lead to an indepth understandingof gene regulatory networks and has implications for cancertreatment in addition the lncrnamirnamrna cernanetwork can predict the prognosis of the disease for examplea lncrnamirnamrna cerna network was established basedon rnaseq data of breast cancer from tcga which consistsof mirnas lncrnas and mrnas multiplexcox regression analyses showed that four of these lncrnasadamts9as1 linc00536 al3914211 and linc00491 havesignificant prognostic value wang identiï¬ed lncrnas mirnas and mrnas from a database toconstruct a lncrnamirnamrna cerna network inthe network a univariate and multivariate cox proportionalhazard regression analysis was used to establish a survivalmodel with target mrnas hoxa9 insr krit1 mybspry2 ube2v1 wee1 and znf711 where auc area undercurve is indicating the sensitivity and speciï¬city ofprognostic prediction however the screening of diï¬erentialgenes in this study was based on leukemia patients andnormal people and did not focus on tumor microenvironmentso far there is no cerna research based on the leukemiamicroenvironment in our research the screening of diï¬erentialgenes was based on the immune score then wgcna wasused to identify the modules most relevantto the amlimmune microenvironment then using wgcna and mirnaprediction websites a lncrnamirnamrna cerna networkconsisting of lncrnas mirnas and mrnaswas constructedsubsequently we built a ppi network predicting theinteraction among the proteins encoded by the degs inthe cerna network tlr8 icam1 tlr6 and il10ra hadhigher degrees tlr8 and tlr6 are members of the tolllike receptor family which is upstream to the transcriptionittransportationthe innate immune system andfactor nfκb and part ofplays an importantin progression of aml roleicam1 is one of the cams a large class of transmembraneproteinsinvolved in the binding of cells to another cellor extracellular matrix and involved in cell proliferationdiï¬erentiation movementandtissue structure the protein encoded by il10ra is areceptor for interleukin which has been shown to mediatethe immunosuppressive signal ofinterleukin and thusinhibits the synthesis of proammatory cytokines and isreported to promote survival of progenitor myeloid cellsthrough the insulin receptor substrate2pi3kakt pathway these results indicated that this novel cerna networkwere closely associated with immune microenvironment andprogression of amlapoptosisfurthermore lncrnas mirnas and mrnas withprognostic signiï¬cance were screened out which could be usedas biomarkers for prognosis among the genes with prognosticsigniï¬cance in our module of immunerelated cerna networkthere were aml related reports about cbx2 ccnd2 ergigf1r larp1 lfng nudt16 pou2f2 ptafr rab3dsiglec7 srsf6 tnfaip2 trib1 zbtb5 and znrf1 themost reported of which were erg ccnd2 and ifg1r ergtranslocation was involved in the occurrence and developmentof aml and high expression of erg was a poor prognosticfactor for patients with normal karyotype aml ccnd2mutations were more common in cbfaml and it was also afrequent mutation event in t aml ccnd2 leadedto increased phosphorylation of retinoblastoma proteins leadingto significant cell cycle changes and increased proliferation ofaml cell lines nicolas chapuis found that igf1spontaneous lesions played a key role in pi3kakt activation ofaml cells providing strong evidence for targeting igf1r as apotential new therapy for aml the functions of agpat3ankrd27 cd300lb cyth1 gdf11 kiaa0513 kiaa0930lpcat1 nrep ppm1h qsox2 ralgps2 slc43a2 tns3and znf70 in aml have not been reported we identiï¬ed lncrnas with clinical signiï¬cance among them only cmahpwas reported to be related to mllpositive aml andother lncrnas have not been reported in leukemia twomirnas including hsamir125b5p and hsamir3383p havebeen reported to be associated with a variety of cancers but no studies have been reported related to aml all theseunreported mrnas mirnas and lncrnas may be potentialnovel biomarkers or therapeutic targets for amlis importantto note that our current research haslimitations we selected the target data from the tcga publicdatabase only through the biological algorithm method weshould further verify the results of this in clinical patientsin further studyconclusionin summary a comprehensive bioinformatics analysis wasperformed on the aml dataset in tcga with an emphasison the immune microenvironment using wgcna andfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlmirna prediction programs an immunerelated lncrnamirnamrna cerna network was established and degs withprognostic value were further identiï¬ed further studies of thesegenes are needed in the clinic and may provide new insights intothe pathogenesis of aml this study increases our understandingof the complex interactions between aml tumor cells and the bmmicroenvironment and may provide novel prognostic factors andtherapeutic targetsdata availability statementall data sets of aml patients were downloaded from the cancergenome atlas tcga database portalgdccancergovauthor contributionsyfl cw and zj conceptualization and design sw dataacquisition and writing original draft sw ly yx and dzmethodology sw and cw data analysis and interpretationyjl and yfl writing review and editing yfl cw and zjproject administration all authors contributed to the andapproved the submitted versionfundingthis work was supported by the national natural sciencefoundation of china grant numbers and u1804191and the henan medical science and technology research projectgrant number acknowledgmentswe thank the tcga database for the availability of the datasupplementary materialthe supplementary materialonline202001579fullsupplementarymaterialfor this can be foundwwwfrontiersins103389foncatreferences ferrara f schiï¬er ca acute myeloid leukaemia in adults lancet doi 101016s0140673612617279 zeng z shi yx samudio ij wang ry ling x frolova o targetingthe leukemia microenvironment by cxcr4 inhibition overcomes resistanceto kinase inhibitors and chemotherapy in aml blood doi 101182blood200805158311 shafat ms gnaneswaran b bowles km rushworth sa the bone marrowmicroenvironmenthome of the leukemic blasts blood rev doi 101016jblre201703004 bullinger l döhner k döhner h genomics of acute myeloid leukemiadiagnosis and pathways j clin oncol doi 101200jco ayala f dewar r kieran m kalluri r contribution of bonemicroenvironment to leukemogenesis and leukemia progression leukemia doi 101038leu2009175 uy gl rettig mp motabi ih mcfarland k trinkaus km hladnik lm a phase study of chemosensitization with the cxcr4 antagonist plerixaforin relapsed or refractory acute myeloid leukemia blood doi 101182blood201110383406 rashidi a uy gl targeting the microenvironmentin acute myeloidleukemia curr hematol malig rep doi 101007s11899 austin r smyth mj lane sw harnessing the immune system in acutemyeloid leukaemia crit rev oncol hematol doi 101016jcritrevonc201604020 yehudairesheï¬ s attiasturgeman s sabbah r gabay t musallam rfridmandror a abnormal morphological and functional nature ofbone marrow stromal cells provides preferential support for survival of acutemyeloid leukemia cells int j cancer doi 101002ijc yoshihara k shahmoradgoli m martÃnez e vegesna r kim h torresgarciaw inferring tumour purity and stromal and immune cell admixture fromexpression data nat commun doi 101038ncomms3612 yan h qu j cao w liu y zheng g zhang e identiï¬cation of prognosticgenes in the acute myeloid leukemia immune microenvironment based ontcga data analysis cancer immunol immunother doi101007s00262019024087 huang s zhang b fan w zhao q yang l xin w identiï¬cation ofprognostic genes in the acute myeloid leukemia microenvironment aging doi 1018632aging102477 ni j wu y qi f li x yu s liu s screening the cancer genome atlasdatabase for genes of prognostic value in acute myeloid leukemia front oncol doi 103389fonc201901509 langfelder p horvath s wgcna an r package for weighted correlationnetwork analysis bmc bioinformatics doi yao y zhang t qi l zhou c wei j feng f integrated analysis of coexpression and cerna network identiï¬es ï¬ve lncrnas as prognostic markersfor breast cancer j cell mol med doi 101111jcmm14721 spiers h hannon e schalkwyk lc smith r wong ccy odonovan mc methylomic trajectories across human fetal brain development genomeres doi 101101gr180273114 liu q jiang c xu j zhao mt van bortle k cheng x genomewide temporal proï¬ling of transcriptome and open chromatin of earlycardiomyocyte diï¬erentiation derived from hipscs and hescs circulat res doi 101161circresaha116310456 salmena l poliseno l tay y kats l pandolï¬ pp a cerna hypothesisthe rosetta stone of a hidden rna language cell doi101016jcell201107014 karreth fa pandolï¬ pp cerna crosstalk in cancer when cebling rivalriesgo awry cancer discov doi 10115821598290cd13 zhang k li q kang x wang y wang s identiï¬cation and functionalcharacterization of lncrnas acting as cerna involved in the malignantprogression of glioblastoma multiforme oncol rep doi103892or20165070 wang jd zhou hs tu xx he y liu qf liu q prediction ofcompeting endogenous rna coexpression network as prognostic markers inaml aging doi 1018632aging101985 ritchie me phipson b wu d hu y law cw shi w limma powersdiï¬erential expression analyses for rnasequencing and microarray studiesnucleic acids res 43e47 doi 101093nargkv007 kanehisa m goto s kegg kyoto encyclopedia of genes and genomes nucleicacids res brenner ak bruserud ø functional tolllike receptors tlrs are expressed
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ORIGINAL Clinical Translational Immunology e1165 101002cti21165wwwwileyonlinelibrarycomjournalcti Elly Marcq1 Bianca von Scheidt2 Ashleigh S Davey2Novel combination immunotherapy for pancreatic cancerpotent antitumor effects with CD40 agonist andinterleukin15 treatmentJonas RM Van Audenaerde12Amanda J Oliver2 Jorrit De Waele1 Delphine Quatannens1 Jinthe Van Loenhout1Patrick Pauwels13 Geert Roeyen4 Filip Lardon1 Clare Y Slaney25Michael H Kershaw25 Phillip K Darcy25 Evelien LJM Smits17 1Center for Oncological Research CORE Integrated Personalized Precision Oncology Network IPPON University of AntwerpWilrijk Belgium2Cancer Immunotherapy and Immune Innovation Laboratory Peter MacCallum Cancer Centre Melbourne VIC Australia3Department of Pathology Antwerp University Hospital Edegem Belgium4Department of Hepatobiliary Endocrine and Transplantation Surgery Antwerp University Hospital Edegem Belgium5Sir Peter MacCallum Department of Oncology The University of Melbourne Parkville VIC Australia6Department of Oncology and Multidisciplinary Oncological Centre Antwerp Antwerp University Hospital Edegem Belgium7Center for Cell Therapy and Regenerative Medicine Antwerp University Hospital Edegem Belgium Marc Peeters16CorrespondenceELJM Smits Center for Oncological ResearchCORE Integrated Personalized PrecisionOncology Network IPPON University ofAntwerp CDE T431 Universiteitsplein Wilrijk BelgiumEmail eveliensmitsuantwerpenbe Contributed equally as senior authorsReceived May Revised July Accepted July 101002cti21165Clinical Translational Immunology e1165AbstractObjectives With the poorest 5yearsurvival of all cancersimproving treatment for pancreatic cancer is one of the biggestchallenges in cancer research We sought to explore the potentialof combining both priming and activation of the immune systemTo achieve this we combined a CD40 agonist with interleukin15and tested its potential in pancreatic cancer Methods Responseto this combination regimen was assessed in pancreatic ductaladenocarcinoma mouse models and a thorough analysis of thetumor microenvironment was performed Results We demonstratedprofound reduction in tumor growth and increased survival ofmice with the majority of mice being cured when both agentswere combined including an unprecedented 8fold dose reductionof CD40 agonist without losing any efï¬cacy RNAseq analysisshowed involvement of natural killer NK cell and Tcellmediatedantitumor responses and the importance of antigenpresentingcell pathways This combination resulted in enhanced ltrationof tumors by both T cells and NK cells as well as a striking increaseT cells over Tregs We also observed ain the ratio of CD8significant increase in numbers of dendritic cells DCs in tumorDCs with crossdraining lymph nodes particularly CD103T cells andpresentation potential A criticalinvolvement of NK cells in the antitumor effect was highlightedImportantly strong immune memory was established with anT cells only when both interleukin15 andincrease in memory CD8the CD40 agonist were combined Conclusion These novelpreclinical data support initiation of a ï¬rstinhuman clinical trialrole for CD8ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alwith this combination immunotherapy strategy in pancreaticcancerKeywords CD40 agonist combination immunotherapy interleukin natural killer cells pancreatic cancer T cellsINTRODUCTIONPancreatic ductal adenocarcinoma PDAC is thethird most lethal cancer worldwide with a 5yearsurvival of barely It is even projected tobecome the second leading cause of cancerrelated death by To date it remains one ofthe most aggressive and challenging malignanciesbecause of a complex tumor microenvironmentincluding a strong desmoplastic reaction4 lowimmunogenicity56 and a molecular signature infavor of the tumor driven by loss of multipletumor suppressor genes7 Despite all efforts madein the past almost no improvement in survival hasbeen achieved rendering PDAC a disease whichrepresents the very deï¬nition of an urgent unmetneed for novel therapeutic approaches to ï¬nallyimprove the outcome of patientsAbout of patients are not eligible for curativesurgical resection because of locally advanced ormetastatic disease at the time of diagnosis Hencepatients are treated with either FOLFIRINOX emcitabinenabpaclitaxel depending on theirphysical ï¬tness Howeverthese treatments areassociated with majortoxicity issues and havelimited impact89 New promising approaches thatare successful in other cancer types such as antiPD1and antiCTLA4 have shown little improvementover treatment with gemcitabine1011 This highlightsthe need for other novel compounds to enter thebattle arena of PDACIn this context the use of CD40 agonists inPDAC has been explored during the past decadeboth as a single modality1213 and in combinationwith current ï¬rstline treatments1415 or othercompounds such as checkpoint inhibitors PD1and CTLA416 Antitumor responses have beenreported in both mice and humans and there is ageneral consensustherapyprovides the necessary immunepriming signals toconverttumormicroenvironmenthotammatory microenvironment17 In addition allstudies demonstrated that combination therapyinvolving CD40 agonists provided more potentresults in terms of tumor growth suppression andthat CD40 agonistimmunogenicdesirablecoldthetoaextended survival1516 These data support furtherinvestigation of combination approaches withother promising candidates to unlock the fullpotentialSinceinterleukin IL15 is an essential cytokine foractivation and maintenance of immune effectorcells there is a strong rationale for combiningimmunepriming agents with this moleculetherapyagonistCD40ofbothTcellproliferationto malignantin maintainingWe have previously shown in vitro that IL15stimulated natural killer NK cells can kill bothPDAC tumor cells and stromal pancreatic stellatecells which are responsible for the poor responseto treatment18 IL15 is a versatile cytokine whichstimulatesandgeneration of cytotoxic T lymphocytes as well asactivation and expansion of natural killer NKcells Furthermore it has the capability to induceTcell memory cells thereby playing a crucialCD8immuneroleresponsespossibletheseprevention offeatures render IL15 a highly attractive cancerimmunotherapeutic as conï¬rmed by its high rankin the NCIs top immunotherapeutic drugs withthe greatest potential for broad usage in cancertherapy22 MoreoverIL15 needs to be transpresented by the IL15Rα on dendritic cells DCsto its target to be effective2023 Since it has beendemonstrated that CD40 agonists also increase theexpression of IL15Rα on DCs we hypothesisedthatinenhanced immune activation and increased antitumor effects24combining both agents mightlonglastingandrelapse19 AlltumorresultcellsIn this we show for the ï¬rst time in micewith pancreatic tumors that when CD40 agonistantibody and IL15 are combined they exhibitsynergistic effectsin terms of enhanced antitumor efï¬cacy resulting in profound increases inlongterm survival with complete cure in themajority of cases Moreover an unprecedentedstriking dose reduction of CD40 agonist waspossible by the addition of IL15 The antitumoreffect was found to be mediated predominantlysupported byby CD8dendritic cells DCincreased amounts of CD103ThewithT cells and NK cellscrosspresentingcapacityunique Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancertumors by both celltypes wasltration ofcommensurate with a reduction in the amount ofregulatory T cells These noveltranslationalpreclinical data provide a solid rationale toinitiate a clinicalinvestigating this novelimmunotherapy combination strategy for patientswith one oftumorsnowadaysthe hardestto treattrialRESULTSCombined IL15 and CD40 agonist therapyresults in increased antitumor efï¬cacyin vivosignificantreduction ofWe sought to investigate whether the combinedtreatment of IL15 and a CD40 agonist antibodymay lead to augmented antitumor responses inPDAC To investigate this C57BL6J mice bearingeither Panc02 or KPC tumors were treated with IL andor a CD40 agonist antibody deliveredintraperitoneally when tumors reached a size of mm2 Figure 1a We demonstrated that thecombination of these agents resulted in decreasedtumor volumes and increased survival of mice inboth the Panc02 and KPC tumor models In theï¬rsttumor model Panc02 we observed asignificant decrease in tumor volume when micewere treated with either single agent Howevertreatment with the combination regimen resultedin a furthertumorvolume and a significant increase in survival with out of mice being completely tumor freeFigure 1b d and f and Supplementary ï¬gure 1AC E and G In the second tumor model KPC weobserved very similar results with reduced tumorvolumes and increased survival of mice with thecombination being significantly betterthanuntreated control or either singleagent therapyIn this experiment seven out of mice werecompletely tumorfree following combinationtreatment Figure 1c e and g and Supplementaryï¬gure 1B D F and H Remarkably while weobserved similar responses between the Panc02and KPC tumor modelsthe dose of CD40agonist used in the Panc02 model ï¬ve doses of µg was eight times lower than in the KPCï¬rst dose µg with four consecutivemodeldoses of µg In summary we show here thatthe combination ofIL15 and CD40 agonisttherapy has profound antitumor activities andcombining both agentsto significantsynergistic effectsleadsDistinct gene expression proï¬les pointtowards combined immune priming andimmune activation following therapyoftheclusteringTo unravel the effects caused by the single agentsRNA sequencing wasand their combination performed on the more resistant KPC tumorsharvested on day in the treatment scheme Firstprincipal component analysis PCA demonstrateda clear distinction between the isotype IL15 andcombination group while the CD40 agonisttreatment revealed a bigger overlap with theisotype control Figure 2a To investigate in moredetail the effect of therapy on immunerelatedgenes we performed a PCA using a veriï¬edNanostring immunerelated gene panel whichdemonstrateddifferenttreatment groups Figure 2b The top up anddownregulated immunerelated genesshowedthat genes involved in antigen presentation Thelper immune type responses and NK cellcytotoxicity were modulated Figure 2cToconï¬rm this gene set enrichment analysis GSEAwas performed showing that gene sets involved inNK cellmediated cytotoxicity Figure 2d and eand Supplementary ï¬gure 2A the IL122 pathwayFigure 2f and g and Supplementary ï¬gure 2Band the CD8 TCR downstream pathway Figure 2hand i and Supplementary ï¬gure 2C were clearlyamong the top upregulated pathwaysin thegroups when IL15 was administered while theCD40promotedantigen processing and presentation pathwaysFigure 2j and k and Supplementary ï¬gure 2DImportantlythese features of both IL15 andCD40 agonisttherapy were retained in thecombination grouptreatmentstronglyagonistT cells are responsible for antitumorCD8efï¬cacy following therapyTo gain more insightinto the immune cellsresponsible for the observed antitumor responseswe depleted several immune cell populations intumorbearing mice in both tumor models usingspeciï¬c depletion antibodies We monitored theeffect the depletions had on both tumor growthkinetics and survival of mice Figure 3ad andSupplementary ï¬gure Upon depletion of CD4T cells we observed in both PDAC models nosignificant difference in survival of mice betweenthe depleted and nondepleted groups indicatingT cells do not play a significant role inthat CD4ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alFigure Combining IL15 with a CD40 agonist results in decreased tumor volume and increased survival C57BL6j mice were injected witheither à Panc02 or KPC cells subcutaneously When tumors reached a size of mm2 mice were randomised and treated withisotype control IL15 CD40 agonist or IL15 CD40 a Treatment scheme showing timing of dosing is indicated for IL15 µg with blackarrows and for CD40 agonist or the corresponding isotype with red arrows ï¬ve doses of µg for Panc02 or ï¬rst dose µg andconsecutive four doses µg for KPC b c Tumor growth kinetics are depicted [n or mice per group representative data of Panc02or KPC independent experiments] Oneway ANOVA with Bonferroni post hoc d e Survival of Panc02 n and KPC n micetreated as indicated Pooled data of Panc02 or KPC independent experiments Survival was determined by tumor size reaching mm2Logrank test f g Waterfall plots showing the change in tumor area relative to baseline after days Panc02 n or days KPCn Pooled data of Panc02 or KPC independent experiments All data represent mean 06 SEM P Ë P ¤ P ¤ P ¤ Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancerFigure RNA proï¬le of KPC tumors KPC tumors were harvested on day of the treatment schedule for subsequent RNA isolation andsequencing a PCA plot showing separation of samples based on all genes b PCA plot showing separation of samples based on immunerelated genes Nanostring mouse immunerelated genes c Volcano plot showing significantly differentially expressed immunerelated genesbetween isotype control group and the combination group dk GSEA plots for differentially expressed genes are showing enrichment for genesinvolved in a n tumorsgroupª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alFigure Immune cell depletion C57BL6j mice bearing Panc02 or KPC tumors were treated with isotype control or the IL15 CD40 agonistcombination regimen alone or with depleting antibodies against CD4 CD8 asialoGM1 NK cell depletion a b Tumor growth kinetics ofPanc02 or KPC tumors either nontreated isotype treated with the combination regimen only no depletion or combination and depletionantibodies Oneway ANOVA with Bonferroni post hoc Data points represent mean 06 SEM n micegroup representative data of twoindependent experiments c d Waterfall plots showing the change in tumor area relative to baseline after days ek Survival of Panc02or KPCbearing mice either nontreated isotype treated with the combination regiment no depletion or the combination and depletionantibodies against CD4 e f CD8 g h asialoGM1 i j or CD8 asialoGM1 k l Data pooled from two independent experiments withn or Panc02 or n KPC LogRank test ns P ¥ P P ¤ P ¤ P ¤ Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic canceroftheeffectantitumorthe antitumor response elicited by the IL15 andCD40 agonist combination treatment Figure 3eT and NK cells wereand f However when CD8depletedthecombination treatment was significantly reducedin both tumor models Figure 3k and l WhenT cells alone were depleted there was aCD8significant increase in tumor growth and reducedsurvival of mice Figure 3g and h Howeverdepletion of NK cells only led to a mixed responseIn the Panc02 tumor model we clearly observed asignificant decrease in survival of mice followingdepletion of NK cells although interestingly therewas no effect on tumor growth In the KPC tumormodelthere was a trend towards decreasedsurvival of mice following NK cell depletionalthough this was notsignificantP In conclusion theseT cells wereexperiments demonstrated that CD8the predominant effector cell type responsible forthe observed antitumor effects with a clearcontribution of NK cells in the Panc02 modelFigure 3i and jstatisticallyCombination therapy increases intratumoral ltration of immune cellsintheKPCutilisedto monitorTo further explore the anticancer effects of thecombination regimen multicolour ï¬ow cytometrytumorltratingwaslymphocytestumor modelSupplementary ï¬gure We observed strikingdifferences between the different treatment armsthat were tested Figure 4a The ï¬rst observationwas that the antitumor effect was not due to ahigher total number of ltrating lymphocytesbut rather caused by significant differences inwhich immune cellsubsets were present Thefrequency of ltrating NK and NKT cells wassignificantly higher in the combination therapygroup compared to the isotype and CD40 agonisttreatment groupsIL15 alone demonstrated anincreased frequency of both cell types althoughthis was not statistically significant compared tocontroltreated miceIn terms of neutrophilnumbers although they were abundantly presentwethetreatment groups For T cells we observed anincreased number offollowingcombination therapy compared to the othertreatment groups This increased number of T cellsT cellscould be attributed to predominantly CD8which werethecombination therapytreated mice compared to alltotal T cellssignificantlydifferenceobservedbetweenhighernoincellsseveralon NK and NKTtreatment arms while there was nootherT cellsdifference in numbers of ltrating CD4Interestingly we also observed a significantdecrease in numbers of regulatory T cells Tregspresent in the tumor when CD40 agonist was partof the treatment We also measured expression ofCD69 on immune cells as an indicator ofactivation We observed clear upregulation of thismarkerfollowingTcombination therapy however not on the CD8cells The results were conï¬rmed by geneexpression analysis ofrelevant genesFigure 4b Here FoxP3 transcription was indeeddownregulated when Tregs were less abundantand the combination regimen demonstratedthe strongest upregulation of NKT cellrelatedgenes such as granzyme A and B The analysisrevealed a strong immune activation signature asIFNγ and CD69 were upregulatedIL12Puttingthecombination treatment of CD40 agonist andIL15 had a more profound antitumor effect asit caused a significant increase in the amountof antitumor immune cells NK NK T and CD8T cells compared to control orsingleagenttreatmentcommensurate with adecrease in immunosuppressive Tregs resulting inan enhanced CD8Treg ratio within the tumorsobservationsthat wastogetherIL18theseCombination therapy results in increasedamounts of CD103crosspresenting DCsDendritic cells are known to play critical roles inantigen processing and presentation and are keyplayers in the activation of both NK and T cellsWe further explored their presence in the KPCtumor model Here we observed a significantincrease in number of DCs in the tumor only inthecombination therapy group Figure 5aDCs theFurthermore the amount of CD103subtype responsible for crosspresentation wasIL15 caused a significantdetermined HereDCs in theincrease in the number of CD103tumor while this was significantly lower in thegroups following treatment with CD40 agonistFigure 5b To further investigate how the DCsbehaved we analysed the presence of these cellsin the tumordraining lymph nodes TDLN andobserved a 3fold increase in number of DCs whenCD40 agonist was administered Figure 5c Thecrosspresenting DCsfrequencyincreasedconditionsFigure 5d suggesting that these DCs capturedoftwofoldCD103undertheseª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alFigure Characterisation of tumorltrating lymphocytes C57BL6j mice bearing KPC tumors were treated with isotype control IL15 CD40agonist or the combination of the latter a Tumors were harvested at day posttreatment initiation Singlecell suspensions were acquired afterenzymatic digestion for ï¬ow cytometry analysis Immune cell populations indicated as fold change of absolute number of cells and CD69T cells Data pooled from three independent experiments n 16group Oneway ANOVA withexpression MFI on NK NKT and CD8Bonferroni P P ¤ P ¤ P ¤ b Heatmap of gene expression of relevant genes for the quantiï¬ed immunesubsets n tumorsgroup Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancerantigens at the tumor site and migrated to theTDLN to activate NK and T cells Furthermoregene signatures showed a higher expression ofCD80 CD83 and CD86 when the combinationtherapy was administeredindicating that likelyantigenpresenting cells like DCs are activated andmaturein mRNA encodingexpression of CXCR3 CXCL9 and CXCL10 notCXCL11 as chemotactic chemokines suggests theirinvolvementin the trafï¬cking of antitumorimmune cells to the tumor site Figure 5eincreaseTheInduction of immune memorythe goals ofimmunotherapy isOne oftheinduction of strong immunological memory toprevent future relapse We observed in our studyan increased number of effector and centralT cells in KPC tumors when treatedmemory CD8with the combination regimencompared toisotypearm treatmentssingleFigure 6aTo investigate whetherfunctional immune memory was induced tumororand bcontrolFigure Characterisation of DCs in tumor and TDLN C57BL6j mice bearing KPC tumors were treated with isotype control IL15 CD40agonist or the combination of the latter Tumors or TDLN were harvested at day posttreatment initiation Singlecell suspensions were acquiredafter enzymatic digestion for ï¬ow cytometry analysis a b DCs or CD103DCs in TDLN Data pooledfrom three independent experiments n 16group Oneway ANOVA with Bonferroni P P ¤ P ¤ e Heatmapof gene expression of relevant genes for the quantiï¬ed immune subsets n tumorsgroupDCs in tumors c d DCs or CD103ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alfree mice from the combination treatment groupwere rechallenged with the same tumor cell typeas they were originally inoculated with Here weobserved clear induction of immune memory inboth PDAC models with out of micebecoming tumor free for the Panc02 tumor modeland all mice becoming tumor free for the KPCtumor model compared to naı¨ve control miceFigure 6cfDISCUSSIONWith high resistance to current ï¬rstline treatmentand failure of numerous clinicaltrials PDACpatients are in desperate need for new treatmentoptions25 There is a general consensus that CD40agonist therapy triggers the necessary immunepriming signalling to convert immunogenic coldcancerenhancementto itstargetthe effects are ofofto hot However when applied intumorsshortpancreaticduration warrantingthistherapy1217 IL15 is a powerful stimulator of NKT cells and induces CD44hi memorycells and CD8T cells This cytokine needs to be transpresentedby the IL15Rα on DCsto beeffective2023 Since it had been demonstrated thatthe CD40 agonist also increases the expression ofIL15Rα on DCs we hypothesised that combiningboth agents may resultin enhanced immuneactivation and increased antitumor effects In thisstudy we demonstrated thatIL15 and CD40agonist administered alone can elicit a powerfulimmune response However when combinedenhanced andthesestronglyimportantly exceeded survivalrates of CD40agonist with gemcitabine and nabpaclitaxel oreffects wereFigure Rechallenge experiments C57BL6j mice cured from Panc02 or KPC tumors after treatment with IL15 CD40 agonist were reinjected with the same tumor type at the contralateral side of the abdomen a b Tumor kinetics and survivallogrank test of micerechallenged with Panc02 tumor cells n c d Tumor kinetics and survival of mice rechallenged with KPC tumor cells n e f FlowEffector or Memory T cells of KPC tumorbearing mice after days following treatment n cytometry quantiï¬cation of intratumoral CD8Oneway ANOVA with Bonferroni P ¤ P ¤ P ¤ Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancerstudy26 A limitation ofCD40 agonist and gemcitabine and nabpaclitaxeltogether with PD1 and CTLA4 blockade in similarpancreatic mouse models1516 In addition whenCD40 agonist was combined with antiangiogenicdrugs blocking VEGFA and angiopoietin survival rates did not equal the ones observed inthese studiesthisincluding ours may be that the mouse models donot completely mimic the human situation sincetumorsubcutaneouslyNevertheless CD40 agonists given as a singlemodality have shown beneï¬cial effect also inhuman clinical trials with PDAC patients131427 andour previous studies have demonstrated that uponIL15 stimulation human NK cells can kill bothpancreatic cancer and stromal stellate cells in anautologous human ex vivo setting18cells wereinjectedThe potential of this combination regimen isnot just limited to PDAC since IL15 and CD40agonist therapy has been tested by others in micebearing established CT26 and MC38 colorectaltumors The authors showed promising resultsalbeit with less surviving mice compared to ourstudy28 This might be due to the fact that wegave in total ï¬ve doses of CD40 agonist instead offour as in the other studies Furthermore resultsof otherinvestigators using this combinationtherapy in a prostate cancer model TRAMPC2demonstrated similar numbers of surviving mice aswe found underscoring the enormous potentialof the combination approach24 Of note bothcolorectal cancer and prostate cancer have asignificant better 5year overall survival of andthesigniï¬cance of our ï¬ndings in pancreatic cancerwith a 5year survival of barely Strikinglyin this study we also demonstrated that IL15potentiates CD40 agonist treatment causing an fold dose reduction in one of the PDAC mousemodels which has not been reported so far Thisimportant dose reduction could be of greattranslationalimportance as lower doses mightsignificantly decrease adverse events in patientsunderscoringrespectivelyWe observed thatthe combination therapyuenced several immune cell types in favor ofincreased antitumor efï¬cacy As also observed byothers in prostate cancer there was an increase innumber of intratumoral effector immune cellsT cells that boththat included NK cells and CD8contributed to enhancing tumor control but thesestudies did not look beyond these immune cells24In our more extensive analysis we also observed areduction in number of Tregs known for theirCD8anTcellsfavorableestablishesreceptor activationimmunosuppressive potential and an increasedfrequency of DCsfor priming T cells andactivating NK cells Especially our ï¬nding ofDCs withstrongly increased amounts of CD103crosspresenting potentialin the tumordraininglymph nodes elucidates on the mechanism behindour observed antitumor effectscompriseimportantcompartment ofthe adaptive immune systemwith wellestablished antitumor effects Upon Tcellthey produce effectorcytokines like IFNγ and kill cancer cells in anantigenspeciï¬c manner via the granzymeperforinsystem3132 Increased numbers of CD8T cell incancer are therefore linked to better outcome andprognosis With our combination strategy weT cellsobserved an increased number of CD8whichtumormicroenvironment for an anticancer response Wefound these cells were the most important playerin our therapy models since depletion of thesecells virtually abrogated the antitumor responseBoth CD40 and IL15 already as single modalitiesTcell numbers butinduced an increase in CD8the combination therapy induced significantlyhigher intratumoral ltration of these effectorcells This might be due to certain chemokinessince our unique gene expression analysis showeda higher expression of CXCR3 and its ligandsCXCL9combinationtreatment These chemokines are known to beresponsible for chemoattraction of both T cellsand NK cells33 Moreover these two chemokinesareandchemotherapeutic efï¬cacy in PDAC in patients34Interestinglyweredownregulated following combination therapyHowever the CCL5CCR5 axis in PDAC has beenshown to correlate with promotion of migrationand invasiveness of the pancreatic cancer cellsand thus downregulation could be actuallythebeneï¬cialCX3CR1 axis is associated with early recurrenceafter surgery with poor patient prognosis36 Theactivation marker CD69 was not shown to beT cells This may be dueupregulated on the CD8to the fact that tumors were harvested at day posttreatment and CD69 is considered to be anearly activation marker which disappears after days37 Among the CD8subsets wememory T cellsfound higher presence of CD8which is linked to immune memoryIL15 hasbeen described to be important for the inductionin our models35 Furthermorecorrelated with increased survivalfollowingandCXCL10TcellCCL5andCX3R1aª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et aland maintenance of these cells and combiningthis modality with for instance PDL1 blockademightincrease the number of CD8memory T cells38furtherNatural killer cells have become an increasinglyimportant target for cancer immunotherapy sincethey have demonstrated to mediate successful andefï¬cient antitumor responses3940 They play animportant role in pancreatic cancer as we haveshown here and although depletion of NK cellsdid not have as drastic an effect on response toT cells it isCD40 and IL15 combination as CD8likely that they still play an important role in thistherapy given the increase in both their activationand numbers in the tumor This is an importantobservation since a higher frequency of NK cells isclearly linked to better survival41 IL15 is a strongstimulator of NK cells23 and its effect is stronglyincreased by the addition of CD40 agonists thatupregulate IL15Rα on DCs28 which is necessaryT cells andfor transpresentation of IL15 to CD8NK cells20 Moreover since expression of IL12 IL and IL18 is upregulated this may lead toin the tumorformation of NK memory cellsmicroenvironment which needsto be furtherexplored42ahighhaveTregsimmunesuppressivepotential43 When CD40 agonist was administeredwe observed a significantreduction in thenumber of Tregs within the tumor as conï¬rmedby others in other solid tumor models2644 Themechanism by which CD40 agonists cause Tregreduction still needs to be elucidated althoughone study indirectly points towards the blockageofmyeloidderivedsuppressor cells and the Tregs45 In addition thehighlyfollowingcombination therapy was very encouraging sincemetaanalysis showed that this is associated withimproved overall survival in cancer patients46 andresponse to therapy4748CD8TreginteractionincreasedbetweenratioFinally reduction of Tregs in PDAC allows DCsimmuneto induce a more potent antitumorT cells49 Ourresponse largely mediated by CD8data demonstrated that DCs in general increasedby 3fold in the tumordraining lymph nodes as aofresultTheirtheimportancein cancer has been extensivelydemonstrated as they function as the generals ofour immune system by capturing tumor antigensand presenting them to T cells thereby elicitingspeciï¬c immune responses5051 A very importantDCs which aresubset of these DCs are the CD103combinationtherapyTbycellstheyactivateconsidered to reside in peripheral tissues Uponthey migrate to the lymph nodesactivationwhereantigenpresentation52 Moreover they have found to bethe only APCs to transport intact antigens totumordraining lymph nodes and prime tumorT cells53 Hence our novel dataspeciï¬c CD8DC in the tumorshowing a doubling in CD103draining lymph nodes support the increased antitumor responses we observ
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"Range 41.684.2 41.684.2 41.681.7 44.282.9 41.684.2 Sex .18 .61 ?Female 44 (54%) 37 (59%) 7 (39%) 12 (60%) 32 (52%) ?Male 37 (46%) 26 (41%) 11 (61%) 8 (40%) 29 (48%) Ethnicity .65 .18 ?Unknown 7 (8%) 5 (8%) 2 (11%) 0 (0%) 7 (11%) ?Non-Hispanic 74 (91%) 58 (92%) 16 (89%) 20 (100%) 54 (89%) Race .73b .75b ?African American 8 (10%) 8 (13%) 0 (0%) 2 (10%) 6 (10%) ?Asian 3 (4%) 2 (3%) 1 (6%) 0 (0%) 3 (5%) ?Pacific Islander 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) ?White 66 (81%) 52 (83%) 14 (78%) 17 (85%) 49 (80%) ?Unspecified 2 (2%) 0 (0%) 2 (11%) 1 (5%) 1 (2%) Histology .06c .60c ?Adeno 52 (64%) 44 (70%) 8 (44%) 14 (70%) 38 (62%) ?Squamous 25 (31%) 17 (27%) 8 (44%) 6 (30%) 19 (31%) ?Large 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Bronchioloalveolar 1 (1%) 0 (0%) 1 (6%) 0 (0%) 1 (2%) ?Other 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) Stage of disease .16 .27 ?IA (<3 cm) 25 (31%) 22 (35%) 3 (17%) 4 (20%) 21 (34%) ?IB (?3 cm) 56 (69%) 41 (65%) 15 (83%) 16 (80%) 40 (66%) Zubrod performance status .11 1.00 ?0 44 (54%) 31 (49%) 13 (72%) 11 (55%) 33 (54%) ?1 37 (46%) 32 (51%) 5 (28%) 9 (45%) 28 (46%) Weight loss (6 mo) 1.00d .31d ?<5% 64 (79%) 49 (78%) 15 (83%) 14 (70%) 50 (82%) ?5-<10% 9 (11%) 7 (11%) 2 (11%) 3 (15%) 6 (10%) ?1020% 4 (5%) 3 (5%) 1 (6%) 2 (10%) 2 (3%) ?>20% 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Unknown 3 (4%) 3 (5%) 0 (0%) 1 (5%) 2 (3%) Smoking status ?Current 33 (41%) 26 (41%) 7 (39%) 8 (40%) 25 (41%) ?Former (quit ?1 y) 39 (48%) 30 (48%) 9 (50%) 10 (50%) 29 (48%) ?Never 9 (11%) 7 (11%) 2 (11%) 1.00e 2 (10%) 7 (11%) 1.00e Abbreviation: Adeno adenocarcinoma. a All P values shown are 2-sided. b White versus all other races. c Adenocarcinoma versus all other histologies. d Weight loss <5% versus ?5%. e Derived using the Freeman-Halton exact test. The distribution of assignment to chemotherapy and observation was 63 patients (78%) and 18 patients (22%) respectively which was not significantly different (P?=?.20 Fisher exact test) from the expected rates of 70% (129 patients) and 30% (55 patients) respectively.16 Based on protein levels in these 81 patients the number of those with low ERCC1 and low RRM1 was 31 patients (38%) 22 patients had low ERCC1 and high RRM1 (27%) 10 patients had high ERCC1 and low RRM1 (12%) and 18 patients had high ERCC1 and RRM1 (22%) which is not significantly different from prior results (P?=?.14 Fisher exact test; 54 of 184 29%; 38 of 184 21%; 37 of 184 20%; and 55 of 1840.3 respectively). We investigated whether treatment arm assignment varied by patients' smoking status histology age and sex. In bivariate comparisons no statistically significant associations were found. However the multivariable logistic model found that patients with adenocarcinoma (P?=?.03) and potentially stage IA disease (P?=?.06) were more likely to be assigned to adjuvant chemotherapy (ie they were more likely to have low levels of ERCC1 RRM1 or both). One of the 18 patients assigned to observation and 19 of the 63 patients assigned to chemotherapy rejected this choice and withdrew consent. There was no statistically significant difference in patient characteristics between those who accepted and those who refused their treatment assignment (). Feasibility The trial achieved its primary feasibility objective with a treatment assignment within the prespecified timeframe in 71 of 81 patients (88%). We successfully determined protein levels in all 85 patients. Ten of the 81 eligible patients did not achieve assignment to treatment versus observation within the 84-day time interval from surgical resection. The time interval from surgery to assignment ranged from 86 days to 105 days in these 10 patients. For 3 patients the specimens were received after the 84-day limit had passed. For the other 7 patients the time interval from receipt to reporting ranged from 7 days to 25 days (median 18 days). For the 71 patients with a successful assignment within the 84-day time interval from surgical resection the time from receipt to reporting ranged from 3 days to 26 days (median 8 days). The reasons for reporting results in excess of 14 days were equipment failure and inadequate expression values in control specimens which required equipment recalibration and a repeat processing of the specimens. Overall the time from receipt of specimens to reporting ranged from 1 day to 27 days (median 11 days; mean 12 days) which is similar to that reported for patients with advanced NSCLC (range 1 day-47 days; median 11 days; mean 12 days).18 Survival and Toxicity Survival analyses were performed on the 61 patients who accepted assignment to treatment (44 patients) or surveillance (17 patients). Patients who rejected their treatment assignment withdrew consent and thus could not be followed for survival. Fourteen patients had DFS events; 2 had died (1 from disease recurrence and the other from cardiac disease without recurrence). The median follow-up among those patients still alive at the time of last follow-up was 27 months (range 3 months-44 months). Six patients had <?24 months of follow-up. The collective 2-year DFS and OS rates were 80% (95% confidence interval [95% CI] 67%-88%) (Fig. 2A) and 96% (95% CI 87%-99%) from the date of registration. The 2-year DFS rate was 83% (95% CI 68%-92%) for patients who received chemotherapy (Fig. 2B) and it was 71% (95% CI 43%-87%) for those observed (Fig. 2C). includes 2-year DFS estimates within each of the 3 gene expression categories in the chemotherapy arm. The median time from surgery to enrollment was 41 days (range 11 days-79 days). The time from surgery was added as a covariate to a Cox regression model and was not found to be significantly related to DFS (P?=?.22) or OS (P?=?.36). Disease-Free Survival Rates Patient Group No. DFS (95% CI) 1-Year 2-Year Accepted assigned treatment 61 88% (77%-94%) 80% (67%-88%) Received chemotherapy 44 95% (83%-99%) 83% (68%-92%) By protein level category (for those that received chemotherapy) ?Low ERCC1/low RRM1 20 95% (69%-99%) 84% (59%-95%) ?Low ERCC1/high RRM1 18 94% (65%-99%) 82% (55%-94%) ?High ERCC1/low RRM1 6 100% (100%-100%) 100% (100%-100%) Abbreviations: 95% CI 95% confidence interval; DFS disease-free survival; ERCC1 excision repair cross-complementing group 1; RRM1 ribonucleotide reductase M1. Kaplan-Meier survival estimates are shown. (A) Collective disease-free survival is shown for patients who accepted adjuvant chemotherapy or observation based on gene expression analysis. (B) Disease-free survival is shown for patients who received adjuvant chemotherapy. (C) Disease-free survival is shown for patients in the observation group. Conf Int indicates confidence interval. A total of 22 patients discontinued chemotherapy because of treatment-related toxicity (50%). None of the patients died because of treatment-related toxicity. Details are provided in . Number of Patients With Grade 3 and Grade 4 Adverse Events Among the 44 Patients Who Received Chemotherapya Level of Severity Adverse Event Grade 3 Grade 4 No. of patients with events 13 14 Type of events ?Neutropenia 11 6 ?Thrombocytopenia 4 4 ?Nausea 4 0 ?Vomiting 4 0 ?Anemia 2 0 ?Anorexia 2 0 ?Fatigue 2 0 ?Febrile neutropenia 1 1 ?Thromboembolism 1 1 ?Dehydration 1 0 ?Hearing impairment 1 0 ?Mucositis 1 0 ?Pleural effusion 1 0 ?Renal failure 1 0 ?Bradycardia (sinus) 1 0 ?Syncope 1 0 ?ALT elevation 1 0 ?Hypokalemia 1 0 ?Hyponatremia 0 2 Abbreviation: ALT alanine aminotransferase. a Adverse events were assessed according to the Common Terminology Criteria for Adverse Events (version 3.0). In Situ ERCC1 and RRM1 Protein Levels RRM1 levels ranged from 2.4 to 234.3 (median 39.7; mean 48.1) which were not significantly different from the expected values (median 40.5; range 8.3-96.2) (P?=?.87).16 ERCC1 protein levels ranged from 4.3 to 211.2 (median 41.9; mean 58.8) and these values were significantly different from the expected values (median 65.9; range 1.9-178.7) (P?=? 0.02). There was a significant correlation noted between ERCC1 and RRM1 levels (correlation coefficient 0.39; P?=?.0003) (Fig. 3) as previously reported.91618 Distribution of excision repair cross-complementing group 1 (ERCC1) and ribonucleotide reductase M1 (RRM1) levels in eligible patients is shown. The median protein levels of ERCC1 in adenocarcinomas squamous cell carcinomas and the other histologies were 34.257.1 and 121.5 respectively. The corresponding median levels of RRM1 were 38.142.6 and 48.9 respectively. Although the levels were higher in squamous cell carcinomas compared with adenocarcinomas the medians were not statistically significant (ERCC1: P?=?.16; RRM1: P?=?.72). DISCUSSION Disease stage is a predictor of benefit from adjuvant chemotherapy in patients with NSCLC. Patients with stage III disease derive the most benefit and those with stage I are reported to derive the least.1241923 Although not statistically significant for patients with stage I disease and a tumor diameter >?3 cm a numerical risk reduction of 7% has been reported and for those with tumors measuring ??3 cm a numerical risk increase of 40% has been reported.23 A significant treatment-related toxicity is febrile neutropenia which has been reported in 7% to 24% of patients.242022 Treatment-related deaths occur in 0.5% to 2% of patients.122022 The inclusion of molecular markers predictive of therapeutic efficacy into adjuvant decision algorithms would greatly improve the clinical benefit and reduce toxicity for patients with NSCLC. This approach is particularly attractive for patients with stage I disease in whom the parameters for weighing risks and benefits are to our knowledge the least well defined. Recent advances in molecular diagnostics have resulted in improved outcomes for patients whose tumors harbor mutations in oncogenic signal transduction molecules that can be inactivated by therapeutic agents. Similarly platinum agents target DNA and gemcitabine targets ribonucleotide reductase; both are unequivocally required not only for cellular proliferation but also for other essential cellular functions. Although to our knowledge specific oncogenic mutations have not been identified to date ERCC1 and RRM1 have emerged as promising predictors of efficacy for cisplatin and gemcitabine respectively. We conducted a phase 2 trial of treatment selection based on the levels of protein expression of ERCC1 and RRM1 for patients with completely resected stage I NSCLC and tumor diameters ??2 cm primarily to establish feasibility but also to evaluate preliminary efficacy as assessed by 2-year survival rates. We achieved our primary goal by demonstrating within a cooperative group environment that treatment assignment can be achieved for >?85% of patients within 84 days (12 weeks) the established timeframe for the initiation of adjuvant therapy from surgery in patients with NSCLC.1242022 At first glance our demonstration of feasibility should not be surprising. However it is important to note that surgical practice has not usually engaged a medical oncologist at the time of initial therapeutic planning but rather after complete recovery which substantially reduces the time available for molecular testing before the initiation of adjuvant treatment. We found no difference (P?=?.20) between academic and community sites in the time elapsed from surgery to the receipt of specimens in the reference laboratory (community sites: 57 patients; median 48 days [range 18 days-90 days]; academic sites: 24 patients; median 53 days [range 20 days-90 days]). The time elapsed from specimen receipt to reporting (median 12 days; range 1 day-27 days) was similar to our previous experience in an international trial of patients with advanced NSCLC (median 11 days; range 1 day-47 days).18 Based on these observations we conclude that the current process for routine specimen procurement handling and shipping to a reference laboratory requires substantial improvements to facilitate implementation of molecularly based therapeutic decision-making. For example a developing National Cancer Institute-sponsored project Adjuvant Lung Cancer Enrichment Marker Identification and Sequencing Trial (ALCHEMIST) which will randomize patients with epidermal growth factor receptor-mutated or anaplastic lymphoma kinase (ALK)-rearranged NSCLC to targeted therapy or not will need to carefully consider these logistical issues. Prior results from adjuvant trials and a retrospective staging project in patients with stage I disease after complete surgical resection have reported 2-year DFS rates of 72% to 74%20 and rates of 68% to 75% for patients with stage IB disease.4 The corresponding 2-year OS rates were 80% to 88% for patients with stage I disease2024 65% to 90% for patients with stage IB disease242225 and 85% for those with stage IA disease.25 Thus our results of a 2-year DFS rate of 80% and OS rate of 96% appear favorable by comparison. However it is prudent to be cautious because we lost 20 of 81 patients from the survival analysis because of consent withdrawal and a direct comparison of outcomes data among trials cannot account for differences in study populations eligibility and staging criteria and provisions for data collection and analysis. The spectrum of protein levels for ERCC1 and RRM1 significant correlation of levels between both molecules and distribution of patients into the 4 gene expression categories in the current study is consistent with previous experience.91213161826 However the current analysis method for biomarker evaluation (ie antibody-based assessment of in situ protein levels) is not suitable for general clinical implementation for several reasons. First ERCC1 has multiple isoforms that cannot be specifically distinguished by the available reagents and only 1 isoform appears to be involved in platinum-induced DNA damage repair.27 Second the monoclonal antibody 8F1 which is consistently used for ERCC1 protein expression analysis detects a second and unrelated protein that shares a common epitope with ERCC1.2830 This observation may account for the highly batch-dependent performance of this antibody1827 which may explain the significantly lower ERCC1 values in the current study compared with prior results.16 Third protein levels for RRM1 in particular and to a lesser degree for ERCC1 appear to be influenced by the specimen processing and handling procedures used at collection sites.26 Finally although the method for immunofluorescence-based quantitative detection of both molecules performs well if all specimens to be analyzed are processed simultaneously there is considerable interassay variability if specimens need to be processed individually over an extended period of time as required for real-time patient decision-making.18 However it is important to note that the biochemical biophysical and cell biological evidence for ERCC1 and RRM1 as predictive molecules for platinum and gemcitabine efficacy remains undisputed.51012273132 A small number of recent clinical trials have used ERCC1 prospectively for therapeutic decision-making. These include 2 randomized phase 3 trials in patients with advanced-stage NSCLC (1 published [NCT00499109]18 and the other terminated and unpublished [NCT00801736]) and 2 adjuvant trials 1 of which was a terminated and not yet published phase 2 trial [TAilored Post-Surgical Therapy in Early Stage NSCLC (TASTE) NCT00775385] and the other an ongoing phase 3 trial [International TAilored Chemotherapy Adjuvant trial (ITACA); EudraCT 2008-001764-36]. Results from the first trial (NCT00499109) demonstrated no improvement in patient survival; however the authors raised the possibility of a false-negative result because of an inexplicably divergent survival in an internal control group.18 The second trial (NCT00801736) and third trial (NCT00775385) were terminated early after the discovery of ERCC1 isoforms27 and specificity problems with the 8F1 antibody.2830 The fourth trial is using ERCC1 and tumor thymidylate synthase mRNA expression levels for treatment assignment compared with a cisplatin-based control treatment with OS as the primary endpoint and a planned accrual of 700 patients. Results from these trials will help to further delineate the feasibility and technical issues mentioned above. The results of the current study demonstrated the feasibility of our biomarker-based decision algorithm in a multiinstitutional cooperative group environment for patients with surgically resected NSCLC. We identified that the current practice of evaluation and treatment for these patients may present an obstacle to rapid molecular-based decision-making. Although encouraging efficacy data emerged from this trial bioassays that specifically measure platinum-induced DNA damage repair must be developed before further clinical trials are launched that seek to tailor the use of these agents. FUNDING SUPPORT Supported by National Cancer Institute grants CA014028 CA016385 CA020319 CA022453 CA027057 CA032102 CA 035090 CA035119 CA035178 CA035261 CA035431 CA 038926 CA042777 CA045377 CA045560 CA045807 CA0 46113 CA046368 CA046441 CA063844 CA063848 CA0 67575 CA067663 CA073590 CA074647 CA076429 CA10 5409 and CA129343. CONFLICT OF INTEREST DISCLOSURES Dr. Bepler has a patent pending for the use of RRM1 and ERCC1 as biomarkers of treatment benefit for therapeutic decision-making in patients with cancer. REFERENCES 1 Arriagada R Bergman B Dunant A Le Chevalier T Pignon JP Vansteenkiste J International Adjuvant Lung Cancer Trial Collaborative Group Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer N Engl J Med 2004 350 351 360 14736927 2 Winton TL Livingston R Johnson D Vinorelbine plus cisplatin versus observation in resected non-small-cell lung cancer N Engl J Med 2005 352 2589 2597 15972865 3 Strauss GM Herndon J Maddaus MA Randomized clinical trial of adjuvant chemotherapy with paclitaxel and carboplatin following resection in stage IB non-small-cell lung cancer: report of the Cancer and Leukemia Group B protocol 9633 [abstract] Proc Am Soc Clin Oncol 2004 23 Page Abstract 7019 4 Strauss GM Herndon JE Maddaus MA Adjuvant paclitaxel plus carboplatin compared with observation in stage IB non-small-cell lung cancer: CALGB 9633 with the Cancer and Leukemia Group B Radiation Therapy Oncology Group and North Central Cancer Treatment Group Study Groups J Clin Oncol 2008 26 5043 5051 18809614 5 Dabholkar M Vionnet J Bostick-Bruton F Yu JJ Reed E Messenger RNA levels of XPAC and ERCC1 in ovarian cancer tissue correlate with response to platinum-based chemotherapy J Clin Invest 1994 94 703 708 8040325 6 Metzger R Leichman CG Danenberg KD ERCC1 mRNA levels complement thymidylate synthase mRNA levels in predicting response and survival for gastric cancer patients receiving combination cisplatin and fluorouracil chemotherapy J Clin Oncol 1998 16 309 316 9440758 7 Lord RV Brabender J Gandara D Low ERCC1 expression correlates with prolonged survival after cisplatin plus gemcitabine chemotherapy in non-small cell lung cancer Clin Cancer Res 2002 8 2286 2291 12114432 8 Olaussen KA Dunant A Fouret P DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy N Engl J Med 2006 355 983 991 16957145 9 Reynolds C Obasaju C Schell MJ Randomized phase III trial of gemcitabine-based chemotherapy with in situ RRM1 and ERCC1 protein levels for response prediction in non-small-cell lung cancer J Clin Oncol 2009 27 5808 5815 19884554 10 Davidson JD Ma L Flagella M Geeganage S Gelbert LM Slapak CA An increase in the expression of ribonucleotide reductase large subunit 1 is associated with gemcitabine resistance in non-small cell lung cancer cell lines Cancer Res 2004 64 3761 3766 15172981 11 Bergman A Eijk P van Haperen V In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as a major determinant Cancer Res 2005 65 9510 9516 16230416 12 Bepler G Kusmartseva I Sharma S RRM1 modulated in vitro and in vivo efficacy of gemcitabine and platinum in non-small-cell lung cancer J Clin Oncol 2006 24 4731 4737 16966686 13 Ceppi P Volante M Novello S ERCC1 and RRM1 gene expressions but not EGFR are predictive of shorter survival in advanced non-small-cell lung cancer treated with cisplatin and gemcitabine Ann Oncol 2006 17 1818 1825 16980606 14 Bepler G Sharma S Cantor A RRM1 and PTEN as prognostic parameters for overall and disease-free survival in patients with non-small-cell lung cancer J Clin Oncol 2004 22 1878 1885 15143080 15 Simon GR Sharma S Cantor A Smith P Bepler G ERCC1 expression is a predictor of survival in resected patients with non-small cell lung cancer Chest 2005 127 978 983 15764785 16 Zheng Z Chen T Li X Haura E Sharma A Bepler G The DNA synthesis and repair genes RRM1 and ERCC1 in lung cancer N Engl J Med 2007 356 800 808 17314339 17 Camp RL Chung GG Rimm DL Automated subcellular localization and quantification of protein expression in tissue microarrays Nat Med 2002 8 1323 1327 12389040 18 Bepler G Williams C Schell MJ Randomized international phase III trial of ERCC1 and RRM1 expression-based chemotherapy versus gemcitabine/carboplatin in advanced non-small-cell lung cancer J Clin Oncol 2013 31 2404 2412 23690416 19 Chemotherapy in non-small cell lung cancer: a meta-analysis using updated data on individual patients from 52 randomised clinical trials Non-Small Cell Lung Cancer Collaborative Group BMJ 1995 311 899 909 7580546 20 Scagliotti GV Fossati R Torri V Randomized study of adjuvant chemotherapy for completely resected stage I II or IIIA non-small-cell lung cancer J Natl Cancer Inst 2003 95 1453 1461 14519751 21 Waller D Peake MD Stephens RJ Chemotherapy for patients with non-small cell lung cancer: the surgical setting of the Big Lung Trial Eur J Cardiothorac Surg 2004 26 173 182 15200998 22 Douillard JY Rosell R De Lena M Adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage IB-IIIA non-small-cell lung cancer (Adjuvant Navelbine International Trialist Association [ANITA]): a randomised controlled trial Lancet Oncol 2006 7 719 727 16945766 23 Pignon JP Tribodet H Scagliotti GV LACE Collaborative Group Lung adjuvant cisplatin evaluation: a pooled analysis by the LACE Collaborative Group J Clin Oncol 2008 26 3552 3559 18506026 24 Waller D Fairlamb DJ Gower N The Big Lung Trial (BLT): determining the value of cisplatin-based chemotherapy for all patients with non-small cell lung cancer. Preliminary results in the surgical setting Lung Cancer 2003 41 54 25 Chansky K Sculier JP Crowley JJ Giroux D Van Meerbeeck J Goldstraw P International Staging Committee and Participating Institutions The International Association for the Study of Lung Cancer Staging Project: prognostic factors and pathologic TNM stage in surgically managed non-small cell lung cancer J Thorac Oncol 2009 4 792 801 19458556 26 Bepler G Olaussen KA Vataire AL ERCC1 and RRM1 in the International Adjuvant Lung Trial by automated quantitative in situ analysis Am J Pathol 2011 179 69 78 21224045 27 Friboulet L Olaussen KA Pignon JP ERCC1 isoform expression and DNA repair in non-small-cell lung cancer N Engl J Med 2013 369 1101 1110 23514287 28 Bhagwat NR Roginskaya VY Acquafondata MB Dhir R Wood RD Niedernhofer LJ Immunodetection of DNA repair endonuclease ERCC1-XPF in human tissue Cancer Res 2009 69 6831 6838 19723666 29 Ma D Baruch D Shu Y Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology BMC Biotechnol 2012 12 88 23171216 30 Vaezi A Bepler G Bhagwat N CCT? is a novel antigen detected by the anti-ERCC1 antibody 8F1 with biomarker value in lung and head and neck squamous cell carcinomas Cancer 31 Reed E Platinum-DNA adduct nucleotide excision repair and platinum based anti-cancer chemotherapy Cancer Treat Rev 1998 24 331 344 9861196 32 Chen Z Zhou J Zhang Y Bepler G Modulation of the ribonucleotide reductase M1-gemcitabine interaction in vivo by N-ethylmaleimide Biochem Biophys Res Commun 2011 413 383 388 21893046 World J Surg Oncol World J Surg Oncol World Journal of Surgical Oncology 1477-7819 BioMed Central 24755441 3999735 1477-7819-12-108 10.1186/1477-7819-12-108 Case Report Surgical treatment of a solitary pulmonary metastasis from eyelid sebaceous carcinoma: report of a case Kaseda Kaoru 1 [email protected] Ohtsuka Takashi 1 [email protected] Hayashi Yuichiro 2 [email protected] Emoto Katsura 2 [email protected] Asakura Keisuke 1 [email protected] Kamiyama Ikuo 1 [email protected] Goto Taichiro 1 [email protected] Kohno Mitsutomo 1 [email protected] 1Department of Surgery Section of General Thoracic Surgery Keio University 35 Shinanomachi Shinjuku-ku Tokyo 160-8582 Japan 2Department of Pathology School of Medicine Keio University Tokyo Japan 2014 23 4 2014 12 108 108 19 2 2014 7 4 2014 Copyright © 2014 Kaseda et al.; licensee BioMed Central Ltd. 2014 Kaseda et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article unless otherwise stated. Background Ocular sebaceous carcinoma is an uncommon aggressive ocular neoplasm with potential for regional and distant metastasis. Case presentation A 77-year-old woman was found to have a solitary pulmonary lesion 6 years after the initial treatment of sebaceous carcinoma of the eyelid. Video-assisted lung wedge resection of an undetermined pulmonary nodule was carried out successfully. Microscopically the tumor showed foamy cytoplasm and atypical nuclei consistent with metastasis of eyelid sebaceous carcinoma. Conclusion This is the first case report of resected solitary pulmonary metastasis of eyelid sebaceous carcinoma. Pulmonary resection is a good option for the treatment and diagnosis of metastatic eyelid sebaceous carcinoma. Sebaceous carcinoma Lung metastasis Solitary metastasis Background Sebaceous carcinoma of the eyelid is a relatively rare malignant tumor and accounts for less than 1% of all eyelid tumors [1]. As well as being a rare tumor sebaceous carcinoma can mimic other benign inflammatory and malignant processes thus errors or delays in diagnosis are not unusual [2-5]. Although local management strategies for this tumor have previously been described [6-10] very few reports have focused on the patterns of metastasis of this tumor and the treatment strategies for such metastases [78]. "
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colorectal carcinoma crc as one of the most commongastrointestinal malignancies has developed the worldsfourth most deadly cancer with a high rate of incidence andmortality [ ] liver metastasis which is the most commonform for crc metastasis is the leading cause of high mortality for this severe malignancy however few clinical prevention and treatment measures could be available for tumormetastasis therefore it is really urgent to develop new biomarkers and explore the underlying mechanism for crcmetastasis and eventually develop new therapeutic strategiesfor crc patientsduring cancer progression metabolic reprograming withincreasing glucose utilization is termed as warburg aï¬ectwhich is accompanied by altered pyruvate and mitochondrialmetabolism the fate of pyruvate is the core manifestationto distinguish normal cells and tumor cells through metabolism in normal cells pyruvate was used for eï¬cient atpproduction directly into mitochondria however pyruvatewas converted into lactate in cytosol despite of normoxicand hypoxic conditions in cancer cells this may be dueto the impaired process of pyruvate from the cytosol intothe mitochondrial matrix which is a critical metabolic steplinking glycolysis and mitochondrial oxidative phosphorylation the mitochondrial pyruvate carriermpc a 0c of immunology researchmultimeric complex containing two distinct proteins mpc1and mpc2 which is located in the inner mitochondrialmembrane is responsible for eï¬cient mitochondrial pyruvate uptake loss of mpc expression or activity blockspyruvate entry into the tca cycle which results in a metabolism switch to increase glycolysis and the compensatoryusage of glutamine [ ]existed studies have reported that mpc1 was related withimmunoregulation stemness metabolism cellular morphology etc [ ] currently the important role of mpc1was uncovered in several tumors in crc and esophagealsquamous cell carcinoma decreased mpc1 results in accelerated aerobic glycolysis and malignant progression [ ] inlung adenocarcinoma mpc1 deï¬ciency accelerates lung adenocarcinoma progression through the stat3 pathway in prostate cancer mpc1 was reported to be involved instemness and metabolism which regulated by couptfii[ ] in renal cell carcinoma hypoxiainduced loss ofmpc1 enhanced the expression of mmp7 and mmp9 to promote cell invasion collectively these data suggestedthat mpc1 maybe serves as a suppressor to disrupt tumormalignancy however whether mpc1 is involved in crcmetastasis and the underlying mechanisms remain to beillustratedin the present study we ï¬gured out the relationshipbetween the mpc1 expression and crc liver metastasiswe identiï¬ed that decreased mpc1 was closely correlatedwith patients metastasis as well as led to poor prognosisfunctionally mpc1 overexpression could attenuate themigration and invasion capacities of crc cells both in vitroand in vivo mechanically mpc1 suppressed crc metastasisthrough mediating the wntcatenin signaling thus ourï¬nding ï¬rstly revealed a critical role of mpc1 in crc livermetastasis materials and methods data mining seven geo datasets gse21510 gse5206gse20916 gse9348 gse89393 gse67675 and gse4183and tcga were used to analyze the mpc1 expression pattern in crc the primary data for tcga datasets weredownloaded wwwcancergov the primary data feo datasets were downloaded at wwwncbinlmnihgovgeo the oncolnc database httpwwwoncolnc was used to detect the prognostic value of mpc1 inthe tcga cohort patients and clinical specimens in our study a total of patients containing paired crc tissues and adjacentnontumor tissues were enrolled from the department of gastrointestinal surgery renji hospital school of medicineshanghai jiao tong university among them cases ofliver metastases were collected and only cases wereavailable with complete followup data for survival analysisinformed consents were signed by all patients the researchwas approved by the research ethics committee of renjihospital and carried out in accordance with ethical standardsas formulated in the helsinki declarationtable sequences of primers used for realtime pcrprimermmp7 forwardmmp7 reverseecadherin forwardecadherin reversesnail1 forwardsnail1 reversemyc forwardmyc reversegapdh forwardgapdh reversesequence ²²gagtgagctacagtgggaacactatgacgcgggagtttaacatatgagtgtcccccggtatcttcacgagcagagaatcataaggcggtatccagagctgtttggaaacattttcctcccaggccatcacagccctcactcacacagattccacaaggtgcctgggctacactgagcaccaagtggtcgttgagggcaatg cell culture and cell transduction human crc celllines hct116 ht29 sw620 rko sw480 and lovoand mouse crc cell line mc38 were gained from the cellbank of the chinese academy of sciences shanghai chinaall cells were cultured in dmem medium supplemented°with fetal bovine serum and antibiotics at c in ahumidiï¬ed incubator with co2mc38 cells were transfected with lentivirus containing aluciferase reporter plasmid stable transfection cells werescreened with μgml blastisidin for days which termedmc38luc in addition one short hairpin rna shrnasequence against mpc1 was packaged as lentivirus andtransfected into mc38luc cells lovo and sw480 were transfected with lentivirus containing fulllength human mpc1cdna or empty vehicle control stable transfection cells werescreened under μgml puromycin for days and veriï¬edby western blot in those assays all lentiviral transfectionswere performed in the presence of μgml polybrene immunohistochemical ihc the protocol of this assayand quantify the mpc1 protein expression level were performed according to previously reported primary antibodies used as follows mmp7 yt2663 immunoway ecadherin cst snail1 ab53519 abcam and mycyp0861 immunoway cellular immunoï¬uorescence assays were performedaccording to the previous description brieï¬y cells wereincubated with antibodies against catenin ab32572abcam and incubated with alexa 488conjugated secondaryantibody the nuclei were stained with ²6diamidino2phenylindole dapi western blotting wholecell lysates or nuclear proteinwas extracted using a protein extraction buï¬er beyotimeshanghai china or nucleoprotein extraction kit sangonbiotech c500009 respectively proteins were resolved bysdspage and transferred onto nitrocellulose nc membranes using standard methods primary antibodies used asfollows mpc1 ab74871 abcam catenin ab32572abcamlamin ac ab8984 abcam and gapdhab9485 abcam speciesspeciï¬c secondary antibodies used 0c of immunology researchtable related enzyme and carrier of pyruvate analyzed in this studygenepdha1pdhbpdk4pdhxmpc2pdk2pdk1pdk3mpc1pcpdp1pdprpdha2pdp2pklrdescriptionpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase lipoamide betapyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase complex component xmitochondrial pyruvate carrier pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme mitochondrial pyruvate carrier pyruvate carboxylasepyruvate dehydrogenase phosphatase catalytic subunit pyruvate dehydrogenase phosphatase regulatory subunitpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase phosphatase catalytic subunit pyruvate kinase liver and rbclocationmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrionas follows irdye goat antimouse igg licor andirdye goat antirabbit igg licorttest was used for comparison between groups p wasconsidered as statistically significantsynthesized using the primescript realtime pcr total rna was extracted using trizoltakara according to the manufacturersinstructionscdna wasreversetranscriptionpolymerasechain reaction rtpcr kittakara the qpcr was performed using sybr green masct method was used to analyze theter mix roche the data and gapdh was used as a loading control the primersequences are listed in table liver metastasis model this study was performed inaccordance with the recommendations in the guide for thecare and use of laboratory animals and relevant chineselaws and regulations the protocol was approved by theinstitutional animal care and use committee iacucof shanghai jiao tong university the mc38 cells transplanting luciferaseexpressing were injected into the spleensof c57bl6n mice n with a concentration of cellsmouse two weeks later the mice were killed andthe liver metastasis tissues were harvested luciferase reporter assay the protocols of this assaywere operated in accordance with previous reported ng top reporter plasmid wntcatenin signalingor ng fop reporter plasmid negative control ofwntcatenin signaling and ng renilla were mixedand transfected into crc cells using lipofectamine dualluciferase reporter assay system promega was usedto detect the ï¬reï¬y and renilla luciferase activities statistical analysesdata are shown as means ± sd spss chicago il usaand graphpad prism software were used to manipulate statistical analyses kaplanmeier method was used to calculatecumulative survival time the chisquare test or students results mpc1 expression was aberrantly decreased in crcpyruvate is a pivotal intermediate in the process of cellmetabolism which connects glycolysis and the tca cycleto determine the potential maladjustment genes involvedin pyruvate metabolism which is located in mitochondriaas shown in table we analyzed the tcga dataset containing crc and their normal counterparts the resultsshowed that multiple genes are significantly upregulated ordownregulated in crc t tissues compared to normal colonn notably mpc1 had a log2 fold change less than figures 1a and 1b as known to us pyruvate translocation from the cytoplasm to the mitochondria is the ï¬rst stepinto the tca cycle which needs mpc1mpc2 heterodimerin the analysis no significant change was found in mpc2therefore mpc1 was selected for further study the expression pattern of mpc1 was further analyzed in ï¬ve independent geo datasetsgse21510 gse5206 gse20916gse9348 and gse4183 consistently we found thatmpc1 was downregulated with statistical diï¬erence in crctissues figure 1c and inï¬ammatory tissue supplementary figure in comparison to their normal counterpartsmeanwhile we found decreased mpc1 expression inhuman crc tissues compared to their normal counterpartsfigure 1d in addition a similar phenomenon wasrevealed in aomdss induced mouse crc modelsfigure 1e then we evaluated the protein level of mpc1used a tissue microarray containing matching cancerand corresponding adjacent nontumortissues whichsubjected ihc staining the expression of mpc1 wasscored as based on the staining area andintensity figure 1f we found that more lower mpc1expression score as and was presented in crc 0c of immunology researcheulavp goltcga t nmpc1mpc2log2 fold changenoisserpxe cpm evitalertcgan normaltumornoisserpxe cpm evitalergeo datasetsgse21510 gse5206 gse20916 gse9348normaltumorabhuman samplecrcnormalaom dssnormalccrcesacesacesacesacdempc1 lower expressionmpc1 higher expressionf egatnecrepromutlamrongfigure expression pattern of mpc1 in crc a volcano plot showed fold changes xaxis and corresponding p values log10 yaxis ofpyruvate metabolismrelated genes located in mitochondria analyzed in the tcga dataset between paired normal and crc samplesstudents ttest b the comparison of mpc1 expression in tumor and matched normal tissues using the tcga dataset students ttest p c expression analysis of mpc1 in tumors and corresponding normal tissue using four independent geo datasetsgse21510 gse5206 gse20916 and gse9348 students ttest p d ihc staining of mpc1 expression in matched crctumor and nontumor tissues scale bar μm e ihc staining of mpc1 expression in the aomdss induced crc mouse models andcontrol animal scale bar μm f representative ihc staining of mpc1 expression in tissue microarray from the ren ji cohortwhich contained crc patients and paired adjacent normal tissue n the tumor tissues were divided into two groups based on theexpression level which scored as scale bar μm g the percentage of tissue displaying diï¬erent expression level ofmpc1 in crc tumor and adjacent nontumor tissues fishers exact test p tissues figure 1g overall these results revealed thatmpc1 was disrupted during crc decreased mpc1 enhanced tumor metastasis capabilityand predicated poor prognosis in crc it is well known thatcrc is one of the most malignant tumors given its strongmetastasis ability so we tried to ï¬gure out whether thempc1 expression was correlated with metastasis we foundthat lower mpc1 expression was closely correlated withmetastasis p lymph node invasion p and tnm stage p which revealed by the analysisbetween the clinical signiï¬cance and mpc1 expression incrc table next to illuminate the expression patternof mpc1 in diï¬erent process of crc data mining was carried out by two independent geo datasets gse21510 andgse89393 which contained normal tissue primary crcand metastatic lesion in the liver mcrc as shown infigures 2a and 2b mpc1 expression was gradually downregulated in patients with an increase in metastasis ability asimilar result was found in mice cells ct26 with high livermetastasis hmct26 or poor liver metastasis pmct26figure 2c which isolated by in vivo selection in an orthotopic mouse model of colon cancer metastasis to the liverfurther analysis showed that mpc1 protein expression was 0c of immunology researchtable clinicopathological correlation of mpc1 expression in theren ji crc cohortclinicopathological featureexpression ofmpc1lowhighp value Ï2testage years¥gendermalefemalemetastasisyesnolymph node invasionyesnotumor size cm¥ cmtnm stageiiiiiiivkras mutationmutationno mutationgradually downregulated in normal tissue primary crc andliver metastasis crc crlm tissues figure 2d furthermore survival analysis showed that patients with lowermpc1 expression had a worse outcome compared to thepatients with higher mpc1 expression using the tcgacohort figure 2e and ren ji cohort figure 2f additionally among patients with metastasis worse prognosiswas emerged in the mpc1 low cases figure 2g while nosignificant association was observed in patients withoutmetastasis figure 2h mpc1 overexpression impaired crc cells motility bothin vitro and in vivo to evaluate the role of mpc1 on themotility of crc cells the transwell assay was performedfirstly we examined low mpc1 protein expression in humanand high mpc1 protein expression in mouse mc38 crccells by western blot figure 3a mpc1overexpressingstable cell lines were established using a lentivirus carrying the mpc1 gene in lovo and sw480 cells and theoverexpression eï¬ciency was conï¬rmed by immunoblotsfigure 3b mpc1 knockdown in lucmc38 cells wereestablished and veriï¬ed by wb figure 3c mpc1 overexpression exhibited significantly weaker migration and invasion ability than the control cells in both lovo figure 3dand sw480 figure 3e cells following the liver metastasismodel of crc was established by spleen orthotopicallyinjecting mc38 cells transplanting luciferaseexpressingwhich would simulate mc38 metastasis to the liver throughsplenic veinportal vein the results revealed that the mpc1knockdown promoted mc38 cells metastasis to the liverthrough detecting the luminescence intensity monitoredby bioluminescence imaging figure 3f notablythenumber of metastatic liver nodules in the mpc1 silencinggroup wasin the control groupfigure 3g histological examination also proved thatmpc1 knockdown decreased the metastatic potential ofcrc in vivo figure 3gthan thatsmaller decreased mpc1 activated the wntcatenin pathwayby promoting nuclear translocation of catenin to furtherexplore the underlying mechanism of mpc1mediated inhibition of crc metastasis the tcga database was used toperform gsea analysis the results indicated that mpc1was involved in the wntcatenin signaling when set themrna expression median as a cutoï¬ figure 4a anddualluciferase reporter gene assay revealed that mpc1 overexpression obviously inhibited the activity of wntcateninpathway figure 4b which conï¬rmed the result abovewe then tested the distribution changes of catenin innuclear and cytoplasmic which was a crucial step inwntcatenin pathway as shown in figure 4c no significant diï¬erence was emerged in the total amount ofcatenin between mpc1 overexpression cells and the control cells figure 4c however obviously decreased distribution of nuclear catenin was presented in mpc1overexpression cells compared to that in the control cellsas revealed by the stronger gray corresponding to cateninfigures 4c and 4d consistently immunoï¬uorescenceif staining showed that mpc1 overexpression weakenednuclear catenin localization in both lovo and sw480 cellswhen compared to control cells figures 4e and 4f asimilar phenomenon was observed in mouse liver metastasistissues by ihc figure 4g following qpcr analyses displayed some downstream target genes of catenin such asmmp7 ecadherin snail1 and myc obviously increasedin ecadherin and decreased in mmp7 snail1 and mycwere observed after the mpc1 overexpression in both lovoand sw480 cells compared to that in the control cellsfigures 4h and 4i meanwhilethe opposite trendwas observed in mc38 cell after mpc1 knockdownfigure 4j and similar results were observed in mouseliver tissue detected by ihc figures 4k4n takentogether the data above indicated that mpc1 mediatedcrc cell metastasis through the wntcatenin pathway discussionaccumulating evidences have shown that reprogrammedenergy metabolism conduces to the tumor malignant propertiesincluding enhanced crc liver metastatic capacity[] mitochondria as the primary site of energy production regulate the pyruvate metabolism under both physiologic and pathologic conditions the ï¬rst step of the tcacycle is mediated by mpc which transports pyruvate into 0cnoisserpxe cpm evitalergse21510normal crc mcrcnoisserpxe cpm evitalernormalacrccrlmesacesac of immunology researchgse67675pmct26 hmct26ctcgan n p hr time daysgse89393normal crcmcrcbnoisserpxe cpm evitaler egatnecreplavivrus llarevolamroncrcmlrclavivrus llarevoren ji cohortn p hr n time monthsfdlavivrus llarevolavivrus llarevocrc with metastasis n n p hr n time monthsmpc1 higher expressionmpc1 lower expressiongecrc without metastasis n n n p hr time monthshfigure decreased mpc1 enhances tumor metastasis and predicts poor prognosis in crc a b data mining showed mpc1 expressionwas gradually decreased in normal tissue primary crc and liver lesion of metastatic crc mcrc from two independent geo datasetsoneway anova a gse21510 p b gse89393 p c data from gse67675 revealed that mpc1 expressionwas lower in high liver metastasis ct26 cells hmct26 than that in poor liver metastasis ct26 cells pmct26 students ttestp d gradually decreased mpc1 expression was presented in normal tissue primary crc and liver metastasis crccrlm tissue scale bar μm n fishers exact test p e kaplanmeier overall survival os curves in the tcgadataset of crc patients according to the mrna expression of mpc1 the lower quartile value of expression was utilized as a cutoï¬logrank test p f kaplanmeier os curve for the mpc1 expression in the ren ji cohort logrank test p gkaplanmeier os curve for the mpc1 expression in patients with metastasis logrank test p h kaplanmeier os curve forthe mpc1 expression in patients without metastasis logrank test p mitochondrial from cytoplasm in the beginning tcgawas used to analyze the relative genes involved in pyruvatemetabolism which is located in mitochondria the resultsrevealed that mpc1 expression was significantly downregulated in crc tissues meanwhile the geo datasets analysisas well as ihc staining on crc patients tissue and mousemodels conï¬rmed this trend these phenomena may indicate that loss of mpc activity enhanced tumorigenic glucoseutilization by blocking mitochondrial pyruvate uptake andoxidation interestingly in the course of data analysis wefound that the expression of mpc1 was decreased at thestage of intestinal inï¬ammation which was not diï¬erentfrom that in tumor tissue mpc1 has been reported to beinvolved in immune regulation of peripheral t cell homeostasis through metabolic regulation and a decrease inmpc1 was found at the earliest stages of crc hence 0c of immunology researchhumanmouselovosw480lovosw480mpc1gapdhmpc1mpc1gapdhmpc1mpc1gapdhwsthwsovolokrcmtchablovorotcevitnelcpmitnelrotcevitnelcpmitneldlefi rep sllec edavnidlefi rep sllec edavnirotcevitnelcpmitneldsw480rotcevitnelcpmitnelcncpmcncpmdlefi rep sllec detargimdlefi rep sllec detargimccncpmcncpmmc38shnc shmpc1¨¯ sp xufl latotcnhscpmhscnhscpmhsradiancepseccm2srfemc38suledon sisatsatemcnhscpmhsgfigure mpc1 overexpression inhibits the motility of crc cells in vitro and in vivo a mpc1 expression in human and mouse crc celllines examined by western blot gapdh serves as loading control b mpc1 overexpression in lovo and sw480 cells c mpc1 silencing by shrnampc1 in mouse mc38 cells d e transwell assays showed that upregulated mpc1 suppressed the invasion and migration ability of lovod and sw480 e cells quantiï¬cation of invaded and migrated cells was performed for ï¬ve randomly selected ï¬elds values are means ± sd frepresentative bioluminescence photograph of mice spleen implanted with luciferaseexpressing mc38 cells treated with shmpc1 or controlvector total ï¬ux was quantiï¬ed by the ivis system to verify the ability of liver metastasis g representative image of liver metastases andquantiï¬ed by the nodules in mice inoculated with mc38 cells treated with shmpc1 or control vector as well as representative images ofhe staining of the liver metastatic lesions scale bar μm students ttest p p p 0c of immunology researchse erocs tnemhcirnewnt signaling pathwaynes p mpc1 highmpc1 lowoitar pof pot evitalerð½cateninlamin acð½cateningapdhmpc1alovosw480csuelcunlatot icanmalnnetacð½ilovoncmpc1bsw480lovosw480ncmpc1dð½catenindapimergeð½catenindapimergeð½catenincpmcnovolnoisserpxe evitaleregnahc doflelovolianscympmmncmpc1nirehdacehcpmcnwsfsw480lianscympmmncmpc1nirehdaceifigure continuedcmcnhscpmhsmc38glianscympmmnirehdaceshncshmpc1jnoisserpxe evitaleregnahc doflnoisserpxe evitaleregnahc dofl 0c of immunology researchmmp7ecadherinsnail1myccmcnhscpmhscmcnhscpmhscmcnhscpmhscmcnhscpmhsklmnfigure decreased mpc1 activates the wntcatenin pathway by promoting nuclear translocation of catenin a gsea analysis ofmpc1 expression in crc using the tcga dataset nes normalized enrichment score b luciferase reporter gene assay of crc cellstreated with mpc1 overexpression or not c the expression of total catenin and nuclear catenin was detected in control and mpc1overexpression crc cells respectively gapdh and lamin ac were used as the loading control of total and nuclear protein respectivelyd the gray value analysis of nuclear catenin in mpc1overexpression cells and control cells e f mpc1 overexpression could inhibitthe nuclear translocation of catenin in crc cells scale bar μm g ihc staining of catenin in mouse liver metastatic lesionsinoculated with mc38 cells treatment with shmpc1 or control vector scale bar μm h i relative mrna expression level of catenin target genes in crc cells with mpc1 overexpression or control vector j relative mrna expression level of catenin targetgenes in mc38 cells with shmpc1 or control vector kn relative protein expression level of catenin target genes in mouse livertissue detected by ihc scale bar μm students ttest p p we suspect that mpc1 is involved in bowel inï¬ammation totumorigenesis and more studies need to be devised to illustrate this processfollowing in the analysis between the clinical signiï¬cance and mpc1 expression in crc we found that mpc1expression was especially correlated with metastasis inspiredby this we detected the mpc1 expression pattern in normaltissue primary crc and metastasis crc by geo datasetsand patients tissue all results revealed that gradually downregulated mpc1 was in patients with an increase in metastasis ability survival analysis indicated that worse outcome waspresented in patients with lower mpc1 expression especiallyin patients with metastasis additionally function assays veriï¬ed that mpc1 overexpression could attenuate the migration and invasion capacities of crc cells in vitro andmpc1 knockdown could enhance the metastasis capacityin vivo and existing studies have revealed that the mpc1was participated in metastatic dissemination of pgc1αtransduced cholangiocarcinoma through elevating reactiveoxygen species ros production besides mpc inhibitor uk5099 treatment could trigger strong invasive capacitythrough blocking pyruvate translocation into the mitochondria so as to attenuate mitochondrial oxidative phosphorylation and trigger aerobic glycolysis these phenomenatogether with our results indicate that mpc1 could act as atumor suppressor through inhibiting tumor metastasis andexisting studies have shown that mpc1 could alter the maintenance and fate of stem cells through regulating cancermetabolism in crc however the relationship betweenthe metabolism and tumor metastasis regulated by mpc1was not being mentioned thus further evidence should bereceived to conï¬rm thissubsequently the underlying mechanism of mpc1 inregulating metastasis was explored for this purpose gseaanalysis was performed the results indicated that mpc1was involved in the wntcatenin signaling the next seriesof experiments also conï¬rmed that mpc1 could mediate thewntcatenin pathway by redistribution of catenin previous reports showed that cytoplasmic catenin phosphorylated in nterminally localized to sites of cellcell contact isassociated with ecadherin and was required for intact cellcell adhesions without any change detected in the levels oftotal catenin [] simultaneously cellcell adhesionbased on cadherin binding with catenin limited wnt signals in addition catenin was reported to interact withusp9x to inhibit the degradation of catenin through thedeubiquitination of catenin in breast cancer a constitutive irs1 and catenin protein interaction activated mycexpression in acute lymphoblastic leukemia cells inhcc catenin was reported to interact with yap1 to leadto rapid tumorigenesis hence it is reasonable to guessthat accumulated cytoplasmic catenin maybe crosstalkwith other genes or involved in other biological processeswhat is more some downstream target genes of cateninsuch as mmp7 ecadherin snail1 and myc were changedin expression as known to us mmp7 is a member of theproteolytic enzyme family which promotes the invasionand metastasis of tumor cells by degrading the basementmembrane and extracellular matrix and previous studies had evidenced for involvement of mmp7 activation incolorectal cancer liver metastases [ ] ecadherin andsnail1 were considered as the epithelialmesenchymal transition emt marker which was involved in metastasis ofmalignant tumor moreover ecadherin was reportedto be involved in cellcell junction to regulate cancer invasionand metastasis [ ] and the gsea analysis also revealedthat mpc1 could aï¬ect the cellcell contacts data notshown as described previously mmp7 could facilitatemorphological transition by cleaving ecadherin thecommunication between the cells is disrupted when ecadherin was shredded leading to destructed cell adhesionand induction of emt followed by increased cell migration inspired by this we assumed that mpc1 could mediatetumor cell motility through aï¬ecting mmp7 activity cellcell 0c of immunology researchcontacts and emt however further studies need to be performed to clarify the detailed underlying mechanisms conclusionsin conclusion we ï¬rstly demonstrated that decreased mpc1was closely correlated with patients metastasis as well as ledto poor outcome moreover mpc1driven nuclear translocation of catenin contributed to crc cell motility thismeans that mpc1 has the potential to be a diagnostic biomarker and therapeutic target for metastasis patientsabbreviationscrc colorectal carcinomampc mitochondrial pyruvate carrieremt epithelialmesenchymal transitiongsea gene set enrichment analysisdata availabilitythe data used to support the ï¬ndings of this study are available from the corresponding author upon requestconflicts of interestall authors declare no conï¬icts of interestauthors contributionsyahui wang and guangang tian conceived and designedthe study chunjie xu and kaixia zhou obtained and anized the data guangang tian analyzed the data zhigangzhang and jianren gu contributed reagentsmaterialsanalysis tools xueli zhang and guangang tian wrote themanuscript guangang tian chunjie xu and kaixiazhou contributed equally to this workacknowledgmentsthis work was supported by the national natural sciencefoundation of china no to zhigang zhangand the natural science foundation of shanghai no18zr1436900 to xuelili zhangsupplementary materialssupplementary figure expression analysis of mpc1 innormal ibd and tumor tissues using the gse4183 datasetoneway anova was used to analyze the statistical diï¬erences between ibd adenoma and crc tissues ns no significance students ttest p p p supplementary materialsreferences h brody colorectal cancer nature vol no r l siegel k d miller s a fedewa et al colorectal cancerstatistics ca a cancer for clinicians vol no pp a j rauckhorst and e b taylor mitochondrial pyruvatecarrier function and cancer metabolism current opinion ingenetics development vol pp m g vander heiden l c cantley and c b thompsonunderstanding the warburg eï¬ect the metabolic requirements of cell proliferation science vol no pp s herzig e raemy s montessuit et al identiï¬cation andfunctional expression of the mitochondrial pyruvate carrierscience vol no pp c yang b ko c t hensley et al glutamine oxidationmaintains the tca cycle and cell survival during impairedmitochondrial pyruvate transport molecular cell vol no pp t bender g pena and j c martinou regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes the embo vol no pp a g ramstead j a wallace sh lee et al mitochondrialpyruvate carrier promotes peripheral t cell homeostasisthrough metabolic regulation of thymic development cellreports vol no pp 2899e6 x zhou z j xiong s m xiao et al overexpression ofmpc1 inhibits the proliferation migration invasion and stemcelllike properties of gastric cancer cells oncotargets andtherapy vol pp d li c wang p ma et al pgc1α promotes cholangiocarcinoma metastasis by upregulating pdha1 and mpc1 expression to reverse the warburg eï¬ect cell death diseasevol no j c schell k a olson l jiang et al a role for the mitochondrial pyruvate carrier as a repressor of the warburg eï¬ectand colon cancer cell growth molecular cell vol no pp y li x li q kan et al mitochondrial pyruvate carrierfunction is negatively linked to warburg phenotype in vitroand malignant features in esophageal squamous cell carcinomas oncotarget vol no pp h zou q chen a zhang et al mpc1 deï¬ciency accelerateslung adenocarcinoma progression through the stat3 pathway cell death disease vol no l wang m xu j qin et al mpc1 a key gene in cancermetabolism is regulated by couptfii in human prostatecancer oncotarget vol no pp y zhong x li d yu et al application of mitochondrialpyruvate carrier blocker uk5099 creates metabolic reprogramand greater stemlike properties in lncap prostate cancer cellsin vitro oncotarget vol no pp x p tang q chen y li et al mitochondrial pyruvatecarrier functions as a tumor suppressor and predicts theprognosis of human renal cell carcinoma laboratory investigation vol no pp g a tian c c zhu x x zhang et al ccbe1 promotesgist developmentthrough enhancing angiogenesis andmediating resistance to imatinib scientiï¬c reports vol no article c xu g tian c jiang et al nptx2 promotes colorectalcancer growth and live
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" child maltreatment leads to enormous adverse short and longterm health outcomes the aim ofthis study is to estimate the burden of disease and the cost of illness attributable to child maltreatment in japanmethods an incidencebased topdown cost of illness analysis was conducted to estimate medical costs andburden of disease attributable to child maltreatment based on a societal perspective the assessment includedshortterm and longterm medical costs and burden of disease measured by disabilityadjusted life years dalysthat generates mortality and morbidities based on several national surveys and systematic review we consideredthe main types of child maltreatment as exposure for which the incidence was obtained from literature reviewbased on population attributable fractions pafs burden of disease of physical and mental health consequencesattributable to child maltreatment were estimated then dalys were converted into monetary value the lifetimeeconomic burden was finally estimated by combining with medical costs and subject to sensitivity analysisresults the lifetime disease burden expressed in dalys was estimated at dalys ci dalys for the cohort victims in based on the incidence according to literature review the overall lifetimeeconomic burden was billion usd equivalent to million times of gross domestic product gdp per capitaamong the total economic burden costs of suffering and pain based on dalys were accounting for theseestimates were times of conservative estimates which used incidence data from official reported casess this study found that the national lifetime cost was huge and equivalent to million gdp percapita and its burden of disease was approximately equal to that of colon and rectum cancers or stomach cancerour findings particularly in terms of revealed the considerable burden of disease in long term and potential effectsof the strengthened maternal and child care as the preventive strategykeywords child maltreatment burden of disease study lifelong health consequences disabilityadjusted life yeardaly costofillness correspondence gairuoyanipssgojp1department of health policy national center for child health anddevelopment tokyo japan3department of empirical social security research national institute ofpopulation and social security research uchisaiwaicho chiyodakutokyo japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cmo bmc public health page of child maltreatment is a raising concern in public healthand social welfare in japan the reported number ofsuspected cases of child maltreatment is increasing from in to in according to theministry of health labour and welfare mhlw ofjapan child maltreatment is categorised into four essential types physical sexual or psychological includingwitnessing domestic violence wdv abuse and neglect exposure to multiple types and repeated episodes ofmaltreatment during childhood is associated with highrisks to enormous adverse health outcomes causing asignificant social and economic burden on individualsfamilies and societies those adverse outcomes duringchildhood include child death injuries and disabilitiesdevelopmental and behavioural problems moreover therelated physical and mental health conditions persistinto adulthood leading to the onset of chronic diseasesdepression drug alcohol misuse and risk sexual behaviour suicide ideation [ ]the number ofthe related analysis ofthe government has introduced a couple of protectivemeasures with increasing public budget [] assessment of costs and burden of disease helps developmentof resource allocation and priority setting in public sector paralleling with growing concerns on child maltreatmenttheprevalence health consequences and economic burdenis increasing so far for the economic burden there aretwo typical research frameworks one is a comprehensively costs evaluation from healthcare social educa[]tional areas and loss in productivity another one is to measure related economic and diseaseburden [ ] wada reported the socialcosts of child abuse in japan included direct costs ofdealing with abuse and the indirect costs related to longterm damage from abuse during the fiscal year onthe other hand the first framework is likely to underestimate longterm deleterious effects of child maltreatmenton which evidence derived from longitudinal studies isless available compared to that on the shortterm counterpart by integrating previous evidence our costofillness study aimed to assess lifetime economic anddisease burden of mortality and morbidities attributed tochild maltreatment based on the later frameworkinorder to address the evidence gap we extended cost calculations for monetary values converted from disabilityadjusted life years dalys covering related mortalityand morbidities methodsan incidencebased victims estimated by incidencetopdown approach or attributable risk approach measuring the proportion of a disease that is due to exposureto risk factor was applied in this study from a societalperspective we employed the following steps to estimate the total economic burden constituted by directand indirect costs population attributable fraction paf wasgenerated to estimate longterm impactscosts attributed to child maltreatment shortterm and longterm direct medical costs wereassessed by using national expenditure databasesindirect costs measured include productivity losscaused by abusive head trauma and economicburden deriving from dalys finally sensitivity analyses were performed for theplausible range of the discount rate and theincidence prevalenceestimating pafin the topdown approach paf for each disease i measured that how health outcomes and their associatedcosts may be attributed to child abuse using the following formula [ ]pafi ¼ p rri °p rri ¾¾ ¾ °p prevalence of child abuse rr the relative risk of theoutcome i in those who experienced child abuse compared with those who did notrisk ratio rr or odds ratio orseveral previous related systematic reviews and metaanalyses summarised the relevant health consequences[ ] as adverse childhood experiences acesoften intertwine with child maltreatment cluster in childrens lives and cumulatively lead to poor health outcomes we pooled the ors from a recent systematicreview and metaanalysis for the effect of multiple aceson health rather than that for each category of childmaltreatmentin japan thethe pooled prevalencea literature review was performed to synthesize the evidence on epidemiological characteristicsthe consequencesreview focused on thosepublished between december and march onmedline pubmed web of science scopus and ciniis japanese literature details of the search strategy search terms used and inclusion and exclusion criteria are provided in the additional file we combinedour review results with those studies in japan includedin an existing systematic review and calculated thesimple sizeweighted mean incidenceprevalenceinthe median value was also calculated toaddition 0cmo bmc public health page of examine the robustness supplementary table theannual incidence rate was obtained by the formula incidence rate ¼prevalenceaverage durationdue to the lack of local data on the average duration we adopted that published in australia theaverage years for physical abuse and years forsexual abuse based on this findingthe weightedaverage of years was used for other categories ofabusedirect medical costsshortterm medical costsfor abusive head trauma aht is the leading causeof death due to child abuse among children youngerthan years old we estimated its hospitalizationcosts as shortterm medical costs by multiplying theincidence of aht under years old the agespecific population in and admission medical fee per case there were two reported incidences one is the possibleincidence considering countable possibility ofaht cases at most and another one is the presumptiveincidence representing victims had intracranial injuriesor intentionalinjuries with certain icd10 code weused the possible incidence for the general calculationand the latter one in sensitivity analysis the total possible aht cases aged under years was about times ofthe presumptive counterparts longterm medical costsfor longterm medical costs we used national healthcare expenditures and patient survey tosimulate disease burden ofrelevant health consequences by sex and age group above and then multiplied with pafs to calculatethe attributable costs in the victim cohort of [ ] on the other hand we did not include selfharm and collective violence because of the limitationto distinguish the two in the reported overallinjurycasesindirect costsin this study we considered differential and loss of earning as a result of human capital depreciation is causedby mortality and morbidities it was presented as a monetary value of dalys and gdp per capita [ ]dalys and its monetary valuethe disease burden indicator daly aggregates yearsof life lost for premature death and years lost due todisability for morbidities related data wereobtained from the who global burden of diseasegbd using the pooled ors as described bykaren we matched each relatedhealth outcome with the cause of disease burdenin the who gbd categories though it was difficultto match some outcomes with the cause of gbdsupplementary table then monetary value was converted from daly attributable to child maltreatment by multiplying dalyand gdp per capita with adjustment of purchasingpower parity in productivity losses due to aht fatal casesproductivity loss due to fatal cases of child maltreatment was calculated based on the reported fatal caseswhich figure was obtained from official data andthe average lifetime income subject to discounting in there were abuserelated deaths reported injapan not including family suicide with the averageonset age of years the discounted lifetime income from to years old was calculated by assuming the longterm growth in labour productivityto be per year dalys losses of survival ahtfor disease burden due to survival aht we considered sequelae such as vision loss brain damage andreduced life span and longterm health consequences as developing diseases in adulthood we calculated the disease burden of aht in bymultiplying average cases and the estimated meanlifetime daly loss per case at different severity mildmoderate and severe longterm dalys losses of other diseasesthen the longterm health consequences were calculatedusing the following formuladaly losses ¼ Ï pafi 03original dalyi°i different child abuse related health outcome°¾¾sensitivity analysesa discount rate of is generally performed which wasrecommended in the domestic guideline for costeffectiveness analysis whereas especially in the usa discount rate of has been selected and applied inthe cost estimate reports of centers for disease controland a best practices for the social return on investmentanalysis recommended by experts and guidelines assuch the parameter potentially affects the finally resultswe adopted a plausible range of to for sensitivityanalysis 0cmo bmc public health page of in addition we also calculated costs and diseaseburden using the incidenceprevalence data based onofficially reported child abuse cases to calculate theconservative incidence of child abuse by categorieswe obtained the official data of victim cases reportedby child consultation facilities in and thendivided them by the total population number in corresponding age data by sex were not availablecocurrentinformation was not available and theoverlapped cases were not considered supplementarytable the initial victim age is assumed to be years old according to an ageweighted incidence calculation based on official reported cases we assumed the probable abuserelated death cases to be times of the reported cases based on the ratio of thepresumptive and the possible incidence of aht casesamong children aged under years resultsthe main results showed in tables were discounted at and conservative estimates were given for sensitivityanalysesthe pooled incidence prevalence and disease burdenthe estimations on different types of child maltreatment incidence draw from literature reviews variedregarding differences between sex except physicalabuse girls suffered more than boys in sexual abuseand witnessing domestic violence table the estimated lifetime disease burden associated with childmaltreatment onset in was considerable dalys with a ci of dalys to dalys table the top causes of totaldisease burden due to child abuse were suicide attempts cardiovascular disease and depression cancercostofillness analysis for child maltreatmenttable demonstrates lifetime costs attributed to childabuse onset in the total direct cost was estimatedtable estimated incidenceprevalence of child abuse in japanestimates aincidence bmalefemalephysical abuse sexual abuse psychological abusewdv c other d prevalencemalefemaleneglect a sample sized weighted mean valueb incidence rate prevalence average durationc wdv witnessing domestic violenced not specified as wdv often expressed as emotionalpsychological abuseto be usd million 95ci million11 million while the total indirect cost was estimatedto be usd million 95ci million52 million accounting for of the total lifetimecosts which were almost million times gdp percapita economic loss initiated from dalys in longterm costs of suffering and pain accounted for ofthe overall estimatessensitivity analysesconservative estimates based on the reported cases incidence showed a tendency similar to that observed in thedisease burden based on the literature review amongwhich psychological abuse including wdv accountedfor the majority of reported child abuse cases however the incidence estimated from the review weremuch higher than those reported by child protectionagencies the conservative estimation leading to about times difference gap on child maltreat burden bydifference discount rate table discussionour results indicated that disease and economic burdenattributable to child maltreatment is substantial in particular that originated from the longterm health consequences accounts for the majoritybased on literature review the pooled incidence ofchild maltreatment in japan is much higher than officially reported which is consistent with the findingsof other studies [ ] because of difficulty toidentify the actual cases and a public attitude to consider child abuse as a private affair in the society theofficially reported cases are likely to represent the tipof an icebergthe fourpsychological abuse including wdv representedthe majority of reported cases the results of the literature review also showed a gender difference in theprevalence oftypes of child abuse sizeweighted mean values girls were found to be morelikely to experience the harmful practices comparedto boys particularly sexual abuse this tendency wasalso observed in other countries in east asia and pacific region comparing those living in othercountries in the east asia and pacific region [ ]japanese children tended to less likely to experiencephysical abuse boys vs girls vsalthough it is difficult to directly compare the results across different study settings due to the different methodologies parameters and target populationsadopted the ingredients of the lifetime economic anddisease burden considered in our studyincludingmedical costs and monetary value of disease burdenare similar to that adopted in previous studies [ 0cmo bmc public health page of table longterm daly lost attributable to child abuse in japandiseases attributed to child abuse asuicide attemptdalys confidence intervalcancercardiovascular diseasedepressionrespiratory diseaseliver or digestive diseaseanxietyproblematic drug useabusive head traumaproblematic alcohol usediabetessexually transmitted infectionsviolence victimisationviolence perpetrationtotaldalys monetary value billion usa simple size weighted mean prevalence at discounted rate] still our results showed that the disease burdenwas about times of the conservative estimationdue to the huge gap of incidence generated from literature and that officially reported the number isconsistent with an australian research that showed awide distribution ofthe annual prevalence rangingfrom to in the conservative lifetimecourse simulation the initial victim age is assumed tobe years old according to an ageweighted incidencecalculation based on official reported cases whichwas also consistent with previous studies our study in particular highlighted dalys in longterm attributable to child maltreatment accountingin the overallfor a relevant proportion lifetime costs the estimation of disease burden attributed to child maltreatment dalys wascomparable to the total dalys due to colon and rectum cancers dalys in or stomachcancer dalys in to our knowledge this is the first study to estimatelifetime economic burden of child maltreatmentinjapan based on an epidemiological model the idea ofthis method is to convert diseaseinduced losses ofwellbeing into economic terms by multiplying theannual number oflost life years due to disease bysubreginal per capita income so far few studies hadever taken this part of costs into account potentiallyleading to an underestimation of health and economictable lifetime costs attributable to child abuse for the first time in ciitems of the costs usd milliondirect costs medical costsshortterm ahtlongterm other diseasesindirect costsabuse death a productivity lossessurvival aht dalys blongterm loss of other diseases btotal costsaht abusive head traumaa we used times of base line data for range costs of child abuseb costs of suffering and pain dalys converted into monetary value by multiplying a gross domestic product per capita million gdp per capita 0cmo bmc public health page of table sensitivity analyses on incidence resource and discounted ratesensitivity analysisliterature based estimation adisease burden in dalys 95cieconomic burden usd million 95cidr dr dr conservative estimation bdr dr dr dr discounted ratea estimated based on literature review simple size weighted average prevalenceb estimated based on the number of consultation cases disposed about child abuse at child guidance centres probable estimate of abuse death was assumedabout times confirmed aht casespossible cases of the costs of conservative estimatechild maltreatmentimpacts ofin addition weadopted conservative calculation methodology in thesensitivity analyses to estimate the burden of childmaltreatment for more reliable range estimationsthere are several limitations to this study first thecooccurrence of multiple types of child abuse isprevalent resulting in difficulties to identify theadverse effects separately in order to minimize possible consequent overestimation we used the pooledors of multiple adverse childhood health experiencesinstead of each types of child maltreatment and itsseverity second we focused on the economic burdendue to the mortality and morbidity of child maltreatment but did not consider nonhealth human capitalaspectslikeother economic burden estimation studies the availability of data on the related medical costs were limited wehealthconsequences and explored their unit costs for the estimates to address the knowledge gap thirdtargeted majorneverthelessthereproductiverecently in japan a continuum ofintensive supports to mothers and childrearing families encompassingcycle has been widelyimplemented in most local authorities such an integral approach serves as an essential preventive strategy against child maltreatment and other harmfulpractices by early detection and intervention of highrisk households in pregnancy postpartum and childrearing periods thisstudy can provide decisionmakers information on the economic burden of childmaltreatment as well as an important input in futureeconomic evaluations costeffectiveness analysis oncurrently ongoing intervention and policy in additionour results hint an emphasis on preventive interventions on suicide attempts and depression which aretop causes of the attributable disease burden due tochild maltreatmentour study demonstrated that lifetime disease and economic burden due to child maltreatment in japan is substantial its disease burden was approximately equal tothe burden of colon and rectum cancers or stomach cancer in particular it is important to include the longterm disease burden in future studies related to diseaseburden and cost of illness for both technical and policyperspectivessupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12889020093978additional file table a1 studies included in the quantitativesynthesis table a2 health outcomes and pooled ors used in this studyaht not included table a3 incidence rate by age and average onsetage based on the number of consultation cases disposed about childabuse at child guidance centersadditional file systematics review 2018520findpossible literature including japanese studies on risk of health outcomesattributable to child maltreatment figure a1 study selection prismaflow diagramabbreviationsdalys disabilityadjusted life years pafs population attributable fractionsgdp gross domestic product mhlw ministry of health labour and welfarewdv witnessing domestic violence aht abusive head traumaicd international classification of diseases gbd global burden of diseaserr risk ratio or odds ratio aces adverse childhood experiencesacknowledgementswe are grateful thank members of health informatics department kyotouniversity of public health school for their kind supportauthors contributionsmx and gr designed the study mx did the calculation and draft themanuscript gr and ty takahashi contributed to the revise ty tachibanatb and nt critically reviewed and provided important intellectual feedbackon the revise all authors have read and approved the manuscriptfundingthis study is granted by health labour sciences research grant japanagency for medical research and development and as part of an ipss 0cmo bmc public health page of project on the realization of japans plan for dynamic engagement of allcitizens the funders did not have any role in the study design datacollection and analysis interpretation of data or in writing the manuscriptavailability of data and materialsall the raw data is publicly accessible from respective official website asreference national healthcare expenditures and patient survey the datasets analysed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare no conflict of interestauthor details1department of health policy national center for child health anddevelopment tokyo japan 2department of health informatics kyotouniversity school of public health kyoto japan 3department of empiricalsocial security research national institute of population and social securityresearch uchisaiwaicho chiyodaku tokyo japan4maternalchild psychiatry department of psychosocial medicine nationalcenter for child health and development tokyo japan 5faculty ofeconomics saitama university sakuraku japanreceived march accepted august referencesgilbert r widom cs browne k fergusson d webb e sjtl j burden andconsequences of child maltreatment in highincome countries lancetnumber of consultation cases disposed about child abuse at child guidancecenters in japan in japanese [httpswwwestatgojpstatsearchfilespage1layoutdatalisttstat000001034573cycle8tclass1000001108815tclass2000001108820second21]definition and present condition of child abuse in japanese [httpswwwmhlwgojpseisakunitsuitebunyakodomokodomo_kosodatedvabouthtml] accessed july currie j spatz widom c longterm consequences of child abuse andneglect on adult economic wellbeing child maltreatment hughes k bellis ma hardcastle ka sethi d butchart a mikton c jones ldunne mp the effect of multiple adverse childhood experiences on healtha systematic review and metaanalysis lancet public health 201728e356fy budget outline for child abuse prevention [httpswwwmhlwgojpfile05shingikai11901000koyoukintoujidoukateikyokusoumuka002_1pdf] accessed july fy budget outline for child abuse prevention [httpswwwmhlwgojpfile06seisakujouhou11900000koyoukintoujidoukateikyoku0000180499pdf] accessed july fy budget outline for child abuse prevention [httpswwwmhlwgojpfile05shingikai12601000seisakutoukatsukansanjikanshitsu_shakaihoshoutantou0000058633pdf] accessed july the economics of child abuse a study of san francisco [httpssafeandsoundwpcontentuploads201709economicsofabuse_report_sfcapc1pdf] accessed july fang x brown ds florence cs mercy ja the economic burden of childmaltreatment in the united states and implications for prevention childabuse negl habetha s bleich s weidenhammer j fegert jm a prevalencebasedapproach to societal costs occurring in consequence of child abuse andneglect child adolesc psychiatry ment health mccarthy mm taylor p norman re pezzullo l tucci j goddard c thelifetime economic and social costs of child maltreatment in australia childyouth serv rev wada i igarashi a the social costs of child abuse in japan child youth servrev miller tr steinbeigle r wicks a lawrence ba barr m barr rgjp disabilityadjusted lifeyear burden of abusive head trauma at ages pediatrics20141346e1545fang x fry da brown ds mercy ja dunne mp butchart ar corso psmaynzyuk k dzhygyr y chen y the burden of child maltreatment inthe east asia and pacific region child abuse negl corso ps fertig ar the economic impact of child maltreatment in theunited states are the estimates credible child abuse negl macroeconomics and health investing in health for economicdevelopment [httpwhqlibdocwhointpublications2001924154550xpdf]accessed july segel je costofillness studiesa primer rtiunc center of excellence inhealth promotion economics jo c costofillness studies concepts scopes and methods clin molhepatol metrics population attributable fraction paf [httpwwwwhointhealthinfoglobal_burden_diseasemetrics_pafen] accessed july norman re byambaa m de r butchart a scott j vos t the longtermhealth consequences of child physical abuse emotional abuse and neglecta systematic review and metaanalysis plos med 2012911e1001349kalmakis ka chandler ge health consequences of adverse childhoodexperiences a systematic review j am assoc nurse pract unicef child maltreatment prevalence incidence and consequences inthe east asia and pacific region new york unicef rothman kj epidemiology an introduction oxford university press joyce t huecker mr pediatric abusive head trauma shaken babysyndrome [updated aug ] in statpearls [internet] treasure islandfl statpearls publishing available from httpswwwncbinlmnihgovbooksnbk499836 yamaoka y fujiwara t fujino y matsuda s fushimi k incidence and agedistribution of hospitalized presumptive and possible abusive head traumaof children under months old in japan j epidemiol httpsdoi102188jeaje20180094japanese population projection [httpwwwstatgojpdatajinsui2016np] accessed july summary of patient survey [httpswwwmhlwgojpenglishdatabasedbhsssps_2014html] accessed july kirigia jm mburugu gn huka gs the indirect cost of disability adjusted lifeyears lost among the elderly in kenya int arch med httpsdoi1038232483 mortality and global health estimates [httpwwwwhointghomortality_burden_diseaseen] accessed july japan gdp gross domestic product [httpscountryeconomycomgdpjapanyear2016] accessed july the results of verification of death cases caused by child abuse threport [httpswwwmhlwgojpstfseisakunitsuitebunya0000173329_00001html] accessed july miller tr steinbeigle r wicks a lawrence ba barr m barr rg disabilityadjusted lifeyear burden of abusive head trauma at ages pediatrics20141346e154550 httpsdoi101542peds20141385shiroiwa t fukuda t ikeda s takura t moriwaki k development of anofficial guideline for the economic evaluation of drugsmedical devices injapan value health moore se scott jg ferrari aj mills r dunne mp erskine he devries kmdegenhardt l vos t whiteford ha burden attributable to childmaltreatment in australia child abuse negl publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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"These studies demonstrate that RRM1 could be a predictive marker of the response to gemcitabine-based chemotherapy in patients with NSCLC [22]. The present study also showed that the DCR was higher in RRM1-positive patients that received docetaxel or vinorelbine rather than gemcitabine-based therapy. In addition docetaxel and vinorelbine each showed a longer PFS than gemcitabine-based therapy. Simon et al. used RRM1 and ERCC1 as molecular determinants and found that RRM1- and ERCC1-tailored selection of first-line therapy could improve response overall survival (OS) and PFS over standard treatments in patients with NSCLC [23]. These studies suggest that responses to cytotoxic chemotherapy vary greatly in patients with NSCLC and individualized therapy based on RRM1 expression may help improve the efficacy of chemotherapeutic agents [24]. Our research was performed retrospectively and this is the major limitation of the study. However the current results provide new information and further insight that can assist clinicians in selecting appropriate and individualized chemotherapy for patients with NSCLC based on RRM1 expression. Several molecular markers have been used as predictive markers of the response to chemotherapy in NSCLC patients. ERCC1 has been used for the prediction of platinum sensitivity in the treatment of NSCLC [6][8]. Park et al. analyzed 217 patients with NSCLC who had received gemcitabine- or taxane-based chemotherapy and found that taxane was associated with a higher response than gemcitabine treatment in patients with EGFR mutations [9]. Another study found that low thymidylate synthase (TS) expression is significantly associated with better clinical outcomes in non-squamous NSCLC patients who were treated with pemetrexed-based chemotherapy [25]. Therefore more prospectively designed studies with combined detection of these markers (RRM1 ERCC1 EGFR and TS) will provide valuable information that will ultimately be used to determine preferable therapeutic approaches for individual patients with NSCLC. In the results of this study suggest that negative RRM1 expression in advanced NSCLC is associated with a higher response rate to gemcitabine-based chemotherapy. Moreover RRM1 may be used as a predictive marker for conventional chemotherapy regimens involving gemcitabine docetaxel and vinorelbine. Additional prospective studies are needed to evaluate the effect of RRM1 expression on the response to various chemotherapeutic regimens in patients with NSCLC. References 1 SchillerJH HarringtonD BelaniCP LangerC SandlerA et al (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med346: 929811784875 2 PfisterDG JohnsonDH AzzoliCG SauseW SmithTJ et al (2003) American society of clinical oncology treatment of unresectable non-small-cell lung cancer guideline: update 2003. J Clin Oncol22: 33035314691125 3 KellyK CrowleyJ BunnPAJr PresantCA GrevstadPK et al (2001) Randomized phase III trial of paclitaxel plus carboplatin versus vinorelbine plus cisplatin in the treatment of patients with advanced non-small-cell lung cancer: a Southwest Oncology Group trial. J Clin Oncol19: 3210321811432888 4 ScagliottiGV De MarinisF RinaldiM Crin²L GridelliC et al (2002) Phase III randomized trial comparing three platinum-based doublets in advanced non small cell lung cancer. J Clin Oncol20: 4285429112409326 5 SchillerJH HarringtonD BelaniCP LangerC SandlerA et al (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med346: 929811784875 6 RothJA CarlsonJJ (2011) Prognostic role of ERCC1 in advanced non-small-cell lung cancer: a systematic review and meta-analysis. Clin Lung Cancer6: 39340121723790 7 PapadakiC"
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"widely usedcancerspecific questionnaire assessing domains of healthrelated quality of life HRQoL Our aim was to facilitatethe interpretation of scores on this questionnaire by providing Austrian normative data based on a general populationsampleMethods The calculation of normative data was based on the EORTC QLQC30 data collected from an Austriangeneral population sample that was part of an international online panel study on the development of Europeannormative data Data reported herein were stratified and weighted by age and sex Normative data were calculated forall HRQoL domains of the EORTC QLQC30 For precise predictions of EORTC QLQC30 scores a regression modelbased on sex age and the presence of health conditions was builtResults The Austrian sample comprised Austrian participants female when weighted by age andsex based on United Nation statistics The mean age was years weighted years and weighted reported at least one health condition Men reported better physical Cohens d and emotional Cohens d functioning as well as less fatigue Cohens d and insomnia Cohens d compared with womenYounger individuals years reported less dyspnea Cohens d and pain Cohens d whereas olderindividuals ¥ years reported better emotional functioning Cohens d Conclusions We present Austrian normative data for the EORTC QLQC30 Differences by age and sex are mostly inline with the findings of other European normative studies The Austrian population sample shows higher HRQoL andlower morbidity compared with other European countries The normative data in this study will facilitate theinterpretation of EORTC QLQC30 scores in oncological practice and research at a national and international levelincluding crosscultural comparisonsKeywords Normative data EORTC QLQC30 Quality of life Oncology Patientreported outcome measures AustriaGeneral population Correspondence jenslehmannimedacat1University Hospital of Psychiatry II Medical University of InnsbruckAnichstrasse Innsbruck AustriaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLehmann Health and Quality of Life Outcomes Page of BackgroundPatientreported outcomes PROs are the gold standardof evaluating the impact of cancer and its treatment fromthe patients perspective The importance of PROs isreflected in their widespread use as study endpoints incancer clinical trials [ ] and in the steadily increasing integration of routine PRO assessments in daily clinicalpractice [ ] Regulatory authorities such as the UnitedStates Food and Drug Administration [] and the European Medicines Agency [] have published guidance documents to foster the collection of highquality PRO datain clinical trials Guidelines from international associationssuch as the European anisation for Research andTreatment of Cancer EORTC [] and the InternationalSociety for Quality of Life Research [] deal with the integration of PRO measures into clinical routine and provideinformation on how to collect process and use PRO dataIn the present study we calculate reference values basedon data assessed with EORTC QLQC30 [] a questionnaire that is among the most widely used PRO measuresin cancer clinical trials [] This questionnaire coversimportant functional health domains eg physical andemotional functioning and key cancer symptoms such asfatigue pain and nauseaIn the literature different methods have been proposedto support the interpretation of results from PRO measuresanalyzing not only individual patients and patient groups ata single time point but also group differences and changesover time One approach is to use minimally important differences to assess or compare changes in PRO results at thegroup or patient level [] To interpret scores obtained ata single time point thresholds for clinical importance canbe used [] Another important approach to interpretPRO data are normative data if comparisons are to bemade with for example a specific disease group or the general population on an individual or grouplevel Whenusing normative data it is important to note that these canvary considerably between countries [] Focusing oncountryspecific values allows for more precise interpretation which becomes even more accurate when regressionmodels that take age and sex distributions into account areused to generate PRO predictionsThe EORTC Quality of Life Group conducted a largescale international panel study that collected normativedata for the EORTC QLQC30 for European countriesCanada and the United States [] While the EORTCQLQC30 has widely been used in studies in Austria eg[] no normative studies have so far been publishedIn the current study we present age and sexspecific normative data for the health domains of the EORTCQLQC30 from the Austrian general populationInaddition we provide a regression model defined by agesex and the presence of health conditions to calculate normative data for specific groupsMethodsSamplingAs part of an international study conducted by Nolte [] data from the Austrian general populationwere collected by GfK SE wwwgfkcom apanel research company which contacted panel members who had voluntarily registered and agreed to participate in panelbased studies An equal number ofparticipants was recruited for each sex and age groupmalefemale ¥ yearsOnce a quota was met for a specific group the recruitment for this group was stopped Response rates topanel studies conducted by GfK are between and as participants are registered voluntarily and usually participate when contactedEORTC QLQC30The EORTC QLQC30 consists of items covering fivefunctioning scales physical social emotional role andcognitive nine symptom scales fatigue nauseavomiting pain dyspnea sleep disturbances appetite loss constipation diarrhea and financial impact and a globalhealth status scale [] Referring to a recall period ofoneweek except for physical function which does notrefer to a recall period at all patients indicate their answers on a 4point Likert scale Linear converted scalescores range from to Higher scores on the functioning scales and on the global health status scale indicate better functioning whereas higher scores on thesymptom scales indicate greater symptom burden fordetails on the scoring and scale structure see [] TheEORTC QLQC30 has been validated in a large European samples and has been widely used in Germanspeaking patients with cancer and the general populationin Germany [ ]Statistical analysisWeights were established following Nolte [] andwere based on official population distribution statisticspublished by the United Nations [] We weighted theresponses to correct for over or underrepresentation ofsex and agestratified subgroups [] We report normative values as means and standard deviations for each subgroup In addition we established a regression model topredict EORTC QLQC30 scores with the following independent predictors sex age age2 a twoway interactionage by sex and the presence of health conditions none orat least one health condition We chose the predictors asthey are linked to HRQoL and have been applied in previous studies [] The basic model can be expressed asfollows Intercept Sex male female Age Age2 Interaction of Age and Sex age sex Presence ofHealth Conditions none at least one 0cLehmann Health and Quality of Life Outcomes Page of ResultsSampleIn April and May we collected online survey datafrom individuals from the Austrian general population with an equal distribution of participants in the predefined age and sex groups Participants were on average years old years when weighted and ofparticipants when weighted reported at least onehealth condition Some participants reported more thanone health condition when weighted reported two and when weighted reportedthree or more health conditions The most frequently reported health conditions were chronic pain when weightedand arthritis whenweighted Regarding relationship status the percentageof respondents in our sample who said that they were ina longterm relationship was and whenweighted The majority of respondents or when weighted reported at least some postcompulsoryeducation and or when weighted obtained auniversity degree ie bachelors or higher The full descriptive data are reported in Table Normative data for the EORTC QLQC30Table shows a summary of mean scores and standarddeviations for the EORTC QLQC30 across all analyzedsexage groups Men and women differed in several domains the three largest differences were observed forthe scales measuring insomnia points for womenvs points for men Cohens d fatigue points for women vs points for men Cohens d and emotional functioning points for womenvs points for men Cohens d The three largest differences between age groups wereobserved for the scales measuring dyspnea pointsfor those aged years vs points for those aged¥ years Cohens d pain points for thoseaged years vs points for those aged ¥years Cohens d and insomnia points forthose aged vs points for those aged years Cohens d See Table for the normativedata stratified by age groupFor functioning scales ceiling effects ie achieving themaximum score were lowest for emotional functioning n and most prevalent for social functioning n Floor effects ie achieving theminimum score were observed most frequently in nauseavomiting n and least common for fatigue n Regression model predicting EORTC QLQC30 scoresTable shows the regression model predicting individual EORTC QLQC30 scores using the weighted normative data This model predicts EORTC QLQC30 scoresusing the individuals sex age and presence of healthconditions An easy to use spreadsheet for the calculation of predicted values for individuals and groups isavailable online see Supplementary For example fora woman aged years with at least one health condition the predicted physical functioning score is calculated as follows Intercept Sex Age Age2 Interactionof Age and Sex Presence of HealthConditions DiscussionIn this study we present age and sexspecific normativedata for the EORTC QLQC30 from a sample of the Austrian general population Men and women differed in several domains most notably insomniafatigue andemotional functioning In general men reported a higherfunctioning except for social functioning and less symptom burden except for diarrhea than women did Olderparticipants ¥ years reported higher symptom burdeneg pain and dyspnea and lower physical and role functioning compared to younger participants We observedboth ceiling effects for functioning scales and floor effects for symptom scales for some scales of the EORTCQLQC30 However floor and ceiling effects were not unexpected considering that we administered a cancerspecific questionnaire to a general population sampleA potentiallimitation of panel studies is that thismethod of data collection may be prone to underrepresenting specific groups of individuals eg those whoare older or less educated Therefore it should be considered whether the assessment of general population datacollected via online surveys are truly representative Acomparison of our data with Statistics Austrias report suggests that the data obtained from the online survey are representative in terms of most basic individualcharacteristics age sex and marital status [] For example regarding the relationship status in our sample of participants stated to be in a longterm relationship Statistic Austria reported a similar rate with ofAustrian adults being in a longterm relationship The unemployment rate of the Austrian sample was whilethe unemployment rate in the report of the StatisticAustria institute was for individuals older than years Furthermore the prevalence rates for commonhealth conditions found in our data match other data onthe Austrian population The prevalence of selfreportedchronic pain in our sample is close to the prevalence of chronic pain in Austria [] as diagnosed by adoctor which is for chronic back pain and for chronic neck pain The percentage of participants withdiabetes in our sample is in line with the prevalence rate of diagnosed diabetes among adults estimated in the latest Austrian diabetes report published by 0cLehmann Health and Quality of Life Outcomes Page of Table Sample characteristics N Sex N Age yearsFemaleMaleM SDMedian IQREducationa N Less than compulsory no or some primary educationCompulsory about years of schoolingSome postcompulsory above years of schooling withoutreaching university entrance certificatePostcompulsory below universityCollege bachelors or equivalent levelUniversity degree Master Doc or equivalentPrefer not to answer aMarital statusa N SingleEmployment statusa N Health statusab N Married or in a steady relationshipSeparated divorced widowedPrefer not to answer aEmployed full timeEmployed part timeHomemakerStudentUnemployedRetiredSelfemployedOtherPrefer not to answer aNo health conditionAt least one health conditionChronic painHeart diseaseCancerDepressionCOPDArthritisDiabetesAsthmaAnxiety disorderObesityDrugalcohol use disorderOtherPrefer not to answerMissingM mean SD standard deviation IQR interquartile rangea For calculating percentages the category prefer not to answer was treated as missing datab The sum of all health conditions is larger than the sample size as respondents could choose multipleUnweighted data Weighted data 0cLehmann Health and Quality of Life Outcomes Page of Table EORTC QLQC30 normative data for the Austrian general population stratified by age and sex N Physical functioningM TotalFemalesAll ¥MalesAll ¥SD Role functioningM SD Emotional functioning M SD Cognitive functioning M SD Social functioningM SD Global healthQoLM FatigueNauseavomitingPainDyspneaInsomniaSD M SD M SD M SD M SD M SD Appetite lossM SD ConstipationM SD DiarrheaM SD Financial difficultiesM M Mean scores SD standard deviation QoL quality of lifeSD the Austrian Health Ministry [] For cancer the prevalence rate was which is only slightly lower than the prevalence rate found in the latest Statistics Austriacancer report [] Notable differences of our data to theStatistics Austria data [] were observed only regardingthe level of education More than onethird of individualstaking part in the online survey reported at least auniversitylevel education while in the Statistic Austriadata only of the sample report a university or comparable degree In our publication of international normative data for the EORTC CAT CORE based on theinternational dataset the relationship between educationand HRQoL scales was investigated in depth [] Highereducation some postcompulsory vsless than postcompulsory education was linked to more favorableHRQoL scores However differences were of low practicalrelevance as indicated by the small effect sizes alleta2 ¤ A strength of our study is the consistent data collectionmode used by Nolte [] which allows comparisonwith other European normative data Comparisons ofHRQoL ratings across country borders can be made providing insight into international differences [ ] Atthe nationaltwo differentstudy offerslevel our 0cLehmann Health and Quality of Life Outcomes Page of Table EORTC QLQC30 normative data for the Austrian general population stratified by age N Physical functioningMTotalRole functioningEmotional functioningCognitive functioningSocial functioningGlobal healthQoLFatigueNauseavomitingPainDyspneaInsomniaAppetite lossConstipationDiarrheaFinancial difficultiesSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMM Mean scores SD standard deviation QoL quality of lifeSD¥approaches for interpreting EORTC QLQC30 data Firstthe normative data allow interpretation and comparisonfor different age and sex groups Second the regressionmodel permits the calculation of expected EORTC QLQC30 normative scores using sex age and the presence ofhealth conditions This regression model can be used to generate more precise predictions than those made throughcomparing different age and sex groups using normative dataCompared with other score interpretation approachesnormative data in particular when relying on regressionmodels offer the advantage of not reducing the amount ofinformation For examplein an alternative approachthresholds for clinical importance are used to condense theinformation into severity categories eg clinically important vs not clinically important which can ease interpretation but also decreases the amount ofinformationconveyed The data we present in this study can be used tocompare HRQoL among patients with cancer and HRQoLin the general population matched by sex and age groupSuch comparisons can be useful at any stage of the cancerjourney as patients HRQoL is likely to be compromised atthe time of diagnosis [] and they can be used to determine whether cancer survivors return to population levelsor whether problems and impairments persist [ ]Ideally such comparisons use countryspecific normativedata which most accurately reflect the average level of 0cLehmann Health and Quality of Life Outcomes Page of Table Regression model for predicting EORTC QLQC30 scores using age sex and the presence of health conditionsPhysical functioningRole functioningEmotional functioningCognitive functioningSocial functioningGlobal healthQoLFatigueNauseavomitingPainDyspneaInsomniaAppetite lossConstipationDiarrheaFinancial difficultiesInterceptSexAge in yearsAge in years squared Agebysex Health conditions yesnoCoding is sex male female age in years above age by sex age in yearssex health conditions no health conditions at least onehealth condition QoL quality of lifeHRQoL within a particular national context Of coursenormative data need a certain amount of currentness to berelevant For the years to come this publication can serveas a reference but data should be updated in due timeSeveral observed results merit in depth discussionThe sex differences observed across various domains especially physical functioning are in line with other normative studies on the EORTC QLQC30 that havereported differences by sex In studies in Germany [] Slovenia [] Denmark [] Sweden [] and theNetherlands [] men tended to report higher functioningand lower symptom burden on most scales than womendid A similar pattern can be observed in the presentedAustrian normative data though both point differencesand effect sizes were rather small Our data allow for better interpretation of these differences among patients withcancer through the comparison of the magnitude of impairment in both groups of individuals patients with cancer and members of the Austrian general population Ashas been discussed previously [] sex differences amongpatients with cancer do not necessarily reflect a sexspecific impact of disease or treatment rather these differences may reflect more general factors including sexspecific response styles that also affect the general population as we observed in the present sampleRegarding age a European sample [] as well assinglecountry studies in Denmark Sweden and Slovenia[ ] report similar deterioration with lower physical and role functioning in older people ¥ or ¥ years compared to younger people or years asfound in our sample Those studies also found emotionalfunctioning to be independent of age [ ] or to evenincrease with age ¥ years [ ] showing a similarpattern of emotional functioning and age as observed inour sample However our sample differed from two studysamples in Germany which did not show an increase inemotional functioning with age and reported much higherdisparity between age groups on the global health statusscale with a difference of up to points oldest vs youngest group in favor of younger people [ ]Compared with the European sample reported byNolte [] fewer respondents in our sample reported health conditions or diseases in the European sample vs in our sample reported having nohealth conditions and our sample showed higher functioning and lower symptom burden on most scales According to the EU Statistics on Income and LivingConditions the Austrian life expectancy is slightly abovethe EU average years vs years and ofthe Austrian adult population report a good or verygood perceived health which ranks higher than mostother European countries measured [] Both of thesefindings support our result of generally high HRQoL interms of fewer health conditions and high functioningamong the Austrian general populationConclusionsThe normative data for the EORTC QLQC30 generatedin this study will ease and foster a more meaningful interpretation of scores obtained from patients with cancer 0cLehmann Health and Quality of Life Outcomes Page of and cancer survivors allowing comparisons of patientlevel and grouplevel data with the sex and agematched general population sample from Austria Usingour regression model precise predictions for individualsbased on sex age and presence of health conditions canbe generatedSupplementary informationSupplementary information accompanies this paper at doi101186s12955020015248Additional file AbbreviationsEORTC European anisation for Research and Treatment of Cancer GfKSE Gesellschaft für Konsumforschung Societas Europaea HRQoL HealthRelated Quality of Life PRO PatientReported OutcomesAcknowledgementsThe authors thank the European anization for Research and Treatment ofCancer for permission to use the data from an EORTC study grant number for this research We thank Jennifer Barrett PhD from EdanzGroup enauthorservicesedanzgroupcom for languageediting adraft of this manuscriptAuthors contributionsJL analyzed the data interpreted the data and wrote the manuscript JMGinterpreted the data and helped to draft the manuscript LMW and MSinterpreted the data and edited the manuscript SN GL and MR generatedthe data and edited the manuscript BH analyzed and interpreted the dataassisted in generating the data and edited the manuscript All authors readand approved the final manuscriptFundingThis research was partly funded by the European anisation for Researchand Treatment of Cancer Quality of Life Group grant number Availability of data and materialsThe datasets analyzed in the study at hand are available upon reasonablerequest from the EORTC Please use the Data Sharing form available throughthe EORTC website wwweortcdatasharingEthics approval and consent to participateNo ethics approval was sought as the study is based on panel dataAccording to the NHS Health Research Authority and the EuropeanPharmaceutical Market Research Association EphMRA panel research doesnot require ethical approval if ethical guidelines are followed The survey wasdistributed via the GfK SE member of EphMRA and obtained informedconsent by each participant before the study All data were collectedanonymously and identification of the respondents through the authors isimpossibleConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1University Hospital of Psychiatry II Medical University of InnsbruckAnichstrasse Innsbruck Austria 2Division of PsychosomaticMedicine Medical Department Charité Universitätsmedizin BerlinCorporate Member of Freie Universität Berlin HumboldtUniversität uu Berlinand Berlin Institute of Health Berlin Germany 3School of Health and SocialDevelopment Population Health Strategic Research Centre DeakinUniversity Burwood VIC AustraliaReceived June Accepted July ReferencesBlazeby JM Avery K Sprangers M Pikhart H Fayers P Donovan J Healthrelated quality of life measurement in randomized clinical trials in surgicaloncology J Clin Oncol Cella D Grunwald V Nathan P Doan J Dastani H Taylor F Bennett BDeRosa M Berry S Broglio K Quality of life in patients with advancedrenal cell carcinoma given nivolumab versus everolimus in CheckMate a randomised label phase trial Lancet Oncol LeBlanc TW Abernethy AP Patientreported outcomes in cancer care hearing the patient voice at greater volume Nat Rev Clin Oncol Austin E LeRouge C Hartzler AL Segal C Lavallee DC Capturing the patientvoice implementing patientreported outcomes across the health systemQual Life Res US Food and Drug Administration PatientReported Outcome Measures Usein Medical Product Development to Support Labeling Claims Guidance forIndustry wwwfdagovregulatoryinformationsearchfdaguidancedocumentspatientreportedoutcomemeasuresusemedicalproductdevelopmentsupportlabelingclaims Accessed Oct European Medicines Agencies Appendix to the guideline on theevaluation of anticancer medicinal products in man The use of patientreported outcome PRO measures in oncology studies wwwemaeuropaeudocumentsotherappendix2guidelineevaluationanticancermedicinalproductsman_enpdf Accessed Feb Wintner LM Sztankay M Aaronson N Bottomley A Giesinger JM GroenvoldM Petersen MA van de PollFranse L Velikova G Verdonckde Leeuw I The use of EORTC measures in daily clinical practicea synopsis of anewly developed manual Eur J Cancer Chan E K H Edwards TC Haywoods K Mikles S Newton L ImplementingPatientReported Outcome Measures in Clinical Practice A CompanionGuide to the ISOQOL Users Guide wwwisoqolwpcontentuploads201909ISOQOLCompanionGuideFINALpdf Accessed Mar Aaronson NK Ahmedzai S Bergman B Bullinger M Cull A Duez NJ FilibertiA Flechtner H Fleishman SB de Haes JC The European anization forResearch and Treatment of Cancer QLQC30 a qualityoflife instrument foruse in international clinical trials in oncology J Natl Cancer Inst Smith AB Cocks K Parry D Taylor M Reporting of healthrelated quality oflife HRQOL data in oncology trials a comparison of the Europeananization for Research and Treatment of Cancer quality of life EORTCQLQC30 and the functional assessment of Cancer therapygeneral FACTG Qual Life Res Howell D Molloy S Wilkinson K Green E Orchard K Wang K Liberty JPatientreported outcomes in routine cancer clinical practice a scopingreview of use impact on health outcomes and implementation factorsAnn Oncol Gnanasakthy A Barrett A Evans E D'Alessio D Romano CD A review ofpatientreported outcomes labeling for oncology drugs approved by theFDA and the EMA Value Health Giesinger JM Blazeby J Aaronson NK Sprangers M Fayers P Sparano FRees J Anota A Wan C Pezold M Differences in patientreportedoutcomes that are most frequently detected in randomized controlled trialsin patients with solid tumors a pooled analysis of trials Value Health Musoro ZJ Hamel JF Ediebah DE Cocks K King MT Groenvold M SprangersMAG Brandberg Y Velikova G Maringwa J Establishing anchorbasedminimally important differences MID with the EORTC qualityoflife measuresa metaanalysis protocol BMJ 20188e019117 Giesinger JM Loth FLC Aaronson NK Arraras JI Caocci G Efficace FGroenvold M van Leeuwen M Petersen MA Ramage J Thresholds forclinical importance were established to improve interpretation of theEORTC QLQC30 in clinical practice and research J Clin Epidemiol Janssen MF Szende A Cabases J RamosGoni JM Vilagut G Konig HHPopulation norms for the EQ5D3L a crosscountry analysis of populationsurveys for countries Eur J Health Econ Fayers PM Interpreting quality of life data populationbased reference datafor the EORTC QLQC30 Eur J Cancer 0cLehmann Health and Quality of Life Outcomes Page of consecutive annual assessments of approximately representativeDutch men and women Acta Oncol Giesinger J Kemmler G Mueller V Zabernigg A Mayrbaeurl B Thaler JOfner D Pegger C Rumpold G Weber B Are genderassociateddifferences in quality of life in colorectal cancer patients diseasespecificQual Life Res Eurostat EU Statistics on Income and Living Conditions EU SILC eceuropaeueurostatdatabrowserviewsdg_03_20defaultmaplangen Accessed Mar Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsSchwarz R Hinz A Reference data for the quality of life questionnaire EORTCQLQC30 in the general German population Eur J Cancer Nolte S Liegl G Petersen MA Aaronson NK Costantini A Fayers PMGroenvold M Holzner B Johnson CD Kemmler G General populationnormative data for the EORTC QLQC30 healthrelated quality of lifequestionnaire based on persons across European countriesCanada and the unites states Eur J Cancer Loth FL Holzner B Sztankay M Bliem HR Raoufi S Rumpold G GiesingerJM Cancer patients' understanding of longitudinal EORTC QLQC30 scorespresented as bar charts Patient Educ Couns Giesinger JM Kuijpers W Young T Tomaszewski KA Friend E Zabernigg AHolzner B Aaronson NK Thresholds for clinical importance for four keydomains of the EORTC QLQC30 physical functioning emotionalfunctioning fatigue and pain Health Qual Life Outcomes Kuijpers W Giesinger JM Zabernigg A Young T Friend E Tomaszewska IMAaronson NK Holzner B Patients' and health professionals' understandingof and preferences for graphical presentation styles for individuallevelEORTC QLQC30 scores Qual Life Res EORTC Quality of Life Group EORTC QLQC30 scoring manual wwweortcappuploadssites2201802SCmanualpdf Accessed July Hinz A Singer S Brahler E European reference values for the quality of lifequestionnaire EORTC QLQC30 results of a German investigation and asummarizing analysis of six European general population normative studiesActa Oncol United Nations Department of Economic and Social Affairs PopulationDivision World population Prospects the revision DVD Edition Liegl G Petersen MA Groenvold M Aaronson NK Costantini A Fayers PMHolzner B Johnson CD Kemmler G Tomaszewski KA Establishing theEuropean norm for the healthrelated quality of life domains of thecomputeradaptive test EORTC CAT Core Eur J Cancer Hjermstad MJ Fayers PM Bjordal K Kaasa S Using reference data on qualityof lifethe importance of adjusting for age and gender exemplified by theEORTC QLQC30 Eur J Cancer Quinten C Coens C Ghislain I Zikos E Sprangers MA Ringash J Martinelli FEdiebah DE Maringwa J Reeve BB The effects of age on healthrelated quality of life in cancer populations a pooled analysis ofrandomized controlled trials using the European anisation for Researchand Treatment of Cancer EORTC QLQC30 involving cancer patientsEur J Cancer Husson O Haak HR Buffart LM Nieuwlaat WA Oranje WA Mols F KuijpensJL Coebergh JW van de PollFranse LV Healthrelated quality of life anddisease specific symptoms in longterm thyroid cancer survivors a studyfrom the populationbased PROFILES registry Acta Oncol Statistik Austria Statistiken Arbeitsmarkt wwwstatistikatweb_destatistikenindexhtml Accessed Mar Bundesministerium für Gesundheit Zivilisationskrankheit DiabetesAusprägungen Lösungsansätze Herausforderungen ÃsterreichischerDiabetesbericht wwwsozialministeriumatdamjcr630d810ce02b4c62a2d95913ae0314edDiabetesbericht202017pdfAccessed Mar Statistik Austria Krebserkrankungen in Ãsterr
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case of a 14year old boy with tumorassociated refractory epilepsy Positron emission tomography imaging demonstrated a region with heterogeneous high 11Cmethionine uptake and a region with homogenous low 18Ffluorodeoxyglucose uptake within the tumor Histopathological and genomic analyses confirmed the tumor as BRAF V600Emutated polymorphous lowgrade neuroepithelial tumor of the young PLNTY Within the highmethionineuptake region we observed increased protein levels of Ltype amino acid transporter LAT1 a major transporter of methionine cMyc and constituents of the mitogenactivated protein kinase MAPK pathway We also found that LAT1 expression was linked to the BRAF V600E mutation and subsequent activation of MAPK signaling and cMyc Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 expression in BRAF V600Emutated PLNTY and glioblastoma cells The BRAF inhibitor dabrafenib moderately suppressed cell viability in PLNTY Collectively our results indicate that BRAF V600E mutationactivated MAPK signaling and downstream cMyc induces specific metabolic alterations in PLNTY and may represent an attractive target in the treatment of the diseaseKeywords PLNTY BRAF V600E mutation Methionine PET LAT1IntroductionPediatric lowgrade neuroepithelial tumors PLGNTs encompass a group of central nervous system neoplasms that longterm epilepsyassociated tumors LEATs such as ganglioglioma and dysembryoplastic neuroepithelial tumor DNT PLGNTs have different characteristics than their adult counterparts and includes Correspondence ktate12yokohamacuacjp Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama JapanFull list of author information is available at the end of the are commonly driven by genomic alterations in the Rasmitogenactivated protein kinase MAPK pathway such as mutations in BRAF and NF1 [ ] Recent largescale genomic studies and genomewide methylation analyses allowed a thorough characterization of PLGNTs [] and cIMPACTNOW the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy currently classifies PLGNTs as distinct disease entities [ ] In Huse et a0al described ten cases of polymorphous lowgrade neuroepithelial tumor of the young PLNTY which were histologically characterized by oligodendrogliomalike cellular components with intense CD34 immunopositivity According to previous publications PLNTYs are indolent tumors The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cTateishi a0et a0al acta neuropathol commun Page of that generally exhibit a benign clinical course and harbor either a BRAF V600E mutation or FGFR2FGFR3 fusion [] Based on its histological and genomic profiles cIMPACTNOW Update recommends PLNTY as a possible future classification for pediatrictype glialglioneuronal tumors However because of their rare etiology only a few PLNTYs have been described to date [ ] and it is unclear how genomic alterations promote the pathogenesis of the disease Herein we present a case of PLNTY with unique metabolic imaging features Using positron emission tomography PET we found regions of heterogeneous high 11Cmethionine uptake and homogenous low 18Ffluorodeoxyglucose FDG uptake within the tumor Activation of the MAPK pathway cMyc and expression of Ltype amino acid transporter LAT1 were increased in the highmethionineuptake area compared with the surrounding cortex lowmethionineuptake Glycolytic metabolites were expressed only weakly in tumor cells Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 and inhibited tumor cell viability suggesting that MAPK signaling and downstream cMyc activates methionine metabolism and inhibition of this pathway induces therapeutic vulnerability in PLNTYMaterials and a0methodsCell viability analysisAM38 and normal human astrocytes was purchased from JCRB Cell Bank and ScienCell Research Laboratories respectively Tumorsphere lines were cultured in serumfree neural stem cell medium as previously described [] Normal human astrocytes were cultured with astrocyte medium ScienCell To assess cell viability primary cultured cells were dissociated into single cells and seeded into 96well plates at a density of cellswell After a0h dabrafenib Selleck and trametinib Selleck were serially diluted and added to the wells Cell viability was measured using the CellTiterGlo Promega assay at day and the results were indicated as viability of the DMSO controlshRNA cell line generationTo knockdown BRAF 293T cells were transfected with lentiviral vector packaging plasmid DNA containing a0 μg of Human BRAF shRNA TRCN0000381693 GP and a0μg of a0pVSVgRev a0with Lipofectamine¢ TRCN0000196844 Sigma Aldrich a0μg of a0pHIVThermo Fisher Scientific YMG62 and AM38 cells were infected with lentivirus in polybrene a0μgmL for a0h Two days later the cells were selected with puromycin a0μgmL for a0days and used for experiments GIPZ nonsilencing lentiviral shRNA Control RHS4348 Horizon Discovery was used as a nonsilencing NS controlImmunohistochemistryTumor tissue specimens were fixed in neutral buffered formalin and embedded in paraffin Hematoxylin and eosin staining was performed using standard procedures For immunohistochemical analysis 5µmthick sections were deparaffinized treated with H2O2 in methanol rehydrated and heated for a0min for antigen retrieval After blocking with serum tissue sections were incubated with primary antibodies against CD34 Novus Biologicals LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology phosphoERK Bethyl Laboratories and cMyc Cell Signaling Technology at a0°C overnight The next day sections were washed with PBS incubated with biotinylated secondary antibody for a0 min at room temperature and then incubated with ABC solution PK6101 PK6102 Vector laboratories for a0 min at room temperature Finally the sections were incubated with DAB Dako and counterstained with hematoxylinWestern blottingcOmplete¢ Mini EDTAfree Protease Inhibitor Cocktail Cells were lysed in RIPA buffer SigmaAldrich with a Roche Fifty micrograms of protein was separated by SDSPAGE gel and transferred to polyvinylidene difluoride membranes Millipore by electroblotting After blocking with or nonfat dry milk in TBST a0mM Tris [pH ] a0 mM NaCl Tween20 membranes were incubated at a0 °C overnight with primary antibodies After washing and incubation with horseradish peroxidaseconjugated secondary antibodies Cell Signaling Technology blots were washed and signals were visualized with chemiluminescent HRP substrate Millipore Primary antibodies against BRAF Gene Tex cMyc Cell Signaling Technology GAPDH Gene Tex LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology a0phosphoERK Bethyl Laboratories and Vinculin Novus Biologicals were used for western blottingCase presentationThis study was performed in accordance with declaration of Helsinki and was approved by the Institutional Review Board Yokohama City University [YCU Yokohama Japan] IRB numbers A1711300006 and B190600002 Written informed consent was obtained from the patient and parents A 14year old boy presented with chronic medial temporal lobe epilepsy for a year Magnetic resonance imaging MRI indicated 0cTateishi a0et a0al acta neuropathol commun Page of See figure on next pageFig Characteristics of a patient with PLNTY a T2weighted left T1weighted middle and contrastenhanced right MR images b Computed tomography CT left 18FfluorodeoxyglucosePETCT middle and 11CmethioninePETCT right images c Video electroencephalography indicating ictal onset in the left temporal lobe with spread to the contralateral temporal lobe d PETCT and MRI merged intraoperative navigation image left and surgical image right showing the highmethionineuptake region and surrounding abnormal lesion on MRIhypointensity on T2weighted images and hyperintensity on T1weighted images with a cystic component in the left temporal lobe Contrastenhanced MRI showed no significant enhancement in the lesion Fig a01a while computed tomography revealed heavy calcification FDGPET showed lower FDG uptake in the tumor while 11CmethioninePET demonstrated increased methionine uptake in the same lesion SUVmax tumornormal tissue ratio Fig a01b Videoelectroencephalographic EEG monitoring indicated ictal onset in the left temporal lobe with subsequent spread to the contralateral temporal lobe Fig a01c We speculated that this abnormal lesion was a LEAT Since we considered this tumor to be completely resectable the patient underwent craniotomy and resection of the neoplasm including the highmethionineuptake region Fig a01d To achieve epileptic control electrocorticography was performed intraoperatively After removal of the highmethionineuptake and T2 hyperintense lesions the surrounding tissue was resected until interictal epileptiform discharge could no longer be detected by electrocorticography The patient became epilepsyfree after lesion removal and MRI indicated complete remission a0months after the surgeryTissue samples of the highmethionineuptake region and surrounding cortex low methionine uptake were collected Hematoxylin and eosin staining indicated diffusely infiltrating growth patterns and presence of oligodendroglialike cellular components Fig a02a Astrocytic and highgrade features were absent with a Ki67 index of less than Chicken wirelike branching capillaries and microcalcification were also found in region Despite lower cellularity oligodendroglialike cells were present in the surrounding tissue Immunohistochemistry revealed extensive CD34 expression and peripherally associated ramified neural elements in the tumor cells Fig a0 2a Targeted DNA sequencing identified a BRAF V600E mutation in the tumor without recurrent mutations in IDH1 IDH2 TERT promoter FGFR1 H3F3A or HIST3H1B Fig a0 2b Chromosome 1p19q codeletion was absent Fig a02c The above histological and genetic features fulfilled the diagnostic criteria for PLNTYTo assess the mechanisms underlying the methionineFDG uptake mismatch indicated by PET we compared the expression of LAT1 glucose transporter GLUT and hexokinase2 HK2 between tissue regions and Notably LAT1 which is a major methionine transporter was more highly expressed in than in Fig a0 3a In contrast GLUT1 and HK2 which is correlated with FDG uptake and lactate dehydrogenase A LDHA expression were weak in either region Additional file a0 Fig a0 S1 LAT1 expression is mediated by cMyc activation and BRAF V600E mutation activates the MAPK pathway and downstream cMyc [ ] Therefore we hypothesized that BRAF V600E mutation promotes LAT1 expression through MAPK signaling and consequent cMyc activation a0 in PLNTY Levels of phosphoMEK phosphoERK and cMyc were higher in tissue region than in Fig a03a suggesting activation of the MAPK pathway and cMyc within the highmethionineuptake lesion To verify whether the BRAF V600E mutation can induce the expression of LAT1 we exposed primary cultured YMG83 PLNTY cells to a BRAF inhibitor dabrafenib As expected the expression of phosphoMEK phosphoERK cMyc and LAT1 was suppressed after dabrafenib treatment in YMG83 cells Fig a0 3b Notably BRAF inhibitor dabrafenibtreated YMG83 cells had lower cell viability compared to normal human astrocytes Fig a03c To confirm the reproducibility of these molecular features we used patientderived YMG62 cells epithelioid glioblastoma with the BRAF V600E mutation which exhibited high 11Cmethionine uptake by PET imaging Additional file a0 Fig a0 S2 and AM38 glioblastoma cells BRAF V600E mutant We found that dabrafenib and a MEK inhibitor trametinib inhibited the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03d and 3e Similarly BRAF knockdown suppressed the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03f Collectively these findings indicated that activation of the MAPK pathway by the BRAF V600E mutation deregulates cMyc and promotes LAT1 expression This oncogenic signaling pathway increases methionine metabolism and tumor maintenance in PLNTYDiscussionThirty cases of PLNTY have been described to date with the first ten reported by Huse et a0al in [ ] BRAF V600E mutation was seen in of the patients and BRAF fusion in patient These BRAF alterations were mutually exclusive with other genomic events including FGFR3TACC3 fusion FGFR3 amplification FGFR2CTNNA3 fusion FGFR2INA fusion 0cTateishi a0et a0al acta neuropathol commun Page of 0cTateishi a0et a0al acta neuropathol commun Page of Fig Histopathologic and genomic features of a patient with PLNTY a Hematoxylin and eosin HE staining top and CD34 immunohistochemistry bottom in the highmethionineuptake and lowmethionineuptake region within tumor tissue Bars μm b Sanger sequencing for detection of BRAF V600E arrow left and IDH1 R132H arrow right mutations c Fluorescence in situ hybridization for detection of 1p311q25 left and 19q1319p13 right chromosomal deletionsFGFR2 KIAA1598 fusion FGFR2 rearrangement and NTRK2 disruption suggesting that the vast majority of PLNTYs are induced by BRAF mutation or FGFR fusion and subsequent MAPK activation Therefore targeting MAPK signaling may become a potential therapeutic strategy in PLNTY Indeed BRAF V600Emutated PLNTY cells were relatively vulnerable to dabrafenib and trametinib in the present study Thus targeted molecular therapy for the MAPK pathway may be particularly useful in PLNTY located in surgically unresectable regions In addition Koh et a0 al reported that the BRAF V600E mutation contributes to the intrinsic epileptogenicity in pediatric brain tumors and that inhibition of BRAF suppressed epileptic seizures [] Thus BRAFMEK inhibitors could exert antiepileptic as well as antitumor effects in PLNTYPET imaging revealed a region with increased methionine uptake and low FDG uptake within tumor tissue in our patient Consistent with this finding previous case reports demonstrated increased methionine uptake but only mild FDG uptake in patients with BRAF V600Emutated PLNTY [ ] Thus excessive 0cTateishi a0et a0al acta neuropathol commun Page of Fig Activating the MAPK pathway induces LAT1 expression in a patient with PLNTY a Immunohistochemistry of indicated proteins in the highmethionineuptake and lowmethionineuptake regions within tumor tissue Bars μm b Western blot analysis of phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG83 PLNTY left cells treated with DMSO and μM BRAF inhibitor BRAFi dabrafenib for h GAPDH loading control c Relative cell viability of dabrafenibtreated left and trametinibtreated right YMG83 cells and immortalized normal human astrocytes NHA P DMSO versus dabrafenib left and trametinib right d Western blot analysis for indicated proteins in YMG62 epithelioid glioblastoma left and AM38 glioblastoma right cells treated with DMSO μM BRAF inhibitor BRAFi dabrafenib and μM MEK inhibitor MEKi trametinib for h GAPDH loading control e Western blot analysis of BRAF phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG62 left and AM38 right cells treated with DMSO and dabrafenib at indicated concentrations Vinculin loading control f Western blot analysis for indicated proteins in nonsilencing NS and BRAF and transduced YMG62 and AM38 cells GAPDH loading controlmethionine uptake and low FDG uptake may be imaging features specific to PLNTY A preclinical study has demonstrated that high uptake of 18FFDG was correlated with increased Glut1 and HK2 expression in human cancers [] Although the diagnostic accuracy is insufficient FDGPET imaging is useful to differentiate highgrade from lowgrade gliomas [] In the present case low FDG uptake and weak expression of Glut1 HK2 and LDHA were observed in tumor tissue suggesting low glycolytic activity in PLNTY On the other hand due to a high signaltonoise ratio 11Cmethionine PET imaging is practical for brain tumors [ ] Several PET imaging studies have demonstrated that methionine uptake was higher in 0cTateishi a0et a0al acta neuropathol commun Page of highgrade adult gliomas than in lowergrade gliomas [ ] In epileptogenic brain tumors however all gangliogliomas and of DNT had increased methionine uptake although these tumors are classified as WHO grade I [ ] implying that methionine uptake may be irrespective of tumor grade in LEATsPrevious studies have reported that methionine uptake was correlated with LAT1 in gliomas [ ] LAT1 plays a major role in the transport of neutral essential amino acids including methionine and is driven by several cancerrelated genes such as MYC [] It has been demonstrated that cMyc which is partly mediated by the MAPK pathway regulates LAT1 expression and MEK inhibitor suppresses LAT1 SLC7A5 transcription [ ] thereby indicating a role of the MAPK pathway and cMyc in the regulation of LAT1 Since RASMAPK pathwayassociated genomic alterations are common in LEATs [] and that the BRAF V600E mutation has been identified in and of gangliogliomas and DNTs respectively [ ] there is a possibility that the BRAF V600E mutation and MAPK pathwayrelated genomic alterations may activate methionine metabolism in LEATs To investigate this hypothesis we evaluated the protein expression of LAT1 and the molecules that are involved in the MAPK pathway As expected levels of phosphoMEK phosphoERK cMyc and LAT1 were higher in the highmethionineuptake area than in the lowmethionineuptake area We also found that genetic andor pharmacological BRAF inhibition suppressed MAPK pathway activation and attenuated LAT1 expression in BRAF V600EmutatedPLNTY cells and glioblastoma cell lines These findings support the hypothesis that the BRAF V600E mutation may upregulate LAT1 and methionine metabolism through cMyc activation for cell survival In addition to LAT1 methionine uptake was correlated with microvascular density MVD in gliomas [] PLNTYs are considered benign brain neoplasms proposed as WHO grade I however in the present case a chicken wirelike MVD which is one of the histopathological characteristics of oligodendroglioma was also observed in the highmethionineuptake tissue region Intriguingly methionine uptake has been reported to be relatively higher in oligodendrogliomas than in astrocytomas [] Thus PLNTY which has an oligodendrogliomalike microvascular structure might show unique metabolic imaging features Further studies are warranted to validate this hypothesis Nonetheless our data indicated that the BRAF V600E mutation induced MAPK pathway activation and downstream cMyc promoted LAT1 expression and methionine metabolism with little effect on glycolytic pathway activation These findings may explain the unique metabolic imaging features of FDGmethionine mismatch in PLNTYSupplementary informationSupplementary information accompanies this paper at https doi101186s4047 Additional file a0 a0Figure S1 Low glycolysis activation in a patient with PLNTY Immunohistochemistry for glucose transporter hexokinase and lactate dehydrogenase A in the highmethionineuptake upper and lowmethionineuptake lower region within tumor tissue A Bars μm Figure S2 Images of the patients glioblastoma with the BRAF V600E mutation Contrastenhanced magnetic resonance left and 11Cmethionine positron emission tomography right images of the YMG62 patientAcknowledgementsWe thank Mrs Emi Hirata and Yasuko Tanaka YCU for technical and administrative assistance We also would like to thank Editage wwweditagecom for English language editingAuthors contributionsKT led the study collected samples designed experiments performed experiments interpreted data and wrote the manuscript JS TH and YM performed experiments NI HM provided tumor samples and associated clinical details TO RM and DU interrupted PET and MRI studies NU and SY performed the histological classification of tumor samples TY designed experiments and interpreted data All authors read and approved the final manuscriptFundingThis work was supported by GrantAid for Scientific Research 19K09488 Princess Takamatsu Cancer Research Fund Takeda Science Foundation SGH Cancer foundation Yokohama Foundation for Advancement of Medical Science and BristolMyers Squibb FoundationCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama Japan Department of Pathology Yokohama City University Hospital Yokohama Japan Department of Radiology Graduate School of Medicine Yokohama City University Yokohama Japan Departmento of Radiology Division of Nuclear Medicine National Center for Global Health and Medicine Tokyo Japan Received June Accepted August References Borbely K Nyary I Toth M Ericson K Gulyas B Optimization of semiquantification in metabolic PET studies with 18Ffluorodeoxyglucose and 11Cmethionine in the determination of malignancy of gliomas J Neurol Sci https doi101016jjns200602015 Chappe C Padovani L Scavarda D Forest F NanniMetellus I Loundou A Mercurio S Fina F Lena G Colin C et al Dysembryoplastic neuroepithelial tumors share with pleomorphic xanthoastrocytomas and gangliogliomas BRAFV600E mutation and expression Brain Pathol https doi101111bpa12048 Chen Y Tian T Guo X Zhang F Fan M Jin H Liu D Polymorphous lowgrade neuroepithelial tumor of the young case report and review focus on the radiological features and genetic alterations BMC Neurol https doi101186s1288 Ellison DW Hawkins C Jones DTW OnarThomas A Pfister SM Reifenberger G Louis DN cIMPACTNOW update diffuse gliomas 0cTateishi a0et a0al acta neuropathol commun Page of characterized by MYB MYBL1 or FGFR1 alterations or BRAFV600E mutation Acta Neuropathol https doi101007s0040 Gupta VR Giller C Kolhe R Forseen SE Sharma S Polymorphous lowgrade neuroepithelial tumor of the young a case report with genomic findings World Neurosurg https doi101016jwneu201908221 Hafliger P Graff J Rubin M Stooss A Dettmer MS Altmann KH Gertsch J Charles RP The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse model J Exp Clin Cancer Res https doi101186s1304 60180907z Hatakeyama T Kawai N Nishiyama Y Yamamoto Y Sasakawa Y Ichikawa T Tamiya T 11Cmethionine MET and 18Ffluorothymidine FLT PET in patients with newly diagnosed glioma Eur J Nucl Med Mol Imaging https doi101007s0025 Hayashi K Jutabha P Endou H Anzai N cMyc is crucial for the expression of LAT1 in MIA Paca2 human pancreatic cancer cells Oncol Rep https doi103892or20121878 Huse JT Snuderl M Jones DT Brathwaite CD Altman N Lavi E Saffery R SextonOates A Blumcke I Capper D et al Polymorphous lowgrade neuroepithelial tumor of the young PLNTY an epileptogenic neoplasm with oligodendrogliomalike components aberrant CD34 expression and genetic alterations involving the MAP kinase pathway Acta Neuropathol https doi101007s0040 Johnson DR Giannini C Jenkins RB Kim DK Kaufmann TJ Plenty of calcification imaging characterization of polymorphous lowgrade neuroepithelial tumor of the young Neuroradiology https doi101007s0023 y Kato T Shinoda J Oka N Miwa K Nakayama N Yano H Maruyama T Muragaki Y Iwama T Analysis of 11Cmethionine uptake in lowgrade gliomas and correlation with proliferative activity AJNR Am J Neuroradiol https doi103174ajnrA1242 Katsanos AH Alexiou GA Fotopoulos AD Jabbour P Kyritsis AP Sioka C Performance of 18FFDG 11Cmethionine and 18FFET PET for glioma grading a metaanalysis Clin Nucl Med https doi101097RLU00000 dysembryoplastic neuroepithelial tumors and other epileptogenic brain neoplasms with [11C]methionine PET Neuro Oncol https doi101093neuon cnou02 Riva G Cima L Villanova M Ghimenton C Sina S Riccioni L Munari G Fassan M Giangaspero F Eccher A Lowgrade neuroepithelial tumor unusual presentation in an adult without history of seizures Neuropathology https doi101111neup12504 Rosenberg DS Demarquay G Jouvet A Le Bars D Streichenberger N Sindou M Kopp N Mauguiere F Ryvlin P [11C]Methionine PET dysembryoplastic neuroepithelial tumours compared with other epileptogenic brain neoplasms J Neurol Neurosurg Psychiatry https doi101136jnnp200405160 Ryall S Tabori U Hawkins C Pediatric lowgrade glioma in the era of molecular diagnostics Acta Neuropathol Commun https doi101186s4047 z Ryall S Zapotocky M Fukuoka K Nobre L Guerreiro Stucklin A Bennett J Siddaway R Li C Pajovic S Arnoldo A et al Integrated molecular and clinical analysis of pediatric lowgrade gliomas Cancer Cell 583e565 https doi101016jccell Salisbury TB Arthur S The regulation and function of the ltype amino acid transporter LAT1 in cancer Int J Mol Sci https doi103390ijms1 Schindler G Capper D Meyer J Janzarik W Omran H HeroldMende C Schmieder K Wesseling P Mawrin C Hasselblatt M et al Analysis of BRAF V600E mutation in nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma ganglioglioma and extracerebellar pilocytic astrocytoma Acta Neuropathol https doi101007s0040 Sumdani H Shahbuddin Z Harper G Hamilton L Case report of rarely described polymorphous lowgrade neuroepithelial tumor of the young and comparison with oligodendroglioma World Neurosurg https doi101016jwneu201903181 Surrey LF Jain P Zhang B Straka J Zhao X Harding BN Resnick AC Storm PB Buccoliero AM Genitori L et al Genomic analysis of dysembryoplastic neuroepithelial tumor spectrum reveals a diversity of molecular alterations dysregulating the MAPK and PI3KmTOR pathways J Neuropathol Exp Neurol https doi101093jnennlz10 Kobayashi K Ohnishi A Promsuk J Shimizu S Kanai Y Shiokawa Y Nagane Tateishi K Nakamura T Yamamoto T Molecular genetics and M Enhanced tumor growth elicited by Ltype amino acid transporter in human malignant glioma cells Neurosurgery discussion https doi10122701neu00003 Koh HY Kim SH Jang J Kim H Han S Lim JS Son G Choi J Park BO Heo WD et al BRAF somatic mutation contributes to intrinsic epileptogenicity in pediatric brain tumors Nat Med https doi101038s4159 10180172x Kracht LW Friese M Herholz K Schroeder R Bauer B Jacobs A Heiss WD Methyl[11C] lmethionine uptake as measured by positron emission tomography correlates to microvessel density in patients with glioma Eur J Nucl Med Mol Imaging https doi101007s0025 Lelotte J Duprez T Raftopoulos C Michotte A Polymorphous lowgrade neuroepithelial tumor of the young case report of a newly described histopathological entity Acta Neurol Belg https doi101007s1376 Louis DN Wesseling P Aldape K Brat DJ Capper D Cree IA Eberhart C FigarellaBranger D Fouladi M Fuller GNet al cIMPACTNOW update new entity and diagnostic principle recommendations of the cIMPACTUtrecht meeting on future CNS tumor classification and grading Brain Pathol https doi101111bpa12832 Okubo S Zhen HN Kawai N Nishiyama Y Haba R Tamiya T Correlation of Lmethyl11Cmethionine MET uptake with Ltype amino acid transporter in human gliomas J Neurooncol https doi101007s1106 Ong LC Jin Y Song IC Yu S Zhang K Chow PK [18F]2deoxydglucose FDG uptake in human tumor cells is related to the expression of GLUT1 and hexokinase II Acta Radiol https doi10108002841 Rheims S Rubi S Bouvard S Bernard E Streichenberger N Guenot M Le Bars D Hammers A Ryvlin P Accuracy of distinguishing between therapeutic targets of pediatric lowgrade gliomas Brain Tumor Pathol https doi101007s1001 Tateishi K Tateishi U Nakanowatari S Ohtake M Minamimoto R Suenaga J Murata H Kubota K Inoue T Kawahara N 62Cudiacetylbis N4methylthiosemicarbazone PET in human gliomas comparative study with [18F]fluorodeoxyglucose and Lmethyl[11C]methionine PET AJNR Am J Neuroradiol https doi103174ajnrA3679 Wakimoto H Kesari S Farrell CJ Curry WT Jr Zaupa C Aghi M Kuroda T StemmerRachamimov A Shah K Liu TC et al Human glioblastomaderived cancer stem cells establishment of invasive glioma models and treatment with oncolytic herpes simplex virus vectors Cancer Res https doi10115800085472CAN083886 Yue M Jiang J Gao P Liu H Qing G Oncogenic MYC activates a feedforward regulatory loop promoting essential amino acid metabolism and tumorigenesis Cell Rep https doi101016jcelre p201712002 Zhang W Liu HT MAPK signal pathways in the regulation of cell proliferation in mammalian cells Cell Res https doi101038sjcr72901 Zhao C Zhang Y Wang J A metaanalysis on the diagnostic performance of 18FFDG and 11Cmethionine PET for differentiating brain tumors AJNR Am J Neuroradiol https doi103174ajnrA3718 Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c'
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"characterize the frequency and clinical features of lungnodules in IgG4 related disease IgG4RD patients as an insight for help with the diagnosis of lung nodulesMethods A retrospective study was carried out in West China Hospital Sichuan University from January toDecember patients with definite IgG4RD were enrolledResults Fifty of patients with definite IgG4RD had radiologically confirmed lung nodules of whom werediagnosed with definite IgG4 related lung disease Lung nodules detected in more than patients were small andsolid always with regular margins Multiple and bilateral distributions was also a major characteristicof these lung nodules Lobulation and speculation were simultaneously detected in patients including patientscombined with pleural indentation Calcification of nodules was detected in only one patient Thirtyseven patientsalso had additional radiological abnormalities of lungs including groundglass opacity thickening of pleura thickening of interlobular septa thickening of bronchial wall pleural effusion mass interstitial changes and mediastinal or hilar lymphadenopathy Most patients were treatedwith glucocorticoids alone or combined with immunosuppressive agents Sixteen patients received a reexamination by chest computed tomography CT scan after treatment of whom showed a decrease in the sizeandor the number of nodulesConclusions The incidence of lung nodules in IgG4RD patients can be high For an IgG4RD patient with lungnodules the possibility that the lung nodules related to IgG4RLD is high It is hard to differentiate IgG4 relatedlung nodules from other lung diseases in particular lung cancer Radiological characteristics and positive responsesto glucocorticoids and immunosuppressive agents can help with the differential diagnosis For these patientsregular followup is also importantKeywords Clinical characteristics IgG4 related disease IgG4 related lung disease Lung nodules RadiologicalcharacteristicsBackgroundClinical presentation of nodules in the lungs of patientscan be a challenge for diagnosis especially when thenodules are small and asymptomatic The nodules maybe difficult to biopsy for histopathology because of size Correspondence liuyihuaxiyiyuan126com Yan Xie and Anji Xiong contributed equally to this work1Department of Rheumatology and Immunology West China HospitalSichuan University Chengdu ChinaFull list of author information is available at the end of the and location for example very small nodules deeply imbedded in the lung The first diagnosis willlikely besome form of neoplasm When that diagnosis is determined to be incorrect many physicians especially respiratory physicians and thoracic surgeons may beunsure of the next step toward diagnosis and treatmentAffected patients of course will be anxious for a confirmed diagnosis since they will likely assume the smallnodules are an early stage of lung cancer A recently recognized diseaseimmunoglobulin G4related disease The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cXie BMC Pulmonary Medicine Page of IgG4RD must be considered for such presentation ofasymptomatic small nodules particularly in the lungsliver or lymph nodes []IgG4RD is a chronic fibrotic inflammation characterized by the involvement of multiple ans The mostcommon manifestations of the disease include swellingof salivary and lacrimal glands lymphadenopathy andtype autoimmune pancreatitis AIP Other anssuch as lung bronchi kidney retroperitoneum thyroidheart mesenterium meninges and skin can also be involved Frequently but not always serum IgG4 levelswill be elevated [] IgG4 is the rarest of the IgG subclasses generally has relatively low antigen affinity andis unable to bind complement component 1q C1q andactivate the complement cascade [] IgG4 can exchangeFab arms by swapping a heavy chain and attached lightchain with a heavylight chain pair from another IgG4molecule which providesthe antiinflammatory activity attributed to IgG4 antibodies [] Akey pathological feature of IgG4RD nodules is a denselymphoplasmacytic infiltrate anized into a storiformpattern which frequently forms a tumefactive mass thatmay destroy an involved an [] When the tumefactive mass occurs in the lungs it may present as a noduleor groundglass opacity on radiology [] IgG4relatedlung disease IgG4RLD is the lung involvement ofIgG4RD which was first described in in a patientwith interstitial pneumonia autoimmune pancreatitisand IgG4positive plasma cells in the interstitium []The IgG4RLD presentation can be heterogeneous andits radiologic manifestations are often extensive evenwhen clinically asymptomatic []the basisforAlthough lung nodules have been described in to of IgG4RLD in a few case reports and reviews todate [ ] no study has employed a systematic analysis of IgG4RD patients with lung nodules Here wepresent retrospective data of the clinical and radiologicalfeatures of IgG4RD patients from a single medical centers experience Our goal is to provide clinical information and insight for the diagnosis of IgG4RD in patientswho present with asymptomatic nonneoplastic smalllung nodulesMethodsPatients informationPatients with definitive IgG4RD were selected from alldepartments of West China Hospital Sichuan Universityfrom January to December The clinical serological and radiographic imaging characteristics andtreatment responses of the patients were analyzedOur study was performed according to the rules of thehospitals medical ethics committee Informed consentwas obtained in accordance with the institutionalguidelinesDiagnosis of IgG4RDThe diagnosis of IgG4RD was made according to thecomprehensive diagnostic criteria for IgG4RD publishedin by Umdehara [] The criteria for definiteprobable and possible IgG4RD are as followsDefinite1 Probable1 Possible1 Diffuse or localized swelling or masses in single ormultiple ans Elevated serum IgG4 mgdl Histopathology of biopsied nodules showsa Marked lymphocyte and plasmacyte infiltrationand fibrosisb Infiltration of IgG4 plasma cells defined as IgG4 plasma among all IgG plasma cells and IgG4 plasma cells per 40X fieldChest CT protocolsCT images were available for all patients involved in thisstudy Because of the retrospective nature of this studychest CT scanning protocols were varied The scanningwas performed on one of the six machines ranging from16detector to 128detector CT scanners Philips Medical Systems Best the Netherlands or Siemens MedicalSystems Erlangen Germany with patients in the supineposition For patients who underwent contrastenhancedexamination an intravenous nonionic contrast mediumIopamidol mgml ml was given and imaging started s later after injection To minimize motion artifacts CT images were acquired during a singlebreathhold The main parameters were shown as follows tube voltage 100120kv tube current mA slice thickness mm section interval mmDefinition of lung nodules and IgG4RLDCT images of patients chests were analyzed by radiologists The lung nodules were defined as rounded or irregular opacity well or poorly defined measuring up to cm in diameter [] Nodules were categorized aslarge or small as follows large cm diameter cmor small diameter cm If the diameter of the lung lesion is larger than cm in the CT images the lesionwas defined as a massIgG4RLD is defined as IgG4RD diagnosed as abovethat includes diffuse or localized swelling or masses inone or both lobes of the lungs with histopathology thatincludes marked lymphocyte and plasmacyte infiltrationand fibrosis andor infiltration of IgG4 plasma cells defined as IgG4 plasma among all IgG plasma cellsand IgG4 plasma cells per à field 0cXie BMC Pulmonary Medicine Page of StatisticsStatistical analyses were performed with GraphPadPrism version GraphPad Software Inc La Jolla CAUSA Descriptive data are reported as median interquartile range and frequencies are percentages Continuous variables without normal distribution wereexpressed as median and interquartile IQR For comparison between groups the unpaired Students ttestwas used for continuous variables and chisquare testwas used for categorical variables All statistical testswere 2sided and results with P values were considered statistically significantResultsFrom January to December a total of patients were diagnosed with definitive IgG4RD in WestChina Hospital Sichuan University of whomhad CTconfirmed lung nodules were included in thisretrospective studyPatient description and clinical presentationDemographic and clinical data for all patients are summarized in Table The median age of the patients withlung nodules was years at diagnosis with amale female ratio of Almost half of the patientshad their initial diagnosis of IgG4RD in the Departmentof Rheumatology The duration of symptoms beforediagnosis averaged months range monthsFortytwo of the patients had no pulmonary infectionor a history of cancer and other chronic pulmonary diseases And more than half of the patients had ahistory of smokingFirst symptomsFor patients with lung nodules the most common firstsymptoms were salivary gland swelling and lacrimal gland swelling Seven patients presented withcough as their first symptom There were several raresymptoms including fever gum swelling low back painand edemaExtrapulmonary ans involvedMost patients had multiple sites or ans involved median number IQR1 Twentyone patients had atleast three extrapulmonary ans or sites affected Themost common involved extrapulmonary ans includedsalivary gland lacrimalgland pancreas and kidney Other sites included liver nasal sinus peritoneum pituitaryskinthyroid glands bile ducts gingiva andpericardiumlymph node Serological characteristicsSerum levels of IgG4 erythrocyte sedimentation rateESR Creaction protein CRP and complements werequantified Serum IgG4 concentrations were measuredin all patients in this study with a median value of mgdl IQR426 for patients with lung nodulescompared to mgdl IQR371 for IgG4RD patients without lung nodules Table Elevated ESR andserum CRP levels were found in and patients respectively among patients with lung nodules versus patients with elevated ESR and patients with elevatedCRP among IgG4RD patients without lung nodulesTwentyfour patients with lung nodules versus without had reduced serum complement Twenty patientswith lung nodules versus without had antinuclearantibody ANA was defined as positive Therewere no statistically significant differences in serologicalmeasurements between IgG4RD patients with and without lung nodules Table Comparison of clinical characteristicsThe general clinical characteristics of patients with lungnodules were compared with those of patients withoutlung nodules As the results indicated in Table significant differences were observed in smoking history between the two groups while no significant differences inage gender ratio duration of symptoms before diagnosis and the number of extrapulmonary involved answere observedRadiological characteristics of the lungsFifty patients included in this study had lung nodules revealed in chest CT images The radiological characteristics of lungs among the patients with lung nodulesare summarized in Table and Table Five patients had large lung nodules Fig 1a with diameters ranging from to cm while almost all patients showed smalllung nodules Fig 1beMost patients had multiple nodules Only patients had single small nodules For the distribution oflung nodules patients had bilateral nodules accounting for more than half of the cases When consideringthe pulmonary lobe involvement the number of patientswith nodules located only in the upper lobe and middleor lower lobe was and respectively The rest patients had nodules randomly distributed in differentlobes at the same timeFor the lesion densities on CT solid nodules can bedetected in almost all patients Only patientsshowed groundglass nodules all of which were smallnodules Nodules in out of the patients showed bothgroundglass and solid patterns Fig 1d Fig 1e Mostpatients showed regular and welldefined nodulemargins Only patients had nodules with irregular or 0cXie BMC Pulmonary Medicine Page of Patients with lung nodulesn Patients without lung nodulesn Table Clinical characteristics of IgG4RD patientsParametersAge medianIQR yearsMenwomen n Smoking history n Duration of disease medianIQR monthsSerum IgG4 medianIQR mgdlElevated ESR n Elevated CRP n Reduced C3 n Reduced C4 n Positive ANA n First symptoms n Salivary gland swellingLymphadenopathyLacrimal gland swellingCoughJaundiceDysuriaFeverAbdominal painGum swellingLow back painEdemaFatigueDiarrheaNumber of extrapulmonary ans involved medianIQRExtrapulmonary an involvement n Salivary glandssubmandibular gland and parotid glandLymph nodeLacrimal glandPancreasKidneyLiverNasal sinusSkinBile ductsPeritoneumPituitaryThyroid glandsGingivaPericardiumBloodP valueData are expressed as medianinterqurtile range for contious variables and frequenciespercentages for categorical variablesIgG4RD IgG4 related disease ESR Erythrocyte sedimentation rate CRP Creactive protein C3 Complement C4 Complement ANA Antinuclear antibody IQRInterquartile range P 0cXie BMC Pulmonary Medicine Page of Table Radiological characteristics of lungs before drugtherapyPatients with indicated lung nodulesSizesinglemultipleSmall nodule onlyLarge nodule onlyBoth small and large noduleDistributionLateralityUnilateralBilateralLobeUpper lobeMiddle or lower lobeRandomTypeGroundglass nodule onlySolid nodule onlyBoth groundglass and solid noduleMarginIrregular or untidyRegularSecond associated featuresLobulationSpiculationPleural retractionCalcificationChanges of nodules after treatmentsn Smaller or disappearNo differenceOther radiological featuresGroundglass opacityThickening of pleuraThickening of interlobular septaThickening of bronchial wallPleura effusionMassConsolidationInterstitial changesn Mediastinal and hilar lymphadenopathypoorly defined margins Some other features of nodulesincluding lobulation spiculation pleuralindentationand calcification were also explored All these signswere rare in our study Lobulation and speculation Fig1c were simultaneously detected in patients including patients combined with pleural indentation Fig 1cCalcification of nodules was detected in only one patientThirtyseven patients including the patients with definite IgG4RLD showed some other radiological abnormality of lungs Fig which included groundglassopacity2150 thickening of pleura950 thickening ofinterlobular septa thickening of bronchial wall3 pleural effusion450 mass350 consolidation1 interstitial changes550 and mediastinal or hilarlymphadenopathy3250Six patients were diagnosed as definite IgG4RLD Thechest CT findings of these patients were shown in Table All of the patients showed mixed patterns in CTchanges Two patients had single large lung nodulesonly while the rest patients showed multiple smalllung nodules only Mass thickening of pleura thickening ofinterlobular septa groundglass opacity andpleura effusion were detected in and patientsrespectively Enlarged mediastinal or hilar lymph nodewas detected in all IgG4RLD patientsTherapy and responsesThe results of therapy and responses are shown in Table and Table For the treatment of the patients withlung nodules patients received prednisone with orwithout additional antiinflammatory or immunosuppressive drugs with prednisone only with prednisone and cyclophosphamide with prednisone andmycophenolate mofetil or methotrexate and withprednisone and azathioprine Three patients received asurgical treatment only and patients with mild diseasereceived symptomatic therapyMost patients improved to some degree aftertreatments during the duration of hospitalization Sixteen patients received a reexamination of chest CT scanafter treatment The durations between CT scans wereat least month Ten patients showed a decrease in thesize andor the number of nodules while patientsshowed no difference between pre and posttherapy CTimagesDiscussionLung nodules are small rounded lesions with at leasttwothirds ofits margins surrounded by lung parenchyma and not associated with atelectasis or lymphadenopathy In this study we focused on the lung nodules ofIgG4RD patients and IgG4RD patients presented with lung nodules in CT images As far as weknow there hasnt been any study concerned about theincidence of lung nodules in IgG4RD patients Previousstudies have reported that lung nodules were incidentallyidentified in approximately ofthe socialdemographically population [ ] Our result revealed 0cXie BMC Pulmonary Medicine Page of Table Radiologic findings in patients with definite IgG4RLDPatientsMass Thickening ofNoGroundglassopacityYespleuraYesSmallnoduleLargenoduleSolitaryThickening ofinterlobular septaPleuraeffusionEnlarged mediastinal or hilarlymph nodeYesSolitaryYesYesMultipleMultipleMultipleMultipleYesYesYesYesYesYesYesYesYesYesYesYesYesYesYesthat the incidence of lung nodules in IgG4RD patientswas much higherComparing the clinical characteristics of patients withlung nodules with that of patients without lung nodulesno significant difference was found in terms of age gender ratio duration of symptoms before diagnosis number of extrapulmonary involved ans and serologicalcharacteristics indicating that these factors have no association with the progress of nodule formation Forclinical symptoms only patients were observed withcough Most patients especially patients with small nodules only were relatively asymptomatic despite substantial burdens of disease within thelung Clinicalsymptoms of lung disease depend on the location andsize of lesions and are often nonspecific for the diagnosisof some lung disease including IgG4RLDIn our study six patients were diagnosed as definiteIgG4RLD All of the patients had lung nodules Consistent with results of previous studies conducted byInoue [] and Sun [] this result revealedthat nodular lesion can be a common manifestation ofIgG4RLD Together with lung nodules some otherchest CT findings including mass solid nodules roundshaped glass opacity thickening of bronchovascular bundles and interlobular septa alveolar interstitial changeslike honeycombing and bronchiectasis lobar or segmental consolidation and lymph node enlargement et alwere often seen in IgG4RLD patients And these CTchanges always present as various mixed patterns [ ] In this study except lung nodules we also detected mass groundglass opacity thickening of pleurapleura effusion mediastinal and hilar lymphadenopathyand thickening of interlobular septa in IgG4RLD patients It is worth noting that IgG4RLD related nodulelesion reported previously are always single solid andlarge type together with or without multiple small nodules [ ] And these nodules are always solidand with spiculated or irregular margins In our studyonly IgG4RLD patients had large nodules while ofthe IgG4RLD patients presented as multiple smallnodules And only one of the patients had noduleswith irregular margins As pulmonary biopsy results areFig Radiological manifestations of lung nodules in IgGRD patients via chest computed tomography scans a A single large solid nodule withirregular margin was detected in the right lung Thickening of pleura and tracheal traction were also noted b Multiple small and solid lungnodules were scattered in both lungs c A single small and solid nodule was shown in the right lung apex Lobulation spiculation and pleuralindentation of the nodule can be noted Thickening of pleura and the bronchial wall can also be observed de Multiple solid and groundglassnodules were shown in both lungs 0cXie BMC Pulmonary Medicine Page of Fig Other radiological manifestations of lungs in IgGRD patients via chest computed tomography scans a Thickening of the left pleura canbe observed A small nodule near the thickened pleura can also be noted bc Thickening of interlobular septa and pleura effusion in the rightlung can be observed d Mass and groundglass opacity were shown ef Small lung nodules combined with groundglass opacity consolidationthickening of interlobular septa and thickening of bronchial wall can be observedhard to get this inconsistency may be caused by a smallsample size of studies related to IgG4RLD Thus morestudies are needed to clarify the CT imaging features ofIgG4RLDAs lung nodules could also be caused by infection malignancy or some other pulmonary disorder we cannotbe certain that in the absence of definitive nodular biopsies all of the lung nodules in our study were related toIgG4RLD For an IgG4RD patient with lung nodulesthe probability of lung nodules related to IgG4RLD isvery high especially in the absence of other lung diseases In a study reported by Tsushima of IgG4RD patients with lung nodules were confirmed tobe IgG4RLD by biopsy [] In our study most patientshad no pulmonary infection or a history of cancer andother chronic pulmonary diseases For these patientsthe probability of lung nodules related to IgG4RLD isvery high Besides CT findings of our study also revealedthat most of the lung nodules in IgG4RD patients weresmall and solid always with regular margins MultipleTable Treatments of IgG4RD patientsTreatmentsPatients with lung nodulesn Pred onlyPredimmunosuppressiveagentsothersPredCYC PredMMF PredMTX PredAZA IgG4RD IgG4 related disease Pred prednisone CYC cyclophosphamide MMFmycophenolate mofetil MTX methotrexate AZA azathioprine Others surgicaltreatment or symptomatic no coticosteriod or immunosuppressive agentswas givenand bilateral distributions was also a major characteristicof these lung nodules Lung nodules in one patient alsoshowed calcification These radiologic features and distribution of lung nodules were usually regarded as benign [ ]IgG4RLD variedIt is noteworthy that three patients with large lunglobulation spiculation andnodules showed signs ofpleuralindentation all of which may predict an increased risk of malignancy [ ] As radiological findings ofthese signs can often beobserved in lung nodules and masses related to IgG4RLD [ ] Besides we also found that the proportionof lung nodules in smokers was significantly higher thanthat in nonsmokers indicating that smoking may be arisk factor of these lung nodules The proportion of elderly in IgG4RD patients was not small Therefore forIgG4RD patients with lung nodules its necessary buthard to differentiate IgG4RLD from lung cancer Asmentioned above the simultaneous presence of otherkinds of lung lesions may provide support for the diagnosis of IgG4RLD and help with the differential diagnosis In our study most patients also had thesimultaneous presence of other CT findings includingthickening of pleura thickening of interlobular septathickening of bronchial wall pleural effusion presenceof an undefined mass interstitial changes consolidationgroundglass opacity and mediastinal or hilar lymphadenopathy which may help to increase the possibilityof IgG4RLDMost IgG4RLD patients have a significant response toglucocorticoid therapy which can help to further distinguish IgG4RD nodules and malignancy [] In ourstudy patients went through a reexamination of CTscan after glucocorticoid therapy with or without 0cXie BMC Pulmonary Medicine Page of immunosuppressive agents Ten patients including patients that had large lung nodules with signs of lobulation spiculation pleural indentation and cavity showedpositive responses A decrease in the size andor thenumber of nodules was observed which helped to further support the diagnosis of IgG4RLDtype oflobectomy forSometimes during a routine medical examinationpatients may present with lung nodules detected without other pulmonary symptoms Since nodules mayresemble bronchoalveolar carcinoma and raise suspicion of malignancy [] case reports indicate that patients with thislung lesions may haveundergone wedge resection orsuspected malignancy only to discover upon tissue examination that the relevant nodules were a consequenceof IgG4RD [ ] In our study a patient with bilateral smalllung nodules was first misdiagnosed astuberculosis and another patient with a single largenodule underwent a middle lobectomy and histological findings were consistent with IgG4RD Thusan understanding of nodules related to IgG4RD isnoteworthy Since IgG4RD may be a multian disease the involvement of other ans and serologicalchange can help with diagnosis in patients with undiagnosed lung nodules In our study only patientshad the pathological proof of IgG4RLD while theother patients were diagnosed with IgG4RD primarilybecause of clinical manifestations in other ansparticularly salivary glandslacrimalgland and pancreas which is consistent with otherstudies [] In Suns study of biopsyproven IgG4RLD patientsinvolvement wasproven in only patient with uveitis mastoiditisSince patients with only lung involvement are moreprone to receive a lung biopsy the uncommon extrapulmonary involvement may be caused by selectionbias [] Once the diagnosis of IgGRD is confirmedthe radiological characteristic and treatment responsecan help with the diagnosis ofthus aregular followup is important for these patientsextrapulmonarylymph nodesIgG4RLDOur study had some limitations First most of the lungnodules involved in our study were not histopathologically proven Therefore we were unable to clarify theexact proportion of lung nodules related to IgG4RLDSecond due to the retrospective features of our studythe scan protocols of CT varied and the number of patients with a reexamination of CT was small whichmay further limit the value of our studyConclusionsThe incidence of lung nodules in IgG4RD patients canbe high For an IgG4RD patient with lung nodules thepossibility that the lung nodules related to IgG4RLD ishigh It is hard to differentiate IgG4 related lung nodulesin particularfrom other lung diseaseslung cancerRadiological characteristics and positive responses toglucocorticoids and immunosuppressive agents can helpwith the differential diagnosis For these patients regularfollowup is also importantAbbreviationsIgG4RD IgG4 related disease AIP Autoimmune pancreatitisC1q Component 1q IgG4RLD IgG4related lung disease CT Computertomographic IQR Interquartile range ESR Erythrocyte sedimentation rateCRP Creaction protein ANA Antinuclear antibody C3 Complement C4 Complement Pred Prednisone CYC CyclophosphamideMMF Mycophenolate mofetil MTX Methotrexate AZA AzathioprineAcknowledgementsNot applicableAuthors contributionsYX and AX equally contributed to study design data collection andinterpretation and drafting and revisions of the manuscript TM contributedto drafting and revision of the manuscript YL contributed to drafting andrevision of the manuscript All authors have read and approved themanuscriptFundingThis work was not funded by any grantsAvailability of data and materialsAll relevant data in this study are freely available to any scientist wishing touse them for noncommercial purposes without breaching participant confidentiality And relevant data can be obtained by contacting the corresponding authorEthics approval and consent to participateThe study was approved by the Ethical Committee of West China HospitalSichuan University According to the rules of the hospitals medical ethicscommittee this study fulfilled the criteria of exception to the requirementsof informed consentConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Rheumatology and Immunology West China HospitalSichuan University Chengdu China 2Department of MicrobiologyImmunology and Biochemistry University of Tennessee Health ScienceCenter Memphis TN USAReceived August Accepted July ReferencesStone JH Zen Y Deshpande V IgG4related disease N Engl J Med Vasaitis L IgG4related disease a relatively new concept for clinicians Eur JIntern Med Doi T Kanatsu K Mayumi M Analisis of IgG immunecomplexes in serofrom patients with membranous nephoropathyrole of IgG4 subclass andlowavidity antibodies Nephron van der Neut KM Schuurman J Losen M Bleeker WK MartÃnezMartÃnez PVermeulen E den Bleker TH Antiinflammatory activity of human IgG4antibodies by dynamic fab arm exchange Science Inoue D Zen Y Abo H Gabata T Demachi H Kobayashi T Yoshikawa J Immunoglobulin G4related lung disease CT findings with pathologiccorrelations Radiology Taniguchi T Ko M Seko S Nishida O Inoue F Kobayashi H Interstitialpneumonia associated with autoimmune pancreatitis Gut 0cXie BMC Pulmonary Medicine Page of Mahajan VS Mattoo H Deshpande V Pillai SS Stone JH IgG4relateddisease Annu Rev Pathol Matsui S Taki H Shinoda K Suzuki K Hayashi R Tobe K Respiratoryinvolvement in IgG4related Mikuliczs disease Mod Rheumatol Fujinaga Y Kadoya M Kawa S Hamano H Udea K Momose M et alCharacteristic findings in images of extrapancreatic lesions associated withautoimmune pancreatitis Eur J Radiol Umehara H Okazaki K Masaki Y Kawano M Yamamoto M Saeki T et alComprehensive diagnostic criteria for IgG4related disease IgG4RD Mod Rheumatol Hansell DM Bankier AA Macmahon H McLoud TC Muller NL Remy JFleischner society glossary of terms for thoracic imaging Radiology Gould MK Tang T Liu IL Lee J Zheng C Danforth KN Recent trendsin the identification of incidental pulmonary nodules Am J Respir Crit CareMed Loverdos K Fotiadis A Kontogianni C Iliopoulou M Gaga M Lung nodulesa comprehensive review on current approach and management AnnThorac Med Sun X Liu H Feng R Peng M Hou M Wang P Biopsyproven IgG4related lung disease BMC Pulm Med Tsushima K Tanabe T Yamamoto H Koizumi T Kawa S Hamano H et alPulmonary involvement of autoimmune pancreatitis Eur J Clin InvestigLin W Lu S Chen H Wu Q Fei Y Li M Clinical characteristics ofimmunoglobulin G4related disease a prospective study of Chinesepatients Rheumatology Ryu JH Sekiguchi H Yi ES Pulmonary manifestations of immunoglobulinG4related sclerosing disease Eur Respir J Zen Y Nakanuma Y IgG4related disease a crosssectional study of cases Am J Surg Pathol MacMahon H Naidich DP Goo JM Lee KS Leung ANC Mayo JR et alGuidelines for management of incidental pulmonary nodules detected onct images from the fleischner society Radiology Masaki Y Kurose N Yamamote M Takahashi H Saeki T Azumi A Cutoffvalues of serum IgG4 and histopathological IgG41 plasma cells for diagnosis ofpatients with IgG4 related disease Int J Rheumatol Okubo T Oyamada Y Kawada M Kawarada Y Kitashiro S Okushiba SImmunoglobulinG4related disease presenting as a pulmonary nodule withan irregular margin Respirol Case Rep 201651e00208 Odaka M Mori S Asano H Yamashita M Kamiya N Morikawa TThoracoscopic resection for a pulmonary nodule with the infiltrate of IgG4positive plasma cells Asian J Endosc Surg Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified dtpa complexes21 the original substance of the modified dtpa dtpamod was synthesized in tomsk polytechnic university preparation of colloid solution dtpamod was produced using the following method a sample of modified dtpa with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of nahco3 solution by heating to a0°c after that the volume was adjusted with the same solvent up to the mark in order to reduce the p size the container with suspension was heated in water to a0°c and treated with ultrasound for a0min 1tomsk polytechnic university lenina avenue tomsk russia 2tomsk national research medical center russian academy of sciences kooperativny street tomsk russia email sadkintpuruscientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorfigure a0 the general scheme for the synthesis of 99mtcdtpamodwhich reduced the average p radius up to a0nm the general scheme for the synthesis of 99mtcdtpamod is shown in fig a0the second type of colloids is iron nanops coated with a carbon shell of fec fig a03a these ps were obtained from the institute of metal physics urb ras ekaterinburg russia in order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates adt onto the surface of these ps has been developed an effective method for the synthesis of adt followed by their application onto the carbon surface of ps was developed at the tomsk polytechnic university22 the general scheme for the synthesis of fec ps and their subsequent interaction with 99mtc is shown in fig a03bin the third type of colloids technetium99m was adsorbed on aluminum oxide powder a powder of lowtemperature cubic modification of gammaoxide al2o3 prepared from aluminum hydroxide powder aloh3 by its calcination in a muffle furnace was used the substance was synthesized in tomsk polytechnic universitya reducing agenttin ii chloride dihydrate was used in order to obtain complexes of 99mtc with colloidsgelatin powdered ph eur uspnf pure pharma grade cas number was purchased from applichem gmbh darmstadt germanymethods method for preparation of 99mtc labeled nanocells the introduction of the radioactive label 99mtc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent sncl22h2o a0mgml in different ratios and then adding a a0ml of eluate of 99mtc a0mbqml to the mixtures the mixtures were incubated for a0min at a temperature of a0°c the preparation is ready for use after cooling at room temperature the reducing agent sncl22h2o was used as a hydrochloric acid solution the amount of a0g of tin chloride ii is added to the vial and a0ml of a0m hydrochloric acid hcl scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cfigure a0 a carbon encapsulated iron nanop b the general scheme for the synthesis of fec psis then added for its preparation after dissolution the volume is adjusted with distilled water to a0ml dissolution was carried out in an inert gas argon mediumdetermination of the size of 99mtc labeled colloidal ps the determination of the size of the labeled nanocolloids was carried out by spectroscopy on a nanophox p size analyzer sympatec gmbh germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity calculations of the yield of products with different p sizes were determined according to the formulas given belowc220 avc a1avc c100 a1 a2a1 c50 a2 a3a2where avc is the activity of the initial suspension prior to filtration a1 is the activity measured after filtration through a a0nm filter a2 is the activity after filtration through a0nm filter a3 is the activity measured after filtration through a0nm filterin parallel determination of the radiochemical purity rcp of preparations by thin layer chromatography method was carried outthinlayer chromatography tlc procedure to determine radiochemical purity of 99mtcnanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip sorbfil russia a0 cm to determine scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0camount of sncl22h2o mga[sn99mtc]a atcviia table change in relative activities of the complex [sn99mtc] and 99mtc viipertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min in this system pertechnetate migrated with the front of the mobile phase rf to determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min in this system the 99mtcnanocolloid migrated with the front of the mobile phase rf stability the stability of 99mtcnanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mtcnanocolloid following by incubation at a0°c for a0h at different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using tlc24determination of the functional suitability of preparations for scintigraphic detection of sln a study to assess the functional suitability of new nanocolloid rps was performed in series of experiments involving white wistar male rats weighing a0g injection of rp in a dose of a0mbq was performed between the first and second fingers of the rats hind paw the animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame a0s in a pixel matrix static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of with a set of pulses scintigraphy of animals was performed on an ecam signature gamma camera siemens germany the results of scintigraphic studies determined the percentage of accumulation of rp in the lymph node and the injection site the maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the european convention for the protection of vertebrates used for experiments or other scientific purposes strasbourg the experimental protocols were approved by cancer research institute biomedical ethics committee protocol number all invasive manipulations with animals were performed using inhalation or drug anesthesiastatistical analysis all mean values are expressed as idg ± sd data were analyzed statistically using methods of general statistics with a commercially available software package statistics for windows statsoft inc version results and discussionto carry out the labeling of colloids 99mtc extracted from a standard generator in the form of pertechnetate ions contained in a nacl solution was used it has a higher degree of oxidation vii in this chemical form and is not prone to complex formation a reducing agenttin ii chloride dihydrate widely used for the preparation of various compounds labeled with 99mtc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 as a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess sncl2·2h2o or the additional formation of a complex of reduced 99mtc with tin26 all this required preliminary experimental studies to establish the necessary and sufficient amount of sncl2·2h2o in the reaction mixtureduring the experiments it was found that the optimal concentration of sn ii in the composition of the reaction mixture when it interacts with 99mtc should be in the range of a0mgml table a0it was found that when the eluate with the preliminarily reduced 99mtc vii was added to the nanops the sn ii concentration introduced in the rp was csn a0mgml almost the entire amount of 99mtc has time to enter the composition of the largesize complex with tin even before its mixing with nanops this means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the sn ii solution into the reaction mixture in this connection the reduction of 99mtc with tin ii was carried out in the presence of the selected substance in this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex the technique of applying of the 99mtc label to the surface of nanosized ps is given in the previous sectionas a result of the studies reagent compositions and conditions for obtaining three nanocolloid rps were determined table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of a0nmproceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mtc vii in the obtained preparations is preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in fig a0scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0ccomposition of the preparation per a0mldtpamod a0mg 99mtc a0mbq sncl22h2o a0mg n fec a0mg 99mtc a0mbq sncl22h2o a0mg n al2o3 a0mg 99mtc a0mbq sncl22h2o a0mg n colloid yield a0nm rcp ± ± ± table composition of reagents for production of technocium99 a0m nanocolloidsfigure a0 distribution of the preparation in the rat when the preparation is administered [al2o3 99mtc sn ii] a immediately after the administration of the drug b a0min after the administration c a0min after the administrationcomposition of preparations per a0mlal2o3 a0mg 99mtc a0mbq sncl22h2o a0mg g a0mg n dtpamod a0mg 99mtc a0mbq sncl22h2o a0mg g a0mg n fec a0mg 99mtc a0mbq sncl22h2o a0mg g a0mg n yield of colloid a0nm rcp ± ± ± table indicators of rcp and the yield of a colloid with a fraction of a0nm after the introduction of gelatin in the reagentsscintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mtc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed gelatin g was introduced into the reaction mixture in this connection to increase the mobility of the labeled ps and increase the speed of their movement through the lymph system matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mtc the results of the experiments showed that the addition of gelatin a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of a0nm table a0in addition the size of these ps was determined on a nanophox p analyzer the obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in fig a0 a b c the average p size diameter is and a0nm respectivelystability test showed that complex 99mtcnanocolloid was stable in normal serum at least for a0h radiochemical purity of the tracer at the end of the experiment was ± a study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes fig a0 table a0 displays the al2o3 99mtc dtpamod 99mtc fec 99mtc biodistribution data at different time points postinjectionthe level of accumulation of preparations in the lymph nodes is of the total injected activityconclusionas a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid rps were determined an experimental dependence of the change in the content of 99mtc vii impurities on the concentration of tin ii was established and its minimum amount a0mgml was determined to reach a rhp greater than in this case the yield of the target colloid with p sizes of ± a0nm is preliminary tests of the developed preparations on experimental animals showed that accumulation of rp in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition a0mgml in addition there was an increase in the yield of the colloid scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 change in the density of the distribution of the number of ps from their size in radiopharmaceuticals a 99mtcal2o3 b 99mtcfec c 99mtcdtpamodwith p sizes of a0nm to with radiochemical purity of the preparations of repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cstomachtime h99mtcal2o399mtcdtpamod99mtcfec ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± figure a0 distribution of the preparation in the rat with injection of suspension [al2o3 99mtc sn ii gelatin] a immediately after the administration of the preparation b a0min after the administration c a0min after the administration d a0min after the administration the numbers indicate lymph node site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± bloodmlheart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± table biodistribution data up to a0h after injection of a0mbq of 99mtc in healthy male rats data represent the average value n accumulation in the sln thus the level of accumulation of rp 99mtcdtpamod and rp 99mtcfecadt in the sln is and respectively at the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityreceived march accepted july references jakobsen j k sentinel node biopsy in urooncology a history of the development of a promising concept urol oncol weixler b et al sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival a prospective singlecenter trial world j surg 101007s0026 beasley g m et al sentinel lymph node biopsy for recurrent elanoma a multicenter study ann surg oncol moser j et al sentinel node biopsy in melanoma a singlecentre experience with consecutive patients br j dermatol 101245s1043 buda a et al optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer comparison of realtime fluorescence with indocyanine green and methylene blue int j gynecol cancer scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0c sahbai s et al pericervical injection of 99mtcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in spectct clin nucl med hoogendam j p et al 99mtcnanocolloid spectmri fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer j nucl med stoffels i leyh j p¶ppel t schadendorf d klode j evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies prospective randomized clinical trial to compare icg99mtcnanocolloid hybrid tracer versus 99mtcnanocolloid eur j nucl med mol imaging beisani m et al initial experience in sentinel lymph node detection in pancreatic cancer rev esp med nucl imagen mol schubert t uphoff j henke r p wawroschek f winter a reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer verification in consideration of the european guidelines bmc urol jaukovic l et al lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions hell j nucl med subramanian s pandey u shah s rangarajan v samuel g an indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection nucl med commun ruizdomnguez j m ibarzservio l garcade manuel g calaf peris© o intraoperative injection of 99mtcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery actas urol schauer a j specific developments in sentinel node labling using 99mtccolloids in the sentinel lymph node concept springer berlin wang y et al gasphase chemistry of technetium carbonyl complexes chem phys oconnor m k et al comparison of tc99m maraciclatide and tc99m sestamibi molecular breast imaging in patients with wang j zhang r evaluation of 99mtcmibi in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer ejnmmi res br j radiol costa p et al scintigraphic imaging with technetium99mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats acta cir bras vera d r wallace a m hoh c k mattrey r f a synthetic macromolecule for sentinel node detection 99mtcdtpamannosyldextran j nucl med hoh c k wallace a m vera d r preclinical studies of [99mtc]dtpamannosyldextran nucl med biol skuridin v et al modified dtpa moleculebased nanocolloid radiopharmaceuticals j radioanal nucl chem filimonov v d et al unusually stable versatile and pure arenediazonium tosylates their preparation structures and synthetic applicability lett lukasz k thin layer chromatography in drug analysis crc press london skuridin v et al radiopharmaceutical drug based on aluminum oxide indian j sci technol 1017485 ijst2015v8i36 sazonova s i et al synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging iran j nucl med skuridin v s et al synthesis and biological characterization of 99mtclabeled ciprofloxacin pharm chem j acknowledgementsthis work was financially supported by ministry of education and science of the russian federation rfmefi57514x0034author contributionsvs conducting experimental research analysis and interpretation of the data final approval for manuscript publication vs development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication en development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication es development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication ar conducting experimental research analysis and interpretation of the data final approval for manuscript publication nv conducting experimental research analysis and interpretation of the data final approval for manuscript publication rz conducting tests of the functional suitability of drugs preparation of the section evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and figures final approval of the manuscript for publication of the manuscriptcompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to vsreprints and permissions information is available at wwwnaturecomreprintspublishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0copen access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate if changes were made the images or other third party material in this are included in the s creative commons license unless indicated otherwise in a credit line to the material if material is not included in the s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0c'
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despite great advances in recent decades in screening diagnosisand curative surgery hepatocellular carcinoma hcc remainsthe second leading cause of cancerrelated mortality worldwidegrandhi siegel epidemiologicalevidence has conï¬rmed that the longterm outcomes of patientswith hcc have notimproved significantly with the rapiddevelopment of surgical techniques madduru moreimportantly because of the limitations of systematic statustumor position and the need to preserve liver function morethan of patients are not eligible for surgical treatment evenafter curative resection prognosis remains unsatisfactory becauseof a high incidence of postoperative recurrence colecchia the initiation and maintenance of hcc is a complexand regulated process involving the accumulation of numerousgenetic changes over decades niu erkekoglu these sequential alterations not only endow normal livercells with neoplastic ability enabling uncontrolled growth butalso provide potential therapeutic targets and biomarkers thusfurther understanding of the initiation and maintenance of hccat the molecular level is crucial to prolonging survival and makingindividual treatment decisionsthe exive development of highthroughput technologyhas provided powerful tools for the molecular study of cancerschmidt and hildebrandt rna sequencing rnaseq and microarraysthe most representative methods ofthis technology are mature enough for use in commercialapplications mantione zhang duringthe past decades the genomewide transcriptional analysis ofgene expression has become critically important to gain betterinsight into the biological processes of hcc and other typesof cancer jin xiong in additionto the aberrant expression of transcripts studies have focusedon diï¬erent molecular levels multilevel omicsincludingcopy number variation epigenetic modiï¬cations nucleotidepolymorphisms and dna methylation especially in hcclee lin evidence obtained from thesestudies clearly demonstrates that hcc is a disease causedby cumulative aberrations at diï¬erentlevels of molecularregulation thus only a highthroughput multiomics analysiscan decipher the complex biology of hcc many previousstudies despite promising results focused only on the aberrantregulation of expression and its biological eï¬ects howeverstructural transcript variation in hcc which is heavily shaped byalternative splicing as has until recently been less well studiedaccording to the manual genome annotation project harrow pruitt there are only about proteincoding genes this number is obviously inconsistent withthe overall cellular complexity which includes at least distinct proteincoding sequences harrow thisdiscrepancy between the numbers of transcripts and proteincoding genes in human cells indicates the existence of anadditional mechanism between the transcriptional and the posttranslational levels that increases the coding capacity of thegenome through the as process a single rna precursorcan be spliced via distinct arrangements to generate rnaslandscape of as in hccwith diï¬erent structures and functions biamonti song this may be one ofthe main causesof cellular complexity and proteome diversity experimentalstudies on the eï¬ects of individual as events suggest that asmay change the biological function of a protein by regulatingits stability controlling its location modifying the mutualinteractions of proteins and even adding or deleting activedomains brett yang these ï¬ndingssuggest that as well as expression abundance the balance ofdiï¬erent as events that result from the same rna precursormust be considered howeverthe latter consideration hasoften been neglected in previous studies in fact emergingdata from genomewide analyses feng kahles indicate that as occurs in more than ofmultiexon genes suggesting thatthe widespread existenceof as is the product of physiological processes rather thantranscription errorsin recent years the diagnostic and the therapeutic role ofas in many human diseases has attracted increasing attentionlargescale screening of as events has been performed usingexpressed sequence tag libraries although this approach isprone to a high rate of false positives venables exonjunction probes provide a higher experimental validation ratelapuk however this method has the disadvantageof being limited to known splice junctions owing to thelimited available techniques complicated mechanisms and hugenumbers involved transcriptomewide as dysregulation and itspotential associations with biological behavior in hcc haveremained uncharacterizedrnaseq not only supports the quantitative measurementof novel as events but also provides deeper coverage higheraccuracy and better resolution li y thus it maybe the most suitable of the currently available approaches foras study in recent years the cancer genome atlas tcgatomczak wang has accumulated a richand publicly available source of rnaseq data and correspondingclinical information this enables the analysis of as dysregulationin hcc at a genomewide level tcga includes rnaseqdata samples obtained from hcc patients together withtheir corresponding clinical information thereby facilitating theclinical analysis of hccrelated as events in a large cohorthowever without reliable and eï¬cient bioinformatical methodsthe advantages of rnaseq in as analysis cannot be fullyexploited spliceseq a recently developed bioinformatics toolcan exactly match rna reads with gene splice graphs and ishelpful for accurately calculating complex or lowfrequency asevents ryan there has been a lack of studies combining largescalernaseq data with the corresponding clinical information tocomprehensively analyze as at singleexon resolution howeverthis is very necessary especially in hcc thereforein thecurrent study we comprehensively analyzed wholegenome asin the tcga hcc cohort to screen out hccrelated asevents and further studied the relationships of these eventswith clinical outcomes our ï¬ndings suggest that certain hccrelated as events including nek2at and troptat havecritical roles in the progression and maintenance of hcc morefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccimportantly these hccrelated as events represent potentialnew therapeutic targetsmaterials and methodstumorlocationinvasioninformation ofdata curationclinicopathologicalthe hcc cohort andcorresponding rnaseq data were retrieved and downloadedfrom tcga1 to ensure appropriate protection of patientprivacy the tcga data were stratiï¬ed according to data typeand level conforming to the publishing guidelines formulatedby tcga wang then the rnaseq data andcorresponding clinicopathological information were mutuallypaired using the unique tcga barcodes only patients whomet the criteria listed below were included grandhi patients with corresponding rnaseq data siegel patients with complete clinicopathological informationincluding localsex age distalmetastasis pathological stage diï¬erentiation grade lymph nodemetastasis and survival information madduru histological diagnosis of hcc and colecchia survival for at least month after the primary pathologicaldiagnosis spliceseq was used to determine rna splicing patternsand produce as proï¬les for each hcc patient as previouslydescribed li y zhu each as eventwas quantiï¬ed using the percent spliced in psi value rangingfrom to a commonly used method to reï¬ect the abundanceof as events in order to remove the eï¬ects of splicing noiseand generate as reliable a set of as events as possible a seriesof strict ï¬lters average psi ¥ percentage of samples withpsi ¥ was applied to the detected as events the interactivesets between the seven types of as were quantitatively analyzedand the results were visualized in upset plots using upsetrversion conway circlize version wasused to generate circos plots to depict the parent genes and theiras events in chromosomes gu the details of thedesign of the present study are illustrated in supplementaryfigure s1 all the methods used in this study were in line withthe relevant guidelines and regulationsidentiï¬cation of deas and enrichmentanalysisto screen the diï¬erentially expressed alternative splicing deasevents between hcc and corresponding normal tissues the psivalue of each as event was determined in the tcga hcccohort hcc tissue samples and paired adjacent normaltissues a generalized linear model was applied to remove thebatch eï¬ects the deas were determined based on adjustedp adj p and associated log2 fold change fc values withadj p ¤ and log2fc ¥ representing as events thatwere downregulated and upregulated respectively biologicalfunction enrichment analysis was performed based on the deasparent genes gene ontology go and kyoto encyclopediaof genes and genomes kegg terms with false discoveryrate less than were considered to be significantly enrichedand were selected for further analysis enrichment analysis wasperformed using the clusterproï¬ler package version yu the parent genes of deas events were importedinto the string database and used to determine proteinprotein interactions ppis a relationship network was thengenerated using cytoscape version su clusteranalysis was conducted using the average linkage agglomerationalgorithm and correlation distance metricsestablishment of hccrelated splicingcorrelation networka total of splicing factors sfs supplementary table s1were identiï¬ed by comprehensive and handcurated screening ofthe literature all the sfs included in the current study had beenexperimentally validated in previous research giulietti and included heterogeneous nuclear ribonucleoproteinsproteins serineargininerich proteins and other proteinsbelonging to the celf fox khdrbs nova and elav familiesthe expression of each sf was obtained from the broadinstitute2 correlations between the psi values of deas and theexpression of sfs were analyzed by weighted gene coexpressionnetwork analysis version langfelder and horvath benjamini and hochberg correlation was used to adjust thepvalues the adjusted pvalues less than were consideredto indicate statistically significant diï¬erences cytoscape version was used to generate the correlation plotssurvival analysisall the included hcc patients were divided into two groupsbased on the psi value of each deas median cut and thetwo artiï¬cial categories were modeled as continuous variables toderive more easily interpretable hazard ratios based on overallsurvival os and diseasefree survival dfs cox regression wasperformed to evaluate the prognostic value of each deas eventlogrank test and kaplanmeier analysis were used to comparepatient survival between subgroups p was consideredas statistically significant the overall survivalrelated deaswere further analyzed in lasso regression to identity the mostpowerful prognostic markers finally a prognostic model wasconstructed for predicting the os in order to quantify the risk ofos a standard form of risk score rs for each colorectal cancercrc patient was calculated combine the levels of the psiι1 psii à lito divide the patients into the high or lowrisk group kaplanmeier curves were used to estimate the survival for patients in thetraining the testing and the validation sets between the highriskand the lowrisk groupspsii and lasso coeï¬cients li risk score pnfunctional experiment of cxcl12splicing variants in hccthe human hcc cell line hepg2 was obtained from the chineseacademy of sciences committee on type culture collectioncell bank shanghai china the cell line was cultured in 1httpsportalgdccancergov2httpgdacbroadinstitutefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccprimers was used asgibco carlsbad ca united states supplemented with fetal bovine serum fbs bi beit haemek israel at ¦c with co2 total cdna from tissues was obtained as described abovethe pcr reaction was carried out using the forward primer5cid48tgcccttcagattgttgcac3cid48 common for allisoforms and theisoformspeciï¬c reverse primers 5cid48gctaactggttagggtaatac3cid48 and5cid48gctagcttacaaagcgccagagcagagcgcactgcg3cid48for np_9546371and np_0010290581 respectively bactin ampliï¬ed usingthe forward 5cid48acactgtgcccatctagcagggg3cid48and reverse 5cid48atgatggagttgaaggtagtttcgtggat3cid48aloading control quantitative realtime pcr was performedin mx3005tm qpcr system with a mxpro qpcr software stratagene la jolla ca united states and sybr greendetection system the adherent hepg2 cells were transfectedwith the corresponding hiscxcl12 construct by the calciumphosphate method and cultured for h at h before collectingthem the cell supernatants were removed and when indicatedbrefeldin a was added to the fresh medium the collectedcells were left untreated or permeabilized with saponin andimmunolabeled with the his mab and a pegoat antiigsecondary antibody and analyzed by ï¬ow cytometry the cellinvasion assay was conducted using matrigelcoated chambers µm pore size corning costar corporation cambridgema united states in brief à cells were plated in theupper chamber coated with matrigel and supplemented withserumfree medium the lower chamber was ï¬lled with a culturemedium containing fbs incubation was carried out for h at ¦c following which the noninvasive cells were scrapedoï¬ with cotton swabs the cells that had successfully translocatedwere ï¬xed with paraformaldehyde stained with crystalviolet and ï¬nally counted using an inverted microscope mttassay colony formation assay and soft agar growth assay wereperformed according to our previously described methods zhou protein structure homology modeling analysis wasperformed as previously described by using the online serverswissmodel waterhouse evaluation of the relationship of asclusters with clinicopathologicalfeaturesbased on the identiï¬ed deas n the tcga hcc cohortin the current study was stratiï¬ed by an unsupervised consensusapproach consensus cluster plus version wilkerson andhayes the optimal number of clusters was determinedby integrating the results of the elbow method and gap statisticthe relationship between clinical outcomes and as clusterswas evaluated using logrank test and kaplanmeier curves asdescribed by xiong 2018aresultslandscape of as event proï¬les in hccto systematically characterize the as events and their clinicalsigniï¬cance in hcc we collected rnaseq libraries andcorresponding clinicalinformation from hcc patientsthe tumor tissues and paired adjacent normal tissues from patients were sequenced simultaneously the includedpatients comprised males and femalesamong which patients developed recurrence and died of hcc the median followup period was months range months the general characteristicsof these hcc patients are fully detailed in supplementarytable s2 rnaseq data were associated with the clinicalthe corresponding patient via the tcgainformation ofbarcodes there were patients with rnaseq data bothfrom tumor tissue and adjacent normal tissue according tothe recommended analysis approach described in a previouslypublished study ryan we identiï¬ed asevents from genes based on their splicing patterns theseas events could be roughly classiï¬ed into seven types alternatepromoter ap mutually exclusive exons me retained intronri exon skipping es alternate acceptor site aa alternateterminator at and alternate donor site ad figure 1ato quantify the detected as events psi values were calculatedthese values measure the proportion of each detected splicingvariation in all of the expressed isoforms the expression ofcertain isoforms was fairly low psi and most of the asevents could not be stably detected in all of the given samplesafter screening average psi ¥ percentage of sampleswith psi ¥ a total of as events from geneswere obtained we further compared the variance in quantityof as events and the genes involved between tumor adjacentpaired normal and unpaired tumor tissues for diï¬erent splicingpatterns there were no significant diï¬erences in quantityvariations however on average one gene might have nearlythree as events figure 1b left panel moreover only annotated genes in this study stably underwent as figure 1bright panel notably diï¬erent as patterns may occur for a singlegene thus upset plots were used to depict the intersectionsbetween as types as demonstrated in figure 1c most of theparent genes only occurred in one type of as event whereascertain parent genes contained up to four types of as eventabout of the parent genes contained two or more asevents the arrangements of diï¬erent as types and as eventsbetween diï¬erent exonsintrons may be the major reason fortranscriptome diversity in order to comprehensively depict asevent proï¬les in hcc circos plots were used to visualize therelationships among as events and the corresponding parentgenes in chromosomes figure 1didentiï¬cation of hccrelated deascomparing the variations in molecular components amongdiï¬erent pathological states using highthroughput techniquesis an eï¬ective way to screen key molecules this approachhas been widely used to identify diseaserelated molecules inprevious research xiong 2018ab it is reasonableto consider that significant diï¬erences in as events betweenprimary hcc tissues and adjacent normal tissues may be relevantto the initiation and maintenance of hcc in this study thetcga barcodes corresponding to tissue samples rnaseq data were analyzed from which as proï¬les of pairednormal and tumor tissues were ï¬nally extracted these pairedfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure landscape of alternative splicing as events in hepatocellular carcinoma hcc a diagrammatic sketch of the seven types of as events in the presentstudy alternate acceptor site aa alternate terminator at alternate promoter ap exon skipping es mutually exclusive exons me alternate donor site adand retained intron ri b number of as events and the corresponding parent genes illustrated according to as type left panel the color bar represents asevents ï¬ltered by criteria the black bar represents the corresponding genes involved in as each as type was divided into four groups based on the tissue sourcen normal tissue t tumor tissue pt paired tumor tissue npt unpaired tumor tissue number of detected as events asrelated genes ï¬ltered as events and thecorresponding genes right panel c intersection of parent genes between different as types n in hcc one gene may incur up to four types ofalternative splicing d circos plots depicting the distribution and the detailed alteration of as events and their parent genes in chromosomesas proï¬les were used to identify deas eventually deaswere identiï¬ed from genes using a threshold of log2fc and adj p including aps ess ats risnine ads aas and one me figure 2a and supplementarytable s3 the top deas are listed in table notablythe proportion of as types between the ï¬ltered as and deaswas inconsistent the es events accounted for of ï¬lteredas but only of deas however the proportion of apevents rose from of ï¬ltered as to of deasfigure 2b these statistical ï¬ndings suggest that as is notthe result of transcription errors but a tightly regulated processmoreover based on the identiï¬ed deas the samples could beclearly separated into normal and tumor groups by unsupervisedhierarchical clustering figure 2c indicating that the deashad been reliably identiï¬ed the psi values of deas eventsin diï¬erent hcc patients are illustrated in figure 2c as amatrix heat map the changes in color gradient intuitively revealthe heterogeneity of hcc a splice graph which representssplice junctions as edges and exons as rectangular nodes wasused to visualize some of the identiï¬ed deas figure 2dfurthermore the diï¬erences in expression of these as eventsbetween primary hcc tissue and corresponding adjacent normaltissues are intuitively depicted in figure 2e taken together theseresults show that a significant variation of as occurred duringhcc initiation and maintenance indicating that the potentialrole of hccrelated as events requires further researchenrichment and interaction analysis ofdeasemerging evidence indicates that as could change a transcribedsequence directly with eï¬ects on expression abundance orprotein function thus the potential biological eï¬ects of deascould be determined by analyzing the corresponding proteinsas shown in supplementary figures s2ac speciï¬c go termsclosely related to liver metabolism including negative regulationof hydrolase activity sterol homeostasis anic acid catabolicprocess and acidic amino acid transport were significantlyenriched by the parent genes of deas in addition certain keggpathways known to be involved in hcc were enriched includingthe cgmppkg signaling pathway the nfκb signaling pathwaythe mrna surveillance pathway and the phosphatidylinositolfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure identiï¬cation of hepatocellular carcinoma hccrelated aberrant alternative splicing as a differences in as events between paired hcc tissue andparacancerous tissue volcano plot of the differentially expressed alternative splicing deas identiï¬ed in hcc the blue and the red points represent the deas withstatistical signiï¬cance logfc ¥ adj p b proportions of different as types among ï¬ltered as and deas c heat map of the deas the horizontal axisshows the clustering information of samples divided into two major clusters adjacent normal tissue n and paired tumor tissue n the left longitudinalaxis shows the clustering information of deas the gradual change of color from green to red represents the alteration of expression of deas from low to highd splice graph of some representative deas the thin exon sections represent untranslated regions and the thick exon sections represent coding regions theexons are drawn to scale and the connecting arcs represent splice paths e differences in percent spliced in values of as events between hcc and pairedadjacent normal tissuessignaling system supplementary figure s2d these resultssuggest that the parent genes of deas are critical in the biologicalregulation of hcc thus aberrant splicing of the transcribedsequences could inï¬uence their translation and change thecharacteristics of the resulting proteins therefore it is essentialto study as events from the perspective of ppi networks basedon the deasrelated genes a ppi network was establishedrepresenting not only normal interactions but also the potentialimpact of as events figure correlation network of sfs andhccrelated asas events are primarily regulated by sfs which attach to themrna precursor and aï¬ect the selection of exons and thechoice of splicing site aberrant as events in tumor tissuemay be orchestrated by a limited number of sfs for thisreason we conjecture that a few key sfs potentially regulatea large proportion of hccrelated as events to validate thisconjecture we ï¬rst identiï¬ed sfs supplementary table s1by comprehensive and handcurated screening of the literatureall of which had been previously experimentally validatedgiulietti then the copy number variation somaticmutations and expression abundance of these sfs in eachhcc patient were investigated using cbioportal figure 4avisualization using oncoprint revealed that each of the sfs harbored at least three molecular alterations figure 4aleft panel the most frequently aï¬ected sf was khdrbs3in which molecular alterations were detected in cases partly owing to the above changes the expressionabundance of the sfs showed a significant heterogeneity atan individual level figure 4a right panel the expressionproï¬les of the sfs in diï¬erent cancer types also showedheterogeneous characteristics figure 4b more importantlythe expression of sfs also diï¬ered between paired normal andcancer tissues of the same hcc patient figure 4c nextcorrelation analyses were performed between the psi value ofeach deas event and the sfs according to the correlationcoeï¬cient ttest p r a splicing regulatorynetwork was established as shown in figure 5a sfs weresignificantly correlated with deas events among which were downregulated and were upregulated several diï¬erentas events in the network were regulated by a single sf insome cases an sf had the opposite regulatory eï¬ect on diï¬erentas events figure 5a we also found that the same bindingsite as event could be competitively bound by diï¬erent sfsthese observations explain at least in part why one gene cangenerate several diï¬erent isoforms representative correlationsfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hcctable the top most different alternative splicing as eventssymbolas typeexonsfrom exonto exonmean nmean tlog fcadjusted pvaluedownregulatedmthfd2ligf2kif22gstz1serpinb5ppargfam3afip1l1tmem59tpm4cdh23mid1gnesamd12kif4afbxw7nek2padi4rnf115vti1aupregulatedrdm1nr1i3psphtmem145nr1i3gpr116piddnr1i3ano1cldn7sardhscp2znf331eno3pemtfn1tnfrsf10carhgef39igf2neil3apapadapatapaaatapapatapapatatapatatesatatatapatriesapriesapapesapatapesatapapat1393e1593e4948e9941e2008e4387e8636e1393e4574e2519e1528e2202e6860e1522e2379e7699e1780e1843e4211e2609e1994e8164e1036e7765e1089e3569e1065e2135e5986e1038e1611e1110e2586e7736e2117e1072e4524e1151e1829e8136ebetween sfs and speciï¬c as events were illustrated using dotplots figures 5bg for example the expression of esrp2 wassignificantly correlated with both es of ceacam1 figure 5cand at of epb41l5 figure 5fassociation of deas with prognosis ofhcc patientsa crossvalidation method was used to evaluate the accuracyof the survival data and the clinical information as shown insupplementary figure s3 stratifying patients according to thetnm stage resulted in separate kaplanmeier curves for bothos and dfs these results conï¬rmed that the survival dataset forthe tcga hcc cohort although it contained censored data wasappropriate and informative for use in further survival analysistheeï¬ect of each deas on survival was determined by coxregression analysis the hcc patients were divided into twogroups according to their psi value median cut of eachdeas event according to univariate analyses a total of to investigate the prognostic signiï¬cance of deasfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure proteinprotein interaction ppi analysis of the identiï¬ed differentially expressed alternative splicing deas interactions of the parent genes affectedby deas these genes were used to construct an intricate ppi network comprising nodes and edges the genes are denoted as nodes in the graph andthe interactions between them are represented as edges the shape size and color of the nodes respectively represent alternative splicing type value of logfcand change patterndeas events were significantly correlated with dfsand deas events were significantly correlatedwith os supplementary table s4 among these prognosisrelated deas events deas events were correlated withboth os and dfs p figure shows some of thedeas events for which the pvalue for both os and dfs waslower than to demonstrate the capability of as eventsfor prognostic prediction we randomly selected two prognosisrelated deas events and used them to draw survival curvesas shown in figure according to the psi value median ofnek2at and troptat the hcc patients could be stratiï¬edto form significant kaplanmeier curves by both os and dfssurvival analysis additionally the deas events that significantlycorrelated with survival in the univariate analysis were furtherassessed by multivariate analysis as shown in supplementaryfigure s4 there were ï¬ve and six deas events that could berecognized as independent prognostic indicators for os and dfsrespectively these ï¬ndings conï¬rm that deas events possessnot only an important biological meaning but also a potentialclinical signiï¬canceconsidering the prognostic value of the aboveidentiï¬ed asa prognostic model integrating multias was established so thatit can be easily applied to clinical practice based on the survivalrelated deas a relative regression coeï¬cient was calculated bylasso analysis by forcing the sum of the absolute value ofthe regression coeï¬cients to be less than a ï¬xed value certaincoeï¬cients were shrunk exactly to zero and the most powerfulprognostic marker of all the hcc survivalassociated deaswas selected including four as supplementary figure 5acombining the relative expression levels of the as in themodels and the corresponding lasso coeï¬cients a riskscore was calculated for each patient obviously patientswith higher rs generally had a significantly worse overallsurvival than those with lower rs p supplementaryfigures 5bc as the majority of events occurred within years timedependent roc curves were used to assess theprognostic power based on os at and years respectivelysupplementary figure 5dclustering hcc patients using deasassociated with prognosisgiven our ï¬ndings of significant heterogeneity among deas atan individual level which could reï¬ect the diï¬erent outcomesfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure multiomics analysis of the splicing factors sfs in hepatocellular carcinoma hcc a cbioportal analysis of the sfs in the cancer genomeatlas hcc patients oncoprint was used to produce a landscape of genomic alterations legend in sfs rows at the individual level columns inframe deletionstruncated mutations and missense mutations with known or unknown signiï¬cance are shown in orange blue green and gray respectively with onethird heightthe copy number variations are annotated with the full height ampliï¬cation is shown in red and copy number loss is in blue heat map matrix shows the variation insfs at expression level the expression abundance from high to low is represented by color gradient from red to blue b expression of the sfs in tumortypes heat map color gradient depicts the normalized expression of sfs between different tumor types c differential expression analysis of representative sf tia1in hcc the expression of tia1 in hcc tissue was significantly higher than that in normal liver tissueof patients with hcc we conjectured that there might existdistinct patterns of as among diï¬erent hcc patients thishypothesis was veriï¬ed using consensus unsupervised analysisbased on the deas the optimal number of clusters wasdetermined by combining the gap statistic and elbow method theoptimal balanced partition as suggested was k figure 8aaccordingly all the hcc patients were divided into four clustersas follows i n ii n iii n and iv n figures 8bc as illustrated bythe heat map the four clusters had a significant interconnectivityamong which cluster ii appeared as a wellindividualized clusterwhereas there was more classiï¬cation overlap among clustersi iii and iv figures 8bc kaplanmeier survival analysisand logrank test were used to evaluate the associations betweenprognosis and the as clusters as illustrated in figures 8dethe stratiï¬cation of hcc patients based on as clusters showeda significant correlation with distinct patterns of survival thevariation tendency that resulted in the as stratiï¬cation betweenfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure speciï¬c regulatory network of hepatocellular carcinomarelated alternative splicing as events a correlation network of splicing factors sfs anddifferentially expressed alternative splicing the shape size and color of nodes respectively represent type as event or sf value of logfc and change patternupregulated or downregulated the breadth of the line represents the interaction strength bg representative dot plots of the correlations between theexpression of sfs and percent spliced in values of as eventsfigure prognostic value of differentially expressed alternative splicing deas in hepatocellular carcinoma part deas events simultaneously associated withoverall survival os and diseasefree survival dfs univariate analysis of deas for os and dfs respectively unadjusted hazard ratios boxes and conï¬denceintervals horizontal lines
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"objectives to describe the benefits and limitations of using individual and combinations of linked english electronic health data to identify incident cancersdesign and setting our descriptive study uses linked english clinical practice research datalink primary care cancer registration hospitalisation and death registration dataparticipants and measures we implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and we calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers we described completeness of grade and stage information in the cancer registration data setresults gold standard cancers were identified positive predictive values of all case definitions were ¥ and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity for case definitions that used cancer registration alone or in combination was ¥ for the four most common cancers and ¥ across all cancer sites except bladder cancer using cancer registration alone for case definitions using linked primary care hospitalisation and death registration data sensitivity was ¥ for the four most common cancers and ¥ for all cancer sites except kidney oral cavity and ovarian cancer when primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancerss ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority strengths and limitations of this study º this is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in england to identify cases of the most common incident cancers º using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions º we described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data º a key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataintroductionthe clinical practice research datalink cprd provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 in the field of cancer epidemiology cprd primary care data linked to hospital episode statistics admitted patient care strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access data hes apc office of national statistics ons mortality and national cancer registration and analysis service ncras cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for ncras data research teams therefore commonly choose not to use all available linked data3 cancer epidemiology studies can also be conducted using ncras and hes apc data provided by national health service nhs digital and public health england phe without linkage to cprd primary care data4 this provides national coverage at the expense of the detailed health data that are available in primary care recordsvalidation studies assessing concordance between cprd gold hes apc and ncras data have estimated high positive predictive values ppvs for cprd gold data and varying proportions of registered cancers that are not captured in cprd gold and hes apc5 the most up to date analysis by arhi et al included the five most common cancers and all papers focussed on concordance between cprd gold only and ncras existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs national data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and clinical commissioning groups10 we aim to describe and compare the benefits and limitations of using different combinations of linked cprd primary care data hes apc ons mortality and ncras cancer registration data for conducting cancer epidemiology studies our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets we have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets we also describe the availability of stage grade and treatment variables over time in the cancer registration data for the cprd linked cohort this reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesmethodsstudy design and settingwe completed a concordance study using linked2 english cprd gold hes apc ons mortality and ncras data cprd gold data were extracted from the january monthly release and the 13th update to cprds linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 malignant neoplasms of urinary organ unspecified was considered consistent with both bladder and kidney cancer for ncras we accessed coded records for the most common cancers we included cancers recorded in the clinical or referral file for cprd gold cancers recorded in any diagnosis field for hes apc and the underlying or strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure gold standard algorithm to identify incident site specific cancers using all data sources hes hospital episode statistics ncras national cancer registration and analysis service ons office of national statisticsmost immediate cancer cause of death in ons mortality datacancer case definitions based on individual sources and combinations of sourceswe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources ncras alone ncras and hes apc all sources cprd gold hes apc and ons mortality cprd gold alone hes apc alone multiple malignant cancers recorded on the index date in cprd gold or hes apc were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the ncras data if available and as multiple site cancer if not for each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upgold standard cancer case definitionwe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure cancers recorded in ncras alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in hes apc alone or gold alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in ncras and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in ncras if there were more data sources in total with records for cancer at that site than data sources with contradictory recordswe used ncras data to identify stage grade and treatment where available in the cancer registry only cohort binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisstatistical analysisfor each cancer site and each individual or combined data source we combined our applied study definitions strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordwe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex we calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourceswe used kaplan meier methods to describe mortality over time for cancers identified using each definition the ons mortality death date was used for these analyseswe used the ncras only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timepatient public involvementpatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsresultsof research quality patients in the cprd gold january build were eligible for linkage to hes ons mortality and ncras data in set were excluded due to unknown sex of the remainder and had at least year of follow up between january and december and were included in the study population using the gold standard algorithm incident cases of cancer were identified the number of patients identified with each cancer is presented in online supplementary appendix table half n82 of these patients were male aged to aged to and aged or olderfigure presents ppvs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm when using ncras data alone to of cancers were confirmed by the algorithm for out of cancer sites the ncras only case definition gave the highest ppv case definitions using data sources not including ncras generally had lower ppvs ranging from to for individual cancer sites for the four most common cancers breast lung colorectal and prostate ppvs were at least for all case definitions minimal differences in ppvs were observed between age groups years and sexes online supplementary appendix figures figure presents sensitivity values for each case definition sensitivity was generally higher for the case definitions that included ncras data ranging from to for individual cancer sites except bladder cancer identified using ncras data alone and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity was also generally high for definitions using a combination of cprd gold hes apc and ons mortality data ranging from to ¥ for the four most common cancers sensitivity was lower for case definitions that used cprd gold alone range to for individual cancer sites or hes apc alone range to sensitivity values for cprd gold alone and hes apc alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures post hoc analysis suggested that the low sensitivity of cprd gold only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary organ cancer codes n1108 in cprd gold rather than contradictory information about the first cancer record n625 these incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 bladder cancers that were not recorded in ncras data n3445 were commonly recorded in both hes apc and cprd gold n2228 or in hes apc only with a subsequent unspecified or bladder cancer record in hes apc within months n995 table describes the number of days median iqr and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used case definitions using ncras alone and combinations of ¥ data sources captured cancers close to the gold standard date median lag ¤ days for all cancer sites whereas median lags were generally longer for the case definitions using cprd gold alone and hes apc alonefigure describes mortality over time following incident cancer diagnoses ascertained from each case definition minimal differences in mortality were observed between cancers identified from different case definitions where variability was observed cancers identified using cprd gold only had the lowest mortality rates eg kidney cancer and cancers identified using hes apc only or ncras only had higher mortality rates eg prostate cancer and bladder cancer respectivelyfigure describes completeness of grade and stage for cancers identified using ncras only recording of grade was highly variable between cancers with gradual increases in completeness over time completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure online supplementary appendix figure describes recording of treatment modalities identified using ncras strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure positive predictive value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service onsoffice of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0celitnecrep hthtrqi inademelitnecreprqi inadem hthtelitnecrep htht inademrqielitnecrep hthtcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l ebatopen access recnac lanoitan sarcn atad erac tneitapdetti mda scitsitats edospei latipsoh cpaseh knil atadhcraeser ecitcarp lacniilc drpc metsys suovren lartnec sncl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iia setis recnac nommoc tsom ruofdcii nosrev sesaesdi fonoitacfissacliscitsitats lanoitan rof ecfifo snoi atadnoitartsgerrecnac ecvres ssyanadna liinoitartsgeri lanoitanretni eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaci snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmun ot ot ot ot ot cl amoeym epitluml ot ci ameakuel ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep hthtsarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure mortality following first ever record of cancer in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service nhl non hodgkin's lymphoma ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure completeness of grade and stage for cancers identified using ncras data only cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites grading information is not applicable to braincns sarcoma or haematological cancers and not required by in the national data standard cosd for prostate cancer core staging is not applicable to haematological and gynaecological cancers other types of staging are recommended by cosd cns central nervous system cosd cancer outcomes and services data set ncras national cancer registration and analysis service nhl non hodgkin's lymphomastrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access only missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordeddiscussionstatement of principal findingswe investigated the use of different sources of electronic health record data to identify incident cancers for all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers combining cprd gold hes apc and ons mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date finally while we observed minimal changes in ppvs and sensitivities between and completeness of ncras cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 completeness of cancer treatment recording was difficult to assess due to the absence of a missing categorystrengths and weaknesses of the studythe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from cprd to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies we have also assessed the value of using ncras cancer registration data to measure stage grade and cancer treatment modalitiesa limitation of the study is that our gold standard algorithm is not validated we feel that we were justified in pre weighting ncras data as more reliable that other data sources as ncras is a highly validated data set that matches merges and quality checks data from multiple sources4 we did not consider ncras to be the outright gold standard as it is plausible that ncras does not identify all tumours diagnosed and treated in primary and secondary care for most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in hes apc cprd gold or ons mortality data but not in ncras these tumours may have been diagnosed and coded as invasive in primary or secondary care but not by ncras been incorrectly coded in hes apc cprd gold or ons mortality data not have been notified to ncras eg tumours treated in private hospitals or be the result of linkage errors between the data sets the proportion of cancers identified in hes apc but not in ncras is particularly high for bladder cancer this is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in ncras which are greatest for bladder cancer4 this explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in ncras compared with other data sources to identify incident cancers we required months of research quality follow up in cprd gold prior to inclusion in the study previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 the identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as ncras data collection started in hes apc in and ons mortality data in and by the inclusion of all diagnostic codes in hes apc assuming that the first ever primary or secondary record identified incident cancer reassuringly ppvs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases requiring internal confirmation within months for cancers recorded in cprd gold alone in our gold standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up our data cut only included ncras data for the top cancers earlier cancers at other sites will have been missed in this studyit is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesstrengths and weaknesses in relation to other studies discussing important differences in resultsthe most up to date study describing concordance between linked cprd gold hes apc and ncras data sets demonstrated that to of the five most common cancers recorded in cprd gold are not confirmed in either hes apc or cancer registration data and to of registered cancers are not recorded in cprd gold8 for cancers recorded in both sources the diagnosis date was a median of to days later in cprd gold than in the cancer registration data using cprd gold alone to identify these strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis use of hes apc only identified a higher proportion of patients with the correct diagnosis date than cprd gold but over represented older patients and those diagnosed through the emergency route the majority of registered cancers were picked up using both cprd gold and hes apc ranging from for lung cancer to for breast cancer previous research demonstrated similar results with substantial differences between cancer types5 additionally a study using data from to found that using hes data in addition to ncras data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancerswe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards levels of data completeness of staging information in the cprd extract in were similar to those reported by the united kingdom and ireland association of cancer registries ukaicr9meaning of the study possible explanations and implications for clinicians and policymakersuse of ncras cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately case definitions based on a combination of cprd gold hes apc and ons mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersthese findings should be considered when deciding which data sources to include in research studies and which sources to use to define cancer exposures outcomes and covariatesunanswered questions and future researchfurther research is required to investigate the validity of cancer recorded in cprd gold and hes apc that are not recorded in the ncras data and to understand differences in cancer data recording with cprd gold and cprd aurum cprds recently launched primary care database based on records from practices that use emis software17 further investigation would be required to confidently identify subtypes of cancer either using codes available in each data set eg colon and rectal cancer or additional information available in hes apc or ncras data use of ncrass recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu
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to the human gut microbiota biochem taxonomic hierarchy of the phylum firmicutes and novel firmicutes species originated from various environments in korea j microbiol front microbiol filippidou s a combination of extreme environmental conditions favor the prevalence of endosporeforming firmicutes bintsis t lactic acid bacteria as starter cultures an update in their metabolism and genetics aims microbiol verma d k bioprocessing technology in food and health potential applications and emerging scope apple academic caulier s overview of the antimicrobial compounds produced by members of the bacillus subtilis group front microbiol lopetuso l r scaldaferri f petito v gasbarrini a commensal clostridia leading players in the maintenance of gut homeopress burlington stasis gut pathogens rood j i general physiological and virulence properties of the pathogenic clostridia in clostridial diseases of animals eds uzal fa prescott jf songer jg and popoff mr wiley wells c l wilkins t d medical microbiology 4th edn university of texas medical branch galveston kho z y lal s k the human gut microbiomea potential controller of wellness and disease front microbiol koliada a association between body mass index and firmicutesbacteroidetes ratio in an adult ukrainian population bmc microbiol yuan j antibacterial compoundsmacrolactin alters the soil bacterial community and abundance of the gene encoding pks front microbiol 103389fmicb scientific reports 101038s41598020706868vol1234567890wwwnaturecomscientificreports 0c romerotabarez m 7omalonyl macrolactin a a new macrolactin antibiotic from bacillus subtilis active against methicillinresistant staphylococcus aureus vancomycinresistant enterococci and a smallcolony variant of burkholderia cepacia antimicrob agents chemother moldenhauer j chen x h borriss r piel j biosynthesis of the antibiotic bacillaene the product of a giant polyketide synthase complex of the transat family angew chem int ed engl 101002anie20070 butcher r a the identification of bacillaene the product of the pksx megacomplex in bacillus subtilis proc natl acad wu l difficidin and bacilysin from bacillus amyloliquefaciens fzb42 have antibacterial activity against xanthomonas oryzae chen xh structural and functional characterization of three polyketide synthase gene clusters in bacillus amyloliquefaciens sci rice pathogens sci rep fzb j bacteriol yang j genomicsinspired discovery of three antibacterial active metabolites aurantinins b c and d from compostassociated bacillus subtilis fmb60 j agric food chem kang h k seo c h park y marine peptides and their antiinfective activities mar drugs 103390md130 sur s romo t d grossfield a selectivity and mechanism of fengycin an antimicrobial lipopeptide from molecular dynamics j phys chem b 101021acsjpcb7b118 knight c a the first report of antifungal lipopeptide production by a bacillus subtilis subsp inaquosorum strain microbiol peypoux f bonmatin j m wallach j recent trends in the biochemistry of surfactin appl microbiol biotechnol grangemard i wallach j magetdana r peypoux f lichenysin a more efficient cation chelator than surfactin appl biores s0025 chem biotechnol zhou m bacillibactin and bacillomycin analogues with cytotoxicities against human cancer cell lines from marine bacillus sp pkuma00093 and pkuma00092 mar drugs hertlein g production of the catechol type siderophore bacillibactin by the honey bee pathogen paenibacillus larvae one e108272 gu q bacillomycin d produced by bacillus amyloliquefaciens is involved in the antagonistic interaction with the plantpathogenic fungus fusarium graminearum appl environ microbiol e0107517 barsby t kelly m t andersen r j tupuseleiamides and basiliskamides mew acyldipeptides and antifungal polyketides produced in culture by a bacillus l aterosporus isolate obtained from a tropical marine habitat j nat prod mthethwa b c comparative analyses of cytochrome p450s and those associated with secondary metabolism in bacillus species int j mol sci 103390ijms1 podust l m sherman d h diversity of p450 enzymes in the biosynthesis of natural products nat prod rep 101039c2np2 0020a greule a stok j e de voss j j cryle m j unrivalled diversity the many roles and reactions of bacterial cytochromes p450 in secondary metabolism nat prod rep 101039c7np0 0063d medema m h minimum information about a biosynthetic gene cluster nat chem biol 101038nchem bio1890 ngwenya m l blooming of unusual cytochrome p450s by tandem duplication in the pathogenic fungus conidiobolus coronatus int j mol sci 103390ijms1 matowane r g in silico analysis of cytochrome p450 monooxygenases in chronic granulomatous infectious fungus sporothrix schenckii special focus on cyp51 biochim biophys acta proteins proteom 101016jbbapa p201710003 qhanya l b genomewide annotation and comparative analysis of cytochrome p450 monooxygenases in basidiomycete biotrophic plant pathogens one e0142100 101371journ alpone01421 kgosiemang i k r syed k mashele s s comparative genomics and evolutionary analysis of cytochrome p450 monooxygenases in fungal subphylum saccharomycotina j pure appl microbiol jawallapersand p cytochrome p450 monooxygenase cyp53 family in fungi comparative structural and evolutionary analysis and its role as a common alternative antifungal drug target one e107209 101371journ alpone01072 syed k shale k pagadala n s tuszynski j systematic identification and evolutionary analysis of catalytically versatile cytochrome p450 monooxygenase families enriched in model basidiomycete fungi one e86683 101371journ alpone00866 suzuki h comparative genomics of the whiterot fungi phanerochaete carnosa and p chrysosporium to elucidate the genetic basis of the distinct wood types they colonize bmc genom 1011861471216413444 akapo o o distribution and diversity of cytochrome p450 monooxygenases in the fungal class tremellomycetes int j mol sci 103390ijms2 sello m m diversity and evolution of cytochrome p450 monooxygenases in oomycetes sci rep 101038srep1 b biol sci hamberger b bak s plant p450s as versatile drivers for evolution of speciesspecific chemical diversity philos trans r soc feyereisen r insect molecular biology and biochemistry elsevier amsterdam senate l m similarities variations and evolution of cytochrome p450s in streptomyces versus mycobacterium sci rep 101038s4159 y mnguni f c more p450s are involved in secondary metabolite biosynthesis in streptomyces compared to bacillus cyanobacteria and mycobacterium int j mol sci 103390ijms1 parvez m molecular evolutionary dynamics of cytochrome p450 monooxygenases across kingdoms special focus on mycobacterial p450s sci rep 101038srep3 syed p r cytochrome p450 monooxygenase cyp139 family involved in the synthesis of secondary metabolites in mycobacterial species int j mol sci 103390ijms2 khumalo m j comprehensive analyses of cytochrome p450 monooxygenases and secondary metabolite biosynthetic gene clusters in cyanobacteria int j mol sci 103390ijms2 kanehisa m sato y kawashima m furumichi m tanabe m kegg as a reference resource for gene and protein annotation nucleic acids res d457d462 101093nargkv10 nelson d r the p450 superfamily update on new sequences gene mapping accession numbers early trivial names of enzymes and nomenclature dna cell biol 101089dna1993121 nelson d r cytochrome p450 nomenclature methods mol biol clifton nj 101385089603 nelson d r cytochrome p450 nomenclature methods mol biol clifton nj 101385159259 scientific reports 101038s41598020706868vol0123456789wwwnaturecomscientificreports 0c de lima procpio r e da silva i r martins m k de azevedo j l de araºjo j m antibiotics produced by streptomyces braz j infect dis munro a w p450 bm3 the very model of a modern flavocytochrome trends biochem sci li h poulos t l the structure of the cytochrome p450bm3 haem domain complexed with the fatty acid substrate palmitoleic acid nat struct biol bm3 biochem soc trans noble m miles c reid g chapman s munro a catalytic properties of key activesite mutants of flavocytochrome p450 lee ds crystallization and preliminary xray diffraction analysis of fattyacid hydroxylase cytochrome p450bsβ from bacillus subtilis acta crystallogr sect d biol crystallogr lee ds substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome p450 from bacillus subtilis crystallographic spectroscopic and mutational studies j biol chem girvan h m structural and catalytic properties of the peroxygenase p450 enzyme cyp152k6 from bacillus methanolicus bower s cloning sequencing and characterization of the bacillus subtilis biotin biosynthetic operon j bacteriol j in biochem cryle m j schlichting i structural insights from a p450 carrier protein complex reveal how specificity is achieved in the p450bioi acp complex proc natl acad sci cryle m j bell s g schlichting i structural and biochemical characterization of the cytochrome p450 cypx cyp134a1 from bacillus subtilis a cyclolleucyllleucyl dipeptide oxidase biochemistry cimermancic p insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters cell tran p n yen m r chiang c y lin h c chen p y detecting and prioritizing biosynthetic gene clusters for bioactive compounds in bacteria and fungi appl microbiol biotechnol s0025 z marchlerbauer a cddsparcle functional classification of proteins via subfamily domain architectures nucleic acids res d200d203 101093nargkw11 syed k mashele s s comparative analysis of p450 signature motifs exxr and cxg in the large and diverse kingdom of fungi identification of evolutionarily conserved amino acid patterns characteristic of p450 family one e95616 101371journ alpone00956 graham s e peterson j a how similar are p450s and what can their differences teach us arch biochem biophys katoh k kuma k toh h miyata t mafft version improvement in accuracy of multiple sequence alignment nucleic acids res 101093nargki19 boc a diallo a b makarenkov v trex a web server for inferring validating and visualizing phylogenetic trees and networks nucleic acids res w573w579 101093nargks48 letunic i bork p interactive tree of life itol v4 recent updates and new developments nucleic acids res w256w259 saeed a i tm4 a free opensource system for microarray data management and analysis biotechniques 10214403342 mt01 weber t antismash 30a comprehensive resource for the genome mining of biosynthetic gene clusters nucleic acids res 101093nargkv43 battistuzzi f u feijao a hedges s b a genomic timescale of prokaryote evolution insights into the origin of methanogenesis phototrophy and the colonization of land bmc evol biol acknowledgementstiara padayachee and nomfundo nzuza thank the department of science and technologynational research foundation dstnrf south africa for masters scholarships grant numbers mnd190619448759 and mnd190626451135 respectively khajamohiddin syed expresses sincere gratitude to the nrf south africa for a research grant grant number and university of zululand grant number c686 the authors want to thank barbara bradley pretoria south africa for english language editingauthor contributionsks designed conceptualized and provided funding for the study all authors were involved in generation analysis and interpretation of data all authors reviewed and approved the manuscriptcompeting interests the authors declare no competing interestsadditional informationsupplementary information is available for this paper at 101038s4159 correspondence and requests for materials should be addressed to drn a0or a0ksreprints and permissions information is available at wwwnaturecomreprintspublishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsopen access this article is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate if changes were made the images or other third party material in this article are included in the articles creative commons license unless indicated otherwise in a credit line to the material if material is not included in the articles creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020706868vol1234567890wwwnaturecomscientificreports 0c'
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"Microwave ablation MWA is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body This study aimed to investigate the effects of MWA on cytokines inpatients who underwent MWA for a hepatic malignancyMethods Patients admitted to the Oncology Department in the First Affiliated Hospital of Soochow Universitybetween June and February were selected Peripheral blood was collected from patients with a hepaticmalignancy treated with MWA The levels of cytokines IL2 IFNÎ TNFα IL12 p40 IL12 p70 IL4 IL6 IL8 IL10and vascular endothelial growth factor VEGF were detected with a Milliplex® MAP Kit The comparison times wereas follows before ablation h after ablation days after ablation and days after ablation Data were analyzedusing a paired sample ttests and Spearmans correlation analysisResults A total of patients with hepatic malignancies were assessed There were significant differences in IL2IL12 p40 IL12 p70 IL1 IL8 and TNFα at h after MWA Significant increases 2fold vs before ablation wereobserved in IL2 IL1 IL6 IL8 IL10 and TNFα after MWA Elevated IL2 and IL6 levels after ablation werepositively correlated with energy output during the MWA procedureConclusions WA treatment for hepatic malignancies can alter the serum levels of several cytokines such as IL2 and IL6Keywords Microwave ablation Hepatic malignancy Cytokines IL2 IL6 ImmunoregulationBackgroundPrimary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwideIn China hepatocellular carcinoma HCC has the secondhighest mortality rate of malignancies [] The treatmentof primary and secondary hepatic malignancies via Correspondence lengbengsudaeducn Jing Zhao Qiang Li and Merlin Muktiali contributed equally to this work2Department of Oncology the First Affiliated Hospital of Soochow UniversitySuzhou China5Division of Neurosurgery City of Hope Beckman Research Institute DuarteCalifornia USAFull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] Recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for HCC and its fiveyear survival rateis similar to that of resection [] Microwave ablationMWA is widely used to treat unresectable HCC and recurrent HCC and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy Immune checkpoint inhibitors ICIs The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhao BMC Cancer Page of such as PD1PDL1 and CTLA4 antibodies have beenwidely applied in several cancers and studies have indicated that ICI treatment could enhance the effect of ablation [] Evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process [] Cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer Several cytokineswhich can arise from either tumor cells or immunocytes[] such as tumor necrosis factor TNFα interleukinIL1 IL6 IL8 IL10 and vascular endothelial growthfactor VEGF have been linked with cancers and can either promote or inhibit tumor development The serumlevels of cytokines differ during cancer development Although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after MWA It is still unknown whether the above cytokines changed before andor after MWA in patientswith hepatic malignancies In this study we investigatedthe effects of MWA on the serum levels of cytokines inpatients with hepatic malignanciesMethodsPatients and samplesThe patient population examined in this study was derivedfrom the First Affiliated Hospital of Soochow UniversityPatients were admitted to the Oncology Department between June and February The total number ofpatients was with liver metastases and primaryliver cancers The inclusion criterion was a tumor locatedat a hepatic site either primary or metastases All patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease SD or partial responsePR for more than days Informed consent for blooddraw and the relevant therapy was obtained from all patients The protocol was approved by the Human EthicsCommittee of the First Affiliated Hospital of SoochowUniversity and was conducted in accordance with theDeclaration of Helsinki All written informed consent wasobtained from all participants and clearly stated Wholeblood mL was drawn into EDTA anticoagulant tubeson days to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesAblation procedureThe ablation procedure used in this research was MWAThe puncture site and pathway were determined underthe guidance of a computed tomography CT scanLocal infiltration anesthesia was achieved by using lidocaine The placement of microwave ablation probeswas guided by a CT scan or ultrasonic device and allprobes were placed at the maximum diameter layerDouble probes were employed when the maximumdiameter of the tumor was up to cm The power andtime of ablation were designed for each patient in therange of W and min respectively basedon the size number and position of the tumor Theboundaries of ablation zones were designed as extended cm upon the tumor siteCytokine detectionA Milliplex MAP Kit with human cytokinechemokinepanels that measured IFNÎ IL2 IL6 IL8 IL10 IL12p40 IL12 p70 IL1 TNFα and VEGF was utilized according to the manufacturers instructions Briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °C washedand then incubated with a biotinylated detection antibodyAfter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a Luminex analyzer Luminex Corporation All samples were measured in duplicate Standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgmL The minimum detectable concentrations were as follows IFNÎ pgmL IL2 pgmLIL12 p40 pgmL IL12 p70 pgmL IL1 pgmL IL6 pgmL IL8 pgmL IL10 pgmL TNFα pgmL and VEGF pgmL All resultsbelow the minimum concentrations were processed as theminimum concentrationsStatistical analysisIBM SPSS Statistics software was used for the statistical analysis along with GraphPad Prism for figurecreations Normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval CI Cytokinesat different times were compared using a onetailedpaired ttest Spearmans correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceResultsClinical characteristics of the enrolled patientsAs shown in Table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed The patients cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0cZhao BMC Cancer Page of Table Clinical characteristics of the patients enrolled n CharacteristicSexmalefemaleAgePathogenesisprimarysecondaryPrimary site For metastatic hepatic malignancesColon rectalPancreasStomacheBreastOthersMaximum tumor length mmAblation probe usedAblation time minAverage power per probe W ± ± ± ± Average energy time à power time à power¼¼ Time and power indicate the time and power respectively ofdifferent probes used during the operation ± IFNÎ IL12 p40 and IL12 p70 were slightly increasedafter MWA treatmentAs shown in Table and Fig the median level ofIFNÎ before the MWA treatment was pgmL CI pgmL at days and days after theMWA treatment there was a slight increase comparedto that preMWA with median levels of pgmL CI pgmL and pgmL CI pgmL respectively The median level of IL p40 before the MWA treatment was pgmL CI pgmL There was a slight increase to pgmL CI pgmL days postMWAThe median IL12 p70 level before the MWA treatmentwas pgmL CI pgmL and increasedto pgmL CI pgmL days afterthe MWA treatment and to pgmL CI pgmL days postMWA No significant alteration in the VEGF median level was detected after theMWA treatmentIL2 IL1 IL6 IL8 and IL10 were elevated over 2foldafter the MWA treatmentAs shown in Table Fig and Fig the median levelof IL2 before the MWA treatment was pgmL CI pgmL There was a significant increase at h postMWA with a median level of pgmL CI pgmL The median level ofIL1 before the MWA treatment was pgmL CI pgmL and a significantincrease wasnoted days after the MWA treatment pgmL CI pgmL The median level of IL6before the MWA treatment was pgmL CI pgmL and significantly increased daysafter the MWA treatment pgmL CI pgmL The median level ofIL8 before theMWA treatment was pgmL CI pgmL and increased significantly to pgmL CI pgmL days after the MWA treatmentThe median level of IL10 before the MWA treatmentwas pgmL CI pgmL and increasedsignificantly days after the MWA treatment pgmL CI pgmL The median level ofTNFα before the MWA treatment was pgmL CI pgmL and increased significantlyto pgmL CI pgmL days afterthe MWA treatmentlevelsElevated IL2 and IL6 levels after ablation were positivelycorrelated with energy output during MWATo further evaluate the relationship between the increased cytokineand MWA treatment weemployed the concept of energy time à power time à power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the MWA procedure As shown in Table and Fig the IL2 levels at h postMWA and the IL levels at days postMWA illustrated significant correlations with energy the relative indexes were and respectivelyDiscussionAs technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used MWA for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed [] A consensus guideline was recently developed to address indications for MWA in these patientsThermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °C Terminal treatment requiresthat a necrotic area surrounds the target site with anadditional 10mm margins [] However in the liverhigh tissue perfusion and large blood vessels can cause aheat sink effect around the ablation zone making itdifficult to achieve terminal ablation [] The heat sink 0cZhao BMC Cancer Page of Table Median levels of cytokines before and after MWACytokineIFNÎIL2preMWA pgmL CI CI CI CI CI CI CI CI CI CI h postMWA pgmL CI CI ¼ CI CI CI CI CI CI CI CI IL12 p40IL12 p70IL1IL6IL8IL10TNFαVEGF p vs preMWA ¼ 2fold vs preMWA days postMWA pgmL CI CI CI CI CI ¼ CI ¼ CI ¼ CI ¼ CI ¼ CI days postMWA pgmL CI CI CI CI CI CI CI CI CI CI effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression LTP [] however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsIn our study significant increases in the secretion ofchemokines IL8 proinflammatory cytokines IL1IL12 IFNÎ and TNFα and antiinflammatory cytokines IL10 were observed after MWA IL8 is mainlyproduced by macrophages the classical biological activity of IL8 is to attract and activate neutrophils whichcan lead to a local inflammatory response However recent studies have indicated that IL8 both macrophageand cancer cellderived can recruit Myeloidderivedsuppressor cells MDSCs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] IL1 is mainlyproduced by macrophages B cells and NK cells couldproduce IL1 under certain circumstances Generallycells can only synthesize and secrete IL1 after beingstimulated by foreign antigens or mitogens IL1 couldpromote the Th1 response promoting the activation ofDendritic cells DCs and Cytotoxic T lymphocytesCTLs IL12 is mainly produced by B cells and macrophages Human IL12 is a heterodimer with two subunits p40 kD and p35 kD which areinactivated in isolated form In general IL12 functionsas a combination of two subunits IL12 p70 while p40alone possesses partial functions of IL12 p70 Its mentionable that IL12 p40 and p35 are not expressed inequal proportions so the amounts of IL12 p40 and IL p70 are different in one cell IL12 can stimulate theproliferation of activated T cells and promote the differentiation of Th0 cells into Th1 cells Moreover IL12could induce the cytotoxic activity of CTLs and NK cellsand promote the secretion of several cytokines such asIFNÎ [] and TNFα [] Previous research indicatedthat TNFα may play a crucial role in MWA in combination with immunotherapy [] Notably our data illustrated that the IL12 results were consistent with thoseof IFNÎ after the ablation operation but not with thoseof TNFα This result indicated that upregulation ofIFNÎ may be a major effect of the IL12 increase afterMWA On the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after MWA IL10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses IL10 was reported to increaseafter thermal ablation in the literature [ ] Strategiesto inhibit IL10induced immunosuppression after thermal ablation treatment would be of interestIL10asAblation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of in situ vaccination [] Moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] Many immunoregulatory cytokineswere released or expressed after thermal ablation Notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle Previously researchers demonstrated that proinflammatory cytokines such as IL1 IL6 IL8 IL18 andTNFα were increased several hours or days after thermalablation [ ] To our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction [] This systemic reaction would becaused by different mechanisms First interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines Second heat injurycould cause acute thermal necrosis in liver and tumor 0cZhao BMC Cancer Page of Fig Levels of cytokines before and after MWA treatment Slightly increased IFNÎ IL12 p40 and IL12 p70 levels after MWA treatment Over fold enhancement of IL2 h postMWA and of IL1 IL6 IL8 IL10 and TNFα d postMWA p 0cZhao BMC Cancer Page of Fig Trends in cytokines significantly altered after MWA treatment The levels of IL2 at h postMWA IL1 at d postMWA IL6 at dpostMWA IL8 at d postMWA and IL10 at d postMWA were elevated over 2fold compared to the levels preMWATable Correlation between the ablation energy and significantly elevated cytokinesEnergyvsIL2 h postMWAEnergyvsIL1 d postMWAEnergyvsIL6 d postMWAEnergyvsIL8 d postMWAEnergyvsIL10 d postMWAEnergyvsTNFα d postMWASpearmans rP value onetailed p 0cZhao BMC Cancer Page of Fig Correlation between the ablation energy and the serum levels of IL2 and IL6 The serum levels of IL2 at h postMWA and IL6 at dpostMWA were positively correlated with energy output during the MWA procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions Generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions Ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions Theseexplanations may be the reason why the cytokine changeslasted different durations Moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the energyindex In our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time Terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction This reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor Our findings indicated that IL2 andIL6 were significantly altered after the ablation procedureand positively correlated with MWA energy IL2 is commonly derived from activated T cells primarily Th1 cellsIL2 can stimulate T cells to proliferate and differentiateactivate natural killer NK cells and macrophages and enhance the functions of cytotoxic T lymphocytes CTLs[] Our data illustrated that IL2 is significantly increased at h after MWA indicating that IL2 may induce a nonspecific immune response after MWA But IL decreased after h postMWA in our study suggesting that the IL2induced immune response may not belong lasting Mentionable many cytokines detected IL8IL1 IL12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellThis result support the theory that MWA could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction Additional cytokines alterationsuch as IL6 after ablation may be no anspecific inliver Evidences indicate that increase of IL6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum IL8 IL1 IL6 IL10 IL12and TNFα were significantly raised after radiofrequencythermal ablation [] Moreover Joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of IL6 and IL10 []Another question remain unveiled was if our result wascancerspecific We checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules[] and adenomyosis [] According to these literatureIL6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of IL6 may be caused by tumour antigenreleased by ablation treatment However the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection To the research about adenomyosis on the other hand experiment design was determined to followup the IL6 at months afterHIFU ablation As our data demonstrated mostly cytokines were return to preMWA level after monthdetection after months may miss the modulation ofIL6 Overall few evidences support that some of thecytokines were altered in a cancerspecific mannerwhile no solid results could confirm that Further animal experiments were required to make a clarifieddata and answer this question 0cZhao BMC Cancer Page of thetumorassociated immuneIn recent years ablationinduced systemic effects suchasresponse haveattracted increased attention [] de Baere T first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis [] Although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled abscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management In it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity [] however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors ICIs [ ] ICIs suchas PD1PDL1 and CTLA4 antibodies are widely applied in several cancers and studies have indicated thatICI treatment could enhance the effect of ablation []Evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process [] However opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders PD1 immunotherapy [] suggesting that ablation treatment may promote tumorprogression Our data demonstrated that IL6 was significantly increased after MWA treatment IL6 is derived from monocytes macrophages DCs Th2 cells andsometimes cancer cells and it plays a key role in T cellproliferation and survival [] The role of IL6 appearsto be rather complex Korn classified IL6 as differentiation factor which could involve in differentiation ofTh17 cells [] However IL6 does not direct the commitment to the Th1 or Th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as TGF TNF or IL21 []For instance IL6 activated STAT3 pathway in naiveCD4 T cells in the presence of the morphogen TGFbpromotes the population expansion of Th17 cells [] Recent evidence indicates that IL6 plays an indispensable role in T cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy [] Incontrast IL6 can increase MDSCs [] inhibit the development and maturation of dendritic cells DCs []and inhibit the polarization of Th1 cells [] eventuallyresulting in negative immunomodulatory effects According to Muneeb Ahmeds work the adjuvant uses ofa nanop smallinterfering RNA siRNA can besuccessfully used to target the IL6mediated locoregional and systemic effects of thermal ablation IL6 knockout via a nanop antiIL6 siRNA in mice coulddecrease the local VEGF level at the ablation site []Therefore how to utilize the positive effect of IL6 whileavoiding the negative effect after MWA needs further investigation Preclinical research indicated that IL6 andPDL1 blockade combination therapy reduced tumorprogression in animal models [ ] Thus an antiIL strategy after ablation should be considered whencombined with ICI therapy Previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation This resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapyConclusionsOur results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionThis reaction could lead to a detectable alteration of cytokine levels Further investigation is required to revealwhether the cytokines altered by MWA treatment couldaffect cancer progression whether positive or negativeAbbreviationsMWA Microwave ablation HCC Hepatocellular carcinoma ICIs Immunecheckpoint inhibitors TNF Tumor necrosis factor IL InterleukinVEGF Vascular endothelial growth factor SD Stable disease PR Partialresponse CT Computed tomography CI Confidence interval LTP Likelihoodof local tumor progression MDSCs Myeloidderived suppressor cellsCTLs Cytotoxic T lymphocytes NK Natural killer siRNA Small interfering RNAAcknowledgementsNot applicableAuthors contributionsJZ Conceptualization data curation writingoriginal draft and writingreview and editing QL Conceptualization and writingreview and editingMM Conceptualization and writingreview and editing BRConceptualization and writingreview and editing and collect samples YHExecute Milliplex assay and collect data DPL Patient enrollment executeMWA ablation and collect samples ZL Execute MWA ablation and collectsamples DML Patient enrollment execute MWA ablation and collectsamples YX Execute Milliplex assay and collect data MT Conceptualizationand writingreview and editing RL Conceptualization data curation formalanalysis visualization writingoriginal draft and writingreview and editingAll authors have read and approved the manuscriptFundingThis work was supported by the National Natural Science Foundation ofChina the Natural Science Foundation ofJiangsu Province of China BK20140295 the Jiangsu GovernmentScholarship for Oversea Studies JS2018179 and the Six one projects forhighlevel health personnel in Jiangsu Province LGY2018077Availability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThe protocol was approved by the Human Ethics Committee of the FirstAffiliated Hospital of Soochow University and was conducted in accordancewith the Declaration of Helsinki Patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0cZhao BMC Cancer Page of research and that their privacy would be maintained All written informedconsent was obtained from all participants and clearly statedConsent for publicationNot applicableCompeting interestsThere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workAuthor details1Department of Radiation Oncology the First Affiliated Hospital of SoochowUniversity Suzhou China 2Department of Oncology the First AffiliatedHospital of Soochow University Suzhou China 3Department of LymphaticHematologic Oncology Jiangxi Cancer Hospital Nanchang China4Department of Interventional Radiology the First Affiliated Hospital ofSoochow University Suzhou China 5Division of Neurosurgery City of HopeBeckman Research Institute Duarte California USAReceived January Accepted August ReferencesFu J Wang H Precision diagnosis and treatment of liver cancer in ChinaCancer Lett Bruix J Han KH Gores G Llovet JM Mazzaferro V Liver cancer approachinga personalized care J Hepatol SupplS144Rognoni C Ciani O Sommariva S Bargellini I Bhoori S Cioni R FacciorussoA Golfieri R Gramenzi A Mazzaferro V Transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis BMC Cancer Nault JC Sutter O Nahon P GanneCarrie N Seror O Percutaneoustreatment of hepatocellular carcinoma state of the art and innovations JHepatol Yin J Dong J Gao W Wang Y A case report of remarkable response toassociation of radiofrequency ablation with subsequent Atezolizumab instage IV nonsmall cell lung cancer Medicine Baltimore 20189744e13112Shi L Chen L Wu C Zhu Y Xu B Zheng X Sun M Wen W Dai X Yang M PD1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor Clin Cancer Res Lippitz BE Cytokine patterns in patients with cancer a systematic reviewLancet Oncol 2013146e218Jin YB Zhang GY Lin KR Chen XP Cui JH Wang YJ Luo W Changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy IMRT PLoS One 2017122e0172264Kim MJ Jang JW Oh BS Kwon JH Chung KW Jung HS Jekarl DW Lee SChange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma Cytokine Gillams A Goldberg N Ahmed M Bale R Breen D Callstrom M Chen MHChoi BI de Baere T Dupuy D Thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans Frontieres meeting Eur Radiol Ahmed M Solbiati L Brace CL Breen DJ Callstrom MR Charboneau JWChen MH Choi BI de Baere T Dodd GD 3rd Imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate Radiology Chiang J Hynes K Brace CL Flowdependent vascular heat transfer duringmicrowave thermal ablation Conf Proc IEEE Eng Med Biol Soc Huang HW Influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors Med Phys Najjar YG Rayman P Jia X Pavicic PG Jr Rini BI Tannenbaum C Ko JHaywood S Cohen P Hamilton T Myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withIntratumoral expression of IL1beta IL8 CXCL5 and Mip1alpha Clin CancerRes Alfaro C Teijeira A Onate C Perez G Sanmamed MF Andueza MP AlignaniD Labiano S Azpilikueta A RodriguezPaulete A TumorproducedInterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps NETs Clin Cancer Res Kundu M Roy A Pahan K Selective neutralization of IL12 p40 monomerinduces death in prostate cancer cells via IL12IFNgamma Proc Natl AcadSci U S A Onishi H Kuroki H Matsumoto K Baba E Sasaki N Kuga H Tanaka MKatano M Morisaki T Monocytederived dendritic cells that capture deadtumor cells secrete IL12 and TNFalpha through IL12TNFalphaNFkappaBautocrine loop Cancer Immunol Immunother Yu Z Geng J Zhang M Zhou Y Fan Q Chen J Treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathOncotarget Yang W Wang W Liu B Zhu B Li J Xu D Ni Y Bai L Liu GImmunomodulation characteristics by thermal ablation therapy in cancerpatients Asia Pac J Clin Oncol 2018145e490Erinjeri JP Thomas CT Samoilia A Fleisher M Gonen M Sofocleous CTThornton RH Siegelbaum RH Covey AM Brody LA Imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 J Vasc Interv Radiol den Brok MH Sutmuller RP van der Voort R Bennink EJ Figdor CG RuersTJ Adema GJ In situ tumor ablation creates an antigen source for thegeneration of antitumor immunity Cancer Res Zerbini A Pilli M Laccabue D Pelosi G Molinari A Negri E Cerioni SFagnoni F Soliani P Ferrari C Radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous NKcell responseGastroenterology
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" in head and neck cancer hnc the relationship between a delay in starting radiotherapy rt andthe outcome is unclear the aim of the present study was to determine the impact of the amount of time beforetreatment intervention tti and the growth kinetics of individual tumors on treatment outcomes and survivalmethods two hundred sixtytwo hnc patients with primary tumors treated with definitive chemo rt wereretrospectively analyzed the tti was defined as the time interval between the date of histopathologic diagnosisand the first day of the rt course volumetric data on tumors were obtained from diagnostic and rt planningcomputer tomography ct scans in order to calculate the tumor growth kinetic parametersresults no significant association between locoregional control or causespecific hazards and tti was found thelog hazard for locoregional recurrence linearly increased during the first days of waiting for rt although this wasnot significant the median tumor volume relative increase rate and tumor volume doubling time was 32dayand days respectively and neither had any impact on locoregional control or causespecific hazards the association between a delay in starting rt and the outcome is complex and does not harm allpatients waiting for rt further research into imagingderived kinetic data on individual tumors is warranted inorder to identify patients at an increased risk of adverse outcomes due to a delay in starting rtkeywords head and neck cancer radiotherapy waiting time treatment delay outcome with new cases and deaths reported in head and neck cancer hnc is the eighth mostcommon and most lethal cancer in men worldwide in addition to surgery and systemic therapy radiotherapy rt is one of the cornerstones for treatment of thiscancer owing to the rising cancer incidence rate in ageing populations and the widening list of indications forirradiation the demands for rt have increased dramatically over the past decades [ ] the increasing complexity of pretreatment diagnostics and rt technology correspondence pstrojanonkoisi1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia4chair of oncology faculty of medicine university of ljubljana ljubljanasloveniafull list of author information is available at the end of the has led to delays in treatment decisionmaking and thereduction in linear accelerator throughputthat hasresulted in a significant imbalance between the demandfor rt and the availability of rt capacities in manypublically funded health systems this is also the case inslovenia [ ]due to obvious ethical reservations the only way tostudy the impact of delays in starting rt on treatmentoutcomes are retrospective observational analyses of cohorts from different institutions or countries intuitively one would expect that the prolongation of thetime taken before treatment intervention tti is harmful to patients both the likelihood of tumor growth andthe acquisition of a metastatic phenotype increases as afunction of time furthermore advanced tumors aremore difficult to treat than smaller tumors the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cžumer radiation oncology page of indeed a systematic review of pertinent literature fromthe period by chen showed an increasein the risk of local hnc recurrence of for everymonth of delay in definitive rt however certainstudies included in chens metaanalysis and also somemore recent reports negated the association betweenthe delay in definitive rt and the increased risk of treatment failure [] several different biases inherent inretrospective analyses either related to the quality ofdiagnostic procedures and treatment or to the inhomogeneity of the studied population as well as a selectionbias ie patients with fasttumor progression or ahigher burden of symptoms receive priority in treatment and significant variability in the kinetics of individual hnc cases may abolish the effect of tti inoutcomes [] however if patients with advancedor fastgrowing tumors have to wait longer the magnitude of this effect may be overestimated furthermore no compelling relationship between treatmentdelay and prognosis was found in some other cancertypes []in order to determine what would be an acceptabletti in hnc patients treated with definitive rt or concurrent chemoradiation we aimed to analyze the impactof tti and growth kinetics of individual tumors on theoccurrence of localregional failure distant metastasisand survival in the present study of a cohort of slovenepatients with hncmethodsin a retrospective study patients with oral cavity oropharyngeal hypopharyngeal or laryngeal squamous cellcarcinoma scc who were treated with curativeintentdefinitive rt with or without concurrent chemotherapybetween january and december at the institute of oncology ljubljana slovenia were includedpatients with t1n0 or t2n0 glottic cancer were left outof this cohort the period was chosen because of fluctuations in the waiting time for irradiationas a result of intensive renovations and expansion in thedepartment of radiotherapy that took place over thistime span from patients medical and rt charts wecollected information on clinical gender age onset ofsymptoms date and type of disease recurrence anddeath tumor histology site of origin tnm stage andtreatment characteristics rt technique regimen anddose duration of rt type of concurrent chemotherapy[cct] and the number of cycles administered thetnm stage was determined according to the 7th editionof the uicc classification systemfor analysis of the impact of tumor growth kinetics ontreatment outcomes the volumes ml of primary tumors and neck nodal metastases as marked on diagnostic and rt planning computer tomography ct scanswere compared patients with the same basic clinicaldisease and treatment characteristics as indicated abovebut with both sets of ct scans available were selectedfor this part of the study diagnostic ct scans were performed through the acquisition of mm thin ct sections whereas planning ct scans had a slice thicknessof mm both with intravenous iodine contrast enhancements sets with extensive artefacts were excluded forthe purposes of rt planning patients were positionedsupine on the flat tabletop and a fivefixation pointthermoplastic mask was used lymph nodes were considered positive if the smallest diameter was more than cm andor the necrotic center or extracapsular extension was seen if available segmentation was guided bymagnetic resonance imaging mri sets and the resulting contours around the primary tumors and metastaticneck nodes represented a consensus between two radiation oncologists and a radiologist volumes of primarytumors and metastatic neck nodes were separately calculated by a computer software program used for rt planning xio computerized medical systems inc stlouis usa eclipse varian medical systems inc paloalto usa the end points in this part of the study werechanges in the tumornodal volume and tnm stage thecalculation of the primary tumor volume doubling timeand their impact on the treatment outcomestatisticsthe study protocol was approved by the republic ofslovenia national medical ethics committee no for retrospective studies a written consentis deemed unnecessary according to national regulationsbasic descriptive statistics were reported with meansstandard deviations and ranges for numerical variablesand as percentages for categorical variables in patientswith two simultaneous hncs some characteristics werereported in regards to patients while others were reported in regards to tumorsthe survival curves were computed using the kaplanmeier estimator and the aalenjohansen estimator wasused to estimate the cumulative probabilities of competing risks the effect of covariates was analyzed using amultiple cox regression analysis with all the analysesthe data were censored at a fiveyear followupwhen focused on the survival of patients the analyseswere completed with patients as the units and the timewas measured from the first day of therapy until deaththe overall survival os regardless of the cause ofdeath and the absolute risk cumulative probability ofdying due to index cancer were reported in the cox regression only the index cancer deaths causespecifichazard csh were considered to be events of interestin the analyses where locoregional control lrc wasof primary interest the calculations were performed in 0cžumer radiation oncology page of regards to tumors excluding the nonresponders to rtie those with residual local or regional tumors at weeks after rt completion for the latter group weconcluded that it is the radioresistance of tumor cellsthat are responsible for the persistence of the diseaseafter therapy and not that patients had to wait for rtthe followup time was calculated from the last day oftherapy the estimated cumulative probability of localandor regional recurrence distant metastases lrcprobability of being still alive and without local andorregional recurrence and diseasefree survivaldfsprobability of being still alive and without events locoregional and distant failure and deaths were the events ofinterest were reported all the analyses were conductedin regards to the tumors as independent units this assumption was checked in the sensitivity analysis andallowed for gamma frailtythe assumptions of the cox regression were checkeda nonlinear effect a spline with degrees of freedomwas allowed for the numerical variables and the proportional hazards assumption was tested using schoenfeldresidualsthe tti was defined as the time interval between thedate of histopathologic diagnosis and the first day of thert course tumor growth kinetics was expressed as thetumor volume relative increase rate per day and as thetumor volume doubling time in daysthe tumor volume relative increase rate per day was 13 calculated as 12v t 2ðþþv t 1ðt2 t1where vt1 gross tumor volume at time t1 ieon diagnostic ct scans and vt2 gross tumor volume at time t2 ie on planning ct scans it was reported as the percentage increase was reported as per day the tumor volume doubling time was calculated asðv t 2ðv t 1ðln 2ð þ t t þþlnþsince a onetoone relationship existed between thetwo only the tumor volume relative increase rate whichrequires no extrapolation was considered for modellingall analyses were performed using r statistical software version and a pvalue below was considered statistically significantresultsimpact of time to treatment initiationbetween and patients with oral cavity oropharyngeal hypopharyngeal or laryngeal primarysccs were treated with definitive chemo radiotherapywith curative intent there were men and women aged years mean the majority oftumors originated in the oropharynx tumors in patients and were tnm stage iv tumors in patients the tti ranges from to days with mean days the distribution of the tti is only slightlyasymmetric with median and interquartile rangeiqr fig patients were irradiated with dimensionaltechnique or 3dimensionalconformal isocentric technique to the median rt dose gy iqr delivered in gy dailyfractions iqr concurrently to rt patientsreceived chemotherapy consisted of platinbased monochemotherapy patients or mitomycinecbleomycin combination patients the characteristics of patients and their tumors are shown in table treatment outcome and survivalclinical andor radiologic assessment at weeksposttherapy confirmed disease persistence locally andor regionally in cases ie in patients and thesepatients were excluded from further analyses of lrcand dfs thus patients with tumors were analyzed and during followup a local andor regional relapse was recorded in cases with a mediantime from treatment completion of months range months the two and fiveyear probability of localrecurrence after the end of treatment was and respectively whereas the regional relapse was and respectively after threeyears posttherapy we only observed a small increase ie inlocal recurrence probability whereas the probability ofregional relapse was stable after the third year followupthe probability of occurrence of distant metastases at years was and at years it was at the second and fifthyear followup the lrc was confidence interval [ci] and ci respectively and the dfs was ci and ci respectivelyin the fifth year posttreatment out of patients had died the index cancer was the reason in of the cases during the first years posttherapythe probability of cancerrelated death steeply increasedfrom zero to but later the changes were less pronounced at the second and fifth year posttreatment theos independent of the cause of death was ci and ci respectivelyeffect of covariatesin the univariate analysis the following parameters weretested for lrc and csh age gender stage of diseasetype of treatment rt vs rt cct and tti for rtas continuous variable results are presented in table 0cžumer radiation oncology page of fig distribution of ttitable clinical characteristics of patients and their tumorscharacteristicsgenderno femalemaleage yearsatumor locationboral cavityoropharynxhypopharynxlarynxtumor stagebstage istage iistage iiistage ivtreatmentbradiotherapyconcurrent chemoradiotherapyrt duration daysatime to treatment interventiona ± ± ± the same parameters were also included in the multivariate model table the occurrence of locoregional recurrence was significantly related to the diseasestage p whereas the relationship with agewas only of marginal significance p higherage had a lower hazard no significant associationwith the type of treatment could be found p table the log hazard for locoregional recurrenceseemed to linearly increase during the first days oftable effect of covariates on locoregional control and indexcancerspecific hazard n patients with tumors anunivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherap cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt a mean ± sd rangeb eleven patients had two simultaneous primary tumorsci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapy 0cžumer radiation oncology page of table impact of tti on locoregional control and indexcancerspecific hazard n patients with tumors amultivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt ci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapywaiting for rt although the association between thehazard and tti wasregardless ofwhether we made an allowance for nonlinearity ornot fig 2ainsignificantthe hazard of dying due to the index cancer ie cshwas found to be favorably associated with a lower disease stage p and the addition of cct to rtp whereas age gender and tti did not reachthe level of statistical significance table fig 2bassumptions and sensitivity analysisproportional hazards for all included variables and thelinearity assumption for age were analyzed and presented graphically some indication of nonproportionalhazards could be found in gender but allowing for nonproportional hazards did not change the interpretationof the other covariates as a part of the sensitivity analysis the assumption of the dependence of the tumorsbelonging to the same individual was relaxed but nochanges in the interpretation of the results could be observed as an additional confounder the timedependentcovariate duration of rt was considered but its effectdid not prove to be importantimpact of tumor growth kineticsdiagnostic and planning ct scans median interval days range days were available from patientsfive of these patients had two primary tumors the majority of patients were males with a meanage of years range and the patients hadprimary tumors located in the oropharynx table when two ct sets were compared the original tstage of the primary tumor was increased ie upgradedin two cases and the nodal stage was increased in six patients due to the limited number of npositive casesn only volumes of primary tumors werecompared between the two ct sets for the calculationof the tumor volume relative increase rate per dayand tumor doubling time the absolute increase intumor volume ranged from to cm3 per daymedian no increase in volume was observed infive patients ct scans in these five patients were takenfig trend of the hazard for locoregional recurrence a and for index cancerspecific death b 0cžumer radiation oncology page of table characteristics of patients with available diagnostic andplanning ct scanscharacteristicsgenderno femalemaleage yearsatumor locationboropharynxhypopharynxlarynxstagebstage istage iistage iiistage ivtherapybradiotherapyconcurrent chemoradiotherapytumor volume relative increase rate dayatumor volume doubling time daysaa mean ± sd rangeb five patients had two simultaneous primary tumors from days to days apart median days the median tumor volume relative increase rate was perday median range and the mediantumor volume doubling time was days range days no difference was observed when these two parameters were compared between different tumor stagestreatment outcome and survivalamong the tumors the time taken for local andorregional relapse and the occurrence of distant metastaseswere assessed for tumors in which treatment resultedin clinicalradiological eradication of the disease at weeks posttherapy local andor regional relapse wasrecorded in nine cases with a median time fromtreatment completion of months range monthsthe two and fiveyear probability of local recurrenceafter the end of the treatment was and respectively whereas for the regional relapse it was and respectively at years posttherapy only anincrease in the local recurrence probability of wasobserved whereas the probability of regional relapseremained stable the two and fiveyear probability ofdistant metastases was at two and years thelrc was ci and ci respectively and the dfs was ci and ci respectivelyin the yearperiod after the start of treatment outof patients died the index cancer was thereason in cases at two and years posttreatment the os rates were ci and ci respectivelyeventseffect of covariatesdue to the low number of events local andor regionalrecurrence ninecancerspecific deaths events only univariate regression models were fittedthe following covariates were tested in the modelsoverall disease stage initial tumor volume tumor doubling time and relative increase rate day of primarytumor volume measures of tumor kinetics did not showany impact on lrc or csh only an inverse relationshipbetween the initial primary tumor volume and csh wasobserved hr for index cancerspecific death perevery cm3 increase in the volume of primary tumorstable there was no difference in the value of dfsbetween patients with a primary tumor volume relativeincrease rate 1day and 1day p fig discussionwhile it is intuitively anticipated and confirmed by theresults of the metaanalysis that a delay in starting rtwould have a negative impact on the treatment of patients with hnc this association was not confirmed inour study we only found a statistically insignificantupward trend in the risk of locoregional recurrence forthe first days of rt delay in addition the differencesin the growth kinetics between individual tumors whichwere studied in a smaller group of patients were considerable but did not appear to be of significance for theprediction of treatment outcomesobviously the relationship between tti and diseaseprognosis in patients with hnc is more complex than itmight seem at first glance the first negative impact ofwaiting for treatment to begin is the risk that the tumorwill increase in size andor metastasize during this timemaking it harder to treat or resulting in it becoming untreatable [ ] however the time for a tumor to growis only one of the factors that determines prognosis andthe absence of a statistically significant association between tti and the treatment outcome in our study andis thus not surprising []many other reportsmoreover no obvious methodological differences couldbe found between these negative studies and the positive studies that confirmed the association between ttiand treatment outcomes [] in both groups thereare individual studies that have similar sample sizestumor sitestage mix and periods covered speaking infavor of comparable quality of diagnostics therapy andstatistics across the studies 0cžumer radiation oncology page of table effect of stage and tumor kinetics on locoregional control and overall survivalparameterstnm stageiv vs iiiivtuper cm3vtu relative increase rateper dayvtu relative increase rate¤ vs 1dayvtu volume of primary tumorlocoregional controlhr cipvaluecause specific hazardhr cipvalueon the contrary in more recently reported analyses ofthe national cancer registries data an adverse effect ofwaiting for radiotherapy was clearly established []however the results from this type of analysis should betaken with caution not only due to the limitations inherent in the tumor registry data unmeasured confounding selection biasincomplete data and codingerrors but also because the effect of delaying rt oncancerspecific outcomes was not evaluated in additionpatients irradiated in postoperative and definitive settings were not analyzed separately overall the researchmethodology and interpretation used in these studieswere criticized and the magnitude of the effect that theysupposedly demonstrated was questioned in the present study a linear increase in the log hazardfor locoregional recurrence was found during the first days of waiting for rt although it was not statisticallysignificant it is possible that unknown confounders thatwere not accounted in our analysis eg tumor growth kinetics reduced the statistical power of the tti the resultsfrom fortin and naghavi who reported on theincreased risk of locoregional failure with tti daysand days respectively [ ] are the most comparable to our own however optimal ttithresholdsfig impact of primary tumor volume relative increase rate to diseasefree survival in patients with no residual disease at weeks posttherapy 0cžumer radiation oncology page of identified in different studies showed considerable variations pointing to the uncertainty of such calculations andtheir dependence on the characteristics of the analyzedpopulation [ ] the possible role of classical prognostic factors such as location of primary tumor diseasestage and addition of cct is not expected to be relevant inthis respect as in our and other similar studies the statistical significance of tti was verified by multivariateanalysisthe effect of tti on treatment outcomes however isnot only conditioned by the duration of waiting for rtbut also on the rate of tumor growth in hnc a vast heterogeneity in tumor cell kinetics has been observed conditioned by the local milieu from which it arises whichdiffers from patient to patient [ ] historicallydifferent methods were explored to evaluate tumor cellkinetics but did not succeed in providing clinically relevant kinetic parameters [ ] more recently a comparison of two sets of imaging data acquired at twodifferent time points was successfully employed for thispurpose usually volumetric data are extracted from diagnostic and rtplanning ct scans for the calculation ofdifferent parameters reflecting the rate of tumor growtheg tumor volume doubling time or absolutepercentagetumor volume increase per day despite some differencesin the calculation methodology across studies a large variation in the individual values of kinetic parameters wasseen in all of them including ours indicating that all ofthe studied populations represent a unique mix of slowand fast growing tumors [] among our patientsfive had no measurable increase in primary tumor volumeeven when the interval between ct scans was up to days the inverse relationship between kinetic parametersdetermined by the comparison of two ct datasets andtreatment outcomes was implied or even confirmed inseveral smaller studies pointing to potential clinical utilityof imagingderived kinetic data [] in our grouphowever no such association was found a small numberof patients and in contrast to other studies the inclusion of different tumor sites could contribute to the negative result as well as differences in the changes in kineticproperties triggered by rtccr in individual tumors tumor radiosensitivity may also influence the effect oftti on outcome while to some extent it may be evaluated before rt begins eg by molecular profiling identification of hypoxic cells and the determination of hpvstatus in oropharyngeal primary tumors in daily clinicalpractice it is usually not considered when planning treatment [ ] in order to diminish the impact of intrinsic tumor radiosensitivity the patients in our study withresidual disease at weeks postrt were excludedfrom the analysis of lrc we hypothesized that the inability to achieve a complete tumor response to chemoradiation was due to the radiobiological characteristicsof the disease and not the delay in starting rt howeverthe exclusion of these patients from the analysis did notaffect the end resultsour study has weaknesses which are mostly due to itsretrospective nature however for obvious ethical reasons this is an inevitable feature of studying delays inthe initiation of cancer therapy we are aware that thereis always some doubt as to the accuracy of the diagnosticprocedures the staging the quality of planning and theimplementation of rt in the case of retrospective research nonetheless the same restraint exists in the caseof other similar studies and even more so in the case ofanalyses of cancer registry datasets an important feature of our data set is that no patients were lost duringthe followup and in our opinion is free from manyhidden biases that larger studies inevitably bring withsince almost half of our patients had oropharyngeal carcinoma missing information regarding the p16 or human papillomavirus hpv tumor status could be ofimportance howeverin the cohort of oropharyngealcancer patients treated at our institution between and only had a hpvrelated tumor thus we reasonably assume that the impact of p16hpv tumor status on the study results was negligible inaddition in hpvpositive cases perni found nosignificant association between tumor growth velocitycalculated from serial pretreatment ct scanslocaland distant control or os and the same was reportedby chu who measured metabolic growth velocityusing pretreatment petct scans [ ]other drawbacks to consider include technical limitationsin ct scan acquisition the accuracy of presentation of actual tumor volume on ct images and the precision of thedelineation of gross tumor volume to minimize errors inthe estimation of comparative tumor volumes only highquality pairs of ct images were selected for the analysis oftumor kinetics in addition physical findings documentedin clinical records and when available diagnostic mriswere used for this purpose and labelled tumor volumes represented a consensus between two experienced radiationoncologists and a radiologist all dedicated to hnc management like many other studies [ ] the comorbidity burden was not registered in our patientsalthough it should be taken into account when assessingsurvival outcomes finallylags in the prebiopsyperiod were not addressed in our study including patientdelay and delays in referral and diagnostics which may addsignificantly to the total tti these are also costly and potentially fatal but can be successfully reduced by effectivecoordination between providers []sthe relationship between tti and treatment outcomesis multifaceted so the controversy of published results is 0cžumer radiation oncology page of not surprising in this study we found that delays in theonset of rt do not harm all patients as tti is a problem in many public health systems further research iswarranted and should focus on two areas evaluatinglarge population surveys with highquality data andtreatmentrelated outcomes not just os and the prognostic relevance of imagingderived kinetic data of individualtumors which appeared promising in severalsmaller and statistically underpowered studies in orderto obtain a tool to identify patients at increased risk oftreatment failure due to delays in starting rt in a situation without clear knowledge to whom waiting for irradiation is harmful the only possible recommendationcould be that the waiting time for rt should be asshort as reasonably achievable asara abbreviationshnc head and neck cancer rt radiotherapy tti time to treatmentintervention scc squamous cell carcinoma cct concurrent chemotherapyct computer tomography mri magnetic resonance imaging os overallsurvival csh causespecific hazard lrc locoregional control dfms distantmetastasisfree survival dfs diseasefree survival iqr interquartile rangeci confidence interval hpv human papillomavirusacknowledgementsthis study was financially supported by the slovenian research agencyprogram no p30307authors contributionsstudy concepts žumer b strojan p study design žumer b pohar perme mstrojan p data acquisition all authors quality control of data strojan p dataanalysis and interpretation pohar perme m strojan p statistical analysispohar perme m manuscript preparation all authors manuscript editingstrojan p manuscript review žumer b pohar perme m strojan p the authors read and approved the final manuscriptfundingthis study was financially supported by the slovenian research agencyprogram no p3availability of data and materialsthe datasets analyzed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study protocol was approved by the republic of slovenia national medicalethics committee no all patients gave consent for using theirdata for study purposes at the start of their treatment for retrospective studies awritten consent is deemed unnecessary according to national regulationsconsent for publicationthe republic of slovenia national medical ethics committee approved thestudy which was conducted in accordance with the ethical standards laiddown in an appropriate version of the declaration of helsinki theneed for consent was waived by the republic of slovenia national medicalethics committeecompeting intereststhe authors declare that they have no competing interestsauthor details1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia 2institute of biostatistics and medicalinformatics faculty of medicine university of ljubljana ljubljana slovenia3department of radiology institute of oncology ljubljana ljubljanaslovenia 4chair of oncology faculty of medicine university of ljubljanaljubljana sloveniareceived may accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin world health anization who life expecta
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inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the p65 and actin cytoskeleton regulatory pathwaysNaze G Avci1 Sadaf EbrahimzadehPustchi1 Yasemin M Akay1 Yoshua Esquenazi2 Nitin Tandon JayJiguang Zhu Metin Akay1Glioblastoma GBM is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy GBM tumors show nuclear factorκB activity that has been associated with tumor formation growth and increased resistance to therapy We investigated the effect of inhibitor BAY with Temozolomide TMZ on the signaling pathways in GBM pathogenesis GBM cells and patientderived GBM cells cultured in 3D microwells were cotreated with BAY and TMZ or BAY and TMZ alone and combined experiments of cell proliferation apoptosis wound healing assay as well as reversephase protein arrays western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells The results revealed that the cotreatment significantly altered cell proliferation by decreasing GBM viability suppressed pathway and enhanced apoptosis Moreover it was found that the cotreatment of BAY and TMZ significantly contributed to a decrease in the migration pattern of patientderived GBM cells by modulating actin cytoskeleton pathway These findings suggest that in addition to TMZ treatment can be used as a potential target to increase the treatments outcomes The drug combination strategy which is significantly improved by inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatmentGlioblastoma multiforme GBM is the most malignant primary brain tumor in the central nervous system Current standard of care therapy includes surgery followed by radiotherapy and concomitant and adjuvant chemotherapy with the alkylating agent Temozolomide TMZ which provides survival benefits for patients with GBM1 However even with the advances in surgical resection combined with TMZ therapy and irradiation the prognosis for newly diagnosed GBM patients remains poor In fact due to its rapid proliferation increased invasion and migration capacity and chemoresistance to the alkylating agents a0the median survival is only a0months with the Stupp regimen radiation with daily TMZ a0weeks followed by cyclic TMZ2 and 5year survival rate is less than which is the lowest longterm survival rate of malignant brain tumors3 TMZ methylates DNA at the O6 positions of guanine and DNA repair enzyme O6methylguanine methyltransferase MGMT removes alkyl groups from O6 position of guanine in DNA making cells resistant to TMZ6 Therefore new therapies are necessary to prevent cell proliferation and induce apoptosis for GBM patientsNuclear factorkappa B NFκB is a regulatory transcription factor of the Rel gene family including p50 cRel RelB or p65 subunits It is involved in the control of tumor cell proliferation migration immune response and apoptosis7 Studies have shown that NFκB gene was involved in the regulation pathways of different cancer types such as thyroid cancer head and neck squamous cell carcinoma and colorectal cancer711 Increased 1Department of Biomedical Engineering University of Houston Cullen Blvd Houston TX USA 2UTHealth Neurosurgery McGovern Medical School Memorial Hermann at Texas Medical Center The University of Texas Health Science Center at Houston Houston TX USA email makayuheduScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cactivation of NFκB has also been identified in GBM tumors where the expression of NFκB was much higher in GBM tissue compared with nonGBM tissue1415 NFκB also promotes chemoresistance to TMZ and regulates MGMT activity in GBM by promoting MGMT gene expression through NFκB binding sites within the MGMT promoter16 NFκB inhibitors such as parthenolide do not completely eradicate tumors therefore they are mostly used in combination with other drugs17 When used in combination with TMZ NFκB inhibitor parthenolide has been shown to activate mitochondrial apoptosis signaling in U87MG and U373 GBM cells which lead to cell death18 and had a combined effect on cell cytotoxicity in LN18 and T98G glioma cells19 NFκB inhibitor CBL0137 has been shown to bind DNA leading the functional inactivation of the Facilitates Chromatin Transcription FACT complex a chromatin remodeling complex regulating transcription replication and DNA repair2021 In a0vitro evaluation of the CBL0137 on FACT p53 and NFκB has been done using U87MG and A1207 GBM cells It was shown that CBL0137 induced loss of chromatinunbound FACT activated p53 and inhibited NFκB dependent transcription21 In a0vivo studies showed that CBL0137 was effective in increasing survival rates in TMZresistant orthotopic mouse models21 Moreover Wang et a0al indicated that NFκB inhibitor BAY suppresses the expression of MGMT and enhances the TMZinduced apoptosis in TMZ resistant U251 cells22 However there is still a lack of characterization of the precise pattern of NFκB activation in combination with TMZ in GBM cell populations that have been a0surgically resected from patientsIn vitro and in a0vivo identifications and validations of molecular targets of GBM are important as they can progress into clinical studies Studies reported that combining multiple gene targets may prevent tumor growth and improve the treatment strategy for GBM23 Both Bay and TMZ exert antitumoral activities individually in different tumor types28 Therefore in this study we aimed to analyze functionally the combined effect of Bay and TMZ in different GBM cells For this purpose first we used our 3D PEGDAbased hydrogel microwell platform31 to provide reliable preclinical models that can recapitulate in a0vivo features of the GBM tumors We cultured GBM cells U87 and LN229 and patientderived GBM cells in 3D microwells for a more precise and personalized treatment approach We then treated GBM cells with Bay and TMZ in combination or alone Our results indicated that the cotreatment of Bay and TMZ significantly reduced cell viability in all three cell lines in correlation with a significant decrease in the spheroid size The levels of NFκB protein and its subunits p65 and p50 were also significantly decreased compared with the control and single drug applications Similar a0decreases in the cell viability and protein levels were observed in all three GBM cells Tumor biopsy samples could give more realistic information about how tumors respond to drugs when they are used for in a0vitro or in a0vivo studies35 Therefore we decided to continue our experiments with only using our patientderived GBM cells We treated patientderived GBM cells with Bay and TMZ or alone and analyzed specific cellular proteins along with their posttranslational modifications via reversephase protein arrays RPPA to elucidate the mechanism of action of the proteins3839 We observed that several cell signaling pathways including cell metabolism proliferation apoptosis were significantly affected by the combination of the drugs which were consistent with the literature4041 Furthermore our RPPA data revealed that there was a significant change in the modulation of actin cytoskeleton and following experiments including western blot analysis for the expression of FAK protein and wound healing assay for cell migration patterns confirmed the RPPA results We observed a significant decrease in both actin fluorescence intensity and migration pattern in the a0cotreated patientderived GBM cells To the best of our knowledge the effect of cotreatment of Bay and TMZ has never been studied previously on the actin modulation of patientderived GBM cells These results suggested that Bay and TMZ induced alteration in the a0actin filament anization by reducing the level of focal adhesion protein which might implicate in cell apoptosis The effect of Bay with TMZ necessitates further exploration to better understand its mechanism of action in GBM and potential therapeutic tools for GBM treatmentResultsCotreatment of Bay and TMZ reduced viability of GBM cells We used our previously a0published data to select the most effective drug concentrations for this study42 We cultured LN229 U87 and patientderived cells in the microwells for a0days where they formed 3D spheroids and we added a0µM of Bay and a0µM of TMZ in combination or alone Then we cultured the spheroids for more days with or without drug Control group did not receive any treatment The cell viability assay was performed on day after drug administration The results showed that the a0cotreatment significantly reduced cell viability of GBM cells LN229 and U87 and patientderived GBM cells cultured in 3D PEGDA microwells respectively as shown in Fig a01ac When they were used alone TMZ reduced cell viability to and p and Bay reduced cell viability to and in LN229 U87 and patientderived GBM cells respectively compared to control groups Fig a01d However when they were used in combination the viability of the cells significantly decreased to and in LN229 U87 and patientderived GBM cells respectively compared to control groups p Fig a01d Tumor cells are generally less sensitive to drug treatments in 3D cultures than in 2D cultures4344 This could reflect reduced compound access or differences in the response to cell death To confirm that cotreatment was more effective compared to single drug use we quantified the size of the spheroids using ImageJ45 Our data showed that after a0days of drug treatment the spheroids sizes were significantly reduced in the cotreatment by and in LN229 Fig a01e U87 Fig a01f and patientderived GBM cells p Fig a01g respectively compared to control group p When we compared the spheroids sizes of the cotreatment with TMZ alone there was a reduction of and in LN229 U87 and patientderived GBM cells respectively p Finally the spheroids sizes of the cotreatment compared with Bay alone showed a decrease of and in LN229 U87 and patientderived GBM cells respectivelyScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Representative images of the GBM tumor cells cultured in the PEGDA microwells ac LN229 U87 and patientderived GBM cells were cultured in the microwells for a0days respectively After day Bay and TMZ were applied either alone or in combination onto the cell spheroids Control group did not receive any treatment The cells were cultured with or without drugs additional more days The images were taken on Day Day and Day after the drug application to observe the disruption in the spheroids Dotted black lines represent the edge of the tumor spheroid Scale bars a0µm d Bar graph showing trypan blue staining for cell viability of LN229 U87 and patientderived GBM cells eg Spheroid size quantification was done using ImageJ for LN229 U87 and Patientderived GBM cells respectively Twotailed ttest followed by Wilcoxon test were done GraphPad Prism v5 Data represent the mean ± SD of three biological replicates p and p Suppression of activity in GBM cells by cotreatment of Bay and TMZ As a readout of NFkB activity after drug treatment we first quantitatively assessed the cytoplasmic activation of phosphorylated NFκB p65 subunit in both treated and untreated groups in all GBM cells NFκB pp65 subunit activity was observed in the control groups of all three GBM cells Fig a02a NFκB pp65 subunit activity decreased to and when TMZ applied alone and and when Bay was applied alone in LN229 U87 and patientderived cells respectively However the decrease in NFκB pp65 subunit was reduced to when LN229 U87 and patientderived cells respectively were cotreated p Fig a02a Bay specifically inhibits NFκB activation by blocking phosphorylation of IκBα46 In independent experiments we analyzed the abundance of phosphorylated NFκB p65 NFκB p50 and IκBα in all three GBM cells Qualitative and quantitative western blot analysis revealed that the exposure to Bay with TMZ significantly downregulated the abundance of NFκB p65 NFκB p50 and IκBα compared with control and Bay or TMZ alone Fig a02b Please note that loading controls were used for each experiment but only the representative loading control for p and tP65 and p and tP50 was presented Fig a02b The cell viability assay cells size and protein expressions in all three GBM cells revealed similar results without any dramatic change Therefore considering the importance of using patientderived tumor cells to elucidate the mechanism of drugs and respective signaling pathways35 we further continued our experiments using patientderived GBM cellsApoptosis was promoted by cotreatment of Bay and TMZ RPPA technology is designed for multiplexed antibodybased relative quantification where each array is tested with a validated antibody specific to a particular protein along with their particular posttranslational modifications47 In the attempt to elucidate the mechanism of action of Bay with TMZ by which NFκB subunits were modulated and to identify downstream signaling molecules we employed RPPA platform using our drug treated or untreated patientderived GBM cells RPPA results showed that many oncogenic pathways were altered by the drug treatments but more specifically by the cotreatment Fig a03a Decreased expression of NFκB was not only associScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 NFkB activity in LN229 U87 and patientderived GBM cell lines a NFkB p65 subunit activity in LN229 U87 and patientderived GBM cell lines respectively The cells cultured with or without drugs for a0days were collected from the microwells and subjected to ELISA Data represent the mean ± SD of three biological replicates p and p b Representative immunoblots LN229 U87 and patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels bottom panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p ated with changes in the a0NFκB pathway but also with apoptosis cell metabolism and proliferation which were confirmed by the analysis of downregulated RPPA proteins in Enrichr KEGG libraries4849 Fig a03c p One of the specific pathways given by RPPA was apoptosis Apoptosis is one of the important mechanisms that regulates cell death and suppress tumorigenesis Studies have demonstrated that Bcl2 family proteins can positively and negatively regulate apoptosis by regulating antiapoptotic protein Bcl2 and proapoptotic protein Bax4050 Our RPPA data using patientderived GBM cells showed that the fold change of Bcl2 relative to control was times higher in cotreated group TMZ alone Bay alone respectively Fig a03b In order to further confirm whether the expression of a0these proteins were downregulated by the cotreatment we performed western blot analysis Our results showed a similar decrease in Bcl2 protein expression in the cotreatment compared with the control and single drug a0treatment Fig a03d In contrast Bax protein fold change relative to control was times higher in cotreated group TMZ alone Bay respectively where we observed a significant increase after the cotreatment of Bay with TMZ compared with the control p Fig a03b Bcl2Bax ratio is a key indicator in susceptibility of the cells to apoptosis Western blot results confirmed the change in Bcl2Bax ratio in the cotreatment compared with the control group and single a0drug treatment Fig a03d Our RPPA data also showed a significant increase in the cleavedcaspase protein Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The effect of Bay and TMZ on signaling pathways in patientderived GBM cells a Heat map presentation of RPPA analysis showing the changes in the protein expression RPPA was performed on lysates treated with Bay and TMZ alone or in combination All relative protein level data points were normalized to the a0control group Red and green indicate up and down regulations respectively in the heat map The samples were run in duplicate n b Fold change of the a0selected proteins relative to the a0control group via RPPA Data represent the mean ± SD of two biological replicates p p Wilcoxon rank sum test c Analysis of downregulated RPPA proteins shows a a0significant activation in numerous Enrichr KEGG pathways The pathways were a0sorted by p value ranking d Representative immunoblot validation of significantly altered proteins involved in different KEGG pathways Patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p fold change relative to control times higher in the cotreatment compared with times higher in TMZ alone and times higher in Bay alone p Fig a03b To confirm if cotreatment triggered apoptosis correlated with caspase activation we performed western blot analysis with procaspase3 cas3 and cleavedcaspase3 Ccas3 We observed that Bay and TMZ induced apoptosis was associated with cas3 Fig a03d Please note that loading controls were used for each experiment but only the representative loading control for Bax cas3 and Ccas3 was presented Fig a03d Moreover another important mechanism of NFκB activation in GBM regulates through AKT phosphorylation of IκB Our RPPA data showed relative fold changes of in the cotreated group TMZ alone and Bay alone respectively p Fig a03b The western blot results also confirmed a significant decrease in the abundance of AKT pT308 Fig a03dTo further investigate whether cotreatment of Bay with TMZ can lead to glioma cell apoptosis and to confirm our RPPA and western blot results we performed apoptosis assay TUNEL The patientderived GBM cells were cotreated with Bay with TMZ or single drug treated and subjected to TUNEL assay to detect DNA damage Fig a04a The results indicated that TUNEL cells in the cotreatment were increased tenfold compared with control and and 24folds compared with TMZ alone and Bay alone respectively p Fig a04b Additionally in some TUNEL cells we observed a typical ring type chromatin aggregation underneath the nuclear membrane which suggested an early stage apoptosis51 Fig a04a red arrows There were also a few TUNEL cells that lacked the typical apoptotic ringlike nuclear structure indicating that they were either at a different stage of apoptosis or alternatively undergoing necrosis52 that we have not investigated furtherCotreatment of Bay with TMZ changed actin anization by inhibiting FAK phosphorylation and cell migration Actin filaments Factin are one of the main components of the cellular cytoskeleton which regulates actin dynamics and migration process in the cells The disruption of the actin cytoskeleton inhibits cell migration and adhesion53 Depolymerization or cleavage of actin lamins and other cytoskeletal proteins have been also found to be involved in cell apoptosis54 To confirm the RPPA results showing changes in the actin modulation pathway and to understand the mechanism that regulates cytoskeletal Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Apoptosis assay TUNEL a Fluorescent images of TUNEL cells in patientderived GBM cells TUNEL assay was performed on cells treated with Bay and TMZ in combination or alone in the microwells Cells were collected from the microwells trypsinized and replated into 8well chamber slides TUNEL cells green with ringlike nuclear stain are indicated with red arrows Nuclei were counterstained with DAPI blue b Numbers of TUNEL cells are presented as mean ± SD of three biological replicates p and p X20 objective Scale bars a0µmanization we treated patientderived GBM cells co treated with Bay with TMZ or single drug treated 3D spheroids collected from the microwells were stained with phalloidin green and DAPI blue Staining cells with fluorescently conjugated phalloidin is considered the most reliable method of accurately labeling Factin in fixed cells57 In the control group intact cells formed finemeshed networks with a distinct Factin anization on both day Fig a05a upper panel and day Fig a05a bottom panel In single drug treated cells actin was still found to be polymerized to filaments as it can be seen by its interaction with phalloidin at both days and However the cells which were cotreated with Bay and TMZ lost their Factin anization and their shape compared with the control and the single drug treated groups at day Fig a05a bottom panel Changes in the a0actin distribution within the cells were quantified by measuring the staining intensity using Fiji Macro ImageJ as described previously5859 At day we observed a a0significant decrease in the fluorescence intensity of phalloidin when the cells were cotreated with Bay and TMZ compared with the a0control and single drug treated groups p Fig a05b To investigate the drug related Factin mechanism we examined the levels of FAK protein following cotreatment or single drug treatment As shown in Fig a05c cotreatment significantly decreased the level of phosphorylated FAK compared with both control and single drug applications p Furthermore we investigated cell migration patterns of the patientderived cells that were cotreated with Bay and TMZ or single drug treated We collected 3D spheroids from microwells after drug treatment and replated them in 24well plate to perform scratch wound healing assay We noted a significant increase in cell density in the scratch area in both control and Bay alone after and a0h of scratch formation p Fig a06a Although compared with the a0control cells both cotreatment and TMZ alone groups showed a decrease in the cell migration into the scratch area after a0h we observed that after a0h the migration rate of the cotreated cells was significantly slower than the cells that were treated with TMZ alone p Fig a06b These results indicated that the disanization of actin microfilaments was concomitant with the cell apoptosis after the a0cotreatment of Bay with TMZDiscussionDespite the increase in the median survival of GBM patients from to months4 the clinical efficacy of standard of care therapy including TMZ chemotherapy combined with surgery and radiotherapy is still limited Due to challenges in treating GBM significant attempts have been made to develop single or combined drug treatments60 However given the cost long time frame and risks of failure associated with developing a new drug repurposing available drugs may be the most effective alternative therapeutic strategy Therefore it is important to evaluate potential drug combinations for GBM treatmentScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Changes in the actin cytoskeleton and migration pattern in patientderived GBM cells cotreated with Bay and TMZ or single drug treated in the microwells a Upper panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin green and DAPI blue Bottom panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin and DAPI Scale bars a0µm b Intensity of staining obtained with phalloidin was measured in each cell using ImageJ and displayed as boxplots with to confidence intervals A twoway ANOVA with Dunnetts multiple comparisons test was performed to determine statistical relevance Three biological replicates n p p c Representative immunoblots show the levels of FAK pTyr397 and total FAK in patientderived GBM cell lysates cotreated with Bay and TMZ or single drug treated for a0days in the microwells The levels of the proteins were quantified using ImageJ right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p Due to the cell repellent property of PEGDA hydrogel tumor cells can form aggregates at the bottom of the microwells and selfassemble into spheroids in each well within a0days following cell seeding313363 Compared with 2D monolayer cell culture 3D spheroids have an important advantage their larger size Thus often drug effects can easily be monitored over time by measuring the size and shape of spheroids4344 Additionally using 3D in a0vitro tumor models can better recapitulate in a0vivo features of the tumors We used PEGDA hydrogelbased microwell platform313363 in order to culture different types of a0GBM cells commercially available GBM cell lines LN229 U87 and a0patientderived GBM cells However we investigated the effect of the drugs on the patientderived GBM cells more in detail since growing tumors from tumor biopsy samples could give very detailed information about how tumors respond to drugs35 Considering the precious nature of the patient samples this platform which requires fewer cells compared with 2D monolayer cultures provides us with a robust tool to recapitulate in vivo features of GBM tumors and to test our drug combinationsNFκB is one of the major transcription factors associated with GBM and responsible for activating a series of cellular responses including cell proliferation survival invasion and apoptosis6465 Previous studies have shown that NFκB can activate Akt and promote cell survival and proliferation by downregulating the expression of phosphatase and tensin homolog deleted on chromosome ten1866 NFκB pathway can inhibit cell apoptosis by inhibiting a stressactivated protein kinase and a mitogenactivated protein kinase signaling pathway67 It can also be activated in response to treatment with cytotoxic drugs such as vinca alkaloids and topoisomerase inhibitors Several studies have demonstrated the activation of NFκB in GBM patientderived stemlike cells cultures96869 Moreover alkylating agents TMZ can activate NFκB through DNA damage pathway activation7071 The combination effect of Bay and TMZ have been showed in our previous study where we determined the most effective drug concentrations on GBM cells using our microfluidics platform42 Another study that investigated the combined effect of NFκB inhibitor BAY with TMZ showed that combined drug application induced TMZ resistant in U251 GBM cells22 However the characterization of the precise pattern of NFκB activation in different GBM cell populations from surgically resected tissues still remains elusive Therefore in this study we investigated the interaction of Bay with TMZ and their effects on the LN299 and U87 GBM cell lines as well as patientderived GBM cells in order to recapitulate NFκB activation as in a0vivo features of the GBM and its signaling pathways We applied a0µM of Bay and a0µM of TMZ3442 in combination or alone for all three GBM cell types First we observed a significant decrease in both cell viability and size of the spheroids in the cotreatment compared with control and single drug application Then we showed quantitatively and Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Cell migration of patientderived GBM cells by wound healing assay a Patientderived cells were cotreated with Bay and TMZ or single drug treated in the microwells trypsinized and replated in 24well plates After they reached to their confluency a scratch wound was formed with a 200μl tip and cells were incubated for the next a0h Images were taken 4x at a0h a0hr and a0hr Scale bars a0µm b The wound width was measured with ImageJ and the average wound width was shown Data represent the mean ± SD of three biological replicates p and p oneway ANOVA with Tukeys post hoc testqualitatively the expression of NFκB in all three GBM cell types a0We noted a significant decrease in the cotreated group compared with control and single drug application Our western blot data also confirmed the decrease in the abundance of pP65 pP50 and pIKBa that Bay has been shown to inhibit its phosphorylation46 However in the cotreated group the decrease was significantly higher compared to both control and single drug application This data showed that cotreatment of Bay and TMZ has more effect on the inhibition of NFκB pathway than Bay or TMZ alone and suggests a a0decreased downstream transcription of oncogenic proteins72 Although there were slight differences in the NFκB expression patterns in three different GBM cell types a0we focused on the patientderived cells in the rest of the study due to their ability to better recapitulate the genomic similarities to primary disease7374Proteins that interact with each other activate multiple pathways which can result in apoptosis according to tissue type and pathological condition Glioblastoma tumors express high levels of antiapoptotic BCL2 family proteins such as Bcl2 and BclxL which may cause glioblastoma cells to resist apoptosis75 The proapoptotic members of Bcl2 family such as Bax and Bak are necessary for their proapoptotic effect Interactions and the ratio between antiapoptotic Bcl2 and proapoptotic Bax are decisive factors in the induction of apoptosis7677 Active NFκB can prevent cells from apoptosis by stimulating the expression of genes and promoting cell proliferation Although patientderived GBM samples have been shown to be highly resistant to apoptosis77 our data revealed changes in the expression of various members of Bcl2 family and NFκB signaling pathway after cotreatment of Bay and TMZ Our RPPA results outlined distinct molecular profiles in which apoptotic P53 signaling and NFκB signaling pathways were significantly affected after the a0cotreatment These results supported that the inhibition of NFκB expression could inhibit the expression of Bcl2 and promote the expression of Bax thus promote apoptosis Our data also suggested the possible interaction between Bcl2 and p53 in Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Proposed schematic of the a0signaling pathways involved in Bay and TMZmediated inhibition in GBM patientderived cells The effect of combined therapy of Bay and TMZ was achieved through the inhibition of SrcFAKVinculin which regulate the cytoskeleton anization through MAPKs JNK and PI3KAKT signaling pathways Exposure to both Bay and TMZ also leads to receptormediated activation of Bax but not Bcl2 in the subsequent inhibition of the downstream NFκB transcription factor Inhibition of NFκB in turn causes cell deathregulating cell survival and death7778 The activation of extrinsic and intrinsic molecular pathways can lead to the proteolytic activation caspases The extrinsic pathway is triggered by proapoptotic ligands that activate cell surface death receptors and procaspase8 which in turn leads to the cleavage of caspase3 and apoptosis79 Our results determined that the a0cotreatment significantly inhibited the expression of caspase3 while the expression of cleaved caspase3 was increased Additionally TUNEL assay which detects DNA strand breaks which could occur as an event in the apoptosis showed a dramatic increase in the TUNEL cells after the cotreatment compared with the a0control and single drug application Altogether these results suggested that the inhibition of cell proliferation Bcl2 and caspase3 by a0the cotreatment of Bay and TMZ may occur through the NFκB mediated apoptosis and they might be tightly coupled8081The literature provides evidence that supports crosstalk between PI3KAktmTOR signaling pathway and NFκB which is downstream of Akt NFκB activation in GBM regulates through AKT phosphorylation of IκB resulting in an activated NFκB that translocates to nucleus8283 Our data showed that when Bay was used with TMZ there was a decrease in the abundance of PI3Kp110 AktpS473 AktpT308 and mTORpS2448 This preliminary data is important to suppo
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"what clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials as a result medical research and care have been centred on male physiology the assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 in the us national institutes of health nih mandated the inclusion of women in nihfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy preclinical research and drug development studies have also predominantly used male animal models and cells4 it is not surprising that a us government accountability office report found that eight of the ten prescription drugs withdrawn from the market between and posed greater health risks for women than for men most funding agencies from europe and north america have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentthis review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences we aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom vol august biological and environmental modifiers of chronic disease ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and mensex as a genetic modifier of biology and diseasesex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an x or a y chromosome resulting in an embryo carrying either xx or xy chromosomes this fundamental difference in chromosome complement eg genes outside the testisdetermining sry gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 first the y chromosome carries genes that exhibit subtle functional differences from their xlinked homologues eg zfy vs zfx and uty vs utx and also carries genes with no homologue at all eg sry in addition in men the x chromosome carries only maternal imprints ie epigenetic modifications made by the parent in generating the sex cellswhich alter the expression of genes in the offspring as women have x chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes random inactivation of one of the x chromosomes in female cells which prevents sex differences in x chromosome gene dosage causes another degree of sex difference in gene expression as some of these xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 sexspecific gene expression due to genomic search strategy and selection criteriawe searched pubmed for papers published in english between jan and june using sex or gender and the name of the disease of interest as search terms although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedlancet diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine and southeast louisiana veterans health care system medical center new orleans la usa prof f mauvaisjarvis md barbra streisand womens heart center cedarssinai smidt heart institute los angeles ca usa prof n bairey merz md national heart lung institute imperial college london london uk prof p j barnes md department of pharmacology and department of neurology college of medicine center for innovation in brain science university of arizona tucson az usa prof r d brinton phd department of medical epidemiology and biostatistics and center for gender medicine karolinska institutet stockholm sweden prof jj carrero phd channing division of network medicine and the division of pulmonary and critical care medicine department of medicine brigham and womens hospital harvard medical school boston ma usa d l demeo md neuroscience institute and department of biology geia state university atlanta ga usa prof g j devries phd department of psychiatry university of colorado school of medicine anschutz medical campus aurora co usa prof c n epperson md division of oncology department of medicine washington university school of medicine st louis mo usa prof r govindan md w harry feinstone department of molecular microbiology and immunology the johns hopkins bloomberg school of public health baltimore md usa prof s l klein phd department of biomedical metabolic and neural sciences university of modena andreview 0creggio emilia azienda ospedalierouniversitaria di modena ospedale civile di baggiovara modena italy prof a lonardo md department of psychiatry department of psychology and department of obstetrics gynecology university of illinois at chicago chicago il usa prof p m maki phd department of neurology mcgovern medical school university of texas health science center houston tx usa prof l d mccullough md berlin institute of gender medicine charituniversittsmedizin berlin berlin germany prof v regitzzagrosek md department of cardiology university hospital z¼rich university of z¼rich switzerland prof v regitzzagrosek center for womens health research divisions of general internal medicine and cardiology university of colorado school of medicine aurora co usaimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 thus fundamental sex differences deriving directly from genetic heterogeneity between the x and y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells these sex differences persist throughout life and are independent of sex hormones figure arguably the greatest source of differences between men and women comes from the y chromosomal sry gene which directs the development of a testis in men the ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 in humans the first surge occurs at the end of the first trimester of pregnancy because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood after this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations after puberty cells with androgen or afemale sexbrandom x chromosomeinactivation and escapexxxxxxxxxxxxxxxxxxxy chromosome complementmale sexsryctesticular testosterone surgefetal testistestosteroneohogenetic diï¬erences of male and female cellsepigenetic programming of male cellsfigure genetic causes of sex differencesa genetic sex differences start with cells carrying either xx or xy chromosome complement eg genes outside the testisdetermining sry gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells b random inactivation of one x chromosome in female cells causes another level of sex differences in gene expression some xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals c the y chromosomal sry gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy the testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling the combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women the combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure gender as a determinant of patients and doctors behaviour and as a modifier of health disease and medicinegender according to the global health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 gender is not a binary term it includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 in transgender people gender identity differs with the sex they were assigned at birth so far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 gender roles represent the behavioural norms applied to men and women in society which influence individuals everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 as such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender as such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom vol august review 0cdiseases this postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 in the genesispraxy prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 although beyond the scope of this review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20 sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 sex influences behaviours eg towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes figure summarises how sex and gender are interrelated in biology and diseasesex and gender differences in major chronic diseaseshaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the usa as an example figure note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 in most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this review focuses on how women differ from men we discuss some key aspects regarding the dimensions of men in a dedicated sectionheart diseaseepidemiology pathogenesis manifestations and diagnosisheart disease is the leading cause of death in the usa in heart disease accounted for · of all deaths for men and for · of all deaths for women figure ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences for example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women the strength of the association with cardiovascular risk factors differ by sex biological sexsex chromosomesepigeneticeï¬ectssex hormonesohohohobehaviour of patients and doctorssocietygender constructslifestylenutritional habitsexerciseperceived stresssmokingdiseasepathophysiologymanifestationresponse to treatmentdisease perceptionhelpseeking behaviouruse of health caredecision makingtherapeutic responsesex and gender diï¬erences in health disease and medicinefigure interrelation between sex and gender in health diseases and medicinebiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment sex also influences behaviours towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex gender constructs determine patients perception of disease helpseeking behaviour and individual use of health care gender constructs also influence decision making and trigger different therapeutic responses from providers biased by gendersystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33 the reasons for this disparity reflect the intersection between sex and gender first biological sex differences exist in the pathogenesis of ischaemic heart disease whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 a metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathprof j g regensteiner phd department of medicine department of paediatrics and department of neuroscience washington university school of medicine st louis mo usa prof j b rubin md center for the study of sex differences in health aging and disease geetown university washington dc usa prof k sandberg phd division of gastroenterology duke university medical center durham nc usa a suzuki md and durham va medical center durham nc usa a suzukicorrespondence to prof franck mauvaisjarvis diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine new orleans la usa fmauvaistulaneeduwwwthelancetcom vol august review 0cmale individualsother·heart disease·protection might disappear after menopause45 by contrast testosterone induces adverse cardiac remod elling in the male heart44chronic liver disease ·inï¬uenza and pneumonia ·suicide ·alzheimers disease ·type diabetes ·stroke ·cpd ·injuries ·female individualsother·septicaemia ·chronic kidney disease ·inï¬uenza and pneumonia ·type diabetes ·injuries ·alzheimers disease ·stroke ·cpd ·cancer·heart disease·cancer·figure percent distribution of the ten leading causes of death by sex usa adapted from heron25 cpdchronic pulmonary diseasesecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39heart failure affects of adults aged years and older and more women than men in absolute numbers4041 heart failure occurs at an older age and with less ischaemic causes in women than in men however hypertension and diabetes predispose older women to heart failure to a greater extent than men heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men by contrast heart failure with reduced ejection fraction affects more men than women women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction under stress premenopausal womens hearts develop less inflammation resulting in less fibrosis than mens hearts4243 this difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 this response to treatmentcompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33 an stelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 this treatment disparity between women and men can be corrected by improving emergency recognition of stelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47guidelines for the treatment of heart failure are similar for women and men24 however evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41cancersepidemiology pathogenesis manifestations and diagnosiscancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure more men develop cancer than women49 with few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than a male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 survival is also shorter for men than women across multiple cancer types the higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 it is unlikely to be the only cause after appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53the universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom vol august review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology sexspecific biology includes genetic differences xx vs xy chromosomes the incomplete xinactivation in female individuals which results in biallelic expression of xencoded female cells54 y chromosomeencoded oncogenes such as the rnabinding motif on y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 these mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 a crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer in glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 after puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer for example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant take colon cancer the second leading cause of cancerrelated death for example although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 this molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksresponse to treatmentin the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches for example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 the molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 in colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968chronic pulmonary diseaseepidemiology pathogenesis manifestations and diagnosischronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure it is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 the female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 the genetic epidemiology of chronic obstructive pulmonary disease copdgene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences future studies should focus on the contribution of maternally inherited factors such as mitochondrial and x chromosome genes to understand disease pathogenesis it is important to consider gender constructs as well smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced womens risk for developing chronic obstructive pulmonary disease73 from a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom vol august review 0casthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently asthma is more prevalent in prepubertal boys than girls regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 from puberty onwards more female than male individuals have asthma with an increased severity in middleaged women and a higher mortality rate76 this phe nomenon could be secondary to gender differences eg symptoms perception or healthseeking behaviours however biological sex plays a crucial role in asthma and sex hormones have a major impact on female asthma symptoms and severity after puberty75 worsening of asthma occurs in women before menstruation and is known as premenstrual asthma premenstrual asthma is more common in women with severe rather than mild asthma obesity rather than normal weight and a long rather than a short duration of asthma77 premenstrual asthma is hypoth esised to be due to a fall in progesterone and patients with a severe disease respond to progestogens78 during pregnancy approximately a third of asthmatic women exhibit a worsening of asthma a third show an improvement and the remainder are unaffected79response to treatmentthe menopausal transition represents a pivotal time of accelerated decline in lung function in women with chronic obstructive pulmonary disease and thus represents a sexspecific window for treatment intensification these observations also suggest that oestrogens protect from chronic obstructive pulmonary disease women exhibit greater expression of m2 over m3 muscarinic receptors and accordingly show greater improvements in lung function than men in response to the muscarinic anticholinergic bronchodilator ipatroprium80 pooled analyses of drug studies also suggest that women experience a greater improvement in quality of life than men after treatment of chronic obstructive pulmonary disease with a β2adrenoreceptor agonist combined with an anti cholinergic drug eg indacaterol and glycopyrronium81unlike chronic obstructiv
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mgat5 knockout ko in hek293 cells induces metabolic changes resulting in increased intracellular udpglcnac increasedglycolysis enhanced spare respiratory capacity and higher citrate ï¬ux from the mitochondria mgat5 ko cells express constitutively high mica mainly regulated onthe transcriptional level through opening of the chromatin at the mica promoter mica expression in mgat5 ko cells is dependent on citrate turnover and histoneacetylation blocking citrate ï¬ux inhibits mica expression in numerous cancer cell lines and we propose that this is a central metabolic regulation of mica andimmune surveillanceintroductionnatural killer nk and cd8 t cells monitor autologouscells for markers of tumorigenesis and stress these immunecells express the nkg2d receptor that recognizes nkg2dligands nkg2dls upregulated on the surface of transformedcells nkg2dl expression is in many ways a doubleedged sword upregulation of nkg2dls on cancer cellsenhance nk cell ltration and promote cancer cytotoxicity conversely numerous cancer cells maintain chronicnkg2dl expression and evade immune elimination bydownmodulating and impairing nkg2d receptor signalingcancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdacisheatshock or shortchain fatty acids scfas that upregulatenkg2dls to date studies have primarily focused ondelineating transient nkg2dl induction whereas not much isknown about regulation of their constitutive expressionmetabolic reprogramming is a central hallmark of cancercancer cells use aerobic glycolysis that was initially believedto be a result of dysfunctional mitochondria howeverlater advances have shown that cancer cells often use aerobicglycolysis alongside mitochondrial oxidative phosphorylationoxphos mitochondria are not merely the powerhouseof the cell but also provide metabolites for anabolic pathwaysnecessary for cell growth citrate can be exported from thetricarboxylic acid tca cycle for biosynthetic purposes inthe cytosol citrate is cleaved by atp citrate lyase acly togenerate acetylcoa and oxaloacetate oaa citrateis an inhibitor of glycolysis thus to maintain high aerobicglycolysis cancer cells require low cytoplasmic citrate moreover conversion of citrate by acly is a critical regulatorof gene transcription by producing acetylcoa for histoneacetylation several of these cancerassociated metabolicfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionproperties are shared with other highly proliferating cells suchas activated t cellsexpression of nkg2dls is associated with hyperproliferation and thus with highly active metabolism two studies havelinked nkg2dl expression to active glycolysis whereasone study reports that inhibition of glycolysis increased basalnkg2dl expression in breast cancer cell lines thesestudies emphasize a link to proliferative cell metabolism andsuggest that the role of glycolysis in nkg2dl regulation iscontextspeciï¬cnkg2dls fall into two groups the ul16 binding protein ulbp16 and the mhc class i chainrelated proteins aand b mica and micb surface expression of each nkg2dlis regulated individually and at all levels of protein biogenesis we have previously shown that surface expression ofspeciï¬c mica alleles depends on nglycosylation nacetylglucosaminyltransferase v mgat5 is an oncoproteincatalyzing the formation of 16branched nglycans thatpromote surface retention of glycoproteins but it is notknown if mgat5 regulates surface expression of mica growthfactor receptors are examples of mgat5 substrates and mgat5overexpression is associated with growth adhesion invasionand metastasis of cancer inhibition of mgat5 reducestumor growth enhances the antitumor responses by cd4 tcells and macrophages and promotes th1 diï¬erentiation in this study we examine the metabolic regulation of thenkg2dl mica we discover that mica was increased aftermgat5 knockout ko in a metabolically dependent way anduse this as a model to investigate the regulatory mechanismsof constitutive mica expression we ï¬nd that glycolysis andmitochondrial export of citrate promotes constitutive micatranscription in mgat5 ko cells a regulation that was alsoshown in several micaexpressing cancer cells in particularincreased mica transcription was associated with alteredchromatin accessibility of the mica promoter our ï¬ndingssuggest that citrate drives a metabolic stress that modulateschromatin accessibility to facilitate basal mica transcription andthereby regulate immune surveillancematerials and methodsanimalsfemale nmri mice to 10weeks old taconic lille skensveddenmark were used and all studies were performed inaccordance with the danish act on animal experimentationwhich implements directive 201063eu on the protection ofanimals in scientiï¬c research the studies were approved by theanimal experimentation inspectorate ministry of environmentand food denmark license no healthmonitoring was carried out in accordance with federation forlaboratory animal science associations guidelinesreagents pharmacological inhibitorsand dna constructspharmacologicalfrom sigmaaldrich werecompoundsnacetyldglucosamine glcnac a3286 pugnac a7229carbonylcyanide2dg d61345aminoimidazole4carboxamide2deoxydglucosetriï¬uoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulï¬de bptes sml0601 potassium hydroxycitrate tribasicmonohydrate hc sodium dihydrogencitrate sodium acetate s5636 oxaloacetic acid oaa o41266mercaptopurine monohydrate 6mp azaserinea4142ribonucleotideaicar a9978 nacetylcysteine nac a9165 sodiumpropionate p1880 sodium butyrate b5887 dmso d2438pbs d8537 etomoxir sodium salt was purchased fromcayman chemicals ann arbor mi united states bristol bms303141 wasunited kingdom the gfpmycmica and mica vectors containingthe coding sequences of mica or mica alleledownstream of a generic leader a gfp cassette and a myctag were provided by dr m wills university of cambridgecambridge united kingdom pgl3basic pgl3bluciferase vector was purchased from promega promegamadison wi united states e1751 micaï¬reï¬y luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c ocallaghan university of oxfordoxford united kingdom from tocris biosciencepuriï¬cation of peripheral bloodlymphocyteshuman peripheral blood mononuclear cells pbmcs wereisolated by histopaque1077 sigmaaldrich st louis mounited states separation from buï¬y coats obtainedfrom healthy blood donors the capital region blood bankcopenhagen university hospital copenhagen denmark toobtain peripheral blood lymphocytes pbls pbmcs weredepleted from monocytes by incubation with dynabeadsinvitrogen carlsbad ca united states as previouslydescribed pbls were activated in rpmi1640 withoutglucose gibco gaithersburg md united states supplemented with dialyzed fetal bovine serumfbs f9665 mm penicillinstreptomycin p4333 mmlglutamine g7513 mm sodium pyruvate s8636 and either mm dglucose g8769 or mm dgalactose g6404all purchased from sigmaaldrich pbls were activated withcd3cd28 beads invitrogen 11132d and 20uml hil2peprotech rocky hill nj united states for dayson day pbls were treated with ngml fr901228 nationalcancer institute bethesda md united states for hline pc3 and the keratinocytederived cellcell line cultivation and proliferationhuman embryonic kidneyderived hek293 cells the prostatecancer celllinehacat were purchased from american type culture collectionatcc manassas va united states nkg2d reporter cellct312 and the 2b4 parental cellline were kindly providedby chiwen chang trowsdale lab cambridge universitylines mdamb231 and mcf7 werethe breast cancer cellprovided by dr jos moreira departmentfor veterinaryfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressiondisease university of copenhagen denmark and henrikleï¬ers the state hospital copenhagen denmark respectivelythe cervical cancer cellline hela was provided by jesperjurlander the state hospital copenhagen denmark themelanoma cells skmel28 fm55m1 fm78 and fm86 andthe human colon adenocarcinoma cell lines ht29 and sw480were provided by dr per thor straten herlev universityhospital denmark hek293 mdamb231 and mcf7 cellswere cultured in dmem with glutamax gibco hela hacat pc3 fm55m1 fm78 fm86 skmel28 andsw480 were cultured in rpmi1640 sigmaaldrich r5886and ht29 were cultured in mccoys 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoys 5a for longterm cell culture inglucosegalactose cells were cultured in dmem medium withoutglucose gibco supplemented with dialyzedfbs mm penicillinstreptomycin mm sodium pyruvate and mm glucosegalactose all cells were kept at culture conditions¦c and co2 and were passaged every daysfor proliferation assay wt and mgat5 ko cells wereseeded in or cellswell for each experimentcells were counted in triplicate wells after and h usingthe biorad tc20 automated cell counter biorad herculesca united statesgene editingmgat5 ko cells were generated by zinc ï¬nger nucleasetargeting in hek293 cells and subsequent cloning and selectionwas performed as described previously hek293cells were transfected with mrna sigmaaldrich or µgof endotoxin free plasmid dna using nucleofection on anamaxa nucleofector lonza copenhagen denmark mgat5ko clones were selected by loss of reactivity with lphaand clones were conï¬rmed to have mgat5 mutations usingpcr and sequencinglentiviralmediated gene transfer was performed with anmgat5 encoding vector constructed by inserting the mgat5sequence generated as a bluntend pcr product from a vectorfrom hw university of copenhagen copenhagen denmarkinto an entry vector system using the pentr directionaltopo cloning kit invitrogen k243520k350020 followingmanufacturers protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were conï¬rmed by sequencing at euroï¬ns mwgoperons luxembourg colonies were ampliï¬ed and plasmidswere puriï¬ed with nucleobond xtra midi kit machereynagelduren germany mgat5 sequences were insertedinto plx302 lentiviral destination vector with lr clonase iienzyme mix invitrogen after proteinase k treatmentconstructs were transformed into dh5α using heatshock andsoc medium selected clones were ampliï¬ed and dna waspuriï¬ed using nucleobond xtra midi kit destination vectorswere checked for insertion using bsrgi digestion at ¦cmgat5coding lentiviral ps were packaged in hek293tcells transfected with a mix of µg pspax2 vector packagingvector µg pcmvvsvg envelope vector µg plx302vector carrying mgat5 and µl cacl2 to a ï¬nal volume of µl the dna mixture was complexed with µl 2x hbsunder constant air ï¬ow and the transfection mix was addeddropwise to hek293t cells in antibioticfree mediumcell culture medium was harvested days after transfection andviral p preparations were prepared by centrifugation at g for min lentiviral ps were added to cells andincubated for h cells were cultivated in puromycin µgmlselection medium for weeks functional mgat5 expressionwas validated by lpha bindingtransient transfectiontransient transfections were performed as described previouslyusing amaxa nucleofector device lonza dna wasintroduced to cells in µl nucleofector solution vlonza vca1003 and pulsed using the nucleofector programq001 for gfpmyctagged mica and micaconstructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbrieï¬y dnarna were prepared in µl cacl2 25mand adjusted to a ï¬nal volume of µl dna mixture wascomplexed with µl 2x hbs hepes nacl na2hpo4and added dropwise to cells scrambled sirnacontrol siidh1 and siidh2 ontarget plus smart poolswere purchased from ge healthcare dharmacon lafayetteco united statesfunctional assaysfor nkg2d downmodulation pbls were isolated as describedabove followed by depletion of cd4 cells using cd4 antibodyebioscience san diego ca united states anddynabeads mouse panigg invitrogen cd4depletedpbls were cultured in rpmi1640 sigmaaldrich r5886supplemented with human serum sigmaaldrich h3667 mm penicillinstreptomycin mm lglutamine and ngmlhil15 peprotech for days to enrich for nkcd8t cells nkg2d downmodulation assay was performed aspreviously described nkg2d ligands on eï¬ector cellshek293 wt or mgat5 ko cells were incubated with blockingnkg2dfc rd systems minneapolis mn united states1299nk or control igg1fc rd systems 110hg µgmlfor min at ¦c eï¬ector cells and target cells nkcd8t cells were mixed at indicated eï¬ectortarget ratios and spundown min g to allow conjugate formation after h cocultivation nkcd8 t cells were analyzed for nkg2d surfaceexpression by ï¬ow cytometry using accuri c6 ï¬ow cytometerbd bioscience franklin lakes nj united statesfor the reporter cell assay the nkg2dreporter cell line2b4ct312 and the parental control 2b4 cell line target cells were mixed with eï¬ector cells wt or mgat5 ko cellsthat were either blocked with nkg2dfc or control igg1fc asdescribed above eï¬ector and target cells were cocultivated atdiï¬erent et ratios for h gfp expression of target cellswas assessed with accuri c6 ï¬ow cytometer for in vivo assaytarget cells were labeled with vybrant did celllabeling solutionfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioninvitrogen v22887 according to manufacturers protocol andinjected intraperitoneally together with wt or mgat5 kocells in a ratio of each mice were usedper group target cells were harvested after approximately hwith peritoneal lavage and nkg2d activation of didpositivereporter cells were assessed as gfp expression with accuric6 ï¬ow cytometerwas assessed by accurate mass and retention time amrt plusfragment identiï¬cation at two collision energies and evdetailed acquisition methodology has been described previously udpglcnacudpgalnac detected peak screened byexpected calculated mass could be of either compound as thesetwo sugars could not be separated chromatographically hencehas been reported as a putative metabolite pending conï¬rmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturers protocolrandox crumlin united kingdom lc2389 reaction andanalysis was performed on an advia chemistry systemsiemens munich germanydntp levels were determined in cells harvested withtrypsinization and pelleted by centrifugation for g for min followed by resuspension of cell pellets in methanolfrozen in liquid nitrogen and boiled at ¦c for min sampleswere evaporated until dryness in a speedvac and whole cell levelsof dttp datp dctp and dgtp were determined using the dnapolymerase assay previously described lchrms metabolite proï¬lingto determine intracellular metabolite levels cell pellets from cells were resuspended in µl of cold methanolafter min sonication samples were prepared by svortex followed by min equilibration at room temperatureafter centrifugation at g for min at ¦c µl supernatants were collected transferred to ultrafreemccentrifugal ï¬lter devices merck millipore ltd cork irelandand centrifuged at g for min at ¦c from this µlwas transferred to lc vials and µl of each sample was pooledto a mixed qc samplelchrms was performed on a nity ii ultrahigh performance liquid chromatography uhplc systemcoupled to a ifunnel quadrupoletime of ï¬ight qtofmass spectrometer equipped with a dual ajs electrosprayionization source agilent technologies santa clara caunited states polar metabolites were separated on a sequantzichilic merck darmstadt germany column mm mm µm p size coupled to a guardcolumn mm mm µm p size and an inlineï¬lter mobile phases consisted of formic acid in water withsolvent a and formic acid in acetonitrile with solvent bthe elution gradient used was as follows isocratic step at bfor min b to b in min and maintained at bfor min then decreasing to b at min and maintainedfor min then returned to initial conditions over min and thecolumn was equilibrated at initial conditions for min the ï¬owrate was mlmin injection volume was µl and the columnoven was maintained at ¦c the acquisition was obtainedwith a mass range of mz for where full scan highresolution data is acquired at three alternating collision energies ev ev and ev positive and negative raw lchrmsï¬les were independently processed with an inhouse developedpcdl library for polar metabolites using proï¬nder version b06agilent technologies identiï¬cation of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular ï¬ux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density cellswell ¼ hbefore the experiment one hour prior to assay run cells wererinsed and switched to xf media agilent technologies with mm sodium pyruvate and mm glucose or galactose andincubated at ¦c co2free incubator for the mitochondrialstress tests ocr was measured under basal conditions andduring sequential injection of µm oligomycin sigmaaldrich µm fccp sigmaaldrich c2920 and µmrotenone rot sigmaaldrich r8875 µm antimycina aa sigmaaldrich a8674 reported basal respiration iscalculated from the third measuring point with ocr after rotand aa subtracted atpcoupled respiration display ocr afteroligomycin subtracted from the third measuring point andmaximal respiration is ocr after fccp with ocr after rotand aa subtractedfor measuring the eï¬ect of hc ocr was assessed h after aninjection of mm hc13c6glucose tracing experiment cells were incubated for h in dmem medium withoutglucose supplemented with fbs mm sodium pyruvateand mm uniformly labeled [u13c]glucose cambridgeisotope laboratories tewksbury ma united states clm incubation medium samples were collected and cleared bycentrifugation g for min cells were washed and detachedsterically intracellular metabolites were extracted in ethanoland centrifuged at g for min ¦c to separatethe soluble extract supernatant from the insoluble componentspellet cell extracts and medium samples were lyophilizedand reconstituted in water for subsequent biochemical analysesextract samples were adjusted to ph with hcl and evaporatedto dryness under nitrogen ï¬ow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modiï¬ed procedure from standards containing unlabeled metabolites of interest andcell extracts were separated and analyzed in a gas chromatographagilent technologies 7820a chromatograph jw gc columnhp5ms parts no 19091s433 coupled to a mass spectrometeragilent technologies 5977e the isotopic enrichment of themetabolites of interest was corrected for natural abundance of 13cusing the unlabeled standards and calculated according to data are presented as labeling of m x where m is the massof the unlabeled molecule and x is the number of labeled catomsin a given metabolite frontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionwestern blottingproteins were extracted using ripa buï¬er thermo scientiï¬cwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientiï¬c for minon ice lysates were sonicated times for s and clearedby centrifugation at rpm for min at ¦c proteinextracts were denatured at ¦c for min in nupage samplebuï¬er and dtt sigmaaldrich proteins were resolvedusing sdspage gels invitrogen and transferred tonitrocellulose membranes invitrogen ib301001 using the iblotdevice invitrogen for total protein stain membranes werewashed in ddh2o and stained with revert protein stainsolution licor biosciences lincoln ne united states according to manufacturers protocol membranes wereblocked in tbst blocking buï¬er licor biosciences probed with primary antibodies in tbs w tween and bsa overnight on a shaker at ¦c and washed intbs tween secondary antibody was from licorlicor biosciences and signals were visualizedby the odyssey fc imaging system licor biosciencesoglcnacylation was detected with rl2 oglcnacylationantibody abcam cambridge united kingdom ab2739 atpcitrate lyase acly was detected with rabbit acly antibodycell signaling and acly phosphorylation with rabbitphosphoacly ser455 antibody cell signaling flow cytometryadherent cells were detached in pbs w mm edta invitrogen or by pipetting cell surface staining was done aspreviously described and cells were analyzed on accuric6 ï¬ow cytometer bd bioscience antibodies used for thisstudy were mica rd systems fab1300a ulbp256 rdsystems fab1298p nkg2d rd systems fab139a ulbp1rd systems fab1380p ulpb3 rd systems fab1517aulbp4 rd systems fab6285a micab bd bioscience icam1 leinco technologies c170 mouse igg1antimyctag merck millipore micb rd systemsmab1599 or igg2b isotype control rd systems mab004detected with secondary antimouse igg biolegend san diegoca united states binding of ï¬uorescently labeledaf647lpha invitrogen l32457 and fitcepha vectorlaboratories burlingame ca united states fl1121 was usedto measure surface levels of complex nglycans all isotypecontrols were purchased from bd biosciencefor staining with mitochondrial probes neutral lipid stainsor 2nbdg uptake cells seeded the day prior toexperiment were washed once in pbs and incubated for minat ¦c and co2 in warm growth medium containing nmtetramethylrhodamine methyl ester perchlorate tmrm sigmaaldrich t5428 nm mitotracker green fm invitrogenm7514 or for h in growth medium with µm 2nbdginvitrogen n13195 bodipy invitrogen d3922 wasdiluted in warm serumfree medium in a dilution andshaken vigorously to solubilize the lipids immediately beforeloading into the cells for min cells were washed twice inpbs fbs and detached sterically prior to analysisthe soluble nkg2dfc receptor 1299nk rd systemsand igg1fc 110hg rd systems were labeled with zenonalexa fluor against human igg1 z25408 invitrogen priorto staining of melanoma cellsin forwardsidescatter plotsdata were acquired with an accuri c6 instrument usingaccuri c6 software and analyzed in flowlogic v721 inivaitechnologies mentone vic australia by gating on viablecellsfollowed bysingle cell gating by areaheightscatter plots fscafsch geometric mean ï¬uorescent intensity mfi values aredisplayed in ï¬gures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and puriï¬ed on directzol spincolumns zymoresearch irvine ca united states according to manufacturersprotocol cdna was generated using superscript cdnasynthesis kit invitrogen under standard pcr conditionsfollowing primersequences were used for quantitativertpcr with brilliant sybr green qpcr master mixkit mica mica_f tggcagacattccatgtttctgmica_r ctcgtcccaactgggtgttg ulbp2 ulbp2_f cagagcaactgcgtgacatt ulbp2_r ggccacaaccttgtcattctidh1 idh1_f ctatgatggtgacgtgcagtcg idh1_r cctctgcttctactgtcttgccidh2 idh2_f agatggcagtggtgtcaaggagidh2_r ctggatggcatactggaagcag glut1 glut1_fctgctcatcaaccgcaac glut1_r cttcttctcccgcatcatct glut2 glut2_f tacattgcggacttctgtgg glut2_r agactttcctttggtttctgg glut3glut3_f cagcgagacccagagatg glut3_r ttggaaagagccgattgtag glut4 glut4_f tgggcttcttcatcttcacc glut4_r gtgctgggtttcacctcctrplp0_fcctcgtggaagtgacatcgt rplp0_r cattcccccggatatgaggc realtime qpcr was performed on biorad cfx96 realtime thermal cycler c1000 touch and alltranscripts were normalized to housekeeping rplp0 transcripthousekeepingand rplp0asgeneluciferase reporter assaycells were transiently transfected using calciumphosphatetransfection as described above with ï¬reï¬y luciferase promotervectors µg and an sv40promoter driven renilla luciferasevector µg cells were harvested and snap frozen hpost transfection pellets were lysed in dualglo luciferasereagent promega e2920 and ï¬reï¬y luciferase activity wasanalyzed by luminometer microbeta ii perkinelmer walthamma united states renilla luciferase activity was recorded bythe instrument after subsequent addition of volume dualglo stop glo promega e2920 to correct for transfectioneï¬ciency ï¬reï¬y luciferase signals were normalized to sv40renilla luciferase signals of corresponding sampleatacseqatacseq was performed as described previously foreach cellline cells were harvested from separatefrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioncultures and used to prepare tagmented chromatin replicatesof wt and replicates of mgat5 ko cell lines samplestotal quality of pcrampliï¬ed sequencing libraries was assessedusing a tapestation instrument with high sensitivity dnascreentapes agilent libraries were sequenced as paired endreads on a single lane of an illumina hiseq4000 ï¬ow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were ï¬ltered toremove low quality duplicate and mitochondrial reads peakswere called using macs2 on merged reads from allsamples and diï¬erential peak accessibility between cell lines wasdetermined using edger with a threshold false discoveryrate of transcription factor binding motifs enriched indiï¬erentially accessible peaks were identiï¬ed using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantiï¬cation and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical signiï¬cance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in ï¬gure legends using unpaired ttest in 1a 1c 1e 3ef3h 5c 7a 7ef paired ttest in 4fg 7d multiple ttest in 1b1d 3d 4ab one sample ttest in 2ac 3c 4c 4e 7g twowayanova in 3a 5df 5hi 6a 6e 7hi or oneway anova in5g level of statistical signiï¬cance was determined by p p and p p resultsmgat5 knockout increases nkg2dlexpression and activates nkg2d in vitroand in vivoregulation of constitutive mica expression remains largelyunknown surface expression of certain mica alleles dependson nlinked glycosylation we questioned whetherthe cancerassociated glycosyltransferase mgat5 is required formica expression to assess the role of mgat5 in regulationof nkg2dl surface expression mgat5 ko clones weregenerated in hek293 cells remarkably mgat5 ko resultedin a permanently increased surface expression of the nkg2dlsmica micb and ulbp256 compared with parental wildtypewt cells figure 1a to conï¬rm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspeciï¬cally to mgat5modiï¬ed nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modiï¬ednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modiï¬cation of mgat5 expressiontherefore associated with substantial changes in constitutiveexpression of several nkg2dlsto verify the functionality of mgat5 koinduced nkg2dlswe tested nkg2d activation in a reporter cell line expressing1httpswwwencodeprojecthuman nkg2d coupled to dap10cd3ζ signaling and nuclearfactor of activated t cells nfatcontrolled gfp ultimatelyexpressing gfp in response to nkg2d activation nkg2dgfp activation was higher after cocultivation with mgat5ko cells than with wt cells figure 1b corresponding to theincreased nkg2dl expression in mgat5 ko cells figure 1athe reporter cells without nkg2d supplementary figure s1aremained inactivated indicating that the activation was nkg2dmediated figure 1b moreover blocking nkg2dls withsoluble nkg2dfc receptor impaired the activation furthervalidating nkg2d speciï¬city supplementary figure s1bto test if mgat5 ko cells could activate nkg2d in vivowe adoptively injected nkg2d reporter cells together with wtor mgat5 ko cells into the peritoneum of nmri mice andmeasured gfp expression in reporter cells in line with ourin vitro data we observed a signiï¬cant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspeciï¬c since the control reporter cellswere unaï¬ected figure 1c these data verify that mgat5 koinduced nkg2dls maintain their functional integrity in vivonkg2d is downmodulated upon activation to furtherexamine the functionality of nkg2dl expression causedby mgat5 ko we assessed nkg2d downregulation afterreceptor activation nkg2d was further downregulated oncd4depleted peripheral blood lymphocytes pbls after cocultivation with mgat5 ko cells than with wt cells and thisdownregulation was abolished by blocking nkg2dls with asoluble nkg2dfc receptor figure 1d combined these dataindicate that ko of mgat5 upregulates mica and ulbp256resulting in nkg2d activation in vitro and in vivoto ensure that the mica upregulation was a result of mgat5ko we stably transfected mgat5 into wt and mgat5 kocells lpha binding was restored within days after transfectionconï¬rming expression of functional mgat5 interestingly ittook multiple passages for mica expression to decrease to wtlevels figure 1e and supplementary figure s1c suggestingthat mica is regulated in response to a longterm adaptation toaltered mgat5 expressionudpglcnac upregulates micaexpressionlongterm mgat5 deï¬ciency willlikely result in aberrantnglycosylation and an accumulation of the mgat5 donorsubstrate udpnacetylglucosamine udpglcnac to addressif mica was regulated by a change in nglycosylation inmgat5 ko cells we assessed the posttranslational regulationof mica by measuring surface expression of transgenicallyexpressed gfpmyctagged mica under a cytomegaloviruscmv promoter the mica alleles mica and micaare distinctly regulated posttranslationally and althoughmica was upregulated in mgat5 ko cells the regulationwas minor and unlikely to account for the profound changein endogenously expressed mica figures 1a 2a micatranscripts on the other hand were highly increased in mgat5ko cells figure 2b as well as ulbp2 mrna supplementaryfigure s2a suggesting that nkg2dls are transcriptionallyfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionfigure mgat5 knockout increases nkg2dl expression and activates nkg2d in vitro and in vivo a surface expression of nkg2d ligands and binding ofï¬uorescently labeled lpha mgat5 modiï¬cations or epha mgat3 modiï¬cations on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by ï¬ow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean ï¬uorescence intensity mfi b in vitro nkg2d activation measured as gfp expression in nkg2d negative reporter cells control and nkg2dexpressing nkg2d reporter cells target cells cocultivated with wt or ko cells effector cells for h at indicated effectortarget et ratios c nkg2dactivation in vivo measured on reporter cells as in b after activation by wt or ko at a ratio in peritoneum of nmri mice for approximately h gfp expressionin didlabeled reporter cells signiï¬es nkg2d activation and is shown as gfp mfi values of cells from foursix mice per group d nkg2d downmodulation wasassessed on nkcd8 t cells target cells after cocultivation for h with wt or ko cells effector cells at indicated effectortarget ratios et nkg2dls on targetcells were blocked with nkg2dfc bl or unblocked with igg1fc un the graph depicts surface expression of nkg2d presented relative to surface nkg2dexpression on target cells alone e mica surface expression left and lphaepha surface binding right after lentiviral introduction of mgat5 into wt or kocells mfi values from antibody staining were corrected for isotype background staining 01mfi statistical analysis was performed by unpaired ttests in ace andmultiple ttest with fdr comparing wt and ko in bd p p p and p regulated in mgat5 ko cells notably we found that themgat5 substrate udpglcnac although indistinguishablefrom udpnacetylgalactosamine udpgalnac tended tobe higher in mgat5
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" the popularization of health and medical informatics yields huge amounts of data extracting clinicalevents on a temporal course is the foundation of enabling advanced applications and research it is a structure ofpresenting information in chronological order manual extraction would be extremely challenging due to thequantity and complexity of the recordsmethods we present an recurrent neural network based architecture which is able to automatically extractclinical event expressions along with each events temporal information the system is built upon the attentionbased and recursive neural networks and introduce a piecewise representation we divide the input sentences intothree pieces to better utilize the information in the sentences incorporates semantic information by utilizing wordrepresentations obtained from bioasq and wikipediaresults the system is evaluated on the thyme corpus a set of manually annotated clinical records from mayoclinic in order to further verify the effectiveness of the system the system is also evaluated on the timebank_dense corpus the experiments demonstrate that the system outperforms the current stateoftheart models thesystem also supports domain adaptation ie the system may be used in brain cancer data while its model istrained in colon cancer data our system extracts temporal expressions event expressions and link them according to actuallyoccurring sequence which may structure the key information from complicated unstructured clinical recordsfurthermore we demonstrate that combining the piecewise representation method with attention mechanism cancapture more complete features the system is flexible and can be extended to handle other document typeskeywords clinical text mining event extraction temporal extraction relation extraction piecewise representationattention mechanism precision medicine is an emerging approach for diseasetreatment and prevention it becomes the whole worldbiomedicine domain the research hot spot which needsthe support of biomedical methods eg data mining it correspondence clixjtueducn zhijing li and chen li contributed equally to this work1school of computer science and technology xian jiaotong universityxian shaanxi china2shaanxi province key laboratory of satellite and terrestrial network techrd xian jiaotong university xian shaanxi chinaassociates with key information extracted from clinicalrecords eg symptoms over a disease course the associations are often statistically concluded from the evidencecollected from the clinical records the medical bigdata mostly exists in an unstructured form eg textwhich could store useful information very well aligningbiomedical events in clinical data along the events actually occurring time is a meaningful and efficient way ofstructuring such complex data the result may assistauxiliary diagnosistreatment scheme determinationepidemic prediction and side effect discovery etc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cli bmc medical informatics and decision making page of many works have been devoted in the study of application in the medical era however large data analysis ofmedicaltreatment needs to map the correspondingmedical events in the clinical records the medical eventswith temporal information are very useful in medical erathese efforts will become the foundation of understanding disease facilitating the analysis of large medical dataas well for example the clinical record in fig may bepresented in a structured manner as the occurring eventsalong with temporal information it is easier for understanding the events and corresponding time point for example using the time point april as a referencethe entity bleeding is before the time point and the entitybolus chemotherapy and nausea is after the time pointin such case the actual events and their occurring consequence becomes clear at a glancein this paper we present a novel system which is builtupon deep neural networks to automatically extractevent expressions and their related temporal expressionsfrom clinical records the system has been evaluated onthe temporal histories of your medical event thyme corpus a corpus developed by a number of professionals according to characteristics of the corpusclinical data contains very long sentences that willundoubtedlythe difficulty of processingtherefore we do not simply use neural networks wewant to make full use of the contextualinformationour proposed method anically combines piecewiserepresentation and attention mechanism by a recurrentneural network rnn and achieves the stateoftheartperformance the results show improvements in automatic extraction of clinical event expressions along witheach events temporal informationincreaserelated workextracting clinical events along with temporal information is a complicated task and the existing systems oftenaccommodate several independent components each ofwhich retrieves different parts eg events and time andassemble them together each component may use a setof hand crafted rules or be based on a pretrained mlmodel velupillai develop the blulab systeminclude the cleartk support vector machine and conditional random fields classification approach and get thefirst place in semeval2015 task clinical tempeval macavaney present the system guir includeconditional random fields and decision tree ensemblesusing lexical syntactic semantic distributional and rulebased features guir receive the best score insemeval2017 task clinical tempeval in the way oftemporal expressions extraction tourille use aneural network based approach and achieve goodperformance for both event and relation extraction insemeval2017 task clinical tempeval lin propose a recurrent neural network with multiplesemantically heterogeneous embeddings within a selftraining framework for clinical temporal relation extraction task they achieve good results for both in andcrossdomainafter event and temporal expression extractionassigning each event with the right temporal expressioninvolves more complicated process some systemsmatch event with temporal expression by a set of syntactic rules crafted by experts wang use syntacticrulebased method for automatic pathway relation information extraction from biomedicalliterature these methods are fast but not flexible enough somefig the example of the medical information extraction the texts marked by the underscores ___ are the temporal expressions the textsmarked by the dash lines _ _ _ are the event expressions 0cli bmc medical informatics and decision making page of existing methods for medical relation information extraction are based on machine learning ml models[]conditional random field crf and supper vectormachine svm are often used in the task of relation extraction lu liu propose an svm model toextract the relations between the potential named entitypairs finkel propose a crfbased informationextraction system to determine relationships deeplearning revives the popularity of neural networkswhich can learn effective relation features from the givensentences withoutengineeringsocher is the first work that employs an rnnmodel to classify relation one early work proposed byluo is based on a recurrent neural network and ableto classify relations from clinical notes compared withthe rule based methods these methods are more flexibleneural network based methods take less time by quicklyscreening out the most unlikely candidate entitypairstherefore some approaches attempt to combine bothhence we think rnn is a good choice and we adopt thernn systemcomplicated featurein recent years attention mechanism has been widelyused in various tasks of nlp based on indepth learning li propose a model that combines abidirectional long shortterm memory network with amultiattention mechanism forrelation extractionzhou propose the attentionbased bidirectional long shortterm memory networksattblstmfor relation classification and the modelresults outperforms most of the existing methods withonly word vectors on the semeval2010 relation classification task so in this paper we also introduce theattention mechanismmethodologythe system consists of three components the first component extracts temporal expressions temporal expressions enable events to be chronologically annotatedthe second component identifies the relevant medicalevents event expressions any situation relevant to thepatients clinical timeline the third component detectsthe relations between the events and the temporalexpressionsannotating clinical records are very expensive frequently only a data of disease specific type is availablebesides the regular mlbased extraction we introducedomain adaption to allow the system to be able to extract the information from one type of disease eg braincancer while it is trained on another type eg coloncancerwe show the pipeline of our system in fig thesystem is built upon an annotating pipeline adoptingthemanagementunstructuredinformationfig the dataprocessing pipeline of the system we first extracttemporal expressions and event expressions respectively frommedical records then extract the relations between themarchitecture uima framework the preprocessingincludestokenization partofspeech tagging andlemmatization which used the stanford corenlp toolkit in both time and event extractions spans oftime and event expressions are represented by the offsets in texts the automatic annotations of event expressions temporal expressions and their relations arebased on three rnn models utilizing lexical syntacticand semantic features [] at the core of deeplearning techniques for nlp lies the vector basedword representation which maps words to an ndimensional space for the choice of the word embeddings corpus we do some research work only entities time and event expressions in the clinical records can be found in the wikipedia in comparisonthe bioasq corpus which is full of biomedical information contains more than of the entities weuse word embeddings from the european projectbioasq obtained by using word2vec on pubmed abstracts [ ] and include the vectorsof distinct words each word is representedas a 200dimensional vector if a word could not befound in the bioasq corpus the embedding is generated from wikipedia by word2vec mikolov as a complement [ ]thestateinternalof rnn can demonstratedynamic timing behavior the hidden state vector can be computed by the following formulaht ¼ f wht þ uxtðþin this formula xt is the input ht is the hidden stateu and w are the weight coefficients f is the nonlinearfunction such as tanh or relu 0cli bmc medical informatics and decision making page of extraction of temporal expressions and event expressionsindependent models are trained for the extractions oftemporal expressions and event expressions figure shows the infrastructure of the system there are twoforms of temporal expressions one is numeric temporalexpressions eg etc and the other is casualtemporal expressions eg day weeks during aperiod etc firstly we generalize all the numeric temporal expressions into for example both and become which can be easily recognizedby the regular expression the regular expression is useddue to the characteristics of the data the numeric temporal expressions are not well recognized by rnn if wedo not use it secondly the casual temporal expressionsare recognized by a rnn model the casual temporalexpressions in the training set are tagged to representthe tokens position in a particular expression there are four tags in our proposed method includingb i o and e which state that the token is at thebeginning on the inside on the outside or at the end ofthe entity respectivelyin this section we propose the system arnn whichis based on a recurrent neural network combining theattention mechanism we need to predict the tokensposition tag of each word before we can train the arnnmodel to predict the type of each temporal expressionwe treat each temporal expression as an entity and eachentity is treated as a unit input we use the average valueof all the word embeddings of an entity in the nextprocess the network in the fig shows the flow chartof arnn network to predict the type of the entitythere is an example sentence from the corpus we willget a ct enterography to rule out crohns disease inthis case the given entity is enterography and thecontext words are we will get a ct to rule outcrohn disease in order to better apply the context information we employ the attention mechanism to learnthe weighted score of each context word related to thegiven entitythe higher weight the higher semantic is bound upwith the given entity 01 00αi exp vti u vhtuv is the parameter that has to be learned from fig we can see that st is the state vector that integrates theother context words information with the given entity attime t st can be computed asst ¼xihαihiwe combine st and h3 to obtain h0sent the given entity which can repre ¼ h3 þ sth0in this process the prediction of entitys type will bepredicted from the given input entity vector the methodof using regular expression are also used to match themissing temporal expressions eg similar to temporal expressions extraction the eventexpressions extraction is built on another rnn unliketime expressions event expressions are all single wordsthere is no need to sign each tokens position we usethe softmax classifier to predict the label y² of the temporal and event expressions the state vector h0 is usedas input and y² could be computed by 10 11py ¼ softmax w h0 ¼ arg maxypyyeventtime relation erer extraction is the most important task in this paperin this section we propose the novel system aprnnwhich is based on a recurrent neural network combiningthe attention mechanism and the piecewise representation the eventtime relation are regarded as a classification problem it is divided into four categories based onsome wellknown communities such as semeval orbionlp the event time relation associates the identifiedevent expressions and temporal expressions and indeedindicates the what and when of a medical event inclinical records the four types are before after beforeoverlap and overlapthe shortestsyntactic path used includespiecewise representationthe dependency parsing of each sentence has beenobtained by utilizing stanford corenlp toolkit thepreviously trained word embeddings which representeach word by a 200dimension word vector and theshortest syntactic paths are fed into the rnn modelof er extraction the word embeddings and shortestsyntactic path are used as features the informationofthewords the poss and the length we add all thesevectors as the entity feature after adding up we stillget a 200dimensional vector for each entity if theentity include severaltemporal expressions we add the vectors of each token and get theaverage vector as the entity vector the whole is divided into sentences as input units we extractall entity pairs based on the annotations given a sentence x x1 x2 ¦ xt the words are projected intoa sequence of word vectors denoted by e1 e2 etwhere t is the number of words in this part wewould like to introduce the piecewise representationtokens eg 0cli bmc medical informatics and decision making page of fig architecture of the rnn model for the extraction of temporal expressions and event expressions we propose the attentionbased rnnmodel to do the entity extraction on the left side of the figure is the details of the attention mechanism the right part of the figure is thernn modelinformation as shown in fig in other wordsthe input sentence is divided intothree parts according to the entity pair we call thisprocess piecewise representation the purpose ofpiecewise representation is to better use of the contextthe examplesentence is divided into three parts according to theentity pair will enterography the whole sentence iswe will get a ct enterography to ruleout crohnsdisease the first part is the sequence before the entity pair we the second part is the sequence between the entity pair get a ct and the third partis the sequence after the entity pair to rule¦ thereare reasons for segmenting the sentence the firstone is that in many cases some studies may choosethe sentence between the entity pair as input insteadof using the whole sentence nevertheless thiscan miss some information and some of them maybe useful only several words cannot supply enoughcontextfeatures segmented sentences can be used to extract the effectiveinformation forextractingrepresentsinformation to the greatest extent of each sequenceand avoid the absence of contextualinformationanother reason comes from the network and our corpus in the corpus the longest sentence contains words however the average length of all sentenceshas words with the rnn structure since the information of a sentence is learned word by word thefeature vector produced at the end of the sentenceactuallysentence althoughrnn has the memory in learning process but thememory time is not long accumulation by recurrentconnectionslongterm informationquickly and the feature vector atthesentence is hard to carry the information of earlysteps in model training there are many longdistancesentences more than words in the training dataso the piecewise representation can help the systembetter use the information of the sentence the syntactic analysis and pos information of the examplesentence are also shown in the fig the end ofentirethetendsto fet 0cli bmc medical informatics and decision making page of fig the flow of our proposed model aprnn on the left side of the figure is the details of the attention mechanism the right part of thefigure is the rnn model which is divided into three partsmodelthe er model contained three parts the first component the feature layer the second component thehidden layer catch the information of word sequenceand produces wordlevel features representations andthen merges wordlevel features into a sentencelevelfeature vector by selecting the most valuable featureinformation among allfeatures weshow the whole process in the fig in this part weproposeattention mechanism to obtain thethe wordleveltherepresentation of the sentence not all the words inthe context describe the er relation each word inthe context has different effects on the given entitypair therefore we introduce the attention mechanism to learn the weighted score of each context wordrelated to the entity pair for the first part the attention mechanism is used to screen the most useful information we use the bilinear operator to computethe attention weight αi for each vector h1 and h2 toreflect how the information relevant to the first entity 0cli bmc medical informatics and decision making page of in the entity pair the current state h3 the calculation method of αi and st is the same as the description in the extraction of temporal expressions andevent expressions sectionwe combine st and h3 to obtain h0 which can represent the sequence before the entity pair for the secondpart we choose the state of the last entity in the sequence ht to represent the whole sequence for thethird part the same method is used as the first part wealso use the same attention mechanism to obtain therepresentation oft the singlesentencelevel feature vector need to be obtained to represent the entire sentence for the relation classificationwe introduce the maxpooling approach as in cnnmodels to obtain the single sentencelevel feature vector the maxpooling is formulated as followsthe sequence h0htf gm ¼ maxtnext the sentencelevel feature vector m is passed tothe output layer furthermore the output layer has classes we use the softmax classifier to predict the labely² from a set of labels y from the sentence the statevector m is used as input therefore y² could be computed bypy ¼ softmax wmð ¼ arg maxypyyþin addition there are two settings in er extraction inorder to better compare our approach the first is basedon our proposed method the second is only utilize thernn network without any piecewise representation orattention mechanism as shows in fig experiment and resultsdatathe major medical data are the data of medical institutions diagnosis and treatment collection of massiveclinical data and laboratory data produced every day atall levels of hospitala golden annotated corpus marked up with temporalexpressions events and relationship between them isneeded to allow us to evaluate by our methods thethyme corpus which has been used since isone of the suitable corpora consisting of clinical andpathological notes of patients with colon cancer andbrain cancer from mayo clinic unlike other datasetsthe events in this dataset are all single words which arevery suitable for our system the notes are manually annotated by the thyme project thymehealthnlpusing an extension of isotimeml for the annotation oftemporal expressions events and temporal relations of the corpus is used for training is forfig the flow of the comparison system this is a commonrnn modeldevelopment and is for testing the developmentset is used for optimizing learning parameters thencombine it with the training set to build the system usedfor reporting results table shows the distribution ofthe thyme corpus the colon cancer data are used astraining data and are tested on both colon cancer andbrain cancer data to demonstrate its effectiveness withtable the distribution of the thyme corpus in this table weshow the different types of data in the corpusdatacolon cancertrain dev testbrain cancertrain testdocumenttemporal expressions event expressions er 0cli bmc medical informatics and decision making page of or without domain adaptation and this can reflect thatour approach is not limited to a particular field inevaluation all methods have access to the same set oftraining and testing datatable the temporal expressions extraction results on braincancer we utilize different methods to do the task the resultsare shown in part the result of previously best system isshown in part resultsthe method has been evaluated on both colon cancerdata and brain cancer data to demonstrate the effectiveness with or without domain adaptation in order to dobetter research several methods are used to de the entityextractionsix methods of temporal expression extraction arulebased method a system based on crf a system based on general rnn without any attention mechanism or context rnn a system based on rnnwith easy attention mechanism but without any contextwords rnnatt our proposed method a rnn system with attention mechanism and context words asystem combines the crf and rnn network all the results are compared part in tables and for therulebased methods firstly we find all the prepositionsaccording to our experience and experimental statisticswe extract five tokens behind their own prepositionsthrough careful observation of data we found thatmany time expressions always show up behind a preposition we then judge whether those five words are relatedto time expressions we define a time dictionary to listthe words which we think can be a part of the time exlike month week day hour maypressionsmonday morning once and so on next we contrastthe five tokens with time dictionary and findwhether it can represent a date or a precise time finallywe extract all the continuous tokens that we thoughtmay relate to the time expressions if there is a definite before those tokens extract it as well there existsome expressions do not after a preposition and onlycontain one word and most of them have the same prefix like pre post peri so we use this prefix rule tofind the remain expressions the major feature we usedfor training the crf and svm classifier is simple lexicaltable the temporal expressions extraction results on coloncancer the part shows the results of six different methodsthat we used to do the temporal expressions extraction thepart shows the result of the previously best systempart methodrulebasedcrfrnnrnnattarnnpart crfarnnblulab run prf1part methodrulebasedcrfrnnrnnattarnncrfarnnpart guirprf1features word embeddings partofspeech tag numericlower case blulab run andtype capital typeguir system are the previously best system mentionedin the section two these results are shown in tables and part in both tables and rule based methods achievethe lowest result the recalls are relatively better thanthe precisions due to the welldefined dictionary theerror analysis shows that some pre post and periare considered as time expressions while they should notbe meanwhile the rulebased method often mistakestwo independent expressions as one if they are adjacentin table the rnn systems performances are lowerthan blulab run 3a cleartk svm pipeline usingmainly simple lexical features along with informationfrom rulebased systems the rulebased information iseffective but it has limitations it can extract rules according to the characteristics of data we do not addany rules to the rnn system the observation on theerror analysis shows that without any attention mechanism and context words rnn is not very effective forsimilar combinations of numbers and letters eg h days etc because the form of the corresponding wordvectors are generated randomly and the time series contains a large number of the above type so the modelcannot learn characteristics of time series so it cannotbe correctly extracted after adding the attention mechanism and context words the arnn system achieve therelatively good results because of the good results ofcrf we combine the crf with the arnn and achievethe best result from table we can see that the rnnoutperforms the guir system which is the current bestsystem it is an ensemble of crf and decision tree withlexical syntactic semantic distributional and rulebasedfeatures the guir system can not extract the previously unseen or atypical date formats very well it is obvious that their rules are not comprehensive enoughthis problem also exists in rnn system however whenadding the attention mechanismit can extract more 0cli bmc medical informatics and decision making page of new and otherwise unknown formats the arnn andcrfarnn system achieve the best results in this partwe have two test data sets one is colon cancer anotherone is brain caner we trained all the models on thesame training data and test them on two different testdata sets except for the different test data the parameters are exactly the same the experimental results provethat our model is effective on other test data setsmeanwhilefive methods of event extraction amethod based on svm a system based on crf asystem based on general rnn without any attentionmechanism or context rnn a system based onrnn with easy attention mechanism but without anycontext words rnnatt our proposed method arnn system with attention mechanism and contextwords all the results are evaluated part in tables and for event extraction the svm and crf modelobtain the relatively good results in colon data and perform poorly in brain colon data compared to the bestsystem limsi however rnn achieves preferably results in the two sets of test data even higher than thebest system limsi as shown in both tables and when adding the attention mechanism and contextwords the results are improvedas for the er extraction which is the key point of thepaper first we compare our proposed model with thefollowing methods a general rnn system withoutany attention mechanism or piecewise representationwe use the sentence between the entity pair as the inputrnn a general rnn system without any attentionmechanism or piecewise representation we use thewhole sentence as the input rnnwhole we can seethe results of rnnwhole is better than the results ofrnn it means that the sentence length can affect theperformance of the system therefore we use the sentence between the entity pair as the input for other system a general rnn system with attention mechanismbut without piecewise representation rnnatt ageneral rnn system without attention mechanism butwith piecewise representation rnnpie our proposed system aprnn but only use the word embeddings trained from wikipedia aprnnwiki ourtable the event extraction results on colon cancer different methods are utilized by us all the results are shown inpar1 the part shows the result of the previously best systemf1methodsvmpart prcrfrnnrnnattpart arnnblulab run table the event extraction results on brain cancer we adopt methods to do the task the results can be compared in part the result of the best system is shown in part part part methodsvmcrfrnnrnnattarnnlimsiprf1proposed system aprnn but only use the word embeddings trained from bioasq aprnnbioasq our proposed system which is based on a recurrentneural network combining the attention mechanism andthe piecewise representation all these results are evaluated part in tables and except for model and the word embeddings for other models are from bothwikipedia and bioasq from the results we can seethat both attention mechanism and piecewise representation are useful they can improve the results to someextent we can directly compare the value of attentionin two groups of experiments result and result result and result the result and result result and result can directly demonstrate the performance with and without segmentation the difference between model and is that model is missing thepiecewise representation and the difference betweenmodel and is without or with the attention mechanism the result has been improved with the piecewiserepresentation the experiment and are aboutlooking at the impact of word embeddings the result and result show that different word embeddings canlead to different results after combining the two corpuswikipedia and bioasq the results increase slightlytable the er classification results on colon cancer part shows the results of the relevant methods we used the otherrelated works which achieved the very good results are shownin part part part methodrnnrnnwholernnattrnnpieaprnnwikiaprnnbioasqaprnnblulab run svmattblstmprf1 0cli bmc medical informatics and decision making page of table the er classification results on brain cancer the resultsof our proposed methods are shown in part part shows theresults of other relatedpart part methodrnnrnn wholernnattrnnpieaprnnwikiaprnnbioasqaprnnlimsisvmattblstmprf1aprnn different factors that may affect the resultsare verified from experimental results eg piecewise representation attention mechanism word embeddings allthese factors are utilized to make better use of contextual informationwe compare our work with other related work thelimsi system which achieves the best score on the ertask in semeval2017 task li rao and zhang proposed the litway which is a system that has adopteda hybrid approach that uses the libsvm classifier with arulebased method for relation extraction theyachieve the best score in the seedev task of bionlpst thus we use their approach as a benchmark forour system the bilstmattention networks proposedby zhou were chosen as another benchmarking model attblstm which outperforms most of theexisting methods they designed a bidirectional attention mechanism to extract wordlevel features from thesentence the features for the attentionbased model include word vector
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" clinical trials have been conducted to clarify the beneficial effects of vd3 1α25dihydroxy vitamind3 also known as calcitriol treatment in prostate cancer however the molecular mechanisms underlying theseeffects are not fully understood recent studies on igfbp3 have indicated its intracellular functions in cell growthand apoptosis the aim of this study was to confirm the benefits of lowdose vd3 treatment and clarify themolecular mechanisms underlying these beneficial effects in prostate cancer cellsmethods the molecular effects of simultaneous treatment of lncap cells and their genetically modified cell lineswith low concentration of docetaxel and vd3 were biologically and biochemically analyzed to further determinethe effects of vd3 treatment on igfbp3 induction system cells were temporarily treated with vd3 in combinationwith a transcriptional inhibitor or protein synthesis inhibitor bcl2 protein and its mrna behavior were also observed inigfbp3 expressionmodified lncap cells to determine the involvement of igfbp3 in the suppression of bcl2 by vd3treatmentresults changes in igfbp3 expression levels in lncap cells indicated that it mediated the inhibition of cell growthinduced by vd3 treatment igfbp3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancercells to vd3 in combination with the anticancer drug we further identified the distinct property of the igfbp3induction system wherein temporal vd3 stimulationinduced prolonged igfbp3 expression and vd3 treatmentinduced increase in igfbp3 expression were optimized based on the protein concentration rather than the mrnaconcentration meanwhile bcl2 expression was downregulated by vd3 treatment in an igfbp3independent manner these findings indicate the molecular mechanisms of igfbp3 induction stimulated by vd3 and igfbp3independent bcl2 suppression by vd3 treatment in prostate cancer cells the results could prompt a reevaluation ofvd3 usage in therapy for patients with prostate cancerkeywords vitamin d nonlinear igfbp3 induction bcl2 suppression prostate cancer treatment correspondence satorusasagawatokushukaijp1molecular biology laboratory research institute nozaki tokushukai hospitaltanigawa daito osaka japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cigarashi bmc cancer page of vitamin d has a central role in calcium and skeletalhomeostasis [ ] its pleiotropic role both in physiological and pathological phenomena such as cell growthimmune function and tumorigenesis has also been examined [] which revealed that exposure of cancercells to vitamin d significantly reduces the cell growthrate in multiple cancer types [] indeed recent epidemiologicalinvestigations have reported that highervitamin d concentration could prevent multiple types oftumorigenesis consistent with such finding for example an increase in colon cancer incidence with lowervitamin d dietary habits has been reported [ ]however suppressive effect on prostate cancer is stillunder discussion [ ]in turntraditional methodscurrently prostate cancer is one of the most commonthecancers in men worldwide clinical use ofprostatespecific antigen psatest dramatically improved the screening sensitivity of prostate cancer compared to that ofthenumber of patients with earlystage prostate cancer hasrapidly increased since the mid1990s [ ] unlikeother cancer types most cases of prostate cancer haveslow progression or have nonprogressive indolentsymptom and are localized in the prostate thus they areunlikely to cause poor physical condition or death therefore patients with prostate cancer need a less burdensome treatment in order to avoid potential harmfrom excessive treatmentalthough the impact of vitamin d as a single agent onprostate cancer has been investigated its significance remains under discussion [ ] meanwhile the synergistic or additive effects of vitamin d and its derivativeswith anticancer drugs on prostate cancer have beenclinically studied and encouraging results have been reported [ ] however the results of larger trials thatevaluated the synergistic effect of vitamin d in combination with docetaxel one of the firstline anticancerdrugs in prostate cancer chemotherapy showed limitedor nonsignificant benefit of vitamin d efficacy in castration or androgen deprivation therapyresistant prostatecancer [ ] furthermore overconsumption of 1α25dihydroxy vitamin d3 vd3 also known as calcitriolthe biologically active form of vitamin d3 from food orprolonged treatment with vd3 derivatives could triggerhypercalcemia resulting in physiological side effects therefore to date vd3 has not been proactivelyused in the treatment of patients with prostate cancerthe biological function of vitamin d is mainly mediatedby vitamin d receptor vdr which acts as a transcriptional factor vitamin d receptor elements vdreon the promoter region of target genes are recognizedand transcriptionally activated by vitamin dcoupledvdr consistent with the diverse physiological functionof vd3 vdre was identified not only in the gene related to calcium and skeletal homeostasis but also in thegene related to fundamental cellular functions includingcell growth igfbp3 is one of the families of sixhigh affinity igfbps and was originally found in plasmaas a stabilizer and transporter of igfs in the bloodstream interestingly vdre was found on the prothe igfbp3 gene and recent studies havemoter ofrevealed that igfbp3 functions inside the cell as wellregulating cell growth and apoptosis [ ]methodsthis study aimed to investigate igfbp3 induction byvitamin d treatment and determine its role in prostatecancer treatment with vitamin d in combination withanticancer drugs in order to provide molecular biologicalevidence of benefit of vitamin d and to suggest effectivevitamin d usage in prostate cancer treatmentchemicals and reagentsdihydrotestosterone dht and calcitriol vd3 purchased from tokyo chemical industry tokyo japanwere resolved in ethanol as a stock solution pei maxmolecular weight was purchased from polysciences pa usa the other chemicals and reagentswere purchased from wako pure chemicalosakajapan and sigmaaldrich st louis mo usaincluding testosteronecharcoal stripping of fetal bovine serum fbsfbs was purchased from gibco waltham ma usato deplete hormonesin fbsdextrancoated charcoal powder was added to theserum and the mixture was incubated with rotation at degree overnight thereafter the mixture was centrifugedto pellet charcoal and the supernatant was filtered througha 022μm polyvinylidene difluoride membrane thecharcoalstripped serum was used for all experimentstotalthe concentrations oftestosterone and totalvitamin d in the serum were determined using a totaltestosterone test kit abbott japan chiba japan and atotal vitamin d test kit roche basel switzerland according to manufacturers instructions the concentrations of total testosterone in the pre and posttreatmentserum were nm and less than nm limit of detection respectively the concentrations of total vitamin d in the pre and posttreatment serum were and nm respectively thus the basal concentrationsin the culture medium supplemented with fbs wereless than nm total testosterone and approximately nm total vitamin dcell culturethe lncap cell line was obtained from american typeculture collection and cultured in dulbeccos modified 0cigarashi bmc cancer page of eagle medium dmem sigmaaldrich supplementedwith 10charcoalstripping fbs the 293ft celllinewas purchased from invitrogen waltham ma usaand cultured in dmem supplemented with fbs mm of lglutamine sodium pyruvate and nonessentialamino acids the cells were cultured at a temperature of °c in co2humidified condition the mycoplasma contamination was routinely checked and confirmed as negativecell growth assaydmem supplemented with charcoalstripping fbs was used for the cell growth assay the cells were seededat à cells per well in a 6well plate the next daythe medium was replaced with ml of fresh mediumand nm dht andor nm vd3 were added the cellculture was continued throughout the indicated periodthe cultured cells were trypsinized and the number ofcells was assessed using an automated cell countercountess iitm fl invitrogen each assay was repeatedat least three times and similar results were obtainedwestern blottingthe antibodies used for western blotting were mouseantiigfbp3 santa cruz biotechnology mouseantiβactin santa cruz biotechnology mouseantibcl2 santacruz biotechnology and horseradish peroxidaseconjugated secondary antibodies jackson immunoresearch laboratories westgrove pa usa the collected cells were resuspendedin ripa buffer supplemented with protease and phosphatase inhibitors roche basel switzerland and lysedusing bioruptortm ii sonicator cosmo bio tokyojapan cell lysates were resolved by nupagegels invitrogen and transferred onto polyvinylidenefluoride membrane millipore burlington ma usathe signals were developed using enhanced chemiluminescence reagent perkinelmer waltham ma usaand luminograph i atto tokyo japan was used forimage capture quantification of band signal wasanalyzed using cs analyzer software atto at leasttwo biological replicates of each experiment were performed with similar resultsrealtime reverse transcriptionpolymerase chain reactionthe total rna from the cultured cells was extractedusing trizol reagent invitrogen according to the manufacturers instruction the rna was reverse transcribedby the primescript rt reagent kit takara bio shigajapan using oligodt quantitative reverse transcriptionpolymerase chain reaction rtpcr reaction wasperformed using tb green premix ex taq ii takaraand genespecific primers supplementary table bycfx96 touch realtimepcr system bioradlaboratories hercules ca usa gene expression datawere normalized against glyceraldehyde 3phosphate dehydrogenase or hprt1 as internal control at leastthree biological replicates of each experiment were performed and similar results were obtainedflow cytometric analysis of cell cycle and apoptosisfor cell cycle analysis and apoptotic cell detection flowcytometric analysis was performed using the guavaeasycyte plus flow cytometry system millipore andguava cell cycle reagent and annexin v fitc apoptosiskit millipore according to manufacturers instructionas previously described at least three biologicalreplicates of each experiment were performed and similar results were obtainedlentiviral construction and transductionbackbone vectors plko1 puro plasmid andplenti cmv puro dest w118 plasmid were provided by drs bob weinberg eric campeau andpaul kaufman respectively via addgene ref pentr1a plasmid and lentiviral packaging mix plp1plp2 and plpvsvg were purchased from invitrogenfulllength igfbp3 was cloned from lncap cell complementary dna cdna using kod fx neo toyoboosaka japan with a specific primer set supplementarytable and inserted into the pentr1a vector thenthe sequence was confirmed the igfbp3 cdna was introduced into the plenti cmv puro dest vector by recombinaseenzymeinvitrogen to generate the lentiviral expression vectorspecific short hairpin rnas shrnas were designedusing invitrogen or biosettia websites the selectedtarget sequence oligos supplementary table wereannealed and inserted into the plko1 puro vector according to addgenes instruction the lentiviral expressionvector or shrna vector was cotransfected with the lentiviral packaging mix into the 293ft cells using pei maxinstead of lipofectamine according to invitrogensinstruction twentyfour hours posttransfectionthemedium was replaced with the culture medium forlncap cells one day later the lentiviruscontaining supernatants were collected and filtrated through a 045mmpolyvinylidene fluoride filter milliporereaction using lr clonaseiilentivirus infectionone day before infection à lncap cells wereplated into a 10cm dish then the culture medium wasreplaced with the lentiviruscontaining supernatant andculture was continued twentyfour hours post infectionreplaced with fresh culturemedium two days later the medium was replaced withfresh culture medium that contained μgml of puromycin and culture was continued until the noninfectedthe medium was 0cigarashi bmc cancer page of control cells were completely killed the puromycinselected cells were then subjected to each assaystatistical analysisresults are presented as mean ± standard deviation unless otherwise specified pairs of groups were comparedusing a twotailed unpaired students ttest onewayanalysis of variance was used for multiplegroup comparisons rather than specifying three a pvalue was considered statistically significant all statistical analyses were performed using excel software microsoftredmond wa usa and statcel3 addin for exceloms publishing tokyo japanresultsvd3 reduces cell growth rateconsistent with a previous report that treatmentwith vd3 inhibits growth of prostate cancer cells ourresults showed that vd3 treatment reduced the cellgrowth rate in a dosedependent manner and nm fig 1a left as shown below igfbp3 induction activity of vd3 was reached to plateau at nm concentration fig 4a on the other hand testosterone hasbeen reported to stimulate the growth of prostate cancer and the results of this study confirmed that dhttreatment from very low concentration nm stimulated cell growth rate and its activity was reached toplateau at less than nm concentration fig 1a centerthe purpose of our study was to investigate the role ofvd3igfbp3 induction system in cell growth inhibitionand to propose the potency oflowdose vd3 usagewhich could evade sideeffect of vd3 treatment such ashypercalcemia in therapy for patients with prostate cancer thus nm of dht and nm of vd3 concentrations were chosen as minimum but stably workingconcentration for following experiment previouslyithas been demonstrated that simultaneous treatmentwith vd3 and dht enhanced the reduction of cellgrowth rate compared to treatment with vd3 alone anda similar result was reproduced with lowdose dht nm and vd3 nm in this study fig 1a right tofurther characterize growth inhibitory effect with lowdose of dht nm and vd3 nm cell cycle andapoptotic analyses were performed with flow cytometrythe cell cycle analysis revealed that there was no significant change in the cell cycle phase distribution amongfig cellular response of lncap cells treated with vd3 and dht a effect of combined treatment of vd3 and dht on cell growth in lncapcells left vd3 treatment reduced cell growth in a dosedependent manner center dht treatment increased cell growth at lowerconcentrations right simultaneous treatment of vd3 and dht enhanced the reduction of cell growth compared to treatment with vd3 aloneb change in cell cycle phase by vd3 or dht treatment neither vd3 nor dht treatment significantly changed the cell cycle phase c induction ofapoptosis by vd3 or dht treatment neither lowdose vd3 nor dht treatment influenced apoptosis at shortterm d induction of igfbp3expression by vd3 or dht treatment vd3 treatmentinduced igfbp3 expression and cotreatment with dht enhanced the expression level ofigfbp3the ratio indicates the density of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium condition the uncropped fulllength blot images are presented in supplementary fig 5a 0cigarashi bmc cancer page of control no treatment dht nm vd3 nm anddhtvd3 treatment conditions fig 1b suggestingthat lowdose of vd3 or dhtvd3 treatment did notarrest the cell cycle at a specific phase previous reportshave shown that longterm vd3 treatment has apoptosisinducible activity and dht has apoptosis inhibitingactivity in a dosedependent manner in lncapcells however it was unclear or controversial whetherlowerconcentration of vd3 and dht at shorttermcould influence apoptosis respectively in lncap cellsthus the apoptosis assay was performed with nm ofdht and nm of vd3 for shortterm and found thatneither lowerdose dht vd3 nor dhtvd3 treatmentfor shortterm influenced apoptosis fig 1c these results suggested that the decrease in the cell number induced by lowdose vd3 or dhtvd3 treatment wasmainly due to a decrease in the cell growth rate to further address what was occurring at the molecular levelduring lowdose dht andor vd3 treatment the genesknown to regulate the cell cycle and inducible by dhtandor vd3 treatment were chosen and messengerrna mrna induction was quantitatively measuredfig 1d upper supplementary fig 1a the quantitativertpcr results showed that igfbp3 mrna inductionwas positively correlated to cell growth suppression inresponse to lowdose vd3 or dhtvd3 treatment andthe expression strength was dramatically sensitive tovd3 or dhtvd3 treatment consistent with thatigfbp3 protein was markedly induced by vd3 treatment and it was enhanced by simultaneous treatment ofvd3 with dht a similar response was observed in theexpression of ar the receptor of dht which wasknown to a one oftarget of vdr supplementaryfig 1bmultiple recent studies have revealed that igfbp3functions in cellular response including cell growth andapoptosisin an insulinlike growth factor igfindependent manner considering these findings we believethat igfbp3 can be a key molecule for vd3 treatmentin prostate cancer cellsigfbp3 was a dominant factor in cell growth suppressionto confirm ifigfbp3 dominantly suppresses cellgrowth in lncap cells we applied the gainoffunctionand lossoffunction approach using a lentivirus systemfirst we generated igfbp3overexpressing lncapcells and found that the expression of igfbp3 mrnawas about higher compared to that by lowdosedhtvd3 treatment fig 2a as an infection controlegfpoverexpressing lncap cells were also generatedand it was confirmed that lentivirus infection per se didnot induce igfbp3 expression using these cell linesthe effect of igfbp3 on cell growth was observed fig2b results showed that the cell number of egfpoverexpressed cells treated with nm of dht and nm of vd3 for days was decreased to comparedwith that of untreated cells and the igfbp3overexpressing cells showed comparable cell growth decreasewithout dhtvd3 treatment next we generatedshrna for igfbp3 shigfbp3expressing lncap cellsthe knockdown of igfbp3 mrna and protein inducedby lowdose dhtvd3 treatment was confirmed inshigfbp3expressing lncap cells fig 2c using thiscell line the effect of lowdose dht and vd3 treatmenton cell growth was observed as we expected the suppressive efficacy of lowdose vd3 on cell growth wasweakening and simultaneous treatment with nm ofdht and nm of vd3 increased cell growth fig 2dtaken together these data indicated igfbp3dominantfactor of cell growth suppression induced by lowdosevd3 treatment in lncap cellsacceleration of anticancer drug effect by vd3as previously reported and we demonstrated abovevd3 alone is not cytotoxic at physiological and pharmacological concentrations meanwhile simultaneous treatment with vd3 has been reported to improve theefficacy of anticancer drugs including docetaxel howeverits molecular mechanisms were remained not fully uncovered here we supposed that igfbp3 might be amediator of vd3induced sensitization to anticancerdrugs in prostate cancer cells to confirm this hypothesis lncap cells were treated with lowdose of dhtvd3 in combination with several concentrations of docetaxel to determine the appropriate concentration ofdocetaxel for evaluating the vd3 effect we first screenedthe concentration of docetaxel based on cytotoxic activity and found that a docetaxel concentration nmkilled bulk of the cells treated fig 3a here ic50 ofdocetaxel was nm in our assay and it was consistent with previous reports nm thus a docetaxel concentration nm was chosen to observe theeffect of combinatoriallowdose dhtvd3 treatmentfor the following assay to evaluate the synergistic effectof dhtvd3 on cytotoxicity by docetaxel lncap cellswere treated with or nm of docetaxel withor without dhtvd3 and results showed that lowdosedhtvd3 with docetaxel reduced the living cell numberat the concentration range of nm but the effectwas masked when nm docetaxel was applied fig 3bsimilarlylowdose dhtvd3 with cisplatin reducedthe living cell number at the concentration range of nm supplementary fig to see if these enhancedcytotoxicity effects were dependent on igfbp3 igfbpoverexpressed or shigfbp3expressed lncap cellswere analyzed in the same manner indeed in igfbp3overexpressed cells the living cell number was reducedby docetaxel without dhtvd3 addition fig 3c 0cigarashi bmc cancer page of fig igfbp3 mediates the effect of vd3 on cell growth in lncap cells a overexpression of igfbp3 in lncap cells lncap cells were infectedwith lentivirus containing the igfbp3 gene and its overexpression was confirmed by quantitative reverse transcriptionpolymerase chainreaction b suppression of cell growth by igfbp3 igfbp3overexpressed cells were cultured as they were and control cells were cultured withdhtvd3 for days and then the cell number was measured c knockdown of igfbp3 in lncap cells lncap cells were infected with lentiviruscontaining shrna for igfbp3 and igfbp3 knockdown was confirmed by quantitative reverse transcriptionpolymerase chain reaction andwestern blotting the ratio indicates the density of igfbp3 band normalized by corresponding βactin band d the igfbp3 knockdown cells weretreated with dht andor vd3 for days and then the cell number was measured the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5bcorrespondingly in the shigfbp3expressed cells reduction of living cell number by dhtvd3 addition wascanceled rather the cell living number was increasedliving cell number despitefig 3d the increase ofdhtvd3 addition in docetaxeltreated shigfbp3expressed cells was assumed by cancelation of vd3 effect and emerging of dht effect on cell growth basedthese findings lowdose dhtvd3induced enhancedcytotoxicity by docetaxel on lncap cells was dependenton igfbp3 expressioncharacterization of the igfbp3 induction mechanismas demonstrated above igfbp3 had a pivotal role inlowdose dhtvd3induced enhanced cytotoxicity byantitumor drugs to further dissect the igfbp3 induction mechanism and to provide the molecular evidenceof vd3 treatment for clinical research mechanisms ofigfbp3 induction by dht and vd3 were analyzed aspreviously reported in prostate cancer cells vd3 treatment induces cyp24a1 as well an enzyme that catalyzes vd3 to its inactive form as a negative feedbackfactor the induced cyp24a1 limits the efficacy of vd3meanwhile activated ar induced by dht treatmentsuppresses cyp24a1 transcription thus cancelling thenegativefeedback loop to inactivate vd3 consistentwith that cyp24a1 induction by vd3 treatment and itssuppression by simultaneous treatment with dht wereconfirmed even when we applied lowdose dht and 0cigarashi bmc cancer page of fig vd3 enhanced cytotoxicity of docetaxel a dosedependent cytotoxicity of docetaxel lncap cells were treated with docetaxel for daysand then the living cell number was measured ic50 was nm b simultaneous treatment of vd3 with dosedependent docetaxelsimultaneous treatment of vd3 with docetaxel reduced the living cell number at nm range c igfbp3 overexpression was conducive todhtvd3 treatment on docetaxel treatment igfbp3overexpressed or control cells were treated with dhtvd3 and nm of docetaxel andcultured for days and then the cell number was measured d igfbp3 knockdown canceled the effect of vd3 on docetaxel treatment in igfbp knockdown cells dhtvd3 treatment increased the cell number compared to that of the notreatment control and the cell number wasalmost equal to that of the dht treated sample the concentration of docetaxel used was nm the all experiments were performed in serumcontaining medium conditionvd3 which were enough to induce and suppresscyp24a1 expression supplementary fig meaningthat h lowdose dhtvd3 treatment could cancel thecyp24a1driven negativefeedback loop to further dissect the mechanism of igfbp3 expression in lncapcells the cells were treated with vd3 alone or dhtfixed in nm and vd3 in a dosedependent manner and nm when treated with vd3alone the induced igfbp3 reached a plateau at to nm in contrast when treated with vd3 together withdht the amount of induced igfbp3 was increased according to the increment of vd3 concentration fig 4athese results indicated that lowdose dht could improve igfbp3 induction activity of vd3 throughcyp24a1 suppressionclinically highdose vd3 or its derivatives for treatment can cause hypercalcemia thus its continual usageshould be carefully monitored to avoid sideeffect ofvd3 here we wondered if continual vd3 treatmentfig 4btop herewould be required for maintaining igfbp3 induction inprostate cancer cells to address this lncap cells weretreated with vd3 alone or lowdose dhtvd3 for or days followed by washout which was done by replacing the culture medium and continuing the culturefor days in totalintracellularigfbp3 protein was observed by western blottinginterestingly 1day treatment of vd3 or dhtvd3 induced stable igfbp3 expression fig 4b bottom although treatment of vd3 alone showed mild igfbp3induction compared to that by dhtvd3 note thatigfbp3 showed similar strength of expression between and days of vd3 or dhtvd3 treatment this resultclearly indicated that temporal vd3 treatment couldinduce prolonged stable igfbp3 expressionthis nonlinear response suggested the presence of aunique molecular property underlying the igfbp3 expression mechanism generally nonlinear cellular response such as sustained protein expression by temporal 0cigarashi bmc cancer page of fig characterization of igfbp3 expression induced by vd3 and dht treatments a effect of dosedependent vd3 treatment with or withoutdht on igfbp3 induction lncap cells were treated with vd3 alone at the indicated concentration or with vd3 and nm dht top westernblotting image of igfbp3 and bottom quantified graph of igfbp3 induction open circles vd3 alone filled circles dht vd3 b effect oftemporal treatment of dhtvd3 on igfbp3 induction top schematic time course of temporal treatment of vd3 and bottom western blottingimage of igfbp3 the ratio indicates the density of igfbp3 band normalized by corresponding βactin band c effect of mrna transcription andprotein synthesis on the stability of igfbp3 expression induced by dhtvd3 lncap cells were treated with vd3dht for day followed byactinomycin d actd μm or cycloheximide chx μm for another day after vd3dht was washed out or left as is top quantification graphof igfbp3 mrna induction middle western blotting image of igfbp3 and the combination of treatments bottom the ratio indicates thedensity of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium conditionthe uncropped fulllength blot images are presented in supplementary fig 5ctriggered by positivefeedback loopstimulation wasmechanismin which protein synthesis and transcriptional regulation was included as hysterical responsedriver thus to further dissect if protein synthesis ortranscriptional regulation or both are involved in nonlinear vd3igfbp3 induction actinomycin d and cycloheximide which are transcriptionalinhibitor andprotein synthesis inhibitor respectively were added withvd3 or dhtvd3 and behavior of igfbp3 proteinand its mrna was observed experimentally actinomycin d or cycloheximide was added day after treatmentwith vd3 alone or dhtvd3 and the culture was continued for another day when interfered with μm ofactinomycin d there was no change in igfbp3 mrnaor protein expression fig 4c by contrast when interfered with μm of cycloheximide the igfbp3 proteinwas immediately reduced howeverthe mrna wasunexpectedly increased several times higher than thosewithout cycloheximide interference fig 4c the unexpected mrna increase became stronger when dhtvd3 washout was performed ahead of the cycloheximideinterference these cellular responses on igfbp3 induction interfered by actinomycin d and cycloheximidesuggested that the cells had a protein abundancebasedpositivefeedback loop to maintain the total amount ofigfbp3 via transcriptional controligfbp3independent bcl2 suppression by vd3as shown above although lowdose dht andor vd3treatment did not induce apoptosis fig 1d vd3 treatment rendered lncap cells sensitive to the antitumordrugs fig 3b and supplementary fig suggesting thatany apoptosisrelated factor might be influenced to addressinvestigated the behavior ofidea wethis 0cigarashi bmc cancer page of apoptosisrelated molecules in response to lowdosevd3dht treatment consistent with previous report although the concentration of vd3 was higher thanthat we used here bcl2 protein an antiapoptotic molecule was downregulated by lowdose vd3 treatmentfig 5a compared to that by vd3 alone it seemed thatbcl2 downregulation by dhtvd3 was not seemed tobe enhanced unlike to igfbp3 expression suggestingthat bcl2 downregulation by vd3 was igfbp3 induction independent manner to see whether bcl2 downregulation was igfbp3dependent or not the behaviorof the expression of bcl2 protein and mrna was observed in shigfbp3expressedcells interestingly despitethe igfbp3 disappearance bcl2 downregulation wasobserved according to vd3 or dhtvd3 treatmentfig 5b bottom and it was not significantly different atinigfbp3expressionenhancedprotein and mrna expression level compared to that inshctrlexpressing cells moreover in order to confirmthatconditionshcyp24a1 expression cells were established in whichvd3 effect on igfbp3 induction was expected tostrengthen the knockdown of cyp24a1 under the vd3treatment condition was confirmed by qrtpcr supplementary fig indeed the amount of igfbp3 protein wasincreased in shcyp24a1expressing cellscompared to that in shctrl cells fig 5c bottom despiteigfbp3 expression downregulation bcl2 protein by lowdose vd3 or dhtvd3treatment was not observed compared to that in shctrlcells fig 5c bottom also the expression of bcl2mrna was not significantly changed fig 5ctopthese results suggested that the downregulation of bclenhancement offig igfbp3independent reduction of bcl2 protein expression induced by vd3 a western blotting image of bcl2 reduction by vd3treatment the ratio indicates the density of bcl2 band normalized by corresponding βactin band b effect of igfbp3 suppression on bcl2reduction by vd3 treatment the cells were infected with lentivirus encoding of shrna for igfbp3 and then treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band c effect of igfbp3 overexpression on bcl2 reduction byvd3 treatment the cells were infected with lentivirus encoding of the cyp24a1 gene then the cells were treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5d e 0cigarashi bmc cancer page of protein by lowdose vd3 treatment was independentof igfbp3 induction besides the mrna of bcl
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"objectives to describe the benefits and limitations of using individual and combinations of linked english electronic health data to identify incident cancersdesign and setting our descriptive study uses linked english clinical practice research datalink primary care cancer registration hospitalisation and death registration dataparticipants and measures we implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and we calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers we described completeness of grade and stage information in the cancer registration data setresults gold standard cancers were identified positive predictive values of all case definitions were ¥ and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity for case definitions that used cancer registration alone or in combination was ¥ for the four most common cancers and ¥ across all cancer sites except bladder cancer using cancer registration alone for case definitions using linked primary care hospitalisation and death registration data sensitivity was ¥ for the four most common cancers and ¥ for all cancer sites except kidney oral cavity and ovarian cancer when primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancerss ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority strengths and limitations of this study º this is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in england to identify cases of the most common incident cancers º using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions º we described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data º a key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataintroductionthe clinical practice research datalink cprd provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 in the field of cancer epidemiology cprd primary care data linked to hospital episode statistics admitted patient care strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access data hes apc office of national statistics ons mortality and national cancer registration and analysis service ncras cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for ncras data research teams therefore commonly choose not to use all available linked data3 cancer epidemiology studies can also be conducted using ncras and hes apc data provided by national health service nhs digital and public health england phe without linkage to cprd primary care data4 this provides national coverage at the expense of the detailed health data that are available in primary care recordsvalidation studies assessing concordance between cprd gold hes apc and ncras data have estimated high positive predictive values ppvs for cprd gold data and varying proportions of registered cancers that are not captured in cprd gold and hes apc5 the most up to date analysis by arhi et al included the five most common cancers and all papers focussed on concordance between cprd gold only and ncras existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs national data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and clinical commissioning groups10 we aim to describe and compare the benefits and limitations of using different combinations of linked cprd primary care data hes apc ons mortality and ncras cancer registration data for conducting cancer epidemiology studies our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets we have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets we also describe the availability of stage grade and treatment variables over time in the cancer registration data for the cprd linked cohort this reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesmethodsstudy design and settingwe completed a concordance study using linked2 english cprd gold hes apc ons mortality and ncras data cprd gold data were extracted from the january monthly release and the 13th update to cprds linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 malignant neoplasms of urinary organ unspecified was considered consistent with both bladder and kidney cancer for ncras we accessed coded records for the most common cancers we included cancers recorded in the clinical or referral file for cprd gold cancers recorded in any diagnosis field for hes apc and the underlying or strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure gold standard algorithm to identify incident site specific cancers using all data sources hes hospital episode statistics ncras national cancer registration and analysis service ons office of national statisticsmost immediate cancer cause of death in ons mortality datacancer case definitions based on individual sources and combinations of sourceswe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources ncras alone ncras and hes apc all sources cprd gold hes apc and ons mortality cprd gold alone hes apc alone multiple malignant cancers recorded on the index date in cprd gold or hes apc were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the ncras data if available and as multiple site cancer if not for each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upgold standard cancer case definitionwe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure cancers recorded in ncras alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in hes apc alone or gold alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in ncras and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in ncras if there were more data sources in total with records for cancer at that site than data sources with contradictory recordswe used ncras data to identify stage grade and treatment where available in the cancer registry only cohort binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisstatistical analysisfor each cancer site and each individual or combined data source we combined our applied study definitions strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordwe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex we calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourceswe used kaplan meier methods to describe mortality over time for cancers identified using each definition the ons mortality death date was used for these analyseswe used the ncras only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timepatient public involvementpatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsresultsof research quality patients in the cprd gold january build were eligible for linkage to hes ons mortality and ncras data in set were excluded due to unknown sex of the remainder and had at least year of follow up between january and december and were included in the study population using the gold standard algorithm incident cases of cancer were identified the number of patients identified with each cancer is presented in online supplementary appendix table half n82 of these patients were male aged to aged to and aged or olderfigure presents ppvs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm when using ncras data alone to of cancers were confirmed by the algorithm for out of cancer sites the ncras only case definition gave the highest ppv case definitions using data sources not including ncras generally had lower ppvs ranging from to for individual cancer sites for the four most common cancers breast lung colorectal and prostate ppvs were at least for all case definitions minimal differences in ppvs were observed between age groups years and sexes online supplementary appendix figures figure presents sensitivity values for each case definition sensitivity was generally higher for the case definitions that included ncras data ranging from to for individual cancer sites except bladder cancer identified using ncras data alone and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity was also generally high for definitions using a combination of cprd gold hes apc and ons mortality data ranging from to ¥ for the four most common cancers sensitivity was lower for case definitions that used cprd gold alone range to for individual cancer sites or hes apc alone range to sensitivity values for cprd gold alone and hes apc alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures post hoc analysis suggested that the low sensitivity of cprd gold only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary organ cancer codes n1108 in cprd gold rather than contradictory information about the first cancer record n625 these incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 bladder cancers that were not recorded in ncras data n3445 were commonly recorded in both hes apc and cprd gold n2228 or in hes apc only with a subsequent unspecified or bladder cancer record in hes apc within months n995 table describes the number of days median iqr and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used case definitions using ncras alone and combinations of ¥ data sources captured cancers close to the gold standard date median lag ¤ days for all cancer sites whereas median lags were generally longer for the case definitions using cprd gold alone and hes apc alonefigure describes mortality over time following incident cancer diagnoses ascertained from each case definition minimal differences in mortality were observed between cancers identified from different case definitions where variability was observed cancers identified using cprd gold only had the lowest mortality rates eg kidney cancer and cancers identified using hes apc only or ncras only had higher mortality rates eg prostate cancer and bladder cancer respectivelyfigure describes completeness of grade and stage for cancers identified using ncras only recording of grade was highly variable between cancers with gradual increases in completeness over time completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure online supplementary appendix figure describes recording of treatment modalities identified using ncras strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure positive predictive value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service onsoffice of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0celitnecrep hthtrqi inademelitnecreprqi inadem hthtelitnecrep htht inademrqielitnecrep hthtcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l ebatopen access recnac lanoitan sarcn atad erac tneitapdetti mda scitsitats edospei latipsoh cpaseh knil atadhcraeser ecitcarp lacniilc drpc metsys suovren lartnec sncl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iia setis recnac nommoc tsom ruofdcii nosrev sesaesdi fonoitacfissacliscitsitats lanoitan rof ecfifo snoi atadnoitartsgerrecnac ecvres ssyanadna liinoitartsgeri lanoitanretni eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaci snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmun ot ot ot ot ot cl amoeym epitluml ot ci ameakuel ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep hthtsarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure mortality following first ever record of cancer in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service nhl non hodgkin's lymphoma ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure completeness of grade and stage for cancers identified using ncras data only cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites grading information is not applicable to braincns sarcoma or haematological cancers and not required by in the national data standard cosd for prostate cancer core staging is not applicable to haematological and gynaecological cancers other types of staging are recommended by cosd cns central nervous system cosd cancer outcomes and services data set ncras national cancer registration and analysis service nhl non hodgkin's lymphomastrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access only missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordeddiscussionstatement of principal findingswe investigated the use of different sources of electronic health record data to identify incident cancers for all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers combining cprd gold hes apc and ons mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date finally while we observed minimal changes in ppvs and sensitivities between and completeness of ncras cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 completeness of cancer treatment recording was difficult to assess due to the absence of a missing categorystrengths and weaknesses of the studythe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from cprd to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies we have also assessed the value of using ncras cancer registration data to measure stage grade and cancer treatment modalitiesa limitation of the study is that our gold standard algorithm is not validated we feel that we were justified in pre weighting ncras data as more reliable that other data sources as ncras is a highly validated data set that matches merges and quality checks data from multiple sources4 we did not consider ncras to be the outright gold standard as it is plausible that ncras does not identify all tumours diagnosed and treated in primary and secondary care for most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in hes apc cprd gold or ons mortality data but not in ncras these tumours may have been diagnosed and coded as invasive in primary or secondary care but not by ncras been incorrectly coded in hes apc cprd gold or ons mortality data not have been notified to ncras eg tumours treated in private hospitals or be the result of linkage errors between the data sets the proportion of cancers identified in hes apc but not in ncras is particularly high for bladder cancer this is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in ncras which are greatest for bladder cancer4 this explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in ncras compared with other data sources to identify incident cancers we required months of research quality follow up in cprd gold prior to inclusion in the study previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 the identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as ncras data collection started in hes apc in and ons mortality data in and by the inclusion of all diagnostic codes in hes apc assuming that the first ever primary or secondary record identified incident cancer reassuringly ppvs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases requiring internal confirmation within months for cancers recorded in cprd gold alone in our gold standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up our data cut only included ncras data for the top cancers earlier cancers at other sites will have been missed in this studyit is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesstrengths and weaknesses in relation to other studies discussing important differences in resultsthe most up to date study describing concordance between linked cprd gold hes apc and ncras data sets demonstrated that to of the five most common cancers recorded in cprd gold are not confirmed in either hes apc or cancer registration data and to of registered cancers are not recorded in cprd gold8 for cancers recorded in both sources the diagnosis date was a median of to days later in cprd gold than in the cancer registration data using cprd gold alone to identify these strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis use of hes apc only identified a higher proportion of patients with the correct diagnosis date than cprd gold but over represented older patients and those diagnosed through the emergency route the majority of registered cancers were picked up using both cprd gold and hes apc ranging from for lung cancer to for breast cancer previous research demonstrated similar results with substantial differences between cancer types5 additionally a study using data from to found that using hes data in addition to ncras data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancerswe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards levels of data completeness of staging information in the cprd extract in were similar to those reported by the united kingdom and ireland association of cancer registries ukaicr9meaning of the study possible explanations and implications for clinicians and policymakersuse of ncras cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately case definitions based on a combination of cprd gold hes apc and ons mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersthese findings should be considered when deciding which data sources to include in research studies and which sources to use to define cancer exposures outcomes and covariatesunanswered questions and future researchfurther research is required to investigate the validity of cancer recorded in cprd gold and hes apc that are not recorded in the ncras data and to understand differences in cancer data recording with cprd gold and cprd aurum cprds recently launched primary care database based on records from practices that use emis software17 further investigation would be required to confidently identify subtypes of cancer either using codes available in each data set eg colon and rectal cancer or additional information available in hes apc or ncras data use of ncrass recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu
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" adenovirus serotype ad5 is a commonly used viral vector for transient delivery of transgenes primarily for vaccination against pathogen and tumor antigens however endemic infections with ad5 produce virus specific neutralizing antibodies nabs that limit transgene delivery and constrain target directed immunity following exposure to ad5 based vaccines indeed clinical trials have revealed the limitations that virus specific nabs impose on the efficacy of ad5 based vaccines in that context the emerging focus on immunological approaches targeting cancer self antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectorsmethods here we evaluated the ability of a chimeric adenoviral vector ad5f35 derived from the capsid of ad5 and fiber of the rare adenovirus serotype ad35 to induce immune responses to the tumor associated antigen guanylyl cyclase c gucy2cresults in the absence of pre existing immunity to ad5 gucy2c specific t cell responses and antitumor efficacy induced by ad5f35 were comparable to ad5 in a mouse model of metastatic colorectal cancer furthermore like ad5 ad5f35 vector expressing gucy2c was safe and produced no toxicity in tissues with or without gucy2c expression importantly this chimeric vector resisted neutralization in ad5 immunized mice and by sera collected from patients with colorectal cancer naturally exposed to ad5s these data suggest that ad5f35 based vaccines targeting gucy2c or other tumor or pathogen antigens may produce clinically relevant immune responses in more ¥ patients compared with ad5 based vaccines inhibitor introductiontherapies immune checkpoint have revolutionized cancer treatment and cancer drug development by engaging the immune system to target various cancers1 despite this success many tumors are immunologically cold characterized by a dearth of immunogenic neoepitopes3 and lack of tumor infiltrating lymphocytes4 and remain refractory to checkpoint inhibitors6 one emerging strategy to modify a cold tumor into one responsive to immunotherapy is through combination with cancer vaccines8 the goal of this strategy is to use cancer vaccines to create a pool of tumor reactive t cells with antitumor activity alone andor in combination with checkpoint therapies however this approach is significantly limited by the paucity of effective vaccine platforms to safely deliver tumor specificassociated antigens to elicit beneficial antitumor immunitythe ability of adenovirus serotype ad5 to mediate gene transfer and induce potent immune responses has made it a popular vector for experimental vaccines infectious diseases10 against cancer and indeed there have been more than clinical trials using the ad5 vector with most trials focused on developing cancer treatments10 however on natural infection the host immune system develops neutralizing antibodies nabs to the ad5 capsid limiting viral spread and blocking reinfection because ad5 infections are endemic in many human populations pre existing nabs present in of the worldwide population limit ad5 based vaccine strategies12 these considerations highlight the need for improved vectors for use in vaccines targeting cancer and pathogen associated antigens that can create therapeutic immune responses in the greatest number of patients importantly while the adenovirus capsid is composed of hexon penton and fiber proteins nabs elicited by natural ad5 infection in humans are directed primarily to the ad5 fiber15 suggesting that strategies to flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access circumvent pre existing immunity to this element may improve ad5 based vaccineshere we sought to overcome pre existing ad5 nabs by replacing the ad5 fiber with that of a rare adenovirus serotype ad35 international seroprevalence to improve antitumor immunity in mouse models expressing the gastrointestinal gi cancer antigen guanylyl cyclase c gucy2c preclinical models demonstrated that an ad5 based gucy2c directed vaccine ad5 gucy2c s1 elicited cd8 t cell and antibody responses without autoimmunity17 further ad5 gucy2c s1 vaccination of mice induced long term t cell mediated protection against metastatic colorectal cancer in lung and liver19 moreover those results were recapitulated in a recent first in human phase i clinical trial nct01972737 demonstrating that a humanized version of the vaccine ad5 gucy2c padre safely induced gucy2c specific cd8 t cell responses in patients with colorectal cancer following conventional therapies21 however patients possessing high pre existing titers of nabs against ad5 failed to generate gucy2c specific immunity following ad5 gucy2c padre vaccination21 to overcome ad5 nabs we generated a chimeric ad5 vector possessing the fiber of ad35 ad5f35 with equivalent safety and antitumor activity to ad5 and resistance to ad5 nabs in mice and humans this chimeric vaccine can be translated to patients with gi cancer to safely induce gucy2c specific immunity not only in those patients with low ad5 immunity but also in those with high pre existing ad5 nabsmaterials and methodsadenovirus vectorsadenovirus containing mouse extracellular domain gucy2c1429 with the influenza ha107119 cd4 t cell epitope known as site s1 was described previously ad5 gucy2c s120 here gucy2c s1 was cloned into pshuttle and subcloned into the e1 region of previously generated replication deficient chimeric adenovirus ad5f35 in which the ad5 fiber was replaced by the ad35 fiber22 to generate ad5f35 gucy2c s1 all adenovirus vaccines used in this study were produced in hek293 cells and purified by cesium chloride ultracentrifugation under good laboratory practices by the baylor college of medicine in the cell and gene therapy vector development lab and certified to be negative for replication competent adenovirus mycoplasma and host cell dna contamination in vitro gucy2c expression experiments doseresponse and timecourse were carried out in a549 american type culture collection atcc cells virus was added to the cultures at the indicated doses and culture supernatants were collected at the indicated time points relative gucy2c levels were quantified in supernatants by western blot using μgml ms7 mouse anti gucy2c monoclonal antibody23 and μgml horseradish peroxidase conjugated goat antimouse secondary antibody jackson immunomice and immunizationseight week old male and female balbcj mice were purchased from the jackson laboratory for experiments animal protocols were approved by the thomas jefferson university institutional animal care and use committee protocol for immunizations mice received or vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or ad5f35 gfp control administered as two μl intramuscular injections one in each hind limb using a ml insulin syringequantifying tcell responses by elispotelispot assays were performed using a mouse interferonÎ ifnÎ single color elispot kit cellular technology according to the manufacturers protocol26 briefly well plates were coated with ifnÎ capture antibody overnight at °c the next day plates were washed with phosphate buffered saline pbs and splenocytes from immunized mice were plated at cellswell with no peptide or μgml gucy2c254262 peptide in dimethyl sulfoxide dmso in ctl test medium cellular technology for hours at °c for t cell avidity studies splenocytes were plated at cellswell with decreasing concentrations of gucy2c254262 peptide μgml to pgml normalized to cellswell26 after incubation cells were removed and development reagents were added to detect ifnÎproducing spot forming cells the number of spot forming cells per well was determined using the smartcount and autogate functions of an immunospot s6 universal analyzer cellular technology gucy2c specific responses were calculated by subtracting mean spot counts of dmso wells from peptide stimulated wells26 tumor studiesgucy2c expressing mouse balbc ct26 colorectal cancer cells were used for in vivo tumor studies17 luciferase expressing cells were generated by transduction with lentiviral supernatants produced by 293ft cells invitrogen with plenti4 v5 gw luciferase28 for tumor experiments balbcj mice were immunized with vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or pbs control days before delivering à ct26 cells into tail veins tumor burden was quantified weekly by subcutaneous injection of mg of d luciferin potassium salt gold biotechnologies in pbs followed by an min incubation and imaging with a s exposure using a caliper ivis lumina xr imaging station perkinelmer total radiance photonssecond was measured using living image in vivo imaging software perkinelmerantibody neutralization assayserum samples were obtained previously from patients before ad5 gucy2c padre nct01972737 approved by the thomas jefferson university institutional review board21 neutralizing antibody titers against ad5 and ad5f35 vectors were quantified as immunization with flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0cdescribed21 briefly dilutions of heat inactivated serum samples were added to well tissue culture plates containing a549 cells atcc and infected with vp of gfp expressing ad5 or ad5f35 virus ad5 cmv egfp or ad5f35 cmv egfp respectively baylor vector development lab following a hour incubation at °c egfp fluorescence nm excitation nm emission was quantified using a polarstar optimate plate reader bmg labtech sample fluorescence was normalized to control wells containing cells and virus neutralization or wells containing cells alone neutralization titers were quantified using non linear regression as the serum dilution producing neutralization prism v8 graphpad softwaread5 neutralizing immunity studiesto induce anti ad5 immunity mice were exposed intranasally to ad5 gfp once or twice at a week interval thirty days after the last exposure ad5 nabs were quantified in sera as described above and mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1biodistribution and toxicology studybalbcj mice were immunized intramuscularly with a single dose of vp of ad5f35 gucy2c s1 three doses of vp of ad5f35 gucy2c s1 at day intervals or pbs control animals were monitored for adverse events once daily with additional evaluations on the day of dosing min hour and hours after dosing on days and designated animals were sacrificed and brain salivary glands stomach small intestine colon heart lungs kidneys liver and injection site were harvested and weighed for histopathological analysis by a blinded pathologist pathology evaluation was performed by idexx bioanalytics and detection of viral dna by quantitative pcr qpcr using the previously described assay for the gucy2c transgene19 also spleens were collected for histopathological analysis and detection of viral dna as described above as well as quantification of gucy2c specific t cell responses by ifnÎ elispot as described abovestatistical analysisstatistical analyses were conducted using graphpad prism software v8 statistical significance was considered as follows nsp p p p and p cohort sizes were powered based on prior studies with β02 and α005 for multiple comparisons of survival outcomes significance thresholds were corrected using the bonferroni method to identify vaccine induced t cell responders and non responders a previously described21 modified distribution free resampling approach was employed and positive t cell responses were defined as à compared with dmso and specific spots106 cells to determine the impact of gender and number of vaccinations on responses log transformed vaccine response magnitude was compared in mice of different genders cohorts and treatment regimens for up to three way interactions with stepwise backward variable selection by akaike information criterion using r29 package mass30open accessresultsad5gucy2cs1 and ad5f35gucy2cs1 vectorswhile ad5 seroprevalence worldwide exceeds in some regions ad35 is and associated with lower titers figure 1a12 thus we constructed a chimeric adenovirus ad5f35 composed of ad5 in which the fiber was replaced by the ad35 fiber and evaluated its ability to induce gucy2c specific immunity and resist ad5 specific immunity in humans and mice ad5 gucy2c s1 is a replication deficient human ad5 expressing the mouse gucy2c extracellular domain fused to the i ed restricted cd4 epitope known as site at its c terminus20 to generate ad5f35 gucy2c s1 the ad5 fiber l5 was replaced with the ad35 fiber figure 1b replication deficient ad5 gucy2c s1 and ad5f35 gucy2c s1 generated in hek293 cells produced dose dependent figure 1c and time dependent figure 1d expression of gucy2c s1 protein in a549 human alveolar basal epithelial cells in vitroad5f35gucy2cs1 induces gucy2cspecific antitumor immunityfollowing in vitro validation of gucy2c expression by ad5f35 gucy2c s1 we confirmed its ability to induce gucy2c specific immune responses after vaccination in vivo balbc mice immunized intramuscularly with vp of ad5f35 gucy2c s1 produced lower gucy2c specific cd8 t cell responses figure 2a and no gucy2c specific antibody responses figure 2b compared with ad5 gucy2c s1 importantly ad5 and ad5f35 vaccines produced gucy2c specific cd8 t cells of comparable avidity figure 2c a critical determinant of the antitumor efficacy of gucy2c targeted vaccines26 in contrast gucy2c specific antibody responses have no detectable antitumor activity20 similarly ad5 and ad5f35 vaccines produced comparable s1 specific cd4 t cell responses figure 2dluciferase this model previous studies revealed that ad5 gucy2c vaccines induced protective antitumor cd8 t cell responses in murine models of metastatic colorectal cancer17 thus balbc mice were immunized with ad5 or ad5f35 expressing gucy2c s1 and challenged days later with ct26 colorectal cancer cells expressing gucy2c and firefly specifically emulates secondary prevention of metastatic disease the clinical setting for which the gucy2c vaccine is being developed21 as previously demonstrated ad5 vaccination nearly eliminated metastatic tumor burden figure 3ab delayed disease progression figure 3c and improved survival figure 3d similarly ad5f35 also reduced tumor burden figure 3ab disease progression figure 3c and prolonged survival figure 3d importantly the efficacy of ad5 based and ad5f35 based gucy2c vaccines in flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure construction of ad5f35 gucy2c s1 and antigen expression a reported international seroprevalence of ad5 and ad3512 b the l5 gene encoding the fiber protein from ad5 was replaced with the l5 gene from ad35 producing the chimeric adenoviral vector ad5f35 recombinant ad5f35 gucy2c s1 was produced by inserting mouse gucy2c s1 into the e1 region of e1e3 deleted ad5f35 c and d the human alveolar basal epithelial cell line a549 was transduced in duplicate with ad5f35 gucy2c s1 at a multiplicity of infection moi from to for hours c or at an moi of for and hours d supernatants from infected cells were analyzed for gucy2c s1 protein expression by immunoblot protein expression was quantified by densitometry and plotted relative to uninfected cells error bars indicate mean±sem ad5 adenovirus serotype reducing tumor burden opposing disease progression and promoting survival was identical figure 3adad5f35 resists ad5directed immunity in mice and humansnabs against ad5 correlated with poor gucy2c specific immune responses in patients receiving ad5 gucy2c padre vaccination and prior exposure of mice to ad5 similarly blunted vaccine induced immunity21 ad5f35 based vaccine resistance to pre existing ad5 immunity was quantified in a model of respiratory pre exposure to ad5 the natural route of infection in patients33 followed by vaccination and quantification of gucy2c specific t cell responses control mice not pre exposed to ad5 naive and those that were pre exposed once à or twice à to intranasal ad5 were vaccinated after weeks with intramuscular ad5 or ad5f35 expressing gucy2c s1 and immune responses were quantified weeks later immunogenicity of ad5 gucy2c s1 and ad5f35 gucy2c s1 ad balbc mice n4 micegroup were figure immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and serum and splenocytes were collected days later gucy2c specific cd8 t cell responses were quantified by interferon gamma ifnÎ elispot a and antibodies were quantified by elisa b c gucy2c specific t cell avidity measurements were analyzed by elispot using non linear regression logagonist versus normalized response with comparisons made using the extra sum of squares f test avidity plots depict the regression line solid with cis dashed d s1 specific cd4 t cell responses were measured by ifnÎ elispot t cell responses in a and d were analyzed by one way analysis of variance values in a b and d indicate individual animals and bars in a and d indicate means tcr t cell receptor ad5 adenovirus serotype flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure antitumor efficacy of ad5 gucy2c s1 and ad5f35 gucy2c s1 ad balbc mice n10 micegroup were immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and challenged days later with a mouse colorectal cancer cell line ct26 expressing gucy2c and luciferase on days and following challenge mice were injected with d luciferin and imaged a to quantify tumor burden day b mice were weighed twice weekly c and monitored for survival d tumor burden b was analyzed by one way analysis of variance and survival comparisons d were analyzed by the mantel cox log rank test in b and d asterisks indicate comparisons of gucy2c vaccines to the control and brackets ] indicate comparisons between ad5 and ad5f35 vaccines ns not significant ad5 adenovirus serotype figure 4a as expected one ad5 pre exposure induced moderate ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses while two pre exposures induced high ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses following ad5 vaccination figure 4b in contrast gucy2c specific t cell responses were reduced only à pre exposure and à pre exposure following ad5f35 vaccination figure 4b importantly ad5f35 produced t cell responses in a substantially greater fraction of the population cohort responses compared with ad5 cohort responses following serial pre exposures to ad5 figure 4cthese observations in mice were recapitulated using sera from patients with colorectal cancer in the ad5 gucy2c padre phase i trial nct0197273721 here nab titers against ad5 and ad5f35 were quantified using an established ad5ad5f35 reporter virus inhibition bioassay in serum samples collected prior to vaccination with ad5 gucy2c padre21 in these patients ad5f35 specific nab titers were substantially lower than ad5 specific titers figure 4d most importantly of patients possessed low ad5 nabs titers figure 4de which closely correlated with a gucy2c specific response rate21 in striking contrast had low ad5f35 nab titers suggesting that the vast majority of patients immunized with ad5f35 based vaccines could produce gucy2c specific responses figure 4e collectively these observations suggest that pre existing viral immunity induced by repeated environmental exposures which neutralizes ad5 delivery platforms may be overcome by the chimeric ad5f35 vector to enhance fractional population vaccine responsessafety biodistribution and toxicity of ad5f35gucy2cs1food and drug administration ind investigational new drug enabling studies quantified the toxicity biodistribution and immunogenicity of ad5f35 gucy2c s1 in balbc mice employing three schemes to examine acute and chronic effects figure 5a cohorts balanced for sex received ad5f35 gucy2c s1 either as a single intramuscular injection or as three intramuscular injections spaced weeks apart monitored daily and sacrificed on day or for analysis as indicated figure 5a there were no signs of acute or chronic toxicity in the in life phase by observation weight changes or survival figure 5bd similarly there were no clinically significant differences in organ weights online supplementary figure s2 or histopathology not shown at necropsy small statistical differences in organ weights were considered clinically insignificant and were unrelated to vaccine exposure dose time online supplementary figure s2 biodistribution quantified by qpcr detected ad5f35 gucy2c s1 at the injection site and in the spleen but not appreciably in other organs after acute and chronic exposures online supplementary figure s3 moreover robust cd8 t cell responses were quantified at day that persisted through day in of mice after a single administration figure 5eg as expected cd8 t cell responses were greater and persisted in more mice at days after three vaccinations figure 5egdiscussionthrough decades of gene therapy trials ad5 has remained a popular vector while high ad5 seroprevalence remains a barrier to universal vaccination33 natural respiratory infection can generate long lived antibodies that neutralize ad5 based vaccines eliminating transgene delivery and potential therapeutic benefit in that context ad5 seroprevalence is across multiple countries12 highlighting an unmet need for alternative vectors here we demonstrate that the chimeric ad5f35 resists pre existing ad5 immunity and induces transgene specific antitumor immunity indeed ad5f35 is less susceptible to neutralization associated with ad5 exposure in mice and humans and generates a substantially higher proportion of vaccine responders in mice pre exposed to ad5 these observations support the suggestion that ad5f35 flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure ad5f35 resists neutralization associated with pre existing anti ad5 immunity in mice and humans ac to generate pre existing immunity to ad5 balbc mice n10 micegroup were exposed intranasally once or twice to vp of ad5 gfp at week intervals four weeks after the final ad5 gfp exposure ad5 exposed and naive mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 b two weeks after immunization gucy2c specific cd8 t cell responses in each group were quantified by interferon gamma ifnÎ elispot and calculated as the of mean responses in naive mice values indicate individual animals and bars indicate means ad5 and ad5f35 were compared by two way analysis of variance c the fraction of animals producing a detectable gucy2c specific cd8 t cell response filled regions in naive à and à ad5 exposed mice was determined from b d and e sera from patients with colorectal cancer collected prior to ad5gucy2c padre vaccination were tested for the ability to neutralize ad5 and ad5f35 vectors and titers were quantified d analyzed by paired t test the dotted line indicates a titer of the threshold for high neutralizing antibody nab titers21 e while subjects had high nab titers against ad5 only had high titers to ad5f35 vector filled regions binomial test ad5 adenovirus serotype will produce a higher proportion of vaccine responders in patient populationsthe extent to which nabs to the ad5 fiber limit reinfection is controversial in some studies replacing the ad5 fiber with that of another serotype circumvents pre existing ad5 immunity34 in contrast other studies suggest that these chimeric adenoviruses do not evade pre existing ad5 nabs suggesting the hexon as the major target of antibody neutralization35 in contrast to those previous studies which generated pre existing ad5 immunity by intramuscular35 or intravenous administration36 here ad5 immunity was induced by intranasal exposure in mice recapitulating natural infection33 moreover natural human respiratory pre existing ad5 nabs in patients with colorectal cancer uniformly produced by repeated respiratory infections33 similarly were overcome by the ad5f35 vector importantly the quality of antibody responses following adenovirus infection is dependent on the route of exposure indeed respiratory infections elicit fiber specific nabs while intramuscular exposure induce capsid specific nabs15 these qualitative differences in nab responses reflecting varying routes of immunization may contribute to observational discrepancies between laboratories the present studies using relevant animal models confirmed and validated with patient samples support the suggestion that ad5f35 based vaccines should produce clinically relevant immune responses in a substantial proportion of patientsflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure safety and immunogenicity of multiple ad5f35 gucy2c s1 administrations ag balbc mice n10 micegroup were immunized intramuscularly with one or three administrations of vp ad5f35 gucy2c s1 or control at week intervals following immunization body weights b female and c male were recorded weekly and mice were monitored for survival d at days and following first immunization mice were euthanized to quantify organ pathology by weight online supplementary figure s2 biodistribution by quantitative pcr online supplementary figure s3 and gucy2c specific cd8 t cell responses by interferon gamma ifnÎ elispot eg g pie charts indicate proportion of responding animals ad5 adenovirus serotype recognizing the pervasive limitations imposed by endemic ad5 immunity in global populations12 there is an emerging interest in alternative serotypes and chimeric constructs as a tractable strategy in vaccine development ad26 ad35 and ad48 vectors have been advanced into phase i clinical trials37 in that regard a comparison of ad5 ad26 ad35 and ad48 immunity among healthy patients revealed that endemic ad35 seropositivity was lowest across global populations12 reinforcing chimeric strategies employed herein similarly the first hexon chimeric adenovirus comprising ad5 and ad48 components was safe and immunogenic in patients39 interestingly ad5 ad35 chimeric vectors more efficiently transduce a variety of human cell types in vitro compared with either parental vector40 these observations underscore the future potential of intelligently designed chimeric adenoviruses strategically constructed to deliver transgenes for replacement therapy or vaccination and targeted precisely to the cellular or disease context40while antitumor efficacy was equivalent cd8 t cell responses were lower and antibody responses were absent for ad5f35 gucy2c s1 compared with ad5 gucy2c s1 however the antitumor efficacy of gucy2c directed immunotherapy is driven primarily by t cell avidity rather than effector t cell quantity26 in that context the functional avidity of gucy2c specific cd8 t cells following ad5 and ad5f35 immunizations were equivalent consistent with their comparable antitumor efficacy quantitative differences in transgene specific immunity between vectors may reflect a variety of factors thus the quantity and persistence of gucy2c s1 transgene following ad5f35 immunization is lower compared with ad519 consistent with prior observations that ad5 transduction efficiency in vivo may be several fold higher than ad5f3541 moreover the ad5 fiber binds to cxadr coxsackievirus and adenovirus receptor42 while the ad35 fiber binds to cd4643 suggesting the two viruses may infect distinct cell typesflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access while checkpoint inhibitors have generated practice shifting results in the clinic and defined immunotherapy as an effective strategy for the treatment of several malignancies they have not been universally successful in that context the dearth of neoepitopes in many cancer types including microsatellite stable colorectal and pancreatic second and third leading causes of cancer mortality respectively makes them insensitive to checkpoint blockade7 indeed examination of neoepitopes presented on the surface of five colorectal cancer specimens revealed a total of three neoepitopes3 thus vaccines targeting cancer associated self antigens have re emerged alone and in combination with checkpoint inhibitors as a strategy to prevent and treat metastases from these cold tumors44 checkpoint inhibitors have become first line therapy in the metastatic setting for some cancers46 while chimeric antigen receptor expressing t cells car t cells are being deployed in patients with metastatic and refractory disease47 in contrast few cancer immunotherapies have been developed for early stage cancer patients with no evidence of disease ned following conventional surgicalradiochemotherapies who are at significant risk of disease recurrence indeed25 of stage ii and of stage iii patients with colorectal cancer recur following surgery and chemotherapy49 while of patients with resectable pancreatic cancer experience recurrence50 vaccines targeting tumor associated antigens such as ad5f35 gucy2c padre may provide safe and effective immunotherapies for the secondary prevention of metastatic disease in patients with ned who are otherwise ineligible to receive checkpoint inhibitors or car t cellsthe present studies suggest that the chimeric adenoviral vector ad5f35 may be preferable to the widely used ad5 vector and warrants further investigation indeed they suggest that ongoing clinical investigations of gucy2c directed immunotherapy in patients with gucy2c expressing cancers including colorectal pancreatic gastric and esophageal could benefit from using the ad5f35 rather than the ad5 vector in that context an upcoming clinical trial will examine the safety immunogenicity and resistance to pre existing immunity of ad5f35 gucy2c padre in patients with gi cancer nct04111172 safe generation of gucy2c targeted immunity in a high proportion of patients will lead to efficacy trials to establish the ability of ad5f35 gucy2c padre to prevent recurrence following standard therapy in patients with gi cancer who represent of all cancer deaths51 and for whom established immunotherapies are ineffectivetwitter adam e snook adamsnookphdacknowledgements the authors thank adrian p gee phd zhuyong mei md deborah lyon and malcolm brenner md phd center for cell and gene therapy baylor college of medicine for assistance in vaccine manufacturingcontributors jcf js bb saw and aes designed the studies jcf js rc el trb jb ec ap jar and jr carried out the studies tz carried out data analysis and statistical analysis in discussion with aes jcf and aes wrote the manuscript and all authors critically reviewed and approved the final version of the manuscriptfunding this work was supported by the national institutes of health nih r01 ca204881 r01 ca206026 and p30 ca56036 the defense congressionally directed medical research program w81xwh17 prcrp ttsa and targeted diagnostic therapeutics to saw aes received a research starter grant in translational medicine and therapeutics from the phrma foundation a degregorio family foundation award and was supported by the defense congressionally directed medical research programs nos w81xwh1710299 w81xw
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netosis is a type of regulated cell death dependent on the formation of neutrophil extracellular traps net where netlike structures of decondensed chromatin andproteases are produced by polymorphonuclear pmn granulocytes these structuresimmobilise pathogens and restrict them with antimicrobial molecules thus preventing theirspread whilst nets possess a fundamental antimicrobial function within the innate immunesystem under physiological circumstances increasing evidence also indicates that netosisoccurs in the pathogenic process of other disease type including but not limited to atherosclerosis airway inflammation alzheimers and stroke here we reviewed the role ofnetosis in the development of an injury including injury to the brain lung heart kidneymusculoskeletal system gut and reproductive system whilst therapeutic agents in blockinginjuries induced by netosis in its primitive stages were also discussed this review providesnovel insights into the involvement of netosis in different an injuries and whilstpotential therapeutic measures targeting netosis remain a largely unexplored area thesewarrant further investigationkey words netosis neutrophil an injury cell death inflammation cell death is commonly segregated into necrosisand apoptosis apoptosis being programmed cell death anaesthetics pain medicine and intensive care department of surgeryand cancer faculty of medicine imperial college london chelsea andwestminster hospital fulham road london sw10 9nh uk department of anesthesiology shanghai fengxian district central hospital shanghai jiao tong university affiliated sixth peoples hospitalsouth campus fengxian district shanghai china to whom correspondence should be addressed at anaesthetics painmedicine and intensive care department of surgery and cancer faculty of medicine imperial college london chelsea and westminsterhospital fulham road london sw10 9nh uk emaildmaimperialacukfor instance during development and physiological cellular turnover whilst necrosis predominantly takesplace in an unregulated manner netosis like necrosis is a mode of cell death that involves the loss ofmembrane integrity during netosis decondensationof chromatin is thought to be initiated by peptidyl arginine deiminase pad4 its subsequent releasetogether with granule contents is vital in the innateimmune response to infection and inflammation recentstudies suggest that net formation is of central topathogenesis of an injury this review will summarise the current understanding of the molecular mechanisms of netosis and the therapeutic approaches underdevelopment targeting netinduced an injury the authors this is an open access publication 0cmolecular mechanism of netformationalthough netosis is closely associated with netformation not all net formation requires the process ofcell death to take place beforehand according to nomenclature committee on cell death the term netosisshould only be used in the context of cell death and notjust based on the presence of net formation two main pathways of net formation have beendescribed and categorised according to their dependenceon the activity of nicotinamide adenine dinucleotide phosphate nadph oxidase pathway fig nadph oxidasedependent net formationthe nadph oxidasedependent molecular pathwayof net formation begins with activation of neutrophilsurface receptors by stimuli derived from pathogenic ornonpathogenic sources such as cholesterol or urate andends with cellular lysis these stimuli trigger calciumrelease from the endoplasmic reticulum er resulting inthe activation of protein kinase c pkc and the assemblyof the nadph oxidase complex generating reactive oxygen species ros following this neutrophil elastasene a protease stored in the cytoplasmic granules migrates to the nucleus in a myeloperoxidase mpodependent manner and cleaves histones to initiate chromatindecondensation this is promoted by the citrullinationof histone arginine residues by peptidylarginine deiminaseiv pad4 finally mixing of the chromatin andgranule proteins takes place as cellular membranes arebroken down interestingly there have been reports ofmitochondrial dna mtdna instead of nuclear dnabeing the source of the dna fibres in nets with observations of mtdna being released from granulocytes inresponse to disease states such as trauma and systemiclupus erythematosus sle [ ] moreover it seemsthat histone citrullination is not always required for netformation as observed by kenny and colleagues in theirstudy of neutrophils activated by the pkc agonist phorbol12myristate 13acetate pma this highlights the diversity of pathways for net formation following their induction degradation of nets can take place through severalpathways for example by dnases or endocytosed bymacrophages factors that influence net formation include phco2 and hco3 levels which modulate neutrophil activation this explains why nets form more readily in thecahilog zhao wu alam eguchi weng and maperiphery of an inflammatory site where the ph is morealkaline this may influence treatment efficacy asnets can seal off the affected area an acidic environmenthas been hypothesised to reduce nadph oxidasedependent net formation by reducing neutrophil glycolysis nadph oxidasedependent net formation also requires neutrophils to be in the cell cycle necessitating theactivation of cyclindependent kinases cdk phosphorylating the retinoblastoma proteinnadph oxidaseindependent net formationthis mechanism of net formation is more relevant inthe context of infection as inducers of netosis here arecalcium ionophore a23187 and the potassium ionophorenigericin which are products of streptomyceschartreusensis and streptomyces hygroscopicus respectively how this pathway leads to net release ispoorly understoodnetosis and inflammationnets under physiological conditions are central topathogen clearance when there is excessive formation orsuboptimal nets are able to initiate further destructivesignalling through interaction with other tissue constituentsand the immune system moreover the antimicrobial histones and peptides within the net structure impose adirect cytotoxic effect on tissues to date there havebeen numerous accounts of netosis being present indiseases of major ans understanding of netosis inpathophysiology may offer unique opportunities for clinical managementnetosis in an injurythere is an expanding body of research describingnetosis in infectious and noninfectious an injurysummarised in fig although it is valid that in thesescenarios nuclear dna released during necrotic cell deathcan contribute to tissue injury and exacerbate the extent ofan damage here we focus on the contribution by aberrant net formation and the implication of understandingits underlying pathogenesis for therapeutic interventions 0crole of neutrophil netosis in an injuryfig type of cell death for neutrophil in an injury during solid an injury neutrophils could be prompted to undergo caspasedependent apoptosiswhich results in controlled dissolution of cell into apoptotic bodies containing cellular debris to prevent immune and inflammatory responses neutrophilextracellular traps nets form via two pathways the first is through lytic netosis a cell death pathway characterised by nuclear delobulation anddisintegration of the nuclear envelope which precedes loss of cellular polarisation chromatin decondensation and plasma membrane rupture the secondmechanism involves the nonlytic form of netosis which is not associated with cellular death but prompts expulsion of nuclear chromatin together withrelease of granule proteins through degranulation these components can assemble in the extracellular space into nets leaving behind enucleated cytoplaststhat continue to ingest microanisms in addition neutrophils could undergo unregulated necrosis that does not involve specific molecular pathwayswith uncontrolled release of cellular debris acting as dangerassociated molecular patterns damps to trigger proinflammatory responsebrainalzheimers disease alzheimers disease ad is acommon disorder of neurodegeneration characterised bygradual loss of cognitive functions in ad patients neutrophils have been observed to invade the brain parenchyma and release nets causing destruction of neural cellsand the bloodbrain barrier stroke it is well known that the adaptive immunesystem is altered after a stroke predisposing patients toinfections [] interestingly netosis has also beendescribed as significantly impaired up until on day inthose with an ischaemic stroke though netosis inhaemorrhagic stroke patients has yet to be documented ithas been noted that the generation of ros a keyrequirement for chromatin decondensation is suppressedin these patients for up to days lungcystic fibrosis it has been well established that chronic infections in cystic fibrosis cf patients are due to thehighly viscous mucus production harbouring microbialgrowth although impaired clearance of mucus has beenprincipally named responsible there is increasing evidencethat the high viscosity is also due vast amounts of freedna found in sputum samples which was in concordance with the high concentration of neutrophil and 0ccahilog zhao wu alam eguchi weng and mafig involvement of netosis in an injury accumulating evidences now point to an important role of netosis in infectious and noninfectious solidan injury neutrophil invasion into brain parenchyma and release of neutrophil extracellular traps nets have been established in the pathophysiology ofalzheimers disease to cause destruction to neural cells and bloodbrain barrier abnormal netosis activity and reactive oxygen species ros response akey element to netosis initiation were observed in stroke patients the degree of neutrophil infiltration net formationcomponent eg cellfreenucleosomes and netosis have been found to correlate with the severity of a range of lung diseases including cystic fibrosis acute lung injury aliacute respiratory distress syndrome ards and lung infection netosis was also shown to be involved in allergic asthma chronic obstructive pulmonarydisease and pulmonary hypertension wherein degree of net formation correlates with disease severity during liver ischaemiareperfusion tolllikereceptordependent net release has been suggested to mediate liver inflammation and injury conversely deficiency in net release was reported indecompensated cirrhotic liver disease and could explain susceptibility to bacterial peritonitis infection in those patients net formation and netosis havefurther been implicated in atherosclerosis and myocardial infarction wherein net was found in thrombi and infarct lesion and correlate with disease severityin rheumatoid arthritis enhanced net release and netosis are observed in synovial tissue rheumatoid nodules and skin whilst proinflammatory cytokinesand autoantibodies further aggravate neutrophil infiltration and netosis neutrophils could also be potently activated by monosodiumurate msu crystals ingout joints and point to a potential role of netnetosis in gout pathogenesis moreover neutrophil activation and net deposition were also observed incolon mucosa of ulcerative colitis excessive neutrophil activation net formation and netosis could also be responsible different pregnancyrelateddisorders including preeclampsia wherein net deposition and netosis in the intervillous space may damage maternal endothelium and impair foetaloxygen exchangenets found in cf lungs the source was believed to befrom necrotic neutrophils but is now considered to besecondary to netosis additionally net productionwas found to be promoted by bacterial infection in cfairways and was defective in clearance of the bacteriapseudomonas aeruginosa nets are also named as a facilitative factor for biofilm formation there is evidencethat surfactant protein d spd responsible foropsonising pathogens and dying cells for clearance byalveolar macrophages is essential for net clearancethrough binding directly to the chromatin within the netsspd levels are decreased in cf patients and the level ofdecrease is proportional to the degree of inflammationthrough accumulation of nets 0crole of neutrophil netosis in an injurylung infection neutrophils migrated into the affectedsite and initiate the cascade of antimicrobial mechanismsincluding net generation to combat microanismsthis happens more readily in the lungs compared with inother tissues with neutrophils found to exist in higherconcentrations in pulmonary vasculature compared withsystemic blood vessels a prominent pathway leadingto net formation in infection is through the interaction oflipopolysaccharide lps with tolllike receptor tlr4found on neutrophils in patients with communityacquired pneumoniacap increased levels of cellfree nucleosomes in serumsamples used as surrogate markers of netosis werefound this was associated with prolonged hospitalisationand a greater 30day allcause mortality this findingsuggests nets could function as a novel marker of prognosis in capacute lung injury and acute respiratory distresssyndrome the degree of neutrophil influx into the lungsand net release during ali and ards positively correlates with disease progression and severity with neutrophil depletion conferring protection in a transfusionrelated ali animal model netosis seems to be akey component of ventilatorinduced lung injury vili as evidenced by detection of citrullinated histone3suggesting that this was a mode of cell death independentfrom apoptosis and necrosis the authors suggestedthat this may be due to increased levels of il1β andhmgb1 which have been both shown to be able to inducenetosisallergic asthma patients with neutrophilic asthmahave a greater severity of disease and reduced response tocorticosteroid treatment compared with the eosinophilictype the increased expression of neutrophilchemoattractant il8 in airway smooth muscle cells couldbe contributing to disease severity through inducingnetosischronic obstructive pulmonary disease netosis hasbeen documented as an integral part of chronic obstructivepulmonary disease copd pathophysiology unlike asthma where neutrophils are important in certain subtypesnetosis has been directly linked to disease severity incopd [ ] tlr4 expression one of the main potentiators for net formation is increased during copd exacerbations pulmonary hypertension nets are also able to potentiate dysregulated angiogenesis as seen in patients withchronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension as plasma levels of dnane and mpo levels are significantly elevated moreovernets also seem to destabilise intercellular junctions andincrease endothelial cell motility through direct contactwith endothelial cells nets were found to induce theactivity of the proinflammatory transcription factor nfκbby approximately 3fold moreover nets increase thesurface expression of von willebrand factor and plateletadhesion thereby producing a prothrombotic state kidneyglomerulonephritides nets have been visualised upon immunostaining renal biopsies from patients with sleand antineutrophil cytoplasmic antibodiesassociated vasculitis aav and may be at least partially responsiblefor activating complement pathways resulting in diseaseexacerbations these autoimmune conditions alsoseem to affect the patients ability to degrade nets amplifying their deleterious inflammatory effects increscentic glomerulonephritis neutrophilmediated glomerular damage is worsened by addition of extracellularmpo which could have been released during netosis netosis could also contribute to the loss of immunetolerance through further externalisation of crucialautoantigens during cell death haemolytic uraemic syndrome hus plasma from affected patients exhibited a greater capacity to undergonetosis compared with healthy patients the ensuingdamage has been linked to the proinflammatory cytokinesil6 and il8 released from glomerular epithelial cellsupon stimulation by nets this potentiates microvasculature inflammation and thrombosis precipitating renal failure liverdecompensated cirrhotic liver disease a deficiency innet release has been demonstrated to play a role in theonset of endstage liver disease as neutrophils incirrhotic patients are found to have defective ros production which commonly triggers net release thismay also partially explain why these have a predispositionto recurrent bacterial infections and increased rates ofdecompensatory complications such as spontaneous bacterial peritonitis sbp this is corroborated by defectivenet release from ascitic fluid neutrophils in cirrhoticpatients compared with controls in vitro cirrhoticpatients with sbp were also found to have an increase in 0cpro and antiinflammatory cytokines in peripheral bloodand ascitic fluid ischaemiareperfusion injury ischaemiareperfusioninjury iri is an inherent consequence of liver transplantation hypovolaemia or trauma and results in the release ofdamageassociated molecular patterns damps which inturn cause net formation in a tlrdependent mannerexacerbating inflammation treatment with apeptidylargininedeiminase pad4 inhibitor ordnase has been shown to be significantly hepatoprotectivefollowing liver iri cardiovascular systematherosclerosis nets are a wellknown constituent ofatherosclerotic lesions mpo has been strongly associated with diminishing the cardioprotective effects ofhighdensity lipoprotein cholesterol hdlc through oxidation reactions and an increased enzymatic activity islinked to increased plaque rupture other proteinsfound in nets such as cathelinrelated antimicrobial peptide cramp have also been shown to contribute todisease progression moreover in vitro studies showthat hypercholesterolemia triggers net formation alargescale study in patients with suspected coronary arterydisease revealed that the markers of netosis such asextracellular dna are independently associated with disease severity coronary specimens from patients afteran acute myocardial infarction mi showed the presenceof nets in both fresh and lytic thrombi therefore suggesting netosis happens in the early stages of thrombusevolution furthermore net burden was shown tobe positively correlated with the infarct size in patientsundergoing primary percutaneous coronary interventionspostmi this is supported by increased levels of mpodna and ne in the lesion site therefore nets couldpotentially be considered as a novel biomarker in atherosclerosis diabetes mellitusinduced vasculopathy it has beenshown that neutrophils form peripheral blood of diabetesmellitus dm patients showed increased spontaneousnetosis interestingly metformin reduces the deleterious effects of netosis in a mechanism independentlyfrom glucose control one recent study showed that month treatment with metformin in predm patients reduced levels of components of nets whereas glycaemiccontrol with other medication such as insulin saw nodifference when compared with placebo controls thiscahilog zhao wu alam eguchi weng and mahas been attributed to a direct effect of metformin oninhibiting the activation of nadph oxidase musculoskeletal systemrheumatoid arthritis neutrophils from the peripheralblood and synovial fluid of patients with rheumatoid arthritisra exhibit increased netosis compared with healthy controls and patients with osteoarthritis the externalisation ofcitrullinated proteins during the process of netosis wasfound to initiate and perpetuate the aberrant immune responsein ra moreover the autoantibodies and inflammatory cytokines commonly seen in ra promote netosis resulting in avicious cycle of tissue destruction gout gout is an inflammatory disease that involvesthe deposition of monosodiumurate msu crystals injoints during acute gout there is increased movement ofneutrophils into the synovial fluid msu is a known neutrophil stimulus and it has been shown that acute gout isassociated with an increase in il1β levels a keyplayer in net formationgutulcerative colitis there is prominent neutrophil infiltration in the colon mucosa in ulcerative colitis uc and this correlates with disease severity in uc the inflammatory environment promotes neutrophil activation andil1β expression in contrast nets do not seem toplay a key role in crohns disease this may explain whymesalazine a known inhibitor of il1β production and thefirstline treatment for uc flareups is not therapeutic incrohns patients per se reproductive systempreeclampsia placentas from women diagnosed withpreeclampsia showed increased neutrophil infiltration andnetosis when compared with nonhypertensive pregnantcontrols [ ] and are probably involved in causingwidespread damage to the maternal endothelium placental and endothelial injury during pregnancy aberrantneutrophil activity during pregnancy is also associated withother severe complications including recurrent foetal loss one recent study indicated neutrophils in pregnant womenseem to have an increased propensity to undergo netosissecondary to an increase in granulocyte colonystimulating 0crole of neutrophil netosis in an injuryfactor during pregnancy progesterone has been shown toattenuate neutrophilmediated ros production whereas 17βestradiol induces intracellular ros generation in a dosedependent manner associated an injury associated with netosis fig examples of recent publications on potential therapeutic compounds targeting netosis are summarised in table netosis as a therapeutic targettargeting critical steps in net formation and degradation is critical for developing treatment strategies for netosistlr inhibitorsdexamethasone dex has been shown in vitro toreduce netosis in neutrophils that are stimulated withstaphylococcus aureus but not in those stimulated withpma the tlrs involved in s aureusinduced net formation seem to be tlr2 and tlr4 as agonists of thesereceptors rescued dex inhibition interestingly althoughfig therapeutic strategies targeting net formation stimulation of neutrophil receptors eg fc γ receptor tolllike receptor by microanisms orsterile signals leads to release of calcium ca2 from the endoplasmic reticulum er cellular ca2 overload results in activation of protein kinase c pkcassembly of the nicotinamide adenine dinucleotide phosphate nadph oxidase complex andor mitochondrial activation thus stimulating reactive oxygenspecies ros production oxidative stress promotes myeloperoxidase mpodependent migration of granular neutrophil elastase ne into the nucleus tocleave histones subsequent activation of peptidylarginine deiminase pad induces histone citrullination to cause chromatin decondensation the last stepinvolves nuclear membrane degradation and extrusion of a mixture of chromatin and granular proteins into extracellular space whereby extracellular dnaseeventually digests and removes neutrophil extracellular traps nets in this regard modulation of critical steps in net formation and degradation shown byblocking arrows might be beneficial for the treatment of inflammatory disorders figure modified and reproduced with permission fcγr fc γ receptortlr tolllike receptor 0cdex reduced net formation it did not significantly affectros production calcineurin inhibitorscalcineurin is a calciumdependent serinethreonineprotein phosphatase that is important for neutrophil activity many stages of netosis induction depend upon calcium mobilisation hence modulators of the calcineurinpathway are potential pharmacological inhibitors of netformation cyclosporine a csa an antagonist of thecalcineurin pathway has been shown to reduce the effectof key physiological activators of neutrophils this effecton netosis may in part explain csas efficacy in ra and steroidresistant uc patients pmainducednetosis seems to be calciumindependent as this wasnot inhibited by csa cahilog zhao wu alam eguchi weng and mapad inhibitorsusing a murine model of atherosclerosis knight andcolleagues have shown that pharmacological inhibition ofpad4 using weeks of daily clamidine injections reduced netinduced vascular damage with delayed plaqueprogression in the carotid arteries the same groupalso showed that pad inhibitors reduce disease activity inmurine models of sle by reducing endothelial dysfunction it is worth mentioning that the possibility of padinhibition as a therapeutic avenue to be pursued in netinduced an damage in glomerulonephritis has beenrecently challenged by the work of gordon and colleagueson murine models on sle with pad4 deletion theyshowed that this did not reduce endan damage asmeasured by proteinuria suggesting that mechanismsother than net formation are implicated in this complexautoimmune conditionros scavengersdnase therapythe mitochondria are a powerful source of ros ros scavengers such as nacetyl cysteine nac reducenet formation and severity of sle in patients troloxand tempol are two antioxidants which have also beenshown to prevent netosis of pmastimulated humanneutrophils in a dosedependent manner and have beenrecommended for treatment of autoimmune and inflammatory pathologies dnase therapy has been proposed to improve outcomes in cf patients through reducing mucous viscosityhowever it appears that recombinant dnase does notreduce the load of dnaprotein complexes seen innetosis one solution to this is to combine elastase withdnase in order to degrade histones and provide dnaseaccess to chromatin this combination has been shown toenhance solubilisation of sputum drug classstudymain findingstable potential therapeutic approaches targeting netosistlr inhibitorswan t et al calcineurin inhibitorsgupta ak et al dexamethasone reduced netosis in neutrophils stimulated with s aureusagonists of tlr2 and tlr4 rescued dexamethasone inhibitionros production was unaffected by dexamethasonecyclosporine a reduced the effect of key physiological stimuli that activate neutrophilssuch as il8 and suppressed netosisros scavengerspatel et al vorobjeva nv and pinegin bvnacetyl cysteine reduced net formation and severity of sle in patientsantioxidants trolox and tempol prevent netosis of in stimulated human neutrophils in apad inhibitorsknight js et al weeks of daily clamidine injections reduced netinduced vascular damage and area ofdosedependent mannerlesions in a murine model of atherosclerosispad inhibition dampens disease activity by reducing endothelial dysfunction in a murinemodel of slednase therapypapayannopoulos v staab delastase combined with dnase therapy enhances solubilisation of sputum in cystic fibrosistetrahydroisoquinolines martinez ne et al tetrahydroisoquinolines selectively target net overproduction at micromolarzychlinsky a patientsconcentrations possibly at multiple stages of net formation without compromisingnormal neutrophil function 0crole of neutrophil netosis in an injurytetrahydroisoquinolinesin contrary to the aforementioned mechanisms ofnetosis inhibitors tetrahydroisoquinolines thiqs area new class of net formation inhibitors that do not targetros formation or granular protein activity as functionalneutrophils are paramount to maintaining immune reactivity this difference offers an advantage to selectively targetnet overproduction without impairing normal functionthe exact molecular mechanisms of thiqs are yet to bedetermined however it is known that thiq inhibition ofnetosis take place at micromolar concentrations and possibly at different stages of net formation conclusionwhen regulated as part of normal physiology netsare antimicrobial and fundamental to the innate immunesystem dysregulated net formation contributes to thepathogenesis of a plethora of diseases this review hassummarised the role of netosis in pathologies of multiplebody systems as well as highlighted the stages of netosisthat has so far been investigated as emerging pharmacological targets these putative strategies seem to hold therapeutic potential and warrant further investigationauthors contributionsdm designed and reviewed the manuscript zc andhz wrote the first draft of the paper all authors readrevised and approved the final manuscriptcompliance with ethical standardscompeting interests the authors declare that they haveno competing interestsethics approval and consent to participate notapplicableconsent for publication not applicableopen access this is licensed under a creativecommons attribution international license whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicateif changes were made the images or other third partymaterial in this are included in the 's creativecommons licence unless indicated otherwise in a creditline to the material if material is not included in the's creative commons licence and your intended useis not permitted by statutory regulation or exceeds thepermitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licencevisit httpcreativecommonslicensesby40references vandenabeele p l galluzzi t vanden berghe and g kroemer molecular mechanisms of necroptosis an ordered cellularexplosion nature reviews molecular cell biology httpsdoi101038nrm2970 lewis hd j liddle je coote sj atkinson md barker bdbax kl bicker rp bingham m campbell yh chen cwchung pd craggs rp davis d eberhard g joberty kelind k locke c maller k martinod c patten o polyakovace rise m rüdiger rj sheppard dj slade p thomas jthorpe g yao g drewes dd wagner pr thompson rkprinjha and dm wilson inhibition of pad4 activity issufficient to disrupt mouse and human net formation naturechemical biology httpsdoi101038nchembio1735 galluzzi lorenzo ilio vitale stuart a aaronson john m abramsdieter adam patrizia agostinis emad s alnemri et al molecular mechanisms of cell death recommendations of the nomenclature committee on cell death cell death and differentiation httpsdoi101038s414180170012 gupta s and mj kaplan the role of neutrophils andnetosis in autoimmune and renal diseases nature reviews nephrology httpsdoi101038nrneph201671 papayannopoulos v neutrophil extracellular traps in immunity and disease nature reviews immunology httpsdoi101038nri2017105 kobayashi sd and fr deleo role of neutrophils ininnate immunity a systems biologylevel approach wiley interdisciplinary reviews systems biology and medicine httpsdoi101002wsbm32 metzler kd c goosmann a lubojemska a zychlinsky andv papayannopoulos a myeloperoxidasecontaining complex regulates neutrophil elastase release and actin dynamics duringnetosis cell reports httpsdoi101016jcelrep201406044 tessarz p and t kouzarides histone core modificationsregulating nucleosome structure and dynamics nature reviewsmolecular cell biology httpsdoi101038nrm3890 yousefi s c mihalache e kozlowski i schmid and husimon viable neutrophils release mitochondrial dna toform neutrophil extracellular traps cell death and differentiation httpsdoi101038cdd200996 wang haiting ting li sheng chen gu yueying and shuang ye neutrophil extracellular trap mitochondrial dna and its 0cautoantibody in systemic lupus erythematosus and a proofofconcept trial of metformin arthritis rheumatology kenny ef a herzig r kruger a muth s mondal prthompson v brinkmann hv bernuth and a zychlinsky diverse stimuli engage different neutrophil extracellular trappathways elife httpsdoi107554elife24437 hakkim a bg furnrohr k amann b laube ua abed vbrinkmann m herrmann re voll and a zychlinsky impairment of neutrophil extracellular trap degradation is associatedwith lupus nephritis proceedings of the national academy of sciences of the united states of america httpsdoi101073pnas0909927107 farrera c and b fadeel macrophage clearance of neutrophil extracellular traps is a silent process journal of immunology httpsdoi104049jimmunol1300436 maueroder c a mahajan s paulus s gosswein j hahn dkienhofer mh biermann et al menageatrois the ratio ofbicarbonate to co2 and the ph regulate the capacity of neutrophilsto form nets frontiers in immunology httpsdoi103389fimmu201600583 behnen m s moller a brozek m klinger and t laskay extracellular acidification inhibits the rosdependent formation ofneutrophil extracellular traps frontiers in immunology httpsdoi103389fimmu201700184 amulic b sl knackstedt u abu abed n deigendesch cjharbort be caffrey v brinkmann fl heppner pw hindsand
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coutard b valle c de lamballerie x canard b seidah ng and decroly e the spike glycoprotein of the new coronavirus 2019ncovcontains a furinlike cleavage site absent in cov of the same clade antiviral res 101016jantiviral2020104742 cao z wu y faucon e and sabatier jm sarscov2 covid19 keyroles of the reninangiotensin systemvitamin d impacting drugand vaccine developments infect disord drug targets 1021741871526520999200505174704 zhang z chen l zhong j gao p and oudit gy ace2ang17 signaling and vascular remodeling sci china life sci s1142701446933 paz ocaranza m riquelme ja garcia l jalil je chiong m santos ras counterregulatory reninangiotensin system incardiovascular disease nat rev cardiol 101038s4156901902448 ziegler cgk allon sj nyquist sk mbano im miao vn tzouanas cn sarscov2 receptor ace2 is an interferonstimulatedgene in human airway epithelial cells and is detected in speciï¬c cell subsets across tissues cell 101016jcell202004035 marrero mb schieffer b paxton wg heerdtt l berkt bc delafontaine p direct stimulation of jakstat pathway by theangiotensin ii at1 receptor j med chem perrotta f matera mg cazzola m and bianco a severe respiratory sarscov2 infection does ace2 receptor matter respir med 101016jrmed2020105996 cheng h wang y and wang g anprotective effect of angiotensinconverting enzyme and its effect on the prognosis of covid19 jmed virol 101002jmv25785 de lang a osterhaus adme and haagmans bl interferon Î and interleukin4 downregulate expression of the sars coronavirusreceptor ace2 in vero e6 cells virology the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 mcgonagle d sharif k oregan a and bridgewood c the role of cytokines including interleukin6 in covid19 induced pneumonia andmacrophage activation syndromelike disease autoimmun rev linkinghubelseviercomretrievepiis1568997220300926101016jautrev2020102537 ye q wang b and mao j the pathogenesis and treatment of the cytokine storm in covid19 j infect linkinghubelseviercomretrievepiis0163445320301651 101016jjinf202003037 behrens em and koretzky ga review cytokine storm syndrome looking toward the precision medicine era arthritis rheumatol 101002art40071 guo xj and thomas pg new fronts emerge in the uenza cytokine storm semin immunopathol s002810170636y ronco c and reis t kidney involvement in covid19 and rationale for extracorporeal therapies nat rev nephrol 101038s4158102002847 wu c chen x cai y xia j zhou x xu s risk factors associated with acute respiratory distress syndrome and death in patientswith coronavirus disease pneumonia in wuhan china jama intern med 101001jamainternmed20200994 jose rj and manuel a covid19 cytokine storm the interplay between ammation and coagulation lancet respir med murakami m kamimura d and hirano t pleiotropy and speciï¬city insights from the interleukin family of cytokines immunity 101016jimmuni201903027 oshea jj schwartz dm villarino av gadina m mcinnes ib and laurence a the jakstat pathway impact on human disease andtherapeutic intervention annu rev med 101146annurevmed051113024537 lawrence t the nuclear factor nf k b pathway in ammation cold spring harb perspect biol catanzaro m fagiani f racchi m corsini e govoni s and lanni c immune response in covid19 addressing a pharmacologicalchallenge by targeting pathways triggered by sarscov2 signal transduct target ther 101038s4139202001911 li q and verma im nfκb regulation in the immune system nat rev immunol 101038nri910 tergaonkar v correa rg ikawa m and verma im distinct roles of iκb proteins in regulating constitutive nfκb activity nat cell biol 101038ncb1296 tergaonkar v nfκb pathway a good signaling paradigm and therapeutic target int j biochem cell biol linkinghubelseviercomretrievepiis1357272506001245 101016jbiocel200603023 correa rg matsui t tergaonkar v rodriguezesteban c izpisuabelmonte jc and verma im zebraï¬sh ikappab kinase negativelyregulates nfkappab activity curr biol 101016jcub200506023 wong et and tergaonkar v roles of nfκb in health and disease mechanisms and therapeutic potential clin sci portlandpresscomclinsci116645168639rolesofnfκbinhealthanddiseasemechanismsand101042cs20080502 morrison dk map kinase pathways cold spring harb perspect biol a011254 101101cshperspecta011254 mizutani t fukushi s saijo m kurane i and morikawa s phosphorylation of p38 mapk and its downstream targets in sarscoronavirusinfected cells biochem biophys res commun 101016jbbrc200405107 grimes jm and grimes kv p38 mapk inhibition a promising therapeutic approach for covid19 j mol cell cardiol 101016jyjmcc202005007 cuadrado a and nebreda ar mechanisms and functions of p38 mapk signalling biochem j 101042bj20100323 han j lee jd tobias ps and ulevitch rj endotoxin induces rapid protein tyrosine phosphorylation in 70z3 cells expressing cd14 jbiol chem jiang y chen c li z guo w gegner ja lin s characterization of the structure and function of a new mitogenactivated proteinkinase p38 j biol chem 101074jbc2713017920 li z jiang y ulevitch rj and han j the primary structure of p38Î a new member of p38 group of map kinases biochem biophys rescommun linkinghubelseviercomretrievepiis0006291x96916629 101006bbrc19961662 jiang y gram h zhao m new l gu j feng l characterization of the structure and function of the fourth member of p38 groupmitogenactivated protein kinases p38δ j biol chem 101074jbc2724830122 zarubin t and han j activation and signaling of the p38 map kinase pathway cell res 101038sjcr7290257 cuevas bd abell an and johnson gl role of mitogenactivated protein kinase kinase kinases in signal integration oncogene httpwwwnaturecoms1210409 101038sjonc1210409 alonso g ambrosino c jones m and nebreda ar differential activation of p38 mitogenactivated protein kinase isoforms depending onsignal strength j biol chem 101074jbcm007835200 brancho d mechanism of p38 map kinase activation in vivo genes dev 101101gad1107303 parker cg hunt j diener k mcginley m soriano b keesler ga identiï¬cation of stathmin as a novel substrate for p38 deltabiochem biophys res commun linkinghubelseviercomretrievepiis0006291x98992506101006bbrc19989250 rouse j cohen p trigon s morange m alonsollamazares a zamanillo d a novel kinase cascade triggered by stress and heatshock that stimulates mapkap kinase2 and phosphorylation of the small heat shock proteins cell linkinghubelseviercomretrievepii0092867494902771 1010160092867494902771 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 tan y rouse j zhang a cariati s cohen p and comb mj fgf and stress regulate creb and atf1 via a pathway involving p38 mapkinase and mapkap kinase2 embo j 101002j146020751996tb00840x xu w wang x tocker am huang p reith mea liuchen ly functional characterization of a novel series of biased signalingdopamine d3 receptor agonists acs chem neurosci 101021acschemneuro6b00221 kotlyarov a neininger a schubert c eckert r birchmeier c volk hd mapkap kinase is essential for lpsinduced tnfαbiosynthesis nat cell biol httpwwwnaturecomsncb0699 10103810061 kontoyiannis d pasparakis m pizarro tt cominelli f and kollias g impaired onoff regulation of tnf biosynthesis in mice lacking tnfaurich elements immunity linkinghubelseviercomretrievepiis1074761300800382101016s1074761300800382 saccani s pantano s and natoli g p38dependent marking of ammatory genes for increased nfκb recruitment nat immunol 101038ni748 dienz o and rincon m the effects of il6 on cd4 t cell responses clin immunol 101016jclim200808018 velazquezsalinas l verdugorodriguez a and rodriguez ll the role of interleukin during viral infections front microbiol zhang c wu z li jw zhao h and wang gq the cytokine release syndrome crs of severe covid19 and interleukin6 receptoril6r antagonist tocilizumab may be the key to reduce the mortality int j antimicrob agents 101016jijantimicag2020105954 matthay ma ware lb and zimmerman ga the acute respiratory distress syndrome j clin invest 101172jci60331 zhang h rostami mr leopold pl mezey jg obeirne sl strulovicibarel y expression of the sarscov2 ace2 receptor inthe human airway epithelium am j respir crit care med 101164rccm2020030541oc tian s xiong y liu h niu l guo j liao m pathological study of the novel coronavirus disease covid19 throughpostmortem core biopsies mod pathol 101038s413790200536x sungnak w huang n b ´ecavin c berg m queen r litvinukova m sarscov2 entry factors are highly expressed in nasalepithelial cells together with innate immune genes nat med 101038s4159102008686 lukassen s chua rl trefzer t kahn nc schneider ma muley t sars cov2 receptor ace and tmprss are primarilyexpressed in bronchial transient secretory cells embo j 1015252embj20105114 leung jm yang cx tam a shaipanich t hackett tl singhera gk ace2 expression in the small airway epithelia of smokersand copd patients implications for covid19 eur respir j 10118313993003006882020 dhawale vs amara vr karpe pa malek v patel d and tikoo k activation of angiotensinconverting enzyme ace2 attenuatesallergic airway ammation in rat asthma model toxicol appl pharmacol 101016jtaap201606026 bradding p richardson m hinks tsc howarth ph choy df arron jr ace2 tmprss2 and furin gene expression in the airwaysof people with asthma implications for covid19 j allergy clin immunol 101016jjaci202005013 vareille m kieninger e edwards mr and regamey n the airway epithelium soldier in the ï¬ght against respiratory viruses clinmicrobiol rev 101128cmr0001410 sabroe i parker lc dower sk and whyte mkb the role of tlr activation in ammation j pathol 101002path2264 frieman m and baric r mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation microbiol mol biolrev 101128mmbr0001508 lafferty ei qureshi st and schnare m the role of tolllike receptors in acute and chronic lung ammation j amm httpwwwjournalammationcomcontent7157 10118614769255757 kawai t and akira s signaling to nfκb by tolllike receptors trends mol med 101016jmolmed200709002 darby wj mcnutt kw and todhunter en niacin nutr rev 101111j175348871975tb05075x yoshikawa t hill te yoshikawa n popov vl galindo cl garner hr dynamic innate immune responses of human bronchialepithelial cells to severe acute respiratory syndromeassociated coronavirus infection plos one e8729101371journalpone0008729 dediego ml nietotorres jl reglanava ja jimenezguardeno jm fernandezdelgado r fett c inhibition ofnfkappabmediated ammation in severe acute respiratory syndrome coronavirusinfected mice increases survival j virol 101128jvi0257613 imai y kuba k neely gg yaghubianmalhami r perkmann t van loo g identiï¬cation of oxidative stress and tolllike receptor signaling as a key pathway of acute lung injury cell 101016jcell200802043 ding y feng q chen j and song j tlr4nfκb signaling pathway gene single nucleotide polymorphisms alter gene expression levelsand affect ards occurrence and prognosis outcomes medicine baltimore e16029 101097md0000000000016029 totura al whitmore a agnihothram s sch ¨afer a katze mg heise mt tolllike receptor signaling via trif contributes to aprotective innate immune response to severe acute respiratory syndrome coronavirus infection mbio 101128mbio0063815 olejnik j hume aj and m ¨uhlberger e tolllike receptor in acute viral infection too much of a good thing plos pathog 101371journalppat1007390 okumura a rasmussen al halfmann p feldmann f yoshimura a feldmann h suppressor of cytokine signaling is aninducible host factor that regulates virus egress during ebola virus infection j virol 101128jvi0173615 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 obrien tr thomas dl jackson ss prokuninaolsson l donnelly rp and hartmann r weak induction of interferon expression bysarscov2 supports clinical clin infect dis 101093cidciaa453 chang yj liu cyy chiang bl chao yc and chen cc induction of il8 release in lung cells via activator protein1 byrecombinant baculovirus displaying severe acute respiratory syndromecoronavirus spike proteins identiï¬cation of two functional regions j immunol 104049jimmunol173127602 wang w ye l ye l li b gao b zeng y upregulation of il6 and tnfα induced by sarscoronavirus spike protein in murinemacrophages via nfκb pathway virus res 101016jvirusres200702007 zhang x wu k wang d yue x song d zhu y nucleocapsid protein of sarscov activates interleukin6 expression throughcellular transcription factor nfκb virology 101016jvirol200704009 toews gb cytokines and the lung eur respir j suppl 101183090319360100266001 russell b moss c rigg a and van hemelrijck m covid19 and treatment with nsaids and corticosteroids should we be limiting theiruse in the clinical setting ecancermedicalscience 103332ecancer20201023 severgnini m takahashi s rozo lm homer rj kuhn c jhung jw activation of the stat pathway in acute lung injury am jphysiol lung cell mol physiol 101152ajplung003492003 zhao j yu h liu y gibson sa yan z xu x protective effect of suppressing stat3 activity in lpsinduced acute lung injury amj physiol lung cell mol physiol l868l880 101152ajplung002812016 pedroza m schneider dj karmoutyquintana h coote j shaw s corrigan r interleukin6 contributes to ammation andremodeling in a model of adenosine mediated lung injury plos one e22667 101371journalpone0022667 goldman jl sammani s kempf c saadat l letsiou e wang t pleiotropic effects of interleukin6 in a twohit murine modelof acute respiratory distress syndrome pulm circ 101086675991 rincon m and irvin cg role of il6 in asthma and other ammatory pulmonary diseases int j biol sci 107150ijbs4874 swanson kv deng m and ting jpy the nlrp3 ammasome molecular activation and regulation to therapeutics nat rev immunol 101038s4157701901650 zhao c and zhao w nlrp3 ammasome a key player in antiviral responses front immunol orzalli mh smith a jurado ka iwasaki a garlick ja kagan jc an antiviral branch of the il1 signaling pathway restrictsimmuneevasive virus replication an antiviral branch of the il1 signaling pathway restricts immuneevasive virus replication mol cell 825e5840e5 101016jmolcel201807009 shi c and nabar nr sarscoronavirus open reading frame8b triggers intracellular stress pathways and activates nlrp3 ammasomescell death discov 101038s4142001901817 siu k yuen k castanorodriguez c ye z yeung m fung s severe acute respiratory syndrome coronavirus orf3a proteinactivates the nlrp3 ammasome by promoting traf3dependent ubiquitination of asc faseb j 101096fj201802418r kusov y tan j alvarez e enjuanes l and hilgenfeld r a gquadruplexbinding macrodomain within the sarsunique domain isessential for the activity of the sarscoronavirus replicationtranscription complex virology 101016jvirol20150601 chang ys ko bh ju jc chang hh huang sh and lin cw sars unique domain sud of severe acute respiratory syndromecoronavirus induces nlrp3 ammasomedependent cxcl10mediated pulmonary ammation int j mol sci 103390ijms21093179 sun dz song cq xu ym and dong xs role of the mapk pathway in human lung epitheliallike a549 cells apoptosis induced byparaquat genet mol biol e20190137 10159016784685gmb20190137 arcaroli j yum hk kupfner j park js yang ky and abraham e role of p38 map kinase in the development of acute lung injury clinimmunol 101006clim20015108 li sw wang cy jou yj yang tc huang sh wan l sars coronavirus papainlike protease induces egr1dependentupregulation of tgf1 via rosp38 mapkstat3 pathway sci rep yoshida k kuwano k hagimoto n watanabe k matsuba t fujita m map kinase activation and apoptosis in lung tissues frompatients with idiopathic pulmonary ï¬brosis j pathol 101002path1208 qian f deng j wang g ye rd and christman jw pivotal role of mitogenactivated protein kinaseactivated protein kinase inammatory pulmonary diseases curr protein pept sci 1021741389203716666150629121324 renda t baraldo s pelaia g bazzan e turato g papi a increased activation of p38 mapk in copd eur respir j 1011830903193600036707 mercer ba and darmiento jm emerging role of map kinase pathways as therapeutic targets in copd int j chron obstruct pulmon dis feng y fang z liu b and zheng x p38mapk plays a pivotal role in the development of acute respiratory distress syndrome clinics 106061clinics2019e509 mizutani t signaling pathways of sarscov in vitro and in vivo mol biol 9783642036835Ë mizutani t signal transduction in sarscovinfected cells ann ny acad sci 101196annals1408006 cheng vcc hung ifn tang bsf chu cm wong mml chan kh viral replication in the nasopharynx is associated withdiarrhea in patients with severe acute respiratory syndrome clin infect dis 101086382681 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 arabi ym alomari a mandourah y alhameed f sindi aa alraddadi b critically ill patients with the middle east respiratorysyndrome a multicenter retrospective cohort study crit care med 101097ccm0000000000002621 wong sh lui rns and sung jjy covid19 and the digestive system j gastroenterol hepatol 101111jgh15047 luo s zhang x and xu h dont overlook digestive symptoms in patients with novel coronavirus disease covid19 clingastroenterol hepatol 101016jcgh202003043 xiao f tang m zheng x liu y li x and shan h evidence for gastrointestinal infection of sarscov2 gastroenterology 1831e31833e3 101053jgastro202002055 young be ong swx kalimuddin s low jg tan sy loh j epidemiologic features and clinical course of patients infected withsarscov2 in singapore jama jamanetworkcomjournalsjamafull2762688 101001jama20203204 xu y li x zhu b liang h fang c gong y characteristics of pediatric sarscov2 infection and potential evidence for persistentfecal viral shedding nat med 101038s4159102008174 tang a tong z wang h dai y li k liu j detection of novel coronavirus by rtpcr in stool specimen from asymptomatic childchina emerg infect dis httpwwwnccdcgoveid266200301 htm 103201eid2606200301 zhang w du rh li b zheng xs yang xl hu b molecular and serological investigation of 2019ncov infected patientsimplication of multiple shedding routes emerg microbes infect 1010802222175120201729071 yeo c kaushal s and yeo d enteric involvement of coronaviruses is faecaloral transmission of sarscov2 possible lancetgastroenterol hepatol 101016s2468125320300480 van doremalen n bushmaker t morris dh holbrook mg gamble a williamson bn aerosol and surface stability ofsarscov2 as compared with sarscov1 n engl j med 101056nejmc2004973 setti l passarini f de gennaro g barbieri p perrone mg borelli m airborne transmission route of covid19 why meters6feet of interpersonal distance could not be enough int j environ res public health 103390ijerph17082932 perlman s and netland j coronaviruses postsars update on replication and pathogenesis nat rev microbiol 101038nrmicro2147 m ¨onkem ¨uller k fry l and rickes s covid19 coronavirus sarscov2 and the small bowel rev esp enferm dig wang d hu b hu c zhu f liu x zhang j clinical characteristics of hospitalized patients with novelcoronavirusinfected pneumonia in wuhan china jama jamanetworkcomjournalsjamafull2761044101001jama20201585 pan l mu m yang p sun y wang r yan j clinical characteristics of covid19 patients with digestive symptoms in hubei chinaam j gastroenterol 1014309ajg0000000000000620 budden kf gellatly sl wood dla cooper ma morrison m hugenholtz p emerging pathogenic links between microbiota andthe gutlung axis nat rev microbiol 101038nrmicro2016142 camargo smr singer d makrides v huggel k pos km wagner ca tissuespeciï¬c amino acid transporter partners ace2 andcollectrin differentially interact with hartnup mutations gastroenterology 872e3882e3linkinghubelseviercomretrievepiis0016508508018933 101053jgastro200810055 hashimoto t perlot t rehman a trichereau j ishiguro h paolino m ace2 links amino acid malnutrition to microbial ecology andintestinal ammation nature 101038nature11228 br ¨oer a juelich t vanslambrouck jm tietze n solomon ps holst j impaired nutrient signaling and body weight control in naneutral amino acid cotransporter slc6a19deï¬cient mouse j biol chem 101074jbcm111241323 ma xm and blenis j molecular mechanisms of mtormediated translational control nat rev mol cell biol 101038nrm2672 bostancklo Ëglu m temporal correlation between neurological and gastrointestinal symptoms of sarscov2 amm bowel dis 101093ibdizaa131 louveau a smirnov i keyes tj eccles jd rouhani sj peske jd structural and functional features of central nervous systemlymphatic vessels nature 101038nature14432 jessen na munk asf lundgaard i and nedergaard m the glymphatic system a beginners guide neurochem res s1106401515816 wu y xu x chen z duan j hashimoto k yang l nervous system involvement after infection with covid19 and othercoronaviruses brain behav immun 101016jbbi202003031 liu k pan m xiao z and xu x neurological manifestations of the coronavirus sarscov2 pandemic j neurol neurosurgpsychiatry 101136jnnp2020323177 xydakis ms dehganimobaraki p holbrook eh geisthoff uw bauer c hautefort c smell and taste dysfunction in patients withcovid19 lancet infect dis s14733099 101016s1473309920302930 moriguchi t harii n goto j harada d sugawara h takamino j a ï¬rst case of meningitisencephalitis associated withsarscoronavirus2 int j infect dis 101016jijid202003062 baig am khaleeq a ali u and syeda h evidence of the covid19 virus targeting the cns tissue distribution hostvirus interaction andproposed neurotropic mechanisms acs chem neurosci 101021acschemneuro0c00122 helms j kremer s merdji h clerejehl r schenck m kummerlen c neurologic features in severe sarscov2 infection nengl j med 101056nejmc2008597 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 panizmondolï¬ a bryce c grimes z gordon re reidy j lednicky j central nervous system involvement by severe acuterespiratory syndrome coronavirus2 sarscov2 j med virol 101002jmv25915 zhou l zhang m wang j and gao j sarscov2 underestimated damage to nervous system travel med infect dis linkinghubelseviercomretrievepiis1477893920301101 101016jtmaid2020101642 panciani pp saraceno g zanin l renisi g signorini l battaglia l sarscov2threesteps infection model and csfdiagnostic implication brain behav immun 101016jbbi202005002 desfes m le coupanec a dubeau p bourgouin a lajoie l dub ´e m human coronaviruses and other respiratory virusesunderestimated opportunistic pathogens of the central nervous system viruses 103390v12010014 fazakerley jk and walker r virus demyelination j neurovirol 10108013550280390194046 murray rs cai gy hoel k zhang jy soike kf and cabirac gf coronavirus infects and causes demyelination in primate centralnervous system virology 101016004268229290757g lane te and buchmeier mj murine coronavirus infection a paradigm for virusinduced demyelinating disease trends microbiol 101016s0966842x97817684 stohlman sa and hinton dr viral induced demyelination brain pathol 101111j175036392001tb00384x li z liu t yang n han d mi x li y neurological manifestations of patients with covid19 potential routes of sarscov2neuroinvasion from the periphery to the brain front med s1168402007865 netland j meyerholz dk moore s cassell m and perlman s severe acute respiratory syndrome coronavirus infection causes neuronaldeath in the absence of encephalitis in mice transgenic for human ace2 j virol 101128jvi0073708 bohmwald k galvez n r´Ä±os m and kalergis am neurologic alterations due to respiratory virus infections front cell neurosci 103389fncel201800386 montalvan v lee j bueso t de toledo j and rivas k neurological manifestations of covid19 and other coronavirus infections asystematic review clin neurol neurosurg 101016jclineuro2020105921 zhou f yu t du r fan g liu y liu z clinical course and risk factors for mortality of adult inpatients with covid19 in wuhanchina a retrospective cohort study lancet linkinghubelseviercomretrievepiis0140673620305663101016s0140673620305663 varga z flammer aj steiger p haberecker m andermatt r zinkernagel as endothelial cell infection and endotheliitis incovid19 lancet 101016s0140673620309375 bostanciklio Ëglu m sarscov2 entry and spread in the lymphatic drainage system of the brain brain behav immun 101016jbbi202004080 li yc bai wz and hashikawa t the neuroinvasive potential of sarscov2 may play a role in the respiratory failure of covid19 patientsj med virol 101002jmv25728 li y bai w hirano n hayashida t taniguchi t sugita y neurotropic virus tracing suggests a membranouscoatingmediatedmechanism for transsynaptic communication j comp neurol 101002cne23171 reichard rr kashani kb boire na constantopoulos e guo y and lucchinetti cf neuropathology of covid19 a spectrum ofvascular and acute disseminated encephalomyelitis ademlike pathology acta neuropathol s00401020021662 kanberg n ashton nj andersson lm yilmaz a lindh m nilsson s neurochemical evidence of astrocytic and neuronal injurycommonly found in covid19 neurology nneurologycontentneurologyearly20200616wnl0000000000010111fullpdf101212wnl0000000000010111 xiao l haack kkv and zucker ih angiotensin ii regulates ace and ace2 in neurons through p38 mitogenactivated protein kinase andextracellular signalregulated kinase signaling am j physiol cell physiol c1073c1079 101152ajpcell003642012 tang n li d wang x and sun z abnormal coagulation parameters are associated with poor prognosis in patients with novel coronaviruspneumonia j thromb haemost 101111jth14768 mehta p mcauley df brown m sanchez e tattersall rs manson jj covid19 consider cytokine storm syndromes andimmunosuppression lancet 101016s0140673620306280 koralnik ij and tyler kl covid19 a global threat to the nervous system ann neurol 101002ana25807 toscano g palmerini f ravaglia s ruiz l invernizzi p cuzzoni mg guillainbarr ´e syndrome associated with sarscov2 nengl j med nejmc2009191 101056nejmc2009191 madjid m safavinaeini p solomon sd and vardeny o potential effects of coronaviruses on the cardiovascular system jama cardiol jamanetworkcomjournalsjamacardiologyfull2763846 epelman s shrestha k troughton rw francis gs sen s klein al soluble angiotensinconverting enzyme in human heartfailure relation with myocardial function and clinical outcomes j cardiac fail 101016jcardfail200901014 wild ps dimmeler s and eschenhagen t an epidemiological study exploring a possible impact of treatment with ace inhibitors orangiotensin receptor blockers on ace2 plasma concentrations j mol cell cardiol 101016jyjmcc202003018 wu z and mcgoogan jm characteristics of and important lessons from the coronavirus disease covid19 outbreak in china jama jamanetworkcomjournalsjamafull2762130 101001jama20202648 zheng yy ma yt zhang jy and xie x covid19 and the cardiovascular system nat rev cardiol 101038s4156902003605 ruan q yang k wang w jiang l and song j clinical predictors of mortality due to covid19 based on an analysis of data of patients from wuhan china intensive care med s0013402005991x the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 cheng r and leedy d covid19 and acute myocardial injury the heart of the matter or an innocent bystander heart 101136heartjnl2020317025 zhu l carretero oa xu j harding p ramadurai n gu x activation of angiotensin ii type receptor suppresses tnfαinducedicam1 via nfκb possible role of ace2 am j physiol heart circ physiol h827h834 101152ajpheart008142014 dzimiri n receptor crosstalk implications for cardiovascular function disease and therapy eur j biochem 101046j14321033200203181x wheelerjones cpd cell signalling in the cardiovascular system an overview heart 101136hrt2005072280 houliston ra pearson jd and wheelerjones cpd agonistspeciï¬c cross talk between erks and p38 mapk regulates pgi synthesis inendothelium am j physiol physiol c1266c1276 101152ajpcell20012814c1266 adam lp franklin mt raff gj and hathaway dr activation of mitogenactivated protein kinase in porcine carotid arteries circ res 10116101res762183 nebreda ar and porras a p38 map kinases beyond the stress response trends biochem sci linkinghubelseviercomretrievepiis0968000400015954 101016s0968000400015954 wang y huang s sah vp ross j brown jh han j cardiac muscle cell hypertrophy and apoptosis induced by distinct membersof the p38 mitogenactivated protein kinase family j biol chem 101074jbc27342161 saurin at martin jl heads rj foley c mockridge jw wright mj the role of differential activation of p38mitogenactivatedprotein kinase in preconditioned ventricular myocytes faseb j 101096fj990671com podewski ek hilï¬kerkleiner d hilï¬ker a morawietz h lichtenberg a wollert kc alterations in janus kinase jaksignaltransducers and activators of transcription stat signaling in patients with endstage dilated cardiomyopathy circulation 10116101cir000005754582749ff patel ab and verma a covid19 and angiotensinconverting enzyme inhibitors and angiotensin receptor blockers what is the evidencejama jamanet
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"objective endoscopic full thickness resection eftr has shown efficacy and safety in the colorectum the aim of this analysis was to investigate whether eftr is cost effective in comparison with surgical and endoscopic treatment alternativesdesign real data from the study cohort of the prospective single arm wall resect study were used a simulated comparison arm was created based on a survey that included suggested treatment alternatives to eftr of the respective lesions treatment costs and reimbursement were calculated in euro according to the coding rules of and eftr r0 resection rate was used as a measure of effectiveness to assess cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were determined calculations were made both from the perspective of the care provider as well as of the payerresults the cost per case was ¬ for the eftr group ¬ for the standard endoscopic resection ser group ¬ for the surgical resection group and ¬ for the pooled alternative treatment to eftr from the perspective of the care provider the acer mean cost per r0 resection was ¬ for eftr ¬ for ser ¬ for surgical treatment and ¬ for all pooled and weighted alternatives to eftr the icer additional cost per r0 resection compared with eftr was ¬ for ser ¬ for surgical resection and ¬ for the pooled rate of alternatives results from the perspective of the payer were similar eftr is cost effective in comparison with surgical and endoscopic treatment alternatives in the colorectumintroductioncolorectal cancer is the third most common type of cancer and the second most common cause of cancer related deaths worldwide1 screening programmes for early detection of premalignant and malignant lesions led summary boxwhat is already known about this subject º endoscopic full thickness resection eftr has shown clinical efficacy and safety in difficult to treat lesions in the colorectum º the cost of the full thickness resection device is higher than the cost of standard endoscopic resection ser devices but lower than surgical devices º cost effectiveness analyses on treatment with eftr compared with treatment alternatives do not existwhat are the new findings º eftr leads to an almost reduction in cost per r0 resection average cost effectiveness ratio compared with surgery º to achieve an additional r0 resection by surgical treatment compared with eftr incremental cost effectiveness ratio an additional cost of ¬ is necessary º these findings are consistent both from the perspective of the care provider as well as the payerhow might it impact on clinical practice in the foreseeable future º in terms of cost effectiveness eftr should be considered first before patients with difficult to treat lesions in the colorectum are sent to surgical treatmentto a significant reduction in cancer related mortality2 with more intense screening more lesions are detected which automatically creates the need for removal standard endoscopic resections ser such as endoscopic mucosal resection emr and endoscopic submucosal dissection esd are well established and sufficient for the vast majority of lesions however ser of non lifting lesions and lesions located at difficult anatomical to cite kuellmer a0a behn a0j beyna a0t et a0al endoscopic full thickness resection and its treatment alternatives in difficult to treat lesions of the lower gastrointestinal tract a cost effectiveness analysis bmj open gastro 20207e000449 101136bmjgast2020000449received may revised july accepted july authors or their employers re use permitted under cc by nc no commercial re use see rights and permissions published by bmjfor numbered affiliations see end of correspondence toarthur schmidt arthur schmidt uniklinik freiburg dekuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access locations eg appendiceal orifice is associated with increased complication rates or incomplete resection3 these types of lesions are therefore often referred to surgery which is associated with significant morbidity and mortality and higher costs6 given the high number of polypectomies performed worldwide this is not only an issue of morbidity and mortality but also a huge economic challengethe efficacy and safety of endoscopic full thickness resection eftr of non lifting and other difficult to treat lesions have been demonstrated in multiple retrospective studies and in one prospective study7 the cost of the device is markedly higher than ser devices but lower than surgical treatment the aim of the present analysis was to evaluate whether eftr is cost effective in comparison with ser as well as surgical treatmentmethodsstudy populationto calculate the cost of eftr we analysed the study cohort of the wall resect trial nct02362126 in this single arm multicentre prospective study patients with difficult to treat adenomas eg non lifting andor challenging anatomical location early adenocarcinomas and subepithelial tumours in the colorectum were treated with eftr the primary endpoints of the study en bloc and r0 resection rate were achieved in and respectively7 written informed consent was obtained from each patient included in the studysimulation second study arm based on a survey of endoscopistswith the wall resect study being single armed a second study arm was created based on treatment simulation in order to compare different treatment modalities a case report form crf was created and sent to each participating centre of the wall resect trial endoscopists at the respective location reviewed the endoscopic images and their case relevant data and decided which treatment modality they would have chosen if eftr were not available treatment alternatives included ser such as emr thermal methods and esd as well as surgical resection the crf was filled out in a pseudonymised fashioninformed consent had already been obtained within the wall resect studydetermination of case costs and reimbursementa certified online it tool the diagnosis related group drg web grouper was used to determine the reimbursement rate for each patient http drg uni muenster de index php therefore the code of the international classification of diseases icd10 and the specific code for the procedure performed operationen und prozedurenschl¼ssel ops code in each patient in both groups were put into the web grouper together with the predefined mean length of hospital stay mittlere grenzverweildauer the drg which accounts for reimbursement was calculatedin the comparison arm the drg codes of were used because this was the year the wall resect study was performed for the eftr arm the drg codes of were used as reimbursement for eftr was increased that yearto calculate the cost per case another certified online it tool g drg report browser was used www g drg de g drg system_ abschlussbericht_ zur_ weiterentwicklung_ des_ g drg systems_ und_ report_ browser this was done by filling in the respective drg icd10 and ops code into the browser the data of the g drg report browser derive from the data that were sent in to inek authority managing the german drg system by certified hospitals kalkulationshuser in grouping was performed following the rules of g drg version the main and secondary diagnoses are shown according to icd10 german modification gm version and the procedures according to ops version g drg report browser inek gmbhas reimbursement for the eftr group was taken from the cost per patient case would ideally also have been calculated from unfortunately these data will be first published by inek in to overcome this problem costs for eftr cases from the university of freiburg between and which were reported to inek were used for the analysiswith the cost of each patient case the mean cost for each treatment modality emr esd laparoscopic surgery transanal endoscopic microsurgery tem eftr could be calculated in the next step the mean cost for each treatment path ser surgical treatment and casemix alternative was determined this was done in the following fashion for ser the mean costs of emr and esd were used for calculation of the surgical treatment laparoscopic surgery and tem were taken together the mean costs of endoscopic and surgical treatments were subsumed as the casemix alternativedetermination of effectivenessthe r0 resection rate was defined as the efficacy parameter to determine cost effectiveness the r0 rate of eftr in the wall resect trial was to determine the efficacy of the therapeutic alternatives to eftr a selective literature review was performed in pubmed and cochrane databases identifying the largest studies comparing resection techniques and r0 rates the respective rates regarding ser found in the literature were for emr and for esd9 for the surgical oncological resection treatment as the gold standard a r0 resection rate was assumed for the tem a rate of had been reported10in order to compare all ser methods emresd all surgical resection methods laparoscopic surgerytem and all alternative methods endoscopic and surgical kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0ctable alternative treatment strategies to eftr with their respective efficacy based on literature review and calculationtreatmentefficacy n n180surgical oncological resection laparoscopictememresdsurgical treatment laparoscopic and temser emresdcasemix alternative assumed arezzo et al fujiya et al arezzo et al calculated calculated calculatedthe combined effectiveness of surgical treatment ser and casemix alternative was calculated by multiplication of the number of patients in each modality eg emr cases for ser with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group overall efficacy of surgical treatment and casemix alternative was performed in the same mannereftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgerycasemix alternative with the eftr procedure a combined effectiveness of each treatment group was calculated this was done by multiplication of the number of patients in each modality eg emr cases with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group using this approach the overall efficacy in the ser group was calculated as overall efficacy of surgical treatment and casemix alternative was performed in the same manner and was calculated as and respectively the respective r0 rates are shown in table calculation of costeffectivenessfor assessment of cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were calculated acer expresses the mean costs for the investigated outcome11 in our study acer describes the mean costs per successful r0 resection in the different treatment modalitiesacer is calculated with the following computational formula acer mean costseffect open accessicer expresses the additional costs of a treatment alternative for improvement in the investigated outcome12 in our study these are the incremental costs for the alternative treatment to eftr required to achieve an r0 resectionicer is calculated with the following computational formula icer mean costs interventionmean costs controleffect intervention effect control the mean costs were the total costs of the respective treatment modality divided by the number of patients in each group for the calculation of cost effectiveness the ser methods emr and esd as well as the surgical resection methods laparoscopic resection and tem were taken together furthermore cost effectiveness was calculated for the casemix alternative to compare eftr with all alternativesresultscomparative study armendoscopist surveyfrom patients of the study cohort responses were included for further analysis in one patient the investigator recommended solely thermal ablation as alternative treatment of choice thus the primary endpoint r0 resection could not be evaluated from the remaining patients the endoscopists recommended surgical treatment in of of cases thereof of were laparoscopic resections and of tem in of of cases an endoscopic resection was proposed thereof of were emr and of were esdcosts from the perspective of the care providercosts per case were derived from the drg report browser which represent costs of the respective treatment alternative for the year the costs for the eftr treatment were derived from university hospital freiburg and represent the mean costs from years to mid2019 the mean cost for eftr was ¬ the cost per surgical treatment laparoscopic surgery and tem was ¬ and for ser ¬ all alternative treatment strategies casemix alternative op laparscopic surgery tem esd and emr were calculated as ¬ per case the results are shown in figure costs from the perspective of the thirdpartyaccording to the german drg system reimbursement for eftr is ¬ for surgical treatment ¬ was calculated the cost per case for ser is ¬ the cost for the casemix alternative is ¬ the results are shown in figure costeffectiveness analysis care provider viewpointaverage costeffectiveness ratiothe mean cost per r0 resection is ¬ in the eftr group and ¬ in the surgical group in the ser group the cost per r0 resection is ¬ in the casemix alternative group including all treatment kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access figure case costs ¬ for the different treatment modalities are shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryfigure incremental cost effectiveness ratio for the different treatment modalities compared with eftr is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryalternatives except eftr the mean cost per r0 resection is ¬ the results are shown in figure the casemix alternative ¬ the results are shown in figure incremental costeffectiveness ratioin comparison with eftr the incremental cost for an additional r0 resection is ¬ if ser is performed the cost for the surgical approach is ¬ and for figure average cost effectiveness ratio ¬ for the different treatment modalities is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgerycosteffectiveness analysis health insurance reimbursement viewpointaverage costeffectiveness ratiofrom the perspective of the health insurance the cost per r0 resection is ¬ in the eftr group in the ser group the cost is ¬ and in the surgical treatment group ¬ in the casemix alternative the cost per r0 resection is ¬ the results are shown in figure incremental costeffectiveness ratiothe icer of ser in comparison with eftr is ¬ the surgical approach costs an additional ¬ for the casemix alternative ¬ is necessary for an additional r0 resection the results are shown in figure discussionwith technical endoscopic progress patient care has constantly improved over the years however as with any technical innovation this is associated with higher costs therefore the efficacy of new methods and devices needs to be evaluated in relation to their costs13 to our knowledge this is the first cost effectiveness analysis cea for eftr our results demonstrate that eftr for difficult to treat lesions in the colorectum is cost effective in comparison with ser as well as surgical therapy furthermore the results are consistent when analysed from the perspective of the care provider as well as of the payerkuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0cfor our analysis a simulated control arm was created this was necessary as to date no randomised controlled trial rct investigating eftr versus alternative treatments has been published in our survey endoscopists proposed surgical treatment as the likely alternative to eftr in the majority of cases as opposed to ser via emr or esd all lesions within the wall resect trial were difficult to resect lesions eg non lifting adenomas exhibiting a high risk of perforation or incomplete resection when treated with ser therefore it may be surprising that ser was suggested in of cases however the suggestions were made by expert endoscopists who might have decided towards an advanced endoscopic procedure more generouslyregarding the costs for each treatment modality it was not surprising that the cost of eftr is above ser ¬ vs ¬ this is due to the cost of the device in germany ¬ plus value added tax however the cost of eftr was roughly one third of the cost of surgery ¬ vs ¬ this reflects the minimally invasive nature of eftr compared with laparoscopic or open surgical operationswhile costs for endoscopic resection and surgical therapy were taken from official and certified tools web grouper and drg report browser the factual costs of the eftr procedure for the year that are determined in a representative cross section of hospitals have not been published yet by inek the administrator of the drg system reimbursement of the procedure changed in thus these data should have been used for calculation to overcome this problem the mean case costs for eftr per case in our home institution university hospital of freiburg germany in the time between and were used as a surrogate in an economic analysis of the cost of eftr in germany presented at the annual conference of the german society for digestive diseases obtained from different endoscopic centres reported ¬ per case as this is only above our number and therefore in the same range our calculated ¬ seems to be a realistic number14in our analysis we chose the r0 rate as a means to detemine effectiveness as this is the most objective parameter to assess curative resection and treatment success furthermore the r0 rate can be compared with the treatment alternatives of eftr as high quality meta analyses and therapeutic success rates exist for those procedures10 the r0 rate for surgical colonic resection was assumed to be however the patient cohorts of these studies are not equal the wall resect study included only difficult to resect lesions mainly non lifting while the studies mentioned above included primarily treatment naive lesions larger studies on ser on non lifting lesions do not exist hence it is reasonable to assume that in these indications real r0 rates of ser would be lower and therefore cost effectiveness would be even worsefor measuring cost effectiveness acer and icer were determined the analysis was performed both open accessfrom the perspective of the care provider hospital as well as the reimbursement authority health insurance acer expresses the mean cost per r0 resection for both investigated perspectives our results reveal that costs are much lower for eftr compared with the surgical alternative although the effectiveness of the surgical approach in terms of radicality can be considered to be higher eftr is cost effective an r0 resection by eftr leads to nearly reduction in costs for the care provider ¬¬ and for the health insurance ¬¬ compared with ser eftr leads to marginally higher costs per r0 resection as explained above comparing eftr with ser has limitations as the investigated difficult lesions in the wall resect study are not well studied for emr and esd however in comparison with all treatment alternatives casemix alternative we calculated and reduction in costs similar to the surgical alternatives figure icer expresses the additional costs for an additional increase in the designated outcome in our analysis it expresses the additional costs that are necessary for an additional r0 resection as shown in figure all alternatives to eftr result in additional costs while ser results in a modest increase ¬ and ¬ additional ¬ and ¬ per r0 resection are required in the surgical group in the casemix alternative group additional costs were ¬ and ¬ respectivelyan absolute threshold at which an icer is thought to be cost effective does not exist16 in the literature the willingness to pay threshold ranges from to and is highly subjective to the investigated outcome and the healthcare system for which the cea is made17 for our analysis we assume that a more invasive treatment that produces at least ¬ more costs for an additional r0 resection cannot be regarded as being cost effectivefor our analysis we did not include costs of follow up endoscopies or further treatment arising from recurrency or from adverse events this was done due to the following reasons first reliable recurrence rates and long term follow up after eftr do not exist follow up in the wall resect trial was only weeks second the lesions of our patient cohort were heterogeneous including adenomas carcinomas and neuroendocrine tumours with different biological features and recurrent rates third treatment of recurrent lesions is not standardised and ranges from re eftr to snare polypectomy to removal with a biopsy forceps leading to highly variable costs fourth management of severe complications and consecutive morbidity differs in every patient and depends on severity of complication patients comorbidities and local expertise we do not have reliable data on costs for such treatment and a hypothetical model would have been highly speculative moreover in the wall resect trial of patients required consecutive surgery due to complications this rate is slightly kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access higher but still grossly comparable with the complication rates of emr and esd on the other hand complications after surgical resection eg anastomotic leakage are much more frequent up to and usually lead to higher morbidity hence even if costs related to complications were added icer is still likely to favour eftr compared with the group of treatment alternativesit is difficult to compare our results with other ceas as this is the first one for this indication the only previous cea on ser compared emr and esd in laterally spreading lesions irrespective of location or lifting sign in most analyses as in the study by bahin and colleagues19 a decision tree model was created to compare different outcome scenarios after each treatment path was filled with probabilities of occurrence costs per predefined outcome were calculated a potential bias of this approach is that the data for the probabilities of occurrence which influence the costs most are taken totally or at least in part from different studies19 22this harbours the risk of resulting in a very heterogeneous study population with uncontrolled confounders this risk can be minimised by deriving data from rcts with well balanced patient cohorts as recently published17 for our analysis we used a different approach than a decision tree factual variables and outcome data derived from the only prospective study on eftr treatment and not from assumptions the simulation of the control arm had to be performed due to the lack of rcts in this setting the strength of our study is that the very same clinician who actually performed the respective eftr could review the different lesions and decide on a solid basis which treatment alternative he or she would have used instead of eftr in our view this approach reflects the clinical situation more precisely than a decision tree modelin most ceas the costs per quality adjusted life years are calculated and taken for healthcare decisions neither survival nor quality of life measurements were part of the wall resect trial in line with most of the recently published cea we calculated costs per defined outcome as the primary endpoint17 our study has several limitations first the comparison arm of the study is based on simulation so there is always a risk of a bias second our analysis is specific to the german healthcare system and may therefore not be fully comparable with different healthcare systems in the world third the estimated r0 rate for the ser methods is very low and likely due to the piecemeal resection in the respective study if efficacy would have been measured as freedom of recurrence efficacy would be higher as proven in the australian colonic endoscopic study24 nonetheless we used the published r0 rate because of the possibility to match this with the endpoint of the wall resect study furthermore as described above an endpoint such as freedom of recurrence cannot be determined reliably as such data do not exist for eftr fourth costs of complications and follow up were not included this is mainly due to lack of an rct and the short follow up period in an ideal cea all treatment related and hospital stay related costs would have been calculatedin our data indicate that eftr for difficult to treat lesions of the colorectum is cost effective compared with surgical and endoscopic treatment alternatives the results are consistent both from a care provider as well as from a third party payer perspective rcts and long term follow up are needed to further assess the cost effectiveness of eftrauthor affiliations1department of medicine ii medical center university of freiburg faculty of medicine university of freiburg freiburg germany2department of gastroenterology klinikum ludwigsburg ludwigsburg baden w¼rttemberg germany3department of gastroenterology evangelisches krankenhaus d¼sseldorf dusseldorf nordrhein westfalen germany4department of internal medicine and gastroenterology elisabeth hospital essen nordrhein westfalen germany5department of medicine ii interventional and experimental endoscopy inexen university hospital wurzburg wurzburg bayern germany6department of gastroenterology university hospital augsburg augsburg bayern germany7department of gastroenterology university hospital ulm ulm baden w¼rttemberg germany8department of gastroenterology klinikum dortmund dortmund nordrhein westfalen germany9department of gastroenterology helios klinikum krefeld krefeld nordrhein westfalen germany10department of gastroenterologyoncology klinikum sindelfingen b¶blingen sindelfingen baden w¼rttemberg germanycontributors as and kc invented and planned the present study and also the underlying wall resect study as assisted with data acquisition and analysis and revised the manuscript jb was responsible for data research and acquisition ak was responsible for data analysis and writing the manuscript kc tb bs am hm hn da mb ap mf tf mg and rt took part in the online survey to create the simulation comparison arm of the study furthermore they carefully read and revised the manuscriptfunding the authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorscompeting interests as and kc received lecture fees and study grants from ovesco endoscopy t¼bingen germany ak jb tb bs am hm hn da mb ap mf tf mg and rt have no conflicts of interest or financial ties to disclosepatient consent for publication not requiredethics approval the wall resect study was approved by the ethical board on january the study protocol conforms to the ethical guidelines of the declaration of helsinki as reflected in a prior approval by the institution's human research committee for the present study an additional approval by the institutional review board was not necessary since no additional personal data were collectedprovenance and peer review not commissioned externally peer revieweddata availability statement all data relevant to the study are included in the or uploaded as supplementary information data were derived from the wall resect trial nct02362126open access this is an open access distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idarmin a0kuellmer http orcid org kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0creferences who colorectal cancer fact sheet the global cancer observatory available http gco iarc fr today data factsheets cancers 10_ 8_ colorectum fact sheet pdf [accessed sep ] zauber ag winawer sj o'brien mj et a0al colonoscopic polypectomy and long term prevention of colorectal cancer deaths n engl j med hong sn byeon js lee b i et a0al prediction model and risk score for perforation in patients undergoing colorectal endoscopic submucosal dissection gastrointest endosc org mizushima t kato m iwanaga i et a0al technical difficulty according to location and risk factors for perforation in endoscopic submucosal dissection of colorectal tumors surg endosc agapov m dvoinikova e factors predicting clinical outcomes of endoscopic submucosal dissection in the rectum and sigmoid colon during the learning curve endosc int open 20142e235 baum p diers j lichthardt s et a0al sterblichkeit und komplikationen nach viszeralchirurgischen operationen dtsch arztebl int schmidt a beyna t schumacher b et a0al colonoscopic full thickness resection using an over the scope device a prospective multicentre study in various indications gut aepli p criblez d baumeler s et a0al endoscopic full
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"elucidate the molecular mechanism underlying the involvement of abnormal DNA methylation in the development of glioma and identify potential newtargets for glioma therapyMethods The GSE79122 chip achieved from the Gene Expression Omnibus GEOdatabase containing glioma samples and normal samples was analyzed Methylationspeciï¬c polymerase chain reaction MSPCR or MSP reverse transcriptionPCR andWestern blot analysis were used to conï¬rm the methylation level and expression level ofthe interleukin receptorassociated kinase IRAK3 gene in glioma cells glioma samplesand the corresponding normal samples In vitro the proliferation apoptosis rate migrationand invasion abilities of glioma cells were detected by Cell Counting Kit8 assay Transwellassay enzymelinked immunosorbent assay and ï¬ow cytometry respectively Besides thexenograft assay of nude mice was used to conï¬rm the effect of the IRAK3 on glioma in vivoResults Microarray analysis showed that the IRAK3 was one of the most hypermethylated genesin glioma and the related mitogenactivated protein kinase MAPK signaling pathway wasactivated More experiments supported the higher methylation level and lower expression levelof the IRAK3 in glioma tissues and cell lines The viability migration and invasion ability ofglioma cells significantly reduced and the apoptosis rate increased with the overexpression anddemethylation of the IRAK3 in vitro Besides treatment with the MAPK signaling pathwayinhibitor PD325901 alone or the overexpression or demethylation of the IRAK3 had a similareffect as the overexpression or demethylation of the IRAK3 alone in glioma cells In vivoxenotransplantation experiments in nude mice conï¬rmed that the overexpression and demethylation of the IRAK3 and suppression of the MAPK signaling pathway inhibited the development ofgliomaConclusion IRAK3 inhibited the development of glioma progression through the MAPKsignaling pathwayKeywords glioma IRAK3 MAPK signaling pathway methylationIntroductionGlioma also known as glioblastoma is one of the most common primary malignant braintumors The average incidence rate of glioma is in individuals1 Despiteimprovements in neurosurgery and radiotherapychemotherapy most patients die within months of diagnosis1 and less than patients survive for years or even more2Recently the molecular mechanisms of glioma have gained increasing attention so as toï¬nd some better methods to defeat this disease Previous studies evaluated that longtermsurvivors of glioma often displayed some favorable molecular characteristics such as thesubmit your manuscript wwwdovepresscomDovePresshttp102147CMARS252772Cancer Management and Research Wu This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphpand incorporate the Creative Commons Attribution Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing the workyou hereby accept the Terms Noncommercial uses of the work are permitted without any further permission from Dove Medical Press Limited provided the work is properly attributed Forpermission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cWu et alDovepresshypermethylation of O6methylguanine DNA methyltransferase MGMT promoter3 which is known as a meaningfulpredictive biomarker for the favorable prognosis of the chemotherapeutic drugs4 Therefore this study proposed that epigenetic regulation might play a key role in the development ofglioma which deserves further research for understanding thiscancerDNA methylation isan epigenetic modiï¬cationinvolved in many biological processes especially geneexpression regulation5 The DNA methylation patterns ofnormal cells are controlled well by a balance betweenmethylation and demethylation However this balance isalways disturbed in cancer cells through ectopic deï¬cientor excessive methylation leading to abnormal biologicalactivities6 Hypermethylation of CpG islands on speciï¬cpromoters inhibiting the transcription of downstreamtumor suppressor genes has been discovered in manycancers which may provide clinicians a new strategy forpatients with cancer7 For instance the promoter region ofSEPT9 is hypermethylated in colorectal cancer Hence theSEPT9 gene methylation assay might become a potentialoption for the early detection and screening of colorectalcancer8 CpG islands also display aberrant hypermethylation at a large number of loci and deï¬ne the subgroup ofglioma910 However the underlying molecular events ofDNA methylation and glioma development remain poorlyunderstoodRecently technical advances in genomewide expression analysis have enabled an improved understanding ofthe diagnosis and prognosis of many types of tumors11Genomewide DNA methylation analysis allows comprehensive DNA methylation proï¬ling of the whole genomehelping in the effective identiï¬cation of novel genes thathave a potential value in clinical practice12 Previous studies have reported many speciï¬c methylation signaturegenes in differenttypes of cancers such as thyroidcancer13 lung squamous cell carcinoma14 hepatocellularcarcinoma15 prostate cancer16 and so on using DNAmethylation analysisThis study aimed to examine the genomewide DNAmethylation proï¬ling of glioma tissuesrevealing thehypermethylation of several genes in glioma Then oneof these hypermethylated genes IRAK3 was selected toconductSubsequentlya relationship between IRAK3 and MAPK signaling pathway was demonstrated by using DNA methyltransferaseinhibitor overexpression of IRAK3 and MAPK signalingpathway inhibitor which can disrupt the development ofcomprehensiveaanalysisgliomas in vitro and in vivo The ï¬ndings might providesome clues for the regulatory role of DNA methylationand the underlying application of targeted treatment ingliomaMethodsTissue SamplesGlioma tissues and adjacent normal tissues were collectedfrom patients with primary glioma n admitted to theZhangye Peoples Hospital Afï¬liated to Hexi UniversitySample collection and use was performed according to theapproval of the ethics committee of the Afï¬liated Hospitalof Shandong University Written informed consent wasprovided by every patient with glioma All samples werefrozen in liquid nitrogen and stored at °C All humanspecimens were obtained with the approval by theInstitutional Ethics Committee of Zhangye PeoplesHospital Afï¬liated to Hexi UniversityCell CultureHuman gliocyte cell line HEB and glioma cell linesSHG44 U251 GOS3 HS683 and SF539 wereobtained from Bena Culture Collection Beijing ChinaHEB U251 HS683 and SF539 cells were grown in highsugar Dulbeccos modiï¬ed Eagles medium DMEMwith fetal bovine serum FBS SHG44 cells weregrown in RPMI1640 medium containing NaHCO3 gL glucose gL and sodium pyruvate gLwith FBS The GOS3 cells were grown with minimum essential medium with Earles Balanced Saltswith FBS and mML glucose All cells were cultured at °C under a humidiï¬ed atmosphere with CO2Cell TransfectionpcDNA31interleukin receptorassociated kinase pcDNA31IRAK3 was obtained from GenePharma ShanghaiChina and 5azadC 5aza2ʹdeoxycytidine 5azadCand signaling pathway inhibitor PD325901 were obtainedfrom SigmaAldrich MO USA U251 cells were seeded insixwell plates at cellswell and cultured at °C andthe cell conï¬uence reached CO2 untilTransfections were executed using Lipofectamine Invitrogen CA USA following the manufacturers protocols The medium was changed with the complete mediumafter h of transfectionsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressTable qRTPCR PrimersWu et alAccession NumberSequence 5ʹ3ʹForwardReverseForwardReverseForwardReverseForwardReverseACCCAAACTAACTGATTTTGCCAAGAGAAATTCCGAGGGCAGGCGACCTCCCATGGCAATTTTAACAGAGACAGGCATGGGAAGCCATCTCGACCAGTCCGTCTAGTTGGTCTGTCTCCGCTAAATACGGACTGCAGCCCTGAGGTCAATGAAGGGGTCGTGeneIRAK3MEK1CfosNM_007199NM_002755NM_005252GAPDHNM_008084GenomeWide DNA MethylationAnalysisFor DNA methylation proï¬ling Inï¬nium HumanMethylation450K BeadChip illuminaga_rnaseqv2100 was obtainedfrom the Gene Expression Omnibus GEO database TheDNA methylation data of chip number GSE79122 whichcontained glioma tissues glioblastomas diffuseastrocytomas and anaplastic astrocytomas and controlbrain tissues were analyzed The Inï¬nium MethylationAbbiotec CA USA and Illumina BeadStudio softwareGenetech Biotech Taipei Taiwan were used to measurethe methylation proï¬les of modiï¬ed DNA and loci CpGThe methylated signal intensity at particular CpG loci and450K BeadChip assay were presented as β values and percentage respectivelyQuantitative RTPCRTotal cellular RNA was extracted using PureLink Invitrogenfollowing the manufacturers protocol RT was performed on µg total cellular RNA using a HighCapacity cDNA ReverseTranscription Kit with RNase Inhibitor Applied Biosystemspurchased from Thermo Fisher Scientiï¬c MA USASubsequently quantitative reverse transcriptionpolymerasechain reaction RTPCR was performed using ArcturusParadise Plus qRTPCR Kit Applied Biosystems ThermoFisher Scientiï¬c Comparative expression values were calculated by the ÎÎCt method GAPDH was used for internalreference All primer sequences involved are listed in Table converted into uracil without changing the state of methylated cytosine The IRAK3 methylation level in glioma wasidentiï¬ed using methylationspeciï¬c PCR MSP The MSPprimers are listed in Table EnzymeLinked Immunosorbent AssayThe human interleukin IL6 enzymelinked immunosorbent assay kit Sangon Biotech Shanghai China was usedto test the IL6 level in the glioma cell culture medium Theglioma cell culture medium was centrifuged at rpm for min to remove cells and polymers The supernatant ï¬uidstored at °C was preserved in the supernatant ï¬uid at °C A normal glioma cell culture medium was used as thecontrolWestern Blot AnalysisLysis buffer RIPA Thermo Fisher Scientiï¬c and NPERThermo Fisher Scientiï¬c were used to extract proteinsfrom glioma cells and tissues respectively Then μg totalprotein per sample was separated using SDSPAGE andtransferred to polyvinylidene ï¬uoride membranes ThermoFisher Scientiï¬c The membranes were probed with primaryantiIRAK3 antibody ab8116 Abcam MA USA antiMEK1 phospho S298 antibody [EPR3338] ab96379 antiERK1 ERK2 antibody [ERK7D8] ab54230 anticFosphospho T232 antibody ab17933 and antiGAPDH antibody [6C5] ab8245 Abcam as control The number ofTable MethylationSpeciï¬c PrimersDNA Methylation AssayGenomic DNA in glioma tissues and cells was extracted andtreated with bisulï¬te using CpGenome DNA Modiï¬cationKit Chemicon CA USA following the manufacturersprotocol All unmethylated cytosine residues in DNA wereGeneIRAK3 forwardIRAK3 reverseIRAK3 forwardIRAK3 reverseSequence 5ʹ3ʹ5ʹTCGGGATAGTGGTTAATATTTC3ʹ5ʹTTTTTTTCGTTTTTCGTAAAA3ʹ5ʹ AGTTTGGGATAGTGGTTAATATTTT 3ʹ5ʹ TTTTTTTCATTTTTCATAAAAAAA 3ʹCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Genomewide methylation data were obtained from the GEO database for available glioma9 adjacent mucosa A Density of methylated DNA intensity byeach sample B Type I and Type II assays showed variant β value distributions The differences between probe types were regulated by normalization procedures whichshowed that represented unmethylated sites while represented fully methylated sites C Multidimensional scaling plot showing differential clustering of control versustumor tissues D Dendrogram produced for probes in normal and tumor tissues E Heatmap of top differentially methylated imprinted CpG sitesAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Distribution of top differentially methylated imprinted CpG sites A Distribution of top differentially methylated imprinted CpG sites according toCpG islands island sea shelf and shore B Distribution of top differentially methylated imprinted CpG sites according to the position relative to genes 1stexon ² UTRs or ² UTRs body IGR TSS1500 and TSS200 C Combined genetic and epigenetic annotation information revealed the distribution of the top differentially methylated imprinted CpG sitesbinding proteins was measured using AlphaEaseFC softwareGenetic Technologies FL USACell Counting Kit8 AssayU251 cells were seeded in 96well plates and allowed toadhere for h at °C Then these cells were transfectedwith pcDNA31IRAK3 and treated with 5azadC orPD325901 After h Cell Counting Kit8 DojindoKumamoto Japan was used to test the cell viability at respective time points To summarize mL of the triazoliumsubstrate was added to each well and coincubated with cellsat °C for h The absorbance was measured at nm andCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure DNA methylation analysis of tumor tissues and normal tissues and analysis of methylation degree for seven CpG sites of IRAK3 A The expression of the top candidate genes was analyzed IRAK3 was hypermethylated significantly in tumor tissues compared with normal tissues B The number of IRAK3methylated CpG islands ineach isosite CI Seven CpG sites for IRAK3 which are presented in the boxplot displayed a decreased methylation in the tumor group The boxplot for cg Ccg D cg E cg F cg G cg H and cg IAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related signaling pathway A The top signaling pathways with the highest and lowest correlation with glioma B The top signalingpathwayrelated genes enriched in the glioma the MAPK signaling pathway was highly expressed C The MAPK signaling pathway was activated in glioma D The status ofthe top enriched signaling pathways in gliomaAbbreviation MG malignant gliomaCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressthe results were normalized to untreated cells at respectivetime pointsCell Migration and Invasion AssayBoth cell migration and invasion were performed using theTranswell assay For cell migration assay U251cells were seeded in a serumfree medium in the upperchamber which contained a polyethylene terephthalatemembrane with mm in diameter and μm in poresize The bottom chamber was prepared with FBS asa chemoattractant After incubating at °C for hnonmigrated cells were scraped from the upper surfaceof the membrane with a cotton swab and the cells migrating to the bottom chamber were ï¬xed with paraformaldehyde and stained with crystal violet Finally the stainedcells were counted under a microscope at magniï¬cation in ï¬ve randomly selected ï¬elds for quantiï¬cationinvasion assay U251 cells weresuspended in mL of serumfree DMEM and thentreated using the same procedure as for the migrationassay following the manufacturers protocol but the chambers mm BD Biosciences San Jose CA USA wereplated with BD BioCoat MatrigelFor cellFlow CytometryEach treatment of U251 cells was washed with phosphatebuffered saline and resuspended in μL of AnnexinV binding buffer After staining with Alexa Fluor Annexin V and 7AAD Viability Staining Solution for min in the dark at room temperature these cells were analyzed using multicolor ï¬ow cytometer Data were analyzedusing Kaluza softwarethe mice were keptXenograft StudiesMale BALBc nude mice weeks were maintained underpathogenfree conditions Allina temperaturecontrolled airconditioned conventional animal house with a h lightdark cycle and given free accessto food and water Besides animal health and behaviour weremonitored every two days All experiments were approvedby the Ethics Committee of Zhangye Peoples Hospital toguarantee that all studies involving experimental animalswere performed in full compliance with National Institutesof Health Guidelines for the Care and Use of LaboratoryAnimals The U251 cells100 μL cells were transfected with pcDNA31IRAK3 and then transferred to micen12 Normal U251 cells were transferred to mice in the5azadC and PD325901 groups and then 5azadC andPD325901 were subcutaneously injected respectively intothe posterior ï¬ank of nude mice After and daysculture the mice were sacriï¬ced and the tumor size wasmeasured The tumor volume was measured using a caliperfollowing the formula length width22Statistical AnalysisAll data were collected from three independent experiments and presented as mean ± standard deviationStatistical analyses were performed using GraphPad software Differences between groups were analyzed usingStudent' ttest or chisquare test Statistical signiï¬cancewas set at P ResultsGenome Methylation Proï¬le in GliomaA total of glioma tissues and adjacent normal sampledata publicly available at GEO were analyzed to revealthe global DNA methylation patterns of glioma Firstdensity plots of β values generated from each samplewere used identifying a poor performance of methylatedsignals for raw data Figure 1A Inï¬nium Type I and TypeII probe assays also showed somewhat different distribution of β value ranging from unmethylated sites to fully methylated sites Figure 1B Therefore these datawere regulated by normalization procedures to reduce thepotential impact later Multidimensional scaling MDSplot and dendrogram exhibited a differential clusteringbetween tumor and normal groups which distinguishedglioma from adjacent normal tissues Figure 1C and DFurther heatmap of the top differentially methylatedCpG sites indicated a visible difference in DNA methylation proï¬les between the tumor and normaltissueand general hypermethylation occurred insamplesglioma Figure 1EDistribution of Genomic Regions forDifferentially Methylated CpG SitesBased on the position relative to gene [1st exon ² untranslated region UTR ² UTRs body transcription start sites bp TSS1500 and TSS200] as well as CpG islands andneighborhood content shores shelves islands and sea the distribution of genomic regions for the differentiallymethylated CpG sites was exhibited More than and methylation differences occurred in bodyIGR and CpGisland sea respectively which was obviously higherthan that in other regions Figure 2A and B From thesubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related gene expression level A Based on the differential genes in the disease data the distribution of upregulated genes anddownregulated genes in the GOrelated pathway was enriched B The expression level of top MAPK signaling pathwayrelated genesAbbreviation MG malignant gliomaperspective of both genetics and epigenetics gene and CpGcontent regions were combined for more analyses whichshowed that CpG sites in genetic bodyisland sea andIGRisland sea were differentially methylated betweenglioma and adjacent normal samples Figure 2CIRAK3 Was Hypermethylated in GliomaThe heatmap was used to present the top hypermethylationgenes in glioma compared with normal tissues so as to ï¬gureout the most characteristic gene with methylation change intumors indicating that the IRAK3 was hypermethylated theCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressmost in glioma Figure 3A Then the methylation differentialdistribution of the IRAK3 was upregulated at each siteFigure 3B Furthermore the β value of CpG sitesIRAK3such as cg cg cg cg cg cg cg andso on on the IRAK3 was significantly higher in the tumorindicating that IRAK3group than in the normal groupwas hypermethylated in the tumor group due to CpGsFigure 3CI The aforementioned results showed that theIRAK3 CpG sites were highly methylated in glioma tissuesthan in normal tissuestissues and the top signaling pathwayrelated genesenriched in the glioma were listed Figure 4A and B Theresults suggested that the MAPK signaling pathway washighly expressed and activated Figure 4C and D Based onthe differential genes in the disease data the distribution ofupregulated genes and downregulated genes in the GOrelatedpathway was enriched Figure 5A The expression level ofMAPK signaling pathwayrelated genes in glioma is shown inFigure 5B The results of Figures and manifested thatIRAK3related MAPK signaling pathway was highly activatedin glioma However whether any correction existed with thedevelopment of glioma remained to be veriï¬edMAPK Signaling Pathway Expression Leveland State in GliomaFor the identiï¬cation of gliomarelated signaling pathwaysthat might be inï¬uenced by aberrant DNA methylation thetop IRAK3related signaling pathways in glioma and normalMethylation and Expression Level of theIRAK3 in Glioma Tissues and CellsThe impact of hypermethylation on the expression of IRAK3in glioma tissues and cells was elucidated The IRAK3 wasFigure Methylation level and expression level of the IRAK3 in glioma tissues and cells A The IRAK3 was highly methylated in glioma tissues compared with adjacent tissues BThe IRAK3 was less expressed in glioma tissues than in adjacent tissues C The IRAK3 in the ï¬ve glioma cells SHG44 U251 HS683 SF539 and GOS3 was hypermethylatedcompared with U251 glioma cells D The IRAK3 was less expressed in the ï¬ve glioma cells than in normal glioma cells The difference was significant P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Effect on glioma cells after the overexpression and demethylation of the IRAK3 A The expression level of the IRAK3 was significantly higher in the glioma U251 cells of thepcDNA31IRAK3 and 5azadC groups than in the control group B and C The protein expression level of the IRAK3 significantly increased in the pcDNA31IRAK3 and 5azadC groupscompared with the control group D The expression level of the inï¬ammatory factor IL6 significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with thecontrol group E The activity significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantlyincreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group H and I The migration capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups compared with the control group J and K The invasiveness capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups than in the the control group Allthe mentioned differences were significant P The IRAK3 had a suppressive effect on glioma cells in vitroCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepresshighly methylated and less expressed in glioma tissues than inadjacent normal tissues Figure 6A and B Next the IRAK3 inï¬ve glioma cell lines SHG44 U251 HS683 SF539 andGOS3 also showed hypermethylation and less expressioncompared with normal gliocytes Figure 6C and D U251cells were selected for subsequent experiments because themethylation level of the IRAK3 in U251 cells was the highestFigure 6COverexpression or Demethylation of theIRAK3 Inhibited the Development of GliomaCellsA systematic test with the overexpression or demethylation ofthe IRAK3 was conducted to understand whether the correctionof abnormal IRAK3 methylation and expression affected thedevelopment of glioma After using pcDNA31IRAK3 tooverexpress or 5azadC to demethylate the IRAK3 the proteinexpression level of the IRAK3 in glioma U251 cells increasedin the tumor group compared with the normal group Figure7AC IL6 an inï¬ammatory factor secreted by the MAPKsignaling pathway [PMID ] so that its level in theculture medium could be used to represent the status ofMAPK signaling pathway activation decreased in thepcDNA31IRAK3 and 5azadC groups compared with thecontrol groups Figure 7D Surprisingly the viability ofglioma cells significantly decreased while the apoptosis rateincreased Figure 7EG and the migration and invasivenesscapability decreased in the pcDNA31IRAK3 and 5azadCFigure Effect on glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The expression level of MAPKsignaling pathwayrelated genes expression level was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group B and C Theexpression level of MAPK signaling pathwayrelated proteins significantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the controlgroup D The level of inï¬ammatory factor IL6 was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group E The activitysignificantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantly increased inthe PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group All the mentioned differences were significant P P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et algroups Figure 7HK which elucidated the associationbetween IRAK3 and the development of glioma The aforementioned results veriï¬ed that the overexpression or demethylation of the IRAK3 inhibited the development of glioma cellsin vitroMAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited the Development ofGlioma CellsThe MAPK signaling pathway inhibitors PD325901pcDNA31IRAK3 and 5azadC were used alone or incombination in vitro to conï¬rm the inï¬uence of the IRAK3and MAPK signaling pathways on glioma In PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901groups the mRNA and protein expression levels of theMAPK signaling pathwayrelated genes such as MEK1ERK and cFos as well as the level of IL6 decreased inglioma Figure 8AD The cell viability significantlythe apoptosis rate increased Figure 8EGdecreasedand the migration and invasiveness capability significantlydecreased in PD325901 pIRAK3PD325901 and 5azadCPD325901 groups Figure 9AC The aforementioned results veriï¬ed that the MAPK signaling pathwaysuppression alone or combined with the overexpression ordemethylation of IRAK3MAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited Glioma in vivoFinally whether MAPK signaling pathway suppression aloneor combined with the overexpression or demethylation of theFigure Effect on the migration and invasiveness capability of glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and5azadC A and B The migration capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group A and C Theinvasiveness capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group All the mentioned differences weresignificant P Cancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Effect on mice after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The tumor in the transplantationPD325901 p IRAK3 PD325901 and 5azadC PD325901 treatment groups after and days B The tumor volume was smaller in the PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901 groups compared with the control group C The tumor weight followed the same trend as the volume D Theexpression level of the IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups compared with thetransplantation control group E and F The protein expression level of IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups than in the transplantation control group All the mentioned differences were significant P P IRAK3 had a suppressive effect on glioma in vivo was studiedAfter transplanting glioma U251 cells treated with PD325901pcDNA31IRAK3 PD325901 or 5azadC PD325901the tumorigenesis significantly decreased compared with thatin the control group mice Figure 10AC Finally mRNAand protein expression levels ofthe MAPK signalingsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alpathwayrelated genes in xenograft glioma were detectedthey were found to be significantly decreased in thePD325901 pcDNA31IRAK3 PD325901 or 5azadC PD325901 group Figure 10DFDiscussionAccording to the World Health anization the overallsurvival of patients with cancer has increased significantlyover the years with the improvement in diagnosis and treatment However glioma is still a highly fatal disease1Therefore this study aimed to elucidate the detail of itsprogression mechanisms and search for new therapeutic strategies One recent study reported that the use of MGMTpromoter methylation test in hospitals had a strong inï¬uenceon the prognosis of glioma17 suggesting a significant clinicalapplication prospect of DNA epigenetic regulation Hencethe main focus of the present study was on the relationshipbetween DNA methylation and glioma IRAK3IRAK3 belongs to the IL receptorassociated kinaseIRAK family involved in inhibiting Tolllike receptorsignaling by changing the activity of other members ofthe IRAK family to decrease inï¬ammatory response1819Increasing evidence supported thatthe expression ofIRAK3 in tumorassociated macrophages led to compromised immune surveillance of cancer cells when it proï¬tably prevented excessive inï¬ammation contributing tocancer development Therefore the growth of transplantable cancer cells could be inhibited by enhancing hostimmune responses in IRAK3deï¬cient mice20 A smallnumber oftumor cellintrinsicIRAK3 could also support the progression of tumor cellsin colorectal and lung cancers depending on the dysfunctional innate immune system indirectly Interestingly theexpression of IRAKM in colorectal and lung cancers correlated with poor prognosisin patients with thesecancers2122 However little is known about the molecularmechanism of the IRAK3 in glioma This study revealedthe downregulation of IRAK3 glioma tissues and cellsthrough DNA methylation analysis which seemed to becontrary to the previous ï¬ndings on other cancers Thisstudy investigated whether a special signaling pathwayexisted that connected IRAK3 and glioma progressionshowed thatstudiesAn ongoing study has validated that all members of theIRAK family mediate the activation of MAPK and nuclearfactorκB NFκB signaling pathways23 MeanwhileIRAK3 a general negative regulator was conï¬rmed toinhibit MAPK and NFκB activation2425 Likewisethese results identiï¬ed that the MAPK signaling pathwaywas highly activated in glioma and its related geneexpression level was downregulated after overexpressionof the IRAK3 In fact upstream genomic events andordifferent extracellular stimuli could activate the MAPKsignaling pathway mediating a wide range of cellularprocesses26 The activation of the MAPK signaling pathway often led to abnormal cell growth and tumorigenesisThe predominant effect relied on the signal intensity andthe context or tissue in which the signal occurred2728Zhang revealed that aberrant DNA methylation inthe promoters of MMPTIMP axis genes upregulated theMAPK signaling pathway promoting the progression oftumor cells Correction of the abnormal DNA epigenotypes attenuated the migration and invasion of tumorcells in vitro and reduced tumorigenicity in vivo29Intriguingly these results were consistent with those ofthe present studyConsidering the reverse expression pattern of IRAK3between glioma and other cancers2122 different epigeneticmodiï¬cation was regarded as the major reason causing thisdistinction In many cancers the DNA methylation patternsbecame aberrant with tissue speciï¬city serving as diagnosticmarkers and therapeutic targets30 Hence based on thelower expression and the tumorigenic function of the IRAK3in glioma its epigen
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Thyroid surgery in children in a single institution from Osama Ibrahim Almosallama Ali Aseerib Ahmed Alhumaida Ali S AlZahranic Saif Alsobhib Saud AlShanafeybFrom the aDepartment of Surgery College of Medicine Qassim University Buraidah Al Qassim Saudi Arabia bDepartment of Surgery King Faisal Specialist Hospital and Research Center Riyadh Saudi Arabia cDepartment of Medicine King Faisal Specialist Hospital and Research Center Riyadh Saudi Arabia Correspondence Dr Osama Ibrahim Almosallam Department of Surgery College of Medicine Qassim University PO Box Buraidah Al Qassim Saudi Arabia osama_iaahotmailcom ORCID orcid0000000290367564 Citation Almosallam OI Aseeri A Alhumaid A AlZahrani AS Alsobhi S AlShanafey S Thyroid surgery in children in a single institution from Ann Saudi Med Received January Accepted May Published August Copyright Copyright Annals of Saudi Medicine Saudi Arabia This is an access under the Creative Commons AttributionNonCommercialNoDerivatives International License CC BYNCND The details of which can be accessed at httpcreativecommons licensesbyncnd40Funding NoneBACKGROUND Data on thyroid surgery in children are scarceOBJECTIVE Analyze outcome data on thyroid surgery in a pediatric populationDESIGN Medical record reviewSETTING Tertiary health care institutionPATIENTS AND METHODS We collected demographic and clinical data on patients years or younger who had thyroid surgery in the period to Descriptive data are presentedMAIN OUTCOME MEASURES Indications for thyroidectomy thyroid pathology complications length of stay and radioactive iodine treatment and recurrencesSAMPLE SIZE RESULTS Of patients who underwent thyroidectomy procedures were females and the mean age at operation was years and were associated with multiple endocrine neoplasia type There was no history of radiation exposure Eightyone patients had fine needle aspiration FNA which correlated with the final histopathology in of cases Sixtysix patients had malignant cancer papillary of patients who had neck dissection had lymph node metastasis and had distant metastases to the lung Procedures included total thyroidectomy hemithyroidectomy completion and subtotal thyroidectomy Twentythree patients developed hypocalcemia permanent and had unilateral recurrent laryngeal nerve injury permanent Patients were followed up for a mean duration of months median months Of patients with thyroid cancer received radioactive iodine and had recurrence Malignancy is the commonest indication for thyroid surgery in children and FNA is highly diagnostic Hypocalcemia and recurrent laryngeal nerve injury are significant complications The recurrence rate in thyroid cancer is LIMITATIONS RetrospectiveCONFLICT OF INTEREST Noneoriginal ANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0cThyroid diseases requiring surgery are relatively uncommon in children compared to adults The prevalence of palpable thyroid nodules in children ranges from to Sporadic welldifferentiated thyroid cancer is the most common endocrine malignancy in children accounting for of pediatric cancers in the prepubertal age group and up to of cancers in adolescents aged year2 The most common indication for thyroid surgery in children varies among published studies but thyroidectomy for malignant conditions is rising38 Data in children throughout the world are relatively scarce The objective of this study was to analyze the clinical data and outcome of thyroid surgery in a large series of children treated at a single center at King Faisal Specialist Hospital and Research Center KFSHRC in RiyadhPATIENT AND METHODS With the approval of the Institutional Review Board IRB at KFSHRC the medical records of all patients years old and younger who underwent a thyroid surgery between and were retrospectively reviewed We elected to include patients up to the year to ensure a reasonable followup period Patients for the study were identified by a search of the operating room log for all procedures involving the thyroid gland for the specified age groupDemographic data clinical features and surgical outcomes were collected Specific data that were obtained included age at operation gender family history presenting symptoms history of radiation exposure presence of multiple endocrine neoplasia type MEN thyroid function test presence and size of thyroid nodules by ultrasound presence of lymph nodes metastasis or distant metastasis fine needle aspiration FNA cytology surgical procedure final histopathology and length of followup Outcomes analyzed were postoperative complications including transient or permanent hypocalcemia transient or permanent recurrent laryngeal nerve paralysis wound infection and hematoma length of stay and radioactive iodine treatment and recurrences Thyroid procedures in this series included hemithyroidectomy subtotal total and completion thyroidectomy Surgeries were performed by either an endocrine adult surgeon or a pediatric surgeon No intraoperative nerve monitoring was used Early in the series procedures were performed by adult endocrine surgeons but lately a combined approach was adopted where pediatric surgeons and adult endocrine surgeons collaborated in such cases proceduresthe normal range in our laboratory regardless of symptoms Transient hypocalcemia was identified if it lasted for less than months while permanent hypocalcemia was considered if the serum calcium level remained below normal range and the patient continued on calcium supplementation after months of the surgery All patients with a family history of MEN underwent genetic testing of the RET protooncogene to confirm the diagnosis All patients who underwent completion thyroidectomy had a preoperative and postoperative vocal cords assessment at the Otolaryngology clinic Descriptive data were generated and comparisons were conducted using the t test for continuous data and the chisquare or Fisher exact tests for proportionsRESULTSBetween and patients underwent surgical procedures patients underwent two procedures for thyroid disease at our institution Eighty patients were females The mean age at operation was years median years range years The most common indication for thyroidectomy was thyroid nodule which was present in of cases Table The mean SD size of thyroid nodules was mm There were cases associated with MEN syndromes The final pathology in two patients with MEN syndrome showed medullary thyroid cancer MTC while the remaining patients had prophylactic procedures before development of MTC None of the patients had a history of radiation exposure Eightyone patients FNA which correlated with the final histopathology in of cases There were three cases of toxic adenoma and one case of Graves disease which did not require FNA The remaining cases underwent FNA at another institution and FNA was not repeated at our institution or they came for completion thyroidectomy with documented pathology for malignancy after they had their first surgery in another hospitalThe most common diagnoses included papillary thyroid cancer and multinodular goiter or colloid Table Indications for thyroidectomy in patients IndicationNodulen MEN prophylaxisHyperthyroidismMultinodular goiterCompletion thyroidectomy Hypocalcemia was defined by calcium levels below Data are number original PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0cnodule Table Surgical procedures included total thyroidectomy hemithyroidectomy completion thyroidectomy and subtotal thyroidectomy Neck dissection was performed in patients Operative complications were observed in patients The most common complication was hypocalcemia transient permanent and Table Thyroid pathology in the patientsPathologyn BenignNormal thyroid tissueColloid noduleCystAdenomaThyroiditisGraves diseaseThyroid cancerPapillaryFollicularMedullaryHurthleAnaplasticTotalData are number Table Benign and malignant lesions in patientsBenignn37Malignantn66 P value Age meanyearsGender malefemalePresence of noduleHypocalcemiaRecurrent laryngeal nerve palsyBleedinghematomaWound infectionTracheal injuryOverall complicationsMean length of stay daysMEN recurrent laryngeal nerve palsy transient permanent all were unilateral Table Of patients with malignant lesions had lymph node metastasis and patients had distant metastases to the lung None of the patients developed postoperative bleeding wound infection or tracheal injury Patients were followed up for a mean of months median range months radioactive iodine treatment was delivered to patients with malignant lesions patients had recurrences were local recurrences and were local and distant recurrences to the lung Three cases received radioactive iodine RAI before and after recurrence One case was low risk before recurrence so did not receive RAI until after recurrence One case had medullary thyroid cancer so did not receive RAI In the remaining five cases there was no clear data whether those patients received RAI before or only after a recurrence All local recurrences underwent resection except for one patient who was lost follow up There was no mortality in this study DISCUSSIONThe most common indication for thyroidectomy in this series was thyroid nodule which correlates with previously published reports in the pediatric population35 Children with thyroid nodules have an estimated fourfold higher risk of developing thyroid cancer compared to adults910 The high incidence of malignancy in this series suggests children with a thyroid nodule should be carefully evaluatedFNA is a valuablemethod for preoperative evaluation of thyroid nodules However there are limitations on the routine use of FNA in children including the need for sedation sampling errors and the limited availability of experienced cytopathologists11 Many previous studies reported high sensitivity and specificity of FNA in evaluating thyroid nodule in children1114 which correlate with our findingsOur data showed lymph node metastasis in of thyroid cancer cases which supports the notion that children with thyroid cancer frequently present with more extensive disease than adults Lymphnode involvement at diagnosis is seen in to of children compared with to of adults with differentiated thyroid cancer1523 Because our hospital is the largest referral center in Saudi Arabia especially for oncology cases this may explain the large number of lymph node and distant metastasis In this cohortThe most common complication reported after thyroidectomy in children is hypoparathyroidism with an incidence ranging between to which original PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0ccorresponds with our results of which are reported as hypocalcemia in Table One study found that total thyroidectomy central and bilateral neck dissection Graves disease and malignancy were risk factors for hypocalcemia after thyroid surgery3 In this cohort postoperative hypocalcemia was noted more in malignant cases but it failed to reach statistical significance Moreover there was no significant difference between benign and malignant cases in terms of mean age gender distribution recurrent laryngeal nerve injury or overall complications a finding that was reported previously26 Multiple studies in recent years have found an inverse relationship between surgeon volume and complication rates2728 but similar data in the pediatric population is lacking One study found that highvolume endocrine surgeons have better outcomes and shorter lengths of stay and lower costs after thyroidectomy and parathyroidectomy in children compared to pediatric surgeons general surgeons or otolaryngologists29 Scheumann and colleagues also concluded that a collaborative approach between pediatric and endocrine surgeons would have better outcomes This has led other authors to suggest that a combined approach with endocrine and pediatric surgeons in addition to pediatric endocrinologists may optimize the care of children with surgical thyroid disease given the low number of pediatric patients4 Our data do not allow for comparisons of different approaches given the late adoption of the combined approach The recurrence rate for thyroid cancer in children after thyroidectomy has varied widely in reported studies ranging from to while it was in this cohort Only a few studies explored the predictors of recurrence Lymph node involvement multiple nodules male gender younger age histologic subtype and advanced tumor stage were risk factors associated with recurrence17233033 In this study of patients with malignant lesions received RAI Although there are conflicting data regarding the indications of postoperative RAI treatment in lowrisk patients the current recommendation is that lowrisk patients can be treated without RAI3436There are some limitations to this study The retrospective nature may affect the validity and quality of the data The small number of cases in some categories did not enable us to compare groups and explore predictors relative to these factors On the other hand this study adds to the scarce data on thyroid surgery in pediatric age group Malignancy is the commonest indication for thyroid surgery in children and FNA is highly diagnostic Hypocalcemia and recurrent laryngeal nerve injury are significant complications Cancerrelated death is extremely rare but recurrence is not uncommon and a significant number of patients with malignant cases received RAI treatmentoriginal PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0cREFERENCES Trowbridge FL Matovinovic J McLaren GD Nichaman MZ Iodine and goiter in children Pediatrics Ries LAG Melbert D Krapcho M Stinchcomb DG Howlader N Horner MJ et al SEER Cancer Statistics Review Bethesda National Cancer Institute Based on November SEER data submission Chen Y[h] Masiakos PT Gaz RD Hodin RA Parangi S Randolph GW et al Pediatric thyroidectomy in a high volume thyroid surgery center Risk factors for postoperative hypocalcemia J Pediatr Surg Aug5081316 Wood JH Partrick DA Barham HP Bensard DD Travers HS Bruny JL et al Pediatric thyroidectomy a collaborative surgical approach J Pediatr Surg May4658238 Scholz S Smith JR Chaignaud B Shamberger RC Huang SA Thyroid surgery at Childrens Hospital Boston a 35year singleinstitution experience J Pediatr Surg Mar46343742 Josefson J Zimmerman D Thyroid nodules and cancers in children Pediatr Endocrinol Rev Sep611423 Hameed R Zacharin MR Changing face of paediatric and adolescent thyroid cancer J Paediatr Child Health LugoVicente H Ortiz VN Irizarry H Camps JI Pagán V Pediatric thyroid nodules management in the era of fine needle aspirationJ Pediatr Surg Mussa A De Andrea M Motta M Mormile A Palestini N Corrias A Predictors of Malignancy in Children with Thyroid Nodules J Pediatr Oct167488692 Amirazodi E Propst EJ Chung CT Parra DA Wasserman JD Pediatric thyroid FNA biopsy Outcomes and impact on management over years at a tertiary care center Cancer Cytopathol Partyka KL Huang EC2 Cramer HM Chen S Wu HH Histologic and clinical followup of thyroid fineneedle aspirates in pediatric patients Cancer Cytopathol Sinha CK Decoppi P Pierro A Brain C Hindmarsh P Butler G et al Thyroid Surgery in Children Clinical Outcomes Eur J Pediatr Surg Oct2554259 Kundel A Thompson GB Richards ML Qiu LX Cai Y Schwenk FW et al Pediatric Endocrine Surgery A 20Year Experience at the Mayo Clinic J Clin Endocrinol Metab February Jiang W Newbury RO Newfield RS Pediatric thyroid surgery and management of thyroid nodulesan institutional experience features and over a 10year period Int J Pediatr Endocrinol Burke JF Sippel RS Chen H Evolution of Pediatric Thyroid Surgery at a Tertiary Medical Center Surg Res AlQahtani KH Tunio MA Al Asiri M Aljohani NJ Bayoumi Y Riaz K et al Clinicopathological treatment outcomes of differentiated thyroid cancer in Saudi children and adults J Otolaryngol Head Neck Surg Nov Kluijfhout WP van Beek DJ Verrijn Stuart AA Lodewijk L Valk GD Van der Zee DC et al Postoperative Complications After Prophylactic Thyroidectomy for Very Young Patients With Multiple Endocrine Neoplasia Type Medicine Baltimore 20159429e1108 Raval MV Browne M Chin AC Zimmerman D Angelos P Reynolds M Total thyroidectomy for benign disease in the pediatric patientfeasible and safe J Pediatr Surg Stavrakis AI Ituarte PH Ko CY Yeh MW Surgeon volume as a predictor of outcomes in inpatient and outpatient endocrine surgery Surgery Sosa JA Bowman HM Tielsch JM Powe NR Gordon TA Udelsman R The importance of surgeon experience for clinical and economic outcomes from thyroidectomy Ann Surg Tuggle CT Roman SA Wang TS Boudourakis L Thomas D Udelsman R et al Pediatric endocrine surgery Who is operating on our children Surgery Dec144686977 Park S Jeong JS Ryu HR Lee C Park JH Kang S et al Differentiated Thyroid Carcinoma of Children and Adolescents27Year Experience in the Yonsei University Health System J Korean Med Sci Palmer BA Zarroug AE Poley RN Kollars JP Moir CR Papillary thyroid carcinoma in children risk factors and complications of disease recurrence J Pediatr Surg Wada N Sugino K Mimura T Nagahama M Kitagawa W Shibuya H et al Pediatric differentiated thyroid carcinoma in stage I risk factor analysis for disease free survival BMC Cancer D Danese Gardini A Farsetti A Sciacchitano S Andreoli M Pontecorvi A Thyroid carcinoma in children and adolescents Eur J Pediatr Astl J Chovanec M Lukes P Katra R Dvorakova M Vlcek P et al Thyroid carcinoma surgery in children and adolescents years experience surgery of pediatric thyroid lymph node metastases carcinoma Int J Pediatr Otorhinolaryngol Chaukar DA Rangarajan V Nair N Nadkarni MS Pai PS Dcruz AK et al Pediatric thyroid cancer J Surg Oncol Dzodic R Buta M Markovic I Gavrilo D Matovic M Milovanovic Z et al Surgical management of welldifferentiated thyroid carcinoma in children and adolescents years of experience of a single institution in Serbia Endocr J Scheumann GF Gimm O Wegener G Hundeshagen H Dralle H Prognostic significance and surgical management of locoregional in papillary thyroid cancer World J Surg Shi RL Qu N Yang SW Tumor size interpretation for predicting cervical lymph node metastasis using a differentiated thyroid cancer risk model Onco Targets Ther Zimmerman D Hay ID Gough IR Goellner JR Ryan JJ Grant CS et al Papillary thyroid carcinoma in children and adults longterm followup of patients conservatively treated at one institution during three decades Surgery Collini P Mattavelli F Pellegrinelli A Barisella M Ferrari A Massimino M Papillary carcinoma of the thyroid gland of childhood and adolescence Morphologic subtypes biologic behavior and prognosis a clinicopathologic study of sporadic cases treated at a single institution during a 30year period Am J Surg Pathol BorsonChazot Causeret S Lifante JC Augros M Berger N Peix JL Predictive factors for recurrence from a series of children and adolescents with differentiated thyroid cancer World J Surg Baumgarten HD Bauer AJ Isaza A MostoufiMoab S Kazahaya K Adzick NS Surgical management of pediatric thyroid disease Complication rates after thyroidectomy at the Childrens Hospital of Philadelphia highvolume Pediatric Thyroid Center Journal of pediatric surgery Oct Kurzawinski TR De Coppi P Thyroidectomy in Children InPediatric Surgery pp Springer Berlin Heidelberg Francis G Waguespack SG Bauer AJ Angelog P Benvenga S et al Management Guidelines for Children with Thyroid Nodules and Differentiated Thyroid Cancer The American Thyroid Association Guidelines Task Force on Pediatric Thyroid Cancer THYROID Volume Number original PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0c'
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glioma initiates from glial cells and contains several types such as astrocytoma and oligodendroglioma1 over a quarter of brain tumors are glioma which causes a large number of cancerrelated deaths every year around the world1 the current clinically therapeutic strategies are surgery combined with chemotherapy and radiotherapy2 however the prognosis of glioma patients remains not well post therapy3 hence it is urgently required to discover new molecular mechanism for glioma therapyboth long noncoding rna lncrna and microrna mirna belong to noncoding rnas which have no proteincoding ability lncrna is characterized with more than nucleotides while mirna is about nucleotides in length4 lncrna and mirna are involved in various cellular processes including cell division invasion and survival5 dysregulation of lncrna or mirna usually causes tumor initiation and progression67 for example lncrna linc00152 upregulation promotes gastric cancer growth and metastasis8 lncrna snhg6 overexpression facilitates lung cancer cell proliferation and metastasis9 mir3405p dysregulation promotes tumorigenesis of esophageal squamous cell carcinoma10 in addition mir126 cancer management and research du this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphpcorrespondence jun wu weiwen qiu email wwwwjjjj924163com weiwenqhotmailcomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars262279 0cdu dovepresssuppresses colon cancer cell survival and induces apoptosis11 besides lncrna has been identified as potential competing endogenous rna cerna for mirna to function in cancer12 the potential roles underlying lncrna and mirna still require much investigation and the relationship between lncrna and mirna also needs to be definedlinc00173 is an oncogene in lung cancer and breast cancer1314 the function of linc00173 in glioma is unclear in the current study we found that linc00173 was upregulated in glioma tissues linc00173 high expression was associated with a low survival rate linc00173 depletion suppressed proliferation migration and invasion of glioma cells linc00173 was discovered to sponge mir765 to elevate nutf2 expression taken together our findings supported that linc00173 plays essential oncogenic roles in glioma through activating mir765nutf2 pathwaymaterials and methodsclinical samplesthirtyseven glioma tissues and normal tissues were collected from lishui city peoples hospital patients received no radiotherapy or chemotherapy before surgery all tissues were stored in liquid nitrogen association between linc00173 expression and clinical characteristics in glioma tissues was analyzed in table written informed consent was obtained from every patient this study was approved by the ethics committee of lishui city peoples hospital no and the table association between linc00173 expression and clinical characteristics in glioma tissuesfeaturesage yearsgendermalefemalegradeiiiiiiivtumor size cmlow n19high n18pvalueexperiments were conducted in accordance with the declaration of helsinkicell culture and treatmentthe normal human astrocyte nha and glioma cell lines were purchased from institute of biochemistry and cell biology of the chinese academy of sciences shanghai china cells were cultured using pmi1640 medium invitrogen carlsbad ca usa supplemented with fetal bovine serum fbs invitrogen shrnas against linc00160 mir6293p mimics mir6293p inhibitors and negative controls were obtained from genepharma and transfected into glioma cells using lipofectamine invitrogen according to the manufacturers instructions efficiency was validated using qrtpcr after hqrtpcrtotal rna was extracted from tissues and cell lines using trizol invitrogen carlsbad ca primescript rt reagent kit rr047a takara holdings inc tokyo japan was used to generate cdna from rna template qpcr was completed through sybr premix ex taq¢ ii takara japan gapdh was the normalized control relative expression was calculated through the δδct methodluciferase reporter assaythe fragment of linc00173 or nutf2 containing indicated mir765 binding site was constructed into pmir report vector for luciferase reporter assay glioma cells were transfected with report vector and mir765 mimics after h the luciferase reporter activity was measured through the dualluciferase reporter assay system promega madison wiwestern blot assaycells were lyzed using radioimmunoprecipitation buffer beyotime shanghai china protein concentration was determined by a bca protein assay kit thermo fisher scientific ma then proteins were separated using sdspage and transferred onto pvdf membranes after blockage using bsa for h the membrane was incubated the primary antibodies at °c overnight after washed times using pbst the membranes were incubated with horseradish peroxidaselabeled second antibody followed by detection the enhanced chemiluminescence reagent emd millipore usathrough submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du cck8 and colony forming assaysproliferation was measured using cck8 and colony formation assay cck8 assay was performed using the cck reagent dojindo kumamoto japan according to the manufacturers instructions and absorbance was determined at nm using a microplate reader biotek winooski vt for colony formation assay cells were seeded into 6well plates and cultured for days then the cells were fixed with methanol and stained with crystal violet for minutesedu assaycells were plated into 96well plates and incubated with edu μl at °c for h followed by detection using facstranswell migration and invasion assaystranswell plates corning ny were used to measure migration and invasion according to the manufacturers instructions in brief cells were suspended into μl serumfree medium and seeded into the upper chamber while the lower chamber was filled with µl of complete medium after incubation for cells in the lower chamber were fixed with methanol and stained with crystal violet for minutes migrated and invaded cells were counted through a light microscopestatistical analysisgraphpad prism graphpad ca usa was used to analyze results data were presented as means±standard deviation sd significant differences were analyzed using students ttest or oneway anova survival rate was analyzed by the kaplanmeier method and log rank test p005 was considered to be significantresultslinc00173 expression is elevated in gliomathe expression of linc00173 was firstly analyzed through qrtpcr we found that linc00173 level was elevated in glioma tissues compared with normal tissues figure 1a besides we found that linc00173 was also upregulated in glioma cell lines compared to nha cells figure 1b then according to the median value of linc00173 glioma tissues were classified into two groups after analysis we found that linc00173 high expression correlated with poor prognosis figure 1ctransfection of linc00173 enhanced glioma cell proliferation migration and invasionto explore the function of linc00173 u87 and u251 cells were chosen after shlinc00173 linc00173 expression was significantly downregulated figure 2a through cck8 assay we observed that linc00173 knockdown suppressed the proliferation capacity of glioma cells figure 2b and c which was validated by edu and colony formation assays figure 2d and e afterwards transwell assay was performed results indicated that linc00173 loss inhibited migration and invasion of glioma cells figure 2f and g thus linc00173 exerted oncogenic roles by affecting proliferation migration and invasionlinc00173 worked as the sponge for mir765linc00173 has been found to serve as cerna for several mirnas such as mir490 and mir2181314 to determine the mechanism of linc00173 in glioma we also figure linc00173 expression is elevated in glioma a the level of linc00173 in glioma tissues was measured b the expression of linc00173 in glioma cell lines and nhas c association between overall survival and linc00173 expression in glioma patients p005cancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 enhanced glioma cell proliferation migration and invasion a qrtpcr analysis of linc00173 expression in u87 and u251 cells be proliferation ability was measured using cck8 edu and colony formation assays f and g migration and invasion capacity was evaluated after linc00173 knockdown in glioma cells p005suppressed the supporting their direct performed bioinformatics analysis using mirdb we identified that mir765 was the most potential candidate because it scored the highest to validate it we constructed luciferase reporters figure 3a followed by luciferase reporter assay results showed that mir765 activity of linc00173wt only figure 3b interaction pulldown assay further demonstrated their interaction figure 3c qrtpcr found that linc00173 overexpression suppressed the level of mir765 figure 3d next bioinformatics analysis using mirdb and targetsan implied that nutf2 is the most potential target of mir765 the corresponding luciferase reporters were further constructed figure 3e luciferase reporter assay also demonstrated the interaction between nutf2 and mir765 figure 3f besides nutf2 expression was suppressed by mir765 mimics figure 3g moreover nutf2 level was decreased after linc00173 knockdown while mir765 inhibitors reversed it figure 3h finally we found that mir765 level was negatively correlated with linc00173 or nutf2 in glioma tissues figure 3i and jlinc00173 promoted glioma progression through mir765nutf2 pathwaywe noticed that nutf2 expression was upregulated in glioma tissues figure 4a and b suggesting an oncogenic role to investigate whether linc00173 regulates glioma progression through mir765nutf2 we restored the expression of nutf2 in shlinc00173 transfected cells cck8 and transwell assays demonstrated that nutf2 restoration successfully rescued the capacities of proliferation migration and invasion in glioma cells transfected with shlinc00173 figure 4cf therefore linc00173 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du figure linc00173 worked as the sponge for mir765 a bioinformatics analysis indicated the binding sites between linc00173 and mir765 b u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter linc00173wt or linc00173mut then relative luciferase activity was determined c rna pulldown assay using biotinlabeled mirnas d relative expression of mir765 after linc00173 knockdown e bioinformatics analysis indicated the binding sites between mir765 and nutf2 f u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter nutf2wt or nutf2mut then relative luciferase activity was determined g qrtpcr analysis for nutf2 expression h western blotting analysis for nutf2 protein level i and j correlation analyses of linc00173 mir765 and nutf2 in glioma tissues using pearsons correlation coefficient p005contributes to glioma progression through mir765nutf2 pathwaydiscussionas the most malignant brain tumor glioma leads to a huge number of deaths patients with glioma display a poor prognosis therefore it is of great significance to reveal the mechanism underlying glioma progression in this study we found that linc00173 was upregulated in glioma tissues and cells linc00173 overexpression predicted a poor prognosis moreover linc00173 knockdown the proliferation migration and invasion of glioma cells linc00173 was also found to inhibit mir765 and promote nutf2 expression summarily our research discovered that linc00173 is an important oncogenic lncrna in gliomasuppressed the potential roles of lncrna in glioma have been researched for a long time many lncrnas have been identified to participate in glioma development for example lncrna nck1as1 enhances growth and metastasis of glioma through targeting mir13823p to activate β catenin signaling2 lncrna ccat2 contributes to glioma progression by activating vegfa pathway15 lncrna linc00467 upregulation promotes glioma development through repressing p53 level16 previous study showed that linc00173 downregulation promotes nonsmall cell lung cancer cell growth and survival17 however another study showed that linc00173 enhances chemoresistance and facilitates tumor progression in small cell lung cancer13 besides linc00173 contributes to breast cancer development14 yet how linc00173 works in glioma remains undermined in our study we found that linc00173 was upregulated in glioma tissues linc00173 knockdown inhibited the proliferation migration and invasion of glioma cells therefore our data discovered that linc00173 is a new oncogene in glioma for the first timecancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 promoted glioma progression through mir765nutf2 pathway a and b nutf2 expression in glioma tissues and normal tissues according to tcga data using gepia tool and qrtpcr c and d proliferation was measured by cck8 assay e and f migration and invasion potential was determined by transwell assay p005lncrna has been found to serve as mirna sponge in tumor cells for instance lncrna ttnas1 sponges to promote breast cancer metastasis18 mir1405p lncrna cdkn2bas1 sponges mir3245p to regulate cellcycle progression in laryngeal squamous cell cancer19 previous studies also revealed linc00173 was a sponge for some mirnas such as mir4903p and mir2181314 in our study we did not observe linc00173 sponges above mirnas however through bioinformatics we identified linc00173 targeted mir765 in glioma we demonstrated their direct interaction and found that linc00173 overexpression inhibited mir765 expression mir765 has important roles in cancers mir765 was found to suppress tongue squamous cell carcinoma development20 mir765 also promotes myeloma and osteosarcoma progression2122 besides mir765 plays oncogenic or anticancer roles in gastric cancer and breast cancer2324 its role in glioma remains unclear our results suggested that mir765 was a tumor suppressor in gliomalncrnamirnamrna regulatory axis is widely observed in cancer for example linc00703mir181a klf6 axis suppresses the development of gastric cancer25 linc00312mir9cdh1 axis was found to promote breast cancer progression26 through bioinformatics we found that mir765 targeted nutf2 in glioma moreover we showed that nutf2 expression was regulated by linc00173mir axis the function of nutf2 in cancer is nearly unknown in our work we found that nutf2 expression was upregulated in glioma tissues compared to normal tissues moreover we found that nutf2 overexpression promoted the proliferation migration and invasion of glioma cells indicating nutf2 was an oncogene in gliomain conclusion our study showed that linc00173 acted as a sponge for mir765 to promote nutf2 expression and linc00173mir765nutf2 axis plays a critical function in promoting glioma progressionfunding this work was supported by zhejiang province analytical testing and experimental animal program lgd19h and zhejiang province welfare technology applied research project 2017c37111 disclosureall authors declare no conflicts of interest in this workreferences ostrom qt cioffi g gittleman h cbtrus statistical report primary brain and other central nervous system tumors diagnosed in the united states in neuro oncol 201921suppl 5v1 v100 101093neuoncnoz150the of glioma huang l li x ye h et al long noncoding rna nck1as1 promotes sponging microrna13823p and activating the trim24wntbetacatenin axis j exp clin cancer res 101186s13046 tumorigenesis through chen w lei c liu p et al progress and prospects of recurrent glioma a recent scientometric analysis of the web of science in world neurosurg 2020134e387e399 101016jwneu20 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du sun b meng m wei j wang s long noncoding rna pvt1 contributes to vascular endothelial cell proliferation via inhibition of mir190a5p in diagnostic biomarker evaluation of chronic heart failure exp ther med 103892etm20208599 feng s yao j chen y functional role of reprogrammingrelated long noncoding rna lincrnaror in glioma j mol neurosci 101007s120310140488z zhang d zhou h liu j mao j long noncoding rna asb16as1 promotes proliferation migration and invasion in glioma cells biomed res int sun l zhao m wang y neuroprotective effects of mir27a against traumatic brain injury via suppressing foxo3amediated neuronal autophagy biochem biophys res commun 101016jbbrc201612001 shi y sun h downregulation of lncrna linc00152 suppresses gastric cancer cell migration and invasion through inhibition of the erkmapk signaling pathway onco targets ther 102147otts217452 li k jiang y xiang x et al long noncoding rna snhg6 promotes the growth and invasion of nonsmall cell lung cancer by downregulating mir1013p thorac cancer wang x gu m ju y zhou j pik3c3 acts as a tumor suppressor in esophageal squamous cell carcinoma and was regulated by mir340 5p med sci monit 202026e920642 1012659msm923909 wei l chen z cheng n microrna126 inhibit viability of colorectal cancer cell by repressing mtor induced apoptosis and autophagy onco targets ther 102147 otts238348 chen y shen z zhi y long noncoding rna ror promotes radioresistance in hepatocellular carcinoma cells by acting as a cerna for microrna145 to regulate rad18 expression arch biochem biophys 101016jabb201803018 zeng f wang q wang s et al linc00173 promotes chemoresistance and progression of small cell lung cancer by sponging mir218 regulate etk expression oncogene to 101038s4138801909842 fan h yuan j li x et al lncrna linc00173 enhances triplenegative breast cancer progression by suppressing mir490 3p expression biomed pharmacother 1010 16jbiopha2020109987 sun sl shu yg tao my lncrna ccat2 promotes angiogenesis in glioma through activation of vegfa signalling by sponging mir424 mol cell biochem 101007 s1101002003712y zhang y jiang x wu z et al long noncoding rna linc00467 promotes glioma progression through inhibiting p53 expression via binding to dnmt1 j cancer 107150 jca41942 yang q tang y tang c diminished linc00173 expression induced mir1825p accumulation promotes cell proliferation migration and apoptosis inhibition via agernfkappab pathway lung cancer am j transl res in nonsmallcell xue j zhang z li x ren q wang q long noncoding rna ttnas1 promotes breast cancer cell migration and invasion via sponging mir1405p oncol lett 1038 92ol201911222 liu f xiao y ma l wang j regulating of cell cycle progression by the lncrna cdkn2bas1mir3245prock1 axis in laryngeal squamous cell cancer int j biol markers 1011771724600819898489 ding j yang c yang s linc00511 interacts with mir765 and modulates tongue squamous cell carcinoma progression by targeting lamc2 j oral pathol med 101111 jop12677 long s long s he h chen g microrna765 is preregulated in multiple myeloma and serves an oncogenic role by directly targeting sox6 exp ther med 103892 etm20197473 lv db zhang jy gao k microrna765 targets mtus1 to promote the progression of osteosarcoma via mediating erkemt pathway eur rev med pharmacol sci 1026355eurrev_201906_18040 jiao y yuan c wu h li x yu j oncogenic microrna765 promotes the growth and metastasis of breast carcinoma by directly targeting ing4 j cell biochem yuan l ma t liu w et al linc00994 promoted invasion and proliferation of gastric cancer cell via regulating mir7653p am j transl res yang h peng m li y zhu r li x qian z linc00703 acts as a tumor suppressor via regulating mir181aklf6 axis in gastric cancer j gastric cancer 105230jgc2019 19e43 chen y qiu f huang l et al long noncoding rna linc00312 regulates breast cancer progression through the mir9cdh1 axis mol med rep 103892mmr201910895cancer management and research publish your work in this journal cancer management and research is an international peerreviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient the manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors dovepress submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalcancer management and research submit your 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"These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown. For example physiologic TF binding studies involve many TFs consequently it is difficult to assign nucleosome reanization to the binding site occupancy of any particular TF. Therefore several aspects remain unclear: does TF binding influence nucleosome (re)anizations locally or impact the chromatin landscape at a more global level; are all or only a fraction of TF binding a result of reanization in nucleosome occupancy and do all TF binding and associated changes in nucleosome occupancy result in altered gene expression? With these in mind following characterization of two states (before and after induction of a single TF of choice) we determined: (i) genomic binding sites of the TF (ii) promoter nucleosome occupancy and (iii) transcriptome profiles. Results demonstrated that promoter-proximal TF binding influenced expression of the target gene when it was coupled to nucleosome repositioning at or close to its binding site in most cases. In contrast only in few cases change in target gene expression was found when TF binding occurred without local nucleosome reanization. cover-date 2 September 2014 INTRODUCTION Although transcription factors (TFs) can recognize cognate binding sites on the nucleosomal surface functional binding is almost always associated with chromatin modification remodeling and finally displacement or compaction of nucleosomes. Specific epigenetic signals determine the site and state of this reanization based on the bound TF leading to cellular differentiation. Modification of the cellular phenotype from a group or community-like (epithelial) state to a more solitary (mesenchymal) form (epithelial to mesenchymal transition EMT) is a basic developmental feature that has been well studied (reviewed in (12)). Metastasis or spreading of cancer from the site of origin is also manifested with enhanced mesenchymal features in tumor cells (34). In this context though gene expression networks for the EMT are well studied regulation of the nucleosomal or chromatin state is poorly understood (5). Though there are several minor variations the overall chromatin architecture of a given transcription unit may be summarized as: a ?1 nucleosome positioned upstream of the transcription start site (TSS) a nucleosome-free region (NFR) followed by a +1 nucleosome downstream of TSS in addition to an array of positioned nucleosomes throughout the gene body (6). This basic anization is stabilized by modified histones or histone variants placed at specific regions within genes (78). anizational change in chromatin such as altered positioning of the +1 and/or ?1 nucleosome with resultant change in the NFR (9) altered inter-nucleosomal spacing due to chromatin compaction (1012) and/or histone variant occupancy near TSS (13) may result in transcriptional response (1416). Together these can modulate accessibility of genomic DNA in vivo toward binding of TFs (6). This model is supported by high-resolution nucleosome maps generated for human promoters by subjecting chromatin to micrococcal nuclease (MNase) and detecting the undigested DNA with tiling arrays (17) or deep sequencing (MNase-Seq) (15). By employing the yeast system nucleosome dynamics during transcriptional changes has been investigated in detail. It was found that nucleosome reanization as a result of physiological perturbations by means of heat-shock induced large-scale transcriptome changes in yeast. However at the nucleosomal level most changes were limited to one or two nucleosomes per promoter (16). Furthermore genome-wide analysis of nucleosome dynamics during meiotic development in yeast revealed dramatic reanization of chromatin (9). A combined analysis of nucleosome architecture transcriptional states and status of DNA binding factors from publicly available data sets in yeast suggested DNA binding factors may control promoter nucleosome architecture (18). Other studies found that nucleosome repositioning events may facilitate TF binding and gene expression upon androgen treatment (19) during differentiation of hematopoietic stem cells to erythrocytes (20) or interferon-beta activation following virus infection (21). Together these suggest a process where nucleosome remodelers general TFs and the transcriptional elongation machinery together orchestrate the nucleosome-positioning pattern in vivo (2224). This understanding prompts further interesting questions regarding both the nature of TF/nucleosome interactions and to what extent this influences transcriptional response. For example in earlier studies induced physiological perturbations activated multiple TFs (1921) it is not clear how unique site-specific TF binding influences nucleosome (re)anizations locally (in close vicinity of their binding sites). Another pertinent question is: do all sites occupied by a TF and associated nucleosome occupancy changes result in altered gene expression? Keeping these in mind we sought to study binding of single TF that would also induce the physiological change. A candidate TF was first identified from analysis of early and advanced lung cancer transcriptome profiles. In vivo binding sites of the TF nucleosome positions and transcriptome profiles of both the metastatic and induced non-metastatic state in lung cancer cells were then determined (see Scheme S1 in Supplementary Information for a summary of the overall design) and using these the correlation between target site occupancy nucleosome reanization and their combined effect on the transcriptome was examined. Results that suggest a model where TF binding coupled with nucleosome reanizations that influence transcription are: (i) almost always associated with nucleosome repositioning that is at close proximity to the TF binding site (TFBS) and (ii) constrained to specific loci and not spread over the whole genome. MATERIALS AND METHODS Chromatin immunoprecipitation and sequencing Chromatin immunoprecipitation (ChIP) assays were performed following the Fast ChIP protocol (25). Briefly two plates were made with 1 105 A549 cells in each on achieving 80% confluency the first plate was transfected with the clone pcDNA3-NME2-Myc to induce non-metastatic 2 (NME2). The second plate was left untreated (un-induced condition). After 48 h ChIP was performed using cells from each plate independently and cells were fixed with 1% formaldehyde for 10 min lysed and sheared (?300 bp) using a Misonix 3000 sonicator. Twenty-five per cent of lysate was used to isolate input chromatin using phenolchloroform and ethanol precipitation. The remaining lysate was pre-cleared using protein-A sepharose beads and further divided into two equal portions: one part was immunoprecipitated using antibody specific to the Myc epitope (clone 9E10 anti-c-Myc monoclonal Sigma) and a negative control of immunoprecipitation was prepared by adding isotypic control (IgG) to the second portion. Immunoprecipitation was done by incubating overnight at 4°C with 2 ?g of antibody. The un-induced experiment was treated in a similar fashion before immunoprecipitation with nm23-h2-L-16 antibody (sc-17587 Santa Cruz Biotechnology Inc.); IgG was used as the respective isotypic control. Immune complexes were collected using herring sperm DNA-saturated protein-A Sepharose washed and eluted using Chelex-100 after proteinase K treatment. Reads were aligned (mapped) to the unmasked human reference genome (NCBI v36 hg18) using the MAQ (Mapping and Assembly with Qualities) (26) after clipping to 24 bp based on quality scores. The sequences were then mapped to the reference human genome (NCBI Build 36 UCSC hg18). Only those that aligned to the reference genome were further considered for peak generation. To accommodate variations relative to the reference genome up to two mismatches were allowed. Commercially available small interfering RNAs (siRNAs) from Dharmacon Inc. USA were used to silence expression of NME2 (transient depletion) in A549 wherever required. Peak generation following ChIP-seq The resulting sequence read distribution was processed with ChIP-Seq peak locator utility CisGenome (26). CisGenome uses a conditional binomial model to identify regions in which the ChIP reads are significantly enriched relative to the control reads. A false discovery rate of 10% was considered while predicting NME2 target regions. In order to filter out low-quality sites we applied two post-processing options boundary refinement and single-strand filtering. Mononucleosome preparation and hybridization to promoter tiling array A549 cells (NME2-induced or -depleted along with respective control cells) were grown in T-75 flasks till 80% confluency in Dulbecco's modified Eagle's medium trypsinized pelleted and washed with ice-cold 1X phosphate buffered saline. Five times the packed pellet volume of 1X hypotonic buffer was added cells homogenized IGEPAL (a non-ionic non-denaturing detergent) was added to final concentration of 0.6% lysate vortexed and centrifuged at 12 000 revolutions per minute. Pellet (nuclei) was resuspended in MNase digestion buffer and incubated on ice. OD (Optical Density) was measured and 1 unit of MNase/OD was added to the suspension. Following incubation at 37ºC for 30 min the reaction was stopped using stop-buffer treated with proteinase K (1 mg/ml) overnight at 37ºC before phenolchloroform purification and ethanol precipitation to pellet down the mononucleosomal DNA. Precipitated DNA was washed with 70% alcohol and dissolved in water. DNase-treated cells were taken as control. Mixture containing 3 ?g of DNA 50 mMTris-Cl 5 mM MgCl2 and 0.3 ?g/?l of random hexamers was incubated at 95ºC for 5 min and chilled to 4ºC. 5 ?l dNTP (1.2 mM dGTP dCTP dATP and 0.25 mM dUTP) and 50 units of Klenow were incubated first at 22ºC for 10 min then 37ºC for 30 min and finally 95ºC for 5 min and chilled as above. Fifty units of Klenow were added again and the mix first kept at 22ºC for 10 min and then 37ºC for 30 min. Reaction was stopped by phenolchloroform followed by purification using Qiagen columns. Purified product was fragmented and labeled as per Affymetrix Chromatin Immunoprecipitation Assay protocol before hybridization to Gene Chip Human Promoter Tiling array 1.0R (Affymetrix) as per manufacturer's protocol. The control DNase-treated cells were also subjected to labeling reactions as above before hybridization. Nucleosome positions were identified using iChIP (Bioconductor package). Nucleosome occupancy analysis A549 cells depleted for NME2 were generated using commercially available short hairpin RNAs (from Origene Inc. USA; catalog no. TR311160) and stable cell clones were selected in presence of puromycin. For analysis of nucleosome repositioning we considered a distance of 300 bp (+/? 150 bases) in other words a nucleosome was denoted as repositioned in the NME2-induced condition when detected beyond 300 bp of a nucleosome found in the cells before NME2 induction. In order to avoid arbitrary assignments in cases where a gene belonged to more than one category (while assigning NME2 nucleosome associations during repositioning analysis (Figure 5)) it was considered in all the respective cases. Figure 1. NME2 binding sites before or after inducing NME2. (A) A Circos plot showing ChIP-seq peak distribution of NME2 on all chromosomes (122 and X and Y); peaks in red represent NME2 binding sites before induction; peaks in blue represent NME2 binding sites after induction (replicates are shown in both cases). Two projections from chromosomes 9 and 15 are shown. (B) Twelve-mer consensus NME2 binding motif identified using Gibbs sampler (upper panel)distribution of the 12-mer motif within NME2 peaks constructed from read counts is shown in the lower panel. Figure 2. Nucleosome positions detected in A549 cells before and after inducing NME2. (A) UCSC browser representation of nucleosome positions with corresponding probe intensity at two loci on chromosome 12. (B) Nucleosome occupancy around TSS and expression level of corresponding genes in cells before (left panels) and after NME2 induction (right panels); gene expression was normalized within respective cases by z-transformation with respect to the mean expression level of the data sets. (C) Frequency of occurrence and location of nucleosomes around TSS. ?1123 +4 denote sequential presence of nucleosomes with respect to their occurrence from TSS; percentage of total number of nucleosomes found in respective cases before or after induction of NME2. (D) Left panel: distribution of repositioned nucleosomes around TSSs in NME2-induced cells relative to the status in cells before NME2 was induced; number of repositioned nucleosomes in a window size of 100 bp is shown on top. Right panel: expression of corresponding genes shown in triplicate before and after NME2 induction. Figure 3. Nucleosome depletion and NME2 occupancy. (A) Schematic representation of possible relationship between nucleosome positions before and after inducing NME2. (B) Nucleosome occupancy is depleted on or near NME2 binding sites on inducing NME2 relative to the condition before NME2 induction. Ratio of number of nucleosomes detected after/before NME2 induction in 300 bp windows is shown; x-axis denotes the distance of nucleosomes from the nearest NME2 binding site in NME2-induced cells. (C) Schematic representation of nucleosome shift between two conditions was represented by ?Ndisplacement (left panel). Percentage of shifted nucleosomes plotted for a given ?Ndisplacement is shown in the right panel (x-axis was plotted to indicate: no shift shift in 100 bp windows and shift exceeding 300 bases). Distribution of the nucleosome shift was also found for the NME2-depleted condition minus A549 cells; significance of the difference in distributions was tested using the Wilcoxon rank sum test (P = 0.00016). (D) Position of the nearest nucleosome with respect to NME2 binding sites in cells before (green) or after inducing NME2 (red; left panels); 791 genes where the nearest nucleosome was within 300 bp and shifted in the NME2-induced condition are shown. Expression level of corresponding genes in triplicate before or after NME2 induction is shown in the right panels. Figure 4. Validation by quantitative real-time PCR (Polymerase Chain Reaction) for NME2 binding and nucleosome occupancy. Nucleosome positions and NME2 binding sites are shown before and after NME2 induction at six different loci in the left panel (genes shown in UCSC browser representation); right panel shows validation at the corresponding loci by quantitative real-time PCR for NME2 binding and nucleosome occupancy in cells before and after inducing NME2. Relative fold change is shown on x-axis. Experiments were performed in triplicate; error bars are for standard deviation (* and ** represent P < 0.05 and P < 0.01 respectively). Figure 5. TF binding and nucleosome repositioning models analyzed in this study. Nucleosome shift in the vicinity of TF binding results in repositioning of the ?1 nucleosome (upper panel) whereas co-occupancy (center panel) or target site binding that has no associated nucleosome repositioning in close vicinity either before or after induction of the TF (lower panel) shows no significant change in ?1 or +1 nucleosome positions. Percentage of nucleosomes is based on genome-wide total for respective cases; to avoid arbitrary assignment genes were considered in more than one category where applicable. Quantitative real-time PCR for validating ChIP-nucleosome positioning For validating individual nucleosome positions quantitative real-time PCRs were performed using ChIPped DNA on ABI's (Applied Biosystems Inc.) 7500 fast platform. Equal concentration of ChIP DNA was taken from the test (from NME2-induced cells) and control (un-induced) samples. The results were analyzed using comparative computed tomography method. Primers were designed against the obtained peak region using ABI's Primer Express software (Supplementary Table S1). Western blotting Western blot for NME2 was done using antibody from Abcam Cambridge MA USA (catalog no. ab60602). "
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" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the eï¬cacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInï¬ammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the global Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationï¬stulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperï¬cialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of years old [] therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societyMoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more eï¬cient at reï¬ecting disease activity []Some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The eï¬cacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the ï¬rst and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study In another study published in AUC values of CI were reported for fecal calprotectin in thediagnosis of colorectal ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and speciï¬city [] Caviglia et al in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with Crohn's disease Asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE ï¬ndings anddiagnosis of Crohn's disease [] In another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren yearsChildren yearsAdultsOver years Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in Crohn's disease [] Given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the ï¬rst studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled Speciï¬cityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and speciï¬city In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modiï¬edBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpeciï¬city CD and UC CD and UC CD and UC and unclassiï¬ed68CD and UC CD and UC and unclassiï¬ed CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSreï¬dbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoeï¬cientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland Modiï¬edCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also conï¬rmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thediï¬culty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRoberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Roberts scoring system [] Theede et al also used themodiï¬ed Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 Sensitivity Speciï¬city andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes
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"assess the antioxidative activity of seleniumenriched ChrysomyiaMegacephala Fabricius C megacephala larvae powder SCML and its impact on the diversity and structure ofintestinal microflora in a mouse model of Dgalactose Dgalinduced oxidative damageMethods Sixty male ICR mice were equally randomized to a normal control NC group a model group a positivegroup a lowdose SCML LSCML group a middose SCML MSCML group and a highdose SCML HSCMLgroup Animals in NC and model groups received water animals in the positive group received mgKg vitamin EVE and those in the three SCML groups received SCML which include and μgKg selenium Serespectively An oxidative damage model induced by subcutaneous injection of Dgal for weeks via the neck wasestablished Serum oxidative stress levels and tissue appearance were evaluated Tissues oxidative stress levels weredetected by commercially available kit Nuclear erythroid 2related factor Nrf2 and gut microbiota weredetermined by western blot and high throughput sequencing 16S rRNA gene respectivelyResults An oxidative damage model was established successfully as represented by a significant elevation ofmalondialdehyde MDA and protein carbonylation and inhibition of the antioxidants including superoxide dismutaseSOD glutathione peroxidase GSHPx total antioxidant capacity TAOC and glutathione GSH It was found thatoxidative damage and histological alterations were attenuated the expression of Kelchlike ECHassociated proteinKeap1 was decreased and the expression of Nrf2 and hemeoxygenase1 HO1 was increased after SCML treatmentIn addition significant changes were observed in the gut microbiota including Proteobacteria and the ratio ofBacteroidetes to Firmicutes at the phylum level as well as Helicobacter Clostridium and Lactobacillus at the genus levelContinued on next page Correspondence jiangzcmueducn Dandan Xie and Liqin Jiang contributed equally to this work1College of Pharmaceutical Science Zhejiang Chinese Medical University Binwen Road Hangzhou ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cXie BMC Complementary Medicine and Therapies Page of Continued from previous pageConclusion SCML exerted an antioxidative effect in vivo probably by increasing the antioxidant activity and reducingthe production of oxidation products via the Nrf2 signaling pathway SCML could also redress the intestinal floraimbalance induced by oxidative stress All these findings suggest that SCML could serve as a functional food andnatural drug additive to protect the human body against oxidative damageKeywords Seleniumenriched Chrysomyia Megacephala Fabricius larvae powder SCML Antioxidant activity Nrf2Intestinal microbiota In vivoBackgroundAging is a natural process that involves the gradual loss ofphysiological functions causing enhanced morbidity andmortality due to various diseases This process is closelyrelated to oxidative stress [] One prevalent theory toexplain aging is the theory of the oxygen free radical []This theory posits that the macromolecules such as nucleic acids lipids sugars and proteins that make up cellsand tissues are subjected to oxidative stress induced bysuperoxide and other free radicals These macromoleculesthen undergo different degrees of oxidation which initiates oxidative damages and ultimately leads to an function impairment and aging [ ] Changes in the level ofoxidative stress affect the microbial environment in the intestine and lead to intestinal flora disorder [] Disorderedintestinal flora may affect the antioxidant activity and lipidmetabolism [] Hence it may be possible to inhibit oxidative stress by regulating the composition and structure ofthe gut flora To prevent oxidative stressassociated cellular damage it is therefore important to keep prooxidantantioxidant balance by supplementation or induction ofcellular antioxidants A high dose of dgalactose is converted to aldose and hydrogen peroxide by dgalactoseoxidase The products then generate reactive oxygen species through oxidative metabolism and glycosylation leading to oxidative stress The accumulation of oxidationproducts further exacerbates the oxidative damage to tissues and cells which then accelerates the aging process[] Therefore dgalactose overload has been used to establish animal models used to conduct aging related metabolic dysfunction and oxidative stress [ ]Selenium Se is an essential trace element for humanbody and other animals The role of Se is reported to beclosely associated with antioxidant activityimmune response and chemoprevention [] Se is mainly presentin the active site of enzymes in the form of selenocysteineMultiple Secontaining proteins such as GSHPx and thioredoxin reductase play important roles in preventing oxidativeimportance of Sesupplementation in boosting up the internal antioxidativedefense has been highlighted in recent years Studies haveshown that anic Se supplements can improve tissue Sedeposition antioxidant level and gene expression whereasSe deficiency may result in cardiac muscular osseous and[] Thereforeinjurytheimmune disturbances [ ] Therefore the healthrelatedbenefits of Se including the type of selenium supplementsand optimal dosage remain to be exploredThe importance of Se has inspired researchers to usebioenrichment to prepare high Se compounds [ ] Cmegacephala larvae is a traditional Chinese medicine witha wide range of pharmacological actions including antioxidant antibacterial and antiinflammatory activitieswhich has been widely applied in agriculture and medicine[] Seenriched C megacephala larvae SCML isgenerated from C megacephala larvae by biological transformation and enrichment of Se Our previous workshowed that SCML was an effective anic Se sourcewith low toxicity and high Se content [] Yet no studyhas reported the antioxidant activity of SCML in vivo andits impact on the gut microbiota which is susceptible toundergo alterations under oxidative stressThe objective of the present study was to evaluate theantioxidant activity of SCML in vivo explore the underlying mechanism as well as evaluate its impact on thegut microbial diversity and structure hoping that the results could provide a scientific basis for a comprehensiveutilization of SCMLMethodsMaterials and chemicalsSCML was provided by Beijing Ershang Biological Technology Co Ltd Beijing China Vitamin E was purchasedfrom Archer Daniels Midland Dictor USA DgalactoseDgal of ¥ purity was purchased from Aladdin Industrial Corporation Shanghai China GSHPx SOD TAOC GSH MDA and protein carbonyl assay kits werepurchased from Nanjin Jiancheng Bioengineering InstituteNanjin China RNA trizol reagent and FastStart Universal SYBR Green Master Rox were purchased from Servicebio Wuhan China The primers for Nrf2 SOD1GSHPx and GAPDH were synthesized and purified byWuhan Servicebio Technology Co LTD Wuhan ChinaThe kits for Revert Aid First Strand cDNA synthesis andHyPure¢Molecular Biology Grade Water were purchasedfrom Thermo Waltham USA and HyClone LoganUSA respectively Keap1 Nrf2 and HO1 polyclonal antibodies were obtained from Proteintech Chicago USARIPA Actin bicinchoninic acid BCA assay kit 0cXie BMC Complementary Medicine and Therapies Page of Western Lightening¢ PlusECL Enhanced chemiluminescence substrate assay kit and the secondary goat antimouse horseradish peroxides HRP were from ServicebioWuhan China All other chemicals and reagents used inthe study were of analytical grade Water used in the experiments was ultrapureDetermination of the compositions of SCMLCompositions of SCML including protein crude fat andmoisture content were analyzed according to methodGB5009 of China National Food Safety StandardSe content was detected by Inductively Coupled PlasmaICP according to Vu with minor modifications[] The results are shown in Table Animal experimentsSixty ICR male mice aged weeks and weighing ± gwere purchased from SinoBritish SIPPRBK Lab Animal Ltd Approval No SCXK HU Theanimal experiments were performed in accordance withthe guidelines of the Laboratory Animal Center of Zhejiang Chinese Medical University Permit No SYSKZHE Allthe experimental procedureswere strictly conducted according to the internationalstandards and nationallegislation on animal care anduse The mice were kept under controlled light conditions h lightdark cycle with free access to food andwater normal light circadian rhythm and 7day adaptivefeeding in a quiet environmentAfter oneweek acclimatization mice were equallyrandomized to six groups normal controlNCgroup model group positive group receiving mgKg·d vitamin E VE group lowdose SCML LSCML group receiving SCML μgKg·d Se middose SCML MSCML group receiving SCML μgKg·d Se and highdose SCML HSCML group receiving SCML μgKg·d Se Except for the mice inNC group animals in the other five groups were givensubcutaneous injection of mgKg·d Dgal for weeksinto the neck to prepare oxidative stress model Animalsin NC and model groups received water and animals inthe other groups as previously described received VE orSCML by intragastric gavage for weeks The experiments were conducted at A certain amountof SCML and gellan gum were weighed precisely anddissolved in purified water heated slightly to a suspension There were three different concentrations and μgmL Se Meanwhile VE was dissolved in purified water containing gellan gum which became aTable Compositions of SCMLsuspension mgmL Dgal was dissolved in physiological saline mgmLThe mice were weighed throughout the experimentThe appearance appetite mental condition and behavioral activity of the mice during the experiment werealso observed and recorded Stool samples were collected at weeks after treatment Blood samples wereobtained from the retrobulbar venous plexus at weeksafter treatment The mice were sacrificed by cervical dislocation and the liver kidney heart brain and caecumwere stripped The dissected ans were divided twoparts one for histological analysis and the other for biochemistry analysis Samples for analysis were thawed onice homogenized with mL cold buffer mM potassium phosphate with mM EDTA pH per gram oftissue and centrifuged at Ãg for min at °CThe supernatants were collected for analysisAnalysis of serum oxidative stress indexesSerum oxidative stress indexes GSHPx SOD and MDAwere determined by using the respective commercial kitsaccording to the manufacturers instructionsAnalysis of tissue oxidative stress indexesThe oxidative stress indexes were determined by measuring GSHPx SOD TAOC GSH MDA and proteincarbonylation ofthe tissue homogenate supernatantusing the commercial kits according to the manufacturers instructionsHistological analysisFor histological analysis the animal tissues were fixed in paraformaldehyde for h dehydrated in alcoholparaffin embedded sliced into μm thick sectionsstained with hematoxylineosin HE and finally photographed under a microscope à objective lensRNA extraction and realtime quantitative PCRexperimentsTotal RNA was extracted from the liver and kidney tissues using Trizol reagent RNA was reverse transcribedinto cDNA using RevertAid First Strand cDNA SynthesisKit Realtime quantitative PCR qRTPCR was performed using FastStart Universal SYBR Green MasterRox and the ABI7900Faxt Sequence Detection systemThe thermal cycle condition was cycle at °C for min followed by cycles of amplification at °C for s and then °C for 30s And the dissolution curvestarted from °C then ascending to °C at °C15ContentProtein g100 gCrude Fat g100 gMoisture g100 gSe μggSCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of s All samples were run in triplicate in each experimentValues were normalized to that for GAPDH The sequences of the primers used are shown in Table Theresults were calculated by using the 2ÎÎCT methodliverandtissueskidneyWestern blot analysisTotal protein and nuclear protein were extracted from mgusing RadioImmunoprecipitation Assay RIPA lysis solution and anuclearcytoplasm protein extraction kit The concentrations of protein lysates were quantified using a BCA protein kit Samples containing an equal amount of protein μg were mixed with the loading buffer containing 2mercaptoethanol heated for min at °C andloaded onto a SDSPAGE gel The proteins fromthe electrophoresing gel were then transferred ontopolyvinylidenethenblocked with milk and Tween in Trisbuffered saline and incubated overnight at °C withantiKeap1 antiNrf2 antiHO1 and actin Then the appropriate horseradish peroxideconjugated secondary antibody wasadded to the membranes at room temperature Finallythe proteins were detected with chemiluminescent substrate Gray semiquantitative analysis was performed byImage J The protein bands were quantified using densitometry Values are expressed as the fold change withrespect to betaactindifluoride membranes whichIntestinal microbiota analysisThe stool samples were sent to BGI Co Ltd WuhanChina for sequencing of the 16S rRNA gene Total genomic DNA of the gut microbiome was extracted and theV3V4 region of the 16S rRNA gene from the sample wassubjected to PCR amplification After normalization of thegenome DNA to ng per PCR reaction V3V4 dualindex fusion PCR primer cocktail and PCR master mixwere added and then a PCR was performed The PCRproducts were purified with Agencourt AMPure XP beadsto remove the unspecific products Pairedend sequencingTable Primers for realtime PCR analysesAccession NoGeneNrf2NM_0109023SOD1GSHPxGAPDHNM_0114341NM_0081606NM_0080842was performed on the Illumina Hiseq platform and theobtained data were subjected to bioinformatics analysisTo obtain clean reads the clean pairedend reads withoverlap were merged to tags using FLASH fast lengthadjustment of short reads v1211 Then the tags wereclustered to operational taxonomic units OTUs at sequence similarity by scripts of software USEARCHv701090 The RDP classifier v22 was used to compare OTUs with the database to comment on the OTUsspecies Finally intestinal microbial diversity and structure were analyzed based on OTUs and taxonomic ranksusing software R v311Statistical analysisAll data are expressed as the means ± SD or means ± SEand analyzed using Statistical Analysis Software SPSS The experimental values were analyzed by oneway ANOVA followed by the Duncans multiplerangetests and Pvalue were considered to be statistically significantResultsEffects of SCML on daily behavior and weight gain inmiceUsual performance of the mice was observed and recorded and no abnormal phenomenon found duringthe experimentincluding antifeeding and vomitingSymptoms such as slow movement and listlessnesswere obviously observed in model groupindicatingthat the oxidative stress model induced by subcutaneous injection of Dgal was successfully establishedHowever the above symptoms receded in varying degrees in VE and SCML groups The weight gain ofthe mice is exhibited in Fig Compared with NCgroup body weight of the mice in model group significantly increased slowly P and increasedsteadily in drug treatment groups MSCML μgKg Se group showed a significant difference compared to the model group P Primer Sequences CTGGCTGATACTACCGCTGTTCAGGTGGGATTTGAGTCTAAGGAGATGTGACTGCTGGAAAGGACGCGCAATCCCAATCACTCCACCCAGGAGAATGGCAAGAATGAGGAAGGTAAAGAGCGGGTGACCTCGTCCCGTAGACAAAATGTGAGGTCAATGAAGGGGTCGTProduct Sizebp bp bp bp bp 0cXie BMC Complementary Medicine and Therapies Page of tissues were decreased significantly compared to NCgroup P except for SOD in the heart as well asGSHPx in the liver and brain After administration ofVE or SCML the activity of GSHPx and SOD as wellas the content of TAOC and GSH were increased gradually As shown in Fig 3a the activity of GSHPx in thekidney and heart was increased significantly comparedto model group P except for the heart in VEgroup and LSCML μgKg Se group The activityof GSHPx in the liver and brain remained unchangedsignificantly compared to model group except for VEgroup in the liver As shown in Fig 3b the activity ofSOD in the kidney and brain was increased significantlycompared to model group P except for the brainin LSCML μgKg Se group However the activityof SOD in the liver and heart remained unchanged significantly compared to model group except for the liverin VE group As seen in Fig 3c the content of TAOCin the liver kidney and heart was increased significantlycompared to the model group P except for theliver in LSCML μgKg Se group The content ofTAOC in the brain was not significantly altered compared to model group except for VE group As shownin Fig 3d the content of GSH in liver kidney and brainof VE group and in the kidney of HSCML μgKgSe and MSCML μgKg Se groups was increasedsignificantly compared to model group P As shown in Fig 3e the MDA level in model group wasincreased significantly compared to NC group P and decreased significantly after VE or SCML treatmentcompared to model group P except for LSCML μgKg Se group in the kidney In Fig 3f the proteincarbonylation level in the liver kidney and brain of modelgroup was increased significantly compared to NC groupP However the level decreased significantly inthe mice treated with SCML or VE except for in the liverand brain of LSCML μgKg Se group compared tomodel group P And compared with model groupFig Percentage of weight gain in mice at weeks Valuesrepresent means ± SD n and evaluated by oneway ANOVAfollowed by the Duncans multiplerange tests Compared with NCP Compared with Model P SCML SeleniumenrichedC megacephala larvae powderEffects of SCML on serum oxidative stress indexes in miceAs shown in Fig the serum antioxidative enzyme activities in model group were decreased significantly andthe MDA content was increased significantly comparedto NC group P As shown in Fig 2a the GSHPxactivities in animals treated with SCML or VE were increased significantly P As shown in Fig 2b theactivities of SOD in MSCML μgKg Se and VEgroups were significantly increased compared to modelgroup P As shown in Fig 2c the MDA levels inanimals treated with SCML or VE were decreased significantly P Effects of SCML on tissue oxidative stress indexes in miceAs illustrated in Fig after 6week subcutaneous injection of Dgal the activity of the antioxidative enzymesand the content of the antioxidants in different miceFig Oxidative stress level indexes of the mice serum a GSHPx activity in the mice serum b SOD activity in the mice serum c MDA content inthe mice serum Values represent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by theDuncans multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig Oxidative stress indexes of the mice tissue a GSHPx activity in the mice tissue b SOD activity in the mice tissue c TAOC content in themice tissue d GSH content in the mice tissue e MDA content in the mice tissue f protein carbonylation content in the mice tissue Valuesrepresent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by the Duncans multiplerangetests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powderFig Optical micrographs of mice tissue sections HE staining à Black arrow derangement of hepatic cord cells Red arrow infiltration ofinflammatory cells White arrow pyknosis Blue arrow cavitation and deformation Orange arrow atrophy and breakage of the villus Yellow arrowthinning of the intestinal wall Scale bar50 μm SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of HSCML μgKg Se group in heart also significantlydecreased in protein carbonylation levelEffects of SCML on histopathological changes in miceThe histopathological results are shown in Fig Normal histological architectures were observed in the tissuesections in NC group However the liver tissue sectionsin model group showed that the number of double nuclei was increased the hepatic cords were disarrangedliver cells expanded widely and infiltration oflargenumbers of inflammatory cells was observed Comparedto NC group kidney histopathology in model groupshowed that the glomeruli became atrophic or even disappeared the number of epithelial cells was reduced therenal proximal tubules were dilated Histologically theheart tissue was seen abnormally structured in modelgroupincluding cavitation and deformation in somemyocardial cells nuclear pyknosis and inflammatory cellinfiltration In model group the brain tissue was alsoseen abnormally structured including nuclear pyknosisand incomplete dissolution of nerve fibers The caecallesions including atrophy and breakage of the villus irregular cell arrangement and thinning of the intestinalwall were observed in model group SCML or VE treatment significantly attenuated these abnormal histologicalchanges of the tissues induced by DgalEffects of SCML on oxidative stress gene expression inmiceThe Nrf2 pathway maintains the redox homeostasis exerts antioxidant activity by regulating its multiple downstream cytoprotective genes thereby plays a vital role incell survival The effect of SCML on oxidative stressgene expression is shown in Fig As shown in Fig 5athe Nrf2 expression in model group in liver was lowerthan that of NC group P Except for LSCML μgKg Se group in the liver the Nrf2 expression in liverwas increased all other drug treatment groups comparedwith model group P The Nrf2 expression was increased in the kidney of in HSCML μgKg Se groupcompared to model group P As shown in Fig 5bthe expression of GSHPx mRNA in the liver of modelgroup was decreased P After SCML treatment theGSHPx mRNA expression in the liver was significantly increased compare to model group P and MSCML μgKg group and HSCML μgKg Se group inkidney was increased significantly compared to model groupP As shown in Fig 5c the expression of SOD1mRNA was decreased in model group especially in the kidney compared to NC group P However the expression was obviously increased in the liver of HSCML group μgKg Se and MSCML group μgKg Se compared to model group P Significant change was alsoobserved in SOD1 mRNA expression in the kidney of HSCML group μgKg Se and MSCML μgKgSe group compared to model group P Effects of SCML on oxidative stress protein expression inmiceTo determine whether Nrf2 activation played a role inSCML protection against Dgal induced oxidative stressthe expression of Keap1 Nrf2 and HO1 in the mouseliver and kidney was detected As shown in Fig compared with NC group the western blot results showedthat the Nrf2 and HO1 protein expression in modelgroup was significantly decreased P while theKeap1 protein expression was increased in model groupAfter SCML or VE treatment the Keap1 expression inthe treatment groups was decreased though the difference was not statistically significant Compared withmodel groupthe Nrf2 expression in the treatmentgroups was increased significantly P except forLSCML μgKg Se group in liver Compared withmodel group the HO1 expression in SCML groups wasincreased especially in the liver of HSCML μgKgSe group P Sequencing depth and diversityA total of sequences from all intestinal microbiota samples were produced averaging sequencesFig The effect of SCML on the expression of Nrf2 SOD1 and GSHPx mRNA in the liver and kidney of the mice a Nrf2 mRNA relativeexpression in the liver and kidney b GSHPx mRNA relative expression in the liver and kidney c SOD1 mRNA relative expression in the liver andkidney Values represent means ± SD from three independent replicates and evaluated by oneway ANOVA followed by the Duncans multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig The effect of SCML on the protein expression of Keap1 Nrf2 and HO1 in the liver and kidney tissue of the mice a Protein strip b Keap1actin relative density c Nrf2actin relative density d HO1actin relative density Values represent means ± SD from three independentreplicates and evaluated by oneway ANOVA followed by the Duncans multiplerange tests Compared with NC P Compared with ModelP SCML Seleniumenriched C megacephala larvae powderclusterper sample These sequences resulted in a mean sequencelength of approximately bp Based on the Clean Tagstheanalysis was processed by USEARCHv701090 The sequences were delineated into operational taxonomic units OTUs at similarity Thevalue of coverage for the observed OTUs was above The species accumulation curves showed clear asymptotes and the curve tended to be flat or reached theplateau stage Fig 7a indicating a nearcomplete sampling of intestinal microbial communities of mice Theboxplot of Shannon index showed that the diversity of theintestinal microbiota was decreased in model group compared to NC group and the diversity of VE group and HSCML μgKg Se group was increased compared tomodel group Fig 7b As shown in Fig 7c the contribution value of PC1 and PC2 for the sample difference was and respectively All intestinal microbiotasamples were presented as three distinct groups Thesefindings indicate that the main components of the intestinal microbiota in model group were different from thoseFig Alpha diversity of the gut microbiota and principal component analysis PCA plots based on abundance of operational taxonomic unitsOTUs a Species accumulation curves b Bacterial diversity estimated by the Shannon index c PCA plots SCML Seleniumenriched Cmegacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of in NC group After VE and SCML treatment the components of the intestinal microbiota were different fromthose in model group while there was an insignificant difference between HSCML μgKg Se group and NCgroup BacteroidetesEffects of SCML on species structures in miceThe species profiling histogram was obtained to knowthe community structural composition of differentgroups at phylum and genus levels Fig As shown inFig 8a the most prevalent phyla in all samples were Firmicutesand Proteobacteria There were otherphyla level bacteria with low abundance in the intestinaltract of mice As shown in Fig 8b species were usedto describe the relative abundance ofthe intestinalmicrobiota at the genus level showing that Prevotella Helicobacter and Clostridium were the most abundant followed byOscillospira Bacteroides andLactobacillus Effects of SCML on intestinal bacteria of differentclassification levels in miceAs shown in Table Proteobacteria were increased significantly at the phylum level in model group comparedto NC group P Proteobacteria were decreasedsignificantly and Bacteroidetes were increased significantly in VE and SCML groups compared to modelgroup P In addition VE group MSCML μgKg Se group and HSCML μgKg Segroup showed significant differences in Firmicutes compared to model group P and MSCML μgKg Se group showed a significant difference in Actinobacteria compared to model group P As shown in Table there was not significant alteration in Bacteroides Lactobacillus Oscillospira Prevotella and Sutterella at the genus level in model groupcompared to NC group Compared with NC group Helicobacter and Clostridium were increased significantlyand Ruminococcus was decreased significantly in modelgroup P Clostridium Helicobacter and Oscillospira were decreased significantly in VE and SCMLgroups compared to model group P while VEgroup and LSCML μgKg Se group showed a significant difference in Bacteroides P In additionLactobacillus in MSCML μgKg Se group and HSCML μgKg Se group Prevotella in VE groupand Sutterella in MSCML μgKg Se group wereall increased significantly compared to model group P There were not significant alterations in Ruminococcus in VE and all SCML groups compared to modelgroupCorrelation analysis of changes in flora abundance andserum biochemical indexesIn order to explain the relationship between the intestinal flora abundance changes of mice and serum biochemicalindexes Spearman correlation analysis wasperformed to analyze correlation between serum biochemical indexes and Clostridium and Helicobacter theabundance of which were significant difference in eachgroup The change of Clostridium abundance was foundto be negatively correlated with GSHPx and SOD andpositively correlated with MDA There was not significant correlation in Helicobacter and serum biochemicalindexes The specific correlation analysis results wereshown in the Table DiscussionThe results of the present study showed that the dailybehaviors of the mice in model group were differentfrom those of the mice in NC group In addition the tissues of the modeled mice underwent significant pathological changes The antioxidant system parametersincluding GSHPx SOD TAOC and GSH in the antissues or serum were decreased while the MDA andcarbonylated protein levels were increased All these results indicated that the Dgalinduced oxidation mousemodel was successfully established in the present studyVE the monomer of which is often used as the positivecontrol for the studies of aging in mice induced by DgalFig Taxonomic composition of the gut microbiome in the mice a Phylumlevel b Specieslevel SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Table Oneway ANOVA test of species differences at the phylum and species level BacteriaNCModelVEPhylumBacteroidetes ± Proteobacteria ± Firmicutes ± GenusActinobacteriaTenericutesBacteroidesClostridiumHelicobacterLactobacillusOscillospiraPrevotellaRuminococcus ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± SCML μgKg Se ± ± ± ± ± ± ± ± ± ± ± ± Sutterella ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Values represent means ± SE n and evaluated by oneway ANOVA followed by the Duncans multiplerange tests Compared with NC P Comparedwith Model P SCML Seleniumenriched C megacephala larvae powder ± ± ± ± [ ] Studiesshowed that mice subcutaneouslyinjected with Dgal in the neck exhibited a significantbody weight declined [] In this study Dgal was foundto significantly inhibit weight gain in mice howeverSCML and VE could increase the body mass in varyingdegrees indicating that SCML and VE could effectivelyenhance the constitution of aging mice Oxidative damage appears in body ans to a large extent Our resultsshowed that Dgalinjection for weeks for mice resulted in severe histopathological changes in the antissues However SCML and VE could alleviate these Dgalinduced pathological damages in an tissues ofmice Recent research work has demonstrated that senescent cells accumulated in various tissues of age anddisease [] Cellular senescence is associated with agerelated phenotypes causally and decreasing senescentcells can retard tissue dysfunction and extend healthspan[] The results suggested SCML c
| 2 |
"ammatory bowel disease ibd is a long life disease with remission and relapse periods ibd arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers in addition ibd has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] ulcerative colitisuc and crohn's diseases cd are known as two main forms of ibdaccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized in this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way for example 5asa which is acommon drug in the treatment of ibd is less eï¬ective on maintainingremission in cd patients on the other hand antibiotic therapy is notrecommended for the treatment uc but it can be eï¬ective on cd patients diï¬erential diagnosis is a serious challenge because cdand uc have significant similarities in terms of their clinical endoscopic and histological features however there are some diï¬erencesbetween uc and cd which are summarized in table1 in addition tointestinal complications uc and cd also have significant extraintestinal manifestations for example it was shown that uc is significantly associated with primary sclerosing cholangitis and cd is alsoassociated with cholelithiasis especially in cases that the ileum is involved furthermore cd can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections both cd and uc can cause several disorderssuch as arthritis erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of ibd the latest statistics showed that the global corresponding author at department of clinical biochemistry and laboratory medicine faculty of medicine tabriz university of medical sciences daneshgahstreet po box tabriz iranemail address vagharimtbzmedacir m vagharitabari101016jcca202008025received july received in revised form august accepted august available online august elsevier bv all rights reserved 0cf khakikhatibi table1clinical endoscopic and histological features of cd and ucclinical featuresfeaturesrectal bleedingabdominal painfevermucus defectionintestinal obstructionperineal diseasepostoperative recurrenceasca positiveanca positiveendoscopic featurescdoccasionallyfrequentlyfrequentlyoccasionallyyesyesyesfrequentlynot commonucfrequentlyoccasionallynot commonfrequentlynonononot commonfrequentlyfeaturescduclocationmucosal involvementdepth of ulcerationï¬stulacobblestone appearanceaphthous ulcerationmucosal friabilityhistological featuresfeaturesgranulomascrypt abscessespatchinessany part of gi tractdiscontinuousdeepyesyesfrequentlynot commoncdfrequentlynot commonfrequentlycolon and rectumcontinuoussuperï¬cialnonooccasionallyfrequentlyucrarefrequentlynot commonprevalence of ibd currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem according to areport published in ibd has the highest prevalence rate ineurope and its prevalence in the newly industrialized countries of asiaafrica and south america also appears to be increased over the pastthree decades unfortunately the peak of the disease is at the young age of years old therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societymoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease the invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage ibd which are unpleasant for patients as well as having thehigh associated costs now the gold standard method for diagnosingibd and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofibd a high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring ibd patients so in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of ibd the role of fecal calprotectin in diagnosis and management ofibdthe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of ibd diagnosis and management have been studied including ibd diï¬erentiation from irritable bowel syndrome ibs evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andclinica chimica acta response to treatment in following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas calprotectin a clinically valuable proteincalprotectin is an antimicrobial protein mainly secreted by neutrophils this protein competes with bacteria over zinc thus kills thebacteria however this is not the only contribution that it has to antimicrobial activity moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsserum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection at theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints in addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker in neonatal sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of neonatal sepsis it has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low crp levels so they appear to be more eï¬cient at reï¬ecting disease activity some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof ibd impressive results have been reported a study onpatients with ibd both uc and cd have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in ibd diagnosis compared to crp and albumin this studyalso indicated that the combination of serum calprotectin with crp oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with cd however no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with cd and uc as well as a slight correlation betweenserum calprotectin level and crp that was observed only in patientswith uc another study showed that the serum level of calprotectin was significantly higher in patients with cd compared to healthyindividuals in addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease the eï¬cacy of salivary calprotectin in the diagnosisof ibd has also been studied which showed that salivary calprotectinsignificantly increased in patients with ibd compared to healthy individuals in this study auc values for unstimulated saliva and stimulated saliva to distinguish ibd patients from healthy individualswere reported to be and respectively however thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of ibd that is discussed in the following 0cf khakikhatibi clinica chimica acta laboratory measurement and reference intervalfecal calprotectin is a stable protein that remains stable for daysat room temperature this property is an excellent advantage for alaboratory marker also it seems that keeping the specimen at refrigerated temperature °c can increase the stability of fecal calprotectin however evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature on the other hand it is not also recommended tokeep samples in the refrigerator for more than days it seemsthat fecal calprotectin remains stable up to one year at °c measurement of fecal calprotectin can be done both qualitatively andquantitatively accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette however in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only it seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom ibd patients rapidly however some studies have shown that it isnot accurate enough in this case as well nevertheless asignificant concordance has been reported between home test resultsibdoc and fecal calprotectin laboratory measurement results whenquantum blue calprotectin elisa kit was used notably the agreements between results were and depending on the selectedcutoï¬s several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin these tests reportpositive results ranged from to µgg there are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin these kits are usually designed in terms of the elisamethod and some have a measurement range between and µgg moreover the chemiluminescence immunoassays cliamethod can also detect values between and µgg fluoro enzyme immunoassays feia and particle enhanced turbidimetric immunoassays petia can also be used for the measurement of fecalcalprotectin in this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals among healthy adults there is asignificant agreement on µgg as an upper limit one study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric ibd table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies according to these reports age can aï¬ect fecal calprotectinlevels fecal calprotectin and ibd diagnosisonly a small percentage of patients complaining of abdominal painand diarrhea have ibd in many cases ibs as a functional gastrointestinal disorder is known as the cause of such clinical symptomspatients with ibs have normal colonoscopy results while ibd patientsindicate abnormal colonoscopy results and have intestinal ulcersunfortunately the significant prevalence of ibs and the overlap between clinical symptoms and ibd can increase the colonoscopy ratetherefore a noninvasive diagnostic marker can be very helpful in thisregard notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of ibd was obtained in the 1990s røseth in proposed a method for measuring calprotectin in stool specimens one of the ï¬rst and most interesting studies regarding fecal calprotectinutility in ibd diagnosis was the study by røseth published in in this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls this study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study in another study published in auc values of ci were reported for fecal calprotectin in thediagnosis of colorectal ammation moreover in a study onchildren with ibd it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with esr levels in astudy published in kolho reported auc values of ci for fecal calprotectin in the diagnosis of pediatric ibd in a study on patients with crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease the results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as crp andesr in the diagnosis of ibd diamanti reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in ibd diagnosis in our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of ibd however oursample size was and the majority of patients were in the active phaseof the disease in another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard in one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withibd from patients without ibd patients with diseases other than ibdpatients with ibs and healthy persons with sensitivity and speciï¬city caviglia in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between ibs and ibd howeversome studies have reported significantly lower values accordingly in astudy on patients with ulcerative colitis kalantari reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with crohn's disease asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting ce ï¬ndings anddiagnosis of crohn's disease in another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city oftable reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesagesmedian levels of fecal calprotectin range µggnumber of subjectsused kitup to monthchildren yearschildren yearsadultsover years bühlmann elisabühlmann elisacalpro® calprotectin elisa test alpphicalphicalreference 0cf khakikhatibi were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in crohn's disease given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of ibd where the small intestine is involvedbesides there are some preanalytical limitations which are explainedin the next sections therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenibd and ibs the probability of ibd was less than at fecal calprotectin values lower than µgg or crp values lower than mgdl therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of ibd in patients with ibslike symptoms aswell as reducing the rate of colonoscopy moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between ibdand ibs it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points hence performing extensive studies indiï¬erent countries on the healthy population and the ibd patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultstable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of ibdfrom ibs and table4 summarizes some metaanalysis results in thisregard as shown in table the most important limitation of the majority of clinical studies conducted to date is the small sample size alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between ibdand nonibd diseases fecal calprotectin and endoscopic and histologic activity evaluationundoubtedly one of the most serious challenges in the managementof ibd is evaluating the endoscopic and histologic activities of thedisease nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withibd as noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations in recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels in addition many studies have been performed inthe last decade all of which cannot be reviewed in this article the ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s in one of the ï¬rst studiesroseth found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis furthermore in another study they observed that ibdpatients who were in remission clinically and had normal fecal calprotectin levels less than mgl had normal colonoscopy results these interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andclinica chimica acta table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdsample sizepooled sensitivitypooled speciï¬cityreferences mucosal healing in ibd patients also these studies were the startingpoint of extensive studies that have been conducted up to now in astudy conducted on patients with crohn's disease sipponen investigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of crohn's disease correspondinglythe researchers used the crohn's disease endoscopic index of severitycdeis scoring system in their study to evaluate the endoscopic activity of crohn's disease as a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of crohn's disease with sensitivity and speciï¬city in another study cdeis and mayo disease activity indexmdai were used to evaluate the endoscopic activity of crohn's diseaseand ulcerative colitis respectively according to the results of thatstudy on ibd patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to therachmilewitz clinical activity index in addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like crp besides one studyfound that fecal calprotectin was more strongly correlated with thesimple endoscopic score for crohn's disease sescd compared to thecrp and even crohn's disease activity index cdai the modiï¬edbaron index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis as a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to crp and clinical activity of the disease in thisregard similar results were also observed in our recent study in whichthe ulcerative colitis endoscopic index of severity uceis and sescdwere used therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of ibd endoscopicactivity the high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring however severalrecent studies have reported some significantly lower values accordingly in a recent study in which mayo endoscopic score [mes] wasused to evaluate the endoscopic activity of ulcerative colitis atable summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdnumber of ibd patientsage grouplocationcut oï¬sensitivityspeciï¬city cd and uc cd and uc cd and uc and unclassiï¬ed68cd and uc cd and uc and unclassiï¬ed cd and uc cd and uc uc cd ucadultsadultsadultsboth adult and pediatricpediatricadultspediatricadultsadultsboth adult and pediatrictaiwanchinaitalyspainfinlandiranitalyirandenmarkindia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggaucreferences spsreï¬dbib60 0cf khakikhatibi clinica chimica acta table summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in ibd patientsage groupstudylocationusedendoscopicactivity indexcorrelationcoeï¬cientrreferencenumberof ibdpatients cd uc uc cd ucadultsadultsadultsadultsadultsfinlandiranswitzerlandswitzerlandswitzerland modiï¬edcdeisuceisrachmilewitzsescd uc cdadultsadults uc cd uc cd cd uc ucadultsadultsadultsadultsadultsadultsadultsbaron scorerachmilewitzsescdgermanyusa andcanadajapanitalyitalybrazilfrancefrancesouth korea uceismattssescdmayo scoresescdcdeismayo score sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactivemes or from mes or in another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating mes ¤ in patients with ulcerative colitis were and respectively overall as presented in table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and ibd endoscopic activity although some of these studies reported a strong correlation some others reported a relativelyweak correlation as noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of ibd undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences however fecal calprotectin does not appear to be a very reliable marker for the predictionof ibd endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy inthis regard further studies are still needed however under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate ibd endoscopic activity can be helpfulpregnant patients with ibd have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy in one study physicianglobal assessment [pga] which is a clinical symptombased criterionwas used to evaluate ibd activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with ibd the results of this study showed a significantcorrelation between fecal calprotectin and pga levels at prepregnancyduring pregnancy and postpartum stages in another study asignificant association was reported between fecal calprotectin levelsand clinical activity of ibd in pregnant women moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of ibd clinical activity at diï¬erent stages ofpregnancy a recently published systematic review has also conï¬rmed the conclusions obtained from these studies according tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith ibd clinical activity during pregnancy therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of ibd fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of ibd endoscopic activity inpregnant women in addition under pandemic conditions fecal calprotectin can be very helpful following the covid19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy therefore noninvasive ibd management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before the combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringcovid19 pandemic therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for ibd endoscopic activity evaluation during pandemic fecal calprotectin appears to be associated with ibd histologic activity as well given thediï¬culty in the evaluation of the histologic activity of crohn's disease some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far correspondingly thesesystems score the disease's histologic activity based on histologic observationstherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory in this regard one of these histologic scoring systems isroberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the roberts scoring system theede also used themodiï¬ed harpaz index and performed some interesting studies in thisregard in one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingauc ci95 sensitivity speciï¬city andcutoï¬ mgkg in another study on patients with endoscopically inactive ulcerative colitis mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg p also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg in a recent study the geboes index has been used toevaluate histologic activity in patients with clinically quiescent ulcerative colitis as a result this study reported relatively low sensitivityand speciï¬city for fecal calprotectin in the prediction of geboesscore sensitivity speciï¬city and cut oï¬ µgg in another recent study the nancy index has been used toevaluate the histologic activity of ulcerative colitis and a high sensitivity and a low speciï¬city were ï¬nally reported for fecalcalprotectin at a cutoï¬ of µgg in the prediction of histologic activity however some studies have reported both high sensitivityand speciï¬city for fecal calprotectin in the prediction of histologicalremission for example one study reported a sensitivity of aswell as a speciï¬city of for fecal calprotectin at a cutoï¬ of µggin the prediction of gs it seems that the cause of theseconï¬icts should be explored in the endoscopic and clinical activity ofthe disease the inclusion and exclusion criteria of these studies andpossibly in the diï¬erent indexes used for the evaluation of the histologicactivity of the disease another notable issue is that all of these studieshave been conducted on a relatively low number of patients with ulcerative colitis so the need for a study with a large sample size is stillstrongly felt moreover a large global study may be helpful in this regard prediction of relapse and response to treatmentas mentioned previously ibd has recurrence and relief periods sopredicting response to treatment and relapse is one of the significant 0cf khakikhatibi clinica chimica acta challenges in ibd management the ï¬rst evidence of the eï¬cacy offecal calprotectin to predict recurrence dates back to the early 2000saccordingly a study published in by tibble is one of the ï¬rststudies in this regard in this study ibd patients in clinical remissionwere followed up for one year for the assessment of recurrence afterpreparing a stool sample to measure calprotectin this study has alsoshown that fecal calprotectin levels were higher in ibd patients withrecurrent disease and it was found that fecal calprotectin had a sensitivity of and a speciï¬city of at a cutoï¬ of mgl to predictibd recurrence in a study published in costa showedthat the sensitivity and speciï¬city of fecal calprotectin to predict ulcerative colitis recurrence are more than that of crohn's disease asensitivity of and a speciï¬city of versus a sensitivity of and a speciï¬city at a cutoï¬ of μgg another studyconducted on patients with ulcerative colitis has also reported appropriate sensitivity and speciï¬city rates for fecal calprotectin in the prediction of relapse a sensitivity of and a speciï¬city at a cutoï¬of μgg however another study was conducted on patientswith ulcerative colitis who have been followedup for year and ï¬nally a lower sensitivity was reported this study have shown that fecalcalprotectin at cutoï¬ of μgg could predict diseas
| 0 |
"Pluripotent stem cellsDirected differentiationIn vitro disease modellingLungAirwayMechanical cuesContentsChronic lung diseases remain major healthcare burdens for which the only curative treatment is lung transplantation In vitro human models are promising platforms for identifying and testing novel compounds to potentiallydecrease this burden Directed differentiation of pluripotent stem cells is an important strategy to generate lungcells to create such models Current lung directed differentiation protocols are limited as they do not recapitulate the diversity of respiratory epithelium generate consistent or sufï¬cient cell numbers for drug discoveryplatforms and establish the histologic tissuelevel anization critical for modeling lung function In this review we describe how lung development has formed the basis for directed differentiation protocols and discussthe utility of available protocols for lung epithelial cell generation and drug development We further highlighttissue engineering strategies for manipulating biophysical signals during directed differentiation such that futureprotocols can recapitulate both chemical and physical cues present during lung development Elsevier BV All rights reservedOverview of key developmental stages Lung anogenesis molecularly deï¬ning lung fate in the embryo Branching morphogenesis and other mechanical cues generated during lung development Introduction Human embryology as a blueprint for lung directed differentiation Directed differentiation of lung epithelia inspired by embryology Mouse embryonic stem cell derived lung epithelia Modeling airway and lung diseases for drug discovery Opportunities to exploit mechanical cues for improving directed differentiation protocols in the future Micropatterning in 2D Stem cell behaviour on substrate topographies Micropatterning in 3D anoid systemsSubstrate textureHuman pluripotent stem cellderived lung epithelia Creation of human proximal lung epithelia Comparisons of proximal airway directed differentiation protocols Creation of human distal lung epitheliaComparisons of distal lung directed differentiation protocols Limitations of current directed differentiation protocols Correspondence to G Karoubi Latner Thoracic Surgery Research Laboratories Toronto General Hospital College St Toronto ON M5G 1L7 Canada Correspondence to A P McGuigan Institute for Biomaterials and Biomedical Engineering University of Toronto College Street Toronto ON M5S 3G9 CanadaEmail addresses golnazkaroubiuhnresearchca G Karoubi alisonmcguiganutorontoca AP McGuigan101016jaddr2020080050169409X Elsevier BV All rights reservedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxAcknowledgementsReferencesFuture outlook IntroductionEndstage lung disease is the third leading cause of morbidity andmortality worldwide [] and produces a significant burden onhealthcare systems due to extensive resource expenditures for diseasemanagement and as lung transplantation is the only curative treatmentoption Such diseases include acute respiratory distress syndromechronic obstructive pulmonary disease cystic ï¬brosis and pulmonaryï¬brosis Chronic pulmonary diseases result in million global deathsper year [] Patients who receive transplants face continued complications associated with chronic immunosuppression and graft rejectionwith the transplant survival rates at and years being and respectively [] Furthermore since lungs function as an important barrier between the internal and the external environments they are a critical site for bacterial and viral infections and disease transmissionparticularly relevant given the current COVID19 pandemic There istherefore a critical need to better elucidate the mechanisms of infectiondisease progression host response and cellular repair in the lung to enable the development of novel targeted therapeutics for lung diseaseTissueengineered models have emerged as a technology to addressthis challenge and shown some success in drug identiï¬cation and toxicology studies For example commercially available airway epithelialmodels such as EpiAirwayTM MatTek Life Sciences serve as convenientplatforms with airliquid interface culture capabilities for assessing theeffect of chemical and physical stimuli [] Other examples includethe Alveolus LungChip and Airway LungChip systems Emulate Incoriginally developed in the Ingber laboratory which mimic theepithelialendothelial interface of the airway and provide a more dynamic platform for testing new antiammatory compounds inasthma [] and new small molecule targets to decrease cancerassociated pulmonary edema [] More complex models have alsobeen reported which involve selfassembly of heterogeneous progenitor cells into 3D structures termed anoids [] These anoidmodels can recapitulate aspects of human lung development in termsof tissue structures and protein expression and therefore present apromising opportunity for drug screening []A challenge in developing such human in vitro lung models to screenfor drugs however is the requirement for large batches of similarhuman cells as a starting population for tissue manufacturing to ensureminimal heterogeneity between test wells [] Achieving this is especially challenging when using primary human lung cells which exhibitconsiderable heterogeneity across donors and have a limited ability togrow and differentiate reliably [] Furthermore primary cells areoften extracted from diseased donors which is not ideal for conductingcontrolled studies due to the wide range of therapeutic and environmental factors these cells have already been exposed to Directed differentiation of pluripotent populations has the potential to create vastnumbers of cells from either healthy or diseased patients It allows introduction of speciï¬c diseaseassociated mutations via CRISPRCas9gene editing to recapitulate and understand pathologies in a controlledmanner As such directed differentiation enables the generation of anattractive cell source for drug screening platforms and personalized disease models that may provide insight into tissue regeneration mechanisms []Directed differentiation protocols to manufacture speciï¬c cell populations from pluripotent stem cells PSCs have been developed to meetthe need for a homogeneous human cell source Older lung directed differentiation protocols from the late 2000s have been proven inefï¬cientdue to the nonstandardized methods through which they derive lungendoderm from embryoid bodies [] A series of more standardizedstepwise protocols have since emerged in the last decade that provideavenues for developing airway and lung epithelia albeit with variableefï¬ciencies [] The ï¬rst uential directed differentiation protocol to produce lung epithelia used human PSCs in [] whichwas further supported by two prominent studies conducted usingmouse PSCs in [] These protocols have continued to be enhanced through adaptations related to the selection of growth factorsand small molecules the chronology of morphogen delivery as wellas innovations in enabling platforms such as cell sorting 3D cultureand singlecell analyses to efï¬ciently derive normal and diseased lungepithelia from human PSCs [] Despite such advancements limitations pertaining to heterogeneity in the resultingpopulations still exist which are likely attributed to variability across directed differentiation trials PSC cell lines or the persistence of contaminating cell populations belonging to other lineages While protocolshave progressed to some degree in differentiating proximal airwayand distal alveolar epithelia they remain limited Overall many unanswered questions remain with regards to the identity maturity andfunctionality of resulting cell types as well as their utility for tissue engineering and drug testing approaches Therefore these protocols mustbe optimized further to reliably produce large numbers of spatially relevant and functional lung and airway epithelial cells that appropriatelyrespond to both chemical and mechanical stimuli in the context of disease modeling and drug discoveryIn this review we discuss the directed differentiation protocols thatattempt to recapitulate lung development and disease and highlightpossible opportunities to enhance these protocols in the future Weï¬rst describe development of native lung tissue and the patterningevents that occur that differentiation models attempt to mimic andhighlight how human lung embryology has served as the blueprint tocreate the common pathway of lung directed differentiation protocolsWe then discuss the evolution of directed differentiation protocols toï¬nd opportunities for creating speciï¬c populations of airway and lungepithelia through targeted manipulation of key signaling pathways in2D and 3D models We further describe how these models have beenused to recapitulate different airway and lung diseases Finally we discuss how tissue engineering and biophysical cues using biomaterialscan be utilized during lung directed differentiation to mimic patterningcues present in development to augment current differentiationprotocols Human embryology as a blueprintdifferentiationforlung directed Overview of key developmental stagesDirected differentiation protocols have been designed to mimicin vivo human lung development [] Indeed in vitro models of lungdevelopment have provided unique insight into human lung development [] As human lung development has been described at greatlength in earlier reviews [] we provide a brief overview as followsschematically represented in Fig During early embryogenesis at days post fertilization a process called gastrulation begins with the appearance of a structure called primitive streak through which cells migrate to form the primary embryonic germ layers deï¬nitive endodermmesoderm and ectoderm [] Deï¬nitive endoderm expandsthereby forming the primitive gut tube comprised of three endodermalregions foregut midgut and hindgut [] This is when lungPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxFig Schematic of human lung development from an epithelial perspectivedevelopment begins at approximately four weeks into embryonic lifewith the outgrowth of foregut endoderm [] and continues througheight years of postnatal life [] There are ï¬ve stages to lungdevelopment Embryonic weeks The future lung buds emerge from the ventral side of the primitive foregut endoderm into the surroundingmesenchyme and develop into embryonic lung buds with early trachea and bronchi [] Pseudoglandular weeks Branching of the airway continuesleading to formation of conducting and terminal bronchioles whilethe proximal airway epithelium begins to develop [] Canalicular weeks Development of the respiratory or gasexchanging airways is initiated primitive alveoli form and the future distal epithelium begins to thin as distal epithelial markers areexpressed [] Saccular weeks Emergence of sacshaped distal airwayswhich develop crests with muscle and elastin to create indentationsThese distal airways extend to form alveoli by weeks [] The developing epithelium and vasculature within the future alveolus continue to merge closer together to facilitate future gas exchange andfurther differentiation of alveolar epithelial cells AEC I and II takesplace Alveolar periods week years True alveoli are seen in week and the majority of alveolarization takes place through sacculeseptation a process by which the sacshaped distal airways changetheir internal architecture and create thin walls intraluminallySeptation leads to an increase in surface area of the gas exchangingportion of the developing lung and prepares the fetus to breath airduring this stage [] Lung anogenesis molecularly deï¬ning lung fate in the embryoDuring the embryonic period early lung is genetically deï¬ned by theexpression of transcription factor NK2 Homeobox NKX21 and Srybox SOX2 [] During human lung development it has beenfound that the lung buds and branches given off during thepseudoglandular period are mostly SOX2SOX9 [] BothSOX2 and SOX9 are individual markers of the early proximal or distal lineage respectively [] Over the course of the canalicular and saccular periods of development weeks these double positivepopulations downregulate one SOX protein and maintain expressionof the other as these cells mature towards proximal or distal lineages[] The proximal airway closer to the mouth is comprised of apseudostratiï¬ed columnar epithelium that is responsible for theconducting airway function debris and pathogen removal ciliatedcells mucus production goblet cells prevention of airway ammation club cells and humidiï¬cation of air as it passes through to the distal lung compartment [] The squamous distal epitheliumcomposed of alveolar epithelial cells AEC I and II facilitates the respiratory function of the lung as air in the epithelial compartment is broughtinto close apposition to blood from the pulmonary vasculature it alsosecretes surfactants which play an immunologic role and decrease thesurface tension present at the airliquid interface thereby preventing alveolar collapse [] In humans a number of cell types are found in theproximal airway each identiï¬ed with speciï¬c markers Table This includes basal cells tumor protein p63P63 keratinKRT5 nerve growthfactor receptorNGFR integrin α6ITGA6 integrin β4ITGB4 ciliatedcells Forkhead BoxJ1FOXJ1 acetylated tubulinAcTUB goblet cellsmucin 5ACMUC5AC mucin 5BMUC5B club cells club cell secretoryproteinCCSP or SCGB1A1 and pulmonary neuroendocrine cellsPNECs synaptophysinSYP chromogranin ACHGA On the otherhand homeodomainonly protein HOPX identiï¬es the distal lungalong with AEC I cells T1α podoplaninPDPN aquaporin 5AQP5while AEC II cells are recognized via surfactant protein B SPC prosurfactant protein C proSPC or SPC and HT2280 []One mechanism by which lung epithelia begin to mature is based onchemokine secretions from the surrounding mesenchyme and the developing heart ï¬eld which are well reviewed here [] Key players including ï¬broblast growth factors FGFs [] WNTs []and bone morphogenetic proteins BMPs [] are known to inducethe differentiation of early lung progenitors in a controlled manner Forexample in mouse it has been found that FGF10 plays a role in bud outgrowth [] and drives lung progenitors towards a distal fate []through canonical WNT signaling [] Proximal epithelia developbecause they are located further away from distally located FGF reservoirs in the mesenchyme in a mechanism that appears dependent onconcentration gradients [] BMP4 plays a key role in lung bud formation from foregut endoderm and establishment of both dorsoventralback to front and proximodistal top to bottom patterning in the nascent lung [] BMP4 is also present at high levels in distal bud tips andepithelia including AEC II cells [] however its inhibition promotesa proximal fate and along with BMP2 inhibition ciliated cell development [] Branching morphogenesis and other mechanical cues generated duringlung developmentWhile the cell fate of early proximal and distal lineages is directedthrough chemical signals the lung epithelium itself undergoes markedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxTable Epithelial populations in native human airways and lungsRegionProximal AirwayDistal LungCell TypeAssociated Markers for Cell CharacterizationCiliated CellGoblet CellClub CellBasal CellAlveolar epithelial cell type I AEC IAlveolar epithelial cell type II AEC IIFOXJ1 AcTUBMUC5AC MUC5BCCSP SCGB1A1 SCGB3A2P63 KRT5 NGFR ITGA6 ITGB4HOPX PDPN AQP5SPB SPC HT2280Cell Proportions in Native Lung [] [] [] [] [] []changes in architecture a process known as branching morphogenesis[] From the simple tube of the anterior foregut endoderm to thecomplex tubular structure of the adult a highly stereotyped mechanismof branching morphogenesis facilitates the outgrowth division placement and structure of lung airway [] Branching morphogenesis ofthe lung is driven by three simple and iteratively used processes domain branching planar and orthogonal bifurcation [] The ï¬rst formof branching is domain branching along a primary branch buds formin a linear and sequential fashion from proximal to distal The nextform of branching is planar bifurcation in which the tip of the formingtube bifurcates to create two new tips which subsequently elongateand bifurcate again creating four tips The last process of branching isknown as orthogonal bifurcation In this process the initial planar bifurcation is followed by a rotation around the planar axis which createstwo new tips through bifurcation A critical gene in this processSprouty has been found to attenuate Erk12 signaling thereby alteringthe orientation of cell division and future tube elongation [] Othercritical genes and regulatory networks associated with FGF signalingalso contribute to controlling the periodicity of the branched network[] Although elements such as domain speciï¬cation bifurcation rotation and branch generation remain largely undetermined [] newtechnologies involving highresolution live imaging tension sensingand forcemapping are opening paths to further explore and explainthe branching morphogenesis phenomenon []The early structure of the lung gives rise to a striking architecturalseparation of future SOX2 proximal lineages and SOX9 distal lineages at least in mice [] The diameter of tube generated duringbranching morphogenesis in the pseudoglandular and canalicularstages has a small degree of variance within each stage as measuredfrom electron micrograph sources of fetal human tissue [] This suggests that the branching program is rigorous in its control of lung structure and that tubes themselves may have instructive potential on thedeveloping epithelia Once the basic an structure has formed thelung continues to be exposed to mechanical cues as it continues to mature In several cases these cues have been shown to be essential forcorrect an function In utero the fetal lung is a secretory an thatonly converts to an absorptive one to prepare for breathing afterbirth through a change in the activity of chloride and sodium channelslate in development Fetal lung secretions result in a static ï¬uid pressureof around cmH2O in the developing terminal sacs of the fetus whichpropels branching morphogenesis outwards into the developing thoracic cavity [] Lack of amniotic ï¬uid in the developing lung alters the expression of distal epithelial markers and consequentlyresults in the creation of smaller than normal lungs pulmonary hypoplasia [] highlighting the importance of this mechanical pressureduring lung development In addition cyclic strain is generated fromfetal breathing movements FBM in utero that prime the airway foruse after birth FBM are detectable from the tenth week of pregnancyand begin as infrequent and erratic activity with long quiescent periodsAs development continues these quiescent periods decrease andsustained periods of fetal breathing occur These breathing movementsvary with the fetal sleep cycle and can be chemically tuned [] andalter the volume of terminal sacs by around [] againhighlighting the importance of mechanical signals uencing lung development Finally a novel FGF10FGFR2dependenttensionalmechanism has been shown by which distal epithelial cells in the lungaccumulate motor proteins at the apex of the cell thereby becoming resistant to compression from increasing ï¬uid pressure within the tubelumen Cells under this tension are more likely to become AEC II cellswhile those under compression become AEC I cells [] Interestinglywhile the above examples highlight the importance of speciï¬c mechanical signals in the growth development and differentiation of the lungPSC directed differentiation protocols of the lung are primarily based onmimicking the sequential chemical changes that occur during lungdevelopment Directed differentiation of lung epithelia inspired by embryologyEarly attempts to create lung epithelia from PSCs began in mouseand did not attempt to mimic the stepwise changes in chemical signaling that occur during development Rather groups focused on applyinglunglike physical cues such as airliquid interface [] These protocols while successful in generating NKX21 positive populationsalso produced contaminating cells expressing pluripotency markersOCT4 NANOG SSEA4 TRA160 TRA181 These early attempts solidiï¬ed that further optimization particularly related to the chemical cuesapplied was needed to reliably create lung progenitors from pluripotentsources without remnant pluripotent contaminating cells More successful directed differentiation protocols were rationalized from the detailed understanding of the chemical changes during lung embryologyIn this section we describe in detail the different differentiation protocols currently available that evolved from this approach Mouse embryonic stem cell derived lung epitheliaAlthough mouse models do not fully recapitulate human lung development they have served as guides for earlier iterations of PSC directeddifferentiation protocols and have identiï¬ed critical chemical cues forlung anogenesis Broadly speaking these protocols begin by drivingstem cells towards a deï¬nitive endoderm fate SOX17 and FOXA2mimicking the preembryonic period of human lung developmentweeks through high doses of the nodal activating molecule ActivinA [] Foregut endoderm is then induced via transforminggrowth factor beta TGFβ inhibition either alone [] or with BMP inhibition [] for a short period a process called anteriorization as duringthe embryonic period of human lung development weeks Thisforegut endoderm FOXA2SOX2 is subsequently induced to generate NKX21 cells putative lung progenitors by stimulating theretinoic acid RA BMP WNT and FGF signaling pathways []These lung progenitors are further matured as demonstrated by increased NKX21 expression through application of corticosteroids[] In brief each protocol begins with PSCs guided through deï¬nitiveendoderm followed by anteriorization to foregut endoderm and subsequent ventralization to generate NKX21 cells [] These protocolsformed the basis and backbone for the creation of human lung epitheliafrom PSCsGiven the structural and cellular complexity of the lung it is reasonable that the earliest protocols focused on mouse However there aresubtle differences that highlight how human models are different interms of structure patterning and differentiation For example thePlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxentire human conducting airway is comprised of a pseudostratiï¬ed epithelium even at diameters less than 05mm [] In contrastconducting airways in mice only exhibit pseudostratiï¬ed epitheliumwith accompanying submucosal glands and cartilage in the most proximal portion of the airway and transition directly into alveolar sacs []This difference in histology affects the residing cell populations as evidenced by the lack of basal cells in the lower portion of the proximal airways of mice [] Similarly mouse models suggest thatSOX2SOX9 progenitors are quite rare and their cell fate is ambiguous [] However evidence from directed differentiation of humanlung epithelia [] which has been conï¬rmed in vivo []reveals that SOX2SOX9 progenitors are common in the developinglung buds and that branch tips of the pseudoglandular staged lunggive rise to both proximal and distal epithelia [] Moreover speciï¬cprotein markers have been found to differ in both timing and locationof expression between human and mouse models proSPC in mouseis expressed early and throughout the developing mouse epithelium[] while in human proSPC is rarely detected early in development and is only robustly found later in distal epithelia [] These examples highlight that while there are similarities development andpatterning of mouse and human lungs is different and these differencesrequire human models to be fully appreciated Human pluripotent stem cellderived lung epitheliaHuman PSC protocols have generally followed the same differentiation chronology as that of mouse directed differentiation wherein deï¬nitive endoderm anterior foregut endoderm and NKX21 lungprogenitors are produced sequentiallyDifferent groups have adhered to their own methods of generatingdeï¬nitive endoderm which primarily involves exposing PSCs to highconcentrations of Activin A Slight variations such as introducing WNTagonism through WNT3a or CHIR99021 prior to [] or alongside[] Activin A or additional exposure to BMP4 and FGF2[] during this stage exist across protocols for differentially inducing primitive streak and its anteriorization towards producing deï¬nitive endoderm In addition the use of embryoid bodies which arelimited by user experience and technique has resulted in a widerange of production efï¬ciencies for achieving this stage from CKITCXCR4EPCAM cells [] to CKIT CXCR4 cells []Recent advances in commercial products have led to development ofstandardized 2D culturebased media STEMdiff Deï¬nitive EndodermKit STEMCELL Technologies which allow reliable derivation of of deï¬nitive endoderm []Similarly generation of both anterior foregut endoderm andtoventralized lung progenitor populations has been subjectmuch investigation and modiï¬cation Earlier work suggested thatSOX2FOXA2 ± in their case anterior foregut endoderm canonly be induced by subjecting deï¬nitive endoderm to TGFβ and BMP inhibition [] Subsequent studies however attempted to anteriorize deï¬nitive endoderm to foregut endoderm through TGFβ inhibition alone SOX2 a combination of endogenous WNT TGFβ and BMP inhibition not quantiï¬ed [] and via Sonic Hedgehog SHH and FGF2signaling FOXA2 EPCAM [] A comparison of the lattertwo strategies demonstrated that SHH and FGF2 are insufï¬cient inproducing reliable NKX21 lung progenitors [] possibly becauseFGF2 is involved in promoting thyroid lineages [] In general TGFβand BMP inhibition [] is the basis for currently applied endodermanteriorization strategies []Factors involved in early versions of ventralization in directed differentiation protocols included WNT3a FGF7 FGF10 BMP4 epidermalgrowth factor EGF and RA have now been reduced based on elimination studies [] As such CHIR99021 CHIR WNT agonist BMP4and RA are necessary and sufï¬cient for producing lung progenitorsfrom anterior foregut endoderm derived from both mouse and humanPSCs [] Despite ï¬nding that FGF7 and FGF10 are nonessential for inducing NKX21 expression they continue to be used forventralization in some protocols [] Although each protocol differsin terms of the duration of each phase NKX21 lung progenitors aregenerally achieved by days with the exception of a study by deCarvalho in which they maintained their cultures for an additional days in FGF7 FGF10 and CHIR99021 to attain NKX21FOXA2 lung progenitors In all cases these lung progenitors are theneither sorted or directly guided towards proximal or distal progeny in2D or 3D culture systems Ideally products of directed differentiationprotocols should mimic the cell proportions present in human airwaysand lungs Table however current protocols have not progressedthat far While these protocols continue to be reï¬ned the percentageof select cell populations generated from these protocols have beensummarized in Table Creation of human proximal lung epitheliaProtocols to create proximal lung epithelia have focused on the production of the four major cells types present ciliated goblet club andbasal cells see Table for a summary of markers for each cell type Motivation for creating proximal epithelia in the ï¬eld has primarily been todevelop patientspeciï¬c cystic ï¬brosis CF models [] andor toproduce epithelia with multiciliated cell populations for protocol validation [] A shift towards human PSCderived CF models hasbeen critical as mouse models do not accurately represent CF diseaseprogression and phenotypes seen in humans [] As such theï¬rst evidence of human PSC proximalization using CF patientderivedPSCs was shown by Mou who exposed anterior foregut endodermto BMP4 GSK3iXV WNT agonist FGF2 and RAsupplemented B27 togenerate NKX21 cells by Day Although contaminatingneuroectodermal and distal lung NKX21SOX9 cells were presentday populations included proximal NKX21SOX2 progenitorsSubcutaneous implantation of this population in immunodeï¬cientmice for days resulted in emergence of NKX21P63 cells howeverno mature epithelial markers for ciliated goblet and club cells werefoundWong employed a longer 2D differentiation approach to produce mature proximal airway epithelia in vitro Through a processthey called proximal speciï¬cation they generated day lung progenitors via low levels of BMP4 mimicking signaling gradients in theairway FGF7 and FGF10 which began expressing proximal genes Further culture with FGF7 FGF10 and FGF18 resulted in upregulated geneexpression of KRT5 P63 FOXJ1 SOX17 cystic ï¬brosis transmembraneconductance regulator CFTR and SCGB1A1 to a lesser extent alongwith low levels of distal SOX9 and SPC by day Protein expressionamounted to NKX21 panKRT P63 FOXJ1 cells These cells were subsequently matured in airliquid interface ALI culture for weeks week of submerged culture withFGF18 followed by weeks of ALI culture to generate CFTRpanKRT FOXJ1 with coexpressing CFTR and CFTRLHS28 cells The resulting epithelium ranged from being squamous to cuboidal with sparse pseudostratiï¬ed regions implying thatthis protocol lacked speciï¬c maturation cues Contaminating thyroidthyroglobulin and PAX9 liver HNF4 and AFP and pancreaticPDX1 lineages were detected through quantitative PCR while percentages of goblet club and basal cell populations barring gene expression analysis were not evaluatedA similar 2D culture approach was employed by Firth to generate proximal lung progenitors which were subsequently matured intomulticiliated epithelia They optimized lower concentrations of BMP4required during the ventralization phase day
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" exosomes are extracellular vesicles containing a variety of biological molecules including micrornasmirnas we have recently demonstrated that certain mirna species are selectively and highly enriched inpancreatic cancer exosomes with mir1246 being the most abundant exosome mirnas have been shown tomediate intercellular communication in the tumor microenvironment and promote cancer progression thereforeunderstanding how exosomes selectively enrich specific mirnas to initiate exosome mirna signaling in cancercells is critical to advancing cancer exosome biologyresults the aim of this study was to identify rna binding proteins responsible for selective enrichment ofexosome mirnas in cancer cells a biotinlabeled mir1246 probe was used to capture rna binding proteins rbpsfrom panc1 cells among the rbps identified through proteomic analysis srsf1 eif3b and tia1 were highlyassociated with the mir1246 probe rna immunoprecipitation rip and electrophoretic mobility shift assay emsaconfirmed the binding of srsf1 to mir1246 lentivirus shrna knockdown of srsf1 in pancreatic cancer cellsselectively reduced exosome mirna enrichment whereas gfpsrsf1 overexpression enhanced the enrichment asanalyzed by next generation small rna sequencing and qrtpcr mirna sequence motif analysis identified acommon motif shared by of srsf1associated exosome mirnas emsa confirmed that shared motif decoysinhibit the binding of srsf1 to the mir1246 sequences we conclude that srsf1 mediates selective exosome mirna enrichment in pancreatic cancer cells bybinding to a commonly shared mirna sequence motifkeywords srsf1 exosome mirna mir1246 pancreatic cancer exosomes are endosomederived extracellular vesiclesevs that can be transferred from cancer cells tostromal cells in the tumor microenvironment [ ]these membrane vesicles are nm in size andcontain proteinsincludinglipids and nucleic acids correspondence weiqundingouhscedu1department of pathology university of oklahoma health sciences centeroklahoma city stanton l young blvd bmsb 401a oklahoma city ok usa6stephenson cancer center university of oklahoma health sciences centeroklahoma city ok usafull list of author information is available at the end of the small rnas such as micrornas mirnas[ ]exosomemediated intercellular communication betweencancer cells endothelial cells [ ] fibroblasts [ ] orimmune cells [ ] can facilitate tumor progressionfurthermore cancer exosomes are released into the circulation and contribute to premetastatic niche formation in distant ans [ ]how cancer exosomes interact with stromal cells topromote tumor progression has been extensively investisignaling eventgated one criticalin the tumormicroenvironmentisthe exosome mirnamediatedintercellular communication [ ] studies haveshown that exosome mirna signaling promotes tumor the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cxu cell communication and signaling page of progression in various model systems [ ] notablyit has been reported that mirnas contained in exosomes are delivered to recipient cells in the tumormicroenvironment or distant ans where they canregulate target gene expression and promote tumorangiogenesis and metastasis [ ]in the context of exosome mirna signaling we andothers have reported that certain mirna species areselectively enriched in cancer exosomes as compared toexosomes derived from normal epithelial cells [ ] results from several studies have also indicated thatselective enrichment of exosome mirnas is relevant totumor progression for example exosome sortingof mir193a was found to promote colon cancer progression likewise mir122 a cancer exosomeenriched mirna [ ] was shown to reprogram glucose metabolism in a premetastatic niche to facilitatemetastasis in a breast cancer model system moreover the exosome enriched mir1246 was reportedto promote tumor invasion in both breast cancer and oral squamous cell carcinoma it seems clearthat selective enrichment of exosome mirnas drivescancer exosome mirna signaling in the tumor microenvironment which in turn reinforces tumor invasiveness and progression however how exosome mirnasare enriched or how exosome mirna signaling is initiated in cancer cells remains largely unknown elucidating the mechanisms ofselective exosome mirnaenrichment in cancer cells may help identify new cancertherapeutic opportunities that are urgently neededrecentreports have indicated that certain rnabinding proteinsrbps are involved in exosomemirna sorting in eukaryotic cells and the type ofrbps involved seems to differ among various modelsystems [ ] suggesting that exosome mirnasorting is a tissue or celltype specific processfurthermore there have been no reports on the identification of rbps that regulate exosome mirna sorting in pancreatic cancer cells we have recentlycharacterized the biogenesis of exosome mir1246 which is the most highly enriched mirna inpancreatic cancer cellderived exosomes the aimof this study was to utilize our established cell modelsystems to identify rbps that are involved in exosomemirna loading in pancreatic cancer cells using alabeled mir1246 probe as bait we fished out several rbpsincludingserine and arginine rich splicing factor srsf1eukaryotic translation initiation factor subunit beif3b and t cellrestricted intracellular antigen tia1 we found that srsf1 a recently claimedoncoproteininregulating exosome mirna enrichment in pancreaticcancer model systemsfrom pancreatic cancer cellspredominantlyinvolved ismethodscell culturelines panc1the human pancreatic cancer celllinemiapaca2 and bxpc3 and breast cancer cellmdamb231 were obtained from the american typeculture collection atcc manassas va usa cellswere cultured following atccs instructions except thatexosomedepleted fetal bovine serum fbs and horseserum wereapplied whenever needed exosomedepleted fbs and horse serum were prepared by pelleting the serum exosomes at g for h at °ccells were routinely incubated in a humidified environment at °c and co2exosome isolationexosomes were isolated from the culture medium utilizing a combination of centrifugation ultracentrifugationand filtration as we recently described [ ] withminor modifications in brief the culture medium ofpanc1 cells was precleared by g centrifugationfor min at °c and the resulting supernatant was filtered through a μm pvdf centrifuge filter thelarge size evs were trapped in the filter and recovered inpbs the filtered supernatant was then applied to a μm pvdf centrifuge filter the medium size evswere trapped in the second filter and resuspended inpbs the small size evs exosomes in the final supernatant were recovered by ultracentrifugation g min at °c the isolated exosomes were verified bywestern blot detecting positive and negative exosomemarker proteins and nanop analysis nanosightns300 system malvern instruments uk measuringboth sizes and concentrations of the isolated exosomesfig mirna binding protein pulldownpulldown experiment was performed using the pierce¢magnetic rnaprotein pulldown kit thermo fisherscientific briefly pmol of biotinlabeled mir1246or polya rna oligonucleotides integrated dna technologies were hybridized to μl streptavidin magnetic beads prod1862766 thermo fisher scientificthe mir1246biotinstreptavidin beads were incubatedwith panc1 lysate for min at °c the lysatebeadmixture was washed three times with washing bufferfrom the abovementioned kit to elute bound proteins μl of elution buffer was applied and a magnetic separator was applied to separate the beads from the elutedprotein following the manufacturers protocol pierce¢magnetic rnaprotein pulldown kit thermo fisherscientific proteins were separated by sdspage beforemass spectrometry ms analysis 0cxu cell communication and signaling page of fig verification of the exosomes derived from panc1 cells a representative western blot analysis of cd63 nonreducing condition cd81flotillin and calnexin in the evs isolated from panc1 cells positive exosome markers are only detected in small evs exosomes b representativenanop tracking analysis of exosomes small evs derived from control and srsf1 knockdown panc1 cells three individual experimentswere performed for both a and bliquid chromatographymass spectrometry lcmsmassspectrometry ms measurementthe experiment was performed by the laboratory formolecular biology and cytometry research core facilityat ouhsc proteins were digested with trypsin according to the fasp protocol briefly the eluate was buffer exchanged in m urea the proteins were reducedwith mm dithiothreitol and then alkylated with mm iodoacetamide the peptides were eluted dried andresuspended liquid chromatography tandem mass spectrometry was performed by coupling a nanaoacquityuplc waters corp manchester uk to a qtofsynapt g2s instrument waters corp manchesteruk each protein digest about ng of peptide wasdelivered to a trap column μm mm nanoacquity uplc nanoease column μm beh c18 waterscorp manchester uk at a flow rate of μlmin in solvent a mm ammonium formate ph inhplc grade water tandem mass spectra were generated in the trapping region of the ion mobility cell byusing a collisional energy ramp from v low massstartend to v high mass startend the pusherionmobility synchronization for the hdmse method wasperformed using masslynx v41 and driftscope v24lockspray of glufibrinopeptideb mz wasacquired every s and lock mass correction was appliedpost acquisitionprotein identificationraw ms data were processed by plgs proteinlynxglobal server waters corp manchester uk for peptide and protein identification msms spectra weresearched against the uniprot human database containing reviewed sequences with the followingsearch parametersfull tryptic specificity up to twomissed cleavage sites carbamidomethylation of cysteineresidues was set as a fixed modification and nterminalprotein acetylation and methionine oxidation were set asvariable modificationssmall rna library preparation and next generationsequencingtotal rna was extracted from cell and exosome pelletsusing the trizol reagent invitrogenlife technologiescarlsbad california the small rna libraries were constructed and run on the illumina miseq platform as werecently described [ ]rna immunoprecipitation assaypanc1 cells or mdamb231 celllysates were prepared using ip buffer mm trishcl ph mmnacl mm edta mm pmsf and triton x the lysate was sonicated for min on ice andinsoluble material was removed by centrifugation supernatants were collected and protein concentrations weremeasured the supernatant was precleared by proteing dynabeads¢thermo fisher scientific and thenmixed with antibody srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 in a ratio of at °c overnight withgentle rotation to capture the antibodyproteinrnacomplexes μl of protein g magnetic beads wereadded and the complexes were rotated for h at °cthe sample was separated by magnetic separation trizol reagent invitrogenlife technologies was appliedto isolated rna from the complex the mirna expression was analyzed by qrtpcr 0cxu cell communication and signaling page of coimmunoprecipitation coipcoimmunoprecipitation coip using panc1 cell lysate and antibody of srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 was performed as described previously and the protein complex was detected by westernblotwestern blot analysiswestern blot was performed as we recently described[ ] primary antibodies raised against srsf1 santacruz sc33652 eif3b santa cruz sc137214 tia1santa cruz sc166247 betaactin a5441 and glyceraldehyde 3phosphate dehydrogenase gapdh santacruz sc47724 were used for detection nuclear andcytoplasmic protein extraction was extracted followingrockland nuclear cytoplasmic extract protocol and verified by histoneh3 cst 4499s andgapdh santa cruz sc47724 detection antibodiesused for exosome marker detection include cd63cd81 santa cruz bio technology inc ca usaflotillin1 and calnexin cell signaling technology incma usaquantitative realtime reverse transcription polymerasechain reaction qrtpcrqrtpcr was performed as we described [ ] withspecific primers cel54 ²gcgcgcccgtaatcttcataatcc3² mir1246 ²gcgcgatggatttttggagcag3² mir320c ²gcaaaagcuggguugagagggu3² and mir320d ²gcgaaaagcuggguugagagga3²srsf1 shrna expression plasmid constructiontarget specific oligonucleotides were designed using online tool rnai codex cold spring harbor laboratoryand were synthesized integrated dna technologieswith the addition of overhangs according to the cuttingsite of bamh1 and ecori the shrna expression plasmid was constructed by annealing the oligonucleotidesto psihh1 vector following the user manual of psihh1 shrna system sbi system bioscience the oligonucleotide sequences for shrna of srsf1 eif3b ortia1 are provided in supplemental table 3rd generation packaging plasmidslentivirus transductionlentiviral ps were produced as previously described using the shrna expression plasmid andthepmd2gaddgene plasmid pmdlrregp addgeneplasmid and prsvrev addgene plasmid the packaging plasmids were cotransfectedwith the lentiviral expression vector into t cellsusing the polyethyleneimine polysciences inc to produce replication deficient lentivirus after transfectionthe supernatant was pooled and filtered with a μmmembrane and concentrated by ultracentrifugation toacquire lentivirus infection was performed by usinglentivirus in the presence of μgml polybrene sigmaaldrich approximately h postinfection cells wereselected by treating with μgml puromycin invivogen san diego cagfpsrsf1 expressionthe gfpsrsf1 expression plasmid was a gift from drmassimo caputi dna transfection was performedusing lipofectamine thermo fisher scientific topanc1 cells and the expression of gfpsrsf1 wasverified by western blotgstsrsf1 protein purificationbl21 thermofisher scientific c600003 competentcells transformed with pgex6psrsf1 dna addgeneplasmid were cultured at °c for hand after od600 reached to bacteria weretreated with mm isopropyl βd1thiogalactopyranoside for h at °c gsttaggedsrsf1 was purifiedwith glutathione sepharose beads ge health carethe purity of the recombinant proteins was determinedby sdspage with coomassie blue stainingelectrophoretic mobility shift assay emsaird800 labeled mir1246 μm integrated dnatechnologies was mixed with μl of gst slurry stsrsf1 in binding buffer tris ph mm kcl mm mgcl2 mm np40 dtt mm glycerol and incubated at room temperature for minavoiding light 5x loading buffer kcl mm tris ph mm glycerol xylene cyanol bromophenol blue was then added and the complexwas separated on a native gel polyacrylamide m tris ph m glycine m edta apstemed at voltage for min the signal was detected using the licor odyssey imaging systemlicor inc usadesign of decoy motif mimicsthe decoy motif mimics were designed by permutationand combination of the identified motif sequences in thelength of nucleotides the secondary structure of thedesigned sequences was analyzed in rnafold webserveruniversityselfcomplementary were selected decoy mimics ²uuggacuaggacuaggau3² decoy mimics ²aggaaggaaggaagga3²sequences withoutof vienna 0cxu cell communication and signaling page of bioinformatics analysisthe mirna motif analysis was performed using memesuite the protein profile analysis for the result ofmass spectrometry was performed using david bioinformatics abcc at saicfrederick inc the rnabinding protein and mirna sequence binding analysiswas performed using the database of rnabindingspecificities rbpdb srsf1 expression in cancertissues was examined using oncomine thecorrelation of gene expression with cancer patient survival was extracted from the human protein atlasscilifelab sweden statisticsstatistical analyses were performed using graphpadprism software graphpad software inc la jolla causa the heatmap was made in rstudio rstudio incwith the ggplot2 package students ttest was applied to determine significant differences among controland experimental groupsresultsidentification of mir1246 associated proteinsbecause rbps are involved in exosome mirna sortingwe first sought to identify proteins that bind to mirnashighly enriched in cancer exosomes mir1246 the mosthighly enriched mirna in pancreatic cancer exosomeswas biotinlabeled and incubated with a cellular lysatefrom panc1 cells the biotinmir1246 probe wascaptured with streptavidincoated magnetic beads biotinlabeled polya mimics were used as control themirnaprotein complexes were eluted and the proteinswere analyzed by liquid chromatographymass spectrometry in triplicate table there were total of proteins specifically pulled down by the mir1246 probeinterestingly about half of the proteins that associatewith mir1246 are vesicleassociated proteins supplement fig 1a based on the intensity of detection rnabinding property and cancer relevance we ranked therbps using the database for annotation visualizationand integrated discovery david this resulted in tencandidate rbps that complex with the mir1246 sequenceexosomestable among them srsf1 also called sfrs1 waspredictedsequencethe mir1246and arerelevantto eukaryotictobindtotable over view of the result of mass spectrometryexperiments conditionpoly a panc1number of proteins detectedpoly a mdamb231mir1246 panc1mir1246 mdamb231table mir1246 rna binding protein candidates obtainedfrom the mass spectrometric analysisprotein full nameprotein symbolsrsf1serineargininerich splicing factor park7eif3bthoc4acocddx5tia1if5a1eif2aimdh2parkinson disease protein eukaryotic translation initiation factor subunit btho complex subunit alyref export factorcytoplasmic aconitate hydrataseprobable atpdependent rna helicase ddx5tcellrestricted intracellular antigen1eukaryotic translation initiation factor 5a1eukaryotic translation initiation factor 2ainosine5²monophosphate dehydrogenase supplement fig by in silico analysis using the database of rnabinding specificities rbpdb levelsverification of srsf1 binding to mir1246rna immunoprecipitation rip was performed to verifythe association of several identified rbps with mir1246including srsf1 eif3b and tia1 igg and gapdhantibody was used as controls for immunoprecipitationas shown in fig 2a mir1246 expression is more than12fold higher in the srsf1precipitants as compared tothat of igg precipitants indicating a specific associationof srsf1 with mir1246 mir1246 expression wasmoderately increased in the tia1precipitants and nearigg controlin the eif3b precipitants coimmunoprecipitation coip experiments were performed to verify the immunoprecipitation proceduresdata not shown to directly determine the binding ofsrsf1 to the mir1246 sequence glutathionestransferase gst conjugated human srsf1 protein wasexpressed in bl21 competent e coli captured by glutathione sepharose beads and eluted by glutathione thepurity of eluted gstsrsf1 protein was shown by sdspage and coomassie blue staining supplement fig the binding of gstsrsf1 to a fluorescenttaggedmir1246 probe was determined by rna emsa asshown in fig 2b binding of the labeled probe was specific to gstsrsf1 but not gst and increased withgreater protein input the specific binding of gstsrsf1 to the mir1246 probe was evident as the unlabeled mir1246 probe effectively competed with thelabeled mir1246 probe in a concentrationdependentmanner fig 2c the detected bands were semi quantified and the kd was calculated from the detected signalsfig 2d these data confirmed the direct binding ofsrsf1 to the mir1246 sequence 0cxu cell communication and signaling page of fig srsf1 binds to mir1246 a qrtpcr detection of mir1246 in igg gapdh srsf1 eif3b and tia1 immunoprecipitants of panc1 lysaten p student ttest bc emsa detection of the srsf1mir1246 complex hot probe ird800 labeled mir1246 mimics cold probemir1246 mimics n direct binding of gstsrsf1 and mir1246 b and concentrationdependent competition between the cold and hotmir1246 probe for binding to gstsrsf1 c d semiquantification of srsf1 and mir1246 binding in c and calculated dissociationconstant n exosome mirna enrichment by srsf1 in cancer cellsbecause srsf1 is a key splicing factor that is essential toeukaryotic cells a knockout model could not beestablished thereforeto determine whether srsf1mirna binding activity is relevant to exosome mirnaenrichment we established a lentivirus srsf1 shrnaconstruct to knockdown srsf1 expression in panc1cells fig 3a interestingly though srsf1 protein wasdetected both in the nucleus and cytoplasm the knockdown was more pronounced in the cytoplasm fig 3bknockdown of srsf1 did not significantly alter the concentration and size distribution of the exosomes releasedby panc1 cells fig 1b cellular and exosome rnafrom control and srsf1shrna cells were isolated andsmall rna sequencing was performed among the highly enriched panc1 exosome mirnas expressionof mirnas was significantly downregulatedin exosomes derived from srsf1shrna panc1 cellsas compared to exosomes derived from control panc1cells fig 3c strongly indicating the involvement ofsrsf1 in exosome mirna enrichment a heatmapshowing the expression of the top mirnas enrichedin panc1 exosomes demonstrates the dramatic dropin expression levels of mirnasin srsf1shrnapanc1 exosomes compared to panc1 exosomesfig 3d notably mir1246 was the highest enrichedexosome mirna data not shown and its expressionin exosomes wassignificantly reduced by srsf1knockdown fig 3d on the other hand among of the mirnas less enriched in exosomes only were expressed at lower levels in exosomesderived from srsf1 knockdown cells as compared toexosomes derived from wild type panc1 cells fig3c suggesting that srsf1 knockdown mainly affectsexosome enriched mirnasto further confirm the effect of srsf1 knockdown onexosome mirna enrichment the expression levels ofseveral representative mirnas were quantified by qrtpcr srsf1 knockdown in panc1 cells significantlyreduced exosome levels of mir1246 mir320c andmir320d confirming the small rna sequencing resultsfig 3e in contrast knockdown of eif3b or tia1 didnot reduce exosome mir1246 expression suggestingthat these rbps may not promote exosome mirna 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig cellular and exosome mirna profiles after srsf1 knockdown in panc1 cells a detection of srsf1 knockdown by shrnas in panc1 cellsb panc1 srsf1 protein levels in nuclear and cytoplasmic fractions nc normal control c venn diagram of overlap of mirnas detected by nextgeneration small rna sequencing in srsf1 knockdown and control panc1 cells and exosomes d heatmap showing the expression of top exosome mirnas in cells and exosomes after srsf1 knockdown e qrtpcr analysis of mir1246 mir320c and mir320d in exosomes derivedfrom control and srsf1 knockdown panc1 cells p students ttest shown are representatives of three independent experiments aeenrichment supplement fig and our observationswere extended to two additional pancreatic cancer celllines miapaca2 and bxpc3 fig 4af in additionexpression of let7c which is less enriched in exosomeswas unchanged in exosomes after srsf1 knockdowndata not shownto verify the involvement of srsf1 in exosomemirna enrichment in cancer cells we also exogenouslyoverexpressed srsf1 in panc1 cells a gfpsrsf1 expression plasmid was introduced into panc1 cells andsrsf1 overexpression was confirmed by western blotfig 5a expression of mir1246 mir320c and mir320d in the exosomes derived from gfpsrsf1 panc1cells was analyzed by qrtpcr fig 5bc as shown infig 5b overexpression of gfpsrsf1 increased exosome expression of mir1246 and rescued mir1246levels in exosomes derived from srsf1shrna cellslevels of mir320c and mir320d were also increased inexosomes derived from the gfpsrsf1 cellsfurthersupporting the involvement of srsf1 in exosomemirna enrichment fig 5cdidentification of rna sequence motifs involved inexosome mirna enrichmentaccording to the rbpdb srsf1 binds specifically to amotif present in the mir1246 sequence supplementfig qrtpcr analysis of mir1246 mir320c mir320d expression in exosomes derived from srsf1 knockdown bxpc3 and miapaca2 cells ac qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown bxpc3 cells n p student ttest df qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown miapaca2 cells n p students ttest 0cxu cell communication and signaling page of fig qrtpcr analysis of exosome enriched mirnas derived from srsf1 overexpression panc1 cells a confirmation of gfpsrsf1overexpression in panc1 cells b qrtpcr detection of mir1246 in exosomes derived from wild type and srsf1 knockdown panc1 cells withgfpsrsf1 overexpression c qrtpcr detection of mir320c in exosomes derived from gfpsrsf1 overexpression panc1 cells d qrtpcrdetection of mir320d in exosomes derived from gfpsrsf1 overexpression panc1 cells p students ttest n for bdfig to understand the contribution of specific rnamotifs involved in exosome mirna enrichment we applied an unbiased approach to identify the rna motifsthat contribute to exosome mirna enrichment for thispurpose we analyzed the rna sequences of the mirnas highly enriched in cancer exosomes and regulatedby srsf1 using the bioinformatics tool meme suite a 6bp length motif was found to be shared in of the exosome enriched mirnasincluding mir fig ac to test whether the binding of srsf1to mir1246 depends on this motif two decoy mimicswere designed according to the shared motif sequencesand their secondary structure determined with thernafold webserver httprnatbiunivieacatcgibinrnawebsuiternafoldcgi the binding of the decoymimics to srsf1 protein was determined by rnaemsa analysis addition of decoy motif did not alterthe binding of srsf1 to the mir1246 probe fig 6dwhereas decoy motif competed with mir1246 binding to srsf1 in a concentrationdependent manner fig6df indicating that srsf1 directly interacts with thissequence motifdiscussionthe role of exosome mirna signaling in promotingcancer progression has been intensely investigated andwell recognized in recent years [ ] the higher enrichment of certain mirnas in cancer exosomes [] indicates that exosome mirna encapsulation isan active cellular process that initiates exosome mirnasignaling in the tumor microenvironment however thespecificselectivecellular processresponsiblefor 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig srsf1associated exosome mirna sequence motif analysis a the motif commonly shared among srsf1associated exosome mirnas bvenn diagram showing the number of srsf1associated exosome mirnas that share the motif c list of mirnas sharing the common motif demsa analysis demonstrating the inhibition of gstsrsf1 binding to mir1246 by rna decoys d1 decoy ²uuggacuaggacuaggau3² d2decoy ²aggaaggaaggaagga3² e concentrationdependent inhibition of gstsrsf1 binding to mir1246 by d2 f semiquantification ofthe detected bands in fig 5e and the calculated dissociation constantexosome mirna enrichment has not been well established in eukaryotic cells the most significant findingfrom the present study is that we have identified srsf1as a mediator of exosome mirna enrichment in pancreatic cancer cells a specific mirna sequence motif wasalso identified that may be involved in the exosomemirna enrichment process these findings provide newinsight into how mirnas are enriched in cancer cellexosomesexosomemediated mirnasignalinginitiatetowe recently reported that exosome mir1246themost highly enriched mirna in pancreatic cancer cellderived exosomes is derived from rnu2 a smallnuclear rna important for mrna splicing alongthis line of our research we sought to determine howthis mirna is enriched in cancer exosomes using ourestablished model systems in the present study we haveprovided severallines of evidence demonstrating thatsrsf1 a vital splicing factor and established oncoprotein is significantly involved in exosome mirnaenrichment in pancreatic cancer cells the first line ofevidence indicating srsf1 involvementin exosomemirna enrichment was obtained from the biotinlabeled mir1246 pulldown experimentfollowed byproteomic analysis among the rbps identified severalwere selected based on their detection intensity relevance to extracellular vesicles and reported connectionsto human cancer including srsf1 eif3b andtia1 of note srsf1 was the only rbp among themthat was also predicted by the rbpdb to bind to a motifin the mir1246 sequence furthermore the direct binding of srsf1 to the mir1246 sequence was verified byrip and rna emsa analysis strongly indicating thephysical interaction of srsf1 and not eif3b or tia1with the mir1246 sequence the most convincing evidence demonstrating the involvement of srsf1 in cancer exosome mirna enrichment was the observationthat knockdown of srsf1 significantly reduces exosomemirna enrichmentfor a majority of the selectivelyenriched exosome mirnas without altering the expression levels of less enriched exosome mirnas these results were based on small rna sequencing andconfirmed by rtpcr analysis the observations werealso extended to additional human pancreatic cancer celllines including miapaca2 and bxpc3srsf1 was initially identified as a splicing factor ineukaryotic cells but srsf1 was later revealed toindependent ofshuttle between the nucleus and cytoplasm to regulate rna metabolism mirna procession and othercellular eventsthe mrna splicingprocess importantly srsf1 is overexpressed indifferent cancer types and is considered a potent oncogene [ ] moreover srsf1 over expression in different types of cancer is associated with worse prognosissupplement fig while the full spectrum of srsf1function remains to be determined our results revealthat srsf1 binds to specific mirnas and is significantlyinvolved in exosome mirna enrichment in cancer cellsthis function is likely independent ofthe splicingprocess as the reduced expression of the detected exosome mirnas after srsf1 knockdown is greater thantheir expression change in the cells because exosomemirna signaling contributes to tumor developmentthrough intercellular communication in the tumormicroenvironmentthe involvement ofsrsf1 in exosome mirna signaling initiation likelyrepresents a part of its oncogenic action which maylead to new therapeutic strategies to intervene withexosome mirna signaling in cancer several rbpshave been previously identified as mediators of exosome mirna sorting in various model systemsincluding major vault protein in colon cancer cells hnrnpa2b1 in t cells and ybx1 in hek293tcells the identification of srsf1 involvement inexosome mirna enrichmentin pancreatic cancercells further supports the notion that the cellular exosome mirna sorting process in eukaryotic cells maydiffer among different cell types[ ]we have also identified a mirna motif commonlyshared by the srsf1associated exosome mirnasusing the meme suite program memesuitethis motif was specifically bound by srsf1 as evidenced by our rna emsa analysis a similar motifalbeit slightly shorter was identified in our recentreport that describes exosome mir1246 enrichmentin pancreatic cancer cells our results reinforcethe concept that specific mirn
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pediatric obesity remains a public health burden and continues to increasein prevalence the gut microbiota plays a causal role in obesity and is a promising therapeutic target speciï¬cally the microbial production of shortchain fatty acids scfa fromthe fermentation of otherwise indigestible dietary carbohydrates may protect against pediatric obesity and metabolic syndrome still it has not been demonstrated that therapies involving microbiotatargeting carbohydrates known as prebiotics will enhance gutbacterial scfa production in children and adolescents with obesity age to yearsold here we used an in vitro system to examine the scfa production by fecal microbiota from children with obesity when exposed to ï¬ve different commercially availableoverthecounter otc prebiotic supplements we found microbiota from all patientsactively metabolized most prebiotics still supplements varied in their acidogenic potential significant interdonor variation also existed in scfa production which 16s rrna sequencing supported as being associated with differences in the host microbiota composition last we found that neither fecal scfa concentration microbiota scfa productioncapacity nor markers of obesity positively correlated with one another together thesein vitro ï¬ndings suggest the hypothesis that otc prebiotic supplements may be unequalin their ability to stimulate scfa production in children and adolescents with obesityand that the most acidogenic prebiotic may differ across individualsimportance pediatric obesity remains a major public health problem in the unitedstates where of children and adolescents are obese and rates of pediatric severeobesity are increasing children and adolescents with obesity face higher health risksand noninvasive therapies for pediatric obesity often have limited success the humangut microbiome has been implicated in adult obesity and microbiotadirected therapiescan aid weight loss in adults with obesity however less is known about the microbiome in pediatric obesity and microbiotadirected therapies are understudied in children and adolescents our research has two important ï¬ndings i dietary prebiotics ï¬ber result in the microbiota from adolescents with obesity producing more scfa andii the effectiveness of each prebiotic is donor dependent together these ï¬ndings suggest that prebiotic supplements could help children and adolescents with obesity butthat these therapies may not be one size ï¬ts allkeywords fermentation microbiome pediatric obesity prebiotics shortchain fattyacidscitation holmes zc silverman jd dressmanhk wei z dallow ep armstrong sc seed pcrawls jf david la shortchain fatty acidproduction by gut microbiota from childrenwith obesity differs according to prebioticchoice and bacterial community compositionmbio 11e0091420 101128mbio0091420invited editor thomas mitchell schmidtuniversity of michiganann arboreditor jose c clemente icahn school ofmedicine at mount sinaicopyright holmes this is anopenaccess distributed under the termsof the creative commons attribution international licenseaddress correspondence to lawrence a davidlawrencedaviddukeedu present address justin d silverman collegeof information science and technologypennsylvania state university state collegepennsylvania usa institute for computationaland data science pennsylvania stateuniversity state college pennsylvania usaand department of medicine pennsylvaniastate university hershey pennsylvania usareceived april accepted july published august julyaugust volume issue e0091420®mbioasm 0cholmes ®approximately of children in the united states have obesity and the prevalence continues to increase among all ages and populations the prevalence ofpediatric obesity is even higher in hispanic and african american populations in theunited states where rates of severe obesity continue to increase children withobesity have an increased risk of adverse health events and incur higher health carecosts despite the severity of the pediatric obesity epidemic current commontreatment strategies centered around lifestyle changes including behavioral dietaryand exercise interventions often fail or have limited success the high prevalence ofpediatric obesity coupled with the low success rate of common interventions highlights the need for more efï¬cacious safe strategies to lower the body mass index bmiin children and adolescentsthe human gut microbiome has emerged as a promising therapeutic target in pediatricobesity over the past decade differences in gut microbial community composition andmetabolic activity between obese and lean individuals have been observed causallinks have also been established fecal transplantation can transfer the obesity phenotypefrom obese donors to lean recipients and recapitulate some key metabolic changes inhuman obesity multiple mechanisms for this link have been proposedincludingincreased energy harvest by obese microbiota activation of enteroendocrine signalingpathways by shortchain fatty acids scfas modulation of glucose and energyhomeostasis through bile acid signaling and increased local and systemic ammation caused by a variety of microbial metabolites recent attention in obesity research has been speciï¬cally drawn to the role ofmicrobially derived scfas scfasprimarily acetate propionate and butyrateareproduced by enteric microbes as end products of anaerobic fermentation of undigested microbially accessible dietary carbohydrates and serve a variety of importantroles in the gut of particular interest is the scfa butyrate which serves as the primarynutrient source for colonocytes and functions as a histone deacetylase inhibitor through its inhibition of nf 242cb signaling in colonocytes butyrate contributesto barrier integrity maintenance and reduces levels of intestinal ammation markers acetate propionate and butyrate also each activate gproteincoupled receptors gprs that modulate key metabolic hormones including peptide yy pyy andglp1 consistent with these mechanistic ï¬ndings mouse studies have shownthat supplementation with acetate propionate butyrate or some mixture of these canprotect against weight gain improve insulin sensitivity and reduce obesityassociatedammation given the experimental evidence for scfa supplementationhaving an antiobesogenic effect in a murine system maintaining high levels of scfasduring a weight loss treatment may improve results if increasing scfa levels is a potential approach to promote weight loss in childrenprebiotic supplementation may provide an effective and lowrisk adjunctive therapyprebiotics are dietary carbohydrates that are indigestible by humanproduced enzymesand thus survive transit to the lower gastrointestinal gi tract once in the colonprebiotics serve as carbon sources for bacterial fermentation which in turn yield scfasas metabolic end products multiple types of prebiotics eg fructooligosaccharides [fos] and inulintype fructans have been tested in children with obesityranging from ages to years old in select cases these treatments have beenassociated with smaller increases in bmi and fat mass and reductions in bodyweight zscores body fat and trunk fat still other prebiotic trials in children whoare overweight have reported no significant beneï¬cial effects interpreting the mixed outcomes of prior prebiotic clinical trials in pediatric obesitythough is complicated by several challenges first in vivo studies in pediatric obesity todate have each used only one prebiotic supplement due to the logistical constraints ofclinical trials trials employing testing only a single type of supplement hinderthe ability to generalize s regarding the efï¬cacy of prebiotics and also makeit challenging to determine whether some prebiotics are inherently more acidogenicthan others second in vivo trials in healthy adults have shown substantial interindividual variation in the single prebiotic effects on stool scfa concentration julyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®variation in the primary and secondary outcomes could be due to differences inmicrobial scfa production or differences in host physiology such as scfa absorptionpotential third while scfa concentrations have been shown to be altered in childrenwho are overweight or obese changes in fecal scfas during dietary interventionhave not been measured in past in vivo studies in pediatric populations if prebioticsmediate their effects through scfas directly tracking scfas could helpdetermine treatment success fourth in vivo studies in adults especially those withobesity may be confounded by the concurrence of chronic disease and the medications a person may be taking to treat chronic diseasein this study we have taken an in vitro approach to address the limitations of priorhuman studies an in vitro approach facilitates more direct comparisons of differentprebiotic supplements the higher throughput of in vitro experiments allows widervariety of prebiotics to be tested and the effects of these supplements can be testedon identical microbiota samples rather than over time within subjects which isconfounded by microbiota drift over time as well as inconsistencies in dietarycomposition taking an in vitro approach to studying the effects of prebiotics on gutmicrobiota allows a more direct investigation of microbial scfa production since wecan study the effects of prebiotic supplementation independent of the effects of hostabsorption using a preclinical in vitro fermentation model and samples fromadolescents with obesity who have not developed longterm complications we pursued three speciï¬c lines of inquiry i whether different types of prebiotics lead todifferences in scfa production by gut microbiota from adolescents with obesity iiwhether the effects of prebiotics are shaped by interindividual differences in gutmicrobiota structure and iii whether fecal scfa production is likely to be associatedwith protection from obesityresultsscfa production capacity to measure scfa production by gut microbiota weadapted the in vitro approach of edwards this method was speciï¬callydesigned to study fermentation of starch in the human lower gi tract and has sincebeen used to measure metabolite production from human stool samples when exposed to prebiotic ï¬ber in brief we homogenized previously frozen feces inreduced phosphatebuffered saline pbs ph «¾ to create a fecal slurry with aï¬nal concentration of gliter fig these fecal slurries were then supplied witheach of ï¬ve prebiotic carbon sources as well as a carbonfree control and allowed toferment at °c in anaerobic conditions for h to approximate colonic transit time after the incubation period the concentrations of scfas in the samples weremeasured by gas chromatography to control for differences in overall cell viability orstool slurry nutrient content between donors we corrected measurements of scfaconcentration by dividing the treatment scfa concentration by the control scfaconcentrationto validate our assay we ran a series of experiments using feces from validationsample sets we veriï¬ed that our controlcorrected scfa production data were notuenced by bacterial abundance p «½ 2433 «½ spearman correlation seefig s1 in the supplemental material absolute not relativized to control scfaconcentrations are supplied in the supplement see fig s2 and s3 since our fermentation experiments used previously frozen fecal samples we veriï¬ed that total scfaproduction was strongly correlated between fresh samples and twice freezethawedsamples p ¬ 2433«½ spearman correlation see fig s4a since we elected tonot provide our fermentation reactions with nutrients in excess of what was containedin the fecal slurries we veriï¬ed that there existed strong correlation in total scfaproduction between pbsgrown and colonic mediumgrown cultures both whensupplied with dextrin and inulin dextrin p «½ 2433«½ inulin p «½ 2433«½ spearman correlations see fig s5 we found that total scfa production over controlwas positively correlated with the ph of starting fecal slurries p «½ 2433 «½ spearman correlation fig 2a a weaker correlation may exist between scfa producjulyaugust volume issue e0091420mbioasm 0cholmes ®fig overview of in vitro fermentation methodstion and the ï¬nal ph of the fermentation vessels p «½ 2433 «½ spearmancorrelation fig 2bwe subsequently applied our assay to fecal microbiota from a cohort of children male female one unknown ranging in age from to years old average age years tanner stages to and a body mass index bmi of to averagebmi see table s1 in the supplemental material one patient provided samplesused in all analyses but was lost to followup before providing clinical metadata thiscohort was a subset of a cohort of patients enrolled in the pediatric obesity microbiomefig relationship between in vitro scfa production and ph a in vitro total scfa production over control is positively correlated with the phof starting fecal slurries p «½ 2433 «½ spearman correlation b relationship between scfa production and the ï¬nal ph of fermentationvessels p «½ 2433 «½ spearman correlationjulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®fig in vitro scfa production by prebiotic a donor b and individually c in a twoway anova of the effects of donor and prebiotic on scfaconcentrationcontrol donor prebiotic and their interaction were all statistically significant p ¬ p ¬ and p ¬ respectively shownis the total scfa concentration of an in vitro culture after h of anaerobic incubation divided by the scfa concentration of the corresponding prebioticfreecontrol culture for each of ï¬ve prebiotic growth conditions across donors black dots gray diamonds are means and gray bars are standard deviationsabsolute scfa concentrations are depicted in fig s3and metabolism study we found all individuals demonstrated a net gain ofscfas relative to the controlin at least one prebiotic treatment which led us toconclude that all tested cultures were viable and metabolically active fig donor and prebiotic both impact scfa production in vitro we next tested thehypothesis that different prebiotics equally promote the production of scfas byjulyaugust volume issue e0091420mbioasm 0cholmes ®fig eighteen genera were found to be credibly associated with scfa production in at least one of our ï¬ve prebiotic growth conditions shownare the mean lambda values and and credible intervals for all genera credibly associated with at least one prebioticgrowth condition plotted on centered logratio clr coordinates red centers denote associations with credible intervals that do not cover lambda represents the strength of the effect of each covariate on each taxa a lambda value of reï¬ects a unit fold change in scfaconcentration over control as being associated with a unit fold change in the clrtransformed relative abundance of the genusperforming statistical analysis of scfa production as a function of the prebiotic typeand individual identity our analysis revealed heterogeneity in the efï¬cacy of prebioticsupplements twoway analysis of variance [anova] p ¬ see table s2 fig 3aranging from inulin which resulted in a mean fold change in total scfas togalactooligosaccharides gos which resulted in mean fold change in total scfasfrequently only two or three of the ï¬ve tested prebiotics resulted in increased totalscfa production within an individual our statistical testing also revealed consistentpatterns between individuals gut microbiota in terms of scfa production twowayanova p ¬ see table s2 fig 3b with mean fold changes in scfas over controlranging from to within individuals the average fold change in scfa concentration in the prebiotic treatments often appeared to be driven by a few stronglyacidogenic prebiotics last our analysis indicated a significant interaction betweenidentity twoway anova p ¬ see table s2prebiotic type and individualfig 3c because our statistical analysis considered technical replicates as separateexperimental conditions this result suggests the presence of consistent prebioticindividual responses across in vitro assay replicate runsnot whether such interactionsare consistent within an individual over timescfa production in vitro predicts the abundance of bacteria in the startingculture if interindividual differences in gut microbiota mediated responses to prebiotictreatment we would expect that speciï¬c bacterial taxa which varied between individuals could also be associated with scfa production to evaluate this hypothesis weused the r package stray to create a bayesian multinomial logistic normal linearregression pibble model that tested for correlations between in vitro scfa productionin response to each prebiotic and 16s rrna community composition of patient stoolused in the fermentations at the genus level this analysis revealed that scfa production from prebiotics was correlated with the relative abundances of differentbacterial genera credible interval not covering fig of the generapositively associated with scfa production are known or likely ï¬ber degraders one akkermansia is often observed to increase in abundance after prebiotictreatment and one methanobrevibacter an archaeon hydrogenotrophic methanojulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesitytable associations between microbial genera and scfa production on ï¬ve different prebiotic substratesassociation with scfa productiondextrin«¹«¹«¹«¹«¹«¹«¹«¹«¹genusakkermansiaruminococcus_2coprostanoligenes_groupparabacteroidesbutyricimonasmethanobrevibactertyzzerella_4tyzzerella_3lachnospiraceae_nk4b4lactobacilluscoprococcus_1collinsellalachnospiraceae_fcs020xosgosfosinulin«¹«¹«¹«¹fiber degrader statussupporterdegraderno evidencedegraderassociatedsupporterdegraderdegraderdegraderdegraderdegraderno evidencedegrader®referencegen is known to increase the efï¬ciency of carbohydrate metabolism by the microbiota table most genera identiï¬ed by stray were associated with scfa production ina limited set of prebiotic treatments one genus lactobacillus is positively associatedwith scfa production on xylooligosaccharides xos but was negatively associatedwith scfa production on gos overall the presence of speciï¬c associations betweenbacterial taxa and different prebiotics supports a model where different individuals varyin their levels of prebiotic degrading gut bacteriametrics of obesity do not appear to correlate with scfa production capacity ofstool finally we tested the hypothesis that in vitro scfa production would beassociated with obesityrelated phenotypes we compared clinical metadata fromindividuals which included bmi insulin and hba1c with average total scfa production across prebiotics and found no significant correlations in our population spearman correlation table fecal microbial scfa production capacity may not be directlyassociated with obesity though because rates of host scfa uptake likely vary and thisvariance may uence host intestinal physiology indeed in support of theidea that scfa absorption rate which was not measured in this study shape metabolichomeostasis and host health we observed a negative association between fecal scfaconcentrations and in vitro scfa production across the range of tested prebioticsfig furthermore if scfa absorption efï¬ciencies varied by individual residual fecalscfa concentrations may not directly reï¬ect the complete effect of bacterial metabolism on obesity consistent with this notion no significant relationships were apparentbetween concentrations of scfa in patient stool and clinical markers of obesitymeasured at enrollment including bmi insulin levels and hba1c table althoughthis may also be explained by uncontrolled patient parametersdiscussionin this study we found that the microbiota of all tested adolescents with obesityincreased total scfa production when exposed in vitro to at least one prebiotic bothdonor and prebiotic were significant factors in determining scfa production in vitro aswas their interaction our modeling revealed distinct associations between speciï¬cmicrobial taxa and scfa production on different prebiotics we interpret this result astable neither average scfa production in vitro nor fecal scfa concentrationcorrelated with metrics of obesity measured in individuals at the time of enrollmentabmiparameteravg net scfa productionfecal scfa concentrationap and 2433 values were determined from spearman correlations 2433pinsulinp 2433hba1cp 2433julyaugust volume issue e0091420mbioasm 0cholmes ®fig spearman correlations between in vitro scfa production and scfa concentration of the starting fecal inoculum scfaproduction is the average of technical replicates with the linear regression line plottedsuggesting that the associated bacteria play a role in the ï¬ber fermenting capacity ofthe community we observed no correlations between either stool scfa concentrationsor in vitro acidogenic capacity of communities and any metrics of obesity table we have recapitulated previous ï¬ndings that both donor and prebiotic areimportant in determining the scfa production from in vitro prebiotic supplementation and we found that not all prebiotics appear equally acidogenic since our in vitro system removes the host as a potential source of variationour data support a gut microbial role for interdonor variation in fecal scfaproduction in addition the strength of the interaction between donor and prebiotic strongly suggests that prebiotics are not one size ï¬ts all rather inconsistentresults from prior studies of prebiotics in pediatric obesity may be dueto variation in the scfa production capacity of individuals gut microbiota acrossthe tested prebiotics future therapeutic efforts involving prebiotics in patients withobesity may beneï¬t from stratiï¬ed or personalized treatments nutritional therapiesthat are personalized to individuals microbiota are already in development murine and in vitro studies show that increased signaling through gpcrs mediatedby acetate propionate and butyrate increases satiety and insulin sensitivity whiledecreasing adipogenesis yet we did not observe associations betweenfecal scfa levels and metrics of obesity the effects of scfa on obesity may be maskedby uncontrolled patient factors such as differences in caloric intake and variation inindividual nutrient harvest and utilization in order to observe the effects of scfa onobesity it would be necessary to control for these variable physiological and lifestyleparameters which we did not attempt these patient factors may also have uencedour inability to observe an association between acidogenic capacity of microbiota andfecal scfa concentrations however this may also be explained by the potentialjulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®uncoupling of fecal scfa production and fecal scfa concentration in vitro increasedluminal concentrations of butyrate have been shown to upregulate the sodiumcoupled monocarboxylate transporter slc5a8 and the addition of physiologicalmixtures of scfa has been shown to upregulate the monocarboxylate transporterslc16a1 both of which uptake acetate propionate and butyrate from the lumensince gut epithelia have the capacity to absorb up to of scfa before excretion increased host scfa uptake triggered by increased gut bacterial production couldtherefore lead to constant or even decreased fecal scfa concentrations this complexrelationship could explain the absence of positive correlations we observed betweenstool scfa levels and the acidogenic capacity of gut microbiota it may be necessary todelve further upstream of fecal scfa concentration by measuring proxies for host scfauptakes such as the expression of scfa transporters slc5a8 and slc16a1 and scfareceptors gpr43 gpr41 and gpr109a the primary limitations of this study involve constraints common to in vitro culturestudies first many factors affecting bacterial scfa production in vivo are difï¬cult toreplicate in vitro including the availability of nutrients such as nitrogen the startingconcentration of scfas the redox state of the environment and the efï¬ciency ofcrossfeeding interactions different metabolic results between prebiotics mayhave occurred if we provided alternative cometabolites or nutrients in addition to thetested prebiotics we chose our culture conditions namely a mediumfree approachthat does not add any nutrients beyond what is present in the stool in an effort toavoid inducing artiï¬cial selective conditions within our cultures prior experimentaldigestion studies have shown that prebiotic response patterns can be recapitulatedacross various culture conditions indeed we found strong correlation in scfaproduction between cultures grown with our mediumfree approach and those grownin a more conventional medium containing added nitrogen vitamins minerals andacetate further this approach allowed us to minimize the uence of the host onmeasurements of microbiota production of scfa we did observe shifts in communitycomposition during the h fermentations fig s6 however we remained able to ï¬ndstatistical associations between scfa production capacity and prefermentation community composition a second set of limitations in this study involves our reliance onpatient collection of stoolinterdonor variation in prebiotic response could haveoriginated in technical variation between how patients exposed stool to aerobicconditions or how they froze their samples which in turn could have affectedthe fraction of viable microbial cells in stool samples still we found a significantcorrelation between in vitro total scfa production from fresh stool and stool that hadbeen frozen and thawed twice variation in donor prebiotic response could also havebiological origins due to physiological differences between people eg efï¬ciency offood digestion consistency of stool [] or differences in diet which can lead tovariation in stool microbial load and nutrient content rather than control for amyriad of different sources of variation whose origins we did not measure we chosethe straightforward approach of standardizing donor samples by employing a consistent concentration of stool slurry [wtvol] stool in pbs in our experimentsfuture work to address these limitations could test multiple stool samples persubject to conï¬rm whether the observed variation in prebiotic response is durablebetween individuals over time future studies could also examine the correlationbetween the metabolic effects of prebiotic supplementation in vitro and in vivo usingrandomized human trials that couple human prebiotic supplementation in vivo measurement of scfa production and in vitro tests of microbiota metabolic activity itwould also be useful for such studies to explore the impact of prebiotic supplementation on host physiology both in vitro and in vivo speciï¬cally the effects of prebioticsupplementation on colonic epithelial barrier integrity scfa receptor gpr41 gpr43and gpr109a expression and scfa transporter mct1 and smct1 expression couldprovide greater insight into the health impacts of prebiotic supplementation as well asexplain why fecal scfa concentrations may not mirror the metabolic capacity of gutmicrobiotajulyaugust volume issue e0091420mbioasm 0c®holmes materials and methodscohort stool was collected from human donors under a protocol approved by the duke healthinstitutional review board duke health irb pro00074547 for a prospective longitudinal cohort studyand biorepository participants whose samples were used in this study were treatmentseeking adolescents with obesity who were newly enrolled in a multidisciplinary weight management program allsubjects received familybased intensive lifestyle modiï¬cation based on clinical necessity some participants also were placed on a lowcarbohydrate diet medications to facilitate weight loss or underwentweight loss surgery see table s3 due to the low number of patients assigned to each treatment armwe did not attempt to base any analyses on patient treatment plan patients were to years old witha bmi ± 95th percentile none had antibiotic use in the month prior to enrollment used medicationsknown to interfere with the intestinal microbiome or had other significant medical problems stoolsamples used in this study were from enrollment 3month 45month and 6month followup visits seetable s3 the clinical metadata used for correlations was collected at enrollment months and months the metadata collected nearest to the stool sample collection date was used in our analysesstool collection patients collected intact stool samples in the clinic or at home using a plastic stoolcollection container fisher scientiï¬c and were asked to immediately store this container intheir home freezer patients then returned the sample by either bringing it to the study team orscheduling a home pickup within h of stooling stool was transported frozen in an insulated containerwith an ice pack upon receipt in the lab samples were placed on dry ice until transferred to a °cfreezer for longterm storage all patient samples were frozen at °c within h of stooling range h to h median h except for one which was stored h after stooling the timebetween stooling and freezing at °c did not have a significant effect on average scfa productionp «½ 2433 «½ «º pearson correlation stool samples for analysis were processed by removingcontainers from °c storage and thawing on ice in a biological safety cabinet until soft enough toaliquot thawed containers of stool were opened to atmosphere for a maximum of min while sampleswere aliquoted after primary aliquoting the remaining stool was transferred to an anaerobic chambercoy laboratory products hydrogen co2 nitrogen and further portioned into ¬2galiquots for this study these aliquots were then stored as solid stool pellets at °c until used for thisstudyin vitro fermentation see fig for an overview of in vitro fermentation methods aliquotedstool was thawed at room temperature in an anaerobic chamber once thawed stool was weighedand placed into a polyethylene ï¬lter bag with 033mm pore size whirlpak b01385 and ml ofanaerobic «» pbs was added for each gram of stool resulting in a wtvol fecal slurry similarto previous studies during our validation experiments a medium designed tosimulate colonic contents was used in place of «» pbs to create stool slurries the ï¬lter bag wasthen closed and placed into a stomacher seward stomacher where the contents were homogenized on the medium speed setting for s the liquid fraction was removed from the downstreamside of the ï¬lter membrane and the solid fraction was discarded a 1ml aliquot of this liquid fractionwas removed for analysis of the scfa concentration to determine the scfa concentration of thestarting stool sample during our validation experiments two separate 1ml aliquots of this liquidfraction were removed one was used to estimate relative bacteria abundance of starting fecalslurries using total extracted dna concentration as has been previously published and theremaining aliquot was used to determine the ph of the starting fecal slurry using a handheld phmeter elite ph spear thermo fisher scientiï¬c the remaining liquid fraction was incubated induplicate across six different treatments either supplemented with inulin now foods inulin powderpart fructooligosaccharides fos cargill part galactooligosaccharides gosbimuno powder xylooligosaccharides xos bionutrition prebiotic with llifeoligo part wheatdextrin beneï¬ber original or unsupplemented for each reaction ml of fecal slurry wasplaced in one well of a 24well cell culture plate each well was then delivered ml of wtvolprebiotic solution in «» pbs or ml of «» pbs without prebiotic during our validation experimentsprebiotics were dissolved in colonic medium instead of «» pbs the resulting fermentationconditions were therefore fecal slurry with prebiotic wtvol a fecal slurry was selectedbecause its fermentative capacity has been previously demonstrated to be insensitive to smallvariations in concentration and is feasible to work with using this method a ï¬nalconcentration of prebiotic in the context of a fecal slurry i
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Patients with resected oral cavity squamous cell carcinoma OCSCC are often treated with adjuvantradiation RT ± concomitant chemotherapy based on pathological findings Standard RT volumes include all surgicallydissected areas including the tumour bed and dissected neck RT has significant acute and longterm toxicitiesincluding odynophagia dysphagia dermatitis and fibrosisThe goal of this study is to assess the rate of regional failure with omission of radiation to the surgically dissectedpathologically node negative pN0 heminecks compared to historical control and to compare oncologic outcomestoxicity and quality of life QoL profiles between standard RT volumes and omission of RT to the pN0 neckMethods This is a multicentre phase II study randomizing patients with T1 N0 OCSCC with at least one pN0hemineck in a ratio between standard RT volumes and omission of RT to the pN0 heminecks Patients will bestratified based on overall nodal status nodal involvement vs no nodal involvement and use of concurrentchemotherapy The primary endpoint is regional failure in the pN0 heminecks we hypothesize that a 2year regionalrecurrence of or less will be achieved Secondary endpoints include overall and progressionfree survival localrecurrence rate of salvage therapy toxicity and QoLDiscussion This study will provide an assessment of omission of RT to the dissected pN0 heminecks on oncologicoutcomes QoL and toxicity Results will inform the design of future definitive phase III trialsTrial registration Clinicaltrialsgov identifier NCT03997643 Date of registration June Current version onJuly Keywords Head and neck cancer Oral cavity Radiotherapy Recurrence Survival Quality of life Randomized controlledtrial Deescalation Correspondence pencillalanglhsconca1Division of Radiation Oncology London Health Sciences Centre Commissioners Rd E London ON N6A 5W9 CanadaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLang Radiation Oncology Page of BackgroundPatients with resected squamous cell carcinoma of the oralcavity OCSCC are at risk of locoregional failure recurrence LRR at either the tumour surgical bed or in theneck Postoperative radiotherapy PORT is often addedafter surgical resection to reduce the risk of local and regional recurrence in patients with highrisk featuresGuidelines generally recommend PORT in patients withmore than one lymph node involved pT3 or pT4 primarydisease lymphovascular invasion LVI perineural invasion PNI close or positive margins extranodal extensionENE and sometimes for patients with lymph node involvement of neck levels IV or V []ENE and positive surgical margins are considered thehighestrisk pathological features whereas the other adverse features are considered intermediate risk Patientswith highrisk features are generally offered concurrentchemotherapy with PORT based on a posthoc analysistwo of randomized trials [] In patients with otheradverse features there is limited randomized evidence ofthe benefits of PORT alone [] Retrospective seriesand comparisons with historical controls have shown reductions in LRR and improvement in overall survivalOS with PORT []RT treatment volumes after surgery generally includethe entire surgical bedincluding the dissected heminecks and may include the contralateral undissectedneck at the discretion of the treating physician []However PORT is associated with significant acute andincluding dysphagia mucositis xerostolate toxicitiesmiavoicechanges ototoxicity and hypothyroidism [ ] LargerRT treatment volumes are associated with increasingtoxicity These large treatment volumes are based onhistorical practice without guidance from randomizedevidence The benefit of treating the nodal regions in thepathologically node negative pN0 neck is unknownosteoradionecrosisdermatitisfibrosisRetrospective studies looking at series of oral cavitycancer patients in whom PORT was omitted altogetherhave also demonstrated low rates of isolated nodal recurrence ranging from to [] In their seriesof patients with mostly T1T2 oral tongue tumourswith a pN0 neck treated with surgery alone PORTto the primary site or PORT to the primary siteand neck So reported a rate of isolatednodal recurrence of in all groups [] Mizrachi described a series of patients with T1T4 disease and pN0 neck with not receiving PORT with aneck recurrence rate of [] In Ganly patients with pT1T2 pN0 oral tongue cancer withoutPORT had an isolated regional recurrence rate of with an additional having a simultaneous locoregional recurrence In this study depth of invasion was predictive of neck recurrence []In a series of patients with a pN1 neck the neck recurrencefree survival was in those receiving PORT vs in thosewithout PORT [] It is difficult to draw comparisonsbetween groups receiving or not receiving PORT inthese retrospective studies since those receiving PORThad more risk featuresRetrospective data from a small number of PORT volume studies also demonstrate good oncologic outcomesin patients where radiation was omitted to the contralateral clinically or pathologically N0 neck [ ] In Vegeer patients with welllateralized oral cavity ororopharynx squamousSCC weretreated with unilateral PORT with of patients N0and N1 or N2 Contralateral metastases developedin only of patients with most successfully receivingsalvage therapy []carcinomacellA recent nonrandomized prospective phase II trialeliminated PORT to the pN0 neck in patients withhead and neck squamous cell carcinoma HNSCCdemonstrating excellent results with no isolated failuresand control in the unirradiated pN0 neck Twentypercent of patients included in the study had oral cavitytumours []Taken together the existing retrospective and prospective data suggests that omitting PORT in patientswith a pN0 neck likely has a recurrence rate less than However no randomized studies have directlyexamined the omission of PORT in the pN0 neck Thereis also a paucity of data examining the effects of omission of PORT on QoL We hypothesize that omittingPORT to the heminecks that have been dissected andshown to be pN0 will be associated with acceptable ratesof regional recurrence and will improve quality of lifeQoL The goal of this randomized phase II study is toassess oncologic outcomesfunctional outcomes andQoL in patients treated with PORT to the historicallystandard volumes usually including the primary site andall dissected neck areas vs PORT only to the primarysite and pathologically involved hemineck omitting radiation to the pN0 heminecksMethods designObjectivesThe objectives of this trial are to Compare regional recurrence rate to historicalcontrols with omission of PORT to the pN0 neck Compare oncologic outcomes toxicity and QoL forPORT ± chemotherapy based on standardtreatment volumes including the primary sitetumour bed dissected neck ± elective nodalregions vs PORT [± chemotherapy] that avoidstreating the dissected pN0 neck 0cLang Radiation Oncology Page of anisation for Research and Treatment of CancerEORTC Quality of Life Cancer Patients generalQLQC30 [] and head neck HN35 scales[] the EuroQOL 5Dimension 5Level EQ5D5L [] and the Neck Dissection Impairment IndexNDII [] 0f QoL measured at treatment completion and and monthsmeasured with MDADI EORTC QLQC30HN35 EQ5D5L and NDII 0f Rate of feeding tube insertion after start of radiationeither gastric gastrojejeunal or nasogastric andrate of feeding tube use at 1year postrandomization 0f Swallowing function at 1year assessed by ModifiedBarium Swallow MBS study and measured by theModified Barium Swallow Impairment ProfileMBSImP¢ score [] the Dynamic ImagingGrade of Swallowing Toxicity DIGEST¢ score []and the Functional Oral Intake Scale FOIS [] 0f Toxicity assessed using the National CancerInstitute Common Toxicity Criteria NCICTCversion 0f Rate of failure in the clinically node negative neck ifapplicable ie the undissected nodenegative neckfor welllateralized tumoursInclusion criteria 0f Age years or older 0f Willing to provide informed consent 0f Eastern Cooperative Oncology Group ECOG 0f Resected OCSCC with at least an ipsilateral selectiveneck dissection The oral cavity includes lips buccalperformance status It is generally accepted that a risk of nodal recurrenceof is sufficient to warrant radiation to a nodalbasin Our hypothesis is that for patients with SCC ofthe oral cavity T1 N0 as per AJCC 8th editionwith at least one surgically dissected pN0 hemineck theregional failure rate will be or less at years whentreated with PORT omitting the pN0 neckStudy designThis is an label phase II multicentre randomizedtrial Patients will be randomized between current standard of care treatment Arm vs omission of radiationto the pN0 dissected heminecks Arm in a ratioFig The required sample size is patients Stratification factors include neck nodal status pN0 vs pNand use of chemotherapy Patients will be recruited fromtertiary care centres full list of participating sites available on clinicaltrialsgov NCT03997643Primary endpoint 0f Regional failure in the pN0 heminecksSecondary endpoints 0f OS 0f Diseasefree survival DFS 0f Local recurrence 0f Regional recurrence 0f Locoregional recurrence 0f Rate of salvage treatment surgery ± radiotherapy inthe pN0 neck and freedom from unsalvageable neckrecurrence 0f QoL at year assessed with the MD AndersonDysphagia Inventory MDADI [] the EuropeanFig Study schema 0cLang Radiation Oncology Page of mucosa oral tongue floor of mouth gingivaretromolar trigone and hard palate 0f Patient has at least one pathological feature that isan indication for PORT positive or close ¤ mmmargin presence of LVI or PNI pT3 or pT4 diseasepositive lymph nodes or ENE 0f PORT is recommended by the treating physician 0f Pathologically lymph node negative in at least onedissected hemineck with at least nodes recovered in each pN0 hemineck after a dissection thatat minimum includes nodal levels in the pN0heminecks 0f Radiation contours have been peerreviewed andapprovedExclusion criteria 0f Patients with an ipsilateral neck dissection only withpositive lymph nodes unless they undergo acontralateral neck dissection that is pN0 0f Patients with bilaterally involved neck nodes 0f Patients with pT3T4 tumours involving midlinewho undergo an ipsilateral neck dissection unless acontralateral neck dissection is performedcontraindications to radiotherapy 0f Serious medical comorbidities or other 0f Prior history of head and neck cancer within years 0f Any other active invasive malignancy except noncavity or neckmelanotic skin cancers lowrisk prostate cancer andStage IIVA papillary or follicular thyroid cancer 0f Prior head and neck radiation at any time 0f Prior oncologic head and neck surgery in the oral 0f Known metastatic disease 0f Locoregional disease recurrence identified followingsurgical resection but prior to start of radiotherapy 0f Inability to attend full course of radiotherapy or 0f Unable or unwilling to complete QoL questionnaires 0f Pregnant or lactating womenfollowup visitsPretreatment evaluation 0f History and physical examination by a radiationoncologist within weeks prior to randomization 0f Staging prior to randomizationCT or MRI of the neck with contrast unlesscontraindicated within weeks ofrandomization This can include the preoperativeCT or the radiation therapy CT or MRI simulation if reviewed by a radiologistª In some instances suspicious lymph nodes arevisible on the scan after surgery In suchinstances recurrence must be ruled outpathologically before enrollment either with aneedle biopsy or resection of these nodesCT of the chest or whole body PETCT usually prior to surgery must be within weeks ofrandomizationedentulous patientswithin weeks of randomization 0f Histological confirmation of SCC 0f Pregnancy test for women of childbearing age 0f Dental evaluation prior to starting treatment except 0f Assessment of dysphagia using NCICTC version 0f Completion of QoL scoring prior to initiation of 0f Prior to randomization radiation contours are to be 0f MBS at baseline prior to initiation of treatment with within weeks of treatment initiationpeerreviewed and approvedtreatmentdocumentation of the MBSIimP¢ DIGEST¢ andthe FOIS scoresresectionTreatment planSurgicalandchemotherapy will be delivered in accordance with National Comprehensive Cancer Network NCCN ClinicalGuidelines []and adjuvantradiotherapyPrimary tumours should be resected en bloc wheneverpossible with the goal of achieving clear margins Patients with midline involvement of the primary tumourshould receive a bilateral neck dissectionAdjuvant cisplatinbased chemotherapy concurrentwith radiotherapy is at the discretion of the treatingmedical oncologist and is recommended for patientswith positive margins or ENE for patients who can tolerate chemotherapy For patients who are deemed unfitor too elderly years of agefor cisplatinbasedchemotherapy the standard dose andor schedule canbe modified alternative systemic therapy regimensmaybe used eg weekly carboplatin Calais regimen orsystemic therapy can be omitted at the discretion of thetreating physicians It is strongly recommended that radiation starts within weeks of the date of surgery andit is mandatory to start no more than weeks after thedate of surgeryDuring treatment supportive care should be in accordance with local standard of care which often includes speech language pathology SLP assessment AnySLP interventions eg providing swallowing exercisesshould be the same in both arms and conform to localstandard of careDosefractionation In both arms a dose of Gy in fractions will be delivered to the operative bed targetvolumes Centres that normally treat dissected nodenegative levels to Gy in fractions will be permitted 0cLang Radiation Oncology Page of to do so if used consistently for all patients on trialAreas of positive margins or ENE should receive Gyin fractions if those areas can be localized Undissected areas that require coverage in the opinion of thetreating radiation oncologist eg low neck retrostyloidspace should receive Gy in fractions Table Immobilization and localization All patients will beimmobilized in a custom thermoplastic shell and willundergo a planning CT simulation with or withoutIV contrast encompassing the head and neck tobelow the clavicles The planning CT will be fusedwith other diagnostic imaging eg MRI scans or preoperative CT where necessary Bite blocks tonguedepressors jaw separators may be used as per institutional protocolthetime of planning CT prior to contour generation andpatient randomizationthese must be determined atRadiotherapy volume definitions A randomization volume will be defined as the nodal volume in the dissectedpN0 heminecks The randomization volume will depend on the laterality of the neck dissection performedipsilateral vs bilateral and whether pathologically involved nodes are present in the neck dissection definedin Fig Patients with bilaterally involved neck nodesare ineligible Patients with a unilateral neck dissectionwith positive lymph nodes are ineligible unless theyundergo a staged neck dissection of the opposite sidethat is pN0Standard treatment volumes Arm If randomizedincluding thecontoured volumesto Arm randomization volume will be treatedallOmission of pN0 neck Arm If randomized toArm the randomization volume will be omitted fromtreatment planningClinical Target Volumes CTVThe following radiation volumes will be contoured forall patients prior to randomization Table The suffixpos denotes CTVs in the nodepositive hemineck andthe suffix neg denotes the CTVs in the nodenegativehemineck Highrisk volume CTV64 regions correspondingto positive margins or ENE if present and if thoseareas can be localized Primary tumour operative bed CTVp60surgically dissected areas corresponding to theresected primary tumour typically including thepreoperative tumour area and any flap reconstructions and clips with a margin Involved necka Surgically dissected involved neck nodalvolume CTVn60pos surgically dissected necklevels in the nodepositive hemineck if applicable In the dissected neck some centres routinely treat only involved nodal areas to Gyand the remainder of the dissected neck to Gy For centres where this is standard this approach is acceptable but all patients enrolledfrom these centres must be treated with this approach The areas treated to Gy would be included in a CTVn54posb Optional uninvolved lowrisk neck on the involved side CTVn54pos lowrisk undissectedneck nodal volume on the involved side if applicable at the discretion of the radiation oncologist This may include the standardlymphatic drainage sites not dissected at thetime of surgery in the node positive heminecksuch as the nodal levels below or above the dissected areas eg level IVb retrostyloid space Randomization Volume corresponding to theUninvolved necka Surgically dissected involved neck nodalvolume CTVn60neg surgically dissectedneck levels in the node negative hemineck Incentres that routinely treat the pN0 neck to Gy that dose is acceptable but all patients mustbe treated with that approach In such centresthese areas treated to Gy would be includedin a CTVn54negb Optional uninvolved lowrisk neck on theuninvolved side CTVn54neg lowrisk undissected neck nodal volume on the uninvolvedside if applicable at the discretion of theTable Radiation treatment volumes and dosesPTV VolumePTV64Areas of positive margin or ENEPTV60Dissected neckPTV54optionalNot surgically dissected elective nodal regionsCTV Volumes IncludedArm CTV64CTVp60 CTVn60posCTVn60negCTVn54posCTVn54negArm CTV64CTVp60 CTVn60posCTVn54posDose infractions Gy Gy Gy 0cLang Radiation Oncology Page of Fig The Randomization Volume corresponds to the pN0 heminecks The neck volumes included in the Randomization Volume dependon whether the patient had an ipsilateral vs bilateral neck dissection and the pathological findings in each heminecks Patients with bilaterallyinvolved neck nodes are ineligible Patients with an ipsilateral neck dissection with positive lymph nodes are ineligible unless they undergo acontralateral neck dissection that is pN0 If randomized to standard treatment volumes Arm all contoured volumes including theRandomization Volume will be treated If randomized to omission of the pN0 neck Arm the Randomization Volume will be omitted fromtreatment planningradiation oncologist In a patient who has had aunilateral neck dissection that was pN0 thismay include the contralateral clinically nodenegative cN0 neck if that is standard institutional practiceIf randomized to omission of PORT to pN0 neckArm the CTVn60neg and CTV54neg contours aredeleted after randomization prior to treatment planningTo prevent bias contours cannot be changed afterrandomization Local peer review of contours must takeplace before randomization occurs An overview of theprotocol timeline is shown in Fig Planning Target Volumes PTVA mm expansion is used around the combinedCTVs Table to create the PTV as per institutionalsetup and protocolRadiotherapy planning Intensity modulated radiotherapy IMRT photon therapy or proton therapy will beused for all patients in this study IMRT can be deliveredusing staticbeam techniques or rotational techniqueseg Tomotherapy or Volumetric Modulated Arc Therapy [VMAT] If protons are used the dose will be reported in Gy relative biological effectiveness [RBE]where the proton dose is multiplied by an RBE of All reported doses in Gy are considered equivalent Centres with proton therapy will use their institutionalstandard planning and delivery techniquesAll plans will be normalized to ensure that ofeach PTV is covered by of the prescription dosefor that volume An exception will be allowed forcentres that normally prescribe in such a manner that of the PTV be covered by of the prescription dose and such centres must prespecify this before enrolling the first patient and all subsequentpatients must be planned in the same manner Amodified PTV cropped mm from the externalcontour for dose evaluation may be used as per institutional guidelines 0cLang Radiation Oncology Page of 0f All dose delivery for intensitymodulated plans including arcbased treatments will be confirmed before treatment by physics staffConebeam CT andor orthogonal xrays will be usedon a daily basis to verify treatment positioning as per institutional standard practiceCredentialling Prior to enrolling patients each centrewill be given a sample CT dataset through secure filetransfer protocol FTP for contouring planning andphysics QA Enrollment can begin once the plan andQA have been approved at the London Regional CancerProgram Centres who have been accredited for ORATOR [] or ORATOR2 [] are exempt from thisrequirementParticipant discontinuation withdrawalParticipants may voluntarily discontinue participation inthe study at any time If a participant is removed fromthe study the clinical and laboratory evaluations thatwould have been performed at the end of the studyshould be obtained If a participant is removed becauseof an adverse event they should remain under medicalobservation as long as deemed appropriate by the treating physicianFollowup evaluationDay of followup will be the first day of radiotherapyFollowup will consist of history and physical examination with laryngopharyngoscopy CT imaging of theneck ± thorax QoL assessments and MBS The followup schedule is shown in Additional file Additional imaging or laboratory investigations should be carried outat the discretion of the oncologist based on findings inthe history or physical examination Additional treatment eg salvage treatment with surgery or furtherradiotherapy is at the discretion of the treating physicians but will be recorded in the case report formMeasurement of outcomes 0f pN0 neck failure measured as time fromrandomization until disease recurrence in theinitially pN0 heminecks Patients with prior orsimultaneous recurrence at the primary site or inthe initially pN hemineck will be censored for thisoutcome as of that timepoint The primary endpointis a comparison of pN0 neck failure in Arm vshistorical controls this endpoint will also be compared between the two arms as a secondaryendpoint 0f OS measured as time from randomization untildeath from any causeFig Flowchart showing timing of randomization with respect topeer review and treatment planning Contours must be finalizedbefore randomization and may not be changed after randomizationThe maximum dose to PTV64 or PTV60 if no PTV64present should not exceed of the prescribed doseand no volume cc outside of these PTVs should receive of the prescription doseans at Risk OAR definitions dose constraints andplanning priorities are adapted from the following protocols RTOG protocols [] Arm and RTOG [] Arm NCICCTG HN6 ORATOR [ ] andORATOR2 [] and are described in Additional file Quality assurance QAIn order to ensure patientsafety and effective treatment delivery a robust QAprotocolis incorporated The following requirementsmust be completed for each patient 0f Prior to randomization each set of contours will bepeerreviewed either by another individual radiationoncologist or at a team head and neck QA rounds 0cLang Radiation Oncology Page of 0f DFS measured as time from randomization toeither recurrence at any location or deathwhichever occurs first New primary tumours willnot count as DFS events 0f Local recurrence measured as time fromrandomization until disease recurrence at theprimary site 0f Regional recurrence measured as time fromrandomization until disease recurrence anywhere inthe neck 0f Locoregional recurrence measured as time fromrandomization until disease recurrence anywhere inthe neck or at the primary site whichever occursfirst 0f Recurrence in the pN0 neck without otherlocoregional recurrence measured as time fromrandomization until disease recurrence in the pN0neck alone without recurrence at the primary site orpN neck 0f Rate of salvage surgery andor radiation in pN0neck measured as time from randomization tosalvage intervention surgery ± radiation in the pN0neck Freedom from unsalvageable neck recurrencewill be reported as the time from randomization todevelopment of a neck recurrence in the pN0 neckthat could not be salvaged 0f Feeding Tube Insertion Rate of feeding tubeinsertion after start of radiation either gastricgastrojejeunal or nasogastric and rate of feedingtube use at 1year postrandomization Patients withfeeding tubes inserted prior to randomization will becensored for this endpoint 0f Rate of failure in the clinically node negativeneck for patients who have unilateral neckdissections the cN0 neck may be treated withradiation or observed at the discretion of thetreating oncologist see section This is measuredas the time from randomization to failure in the cN0neck and will be reported for the whole group ofpatients who had unilateral dissections and alsostratified by whether radiation was delivered to thatareaEnrollment randomization and allocationPatients will be enrolled by dedicated clinical trials staffandor the investigator at each participating institutionPatients will randomized in a ratio to standard radiation volumes Arm vs omission to the pN0 neckArm A permuted block design with two stratification factors will used with the size of the blocks knownonly to the statistician stratified based upon overall necknodal status pN0 vs pN and use of concurrentchemotherapy yes vs no Randomization sequences aregenerated for each strata separately with a randomnumber generator based on permuted block design Thisgets formatted as a CVS file which gets uploaded intoREDCap []Statistical considerationsSample size The primary endpoint is the rate of relapsein the pN0 neck ie the regional relapse rate in the pN0neck in Arm compared to historical controls Allother endpoints are a comparison between Arm andArm It is generally accepted that a risk of nodal recurrenceof is sufficient to warrant radiation to a nodalbasin and therefore we wish to exclude a risk of regionalrecurrence in the pN0 neck of at 2years Using aonesided onesample binomial test allowing for dropout a sample size in Arm of patients provides power at a significance level to detect a regional control rate in the pN0 neck of compared toan unacceptable level of This sample size in Arm and in Arm alsoallows for power to detect a 10point difference inthe total MDADI score at year a secondary endpointassuming the scores are normally distributed with astandard deviation of in each arm It is generally believed that a 10point difference in standardized QoLscores represents a clinically significant difference inQoL [] It is assumed that the QoL scores will be normally distributed with a standard deviation of in eacharm The MDADI will be calculated as the compositescore but will also be reported for each of the subscalesAnalysis planPatients will be analyzed in the groups to which they areassigned intentiontotreat Oncologic outcomes andOS will be calculated from date of randomization usingthe KaplanMeier method A onesided onesample binomial test will be used for the primary endpoint as described above For actuarial comparisons between armsthe stratified logrank test will be used stratified bystratification factors An twosample Ttest will be usedto compare QoL scores at 1year The percentage of patients in each arm who experience a clinically significantQoL decline on the MDADI points will also bereportedMBSImP¢ scores will be compared at 1year including the total score and the scores on the oral andpharyngeal subscales using a twosample Ttest or aWilcoxon rank sum test as appropriate DIGEST¢ andFOIS scores will be compared using the Chisquare testPreplanned subgroup analysis will occur based on thestratification variables nodal status [pN0 vs pN] anduse of concurrent chemotherapy as well as based onthe neck dissection performed unilateral vs bilateral 0cLang Radiation Oncology Page of and Tstage T1 vs T3 There will also be a preplanned analysis based on the extent of nodes harvestedin the pN0 neck vs or more depth of invasionof the primary tumour mm vs mm or more andphoton versus proton treatmentMultivariable Cox proportional hazards or logistic regression analysis as appropriate will be used to determine baseline and pathologic factors predictive of pN0neck failure DFS OS locoregional recurrence and salvage therapy For the secondary endpoints involvingQoL scales linear mixed effects models will be used forthe MDADI the total scores will be compared betweenthe two arms whereas for the EORTC QLQC30 andHN35 scales each of the subscales eg pain swallowing etc will be compared between the two armsRates of grade ¥ toxicity and use of feeding tubes willbe compared between arms using the Chisquare orFishers exact test as appropriateUtilities will be calculated from the EQ5D5L whichwill be administered at baseline and at month intervalsQuality adjusted life years QALYs will be assessed asthe area under the preferenceweighted survival curveOverall costs of each treatment strategy will be abstracted from the available literature The incrementalcost effectiveness ratios ICERs between treatment armswill be compared through the standard method of ratiobetween differences in costs and QALYs Point estimatesfor these differences can be derived from multivariablegeneralized estimating equations GEE or generalizedlinear model GLM analysesData safety monitoring committeeThe Data Safety Monitoring Committee DSMC consisting of at least one radiation oncologist and one medical oncologist not involved in the study will meet every months after study initiation Toxicity outcomes willbe monitored but since the experimental arm involvessmaller radiation volumes it is extremely unlikely thattoxicity would be higher in the experimental arm andtherefore no stopping rules for toxicity are includedInterim analysis The DSMC will conduct one interimanalysis once patients have been accrued and completed the 6month QoL questionnaires For this analysis the DSMC will be blinded to the identity of eachtreatment arm but QoL recurrence in the pN0 neckOS and DFS estimates at 2years will be presented foreach armThe DSMC will recommend stopping the trial if thereis an OS difference that is statistically significant with athreshold of p using the stratified logrank testbased on the HaybittlePeto stopping rule this retainsan overall alpha of Ethical considerationsThe Principal Investigator will obtain ethical approvaland clinical trial authorization by competent authoritiesaccording to locallaws and regulations The WorldHealth anization WHO Trial Registration Data Setis shown in Additional file Institutional review board IRB research ethicsboard REB The protocol and any
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collagen triple helix repeat containing1 cthrc1 anextracellular matrix ecm protein was identiï¬ed in thescreening of diï¬erentially expressed sequences between balloon injury and normal arteries the evolution of cthrc1can be traced back to at least million years ago and theconserved genes were not found in invertebrates cthrc1has complicated interactions with various intracellular andextracellular matrices in diï¬erent ways of secretion [ ]cthrc1 increases the activity of collagen promoter throughbinding to ligands and could contribute to vascular remodeling by limiting collagen matrix deposition and promoting cellmigration cthrc1 promotes the recruitment of m2macrophages and regulates tgf and notch pathways toaccelerate wound healing in a mouse model of acute woundhealing as a coupling factor cthrc1 can be secretedby osteoclasts and uence bone formation and remodelingby acting on osteoblasts and osteocytes [ ] cthrc1 maypromote il1induced apoptosis of chondrocytes by activating the jnk12 pathway the antiammatoryeï¬ect of cthrc1 expressed on activated synovial cellswas also found in a collagen antibodyinduced arthritismodel besides cthrc1 can regulate physiologicalfunctions such as fat and glycogen synthesis and promoteautonomous activity [ ]therefore as a secreted protein cthrc1 is involved inmultiple pathophysiologies a remarkable eï¬ect is that thehigh expression of cthrc1 promotes tumorigenesis anddevelopment through positive regulation of tumor spreadinvasion migration adhesion and metastasis cthrc1exerts its eï¬ects through several signaling pathways such as 0cmediators of ammationgpa gvp grd gsp gan gip gtp gip grd gfk gek gechydrophobic regiongxy repeatcysteinesnglycosylationnh2signal peptidecollagendomainc c c cc cc ccoohfigure the structure of the cthrc1 protein the construct of cthrc1 contains an nh2terminal peptide for extracellular secretion ashort collagen triple helix repeat of amino acids and a coohterminal globular domain the prolinerich hydrophobic domain liesbetween the 1st and 30th amino acids and serves as a signal peptide for transport to the endoplasmic reticulum cthrc1 comprises acollagen domain between amino acids and and the protein contains cysteine residues corresponding to about cysteine inthe ï¬nal protein what is more its only amino acid posttranslational modiï¬cation is the glycosylation of asparagine at position integrin faktgf wntsrcfak mekerkpi3kakterk hif1α and pkcδerk signaling pathways in this we focus on the advances in the signalingpathways mediated by cthrc1 in tumors the structural characteristics andexpression of cthrc1 the structural features of cthrc1 the cthrc1 geneis located at chromosome 8q223 and it contains ï¬ve exonsin humans and four exons in mice it covers kb onthe direct strand and can be transcribed into kb mrnathe amino acid sequence identity between human and ratcthrc1 proteins was and no homologs were foundin lower species [ ]protein nglycosylationsecreted cthrc1 exists primarily as a dimer kdaand a trimer kda as well as multimers of the trimericcthrc1 kda and kda the construct of cthrc1contains an nh2terminal peptide for extracellular secretiona short collagen triple helix repeat of amino acids and acoohterminal globular domain [ ] similar molecularweight and structural characteristics to adiponectin alsoexplain why cthrc1 can form high molecular weightcomplexes the biological activity of cthrc1 isrestricted to the highly conserved amino acids at thecterminal region and the cterminal region of cthrc1contains a putative nglycosylation site that stabilizescthrc1promotescthrc1 to tether to the cell membrane which promotesactin polymerization and cell polarity a short collagen motif with glyxy repeats presents in c1qtumornecrosis factorαrelated proteins ctrps which appearsto be responsible for the trimerization of protein and renders molecule susceptible to cleavage by collagenase seefigure however dimeric cthrc1 would not be susceptible to cleavage by collagenase [ ] the molecularweight of secreted cthrc1 kda appears to be largerthan that of cellular cthrc1 kda cthrc1 has fourdiï¬erentabout kda to kda the fulllength of cthrc1 accountsfor both secreted and cellular cthrc1 glycosylatedprotein cthrc1 with a signal sequence is related to ecmisoforms with molecular weights ofalsoand contains a variable short collagenlike motif intriguingly cthrc1 plays a role in inhibiting structural proteins unlike other members of the collagen family moreover leclair found that cthrc1 cleaved atthe nterminus by plasmin shows better inhibition of collagen synthesis compared to fulllength cthrc1 in thepac1 cell line these studies suggested that cthrc1might obtain biologicalthrough proteolyticprocessingactivity the expression of cthrc1 cthrc1 is transientlyexpressed by ï¬broblasts in remodeling adventitia and bysmooth muscle cells in the neointima of injured tissuehowever cthrc1 is not detected in normal arteries ininjured arteries and skin the expression of cthrc1 isassociated with myoï¬broblasts and locates in the sites ofcollagen matrix deposition in micethe ï¬rst exon ofcthrc1 was targeted to be replaced with a galactosidase expression construct which demonstrated the expression of cthrc1 in inner ear hair cells there iscthrc1 expression in many mesenchymalderived cellsduring body growth and tissue repair in mouseembryos cthrc1 is expressed in visceral endodermnotochord neuraltube developing kidney and heartabundant expression of cthrc1 is observed in developincluding cartilage primordia growth plateing skeletoncartilagein adultscthrc1 is expressed only in bone matrix and periosteum cthrc1 is also found in the matrix of calcifyingatherosclerotic plaques and mineralized bone of skeletaltissues in humans in other tissues the sites of cthrc1expression overlap considerably with interstitial collagensand transforming growth factor tgf family membersparticularly bone morphogenetic proteins bmps the sitesof cthrc1 expression are characterized by the presence ofactive tgf and abundant collagen synthesis cthrc1mrna expression levels are increased in response to bmp4bmp2 and tgf furthermore tgf signaling could leadto a significant increase in neointimal lesion formation the expression of cthrc1 is also positively correlatedwith tumor lymph node metastasis tumornodemetastasistnm stage and disease prognosis however its potentialand periosteumbone matrix 0cmediators of ammationregulatory mechanisms in the tumor environment have notyet been elucidated the molecules that regulate theexpression of cthrc1cthrc1 is abnormally expressed in several solid tumorsespecially in gastric cancer pancreatic cancer hepatocellularcarcinoma keloid breast cancer colorectal cancer crcepithelial ovarian cancer esophageal squamous cell carcinoma escc cervical cancer nonsmallcell lung carcinoma nsclc melanoma and so on [ ] andmolecules that regulate the expression of cthrc1 includemirnas lncrnas waif1 and dpagt1 mirnas microribonucleic acids mirnas which canregulate gene expression are a class of noncoding singlestranded small rnas of about nucleotides that can inhibitthe mrna translation process by exclusively promoting thedegradation of several mrnas in many tumors mirnas such as mir30c mir9 mir520d5p mir1555pmir98 let7b mir155 mir101 and mir217 can regulate the expression of cthrc1mir30c could regulate cthrc1 at a posttranscriptionallevelin breast cancer it downregulates the cthrc1mediated gsk3catenin signal and inhibits tumor cellproliferation invasion and migration in addition mir30ccan also upregulate baxcaspase9caspase3 a downstreamsignal of cthrc1 inhibiting apoptosis in hepatocellular carcinoma cthrc1 downregulates mir1555p throughthe activation of gsk3involved wntcatenin signalingto promote tumor formation and mir98 dramaticallydownregulates cthrc1 by directly targeting the ²utr ofcthrc1 suppressing hepatocellular carcinoma formation mir9 could inhibit the migration of schwann cell bytargeting cthrc1 following sciatic nerve injury therebyinactivating downstream rac1 gtpase mir520d5pis significantly downexpressed and suppresses cell proliferation migration and invasion by targeting cthrc1 in crc as the second mirna following lin4 in caenorhabditiselegans let7b may directly target cthrc1 and function asa tumor suppressor gene in gastric cancer [ ] in esccmir101 and mir217 could inhibitthe expression ofcthrc1 mir30 could downregulate the expressionof cthrc1 and downstream signal molecules such as mmp and mmp2 to inhibit the invasion and migration ofnsclc cells a recent study found that mir155 downregulation and cthrc1 upregulation were observed incrc moreover overexpression of mir155 can silencedownstream cthrc1 thereby inhibiting cell proliferationand inducing apoptosis of cells to prevent tumor progressionand metastasis in conclusion the negative regulation ofcthrc1 by mirna has the potential to become a noveldirection for cancer treatment in the futurelncrnas metastasisassociated lung adenocarcinomatranscript i malat1 is a large infrequently spliced longnoncoding rna lncrna which could genetically increasecthrc1 activity to regulate lung cancer cell migration the silence of malat1 could also inhibit the expression ofcthrc1 which is a positive regulator of escc furtheranother lncrna named nonmmug014387 could also regulate cthrc1 and activate the wntpcp pathway to promote schwann cell proliferation at the site of injury waif1 wntactivated inhibitory factor waif1 issilenced by promoter hypermethylation in various cancers[ ] lcmsms analysis using liquid chromatographyand mass spectrometry analysis of samples of cthrc1binding membrane proteins indicates that the largest partof cthrc1 binds the waif1 receptor recent researchsuggests that waifi expression is activated by suppressingmethylation of its promoter activated waif1 downregulates the expression of wntcatenin target genes to inhibitthe development of endometrial adenocarcinoma thebinding of cthrc1 to waif1 could promote osteoblast differentiation therefore cthrc1waif1 interactioncan be a potential therapeutic target in the futurepromoter hypomethylation dpagt1 nglycosylation is essential for the migrationpattern of immune cells and its dysregulation is related tovarious diseases including cancer in human escc the overexpression of cthrc1 is associated with hyperglycosylationandincreased nglycosylation is associated with preferential localization ofcthrc1 in wound cells and nglycosylation facilitatesthe promigratory function of cthrc1 dolichylphosphatenacetylglucosaminephosphotransferase dpagt1 thegene that encodes the ï¬rst enzyme and ratelimiting enzymein the assembly of lipidlinked oligosaccharide precursors inthe endoplasmic reticulum is related to the formation ofmature intercellular adhesion complexes as anupstream regulator of nglycosylation status of ecadherindpagt1 could upregulate cthrc1 by increasing proteinturnover indicating that nglycosylation can also stabilizecthrc1 besides tgf and fak could also regulate the expression of cthrc1 in diï¬erent signaling pathways it should behighlighted that cthrc1 not only is the result of tumor progression but also plays a predominant regulatory role in theprogression and metastasis of many solid tumors [ ]in summary many molecules can regulate the expressionand activity of cthrc1 and together with cthrc1 as novelantitumor molecular targets for the treatment of cancer inthe future signaling pathways mediated by cthrc1involved in the progression andmetastasis of tumorthe uence of cthrc1 on various events in tumor progression is based on its regulation of various signaling pathways such as tgf wntintegrin fak srcfakmekerk pi3kakterk hif1α and pkcδerk signaling pathways see figure these properties pathwaysaï¬ected by cthrc1 play an essential role not only in tissueremodeling after injury regulation of ossiï¬cation and other 0cmediators of ammationcthrc1tgfð½tð½r2wnt3alrp56cthrc1wnt5aror12cthrc1cthrc1ð½ð¼ð¼ð½integrincxrc4tð½r1p ppsmad23smad4cthrc1dvlpdvlaxinapcð½cateninck1ð¼gsk3dpagt1stabilizationð½cateninpkcð¿daamrac1rhoajnkrockpsrcfakpaxgrb2rasrafmekerkpfakpi3kaktmtorfra1crebmmpmekerkhifð¼vegfecminvasionpsmad23smad4ð½catenintcflefcjunap1snailcyclin d1g1mg2smetastasisangiogenesisproliferationfigure signaling pathways mediated by cthrc1 involved in the progression and metastasis of tumor tgf signaling pathway isquite complex especially in terms of its eï¬ects which are often contradictory depending on location and time there exists a criticalnegative feedback regulatory loop between tgfsmad23 signaling pathway and cthrc1 wnt signaling includes wntcatenincanonical pathway and cateninindependent noncanonical pathway in the canonical wnt signaling fzd receptor and lrp5lrp6coreceptor are transduced to catenin signaling cascade for the maintenance of stem and progenitor cells in the noncanonical wntsignaling fzd receptor and ror2ptk7ryk coreceptor are transduced to rhoa jnk signaling cascades for the control of tissuepolarity cell adhesion or cell movement the downstream molecules of the wntpcp pathway mainly include the small gtpase familysuch as rac1 rhoa and jnk which play essential roles in cancer cell migration and invasion cthrc1 signal via waif1 canactivate pkcδ which is an essential component of the wntpcp pathway furthermore pkcδ is responsible for the activation of thecthrc1induced erk signaling pathway in cthrc1integrin signaling pathway the upregulation of cthrc1 is related to theprogression and metastasis of several cancers through the activation of several key signaling molecules including src fak paxillin mekerk and rac1 fak promotes cancer cell migration by regulating focal adhesion formation and turnover which involve activation of srcand paxillin fra1 is activated by cthrc1 through the mapkmekerk signaling which leads to the upregulation of cyclin d1 andthat promotes cell proliferation fra1 also induces snail1mediated mmp14 expression to facilitate escc cell invasion migration andmetastasis pi3kakt signaling pathway induces emt change and mmp2mmp9 expression hif1α and vegf are activated bycthrc1 through activating the pi3kaktmtor signaling pathway which promotes tumor angiogenesis cthrc1 also participates intumor cell migration and invasion through hif1αcxcr4 signalsphysiological processes but also in the development of cancerand metastasis negative feedback regulation of cthrc1 and cell typespeciï¬c tgf signaling pathway as the most potentgrowth factor involved in wound healing tgf is releasedby platelets at the site of injury uencing ammatoryresponse angiogenesis reepithelialization ecm and remodeling tgf superfamily members include tgf activin and bmps smad158 mediates bmp signaling whilesmad23 mediates tgf and activin signalingcthrc1 has been reported to have a relationship withthe tgf family since its discovery as their expression sitesoverlap significantly tgf1 and bmp4 can induce thetranscription and expression of cthrc1 in nih3t3 cells cthrc1 can activate tgf signaling via an elevationin smad2smad3 phosphorylation activated smad23 formsa complex with smad4 and accumulates in the nucleus causing an increase in collagen type i deposition during vascularremodeling [] there exists a critical negative feedbackregulatory loop between tgf1 and cthrc1 the conserved region of amino acids in cthrc1 proteincan bind to phosphosmad3 cthrc1 is induced by tgf1 via phosphosmad3 binding to the promoter with subsequenttranscription activation and in turn cthrc1inhibits tgf1 signaling by accelerating proteasomal degradation of phosphosmad3 which inhibits collagen deposition tgf can enhance the migration and invasioncharacteristics of endothelial cells by regulating the secretionand expression of mmp2 and mmp9 therefore inhibiting cthrc1mediated tgf signaling pathways mayeï¬ectively suppress the invasion and angiogenesis of cancercells [ ]however the mechanism of tgf involved in tumorprogression is very complex even in the same tumor typetgf has many diï¬erent roles in tumor progression forexample the activation of nuclear factor of activated tcellsnfats can drive the switch of the tumorsuppressive function of tgf towards tumor progression [ ] tgfincreases the level of cthrc1 in crc cells highly 0cmediators of ammationexpressed cthrc1 promotes epithelialmesenchymal transition emt and tumor metastasis through the smad2smad3 activation of tgf pathway cthrc1 can alsoinhibit the tgfsmad pathway and yap nuclear translocation thereby inhibiting type i collagen synthesis metabolites such as bile acid may induce cthrc1 to activatethe tgfsmad2smad3 pathway to mediate liver ï¬brosisand may progress towards hepatocellular carcinoma in the polyvinyl alcohol sponge model cthrc1 activates tgf and notch pathways to promote the recruitment of m2 macrophages however cthrc1 maydownregulate tgf expression during the late remodelingphase of wound healing tgf regulates the expressionof cthrc1 in a concentrationdependent manner inkeloids and excess cthrc1 reverses collagen synthesis therefore these results of the regulation betweencthrc1 and tgf are not contradictory other than thatcthrc1 has no inhibitory eï¬ect on tgf signaling inendothelial cells these results indicate that the regulation of tgf by cthrc1 may play a role in other interstitial cells of the tumor microenvironment and that thisregulation is cell typespeciï¬c the further exploration ofdetailed molecular mechanism by which cthrc1 activatesthe tgf pathway may resolve these disputes mutual regulation between cthrc1 and wnt pathwaysto promote tumor progression and metastasis wnt familyare secreted glycoproteins include wnt1 wnt1 wnt3awnt4 wnt5a wnt5b wnt6 wnt7a and wnt7b andparticipate in the process of numerous oncogenic and development progress [] wnt5a is a member of the wntprotein family and plays an essential role in the pathologicalprocess of neuropathy and malignant tumors [] wntproteins activate the wntcatenin canonical pathway andcateninindependent noncanonicalamongwhich the planar cell polarity pcp pathway and wntcalcium ca2 pathway are the most widely studied []current reports indicate that cthrc1 is mainly involvedin tumor progression through the canonical wntcateninand noncanonical wntpcp pathwayspathway wntcatenin canonical signaling pathway in thewntcatenin canonical pathway wnt proteins bind tofrizzled fzd receptor and lipoprotein receptorrelatedprotein lrp56 coreceptor in the absence of wntsignaling the cytoplasmic catenin form the destructioncomplex composed with the casein kinase 1α ck1αglycogen synthase kinase 3 gsk3 adenomatous polyposis coli apc and axin which activates the emt topromotethroughcthrc1wntcatenin [ ] the level of cateninis maintained as low by the series of eventsincludingpriming phosphorylation by ck1α at ser45 and subsequently at thr41 ser37 and ser33 by gsk3 [ ]when secreted wnt ligands are accumulated wnt combines with fzd receptor and lrp56 coreceptors lead toactivation of dishevelled dvl protein the activateddvl is phosphorylated and translocated to the fzd recepthe catenintorcausing the dissociation ofand metastases cancerinvasiondestruction complex and the cytosolic accumulation ofcatenin as the cytosolic catenin accumulates rasproteins are accumulated due to the absence of gsk3mediated phosphorylation the stabilized ras proteins atthe plasma membrane activate rafmitogenactivatedprotein kinase mekextracellular signalregulated kinaseerk cascade besides cytosolic catenin subsequently translocates into the nucleus and forms a complexwith the tcell factor tcf or lymphoid enhancer factorlef the complex activates the expression oftargetgenes involving proliferation and transformation such ascmyc cjun ccnd1 gene encoding cyclin d1 epidermal growth factor receptor egfr cd44 cd133 andleucinerich repeatcontaining g proteincoupled receptor lgr5 []the wntcatenin signaling pathway plays an indispensable role in the occurrence and development of manytypes of cancer cthrc1induced nuclear translocation ofcatenin was observed in nclh23 cells and luciferaseassay showed that catenintcf transcriptional activitywas enhanced in contrast the knockdown of cthrc1reduced the catenintcf transcriptional activity whichshows that cthrc1 regulates the invasiveness of nsclccells through the wntcatenin pathway similarlycthrc1 activates snail1 through the wntcatenin signaling pathway to promote emt in epithelial ovarian cancer during the development and metastasis of crccthrc1 may promote the activation of the wnt signalingpathway through anos1 it can also participate in thewntcatenin pathway to regulate the malignant behaviorof hepatocellular carcinoma with gsk3 many cancers usually metastasize to bone in advanced stages cthrc1secreted by osteoclasts promotes basic ï¬broblast growth factor bfgf expression in osteoblasts by stimulating wntcatenin signaling which may induce the development of cancerous bone lesions but not mediate vascular production the constitutive activation of the wntcatenin pathway leads to carcinogenesis in tumors cthrc1 promotes catenin nucleartranslocation and inducestranscription of downstream target genes such as cmycand cyclin d1 in the nucleus reduces cell adhesion and promotes cell proliferation subsequently tumor cell invasion and metastasis occurredinterestingly another reported that catenincould act on the cthrc1 promoter region and promotetranscription nglycosylation stabilizes cthrc1 in oralsquamous cell carcinoma oscc specimens by reducingprotein turnover rate and cthrc1 is positively feedbackregulated by the dpagt1canonical wnt pathway therebyactivating noncanonical wnt pathways to drive tumor cellmigration and invasion in contrast the overexpressionof cthrc1 in hek293t cell and gastrointestinal stromaltumor gist cell significantly inhibited the canonical wntpathway but activated the noncanonical wntpcp pathway[ ] based on the evidence reviewed above it can be indicated that crosstalk between the canonical wntcateninpathway and noncanonical wntpcp pathway and themutual regulation of wntcatenin and cthrc1 acceleratethe process of tumor progression 0cmediators of ammation wntpcp noncanonical signaling pathway earlyreports suggest that cthrc1 activates the pcp pathwayduring inner ear development cthrc1 can interactwith multiple extracellular components of wnt signalingfzd proteins and wntpcp coreceptor ror2 the components form a cthrc1wntfzdror2 complex to activatethe wntpcp pathway selectively and transmit signals fromthe cellsurface complex to the nucleus by dvlrhoarac1jnkatf2cjun cascade promoting cancer cell protrusionsproliferation migration and invasion [ ]cthrc1 is capable of coordinating three small rho gtpasesrac1 rhoa and cdc42 which are the leading performers ofwntpcp to promote cell migration in cervical cancercthrc1 cooperates with e6e7 human papillomavirushpv to activate the noncanonical wntpcp pathway whichaggravates tumor malignancy in pancreatic cancer andhuman urothelial carcinoma wnt5aror2 signaling is associated with emt and promotes tumor cellinvasion andmetastasis [ ] in gist cthrc1 appears to activatethe wntpcp pathway in a dosedependent manner andwnt5apcprho axis determinesinvasionpromoting activity of cthrc1 a recent study demonstrated that cthrc1 could promote erk and jnkphosphorylation by activating pcp signaling pathways inhuman umbilical vein endothelial cells huvecs and promote tumor angiogenesis whatit wasobserved that the paracrine cthrc1 controls the expression of ang2 via noncanonical wnt pathway activationof erkdependent ap1 in huvecs hence overand above that associated with the canonical wntcateninpathway noncanonical wnt signaling pathways interactwith other signaling pathwaysis moretumorthe cthrc1 binds integrin and triggers a series ofsignaling cascades to promote tumor progressionand metastasis integrin faksrc signaling pathway integrins aretransmembrane receptors that promote cellecm adhesion with two subtypes of α and it can participate ina variety of physiological activities such as tumor progression and migration cthrc1 promotes hepatocellular carcinoma cell invasion by activating the rhoarhoassociated kinase rock pathway and facilitates adhesionof hepatocellular carcinoma cells to ecm through induction of integrin 1 expression and activation of focal adhesion kinase fak another study of hepatocellularcarcinoma suggests that cthrc1 inhibits anoikis andincreases tumor cell survival by activating integrin expression cell adhesion to ï¬bronectin is mediatedby integrin 1 previous researches have demonstrated that targeting the integrin 3fak signaling couldenhance the antitumor activity and attenuate cancermetastasiscancernsclc and escc [] guo found that phosphorylated fak was significantly reduced in mice witheoc xenograft tumors and inhibition of fak did notinterfere with integrin 3 expression in vivo howeverthe overexpression of cthrc1 leads to the upregulationincluding melanomaendometrialof integrin 3 in model mice proving that cthrc1 interacts with integrin 3 and promotes fak phosphorylationat tyr397 thereby promoting ovarian cancer cell adhesionmigration and invasion the high level of cthrc1 is connected with the progression and metastasis of pancreatic cancers through the activation of several key signaling molecules including the steroidreceptor coactivator src fak paxillin mek erk andrasrelated c3 botulinum toxin substrate rac1 srcfak signaling cascade takes a regulative role in regulating the formation of protein complexes at focal adhesionsin the migration and metastasis of cancer cells srccan correspond to integrinecm interaction and is recruitedto form the srcfak complex which permits fak to beactive [ ] then fak activates src and paxillin by regulating focal adhesion formation and turnover paxillin a focaladhesion adaptor molecule serves as a scaï¬oldfor the organization and the activation of raf mek anderk [ ] furthermore paxillin can stimulate rac1which is a ras superfamily member of small guanosine triphosphatase gtpase and a critical factor in cytoskeleton reorganization regulation of gene expression and cell proliferationand cellular transformation []erk2mediated paxillin phosphorylation promotes fakadhesion to focal adhesions additionally the inhibitionof fakpaxillin interaction results in decreased phosphorylation of fak and its targets which in turn changes cell adhesion and migration thisinspired thedevelopment of anticancer drugs targeting fak faksrc complex plays essential functions in tgfinduced hepatocyte emt models such as upregulating mmp9 and ï¬bronectin and downregulating ecadherin evidence has mekerk and pi3kakterk signaling pathwaycthrc1 interacts with integrin to trigger a series of signalcascades because src can phosphorylate other fak sites itcan recruit proteins containing src homology sh2domains such as grb2 subsequentlythe downstreamrasmapk pathway and the phosphatidylinositol kinase pi3k akt cascades are activated to participatein cellular response cthrc1 activates fosrelatedantigen1 fra1 through mapkmekerk signalingwhich leads to the upregulation of cyclin d1 and promotescell proliferation fra1 a fos family transcription factoralso induces snail1mediated mmp14 expression to facilitate escc cellinvasion migration and metastasis snail1 transcriptionaltriggeringemt and inducing tumor cell invasion knockingdown cthrc1 will change the phosphorylation level oferk12 and thus regulate the pathological process of endometriosisfrequent upregulation ofcthrc1 observed in human colon cancer cells may bedue to a cpg demethylation event in the exon regionof the gene kim tested the luciferase reporter geneusing the erkresponsive elk1 promoter proposing thatcthrc1 upregulates mmp9 through erk activation further treatment with mek12 inhibitors can reduce tumorcell invasion and erk activation and aggressiveness arereduced by knocking down cthrc1 theis essentialfactorforem 0cmediators of ammationcthrc1 promotes invasiveness and metastasis of hepatocellular carcinoma through the activation of pi3kproteinkinase b pkbakterkcamp responseelementbinding protein creb signaling pathway which inducesemt change and mmp2mmp9 expression cthrc1is highly expressed in hepatitis b virus hbv associatedhepatocellular carcinoma hbv activates nuclear factorkappa b nfκb and creb through the erkcjun nterminal kinase cjnk pathway to stimulate cthrc1expression in addition hypoxiainducible factor 1α hif1α and vascular endothelial growth factor vegf are activated by cthrc1 through activating the pi3kaktmammalian target of rapamycin mtor signaling pathwaywhich promotesis morecthrc1 enhances colony formation migration and invasion of hepatocellular carcinoma cells by downregulatingtumor suppressor p53 and stimulating invasionassociatedfactor mmp9 tumor angiogenesis whatstudies on myocardial infarction mi have also foundthat activation of infarct repair cardiac ï¬broblasts ircfinvolves cthrc1 expression and pi3kakt signaling pathway in ovarian cancer cells gene silencing cthrc1 doesnot alter mmp9 expression or phosphorylate mek theinvasionpromoting eï¬ect of cthrc1 on eoc cells dependson downstream pi3kakt and erk12 signaling dominatedby egfr besides the invasion and metastasis of endometrial cancer are closely related to the upregulation ofcthrc1mediated cx3cr1 in macrophages this processregulates the integrin 3pi3kakt pathway which alsopromotes the recruitment of m2like macrophages cthrc1 activates hifα pathway and contributes totumor angiogenesis as mentioned above cthrc1 in hepatocellular carcinoma can induce hif1α to promote tumorangiogenesis and regulate downstream mmps and tumorsuppressor gene p53 by activating the pi3kakt signalingpathway in human squamous cell carcinoma hif1αoverexpression stimulates vegfc upregulation and induceslymphangiogenesis and tumor cell invasion ding pinpointed that cthrc1 and hif1α were upregulated inthe nucleus of cthrc1 overexpressed gc cells hif1αinhibitors reduced cthrc1induced cxcr4 expressionfurthermore it was found that inhibition of hif1α expression and inhibition of cxcl12cxcr4 signals all alleviatetumor cell migration and invasion therefore cthrc1 canparticipate in tumor cell migration and invasion throughhif1αcxcr4 signals in gc in short cthrc1 canaï¬ect the expression of hif1α which is not only related tolymphangiogenesis but also closely related to tumor progression and invasion a novel signaling pathway the potential role of pkcδerk in tumors protein kinase c δ pkcδ has beenimplicated in various epithelial tumors such as prostatebreast and crc [ ] activated pkcδ causes angiogenesis and tumor growth of prostate tumors by increasingnadph oxidase activity and hif1α expression levels pkcδ can also inhibit the wntcatenin pathwayin colon cancer cells however a recent study illustrated that mek and pkcδ inhibitors could blockcthrc1induced erk phosphorylation and that pkcδphosphorylation was not inhibited by mek inhibition surprisingly inhibition of plc a membraneassociated enzymethat activates pkcδ to promote bone formation in noncanonical wnt signals did not inhibit cthrc1induced alkaline phosphatase alp activity therefore waif1 bound bycthrc1 activates pkcδ and erk to stimulate osteoblastdiï¬erentiation which is a novel signaling pathway unrelatedto the noncanonical wnt pathway therefore pkcδsignal may explain the role of cthrc1 in tumor progressions such as angiogenesis and bone metastasisto put it brieï¬y cthrc1 may be involved in manyother signaling pathways including mirna and lncrnawhich interact with or crosstalk with the tgf wnt andintegrin erk pathways and jointly participate in tumordevelopment and metastasis see table conclusiontumor development and metastasis a complex processinvolving cell adhesion and proteolytic degradation of theecm depends not only on the cancer cells but also on theinteraction between the cancer cells and their microenvironment complementary dna microarray analysis also demonstrated that the cthrc1 gene is expressed in mosthuman solid cancers as we all know cthrc1 is a secretedecm protein which can inhibit collagen deposition and participate in tumor invasion and metastasis even thoughcthrc1 was ï¬rst discovered more than a decad
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" preproof 0c highlights f0b7 propolis made by bees from bioactive plant resins has antiviral activity f0b7 propolis potentially can interfere with host cell invasion by sarscov2 f0b7 propolis blocks proinflammatory pak1 a kinase highly expressed in covid19 patients propolis a resinous material produced by honey bees from plant exudates has long been used in traditional herbal medicine and is widely consumed as a health aid and immune system booster the covid19 pandemic has renewed interest in propolis products worldwide fortunately various aspects of the sarscov2 infection mechanism are potential targets for propolis compounds sarscov2 entry into host cells is characterized by viral spike protein f0b7 propolis is a safe widely consumed functional food with medicinal properties f0b7 standardized propolis has consistent properties for lab and clinical research preprooffactor in advanced covid19 disease propolis has also shown promise as an aid in the treatment of various of the comorbidities that are particularly dangerous in covid19 patients including induced lung inflammation fibrosis and immune system suppression propolis components have inhibitory effects on the ace2 tmprss2 and pak1 signaling pathways in addition antiviral activity has been proven in vitro and in vivo in preclinical studies propolis promoted α this immunoregulation involves monocytes and macrophages as well as jak2stat3 nfkb and inflammasome pathways reducing the risk of cytokine storm syndrome a major mortality interaction with cellular angiotensinconverting enzyme ace2 and serine protease tmprss2 this mechanism involves pak1 overexpression which is a kinase that mediates coronavirusimmunoregulation of proinflammatory cytokines including reduction in il6 il1 beta and tnfrespiratory diseases hypertension diabetes and cancer standardized propolis products with consistent bioactive properties are now available given the current emergency caused by the covid19 pandemic and limited therapeutic options propolis is presented as a promising and 0crelevant therapeutic option that is safe easy to administrate orally and is readily available as a natural supplement and functional food keywords propolis sarscov2 covid19 antiviral antiinflammatory pak1 blocker introduction the covid19 pandemic is of grave concern due its impact on human health and on the deadly disease most require more robust data in clinical trials before they can be widely and safely used isolation and stayathome measures do not effectively protect essential workers economy it is much more deadly than influenza and other types of diseases that recently have had worldwide impact forcing countries to take unusual measures such as limiting travel closing schools businesses and other locations where many people can come into contact with each other various public healthcare strategies have been adopted in an attempt to reduce the impact of the disease but with limited effectiveness as the virus continues to spread often through asymptomatic patients unfortunately tests to determine if people are infectious or were previously infected are not widely available often are costly and frequently do not provide timely and accurate results various therapeutic alternatives have been proposed and tested however preproofnonessential professions return to the workplace they become more at risk for infection in this scenario any options that could help ameliorate disease progression and its consequences even marginally would be useful the world needs safe alternatives to help reduce the impact of this especially health care personnel who have become infected and are dying at alarming rates economic and other necessities limit how well and how long these isolation measures can be maintained especially in poor countries and in poor communities such as slums and favelas [ ] as populations gradually try to get back to normalcy reducing social distancing and people in natural products which have historically been widely used to help avoid and alleviate diseases are among the options being considered as an adjuvant treatment for sarscov2 infection because they are generally inexpensive widely available and rarely have undesirable side effects some have proven antiviral activity an important advantage of using natural remedies is that people who have other health problems or who have mild flurelated 0c infection infection by sarscov2 the virus that causes covid19 is characterized by binding symptoms but do not have the means or courage to visit an already overcrowded medical facility could take simple and inexpensive measures to help reduce the impact of infection with sarscov2 considering the large number of deaths and other types of damage that the covid19 pandemic is causing there is an urgent need to find treatments that have been approved as safe and potentially able to inhibit the new coronavirus reduce its infectivity andor alleviate the symptoms of infection [ ] along this line propolis and its components emerge as potential candidate materials that could help to reduce the pathophysiological consequences of sarscovfactors increased pak1 levels also suppress the adaptive immune response facilitating viral replication [ ] sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory impairment and pulmonary failure immunologicalinflammatory between viral spike proteins and angiotensinconverting enzyme ace2 activation of the spike protein is mediated through proteases such as tmprss2 which play important roles in the viral infection after entry followed by endocytosis coronavirus infection causes pak1 upregulation a kinase that mediates lung inflammation lung fibrosis and other critical mortality preproofphenomena such as cytokine release syndrome have been shown to be important in the spectrum of sarscov2 infection these mechanisms are associated with an dysfunction more than the viral load per se along this line a retrospective observational study found higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα in patients with severe inflammatory diseases by affecting various metabolic cycles recently several studies have shown that propolis extract and some of its components act against several important targets in the pathophysiological context of the disease caused by sarscov2 such as reducing tmprss2 expression and reducing ace2 anchorage which would otherwise facilitate entry of covid19 compared to individuals with mild disease there is considerable evidence that propolis can reduce and alleviate the symptoms of the virus into the cell this is in addition to immunomodulation of monocytes macrophages reducing production of and eliminating il1 beta and il6 reduction of the transcription factors 0cviral replication and antiinflammatory action [ ] propolis and its properties are particularly relevant to sarscov2 infection such as immune system fortification reduced nfkb and jak2 stat3 and blocking pak1 which determine inflammatory activities and fibrosis caused by covid19 various comorbidities have been associated with severe covid19 symptoms and a greater chance of patients requiring intensive care these include hypertension and diabetes also mortality rates of covid19 patients are much higher in those with cardiovascular disease chronic respiratory disease and diabetes [ ] there is considerable evidence that these conditions could be alleviated by treatment with propolis this includes research in animal models of diabetes [ ] hypertension [ ] and cardiovascular disease [ ] propolis has properties that preproofthese plant products to make propolis which they use to protect the colony the production and use of propolis by honey bees evolved to the point that these social bees have considerably fewer immune genes than solitary insect species bees in colonies that produce more propolis are healthier and live longer and propolis consumption by the bees augments their immune [ ] as this variability can affect their medicinal properties standardized propolis products have been developed to help meet the need for a product that does not vary in the main bioactive components and is safe with minimal interaction with pharmaceutical drugs and proven efficacy in clinical trials in recent decades it has been shown to have antimicrobial including the composition of propolis varies according to the plant species available in each region propolis is a product derived from resins and plant exudates plants defend themselves from pathogens mainly by producing phytochemicals many of which have been extracted and used in medicine plant defense substances collected by bees include phenols and terpenoids phytochemical compounds that show promise for the inhibition of coronavirus in humans include quercetin myricetin and caffeic acid all components of propolis honey bees and many other species of social bees recognize these antimicrobial properties and selectively collect and process response to bacterial challenge antiviral antiinflammatory immunomodulatory antioxidant and anticancer properties propolis has historically been widely used to alleviate various diseases [ ] it also has been considered among other natural alternatives as an adjuvant treatment for sarscov2 infection 0ccompared to the propolis that the bees make from these plant materials because it is generally inexpensive widely available and rarely causes undesirable side effects some types of propolis that are highly valued for their medicinal properties such as brazilian green propolis are mainly produced by bees from materials they collect from specific plants in this case baccharis dracunculifolia after the botanical origin of the propolis has been identified extracts of the plant can be made to develop useful products such as a medicinal mouthwash however the medicinal properties of these plant extracts are often inferior can be safely consumed these propolis products and the raw material for their manufacture are extensively exported by brazilian companies especially to asian countries including japan south among natural medicine alternatives propolis has been widely studied and is already extensively consumed in many countries [ ] for example propolis products such as throat sprays and extracts are produced by hundreds of companies in brazil and are sold as a health aid in nearly every pharmacy throughout the country demonstrating on a practical basis that they preproofin fact propolis has a wide spectrum of pharmacological properties and is a dietary supplement that is commonly consumed by both healthy and sick people as a preventative precaution and for treatment it is also used in veterinary medicine due its antibacterial antifungal antiviral korea and china [ ] the importance of propolis in chinese japanese russian and korean medicine is reflected in the number of patents for propolis containing products registered by including about by china and each for japan russia and korea since about new propolisrelated patents were applied for in the us patent office it is a key ingredient in traditional chinese medicine japanese scientists have isolated and patented various brazilian propolis components for cancer treatment demonstrating their usefulness why propolis may be a good fit for dealing with covid19 antiparasitic hepatoprotective and immunomodulatory activities in the wake of the coronavirus outbreak south korea has seen a boon in the use of functional foods according to their ministry of food and drug safety health functional foods are nutrients that have been proven to be beneficial to health in march of this year in response to the coronavirus pandemic the ministry eased regulations for propolis which is considered a 0ccould help fight against the covid19 pandemic active site of this enzyme is a relevant target for drug discovery functional food and allowed new oral formulations however despite considerable evidence that propolis can reduce and alleviate disease symptoms its acceptance as a healthpromoting supplement in human medicine has been limited in many countries such as the usa because of a relevant criticism that propolis products are not standardized and vary in their components and biological activity in part this is because propolis varies with the species of plants available in each region from which the bees collect resins to produce it [ ] however standardized propolis products have recently become available to help fill the need for a product that does not vary in the main bioactive components and effectiveness [ ] one such option a standardized brazilian propolis extract blend has been tested for safety and effectiveness in clinical trials for treating kidney disease and diabetes denture stomatitis and burn patients therefore propolis as a nutraceutical or functional food should be considered as a resource that preproofcaffeic acid phenethyl ester cape galangin chrysin and caffeic acid substances found in several different types of propolis around the world appeared as potential drugs against this viral target table specifically cape was predicted to interact with sarscov2 mpro in a potential targets in order to inactive the virus and reduce the damage that it causes the main protease of coronavirus sarscov2 mpro 3chymotrypsinlike cysteine enzyme is essential similar study therefore although it will be necessary to run in vitro assays to evaluate the potential antisarscov2 effects of propolis andor its constituents these in silico results are along this line hashem evaluated various natural compounds with an in silico approach molecular docking to try to find useful options for treating sarscov2 infection curiously for coronavirus processing of polyproteins and for its life cycle and therefore inhibition of the some propolis compounds can potentially interact with sarscov2 mpro the research community has examined the genetic code of coronavirus and the mechanisms underlying the damages caused by sarscov2 to help search for drugs andor well boding propolis can interact with ace and tmprss2 potentially blocking or reducing sarscov2 invasion of the host cell 0c sarscov2 strongly binds to angiotensinconverting enzyme ace2 using this enzyme as a receptor for invasion and replication in the host cell [ ] causing damage and increasing interpersonal transmission [ ] consequently ace inhibitors have been considered as useful drug alternatives however potential deleterious effects on users of angiotensinconverting enzyme ace inhibitors and angiotensin receptor blockers arbs have tool against potential cardiovascular events inhibition of ace2 enzyme is an important target for treatment against sarscov2 myricetin caffeic acid phenethyl ester hesperetin and pinocembrin rutin interacts with zinc fingers of the active sites of ace2 a metalloprotease that presents the same zinc finger in ace1 emerged as a concern for treatment of covid19 patients an observational study involving patients did not confirm this suspicion and therefore these classes of drugs remain an important infection [ ] güler et al prepared an alcoholic extract of propolis and identified some hydroxycinnamic acids caffeic acid pcoumaric acid tcinnamic acid and cape the flavanons rutin and myricetin and the flavones hesperidin chrysin and pinocembrin using molecular docking evaluations they found that rutin had the highest binding energy to ace2 followed by preproofvarious characteristics including inhibition of ace they found strong inhibition for most of the propolis types they studied with higher than ace inhibition the best results were found in addition to the in silico evidence osés et al evaluated several types of propolis for with the propolis components catechin and pcoumaric acid ace2 and tmprss2 transmembrane serine protease on the surface of host cells are used by sarscov2 via interaction with spike glycoproteins in order to proceed with invasion and replication vardhan sahoo studied several molecules commonly found in medicinal herbs using molecular docking procedures with relevant targets such as rnadependent rna polymerase rdrp ace2 and spike glycoproteins and compared the resulting scores with those of hydroxychloroquine limonin was the most active compound however quercetin and kaempferol also propolis compounds gave high docking scores kaempferol was studied in prostate cancer models and the expression of tmprss2 was reduced showing a potential mechanism of action for an antitumoral effect kaempferol could be an important propolis component for use against covid19 since it is involved in the inhibition of tmprss2 0c potentially interacting with ace2 rdrp and spike glycoprotein sgp besides its antiviral activity table propolis blocks pak1 potentially avoiding lung fibrosis and restoring a normal immune response among the possible targets for controlling covid19 damage the major pathogenic contributes to their suppression pak1 inhibitors can both help combat the virus and restore a normal immune response kinase pak1 is key it is an essential component in malaria and viral infections but it is also involved in a wide variety of other diseases and disease conditions including cancer inflammation and immunosuppression when abnormally activated consequences of pak1 activation include lung fibrosis which is an aggravating factor in covid19 pak1 is activated by rac xu et al demonstrated that caffeic acid and its ester cape components of propolis can inactivate rac consequently inhibiting pak1 the inactivation of pak1 directly or upstream can potentially attenuate coronavirus pathogenesis bcells and tcells are lymphocytes that produce specific antibodies against viruses and other intruders and pak1 preproofimprove the immune response its components increase neutralizing antibody titers activate phagocytosis and increase ifnγ levels and the number of lymphocytes an increase in ifnγ levels was also detected by shimizu et al who evaluated the mechanisms involved in the cape caffeic acid phenethyl ester is a potent inhibitor of activation of nfkb in myelomonocytic cells anse et al demonstrated that propolis cape quercetin hesperidin and some other propolis flavonoids can inhibit the cytokine production of th1 and th2 type t cells while increasing tgfbeta an important antiinflammatory cytokine moreover cape can attenuate oxidative stress and inflammation through downregulation of jak2stat3 signaling propolis from europe and temperate asia usually made by bees from resins collected from poplar trees has predominantly flavonoid compounds while green propolis from baccharis dracunculifolia a propolis exclusively found in brazil has various kinds of flavonoids and prenylated phenylpropanoids such as artepellin c baccharin and drupanin these and all other types of propolis can inactivate pak1 artepillin c selectively inhibits pak1 table some studies have shown that propolis can act as an immunostimulant with the ability to effects of some types of propolis in a herpes simplex animal model 0c as well as having an immunomodulatory effect in which cape inhibits il6 phosphorylation and stat3 which are important for proinflammatory th17 development besides the antiinflammatory effect of cape and kaempferol paulino et al evaluated the antiinflammatory effect of artepellin c in rat paw edema and in cell cultures demonstrating that the activity was at least in part mediated by prostaglandin e2 and no inhibition through nfkb modulation artepillin c is an important biomarker of brazilian green propolis botanical source baccharis dracunculifolia immune modulation is desirable since coronavirus infection dysregulates the immune response in the initial phases of infection which facilitates viral replication however in later stages of covid19 the body develops an exaggerated inflammatory response which can greatly damage the lungs and other ans propolis different from typical immunosuppressants can help avoid immunosuppression during the initial phases of disease and in later stages reduce an exaggerated host inflammatory response inhibiting excess il6 il2 and jak signaling cape a propolis component is also known as an immunemodulating agent and should be considered as an alternative to help reduce an exaggerated inflammatory response in a mouse model propolis had immunomodulatory action in vivo on tolllike receptor expression and on preproofreplication and infectivity potentially decreasing lung inflammation due to antiinflammatory properties while promoting immune system fortification these are useful properties that could there is ample evidence for interference of propolis andor its components with viral propolis has been tested against various viral disease anisms initial successes have prompted research to determine the most useful components which may be modified to produce more active and specific pharmaceuticals viruses that were controlled by propolis in animal models with suggestion for control in humans include influenza [ ] herpes simplex virus type and hiv [ ] shimizu et al evaluated three different types of propolis in ethanol extracts using a murine model of herpes simplex virus type despite the chemical differences proinflammatory cytokine production help minimize the symptoms and deleterious effects of covid19 figure figure propolis as an antiviral substance 0cdue to the different plant origins of the resins the bees used to produce the propolis baccharis dracunculifolia baccharis eriodata and myrceugenia euosma all three propolis extracts not only had direct antihsv1 effects but also stimulated immunological activity against intradermal hsv1 infection in mice antiviral activity of propolis has been reported for dna and rna viruses poliovirus herpes simplex virus and adenovirus in an in vitro model cultured cells the best results were obtained against poliovirus and herpes virus with inhibition of the latter at a propolis concentration of ugml the propolis components chrysine and kaempferol caused a concentrationdependent reduction of intracellular replication of herpesvirus strains when host cell monolayers were infected and subsequently cultured in a drugcontaining medium quercetin another propolis component had the same effect but only at the highest concentrations tested ugml against various human herpes simplex virus strains with a intracellular replication reduction of approximately while it reduced the infectivity of bovine herpes virus human adenovirus human coronavirus and bovine coronavirus about the reduction was in the preproofimpairment and pulmonary failure inflammatory response to infection sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory immunologicalinflammatory phenomena such as cytokine release syndrome have been shown to be important mortality factors in sarscov2 infection higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα are found in patients with severe covid compared to those of individuals with mild disease molecular mechanisms involved in this immune process are the targets of various synthetic medicines being tested in patients including ciclesonide hydroxy chloroquine ivermectin and ketorolac which are pak1 blockers pak1 raccdc42activated kinase is overexpressed in the lung in response to sarscov2 infection and is a critical mediator of the cytokine storm that frequently results in mortality the most critical cases of covid19 which require ventilatorassisted intensive care and often result in prolonged ventilator dependency and death are a result of an exaggerated case of rotavirus antiinflammatory and immunomodulatory properties of propolis 0cin hospitalized patients fortuitously propolis components are effective pak1 blockers table there is considerable evidence that propolis can reduce and alleviate the symptoms of inflammatory diseases and has immunomodulatory properties [ ] however these properties can vary according to the plant origin of the propolis as well as the extraction processsolvent used and the inflammatory protocol cell culture animal models induction by lipopolysaccharides when the propolis extracts are tested tests with animal models have shown that propolis can reduce the levels of il6 and tnfalpha which are key proinflammatory mediators and increase the levels of the regulatory cytokine il10 kaempferol a propolis component reduces il6 tnfalpha and vegf vascular endothelial growth factor via the preproofifnγ in serum fernandes et al found that propolis exerted a positive adjuvant effect on vaccines that were developed against canine coronavirus they assayed ifnγ which is an effective way to measure the cellular response induced by a vaccine in a mouse model propolis added as an adjuvant to inactivated swine herpesvirus type vaccine stimulated increased cellular propolis is considered a safe immunostimulant and a potent vaccine adjuvant propolis has been widely tested as a vaccine adjuvant because it induces an earlier immune response and provides a longer protection period it is also included as an adjuvant ingredient in traditional chinese medicine propolis flavonoids have potential as adjuvants enhancing igg il4 and propolis has potential as a vaccine adjuvant erknfkbcmycp21 pathway table tests on macrophage cell cultures also demonstrated that propolis inhibits the production of il1 beta an important component of the inflammasome inflammatory pathway in diseases such as rheumatoid arthritis lupus and other autoimmune diseases although the mechanisms of action are not well elucidated these propolis components have potential as complementary supplements in the preventive treatment of chronic inflammatory diseases and humoral responses increasing ifnγ [ ] propolis enhanced the immune response to inactivated porcine parvovirus vaccine in guinea pigs when added to a trichomonas vaginalis protein vaccine propolis increased the igg antibody response times in mice 0ccancer is considered a relevant comorbidity factor for covid19 cancer patients have a patients cancer patients compared to the protein alone propolis was also effective as an adjuvant in the immunization of cattle with bovine herpesvirus it improved the humoral and cellular responses in mice inoculated with inactivated virus vaccines propolis as an adjuvant gave a similar immune response increasing ifnγ levels to alum and freunds adjuvant in mice vaccinated with an hiv1 polytope vaccine candidate with less risk of undesirable side effects comorbidities and evidence of how propolis can help reduce their impact in covid19 to risk a visit to a clinic or hospital to determine if they have cancer alternative therapies could help retard cancer or reduce the impact of cancer and cancer treatment in covid19 times higher risk of progressing to severe covid19 disease than patients without comorbidities also the hospital environment during the coronavirus pandemic can interfere with or delay the treatment that cancer patients should receive patients with symptoms may choose not preproofacid cape quercetin and chrysin propolis and its components normally have little impact on normal cells displaying differential cytotoxicity in liver cancer melanoma and breast cell carcinoma cell lines [ ] propolis enhances the activity of tumor various types including bladder blood brain breast colon head and neck kidney liver pancreas prostate and skin cancers propolis could help prevent cancer progression in various parts of the world it is considered an alternative therapy for cancer treatment propolis extracts have been found to inhibit tumor cell growth both in vitro and in vivo including inhibition of angiogenesis demonstrating potential for the development of new anticancer drugs various components of propolis have been shown to inhibit cancer cell growth including cinnamic propolis has potential as a complementary therapy for cancer it has shown efficacy against necrosis factor related apoptosis inducing ligand trail in cancer cells hypertension and cardiovascular disease 0c hypertension and cardiovascular disease are considered relevant comorbidities for covid19 propolis has demonstrated antihypertensive effects in rat models [ ] in cameroon it is used in popular medicine to treat various ailments including high blood pressure propolis has been widely used as a dietary supplement for its health benefits including cardiovascular protective effects [ ] in a human trial consumption of propolis improved critical blood parameters including hdl gsh and tbars levels demonstrating that obesity a natural antiobesity agent it could contribute to a reduced risk for cardiovascular disease obesity is a major comorbidity and predictor of increased mortality in covd19 patients obesity and sarscov2 both induce an inflammatory process exacerbating sarscov2 infection in the obese propolis reduced inflammation and prevented hyperlipidemia and metabolic syndromes in highly caloric diet induced obesity in mice body weight gain visceral adipose tissue liver and serum triglycerides cholesterol and nonesterified fatty acids were all reduced in the propolis fed mice [ ] caffeic acid phenethyl ester a propolis component is preproofplatelet aggregation was reduced by propolis in tests on human blood in vitro and in other in vitro tests caffeic acid phenethyl ester cape a wellstudied bioactive propolis component inhibits collagen induced platelet activation thromboembolism thrombosis and microthrombosis microthrombosis disseminated intravascular coagulation and consequent multian failure are common in severely affected covid19 patients with associated high mortality rates anticoagulants are sometimes prescribed to such patients because they can reduce mortality tang et al an elevated level of plasminogen activator inhibitor1 pai1 is a biomarker and risk factor for thrombosis and atherosclerosis [ ] various types of evidence demonstrate that propolis can reduce platelet aggregation and other thrombosisrelated parameters propolis decreased thrombotic tendencies in mice by suppressing lipopolysaccharideinduced increases in pai1 levels [ ] propolis downregulated plateletderived growth factor and platelet endothelial cell adhesion molecules in lowdensity lipoprotein knockout mice 0c old age the elderly are more often affected by chronic inflammation characterized by systemically increased levels of proinflammatory cytokines which can contribute to development of a cytokine storm a major cause of covid19 mortality propolis has antioxidant properties which could help retard o
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continuously increasing with development of the economy and the environment [] the prognosis for hcc patients remains extremely poor although significant progress has been achieved strategies for early diagnosis are urgently needed because the majority of patients with hcc are diagnosed in very late stages however the molecular mechanism of hcc has not been clearly defined circular rnas circrnas are a new class of rna molecules that have functions as regulators of parental gene transcription in alternative splicing and as mirna sponges through use of rna deep sequencing gtechnology numerous circrnas have been identified as the predominant regulatory elements in diseases moreover accumulating evidence shows that circrnas play pivotal roles in many diseases in particular abnormally expressed circrnas are involved in tumor progression including cell proliferation migration and invasion [] in addition some research indicates that circrnas level are closely correlated wit specific phenotypes and tumorigenesis in hcc [] nevertheless the research concerning circrnas is frankly in its infancy which greatly hinders the application of circrnas as biomarkers for diagnosis of hcc in clinicsrelated research shows that circrnas possess great potential to be used for diagnosis of hcc recent studies have found that hsa_circ_0067934 plays oncogenic roles by accelerating cell proliferation and metastasis in glioblastoma gbm circsmarca5 was significantly elevated and thereby suppressed cell apoptosis and arrested cell cycle in prostate cancer in addition previous studies have shown that downregulation of hsa_circ_0005986 facilitated cell proliferation by promoting the g0g1 to s phase transition in hcc similarly alteration in expression of circrnas correlated with development and metastasis of malignant tumors these data suggest that circrnas may be of greater benefit in clinical diagnosis of hcc however reliable circrna biomarkers for hcc are still lacking therefore this review synthetically integrates available data on the role of circrna in hcc progression and attempts to provide crucial clues for investigating the molecular mechanism regarding hccoverview of circrnacircrnas are a category of singlestranded closedcircle molecules which take part in multifaceted biological regulation recently research has verified that the majority of circrnas are synthesized by backspliced exons and that others are formed from intron intergenic and untranslated regions utr therefore biogenesis of circrnas can be divided into eicirnas exonintron circrnas ecircrnas circular exonic rnas and cirnas circular intronic rnas meanwhile over circrnas have been identified and this type of transcript has been considered a new form of gene expression generally the structure of the transcription is inverted and the order of genomic exons is altered and these exons are spliced over time the biological functions of circrnas gradually have been recognized including roles in embryonic development maintainenance of homeostasis and promotion of tumor progression figure properties of circrnascircrnas recently have attracted great attention related to their pathological role in disease development compared with linear rnas circrnas have special properties including biological roles and clinical use circrnas are mainly enriched in certain body fluids comprising blood saliva and urine they are covalently closed loop structures degradation of most rna is highly dependent on rna exonuclease or rnase hence circrnas remain highly stable based on their high resistance to enzyme degradation moreover studies have shown that expression of circrnas is tissuespecific and correlated with different phases of development and they exhibit different expression patterns at different developmental stages roles of circrnasaccumulating evidence shows that circrnas play a crucial role in the pathogenesis of diseases as a result of their complex biological functions generally the molecular functions of circrnas mainly include being sponges of mirna acting as rnabinding proteins performing alternative splicing of premrnas regulating transcription and translation and potentially encoding proteins these properties are described in detail belowsponges of mirnathe different types of circrnas have different mirna binding sites some circrnas negatively regulate mirnas by absorbing and specifically binding to mirnas then decreasing mirna activity and elevating expression of mirnarelated target genes researchers have shown that cirs7 inhibits mir7 function and positively mediates mir7 target genes acting as a molecular sponge in addition functional analyses have indicated that circrnas constitute an entire molecular regulatory network which specifically regulates degradation of mirnas as mirna sponges this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238322indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832premrnae1e2abcbasepairinge3dguriche4ecrichpretrnarna bindingproteinsrbpgnicilpskcablariat splicingaecircrnaelcirnacirnatrnatricrnafigure1 biogenesis of circular rnas a lariatdriven circularization the hydroxyl of the upstream exon reacts with the phosphate of the downstream exon to form a covalent linkage then producing a lariat including exons and introns the hydroxyl of the intron interacts with the phosphate of the intron to form an ecircrna following an interaction between the hydroxyl of the exon and the phosphate of the exon b rnabinding protein rbpdriven circularization rbps accelerate interaction of the downstream intron and upstream intron thereby promoting formation of ecircrna c basepairingdriven circularization the downstream introns and upstream introns are paired depends on inverserepeatingcomplementary sequences formation of ecircrnaeicirna was derived from the introns are removedretained d biosynthesis of cirna formation of cirnas mainly based on a 7nt gurich element and an 11nt crich element to escape debranching and exonucleolytic degradation e formation of tricrna trna splicing enzymes divide pretrna into two parts tricrnas are generated by a phosphodiester bond and the other part generates trnascircrnasbinding proteinsrna binding proteins rbps are a broad class of proteins involved in gene transcription translation and interaction studies suggest that distribution of rbps is widespread in many tissue types furthermore rbps participate in development of disorders by regulating posttranscriptional regulation of rnas rbps assemble ribonucleoprotein complexes to bind rna sequences thereby affecting the function of the target rnas previous research has shown that circrnas serve as protein decoys to harbor binding sites of specific proteins and block protein activity circfoxo3 induces cell cycle arrest resulting in defective cdk2 gene function by binding to p21 and cdk2 moreover circrna ciacgas binds to cgas protein and suppresses enzymatic activity of cgas thereby preventing cgas from recognizing selfdna e9238323indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832circrnas regulate alternative splicing transcription and translationcellular localization of most circrnas is cytoplasmic which is the basis for the biological function of mirna and protein decoys several studies have suggested that circrnas participate in rna splicing assembly and biosynthesis recently research has shown that circrnas may play pivotal roles in regulating alternative splicing transcription and translation in addition the exon of the splicing factor may form a circrna by affecting formation of linear rna eicirnas interact with the u1 small nuclear ribonucleoproteinsnrnp thereby regulating parental gene transcription by binding to rna polymerase ii interestingly translation of circrnas is mediated by ires and n6methyladenosine m6a and translation efficiency of circrna is regulated by the level of m6a modification moreover circfbxw7 effectively inhibits glioma proliferation and cell cycle progression by antagonizing usp28induced cmyc stabilization potential to encode proteinscircrnas are implicated in numerous physiological processes and pathogenesis of diseases strong evidence indicates that circrnas can encode proteins by mimicking dna rolling circle amplification related studies indicate that circrna circppp1r12a plays a key molecular role by encoding a functional protein circppp1r12a73aa which promotes proliferation migration and invasion of colon cancer circanril interacts with pescadillo zebrafish homolog pes1 to mediate ribosome biogenesis and prerrna processing in vascular macrophages and smooth muscle cells these studies have significantly increased the knowledge base about the biological functions of circrnascircrnas in diseasescircrnas are involved in processes that lead to development of various disorders such as neuronal and cardiovascular diseases and cancers circrnas participate in regulating gene transcription and protein expression and are indirectly and directly associated with time and regionspecific variations as mentioned previously abnormal expression of circrnas is implicated in neurological disorders atherosclerosis and ribosomal rna maturation reportedly are regulated by circanril simultaneously some studies have suggested that circrnas upregulation significantly affects sprouting and proliferation of vascular endothelial cells and elicits vascular dysfunction recently several experiments have implicated circrnas in pathogenesis of cancer via activation of a series of cascade reactions however the underlying mechanism for the effect of circrnas in initiation and progression of tumors has not been fully clarified to date related studies have revealed that certain circrnas are highly expressed in tumor tissues and overexpression of circrnas promotes tumor proliferation and deterioration an investigation revealed that hsa_circ_002059 was downregulated in gastric cancer while hsa_circ_0004018 was upregulated in hcc meanwhile tumorspecific circrnas candidates were screened in lung adenocarcinoma tissue by microarrays and circrnas were identified downregulated and upregulated of the circrnas hsa_circ_0013958 clearly was positive correlated with lymph node metastasis and tnm stage these findings indicate that circrnas have important roles in tumor progression and may have potential for broad applicatoins in medicine scienceoverview of hcchcc is one of the most prevalent tumors worldwide with diagnoses and approximately deaths annually epidemiological survey data indicate that morbidity and mortality from hcc are gradually increasing risk factors for hcc include diabetes mellitus obesity smoking alcohol consumption older age male sex chronic hbv liver cirrhosis and chronic hepatitis c virus hcv the primary risk factors include liver cirrhosis viral hepatitis alcohol intake and obesity worldwide approximately hcc patients are infected with hepatitis b virus hbv or hcv in addition alcohol abuse is a crucial factor for onset of hcc [] obesity hypertension and diabetes are closely linked with development of hcc but specific correlations remain unknown moreover regular screening has been widely applied for early detection and to ensure effective treatment of hcc most commonly good results have been achieved with regular screening with ultrasonography blood alphafetoprotein content testing mri and ct generally surgical resection and chemotherapy are mainstays of therapy in patients with hcc yet some tumors cannot be fully removed which results in tumor growth invasion and metastasis local and systemic metastases are the main reasons for the unsatisfactory prognosis in patients with hcc therefore more effective therapeutic approaches need to be developedroles of circrnas in hccnumerous studies have documented the important role that circrnas play in tumorigenesis metastasis and invasion research has shown that circrnas are localized in the nucleus and interfere with transcription and promote alternative this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238324indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832hsa_circ_0001649circzkscan1circitchwntbetacatenincircmto1mir9p21hsa_circ_00059836mir1295phmgb1 ragenfκbmir7hsa_circ_101368hsa_circ_001569cdr1ashsa_circ_0000673figure the function of circrnas in hcc carcinogenesis this graph demonstrates the role of circrnas in hcc carcinogenesis including positive and negative effects respectivelytable brief summary of circrnas as biomarkers for hccnamediseaseconclusiondoicirs7hsa_circ_0003570hsa_circ_0005075hepatocellular carcinomahepatocellular carcinomahepatocellular carcinomacirs7 was one of the independent factors and may be a promising biomarker for hepatic mvi and a novel therapy target for restraining mvi101007s0043201622567hsa_circ_0003570 expression levels were associated with hcc clinicopathological characteristics101002jcla22239hsa_circ_0005075 promotes proliferation migration and invasiveness of hcc via mir431 regulation101016jbiopha201801150splicing circpvt1 is overexpressed in gastric cancer tissues compared with nontumor tissues and circpvt1 acts as an oncogene to mediate expression of mir4975p however studies concerning the role of circrnas in development and progression of hcc remain in their infancytumor inhibitioncurrently circrnas are considered promising diagnostic biomarkers and ideal therapeutic targets for hcc studies have revealed that circitch inhibits tumor proliferation by suppressing the wntbetacatenin pathway expression of circitch has been positively correlated with good survival outcome in patients with hcc analysis of the circrnas expression profile in human hcc tissues showed that circmto1 was markedly decreased in hcc tissues and that expression of circmto1 was positively correlated with survival rate circmto1 reportedly inhibits hcc progress by sponging mir9 and thereby increasing p21 expression meanwhile overexpression of hsa_circ_0001649 negatively affects invasion and proliferation and promotes apoptosis of hcc cells downregulation of zkscan1 and circzkscan1 enhances cell proliferation and promotes progression of hcc tumor promotionin patients with hcc cdr1was more abundant in tumor specimens than in adjacent normal tissues cdr1as effectively suppresses the invasion and proliferation of hcc cells by targeting mir7 some reports have shown that hsa_circ_0000673 is significantly upregulated in hcc tissues and hsa_circ_0000673 downregulation markedly inhibits proliferation and invasion of hcc cells in vitro meanwhile a positive correlation was found between circ_001569 expression level and tumor size advanced tnm stages and unfavorable prognosis in patients with hcc circrna101368 was abundantly expressed in hcc tissue which correlated with poorer prognosis in addition circrna101368 inhibited cell migration by reducing protein levels in nfkb rage and hmgb1 figure e9238325indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832biomarkerconclusionsprevious studies have shown that circrnas are closely related to development of tumors clinicopathological features in patients with hcc are correlated to with levels of expression of cirs7 and its targeted mrnas global circrna expression profile analysis showed that hsa_circ_0005075 exhibited significant differences in tumor tissue versus adjacent tissues in patients with hcc expression of hsa_circ_0005075 also was related to tumor proliferation and metastasis therefore an increasing number of circrnas have been identified as diagnostic markers as summarized in table given the high incidence and mortality fo hcc worldwide it is one of the most serious diseases threatening human health increasing attention is being paid due to this serious situation evidence is increasing to support the close association between circrnas progression of hcc circrnas may play an important role in the occurrence and development of tumors however the molecular mechanism underlying the relationship between circrnas and hcc has not been fully elucidated therefore indepth research is needed on the potential regulatory relationships and to uncover regulatory patterns between circrnas and hcc so that new diagnostic markers for hcc can be developedreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries cancer j clin feng rm zong yn cao sm xu rh current cancer situation in china good or bad news from the global cancer statistics cancer commun lond jemal a bray f center mm global cancer statistics cancer j clin li r jiang j shi h circrna a rising star in gastric cancer cell mol life sci salzman j gawad c wang pl circular rnas are the predominant transcript isoform from hundreds of human genes in diverse cell types plos one e30733 lukiw wj circular rna circrna in alzheimers disease ad front genet liu y yang y wang z insights into the regulatory role of circrna in angiogenesis and clinical implications atherosclerosis zhao y alexandrov pn jaber v lukiw wj deficiency in the ubiquitin conjugating enzyme ube2a in alzheimers disease ad is linked to deficits in a natural circular mirna7 sponge circrna cirs7 genes basel shen f liu p xu z circrna_001569 promotes cell proliferation through absorbing mir in gastric cancer j biochem song t xu a zhang z circrna hsa_circrna_101996 increases cervical cancer proliferation and invasion through activating tpx2 expression by restraining mir8075 j cell physiol min l wang h zeng y circrna_104916 regulates migration apoptosis and epithelialmesenchymal transition in colon cancer cells front biosci landmark ed verduci l strano s yarden y blandino g the circrnamicrorna code emerging implications for cancer diagnosis and treatment mol oncol wei j wei w xu h circular rna hsa_circrna_102958 may serve as a diagnostic marker for gastric cancer cancer biomark li p chen s chen h using circular rna as a novel type of biomarker in the screening of gastric cancer clin chim acta xin j zhang xy sun dk upregulated circular rna hsa_circ_0067934 contributes to glioblastoma progression through activating pi3kakt pathway eur rev med pharmacol sci kong z wan x zhang y androgenresponsive circular rna circsmarca5 is upregulated and promotes cell proliferation in prostate cancer biochem biophys res commun fu l chen q yao t hsa_circ_0005986 inhibits carcinogenesis by acting as a mir1295p sponge and is used as a novel biomarker for hepatocellular carcinoma oncotarget zhu x wang x wei s hsa_circ_0013958 a circular rna and potential novel biomarker for lung adenocarcinoma febs j zhang q wang w zhou q roles of circrnas in the tumour microenvironment mol cancer qu z jiang c wu j ding y exosomes as potent regulators of hcc malignancy and potential biotools in clinical application int j clin exp med memczak s jens m elefsinioti a circular rnas are a large class of animal rnas with regulatory potency nature cocquerelle c mascrez b hetuin d bailleul b missplicing yields circular rna molecules faseb j zhao x cai y xu j circular rnas biogenesis mechanism and function in human cancers int j mol sci qu s yang x li x circular rna a new star of noncoding rnas cancer lett bahn jh zhang q li f the landscape of microrna piwiinteracting rna and circular rna in human saliva clin chem hsu mt cocaprados m electron microscopic evidence for the circular form of rna in the cytoplasm of eukaryotic cells nature yu x odenthal m fries jw exosomes as mirna carriers formationfunctionfuture int j mol sci hanan m soreq h kadener s circrnas in the brain rna biol constantin l circular rnas and neuronal development adv exp med biol van rossum d verheijen bm pasterkamp rj circular rnas novel regulators of neuronal development front mol neurosci ebert ms sharp pa microrna sponges progress and possibilities rna ebert ms neilson jr sharp pa microrna sponges competitive inhibitors of small rnas in mammalian cells nat methods hansen tb jensen ti clausen bh natural rna circles function as efficient microrna sponges nature hsiao ky lin yc gupta sk noncoding effects of circular rna ccdc66 promote colon cancer growth and metastasis cancer res janga sc mittal n construction structure and dynamics of posttranscriptional regulatory network directed by rnabinding proteins adv exp med biol du ww yang w liu e foxo3 circular rna retards cell cycle progression via forming ternary complexes with p21 and cdk2 nucleic acids res xia p wang s ye b a circular rna protects dormant hematopoietic stem cells from dna sensor cgasmediated exhaustion immunity 701e7 li z huang c bao c exonintron circular rnas regulate transcription in the nucleus nat struct mol biol this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238326indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832 yang y fan x mao m extensive translation of circular rnas driven by n6methyladenosine cell res yang y gao x zhang m novel role of fbxw7 circular rna in repressing glioma tumorigenesis j natl cancer inst abe n hiroshima m maruyama h rolling circle amplification in a prokaryotic translation system using small circular rna angew chem int ed engl zheng x chen l zhou y a novel protein encoded by a circular rna circppp1r12a promotes tumor pathogenesis and metastasis of colon cancer via hippoyap signaling mol cancer holdt lm stahringer a sass k circular noncoding rna anril modulates ribosomal rna maturation and atherosclerosis in humans nat commun gokul s rajanikant gk circular rnas in brain physiology and disease adv exp med biol idda ml munk r abdelmohsen k gorospe m noncoding rnas in alzheimers disease wiley interdiscip rev rna luo q chen y long noncoding rnas and alzheimers disease clin interv aging li cy ma l yu b circular rna hsa_circ_0003575 regulates oxldl induced vascular endothelial cells proliferation and angiogenesis biomed pharmacother chen j cui l yuan j circular rna wdr77 target fgf2 to regulate vascular smooth muscle cells proliferation and migration by sponging mir biochem biophys res commun kristensen ls hansen tb veno mt kjems j circular rnas in cancer opportunities and challenges in the field oncogene fu l yao t chen q screening differential circular rna expression profiles reveals hsa_circ_0004018 is associated with hepatocellular carcinoma oncotarget massarweh nn elserag hb epidemiology of hepatocellular carcinoma and intrahepatic cholangiocarcinoma cancer control salem r gilbertsen m butt z increased quality of life among hepatocellular carcinoma patients treated with radioembolization compared with chemoembolization clin gastroenterol hepatol 65e1 ozer ed suna n boyacioglu as management of hepatocellular carcinoma prevention surveillance diagnosis and staging exp clin transplant 15suppl lou w liu j ding b identification of potential mirnamrna regulatory network contributing to pathogenesis of hbvrelated hcc j transl med yang t hu ly li zl liver resection for hepatocellular carcinoma in nonalcoholic fatty liver disease a multicenter propensity matching analysis with hbvhcc j gastrointest surg nishibatake km minami t tateishi r impact of directacting antivirals on early recurrence of hcvrelated hcc comparison with interferonbased therapy j hepatol toyoda h kumada t tada t the impact of hcv eradication by directacting antivirals on the transition of precancerous hepatic nodules to hcc a prospective observational study liver int zhao j oneil m vittal a prmt1dependent macrophage il6 production is required for alcoholinduced hcc progression gene expr vandenbulcke h moreno c colle i alcohol intake increases the risk of hcc in hepatitis c virusrelated compensated cirrhosis a prospective study j hepatol fabris c toniutto p falleti e mthfr c677t polymorphism and risk of hcc in patients with liver cirrhosis role of male gender and alcohol consumption alcohol clin exp res vernon g baranova a younossi zm systematic review the epidemiology and natural history of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in adults aliment pharmacol ther bruix j reig m sherman m evidencebased diagnosis staging and treatment of patients with hepatocellular carcinoma gastroenterology zhang bh yang bh tang zy randomized controlled trial of screening for hepatocellular carcinoma j cancer res clin oncol verduci l ferraiuolo m sacconi a the oncogenic role of circpvt1 in head and neck squamous cell carcinoma is mediated through the mutant p53yaptead transcriptioncompetent complex genome biol yu j xu qg wang zg circular rna csmarca5 inhibits growth and metastasis in hepatocellular carcinoma j hepatol wang m yu f li p circular rnas characteristics function and clinical significance in hepatocellular carcinoma cancers basel guo w zhang j zhang d et al polymorphisms and expression pattern of circular rna circitch contributes to the carcinogenesis of hepatocellular carcinoma oncotarget han d li j wang h circular rna circmto1 acts as the sponge of microrna9 to suppress hepatocellular carcinoma progression hepatology qin m liu g huo x hsa_circ_0001649 a circular rna and potential novel biomarker for hepatocellular carcinoma cancer biomark yao z luo j hu k zkscan1 gene and its related circular rna circzkscan1 both inhibit hepatocellular carcinoma cell growth migration and invasion but through different signaling pathways mol oncol xu l zhang m zheng x the circular rna cirs7 cdr1as acts as a risk factor of hepatic microvascular invasion in hepatocellular carcinoma j cancer res clin oncol yu l gong x sun l the circular rna cdr1as act as an oncogene in hepatocellular carcinoma through targeting mir7 expression plos one e0158347 jiang w wen d gong l circular rna hsa_circ_0000673 promotes hepatocellular carcinoma malignance by decreasing mir7673p targeting set biochem biophys res commun liu h xue l song c overexpression of circular rna circ_001569 indicates poor prognosis in hepatocellular carcinoma and promotes cell growth and metastasis by sponging mir4115p and mir4325p biochem biophys res commun li s gu h huang y circular rna 101368mir200a axis modulates the migration of hepatocellular carcinoma through hmgb1rage signaling cell cycle shang x li g liu h comprehensive circular rna profiling reveals that hsa_circ_0005075 a new circular rna biomarker is involved in hepatocellular carcinoma development medicine baltimore e3811 yao t chen q shao z circular rna as a new biomarker for hepatocellular carcinoma metastasis j clin lab anal e22572e9238327indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0c'
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Neurogenesis From Neural CrestCells Molecular Mechanisms in theFormation of Cranial Nerves andGangliaKarla MndezMaldonado12 Guillermo A VegaLpez34 Manuel J Aybar34 andIv¡n Velasco15 Instituto de Fisiologa Celular Neurociencias Universidad Nacional Autnoma de Mxico Ciudad de Mxico Mexico Departamento de Fisiologa y Farmacologa Facultad de Medicina Veterinaria y Zootecnia Universidad Nacional Autnomade Mxico Ciudad de Mxico Mexico Instituto Superior de Investigaciones Biolgicas INSIBIO CONICETUNT SanMiguel de Tucum¡n Argentina Instituto de Biologa Dr Francisco D Barbieri Facultad de Bioqumica Qumica yFarmacia Universidad Nacional de Tucum¡n San Miguel de Tucum¡n Argentina Laboratorio de Reprogramacin CelularInstituto Nacional de Neurologa y Neurociruga Manuel Velasco Su¡rez Ciudad de Mxico MexicoThe neural crest NC is a transient multipotent cell population that originates in thedorsal neural tube Cells of the NC are highly migratory as they travel considerabledistances through the body to reach their ï¬nal sites Derivatives ofthe NC areneurons and glia of the peripheral nervous system PNS and the enteric nervoussystem as well as nonneural cells Different signaling pathways triggered by BoneMorphogenetic Proteins BMPs Fibroblast Growth Factors FGFs Wnt proteins Notchligands retinoic acid RA and Receptor Tyrosine Kinases RTKs participate in theprocesses of induction speciï¬cation cell migration and neural differentiation of the NCA speciï¬c set of signaling pathways and transcription factors are initially expressed inthe neural plate border and then in the NC cell precursors to the formation of cranialnerves The molecular mechanisms of control during embryonic development havebeen gradually elucidated pointing to an important role of transcriptional regulatorswhen neural differentiation occurs However some of these proteins have an importantparticipation in malformations of the cranial portion and their mutation results in aberrantneurogenesis This review aims to give an overview of the role of cell signaling and of thefunction of transcription factors involved in the speciï¬cation of ganglia precursors andneurogenesis to form the NCderived cranial nerves during anogenesisKeywords cranial nerve peripheral nervous system hindbrain cell signaling transcriptional regulatory networktrigeminal nerve facial nerve vagus nerveAbbreviations BMP bone morphogenetic proteins CN cranial nerve CNS central nervous system DRG dorsal rootganglia FP ï¬oor plate hESCs human embryonic stem cells Msx Muscle segmentrelated homeobox NC neural crestNCCs neural crest cells NT neural tube PA pharyngeal arches Pax Paired box PNS peripheral nervous system rrhombomere RA retinoic acid RTK receptor tyrosine kinase Shh Sonic hedgehog Sox Srybox VSMC vascular smoothmuscle cells Wnt Wingless and Int1 WRPW TrpArgProTrp Zic Zinc ï¬nger protein of cerebellumEdited byMichael PiperThe University of QueenslandAustraliaReviewed byRoberto MayorUniversity College LondonUnited KingdomRebecca McLennanStowers Institute for MedicalResearch United StatesCorrespondenceManuel J AybarmanuelaybarfbqfunteduarIv¡n Velascoivelascoifcunammx These authors have contributedequally to this workSpecialty sectionThis was submitted toStem Cell Researcha section of the journalFrontiers in Cell and DevelopmentalBiologyReceived April Accepted June Published August CitationMndezMaldonado KVegaLpez GA Aybar MJ andVelasco I Neurogenesis FromNeural Crest Cells MolecularMechanisms in the Formationof Cranial Nerves and GangliaFront Cell Dev Biol 103389fcell202000635Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alINTRODUCTIONDuring the embryonic development of vertebrates one of themain events after the gastrulation process is neurulation whichallows the formation of the neural tube NT The neuralectoderm generates not only the central nervous system CNSbut also another set of cells between the NT and the nonneural ectoderm located in the most dorsal part of the NT calledthe neural crest NC Hall SimµesCosta This versatile and plastic cell population was ï¬rst described byWilhelm His years ago Hall The NC is one of themost important features that separate vertebrates from otherchordate anisms It arises at the posterior and lateral bordersof the neural and nonneural ectoderm the neural plate borderFigure Cerrizuela the embryo NCCs are divided into cranialNC cells NCCs are multipotent and give rise to several celltypes depending on the site of origin along the anteroposterioraxis oftrunkincluding cardiac vagal and sacral A Minouxand Rijli SimµesCosta and Bronner VegaLopez Cranial nerves CN transmit sensory and motorinformation between the brain and tissues of the head andcervical region The CN are formed from the contribution oftwo specialized embryonic cell populations cranial NC andectodermal placodesOrigin of the Neural CrestNCCs which are multipotent delaminate from their origin andmigrate throughout the body to diï¬erentiate into several celltypes including cells of the peripheral nervous system PNSmelanocytes cranial cartilage and bone neuroendocrine cellsand several other phenotypes B In humans at least cell types have been deï¬ned as NC derivatives Vickaryousand Hall Proper NC migration relies on environmentalcues such as EphEphrins Smith Semaphorin3F Gammill Versican Szab thechemokine Stromal cellderived factor Theveneau or Robo2 Shiau The migration patternsof NCCs have been clearly described for model anisms likebirds frogs and mice In all vertebrates cranial NCCs emergefrom the forebrain midbrain and hindbrain regions Coulyand Le Douarin Serbedzija Depending ontheir axial origin cranial NCCs will either migrate through thefacial mesenchyme and into the frontonasal process or willpopulate the branchial arches Noden Lumsden Serbedzija The sensory module of the PNSin the cranial region is composed of an array of paired gangliaadjacent to the hindbrain that transduce the perception of touchpain temperature position and special sensory informationfrom the periphery to the CNS Cranial NCCs migrate to formsensory ganglia such as the trigeminal V the facial VII theglossopharyngeal IX the vagus X CN and also to form themotor ganglia for the oculomotor III and accesory XI CNTable and Figures acomplex and multistep processinitially directed by cell signaling molecules including BoneMorphogenetic Proteins BMPs Wnts Wingless and Int1NC formation isNeural CrestDerived Cranial NervesFibroblast Growth Factor FGF and retinoic acid RA Thesesignals reveal the tissue interactions into the ectodermal cellpopulations the neural plate the nonneural ectoderm and theunderlying mesoderm in a highly coordinated manner VegaLopez It has been proposed that NC speciï¬cationoccurs during gastrulation as a consequence of the action oftwo successive gradients of secreted signals A combinationof intermediate levels of activity of BMP and Wnt signalingacting on the ectoderm to induce and specify NC precursorsat the neural plate border and a subsequent requirement ofboth signals is needed for maintenance of speciï¬cation duringneurulation Aybar and Mayor Steventon Inchick embryos it was shown that NCCs are speciï¬ed as earlyas the blastula stage Prasad It was demonstratedthat during gastrulation Pax7 expression is restricted to cellslocated in a region in the medial epiblast which are NCfated andcontribute to the neural folds and later to migrating NCCs Basch The inhibition of Pax7 function in chicks inhibitedthe expression of key NC markers such as Snai2 OMIM Sox9 Srybox OMIM Sox10 OMIM andHNK1 beta13glucuronyltransferase like OMIM Basch and BronnerFraser This evidence suggests thatthe neural plateprospective ectoderm interaction at the neuralplate border might not be a requisite for NC speciï¬cation orinduction and that neural plate border formation and NCinduction might be separable eventsThe variousresearch works carried outto study theorigin of NCCs have identiï¬ed genes anized into a generegulatory network that participate in and control the inductionspeciï¬cation and diï¬erentiation of NC SimµesCosta An example of this are the transcription factors involvedin induction such as FoxD3 Forkhead Box D3 OMIM Snai2 and Sox9 SaukaSpengler and BronnerFraser GarcaCastro and coworkers identiï¬ed a novel preneuralborder state characterized by early Wntβcatenin signalingtargets that displayed diï¬erent responses to BMP and FGFsignaling from the neural border genes in human cells Leung These preborder genes Gbx2 Gastrulation brainhomeobox OMIM SP5 OMIM Zic3 OMIM and Zeb2 OMIM had their induction andpeak of expression before the classical neural plate borderspeciï¬er genes such Msx12 Muscle segmentrelated homeobox OMIM Pax37 OMIM and Zic1 OMIM Such speciï¬er genes together withsignaling molecules direct the expression of NCspeciï¬c geneslike AP2 OMIM FoxD3 Snai2 Sox9 and Sox10Speciï¬ers regulate NC eï¬ector genes involved in migrationSox9 Sox10 Cad7 and diï¬erentiation [Col1a Collagen typeI alpha OMIM Ngn1 Neurogenin OMIM MitfMicrophthalmiaassociated transcription factor OMIM] in human NC development Betters The NC population migrates to diï¬erent regions of the mouseembryo from the NT after the epithelialmesenchymal transitionmaintaining its multipotential character until completingdiï¬erentiation in its ï¬nal destination Baggiolini To study the ontogeny of the NC diï¬erent model anismsboth in vivo and in vitro have been used Several proteinsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Neural crest origin regions in human and mouse embryos and some of its cranial derivatives A The topleft part of the scheme shows the origin of theneural crest cells green that migrate through the embryo On the topright side the level of axial origin see axial color key of different regions of the neural crest isrepresented in developing mouse or equivalent human embryos the migration of neural crest is represented in green inside the embryos and the direction ofmigration is marked with black arrows Depending on their axial level of origin and migratory pathways neural crest cells adopt different fates and contribute tovarious tissues and ans B The main cranial derivatives labeled in green are shown Abbreviations d days E mouse embryonic stage NCCs Neural CrestCells s somite St human stage VSMC vascular smooth muscle cellsincluding transcription factors as well as epigenetic modiï¬ersthat take part in the speciï¬cation and diï¬erentiation of the NChave been described The study of transcription factors and ofthe signaling pathways in which they participate is importantto understand the diï¬erentiation programs and how thesemultipotent cells are committed to a speciï¬c destination On theother hand transcriptome analysis during the development of theNC from speciï¬cation to migration Meulemans and BronnerFraser and a more recent study covering the migrationto the diï¬erentiation of the NC show the importance of theinteraction between the diï¬erent transcription factors and thesignaling pathways at every stage of NC development SimµesCosta However these authors acknowledge thatit is diï¬cult to have a complete global map since only a fewtranscriptional regulators have been characterized and little isknown about the function of the products of the eï¬ector genesacting on NC migration Betancur SimµesCosta andBronner VegaLopez NC and cranial placodes are thought to appear togetherduring the evolution of vertebrates to give rise to speciï¬c sensorystructures of the head Northcutt and Gans Northcutt The components of the sensory nervous system of the headare derived from the NC and from an embryonic cell populationdeveloping in close proximity the cranial sensory placodes theolfactory lens otic trigeminal epibranchial and paratympanicplacodes A series of events induce develop and anizethese cell precursors which through reciprocalinteractionswith NCCs build the functional sensory system in vertebratesSteventon Singh and Groves Migrating NCCsarrive ï¬rst at the site of ganglia development ie the trigeminalFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cFrontiersinCellandDeveopmentallliBoogywwwfrontiersniAugustlVoumeAlrticeTABLE Contributions of neural crest cells and placodes to ganglia and cranial nervesCranial nerveGanglion and typeOrigin of neuronsReferencesCNI Olfactory Ensheating gliaof Olfactory nervesCNIII Oculomotor mCiliary visceral efferentCNV Trigeminal mixTrigeminal general afferentCNVII Facial mixSuperior general and special afferentCNVIII Vestibulocochlear sCNIX Glossopharyngeal mixCNX Vagus mix Superiorlaryngeal branch and recurrentlaryngeal branchCNXI Accessory mInferior geniculate general and special afferentSphenopalatine visceral efferentSubmandibular visceral efferentAcoustic cochlear special afferent and Vestibularspecial afferentSuperior general and special afferentInferior petrosal general and special afferentOtic visceral efferentSuperior general afferentInferior nodose general and special afferentVagal parasympathetic visceral efferentNo ganglionTelencephalonolfactory placode NCCs at forebrainNCCs at forebrainmidbrain junction caudal diencephalonand the anterior mesencephalonNCCs at forebrainmidbrain junction from r2 into 1st PAtrigeminal placodeHindbrain NCCs from r4 into 2nd PA 1st epibranchialplacode1st epibranchial placode geniculateHindbrain NCCs 2nd PAHindbrain NCCs 2nd PAOtic placode and hindbrain from r4 NCCsHindbrain NCCs from r6 into 3rd PA2nd epibranchial placode petrosalHindbrain NCCs from r6 into 3rd PAHindbrain NCCs from r7r8 to 4th and 6th PAHindbrain NCCs 4th and 6th PA 3rd nodose and 4thepibranchial placodesHindbrain NCCs 4th and 6th PAHindbrain from r7r8 to PA NCCs 4th PABoyd Muller ORahilly andM¼ller Barraud Noden Couly Wahl Lee dAmicoMartel and Noden Forbes and Welt DamicoMartel and Noden DamicoMartel and Noden Lumsden Barlow and Northcutt Begbie andGraham Barlow Krimm Sandell Narayanan and Narayanan DamicoMarteland Noden ORahilly and M¼ller Barlowand Northcutt Narayanan and Narayanan DamicoMarteland Noden Muller and ORahilly ORahilly and M¼ller Abbreviations CN Cranial Nerve m purely motor nerve mix mixed nerve sensory and motor NC neural crest PA pharyngeal branchial arch r rhombomere s purely sensory nerve There is no known ganglionof the accessory nerve The cranial part of the accessory nerve sends occasional branches to the superior ganglion of the vagus nerveMndezMadonadoletalNeuralCrestDerivedCranailNerves\x0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Contribution of neural crest cells to the formation of cranial nerves I III V VII VIII IX X and XI These selected cranial nerves are formed by thecontribution of cranial placodes and neural crest cells indicated in green Neural crestderived Schwann cells produce peripheral myelination of cranial nerves IIIXIIThe sensory nerves are the olfactory I the optic II and the vestibulocochlear VIII The motor nerves are the oculomotor III the trochlear IV the abducens VIand the accessory XI The remaining are mixed nervesganglion but the diï¬erentiation of these cells is delayed untilthe migration and diï¬erentiation of the corresponding placodalcells in chicks Covell and Noden Placodal speciï¬cationand development as well as its contribution to the assembly ofplacodal derivatives is a complex and wideranging topic thatis beyond the scope of this review We will focus on discussingthe main signaling pathways and relevant transcription factorsinvolved in the speciï¬cation of cranial NCCs precursors theirdiï¬erentiation to form CNs and ganglia that are exclusively NCderived and the alterations caused by the mutations of certaingenes that are important for the neurogenesis of NC derivativesSIGNALING PATHWAYS INVOLVED INCRANIAL NEURAL CRESTDEVELOPMENTareseveralsignaling pathwaysThereand transcriptionfactors that are known to regulate NC and CN formationduring development We discuss some important pathwaysinvolved in cranial NCCs induction and speciï¬cation in closerelationship with the cranial ganglia and nerves derived from theNC Figure BMPsBone morphogenetic proteins are proteins that control severalimportant steps in the formation and diï¬erentiation of the CNSof vertebrates These proteins act in diï¬erent regions of theCNS to regulate fate proliferation and diï¬erentiation Aftergastrulation the presence of BMPs and the activation of thissignaling pathway are essential for the diï¬erentiation of thenonneural ectoderm whereas the inhibition of this pathway isrequired for the proper formation of the neural plate It has beenproposed that the later activation of BMPs receptors participatesin the induction of the NC through a very ï¬ne regulation wherethe presence of BMPs at a speciï¬c time will give rise to the NCin mouse and human Embryonic Stem Cells ESCs Figure 3BMizuseki Leung Seminal studies in Xenopus have shown that there is an activitygradient of BMPs controlled by their antagonists and that anintermediate level is needed to induce the formation of the NCLaBonne and BronnerFraser Marchant Barth Tribulo Thus the BMP antagonistsChordin OMIM and Noggin OMIM areexpressed in a spatiotemporal manner thatinï¬uences theformation of the NC In mouse at embryonic day E Nogginis expressed in the neural folds and in the dorsal region afterthe closure of the NT The expression of Chordin is low atthe level of the neural plate and in the paraxial mesodermThese antagonists participate in the induction of NC as wellas in delamination but also protect from apoptosis induced byBMP during migration and diï¬erentiation of NCCs Importantlyit was observed that the decrease in the expression of theseBMP antagonists alters the PNS derived from the NC andcraniofacial skeletal elements Noggin knockout mice presentedall cranial nerves but the vagus X and glossopharyngeal IX aredisanized and fused Doubleknockout mice of Noggin andChordin lack CN and only a structure similar to the trigeminalganglion V is present Anderson In the chickembryo the activity of BMP signaling during the formation ofNC precursors is modulated by CKIPSmurf factors throughthe regulation of Smad degradation resulting in intermediateFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Gene regulatory network involved in neural crest contribution to the formation of cranial nerves The cranial ganglia and cranial nerves are formed inprecise positions along the dorsoventral and anteroposterior axes of the midbrainhindbrain region A The drawing represents a human embryo at stage days somites equivalent to mouse day E9510 somites and chick stage h somites The cell signaling pathways that providedevelopmental cues to neural crest precursors are colorcoded when these factors diffuse the target regions are indicated with arrows with the same color In panelB an idealized scheme of the hindbrain shows the cell signaling gradients and the genes that establish the dorsoventral pattern C The illustration of the human days stage and chick stage hindbrain rendered ï¬at to eliminate cerebral ï¬exures The levels of origin of the neural crest cells NCCs and placodeswhich contribute to the formation on cranial nerves are indicated on the left NCCs from the corresponding rhombomeres also populate other embryo structures in asegmental fashion and generate different craniofacial derivatives The positions of the cranial ganglia and the otic vesicles are indicated on the right side thecontribution of NCCs is indicated in green The segmental nested expression of HOX genes is colorcoded On the right signaling pathways and the expression oftranscription factors involved in cranial nerve CN formation are indicated Adapted from Lumsden and Keynes Noden Yamamoto and Schwarting BallyCuif and Wassef Takahashi and Osumi and M¼ller and ORahilly Abbreviations CN cranial nerve FP ï¬oor plate Mmesencephalon NCCs neural crest cells OV otic vesicle r rhombomere PA pharyngeal archeslevels of BMP activity required for proper NC formationPiacentino and Bronner In contrast placode progenitorshave diï¬erential BMP signaling requirements as they can bespeciï¬ed under low or no BMP signaling Thiery A study of human ESCs hESC showed that if BMPs areblocked with Noggin for h on days or of thediï¬erentiation protocol there is a dramatic decrease in theinduction of human NCCs However if the inhibition is madeon day the inhibition is partial so the participation of BMPsat the beginning of the induction of the NC is very importantwhile the inhibition of this pathway promoted the expressionof neural genes such as SOX1 OMIM HES5 OMIMFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial Nerves and SOX2 OMIM Leung Thisprotocol produced sensory peripheral neurons and it will beof interest to investigate if such neurons can contribute to thesensory CN after grafting them in experimental animals aswell as the eï¬ect of modulating BMPs on peripheral neurondiï¬erentiation Interestingly BMP antagonism upregulates theseneural stem cell markers but several reports indicated that Sox1Hes5 and Sox2 are involved in the suppression of neuronaldiï¬erentiation by maintaining neural stem and progenitorcells in an undiï¬erentiated state in mammalian cells Kan BaniYaghoub The generation ofneurons from stem cells depends on the decrease of Sox13expression caused by proneural proteins However if Sox13target genes were repressed independently of proneural activityneural progenitor cells diï¬erentiated prematurely and someneuronal features emerged These results demonstrate a dualrole of proneural proteins in the acquisition of a deï¬nitiveneuronal fate and indicate that the proneural proteindirectedrepression of Sox13 expression is a required and irreversiblestep in the commitment to neuronal diï¬erentiation in severalspecies including mammals Guillemot Farah Bylund BMP4 OMIM and Smad proteins have beeninvolved in an interesting mechanism called retrograde signalingin trigeminal ganglia from rats Ji and Jaï¬rey Thismechanism elicits a speciï¬c transcriptionalresponse thatcontributes to the speciï¬cation of diï¬erent subpopulations ofsensory neurons in the trigeminal ganglia CN V As axonsfrom the neurons oftrigeminal ganglia grow and extendinto their three main peripheral axonal branches ophthalmicmaxillary and mandibular that innervate the correspondingregions of the face they encounter BMP4 which results in aretrograde signal that leads to transport back transcription factorsSMAD1 and from axons to the somata where nuclearaccumulation of the phosphorylated and transcriptionally activeSmad forms contributes to neuronal speciï¬cation and gangliapatterning Nohe Ji and Jaï¬rey BDNF BrainDerived Neurotrophic Factor OMIM signaling wasalso found to regulate axonal levels of SMAD1 and inconcert with BMP4 for patterning of the trigeminal gangliaJi and Jaï¬rey Hippo PathwayGenetic studies have demonstrated that Hippo signaling is crucialin an size regulation controlling cell number by modulatingcell proliferation and apoptosis processes Huang Hippo is a criticalfactor for proliferation and epithelialmesenchymal transition during embryonic development andcancer In the neural tube of the mouse chicken and frogYAP YesAssociated Protein OMIM is expressed inthe ventricular zone progenitor cells and colocalizes with theneural progenitor cell marker Sox2 Milewski Cao It has been observed that the ectopic expressionof one ofthis pathwayTAZ Transcriptional Coactivator With PDZBinding MotifOMIM in mammalian cells stimulates cell proliferationthe transcriptional regulators ofreduces the inhibition by contact and promotes the epithelialmesenchymal transition Lei A relationship between this signaling pathway and the classicalNC genes such as interaction with Pax3 is through TAZ andthe phosphoprotein YAP65 These proteins participate as coactivators of Pax3 It has been suggested using transgenicmice that Tead2 TEA Domain Family Member OMIM is an endogenous activator of Pax3 in NCCs Milewski Through expression assays Pax3 and Yap65were colocalized in the nucleus of NC progenitors in thedorsal region offorSchwann cell proliferation and diï¬erentiation in a stagedependent manner Nuclear TAZYAP complexes activate cellcycle regulators to promote Schwann cell proliferation whiledirecting diï¬erentiation regulators in cooperation with Sox10 formyelination in rodents Deng the NT HippoTAZYAP are criticalNeuroï¬bromatosis Nf2 OMIM is a tumorsuppressor that inhibits YAP during dorsal root ganglia DRGdevelopment Merlin encoded by the NF2 tumorsuppressivegene was identiï¬ed through genetic studies in mouse embryosand proved to be an important upstream regulator of theHippoYap pathway Neuroï¬bromatosis is an inherited diseasecharacterized by the development of bilateral Schwann celltumors originated from CN VIII Mouse with speciï¬c Schwanncellinactivated Nf2 alleles developed schwannomas and SChyperplasia McClatchey Giovannini Merlin has also been shown to act as a suppressor ofmouse neural progenitor proliferation by inhibiting TAZYAPpathway activity Lavado The mechanism bywhich Merlin regulates YAP activity might involve p21 Proteinactivated kinase PAK1 OMIM activation whichinduces phosphorylation of Merlin thus abrogating its scaï¬oldfunction for YAP and LATS12 OMIM andthereby attenuates YAP phosphorylation by LATS12 in mousecells Sabra it has been suggested that nuclearexport signals of Merlin mediate YAP nuclear export in epithelialmammalian cells Furukawa Hindley and coworkers investigated the role of HippoYAPsignaling in NC development and neural diï¬erentiation Theyshowed thatthe activity of YAP promotes an early NCphenotype accompanied by premature migratory behavior andthat HippoYAP interacts with RA signaling in hESCs Hindley A recent study demonstrates that YAP is necessaryfor the migration of a premigratory pool of NCCs sincethey incorporated YAP signaling into a BMPWntdependentmolecular network responsible for the migration of trunklevelNC in avians Kumar Notch SignalingNotch is a family of conserved receptors whose activation isinduced by speciï¬c ligands Delta1 OMIM Delta OMIM Delta4 OMIM Jagged1 OMIM and Jagged2 OMIM through interactionwith four possible receptors Notch14 Perdigoto and Bardin Once the Notch receptors are activated through thecellcell interaction proteolytic cuts are carried out resultingin the release ofthe Notch Intracellular Domain NICDFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesMumm NICD translocate to the nucleus andforms a transcriptional complex together with the DNA bindingprotein CBF1 C promoter binding factor OMIM This complex recognizes the speciï¬c sequence CTGTGGGAAin itstarget genesfor example Hes1 OMIM Kageyama Notch1 receptor is present during development oftherhomboencephalon at E95 in mice showing strong expressionwithin the hindbrainincluding the trigeminal geniculatepetrosum and nodose placodes which give rise to CN VVII IX and X respectively and is also expressed in the oticand olfactory vesicle Reaume A study wherehuman induced pluripotent stem cells were induced toward NCdiï¬erentiation showed that when Notch signaling is blockedusing a Îsecretase inhibitor DAPT or shRNA for JAGGED the genes specifying NC [DLX5 Distalless homeobox OMIM PAX3 SNAI2 SOX10 and TWIST1 OMIM] are downregulated However the ectopic expression ofNICD1 increased its expression demonstrating that Notch alsoparticipates signiï¬cantly in NC induction Noisa Mead and Yutzey evaluated the function of Notch signaling inmurine NCderived cell lineages in vivo They demonstratedthat cellautonomous Notch has an essential role in properNCCs migration proliferation and diï¬erentiation with criticalimplications in craniofacial cardiac and neurogenic developmentMead and Yutzey Sonic HedgehogSonic Hedgehog Shh signaling is involved in the correctdevelopment of NC and therefore in the generation of its cellularderivatives Figure 3B Shh is a member of the family ofthe secreted Hedgehog proteins Sonic Shh OMIM Indian Ihh OMIM and Desert Hedgehog Dhh OMIM Shh regulation during NC diï¬erentiation is crucialduring head and face morphogenesis Mutant mice and humanslacking Shh present holoprosencephaly and cyclopia due to thelack of separation of the forebrain lobes Chiang It is suggested that Shh inhibition maintains Pax3 expressionso the lack of Shhmediated regulation for Pax3 inductionpromotes the constitutive induction of NC generating theaforementioned phenotypes A subset of Fox genes regulated byShh signaling is important during lip morphogenesis in miceEither Shh addition or Foxf2 OMIM overexpressionwas shown to be suï¬cient to induce cranial NCCs proliferationEverson On the other hand enhanced Shh signaling in mousemediated by lossoffunction Ptch1WigWig of the Shh receptorPatched1 Ptch1 OMIM suppressed canonical Wntsignaling in the CN region This critically aï¬ected the survivaland migration of cranial NCCs and the development ofplacodes as well as the integration between NC and placodesKurosaka Ptch1WigWig mutants exhibited severelydisanized trigeminal CNV and facial nerves CNVII thatdid not develop properly and failed to project to their appropriatetargettissues Kurosaka High levels of Shhsignaling have been correlated with Moebius Syndrome whichis characterized by cranial nerve defects including trigeminalabducens CNVI and facial alterations concurrent with othercraniofacial defects Verzijl VegaLopez NCCs migration is particularly sensitive to Shh levels since inmice lacking Shh these cells continue their migration beyondthe normal position and fuse medially condensing into a singlemidline ganglion Fedtsova Mutation in the mouseHedgehog acyltransferase Hhat OMIM gene producedhypoplasia and aberrant fusion of cranial ganglia CN V VII IXand X and aï¬ected NC and placode gene markers expressionsuggesting that a regionalized action of the Hedgehog signalingis required for proper cranial ganglia and nerve developmentand patterning Dennis In vitro analyses showedthat Shh increased the number of cranial NC progenitors fromquail embryos yielding neural and mesenchymal lineages Shhcan decrease the neuralrestricted precursors without aï¬ectingsurvival or proliferation These data also suggestthemesenchymalneural precursor was able to yield both the PNSand superï¬cial skeleton Calloni thatReceptor Tyrosine Kinase RTK FamilyHumans have known RTKs which fall into subfamiliesA few years ago a systematic work summarized the contributionof the mouse model to the understanding of the role of a subsetof RTKs in regulating the activity of NCCs in developmentFantauzzo and Soriano With respect to its downstreamsignaling RTKs induce the activation of various pathwaysincluding PLCÎ PI3K MAPK JNK Shc Erk and the JAKSTATpathways In this section we discuss insights pointing tomechanisms of action of some RTK families in relation tothe development of the cranial NC that have emerged fromrecent evidenceEph ReceptorsEphrin ligands and Eph erythropoietinproducing humanhepatocellular carcinoma receptors comprise an increasinglywell studied family of signaling molecules Ephrins bind totwo families of transmembrane tyrosine kinase receptors EphAand EphB While Atype Ephrins preferentially bind to EphAreceptors Btype Ephrins do so to EphB receptors In Xenopusthe streams of NCCs going to the second branchial arch expressEphrinB2 whereas cells reaching the third arch express EphB1disruption of EphEphrin signaling results in aberrant migrationof NCCs causing mixing of the streams in the branchial pouchesSmith Eph receptor functions are best characterizedin the mouse nervous system where they are involved inneuronal development and axon guidance Wilkinson Xuand Henkemeyer migration and proliferation Conover H
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"dramatic spread of Coronavirus Disease COVID19 has profound impacts on every continent and life Due to humantohuman transmission of COVID19 nuclear medicine staffs also cannot escape the risk of infection from workplaces Everystaff in the nuclear medicine department must prepare for and respond to COVID19 pandemic which tailored to the characteristics of our profession This provided the guidance prepared by the Korean Society of Nuclear Medicine KSNM incooperation with the Korean Society of Infectious Disease KSID and Korean Society for HealthcareAssociated InfectionControl and Prevention KOSHIC in managing the COVID19 pandemic for the nuclear medicine department We hope that thisguidance will support every practice in nuclear medicine during this chaotic periodKeywords Coronavirus COVID19 Nuclear medicine Prevention and control Practice guideline HoYoung Leedebobkrgmailcom Department of Nuclear Medicine CHA Bundang Medical CenterCHA University of Medicine Professor Pocheon Republic ofKorea Department of Nuclear Medicine Seoul National UniversityBundang Hospital Professor Seongnam Gyeonggido Republic ofKorea Department of Nuclear Medicine Samsung Medical CenterSeoul Republic of Korea Department of Nuclear Medicine Seoul National UniversityHospital Seoul Republic of Korea Department of Nuclear Medicine Chosun University HospitalGwangju Republic of Korea Department of Nuclear Medicine Korea University Guro HospitalSeoul Republic of Korea Department of Nuclear Medicine Hanyang University Guri HospitalSeoul Republic of Korea Department of Nuclear Medicine National Cancer CenterGoynag Republic of Korea Department of Nuclear Medicine Seoul Medical CenterSeoul Republic of Korea Division of Nuclear Medicine Department of RadiologyEunpyeong St Marys Hospital College of Medicine The CatholicUniversity of Korea Seoul Republic of Korea Department of Nuclear Medicine Soonchunhyang University SeoulHospital Bucheon Republic of Korea Department of Nuclear Medicine Inje University Haeundae PaikHospital Busan Republic of Korea Department of Nuclear Medicine Keimyung University DongsanMedical Center Daegu Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityCheonan Hospital Cheonan Republic of Korea Department of Nursing Soonchunhyang University BucheonHospital Bucheon Republic of Korea Division of Infectious Disease Department of Internal MedicineKangdong Sacred Heart Hospital Hallym UniversityChuncheon Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityBucheon Hospital Bucheon Republic of Korea Department of Nuclear Medicine Korea University Anam Hospital Korean Society of Nuclear Medicine Quality Control CommitteeSeoul Republic of KoreaBucheon Republic of Korea 0cIntroductionSince the first reports of Coronavirus Disease COVID in Wuhan China the infection had spread worldwiderapidly and COVID19 has reached pandemic levels InSouth Korea since its outbreak in February COVID has affected profoundly every aspect of communities Thehumantohuman transmission of COVID19 provides challenges for all healthcare facilities and healthcare providersIn the face of the COVID19 pandemic the Korean Societyof Nuclear Medicine KSNM Korean Society of InfectiousDisease KSID and Korean Society for HealthcareAssociated Infection Control and Prevention KOSHIC haveprepared the guidance for the nuclear medicine department tominimize confusion and ensure that nuclear medicine physicians and technicians continue to provide their services whileprotecting the patients and workers and preventing the transmission of the virus The Quality Control Committee ofKSNM reviewed several reports and recommendations previously published by the European Association of NuclearMedicine EANM [] Society of Nuclear Medicine andMolecular Imaging SNMMI American Society of NuclearCardiology ASNC [] International Atomic Energy AgencyIAEA and others [] This guidance is basically in compliance with the COVID19 guidelines of the Korea Centersfor Disease Control and Prevention KCDC [] Finallythis document was prepared in cooperation with KSID andKOSHIC KSNM emphasize that this guidance must be considered in the context of following the state and hospital infection control policies and flexibly applied according tochanges in circumstances and evidenceGeneral Principles During COVID19PandemicIn a pandemic situation such as COVID19 if necessarythe condition of the scheduled patient can be checked inadvance to adjust the examination schedule Nonurgent elective studies or therapy should be postponed in COVID19confirmed or COVID19suspectedpatients Rescheduling the studiestherapy must be donein a discussion with the referring clinicians Only urgent studies or therapy could be performed inCOVID19confirmed or COVID19suspected patientswhenever clinically appropriate The priority of studytherapy should be based on a casebycase indepth discussion between nuclear medicine physicians and referring clinicians In case of performing the urgent studiestherapy consult with the infection control offices of eachinstitution to comply with the infection control rules ofownNucl Med Mol Imaging COVID19suspected patients should undergo COVID testing before performing the studiestherapy Lung ventilation scan should not be performed in anyCOVID19confirmed or COVID19suspected patients Lowdose radioiodine therapy may be considered in caseof acute hyperthyroidism patients who are unable to tolerate antithyroid medications As lowdose radioiodinetherapy lower than GBq of I131 can be performedin an outpatient setting in South Korea COVID19infected patient can be administrated lowdoseradioiodine in the isolation room or negative pressureroom without any additional monitoring related toradioiodine therapyConsideration During the StudyTherapy Patient transportationScheduling COVID19confirmed or COVID19suspected patient as last study of the day to preventcrossinfection in the nuclear medicine department Ensure that other patients or caregivers should notaccess the nuclear medicine department to minimizethe exposure to COVID19 patient during the studytherapy Transfer the COVID19infected patient to the nuclear medicine department using negative pressuretransport bag to minimize exposure and contact todroplet COVID19 patients should wear masks at all timesof procedures If necessary add gowns gloves etc Devices and scanner management Mainly use disposable instruments or items Do notreuse disposable items such as oxygen masks nasalprongs suction tubes or suction lines The protocolfor reusable devices is as follows Cleaning After use the equipment contaminated with blood bodyfluids secretions and feces should be delivered to awashing room with care not to contaminate the surrounding environment The washing place should be separated from the spaceused for cleaning other items or other patients After immersing the contaminated equipment in a washing spacewash the product carefully to avoid splashing Wash enough to remove blood body fluids secretionsand feces from remaining 0cNucl Med Mol Imaging Staff undertaking cleaning should wear KF94 or N95masks longsleeved waterproof gowns goggles or faceshields hats shoe covers or rubber boots and doublegloves outer gloves are rubber gloves Disinfection and sterilization Depending on the risk level of the device according tothe Spaulding Classification of medical equipmentdevices noncritical devices require lowlevel disinfectionsemicritical devices require highlevel disinfectionsterilization and critical devices must be sterilized Disinfectants and sterilization methods by device classification should be followed in accordance with the notificationof the Ministry of Health and Welfare Be sure to check the disinfectant manufacturers recommendations The recommended disinfection process suchas dilution and application time of disinfectant and theeffective period and concentration of disinfectant arestrictly followed Laboratory and scan room management Only the minimum number of staffs should be placedin the nuclear medicine department All participatingstaffs should wear appropriate personal protectiveequipment PPE eye protection with goggles or faceshield medical protective masks N95KF94 or equivalent respirator disposable latex gloves disposablegown disposable shoe covers etc Cover the scanner couch or other equipment with aplastic cover to prevent contamination Every effort should be made to minimize theCOVID19 exposure to medical staff during injection of radiopharmaceuticalsSelect the protocol with the shortest duration of uptake time and scan time to minimize the time spentby the COVID19 patient in the departmentIn case of studies requiring an uptake phaseCOVID19 patients should be waiting in separatespace If possible COVID19 patients wait in negative pressure transport bag If negative pressuretransport bag is not available use bed or stretcherin waiting room with disposable cover Considerusing standard radiopharmaceutical dose to shortenthe procedure time After the completion of image acquisition the scanroom and patients space area should be disinfectedaccording to the standard protocol After image acquisition remove the plastic cover ofthe scanner and disinfect the scanner surface Remove and discard PPE adequately when leavingthe camera room or care area and immediately perform hand hygieneIn case of performing the radiolabeling of theCOVID19 patients blood products every processwith infectious materials openingstirringmixingdispensing COVID19 patients blood sampleradiolabeling etc should be done in class II biosafety cabinet according to the Biosafety Level Regulation Disinfection of laboratory with properdisinfectants ethanol hydrogen peroxide or ppm sodium hypochlorite should bedone Used PPE and disposable covers are removed withcaution not to contaminate the clean area and disposed in a container for biosafety waste Employee management All employees should be trained in the preventionand management of COVID19 infection and adhereto the rules of infection prevention Considering the skill level fatigue etc of the working staff sufficient personnel are allocated to securethemPriority from exemption is given to employees withhighrisk underlying diseases such as diabetesmellitus chronic obstructive pulmonary diseaseCOPD endstage renal disease ESRD chroniccardiac disease etc or pregnant women Cleaning and environmental management General principle Personnel responsible for cleaning or disinfectionshould complete the infection preventioneducation Employees should wear PPE KF94 or N95 respirators fullbody protective clothing or aprons goggles or face shields shoe covers or rubber bootsdouble gloves outer gloves are rubber gloveswhen cleaning or disinfectingIf there are organic substances on the surface of theenvironment it cannot be properly disinfectedTherefore wipe the surface before disinfecting theenvironmentIn order to prevent the possibility of microbialspraying cleaning should be performed using acleaning solution or a mop moistened with a disinfectant rather than a cleaning method using abroom or a vacuum cleanerInstead of spraying disinfectants thoroughly cleanthe surface of the environment using a clean towelmoistened with the disinfectant or a commerciallyavailable disinfecting tissue towel 0cNucl Med Mol Imaging Use cleaning tools as disposable as possible or exclusively However when the cleaning tool isreused the used cleaning tool is sterilized usingan appropriate disinfectant and then dried andstored Disinfection of a patients space areaIn the case of the space area used by the patientmark the place where contamination was confirmedbefore cleaning and disinfecting the surface andseal the contaminated object to prevent others frombeing exposed Ventilation before during and after cleaningdisinfection disinfection after ventilation for hbased on air cycles per hour Wear PPE Wipe with a cloth cloth etc wet withthe diluted disinfectant Wipe the touched wall surface and all frequently used areas and keep it for atleast min After then wipe the surface with acloth dampened with clean water cloth etcResumption of use Consider the characteristics ofeach type of disinfectant used and the purpose of thefacility After disinfection the virus is killed but thedecision at the time of resumption of use cannot beapplied in batch due to different characteristics ofdisinfectants so it is necessary to consider the precautions for each productFor details on disinfecting methods such as surfacedisinfection and washing refer to DisinfectionGuidelines to Prevent the Spread of COVID19 atPublic and Multipurpose Facilities 3rd editionRefer to the method of disinfecting the patient spaceareaSelect an environmental disinfectant Select an approved or declared disinfectant by the Ministry ofEnvironment and follow the usage usage and precautions for each product Disinfectant list of the Ministry of Environmenthttpecolifemegokr Precautions when using environmentaldisinfectants Select the disinfectant after confirming informationsuch as approval from the Ministry of Environmentand Environment When using environmental sterilizers make sure tofollow the manufacturers recommendations suchas checking the expiration date safe usage for eachproduct and precautions and preparing the diluentaccording to the manufacturers instructions The disinfecting method of sprayinginjecting disinfectant is not applied to surface disinfectionbecause it causes aerosol infection increased riskof inhalation and the range of contact between thedisinfectant and the surface is insufficient so thedisinfecting effect is insufficient Disinfectant hazard information must be checkedand used carefully Do not mix different disinfectants Do not placenear flammable materials Disinfectant should beused in a wellventilated area As the disinfection effect may decrease over timedilute as much as necessary and use it immediatelyDo not store the remaining amount and discard itimmediately Laundry managementStore clean laundry in a separate space Employees handling laundry should be trained toprevent infection Employees handling contaminated laundry shouldwear PPE N95 masks or equivalent respiratory protection gowns gloves overshoes etc and performhand hygiene after removing PPE The laundry used for the patient is disposed of according to the relevant regulations see WasteManagement Act Medical Institution LaundryManagement Rules etc Thoroughly ensure that pathogens are not exposed topersonnel handling the laundry or surrounding environment during the entire process of collectingtransporting and washing laundry Waste management Waste related to COVID19 patients is managed bythe rules of hospital infectious control policySharp tools such as needles or blades are collectedin containers for impervious and nonpermanentwaste and containers should be stored in the placewhere the items are usedSimple infectious waste contaminated or possiblycontaminated with COVID19 patients sample isautoclaved and discarded Radioactive waste shouldbe discarded in compliance with national regulationwith caution not to contaminate the staff or areaConclusionConsidering that outbreaks of novel viruses have been periodically appearing these days nuclear medicine staffs should getused to guidance and policies for infectious disease in working 0cNucl Med Mol Imaging place to protect patients worker themselves and furthermorevaluable medical resources Basically this guidance can beapplied in case of any other humantohuman transmissiondisease for operating the nuclear medicine department Alsoalways bear in mind the rapid change in the situation thisguidance should be used in conjunction with the currentgovernment and local hospital policiesCompliance with Ethical StandardsConflict of InterestJiIn Bang HoYoung Lee Young Seok ChoHongyoon Choi Ari Chong Jae Sun Eo Ji Young Kim Tae SungKim HyunWoo Kwon Eun Jeong Lee Eun Seong Lee Hye LimPark Soo Bin Park Hyekyung Shim BongIl Song Ik Dong YooKyung Jae Lee Hong Jae Lee Su Ha Han Jin Seo Lee Jung Mi Parkand Sung Hoon Kim declare that they have no conflict of interestEthical Approval This work does not contain any studies with humanparticipants or animals performed by any of the authorsInformed Consent Not applicableReferences Paez D Gnanasegaran G Fanti S Bomanji J Hacker M SathekgeM et al COVID19 pandemic guidance for nuclear medicine departments Eur J Nucl Med Mol Skali H Murthy VL AlMallah MH Bateman TM Beanlands RBetter N et al Guidance and best practices for nuclear cardiologylaboratories during the coronavirus disease COVID19 pandemic an information statement from ASNC and SNMMI J NuclCardiol httpsdoiorg101007s12350020021232 Huang HL Allie R Gnanasegaran G Bomanji J COVID19nuclear medicine departments be prepared NuclMedCommun MossaBasha M Medverd J Linnau K Lynch JB Wener MHKicska G et al Policies and guidelines for COVID19 preparedness experiences from the University of Washington Radiology httpsdoiorg101148radiol2020201326 Zhang X Shao F Lan X Suggestions for safety and protectioncontrol in Department of Nuclear Medicine during the outbreak ofCOVID19 Eur J Nucl Med Mol Buscombe JR Notghi A Croasdale J Pandit M O'Brien J GrahamR et al COVID19 guidance for infection prevention and controlin nuclear medicine Nucl Med Commun Standard guideline for healthcareassociated infection control andprevention Korean Center for Disease Control and Prevention andKorean Society for HealthcareAssociated Infection Control andPrevention httpcdcgokrCDCcmscontentmobile2675626_viewhtml Accessed 2nd Jun Korean Society for HealthcareAssociated Infection Control andPrevention Korean Center for Disease Control and Preventionhttpwwwcdcgokrboardesmida20507020000bid0019actviewlist_no366579 Accessed 2nd Jun Guidelines in response to coronavirus disease for local governmentKorea Centers of Disease Control and Prevention2020 httpswwwcdcgokrboardboardesmida20507020000bid0019actviewlist_no367279tagnPage1 Accessed 2ndJun Disinfection guidelines to prevent the spread of COVID19 at public and multipurpose facilities Korea Centers of Disease Controland Prevention httpswwwcdcgokrboardboardesmida20507020000bid0019 Acessed 15th Jun Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c"
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" accurate detection of patients with minimal residual disease mrd after surgery for stage ii coloncancer cc remains an urgent unmet clinical need to improve selection of patients who might benefit formadjuvant chemotherapy act presence of circulating tumor dna ctdna is indicative for mrd and has highpredictive value for recurrent disease the medocccreate trial investigates how many stage ii cc patients withdetectable ctdna after surgery will accept act and whether act reduces the risk of recurrence in these patientsmethodsdesign medocccreate follows the trial within cohorts twics design patients with colorectal cancercrc are included in the prospective dutch colorectal cancer cohort plcrc and give informed consent forcollection of clinical data tissue and blood samples and consent for future randomization medocccreate is asubcohort within plcrc consisting of stage ii cc patients without indication for act according to currentguidelines who are randomized into an experimental and a control armin the experimental arm postsurgery blood samples and tissue are analyzed for tissueinformed detection ofplasma ctdna using the pgdx elio¢ platform patients with detectable ctdna will be offered act consisting of cycles of capecitabine plus oxaliplatin while patients without detectable ctdna and patients in the control groupwill standard followup according to guidelinethe primary endpoint is the proportion of patients receiving act when ctdna is detectable after resection themain secondary outcome is 2year recurrence rate rr but also includes 5year rr disease free survival overallsurvival time to recurrence quality of life and costeffectiveness data will be analyzed by intention to treatcontinued on next page correspondence mkoopman6umcutrechtnlsj schraa and kl van rooijen are shared first authorrja fijneman gr vink and m koopman are shared last author1department of medical oncology university medical center utrechtutrecht university heidelberglaan cx utrecht the netherlandsfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cschraa bmc cancer page of continued from previous pagediscussion the medocccreate trial will provide insight into the willingness of stage ii cc patients to be treatedwith act guided by ctdna biomarker testing and whether act will prevent recurrences in a highrisk populationuse of the twics design provides the opportunity to randomize patients before ctdna measurement avoidingethical dilemmas of ctdna status disclosure in the control grouptrial registration netherlands trial register nl6281ntr6455 registered may wwwtrialregisternltrial6281keywords colon cancer circulating tumor dna ctdna adjuvant chemotherapy twics in patients with stage ii colon cancer cc the recurrence rate rr after surgery is approximately disease management after surgical resection in stageii cc is still under debate because the overall survivalos benefit of adjuvant chemotherapy act in thisgroup of patients varies between and only [ ]moreover offering act in a lowrisk population induces an important amount of overtreatment with unnecessary but sometimes severe toxicity and costsseveral prognostic characteristics of stage ii cc havebeen identified to provide better selection of patientsthat might benefit from act patients with presence ofat least one of the following characteristics are classifiedas being at high risk of disease recurrence poorly differentiated histology pt4 lesions inadequately less than sampled lymph nodes lymphovascular or perineuralinvasion or tumor presentation with perforation or obstruction in contrast patients with a deficient mismatch repair dmmr status in stage ii cc have a low risk ofrecurrence and act is not considered beneficial irrespective of the presence of other risk factors [ ]other known prognostic factors in cc like gene expression profiles or braf v600e and ras mutations have been investigated but do not adequatelyidentify the patients that will benefit from act []despite the definition of high and low risk subgroupsof stage ii cc patients retrospective analyses demonstrated that improved survival after administration ofact was not observed in high risk patients or exclusivelyin patients with a pt4 tumor [] therefore in thenetherlands act is currently only recommended in stageii cc patients with a pt4 tumor without dmmrunfortunately also pt4 is not an absolute predictorfor disease recurrence in stage ii patients in a retrospective analysis of stage ii cc patients with pt4tumors the 3year diseasespecific survival rate aftersurgery was in patients who received act and in patients who did not receive act whichmeans that of these patients are exposed to actunnecessarily considering nonpt4 stage ii patients a population registry analysis of patientsshowed that in this group of patients sufferedfrom recurrences these data demonstrate thatusing pt4 as a prognostic factor results in significantunder and overtreatmentminimal residual disease mrd is defined as the presence of tumor cells in the blood bone marrow or lymphnodes not detected by conventional staging procedures patients who have mrd after surgery are not completely cured and therefore at high risk of developingdisease recurrence development of a highly specific andsensitive biomarker testindicative for mrd wouldallow identification of the subset of patients likely to experience recurrence of disease thereby improving the selection of patients who may benefitfrom adjuvanttreatment in adjuvant trials this would solve problemsof high numbers needed for inclusion and dilution of effectiveness of adjuvant treatment by inclusion of manyalready cured participants cellfree circulating tumor dna ctdna has a strongpotential for being this sensitive and yet specific biomarker ctdna consists of smallfragments usually bp of tumorderived dna containing tumorspecific mutations which can be detected in liquid biopsies such as blood samples [] because of the shorthalflife of ctdna estimates ranging from to min the presence of ctdna in blood samples taken several days after surgery presumably reflects a state ofmrd [] patients with mrd have the highest riskfor disease recurrencerecently the presence of ctdna after tumor resection demonstrated a very strong prognostic value fordisease recurrence in stage ii cc with a 2years rrof versus in patients with and without detectable ctdna after surgery respectively in thisstudy the univariate prognostic value of ctdna was muchhigher than that of pt4 status hazard ratio of versus respectively there are several ongoing trials that usectdna in prognostication nct03637686 nct03737539nct03416478 nct03312374 nct02842203 nct0361 and treatment nct03748680 actrn12615000381 nrggi of nonmetastatic cc but to date thereare no results available of randomized controlled trialsrcts that use ctdna for selection of act treatment 0cschraa bmc cancer page of the accumulating evidence for the strong prognosticvalue of ctdna raises an important ethical dilemma forrandomization of patients when designing a conventional rct in which patients with detectable ctdna arerandomized into act treatment or standard of carefollowup while disclosing ctdna status to the controlgroup indeed the knowledge of having a very highchance of disease recurrence will be a big burden for patients with detectable ctdna in the control group andtheir caregivers as they are not being offered any additional therapy this warrants an innovative trial designlike the trialdifferent from the conventional rctwithin cohorts twics design [] the twics design enables an experimental group in which ctdna status is disclosed and a control group that is unaware oftheir ctdna statusthe medocccreate trial is designed as a multicenter twics study with two parallel groups in whichwe will investigate whether stage ii cc patients with detectable ctdna after resection are willing to receiveact and whether act reduces the rr in these ctdnapositive patientsmethodsdesignaimthis study investigates the willingness of patients to receive act after detection of ctdna postsurgery andthe effect of ctdnaguided act on the rr in stage iicc patientsstudy designthe medocccreate trial follows the twics designand is performed within the prospective dutch colorectal cancer cohort plcrc wwwplcrcnl plcrc is set up by the dutch colorectal cancer groupdccg and collects clinical data and patient reportedoutcome measures proms at baseline and at multipletime points during followup fig at enrollment patients give informed consent for use of their clinical dataand optionally for receiving quality of life questionnairesfig schematic presentation of medocccreate using the trial within cohort twics design a plcrc is a nationwide cohort study in thenetherlands with inclusion of crc patients all stages by optional informed consent regarding collection of biomaterials and futurerandomization observational as well as interventional trials can be performed within the cohort b nonmetastatic crc patients are included inmedocc when the patient signs informed consent for plcrc including additional blood sampling blood samples are withdrawn beforeresection days after resection and every months during the first years of followup c eligible stage ii colon cancer patients arerandomized following the twics design in the experimental group informed consent is being asked for immediate ctdna analysis of theblood sample obtained after resection if ctdna is detectable patients are offered adjuvant chemotherapy the control group is not informedabout medocccreate and will receive standard of care 0cschraa bmc cancer page of collection of biomaterials for research additional sequential blood sampling and for being approached forfuture studies conducted within the infrastructure ofthe cohort either in accordance with the twics design or notpatients with pt4 tumors will be offered act therefore we will include eligible patients with pt4 tumors without a recommendation for act according totheir treating physician and use pt4 status as a stratification factorpatient selection and recruitmentpatients will be recruited in both academic and nonacademic hospitals in the netherlands that are participating in plcrc nonmetastatic colorectal cancercrc patients that give informed consent for plcrcincluding consent for additional blood sampling at enrollment will be included in the observational plcrcsubstudy medocc molecular early detection of coloncancer before surgery the participants are eligible forthe current medocccreate trial if they meet the following criteria after surgery histopathological confirmed and radically resected stage ii cc age ¥ years informed consent for plcrc and medoccincluding consent for randomization in future trials anduse of tissue physical condition allows treatmentwith combination chemotherapy consisting of a fluoropyrimidine and oxaliplatin and no indication foract according to the treating physician andor multidisciplinary board patients who are pregnant have hadanother malignancy in the previous years except forcarcinoma in situ or patients with contraindications forfluoropyrimidines andor oxaliplatin will be excludedcurrently the dutch guidelines recommend act forpatients with pt4 tumors however there is large ageand hospital dependent variation in administration ofact in this group and in clinical practice not all stage iiblood sample collectionblood samples are collected before and days aftersurgery for all patients included in the medocc clinicalstudy predominantly comprising stage i ii and iii ccpatients table blood samples two tubes of mlper timepoint are collected in cell free dna streckblood collection tubes for various research purposesamong which the medocccreate trialrandomizationabout week after surgery when the histopathologicalreport is finished medocc patients who are eligiblefor medocccreate will be randomized to theintervention or control arm using slim an onlineplatform to manage patientinclusion including arandomizationgeneratedcomputerservice therandomization schedule is stratified by tstage and usespermuted blocks of random sizes allocation concealment will be ensured as the service will not release therandomization code only patients randomized to theintervention arm will be informed about medocccreate according to the twics design experimental armafter randomization only patients randomized to theexperimental arm will be asked separate informedtable standard protocol items for intervention trials spirit schedule of enrollment interventions and repeated measurementsact adjuvant chemotherapy ctdna circulating tumor dna qol quality of life intervention group only intervention grouponly if ctdna is positive 0cschraa bmc cancer page of consent for the immediate analysis of ctdna status ofthe postsurgery sample a small proportion of patientsestimated approximately will have detectablectdna in their blood these patients will be offeredact patients decide whether they accept or refuse thistreatment patients without detectable ctdna will receive routine standard of careact will consist of months of capecitabine and oxaliplatin capox or months of fluorouracil leucovorinand oxaliplatin folfox treatment starts preferablywithin weeks and not beyond weeks after surgeryduring and after completing act routine followupwill consist of regular visits at the surgical outpatient department blood withdrawals for analysis of carcinoembryonic antigen cea and imaging standard ultrasoundofthe liver according to current guidelines in thenetherlands no additional imaging will be performed toprevent detection biascontrol armin the control arm patients will not be informed aboutthe medocccreate trial and receive routine followup care consisting of cea tests every months for thefirst years and abdominal ultrasound or ct every months in the first year and once a year afterwards oneyear after surgery a colonoscopy is performed postsurgery blood samples will not be tested for ctdna immediately but will be analyzed batchwise after severalmonths without result disclosure to patients and theirtreating physiciansfollowupblood samples will be collected at 6monthly intervalsfor the first years after surgery for both patients in theexperimental arm and the control arm conform themedocc study protocol these samples will not be analyzed for ctdna immediately and results will not bedisclosed to patients and treating physicianstumor tissueinformed ctdna analysisafter surgery the local pathologist will send a formalinfixed paraffinembedded ffpetissue block to thecentral laboratory where dna will be isolated for further analysisthe postsurgery blood sample is drawn between and days after surgery the sample is not withdrawnbefore day to reduce the risk of falsenegative ctdnatests due to the relatively large amount of cell free dnacfdna released due to cell damage after surgery theblood is taken no later than days after surgery to beable to start chemotherapy within weeks after surgerysamples are kept at room temperature and sent by regular mail to the central laboratory within days wherectdna will be isolated for further analysistumor tissue dna will be analyzed by targeted nextgeneration sequencing of a panel of more than genes using the pgdx elio¢ tissue complete assay frompersonal genome diagnostics pgdx baltimore mdusa plasma ctdna will be analyzed by targeted nextgeneration sequencing of a panel of more than genesusing the pgdx elio¢ plasma resolve assay from pgdxbaltimore md usa both panels include the mostcommonly mutated genes in cc including apc tp53kras and braf tumor tissue dna mutations are usedas input information for plasma ctdna mutation callingthereby increasing both sensitivity and specificity of thectdna testprimary endpointthe primary endpoint is the proportion of patients starting with act after detection of ctdna in the postsurgery samplesecondary endpointsthe most important secondary endpoint is 2year rr inpatients with detectable ctdna in their blood expressedas the proportion of patients that experience a recurrence within years after surgery detection of recurrences in months after surgery will occur by standardfollowup investigations including monthly bloodsampling of tumor marker cea and monthly imagingwith ultrasound liver or ct abdomen and when indicated by symptoms radiological andor histopathological evidence is used to confirm the recurrence thedate of the said investigation is considered the date ofrecurrencedata about followup recurrences and survival areroutinely collected within plcrc using the netherlandscancer registry ncr managed by the netherlandscomprehensive cancer anisation iknl to provideinsight in the characteristics and magnitude of cancer inthe netherlands other secondary endpoints include 2year rr in aperprotocol analysis 5year rr intentiontotreatand perprotocol analysis time to recurrence ttr and 5year disease free survival dfs rate and7year diseaserelated os rate and 5year rr inpatients with undetectable ctdna after surgery quality of life qol and costeffectiveness of the ctdnaguided strategytimetoevent outcomesos rate is expressed as proportion of patients that arealive and years after surgery dfs rate is expressed asproportion of patients that did not experience disease recurrence a second primary cc or death within and years after surgery ttr is expressed as time monthsbetween surgery and detection of disease recurrence 0cschraa bmc cancer page of patients will be censored at the last date of followup if adate of death is not recorded and at the date of death ifthe cause of death is not due to ccquality of lifeqol is measured within the cohort at regular intervalsin patients who gave consent to send questionnaires nationally and internationally validated questionnaires areused among which the european anisation for research and treatment of cancer quality of life questionnaire core and the colorectal cancer moduleeortcqlqc30 and cr29 the work ability indexwai the euro quality of life5 dimensions eq5dthe multidimensional fatigue inventory20 mfi20and the hospital anxiety and depression score hadscosteffectiveness of the ctdnaguided treatmentthe costeffectiveness analysis will be carried out from asocietal perspectiveincluding both direct health carecosts as well as indirect costs from productivity loss thehealth outcome measure in the costeffectiveness analysis will be the total quality adjusted life years qalyper group for analysis offactors related to qalysquestionnaires are used provided within plcrcsample size considerationsthe primary endpoint is the proportion of ctdna positive patients starting with act however 2year rr inthe ctdna positive patients after surgery is an importantsecondary endpoint and the power calculation is performed for this secondary endpoint we estimate thatsimilar to effectiveness in stage iii cc patients act inctdnabased highrisk stage ii cc patients will lead toa absolute reduction of recurrences within yearsafter surgery in the observational trial of patientswith detectable ctdna experienced disease recurrencewithin years after resection with a power of and an alpha of patientswith detectable ctdna need to be included in botharms assuming a prevalence of ctdna after surgery of and adjustment for loss to followup and rejection ofadjuvant therapy in the intervention arm of a totalsample size of patients is calculated in eacharm we expect few patients with detectable ctdna inthe intervention group to refuse act because patientsare selected upfront for being in a physical condition toreceive act and the established prognostic value of detectable ctdna is highwe assume that crossover from the control arm tothe intervention arm will not occur because only eligiblepatients randomly selected in the cohort and allocatedto the intervention arm will be informed about the trialand have the opportunity for immediate analysis ofctdna patients in the control group will not be informed about the trial or their ctdna statuswe assume that of patients in the interventionarm with detectable ctdna will be treated with actthe proportion of patients starting with chemotherapythe primary endpoint can in that instance be determined with a margin of error width of the confidence interval of we expect to complete recruitment of patients within years with more than participating dutchhospitalsdata analysisdata will be analyzed according to the intentiontotreatprinciple for the primary endpoint and the secondaryendpoint of 2year rr in patients with detectable ctdnaafter surgery in this analysis we expect to compare patients with detectable ctdna who received act inthe intervention arm with patients with detectablectdna in the control arm ie based on ctdna analysisperformed retrospectively at least months after surgery and not disclosed to patients and treating physicians the proportion of patients that experience arecurrence in both arms will be compared by means of achisquare test in addition for other secondary endpoints and exploratory analyses we will analyze timetoevent outcomes in patients in both arms with detectablectdna after surgery differences in timetoevent outcomes will be analyzed by standard survival methodseg kaplanmeier curves compared by logrank testscoxs proportional hazards models will be used for multivariable analysiscomparison of qol of the ctdna positive patients inboth study arms will be done using repeated measurements methods and including act as factor qol willalso be analyzed for the whole population in both armsof the study treatment differences at each qol assessment time point will be compared by means of thewilcoxon rank sum testa lifetime horizon will be applied forthe costeffectiveness analysis parametric survival functions willbe used to extrapolate dfs and os curves beyond yearsresponsibilitiesprotocol modifications will be submitted as amendmentto the medical ethical committee by the study coordinator the local principle investigator of each participating hospital is responsible for patient inclusion logisticsof biomaterials to the centrallaboratory and patientfollowup to ensure quality of data study integrity andcompliance with the protocol and the various applicableregulations and guidelines a data monitor of the iknlhas been appointed to conductto thesite visits 0cschraa bmc cancer page of participating centers and randomly check patient datathe study coordinator together with the principle investigator will have access to the final dataset and is responsible for publishing study results the results will besubmitted to a peerreviewed journaldiscussionmedocccreate is the first clinical trial using thetwics design to investigate ctdnaguided strategies instage ii cc taking an important step towards clinicalimplementation of ctdna in cancer diagnostics andcarewithctdnadetectablea few other trials with the aim to reduce recurrencesin cc by use of a ctdnaguided approach are in preparation or recently started the improveit trial adanish study started in october uses a classicalrct in stage i and ii crc patients randomizing between months of act or intensified followup for patientspostsurgerynct03748680 four hundred fifty stage ii crc patients are being included in the australian dynamicstudy and randomized to be treated according to thectdna result with to months of act or accordingto standard of care actrn12615000381583 thecobra study in the united states and canada has asimilar rct approach nrggi also several trialsin stage iii crc patients started recently dynamiciii actrn12617001566325 in the near future thesestudies will provide deeper understanding and lead toimplementation of ctdnaguided strategies in clinicalpracticein the current era of rapidly emerging new diagnosticand treatment strategies the classical rct is challengedbecause of inefficient and therefore timeconsuming recruitment of eligible patients main reasons for patientsto refrain from participation in rcts are preference forone ofthe treatment arms anxiety or aversion torandomization and difficulties understanding the concept of an rct resulting in a delay of availability ofpotential beneficial treatments modern trial designsare being adopted to avoid thistimeconsuming and costly way of conducting trials with highrates of unfinished studies therefore the medocccreate trial uses the modern twics design the twicsdesign has shown to have a positive impact on trialefficiency also by enrolling higher proportions of eligible patients generalizability to daily clinical practiceimproves inefficientthisseveralstudy design hasstrengths firstmedocccreate is nested within the large nationwide plcrc cohort study with currently almost included crc patients the infrastructure of this cohortin which clinical data and biomaterials are collected afterbroad informed consent of participating patients allowsinnovative and efficientcomprehensiveresearch incrc using this infrastructure the study can be quicklyimplemented in many participating hospitals savingcosts and complicated logistics several studies accordingto the twics design are performed within this or comparable cohorts therefore experience with this trialdesign has been gained and this will contribute to execution of the medocccreate study [ ]secondly a difficult ethical dilemma in an rct analyzing ctdna presence postsurgery is avoided by thetwics design with the current knowledge about thestrong association with recurrent disease disclosingctdna status to all participants would be a great burdenfor patients with detectable ctdna and their treatingphysicians in the control group because of disappointment bias in the control group we would expect highdropout and contamination due to crossover when aclassical rct design would be applied making accrualand interpretation of results unfeasible in thistwics study all participants already have blood withdrawn after surgery for research purposes and only theeligible patients allocated to the intervention arm willhave the opportunity to obtain a ctdna test result andact if ctdna is detected patients in the control armtreated according to current guidelines will not be informed about randomization and their blood sampleswill be analyzed at a later point in time beyond the window of act treatmentthis study has also potentiallimitations and challenges the twics design is potentially susceptible tolow statistical power and internal validity biases levelsof participants eligibility and consent should be substantial to achieve valid and reliable results and measurements taken in the control group should be sufficient foradequate comparisons to be made therefore thetwics design is not appropriate for every experimentalintervention in case of the medocccreate studywe argue that eligibility and also consent will be substantial because of the high incidence of cc the large cohortwith high inclusion rates and the assumption that eligible patients in the intervention group are willing toaccept act because of the very strong association of thepresence of ctdna with recurrent diseaseanother limitation is the small sample size for primaryoutcome analysis eventually only patients in botharms of the trial are expected to have detectable ctdnaafter surgery based on previous data relapses areexpected within years and with a high event rate smallnumbers are sufficient capecitabinewe recommend a 6month duration of act consistand oxaliplatin capox oring offluorouracilleucovorin and oxaliplatin folfox forpatients with detectable ctdna after surgery the firstadjuvant cc trials investigating the combination of a 0cschraa bmc cancer page of fluoropyrimidine and oxaliplatin reported results for month duration of act in the idea trialfound a large reduction in toxicity for months treatment compared to months treatment although thistrial could not confirm noninferiority for monthstreatment for all patients treated with capox or folfox in stage iii crc the small difference limits clinicalrelevance besidesit did show noninferiority of theshorter regimen in patients treated with capox consequently dutch guidelines recommend months of actfor cc since however among patients withhighest risk of recurrence t4 n2 or both superiorityof 6month duration of therapy was found additionalideafrance resultsthe esmocongress showed the worst prognosis for ctdnapositive patients who only received months of act therefore in this study we recommend 6monthsact for patients with a very high risk of disease recurrence due to the presence of ctdna after surgerypresentedatliquid biopsy ctdna detection has become a promising technology with multiple putative clinical applications including its potential use as a biomarker for earlydiagnosis prognosis prediction and monitoring of treatment response driven by the excitement of its possibilities the field of technology of ctdna detection andanalysis is rapidly evolving yet the clinical utility ofctdna testing still needs to be proven when to applywhat technology to address which unmet clinical need isa key question that remains to be addressed applying ctdna detection as a biomarker for mrd isa challenging task biologically only a very low amountof ctdna is present in postsurgery patients with mrdstochastically by looking at mutations in a panel ofgenes chances increase that in a given blood sample atleast in one of the genes a mutation can be reliably detected test sensitivity can be further increased by making use of dna mutation information from tumortissue because the stringency in the calling of plasmactdna mutations can be reduced once you know whatmutations to look for tissueinformed ctdna analysisalso increases the ctdna test specificity recent observations showed that ctdna mutation detection can beconfounded by mutations that are present in clonalhematopoiesisincluding mutations in genes that arecommonly affected in cc such as tp53 these confounding mutations can be filtered by applying tissueinformed ctdna analyses as suchtechnically themedocccreate trial makes use of a ctdna test thatis wellsuited for mrd detection clinically howeverthe medocccreate trial needs to resolvewhether a positive ctdna test also allows to select forpatients who truly benefit from act treatment a requirement for clinical implementation to further support clinical implementation of ctdna analyses in thenetherlands the dutch coin initiative aims to providea validation framework for clinicalimplementation ofctdna analyses in the netherlands zonmw projectnumber in conclusion the medocccreate study is thefirst study using the modern and innovative twics design to study ctdnaguided administration of act instage ii cc patients the study aims to answer the important clinical question whether ctdna has prognosticas well as predictive value if this study demonstrates asignificant and substantial difference in disease recurrence in the intervention group compared to the controlgroup ctdna analysis and ctdnaguided treatmentshould be implemented into clinical practice to improvethe prognosis of stage ii cc patientsabbreviationsact adjuvant chemotherapy cc colon cancer cea carcinoembryonicantigen crc colorectal cancer ctdna circulating tumor dnadfs disease free survival dmmr deficient mismatch repair eortcqlqc30 and cr29 european anisation for research and treatment ofcancer quality of life questionnaire c30 and colorectal cancer module eq5d euro quality of life5 dimensions ffpe formalinfixed para
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" new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease In addition to the personto person transmission dynamic of the novel respiratory virus it has been recently studied the role of environmental factors in accelerate SARSCoV2 spread and its lethality The time being air pollution has been identified as the largest environmental cause of disease and premature death in the world It affects bodys immunity making people more vulnerable to pathogens The hypothesis that air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography can acts both as a carrier of the infection and as a worsening factor of the health impact of COVID19 disease has been raised recently With this review we want to provide an update state of art relating the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality The Authors who first investigated this association often used different research methods or not all include confounding factors whenever possible In addition to date incidence data are underestimated in all countries and to a lesser extent also mortality data For this reason the cases included in the reviewed studies cannot be considered conclusive Although it determines important limitations for direct comparison of results and more studies are needed to strengthen scientific evidences and support firm s major findings are consistent highlighting the important contribution of PM25 and NO2 as triggering of the COVID19 spread and lethality and with a less extent also PM10 although the potential effect of airborne virus exposure it has not been still demonstrated Introduction A new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease The scientific community has come together to implement pharmaceutical and nonpharmaceutical intervention measures designed to contain SARSCoV2 global spread Nevertheless on March 11th WHOs Directeneral announced that COVID19 can be characterized as a pandemic SARSCoV2 is primarily transmitted from persontoperson through close contact approximately m by aerosol respiratory droplets smaller than μm in diameter wwwwhoint Indoor environments might be especially hazardous because of their reduced ventilation Morawska lack ultraviolet light which rapidly inactivates the virus and because it can become less diluted than it would in outdoor environments Schuit It is also known how the virus can survive and being infectious in aerosols for hours and on surface up to days van Doremalen et al similarly with transmission dynamics known for SARSCoV1 associated with nosocomial spread and superspreading events Chen et al 2020ab Beyond the causality it is uncertain even if certain demographics of the population are more vulnerable to SARSCoV2 infection Based on recent reports male gender advancing age and comorbidities seem to be correlated with death and severe illness Harris et al Furthermore COVID19 seems to be associated with an increasing rate of thromboembolic events in hospitalized patients Llitjos Mechanisms of social and economic interactions are additionally supposed to be involved in the diffusion dynamic of COVID19 in the diverse parts of the world or of the same country such as the living conditions the healthrelated behaviour KhalatbariSoltani et al Corresponding author Email address ccopatunictit C Copat 101016jenvres2020110129 Received July Received in revised form August Accepted August EnvironmentalResearch1912020110129Availableonline24August2020001393512020ElsevierIncAllrightsreserved 0cC Copat the commercial exchanges Bontempi 2020a or the migration scale index H Chen It seems that these diffusion dynamics have particularly affected the COVID19 spread at the early stage Among the environmental parameters some climate condition such as temperature humidity sunlight and wind revealed a reduction of the COVID19 spread S Chen Coccia 2020a and air pollution seems to have a role in airborne transmission of SARSCoV2 and severity of COVID19 Domingo Nevertheless to better understand COVID19s diffusion patterns an interdisciplinary multidimensional approach should be encourage in order to develop firm s Bontempi Air pollution has been identified as the largest environmental cause of disease and premature death in the world GBD Ambient particulate matter PM induces its proinflammatory and thrombogenic effects through the generation of oxidative stress by its chemical compounds and metals Li Signorelli The recent identification of environmentally persistent free radicals EPFRs in the PM resulting from a mixture of combustion sources theorize its role in the increase of disease severity of lower respiratory tract infections LRTI Jaligama Scientific evidences support that short and longterm exposures to ambient air pollutants are associated with a broad of adverse health outcomes Ferrante and Conti Fiore such as higher mortality rates greater hospital admissions and increased outpatient visits Bremner Cohen Dehghani Dockery It has markedly detrimental consequences on asthma bronchitis pneumonia and COPD Dick Perng and Chen Raji Vignal Yarahmadi et al where bacteria and viruses are the most accepted causative factors that harm airway stability driving to infection exacerbation Furthermore air pollution represents an aggravating factor for infection diseases caused by some viral infections Domingo and Rovira such as respiratory syncytial virus RSV influenza A and B para influenza virus type pneumonia and influenzalike illness Carugno Croft Fukuda Huang Huh Liang Lin Silva Somayaji It determines an increase in the rate of hospitalizations and access to emergency department visits Studies related to the epidemic of SARSCoV coronavirus identified in November from the Guangdong province of southern China reported similar associations Cai Cui Kan Several Authors suggest that outdoor air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography could operate both as a carrier of the infection and as a worsening factor of the COVID19 severity Conticini Frontera Isaifan Martelletti and Martelletti This association is getting stronger thanks to the results of the numerous studies that have been launched all over the world and summarized with this review Most of the reviewed studies support that chronic exposure to air pollution might led people more susceptible to COVID19 disease leading to widespread COVID19 spread and lethality Nevertheless as suggested by Bontempi 2020b the potential effect of airborne virus exposure due to PM10 remain unclear With this review we want to provide an updated state of art of the recently epidemiological studies dealing with understanding the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality Fig PRISMA Flow Diagram of identification screening and inclusion of studies EnvironmentalResearch19120201101292 0cC Copat Method We have conducted a systematic review of the literature concerning the relationship between some air pollutants PM25 PM10 and NO2 and COVID19 outbreak The research was performed in compliance with the PRISMA criteria Preferred Reporting Items for Systematic Reviews and MetaAnalyses and the Flow Diagram is showed in Fig The research was conducted between April and July 6th in PubMed database It was used the Advanced Search Builder and the keywords were searched in [Title OR Abstract] We have filtered only research articles published in English language and selected the following keywords Air pollution and COVID19 or SARSCOV2 Particulate matter or PM and COVID19 or SARSCOV2 Nitrogen dioxide or NO2 and COVID19 or SARSCOV2 We choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported COVID19 cases andor deaths and air pollution data related to PM25 PM10 and NO2 thus excluding any Letter Opinion Commentary Review or nonrelevant articles We obtained a total of eligible published research articles in their final version and paper in its preprint version For some of them we chose to include only principal findings that clearly fit the aim this review Particulate Matter and COVID19 Atmospheric particulate matter PM is originated by a wide range of anthropogenic and natural sources Kim It consists of a heterogeneous mixture of solid and liquid particles suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals WHO It has been associated with increased respiratory morbidity and mortality Liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis Li Rhee In vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections Becker and Soukup Recently the research group of Setti gave first preliminary evidence that SARSCoV2 RNA can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of PM it could represent a potential early indicator of COVID19 although it does not give information regarding COVID19 progression or severity Several observations report a significant association between ambient concentrations of PM25 Adhikari and Yin Bashir Fattorini and Regoli Frontera Jiang Li VasquezApestegui Wu Yao Zhu Zoran 2020a and PM10 Bashir Coccia 2020b Fattorini and Regoli Jiang Li Yao Zhu Zoran 2020a with COVID19 pandemic across the most affected countries China Italy and USA see Table First evidences on the temporal association between air pollution and COVID19 were reported in China where the outbreak was first identified Zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in China The Authors included over of dailyconfirmed new cases in the whole of China between January 23rd and February 29th They applied a Generalized Additive Model GAM to examine the effects of meteorological factors and air pollution on COVID19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders They observed that the effect of PM25 on daily confirmed cases was greater than PM10 In particular they found that a 10μgm3 increase lag0 in PM25 and PM10 was associated with a CI to and CI to increase in the daily counts of COVID19 confirmed cases respectively Jiang focused their attention on three most affected cities of China Wuhan XiaoGan and HuangGang collecting data of daily cases and ambient air pollutant from Jan 25th to Feb 29th The Authors by applying a multivariate Poisson regression revealed a significant temporal association between PM25 increased and COVID19 incidence in all the considered cities especially in HuangGang Wuhan RR CI XiaoGan RR CI HuangGang RR CI Conversely an increase in PM10 concentrations was associated with a decrease of COVID19 incidence These results were partially confirmed by findings of Li who conducted a simple linear regression to compare COVID19 incidence with PM concentrations in Wuhan and XiaoGan from Jan 26th to Feb 29th in They found that an increase in PM25 was correlated with an increase of COVID19 incidence in both cities Wuhan R2 p XiaoGan R2 p while for PM10 only in XiaoGan R2 p The spatial distribution of particulate matter and case fatality rate CFR of COVID19 was studied by Yao in cities of China including Wuhan collecting data up to March 22nd First they found a significantly positive global spatial autocorrelation of COVID19 CFR Global Morans index I p highlighting a high CFR clustering located in Hubei Province With a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product GDP per capita hospital beds per capita local indicators of spatial association LISA map values city size and population or proportion of people older than years It was found that for every μgm3 increase in PM25 and PM10 the CFR increased by and respectively and the risk estimates increased to and with every μgm3 increase in average concentrations of PM25 and PM10 in respectively Some studies describe the association between air pollution and COVID19 across Italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other European countries The 28th of July Italy recorded more than total confirmed cases and deaths WHO most of which were distributed in the regions of Northern Italy especially the Lombardy It is recognized as one the most air polluted areas of Europe EEA where the frequent PM10 annual exceedances of the WHO threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year Baccini Bontempi 2020bfocused the attention on two of the most affected regions of Northern Italy Lombardy and Piedmont The Authors based on PM10 daily exceedances and COVID19 confirmed cases on March 12th thus before the Italian sanitary crisis observed that PM10 concentration was exceeded only few times among the Lombard cities that at the beginning of the epidemic were most affected On the contrary among some Piedmont cities suffering of severe PM10 pollution events COVID19 incidence was lower Based on their results the Authors concluded that COVID19 diffusion by airborne PM10 is hard to demonstrate nevertheless several research article revealed how PM in particular PM25 could had a role in accelerate and vast diffusion of COVID19 in Northern Italy For example Coccia 2020b by analyzed data on Italian province capitals and data of infected individuals up to April 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for PM10 in previous years and COVID19 diffusion In particular cities with more than days of PM10 exceedances showed a very high average number of infected individual about infected individuals on 7th April whereas cities having less than days of PM10 exceedances showed a lower average number of infected about infected individuals Frontera gave also evidences on the role of PM25 as a contributing factor of COVID19 outbreak in Northern Italy where EnvironmentalResearch19120201101293 0cC Copat Table Summary table reporting reviewed results on the association between COVID19 casesdeaths and air pollution PM25 PM10 and NO2 References Zhu Data analysis Generalized Additive Model GAM Aim Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Spatial association between fatality rate and air pollution PM25 and PM10 Spatial association between deaths counts and air pollution NO2 Temporal association between total cases daily confirmed cases and total deaths and air pollution PM25 and PM10 Temporal association between total cases daily confirmed cases and total deaths and air pollution NO2 Spatial description of PM10 exceedances versus COVID19 cases Multivariate Poisson regression Simple linear regression Multiple linear regression Descriptive analysis percentage of deaths in three NO2 μmol m2concentration range Pearson coefficient correlation Pearson coefficient correlation Descriptive analysis Number of days of PM10 exceeding μgm3 and COVID19 incidence Area of Study cities of China Period From Jan 23rd to Feb 29th Jiang Li Yao Ogen Zoran 2020a Zoran 2020b Bontempi 2020b From Jan 25th to Feb 29th From Jan 26th to Feb 29th in Data up to March 22nd Data up to the end of Feb From Jan 1st to Apr 30th From Jan 1st to Apr 30th From Feb 10th to March 12th Wuhan XiaoGan and HuangGang China Wuhan and XiaoGan cities of China administrative regions in Italy Spain France and Germany Milan Italy Milan Italy provinces of Lombardy Italy provinces of Piedmont Italy Coccia 2020b Data up to April 7th Italian Provinces Fattorini and Regoli Data up to April 27th Italian provinces PM25 A 10μgm3 PM25 increase lag0 was associated with a increase of daily confirmed new cases PM10 A 10μgm3 PM10 increase lag0 was associated with a increase of daily confirmed new cases Wuhan RR CI1032 XiaoGan RR CI HuangGang RR CI Wuhan R2 p XiaoGan R2 p Wuhan RR CI XiaoGan RR CI HuangGang RR CI Wuhan R2 p XiaoGan R2 p Ï2 p A μgm3 increase in PM25 was associated with a increase in fatality rate Ï2 p A μgm3 increase in PM10 was associated with a increase in fatality rate NO2 A 10μgm3 NO2 increase lag0 was associated with a increase in daily confirmed new cases Wuhan RR CI XiaoGan RR CI HuangGang no association found Wuhan R2 p XiaoGan R2 p of fatality cases are associated with NO2 μmolm2 R cid0 R R cid0 R cid0 R R cid0 R cid0 R cid0 R cid0 Lombardy PM10 exceeding between and COVID19 incidence between and Piedmont PM10 exceeding between and COVID19 incidence between and COVID19 in North Italy has a high association with air pollution of cities measured with days exceeding the limits set for PM10 R2 p R2 p continued on next page Hierarchical multiple regression model Pearson regression coefficient analysis R2 p Spatial association between confirmed cases and air pollution PM10 Spatial association between total confirmed cases and air pollution PM25 PM10 and NO2 EnvironmentalResearch19120201101294 0cC Copat Table continued References Frontera Frontera Wu Adhikari and Yin Bashir Bashir VasquezApestegui VasquezApestegui VasquezApestegui Period Data up to 31st March Data up to 31st March Data up to April 04th From March 1st to Apr 20th From March 4th to April 24th From March 4th to April 24th Data up to June 12th Data up to June 12th Data up to June 12th Area of Study Italian regions Italian regions counties in the USA Queens county New York USA California California districts of Lima Perù districts of Lima Perù districts of Lima Perù Aim Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Prediction of risk of COVID19 deaths in the long term average exposure to fine particulate matter PM25 Temporal association between daily confirmed cases and total deaths and air pollution PM25 Association between confirmed cases and air pollution PM25 PM10 and NO2 Association between deaths and air pollution PM25 PM10 and NO2 Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Spatial association between case fatality rate and air pollution PM25 Data analysis Pearson regression coefficient analysis PM25 R2 p PM10 Pearson regression coefficient analysis R2 p Longterm exposure increase of μgm3 in PM25 is associated with a increase in the COVID19 death rate Estimate on cases values cid0 CI Estimate on deaths value cid0 CI Kendall r cid0 Spearman r cid0 Zeroinflated negative binomia models Negative binomial regression model Spearman and Kendall correlation tests Spearman and Kendall correlation tests NO2 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient cid0 p mortality was found significantly higher than less polluted Italian regions By collecting data up to March 31st for all Italian regions and performing a Pearson correlation analysis they found a strong positive association both with the total number of confirmed cases R and deaths R other than with hospitalized cases R The Italian situation was further highlighted by the study of Fattorini and Regoli in Italian provinces They explored the spatial association between air pollution and COVID19 cases with data up to April 27th By applying the Pearson regression coefficient analysis they revealed a positive association both with PM25 and PM10 R2 p and R2 p respectively A focus on the most affected city of Italy Milan was conducted by Zoran 2020a This city is located in the Po valley basin known hotspot for atmospheric pollution at the continental scale EEA The Authors performed a temporal association between COVID19 Total cases Daily New positive cases and Total Deaths and particulate matter from Jan 1st and Apr 30th by applying a Person correlation In accordance with other studied they found a positive association between daily confirmed cases and PM25 R and PM10 R although they did not consider any delay time from infection to COVID19 onset Nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships To date the USA have more than million confirmed cases and thousand deaths WHO Here ambient concentrations of PM and O3 were found responsible to cause between and premature deaths Fann The association between air pollutants and COVID19 cases and deaths was studied by Bashir in the state of California from March 4th to April 24th corresponding to the beginning of the COVID19 outbreak in USA Based on their significant correlation found the Authors state that a limited human exposure to these pollutants will contribute to defeating COVID19 This seems unclear because they found a negative correlation with PM25 and PM10 EnvironmentalResearch19120201101295 0cC Copat by applying both the Kendall rank correlation and Spearmans one and it is not clear if they normalized COVID19 cases by population size and if they performed a day by day association or a spatial association across the country A focus on the Queen county New York USA was provided by Adhikari and Yin They retrieved data of PM daily concentrations from two ground monitoring stations and collected data of confirmed COVID19 cases and numbers of related deaths from USAFacts in the period from March to April The Authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of PM25 on disease outcomes over the past days They found a significant negative association among PM25 and new daily confirmed COVID19 cases cid0 CI and deaths cid0 CI Low PM concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as Italy where PM25 monthly concentrations reached values higher than μgm3 Fattorini and Regoli Frontera or in China where PM25 monthly concentrations reached values higher than μgm3 Zhu Jiang As said by the Authors other gaseous pollutants such as NO2 and SO2 could have influenced transmission and pathogenesis of COVID19 In the United States Wu investigated whether longterm average exposure to fine particulate matter PM25 increases the risk of COVID19 deaths by considering approximately counties in the United States of the population With an exposure prediction model the Authors calculated the county level longterm exposure to PM25 averaged for to and collected COVID19 deaths counts up to April 04th They conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors They found that a small longterm exposure increase of only μgm3 in PM25 is associated with a increase in the COVID19 death rate confidence interval CI VasquezApestegui recently reported first evidences on the spatial relationship between particulate matter and COVID19 outbreak from Latin America The Authors described the situation occurred in districts of Lima located in the second most affected country of Latin America Peru In particular by applying a multivariate regression model they evaluated the association between the population exposure to PM25 concentrations in the previous years and cases deaths and casefatality rates of COVID19 with data up to June 12th A significant association has been found both with cases and deaths Crude coefficient with p and with p respectively but not with case fatality rate All these studies highlight the role of PM in triggers of the COVID19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems Nitrogen dioxide NO2 and COVID19 induced lung damage Hence viral infection becomes more common after exposure to NO2 Zhu Furthermore NO2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children To increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation Bahrami Asl Kowalska increase of chronic obstructive pulmonary disease COPD Ghanbari Ghozikali Pfeffer and increase of pulmonary heart disease related mortality Chen A recent study explored the possible role of NO2 in interference in Angiotensin converting enzyme ACE2 The expression of ACE2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of COVID19 Alifano First observations report an association between ambient concentrations of NO2 and COVID19 pandemic across Europe China and USA Bashir Fattorini and Regoli Jiang Li et al Ogen Zhu et al Zoran et al 2020b Conversely to the other papers findings of Zoran 2020b and Bashir provides different findings reporting no association or a negative one between NO2 and daily deaths counts In China Zhu by applying the same method explained for PM observed that a 10μgm3 increase lag0 in NO2 is associated with a CI increase in the daily counts of COVID19 confirmed cases in cities of China These findings are confirmed by Jiang and Li et a who applied the same method described for PM Jiang revealed a significant positive association between NO2 and COVID19 both in Wuhan and XiaoGan Wuhan RR CI1053 XiaoGan RR CI but did not found any significant association in HuangGang Li found a significant linear correlation both in Wuhan R2 p and XiaoGan R2 p Ogen presented evidences on the relationship between exposure to NO2 including the months of January and February shortly before the COVID19 spread in Europe and novel coronavirus fatality in the most affected European countries concluding that longterm exposure to NO2 may be a potential contributor to mortality caused by SARSCoV2 He collected data concerning the number of fatality cases from administrative regions in Italy Spain France and Germany and correlated mortality with tropospheric NO2 concentrations measured by the Sentinel5 Precursor spaceborne satellite The major tropospheric NO2 hotspot identified was located in the Northern Italy In all European regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards Results showed that out of the fatality cases by March were in five regions located in north Italy and central Spain Furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum NO2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 The methodology used by Ogen cannot support a longterm exposure investigation Surely a validation of the satellite measure with those of the ground ones the adjustment of the results according to the different population size of each country could have made their results more robust Nevertheless the study provide new insights for future investigation The Italian situation was further studied by Fattorini and Regoli who collected data of COVID19 incidence up to April 27th from Italian provinces They revealed a strong spatial corr
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"Ovarian cancer is the second most common gynecologic cancer with high mortality rate andgenerally diagnosed in advanced stages The 5year diseasefree survival is below MicroRNAs subset of thenoncoding RNA molecules regulate the translation in post transcriptional level by binding to specific mRNAs topromote or degrade the target oncogenes or tumor suppressor genes Abnormal expression of miRNAs were foundin numerous human cancer including ovarian cancer Investigating the miRNAs derived from the peripheral bloodsamples can be used as a marker in the diagnose treatment and prognosis of ovarian cancer We aimed to findbiological markers for early diagnosis of ovarian cancer by investigating BRCA1 gene mutation carrier monozygoticdiscordant twins and their high risk healthy family individuals miRNAsMethods The study was conducted on monozygotic twins discordant for ovarian cancer and the liquid biopsyexploration of miRNAs was performed on mononuclear cells that were isolated from the peripheral blood samplesThe miRNA expression profile changes in the study were found by using microarray analysis miRNA isolationprocedure performed from the lymphocyte in accordance with the kit protocol The presence and quality of theisolated miRNAs screened by electrophoresis Raw data logarithmic analysis was studied by identifying thethreshold normalization correlation mean and median values Target proteins were detected for each miRNA byusing different algorithmsResults After the comparison of monozygotic discordant twins for epithelial ovarian carcinoma upregulation of the miRNAs miR6131 miR1305 miR1973p miR3651 and downregulation of miRNAs miR3135b miR4430 miR664b5p miR7663p were found statically significantContinued on next page Correspondence hy2188istanbuledutrDepartment of Cancer Genetics Istanbul Faculty of Medicine OncologyInstitute Istanbul University Istanbul Turkey The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cTuncer Journal of Ovarian Research Page of Continued from previous pageConclusions The detected miRNAs out of miRNAs might be used in the clinic as new biological indicatorsin the diagnosis and follow up of epithelial ovarian cancer with complementary studies The miRNA expressionprofiles were identified to be statistically significant in the evaluation of ovarian cancer etiology BRCA1 mutationstatus and ovarian cancer risk in accordance with the obtained dataThere is a need for validation of the miRNAs which were particularly detected between monozygotic twins and itsassociation with ovarian cancer was emphasized in our study in wider cohorts including ovarian cancer patientsand healthy individualsKeywords Monozygotic twins miRNA expression profiles BRCA1 and BRCA2 BiomarkersBackgroundOvarian cancer is a significant cause of mortality in gynecologic cancers and one ofthe leading cause ofcancerassociated mortality [] In Turkey ovarian cancer is the 7th most common type of cancer in women inaccordance with worldwide Globocan datas showthat each year more than women are diagnosedwith ovarian cancer OC worldwide and approximately women die from it The data of Globocan for Turkey shows that annually women are diagnosed with ovarian cancer and women die fromthis malignancy The 5year survival rate was given as These data revealed that ovarian cancer is an important reason of gynecologic cancer associated mortality rate [] The epithelial ovarian cancersEOCoriginating from the ovarian surface epithelium constitutes approximately of ovarian malignancy [] Themajority of EOC patients are diagnosed in advanced stages Stage III and IV and year freesurvivalrate is below [] The standard treatment for newlydiagnosed ovarian cancer is the combination of cytoreductive surgery and platinbased chemotherapy Significant advances in radical surgery and chemotherapystrategies have improved clinical outcomes but unfortunately no progress has been made with relapse andtreatment resistance [] Ninety percent of ovarian cancer occurs sporadically in the population whereas hereditary type appears of ovarian cancer patientsBRCA1 and BRCA2 genes are the most common breastovarian cancer syndrome associated genes Both BRCA1and BRCA2 have roles in the control of the genomic stability cell cycle and apoptosis The mutations occurringin these genes result with the inability of DNA repairand therefore results in the accumulation of the mutations in the cell The rate of the breast cancer susceptibility of women with BRCA1 gene mutation until theage of years was and the rate of ovarian cancersusceptibility rate is breast cancer susceptibilitywomen with BRCA2 gene mutation until the age of years is and ovarian cancer development risk is [] Twin studies became important on genetics by theendandcentury Geneticnineteenthofthethe differentiated genesepidemiologic studies with monozygotic twins were accepted as highly useful investigation models in the pastdecades and have been used recently [] When a similarity for a disease or a quantitative feature betweenmonozygotic and dizygotic twins is compared variationsare excluded according to studies conducted in thepopulation and thereforeit is easier to identify andmake etiological differences visible via twin studies Because the affected siblings and dizygotic twins share theapproximately ofthephenotypic differences between twins are known to beassociated with the genetic variation In addition diversity may be revealed with a very limited patient population Therefore the results of the twin studies can beapplied to the population and can make valuable contributions to the genetic studies Monozygotic twins aregenetically similar and generally expected to be compatible for congenital malformations chromosomal abnormalities and Mendelian disorders There are numerousstudies conducted via discordant monozygotic twins revealing the genetic contribution [] Therefore investigating the genetic variability in monozygotic twins ishighly important and the majority of the human geneticsassociated research focus on finding genetic variability indiscordant monozygotic twins Phenotypically discordantmonozygotic twins are used as the model systems inidentification of the variable in understanding the pathogenesis of a disease The most striking study is the oneconducted with monozygotic twins in Canada and evidencing that multiple sclerosis MS was a genetic disease [] MicroRNAs are one of the subset of the noncoding RNAs generally consisting of single strand in nucleotide length not transformed to protein havingroles in post transcriptional regulation or suppression oftranslation of the target mRNAs [ ] The regulatoryroles of miRNAs were demonstrated to occur in tumorigenesis cell differentiation proliferation and apoptosis[] miRNA genes are known to locate in thechromosomal breaks This DNA breaks cause chromosomal abnormalities frequently associated with cancersusceptibility and tumor development [] The noninvasive biologicalindicators have been used for the 0cTuncer Journal of Ovarian Research Page of treatment resistance of ovarian cancer The most common of this indicators are the cancer antigen125 CA and cancer antigen153 CA153 These biological indicators can be used in the followup of thetreatment response in the diagnosed patients but cannotbe used in the early diagnosis and in differentiation ofthe malignant disease [] Therefore there is a need forspecial therapeutic agents customized for patients thatmay be used target specific therapies and in the earlydiagnosis of the ovarian cancer in identification of theefficacy of therapy and in the follow up period Thusstudies investigating the target molecules and biologicalindicators are required that will enable the early diagnosis and in the development of the better therapy optionsDifferentially synthesize miRNAs such as miR miR141 miR125b miR2223p or let7 family has beenshown in studies with ovarian cancer patients [] However the use of these miRNAs as a biomarker in ovariancancer is not yet available In order to clearly define therole of miRNAs in the pathogenesis of ovarian cancerwe planned to investigate the BRCA mutant monozygotic twins with the same genetic profile but with discordant for ovarian malignant transformation In thisstudy miRNAs which are thought to have the potential biological indicator role were studied from bloodsamples of both discordant monozygotic twins andBRCA wild type healthy siblingsMethodsPatients recruitmentThe peripheral blood lymphocytes of monozygotic twinsdiscordant for ovarian cancer and healthy individuals inthe same family were used in the study The patient diagnosed with ovarian cancer and all family members applied to the Cancer Genetics Clinic of OncologyInstitute of Istanbul University for BRCA breast cancersusceptibility gene testing were examined for BRCAgene mutation All family members in the study consisted of highrisk individuals with Hereditary Breast andOvarian Cancer HBOC syndrome and the people included in the study were given as BR codes according topatient file number The monozygotic ovarian cancer patient healthy monozygotic twin healthy sisters and niece were found to have BRCA1 gene mutation c5266dupC pGln1756Profs74 rs397507247 on exon Thepatients brother and daughter were found negative forBRCA1BRCA2 gene mutations In this study lymphocyte cells separated from peripheral blood belonging tototal of cases including younger age ovarian cancer patient and healthy monozygotic twin a patients daughter elder sisters a younger sister a nephew and a brotherwere examined by miRNA microarray method Thepedigree of the family included in the study and theirhierarchical cluster analaysis via Euclidean method isshown on Fig The study was approved by the Ethics Committee ofthe Istanbul Faculty of Medicine Following InstitutionalEthics Committee approval informed consents were obtained from all participants before enrollment into thestudy Ethics Committee Approval Number atstoredthey wereLymphocyte and miRNA isolationFicoll SigmaAldrich Darmstadt Germany density gradient was used to separate white blood cells mononuclear cells from other blood components miRNAisolation procedure was performed from the lymphocytein accordance with the kit protocol using the miRNeasyMini Kit Qiagen cat NoID The proceduresteps in accordance with the protocol are as follows μL QIAzol solution was included on the cells storedin nitrogen tank Cell fractionation was enabled by mixturing using the vortex For complete nucleoproteinfractionation °C roomtemperature for min By adding μL chloroformthey were shacked and mixed on hand The tubes wereincubated for min at °C room temperature thenwere centrifuged for min at °C and g Thesupernatant formed after centrifugation was transferredto collection tube using a pipette and was mixed usingvortex by inclusion of μL ethanol The supernatant formed after the ethanol centrifuging was transferred to collection tube was removed with a pipettewas mixed with vortex by including μL ethanol Seven hundred microliters was taken from the obtained mixture and was transferred to the RNeasyMiniElute spin colon placed on mL collection tubeThe tubes were centrifuged for s at g at °Croom temperature Seven hundred microliters RWT buffer was added to spin colons and the colons werewashed by centrifuging at g for sThe centrifuging procedure was repeated by including μL RPE buffer twice consecutively to colons The colons placed into clean tubes with mL were dried bycentrifuging min in maximum speed The colonsplaced in mL sterile tubes were included μL distilled water by centrifuging at g in min and themiRNAs were collectThe quality control of the miRNAsThe presence and quality of the isolated miRNAs werescreened by electrophoresis at V on Agarose gelThen the purity and concentrations of the miRNAswere measured on Thermo Scientific NanoDrop spectrophotometer NanoDrop Technologies Wilmington DE USA device The miRNA purity for each persondevicemeasurement result were obtained with the comparisonaccordance withthe NanoDropin 0cTuncer Journal of Ovarian Research Page of Fig The pedigree of the family included in the study and their hierarchical cluster analysis Legend The pedigree of the family and using thecorrelations between samples plotted a dendrogram for sample grouped by Hierarchical clustering Euclidean distance Complete Linkage BR Healthy Brother BRCA1 negative nonBRCA1 mutation carrier BR Healthy Niece BRCA1 positive BRCA1 mutation carrier BR Healthy Daughter BRCA1 negative BR Healthy Monozygotic twin BRCA1 positive BRCA1 mutation carrier BR Monozygotic twindiagnosed with ovarian cancer BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrierof the measurements at spectrophotometrically at nm and nm wave lengths The measurement rates at nm wave lengths is a sign of quality of the purity of the samples therefore the samples in the idealvalue interval of and for RNA measurementswere included in the study The purity of miRNAs wereevaluated using a Bioanalyser device BioanalyserAgilent Technologies Santa Clara CA USA AgilentRNA Nano Kit Agilent Technologies Santa ClaraCA USA for confirming whether the miRNAs were appropriate and in adequate level for microarray analysisThe evaluated sample concentrations and results wereanalyzed The samples with RNA concentrations between ngμL and rRNA rate over and RNA integrity number values between and were evaluated asthe appropriate samples for array study 0cTuncer Journal of Ovarian Research Page of Microarray trial protocolMicroarray protocol was performed by preparing theSpikein solution sample marking hybridization sampledephosphorylation sample denaturation sample ligationhybridization of the samples slide loading preparationof the hybridization unit and elution and scanning ofslides The slide scanning procedure was performedusing the Agilent Microarray Scanner Agilent Microarray Scanner with Surescan High Resolution Technology Agilent Technologies Santa Clara CA USAdevice The scanning procedure of the slides were performed on SurePrint G3 Human miRNA MicroarrayRelease 8x60K Agilent Inc Santa Clara CA platform and using the Agilent Technologies G2600D scanning protocol The analysis of the TIFF Tagged ImageFile Format extensioned files obtained after scanningprocedure was performed using the Agilent Feature Extraction v11011 programThe success levels of stages developed in all experiment process with this analysis program the quality ofthe levels the process were monitored and evaluatedThen Bioinformatic Analysis procedure was performedData analysisRaw data logarithmic analysis was studied by identifyingthe threshold normalization correlation mean and median values Then the miRNAs demonstrating differentexpression profile among the samples were filteredUsing the Fold change rates and independent twosample T test the possible difference between the compared groups were evaluated All evaluations were performed to enable the cutoff values as the foldchangerates FC ¥ and pvalue Hierarchical clusteranalysis was performed using the Euclidean method Fig and Complete Linkage cluster method The control ofthe experimental errors and the detection of the erroneous finding rate were identified using the HochbergmethodBioinformatic analysisTarget proteins were detected for each miRNA by usingtwo algorithms Targetscan71 httpswwwtargetscanvert_71 and MirdbV5 httpsmirdbmiRDBThe targeted genes thought for each miRNA wereconfirmed by also both algorithms and the miRNAtarget relations were also experimentally confirmed mirTarbase70httpsmirtarbasembcnctuedutwphpindexphp databaseComparison groupsIn the study miRNA analysis was performed at the genome level withwithout mutation in cases withwithoutovarian cancer The miRNA data was evaluated by comparing different groups in order to investigate the effectof BRCA mutation in ovarian malignancy developmentand determine the miRNAs that can be important in theovarian cancer pathogenesis In Group the monozygotic twins discordant for ovarian cancer were comparedin order to find the effects of miRNAs in the formationof ovarian cancer In Group the family members withBRCA1 mutation were compared with family memberswithout BRCA1 mutation to identify the changes ofmiRNAs expression levels according to BRCA positivityIn Group the monozygotic ovarian cancer patient withBRCA1 mutation carrier and the other healthy familymembers with mutation carrier were compared for investigate the effects of both ovarian cancer developmentand BRCA positivity on miRNAs expression level InGroup all family members were compared with ovarian cancer monozygotic twin in order to find the miRNAs that might be important in the predisposition ofovarian cancer The comparison groups also showed inTable ResultsWe identified differentially expressed comparisonof miRNAs between the groups The raw data obtainedafter experimental studies were filtered before the comparisons between the groups The upregulated or downregulated miRNAs expression levels more than foldFC and smaller than the p value p wereconsidered in evaluation and the comparisons betweenthe groups were performed based on these values Allthese comparisons were evaluated for ovarian cancer etiology BRCA1 mutation carriage and the ovarian cancerrisk Hierarchical cluster analysis of the expression of miRNAs represents sharp separations of upregulatedyellow from downregulated blue in Fig miRNAs total of miRNAs were found statistically different after the comparison of phenotypicallydiscordant monozygotic twin siblings The miRNAsmiR1273 g3p miR1305 miR1973p miR3651 miR and miR92a3p expressions were found to haveupregulated and the other miRNAs let7i 5p miR125a5p miR15b5p miR22 3p miR3135b miRTable Comparison groups and cases in the groupsCaseGroup BR1639ControlBR1447Group BR1639BR1447BR1547BR2030BR1546BR1861BR1850BR2028Group BR1639Group BR1639BR1447BR1447BR1547BR2030BR1546BR1861BR1547BR2030BR1546BR1861BR1850BR2028 0cTuncer Journal of Ovarian Research Page of Fig Hierarchical cluster analysis of the expression of miRNAs Legend BR Healthy Brother BRCA1 negative nonBRCA1 mutationcarrier BR Healthy Niece BRCA1 positive BRCA1 mutation carrier BR Healthy Daughter BRCA1 negative BR HealthyMonozygotic twin BRCA1 positive BR Monozygotic twin diagnosed with ovarian cancer BRCA1 positive BR Healthy Sister BRCA1positive BR Healthy Sister BRCA1 positive BR Healthy Sister BRCA1 positive The miRNAs that may be effective in the etiology ofovarian cancer were identified after the comparison of monozygotic twins who were phenotypically discordant for ovarian cancer diagnosis ingroup 320d miR3423p miR4430 miR451a miR664b5pand miR7663p expressions were found to have downregulated After the bioinformatic analysis a total of upregulated and downregulated statistically significantmiRNAs and their target molecules are given in Table and Fig Different miRNAs level were compared between group in order to determine the effect of BRCA1 gene mutation Group was consisted after the comparison of theBRCA1 gene mutation carrier family members and individuals not carrying BRCA12 gene mutation accordingto miRNAs expression profiles After the comparisonsdownregulated and upregulated miRNAsrelated toBRCA1 gene mutation carrier were determined The expression of a total of miRNAs including miR4449miR46533p miR4865p miR5739 miR6165 andmiR 8743p associated with the BRCA1 gene mutationcarrying were upregulated and the expression of a totalof miRNAs including miR1263p miR320a miR320b miR320c miR320d miR320e miR3243p miR miR miR4428 miR4516 miR4741 miR miR564 miR6089 miR68695p miR68915pmiR71075p and miR78473p were found downregulated After the bioinformatic analysis a total of upregulated and downregulated statistically significantmiRNAs and their target molecules are shown in Table and Fig AftercomparisonDifferent miRNA levels were compared between group in order to determine the relation with ovarian cancerdevelopment and BRCA positivity Group consists ofcomparison of miRNAs of BRCA1 positive ovarian cancer patient with all other BRCA1 positive healthy individualsanddownregulated miRNAs related to mutation carriage inBRCA1 gene and epithelial ovarian cancer etiology weredetermined The expression of miRNAs includingmiR1260a miR1260b miR165p miR175p miR181b5p miR 26b5p miR4281 miR4286 miR5100miR68403p miR71145p miR7975 and miR7977were found to have upregulated and the expression of miRNAs including miR12255p miR1423p miR 26a5p miR miR29a3p miR30d5p miR3196upregulated 0cTuncer Journal of Ovarian Research Page of Table Ovarian cancer etiology related upregulated and downregulated miRNAs and target proteins in monozygotic twinsmiRNAsSequence of miRNAmiRNAStatusTarget genesFold change FCvaluesACCACUGCACUCCAGCCUGAGUpregulatedZNF138 TMEM239 BMP3UUUUCAACUCUAAUGGGAGAGAUpregulatedPTPN4 PRKAA1 PAPD7FRAT2 DEPDC1 FBXO41UUCACCACCUUCUCCACCCAGCUpregulatedCD82 PMAIP1 MTHFD1 CHECK1 AGO1 CASP10CAUAGCCCGGUCGCUGGUACAUGA UpregulatedGGCUGGUCAGAUGGGAGUGUpregulatedRACGAP1 OLA1 TEX261PTGS1 NFIC ZNF200PAGR1 IGF2BP1 CACNG8UAUUGCACUUGUCCCGGCCUGUUGAGGUAGUAGUUUGUGCUGUUUCCCUGAGACCCUUUAACCUGUGA Downregulated ERBB3 CDKN1A TP53 ERBB2 EGFR STAT3MYCSTAT3 PTEN ATM NOTCH2 CDH1 NFKB1UpregulatedDownregulated TLR4 BMP4 EIF2C1 NEUROG1 SOCS1 IGF1VEGFAmiR1273 g3pmiR1305miR1973pmiR3651miR6131miR92a3plet7i5pmiR125a5pmiR15b5pUAGCAGCACAUCAUGGUUUACAmiR223pAAGCUGCCAGUUGAAGAACUGUDownregulated BCL2 VEGFA CCND1 CCNE1 CDK1 CDK4 CDK6 E2F3MAPK1Downregulated CDKN1A WNT1 ERBB3 MYCBP HMGB1 E2F2 PTENPOTED SOD2miR3135bmiR320dmiR3423pmiR4430miR451amiR664b5pGGCUGGAGCGAGUGCAGUGGUGAAAAGCUGGGUUGAGAGGAUCUCACACAGAAAUCGCACCCGUAGGCUGGAGUGAGCGGAGAAACCGUUACCAUUACUGAGUUUGGGCUAAGGGAGAUGAUUGGGUADownregulated BIRC5 ABL2 MAPK1 MYCNDownregulated DCTN5 SYNCRIP FBXO28Downregulated GEMIN4 DNMT1 ID4 SREBF1SREBF2 BMP7Downregulated ZNF485ABL2 MAPK1 MSH5 PTENDownregulated CPNE3 RAB5A IL6R AKT1 MMP2Downregulated CD55MSN RHOBTB3 PLAG1miR7663pACUCCAGCCCCACAGCCUCAGCDownregulated COX1 MAPK1 NF2 RAD51 STK4 STK24 VEGFCFig The upregulated and downregulated miRNAs and foldchanges associated with ovarian cancer etiology in monozygotic twins 0cTuncer Journal of Ovarian Research Page of Table BRCA1 gene mutation positivity related upregulated and downregulated miRNAs and target molecules An additional tablefile shows this in more detail [see Additional file ]miRNAsSequence of miRNATarget genesmiRNAStatusFold changeFC valuesmiR4449miR46533pmiR4865pmiR5739miR6165miR8743pmiR1263pmiR320amiR320bmiR320cmiR320dmiR320emiR3243pmiR3656miR4284miR4428miR miR4741miR484miR564miR miR6869 5pmiR68915pmiR71075pmiR7847 3pCGUCCCGGGGCUGCGCGAGGCAUpregulatedZFHX3UGGAGUUAAGGGUUGCUUGGAGAUpregulatedATG2A CREBL2 MAT2A FRS2 TMED4 UBN2UCCUGUACUGAGCUGCCCCGAGGCGGAGAGAGAAUGGGGAGCCAGCAGGAGGUGAGGGGAGCUGCCCUGGCCCGAGGGACCGAUpregulatedUpregulatedUpregulatedUpregulatedOLFM4CD40ARHGAP5 IGF1R DOCK3 CADM1DLX6CD207CHIC1PPL2A PLXDC1PER1TFAP2AFADS1 AMER1LUZP1 COX6B1HDAC1 AQP3 STAT3 CDK9UCGUACCGUGAGUAAUAAUGCGDownregulatedTOM1 CRK VEGFA SOX2 TWF1 PITPNC1 IGFBP2 KRASAAAAGCUGGGUUGAGAGGGCGADownregulatedMCL1 BANP ITGB3 BMI1 NRP1 NFATC3 TRPC5AAAAGCUGGGUUGAGAGGGCAADownregulatedCDK6DCTN5SYNCRIPARF1 BCL9L ZNF600AAAAGCUGGGUUGAGAGGGUAAAAGCUGGGUUGAGAGGAAAAGCUGGGUUGAGAAGGACUGCCCCAGGUGCUGCUGGGGCGGGUGCGGGGGUGGDownregulatedDownregulatedSYNCRIPFBXO28SMARCC NPM3DCTN5 SYNCRIP FBXO28DCTN5 NPM3 ZNF275 DDX19A NCAPD2 TXNL1DownregulatedDownregulated WNT9B CREBBP DVL2 WNT2BDownregulatedMRPL12 LSP1 MNT PRDM2ZNF770 CECR1GGGCUCACAUCACCCCAUDownregulatedBCL2L11RBBP5HNRNPA1 ZNF264 TRIB3 CRTAPCAAGGAGACGGGAACAUGGAGCDownregulatedMSL1MAPRE3MYH14CASP2 CCND2 CDK14TP63GGGAGAAGGGUCGGGGCDownregulatedSTAT3M6PRGPR137CCCND2 CCNT1 CDKN1A SCOC TP53CGGGCUGUCCGGAGGGGUCGGCUDownregulatedDDX39BMAPK1 ZBTB39 HMGA1UCAGGCUCAGUCCCCUCCCGAUDownregulatedFIS1 PAGR1 ZEB1 SLC11A2SMAD2 ANAPC7 TBRG1AGGCACGGUGUCAGCAGGCDownregulatedGID4 CNBP E2F3 RCAN3 AKT2 APPL1 SLC1A2 GPR155GGAGGCCGGGGUGGGGCGGGGCGGDownregulatedNKX2 TPT1 KCTD5 BBX SGCD CDH7 CCNB1GUGAGUAGUGGCGCGCGGCGGCDownregulatedTUBB2AMAPK1NRBF2 WEE1HMGA2 MAPK1 STAG2UAAGGAGGGGGAUGAGGGGDownregulatedCHD4 CD207 DDX6 CHRDL1CCND2 TP63UCGGCCUGGGGAGGAGGAAGGGDownregulatedVAV3 CASP16 CCND1 CASP16 MAPK14CGUGGAGGACGAGGAGGAGGCDownregulatedHAVCR1 POTED DNAJC10 SOD2 M6PR CDK19miR3423p miR3665 miR3960 miR4466 miR4530miR46873p miR47875p miR4943p miR50015pmiR50065p miR5787 miR6068 miR6087 miR miR6090 miR6124 miR6125 miR638 miR65105p miR68005p miR7704 miR8063 and miR were found to have downregulated After bioinformatic analysis a total of upregulated and downregulated statistically significant miRNAs and the targetmolecules are given in Table and Fig For identifying the ovarian cancer predisposition allfamily members were compared with ovarian cancermonozygotic twins in group The upregulated ordownregulated miRNAs in association with the epithelialovarian cancer risk were identified after the comparisonThe expression of the miRNAs consisting of let7a5plet7b5p miR181a5p miR1973p miR215pmiR2233p miR23a3p miR27a3p miR36533pmiR4255p miR572 miR5745p miR6127 and miR were detected to be upregulated The expression ofthe miRNAs consisting of let7i5p miR 125a5pmiR15b5p miR1505p miR22 3p miR3283pmiR4430 miR451a miR46975p miR664b5p andmiR7663p were detected to have downregulated Atotal of upregulated and downregulated statisticallysignificant miRNAs and the target molecules are givenin Table and Fig DiscussionWomen are diagnosed with ovarian cancer at an advanced stage due to limited number of biologicalmarkers for ovarian cancer patients Although existingovarian cancer biomarkers cancer antigen125 and cancer antigen153 CA125 CA15 are sensitive in thefollowup of diagnosed gynecological cancers they have 0cTuncer Journal of Ovarian Research Page of Fig The upregulated and downregulated miRNAs and foldchanges associated with BRCA1 gene mutation positivityofearlysensitivityin the diagnosislessstagegynecological cancers and separation of malignant tumorformations from benign formations [] Therefore tounderstand the underlying mechanisms of ovarian cancer and to explore targeting drugs and to improve newtreatment protocols for ovarian malignancy revealingsignificant genetic changes is necessary The genetic andepidemiologic studies conducted on monozygotic twinsare known to provide accurate and direct informationabout the gene and environment interaction with thedisease occurrence mechanism [] The changes in genesthat result in the occurrence of tumors such as miRNAexpression level among the monozygotic twins providesinformation on the etiology of disease and may have arole as a biological indicator in identifying the early stagedisease and in the follow up of the prognosis We aimedto identify the noninvasive biological markers that maybe used in the early diagnosis of ovarian cancer throughinvestigating the miRNAs in the peripheral blood ofmonozygotic twin siblings discordant for ovarian cancerwith the miRNA molecules of the other healthy members in the family Thus that may cause less bias thanthe controls to be selected from the population Ninetynine different miRNA molecules presented in the studywere detected after the comparison of monozygotic twinsiblings who were discordant for ovarian cancer andwith the other healthy individuals Seventeen differentmiRNAs were found that could be used for detectingearly diagnosis and prognosis of ovarian cancer betweenthe monozygotic twin siblings who were discordant forovarian cancer in our study The association between out of miRNAs and ovarian carcinoma is beingreported for the first time in this study Due to the highnumber of newly detected miRNAs in our study the discussion and comparison were only made between thecandidate miRNAs Although miR1973p miR1305miR6131 miR3651 miR3135b miR4430 miR664b5p and miR7663p have not been shown to be associated with ovarian cancer in literature but limited number of studies have suggested the association with othercancersWang found the elevated level of miR1973pinthe same way as we do The upregulated miR1973p expression level was shown to promote the cellular invasion and metastasis in bladder cancer in that studyResearchers reported that LINC00312 gene was responsible for invasion and metastasis mechanisms and thisgene inhibited the cellular migration and invasion bysuppressing the miR1973p expression Similar resultswere detected in thyroid cancer in the study of Liu et al[ ] Jin reported that increased expressionlevel of miR1305 caused pluripotent stem cells to accelerate the cell cycle G1S transfer in addition to causingthe cellular differentiation with the increased miR1305expression [] The expression levels ofreducedmiRNA125a5p and let7i5p found in the scope of ourstudy have been shown to parallel with other studies inthe literature Langhe suggested thatlet7i5pmight be described as a diagnostic indicator in ovariancancer [] The miRNA125a5p expression was upregulated to inhibit the cancer proliferation and migrationin the in vitro study of Qin in human cervical carcinomas [] and miR125a5p upregulated expressionlevel was demonstrated to inhibit the cervical cancer 0cTuncer Journal of Ovarian Research Page of Table BRCA1 mutation carriage and epithelial ovarian cancer etiology related upregulated and downregulated miRNAs targetmolecules An additional table file shows this in more detail [see Additional file ]miRNAsSequence of miRNAmiRNAStatusTarget genesAUCCCACCUCUGCCACCAAUCCCACCACUGCCACCAUUAGCAGCACGUAAAUAUUGGCGUpregulatedUpregulatedUpregulatedCAAAGUGCUUACAGUGCAGGUAGUpregulatedPSAT1UNC13A RPS27 BRD7SFRP1 DKK2 SMAD4 PSAT1UNC13A RPS27CCNE1 ARL2 BCL2 HMGA1 CDK6 CCND1VEGFA RECK PRDM4TGFBR2 PTEN CDKN1A BCL2L11E2F1TP53STAT3AACAUUCAUUGCUGUCGGUGGGUUpregulatedTCL1A TIMP3 PLAG1 BCL2RNF2VSNL1 ATMFold changeFC valuesmiR1260amiR1260bmiR16 5pmiR175pmiR181b5pmiR26b5pmiR4281miR4286miR5100UUCAAGUAAUUCAGGAUAGGUUpregulatedGGGUCCCGGGGAGGGGGGACCCCACUCCUGGUACCUpregulatedUpregulatedUUCAGAUCCCAGCGGUGCCUCUUpregulatedPTGS2 EPHA2 CHORDC1 EZH2CCNE1ABCA1 GATA4NCDN CDKN1A BCL3LDLR ZNF354B NSD1 RABGAP1TAOK1 MKNK2COX10 DEK KCNN3 RAB11FIP1DYNLT1 NOTCH2SLFN12L CTC1 GXYLT2GDE1FADS1PER1 ATG9AmiR68403pGCCCAGGACUUUGUGCGGGGUGUpregulatedmiR71145pUCUGUGGAGUGGGGUGCCUGUUpregulatedM6PR HNRNPUL1 SHMT1 ZNF529ACVR2B PAICS TAF8miR7975miR7977AUCCUAGUCACGGCACCAUUCCCAGCCAACGCACCAUpregulatedUpregulatedmiR12255pGUGGGUACGGCCCAGUGGGGGGDownregulatedKBTBD8GULP1 CASZ1 RAD51HSPA1B ZNF703 TMEM185BSF3B3COX6B1 CCDC9 CDH7ORC4 ODF2L MTRNR2L7PSMG2 MTRNR2L3miR1423pmiR26a5pmiR2861miR29a3pmiR30d5pmiR3196miR 3423pmiR3665miR3960miR4466miR4530miR46873pmiR47875pmiR4943pmiR50015pmiR50065pmiR miR6068miR6087miR6088miR6090miR6124UGUAGUGUUUCCUACUUUAUGGADownregulatedARNTLTGFBR1 RAC1 ROCK2 CCNT2 TAB2 PTPN23UUCAAGUAAUCCAGGAUAGGCUDownregulatedEZH2RB1ADAM17 HMGA2CCND2 CPEB3 DNMT3BGGGGCCUGGCGGUGGGCGGUAGCACCAUCUGAAAUCGGUUAUGUAAACAUCCCCGAC
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glioma initiates from glial cells and contains several types such as astrocytoma and oligodendroglioma1 over a quarter of brain tumors are glioma which causes a large number of cancerrelated deaths every year around the world1 the current clinically therapeutic strategies are surgery combined with chemotherapy and radiotherapy2 however the prognosis of glioma patients remains not well post therapy3 hence it is urgently required to discover new molecular mechanism for glioma therapyboth long noncoding rna lncrna and microrna mirna belong to noncoding rnas which have no proteincoding ability lncrna is characterized with more than nucleotides while mirna is about nucleotides in length4 lncrna and mirna are involved in various cellular processes including cell division invasion and survival5 dysregulation of lncrna or mirna usually causes tumor initiation and progression67 for example lncrna linc00152 upregulation promotes gastric cancer growth and metastasis8 lncrna snhg6 overexpression facilitates lung cancer cell proliferation and metastasis9 mir3405p dysregulation promotes tumorigenesis of esophageal squamous cell carcinoma10 in addition mir126 cancer management and research du this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphpcorrespondence jun wu weiwen qiu email wwwwjjjj924163com weiwenqhotmailcomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars262279 0cdu dovepresssuppresses colon cancer cell survival and induces apoptosis11 besides lncrna has been identified as potential competing endogenous rna cerna for mirna to function in cancer12 the potential roles underlying lncrna and mirna still require much investigation and the relationship between lncrna and mirna also needs to be definedlinc00173 is an oncogene in lung cancer and breast cancer1314 the function of linc00173 in glioma is unclear in the current study we found that linc00173 was upregulated in glioma tissues linc00173 high expression was associated with a low survival rate linc00173 depletion suppressed proliferation migration and invasion of glioma cells linc00173 was discovered to sponge mir765 to elevate nutf2 expression taken together our findings supported that linc00173 plays essential oncogenic roles in glioma through activating mir765nutf2 pathwaymaterials and methodsclinical samplesthirtyseven glioma tissues and normal tissues were collected from lishui city peoples hospital patients received no radiotherapy or chemotherapy before surgery all tissues were stored in liquid nitrogen association between linc00173 expression and clinical characteristics in glioma tissues was analyzed in table written informed consent was obtained from every patient this study was approved by the ethics committee of lishui city peoples hospital no and the table association between linc00173 expression and clinical characteristics in glioma tissuesfeaturesage yearsgendermalefemalegradeiiiiiiivtumor size cmlow n19high n18pvalueexperiments were conducted in accordance with the declaration of helsinkicell culture and treatmentthe normal human astrocyte nha and glioma cell lines were purchased from institute of biochemistry and cell biology of the chinese academy of sciences shanghai china cells were cultured using pmi1640 medium invitrogen carlsbad ca usa supplemented with fetal bovine serum fbs invitrogen shrnas against linc00160 mir6293p mimics mir6293p inhibitors and negative controls were obtained from genepharma and transfected into glioma cells using lipofectamine invitrogen according to the manufacturers instructions efficiency was validated using qrtpcr after hqrtpcrtotal rna was extracted from tissues and cell lines using trizol invitrogen carlsbad ca primescript rt reagent kit rr047a takara holdings inc tokyo japan was used to generate cdna from rna template qpcr was completed through sybr premix ex taq¢ ii takara japan gapdh was the normalized control relative expression was calculated through the δδct methodluciferase reporter assaythe fragment of linc00173 or nutf2 containing indicated mir765 binding site was constructed into pmir report vector for luciferase reporter assay glioma cells were transfected with report vector and mir765 mimics after h the luciferase reporter activity was measured through the dualluciferase reporter assay system promega madison wiwestern blot assaycells were lyzed using radioimmunoprecipitation buffer beyotime shanghai china protein concentration was determined by a bca protein assay kit thermo fisher scientific ma then proteins were separated using sdspage and transferred onto pvdf membranes after blockage using bsa for h the membrane was incubated the primary antibodies at °c overnight after washed times using pbst the membranes were incubated with horseradish peroxidaselabeled second antibody followed by detection the enhanced chemiluminescence reagent emd millipore usathrough submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du cck8 and colony forming assaysproliferation was measured using cck8 and colony formation assay cck8 assay was performed using the cck reagent dojindo kumamoto japan according to the manufacturers instructions and absorbance was determined at nm using a microplate reader biotek winooski vt for colony formation assay cells were seeded into 6well plates and cultured for days then the cells were fixed with methanol and stained with crystal violet for minutesedu assaycells were plated into 96well plates and incubated with edu μl at °c for h followed by detection using facstranswell migration and invasion assaystranswell plates corning ny were used to measure migration and invasion according to the manufacturers instructions in brief cells were suspended into μl serumfree medium and seeded into the upper chamber while the lower chamber was filled with µl of complete medium after incubation for cells in the lower chamber were fixed with methanol and stained with crystal violet for minutes migrated and invaded cells were counted through a light microscopestatistical analysisgraphpad prism graphpad ca usa was used to analyze results data were presented as means±standard deviation sd significant differences were analyzed using students ttest or oneway anova survival rate was analyzed by the kaplanmeier method and log rank test p005 was considered to be significantresultslinc00173 expression is elevated in gliomathe expression of linc00173 was firstly analyzed through qrtpcr we found that linc00173 level was elevated in glioma tissues compared with normal tissues figure 1a besides we found that linc00173 was also upregulated in glioma cell lines compared to nha cells figure 1b then according to the median value of linc00173 glioma tissues were classified into two groups after analysis we found that linc00173 high expression correlated with poor prognosis figure 1ctransfection of linc00173 enhanced glioma cell proliferation migration and invasionto explore the function of linc00173 u87 and u251 cells were chosen after shlinc00173 linc00173 expression was significantly downregulated figure 2a through cck8 assay we observed that linc00173 knockdown suppressed the proliferation capacity of glioma cells figure 2b and c which was validated by edu and colony formation assays figure 2d and e afterwards transwell assay was performed results indicated that linc00173 loss inhibited migration and invasion of glioma cells figure 2f and g thus linc00173 exerted oncogenic roles by affecting proliferation migration and invasionlinc00173 worked as the sponge for mir765linc00173 has been found to serve as cerna for several mirnas such as mir490 and mir2181314 to determine the mechanism of linc00173 in glioma we also figure linc00173 expression is elevated in glioma a the level of linc00173 in glioma tissues was measured b the expression of linc00173 in glioma cell lines and nhas c association between overall survival and linc00173 expression in glioma patients p005cancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 enhanced glioma cell proliferation migration and invasion a qrtpcr analysis of linc00173 expression in u87 and u251 cells be proliferation ability was measured using cck8 edu and colony formation assays f and g migration and invasion capacity was evaluated after linc00173 knockdown in glioma cells p005suppressed the supporting their direct performed bioinformatics analysis using mirdb we identified that mir765 was the most potential candidate because it scored the highest to validate it we constructed luciferase reporters figure 3a followed by luciferase reporter assay results showed that mir765 activity of linc00173wt only figure 3b interaction pulldown assay further demonstrated their interaction figure 3c qrtpcr found that linc00173 overexpression suppressed the level of mir765 figure 3d next bioinformatics analysis using mirdb and targetsan implied that nutf2 is the most potential target of mir765 the corresponding luciferase reporters were further constructed figure 3e luciferase reporter assay also demonstrated the interaction between nutf2 and mir765 figure 3f besides nutf2 expression was suppressed by mir765 mimics figure 3g moreover nutf2 level was decreased after linc00173 knockdown while mir765 inhibitors reversed it figure 3h finally we found that mir765 level was negatively correlated with linc00173 or nutf2 in glioma tissues figure 3i and jlinc00173 promoted glioma progression through mir765nutf2 pathwaywe noticed that nutf2 expression was upregulated in glioma tissues figure 4a and b suggesting an oncogenic role to investigate whether linc00173 regulates glioma progression through mir765nutf2 we restored the expression of nutf2 in shlinc00173 transfected cells cck8 and transwell assays demonstrated that nutf2 restoration successfully rescued the capacities of proliferation migration and invasion in glioma cells transfected with shlinc00173 figure 4cf therefore linc00173 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du figure linc00173 worked as the sponge for mir765 a bioinformatics analysis indicated the binding sites between linc00173 and mir765 b u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter linc00173wt or linc00173mut then relative luciferase activity was determined c rna pulldown assay using biotinlabeled mirnas d relative expression of mir765 after linc00173 knockdown e bioinformatics analysis indicated the binding sites between mir765 and nutf2 f u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter nutf2wt or nutf2mut then relative luciferase activity was determined g qrtpcr analysis for nutf2 expression h western blotting analysis for nutf2 protein level i and j correlation analyses of linc00173 mir765 and nutf2 in glioma tissues using pearsons correlation coefficient p005contributes to glioma progression through mir765nutf2 pathwaydiscussionas the most malignant brain tumor glioma leads to a huge number of deaths patients with glioma display a poor prognosis therefore it is of great significance to reveal the mechanism underlying glioma progression in this study we found that linc00173 was upregulated in glioma tissues and cells linc00173 overexpression predicted a poor prognosis moreover linc00173 knockdown the proliferation migration and invasion of glioma cells linc00173 was also found to inhibit mir765 and promote nutf2 expression summarily our research discovered that linc00173 is an important oncogenic lncrna in gliomasuppressed the potential roles of lncrna in glioma have been researched for a long time many lncrnas have been identified to participate in glioma development for example lncrna nck1as1 enhances growth and metastasis of glioma through targeting mir13823p to activate β catenin signaling2 lncrna ccat2 contributes to glioma progression by activating vegfa pathway15 lncrna linc00467 upregulation promotes glioma development through repressing p53 level16 previous study showed that linc00173 downregulation promotes nonsmall cell lung cancer cell growth and survival17 however another study showed that linc00173 enhances chemoresistance and facilitates tumor progression in small cell lung cancer13 besides linc00173 contributes to breast cancer development14 yet how linc00173 works in glioma remains undermined in our study we found that linc00173 was upregulated in glioma tissues linc00173 knockdown inhibited the proliferation migration and invasion of glioma cells therefore our data discovered that linc00173 is a new oncogene in glioma for the first timecancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 promoted glioma progression through mir765nutf2 pathway a and b nutf2 expression in glioma tissues and normal tissues according to tcga data using gepia tool and qrtpcr c and d proliferation was measured by cck8 assay e and f migration and invasion potential was determined by transwell assay p005lncrna has been found to serve as mirna sponge in tumor cells for instance lncrna ttnas1 sponges to promote breast cancer metastasis18 mir1405p lncrna cdkn2bas1 sponges mir3245p to regulate cellcycle progression in laryngeal squamous cell cancer19 previous studies also revealed linc00173 was a sponge for some mirnas such as mir4903p and mir2181314 in our study we did not observe linc00173 sponges above mirnas however through bioinformatics we identified linc00173 targeted mir765 in glioma we demonstrated their direct interaction and found that linc00173 overexpression inhibited mir765 expression mir765 has important roles in cancers mir765 was found to suppress tongue squamous cell carcinoma development20 mir765 also promotes myeloma and osteosarcoma progression2122 besides mir765 plays oncogenic or anticancer roles in gastric cancer and breast cancer2324 its role in glioma remains unclear our results suggested that mir765 was a tumor suppressor in gliomalncrnamirnamrna regulatory axis is widely observed in cancer for example linc00703mir181a klf6 axis suppresses the development of gastric cancer25 linc00312mir9cdh1 axis was found to promote breast cancer progression26 through bioinformatics we found that mir765 targeted nutf2 in glioma moreover we showed that nutf2 expression was regulated by linc00173mir axis the function of nutf2 in cancer is nearly unknown in our work we found that nutf2 expression was upregulated in glioma tissues compared to normal tissues moreover we found that nutf2 overexpression promoted the proliferation migration and invasion of glioma cells indicating nutf2 was an oncogene in gliomain conclusion our study showed that linc00173 acted as a sponge for mir765 to promote nutf2 expression and linc00173mir765nutf2 axis plays a critical function in promoting glioma progressionfunding this work was supported by zhejiang province analytical testing and experimental animal program lgd19h and zhejiang province welfare technology applied research project 2017c37111 disclosureall authors declare no conflicts of interest in this workreferences ostrom qt cioffi g gittleman h cbtrus statistical report primary brain and other central nervous system tumors diagnosed in the united states in neuro oncol 201921suppl 5v1 v100 101093neuoncnoz150the of glioma huang l li x ye h et al long noncoding rna nck1as1 promotes sponging microrna13823p and activating the trim24wntbetacatenin axis j exp clin cancer res 101186s13046 tumorigenesis through chen w lei c liu p et al progress and prospects of recurrent glioma a recent scientometric analysis of the web of science in world neurosurg 2020134e387e399 101016jwneu20 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du sun b meng m wei j wang s long noncoding rna pvt1 contributes to vascular endothelial cell proliferation via inhibition of mir190a5p in diagnostic biomarker evaluation of chronic heart failure exp ther med 103892etm20208599 feng s yao j chen y functional role of reprogrammingrelated long noncoding rna lincrnaror in glioma j mol neurosci 101007s120310140488z zhang d zhou h liu j mao j long noncoding rna asb16as1 promotes proliferation migration and invasion in glioma cells biomed res int sun l zhao m wang y neuroprotective effects of mir27a against traumatic brain injury via suppressing foxo3amediated neuronal autophagy biochem biophys res commun 101016jbbrc201612001 shi y sun h downregulation of lncrna linc00152 suppresses gastric cancer cell migration and invasion through inhibition of the erkmapk signaling pathway onco targets ther 102147otts217452 li k jiang y xiang x et al long noncoding rna snhg6 promotes the growth and invasion of nonsmall cell lung cancer by downregulating mir1013p thorac cancer wang x gu m ju y zhou j pik3c3 acts as a tumor suppressor in esophageal squamous cell carcinoma and was regulated by mir340 5p med sci monit 202026e920642 1012659msm923909 wei l chen z cheng n microrna126 inhibit viability of colorectal cancer cell by repressing mtor induced apoptosis and autophagy onco targets ther 102147 otts238348 chen y shen z zhi y long noncoding rna ror promotes radioresistance in hepatocellular carcinoma cells by acting as a cerna for microrna145 to regulate rad18 expression arch biochem biophys 101016jabb201803018 zeng f wang q wang s et al linc00173 promotes chemoresistance and progression of small cell lung cancer by sponging mir218 regulate etk expression oncogene to 101038s4138801909842 fan h yuan j li x et al lncrna linc00173 enhances triplenegative breast cancer progression by suppressing mir490 3p expression biomed pharmacother 1010 16jbiopha2020109987 sun sl shu yg tao my lncrna ccat2 promotes angiogenesis in glioma through activation of vegfa signalling by sponging mir424 mol cell biochem 101007 s1101002003712y zhang y jiang x wu z et al long noncoding rna linc00467 promotes glioma progression through inhibiting p53 expression via binding to dnmt1 j cancer 107150 jca41942 yang q tang y tang c diminished linc00173 expression induced mir1825p accumulation promotes cell proliferation migration and apoptosis inhibition via agernfkappab pathway lung cancer am j transl res in nonsmallcell xue j zhang z li x ren q wang q long noncoding rna ttnas1 promotes breast cancer cell migration and invasion via sponging mir1405p oncol lett 1038 92ol201911222 liu f xiao y ma l wang j regulating of cell cycle progression by the lncrna cdkn2bas1mir3245prock1 axis in laryngeal squamous cell cancer int j biol markers 1011771724600819898489 ding j yang c yang s linc00511 interacts with mir765 and modulates tongue squamous cell carcinoma progression by targeting lamc2 j oral pathol med 101111 jop12677 long s long s he h chen g microrna765 is preregulated in multiple myeloma and serves an oncogenic role by directly targeting sox6 exp ther med 103892 etm20197473 lv db zhang jy gao k microrna765 targets mtus1 to promote the progression of osteosarcoma via mediating erkemt pathway eur rev med pharmacol sci 1026355eurrev_201906_18040 jiao y yuan c wu h li x yu j oncogenic microrna765 promotes the growth and metastasis of breast carcinoma by directly targeting ing4 j cell biochem yuan l ma t liu w et al linc00994 promoted invasion and proliferation of gastric cancer cell via regulating mir7653p am j transl res yang h peng m li y zhu r li x qian z linc00703 acts as a tumor suppressor via regulating mir181aklf6 axis in gastric cancer j gastric cancer 105230jgc2019 19e43 chen y qiu f huang l et al long noncoding rna linc00312 regulates breast cancer progression through the mir9cdh1 axis mol med rep 103892mmr201910895cancer management and research publish your work in this journal cancer management and research is an international peerreviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient the manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors dovepress submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalcancer management and research submit your 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the diagnostic platform utilizing the detection of biomarkers in various body ï¬uids called liquidbiopsy can revolutionize precision medicine precision medicine is aimed at attaining betterpersonalized care by the development of the latest diagnostic and prognostic methods thatconsider individual variability kaur liquid biopsy is being utilized for noninvasiveabbreviations 5hmc 5hydroxy methyl cytosine ccfdnas circulating cellfree deoxyribonucleic acids ccffetalnascirculating cellfree fetal nucleic acids ccfmirnas circulating cellfree mirnas ccfnas circulating cellfree nucleic acidsccfrnas circulating cellfree ribonucleic acids mtdna mitochondrial dnaedited byrui henriqueportuguese oncology instituteportugalreviewed bynaoko hattorinational cancer center researchinstitute japanigor kovalchukuniversity of lethbridge canadacorrespondencejyotdeep kaurjyotdeep2001yahoocoinspecialty sectionthis was submitted toepigenomics and epigeneticsa section of the frontiers in geneticsreceived february accepted july published august citationrahat b ali t sapehia dmahajan a and kaur j circulating cellfree nucleic acids asepigenetic biomarkers in precisionmedicine front genet 103389fgene202000844frontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersprognostic and predictive purposes eï¬cient and reliable markerswithin the body ï¬uids can help in personalized treatmentdecisions for monitoring disease and survival ccfnas haveemerged as such markers for screening diagnosis prognosismanagement and treatment of various cancers autoimmuneneurological and mitochondrial diseases prenatal diagnosisdiagnosis of pregnancyrelated complications pos diabetes ammation rheumatoid arthritis stroke and traumaswarup and rajeswari an increased amount of ccfnasis observed in these disorders making liquid biopsies moresensitive rapid accurate and preferable alternatives for variousinvasive diagnostic methods pos ccfnas present in blood circulation include cellfree genomicdnas ccfdnas and cellfree mtdna kohler thierry and cellfree rnas ccfrnas includingproteincoding messenger rna mrna regulatory noncodingrnas like micrornas mirnaslong noncoding rnaslncrnas circular rnas and rnas involved in translationlike transfer rnas trnas and ribosomal rnas rrnaspos the ccfnas dnas and rnas are generally released into theblood circulation either by apoptosis necrosis or active secretionin healthy persons the origin of ccfnas is mainly attributed tolymphoid and myeloid tissues snyder while in thecase of various clinical conditions the associated or the aï¬ectedtissues would release the extra amount of ccfnas into bloodswarup and rajeswari devonshire in a patternspeciï¬c to the pathophysiological condition hunter noferesti various genetic as well as epigenetic biomarkers havebeen explored for ccfnabased liquid biopsy as geneticbiomarkers are less consistent and provide more variabilityacross studies epigenetic markers which are more generalizedbetween samples present as a promising alternative for earlydiagnosis and monitoring of the diseases these epigeneticmarks are tissue speciï¬c and reï¬ect the pattern of diseaseprogression zeng furthermore epigeneticbiomarkers are dynamic with most techniques required foranalysis ofthese biomarkers that are already available inclinical laboratoriescare thein future precise patientthe use of epigenetic marks has revolutionized the ï¬eldof noninvasive molecular diagnosisreplacing traditionalscreening and treatment methods these assays have greatpotentialepigeneticmarks for ccfnas reï¬ect the pattern speciï¬c for the tissuecontributing to these ccfnas therefore the use of epigeneticmarkers can help in the diagnosis of various diseases evenbefore the onset of actual symptoms and hence help inbetter management ofthe disease precision medicine hasimproved health care by theidentiï¬cation of diï¬erentstagessubsets of diseases precise diagnosis and treatmentfurthermore the development of advanced analytical softwaretechniques like machine learning and artiï¬cialintelligencecan enhance precision medicine ahlquist beltrangarcia these are used in combination withnextgeneration sequencing to identify novel ccfnabasedepigenetic markersepigenetic biomarkers in ccfnasreliable markers are required to guide personalized treatmentdecisions for monitoring disease progression and survivalthe presence of epigenetic marks on ccfnas speciï¬c toa particular clinical condition is widely being explored toadvance personalized medicine a perfect epigenetic markerfor precision medicine should be able to detect the diseasewith high sensitivity predict the risk of disease developmentand its progression and monitor the therapeutic responseofbeltrangarcia ccfdnas areassociated with various epigenetic marks schwarzenbach like dna methylation hydroxymethylcytosine 5hmcand posttranslational modiï¬cations of histones in additionnucleosome positioning and occupancy on ccfdnas haveexhibited high sensitivity and speciï¬city in liquid biopsybasedmethods for disease detection and classiï¬cationthe patientthe5methylcytosine5mc modiï¬cationat cpgdinucleotides is the most abundant form of dna methylationit plays an important role in the regulation of gene expressionand is widely used as an epigenetic biomarker for ccfdnabasedassays dna methylation has replaced many genetic mutationor proteinbased markers these 5mc biomarkers are alsovaluable in identifying tissuespeciï¬c methylation to estimatetumor burden and tissue of origin in ccfdnas in additionto 5mc 5hydroxymethylcytosine 5hmc is also used as anepigenetic mark on ccfdnas zeng 5hmc is createdby the oxidation of 5mc by translocation tet proteinsalthough 5hmc is far less abundant compared to 5mc it ismore distinctly distributed among diï¬erent transcriptionallyactive regions which emphasizes its potential as a diagnosticmarker genomewide analysis of 5hmc pattern can providemore information about the potential of this epigenetic markerfor ccfdnas zeng nucleosome positioning has emerged as a recent biomarkerto distinguish the tissue of origin of ccfdna based onderived nucleosome maps snyder performed deepsequencing on ccfdnas and observed a distinct pattern ofnucleosome positioning between healthy persons and cancerpatients correlating with the tissues of origin snyder this emphasizes the use of nucleosome maps whichconsist of occupancy of transcription factor and nucleosomeas the epigenetic marks to distinguish normal versus cancerccfdnas hence nucleosome positioning can also be used toidentify various cancers that generally require invasive biopsiesfor deï¬nitive diagnosis moreover genomewide nucleosomepositioning of ccfdnas is utilized to infer pathological statesof multiple disease types a comprehensive public databasecalled cellfree epigenome atlas cfea provides the epigenomeproï¬le of ccfdnas from various human diseases and canhelp in a better understanding of collected data yu ccfdna are generally associated with nucleosomesand histone proteins histone proteins are posttranslationallymodiï¬ed at amino acid residues located on their n andcterminal tails these modiï¬cations act as epigenetic marksthat can speciï¬cally distinguish diseaserelated ccfdnas in bloodsamples various types of histone modiï¬cations are associatedfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersarewith the development and pathogenesis of human diseaseszhao and shilatifard in addition to dna markers rna markers like mrnasmirnas lncrnas and circrnas are also getting attention in thefocus of clinical research pos most of the currently available diagnostic tests based onccfnas use either dna methylation markers or the diï¬erentialexpression of mirnas these biomarkersrelativelyeasily detected and estimated using accessible techniqueslike methylight methylspeciï¬c pcr methylationsensitivehighresolution melting and pyrosequencing garcagimnez dna methylation speciï¬c to fetal and tumor dnahas been reported in pregnant women and cancer patientsrespectively wong poon the pattern ofthe methylation in these ccfdnas has been traced back to theirtissue of origin lun sun diï¬erentiallymethylated markers have been reported in ccfdnas like inspromoter in diabetes and reg1a and cux2 genes in pancreaticcancer lehmannwerman promoter methylationof serpinb5 rassf1a and stat5a act as epigenetic fetalmarkers in maternal blood chim chan rahat 2016atumor dna depending on the copy number mostly targetedmethylation sequencing is carried out in such cases which hasa greater potential for the detection of lower levels of ccfna inpatients with earlystage diseasechromatinbased chipseq experiments are revolutionizingour understanding of the complexes associated with chromatindynamics ongoing advances such as nanochipseq allow chipseq to be analyzed from far fewer cells necessary for embryologyand development studies nakato and shirahige theemergence of chipexo that digests the ends of dna fragmentsnot bound to protein is quite promising furey howeverthe application of these techniques to identify biomarkers islimited due to the expertise and cost associatedcriticalchipseq also providesinformation on otherchromatin modiï¬ers such as histone marks and the enzymesthat modify these marks in diseases such as cancer chipseq has identiï¬ed the role of aberrant h3k79 methylationby the methyltransferase dot1l in mixed lineage leukemiamllrearranged leukemias bernt in additionto chipseq diï¬erentlike chippcr elisabased assays or mass spectrometry are used to detect andquantify histone modiï¬cations on ccfnas in serum or plasmaadli and bernstein techniquesdiagnostic approach forepigenetic modifications in ccfnathe various diagnostic approaches to study the epigeneticmodiï¬cations in the nucleic acids include methylated cpgisland recovery assay mira and methylcap that rely onmethylcpgbinding domains mbd to capture methylateddna after dna fractionation either by restriction digestion orsonication mitchell these methods can also becombined with microarray or ngs technologies methylcapseq to identify biomarkers for cancer diagnosis and dnamethylation maps of cancer genomes simmer reduced representation bisulï¬te sequencing rrbs meissner is an eï¬cient method for absolute quantiï¬cation ofthe methylation status of more than one million cpg sites atsingle basepair resolution covering regions of moderate to highcpg density lee new techniques such as wholegenome bisulï¬te sequencing wgbs allows for an unbiasedassessment of dna methylation at singlebase resolution withfull coverage of more than million cpg sites in the humangenome and by using this technique in the clinical settingsrelevant biomarkers were identiï¬ed in colorectal and breastcancers and certain types of leukemia berman some of the techniques are used in clinical settingslikeparallel shotgun sequencing and targeted sequencing norwitzand levy for noninvasive prenatal testing wgs for fetalgene detection lo and cancer personalized proï¬lingby deep sequencing cappseq to quantify circulating tumordna newman despite the advancement of the techniques to study epigeneticmodiï¬cations the use of epigenetic biomarkers present onccfnas is limited due to their lower levels in the blood circulationin the case of cancer wgs is applied to only of cellfreeccfnas as epigenetic biomarkersin various diseasesthe detection and quantiï¬cation of ccfnas viz rna dnafetal dna fetal rna mtdna and mitochondrial rna andmirna levels in body ï¬uids are of clinical signiï¬cance theseccfnas have the potential to act as biomarkers for diagnosisas well as prognosis of various diseases fleischhacker andschmidt breitbach such as diï¬erent cancersobstetric autoimmune neurological and mitochondrial diseasesas well as prenatal diagnosis etc kandel shaw although the most studied area of epigenetics is dnamethylation yet in the clinical setting there are only a fewmethylation markers various blood or tissuebased cohortwellpowered studies have recently shown that changes in thedna methylation are not only observed frequently in cancersbut also in other broad range of complex diseases includingneurodegenerative metabolic autoimmune and ammatorydiseases although at a lower frequency tost dna methylation analysis of ccfdna might provide avaluable option in some cases when the bloodbrain barrieris temporarily disrupted it was recently demonstrated by thedetection of unmethylated fragments of mbp3 and wm1 speciï¬cfor oligodendrocytes in about of patients with relapsingmultiple sclerosis zachariah cfrnas are also presentin the patients serumplasma in addition to ccfdnas higherlevels of circulatory rnases were observed in cancers and variousdiseases like cerebral attack preeclampsia etc and surprisinglyrna found in the circulation was found to be stable umu changes in the expression of intracellular mirnahave been causally linked with many diseases that include canceresquelakerscher and slack cardiovascular diseasesfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersnavickas neurodegenerative diseases gupta etc such changes in expression of mirna are eithersimilar or distinct in the serum of a particular set of patientsthus enabling mirna detection in serum as biomarkers ofhuman diseases backes therefore ccfnas playa prominent role in the pathogenesis and diagnosis of variousdiseases further research is required in this ï¬eld to ensure thewidespread application of these markers in clinical settingsccfnas in cancerevery year about million new cases of cancer are reportedexcluding skin cancer other than melanoma that cause about million deaths accounting for of deaths in a yearferlay an estimated number of more than million new cancer cases are likely to be diagnosed and cancer deaths are expected in the united states in whichdeciphers almost deaths per day siegel thesix major hallmarks of cancer hanahan and weinberg are uncontrolled cell growth and division programmed cell deathavoidance invasion metastasis and angiogenesis the diagnosisof cancer usually occurs following the appearance of signs orsymptoms or through screening and investigations like xraysblood tests endoscopy ct scans etc biopsy tissue examinationindicates the type of proliferating cells genetic abnormalitiesand histological grade and other characteristics thereforeadvanced measures such as estimating prognosis risk assessmentfor early diagnosis biomarkers and observing the response totherapy can lead to successful treatment positive outcomes andimprovement of the quality of life for patients the tissue biopsymatched ccfdna is considered as surrogate marker due to itsrelease from the tumor sites de mattosarruda it is proven to be a noninvasive rapid and sensitive markerfor diagnosis prognosis and therapy response monitoring indiï¬erent cancers volik in addition the integrityof ccfdna extent of ccfdna fragmentation may be utilizedas a promising biomarker for diagnosis and prognosis of cancermadhavan ccfnas as diagnostic and prognosticbiomarkers for cancerserum or plasma ccfna serves as a liquid biopsy which is usefulfor various applications in diagnostics and avoids the necessityfor biopsy of tumor tissue the levels of ccfna in blood andlymphatic circulation are aï¬ected by degradation clearance andvarious other physiological events liver and kidney clear nucleicacids from the blood and they have a halflife of diï¬erent timeintervals in the circulation that varies from min to severalhours fleischhacker and schmidt mirnas appear to beextremely stable but their rate of clearance from the blood is notwell studied in cancer patients thus owing to the uniqueness ofthis research areaccfdnas in cancerccfdnas consists of both genomic dna gdna as well asmtdna there is a production of longer uneven fragments ofdna by necrosis in cancer patients and shorter dna fragmentsfrom apoptosis hence increased levels of longer dna fragmentsin the bloodstream have been targeted as a potential marker forthe presence of malignant tumor dna arkoboham tumor cells are the origin of ccfdna in the blood of cancerpatients stroun aberrations speciï¬c to tumors likeoncogene and tumor suppressor gene mutations wang methylation of dna fujiwara and instabilityof microsatellite dna shaw were recognized inccfdna tumorigenesis and its progression are monitored bythe change in various epigenetic modiï¬cations patients withdiï¬erent types of malignancies have methylated dna in theirserum or plasma one of the most important methods foranalyzing malignancy is by detecting the presence of methylatedccfdna in cancer patientsfor early diagnosis of colorectal cancer crc analysisof promoter hypermethylation in blood and fecal dna hasthe potential to be used as a noninvasive test and eï¬ortsare made for clinical application of these molecular markersvarious studies have observed mgmt rassf2a wif1 ngfrand sept9 as aberrantly methylated genes used as diagnosticbiomarkers in patients with crc lee powrozek several potential methylation biomarkers have beenfound that diï¬erentiate plasma from breast cancer patients andthat from control subjects hoque remarkablytwo independent studies recognized cst6 as being methylateddiï¬erentially between breast cancer and control plasma samplesradpour chimonidou for lungcancer an early focus was to search methylated cdkn2a as aplasma diagnostic biomarker studies observed hypermethylationof cdkn2a in the plasma of patients with lung cancer ascompared to cancerfree controls zhang shox2was identiï¬ed as a potential biomarker in a retrospective studydone by researchers from the theracode a diagnostic ï¬rmkneip a recent study by a group as part ofthe australian pancreatic cancer genome initiative apgihas observed elevated levels of aberrant methylation in theimportant cell signaling pathways thus suggesting its possibilityas a disease biomarker they worked on a group of sixcandidate genes nptx2 sarp2 uchl1 ppenk cdkn2aand rassf1a and observed diï¬erential methylation in thepromoters of all the genes in pancreatic cancer and healthycontrols except in cdkn2a promoter which was methylateddiï¬erentially between pancreatic cancer patients and thosehaving chronic pancreatitis park epigenetic eventsin the progression of cancer include the promoter regionthe genes piclass gstp1 and apchypermethylation ofwhich are the most common somatic genome abnormalities incolorectal and prostate cancer ellinger rassf1ararb sept9 esr1 and cdkn2a are the important methylatedgenes that have shown utility in prognosis using ccfdnaassays in many patients methylation of histones is an activeprocess with vital roles in diï¬erentiation and developmenttumorigenesis also occurs due to aberrant levels of histonemethylation the promoters associated with h3k4 are primarilytrimethylated by set1a and set1b set1a plays a vitalrole in oncogenic function in breast cancer metastasis lungfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerscancer and colorectal cancer zhao and shilatifard table presents the frequently hypermethylated genes invarious cancer typesccfmirnas in cancerin various cancers mirna expression dysregulation has beenobserved that suggests its role in many processes necessaryfor the progression of cancer like proliferation cell deathmetastasis and resistance to treatmentiorio and croce during the development of the liver mirna expressionchanges dynamically mir500 is one such oncofetal mirnathat is important for the diagnosis of hepatocellular carcinomayamamoto lately in nonsmall cell lung cancernsclc mir1246 and mir1290 were recognized as tumorinitiating and cellspeciï¬c mirnas zhang mir was found to be a signiï¬cant prognostic factor for osccpatients based on cox regression analysis in addition mir could serve as a valuable biomarker in oscc patientsto predict the clinical response to chemoradiotherapy lin a study by alhasan showed a serumsignature of 5mirnas mir135a mir106a mir200c mir and mir433 predicted a very highrisk prostate canceralhasan expression levels of mir21 mir23bmir200c and mir200b were upregulated in metastatic breastcancer when compared to early breast cancer patients thereforesupporting the notion that ccfmirnas presents a tool with thecrucial diagnostic and prognostic implication in breast cancerpapadaki furthermore a study discovered thatincreased mir122 expression was signiï¬cantly associated witha reduction in the overall survival as well as progressionfreesurvival in breast cancer patients saleh elevationin the levels of serum mir29 mir122 mir155 and mir was observed in cholangiocarcinoma although mirnaslevels before surgery were inappropriate as survival prognosticmarker however postsurgery decrease in the serum mir122levels was signiï¬cantly linked with better patient prognosisloosen ccfnas in treatment and cancerprogressionccfdna analysis is a noninvasive process that allows day to daypatient followup and monitoring of response toward treatmentges both genetic and epigenetic changes areexhibited by ccfdna stroun the study of thesechanges might provide valuable information to mold the choiceof treatment by clinicians given the limitations of the noveltargeted therapiesabnormal hypermethylation at cpg islands occurs rarely innonmalignant and normally diï¬erentiated cells so the releaseof dna from tumor cells can be found with a prominentextent of sensitivity even when the excess of dna is releasedfrom normal cells and this characterizes its potential clinicalapplication wong in this context promoter regionhypermethylation of ink4a occurs very early in the progressionof hepatocellular carcinoma hcc and henceit serves asa valuable biomarker for noninvasive diagnosis as well asprediction of response to therapy huang isatherapyimmunotherapyidentiï¬cation ofrapidly developingsigniï¬cant mirnasinmany cancers because of various advantages over standardchemotherapythatprovides a foresight of response in cancer immunotherapy wouldenable better patient selection and enhancement of therapeuticeï¬cacy and provide a novel target antonia chen mirna21 is a cellfree oncogenic mirna whichhas been known as a potential regulator of stat3 and thusit could be detected in various tumors ji thuscirculating mirna21 can act as a biomarker for response incancer immunotherapy wu in the mycnampliï¬ed neuroblastoma progression mycnis detected in circulating dna this phenomenon was found tobe associated strongly with the quick progression of tumors andpoor outcomes combaret loss of heterozygosityloh and abnormal methylation at the promoter region ofmycn were detected using ccfdna which showed elevatedlevels in patients of highgrade glioma detection of promoterregion hypermethylation of myod1 in serum may serve asa potential prognostic marker for discriminating patients ofcervical cancer at high risk for lymph node metastasis or relapsewidschwendter moreover the investigation of circulating mirnas presentsgreat potentialin revealing new insights into their role intherapy and diagnosis mirna serum signatures mir345 5pmir330 3p and mir9 3p were found to be signiï¬cantlyupregulated in patients of prostate cancer pca when comparedto healthy individuals the role of mir3455p to act as anoncomir through cdkn1a targeting makes it a potential targetfor pca therapeutically tinay ccfdnasin glioma wereassociated with diï¬erentialmethylation levels of mgmt cyclindependent kinase inhibitor2a multiple tumor suppressor p16ink4a p73 and retinoicacid receptor beta rarb balana weaver wakabayashi all these studies propose acrucial role of epigenetic marks in ccfnas in cancertargetedtherapy as well as pathogenesisccfnas in cancer precision medicineprecision oncology is an approach that includes the molecularproï¬ling of tumors to identify eï¬ective therapeutic strategiesa clinical research program initiated by the englander institutefor precision medicine eipm in uses wholeexomesequencing of metastatic and primary tumors to identifyindividualized therapeutic options and to help guide clinicaldecision making by prospective followup of patients rennert oncology is the obvious choice for heightening theimpact of precision medicine several targeted therapies havebeen developed that have shown profound beneï¬ts recentlynovel immunological approaches produced insightful responsessnyder in addition the identiï¬cation of epigenetic biomarkers leadsto more precise disease prognosis especially in therapeutic areasthat are linked with a high degree of variability concerningsurvival van neste research carried out in severalcancers like glioblastoma reveals that levels of 5hmc are criticalin the regulation of genes having a crucial role in disease andfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerstable frequently hypermethylated genes in various cancer typesgenecancer typereferencesitih5 dkk3 brca1 erbeta apc gstp1 esrbrassf1ap16arf bax bcl2 cdh1 dapk ednrb eomes faddpcdh17 pou4f2sept9 hltf nell1 cea tac1vhlrbtmeff2 prdm13ost2 mgmtapc gstp1st6galnac3 znf660brca1 rassf1a rassf2ahtertp16ink4a timp3 thbs1breast cancerprostate canceresophageal liver and pancreasbladder cancerkloten cheuk vu liu house abern wang colorectal cancerkidney tumorsretinoblastomalung cancerrenal cell carcinomaprostateovarian cancerleptomeningeal carcinomatosis in csfgliomatham semaan ma ohtanifujita palmisano lee su hauser haldrup giannopoulou lonning bougel liu show that global reduction in 5hmc over the genome leads topoor clinical outcomes in these patients johnson epigenetic changes introduced common genetic mutations inan in vitro model of lung cancer vaz epigeneticbased diagnostics can detect early disease signals and thuscan provide possibilities for clinicalintervention before theprogression of symptomsthe detection of ccfnas could be exploited by targetedtherapies approved lately and eventually beneï¬t the patientsscrutinizing cancers by analyzing ccfna dynamics in blood orserum is an innovative and emerging research area as far as theexisting research advancement and the growth of the medicalindustry are concerned we consider that ccfna assays may beemployed for realtime personalized treatments in the future forcancer patients based on their ccfnas or ccfdna methylationlevels for diagnosis and prognosis nevertheless there is muchscope for improvement before the application of this technologyin clinical settingsuse of ccffetalnas in prenataldiagnosis and pregnancyrelateddisordersthe apoptosisnecrosis ofduring pregnancytrophoblastsarising from syncytiotrophoblast is the prime source of therelease of ccffetalnasinto the maternal blood litton the presence of ccffetalnas has pavedthe way for noninvasive prenatal diagnosisand earlylo prediction of pregnancyrelated complications the use of ccffetalnas has gradually replacedinvasive techniques like amniocentesis or chorionic villussampling serr ccffetaldna comprises ofthe maternal ccfdna wang andcan be eï¬ciently detected atthe ï¬fth week of gestationguibert the amount of ccffetaldna inmaternal blood increases progressively throughout pregnancybirch ccfnas in prenatal diagnosisprenatal diagnosis is an established practice for the managementof pregnancy as well as avoidance of prenatalneonatal deathsthe leading causes for such deaths are genetic disorderbirth defects congenital malformations and chromosomalabnormalities like trisomy downs syndrome edwardssyndrome and patau syndrome and sex chromosomeaneuploidies like monosomy x turner syndrome carlson andvora therefore successful management of pregnancydemands eï¬cient and timely prenatal diagnosis to determine theoutcome of pregnancy timely detection of neural tube defectsis already providing early prenatal treatment resulting in betterneonatal outcomes adzick ccffetaldna is clinically used for the detection of fetal sexand multiple anomalies based on paternally inherited mutationsbianchi recent studies have discovered many fetalepigenetic biomarkers for ccffetalnabased liquid biopsies inclinical samples that have demonstrated high clinical potentialin disease diagnosis prognosis and pregnancy managementthese epigenetic modiï¬cations are speciï¬c to the fetus and helpto distinguish fetal nucleic acids from maternal nucleic acidsjones and takai clinical testing of recently developedfetal epigenetic markers can help in the proper managementof personalized care the ï¬rst reported use of fetalderivedepigenetic marker in maternal body ï¬uids had come frompoon who utilized an imprinted h19igf2 locusbased on parentoforiginspeciï¬c methylation and the maternaland the paternal copies of the gene were distinguished inmaternal blood poon based on the placental originof ccffetaldna having placentaspeciï¬c dna methylationpattern the genomic regions that show diï¬erential methylationbetween the placenta and the maternal blood cells can actas a marker for fetal dna in maternal blood the promoterregion of maspin serpinb5 is the ï¬rst such reported universalfetal dna marker with detectable hypomethylationin thebackground of hypermethylated maternal sequences the fetalorigin of these hypomethylated maspin has been conï¬rmedby the clearance of these sequences within h of deliveryfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerschim the clinical use of hypomethylated maspinis limited by the required bisulï¬te treatment of ccffetaldna as this treatment can degrade around of the dnagrunau thus decreasing the amount of alreadylow levels of fetal dna in maternal blood such limitationwas overcome by the detection of fetalderived hypermethylatedrassf1a in maternal blood for prenatal diagnosis chan hyland tounta 2011b the maternalhypomethylated rassf1a ccfdna can be removed by treatmentwith methylationsensitive restriction enzyme digestion leavingbehind fetal hypermethylated rassf1a ccffetaldna chan various other fetalderived diï¬erentially methylatedsequences have also shown a similar potential to act as fetal dnaepigenetic markers in maternal blood table ccffetaldna methylation markers have the potential ofbeing used as both quantitative as well as qualitative markersin prenatal diagnosis as qualitative markers these are used toestimate the false positives during the determination of fetalgender rh status and paternally inherited polymorphisms chan while as quantitative markers these can estimate thelevels of ccffetaldna in maternal plasma such an applicationof ccffetaldna ï¬nds its use in the detection of chromosomalaneuploidies lun based on the location of themaspin gene on chromosome hypomethylated fetal maspinhas been used to calculate the allelic ratio to diagnose trisomy with sensitivity tong fetal trisomy was detected by analyzing chromosomal dosage via targetingof fetal hypermethylated hlcs sequences in the combinationof microï¬uidics digital pcr rassf1a on chromosome and zfy on the y chromosome were used as referencestong fragmentation pattern of ccffetaldnain maternal plasma has been successfully used for enrichmentmethod in size separation manner on agarose gel electrophoresisramezanzadeh various nextgeneration sequencing and highthroughputtechniques have catalyzed the identiï¬cation of newer and novelfetal epigenetic markers further advancing noninvasive prenataldiagnosis the microarraybased approach has identiï¬ed manyfetal epigenetic markers with diï¬erential methylation betweenchorionic villus samples and maternal blood on chromosomes and for aneuploidy detection chu combining highresolution tiling oligonucleotide array withmethylated dna immunoprecipitation medip has helped ina genomewide screen for detecting the diï¬erential methylatedsites between placental tissue and maternal blood cells it hasdetected various new fetal epigenetic markers on chromosomes and
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"Validation of samples The primary aim of this scheme was to develop a flexible scalable EQA scheme designed to assess issues related to techniques and minimum detection limits used in standard laboratory practice focusing exclusively on the analytical (that is sample processing genotyping) and reporting phases (interpretation of the results in relation to the clinical context). To enable this and to avoid the significant challenges of sample heterogeneity in real tissue samples 20 artificial materials were used composed of formalin-fixed paraffin-embedded (FFPE) cell line samples. These EQA materials were designed to mimic real tissue samples as closely as possible and contained homogenous mixtures of mutant vs wild-type cell lines at a range of different allelic ratios. The paraffin blocks were cut and 10??m sections placed in eppendorf tube at the Pathology department of the VU University Medical Centre in Amsterdam The Netherlands by Dr Erik Thunnissen. H&E (4??m) sections were used to estimate the number of tumour cells. In each EQA sample section at least 200 nuclei were present (usually >300) roughly mimicking the amount of cells from a small NSCLC biopsy. For each EQA sample one 10-?m-thick section was sent by EMQN to each of the three validating laboratories for mutational analysis in a blinded fashion. Different sections from the block were analysed for EGFR mutation status to ensure that the mutation was homogeneously represented within each block. The validating laboratories independently analysed the samples by using three different approaches: direct sequencing of the PCR product for exons 1821 mutations; fragment analysis for exon 19 deletions and an allelic discrimination-based real-time PCR assay for the L858R mutation in exon 21; and the Therascreen EGFR RGQ kit (Qiagen Hilden Germany) reporting the results directly to the EMQN. The allelic ratios of mutations in each sample used in rounds 2 and 3 were accurately quantified by a commercial sponsor (Horizon Diagnostics Cambridge UK) using droplet digital PCR (ddPCR) on a BioRad QX100 (Hercules CA USA) platform. Genomic DNA (gDNA) was extracted from FFPE sections on the Promega (Madison WI USA) Maxwell System using the Maxwell 16 FFPE Plus LEV DNA purification kit according to the manufacturer's protocol. Quantification was performed using a Promega QuantiFluor dsDNA assay kit according to the manufacturer's protocol. ddPCR was performed using Taqman custom SNP 40 primer/probe assays (Life Technologies Carlsbad CA USA) to assess the frequency of each mutation with the exception of the p.(E746_A750) assay which was designed in-house. DNA (40?ng) was added to each ddPCR reaction. Reactions were performed in quadruplicate and droplets were generated using a Droplet Generator according to the manufacturer's instructions. PCR was performed on a standard thermocycler using previously optimised assay-specific cycling conditions. Droplets were analysed using a QX100 Droplet Reader as described in the manufacturer's instructions. Data from at least 45?000 useable droplets were collected for each sample. Formalin-fixed paraffin-embedded reference standards (Horizon Diagnostics) were included as assay controls. Registration of participant laboratories and shipment of samples Laboratories that performed EGFR mutational analysis were invited to participate in the EQA via an open call from the EMQN in conjunction with the ESP ETOP and ESMO. Participating laboratories registered via the EMQN website (European Molecular Genetics Quality Network (EMQN) 2014) and were requested to perform DNA extraction and analysis using their routine method. In each round 10 samples (one 10-?m-thick section for each) with accompanying mock clinical referral information were sent to participating laboratory. Each laboratory was identified only by a unique EMQN ID code to avoid exchange of information between participants and minimise bias in the results' interpretation process. The laboratories were given 8 weeks to complete their analyses and to submit the results of genotyping to the EMQN website. The centres were requested to provide information on the technique used for mutational analysis and metrics relating to their experience of performing EGFR mutational analyses. Evaluation of results The scheme included three rounds: the first was restricted to a maximum of 30 labs to establish proof of principle and validate the materials. A subsequent second round of the scheme was anised with no restriction on participation. Laboratories that failed the second round were provided with another set of samples in a restricted third round. The steering group evaluated the results according to a pre-defined scoring system. The scoring system assigned two points to correct genotype and zero points to false-positive or -negative results (). Errors in mutation nomenclature that might lead to misinterpretation of the results (for example stating deletion' without specifying the exon in which the deletion occurs) were assigned 1.50 points. This deduction was applied only once for each center generally to the first sample for which the error was found. One point was awarded for cases in which the genotype was mispositioned or miscalled: this error sometimes occurs with exon 19 deletions for which it might be difficult to define the precise base or amino acid in which the deletion starts or ends. If a test failed giving no result on the sample (analytical failure) then the lab received 1.00 point for that sample. The threshold to pass the EQA was set at a total score for the 10 samples of ?18 out of 20 (Thunnissen et al 2011) laboratories with a genotyping score <18 were classified as poor performers (applied to rounds 2 and 3 only). Performance in the assessment of clinical interpretation and reporting did not contribute to poor performance. Results Selection of the samples for the EQA The first step of the EQA scheme was the selection and the validation of the samples. Twenty materials were manufactured by Dr Thunnissen by mixing four lung cancer cell lines (A549 EGFR wild type) H1650 (EGFR p.(E746_A750del) H1975 (EGFR p.(T790M) p.(L858R)) and SW48 (p.G719S). Cell lines with mutations were serially diluted into A549 or SIHA cells at different ratios relevant to establishing the analytical sensitivity of the tests used by labs. Each material was validated in three different reference laboratories using different techniques to confirm the genotype and the results showed that the mutations were detectable at all the designated ratios dependent on the technology used (). A good yield of gDNA was obtained from all the samples. In addition there was complete concordance on the EGFR mutational status of the selected specimens and therefore all were selected for use in the quality assessment scheme with samples A1A10 used for the pilot and B110 and C1C10 in subsequent rounds 2 and 3. To accurately establish quantitative measurements of the allelic frequencies of the EGFR mutations all 10 EQA samples (; samples B/C1B/C10) used in rounds 2 and 3 were analysed on a ddPCR platform (BioRad QX100). Three of the samples had allelic frequencies higher than expected (C3 C8 and C9) two were lower (C2 and C10) and in one (C5) it was not possible to establish the true value due to insufficient availability of sample material (). First round proof of principle pilot scheme Twenty-nine laboratories registered from 13 countries and 25 participated in the pilot EQA scheme (4 labs withdrew due to customs sample importation problems) which was run in fourth quarter of 2011. A set of 10 samples were sent to the laboratories (; samples A1A10). All the participating laboratories submitted results within the 8-week time frame. The main methodology used by the participants was PCR/sequencing (n=10 laboratories; 34%) and real-time PCR (n=10; 34%) (). Two analytical errors (false-negative results) were observed. A further five laboratories made process errors (sample swaps) that resulted in an additional 24 genotype errors. In all cases the genotypes were correct but reported for the wrong sample. Therefore 92% of the false-negative results were concentrated in five laboratories. No false-positive results were reported. The materials performed well and there were no analytical test failures."
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pediatric obesity remains a public health burden and continues to increasein prevalence the gut microbiota plays a causal role in obesity and is a promising therapeutic target speciï¬cally the microbial production of shortchain fatty acids scfa fromthe fermentation of otherwise indigestible dietary carbohydrates may protect against pediatric obesity and metabolic syndrome still it has not been demonstrated that therapies involving microbiotatargeting carbohydrates known as prebiotics will enhance gutbacterial scfa production in children and adolescents with obesity age to yearsold here we used an in vitro system to examine the scfa production by fecal microbiota from children with obesity when exposed to ï¬ve different commercially availableoverthecounter otc prebiotic supplements we found microbiota from all patientsactively metabolized most prebiotics still supplements varied in their acidogenic potential significant interdonor variation also existed in scfa production which 16s rrna sequencing supported as being associated with differences in the host microbiota composition last we found that neither fecal scfa concentration microbiota scfa productioncapacity nor markers of obesity positively correlated with one another together thesein vitro ï¬ndings suggest the hypothesis that otc prebiotic supplements may be unequalin their ability to stimulate scfa production in children and adolescents with obesityand that the most acidogenic prebiotic may differ across individualsimportance pediatric obesity remains a major public health problem in the unitedstates where of children and adolescents are obese and rates of pediatric severeobesity are increasing children and adolescents with obesity face higher health risksand noninvasive therapies for pediatric obesity often have limited success the humangut microbiome has been implicated in adult obesity and microbiotadirected therapiescan aid weight loss in adults with obesity however less is known about the microbiome in pediatric obesity and microbiotadirected therapies are understudied in children and adolescents our research has two important ï¬ndings i dietary prebiotics ï¬ber result in the microbiota from adolescents with obesity producing more scfa andii the effectiveness of each prebiotic is donor dependent together these ï¬ndings suggest that prebiotic supplements could help children and adolescents with obesity butthat these therapies may not be one size ï¬ts allkeywords fermentation microbiome pediatric obesity prebiotics shortchain fattyacidscitation holmes zc silverman jd dressmanhk wei z dallow ep armstrong sc seed pcrawls jf david la shortchain fatty acidproduction by gut microbiota from childrenwith obesity differs according to prebioticchoice and bacterial community compositionmbio 11e0091420 101128mbio0091420invited editor thomas mitchell schmidtuniversity of michiganann arboreditor jose c clemente icahn school ofmedicine at mount sinaicopyright holmes this is anopenaccess distributed under the termsof the creative commons attribution international licenseaddress correspondence to lawrence a davidlawrencedaviddukeedu present address justin d silverman collegeof information science and technologypennsylvania state university state collegepennsylvania usa institute for computationaland data science pennsylvania stateuniversity state college pennsylvania usaand department of medicine pennsylvaniastate university hershey pennsylvania usareceived april accepted july published august julyaugust volume issue e0091420®mbioasm 0cholmes ®approximately of children in the united states have obesity and the prevalence continues to increase among all ages and populations the prevalence ofpediatric obesity is even higher in hispanic and african american populations in theunited states where rates of severe obesity continue to increase children withobesity have an increased risk of adverse health events and incur higher health carecosts despite the severity of the pediatric obesity epidemic current commontreatment strategies centered around lifestyle changes including behavioral dietaryand exercise interventions often fail or have limited success the high prevalence ofpediatric obesity coupled with the low success rate of common interventions highlights the need for more efï¬cacious safe strategies to lower the body mass index bmiin children and adolescentsthe human gut microbiome has emerged as a promising therapeutic target in pediatricobesity over the past decade differences in gut microbial community composition andmetabolic activity between obese and lean individuals have been observed causallinks have also been established fecal transplantation can transfer the obesity phenotypefrom obese donors to lean recipients and recapitulate some key metabolic changes inhuman obesity multiple mechanisms for this link have been proposedincludingincreased energy harvest by obese microbiota activation of enteroendocrine signalingpathways by shortchain fatty acids scfas modulation of glucose and energyhomeostasis through bile acid signaling and increased local and systemic ammation caused by a variety of microbial metabolites recent attention in obesity research has been speciï¬cally drawn to the role ofmicrobially derived scfas scfasprimarily acetate propionate and butyrateareproduced by enteric microbes as end products of anaerobic fermentation of undigested microbially accessible dietary carbohydrates and serve a variety of importantroles in the gut of particular interest is the scfa butyrate which serves as the primarynutrient source for colonocytes and functions as a histone deacetylase inhibitor through its inhibition of nf 242cb signaling in colonocytes butyrate contributesto barrier integrity maintenance and reduces levels of intestinal ammation markers acetate propionate and butyrate also each activate gproteincoupled receptors gprs that modulate key metabolic hormones including peptide yy pyy andglp1 consistent with these mechanistic ï¬ndings mouse studies have shownthat supplementation with acetate propionate butyrate or some mixture of these canprotect against weight gain improve insulin sensitivity and reduce obesityassociatedammation given the experimental evidence for scfa supplementationhaving an antiobesogenic effect in a murine system maintaining high levels of scfasduring a weight loss treatment may improve results if increasing scfa levels is a potential approach to promote weight loss in childrenprebiotic supplementation may provide an effective and lowrisk adjunctive therapyprebiotics are dietary carbohydrates that are indigestible by humanproduced enzymesand thus survive transit to the lower gastrointestinal gi tract once in the colonprebiotics serve as carbon sources for bacterial fermentation which in turn yield scfasas metabolic end products multiple types of prebiotics eg fructooligosaccharides [fos] and inulintype fructans have been tested in children with obesityranging from ages to years old in select cases these treatments have beenassociated with smaller increases in bmi and fat mass and reductions in bodyweight zscores body fat and trunk fat still other prebiotic trials in children whoare overweight have reported no significant beneï¬cial effects interpreting the mixed outcomes of prior prebiotic clinical trials in pediatric obesitythough is complicated by several challenges first in vivo studies in pediatric obesity todate have each used only one prebiotic supplement due to the logistical constraints ofclinical trials trials employing testing only a single type of supplement hinderthe ability to generalize s regarding the efï¬cacy of prebiotics and also makeit challenging to determine whether some prebiotics are inherently more acidogenicthan others second in vivo trials in healthy adults have shown substantial interindividual variation in the single prebiotic effects on stool scfa concentration julyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®variation in the primary and secondary outcomes could be due to differences inmicrobial scfa production or differences in host physiology such as scfa absorptionpotential third while scfa concentrations have been shown to be altered in childrenwho are overweight or obese changes in fecal scfas during dietary interventionhave not been measured in past in vivo studies in pediatric populations if prebioticsmediate their effects through scfas directly tracking scfas could helpdetermine treatment success fourth in vivo studies in adults especially those withobesity may be confounded by the concurrence of chronic disease and the medications a person may be taking to treat chronic diseasein this study we have taken an in vitro approach to address the limitations of priorhuman studies an in vitro approach facilitates more direct comparisons of differentprebiotic supplements the higher throughput of in vitro experiments allows widervariety of prebiotics to be tested and the effects of these supplements can be testedon identical microbiota samples rather than over time within subjects which isconfounded by microbiota drift over time as well as inconsistencies in dietarycomposition taking an in vitro approach to studying the effects of prebiotics on gutmicrobiota allows a more direct investigation of microbial scfa production since wecan study the effects of prebiotic supplementation independent of the effects of hostabsorption using a preclinical in vitro fermentation model and samples fromadolescents with obesity who have not developed longterm complications we pursued three speciï¬c lines of inquiry i whether different types of prebiotics lead todifferences in scfa production by gut microbiota from adolescents with obesity iiwhether the effects of prebiotics are shaped by interindividual differences in gutmicrobiota structure and iii whether fecal scfa production is likely to be associatedwith protection from obesityresultsscfa production capacity to measure scfa production by gut microbiota weadapted the in vitro approach of edwards this method was speciï¬callydesigned to study fermentation of starch in the human lower gi tract and has sincebeen used to measure metabolite production from human stool samples when exposed to prebiotic ï¬ber in brief we homogenized previously frozen feces inreduced phosphatebuffered saline pbs ph «¾ to create a fecal slurry with aï¬nal concentration of gliter fig these fecal slurries were then supplied witheach of ï¬ve prebiotic carbon sources as well as a carbonfree control and allowed toferment at °c in anaerobic conditions for h to approximate colonic transit time after the incubation period the concentrations of scfas in the samples weremeasured by gas chromatography to control for differences in overall cell viability orstool slurry nutrient content between donors we corrected measurements of scfaconcentration by dividing the treatment scfa concentration by the control scfaconcentrationto validate our assay we ran a series of experiments using feces from validationsample sets we veriï¬ed that our controlcorrected scfa production data were notuenced by bacterial abundance p «½ 2433 «½ spearman correlation seefig s1 in the supplemental material absolute not relativized to control scfaconcentrations are supplied in the supplement see fig s2 and s3 since our fermentation experiments used previously frozen fecal samples we veriï¬ed that total scfaproduction was strongly correlated between fresh samples and twice freezethawedsamples p ¬ 2433«½ spearman correlation see fig s4a since we elected tonot provide our fermentation reactions with nutrients in excess of what was containedin the fecal slurries we veriï¬ed that there existed strong correlation in total scfaproduction between pbsgrown and colonic mediumgrown cultures both whensupplied with dextrin and inulin dextrin p «½ 2433«½ inulin p «½ 2433«½ spearman correlations see fig s5 we found that total scfa production over controlwas positively correlated with the ph of starting fecal slurries p «½ 2433 «½ spearman correlation fig 2a a weaker correlation may exist between scfa producjulyaugust volume issue e0091420mbioasm 0cholmes ®fig overview of in vitro fermentation methodstion and the ï¬nal ph of the fermentation vessels p «½ 2433 «½ spearmancorrelation fig 2bwe subsequently applied our assay to fecal microbiota from a cohort of children male female one unknown ranging in age from to years old average age years tanner stages to and a body mass index bmi of to averagebmi see table s1 in the supplemental material one patient provided samplesused in all analyses but was lost to followup before providing clinical metadata thiscohort was a subset of a cohort of patients enrolled in the pediatric obesity microbiomefig relationship between in vitro scfa production and ph a in vitro total scfa production over control is positively correlated with the phof starting fecal slurries p «½ 2433 «½ spearman correlation b relationship between scfa production and the ï¬nal ph of fermentationvessels p «½ 2433 «½ spearman correlationjulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®fig in vitro scfa production by prebiotic a donor b and individually c in a twoway anova of the effects of donor and prebiotic on scfaconcentrationcontrol donor prebiotic and their interaction were all statistically significant p ¬ p ¬ and p ¬ respectively shownis the total scfa concentration of an in vitro culture after h of anaerobic incubation divided by the scfa concentration of the corresponding prebioticfreecontrol culture for each of ï¬ve prebiotic growth conditions across donors black dots gray diamonds are means and gray bars are standard deviationsabsolute scfa concentrations are depicted in fig s3and metabolism study we found all individuals demonstrated a net gain ofscfas relative to the controlin at least one prebiotic treatment which led us toconclude that all tested cultures were viable and metabolically active fig donor and prebiotic both impact scfa production in vitro we next tested thehypothesis that different prebiotics equally promote the production of scfas byjulyaugust volume issue e0091420mbioasm 0cholmes ®fig eighteen genera were found to be credibly associated with scfa production in at least one of our ï¬ve prebiotic growth conditions shownare the mean lambda values and and credible intervals for all genera credibly associated with at least one prebioticgrowth condition plotted on centered logratio clr coordinates red centers denote associations with credible intervals that do not cover lambda represents the strength of the effect of each covariate on each taxa a lambda value of reï¬ects a unit fold change in scfaconcentration over control as being associated with a unit fold change in the clrtransformed relative abundance of the genusperforming statistical analysis of scfa production as a function of the prebiotic typeand individual identity our analysis revealed heterogeneity in the efï¬cacy of prebioticsupplements twoway analysis of variance [anova] p ¬ see table s2 fig 3aranging from inulin which resulted in a mean fold change in total scfas togalactooligosaccharides gos which resulted in mean fold change in total scfasfrequently only two or three of the ï¬ve tested prebiotics resulted in increased totalscfa production within an individual our statistical testing also revealed consistentpatterns between individuals gut microbiota in terms of scfa production twowayanova p ¬ see table s2 fig 3b with mean fold changes in scfas over controlranging from to within individuals the average fold change in scfa concentration in the prebiotic treatments often appeared to be driven by a few stronglyacidogenic prebiotics last our analysis indicated a significant interaction betweenidentity twoway anova p ¬ see table s2prebiotic type and individualfig 3c because our statistical analysis considered technical replicates as separateexperimental conditions this result suggests the presence of consistent prebioticindividual responses across in vitro assay replicate runsnot whether such interactionsare consistent within an individual over timescfa production in vitro predicts the abundance of bacteria in the startingculture if interindividual differences in gut microbiota mediated responses to prebiotictreatment we would expect that speciï¬c bacterial taxa which varied between individuals could also be associated with scfa production to evaluate this hypothesis weused the r package stray to create a bayesian multinomial logistic normal linearregression pibble model that tested for correlations between in vitro scfa productionin response to each prebiotic and 16s rrna community composition of patient stoolused in the fermentations at the genus level this analysis revealed that scfa production from prebiotics was correlated with the relative abundances of differentbacterial genera credible interval not covering fig of the generapositively associated with scfa production are known or likely ï¬ber degraders one akkermansia is often observed to increase in abundance after prebiotictreatment and one methanobrevibacter an archaeon hydrogenotrophic methanojulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesitytable associations between microbial genera and scfa production on ï¬ve different prebiotic substratesassociation with scfa productiondextrin«¹«¹«¹«¹«¹«¹«¹«¹«¹genusakkermansiaruminococcus_2coprostanoligenes_groupparabacteroidesbutyricimonasmethanobrevibactertyzzerella_4tyzzerella_3lachnospiraceae_nk4b4lactobacilluscoprococcus_1collinsellalachnospiraceae_fcs020xosgosfosinulin«¹«¹«¹«¹fiber degrader statussupporterdegraderno evidencedegraderassociatedsupporterdegraderdegraderdegraderdegraderdegraderno evidencedegrader®referencegen is known to increase the efï¬ciency of carbohydrate metabolism by the microbiota table most genera identiï¬ed by stray were associated with scfa production ina limited set of prebiotic treatments one genus lactobacillus is positively associatedwith scfa production on xylooligosaccharides xos but was negatively associatedwith scfa production on gos overall the presence of speciï¬c associations betweenbacterial taxa and different prebiotics supports a model where different individuals varyin their levels of prebiotic degrading gut bacteriametrics of obesity do not appear to correlate with scfa production capacity ofstool finally we tested the hypothesis that in vitro scfa production would beassociated with obesityrelated phenotypes we compared clinical metadata fromindividuals which included bmi insulin and hba1c with average total scfa production across prebiotics and found no significant correlations in our population spearman correlation table fecal microbial scfa production capacity may not be directlyassociated with obesity though because rates of host scfa uptake likely vary and thisvariance may uence host intestinal physiology indeed in support of theidea that scfa absorption rate which was not measured in this study shape metabolichomeostasis and host health we observed a negative association between fecal scfaconcentrations and in vitro scfa production across the range of tested prebioticsfig furthermore if scfa absorption efï¬ciencies varied by individual residual fecalscfa concentrations may not directly reï¬ect the complete effect of bacterial metabolism on obesity consistent with this notion no significant relationships were apparentbetween concentrations of scfa in patient stool and clinical markers of obesitymeasured at enrollment including bmi insulin levels and hba1c table althoughthis may also be explained by uncontrolled patient parametersdiscussionin this study we found that the microbiota of all tested adolescents with obesityincreased total scfa production when exposed in vitro to at least one prebiotic bothdonor and prebiotic were significant factors in determining scfa production in vitro aswas their interaction our modeling revealed distinct associations between speciï¬cmicrobial taxa and scfa production on different prebiotics we interpret this result astable neither average scfa production in vitro nor fecal scfa concentrationcorrelated with metrics of obesity measured in individuals at the time of enrollmentabmiparameteravg net scfa productionfecal scfa concentrationap and 2433 values were determined from spearman correlations 2433pinsulinp 2433hba1cp 2433julyaugust volume issue e0091420mbioasm 0cholmes ®fig spearman correlations between in vitro scfa production and scfa concentration of the starting fecal inoculum scfaproduction is the average of technical replicates with the linear regression line plottedsuggesting that the associated bacteria play a role in the ï¬ber fermenting capacity ofthe community we observed no correlations between either stool scfa concentrationsor in vitro acidogenic capacity of communities and any metrics of obesity table we have recapitulated previous ï¬ndings that both donor and prebiotic areimportant in determining the scfa production from in vitro prebiotic supplementation and we found that not all prebiotics appear equally acidogenic since our in vitro system removes the host as a potential source of variationour data support a gut microbial role for interdonor variation in fecal scfaproduction in addition the strength of the interaction between donor and prebiotic strongly suggests that prebiotics are not one size ï¬ts all rather inconsistentresults from prior studies of prebiotics in pediatric obesity may be dueto variation in the scfa production capacity of individuals gut microbiota acrossthe tested prebiotics future therapeutic efforts involving prebiotics in patients withobesity may beneï¬t from stratiï¬ed or personalized treatments nutritional therapiesthat are personalized to individuals microbiota are already in development murine and in vitro studies show that increased signaling through gpcrs mediatedby acetate propionate and butyrate increases satiety and insulin sensitivity whiledecreasing adipogenesis yet we did not observe associations betweenfecal scfa levels and metrics of obesity the effects of scfa on obesity may be maskedby uncontrolled patient factors such as differences in caloric intake and variation inindividual nutrient harvest and utilization in order to observe the effects of scfa onobesity it would be necessary to control for these variable physiological and lifestyleparameters which we did not attempt these patient factors may also have uencedour inability to observe an association between acidogenic capacity of microbiota andfecal scfa concentrations however this may also be explained by the potentialjulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®uncoupling of fecal scfa production and fecal scfa concentration in vitro increasedluminal concentrations of butyrate have been shown to upregulate the sodiumcoupled monocarboxylate transporter slc5a8 and the addition of physiologicalmixtures of scfa has been shown to upregulate the monocarboxylate transporterslc16a1 both of which uptake acetate propionate and butyrate from the lumensince gut epithelia have the capacity to absorb up to of scfa before excretion increased host scfa uptake triggered by increased gut bacterial production couldtherefore lead to constant or even decreased fecal scfa concentrations this complexrelationship could explain the absence of positive correlations we observed betweenstool scfa levels and the acidogenic capacity of gut microbiota it may be necessary todelve further upstream of fecal scfa concentration by measuring proxies for host scfauptakes such as the expression of scfa transporters slc5a8 and slc16a1 and scfareceptors gpr43 gpr41 and gpr109a the primary limitations of this study involve constraints common to in vitro culturestudies first many factors affecting bacterial scfa production in vivo are difï¬cult toreplicate in vitro including the availability of nutrients such as nitrogen the startingconcentration of scfas the redox state of the environment and the efï¬ciency ofcrossfeeding interactions different metabolic results between prebiotics mayhave occurred if we provided alternative cometabolites or nutrients in addition to thetested prebiotics we chose our culture conditions namely a mediumfree approachthat does not add any nutrients beyond what is present in the stool in an effort toavoid inducing artiï¬cial selective conditions within our cultures prior experimentaldigestion studies have shown that prebiotic response patterns can be recapitulatedacross various culture conditions indeed we found strong correlation in scfaproduction between cultures grown with our mediumfree approach and those grownin a more conventional medium containing added nitrogen vitamins minerals andacetate further this approach allowed us to minimize the uence of the host onmeasurements of microbiota production of scfa we did observe shifts in communitycomposition during the h fermentations fig s6 however we remained able to ï¬ndstatistical associations between scfa production capacity and prefermentation community composition a second set of limitations in this study involves our reliance onpatient collection of stoolinterdonor variation in prebiotic response could haveoriginated in technical variation between how patients exposed stool to aerobicconditions or how they froze their samples which in turn could have affectedthe fraction of viable microbial cells in stool samples still we found a significantcorrelation between in vitro total scfa production from fresh stool and stool that hadbeen frozen and thawed twice variation in donor prebiotic response could also havebiological origins due to physiological differences between people eg efï¬ciency offood digestion consistency of stool [] or differences in diet which can lead tovariation in stool microbial load and nutrient content rather than control for amyriad of different sources of variation whose origins we did not measure we chosethe straightforward approach of standardizing donor samples by employing a consistent concentration of stool slurry [wtvol] stool in pbs in our experimentsfuture work to address these limitations could test multiple stool samples persubject to conï¬rm whether the observed variation in prebiotic response is durablebetween individuals over time future studies could also examine the correlationbetween the metabolic effects of prebiotic supplementation in vitro and in vivo usingrandomized human trials that couple human prebiotic supplementation in vivo measurement of scfa production and in vitro tests of microbiota metabolic activity itwould also be useful for such studies to explore the impact of prebiotic supplementation on host physiology both in vitro and in vivo speciï¬cally the effects of prebioticsupplementation on colonic epithelial barrier integrity scfa receptor gpr41 gpr43and gpr109a expression and scfa transporter mct1 and smct1 expression couldprovide greater insight into the health impacts of prebiotic supplementation as well asexplain why fecal scfa concentrations may not mirror the metabolic capacity of gutmicrobiotajulyaugust volume issue e0091420mbioasm 0c®holmes materials and methodscohort stool was collected from human donors under a protocol approved by the duke healthinstitutional review board duke health irb pro00074547 for a prospective longitudinal cohort studyand biorepository participants whose samples were used in this study were treatmentseeking adolescents with obesity who were newly enrolled in a multidisciplinary weight management program allsubjects received familybased intensive lifestyle modiï¬cation based on clinical necessity some participants also were placed on a lowcarbohydrate diet medications to facilitate weight loss or underwentweight loss surgery see table s3 due to the low number of patients assigned to each treatment armwe did not attempt to base any analyses on patient treatment plan patients were to years old witha bmi ± 95th percentile none had antibiotic use in the month prior to enrollment used medicationsknown to interfere with the intestinal microbiome or had other significant medical problems stoolsamples used in this study were from enrollment 3month 45month and 6month followup visits seetable s3 the clinical metadata used for correlations was collected at enrollment months and months the metadata collected nearest to the stool sample collection date was used in our analysesstool collection patients collected intact stool samples in the clinic or at home using a plastic stoolcollection container fisher scientiï¬c and were asked to immediately store this container intheir home freezer patients then returned the sample by either bringing it to the study team orscheduling a home pickup within h of stooling stool was transported frozen in an insulated containerwith an ice pack upon receipt in the lab samples were placed on dry ice until transferred to a °cfreezer for longterm storage all patient samples were frozen at °c within h of stooling range h to h median h except for one which was stored h after stooling the timebetween stooling and freezing at °c did not have a significant effect on average scfa productionp «½ 2433 «½ «º pearson correlation stool samples for analysis were processed by removingcontainers from °c storage and thawing on ice in a biological safety cabinet until soft enough toaliquot thawed containers of stool were opened to atmosphere for a maximum of min while sampleswere aliquoted after primary aliquoting the remaining stool was transferred to an anaerobic chambercoy laboratory products hydrogen co2 nitrogen and further portioned into ¬2galiquots for this study these aliquots were then stored as solid stool pellets at °c until used for thisstudyin vitro fermentation see fig for an overview of in vitro fermentation methods aliquotedstool was thawed at room temperature in an anaerobic chamber once thawed stool was weighedand placed into a polyethylene ï¬lter bag with 033mm pore size whirlpak b01385 and ml ofanaerobic «» pbs was added for each gram of stool resulting in a wtvol fecal slurry similarto previous studies during our validation experiments a medium designed tosimulate colonic contents was used in place of «» pbs to create stool slurries the ï¬lter bag wasthen closed and placed into a stomacher seward stomacher where the contents were homogenized on the medium speed setting for s the liquid fraction was removed from the downstreamside of the ï¬lter membrane and the solid fraction was discarded a 1ml aliquot of this liquid fractionwas removed for analysis of the scfa concentration to determine the scfa concentration of thestarting stool sample during our validation experiments two separate 1ml aliquots of this liquidfraction were removed one was used to estimate relative bacteria abundance of starting fecalslurries using total extracted dna concentration as has been previously published and theremaining aliquot was used to determine the ph of the starting fecal slurry using a handheld phmeter elite ph spear thermo fisher scientiï¬c the remaining liquid fraction was incubated induplicate across six different treatments either supplemented with inulin now foods inulin powderpart fructooligosaccharides fos cargill part galactooligosaccharides gosbimuno powder xylooligosaccharides xos bionutrition prebiotic with llifeoligo part wheatdextrin beneï¬ber original or unsupplemented for each reaction ml of fecal slurry wasplaced in one well of a 24well cell culture plate each well was then delivered ml of wtvolprebiotic solution in «» pbs or ml of «» pbs without prebiotic during our validation experimentsprebiotics were dissolved in colonic medium instead of «» pbs the resulting fermentationconditions were therefore fecal slurry with prebiotic wtvol a fecal slurry was selectedbecause its fermentative capacity has been previously demonstrated to be insensitive to smallvariations in concentration and is feasible to work with using this method a ï¬nalconcentration of prebiotic in the context of a fecal slurry i
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" mirnas regulate a multitude of cellular processes and their aberrant regulation is linked to humancancer however the role of mir4255p in lung cancer lca is still largely unclear here we explored the role ofmir4255p during lca tumorigenesismethods cell proliferation was evaluated by cell counting kit8 and colony formation assay western blot and realtime pcr were accordingly used to detect the relevant proteins mirna and gene expression luciferase reporterassays were used to illustrate the interaction between mir4255p and ptenresults we demonstrate that mir4255p is overexpressed in lca tissue and enhances the proliferative and colonyformation capacity of the lca cell lines a549 and ncih1299 through predictive binding assays pten wasidentified as a direct gene target and its exogenous expression inhibited the procancer effects of mir4255pthrough its ability to downregulate pten mir4255p activated the pi3kakt axis we conclude that mir4255p promotes lca tumorigenesis through ptenpi3kakt signalingkeywords mir4255p lung cancer pten pi3kakt signaling pathways lung cancer lca is a leading cause of cancer relatedmortality across the globe lca is prevalent in males and asymptomatic during early disease stages as manyas in every cases are at an advanced stage iii or ivwhen diagnosed and the 5year survival rates remainlow particularly for those with metastatic lca improved lca therapeutics is thus urgently requiredmicrornas mirnas regulate many cell processesincluding differentiation metabolism and tumorigenesis[] emerging evidence suggests that mirnas are keyplayers during lca tumorigenesis [] the aberrantexpression of mir4255p is linked to hepatocellular carcinoma hcc gastric cancer gca and colorectal correspondence kjhhev163comcardiothoracic surgery the fourth affiliated hospital zhejiang universityschool of medicine shangchen road no1 of yiwu zhejiang chinacancer crc [] here we report the upregulationof mir4255p in lca and highlight its contribution tolca development we further identify pten as a novelmir4255p target that is inhibited in lca to promoteptenpi3kakt signalingmethodspatient specimenslca samples n and adjacent healthy tissue at least cm from the resection margin were collected from thefourth affiliated hospital zhejiang university school ofmedicine the study was fully supported by the institutional review board of the fourth affiliated hospitalzhejiang university school of medicine no2015009all participants provided consent for sample analysisand anything about their identities will not be includedin the data the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhou bmc pulmonary medicine page of lca cell lines cell culture and cell transfectionhuman lung cancer cell lines a549 ncih1299 ncih460 hcc827 and normal human lung epithelial cellline beas2b were obtained from the cell bank of thechinese academy of sciences shanghai china a549 hcc827 cells were cultured in dmem plus fbsand penstrep ncih1299 ncih460 cells weregrown in rpmi1640 plus fbs at °c in co2beas2bs were cultured in clonetics¢ mediathe mir4255p mimics and negative control mirnanc were chemically synthesized by shanghai genepharma co ltd songjiang shanghai china lipofectamine invitrogen eugene or usa was usedfor transfection according to the manufacturers protocol pi3k activity was assayed as previously described pi3k inhibitor ly294002 was obtained from abcamto analyze the effects of mir4255p on pi3kakt indicated lca cells were cultured in the presence or absence ofthe workingconcentrations of ly294002 were μm for experiments with ly294002 treatmentsindicated lca cellswere pretreated with ly294002 for h prior to exposure to proteasome inhibitorsthe drugs for h at °ccell growth assayslca viability was assessed using cell counting kit8from shanghai haling biotechnology co ltd shanghai china as per the manufacturers protocols brieflyafter incubating the transfected cells for one full daythey were collected after trypsinization and seeded cellswell into 96well plates ten microliters ofcck8 solution were added per well and kept for h at °c the absorbance of the mixture was estimated in amicroplate readerinchercules usa at nmfrom biorad laboratoriescolony formation assaythe colony formation assays were performed as previous each group of treated cells per well wasseeded into cm culture dish and cultured for weeksfinally colonies were stained using crystal violet andthe number of cell colonies was countedqrtpcr analysistotal rna was isolated by trizol® reagent from invitrogen thermo fisher scientific inc and a nanodropnanodrop technologies thermo fisher scientific incwas used to estimate its quality and concentration theexpression of mir4255p was done by reverse transcription using the mirx¢ mirna firststrand synthesis kitfrom takara biotechnology co ltd dalian chinaand quantitative evaluation of the synthesized cdnawas done by quantitative pcr rtqpcr using themirx¢ mirna qrtpcr tb green® kit from takaraforwardbiotechnology as an endogenous control the small nuclear rna u6 normalized the expression of mir4255pthe δδcq system was used to evaluate all genes expressions and the primer sequences were shown as fol² ggggagttaglows mir4255pgattaggtc3² reverse ² tgcgtgtcgtggagtc3² u6 forward ²ctcgct tcggcagcaca² reverse ²aac gct tca cga att tgc gt3²pten forward ² tggattcgacttagacttgacc ² reverse ² aggatattgtgcaactctgcaa² gapdh forward ²catcaccatcttccaggagcg3² reverse tgaccttgcccacagcctt3²to getdual luciferase assaysthe design and synthesis of pten fragments containingbinding sites for wt wildtype and mut mutant onmir4255p was done by shanghai genepharma thesewere cloned into the target expression vector pmirglo dualluciferase from promega corporation wiusathe reporter plasmids wtpten andmutpten one night prior to transfection seeding ofcells confluence was done in plates with wells transfection of these cells was done with reporterplasmids harboring wt or mut in the presence ofmirnc or mir4255p mimic post h of transfectionluciferase activity of the cells was estimated as per instructions using the dualluciferase reporter assay system from promega corporation promega fitchburgwi usa the data normalization was done by the activity of renilla luciferasewbcell lysates ripa lysates were resolved on sds pageand transferred to pvdf membranes membraneswere blocked for h in milk plus tbstincubated with primary antibodies against pten dilution cat no ab170941 abcam pi3k dilution cat no ab32089 abcam pakt dilution cat no ab81283 abcam akt dilution cat no ab32505 abcam pakt dilution cat no ab81283 abcam actin dilution cat no ab8226 abcam at °c overnights andlabeled with hrpconjugated secondarys for h atroom temperature bands werevisualized usingchemiluminescent hrp substrate and analyzed usingimage lab tm softwarestatistical analysesdata analysis was performed using spss treatmentgroups were compared using a oneway anova pvalues were taken as significant experiments wereperformed on at least three occasions data representthe mean ± sd 0czhou bmc pulmonary medicine page of resultsmir4255p is upregulated in lcawe compared mir4255p levels in paired lcaand normal lung tissue samples by qrtpcr analysismir4255p was upregulatedspecimensfig 1a and expressed to high levels in a549 ncih1299 ncih460 and hcc827 cells compared tonormal human lung epithelial cellline beas2bfig 1b consistent with previous findings in othercancer types previous results indicated that mir4255p is upregulated in lcain lcamir4255p enhances proliferation and inhibits apoptosisin lca cellsto dissect the role of mir4255p in lca its expressionwas manipulated using mir4255p mimics figure 2acshows that mir4255p upregulation enhanced cell survival meanwhile enhanced cell colony formation abilityfig 2d and e taken together above results indicatedmir4255p is thus an oncogene in lca cellsmir4255p targets ptenfrom targetscan pten was identified as a predictedmir4255p target fig 3a in pten ²utr reporterassays mir4255p suppressed wt pten expressionfig 3bc but had little effect on mutated pten ²utr fragments fig 3bc the levels of pten werelower in cells transfected with mir4255p mimics fig3de mir4255p also negatively related pten mrnalevels in lca tissue p r2 fig 3f thesedata implicate pten as a cellular target of mir4255pmir4255p promotes lca via ptento define whether mir4255p regulate pten in lcawe firstly overexpression pten in lca fig 4a wbanalysis demonstrated that mir4255p reduces ptenlevels which could be recovered by exogenous expression fig 4b cell viability assays showed that mir4255p enhances lca proliferation which could be reversedby pten transfection fig 4cd suggesting mir4255pmediates its activities via pten this indicates that mir4255p targets pten to mediate its protumor effectsmir4255p activates ptenpi3kakt signalingit has been reported ptenpi3kakt signaling wasclosely related to cell proliferation and apoptosis [] next we explored the effect of mirna4255p onptenpi3kakt signaling as shown in fig 5aincomparison to nc groups pten was down regulated inresponse to mir4255p mimics whilst pi3k and paktlevels increased in addition nsclc cells were treatedwith the pi3kakt inhibitor ly294002 or ly294002 mir4255p mimics mimic fig 5b kinase activity assays further showed that pi3k activity in nsclc transfected with ly294002 was significantly lower than thattransfected with mimic control p and pi3k activity in nsclc cells transfected with both ly294002and mir4255p mimic was significantly higher than thattransfected with ly294002 p take together thisimplicates ptenpi3kakt signaling in the protumorigenic effects of mir4255pdiscussionthe poor prognosis of lca highlights the need for urgent therapeutic strategies mirnas are novel targets forfig mir4255p in highly expressed in lca a qrtpcr of mir4255p b qrtpcr of a549 ncih1299 ncih460 hcc827 and beas2b cellsdata mean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca a mir4255p expression in a549 and ncih1299 cells b c cck8 assays in mir4255p mimic transfectedcells d e colonyforming assay in mir4255p mimic transfected cells the raw data of all colony formation experiments was listed insupplemental table data mean ± sd p p p fig mir4255p targets pten in lca a predicted binding of mir4255p and pten b c relative luciferase activity of ptenwt ptenmut inlca cells expressing mir4255p mimic d pten mrna levels are significantly lower after transfection with mir4255p mimic e pten expressionassessed by wb fulllength blotsgels are presented in supplementary figure f mir4255p and pten negatively correlate in lca tissue datamean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca growth by targeting pten a qrtpcr of pten in indicated lca cell lines b wb analysis of pten expression fulllength blotsgels are presented in supplementary figure c d cck8 analysis of cell viability in the indicated cell lines with empty vector mir4255p mimic andor pten in a549ncih1299 cells data mean ± sd p p p fig effects of mir4255p on ptenpi3kakt a wb analysis of pten pi3k pakt and akt in lca cells fulllength blotsgels are presented insupplementary figure b the pi3k kinase activity was determined in nsclc cells transfected with ly294002 was significantly lower than thattransfected with mimic control and the pi3k kinase activity in nsclc transfected with both ly294002 and mir4255p mimic was significantlyhigher than that transfected with ly294002 data mean ± sd p p p 0czhou bmc pulmonary medicine page of cancer therapies and their dysregulation occurs in lcatissue [] duan showed that mir203 bindsto zeb2 to suppress emt yuan showed thatmir30a inhibits eya2 migration and invasion andli showed that mir1304 inhibits lca cell divisionthrough heme oxygenase1 the cellular roles ofmir4255p in lca are poorly understoodin thepresent work we further explore the underlying mechanisms of mir4255pinduced lca cell progressionin the present study we confirmed that mir4255p isoverexpressed in lca cell lines and tissues implicating arole in lca tumorigenesis upregulating mir4255plevels enhanced the cell survival and colony formationability of lca cells in vitro implicating it as a novel lcaoncogene in the mechanism using the algorithms targetscan website tools we identified pten as the potential target of mir4255p furthermore we performedluciferase reporter assays and the results showed thatmir4255p may directly target pten3utr the resultof qrtpcr and western blot also confirmed that overexpression of mir4255p could suppress the expressionlevel of pten all the above suggested that pten was apotentialtarget of mir4255p moreovermir4255p also negatively related pten mrna levelsin lca tissue rescue experiment indicated that exogenous pten expression inhibited the procancer effects ofmir4255p pten was downregulated in lca tissuepten is a tumor suppressor with wellcharacterizedphosphatase activity pi3kakt promotes cell cycleprogression inhibits apoptosis and is known to be overactive in a multitude of human cancers [ ] ptencan suppress pi3kakt signaling and thus displays anticancer effects upon assessment of the molecularmechanisms of mir4255p in lca cells its procancereffects were found to be mediated through manipulationof the ptenpi3kakt signaling axisfunctionalin we highlight mir4255p as an oncogenein lca that promotes an oncogene phenotype by inhibiting pten these findings enhance our knowledge of therole of mir4255p and reveal new therapeutic strategiesfor the diagnosis and treatment of lca angiogenesisand metastasis biological experiments will clarify thefunctions and roles of mir4255p in lcasupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12890020012610additional file supplement figure s1 uncropped images of blotsand gels related to fig supplement figure s2 uncropped imagesof blots and gels related to fig supplement figure s3 uncroppedimages of blots and gels related to fig table s1 the raw data of allcolony formation experimentsabbreviationslca lung cancer pten gene of phosphate and tension homology deletedon chromsome ten pi3k phosphatidylinositol 3kinase nsclc nonsmallcell lung cancer gca gastric cancer crc colorectal cancerhcc hepatocellular carcinoma emt epithelialmesenchymal transitionacknowledgementsnot applicableauthors contributionsjsz zsy syc and qf did patient recruitment and assessment andperformed the experiments jhy and cjh were responsible for statisticalanalysis jsz zsy and syc interpreted the data and wrote the manuscriptall authors contributed to the subsequent drafts and approved the finalversionfundingthis research was funded by zhejiang provincial natural science foundationof chinaexploration program lq19h020010 core talent program ofdepartment of health of zhejiang province 2013rca018 zhejiangprovincial natural science foundation of chinageneral programly15ho2004 the funders had no role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participateethics approval for this study was obtained from the fourth affiliatedhospital zhejiang university school of medicine no2015009 all patientsgave written informed consent for participation in the studyconsent for publicationnot applicablecompeting interestsall authors declare no conflicts of interestreceived march accepted august referenceschen w zheng r zuo t zeng h zhang s he j national cancer incidenceand mortality in china chin j cancer res aberle dr berg cd black wc the national lung screening trialoverview and study design radiology ni js zheng h huang zp microrna1973p acts as a prognosticmarker and inhibits cell invasion in hepatocellular carcinoma oncol lettzhang tj wang yx yang dq downregulation of mir186 correlateswith poor survival in de novo acute myeloid leukemia clin lab wang b teng y liu q microrna153 regulates nrf2 expression and isassociated with breast carcinogenesis clin lab lin h huang zp liu j mir4943p promotes pi3kakt pathwayhyperactivation and human hepatocellular carcinoma progression bytargeting pten sci rep yang j li j le y zhou c zhang s gong z pfklmir128 axis regulatesglycolysis by inhibiting akt phosphorylation and predicts poor survival inlung cancer am j cancer res wang r chen x xu t mir326 regulates cell proliferation andmigration in lung cancer by targeting phox2a and is regulated by hotairam j cancer res qin q wei f zhang j wang x li b mir134 inhibits nonsmall cell lungcancer growth by targeting the epidermal growth factor receptor j cellmol med hu h xu z li c mir145 and mir203 represses tgfbetainducedepithelialmesenchymal transition and invasion by inhibiting smad3 in nonsmall cell lung cancer cells lung cancer 0czhou bmc pulmonary medicine page of fang f song t zhang t cui y zhang g xiong q mir4255p promotesinvasion and metastasis of hepatocellular carcinoma cells through scaimediated dysregulation of multiple signaling pathways oncotarget cristobal i madozgurpide j rojo f garciafoncillas j potential therapeuticvalue of mir4255p in metastatic colorectal cancer j cell mol med zhang z wen m guo j clinical value of mir4255p detection and itsassociation with cell proliferation and apoptosis of gastric cancer pathol respract huang z xing s liu m deng w wang y huang z huang y huang x wuc guo x pan x jiang j feng f li t mir26a5p enhances cellsproliferation invasion and apoptosis resistance of fibroblastlikesynoviocytes in rheumatoid arthritis by regulating ptenpi3kakt pathwaybiosci rep meng j liu gj song jy chen l wang ah gao xx wang zj preliminaryresults indicate resveratrol affects proliferation and apoptosis of leukemiacells by regulating ptenpi3kakt pathway eur rev med pharmacol sciliu hy zhang yy zhu bl feng fz yan h zhang hy zhou b mir21regulates the proliferation and apoptosis of ovarian cancer cells throughptenpi3kakt eur rev med pharmacol sci yu g wang c song x liu s zhang y fan l yang y huang y song jformaldehyde induces the apoptosis of bmcs of balbc mice via the ptenpi3kakt signal transduction pathway mol med rep sun xh wang x zhang y hui j exosomes of bonemarrow stromal cellsinhibit cardiomyocyte apoptosis under ischemic and hypoxic conditions viamir4865p targeting the ptenpi3kakt signaling pathway thromb res mou x liu s mir485 inhibits metastasis and emt of lung adenocarcinomaby targeting flot2 biochem biophys res commun su tj ku wh chen hy oncogenic mir137 contributes to cisplatinresistance via repressing casp3 in lung adenocarcinoma am j cancer resliu y wang f xu p mir590 accelerates lung adenocarcinoma migrationand invasion through directly suppressing functional target olfm4 biomedpharmacother yang l luo p song q fei x dnmt1mir200agolm1 signaling pathwayregulates lung adenocarcinoma cells proliferation biomed pharmacother duan x fu z gao l direct interaction between mir203 and zeb2suppresses epithelialmesenchymal transition signaling and reduces lungadenocarcinoma chemoresistance acta biochim biophys sin shanghai yuan y zheng s li q overexpression of mir30a in lungadenocarcinoma a549 cell line inhibits migration and invasion via targetingeya2 acta biochim biophys sin shanghai li cg pu mf li cz microrna1304 suppresses human nonsmall celllung cancer cell growth in vitro by targeting heme oxygenase1 actapharmacol sin li dm sun h ptenmmac1tep1 suppresses the tumorigenicity andinduces g1 cell cycle arrest in human glioblastoma cells proc natl acad sciu s a bellacosa a chan to ahmed nn akt activation by growth factors is amultiplestep process the role of the ph domain oncogene li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep qin y huo z song x chen x tian x wang x mir106a regulates cellproliferation and apoptosis of colon cancer cells through targeting theptenpi3kakt signaling pathway oncol lett publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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"Cowdens syndrome is an autosomal dominant disease with variable penetrance involving the tumorsuppressor phosphatase and tension homolog gene located on chromosome 10q2223 responsible for cellproliferation migration and cellular apoptosis Its clinical presentation encompasses mucocutaneous lesions whichare present around of the time macrocephaly and cognitive impairment and it precedes the appearance ofneoplasms such as thyroid carcinoma breast cancer among othersIn addition to these malformations arteriovenous malformations of the brain and spine endocrine abnormalitiesskeletal defects and cardiopulmonary lesions may also be foundThe relevance of the case is due to the fact that through a certain phenotype the patients genotype can beinferred and thus followed up closelyCase representation The clinical case concerns a 28yearold Caucasian and Portuguese woman with palmar pitsmacrocephaly and cognitive impairment She was diagnosed with papillary thyroid carcinoma at years of ageand proposed total thyroidectomyAt age a pregnancy was diagnosed with a Breast ImagingReporting and Data System 2rated breast lump Afterthe histological verification it was concluded that it was a high metastatic breast sarcoma opting for palliativemastectomy A genetic evaluation confirmed alteration in the phosphatase and tension homolog gene confirmingCowdens syndrome The patient died at age due to neoplastic pathologyConclusion This report aims to alert to the clinical signs of this entity and the clinical supervision and followup ofthese patients In order to prevent premature deaths and to improve patients quality of life genetic diseases withcancer impact should be diagnosed as early as possibleKeywords Cowdens syndrome PTEN Papillary carcinoma Breast sarcomaBackgroundCowdens syndrome CS was first described by Lloydand Dennis in in a 20yearold patient RachelCowden who had multiple deformations such as scrotaltongue syndrome papillomatous papules thyroid adenomas fibrocystic breast disease with malignant degeneration central nervoussystem changes and familymembers with a mild form of the disease [] Correspondence sofiamiguelotehotmailcom1Faculty of Medicine University of Porto Porto PortugalFull list of author information is available at the end of the Only in was the susceptible zone of SC 10q2223mapped [] and the tumor suppressor gene phosphataseand tension homolog PTEN that regulates the PI3KAktmTOR pathway was identified which is responsible forthe proliferation migration and cellular apoptosis []adultLhermitteDuclosPTEN hamartoma tumor syndrome PHTS whichcomprises CSdiseaseLDD BannayanRileyRuvalcaba syndrome BRRSand Proteuslike syndrome represents a spectrumof hamartomatous overgrowth manifestations associated with germline mutations in the PTEN gene[] The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMiguelote World Journal of Surgical Oncology Page of LDD is a rare slowgrowing hamartoma which is usually diagnosed when patients are in their twenties orthirties While it appears to be clearly associated withPTEN mutations the incidence of LDD in patients withCS is unknown Vascular venous or arterial anomaliesare reportedly common in BRRS and CS particularly inpatients with a BRRS diagnosis A number of case reports support the association of arteriovenous malformations in clinically diagnosed BRRS patients []Itis importantto recognize thatin addition toLLD other intracranial findings such as multiple venous anomalies meningiomas greater than expectedwhite matter signal abnormality prominent perivascular spaces and cortical malformations may warrant athorough evaluation for CS in the appropriate clinicalsetting [ ]BRRS is typically diagnosed in childhood and ischaracterized by macrocephaly hamartomas including lipomas or intestinal polyps penile freckling inmales and developmental delaysincluding an increased risk of autism spectrum disorder ASD []Other reported features include developmental delayvascular anomalieslarge birth weight and jointhyperextensibility []CS is an autosomal dominant genodermatosis withvariable expressiveness with over germline mutations in the PTEN gene already described but no significant correlation between mutation type and tumortypes and in of the patients no mutations werefound [] Mucocutaneous lesions including trichilemmoma acral keratosis and oral papillomatosis plusmacrocephaly are the most frequent features describedin more than of cases after the third decade []Multiple extramucocutaneous manifestations can alsooccur in CS The skeletal system may form a higharched palate scoliosis or macrocephaly []The estimated prevalence is with a slightpredominance in females and Caucasians and penetrance of up to in the second decade [] The diagnosis normally is based on clinical criteria periodicallyupdated by the National Comprehensive Cancer Network® NCNN® [] though confirmatory genetic testing is frequently usedCase representation sectionThe case being studied was reported at the S£o Jo£oHospital which is part of the Porto Medical School University of Porto in the North of PortugalPatient female Caucasian born on September having concluded year and unemployed at the timeFamily history mother with oligophrenia and maternalaunt died of thyroid carcinomaPersonal history of childhood hydrocephalus menarche at years with irregular catamenia that have beencorrected after the introduction of oral contraceptive In the patient was directed to psychiatry for suspected cognitive impairmentIn she had severe microcytic and hypochromicanemia and was treated with saccharized ferric oxideGynecological ultrasound without major changes andoccultstool was negative Afterhematological research clinical research von Willebrands disease was excludedin thebloodIn she was admitted to endocrinology due tochanges in thyroid analytical parameters TSH and T4and 5cm thyroid nodule in the left lobe An ultrasoundwas performed which identifies a 56cm hypoechogenicnodule and anecogenic areas the largest with cmSubsequently she performed a fineneedle biopsy andwas diagnosed with follicular lesion of undetermined significance FLUS Bethesda category III After a multidisciplinary meetingit was decided to intervene thepatient with total thyroidectomyIn September she underwent g total thyroidectomy and was diagnosed with papillary carcinomasolidtrabecular variant Fig Concomitantly she performs ablative therapy with MBq mCi of I131and is treated with levothyroxine and calciumIn January research scintigraphy after 24h oralI131 wasadministration of MBq mCi ofFig Solidtrabecular variant of papillary carcinoma Welldefined tumor with trabecular pattern a composed by follicular cells with nuclearfeatures of papillary carcinoma b and expression of TTF1 c 0cMiguelote World Journal of Surgical Oncology Page of Fig a Right breast lump b Radiological image of the breast lesionconducted revealing anterior cervical uptake focus compatible with functioning thyroid tissue and no otherpathological fixation fociAfter year in routine ultrasound microcalcificationswere detected in the jugulocarotid chain possibly relatedto a secondary injury to papillary thyroid carcinomaIn November she was referred to the center ofthe breast by right breast lump in the upper left quadrant At that time she was weeks pregnant and wasbeing followed in obstetrics with a diagnosis of hydramnios fetal hypothyroidism and suspected fetal macrosomia opting for childbirth this weekThe breast ultrasound identified a wellcircumscribed3cm polylobular nodule classified as Breast ImagingReporting and Data System BIRADS2In May the breast ultrasound revealed a 4cmnodular lesion and FNB was performed The result revealed a heterogeneous mass of about cm classifyingas BIRADS4C Mammarycomputed tomographyshowed multilobulated contours with gross calcificationsFig In the histological study of the microbiopsy of thebreast lesion a malignant neoplasm compatible withhighgrade sarcoma with areas of necrosis was observedThe neoplasm consists of spindle cells atypical and withfrequent figures of mitosis The presence of osteosarcoma and rhabdomyosarcoma components is shown inFig It was proposed to the patient to undergo palliativeright total mastectomy and the recommended therapywas doxorubicin and ifosfamide After explaining theprecautions and warnings of the drugs the patient choseto take doxorubicin onlyIn August she performs bone scintigraphy revealing no image suggestive of focal metastatic andortumoral bone pathology with hyperfixation in rightbreast dependence compatible with heterotopic fixationby the breast lesion studied For staging she underwentcomputed tomography where nodules suggestive of metastases were found in the different lobes of both lungfields all with a diameter less than cm Fig Concomitantly she was admitted for a genetic consultation showing clinical macrocephaly cm roundkeratotic palmar pits and wart vulgaris on the face andscalp Fig The genetic evaluation included the genes PTENTP53 BRCA1 BRCA2 ATM CHEK2 and PALB2PTEN gene alteration was found The result coincideswith a change in heterozygote pathogenic variantc405dupA p Cys136Metfs in the PTEN geneFig Highgrade sarcoma of the right breast composed by atypical spindle cells with rhabdomyoblastic a b and osteosarcomac components 0cMiguelote World Journal of Surgical Oncology Page of Fig Computed axial tomography with evidence of metastasis white arrowThis variant leads to the formation of a premature stopcodon and potentially the appearance of a truncatedproteinShe has stepbrothers and stepsister but she has norelationship with them The mother and uncles do notwant to carry out the genetic test and the siblings areunreachable Genetic tests were not performed on thepatients limbs because they do not want to knowIn she died due to extensively metastatic breastcancer Table Since the diagnosis of papillary carcinoma at her death only years have passed which demonstrates that the diagnosis made correctly and the timecould have dictated another pathDiscussion and conclusionCS presents itself with multiple hamartomas and in of cases its initial presentation is mucocutaneous lesionsThese precede by a few years the neoplasms most at riskfor this syndrome such as renal cell neoplasia and thyroidand breast carcinoma [] Table The importance of early diagnosis of this entity is related to the increase risk for certain cancersWhen the clinical phenotype raises suspicion a screening test httpswwwlernerccfgmiccscore can becarried out which takes into account the individuals ageand phenotype In adults a clinical threshold score of ¥ leads to a recommendation for referral to a genetics professional to consider PHTS In children macrocephalyand ¥ of the following leads to the consideration ofPHTSdevelopmentaldelayautism ordermatologic features including lipomas trichilemmomasoral papillomas or penile freckling vascular featuressuch as arteriovenous malformations or hemangiomasand gastrointestinal polyps []The diagnosis is mostly clinical and the diagnostic criteria created by Eng have been developed and arereviewed annually by the National Comprehensive Cancer Network® NCCN® Eng Table Confirmation ofthe diagnosis of CS is confirmedwhen it presents Pilarsky Three or more major criteria but one must includemacrocephaly LDD or gastrointestinalhamartomas or Two major and three minor criteriaWhen there is a family history of CS or PTEN mutation CS is considered in the individual presentingPilarsky a Two major criteria with or without minor criteria orb One major criterion and two minor criteria orc Three minor criteriaThe patient had several characteristic CS findings thatsatisfied three major criteria macrocephaly breast cancer and mucocutaneous lesions and two minor criteriafollicular variant of papillary thyroid cancer and intellectual disability so she could be clinically diagnosedwith CSFig Mucocutaneous lesion on the face and scalp 0cMiguelote World Journal of Surgical Oncology Page of Table Clinical condition summaryANOIdadeDIAGNSTICOOligophreniaTable Diagnostic criteria for Cowden syndromeCriteria minorCriteria majorColon cancerBreast cancerMicrocytic and hypochromic anemiaFollicular thyroid cancerEsophageal glycogenicacanthoses ¥ Lipomas ¥ Thyroid cancer papillary orfollicular variant of papillaryRenal cell carcinomaThyroid structural lesions egadenoma nodules goiterIntellectual disability QI ¤ Autism spectrum disorderTesticular lipomatosisVascular anomalies includingmultiple intracranialdevelopmental venousanomaliesEndometrial cancerMultiple gastrointestinal hamartomasLhermitteDuclos disease adultMacrocephaly P ¥ Macular pigmentation of glans penisMucocutaneous lesions Trichilemmoma ¥ biopsy proven Acral keratoses ¥ palmoplantarkeratotic pits andor acralhyperkeratotic papules Oral papillomas particularlyon tongue and gingiva multiple¥ or biopsyproven ordermatologistdiagnosedmucocutaneous neuromas¥ Papillary thyroid carcinomaBreast sarcomaCowdens syndrome diagnoseSarcoma metastasisDeathNCCN recommends for woman an annual mammography of tomosynthesis starting at age or years before the earliest known breast cancer in the family Given the risk of developing breast cancer thepossibility of total mastectomy can be considered Counseling should include a discussion regarding the degreeof protection reconstruction options and risks Becauseendometrial cancer can often be detected early based onsymptoms women should be educated regarding the importance of prompt reporting and evaluation of any abnormal uterine bleeding or postmenopausal bleedingThe evaluation of these symptoms should include endometrial biopsy every yearsFor both women and men NCCN advocates annualthyroid ultrasound starting at the time of CSPHTS diagnosis including childhood and colonoscopy starting at age years unless symptomatic or if the close relative withcolon cancer before age then start years beforethe earliest known colon cancer in the family Colonoscopyshould be done every years or more frequently if the patient is symptomatic or polyps are found Renal ultrasoundshould be considered starting at age and then every years Dermatological evaluation and treatment may benecessary for some patients Children should have a thorough psychomotor assessment and brain symptomsshould be assessed with magnetic resonance imagingThe differential diagnosis of entities related to PTENhamartoma must be considered Some characteristics ofCS are similar to BRRS such as macrocephaly gastrointestinal hamartomas cognitive impairment and pigmentedmacules on the penis The mutational prevalence in PTENAdapted from httpswwwnccn Copyright2019is around however specific diagnostic criteria for thisentity have not yet been crated Regarding LDD in whichthe prevalence of the affected PTEN is around theclinical findings are fundamentally based on slowly growing hamartoma of the cerebellum and usually diagnosedwhen patients are in their twenties or thirties []When the trio of thyroid breast and renal cell carcinoma arises not only the PTEN gene but also the SDHBCD gene should be screened []The clinical evaluation and followup of these patientsshould be thorough so that the evolution of certain malignancies is detected in a timely manner and their diagnosis and treatment as appropriate as possibleWhen diagnosed patients should be instructed to beaware of the signs and symptoms of cancers with ahigher incidence in this disease Sirolimus is in phase IIfor PHTS and other drugs are being studied for solidneoplasms in individuals with CS that act on the pathway PI3KaktmTOR BGT226 and BEZ235 in phase IITable Neoplasic risk and age of onsetRisk to life TumorBreastBeginning ageAbbreviationsCS Cowdens syndrome PTEN Phosphatase and tension homologLLD LhermitteDuclos disease BRRS BannayanRileyRuvalcaba syndromeBIRADS Breast ImagingReporting and Data SystemThyroidKidney cellsEndometriumGastrointestinalMelanomaAdaptedfrom httpsrarediseasesrarediseasesptenhamartomatumorsyndromeCopyright2019AcknowledgementsNot applicableAuthors contributionsSM was the main contributor to the writing of the manuscript RHperformed the histological examination of the thyroid and breast LFprovided some patient data and images LE analyzed and interpreted thepatient data JAT is the first authors advisor for the elaboration of themasters thesis in medicine The authors read and approved the finalmanuscript 0cMiguelote World Journal of Surgical Oncology Page of FundingNo financial sourcesAvailability of data and materialsThe datasets generated and analyzed during the current study are not publiclyavailable due to belonging to the clinical entity S£o Jo£o University HospitalCenter but are available from the corresponding author on reasonable requestEthics approval and consent to participateAll clinical information whether imaging or histological was provided by thebioethics department of the University Hospital Center of S Jo£o for theelaboration of the masters thesis in medicine The respective authorizationsof the hospital ethics committee are in the supplementConsent for publicationInformed consent was provided by the patients hospitalCompeting interestsThe authors declare that they have no competing interestAuthor details1Faculty of Medicine University of Porto Porto Portugal 2University HospitalCenter of S Jo£o Porto Portugal 3Pathological Anatomy DepartmentUniversity Hospital Center of S Jo£o Porto Portugal 4Breast CenterUniversity Hospital Center of S Jo£o Porto Portugal 5Surgery DepartmentUniversity Hospital Center of S Jo£o Porto PortugalReceived March Accepted July ReferencesYehia L Eng One gene many endocrine and metabolic syndromes PTENopathies and precision medicine Endocrine Related Cancer Nelen MR Padberg GW Peeters EA Lin AY van den Helm B Frants RR CoulonV Goldstein AM Van Reen MM Easton DF Localization of the gene forCowden disease to chromosome 10q2223 Nature Genetics Shenandoah R Alan RC Cowden disease and LhermitteDuclos disease anupdate Case report and review of the literature Neurosurg Focus 2016201E6Ueno Y Enokizono H Ohto T Imagawa K Tanaka M Sakai A Suzuki HUehara T Takenouchi T Kosaki K Takada H A novel missense PTENmutation identified in a patient with macrocephaly and developmentaldelay Human Genome Variation Pilarski R PTEN hamartoma tumor syndrome a clinical overview Division ofHuman Genetics Department of Internal Medicine and ComprehensiveCancer Center Ohio State University Columbus OH USA Dhamija R Weindling SM Porter AB Hu LS Wood CP HoxworthNeuroimaging abnormalities in patients with Cowden syndromeretrospective singlecenter study Neurol Clin Pract Kimura F Ueda A Sato E Akimoto J Kaise H Yamada K Hosonaga M KawaiY Teraoka S Okazaki M Ishikawa T Hereditary breast cancer associated withCowden syndrome related PTEN mutation with LhermitteDuclos diseaseSurgical Case Report Porto AC Roider E Ruzicka T Cowden syndrome report of a case and briefreview of literature An Bras Dermatol NovDec Suppl Cavaill© M PonelleChachuat F Uhrhammer N Viala S GayBellile M Privat MBidet Y Bignon Y Early onset multiple primary tumors in atypical presentation ofCowden syndrome identified by wholeexomesequencing Front Genet Garofola C Gross G Cowden disease multiple hamartoma syndrome NCBIBookshelf Farooq A Walker LJ Bowling J Audisio RA Cowden syndromeCancer Treat Rev National Comprehensive Cancer Network NCCN Cowden syndromePTENhamartoma tumor syndrome Eng C PTEN hamartoma tumor syndrome PHTS GeneReviews Updated Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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" apolipoprotein l1 apol1 is a trypanosome lytic factor present in human and certain other primatesapol1 gene variants present in individuals of recent subsaharan african descent increase risk for glomerulardisease and associate with the disease progression but the molecular mechanisms have not been definedobjectives we focus on the mechanism how apol1 variant proteins enhance podocyte injury in the stressedkidneymethods first we investigated the expression of apol1 protein isoform and the localization of apol1 protein inthe kidney next we examined the role of apol1 in the podocyte stress and the inflammatory signaling in thekidney after heminephrectomyresults we identified a novel rna variant that lacks a secretory pathway signal sequence and we found that thepredicted apol1b3 protein isoform was expressed in human podocytes in vivo and by bacapol1 transgenicmice apol1b3g2 transgenic mice carrying a renal risk variant manifested podocyte injury and increased proil1 mrna in isolated glomeruli and increased il1 production in the remnant kidney after uninephrectomyapol1b3 interacted with nlrp12 a key regulator of tolllike receptor signalings these results suggest a possible mechanism for podocyte injury by which one of the apol1 proteinisoforms apol1b3 and its renal risk variants enhances inflammatory signalingkeywords apol1 protein isoform interleukin1 beta nlrp12 podocytes kidney chronic kidney diseaseinflammatory stresstermed g1 and g2resistance antigen sra which binds apol1 and prevents pore formation the human apol1 coding variantsto the morewidespread g0 isoform elude binding by sra and killt b rhodesiense the g1 variant has two nonsynonymous coding variants s342g and i384m and in theg2 variant n388 and y389 are deletedin contrastintroductionafrican americans have increased rates of chronic kidney disease and a major cause of this health disparityare apol1 genetic variants present exclusively in individuals of african descent apol1 kill trypanosomabrucei likely via formation of pores that serve as ionchannels t b rhodesiense possesses a protein serum correspondence jbkoppnihgov1kidney disease section niddk nih kdb center dr 3n116 bethesdamd usafull list of author information is available at the end of the apol1 gene variants are strongly associated with riskfor focal segmental glomerulosclerosis fsgs hivassociated nephropathy hivan and hypertension attributed kidney disease arterionephrosclerosis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwakashin bmc nephrology page of apol1 risk allele status in deceased donor kidneys affects transplant kidney survival while the apol1 riskallele status of the recipient does not affect transplantkidney survival these data suggest that intrarenalapol1 expression rather than circulating apol1 is associated with kidney disease as most apol1 nephropathies are either podocyte diseasessegmentalglomerulosclerosis hivassociated nephropathy lomerular and vascular diseases arterionephrosclerosisit appears that apol1 risk variant expression inpodocytes and possibly renal vascular cells contributesto kidney diseasefocalchronic inflammation contributes to progression ofglomerular diseases in an experimental model datafrom the neptune study of glomerular disease patientsindicated a role for apol1 variants in enhancing glomerular expression of cxcl9 and cxcl11 chemokinesinduced by proinflammatory cytokines such as tnf andil1 nodlike receptors nlr are pattern recognition receptors that sense intracellular pathogens and hostderived danger signals particular nlrs may amplify orsuppress the inflammatory response nlrp3 is a component of the inflammasome which activates caspase1and in turn produces mature il1 proinflammatorysignaling via both nlrp3 inflammasome and nfκbpathways is involved in the pathogenesis of experimentalglomerular disease [ ] nlrp12 is a negative regulator of the proinflammatory signaling pathway inducedby tolllike receptors tlr and the tumor necrosis factor tnf receptor nlrp12 which contains pyrinnacht and leucinerich repeat domains associates withinterleukin1 receptor associated protein kinase irak1in canonical nfκb signaling and associates withtnf receptorassociated factor traf3 and nfκb inducing kinase in noncanonical nfκb signaling [ ]nlrp12 negatively regulates the nfκb and mapk pathways as shown in an experimental colitis model nlrp12 is also a negative regulator of nfκb signaling intcells and absence of nlrp12 enhances severity of experimental autoimmune encephalomyelitis seven exonsand isexpressed in most ans including the kidney []after cleavage of the secretory signal sequence locatedin exon mature apol1 is secreted and binds to hdlps isoform c lacking a signal sequence ispredicted to be retained within the cell the expressionpattern of specific apol1 splice variants in ans andcells is unknownapol1 mrna compriseswe have examined whether apol1 regulates proinflammatory signaling in glomerular cells we identified anovelintracellular protein isoform apol1b3 andfound that this isoform associated with nlrp12 further apol1b3g2 enhanced podocyte damage inmurine nephropathy this proinflammatory pathwayrepresents a possible therapeutic targetfor apol1nephropathymethodsethics approvalsparaffinembedded autopsy kidney sections were usedfor immunostaining human subjects gave written consent to participate under research protocols approved inadvance by the niddk institutional review boardcell cultureconditionally immortalized human podocytes a generous gift from moin saleem bristol uk were cultured at °c and differentiated at °c with rpmi completemedium with fetal bovine serum insulin transferrin selenium penicillin and streptomycin helacells were cultured at °c with dmem completemedium with fetal bovine serum penicillin andstreptomycinantibodiesapol1 was detected using the mouse monoclonal antibody clone cl0171 rabbit polyclonal antibody fromsigma aldrich hpa st louis mo and rabbitmonoclonal antibody clone rpr2907 from abcam allof which are predicted to recognize isoforms a b and cand by rabbit polyclonal antibody produced at niddkthat is specific for isoform b3 affinity purified with immunizing peptides using sulfolink immobilization kitfor peptides thermofisher carlsbad ca antibody details are provided in the supplementary tablequantitative analysis of p57 staining of mouse kidneycortexformalinfixed paraffinembedded tissue sections werestained with recombinant antip57 kip2 rabbit monoclonal antibody sigma sab5500158 which highlightspodocyte nuclei with dab detection on an automatedstainer lab vision autostainer grand island nysections were counterstained with periodic acid schiffpas podocyte numbers were assessed as follows usingwhole slide scans nanozoomer hamamatsu bridgewater nj a glomeruli were randomly selected in eachcortical profile and p57 positive podocytes were enumerated b using the pas stain as the delimiter of theglomerular area glomerular area was measured usingndpiview2 hammamatsu and c the podocyte number was indexed to the glomerular area μm2 the intensity of podocyte staining was evaluated using thequpath qupathgithubio digitalimage analysis platform which permits quantification of the stained tissueas follows a at least glomeruli were selected in eachcortical region b the software identified the podocytes 0cwakashin bmc nephrology page of and graded the diaminobenzidine dab chromogen intensity for each stained pixel on a continuous scale alonga predefined spectrum considered as positive stain bythe user c multiple thresholds were tested to explore arange of sensitivities and specificities and the finalthresholds were defined based on the highest accuracyto find the region of interest across all the images whichpermits the comparison of the relative intensities ofstains between the images d the average dab intensityfor all pixels inside a podocyte was used to compare thestaining intensity of the p57positive podocytes in theglomerulus across different imagesthe locustransgenic micehuman apol1 gene locustransgenic mice bacapol1 mice were generated using a bacterial artificialchromosome bac which containsforapol1 resulting in apol1g0 g1 and g2 mice a kb human dna encompassing only the apol1gene with ² and ² flanking regions including exons and of apol2 and ² region including exons ofpart of myh9 gene was isolated and subcloned fromhuman bac clone enst00000397278 which corresponds to nm_003661 bac subclone was injected into129svjb6n f1 embryos and the founders were subsequently backcrossed into 129svj the subclone was sequenced and found to be identical to the referencegenome sequences in the ncbi and ensembl databasesexcept for the g1 and g2 polymorphisms for cagapol1b3 transgenic mice linear dna encoding cagpromotorapol1b3 g0 or g2 flagsv40 polya signal was introduced into fvbn mouse embryos by pronuclear injectionmouse nephropathy model experimental procedure weeks old and independent lines of apol1b3g0transgenic mice and lines of apol1b3g2 transgenicmice were studied details in supplementary tables following unilateral nephrectomy random urine was harvested before and at days after the surgery glomeruliwere isolated from the remnant kidney at days after thesurgery and from intact kidney by using dynabeads m tosylactivated invitrogen animals wereeuthanized by exposure to compressed carbon dioxide peran nihapproved animal care and use protocol and peranimal facility standard operating proceduresresultskidney cells expressed apol1 splice variants in vitro andin vivoapol1 has several mrna splice variants predicted toencode diverse protein isoforms it has not been fullyclarified which apol1 splice variants are expressed inthe kidney and which of those contribute to kidney disease first we examined the expression of apol1mrna splice variants in immortalized human podocytesby tacloning we identified six mrna variants fourof them have been described as v1 a v3 a v2b1 and v4 c with the protein isoform precursor inparentheses fig 1a we identified two novel mrnavariants containing exon which we have named v2 b2 and v2 b3 with regard to v2 b2 we isolated an mrna encoding exon to exon and partiallysharing exon to exon with a variant that has beendescribed in ncbi aceview database ncbi aceviewapol1 variant l aug10 further the protein isoformshave predicted variants as shown by the first numberafter the letter v exon is unique to the v2 variantsthe apol1b3 and apol1c protein isoforms are derived from isoform precursors of b3 and c respectivelyand the localization of these proteins are different fromisoform a as they lack a cleavable signal sequencethus apol1 mrna splice variants are predicted toproduce three different proteins apol1a apol1b3and apol1c fig 1bnext we examined the expression of apol1 splice variants in human tissues by rtpcr using caucasian cdnasamples with no disease in kidney we observed foursplice variants v1 v4 fig 1c v2 and v2 whilelung also expressed v2 fig 1d we confirmed thepresence of apol1 v2 in human kidney by pcr usingthe v2 specific primer pair supplementary figure 1aand confirmed the dna sequence of v2 pcr productsupplementary figure 1c we then asked whetherapol1 mrna splicing could also be observed in themouse kidney we generated bacapol1 transgenicmice that carry the human apol1 gene and upstreamnoncoding regions and we examined the expression ofapol1 splicing variants in the kidneys of the mouse asshown in supplementary figure 1b apol1 v1 v2 andv2 mrna splice variants were expressed in the kidneyand by using pcr with the v2 and v2 specificprimers we confirmed the presence of respective dna sequences supplementary figure 1cstatisticsin vivo data were analyzed by the kruskalwallis testwith posttesting of selected pairs of data sets by dunnett multiple comparison test prism graphpad sandiego ca in the figures group medians are shown ap was accepted as significantapol1b3 was expressed in kidney cells but was absentin human serumhuman kidney expressed apol1 v1 v2 and v2the circulating form which ismrnas apol1amainly expressed in and secreted from liver tissueispresent in human serum and is part of the trypanosome 0cwakashin bmc nephrology page of fig kidney tissue expressed apol1 splice variants a b apol1 splice variants were cloned from differentiated immortalized human podocytesby tacloning the exons of each of the six mrna splice variants are shown a as are the three predicted protein isoforms following signalsequence cleavage b exon lengths are not drawn to actual size c d apol1 splice variant expression in human tissues was analyzed by using ahuman cdna library rtpcr was performed with two sets of primers as shown by arrows in the above exon schema apol1 splice variant tavectors were used for pcr positive controls the major splice variant v1 encoding isoform a is seen in all examined tissues c human lung andkidney expressed the apol1 splice variant v2 d lower band encoding protein isoform b3lytic factor howeverlocalization and function ofapol1b3 were unknown to investigatecellularlocalization of apol1b isoforms in kidney tissue wetherefore produced a polyclonal rabbit antibody againsta amino acid sequence mrfkshtvelrrpcsd thatis encoded by exon and exon is specific to apolbisoforms and is absent from isoforms apol1a andapol1c apol1b antibodies were affinity purifiedfrom the serum of immunized rabbits we then characterized the specificity of apol1b antibody by probingit against the immunizing peptides and apol1b protein isoforms for that we transfected human podocyteswith 3x flagtagged flagtagged expression constructs encoding b1 b2 and b3 isoforms apol1protein isoforms were then immunoprecipitated withantiflag antibodies and analyzed by immunoblottingusing apol1b and mouse antiapol1 cterminalantibodies clone cl0171 a signal for apol1b wasnot observed when the apol1b antibody was first preincubated with immunizing peptides this demonstratedthat the apol1b antibody was specific for the immunizing peptides supplementary figure 2a top all threeproteins were recognized by the antiapol1 cterminalantibody but only apol1b3 was detected by bothapol1b and apol1c antibodiessupplementaryfigure 2a second from top this suggests that apol1b1 and apol1b2 were cleaved probably in the signalsequence encoded by exon and consequently lost exon 0cwakashin bmc nephrology page of apol1b3 lacked exon and therefore retained exon thus we concluded that the apol1b antibody specifically recognized apol1b3we examined the cellular localization of apol1b3in vivo using apol1b and mouse antiapol1 cterminal antibodies cl0171 in human serum apol1was not detected by the apol1b antibody supplementary figure 2b but was detected with an apol1 antibody thatis predicted to recognize the cterminalregion of apol1 a b and c protein isoforms supplementary figure 2c apol1 was originally identified inhuman serum as two species polypeptides with apparentmolecular weights of kda and kda aminoacid sequence analysis had shown thatthe kdaapol1 was the result of cleavage at exon which encodes for the apol1 signal peptide the kda apol1species was amino acids shorter at the n terminusthan the kda form and glycosylated the lowerapol1b3 protein band was not recognized by apol1b antibody supplementary figure 2a suggesting it wascleaved similar to isoform a the biological significanceof this species is undeterminednext we examined the localization of apol1b3 inkidney tissue we first selected wildtype mouse andbacapol1 transgenic mouse kidneys for these studiessince mice do not express apol1 and thus wildtypemice can be used as a negative control bacapol1g0mouse kidneys which expressed v2 mrna weresubjected to immunohistochemical analysis using therabbit polyclonal apol1b antibody and the rabbit antiapol1 monoclonal antibody clone epr2907 in bacapol1 mouse kidney we found apol1 immunoreactive signalin podocytes and tubular cells using themonoclonal apol1 antibody epr2907 supplementaryfigure 2d and also with the polyclonal apol1b antibody supplementary figure 2e suggesting that bothpodocytes and tubular cells expressed apol1b3 theseantibodies did not stain glomeruli in wildtype mousekidney fig 2d e the polyclonal apol1b antibodyproduced staining of tubular cells in bothwild type and transgenic mice next we examined theexpression of apol1 in autopsy samples from humankidneys similar to the pattern seen in the bacapol1mouse kidney in human kidney both the monoclonalapol1 antibody epr2907 fig 2a and the polyclonalapol1b antibody fig 2b stained the cytoplasm ofpodocyte and tubular cells while the antibody againstthe podocyte marker wt1 stained podocyte nucleirabbit igg was used as negative control fig 2c themonoclonal apol1 antibody epr2907 stained vascularwallin the autopsy kidney in summary our resultsgathered from kidneys of transgenic mice and humansindicated that podocyte and tubular cells expressedapol1b3 in vivoapol1 mrna expression was enhanced by il1it has been shown that apol1 mrna expression increases upon proinflammatory stimulation [ ]we examined the expression of apol1 mrna variantsin human podocytes that were differentiated for threedays quantitatively using v2 and v1 specific primerswe observed that expression of both v2 and v1 variants was increased following il1 stimulation fig this was consistent with a previous report [ ]however the role of apol1 in the regulation of inflammatory signaling is unknownexpressedflagtagged apol1b3g0cagapol1b3 transgenic mice expressed apol1b3 inpodocytes and tubular cellsfollowing the results shown in fig we asked whetherthe apol1b3 renal risk variant induces podocyte damage by promoting inflammatory stress in vivo for thispurpose we generated cagapol1b3 transgenic micewhichorapol1b3g2 expression of apol1b3 protein in themouse kidney was confirmed by western analysisapol1b3 protein from whole kidney was immunoprecipitated with antiflag antibody and characterizedusing apol1b and antiflag antibodies showing thatapol1b3 was expressed in the transgenic mouse kidney fig 4a in the immunoblot the lower flag signalband corresponded to truncated apol1b3 as discussedaboveantiapol1 monoclonalwe examined the cellular localization of apol1b3 inmouse kidney sections by immunohistochemistry usingrabbitcloneepr2907 immunoreactive signal was present in thepodocytes and tubular cells of apol1b3g0 and g2expressing mice fig 4b there were no remarkablepathologicalfindings in the kidneys from 8weekoldapol1b3 transgenic mice as judged by light microscopy supplementary figure antibodyapol1b3g2 transgenic mice manifested enhancedalbuminuria and il1 production followinguninephrectomywe hypothesized that apol1b3g2 might be involvedin podocyte damage we evaluated podocyte damageand glomerular proil1 mrna levelin apol1b3mice after uninephrectomy three days following uninephrectomy acr was significantly higher in apol1b3g2 mice compared to wildtype and apol1b3g0 micefig 5a to elucidate mechanisms of podocyte injurywe isolated glomeruli from the remnant kidney and bonemarrow cells as a positive control glomerular apol1mrna levels did not differ among mouse lines supplementary figure 4aexpressednephrin that was as expected absent in isolated bonemarrow cells and both glomerular[add] cells and boneisolated glomeruli 0cwakashin bmc nephrology page of fig human kidney cells expressed apol1b3 in vivo ac to examine the presence of apol1 isoforms in human kidney human autopsy kidneyspecimens were used for immunohistochemical analysis using apol1b antibody and rabbit monoclonal antibody rpr2907 podocytesexpressed apol1 as shown by staining with rpr2907 antibody a and with apol1b antibody recognizing apol1b3 b control rabbit igg didnot stain podocytes c wilms tumor1 wt1 was used as a podocyte marker all glomeruli were derived from identical kidney scale bar indicates μm in regular and μm in high magnification images arrow and arrowhead indicate podocytes and tubular cell respectively 0cwakashin bmc nephrology page of fig apol1b3 regulated proinflammatory signaling in vitro immortalized human podocytes were stimulated by il1 ng ml h theexpression of the apol1 splice variant was examined by qpcr using v2 primers and v1 primers as indicated by arrows in the above exonschema the graph shows the relative expression of il1 to gapdh students t test p fig cagapol1b3 transgenic mice expressed apol1b3 in podocytes and tubular cells a cag chicken Ãactin promoter apol1b3flagtransgenic mice were generated in a fvbn mouse to confirm the expression of apol1b3 in the kidney extracts from wholekidneys were subjected to western analysis using indicated antibodies showing that apol1b3 was expressed b localization of apol1b3 in thekidney was determined by immunohistochemistry using rabbit monoclonal antibody epr2907 podocalyxin was used as a podocyte markerscale bar shows μm 0cwakashin bmc nephrology page of fig apol1b3g2 enhanced podocyte damage and il1 production in vivo a three days after uninephrectomy the urine acr was increasedin apol1b3g2 mice compared to wildtype mice and apol1b3g0 mice b following uninephrectomy proil1 mrna from isolatedglomeruli was upregulated in apol1b3g2 but not in apol1b3g0 mice data shown as expression of proil1 relative to actin c il1 levelsin the urine standardized by urine creatinine cr concentration were found to be significantly higher in apol1b3g2 transgenic mice comparedto baseline data shown as il1 pgmlcr mgdl bars are shown median asterisk shows p marrow cells expressed tlr4 mrna supplementaryfigure 4b tlr4 signaling has been associated with proteinuria and podocyte tlr4 signaling contributesto podocyte injury in experimental animal models further podocyte nfκb pathway activation has beenassociated with proteinuria in a glomerulonephritismodel [ ] glomerular expression of tlr4 mrnawas significantly upregulated in apol1b3g2 mice following uninephrectomy supplementary figure 4c andproil1 mrna levels were higher at day in apol1b3g2 mice than in the other groups fig 5b moreover il1 in the urine was increased following uninephrectomy and the change reached significance inapol1b3g2 mice suggesting enhancement of il1processing in the kidney fig 5c these data indicatedthat the uninephrectomy was associated with inductionof tlr4 signaling proil1 mrna expression and il1 production in the remnant kidney possibly involvingan activation of the inflammasomewe further investigated the effects of apol1b3g0and apol1b3g2 expression on podocytes in kidneysections of mice prior to and following uninephrectomywe quantitated podocyte numbers with p57 stainingand determined p57 staining intensity as a surrogate forpodocyte injury supplementary figure 4d i we observed that the number of podocytes differed little whencomparing pre and postuninephrectomy kidney samples for wildtype fvb mice and apolb3g0 micesupplementary figure 4e in contrast for the risk variant apol1b3g2 mice podocyte number decreased by following nephrectomya difference whichreached statistical significance p we next measured glomerular areas supplementary figure 4f inapol1b3 variant samples glomerular areas changedlittle and without reaching statistical significance incontrast in wildtype samples there was a increasein glomerular area p however the ratio of thepodocyte number per glomerular area did not changesignificantly for any group supplementary figure 4gfinally we investigated p57 staining intensities as a surrogate for podocytespecific changes in cellular statussuch as observed during podocyte injury 0cwakashin bmc nephrology page of supplementary figure 4h i when comparing preand postuninephrectomy samples mean p57 stainingintensities declined both per glomerular area and per podocyte only for the apol1b3g2risk variant p differences were not significantfor p57 staining intensities when calculated per glomerulus for wildtype and apol1b3g0 mice howeverp57 intensity values increased for both wildtype andapol1b3g0 mice and respectively significantly when evaluated per podocyte p andp respectively this observation that podocytenumbers and podocyte p57 expression were lower inapol1b3g2 mice following uninephrectomy is consistent with the presence of greater microalbuminuriasuggesting increased podocyte injuryin summaryapol1b3g2 expression aggravated podocyte damageand enhanced the proinflammatory response in glomerular cells after uninephrectomyapol1b3 associated with nlrp12we wished to determine whether apol1b3 modulatesproinflammatory signaling via either the il1 receptor ortlr4 we investigated apol1b3 binding partners byusing mass spectrometry analysis of proteins that coprecipitated with apol1b3flag from apol1b3flagstably transfected hela cells we identified a 12aminoacid sequence tvvmqgaagigk specific to nlrp12suggesting an interaction between apol1b3 andnlrp12 to identify apol1 isoformsthat bindnlrp12 we cotransfected nlrp12 and flagtaggedapol1 isoforms into hela cells and immunoprecipitated apol1 with antiflag antibodies transfectednlrp12 was coprecipitated with apol1b3flag andapol1cflag but not with apol1b1flag fig or apol1aflag supplementary figure 5a thesedata indicated that nlrp12 specifically associated withthe predicted intracellular forms of apol1 namely b3and c but not the secreted forms of apol1 namely b1or a in concordance with the notion that presence orabsence of a signal peptide determines the localizationof apol1 isoforms and hence their cellular interactionsnext we examined the interaction of apol1b3 renalrisk variants with nlrp12 all wildtype and renal riskvariants of apol1b3 associated with nlrp12 as shownin pulldown experiments supplementary figure 5bhowever the reason for the differences in the observeddegree of coprecipitation of nlrp12 by the apol1variants remains to be determinedfinally we examined which domains of apol1b3were responsible for the interaction with nlrp12 wefig apol1b3 and c isoforms uniquely interacted with nlrp12 shown are the structure of flagtagged apol1 isoforms expression vectorsalso shown is the design of the nlrp12 expression vector highlighting the three major domains pyrin domain pyd nacht domain and leucinerich repeat lrr of this protein the constructs were transiently transfected into hela cells apol1b3 and apol1c isoforms coimmunoprecipitated with nlrp12 while the secreted apol1b1 isoform did not apol1b3 interacted with nlrp12 but not apol1b1 which isidentical to apol1a in its amino acid sequence 0cwakashin bmc nephrology page of made apol1b3g0 constructs in which the cterminalsra binding domain cdel orthe membraneaddressing domain mdel was deleted nlrp12 wascoprecipitated with fulllength apol1 and both domain deleted apol1b3 polypeptides the cterminaldomain seemed to be dispensable for association withnlrp12 supplementary figure 5cis not clearwhether apol1b3 and nlrp12 interacted directly itremains to be shown how renal risk variants located inthe cterminal affect binding to and to inhibition ofnlrp12ittoemptyapol1b3 regulated nlrp12 functionwe addressed the consequences of apol1b3 association with nlrp12 in hela cells stably transfected withempty vector apol1b3g0 or apol1b3g2 constructs we examined the effect of apol1b3enhancedil1 receptor signaling in the setting of nlrp12 overexpression achieved by transient transfection of hela cellscompared to control hela cells apol1b3 transfectedcells g0 and g2 both showed enhanced erk phosphorylation at min after stimulation with il1 comparedoverexpressiondownregulated phosphorylation of erk in each groupempty vector apol1b3g0 and apol1b3g2 stablytransfectedhela cells as expected for a negative regulator of the immune process supplementary figure these datatransfectednlrp12 attenuated il1 receptor signaling and thatapol1b3 g0 and g2 did not overcome this inhibitory function at supraphysiologic levels of nlrp12 inthese data the effect of overexpressed apol1b3 onsignaling was similar for the g0 and g2 variants in helacellsvector nlrp12indicated thatexogenousour data suggest that overexpressed apol1b3 g0and g2 functionally antagonized endogenous nlrp12in the early phase of il1 receptor activation in vitrohow apol1b3g2 and nlrp12 are functionally linkedin the kidney remains unclear as we did not investigatethe consequences of overexpression of nlrp12 in thekidney of apol1b3 transgenic micediscussionwe report an apol1 isoform apol1b3 that regulatesproinflammatory signaling and associates with nlrp12a tolllike receptor signaling regulator as previously reported apol1 has multiple mrna splice variants and we confirmed the expression of these variants in immortalized human podocytes and in human and bacapol1 mouse kidney we identified a novel mrnavariant apol1v2 that lacks the signal sequencewhich suggests that apol1b3 is a cytoplasmic proteinand not secreted as is the case for the apol1a isoform khatua reported that immortalized humanpodocytes express apol1 mrna which include splicevariants thatlack exon and which correspond toapol1b3 and apol1c kidney transplant studies suggest that kidney injury isthe result of kidneyexpressed apol1 and not that ofcirculating apol1 [ ] kidneyexpressed apol1mrna v1 a v2 b1 v2 b3 and v4 c arepredicted to produce apol1 isoform a b3 and c priorreports [ ] on podocyte apol1 expression haveused antibodies that were not specific for individualapol1 splice isoforms a b3 and c using anapol1b3 specific antibody we showed that apol1b3is present in human podocytes human and bacapol1mouse kidneywe demonstrated that transgenic apol1b3g2 expression enhanced proinflammatory response in theglomeruli and albuminuria following uninephrectomynot only in apol1b3g2 glomeruli but also in wildtype and apol1b3g0 mice glomeruli tlr4 mrnawas numerically upregulated after uninephrectomy butproil1 mrna was enhanced only in apol1b3g2this suggest that apol1b3g2 might interfere withnegative regulation of tlr4 signaling the resulting prolonged tlr4 signaling and cellular inflammation wouldpromote progressive podocyte dysfunction and fibrosisthis agrees with the observation that apol1 gene variants are associated with glomerular disease which tendsto manifest in a more rapid progression to endstagekidney disease tlr4 signaling has been implicatedin the pathogenesis of glomerular diseaseincludingcryoglobulinemia in mice and diabetic nephropathyin humans data from the neptune study ofglomerular disease supports a role for apol1 variantsin enhancing inflammatory signaling thus two of thethree glomerular transcripts that are upregulated inapol1 highrisk genotype subjects compared to lowrisk subjectschemokinescxcl9 and cxcl11 which are enhanced by tnfil1 and tlr signaling [ ]the proinflammatoryareit is wellestablished that nephrectomy activates thereninangiotensin system in addition studies have shownthat angiotensin ii induces tlr4 expression []here we showed that glomerular proil1 mrna andurine il1 levels were increased in apol1b3g2 miceapol1b3g2 promoted podocyte damage followinguninephrectomy possibly by increasing glomerular synthesis and processing of proil1 however we have notevaluated the contribution of podocytederived il1 inpodocyte damage in this settingstudyin thisconstitutivelyexpressedtransgenicapol1b3 did not induce glomerulosclerosis at steadystate however doxycyclineinduced renal riskapol1overexpression in podocytes caused severe podocytedamage glomerulosclerosis and resulted in enhanced 0cwaka
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"We used a total of 10 samples for each round of the scheme as has been suggested as the adequate number of cases for proficiency testing in this area of pathology (Thunnissen et al 2011). Overall the results of the EQA suggest that there is more work to be done if laboratories are to ensure that the quality of their EGFR mutation testing meets an acceptable standard as only 72 out of 91 (72%) of the laboratories passed the EQA and is in line with the findings of other similar EQA schemes (Deans et al 2013). The threshold that was used (?18 points) is comparable with that of the majority of other EQA schemes in this field (van Krieken et al 2013). We weighted the marking of the errors' type differently depending on its implications for the patient. Errors resulting in no diagnosis (analytical failures) or no change in diagnosis (different mutation) were marked more leniently (deduction of 1.00 mark) when compared with those that resulted in the wrong diagnosis for the patient (false-negative or false-positive results; deduction of 2.00 marks). Given the potentially significant clinical consequences for the patient of any error we propose to change the threshold criteria to >18 points (Normanno et al 2013) in future schemes to ensure that any significant errors are picked up in the performance data. The materials we used in the scheme allowed us to control for some of the causes of variation seen in other EQA schemes such as influence of fixative on DNA quality (Bellon et al 2011). However our results show that not all laboratories are able to produce EGFR mutation test results to a high standard. At least 1 in 10 samples is genotyped incorrectly in >19% of laboratories (round 2) although there is evidence to show that this does improve with continued participation (round 3). In the first round proof of principle EQA we identified five laboratories that made systematic errors in their pre or post-analytical processes. The way the sample blocks were labelled may have contributed to these errors but it is impossible to identify the true root cause from our data. However the outcome of these errors was that the correct results were reported for the wrong patients indicating a failure of their quality management system. False-negative and false-positive results were the main sources of genotyping error in the scheme. Both these results are extremely harmful for NSCLC patients. In fact a negative result will lead to treatment of an EGFR-mutant patient with chemotherapy as first line of therapy which is less effective as compared with EGFR TKIs in this subgroup of patients (Mok et al 2009). On the other hand false-positive findings will lead to treatment of EGFR wild-type patients with EGFR TKIs that have been shown to be detrimental as first-line treatment in this subgroup of patients who benefit more from first-line chemotherapy (Mok et al 2009; Fukuoka et al 2011). False-negative results accounted for 85% of all the genotype errors made in the scheme and might be due to the low sensitivity of the method used for mutational analysis. PCR/sequencing was the most common method used in the scheme for scanning to detect point mutations. The major disadvantage of sequencing is that it is not very sensitive (Angulo et al 2010) especially in samples with low tumour cell content. Real-time allele-specific tests such as Qiagen's Therascreen EGFR kits are much more sensitive and specific but only test for a subset of common mutations (Lopez-Rios et al 2013). However it is difficult for us to draw strong s from this scheme about the errors made using the different technologies due to the lack of detailed data provided by the labs on exactly which methods were used for each sample except for samples B2/C3 and B8/C10 that gave a disproportionately high error rate compared with the other samples used. We hypothesised that our estimates of mutational allelic frequency in the samples used in rounds 2 and 3 were inaccurate possibly due to EGFR gene copy number variation. We therefore undertook further quantitative validation on these samples using ddPCR to establish the true allelic frequency (Hindson et al 2011). This innovative approach enabled us to establish that in three of the samples the true value was higher than expected and for the other two samples lower than expected. However crucially for two samples (B2/C3 and B8/C10) the value established by ddPCR is very close to the expected minimum level of methodological sensitivity (for example 15% for Sanger sequencing and 5.43% for the p.(G719S) mutation as defined in version 1 of the Qiagen Therascreen kit packaging insert). We speculate that latent problems with the pre-analytical processes used by these labs (for example poor recovery of DNA inaccurate DNA quantification) resulted in false-negative results due to suboptimal analytical conditions. These include insufficient method validation misinterpretation of raw data lack of awareness of assay limitations sample contamination and poor in-house assay design. Nevertheless all of the mutations were identified by the validating laboratories using a range of different methodologies used by the participant laboratories. These findings confirm that every laboratory should be undertaking an appropriate test validation or verification to define the limits of detection and measurement uncertainty of the techniques they are using. This is a requirement for all labs that are accredited to the ISO 15189:2012 standard (International anisation for Standardization (ISO) 2010). A fundamental aspect of all diagnostic testing is the accurate reporting of the results. The quality of reports submitted was acceptable with a large proportion being comprehensive stand-alone documents containing most of the basic core elements. However the report is meaningless if the referring clinician cannot easily extract the relevant information. Therefore it is essential that that the report is clear concise and easy to read. Many of the reports obscured the take home message and there was often a lack of clarity and balance between the test information and the clinical context. Standardisation of the reporting and naming of mutations is also important and we assessed labs against the nomenclature guidelines from the Human Genome"
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cancer is the second leading cause of death in the united states cancer screenings candetect precancerous cells and allow for earlier diagnosis and treatment our purpose was tobetter understand risk factors for cancer screenings and assess the effect of cancer screenings on changes of cardiovascular health cvh measures before and after cancer screenings among patientsmethodswe used the guideline advantage tgaamerican heart association ambulatory qualityclinical data registry of electronic health record data n patients to investigateassociations between timeseries cvh measures and receipt of breast cervical and coloncancer screenings long shortterm memory lstm neural networks was employed to predict receipt of cancer screenings we also compared the distributions of cvh factorsbetween patients who received cancer screenings and those who did not finally we examined and quantified changes in cvh measures among the screened and nonscreenedgroupsresultsmodel performance was evaluated by the area under the receiver operator curve aurocthe average auroc of curves was for breast for cervical and for coloncancer screening distribution comparison found that screened patients had a higher prevalence of poor cvh categories cvh submetrics were improved for patients after cancerscreeningsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation guo a drake bf khan ym langabeer iijr foraker re timeseries cardiovascularrisk factors and receipt of screening for breastcervical and colon cancer the guidelineadvantage one e0236836 101371 pone0236836editor antonio palazo´nbru universidad miguelhernandez de elche spainreceived april accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236836copyright guo this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement the data are ownedby a third party and the authors do not havepermission to share the data requesting access tothe guideline advantage tga data must be done one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsby contacting the american heart association viaemail qualityresearchheart the python coderelated to the analyses can be found in githubrepository githubcomaixiaguopythoncodefunding the authors received no specificfunding for this workcompeting interests the authors have declaredthat no competing interests existdeep learning algorithm could be used to investigate the associations between timeseriescvh measures and cancer screenings in an ambulatory population patients with moreadverse cvh profiles tend to be screened for cancers and cancer screening may alsoprompt favorable changes in cvh cancer screenings may increase patient cvh healththus potentially decreasing burden of disease and costs for the health system eg cardiovascular diseases and cancersintroductioncancer is the second leading cause of death for both men and women in the united statesus breast cancer is the second leading cause of cancer death among women colorectal cancer ranks second among men and third among women while cervical cancer ranksas a major cause of cancer death among women regular cancer screenings for breast cervical and colorectal cancers can help to diagnose cancers early and reduce cancer deaths for example in the past years the number of deaths caused by cervical cancer has significantly decreased thanks to pap tests which can find abnormal cervical cells before they turn tocancer similarly colonoscopy removes noncancerous colon polyps before becomingmalignant and regular mammography screening can identify breast cancer in an earliermore treatable stage thus breast cancer screening bcs cervical cancer screening cecsand colorectal cancer screening cocs are very important for early detection and treatmentfactors associated with cancer screenings include demographic factors health insurancecoverage education level smoking status obesity and cholesterol testing for example receiptof mammography is associated with modifiable factors such as weight smoking and other lifestyle factors [] receipt of cecs is associated with healthier weight lower cardiovascular disease occurrence and lower cholesterol some studies suggest that smokingsedentary lifestyle high body mass index and high comorbidity are associated with a higherpercentage of cocs participation [] traditionally data for such studies originate fromquestionnaires claims data and telephone surveys and statistical analysis methods such aslogistic regression models are applied to examine the associations between the risk factors andcancer screenings electronic health records ehr contain longitudinal healthcare information and data including diagnoses medications procedures lab tests and images andtherefore can be used to discover new patterns and relationships from the rich data deeplearning algorithms have been widely and successfully used in bioinformatics and healthcarefields as they can effectively capture features and patterns in longitudinal data in this study we investigated associations between longitudinal cvh risk factors and thereceipt of cancer screenings using ehr data by the long shortterm memory lstm model we then studied the distribution of cvh factors between patients who did and did notreceive cancer screenings to further investigate the associations finally we compared measures of cvh longitudinally within those who did and did not receive screening to betterunderstand the effect of cancer screenings on cvh measuresmaterials and methodsethics statementall the data were fully anonymized before we accessed them our study was approved by theinstitutional review board at the washington university school of medicine in st louis we one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsobtained a written acknowledgement of proprietary rights and nondisclosure and data useagreement from the american heart association the washington university_nda_dua_contractid 158065_20190426_kdata source and study populationthe guideline advantage tga is a clinical data registry established in by the americancancer society the american diabetes association and the american heart associationaha ehr data has been collected from over clinics across the us by the tga totrack and monitor disease management and outpatient preventative care we used longitudinal tga data to predict three types of cancer screenings among unique patientswe used a 6year range to identify female patients in the year oldage group who received bcs female patients in the year old age group who receivedcecs and patients in the year old age group who received cocs if patientsreceived multiple types of cancer screening we only considered the first using the same criteria for gender and age we randomly selected a comparison group of patients who did notreceive cancer screenings for bcs for cecs and for cocswe utilized the following cvh measures defined by the aha smoking status body massindex bmi blood pressure bp hemoglobin a1c a1c and cholesterol lowdensitylipoprotein ldl in our dataset we then classified them into three categories ideal intermediate or poor according to table we utilized the multum drug database as a template to convert the drug names in our dataset to their corresponding drug classes thelevenshtein distance algorithm was employed for the conversion by comparing the drugnames in our dataset to the multum drug database template the conversion was consideredsuccessful and medications were considered as treatments for bp a1c or ldl table if thedistance between the two compared strings was less than five all cvh measurements prior tothe date of cancer screening were considered in the analysis for those who received screeningand all cvh measurements in the data set were considered in the analysis for those who didnot receive screeningfor the primary analysis we selected patients who had at least one measure of cvh for bcs for cecs and for cocs in the comparison groups there were availabledata for bcs cecs and cocsstatistical analysiswe first studied the lstm prediction of cancer screening from timeseries cvh factors wedivided each cvh factor into its submetric of ideal intermediate or poor according totable for example if a patient had a measure of ideal blood pressure then that featuretable measures of cvh which are available in the tga adapted from lloydjones poor healthintermediate healthideal healthhealth behaviorssmoking statusyesformer � monthsbody mass index� kgm2 kgm2health factorsnever or quit months kgm2ldl� mgdl mgdl or treated to goal mgdlblood pressurefasting plasmaglucosesystolic � mm hg or diastolic � mmhgsystolic mm hg or diastolic mm hg or treated togoalsystolic mm hgdiastolic mm hg� mgdl mgdl or treated to goal mgdl101371 pone0236836t001 one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningswas called blood pressure ideal all features were then embedded to a 32dimensional vectorspace by word2vec for each type of cancer screenings the python genism word2vecmodel used the following hyperparameters size embedding dimension was window themaximum distance between a target word and all words around it was min_count theminimum number of words counted when training the model was sg the training algorithm was cbow the continuous bag of words time information for each measure wasadded and was calculated by the difference in days between each visit date and the most recentvisit date thus each feature was associated with its own time point in the unit of daysthe resulting embedded vectors and associated time points were fed to the lstm modeldue to the comparison group being much larger than the number of patients with cancerscreening we randomly selected patients for bcs patients for cecs and patientsfor cocs and repeated this process for times to account for the imbalance betweenscreened and unscreened groups each time the data set for each type of cancer screening wassplit into a training data set and a test data set we trained the lstm model onthe training data and tested the trained model on the test data we utilized the average of thearea under the receiver operator curve auroc to evaluate the performance of our lstmmodel for each type of cancer evaluatedour lstm model comprised an input layer one hidden layer with dimensions andan output layer the hyperparameter used in the model was as follows a sigmoid function wasused as the activation function in the output layer a binary crossentropy was used as the lossfunction adam optimizer was used to optimize the model with a minibatch size of sampleswe then investigated whether distributions of cvhcounts and percentages for each submetricdiffered between patients who did and who did not receive cancer screenings by chisquared test finally we studied changes in cvh factors within screening group for the samepatients who received screening and for those who did not within screening group we compared cvh measures from before and on the day of the screening to the cvh measures collected after the screening for the patients who did not receive screening we compared cvhmeasures before and after the midpoint of the visit dates if patients only had a single visitthen they were not included in the before and after analysis analyses were conducted by usingthe libraries of scikitlearn scipy matplotlib with python version in resultsthe majority of our study population was white with a mean of age of approximately yearsfor bcs years for cecs and years for cocs table the nonwhite study populationwas predominantly africanamerican the average number of measures avg amongpatients who received screening was higher than that of patients who were not screened forexample the average number of bp measurements for patients with bcs was for cecsand for cocs compared to for bcs for cecs and for cocs for patients who werenot screenedfig displays the performance of lstm cancer screening predictions in terms of repeated aurocs for each type of screening the average auroc of curves was forbcs for cecs and for cocstable lists the numbers and proportions of patients in ideal intermediate and poor categories for each submetric for the comparison between patients who received cancer screeningand those who did not we applied a chisquared test to check if the frequencies herepercentages between screening groups were significantly different from one other within eachcvh submetric as shown in table patients who received cancer screening had a higher one 101371 pone0236836 august one 0ctable characteristics [mean sd or n ] of the study population by receipt of cancer screeningcardiovascular risk factors and receipt of cancer screeningscancer screeningsbcsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cecsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cocsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n n n n yesnon � n n avg avg avg avg avg avg � the percentages may not add up to due to rounding101371 pone0236836t002prevalence of poor a1c for bcs for cecs and for cocs compared topatients who did not receive screening for bcs for cecs and for cocsfig shows changes in cvh submetrics within the same patient screening groups fig a2c show the changes in cvh submetrics for the patients who were screened while fig2e and 2f show the changes in cvh for patients who were not screened one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the area under the curve auc are shown for lstm cancer screening predictions from timeseries cvh factors which were repeated times withdifferent comparison patients for bcs a cecs b and cocs c101371 pone0236836g001from the first column of fig we can see that the prevalence of poor submetricsdecreased after cancer screenings for example all five submetrics improved after bcs fig a while bp and a1c improved after cecs fig 2b and bp a1c and smoking improvedafter cocs fig 2c notably for the prevalence of poor a1c decreased for all patients whoreceived cancer screenings in bcs in cecs and in cocs on the other handfrom the second column of fig we can see that the prevalence of poor a1c increased forall comparison patientsdiscussionin this study we demonstrated associations between timeseries cvh risk factor measuresand receipt of three types of cancer screenings ie breast cervical and colon cancer screenings by using a nationally representative datasettga data the tga data enabled us toexamine multiple sites cvh submetrics and types of cancer screenings using advanced deeplearning models an advantage of our study was that all cvh submetrics were investigatedsimultaneously for an association with different cancer screenings on a unique nationallyrepresentative dataset of patients ie the large tga data set which contains longitudinaltable comparison cvh factors between patients with cancer screening or without [n ]patients with bcs n chisquared pvalueidealintermediatepoorpatients without bcs n idealintermediatepoorpatients with cecs n chisquared pvalueidealintermediatepoorpatients without cecs n idealintermediatepoorpatients with cocs n chisquared pvalueidealintermediatepoorpatients without cocs n idealintermediatepoorbmi bmi bmi 101371 pone0236836t003bp bp bp a1c a1c a1c ldl ldl ldl smoking smoking smoking one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the plots of percentages for poor cvh factors for the same patients before and after time points of cancer screening for patients with screeningsac and before and after middle time points for patients without cancer screenings df the first row is for bcs second row is for cecs andthe third is for cocs101371 pone0236836g002cvh measurements and cancer screening patterns from more than different clinics in theusthe comparison of different cvh measure distributions between patients who receivedcancer screenings and those who did not showed that patients with poorer cvh especiallypoor a1c were more likely to receive cancer screenings specifically patients with poorera1c were more likely to receive cancer screenings some recent studies have showed that individuals with diabetes had higher incidence of certain cancers and also were more likely tobe diagnosed with advancedstage tumors [] thus providers might be more likely torecommend patients with diabetes to uptake cancer screenings for early prevention of developing cancers which may lead to more individuals with diabetes to participate in cancerscreeningsmoreover we investigated the effects of cancer screenings on the changes of cvh measuresof the patients to better understand if the screenings had potential associations with theimprovement of cvh measures our results indicated that patients who received cancerscreenings appeared to have better control of cvh factors especially a1c than patients whodid not receive cancer screenings specifically a1c levels were improved after patientsreceived any type of screening while a1c levels worsened among patients who did not receivecancer screening a similar trend could be observed for bmi it became better after patientsreceived any type of screening while bmi became worse among patients without bcs orcocs levels of bp were improved after patients received bcs or cocs screenings and worsened among patients without bcs or cocs poor levels of ldl decreased among patientsafter receipt of bcs and among those without bcs however ldl improvements were muchgreater among patients after receipt of bcs decrease in ldl than those without bcs decrease in ldl after receipt of bcs and cocs current smoking declined comparedto the increase observed among those without the screeningsin summary our analyses showed that patients with poor cvh measures were more likelyto receive cancer screenings patients with receipt of cancer screenings appeared to haveimproved cvh measures after the screening as compared to before one possible reason forthis was that patients might receive more attention and through care from providers to detectand manage cvh by virtue of reviewing cancer screening and other risk factor data at thepopulation level better cvh is associated with a lower risk of cardiovascular disease cvdand cancers thus cancer screenings may indirectly decrease burden and cost on thehealth system eg cvd and cancers by improving patient cvh healthlimitationsthere were some limitations in our analyses we used values of auroc to evaluate associations between timeseries cvh measurements and receipt of cancer screenings higherauroc values indicated stronger associations between predictors and the binary outcomes however our observed auroc values were relatively low and thus have limited clinicalutility at this time cancer screenings are potentially affected by cvh and other factors weacknowledge that we had relatively few patients with receipt of cancer screening specificallythere were relatively few patients who received cancer screenings compared to patients whodid not within the same age and gender groups this limitation likely affected the accuracy of one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsour prediction models the prediction accuracy of our models could be improved if morepatients in our data set had received cancer screeningswe demonstrated that deep learning lstm models can effectively predict the associationsbetween timeseries cvh measures and receipt of cancer screening poor cvh especiallypoor a1c may prompt providers to recommend cancer screening for their patients andpatients who received cancer screening may also receive better care for andor have improvedselfmanagement of cvh especially a1c overall these findings suggest that unhealthierpatients are screened for cancers and that cancer screening may also prompt favorablechanges in cvhauthor contributionsconceptualization randi e forakerformal analysis aixia guosupervision randi e forakerwriting original draft aixia guowriting review editing bettina f drake yosef m khan james r langabeer ii randi eforakerwwwmedicalnewstodaycoms282929phpreferences humphrey ll helfand m chan bks woolf sh breast cancer screening a summary of the evidencefor the us preventive services task force annals of internal medicine 107326000348191375_part_120020903000012 american cancer society cancer facts figures am cancer soc 10 pmid wwwcdcgovcancercervicalstatisticsindexhtmwwwcdcgovcancerdcpcpreventionscreeninghtmwwwcdcgovcancercervicalstatisticsindexhtm1 edwards qt li ax pike mc kolonel ln ursin g henderson be ethnic differences in the use ofregular mammography the multiethnic cohort breast cancer res treat 101007s1054900800497 pmid bynum jpw braunstein jb sharkey p haddad k wu aw the influence of health status age andrace on screening mammography in elderly women arch intern med 101001archinte165182083 pmid lipscombe ll hux je booth gl reduced screening mammography among women with diabetesarch intern med 101001archinte165182090 pmid berz d sikov w colvin g weitzen s weighing in on screening mammography breast cancer restreat 101007s105490080037y pmid cook nr rosner ba hankinson se colditz ga mammographic screening and risk factors for breastcancer am j epidemiol 101093ajekwp304 pmid fontaine kr heo m allison db body weight and cancer screening among women j womenshealth gend based med 101089152460901300233939 pmid hsia j kemper e kiefe c zapka j sofaer s pettinger m the importance of health insurance asa determinant of cancer screening evidence from the womens health initiative prev med baltim 101006pmed20000697 pmid hueston w stiles m the papanicolaou smear as a sentinel screening test for health screening inwomen arch intern med pmid one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings robb ka miles a wardle j demographic and psychosocial factors associated with perceived risk forcolorectal cancer cancer epidemiology biomarkers and prevention robb ka miles a wardle j perceived risk of colorectal cancer sources of risk judgments cancer epidemiol biomarkers prev 10115810559965epi060151 pmid gimeno garca az factors influencing colorectal cancer screening participation gastroenterologyresearch and practice 1011552012483417 pmid jensen pb jensen lj brunak s mining electronic health records towards better research applications and clinical care nature reviews genetics 101038nrg3208 pmid goodfellow i bengio y courville a deep learning mit press 101533 rajkomar a oren e chen k dai am hajaj n hardt m scalable and accurate deep learning withelectronic health records npj digit med 101038s4174601800291 pmid hochreiter s s long shortterm memory neural comput bufalino v bauman ma shubrook jh evolution of the guideline advantage lessons learnedfrom the front lines of outpatient performance measurement ca cancer j clin 103322caac21233 pmid wwwscrippssparkleassetsdocumentsheart_rhythm_factspdf shickel b tighe pj bihorac a rashidi p deep ehr a survey of recent advances in deep learningtechniques for electronic health record ehr analysis ieee j biomed heal informatics 101109jbhi20172767063 pmid levenshtein vi binary codes capable of correcting deletions insertions and reversals sov phys dokl citeulikeid311174lloydjones dm hong y labarthe d mozaffarian d appel lj van horn l defining and settingnational goals for cardiovascular health promotion and disease reduction circulation 101161circulationaha109192703 pmid mikolov t corrado g chen k dean j word2vec proc int conf learn represent iclr kingma dp ba j adam a method for stochastic optimization corr 2015abs14126 pearson k on the criterion that a given system of deviations from the probable in the case of a correlated system of variables is such that it can be reasonably supposed to have arisen from random sampling philos mag tsilidis kk kasimis jc lopez ds ntzani ee ioannidis jpa type diabetes and cancer umbrellareview of metaanalyses of observationlal studies bmj online 101136bmjg7607 pmid lipscombe ll fischer hd austin pc fu l jaakkimainen rl ginsburg o the associationbetween diabetes and breast cancer stage at diagnosis a populationbased study breast cancer restreat 101007s1054901533235 pmid bhatia d lega ic wu w lipscombe ll breast cervical and colorectal cancer screening in adults withdiabetes a systematic review and metaanalysis diabetologia 101007s00125 pmid wilkinson je culpepper l associations between colorectal cancer screening and glycemic control inpeople with diabetes boston massachusetts prev chronic dis wang yq wang cf zhu l yuan h wu lx chen zh ideal cardiovascular health and the subclinicalimpairments of cardiovascular diseases a crosssectional study in central south china bmc cardiovasc disord 101186s1287201706979 pmid foraker re abdelrasoul m kuller lh jackson rd van horn l seguin ra cardiovascularhealth and incident cardiovascular disease and cancer the womens health initiative am j prev med 101016jamepre201507039 pmid yin j using the roc curve to measure association and evaluate prediction accuracy for a binary outcome biometrics biostat int j 1015406bbij20170500134 one 101371 pone0236836 august one 0c'
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" new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease In addition to the personto person transmission dynamic of the novel respiratory virus it has been recently studied the role of environmental factors in accelerate SARSCoV2 spread and its lethality The time being air pollution has been identified as the largest environmental cause of disease and premature death in the world It affects bodys immunity making people more vulnerable to pathogens The hypothesis that air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography can acts both as a carrier of the infection and as a worsening factor of the health impact of COVID19 disease has been raised recently With this review we want to provide an update state of art relating the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality The Authors who first investigated this association often used different research methods or not all include confounding factors whenever possible In addition to date incidence data are underestimated in all countries and to a lesser extent also mortality data For this reason the cases included in the reviewed studies cannot be considered conclusive Although it determines important limitations for direct comparison of results and more studies are needed to strengthen scientific evidences and support firm s major findings are consistent highlighting the important contribution of PM25 and NO2 as triggering of the COVID19 spread and lethality and with a less extent also PM10 although the potential effect of airborne virus exposure it has not been still demonstrated Introduction A new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease The scientific community has come together to implement pharmaceutical and nonpharmaceutical intervention measures designed to contain SARSCoV2 global spread Nevertheless on March 11th WHOs Directeneral announced that COVID19 can be characterized as a pandemic SARSCoV2 is primarily transmitted from persontoperson through close contact approximately m by aerosol respiratory droplets smaller than μm in diameter wwwwhoint Indoor environments might be especially hazardous because of their reduced ventilation Morawska lack ultraviolet light which rapidly inactivates the virus and because it can become less diluted than it would in outdoor environments Schuit It is also known how the virus can survive and being infectious in aerosols for hours and on surface up to days van Doremalen et al similarly with transmission dynamics known for SARSCoV1 associated with nosocomial spread and superspreading events Chen et al 2020ab Beyond the causality it is uncertain even if certain demographics of the population are more vulnerable to SARSCoV2 infection Based on recent reports male gender advancing age and comorbidities seem to be correlated with death and severe illness Harris et al Furthermore COVID19 seems to be associated with an increasing rate of thromboembolic events in hospitalized patients Llitjos Mechanisms of social and economic interactions are additionally supposed to be involved in the diffusion dynamic of COVID19 in the diverse parts of the world or of the same country such as the living conditions the healthrelated behaviour KhalatbariSoltani et al Corresponding author Email address ccopatunictit C Copat 101016jenvres2020110129 Received July Received in revised form August Accepted August EnvironmentalResearch1912020110129Availableonline24August2020001393512020ElsevierIncAllrightsreserved 0cC Copat the commercial exchanges Bontempi 2020a or the migration scale index H Chen It seems that these diffusion dynamics have particularly affected the COVID19 spread at the early stage Among the environmental parameters some climate condition such as temperature humidity sunlight and wind revealed a reduction of the COVID19 spread S Chen Coccia 2020a and air pollution seems to have a role in airborne transmission of SARSCoV2 and severity of COVID19 Domingo Nevertheless to better understand COVID19s diffusion patterns an interdisciplinary multidimensional approach should be encourage in order to develop firm s Bontempi Air pollution has been identified as the largest environmental cause of disease and premature death in the world GBD Ambient particulate matter PM induces its proinflammatory and thrombogenic effects through the generation of oxidative stress by its chemical compounds and metals Li Signorelli The recent identification of environmentally persistent free radicals EPFRs in the PM resulting from a mixture of combustion sources theorize its role in the increase of disease severity of lower respiratory tract infections LRTI Jaligama Scientific evidences support that short and longterm exposures to ambient air pollutants are associated with a broad of adverse health outcomes Ferrante and Conti Fiore such as higher mortality rates greater hospital admissions and increased outpatient visits Bremner Cohen Dehghani Dockery It has markedly detrimental consequences on asthma bronchitis pneumonia and COPD Dick Perng and Chen Raji Vignal Yarahmadi et al where bacteria and viruses are the most accepted causative factors that harm airway stability driving to infection exacerbation Furthermore air pollution represents an aggravating factor for infection diseases caused by some viral infections Domingo and Rovira such as respiratory syncytial virus RSV influenza A and B para influenza virus type pneumonia and influenzalike illness Carugno Croft Fukuda Huang Huh Liang Lin Silva Somayaji It determines an increase in the rate of hospitalizations and access to emergency department visits Studies related to the epidemic of SARSCoV coronavirus identified in November from the Guangdong province of southern China reported similar associations Cai Cui Kan Several Authors suggest that outdoor air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography could operate both as a carrier of the infection and as a worsening factor of the COVID19 severity Conticini Frontera Isaifan Martelletti and Martelletti This association is getting stronger thanks to the results of the numerous studies that have been launched all over the world and summarized with this review Most of the reviewed studies support that chronic exposure to air pollution might led people more susceptible to COVID19 disease leading to widespread COVID19 spread and lethality Nevertheless as suggested by Bontempi 2020b the potential effect of airborne virus exposure due to PM10 remain unclear With this review we want to provide an updated state of art of the recently epidemiological studies dealing with understanding the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality Fig PRISMA Flow Diagram of identification screening and inclusion of studies EnvironmentalResearch19120201101292 0cC Copat Method We have conducted a systematic review of the literature concerning the relationship between some air pollutants PM25 PM10 and NO2 and COVID19 outbreak The research was performed in compliance with the PRISMA criteria Preferred Reporting Items for Systematic Reviews and MetaAnalyses and the Flow Diagram is showed in Fig The research was conducted between April and July 6th in PubMed database It was used the Advanced Search Builder and the keywords were searched in [Title OR Abstract] We have filtered only research articles published in English language and selected the following keywords Air pollution and COVID19 or SARSCOV2 Particulate matter or PM and COVID19 or SARSCOV2 Nitrogen dioxide or NO2 and COVID19 or SARSCOV2 We choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported COVID19 cases andor deaths and air pollution data related to PM25 PM10 and NO2 thus excluding any Letter Opinion Commentary Review or nonrelevant articles We obtained a total of eligible published research articles in their final version and paper in its preprint version For some of them we chose to include only principal findings that clearly fit the aim this review Particulate Matter and COVID19 Atmospheric particulate matter PM is originated by a wide range of anthropogenic and natural sources Kim It consists of a heterogeneous mixture of solid and liquid particles suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals WHO It has been associated with increased respiratory morbidity and mortality Liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis Li Rhee In vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections Becker and Soukup Recently the research group of Setti gave first preliminary evidence that SARSCoV2 RNA can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of PM it could represent a potential early indicator of COVID19 although it does not give information regarding COVID19 progression or severity Several observations report a significant association between ambient concentrations of PM25 Adhikari and Yin Bashir Fattorini and Regoli Frontera Jiang Li VasquezApestegui Wu Yao Zhu Zoran 2020a and PM10 Bashir Coccia 2020b Fattorini and Regoli Jiang Li Yao Zhu Zoran 2020a with COVID19 pandemic across the most affected countries China Italy and USA see Table First evidences on the temporal association between air pollution and COVID19 were reported in China where the outbreak was first identified Zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in China The Authors included over of dailyconfirmed new cases in the whole of China between January 23rd and February 29th They applied a Generalized Additive Model GAM to examine the effects of meteorological factors and air pollution on COVID19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders They observed that the effect of PM25 on daily confirmed cases was greater than PM10 In particular they found that a 10μgm3 increase lag0 in PM25 and PM10 was associated with a CI to and CI to increase in the daily counts of COVID19 confirmed cases respectively Jiang focused their attention on three most affected cities of China Wuhan XiaoGan and HuangGang collecting data of daily cases and ambient air pollutant from Jan 25th to Feb 29th The Authors by applying a multivariate Poisson regression revealed a significant temporal association between PM25 increased and COVID19 incidence in all the considered cities especially in HuangGang Wuhan RR CI XiaoGan RR CI HuangGang RR CI Conversely an increase in PM10 concentrations was associated with a decrease of COVID19 incidence These results were partially confirmed by findings of Li who conducted a simple linear regression to compare COVID19 incidence with PM concentrations in Wuhan and XiaoGan from Jan 26th to Feb 29th in They found that an increase in PM25 was correlated with an increase of COVID19 incidence in both cities Wuhan R2 p XiaoGan R2 p while for PM10 only in XiaoGan R2 p The spatial distribution of particulate matter and case fatality rate CFR of COVID19 was studied by Yao in cities of China including Wuhan collecting data up to March 22nd First they found a significantly positive global spatial autocorrelation of COVID19 CFR Global Morans index I p highlighting a high CFR clustering located in Hubei Province With a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product GDP per capita hospital beds per capita local indicators of spatial association LISA map values city size and population or proportion of people older than years It was found that for every μgm3 increase in PM25 and PM10 the CFR increased by and respectively and the risk estimates increased to and with every μgm3 increase in average concentrations of PM25 and PM10 in respectively Some studies describe the association between air pollution and COVID19 across Italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other European countries The 28th of July Italy recorded more than total confirmed cases and deaths WHO most of which were distributed in the regions of Northern Italy especially the Lombardy It is recognized as one the most air polluted areas of Europe EEA where the frequent PM10 annual exceedances of the WHO threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year Baccini Bontempi 2020bfocused the attention on two of the most affected regions of Northern Italy Lombardy and Piedmont The Authors based on PM10 daily exceedances and COVID19 confirmed cases on March 12th thus before the Italian sanitary crisis observed that PM10 concentration was exceeded only few times among the Lombard cities that at the beginning of the epidemic were most affected On the contrary among some Piedmont cities suffering of severe PM10 pollution events COVID19 incidence was lower Based on their results the Authors concluded that COVID19 diffusion by airborne PM10 is hard to demonstrate nevertheless several research article revealed how PM in particular PM25 could had a role in accelerate and vast diffusion of COVID19 in Northern Italy For example Coccia 2020b by analyzed data on Italian province capitals and data of infected individuals up to April 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for PM10 in previous years and COVID19 diffusion In particular cities with more than days of PM10 exceedances showed a very high average number of infected individual about infected individuals on 7th April whereas cities having less than days of PM10 exceedances showed a lower average number of infected about infected individuals Frontera gave also evidences on the role of PM25 as a contributing factor of COVID19 outbreak in Northern Italy where EnvironmentalResearch19120201101293 0cC Copat Table Summary table reporting reviewed results on the association between COVID19 casesdeaths and air pollution PM25 PM10 and NO2 References Zhu Data analysis Generalized Additive Model GAM Aim Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Spatial association between fatality rate and air pollution PM25 and PM10 Spatial association between deaths counts and air pollution NO2 Temporal association between total cases daily confirmed cases and total deaths and air pollution PM25 and PM10 Temporal association between total cases daily confirmed cases and total deaths and air pollution NO2 Spatial description of PM10 exceedances versus COVID19 cases Multivariate Poisson regression Simple linear regression Multiple linear regression Descriptive analysis percentage of deaths in three NO2 μmol m2concentration range Pearson coefficient correlation Pearson coefficient correlation Descriptive analysis Number of days of PM10 exceeding μgm3 and COVID19 incidence Area of Study cities of China Period From Jan 23rd to Feb 29th Jiang Li Yao Ogen Zoran 2020a Zoran 2020b Bontempi 2020b From Jan 25th to Feb 29th From Jan 26th to Feb 29th in Data up to March 22nd Data up to the end of Feb From Jan 1st to Apr 30th From Jan 1st to Apr 30th From Feb 10th to March 12th Wuhan XiaoGan and HuangGang China Wuhan and XiaoGan cities of China administrative regions in Italy Spain France and Germany Milan Italy Milan Italy provinces of Lombardy Italy provinces of Piedmont Italy Coccia 2020b Data up to April 7th Italian Provinces Fattorini and Regoli Data up to April 27th Italian provinces PM25 A 10μgm3 PM25 increase lag0 was associated with a increase of daily confirmed new cases PM10 A 10μgm3 PM10 increase lag0 was associated with a increase of daily confirmed new cases Wuhan RR CI1032 XiaoGan RR CI HuangGang RR CI Wuhan R2 p XiaoGan R2 p Wuhan RR CI XiaoGan RR CI HuangGang RR CI Wuhan R2 p XiaoGan R2 p Ï2 p A μgm3 increase in PM25 was associated with a increase in fatality rate Ï2 p A μgm3 increase in PM10 was associated with a increase in fatality rate NO2 A 10μgm3 NO2 increase lag0 was associated with a increase in daily confirmed new cases Wuhan RR CI XiaoGan RR CI HuangGang no association found Wuhan R2 p XiaoGan R2 p of fatality cases are associated with NO2 μmolm2 R cid0 R R cid0 R cid0 R R cid0 R cid0 R cid0 R cid0 Lombardy PM10 exceeding between and COVID19 incidence between and Piedmont PM10 exceeding between and COVID19 incidence between and COVID19 in North Italy has a high association with air pollution of cities measured with days exceeding the limits set for PM10 R2 p R2 p continued on next page Hierarchical multiple regression model Pearson regression coefficient analysis R2 p Spatial association between confirmed cases and air pollution PM10 Spatial association between total confirmed cases and air pollution PM25 PM10 and NO2 EnvironmentalResearch19120201101294 0cC Copat Table continued References Frontera Frontera Wu Adhikari and Yin Bashir Bashir VasquezApestegui VasquezApestegui VasquezApestegui Period Data up to 31st March Data up to 31st March Data up to April 04th From March 1st to Apr 20th From March 4th to April 24th From March 4th to April 24th Data up to June 12th Data up to June 12th Data up to June 12th Area of Study Italian regions Italian regions counties in the USA Queens county New York USA California California districts of Lima Perù districts of Lima Perù districts of Lima Perù Aim Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Prediction of risk of COVID19 deaths in the long term average exposure to fine particulate matter PM25 Temporal association between daily confirmed cases and total deaths and air pollution PM25 Association between confirmed cases and air pollution PM25 PM10 and NO2 Association between deaths and air pollution PM25 PM10 and NO2 Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Spatial association between case fatality rate and air pollution PM25 Data analysis Pearson regression coefficient analysis PM25 R2 p PM10 Pearson regression coefficient analysis R2 p Longterm exposure increase of μgm3 in PM25 is associated with a increase in the COVID19 death rate Estimate on cases values cid0 CI Estimate on deaths value cid0 CI Kendall r cid0 Spearman r cid0 Zeroinflated negative binomia models Negative binomial regression model Spearman and Kendall correlation tests Spearman and Kendall correlation tests NO2 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient cid0 p mortality was found significantly higher than less polluted Italian regions By collecting data up to March 31st for all Italian regions and performing a Pearson correlation analysis they found a strong positive association both with the total number of confirmed cases R and deaths R other than with hospitalized cases R The Italian situation was further highlighted by the study of Fattorini and Regoli in Italian provinces They explored the spatial association between air pollution and COVID19 cases with data up to April 27th By applying the Pearson regression coefficient analysis they revealed a positive association both with PM25 and PM10 R2 p and R2 p respectively A focus on the most affected city of Italy Milan was conducted by Zoran 2020a This city is located in the Po valley basin known hotspot for atmospheric pollution at the continental scale EEA The Authors performed a temporal association between COVID19 Total cases Daily New positive cases and Total Deaths and particulate matter from Jan 1st and Apr 30th by applying a Person correlation In accordance with other studied they found a positive association between daily confirmed cases and PM25 R and PM10 R although they did not consider any delay time from infection to COVID19 onset Nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships To date the USA have more than million confirmed cases and thousand deaths WHO Here ambient concentrations of PM and O3 were found responsible to cause between and premature deaths Fann The association between air pollutants and COVID19 cases and deaths was studied by Bashir in the state of California from March 4th to April 24th corresponding to the beginning of the COVID19 outbreak in USA Based on their significant correlation found the Authors state that a limited human exposure to these pollutants will contribute to defeating COVID19 This seems unclear because they found a negative correlation with PM25 and PM10 EnvironmentalResearch19120201101295 0cC Copat by applying both the Kendall rank correlation and Spearmans one and it is not clear if they normalized COVID19 cases by population size and if they performed a day by day association or a spatial association across the country A focus on the Queen county New York USA was provided by Adhikari and Yin They retrieved data of PM daily concentrations from two ground monitoring stations and collected data of confirmed COVID19 cases and numbers of related deaths from USAFacts in the period from March to April The Authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of PM25 on disease outcomes over the past days They found a significant negative association among PM25 and new daily confirmed COVID19 cases cid0 CI and deaths cid0 CI Low PM concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as Italy where PM25 monthly concentrations reached values higher than μgm3 Fattorini and Regoli Frontera or in China where PM25 monthly concentrations reached values higher than μgm3 Zhu Jiang As said by the Authors other gaseous pollutants such as NO2 and SO2 could have influenced transmission and pathogenesis of COVID19 In the United States Wu investigated whether longterm average exposure to fine particulate matter PM25 increases the risk of COVID19 deaths by considering approximately counties in the United States of the population With an exposure prediction model the Authors calculated the county level longterm exposure to PM25 averaged for to and collected COVID19 deaths counts up to April 04th They conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors They found that a small longterm exposure increase of only μgm3 in PM25 is associated with a increase in the COVID19 death rate confidence interval CI VasquezApestegui recently reported first evidences on the spatial relationship between particulate matter and COVID19 outbreak from Latin America The Authors described the situation occurred in districts of Lima located in the second most affected country of Latin America Peru In particular by applying a multivariate regression model they evaluated the association between the population exposure to PM25 concentrations in the previous years and cases deaths and casefatality rates of COVID19 with data up to June 12th A significant association has been found both with cases and deaths Crude coefficient with p and with p respectively but not with case fatality rate All these studies highlight the role of PM in triggers of the COVID19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems Nitrogen dioxide NO2 and COVID19 induced lung damage Hence viral infection becomes more common after exposure to NO2 Zhu Furthermore NO2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children To increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation Bahrami Asl Kowalska increase of chronic obstructive pulmonary disease COPD Ghanbari Ghozikali Pfeffer and increase of pulmonary heart disease related mortality Chen A recent study explored the possible role of NO2 in interference in Angiotensin converting enzyme ACE2 The expression of ACE2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of COVID19 Alifano First observations report an association between ambient concentrations of NO2 and COVID19 pandemic across Europe China and USA Bashir Fattorini and Regoli Jiang Li et al Ogen Zhu et al Zoran et al 2020b Conversely to the other papers findings of Zoran 2020b and Bashir provides different findings reporting no association or a negative one between NO2 and daily deaths counts In China Zhu by applying the same method explained for PM observed that a 10μgm3 increase lag0 in NO2 is associated with a CI increase in the daily counts of COVID19 confirmed cases in cities of China These findings are confirmed by Jiang and Li et a who applied the same method described for PM Jiang revealed a significant positive association between NO2 and COVID19 both in Wuhan and XiaoGan Wuhan RR CI1053 XiaoGan RR CI but did not found any significant association in HuangGang Li found a significant linear correlation both in Wuhan R2 p and XiaoGan R2 p Ogen presented evidences on the relationship between exposure to NO2 including the months of January and February shortly before the COVID19 spread in Europe and novel coronavirus fatality in the most affected European countries concluding that longterm exposure to NO2 may be a potential contributor to mortality caused by SARSCoV2 He collected data concerning the number of fatality cases from administrative regions in Italy Spain France and Germany and correlated mortality with tropospheric NO2 concentrations measured by the Sentinel5 Precursor spaceborne satellite The major tropospheric NO2 hotspot identified was located in the Northern Italy In all European regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards Results showed that out of the fatality cases by March were in five regions located in north Italy and central Spain Furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum NO2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 The methodology used by Ogen cannot support a longterm exposure investigation Surely a validation of the satellite measure with those of the ground ones the adjustment of the results according to the different population size of each country could have made their results more robust Nevertheless the study provide new insights for future investigation The Italian situation was further studied by Fattorini and Regoli who collected data of COVID19 incidence up to April 27th from Italian provinces They revealed a strong spatial corr
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" recent impressive advances in cancer immunotherapy have been largely derived from cellular immunity the role of humoral immunity in carcinogenesis has been less understood based on our previous observations we hypothesize that an immunoglobulin subtype igg4 plays an essential role in cancer immune evasionmethods the distribution abundance actions properties and possible mechanisms of igg4 were investigated with human cancer samples and animal tumor models with an extensive array of techniques both in vitro and in vivoresults in a cohort of patients with esophageal cancer we found that igg4 containing b lymphocytes and igg4 concentration were significantly increased in cancer tissue and igg4 concentrations increased in serum of patients with cancer both were positively related to increased cancer malignancy and poor prognoses that is more igg4 appeared to associate with more aggressive cancer growth we further found that igg4 regardless of its antigen specificity inhibited the classic immune reactions of antibody dependent cell mediated cytotoxicity antibody dependent cellular phagocytosis and complement dependent cytotoxicity against cancer cells in vitro and these effects were obtained through its fc fragment reacting to the fc fragments of cancer specific igg1 that has been bound to cancer antigens we also found that igg4 competed with igg1 in reacting to fc receptors of immune effector cells therefore locally increased igg4 in cancer microenvironment should inhibit antibody mediated anticancer responses and help cancer to evade local immune attack and indirectly promote cancer growth this hypothesis was verified in three different immune potent mouse models we found that local application of igg4 significantly accelerated growth of inoculated breast and colorectal cancers and carcinogen induced skin papilloma we also tested the antibody drug for cancer immunotherapy nivolumab which was igg4 in nature with a stabilizing s228p mutation and found that it significantly promoted cancer growth in mice this may provide an explanation to the newly appeared hyperprogressive disease sometimes associated with cancer immunotherapy there appears to be a previously unrecognized immune evasion mechanism with igg4 playing an essential role in cancer microenvironment with implications in cancer diagnosis and immunotherapyintroductionwhile new immune therapy for cancer focuses mostly on manipulating cellular immunity humoral immunity also holds great promise for cancer therapy1 igg4 is a unique antibody that has the lowest concentration among igg subtypes in healthy individuals and its function has not been well understood3 igg4 was regarded as a blocking antibody because of its reduced ability to trigger effector immune reactions6 therefore whatever molecules igg4 reacts to the subsequent immune reaction was subdued8 igg4 has a unique structure of fab arm exchange fae in which the two heavy and light chains of two different antibodies with different specificities are exchanged resulting in an asymmetric bispecific antibody with reduced ability to bind to antigen and to form immune complexes9 another unique feature of igg4 is that it can react to other iggs via its fc fragment and the significance of this property has not been well understood evidence suggests that fae and fc fc reactivity may involve the same molecular structure on igg4 molecule10 davies et al11 resolved the crystal structure of igg4 fc fragment revealing a unique molecular conformation supporting its fc binding property recent interests in igg4 related diseases unveiled a wide range of pathologies with a common phenomenon of often increased igg4 concentration in the serum and igg4 postive plasma cells in the affected ans accompanied by local inflammation and fibrosis but its pathogenic mechanism is still poorly understood13to cite wang a0h xu a0q zhao a0c et a0al an immune evasion mechanism with igg4 playing an essential role in cancer and implication for immunotherapy for immunotherapy of cancer 20208e000661 101136jitc2020000661 º additional material is published online only to view please visit the online http dx jitc hw qx cz and zz are joint first authorsaccepted july authors or their employers re use permitted under cc by nc no commercial re use see rights and permissions published by bmjfor numbered affiliations see end of correspondence toprofessor jiang gu qq comwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access the possible functions of igg4 in cancer and also in the immune system have not been well elucidated increases of igg4 positive plasma cells were reported in gastrocarcinoma16 extrahepatic cholangiocarcinoma17 and melanoma18 the above studies were performed on limited number of cases without convincing explanation of mechanism or significance wu 19 reported that serum igg4igg ratio could predict recurrence of hepatocellular carcinoma after surgery the most extensive study on igg4 and cancer was performed by karagiannis 20 who investigated the possible effect of cancer specific igg4 in inhibiting cancer immunity in melanomas and suggested competition between cancer specific igg4 and igg1 in binding to cancer antigens as the cause for the inhibition a recent report raised the concept of cancer educated b cells and toxic igg produced by these cells in facilitating lymph node metastasis for breast cancer in a mouse model21 we performed a multidimensional investigation of igg4 in a wide array of patients with cancer and tissues with both in vitro and in vivo experiments extensive new evidence led us to hypothesize that there is a potent humoral immune editing mechanism in cancer microenvironment with igg4 playing an essential role we propose that fc fc reaction could be the basic mechanism of this immune inhibition we validated this in three immune potent animal models this property was also found applicable to cancer immunotherapy drug nivolumab which was igg4 in nature our study points to a so far little appreciated mechanism of immune evasion in cancermaterials and methodskey resourcesdetailed information about key resources including antibodies biological samples chemicals assay kits cell lines and software are shown in online supplementary table experimental model and subject detailspatients and healthy volunteerswe collected tissue and blood samples from over patients with cancer in shantou university affiliated tumor hospital and the east guangdong provincial pathological consultation center details of the human samples are shown in online supplementary table immunohistochemistrydetails of the protocols and the antibodies are shown in online supplementary datasds techniquethe expressions of igg1 igg2 igg3 and igg4 in esophageal cancer were detected at the histological level the stain decolorize stain sds technology22 was performed on the same slide sequentially with four different antibodies to demonstrate the four antigens the distribution pattern abundance and relationship of the four antigens were processed with an image software photoshop to achieve an integrated figure the proximity of different cell types on the tissue sections was measured with an image analyzing software image pro plus v60 details of the protocol are presented in the online supplementary dataimmunofluorescence double stainingtwo antibodies from different species were incubated on the same tissue section primary antibodies were detected with goat antimouse 555antirabbit igg or goat antimouse 488antirabbit igg alexa fluor life sciences and immunoreactivity was visualized with a fluorescence microscope antigens were detected and demonstrated with two fluorescence signals in red and green and the blue signal of ' diamidino2 phenylindole dapi as for cell nuclei images were acquired with the evos fl fluorescence microscope life technologies usaigg4 immunohistochemistryto verify that igg4 could react to igg1 that had been immobilized to tissue sections we used human pancreas and brain and antibodies to insulin glucagon and neurofilament as models detailed protocol of this experiment is shown in the online supplementary dataimmunoglobulin preparationsfab and fc fragments of igg igg1 and igg4 were prepared with papain digestion in the presence of cysteine iggs were cleaved at a position above the hinge region by papain at °c for hours after purification with protein a affinity chromatography pure fab and fc fragments were isolated with elution buffer igg fc fragment was washed down from protein a column after centrifugation and concentration measurement igg preparations were collected and stored at °c before usewestern blotigg subclasses were resolved on sodium dodecyl sulfate polyacrylamide gels under reducing conditions and then transferred onto nitrocellulose membranes ge healthcare life sciences the membranes were blocked for hour with bovine serum albumin bsa in tris buffered saline with tween20 ph and then incubated with primary antibody biotin labeling kit anaspec overnight at °c it was then incubated with secondary antibody for hour at room temperature finally reaction density was measured with odyssey western at nm and nm detection channelserum igg4 and igg assessmentsera samples collected from patients with esophageal cancer and healthy volunteers were sent to golden field medical test company guangzhou china and roche immune turbidimetry method was used to wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cmeasure serum igg4 and total igg concentrations all serum samples were stored at °c freezer immediately before analysis all quantitative data were treated statisticallymeasurement of igg4 concentrations in tumor and tumoradjacent normal tissues with elisaconcentrations of igg4 in pairs of esophageal cancer and adjacent normal tissues cm from the edge of the cancer mass were measured with elisa detailed protocol of this experiment is shown in the online supplementary datacell culture for fc receptor binding assaysu937 cell line was bought from procell life science technology china cl0239 and cells were cultured in rpmi gibco c22400500bt supplemented with fetal bovine serum gibco and uml penicillin streptomycin gibco at Ã106ml in ml cell culture bottleprotein preparationsdetails of the protocols for protein preparation separation and papain digestion are shown in online supplementary datafluorescence activating cell sorter facs for fc receptor assaysdetails of the relevant protocols are shown in online supplementary dataadcc adcp and cdc teststhe classic antibody dependent cell mediated cytotoxicity adcc antibody dependent cellular phagocytosis adcp and complement dependent cytotoxicity cdc tests were performed based on previously reported protocols23 non specific igg4 instead of cancer specific igg4 was used to inhibit these reactions igg1 was used as control details of the protocols are shown in online supplementary dataanimal modelsbreast cancer and colon cancer modelsbalbc mice were obtained from vital river technical beijing china mice aged between and weeks and weighed ± g were used in all experiments all mice were inoculated with 4t1 mouse breast cancer cells or ct26 mouse colon cancer cells subcutaneously under the left forearm à 4t1 cells per mouse to build cancer models the mice were divided into different groups and were treated with igg1 or igg4 derived from intravenous igg ivig ivig without igg4 nivolumab and fc of nivolumab respectively details of the protocols are shown in online supplementary datacarcinogeninduced skin tumor modelwe employed a well established carcinogen induced skin tumor model to study the effect of igg4 and ivig without igg4 in comparison with controls phosphate buffered open accesssaline pbs detailed protocol is shown in online supplementary dataquantification and statistical analysisdata were analyzed in graphpad prism all reported p values were derived from two sided comparisons with values of less than considered to be statistically significantresultsigg4 was significantly increased in the serum of patients with cancer and this increase was related to cancer stage and patient prognosiswe first measured the concentrations of igg1 igg2 igg3 and igg4 in sera of patients with esophageal cancer n82 igg4 was significantly increased in patients with cancer in comparison with normal healthy subjects n70 the concentration of igg4 was increased from about to the ratio of igg4iggtotal was also significantly increased the statistical significance of both reached p00001 the increase of igg4 was also positively correlated to the stages of cancer with late stage cancers having more obvious increases higher igg4 serum concentrations were associated with worse prognosis figure 1adigg4containing lymphocytes and igg4 concentration were significantly increased in cancer microenvironment and this increase was associated with cancer cell infiltrationigg4 positive lymphocytes were significantly increased in cancer microenvironment in comparison with tissue more distant to the cancer mass igg4 positive lymphocytes were barely detectable in tumor adjacent normal tissue figure 1h on the same tissue sections employing the sds technique22 to simultaneously visualize the four subtypes of igg with four distinct colors we found that one plasma cell only contained one igg subtype that is igg1 igg2 igg3 and igg4 were all contained in their own plasma cell populations separately figure 1i the marked increase of igg4 containing plasma cells in comparison with other subtypes was clearly visualized on cancer tissue sections igg4 positive plasma cells appeared to accumulate more in tissues with extensive cancer cell infiltration than in other areas figure 1eh in addition igg4 positive cells are often in close proximity to cells containing igg1 and igg2 but not to igg3 this phenomenon was not seen among other igg subtypes apart from igg4 quantitative data of the proximity of different cell types are presented in online supplementary figure s2 with the multiple immunostaining method we also demonstrated that igg1 containing and igg4 containing lymphocytes were distinct from cd3 positive t lymphocytes in the same region of the cancer tissue figure 1j in addition we measured the concentrations of igg4 in cancer tissue and cancer adjacent normal tissue n46 pairs the average concentration of igg4 in cancer tissue was about four times higher than that in the adjacent wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access figure significant increase of igg4 and igg4iggtotal in serum and igg4 positive b lymphocytes in esophageal cancer a igg4 in serum of esophageal squamous cell cancer escc n82 was significantly higher when compared with healthy controls n70 p00001 b igg4iggtotal ratio in escc n82 was significantly higher than that in matching healthy adults n70 p00001 c igg4 in stage
³ n18 was significantly higher than those in stages
° and
± n16 p001 d igg4iggtotal in serum of stage iv escc n18 was significantly higher than those in stages
° and
± n16 p005 e scatter diagram of igg4 positive cell numbers in cancer cancer adjacent normal tissue adjacent and normal tissues igg4 positive lymphocytes in and around the esophageal cancer mass n110 are significantly more abundant than those in the adjacent normal tissue n60 and normal lymphoid tissues n63 p0001 for both increases of igg4 positive lymphocytes were most abundant in areas of cancer cell proliferation f the increase of igg4 positive cell numbers was related to the prognoses of the patients more igg4 positive cells were associated with worse outcome p005 g igg4 concentration in cancer tissue n46 was significantly higher than that in adjacent normal tissue n46 p001 h immunohistochemistry of igg4 in esophageal cancer tissues from left to right are igg4 in cancer tissues cancer adjacent tissue and normal lymphoid tissue tonsil it clearly demonstrates that igg4 positive lymphocytes red were markedly increased left in comparison with normal lymphoid tissue right and with tumor adjacent normal tissue middle scale bar µm i demonstration of four subpopulations of igg containing plasma cells with multiple immunostaining sds method in cancer each subclass has its own distribution pattern and one plasma cell only produces one subclass of igg igg1 yellow igg2 green igg3 purple igg4 red j on the same tissue section a triple immunostaining was performed with the sds method to demonstrate the distribution and relationship among cd3 positive t cells yellow igg1 positive b cells greens and igg4 positive b cells red each cell type has its own distribution and no overlap between different cell types is observed sds stain decolorize stainwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cnormal tissue and the difference between these two groups was statistically significant p001 figure 1gigg1 extracted from patients with cancer reacted to cancer cells of the same patients but igg4 extracted from the same patients did notwe extracted igg1 and igg4 from the serum of patients with esophageal cancer with breast cancer and with colon cancer with respective specific antibody columns we then labeled the antibodies with biotin and tested the reactivity of these extracted antibodies to cancer tissue sections of the same patients in all cases igg1 reacted to cancer cells from the same patients but igg4 did not figure 2a it appeared that the increased igg4 in cancer microenvironment and in the patients serum was not reactive to cancer antigens while igg1 extracted from the same patients reacted to the cancer antigensigg4 reacted to cancerspecific igg1 that was bound to cancer cellsalthough igg4 extracted from patients with cancer did not react to cancer antigen it did react to cancer specific igg1 that was bound to cancer antigen on tissue sections as shown in figure 2b when cancer specific igg1 that was not labeled with biotin was applied to cancer tissue sections followed by biotin labeled igg4 the cancer cells became positive while when the same biotin labeled igg4 was applied to the same cancer tissue section without prior application of igg1 it did not react this reaction of igg4 to cancer specific igg1 was not via the antigen specific variable region of igg4 as such igg4 was neither specifically against igg1 nor was it specifically against cancer antigen with its antigen recognizing fab variable region as shown in figure 2a the only explanation was that igg4 reacted to igg1 through its fc region this was validated by subsequent experiments with western blot analysis as shown in figure 3aein western blot igg4 was found to react to other iggs igg1 igg2 igg3 and igg4 via an fcfc mechanism and this reaction was across species but igg4 did not react to other ig subtypes igm ige iga or igdto test if and how igg4 could react to igg1 we performed western blot with igg4 from different sources extracted from patients with cancer from normal adults and commercially purchased igg4 was found to react to igg1 igg2 igg3 and igg4 at the molecular weight mw of about kd and this reaction was not seen when igg1 igg2 or igg3 was used as the antibody and igg4 as the target molecule running on the gel the above phenomenon was observed in western blot of both reducing and non reducing conditions figure 3a online supplementary figure s3 however human igg4 did not react to human igm iga igd or ige online supplementary figure s4 nevertheless we found that this reaction was across species that is human igg4 reacted to iggs of human mouse rabbit and goat online supplementary figure s5 we open accessigg4 extracted from a patient with cancer reacted figure to cancer bound igg1 and blocked antibody mediated cancer immunity a upper panel these photos serve as an example of the reactivity of igg1 and igg4 extracted from patients with cancer igg1 from the serum of a patient with breast cancer was labeled with biotin and stained a frozen cancer tissue section of the same patient cancer cells were positively stained by igg1 left the cancer cells were confirmed by their characteristic histopathology of he staining middle igg4 from serum of the same patient labeled with biotin and applied on the same cancer on a consecutive section was completely negative right lower panel another breast cancer positively stained by igg1 from the patients serum left the cancer cells were identified by positive immunostaining of cytokeratin ck on a consecutive section middle igg4 from the same patient was not reactive to the same cancer on a consecutive section right b the upper panel illustrates the principle of the experimental reactions and the middle and lower panels show staining results from two patients with breast cancer left igg1 from a patient with cancer positively reacted to frozen cancer tissue of the same patient brown cells middle igg4 from the same patient with cancer applied to consecutive sections but did not react to the same cancer right however when unlabeled igg1 was applied to the same cancer tissue section followed by biotin labeled igg4 the cancer cells were positively stained brown cellsfurther digested human igg4 and igg1 into fab and fc fragments with papain it was found that it was the fc fragment of igg4 reacting to fc of igg1 figure 3be this reaction was easy to occur as only min incubation wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access igg4 reacted to igg1 in western blot and tissue figure sections in an fc fc fashion a in western blot non cancer specific igg4 from a patient with breast cancer reacted to igg1 and igg4 from the same patient with cancer right panel arrows however when igg1 and igg4 were run on the gel and biotin labeled igg1 was used as the primary antibody no band was seen left panel these are the same antibodies used in figure a02ab providing support to explain the reaction between igg4 and igg1 seen on cancer tissue b western blot demonstrated that igg4 reacted with igg1 igg2 igg3 and igg4 c igg4 reacted with igg fc fragment but not with fab arm d igg4 reacted with igg1 fc fragment but not with fab arm e biotin labeled igg4 fc fragment reacted to igg1 and igg4 fc fragments but not with igg1 or igg4 fab biotin labeled igg4 fab did not react to igg1 or igg4 fc or fab these results demonstrate that it is the fc region of igg4 that reacted to fc of igg1resulted in a clear band therefore the positive reaction obtained by sequential applications with cancer specific igg1 followed by non cancer specific igg4 on cancer tissue figure 2b had to take place between the fc of igg4 and the fc of igg1 as shown in figure the fcfc reaction between igg4 and igg1 bound to tissue sections was further tested and validated with a number of antibodies and tissue types apart from cancerfollowing the same logic we tested the reactivity between the fc fragments of igg4 and igg1 already demonstrated in western blot on tissue sections we used igg1 primary antibodies to insulin and glucagon in normal human pancreas and antibody to neurofilament in human brain for this test the same principle was established with these normal tissues detailed results and figures are shown in online supplementary figure s6igg4 competed with igg1 to bind to fc receptors of pbmc and macrophageswe performed immunoglobulin and fc receptor binding assays with peripheral blood monocytes pbmc and with a human monocyte cell line u937 procell life science technology china cl0239 igg1 and igg4 were extracted from the serum of patients with cancer and pbmcs were isolated from the same human subjects the extracted and purchased igg1 and igg4 were labeled with biotin or fitc in the igg1 and igg4 binding assay we found that igg1 and igg4 competed with one another in binding to pbmc and this reaction could be completely blocked by fc receptor blocker this competition was concentration dependent that is as the concentration of igg4 increased more igg1 bound to pbmc was replaced the reverse was also true that is igg1 could also replace igg4 in this competition assay this phenomenon was demonstrated on cytospin slides of pbmc preparation online supplementary figure s7in addition flow cytometry was performed to examine the binding properties of igg1 and igg4 to fc receptors of monocytes the ability of igg1 and igg4 to bind to all three subtypes of fc gamma receptor fcγrfcγr
° cd64 fcγr
± cd32 and fcγr
² cd16was examined with corresponding antibodies we found that igg4 could compete with igg1 in binding to the fc receptor of monocytes u937 we further found that the binding force of igg1 was about twice as strong as that of igg4 for individual receptor subtypes igg1 could bind to all three receptor subtypes that is fcγr
° cd64 fcγr
± cd32 fcγr
² cd16 in contrast igg4 could only bind to fcγr
° cd64 although their binding sites were different igg4 could completely block igg1 we also found that it was necessary for a relatively high concentration of igg4 to be present in the solution in order to compete with igg1 in binding to fc receptors online supplementary figure s8igg4 inhibited the classic immune reactions of adcc adcp and cdc mediated via cancerspecific igg1 and effector immune cellscomplementswe first verified that non cancer specific igg4 indeed reacted to igg1 cetuximab used in adcc adcp and cdc figure 4a we then found that igg4 inhibited adcc elicited cytotoxicity with cancer specific igg1 antibody and pbmc figure 4b the igg4 used in our test was not directed against cancer antigen or to lymphocytes non cancer specific igg1 could also inhibit adcc but to a much lesser extent also reached statistical significance we obtained evidence to show that inhibition of adcc appeared to take place at the site of the cancer specific antibody that is igg4 reacted to the igg1 antibody bound to cancer cells figure 2b and the site of immune effector fc receptors the latter effect could wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen accessfigure non cancer specific igg4 inhibited classic adcc adcp and cdc reactions against cancer but had no direct effect on cancer cell growth a on western blot the chimeric antibody cetuximab igg1 against egfr was run on the gel and igg4 and igg1 at concentrations of and µgml were used as the primary antibodies igg4 reacted to cetuximab at a concentration dependent manner but igg1 did not react b left in a classic adcc experiment cetuximab igg1 was incubated with an egfr expressing lung cancer cell line a549 and then with pbmc from normal healthy adult cancer cell activity was significantly reduced n12 non cancer specific igg1 and hsa were used as controls showing that they had no direct effect on the cancer cells n12 middle when non cancer specific igg4 was added to the mixture the effect of cetuximab was significantly reversed demonstrating an inhibitory effect of igg4 in adcc n12 non cancer specific igg1 had a much smaller but also significant effect in inhibiting adcc action n12 right when fc receptor blocker was incubated with pbmc the effect of cytotoxicity was blocked ce adcp was performed with a lung cancer cell line a549 expressing egfr as the targets human peripheral monocyte derived macrophages as the effector cells and the antibody cetuximab igg1 against egfr as the mediating antibody the tumor cells were stained with cfda se fluorescence probes green macrophages derived from pbmc were stained with dii fluorescent probes orange blue fluorescence is the nuclei stained with dapi d higher magnification of c the orange colored macrophages were in close contact with green tumor cells tumor debris ingested by macrophages appeared yellow in the cytoplasm of macrophages e bar chart showing the effect of adcp and its inhibition by igg4 f left in µgml rituximab mediated adcp model giemsa staining results of phagocytosis of raji cells by macrophages after the addition of µgml igg1 and igg4 respectively right igg4 significantly inhibited rituximab mediated adcp in phagocytosis by macrophage but igg1 could not inhibit the adcp effect scale bar30 µm p005 p001 p0001 g in a classic cdc experiment cetuximab anti egfr antibody was incubated with an egfr expressing lung cancer cell line a549 and then with complement co from serum of a normal healthy adult the cancer cell activity was significantly reduced h when non cancer specific igg4 was added to the mixture in the above cdc experiment the effect of cetuximab was significantly reversed i igg4 and igg1 were incubated with kyse150 for hours and no effect on cell growth was found adcc antibody dependent cell mediated cytotoxicity adcp antibody dependent cellular phagocytosis cdc complement dependent cytotoxicity cfse da carboxyfluorescein diacetate succinimidyl ester dapi ' diamidino2 phenylindole egfr epidermal growth factor receptor hsa human serum albumin pbmc peripheral blood mononuclear cellwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access be abolished with the addition of fc receptor blocker human trustain fcx biolegend china to the pbmcwe also performed an adcp experiment employing human monocyte derived macrophages and esophageal cancer cells cetuximab igg1 was used as the mediating antibody this was performed employing a coculture and cell counting method and fitc labeled antibody flow cytometry two models were employed and both methods showed that non cancer specific igg4 was able to reduce the effect of adcp mediated by cancer specific igg1 figure 4cfin a classic cdc assay we used cancer cell line a549 atcc usa c4215 as the target cancer cells cetuximab as the cancer specific igg1 mediating antibody and human plasma as the source of complements and demonstrated the destructive effect on cancer cells we then used non cancer specific igg4 or igg1 to inhibit the effect we found that the cdc effect was partially inhibited by non cancer specific igg4 but not by igg1 figure 4ghfor comparison we added igg4 or igg1 at various concentrations in cancer cell culture for different periods of time and found no direct effect of these proteins on cancer cell growth figure 4iigg4 including nivolumab significantly accelerated breast cancer cell and colon cancer cell growth in two immune potent mouse models in vivothe above results point to a mechanism that igg4 plays an important role in local immune evasion by blocking immune responses mediated by cancer specific igg antibodies to further examine this mechanism mediated by such antibodies we performed in vivo studies to verify this hypothesis with immune competent mouse models in one model we injected non cancer specific igg4 into a location where breast cancer cells were inoculated subcutaneously in this group of mice cancer cell growth was significantly increased resulting in a much larger cancer mass by days in comparison with other groups injections of pbs or igg without igg4 figure 5ab as there is no direct effect of igg4 on cancer cell growth figure 4i these results unequivocally confirmed that igg4 can inhibit local immune reaction and thereby promote cancer growth in vivo through immune evasionin a separate but similar experiment of a colon cancer mouse model we injected antibody to programmed cell death1 pd1 nivolumab which is a widely used drug in cancer immune therapy and is also an igg4 isotype with s228p mutation which replaces a serine residu
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role of miR1179 in the development of cancer has been proved by different studies However the expression profile and role of miR1179 is yet to be explored in human oral cancer Consistently this study was undertaken to explore the molecular role of miR1179 in regulation of the human oral cancer development and progression The results showed miR1179 to be significantly p overexpressed in all the oral cancer cell lines relative to normal cells The repression of miR1179 transcript levels not only suppressed the proliferation of oral cancer cells but also increased their sensitivity to vincristine The decline in proliferative rates was attributed to induction of autophagy in oral cancer cells as confirmed by transmission electron microscopic analysis Western blot analysis showed that the expression of LC3BII increased and that of beclin decreased while LC3BI expression remained constant upon miR1179 inhibition Inhibition of miR1179 caused significant decrease in the migration and invasion of the oral cancer cells The migration and invasion found to be and for SCC9 and and for SCC25 cells upon miR1179 inhibition At molecular level the miR1179 was shown to exert its anticancer effects via deactivation of MEKERK and PI3KAKT signalling cascades In the findings point towards the potential of miR1179 in the treatment of oral cancerKeywords Oral cancer Micro RNA Proliferation Autophagy MetastasisIntroductionDespite advancement in science and technology the current strategies employed for the treatment of human oral cancer are still far from descent As such presently the oral cancer is still ranked 6th most prevalent type of cancer globally Peers et a0al The survival rates of oral cancer are comparatively lower than breast and prostate cancers The overall 5year survival rate of oral Correspondence MonicaHendrixaxoyahoocom Yanmei Gao and Hanmei Xu contributed equally to this workDepartment of Stomatology Affiliated Hospital of Jilin Medical University NO of Road Huashan Fengman Area Jilin Chinacancer is only as against and for breast and oral cancers respectively Gupta et a0al Additionally the recurrence of human oral cancer further adds to the problem Borsetto et a0al Hence it is need of the hour to look for the alternative approaches for the management of oral cancer As revealed by the recent research reports the molecular regulators of human cancers have emerged as vital targets to be studied for their usage against these deadly ailments Cao et a0 al Among these molecular agents the small RNA entities called microRNAs miRs which do not code for proteins but have molecular regulatory roles have been shown to be involved in the development and tumorigenesis The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 0cGao a0et a0al AMB Expr Page of of number of human cancers including the human oral cancer Gaezon et a0 al Wu et a0 al The miRs have not only been shown to regulate the proliferation of human cancers but have also been shown to have influence the metastasis to neighbouring tissues Peng et a0al MicroRNA1179 miR1179 has been implicated in the regulation of proliferation and metastasis of different human cancers Jiang et a0al Krutovskikh et a0al Song et a0 al Lin et a0 al The miR1179 has been shown to inhibit the growth of the glioblastoma cells by inducing cell cycle arrest Xu et a0al The inhibition of metastasis of hepatocellular carcinoma has been reported to be due to interaction of miR365 with ZEB2 Gao et a0al In yet another study miR targets HMGB1 to suppress the proliferation of gastric cancer cells Li and Qin Nonetheless the role and therapeutic implications of miR1179 has not be reported This study was therefore designed to investigate the role of miR1179 in human oral cancer cells via modulation of the MEKERK and PI3KAKT signalling pathwaysMaterials and a0methodsCell lines and a0culture conditionsThe oral cancer cell lines SCC4 SCC9 SCC15 and SCC25 along with normal EBTr cell line were procured from ATCC USA The culturing of cell lines was performed using Dulbeccoʼs modified Eagleʼs medium DMEM Thermo Scientific The cell lines were maintained in a CO2 incubator at a0°C with CO2 concentration and relative humidity of TransfectionTo stably transfect the oral cancer cells with miRNC and miRinhibitor Lipofectamine Thermo Scientific was used and the procedure was carried as per the manufacturer guidelinesExpression analysisThe RNeasy Mini Kit Qiagen and miScript Reverse Transcription Kit Qiagen were respectively used to isolate the RNA from cancer and normal cell lines and for cDNA synthesis The SYBR Green mix Thermo Scientific was used for performing the expression analysis of miR1179 through quantitative realtime polymerase chain reaction qRTPCR on QuantStudio real time PCR Thermo Scientific The cycling conditions were as follows °C for a0 s followed by cycles of °C for a0s and °C for a0min The expression values were quantified using ddCt method The real primer sequences were miRF ² GCG GAA GCA TTC TTT CAT ² and miRR ² CAA GGG CTC GAC TCC TGT ²Cell viability assayTo assess the proliferation rates of the oral cancer cells transfected with miRNC and miRinhibitor for a0 h and administered withwithout a0 µM vincristine the cells were cultured in 96well plate for a0h a0h or a0h at °C Following this the 345dimethylthiazol2yl25diphenyl tetrazolium bromide MTT reagent Thermo Scientific was added at final concentration of After a0 h a0 µl dimethyl sulfoxide DMSO was added for dissolving the crystals of formazan Absorbance at a0 nm was taken using spectrophotometer to determine the proliferation rates of oral cancer cellsElectron microscopyFor the assessment of induction of cell autophagy the cancer cells transfected with miRNC or miRinhibitor for a0 h were examined using Transmission electron microscope TEM Trypsin treatment was used to make the collection of cancer cells which were then washed and then fixed with glutaraldehyde in phosphate buffer a0m The Osmium tetroxide was then used for postfixing of cells The cells were then treated with ethanol and embedded in resin The ultramicrotome was used for section cutting which was followed by electron microscopyTranswell chamber assayThe migration and invasion of oral cancer cells transfected with miRNC or miRinhibitor were determined by using Transwell chamber without or with matrigel coating Here the cell suspension containing approximately cells was poured into the upper portion of transwell chamber and lower portion was supplemented with a0µl DMEM medium with fetal bovine serum FBS Following a0 h incubation at a0 °C and CO2 concentration the cancer cells from the surface of membranes upper side were swabbed away with cotton swabs while the cells sticking to lower surface of membrane were fixed with ethanol and the stained with crystal violet The cells were then examined and photographs were taken using light microscopeWestern blottingFor the estimation of protein concentrations the western blotting technique was used Precisely after transfection with miRNC or miRinhibitor the oral cancer cells were cultured for a0h at a0°C Centrifugation was used to collect the cells which were then washed using PBS buffer This was followed by treatment with RIPA buffer containing a0 mM TrisHCl pH a0 mM NaCl Triton X100 SDS a0mM EDTA a0mM Na2HPO4 a0mM NaF a0mM NaVO4 a0mM phenyl 0cGao a0et a0al AMB Expr Page of methylsulfonyl fluoride and protease cocktail inhibitor The total protein concentrations were estimated using Bradford assay Equal protein concentrations were loaded on the Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDSPAGE from each sample The gel was blotted to PVDF membrane followed by treatment with primary antibodies Santa Cruz Biotechnology Inc Dallas TX USA overnight at °C The blots were washed in tris buffered saline TBS incubated with horseradish peroxidaseconjugated secondary antibody Santa Cruz Biotechnology Inc for a0h at °C washed again three times with TBS and chemiluminescence was captured on hyperfilm following incubating the blots in enhanced chemiluminescence reagentStatistical analysisThe experiments were performed in triplicate and expressed as mean ± standard deviation SD The Students ttest was performed through GraphPad Prism software The pvalues were taken as statistically significant differenceResultsmiR is a0upregulated in a0oral cancer cellsThe qRTPCR analysis showed miR1179 to be significantly upregulated in all the oral cancer cell lines studied SCC4 SCC9 SCC15 and SCC25 relative to normal oral cell line EBTr The maximum transcript upregulation of miR1179 was observed in SCC9 oral cancer cells 65fold and SCC25 cells 45fold relative to normal EBTr cells Fig a01a As such SCC9 and SCC25 cell lines were taken for further experimentationmiR inhibition suppresses the a0proliferation and a0enhances chemosensitivity of a0oral cancer cellsTo reveal the molecular functionality of miR1179 in oral cancer the suppression of miR1179 was achieved by transfecting the SCC9 and SCC25 cancer cells with miR1179 inhibitor Using miRNC transfected as control the repression of miR1179 transcript levels was confirmed by qRTPCR method which showed significant p decrease of miR1179 expression in SCC9 and SCC25 cells Fig a01b Additionally the results showed that suppression of miR1179 resulted in remarkable decline of proliferation rates of SCC9 and SCC25 cancer cells Fig a01c Interestingly the antiproliferative effects of vincristine were shown to be greatly enhanced when SCC9 and SCC25 cancer cells were transfected with miRinhibitor construct Fig a0 Together the results clearly indicate that miR1179 inhibition suppresses proliferation and enhances the chemosensitivity of the human oral cancer cellsFig miR1179 regulates the proliferation of human oral cancer cells a Expression of miR1179 in human oral cancer and normal cells b Expression of miR1179 in miRNC and miR1179 inhibitor transfected SCC9 and ACC25 cells c Cell viability of miRNC and miR1179 inhibitor transfected SCC9 and ACC25 cells Individual experiments were performed in triplicate and expressed as mean ± SD P 0cGao a0et a0al AMB Expr Page of Fig Inhibition of miR1179 enhances the Vincristine sensitivity of the human SCC9 and SCC25 oral cancer cells Individual experiments were performed in triplicate and expressed as mean ± SD P by the western blotting of LC3BI LC3BII and beclin autophagy related proteins The LC3BI protein levels remined constant beclin protein levels declined and the LC3BII protein levels increased in miR1179 inhibitor transfected SCC9 and SCC25 oral cancer cells Fig a03b Hence it is evident that the transcript repression of miR1179 in human oral cancer cells declines the cell proliferation rates via induction of cell autophagymiR suppression reduces oral cancer cell metastasisThe assessment of migration and invasion capabilities of SCC9 and SCC25 oral cancer cells transfected with miR inhibitor or miRNC constructs by transwell chamber assay showed that the miR1179 inhibitor mediated suppression of miR1179 expression resulted in significant decline in the migratory and invasive potential of SCC9 and SCC25 cancer cells Fig a0 The migration and invasion found to be and for SCC9 and and for SCC25 cells upon miR1179 inhibition Taken together the results indicate that miR1179 has a regulatory role on cancer cell metastasismiR modulates MEKERK and a0PI3KAKT pathways in a0oral cancerIn order to analyse the molecular mechanics of miR1179 in oral cancer the repression of miR1179 was made in SCC9 and SCC25 oral cancer cells The assessment of MEKERK signalling pathway revealed that the decline in miR1179 expression reduced the phosphorylatedMEK and ERK pMEK and pERK protein levels Fig a0 5a However the protein levels of nonphosphorylated versions of MEK and ERK remained almost unchanged Similarly the reduction in phosphorylated protein versions of PI3K and Akt proteins pPI3K and pAkt was Fig Inhibition of miR1179 induces autophagy in oral cancer cells a Ultrastructural analysis of the miRNC and miR1179 transfected SCC9 and SC25 cells b Western blot analysis of miRNC and miR1179 transfected SCC9 and SCC25 cells showing the expression of LC3B I II and Beclin Individual experiments were performed in triplicateInhibition of a0miR induces autophagy in a0the a0oral cancer cellsThe electron microscopic examination of SCC9 and SCC25 human oral cancer cells transfected with miR inhibitor or miRNC clearly revealed the presence of autophagy vesicular structures in miRinhibitor transfected cancer cells Fig a0 3a However such structures were totally lacking from the cancer cells transfected with miRNC The results were further supported 0cGao a0et a0al AMB Expr Page of Fig Inhibition of miR1179 inhibits the migration and invasion of miRNC and miR1179 transfected SCC9 and SC25 cells Individual experiments were performed in triplicate and expressed as mean ± SD P observed under miR1179 repression Fig a05b The protein level of PI3K and Akt remained unchanged Taken together the results reveal that the suppression of miR expression in oral cancer results in the blocking of phosphorylation of MEK ERK PI3K and Akt proteinsDiscussionOral cancer is one of the lethal human disorders and this malignancy is responsible for a considerable level of human mortality and morbidity Warnakulasuriya Peterson Overall the oral cancer ranks 6th in terms of its incidence rates globally One more worrying fact about human oral cancer is its recurrence and development of drug resistance Silva et a0 al So an urgency is felt in the scientific community to devise more robust measures for the oral cancer management Recently miRs have emerged as potent regulators of various human cancers as they have been seen to have profound role in human physiology and disease development Gong et a0al The deregulation of miR1179 has been implicated to be associated with a number of human cancers Peng et a0al In the present study miR1179 was found to be upregulated in oral cancer cells which is in accordance with several previous studies Peng et a0 al Krutovskikh et a0 al Further miR1179 downregulation was previously been shown to decline the viability of ovarian cancer cells Zhihong et a0al Similar observations were made from the results of the current study The miR1179 repression was found to inhibit proliferation and enhance the drug sensitivity of oral cancer cells to vincristine Such implications have been drawn also previously Zhihong et a0 al The characterization of miR1179 in oral cancer in this study revealed that autophagy is induced in oral cancer cells when miR1179 transcript levels are repressed The autophagy was confirmed by the presence of autophagy vesicles which was further implicated by the enhancement of LC3II conjugated protein expression level and declining of LC3I and Beclin expression The miR repression in oral cancer cells further declined the migration and invasion rates of cancer cells which is in conformity with the previous studies on miR1179 Jiang et a0al Lastly among the most important molecular events of cancer progression the activation of MEKERK 0cGao a0et a0al AMB Expr Page of upregulated in human oral cancer cells Inhibition of miR1179 overexpression resulted in decline of proliferation and metastasis and enhancement of chemosensitivity of human oral cancer cells Additionally miR1179 also resulted in the blockage of the MEKERK and PI3KAKT signalling pathway pointing towards the therapeutic implications of miR1179 in oral cancer treatmentAcknowledgementsWe acknowledge the Affiliated Hospital of Jilin Medical University Jilin China Shandong China for providing necessary laboratory faciality and supportAuthors contributionsYG and TP designed the protocol of the study YG and HX performed the experimental work and collect the data for presented study YG and HX involve in the statistical analysis TP supervised the work and drafted the manuscript although all author contributes for the preparation of manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsNot applicableEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived June Accepted August ReferencesBorsetto D Higginson JA Aslam A AlQamachi L Dhanda J Marioni G Franchella S Frigo A Praveen P Martin T Parmar S Factors affecting prognosis in locoregional recurrence of oral squamous cell carcinoma J Oral Pathol Med Cao M Tang Y Tang Y Liang XH Noncoding RNAs as regulators of lymphangiogenesis in lymphatic development inflammation and cancer metastasis Front Oncol Gao HB Gao FZ Chen XF MiRNA1179 suppresses the metastasis of hepatocellular carcinoma by interacting with ZEB2 Eur Rev Med Pharmacol Sci Garzon R Calin GA Croce CM MicroRNAs in cancer Ann Rev Med Gong H Liu CM Liu DP Liang CC The role of small RNAs in human diseases potential troublemaker and therapeutic tools Med Res Rev Gupta N Gupta R Acharya AK Patthi B Goud V Reddy S Garg A Singla A Changing Trends in oral cancera global scenario Nepal J Epidemiol Huang M Huang B Li G Zeng S Apatinib affect VEGFmediated cell proliferation migration invasion via blocking VEGFR2RAFMEKERK and PI3KAKT pathways in cholangiocarcinoma cell BMC Gastroenterol Jiang L Wang Y Rong Y Xu L Chu Y Zhang Y Yao Y miR1179 promotes cell invasion through SLIT2ROBO1 axis in esophageal squamous cell carcinoma Int J Clin Exp Pathol Krutovskikh VA Herceg Z Oncogenic microRNAs OncomiRs as a new class of cancer biomarkers Bioessays Fig Inhibition of miR1179 blocks MEKERK and PI3K and AKT signalling pathways a Western blots showing the effects of miR1179 inhibition on expression of MEK pMEK ERK and pERK proteins in SCC9 and SCC25 cells b Western blots showing the effects of miR1179 inhibition on expression of PI3K pPI3K AKT and pAKT proteins in SCC9 and SCC25 The experiments were performed in triplicateand PI3KAKT pathways hold a prime essence as these pathways enable the transcription of various downstream regulators of cancer growth and proliferation Huang et a0al These pathways have been shown to be aberrantly activated in different cancer types For example PI3KAKT pathway has been shown to be activated in pancreatic cancer Lan et a0 al In thyroid cancer cells PI3KAKT and MAPKERK pathway has been shown to significantly overexpressed Su et a0 al Similarly MEKERK pathway has shown to be activated in ovarian cancer Zhang et a0al As such the blockage of these signalling pathways is a vital asset to keep the cancer progression at check Zhihong et a0 al The miR1179 suppression renders the MEKERK and PI3KAkt pathways to proceed at fairly diminished rates by preventing the phosphorylation of crucial signalling components like MEK ERK PI3K and AKT Summing up the current study deduced the molecular functionality of miR1179 and implicated its molecular targeting in the management of human oral cancer To conclude the present study showed miR1179 to be significantly 0cGao a0et a0al AMB Expr Page of Lan CY Chen SY Kuo CW Lu CC Yen GC Quercetin facilitates cell death and chemosensitivity through RAGEPI3KAKTmTOR axis in human pancreatic cancer cells J Food Drug Anal Lin C Hu Z Yuan G Su H Zeng Y Guo Z Zhong F Jiang K He S MicroRNA1179 inhibits the proliferation migration and invasion of human pancreatic cancer cells by targeting E2F5 Chem Biol Interact Li Y Qin C MiR1179 inhibits the proliferation of gastric cancer cells by targeting HMGB1 Hum Cell Peng X Guo W Liu T Wang X Tu XA Xiong D Chen S Lai Y Du H Chen G Liu G Identification of miRs143 and145 that is associated with bone metastasis of prostate cancer and involved in the regulation of EMT PLoS ONE 275e20341Peres MA Macpherson LM Weyant RJ Daly B Venturelli R Mathur MR Listl S Celeste RK GuarnizoHerre±o CC Kearns C Benzian H Oral diseases a global public health challenge Lancet Petersen PE Oral cancer prevention and controlthe approach of the World Health anization Oral Oncol Silva SD Hier M Mlynarek A Kowalski LP AlaouiJamali MA Recurrent oral cancer current and emerging therapeutic approaches Front Pharmacol Song L Dai Z Zhang S Zhang H Liu C Ma X Liu D Zan Y Yin X MicroRNA1179 suppresses cell growth and invasion by targeting spermassociated antigen 5mediated Akt signaling in human nonsmall cell lung cancer Biochem Biophy Res Co Su X Shen Z Yang Q Sui F Pu J Ma J Ma S Yao D Ji M Hou P Vitamin C kills thyroid cancer cells through ROSdependent inhibition of MAPKERK and PI3KAKT pathways via distinct mechanisms Theranostics Warnakulasuriya S Global epidemiology of oral and oropharyngeal cancer Oral Oncol Wu BH Xiong XP Jia J Zhang WF MicroRNAs new actors in the oral cancer scene Oral Oncol Xu X Cai N Zhi T Bao Z Wang D Liu Y Jiang K Fan L Ji J Liu N MicroRNA1179 inhibits glioblastoma cell proliferation and cell cycle progression via directly targeting E2F transcription factor Am J Cancer Res Zhang R Shi H Ren F Feng W Cao Y Li G Liu Z Ji P Zhang M MicroRNA3383p suppresses ovarian cancer cells growth and metastasis implication of Wntcatenin beta and MEKERK signaling pathways J Exp Clin Cancer Res Zhihong Z Rubin C Liping L Anpeng M Hui G Yanting W Zhenxiu S MicroRNA1179 regulates proliferation and chemosensitivity of human ovarian cancer cells by targeting the PTENmediated PI3KAKT signaling pathway Arch Med Sci Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c'
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"EPMA celebrated its 10th anniversary at the 5th World Congress in Pilsen Czech Republic The history of theInternational Professional Network dedicated to Predictive Preventive and Personalised Medicine PPPM 3PM is rich inachievements Facing the coronavirus COVID19 pandemic it is getting evident globally that the predictive approach targetedprevention and personalisation of medical services is the optimal paradigm in healthcare demonstrating the high potential to savelives and to benefit the society as a whole The EPMA World Congress Supplement highlights advances in 3P medicineIntroductionEuropean Association for Predictive Preventive and PersonalisedMedicine has been created in In the historical 1stEPMA World Congress took place in Bonn GermanyIn the EPMA celebrated its 10th anniversary at the 5thWorld Congress in Pilsen Czech Republic The decadeoldprofessional history of the EPMA is rich in achievementsHerewith we briefly highlight some of themGeographic distribution of the 3PMrelevant expertise underthe EPMAumbrella started with approximately countries in currently the EPMA is represented in countries University Hospital in Pilsen Medical Faculty in Pilsen CharlesUniversity Prague Czech RepublicEuropean Medical Association Brussels BelgiumEuropean Association for Predictive Preventive and PersonalisedMedicine EPMA Brussels BelgiumThe historical 1st EPMA World Congress in former Bundestag BonnGermany September Declarations The authors declare that they have no competing interest Permissions by responsible ethic commissions for correspondingcontributions have been received and thoroughly check prior topublishing the EPMA World Congress Supplement Research involving human participants andor animals was performedin accordance with international regualtions All the patient investigations conformed to the principles outlined in theDeclaration of Helsinki Informed consent was obtained from all individual participants included in the corresponding study Olga GolubnitschajaOlgaGolubnitschajaukbonndePredictive Preventive and Personalised 3P Medicine Departmentof Radiation Oncology University Hospital Bonn RheinischeFriedrichWilhelmsUniversitt Bonn VenusbergCampus Bonn Germany 0cworldwide who actively promote 3PM concepts in biomedicalsciences and practical medicine strongly benefiting patients andhealthcare systemsThe first issue of the EPMA Journal Springer Nature wasreleased in March In the journal received its firstIF in it reached Nowadays the EPMA J is ahighly recognised international forum for 3P medicine operating in a hybrid subscription access modus ScopusCiteScore of the EPMA J is wwwscopuscomsourceid19700201201originsourceInfozonerefpointranktabs1 thereby Scopus ranks the EPMA Jamongst the top in the category Health Policy due tohighly requested and wellcited strategic papers created bymultiprofessional groups of EPMA experts such as General report recommendations in predictive preventive and personalised medicine white paper ofthe European association for predictive preventive andpersonalised medicine 1011861878 Medicine in the early twentyfirst century paradigm andanticipation EPMA position paper 101186s1316701600724SCImago topranks the EPMA J in all three categoriesnamely Health policy Medical Biochemistry and Drugdiscoverywwwscimagojrcomjournalsearchphpq19700201201tipsidIn Springer Nature awarded the belowmentioned the status of an with a potential to change thew o r l d i n t h e c a t e g o r y M e d i c i n e a n d P u b l i cHealth wwwspringernaturecomgpresearcherscampaignschangetheworldmedicinepublichealthPregnancy Associated Breast Cancer The Risky StatusQuo and New Concepts of Predictive Medicine EPMA J s1316701801297Advances in Predictive Preventive and PersonalisedMedicine is a very successful EPMASpringer Nature bookseries which educates both professionals and the general population in 3P medicine Since book volumes havebeen released dedicated to a whole spectrum of PPPM relatedaspects such as digital health information technology framework application of artificial intelligence in healthcare drugdelivery systems liquid biopsy and multilevel diagnosticsamongst othersHorizon is the main European Scientific Programmewhich EPMA experts have contributed to with 3PMrelatedprotocols as well as with the topexpertise provided byRepresentatives and Members of the association involved inthe evaluation panelsEPMA JournalEPMA AWARD for EXCELLENCE in BIOMEDICALSCIENCES was created in and the 1st EPMA awardwas given to Prof Dr Josef Flammer University of Basel forphenotyping of the Flammer Syndrome which the EPMAinternational jury panel valued as being of great clinical utilityYoung professionals in PPPM Award was created bythe EPMA in Atthe international workshopslinked to the biannual EPMA World Congresses thepresentations made by young professionals get evaluated by an international jury panel The best presentations and smart 3PM concepts receive awards thateffectively promote the careers of young professionalsin innovative biomedical fieldsEPMA World Congress in Pilsen Czech Republicattracted 3PM experts from countries The congress wasdedicated to innovation in a broad spectrum of biomedicalfields with a specific focus on the concepts of predictive diagnostics targeted prevention and personalisation of medical services in Cancer Metabolic DisordersCardiovascular Disease Neurological Neurodegenerativeand Neuropsychiatric Disorders Inflammatory DisordersDentistry Biobanking and Screening ProgrammesMultiomics Microbiome Immune Pre andProbiotics and Innovative Technologies among othersFurther there were several new topics presented at the congress among others these were Implementation of 3PMConcepts in Plastic Surgery Application of ArtificialIntelligence in Medicine 3PM strategies and MedicalUse of Cannabis The latter topic was discussed in the EUParliament in and the EPMA position has been elucidated by the EPMA Representatives for more information seethe below linkhttpwwwepmaneteulatestevents2019epmapositiononmedicaluseofcannabispresentedattheeuparliamentOral and poster presentations provided valuable information regarding pilot projects towards personalisedhealthcare eg awarded by ICPerMedindividualisedpatient profiles multilevel biomarker panels patientstratification creation and application of innovative ITtools ethical issues doctorpatient collaboration optimalthe modern hospitalstructure and anisation ofambitioned to practically implementthe paradigmchange from reactive to predictive preventive andpersonalised medicineWorld First 3P Medical Unitthe historically first worldwide unitIn March dedicated to Predictive Preventive and Personalised3P Medicine led by SecretaryGeneral of the EPMAProf Dr Olga Golubnitschaja was created in Germanyatthe Department of Radiation Oncology UniversityHospital Rheinische FriedrichWilhelmsUniversittBonn 0cEPMA Journal3PM vision and strategiesPPPM for Twentyfirst Century Biosensing PainlessPersonalised PointofCare Monitoring with Wearableand Implantable DevicesAndrews RACorresponding author Nanotechnology Smart Systems NASA Ames Research Center Moffett Field CA USA email rjarusselljandrewsKeywords Artificial intelligence Biosensors Blood pressuremonitoring BraincomputerBrainmachine interfaceContinuous monitoring Diabetes ElectrocardiogramElectroencephalogram Epilepsy Fall detection Gait disorders Glucose monitoring Implantable sensors Ingestible sensors Internet of things Iontophoresis Interstitial fluidNanosensors Neurotechnology Pressure monitoring Salivamonitoring Seizure detection Smart contact lenses Smartmouthguards Smart patches Smart skin Smart watchesSmartphone apps Skin patches Sweat monitoring Tear monitoring Temperature monitoring Tissuedevice interfaceWearable sensors Wireless monitoringIntroductionMany people do not realize they already have adopted wearable devices for medical monitoringsmartwatches Typicalstories of smartwatches providing lifesaving diagnostic information include the following A smartwatch alarming allnight regarding abnormal heartrate alerted the wearer to seekmedical attention for what proved to be atrial fibrillation [] A hikerlost as nightfall approachedstumbled and fellon difficult terrain Unbeknownst to the hiker the fall triggered his smartwatch to automatically call the emergencyphone number in the USA thereby avoiding what couldhave been a tragic outcome Smartphones with accelerometerand GPS capabilities have apps for people with epilepsy whomay require emergency medical assistance []Medical monitoring has not always been so painless persistent and unobtrusive Atrial fibrillation required attachingelectrodes to the skin with a conductive gel in turn connectedto a devicepossibly portable but certainly obtrusiveMonitoring of blood glucose by diabetic patients required repeated fingerstickspainful intermittent and obtrusivePhases of Biofluid MonitoringDiagnostic techniques for biofluids eg blood urine salivaand cerebrospinal fluid CSF have evolved over the pastseveral decades Fig [] The first phaseextendingfrom the twentieth century to the presententailsobtaining a sample from the patient an invasive procedurefor blood and CSF and sending it to a laboratory for analysisResults are not available for hours to days for samples obtained from outpatients and minutes to hours for inpatientsFig The four technological waves of biochemical monitoring reference []with permissionThe second phase began about two decades ago with pointofcare POC monitoring where the laboratory comes to thepatient ie to the recently obtained sample rather thantransporting the sample to the laboratoryThe third phase more recently available consists of wearabledevices This is the epitome of POC monitoring since the patientand the device are inseparable Smartwatches can do this forpulse and blood pressure patches applied to the skin for continuous blood glucose monitoring The patches eg for glucosemonitoring typically monitor the analyte concentration in interstitial fluid ISF which closely parallels blood glucose []The line between the third and fourth phaseswearable andimplantable devicesis blurred Part of this is due to expansion of the fluids monitoring from blood or ISF to sweatsaliva and tears Most would call a mouthguard to monitor salivaa wearable devicebut what about a smart contact lens tomonitor tears Truly implantable devices eg inserted subcutaneously by a minor surgical procedure can monitor analytes suchas glucose for months potentially longer rather than the days toa week or so of most patches []Power to the PatientDigitizing Biofluid MonitoringDuring the sampletolab and POC phases urine was the idealbiofluidnoninvasively obtained and relatively easilytransported Blood required an invasive procedure a needlestickSweat and tears were not easily obtained in a manner guaranteeing uniformity and saliva could vary greatly depending on timeof sampling eg after a drink or a mealWearable devices have transformed those problems into oneconsisting of a tissuedevice interface TDI challenge 0cEPMA JournalContinuous biofluid monitoring is a reality sampling urineblood or other biofluids continuously was not practical previously outside a hospital setting with an indwelling catheterfor urine or blood or even CSF A second problem in phasesone and two was obtaining continuous diagnostic informationfrom the biofluidThe smartphone and smartwatches plus machine learning andartificial intelligence AI have allowed not only continuousbiofluid monitoring but also continuous realtime interpretation of that monitoring information in a precise and personalized mannerdigital biomarkers [] This can answer thequestionWhat does this biofluid monitoring value mean for this particular patient at this precise momentOnce answered that information can guide realtime precisepersonalized treatment eg continuous feedbackguided orclosedloop insulin release in diabetes The patient if desiredcan have control over when the biofluid monitoring information is gathered or processed or transmitted eg to adatabank The patient can remove the patch or the smartwatchor turn off the smartphone containing the app transferring thedataBlood Sweat Tears and SalivaAlthough the primary target has been a wearable monitor ofblood glucose for diabetic patients other biological signalsthat can be measured through the skin include chemicals beyond glucosepotassium chloride lactate electrical electrocardiogram ECG electroencephalogram EEG electromyogram EMG and physical temperature pressure lightsound [] Additionally non or minimally invasive monitoring has included measures ranging from respiratory rate tojoint movement to gait [ ] This review is primarilylimited to the TDI for biofluidsWearable skin patches depend on knowledge of thestructure of human skin [ ] Smart skin exhibitsmany technological advances as illustrated in Fig [] Skin patches usually monitor ISF concentrations of thechemical of interest relatively straightforward for ISF glucoseas a surrogate for blood glucose Sweat however poses adifferent problem since sweat is not continuously availablefor monitoring In the typical skin patch for sweat the patchincorporates an electrode to deliver a cholinergic agent such ascarbacholinto the skin for stimulation of sweationtophoresis [ ]Fig Recent research trends in smart skin from four viewpoints First the structures of smart skins are advancing from stretchable toultrathin to breathable sensors resulting in enhancement of biocompatibility and reduced burden of sensor attachment Second multimodality is expanding from electrical to physical to chemical sensors Third more advanced functions such as stimulation drug deliveryand displays are being incorporated in addition to sensing functions Fourth novel materials such as selfhealing conductors intrinsicallystretchable semiconductors and photoactive materials are being developed reference [] with permission 0cEPMA JournalMonitoring tears is challenging the rate of tearing is notuniform the device must be acceptable to the patientTearbased biofluid sensors include smart contact lenses anddevices placed in the lower eyelid Fig 3A [ ]Fig 3A Tearbased biosensors a Contact lens sensor previously under development by Google and Novartis to measure tear glucose concentrationPrototype platform contained integrated electronics for sensor response processing and wireless transmission b Multifunctional wearablelens for monitoring both glucose in tears and intraocular pressure using enzymesmart sensor system incorporated onto a contactfunctionalized graphenesilver nanowire hybrid nanostructures c A wireless glucose sensor incorporated into a contactlens platformwith wireless power transfer circuitry and display pixels for a fully integrated and transparent platform that does not hinder vision dWearable contact lens tear glucose biosensor applied to an artificial eye with schematic representation of smartphonebased quantification of glucose levels through reflection of incident light by the photonic microstructure within the lens The smart contact lens systemintegrated with a glucose sensitive hydrogel monitors changing glucose concentrations in vitro without complicated fabrication proceduresand allows rapid response time for continuous measurements e NovioSense electrochemical tear glucose sensor A small springlikesensing device is designed to be placed within the conjunctival fornix for continuous access to tear glucose reference [] withpermission Saliva is readily available but suffers from analyte variabilityeg temperature and concentration resulting from the presence of liquids of varying temperatures over time in the oralcavity hot vs cold drinks [] Patient acceptance of a devicein the oral cavitygiven that some saliva biofluid sensors aremouthguards or otherwise bulkyobtrusiveis another issueFig 3B [] 0cEPMA Journal Fig 3B Salivabased biosensors a Mouthguardbased wearable salivary uric acid biosensing platform with integrated wireless electronics andanalysis of salivary uric acid concentrations b Mouthguardbased sensor for glucose monitoring in saliva with onbody applicationand analysis of increasing glucose concentrations c Onbody depiction and crosssectional configuration of radio frequency trilayertoothmounted sensor for wireless monitoring of food consumption This dielectric sensor fabricated with biocompatible materials iscapable of being mounted onto tooth enamelto detect foods and fluids during ingestion when functionalized with analytesensitivelayers Projected uses were for detection of sugars alcohol salinity pH and temperature d Operational principles and electronicsconfiguration of a wireless usercomfortable sensing platform for longrange oral monitoring of sodium intake during hypertensionmanagement reference [] with permissionSometimes It Takes Guts to MonitorConfirmation of ingestion of prescribed medications particularly in unreliable patients eg dementia is another biosensing challenge One solution is the smart pilla capsulecontaining a microsensor that is swallowed monitoringwhether the medication is present in the stomach [] Thesmart pill communicates with a skin patch which not onlydocuments that the pill was swallowed and when but also ifdesired blood pressure pH and temperatureFor continuous monitoring a sensor can be stationed in thegut most likely the stomach Such monitoring could includemedication ingestion pH controlled drug delivery and imaging of the gut lining An ingestible sensor that is selfpowered by stomach acid in contact with zinc and copper electrodes on the sensor surface is being developed[] Another ingestible capsule under development usesa microneedle that inserts into the stomach wall to deliver a drug eg insulin [] 0cEPMA JournalWear Your Heart on Your Sleeve Wear Your Brain onYour HatThe topic of brain biomonitoringfrom EEG to nextgeneration brainmachine interfaces BMIsis beyond the scope of this but has been recentlyreviewed [] An area of concern regarding brainbiomonitoring is directtoconsumer DTC marketingof devices that are of undocumented value or possiblerisk [] Brain biomonitoring information obtained through DTC marketing raises questions of bothpersonal privacy and ultimate use of such data bymarketers Increasing DTC availability of brain electrical stimulation eg via a skullcap notably transcranial direct current alternating current and randomnoise stimulation techniques raises questions of safety [] Ethical considerations regarding DTC brainbiomonitoring and biostimulating remain unresolved[]Conclusions and Expert RecommendationsThe field of wearable and implantable biosensors is evolvingso rapidly that no review truly reflects the stateoftheartAdvances in the TDI and AI promise that such devices willnot only enhance diagnostic capabilities but also provide awealth of information for improved treatmentsSpecific recommendationsIncorporating the latest technology into biosensorsfromnanotechniques to microfluidicsis essential Asmartphone from ten years ago would be unacceptablein the consumer marketplace outdated diagnostic techniques in medicine are similarly unwarranted Similarly the latest AI is necessary to analyze the hugeamounts of data that wearable and implantable biosensorsprovide The consumerpatient must be involved in device development from the outset What may appear wonderful inthe lab or the boardroom may prove a failure in the marketplace and social media Consumerpatient acceptanceCPA is crucial for widespread adoption Flexibility is key Some patients may prefer a patch forcontinuous glucose monitoring others a smart contactlens and others an implanted device requiring a minorprocedure for implantation but not frequent replacementWhat works in a highincome country such as Belgiummay not work in a lowincome country such as BurkinaFaso Legislation and safeguards regarding the huge amounts ofpersonal medical data generated by wearable and implantable biosensors is essential since data collection and storage systems can be hacked This is especially crucial withregard to biomodulating devices eg cardiac pacemakers brain stimulation and controlled drug deliverysystems Given the vulnerability to hacking wearable and implantable biosensors require the same caution as other widespread threats to population health eg toxins both liquid and aerosol biological warfare agents and radiationReferences Weichert W My watch kept on alarming all night aboutmy heart rate Oxford Med Case Rep Seizario detecting seizures and falls Available seizariohealthhappycom [Accessed Apr ] Heikenfeld J Jajack A Feldman B Granger SWGaitonde S Begtrup G et al Accessing analytes inbiofluids for peripheral biochemical monitoring NatBiotechnol Guk K Han G Lim J Jeong K Kang T Lim EK et alEvolution of wearable devices with realtime diseasemonitoring for personalized healthcare Nanomaterials Khan S Ali S Bermak A Recent developments in printable flexible and wearable sensing electronics forhealthcare applications Sensors Kim J Campbell AS EstebanFernandez de Avila BWang J Wearable biosensors for healthcare monitoringNat Biotechnol Someya T Amagai M Toward a new generation ofsmart skins Nat Biotechnol Waltz E Sweet sensation Nat Biotechnol McCarthy A The biomarker future is digital ClinicalOMICS 2020JanFeb24 Massaroni C Nicolo A Lo Presti D Sacchetti MSilvestri S Schena E Contactbased methods for measuring respiratory rate Sensors Faisal AI Majumder S Mondal T Cowan D Naseh SDeen MJ Monitoring methods of human body jointsstateoftheart and research challenges Sensors McDonnell S Ingestible sensors powered by stomachacid Tech Briefs 2018Aug45Jarchum I To the stomach and beyond Nat Biotechnol Frank JA Antonini MJ Anikeeva P Nextgenerationinterfaces for studying neural function Nat BiotechnolIenca M Haselager P Emanuel EJ Brain leaks and consumer neurotechnology Nat Biotechnol 0c Wexler A Separating neuroethics from neurohype NatBiotechnol Jarchum I The ethics of neurotechnology NatBiotechnol The Navarra Genomes Project NAGEN Benefits for Predictive Preventive and PersonalizedMedicinePasalodos S1 Salgado J1 Miranda M1 Maillo A1Matalonga L2 Beltr¡n S2 Carmona R3 P©rezFlorido J3Etayo G4 Lasheras G4 Bernad T4 G³mezCabrero D1Angel Gonz¡lez L5 Brennan P6 Gut I2 Dopazo J3Pinillos I4 Lasa I1 Alonso A11Navarrabiomed Complejo Hospitalario de NavarraUniversidad Pºblica de Navarra UPNA IdiSNAPamplona Spain2Centro Nacional de An¡lisis Gen³mico CNAGCRGCenter for Genomic Regulation Barcelona Institute ofScience and Technology BIST Barcelona Spain3rea de Bioinform¡tica Fundaci³n Progreso y Salud Nodode Gen³mica Funcional INBELIXIRes Bioinform¡ticade ER BiERCIBERER CDCA Hospital Virgen delRoco Sevilla Spain4Navarra de Servicios y Tecnologa NASERTIC Spain5AVANTIA Pyramide Asesores Spain6NENC NHS Genomic Medicine Centre Newcastle uponTyne UKCorresponding author Dr Angel Alonso GenomicMedicine Unit Navarrabiomed Complejo Hospitalario deNavarra Universidad Pºblica de Navarra UPNA IdiSNAEPMA JournalCIrunlarrea Pamplona Spain emailangelalonsosancheznavarraesKeywords predictive preventive personalized medicinegenomics next generation sequencing NGS whole genome sequencing WGS rare diseases eHealth bioinformatics big data ICPerMed multiomicsBackgroundIn the past few years extraordinary developments in the fieldof next generation sequencing NGS technologies such aswhole genome sequencing WGS have made it possible forclinicians to have access to a huge amount of biological information which could potentially explain complex genetic diagnoses genetic predisposition to severe diseases reproductiverisks and inappropriate responses to certain medicationsThese advances herald a new era of predictive preventive personalized medicine PPPM although incorporation into clinical practice has proved to be challenging [] NAGEN is a Spanish regional pilot study to implement recentadvances of cutting edge genomic research technology intoreal clinical practiceGoal materials and methodsNAGEN s main goal is the implementation of the wholegenome sequencing WGS derived information as a clinicaltool for the development of PPPM in the Public Health ServiceA scientific implementation approach was used to identify andcategorize both the local barriers and facilitators to acceleratethe incorporation of translational genomics intohealthcare see Fig Fig Local barriers for genomic medicine implementation in Navarra NAGEN project 0cEPMA JournalKey Actions for this implementation Subjects NAGEN is recruiting patients and theirrelatives affected with one condition from a list of nearly rare diseases RD Albeit rare joint RDs prevalence ishigh with a very high social impact wide multidisciplinary medical coverage and a high rate of identifiablegenetic causes These features make it possible to involve themedical community raise population awareness and offergood support to evidencebased medicine practice The rateof genome per inhabitants facilitates a wide participation from patients and health professionals Results and incidental findings Pertinent findingsexplaining the referral condition secondary findings onpersonal and reproductive risks of severe inherited diseases and pharmacogenomic variants determining drugsdose and toxicity are reported based on patients choiceproviding the necessary evidence of the effectiveness ofmedical interventions based on genomic medicine Newgenetic counselling interventions variant validation andreporting pathways have been put in place for the bestprovision of services Electronic health record EHR adaptation The existingEHR has been modified to host a newly designed recruitment tool which enables and guides the identification andimmediate referral of patients from any point in theNavarre health system network An additional development also makes it possible that clinically actionable genomic results are available for participants doctors withall other clinical information across the system Clinical research A number of new exciting genomicresults potentially providing new insights into the geneticbasis of RDs and additional information on populationgenomics are being produced by NAGEN offeringexceptional material to support new research It is a maingoal of the Project to ensure an adequate data harmonization which enables data sharing for research under anappropriate regulatory and legal framework Optimized use of preexisting public infrastructures Inorder to overcome the lack of local facilities NAGEN externalizes WGS sequencing services to CNAGCRG the Spanish world leader public centre for genomicanalysis Bioinformatic analysis also relies primarily onCNAGCRG through the RDConnect GenomePhenome Analysis Platform which was deployed for theproject to store analyse and interpret the genomic datamaking use of the phenotypes encoded with the HumanPhenotype Ontology HPO and the experts from theBioinformatics Platform of the Rare Diseases Spanishnetwork CIBERer through the Interactive VariantAnalysis IVA tool based on the genome browserGenome Maps but expertise in this field has graduallybeen transferred to the newly created local TranslationalBioinformatics Unit during the course of the ProjectICT New ICT solutions have been adopted for NAGEN allowing the storage and high performance managing of massive genomic data through an innovative partnership with NASERTIC a local company providing dataanalysis infrastructures such as the new IBM POWER processor which build on crossdisciplinary collaborationin research and development with the local industry ELSI While genetic data protection is widely regulated forclinical and research purposes within the NAGEN project the local Health Research Authority has specificallyresolved that the massive genomic information resultingfrom WGS will also be part of the patients medical recordand it will accordingly be protected and stored In order toenable the use of genomic data for research the constitutionof a Genomic Library has been proposed which wouldaccept specific research enquiries on anonymized genomicsequences upon pertinent EC approval This scenario requires a new regulatory legal framework which has alsobeen explored through a specific partnership with Avantia from the Pyramide group a local consulting companywith wide experience in data protectionResultsKey results to date Clinical and preclinical results Around patients have todate followed through the aboveoutlined pathway and of the families have now found the longawaited geneticcause for their previously unexplained condition and nowhave hope of an improvement of their clinical care based onthese findings Remarkably of these diagnoses wereattributed to genes previously unknown to cause a humandisease or causing different phenotypes than those previously described Fig Additionally of participants carriedgenetic predispositions to severe diseases had reproductive risks and had pharmacogenomic actionable variants influencing prescription Table Further candidategenomic variants potentially explaining patients diseaseshave been identified in an additional of participatingfamilies which provides an extended base for new collaborative research projects Interestingly about differentmedical specialities have referred patients to NAGEN indicating a desirable multidisciplinary involvementin this implementation initiative Healthcare workforce education and public empowerment Monographic NAGEN symposiums hospitalbriefings clinical sessions and face to face meetings havebeen anized ing the participation to all medicalprofessionals in the region Moreover the designatedspecialities physician champions especially commissioned to facilitate recruitment help with the clinical 0cinterpretation of genomic variants and to spread the wordreceived category and CME credits from a NAGEN tailored genomics education programme Public involvement has also been possible through a press conference which was widely covered by national general andmedical press and social media conferences at theScience Week and Rare Diseases Day a specificwebsite wwwnagen1000navarraes and communicationsto national and international congresses Sustainability After deducting marginal costs due to theTranslational Bioinformatics Unit establishment and ICTinfrastructures the costeffectiveness analysis CEA recEPMA Journalognized a full running costs of ¬ per RD diagnosisprior to familial cascade genetic testing and including duoand trio studies costs when necessary compared with¬ average cost per diagnosis estimated for thestandard of care pathway [] Considering that costbenefit analysis CBA outperforms CEA for RDwe conducted a survey of all participants whichshowed that more than of them would be willing to pay more than ¬ for the genomic information they received after their participation inNAGEN regardless of whether their diagnosiswas ultimately achieved or notFig Pertinent clinical findings a Piechart showing the performance of genomic diagnoses achieved by NAGEN and of strong and mild candidates genomic variants b Table listing OMIM codes and diagnoses in red cases with no OMIM codificationTable Clinical Actionable Incidental FindingsClinical Actionable Incidental FindingsTypeConsentN of patients of casesDisease PredispositionReproductive RiskPharmacogenomicpatients and it was awarded as the Best Practice in PersonalisedMedicine by ICPerMed in Significantly it resulted in setting the new Genomic Medicine Unit of Navarrabiomed and itsNAGEN strategy which has now raised ¬ million for RDprojects on PM over the past years NAGEN is an exemplarpractice for the Spanish Senate Initiative for a National Strategyon Genomics and PM and has given rise to the launch ofthe Navarra Government Strategy on Personalised Medicine announced in November Conclusions and expected impactsGenomics has become a major contributor to multiomics andPPPM related approaches in management of major and fatal pathologies such as cancer diabetes and stroke [] NAGEN illustrates how translational research and innovation in thefield of genomics and PPPM is already delivering real benefits toAcknowledgements This study will always be in debt to all participa
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purpose to explore a new therapeutic option for patients with hepatocellular carcinoma hcc the efficacy and safety of a group of traditional chinese medicines banxia xiexin recipe as monotherapy for patients with advanced hcc was studied materials and methods the study included patients with advanced hcc from august to august for analysis these eligible patients received treatment with banxia xiexin recipe for at least month the primary endpoints were progressionfree survival pfs and overall survival os the secondary efficacy endpoints included objective response rate orr and disease control rate dcr in addition safety was also assessed results the median treatment duration of these patients was months range months and followup is still ongoing the median pfs was months confidence interval [ci] months and the median os was months ci months the orr was and the dcr was in the subgroup analysis the median os in the transcatheter arterial chemoembolization tace group was not reached and the median os in the no tace group was months ci months in addition no drugrelated serious adverse events were observed during the study this is the first clinical analysis of traditional chinese medicine as a single treatment for advanced hcc the obtained results are encouraging as they suggest that this panel of chinese herbs is safe and it may be effective for patients with advanced hcc in a realworld clinical settingkeywordshepatocellular carcinoma herbs tace banxia xiexin recipe clinical researchsubmitted december revised june accepted june introductionliver cancer is one of the most frequent malignant tumors and the third leading cause of cancerrelated deaths worldwide both the incidence and mortality rates for liver cancer are increasing while the overall incidence and mortality for all cancers combined tend to decline12 hepatocellular carcinoma hcc is the most common and deadly form of liver cancer in china chronic hepatitis b virus hbv infection and subsequent liver fibrosis and cirrhosis are the 1affiliated hospital of chengdu university of traditional chinese medicine chengdu sichuan province chinathese authors contributed to the work equally and should be regarded as cofirst authorscorresponding authorshaoquan xiong department of medical oncology affiliated hospital of chengdu university of traditional chinese medicine chengdu sichuan province china email xsquan106163comcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies leading causes of liver cancer due to the lack of effective treatment options as well as the high recurrence and metastasis rates hcc is associated with an extremely poor prognosis at present surgical resection and liver transplantation are effective treatments for liver cancer but only a few patients are surgical candidates due to tumor extension poor hepatic functional reserve or underlying liver cirrhosis34 even for the few lucky patients who have completed the surgery the recurrence rates at years following liver resection have been reported to exceed for patients with advanced liver cancer it is essential to improve general wellbeing promote quality of life and prolong survival time it is well known that chemotherapy effects in patients with advanced liver cancer are not outstanding67 some patients can consider local treatment such as transarterial chemoembolization tace the progressionfree survival pfs of patients receiving tace treatment is reported to be months8 molecular targeted drugs are also one of the options for patients with advanced liver cancer such as sorafenib and lenvatinib however according to reports on sorafenib the only approved molecular targeted therapy for advanced hcc the median pfs of patients treated with sorafenib was only months in the asiapacific region9 in recent years immunotherapy which has been attracting worldwide attention is also becoming an important treatment method for example nivolumab and pabolizumab which have been approved by the food and drug administration for secondline treatment of advanced hcc can bring certain benefits to patients with advanced liver cancer but are expensive and difficult to afford for many patientsin china traditional chinese medicine tcm therapies which can date back more than years are widely used in cancer treatment10 banxia xiexin decoction was created by the ancient doctor zhang zhongjing it is mainly used to treat digestive diseases such as bloating and abdominal pain it has a history of more than years in a previous retrospective study it was found that banxia xiexin decoction can bring survival benefits to patients with advanced liver cancer and one of them achieved complete response cr11 as a tcm hospital many patients with advanced liver cancer come to our hospital to undergo chinese medicine for treatment therefore we designed a prospective study of banxia xiexin decoction as monotherapy for advanced liver cancer and analyzed its treatment effect and safetymethodsstudy design and patient enrollmentin this prospective openlabel study patients from the oncology department of the affiliated hospital of the chengdu university of traditional chinese medicine chengdu china were recruitedaccording to the american association for the study of liver diseases criteria eligible patients were adults with advanced hcc confirmed by pathological assessment or noninvasive assessment12 these patients had not received any cancerrelated treatment within weeks before the start of study treatment and the disease had progressed after the prior treatment in addition these patients had to have at least one measurable lesion defined by modified response evaluation criteria in solid tumors for hcc mrecist and recist version inclusion criteria also included eastern cooperative oncology group ecog performance status of to an estimated life expectancy of months childpugh liver function class a or b adequate an function such as white blood cell count ¥ cellsl neutrophils ¥ cellsl and platelets ¥ cellsl and the absence of any serious comorbidities such as significant cardiac cerebrovascular hepatic or nephritic abnormities or other important an dysfunctionskey exclusion criteria included patients with low compliance or use of other antitumor treatments at the same time diagnosis of other malignant tumors and hcc with brain metastasisthe protocol was approved by the ethics committee of the affiliated hospital of chengdu university of traditional chinese medicine no 2015bl003 all patients provided written informed consent before study entrystudy treatment and proceduresthe enrolled patients received treatment with banxia xiexin recipe in the form of decoction the herbal components and their doses in the banxia xiexin recipe decoction are listed in the appendix with the use of an automatic herbboiling machine the decoction volume of each prescription was about ml patients were instructed to take ml each time times dailytreatment was continued until disease progression death development of unacceptable toxicity patients refusal to continue or at the discretion of the investigator when the disease progressed treatment could also be continued for month to assess tumor response as long as the patient agreed to do sofollowing an initial baseline assessment within days of the start of study treatment the investigator assessed tumor response by using computed tomography ct scan after to months of treatment subsequently followup assessments were done every months until the patient died adverse events were recorded continuously from enrollment to the end of the final study visitoutcomesthe primary endpoints were pfs time from enrollment to radiological disease progression or death and overall survival os time from enrollment to death due to any cause the secondary efficacy endpoints included the proportion 0cwang et al of patients who had an objective response complete or partial response [pr] according to recist version namely objective response rate orr and disease control rate dcr the proportion of patients who had a cr pr or stable disease besides safety was also assessed by the incidence and nature of adverse eventsstatistical analysisthe pfs and os were estimated using the kaplanmeier method all statistical analyses were performed using graphpad prism software version graphpad software inc and logrank testresultsfrom august to august a total of patients with advanced hcc were enrolled from the oncology department of the affiliated hospital of chengdu university of traditional chinese medicine however only the results of the mediumterm analysis are presented here since patients were still on treatment and had not yet reached the assessment time point therefore patients were finally included in the current analysis meaningful results were found to report in this midterm analysis of course we will update the results according to the latest followup data in the future the baseline characteristics of the included patients are shown in table notably patients were without any treatment in this study all patients who were previously treated with surgery or tace had progressive disease which was similar to the clinical trials of sorafenib9 the number of patients receiving chemotherapy and molecular targeted therapy is and respectively patients who had received previous systemic therapy were excluded in the clinical trials of sorafenib but these patients had progressive disease after the prior treatment according to recist version in this studythe data cutoff date for the current analysis was august the median treatment duration of these patients was months range months and followup is still ongoing since if the disease progressed treatment could also be continued for month to assess tumor response as long as the patient agreed to do so or if thepatient would like to use it as an adjuvant treatment to other treatment such as tace moleculartargeted therapy and so onin the primary analysis the median pfs was months confidence interval [ci] figure and the median os was months ci figure thirtysix patients died all due to disease progression the 1year survival rate was notably most of these patients received prior treatments such as tace and targeted molecular therapy before enrollment in order to explore the potential effect of previous treatment we performed a subgroup analysis the median pfs months ci was longer in the subgroup of patients who had previously received tace compared with the median pfs months ci who had not previously received tace thus the treatment effect in this type of patient seems to be better p figure the median os of advanced hcc patients who had previously received tace was not reached and the median os of subgroups who had not received tace was months ci as shown in figure in terms of tumor response after treatment with banxia xiexin recipe patients achieved a pr had stable disease the orr was and the dcr was table particularly the imaging data of typical cases were presented as follows the first patient was a 58yearold male who was diagnosed with advanced hcc with metastasis to the right kidney on august at baseline the ct scan showed that the size of the target lesion in the liver was cm cm cm figure 5a blood tests revealed elevated αfetoprotein µgml and the ecog performance status score was this patient had not received any anticancer treatment previously after months treatment with banxia xiexin recipe the ct scan showed that the target lesion in the liver shrank to cm cm figure 5b and reached a pr according to recist version the ecog performance status score improved from to the patient is still currently in treatment and has not yet had progressive diseaseone 54yearold male patient was first diagnosed to have hcc in december he was treated by surgical resection on january no antineoplastic treatment was done thereafter when a regular review was conducted on september it showed intrahepatic recurrence and right lung metastasis and then he received treatment with chinese herbs at baseline the ct scan showed that the size of the target lesion in the liver was cm cm cm figure 6a blood tests revealed elevated αfetoprotein µgml and the ecog performance status score was after a 32month treatment with modified xiexin recipe the ct scan showed that the target lesion in the liver shrank to cm figure 6b and reached a pr according to recist version the duration of the pr was months he continued to insist on treatment after the disease progressed and he died in october concerning safety no adverse events were observed except for the occurrence of mild nausea three patients reported mild nausea after taking pills but the symptom was relieved spontaneously after week without any 0c integrative cancer therapies table the clinical characteristics of the patients with advanced hepatocellular carcinomavariablesnumber of patientspercentageage years ¥sex male femaleecogps childspugh class a bhepatitis b virus infection yes noliver cirrhosis yes noafp µgl ¥ tumor size cm ¥pvtt yes noextrahepatic spread yes noinvolved disease sites per patient ¥previous treatment no sur tace rf che mttajcc stage iiibivaivbabbreviations ecogps eastern cooperative oncology groupperformance status pvtt portal vein tumor thrombus sur surgical resection tace transcatheter arterial chemoembolization rf radiofrequency che chemotherapy mtt molecular targeted therapy ajcc american joint committee on cancerfigure kaplanmeier analysis of progressionfree survivalfigure kaplanmeier analysis of overall survivalfigure kaplanmeier subgroup analysis of progressionfree survival stratified by prior tace treatmenttreatment in addition no serious adverse events such as liver or kidney damage were detected during the followup period thus the tcm treatment was safe and welltolerated in these patients with advanced hccaccording to the international tumor chemotherapy adverse drug evaluation systemctcae v4014 patients included in this study were evaluated for safety during the followup the adverse reactions were nausea 0cwang et al control effect of tcm as monotherapy for patients with liver cancer banxia xiexin decoction has been used for treating digestive diseases for more than years in our previous retrospective analysis it was found that banxia xiexin decoction can relieve the symptoms of patients with advanced liver cancer inhibit tumor growth and prolong survival one of the patients tumors bulk was significantly reduced to reach cr status this state has been maintained for up to months11 therefore it is necessary to carry out further scientific researchto the authors best knowledge this is the first prospective study of chinese herbs alone for the treatment of advanced hcc that used the widely accepted endpoints of pfs os orr and dcr according to recist version in the present study the median pfs was months in clinical studies of sorafenib an oral multikinase inhibitor which is the first molecularly targeted drug approved by multiple countries around the world for firstline treatment of advanced hcc the median pfs was months in western countries19 and only months in the asiapacific region9 in current research work all patients were from china and in particular of patients had been diagnosed with liver cirrhosis when enrolled table the manifestations of liver decompensation such as splenomegaly and portal hypertension had appeared however the childpugh class was a or b it had been reported that hcc patients with liver cirrhosis showed significantly worse os than noncirrhotic hcc patients months vs months p even so the median pfs reached months and the median os reached months in a realworld clinical setting these findings suggest that the efficacy of the banxia xiexin recipe may be superior to that of sorafenib in patients with advanced hcc although this was not a headtohead comparison a further study directly comparing chinese herbs versus sorafenib for advanced hcc will be conducted in the futurein the reflect trial21 a subgroup analysis of the chinese population found that the os of the lenvatinib group was significantly longer than that of the sorafenib group by months with statistical differences in addition when combined with hbv infection the survival benefit was significantly different between the lenvatinib group and the sorafenib group os months vs months therefore it was approved by the china food and drug administration on september and approved for domestic listing it is important to note that the experimental conditions for entering the reflect trial were strict excluding ecog performance status patients with a score of or and a large liver mass however in patients enrolled in the present study had liver masses cm and had ecog scores of to nevertheless the median os of this study was figure kaplanmeier subgroup analysis of overall survival stratified by prior tace treatmenttable tumor response according to recist version variablesnumber of patientspercentagecrprsdpdorrdcrabbreviations cr complete response pr partial response sd stable disease pd progressive disease orr objective response rate dcr disease control ratevomiting diarrhea abdominal distension oral ulcers the painless mass of the scalp and so on the overall adverse reaction rate was all of which were grade i to ii adverse reactions and responded to symptomatic treatment there was no adverse reaction of grade iii or above and no drugrelated lethal events occurred specific adverse reaction events and their incidence rates are shown in table discussionit is wellknown that hcc is one of the leading causes of cancerrelated mortality worldwide the longterm survival rate remains unsatisfactory due to the high recurrence and metastasis rates after conventional treatment to improve the outcome of patients with hcc there is an urgent need for more effective therapies at present tcm is often used as an adjuvant therapy to conventional treatments such as tace in treating patients with liver cancer1518 to reduce the toxicities of other treatments relieving symptoms and improving quality of life tcm has only been considered as a complementary or alternative treatment for cancer patients and there is a paucity of data regarding the tumor 0c integrative cancer therapies figure a b the imaging data of one partial response patient diagnosed as advanced hepatocellular carcinoma with metastasis to the right kidneyfigure a b the imaging data of another partial response patient diagnosed as advanced hepatocellular carcinoma with intrahepatic recurrence and right lung metastasismonths therefore for patients with worse economic and physical conditions banxia xiexin decoction may be a better choice for many patients with advanced liver cancer in addition some other new drugs have been tried for the treatment of hcc unfortunately all phase trials assessing novel systemic drugs2224 have failed to improve outcomes over sorafenibin immunotherapy immune checkpoint inhibitors represented by pd1pdl1 monoclonal antibodies have made rapid progress the results of the checkmate040 study25 showed that nivolumab achieved an os of months in newly diagnosed patients and a 150month survival benefit in patients who had undergone sorafenib treatment pabrizumab which is also a pd1 immunosuppressant used in the keynote224 study26 for patients with advanced hcc who had previously progressed with sorafenib achieved a pfs of months median os of months compared with the enrollment conditions for keynote224 the condition of the patients enrolled in the present study are more complicated the largest diameter of the mass in of patients is ¥ cm of patients are in stage iv of patients had hbv infection and of patients had hepatic cirrhosis even so this study still achieved a pfs of months higher than the above chemotherapy and immune drugs it can be seen that the efficacy of banxia xiexin decoction for the treatment of advanced liver cancer may be better than the abovementioned immune drugs and it has the potential to become one of the important methods for liver cancer treatmentfurther subgroup analysis revealed that previous treatment might affect the efficacy of chinese herbs in the current analysis patients had been treated with tace previously whose outcome was slightly better compared with those who had not been treated with tace previously pfs vs months p in terms of os the difference is more obvious os undefined vs months p subsequent treatment with banxia xiexin decoction seems to have a greater benefit for patients with advanced liver cancer who have progressed with interventional therapyoverall the orr was and the dcr was in this study in comparison with the data reported in 0cwang et al table adverse reactionsgrade igrade iigrade iiigrade ivany grade grade iii nauseavomitingdiarrheabloatingulcersuperficial painless mass related literature the orr and dcr were respectively and in the orient trial of sorafenib919 it should be noted that the ecogps score of the included patients was to in our study but to in the orient trial indicating that tcm treatment may have a wider scope of application and may benefit more patients compared with targeted therapyin terms of safety no serious adverse events such as liver or kidney damage were observed during the followup period thus banxia xiexin recipe was generally safe and welltolerated in patients with advanced hccas for the antitumor mechanism of tcm it has been reported that tcm or its extracts have direct antitumor effects such as pinellia27 ginseng2829 coptis30 glycyrrhiza31 atractylodes macrocephala32 tangerine peel33 and scutellaria34 the effect of inducing apoptosis is also noted such as with berberine contained in coptis3537 the effect of regulating immunity is noted such as with pinellia27 ginseng3839 atractylodes macrocephala40 and milkvetch root41 antitumor vascular growth such as that of scutellarin4243 can inhibit protein kinase b akt phosphorylation downregulate vegf and inhibit tumor angiogenesis thus playing a role in inhibiting tumor growth and metastasis also it has been reported that the imbalance of intestinal flora not only affects the occurrence and development of intestinal cancer and irritable bowel syndrome but also is closely related to the occurrence of breast cancer lung cancer liver cancer and other malignant tumors4446 however atractylodes macrocephala scutellaria baicalensis and coptis4047 can indirectly inhibit tumors by regulating intestinal flora it is worth mentioning that much basic research has found that pinellia48 coptis4950 ginseng51 scutellaria52 atractylodes macrocephala53 and astragalus54 have antihepatoma effects for example berberine4950 a constituent of the extract of coptis in banxia xiexin decoction can inhibit the pi3kakt pathway of liver cancer downregulate mhcc97h and hepg2 cells phosphorylate the expression of akt and pi3k and inhibit the growth of liver cancer cells in a dosedependent manner besides it can induce cell cycle arrest and promote apoptosis to treat hccthere are only a few patients with tumor shrinkage in the present work it is believed that the direct antitumor effect of banxia xiexin decoction is not outstanding and there may be other mechanisms after analysis it was observed that the more prominent effect of banxia xiexin decoction on liver cancer is reflected in os which is similar to the efficacy characteristics of pd1 immune checkpoint inhibitors banxia xiexin decoction contains pinellia27 ginseng3839 atractylodes40 and astragalus41 medicinal herbs with immunomodulatory effects for example ginseng38 could relieve immunosuppression by increased viability of natural killer cells enhanced immune an index improved cellmediated immune response increased content of cd4 and ratio of cd4cd8 and recovery of macrophage function therefore it was speculated that liver cancer patients in this study may benefit from indirect antitumor mechanisms such as antivascular immunomodulation and others in addition some studies5556 confirmed that some ingredients of banxia xiexin decoction could improve liver function and regulate gastrointestinal function these comprehensive effects can also partially explain why liver cancer patients in this study seem to obtain longer pfs and osin china most patients with hcc have liver diseases such as hepatitis and cirrhosis among the patients enrolled in this study patients had a history of hepatitis b and patients had liver cirrhosis hcc and basal liver disease often affect each other and form a vicious circle the scutellaria baicalensis ginseng licorice tangerine peel and astragalus in banxia xiexin decoction all have liverprotective effects and licorice has a direct antihbv and hepatitis c virus effects5758 from this point of view this is also one of the reasons why banxia xiexin decoction may be effective in treating liver cancer although much basic research has confirmed the antitumor effects of single herbal extracts in banxia xiexin decoction a single herb may not have a good antitumor effect in clinical use even though the decoction affects liver cancer after being prescribed still each herb may play its respective role whether it causes a new antitumor effect or through compatibility with the effects of other herbs the antitumor mechanism is unclear and needs further studylimitationsthe report is limited by its singlearm research design and cannot be directly compared with standard chemotherapy 0c integrative cancer therapies targeted therapy or immunotherapy at the same time the small number of cases in this study is also one of the disadvantages but this study is still continuing and there will be more data analysis in the future due to the willingness of patients to choose tcm for treatment in tcm hospitals it is difficult to avoid certain limitations when we adopt the single treatment of tcm although oral chinese medicine decoction is difficult to take according to scientific equal dosage and concentration it is in line with the use habits of tcmsin this is the first prospective study of chinese herbs as monotherapy for the treatment of advanced hcc that used the internationally accepted endpoints of pfs os orr and dcr the findings are encouraging as they suggest that this panel of chinese herbs is safe and may be effective for patients with advanced hcc in a realworld clinical setting further studies are required to assess the comparative efficacy of tcm treatment versus other antitumor therapies in this patient populationappendixcomponents and their doses in the modified xiexin recipe decoction chinese latin and english nameschinese namelatin nameenglish nameplace of origin production batch dose gbanxiahuangqinganjiangrenshenhuangliandazaogancaobaizhuchenpihuangqirhizoma pinelliaeradix scutellariaerhizoma zingiberisradix ginsengrhizoma coptidisfructus jujubaeradix glycyrrhizaerhizoma atractylodis macrocephalaepericarpium citri reticulataeradix astragalipinellia tuberbaikal skullcap rootdried gingerginsengcoptisjujubae chinese datelicorice rootlargehead atractylodes rhizometangerine peelmilkvetch rootsichuanshanxisichuanjilinsichuanxinjiangsichuanzhejiangsichuangansuacknowledgmentsthe authors acknowledge the contributions of the other investigators in this trialauthor contributionslijuan wang jianlong ke and shaoquan xiong conceived and designed the study lijuan wang jianlong ke cui wang yaling li qiuyue luo rui cai qian ding panpan lv and tingting song collected the data lijuan wang jianlong ke and cui wang processed the data lijuan wang and jianlong ke wrote the manuscript all authors have participated in the drafting review and approval of the report and the decision to submit for publicationdeclaration of conflicting intereststhe authors declared no potential conflicts of interest with respect to the research authorship andor publication of this fundingthe authors disclosed receipt of the following financial support for the research authorship andor publication of this this research was supported by the national natural science foundation of china no and sichuan provincial science and technology department project no2017jy0327orcid idslijuan wang jianlong ke panpan lv orcid0000000317048847 orcid0000000150587581 orcid0000000271727659references torre la bray f siegel rl ferlay j lortettieulent j jemal a global cancer statistics ca cancer j clin doi103322caac21262 chen w zheng r baade pd et al cancer statistics in china ca cancer j clin doi103322caac21338 hung h treatment modalities for hepatocellular carcinoma curr cancer drug targets doi102174 vitale a trevisani f farinati f cillo u treatment of hepatocellular carcinoma in the precision medicine era from treatment stage migration to therapeutic hierarchy hepatology published online february doi101002hep31187 bruix j sherman m practice guidelines committee american association for the study of liver diseases management of hepatocellular carcinoma hepatology doi101002hep20933 lee dw lee kh kim hj et al a phase ii trial of s1 and oxaliplatin in patients with advanced hepatocellular carcinoma bmc cancer doi101186s1288501840399 0cwang et al zaanan a williet n hebbar m et al gemcitabine plus oxaliplatin in advanced hepatocellular carcinoma a large multicenter ageo study j hepatol doi101016jjhep201209006 wang y cui w raltitrexed based transcatheter arterial chemoembolization tace for unresectable hepatocellular carcinoma a singlecenter randomized controlled study chemotherapy doi104172216777001000192 cheng al kang yk chen z et al efficacy and safety of sorafenib in patients in the asiapacific region with advanced hepatocellular carcinoma a phase iii randomised doubleblind placebocontrolled trial lancet oncol doi101016s1470204508702857 anonymous miraculous pivot ling shu jing originally published during warring states period to bc peoples health press shaoquan x lijian w qiuyue l et al retrospective analysis of modified xiexin recipe combined retrospective analysis of modified xiexin recipe combined chin j integr tradit western med bruix j sherman m american association for the study of liver diseases management of hepatocellular carcinoma an update hepatology doi101002hep24199 lencioni r llovet jm modified recist mrecist assessment for hepatocellular carcinoma semin liver dis doi101055s00301247132 gao wj liu yy yuan cr international evaluation system for adverse events of chemotherapeutic drugs in cancer treatment ctcae v40 tumor meng mb cui yl guan ys et al traditional chinese medicine plus transcatheter arterial chemoembolization for unresectable hepatocellular carcinoma j altern complement med doi101089acm20080060 xu h deng y zhou z huang y chinese herbal medicine chaihuhuaji decoction alleviates postem
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since leung reported a single case of lowdose aspirin lda induced multiple smallintestinal ulcers in many investigators have described ldainducedsmall intestinal mucosal injuries watanabe reportedthat all of their patients who used lda had small intestinallesions that were detected using capsule endoscopy 0cgastroenterology research and practicece iwamoto investigated patients whounderwent ce for occult bleeding and erosions wereobserved in cases of whom were taking lda ornonlda nonsteroidal antiammatory drugs endo described small intestinal lesions in of subjects who took aspirin mg for weeks shiotani performed ce on young healthy individuals beforeand after medium doses of enteric aspirin were administered for days and rabeprazole mg was administered for week and found large erosions that included small intestinal ulcers in of the subjects recently direct oral anticoagulants doacs have beenadministered as alternatives to warfarin dabigatran is adirect thrombin inhibitor and rivaroxaban apixaban andedoxaban are factor xa inhibitors doacs have significantlyfewer side eï¬ects than warfarin including intracranial hemorrhage hence the number of patients taking doacs isgradually increasing [] howeverthe ï¬ndings frommetaanalyses have shown that compared with warfarinthe incidence of gastrointestinal gi bleeding is higher inassociation with doacs [ ] the causes of gi bleedingin association with doacs were bleeding from colon astric cancers and diverticular hemorrhages comparedwith warfarin dabigatran and rivaroxaban are associatedwith higher risks of gi bleeding depending on their dosesand the risk of gi bleeding associated with apixaban iscomparable edoxaban is associated with significantlyless gi bleeding at low doses mg once daily comparedwith warfarin but the risk is significantly greater at highdoses mg once daily but unlike western countries the rate of gi bleeding for both dabigatran and rivaroxaban is equivalent to warfarin in asian countries furthermore edoxaban tends to cause less digestive tractbleeding than warfarin esophageal ulcers caused bydabigatran have been described by toya kasai and okada and okada the tartaric acidcoating on dabigatran causes esophageal mucosal disorders because dabigatran persists in the midesophagus ifit is consumed without water however there havebeen no reports of smalllesions in patientswho receive doacs here we aimed to evaluate smallintestinal mucosal injuries in patients taking doacs usingvideo capsule endoscopy vceintestinal methodsthis study was a prospective openlabel nonblinded multicenter and observational study from september tomarch pat5ents taking doacs namely dabigatranrivaroxaban and apixaban for atrial ï¬brillation at saitamamedical university hospital keio university hospital saitama medical center and yokohama municipal citizenshospital were enrolled patients with severe comorbiditiesincluding severe anemia and exacerbations of heart failurethat required blood transfusion crohns disease and ileuswere excludedthe hemoglobin hb and serum ferritin levels the esophagogastroduodenoscopy egd ï¬ndings and colonoscopicï¬ndings were examined vce pillcam sb2 given imagingfigure rednessfigure erosionltd yoqneam israel was performed to examine small intestinal lesions according to the doac used redness erosionulcer and angioectasia were checked redness was a red spotfigure and erosions were deï¬ned as small and superï¬cialmucosal disruptions denuded of villi figure ulcers weredeï¬ned as large submucosal disruptions with a central areacovered with exudate and angioectasia was a patchy erythematous lesion figure the images were analyzed using theproprietary rapid software by an expert n h whohad performed more than vce examinations blindlythe type and location of smallbowel lesions were registeredalso the proportion of lesions detected between types ofdoac was evaluated and the hemoglobin hb and serumferritin levels were compared between patients with and without smallbowel lesionsthe study protocol accorded with the tenets of therevised declaration of helsinkiand it wasapproved by the institutional review boards at our institutions written informed consent was obtained from all ofthe patients this study was registered with the universityhospital medicalinformation network clinical trialsregistry umin000011527 october 0cgastroenterology research and practicetable patients characteristicsparametermean age years rangesex n malefemalecomorbid disease natrial ï¬brillationparoxysmal atrial ï¬brillationhypertensionhyperlipidemiacerebral infarctiondoac n years dabigatranrivaroxabanapixabanbayaspirin ncelecoxib nppi nh2 blocker n mean hb gdl rangedoac direct oral anticoagulant ppi proton pump inhibitor hbhemoglobinlower portion in patients table erosions wereobserved in patients and they were present in theupper portion in in the middle portion in and in the lower portion in patientstable erosions tended to occur less frequently in themiddle portion however the diï¬erence was not significantp compared with the upper portion p compared with the lower portion angioectasia was observedin patients and was present in the upper portionin patients and in the middle portion in patient and was absent from the lower portion table there were no ulcers in any patients erosions tended to bemore frequent in patients taking dabigatran or apixaban thanin patients taking rivaroxaban this diï¬erence was not significant p table no significant diï¬erences wereobserved regarding angioectasia among the patients takingthe diï¬erent doacs none of these patients had activebleeding from small intestinal lesionsthe mean hb concentrations in the patients with andlesions were gdl and gdlwithout smallbowelrespectively a diï¬erence which was not significant p the mean ferritin levels in the patients with and withoutsmallbowel lesions were mgdl and mgdl respectively a diï¬erence which was not significant p discussionthis studys ï¬ndings showed that of the patients who tookdoacs had redness had erosionsor small ulcers had angioectasia and had no abnormalities in their small bowel smallbowellesions were observed in of patients thereforethere was a high incidence of smallbowel lesions in patientsfigure angioectasiaibm®spss® statistical software version ibm corporation armonk ny usa was used for the statisticalanalyses the data were analyzed using ttests and fishersexact test resultsthirtythree patients were enrolled to participate in thisstudy but patients withdrew their consent and vce wasperformed on patients the patients mean age was years range years and males and females participated in this study twenty patients had atrial ï¬brillation had paroxysmal atrial ï¬brillation had hypertension hadhyperlipidemia and had cerebral infarction eight patientstook dabigatran took rivaroxaban and took apixabanthe mean duration of doac use was months months additionally patient took bayaspirin patienttook celecoxib patients took proton pump inhibitorsppis and patients took h2 receptor antagonists theaverage hb concentration was gdl range gdl table twentytwo patients underwent egdand atrophic gastritis was present in patients hiatal hernias in patients gastric polyps in patients erosive gastritisin patients gastric ulcer or ulcer scar in patients reï¬uxesophagitis in patients endoscopic submucosal dissectionscar for early gastric cancer in patients and an esophagealulcer in patient nineteen patients underwent colonoscopyand colonic polyps were present in patients colonic diverticulum were present in patients and a rectal ulcer waspresent in patient none of these lesions detected by egdand colonoscopy had active bleedingthe patients evaluated with vce was redness in thelower esophagus was present in patient gastric erosionswere present in patients and gastric redness was presentin patient table the patient who had redness in thelower esophagus was taking apixaban smallbowel transitwas complete in of patients smallbowellesions were observed in of patients redness was observed throughout the small intestine in patients and it was present in the upper portionin in the middle portion in and in the 0cgastroenterology research and practicetable capsule endoscopy ï¬ndingsentire small intestine observation rate n detection rate n esophagusstomachupper small intestinemiddle small intestinelower small intestineentire small intestineredness lower n erosions n redness n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions nangioectasia nno abnormalities n table findings at each small intestinal site according to the directoral anticoagulant useddabigatranrivaroxabanapixabansiteï¬ndingupper small intestineredness nerosions nangioectasia nmiddle small intestineredness nerosions nangioectasialower small intestineredness nerosions nangioectasia nentire small intestineredness nerosions nangioectasia ntaking doacs however none of these patients had activebleeding and most of the lesions were mild patients withsevere anemia or active bleeding were excluded from thisstudy hence only patients with mild symptoms wereincluded ldainduced lesions cause redness erosions andulcers [] previous studies ï¬ndings that describe the characteristics of smallbowel injuries associated with chroniclda use suggest that ulcers are observed mainly in the distalpart of the small bowel [] in this study erosionstended to be observed less frequently in the middle portionof the small bowel in the patients taking doacs howeverthere were no significant diï¬erences regarding the distributions of the lesions there were no ulcers in any patientstherefore the intake of doac might not be related withsevere ulcers in the small intestinein this study angioectasia was observed in the upper andmiddle portions but not in the lower portion of the smallbowel kaufman used a transit timebased quartilemethod to evaluate patients with angioectasia whounderwent ce and found that most lesions were inthe ï¬rst quartile igawa reported that while therewere no diï¬erences regarding the location of type 1a angioectasia among patients with occult gastrointestinal bleedingtype 1b angioectasia was relatively less frequent in the lowerportion compared with that in the upper and middle portionsof the small bowel the data reported before thereforeangioectasia might not be aï¬ected by the intake of doacscomparison of vce ï¬ndings before and after the administration of d719acs is neededno significant relationships were determined in relationto the presence of the hb level or the serum ferritin levelbetween the patients with and without smallbowel lesionsin this study despite detecting abnormal ï¬ndings in thesmall bowel no active bleeding was seen by vce and therewas no severe anemia in any patients in this study furthermore compared with warfarin the incidence of gi bleedingis higher in association with doacs the causes of gi bleeding in association with doacs are bleeding from colon astric cancers and diverticular hemorrhages however noneof these lesions detected by egd and colonoscopy had activebleeding doacs did not aï¬ect bleeding from the upper gitract and the colon in this studyno significant diï¬erences were observed among thedoacs in relation to smallbowel lesions the ï¬ndings fromthe randomized evaluation of longterm anticoagulationtherapy trial of dabigatran showed that in the warfaringroup patients with gi tract bleeding had gastric canceror colonic cancer and that in the dabigatran group patients with gi tract bleeding had colonic cancer and patient had gastric cancer hence dabigatran mightinduce gi tract bleeding from colon cancer rivaroxaban apixaban and edoxaban compete directly with the s1pocket of factor xa and inhibit factor xa activity whereasdabigatran is a prodrug that is activated in the presence ofesterase in the gi tract plasma and liver the causes of themucosal damage by dabigatran were thought to be due todirect acting at the local area where it is absorbed in addition tartaric acid coats dabigatran tablets and the tabletscan cause mucosal damage if they are retained within theesophagus while we expected an increase in the frequencyof intestinal mucosal injury among the patients who tookdabigatran as a consequence of the tartaric acid coating this 0cgastroenterology research and practicestudys ï¬ndings did not demonstrate a higher rate of smallbowel lesions associated with the use of this doac themechanisms underlying mucosal injuries caused by doacsother than dabigatran remain unclear smallbowel lesionsincluding redness erosions and angioectasia might be moreeasily detected by performing ce on patients who takedoacs because the doacs might cause bleeding thatcould facilitate the detection of the lesionsthis study has several limitations while this was a multicenter study the sample size was small hence more patientsshould be accrued and investigated in the near future moreover this study only included data that described the patientsï¬ndings after the administration of the doacs and datadescribing the ï¬ndings before their administration wereabsent therefore it remains unclear whether small intestinallesions are directly induced by doacs studies of patientsï¬ndings before and after the administration of doacs areneeded in the near future furthermore patients with severeanemia and overt bleeding were excluded from this studyand most of the enrolled patients did not have bleeding orhad minor bleeding patients taking edoxaban were notincluded in this study because edoxaban was not available injapan when this study began conclusionlesionssmallbowelincluding redness erosions smallulcers and angioectasia were detected in of patientswho took doacs more patients using doacs should beinvestigated using ce in the near futureabbreviationsdoac direct oral anticoagulantvideo capsule endoscopyvceldalowdose aspirincapsule endoscopycegastrointestinalgihemoglobinhbegdesophagogastroduodenoscopyproton pump inhibitorppidata availabilitythis manuscript describes a study that was aimed at evaluating direct oral anticoagulant doac related to smallbowellesions using video capsule endoscopy we believe that ourstudy makes a significant contribution to the literaturebecause its ï¬ndings showed that many patients takingdoacs had smallbowel lesions however most lesions wererelatively mild and they did not cause bleedingconflicts of interestthe authors have no conï¬icts of interest to disclose that arerelevant to this studyauthors contributionshiroyuki imaeda was responsible for the conception anddesign and ï¬nal approval of the article minoru yamaokahiroyuki imaeda naoki hosoe kazuaki yoneno ryukanno hisashi mitsufuji takahiro sasaki and keijiyamamoto were responsible for enrollment of patientshiroyuki imaeda and naoki hosoe were responsible foranalysis and interpretation of the data minoru yamaokaand hiroyuki imaeda were responsible for drafting of thearticle naoki hosoe was responsible for the critical revision of the article for important intellectual content takanori kanai toshimasa yamamoto toshihide mimuraharuhiko ogata nobuo araki and hidetomo nakamotowere the supervisorsreferences w k leung i bjarnason v w s wong j j y sung andf k l chan small bowel enteropathy associated withchronic lowdose aspirin therapy lancet vol no t watanabe s sugimori n kameda small bowelinjury by lowdose entericcoated aspirin and treatment withmisoprostol a pilot study clinical gastroenterology andhepatology vol no pp j iwamoto y mizokami y saito smallbowel mucosalinjuries in lowdose aspirin users with obscure gastrointestinalbleeding world journal of gastroenterology vol no pp h endo k hosono m inamori incidence of smallbowel injury induced by lowdose aspirin a crossover studyusing capsule endoscopy in healthy volunteers digestionvol no pp a shiotani k haruma r nishi randomized doubleblind pilot study of geranylgeranylacetone versus placebo inpatients taking lowdose entericcoated aspirin lowdoseaspirininduced small bowel damage scandinavian journalof gastroenterology vol no pp s j connolly m d ezekowitz s yusuf dabigatranversus warfarin in patients with atrial ï¬brillation the newengland journal of medicine vol no pp m r patel k w mahaï¬ey j garg rivaroxaban versuswarfarin in nonvalvular atrial ï¬brillation new england journal of medicine vol pp c b granger j h alexander j j v mcmurray apixaban versus warfarin in patients with atrial ï¬brillation thenew england journal of medicine vol no pp c t ruï¬ r p giugliano e braunwald comparison ofthe eï¬cacy and safety of new oral anticoagulants with warfarinin patients with atrial ï¬brillation a metaanalysis of randomised trials lancet vol no pp c s miller a dorreen m martel t huynh and a n barkun risk of gastrointestinal bleeding in patients taking nonvitamin k antagonist oral anticoagulants a systematic reviewand metaanalysis clinical gastroenterology and hepatologyvol no pp 1683e3 d caldeira m barra a ferreira systematic reviewwith metaanalysis the risk of major gastrointestinal bleeding 0cgastroenterology research and practicewith nonvitamin k antagonist oral anticoagulants alimentary pharmacology therapeutics vol no pp t yamashita y koretsune y yang edoxaban vs warfarin in east asian patients with atrial ï¬brillationan engageaftimi subanalysis circulation journal vol no pp m hori m matsumoto n tanahashi rivaroxaban vswarfarin in japanese patients with atrial ï¬brillation circulation journal vol no pp y toya s nakamura k tomita dabigatraninducedesophagitis the prevalence and endoscopic characteristicsjournal of gastroenterology and hepatology vol no pp k kasai e ishida and y kobayashi two cases of esophagealulcer caused by dabigatran gastroenterological endoscopyvol pp m okada and k okada exfoliative esophagitis and esophageal ulcer induced by dabigatran endoscopy vol supplement pp e23e24 t vanassche j hirsh j ginsberg and j eikelboom anspeciï¬c bleeding patterns of anticoagulant therapy lessonsfrom clinical trials thrombosis and haemostasis vol pp h endo k hosono m inamori characteristics ofsmall bowel injury in symptomatic chronic lowdose aspirinusers the experience of two medical centers in capsule endoscopy journal of gastroenterology vol no pp i watari s oka s tanaka comparison of smallbowelmucosalinjury between lowdose aspirin and nonaspirinnonsteroidal antiammatory drugs a capsule endoscopystudy digestion vol no pp h endo k hosono t higurashi quantitative analysisof lowdose aspirinassociated small bowel injury using a capsule endoscopy scoring index digestive endoscopy vol no pp d kaufman g leslie n marya smallintestinalangioectasia characterization risk factors and rebleedingjournal of clinical gastroenterology vol no pp a igawa s oka s tanaka major predictors and management of smallbowel angioectasia bmc gastroenterologyvol no 0c'
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"In this model the lymphangiogenesis induced by PDGF-BB could not be restricted by blocking interaction of VEGF-C with VEGFR-3 suggesting that PDGF-BB exerts its effect via an independent pathway that may involve PDGF receptors on lymphatic vessels [34]. Another study showed that VEGF-C is an essential regulator determining PDGF-BB expression for vascular stabilization via a paracrine mode of action [22]. The stimulation of proliferation of lymphatic endothelial cells by platelets seems to be induced in a time and dose dependent manner mainly by VEGF-C and PDGF-BB which are secreted by platelets. Blocking the experiments indicate a predominant role of VEGF-C in this process [35]. All those results suggested that both factors play complicated roles in tumor lymphangiogenesis. However the overlapping biological effects of these two factors have not been clarified clearly in human cancers. In this study overexpression of both PDGF-BB and VEGF-C significantly correlated. LMVD. Those cases were also younger and had larger tumor size more likely lymph node metastasis worse histological differentiation and poorer OS. In addition a significant association between VEGF-C overexpression alone and worse histological differentiation was found. For the rest however PDGF-BB or VEGF-C alone was not linked to any other clinical feature including LMVD. The results indicated NSCLC patients who had overexpression of both PDGF-BB and VEGF-C might present with more rapid growth and higher potential for invasion due to their lymphangiogenesis. Thereby these patients had poorer OS which was consistent with the results in patients with esophageal squamous cell carcinoma those with positive expressions of PDGF-BB and VEGF-C have been shown to possess a worse prognosis compared to those with negative expressions [23]. Also those results suggested that poorly differentiated cancer cells might be more capable to secrete VEGF-C and PDGF-BB which induced lymphangiogenesis thereby promoting disease progression in NSCLC. The secretion of VEGF-C or PDGF-BB by tumor could induce the activation of their receptors on the vascular endothelium and thereby inducing the formation of new lymphatic vessels [36]. However little is currently known about the interplay among these lymphangiogenic factors. In this study a significant positive correlation between PDGF-BB and VEGF-C protein expression of tumor cells was seen in NSCLC suggesting a lymphangiogenesis pathway that one factor (PDGF-BB or VEGF-C alone) may up-regulate the other factor expression in the same cells. Therefore we suspected that PDGF-BB and VEGF-C could synergistically promote NSCLC lymphangiogenesis and enhance the tumor growth and lymph node metastasis. Combined targeting both PDGF-BB and VEGF-C may become a promising strategy for the treatment of NSCLC. Conclusions We found for the first time that compared with the overexpression of PDGF-BB or VEGF-C alone both PDGF-BB and VEGF-C overexpression in primary human NSCLC was significantly associated with lymphangiogensis and poor outcome. Furthermore our data suggested that PDGF-BB and VEGF-C expression might have a correlative dependence and interplay not only in NSCLC lymphangiogenesis but also in cancer progression. Based on the expression of PDGF-BB and VEGF-C we speculated the therapy targeting VEGF-C expression in combination with targeting PDGF-BB might be an important approach for control the cancer growth in patients with NSCLC having high expression of both PDGF-BB and VEGF-C. Competing interests All authors declare they have no actual or potential competing financial interests. Authors contributions All authors read and approved the final manuscript"
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acute myeloid leukemia aml is the most common type ofacute leukemia in adults caused by the clonal expansion ofundiï¬erentiated myeloid progenitor cells although mostpatients with aml can achieve complete remission by inductionchemotherapy the recurrence rate remains high and thus is themain factor aï¬ecting the outcomes of aml patients relapseoften develops from minimal residual disease in the protectivebone marrow bm microenvironment a comprehensiveunderstanding of the bm microenvironment is conducive to thediagnosis and personalized treatment of aml leukemic cellscytogenetics and molecular aberrations are the main factorsfor risk stratiï¬cation in patients with aml in additionthe bm microenvironment also plays a very important role thein the homing and survival ofbm microenvironment contains various componentssuchas immune cells stromal cells endothelial progenitor cellsextracellular matrix growth factors and cytokines theinteraction between leukemic cells and bm microenvironmentaï¬ects resistance to chemotherapy in aml the modulationof bm microenvironment in aml is currently undergoingpreclinical research and early clinical trials molecular inhibitorssuch as cxcr4 inhibitors vla4 inhibitors and eselectininhibitors are currently undergoing clinical trials immunecells and stromal cells are important components of the bmmicroenvironment and are also the main uencing factorsof leukemia development the estimate program isa common method to explore the microenvironment of manytumors recently it has also been used to explore theprognostic genes in the microenvironment of aml patients most studies have focused on diï¬erentially expressedgenes degs however the interaction and relationship betweengenes are open to investigate moreover the coding genes wereextensively explored but regions that encoded lncrnas andmirnas were less wellstudiedweighted gene coexpression network analysis wgcnais an algorithm commonly used in systems biology toexplore the correlation between gene sets and the clinic functionalization is achieved by constructing a freescalecoexpression gene network wgcna can identify highlyrelated genes and aggregate them into the same genetic modulecurrently wgcna is used in multiple ï¬elds such as cancer andnervous system or to identify potential biomarker candidates ornew therapeutic targets from genetic data the competition endogenous rna cerna hypothesis was anew regulatory mechanism between noncoding rna ncrnaand messenger rna mrna proposed by salmena in in this theory crosstalk between cernas is achieved byabbreviations aml acute myeloid leukemia auc area under curve bmbone marrow cam cell adhesion molecules cerna competing endogenousrnas deg diï¬erentially expressed gene david the database for annotationvisualization and integrated discovery go gene ontology icam1 intercellularadhesion molecule il10ra interleukin receptor subunit alpha keggkyoto encyclopedia of genes and genomes ppi proteinprotein interactiontcga the cancer genome atlas tlr6 toll like receptor tlr8 toll likereceptor tom topological overlap matrix wgcna weighted correlationnetwork analysisimmunerelated cerna network of amlcompetitively combining shared mirnas in recent yearsthe cernas hypothesis has attracted widespread attention in thestudy of molecular and biological mechanisms of tumorigenesisand development for example previous studies have foundthat lncrnarelated cernas were involved in the biologicalprocesses of glioblastoma and breast cancer theresearch on cernas of leukemia was generally based on thediï¬erential genes screened by leukemia and normal controls but no known module based on cernas network related tomicroenvironment in leukemia has been set upinformation ofthe aml cohortin this study mrnas mirnas and lncrnas data andclinicalfrom the cancergenome atlas tcga database were used to calculate theimmune and stromal scores of these aml cases using theestimate algorithm diï¬erentially expressed mrnas andlncrnas were applied to wgcna to identify the modulesmost relevant to the aml immune microenvironment thenthe immunerelated lncrnamirnamrna cerna networkwas established to screen genes with clinical signiï¬cance theseï¬ndings will help to better understand the role oftumormicroenvironment in aml and shed light on the developmentand progression of amlmaterials and methodsdata acquisitionall data sets of aml patients were downloaded from tcgadatabase1 the data used in this study met the following criteria excluding samples combined with other malignancies samples with lncrnas and mirnas and mrnas detection datafinally all lncrnas mrnas and mirnas expression proï¬lesof aml specimens and the corresponding clinical followupdata were downloadedidentiï¬cation of differentially expressedgenesthe estimate algorithm2 was used to calculate the immunescores and stromal scores of aml samples diï¬erentiallyexpressed genes degs such as lncrnas mirnas and mrnaswere identiï¬ed between high and low score groups stratiï¬ed bythe median value of immune scores and stromal scores usinglimma package all q values use fdr to correct the statisticalsigniï¬cance of the multiple test lncrnas and mrnas withlog fc and fdr were regarded as diï¬erentiallyexpressed while mirnas with log fc and fdr were regarded as diï¬erentially expressed then all the degs wereentered into r version auckland nz united states forcluster analysis based on the expression value of each sample inits respective data set the results were expressed in a clustergrameach column represents a sample and each row represents theexpression level of a given gene1portalgdccancergov2sourcefenetprojectsestimateprojectfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amldavid3 wasgo and pathway enrichment analysesthe database for annotation visualization and integrateddiscoveryenrichedbiological themes of degs functions particularly go geneontology terms and kegg kyoto encyclopedia of genes andgenomes pathway enrichment p was set as thecutoï¬ criterionapplied to analyzeweighted gene coexpression networkanalysiswgcna is an algorithm for identiï¬cation of gene coexpressionnetworksthrough highthroughput expression proï¬les ofmrnas or lncrnas with diï¬erent characteristics pairwisepearson correlation analysis was used to evaluate the weightedcoexpression relationship between all datasettopics in theadjacency matrix in this study wgcna was used to analyzemrnas and lncrnas to obtain the mrnas or lncrnas mostrelevant to aml immune microenvironmentcerna network construction andanalysisaccording to the results of wgcna we selected all mrnasand lncrnas in the most relevant module turquoise anddiï¬erentially expressed mirnas to construct a cerna networkbrieï¬y the associated cerna network in aml was constructedfollowing three stages prediction of lncrnamirna inorder to make lncrnas and mirnas map into the interactionssuccessfully we used the miranda4 and pita5 to get targetedlncrnas that mirnas may regulate prediction of mirnamrna three online databases miranda4 targetscan6 andmirwalk7 were used simultaneously for target mrna prediction construction of lncrnamirnamrna cerna networkthe cerna network was constructed based on the negativelyregulating target relationships of mirnamrna and mirnalncrna correlation pairsppi network constructionthe retrieval of interacting genes string database8 wasutilized to construct a proteinprotein interaction ppi networkof the degs identiï¬ed in the cerna network the interactingpairs with a conï¬dence score greater than were considered assignificant and were retained the degree represents the numberof interaction partnerssurvival analysiskaplanmeier plots weretherelationship between the overall survival of aml patientsand the expression level of mrnas lncrnas and mirnasthe statistical signiï¬cance of the correlation was tested by theconstructed to illuminatelogrank test and p was considered significant theanalysis was conducted using the r package of survivalstatistical analysisgraphpad prismtm san diego ca united states or rversion auckland nz united states software was usedfor all data analyses diï¬erences across groups were comparedusing kruskalwallis test for continuous variables diï¬erenceswere considered significant when p resultsimmune and stromal scores areassociated with aml clinicalparameterswe obtained gene expression proï¬les and clinical informationof aml patients from tcga database supplementarymaterial among them were male and were female with a median age range years oldaccording to the fab classiï¬cation there were cases of m0 m1 cases m2 cases m3 cases m4 cases m5 cases m6 cases and m7 case the estimate algorithm was used tocalculate the immune scores and stromal scores of aml patientsthe median immune score was range from to and the median stromal score was range from to we analyzed the relationship between immune scores andstromal scores and clinical parameters of aml patients caseswith m4 subtype aml had the highest immune scores while caseswith m3 subtype had the lowest immune scores p figure 1a similarly m4 cases had the highest stromal scoreswhereas m0 subtypes had the lowest p figure 1baccording to cytogenetics aml patients were divided intothree groups favorable intermediatenormal and poor therewas an obvious correlation between the cytogenetic risk andthe immune scores p figure 1cfavorable vsintermediate p favorable vs poor p intermediate vs poor p but no significant correlationbetween the cytogenetic risk and the stromal scores was observedp figure 1dscoreand high immunestromalusing the median immune or stromal score as a thresholdaml patients were divided into two groups with lowimmunestromalscoresurvival analysis showed that the survival rate of aml patientswith low immune scores was significantly higher than that ofpatients with high immune scores p figure 1ehowever there was no significant diï¬erence in survival betweenpatients with low stromal scores and those with high stromalscores p figure 1f3httpdavidncifcrfgov4httpwwwmicrorna5genieweizmannacilpubsmir07mir07_exehtml6httpwwwtargetscan7http12920671508tringdbidentiï¬cation of differentially expressedgenes based on immune scoresand stromal scoressetting the cutoï¬ criteria as log fc and fdr we identiï¬ed mrnas figure 2a and lncrnasfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure immune and stromal scores are associated with aml clinical parameters ab distribution of immune scores a and stromal scores b for aml fabsubtypes cd the correlation between immune scores c and stromal scores d and aml cytogenetic risk ef kaplanmeier survival curve based on immunese and stromal scores f aml acute myeloid leukemiafigure heatmap of differentially expressed genes in the high and low immunestromal score groups a mrnas b lncrnas and e mirnas based onimmune scores c mrnas d lncrnas and f mirnas based on stromal scoresfigure 2b based on immune scores and mrnasfigure 2c and lncrnasfigure 2d based onstromal scores setting the cutoï¬ criteria as log fc and fdr we identiï¬ed and mirnas based onimmune scores figure 2e and stromal scores figure 2frespectively the degs oflow vs high immunethefrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top go terms in each of biological process were performed for functional enrichment clustering analysis a top25 significant go terms based onupregulated genes in immune scores b top25 significant go terms based on upregulated genes in stromal scores c top25 significant go terms based ondownregulated genes in immune scores d top25 significant go terms based on downregulated genes in stromal scores go gene ontologyscore or stromal score groups were illustrated in the heatmap figure aml and thus require further research to determine theirbiological contributionfunctional enrichment analysis of degsbased on the david the databasefor annotationvisualization and integrated discovery gene annotation tool weperformed go analyses of both upregulated and downregulateddegs the top go biological process indicated that theupregulated degs based on immune or stromal scores wereprimarily enriched in neutrophil degranulation regulationof immune response signal transduction and ammatoryresponse figures 3ab while the downregulated degs basedon immunestromal scores were primarily enriched in rrnaprocessing regulation of translation regulation of transcriptionand cell diï¬erentiation figures 3cd subsequently weperformed kegg kyoto encyclopedia of genes and genomespathway enrichment and interrelation analysis kegg analysisrevealed that the upregulated degs were mainly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingpathway hematopoietic cell lineage and cell adhesion moleculescams pathways figures 4ab while the downregulateddegs were mainly enriched in ribosome metabolism pi3kaktsignaling pathway transcriptional dysregulation in cancer andmirnas in cancer figures 4cd above analyses revealedthatthese degs play a vital role in the development ofconstruction of weighted correlationnetwork analysis and identiï¬cation ofkey modulesbased on the results of survival analysis degs based on immunescores were selected for subsequent analysis the best β valuein the lncrnamrna coexpression network was which wascalculated using the diï¬erential lncrnas diï¬erentialmrnas and their expression data in leukemia samples nextthe method of dynamic tree cutting was used to producecoexpression modules finally modules of lncrnamrnacoexpression networks were generated and the heat map plot oftopological overlap matrix tom was shown figure 5a eachmodule was calculated and plotted with its corresponding clinicalcharacteristics correlation analysis showed that turquoisemodule displayed the highest relationship with aml immunescores r which included mrnas and lncrnasfigure 5b these mrnas were further used to performthe gene enrichment analysis the genes were most relatedto neutrophil degranulation immune response ammatoryresponse signal transduction and tolllike receptor signalingpathway figure 5c in addition genes were highly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top kegg pathway analysis were performed for functional enrichment clustering analysis a top25 significant kegg pathways based onupregulated genes in immune scores b top25 significant kegg pathways based on upregulated genes in stromal scores c top25 significant kegg pathwaysbased on downregulated genes in immune scores d top25 significant kegg pathways based on downregulated genes in stromal scores kegg kyotoencyclopedia of genes and genomespathway metabolic pathways and hematopoietic cell lineage bykegg analysis figure 5dcerna network constructionsince the turquoise module showed the highest relationshipwith aml immune scores we selected lncrnas and mrnas inthe turquoise module and diï¬erentially expressed mirnasbased on immune scores to construct a cerna network firstlybased on the pita and mircode online database that matchespotential mirnas with lncrnas a total of lncrnamirnapairs contained lncrnas and mirnas then we searchedfor the mrnas targeted by the diï¬erentially expressed mirnasusing three target gene prediction websites miranda mirwalkand targetscan using these websites we detected and target mrnas respectively based on the venn intersectionanalysis target mrnas were selected subsequently wematched the predicted target genes with the mrnas in theturquoise module then we constructed the cerna networkby integrating the mirnalncrnamrna interactions at lasta ï¬nal lncrnamirnamrna cerna network was constructedwith lncrnas mirnas and mrnas figure 6aproteinprotein interaction ppinetwork analysisto further explore the interplay among the mrnas in cernawe constructed a ppi network based on the string theretrieval of interacting genes online database figure 6b inthe network tlr8 toll like receptor icam1 intercellularadhesion molecule tlr6 toll like receptor and il10rainterleukin receptor subunit alpha had higher degrees and respectively supplementary table the genes encoding these proteins have been conï¬rmed tobe associated with immune microenvironment and leukemiaprogression association between mrnas mirnasand lncrnas in cerna and overall amlsurvivalwe further analyzed the prognostic values of mrnas mirnasand lncrnas in the cerna network subjects were dividedinto highexpression and lowexpression cohorts accordingto the median value ofthese genes for overall survivalfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure wgcna was used to analyze genetic modules a cluster diagram of coexpression network modules based on topological overlap in mrna andlncrna b study the relationship between each module with their corresponding clinical characteristics c go analysis showed the gene symbols and geneinteractions in the turquoise module d kegg analysis was used to study the pathway enrichment in the turquoise module wgcna weighted correlation networkanalysis go gene ontology kegg kyoto encyclopedia of genes and genomesthrough the package survivalthe highexpression and lowexpression cohorts were splitfor the logrank testin rsoftware out of lncrnas ac0099485 cmahp cta331p31 fcgr2c grk6p1 linc00539 linc01272 mipepp3psmb8as1 rp11266l95 rp11320g101 rp11421f163rp11439e1910 rp11792a83 and stag3l2 out of mirnas hsamir125b5p and hsamir3383p and out of mrnas agpat3 ankrd27 cbx2 ccnd2cd300lb cyth1 erg gdf11 igf1r kiaa0513 kiaa0930larp1 lfng lpcat1 nrep nudt16 pou2f2 ppm1hptafr qsox2 rab3d ralgps2 siglec7 slc43a2 srsf6tnfaip2 tns3 trib1 zbtb5 znf70 and znrf1 wereassociated with overall survival according to the logrank testp representative genes are shown in figure age ¥ years vs years and risk group favorablevsintermediatenormal vs poor were also associated withoverall survival according to the logrank test p and respectively the above mentioned lncrnas mrnas and mirnas were brought into further multivariatecox proportional hazard regression analysis with age and riskgroup finally mrnas ccnd2 erg lpcat1 nudt16ralgps2 tnfaip2 and znf70 lncrnas cmahp fcgr2cpsmb8as1 rp11266l95 rp11320g101 rp11792a83 andstag3l2 and mirnas hsamir125b5p and hsamir3383p were independently associated with overall survival table discussionin recent years studies about the roles of gene mutationsand chromosomalin the occurrence anddevelopment of aml and their prognostic values have madesignificant progress however the bm microenvironmentwhich also plays an important role in the pathophysiologytranslocationsfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure cerna network construction and proteinprotein interaction network a a lncrnamirnamrna cerna network was constructed by lncrnas mirnas and mrnas b a ppi network was constructed based on the string database the rectangle represents micrornas the circle represents mrnasand the triangle represents lncrnas the red represents upregulation in ishigh and the green represents downregulation in ishigh the size of the dot representsthe regulatory capacity of the mrna and larger points indicate stronger regulatory capability cerna competing endogenous rnas ppi proteinprotein interactionprocess in aml are poorly understood therefore mosttreatments previously targeted only tumor cells butfewtargeted the tumor microenvironmentindepth researchon the microenvironment of leukemia will help to furtherunderstand the mechanism of leukemia development and mayï¬nd new targets for microenvironment treatment this studyscreened microenvironmentrelated genes based on the tcgadatabase and further established microenvironmentrelatedlncrnamirnamrna cerna networks through wgcnafirst we calculated the immune scores and stromal scores ofaml patients based on the estimate algorithm and found thatthese scores were related to the fab typing of aml in additionimmune scores were significantly correlated with cytogenetic riskand overall survival estimate is a new algorithm to inferthe level of stromal and immune cells in tumor tissues andtumor purity using gene expression data high immunescores in the bm samples from patients with poor prognosisindicated that more immune cells were recruited in their bmfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure correlation between mrnas mirnas and lncrnas in cerna and overall aml survival in tcga kaplanmeier survival curves with the logrank testwere performed for the representative mrnas mirnas and lncrnas cerna competing endogenous rnas aml acute myeloid leukemia tcga the cancergenome atlasmicroenvironment this may be due to that aml cells activelyshape the bm environment and immune cells to promote diseaseprogression through cellular structural and functional changes however there was no significant correlation betweenstromal scores and cytogenetic risk or survival of aml patientssuggesting that the proportion of stromal cells were comparablein diï¬erent group possible explanation may be that stromal cellsplay an important role in solid tumors while its role inleukemia is not as strong as in solid tumors then we identiï¬ed diï¬erentially expressed mrnas mirnasstromaland lncrnas based on the immune scores orscores functional enrichment analysis indicated thatthesedegs were mainly involved in immune and ammatoryresponses consistent with these results previous studies haveshown that the biology of the immune system is essentialfor the formation of a complex bm microenvironment in recent yearsthe immunologicalcharacteristics of aml has increased and the developmentof eï¬ective aml immunotherapy strategies has attractedwidespread attention the understanding ofin recent years important advances in cerna coexpressionnetwork research have developed rapidly the disruption offrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amltable multivariate cox proportional hazard regression analysis of lncrnas mrnas and mirnasgenesccnd2erglpcat1nudt16ralgps2tnfaip2znf70cmahpfcgr2cpsmb8as1rp11266l95rp11320g101rp11792a83stag3l2hsamir125b5phsamir3383phr 95ci pthe cerna network balance is a major cause for tumorigenesis therefore understanding the complex interactions betweendiï¬erent cerna networks will lead to an indepth understandingof gene regulatory networks and has implications for cancertreatment in addition the lncrnamirnamrna cernanetwork can predict the prognosis of the disease for examplea lncrnamirnamrna cerna network was established basedon rnaseq data of breast cancer from tcga which consistsof mirnas lncrnas and mrnas multiplexcox regression analyses showed that four of these lncrnasadamts9as1 linc00536 al3914211 and linc00491 havesignificant prognostic value wang identiï¬ed lncrnas mirnas and mrnas from a database toconstruct a lncrnamirnamrna cerna network inthe network a univariate and multivariate cox proportionalhazard regression analysis was used to establish a survivalmodel with target mrnas hoxa9 insr krit1 mybspry2 ube2v1 wee1 and znf711 where auc area undercurve is indicating the sensitivity and speciï¬city ofprognostic prediction however the screening of diï¬erentialgenes in this study was based on leukemia patients andnormal people and did not focus on tumor microenvironmentso far there is no cerna research based on the leukemiamicroenvironment in our research the screening of diï¬erentialgenes was based on the immune score then wgcna wasused to identify the modules most relevantto the amlimmune microenvironment then using wgcna and mirnaprediction websites a lncrnamirnamrna cerna networkconsisting of lncrnas mirnas and mrnaswas constructedsubsequently we built a ppi network predicting theinteraction among the proteins encoded by the degs inthe cerna network tlr8 icam1 tlr6 and il10ra hadhigher degrees tlr8 and tlr6 are members of the tolllike receptor family which is upstream to the transcriptionittransportationthe innate immune system andfactor nfκb and part ofplays an importantin progression of aml roleicam1 is one of the cams a large class of transmembraneproteinsinvolved in the binding of cells to another cellor extracellular matrix and involved in cell proliferationdiï¬erentiation movementandtissue structure the protein encoded by il10ra is areceptor for interleukin which has been shown to mediatethe immunosuppressive signal ofinterleukin and thusinhibits the synthesis of proammatory cytokines and isreported to promote survival of progenitor myeloid cellsthrough the insulin receptor substrate2pi3kakt pathway these results indicated that this novel cerna networkwere closely associated with immune microenvironment andprogression of amlapoptosisfurthermore lncrnas mirnas and mrnas withprognostic signiï¬cance were screened out which could be usedas biomarkers for prognosis among the genes with prognosticsigniï¬cance in our module of immunerelated cerna networkthere were aml related reports about cbx2 ccnd2 ergigf1r larp1 lfng nudt16 pou2f2 ptafr rab3dsiglec7 srsf6 tnfaip2 trib1 zbtb5 and znrf1 themost reported of which were erg ccnd2 and ifg1r ergtranslocation was involved in the occurrence and developmentof aml and high expression of erg was a poor prognosticfactor for patients with normal karyotype aml ccnd2mutations were more common in cbfaml and it was also afrequent mutation event in t aml ccnd2 leadedto increased phosphorylation of retinoblastoma proteins leadingto significant cell cycle changes and increased proliferation ofaml cell lines nicolas chapuis found that igf1spontaneous lesions played a key role in pi3kakt activation ofaml cells providing strong evidence for targeting igf1r as apotential new therapy for aml the functions of agpat3ankrd27 cd300lb cyth1 gdf11 kiaa0513 kiaa0930lpcat1 nrep ppm1h qsox2 ralgps2 slc43a2 tns3and znf70 in aml have not been reported we identiï¬ed lncrnas with clinical signiï¬cance among them only cmahpwas reported to be related to mllpositive aml andother lncrnas have not been reported in leukemia twomirnas including hsamir125b5p and hsamir3383p havebeen reported to be associated with a variety of cancers but no studies have been reported related to aml all theseunreported mrnas mirnas and lncrnas may be potentialnovel biomarkers or therapeutic targets for amlis importantto note that our current research haslimitations we selected the target data from the tcga publicdatabase only through the biological algorithm method weshould further verify the results of this in clinical patientsin further studyconclusionin summary a comprehensive bioinformatics analysis wasperformed on the aml dataset in tcga with an emphasison the immune microenvironment using wgcna andfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlmirna prediction programs an immunerelated lncrnamirnamrna cerna network was established and degs withprognostic value were further identiï¬ed further studies of thesegenes are needed in the clinic and may provide new insights intothe pathogenesis of aml this study increases our understandingof the complex interactions between aml tumor cells and the bmmicroenvironment and may provide novel prognostic factors andtherapeutic targetsdata availability statementall data sets of aml patients were downloaded from the cancergenome atlas tcga database portalgdccancergovauthor contributionsyfl cw and zj conceptualization and design sw dataacquisition and writing original draft sw ly yx and dzmethodology sw and cw data analysis and interpretationyjl and yfl writing review and editing yfl cw and zjproject administration all authors contributed to the andapproved the submitted versionfundingthis work was supported by the national natural sciencefoundation of china grant numbers and u1804191and the henan medical science and technology research projectgrant number acknowledgmentswe thank the tcga database for the availability of the datasupplementary materialthe supplementary materialonline202001579fullsupplementarymaterialfor this can be foundwwwfrontiersins103389foncatreferences ferrara f schiï¬er ca acute myeloid leukaemia in adults lancet doi 101016s0140673612617279 zeng z shi yx samudio ij wang ry ling x frolova o targetingthe leukemia microenvironment by cxcr4 inhibition overcomes resistanceto kinase inhibitors and chemotherapy in aml blood doi 101182blood200805158311 shafat ms gnaneswaran b bowles km rushworth sa the bone marrowmicroenvironmenthome of the leukemic blasts blood rev doi 101016jblre201703004 bullinger l döhner k döhner h genomics of acute myeloid leukemiadiagnosis and pathways j clin oncol doi 101200jco ayala f dewar r kieran m kalluri r contribution of bonemicroenvironment to leukemogenesis and leukemia progression leukemia doi 101038leu2009175 uy gl rettig mp motabi ih mcfarland k trinkaus km hladnik lm a phase study of chemosensitization with the cxcr4 antagonist plerixaforin relapsed or refractory acute myeloid leukemia blood doi 101182blood201110383406 rashidi a uy gl targeting the microenvironmentin acute myeloidleukemia curr hematol malig rep doi 101007s11899 austin r smyth mj lane sw harnessing the immune system in acutemyeloid leukaemia crit rev oncol hematol doi 101016jcritrevonc201604020 yehudairesheï¬ s attiasturgeman s sabbah r gabay t musallam rfridmandror a abnormal morphological and functional nature ofbone marrow stromal cells provides preferential support for survival of acutemyeloid leukemia cells int j cancer doi 101002ijc yoshihara k shahmoradgoli m martÃnez e vegesna r kim h torresgarciaw inferring tumour purity and stromal and immune cell admixture fromexpression data nat commun doi 101038ncomms3612 yan h qu j cao w liu y zheng g zhang e identiï¬cation of prognosticgenes in the acute myeloid leukemia immune microenvironment based ontcga data analysis cancer immunol immunother doi101007s00262019024087 huang s zhang b fan w zhao q yang l xin w identiï¬cation ofprognostic genes in the acute myeloid leukemia microenvironment aging doi 1018632aging102477 ni j wu y qi f li x yu s liu s screening the cancer genome atlasdatabase for genes of prognostic value in acute myeloid leukemia front oncol doi 103389fonc201901509 langfelder p horvath s wgcna an r package for weighted correlationnetwork analysis bmc bioinformatics doi yao y zhang t qi l zhou c wei j feng f integrated analysis of coexpression and cerna network identiï¬es ï¬ve lncrnas as prognostic markersfor breast cancer j cell mol med doi 101111jcmm14721 spiers h hannon e schalkwyk lc smith r wong ccy odonovan mc methylomic trajectories across human fetal brain development genomeres doi 101101gr180273114 liu q jiang c xu j zhao mt van bortle k cheng x genomewide temporal proï¬ling of transcriptome and open chromatin of earlycardiomyocyte diï¬erentiation derived from hipscs and hescs circulat res doi 101161circresaha116310456 salmena l poliseno l tay y kats l pandolï¬ pp a cerna hypothesisthe rosetta stone of a hidden rna language cell doi101016jcell201107014 karreth fa pandolï¬ pp cerna crosstalk in cancer when cebling rivalriesgo awry cancer discov doi 10115821598290cd13 zhang k li q kang x wang y wang s identiï¬cation and functionalcharacterization of lncrnas acting as cerna involved in the malignantprogression of glioblastoma multiforme oncol rep doi103892or20165070 wang jd zhou hs tu xx he y liu qf liu q prediction ofcompeting endogenous rna coexpression network as prognostic markers inaml aging doi 1018632aging101985 ritchie me phipson b wu d hu y law cw shi w limma powersdiï¬erential expression analyses for rnasequencing and microarray studiesnucleic acids res 43e47 doi 101093nargkv007 kanehisa m goto s kegg kyoto encyclopedia of genes and genomes nucleicacids res brenner ak bruserud ø functional tolllike receptors tlrs are expressed
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coutard b valle c de lamballerie x canard b seidah ng and decroly e the spike glycoprotein of the new coronavirus 2019ncovcontains a furinlike cleavage site absent in cov of the same clade antiviral res 101016jantiviral2020104742 cao z wu y faucon e and sabatier jm sarscov2 covid19 keyroles of the reninangiotensin systemvitamin d impacting drugand vaccine developments infect disord drug targets 1021741871526520999200505174704 zhang z chen l zhong j gao p and oudit gy ace2ang17 signaling and vascular remodeling sci china life sci s1142701446933 paz ocaranza m riquelme ja garcia l jalil je chiong m santos ras counterregulatory reninangiotensin system incardiovascular disease nat rev cardiol 101038s4156901902448 ziegler cgk allon sj nyquist sk mbano im miao vn tzouanas cn sarscov2 receptor ace2 is an interferonstimulatedgene in human airway epithelial cells and is detected in speciï¬c cell subsets across tissues cell 101016jcell202004035 marrero mb schieffer b paxton wg heerdtt l berkt bc delafontaine p direct stimulation of jakstat pathway by theangiotensin ii at1 receptor j med chem perrotta f matera mg cazzola m and bianco a severe respiratory sarscov2 infection does ace2 receptor matter respir med 101016jrmed2020105996 cheng h wang y and wang g anprotective effect of angiotensinconverting enzyme and its effect on the prognosis of covid19 jmed virol 101002jmv25785 de lang a osterhaus adme and haagmans bl interferon Î and interleukin4 downregulate expression of the sars coronavirusreceptor ace2 in vero e6 cells virology the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 mcgonagle d sharif k oregan a and bridgewood c the role of cytokines including interleukin6 in covid19 induced pneumonia andmacrophage activation syndromelike disease autoimmun rev linkinghubelseviercomretrievepiis1568997220300926101016jautrev2020102537 ye q wang b and mao j the pathogenesis and treatment of the cytokine storm in covid19 j infect linkinghubelseviercomretrievepiis0163445320301651 101016jjinf202003037 behrens em and koretzky ga review cytokine storm syndrome looking toward the precision medicine era arthritis rheumatol 101002art40071 guo xj and thomas pg new fronts emerge in the uenza cytokine storm semin immunopathol s002810170636y ronco c and reis t kidney involvement in covid19 and rationale for extracorporeal therapies nat rev nephrol 101038s4158102002847 wu c chen x cai y xia j zhou x xu s risk factors associated with acute respiratory distress syndrome and death in patientswith coronavirus disease pneumonia in wuhan china jama intern med 101001jamainternmed20200994 jose rj and manuel a covid19 cytokine storm the interplay between ammation and coagulation lancet respir med murakami m kamimura d and hirano t pleiotropy and speciï¬city insights from the interleukin family of cytokines immunity 101016jimmuni201903027 oshea jj schwartz dm villarino av gadina m mcinnes ib and laurence a the jakstat pathway impact on human disease andtherapeutic intervention annu rev med 101146annurevmed051113024537 lawrence t the nuclear factor nf k b pathway in ammation cold spring harb perspect biol catanzaro m fagiani f racchi m corsini e govoni s and lanni c immune response in covid19 addressing a pharmacologicalchallenge by targeting pathways triggered by sarscov2 signal transduct target ther 101038s4139202001911 li q and verma im nfκb regulation in the immune system nat rev immunol 101038nri910 tergaonkar v correa rg ikawa m and verma im distinct roles of iκb proteins in regulating constitutive nfκb activity nat cell biol 101038ncb1296 tergaonkar v nfκb pathway a good signaling paradigm and therapeutic target int j biochem cell biol linkinghubelseviercomretrievepiis1357272506001245 101016jbiocel200603023 correa rg matsui t tergaonkar v rodriguezesteban c izpisuabelmonte jc and verma im zebraï¬sh ikappab kinase negativelyregulates nfkappab activity curr biol 101016jcub200506023 wong et and tergaonkar v roles of nfκb in health and disease mechanisms and therapeutic potential clin sci portlandpresscomclinsci116645168639rolesofnfκbinhealthanddiseasemechanismsand101042cs20080502 morrison dk map kinase pathways cold spring harb perspect biol a011254 101101cshperspecta011254 mizutani t fukushi s saijo m kurane i and morikawa s phosphorylation of p38 mapk and its downstream targets in sarscoronavirusinfected cells biochem biophys res commun 101016jbbrc200405107 grimes jm and grimes kv p38 mapk inhibition a promising therapeutic approach for covid19 j mol cell cardiol 101016jyjmcc202005007 cuadrado a and nebreda ar mechanisms and functions of p38 mapk signalling biochem j 101042bj20100323 han j lee jd tobias ps and ulevitch rj endotoxin induces rapid protein tyrosine phosphorylation in 70z3 cells expressing cd14 jbiol chem jiang y chen c li z guo w gegner ja lin s characterization of the structure and function of a new mitogenactivated proteinkinase p38 j biol chem 101074jbc2713017920 li z jiang y ulevitch rj and han j the primary structure of p38Î a new member of p38 group of map kinases biochem biophys rescommun linkinghubelseviercomretrievepiis0006291x96916629 101006bbrc19961662 jiang y gram h zhao m new l gu j feng l characterization of the structure and function of the fourth member of p38 groupmitogenactivated protein kinases p38δ j biol chem 101074jbc2724830122 zarubin t and han j activation and signaling of the p38 map kinase pathway cell res 101038sjcr7290257 cuevas bd abell an and johnson gl role of mitogenactivated protein kinase kinase kinases in signal integration oncogene httpwwwnaturecoms1210409 101038sjonc1210409 alonso g ambrosino c jones m and nebreda ar differential activation of p38 mitogenactivated protein kinase isoforms depending onsignal strength j biol chem 101074jbcm007835200 brancho d mechanism of p38 map kinase activation in vivo genes dev 101101gad1107303 parker cg hunt j diener k mcginley m soriano b keesler ga identiï¬cation of stathmin as a novel substrate for p38 deltabiochem biophys res commun linkinghubelseviercomretrievepiis0006291x98992506101006bbrc19989250 rouse j cohen p trigon s morange m alonsollamazares a zamanillo d a novel kinase cascade triggered by stress and heatshock that stimulates mapkap kinase2 and phosphorylation of the small heat shock proteins cell linkinghubelseviercomretrievepii0092867494902771 1010160092867494902771 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 tan y rouse j zhang a cariati s cohen p and comb mj fgf and stress regulate creb and atf1 via a pathway involving p38 mapkinase and mapkap kinase2 embo j 101002j146020751996tb00840x xu w wang x tocker am huang p reith mea liuchen ly functional characterization of a novel series of biased signalingdopamine d3 receptor agonists acs chem neurosci 101021acschemneuro6b00221 kotlyarov a neininger a schubert c eckert r birchmeier c volk hd mapkap kinase is essential for lpsinduced tnfαbiosynthesis nat cell biol httpwwwnaturecomsncb0699 10103810061 kontoyiannis d pasparakis m pizarro tt cominelli f and kollias g impaired onoff regulation of tnf biosynthesis in mice lacking tnfaurich elements immunity linkinghubelseviercomretrievepiis1074761300800382101016s1074761300800382 saccani s pantano s and natoli g p38dependent marking of ammatory genes for increased nfκb recruitment nat immunol 101038ni748 dienz o and rincon m the effects of il6 on cd4 t cell responses clin immunol 101016jclim200808018 velazquezsalinas l verdugorodriguez a and rodriguez ll the role of interleukin during viral infections front microbiol zhang c wu z li jw zhao h and wang gq the cytokine release syndrome crs of severe covid19 and interleukin6 receptoril6r antagonist tocilizumab may be the key to reduce the mortality int j antimicrob agents 101016jijantimicag2020105954 matthay ma ware lb and zimmerman ga the acute respiratory distress syndrome j clin invest 101172jci60331 zhang h rostami mr leopold pl mezey jg obeirne sl strulovicibarel y expression of the sarscov2 ace2 receptor inthe human airway epithelium am j respir crit care med 101164rccm2020030541oc tian s xiong y liu h niu l guo j liao m pathological study of the novel coronavirus disease covid19 throughpostmortem core biopsies mod pathol 101038s413790200536x sungnak w huang n b ´ecavin c berg m queen r litvinukova m sarscov2 entry factors are highly expressed in nasalepithelial cells together with innate immune genes nat med 101038s4159102008686 lukassen s chua rl trefzer t kahn nc schneider ma muley t sars cov2 receptor ace and tmprss are primarilyexpressed in bronchial transient secretory cells embo j 1015252embj20105114 leung jm yang cx tam a shaipanich t hackett tl singhera gk ace2 expression in the small airway epithelia of smokersand copd patients implications for covid19 eur respir j 10118313993003006882020 dhawale vs amara vr karpe pa malek v patel d and tikoo k activation of angiotensinconverting enzyme ace2 attenuatesallergic airway ammation in rat asthma model toxicol appl pharmacol 101016jtaap201606026 bradding p richardson m hinks tsc howarth ph choy df arron jr ace2 tmprss2 and furin gene expression in the airwaysof people with asthma implications for covid19 j allergy clin immunol 101016jjaci202005013 vareille m kieninger e edwards mr and regamey n the airway epithelium soldier in the ï¬ght against respiratory viruses clinmicrobiol rev 101128cmr0001410 sabroe i parker lc dower sk and whyte mkb the role of tlr activation in ammation j pathol 101002path2264 frieman m and baric r mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation microbiol mol biolrev 101128mmbr0001508 lafferty ei qureshi st and schnare m the role of tolllike receptors in acute and chronic lung ammation j amm httpwwwjournalammationcomcontent7157 10118614769255757 kawai t and akira s signaling to nfκb by tolllike receptors trends mol med 101016jmolmed200709002 darby wj mcnutt kw and todhunter en niacin nutr rev 101111j175348871975tb05075x yoshikawa t hill te yoshikawa n popov vl galindo cl garner hr dynamic innate immune responses of human bronchialepithelial cells to severe acute respiratory syndromeassociated coronavirus infection plos one e8729101371journalpone0008729 dediego ml nietotorres jl reglanava ja jimenezguardeno jm fernandezdelgado r fett c inhibition ofnfkappabmediated ammation in severe acute respiratory syndrome coronavirusinfected mice increases survival j virol 101128jvi0257613 imai y kuba k neely gg yaghubianmalhami r perkmann t van loo g identiï¬cation of oxidative stress and tolllike receptor signaling as a key pathway of acute lung injury cell 101016jcell200802043 ding y feng q chen j and song j tlr4nfκb signaling pathway gene single nucleotide polymorphisms alter gene expression levelsand affect ards occurrence and prognosis outcomes medicine baltimore e16029 101097md0000000000016029 totura al whitmore a agnihothram s sch ¨afer a katze mg heise mt tolllike receptor signaling via trif contributes to aprotective innate immune response to severe acute respiratory syndrome coronavirus infection mbio 101128mbio0063815 olejnik j hume aj and m ¨uhlberger e tolllike receptor in acute viral infection too much of a good thing plos pathog 101371journalppat1007390 okumura a rasmussen al halfmann p feldmann f yoshimura a feldmann h suppressor of cytokine signaling is aninducible host factor that regulates virus egress during ebola virus infection j virol 101128jvi0173615 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 obrien tr thomas dl jackson ss prokuninaolsson l donnelly rp and hartmann r weak induction of interferon expression bysarscov2 supports clinical clin infect dis 101093cidciaa453 chang yj liu cyy chiang bl chao yc and chen cc induction of il8 release in lung cells via activator protein1 byrecombinant baculovirus displaying severe acute respiratory syndromecoronavirus spike proteins identiï¬cation of two functional regions j immunol 104049jimmunol173127602 wang w ye l ye l li b gao b zeng y upregulation of il6 and tnfα induced by sarscoronavirus spike protein in murinemacrophages via nfκb pathway virus res 101016jvirusres200702007 zhang x wu k wang d yue x song d zhu y nucleocapsid protein of sarscov activates interleukin6 expression throughcellular transcription factor nfκb virology 101016jvirol200704009 toews gb cytokines and the lung eur respir j suppl 101183090319360100266001 russell b moss c rigg a and van hemelrijck m covid19 and treatment with nsaids and corticosteroids should we be limiting theiruse in the clinical setting ecancermedicalscience 103332ecancer20201023 severgnini m takahashi s rozo lm homer rj kuhn c jhung jw activation of the stat pathway in acute lung injury am jphysiol lung cell mol physiol 101152ajplung003492003 zhao j yu h liu y gibson sa yan z xu x protective effect of suppressing stat3 activity in lpsinduced acute lung injury amj physiol lung cell mol physiol l868l880 101152ajplung002812016 pedroza m schneider dj karmoutyquintana h coote j shaw s corrigan r interleukin6 contributes to ammation andremodeling in a model of adenosine mediated lung injury plos one e22667 101371journalpone0022667 goldman jl sammani s kempf c saadat l letsiou e wang t pleiotropic effects of interleukin6 in a twohit murine modelof acute respiratory distress syndrome pulm circ 101086675991 rincon m and irvin cg role of il6 in asthma and other ammatory pulmonary diseases int j biol sci 107150ijbs4874 swanson kv deng m and ting jpy the nlrp3 ammasome molecular activation and regulation to therapeutics nat rev immunol 101038s4157701901650 zhao c and zhao w nlrp3 ammasome a key player in antiviral responses front immunol orzalli mh smith a jurado ka iwasaki a garlick ja kagan jc an antiviral branch of the il1 signaling pathway restrictsimmuneevasive virus replication an antiviral branch of the il1 signaling pathway restricts immuneevasive virus replication mol cell 825e5840e5 101016jmolcel201807009 shi c and nabar nr sarscoronavirus open reading frame8b triggers intracellular stress pathways and activates nlrp3 ammasomescell death discov 101038s4142001901817 siu k yuen k castanorodriguez c ye z yeung m fung s severe acute respiratory syndrome coronavirus orf3a proteinactivates the nlrp3 ammasome by promoting traf3dependent ubiquitination of asc faseb j 101096fj201802418r kusov y tan j alvarez e enjuanes l and hilgenfeld r a gquadruplexbinding macrodomain within the sarsunique domain isessential for the activity of the sarscoronavirus replicationtranscription complex virology 101016jvirol20150601 chang ys ko bh ju jc chang hh huang sh and lin cw sars unique domain sud of severe acute respiratory syndromecoronavirus induces nlrp3 ammasomedependent cxcl10mediated pulmonary ammation int j mol sci 103390ijms21093179 sun dz song cq xu ym and dong xs role of the mapk pathway in human lung epitheliallike a549 cells apoptosis induced byparaquat genet mol biol e20190137 10159016784685gmb20190137 arcaroli j yum hk kupfner j park js yang ky and abraham e role of p38 map kinase in the development of acute lung injury clinimmunol 101006clim20015108 li sw wang cy jou yj yang tc huang sh wan l sars coronavirus papainlike protease induces egr1dependentupregulation of tgf1 via rosp38 mapkstat3 pathway sci rep yoshida k kuwano k hagimoto n watanabe k matsuba t fujita m map kinase activation and apoptosis in lung tissues frompatients with idiopathic pulmonary ï¬brosis j pathol 101002path1208 qian f deng j wang g ye rd and christman jw pivotal role of mitogenactivated protein kinaseactivated protein kinase inammatory pulmonary diseases curr protein pept sci 1021741389203716666150629121324 renda t baraldo s pelaia g bazzan e turato g papi a increased activation of p38 mapk in copd eur respir j 1011830903193600036707 mercer ba and darmiento jm emerging role of map kinase pathways as therapeutic targets in copd int j chron obstruct pulmon dis feng y fang z liu b and zheng x p38mapk plays a pivotal role in the development of acute respiratory distress syndrome clinics 106061clinics2019e509 mizutani t signaling pathways of sarscov in vitro and in vivo mol biol 9783642036835Ë mizutani t signal transduction in sarscovinfected cells ann ny acad sci 101196annals1408006 cheng vcc hung ifn tang bsf chu cm wong mml chan kh viral replication in the nasopharynx is associated withdiarrhea in patients with severe acute respiratory syndrome clin infect dis 101086382681 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 arabi ym alomari a mandourah y alhameed f sindi aa alraddadi b critically ill patients with the middle east respiratorysyndrome a multicenter retrospective cohort study crit care med 101097ccm0000000000002621 wong sh lui rns and sung jjy covid19 and the digestive system j gastroenterol hepatol 101111jgh15047 luo s zhang x and xu h dont overlook digestive symptoms in patients with novel coronavirus disease covid19 clingastroenterol hepatol 101016jcgh202003043 xiao f tang m zheng x liu y li x and shan h evidence for gastrointestinal infection of sarscov2 gastroenterology 1831e31833e3 101053jgastro202002055 young be ong swx kalimuddin s low jg tan sy loh j epidemiologic features and clinical course of patients infected withsarscov2 in singapore jama jamanetworkcomjournalsjamafull2762688 101001jama20203204 xu y li x zhu b liang h fang c gong y characteristics of pediatric sarscov2 infection and potential evidence for persistentfecal viral shedding nat med 101038s4159102008174 tang a tong z wang h dai y li k liu j detection of novel coronavirus by rtpcr in stool specimen from asymptomatic childchina emerg infect dis httpwwwnccdcgoveid266200301 htm 103201eid2606200301 zhang w du rh li b zheng xs yang xl hu b molecular and serological investigation of 2019ncov infected patientsimplication of multiple shedding routes emerg microbes infect 1010802222175120201729071 yeo c kaushal s and yeo d enteric involvement of coronaviruses is faecaloral transmission of sarscov2 possible lancetgastroenterol hepatol 101016s2468125320300480 van doremalen n bushmaker t morris dh holbrook mg gamble a williamson bn aerosol and surface stability ofsarscov2 as compared with sarscov1 n engl j med 101056nejmc2004973 setti l passarini f de gennaro g barbieri p perrone mg borelli m airborne transmission route of covid19 why meters6feet of interpersonal distance could not be enough int j environ res public health 103390ijerph17082932 perlman s and netland j coronaviruses postsars update on replication and pathogenesis nat rev microbiol 101038nrmicro2147 m ¨onkem ¨uller k fry l and rickes s covid19 coronavirus sarscov2 and the small bowel rev esp enferm dig wang d hu b hu c zhu f liu x zhang j clinical characteristics of hospitalized patients with novelcoronavirusinfected pneumonia in wuhan china jama jamanetworkcomjournalsjamafull2761044101001jama20201585 pan l mu m yang p sun y wang r yan j clinical characteristics of covid19 patients with digestive symptoms in hubei chinaam j gastroenterol 1014309ajg0000000000000620 budden kf gellatly sl wood dla cooper ma morrison m hugenholtz p emerging pathogenic links between microbiota andthe gutlung axis nat rev microbiol 101038nrmicro2016142 camargo smr singer d makrides v huggel k pos km wagner ca tissuespeciï¬c amino acid transporter partners ace2 andcollectrin differentially interact with hartnup mutations gastroenterology 872e3882e3linkinghubelseviercomretrievepiis0016508508018933 101053jgastro200810055 hashimoto t perlot t rehman a trichereau j ishiguro h paolino m ace2 links amino acid malnutrition to microbial ecology andintestinal ammation nature 101038nature11228 br ¨oer a juelich t vanslambrouck jm tietze n solomon ps holst j impaired nutrient signaling and body weight control in naneutral amino acid cotransporter slc6a19deï¬cient mouse j biol chem 101074jbcm111241323 ma xm and blenis j molecular mechanisms of mtormediated translational control nat rev mol cell biol 101038nrm2672 bostancklo Ëglu m temporal correlation between neurological and gastrointestinal symptoms of sarscov2 amm bowel dis 101093ibdizaa131 louveau a smirnov i keyes tj eccles jd rouhani sj peske jd structural and functional features of central nervous systemlymphatic vessels nature 101038nature14432 jessen na munk asf lundgaard i and nedergaard m the glymphatic system a beginners guide neurochem res s1106401515816 wu y xu x chen z duan j hashimoto k yang l nervous system involvement after infection with covid19 and othercoronaviruses brain behav immun 101016jbbi202003031 liu k pan m xiao z and xu x neurological manifestations of the coronavirus sarscov2 pandemic j neurol neurosurgpsychiatry 101136jnnp2020323177 xydakis ms dehganimobaraki p holbrook eh geisthoff uw bauer c hautefort c smell and taste dysfunction in patients withcovid19 lancet infect dis s14733099 101016s1473309920302930 moriguchi t harii n goto j harada d sugawara h takamino j a ï¬rst case of meningitisencephalitis associated withsarscoronavirus2 int j infect dis 101016jijid202003062 baig am khaleeq a ali u and syeda h evidence of the covid19 virus targeting the cns tissue distribution hostvirus interaction andproposed neurotropic mechanisms acs chem neurosci 101021acschemneuro0c00122 helms j kremer s merdji h clerejehl r schenck m kummerlen c neurologic features in severe sarscov2 infection nengl j med 101056nejmc2008597 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 panizmondolï¬ a bryce c grimes z gordon re reidy j lednicky j central nervous system involvement by severe acuterespiratory syndrome coronavirus2 sarscov2 j med virol 101002jmv25915 zhou l zhang m wang j and gao j sarscov2 underestimated damage to nervous system travel med infect dis linkinghubelseviercomretrievepiis1477893920301101 101016jtmaid2020101642 panciani pp saraceno g zanin l renisi g signorini l battaglia l sarscov2threesteps infection model and csfdiagnostic implication brain behav immun 101016jbbi202005002 desfes m le coupanec a dubeau p bourgouin a lajoie l dub ´e m human coronaviruses and other respiratory virusesunderestimated opportunistic pathogens of the central nervous system viruses 103390v12010014 fazakerley jk and walker r virus demyelination j neurovirol 10108013550280390194046 murray rs cai gy hoel k zhang jy soike kf and cabirac gf coronavirus infects and causes demyelination in primate centralnervous system virology 101016004268229290757g lane te and buchmeier mj murine coronavirus infection a paradigm for virusinduced demyelinating disease trends microbiol 101016s0966842x97817684 stohlman sa and hinton dr viral induced demyelination brain pathol 101111j175036392001tb00384x li z liu t yang n han d mi x li y neurological manifestations of patients with covid19 potential routes of sarscov2neuroinvasion from the periphery to the brain front med s1168402007865 netland j meyerholz dk moore s cassell m and perlman s severe acute respiratory syndrome coronavirus infection causes neuronaldeath in the absence of encephalitis in mice transgenic for human ace2 j virol 101128jvi0073708 bohmwald k galvez n r´Ä±os m and kalergis am neurologic alterations due to respiratory virus infections front cell neurosci 103389fncel201800386 montalvan v lee j bueso t de toledo j and rivas k neurological manifestations of covid19 and other coronavirus infections asystematic review clin neurol neurosurg 101016jclineuro2020105921 zhou f yu t du r fan g liu y liu z clinical course and risk factors for mortality of adult inpatients with covid19 in wuhanchina a retrospective cohort study lancet linkinghubelseviercomretrievepiis0140673620305663101016s0140673620305663 varga z flammer aj steiger p haberecker m andermatt r zinkernagel as endothelial cell infection and endotheliitis incovid19 lancet 101016s0140673620309375 bostanciklio Ëglu m sarscov2 entry and spread in the lymphatic drainage system of the brain brain behav immun 101016jbbi202004080 li yc bai wz and hashikawa t the neuroinvasive potential of sarscov2 may play a role in the respiratory failure of covid19 patientsj med virol 101002jmv25728 li y bai w hirano n hayashida t taniguchi t sugita y neurotropic virus tracing suggests a membranouscoatingmediatedmechanism for transsynaptic communication j comp neurol 101002cne23171 reichard rr kashani kb boire na constantopoulos e guo y and lucchinetti cf neuropathology of covid19 a spectrum ofvascular and acute disseminated encephalomyelitis ademlike pathology acta neuropathol s00401020021662 kanberg n ashton nj andersson lm yilmaz a lindh m nilsson s neurochemical evidence of astrocytic and neuronal injurycommonly found in covid19 neurology nneurologycontentneurologyearly20200616wnl0000000000010111fullpdf101212wnl0000000000010111 xiao l haack kkv and zucker ih angiotensin ii regulates ace and ace2 in neurons through p38 mitogenactivated protein kinase andextracellular signalregulated kinase signaling am j physiol cell physiol c1073c1079 101152ajpcell003642012 tang n li d wang x and sun z abnormal coagulation parameters are associated with poor prognosis in patients with novel coronaviruspneumonia j thromb haemost 101111jth14768 mehta p mcauley df brown m sanchez e tattersall rs manson jj covid19 consider cytokine storm syndromes andimmunosuppression lancet 101016s0140673620306280 koralnik ij and tyler kl covid19 a global threat to the nervous system ann neurol 101002ana25807 toscano g palmerini f ravaglia s ruiz l invernizzi p cuzzoni mg guillainbarr ´e syndrome associated with sarscov2 nengl j med nejmc2009191 101056nejmc2009191 madjid m safavinaeini p solomon sd and vardeny o potential effects of coronaviruses on the cardiovascular system jama cardiol jamanetworkcomjournalsjamacardiologyfull2763846 epelman s shrestha k troughton rw francis gs sen s klein al soluble angiotensinconverting enzyme in human heartfailure relation with myocardial function and clinical outcomes j cardiac fail 101016jcardfail200901014 wild ps dimmeler s and eschenhagen t an epidemiological study exploring a possible impact of treatment with ace inhibitors orangiotensin receptor blockers on ace2 plasma concentrations j mol cell cardiol 101016jyjmcc202003018 wu z and mcgoogan jm characteristics of and important lessons from the coronavirus disease covid19 outbreak in china jama jamanetworkcomjournalsjamafull2762130 101001jama20202648 zheng yy ma yt zhang jy and xie x covid19 and the cardiovascular system nat rev cardiol 101038s4156902003605 ruan q yang k wang w jiang l and song j clinical predictors of mortality due to covid19 based on an analysis of data of patients from wuhan china intensive care med s0013402005991x the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science 101042cs20200904 cheng r and leedy d covid19 and acute myocardial injury the heart of the matter or an innocent bystander heart 101136heartjnl2020317025 zhu l carretero oa xu j harding p ramadurai n gu x activation of angiotensin ii type receptor suppresses tnfαinducedicam1 via nfκb possible role of ace2 am j physiol heart circ physiol h827h834 101152ajpheart008142014 dzimiri n receptor crosstalk implications for cardiovascular function disease and therapy eur j biochem 101046j14321033200203181x wheelerjones cpd cell signalling in the cardiovascular system an overview heart 101136hrt2005072280 houliston ra pearson jd and wheelerjones cpd agonistspeciï¬c cross talk between erks and p38 mapk regulates pgi synthesis inendothelium am j physiol physiol c1266c1276 101152ajpcell20012814c1266 adam lp franklin mt raff gj and hathaway dr activation of mitogenactivated protein kinase in porcine carotid arteries circ res 10116101res762183 nebreda ar and porras a p38 map kinases beyond the stress response trends biochem sci linkinghubelseviercomretrievepiis0968000400015954 101016s0968000400015954 wang y huang s sah vp ross j brown jh han j cardiac muscle cell hypertrophy and apoptosis induced by distinct membersof the p38 mitogenactivated protein kinase family j biol chem 101074jbc27342161 saurin at martin jl heads rj foley c mockridge jw wright mj the role of differential activation of p38mitogenactivatedprotein kinase in preconditioned ventricular myocytes faseb j 101096fj990671com podewski ek hilï¬kerkleiner d hilï¬ker a morawietz h lichtenberg a wollert kc alterations in janus kinase jaksignaltransducers and activators of transcription stat signaling in patients with endstage dilated cardiomyopathy circulation 10116101cir000005754582749ff patel ab and verma a covid19 and angiotensinconverting enzyme inhibitors and angiotensin receptor blockers what is the evidencejama jamanet
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pediatric obesity remains a public health burden and continues to increasein prevalence the gut microbiota plays a causal role in obesity and is a promising therapeutic target speciï¬cally the microbial production of shortchain fatty acids scfa fromthe fermentation of otherwise indigestible dietary carbohydrates may protect against pediatric obesity and metabolic syndrome still it has not been demonstrated that therapies involving microbiotatargeting carbohydrates known as prebiotics will enhance gutbacterial scfa production in children and adolescents with obesity age to yearsold here we used an in vitro system to examine the scfa production by fecal microbiota from children with obesity when exposed to ï¬ve different commercially availableoverthecounter otc prebiotic supplements we found microbiota from all patientsactively metabolized most prebiotics still supplements varied in their acidogenic potential significant interdonor variation also existed in scfa production which 16s rrna sequencing supported as being associated with differences in the host microbiota composition last we found that neither fecal scfa concentration microbiota scfa productioncapacity nor markers of obesity positively correlated with one another together thesein vitro ï¬ndings suggest the hypothesis that otc prebiotic supplements may be unequalin their ability to stimulate scfa production in children and adolescents with obesityand that the most acidogenic prebiotic may differ across individualsimportance pediatric obesity remains a major public health problem in the unitedstates where of children and adolescents are obese and rates of pediatric severeobesity are increasing children and adolescents with obesity face higher health risksand noninvasive therapies for pediatric obesity often have limited success the humangut microbiome has been implicated in adult obesity and microbiotadirected therapiescan aid weight loss in adults with obesity however less is known about the microbiome in pediatric obesity and microbiotadirected therapies are understudied in children and adolescents our research has two important ï¬ndings i dietary prebiotics ï¬ber result in the microbiota from adolescents with obesity producing more scfa andii the effectiveness of each prebiotic is donor dependent together these ï¬ndings suggest that prebiotic supplements could help children and adolescents with obesity butthat these therapies may not be one size ï¬ts allkeywords fermentation microbiome pediatric obesity prebiotics shortchain fattyacidscitation holmes zc silverman jd dressmanhk wei z dallow ep armstrong sc seed pcrawls jf david la shortchain fatty acidproduction by gut microbiota from childrenwith obesity differs according to prebioticchoice and bacterial community compositionmbio 11e0091420 101128mbio0091420invited editor thomas mitchell schmidtuniversity of michiganann arboreditor jose c clemente icahn school ofmedicine at mount sinaicopyright holmes this is anopenaccess distributed under the termsof the creative commons attribution international licenseaddress correspondence to lawrence a davidlawrencedaviddukeedu present address justin d silverman collegeof information science and technologypennsylvania state university state collegepennsylvania usa institute for computationaland data science pennsylvania stateuniversity state college pennsylvania usaand department of medicine pennsylvaniastate university hershey pennsylvania usareceived april accepted july published august julyaugust volume issue e0091420®mbioasm 0cholmes ®approximately of children in the united states have obesity and the prevalence continues to increase among all ages and populations the prevalence ofpediatric obesity is even higher in hispanic and african american populations in theunited states where rates of severe obesity continue to increase children withobesity have an increased risk of adverse health events and incur higher health carecosts despite the severity of the pediatric obesity epidemic current commontreatment strategies centered around lifestyle changes including behavioral dietaryand exercise interventions often fail or have limited success the high prevalence ofpediatric obesity coupled with the low success rate of common interventions highlights the need for more efï¬cacious safe strategies to lower the body mass index bmiin children and adolescentsthe human gut microbiome has emerged as a promising therapeutic target in pediatricobesity over the past decade differences in gut microbial community composition andmetabolic activity between obese and lean individuals have been observed causallinks have also been established fecal transplantation can transfer the obesity phenotypefrom obese donors to lean recipients and recapitulate some key metabolic changes inhuman obesity multiple mechanisms for this link have been proposedincludingincreased energy harvest by obese microbiota activation of enteroendocrine signalingpathways by shortchain fatty acids scfas modulation of glucose and energyhomeostasis through bile acid signaling and increased local and systemic ammation caused by a variety of microbial metabolites recent attention in obesity research has been speciï¬cally drawn to the role ofmicrobially derived scfas scfasprimarily acetate propionate and butyrateareproduced by enteric microbes as end products of anaerobic fermentation of undigested microbially accessible dietary carbohydrates and serve a variety of importantroles in the gut of particular interest is the scfa butyrate which serves as the primarynutrient source for colonocytes and functions as a histone deacetylase inhibitor through its inhibition of nf 242cb signaling in colonocytes butyrate contributesto barrier integrity maintenance and reduces levels of intestinal ammation markers acetate propionate and butyrate also each activate gproteincoupled receptors gprs that modulate key metabolic hormones including peptide yy pyy andglp1 consistent with these mechanistic ï¬ndings mouse studies have shownthat supplementation with acetate propionate butyrate or some mixture of these canprotect against weight gain improve insulin sensitivity and reduce obesityassociatedammation given the experimental evidence for scfa supplementationhaving an antiobesogenic effect in a murine system maintaining high levels of scfasduring a weight loss treatment may improve results if increasing scfa levels is a potential approach to promote weight loss in childrenprebiotic supplementation may provide an effective and lowrisk adjunctive therapyprebiotics are dietary carbohydrates that are indigestible by humanproduced enzymesand thus survive transit to the lower gastrointestinal gi tract once in the colonprebiotics serve as carbon sources for bacterial fermentation which in turn yield scfasas metabolic end products multiple types of prebiotics eg fructooligosaccharides [fos] and inulintype fructans have been tested in children with obesityranging from ages to years old in select cases these treatments have beenassociated with smaller increases in bmi and fat mass and reductions in bodyweight zscores body fat and trunk fat still other prebiotic trials in children whoare overweight have reported no significant beneï¬cial effects interpreting the mixed outcomes of prior prebiotic clinical trials in pediatric obesitythough is complicated by several challenges first in vivo studies in pediatric obesity todate have each used only one prebiotic supplement due to the logistical constraints ofclinical trials trials employing testing only a single type of supplement hinderthe ability to generalize s regarding the efï¬cacy of prebiotics and also makeit challenging to determine whether some prebiotics are inherently more acidogenicthan others second in vivo trials in healthy adults have shown substantial interindividual variation in the single prebiotic effects on stool scfa concentration julyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®variation in the primary and secondary outcomes could be due to differences inmicrobial scfa production or differences in host physiology such as scfa absorptionpotential third while scfa concentrations have been shown to be altered in childrenwho are overweight or obese changes in fecal scfas during dietary interventionhave not been measured in past in vivo studies in pediatric populations if prebioticsmediate their effects through scfas directly tracking scfas could helpdetermine treatment success fourth in vivo studies in adults especially those withobesity may be confounded by the concurrence of chronic disease and the medications a person may be taking to treat chronic diseasein this study we have taken an in vitro approach to address the limitations of priorhuman studies an in vitro approach facilitates more direct comparisons of differentprebiotic supplements the higher throughput of in vitro experiments allows widervariety of prebiotics to be tested and the effects of these supplements can be testedon identical microbiota samples rather than over time within subjects which isconfounded by microbiota drift over time as well as inconsistencies in dietarycomposition taking an in vitro approach to studying the effects of prebiotics on gutmicrobiota allows a more direct investigation of microbial scfa production since wecan study the effects of prebiotic supplementation independent of the effects of hostabsorption using a preclinical in vitro fermentation model and samples fromadolescents with obesity who have not developed longterm complications we pursued three speciï¬c lines of inquiry i whether different types of prebiotics lead todifferences in scfa production by gut microbiota from adolescents with obesity iiwhether the effects of prebiotics are shaped by interindividual differences in gutmicrobiota structure and iii whether fecal scfa production is likely to be associatedwith protection from obesityresultsscfa production capacity to measure scfa production by gut microbiota weadapted the in vitro approach of edwards this method was speciï¬callydesigned to study fermentation of starch in the human lower gi tract and has sincebeen used to measure metabolite production from human stool samples when exposed to prebiotic ï¬ber in brief we homogenized previously frozen feces inreduced phosphatebuffered saline pbs ph «¾ to create a fecal slurry with aï¬nal concentration of gliter fig these fecal slurries were then supplied witheach of ï¬ve prebiotic carbon sources as well as a carbonfree control and allowed toferment at °c in anaerobic conditions for h to approximate colonic transit time after the incubation period the concentrations of scfas in the samples weremeasured by gas chromatography to control for differences in overall cell viability orstool slurry nutrient content between donors we corrected measurements of scfaconcentration by dividing the treatment scfa concentration by the control scfaconcentrationto validate our assay we ran a series of experiments using feces from validationsample sets we veriï¬ed that our controlcorrected scfa production data were notuenced by bacterial abundance p «½ 2433 «½ spearman correlation seefig s1 in the supplemental material absolute not relativized to control scfaconcentrations are supplied in the supplement see fig s2 and s3 since our fermentation experiments used previously frozen fecal samples we veriï¬ed that total scfaproduction was strongly correlated between fresh samples and twice freezethawedsamples p ¬ 2433«½ spearman correlation see fig s4a since we elected tonot provide our fermentation reactions with nutrients in excess of what was containedin the fecal slurries we veriï¬ed that there existed strong correlation in total scfaproduction between pbsgrown and colonic mediumgrown cultures both whensupplied with dextrin and inulin dextrin p «½ 2433«½ inulin p «½ 2433«½ spearman correlations see fig s5 we found that total scfa production over controlwas positively correlated with the ph of starting fecal slurries p «½ 2433 «½ spearman correlation fig 2a a weaker correlation may exist between scfa producjulyaugust volume issue e0091420mbioasm 0cholmes ®fig overview of in vitro fermentation methodstion and the ï¬nal ph of the fermentation vessels p «½ 2433 «½ spearmancorrelation fig 2bwe subsequently applied our assay to fecal microbiota from a cohort of children male female one unknown ranging in age from to years old average age years tanner stages to and a body mass index bmi of to averagebmi see table s1 in the supplemental material one patient provided samplesused in all analyses but was lost to followup before providing clinical metadata thiscohort was a subset of a cohort of patients enrolled in the pediatric obesity microbiomefig relationship between in vitro scfa production and ph a in vitro total scfa production over control is positively correlated with the phof starting fecal slurries p «½ 2433 «½ spearman correlation b relationship between scfa production and the ï¬nal ph of fermentationvessels p «½ 2433 «½ spearman correlationjulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®fig in vitro scfa production by prebiotic a donor b and individually c in a twoway anova of the effects of donor and prebiotic on scfaconcentrationcontrol donor prebiotic and their interaction were all statistically significant p ¬ p ¬ and p ¬ respectively shownis the total scfa concentration of an in vitro culture after h of anaerobic incubation divided by the scfa concentration of the corresponding prebioticfreecontrol culture for each of ï¬ve prebiotic growth conditions across donors black dots gray diamonds are means and gray bars are standard deviationsabsolute scfa concentrations are depicted in fig s3and metabolism study we found all individuals demonstrated a net gain ofscfas relative to the controlin at least one prebiotic treatment which led us toconclude that all tested cultures were viable and metabolically active fig donor and prebiotic both impact scfa production in vitro we next tested thehypothesis that different prebiotics equally promote the production of scfas byjulyaugust volume issue e0091420mbioasm 0cholmes ®fig eighteen genera were found to be credibly associated with scfa production in at least one of our ï¬ve prebiotic growth conditions shownare the mean lambda values and and credible intervals for all genera credibly associated with at least one prebioticgrowth condition plotted on centered logratio clr coordinates red centers denote associations with credible intervals that do not cover lambda represents the strength of the effect of each covariate on each taxa a lambda value of reï¬ects a unit fold change in scfaconcentration over control as being associated with a unit fold change in the clrtransformed relative abundance of the genusperforming statistical analysis of scfa production as a function of the prebiotic typeand individual identity our analysis revealed heterogeneity in the efï¬cacy of prebioticsupplements twoway analysis of variance [anova] p ¬ see table s2 fig 3aranging from inulin which resulted in a mean fold change in total scfas togalactooligosaccharides gos which resulted in mean fold change in total scfasfrequently only two or three of the ï¬ve tested prebiotics resulted in increased totalscfa production within an individual our statistical testing also revealed consistentpatterns between individuals gut microbiota in terms of scfa production twowayanova p ¬ see table s2 fig 3b with mean fold changes in scfas over controlranging from to within individuals the average fold change in scfa concentration in the prebiotic treatments often appeared to be driven by a few stronglyacidogenic prebiotics last our analysis indicated a significant interaction betweenidentity twoway anova p ¬ see table s2prebiotic type and individualfig 3c because our statistical analysis considered technical replicates as separateexperimental conditions this result suggests the presence of consistent prebioticindividual responses across in vitro assay replicate runsnot whether such interactionsare consistent within an individual over timescfa production in vitro predicts the abundance of bacteria in the startingculture if interindividual differences in gut microbiota mediated responses to prebiotictreatment we would expect that speciï¬c bacterial taxa which varied between individuals could also be associated with scfa production to evaluate this hypothesis weused the r package stray to create a bayesian multinomial logistic normal linearregression pibble model that tested for correlations between in vitro scfa productionin response to each prebiotic and 16s rrna community composition of patient stoolused in the fermentations at the genus level this analysis revealed that scfa production from prebiotics was correlated with the relative abundances of differentbacterial genera credible interval not covering fig of the generapositively associated with scfa production are known or likely ï¬ber degraders one akkermansia is often observed to increase in abundance after prebiotictreatment and one methanobrevibacter an archaeon hydrogenotrophic methanojulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesitytable associations between microbial genera and scfa production on ï¬ve different prebiotic substratesassociation with scfa productiondextrin«¹«¹«¹«¹«¹«¹«¹«¹«¹genusakkermansiaruminococcus_2coprostanoligenes_groupparabacteroidesbutyricimonasmethanobrevibactertyzzerella_4tyzzerella_3lachnospiraceae_nk4b4lactobacilluscoprococcus_1collinsellalachnospiraceae_fcs020xosgosfosinulin«¹«¹«¹«¹fiber degrader statussupporterdegraderno evidencedegraderassociatedsupporterdegraderdegraderdegraderdegraderdegraderno evidencedegrader®referencegen is known to increase the efï¬ciency of carbohydrate metabolism by the microbiota table most genera identiï¬ed by stray were associated with scfa production ina limited set of prebiotic treatments one genus lactobacillus is positively associatedwith scfa production on xylooligosaccharides xos but was negatively associatedwith scfa production on gos overall the presence of speciï¬c associations betweenbacterial taxa and different prebiotics supports a model where different individuals varyin their levels of prebiotic degrading gut bacteriametrics of obesity do not appear to correlate with scfa production capacity ofstool finally we tested the hypothesis that in vitro scfa production would beassociated with obesityrelated phenotypes we compared clinical metadata fromindividuals which included bmi insulin and hba1c with average total scfa production across prebiotics and found no significant correlations in our population spearman correlation table fecal microbial scfa production capacity may not be directlyassociated with obesity though because rates of host scfa uptake likely vary and thisvariance may uence host intestinal physiology indeed in support of theidea that scfa absorption rate which was not measured in this study shape metabolichomeostasis and host health we observed a negative association between fecal scfaconcentrations and in vitro scfa production across the range of tested prebioticsfig furthermore if scfa absorption efï¬ciencies varied by individual residual fecalscfa concentrations may not directly reï¬ect the complete effect of bacterial metabolism on obesity consistent with this notion no significant relationships were apparentbetween concentrations of scfa in patient stool and clinical markers of obesitymeasured at enrollment including bmi insulin levels and hba1c table althoughthis may also be explained by uncontrolled patient parametersdiscussionin this study we found that the microbiota of all tested adolescents with obesityincreased total scfa production when exposed in vitro to at least one prebiotic bothdonor and prebiotic were significant factors in determining scfa production in vitro aswas their interaction our modeling revealed distinct associations between speciï¬cmicrobial taxa and scfa production on different prebiotics we interpret this result astable neither average scfa production in vitro nor fecal scfa concentrationcorrelated with metrics of obesity measured in individuals at the time of enrollmentabmiparameteravg net scfa productionfecal scfa concentrationap and 2433 values were determined from spearman correlations 2433pinsulinp 2433hba1cp 2433julyaugust volume issue e0091420mbioasm 0cholmes ®fig spearman correlations between in vitro scfa production and scfa concentration of the starting fecal inoculum scfaproduction is the average of technical replicates with the linear regression line plottedsuggesting that the associated bacteria play a role in the ï¬ber fermenting capacity ofthe community we observed no correlations between either stool scfa concentrationsor in vitro acidogenic capacity of communities and any metrics of obesity table we have recapitulated previous ï¬ndings that both donor and prebiotic areimportant in determining the scfa production from in vitro prebiotic supplementation and we found that not all prebiotics appear equally acidogenic since our in vitro system removes the host as a potential source of variationour data support a gut microbial role for interdonor variation in fecal scfaproduction in addition the strength of the interaction between donor and prebiotic strongly suggests that prebiotics are not one size ï¬ts all rather inconsistentresults from prior studies of prebiotics in pediatric obesity may be dueto variation in the scfa production capacity of individuals gut microbiota acrossthe tested prebiotics future therapeutic efforts involving prebiotics in patients withobesity may beneï¬t from stratiï¬ed or personalized treatments nutritional therapiesthat are personalized to individuals microbiota are already in development murine and in vitro studies show that increased signaling through gpcrs mediatedby acetate propionate and butyrate increases satiety and insulin sensitivity whiledecreasing adipogenesis yet we did not observe associations betweenfecal scfa levels and metrics of obesity the effects of scfa on obesity may be maskedby uncontrolled patient factors such as differences in caloric intake and variation inindividual nutrient harvest and utilization in order to observe the effects of scfa onobesity it would be necessary to control for these variable physiological and lifestyleparameters which we did not attempt these patient factors may also have uencedour inability to observe an association between acidogenic capacity of microbiota andfecal scfa concentrations however this may also be explained by the potentialjulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®uncoupling of fecal scfa production and fecal scfa concentration in vitro increasedluminal concentrations of butyrate have been shown to upregulate the sodiumcoupled monocarboxylate transporter slc5a8 and the addition of physiologicalmixtures of scfa has been shown to upregulate the monocarboxylate transporterslc16a1 both of which uptake acetate propionate and butyrate from the lumensince gut epithelia have the capacity to absorb up to of scfa before excretion increased host scfa uptake triggered by increased gut bacterial production couldtherefore lead to constant or even decreased fecal scfa concentrations this complexrelationship could explain the absence of positive correlations we observed betweenstool scfa levels and the acidogenic capacity of gut microbiota it may be necessary todelve further upstream of fecal scfa concentration by measuring proxies for host scfauptakes such as the expression of scfa transporters slc5a8 and slc16a1 and scfareceptors gpr43 gpr41 and gpr109a the primary limitations of this study involve constraints common to in vitro culturestudies first many factors affecting bacterial scfa production in vivo are difï¬cult toreplicate in vitro including the availability of nutrients such as nitrogen the startingconcentration of scfas the redox state of the environment and the efï¬ciency ofcrossfeeding interactions different metabolic results between prebiotics mayhave occurred if we provided alternative cometabolites or nutrients in addition to thetested prebiotics we chose our culture conditions namely a mediumfree approachthat does not add any nutrients beyond what is present in the stool in an effort toavoid inducing artiï¬cial selective conditions within our cultures prior experimentaldigestion studies have shown that prebiotic response patterns can be recapitulatedacross various culture conditions indeed we found strong correlation in scfaproduction between cultures grown with our mediumfree approach and those grownin a more conventional medium containing added nitrogen vitamins minerals andacetate further this approach allowed us to minimize the uence of the host onmeasurements of microbiota production of scfa we did observe shifts in communitycomposition during the h fermentations fig s6 however we remained able to ï¬ndstatistical associations between scfa production capacity and prefermentation community composition a second set of limitations in this study involves our reliance onpatient collection of stoolinterdonor variation in prebiotic response could haveoriginated in technical variation between how patients exposed stool to aerobicconditions or how they froze their samples which in turn could have affectedthe fraction of viable microbial cells in stool samples still we found a significantcorrelation between in vitro total scfa production from fresh stool and stool that hadbeen frozen and thawed twice variation in donor prebiotic response could also havebiological origins due to physiological differences between people eg efï¬ciency offood digestion consistency of stool [] or differences in diet which can lead tovariation in stool microbial load and nutrient content rather than control for amyriad of different sources of variation whose origins we did not measure we chosethe straightforward approach of standardizing donor samples by employing a consistent concentration of stool slurry [wtvol] stool in pbs in our experimentsfuture work to address these limitations could test multiple stool samples persubject to conï¬rm whether the observed variation in prebiotic response is durablebetween individuals over time future studies could also examine the correlationbetween the metabolic effects of prebiotic supplementation in vitro and in vivo usingrandomized human trials that couple human prebiotic supplementation in vivo measurement of scfa production and in vitro tests of microbiota metabolic activity itwould also be useful for such studies to explore the impact of prebiotic supplementation on host physiology both in vitro and in vivo speciï¬cally the effects of prebioticsupplementation on colonic epithelial barrier integrity scfa receptor gpr41 gpr43and gpr109a expression and scfa transporter mct1 and smct1 expression couldprovide greater insight into the health impacts of prebiotic supplementation as well asexplain why fecal scfa concentrations may not mirror the metabolic capacity of gutmicrobiotajulyaugust volume issue e0091420mbioasm 0c®holmes materials and methodscohort stool was collected from human donors under a protocol approved by the duke healthinstitutional review board duke health irb pro00074547 for a prospective longitudinal cohort studyand biorepository participants whose samples were used in this study were treatmentseeking adolescents with obesity who were newly enrolled in a multidisciplinary weight management program allsubjects received familybased intensive lifestyle modiï¬cation based on clinical necessity some participants also were placed on a lowcarbohydrate diet medications to facilitate weight loss or underwentweight loss surgery see table s3 due to the low number of patients assigned to each treatment armwe did not attempt to base any analyses on patient treatment plan patients were to years old witha bmi ± 95th percentile none had antibiotic use in the month prior to enrollment used medicationsknown to interfere with the intestinal microbiome or had other significant medical problems stoolsamples used in this study were from enrollment 3month 45month and 6month followup visits seetable s3 the clinical metadata used for correlations was collected at enrollment months and months the metadata collected nearest to the stool sample collection date was used in our analysesstool collection patients collected intact stool samples in the clinic or at home using a plastic stoolcollection container fisher scientiï¬c and were asked to immediately store this container intheir home freezer patients then returned the sample by either bringing it to the study team orscheduling a home pickup within h of stooling stool was transported frozen in an insulated containerwith an ice pack upon receipt in the lab samples were placed on dry ice until transferred to a °cfreezer for longterm storage all patient samples were frozen at °c within h of stooling range h to h median h except for one which was stored h after stooling the timebetween stooling and freezing at °c did not have a significant effect on average scfa productionp «½ 2433 «½ «º pearson correlation stool samples for analysis were processed by removingcontainers from °c storage and thawing on ice in a biological safety cabinet until soft enough toaliquot thawed containers of stool were opened to atmosphere for a maximum of min while sampleswere aliquoted after primary aliquoting the remaining stool was transferred to an anaerobic chambercoy laboratory products hydrogen co2 nitrogen and further portioned into ¬2galiquots for this study these aliquots were then stored as solid stool pellets at °c until used for thisstudyin vitro fermentation see fig for an overview of in vitro fermentation methods aliquotedstool was thawed at room temperature in an anaerobic chamber once thawed stool was weighedand placed into a polyethylene ï¬lter bag with 033mm pore size whirlpak b01385 and ml ofanaerobic «» pbs was added for each gram of stool resulting in a wtvol fecal slurry similarto previous studies during our validation experiments a medium designed tosimulate colonic contents was used in place of «» pbs to create stool slurries the ï¬lter bag wasthen closed and placed into a stomacher seward stomacher where the contents were homogenized on the medium speed setting for s the liquid fraction was removed from the downstreamside of the ï¬lter membrane and the solid fraction was discarded a 1ml aliquot of this liquid fractionwas removed for analysis of the scfa concentration to determine the scfa concentration of thestarting stool sample during our validation experiments two separate 1ml aliquots of this liquidfraction were removed one was used to estimate relative bacteria abundance of starting fecalslurries using total extracted dna concentration as has been previously published and theremaining aliquot was used to determine the ph of the starting fecal slurry using a handheld phmeter elite ph spear thermo fisher scientiï¬c the remaining liquid fraction was incubated induplicate across six different treatments either supplemented with inulin now foods inulin powderpart fructooligosaccharides fos cargill part galactooligosaccharides gosbimuno powder xylooligosaccharides xos bionutrition prebiotic with llifeoligo part wheatdextrin beneï¬ber original or unsupplemented for each reaction ml of fecal slurry wasplaced in one well of a 24well cell culture plate each well was then delivered ml of wtvolprebiotic solution in «» pbs or ml of «» pbs without prebiotic during our validation experimentsprebiotics were dissolved in colonic medium instead of «» pbs the resulting fermentationconditions were therefore fecal slurry with prebiotic wtvol a fecal slurry was selectedbecause its fermentative capacity has been previously demonstrated to be insensitive to smallvariations in concentration and is feasible to work with using this method a ï¬nalconcentration of prebiotic in the context of a fecal slurry i
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"diagnostic performance of intravoxel incoherent motion diffusionweightedimaging IVIMDWI in the differential diagnosis of pulmonary tumors remained debatable among published studiesThis study aimed to pool and summary the relevant results to provide more robust evidence in this issue using ametaanalysis methodMaterials and methods The researches regarding the differential diagnosis of lung lesions using IVIMDWI weresystemically searched in Pubmed Embase Web of science and Wangfang database without time limitation ReviewManager was used to calculate the standardized mean difference SMD and confidence intervals ofapparent diffusion coefficient ADC tissue diffusivity D pseudodiffusivity D and perfusion fraction f Stata was used to pool the sensitivity specificity and area under the curve AUC as well as publication bias andheterogeneity Fagans nomogram was used to predict the posttest probabilitiesResults Eleven studies with malignant and benign lung lesions were included Most include studies showed alow to unclear risk of bias and low concerns regarding applicability Lung cancer demonstrated a significant lower ADCSMD P D SMD P and f values SMD P than benign lesions except Dvalue SMD P D value demonstrated the best diagnostic performance sensitivity specificity AUC and highest posttest probability and for D ADC f and D values in the differential diagnosisof lung tumors followed by ADC sensitivity specificity AUC f sensitivity specificity AUC and D values sensitivity specificity AUC Continued on next page Correspondence 849049724qqcom wuypsysucccnhenisysucccn Jianye Liang Jing Li and Zhipeng Li contributed equally to this work2Department of Radiology Maoming Peoples Hospital Maoming Guangdong China1Department of Medical Imaging Sun Yatsen University Cancer Center StateKey Laboratory of Oncology in South China Collaborative Innovation Centerfor Cancer Medicine No651 Dongfeng Road East Guangzhou Guangdong China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLiang BMC Cancer Page of Continued from previous pageConclusion IVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lungtumors based on the tumor cellularity and perfusion characteristics and D value demonstrated better diagnosticperformance compared to monoexponential ADCKeywords IVIMDWI Posttest probability Diagnostic performance Lung neoplasm Magnetic resonance imaging MetaanalysisIntroductionLung cancer is the most commonly diagnosed cancer of the total cases and the leading cause of cancerdeath of the total cancer deaths in aroundthe world [] The incidence and mortality of lung cancer still increased in recent years Accurate and earlydiagnosis is help to select optimal treatment strategy andimprove the outcome of patients with lung cancerlungtumorsandefficacyComputed tomography CT is the main imaging modality for lung lesions largely based on morphologicaland enhanced characteristics However the relativelylow specificity and administration of contrast agent limitits wide use in clinical practice Magnetic resonance imaging MRI was rarely used in detecting lung lesionspreviously due to the obvious cardiac and respiratorymotionlow signaltonoise ratio from the inherentlylow lungproton density and magnetic susceptibilityartifact of airfilled pulmonary tissue subjected to highfield strength [] With the development of MRI hardwares and various rapid imaging technologies such asimproved gradient performance parallel imaging techniques and freebreathing acquisition MRI has been inidentification of benign andcreasingly used formalignantevaluationDiffusionweighted imaging DWI is a radiationfreeand contrastfree functional imaging sequence which allows measurement of water molecular movement usingapparent diffusion coefficient ADC and demonstratespotential to differentiate malignant from benign lung lesions A previous metaanalysis even reported a higherdiagnostic performance with a pooled sensitivity specificity and areas under the curve AUC of and in DWI compared to PETCT whose sensitivityspecificity and AUC were and respectivelyThe monoexponential model is expressed as SI SI0 expb·ADC where SI0 refers to the mean signal intensity SI of the region of interest for b smm2 while SIrefers to the signal intensity for higher b values However the monoexponential model cannot separate thepseudodiffusion from pure molecular diffusion andADC calculated from the monoexponential modelmixesthe conventionalmonoexponential model cannot accurately reflect thetrue diffusivity owing to the influence of microcirculation perfusion []the two effects Thereforechangesthe microenvironmentIntravoxel incoherent motion IVIM is an advancedimaging technique which was first proposed by Le Bihan [] It can separate the incoherent motion of watermolecules within the capillaries from molecular diffusionin the extravascular space [] The true diffusion coefficient D value pseudodiffusion coefficient D valueand perfusion fraction f value were generated using abiexponential model with multiple bvalues expressed asSI SI0 f · expbD f · expbD The IVIMmodel can separate the pseudodiffusion from pure molecular diffusion and independently reflect the microcirculation perfusion D and tumor cellularity D basedon that equation [] This model provides more detailedand accurate information and can make a better interpretation forandcharacterization of tumor grades As such these parameters are important to be analyzed Several studies hadapplied IVIMDWI to discriminate lung cancer from benign lesions and demonstrated better or comparablediagnostic performance compared with traditional ADCvalue [] However the diagnostic performances ofIVIMDWI derived parameters in the differentiation oflung tumors were not consistent and the application stillremained debatable in the lung For example severalstudies indicated that lung cancer had a higher D valuethan benign lesion [] while some studies reportedadverse [ ] or insignificant results [ ] Theoretically the true diffusitivity should have better diagnostic performance than ADC in distinguishing lunglesions but some studies indicated a much lower areaunder the curve AUC or accuracy in D value comparedto ADC [ ] Cancerous tissue generally has activeangiogenesis and rich blood supply compared to benignlesions but most studies indicated a lower f value inlung cancer the results of which should be further confirmed The sample sizes in most studies were still notenough to draw a robust for its performancethe application of IVIMDWI in the lung has not yetformed a clinical guideline or become a routine sequence in the MRI protocol Therefore we attempted topool all the published results about the diagnostic performance of IVIMDWI in the differentiation of malignant and benign lung lesions using a metaanalysismethod Besides the diagnostic performance of IVIMDWI was compared to conventional DWIderived ADC 0cLiang BMC Cancer Page of this study provides additionalvalue to determine the suitability for clinical applicationThe controversialissues between different researcheswill also be addressed with more reliable evidence Furthermoreinformationabout technical feasibility on lung MRI and the functional changes oflung lesions with IVIMDWI Thisstudy may further attract the researchers to perform thelung studies using noninvasive MR imaging by solvingthe technical issues on Lung MRIMaterials and methodsData sourcesThe studies regarding the differential diagnosis of lungtumors using IVIMDWI parameters were systemicallyretrieved by two senior librarians in PubMed EmbaseWeb of science and Wangfang database without timelimitation A searching formula was formed with different combinations of the medical subject headings or keywords from IVIM intravoxel incoherent motion multiple bvalue DWI biexponential and lung or pulmonarylesion cancer carcinoma neoplasm The primarysearches were limited in the titles and abstracts We alsoperformed a manual retrieval of the reference lists fromincluded studiesbStudies selectionStudies met the following criteria were included a theresearch purpose was to differentiate lung cancer frombenign lesions using IVIMDWI parametersthemean and standard deviation SD of each parameterwas provided c their diagnostic performance aboutsensitivity and specificity or truepositive TP falsenegative FN falsepositive FP and truenegative TNwere reported d the lung cancer should be confirmedby pathology after initial MRI examination Exclusioncriteria mainly included a duplication from the sameauthors or institutions b metaanalysis conference abstract review or any unpublished results and c animalexperiments or nonlung researchesData extractionA spreadsheet was used to extract the mean values andSD as well as the diagnostic performance of ADC D Dand f values with threshold value AUC sensitivityand specificity in respective study by one author andreviewed by another one Other information includedthe first author publication years field strength and vendors b values patient ages tumor sizes and numbers ofmalignant and benign lesions TP FN FP and TN canbe calculated when only the amount of malignant andbenign lesions as well as sensitivity and specificity or receiver operating curve was providedQuality assessmentThe quality of studies and likelihood of bias were evaluated using Review Manager software Cochrane Collaboration Oxford UKreferring to the QualityAssessment of Diagnostic Accuracy Studies [] Weassessed the risk of bias and applicability in four domains including patient selection index tests referencestandard flow and timing []Publication bias and heterogeneity evaluationAs two parts of data were pooled in our study includingquantitative values and diagnostic performance of eachparameter funnel plots and Beggs test were used tovisually and quantitatively assess the publication bias forthe continuous variables and Deeks plot assessed thepublication bias of sensitivity and specificity using Stataversion StataCorp LP College Station TX Anasymmetric or skewed funnel plot P of Beggs testor Deeks test indicated the potential of publication bias[] Inconsistency index I2 and Cochrans Q tests wereused to explore the heterogeneity of included studieswith I2 or P for Cochran Q test suggestedstatistically significant heterogeneity and a randomeffect model was applied in subsequent pooling or afixedeffect model when I2 []Evidence synthesisWe constructed the forest plots for continuous variablesand calculated the standardized mean difference SMDbetween lung cancer and benign lesions using ReviewManager software We used the bivariate regressionmodel to pool the diagnostic performance with sensitivity specificity positive likelihood ratio PLR negativelikelihood ratio NLR diagnostic odds ratio DOR andAUC using Stata version The summary receiveroperating characteristic curves and Fagans nomogramswere also plotted to determine the diagnostic values andpredict the posttest probabilities of ADC D D and fvalues in the differential diagnosis of lung tumorsResultsLiterature search and selectionBy searching the key words in the titles and abstracts atotal of potential studies were obtained from multiple databases A total of studies regarding metaanalysis conference abstract case report and reviewwere excluded after screening the titles and abstractsAnimal studies nonlung researches and duplicationfrom the same authors or institutions led to further exclude studies We scrutinized the fulltexts of theremaining studies in detail and excluded an additional studies for the following reasons a lack ofsufficient data to be pooled b low quality assessmentcIVIMDWI was interfered by treatment and d 0cLiang BMC Cancer Page of cancer was not confirmed by pathology Eventually eligible studies with malignant and benign lunglesions were included for analysis The flowchart detailing the process of study selection was provided in Fig Basic information and diagnostic performance for eachincluded study was detailed in Table and Table Inother to include every potential we did not set acriterion on the field strength T or T FromTable there are three studies using T and eightstudies using T for imaging Although field strengthof T is better for image quality the results from Tscanner are also acceptable Therefore studies with either of field strengths are included for analysisQuality assessmentThe distribution of Quality Assessment of DiagnosticAccuracy Studies scores for risk of bias and applicability concerns were shown in Fig The overall qualityof included studies was acceptable Regarding patient selection four studies were marked unclear risk of bias dueto ambiguity for consecutive enrollment and prospectivedesign or not The applicability concerns remainedunclear concern as the tumor types were inconsistentbetween malignant and benign tumors from two studiesTwo studies were marked unclear and high risk of biaswith unclear concern of applicability for index test asthe threshold values for D and f values were not provided Three studies showed unclear risks of bias for reference standard because some of the benign lesionswere diagnosed through a long time followup Threestudies were marked unclear and high risk of bias in patient flow and timing domain because the time intervalbetween MR examination and pathological confirmationwas not reportedQuantitative analysisADC used for diagnosis of lung tumorNine studies regarding ADC used in differentiating lungtumors were included for analysis The Ï2 andP of heterogeneity test with I2 suggestedmoderate heterogeneity among included studies Theforest plot in Fig showed the distribution of ADC between lung cancer and benign lesions A randomeffectsmodel generated a SMD of P Fig Flowchart detailing the study selection process Eleven studies that met the inclusion criteria were included FN false negative FP falsepositive TN true negative TP true positive 0cLiang BMC Cancer Page of Table Basic information for each included studyAuthorDeng []Machine type T PhilipsYearb values smm2Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Koyama []NA Not available T GE T Siemens T GE T Philips T Siemens T Philips T Siemens T GE T GE T PhilipsAge years ± ± ± NA ± ± ± Tumor size cm Malignant ± BenignNA ± NA ± NA ± Table The diagnostic performance for each included studyIndicatorADCAuthorDeng []ThresholdYearHuang []Jiang []Wan []Wang Y []Yuan []Zhou []Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Deng []Huang []Jiang []Wan []Yuan []Zhou []Wang XH []Deng []Huang []Wan []Wang LL []Yuan []NANANADDfAUCNANANANANANASensitivitySpecificityTPFPFNTNWang XH []NA Not available ADC Apparent diffusion coefficient D Tissue diffusivity D pseudodiffusivity f Perfusion fraction AUC Area under the curve FNFalse negative FP False positive TN True negative TP True positive Threshold values of ADC D and D are factors of mm2s 0cLiang BMC Cancer Page of Fig The distribution of risk of bias and applicability concerns for each included study using QUADAS2 a and a summary methodologicalquality b between lung cancer and benign lesions forADC A basically symmetric funnel plot in Fig andP of Beggs Test suggested no publication biasin ADCD value used for diagnosis of lung tumorEleven studies regarding D value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studies Theforest plot in Fig showed the distribution of D value between lung cancer and benign lesions A randomeffectsmodel generated a SMD of P between lung cancer and benign lesions for D value A basically symmetric funnel plot in Fig and P of BeggsTest suggested no publication bias in D value 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of apparent diffusion coefficient ADC between lung cancer and benign lesions The standardized meandifferences indicated that lung cancers had a significantly lower ADC than benign lesionsFig Funnel plot of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f Thebasically symmetric funnel plots indicated no publication bias in these parameters 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of tissue diffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower D value than benign lesionsD value used for diagnosis of lung tumorTen studies regarding D value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested obvious heterogeneity among included studiesThe forest plot in Fig showed the distribution of Dbetween lung cancer and benign lesions A randomeffects model generated a SMD of P between lung cancer and benign lesions forD A basically symmetric funnel plot in Fig and P of Beggs Test suggested no publication bias in Df value used for diagnosis of lung tumorEleven studies regarding f value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studiesThe forest plot in Fig showed the distribution off value between lung cancer and benign lesions Arandomeffects model generated a SMD of P between lung cancer andbenign lesions for f value A basically symmetricfunnel plot in Fig and P of Beggs Testsuggested no publication bias in f valueDiagnostic performanceThe Diagnostic performance with pooled sensitivity specificity PLR NLR DOR and AUC of ADC D D and fvalues were listed in Table Deeks funnel plots in Fig and asymmetry tests indicated no obvious publicationbias in ADC D D and f values P and for ADC D D and f values respectively Fig plotted the summary receiver operating characteristiccurves of ADC D D and f values D value demonstrated the best diagnostic performance sensitivity specificity AUC in the differentialdiagnosis of lung tumors followed by ADC sensitivity specificity AUC f sensitivity Fig Forest plot of the mean value of pseudodiffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that the difference of D value between lung cancers and benign lesions were insignificant 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of perfusion fraction f between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower f value than benign lesionsspecificity AUC and D values sensitivity specificity AUC Posttest probabilitiesLikelihood ratio and posttest probability were also important for diagnosing a disease [] which provided alikelihood that a patient was diagnosed with a certaindisease or not using the MRI parameters Fig plottedthe Fagans nomograms of ADC D D and f values forpredicting posttest probabilities All the pretest probabilities were set at by default We regarded thediagnosis of lung cancer as a positive event corresponding to a lower ADC D and f values Similarly the noncancerous tissues with a higher ADC D and f valueswere regarded as a negative event The posttest probability increased to from a pretest probability of with a PLR of and decreased to with a NLRof with the prompt of ADC This indicated that thediagnostic preference to lung cancer will be obviouslyenhanced with the help of ADC a lower ADC compared with the condition without the prompt of ADCwhose diagnostic probability was set at beforehandIn contrast the probability of diagnosing lung cancerwill significantly drop from to when a negativeevent occurs a higher ADC Similarly the posttestprobability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of using D for guiding The posttest probability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of in the help of fvalue These data indicated that both ADC and IVIMparameters helped to enhance the accuracy for diagnosing lung cancerDiscussionIVIMDWI is a noninvasive technique that shows superiority in reflecting tumor cellularity and perfusion without the need of contrast agent It had already beenapplied in the differentiation of thyroid nodules []breast [] liver [] and brain tumors [] with gooddiagnostic performance To our best knowledge there isstill no pulmonary study with large sample size to settledown the value of IVIM for quantitatively distinguishinglung cancer from benign tissues in the background ofIVIM becoming a research hotspot in the wholebodytumors Our study provided a timely summary in thisissue through pooling all published evidence with strictinclusion criteria and quality assessment The resultsdemonstrated IVIM model had a good diagnostic performance in distinguishing lung lesionsTable Pooled estimates and heterogeneity measures for ADC D D and f valuesDORIndexSpecificitySensitivityNLRPLRAUCADC DD I2 SensitivitySpecificity fADC Apparent diffusion coefficient D Tissue diffusivity D Pseudodiffusivity f Perfusion fraction PLR Positive likelihood ratio NLR Negative likelihood ratio DORDiagnostic odds ratio AUC Area under the curve I2 inconsistency index 0cLiang BMC Cancer Page of Fig Deeks funnel plots regarding diagnostic performance for a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivityD and d perfusion fraction f No publication bias was indicated in the four parameters P In this metaanalysis the SMDs suggested that lungcancer demonstrated a lower ADC and D values thanbenign lesions The lung cancer usually has dense cellularity and nucleoplasm ratio with active proliferativecapacity which may reduce the extracellular space andrestrict the movement of water molecules causing a reduction in diffusion coefficient The pooled results alsosuggested an excellent diagnostic performance with ahigh sensitivity specificity AUC and increased posttestprobability in both ADC and D values followed by fvalue Monoexponential modelancannot provideindependent perfusionrelated parameter and may miscalculate the water molecule movement due to a mixwith microcirculation perfusion and therefore resultedin an overestimated ADC value in a certain extent []Therefore the best diagnostic performance was observedin D value instead of ADC valueInterestinglylung cancer demonstrated a significantlower f value but insignificant D value compared withbenign lesions F value refers to vascular volume ratioand reflects the microcirculation perfusion in the capillaries F value increases with increased tissue perfusion 0cLiang BMC Cancer Page of Fig Summary receiver operating characteristic SROC curve of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f in the diagnosis of lung lesions D value demonstrated the highest area under the curve followed byADC f and D valuesinflammatoryconsistHigher f value is supposed to be observed in malignanttumors due to neovascularization compared to benignlesions However these results are not unreasonable because the benign lesions occurring in the lung are generallyoftuberculosisinfectiongranuloma or bloodrich tumor such as inflammatorypseudotumor They are usually featured by marked vascular changesincreased bloodflow and enhanced vessel permeability which generallyincluding vasodilationinfections whichanic pneumoniafungaloccur at the capillary network [] A perfusion studyusing CT with exogenous contrast indicated active infectious nodules had comparable or even higher perfusionpeak enhancement increment and blood volume withsteeper time to peak than malignant nodules [] Theresults were in good agreement with our study in another aspect However the diagnostic performance of fvalue was relatively low with the sensitivity specificityand AUC of and only F value is also associated with echo time relaxation effects and T2 0cLiang BMC Cancer Page of Fig Fagans nomogram of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction fD and ADC demonstrated similar and highest posttest probability among the four parameterscontribution [] which may reduce its diagnostic accuracyperformance to a certain extentD value is proportional to the average blood velocityand mean capillary segment length [] D value wasnot statistically significant in differentiating benign andmalignant lung lesions in this metaanalysis A poormeasurement reproducibility of D was indicated by thehuge standard deviations in the included studies Theoretically the more bvalues are selected the higher theaccuracy of model fitting will be Besides measurementat lower bvalue had been reported to be less reproducible and stable compared with measurement at higherbvalue and previous studies suggested measurements ata larger number of lower bvalue should be obtained forreducing measurement errors and signalto noise variation [ ] However a larger number of bvalue applied in IVIM model will significantly prolong thescanning times and introduce obvious motion and susceptibility artifacts especially in the pulmonary MRITherefore D value is still not adequate to differentiatelung lesions due to the low reliability stability and accuracy as indicated in our metaanalysisADC D D and f values all demonstrated moderate toobvious heterogeneity which should be explored Firstboth T and T MR scanners with various combinations of bvalue were used to perform IVIMDWI inthese studies which may influence the accurate calculations of diffusion and perfusion coefficients and decrease the diagnostic performance compared to monoexponential ADC Second the lesion sizes and density oflung cancer such as ground glass opacity on initial CTvaried from studies to studies which may perform different biological characteristics and also lead to themeasurement variability in ADC and IVIM parametersindicated by Weller [] and Jiang []Third the benign lesions consisted of a variety of inflammatory infections and benign tumors which mayintroduce significant heterogeneity in these parameterswhen compared with lung cancer Last most studies delineated the regions of interest on the largest slice instead of the entire tumors which may lead to someselection bias owing to tumor heterogeneity Histogramanalyses for the whole lesions which can reduce themeasurement variability may be a more promisingmethod for assessing lung nodules in the future studyThere were several limitations First as the sensitivityof detecting pure ground glass opacity or small lesionsare quite low on conventional DWI or IVIMDWI theselesions were inevitably excluded from the original studies which may decrease the availability of IVIM in theclinical application to a certain extent Second we hadnot performed a direct comparison with dynamic contrast enhancedCTMRI or Fluorine 18FDG PETCTwhich was also commonly used in the diagnosis of lungcancer The issue about whether IVIMDWI addedvalues to multiparametric MRI or CT in a large samplesize was still not clearConclusionsIVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lung tumors and D value demonstrated better diagnosticperformance compared to monoexponential ADC Fvalue can differentiate the perfusion difference betweenlung cancer and benign lesions The application ofIVIMDWI will further help the clinicians make a bettermanagement for cancer treatment and prognosis evaluation based on the tumor cellularity and perfusion characteristics detected by IVIM technique 0cLiang BMC Cancer Page of AbbreviationsAUC Area under the curve ADC Apparent diffusion coefficient D Tissuediffusivity D Pseudodiffusivity IVIMDWI Intravoxel incoherent motiondiffusionweighted imaging SMD Standardized mean differenceI2 Inconsistency index PLR Positive likelihood ratio NLR Negative likelihoodratio DOR Diagnostic odds ratioAcknowledgementsNot applicableAuthors contributionsNH was the guarantor of this metaanalysis and had full access to all the datain the study and took responsibility for the integrity of the data and the accuracy of the data analysis NH YW and XL conceived the study and revisedthe manuscript JL ZL and TM drafted the manuscript JC and WM searchedthe databases and acquired the data WM and SC performed data analysisand interpretation Jing Li substantively revises the manuscript based on thecomments and provides language proofreading for the revised version Allauthors had read and approved the manuscriptAuthors informationNot applicableFundingThe Highlevel Hospital Construction Research Project of Maoming PeoplesHospital supported the consultation fee from a statistician for checking thecorrectness of the statistical methods the National Key Research and Development Program of China grant no 2017YFC0112605 and the Medical Science Research Foundation of Guangdong Province of China grant no supported the fee for language editing and processingcharge for accessAvailability of data and materialsAll the original data were provided in the main document as well as thetables and figures They can also be obtained from the Internet databasesEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors have stated explicitly that there are no conflicts of interest inconnection with this Received May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin httpsdoi103322caac21492Koyama H Ohno Y Seki S Nishio M Yoshikawa T Matsumoto S Maniwa YItoh T Nishimura Y Sugimura K Value of diffusionweighted MR imagingusing various parameters for assessment and characterization of solitarypulmonary nodules Eur J Radiol httpsdoi101016jejrad201411024Le Bihan D Turner R The capillary network a link between IVIM andclassical perfusion Magn Reson Med httpsdoi101002mrm1910270116Le Bihan D Breton E Lallemand D Grenier P Cabanis E LavalJeantet MMR imaging of intravoxel incoherent motions application to diffusion andperfusion in neurologic disorders Radiology httpsdoi101148radiology16123763909Liang J Ma R Chen H Zhang D Ye W Shi C Luo L Detection ofHyperacute reactions of Desacetylvinblastine Monohydrazide in a Xenograftmodel using Intravoxel incoherent motion DWI and R2 mapping AJR Am JRoentgenol httpsdoi102214AJR1820517Liang J Cheng Q Huang J Ma M Zhang D Lei X Xiao Z Zhang D Shi CLuo L Monitoring tumour microenvironment changes during antiangiogenesis therapy using functional MRI Angiogenesis httpsdoi101007s10456019096704Deng Y Li X Lei Y Liang C L
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" many suggest that shared decisionmaking sdm is the most effective approach to clinical counseling itis unclear if this applies to surgical decisionmakingespecially regarding urgent highlymorbid operations in thisscoping review we identify s that address patient and surgeon preferences toward sdm in surgerymethods we used the preferred reporting items for systematic reviews and metaanalyses extension for scopingreviews prismascr to develop our protocol medline embase and cochrane databases were searched frominception through title review identified peerreviewed empirical s that addressed patientsurgeon preferences toward sdm in surgery identified s underwent full review by two independentinvestigators we addressed the following questions what is known from existing empirical evidence aboutpatients andor surgeons surgical decisionmaking preferences why might patients andor surgeons prefer sdm does acuity of intervention impact surgical decisionmaking preferences outcome measures included studymethods surgical specialty diagnosis study locationsetting typenumber of subjects acuity of intervention surgeonpatient decisionmaking preferences and factors associated with favoring sdm data was analyzed in microsoft excelresults s were identified with duplicates yielding s for title review swere included in final analysis of s discussed oncologic decisionmaking of these focused on breastcancer of s included patients included surgeons of s found surgeons favored sdm demonstrated surgeons favored surgeon guidance of s demonstrated patients favored sdm showedpatients favored surgeon guidance showed patients preferred independent decisionmaking the most commonfactors for patients favoring sdm included female gender higher education and younger age for surgeons the mostcommon factors for favoring sdm included limited evidence for a given treatment plan multiple treatment optionsand impact on patient lifestyle no s evaluated decisionmaking preferences in an emergent settings there has been limited evaluation of patient and surgeon preferences toward sdm in surgical decisionmaking generally patients and surgeons expressed preference toward sdm none of the s evaluated decisionmaking preferences in an emergent setting so assessment of the impact of acuity on decisionmaking preferences islimited extension of research to complex emergent clinical settings is neededkeywords surgery shared decision making ethics correspondence ericacarlisleuiowaedu1program in bioethics and humanities university of iowa carver college ofmedicine iowa city usa3department of surgery university of iowa hospitals and clinics iowa cityusafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cshinkunas bmc medical informatics and decision making page of over the past several decades physician paternalism hasbeen systematically rejected and respect for patient autonomy has emerged as a leading ethical priority in clinical counseling shared decisionmaking sdm aprocess by which physicians and patients actively worktogether to integrate care plans that are responsive topatient goals and values has been advocated as a clinicalcounseling approach that promotes patient autonomy byencouraging patients to participate in clinical decisionmaking [] along with its presumed promotion ofpatient autonomy data suggesting that sdm reduceshealth care costs and improves quality of care have ledto relatively widespread incorporation of sdm intohealth policy despite this implicit acceptance ofsdm relatively limited data exist regarding patient orphysician preferences toward sdm such data seem tobe especially lacking in surgical decisionmakingby supporting patient autonomy sdm places somelimits on the extent to which a physicians influenceguides a patients decisions some ethicists have arguedthat such prioritization of patient autonomy is criticallyimportant and that even subtle attempts by a physicianto sway a patient toward a particular decision violates respect for patient autonomy however others have argued that if attempts to promote patient autonomy aretoo strong or rigid the emphasis on selfdeterminationmay be inconsistent with patients wishes for more professional guidance [ ] in fact there is an emergingbody of literature that suggests that patients may prefermore physician guidance during medical decision making [ ] these findings prompt one to questionwhether autonomyheavy approaches to sdm in clinicalcounseling are always consistent with patient preferencesor whether patients would at least sometimes prefer aless autonomous and more guided approach to clinicalcounselingwith respect to the physicians perspective it is important to note that studies have shown physicians to besomewhat reluctant to incorporate sdm into clinicalpractice one reason for this may be a sense thatwhen a physician overly prioritizes patient autonomythere is lessening of the physicians role such that the fiduciary nature of the patientphysician relationship isundermined prioritization of patient autonomy and integration of sdm into clinical counseling has left somephysicians feeling that their role has become one ofmerely offering patients the information necessary tomake their own informed decisions rather than trulyengaging in a fiduciary relationship with the patient this is illustrated in a recent narrative that describes anencounter in which a physician reviewed all options fortreatment of nonischemic cardiomyopathy with her patient but was stopped by the patient before she couldmake a recommendation with the request that the patient be allowed time to independently reflect and makea decision that was best for him in the physicians reflection on the encounter she notes since the decisionwas his it was no longer mine i had informed him buthad i been his doctor perhaps such efforts to assure patient autonomy and sdm limit the role of thephysician in patient counseling these types of reportscall for further investigation so we can better understandphysician preferences toward shared decision makingconcerns about the appropriateness of sdm may beparticularly pronounced in surgical decision making giventhe often dramatic and irreversible outcomes associatedwith surgery these concerns may further escalate whenconsidering emergent highly complex operations that areassociated with a high risk of mortality or morbidity in aninitial effortto better understand preferences towardsdm in surgical decision making we reviewed the literature regarding parent and surgeon preferences towardsdm in pediatric surgery we found that there wasmarkedly limited data available of the existing sthe predominant focus was on parent preferences towarddecision making in elective nonurgent procedures therewas limited data regarding surgeon preferences and virtually no discussion of preferences for decision making inmore urgent settings the purpose of this review is to gain a more thorough understanding of patient and surgeon preferences toward sdmin adult surgery we chose to conduct a scoping review because there is limited published data on patient and surgeondecision making preferences particularly when surgery isconsidered urgent or emergent scoping reviews are a valuable methodology because they allow for the mapping of important concepts and research gaps in a defined area ofstudy by comprehensively identifying reviewing and summarizing the existing information from the literature specific research questions addressed in our scoping reviewincluded what is known from existing empirical evidence about patients andor surgeons surgical decisionmaking preferences why might patients andor surgeonsprefer sdm does acuity of intervention impact surgicaldecisionmaking preferencesmethodsprotocol designour scoping review protocol follows arksey and omalleys methodological framework as well as the preferred reporting items for systematic reviews andmetaanalyses extension for scoping reviews prismascr this protocol has not been registeredidentifying relevant studiesafter ascertaining our research questions we worked inconjunction with an experienced medicallibrarian to 0cshinkunas bmc medical informatics and decision making page of identify relevant studies we followed the preferredreporting items for systematic reviews and metaanalyses prisma guidelines for reporting the identified screened eligible and included studies fig after drafting refining and finalizing our search strategies we searched three bibliographic databases from inception through november medline embaseand cochrane databases the final search strategies forall three databases are outlined in additional file thefinal search results were imported into endnote versionx91 and yielded sstudy designeligibility criteriainclusion and exclusion criteria were defined a priorireview was limited to english language no translatorsavailable peerreviewed published literature only empirical studies were included review was limited todecision making preferences of surgeons andor adultpatients decision making preferences were loosely defined and included preferred role perceived role expectationsdesires and satisfaction with actualdecisionmaking role s in the following categories were excluded reviews letters to the editor editorialssuggested models of care patient educationhandouts decision making tools animal studies and s related to pediatric surgery in addition we excluded s without accessible full textliterature reviewafter duplicates were removed by the primary authorlas we were left with s to screen twoof the authors las and emc independently reviewedall titles and s and jointly decided to exclude s based on the eligibility criteria theremaining s were selected for full text reviewfig flow diagram of study selection 0cshinkunas bmc medical informatics and decision making page of following full text review additional s were excluded because they either did not pertain to an adultsurgical population or to decisionmaking preferences inthe surgery setting disagreements were resolved by discussion between the two authorssubjects gender acuity ofsurgeryothercharting the datafor each of the included s two of the authorslas and cjk independently ed the followingoutcome measures study methods quantitativequalitativemixed methods surgical specialty cancer diagnosisyesnounclear study location usnonus study setting inpatientoutpatient type of subject patientsurgeon number oftheintervention electiveurgentemergentunclear surgicaldecision to be made surgery v nonoperative managementchoice among different surgical proceduresdecision on timing ofsurgeonpatientdecision making preferences shared decision makingsurgeon guided decision making independent decisionmaking and surgeonpatient factors associated with favoring sdmacuity ofthe intervention was defined as followsemergent immediate need for surgery to preserve lifeurgentsurgery is required within the next days orweeks and elective surgery is not required notablycancer resections were considered urgent however subsequentreconstruction was considered elective iebreast cancer resection with subsequent reconstructionthe control preferences scale which is a fivepointmeasure used to gauge preferred involvement in medicaldecision making was adapted to define surgeonpatient preferences as follows shared decision makingsdm the patient and surgeon prefer to make the decision regarding surgery together surgeon guided decisionmaking sg the preference is for the surgeon to guidedecision making either entirely or in part while the patient takes a more passive role independent decisionmaking idm the preference is for the patient to take amore active role in decision making either partly orentirely independent from the surgeonthe data ion form was a modified version ofthe one we used for a literature review we conducted ondecision making preferences in the pediatric surgical setting discordant opinions were discussed at weeklymeetings the third author emc was available to mediate if consensus could not be reached data was analyzed in microsoft excel resultssummarizing collating and reporting the resultssummarizing the results s were identified medline n embase n cochrane n duplicateswere removed and s underwent title review seventyfour s were included in thefinal analysis because they specifically addressed existingempirical evidence about patient andor surgeon decision making preferences toward sdm in adult surgerysetting table provides a summary of allincludedscollating and reporting the resultstable provides frequencies for the characteristics of allincluded s over half of the s were quantitative n and performed outside of the us n sixtyseven included outpatient surgeries fourteen surgical subspecialties were representedwith the most s originating from surgical oncology n general surgery n orthopedic surgery n and urology n fifty s discussed decision making for patients with cancer and of these focused onbreast cancer most s assessed a choice betweenoperative and nonoperative management n or an option among different surgical procedures n sixtyeight of the s included patients ofthese demonstrated that patients preferredsdm showed that patients favored a surgeonguided approach and revealed a patient preference for independent decision making the most common factors for patients favoring sdm included femalegender higher education and younger ageonly of the s assessed surgeons preferences of these found that surgeons preferredsdm while demonstrated that surgeons favoreda more surgeonguided decisionmaking approach thefactors most commonly listed for surgeons favoringsdm included limited evidence for a given treatmentplan multiple treatment options and impact on patientlifestylenone of the s evaluated patient decisionmakingpreferences in an emergent setting out of the sthat assessed patient decisionmaking preferences in theelective surgery setting preferred sdm threeout of four of the s assessing surgeon decision making preferences in the elective surgery settingreported that surgeons preferred sdm in six out of nine of the s surgeons also preferred sdm in theurgent surgery setting in s patients were fairlysplit on their decisionmaking preference when it cameto urgent surgeries with desiring sdm and favoring a more surgeonguided approachonly s looked at both patient and surgeon decision making preferences in a little over half ofthese s n there was discordance between patient and surgeon decision making preferences 0cshinkunas bmc medical informatics and decision making page of table characteristics of included sacuity of andthedate ofpublicationinterventionstudy populationmajor findings related to decisionmaking dm preferencesalmyroudi ananian andersen asghari ashraf avis ballinger breast cancerpatients breast cancerpatientsurgentelective breast cancersurvivorsurgent hospitalized patients on surgical wardsunclear patients undergoingeither immediate ordelayed breastreconstructionelective hernia repair patientselective breast cancerpatientsurgentbeaver colorectal cancerpatientsurgentbeaver belue health professionalscaring for colorectalcancer patients weresurgeons cardiologists making adecision about surgery patients with coronaryartery diseaseblumenthalbarby left ventricular assistdevice patients andcandidatesburton older breast cancerpatientsbutow patient advocates breast cancersurgeonsurgenturgenturgenturgenturgent preferred a passive role acollaborative role46 an active role of women choosing breastreconstruction decided with surgeon of these patients were satisfied withthe information receivedon average reported being veryinvolved i made all the decisions myself were content with dm rolestrongly desire to receive informationand participate in decisionmaking were in the informedconsumeristgroup when it came to actual dm of these patients were satisfied with theinformation receivedexpectations of participation can besummarized as being told and going into get it fixed felt their healthcare professionalshad surgical preferences for thembelieved that clinical issues determinedthese preferences but still knew thechoice was theirswanted to be well informed andinvolved in the consultation process butdid not necessarily want to use theinformation they received to makedecisionsshared decision making was favored byhealth professionalsphysicians prefer patients whoactively participate in the decisionpatients prefer the physician tomake the decision sdm prefer to make the decision on theirowndeferred heavily to clinicians preferred patientcentred doctorcentred sdm of surgeons and of patientadvocates preferred sdm breast cancer survivors urgentcampesino cohen patients with localizedprostate cancerurgentcorriere patients undergoingelectivespanishspeaking latinas preferredphysician treatment recommendationsenglishspeaking latinas and africanamericans preferred sdmmost viewed the surgeonguided approach as appropriate and welcome preferred choosing together withdm themerelated tomajorfindingsaptsgsurgfactors associated with favoringsdmyounger age higher educationsdm type of procedureidmyounger age level of educationincomesdm female level of educationidmsgsdm sgsdmyounger patient agesgsdmsgsgidmsdm sdmsdmsgsgenglishspeakingsdm multiple treatment options type 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferencesfactors associated with favoringsdmdm themerelated tomajorfindingsaptsurg elective vascularprocedures prostate cancersurvivors hand surgeons patients with triggerfinger breast cancerpatientsurgentelectiveurgentcuypers doring durifbruckert gainer the provider preferred having theprovider choose for them preferred a collaborative role an active role a passive roleof proceduresdm higher education younger agehigher sespatients preferred to decide forthemselves surgeons preferred sdmidmsdmwanted to participate in decisions butperceived sdm as an obligation becauseit did not seem to fit with their idea of aproper doctorpatient relationshipsgtrust in surgeon support fromfamily written information fromsurgeon frail and olderpatients care teammembers includessurgeonsunclearboth patients and care team memberssupported a formal approach to sdmsdm sdmghane general surgerypatientsgolden clinicians weresurgeonsgong patients with carpaltunnel syndromeelectiveurgentpreferred relatively high levels ofdecisional control on averagem out of sd most felt that they practiced sdm eventhough they did not tend to distinctlyprompt patient dm preferencesidmmale good health high healthliteracysdmelective preferred sdmsdm hack breast cancerpatientshageman hand surgeons patients with carpaltunnel syndromehawley breast cancerpatientsheggland hausken health professionalsfrom surgical wards patients who underwentsurgical treatmenturgentelectiveurgentelectiveheggland hausken surgical patients surgeonselectiveurgentheggland [ ]henderson shum physicians workingin surgical wardsunclear surgical and medicalpatientselectiveurgent preferred a collaborative role an active role a passive rolesurgeons preferred patient andprovider make a shared decisionpatients preferred that the patientdecidesactual dm role sdm patientbased surgeonbased preferreddm role content with level of dminvolvementhealth professionals majority preferred ashared or informed model patientsabout half preferred a shared orinformed model and the other halfpreferred a paternalistic modelsurgeons the majority preferred aninformed model ¦ patient is giveninformation and left to make thedecision patients preferred apaternalistic model and preferredsharedphysicians on average rated decisionmaking control a which means thatphysicians were not reluctant to involvepatients in decisionmaking processeswhere active role shared and passive the mean dm value for thesevere scenario was moderatescenario was mild scenario was history of surgical procedureimportance of family memberopinions having privateinsuranceage nonwidowed longerduration postopsdm idmsdmsdmidmsdmfemalesdmsgsgidmsdmsdm younger age noncriticalcondition 0cdm themerelated tomajorfindingsaptsdm surgfactors associated with favoringsdmfemale higher educationsgsgfemale no stomasdmmore years in practice morecomfort in pulmonary nodulemanagementcollege degree higher selfefficacysdm sdm sgsdm females indicated that they would like tohave more input in the decisionmakingprocess than the males v onthe controlled preferences scaleguidance by the clinician was identifiedas most important active role of patientin treatment decision making regardedas less important preferred a passive role sdm an active roleperceived role share decisionsequally with the patient allowthe patient to decide decide forthemselves after considering the patientsopinionmost patients wanted to decide on theirtreatment options together with theirphysician preferred sdm preferred tomake the decision with physician input wanted their surgeons to make arecommendation and when given followed the recommended treatmentplan preferred a collaborative role inpatients in twosurgical unitsunclear lung cancer patientsurgenthenderson hopmans hou colorectal cancerpatientsiaccarino clinician members ofthe american thoracicsocietyurgenturgentihrig prostate cancerpatientsjanz breast cancerpatients newly diagnosedbreast cancer patientsurgenturgenturgent breast cancerpatientsurgentjohnson keating keating lally shinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferences lung or colorectalcancer patientsurgent patient controlled sdm physician controlledsdm married better prediagnosishealth status caucasian strongevidence for procedure breast cancer patients urgentwomens lack of sharing theirpreferences with their surgeons and thesurgeons lack of making treatmentrecommendations resulted in what wasmore likely informed than shared decisionmaking preferred sdm preferred thechoice to be their own preferred todelegate the decisionactual role sdm madedecision with surgeon input weresatisfied with dm levelactual dm joint patientdoctor decision doctor advocated patient asked preferred dm contentwith level of dm involvementthe surgical group showed a morepassive role in both their preferred andactual dm rolesurgeons recommendationand fear of dying from cancer played themost important role in dmin surgery preferred a directivecommunication style a nondirective communication styleidmsdm younger agesdm sdm femalesgsgsdmsglam breast cancerpatientslantz breast cancerpatientsurgenturgentlarsson patients scheduledfor invasive surgeryelectiveurgenturgentlee patients with earlygastric cancermarkovic martinez newly diagnosedgynecologic cancerpatients newly diagnosedbreast cancer patientsurgent 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferences surgeonsunclearmcguire mendick breast cancer patients surgeonsurgentmeredith surgical patients surgeonsunclear older breast cancerpatientsurgent patients withirritable bowel diseaseelective breast cancer patients urgentman morishige moumjid nam patients with carpaltunnel syndromeelectiveomar op dendries consecutive patientsbeing seen in a multidisciplinary stone clinicelective liver transplantcandidates and recipientsurgentorsino end stage renaldisease patientselectivemany physicians saw their role as anexpert who educates the patient butretains control over the decisionmakingprocessothers took a more collaborativeapproach encouraging patients toassume decisional prioritysurgeons made most decisions forpatients patients generally lacked trustin their own decisions and usually soughtsurgeons guidancepatients majority agreed that thesurgeon should supply them with thepros and cons of all measures toaddress the problem and it was for themultimately to decide what was right forthem surgeons not enthusiastic at theprospect of devoting more time todiscussing surgical alternatives risks andcomplications and outlook indicators fortheir patients benefitin surgery preferred sdm adoctorcentered approach apatientcentered approach thought having a physician involvethem in the decisions concerning theirtreatment was very importantmost were satisfied with the informationgiven and the possibility of participatingto the treatment decisionmakingprocessi prefer that my doctor and i shareresponsibility i prefer that mydoctor makes the final decision aboutwhich treatment will be used butseriously considers my opinion would rely on the physiciansrecommendation wished to be involved in makingthe decision to accept or not accept aliver for transplantation preferred equal responsibility an autonomous role adecision driven by the health care teamdm themerelated tomajorfindingsaptsurgsdmsgfactors associated with favoringsdmmultiple treatment optionsincreased risk impact ofprocedure on patient lifestylemoral contentsgsgsdm sgpatients strong evidence forprocedure surgeons multipletreatment options impact ofprocedure on patient lifestylesdm older agecomorbidities surgical historyuse of biologics treated at anacademic hospital being marriedsdm sdm sdm sgsdm sdm younger agepieterse ramfelt rectal cancer patients surgical oncologistsurgentthe majority of patients and clinicianspreferred sdmsdm sdmpatients female highereducation rectal or colon cancerpatientsurgent of rectal cancer patients ofcolon cancer patients preferred acollaborative rolesdm younger ageratsep patients with lumbardisc herniationelective preferred sdmsalkeld rectal or coloncancer patientsurgent preferred a surgeonguided approach sdm a more independent dm rolesdm sgdesire for more disease specificinformationfemale younger age history ofradiation 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferencessantema patients with eitherabdominal aorticaneurysm or peripheralarterial occlusive diseaseelective preferred sdmseror young breast cancerpatientsurgentsidana young prostatecancer patientsurgentsnijders stiggelbout kiebert gi surgeonsurgent cancer patients surgical patientsunclearpreferred a more passive approach preferred fully passive and preferred fairly passive preferred sdm an informeddecision made by myself based oninformation a passive rolemost patients were offered only onetreatment option and little sdm wasseenthe physician should make the decisionsbut strongly consider my opinion wasselected most frequentlyfactors associated with favoringsdmtrust in doctor doctor has aclear communication styledoctor listens enough time forconsultationhigher education type ofproceduredm themerelated tomajorfindingsaptsdm surgsgsdm sgsgyounger age femalesung patients with pelvicfloor disordertyler ellis newly diagnosedrectal cancer patientsuldry patients undergoingelective gi surgeryvogel breast cancerpatientswang breast cancerpatientsurgenturgent spine clinic patientselectiveweiner essis wilson patients undergoingmajor thoracicabdominal operationsurgentelectivewoltz patients withdisplaced midshaftclavicular fractureelectiveurgent preferred a collaborative role an active role a passive rolesdm of total mesorectal excision patientsand of local excision patientspreferred sdmsdm higher education younger ageelective preferred an active roleidmyounger age male level ofeducationhigher anxiety scores multipletreatment options preferred a passive role anactive role sdm preferred a collaborative role a passive role an active rolethe majority of patients felt that thephysician rather than the patient shouldmake the basic treatment decision preferred a patientdriven role sdm a surgeondriven rolesgsdm sgidm preferred sdm autonomousrole a passive rolesdm ovarian cancerpatientsziebland adecision making preference dm decision making sg surgeonguided sdm shared decision making idm independent decision makingdx diagnosis pt patient surg surgeonpreferred their medical team to decideon their behalf or going along withtheir doctors recommendationurgentsgout of these s three focused on elective surgeriesin orthopedics one on urgent surgeries in cardiac surgery one on both elective and urgent surgeries in general surgery and one was unclear on the acuity of theinvention but occurred in general surgerydiscussionshared decision making has been highlighted as a desirable approach to clinical counseling however it isunclear if this applies to surgical decision making particularly when considering surgical counseling in settingsof emergent complex highlymorbid operations inour scoping review of the adult surgical literature wefound relatively few studies that address patient and surgeon preferences toward sdm in surgery we found thata large proportion of existing s on preferences toward sdm address elective outpatient procedureswhile patients did seem to prefer sdm in these controlled settings it is possible that patients and surgeonsmay prefer more surgeon guidance when discussingemergent complex operations that have a high risk ofmorbidity or mortality further studies that specifically 0cshinkunas bmc medical informatics and decision making page of table frequencies for characteristics of all included sn variablesurgical specialtyastudies noncologygeneral surgeryorthopedicsurologygynecologycolorectalthoraciccardiacplastic surgerytransplantationvascularneurosurgeryentotolaryngologyophthalmologycancer diagnosisyesnounclearstudy methodsqualitativequantitativemixed methodsstudy locationusnonusstudy settinginpatientoutpatientbothtype of subjectspatients onlysurgeons onlybo
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to machine learning with python a guide for data scientists oreilly media inc california demÅ¡ar j statistical comparisons of classifiers over multiple data sets j mach learn res yates a the ensembl rest api ensembl data for any language bioinformatics kim e r chang d k colorectal cancer in inflammatory bowel disease the risk pathogenesis prevention and diagnosis world j gastroenterol diovasc dis schulte d small dense ldl cholesterol in human subjects with different chronic inflammatory diseases nutr metab car smedley d biomartbiological queries made easy bmc genom quinlan a r hall i m bedtools a flexible suite of utilities for comparing genomic features bioinformatics 101093bioin forma ticsbtq03 rom¡n j evaluation of responsive gene expression as a sensitive and specific biomarker in patients with ulcerative colitis inflamm bowel dis 101002ibd23020 song r identification and analysis of key genes associated with ulcerative colitis based on dna microarray data medicine baltimore e10658 101097md00000 schwegmann k detection of early murine colorectal cancer by mmp29guided fluorescence endoscopy inflamm bowel dis 101097mib00000 oliveira l g d positive correlation between disease activity index and matrix metalloproteinases activity in a rat model of colitis arq gastroenterol 101590s0004 shin js antiinflammatory effect of a standardized triterpenoidrich fraction isolated from rubus coreanus on dextran sodium sulfateinduced acute colitis in mice and lpsinduced macrophages j ethnopharmacol 158pt a 101016jjep201410044 owens d w lane e b keratin mutations and intestinal pathology j pathol 101002path1646 macfie t s duox2 and duoxa2 form the predominant enzyme system capable of producing the reactive oxygen species h2o2 in active ulcerative colitis and are modulated by 5aminosalicylic acid inflamm bowel dis 10109701mib00004 0e palmer n p concordance between gene expression in peripheral whole blood and colonic tissue in children with inflammatory bowel disease one e0222952 101371journ alpone02229 wei z large sample size wide variant spectrum and advanced machinelearning technique boost risk prediction for inflammatory bowel disease am j hum genet amrhein v greenland s mcshane b scientists rise up against statistical significance nature wasserstein r a0l schirm a a0l lazar n a0a moving to a world beyond p maeda y fully automated diagnostic system with artificial intelligence using endocytoscopy to identify the presence of histologic inflammation associated with ulcerative colitis with video gastrointest endosc 101016jgie201809024 acknowledgementsthis research was partially supported by grants from the natural sciences and engineering research council of canada nserc to hu grant number rgpin and to lpc grant number rgpin hmk was partially supported by funding from memorial universitys school of graduate studiesauthor a0contributionsconceptualization hu and lpc methodology hmk hu and lpc analysis hmk and lpc writing hmk hu and lpc experiments hmk supervision hu and lpccompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to hu a0or a0lpcreprints and permissions information is available at wwwnaturecomreprintspublishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020705830vol1234567890wwwnaturecomscientificreports 0copen access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate if changes were made the images or other third party material in this are included in the s creative commons license unless indicated otherwise in a credit line to the material if material is not included in the s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020705830vol0123456789wwwnaturecomscientificreports 0c'
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" variability in the health effects of dietary fiber might arise from interindividual differences in the gutmicrobiotas ability to ferment these substrates into beneficial metabolites our understanding of what drives thisindividuality is vastly incomplete and will require an ecological perspective as microbiomes function as complexinterconnected communities here we performed a parallel twoarm exploratory randomized controlled trial in adults with overweight and classi obesity to characterize the effects of longchain complex arabinoxylan n at high supplementation doses female gday male gday on gut microbiota composition and shortchainfatty acid production as compared to microcrystalline cellulose n nonfermentable control and integratedthe findings using an ecological frameworkresults arabinoxylan resulted in a global shift in fecal bacterial community composition reduced αdiversity andthe promotion of specific taxa including operational taxonomic units related to bifidobacterium longum blautiaobeum and prevotella copri arabinoxylan further increased fecal propionate concentrations p friedmanstest an effect that showed two distinct groupings of temporal responses in participants the two groups showeddifferences in compositional shifts of the microbiota p permanova and multiple linear regression mlranalyses revealed that the propionate response was predictable through shifts and to a lesser degree baselinecomposition of the microbiota principal components pcs derived from community data were better predictors incontinued on next page correspondence jenswalteruccie nguyen k nguyen and edward c deehan contributed equally to this work1department of agricultural food nutritional science university of albertaedmonton ab t6g 2e1 canada13department of biological sciences university of alberta edmonton abt6g 2e1 canadafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cnguyen microbiome page of continued from previous pagemlr models as compared to single taxa indicating that arabinoxylan fermentation is the result of multispeciesinteractions within microbiomes this study showed that longchain arabinoxylan modulates both microbiota composition and theoutput of healthrelevant scfas providing information for a more targeted application of this fiber variation inpropionate production was linked to both compositional shifts and baseline composition with pcs derived fromshifts of the global microbial community showing the strongest associations these findings constitute a proofofconcept for the merit of an ecological framework that considers features of the wider gut microbial community forthe prediction of metabolic outcomes of dietary fiber fermentation this provides a basis to personalize the use ofdietary fiber in nutritional application and to stratify human populations by relevant gut microbiota features toaccount for the inconsistent health effects in human intervention studiestrial registration clinicaltrialsgov nct02322112 registered on july keywords arabinoxylan dietary fiber gut microbiota interindividual variability overweight adults shortchainfatty acids epidemiologic studies consistently associate dietary fiberconsumption with a reduced incidence of obesityassociated pathologies [ ] in largescale observationalstudies whole grains and cerealderived fibers eg arabinoxylan and glucan showed stronger associationswith reduced risk of developing cardiovascular diseasetype ii diabetes gastrointestinal cancers and of allcausemortality when compared to other fiber sources [ ] asubstantial body of animal research further consolidatedthe mechanisms by which fiber reduces metabolic pathologies despite these convincing associations findings obtained from human dietary intervention trialsaimed to improve metabolic risk markers by supplementing isolated dietary fibers remain inconsistent possibly due to an individualized clinical response [ ]owing to their chemical structure dietary fibers resistdigestion in the smallintestine and reach the colonwhere they become substrates for the gut microbiotathe microbial fermentation of fiber to shortchain fattyacids scfas has been implicated in the prevention ofobesityassociated pathologies propionate and butyrate are two scfas that are especially relevant as theyhave been linked to beneficial immunological and metabolic effects intervention studies with arabinoxylanisolated from wheat endosperm for instance have demonstrated increased fecal concentrations of both butyrateand propionate dietary fibers can further modulategut microbiota composition in a structuredependentway through the enrichment of bacterial taxa that utilizethe substrate and tolerate or benefit from the environmental changes caused by fiber fermentation [ ] forexample dietary interventions with shortchain fractionsof arabinoxylan resulted in an enriched abundance ofbacterial species that can either utilize arabinoxylan oliaxos directly eg bifidobacteriumgosaccharidesadolescentis and bifidobacterium longum or benefitfrom metabolic byproducts released during axos degradation eg anaerobutyricum hallii and faecalibacterium prausnitzii although fiberinduced alterationsto the gut microbiota are significant the effects are alsohighly individualized and this variability might haveclinical ramifications that could explain the individualized clinical responses to understand the individualized response of the gutmicrobiota to dietary fiber an ecological perspective isrequired as fiber fermentation is determined by complexinterspecies interactions between members of the gutmicrobiota the process is often based on a crossfeeding cascade where primary degraders that access thefiber provide breakdown products oligosaccharides disaccharides and monosaccharides to other microbesand metabolites that result from the fermentation ofthese products also serve as substrates interindividual variation in gut microbiota composition mayresult from the absence of keystone species that initiatethe degradation of recalcitrant fibers differences inunrelated species with similar ecological functions thatcompete for the same substrate or variation instrains of the same species that differ in their capacity tometabolize the substrate these compositional variations likely determine both the competitive and cooperative relationships between community membersthat form trophic networks some of which anize intoecological guilds that collaborate to degrade complexfibers although interindividual variation in the response of the gut microbiota to fiber can influence metabolite outputs relevant to health ie propionate orbutyrate this topic and the underlying ecologicalprinciples have received little attentionthe objective of this study was to apply an ecologicalframework to characterize the compositional and metabolic responses of the human gut microbiota to a longchain arabinoxylan isolated from corn bran compared to 0cnguyen microbiome page of a fiber that is not fermented by the gut microbiotamicrocrystalline cellulose mcc we further assessedwhether nutritional and microbiotarelated factors couldexplainamongindividualsresponsesobservedthevariableresultssubject characteristics and protocol adherenceto compare the effects of arabinoxylan and mcc weconducted a 6week parallel twoarm exploratory randomized controlled trial in individuals with overweightand classi obesity where females received gday andmales gday of either fiber fig of the subjectsenrolled and randomized to an intervention arm sevenwithdrew from the study in the arabinoxylan groupthree experienced challenges consuming the supplementand one reported constipation in the mcc group twowithdrew due to personal reasons and one due to constipation and weretherefore excluded from analysesadditional file fig s1 subjects that completed thestudy protocol n included females and males aged ± years with a body mass indexbmi of ± kgm2 no differences in age sex orbmi were detected between the intervention groups atbaseline additional file table s1 overall protocoladherence assessed by the amount weight of returnedsupplement was ± and ± in the arabinoxylan and mcc arms respectivelyeffect on the composition of the fecal microbiotafecal microbiota diversitynonmetric multidimensional scaling analysis of euclidean distances between subjects based on centered logratio clrtransformed operationaltaxonomic unitotu data showed that the two treatment groups harbored bacterialcould not becommunitiesthatdifferentiated at baseline p permutational multivariate analysis of variance [permanova] fig 2aoneweek supplementation with arabinoxylan alteredthe global fecal bacterial community which became significantly different from the fecal microbiota of subjectsreceiving mcc p this effect was maintaineduntil the end of the fiber intervention p thesechanges occurred by arabinoxylan inducing temporalshifts in fecal microbiota composition determined as theaverage diversity between the individuals treatmentand baseline samples which were significantly largerwhen compared to the mcc group p mannwhitney test fig 2b in addition while mcc increasedinterindividual differences diversity between subjectsp generalized estimated equation [gee] modelarabinoxylan reduced it p fig 2canalysis of αdiversity showed that arabinoxylan reduced fecal bacterial diversity shannons index p gee model fig 2d but not richness total otusafter weeks of supplementation overall these findingsshowed that while the nonfermentable mcc had no detectable effects on measures of bacterial diversity arabinoxylan altered the global bacterial community within week inducing temporal shifts in composition and a reduction of both interindividual variation and αdiversityeffect on the relative abundance of bacterial taxa and coabundance response groupsneither arabinoxylan nor mcc altered microbiota composition at the phylum level at lower taxonomic levelschanges in the relative abundance of two bacterial families were detected at weeks of arabinoxylan relative tobaseline and mcc namely an increase in bifidobacteriaceae q wilcoxon test fig 2e additional file table s2 and a decrease in erysipelotrichaceae q fig study design shaded study week blocks indicate a scheduled clinic visit the x indicates the task was completed during the study weekcdhq ii canadian diet history questionnaire ii stool characteristics selfreported stool consistency and bowel movement frequency 0cnguyen microbiome page of fig arabinoxylan alters the global composition of fecal bacterial communities and induces distinct shifts in taxa a nonmetricmultidimensional scaling nmds plot based on euclidean distance metrics of arabinoxylan and microcrystalline cellulose groups at each timepoint intersubject diversity showing changes in the distance between subjects over time euclidean distances b between fecal microbiotas ofsubjects at each study time point intersubject and c between each subjects fecal microbiota at baseline and during w1 and w6 of treatmentintrasubject d αdiversity displayed as shannon index and total otus of the fecal microbiotas of subjects at each time point e absolutechange δw6bl in relative abundance of bacterial taxa affected by the dietary intervention data analyzed using permanova for a gee modelswith bonferroni correction for b and d and mannwhitney tests for c for e data were analyzed using either wilcoxon tests to assess withingroup changes relative to baseline or mannwhitney tests to assess betweengroup changes ie ax vs mcc with fdr correction diversityand compositional data were reported as mean ± sd and centered logratio transformed prior to the statistical analyses bl baseline otuoperational taxonomic unit w1 week w6 week at the genus level arabinoxylan increased thegenera bifidobacterium and prevotella when comparedto both baseline and mcc and enriched blautia whencompared to mcc otu level analysis revealed that otus changed during arabinoxylan treatment relative tobaseline henceforth referred to as significant otusin particular otus related to bifidobacterium longumotu4 prevotella copri otu6 bacteroides plebeiusotu53 bacteroides sp otu56 bacteroides ovatussuccinatutensotu26otu38 blautia obeum otu85 subdoligranulum spotu11 clostridium leptum otu46 mollicutesotu32 and muribaculaceae otu79q phascolarctobacteriumbecame enriched while otus related to ruminococcusbromii otu5 eubacterium oxidoreducens otu41bacteroides uniformis otu7 and faecalibacillus sppotu21 declined in relative abundance supplementation with mcc only increased the family lachnospiraceaegenus parasutterella q numerically the dominant compositional effects of arabinoxylan were to a large degree specific to b longumotu4 and p copri otu6 as these taxa increased inrelative abundance by an average of 46fold and 4fold while other otus increased by and thein an attempt to identify groups of cooperating species that could function as ecological guilds in the 0cnguyen microbiome page of degradation of arabinoxylan we adapted a clustering approach conceptually similar to that described by tong instead of absolute proportions of taxa weused arabinoxylaninduced shifts to identify clusters ofspecies whose responses were intercorrelated this analysis revealed a total of seven coabundance responsegroups cargs fig 3a five of which showed statistically significant responses to arabinoxylan while noneresponded to mcc additional file table s2 thecarg that showed the largest increase in relative abundance was carg1 p wilcoxon test whichconsisted of six out of the eleven otus that increasedthrough arabinoxylan fig 3b among those six otusbshift andshowed significant connections to all but one member ofcarg1 rs q spearmans correlations usingpermutation tests suggesting arabinoxylan may be degraded through cooperative interactions between theselongum otu4 exhibited the largesttaxa in carg6 p copri otu6 exhibited the largestresponse but only showed one strong connection withanother member of the carg bacteroides massiliensisotu98 rs q which suggests that pcopri might act to a larger degree independently to degrade arabinoxylan fig 3b the majority of taxa thatdecreased during arabinoxylan consumption particularlyb uniformisotu7 clustered within carg7 andshowed negative correlations with taxa of carg1carg2 and carg6 suggesting competitive or antagonistic interactionstemporal responses of otus and cargsto determine if short and longterm treatment witharabinoxylan and mcc differed in their effects on thefecal microbiota we compared shifts from baseline toweek w1 with those from baseline to week w6however there were no detectable differences betweenfig identification of coabundance response groups cargs during arabinoxylan supplementation a heatmap shows the change δw6bl inrelative abundance of otus affected by arabinoxylan p wilcoxon test the hierarchical dendrogram shows clustering of centered logratio clr transformed otus rows based on spearmans correlation distances by the completelinkage clustering algorithm and then groupedon the dendrogram into seven cargs by permanova p subjects columns clustered based on euclidean distances colors from blue tored indicate the direction and magnitude of change b coresponse network analysis each node represents an otu where the size isproportional to the change δw6bl in relative abundance the shape indicates direction of change positive circle negative square and thecolor references the respective carg to which it was clustered lines between nodes represent significant positive red line or negative blueline spearmans correlations rs values ¥ or and q values bl baseline otu operational taxonomic unit w6 week 0cnguyen microbiome page of the two time frames q wilcoxon test data notshown in addition comparison of baseline w1 andw6 values by friedmans test indicated that the effectsof arabinoxylan occur rapidly within week with nofurther detectable changes at weeks fig 4a considering these findings analyses on compositional changeswere performed with w6 data unless otherwise statedinterindividual variation in responses to arabinoxylanbacterial shifts in response to arabinoxylan and theirmagnitude were highly individualized fig 4b for instance absolute increases in relative abundance rangingfrom to to 429fold change were detected inseven subjects for the otu classified as blongumotu4 while other subjects showed either a muchsmaller increase a decrease or the species was undetectable otus related to b obeum otu85 subdoligranulum sp otu11 b ovatus otu26 and c leptumotu46 were enriched by arabinoxylan in around twothirds of the subjects less frequently enriched wereotus classified as potu6 b plebeiusotu53 and bacteroides sp otu56 p copri otu6responded in only four subjects but effects were largewith the species expanding beyond to 7foldchange ofthe total bacterial community in threesubjectscoprito determine drivers of these individualized responseswe used multiple linear regression mlr analyses totest if responses in otus that showed numerically thelargest shifts p copri b longum b obeum and subdolifig temporal and individualized responses of the otus and cargs affected by arabinoxylan and microcrystalline cellulose a plots show thetemporal response of the ten most abundant otus detected in of subjects and the seven cargs centered logratio transformed datawere analyzed by friedmans test with dunns correction to assess withingroup changes between time points ie δw1bl and δw6w1 bbubble plot shows individualized differences δw6bl in relative proportions of the ten most abundant otus percentage of total microbiotacomposition and cargs sum of otus detected after weeks of arabinoxylan and microcrystalline cellulose supplementation the size of thebubble is proportional to the change in abundance relative to baseline while the color of the bubble represents the direction of the change redincrease black decrease the x indicates that the otu was either undetected or the change was relative abundance bl baseline cargcoabundance response group otu operational taxonomic unit w1 week w6 week 0cnguyen microbiome page of granulum sp and in cargs with significant responsescargs and could be predicted by baselinediet or microbiota composition baseline microbiota allotus and significant otus and diet variables werefirst reduced in their dimensionality by principal component analysis pca and then treated as predictors thisanalysis revealed that individualized responses of bacterial taxa and cargs to arabinoxylan and mcc could notbe predicted by baseline diet or microbiota compositionq additional file fig s2effect on stool characteristics and bowel movementswhile fecal moisture content was not changed by eitherfiber q wilcoxon test additional file table s3subjects consuming arabinoxylan reported softer stoolconsistencies when compared to subjects consumingmcc treatment effect p gee model additional file fig s3a both arabinoxylan and mcc ledto an increase in bowel movements relative to baselinep gee model additional file fig s3b withno difference detected between treatment groups treatment effect p effect on fecal ph and scfasfecal ph and scfa concentrations did not change after weeks of either fiber treatment q wilcoxon testadditional file table s3 considering that absoluteconcentrations of fecal scfas are affected by their absorption in the gut we additionally assessedchanges in the percentages of acetate propionate andbutyrate relative to total scfa concentrations at w6which has been previously shown to vary little across colonic regions this analysis revealed an increase inthe percentage of propionate produced through arabinoxylan when compared to mcc q mannwhitney test and a reduction in the percentage ofbutyrate relative to baseline q wilcoxon test although differences in butyrate were not detected whencompared to mcc q further investigation ofthe ratio between propionate and butyrate showed an increase in propionate relative to butyrate when comparedto baseline q and mcc q suggestingthat arabinoxylan supplementation directed the outputof scfas in favor of propionatecharacterization of the temporal response in the threeprimary scfas also showed an increase in fecal propionate concentrations by arabinoxylan at w1 p friedmans test fig 5a although propionate concentrations remained elevated at w6 this increase was notstatistically significant when compared to baseline p this loss of significance was caused by an increasein the interindividual variation at w6 fig 5b visualevaluation of the individualized temporal response ofpropionate to arabinoxylan revealed clear separation offig temporal and individualized output of fecal scfas in response to arabinoxylan and microcrystalline cellulose supplementation a line plotsshow the temporal response of acetate propionate and butyrate reported as mean ± sd b individualized temporal propionate response of w6responders red and w6nonresponder black grouped based on δw6w1 data analyzed for a and b using friedman test with dunnscorrection to assess withingroup changes between time points and for b using mannwhitney tests to assess differences betweengroup ateach time point bl baseline carg coabundance response group otu operational taxonomic unit scfa shortchain fatty acid w1 week w6week 0cnguyen microbiome page of subjects into two distinct patterns fig 5b based on thedirection of change from w1 to w6 ie positive ornegative subjects were grouped into w6respondersδ w6w1 and w6nonresponders δ w6w1 in general w6responders showed a higher outputof propionate at w6 p friedmans test butnot at w1 while the opposite isseen in w6nonresponders p the two groups differed bypropionate concentrations at w6 p mannwhitney testw6propionate responders and nonresponders differ intheir microbiota response to arabinoxylanmicrobiota compositionalbaseline and shifts anddiet data were ordinated using pca and then differencesandbetween w6propionaterespondersnonresponders were tested using permanova thisthe bacterial communities ofanalysis revealed thatw6responders wereindistinguishablefrom w6nonresponders at baseline but differed in their response to arabinoxylan δw6baseline fig thiswas detected if analysis was based on all otus p the significant dietresponsive otus p or the seven cargs p in contrastneither baseline microbiota composition fig nordietary factors additional file fig s4a separatedaccording to w6 response p in addition comparing w6respondersinterms of their baseline total grain whole grain andtotal fiber consumption or their stool consistency andbowel movement frequency during treatment did notreveal any differences either p mannwhitneyand w6nonrespondersfig the individualized temporal propionate response to arabinoxylan associates with compositional responses in the fecal microbiota principalcomponent analysis plots based on euclidean distance comparing the relative abundance of fecal microbiota both at baseline and arabinoxylaninduced shifts δw6baseline between w6responders red and w6nonresponders black microbiota variables ie otu or carg thatcontributed the most to intersubject variation were shown as vectors on the plot when statistical significances were determined by permanovap carg coabundance response group otu operational taxonomic unit w1 week w6 week 0cnguyen microbiome page of test additional files and fig s3c and fig s4btogether these findings indicate that the temporal response in fecal propionate concentrations is primarilyassociated with the shifts in the microbiota and notbaseline microbiota composition or dietindividualized scfa responses can be predicted by gutmicrobiota featuresas with compositional responses gut microbiota functional responses to fiber interventions have been shownto be individualized [ ] but what drives this variation is poorly understood we applied mlr to determine whether fecal scfa responses could be explainedby stool consistency and bowel movement frequencydiet or microbiotarelated factors and then comparedthe quality ofthe models using corrected akaikeinformation criterion aicc values where lower valuesmean higher quality these analyses revealed that thew6 scfa response to arabinoxylan could be predictedby the fecal microbiota fig additional file fig s5but not by baseline diet stool consistency or bowelmovement frequency reported during treatment additional file fig s6a and fig s6b the best modelswere achieved for propionate especially when principalcomponents pcs generated from w6 shifts of all otuswere used as predictors fig 7a additional file tables4 models were of lower quality when w6 shifts of significant otus cargs pcs of cargs or single otuswere used suggesting that global community measuresexhibited stronger linear relationships with the propionate response than single or groups of taxa although themodels that used baseline and w1 shifts of otus asfig individualized arabinoxylaninduced propionate responses could be explained by baseline gut microbiota composition and microbiotashifts a heatmap shows the linear associations between the individualized propionate response δw6bl dependent variable columns andmicrobiota profiles bl δw1bl δw6bl predictors rows cells represent individual multiple linear regression models with fdr correction thatassess whether the predictors explain the individualized propionate response multivariate microbiota data were simplified into principalcomponent pc variables pc1 pc2 and pc3 prior to analysis each model contained the best one or two predictors of pcs individual cargs orsignificant otus selected by stepwise regression all models were adjusted by fiber dosesex colors from white to red indicate relative aicchighest aicc value x lower aicc values red indicate higher quality models b scattercorrected akaike information criterion values calculated byplots show the linear relationship between propionate responses δw6bl and either the baseline contribution of all otus to pc1 or the shiftsof carg1 color and size of each point indicate propionate response magnitude and the shaded area specifies the confident interval thetop six otus that contributed the most to either pc1 of all otus or carg1 are further provided ax arabinoxylan bl baseline carg coabundance response group mcc microcrystalline cellulose otu operational taxonomic unit w1 week w6 week aicc value 0cnguyen microbiome page of predictors were of lower quality than those based on w6shifts they are still valid showing q values less than after benjaminihochbergs false discovery rate fdrcorrection linear relationships between propionate responses and significant predictors using baseline pc1 ofall otus and shifts carg1 were further visualizedusing scatter plots fig 7b reaffirming the quality ofthe analysis as a majority of subjects fall within the confidence regionssignificant models could also be designed for acetateand butyrate responses to arabinoxylan additional file fig s5 interestingly in contrast to propionate the bestmodels to predict butyrate responses were achieved usingshifts of a single otu e oxidoreducens otu41 aknown butyrate producer however overallthemodels for acetate and butyrate were of much lower quality than those for propionate in summary while individualized responses in scfas showed no association withdiet they could be predicted by microbiota shifts andbaseline composition in contrast to the analysis of the effects of arabinoxylan not one single mlr model wasfound to be significant for mcc indicating that the statistical approach based on mlr models did not detect anyassociations independent of fiber fermentationdetermining the role of bacterial taxa in propionateresponsemlr analyses were applied to determine connectionsbetween arabinoxylan responding otus within cargs and and fecal propionate concentrations fig 8athis analysis revealed that shifts in p copri otu6 didnot predict propionate responses while blongumotu4 and correlated taxa in carg1 showed strongerlinear relationships the highest quality models were obtained with b obeum otu85 b plebeius otu53and p succinatutens otu38 all of which encodemetabolic pathways for propionate production such analysis provides a potential explanation for themetabolic interactions between proposed primary degraders secondary fermenters and metabolite utilizersthat result in the promotion of propionate in responseto arabinoxylan fig 8bdiscussionin the present study we characterized the impact of a6week highdose corn bran arabinoxylan supplementation on the composition and function of the fecalbacterial community in healthy adults with overweightand classi obesity arabinoxylan treatment changedfig relationship between propionate responses to arabinoxylan and proposed primary degraders secondary fermenters and metaboliteutilizers a individual multiple linear regression models determine otu responses δw6bl that predict the fecal propionate response δw6blyaxis shows the coefficient for each predictor as in the average propionate response when otu relative abundance increases xaxis showsthe p value for each predictor all models were adjusted by fiber dosesex where bubble size represents the adjustedr2 b proposed model ofbacterial crossfeeding in the gut during degradation of complex soluble arabinoxylans otu operational taxonomic unit 0cnguyen microbiome page of showing two distinctcommunity structure and induced specific shifts inthe composition of the gut microbiota that manifestedthemselves after week of treatment without furtherchanges at w6 arabinoxylan induced increases inpropionate output both compositional and functionalresponses were highly individualized with propionateresponsestemporal patternscompositional responses to arabinoxylan could not bepredicted and functional responses were independentof stool consistency bowel movement frequency andbaseline diet however baseline microbiota composition and especially the compositional shifts correlatedwith propionateresponses the nonfermentablemcc showed virtually no effect on gut microbiotacomposition or functionan understanding of compositional and functional responses of the gut microbiota to changes in diet requiresan ecological framework arabinoxylan supplementation provides resources that can be used by microbesthat possess the traits to either access the chemicalstructures directly or utilize public goods released duringarabinoxylan degradation in our study the dominant effects of arabinoxylan were directed toward twobacterial species b longum and p copri while nine additional otus showed smaller increases including threebacteroides species eg b ovatus b plebeius and bacteroides sp this high degree of specificity toward blongum over other bifidobacterium species is in agreement with other studies testing longchain arabinoxylans[] and genomic analyses that showed that genesencoding arabinoxylandegrading glycosidase eg xylosidase and αarabinofuranosidase are conservedonly among b longum strains [ ] in contrast to thespeciesspecific enrichment of b longum arabinoxylanenriched several species within the phylum bacteroidetesthat possess the genetic and functional traits necessaryfor accessing arabinoxylan [] although arabinoxylan utilization is not universally conse
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"MALAT1 also known as NEAT2 (nuclear-enriched abundant transcript 2) is a highly conserved nuclear lncRNA and a predictive marker for metastasis development in lung cancer [23]. In this study we found that the expression of another lncRNA BANCR was significantly downregulated in NSCLC tissues. Specifically BANCR expression was significantly lower at the later stages of tumor development and in tumors that had undergone extensive metastasis. Moreover the overall survival time of patients with lower BANCR expression levels was significantly shorter than that for patients with higher BANCR expression levels. Our results indicate that BANCR expression provided a significant independent predictive value for TNM stage (P?=?0.038). We demonstrated that upregulation of BANCR expression led to the significant inhibition of cell viability migration invasion and promotion of apoptosis. Knockdown of BANCR expression promoted cell migration and invasion. BANCR induced cell apoptosis may be partly via P53 which could contribute to the less cells in migration and invasion; however the impaired migration and invasion ability is the main reason which could be supported by wound-healing assay. Moreover increased BANCR expression levels resulted in a significant reduction in the number of metastatic nodules on the lungs in vivo. These findings suggest that BANCR plays a direct role in the modulation of cell metastasis and NSCLC progression and may be useful as a novel prognostic or progression marker for NSCLC. Tumor development and progression is precisely regulated by several subsets of genes that act by either silencing tumor suppressor genes or activating oncogenes [24]. Tumor suppressor genes can negatively regulate cell proliferation by inducing growth arrest and inhibiting cell invasion. In cancer cells tumor suppressor genes are usually silenced by genetic or epigenetic alterations [25]. Whether epigenetic regulatory factors such as histone acetylation or DNA methylation manipulate the expression of lncRNAs remains unclear. Hypermethylation of the promoter or the intergenic differentially methylated region has been found to contribute to reduced lncRNA MEG3 expression in tumors indicating that epigenetic regulation is also involved in the expression of these genes [2627]. Our findings highlight that histone acetylation is a key factor in controlling lncRNA BANCR expression. These results along with those from a recent study [28] highlight the role of epigenetics in regulating lncRNA transcription. To explore the molecular mechanism through which BANCR contributes to the invasion and metastasis of NSCLC we investigated potential target proteins involved in cell motility and matrix invasion. Hallmarks of EMT are the loss of E-cadherin expression and aberrant expression of N-cadherin and Vimentin [29-32]. Therefore we determined the protein levels of these EMT-induced markers following BANCR overexpression. Our results indicated that inhibitory effects on cell migration and invasion were associated with EMT. Matrix metalloproteases (MMPs) are also important to many aspects of biology ranging from cell proliferation differentiation and remodeling of the extracellular matrix (ECM) to vascularization and cell migration. Upregulation of BANCR expression in NSCLC cells led to a significant decrease in MMP2 protein levels. Our findings demonstrated that BANCR mediated NSCLC cell migration invasion and metastasis suppression which possibly also affected EMT. As a central differentiation process EMT allows for remodeling of tissues during the early stages of embryogenesis and is implicated in the promotion of tumor cell invasion and metastasis [733]. It has been proposed and supported by many studies that EMT could be a potent mechanism for promoting the detachment of cancer cells from primary tumors. A characteristic of cells that undergo EMT is increased expression levels of N-cadherin and Vimentin and a loss of E-cadherin expression. Importantly EMT has been reported to be associated with poor clinical outcome in NSCLC [3435]. Therefore lncRNAs as regulators of EMT might be suitable candidates for intervention in the treatment of cancer. Although only a small number of functional lncRNAs have been well characterized to date they have been shown to regulate gene expression at various levels including chromatin modification transcription and post-transcriptional processing. Hox transcript antisense intergenic RNA (HOTAIR) is one of the most studied lncRNAs involved in chromatin modification which can target PRC2 genome-wide to alter H3K27 methylation and gene expression patterns [22]. A muscle-specific lncRNA linc-MD1 may function as competing endogenous RNAs (ceRNAs) to sponge miRNAs thereby modulating the derepression of miRNA targets and impose an additional level of post-transcriptional regulation [36]. Here although we observed BANCR overexpression induced NSCLC cells apoptosis and regulate EMT phenotype the possible mechanisms that underlie such regulatory behaviors still remain to be fully understood. Further investigation of BANCR molecular and biological functions in controlling EMT will undoubtedly be important in understanding the molecular biology of NSCLC metastasis and progression. Conclusions The expression of BANCR was significantly decreased in NSCLC tissues suggesting that its downregulation may be a negative prognostic factor for NSCLC patients and indicative of poor survival rates and a higher risk for cancer metastasis. We showed that BANCR possibly regulates the invasive and metastatic ability of NSCLC cells partially through regulation of EMT. Our findings further the understanding of NSCLC pathogenesis and development and facilitate the development of lncRNA-directed diagnostics and therapeutics against cancers. However the molecular mechanisms through which BACNR regulates EMT requires further investigation."
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t he gut is the largest immune an of the humanbody and also an important target an for variouskinds of stress caused by severe insults such as infectiontrauma and shock12 these stresses are considered to haveinstitution at which the work was performed department oftraumatology and acute critical medicine osaka universitygraduate school of medicine yamadaoka suita osaka japancorresponding yasutaka nakahori md division of trauma andsurgical critical care osaka general medical center bandaihigashi sumiyoshi ward osaka japan emailp_olysyahoocojpreceived apr accepted jul funding informationno funding information providedan important role in promoting infectious complications andmultiple an dysfunction syndrome from the viewpointsof deteriorated intestinal epithelium the immune systemand commensal bacteria gut dysfunction is now recognizedas a cause for the promotion of diseases34lipids have been a focus not only as energy sources butalso as immunemodulating substrates5 unlike long andmediumchain fatty acids shortchain fatty acids scfaswhich mainly consist of acetate propionate and butyratewith two to four carbon atoms are principally derived fromthe fermentation of carbohydrates and amino acids by anaerobic microanisms shortchain fatty acids are usedmainly by intestinal epithelial cells as energy substrates andthey uence the motility of the intestinal tract and increaseintestinal blood ï¬ow we previously reported on altered gutï¬ora and fecal anic acids in critically ill patients andshowed that these patients had significantly lower levels of the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicinethis is an open access under the terms of the creative commons attributionnoncommercial licensewhich permits use distribution and reproduction in any medium provided the original work is properly cited andis not used for commercial purposes of 0c of y nakahori acute medicine surgery 20207e558fecal anic acids especially scfas than healthy volunteers67despite the evidence implicating the importance ofscfas in the gut the effect of scfas on prognosis in critically ill patients has not yet been clariï¬ed in this study wemeasured eight kinds of fecal anic acidsincludingscfas in critically ill patients and investigated the effect ofscfas on their prognosismethodspatientst his retrospective study enrolled patientswho i fulï¬lled the criteria of systemic ammatoryresponse syndrome sirs according to the american college of chest physicians and the society of critical caremedicine8 ii had a serum creactive protein crp levelgreater than mgdl iii were admitted to the departmentof traumatology and acute critical medicine osakauniversity graduate school of medicine osaka japan during the period november january iv were treated in the intensive care unit for more than days we usedcrp ¥ mgdl to focus on cases with severe ammationbecause crp is mainly used as a marker of ammationwe excluded patients from whom we could not collect anyfecal samples during hospitalization continuous enteralfeeding was initiated with kcalday and graduallyincreased depending on the patients condition we usedimpact novartis nutrition minneapolis mn usa as anenteral nutritional product for all patients in our intensivecare unit and used kcalkg ideal body weight as thegoal for the administered dose fourteen healthy volunteersprovided fecal samples as controls for examinations of fecalanic acids concentrations this study was approved bythe institutional review board of osaka university approval no and informed consent was obtained fromeach patients familyfecal samples and determination of fecalanic acid concentrationswe collected fecal samples serially from all patients thefecal samples were transported at 00°c to the yakult central institute tokyo japan we measured eight kinds ofanic acid in the feces by highperformance liquid chromatography feces were homogenized in ml distilledwater the homogenate was then placed in an eppendorftube and centrifuged at g for min at °c a mixture of ml of the resulting supernatant and ml of moll perchloric acid was mixed well in a glass tubeand allowed to stand at °c for h the suspension wasthen passed through a ï¬lter with a pore size of lmnihon millipore tokyo japan the sample was analyzedfor anic acids by highperformance liquid chromatography as previously described9 using a waters conductivity detector waters milford ma usa equipped withtwo columns shodex kc811 showa denko tokyojapan the concentrations of fecal anic acids were calculated with the use of external standards the reproducibilityand stability of these measurements were shown previously10 we retrospectively evaluated the minimum andmaximum value of each fecal anic acid measured duringhospitalization and determined prognostic factors by classiï¬cation and regression tree cart analysissurveillance and deï¬nition of complicationsbacterial infection was diagnosed in accordance with thedeï¬nitions of the centers for disease control and prevention11 body temperature was measured continuouslysurveillance cultures from blood and sputum were undertaken routinely for each patient in cases of suspected infectionand computedtomography scanning were carried out as necessary bacteremia was deï¬ned as a positive blood culturelaboratory testingchest xraystatistical analysiscontinuous variables expressed as the median 25th and 75thpercentiles were compared by the mannwhitney utestcategorical variables expressed as number were comparedby the v2test unless the expected counts in any of the cellswere below in which case the fisher exact test was usedwe used cart analysis which is a binary recursive partitioning using nonparametric approaches to identify keyfecal anic acids and their cutoff values for mortality12we also used multivariate logistic regression analysis toquantitatively evaluate the effect of the covariates that weresuggested by cart analysisthe most important prognostic factors were selected anda predictive model was developed as follows first the minimum and maximum values of each fecal anic acid wereselected from all patients the maximum value was the highest value and the minimum value was the lowest value measured during hospitalization second the mannwhitney utest was applied to discover variables with potential prognostic value and covariates with p were excludedfinally cart was used to create a tree using the minimumand maximum data values of all fecal anic acids theeffects of the minimum and the maximum values of the fecalanic acids that were selected as key prognostic factors by the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of table characteristics of critically ill patients included in this studytotal n survivors n nonsurvivors n pvalueage yearssex maleicu stay daysapache ii score on admissionorigins of sirssepsistraumaburnunknownbacteremianumber of antibiotic typesduration of antibiotic use days categorical values are expressed as number continuous values are expressed as median 25th75th percentiles bold values indicatestatistical signiï¬canceapache acute physiology and chronic health evaluation icu intensive care unit sirs systemic ammatory response syndromecart analysis and age sex and acute physiology andchronic health evaluation apache
± score on admission were analyzed with the multivariable logistic regressionmodelsall reported pvalues were twosided and a pvalue of was considered to indicate statistical signiï¬cance allstatistical analyses were undertaken using ibm spss statisticsversion armonk ny usaresultspatient characteristicsc haracteristics of the patients are shown intable there were survivors and nonsurvivors the two groups did not differ significantly in termsof sex or apache ii score age in the nonsurvivors wassignificantly older than that in the survivors p the principal origin of sirs was sepsis in of the patients in the two groups significantly more types ofantibiotics were used in the nonsurvivors than in the survivors during hospitalization p and the durationof antibiotic use in the nonsurvivors was significantlylonger than that in the survivors p the incidenceof bacteremia was significantly higher in the nonsurvivorsthan that in the survivors vs p anic acid proï¬lestiming of fecal sample collection is shown in table wecollected fecal samples for scfa analysis fecalsamples were collected evenly throughout hospitalization inboth the survivors and the nonsurvivors the minimum andmaximum values of each fecal anic acid and the resultsof univariate analysis are shown in tables and in termsof the minimum values total anic acids acetate propionate and isovaleric acid were significantly decreased in thenonsurvivors in terms of the maximum values lactate formic acid and succinic acid were significantly increased inthe nonsurvivors otherwise isovaleric acid and acetatewere significantly decreased in the nonsurvivorsadditionally cart and multivariate logistic regressionanalyses of the predominant covariates were carried out fortable timing of fecal sample collection in critically illpatientssurvivorsnonsurvivorstotalweek week week week week week week week week week weekstotal the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558table minimum values of fecal anic acids in eachgroup of critically ill patientssurvivorsnonsurvivorspvaluetotal anic acidsacetatepropionatebutyratesuccinic acidlactateformic acidisovaleric acidvaleric acidvalues are expressed as mean 06 se lmolg of feces bold val 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 ues indicate statistical signiï¬cancetable maximum values of fecal anic acids in eachgroup of critically ill patientssurvivorsnonsurvivorspvalue 06 06 06 06 06 06 06 06 06 total anic acidsacetatepropionatebutyratesuccinic acidlactateformic acidisovaleric acidvaleric acidvalues are expressed as mean 06 se lmolg of feces bold val 06 06 06 06 06 06 06 06 06 ues indicate statistical signiï¬cancethe minimum and maximum values of the fecal anicacids for the minimum values the primary split variablewas determined to be the minimum value of propionate andthe cutoff value was lmolg of feces the secondarysplit variable was determined to be the minimum value ofacetate and the cutoff value was lmolg of feces themortality in each partition and the diagnostic characteristicsof this tree are shown in figure for the maximum values of fecal anic acids the primary split variable was determined to be the maximumvalue of lactate and the cutoff value was lmolg offeces the secondary split variable was determined to bethe maximum value of formic acid and the cutoff valuewas lmolg of feces the mortality in each partitionand the diagnostic characteristics of this tree are shown infigure the diagnostic characteristics of the tree for minimumvalue showed a sensitivity of and speciï¬city of whereas those ofthe tree for maximum valueshowed a sensitivity of and speciï¬city of for the minimum values of fecal anic acids multivariate logistic regression analysis revealed that age and theminimum values of propionate and acetate were significantprognostic factors for mortality table for the maximumvalues age and maximum lactate were identiï¬ed as significant prognostic factors for mortality table discussiont his study provides the ï¬rst in vivo evidence toour knowledge that the altered balance of fecal anicacids is associated with mortality in critically ill patients arecent study has shown that scfas particularly acetate andpropionate bind the gproteincoupled receptor gpr43in adipose tissue stimulation of gpr43 by scfas was necessary for the antiammatory responses because gpr43deï¬cient mice showed exacerbated ammation in modelsof colitis arthritis and asthma the scfagpr43 interactions profoundly affected intestinal and systemic ammatory responses13 the low concentrations of acetate andpropionate observed in nonsurvivors in the present studycould reduce the antiammatory effect fukuda 14showed that acetate produced by protective biï¬dobacteriaimproves intestinal defense mediated by epithelial cells andthereby protects the host against lethal infection furthermore asahara 15 showed a positive correlation betweenfecal acetic acid level and tightjunctionrelated gene expression in the intestinal epithelium in their experiment using aninfected mouse modelthe maximum values of lactate and formic acid were alsoselected as prognostic factors fig our group previouslyshowed that when patients had severely acidic feces fecallactate succinic acid and formic acid were significantlyincreased over those of patients with normal feces we alsoshowed that abnormal fecal ph was associated with the incidence of bacteremia and mortality16 the increase of lactateand formic acid can make feces severely acidic and severelyacidic feces could injure intestinal epithelial cells and worsen the patients condition the present results indicate thatnot only a lack of scfas but also an accumulation of lactateand formic acid might be associated with disruption of thegastrointestinal environmentin critically ill conditionswhich leads to bacterial translocationthere was a significant difference between the two groupsin age number of antibiotic types and duration of antibioticuse in this study previous studies have suggested thatchanges in the composition of bacterial species and genes in the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of fig charts showing mortality in critically ill patients partitioned by the minimum value of fecal anic acids using classiï¬cationand regression tree analysis min minimumfig charts showing mortality in critically ill patients partitioned by the maximum value of fecal anic acids using classiï¬cationand regression tree analysis max maximum the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558table results of multivariate logistic regression analysis in critically ill patients using the minimum value of fecal anic acidscoeff bsepvalueodds ratio conï¬dence intervallower limitupper limitmortalityagesexapache iipropionate minacetate min 00 00 00bold values indicate statistical signiï¬canceapache acute physiology and chronic health evaluation coeff b coefï¬cient min minimum se standard errortable results of multivariate logistic regression analysis in critically ill patients using the maximum value of fecal anic acidscoeff bsepvalueodds ratio conï¬dence intervallower limitupper limitmortalityagesexapache iilactate maxformic acid max 00bold values indicate statistical signiï¬canceapache acute physiology and chronic health evaluation coeff b coefï¬cient max maximum value se standard errorthe intestinal ï¬ora occur with aging17 it is also reported thatthe genes related to scfa production were lost from theintestinal ï¬ora and that glycolytic ability was decreased withaging18 these changes could be one of the reasons whyaging was detected as an important factor related to mortality in the present study there is no doubt that antibiotics areimportant butthe intestinal ï¬orasome systemically administered antibiotics are excreted inthe bile and secreted in the upper gastrointestinal tract andupon reaching the colon they cause serious damage to theintestinal ï¬ora19 shortchain fatty acids could also bereduced with the destruction of the intestinal ï¬ora whichmight have worsened the prognosis20they adversely affectshortchain fatty acids are mainly used by intestinalepithelial cells as energy substrates and some are absorbedinto the portal ï¬ow to the liver and used as systemic energysources they can also uence the motility of the intestinaltract absorption of electrolytes and pancreatic secretion andcan increase intestinal blood ï¬ow in the present study totalanic acids including scfas were significantly decreasedin the critically ill patients compared with those in thehealthy controls data not shown one reason would be thealtered gut ï¬ora that produce scfas the commensal gutï¬ora in humans plays an essential role in homeostasis andprotection from injury in the gut2122 under critically illconditions it is difï¬cult to maintain normal gut ï¬ora6 notonly does the stress derived from diseases such as traumaand burns affect the balance of the gut microbiota but alsothe stress caused by the treatment with various therapeuticagents such as histamine h2 receptor blockers catecholamines and broadspectrum antibiotics can uence the gutï¬ora we reported that critically ill patients had to times fewer total anaerobes biï¬dobacterium andlactobacillus and times greater staphylococcus whencompared with counts in healthy volunteers6 these dataindicate that the balance of gut ï¬ora is significantly disturbed in critically ill patients the concentrations of fecalanic acids decreased significantly in the nonsurvivors asshown in table and we believe thatthis was likelybecause the number of good bacteria biï¬dobacterium andlactobacillus had decreased similarly the cause of thereduction of shortchain fatty acids in the present study may the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of have been the reduction of the scfaproducing bacteriasuch as clostridium eubacterium peptococcusandfusobacterium2324synbiotics are the combination of probiotics and prebiotics the clinical effects of synbiotics have been reported inthe ï¬elds of pediatric surgery abdominal surgery and intensive care25 in our preliminary report critically ill patientstreated with synbiotics maintained the necessary amounts offecal anic acids including scfas and had fewer incidences of enteritis pneumonia and bacteremia than thosewithout synbiotics26 these reports suggest that the maintenance of gut ï¬ora and scfas would be a promising intestinal therapy synbiotics were given to patients in thisstudy in the synbioticstreated group maximum values offecal anic acids acetic acid propionic acid succinicacid lactic acid and formic acid increased significantly butminimum values did not increase only the minimum valueof lactic acid decreased significantly however there wasno ï¬xed protocol for the treatment with synbiotics in thisstudy the treatment start date was variable and the averagehospital day of starting treatment was day so itwas difï¬cult to evaluate the effects of synbiotics in thisstudy evaluating the impact of synbiotics on fecal anicacids and prognosis in critically ill patients will be a futuretaskour study has some limitations this was a retrospectivestudy and thus the timing of feces sampling was differentbetween patients the number of samples was too small tomake conclusions about the temporal patternsin conclusion the minimum values of fecal acetic acidand propionate in the nonsurvivors were significantly lowerthan those in the survivors the altered balance of fecalanic acids was associated with mortality in critically illpatients thus the maintenance of scfas could be a targetfor future intestinal therapyacknowledgementsw e thank all the staff of the department of traumatology and acute critical medicine osakauniversity school of medicinedisclosureapproval of the research protocol this study was approvedby the institutional review board of osaka universityinformed consent informed consent was obtained from eachpatients familyregistry and the registration no of the studytrial naanimal studies naconï¬ict of interest nonereferences meddings j the signiï¬cance of the gut barrier in disease gut macfie j oboyle c mitchell cj buckley pm johnstoned sudworth p gut origin of sepsis a prospective studyinvestigating associations between bacterialtranslocationgastric microï¬ora and septic morbidity gut clark ja coopersmith cm intestinal crosstalk a new paradigm for understanding the gut as the motor of critical illness shock deitch ea bacterial translocation or lymphatic drainage oftoxic products from the gut what is important in humanbeings surgery kreymann kg berger mm deutz ne espen guidelines on enteral nutrition intensive care clin nutr shimizu k ogura h goto m altered gut ï¬ora andenvironment in patients with severe sirs j trauma yamada t shimizu k ogura h rapid and sustainedlongterm decrease of fecal shortchain fatty acids in criticallyill patients with systemic ammatory response syndromejpen j parenter enteral nutr american college of chest physicianssociety of criticalcare medicine consensus conference deï¬nitions for sepsisand an failure and guidelines for the use of innovative therapies in sepsis crit care med sugawara g nagino m nishio h perioperative synbiotic treatment to prevent postoperative infectious complications in biliary cancer surgery a randomized controlled trialann surg kikuchi h yajima t correlation between waterholdingcapacity of different types of cellulose in vitro and gastrointestinal retention time in vivo of rats j sci food agr horan tc andrus m dudeck ma cdcnhsn surveillancedeï¬nition of health careassociated infection and criteria forspeciï¬c types of infections in the acute care setting am jinfect control shimizu k ogura h hamasaki t altered gut ï¬ora areassociated with septic complications and death in critically illpatients with systemic ammatory response syndrome digdis sci maslowski km vieira at ng a regulation of ammatory responses by gut microbiota and chemoattractantreceptor gpr43 nature fukuda s toh h hase k biï¬dobacteria can protectfrom enteropathogenic infection through production of acetate nature asahara t takahashi a yuki n kaji r takahashi tnomoto k protective effect of a synbiotic against multidrug the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558resistant acinetobacter baumanniimodel antimicrob agents chemother in a murine infection guarner f malagelada jr gut ï¬ora in health and diseaselancet osuka a shimizu k ogura h prognostic impact of gill sr pop m deboy rt metagenomic analysis of thefecal ph in critically ill patients crit care r119human distal gut microbiome science otoole pw jeffery ib gut microbiota and aging science macfarlane s macfarlane gt regulation of shortchain fatty rampelli s candela m turroni s functional metagenomic proï¬ling of intestinal microbiome in extreme ageingaging albany ny de gunzburg j ghozlane a ducher a protection ofthe human gut microbiome from antibiotics j infect dis p 13erezcobas ae gosalbes mj friedrichs a gutmicrobiota disturbance during antibiotic therapy a multiomic approach gut acid production proc nutr soc pryde se duncan sh hold gl stewart cs flint hj themicrobiology of butyrate formation in the human colonfems microbiol lett shimizu k ogura h asahara t probioticsynbiotictherapy for treating critically ill patients from a gut microbiotaperspective dig dis sci shimizu k ogura h goto m synbiotics decrease theincidence of septic complications in patients with severesirs a preliminary report dig dis sci the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c'
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despite great advances in recent decades in screening diagnosisand curative surgery hepatocellular carcinoma hcc remainsthe second leading cause of cancerrelated mortality worldwidegrandhi siegel epidemiologicalevidence has conï¬rmed that the longterm outcomes of patientswith hcc have notimproved significantly with the rapiddevelopment of surgical techniques madduru moreimportantly because of the limitations of systematic statustumor position and the need to preserve liver function morethan of patients are not eligible for surgical treatment evenafter curative resection prognosis remains unsatisfactory becauseof a high incidence of postoperative recurrence colecchia the initiation and maintenance of hcc is a complexand regulated process involving the accumulation of numerousgenetic changes over decades niu erkekoglu these sequential alterations not only endow normal livercells with neoplastic ability enabling uncontrolled growth butalso provide potential therapeutic targets and biomarkers thusfurther understanding of the initiation and maintenance of hccat the molecular level is crucial to prolonging survival and makingindividual treatment decisionsthe exive development of highthroughput technologyhas provided powerful tools for the molecular study of cancerschmidt and hildebrandt rna sequencing rnaseq and microarraysthe most representative methods ofthis technology are mature enough for use in commercialapplications mantione zhang duringthe past decades the genomewide transcriptional analysis ofgene expression has become critically important to gain betterinsight into the biological processes of hcc and other typesof cancer jin xiong in additionto the aberrant expression of transcripts studies have focusedon diï¬erent molecular levels multilevel omicsincludingcopy number variation epigenetic modiï¬cations nucleotidepolymorphisms and dna methylation especially in hcclee lin evidence obtained from thesestudies clearly demonstrates that hcc is a disease causedby cumulative aberrations at diï¬erentlevels of molecularregulation thus only a highthroughput multiomics analysiscan decipher the complex biology of hcc many previousstudies despite promising results focused only on the aberrantregulation of expression and its biological eï¬ects howeverstructural transcript variation in hcc which is heavily shaped byalternative splicing as has until recently been less well studiedaccording to the manual genome annotation project harrow pruitt there are only about proteincoding genes this number is obviously inconsistent withthe overall cellular complexity which includes at least distinct proteincoding sequences harrow thisdiscrepancy between the numbers of transcripts and proteincoding genes in human cells indicates the existence of anadditional mechanism between the transcriptional and the posttranslational levels that increases the coding capacity of thegenome through the as process a single rna precursorcan be spliced via distinct arrangements to generate rnaslandscape of as in hccwith diï¬erent structures and functions biamonti song this may be one ofthe main causesof cellular complexity and proteome diversity experimentalstudies on the eï¬ects of individual as events suggest that asmay change the biological function of a protein by regulatingits stability controlling its location modifying the mutualinteractions of proteins and even adding or deleting activedomains brett yang these ï¬ndingssuggest that as well as expression abundance the balance ofdiï¬erent as events that result from the same rna precursormust be considered howeverthe latter consideration hasoften been neglected in previous studies in fact emergingdata from genomewide analyses feng kahles indicate that as occurs in more than ofmultiexon genes suggesting thatthe widespread existenceof as is the product of physiological processes rather thantranscription errorsin recent years the diagnostic and the therapeutic role ofas in many human diseases has attracted increasing attentionlargescale screening of as events has been performed usingexpressed sequence tag libraries although this approach isprone to a high rate of false positives venables exonjunction probes provide a higher experimental validation ratelapuk however this method has the disadvantageof being limited to known splice junctions owing to thelimited available techniques complicated mechanisms and hugenumbers involved transcriptomewide as dysregulation and itspotential associations with biological behavior in hcc haveremained uncharacterizedrnaseq not only supports the quantitative measurementof novel as events but also provides deeper coverage higheraccuracy and better resolution li y thus it maybe the most suitable of the currently available approaches foras study in recent years the cancer genome atlas tcgatomczak wang has accumulated a richand publicly available source of rnaseq data and correspondingclinical information this enables the analysis of as dysregulationin hcc at a genomewide level tcga includes rnaseqdata samples obtained from hcc patients together withtheir corresponding clinical information thereby facilitating theclinical analysis of hccrelated as events in a large cohorthowever without reliable and eï¬cient bioinformatical methodsthe advantages of rnaseq in as analysis cannot be fullyexploited spliceseq a recently developed bioinformatics toolcan exactly match rna reads with gene splice graphs and ishelpful for accurately calculating complex or lowfrequency asevents ryan there has been a lack of studies combining largescalernaseq data with the corresponding clinical information tocomprehensively analyze as at singleexon resolution howeverthis is very necessary especially in hcc thereforein thecurrent study we comprehensively analyzed wholegenome asin the tcga hcc cohort to screen out hccrelated asevents and further studied the relationships of these eventswith clinical outcomes our ï¬ndings suggest that certain hccrelated as events including nek2at and troptat havecritical roles in the progression and maintenance of hcc morefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccimportantly these hccrelated as events represent potentialnew therapeutic targetsmaterials and methodstumorlocationinvasioninformation ofdata curationclinicopathologicalthe hcc cohort andcorresponding rnaseq data were retrieved and downloadedfrom tcga1 to ensure appropriate protection of patientprivacy the tcga data were stratiï¬ed according to data typeand level conforming to the publishing guidelines formulatedby tcga wang then the rnaseq data andcorresponding clinicopathological information were mutuallypaired using the unique tcga barcodes only patients whomet the criteria listed below were included grandhi patients with corresponding rnaseq data siegel patients with complete clinicopathological informationincluding localsex age distalmetastasis pathological stage diï¬erentiation grade lymph nodemetastasis and survival information madduru histological diagnosis of hcc and colecchia survival for at least month after the primary pathologicaldiagnosis spliceseq was used to determine rna splicing patternsand produce as proï¬les for each hcc patient as previouslydescribed li y zhu each as eventwas quantiï¬ed using the percent spliced in psi value rangingfrom to a commonly used method to reï¬ect the abundanceof as events in order to remove the eï¬ects of splicing noiseand generate as reliable a set of as events as possible a seriesof strict ï¬lters average psi ¥ percentage of samples withpsi ¥ was applied to the detected as events the interactivesets between the seven types of as were quantitatively analyzedand the results were visualized in upset plots using upsetrversion conway circlize version wasused to generate circos plots to depict the parent genes and theiras events in chromosomes gu the details of thedesign of the present study are illustrated in supplementaryfigure s1 all the methods used in this study were in line withthe relevant guidelines and regulationsidentiï¬cation of deas and enrichmentanalysisto screen the diï¬erentially expressed alternative splicing deasevents between hcc and corresponding normal tissues the psivalue of each as event was determined in the tcga hcccohort hcc tissue samples and paired adjacent normaltissues a generalized linear model was applied to remove thebatch eï¬ects the deas were determined based on adjustedp adj p and associated log2 fold change fc values withadj p ¤ and log2fc ¥ representing as events thatwere downregulated and upregulated respectively biologicalfunction enrichment analysis was performed based on the deasparent genes gene ontology go and kyoto encyclopediaof genes and genomes kegg terms with false discoveryrate less than were considered to be significantly enrichedand were selected for further analysis enrichment analysis wasperformed using the clusterproï¬ler package version yu the parent genes of deas events were importedinto the string database and used to determine proteinprotein interactions ppis a relationship network was thengenerated using cytoscape version su clusteranalysis was conducted using the average linkage agglomerationalgorithm and correlation distance metricsestablishment of hccrelated splicingcorrelation networka total of splicing factors sfs supplementary table s1were identiï¬ed by comprehensive and handcurated screening ofthe literature all the sfs included in the current study had beenexperimentally validated in previous research giulietti and included heterogeneous nuclear ribonucleoproteinsproteins serineargininerich proteins and other proteinsbelonging to the celf fox khdrbs nova and elav familiesthe expression of each sf was obtained from the broadinstitute2 correlations between the psi values of deas and theexpression of sfs were analyzed by weighted gene coexpressionnetwork analysis version langfelder and horvath benjamini and hochberg correlation was used to adjust thepvalues the adjusted pvalues less than were consideredto indicate statistically significant diï¬erences cytoscape version was used to generate the correlation plotssurvival analysisall the included hcc patients were divided into two groupsbased on the psi value of each deas median cut and thetwo artiï¬cial categories were modeled as continuous variables toderive more easily interpretable hazard ratios based on overallsurvival os and diseasefree survival dfs cox regression wasperformed to evaluate the prognostic value of each deas eventlogrank test and kaplanmeier analysis were used to comparepatient survival between subgroups p was consideredas statistically significant the overall survivalrelated deaswere further analyzed in lasso regression to identity the mostpowerful prognostic markers finally a prognostic model wasconstructed for predicting the os in order to quantify the risk ofos a standard form of risk score rs for each colorectal cancercrc patient was calculated combine the levels of the psiι1 psii à lito divide the patients into the high or lowrisk group kaplanmeier curves were used to estimate the survival for patients in thetraining the testing and the validation sets between the highriskand the lowrisk groupspsii and lasso coeï¬cients li risk score pnfunctional experiment of cxcl12splicing variants in hccthe human hcc cell line hepg2 was obtained from the chineseacademy of sciences committee on type culture collectioncell bank shanghai china the cell line was cultured in 1httpsportalgdccancergov2httpgdacbroadinstitutefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccprimers was used asgibco carlsbad ca united states supplemented with fetal bovine serum fbs bi beit haemek israel at ¦c with co2 total cdna from tissues was obtained as described abovethe pcr reaction was carried out using the forward primer5cid48tgcccttcagattgttgcac3cid48 common for allisoforms and theisoformspeciï¬c reverse primers 5cid48gctaactggttagggtaatac3cid48 and5cid48gctagcttacaaagcgccagagcagagcgcactgcg3cid48for np_9546371and np_0010290581 respectively bactin ampliï¬ed usingthe forward 5cid48acactgtgcccatctagcagggg3cid48and reverse 5cid48atgatggagttgaaggtagtttcgtggat3cid48aloading control quantitative realtime pcr was performedin mx3005tm qpcr system with a mxpro qpcr software stratagene la jolla ca united states and sybr greendetection system the adherent hepg2 cells were transfectedwith the corresponding hiscxcl12 construct by the calciumphosphate method and cultured for h at h before collectingthem the cell supernatants were removed and when indicatedbrefeldin a was added to the fresh medium the collectedcells were left untreated or permeabilized with saponin andimmunolabeled with the his mab and a pegoat antiigsecondary antibody and analyzed by ï¬ow cytometry the cellinvasion assay was conducted using matrigelcoated chambers µm pore size corning costar corporation cambridgema united states in brief à cells were plated in theupper chamber coated with matrigel and supplemented withserumfree medium the lower chamber was ï¬lled with a culturemedium containing fbs incubation was carried out for h at ¦c following which the noninvasive cells were scrapedoï¬ with cotton swabs the cells that had successfully translocatedwere ï¬xed with paraformaldehyde stained with crystalviolet and ï¬nally counted using an inverted microscope mttassay colony formation assay and soft agar growth assay wereperformed according to our previously described methods zhou protein structure homology modeling analysis wasperformed as previously described by using the online serverswissmodel waterhouse evaluation of the relationship of asclusters with clinicopathologicalfeaturesbased on the identiï¬ed deas n the tcga hcc cohortin the current study was stratiï¬ed by an unsupervised consensusapproach consensus cluster plus version wilkerson andhayes the optimal number of clusters was determinedby integrating the results of the elbow method and gap statisticthe relationship between clinical outcomes and as clusterswas evaluated using logrank test and kaplanmeier curves asdescribed by xiong 2018aresultslandscape of as event proï¬les in hccto systematically characterize the as events and their clinicalsigniï¬cance in hcc we collected rnaseq libraries andcorresponding clinicalinformation from hcc patientsthe tumor tissues and paired adjacent normal tissues from patients were sequenced simultaneously the includedpatients comprised males and femalesamong which patients developed recurrence and died of hcc the median followup period was months range months the general characteristicsof these hcc patients are fully detailed in supplementarytable s2 rnaseq data were associated with the clinicalthe corresponding patient via the tcgainformation ofbarcodes there were patients with rnaseq data bothfrom tumor tissue and adjacent normal tissue according tothe recommended analysis approach described in a previouslypublished study ryan we identiï¬ed asevents from genes based on their splicing patterns theseas events could be roughly classiï¬ed into seven types alternatepromoter ap mutually exclusive exons me retained intronri exon skipping es alternate acceptor site aa alternateterminator at and alternate donor site ad figure 1ato quantify the detected as events psi values were calculatedthese values measure the proportion of each detected splicingvariation in all of the expressed isoforms the expression ofcertain isoforms was fairly low psi and most of the asevents could not be stably detected in all of the given samplesafter screening average psi ¥ percentage of sampleswith psi ¥ a total of as events from geneswere obtained we further compared the variance in quantityof as events and the genes involved between tumor adjacentpaired normal and unpaired tumor tissues for diï¬erent splicingpatterns there were no significant diï¬erences in quantityvariations however on average one gene might have nearlythree as events figure 1b left panel moreover only annotated genes in this study stably underwent as figure 1bright panel notably diï¬erent as patterns may occur for a singlegene thus upset plots were used to depict the intersectionsbetween as types as demonstrated in figure 1c most of theparent genes only occurred in one type of as event whereascertain parent genes contained up to four types of as eventabout of the parent genes contained two or more asevents the arrangements of diï¬erent as types and as eventsbetween diï¬erent exonsintrons may be the major reason fortranscriptome diversity in order to comprehensively depict asevent proï¬les in hcc circos plots were used to visualize therelationships among as events and the corresponding parentgenes in chromosomes figure 1didentiï¬cation of hccrelated deascomparing the variations in molecular components amongdiï¬erent pathological states using highthroughput techniquesis an eï¬ective way to screen key molecules this approachhas been widely used to identify diseaserelated molecules inprevious research xiong 2018ab it is reasonableto consider that significant diï¬erences in as events betweenprimary hcc tissues and adjacent normal tissues may be relevantto the initiation and maintenance of hcc in this study thetcga barcodes corresponding to tissue samples rnaseq data were analyzed from which as proï¬les of pairednormal and tumor tissues were ï¬nally extracted these pairedfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure landscape of alternative splicing as events in hepatocellular carcinoma hcc a diagrammatic sketch of the seven types of as events in the presentstudy alternate acceptor site aa alternate terminator at alternate promoter ap exon skipping es mutually exclusive exons me alternate donor site adand retained intron ri b number of as events and the corresponding parent genes illustrated according to as type left panel the color bar represents asevents ï¬ltered by criteria the black bar represents the corresponding genes involved in as each as type was divided into four groups based on the tissue sourcen normal tissue t tumor tissue pt paired tumor tissue npt unpaired tumor tissue number of detected as events asrelated genes ï¬ltered as events and thecorresponding genes right panel c intersection of parent genes between different as types n in hcc one gene may incur up to four types ofalternative splicing d circos plots depicting the distribution and the detailed alteration of as events and their parent genes in chromosomesas proï¬les were used to identify deas eventually deaswere identiï¬ed from genes using a threshold of log2fc and adj p including aps ess ats risnine ads aas and one me figure 2a and supplementarytable s3 the top deas are listed in table notablythe proportion of as types between the ï¬ltered as and deaswas inconsistent the es events accounted for of ï¬lteredas but only of deas however the proportion of apevents rose from of ï¬ltered as to of deasfigure 2b these statistical ï¬ndings suggest that as is notthe result of transcription errors but a tightly regulated processmoreover based on the identiï¬ed deas the samples could beclearly separated into normal and tumor groups by unsupervisedhierarchical clustering figure 2c indicating that the deashad been reliably identiï¬ed the psi values of deas eventsin diï¬erent hcc patients are illustrated in figure 2c as amatrix heat map the changes in color gradient intuitively revealthe heterogeneity of hcc a splice graph which representssplice junctions as edges and exons as rectangular nodes wasused to visualize some of the identiï¬ed deas figure 2dfurthermore the diï¬erences in expression of these as eventsbetween primary hcc tissue and corresponding adjacent normaltissues are intuitively depicted in figure 2e taken together theseresults show that a significant variation of as occurred duringhcc initiation and maintenance indicating that the potentialrole of hccrelated as events requires further researchenrichment and interaction analysis ofdeasemerging evidence indicates that as could change a transcribedsequence directly with eï¬ects on expression abundance orprotein function thus the potential biological eï¬ects of deascould be determined by analyzing the corresponding proteinsas shown in supplementary figures s2ac speciï¬c go termsclosely related to liver metabolism including negative regulationof hydrolase activity sterol homeostasis anic acid catabolicprocess and acidic amino acid transport were significantlyenriched by the parent genes of deas in addition certain keggpathways known to be involved in hcc were enriched includingthe cgmppkg signaling pathway the nfκb signaling pathwaythe mrna surveillance pathway and the phosphatidylinositolfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure identiï¬cation of hepatocellular carcinoma hccrelated aberrant alternative splicing as a differences in as events between paired hcc tissue andparacancerous tissue volcano plot of the differentially expressed alternative splicing deas identiï¬ed in hcc the blue and the red points represent the deas withstatistical signiï¬cance logfc ¥ adj p b proportions of different as types among ï¬ltered as and deas c heat map of the deas the horizontal axisshows the clustering information of samples divided into two major clusters adjacent normal tissue n and paired tumor tissue n the left longitudinalaxis shows the clustering information of deas the gradual change of color from green to red represents the alteration of expression of deas from low to highd splice graph of some representative deas the thin exon sections represent untranslated regions and the thick exon sections represent coding regions theexons are drawn to scale and the connecting arcs represent splice paths e differences in percent spliced in values of as events between hcc and pairedadjacent normal tissuessignaling system supplementary figure s2d these resultssuggest that the parent genes of deas are critical in the biologicalregulation of hcc thus aberrant splicing of the transcribedsequences could inï¬uence their translation and change thecharacteristics of the resulting proteins therefore it is essentialto study as events from the perspective of ppi networks basedon the deasrelated genes a ppi network was establishedrepresenting not only normal interactions but also the potentialimpact of as events figure correlation network of sfs andhccrelated asas events are primarily regulated by sfs which attach to themrna precursor and aï¬ect the selection of exons and thechoice of splicing site aberrant as events in tumor tissuemay be orchestrated by a limited number of sfs for thisreason we conjecture that a few key sfs potentially regulatea large proportion of hccrelated as events to validate thisconjecture we ï¬rst identiï¬ed sfs supplementary table s1by comprehensive and handcurated screening of the literatureall of which had been previously experimentally validatedgiulietti then the copy number variation somaticmutations and expression abundance of these sfs in eachhcc patient were investigated using cbioportal figure 4avisualization using oncoprint revealed that each of the sfs harbored at least three molecular alterations figure 4aleft panel the most frequently aï¬ected sf was khdrbs3in which molecular alterations were detected in cases partly owing to the above changes the expressionabundance of the sfs showed a significant heterogeneity atan individual level figure 4a right panel the expressionproï¬les of the sfs in diï¬erent cancer types also showedheterogeneous characteristics figure 4b more importantlythe expression of sfs also diï¬ered between paired normal andcancer tissues of the same hcc patient figure 4c nextcorrelation analyses were performed between the psi value ofeach deas event and the sfs according to the correlationcoeï¬cient ttest p r a splicing regulatorynetwork was established as shown in figure 5a sfs weresignificantly correlated with deas events among which were downregulated and were upregulated several diï¬erentas events in the network were regulated by a single sf insome cases an sf had the opposite regulatory eï¬ect on diï¬erentas events figure 5a we also found that the same bindingsite as event could be competitively bound by diï¬erent sfsthese observations explain at least in part why one gene cangenerate several diï¬erent isoforms representative correlationsfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hcctable the top most different alternative splicing as eventssymbolas typeexonsfrom exonto exonmean nmean tlog fcadjusted pvaluedownregulatedmthfd2ligf2kif22gstz1serpinb5ppargfam3afip1l1tmem59tpm4cdh23mid1gnesamd12kif4afbxw7nek2padi4rnf115vti1aupregulatedrdm1nr1i3psphtmem145nr1i3gpr116piddnr1i3ano1cldn7sardhscp2znf331eno3pemtfn1tnfrsf10carhgef39igf2neil3apapadapatapaaatapapatapapatatapatatesatatatapatriesapriesapapesapatapesatapapat1393e1593e4948e9941e2008e4387e8636e1393e4574e2519e1528e2202e6860e1522e2379e7699e1780e1843e4211e2609e1994e8164e1036e7765e1089e3569e1065e2135e5986e1038e1611e1110e2586e7736e2117e1072e4524e1151e1829e8136ebetween sfs and speciï¬c as events were illustrated using dotplots figures 5bg for example the expression of esrp2 wassignificantly correlated with both es of ceacam1 figure 5cand at of epb41l5 figure 5fassociation of deas with prognosis ofhcc patientsa crossvalidation method was used to evaluate the accuracyof the survival data and the clinical information as shown insupplementary figure s3 stratifying patients according to thetnm stage resulted in separate kaplanmeier curves for bothos and dfs these results conï¬rmed that the survival dataset forthe tcga hcc cohort although it contained censored data wasappropriate and informative for use in further survival analysistheeï¬ect of each deas on survival was determined by coxregression analysis the hcc patients were divided into twogroups according to their psi value median cut of eachdeas event according to univariate analyses a total of to investigate the prognostic signiï¬cance of deasfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure proteinprotein interaction ppi analysis of the identiï¬ed differentially expressed alternative splicing deas interactions of the parent genes affectedby deas these genes were used to construct an intricate ppi network comprising nodes and edges the genes are denoted as nodes in the graph andthe interactions between them are represented as edges the shape size and color of the nodes respectively represent alternative splicing type value of logfcand change patterndeas events were significantly correlated with dfsand deas events were significantly correlatedwith os supplementary table s4 among these prognosisrelated deas events deas events were correlated withboth os and dfs p figure shows some of thedeas events for which the pvalue for both os and dfs waslower than to demonstrate the capability of as eventsfor prognostic prediction we randomly selected two prognosisrelated deas events and used them to draw survival curvesas shown in figure according to the psi value median ofnek2at and troptat the hcc patients could be stratiï¬edto form significant kaplanmeier curves by both os and dfssurvival analysis additionally the deas events that significantlycorrelated with survival in the univariate analysis were furtherassessed by multivariate analysis as shown in supplementaryfigure s4 there were ï¬ve and six deas events that could berecognized as independent prognostic indicators for os and dfsrespectively these ï¬ndings conï¬rm that deas events possessnot only an important biological meaning but also a potentialclinical signiï¬canceconsidering the prognostic value of the aboveidentiï¬ed asa prognostic model integrating multias was established so thatit can be easily applied to clinical practice based on the survivalrelated deas a relative regression coeï¬cient was calculated bylasso analysis by forcing the sum of the absolute value ofthe regression coeï¬cients to be less than a ï¬xed value certaincoeï¬cients were shrunk exactly to zero and the most powerfulprognostic marker of all the hcc survivalassociated deaswas selected including four as supplementary figure 5acombining the relative expression levels of the as in themodels and the corresponding lasso coeï¬cients a riskscore was calculated for each patient obviously patientswith higher rs generally had a significantly worse overallsurvival than those with lower rs p supplementaryfigures 5bc as the majority of events occurred within years timedependent roc curves were used to assess theprognostic power based on os at and years respectivelysupplementary figure 5dclustering hcc patients using deasassociated with prognosisgiven our ï¬ndings of significant heterogeneity among deas atan individual level which could reï¬ect the diï¬erent outcomesfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure multiomics analysis of the splicing factors sfs in hepatocellular carcinoma hcc a cbioportal analysis of the sfs in the cancer genomeatlas hcc patients oncoprint was used to produce a landscape of genomic alterations legend in sfs rows at the individual level columns inframe deletionstruncated mutations and missense mutations with known or unknown signiï¬cance are shown in orange blue green and gray respectively with onethird heightthe copy number variations are annotated with the full height ampliï¬cation is shown in red and copy number loss is in blue heat map matrix shows the variation insfs at expression level the expression abundance from high to low is represented by color gradient from red to blue b expression of the sfs in tumortypes heat map color gradient depicts the normalized expression of sfs between different tumor types c differential expression analysis of representative sf tia1in hcc the expression of tia1 in hcc tissue was significantly higher than that in normal liver tissueof patients with hcc we conjectured that there might existdistinct patterns of as among diï¬erent hcc patients thishypothesis was veriï¬ed using consensus unsupervised analysisbased on the deas the optimal number of clusters wasdetermined by combining the gap statistic and elbow method theoptimal balanced partition as suggested was k figure 8aaccordingly all the hcc patients were divided into four clustersas follows i n ii n iii n and iv n figures 8bc as illustrated bythe heat map the four clusters had a significant interconnectivityamong which cluster ii appeared as a wellindividualized clusterwhereas there was more classiï¬cation overlap among clustersi iii and iv figures 8bc kaplanmeier survival analysisand logrank test were used to evaluate the associations betweenprognosis and the as clusters as illustrated in figures 8dethe stratiï¬cation of hcc patients based on as clusters showeda significant correlation with distinct patterns of survival thevariation tendency that resulted in the as stratiï¬cation betweenfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure speciï¬c regulatory network of hepatocellular carcinomarelated alternative splicing as events a correlation network of splicing factors sfs anddifferentially expressed alternative splicing the shape size and color of nodes respectively represent type as event or sf value of logfc and change patternupregulated or downregulated the breadth of the line represents the interaction strength bg representative dot plots of the correlations between theexpression of sfs and percent spliced in values of as eventsfigure prognostic value of differentially expressed alternative splicing deas in hepatocellular carcinoma part deas events simultaneously associated withoverall survival os and diseasefree survival dfs univariate analysis of deas for os and dfs respectively unadjusted hazard ratios boxes and conï¬denceintervals horizontal lines
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" vibrio cholerae are oxidasepositive bacteria that are classified into various serotypes based on the osurface antigen v cholerae serotypes are divided into two main groups the o1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v choleraenovc can cause choleralike diarrhea a pubmed search revealed that only cases of necrotizing fasciitis causedby novc have been recorded in the scientific literature to date we report the case of a japanese woman whodeveloped necrotizing fasciitis caused by novc after traveling to taiwan and returning to japancase presentation a 63yearold woman visited our hospital because she had experienced left knee pain for thepast days she had a history of colon cancer stage iv t3n3 m1a and had received chemotherapy she hadvisited taiwan days previously where she had received a massage she was diagnosed with septic shock owingto necrotizing fasciitis she underwent fasciotomy and received intensive care she recovered from the septic shockhowever after weeks she required an aboveknee amputation for necrosis and infection her condition improvedand she was discharged after weeks in the hospitals with the increase in tourism it is important for clinicians to check patients travel history cliniciansshould be alert to the possibility of necrotizing fasciitis in patients with risk factors necrotizing fasciitis caused bynovc is severe and requires early fasciotomy and debridement followed by intensive postoperative carekeywords necrotizing fasciitis vibrio cholerae taiwan massage septic shock polymyxin b correspondence kei610805gmailcom1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctsuruta bmc infectious diseases page of vibrio cholerae are curved gramnegative rod gnrbacteria that are oxidase positive they are classified intovarious serotypes based on the o surface antigen vcholerae serotypes are divided into two main groups theo1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v cholerae novccan cause choleralike diarrhea novc are found as autochthonous microbes in coastal and marine environments outbreaks of choleralike illness caused bynovc have been reported in the united states o141and o75 former czechoslovakia o37 sudan o37peru o10 o12 and mexico o14 [] moreovernovc can cause a range of extraintestinal infectionsincluding bacteremia meningitis pneumonia peritonitischolangitis salpingitis and softtissue infection seafood including oysters fishes shrimps clams musselsand apple snail is the most common source of infection a pubmed search revealed that only cases of necrotizing fasciitis caused by novc have beenreported in the scientific literature to date we reportthe case of a patient who developed necrotizing fasciitisand septic shock caused by novc which necessitatedan aboveknee amputation of her left legcase presentationa 63yearold woman visited minaminara general hospital in nara japan because she had experienced leftknee pain for days prior to her visit she had been diagnosed with colon cancer stage iv t3n3 m1a years and months previously and had undergone surgery and received chemotherapy her most recent doseof chemotherapy was administered days before herinitial consultation she had visited taiwan days previously where she had received a massage after themassage she developed gradually worsening pain in herlower left leg on presentation she was able to walkunaided and she reported her history of colon cancerand recent travel as we suspected that the pain in herleg could be due to necrotizing fasciitis we requestedmagnetic resonance imaging mri of her left lowerleg the images showed a swollen soleus muscle andposterior tibial muscle and the t2weighted imageshowed hyperintensity of the muscle tissue fig after the mri our patients condition deteriorated andthe following vital signs were observed blood pressurebp mmhg heart rate beatsmin respiratory rate breathsmin and temperature °cthe results of arterial blood gas analysis were as fol mmhglows ph paco2 mmhg hco3base excess be meql and lactate mmoll the patients laboratory test results were as followscreactive protein crp mgdl blood ureanitrogen bun mgdl creatinine mgdlprocalcitonin ngml nterminal probrain natriuretic peptide ntprobnp pgml and fibrinfibrinogen degradation products fdp μgmlinitiatedadministeredlowvenovenoushemodiafiltrationsurgery her blood pressure wasintravenous infusion of meropenem and noradrenaline wasand the patient underwentemergency surgery before the surgery the compartment pressure of her left leg was measured by simpleneedle manometry the pressures were as follows mmhg mmhg mmhg and mmhg in theanterior lateral superficial posterior and deep posterior compartments respectively some muscle tissuesin the anterior and deep posterior compartments werenecrotic for double incision fasciotomy a relaxationincision was made on her left knee and theaffected area was irrigated and debrided fig aftertheandtherefore wepolymyxin b directhemoperfusion pmxdhp to trap endotoxins andcontinuoususinghemofeel ch13 w toray medical co ltdurayasu japan as a slightly curved gnr that wasoxidase positive was detected in her blood we diagnosed her with necrotizing fasciitis and septic shockcaused by vibrio species we changed the antibioticsfrom meropenem to ceftriaxonelevofloxacin andminocycline we used the pmxdhp once again andtapered the dose of noradrenalin gradually wediscontinued noradrenalin on day postoperativelyon day postoperatively the anism was identifiedas novc theantibiotics wasconfirmed postoperatively on day and we discontinued levofloxacin table although the patientsgeneral condition improved there was a discharge ofpus from the postoperative wound on day postoperatively a second debridement was performedseveral muscles in the patients left leg including theanterior tibial muscle had become necrotic and thenecrosis had spread to her knee on day postoperatively an aboveknee amputation was performedher vital signs and laboratory data obtained since admission are shown in fig her condition improvedand she was discharged weeks after admissionsusceptibility ofdiscussion and sixteen cases of necrotizing fasciitis caused by novchave been previously reported table [] themajority of patients were exposed to seawater or hadan injury in rare cases vigorous massage is one ofthe risk factors of necrotizing fasciitis howeverthe patient in the present case had a risk of novcinfection because of colon cancer and immunosuppression due to chemotherapy as she received chemotherapy within a month thusthein this case 0ctsuruta bmc infectious diseases page of fig t2weighted magnetic resonance images of the patients lower legs a coronal image b axial image these images show that the soleusand posterior tibial muscles on the left lower leg indicated by red arrows are swollen and inflamedfig photographs of lesions in the patients leg the patients leg before surgery shows multiple large blisters 0ctsuruta bmc infectious diseases page of table susceptibility of antibioticsantibioticsampicillinminimal inhibitory concentrations piperacillinceftazidimeimipenemcilastatinamoxicillinclavulanategentamicinminocyclinechloramphenicolsulfamethoxazoletrimethoprimlevofloxacinfosfomycins s s ¦s s s ¦s ¦s ¦s ¦r the novc remains unknown assource ofthepatient did not report any exposure to sea water oreating seafood the only potential cause of injury toher left leg was the massage she received thereforewe speculate that the massage might have been thesource ofthe novc based on the circumstantialevidence we administered blood purification therapyusing pmxdhp and venovenous hemodiafiltrationfor septic shock although no previous studies havereported the use of pmxdhp for novc a studyreported the use of pmx for v vulnificus thirdgenerationtetracyclineandfluoroquinolone were used for severe vibrio infections tetracycline combined with the fluoroquinoloneorcephalosporinfollowed by oral fluoroquinolones or doxycycline wasrecommended for invasive novc infections [ ]an in vitro study revealed that cefotaxime and minocycline have a synergistic effect in the treatment forcephalosporinsaparenteralthirdgenerationfig change of vital signs and laboratory data during the hospital admission a changes in the patients vital signs during days ofhospitalization b changes in patients blood biochemistry during days of hospitalization atiii antithrombin iii crp creactive proteinfdp fibrinfibrinogen degradation products map mean arterial pressure nad noradrenaline 0ctsuruta bmc infectious diseases page of age sex underlying diseasestable clinical characteristics of patients with nono vibrio cholerae necrotizing fasciitisyear ofreportsourcesurgery amputation multiple debridementand antibiotics ticarcillinclavulanate imipenemgentamicin clindamycinrisk factors mdiabetes mellitussurvived usatreatmentoutcome country oantigen epidemiologicexposureexposure of achronic plantar ulcerto sand in abathhouse mcirrhosissurgery cefotaxime minocycline cefotaximesurvivedtaiwan f mcirrhosis congestiveheart failurecirrhosis diabetesmellitus mhepatitis csurgery ceftriaxonediedtaiwansurgical debridement ceftazidime doxycyclinediedtaiwan o56surgery antibiotics thirdgeneration cephalosporindoxycyclinediedtaiwanhandling seafood mhepatitis steroidssurgery antibiotics thirdgeneration cephalosporindoxycyclinesurvivedtaiwan mcirrhosissurgery clindamycin ceftazidime tetracyclinesurvivedtaiwansurgery antibioticssurgery antibioticsdiedtaiwandiedtaiwan m m mcirrhosis hepatitis cdiabetes mellituscirrhosis hepatitis bhepatitis c diabetesmellituscirrhosis diabetesmellitusexposure to seawaterprobable woundinfectionconsumption of rawseafoodseawater exposureinsect bite woundinfectionminor abrasionexposed to seawaterseawater exposuresurgery antibioticsdiedtaiwanseawater exposure mcirrhosissurgery antibioticssurvivedtaiwanseawater exposure mcopdsurgery antibioticssurvivedtaiwan mhiv hepatitis ccirrhosis mdiabetes mellitus michthyosis cellulitisnone mcopd chronic constructive pulmonary diseasesurgical debridement daptomycin levofloxacinsurviveditalyo137surgical debridement piperacillintazobactamfosfomycinsurgical debridement piperacillintazobactamtigecycline metronidazolesurgical debridementpenicillin gentamicin metronidazolesurvived austriadiedaustriaseawater exposuresurvived croatia o8seawater exposurev cholerae infections as patients with novcbacteremia require antibiotic treatment for at least month we administered ceftriaxone and minocycline for month necrotizing softtissue infectionscaused by novc are more lethal than those causedby v vulnificus to conclude we treated a woman with necrotizingfasciitis and septic shock caused by novc this caseillustrates that early fasciotomy and debridement arenecessary forsevere necrotizing fasciitis caused bynovc and prolonged intensive care may be requiredafter surgeryo139 vibrio cholerae ntprobnp nterminal probrain natriuretic peptidepmxdhp polymyxin b direct hemoperfusionacknowledgementsnoneauthors contributionskt tu tw kn and ku treated the patient kt tu hf reviewed the literatureand mainly wrote this report kn tw ku reviewed the literature andmodified this paper based on specialty orthopedics emergency departmentinfectious disease all authors have read and approved the manuscriptfundingnoneabbreviationsbun blood urea nitrogen fdp fibrinogen degradation productsgnr gramnegative rod mri magnetic resonance imaging novc nono1availability of data and materialsall data are included in this published 0ctsuruta bmc infectious diseases page of ethics approval and consent to participatethis case report was approved by the ethics review committee atminaminara general hospital and was conducted in accordance with thedeclaration of helsinki consent for participation is not applicableconsent for publicationwritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images a copy of the writtenconsent is available for review by the editor of this journalcompeting intereststhe authors declare that they have no competing interestsauthor details1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japan 2orthopedic departmentminaminara general hospital nara japan 3infectious diseases departmentminaminara general hospital nara japan 4department of emergency andcritical care medicine nara medical university nara japanreceived march accepted august referencesgardner ad venkatraman kv the antigens of the cholera group of vibriosj hyg lond hirk s huhulescu s allerberger f lepuschitz s rehak s weil s necrotizing fasciitis due to vibrio cholerae nono1nono139 after exposureto austrian bathing sites wien klin wochenschr dobroviÄ k rudman f ottaviani d crnek sÅ¡ leoni f Å¡krlin j a rare case ofnecrotizing fasciitis caused by vibrio cholerae o8 in an immunocompetentpatient wien klin wochenschr jain akc varma ak mangalanandan kh kumar h bal a surgical outcome ofnecrotizing fasciitis in diabetic lower limbs j diab foot comp ikeda t kanehara s ohtani t furukawa f endotoxin shock due to vibriovulnificus infection eur j dermatol su ba tang hj wang yy liu yc ko wc liu cy in vitro antimicrobialeffect of cefazolin and cefotaxime combined with minocycline against vibriocholerae nono1 nono139 j microbiol immunol infect tsai yh huang tj hsu rww weng yj hsu wh huang kc necrotizing softtissue infections and primary sepsis caused by vibriovulnificus and vibrio cholerae nono1 j trauma publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations morris jg nono group vibrio cholerae a look at the epidemiology of anoccasional pathogen epidemiol rev li m shimada t morris jg jr sulakvelidze a sozhamannan s evidence forthe emergence of nono1 and nono139 vibrio cholerae strains withpathogenic potential by exchange of oantigen biosynthesis regions infectimmun dalsgaard a albert mj taylor dn shimada t meza r serichantalergs o characterization of vibrio cholerae nono1 serogroups obtained froman outbreak of diarrhea in lima peru j clin microbiol isaacmárquez ap lezamadávila cm eslavacampos c navarroocaña acraviotoquintana a serotypes of vibrio cholerae nono1 isolated fromwater supplies for human consumption in campeche mexico and theirantibiotic susceptibility pattern mem inst oswaldo cruz hughes jm hollis dg gangarosa ej weaver re noncholera vibrioinfections in the united states clinical epidemiologic and laboratoryfeatures ann intern med deshayes s daurel c cattoir v parienti jj quilici ml de la blanchardière anono1 nono139 vibrio cholerae bacteraemia case report and literaturereview springerplus mubarak sj owen ca doubleincision fasciotomy of the leg fordecompression in compartment syndromes j bone joint surg am wagner pd evans sd dunlap j ballonlanda g necrotizing fasciitis andseptic shock caused by vibrio cholerae acquired in san diego californiawest j med ko w chuang y huang g hsu sy infections due to nono1 vibrio choleraein southern taiwan predominance in cirrhotic patients clin infect dis cheng nc tsai jl kuo ys hsueh pr bacteremic necrotizing fasciitis causedby vibrio cholerae serogroup o56 in a patient with liver cirrhosis j formosmed assoc tsai yh hsu rww huang kc chen ch cheng cc peng kt systemicvibrio infection presenting as necrotizing fasciitis and sepsis a series ofthirteen cases j bone joint surg am changchien ch bacteraemic necrotizing fasciitis with compartmentsyndrome caused by nono1 vibrio cholerae j plast reconstr aesthetic surg maraki s christidou a anastasaki m scoulica e nono1 nono139 vibriocholerae bacteremic skin and soft tissue infections infect dis lond ottaviani d leoni f rocchegiani e canonico c masini l pianetti a unusual case of necrotizing fasciitis caused by vibrio cholerae o137 j clinmicrobiol 0c"
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"melatonin is a sleepregulating hormone created by the pineal glandand is released at night it has been found to have biological activityin almost all living anisms including plants animals and microbes itcan quickly enter cells through the bilipid bilayer and exhibit scavenging activity towards oxygen free radicals as well as antioxidant properties due to its low molecular weight and amphiphilic nature peripheral tissues have been found to show a high afï¬nity towardsthis hormone melatonin and hence act on receptors and bindingsites studies reported by tan show that melatonin can alsobe produced in the mitochondria and hence tissue melatonin levelsare more than that of serum levels hence can be used as a moleculethat targets mitochondria the main physicochemical and biologicalproperties of melatonin are sleepinducing effects antioxidant behavior [] antiammatory activity [] antiapoptotic effects and neuroprotective effects it also regulates various corresponding authoremail address renjithsbcollegeacin r thomas101016jmolliq2020114082 elsevier bv all rights reservedphysiological functions of the brain as melatonin can diffuse quicklythrough the bloodbrain barrier it is effectively used in the treatmentof brain injuries rapid eyemovement sleepbehavior disorder patients are managed by a combination treatment of melatoninand clonazepam [] the sensing of bacteria through tolllikereceptor4 and regulation of bacteria through altered goblet cells andantimicrobial peptides are all involved in the anticolitic effects of melatonin in ammatory bowel disease melatonin is involved in theaging process growth towards puberty and modulation of blood pressure this versatile compound blocks proangiogenic andantiangiogenic effects caused by docetaxel and vinorelbine which areantitumor drugs and it enhances their tumorï¬ghting behavior this molecule can modify the redox state of the rat pancreaticstellate cellmelatonin is an endogenous hormone that is involved in circadianrhythm control it is inexpensive and safe as it has a significant effectas an antioxidant and antiammatory melatonin chemically nacetyl5methoxytryptamine is a tryptophan derivative has multiplephysiological effects and can be used to treat many diseases related tovirus infections especially respiratory diseases in covid19 patientswith digestive complications melatonin has positive effects 0cn alzaqri of molecular liquids melatonin can thus be used as an adjunctive or even as a regular therapyas no antiviral treatment is currently available electrochemical measurements of melatonin overï¬ow demonstrate that melatonin secretiondecreases with age melatonin treatments result in the enhancement of essential oil production in salvia species in the context of the recent covid pandemic melatonin can beresearched as a potential molecule to control the dangerous effect ofthis disease rising patients' tolerance and decreasing the mortality infatal virus infections would control the innate immune response and reduce ammation during this period melatonin is a molecule with respective properties as it decreases the overreaction of the innateimmune response and overshoots ammation but also facilitatesadaptive immune function even though melatonin is a critical biomolecule few works havebeen reported on the electronic structure and reactivity of this moleculeexcept for a preliminary work reported by turjanski and coworkers in using semiempirical methods in this manuscript we describe a detailed investigation into the quantum mechanical propertiesof melatonin its spectral features reactivity preferences and the resultsof docking studies with three known structural protein receptors of thenovel coronavirus2 we found that melatonin docks strongly with thethree proteins we hypothesise that this compound can be used as anadjuvant medicine for the treatment of covid19 also significant restby a person peacefully sleeping in dark surroundings will enhance theproduction of this hormone which could help in the management ofcurrent patients or as a preventive measure in the vulnerablepopulation material and methodsthe melatonin molecule was optimised using the gaussian09 software package with the dftb3lyp functional and the 6311g 2dp basis set b3lyp is a commonly used functional and 63112dpbasis set is mediumsized basis set with diffused functions over heavyatoms and polarization functions to bring accuracy we performed frequency calculations to ensure that no imaginary frequency exists suchthat the geometry determined would correspond to a global minimumfor reaching the optimised geometry we used the same geometry for calculating frontier molecular analysis natural bonding orbitals and nonlinear optical studies for uvvisible spectrum simulation we usedtimedependent density functional theory tddft with longrangecorrected camb3lyp functionals with 6311g 2dp as the basis setbecause electronic transitions are timedependent phenomena tddftcalculations are done using the optimised geometry obtained fromb3lyp6311g2dp simulations the frontier molecular orbitals wereviewed from the checkpoint ï¬le generated during the optimisation calculations a wavefunction ï¬le was generated during a single point groundstate calculation job using which the subsequent analysis performedthe melatonin molecule has more than two reaction sites for examplemethoxy carbonylamide and purine ring reaction sites of melatonincalculated using the multiwavefunction software for calculatingtotal electrostatic potential average localised ionization energies and noncovalent interactions as it is reported that melatonincan be used as an adjuvant therapeutic material to ï¬ght covid19 we decided to dock the molecule with three ncov2019 protein's rcsb site melatonin is effective in critical care patients by decreasing vessel permeability anxiety use of sedation and increasing the quality ofsleep which may also be beneï¬cial to covid19 patients for improvedclinical outcome melatonin especially has a high health proï¬le significant data indicate that melatonin reduces virusrelated diseases and willpossibly also be effective in patients with covid19 the target proteinswere downloaded cleaned removed alien atoms and molecules andthen used for docking the energy received from the swissdock software and the score values received from patchdock as well as thedocked results collected from biodiscovery studio software arepresented results and discussion geometry of melatoninthe geometry of the molecule explains its rigid structure thestructure of melatonin can be explained based on its physical parametersof bond lengths and bond angles between important atoms or groups asshown in fig the bond lengths of and arebonds of 1c14n 7c14n and 12h14n respectively and the corresponding bond angles are1090343° ° and ° for 1c14n7c 1c14n12h and 7c14n12h respectively the bond angle of° for 4c25o26c having bond lengths of and for 4c\\\\25o and 25o\\\\26c respectively the bond lengths of 18c21n21n22h 21n23c 23c\\\\24o and 23c\\\\30c are and respectively with the corresponding bondangles of ° ° ° ° ° and° for 18c21c22h 18c21c23c 22h22n23c 21n23c24o21n23c30c and 24o23c30c respectively frontier molecular orbital fmo properties of melatoninthe frontier molecular orbitals are highly reactive orbitals of othermolecules and some chemical descriptors are shown in table the higher occupied molecular orbital homo lower unoccupied molecular orbital lumo and energy gap of melatonin are and kcalmol respectively the energygap is significant indicating that the molecule is inherently stable theionization energy electron afï¬nity hardness softness chemical potential electronegativity electrophilicity index and nucleophilicity indexof melatonin are and kcalmol respectively interactionof the melatonin with the biological target can be explained by the softness value the softness value is high kcalmol indicatingthat the molecule can positively interact with biological systems andshow the desired effect electron transition study and excitedstate properties of melatonin insolutionthe electron transition study explains electrontransfer excitedstates we used the tddft formalism using camb3lyp functionalsand 6311g 2dp basis sets in an implicit solvation atmosphere ofmethanol using the iefpcm model as transmission occurs some energyis also emitted melatonin electron transitions to homo having a pyrrole ring and oxygen in methoxy homo1 over the pyrrole ring andethyl carbons and homo2 over acetamide oxygen nitrogen andethyl carbons with energies of and ev respectively melatonin electron transitions to lumo which is over the pyrrolering lumo which is over the pyrrole ring oxygen in methoxyethyl carbons and acetamide nitrogen and carbon and lumo2 having acetamide carbons acetamide nitrogen and carbons with energiesof and ev respectively the electronic spectral datausing tddft simulations indicate a significant λmax of nm in amethanol solvent the transitions are due to the movement of electronsfrom homo1 to lumo and homo to lumo with an oscillator strength of the electronic transitions are due to chargetransfer transitions from one region of the molecule to another whichindicates its inherent stability due to electronic excitations nonlinear optical behavior of melatoninscientists and technologists working in the molecular electronics ï¬eldare continuously searching for compounds with substantial nonlinearoptical nlo activity such compounds ï¬nd immense application in electronic displays surveillance equipment and consumer electronic gadgetscomputationally the ability of a molecule to act as an nlo material can be 0cn alzaqri of molecular liquids fig geometry of melatonindetermined from the polarizability and hyperpolarizability data []the nlo properties of melatonin are shown in table this is an essentialbehavior of melatonin that has a light absorption nature movement ofelectrons or protons as compared with a standard nlo material such asurea the dipole moment of melatonin is d which is times greater than urea hyperpolarizability mean polarizabilityand anisotropy of the polarizability of melatonin are and esu and which are and times greater than urea respectively the compound is not centrosymmetric hence generates secondorder spherical harmonics and betahyperpolarizability functions this compound can hence be used as an anic nonlinear optically active substance in anic electronicappliances nature of nbo study of melatonina molecule especially one with profound biological activity may havemany intramolecular electron delocalisation and hyperconjugativestabilisation regions natural bond orbital analysis which is a quantummechanical method is useful for this type of study the molecular orbitaltable frontier molecular orbital properties for melatoninchemical descriptorshomolumoionization energy i Éhomo homoelectron afï¬nity a Élumo lumoenergy gap homo lumoglobal hardness η i a global softness s ηchemical potential μ i a electronegativity Ï Î¼electrophilicity index Ï Î¼2 2ηnucleophilicity index n Ïenergy in kcalmolproperties of melatonin for the occupancy of the natural orbitals wereperformed by the nbo suite embedded in the gaussian softwarefrom donorbonding orbital Ï c1c2 with occupancy is toacceptor antibonding orbitals Ï c3c4 Ï c5c6 and Ï c7c8exhibiting the transition the energies are and kcalmol respectively from Ï c3c4 with occupancy is to Ï c5c6 and the rydberg orbital r c30 with the energies are and kcalmol respectively from Ï c5c6 having an occupancy is to Ï c3c4 r and r h33 with the energies are and kcalmol respectively from Ï n21c23 has occupancy to rydberg orbital r c30 and r h33 with the energies are and kcalmol respectively from Ï c23o24 having the occupancy is to Ï c1c2 Ï c2c3 r c23 and r h33 withthe energies are and kcalmol respectivelyfrom Ï c23c30 has occupancy is to r h17 r c18 rh19 r c23 r o24 r c30 r h33 Ï c1c2 Ï c1c6Ï c2c3 Ï c26h27 Ï c30h31 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectively from Ï o25c26 having occupancy is to r c30 andÏ c1c2 with the energies are and kcalmol respectivelyfrom Ï c26h27 to Ï c1c2 having the energy kcalmol withthe occupancy is from Ï c30h31 with the occupancy is to r h17 r c18 r h19 r c23 r o24 r c30 rh33 Ï c1c2 Ï c2c3 Ï c23o24 Ï c30h31 and Ïc30h32 having the energies are and kcalmol respectively from Ï c30h32 with occupancy is to r h17 rc18 r h19 r c23 r o24 r c30 r h33 Ï c1c2 Ïc2c3 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectively and from Ï c30h33 with the occupancy is to r h17 r c18 r c19 r c23 r c24 ro25 r c26 r h28 r c30 r h33 Ï c1c2 Ï c1c6Ï c2c3 Ï c2c4 Ï c18h19 Ï c23o24 Ï c26h27 Ï 0cn alzaqri of molecular liquids table nonlinear optics property for melatoninnonlinear propertydipole moment μhyperpolarizability βmean polarizability α0anisotropy of the polarizability δαmelatonin d esu esu esuurea d esu esu esucomparison of melatonin with urea times greater than urea times greater than urea times greater than urea times greater than ureac30c31 Ï c30h32 and Ï c30h33 having the energies are and kcalmol respectivelyfrom core bonding orbital c c23 which has the occupancy electrons move to antibonding r c23 r c30 and r h33 withthe transition energies are and kcalmol respectively from c c23 with the occupancy is to r h33 Ï c2c8 and Ï c26h28 having the energies are and kcalmol respectively from c o24 to Ï c26h29 has the energy is kcalmol with occupancy is and from c c30 having the occupancy is to r h17 r c23 r c30 r h33 Ïc2c8 Ï c26h28 Ï c30h31 and Ï c30h32 with the energies are and kcalmol respectively from lone pair orbital n n14 with the occupancy is to Ï c7c8 with the energy kcalmol from nn21 has the occupancy is to Ï c23o24 having the energy kcalmol and from n o24 having the occupancy is to rc23 r c30 r h33 Ï c1c2 and Ï c2c3 with the energiesare and kcalmol respectively fromantibonding orbital Ï c1c2 having the occupancy to rc30 and Ï c2c3 with the energies are and kcalmolrespectively from Ï c5c6 has occupancy is to r c30 withthe energy is kcalmol and from Ï c23o24 has occupancy is to r c30 with the energy is kcalmol the inherentstabilisation offrom the series ofhyperconjugative interactions presented above these interactions canalso be between the melatonin and the surrounding solvent moleculeswhich reveals its stabilisation in biological medium and also betweenthe molecule and the target proteins used in the dockingthe molecule is evident total electrostatic potentials esp and average localised ionization energy alie of melatoninthe electrostatic potential explains how reactive sites canundergo nucleophilic or electrophilic addition or substitution reactionsof melatonin shown in fig within bohr and a color changefrom blue to red indicates charges on elements from to the blue color appears in both methoxyoxygen and acetamideoxygen these are electronrich sites and electrophiles can quickly attack them the red color appears on all of the hydrogens these areelectronpoor sites and nucleophiles can quickly attack themthe alie clariï¬es the stability of any molecule based on saturatedand unsaturated bond electron movements which are localised ordelocalised the number of the resonance structure is proportionalto the stability of the molecule the alie of melatonin shown in fig iswithin the range of ± bohr color is from indigo to red and the numerical value is from to the blue color of protons in themethoxy group three protons in the sixmembered ring in the indolegroup methyl protons and both adjacent carbons in the acetamidegroup are all sites that act as electrophiles the red color ofacetamidecarbon conjugated carbon with oxygen atoms and bothacetamideamide and methoxy groups are all sites that act as nucleophiles these blue and red regions represent saturated bonds thebluishgreen regions are on indole rings to methoxy carbons via oxygenand acetamide chains this indicates that delocalised electrons and unsaturated bonds lead to several resonance structures and explains thestability of melatonin the electrophilic and nucleophilic reactive centres identiï¬ed above interact with the covid virus proteins and providevarious electrostatic and noncovalent interactions and increases drugafï¬nity noncovalent interaction nci properties of melatoninnoncovalent interactions are a valuable biological property of molecules and are nonbonded directly but are bound by some forces suchas hydrogen bonding van der waals bonding andor steric constraintsnoncovalent interactions of melatonin are shown in fig plotted as agraph with energy plotted versus a reduced density gradient hydrogen bonds appear from to au between oxygenand protons from the acetamide group the van der waals force rangesfig electrostatic potentials of melatoninfig average localised ionization energy for melatonin 0cn alzaqri of molecular liquids fig noncovalent interactions of melatoninfrom to au between acetamideoxygen and its adjacentprotons in both methyl and methoxy groups the steric force rangesfrom to au between the indole ring the methyl groupand the carbonylamide group noncovalent interactions are a groupof interactions like hydrogen bond pistacking hydrophobic interactions van der waal's forces iondipole interactions and dipoledipoleinteractions responsible for the stabilisation of the molecule and thedocking between melatonin and the covid proteins molecular dockingmolecular docking is one of the essential functions of biologically active molecules this is the theoretical evidence to design the structureand reactivity relationship of a molecule at present the covid19 pandemic caused by a new strain of the coronavirus is creating havocthroughout the world we made efforts to dock the melatonin withthe three proteins isolated from the virus represented through thepdb id 6lu7 6m03 and 6w63 were deposited in the database as mentioned in the methodology sectionwith the rapid spread of the novel coronavirus globally the designof vaccines is of great importance sarscov2 is an enveloped nonsegmented and single stranded positive sense rna virus the bestdrug target among coronaviruses is the main protease mpro also called3cl protease this is a key coronavirus enzyme and plays a vitalrole in mediating viral replication and transcription it is identiï¬ed ashaving a mechanismbased inhibitor the main targeting protease protein pdb 6lu7 is widely studied a series of frontier molecular orbital based interaction analyseswere performed on the complex between the main protease ofcovid19 and the peptidelike inhibitor whose fundamental structure was obtained from the protein pdb 6lu7 anothertargeted protease protein in an apo form pdb 6m03 shows themost stable form after binding with the selected drug threonine residue with the help of several covalent bond interactionwith a kcalmol docking afï¬nity lasinavir brecanavirtelinavir rotigaptide 13bis2ethoxycarbonylchromon5yloxy2lysyloxypropane and pimelautide can be consideredas the main protease inhibitors of covid19 by docking them tothe binding cavities of apo pdb 6m03 and holo pdb 6lu7 another protease protein pdb 6w63 is a reversible inhibitorthe ï¬avonoid narcissoside is reported to have a high afï¬nity towards the protease protein pdb 6w63 according to moleculardocking studies thus these three protease proteins pdb 6lu7pdb 6m03 and pdb 6w63 can be included in the category ofnonstructural proteins in the structure of sarscov2 from table the result from swissdock explains the biological activity of melatonin with coronavirus proteins pdb id 6lu7 6m03and 6w63 in general the total δg is more than kcalmol isright active luckily melatonin has a total δg of and kcalmol with coronavirus2 proteins pdb id 6lu7 6m03 and6w63 respectively and the total δg is directly proportional to the fullï¬tness energy values which are and kcalmol respectively it can also be seen from this table theinterfull ï¬tness intrafull ï¬tness full solvent ï¬tness full surface ï¬tnessδg complex polar solvent δg complex nonpolar solvent δg proteinpolar solvent δg protein nonpolar solvent δg ligand polar solventδg ligand nonpolar solvent δg van der waals force and δg electricforce relationships between melatonin and coronavirus2 proteins asreferredthe result from patchdock are as follows the score values are and total surface interacting area and and the minimum atomic contact energies are and kcalmol for melatonin with coronavirus2 proteins pdb id 6lu7 6m03 and 6w63 respectively figs and s1show the skeletal structure and protein residue interactions betweenmelatonin and coronavirus2 protein pdb id 6lu7 6m03 and 6w63table explains what protein residues are interacting with melatoninand details the residue names labels hydrophobic values pka valuesaverage isotropic displacements secondary structures residue solventaccessibility sidechain solvent accessibility percent solvent accessibility and percent sidechain solvent accessibility values of coronavirus2proteinstable and fig s2 show the residue structure of the favorable nonbond interactions between melatonin and coronavirus2 proteinstable lists favorable nonbond interactions of 6lu7 having conventional hydrogen bonds carbonhydrogen bonds pidonor hydrogenbonds pisulfur and pialkyl with melatonin 6m03 has pisigmapipi tshaped and pialkyl with melatonin while 6w63 has pipi tshaped pialkyl and pialkyl with melatonin along with the bond distance from chemistry fig s2 and table show the residue structure 0cn alzaqri of molecular liquids table swissdock result for melatonin with coronovirus2 proteins pdb id 6lu7 6m03 and 6w63energysimple ï¬tnessfull ï¬tnessinter full ï¬tnessintra full ï¬tnesssolvent full ï¬tnesssurface full ï¬tnessextra full ï¬tnessδg complex solvent polarδg complex solvent nonpolarδg protein solvent polarδg protein solvent nonpolarδg ligand solvent polarδg ligand solvent nonpolarδg van der waals forceδg electric forcetotal δg6lu7 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol 6m03 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol6w63 kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmol kcalmolof the unfavorable nonbond interactions between melatonin and coronavirus2 proteins protein 6lu7 does not have any unfavorablestericinteractions protein 6m03 having three unfavorable nonbond interactions and protein 6w63 having unfavorable bumpnonbond interactions fig s2 and table show unsatisï¬ed bonds within melatonininteracting with coronavirus proteins when interacting protein 6lu7has one hydrogen donor and one oxygen acceptor protein 6m03 hastwo hydrogen donors and two oxygen acceptors and protein 6w63has one hydrogen donor and two oxygen acceptors with melatonin table shows noncovalent interactions between melatonin and coronavirus2 proteins hydrophobic groups of protein 6lu7 residues areahis41 aleu141 acys145 ahis164 amet165 and aglu166those of protein 6m03 residues are ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu167 and those ofprotein residues 6w63 are ahis41 acys44 amet49 aleu50 amet165 aglu166 aleu167 and agln189 with melatonin as shownin fig s2 and table the hydrophilic groups of protein 6lu7 residuesare ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189 those of protein 6m03 residues are ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189 andthose of protein 6w63 residues are ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192 with melatonin as shownin fig s3 and table neutral groups of protein 6lu7 residues are atyr54 agly143 and aser144 those of protein 6m03 residues are agly143 aser144 and apro168 and those of protein 6w63 residuesare apro52 atyr54 aarg188 and athr190 with melatonin asshown in fig s4 and table acidic groups of protein 6lu7 residuesare aglu166 and aasp187 that of protein 6m03 residue is aglu166 and protein 6w63 residues are aglu166 and aasp187 withmelatonin as shown in table and fig s5 basic group interactions ofprotein 6lu7 residues are ahis41 ahis164 ahis172 and aarg188those of protein 6m03 residues are ahis41 ahis163 ahis164 andahis172 and those of protein 6w63 residues are ahis41 ahis164and aarg188 as shown in table and fig s6 tan etal has shownthat melatonin and derivatives has excellent biological responses likeacting against oxidative stress and free radical scavenging []our studies show that melatonin molecule can interact with differentproteins present in the ncov19 virus and inhibit their proliferationthese results need further clinical follow up and could assist in the management of covid pneumonia conclusionswe conducted a detailed quantummechanical investigation of thehormone melatonin and regulation of the sleepwake cycle naturalbonding orbital studies revealed the intensity of several intramolecularinteractions the various frontier molecular orbital data explain the nature and physical parameters of melatonin and the nonlinear opticalproperties are compared with urea which is a standard materialfig skeletal structure of interactions between melatonin and 6lu7 a 6m03 b and 6w63 c coronavirus2 protein residues 0cn alzaqri of molecular liquids table interactions between melatonin and coronavirus2 protein residuesnamelabelhydrophobicitypkapdbidsavg isotropicdisplacementsecondarystructureresidue solventaccessibilitysidechain solventaccessibilitypercent solventaccessibilitypercent sidechainsolvent accessibility6m03 histidine6lu7 histidineahis41amet49methionineatyr54tyrosinealeu141leucineaasn142asparagineagly143glycineaser144serineacys145cysteineahis164histidinemethionineamet165glutamic acid aglu166ahis172histidineaasp187aspartic acidaarg188arginineglutamineagln189ahis41methionineamet49phenylalanine aphe140aleu141leucineaasn142asparagineglycineagly143aser144serineacys145cysteineahis163histidineahis164histidinemethionineamet165glutamic acid aglu166aleu167leucineapro168prolineahis172histidineagln189glutamine6w63 histidineahis41cysteineacys44methionineamet49leucinealeu50prolineapro52tyrosineatyr54histidineahis164methionineamet165glutamic acid aglu166aleu167leucineapro168prolineaasp187aspartic acidarginineaarg188agln189glutamineathr190threonineglutamineagln192helixturnhelixcoilturnturncoilturnsheetsheetsheetsheetcoilcoilcoilhelixhelixcoilcoilturnturncoilturnsheetsheetsheetsheetcoilturnsheetcoilhelixcoilcoilcoilcoilhelixsheetsheetsheetcoilturncoilcoilcoilcoilcoilwavefunction studies gave information about electrostatic potentialsaverage localised ionization and noncovalent interactions these datahelped to predict the reactivity and identify the active site of the reactivity of the molecule melatonin docks with novel coronavirus proteinsand shows a variety of interactions with an excellent docking scorewhich leads to inhibition of the virus proteins leading to its destructionhence clinicians can consider incorporating melatonin also in thecovid19 treatment regime after further studiestable favorable nonbond interactions between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63hydrogen bondhydrogen bondhydrogen bondhydrogen bond otherhydrophobichydrophobichydrophobichydrophobichydrogen bondhydrophobichydrophobichydrophobicconventional hydrogen bondconventional hydrogen bondcarbon hydrogen bondpidonor hydrogen bond pisulfurpialkylpisigmapipi tshapedpialkylconventional hydrogen bondpipi tshapedpialkylpialkylaglu166nunk0hunk0hacys145sgunk0amet165caahis41unk0unk0hahis41unk0unk0hdonorhdonorhdonorhdonor sulfurpiorbitalschpiorbitalspiorbitalshdonorpiorbitalspiorbitalspiorbitalsunk0oahis164oaglu166ounk0acys145unk0unk0acys145aglu166ounk0amet165amet165hacceptorhacceptorhacceptorpiorbitals piorbitalsalkylpiorbitalspiorbitalsalkylhacceptorpiorbitalsalkylalkyl 0cn alzaqri of molecular liquids table unfavorable nonbond between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63nilunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorable bumpunfavorable bumpunfavorable bump carbon hydrogen bondunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpacys145sgaglu166ounk0hagln189caagln189caagln189cbagln189cgagln189cgagln189cgagln189cgagln189cdagln189cdagln189cdagln189cdagln189oe1agln189oe1agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189haagln189haagln189haagln189hg1agln189hg2agln189hg2agln189he21agln189he21agln189he21agln189he21agln189he22agln189he22agln189he22stericstericsteric hdonorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericunk0cunk0caglu166ounk0cunk0hunk0cunk0cunk0hunk0nunk0hunk0cunk0cunk0hunk0hunk0cunk0hunk0nunk0cunk0ounk0cunk0hunk0hunk0hunk0cunk0hunk0hunk0hunk0cunk0hunk0nunk0cunk0cunk0hunk0cunk0cunk0hstericstericsteric hacceptorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericsterictable unsatisï¬ed bonds in melatonin with coronavirus2 proteinspdb ids6lu76m036w63nameunk0hunk0ounk0hunk0hunk0ounk0ounk0hunk0ounk0oatomunsatisï¬ed typehohhoohoodonoracceptordonordonoracceptoracceptordonoracceptoracceptorrenjith thomas conceptualization formal analysis investigation methodology project administration resources softwaresupervision validation visualization writing review editingdeclaration of competing interestthe authors declare that they have no known competing ï¬nancialinterests or personal relationships that could have appeared to uence the work reported in this paperacknowledgementsresearchers supporting project number rsp202078 king saudcredit authorship contribution statementuniversity riyadh saudi arabianabil alzaqri cenceptualization funding acquisition tpooventhiran investegation methodology original draft alialsalme investegation original draft ismail warad resourcesreview methods athira m john methodology writing draftappendix a supplementary datasupplementary data to this article can be found online at 101016jmolliq2020114082table noncovalent interactions between melatonin and coronavirus2 proteinshydrophobicityhydrophilicityneutral groupacidic groupbasic grouppdbids6lu7ahis41 aleu141 acys145 ahis164 amet165 and aglu1666m03 ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu1676w63 ahis41 acys44 amet49 aleu50 amet165aglu166 aleu167 and agln189ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192atyr54 agly143 andaser144agly143 aser144 andapro168apro52 atyr54 aarg188 and athr190aglu166 andaasp187aglu166aglu166and aasp187ahis41 ahis164 ahis172 and aarg188ahis41 ahis163 ahis164 and ahis172ahis41 ahis164 andaarg188
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" african americans aa are at high risk for colorectal cancer crc studies report a increasein crc risk with physical inactivity obesity and metabolic syndrome activation of the wntcatenin ctnnb1signaling pathway plays a critical role in colorectal carcinogenesis accumulating evidence also indicates a role ofwntctnnb1 signaling in obesity and metabolic diseasesaim to examine the association between obesity catenin expression and colonic lesions in african americansmethods we reviewed the pathology records of colorectal specimens from to crcs advanced adenomas and normal colon tissues tissue microarrays tma were constructed from these samplesimmunohistochemistry ihc for ctnnb1 catenin clone catenin1 was performed on the constructed tmasthe ihc results were evaluated by pathologists and the nuclear intensity staining was scored from to bmisex age location of the lesion and other demographic data were obtainedresults positive nuclear staining in normal advanced adenoma and crc was and respectively p crc was asso ciated with positive status for nuclear ctnnb1 intensity adjusted or 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenoma nuclearstaining percentage has a fair diagnostic ability for crc with an auc of 95ci overweightobese patients bmi did not show a significant difference in p nuclear ctnnb1 staining positive in normal weight vs positive in overweightobese the association between nuclear intensityand crc was not different between normal and overweight patients p for interaction the positive nuclearctnnb1status in crc stage iii and iv of all crc was not different from stage i and ii vs respectively p continued on next page correspondence b_shokranihowardedu hashktorabhowardedu1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cshokrani bmc gastroenterology page of continued from previous page in our study advanced adenoma and crc were associated with activation of catenin in physically fitoverweight and obese patients thus obesity and wntcatenin pathway seem to be independent in africanamerican patients wntcatenin signaling pathway has a potential to be used as an effector in coloncarcinogenic transformation whether or not bmi is a modifier of this pathway needs to be investigated furtherkeywords catenin colorectal cancer advanced adenoma african americans colorectal cancer crc is one of the most commoncancers in the industrialized world lifestyle and epidemiological factors associated with an increased risk ofcrc include physical inactivity obesity and metabolicsyndrome in the united states approximately twothirds of the adult population are overweight or obesewhich represents a putative risk factor for multiple target an malignancies including crc there is evidence to suggest that excess adiposity is associated with up to greater risk of crc comparedwith normal weight individuals and that physical activity may decrease colorectal cancer risk althoughexcessive accumulation of white adipose tissue wat isthe key feature of adiposity obesity is clinically definedby a bmi over kgm2 which does not take fat contentinto account it is also known that most crcs arise froma genetic and morphological adenoma to carcinomatransition also it is widely accepted that both crcsand colorectal adenomas cras share similar etiologicalcauses which explains why cras which are amongstthe mostfindings in all crcscreening participants are present in more than ofgeneral asymptomatic populations consequentlyrisk algorithms have been applied to use bmi as a predictor variable to stratify individuals according to theirrisk of colorectal neoplasia however the underlyingmechanisms that might explain the association and themagnitude ofthe connection between excess bodyweight and crc remain unclearfrequent pathologicalin the obesitycancer relationship multiple biologicalprocesses including insulininsulinlike growth factorigf1 insulin resistance sexual hormones estrogensand proinflammatory cytokines tnfα il6 and crpactively participate all these elements create a favorable environment for carcinogenesis and a decrease inapoptosisas a separate molecular pathway activation of thewnt signaling pathway plays a critical role in colorectalcarcinogenesis wnt ligands are a family of proteinsfor normal cell development that are importantcatenin ctnnb1 is a major mediator of the wntpathway that is traditionally classified into canonical catenindependent and noncanonical cateninindependent wnt canonical pathway utilizes a group ofcell surface receptors called frizzled frz to activateseveral pathways the most important one involving catenin and apc in the absence of wnt signalingapc causes degradation of catenin preventing its accumulation in the cytoplasm by forming a complex withcatenin which leads to the phosphorylation and eventually destruction of catenin by the proteasome signaling by wnt blocks this process allowing cateninto migrate from the cytoplasm to the nucleus once inthe nucleus catenin upregulates cmyc cyclin d1and other genes which increase cellular proliferation therefore continuous wnt signaling can be seenin cells with loss of apc metabolic syndromeassociated conditionssuch asobesity and type ii diabetes are influenced by geneticand functional variations in the wnt signaling pathway wnt signaling when activated represses the terminal differentiation during adipogenesis whereby preadipocytes take on the characteristics of mature adipocytes a cascade of transcriptional events like the induction of catenin ensues which in turn inducesenhancer binding proteinα cebpa and peroxisomeproliferatoractivated receptorÎ pparg the excessive accumulation of wat features adiposity butobesity does not take fat content into account recently genetic factors linked to fat mass and adipositywere reported to be associated with increased obesityrisk in young obese individuals wholeexome sequencing revealed rare gainoffunction mutations inctnnb1catenin the cateninregulated transcription of an adipocytederived chemokine calledserum amyloid a3 saa3 leads to the formation of a catenintcf complex in mature adipocytes that promotes the proliferation of preadipocytes in wat andthereby increases obesity and the risk for metabolic syndrome other data also suggest that obesity and lack ofphysical activity are associated with a higher risk forcolorectal cancer [ ] these findings have importantimplications especially in the obese and physically inactive african american population that may haveunderlying predisposing mutations to colorectal cancer the aim of this study is to assess the catenin expression profile in colorectal premalignant and malignant lesions in correlation with obesity as determined by 0cshokrani bmc gastroenterology page of body mass index bmi or waist circumference wc inafrican american populationmethodspatients and clinical datacolorectaltissue samples submitted to the surgicalpathology laboratory at howard university hospitalfrom january to december were retrieved from the pathology archive system powerpath¢a total of samples were included in the study consisting of tissue samples from crc n advancedadenoma n and normal colon n patientsdata included age sex height weight and waist circumference body mass index bmi was calculated in thestudy table the protocol of this study was approvedby the howard university institutional review boardirbtissue microarray tma constructionhematoxylineosin stained slides he slides weremade from paraffinembedded blocks the he slideswere reviewed by two pathologists to confirm the pathological diagnosis and to mark areas of interest multipleareas from more than one block were marked to ensurea good representability of the sample on the tma fivetma paraffin blocks each containing cores of mmin diameter each and mm distance from each otherwere constructed tissuespecific and an system controls were included in each tmaimmunohistochemical ihc analysis of ctnnb1 cateninthe constructed tmas were stained for catenin theimmunostaining was carried out as follows dako monoclonal mouse antihuman betacatenin cateninc1intended for laboratory application to identify qualitatively by light microscopy catenin positive cells in normal and neoplastic tissues was used at a dilution of using the envision dab code k4006 detectionsystem the deparaffinized tmas were treated prior tothe ihc staining procedure target retrievalinvolvedimmersion of tissue sections in a preheated buffer solution and maintaining heat in a water bath °cfor greater adherence of tissue sections to glass slidessilanized slides dako code s3003 were used targetretrieval solution code s1700 or 10x concentratecode s1699 using a 20min heating protocol was usedthe cellular staining pattern of anticatenin is mainlymembranous especially at the cellcell boundaries positive nuclear staining and diffuse cytoplasmic staining arealso reported in cancer cells fig evaluation and assessment of the catenin expressiontwo pathologists interpreted the ihc slides cateninexpression status was assessed based on the pattern ofstaining nuclear cytoplasmic and membranous intensity to and percentage of staining to the staining would be considered negative if there wasweak or no nuclear expression or positive if there wasmoderate or strong nuclear expressionstatistical analysisdistribution of continuous and categorical variables weretested by kruskalwallis and chisquare test betweendifferent groups respectively we used logistic regression analysis to test association between the staining andrisk of crc after adjusting for age and gender areaunder the curve auc was calculated for variables withsignificant association with crc using receiver operative characteristics curve all statistical analyses wereperformed by stata statacorp college station txresultsepidemiological characteristics and bmi in normaladvanced adenoma and crcthe bmi was calculated for individual patients and normal subjects as represented in table crc patientswere older p while our healthy normal population was mostly overweight higher bmi was moreclosely associated with advanced adenoma and crchowever the differences were not significant table advanced adenomas and crcs were associated withpositive nuclear ctnnb1we assessed whether alterations in wntctnnb1 catenin signaling plays any role colon carcinogenesispositive catenin nuclear stains were seen in normaladvanced adenomas and crcs were and respectively p table based on the designationtable epidemiological characteristics and bmi in normal advanced adenoma and crc patientsage median iqrmale n bmi kgm2 median iqroverweight n normaln advanced adenoman crcn p value 0cshokrani bmc gastroenterology page of fig immunostain for catenin in three individuals normal a à advanced adenoma b à and cancer c d à and à respectively showing membranous staining in the normal cytoplasmic and membranous staining in adenoma with no evidence of nuclearexpression arrow showing lack of nuclear staining and nuclear and cytoplasmic staining in cancer arrow showing nuclear stainingof n intensity which is associated with higher risk ofcancer crcs were associated with positive status fornuclear ctnnb1 intensity age gender adjusted or 95ci p for positive nuclearstaining compared to noncrc samples normal or advanced adenoma fig nuclear staining percentagehas also a fair diagnostic ability for crc with an auc of 95ci table fig overweight and obese patients show a trend withpositive nuclear ctnnb1 expressionpositive nuclear ctnnb1 staining was in normalweight and in overweightobese bmi patientsthis difference pointed to trend that was not statisticallysignificantassociation between nuclear intensity and crc in normaland overweight patientsthe association between nuclear intensity and crcwas not statistically significant different between normal weight and overweight patientsinteraction tables and the positive nuclearctnnb1 status in crc stage iii and iv of allcrc was not different from stage i and ii vs respectively p p fortable catenin nuclear and cytoplasmic expression tabulated as intensity and percentage in normal advanced adenoma andcrcnormaln cnc intensity n intensity advanced adenoma n crcn c cytoplasmic n nuclearc intensity and n intensity mean intensity and aboveoverall p valuep value for advanced adenoma vs normalp value for crc vs other 0cshokrani bmc gastroenterology page of fig catenin nuclear and cytoplasmic expression in normal advanced adenoma and crcdiscussionone of the important risk factors in colorectal cancer isobesity [ ] catenin is an ecadherin binding proteinthat mediates cellcell adhesion and plays a role inthe canonical wnt signaling pathway that controls thecoordinated expansion and differentiation of the intestinal crypt stem cells degradation of catenin byphosphorylation followed by alteration of destructioncomplex apc gsk3 and axin results in inactivation if wnt pathway in our study we found thatwas associated with an increased adjusted or of 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenomathe gatekeeper gene apc is a negative regulator of catenin and is mutated in approximately of sporadic and hereditary colon cancers there are severalmutations that can cause an accumulation of cateninin tumor cells such as mutations of the apc gene pointmutations in gsk3 or mutations in catenin gene itself []our positive nuclear staining in crc and its association with the positive status for nuclear ctnnb1intensity compared to noncrc samples are in contrastto a study by brabletz which showed that catenin is localized in the cytoplasm and membrane ofthe tumor cells whereas in our study it was mainly concentrated in the cytoplasm and the nucleus they alsomentioned that there was positive nuclear staining at theinvasive front as catenin is involved in tumor progression such is not the case in our studyindeed evenwhen considering nuclear staining in our specimensthere was no statistically significant differences betweenstage iiiiv cases staining versus stages iii crc caseslevels of staining the fact that catenin is expressedearly in the african american specimens analyzed heremight partially explain the aggressive nature of crc inthis population in addition we showed that there is uniform membranous staining in normal and increasingcytoplasmic and nuclear staining in advanced adenomasand crcs this confirms that the decrease in membranous staining begins with dysplastic changes leading to atable association of bmi with catenin nuclear intensity in advanced adenoma casesadvanced adenoma with catenin expression n in intensity and no nuclear intensity n bmi median interquartileoverweight n nuclear intensity negativen nuclear intensity n p value 0cshokrani bmc gastroenterology page of table association of bmi with catenin nuclear intensity in crc casescrc with catenin expression with high nuclear intensity and without negativebmi median interquartileoverweight n nuclear intensity negativen nuclear intensity n p valueprogressive disappearance atcrcsthe membrane levelinas we mentioned above a major risk factor for crc isobesity which continues to expand as a pandemicworldwide the american cancer society cancerprevention study ii states that there is an increased incidence of crc esophageal adenocarcinoma and othercancers with obesity in our study we showed that of advanced adenoma patients and of crc patients were overweight with bmi in comparison toadvanced adenoma the percentage was lower in cancerperhaps due to the late stage of cancer and weight lossin the interim table there are several mechanismsby which obesity is believed to promote crc this includes increase in leptin levels that cause an increase ingrowth and proliferation of colon cancer cells altered adipokine levels altered gut microbiome apartfrom increased steroid hormones and growth factors insulin is however the established biochemical linkand the main pathway involved is pik3aktmtorpathway elevated igf1 and insulin act through the insulin receptors and phosphotidylinositide3 kinase in addition to the above findings we also found thatoverweight and obese patients bmi did not showa significantly increased expression p of nuclearctnnb1 positive in normal weight vs positive in overweightobese morikawa found that inobesepositivity wasnuclear ctnnb1patientsassociated with significantly better cancerspecific survival suggesting that wnt signaling acts as a switch andwhen it is on adipogenesis is repressed kennell demonstrated that activated frz1 frizzled promotes catenin stabilityinhibits apoptosis and adipogenesisross also showed that wnt signaling acts as a molecular switch that controls adipogenesis upregulationof wnt signaling maintains preadipocytes in an undifferentiated state and when wnt signaling is prevented theydifferentiate into adipocytes [ ]although in our study there was no association between nuclear intensity and crc between normal andoverweight patients p for interaction there is accumulating evidence to show that the state of chronicinflammation incited by obesity might play a role in promoting colorectal carcinogenesis [ ] of the manymarkers tnfα is important [ ] as it activateswnt signaling through the induction of gsk3 phosphorylation resulting in increased nuclear localization ofcatenin in addition to tnfα other humoralagents associated with obesity might also be contributingto the activation of wnt signaling like il1 and adiponectin which is decreased in the obese state and is notan inflammatory cytokine that can modulate gsk3catenin signaling pathway although multiplemechanisms may be operating in parallel and contributing to the protumorigenic milieu wnt is a pivotaltumorigenic pathway aberrations of which isfig the putative relationship between obesity and colorectal cancer evolution pathways by cellular ctnnb1 status based on the data by thecurrent study our study suggests that there is no association between obesity and ctnnb1 expression 0cshokrani bmc gastroenterology page of important in the evolution of most sporadic crc insummary there is positive nuclear staining in crcs which was associated with the positive status fornuclear ctnnb1 intensity adjusted or 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenoma this shows that advanced adenomas and crcswere associated with activation of catenin in physically fit overweight and obese patients fig advanced adenoma and crc were associated with activation of catenin in physically fit overweight andobese patients thus participation of obesity and wntpathway seem to be independent crc factors in africanamerican patientsinflammationdriven activation ofwnt signaling as a potential pathway linking obesity tothe development of crc needs to be investigated furtherin the african american population this might provideinsights into the identification of new therapeutic targetsto reduce the burden of obesityassociated crcabbreviationscrc colorectal cancer wat white adipose tissue aa african americansacknowledgementsnot applicableauthors contributionsconceived and designed experiments bs ha performed experiments bsel ha hb analyzed data ha hb mn th aa and zs contributed reagentsmaterialsanalysis tools zs ha hb bs and el wrote and edited manuscriptbs and ha provided statistical analysis mn all authors have read andapproved the manuscriptfundingthis project was supported in part by grant from the national institute onminority health and health disparities of the national institutes of healthunder award numbersg12md007597 the funder had no role in designing or execution of thisstudyavailability of data and materialsall data generated or analyzed during this study are included in thispublished ethics approval and consent to participatethis retrospective and chart review study was conducted according to theworld medical association declaration of helsinki and was approved by theinternal review board of howard university since the chart review was donethrough unidentifiable approach no consent form needed for this studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests related to thismanuscriptauthor details1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usa 2division of pulmonary allergy and criticalcare medicine university of pittsburg pittsburg pa usareceived april accepted august referencesresearch iafco estimated cancer incidence mortality and prevalenceworlwide in lyon globocan haggar fa boushey rp colorectal cancer epidemiology incidencemortality survival and risk factors clin colon rectal surg oxentenko as body size and incident colorectal cancer a prospectivestudy of older women cancer prev res phila ma y obesity and risk of colorectal cancer a systematic review ofprospective studies plos one 201381e53916slattery ml physical activity and colorectal cancer sports med wong mc lam ty tsoi kk hirai hw chan vc ching jy chan fk sung jja validated tool to predict colorectal neoplasia and inform screening choicefor asymptomatic subjects gut sung jjy wong mcs lam tyt tsoi kkf chan vcw cheung w ching jyla modified colorectal screening score for prediction of advanced neoplasiaa prospective study of subjects jgastroenterolhepatol liu z dietinduced obesity elevates colonic tnfalpha in mice and isaccompanied by an activation of wnt signaling a mechanism for obesityassociated colorectal cancer j nutr biochem najdi r holcombe rf waterman ml wnt signaling and coloncarcinogenesis beyond apc j carcinog macdonald bt tamai k he x wntbetacatenin signaling componentsmechanisms and diseases dev cell denysenko t wntbetacatenin signaling pathway and downstreammodulators in low and highgrade glioma cancer genomics proteomics willert k nusse r betacatenin a key mediator of wnt signaling curr opingenet dev vella a camilleri m pharmacogenetics potential role in the treatment ofdiabetes and obesity expert opin pharmacother christodoulides c adipogenesis and wnt signalling trendsendocrinol metab world health anization [httpwwwwhointmediacentrefactsheetsfs311en] chen m lu p ma q cao y chen n li w zhao s chen b shi j sun yshen h sun l shen j liao q zhang y hong j gu w liu r ning g wangw wang j ctnnb1betacatenin dysfunction contributes to adiposity byregulating the crosstalk of mature adipocytes and preadipocytes sci adv20206eaax9605 morikawa t kuchiba a yamauchi m meyerhardt ja shima k nosho kchan at giovannucci e fuchs cs ogino s association of ctnnb1 betacatenin alterations body mass index and physical activity with survival inpatients with colorectal cancer jama morikawa t prospective analysis of body mass index physical activityand colorectal cancer risk associated with betacatenin ctnnb1 statuscancer res ashktorab h kupfer ss brim h carethers jm racial disparity ingastrointestinal cancer risk gastroenterology kaler p augenlicht l klampfer l activating mutations in betacatenin incolon cancer cells alter their interaction with macrophages the role of snailplos one 201279e45462enzo mv the wntbetacatenin pathway in human fibroticlikediseases and its eligibility as a therapeutic target mol cell ther stamos jl weis wi the betacatenin destruction complex cold springharb perspect biol 201351a007898 chung gg tissue microarray analysis of betacatenin in colorectalcancer shows nuclear phosphobetacatenin is associated with a betterprognosis clin cancer res miyoshi y activation of the betacatenin gene in primaryhepatocellular carcinomas by somatic alterations involving exon cancerres fukuchi t betacatenin mutation in carcinoma of the uterineendometrium cancer res brabletz t variable betacatenin expression in colorectal cancersindicates tumor progression driven by the tumor environment proc natlacad sci u s a 0cshokrani bmc gastroenterology page of berger na obesityassociated gastrointestinal tract cancer from beginningto end cancer calle ee the american cancer society cancer prevention study iinutrition cohort rationale study design and baseline characteristicscancer frezza ee wachtel ms chirivainternati m influence of obesity on the riskof developing colon cancer gut berger na obesity and cancer pathogenesis ann n y acad sci guo s insulin signaling resistance and the metabolic syndrome insightsfrom mouse models into disease mechanisms j endocrinol 20142202t1t23 ross se inhibition of adipogenesis by wnt signaling science kennell ja macdougald oa wnt signaling inhibits adipogenesis throughbetacatenindependent and independent mechanisms j biol chem liu z yingka y inflammation driven activation of wnt pathway a potentialmechanism responsible for obesity associated colorectal cancer obes resopen j tilg h moschen ar adipocytokines mediators linking adipose tissueinflammation and immunity nat rev immunol fischerposovszky p wabitsch m hochberg z endocrinology of adiposetissue an update horm metab res oguma k activated macrophages promote wnt signalling throughtumour necrosis factoralpha in gastric tumour cells embo j wang y adiponectin modulates the glycogen synthase kinase3betabetacatenin signaling pathway and attenuates mammary tumorigenesis ofmdamb231 cells in nude mice cancer res renehan ag roberts dl dive c obesity and cancer pathophysiologicaland biological mechanisms arch physiol biochem publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the eï¬cacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInï¬ammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the global Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationï¬stulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperï¬cialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of years old [] therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societyMoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more eï¬cient at reï¬ecting disease activity []Some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The eï¬cacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the ï¬rst and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study In another study published in AUC values of CI were reported for fecal calprotectin in thediagnosis of colorectal ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and speciï¬city [] Caviglia et al in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with Crohn's disease Asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE ï¬ndings anddiagnosis of Crohn's disease [] In another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren yearsChildren yearsAdultsOver years Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in Crohn's disease [] Given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the ï¬rst studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled Speciï¬cityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and speciï¬city In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modiï¬edBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpeciï¬city CD and UC CD and UC CD and UC and unclassiï¬ed68CD and UC CD and UC and unclassiï¬ed CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSreï¬dbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoeï¬cientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland Modiï¬edCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also conï¬rmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thediï¬culty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRoberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Roberts scoring system [] Theede et al also used themodiï¬ed Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 Sensitivity Speciï¬city andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes
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" natural orifice specimen extraction surgery is a novel technique of minimally invasive surgery thepurpose of this study was to compare the safety of laparoscopic anterior resection with natural orifice specimenextraction noselar and abdominal incision specimen extraction aiselar for sigmoid or rectum tumorsmethods medline pubmed embase central cochrane central register of controlled trials scopus andclinicaltrials databases were systematically searched for related s up to august the primary outcomesincluded postoperative complications overall postoperative complication incisionrelated complicationanastomotic fistula and severe complication and pathologic results lymph nodes harvested proximal resectionmargin and distal resection edge the statistical analysis was performed on stata softwareresults ten studies comprising patients were used for metaanalysis compared with aiselar noselar hadmore advantages in terms of overall postoperative complication odds ratio or ci to p incisionrelated complication or ci to p distal resection edge weighted meandifference wmd cm ci to cm p recovery of gastrointestinal function wmd day ci to day p pain scores in postoperative day wmd ci to p additional analgesics usage or ci to p and hospital stay wmd day ci to day p while the operation time of noselar was prolonged wmd min ci to min p the anastomotic fistula severe complication lymph nodes harvested proximalresection margin intraoperative blood loss and longterm outcomes in noselar were comparable with aiselars the safety of noselar was demonstrated and it could be an alternative to conventional surgery inlaparoscopic anterior resection for sigmoid and rectal tumors however further randomized and multicenter trials are requiredkeywords natural orifice specimen extraction nose laparoscopic anterior resection rectal tumor sigmoid tumor metaanalysis correspondence guangenyang163com1department of colorectal surgery hangzhou third hospital hangzhou zhejiang peoples republic of chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0che world of surgical oncology page of introductionover the past three decades laparoscopic surgery hasevolved incessantly especially in the field of colorectalsurgery it has been widely accepted by surgeons and patients in light of the better perioperative outcomes andanalogical longterm effectiveness compared with opensurgery for colorectal cancers []for conventional laparoscopic colorectal operation asmall laparotomy in the abdomen is required for specimen harvested and colorectal anastomosis because ofthe miniincision it causes many undesirable outcomessuch as incision pain wound infection scar and even incision hernia and the advantages of laparoscopic surgeryare reduced [] to minimize those drawbacks anovel surgical variant known as natural orifice specimenextraction nose surgery with the features of naturalorifice specimen extraction and totalintraperitonealanastomosis has been introduced and is becoming ahotspot [] some studies have reported the oncology and safety outcomes between nose surgery andconventional laparoscopic surgery are comparable [] and the nose surgery therefore is supposed tohave a progress of minimally invasive surgeryrecently some metaanalysis studies had comparednatural orifice specimen extraction nose with abdominal incision specimen extraction aise in laparoscopiccolorectum resection for the colorectal disease [ ]however colorectum resection comprises right hemicolectomyleft hemicolectomy and anterior resection procedures among those surgeries were largely different and the surgical procedures and the excision extension between sigmoid and rectum resection aresimilar in addition several studies compared laparoscopic anterior resection with natural orifice specimenextraction noselar with abdominal incision specimen extraction aiselar for sigmoid or rectum tureleased [] hence wemors wereconducted this metaanalysis to evaluate the safety ofnoselar in sigmoid and rectal tumorsrecentlymethodsstudy design and inclusion criteriathis metaanalysis followed the preferred reportingitems for systematic reviews and metaanalysis prisma statements the inclusion and selection criteria were determined before starting a literature searchonly when studies with fulltext on sigmoid or rectaltumors that compared noselar and aiselar andreported at least one of the endpoints of focus were retrieved and analyzed the most comprehensive researchwas recruited when overlapping researches was conducted by the same team no language restriction wasapplied conference s case reportsreviewsrobotic surgery and singleportwere not consideredlaparoscopic surgeryselection criteria conformed to the framework ofpico participant intervention comparison and outcome patients diagnosed with sigmoid or rectal tumorsbenign and malignant tumors requiring surgery wereincludedinterventions consisted of noselar andaiselar noselar was compared with aiselarin all eligible studies primary endpoint outcomes werepostoperative complications overall postoperative complication abdominal incisionrelated complication anastomotic leak and severe complication claviendindoclassification ¥ iii and pathologic results retrievedlymph nodes proximal resection edge distal resectionedge secondary outcomes included operation timeblood loss pain score numeric rating scale score additional analgesics gastrointestinalfunction recoveryhospitalization duration 5year diseasefree survivaldfs and 5year overall survival os search strategythe following databases had been searched up to august medline pubmed central cochrane central register of controlled trials embase scopus andclinicaltrials for a more accurate search the followingkeywords andor mesh terms were used sigmoid neoplasms rectal neoplasms colorectal neoplasmslaparoscopynatural orifice specimen extractiontransvaginal specimen extraction transanal specimenextraction and transrectal specimen extraction thespecific search strategies among databases existed differences the search strategy of pubmed was presented inadditional text reference s of the eligible studies were reviewed to find the potentially relevant studiesstudy selection and quality assessmentretrieved studies were independently assessed for relevance by reviewers changjian wang and jinmingchen by screening the title and in order to enhance sensitivity studies were removed only when bothreviewers excluded the study subsequently a fulltextassessment was performed on the initial screening included studies the risk of bias was assessed by thenewcastleottawa scale nos for observational studies and studies achieving five or more stars were eligible cochrane collaborations tool for assessing risk forbias was used for randomized controlled trials [ ]all discrepancies were discussed before a final decisionwas madedata collection and statistical analysisdata from the recruited studies were extracted by tworeviewers changjian wang and jinming chen andused formeananalysis outcome valuesfurther 0che world of surgical oncology page of standard deviation and median interquartile rangewere extracted from each study considering potentialheterogeneity among studies we pooled the results byusing a randomeffects model the weighted mean difference wmd and confidence intervals cis wereapplied for continuous variables and the odds ratioor and cis were used for dichotomous variablesthe continuous outcomes were adopted the inverse variance method and dichotomous outcomes were adoptedthe mantelhaenszel statistical method when a studymerely offered the outcomes with median and interquartile range an estimation based on formulas designed byhozo was performed if a study did not provide the hazard ratio hr and cis of 5year dfsorand os the methods presented by tierney wereused for data extraction from survival curves thechisquare test and isquared value were used for measuring heterogeneity and i2 p was definedas significant heterogeneity sensitivity analyses basedon nos score ¥ and the sample size of noselargroup ¥ were conducted to assess the potentialsource of heterogeneity and the robustness of the resultspublication bias was examined with a funnel plot andharbord test p was considered statistically significant the statistical analysis was performed onstata softwareresultsliterature selection and characteristicsthe initial database search identified s ofwhich were removed based on the title and assessment the rest of the literature were evaluated byfulltext assessment and studies were excludedcharacteristics of the excluded studies were presented inadditional table ten studies were finally included forfurther qualitative and quantitative synthesis [ ] all of these were retrospective studies the process of the search and selection wasshown in fig a total of patients were recruitedin those studies with patients in the noselargroup and patients in the aiselar group themain characteristics of studies and patients were presented in table and details were shown in additionalfig flow chart of studies included in the metaanalysis 0che world of surgical oncology page of sunasunasunasunasunasunarosunaiangavsunasunasunarorororomutcermutceridomgsimutceridomgsimutceridomgsimutceridomgsiralesaipurnsonerocsetisinemcepsnoitcartxenoitacolromutleamefeamlrednegmutceridomgsimutcermutcermutcerralesonpuralesaipuraeyaegaralesonpuralesaipuralesonpugnitnuocisetapcitrapngisedydutsinogerraeyydutsisedutsdeducnliehtfoscitsiretcarahcinamelbatevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorternapajlateadasihianhclateuhianhclategnianhclategnahzianhclateuohzianhclategnxiianhclateuliydutsaissurydutsevitcepsorternawatihbaruaslateevitcepsorterecnarflatetsonedevitcepsorterydutsydutsianhclategnawrnleacsawattoeltsacwensonnoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaegnarinademronoitavedidradnats±naemsadrocertondetropera 0che world of surgical oncology page of euavlpytienegoretehieuapvldmwstluserldeoopicrororalesairalesonstneitapfoonfoonisedutsnoitacilpmocsemoctuollafostluserldeoopehtelbatsemoctuoyramirpsemoctuoevitarepotsopllarevonoitacilpmocnoitacilpmocdetaerlnoisicnilautsifcitomotsananoitacilpmocerevesdetsevrahsedonhpmylsemoctuoilcgoohtapingramnoitceserliamxorpegdenoitceserlatsidnoitcnufsemoctuoyradnocesemitnoitarepossoldooblevitarepoartnilanitsetnortsagifoyrevocerdopinapevitarepotsopegasusciseganallanoitiddayatslatipsohsoraeyevfisfdraeyevfiecnereffidnaemdethgewdmwinoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaliavvruseerfesaesidsfdliavvrusllarevosoyadevitarepotsopdopoitarsddoro 0che world of surgical oncology page of table the results of the pooled outcomes were summarized in table primary outcomesall included studies reported the overall postoperativecomplication the pooled data revealed that the postoperative complication in of patients whotreated with noselar and of patientswho treated with aiselar the or was ci to p with low heterogeneity i2 fig 2a among the studies eight studies reportedthe incisionrelated complication in of patients who underwent noselar and of patients who underwent aiselar the or was ci to p with no heterogeneity i2 fig 2b nine studies reported the anastomoticfistula of which ng reported zero events in bothgroups the remaining eight studies recorded anastomotic fistula in of patients sufferednoselar and of patients suffered aiselar or was ci to p withno heterogeneity i2 fig 2c a severe complication was defined based on the claviendindo classification two of the included studies recorded severecomplication claviendindo classification ¥ iii and thesevere complication in of patients withnoselar and of patients with aiselar or was ci to p withsignificant heterogeneity i2 fig 2da total of nine studies reported lymph node harvestthere was no significant difference in lymph node harvestbetween the two groups wmd ci to p no significant heterogeneity was observed i2 fig 3a the mean number of dissectedlymph nodes in the noselar group was and theaiselar group was the proximal resection marginwas reported in studies and the wmd in the upper resection edge was cm ci to cm p with no heterogeneity i2 fig 3b the distalresection margin was reported in studies and the wmdin the inferior resection edge was cm ci to cm p with no heterogeneity i2 fig3c the length of the distal resection margin in the twogroups was cm noselar group and cmaiselar groupsecondary outcomesa total of nine studies recorded operation time and intraoperative blood loss the pooled data revealed thatthe wmd of operative duration was min ci to min p heterogeneity i2 fig 4a the wmd of blood loss was ml ci to ml p heterogeneity i2 fig 4bfig forest plot comparing postoperative complications in thenoselar group and aiselar group a overall postoperativecomplication b incisionrelated complication c anastomotic fistulaand d severe complication 0che world of surgical oncology page of fig forest plot comparing pathologic outcomes in the noselar group and aiselar group a lymph nodes harvested b proximal resectionmargin and c distal resection edgesix studies provided data about the recovery of gastrointestinal function the wmd of bowel movement was day ci to day p heterogeneity i2 fig 5a the postoperative painwas recorded in studies and studies hisada and wang recorded the postoperative pain periodand the remaining reported the pain scores in postoperative day pod the wmd of pain score in pod 0che world of surgical oncology page of fig forest plot comparing intraoperative outcomes in the noselar group and aiselar group a operation time and b blood loss was ci to p heterogeneity i2 fig 5b the additional analgesicusage rate was also reported in those studies and theor of additional analgesics usage was ci to p heterogeneity i2 fig 5c theduration of hospital stay was reported in nine studiesthe wmd of hospital stay was day ci to day p heterogeneity i2 fig 5dfiveyear diseasefree survival dfs and 5year overallsurvival os were available in two studies the hazardratio hr in the 5year dfs was ci to p heterogeneity i2 fig 6a and thehr in the 5year os was ci to p heterogeneity i2 fig 6bsensitivity analysissensitivity analysis results based on the nos score ¥ and the sample size of noselar group ¥ were presented in additional table it showed a slight inconsistency in distal resection edge operation time andrecovery of gastrointestinal function and all the otheroutcomes showed a similar trend of results between thetwo groupspublication biasa funnel plot of overall postoperative complication wasperformed to detect publication bias it showed that allthe inclusive studies were within the confidenceinterval and no publication bias was found fig inaddition a harbord test confirmed there was no publication bias p discussionas a technique used for clinical treatment the safety ofnoselar should be efficiently proved morbidity isone of the most efficient indicators for assessing thesafety of an emerging technique postoperative complications may not only lead to failures of surgery but alsothreaten lives the overall postoperative complicationrate in noselar was lower than that in aiselar severe morbidity claviendindo ¥ iiiamong the two techniques was not a significant difference the operation involving digestive tract reconstruction anastomotic leakage is a potential risk once itoccurs reoperation is usually inevitable the incidence of anastomotic leakage in noselar wassimilar with that in aiselar in addition theincidence ofincisionrelated complications in noselar was significantly lower than that in theaiselar group obviously the reduction ofcomplications in noselar has largely attributed tothe decrease of incisionrelated complications althoughthe complications in noselar were reduced the riskof bacteria contamination in the peritoneal cavity shouldnot be neglected costantino had reported the peritoneal contamination in the nose group was higherthan that of the conventional group hence measures such as bowel preparation prophylactic antibioticsperitonealtransluminalwound retractor and abdominal drains are recommended to avoid the contamination of the peritonealcavity irrigationtransanallavagethe postoperative pathology results to some extentalso reflect the safety of a surgery this metaanalysisshowed lymph nodes harvested between the two groupswas comparable and it also conformed to the minimumrequirement of the guideline retrieved more than nodes in our metaanalysis the proximal marginin the noselar group was similar with the conventional group however the distal margin in the nosegroup was longer than that of the aiselar group the 0che world of surgical oncology page of potential cause of this difference was the use of transanalspecimen eversion and extraabdominal resection technique in the nose group [ ] because of thisprocedure the distal rectal resection is performed extraabdominally under direct vision moreover circumferential resection margin crm between the two groupshave no difference [ ] in addition accordingto our metaanalysis the longterm outcomes 5yeardfs and 5year os were comparable all of those indicated the nose technique was a safety procedure in thetreatment of sigmoid and rectal cancers nevertheless aconcern about tumor seeding was raised during the procedure of enterotomy and specimen extraction it is necessary to apply several measures such as the use ofprotection devices sterile specimen bags and avoidingoverpulling and compression during specimen extraction as a minimally invasive surgery noselar had moreadvantages in alleviating patients distress the reductionof pain scores in postoperative day pod was observed and this reduction could be attributed to thetrauma in noselar being further reduced owingto less pain the need for additional analgesics was alsoreduced in addition accelerating postoperative recoverywas also observed the recovery of gastrointestinal function and hospital duration in patients who sufferednoselar was much shorter besides some scholarsmay doubt if there have alterations in sexual urinary ordefecation function in the groups according to the included studies there were no differences in functionaloutcomes such as sexual urinary or defecation betweentwo groups [ ] even though a small part of patientsexperienced function alteration and the alternation wasreversible [ ] those all demonstrated thatnoselar was a safety surgery and to some extent ithad advantages in postoperative recoverynevertheless our study has several limitations firstlyintersphincteric resections were mixed with coloanalanastomoses with sigmoid cancer in our studies although there exist some differences we mixed the twotechniques and mainly considered there existing common procedures between sigmoid and rectum resectionin laparoscopic anterior resection and some studies didnot record the methods of outcome evaluation such asblood loss evaluation to some extent it reduces thecomparability of outcomes secondly the present metaanalysis relied solely on retrospective studies and someoriginal studies not presented how patients were selectedto be candidates for one technique or another the quality of all included studies was regarded as fair or good however this type of study cannot be comparedwith a randomized controlled trial and potential biascannot be ruled out thirdly this study only recruitedone multicenter research and some outcomes includedfig forest plot comparing postoperative recovery in the noselar group and aiselar group a recovery of gastrointestinalfunction b postoperative pain pod c additional analgesicsusage and d hospital stay 0che world of surgical oncology page of fig forest plot comparing longterm outcomes in the noselar group and aiselar group a 5year dfs and b 5year oslimited studies so further multicenter randomized controlled and more comprehensive studies containing adequate outcomes are needed fourthly the results ofsome pooled results among studies existed heterogeneity the sensitivity analysis could not be detected as thecause of heterogeneity although some results existedheterogeneity the major results were homogeneity andthe heterogeneity of outcomes such as operation duration blood loss and hospital stay can be explained byclinical heterogeneity such as the difference of patientssurgeons patient management and differences in surgical proficiency in nose technology in addition the results of the major parameters were robust all in all theresults of this analysis are convincedaccording to our metaanalysisthe advantages ofnose are reduced overall complications especiallyincisionrelated complications increased distal resectionedge enhanced recovery of gastrointestinal function reduced postoperative pain reduced additional analgesicsusage and shortened hospital stay and without an auxiliary patients operated by the nose technique achievebetter aesthetics however the operative time is prolonged although the nose technique has many advantagesthere are many requirements that should befollowed before the application of this technique in colorectal surgery firstly the nose should be operated byexperienced surgeons with conventionallaparoscopiccolorectal surgery secondly the indication of noseshould follow the indication of conventionallaparoscopic colorectal resection the depth of tumor invasionshould be within t3 and body mass index bmi shouldbe less than kgm2 for transanalnose and less than kgm2 for transvaginalnose transanal nose suitsfor male or female patients and the tumor diameter isfig funnel plot of the overall postoperative complications 0che world of surgical oncology page of recommended less than cm while transvaginal noseis only applied for female patients and the tumor diameter is limited within cm and the emergent conditionssuch as bowel obstruction perforation and massivebleeding are excluded all in all as surgeons follow appropriate indicationsthe noselar for sigmoid or rectal tumors is a safesurgery and the longterm outcomes between twooperations have no difference and the benefits of thenoselar in shortterm outcomes are noticeablethese findings promote enthusiasm in support ofnose surgery as an alternative approach forthetreatment of sigmoid and rectal tumorssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s1295702001982wadditional file additional file text the search strategy of pubmedadditional file additional table characteristics of the excludedstudiesadditional file additional table summary of the included studiesadditional file additional table the results of sensitivity analysisadditional file additional table quality assessment based on thenos for retrospective studiesabbreviationsnoselar laparoscopic anterior resection with natural orifice specimenextraction aiselar laparoscopic anterior resection with abdominal incisionspecimen extraction wmd weighted mean difference or odds rationos newcastleottawa scale dfs diseasefree survival os overall survivalcis confidence intervals hr hazard ratio pod postoperative day acknowledgementswe wish to thank the timely help given by junfeng hu in statistic analysisand mengdan zhou in language editingauthors contributionsguangen yang and zhong shen contributed to the conception and designof the study jun he performed the literature search and the writing of themanuscript changjian wang and jinming chen performed the data extraction haibo yao performed the data analysis qinyan yang and jianmingqiu participated in the writing of the manuscript guangen yang and haibo yao helped to revise the intellectual content the authors read and approved the final version of the manuscriptfundingthis work was funded by the zhejiang natural science foundation grantno lq19h160013 and the zhejiang medical health science and technologyproject grant no and the hangzhou health science andtechnology project grant no 2017a26availability of data and materialsall the data analyzed in this study was obtained from the included originals or related authorsethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of colorectal surgery hangzhou third hospital hangzhou zhejiang peoples republic of china 2departments ofgastroenterology pancreatic surgery zhejiang provincial peoples hospitalhangzhou zhejiang peoples republic of chinareceived february accepted july references wang cl qu g xu hw the short and longterm outcomes oflaparoscopic versus open surgery for colorectal cancer a metaanalysisint j color dis ishibe a ota m fujii s suwa y suzuki s suwa h momiyama m watanabej watanabe k taguri m midterm followup of a randomized trial ofopen surgery versus laparoscopic surgery in elderly patients withcolorectal cancer surg endosc allaix me giraudo g mistrangelo m arezzo a morino m laparoscopicversus open resection for colon cancer 10year outcomes of aprospective clinical trial surg endosc lee l aboukhalil m liberman s boutros m fried gm feldman lsincidence of incisional hernia in the specimen extraction site forlaparoscopic colorectal surgery systematic review and metaanalysissurg endosc hennessey db burke jp nidhonochu t shields c winter dc mealy k riskfactors for surgical site infection following colorectal resection a multiinstitutional study int j color dis goto s hasegawa s hata h yamaguchi t hida k nishitai r yamanokuchis nomura a yamanaka t sakai y differences in surgical site infectionbetween laparoscopic colon and rectal surgeries subanalysis of amulticenter randomized controlled trial japanmultinational trialanization prev int j color dis park js choi gs lim kh jang ys kim hj park sy jun sh clinical outcomeof laparoscopic right hemicolectomy with transvaginal resectionanastomosis and retrieval of specimen dis colon rectum ooi bs quah hm fu cw eu kw laparoscopic high anterior resection withnatural orifice specimen extraction nose for early rectal cancer techcoloproctol franklin mj kelley h kelley m brestan l portillo g torres j transvaginalextraction of the specimen after total laparoscopic right hemicolectomywith intracorporeal anastomosis surg laparosc endosc percutan tech wolthuis am de buck voa d'hoore a laparoscopic natural orificespecimen extractioncolectomy a systematic review world j gastroenterol xingmao z haitao z jianwei l huirong h junjie h zhixiang z totallylaparoscopic resection with natural orifice specimen extraction nose hasmore advantages comparing with laparoscopicassisted resection forselected patients with sigmoid colon or rectal cancer int j color dis leung al cheung hy fok bk chung cc li mk tang cn prospectiverandomized trial of hybrid notes colectomy versus conventionallaparoscopic colectomy for leftsided colonic tumors world j surg hisada m katsumata k ishizaki t enomoto m matsudo t kasuya ktsuchida a complete laparoscopic resection of the rectum using naturalorifice specimen extraction world j gastroenterol ma b huang xz gao p zhao jh song yx sun jx chen xw wang znlaparoscopic resection with natural orifice specimen extraction versusconventional laparoscopy for colorectal disease a metaanalysis int j colordis liu rj zhang cd fan yc pei jp zhang c dai dq safety and oncologicaloutcomes of laparoscopic nose surgery compared with conventionallaparoscopic surgery for colorectal diseases a metaanalysis front oncol zhou s wang x zhao c pei w zhou h liu q liang j zhou z wang xcomparison of shortterm and survival outcomes for transanal natural 0che world of surgical oncology page of orifice specimen extraction with conventional minilaparotomy afterlaparoscopic anterior resection for colorectal cancer cancer manag res xing j zhang c yang x wang h wang h yu e fu c comparison of shortterm outcomes of transrectal specimen extraction during laparoscopicsigmoid radical resection versus conventional laparoscopically assistedprocedure zhonghua wei chang wai ke za zhi wang r wei z liu q li w xiao l han hf yang s transanal versustransabdominal specimen extraction in laparoscopic rectal cancer surgery aretrospective analysis from china wideochir inne tech maloinwazyjne ng hi sun wq zhao xm jin l shen xx zhang zt wang j outcomes oftransanal natural orifice specimen extraction combined with laparoscopicanterior resection for sigmoid and rectal carcinoma an observational studymedicine baltimore 20189738e12347liu z efetov s guan x zhou h tulina i wang g tsarkov p wang x amulticenter study evaluating natural orifice specimen extraction surgery forrectal cancer j surg res hu jh li xw wang cy zhang jj ge z li bh lin xh shortterm efficacy ofnatural orifice specimen extraction surgery for low rectal cancer world jclin cases moher d liberati a tetzlaff j altman dg preferred reporting items forsystematic reviews and metaanalyses the prisma statement ann internmed w64 hawker ga mian s kendzerska t french m measures of adult pain visualanalog scale for pain vas pain numeric rating scale for pain nrs painmcgill pain questionnaire mpq shortform mcgill pain questionnaire sfmpq chronic pain grade scale cpgs short form36 bodily pain scalesf36 bps and measure of intermittent and constant osteoarthritis painicoap arthritis care res 201163suppl 11s240 higgins jp altman dg gotzsche pc juni p moher d oxman ad savovic jschulz kf weeks l sterne ja the cochrane collaborations tool forassessing risk of bias in randomised trials bmj 2011343d5928 ga wells bsdo the newcastleottawa scale nos for assessing the qualityof nonrandomised studies in metaanalyses hozo sp djulbegovic b hozo i estimating the mean and variance from themedian range and the size of a sample bmc med res methodol tierney jf stewart la ghersi d burdett s sydes mr practical methods forincorporating summary timetoevent data into metaanalysis trials denost q adam jp pontallier a celerier b laurent c rullier elaparoscopic total mesorectal excision with coloanal anastomosis for rectalcancer ann surg saurabh b chang sc ke tw huang yc kato t wang hm tzuliang cwfingerhut a natural orifice specimen extraction with single stapling colorectalanastomosis for laparoscopic anterior resection feasibility outcomes andtechnical considerations dis colon rectum dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg almazrou am suradkar k mauro cm kiran rp characterization ofreadmission by day of rehospitalization after colorectal surgery dis colonrectum costantino fa diana m wall j leroy j mutter d marescaux j prospectiveevaluation of peritoneal fluid contamination
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"pd1pdl1 blockade therapy is a promising cancer treatment strategy which has revolutionized the treatmentlandscape of malignancies over the last decade pd1pdl1 blockade therapy has been trialed in a broad range ofmalignancies and achieved clinical success despite the potentially curelike survival benefit only a minority ofpatients are estimated to experience a positive response to pd1pdl1 blockade therapy and the primary oracquired resistance might eventually lead to cancer progression in patients with clinical responses accordingly theresistance to pd1pdl1 blockade remains a significant challenge hindering its further application to overcomethe limitation in therapy resistance substantial effort has been made to improve or develop novel antipd1pdl1based immunotherapy strategies with better clinical response and reduced immunemediated toxicity in thisreview we provide an overview on the resistance to pd1pdl1 blockade and briefly introduce the mechanismsunderlying therapy resistance moreover we summarize potential predictive factors for the resistance to pd1pdl1blockade furthermore we give an insight into the possible solutions to improve efficacy and clinical response inthe following research combined efforts of basic researchers and clinicians are required to address the limitation oftherapy resistancekeywords pd1pdl1 blockade cancer immunotherapy resistance immunotherapy is a validated and significant cancertreatment strategy which eliminates tumors by normalizing the antitumor immune responses [ ] over thelast decade cancer immunotherapy has revolutionizedthe treatment landscape of malignancies and achievedclinical success especially in immune checkpoint inhibitors correspondence 189whueducn lschrjjs163com jinyu sun and dengke zhang are cofirst authors4department of general surgery the first affiliated hospital of nanjingmedical university nanjing china2key laboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui chinafull list of author information is available at the end of the signalsandprogrammed death1 pd1 is a class of receptorexpressed on the t cell surface which could downregulate the immune system by abrogating t cellreceptorinducedantigenmediated t cell activation the interaction betweenpd1 and its ligand programmed deathligand pdl1 plays an essential role in maintaining selftoleranceand avoiding autoimmune diseases however pd1pdl1 could also prevent the activation of t cells in thetumor and thus result in immune resistance preventingpd1pdl1 blockade is a breakthrough in cancerimmunotherapy and it has been trialed in a broadrange of malignancies in the preclinical or clinicalincluding melanoma hodgkins lymphomastage breast cancer [ ] nonsmall celllung cancer as well as hepatocellular carcinomansclc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun biomarker research page of [ ] despite the longterm potentially curelikeclinical benefits therapy resistance remains a significant challenge for the further application of pd1pdl1 blockade therapy only a minority of patientsin general are estimated to experience apositive response to pd1pdl1 blockade therapy[] and the primary or acquired resistance mighteventually lead to cancer progression in patients withclinical response [ ]in this review we provide an overview on the resistance to pd1pdl1 blockade and its underlying mechanisms moreover we summarize potential predictivefactors for the resistance to pd1pdl1 blockade furthermore we give an insight into the possible solutionsto improve efficacy and clinical response of pd1pdl1blockade therapyresistance to pd1pdl1 blockade therapycheckpoint inhibitors targeting pd1 or pdl1 coulddisturb the interaction between pd1 and pdl1 whichwould preserve antitumor properties of t cells withdraw immune escape and normalize their ability to induce tumor cell death currently pd1pdl1 blockadehas shown sustained survival benefits in multiple malignancies and is at the forefront of cancer immunotherapy howeverjust as tumor cells can avoid immuneevasion several cancers may evolve to resist pd1pdl1 blockade therapy clinical evidence indicated thateven for patients with tumors highly positive for pdl1more than of them might not respond to pd1pdl1 blockade due to tumor heterogeneity and manyother reasons clinical responses vary largely across different tumor entities the objective response rate was in melanoma in nsclc in head and neck carcinoma and in kidneycancer besides for most patients experiencing initial clinical response acquired resistance remains another problem which would lead to cancer progressionor relapse after a few years [ ]many studies have demonstrated that antipd1therapy can significantly improve survival outcomes forpatients with metastatic or unresectable melanoma however only a small number of patients approximately could achieve a complete response in arecent phase i trial of atezolizumab antipdl1 involving patients with metastatic melanoma the overall response rate was among efficacy evaluablepatients and the median response duration was months moreover in another study on the longtermoutcomes of melanoma patients receiving antipd1therapy complete responses were only observed in of patients after a median followup of months of patients were alive without additional melanoma therapy additionally in the retreatedpatients after disease progression the response was onlyobserved in retreated patients receiving singleagent pd1 blockade therapy and of patientsescalated to pd1 blockade plus ipilimumab therapy inthis cohort most complete responses were durable withthe treatment failure rate of at three years whilethe response to retreatment remained relatively infrequent with a response rate of for patients withsingleagent pd1 blockade therapy moreover in aphase ii study of pembrolizumab on patients withadvancedobjectiveresponse was observed in of patients with a diseasecontrol rate of after a median followup of months adrenocorticalcarcinomatheinterestingly the response rate of some malignanciesis relatively high in hematological malignancies for example for patients with relapsed or refractory classicalhodgkin lymphoma tislelizumab antipd1 achievedan objective response rate of and a completeresponse of in a phase ii singlearm multicenterstudy similarly the complete response rate of camrelizumab antipd1 was with a partial remission rate of mechanisms underlying the resistance to pd1pdl1 blockadesince therapy resistance remains a significant limitationof pd1pdl1 blockade in clinical practice interest isgrowing in understanding the mechanisms underlyingthe resistance the response to pd1pdl1 blockaderelies on a preexisting immune response and determinants of adaptive immunity currently multiple factorshave been discovered to be involved in the efficacy ofpd1pdl1 blockade therapy such as tumor immunogenicity t celltumormicroenvironment and so forthfunction pdl1 expressionthe lack of tumor antigensthe genetic alterations are centralin the oncogenicprocess which could lead to tumor immunogenicity andprovide an opportunity for cancer immunotherapy tumor immunogenicity is positively associated with theability of the t cell to recognize tumor cells which isessential for the antitumor effect of pd1pdl1 blockade however the lack of tumor antigen will significantlyimpede the recognition ability of t cells and eventuallyresult in the failure of immunotherapymicrosatellites are prone to dna replication errorswhich will usually be repaired in normal cells however in tumors with mismatch repair mmr deficiencythese errors will accumulate which eventually result in alarge number of mutations and lead to microsatellite instability msi importantly high msi positivelycontributes to increased neoantigen production greater 0csun biomarker research page of immunogenicity and a more robust immune response moreoverthe resultant high tumor mutationburden would contribute to tumor immunogenic andenhance the response to pd1pdl1 blockade therapy[ ]multiple studies have demonstrated that the tumormutation burden is positively correlated with neoantigenburden as well as response to immunotherapy [ ]for example in colorectal cancer with mmr deficiencywhich usually exhibits a high tumor mutation burdenantipd1 therapy showed a higher response rate andbetter survival outcome compared to other subtypeswith mmr proficiency [] yarchoan analyzed the objective response rates of pd1pdl1blockade therapy for the corresponding tumor mutationburden in various cancers and their results showed thatthe mutation burden was closely associated with the objective response rate moreover pancreatic cancer generally exhibits a lowermutation load compared with other solid tumors andtherefore pd1pdl1 blockade is usually ineffective forthose patients and fails to improve their survival outcomes nevertheless in pancreatic cancer patients harboring an mmr deficiency they appear to be responsiveto pd1pdl1 blockade therapy mmr deficiency significantly increases the somatic mutation rate whichcould be translated into neoantigens and recognized bythe immune system thus making these patients responsive to pd1pdl1 blockade therapy [ ] accordingly pembrolizumab has been approved for selectedcancer patients with mmr deficiencyt cell dysfunctioneffective pd1pdl1 blockade therapy relies on the tcell function and any disruption in the processes of tcell immune function will result in the failure of pd1pdl1 blockade therapy a recent review by ren has provided an indepth insight into the mechanisms underlying the t cell dysfunctionmediated resistance with a focus on t cell recognition activationdifferentiation infiltration depletion as well as chemotaxis identification byantigen presentation is a critical process for the tumorantigensinitial t cells beta2microglobulin b2m is a significant hla1 moleculewhose mutation will hinder tumor antigen presentationand result in therapy resistance [] zaretsky analyzed biopsy samples from patients with metastatic melanoma receiving pembrolizumab who exhibited disease progression after an initial tumor regressionand they found a truncating mutation in the b2m genein the following research gettinger identifiedacquired homozygous loss or downregulation of b2m inlung patients with resistance to pd1pdl1 blockadeto further explore the role of b2m in mediating resistance they knocked out the b2m gene in immunocompetent lung cancer mice by crispr technology and theloss of b2m resulted in the resistance to pd1pdl1blockade additionally b2m mutationinducedresistance primarily occurred in an environment ofactivated pd1 positive t cellinfiltration whichresistance to pd1pdl1 blockadesuggested thattherapy might be particularly common in patients withhigh pd1 positive t cell for example t cellmoreover t cell activation is another critical processfor pd1pdl1 blockade therapy after blocking pd1pdl1 tumor cells can still counteract the activity ofimmune checkpoints and activate additional inhibitorypathways via expression of other immune checkpointsand their ligands within the tumor immune microenvironmentimmunoglobulinmucin3 tim3 is another type of immune checkpointreceptor expressed on tumorinfiltrating lymphocytes inhuman head and neck squamous cell carcinoma tumorinfiltrating lymphocytes pd1 blockade was demonstrated to upregulate tim3 expression which inhibitedt cells activation and contributed to tim3mediatedescape from pd1 blockade in the tumor microenvironment via pi3kakt pathway pd1 or pdl1physiologicallyinteractions between pd1 and pdl1block t cell activation pathways related to the immuneresponse against specific antigens and the expression ofpd1 or pdl1 has gained importance as a significantplayerin regulating the response to pd1pdl1blockade therapy pd1 and pdl1 are upregulated inthe tumor immune microenvironment of various malignancies which is considered as a strategy to evadeimmunosurveillance and imposes a significant barrier ofthe antitumor immune response importantly pdl1 primarily exhibits two distinct expression patternson tumor cells or on tumorinfiltrating immune cellspdl1 expression on immune cells reflects the adaptiveregulation meditated by ifnγ which is accompanied byincreased effector t cells as well as tumorinfiltratinglymphocytes effector t cells differently the expressionof pdl1 on tumor cells is less prevalent and it indicates the epigenetically dysregulated pdl1 gene whichis correlated with reduced immune infiltration scleroticor desmoplastic stroma as well as mesenchymal molecular features multiple studies have revealed a significantly higherobjective response rate in tumor pdl1 positive patientsthan pdl1 negative subgroups together with an improved progressionfree and overall survival [ ]kowanetz observed that atezolizumab antipdl1 achieved an objective response rate of in 0csun biomarker research page of patients with high pdl1 levels on tumor cells alone andof in those with a high expression on immune cellsalone although these observations indicated that thefunctional importance of pdl1 expression in regulatingpd1pdl1 blockadeinduced t cellthemechanistic significance of pdl1 on tumor cells or immune cells remains vagueresponsenoncoding rnasa large amount of micrornas mirnas and some longnoncoding rnas lncrnas have emerged as players inregulating tumor immunity [] and resistance topd1pdl1 blockade therapy recently huber identified a panel of circulating mirnas mir146a mir155 mir125b mir let7e mir125a mir146b mir99b which wereassociated with phenotypic and functional features ofmyeloidderived suppressor cells mdscs in melanomapatients importantly mdscs are a subclass of immature myeloid cells pathologically associated with cancerand play an inhibitory role against antitumor t cell immunity the transcriptional analysis showed thatthese mirnas could facilitate the conversion of monocytes into mdscs by melanoma extracellular vesiclesand the expression level ofthese mirna was upregulated in circulating cd14 monocytes and tumorsamples which was associated with myeloid cell infiltration and could predict the resistance to pd1 blockadetherapy moreover hu revealed the role of oncogeniclncrna for kinase activation linka in losing antigenicity and evading immune checkpoints and demonstrated lncrnadependent antigenicity downregulationsuppression for patients withand intrinsic tumortriplenegative breast cancer and resistantto pd1blockade therapythey showed upregulated linkalevels and downregulated peptideloading complex components the analysis suggested that linka expressioncould attenuate protein kinase amediated phosphorylation of the e3 ubiquitinprotein ligase trim71 via facilitating the crosstalk between phosphatidylinositol []trisphosphate and inhibitory gproteincoupled receptor pathways consequently linka could contribute to the degradation of the antigen peptideloadingcomplex and upregulate intrinsic tumor suppressors gut microbiomethe gut microbiome is a complex system composed ofmore than trillion microanisms which has beendemonstrated to regulate the efficacy and toxicity ofcancer immunotherapy many studies have reported theinfluence of the gut microbiome on cancer immunotherapy and the therapeutic response of pd1pdl1blockade therapy can be improved or diminished via gutmicrobiome modulationin mice models with distinct microbiome a significantly different response to pd1pdl1 blockade therapy was observed for example melanoma mice with anincreased bifidobacterium species in the gut microbiomeexhibited an effective response to pd1 blockade therapy similarly antibiotic administration was reported toreduce the diversity and aggravate dysbiosis of the gutmicrobiome thus influencing the clinical response topd1pdl1 blockade in tumorbearing mice as well ascancer patients [] compared to patients withoutantibiotic treatment the oral antibiotic application couldsignificantly diminish the clinical benefit of pd1pdl1blockade therapy and decrease progressionfree survivaland overall survival therefore dysbiosis of the gut microbiome is considered as one of the putative mechanisms underlying poorresponse to pd1pdl1 blockade therapy and thedualdirectional modulation of the gut microbiome oncancer immunotherapy is increasingly revealed howeverit is still unclear how gut microbiome regulatestherapy response and whether a specific bacterial taxaor gut microbiome as a whole plays a primary role remains largely unclear further research is required toprovide a more indepth understanding of the underlying mechanismspredictive factors for pd1pdl1 blockadetherapydespite the clinical success achieved in pd1pdl1blockade across multiple cancers the knowledge concerning therapy selection criteria is relatively limitedconsidering the potential adverse events and high costof immune checkpoint inhibitor agents there is a substantial need to identify predictive factors to select patients likely to benefit from this therapy currently apartfrom the functional status of immune cells [] ortumor infiltrating lymphocytes multiple factorshave been identified to predict the response to pd1pdl1 blockade therapy such as pd1pdl1 expression antigen recognition gut microbiome and so forthtable pd1 or pdl1 expressioninhibiting the pd1 pathwaymediated immune suppression is the basis and premise of pd1pdl1 blockadetherapy accumulating research has suggested that pdl1 is a biomarker to predict therapeutic response to pd1pdl1 blockade across multiple tumor types forexample atezolizumab achieved overall survival benefitacross all pdl1 expression subgroups in nsclc patients while those with high pdl1 expression experienced a more substantial survival benefit currently 0csun biomarker research page of table predictive factors for pd1pdl1 blockade therapytumor typenonsmall cell lung canceragentatezolizumabmultiple cancerscolorectal cancerurothelial carcinomaurothelial carcinomaurothelial cancermelanomamelanomamelanomapembrolizumabnivolumabatezolizumabatezolizumabatezolizumabantipd1 therapyantipd1 therapyantipd1 therapymmr mismatch repair msi microsatellite instability tmb tumor mutation burdenpredictive factorpdl1pdl1mmr msitmbtmbtmbgut microbiomegut microbiomegut microbiomereference pdl1 testing is recommended as a predictive test fornsclc urothelial carcinoma or head andneck cancers and so forthott assessed the predictive value of pdl1expression in patients with advanced solid tumors receiving pembrolizumab and the analysis showed that tumors with higher pdl1 expression and tumor mutationburden were significantly associated with higher response rate and more prolonged progressionfree survival heat map analysis revealed a close correlationbetween pdl1 expression and a broader pattern ofcoregulated gene expression which involved cytokine recruitment of t cells t cell activation markers as well asantigen presentation also the regression metaanalysisdemonstrated that pdl1 expression level was positivelyassociated with objective response rate p aswell as progressionfree survival p moreover nct02853305 and nct02807636 evaluated the efficacy of pembrolizumab or atezolizumab asfirstline treatment and the current data showed reduced survival in patients with low expression of pdl1accordingly it is advised that pembrolizumab or atezolizumab should be used for adult patients with a relativelyhigh pdl1 expression pdl1 expression of ¥ foratezolizumab and a combined positive score of ¥ forpembrolizumab however the efficacy of pd1pdl1blockade therapy as firstline therapy for advancedurothelial carcinoma still remains unclear [ ]importantly pdl1 positive only is not a predictivefactor for the response to pd1pdl1 blockade sincemultiple factors are involved in the pd1pdl1 blockade therapy in a study on patients with metastaticmelanoma receiving pembrolizumab preexisting cd8t cells were demonstrated as a prerequisite for thetumor regression after pd1pdl1 blockade therapy besidesin advanced adrenocortical carcinomatumor pdl1 expression status was not associated withtherapy response additionally it was reported thatpdl1 expression on tumor cells was not associatedwith therapy response in resected head and necksquamous cell cancer additionalinvestigation isrequired to illustrate the mechanisms accounting for thedifferenceantigen recognitionantigen recognition plays a vital role in initiating theadaptive immune response while the lack of tumor antigens significantly impedes the response to pd1pdl1blockade therapycurrently the fda has approved pembrolizumab totreat unresectable solid tumors with high msi or mmrdeficiency in a study on recurrent or metastaticcolorectal cancer patients with mmr deficiency or highmsi nivolumab showed an objective response rate of and of the patients had a disease control rateof ¥ weeks which indicated that patients with highmmr deficiency or high msi might exhibit better responses to pd1pdl1 blockade therapy [ ] interestingly the responses of tumors with mmrdeficientare highly variable and approximately half are resistantto pd1pdl1 blockade therapy mandal revealed that msi and the resultant mutation load wereresponsible for the variable response to pd1 blockadetherapy in mmrdeficiency tumors and the responsedegree was significantly correlated with the degree ofinsertiondeletion mutation loadseveral studies have revealed the association betweentumor mutation burden and the response to pd1pdl1blockade therapy [ ] mariathasan examined samples from patients with metastatic urothelial cancer receiving atezolizumab treatment and identified highneoantigen and tumor mutation burden as major determinants of clinical outcome their results showed that thetumor mutation burden was closely correlated with the response in the excluded and inflamed phenotypes 0csun biomarker research page of gut microbiome compositionclinical experiments on the human gut microbiomehave identified several specific bacteria genres that playimportant roles in human immunity and can be used asprognostic biomarkers for clinical response to pd1pdl1 blockade therapy based on the gut microbiome analysis of melanomapatients receiving pd1 blockade gopalakrishnan found that patients with prolonged progressionfree survival showed a higher multiplicity of bacteriaand clostridiales ruminococcaceae and faecalibacterium were abundant in therapy responders moreovermatson evaluated the baseline stool samplesfrom patients with metastatic melanoma before pd1pdl1 blockade treatment and the results showed thatcommensal microbial composition was significantly associated with the clinical response bifidobacteriumlongum collinsella aerofaciensand enterococcusfaecium were more abundant in responders similarlyin patients with epithelial tumors routy revealed that akkermansiacea muciniphila and enterococcus hirae were significantly abundant in those withbetter clinical response progressionfree survival months all these results indicate that gut microbiomecomposition may be a potential determinant of therapyresponse and might be used as a predictive factor inthe following research more studies are needed to validate the predictive value of gut microbiome in largercohorts and explore their efficiency in the context ofvarious types of tumorsstrategies and it hasfuture perspectivesimmunotherapy is one of the most promising cancertreatmentrevolutionized thelandscape of cancer management over the last decadehowever together with the costly and timeconsumingtrialanderror approach the limited therapy responseremains a tricky problem which hinders the furtherapplication of pd1pdl1 blockade to overcome therapy resistance and potential adverse events substantialeffort has been made on developing novel antipd1pdl1 based immunotherapy strategies with better clinical response and limited immunemediated toxicityfigs tobetterclinicallikelyachievesystem issince the interaction between cancer and the immunecomplex and involves multiplefactors strategies in combination with multiple agentsareoutcomescompared with singleagent administration a largenumber ofcombinedtherapy is an effective therapeutic strategy againstcancers for example transforming growth factor βtgfβblocking agents concomitantly with combinedpd1pdl1 blockade combined provides a clinicallyrevealed thatstudies haveexperimentson mice withfeasible strategy to improve efficacy and reduce toxicity mariathasan revealed that metastaticurothelial cancer with upregulated tgfβ signalingbefore treatmentresponded poorly to pd1pdl1blockade therapy the tumors with dense collagenfibrils could trap t cells in the stromal compartmentthus preventing them from playing their functions inpreclinicalimmuneexcluded phenotype they demonstrated that the coadministration of pdl1 blockade and tgfβblockingagents could reduce tgfβ signaling facilitate t cellinfiltration and achieve active antitumor immunityand tumor regression similarly the combination ofpd1pdl1 blockade with tumor necrosisfactorinhibitor [ ] metformin antivegf agents or otherinhibitors egcxcr4 has been demonstrated as a clinicallyfeasible strategy with improved antitumor efficacyand reduced toxicityimmune checkpointinhibitor agentspd1pdl1 blockade usually acts on the whole hostimmune system instead ofsitespecifically targetingtumorspecific immune cells while nanomedicine technology provides a powerful tool to selectively deliverimmune checkpointto tumors orlymphoid ans using drugloaded nanops usually to nm in diameter recent studies suggest that the pd1pdl1 antibody could be conjugatedor modified on the surface of nanops which couldmaintain their stability enhance efficiency and minimizethe toxicity of pd1pdl1 blockade [ ] forexamplein gastric cancer cells the pdl1 blockadeconjugated nanops contributed to significantlyhigher cellular uptake and achieved more effective inhibition of pdl1 expression compared with the controlgroups moreover in patients with metastatic triplenegative breast cancer the coadministration of nabpaclitaxelatezolizumabprolonged progressionfree survival owing to thesuccess in previous research clinical trials on nanoimmunotherapysuch asnct03589339 and nct03684785 these clinical trialsshould provide substantial evidence for the combinationof nanomedicine and pd1pdl1 blockade in the nextfew yearscurrently underwayblockadepdl1plusarethe manipulation offurthermore accumulating evidence has demonstrated that gut microbiome significantly impacts theefficacy of cancer immunotherapy which in turn indithe gut microbiomecates thatcould latently affectthe response to pd1pdl1blockade therapy [] currently antibiotic applicationfecal microbiota transplantation fmt anddiet regulation are considered as practical approachesto manipulate gut microbiome for example fmtfrom patients with a positive response to germfree or 0csun biomarker research page of fig overview on the strategies to improve the resistance to pd1pdl1 blockade therapy multiple strategies have been proposed toimprove the resistance to pd1pdl1 blockade therapy including combined therapy nanoimmunotherapy gut microbiome manipulation andso forthin contrastantibiotictreated mice could improve tumor controlaugment t cell responses and ameliorate the antitumor effects of pd1 blockadethetransplantation from resistant patients did not resultin improvement similarly responses to pdl1blockade are distinctin mice with different commensal microbes and the positive response of micewith advantageous gut microbiome can be transplanted to mice with negative responses by fmt orcohousing conclusionsdespite the success across multiple types of cancersonly a minority of patients are estimated to exhibit apositive response to pd1pdl1 blockade therapy andthe primaryacquired resistance might eventually leadto progression in patients with clinical responses thelimitation in clinical response impairs the efficacy andhinders its further application since the understandingof the mechanisms underlying therapy resistance remains vague only a few therapeutic options areavailable for those patients currently illustrating thedeterminants of response or resistance is significant toaccelerate improving survival outcomes and developingimproved treatment options for cancer patients tobetter realize the therapeutic potential of pd1pdl1blockade therapyit is essential to identify predictivebiomarkers for therapy response develop novel therapeutic strategies and improve therapeutic strategies incombination with other agents in the following research combined efforts of basic researchers and clinicians are required to address the pd1pdl1 blockadetherapy resistanceabbreviationspd1 programmed death1 pdl1 programmed deathligand nsclc nonsmall cell lung cancer mmr mismatch repair msi microsatelliteinstability b2m beta2microglobulin tim3 t cell immunoglobulin mucin3mirnas micrornas lncrnas long noncoding rnas mdscs myeloidderived suppressor cells tgfβ transforming growth factor β fmt fecalmicrobiota transplantationacknowledgmentsnot applicableauthors contributionsjys dk z mx and xz wrote original draft preparation sq w jsj and xjprovided critical revision all authors read and approved the final manuscriptfundingthis study was supported by national key research and developmentprojects intergovernmental cooperation in science and technology of chinano 2018yfe0126900 to jiansong ji the key research and developmentproject of zhejiang province no 2018c03024 to jiansong ji the nationalnatural science foundation of china to xl 0csun biomarker research page of availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1the first college of clinical medicine the first affiliated hospital of nanjingmedical university nanjing medical university nanjing china 2keylaboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui china 3college of medicine lishui college lishui china 4department of general surgery the first affiliated hospitalof nanjing medical university nanjing china 5department of radiologyaffiliated lishui hospital of zhejiang university lishui chinareceived april accepted august referenceshellmann md pazares l bernabe caro r zurawski b kim sw carcerenycosta e nivolumab plus ipilimumab in advanced nonsmallcell lungcancer n engl j med niglio sa jia r ji j ruder s patel vg martini a programmed death1or programmed death ligand1 blockade in patients with platinumresistant metastatic urothelial cancer a systematic review and metaanalysis eur urol sun jy lu xj cancer immunotherapy current applications and challengescancer lett andrews lp yano h vignali daa inhibitory receptors and ligands beyondpd1 pdl1 and ctla4 breakthroughs or backups nat immunol prestipino a zeiser r clinical implications of tumorintrinsic mechanismsregulating pdl1 sci transl med betof warner a palmer js shoushtari an goldman da panageas ks hayessa et
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Pralatrexate is a folate analogue inhibitor of dihydrofolate reductase exhibiting high affinity for reduced folate carrier1 with antineoplastic and immunosuppressive activities similar to methotrexate Despite advances in multimodality treatment strategies the survival rates for children with highrisk neuroblastoma have failed to improve Therefore the intense research continues in order to identify the ideal novel agent or combination of chemotherapy drugs to treat highrisk neuroblastomaMaterials and Methods Four human neuroblastoma cell lines were used to determine IC50 values of select chemotherapy agents Antiproliferative effects of pralatrexate were assessed by adherent and nonadherent colony formation assays Cell cycle arrest and apoptosis were measured by flow cytometry and immunoblotting PDX tissue culture was used to assess ex vivo efficacyResults Treatment with pralatrexate in all four neuroblastoma cell lines blocked cell growth in 2D and 3D culture conditions in a timedependent manner The potency of pralatrexate was tenfold stronger than methotrexate as measured by IC50 Pralatrexateinduced apoptosis was confirmed by caspase3 activation and PARP cleavage MYCN and SLC19A1 mRNA expressions were decreased with pralatrexate in MYCNamplified neuroblastoma cellsConclusions Pralatrexate demonstrated effective inhibition of cell growth and viability The higher potency of pralatrexate compared to methotrexate a drug with high levels of toxicity suggests pralatrexate may be a safer alternative to methotrexate as an effective chemotherapeutic agent in the treatment of patients with highrisk neuroblastomaINTRODUCTIONNeuroblastoma is a pediatric tumor derived from neural crest cells It is the most common pediatric solid tumor accounting for approximately of pediatric cancer deaths [] and it typically presents as a painless abdominal mass in infants and toddlers of to months of age [] Poor prognostic factors in children with neuroblastoma include age months at time of diagnosis unfavorable histology increased vascularization and MYCN amplification [] Despite intense research focused on the biology of neuroblastoma it remains one of the most enigmatic pediatric cancers in terms of its underlying molecular pathogenesis There has been only incremental improvement in the overall survival of children with highrisk neuroblastoma necessitating the search for a novel agent or combination of chemotherapy drugs []is key Altered metabolism to cancer cell proliferation Among the various metabolic pathways that are affected folate metabolism plays an important role Folate is essential for DNA synthesis and cell growth especially in rapidly dividing cells Inhibition of folate metabolism is the basis for many chemotherapy drugs In neuroblastoma folate mediated onecarbon metabolism is associated with aggressiveness and MYCN amplification Oncotargetwwwoncotargetcomwwwoncotargetcom Oncotarget Vol No pp 0cMethotrexate is a widely used [] A study by Lau in demonstrated higher folate requirements in MYCN amplified neuroblastoma cells compared to nonMYCN amplified cells [] They also showed that the increased folate uptake is mediated by reduced folate carrier1 RFC1 which is encoded by the gene SLC19A1 [] Previous studies have demonstrated that SLC19A1 is associated with MYCN amplification in neuroblastoma and that SLC19A1 is a direct transcriptional target of Nmyc [] The association between MYCN amplification and folate metabolism suggests the potential role of antifolate drugs in the treatment of neuroblastomasinhibitor of folate metabolism It inhibits dihydrofolate reductase DHFR and therefore disrupts purine and thymidylate biosynthesis leading to inhibited DNA replication and cell death However in the 1970s methotrexate was found to have high levels of toxicity combined with low treatment response rates in neuroblastoma patients and therefore it has not been clinically used for neuroblastoma treatment [] Pralatrexate is a folate analogue inhibitor of DHFR that exhibits high affinity for RFC1 [] and folylpolyglutamate synthetase FPGS Pralatrexate demonstrates antineoplastic and immunosuppressive properties that are similar to methotrexate It was FDA approved in the United States for treatment of relapsed or refractory peripheral Tcell lymphoma in [] A study by Serova in demonstrated decreased mRNA expression of SLC19A1 and SLC25A32 a mitochondrial folate carrier with pralatrexate treatment in several cancer cell lines [] As previously discussed SLC19A1 is downstream target of Nmyc in neuroblastoma The high affinity of pralatrexate for the SLC19A1 encoded RFC1 protein may demonstrate a potential role in the treatment of MYCNamplified neuroblastomaThe development of a new chemotherapeutic regimen is a long process that can take years to enter clinical trials and subsequently into bedside therapy Identifying alterative applications for previously FDAapproved drugs is a method that allows for quicker use in clinical practice [] Therefore we sought to evaluate current FDA approved antineoplastic drugs as potential novel treatment strategies for highrisk neuroblastoma and set out to assess the inhibitory role of pralatrexate on neuroblastoma cellsRESULTSThe IC50 of pralatrexate is tenfold less than methotrexateFour human neuroblastoma cell lines including MYCNamplified BE2C CHP212 and LAN1 as well as the nonMYCN amplified cell line SKNAS were treated with methotrexate or pralatrexate Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM µM measured by Cell Titer Glo¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments As shown in A in all four cell lines the IC50 of pralatrexate was approximately tenfold less than the IC50 of methotrexate These data demonstrate that highrisk neuroblastoma cells have enhanced pralatrexate sensitivity compared to methotrexate and that pralatrexate inhibits both MYCN amplified and nonMYCN amplified neuroblastoma growth in the low nanomolar range in vitroPralatrexate inhibited neuroblastoma cell growthTo demonstrate the timing of growth inhibition we treated neuroblastoma cells with pralatrexate or nM and measured cellular viability over a time course of days B Significant cell growth inhibition was first noted by day in SKNAS and CHP212 cells and by day in LAN1 and BE2C cells This indicates that pralatrexate effectively inhibits the proliferative potential of neuroblastoma cells We further validated the effects of pralatrexate on neuroblastoma cells in vitro by quantifying colony growth in a 3D matrix hydrogel where cells grow and selfassemble into clusters 3D cultures are more physiologically relevant and better represent in vivo tissue BE2C and LAN1 cells are high colonyforming neuroblastoma cell lines [] Concurrent treatment with pralatrexate completely abolished the ability of BE2C and LAN1 cells to develop colonies in gel drops A Both BE2C and LAN1 cell lines treated with pralatrexate demonstrated a decreased colony count and a decrease in colony size compared to cells treated with DMSO Figure 2B and 2C The colonies were counted from three separate microscopic fields and their size was measured using the scale bar on each image using Image J These findings suggest that pralatrexate represses the tumorigenesis potential and tumor progression of neuroblastomaPralatrexate induced G1 phase cell cycle arrest apoptosis and decreased Nmyc expressionTo further test whether pralatrexate directly altered neuroblastoma cell proliferation we evaluated the cell cycle distribution of treated cells compared with control Cell cycle analysis was performed in BE2C cells treated with pralatrexate nM or control at days and after treatment We observed a significant but modest increase in the G1 phase of the cell cycle ranging from a increase in the G1 cell population demonstrating induction of G1 cell cycle arrest Figure 3A Given the dramatic decrease in cell viability observed between days and of pralatrexate treatment Figure 1B we hypothesized that pralatrexate may also induce apoptosis in neuroblastoma cells To confirm apoptosis Oncotargetwwwoncotargetcom\x0cin cells treated with pralatrexate Western blotting was performed BE2C and CHP212 cells were treated with increasing doses of pralatrexate and nM The protein expression of total and cleaved caspase3 as well as total and cleaved PARP were examined at each increasing dose of pralatrexate Figure 3B confirming the induction of apoptosis Apoptosis was also seen secondary to pralatrexate treatment in nonMYCN amplified cells SKNAS SKNSH and SHSY5Y Supplementary Figure Given the proliferating role of Nmyc in neuroblastoma tumorigenesis we next evaluated whether pralatrexate could alter the Nmyc expression Interestingly we found decreased Nmyc expression with increasing doses of pralatrexate in both BE2C and CHP212 cells Figure 3B demonstrating persistent defects in proliferative potential induced by pralatrexate in neuroblastomaPralatrexate decreased MYCN and SLC19A1 gene expressions compared to SLC25A32Previous studies demonstrated that the expression of SLC19A1 the gene encoding the RFC1 receptor is associated with MYCN amplification in neuroblastoma [] Pralatrexate is a folate analogue inhibitor with high affinity for RFC1 [] Therefore we sought to examine the effects of pralatrexate treatment on MYCN and SLC19A1 gene expression in BE2C and CHP212 cell lines compared to the effects in nonMYCN amplified cells BE2C and CHP212 cells were treated with and nM pralatrexate or DMSO and qPCR was performed As expected from the finding in Figure 3B pralatrexate treatment resulted in decreased MYCN expression in both BE2C and CHP212 cells Figure 4A and 4B Interestingly we also found that both BE2C and CHP212 cells treated with pralatrexate demonstrated decreased SLC19A1 expression but no difference in SLC25A32 expression compared to control cells Figure 4A and 4B Treatment of nonMYCN amplified cells SKNAS SKNSH and SHSY5Y did not affect SLC19A1 or SLC25A32 expression Supplementary Figure These findings may support the previous studies [ ] that SLC19A1 is a direct transcription target of MYCN in neuroblastomas and pralatrexate treatment affects SLC19A1 expression in MYCN amplified cells but not SLC25A32 expression In addition in both BE2C and CHP212 cells pralatrexate did not decrease FPGS mRNA expression Figure 4C and 4D Neither SLC25A32 or FPGS expression was affected in nonMYCN amplified cells treated with pralatrexate Supplementary Figure Interestingly in the BE2C cells and the nonMYCN amplified cells pralatrexate treatment led to an increase in DHFR expression This may imply treatment with pralatrexate selected for cells with increased DHFR expression and inherent pralatrexate resistance or a resultant upregulation of the DHFR gene with DHFR protein inhibition These findings would Figure IC50 of human neuroblastoma cell lines BE2C CHP212 and LAN1 MYCNamplified and SKNAS nonMYCNamplified A The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with methotrexate were and µM respectively The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with pralatrexate were and µM respectively Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM µM measured by Cell Titer Glo¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments B Cell viability was assessed using the Cell Counting Kit8 assay Pralatrexate significantly inhibited cell viability in all four neuroblastoma cell lines as compared to DMSO control groupOncotargetwwwoncotargetcom\x0cbe consistent with findings by Serova et al in which pralatrexateresistant cells demonstrated increased DHFR protein expression []Pralatrexate treatment response in ex vivo neuroblastoma growthGiven these persistent in vitro findings we next evaluated whether pralatrexate could be evaluated ex vivo to guide treatment decisions for individual patients We used a neuroblastoma PDX model where tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin The size of these fragments is comparable to the size of a standard clinical tumor biopsy specimen The fragments were then cultured for days in the presence of pralatrexate nM using standard cell culture conditions Notably tumor tissues were significantly affected by ex vivo pralatrexate treatment and showed decreased Ki67 staining compared to tissues cultured in vehicle control treatment Figure These results suggest that a simple shortterm ex vivo treatment assay of a viable tumor specimen may aid in identifying neuroblastoma patients who are likely to gain benefit from pralatrexate treatment options in the futureDISCUSSIONHighrisk neuroblastoma remains quite difficult to cure necessitating the discovery of new chemotherapy agents to be used alone or in combination therapy Previous studies have reported an increased folate in MYCNamplified neuroblastoma cells demand mediated by the RFC1 receptor [] Additionally the gene encoding the RFC1 receptor SLC19A1 is a direct transcriptional target of Nmyc in neuroblastoma cells suggesting a role for antifolate drugs in the treatment of neuroblastoma Methotrexate has previously been studied in neuroblastoma however it was found to have a prohibitive toxicity and has not been used in neuroblastoma clinically In contrast pralatrexate a folate analogue inhibitor is similar to methotrexate with a more favorable side effect profile suggesting a potential role for the use of pralatrexate as a chemotherapeutic agent against neuroblastomaThe present study sought to determine the effects of treatment with pralatrexate on in vitro and ex vivo cell growth in four human neuroblastoma cell lines The IC50 of pralatrexate was found to be 10fold less than that of methotrexate This tenfold difference between pralatrexate and methotrexate was also found in colon breast and thyroid cancer cells by Serova et al in [] The decreased IC50 of pralatrexate allows for treatment with lower doses and a more tolerable sideeffect profile compared to methotrexate independent of MYCN amplificationPralatrexate not only induced celldeath via apoptosis but it also successfully inhibited neuroblastoma in vitro cell growth and proliferation in 2D and 3D cell cultures as well as in our PDX exvivo model By inhibiting the RFC1 receptor pralatrexate decreased the amount of folate entering cells and in turn decreased Figure Pralatrexate inhibited neuroblastoma colony growth A Representative images of light microscopy à magnification for BE2C and LAN1 cells after days of treatment with pralatrexate versus control Pralatrexate treatment decreased cell growth in both BE2C and LAN1 cells compared to control scale bar μm B Colony count and colony size for BE2C cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drug C Colony count and colony size for LAN1 cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cDNA synthesis This was demonstrated by the increased time spent in the G1phase of the cell cycle in cells treated with pralatrexate PDXs have been shown to parallel clinical outcome in various tumor types [] The major applications of neuroblastoma PDXs would be related to drug testing exploration of treatment resistance and biomarker discovery Combining PDXs and ex vivo culture will incorporate human tumor tissue in its native Figure Effects of pralatrexate treatment on caspase3 PARP and Nmyc protein expression A Cell cycle analysis with propidium iodide demonstrates enhanced G1 cell cycle arrest at and h following treatment Cell cycle analysis was completed with events per replicate mean ± SD p for nM pralatrexate treatment vs no drug B Treatment with increasing doses of pralatrexate induced apoptosis in BE2C and CHP212 cells Cells treated with pralatrexate demonstrated cleaved caspase3 protein expression when treated with and nM doses Cleaved PARP expression was noted after treatment with nM Treatment with pralatrexate decreased Nmyc protein expression in BE2C and CHP212 cells βactin was used as an internal controlOncotargetwwwoncotargetcom\x0c3D state and enable dynamic manipulation of the system minimizing animal experiments and costLau has shown that SLC19A1 is a downstream direct transcriptional target of Nmyc in neuroblastoma cells and that MYCNamplified cells have an increased folate dependence [] In our study pralatrexate also led to a decrease in expression of the RFC1 genes SLC19A1 and SLC25A32 The decrease in SLC19A1 was more pronounced compared to the mitochondrial folate receptor gene SC25A32 suggesting pralatrexate may be more specific to the cytosolic RFC1 receptor compared to the mitochondrial RFC1 receptor However further studies are necessary to investigate this relationship Meanwhile pralatrexate treatment led to a marked increase in DHFR expression in BE2C cells and a slight increase in CHP cells This is unlikely an upregulation of DHFR and more indicative of increased DHFR mRNA being harvested from pralatrexate resistance cells Similar results were found in a previous study on colon breast and thyroid cancer cells lines Serova found that pralatrexateresistant cells had increased DHFR protein expression [] The increase in DHFR expression may lead to an increase in the amount of DHFR protein requiring more than nM of pralatrexate to inhibit cell growth and proliferation However further studies surrounding the dose of pralatrexate and its relationship to DHFR gene expression are neededNeuroblastoma is a heterogenous tumor and further studies are needed to examine the effects of pralatrexate on additional cells lines Additionally the remaining cells that survived after pralatrexate treatment may represent pralatrexate resistant cells Future studies are needed to elucidate potential mechanisms of pralatrexate resistance such as increased DHFR gene expression as well as the relationship between pralatrexate and SLC19A1 versus other folate synthesis enzyme expressions Given pralatrexate is already an FDAapproved and in clinical use future clinical studies are needed to investigate the effects of pralatrexate treatment on neuroblastoma in vivoMATERIALS AND METHODSCells antibodies and reagentsThe neuroblastoma cell line LAN1 was a gift from Dr Robert C Seeger University of Southern California Los Angeles CA All other neuroblastoma cell lines BE2C CHP212 SKNAS SKNSH and SHSY5Y Figure Effects of pralatrexate treatment on MYCN SLC19A1 and SLC25A32 gene expressions After day of treatment the mRNA expression of MYCN SLC19A1 and SLC25A32 were measured by qPCR in A BE2C cells treated with nM of pralatrexate when compared with DMSO treated cells and in B CHP212 cells treated with nM of pralatrexate C D qPCR was performed on BE2C and CHP212 cells treated with pralatrexate to assess for mRNA expression of two keyenzymes in folate synthesies DHFR and FPGS in the treatment mean ± SD p for pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cwere purchased from the American Type Culture Collection ATCC Manassas VA Cells were maintained in RPMI with glutamine and FBS at °C in a humidified atmosphere consisting of CO2 and air Primary antibodies for Caspase3 Cat No PARP Cat No Nmyc Cat No were purchased from Cell Signaling Technology Danvers MA Ki67 Cat No was from Abcam Cambridge MA and βactin Cat No A2066 was from SigmaAldrich St Louis MO Methotrexate and pralatrexate were obtained from National Cancer InstituteDivision of Cancer Treatment and DiagnosisDevelopmental Therapeutics Program httpdtpcancergov and dissolved in dimethyl sulfoxide DMSO and further diluted in culture media to desired concentrations Neuroblastoma COGN415à patientderived xenograft PDX cells were obtained from the Childhood Cancer Repository maintained by the Childrens Oncology Group COG and Xenograft RepositoryDrug sensitivity and dose responsive curve assayFor cell viability screening cells [BE2C1500well LAN1 3000well CHP2124000well SKNAS 3000well] were plated and treated the following day with methotrexate and pralatrexate Cell growth was determined after h of continuous exposure to nM µM of methotrexate or pralatrexate using Cell Titer GloTM reagent Promega with luminescence measured using an EnVision multilabel plate reader PerkinElmer IncCell viability assayNeuroblastoma cells were seeded onto 96well plates permitted to attach overnight and were treated with either pralatrexate or nM or DMSO for days Cell viability measurements using the Cell Counting Kit8 Dojindo Molecular Technologies Inc Rockville MD were obtained daily3D colony formation assayBE2C or LAN1 cells were trypsinized embedded in μl of Cultrex® RGF BME Type matrix hydrogel Trevigen Gaithersburg MD and seeded in 48well plates cellswell RPMI medium containing FBS was added with pralatrexate treatment and incubated for days Colonies were photographed and the number and size were quantified The colonies were counted from three separate microscopic fields and their size was measured by the scale bar on each image using Image JCell cycle analysisCell cycle distribution was analyzed using flow cytometry with propidium iodide Sigma Aldrich BE2C cells were plated at equal numbers à cells and treated with either pralatrexate nM or DMSO At day and after treatment cells were washed and fixed in ethanol Fixed cells were incubated with mgmL RNAase for minutes at °C stained with propidium iodide mg mL and analyzed on a BD FACSCalibur BD Biosciences San Jose CAqPCR and immunoblottingTotal RNA was isolated and purified using a TRIzol® Reagent Thermo Scientific cDNA was synthesized using the qScript cDNA SuperMix QuantaBio RealFigure Ex vivo tissue culture model recapitulated antitumor response to pralatrexate Representative HE and Ki67 immunohistochemistry staining sections were obtained from a neuroblastoma PDX COGN415à treated ex vivo with nM pralatrexate or vehicle control for days and demonstrated poorly differentiated neuroblastoma cells and decreased Ki67 staining in pralatrexatetreated tumor compared to vehicle control à magnification scale bar μmOncotargetwwwoncotargetcom\x0creverse reverse time PCR and data collection were performed on a CFX96 instrument BioRad Data were normalized to an endogenous control βactin Specific target primers are MYCN forward 5ʹGCTTCTACCCGGACGAAGATG3ʹ reverse 5ʹCAG CTCGTTCTCAAGCAGCAT3ʹ SLC19A1 forward 5ʹAACAGGTCTGGGTTTTGTGC3ʹ 5ʹGTGCAGTATCATGCCCTGTG3ʹ SLC25A32 forward 5ʹATTGGTGGAAGCTGATTTGC3ʹ 5ʹTGGTCTGGATTTGGTCAACA3ʹ DHFR forward 5ʹCTCAAGGAACCTCCACAAGG3ʹ reverse 5ʹGTTTAAGATGGCCTGGGTGA3ʹ FPGS forward 5ʹGGGTGACCCTCAGACACAGT3ʹ reverse 5ʹGTCTTCAGGCCATAGCTTCG3ʹ Amplification was performed for cycles of s at °C s at °C and s at °C Whole cell lysates were collected using cell lysis buffer and equal amounts of protein were loaded on a NuPAGE BisTris gel followed by transfer onto PVDF membranes BioRad Hercules CA USA and probed with antibodiesEx vivo culture and immunohistochemistryCOGN415à patientderived xenograft cells were obtained from the Childhood Cancer Repository maintained by COG Clinical and genomic features of the tumors were detailed in a study by Harenza in [] Cells were suspended in Matrigel diluted with PBS and à cells were injected into the flank of NOD scid gamma mice at weeks of age UTSW Mouse Breeding Core All studies were approved by the Institutional Animal Care and Use Committee at University of Texas Southwestern Medical Center To keep cost down we used only female mice for passing PDX Mice were euthanized once tumors reached mm and tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge Johnson and Johnson in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin SigmaAldrich Tissues were cultured at °C for days with either pralatrexate nM or vehicle control then formalinfixed and paraffin embedded COGN415à tissue sections were stained with hematoxylin and eosin or with an antibody against Ki67Statistical analysisAll results are shown as the mean value ± SD statistical analyses were performed using student ttest for comparisons between the groups A p value of was considered significant GraphPads Prism software was used for the statistical analysisAbbreviationsCOG Childrens Oncology Group DHFR dihydrofolate reductase DMSO dimethyl sulfoxide FPGS folylpolyglutamate synthetase PDX patientderived xenograft RFC1 reduced folate carrier1Author contributionsStudy concept and design RAC SL JQ DHC Acquisition of data RAC SL JQ Analysis and interpretation of data RAC SL JQ DHC Drafting of manuscript RAC SL JQ DHC Critical revision RAC SL JQ DHC RAC and SL contributed equally to this workACKNOWLEDGMENTSWe thank Karen Martin for her assistance in manuscript preparationCONFLICTS OF INTERESTThe authors declare no conflicts of interestFUNDINGThis work was supported by a grant from the National Institutes of Health R01 DK61470REFERENCES Colon NC Chung DH Neuroblastoma Adv Pediatr httpsdoiorg101016jyapd201103011 [PubMed] Park JR Eggert A Caron H Neuroblastoma biology prognosis and treatment Hematol Oncol Clin North Am httpsdoiorg101016jhoc200911011 [PubMed] Matthay KK Maris JM Schleiermacher G Nakagawara A Mackall CL Diller L Weiss WA Neuroblastoma Nat Rev Dis Primers httpsdoiorg101038nrdp201678 [PubMed] Sidarovich V De Mariano M Aveic S Pancher M Adami V Gatto P Pizzini S Pasini L Croce M Parodi F Cimmino F Avitabile M Emionite L A HighContent Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy Mol Cancer Ther httpsdoiorg10115815357163MCT170841 [PubMed] Schramm G Wiesberg S Diessl N Kranz AL Sagulenko V Oswald M Reinelt G Westermann F Eils R Konig R PathWave discovering patterns of differentially regulated enzymes in metabolic pathways Bioinformatics httpsdoiorg101093bioinformaticsbtq113 [PubMed] Lau DT Flemming CL Gherardi S Perini G Oberthuer A Fischer M Juraeva D Brors B Xue C Norris MD Marshall Oncotargetwwwoncotargetcom\x0cGM Haber M Fletcher JI et al MYCN amplification confers enhanced folate dependence and methotrexate sensitivity in neuroblastoma Oncotarget httpsdoiorg1018632oncotarget3732 [PubMed] OConnor OA Amengual J Colbourn D Deng C Sawas A Pralatrexate a comprehensive update on pharmacology clinical activity and strategies to optimize use Leuk Lymphoma httpsdoiorg101080 [PubMed] Foss FM Evaluation of the pharmacokinetics preclinical and clinical efficacy of pralatrexate for the treatment of Tcell lymphoma Expert Opin Drug Metab Toxicol httpsdoiorg10151717425255201159540 [PubMed] Visentin M Unal ES Zhao R Goldman ID The membrane transport and polyglutamation of pralatrexate a newgeneration dihydrofolate reductase inhibitor Cancer Chemother Pharmacol httpsdoiorg101007s0028001322319 [PubMed] Serova M Bieche I Sablin MP Pronk GJ Vidaud M Cvitkovic E Faivre S Raymond E Single agent and combination studies of pralatrexate and molecular correlates of sensitivity Br J Cancer httpsdoiorg101038sjbjc6606063 [PubMed] Walton JD Kattan DR Thomas SK Spengler BA Guo HF Biedler JL Cheung NK Ross RA Characteristics of stem cells from human neuroblastoma cell lines and in tumors Neoplasia httpsdoiorg101593neo04310 [PubMed] Hidalgo M Amant F Biankin AV Budinska E Byrne AT Caldas C Clarke RB de Jong S Jonkers J Maelandsmo GM RomanRoman S Seoane J Trusolino L et al Patientderived xenograft models an emerging platform for translational cancer research Cancer Discov httpsdoiorg10115821598290CD14 [PubMed] Harenza JL Diamond MA Adams RN Song MM Davidson HL Hart LS Dent MH Fortina P Reynolds CP Maris JM Transcriptomic profiling of commonlyused neuroblastoma cell lines Sci Data httpsdoiorg101038sdata201733 [PubMed]Oncotargetwwwoncotargetcom\x0c'
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"Sleep disturbance is an issue reported by caregivers Waking at night is a feature of dementia and byproxy sleep disturbance among caregivers is reported to be high Little is known about the characteristics ofdementia caregivers sleep and the factors that may influence sleep disruptionThe purpose of this study was to investigate the sleep characteristics and disturbances of Australian caregivers of aperson living with dementia In addition it evaluated the psychological wellbeing of caregivers by evaluatingassociations between mood and sleep in this populationMethods This study used a crosssectional descriptive correlation design Participants were recruited with theassistance of Alzheimers Australia Dementia Australia and targeted social media advertising In total adultprimary informal caregivers of people with dementia participated completing a questionnaire on demographiccharacteristics the Depression Anxiety and Stress Scale DASS21 and the Pittsburgh Sleep Quality Index PSQIResults In this study of caregivers were female who had been caring for someone living with dementia onaverage for years of participants had two or more comorbidities namely cardiovascular disease osteoarthritisand diabetes of participants were poor sleepers with with difficulty initiating sleep and reporting havingdifficulty maintaining sleep Overall psychological distress was common with high levels of moderate to severedepression anxiety and stress Global PSQI scores were significantly positively associated with depression and anxietywith the strongest correlation seen with stress scores Depression scores were also moderately associated with daytimedysfunction Stress was identified as a significant predictor of overall sleep qualityConclusions Sleep problems are common within the population of dementia caregivers Due to the nature andduration of caregiving and the progression of dementia of the care recipient there is the potential for a decline in thecaregivers mental and physical health Caregivers of those living with dementia are more likely to have comorbiditiesdepression anxiety and stress Sleep quality is correlated with emotional distress in dementia caregivers although thedirection of this association is unclear Therefore sleep and psychological wellbeing may be intertwined withimprovements in one aspect resulting in a positive impact in the otherKeywords Carers Caregivers Sleep Mood Psychological wellbeing Dementia Correspondence aislingsmythecueduau1School of Nursing Midwifery Edith Cowan University JoondalupDrive Joondalup WA AustraliaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cSmyth BMC Geriatrics Page of BackgroundDementia is an inclusive term used to describe a numberof neurological conditions resulting in cognitive impairment and can include Alzheimers Disease senile dementia frontotemporal dementia vascular dementia LewyBody dementia Korsakoff syndrome alcohol related braininjury and younger onset dementia [] Globally thenumber of individuals with a formal dementia diagnosisrose from million in to million in []A further million new cases of dementia worldwideare predicted each year [] as populations around theglobe continue to live longer [] These figures could bevery much under estimated as it is widely accepted thatbetween and of people affected have no formaldementia diagnosis [] This growing epidemic effects notonly the individuals living with dementia but also theirfamilies caring for them the communities they live in andthe health care systems they rely uponFamily or friends are often key informal caregivers forpeople living with dementia [ ] Globally over billion hours ofinformal care is provided to peopleliving with dementia [ ] Whilst providing this carecan be highly rewarding it has also been described as achronic stressor with caregivers reporting low qualityand quantity of sleep [] and high levels of stress anddepression [] In fact sleep disruption is prevalentamongst dementia caregivers with over of caregiversexperiencing sleep disturbances []The National Sleep Foundation recommend that olderadults ¥ years require seven to h sleep for optimalphysical and psychological wellbeing [] Yet numerousstudies have illustrated that dementia caregivers sleepsignificantly less than that with estimates reporting mostcaregivers sleep less than h per night [] Not only arecaregivers sleeping less than they should the sleep theydo get is of significantly lower quality than their noncaregiver counterparts []Poor sleep is associated with a myriad of negative physical and psychological outcomes including hypertensionobesity mood disorders and dementia [] A disruptedsleep pattern is also recognised as a significant factor inpredicting caregiver strain [ ] and perhaps more importantly in predicting placing an individual into long termcare [] Enabling people living with dementia to stay athome rather than transfer to longterm care is the optimaloutcome for many families However this cannot be to thedetriment of the caregivers own physical andor psychological wellbeing Therefore in order to support the personliving with dementia PLWD to remain in the communitymaintaining caregiver health is vital Given the pivotal rolesleep has in a myriad of physiological processes it is essential to optimise and preserve caregivers sleepDespite the important role sleep plays in dementiacaregiver health it remains an understudied populationparticularly within the Australian context In fact arecent report by Carers Australia identified noAustralian studies on sleep disruption in dementia caregivers Only sixteen international studies were identifiedwhich subjectively measured sleep in the dementiacaregivers population with the majority of studies notreporting on the causes or consequences of disturbedsleep []In order to address this paucity of Australian data thepurpose of this study was to elucidate the sleep characteristics and disturbances of Australian caregivers ofPLWD Furthermore this study will determine whetherthere is a relationship between sleep and psychologicalwellbeing among caregivers of communitydwellingpeople living with dementia Lastly we will aim to identify significant predictors of poor sleep which in turncould offer a therapeutic target of poor sleep in dementia caregiversMethodsRecruitmentThis study used a crosssectional descriptive correlationdesign One hundred and four informal caregiversof people with dementia living in the community wereenrolled in the study Participants were invited via anumber of anisations including Alzheimers AustraliaDementia Australia and targeted social media advertising Mail outs were conducted and online questionnairesdistributed The inclusion criterion required the participant be an adult years or older primary informalcaregiver of a communitydwelling person living withdementia Power analysis was undertaken to computeminimum number of sample size required A samplesize of at least participants is necessary to detect amedium to large effect with power assuming atwotailed ttestEthicsThis study was granted ethical approval by Edith CowanUniversity Human Research Ethics Committee No Informed written consent was obtained from allparticipants prior to participation Participants receivedno incentive for taking partMaterialsThe survey collected demographic characteristics including gender age body mass index BMI as well as information about preexisting medical history caregivinghistory and use of respite The survey also included the questions from the Depression Anxiety and StressScale DASS21 [] and the Pittsburgh Sleep QualityIndex PSQI []The DASS21 is a selfreport questionnaire assessinglevels of caregiver stress anxiety and depression over the 0cSmyth BMC Geriatrics Page of previous seven [] day period The results provide anindication of the individuals perception of their experience of depression anxiety and stress The DASS21 isoften used as a screen for high levels of distress whenthe depression andor anxiety scores are high and as ascreen for the presence of a significant life event orproblem if the stress score is elevated DASS scorescorrespond to ranges of severity normal mild moderate severe extremely severe for depression anxiety andstress individually []The PSQI [] is a selfreport questionnaire assessinglevels of perceived quality and patterns of sleep over theprevious month across seven components subjectivesleep quality sleep latency sleep duration habitual sleepefficiency sleep disturbances use of sleep medicationand daytime dysfunction Each component has a potential score of three with a higher score indicating poorersleep related performance The total score for the sevencomponents creates a global score range to wherea score greater or equal to five ¥ indicates that theperson is a poor sleeper with severe difficulties in atleast two of the seven components or moderate difficulties in three or more components []Presence of comorbidities was determined by participantshaving one or more chronic conditions As per WorldHealth anisation WHO chronic conditions wereidentified as those requiring ongoing management over aperiod of years or decades covering a wide range of healthproblems such as heart disease diabetes asthma immunodeficiency disorder depression and schizophreniaStatistical analysesData analysis was undertaken using IBM SPSS Version and GraphPad Prism Descriptive statistics weregenerated for demographic characteristics DASS21 andPSQI scales Spearmans rho r correlation analysis wasused to assess the relationship between categorical components of DASS21 depression anxiety and stress andboth PSQI global scores and individual componentscores subjective sleep quality sleep latency sleep duration sleep efficiency sleep disturbances use of sleepingmedication and daytime dysfunction For logistic regression all variables were categorical age in years genderBMI length of care in months comorbidities DASS21component scores An r of was considered a mediumcorrelation and an r of was considered a large correlation [] Nonpaired ttests were used to determine ifthese values were statistically significant Results wereconsidered statistically significant if p Multivariatestepwise regression analysis was undertaken to determinethe predictive factors of global PSQI scores and identify variables significantly associated with sleep quality Independentvariables included in multivariate stepwise regression analyses were age gender BMI length of care comorbiditiesand DASS21 subscale scores Subjects with missing data oneither PSQI or DASS21 were excluded from inclusion incorrelation and regression analysesResultsCharacteristics of the caregiversOne hundred and four n surveys were completedin either hardcopy n or online n Table As the surveys were widely distributed by service providers and links made available online the response ratecannot be determinedParticipating caregivers were predominantly femalen with a mean age of years Range years The average BMI for respondents was kgm2range kgm2 Table which lies within theoverweight category While half of the participants n had a BMI within the healthy weight range twenty two participants were classified as underweight BMI less than and participants were classified as overweight The averagelength of time in the caregiving role in this study was months years ranging from months to yearsOnly n of participants reported using formalrespite services and all were female n of participants reported no comorbidities of participants n reported one comorbidity and n reported two or more comorbidities Themost commonly reported comorbidities were cardiovascular disorders n mainly hypertension and hypercholesterolemia bone and joint disorders n mainlyTable Demographic of participantsCharacteristicGendern or mean SDMaleFemaleAllAgeBMI kgm2Caregiver role MonthsComorbiditiesNoneOneTwo TotalUse of RespiteYesNoTotal99a94b96c ± ± ± a n missingb n missingc n missing 0cSmyth BMC Geriatrics Page of osteoporosis and arthritis and endocrine disorders n mainly prediabetes diabetes and thyroid dysfunctionTable Characteristics of Caregivers sleepMeasuren Subjective Sleep qualityThe majority of participants n reported aglobal sleep score equal or greater than which indicated they had clinically significant sleep issues in thepreceding month with a mean global score of ± Table The highest scoring individual subcomponentsincluded sleep latency ± subjective sleep quality ± and sleep disturbances ± n of participants had a sleep latency period greater than min indicative of issues initiating sleep Table ofparticipants had sleep efficiencies less than suggestingissues with maintaining sleep while in bed of participants had taken sleep medication at least once a weekover the previous month Participants were invited to addany additional relevant comments to their PSQI sleep assessment Forty participants provided additional information regarding their sleeping issues and identified issueswhich fell broadly into three themes Sleep disruption dueto caregivers physical needs such as pain and restless legs caregivers emotional distress such as stress anxietyand worrying and responding to care recipientneeds Depression anxiety and stress in caregivers of participants reported mild depression scores of participants reported mild anxiety scores and of participants reported mild stress scores Over athird of respondents reported moderate to extremelysevere stress levels n and depression n and more than a quarter n of respondents reported moderate to extremely severe anxietylevels Table In the bivariate analyses numerous subcomponents ofthe PSQI scale were significantly associated with measures of depression anxiety and stress Table Theglobal PSQI score is significantly positively associatedwith depression r anxiety r and stressscores r Stress scores also significantly correlatedwith other PSQI subcomponents including subjectivesleep quality r sleep latency r and daytimedysfunction r Depression scores were moderatelyassociated with daytime dysfunction r Global ScoreSubjective Sleep QualityVery GoodFairly GoodFairly BadVery BadSleep Latency¤ mins min min minsSleep duration h h h hSleep Efficiency¥ Sleep DisturbancesDaytime DysfunctionFrequency of SleepingMedicationNeverOnce per weekTwice per weekThree per week PSQI ComponentMean SD ± ± ± ± ± ± ± ± Predictors of Sleep quality in caregiversTo understand the relationship between the predictorsand global PSQI scores multivariate stepwise regressionanalyses were conducted to assess variables significantlyassociated with sleep quality The dependent variablewas the global PSQI score and the independent variableswere age gender BMI use of respite length of carecomorbidities and scores on the subscales of DASS21scale Depression Anxiety and Stress Stress was theonly significant covariate of global PSQI scores Stressscores could statistically significantly predict PSQIGlobal scores accounting for of variance Thestandardised coefficient was which was statisticallysignificant p as was the overall model F p All other variables were excluded from themodel due to nonsignificance Forward stepwise regression 0cSmyth BMC Geriatrics Page of Table Depression anxiety and stress in caregivers DASS21Clinical classificationTotalDepression n Anxiety n Stress n NormalMildModerateSevereExtremely Severe analyses were then undertaken with global PSQI scores asthe dependent variable and stress scores as a predictorwhile adjusting for the following confounders gender agecomorbidities of the caregiver and length of care Whilethese confounders were not statistically significant theywere retained within the model to adjust the effects ofstress on PSQI After adjusting for these factors that modelremains statistically significant F p andaccounts for of the adjusted variance in sleep scoresFor every one unit increase in stress scores PSQI Globalscores increases by a score of t p Thisregression model confirms that stress is a key significantcovariate of selfreported sleep issuesDiscussionA recent Australian report highlighted a significant gapin the literature around Australian caregivers of a PLWD[] Our study provides a comprehensive overview ofAustralian dementia caregiverssleep characteristicsassociations between psychological wellbeing and sleepand highlights a predictive role for stress in sleep qualityAll previous international studies identified an averagePSQI in dementia caregiver studies [] highlightingthe prevalence of the issue As expected and consistentwith previous studies [] Australian caregivers ofPLWD have a high prevalence of poor sleep with ofparticipants classified as poor sleepers The PSQI globalscore ± was higher than those found in somedementia caregiver studies [ ] but comparable withTable Correlation between PSQI subcomponent scores andDASS21 subcomponent score in dementia caregiversPSQI componentDASS21DepressionDASS21AnxietyDASS21StressPSQI Global ScoreSubjective sleep qualitySleep latencySleep durationSleep efficiencySleep disturbancesUse of sleeping medicationDaytime dysfunctionp p others [] The PSQI subcomponents which greatestcontributed to the overall score were sleep latency timetaken to fall asleep sleep quality overall subjectivequality and sleep disturbances Sleep latency and sleepdisturbances have previously been identified as the mostcommon contributors to sleep quality in caregivers ofPLWD in a recent systematic review [] Given thedearth of Australian specific data this study identifiedimportant depth of detail around caregiverssleepcharacteristics of caregivers took longer than therecommended min to fall asleep only of caregivers slept more than h of caregivers had sleepefficiency lower that the recommended and ofcaregivers used sleep aiding medication in the previousmonth Taken togetherfindingspresent a novel and ominous overview of the poor sleephealth conditions that Australian dementia caregiversexperiencethese descriptiveCaregivers of community dwelling people living withdementia reported poor sleep quality and high levels ofdepression anxiety and stress This study found that poorsleep was correlated with subjective feelings of depressionanxiety and stress which were in keeping with previousliterature around caregiving and psychological distressMore than half of the caregivers surveyed reported symptoms of depression and stress and respectivelyand reported anxiety These findings are in keepingwith that of a recent study of dementia caregivers in ruralVictoria where of caregivers reported depression orstress and reported anxiety []Previous work has identified an association betweendepression and poor sleep among dementia caregivers[ ] Reduced quality of sleep and depression arehigher among caregivers of people living with dementia[] Furthermore caregivers of people living withdementia who were also depressed experience a greatervariation in sleep patterns []This study reveals sleep scores were significantly associated with measures of depression anxiety and stressThese findings illustrate the interplay between sleepquality and quantity and psychological wellbeing incaregivers providing care for an individual living withdementia Further exploration of predictive factors incaregiverssleep quality identified stress as a keypredictor of poor overall sleep quality whilst adjustingfor age gender comorbidities and length of care This isa novel finding and represents a potential therapeutictarget to improve sleep quality in dementia caregiversDespite the high prevalence of selfidentified poorsleep overnight respite was only used by of participants which is in keeping with previous research thatcites of Australian caregivers have never usedrespite services [] Access to respite care remains oneof the major means of easing caregiver burden and is 0cSmyth BMC Geriatrics Page of frequently identified as needed by caregivers yet remainsunderutilised Whilst a high proportion of dementiacaregivers report a need for respite services there isinsufficient awareness of and access to respite servicesfor caregivers [] Respite service availability can beinvaluable with caregivers reporting lower stress levelsand improved health after use [] Sleep disturbanceshave also been shown to be partially reversed for caregiversduring periods of respite care [] perhaps due to reversalof hyper stressed state Respite services have been cited asan important measure to allow caregivers time to attend totheir own health imperative to supporting the caregiver tocontinue in providing care for their loved one []A recent Australian parliamentary inquiry into sleephealth identified sleep as a foundation of positive healthand wellbeing alongside diet and exercise [] Furthermore the report urged government to prioritise sleephealth as a national priority given its pivotal role inmaintaining health It is clear that caregivers of PLWDhave suboptimal sleep which is associated with poorerpsychological wellbeing and potential increased risk ofdeveloping chronic health conditions such as diabetesand cardiovascular disease [] Interestingly cardiovascular disease and endocrine disease such as diabeteswere the most prevalent comorbidities in our studypopulation Previous studies have also identified highblood pressure diabetes and arthritis as the most prevalent chronic disease in the dementia caregiver groupwhich is in keeping with our findings []Managing sleep and its associated mediators will havedirect impact on both the caregivers own health as wellas the caregiver care recipient relationship It is imperative to provide educational support around sleep considerpractical interventions such as overnight respite and toaddress stress management interventions for caregivers inturn reinstating and preserving sleep of this critical population of informal caregiversLimitationsA limitation of this study was the small sample size despitenumerous attempts to recruit participants and involvementof national anisations Although over participantsprovided demographic details only participants completed all questionnaires and were included in statisticalanalyses Challenges related to recruiting caregivers andparticularly caregivers of people living with dementia hasbeen described in numerous publications [ ]Caregivers of people with dementia living in the community are more likely to be female and this was representedin our population Furthermore caregivers who are underthe greatest stress may also be those least likely to participate However this remains one of the larger studies ofAustralian dementia caregivers sleep Another limitationwithin this study was that no data was collected regardingthe severity of dementia or behavioural disturbances whichmay impact sleep of the caregiverConclusionSleep problems are widespread within the population ofdementia caregivers Given that the majority of peopleliving with dementia are reliant on family caregiversminimising health and psychologicalimpact on caregivers should be of major concern Furthermore sleepquality is correlated with emotional distress in dementiacaregivers so improving the sleep of caregivers may inturn improve their psychological wellbeing Converselystress is a significant predictor of poor sleep so managing stress may have a positive impact on sleepDementia caregivers are often older with coexistingmorbidities and high levels of psychological distress Withprolonged caregiving and the progression of dementia ofthe care recipient there is the potential for a concurrent decline in the caregivers mental and physical health In orderto support the caregiver in their role it is of the upmost importance that we promote and maximises their health andwellbeing By managing and minimising the negative factorsassociated with caregiving we can enhance caregiving satisfaction and gratification In turn this can ensure optimalcaregivercare recipient relationship supporting the personliving with dementia to remain at home as long as possibleAbbreviationsDASS21 Depression Anxiety and Stress Scale21 PLWD PersonPeopleliving with dementia PSQI Pittsburgh Sleep Quality Index WHO WorldHealth anisationAcknowledgementsThe authors acknowledge the statistical advice of Dr Mark Jenkins and DrPeter PalamaraAuthors contributionsAS was involved in study design data collection data interpretation andmanuscript preparation LW supported study design data interpretation andediting of the manuscript CV supported study design and manuscriptpreparation EQ advised on use of psychological tools interpretation of theirdata and manuscript preparation LE was involved in data collection andmanuscript preparation All authors have read and approved the manuscriptFundingThis research was supported by an Edith Cowan University ECU Early CareerResearcher Grant awarded to Dr Aisling Smyth ECU had no role in thedesign data collection and data interpretation of the studyAvailability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThis study was granted ethical approval by Edith Cowan University HumanResearch Ethics Committee No Informed written consent wasobtained and participants received no incentive for taking partConsent for publicationNot applicableCompeting interestsThe authors state that they have no competing interests 0cSmyth BMC Geriatrics Page of Cohen J Statistical power analysis for the behavioral sciences 2nd edHillsdale NJ Erlbaum Wilcox S King AC Sleep complaints in older women who are familycaregivers J Gerontol B Psychol Sci Soc Sci 1999543P189Simpson C Carter P Dementia behavioural and psychiatric symptomseffect on caregiver's sleep J Clin Nurs Kochar J Fredman L Stone KL Cauley JA Sleep problems in elderlywomen caregivers depend on the level of depressive symptoms resultsof the caregiverstudy of osteoporotic fractures J Am Geriatr Soc Lee D Heo S Yoon S Chang D Lee S Rhee H Sleep disturbances andpredictive factors in caregivers of patients with mild cognitive impairmentand dementia J Clin Neurol Johnson E Respite reconsidered a discussion of key issues and futuredirections for carer respite Sydney Carers Australia Phillipson L Johnson K Cridland E Hall D Neville C Fielding E et alKnowledge helpseeking and efficacy to find respite services an exploratorystudy in helpseeking carers of people with dementia in the context ofaged care reforms BMC Geriatr Oconnell B Hawkins M Ostaszkiewicz J Millar L Carers perspectives ofrespite care in Australia an evaluative study Contemp Nurse Lee D Man K Lindesay J Effect of institutional respite care on the Sleepof people with dementia and their primary caregivers J Am Geriatr Soc Parliament of the Commonwealth of Australia Bedtime Reading inquiry intoSleep health awareness in Australia Canberra Commonwealth of Australia [Available from httpsparlinfoaphgovauparlInfodownloadcommitteesreportrep024220toc_pdfBedtimeReadingpdffileTypeapplication2Fpdf] Accessed Mar Moon H DilworthAnderson P Baby boomer caregiver and dementiacaregiving findings from the National Study of caregiving Age Ageing Bristow M Cook R Erzinclioglu S Hodges J Stress distress and mucosalimmunity in carers of a partner with frontotemporal dementia Aging MentHealth McConaghy R Caltabiano ML Caring for a person with dementia exploringrelationships between perceived burden depression coping and wellbeing Nurs Health Sci Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsAuthor details1School of Nursing Midwifery Edith Cowan University JoondalupDrive Joondalup WA Australia 2School of Arts and HumanitiesPsychological Services Centre Edith Cowan University Joondalup AustraliaReceived April Accepted August ReferencesAlzheimer's Association Types of dementia [Available from httpswwwalzalzheimersdementiawhatisdementiatypesofdementia]Ozcare Types of Dementia The six most common forms of dementia [Available from httpswwwozcareaudementiacareunderstandingdementiatypesofdementia]Dementia Australia Types of dementia [Available from httpswwwdementiaauinformationaboutdementiatypesofdementia]Global Burden of Disease Dementia Collaborators Global regional andnational burden of Alzheimer's disease and other dementias asystematic analysis for the Global Burden of Disease Study LancetNeurol Alzheimer's Disease International The Global Voice on Dementia2020 Availablefrom httpswwwalzcoukresearchstatistics] Accessed Mar James BD Bennett DA Causes and patterns of dementia an update inthe era of redefining Alzheimer's disease Annu Rev Public Health Ahern S Cronin J Woods N Brady NM O'Regan NA Trawley S et alDementia in older people admitted to hospital an analysis of length of stayand associated costs Int J Geriatr Psychiatr Allen AP Buckley MM Cryan JF Nà Chorcoráin A Dinan TG KearneyPM et alInformal caregiving for dementia patients the contribution ofpatient characteristics and behaviours to caregiver burden Age AgeingBevans M Sternberg EM Caregiving burden stress and health effectsamong family caregivers of adult Cancer patients JAMA Alzheimer's Disease International Global estimates of informal care LondonUK 2018Available from httpswwwalzcoukadipdfglobalestimatesofinformalcarepdf Accessed Mar Gao C Chapagain NY Scullin MK Sleep Duration and Sleep Quality inCaregivers of Patients With Dementia A Systematic Review and Metaanalysis JAMA Netw 201928e199891Ervin K Pallant J R C Caregiver distress in dementia in rural VictoriaAustralasian J Ageing Peng HL Lorenz RA Chang YP Factors associated with sleep in familycaregivers of individuals with dementia Perspect Psychiatric Care Hirshkowitz M Whiton K Albert SM Alessi C Bruni O DonCarlos L et alNational Sleep Foundation's updated sleep duration recommendations finalreport Sleep Health Jay S Vincent G Kovac K Dorrian J Thomas M Reynolds A Ferguson SReport Reducing Sleep Disruption in Carers Australia Carers Australia httpswwwsleephealthfoundationaufilesResearch_GrantsSpecial_reportsReport_Carers_Australia_FINALv2pdf Accessed Mar2020 Worley SL The extraordinary importance of Sleep the detrimental effects ofinadequate Sleep on health and public safety drive an explosion of Sleepresearch P T Rowe MA McCrae CS Campbell JM Benito AP Cheng J Sleep patterndifferences between older adult dementia caregivers and older adultnoncaregivers using objective and subjective measures J Clin Sleep MedSchulz R Belle SH Czaja SJ McGinnis KA Stevens A Zhang S Longtermcare placement of dementia patients and caregiver health and wellbeingJAMA Gaugler JE Yu F Krichbaum K Wyman JF Predictors of nursing homeadmission for persons with dementia Med Care Lovibond SH Lovibond PF Manual for the Depression Anxiety Stress ScalesSydney Smyth C The Pittsburgh Sleep quality index PSQI Insight Buysee DJ Reynolds CF Monk TH Berman SR Kupfer DJ The PittsburghSleep Qulaity index a new instrument for psychiatric practice and researchPsychiatry Res
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ShortTerm Consequences ofPediatric Anticancer TreatmentRegarding Blood Pressure MotorPerformance Physical Activity andReintegration Into Sports StructuresTina Keiser Dominik Gaser Christiane Peters Renate OberhofferFritz Sabine Kesting and Irene von Luettichau Edited byKirsten K NessSt Jude Childrens Research Hospital Department of Sports Medicine and Exercise JustusLiebig University Gieen Gieen Germany Department of Sport andHealth Sciences Institute of Preventive Pediatrics Technical University of Munich Munich Germany Department ofPediatrics and Childrens Cancer Research Center Kinderklinik M¼nchen Schwabing TUM School of Medicine TechnicalUnited StatesReviewed bySeth E KarolSt Jude Childrens Research HospitalUnited StatesJacques GrillInstitut Gustave Roussy FranceCorrespondenceSabine KestingsabinekestingtumdeIrene von LuettichauIreneTeichertvonLuettichaumritumde These authors sharesenior authorshipSpecialty sectionThis was submitted toPediatric Oncologya section of the journalFrontiers in PediatricsReceived January Accepted July Published August CitationKeiser T Gaser D Peters COberhofferFritz R Kesting S and vonLuettichau I ShortTermConsequences of PediatricAnticancer Treatment RegardingBlood Pressure Motor PerformancePhysical Activity and ReintegrationInto Sports StructuresFront Pediatr 103389fped202000463University of Munich Munich GermanyBackground Cardiovascular diseases in childhood cancer survivors are knownlate sequelae following treatment Arterial stiffness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictors to assess the statusof cardiovascular health Frequent inpatient stays and reduced physical activity PAduring treatment can lead to noticeable impairments regarding motor skills and physicalperformance The present study examined parameters of cardiovascular health motorperformance and the status of integration into sports structures shortly after cessationof treatmentMethods A crosssectional monocentric study was conducted from April to June Participants yrs mixed cancer entities during maintenance therapy andfollowup care were recruited Peripheral and central systolicdiastolic blood pressurepSBP pDBP cSBP and PWV were assessed using the MobilOGraph® The MOONtest MOtor performance in pediatric ONcology was used to scale motor performancePA levels and status ofintegration into sports structures were assessed with aquestionnaire referring to the KiGGS study All measured data were compared topublished reference valuesResults Forty participants ± years female were recruited ± years posttreatment PSBP zscore ± p pDBP ± p and cSBP ¥ years ± p were significantly increasedcompared to reference values PWV was also elevated but not significantly Motorperformance was reduced in almost all motor abilities Thirtysix percent of the examinedgroup did not participate in physical education at school to the full extent Only reported hour of daily moderatetovigorous PA as recommended for children andadolescents by the World Health anization Half of the participants were active sportsclub members before treatment but one third did not resume their former membershipFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerConclusionIncreased cardiovascular parameters and impaired motor performanceshortly after cessation of treatment physical inactivity and low rates of integration intoregular sports programs highlight the support needed Young cancer patients shouldreceive early support in coping with physical limitations preferably soon after diagnosisMotor deï¬cits could be reduced by applying targeted interventions Furthermorea regular sports therapy program during in and outpatient care could increaseengagement in PA to possibly counteract risk factors and improve cardiovascular healthKeywords childhood cancer cardiovascular health motor performance physical activity sports reintegrationblood pressure arterial stiffnessINTRODUCTIONExtensive research and optimized treatment regimens resulted inan increase of the 5year survival rate to in the USA and of the 15year survival rate to for patients under theage of in Germany However childhood cancer is a raredisease It contributes only around to all malignant diseasesin developed countries Worldwide children underthe age of and adolescents aged between and are diagnosed with cancer every year As a consequence ofthe success in the treatment of childhood cancer the importanceof survival quality and prevention of late sequelae have receivedmore attention during the last yearsKnown negativelongterm consequences ofintensivetreatment for childhood and adolescent cancer patients ofteninclude adverse eï¬ects on the cardiovascular system Cardiovascular diseases are the most frequently reported causesof death in childhood cancer survivors following secondarytumors Arterial stiï¬ness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictorsfor cardiovascular diseases frequently investigated in medicalresearch to evaluate the status of a patients cardiovascularhealth PWV describes the velocity of the pressure wave in the aortawhich spreads from the left ventricle through the arterial vascularsystem during systole Noninvasive investigation of the PWVvia ultrasound or oscillometric methods provides informationon the elasticity ofthe vascular system and enables earlyrecognition of damages in the vessels Thus in order to detectpotential structural modiï¬cations in the vascular system andindicators for arterial stiï¬ness at an early stage this subclinicalparameter should be surveyed continually Previous data indicatea positive correlation of PWV with arterial vascular stiï¬ness Moreover elevated PWV reï¬ecting subclinical vasculardamage was shown in pediatric patients after hematopoieticstem cell transplantation On the contrary another studyinvestigated elevated blood pressure levels but no statisticallyAbbreviations PWV Pulse Wave Velocity WHO World Health anizationcSBP central systolic blood pressure pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure BMI Body mass index MOONMotor performance in pediatric oncology KiGGS German Health Interviews andExamination Survey for Children and Adolescentssignificant variation for PWV in pediatric cancer survivorscompared to healthy children and adolescents In addition to the abovementioned late sequelae severalproblems already arise during treatment and often persistthroughout survivorship For instancelongterminpatient stays and reduced physical activity during treatmentcan lead to noticeably reduced physical performance ofsurvivors and reintegration into sportschildhood cancerstructures mightberehabilitationprocess throughoutfrequentaï¬ectedIn healthy populations reduced physical activity leadsto negative consequencesfor cardiovascular health Additionally the necessary use of anthracyclines in almost ofapplied therapy regimens in childhood cancer increases the riskof cardiovascular morbidity and mortality eightfold comparedto agematched patients not receiving anthracyclines indicatingthe importance of reducing such longterm consequences Due to a poor state of health and impaired immune functionsports options such as physical education at school engagementin sports clubs or recreational sports are no longer feasibleduring therapy Consequently reintegration after cessation oftreatment is associated with even more barriers due to diseaseand treatmentrelated impairments Circumstances ofanticancer treatment can lead to inactivity resulting in deï¬citsof ï¬ne and gross motor skills reduced muscle strength andpoor physical ï¬tness following treatment Especiallymotor performance in pediatric bone tumor patients oftenremains reduced until at least years after cessation of treatment Impairments of physical performance have been shown topersist throughout survivorship which may complicate thesurvivors reintegration into both social and sports structures aswell as the development of a longterm active lifestyle According to a questionnairebased study childhood cancersurvivors reintegration rate into physical education at school isvery low especially after treatment for bone tumors The lackof comprehensive oï¬ers of physical activity promotion and motordevelopment might exacerbate motor impairments and problemsof reintegration into sports structures Von Korn et al examined motor performance usingthe Fitnessgram Rcid13 as well as peripheral blood pressure centralblood pressure and PWV using the MobilOGraph Rcid13 in childrenafter treatment for childhood cancer n aged ± years ± years postdiagnosis Their results show reducedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood Cancermotor performance of childhood cancer survivors compared toreference values of healthy children However no correlationcould be drawn regarding cardiovascular parameters and motorperformance The present crosssectional study aimed atinvestigatingvarious parameters of cardiovascular health motor performanceand status of physical activity in children and adolescentsshortly after cessation of anticancer treatment or during ongoingoral maintenance therapy The collection of such data isof considerable importance for the early detection of healthimplications related to both disease and treatment Moreoverthe ï¬ndings will help to support the development of preventivestrategies regarding the health of children and adolescents treatedfor cancer Appropriate strategies during primary and secondaryprevention and following cancer treatment tertiary preventionneed improvementsMATERIALS AND METHODSDesignThe crosssectional monocentric study was performed over aperiod of months AprilJune at our institution Theassessment of cardiovascular parameters using the MobilOGraph Rcid13 was followed by the MOON test MOtor performance inpediatric ONcology to evaluate motor performance Finally theparticipants completed a standardized questionnaire referring tothe KiGGS study German Health Interview and ExaminationSurvey for Children and Adolescents to collect data regardingtheir current level of physical activity and status of integrationinto sports structures The Ethics Committee of the School ofMedicine of the Technical University of Munich approved thestudy project number SSR Participation was voluntaryand informed written consent was signed by each participant aswell as by his or her legal guardian All data was collected encodedpseudonym and in accordance with privacy policy standardsParticipantsPrior to addressing the participants all eligible children andadolescents were screened using the electronic patient recordSAP Rcid13 ERP Patients were recruited during routine followupvisits The following inclusion criteria were applied childrenand adolescents during maintenance therapy and followupcare of a pediatric oncological disease and currently agedbetween and years No restriction was applied regardingthe period posttreatment Exclusion criteria were medicalcontraindications such as fever acute infection orthopedicrestrictions and mental retardation insuï¬cient knowledgeof the German language and absence of written informedconsent The attending physician conï¬rmed participation forall recruited children and adolescents Following these inclusioncriteria children and adolescents were initially found eligibleFigure Outcome MeasuresAnamnestic and Anthropometric DataAnamnestic and clinical data ie type of cancer treatmentregime end of therapy was obtained from the electronic patientrecord SAP Rcid13 ERP The nursing staï¬ assessed anthropometricdata height and weight during routine medical examinationseca electronic column scaleseca mechanicalmeasuring rod Body mass index BMI was calculated as aratio of body weight kg per square body height m2 By usingthe reference values of a healthy German cohort BMI wasconverted into percentiles and classiï¬ed in underweight 10thpercentile normal weight 10th90th percentile and overweight90th percentile Cardiovascular ParametersPrior to the measurement the participants had to rest for atleast min in a supine position PWV central blood pressureand peripheral blood pressure were assessed using the MobilOGraph Rcid13 IEM GmbH Stolberg Germany and HMS ClientServer Version an oscillometric noninvasive methodMeasurements were performed on the left upper arm The cuï¬was inï¬ated twice with a rest of s in between Cuï¬ sizewas chosen according to the circumference of the participantsleft upper arm An ARCSolver Algorithm calculated the cSBPindirectly as well as the PWV from recorded brachial pulseRaw data was transformed into zscores and compared by usingzscores of a healthy reference cohort PSBP and pDBPwere compared to references from the national cohort of children and adolescents KiGGS study For the assessmentof PWV and cSBP values references from Elmenhorst et al of healthy children and young adults were used To evaluatethe results of the parameters cSBP and PWV the examinedparticipants were separated into two groups years and¥ years According to the age distribution of the referencevalues participants years were compared to heightmatchedreference values and participants ¥ years were compared toagematched referencesThe measurement using the MobilOGraph Rcid13 was previouslyused several times in pediatric patients and was alsosuccessfully applied in childhood cancer survivors Motor PerformanceTo quantify motor performancethe MOON test MOtorperformance in pediatric ONcology was applied Thetest assesses motor abilities coordination speed ï¬exibility andstrength and consists of eight test items eyehand coordinationinserting pins static balance static stand upper extremitycoordination throwing at a targetspeed reaction testmuscular endurance sittostand ï¬exibility stand and reachhand grip strength handheld dynamometry and muscularexplosive strength medicine ball shot The test lasts min onaverage Data of each item was compared to published age andsexmatched reference values of a healthy population within anage range of to years Data of participants older than years was compared to reference values of healthy 17yearoldssince reference values of healthy 18yearolds are not availableCalculation of a total score is not possible within this toolInstead each item was analyzed individually and the percentagedeviation to reference values was computedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Flow chart of recruitment Out of participants eligible could not be addressed due to several medical examinations in different departments of thehospital and resulting in missing time slots Longer periods posttreatment are associated with fewer appointments for followups and more medical examinationstake place in day This does not necessarily mean that the children who could not be included in the study due to missing time slots are medically more complexTen participants refused participation Thus the sample included participantsPhysical Activity and Reintegration Into SportsStructures After Acute TreatmentPhysical activity levels and status of integration into sportsstructures were assessed with a standardized selfreportingreferring to the KiGGS study Thequestionnairequestionnaire wassupplemented by several disease andtreatmentrelated aspects in accordance with the study ofKesting et al to investigate potential barriers regardingreintegration and participation in sports activities eg barrierswith respect to exemption from physical education at school ornonparticipation in sports clubs sports therapy oï¬ers duringtreatment The KiGGS study oï¬ers the reference values ofhealthy children and adolescents n for comparison ofour dataData AnalysisCardiovascular parameters were analyzed and compared to thehealthy reference population with the one sample ttest Motorperformance was analyzed using the Wilcoxon signedranktest in comparison to age and sexmatched reference valuesPearson correlation was applied to calculate possible associationsbetween motor performance and cardiovascular parametersBMI and the period posttreatment The MannWhitneyUtestwas performed to evaluate anthracyclinemediated eï¬ects oncardiovascular health as well as diï¬erences within subgroupsregarding diï¬erent entities and levels of physical activity motorperformance physical education at school and achievement ofphysical activity recommendationsExplorative twosided statistical tests were conducted andp ¤ was considered statistically significant No adjustmentfor multiple comparison was conducted Correlations coeï¬cientÏ were classiï¬ed according to Cohen Descriptive statistics were calculated with Microsoft Excelversion for demographic characteristics and medicaldata GraphPad Prism version was used to perform allfurther statistical analyses Data analysis was performed inconsultation with the Institute of Medical Informatics Statisticsand Epidemiology of the Technical University of MunichRESULTSParticipantsOut of eligible children and adolescents who met the inclusioncriteria a total of participants female with variouscancer entities were recruited and examined Table Performed AssessmentsFor various reasons not alltests could be realized withall participants In participants cardiovascularparameters could not be evaluated due to missing time slotsFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Anthropometric and medical characteristics of the participants n CharacteristicsN Mean ± SD MedianRangeAge at diagnosis years ± Age at assessment years ± years¥ years Period postdiagnosis years ± Period posttreatment years ± year years yearsLeukemiaLymphomaBone tumorBrain tumorOther solid tumors Body mass index¢ kgm2 ± UnderweightNormal weightOverweightChemotherapyAnthracycline applicationCumulative dose mgm2RadiotherapyChestdirected radiation Anthracycline chest radiation Surgical tumor resectionRelapse ± FIGURE Cardiovascular parameters shown in zscores and compared topublished reference values pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure PWV pulse wave velocity cSBPcentral systolic blood pressure Signiï¬cant values p ¤ Results are given in Mean ± SD M median RangeOther solid tumors alveolar rhabdomyosarcoma n carcinoid tumor of the appendixn nephroblastoma n focal nodular hyperplasia liver n mature cysticteratoma ovary n thoracic ganglioneuroma n papillary thyroid carcinoman neuroblastoma n ¢BMI was converted into percentiles and classiï¬ed in underweight 10th percentilenormal weight 10th90th percentile and overweight 90th percentile Thirteen participants received a combination of anthracyclines mainly the combination ofDoxorubicin and Daunorubicinduring routine appointment and a lack of willingness to prolongthe outpatient visit Six of the participants could not performthe MOON test due to medical limitations current orthopedicrestrictions n examinationrelated limitations ie a draintube in the crook of the arm for followup MRT n andabandonment due to lack of time n The central venousdevice has already been explanted in all participants prior toour studyOf the remaining participants not everyone performedevery test item Two participants could not perform the testitem speed due to a drain tube in the crook of the armFour participants could not accomplish the test item muscularexplosive strength due to orthopedic restrictions as well asexaminationrelated limitations Three participants did notperform the item muscular endurance of the legs n hadcrutches n severe muscular deï¬cit in the legs n lackof time Two participants could not perform the test item handgrip strength with both hands due to lack of time n andinfusion needle in the crook of the arm n The test itemupper extremity coordination was measured in n participantsbecause reference values are provided for children between and years onlyCardiovascular HealthIn participants all parameters were assessed Based on theunderlying reference values for cSBP and PWV of Elmenhorstet al the group was separated into participants aged years heightmatched reference values and ¥ years agematched reference values PSBP zscore ± p pDBP zscore ± p as well as cSBP values¥ years zscore ± p were significantlyincreased compared to reference values of healthy children andadolescents Figure PWV was elevated but not significantly years zscore ± p ¥ years zscore ± p Comparison of cardiovascular parameters of participantswho received anthracyclines during intense therapy with acumulative dose of ± mgm2 and subjects whodid not receive cardiotoxic agents did not show statisticallysignificant diï¬erencesMotor PerformanceThe participants n motor performance wasreduced in almost all motor abilities compared to the referencevalues of healthy children and adolescents Table Signiï¬cantimpairments became obvious in the following dimensionsmuscular explosive strength p upper extremitycoordination p muscular endurance of the legs p Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Results of the MOONtest compared to reference values n Motor abilityTest itemEyehand coordinationStatic balanceÏSpeedInserting pins timeStatic stand contactsReaction test timeUpper extremity coordinationθFlexibilityThrowing at a target pointsStand and reach cmMuscular explosive strengthMedicine ball shot meterMuscular endurance legsSittostand secHand grip strengthHandheld dynamometry kgRightLeftNMean ± SD of differenceMedian pvalueto reference values ± ± ± ± ± ± ± ± ± All results were compared to the reference values of each single test item Speed could only be measured in participants muscular explosive strength in participants andhand grip strength in right and left participants due to lack of time orthopedic restrictions or drain tube in the crook of the armFor the test items static balance and ï¬exibility the absolute differences were used as the measured values would have ï¬uctuated around zero and would have givenoversized percentagesÏ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsθ Although all participants completed this test item reference values are provided for children between and years onlyM median abs indicates absoluteBold numbers indicate significant values p ¤ TABLE Results of the MOONtest comparing participants treated for leukemialymphoma and participants treated for brain tumors compared to reference valuesLeukemiaLymphoma n Brain tumor n Motor abilityEyehand coordinationStatic balanceÏSpeedFlexibilityMuscular explosive strengthMuscular endurance legsHand grip strength rightHand grip strength leftNMean ± SD Median ± ± ± ± ± ± ± ± NMean ± SD Median pvalue ± ± ± ± ± ± ± ± All results were compared to the reference values of each single test item Ï Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsFor the test items static balance and ï¬exibility the absolute differences were used as the measured values would have ï¬uctuated around zero and would have givenoversized percentagesDue to insufï¬cient number of participants the comparison regarding the test item upper extremity coordination was disregarded reference values only provided for children aged and yearsM median abs indicates absoluteBold numbers indicate significant values p ¤ and hand grip strength on the right hand p The performance of eyehand coordination speed ï¬exibility andhand grip strength on the left hand were also reduced but notsignificantly In the test item static stand the study participantsperformed slightly better compared to the reference populationFor upper extremity coordination throwing at a target onlyreference values from children aged years are availableTherefore comparison of the collected data was only possiblewith the same age group n Data for olderparticipants was not collectedComparing motor performance of participants diagnosedwith leukemialymphoma n and participantsn diagnosed with brain tumorsrevealedsome diï¬erences Table Children and adolescents treatedfor brain tumors performed significantly worse in eyehandcoordination than participants treated for leukemialymphomap Moreover the performances in all other testedmotoric dimensions of participants diagnosed with braintumor were deteriorated compared to participants treated forleukemialymphomas but not significantlyTo determine inï¬uencing factors on motor performance thecorrelation between motor performance results and BMI as wellas the period posttreatment was performed With increasingBMI values of static balance deteriorated significantly Ï Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Pearson correlation between static balance and BMI kgm2¢ leukemialymphoma ¦ bone tumor ³ brain tumor cid7 other solid tumorsHorizontal line indicates the reference values The absolute difference fromreference values is given number of contacts to the ground A negativedifference means fewer ground contacts and therefore better performanceThirty four participants performed the testp which corresponds to a moderate to high correlationFigure A negative diï¬erence to reference values means fewercontacts to the ground and therefore better performance in staticbalance Furthermore some nonsignificant correlations werefound Deteriorated eyehand coordination Ï p and ï¬exibility Ï p were also associatedwith a higher BMI However superior values in upper extremitycoordination Ï p muscular explosive strengthÏ p hand grip strength right Ï p left Ï p and muscle endurance ofthe legs Ï p were associated with increasedBMI The test item speed showed no association with BMIÏ p A longer period posttreatment was significantly associatedwith decreased eyehand coordination Ï p corresponding to a moderate correlation Figure Especially participants treated for a brain tumor with a longerperiod posttreatment showed deteriorated values in eyehandcoordination Speed performance was deteriorated in participantswith longer posttreatment period Ï p Physical Activity and Reintegration IntoSports StructuresAccording to the selfreported questionnaire n did not participate in physical education at school to full extend n were not admitted to school sports activitiesand n were partly excluded Table Neitherof the two participants treated for bone tumor was takingpart in physical education at school n whereaschildren with other tumors participated to a notably higherrate Treatmentrelated muscular deï¬cits n andosteonecrosis n were the most common reasons forparticipants not taking part in physical education at schoolFIGURE Pearson correlation between eyehand coordination and periodposttreatment months ¢ leukemialymphoma ¦ bone tumor ³ brain tumorcid7 other solid tumors Horizontal line indicates the reference values Thirtyfour participants performed the test itemTABLE Participation in physical education at school subdivided into entitiesn Participation Partial exemption Full exemptionN N N Entire group n LeukemiaLymphoma n Bone tumor n Brain tumor n Other solid tumors n Abs N number Four out of children were still attending kindergarten and could notanswer the question regarding participation in physical education at schoolOnly n reported moderatetovigorous physicalactivity for min daily as generally recommended by the WHOfor healthy children and adolescents Table This percentageis comparable to the achievements in the healthy referencepopulation n Every second participant questioned was an active memberin a sports club whereas n did not return to asports club following cancer treatment Almost onethird n has never been a sports club member Reasons fornot engaging in sports club activities of participants wereno interestfun n physical weakness n no time n anxiety n andphysicianbased prohibition due to clear medical reasons n Nearly all participants n were active inrecreational sportsFurther analyses pointed toward diï¬erences in physicalactivity and sports club participation especially betweenparticipants with brain tumors and leukemialymphomas Thenumber of participants in recreational sports was reported highin both groups leukemialymphoma and brain tumor In contrast a diï¬erence was found in sports club activitySixtysix percent of leukemialymphoma patients were membersFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Physical activity and engagement in sports club and recreationalsportsEntire groupReference populationn n Physical activity guidelinesWHO mindayDaily physical activityDaily walking distance kmDaily walking distance kmDaily walking distance kmSports club activity currentlyFormer membershipRecreational sports activityAbs N number Reference values derived from the national cohort of healthy childrenand adolescents in the KiGGS study German Health Interviews and Examination Surveyfor Children and Adolescents of a sports club whereas only of participants with a braintumor were active in a sports club On the other hand almosthalf of the children treated for brain tumor and of thechildren treated for leukemialymphoma were former membersConcerning possible correlations between motor abilities andphysical education at school participation in sports clubs orrecreational sports deï¬ned as activeinactive as well as meetingthe physical activity recommendations no significant associationscould be determined Likewise the comparison of motor abilitiesof participants receiving sports therapy during treatment didnot show any correlation Almost half of the participants n took part in a sports therapy programme duringtreatment which was mainly oï¬ered as care and varied greatlyin terms of training interventions without any standardizationDISCUSSIONtreatmentThe results of our study clearly present evidence for deterioratedcardiovascular function in children and adolescents shortlyafter cessation of cancerIncreased pSBP andincreased pDBP are risk factors for cardiovascular diseasesregarding guidelines for arterial hypertension Potentialcardiovascular consequences such as stroke sudden deathheart failure and peripheral artery disease due to elevatedblood pressure values are described in the aforementionedguidelines as well as in the literature Accordinglychildhood cancer survivors with elevated blood pressure are atrisk to experience such cardiovascular late eï¬ects Regarding10years survivors of childhood cancer a higher prevalenceof hypertension is assumed and cardiovascular diseaserelated deaths are eighttimes more likely in childhoodcancer survivors compared to the general population Our ï¬ndings support previous study results which depictcomplications such as increased blood pressure prehypertensionand hypertension in children and adolescents treated for cancer This study aimed at investigating speciï¬c parameters thatcould serve as early predictors for potential damage to thecardiovascular system Recent evidence of cSBP as a suitableparameter to determine the elasticity of blood vessels suggeststhat cSBP is more closely related to cardiovascular events in thefuture than brachial blood pressure Increased cSBP inparticipants in our study may result from early changes in arterialwall stiï¬ness As a further parameter to detect early impairmentsin elasticity of the vascular system PWV was investigated Incontrast to prior studies no decisive change was observedin PWV While anthracycl
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" ovarian cancer is the leading cause of cancerrelated death among women complete cytoreductivesurgery followed by platinumtaxene chemotherapy has been the gold standard for a long time variouscompounds have been assessed in an attempt to combine them with conventional chemotherapy to improvesurvival rates or even overcome chemoresistance many studies have shown that an antidiabetic drug metforminhas cytotoxic activity in different cancer models however the synergism of metformin as a neoadjuvant formulaplus chemotherapy in clinical trials and basic studies remains unclear for ovarian cancermethods we applied two clinical databases to survey metformin use and ovarian cancer survival rate the cancergenome atlas dataset an l1000 microarray with gene set enrichment analysis gsea analysis western blotanalysis and an animal model were used to study the activity of the aktmtor pathway in response to thesynergistic effects of neoadjuvant metformin combined with chemotherapyresults we found that ovarian cancer patients treated with metformin had significantly longer overall survival thanpatients treated without metformin the protein profile induced by low concentration metformin in ovarian cancerpredominantly involved the aktmtor pathway in combination with chemotherapy the neoadjuvant metforminprotocol showed beneficial synergistic effects in vitro and in vivos this study shows that neoadjuvant metformin at clinically relevant dosages is efficacious in treatingovarian cancer and the results can be used to guide clinical trialskeywords neoadjuvant metformin ovarian cancer clinically relevant dosage aktmtor pathway synergisticeffects correspondence syeungmdanderson limh33mailsysueducn kuochang wen and pilin sung share first authorship9department of emergency medicine division of internal medicine theuniversity of texas md anderson cancer center houston tx usa10guangdong research institute of gastroenterology the sixth affiliatedhospital of sun yatsen university yuancun erheng rd guangzhou pr chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwen of ovarian research page of ovarian cancer is the fifth leading cause of mortality in developed countries in the united states an estimated women were diagnosed with ovarian cancer in and deaths due to ovarian cancer occurred complete cytoreductive surgery followed by standard firstline platinumtaxene chemotherapy has been shown to improve the survival rate however the majority of patientsexperience relapse and the 5year survival rate is approximately chemoresistance to platinumbased treatment remains a major challenge in the successful treatmentof ovarian cancer and the mechanisms underlying platinum resistance are multifactorial various cellular processes are observed in resistant cells and activation of thepi3kakt pathway is believed to be a determinant of resistance in ovarian cancer [ ] thus the development ofan improved treatment to overcome acquired resistance incancer cells or decrease the side effects of platinumbasedtreatment is needed to treat ovarian cancermetformin n0n0dimethylbiguanide a biguanide is anoral hypoglycemia agent that is widely used as an antidiabeticdrug to treat type diabetes mellitus dm it is also widelyused to treat polycystic ovarian syndrome metformin hasbeen shown to reduce cancer development in type dm patients and inhibit growth in several cancer models [ ] either alone or in combination with cytotoxic agents [ ]the major target of metformin in cancer cells is the tumorsuppressor lkb1ampactivated protein kinase ampkpathway which serves as a metabolic checkpoint to arrestcell growth when intracellular atp levels are low such as innutrientpoor conditions after activating ampk metformin phosphorylates tuberous sclerosis complex tsc2and then binds with its obligate partner tsc1 tsc2 leadsto the accumulation of rhebgdp and the inhibition ofmtorc1 which influence eukaryotic translation initiationfactor 4ebinding protein 4ebp1 and ribosomal s6 kinases6k1 respectively shank showed that metformincan restrict the growth and proliferation of ovarian cancerstem cells yasmeen revealed that metformininduces apoptosis in ovarian cancer cell lines in an ampkindependent manner by activating caspases downregulating bcl2 and bclxl expression and upregulating baxand bad expression which results in cell cycle arrest in the sand g2m phases rattan identified metformin asan antiproliferative therapeutic that can act through bothampkdependent and independent pathways via thesepathways metformin inhibited cell proliferation in bothwildtype and ampk null mouse embryo fibroblasts as wellas in ampksilenced ovarian cancer cells in addition metformin has been shown to inhibit pi3kaktmtor signaling in lung cancer [ ] breast cancer pancreaticcancer and hepatic cancer however most studies showing that metformin alleviates cancer have used higher doses in vitro than thoseused in diabetic patients these high concentrationsmay directly cause the death of tumor cells in thepresent study we tested a low concentration as the effective dose which was a clinically relevant dose the effects of lowconcentration metformin on aktmtorsignaling in ovarian cancer remain unclearthe aim of the present study was to examine the effectsof a combination of metformin at clinically relevant dosages and chemotherapy on ovarian cancer via the aktmtor pathway we found that metformin reduced ovarian cancer death in two clinical datasets and predicted thatthe effect of metformin in ovarian cancer was mediated bythe aktmtor pathway using a bioinformatics modelthen we demonstrated that the low concentration ofmetformin inhibited the growth of a mouse ovarian surface epithelial cell line mosec and that it had a synergistic effect in combination with chemotherapy via theaktmtor pathway neoadjuvant application of metformin plus chemotherapy yielded beneficial synergistic effects both in vitro and in vivo the results provide insightinto the potential of neoadjuvant metformin to augmentthe efficacy of existing cancer therapeuticsresultsthe effect of metformin on survival in ovarian cancerpatientswe investigated the impact of metformin on humanovarian cancer by analyzing a clinical dataset in total patients were diagnosed with primary ovarian cancerin the department of gynecology and obstetrics taipeiveteran general hospital from to after theirclinical and drug histories were reviewed patientswho underwent complete surgery and were treated withcarboplatin were included in the analysis thirtytwo ofthese patients took metformin either during admissionor in the outpatient clinic os was measured from thedate of diagnosis to death or was censored at the date ofthe last followup the os of patients with metformintreatment n was significantly higher than that ofpatients without metformin n p fig 1atable figure 1b shows the ovarian cancerfree incidence of female dm patients n fromthe national health insurance taiwanese dataset ovarian cancer was less frequent among metformininsuthan among metforminlin users n insulin users n p the use of metformin or insulin may help prevent ovarian cancer in female dm patients we next investigated the cellulareffects of metformin on ovarian cancer the expressionprofiles of differentially expressed genes in response totreatment with metformin chemotherapy or both wereretrieved from the l1000 study gsea was performedusing the up and down gene expression datasets froml1000 gsea revealed that the gene expression induced 0cwen of ovarian research page of fig the effect of metformin on survival in ovarian cancer patients a kaplanmeier os of ovarian cancer patients with n or without n metformin use b the ovarian cancerfree incidence in female dm patients metformin ever usedinsulin ever used users n and metformin never usedinsulin never used users n from the national health insurance taiwanese dataset c tumorproliferation cell number 2dcolony formation and western blot analyses of the indicated proteins in the aktmtor pathway of mouse ovariancancer cells treated with different concentrations of metformin d comparison of the mortality rates between groups with low and high proteinexpression by the halfdivision approach kaplanmeier analysis assessed the correlations of the indicated proteins akt [total and pser473] mtor[total and pser2448] with the overall survival of patients data from the cbioportal tcga database tcga provisional ovarian cancer genomicsn a logrank pvalue less than indicated a significant difference in overall prognosis p p p by control and metformin treatment was similar to thatof the kegg pancreatic cancer pathway httpwwwgenomejpdbgetbinwww_bgetpathwayhsa05212fig s1 which predominantly involved the aktmtorpathway based on the proteomic and viability investigation metformin may involve the alternation of phosphorylation in the aktmtor pathway accompanyingcell retardation in ovarian cancer without affecting thetotal amount of protein fig 1c metformin inhibitedthe aktmtor pathway in a dosedependent manneras shown by western blot analysis the phenomenonwas found at both high mm and low mmdoses of metforminlow doses were closer to theclinically relevant concentration fig s2 at low dosesof metformin the alternation of phosphoampk wasnot as obvious as that in the aktmtor pathway wefurther investigated the clinical role of the aktmtorpathway using the tcga ovarian cancer dataset thepatients with upregulated phosphorprotein akt_pser473 or mtor_pser2448 had significantly poor osthe expression of total protein was not associated withclinicalimportance in ovarian cancer fig 1d thesefindings highlight the value of metformin in inhibitingovarian tumor cells via phosphorylation of the aktmtor pathway which indeed plays an essential role inthe prognosis of ovarian cancer 0cwen of ovarian research page of table the clinical characteristics of the study populationcharacteristicp valueno metformin usen metformin usen diabetes ratestageaearly i ii late iii iv histologybepithelialother typesc ± ± ± ± ca125ddeath rateoverall survivale§afigo stage international federation of gynecology and obstetrics surgicalstaging of ovarian cancer missing value not included in statistical testbmissing value not included in statistical testcincluding primary peritoneal serous carcinoma ppscdbefore surgery mean ± sd umleoverall survival months mean ± sdcalculated by chisquare testcalculated by students ttest§calculated by kaplanmeier logrank testmetformin at a clinically relevant dosage inhibits ovariancancer growth through the aktmtor pathwayto mimic the clinically relevant dosages in the humanbody we used lowdose concentrations of metformin inthe study we assessed the cell growth of metformin in mmtreated ovarian cancer cell lines to evaluate thegrowthinhibitory effect of metformin as shown infig 2ab mm metformin can reduce colony formation and cell growth both mouse and human ovariancancer cell lines manifested significantly reduced proliferation after treatment with clinically relevant doses ofmetformin fig s3a furthermore mm metformintreatment beginning from day to day reduced cellviability and cell viability recovered from day to day after discontinuing treatment fig 2c the inhibitory effect of lowdose metformin on the aktmtor pathwaywas shown during metformin treatment day to day the phosphoprotein expression was inhibited duringmetformin treatment but these expression levels wererestored when suspending metformin fig 2d theaforementioned data suggested that metformin at lowdose concentrations could inhibit cell growth in ovariancancer through inhibition of the aktmtor pathwayas supported by the gseathe synergistic effects of metformin and chemotherapyon ovarian canceralthough standard firstline platinumbased protocolsimprove survival in ovarian cancer strengthening thesechemotherapy regimens is warranted we evaluated theantiproliferative effects of different protocols beforeduring or after and doses of metformin in combinationwith chemotherapy fig 3aleft panel as shown infig 3a concurrent combination of metformin andchemotherapy yielded the strongest inhibition fortyeighthour exposure to concurrent metformin andchemotherapy resulted in a clear synergistic effect withnegative log ci values in ovarian cancer cells fig 3aright panel regarding human ovarian cancer cellslower concentrations of carboplatin μm but nothigher concentrations μm show synergy with metformin with negative log ci values fig s3b the activated forms of akt are key intracellular mediators ofgrowth cell survival and platinum response determining the activation state of the aktmtor pathway isimportant for understanding the synergistic mechanismof action of lowdose metformin combined with chemotherapy in ovarian cancer western blot analysis demonstrated that both chemotherapy alone and metforminalone reduced the levels of phosphorylated aktmtorwithout affecting the total amount of aktmtor protein fig 3b the combination of metformin and chemotherapy produced a stronger inhibition of pakt and theakt downstream effectors pmtor ser2448 ps6 kinaseser235236 and p4ebp1 thr3746 compared withmetformin or chemotherapy alone in contrast the totalamounts of mtor s6 kinase and 4ebp1 were unaffectedby treatment and ampk phosphorylation was not reduced by treatment with lowdose metformin or chemotherapy either alone or in combinationsome dm patients treated with metformin cannotachieve good blood sugar control and require other medications we investigated the cellular effects of metformintreatment on tumors in patients with poor blood sugarcontrol fig 3c left panel as shown in fig 3c middlepanel mtt assays indicated that cells cultured in highglucose medium mgl showed accelerated cell proliferation relative to that of cells in control medium mgl however metformin treatment diminished this effect induced by high glucose the combined effect of metformin and chemotherapy in a highglucose medium wasantagonistic with a positive log ci fig 3c right panelto determine whether high glucose induces the aktmtor pathway in mosecs we treated the cells with metformin alone chemotherapy alone or a combination inhighglucose medium or control medium for h fig s4highglucose medium resulted in marked increases inpakt pmtor ps6 kinase and p4ebp1 levels comparedwith the control medium treatment with metformin andcarboplatin in the highglucose medium reduced the protein level of pakt but not the protein levels of pmtorps6 kinase and p4ebp1 relative to the levels in the controlmedium these results revealed that high glucose or poorblood sugar control may diminish the antitumor effects ofmetformin and chemotherapy although they still have effects on the paktmtor pathway 0cwen of ovarian research page of fig see legend on next page 0cwen of ovarian research page of see figure on previous pagefig metformin at a clinically relevant dosage inhibits ovarian cancer growth through the aktmtor pathway ab growth of mouse ovariancancer cell line mosec in cells incubated for serial days with lowconcentration metformin mm met metformin p by twowayanova c and d cell viability and protein analyses of the aktmtor pathway under treatment with metformin mm from day to day andwhen treatment was suspended from day to day the beneficial synergistic effects of neoadjuvantmetformin under combination treatmentwe further investigated the synergistic effects of lowconcentrations of metformin and chemotherapy underdifferent treatment regimens commonly used in clinicalsettings as shown in fig 4a synergistic effects were obviously observed in both protocol neoadjuvant metformin and protocol concurrent metformin but notin protocol adjuvant metformin western blot analysis indicated that protocols reduced the activitiesof pakt pmtor ps6 kinase and p4ebp1 with protocols and having superior effects compared withprotocol fig 4b these results indicate that metformin should be used before or at least concurrent withchemotherapy to enhance the antitumor effect neoadjuvant metformin combined with chemotherapy was betterthan the combination protocolwe further investigated the in vivo antitumor activityof neoadjuvant metformin in b6 mice bearing mosecsthat were grown subcutaneously as tumor xenograftstreatment with metformin or chemotherapy as singleagents caused a decrease in tumor size relative to that ofcontrol untreated mice treatment with the neoadjuvantmetformin combined with chemotherapy significantlyreduced tumor growth fig 4c finally we selected thecases with chemotherapy from fig 1a to determine theclinical impact of metformin in chemotherapy metformin before or during chemotherapy indeed showed atrend toward better overall survival compared with single chemotherapy although this trend did not reach statistical significance fig 4dschematics of the intracellular effects of treatmentwith metformin chemotherapy or their combination onovarian cancer are shown in fig 4e neoadjuvant metformin combined with chemotherapy produced bettersynergistic effects inhibiting akt and its downstreampathway a highglucose environment such as that inpoorly controlled type dm patients may increase activated aktmtor signaling thusfor patients withpoor glucose control the combination of metformin andchemotherapy may be slightly superior to chemotherapyalone and not as efficacious as that in patients with goodglucose controldiscussiondiabetes is strongly associated with an increased incidence of cancer many studies have shown thatmetformin can reduce the risk of cancerincludingbreast colon liver and pancreatic cancers and improveoutcomes over those obtained with other antidiabetictreatments sulfonylurea insulin in diabetic patients whether metformin can reduce the risk of ovarian cancer has been investigated [] but few studies havefocused on the effects of metformin combined withcommonly used firstline chemotherapeutic drugs suchas carboplatin and the underlying mechanisms neoadjuvant metformin combined with other therapieshave been administered to treat erpositive breast cancer in a phase ii clinical trial clinicaltrialsgov identifier nct01589367 in this study we evaluated thesynergistic effects of neoadjuvant metformin combinedwith chemotherapy in ovarian cancer via the aktmtor pathway both in vitro and in vivo and found thatpoor glucose control diminished the synergistic antitumor effects of combination treatmenta previous casecontrol study that used the ukbasedgeneral practice research database revealed thatthe adjusted odds ratio of metformin use vs nonuse forovarian cancer incidence was not significant in nondiabetic patients but was significant in diabetic patientsin another recent metaanalysis which included one observational study and two clinical trials the pooled oddsratio ci of metformin use for ovarian cancer incidence was recently a study wasperformed using reimbursement databases of the national health insurance nhi to evaluate metforminuse in taiwanese women with type dm metformindecreased the incidence of ovarian cancer and the overall fully adjusted hazard ratio ci for everusersversus neverusers was in thepresent study the results were similar to those of a previous report in which ovarian cancer was less likelyto occur in metformininsulin users n than in metformininsulin users n thisprevious study similarly used reimbursement databasesof the nhi although the data were from a different timeperiod the hospital cohort data showed that ovariancancer patients treated with metformin had a significantly longer os compared with patients not treatedwith metformin this finding is similar to that of another casecontrol studyin which an association between metformin use and improved survival of ovariancancer patients was identified these results indicated that metformin may reduce ovarian cancer incidence in type dm patients and improve survival aftera diagnosis of ovarian cancer 0cwen of ovarian research page of fig the synergistic effects of metformin and chemotherapy on ovarian cancer a synergistic effects of metformin combined withchemotherapy carboplatin at different concentrations and different combination protocols concentration of metformin and mmconcentration of carboplatin and μm met metformin chemo chemotherapy b the effects of metformin alone mmchemotherapy carboplatin μm alone or combined treatment assessed by the indicated antibodies in western blot analysis c mtt assaysshowed that cells cultured in a highglucose medium mgl exhibited greater growth than those in control medium mgl greensquares represent cells cultured in highglucose medium and red solid circles represent those cultured in control medium concentration ofmetformin and mm concentration of carboplatin and μmmost studies examining the effects of metformin oncancer have used doses mm higher than those usedclinically for diabetic patients [ ] yielding metforminplasma concentrations between and μmoll fewstudies have focused on the impact of metformin at clinically relevant dosages although dr hu and colleagues 0cwen of ovarian research page of fig see legend on next page 0cwen of ovarian research page of see figure on previous pagefig the beneficial synergistic effects of neoadjuvant metformin under combination treatment a and b protocol a neoadjuvant protocolmosecs treated with metformin alone for day and then with a combination of metformin and carboplatin for days protocol a concurrentprotocol mosecs treated with both metformin and carboplatin from day for days protocol an adjuvant protocol mosecs treated withcarboplatin alone from day for day and then with a combination of metformin and carboplatin for day pink circles represent log ci valuesunder protocol green squares represent those under protocol and yellow triangles represent those under protocol concentration ofmetformin and mm concentration of carboplatin and μm met metformin chemo chemotherapy imagej analysis for relativeintensity of protein bands c mosecs were injected into b6 mice n which were divided into groups and subcutaneous tumor size wasmeasured after different treatments control metformin or carboplatin alone neoadjuvant metformin from monday combined with carboplatinfrom wednesday subcutaneous tumors were assessed at the end of the experiment control vs met p control vs chemo andmet chemo p d kaplanmeier os of ovarian cancer patients with n or without n metformin in before or duringchemotherapy e the model of synergistic inhibitory effects by neoadjuvant metformin combined with chemotherapy in the aktmtor pathwayadministered lowdose metformin mm or mmto suppress ovarian and breast cancer cell growth and survival in vitro the aim of their study was to investigatewhether lowdose metformin reprograms these cancercells into noncancerous cells in a foxo3dependentmanner which may allow patients to successfully overcome these cancers with minimal side effects howeverthey did not compare the inhibitory effects or therapyprotocol of metformin combined with chemotherapy inaddition they administered metformin via intravenous injection of metformin mgkg bw in mice which contrasts the more common oral route used in clinicalapplications for example diabetes mellitus in thepresent study we tested a low concentration of mm μmoll as the effective dose the cellular events observed in vitro suggest that this dose is safe and can betranslated to in vivo conditions the dosage of metformingiven to mice was mgkgday which is equivalent to mgday for a 60kg person according to a formulasuggested by the national institute of health usa this equivalent dosage is times lower than the maximum safe dosage of mgday recommended in thephysicians desk referencemetformin activates ampk via lkb1 which leads tothe inhibition of mtor signaling and its major downstream effectors the 4ebps and p70s6ks and the inhibition of global protein synthesis and proliferation invarious cancer cell lines [ ] in previous studies metformin tested on ovarian cancer was found to induce cellcycle arrest apoptosis angiogenesis and decreasedpmtor expression p38 mapk pathway activity and cancer stem cell activity in this study weshowed for the first time the effects of metformin on geneexpression patterns using gesa and the l1000 systemand found that the effects of metformin on gene expression in ovarian cancer are similar to those following activation of the kegg pancreatic cancer pathway fig s1in previousfollowing metformin treatmentphosphoakt levels decreased in two pancreatic cancercell lines a549 and panc1 we analyzed tcga dataand found that the phosphoaktmtor pathway is a determinant of clinical survival in ovarian cancer [ ] westudiesalso demonstrated for the first time the effects of metformin in combination with carboplatin a firstline chemotherapy drug and showed that the synergism of thesedrugs is due to the inhibition of the aktmtor pathwaywhich is independent of ampk at micromolar concentrations of metformin mm these results are consistentwith those of rattan who showed that metformin as an antiproliferative therapeutic can act throughboth ampkdependent and independent pathways metformin can be a veryveryvery inexpensive drug compared with aktmtor inhibitors and it may also haveantitumor effects knowledge of this mechanism may beuseful in clinical trials to adjust the dosage of chemotherapy or further overcome the chemoresistance to platinumin the futuremetformin can be added at different times beforeduring and after adjuvant chemotherapy and the effectsof these addition time points need to be investigated inthe present study a synergistic effect ci was observed in the neoadjuvant protocol and concurrentprotocol protocols but not in protocol adjuvantneoadjuvant metformin protocol was better than theconcurrent regimen these results are similar to those oferices although they did not assess the adjuvant use of metformin the optimal time frame of neoadjuvant metformin before chemotherapy should bedetermined in future studies or clinical trials althoughwe observed an inhibitory effect of neoadjuvant metformin day prior to chemotherapy in vitro and daysprior to chemotherapy in vivo furthermore the ovariancancer patients have a better prognosis with metformincombined with before or during chemotherapy compared with chemotherapy alone due to the small casenumber and retrospective analysis our results are notsignificant the optimal time frame for neoadjuvant metformin administration before chemotherapy will allow forthe acquisition of the most ideal results in basic experiments and clinical analyses these findings provide beneficial evidence that can guide the design of clinical trialsthe differences among protocols observed in this studymay be related to the cytotoxic effects of metformin oncancer stem cells which can enhance the efficacy of 0cwen of ovarian research page of neoadjuvant and concurrent chemotherapy by preventingthe establishment of chemoresistant clonesreducesome patients treated with metformin continue to showpoor blood sugar control and high blood glucose may diminish the antitumor effect of metformin karnevi reported that metformin can significantlytheproliferation of several pancreatic cancer cell lines under normal glucose conditions however they found that hyperglycemia reduced metformininduced growth inhibition byenhancing the igfi response and activating akt whichstimulated ampkser485 phosphorylation and impairedampkthr172 zhuang obtained similar results inbreast cancer and ovarian cancer cell lines reporting that cancer cells became less responsive to metformin when glucosewas increased to mm in a breast cancer cell line underlowglucose conditions metformin significantly decreased thephosphorylation of akt and various targets of mtorwhereas phosphoampk was not significantly altered in thepresent study we used an ovarian cancer cell line and demonstrated that a highglucose medium decreased the response tometformin the synergistic effects of carboplatin and metformin were abolished the phosphorylation of akt in lowglucose conditions mgl was substantially reduced bymetformin and phosphorylation levels of targets of mtors6k and 4ebp1 were decreased relative to those in highglucose conditions mm in ovarian cancer phosphoampk was not significantly altered the response to metformin was substantially altered in lowglucose conditions basedon previous studies and our observations we hypothesize thathigh glucose fuels glycolytic metabolism which maintains cellular atp levels when metformin blocks mitochondrial function when glucose is limiting cancer cells lack sufficient fuelto maintain glycolytic metabolism additionally mtor signaling is blocked in an ampkindependent manner enhancingmetabolic deficiency cellular atp is depleted leading to energy collapse and cell death sin lowconcentration metformin treatment ofpatients with ovarian cancer may have antitumor effectsand synergistic effects when used in combination withchemotherapy through the aktmtor pathway neoadjuvant metformin is a more preferable protocol than theconcurrent regimen future prospective clinical trials inpatients with ovarian cancer are required to investigatethe beneficial effects of neoadjuvant metformin in augmenting the efficacy of existing cancer therapeuticsmethodspatient samples from taipei veteran general hospitalmedical centera total of patients were diagnosed with primaryovarian cancer in the department of gynecology andobstetrics taipei veteran general hospital from to after a review of the patients clinical and drughistories patients who underwent complete surgeryand were treated with platinumbased therapy plus paclitaxel were included in the analysis of these patients were identified as having taken metformin eitherduring admission or in the outpatient clinic the overallsurvival os was measured from the date of diagnosisto death or was censored at the date of the last followup all documents were collected under protocols approved by the institutional review board of the hospitalpatient samples from the national health insurancetaiwanese datasetthe reimbursement data of taiwanese female patientswith a new diagnosis of type dm between and n were retrieved from the nationalhealth insurance database among these patients noneused only insulin or only metformin therefore we compared two groups those who received metforminand insulin n and those who received neiinsulin n then wether metformin norfollowed the two groups for newly diagnosed ovariancancer from to thirtyseven patients acrossthe two groups were diagnosed with ovarian cancermicroarray analysisthe microarray experiments were conducted following thel1000 operating procedure l1000 sop briefly thehuman ovarian cancer cell line es2 was left untreated control or treated with micromolar concentrations of metformin mm μm carboplatin or a combination in amicroplate after h of drug treatment the medium was removed and lysis buffer was added included in the l1000kit to the wells for min after cell lysis the lysate wasstored at °c for at least one night before being transferred to a 384well plate which was performed using theprotocol avai
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" phytolaccaceae species in china are not only ornamental plants but also perennial herbs that areclosely related to human health however both largescale fulllength cdna sequencing and reference genevalidation of phytolaccaceae members are still lacking therefore singlemolecule realtime sequencing technologywas employed to generate fulllength transcriptome in invasive phytolacca americana and noninvasive exotic picosandra based on the transcriptome data rtqpcr was employed to evaluate the gene expression stability in thetwo plant species and another indigenous congener p acinosaresults total of gb and gb clean reads of p americana and p icosandra were generated including and full length nonchimeric flnc reads respectively transcript clustering analysis of flnc readsidentified and consensus isoforms including and highquality ones after removingredundant reads and transcripts were obtained based on structure analysis total and alternative splicing variants and simple sequence repeats ssr as well as and completecoding sequences were detected separately furthermore and lncrna were predicted and and transcripts were annotated respectively subsequently seven reference genes in the two plant species and anative species p acinosa were selected and evaluated by rtqpcr for gene expression analysis when tested indifferent tissues leaves stems roots and flowers 18s rrna showed the highest stability in p americana whetherinfested by spodoptera litura or not ef2 had the most stable expression in p icosandra while ef1α was the mostappropriate one when attacked by s litura ef1α showed the highest stability in pacinosa whereas gapdh wasrecommended when infested by s litura moreover ef1α was the most stable one among the three plant specieswhenever germinating seeds or flowers only were consideredcontinued on next page correspondence yiwangynueducn1yunnan key laboratory of plant reproductive adaption and evolutionaryecology yunnan university kunming china2laboratory of ecology and evolutionary biology state key laboratory forconservation and utilization of bioresources in yunnan yunnan universitykunming chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cliu bmc plant biology page of continued from previous page fulllength transcriptome of p americana and p icosandra were produced individually based on thetranscriptome data the expression stability of seven candidate reference genes under different experimentalconditions was evaluated these results would facilitate further exploration of functional and comparative genomicstudies in phytolaccaceae and provide insights into invasion success of p americanakeywords phytolaccaceae smrt sequencing fulllength transcriptome analysis reference gene evaluation rtqpcr phytolacca americana is a member of the phytolaccaceae family and is native to northeast america becauseof its ornamental and medicinal applications it was introduced into china in unfortunately it hasevolved into an invasive species and spread to mostareas of the country especially in central and southernchina compared to noninvasive exotic congener p icosandra and native congener p acinosa p americana isof interest because it exhibits multiple biological activities such as plant pesticides antimicrobial propertyheavy metal accumulation capacity []in order to investigate the mechanisms of various bioactivities of p americana further transcriptomewidestudy is necessary to facilitate reports have showed thatjasmonic acidinduced and cadmiumtreated transcriptome data of p americana have been obtained by illumina hiseq and illumina hiseq platformrespectively [ ] howeverthese data were bothachieved by second generation sequencing sgs whichcould not produce fulllength transcripts genomic dataof p americana was available at the sra under projectprjna544344 but its raw reads without coding sequences prediction and functional annotation third generation sequencing tgs is known for itskilobasesized long reads and is an outstanding strategyfor better understanding rna processing for exampleit can be used to analyze different transcript isoformsregulated by alternative splicing which greatly increasesthe repertoire of proteins lead to genetic and functionaldiversity and is prevalent in most eukaryotic anisms the long reads could also provide sequence information on genecoding regions for functional analysis atthe transcriptional level and thus can be applied to refine an assembled genome for better annotation however tgs could not quantify gene expression forthe moment and have a relatively high error rate thansgs the combination of tgs and sgs are able to solvethis problem and are highly recommended by most researchers with the transcriptome and genome data availablefunctional genomics research is being performed whichrelied heavily on gene expression analysis reversetranscription quantitative real time pcr rtqpcr hasbeen reported to be a very sensitive and accurate technique to analyze gene expression level but it requiresappropriate reference gene as an internal control tonormalize mrna levels between different samples foran exact comparison of gene expression [ ] anideal reference gene should be expressed at a constantlevel across various experimental conditions howeverstudies have shown that no single reference gene is universal for all experimental conditions [] therefore its necessary to estimate the stability of referencegenes under particular experimental condition beforeusing them for gene expression analysisin the present study to provide highquality and morecomplete assemblies in genome and transcriptome studies of phytolaccaceae a hybrid sequencing approachcombining the sgs and tgs technologies was carriedout first fulllength transcriptome of the invasive plantspecies of pameracana and an noninvasive exotic conicosandra was generated by singlemoleculegener prealtimesmrtsplicingevents simple sequence repeats ssr coding sequencesprotein annotations and long noncoding sequenceswere analyzed respectively at transcription level furtherthe stability of reference genes was evaluated in two phytolaccaceae species mentioned above and one nativecongener p acinosa by rtqpcr in order to facilitatefuture research on functional gene expressionsequencing alternativeresultsto classify the plant species these three phytolaccaceaemembers p americana p icosandra and pacinosa wereidentified by pcr and followed by sequences alignmentbased on sequences of second internal transcribed spacer its2 and the intergenic spacer of photosystem iiprotein d1 gene and trnahis gene of chloroplast genome psbatrnh table s1 the sequences of its2 andpsbatrnh in p americana that we employed had theidentity of with the sequences reported by chen in p icosandra the sequences of its2 had identity and the sequences of psbatrnh had identity with the results of chens in pacinosa 0cliu bmc plant biology page of similarity of its2 and identify of psbatrnh werefound isoforms afterremoving redundantsmrt sequencing data outputusing the pacific biosciences smrt sequencing protocol gb clean reads of invasive species p americana were obtained after preprocessing on the basis offull passes and sequence quality circular consensus sequences ccs with fulllength rate were obtained including fulllengthnonchimeric flnc sequences and highqualityconsensussequences from the high quality consensus isoforms transcripts alternative splicing events ssr complete coding sequences lnc rnasand annotated transcripts in p americana wereachieved similarly gb clean reads in p icosandrawere identified and ccs with fulllength rate flnc sequences as well as highquality consensus isoforms were filtered subsequently transcripts and alternative splicingevents were obtained whats more ssrs complete coding sequences lnc rnas and annotated transcripts were identified in picosandratable transcriptome analysisbased on the structure of achieved transcripts and alternative splicing events were identified in pamericana and p icosandra respectively transcripts of bp in total in p americana wereemployed for ssr analysis based on the sequence lengththat was more than bp including ssrs and ssrcontaining sequences similarly transcripts bp in total in picosandra wereemployed for ssr analysis and ssrs togetherwith ssrcontaining sequences were identifiedtable summary of fulllength transcriptome sequencingclean reads gbccsflncflnc flncccsconsensus isoformhigh quality consensus isoformtranscriptsalternative splicingssrcomplete coding sequenceslncrnaannotated transcriptsp americanap icosandrathe detail information about the number of sequencescontaining more than one ssr the number of ssrspresent in compound formation and the number of different types of ssrs were shown in table in additiontotal of complete coding sequences cds in pamericana and cds in p icosandra were identified by using transdecoder the length distribution ofpredicted proteins was shown in fig s1in pdatabasespecifically nrwith the eight protein databases sequence alignmentswere performed to annotate predicted proteins in total transcripts in p americana and tranicosandra were annotated separatelyscriptstable the number of annotated protein sequencesin p americana was similar with p icosandra under aparticularncbi nonredundant protein analysis revealed that approximately transcripts in p americana and transcripts in picosandra showed the highest sequencesimilarity with beta vulgaris fig go gene ontology assignment also suggested that similar amount ofsequences in the two plant species belonged to the sameterm and many were classified into cell part and cellterm of cellular component catalytic activity and binding of molecular function and metabolic process andcellular process of biological process fig cogclusters of orthologous groups of proteins annotationshowed that a large number of predicted proteins in thetwo plant species were linked to functional class r general function prediction only j translation ribosomalstructure and biogenesis t signal transduction mechanisms g carbohydrate transport and metabolism ando posttranslational modification protein turnoverchaperones fig s2 the result of eggnog evolutionary genealogy of genes nonsupervised orthologousgroup annotation indicated that most of the annotatedproteins in the two plant species were belonging to thefunctional class s function unknown fig s3 kogeukaryotic ortholog groups functional classificationsuggested that r general function prediction only ando posttranslational modification protein turnover andchaperones were the most abundant functional categories in the two plant species fig s4 these results indicated that most of the sequences obtained were trulyfunctional proteins and had a similar functional classification in p americana and its congener picosandraeven though more work is needed to identify sequencesthat regulated or involved in the invasion success of pamericana the annotation of predicted proteins provided necessary information for further studiesbesides the transcripts encoding proteins long noncoding rnas lncrnas were achieved lncrnas arereported to be key regulators in plant biological prolncrna in pameracana andcesses the number ofpicosandra was predicted by cpc coding potential 0cliu bmc plant biology page of table ssrs obtained from transcripts with more than bpsearching itemtotal number of sequences examinedtotal size of examined sequences bptotal number of identified ssrsnumber of ssr containing sequencesnumber of sequences containing more than ssrnumber of ssrs present in compound formationnumber of mono nucleotide ssrnumber of di nucleotide ssrnumber of tri nucleotide ssrnumber of tetra nucleotide ssrnumber of penta nucleotide ssrnumber of hexa nucleotide ssrcalculator cnci codingnoncoding index pfamand cpat coding potential assessment tool respectively in total lncrna in pamericana and lncrna in picosandra were predicted by all these fourmethods fig subsequentlytranscription factorstfs that are key components involved in the transcriptional regulatory system were predicted in p americana tfs of types were filtered and in picosandra tfs of types were predicted thesetwo plant species shared the first types of tfs butthe number of each type tf was not similar especiallyrlkpelle_dlsv c3h snf2 and camk_camklchk1 indicating the particular functions on transcriptregulation fig amplification performance of rtqpcrprimers designed for rtqpcr were evaluated by pcrfirst the primers which produced single ampliconwithout primer dimer were chosen for melting curveanalysis only primers which produced a single fragmentefficiencywereqpcr amplificationchosenforp americanap icosandraassessment the qpcr efficiency of each primer pair wasgenerated from a 10fold serial dilution of pooled cdnaand was shown in table the threshold cycle ct values of each reference genewere employed to evaluate expression level under different experimental conditions fig average ct valuesfor all the seven candidate reference genes ranged from to in which ef1α showed the highest expression level and 28s rrna had the lowest expression levelit was also suggested that ct values of βactin and tubulin fluctuated significantly across all the experimentalsamplesstability of candidate reference genesforto determine the appropriate reference genesnormalization in different experimental conditions theexpression data was analyzed by genorm normfinderand bestkeeper respectively table s2when expression stability of reference genes wereanalyzed in different tissues leaves stems roots andflowers of p americana 18s rrna and ef2 oftable number of proteins annotated via differential protein databasedatabasesp americanaannotated number ¤ length length ¥ p icosandraannotated number ¤ length length ¥ coggokeggkogpfamswissproteggnognrall 0cliu bmc plant biology page of fig homologous species distribution of p americana and p icosandra annotated based on the nr database a p americana b p icosandrapamericana were identified as the most suitable reference genes by genorm and normfinder and 18s rrnawas also suggested by bestkeeper pairwise variation valueof v23 was below the cutoff value of which meansthe combination of two reference genes were most suitable for gene expression normalization fig whentested in picosandra ef1α was recommended for normalizing gene expression analysis not only by genorm butalso by normfinder ef2 was also suggested by genormand bestkeeper in pacinosa ef1α was the best reference gene suggested by genorm and bestkeeper but 18srrna was recommended by normfinder the use of tworeference genes was suitable because pairwise variationvalue of v23 was below when pooled the data of different tissues from pamericana and picosandra togetheref2 was shown to be the most stable gene by all the threemethods when investigated the expression stability ofreference genes in different tissues of pamericana andpacinosa 18s rrna showed the best expression stabilityby genorm and normfinder while ef2 was referred asthe most stable one by bestkeeper however the combination of five reference genes was recommended bygenorm for v56 which was less than when thedata of different tissues from picosandra and pacinosawas put together ef1α was identified as the best oneby genorm and normfinder whereas ef2 was suggested to be the best stability reference gene by bestkeeper when set the data of these three plant speciesas a pool ef1α was suggested to be the most stableone by genorm and normfinder while ef2 was alsorecommended by genorm and bestkeeper accordingto these results it is very important to select the appropriate reference gene when analyze the gene expressionlevel among plant species 0cliu bmc plant biology page of fig classification of the transcripts annotated by the gene ontology gowhen analyzed the data among germinating seeds28s rrna and ef1α were identified as the best reference genes by genorm while 18s rrna was recommended by normfinder and gapdh was suggested bybestkeeper three reference genes were sufficient tonormalize gene expression for v34 was below inflowers only of these three plant species ef1α was confirmed by all the three methods the genorm analysisshowed that the value of v45 was below so fourreference genes in combination were suggested thesefig venn diagram of the number of lncrnas predicted by cpc cnci cpat and pfam a p americana b p icosandra 0cliu bmc plant biology page of fig classification of predicted transcription factorsresults indicated that when focusing on particular tissuesof different plant species the selection of reference genewas also very essentialwhen plants were infested by slitura 18s rrnashowed the most expression stability suggested by genorm and normfinder in different tissues of pamericanawhile ef1α wasby bestkeeper therevealedcombination of two reference genes was suggested bygenorm due to the value of v23 was less than 18srrna was also recommended by genorm in s liturainfested picosandra and ef1α was shown to be themost stable one by normfinder and bestkeeper fourreference genes in combination were recommended bygenorm 18s rrna was also identified as the besttable primers for rtqpcr analysisgene nametubulingene descriptiontubulin ef1αelongation factor 1alphaprimer sequence ²²f gtaaggaagccgagaattgr tcaacaacagtgtcagagaf tgaagaaggtcggatacaatr gtagacatcctggagtgggaphdglyceraldehyde3phosphate dehydrogenasef tggtgctaagaaggttattatcef2elongation factor 18s rrna18s rrnaβactinactin728s rrna28s rrnar2 linear regression coefficientr gagtgaacggtggtcataf gtatcaccatcaagtcaactgr acaatcaaccacaacaaggf acttcctcttctcgtatcattr tgttcagcatagactgtgaf atgctatccttcgtctggr tactcttggctgtctctgf tacgattggttacggacatr ttctcatcaacaacagcatatlength bppcr efficiency r2 0cliu bmc plant biology page of together 18s rrna showed the best stability in genormand normfinder while the expression stability of ef1αwas suggested by bestkeeper in pamericana and picosandra 18s rrna was identified as the best referencegene by all the three algorithms in pamericana andpacinosa 18s rrna and βactin were suggested bygenorm in picosandra and pacinosa while gapdhand ef2 were recommended by normfinder and bestkeeper respectively when take all the data of s liturainfested plant species into account 18s rrna exhibitedthe most stable expression suggested by genorm andnormfinder while ef2 was the gene with the most constant expression identified by bestkeeperdiscussionfulllength transcripts are fundamental resources forstructuralfunctional and comparative genomics research [ ] smrt sequencing has been acknowledged by enabling the generation of multikilobasesequences to improve genome and transcriptome assembly the fulllength cdna sequences generated areable to characterize the posttranscriptional processsuch as alternative splicing lncrna prediction and coding sequences for further gene functional studies basedon the fulllength transcriptome data generated about gb of clean data were obtained for pamericana andpicosandra respectively table accordinglythenumber of ccs flnc consensus isoforms highqualityfig rna transcription levels of seven candidate reference genesin p americana picosandra and p acinosa the expression level ofcandidate reference genes in total samples n was presentedas cycle threshold number ctvalue and explained by box andwhisker plots the asterisks represented the minimum and maximumct value the squares indicated the 25th and 75th percentiles andthe median was represented by a bar across the squarereference gene by genorm in plant species pacinosawhile tubulin was suggested by normfinder and 28srrna was recommended by bestkeeper the combination of three reference genes was appropriate by genorm when analyzed the data oftwo plant speciesfig pairwise variation analyzed by genorm to determine the optimal number of reference genes for accurate normalization a threshold valueof was suggested for valid normalization if the value of vnn pairwise variation is less than then n reference genes in combinationare recommended for gene normalization if the value of vnn is more than then vn 1n should be taken into account pam pamericana pic p icosandra pac p acinosa lsrf different tissues of leaves stems roots and flowers gs germinating seeds of these three plantspecies f flowers of these three plant species lsr different tissues of leaves stems and roots i infested by s litura of third instar 0cliu bmc plant biology page of isoforms transcripts alternative splicing events ssrscomplete coding sequeces lncrnas and annotated transcripts were analyzed providing basic transcriptomic information for further studiesreports have showed that fulllength transcriptome ofzea mays have greatly helped in refining gene annotation and revealed the complexity of gene expression inmaize similar analysis has also been conducted inshum bicolor whats more the world expansioncapability of cydia pomonella has been informed according to its genome information molecularmechanism of rapid growth and invasive adaptation ofan invasive species mikania micrantha has also been investigated according to itsreference genome therefore the fulllength transcriptome data of pamericana and picosandra will contribute to the genomic research and provide insights into invasive mechanism ofpamericana through comparative genomics study inphytolaccaceae speciesgenereliesonanalysisexpressionaccurate relative quantification of rtqpcr for furtherrobustnormalization by stably expressed reference genes tominimize error in the experimental process therefore suitable reference genes for the normalization ofrelative gene expression data in three phytolaccaceaespecies pamericana picosandra and pacinosa weresought under a diverse set of conditions these resultsdemonstrated the importance of validating referencegenes under the relevant experimental conditions forexamplein different tissues leaves stems roots andflowers of pamericana 18s rrna and ef2 were recommended to be the bestsuited reference genes and similar results were found in s liturainfested pamericanahowever even though the appropriate reference genesin picosandra were ranked according to the analyzed results of the three methods all the pairwise variationvalues were above the cutoff value of while thecombination of 18s rrna βactin ef1α and ef2 weremost suitable in s liturainfested picosandra ef2 andef1α have been considered as the ideal reference genesin pacinosa whereas the combination of 18s rrna βactin and gapdh were recommended after s litura infestation researches have also showed that no singlereference gene is stably expressed among different tissues of an anism such as the reference gene selectionin amygdalus persica solanum lycopersicum and glycine max [ ] whats more our results alsosuggested that reference genes identified based on transcriptome data should be confirmed by experimentalevidence in jainduced transcriptome of p americana28s rrna showed stable expression between exogenousjatreated and control plants ja signal pathway ofplants can be induced by lepidopteran herbivores infestation however 18s rrna and ef2 were identifiedas the most stable expression reference genes in pamericana after s litura infestationin order to conductthe gene expression analysisamong different plant species of phytolaccaceae the dataof the three plant species were also compared togetherwhen compared the data in germinating seeds of threeplant species various genes were recommended by thethree methods the combination of plant species underother experimental conditions showed that the pairwisevalues of almost all the combination were higher thanthe cutoff value of exceptthe combination ofpamericana and pacinosa where five reference geneswere recommended for data normalization as well as thecombination of sliturainfested pamericana and sliturainfested picosandra where three reference geneswere suggested these results indicated that no particular gene was expressed constantly across different plantspecies even though these plants are congeners therefore reference genes should be employed appropriatelyunder the relevant experimental conditionsthe research has provided transcriptomewide fulllength isoforms of pamericana and picosandraproviding insights into invasive success of pamericanaguidelines for selecting appropriate reference genesunder different tissues in one plant species or amongvaried plant species were recommended further no particular gene was expressed constantly under differentexperimental conditions indicating the necessity of reference gene identification these results would facilitatethe exploration of functional and comparative genomicsstudies in phytolaccaceae to better understand plantbiologymethodsplant and insect materialsplants of p americana °²n °²e p icosandra°²n °²e and p acinosa °²n °²eused in this study which was named m k and q firstwere collected in yunnan china sampling was permitted when conducted complying with locallegislationthe formal identification of the samples were conductedby chao chen botany major of laboratory of ecologyand evolutionary biology state key laboratory for conservation and utilization of bioresources in yunnanyunnan university according to flora of china vol5 flora of north america vol43 chinese virtual herbarium httpwwwcvhaccn and global plants on jstor httpplantsjstor dna identification was also employed according tothe its2 region of nuclear ribosomal dna one of themost widely used dna fragments in plant molecularsystematics at the generic and species levels and the 0cliu bmc plant biology page of chloroplast psbatrnh intergenic region all voucher specimens were maintained at an experimental fieldof laboratory of ecology and evolutionary biology statekey laboratory for conservation and utilization of bioresources in yunnan yunnan universitytissues of leaves stems roots and flowers from oneindividual plant of p americana or p icosandra werecollected individually from the wild in yunnan provinceand no permission is needed for collecting theses samples each sample was flash frozen in liquid nitrogen andstored at °c for further experimentsshop101732681taobaocomthird instar larvae of spodoptera litura were purchased from henan jiyuan baiyun industry co ltdchinaand then werereared on artificial diet in a climate chamber h at °c with light and h at °c without light for further usefor reference gene evaluation seeds of p americanap icosandra and p acinosa were collected first from thewild in yunnan province and no permission is neededthe seeds were sown separately in agar plates andcultivated in the climate chamber after d five germinating seeds of one plant species were collected togetheras one sample for subsequent experiments each plantspecies have three replications two weeks later othergerminating seeds of each species were transplanted intoplastic pots cm diameter and cm height withsoil jiangsu peilei matrix technology development coltd china and cultivated with adequate water in artificial chambers with same conditions as described abovefour months later leaves stems roots and flowers ofeach plant species were collected individually simultaneously six larvae s litura of third instar were employedto infest on p americana p icosandra or p acinosawith one insect per leaf control treatments were herbivore free after h infestation leaves stems and rootsof these three plant species were harvested individuallyall samples collected were flash frozen in liquid nitrogenand stored in °c for subsequent assays and threereplicates were conducted for each treatmentnucleic acid extraction and assaysgenomic dna was isolated from the leaves of differentplant species following protocols provided by dnaquickplant system tiangen biotech co ltd beijing chinathen it was employed as the pcr template for plantspecies identificationpurekitplanttotal rnas from different tissues was prepared usingrnapreppolysaccharides polyphenolicsrich tiangen biotech co ltd beijingchina according to the manufacturers instructionsthe rna quality and purity were measured by using ananophotometer n60 implen germany and the agilent bioanalyzer system agilent technologies causa samples only with a ratio of to a ratio between and and a rin value morethan were chosen for the sequencing library construction an equal amount of total rnas from four different tissues of the same plant species were mixed asone sample for fulllength transcriptome sequencingtotal rnas from the samples collected for referencegene evaluation was also extracted individually as described above for each sample cdna was prepared byusing μg of total rna following the recommendedinstructions of fastquant rt kit with gdnase tiangenbiotech co ltd beijing chinapacbio cdna library preparation and smrt sequencingfulllength cdna was synthesized by using the smarter¢ pcr cdna synthesis kit clontech ca usathe generated cdna was then reamplified using pcrafter end repairing smrt adaptor with a hairpin loopstructure was ligated to the cdna via exonucleasedigesting the cdna library was constructed after quality measurement of the cdna library smrt sequencingwas performed using the pacific bioscience sequel platform following the provided protocolillumina cdna library construction and secondgenarationsequencingthe extracted mrna was purified using oligo dtattached magnetic beads fragmentation was conducted inthe nebnext first strand synthesis reaction bufferfirststrand cdna was acquired based on the randomhexamers and then the secondstrand cdna was synthesized with dntps rnase h and primestar gxldna polymerase the synthesized cdna was purifiedwith ampure xp beads after end repairing adding polya and adaptor ligation ampure xp beads were used forsize selection the generated cdna was then amplifiedfor building cdna libraries the qualified libraries werepair end sequenced on illumina nova platformquality filtering and error correction of long readsraw smrt sequencing reads were filtered by removingpolymerase reads less than bp and sequence accuracyless than after removing adaptor subreads were obtained clean data was produced with subreads morethan bp ccss were produced from clean data withparameters of full passes and accuracy over after examining the coexistence of ² and ² adaptorsand poly a tail fulllength transcripts were selectedduring the processes of library preparation the chimericsequences formed by the direct linkage of two cdnatemplate strands due to the low concentrations ofadaptor or smrtbell are called artificial chimeric sequences the nonchimeric sequences in the fulllength 0cliu bmc plant biology page of transcripts are the fulllength nonchimeric flncsequencesas smrt sequencing generates a high error rate it isnecessary to perform error correction iterative clustering was used first to obtain consensus isoforms and thefulllength consensus sequences from iterative clusteringfor error correction were refined using quiver [ ]moreover the raw illumina sgs reads were filtered toremove adaptor sequences and low quality reads anderror correction of lowquality isoforms was conductedusing the sgs reads with the software proovread inbriefly the short reads of illumina rnaseq data weremapped to the low quality isoforms and then the basein the low quality isoform was replaced by the particularbase that had the maximum number
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mgat5 knockout ko in hek293 cells induces metabolic changes resulting in increased intracellular udpglcnac increasedglycolysis enhanced spare respiratory capacity and higher citrate ï¬ux from the mitochondria mgat5 ko cells express constitutively high mica mainly regulated onthe transcriptional level through opening of the chromatin at the mica promoter mica expression in mgat5 ko cells is dependent on citrate turnover and histoneacetylation blocking citrate ï¬ux inhibits mica expression in numerous cancer cell lines and we propose that this is a central metabolic regulation of mica andimmune surveillanceintroductionnatural killer nk and cd8 t cells monitor autologouscells for markers of tumorigenesis and stress these immunecells express the nkg2d receptor that recognizes nkg2dligands nkg2dls upregulated on the surface of transformedcells nkg2dl expression is in many ways a doubleedged sword upregulation of nkg2dls on cancer cellsenhance nk cell ltration and promote cancer cytotoxicity conversely numerous cancer cells maintain chronicnkg2dl expression and evade immune elimination bydownmodulating and impairing nkg2d receptor signalingcancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdacisheatshock or shortchain fatty acids scfas that upregulatenkg2dls to date studies have primarily focused ondelineating transient nkg2dl induction whereas not much isknown about regulation of their constitutive expressionmetabolic reprogramming is a central hallmark of cancercancer cells use aerobic glycolysis that was initially believedto be a result of dysfunctional mitochondria howeverlater advances have shown that cancer cells often use aerobicglycolysis alongside mitochondrial oxidative phosphorylationoxphos mitochondria are not merely the powerhouseof the cell but also provide metabolites for anabolic pathwaysnecessary for cell growth citrate can be exported from thetricarboxylic acid tca cycle for biosynthetic purposes inthe cytosol citrate is cleaved by atp citrate lyase acly togenerate acetylcoa and oxaloacetate oaa citrateis an inhibitor of glycolysis thus to maintain high aerobicglycolysis cancer cells require low cytoplasmic citrate moreover conversion of citrate by acly is a critical regulatorof gene transcription by producing acetylcoa for histoneacetylation several of these cancerassociated metabolicfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionproperties are shared with other highly proliferating cells suchas activated t cellsexpression of nkg2dls is associated with hyperproliferation and thus with highly active metabolism two studies havelinked nkg2dl expression to active glycolysis whereasone study reports that inhibition of glycolysis increased basalnkg2dl expression in breast cancer cell lines thesestudies emphasize a link to proliferative cell metabolism andsuggest that the role of glycolysis in nkg2dl regulation iscontextspeciï¬cnkg2dls fall into two groups the ul16 binding protein ulbp16 and the mhc class i chainrelated proteins aand b mica and micb surface expression of each nkg2dlis regulated individually and at all levels of protein biogenesis we have previously shown that surface expression ofspeciï¬c mica alleles depends on nglycosylation nacetylglucosaminyltransferase v mgat5 is an oncoproteincatalyzing the formation of 16branched nglycans thatpromote surface retention of glycoproteins but it is notknown if mgat5 regulates surface expression of mica growthfactor receptors are examples of mgat5 substrates and mgat5overexpression is associated with growth adhesion invasionand metastasis of cancer inhibition of mgat5 reducestumor growth enhances the antitumor responses by cd4 tcells and macrophages and promotes th1 diï¬erentiation in this study we examine the metabolic regulation of thenkg2dl mica we discover that mica was increased aftermgat5 knockout ko in a metabolically dependent way anduse this as a model to investigate the regulatory mechanismsof constitutive mica expression we ï¬nd that glycolysis andmitochondrial export of citrate promotes constitutive micatranscription in mgat5 ko cells a regulation that was alsoshown in several micaexpressing cancer cells in particularincreased mica transcription was associated with alteredchromatin accessibility of the mica promoter our ï¬ndingssuggest that citrate drives a metabolic stress that modulateschromatin accessibility to facilitate basal mica transcription andthereby regulate immune surveillancematerials and methodsanimalsfemale nmri mice to 10weeks old taconic lille skensveddenmark were used and all studies were performed inaccordance with the danish act on animal experimentationwhich implements directive 201063eu on the protection ofanimals in scientiï¬c research the studies were approved by theanimal experimentation inspectorate ministry of environmentand food denmark license no healthmonitoring was carried out in accordance with federation forlaboratory animal science associations guidelinesreagents pharmacological inhibitorsand dna constructspharmacologicalfrom sigmaaldrich werecompoundsnacetyldglucosamine glcnac a3286 pugnac a7229carbonylcyanide2dg d61345aminoimidazole4carboxamide2deoxydglucosetriï¬uoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulï¬de bptes sml0601 potassium hydroxycitrate tribasicmonohydrate hc sodium dihydrogencitrate sodium acetate s5636 oxaloacetic acid oaa o41266mercaptopurine monohydrate 6mp azaserinea4142ribonucleotideaicar a9978 nacetylcysteine nac a9165 sodiumpropionate p1880 sodium butyrate b5887 dmso d2438pbs d8537 etomoxir sodium salt was purchased fromcayman chemicals ann arbor mi united states bristol bms303141 wasunited kingdom the gfpmycmica and mica vectors containingthe coding sequences of mica or mica alleledownstream of a generic leader a gfp cassette and a myctag were provided by dr m wills university of cambridgecambridge united kingdom pgl3basic pgl3bluciferase vector was purchased from promega promegamadison wi united states e1751 micaï¬reï¬y luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c ocallaghan university of oxfordoxford united kingdom from tocris biosciencepuriï¬cation of peripheral bloodlymphocyteshuman peripheral blood mononuclear cells pbmcs wereisolated by histopaque1077 sigmaaldrich st louis mounited states separation from buï¬y coats obtainedfrom healthy blood donors the capital region blood bankcopenhagen university hospital copenhagen denmark toobtain peripheral blood lymphocytes pbls pbmcs weredepleted from monocytes by incubation with dynabeadsinvitrogen carlsbad ca united states as previouslydescribed pbls were activated in rpmi1640 withoutglucose gibco gaithersburg md united states supplemented with dialyzed fetal bovine serumfbs f9665 mm penicillinstreptomycin p4333 mmlglutamine g7513 mm sodium pyruvate s8636 and either mm dglucose g8769 or mm dgalactose g6404all purchased from sigmaaldrich pbls were activated withcd3cd28 beads invitrogen 11132d and 20uml hil2peprotech rocky hill nj united states for dayson day pbls were treated with ngml fr901228 nationalcancer institute bethesda md united states for hline pc3 and the keratinocytederived cellcell line cultivation and proliferationhuman embryonic kidneyderived hek293 cells the prostatecancer celllinehacat were purchased from american type culture collectionatcc manassas va united states nkg2d reporter cellct312 and the 2b4 parental cellline were kindly providedby chiwen chang trowsdale lab cambridge universitylines mdamb231 and mcf7 werethe breast cancer cellprovided by dr jos moreira departmentfor veterinaryfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressiondisease university of copenhagen denmark and henrikleï¬ers the state hospital copenhagen denmark respectivelythe cervical cancer cellline hela was provided by jesperjurlander the state hospital copenhagen denmark themelanoma cells skmel28 fm55m1 fm78 and fm86 andthe human colon adenocarcinoma cell lines ht29 and sw480were provided by dr per thor straten herlev universityhospital denmark hek293 mdamb231 and mcf7 cellswere cultured in dmem with glutamax gibco hela hacat pc3 fm55m1 fm78 fm86 skmel28 andsw480 were cultured in rpmi1640 sigmaaldrich r5886and ht29 were cultured in mccoys 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoys 5a for longterm cell culture inglucosegalactose cells were cultured in dmem medium withoutglucose gibco supplemented with dialyzedfbs mm penicillinstreptomycin mm sodium pyruvate and mm glucosegalactose all cells were kept at culture conditions¦c and co2 and were passaged every daysfor proliferation assay wt and mgat5 ko cells wereseeded in or cellswell for each experimentcells were counted in triplicate wells after and h usingthe biorad tc20 automated cell counter biorad herculesca united statesgene editingmgat5 ko cells were generated by zinc ï¬nger nucleasetargeting in hek293 cells and subsequent cloning and selectionwas performed as described previously hek293cells were transfected with mrna sigmaaldrich or µgof endotoxin free plasmid dna using nucleofection on anamaxa nucleofector lonza copenhagen denmark mgat5ko clones were selected by loss of reactivity with lphaand clones were conï¬rmed to have mgat5 mutations usingpcr and sequencinglentiviralmediated gene transfer was performed with anmgat5 encoding vector constructed by inserting the mgat5sequence generated as a bluntend pcr product from a vectorfrom hw university of copenhagen copenhagen denmarkinto an entry vector system using the pentr directionaltopo cloning kit invitrogen k243520k350020 followingmanufacturers protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were conï¬rmed by sequencing at euroï¬ns mwgoperons luxembourg colonies were ampliï¬ed and plasmidswere puriï¬ed with nucleobond xtra midi kit machereynagelduren germany mgat5 sequences were insertedinto plx302 lentiviral destination vector with lr clonase iienzyme mix invitrogen after proteinase k treatmentconstructs were transformed into dh5α using heatshock andsoc medium selected clones were ampliï¬ed and dna waspuriï¬ed using nucleobond xtra midi kit destination vectorswere checked for insertion using bsrgi digestion at ¦cmgat5coding lentiviral ps were packaged in hek293tcells transfected with a mix of µg pspax2 vector packagingvector µg pcmvvsvg envelope vector µg plx302vector carrying mgat5 and µl cacl2 to a ï¬nal volume of µl the dna mixture was complexed with µl 2x hbsunder constant air ï¬ow and the transfection mix was addeddropwise to hek293t cells in antibioticfree mediumcell culture medium was harvested days after transfection andviral p preparations were prepared by centrifugation at g for min lentiviral ps were added to cells andincubated for h cells were cultivated in puromycin µgmlselection medium for weeks functional mgat5 expressionwas validated by lpha bindingtransient transfectiontransient transfections were performed as described previouslyusing amaxa nucleofector device lonza dna wasintroduced to cells in µl nucleofector solution vlonza vca1003 and pulsed using the nucleofector programq001 for gfpmyctagged mica and micaconstructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbrieï¬y dnarna were prepared in µl cacl2 25mand adjusted to a ï¬nal volume of µl dna mixture wascomplexed with µl 2x hbs hepes nacl na2hpo4and added dropwise to cells scrambled sirnacontrol siidh1 and siidh2 ontarget plus smart poolswere purchased from ge healthcare dharmacon lafayetteco united statesfunctional assaysfor nkg2d downmodulation pbls were isolated as describedabove followed by depletion of cd4 cells using cd4 antibodyebioscience san diego ca united states anddynabeads mouse panigg invitrogen cd4depletedpbls were cultured in rpmi1640 sigmaaldrich r5886supplemented with human serum sigmaaldrich h3667 mm penicillinstreptomycin mm lglutamine and ngmlhil15 peprotech for days to enrich for nkcd8t cells nkg2d downmodulation assay was performed aspreviously described nkg2d ligands on eï¬ector cellshek293 wt or mgat5 ko cells were incubated with blockingnkg2dfc rd systems minneapolis mn united states1299nk or control igg1fc rd systems 110hg µgmlfor min at ¦c eï¬ector cells and target cells nkcd8t cells were mixed at indicated eï¬ectortarget ratios and spundown min g to allow conjugate formation after h cocultivation nkcd8 t cells were analyzed for nkg2d surfaceexpression by ï¬ow cytometry using accuri c6 ï¬ow cytometerbd bioscience franklin lakes nj united statesfor the reporter cell assay the nkg2dreporter cell line2b4ct312 and the parental control 2b4 cell line target cells were mixed with eï¬ector cells wt or mgat5 ko cellsthat were either blocked with nkg2dfc or control igg1fc asdescribed above eï¬ector and target cells were cocultivated atdiï¬erent et ratios for h gfp expression of target cellswas assessed with accuri c6 ï¬ow cytometer for in vivo assaytarget cells were labeled with vybrant did celllabeling solutionfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioninvitrogen v22887 according to manufacturers protocol andinjected intraperitoneally together with wt or mgat5 kocells in a ratio of each mice were usedper group target cells were harvested after approximately hwith peritoneal lavage and nkg2d activation of didpositivereporter cells were assessed as gfp expression with accuric6 ï¬ow cytometerwas assessed by accurate mass and retention time amrt plusfragment identiï¬cation at two collision energies and evdetailed acquisition methodology has been described previously udpglcnacudpgalnac detected peak screened byexpected calculated mass could be of either compound as thesetwo sugars could not be separated chromatographically hencehas been reported as a putative metabolite pending conï¬rmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturers protocolrandox crumlin united kingdom lc2389 reaction andanalysis was performed on an advia chemistry systemsiemens munich germanydntp levels were determined in cells harvested withtrypsinization and pelleted by centrifugation for g for min followed by resuspension of cell pellets in methanolfrozen in liquid nitrogen and boiled at ¦c for min sampleswere evaporated until dryness in a speedvac and whole cell levelsof dttp datp dctp and dgtp were determined using the dnapolymerase assay previously described lchrms metabolite proï¬lingto determine intracellular metabolite levels cell pellets from cells were resuspended in µl of cold methanolafter min sonication samples were prepared by svortex followed by min equilibration at room temperatureafter centrifugation at g for min at ¦c µl supernatants were collected transferred to ultrafreemccentrifugal ï¬lter devices merck millipore ltd cork irelandand centrifuged at g for min at ¦c from this µlwas transferred to lc vials and µl of each sample was pooledto a mixed qc samplelchrms was performed on a nity ii ultrahigh performance liquid chromatography uhplc systemcoupled to a ifunnel quadrupoletime of ï¬ight qtofmass spectrometer equipped with a dual ajs electrosprayionization source agilent technologies santa clara caunited states polar metabolites were separated on a sequantzichilic merck darmstadt germany column mm mm µm p size coupled to a guardcolumn mm mm µm p size and an inlineï¬lter mobile phases consisted of formic acid in water withsolvent a and formic acid in acetonitrile with solvent bthe elution gradient used was as follows isocratic step at bfor min b to b in min and maintained at bfor min then decreasing to b at min and maintainedfor min then returned to initial conditions over min and thecolumn was equilibrated at initial conditions for min the ï¬owrate was mlmin injection volume was µl and the columnoven was maintained at ¦c the acquisition was obtainedwith a mass range of mz for where full scan highresolution data is acquired at three alternating collision energies ev ev and ev positive and negative raw lchrmsï¬les were independently processed with an inhouse developedpcdl library for polar metabolites using proï¬nder version b06agilent technologies identiï¬cation of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular ï¬ux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density cellswell ¼ hbefore the experiment one hour prior to assay run cells wererinsed and switched to xf media agilent technologies with mm sodium pyruvate and mm glucose or galactose andincubated at ¦c co2free incubator for the mitochondrialstress tests ocr was measured under basal conditions andduring sequential injection of µm oligomycin sigmaaldrich µm fccp sigmaaldrich c2920 and µmrotenone rot sigmaaldrich r8875 µm antimycina aa sigmaaldrich a8674 reported basal respiration iscalculated from the third measuring point with ocr after rotand aa subtracted atpcoupled respiration display ocr afteroligomycin subtracted from the third measuring point andmaximal respiration is ocr after fccp with ocr after rotand aa subtractedfor measuring the eï¬ect of hc ocr was assessed h after aninjection of mm hc13c6glucose tracing experiment cells were incubated for h in dmem medium withoutglucose supplemented with fbs mm sodium pyruvateand mm uniformly labeled [u13c]glucose cambridgeisotope laboratories tewksbury ma united states clm incubation medium samples were collected and cleared bycentrifugation g for min cells were washed and detachedsterically intracellular metabolites were extracted in ethanoland centrifuged at g for min ¦c to separatethe soluble extract supernatant from the insoluble componentspellet cell extracts and medium samples were lyophilizedand reconstituted in water for subsequent biochemical analysesextract samples were adjusted to ph with hcl and evaporatedto dryness under nitrogen ï¬ow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modiï¬ed procedure from standards containing unlabeled metabolites of interest andcell extracts were separated and analyzed in a gas chromatographagilent technologies 7820a chromatograph jw gc columnhp5ms parts no 19091s433 coupled to a mass spectrometeragilent technologies 5977e the isotopic enrichment of themetabolites of interest was corrected for natural abundance of 13cusing the unlabeled standards and calculated according to data are presented as labeling of m x where m is the massof the unlabeled molecule and x is the number of labeled catomsin a given metabolite frontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionwestern blottingproteins were extracted using ripa buï¬er thermo scientiï¬cwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientiï¬c for minon ice lysates were sonicated times for s and clearedby centrifugation at rpm for min at ¦c proteinextracts were denatured at ¦c for min in nupage samplebuï¬er and dtt sigmaaldrich proteins were resolvedusing sdspage gels invitrogen and transferred tonitrocellulose membranes invitrogen ib301001 using the iblotdevice invitrogen for total protein stain membranes werewashed in ddh2o and stained with revert protein stainsolution licor biosciences lincoln ne united states according to manufacturers protocol membranes wereblocked in tbst blocking buï¬er licor biosciences probed with primary antibodies in tbs w tween and bsa overnight on a shaker at ¦c and washed intbs tween secondary antibody was from licorlicor biosciences and signals were visualizedby the odyssey fc imaging system licor biosciencesoglcnacylation was detected with rl2 oglcnacylationantibody abcam cambridge united kingdom ab2739 atpcitrate lyase acly was detected with rabbit acly antibodycell signaling and acly phosphorylation with rabbitphosphoacly ser455 antibody cell signaling flow cytometryadherent cells were detached in pbs w mm edta invitrogen or by pipetting cell surface staining was done aspreviously described and cells were analyzed on accuric6 ï¬ow cytometer bd bioscience antibodies used for thisstudy were mica rd systems fab1300a ulbp256 rdsystems fab1298p nkg2d rd systems fab139a ulbp1rd systems fab1380p ulpb3 rd systems fab1517aulbp4 rd systems fab6285a micab bd bioscience icam1 leinco technologies c170 mouse igg1antimyctag merck millipore micb rd systemsmab1599 or igg2b isotype control rd systems mab004detected with secondary antimouse igg biolegend san diegoca united states binding of ï¬uorescently labeledaf647lpha invitrogen l32457 and fitcepha vectorlaboratories burlingame ca united states fl1121 was usedto measure surface levels of complex nglycans all isotypecontrols were purchased from bd biosciencefor staining with mitochondrial probes neutral lipid stainsor 2nbdg uptake cells seeded the day prior toexperiment were washed once in pbs and incubated for minat ¦c and co2 in warm growth medium containing nmtetramethylrhodamine methyl ester perchlorate tmrm sigmaaldrich t5428 nm mitotracker green fm invitrogenm7514 or for h in growth medium with µm 2nbdginvitrogen n13195 bodipy invitrogen d3922 wasdiluted in warm serumfree medium in a dilution andshaken vigorously to solubilize the lipids immediately beforeloading into the cells for min cells were washed twice inpbs fbs and detached sterically prior to analysisthe soluble nkg2dfc receptor 1299nk rd systemsand igg1fc 110hg rd systems were labeled with zenonalexa fluor against human igg1 z25408 invitrogen priorto staining of melanoma cellsin forwardsidescatter plotsdata were acquired with an accuri c6 instrument usingaccuri c6 software and analyzed in flowlogic v721 inivaitechnologies mentone vic australia by gating on viablecellsfollowed bysingle cell gating by areaheightscatter plots fscafsch geometric mean ï¬uorescent intensity mfi values aredisplayed in ï¬gures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and puriï¬ed on directzol spincolumns zymoresearch irvine ca united states according to manufacturersprotocol cdna was generated using superscript cdnasynthesis kit invitrogen under standard pcr conditionsfollowing primersequences were used for quantitativertpcr with brilliant sybr green qpcr master mixkit mica mica_f tggcagacattccatgtttctgmica_r ctcgtcccaactgggtgttg ulbp2 ulbp2_f cagagcaactgcgtgacatt ulbp2_r ggccacaaccttgtcattctidh1 idh1_f ctatgatggtgacgtgcagtcg idh1_r cctctgcttctactgtcttgccidh2 idh2_f agatggcagtggtgtcaaggagidh2_r ctggatggcatactggaagcag glut1 glut1_fctgctcatcaaccgcaac glut1_r cttcttctcccgcatcatct glut2 glut2_f tacattgcggacttctgtgg glut2_r agactttcctttggtttctgg glut3glut3_f cagcgagacccagagatg glut3_r ttggaaagagccgattgtag glut4 glut4_f tgggcttcttcatcttcacc glut4_r gtgctgggtttcacctcctrplp0_fcctcgtggaagtgacatcgt rplp0_r cattcccccggatatgaggc realtime qpcr was performed on biorad cfx96 realtime thermal cycler c1000 touch and alltranscripts were normalized to housekeeping rplp0 transcripthousekeepingand rplp0asgeneluciferase reporter assaycells were transiently transfected using calciumphosphatetransfection as described above with ï¬reï¬y luciferase promotervectors µg and an sv40promoter driven renilla luciferasevector µg cells were harvested and snap frozen hpost transfection pellets were lysed in dualglo luciferasereagent promega e2920 and ï¬reï¬y luciferase activity wasanalyzed by luminometer microbeta ii perkinelmer walthamma united states renilla luciferase activity was recorded bythe instrument after subsequent addition of volume dualglo stop glo promega e2920 to correct for transfectioneï¬ciency ï¬reï¬y luciferase signals were normalized to sv40renilla luciferase signals of corresponding sampleatacseqatacseq was performed as described previously foreach cellline cells were harvested from separatefrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioncultures and used to prepare tagmented chromatin replicatesof wt and replicates of mgat5 ko cell lines samplestotal quality of pcrampliï¬ed sequencing libraries was assessedusing a tapestation instrument with high sensitivity dnascreentapes agilent libraries were sequenced as paired endreads on a single lane of an illumina hiseq4000 ï¬ow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were ï¬ltered toremove low quality duplicate and mitochondrial reads peakswere called using macs2 on merged reads from allsamples and diï¬erential peak accessibility between cell lines wasdetermined using edger with a threshold false discoveryrate of transcription factor binding motifs enriched indiï¬erentially accessible peaks were identiï¬ed using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantiï¬cation and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical signiï¬cance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in ï¬gure legends using unpaired ttest in 1a 1c 1e 3ef3h 5c 7a 7ef paired ttest in 4fg 7d multiple ttest in 1b1d 3d 4ab one sample ttest in 2ac 3c 4c 4e 7g twowayanova in 3a 5df 5hi 6a 6e 7hi or oneway anova in5g level of statistical signiï¬cance was determined by p p and p p resultsmgat5 knockout increases nkg2dlexpression and activates nkg2d in vitroand in vivoregulation of constitutive mica expression remains largelyunknown surface expression of certain mica alleles dependson nlinked glycosylation we questioned whetherthe cancerassociated glycosyltransferase mgat5 is required formica expression to assess the role of mgat5 in regulationof nkg2dl surface expression mgat5 ko clones weregenerated in hek293 cells remarkably mgat5 ko resultedin a permanently increased surface expression of the nkg2dlsmica micb and ulbp256 compared with parental wildtypewt cells figure 1a to conï¬rm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspeciï¬cally to mgat5modiï¬ed nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modiï¬ednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modiï¬cation of mgat5 expressiontherefore associated with substantial changes in constitutiveexpression of several nkg2dlsto verify the functionality of mgat5 koinduced nkg2dlswe tested nkg2d activation in a reporter cell line expressing1httpswwwencodeprojecthuman nkg2d coupled to dap10cd3ζ signaling and nuclearfactor of activated t cells nfatcontrolled gfp ultimatelyexpressing gfp in response to nkg2d activation nkg2dgfp activation was higher after cocultivation with mgat5ko cells than with wt cells figure 1b corresponding to theincreased nkg2dl expression in mgat5 ko cells figure 1athe reporter cells without nkg2d supplementary figure s1aremained inactivated indicating that the activation was nkg2dmediated figure 1b moreover blocking nkg2dls withsoluble nkg2dfc receptor impaired the activation furthervalidating nkg2d speciï¬city supplementary figure s1bto test if mgat5 ko cells could activate nkg2d in vivowe adoptively injected nkg2d reporter cells together with wtor mgat5 ko cells into the peritoneum of nmri mice andmeasured gfp expression in reporter cells in line with ourin vitro data we observed a signiï¬cant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspeciï¬c since the control reporter cellswere unaï¬ected figure 1c these data verify that mgat5 koinduced nkg2dls maintain their functional integrity in vivonkg2d is downmodulated upon activation to furtherexamine the functionality of nkg2dl expression causedby mgat5 ko we assessed nkg2d downregulation afterreceptor activation nkg2d was further downregulated oncd4depleted peripheral blood lymphocytes pbls after cocultivation with mgat5 ko cells than with wt cells and thisdownregulation was abolished by blocking nkg2dls with asoluble nkg2dfc receptor figure 1d combined these dataindicate that ko of mgat5 upregulates mica and ulbp256resulting in nkg2d activation in vitro and in vivoto ensure that the mica upregulation was a result of mgat5ko we stably transfected mgat5 into wt and mgat5 kocells lpha binding was restored within days after transfectionconï¬rming expression of functional mgat5 interestingly ittook multiple passages for mica expression to decrease to wtlevels figure 1e and supplementary figure s1c suggestingthat mica is regulated in response to a longterm adaptation toaltered mgat5 expressionudpglcnac upregulates micaexpressionlongterm mgat5 deï¬ciency willlikely result in aberrantnglycosylation and an accumulation of the mgat5 donorsubstrate udpnacetylglucosamine udpglcnac to addressif mica was regulated by a change in nglycosylation inmgat5 ko cells we assessed the posttranslational regulationof mica by measuring surface expression of transgenicallyexpressed gfpmyctagged mica under a cytomegaloviruscmv promoter the mica alleles mica and micaare distinctly regulated posttranslationally and althoughmica was upregulated in mgat5 ko cells the regulationwas minor and unlikely to account for the profound changein endogenously expressed mica figures 1a 2a micatranscripts on the other hand were highly increased in mgat5ko cells figure 2b as well as ulbp2 mrna supplementaryfigure s2a suggesting that nkg2dls are transcriptionallyfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionfigure mgat5 knockout increases nkg2dl expression and activates nkg2d in vitro and in vivo a surface expression of nkg2d ligands and binding ofï¬uorescently labeled lpha mgat5 modiï¬cations or epha mgat3 modiï¬cations on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by ï¬ow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean ï¬uorescence intensity mfi b in vitro nkg2d activation measured as gfp expression in nkg2d negative reporter cells control and nkg2dexpressing nkg2d reporter cells target cells cocultivated with wt or ko cells effector cells for h at indicated effectortarget et ratios c nkg2dactivation in vivo measured on reporter cells as in b after activation by wt or ko at a ratio in peritoneum of nmri mice for approximately h gfp expressionin didlabeled reporter cells signiï¬es nkg2d activation and is shown as gfp mfi values of cells from foursix mice per group d nkg2d downmodulation wasassessed on nkcd8 t cells target cells after cocultivation for h with wt or ko cells effector cells at indicated effectortarget ratios et nkg2dls on targetcells were blocked with nkg2dfc bl or unblocked with igg1fc un the graph depicts surface expression of nkg2d presented relative to surface nkg2dexpression on target cells alone e mica surface expression left and lphaepha surface binding right after lentiviral introduction of mgat5 into wt or kocells mfi values from antibody staining were corrected for isotype background staining 01mfi statistical analysis was performed by unpaired ttests in ace andmultiple ttest with fdr comparing wt and ko in bd p p p and p regulated in mgat5 ko cells notably we found that themgat5 substrate udpglcnac although indistinguishablefrom udpnacetylgalactosamine udpgalnac tended tobe higher in mgat5
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among synchronous colorectal cancers scrcs reported previously the incidence of quadruple advanced scrcs is very rarewe present the case who underwent laparoscopic twosegment resection of the colon requiring two anastomoses that wasperformed for quadruple advanced cancers and four tumors were curatively removed there were no signs of recurrence at months after surgery laparoscopic surgery provided less invasiveness even for quadruple advanced scrcs in terms of earlyrecovery with an acceptable longterm outcomeintroductionsynchronous colorectal cancers scrcs are characterized bythe simultaneous occurrence of multiple primary tumors inthe same patient synchronous malignancies most commonlyoccur in the colon among other ans [] the occurrence ofadvanced scrcs is rare and may be identified at any locationwithin the large intestine the prevalence of scrcs is reportedto range from to among these however the incidence of quadruple advanced scrcs is extremely rare accounting for of all scrcs surgical resection is considered thestandard treatment for scrcs as a surgical approach laparoscopic surgery has significant advantages in terms of shortterm outcomes including early recovery and no disadvantageouslongterm outcomes according to recent reports laparoscopicsurgery has been used in scrcs but these reports noted thatcontroversy remains concerning operative procedures for multiple segmental resections and for total or subtotal colectomywe report the case who presented with quadruple synchronousadvanced cancers arising from the colon which were successfully treated with laparoscopic twosegment colectomyreceived may accepted june published by oxford university press and jscr publishing ltd all rights reserved the authors this is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0cj ma figure colonoscopy images showing four tumors a one cauliflowerlike tumor with lumen stenosis is located in the ascending colon b another cauliflowerliketumor is located in the descending colon the third c and fourth d tumors are located in the sigmoid coloncase presentationa 70yearold man who was positive for a blood stool testvisited our hospital colonoscopy computed tomography ctand barium enema indicated quadruple concurrent locallyadvanced cancers the firsttumor with observed lumenstenosis was located in the ascending colon the secondtumor was located in the descending colon and the third andfourth tumors were located in the sigmoid colon fig ctrevealed marked intestinal wall thickness in the ascendingdescending and sigmoid colon fig preoperative precisesimulation using 3d angiography was performed to determineadequate lymph node dissection along the arteries feedingthe tumors and appropriate resection to avoid anastomoticleakageswe planned the appropriate placement of trocars as shownin figure because we wanted to create a single minilaparotomy for specimen retrieval and extracorporeal reconstruction after lymph node dissection and mobilization of thecolonduring the operation laparoscopic exploration confirmed thepresence of known four tumors with no invasion of the serosasubsequently a right hemicolectomy and sigmoid colectomywere performed laparoscopically the right half of the colon wasseparated and a sidetoside anastomosis between the jejunumand transverse colon was performed followed by the sigmoidcolon and a colorectal anastomosis between the descendingcolon and rectum was performed the resected tissue specimensrevealed four tumors fig histological examination showedthat the first tumor in the ascending colon the second tumor inthe descending colon and the third tumor in the sigmoid colonhad invaded up to the subserosa whereas the fourth tumor inthe sigmoid colon had invaded up to the muscularis propriafig according to the american joint committee on cancertumornodemetastasis staging system the pstage was iiia t3n1m0the patient was discharged days after surgery for adjuvantchemotherapy the patient chose to take an oral fluoropyrimidine agent for months fortunately there have been no signsof metastasis or recurrence after the operation at months offollowupdiscussionwe reported a rare case of quadruple scrcs all four tumorswere removed curatively by laparoscopic surgery with d3 lymphnode dissection we planned a strategy for quadruple scrcsbased on preserving the remnant large intestine and sufficientd3 lymph node dissection through a laparoscopic approachwe believe that laparoscopic surgery can be a safe even forquadruple scrcs this is the first case report of laparoscopicsurgery with d3 lymph node dissection for quadruple advancedscrcsthe incidence of malignant scrc with four or five synchronous lesions is extremely rare with a rate of beingreported this is a quite rare case of quadruple synchronous 0cquadruple advanced synchronous colorectal cancersfigure placement of trocars and miniincision in the present casethan the index cancer however all of the scrcs in our patienthad the same histological grade and t staging pstage iiiawith the tumor locations being in the ascending descending andsigmoid colonsurgical management of scrcs needs to be tailored tothe individual based on tumor location invasion status andthe patients health condition some studies have suggestedtotal or subtotal colectomy to remove any potential existingsynchronous tumors or polyps that have not been detected however other studies recommend a more conservativesurgical approach it is thought that the removal of the entirecolon will prevent the development of metachronous tumorsand a previous study indicated that subtotal colectomy mayincrease defecation frequency as the normal colon cannot bepreserved we successfully performed laparoscopic surgerycombining twosegment resection of a right hemicolectomyand sigmoid colectomy with no intra or postoperative adverseevents in our patient we tried preserving as much colonas possible considering the patients quality oflife aftersurgery in addition to performing sufficient d3 dissection ofcourse the meaning of preserving colon in terms of patientpostoperative quality of life needs to be more clearly assessed infuturewe encountered a rare case of advanced quadruple scrcsfor which we achieved a curative resection that required twoanastomoses through a laparoscopic approach we suggest thatlaparoscopic surgery that requires multiple anastomoses foradvanced scrcs can be a safe procedure even if the number ofcolorectal cancers is multiplefigure abdominal ct scan revealing a tumor of the ascending colon aarrowhead a tumor in the descending colon b arrowhead and two tumorsin the sigmoid colon are also visible c d arrowheadadvanced cancer arising from the ascending descending andthe sigmoid colon it was reported that scrcs often occur inthe same or adjacent segment of the large intestine and thatother smaller colorectal cancers in the patients with scrcs wereusually smaller and of lower pathological grade and t stagingconflict of interest statementnone declared 0cj ma figure the surgical specimens of the ascending colon cancer a descending colon cancer b and the two sigmoid colon cancers c and dfigure histopathological examination of the tissue specimens revealed four tumors showing cancerous cells arranged in a tubular pattern 0cfundingnonereferencesjiang x xu c tang d wang d laparoscopic subtotal colectomy for synchronous triple colorectal cancer a case reportoncol lett yang j peng jy chen w synchronous colorectal cancersa review of clinical features diagnosis treatment and prognosis dig surg aky l synchronous colorectal cancer clinical pathological and molecular implications world j gastroenterol fukatsu h kato j nasu ji kawamoto h okada h yamamotoh clinical characteristics of synchronous colorectalcancer are different according to tumour location dig liverdis holubar sd wolff bg poola vp soop m multiple synchronous colonic anastomoses are they safe colorectal disquadruple advanced synchronous colorectal cancers li z wang d wei y liu p xu j clinical outcomes oflaparoscopicassisted synchronous bowel anastomoses forsynchronous colorectal cancer initial clinical experienceoncotarget nosho k kure sirahara n shima k baba yspiegleman d a prospective cohort study showsunique epigenetic genetic and prognostic features ofsynchronous colorectal cancers gastroenterology 20e1 lam ak carmichael r gertraud buettner p gopalan dho yh siu s clinicopathological significance of synchronous carcinoma in colorectal cancer am j surg easson am cotterchio m crosby ja sutherland h dale daronson m a populationbased study of the extent ofsurgical resection of potentially curable colon cancer annsurg oncol tsantilas d ntinas apetrasp zambas n aimogrambi s frangandreas g metachronouscolorectals202adenocarcinomastech coloproctol 0c'
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"autophagy is an evolutionary cellular program that serves for thebreakdown of cytoplasmic components within lysosomes [] inidentified autophagy in's electron microscopic studies firstmammalian cells but the molecular pathways were not understooduntil the discovery of autophagy genes atgs in yeast by performinggenetic screening it is a cytoprotective rather than a selfdestructive process it is extensively accepted as a main regulator of innateand adaptive immune mechanisms the change in which completelyimpact the pathogenesis of disease and the processes that are influencedby autophagy includes the regulation of inflammation antigen presentation and bacterial clearance moreover autophagy aids in themaintenance of fundamental anelle populations such as mitochondria which is necessary for cellular bioenergetics and homeostasis homeostasis of the cell is been accomplished by maintaining the biosynthesis and turnoverthere are two broader protein degrading systems in eukaryoticcells first is the ubiquitinproteasome system responsible for the selective breakdown of most shortlived proteins and second is the lysosomalsystem the primary anelle called lysosome ineukaryotes is known for degradation through its acid hydrolases inunfavourable nutrient deprivation condition autophagy arbitrates aregulated phagocytosis via lysosomes autophagy is mediated byautophagosome that is thought to be an on selective degradation systemas it engulfs some of the cytoplasmic contents the ubiquitinproteasome system concedes only ubiquitinated proteins for degradation andit marks a remarkable contrast to autophagosome process autophagosomes a doublemembered vesicle that engulfs durableproteins impaired anelles intracellular pathogenic anisms andtransports it to the lysosomes that are fused to form autolysosome andthe inner vesicle along with its cargo is been degraded at the time ofstarvation the remaining macromolecules are again recycled to thecytosol for reuse the precise mechanism in cargo recognition isuncertain but this process involves ubiquitination autophagicprocess is separated into distinct steps which includes induction recognition selection of cargo formation of vesicle then occurs the fusion of autophagosomevacuole followed by the degradation of thecargo and release into the cytosol various atg proteins are indulged inthis process and it consists of the central autophagic machinery autophagy encompasses different process by which cells delivercytoplasmicaredegradation theylysosomalsubstratesfor corresponding author at department of biotechnology psg college of arts and science civil aerodrome post coimbatore indiaemail address rashmiffrajangmailcom rr rasmi all authors deserve contribution equally101016jlfs2020118308received june received in revised form august accepted august available online august elsevier inc all rights reserved 0cs vishnupriya life sciences macroautophagy chaperonemediated autophagy cma and microautophagy all the three processes of autophagy are morphologically distinct in microautophagy the lysosomal membrane invaginations or protrusions are needed to seize cargo once thecargo is captured the uptake directly happens at the limiting membrane of the lysosome that includes intact anelles cma uses chaperones to sequester cargo proteins that have a pentapeptide motifthese proteins are unfolded and translocated directly across the lysosomal membrane via lamp2a receptor macroautophagy involves requisition of cargo vesicle formation and its subsequenttransport to the lysosome in recent years deletion of the autophagy related genes atg invarious model anisms has proved that autophagy plays decisive rolein adaptive responses to stress cellular differentiation and development an oncogenic event may establish by the partial minimization inthe autophagic capacity atg6beclin one of the phylogeneticallypremeditated autophagy genes is often subdued at one locus in humancancers studies in mice have shown that beclin is a haplo insufficienttumor suppressor autophagic programmed cell death was primarily depicted in actively developing tissues several conjugationsystems comprising of the atg genes are available that take part inautophagasomal elongation one such system is the lc3 microtubule associated protein light chain and atg8 conjugation system lc3 is the mammalian conjugative protein ortholog of yeastprotein atg8 the lipid derivative phosphatidylethanolamine bindsto lc3 to form lc3ii an important molecular marker of autophagylc3ii remains on the mature autophagosome until it fuses with thelysosomes fig the beclin complex gives rise to an incipientautophagosome membrane and it assemble around cargo in a vesiclethat combines with a lysosome forming autolysosome that is degradedby acid hydrolases present in the lysosomes lung injury clinical implicationsthe primary an of gas exchange is the pair of lungs theymediate inspiration of oxygen and elimination of mono and dioxides ofcarbon lung also serves as an attractive target for the entry of thepathogens regulation of the pulmonary functions is mediated by intricate cells of endothelial and epithelial lining dendritic cells alveolarmacrophages and fibroblasts all these pulmonary cells are highlyheterogeneous in nature they together in association respond to thelung injury by provoking inflammatory and immune responses airway epithelial cells express pattern recognition receptors prrsalong with toll like receptors ctype lectins rigi and inflammasome components which are involved in the innate response against microbes microbial cell wall constituents likelipopolysaccharide are sensed by prrs and induce an inflammatoryresponse alveolar epithelium comprises type i and type ii alveolarcells apart from these the mucus layer and the physical barrier madeby the epithelium contribute to the first line of defense lung injury is described as any damage to the associated tissues and compartments of the pulmonary system the concept of lung injury influenced by the microenvironment can be demonstrated via series ofchanges in cell deformability and manifestation of intercellular adhesive molecules the major causes of lung injury are atelectasisalveolar instability volutrauma barotrauma infections and oxygentoxicity the pathogenicity of acute and chronic lung injury iscorrelated with the release of proteases free radicals and growth regulatory proteins by the alveolar macrophages neutrophil takescare of the extreme ranges of lungs searching for pathogens therebyfig mechanism of autophagy ulk1pi3kmtor signalling pathway binds to endoplasmic reticulum and activates dcedp1 that initiates beclin and atg512conjugation system which forms isolation membrane this is followed by the sequestration process that leads to autophagosomal formation the autophagosomefuses with lysosomes to form autolyososome that leads to degradation 0cs vishnupriya eliminating via phagocytosis they undergo transendothelial andtransepithelial migration primary host defense mechanism oflungs include the immunity conferred by surfactant proteins spnamely spa and spd they link both innate and adaptive immunity byregulating the responses of innate immune cells antigen presentingcells apcs and tcells they can act as potential biomarkers of lunginjury the implication of autophagy over lung injury is tortuousas its function varies highly with the cell types specific to the pulmonary disorders it provides both defensive and injurious outcomes of lung injury lung injury types and associated pathologies acute lung injury ali and acute respiratory distress syndromeardsacute lung injury and acute respiratory distress syndrome arecommon grievous diseases among critically illpatients and are evidential sources of mortality and morbidity they are expressed alongwith hypercapnia and hypoxemia ards is outlined as the most seriousform of ali ali is characterized by inflammation of lung surfacesresulting in the disruption of alveolarcapillary membrane followed bytransmigration of neutrophils significant event in the procession of aliand ards and outbreak of cytotoxic mediators immune responsemediated by several components like tcells macrophages naturalkiller nk cells chemokines and proinflammatory cytokines also has apredominant role in the progression of the acute injury and results inimmune reconstitution inflammatory syndrome iris immune cellsinvolved in the immune response like macrophages and monocytes arethought to be involved in the pathophysiology of iris elevatedlevels of cd14 cd16 monocyte population and an resulting increase in the proinflammatory cytokines il6 tnfα and c reactiveprotein crp are also observed in tuberculosis tbiris patients lung endothelial biomarkers like vwf and epithelial biomarkers likespd are the diagnostic targets of ali sepsis pancreatitis pneumoniatrauma transfusions aspiration and inhalation of toxic gases are someof the clinical factors of ali and ards histological patterns ofali and ards have the chances of demonstrating diffuse alveolar damage alveolar haemorrhage eosinophilic pneumonia and acute fibrinous pneumonia associated with ards may also result frompathogens like fungal viral bacterial and parasitic most commonpathogen strains include streptococcus pneumoniae respiratory virusesstaphylococcus aureus fungal pathogens like pneumocystis jirovecii andaspergillus fumigatus legionella pneumophila and enteric gramnegativeanisms among bacteria the virulence capability of pseudomonas aeruginosa is one of the prime determinant of the severity of thelung injury disease progress takes place in three degrees namelyacute phase exudative subacute phase proliferative and chronicphase fibrotic radiological features during the acute phasesignify twosided patchy groundglass densities relating to interstitialedema and hyaline membranes the important constituent of innateimmune system the pattern recognition receptors prrs are affiliatedwith the advancement of ali and ards the ligands of prrs includepathogenassociated molecular patterns pamps which induce inflammatory signalling events and the damageassociated molecularpatterns damps which induce neutrophilmediated tissue damageincreased incidences of mitochondrial damps result in high mortalityrates a common type of ali is the transfusionrelated acute lunginjury trali trali is defined as adult respiratory distress syndromeoccurring with transfusionsis manifested by pulmonary insufficiency severe hypoxia and dyspnea it is often reported as theneutrophil pmnmediated syndrome a particular study has reported that the potential risk factors for ali and ards may be associated with larger tidal volume vt and higher airway pressure pawduring one lung ventilation olv in postpneumonectomy aliardspatients and also in patients who developed aliards longtermeffects after acute lung injury are related to neurological impairmentsitlife sciences namely neuromuscular dysfunction neurocognitive dysfunction andneuropsychological dysfunction some of the minor impacts includeheterotopic ossification stiae and frozen joints chronic lung injury cli and bronchopulmonary dysplasia bpdchronic lung injury is characterized by a condition called pleuroparenchymal fibroelastosis ppfe in which a rapid multiplication ofsubpleural intestinal elastic fibres majorly in the upper lobes predominates along with other clinicopathological conditions following chronic lung injury cells undergoing apoptosis is cleared by aprocess called efferocytosis it is a type of phagocytosis confined only tothe cells that undergo apoptosis to maintain the cellturnover in pulmonary airways carried out mainly by the macrophages while immature dendritic cells mesenchymal cells and epithelial cells can alsoperform efferocytosis recently sarscov2 manifested as severerespiratory infection resulted in number of deaths worldwide commonrisk factors associated with death in sarscov2 patients identified arehypertension diabetes cardiovascular disease or chronic lung disease the most prevailing chronic lung injury ofinfants is thebronchopulmonary dysplasia bpd occurs when preterm infants suffering from various respiratory syndromes like meconium aspirationsyndrome and neonatal pneumonia are subjected to treatment withsupplemental oxygen and extensive mechanical ventilators it is proventhat the common risk factors of bpd increases with a fall in birthweight prematurity and gestational period apart from theabove other perinatal risk factors with which bpd is associated with areintrauterine growth restrictions race or ethnicity chorioamnionitis and genetic risk factors it is characterized bysaccular formation and elastic fibre aggregation of distal air spacesinflation and edema barotrauma and pulmonary oxygen toxicity arepathologies of bpd bpd is multifactorial in nature studiesdeclare that surviving patients of bpd have established pulmonarydysfunction incidence adults with bpd history are strictly prohibitedto cigarette smoking another form of bpd called the new bpdwhen surfactants are used as treatment new bpd is depicted by pulmonary hypertension and abnormalities in vasculopulmonary development glucocorticoids and tgfbeta are the efficient modulators thatinitiate bpd injury bpd infants have significantly lower levels ofvascular endothelial growth factor and platelet endothelial cell adhesion molecules chronic obstructive pulmonary disease copd and asthmacopd is a general illness worldwide it is defined as the completeirreversible state of airflow limitation characterized by weak inflammatory response of the lungs emphysema chronic obstructivebronchiolitis fibrosis blocking and narrowing of airways deprivationof lung parenchyma and elasticity immune cells like tlymphocyteswith cd8 dominance blymphocytes macrophages and neutrophilsare the regulators of copd acute provocation of copd is definedas the continuous deterioration from steady state facilitating an alteration in typical medication for rudimentary copd systemicinflammation responses are a result of leukotriene b4 tnfalpha interleukin il8 and proteases a decrease in the ratio of cd4 to cd8is a typical feature of pulmonary inflammatory responses additionalimpacts of copd are cardiovascular diseases nervous effects and osteoskeletal effects smoking is an important cause of systemicoxidative stresses immoderate inflammatory responses and emphysema as manifested as copd implications copd being an hetergoenousdisease patients with exacerbations are found to be associated withbacterial infections like moraxella catarrhalis streptococcus pneumoniaand haemophilus influenza apart from bacteria other microbes likevirus and fungi also comtribute to the lung microbiota and pathogenesisof lung microbiota through initiation of chronic inflammation it hasbeen found that bacteria and viruses fungi can promote local andsystemic inflammation that may contribute to the pathogenesis ofcopd specific biomarkers of copd observed are creactive 0cs vishnupriya protein crp il6 il10 and ccl18parc skeletal muscle losstakes place in copd wasting of the cell mass is evidenced to be theresult of tnfα participation in the pathogenesis of copd muscleglutamate reduction is linked with lactic acidosis in copd patientsending in muscle wastage similar to copd asthma is characterized by pulmonary obstruction and similar immune responses onlyvariation from copd is that the airway obstruction in asthma is reversible and it does not affect lung parenchyma dendritic cells are themodulators of th2 cells playing an important immune response ofasthma severe asthma is equal to the effects of copd illustrated by theincrease in neutrophils tumor necrosis factor tnf cxcl8 and decreased reception to corticosteroids while reversible copd is potentially to have subsequent asthma and copd ventilatorinduced lung injury vili and ventilatorassociated lunginjury valithe prevailing lung injury cases with ards when given ventilatorassistance mechanically in a clinical setup may develop additional lunginjuries ending up in ventilatorassociated lung injury vali similarlyin experimental models lung injury can be provoked by external application of injurious ventilation procedures contributing to ventilatorinduced lung injury vili thus vali can adversely aggravate thehealth of the ards patients at low lung volumes of ventilation atelectrauma occurs at high lung volumes of ventilation barotrauma andpulmonary edema occur biomarkers studied via experimental modelsof vali include several proinflammatory molecules like tnfα il1βil8 and antiinflammatory molecules like il10 il6 and stnfr1 respectively [] positive endrespiratory pressure peep and tidalvolume applied have direct influence on vali and are to be studiedspecifically during ali and edema it is reported that peep when provokes overinflation the extent of edema also increases mechanism ofsurfactant inactivation is seen in alveolar microvessels with an increasein fluid filtration further greater the lung volume higher is thetransmural pressure in them a retrospective cohort study revealedthat height and gender of the patients should be taken into considerations along with establishing limitations to large tidal volumes beforesetting up a ventilator experiments explain that decrease in thepeak pulmonary arterial pressure or respiratory frequency can lessenthe grimness effects of vali it is proven that hypercapnic acidosisaffords protection to vili addressable implications of vali arebarotrauma volutrauma atelectrauma biotrauma and oxytoxic effects cellular pathology includes physical disruption of cells and tissuesand activation of cytotoxic responses leucocytes are raised to likelyinteract with the endothelium as the increasing intraalveolar pressurefastens the transit time of them inferences for current medicalpractices involve lungprotective ventilation survival of patients can beencouraged by prone positioning future clinical practices involveprecisioned ventilationindividualized tidal volumes using drivingpressure individualized peep and extracorporeal strategies pulmonary fibrosis pf and cystic fibrosis cfidiopathicpulmonary fibrosis ipf is a degenerative interstitial fibrosing disease prevalent worldwide it is also termed as cryptogenicfibrosing alveolitis a typical honeycomblike structure of asymmetricairspaces covered by dense fibrosis is observed ipf is followed by interstitial pneumonia which is featured by deficit inflammation and withabsence of homogeneous participation of lung tissues studies havebeen made since ages which revealed several inherited forms of pulmonary fibrosis mutations in various genes were associated to pfnamely sftpc sftpa2 tert terc and muc5b incidence withrheumatoid arthritis and scleroderma are more likely to form pf thereis a chance of clearance of alveolar basement membrane and occurrenceof hyperplastic epithelial cells ipf ensures migration of fibroblastsinto the fibrinrich exudates thus a chemoattractant activity is createdin the airspaces after lung injury this process is proven to be regulatedthe proby lysophosphatidic acid experiment proves thatlife sciences inflammatory cytokines il1β directly regulates the initiation of acuteand chronic inflammation making it a valuable target of ipf cystic fibrosis is characterized as a most common autosomal recessivegenetic disorder caused by the mutation in cftr gene transmembraneprotein genecystic fibrosis transmembrane conductance receptor pathology of the disease involves bronchiolitis obstruction of pathways endobronchiolar infection impaired ciliary actions atelectasisfinally leading to secondary alveolar injury bacterial infections of saureus p aeruginosa and h influenzae causes cf it is the pulmonarymacrophages and polymorphonuclear neutrophils that forms the defense against infections while lymphocytemediated mucosal injury isobserved in cf patients cf patients have increased flow of tnfαil8 and il1β submucosal glands of the lungs are the crucialhosts of cftr genes as the number expressed are high hence cfcontributes to epithelial airway lining abnormalities with respect to thechanges taking place in the submucosal glands respectively the macromolecular secretions of the submucosal glands have changes in theircomposition viscosity and greatly impacts on the mucociliary clearance radiationinduced lung injury riliseveral complications of the lung malignancies require treatmentsinvolving radiation therapy rt extensive reports claim that rt maylead to a state of rili the threedimensional dosimetric predictors canbe emphasized for the risk of symptomatic rili the alveolarcapillary subunit forms the most radiosensitive complex of the lungs thusthe rili is also called as the diffuse alveolar damage radiation exposure provokes the production of reactive oxygen species creatinghigh toxicity levels in the lung parenchyma this may ultimately lead tolung fibrosis which develops one to six months after rt accompaniedby dyspnea in addition to fibrosis rili also develops radiationinduced pneumonitis gradually after months to years almost all thepatients undergoing rt have the chance of developing fibrosis radiationinduced pneumonitis is characterized by cough occasional fevernonspecific symptoms of dyspnea and chest pain with or without deformities in pulmonary functional tests while radiationinduced pulmonary fibrosis is characterized by cough differential levels of dyspnea chest pain or symptomless and stable scarring of the lung tissueswhen detected radiographically it has been stated that the incidence and occurrence of rili is directly influenced by dose and volume determinants of rt several studies provide information onrili that result in provoking inflammatory responses a biphasicmanifestation of cytokines is observed in the lung tissues once exposedto rt one such study carried out to assess the cytokine productionin c3hhen irradiated mice revealed a spatial change in the expression of proinflammatory cytokines among various cellular compartments of the lungs further it was noted that the bronchoalveolar lavage cells responded immediately while the interstitial cells contributedonly in the later stages during pneumonitis profiling of cytokinesnamely the interleukins interferons monocyte chemotactic protein1tumor necrosis factors macrophage inflammatory proteins and granulocyte colonystimulating factors can be performed to analyse the developmental risks of symptomatic radiationinduced lung injury in vitro studies experiments state that the application of melatonin andcarnosine compounds reduced the reactive oxygen species and inflammatory cytokines produced following rili regulators of autophagy in lung injurynumerous regulators of autophagy play a vital part in the development of lung injury with regard to autophagy the following are theimportant regulators fig the mechanism by which these autophagy regulators act is shown in the table lc3biithe microtubuleassociated protein light chain lc3 is the 0cs vishnupriya life sciences fig figure depicts the autophagy regulators in regard to lung injury various conditions like hypoxia starvation environmental stress and cigarette smoke leadsto autophagy that in turn causes lung injury activation of class ipi3kmtor pathway takes place under hypoxic and starvation conditions while environmentalstress results in provoking beclin 1vps34 pathway that induces the sequestration of the phagophore formation cigarette smoke induces ros accumulation which inturn causes egr1 e2f signalling to activate lc3ii along with sqtm1p62 and atg512 conjugation system the ros generated may also leads to apoptosis theautophagosome fuses with lysosomes and forms autophagolysosome that causes pulmonary arterial hypertension and lung injuryprinciple autophagic protein expressed on the doublemembraned autophagosome nearly eight homologues of lc3 proteins are studied inmammals the amino acid composition of these homologues classifiesthem into two subfamilies first one comprising of lc3a splicingvariants lc3b and lc3c taking part in autophagosomal membraneelongation while the second one comprising of gabarap gabarapl1 gabarapl2 and gabarapl3 taking part in the maturation ofautophagosome generally lc3b occurs in cytosol as lc3bi by theproteolytic cleavage of cterminal of lc3b which then unites withphosphatidylethanolamine to form lc3bii to assemble on the autophagosomal membrane conjugation of phosphatidylethanolamine withlc3bii can be revoked by the activity of atg4 thus it is clear thatoccurrence of lc3bii is crucial in the process of autophagy lc3bii levels are analysed through immunoblotting while limitationsare degradation of lc3bii by autophagy itself and the nonindication ofautophagic flux at distinct time points this can be overcome by comparison studies with lc3bi and using lysosomal protease inhibitors antilc3b antibodies can be applied to detect autophagy invarious cell types application of antilc3b to glioblastoma tissuesdemonstrated a positive detection of lc3b levels both in vitro and invivo suggesting a latent monitoring system of lc3b severalstudies state that hyperoxic conditions can result in ali and ards thisfurther triggers the morphological biomarker of autophagy lc3bii toaccumulate thereby determining the fate of cell clearance experimentsperformed in hyperoxiainduced human bronchial epithelial cells andcultured epithelial cells beas2b clearly stated that the expression oflc3bii was high mediated by the apoptotic regulators similarexperiment carried out in the hyperoxiainduced lung injury in c57bl mice inferred the involvement of apoptotic pathways in the activationof lc3bii interaction of lc3bii with fas proteins was observedmarking the importance of lc3bii in the management of ali pulmonary hypertension is a main cause of copd manifestation inlungs that affects vascular architecture when chronic hyperoxia wasinduced in the lung tissue extracts of patients with pulmonarytable regulators of autophagy in lung injury and their mechanismregulatorslc3biibeclin p62hif1bnip3mtormechanismelongation of autophagosome and its maturation requires atg8map1lc3 protein lc3i combines with phosphatidylethanolamine forming lc3ii essential for autophagosome formationthe initiation of the isolation membrane that forms autophagosome after the sequestration process is regulated by beclin and pi3kp62 along with sqstm1 is the receptor for polyubiquitinated substrates it helps in the transportation of cargo into the autophagosome by bindingwith lc3ii for degradationduring hypoxic conditions hif1 induces bnip3 that in turn brings about cell survival through autophagy and provoke cell death by apoptosisthe mammalian homologue atg13 is phosphorylated by mtor that binds ulk1 proteins fip200 phosphorylates ulk and initiates the isolationmembrane formation in autophagyreference no 0cs vishnupriya hypertension the upregulation of lc3b prevailed indicating the regulatory role of lc3b in vascular cell proliferation and mediatingadaptive cellular responses smoking results in various implications of lung injury in due course a multimeric protein complexcomprising of lc3bcaveolin1fas occurs under basal state extensivestudies reveal that smoking triggers lc3b to initiate the dissociation ofcaveolin1 from fas protein thus facilitating apoptotic pathways accordingly emphysema a destructive expression of copd isworsened by cigarette smoking in vitro studies in lung tissues of miceon exposure to cigarette smoking ensured the driving role of lc3b inregulating apoptotic mechanisms and finally developing emphysemarespectively all these experiments prove the comprehensive bridgebetween autophagy and apoptosis highly regulated by the expressionlc3b lc3bi and lc3bii bind to microtubule associated protein1b map1b wherein overexpression of map1b decreases the levels oflc3bii protein kinase c is known to cease autophagy by interferingwith autophagosomal formation both in vitro and in vivo studiesconfirmed that the lc3b phosphorylation by protein kinase c takesplace consistently in lungs the emergence of autophagy either asa protective role or maladaptive response due to sepsis was studied in acecal ligation and puncture clp induced septic mice it was ensuredautophagy to be a protective response yet an overexpression of lc3bii in the later stages of sepsis leads to ali describing a maladaptive role lung injury can be provoked by ischemiareperfusion in whichautophagy is stated as the safeguarding mechanism by moderatelymaintaining the level of lc3bii also the ischemiareperfusioninduced lung injury is positively governed by the erk12 signallingpathway that regulates the cellular expression of lc3bii respectively nanoparticles of zinc oxide on exposure to lungs may induceali further zinc oxide nanoparticles resulted in the raise of autophagosomal structures followed by the accumulation of lc3bii proteinsthus zno nanoparticlesinduced ali is autophagy dependent 3methyladenine a classical autophagy inhibitor reduced the manifestationof lc3bii and lowered the release of zinc particles thereby stoppingzno nanoparticlesrelated toxicity of lungs similarly the artificially synthesized polyamidoamine dendrimers pamam used as aneffective drugdelivery system may sometimes result in pamam nanoparticlesinduced ali the levels of lc3bii biomarkers were highindicating the autophagic responses beclin beclin was first identified by beth levine as becn1atg6 inchromosome 17q21 in the year and it is the major autophagyregulating gene it is a coiledcoil protein of molecular weight kda comprising of amino acids and acts together with bcl2an antiapoptotic protein beclin is an indispensable autophagypromoting gene that is homologue to the mammalian yeastatg6 gene which regulates cell survival of different types and is involved in the constitution of autophagosomes the initiation ofthe anization of autophagosomes is regulated by class iii phsophoinositide 3kinase pi3k and autophagy related gene beclin beclin has got a novel bcl2 homology region3 bh3 domainthe bh3 domain in beclin1 can bind to bcl family proteins that initiateapoptotic signalling and prevents the beclin 1mediated autophagy byremoving beclin from hvps34 either phosphorylation or ubiquitination of beclin or bcl2 can disunite bcl2 from beclin andincrease the activity of vps34 kinase which brings about increase infunction during autophagy autophagyassociated protein beclin1 binds to lc3i that adapts to its membranebound form lc3iiand it cooperates with the ubiquitinbinding protein p62sequestosome sqstm1 the first an that fails during sepsis is lungs thefamiliar complications of sepsis are ali and ards ali activated various autophagy related proteins like lc3ii beclin and lysosomerelated protein lamp2 and rab7 expressions in sepsisinduced ali to evaluate the function of autophagy in severe sepsis an experiment is carried out using endotoxemia that frequently uses septiclife sciences shock and clp which is a clinical polymicrobial sepsis model herebecn1 mice was susceptible to clpinduced sepsis in cysticfibrosis transmembrane conductance regulator cftr autophagy bybeclin overexpression cystamine or antioxidants and the restorationof beclin recovers the localization of beclin to endoplasmic reticulum and regresses the cf airway phenotype both in vitro and in vivoin scnn1btransgenic and cftr f508del homozygous mice and also inhuman cf nasal biopsies in lpsinduced ali there are threedistinct complexes of beclin1vps34 have been identified the firstcomplex contains beclin1 vps34 vps15 and atg14l second complexcontains beclin1 vps34 vps15 and ultraviolet irradiation resistanceassociated gene uvarag and the final complex contains beclin1vps34 vps15 uvrag and rubicon among these complex the onethat contains atg14l is concerned in the formation of autophagosomewhile others are in the autophagosome and endosome maturationbeclin forms the bridge in the recruitment of inducers and suppressorsof autophagy and simultaneously behaves as a key modulator in autophagosome formation mesenchymal stem cells mscs increases the translation level of beclin but not its transcription ratemscs might alleviate lpsali via downregulation of mir142a5p thatpermits pulmonary epithelial cells pecs to proceed with beclin mediated cell autophagy in lung disease the bacterial stu
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" exosomes are extracellular vesicles containing a variety of biological molecules including micrornasmirnas we have recently demonstrated that certain mirna species are selectively and highly enriched inpancreatic cancer exosomes with mir1246 being the most abundant exosome mirnas have been shown tomediate intercellular communication in the tumor microenvironment and promote cancer progression thereforeunderstanding how exosomes selectively enrich specific mirnas to initiate exosome mirna signaling in cancercells is critical to advancing cancer exosome biologyresults the aim of this study was to identify rna binding proteins responsible for selective enrichment ofexosome mirnas in cancer cells a biotinlabeled mir1246 probe was used to capture rna binding proteins rbpsfrom panc1 cells among the rbps identified through proteomic analysis srsf1 eif3b and tia1 were highlyassociated with the mir1246 probe rna immunoprecipitation rip and electrophoretic mobility shift assay emsaconfirmed the binding of srsf1 to mir1246 lentivirus shrna knockdown of srsf1 in pancreatic cancer cellsselectively reduced exosome mirna enrichment whereas gfpsrsf1 overexpression enhanced the enrichment asanalyzed by next generation small rna sequencing and qrtpcr mirna sequence motif analysis identified acommon motif shared by of srsf1associated exosome mirnas emsa confirmed that shared motif decoysinhibit the binding of srsf1 to the mir1246 sequences we conclude that srsf1 mediates selective exosome mirna enrichment in pancreatic cancer cells bybinding to a commonly shared mirna sequence motifkeywords srsf1 exosome mirna mir1246 pancreatic cancer exosomes are endosomederived extracellular vesiclesevs that can be transferred from cancer cells tostromal cells in the tumor microenvironment [ ]these membrane vesicles are nm in size andcontain proteinsincludinglipids and nucleic acids correspondence weiqundingouhscedu1department of pathology university of oklahoma health sciences centeroklahoma city stanton l young blvd bmsb 401a oklahoma city ok usa6stephenson cancer center university of oklahoma health sciences centeroklahoma city ok usafull list of author information is available at the end of the small rnas such as micrornas mirnas[ ]exosomemediated intercellular communication betweencancer cells endothelial cells [ ] fibroblasts [ ] orimmune cells [ ] can facilitate tumor progressionfurthermore cancer exosomes are released into the circulation and contribute to premetastatic niche formation in distant ans [ ]how cancer exosomes interact with stromal cells topromote tumor progression has been extensively investisignaling eventgated one criticalin the tumormicroenvironmentisthe exosome mirnamediatedintercellular communication [ ] studies haveshown that exosome mirna signaling promotes tumor the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cxu cell communication and signaling page of progression in various model systems [ ] notablyit has been reported that mirnas contained in exosomes are delivered to recipient cells in the tumormicroenvironment or distant ans where they canregulate target gene expression and promote tumorangiogenesis and metastasis [ ]in the context of exosome mirna signaling we andothers have reported that certain mirna species areselectively enriched in cancer exosomes as compared toexosomes derived from normal epithelial cells [ ] results from several studies have also indicated thatselective enrichment of exosome mirnas is relevant totumor progression for example exosome sortingof mir193a was found to promote colon cancer progression likewise mir122 a cancer exosomeenriched mirna [ ] was shown to reprogram glucose metabolism in a premetastatic niche to facilitatemetastasis in a breast cancer model system moreover the exosome enriched mir1246 was reportedto promote tumor invasion in both breast cancer and oral squamous cell carcinoma it seems clearthat selective enrichment of exosome mirnas drivescancer exosome mirna signaling in the tumor microenvironment which in turn reinforces tumor invasiveness and progression however how exosome mirnasare enriched or how exosome mirna signaling is initiated in cancer cells remains largely unknown elucidating the mechanisms ofselective exosome mirnaenrichment in cancer cells may help identify new cancertherapeutic opportunities that are urgently neededrecentreports have indicated that certain rnabinding proteinsrbps are involved in exosomemirna sorting in eukaryotic cells and the type ofrbps involved seems to differ among various modelsystems [ ] suggesting that exosome mirnasorting is a tissue or celltype specific processfurthermore there have been no reports on the identification of rbps that regulate exosome mirna sorting in pancreatic cancer cells we have recentlycharacterized the biogenesis of exosome mir1246 which is the most highly enriched mirna inpancreatic cancer cellderived exosomes the aimof this study was to utilize our established cell modelsystems to identify rbps that are involved in exosomemirna loading in pancreatic cancer cells using alabeled mir1246 probe as bait we fished out several rbpsincludingserine and arginine rich splicing factor srsf1eukaryotic translation initiation factor subunit beif3b and t cellrestricted intracellular antigen tia1 we found that srsf1 a recently claimedoncoproteininregulating exosome mirna enrichment in pancreaticcancer model systemsfrom pancreatic cancer cellspredominantlyinvolved ismethodscell culturelines panc1the human pancreatic cancer celllinemiapaca2 and bxpc3 and breast cancer cellmdamb231 were obtained from the american typeculture collection atcc manassas va usa cellswere cultured following atccs instructions except thatexosomedepleted fetal bovine serum fbs and horseserum wereapplied whenever needed exosomedepleted fbs and horse serum were prepared by pelleting the serum exosomes at g for h at °ccells were routinely incubated in a humidified environment at °c and co2exosome isolationexosomes were isolated from the culture medium utilizing a combination of centrifugation ultracentrifugationand filtration as we recently described [ ] withminor modifications in brief the culture medium ofpanc1 cells was precleared by g centrifugationfor min at °c and the resulting supernatant was filtered through a μm pvdf centrifuge filter thelarge size evs were trapped in the filter and recovered inpbs the filtered supernatant was then applied to a μm pvdf centrifuge filter the medium size evswere trapped in the second filter and resuspended inpbs the small size evs exosomes in the final supernatant were recovered by ultracentrifugation g min at °c the isolated exosomes were verified bywestern blot detecting positive and negative exosomemarker proteins and nanop analysis nanosightns300 system malvern instruments uk measuringboth sizes and concentrations of the isolated exosomesfig mirna binding protein pulldownpulldown experiment was performed using the pierce¢magnetic rnaprotein pulldown kit thermo fisherscientific briefly pmol of biotinlabeled mir1246or polya rna oligonucleotides integrated dna technologies were hybridized to μl streptavidin magnetic beads prod1862766 thermo fisher scientificthe mir1246biotinstreptavidin beads were incubatedwith panc1 lysate for min at °c the lysatebeadmixture was washed three times with washing bufferfrom the abovementioned kit to elute bound proteins μl of elution buffer was applied and a magnetic separator was applied to separate the beads from the elutedprotein following the manufacturers protocol pierce¢magnetic rnaprotein pulldown kit thermo fisherscientific proteins were separated by sdspage beforemass spectrometry ms analysis 0cxu cell communication and signaling page of fig verification of the exosomes derived from panc1 cells a representative western blot analysis of cd63 nonreducing condition cd81flotillin and calnexin in the evs isolated from panc1 cells positive exosome markers are only detected in small evs exosomes b representativenanop tracking analysis of exosomes small evs derived from control and srsf1 knockdown panc1 cells three individual experimentswere performed for both a and bliquid chromatographymass spectrometry lcmsmassspectrometry ms measurementthe experiment was performed by the laboratory formolecular biology and cytometry research core facilityat ouhsc proteins were digested with trypsin according to the fasp protocol briefly the eluate was buffer exchanged in m urea the proteins were reducedwith mm dithiothreitol and then alkylated with mm iodoacetamide the peptides were eluted dried andresuspended liquid chromatography tandem mass spectrometry was performed by coupling a nanaoacquityuplc waters corp manchester uk to a qtofsynapt g2s instrument waters corp manchesteruk each protein digest about ng of peptide wasdelivered to a trap column μm mm nanoacquity uplc nanoease column μm beh c18 waterscorp manchester uk at a flow rate of μlmin in solvent a mm ammonium formate ph inhplc grade water tandem mass spectra were generated in the trapping region of the ion mobility cell byusing a collisional energy ramp from v low massstartend to v high mass startend the pusherionmobility synchronization for the hdmse method wasperformed using masslynx v41 and driftscope v24lockspray of glufibrinopeptideb mz wasacquired every s and lock mass correction was appliedpost acquisitionprotein identificationraw ms data were processed by plgs proteinlynxglobal server waters corp manchester uk for peptide and protein identification msms spectra weresearched against the uniprot human database containing reviewed sequences with the followingsearch parametersfull tryptic specificity up to twomissed cleavage sites carbamidomethylation of cysteineresidues was set as a fixed modification and nterminalprotein acetylation and methionine oxidation were set asvariable modificationssmall rna library preparation and next generationsequencingtotal rna was extracted from cell and exosome pelletsusing the trizol reagent invitrogenlife technologiescarlsbad california the small rna libraries were constructed and run on the illumina miseq platform as werecently described [ ]rna immunoprecipitation assaypanc1 cells or mdamb231 celllysates were prepared using ip buffer mm trishcl ph mmnacl mm edta mm pmsf and triton x the lysate was sonicated for min on ice andinsoluble material was removed by centrifugation supernatants were collected and protein concentrations weremeasured the supernatant was precleared by proteing dynabeads¢thermo fisher scientific and thenmixed with antibody srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 in a ratio of at °c overnight withgentle rotation to capture the antibodyproteinrnacomplexes μl of protein g magnetic beads wereadded and the complexes were rotated for h at °cthe sample was separated by magnetic separation trizol reagent invitrogenlife technologies was appliedto isolated rna from the complex the mirna expression was analyzed by qrtpcr 0cxu cell communication and signaling page of coimmunoprecipitation coipcoimmunoprecipitation coip using panc1 cell lysate and antibody of srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 was performed as described previously and the protein complex was detected by westernblotwestern blot analysiswestern blot was performed as we recently described[ ] primary antibodies raised against srsf1 santacruz sc33652 eif3b santa cruz sc137214 tia1santa cruz sc166247 betaactin a5441 and glyceraldehyde 3phosphate dehydrogenase gapdh santacruz sc47724 were used for detection nuclear andcytoplasmic protein extraction was extracted followingrockland nuclear cytoplasmic extract protocol and verified by histoneh3 cst 4499s andgapdh santa cruz sc47724 detection antibodiesused for exosome marker detection include cd63cd81 santa cruz bio technology inc ca usaflotillin1 and calnexin cell signaling technology incma usaquantitative realtime reverse transcription polymerasechain reaction qrtpcrqrtpcr was performed as we described [ ] withspecific primers cel54 ²gcgcgcccgtaatcttcataatcc3² mir1246 ²gcgcgatggatttttggagcag3² mir320c ²gcaaaagcuggguugagagggu3² and mir320d ²gcgaaaagcuggguugagagga3²srsf1 shrna expression plasmid constructiontarget specific oligonucleotides were designed using online tool rnai codex cold spring harbor laboratoryand were synthesized integrated dna technologieswith the addition of overhangs according to the cuttingsite of bamh1 and ecori the shrna expression plasmid was constructed by annealing the oligonucleotidesto psihh1 vector following the user manual of psihh1 shrna system sbi system bioscience the oligonucleotide sequences for shrna of srsf1 eif3b ortia1 are provided in supplemental table 3rd generation packaging plasmidslentivirus transductionlentiviral ps were produced as previously described using the shrna expression plasmid andthepmd2gaddgene plasmid pmdlrregp addgeneplasmid and prsvrev addgene plasmid the packaging plasmids were cotransfectedwith the lentiviral expression vector into t cellsusing the polyethyleneimine polysciences inc to produce replication deficient lentivirus after transfectionthe supernatant was pooled and filtered with a μmmembrane and concentrated by ultracentrifugation toacquire lentivirus infection was performed by usinglentivirus in the presence of μgml polybrene sigmaaldrich approximately h postinfection cells wereselected by treating with μgml puromycin invivogen san diego cagfpsrsf1 expressionthe gfpsrsf1 expression plasmid was a gift from drmassimo caputi dna transfection was performedusing lipofectamine thermo fisher scientific topanc1 cells and the expression of gfpsrsf1 wasverified by western blotgstsrsf1 protein purificationbl21 thermofisher scientific c600003 competentcells transformed with pgex6psrsf1 dna addgeneplasmid were cultured at °c for hand after od600 reached to bacteria weretreated with mm isopropyl βd1thiogalactopyranoside for h at °c gsttaggedsrsf1 was purifiedwith glutathione sepharose beads ge health carethe purity of the recombinant proteins was determinedby sdspage with coomassie blue stainingelectrophoretic mobility shift assay emsaird800 labeled mir1246 μm integrated dnatechnologies was mixed with μl of gst slurry stsrsf1 in binding buffer tris ph mm kcl mm mgcl2 mm np40 dtt mm glycerol and incubated at room temperature for minavoiding light 5x loading buffer kcl mm tris ph mm glycerol xylene cyanol bromophenol blue was then added and the complexwas separated on a native gel polyacrylamide m tris ph m glycine m edta apstemed at voltage for min the signal was detected using the licor odyssey imaging systemlicor inc usadesign of decoy motif mimicsthe decoy motif mimics were designed by permutationand combination of the identified motif sequences in thelength of nucleotides the secondary structure of thedesigned sequences was analyzed in rnafold webserveruniversityselfcomplementary were selected decoy mimics ²uuggacuaggacuaggau3² decoy mimics ²aggaaggaaggaagga3²sequences withoutof vienna 0cxu cell communication and signaling page of bioinformatics analysisthe mirna motif analysis was performed using memesuite the protein profile analysis for the result ofmass spectrometry was performed using david bioinformatics abcc at saicfrederick inc the rnabinding protein and mirna sequence binding analysiswas performed using the database of rnabindingspecificities rbpdb srsf1 expression in cancertissues was examined using oncomine thecorrelation of gene expression with cancer patient survival was extracted from the human protein atlasscilifelab sweden statisticsstatistical analyses were performed using graphpadprism software graphpad software inc la jolla causa the heatmap was made in rstudio rstudio incwith the ggplot2 package students ttest was applied to determine significant differences among controland experimental groupsresultsidentification of mir1246 associated proteinsbecause rbps are involved in exosome mirna sortingwe first sought to identify proteins that bind to mirnashighly enriched in cancer exosomes mir1246 the mosthighly enriched mirna in pancreatic cancer exosomeswas biotinlabeled and incubated with a cellular lysatefrom panc1 cells the biotinmir1246 probe wascaptured with streptavidincoated magnetic beads biotinlabeled polya mimics were used as control themirnaprotein complexes were eluted and the proteinswere analyzed by liquid chromatographymass spectrometry in triplicate table there were total of proteins specifically pulled down by the mir1246 probeinterestingly about half of the proteins that associatewith mir1246 are vesicleassociated proteins supplement fig 1a based on the intensity of detection rnabinding property and cancer relevance we ranked therbps using the database for annotation visualizationand integrated discovery david this resulted in tencandidate rbps that complex with the mir1246 sequenceexosomestable among them srsf1 also called sfrs1 waspredictedsequencethe mir1246and arerelevantto eukaryotictobindtotable over view of the result of mass spectrometryexperiments conditionpoly a panc1number of proteins detectedpoly a mdamb231mir1246 panc1mir1246 mdamb231table mir1246 rna binding protein candidates obtainedfrom the mass spectrometric analysisprotein full nameprotein symbolsrsf1serineargininerich splicing factor park7eif3bthoc4acocddx5tia1if5a1eif2aimdh2parkinson disease protein eukaryotic translation initiation factor subunit btho complex subunit alyref export factorcytoplasmic aconitate hydrataseprobable atpdependent rna helicase ddx5tcellrestricted intracellular antigen1eukaryotic translation initiation factor 5a1eukaryotic translation initiation factor 2ainosine5²monophosphate dehydrogenase supplement fig by in silico analysis using the database of rnabinding specificities rbpdb levelsverification of srsf1 binding to mir1246rna immunoprecipitation rip was performed to verifythe association of several identified rbps with mir1246including srsf1 eif3b and tia1 igg and gapdhantibody was used as controls for immunoprecipitationas shown in fig 2a mir1246 expression is more than12fold higher in the srsf1precipitants as compared tothat of igg precipitants indicating a specific associationof srsf1 with mir1246 mir1246 expression wasmoderately increased in the tia1precipitants and nearigg controlin the eif3b precipitants coimmunoprecipitation coip experiments were performed to verify the immunoprecipitation proceduresdata not shown to directly determine the binding ofsrsf1 to the mir1246 sequence glutathionestransferase gst conjugated human srsf1 protein wasexpressed in bl21 competent e coli captured by glutathione sepharose beads and eluted by glutathione thepurity of eluted gstsrsf1 protein was shown by sdspage and coomassie blue staining supplement fig the binding of gstsrsf1 to a fluorescenttaggedmir1246 probe was determined by rna emsa asshown in fig 2b binding of the labeled probe was specific to gstsrsf1 but not gst and increased withgreater protein input the specific binding of gstsrsf1 to the mir1246 probe was evident as the unlabeled mir1246 probe effectively competed with thelabeled mir1246 probe in a concentrationdependentmanner fig 2c the detected bands were semi quantified and the kd was calculated from the detected signalsfig 2d these data confirmed the direct binding ofsrsf1 to the mir1246 sequence 0cxu cell communication and signaling page of fig srsf1 binds to mir1246 a qrtpcr detection of mir1246 in igg gapdh srsf1 eif3b and tia1 immunoprecipitants of panc1 lysaten p student ttest bc emsa detection of the srsf1mir1246 complex hot probe ird800 labeled mir1246 mimics cold probemir1246 mimics n direct binding of gstsrsf1 and mir1246 b and concentrationdependent competition between the cold and hotmir1246 probe for binding to gstsrsf1 c d semiquantification of srsf1 and mir1246 binding in c and calculated dissociationconstant n exosome mirna enrichment by srsf1 in cancer cellsbecause srsf1 is a key splicing factor that is essential toeukaryotic cells a knockout model could not beestablished thereforeto determine whether srsf1mirna binding activity is relevant to exosome mirnaenrichment we established a lentivirus srsf1 shrnaconstruct to knockdown srsf1 expression in panc1cells fig 3a interestingly though srsf1 protein wasdetected both in the nucleus and cytoplasm the knockdown was more pronounced in the cytoplasm fig 3bknockdown of srsf1 did not significantly alter the concentration and size distribution of the exosomes releasedby panc1 cells fig 1b cellular and exosome rnafrom control and srsf1shrna cells were isolated andsmall rna sequencing was performed among the highly enriched panc1 exosome mirnas expressionof mirnas was significantly downregulatedin exosomes derived from srsf1shrna panc1 cellsas compared to exosomes derived from control panc1cells fig 3c strongly indicating the involvement ofsrsf1 in exosome mirna enrichment a heatmapshowing the expression of the top mirnas enrichedin panc1 exosomes demonstrates the dramatic dropin expression levels of mirnasin srsf1shrnapanc1 exosomes compared to panc1 exosomesfig 3d notably mir1246 was the highest enrichedexosome mirna data not shown and its expressionin exosomes wassignificantly reduced by srsf1knockdown fig 3d on the other hand among of the mirnas less enriched in exosomes only were expressed at lower levels in exosomesderived from srsf1 knockdown cells as compared toexosomes derived from wild type panc1 cells fig3c suggesting that srsf1 knockdown mainly affectsexosome enriched mirnasto further confirm the effect of srsf1 knockdown onexosome mirna enrichment the expression levels ofseveral representative mirnas were quantified by qrtpcr srsf1 knockdown in panc1 cells significantlyreduced exosome levels of mir1246 mir320c andmir320d confirming the small rna sequencing resultsfig 3e in contrast knockdown of eif3b or tia1 didnot reduce exosome mir1246 expression suggestingthat these rbps may not promote exosome mirna 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig cellular and exosome mirna profiles after srsf1 knockdown in panc1 cells a detection of srsf1 knockdown by shrnas in panc1 cellsb panc1 srsf1 protein levels in nuclear and cytoplasmic fractions nc normal control c venn diagram of overlap of mirnas detected by nextgeneration small rna sequencing in srsf1 knockdown and control panc1 cells and exosomes d heatmap showing the expression of top exosome mirnas in cells and exosomes after srsf1 knockdown e qrtpcr analysis of mir1246 mir320c and mir320d in exosomes derivedfrom control and srsf1 knockdown panc1 cells p students ttest shown are representatives of three independent experiments aeenrichment supplement fig and our observationswere extended to two additional pancreatic cancer celllines miapaca2 and bxpc3 fig 4af in additionexpression of let7c which is less enriched in exosomeswas unchanged in exosomes after srsf1 knockdowndata not shownto verify the involvement of srsf1 in exosomemirna enrichment in cancer cells we also exogenouslyoverexpressed srsf1 in panc1 cells a gfpsrsf1 expression plasmid was introduced into panc1 cells andsrsf1 overexpression was confirmed by western blotfig 5a expression of mir1246 mir320c and mir320d in the exosomes derived from gfpsrsf1 panc1cells was analyzed by qrtpcr fig 5bc as shown infig 5b overexpression of gfpsrsf1 increased exosome expression of mir1246 and rescued mir1246levels in exosomes derived from srsf1shrna cellslevels of mir320c and mir320d were also increased inexosomes derived from the gfpsrsf1 cellsfurthersupporting the involvement of srsf1 in exosomemirna enrichment fig 5cdidentification of rna sequence motifs involved inexosome mirna enrichmentaccording to the rbpdb srsf1 binds specifically to amotif present in the mir1246 sequence supplementfig qrtpcr analysis of mir1246 mir320c mir320d expression in exosomes derived from srsf1 knockdown bxpc3 and miapaca2 cells ac qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown bxpc3 cells n p student ttest df qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown miapaca2 cells n p students ttest 0cxu cell communication and signaling page of fig qrtpcr analysis of exosome enriched mirnas derived from srsf1 overexpression panc1 cells a confirmation of gfpsrsf1overexpression in panc1 cells b qrtpcr detection of mir1246 in exosomes derived from wild type and srsf1 knockdown panc1 cells withgfpsrsf1 overexpression c qrtpcr detection of mir320c in exosomes derived from gfpsrsf1 overexpression panc1 cells d qrtpcrdetection of mir320d in exosomes derived from gfpsrsf1 overexpression panc1 cells p students ttest n for bdfig to understand the contribution of specific rnamotifs involved in exosome mirna enrichment we applied an unbiased approach to identify the rna motifsthat contribute to exosome mirna enrichment for thispurpose we analyzed the rna sequences of the mirnas highly enriched in cancer exosomes and regulatedby srsf1 using the bioinformatics tool meme suite a 6bp length motif was found to be shared in of the exosome enriched mirnasincluding mir fig ac to test whether the binding of srsf1to mir1246 depends on this motif two decoy mimicswere designed according to the shared motif sequencesand their secondary structure determined with thernafold webserver httprnatbiunivieacatcgibinrnawebsuiternafoldcgi the binding of the decoymimics to srsf1 protein was determined by rnaemsa analysis addition of decoy motif did not alterthe binding of srsf1 to the mir1246 probe fig 6dwhereas decoy motif competed with mir1246 binding to srsf1 in a concentrationdependent manner fig6df indicating that srsf1 directly interacts with thissequence motifdiscussionthe role of exosome mirna signaling in promotingcancer progression has been intensely investigated andwell recognized in recent years [ ] the higher enrichment of certain mirnas in cancer exosomes [] indicates that exosome mirna encapsulation isan active cellular process that initiates exosome mirnasignaling in the tumor microenvironment however thespecificselectivecellular processresponsiblefor 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig srsf1associated exosome mirna sequence motif analysis a the motif commonly shared among srsf1associated exosome mirnas bvenn diagram showing the number of srsf1associated exosome mirnas that share the motif c list of mirnas sharing the common motif demsa analysis demonstrating the inhibition of gstsrsf1 binding to mir1246 by rna decoys d1 decoy ²uuggacuaggacuaggau3² d2decoy ²aggaaggaaggaagga3² e concentrationdependent inhibition of gstsrsf1 binding to mir1246 by d2 f semiquantification ofthe detected bands in fig 5e and the calculated dissociation constantexosome mirna enrichment has not been well established in eukaryotic cells the most significant findingfrom the present study is that we have identified srsf1as a mediator of exosome mirna enrichment in pancreatic cancer cells a specific mirna sequence motif wasalso identified that may be involved in the exosomemirna enrichment process these findings provide newinsight into how mirnas are enriched in cancer cellexosomesexosomemediated mirnasignalinginitiatetowe recently reported that exosome mir1246themost highly enriched mirna in pancreatic cancer cellderived exosomes is derived from rnu2 a smallnuclear rna important for mrna splicing alongthis line of our research we sought to determine howthis mirna is enriched in cancer exosomes using ourestablished model systems in the present study we haveprovided severallines of evidence demonstrating thatsrsf1 a vital splicing factor and established oncoprotein is significantly involved in exosome mirnaenrichment in pancreatic cancer cells the first line ofevidence indicating srsf1 involvementin exosomemirna enrichment was obtained from the biotinlabeled mir1246 pulldown experimentfollowed byproteomic analysis among the rbps identified severalwere selected based on their detection intensity relevance to extracellular vesicles and reported connectionsto human cancer including srsf1 eif3b andtia1 of note srsf1 was the only rbp among themthat was also predicted by the rbpdb to bind to a motifin the mir1246 sequence furthermore the direct binding of srsf1 to the mir1246 sequence was verified byrip and rna emsa analysis strongly indicating thephysical interaction of srsf1 and not eif3b or tia1with the mir1246 sequence the most convincing evidence demonstrating the involvement of srsf1 in cancer exosome mirna enrichment was the observationthat knockdown of srsf1 significantly reduces exosomemirna enrichmentfor a majority of the selectivelyenriched exosome mirnas without altering the expression levels of less enriched exosome mirnas these results were based on small rna sequencing andconfirmed by rtpcr analysis the observations werealso extended to additional human pancreatic cancer celllines including miapaca2 and bxpc3srsf1 was initially identified as a splicing factor ineukaryotic cells but srsf1 was later revealed toindependent ofshuttle between the nucleus and cytoplasm to regulate rna metabolism mirna procession and othercellular eventsthe mrna splicingprocess importantly srsf1 is overexpressed indifferent cancer types and is considered a potent oncogene [ ] moreover srsf1 over expression in different types of cancer is associated with worse prognosissupplement fig while the full spectrum of srsf1function remains to be determined our results revealthat srsf1 binds to specific mirnas and is significantlyinvolved in exosome mirna enrichment in cancer cellsthis function is likely independent ofthe splicingprocess as the reduced expression of the detected exosome mirnas after srsf1 knockdown is greater thantheir expression change in the cells because exosomemirna signaling contributes to tumor developmentthrough intercellular communication in the tumormicroenvironmentthe involvement ofsrsf1 in exosome mirna signaling initiation likelyrepresents a part of its oncogenic action which maylead to new therapeutic strategies to intervene withexosome mirna signaling in cancer several rbpshave been previously identified as mediators of exosome mirna sorting in various model systemsincluding major vault protein in colon cancer cells hnrnpa2b1 in t cells and ybx1 in hek293tcells the identification of srsf1 involvement inexosome mirna enrichmentin pancreatic cancercells further supports the notion that the cellular exosome mirna sorting process in eukaryotic cells maydiffer among different cell types[ ]we have also identified a mirna motif commonlyshared by the srsf1associated exosome mirnasusing the meme suite program memesuitethis motif was specifically bound by srsf1 as evidenced by our rna emsa analysis a similar motifalbeit slightly shorter was identified in our recentreport that describes exosome mir1246 enrichmentin pancreatic cancer cells our results reinforcethe concept that specific mirn
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Costello syndrome is an autosomal dominant disorder that is caused by germline HRAS mutations Patients withCostello syndrome present craniofacial abnormalities cardiac defects and cancer predisposition as well as skinabnormalities including papillomas keratosis pilaris and eczematous dermatitis However the mechanisms underlyingthe dermatological abnormalities remain unclear Here we demonstrated that knockin mice expressing an Hras G12Smutation HrasG12S mice are susceptible to develop atopic dermatitis ADlike skin lesions including eczemapruritus elevated serum IgE levels acanthosis and the ltration of mast cells basophils and type2 innate lymphoidcells in the dermis after stimulation with house dust mite allergens Dermatophagoides farinae Dfb Reduced skinbarrier function increased proliferation of phosphorylated ERK pERKpositive epidermal cells and increased Th2typecytokines as well as epithelial cellderived cytokines including IL33 were observed in the skin tissue of HrasG12Smice compared with Hras mice Cultured HrasG12S keratinocytes exhibited increased IL33 expression after Dfbstimulation PD0325901 an MEK inhibitor ameliorated ADlike symptoms in HrasG12S mice showing decreasedproliferation of pERKpositive epidermal cells and decreased expression of IL33 Our ï¬ndings indicate that theepidermis of HrasG12S mice stimulated by Dfb strongly induced IL33 expression and type2 innate lymphoid cellsresulting in ADlike skin lesions These results suggest that the epidermis of HrasG12S mice are prone to developmentof eczematous dermatitis stimulated with house dust mite allergensIntroductionThe skin is a stratiï¬ed epithelium consisting of severallayers of cells in various stages of differentiation In orderto maintain normal skin homeostasis the proliferationdifferentiation and response of epidermal cells to externalstimuli must be tightly regulated1 The RASMAPK signaling pathway plays a crucial role in cell proliferationdifferentiation and apoptosis23 A strong activation of theRASMAPK pathway in skin is known to resultinCorrespondence Yoko Aoki aokiymedtohokuacjp1Department of Medical Genetics Tohoku University Graduate School ofMedicine Sendai Japan2Department of Pediatrics Tohoku University Graduate School of MedicineSendai JapanFull list of author information is available at the end of the Edited by E Candiepithelial cancers and melanoma45 Pigmented lesionshyperkeratosis pruritus curly hair and hyperplasia havealso been observed in vemurafenib a BRAF inhibitortreated patients6 The balance of the RASMAPK signaling pathway could be particularly important for epidermalhomeostasisCFCNoonan syndrome Costello syndrome and cardiofaciocutaneoussyndrome are phenotypicallyoverlapping genetic disorders characterized by craniofacial dysmorphia congenital heart defects and psychomotor retardation7 These syndromes are commonlycaused by germline mutations in components of the RASMAPK pathwaytermed RASopathies which constitutively activate the RASMAPK pathway89 Of thesesyndromes Costello syndrome is characterized by short The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of stature craniofacial abnormalities congenital heart diseases hypertrophic cardiomyopathy and intellectual disability10 Approximately patients with Costellosyndrome develop malignant tumors including rhabdomyosarcoma and bladder carcinoma In we identiï¬ed germline HRAS mutations in patients with Costellosyndrome11 A nucleotide change that cause the substitution of glycine at codon to serine pG12S in oneallele of HRAS has been observed in of Costellosyndrome patients The G12S mutation which has beenidentiï¬ed in somatic cancer is an oncogenic mutationthat activates the downstream pathway Patients withCostello syndrome develop a variety of skin abnormalitiesincluding palmoplantar keratoderma acanthosis nigricans eczema loose skin cutis laxa darker skin colorand papillomata around nose and anus However thepathogenesis of dermatological abnormalities remainsunclearWe have recently generated a strain of knockin miceexpressing an Hras G12S mutation the most frequentmutation in Costello syndrome12 which exhibited facialdysmorphia cardiomyocyte hypertrophy and kidneyanomalies Impaired mitochondrial fatty acid oxidationwas observed in HrasG12S mice fed a highfat diet13Skin abnormalities including papillomas have not beenobserved in young HrasG12S mice weeks old underspeciï¬c pathogenfree conditions In contrast HrasG12Smice over weeks of age or highfat diet fedHrasG12Smice had cutaneous lesions due to scratching Supplementary Fig 1a under the same pathogenicfree condition Although we have not analyzed the histology of skinof HrasG12S mice over weeks of age gross appearances of the skin lesions and scratching behavior suggestthat they are atopic dermatitislike In the current studywe tested to generate experimentally induced dermatitisin HrasG12S mice and found that HrasG12S micedeveloped more severe atopic dermatitis ADlike lesionsthan Hras mice after treatment with house dust miteallergens Dermatophagoides farinae Dfb Furthermorethese ADlike skin lesions in HrasG12S mice werereversedbyan MEK inhibitorPD0325901treatment withResultsDfb ointment induces ADlike skin lesions in HrasG12SmiceWe ï¬rst tested the effect of picryl chloride whichinduce contact dermatitis and imiquimod which inducepsoriasis on the skin of Hras and HrasG12S miceSupplementary Fig 1b c but no difference in skinlesions was observed between them Supplementary Fig1d In contrast the treatment with Dfb ointment developed severe dermatitisincluding severe erythemaOfï¬cial journal of the Cell Death Differentiation Associationhemorrhage scarring and eczema in the dorsal skin ofHrasG12S mice but not in Hras mice Fig 1a andSupplementary Fig 2a The ears of HrasG12S micebecame thick with edema erosion and excoriationFig 1b The dermatitis score was significantly higher inDfbtreated HrasG12S mice than in any other group ofmice SDStreated control Hras mice DfbtreatedHras mice and SDStreated control HrasG12Smice on day Fig 1c and Supplementary Table Other dermatitis parameters including the ear swellingFig 1d and the scratching behavior Fig 1e increasedsignificantly in Dfbtreated HrasG12S mice comparedwith Dfbtreated Hras mice Serum IgE levels weresignificantly higher in HrasG12S mice compared toHras in nontreated baseline ± ngmL vs ± ngmL P Supplementary Fig Although the difference was not statistically significant in SDS treatment groups IgE levels were higher inHrasG12S mice compared to Hras ± ngmLvs ± ngmL P Fig 1f as well as in theDfb treatment groups ± ngmL vs ± ngmL P Fig 1f These symptoms werealso seen in Hras mice but skin lesions are moresevere in HrasG12 mice In both groups of mice the IgEelevations were triggered by Dfb ointmentWe next examined if Dfbinduced dermatitisinHrasG12S mice is caused by the same pathology as inHras miceHistological analysis revealed hyperkeratosis and epidermal hyperplasia in the dorsal skin of DfbtreatedHrasG12S mice Fig 2a The epidermis of HrasG12Smice became thicker than that of Hras mice althoughDfb treatment increased the epidermal thickness in bothHras and HrasG12S mice Supplementary Fig 2b Inthe ADlike skin lesions Dfbtreated HrasG12S micedisplayed increased number of mast cells toluidine blueand tryptase 1 a marked increase in the numbersof T cells CD4 and dendritic cells MHC class IIFig 2a b and Supplementary Fig 2c e Western blottinganalysis revealed that the levels of CD4 protein weresignificantly increased in Dfbtreated mice compared withcontrol mice Supplementary Fig 2d In line with theacanthosis of Dfbtreated HrasG12S mice an increasednumber of phosphohistone H3positivecells wereobserved in the suprabasal epidermis layers of HrasG12Smice Fig 2b Although phosphorylated ERK pERKpositive cells were also increased in the epidermis of Dfbtreated Hras and HrasG12S mice the immunostainedarea in HrasG12S mice was significantly larger than thatin Hras mice Fig 2b and Supplementary Fig 2f g Inaddition we examined the expression of ï¬laggrin andclaudin1 as epidermal barrier markers in AD14 Adecreased expression of claudin1 was observed in Dfb 0cKatata Cell Death and Disease Page of Fig Dfb treatment induces atopic dermatitislike skin lesions in HrasG12S mice a b Skin and ear features of mice on day after treatmentof Dfb ointment HrasG12S mice showed dermatitis by repeated application of Dfb a The severity of ear swelling responses to Dfb was strongerin HrasG12S than Hras mice b c Dermatitis scores of only SDStreated control Hras and HrasG12S and Dfbtreated Hras andHrasG12S mice n per group d Time course of ear thickness from Hras and HrasG12S mice after treatment with SDS or Dfb n pergroup e The number of scratching bouts per min assessed by the video n per group f Serum IgE levels in Hras and HrasG12S mice aftertreatment with SDS or Dfb n per group Data are presented as mean ± SD Signiï¬cance in c d and f was analyzed by oneway ANOVAand the TukeyKramer method P P P and P HrasG12S Dfb vs Hras Dfb P P P and P HrasG12S Dfb vs HrasG12S SDS P P P and P Hras Dfb vs Hras SDSSigniï¬cance in e was analyzed by twotailed Students t test P treated HrasG12S mice compared with control HrasG12Smice Fig 2c d Together these results indicate that Dfbapplied HrasG12S mice exhibited more severe ADlikeskin lesions than Hras mice including acanthosis withhyperproliferation of pERKpositive cells in the epidermisas well as increased ammatory cells and reducedclaudin1 expressionThe skin of Dfbapplied HrasG12S mice shows an increaseof itchassociated factors and ammatory cytokinesTo further characterize the ADlike skin lesions weevaluated the levels of the itchassociated factors andammatory cytokinesin the skin of DfbtreatedHrasG12S mice Itchrelated neuronal markers including skin Tac1 Klk7 and Klk14 mRNA levels or PAR2 andOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see ï¬gure on previous pageFig Histological analysis reveals acanthosis with hyperproliferation of pERKpositive epidermal cells increased ammatory cells andreduced claudin1 expression in the dorsal skin of Dfbtreated HrasG12S mice a Skin tissue stained with HE and TB b c Immunohistochemistryof CD4 tryptase 1 pERK pHH3 and claudin1 in the skin ac Scale bars μm d Lysates from the skin were immunoblotted with antiClaudin1antibody Band intensities were quantiï¬ed and compared among the four groups The expression levels were normalized to GAPDH n in eachgroup Data are presented as mean ± SD Signiï¬cance was analyzed by oneway ANOVA and the TukeyKramer method P P twotailedStudents t testIl1 proammatory cytokineEndothelin proteins17 were significantly higher thanthose in control HrasG12S and Dfbtreated Hras miceFig 3ac Regarding ammatory cytokines the skinIl4mRNA levels ofTh2related cytokine and epidermalderived cytokinesIl33 and thymic stromal lymphopoietin Tslp were significantly elevated in Dfbtreated HrasG12S mice compared with control HrasG12S and Dfbtreated Hrasmice Fig 3d IL33 leads to the activation of type2innate lymphoid cells ILC2s through ST2 IL33 receptor21 In Dfbtreated HrasG12S mice the skin mRNAlevels of St2 were also significantly increased Fig 3dImmunohistochemistry analysis revealed that DfbtreatedHrasG12S mice showed enhanced expression levels ofIL33 and TSLP in the basal epidermal layers and thesurface of epidermis respectively Fig 3e Likewise theIL33 protein levels were significantly higher in the skin ofDfbtreated HrasG12S mice than in Dfbtreated Hrasmice Fig 3fIncreased numbers of ILC2 and increased IL33 expressionwere observed in the epidermis of HrasG12S miceTo investigate when and how ammatory cytokinesare induced in HrasG12S mice we performed ï¬owcytometry on skin and ear samples days after Dfbapplication that is once the ADlike skin lesions hadbegun to appear Fig 1c The accumulation of basophilsand ILC2s was observed on skin samples of DfbtreatedHrasG12S mice days after Dfb application Fig 4a Asignificant increase in mast cells eosinophils basophilsand ILC2s was also observed in the ears of HrasG12Smice Fig 4b On the other hand these immune cellswere hardly detected in Hras and HrasG12S mice days after SDS application Supplementary Fig The mRNA levels of ammatory cytokines in the skinfrom HrasG12S mice days after Dfb applicationshowed that Il1 and Il33 were significantly elevated inthe skin of Dfbtreated HrasG12S mice compared withDfbtreated Hras mice Fig 5aAtopic dermatitis is characterized by increased serumIgE acanthosis loss of skin barrier function and ltration of immune cells including Th2 cells dendriticcells eosinophils basophils and mast cells2223 On thataccount we next examined the response of immune cellsin these mice HRAS is known to be highly expressed inOfï¬cial journal of the Cell Death Differentiation Associationthe epidermal cells but not in immune cells2425 Indeedno significant difference was found in the population ofimmune cells from the spleen and lymph node LN Theproliferation of naive CD4 T cells and Th2 immuneresponse was comparable between Hras and HrasG12Smice at weeks of age suggesting that ADlike dermatitismay not be caused by different response of immune cellsFig 6a b Then we examined whether an increasedproduction of IL33 and TSLP was observed in primaryepidermal keratinocytes in response to Dfb Six hoursafter Dfb stimulation the mRNA level of Il1 not Tslpwas significantly elevated in cultured epidermal keratinocytes of Hras and HrasG12S mice Fig 5b NotaIl33 in the Dfbstimulatedblykeratinocytes of HrasG12S mice were significantlyincreased compared with those of nonstimulatedHrasG12S and Dfbstimulated Hras keratinocytessuggesting that epidermal keratinocytes with Hras G12Smutation have increased IL33 expression after stimulation with Dfb Fig 5bthe mRNA levels ofPD0325901 reduces skin lesions in DfbstimulatedHrasG12S miceamelioratesTreatment with MEK inhibitorstheabnormalities observed in RASopathyrelated modelmice26 We investigated the effects of an MEK inhibitor PD0325901 on skin lesionsin DfbtreatedHrasG12S mice Supplementary Fig 5a Ten days oftreatment with PD0325901 or saline resulted in a significant improvement of the dermatitis score and earswelling in HrasG12S mice Fig 7a b PD0325901treatment resulted in a marked reduction of epidermalthickness mast cell numbers and pERKpositive epidermal cells as well as recovered expression levels ofclaudin1 Fig 7c d and Supplementary Fig 5b c ThemRNA levels of Il1 Il4 Il33 St2 and Klk14 were significantly lowerin the skin of PD0325901treatedHrasG12S mice than that of vehicle salinetreatedHrasG12S mice Fig 7e and Supplementary Fig 5dThese results suggest that ERK inhibition partially ameliorates Dfbinduced skin lesions in HrasG12S miceDiscussionHere we demonstrated that mice expressing a germlineHras G12S mutation but not Hras mice developed 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see ï¬gure on previous pageFig Expression of itchassociated factors and ammatory cytokines is enhanced in Dfbinduced skin lesions in HrasG12S mice a c dRelative mRNA expression related to neuronal factors a Tac1 and Ngf skin proteases c Klk5 Klk7 and Klk14 and ammation d Il1 Il4 Tslp Il33and St2 in the dorsal skin mRNA levels were normalized to those of 18s rRNA n per group b f Protein extracts from dorsal skin wereimmunoblotted with antiPGP95 antiPAR2 antiEndothelin and antiIL33 antibody n in each group GAPDH same data as in Fig 2d f Bandintensities were quantiï¬ed and compared among the four groups Expression levels were normalized to GAPDH e Immunohistochemistry of IL33and TSLP Scale bars μm Data are presented as mean ± SD Signiï¬cance was analyzed by oneway ANOVA and the TukeyKramer method P P P and P P twotailed Students t testADlike skin lesions under conditions of Dfb exposureThe levels of IL1 and epithelial cellderived cytokinesIL33 and TSLP were also increased in DfbtreatedHrasG12S mice In addition an increased production ofIL1 IL4 and IL33 as well as ammatory cellsbasophils and ILC2s was observed in the dorsal skin ofHrasG12S mice before the development of ADlike skinlesions Analysis of the underlying mechanism revealedthat Dfbstimulated keratinocytes in HrasG12S miceinduced IL33 production while in naive CD4 T cellsfrom the spleen the Th2 immune response was comparable between Hras and HrasG12S mice Finallythe inhibition of ERK activation by PD0325901 treatmentameliorated the ADlike skin lesions and IL33 production Together these data indicate that germline HrasG12S activating mutation causes ADlike skin lesions viathe ERKIL33 axis Fig cellsepidermalIn the present study after repeated stimulation withDfb HrasG12S mice showed hyperproliferation of pERKpositiveand increased IL33expression in the dorsal skin Increased IL33 expressionwas also observed in primary epidermal keratinocytesfrom HrasG12S mice after Dfb stimulation which issimilar to the reports that the activation of RASMAPKsignaling was associated with increased IL33 expressionin cancer cells2930 Recently IL33 was found to inducethe Th2 ammatory response in allergic diseasesespecially AD31 Excess IL33 is also associated with skinbarrier dysfunction and ILC2 functions which is partiallyregulated by the RASMAPK signaling pathway3233Epithelialspeciï¬c IL33 transgenic mice have been foundto develop ADlike dermatitisincluding acanthosispruritus increased IgE serum levels reduced claudin1expression and increased production of eosinophils mastcells and ILCs3334 Of note IL33 and its receptor ST2are highly expressed in the skinderived ILC2s of ADpatients and lung ILC2s of patients with allergic airwaydiseases respectively3536 Importantly consistent with thephenotype of IL33 transgenic mice and AD patients Dfbtreated HrasG12S mice showed ADlike skin lesionsreduced claudin1 expression increased IL33 expressionhyperproliferation of pERKpositive epidermal cells andincreased ILC2 production Collectively excess IL33could lead to ERK activation resulting in an increase ofILC2s and impaired skin barrier Fig Ofï¬cial journal of the Cell Death Differentiation AssociationThe pathogenesis of skin lesions observed in Costellosyndrome includes dermal connective tissue abnormalities cutis laxa and deep palmer and plantar creaseshyperproliferative skin disease palmoplantar keratoderma cutaneous papilloma and acanthosis nigricansand ammatory skin abnormalities sensitive skineczema and pruritus A previous study showed that inthe skin ï¬broblasts of Costello syndrome elastic ï¬berswere not assembled due to a functional deï¬ciency of theelastinbinding protein as a result of an unusual accumulation ofchondroitin sulfatebearing proteoglycans3738 Regarding the hyperproliferative skin lesions ithas been reported that the root cause of papillomashyperkeratosis and epidermal hyperplasia such as psoriasis is the activation of the RASMAPK pathway39However the pathophysiological mechanism of ammatory skin abnormalities in Costello syndrome remainsunclear In the present study Dfbtreated HrasG12S micedisplayed pruritus and eczema Recently mice withepidermisspeciï¬c BRAFRAF1 deï¬cient also showed ADlike dermatitis which is characterized by increased serumIgE levels and a Th2 response43 The elevated IgE levelshave not been systematically examined or reported inCostello syndrome patients However it is possible thatthe allergic reaction stimulated by house dust mites couldbe involved in the development of ammatory skinabnormalities in Costello syndrome patientsincludingsensitive skin pruritus and eczemaimprove HRASdrivenAt present acitretin has been reported to treat palmoplantar keratosis in patients with Costello and CFC syndrome4445 Several reports have demonstrated that MEKtumorigenesis46inhibitorsimpaired enamel formation in the teeth27 and longtermdepression in the hippocampus in Hras G12V knockinmice28 as well as hyperkeratosis and hyperplasia in theforestomach of BrafQ241R mice26 In the present studyPD0325901 treatment of the ADlike skin lesions inHrasG12S mice was found to reverse these lesions byreducing hyperproliferation of pERKpositive epidermalcells and the production of ammatory cells and cytokines including IL1 IL4 and IL33 Treatment withU0126 an MEK inhibitor in human keratinocytes hasalso been found to restore the reduced expression levels ofclaudin1 and ï¬laggrin and increase ERK activationthrough excess IL333347Indeed reduced claudin1 0cKatata Cell Death and Disease Page of Fig An increase in ILC2s and basophils observed in the skin and ear of Dfbtreated HrasG12S mice a b Flow cytometric analysis of skin aand ear b cells from Hras and HrasG12S mice collected days after Dfb application Eosinophils CD45 SSChi siglecF Basophils CD45siglecF FcεRIα DX5 Mast cells CD45 siglecF FcεRIα DX5 ILC2 CD45 Lin CD3ε CD4 CD8a CD11c FcεRIα NK11 CD19 Ter119 CD5 F4 Gr1 Sca1 GATA3 n in each group Data are presented as mean ± SD Signiï¬cance was analyzed by twotailed Students t test P P expression was improved in Dfbtreated HrasG12S miceafter PD0325901 treatment Recently hypertrophic cardiomyopathy in Noonan syndrome patients were treatedby MEK inhibitor48 So MEK inhibitors could be effectivein patients with RASopathies The most common sideeffect of trametinib MEK inhibitor in human patients isOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig Expression of ammatory cytokines is enhanced in the skin of Dfbtreated HrasG12S mice in the early stage of dermatitisa Relative mRNA expression related to ammation including Il1 Il4 Il13 Tslp Il33 and St2 in the dorsal skin of Hras and HrasG12S mice on day after treatment of Dfb mRNA levels were normalized to those of 18s rRNA n per group b Relative mRNA expression of Il1 Tslp and Il33 inDfbstimulated ngμl h or vehicletreated PBS h keratinocyte from Hras and HrasG12S mice mRNA levels were normalized to those ofGapdh Results represent ï¬ve independent experiments Data are presented as mean ± SD Signiï¬cance was analyzed by oneway ANOVA and theTukeyKramer method P P and P skin rush and common toxicity associated with vemurafenib BRAF inhibitor is cutaneous abnormalities suchas keratoacanthoma and squamous cell carcinoma by themechanism of paradoxical MAPK pathway activation49Therefore the balance of RASMAPK signaling plays animportant role in the emersion of skin abnormalitiesAdjusting dosage of MEK inhibitors may be effective onskin lesions of patients with Costello syndromeHere Dfbtreated HrasG12S mice exhibited increasedIL33 expression through hyperproliferation of pERKpositive epidermal cells Additionally we show thatPD0325901 treatment ameliorated the ADlike skinlesions in HrasG12S mice under conditions of exposureto Dfb Thus it will be interesting to investigate whethertreatment with IL33 antibody reduces the ADlike skinlesions in HrasG12S mice Our ï¬ndings provide additional perspective that HrasG12S mice will serve as avaluable model to study pathophysiology and potentialtherapeutic approaches in ADMaterials and methodsMiceHrasG12S mice on a C57BL6J background have beendescribed previously13 Male mice were analyzed inthis studyGenotypingThe genomic DNA was extracted from the tail tissueusing a Maxwell Mouse Tail DNA Puriï¬cation KitPromega Madison WI USA or the alkaline lysismethod For the alkaline lysis method a small piece ofeach tail mm was incubated in mM NaOH for min at °C After the addition of M TrisHClpH the extracts were used for PCR Genotyping ofOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig No significant difference was found in Th2 cytokine responses and the proliferation of naive CD4 T cells between Hras andHrasG12S mice a Naive CD4 T cells were isolated from the spleen of Hras and HrasG12S mice at weeks of age After that the cells werestained with CSFE and cultured with mouse antiCD3 monoclonal antiCD28 monoclonal recombinant mouse IL2 recombinant mouse IIL4 andantiIFNγ antibodies for days After incubation the cells were ï¬xed permeabilized and stained with IL5 and IL13 antibodies Flow cytometricanalysis was performed n in each group b After days culture of puriï¬ed naive T cells from spleen of Hras and HrasG12S mice theproliferation index was measured by ï¬ow cytometry n in each group Data are presented as mean ± SD Signiï¬cance was analyzed by twotailedStudents t testHras and HrasG12S mice was performed by PCRusing KOD FX Neo TOYOBO Osaka Japan The primers used for PCR have been described previously1350Induction of dermatitisAtopic dermatitislike skin lesions were induced in malemice at weeks of age according to the manufacturersinstructions The mice were anesthetized with isoï¬uraneand their dorsal hair was removed using an electric clipper2000AD Natsume Seisakusho Tokyo Japan For sensitization mg of Biostir AD Biostir Inc Kobe Japanan ointment of Dfb extract was applied to the shaveddorsal skin and ears Three or four days later hair growthwas removed with an electric clipper and μl of SDS SigmaAldrich St Louis MO USA was applied tothe shaved dorsal skin and ears to disrupt the skin barrierAfter h mg of Biostir AD was applied to theirshaved dorsal skin and ears to induce ADlike skin lesionsThese procedures were repeated twice a week for daysThe mice were sacriï¬ced on day to collect skin and earsamples Supplementary Fig 2a To assess the effect ofDfb ointment to Hras and HrasG12S mice they wererandomly divided into four groups SDStreatedHras SDStreated HrasG12S Dfbtreated Hrasand Dfbtreated HrasG12S using single blinding testEvaluation of dermatitis and ear thicknessThe dermatitis scores were evaluated twice a weekaccording to the development of four symptoms erythemahemorrhage of dorsal skin scarringdryness ofdorsal skin edema of ear and excoriationerosion ofear51 The total dermatitis score maximum score wasdeï¬ned as the sum of individual scores none mild moderate severe for each symptom SupplementaryTable Ear thickness was measured with a digimaticmicrometer CLM115QM Mitutoyo Kanagawa JapanMeasurement of scratching behaviorOn day scratching behavior was monitored by videoGZHM890 JVC Kanagawa Japan for min TheOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see ï¬gure on previous pageFig MEK inhibitor reduces the clinical severity of dermatitis in Dfbstimulated HrasG12S mice a b Gross appearances dermatitis score andear thickness in vehicle saline or PD0325901treated Hras and HrasG12S mice after days of daily injections n per group All of the micein these ï¬gures were treated with Dfb The circle and square show vehicle group without MEKi with same amount of saline c Skin tissuestained with HE and TB in Hras and HrasG12S mice with PD0325901 or vehicle treatment n per group d Immunohistochemistry of pERKand claudin1 in skin c d Scale bars μm e Relative mRNA expression of Il1 Il4 and Il33 in dorsal skin mRNA levels were normalized to those of18s rRNA vehicle group n PD0325901 group n Data are presented as mean ± SD Signiï¬cance was analyzed by oneway ANOVA and theTukeyKramer method P P and P HrasG12S PD0325901 vs HrasG12S vehicle P HrasG12S vehicle vs Hrasvehicle P onetailed Students t testFig Germline HrasG12S mutation causes ADlike skin lesions via ERKIL33 axis Exposure to Dfb allergen induces ADlike skin lesionsincluding eczema acanthosis and pruritus in HrasG12S mice Dfbtreated HrasG12S keratinocytes show increased IL33 expression throughhyperproliferation of pERKpositive epidermal cells Excess IL33 activates basophil and ILC2containing ST2 receptors These activated immune cellsinduce the production of type2 ammatory cytokines such as IL4 Furthermore excess IL33 can activate ERK signaling resulting in reducedclaudin1 expression and skin barrier dysfunction DC dendritic cellsnumber of scratching bouts was assessed by replaying thevideo Each incidence of scratching behavior was deï¬nedas rubbing of ears and dorsal skin with forepaws hindpaws and mouthHistology and immunohistochemistryThe dorsal skins were ï¬xed in neutral bufferedformalin for parafï¬nembedded specimen and paraformaldehyde for frozen specimen Embedded tissueswere sectioned at μm parafï¬nembedded specimensor μm frozen specimens Parafï¬nembedded specimens were stained with hematoxylin and eosin HEand toluidine blue TB Epidermalthickness wasmeasured in ï¬ve randomly selected areas à μmof each HEstained sample Mast cells were counted inten randomly selected areas à μm of each TBstained sample For immunohistochemistry the parafï¬nembedded sections were deparafï¬nized using xylene andrehydrated with ethanol Frozen specimens were driedsufï¬ciently with a dryer Antigens were activated using aHistoï¬ne simple stain kit Nichirei Bio Sciences TokyoJapan The antibodies used are described in Supplementary Table Signals were visualized with a DABSubstrate KitNichirei Bio Sciences Sections werecounterstained with hematoxylin pERK immunostainedareaepidermis was measured in ï¬ve randomlyOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of selected areas à μm of each pERKstainedsampleSpleen and LN immune cell preparation and ï¬owcytometryQuantitative reverse transcriptionPCRTotal RNA extraction and puriï¬cation of keratinocyteswas performed according to the standard proceduresusing an RNeasy Mini Kit Qiagen Hilden Germany andQIAshredder Qiagen The extraction and puriï¬cation ofthe total RNA from the dorsal skin and cDNA synthesiswere performed as previously described26 Quantitativerealtime PCR was performed using THUNDERBIRDSYBR qPCR MIX TOYOBO in a StepOnePlus ThermoFisher Scientiï¬c Waltham MA USA The ampliï¬cationprimers are described in Supplementary Table Eachsample was run in duplicateWestern blottingSkin tissue was homogenized in lysis buffer mM TrisHCl pH and SDS containing phosphatase and protease inhibitor P5726 and P8340 SigmaAldrich Supernatants were collected after centrifugation and the proteinconcentration was determined using a BioRad ProteinAssay BioRad Laboratories Hercules CA Western blotanalyses were performed as previously described26 Brieï¬ythe proteins were transferred onto nitrocellulose membranesand blocked with nonfat milk in Trisbuffered salinewith Tween20 mmolL TrisHCl pH mmolLNaCl and Tween20 for h at room temperature Themembranes were incubated overnight at °C with theantibodies described in Supplementary Table All membranes were visualized using the Western Lightning ECLPlus Kit PerkinElmer Waltham MA USA The bandintensities were quantiï¬ed using NIH ImageJ softwareELISASerum IgE was determined using LBIS Mouse IgEELISA Kit FUJIFILM Wako Shiba
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" trophoblast cell surface antigen trop2 is overexpressed in many squamous cell carcinomas andpromotes tumor development and invasion the association between trop2 expression and occurrence anddevelopment of oral squamous cell carcinoma oscc remains to be understoodmethods we investigated the role of trop2 in oscc patients using a combination of biophysical approaches atotal of oscc patient specimens with varying degrees of differentiation were subjected to hematoxylin andeosin staining immunohistochemistry kaplanmeier survival curve analysis and atomic force microscopy to analyzetrop2 expression morphology and mechanical properties of oscc tissuesresults trop2 was overexpressed in of poorly differentiated oscc samples high levels of trop2 wereassociated with survival rate lower than and patient age odds ratio [or] p confidence interval [ci ] tumor size or p ci [] and tnm stageor p ci [] average surface roughness of low medium and highly differentiatedoscc tissues were ± ± and ± nm respectively the pearson coefficient revealed anegative association between tumor stiffness and trop2 expression r p overexpression of trop2 negatively associated with patient survival degree of tumor differentiationand tissue mechanics taken together our findings demonstrated that trop2 may be an indicator of osccdifferentiation leading to the altered mechanical properties of oscc tissueskeywords oral squamous cell carcinoma trop2 tissue stiffness differentiation survival oral squamous cell carcinoma oscc is a commonsubtype of head and neck and other malignant tumors[ ] the past few decades have shown increased incidence of oscc that is expected to rise further in the future thereforeimperative to determineisit correspondence zhangkllzu163com baoping zhang shuting gao and ruiping li contributed equally to thiswork1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu chinafull list of author information is available at the end of the biological factors associated with the early diagnosis andtreatment of oscchuman trophoblast cell surface antigen trop2 alsocalled tumorassociated calcium signaltransduction2tacstd2 is a surface glycoprotein encoded by tacstd that has extracellular domains a single transmembrane domain and a short tail [ ] trop2 is overexpressed in many human cancers including ovarian [ ]gastric [ ] colorectal pancreatic and laryngealcancers inhibiting trop2 expression has shownpromise in clinical applications [ ] trop2 regulates the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang bmc cancer page of tumorigenic properties including cancer cell adhesion invasion and migration tang have recentlyshown that trop2 impacts growth and metastasis byactivating pi3kakt signaling this phenomenon hasalso been observed in gallbladder cancer amongtheinvolved intumorigenesis the role of catenin has been studiedextensively [ ] this has shed light on the biological functions of trop2 and its use as a prognostic biomarker for osccvarious biochemical mechanismsatomic force microscopy afm is a powerful toolthat generates surface topographical images with magnifications that range between macro and nanoscalesafm has been used to determine the mechanical properties of tumor tissues in a variety of cancers such asthose of the breast liver and lung parameters for tissue stress such as mechanical phenotypeindex correlate with cancer development and invasion advancements in technology used for determiningbiophysical properties have facilitated the nanolevelanalysis of tumor tissuesthis study aims at investigating the correlation between trop2 expression and clinicopathological characteristics of oscc we have demonstrated the tissuemorphology and mechanics of oscc samples duringtumor development using afm we believe our findingswill help develop trop2 in accurately diagnosing osccin tumors with different grades of differentiationmethodstissue preparationthe protocols in this study were approved by the researchethics committee of lanzhou university tumor sampleswere collected from patients after obtaining written informed consent a total of patients with oral squamous cell carcinoma oscc were registered atthesecond hospital of lanzhou university between january and march among these samples sampleseach showed high moderate and low levels of differentiation the experimental group comprised males and females aged years average years all patientswere diagnosed with oscc based on surgery andfig paraffin pathological sections of tissues a d g 4fold b e h 10fold c f i 40fold 0czhang bmc cancer page of fig immunohistochemical staining was performed to detect the expression of trop2 at different stages of osccpathology patients did not undergo radiotherapy chemotherapy or immunotherapy before surgery pathologicalanalysis after tumor biopsy was performed by two experienced pathologists after which the diagnosis of other diseases including inflammation at other sites and secondarytumors were excluded cancer and cervical lymph nodetissues were collected after maxillofacial surgery all specimens were sampled from typical areas of the lesion andfixed with neutralbuffered formalin followed by conventional paraffin embedding among them and fig average optical density of trop2 poorly differentiated squamous cell carcinoma showed high expressionp 0czhang bmc cancer page of table correlation between trop2 expression and clinicopathological characteristicscharactersntrop2 expressionlow or nototalgendermalefemaleage¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 cm cmt2 cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvascular invasionnoyespearson x2p value highpatients exhibited no and cervical lymph node metastasesrespectively clinical tnm staging was performed according to the 7th edition tnm staging classification standardjointly developed by the international union for cancercontrol and american joint committee on cancer and world health anization guidelines hematoxylin and eosin he stainingoscc tissues were fixed overnight using neutralformalin solarbio beijing china paraffin embeddedsliced into 4μm thick sections dewaxed using xyleneand rehydrated using different concentrations of ethanol the sections were stained with hematoxylin for min and hydrochloric acidethanol and eosin for min followed by gradient dehydration transparentizationresin sealing solarbiobeijing china sections were visualized and imagedusing the olympus bx53 at magnifications of and sealing and neutral 0czhang bmc cancer page of immunohistochemistryhe sections were subjected to the sp method to detecttrop2 expression the sections were incubated overnight with the primary antibody against trop2 abcam usa at °c followed by incubation withbiotinlabeled goat antirabbitigg abcamusa at °c for h the sections were then developed using dab beijing zhongshan golden bridgebiotechnology china dehydrated transparentizationand film and neutral resin sealed the prepared sections were visualized using microscopy olympusbx53 japanafmfixed tissues were placed in petri dishes containingphosphatebuffered saline all analyses for mechanical properties were performed using the biologicalatomic force microscope bioafm nanowizard iiibruker usa silicon afm probes from the pointprobe®constant of nmcoating nanoworld usa werecontrreflexused the spring constant ofthe probe was calibrated using builtin thermal vibration before measuringandthickness of μm afm was performed using theseries with a khzforcetheresonancefrequencyofcontact model and a scan rate of hzs in airforcedistance curves are generated when the probecontacts the tissue following whichthe structuremorphology and mechanical properties of samplesare measured at μms six random sites wereselected for each sample and each site was measured times we used the modified hertz contact modelto analyze forcedistance curves and calculateyoungs modulus and roughness of oscc tissueswith varying differentiationstatistical analysisstatistical analyses were performed using spss statistical product and service solutions ibm forcespectrum data were expressed as mean ± standard errorand statistical comparisons were performed using oneway analysis of variance followed by the tukeykramerhsd test for pairwise comparisons pearson chisquaretest was used to analyze clinical features and trop2 expression based on the calculated odds ratios ors and confidence intervalci survival was evaluatedusing kaplanmeier curves and the difference was analyzed using the logrank test p was consideredstatistically significantfig trop2 total survival curve using kaplanmeier survival curves low blue line high green line 0czhang bmc cancer page of resultstissue morphology and trop2 expression across theclinical stages of oscctumor cells from poorly differentiated oscc samples exhibited characteristic atypia poor differentiation and irregular morphology fig howeverthe number volume atypia nuclear pyknosis andmitotic structures decreased in tumor cellsfromhighly differentiated oscc as compared to those inpoorly differentiated cells trop2 primarily localizedin the cytoplasm of tumor cells but not in adjacentnormal epithelial cells we observed that low differentiation and high malignancy of oscc was associated with higher trop2 expression fig theaverage optical density of trop2 among the lowmedium and highly differentiated oscc tissues were ± ± and ± respectively fig table trop2 expression risk factors with clinicopathological featurescharacteristicsntrop2 expressionlow or nototalgendermalefemaleage¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 cm cmt2 cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvscular invasionnoyesnote a well vs moderate b moderate vs poor c well vs poorp valueor cihigh 005a 0001b 0001c 0czhang bmc cancer page of association between trop2 expression and clinicalcharacteristics of osccwe analyzed the clinicopathological characteristics of patients with oscc with varying degree of differentiationdifferential expression of trop2 was associated with patient age tumor differentiation tumor size tnm stagepercutaneous nerveinvasiontable p patients with poorly differentiated tumors were more likely than patients with well and moderate differentiated tumors to have high trop2 expressionp however there was no association between theexpression of trop2 and patient gender tumor locationlymph node metastasis or distant metastases p and vascularfiltrationtrop2 expression and patient survivalusing kaplanmeier survival curves we observed that anincrease in trop2 expression negatively correlated withthe overall survival of patients fig and lowno oftrop2 expression groups 3years survival rate was a for high expression group and 5years ratewere and respectively trop2 expression wasassociated with patient age p or ci[] tumor differentiation well vs moderatep or ci [] moderate vspoor p or ci []well vs poor p or ci [] tumor size p or ci[] tnm stage p or ci[] vascular invasion p or ci [] and peripheral nerve invasionp or table high trop2 expressionwas detected in older patients with low degree of differentiation larger tumor volume higher tnm staging andvascular and peripheral nerve invasion thereby resultingin lower overall survival thus trop2 may be a prognostic indicator for survival in oscc patientsfig surface morphology of oscc tissue sections via afm detection irregular morphology appeared in the low differentiation 0czhang bmc cancer page of surface morphology and roughness of oscc tissuesthe surface morphologies of oscc tissues with varying degrees of differentiation were analyzed directtopographical imaging using bioafm figure showsthe representative image from each tissue acquiredduring the cantileverbased afm nano indentationtest the tissue interface varied with tumor differentiationindicating that highly differentiated oscc tissues had a regular and flat morphology oscc tissueswith low differentiation exhibited an overall irregularmorphology with distinct modulation and loose tissueanization figure summarizes the roughness ofoscc tissues with varying differentiation the average surface roughness of low medium and highly differentiated oscc tissues were ± ± and ± nm respectively roughness ofthe tissue surface was enhanced with increasing differentiation of oscc tissuesyoungs modulus of oscc tissueswe used bioafm to determine youngs modulusbased on the mechanical properties of oscc tissues with varying degrees of differentiation we randomly selected six contact points from each slice andeach contact point was measured times forcedistance curves were generated for each slice and thejpk data processing software version was usedto calculate youngs modulus figure shows theaverage variation in stiffness within individual tissuesin the range of kpa in the low differentiationsamples we observed low stiffness as compared tothat in high or medium differentiation samples p fig surface roughness results are express as mean ± sem nm 0czhang bmc cancer page of fig afm test average tissue stiffness youngs modulus e was thus confirmed to be a parameter of cell hardness for various cells and tissuepa p thus tissue differentiation was positively associated with its stiffness fig association between mechanical properties and trop2expression in osccthe pearson coefficient showed a negative associationbetween the stiffness of oscc tissues and trop2 expression fig r p thus we detectedan increase in stiffness with varying differentiation in thetumor samplesdiscussiontrop2 belongs to the family of genes involved in calcium signaling associated with tumorigenesis and foundin human trophoblast and chorionic cell lines studieshave shown that overexpression of trop2 is associatedwith tumorigenesis and malignancy []in thisstudy trop2 expression was observed to be a highlysensitive and specific marker of tongue squamous cellcarcinoma and tissue stiffness the relative thickness ofsamples helped accurately diagnose and determine thestaging of tongue squamous cell carcinomaimmunohistochemical analysis revealed that the expression of trop2 in poorly differentiated oscc tissueswas significantly higher than that in welldifferentiatedoscc tissues additionally trop2 upregulation wascorrelated with tumors of advanced tnm iii iv staging and poor differentiation than that in tumors withlow tnm i ii staging thus the abnormal expressionof trop2 may be associated with the occurrence anddevelopment of tongue malignancies furthermore hightrop2 expression predicted low survival as comparedto that in the tumors with low trop2 expression previous research has also demonstrated the correlation between shorter patient lifespan and high levels of trop2as compared to that in patients with laryngeal squamouscell carcinoma and low levels of trop2 trop2possesses sites for tyrosineserine phosphorylation thatregulate signal transduction or its expression and activity thereby rendering cancer cells resistant to apoptosis upregulated trop2 correlates with the poor prognosis of thyroid papillary carcinoma colon cancer liver cancer and other malignanciesthere have been an increasing number of studies on thebiological role of trop2 at the molecular level trop2induces the downregulationloss of pten thereby stimulating pi3kakt signaling and tumor development pten is a wellknown tumor suppressor that is a phosphatase and affects the pi3kpkbakt signaling axisduring the dephosphorylation of pip2 and pip3 pi3k signaling is important in regulating tumor cell proliferation migration and invasion [ ] thus pten is anegative regulator of cancer [ ] li have shownthat trop2 activates epithelialmesenchymal transitionvia pi3kakt signaling thereby promoting proliferationmigration and metastasis in gallbladder cancer similarly trop2 expression stimulates the proliferation migration and invasion of osteosarcoma cells hou demonstrated that trop2 regulates jak2stat3 signaling in glioblastoma cells 0czhang bmc cancer page of fig correlation analysis between changes in mechanical stiffness of oscc tissues and trop2 expression note changes have statisticalsignificance p and show a certain negative correlation r functional differentiation oftissues influences themicromorphology and mechanical stiffness of oscccells we detected low surface roughness on oscc tissues with loose structure reduced hardness and enhanced cell adhesion migration and invasion poorlydifferentiated oscc tissues are softer than highly differentiated oscc tissues pi3k is an important celladhesion molecule trop2 triggers the synthesis of proteins with homologous domains such as pleckstrinrac tiam and vav tiam and vav activate rac thatleads to reanization of the actin cytoskeleton cellrecognition and adhesion the underlying mechanisms involved in the alterationof micromechanical properties of oscc samples and occurrence development metastasis and invasion ofoscc tumors remain to be elucidated he staining isthe gold standard for tumor diagnosis with the development of biomechanics in the past two decades the mechanical properties of tissues need to be investigated based on biomedical and physical parametersin this study we have assayed the changes in mechanicalproperties at the micronanometer level using afm anddetermined the association between the tnm grademetastasis and stiffness of tumor samplesin we have demonstrated the association between differential expression of trop2 and patient agetumor differentiation tumor size tnm stage percutaneousnerve filtration and vascular invasion moreover high levelsof trop2 correlated with poor overall survival in patientshighly differentiated cancer tissues exhibited increasedsurface roughness and stiffness lastly high trop2 expression resulted in reduced tumor stiffness however thisstudy had some limitations first the cohort used in thisstudy was relatively small second we did not employ molecular methods of analysis such as western blotting orenzymelinked immunosorbent assay thus using a largerpatient cohort and multiple techniques in molecular andcell biology will help validate our findings and developtrop2 as a specific and efficient prognostic biomarker forosccthese findings could promote new methods for the earlyoscc diagnosis depend on the stage of cancer and developing screening methods with high sensitivity andspecificity more detailed studies are needed to determine the feasibility and therapeutic benefit of testing tissue stiffness in human diseaseabbreviationsoscc oral squamous cell carcinoma trop2 trophoblast cell surfaceantigen afm atomic force microscopyacknowledgementswe thank the individual who participated in this studyauthors contributionsbz sg and rpl are responsible for conception and design data wascollected by ytl rc jyc and ymg data was analyzed by ew and yh klzrevised the all authors have read and approved the manuscriptfundingthis work was supported by the fundamental research funds for thecentral universities no lzujbky2020cd03 baoping zhang doctoralmaster 0czhang bmc cancer page of students of the second hospital of lanzhou university sdkygg17 lan yangand key laboratory of mechanics on disaster and environment in westernchina the ministry of education of china no kailiang zhangavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatewritten informed consent was obtained from each participant before samplecollection the study was approved by the committee for ethical affairs ofschool of stomatology lanzhou universityconsent for publicationnot applicablecompeting intereststhe authors have no conflicts of interestauthor details1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu china 2institute of biomechanics andmedical engineering lanzhou university lanzhou chinareceived april accepted august referencesiyer s thankappan k balasubramanian d early detection of oral cancerscurrent status and future prospects curr opin otolaryngol head neck surgcaldeira pc soto aml de aguiar mcf martins cc tumor depth of invasionand prognosis of earlystage oral squamous cell carcinoma a metaanalysisoral dis online ahead of printkim y kim jh increasing incidence and improving survival of oral tonguesquamous cell carcinoma sci rep mcdougall ar tolcos m hooper sb cole tj wallace mj wallace trop2from development to disease dev dyn guan gf zhang dj wen lj yu dj zhao y zhu l prognostic value oftrop2 in human nasopharyngeal carcinoma int j clin exp pathol stewart d cristea m antibodydrug conjugates for ovarian cancer currentclinical development curr opin obstet gynecol liu j yang d yin z gao m tong h su y a novel human monoclonaltrop2igg antibody inhibits ovarian cancer growth in vitro and in vivobiochem biophys res commun zhao w jia l kuai x tang q huang x yang t the role andmolecular mechanism of trop2 induced epithelialmesenchymal transitionthrough mediated betacatenin in gastric cancer cancer med zhao w jia l zhang m huang x qian p tang q the killing effect ofnovel bispecific trop2pdl1 cart cell targeted gastric cancer am jcancer res jordheim lp chettab k crosperrial e matera el dumontet c unexpectedgrowthpromoting effect of oxaliplatin in excision repair crosscomplementation group transfected human colon cancer cellspharmacology nishimura t mitsunaga m sawada r saruta m kobayashi h matsumoto n photoimmunotherapy targeting biliarypancreatic cancer withhumanized antitrop2 antibody cancer med wang xd wang q chen xl huang jf yin y da p trop2 inhibitionsuppresses the proliferation and invasion of laryngeal carcinoma cells viathe extracellular signalregulated kinasemitogenactivated protein kinasepathway mol med rep wanger tm dewitt s collins a maitland nj poghosyan z knauper vdifferential regulation of trop2 release by pkc isoforms through vesiclesand adam17 cell signal tang g tang q jia l chen y lin l kuai x trop2 increasesgrowth and metastasis of human oral squamous cell carcinomathrough activation of the pi3kakt signaling pathway int j mol medtrerotola m li j alberti s languino lr trop2 inhibits prostate cancer celladhesion to fibronectin through the 1 integrinrack1 axis j cell physiolli t su y yu x mouniir dsa masau jf wei x trop2 guaranteescardioprotective effects of cortical bonederived stem cells on myocardialischemiareperfusion injury cell transplant stoyanova t goldstein as cai h drake jm huang j witte on regulatedproteolysis of trop2 drives epithelial hyperplasia and stem cell selfrenewalvia betacatenin signaling genes dev sun x xing g zhang c lu k wang y he x knockdown of trop2 inhibitsproliferation and migration and induces apoptosis of endometrial cancercells via aktcatenin pathway cell biochem funct lee h jang y seo j nam jm char k nanopfunctionalized polymerplatform for controlling metastatic cancer cell adhesion shape and motilityacs nano kruse sa smith ja lawrence aj dresner ma manduca a greenleaf jf tissue characterization using magnetic resonance elastographypreliminary results phys med biol kaneko ts pejcic mr tehranzadeh j keyak jh relationships betweenmaterial properties and ct scan data of cortical bone with and withoutmetastatic lesions med eng phys goetz jg minguet s navarrolerida i lazcano jj samaniego r calvo e biomechanical remodeling of the microenvironment by stromalcaveolin1 favors tumor invasion and metastasis cell edge sb compton cc compton the american joint committee on cancerthe 7th edition of the ajcc cancer staging manual and the future of tnmann surg oncol barnes l eveson jw reichart p sidransky d pathology genetics headand neck tumours lyon barness p zhang b li l li z liu y zhang h wang j carbon ionirradiated hepatomacells exhibit coupling interplay between apoptotic signaling andmorphological and mechanical remodeling sci rep yan jf huang gy a doublehertz model for adhesive contact betweencylinders under inclined forces philos trans a math phys eng sci kowalsky ca faber ms nath a dann he kelly vw liu l rapid fineconformational epitope mapping using comprehensive mutagenesis anddeep sequencing j biol chem zeng p chen mb zhou ln tang m liu cy lu ph impact of trop2expression on prognosis in solid tumors a systematic review and metaanalysis sci rep calvo a xiao n kang j best cj leiva i emmertbuck mr alterationsin gene expression profiles during prostate cancer progression functionalcorrelations to tumorigenicity and downregulation of selenoproteinp inmouse and human tumors cancer res ju x jiao x ertel a casimiro mc di sante g deng s vsrc oncogeneinduces trop2 proteolytic activation via cyclin d1 cancer res cubas r li m chen c yao q trop2 a possible therapeutic target for latestage epithelial carcinomas biochim biophys acta zargari n mokhtari m evaluation of diagnostic utility ofimmunohistochemistry markers of trop2 and hbme1 in the diagnosis ofthyroid carcinoma eur thyroid j zhao p zhang z tnfα promotes colon cancer cell migration and invasionby upregulating trop2 oncol lett sin stk li y liu m yuan yf ma s guan xy downregulation of trop2predicts poor prognosis of hepatocellular carcinoma patients hepatolcommun zhang y zhang r luo g ai k long noncoding rna snhg1 promotes cellproliferation through pi3kakt signaling pathway in pancreatic ductaladenocarcinoma j cancer sai j owens p novitskiy sv hawkins oe vilgelm ae yang j pi3kinhibition reduces mammary tumor growth and facilitates antitumor immunityand antipd1 responses clin cancer res chen x pang b liang y xu sc xin t fan ht overexpression of zhang xr wang sy sun w wei c isoliquiritigenin inhibits proliferation andepcam and trop2 in pituitary adenomas int j clin exp pathol metastasis of mkn28 gastric cancer cells by suppressing the pi3kaktmtor signaling pathway mol med rep 0czhang bmc cancer page of wise hm hermida ma leslie nr prostate cancer pi3k pten and prognosisclin sci lond yuan b zou m zhao y zhang k sun y peng x upregulation of mir130b3p activates the ptenpi3kaktnfκb pathway to defense againstmycoplasma gallisepticum hs strain infection of chicken int j mol sci pii e2172li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep li x teng s zhang y zhang w zhang x xu k trop2 promotesproliferation migration and metastasis of gallbladder cancer cells byregulating pi3kakt pathway and inducing emt oncotarget gu qz nijiati a gao x tao kl li cd fan xp trop2 promotes cellproliferation and migration in osteosarcoma through pi3kakt signalingmol med rep hou j lv a deng q zhang g hu x cui h trop2 promotes theproliferation and metastasis of glioblastoma cells by activating the jak2stat3 signaling pathway oncol rep rivard n phosphatidylinositol 3kinase a key regulator in adherens junctionformation and function front biosci landmark ed pankova d jiang y chatzifrangkeskou m vendrell i buzzelli j ryan a rassf1a controls tissue stiffness and cancer stemlike cells in lungadenocarcinoma embo j 20193813e100532 wullkopf l west av leijnse n cox tr madsen cd oddershede lb cancer cells' ability to mechanically adjust to extracellular matrix stiffnesscorrelates with their invasive potential mol biol cell publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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