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+{"text": "Methotrexate (MTX) was linked, via an active ester intermediate, to a purified IgG fraction of rabbit polyclonal antiserum raised against a cell membrane preparation from the human prostatic cell line PC3. The resulting conjugates contained an average of 0.044 mg of MTX per mg of antibody with acceptable losses in both the binding activity of the immunoglobulin (27.5%) and the enzyme inhibitory activity of the drug (32% at a MTX concentration of 3 x 10(-7) M). Using cultures of PC3 cells the antibody-MTX (Ab-MTX) conjugates were observed to be as effective as free drug in causing cell death and more effective than non-immune IgG-MTX (NIgG-MTX) conjugates. When athymic nude mice bearing PC3 tumours were administered with Ab-MTX conjugates, significant reductions in tumour growth rates were observed compared to animals given saline, MTX alone or NIgG-MTX conjugates . Furthermore, the accumulation of radioactive MTX in the tumour tissue of animals injected with these Ab-MTX conjugates was 16-fold greater than those given free drug and 8.6-fold greater than those administered with NIgG-MTX conjugates. Uptake by the reticuloendothelial system, however, was not significantly different when animals from each treatment group were compared."}
+{"text": "Several Methotrexate (MTX)-resistant sublines of the osteogenic sarcoma cell line 791T were derived by continuous selection in the presence of MTX and 12-O-tetradecanoylphorbol-13-acetate (TPA). Studies including assays of the uptake and binding of [3H]MTX and fluoresceinated-MTX, determined that these sublines showed diminished MTX transport, and that none of them appeared to overproduce the MTX-target enzyme dihydrofolate reductase. Conjugates of the anti-791T monoclonal antibody 791T/36 linked to MTX via human serum albumin (HSA) were prepared by Dr M.C. Garnett. These were cytotoxic selectively for cells bearing the 791T/36-defined antigen (gp72), and were found to be as cytotoxic to most of the MTX-resistant 791T sublines as they were to parental 791T cells. Furthermore, an anti-MTX/anti-gp72 bispecific antibody 516 augmented the cytotoxicity of HSA-MTX conjugate to the MTX-resistant 791T variant R120 apparently as efficiently as for parental 791T cells. It is suggested that acquired drug resistance caused by deficient transport mechanisms may be partially or wholly overcome by targeting the drug to a readily-internalised cell surface antigen."}
+{"text": "The cytotoxicity of methotrexate (MTX) on representative human tumour cell lines  was evaluated to: (1) examine the optimal time interval between MTX and folinic acid (FA) administration; (2) determine the critical FA/MTX concentration ratios; and (3) compare the relative effects of the equimolar mixture d,I-FA and I-FA. The cytotoxic effects of MTX were assessed by the MTT semi-automated test. For all of the cell lines tested, a significant inverse relationship was noted between the degree of MTX cytotoxicity reversal and the duration of the time interval between MTX and FA administration. Overall an 18-24 h interval between MTX and FA represented a time-threshold after which MTX effects could not efficiently be reversed by FA in most cell lines. With shorter time intervals between MTX and FA, MTX cytotoxicity could be partially on even totally reversed by FA; the intensity of reversal varied among the cell lines tested, and depended on the FA/MTX ratio. Regardless of the interval between MTX and FA, analysis of the various FA/MTX ratios revealed a significant direct relationship between this ratio and the percentage of recovery. Presence of the d-form had no influence on the MTX rescue capacity of the I-form; this suggests that the presence of the d-FA is unlikely to have any significant clinical consequences."}
+{"text": "Severe methotrexate (MTX) toxicity is a proven complication of associations of MTX and non-steroidal anti-inflammatory drugs (NSAIDs). This study investigated the interaction between MTX (50 or 100 mg kg-1) and ketoprofen (KP)  in the rabbit. The drug association induced a reversible increase in blood urea and creatinine. The severity degree of renal dysfunction was significantly related to the MTX dose; it was not modified by prolonged exposure to KP after MTX administration. The biological markers of haematopoietic and hepatic functions were unchanged. Pretreatment by KP induced a marked reduction (70%) in the urinary excretion of the prostaglandin 6-keto-PGF1 alpha. MTX dose-related alterations in MTX pharmacokinetics were also observed with the drug association: at a MTX dose of 100 mg kg-1, the presence of KP significantly reduced the total body clearance, the renal clearance and the fraction of MTX eliminated in urine as compared to controls. An appreciable reduction in the plasma binding of MTX was also noted in vivo when KP was associated. This experimental study confirms the existence of an interaction between MTX and KP and demonstrates its renal origin."}
+{"text": "A bispecific monoclonal antibody, reactive with methotrexate (MTX) and a tumour associated antigen (gp72) has been produced by fusing spleen cells from MTX immunised mice with 791T/36/3 (anti-gp72) hybridoma. The hybrid antibody was purified from anti-MTX and anti-gp72 antibodies present in the hybridoma culture supernatant by combinations of affinity chromatography on a MTX-agarose immunoabsorbent and stepwise acid elution from Sepharose-Protein A. A particular feature of the present antibody is that it reacts with conjugated MTX; this would allow in vivo targeting of conjugates, increasing many fold the number of molecules of drug carried or localising to pre-targeted antibody. Dual binding between tumour cell surface antigen and MTX was demonstrated by the ability of the hybrid antibody to bridge between tumour cells and MTX as MTX-HSA conjugate, reaction here being detected by flow cytofluorimetry. Purified hybrid antibody specifically enhanced the in vitro cytotoxicity of MTX-HSA for gp72 positive tumour cells."}
+{"text": "While viable animals were derived in both Sirt1\u2212/\u2212 X wild type and Sirt1\u2212/\u2212 X Sirt1\u2212/\u2212 crosses, the efficiency of producing both 2-cell zygotes and viable offspring was diminished when IVF was performed with Sirt1\u2212/\u2212 sperm and/or oocytes. Together, these data support an important role for Sirt1 in spermatogenesis, including spermatogenic stem cells, as well as germ cell function.In mammals, Sirt1, a member of the sirtuin family of proteins, functions as a nicotinamide adenine dinucleotide-dependent protein deactylase, and has important physiological roles, including the regulation of glucose metabolism, cell survival, and mitochondrial respiration. The initial investigations of Sirt1 deficient mice have revealed a phenotype that includes a reduced lifespan, small size, and an increased frequency of abnormal sperm. We have now performed a detailed analysis of the molecular and functional effects of Sirt1 deficiency in the germ line of Sirt1 knock-out (\u2212/\u2212) mice. We find that Sirt1 deficiency markedly attenuates spermatogenesis, but not oogenesis. Numbers of mature sperm and spermatogenic precursors, as early as d15.5 of development, are significantly reduced  in numbers in Sirt1\u2212/\u2212 mice, whereas Sirt1 deficiency did not effect the efficiency oocyte production following superovulation of female mice. Furthermore, the proportion of mature sperm with elevated DNA damage  was significantly increased in adult Sirt1\u2212/\u2212 males. Analysis of global gene expression by microarray analysis in Sirt1 deficient testis revealed dysregulated expression of 85 genes, which were enriched (P<0.05) for genes involved in spermatogenesis and protein sumoylation. To assess the function of Sirt1 deficient germ cells, we compared the efficiency of generating embryos and viable offspring in  Sir (Silent Information Regulator) genes were originally discovered in yeast (Saccharomyces cerevisiae) where they were shown to affect expression of genes, due to their ability to promote heterochromatin, at the HML and HMR mating type loci The sWld) mice has been shown to be dependent on SirT1 Recent studies have demonstrated that Sirt1 has a number of important physiological roles in mammals. Most notably, it plays an important role in the regulation of glucose metabolism. Sirt1 has been shown to promote gluconeogenesis in the liver Sirt1 deficient (knock-out) mouse strains have now been developed in a number of independent studies In the present study, we have performed a detailed analysis of the effect of Sirt1 deficiency on the function of male and female germ cells at the cellular and molecular level. We show that the low number of spermatozoa and atrophied testis in male Sirt1 deficient mice is due to effects that manifest at least as early as d15.5 of development, and correspond with elevated frequency of DNA damage in mature sperm in adult mice. Microarray analysis of global gene expression in testis from Sirt1 deficient mice reveals aberrant expression of a number of genes that have important roles in spermatogenesis, as well as genes involved in sumoylation. Furthermore, using in vitro fertilization (IVF), we show that both male and female gametes from Sirt1 deficient mice have a reduced efficiency at generating viable zygotes, although these zygotes are fully capable of developing to term following embryo transfer to pseudo-pregnant females.The Sirt1 knock-out (\u2212/\u2212) strain For analysis of epididymal sperm, male mice were sacrificed using cervical dislocation and the caudal epididymis was dissected out and cut. Epididymal sperm was extracted using forceps and placed in a droplet of HTF media overlayed with mineral oil. After capacitation, an aliquot near the top of the droplet was removed, and sperm were counted using a hemocytometer.To assess the effect of Sirt1 deficiency on early spermatogenic stem cells, we bred Sirt1+/\u2212 mice homozygous for the Oct4-GFP transgene to generate Sirt\u2212/\u2212,Oct4-GFP embryos (see above), which are easily distinguished from Sirt1+/\u2212,Oct4-GFP or Sirt1+/+,Oct4-GFP embryos on the basis of size . Gonads were then isolated from d15.5 male embryos, and single cell suspensions for FACS analysis of stem cell numbers were generated as previously described Tissues were removed from mice and fixed in Bouin's solution overnight. The following morning, samples were washed in 70% ethanol, dehydrated, and embedded in paraffin. Six-micrometer sections were prepared on glass slides, cleared in xylenes, and stained with fresh hematoxylin for 10 minutes, washed, and then immediately stained with eosin. Following staining, slides were dehydrated in ethanol, air-dried, then mounted in balsam for microscopic analysis.In vitro fertilization (IVF) experiments were performed using established protocols Following IVF, surrogate mothers were prepared by mating female CD-1 mice in estrus with vasectomized CD-1 male mice. Twenty-four hours later, the pseudopregnant CD-1 females were anesthetized and 2-cell embryos were injected into each oviduct and allowed to develop to term.Epididymal sperm was isolated and re-suspended in HTF media. Each sperm sample was then promptly pelleted, re-suspended in low melt agarose at 37C, and applied as a uniform layer on glass slides. Alkaline comet assays were performed according to the manufacturers' protocol  with minor revisions. Specifically, 40 mM DTT was added to the lysis solution during initial incubation, without Proteinase K, for one hour. Slides were then further incubated in lysis solution containing 10ug/mL of Proteinase K for an additional 2.5 hours at 37\u00b0C. Following electrophoresis, slides were air dried over night and then stained in Sybr- Green before microscopic analysis. Only sperm with clearly extended Comet tails (at least 2-fold greater than the average comet tail size) were scored as positive.Whole testis were isolated, fixed in 4% paraformaldehyde/PBS solution at 4C over-night solution. Six micrometer thick sections were mounted on silanized glass slides and processed using the Apoptag Plus Peroxidase Apoptosis Detection Kit  according to manufacturers' protocol.Testis were snap frozen in liquid nitrogen and then homogenized using mortar and pestle. Telomerase extracts were prepared using CHAPS lysis buffer, and protein concentration was determined using the Bradford assay. The TRAP assay was performed as previously described Whole testis were snap frozen in liquid nitrogen, ground with mortar and pestle, followed by extraction of total RNA with Trizol reagent. Quality of RNA for all samples was assessed on 1% agarose gels. Labeled cDNA was generated using the MicroMax Direct Labeling kit (Perkin Elmer). Briefly, 20ug of each RNA sample was used as template in reverse transcription (RT) reactions using an oligo dT primer. Each RT reaction included either Cy3 or Cy5 conjugated nucleotides to label the cDNA product. Input RNA was hydrolyzed after each RT reaction using NaOH, and cDNA was purified by isopropanol precipitation. The cDNA samples from sibling Sirt1\u2212/\u2212 and Sirt1+/\u2212 mice (each labeled with a different fluorophor) were then washed briefly, re-suspended in hybridization buffer (see below), combined together, and immediately used in the hybridization step.After a brief rinse in 1\u00d7 SSC buffer, each array slide was immersed in pre-hybridization buffer  for 30 minutes at 42C. Slides were then rinsed in water, then 95% ethanol, and finally spun dry prior to hybridization. The labeled cDNA probe mixture, re-suspended in hybridization buffer , was heated to 95C for 2 minutes just prior to applying to the slide. All hybridizations were performed at 42C over night in sealed hybridization chambers immersed in a water bath. After hybridization, slides were washed in the following solutions, in order, for 15 minutes each at room temperature- 0.5\u00d7 SSC, 0.01%SDS, 0.06\u00d7 SSC, 0.01% SDS, 0.06\u00d7 SSC.After the final wash step, slides were immediately scanned using a GenePix 4000b array scanner and GenePix Pro 6.0 software, at settings which produced equivalent signal in both Cy3 and Cy5 channels and low background. Array data was then transferred to Acuity 4.0 software for normalization and analysis.Real time RT PCR was performed using a Roche Light-Cycler, as previously described 18. In addition, analysis of the frequency of apoptotic cells in the testis of Sirt1\u2212/\u2212 and Sirt1+/\u2212 mice was elevated  experiments to examine the relative efficiency of developing zygotes as well as development to term following embryo transfer to surrogate mothers. We find that both sperm and oocytes from Sirt1\u2212/\u2212 mice are capable of fertilization, although the efficiency of developing to the 2-cell stage is substantially compromised for both sperm and oocytes from Sirt1\u2212/\u2212 mice as compared to gametes from Sirt1+/\u2212 mice (We and others +/\u2212 mice . NeverthWe and others have previously shown that the Oct4-GFP transgene is a useful marker for the identification and purification of primitive germ stem cells from mouse embryos Since Sirt1 has been implicated in the regulation of expression of telomerase reverse transcriptase (Tert) Recent studies have implicated that Sirt1 may play a role in maintaining genomic integrity To assess the effect of Sirt1 deficiency on spermatogenesis in more detail, we compared global gene expression for Sirt1\u2212/\u2212 and Sirt1+/\u2212 testis using spotted oligonucleotide microarray technology . In this analysis, we performed intriplicate analysis of global gene expression, including dye swap analysis, for 2 pairs of sibling Sirt1\u2212/\u2212 and Sirt1+/\u2212 mice. We identified a total of 85 differentially expressed genes, with a >1.7 fold change in gene expression, either up or down, in at least 5 of the 6 arrays analyzed  by qRT-PTo objectively assess which genes were differentially regulated, we used the gene classification tool Gene Ontology (GO) Sirt1 has been shown to have a significant role in the regulation of glucose metabolism and the promotion neural cell survival. In this study, we have examined in detail the role of Sirt1 in germ cell function and gametogenesis using a Sirt1 knock-out mouse strain. We find that both mature sperm and spermatogenic stem cell numbers are significantly reduced in Sirt1 deficient mice and embryos respectively, whereas oocyte numbers are not affected. While the fertility of Sirt1 deficient gametes is markedly attenuated, we were able to generate F1 Sirt1\u2212/\u2212 mice which survived to adulthood by performing IVF with gametes from Sirt1\u2212/\u2212 mice. The deleterious effect of Sirt1 deficiency on sperm numbers is at least in part accounted for by elevated levels of DNA damage, as assessed using the Comet assay. Microarray analysis of global gene expression in the testis from Sirt1 deficient mice revealed aberrant expression of a number of genes involved in spermatogenesis. Together, these data demonstrate that Sirt1 has important roles in spermatogenesis and germ cell function.While we did not observe an affect of Sirt1 deficiency on the ability to produce mature oocytes, we did notice a marked decrease in efficiency in generating both 2-cell embryos and live-offspring when performing IVF with oocytes from Sirt1\u2212/\u2212 females . We haveThe observation that Sirt1 deficiency causes a reduction not only in the numbers of mature sperm in adult mice  but alsoGlobal analysis of gene expression revealed an over-representation of genes in the GO categories of spermatogenesis and protein sumoylation. The former observation is in agreement with the reduced sperm count and increased frequency of abnormal sperm and DNA damage in sperm from Sirt1 deficient mice. Most of the differentially expressed genes involved in spermatogenesis were over-expressed in the testis of Sirt1 deficient mice. Sirt1 has the potential to promote transcriptional silencing, for example by deacetylating and inactivating the transcription factors p300 and FOXO proteins Interestingly, genes involved in the sumoylation of proteins, the addition or removal of small ubiquitin-like modifiers (SUMO) to proteins, are also over-represented in the genes differentially expressed in Sirt1 deficient testis. Recent studies have suggest that Sirt1 can affect the sumoylation of certain proteins via a mechanism where the deacetylation of specific Lys residues by Sirt1 allows the subsequent addition of SUMO by other proteins Results from recent studies on the ability of Sirt1\u2212/\u2212 mice to successfully mate and produce offspring with other mice are incongruent. A study by McBurney et al Figure S1Numbers of apoptotic cells is increased in Sirt1 testis. Testis from Sirt1+/\u2212 and Sirt1\u2212/\u2212 mice were dissected, fixed, embedded in paraffin, and sectioned. The sections were then stained for apoptotic cells using the ApoTag kit (Chemicon- CHECK). A. Sample images of stained sections from Sirt1\u2212/\u2212 and Sirt1+/\u2212 mice are shown. Apoptotic cells are indicated by arrowheads. B. Quantitative analysis of the number of apoptotic cells per seminiferous tubule. The number of tubules in Sirt1\u2212/\u2212 testis with no detectable apoptotic cells was <1%.(3.16 MB TIF)Click here for additional data file.Figure S2Telomerase activity is not affected by Sirt1 deficiency in the testis. Extracts were prepared from testis from Sirt1\u2212/\u2212 and Sirt1+/\u2212 mice and telomerase activity was measured using the TRAP assay according to manufacturers' protocol . A. Sample blot showing TRAP assay results for testis samples from sibling Sirt1\u2212/\u2212 and Sirt1+/\u2212 mice. The internal control PCR product is indicated by the arrowhead. B. Quantitative analysis of telomerase activity. The level of telomerase activity was assessed for Sirt1\u2212/\u2212 and Sirt1+/\u2212 testis samples according to manufacturers' protocol. The bars represent average results from intriplicate analyses of testis samples from 3 pairs of sibling Sirt1\u2212/\u2212 and Sirt1+/\u2212 mice . Error bars representing standard deviation are shown.(0.65 MB TIF)Click here for additional data file.Table S1Top 10 up and down regulated differentially expressed genes in testis(0.03 MB DOC)Click here for additional data file."}
+{"text": "The Neighborhood Environment Walkability Scale (NEWS) and its abbreviated form (NEWS-A) assess perceived environmental attributes believed to influence physical activity. A multilevel confirmatory factor analysis (MCFA) conducted on a sample from Seattle, WA showed that, at the respondent level, the factor-analyzable items of the NEWS and NEWS-A measured 11 and 10 constructs of perceived neighborhood environment, respectively. At the census blockgroup (used by the US Census Bureau as a subunit of census tracts) level, the MCFA yielded five factors for both NEWS and NEWS-A. The aim of this study was to cross-validate the individual- and blockgroup-level measurement models of the NEWS and NEWS-A in a geographical location and population different from those used in the original validation study.A sample of 912 adults was recruited from 16 selected neighborhoods (116 census blockgroups) in the Baltimore, MD region. Neighborhoods were stratified according to their socio-economic status and transport-related walkability level measured using Geographic Information Systems. Participants self-completed the NEWS. MCFA was used to cross-validate the individual- and blockgroup-level measurement models of the NEWS and NEWS-A.The data provided sufficient support for the factorial validity of the original individual-level measurement models, which consisted of 11 (NEWS) and 10 (NEWS-A) correlated factors. The original blockgroup-level measurement model of the NEWS and NEWS-A showed poor fit to the data and required substantial modifications. These included the combining of aspects of building aesthetics with safety from crime into one factor; the separation of natural aesthetics and building aesthetics into two factors; and for the NEWS-A, the separation of presence of sidewalks/walking routes from other infrastructure for walking.This study provided support for the generalizability of the individual-level measurement models of the NEWS and NEWS-A to different urban geographical locations in the USA. It is recommended that the NEWS and NEWS-A be scored according to their individual-level measurement models, which are relatively stable and correspond to constructs commonly used in the urban planning and transportation fields. However, prior to using these instruments in international and multi-cultural studies, further validation work across diverse non-English speaking countries and populations is needed. IPEN uses common study design and measurement to produce more reliable and valid effect size estimates.Ecological models postulate that health behavior changes are a function of psychological, social, policy, and physical environmental factors ,2. NumerThe Neighborhood Environment Walkability Scale, (NEWS)  and its Two recent studies examined the measurement models of the original and Australian versions of the NEWS ,16. The To maximize the variability in environmental attributes, both studies adopted a stratified two-stage cluster sampling strategy whereby participants were recruited from specific areas  selected according to their objectively-measured walkability and socio-economic status . Stratification by SES likely enhanced the representativeness of the sample because, otherwise, low SES respondents might have been underrepresented . Two disThe individual-level measurement models were based on differences in responses between study participants living in the same blockgroups and described the way perceived environmental attributes (represented by the NEWS items) covaried within census blockgroups. The differences in responses may have resulted from actual environmental differences within a blockgroup (e.g. differences in traffic load or aesthetics across locations within a blockgroup), response biases (e.g. tendency to provide extreme ratings), and/or perceptual biases (e.g. anxious respondents' tendency to overestimate the risk of crime in their neighborhood) . In contThe authors recommended scoring the NEWS according to the individual-level measurement model for three main reasons: (1) the individual-level factors more accurately represented constructs commonly used in the urban planning and transportation fields ; (2) theThe measurement model of the Australian NEWS mostly resembled that of the original version, but differed in significant ways . For insSpecifically, according to the 2003 American Community Survey, Seattle was the most educated larger city in the USA, with 52% of residents aged 25 and over having attained at least a bachelor's degree . In contWe hypothesized that the individual-level factor structures of the NEWS and NEWS-A, derived from the original validation sample , would show a sufficient level of fit to the data from the cross-validation sample  due to them being in part a function of psychological principles that apply across diverse subgroups. We also hypothesized that the original blockgroup-level factor structures of the NEWS and NEWS-A would show poorer fit to the data from the cross-validation sample than their individual-level counterparts due to them reflecting patterns of associations between objective environmental factors, which likely vary across geographical locations.This study used cross-sectional survey data from the Neighborhood Quality of Life Study (NQLS) conducted in the Baltimore, MD \u2013 Washington, DC region. Neighborhoods, defined as clusters of blockgroups, were selected to vary in walkability characteristics and socio-economic status (SES). Median household income was used to define blockgroup SES, while data within a Geographic Information System (GIS) on residential density, street connectivity, land-use mix, and retail floor area ratio were used to operationalize blockgroup walkability . BlockgrHouseholds within selected blockgroups were identified by a marketing firm, sent an invitation letter, and then called within 2 weeks of the expected receipt of this letter. An adult in the household was asked about interest and study eligibility. A sample of 912  English-speaking adults, aged 20\u201365, able to walk without assistance, and living in private dwellings, was recruited. Participants' socio-demographic characteristics are shown in Table Participants self-reported gender, age, educational attainment, annual household income, marital status, and number of children (\u2264 18 years old) in the household.[The NEWS and NEWS-A (abbreviated version) consist of 67 and 54 items, respectively . These a. The weiOne interested and eligible adult per household was sent the consent form and, upon its return, was sent questionnaires with instructions and postage paid return envelope or was sent a link via e-mail to complete the survey online. The study was approved by the ethics committee of participating research institutions.Multilevel confirmatory factor analysis (MCFA) was employed to estimate the individual- and blockgroup-level measurement models of the factor-analyzable items  of the NEWS and NEWS-A. The analyses were multilevel because the study adopted a two-stage cluster sampling design and substantial intraclass correlation coefficients  were observed at the blockgroup level. The average ICC was .22 (range: 0.02 to 0.42).MCFA was conducted using Bentler and Liang's Maximum Likelihood Estimation (MLE) method, applicable to multilevel samples with clusters (e.g. blockgroups) varying in size . EmpiricRe-specification of the a priori models was based on J\u00f6reskog and S\u00f6rbom's iterative model-generating approach , wherebyThe measures of model fit included the Bentler-Liang likelihood ratio (LR) statistic, the Goodness-of-Fit Index (GFI), the Root Mean Square Error of Approximation (RMSEA), the Non-Normed Fit Index (NNFI), the Comparative Fit Index (CFI), and the Standardized Root Mean Squared Residual (SRMS) ,28. We aAll but two items had acceptable values of univariate skewness (< 2.0) and kurtosis (< 7.0) for the use of maximum likelihood estimation . The aveTable The individual-level models of the NEWS and NEWS-A yielded acceptable standardized factor loadings for all but one item  indicated a better fit of the re-specified than the a priori model to the data , the data in the present study supported the existence of six correlated factors Table . Items dSimilar to the model of the NEWS, an analysis of standardized factor loadings indicated a certain degree of misfit at the blockgroup-level of the NEWS-A model, whereby two items 16 and 25) had unacceptably low loadings (-.10 and -.21). An analysis of indices of poor model fit led to several modifications of the blockgroup-level model. The re-specified model showed excellent fit, with all indices meeting the goodness-of-fit criteria . It is noteworthy that the same item had a relatively low loading in the original validation study of the NEWS (0.38)  and did It appears that the separation of sidewalks from traffic by parked cars may be indicative of access to destinations (i.e. parked cars at nearby services) as well as infrastructure for walking . However, this environmental characteristic is less likely to be associated with pedestrian safety. Cars parked along sidewalks are a sign of local motorized traffic that may pose problems to pedestrians wishing to cross a road, especially in the absence of crosswalks. This may explain why this particular environmental characteristic weakly or inconsistently loaded on the infrastructure and safety dimension. At present, before more information on the factorial validity of the NEWS across various geographical locations is gathered, it is suggested the item 'Sidewalks are separated from road/traffic in my neighborhood by parked cars' be considered part of the infrastructure and safety factor, particularly given that both item and factor (excluding and including the item) were found to be significantly positively related to weekly minutes of walking for transport ,16 and, In this study, the a priori blockgroup-level models of the NEWS and NEWS-A did not show a sufficient level of fit to the data. The main differences between the poorly-fitting a priori and the well-fitting re-respecified blockgroup level models were (1) the separation of natural  from building aesthetics; (2) a stronger association between building aesthetics and crime; (3) and the separation of sidewalks from other infrastructure for walking .As noted earlier, blockgroup-level factors were expected to be less stable and generalizable across locations than individual-level factors for two main reasons. First, blockgroup-level associations depend on the criteria for the selection of study areas. Second, blockgroup-level factors are more likely to represent patterns of associations between objective environmental factors, which can substantially vary across geographical locations. For example, high levels of household density and access to services may, in certain urban environments, be associated with lower socio-economic status and higher crime , while iIn contrast, individual perceptions of environmental characteristics are likely to be in part a function of psychological principles that apply across diverse subgroups. Experimental studies indicate that the evaluation of concepts believed to be related substantially influence perceptions of these concepts . People The relatively low response rate is one of the main limitations of the study. This is likely due in part to the extensive measurement protocol, including surveys and accelerometer monitoring on two occasions. Because recruitment rates did not differ by walkability/income quadrants, differential selection bias seems unlikely. However, the fact that similar individual-level measurement models were observed across three geographical locations  assuages concerns about sample bias effects. Another limitation pertains to the adopted sampling design that, while facilitating the recruitment of a socio-economically balanced sample, precluded the derivation of blockgroup-level measurement models of the NEWS and NEWS-A representative of the geographical locations. It is possible that a random sample of blockgroups might have resulted in greater similarities between the individual- and blockgroup-level measurement models and higher levels of generalizability of the blockgroup-level measurement model across geographical locations. This is because the procedure for the selection of blockgroups adopted in the three validation studies of the NEWS might have artificially inflated the blockgroup-level correlation between certain environmental features (street connectivity and land use mix \u2013 access).It is important to note that all three validation studies of the NEWS were conducted in the USA and Australia, two countries with similar cultures and language, as well as a preponderance of low-density land uses. This may have in part contributed to the observed similarities among the individual-level measurement models. It is yet to be seen whether the current measurement model of the NEWS can be replicated in populations outside Australia and the USA, although we hypothesize that it is likely to be sufficiently generalizable to other countries and cultures. The reason for this is that most the items of the NEWS depict tangible, physical neighborhood attributes, with the exception of safety-related items. We believe that the interpretation of items dealing with physical attributes is less likely to differ across cultures than is that of items gauging socio-cultural and psychological concepts. Yet, empirical confirmation for our hypothesis is needed.A limitation on generalizability is likely to be incomplete assessment by the NEWS and NEWS-A of environmental attributes found in other geographic locations. Different forms of mixed use, different pedestrian and bicycling infrastructure, and different public transit access and facilities in other countries should be reflected in modified versions of the NEWS and NEWS-A. Efforts to make such modifications are currently ongoing.This study provided further support for the factorial validity of the NEWS and its abbreviated version. At present, it is recommended that the NEWS and NEWS-A be scored according to the individual-level model comprising eight multi-item subscales and five (for the NEWS) or four (for the NEWS-A) single-item subscales. As these subscales are clearly related to constructs used in urban planning and transportation, findings based on these subscales can inform policies and interventions that may improve the activity-friendliness of a neighborhood. Given that all factorial-validation studies of the NEWS and NEWS-A were conducted on English-speaking populations, before they can be 'comfortably' used in global or multi-cultural studies further validation work across diverse populations is needed.The authors declare that they have no competing interests.EC analyzed the data, conceptualized and wrote all drafts of the manuscript. BES, LDF and JFS designed, organized and conducted the Neighborhood Quality of Life Study (NQLS). TLC contributed to the coordination of NQLS, data management, and preliminary data analyses for this paper. TLC, BES, LDF, and JFS critically reviewed and edited all drafts of the manuscript. All authors approved the final version of the manuscript."}
+{"text": "DAMPA is thought to be an inactive metabolite of MTX because it is not an effective inhibitor of the MTX target enzyme dihydrofolate reductase. DAMPA is eliminated more rapidly than MTX in these patients, which suggests a nonrenal route of elimination. In a phase II study (May 1997\u2013March 2002), CPDG2 was administered intravenously to 82 patients at a median dose of 50\u2009U\u2009kg\u22121 (range 33\u201360\u2009U\u2009kg\u22121). Eligible patients for this study had serum MTX concentrations of >10\u2009\u03bcM at 36\u2009h or >5\u2009\u03bcM at 42\u2009h after start of MTX infusion and documented renal failure . Immediately before CPDG2 administration, a median MTX serum level of 11.93\u2009\u03bcM (range 0.52\u2013901\u2009\u03bcM) was documented. Carboxypeptidase G2 was given at a median of 52\u2009h (range 25\u2013178\u2009h) following the start of an MTX infusion of 1\u201312\u2009g\u2009m\u22122\u20094\u201336\u2009h\u22121 and resulted in a rapid 97% (range 73\u201399%) reduction of the MTX serum level. Toxicity related to CPDG2 was not observed. Toxicity related to MTX was documented in about half the patients; four patients died despite CPDG2 administration due to severe myelosuppression and septic complications. In conclusion, administration of CPDG2 is a well-tolerated, safe and a very effective way of MTX elimination in delayed excretion due to renal failure.The methotrexate (MTX) rescue agent carboxypeptidase G It may cause acute nephrotoxicity, which is presumed to result from its precipitation or its relatively insoluble metabolites in acidic urine. This nephrotoxicity leads to delayed MTX elimination, ineffective rescue and a marked increase of other nonhaematological and haematological toxicities associated with MTX, such as myelosuppression, oro-gastrointestinal mucositis and dermatitis. In early studies using HD-MTX, severe toxicity occurred in approximately 10% of patients and there was a 6% mortality rate . However\u03bcM, high doses of LV are not likely to completely reverse the toxicity of MTX  was originally extracted from Pseudomonas stutzeri. During the 1970s, CPDG1 was administered successfully to a small number of patients with brain tumours and to one patient with pre-existing renal failure after MTX administration. However, the bacterial source for CPDG1 is no longer available methyl]-methylamino]-benzoic acid) and glutamate. In contrast to CPDG1, CPDG2 has a higher affinity for MTX (Km 8\u2009\u03bcM) than LV (Km 120\u2009\u03bcM) and 5-methyltetrahydrofolate (Km 35\u2009\u03bcM), the primary circulating metabolite of LV. In studies performed in rhesus monkeys, a single CPDG2 dose of 50 units (U)\u2009kg\u22121 lowered serum MTX steady-state concentration from 10\u2009\u03bcM to nontoxic levels of <0.05\u2009\u03bcM within 30\u2009min  resulted in a rapid 95\u201399% reduction of plasma levels of MTX in patients with renal dysfunction after HD-MTX .The study was a prospective, open, nonrandomised, multicentre trial. The primary study centres were in Bonn, Berlin and Vienna. Their task consisted of consultation and emergency dispatch of CPDG\u03bcM at 36\u2009h or of >5\u2009\u03bcM at 42\u2009h or of >3\u2009\u03bcM at 48\u2009h after start of MTX infusion, and (2) decreased diuresis (less than 50% excretion of the input hydration) or (3) a serum creatinine (CreaS) >1.5 times the upper limit of normal and documented increase of CreaS during the infusion period.Patients of any age were eligible if they had (1) serum MTX concentration of >10\u20092 was manufactured by the Centre for Applied Microbiology and Research  and supplied in lyophilised form with vials containing 1000\u2009U of enzyme activity. The vials were each resuspended in 1\u2009ml of sterile isotonic saline; further dilution with sterile isotonic saline (1\u2009:\u20095 or 1\u2009:\u200910) was recommended. Carboxypeptidase G2 was administered at a dose of 50\u2009U\u2009kg\u22121 over 5\u2009min intravenously by an infusion pump or by bolus injection. Following each dose of CPDG2, serum MTX concentrations were determined. Patients who experienced a decrease in serum MTX concentration greater than one logarithm following CPDG2 administration but still had serum MTX concentrations >1\u2009\u03bcM might receive additional doses of CPDG2 with the approval of the principal investigator of the study.Recombinant CPDG2 and readministered 1\u2009h following enzyme injection. The recommended LV dose during the first 24\u2009h after the CPDG2 injection was 100\u2009mg\u2009m\u22122 every 6\u2009h followed by an increased rescue for 5 days according to the scheme recommended in the ALL BFM 95 protocol  were determined according to National Cancer Institute Common Toxicity Criteria (CTC) version 2.0.Patients were evaluated daily for signs and symptoms of MTX toxicity. Complete blood counts with differential, bilirubin and ALT, AST were determined at least twice weekly. Recovery of renal function was documented daily by serum creatinine. The nonhaematological  and haematological toxicity of MTX and side effects of CPDG2, and then once daily until recovery of kidney function and decrease of MTX serum levels to less than 0.1\u2009\u03bcM. Most of the local hospitals determined the MTX level immediately with the methods such as fluorescence polarisation immunoassay (TDX) or enzyme-multiplied immunotechnique (EMIT). In a few centres, additional blood samples for analysis by high-pressure liquid chromatography (HPLC) were placed on ice, and serum was rapidly separated by centrifugation. To inactivate CPDG2, the serum samples were heated to greater than 80\u00b0C for 5\u2009min in a water bath or treated with 1\u2009N HCl to give a final concentration of 0.1\u2009N hydrochloric acid. If the enzyme activity in the samples was not fully stopped, MTX in the sample might continue to be degraded after collection. Serum was stored at \u221220\u00b0C until time of analysis. All serum samples were sent on dry ice to a central laboratory where they were stored at \u221220\u00b0C until analysis by HPLC.EDTA samples for MTX determination were obtained immediately before and 15, 30, 60 and 120\u2009min following the dose of CPDG2 action on MTX, is known to crossreact with MTX in most commercial immunological MTX assays ), samples were injected onto a 3\u2009\u03bcm, Supelcosil\u2122 LC-18-DB 15\u2009cm \u00d7 4.6\u2009mm  radial compression analytical column with a Supelquard\u2122 LC-18-DB 5\u2009\u03bcm  guard column and eluted isocratically with 80\u2009:\u200920 (v\u2009v\u22121) 0.1\u2009mol\u2009l\u22121 sodium phosphate (pH 6.8)\u2009:\u2009methanol (Lichrosolv\u00ae) at a flow rate of 1.2\u2009ml\u2009min\u22121. Eluent was monitored using a Waters 490 UV adjustable wavelength detector at a wavelength of 310\u2009nm .Serum MTX was measured by HPLC using a modification of a previously reported method methyl]amino]-benzoic acid (Dm-APA) were approximately 5.6, 9.5 and 3.5\u2009min, respectively. The peak areas were evaluated using the Chromquest software . Methotrexate and DAMPA were corrected by means of an internal standard in combination with a calibration curve.2 and (3) for the toxicity of MTX due to complete data documentation. Data were evaluated using descriptive statistical methods .Patient data were collected consecutively, recorded on data sheets prepared centrally beforehand, checked by the principal investigator and recorded in a protected database at the University of Bonn. The biometric analysis was performed for all eligible patients who were evaluable (1) for response to the study medication, (2) for the toxicity of CPDG2 and had given their written informed consent.The study was conducted in accordance with the updated declaration of Helsinki  and approved by the local ethics committees in Bonn, Berlin and Vienna. Prior to enrolment in the study, the patient's parents/legal representatives and/or the patients themselves were informed of the investigational character of the study and the emergency use of CPDGn=26), non-Hodgkin's lymphoma , osteosarcoma (n=12), brain tumours (n=3), Hodgkin's lymphoma (n=2) and pleural mesothelioma (n=1). The patient's characteristics and the different MTX regimens are listed in \u03bcmol\u2009l\u22121, all patients had an age-dependent normal value of CreaS before treatment . All patients received intravenous hydration and alkalinisation before, during and after the MTX infusion.A total of 82 patients from institutions in Europe were enrolled in this compassionate and emergency use protocol between May 1997 and March 2002. Complete or nearly complete data were available from 65 patients. For 17 patients, no data are available despite repeated telephone and written efforts. The evaluable 65 patients aged 0.9\u201371.8 years (median 15.4 years) suffered from acute lymphoblastic leukaemia (\u03bcM (median 18.0\u2009\u03bcM) (n=65). Two patients were included in the study with MTX levels of 0.52\u2009\u03bcM at 94\u2009h (case 1) and 1\u2009\u03bcM at 142\u2009h (case 2) after start of MTX and a serum creatinine of six-fold of the initial value. A total of 12 patients had no documented increase of serum creatinine at the time of contact in contrast to the inclusion criteria. Despite the absent renal failure, the principal investigator (UB) decided to give CPDG2 due to very high MTX levels at a late time of contact.Contact with the study centres was established by telephone at a median of 47.5\u2009h (range 19\u2013142\u2009h) after start of the MTX infusion. The MTX levels at the time of contact were between 0.52 and 1082\u20092 administration are documented in Methotrexate levels and serum creatinine at the first contact and before CPDG2 administration, a median MTX serum level of 11.93\u2009\u03bcM (range 0.52\u2013901\u2009\u03bcM) was documented (n=58).Immediately before CPDG2 per kg body weight was intended. A vial CPDG2 contains 1000\u2009U dry frozen. In all, 57 of 58 evaluable patients received a dose of between 33 and 60\u2009U CPDG2 per kg body weight (median 50\u2009U\u2009kg\u22121). One patient received only a dose of 17\u2009U CPDG2\u2009kg\u22121, although the correct amount was available. Carboxypeptidase G2 was administered at a median of 52\u2009h (range 25\u2013178\u2009h) following the start of an MTX infusion of 1.5\u201312\u2009g\u2009m\u22122 over 4\u201336\u2009h and was well tolerated. At 15\u2009min after CPDG2 administration, the MTX serum level of these patients was reduced by around 87%.A single dose of 50\u2009U CPDG2. One patient received an additional third dose of CPDG2. Six of these nine patients suffered from an osteosarcoma and received an MTX therapy of 12\u2009g\u2009m\u22122 over 4\u2009h. An additional three patients suffered from ALL and NHL and received MTX infusion of 5\u2009g\u2009m\u22122\u200924\u2009h\u22121 (n=2) or 8\u2009g\u2009m\u22122\u20096\u2009h\u22121 (n=1), respectively. The median time point of the first CPDG2 dose in these patients was 37.5\u2009h after the start of MTX infusions. The second dose was given at a median of 6\u2009h (range 1.5\u201390\u2009h) after the first dose. One patient received a second dose of CPDG2 1.5\u2009h after the first dose of CPDG2 due to logistics.Nine patients received a second dose of CPDG2, side effects described by two patients included flushing (n=2) and shaking (n=1). Both had stable vital signs and the symptoms resolved completely without any intervention.After the administration of CPDG2 resulted in a rapid reduction of serum MTX concentrations in all patients. The MTX concentration determined with immunological MTX detection such as TDX or EMIT showed a median decrease of the MTX level of about 87% (range 70\u201399%) 15\u2009min after CPDG2 administration. Subsequent samples also showed an ongoing decrease of MTX concentrations. Additionally, the MTX concentrations after CPDG2 administration were determined by the HPLC method. The MTX levels estimated by HPLC 15\u2009min after the first CPDG2 injection decreased by a median of 97% (range 73\u201399%)  . Almost 2, because their serum MTX concentrations determined by immunological methods remained higher than 1\u2009\u03bcM after the first dose of CPDG2. The first dose of CPDG2 resulted in a median decrease of 88% (2 did not result in a significant further decline in the serum MTX concentration. Five patients with an MTX level at a median of 8.9\u2009\u03bcM (range 3.1\u201377.6\u2009\u03bcM) before the second CPDG2 injection showed a second decrease at a median of 26% (range 5\u201370%). An additional two patients showed an increase after second CPDG2 injection of 2 and 26%. In one patient, the MTX level after the second dose of CPDG2 is not known and in one case the time between the first and second injections was only 1.5\u2009h and there was no MTX level within the documented level. All patients who received a second dose showed a clearly limited kidney function with median CreaS of 274.4\u2009\u03bcmol\u2009l\u22121 (range 168.2\u2013654.9\u2009\u03bcmol\u2009l\u22121) before the second administration.Nine patients received an additional dose of CPDGe of 88% . The secIn all, 50% of the patients had a reduced Karnowsky index (\u2a7d60%) due to the MTX intoxication. Gastrointestinal complaints with nausea and diarrhoea were frequent. Severe stomatitis  occurred in 33% of the patients. A total of 66% of the patients showed an increase of serum creatinine of more than 1.5 times of the initial value before MTX therapy. Increased liver values based on increased GPT/GOT were documented in 58% of the patients with CTC scale \u2a7e2. Only a small number of patients suffered high-grade toxicity of lung and heart/circulation. In five and six patients, respectively, a mild peripheral and/or central neurotoxicity was observed. Two patients suffered from severe central neurotoxic side effects, a female patient additionally from severe peripheral neurotoxic side effects . Further data are listed in 2 insufficiently. Thus, progressive renal failure and fatal septic complications were observed. In an additional patient (case 3), the MTX concentration was lowered by CPDG2 adequately without progressive renal failure. This patient died due to prolonged myelosuppression and its complications (pulmonary haemorrhage due to pulmonary aspergillosis), which were caused by the MTX intoxication and the progressive underlying disease (T-cell ALL) too.Four patients died due to severe myelosuppression and septic complications 7\u201322 days following the MTX infusion. Three patients  and marked (>97%) decrease in serum MTX concentration (determined by HPLC).After the initial dose of CPDG2 not resulting in a further marked decrease of the MTX concentration. This could be the result of high DAMPA concentrations inhibiting the hydrolysis of MTX by CPDG2  of the initial values 15\u2009min after CPDG2 injection.In addition, substantial crossreactivity of the MTX antibodies with DAMPA used in immunoassays results in overestimation of serum MTX concentrations. Therefore, the MTX and DAMPA serum concentration after CPDG2 is restricted to the extracellular compartment, and the intracellular MTX concentration is initially unaffected by its use. In time, the changed equilibrium between intracellular and extracellular MTX will result in the efflux of intracellular MTX back into the serum, resulting in a rise of serum MTX levels some hours after CPDG2 administration, but this is a relatively slow process. For this reason, rescue with LV must be continued following the CPDG2 administration.Due to its molecular size, CPDGIn this study, six of 65 patients were treated additionally by haemofiltration and/or haemodialysis. These methods must be repeated continuously or daily, in order to reduce the MTX level to nontoxic concentrations. A further risk of these methods results from the necessary vascular entrance, as well as the risk of bleeding secondary to heparinisation and thrombocytopenia. The effectiveness of these procedures cannot be evaluated from our data. Three patients deceased despite these approaches.2 injection due to severe myelosuppression and septic complications 7\u201322 days following the MTX infusion. The probable reasons for the lethal complications during a prolonged myelosuppression were (1) very high MTX level with an inadequate decrease of MTX levels after the second CPDG2 injection, (2) delayed CPDG2 injection (143.5\u2009h after the start of MTX infusion), (3) a progressive underlying disease (T-cell ALL) and (4) inadequate treatment of severe anaemia in a patient who was a Jehovah witness and refused treatment.In total, four patients died despite CPDG2 followed by long-term LV is highly effective in rescuing patients at high risk of life-threatening MTX toxicity. Carboxypeptidase G2 is safe and well tolerated. No severe side effects were observed in this study. Administration of CPDG2 is most beneficial for patients with MTX-induced renal failure, especially if it is administered within 48\u201372\u2009h after the start of MTX infusion.In summary, systemic administration of CPDG2 did not result in a significant further decrease of MTX levels, one initial high dose of 100\u2009U\u2009kg\u22121 CPDG2 for patients with extremely high MTX concentrations  may be more effective. For the patients with moderately high MTX levels and renal failure, the application of 50\u2009U\u2009kg\u22121 seems to be sufficient. Due to the high costs of the drug, it may be discussed whether an application of 25\u2009U\u2009kg\u22121 CPDG2 would not be sufficient to lower the MTX levels to nontoxic concentrations in patients with marginal high MTX concentrations and renal failure.For the future, it might be helpful to differentiate between patients with extremly high MTX concentrations and severel renal failure and patients with moderately high MTX concentration and renal failure. Since additional injections at CPDG2 should always be accompanied by a sufficient hydration, alkalisation and a long-term LV therapy, in order to counterbalance the intracellular MTX. Haemofiltration or haemodialysis might be helpful in oliguric or anuric renal failure. In individual cases with renal failure, severe and lethal courses may occur despite a CPDG2 application, in particular if delayed CPDG2 administration did not prevent overt toxicity.The therapy with CPDG"}
+{"text": "Diseases associated with Epstein-Barr virus (EBV) infections, such as infectious mononucleosis (IM), EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and chronic active EBV infection (CAEBV) are not rare in Chinese children. The association of type 1 or type 2 EBV and variants of the EBV BZLF1 promoter zone (Zp) with these diseases is unclear.P \u2264 0.01). The frequency of co-infection with Zp variants was higher in patients with CAEBV and EBV-HLH, compared with IM and healthy controls, mostly as Zp-P+V3 co-infection. Type 1 EBV was predominant in all categories (81.3-95%) and there was no significant difference in the frequency of the EBV types 1 and 2 in different categories (P > 0.05).The objective of this study was to investigate the relationship between EBV genotypes (Zp variants and EBV type 1 and 2) and the clinical phenotypes of EBV-associated diseases in Chinese children. The Zp region was directly sequenced in 206 EBV-positive DNA samples from the blood of patients with IM, EBV-HLH, CAEBV, and healthy controls. Type 1 or type 2 EBV was examined by PCR for EBNA2 and EBNA3C subtypes. Four polymorphic Zp variants were identified: Zp-P, Zp-V3, Zp-P4 and Zp-V1, a new variant. The Zp-V3 variant was significantly associated with CAEBV (Type 1 EBV and BZLF1 Zp-P of EBV were the predominant genotypes in nonmalignant EBV associated diseases in Chinese children and Zp-V3 variant may correlates with the developing of severe EBV infection diseases, such as CAEBV and EBV-HLH. Epstein-Barr virus (EBV) is a member of the Lymphocryptovirus genus, Gammaherpesvirinae subfamily of the Herpesviridae family of viruses. This virus is associated with a wide variety of diseases, both benign and malignant, which ubiquitously infect humans and persist for the lifetime of the individual. During its life cycle, EBV has latent and productive (lytic) phases. The latent phase maintains the virus long-term in its host and can lead to the productive phase where virus is reactivated and produced allowing it to be transmitted. During the two phases, EBV expresses a set of viral gene products in its life cycle and some of these genes were proved to possess the potential to cause changes in the interactions between the virus and the host's immune system [e system ,2.BamHI fragment Z. EBV genotypes can be categorized as type 1 or type 2 on the basis of marked allelic polymorphisms within the EBNA2, 3A, 3B, and 3C genes [The biology and pathogenesis of EBV has been the focus of many studies but the clinical management of the disease is poorly understood. Whether certain EBV genotypes are involved in the pathogenesis of specific EBV-related diseases has been the subject of investigation in recent years. Several viral variants can be distinguished according to polymorphisms in EBV genes, such as EBV nuclear antigen (EBNA) and BZLF1, a potent regulator of the switch from latency to lytic phases encoded by the EBV 3C genes . Both EB3C genes -7. Seque3C genes ,9.Childhood EBV infection is typically asymptomatic but can also induce three types of non-malignant disorders, including infectious mononucleosis (IM), EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and chronic active EBV infection (CAEBV). Certain linkages exist between these diseases where IM, usually a benign self-limiting disease, can develop to EBV-HLH and CAEBV in some patients. Likewise, EBV-HLH progresses very rapidly and becomes a life-threatening disease without immunosuppressive therapy, which occurs during the process of CAEBV sometimes or in association with fulminant IM-12. CAEBIn this study, EBV DNA from blood samples of 206 patients with IM, EBV-HLH, CAEBV, and healthy controls was examined by PCR for EBNA2 and EBNA3C subtypes (EBV type 1 and type 2) and Zp variants. This case-control study is the first investigation to explore the association between EBV subtypes and BZLF1-Zp variants and EBV infection in the China children population.P > 0.05) in the frequency of the EBV type 1 and type 2 between categories. The remaining four cases (1.9%) displayed co-infection with both type 1 and type 2 EBV and were all from the CAEBV group.The frequency of type 1 or type 2 EBV infection was determined for all samples (Table P \u2264 0.01) and relatively high in EBV-HLH cases. The Zp-V1 variant, however, was only found in IM and control cases, while the V4 variant was not detected in any CAEBV cases.Sequence differences identified within the major regulatory Zp domains (nucleotides -211 to +12) of EBV infected individuals can be grouped into four variant forms , while Zp-P was predominant in all categories except CAEBV. This suggests that the Zp-P variant EBV was the most common variant found in China and that infection by Zp-V3 is strongly correlated to CAEBV. The Zp-V3 variant is significantly associated with malignancy in both immunocompetent and immunocompromised patients [This case-control study is the first investigation to explore the association between EBV subtypes and BZLF1-Zp variants and EBV infection in the China children population. In this study, statistical analysis determined that differences in the distribution of Zp variants were significant in the four patient categories. The frequency of the Zp-V3 variant in the CAEBV group was statistically higher than for other categories  was detect in this study in only one patient with EBV-HLH. Due to the infrequent isolation of this variant, we did not include this data in correlations with disease. Previously described Zp variants, Zp-PV, Zp-V1-104, Zp-V1-105 or Zp-V1-119  were notSimilar to other studies that reported that type 1 EBV was predominant in Asian nasopharyngeal carcinoma (86.5-96%) ,18, the The prevalence of co-existence EBV Zp variants within the four categories studied ranged from 14.6 to 37.5%. The majority of these co-existence viruses occurred in patients with CAEBV and EBV-HLH and always was found associated with Zp-P and not other variants. It is likely that the majority of people are first infected with a more prevalent variant like Zp-P, the predominant variant found in this study, but does not rule out the possibility that new point mutations are likely to be arised during EBV replication of in its hosts from pre-existing variant. In this way, the balance of one pre-existing virus variant which could be controlled by its host, may be disturbed by a specific new variant. Thus, virus replication, tropism, or immune evasion in its hosts could be greatly enhanced after acquiring this new variant. As the Zp-V3 variant was associated with severe diseases in this study, the Zp-V3 type point mutations derived from Zp-P are likely to be associated with a more invasive capacity than Zp-V1 or Zp-V4 variants. Taken together, superinfection by multiple strains of EBV, especially the presence of the Zp-V3 variant, may be a contributing factor in the development of severe EBV infections in children. Thus, these findings may give some prospect to explore the differential distribution of Zp variants in susceptible populations and their association with severe or even fatal EBV diseases. A close dynamic follow-up on patients carrying EBV from an early stage of infection may help us understand how the host immune response allows such mutations to occur.Just how an individual acquires such mixtures of Zp variants is unknown. This could occur by simultaneous acquisition or by the serial accumulation from exposure to different variant carriers. It seems implausible that such co-infections can be co-acquired from a carrier who was shedding multiple variants in saliva, because it is unclear how the source can accumulate multiple infections before transmitting those orally shedding multiple EBV variants to the next. As infection by EBV with the Zp-P variant was a prerequisite for co-existence in this study, it is possible that an individual is more likely to acquire a prevalent variant, such as Zp-P, at first exposure to the virus, and then the host immunity to this variant is developed. However, part of hosts may fall short in resisting another different variant the next time. It is more likely that some instances of co-existence are the result of serial acquisition over time from independent sources. The host could gain multiple variants dynamically in the light of this hypothesis, but this cannot explain why co-infections harbor no more than two variants. It is plausible that the co-existence variants observed in this study may be new variants that were generated from point mutations of a pre-existing variant. However, the complex relationship between EBV variants and the host requires further investigation of other EBV-related diseases, various ethnic groups, different tissues or a study including a larger sample size.In conclusion, this study described the EBV genotype profiles found in children with different EBV-related diseases. The most prevalent EBV genotypes found in Chinese children were type 1 EBV and the BZLF1 Zp-P variant. The results show that patients with the Zp-V3 variant may have a higher risk of developing severe EBV-associated diseases.A total of 206 whole blood samples were obtained from inpatients at Beijing Children's Hospital from 2006-2008 with an age range from 13 months to 12 years old. Blood was collected from 40 healthy control cases, 88 individuals diagnosed with IM, 46 with EBV-HLH and 32 with CAEBV based on their respective diagnostic criteria in previously literatures ,11,20. GAll individuals in the present study are of Han descents and provided informed written consent to perform the study. All procedures were approved by the Committee of Human Studies at the Beijing Children's Hospital affiliated with the Capital University of Medical Sciences.The determination of EBV type 1/type 2 infections was performed using a standard PCR assay across type-specific regions of EBNA2 and EBNA3C gene using primers for EBNA2: 5'-AGGCTGCCCACCCTGAGGAT-3' and 5'-GCCACCTGGCAGCCCTAAAG-3' and primers for EBNA3C: 5'-AGAAGGGGAGCGTGTGTTGT-3' and 5'-GGCTCGTTTTTGACGTCGGC-3', as previously described ,22. The The Zp genotype was investigated based on the sequence diversity between positions -211 to +12 (with reference to the transcription start site) of the BZLF1 promoter zone (Zp) by a single step PCR. Oligonucleotide primers (5'-AGCATGCCATGCATATTTC-3' and 5'-TTGGCAAGGTGCAATGTTT-3') were used to amplify genomic DNA extracted from PBL samples by PCR, as previously described before . The reaP-values less than 0.05 were considered statistically significant.Statistical analysis was performed using the SPSS  software package. The association of each polymorphic Zp with type 1 and 2 EBV and the distribution of the variants within different categories were analyzed using the chi-square analysis. All statistical tests were two-sided and CA: capsid antigens; CAEBV: chronic active EBV infection; EBV: Epstein-Barr virus; EBNA: EBV nuclear antigen; EBV-HLH: EBV-associated hemophagocytic lymphohistiocytosis; IM: infectious mononucleosis; PCR: polymerase chain reaction.The authors declare that they have no competing interests.YKJ carried out most of the studies and drafted the manuscript. GL and SY participated parts of the studies and writing. ZDX and KLS provided consultation and preparation of the final report. All authors read and approved the final manuscript."}
+{"text": "The response of drug-resistant patients with acute leukaemia and Burkitt's lymphoma to treatment with a 24 h infusion of methotrexate (MTX) followed, in some cases,by cytosine arabinoside was correlated with in vitro measurements of total intracellular MTX, exchangeable intracellular MTX, and suppressibility of deoxyuridine (UdR) incorporation in isolated marrow blast cells at extracellular MTX concentrations of 10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M. Total intracellular MTX levels and exchangeable intracellular MTX levels were not significantly different in responding or non-responding patients at any MTX concentration, but increased four-fold for every ten-fold concentration increment studied. Extracellular MTX levels in excess of 10(-7)M appeared necessary to allow accumulation of exchangeable intracellular MTX. UdR incorporation at 10(-6)M and 10(-5)M differed significantly between responding and non-responding patients, with responders having less than 20% of control values and non-responders having greater than 40% of control values. Further, increasing the extracellular MTX concentration from 10(-6)M to 10(-5)M produced no significant decrease in UdR incorporation in either group. The therapeutic implications of this apparent threshold are discussed."}
+{"text": "Increased eukaryotic translation initiation factor 4E (eIF4E) expression occurs in many cancers, and makes fundamental contributions to carcinogenesis by stimulating the expression of cancer-related genes at post-transcriptional levels. This key role is highlighted by the facts that eIF4E levels can predict prognosis, and that eIF4E is an established therapeutic target. However, eIF4E activity is a complex function of expression levels and phosphorylation statuses of eIF4E and eIF4E-binding proteins (4E-BPs). Our hypothesis was that the combined analyses of these pathway components would allow insights into eIF4E activity and its influence on cancer. We have determined expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 within 424 breast tumours, and have carried out analyses to combine these and relate the product to patient survival, in order to estimate eIF4E activity. We show that this analysis gives greater prognostic insights than that of eIF4E alone. We show that eIF4E and 4E-BP expression are positively associated, and that 4E-BP2 has a stronger influence on cancer behaviour than 4E-BP1. Finally, we examine eIF4E, estimated eIF4E activity, and phosphorylated 4E-BP1 as potential predictive biomarkers for eIF4E-targeted therapies, and show that each determines selection of different patient groups. We conclude that eIF4E's influence on cancer survival is modulated substantially by 4E-BPs, and that combined pathway analyses can estimate functional eIF4E. The eukaryotic translation initiation factor 4E (eIF4E) has key roles in carcinogenesis . eIF4E iThe importance of eIF4E in cancer has been underlined by the fact that eIF4E expression levels can be used to determine prognosis. Cases in which eIF4E is highly overexpressed tend to have poor prognoses  Supplem. ActivitEthical approval was obtained (Leeds East 05/Q1206/136). Archival cancer tissue and data were obtained for 424 patients diagnosed at LTH NHS Trust from 1983\u20132006. Tissue microarrays (TMAs) were constructed containing 0.6\u2009mm cores selected from representative tumour areas as determined by a consultant breast histopathologist (AMS) from H&E stained sections. Survival periods \u2013 overall: initial diagnosis to death; disease-free: initial diagnosis to the diagnosis of recurrence/metastasis; disease-specific: initial diagnosis to death after recurrence or metastasis (cancer-specific death confirmed in most cases).\u03bcm were dewaxed and blocked in hydrogen peroxidase block (20\u2009min). Antigens were retrieved and stained as described in MCF7 and MDA-MB-231 cells were cultured/transfected as earlier  (termed p4E-BP1). We have not examined 4E-BP3 because it is not thought to have a role in breast . In addiTissue micro-arrays containing samples from 424 breast tumours were established, supported by detailed clinicopathological data . The coh\u03b1 status and eIF4E expression  and 4E-BP2 , but not with 4E-BP1 or p4E-BP1. Strong correlations between expression of markers and tumour grade were found. eIF4E expression  was positively associated with grade , whereas expression of both 4E-BPs was negatively associated with grade . p4E-BP1 was positively associated with grade (P=0.012). A positive association between eIF4E expression and grade has been reported earlier , 1.3 (P=0.008) and 1.33 (P=0.005) for OS, DFS and DSS, respectively. Thus individuals with scores of 7 have DFS HRs of 6.15  as compared with individuals with scores of 0. The prognostic value of eIF4E has been reported earlier as independent of grade/nodal status in breast cancer , DFS  and DSS .Kaplan\u2013Meier survival analyses were used to determine survival with respect to eIF4E. Analyses were carried out with expression divided into IHC scores, although scores of 0, 2 and 3 were combined as each individual group was small, for overall survival (OS), disease-free survival (DFS) and disease-specific survival (DSS) . High eIKaplan\u2013Meier survival analyses were also used to determine survival with respect to the other markers. We present data for DFS , and forP=0.011), 1.24 (P=0.035) 1.27 (P=0.02) for OS, DFS and DSS, respectively, for each increase of 1 in eIF4E score. We were unable to construct significant Cox models on the basis of combinations of 4E-BPs without including eIF4E, suggesting that eIF4E is their critical effector. We found models combining expression of 4E-BP1 or p4E-BP1  with eIF4E, provided little additional prognostic value over that found with eIF4E alone. However, including 4E-BP2 in a model for OS enhanced the model significantly with hazard increasing by 1.28 with each point increase in eIF4E score (P=0.005) and decreasing 0.11 with each increase in 4E-BP2 score (P=0.02). We refined this by combining eIF4E and 4E-BP2 scores into a single non-linear variable in which high levels of eIF4E or 4E-BP2 act to increase or decrease the value respectively , where X and B2 represent eIF4E and 4E-BP2 scores). This variable, termed \u2018y\u2019, predicted survival more accurately than examination of eIF4E alone; each increase of 1 in y carried HRs of 1.32 (P=0.0003), 1.32 (P=0.013) and 1.36 (P=0.006) for OS, DFS and DSS, respectively. We also investigated Cox models including expression of all four antigens. In order to combine terms, we considered models that included each variable separately and found their relative effect on HRs using maximum likelihood estimation. Although individual components were not statistically significant and had only little effects on likelihoods (with the exception of eIF4E), a combination gave improved prognostic power. This variable termed \u2018z\u2019 can be described as X\u2013B1/4+PB1/2-B2/4, where B1 and PB1 represent 4E-BP1 and p4E-BP1. Each increase of 1 in z gave HRs of 1.15 (P=0.006), 1.26 (P=0.002) and 1.28 (P=0.0008) for OS, DFS and DSS, respectively. This variable has a highly significant relationship with survival but this should be treated with caution because the constants were determined using regressions for OS, and, thus to an extent, significance is self fulfilling, at least for OS. The utility of z, however, is supported by the fact that its relationship with survival is more significant with DFS and DSS than OS, a result not predetermined by the approach. We also examined whether y or z give prognostic insights independently of NPI using multivariate analyses. NPI and either y or z remain significant in models for DFS  and DSS .Next we examined influences of 4E-BPs in the context of eIF4E expression. We necessarily restricted these analyses to the 282 patients for whom scores of all four antigens were available. Using this dataset, models including solely eIF4E expression gave HRs of 1.21 , as it is in this context that differential expression of 4E-BPs would be most relevant. Patients with eIF4E scores 6 or 7 have a relatively poor prognosis (y was applied to this cohort some discrimination occurred with improved prognosis for patients whose y scores were lowered by 4E-BP2 (z was applied to this cohort further discrimination occurred (z was applied (z), or relatively good prognosis (low z). In this case, z discriminated into statistically significantly different groups (P=0.039) when use of eIF4E alone was not significant (P=0.15). In this case y was substantially less successful as a prognostic indicator (P=0.5) showing the importance of combining all four components.The statistical significance of relationships of rognosis , but no rognosis  and 3A. y 4E-BP2 , althougoccurred  showing occurred . These poccurred  into sepoccurred . As befo applied  further P<0.0001), although weak positive/no association with p4E-BP1. Expression of 4E-BPs was also moderately positively associated with each other (P=0.02). Expression of p4E-BP1 was positively associated with 4E-BP1 (P<0.0001) (expected as 4E-BP1 must be expressed to be phosphorylated).Although including assessments of 4E-BPs provided additional prognostic information over that from only eIF4E, we were surprised that influences of 4E-BPs, especially of 4E-BP1, were relatively weak. One explanation for this was that expression levels of eIF4E and 4E-BPs were not independent of each other. This would mean that that at a given eIF4E level, differential expression of 4E-BPs \u2013 therefore differential modification of eIF4E activity \u2013 would be relatively rare, thereby minimising apparent influences of 4E-BPs in our analyses. Associations between marker expressions were examined using Spearman's correlation tests . eIF4E ez values) had large overlaps with both other groups (46% of group 1 and 81% of group 3). In addition, group 2 included all individuals with high eIF4E and p4E-BP1  reflecting the fact that z successfully takes account of both eIF4E and p4E-BP1, thereby supporting its utility as a potential predictive marker.The eIF4E pathway is a target for cancer therapy with drugs that either inhibit eIF4E activity indirectly (mTOR inhibitors that reduce 4E-BP1 phosphorylation thereby inhibiting eIF4E ), or dirExpression of eIF4E in cancer has been studied extensively, however, expression does not equate to activity; therefore, interpretation of its influence is more complex than simply assessing expression. Our hypothesis was that combined examination of eIF4E and its regulators would allow greater insights into eIF4E's influence on cancer. Therefore, we determined the expression levels of eIF4E and its most well-established regulatory proteins 4E-BP1, 4E-BP2 and p4E-BP1 within tumour cells of a large cohort of cancer patients, and have combined these data into an improved measure of prognosis and estimate of eIF4E activity. In common with initial publications on eIF4E's role in cancer , the conclusion that high eIF4E levels are associated with poor prognosis is well established . We havez\u2019 function; text, z, rather these were defined by the best fit with the data; the fact that the relationships reflect our expectations from understanding the pathway supports the view that z is a true estimate of eIF4E activity. Third, we showed differential expression of 4E-BP2 in cancer to be more influential in terms of survival than 4E-BP1. This was shown by the observations that expression of 4E-BP2, but not 4E-BP1, showed a trend towards being a prognostic factor alone (y), and was the most statistically significant component of z after eIF4E itself. This observation may relate to the fact that 4E-BP2 binds, and therefore inhibits eIF4E more strongly than 4E-BP1  (group 2) and high p4E-BP1 levels (group 3) should provide good candidates for treatment. The former group has a particularly poor prognosis (Clinical trials of the efficacy and safety of cancer therapeutics that target eIF4E have been carried out . Patientrognosis , and the"}
+{"text": "Methotrexate (MTX) shows consistent cytotoxicity for melanoma cells in vitro but it is ineffective in clinical use at equivalent concentrations in vivo. This apparent paradox has been investigated by cell culture techniques and results quantified by cell number. In an in vitro model of high dose MTX therapy followed by leucovorin rescue (HD-MTX-LCR) there was survival of both melanoma and choriocarcinoma cell lines but not of an acute lymphocytic leukaemia cell line. The 70H metabolite of MTX was identified by HPLC in plasma samples of melanoma patients treated by HD-MTX-LCR, in which MTX concentrations approximately 10(-5) M were maintained for 24 h. However, metabolism per se is unlikely to account for the lack of response to MTX clinically. In vitro 70H MTX (10(-7) - 10(-6) M) was two orders of magnitude less cytotoxic for melanoma than MTX (10(-9) - 10(-8) M). The cellular accumulation of [3H]-MTX, using a rapid gradient centrifuge technique for separation of melanoma cells from medium, was reduced in the presence of 70H-MTX. The results suggest that reduced cellular uptake of MTX combined with biochemical rescue of tumour cells may partially explain the paradoxical lack of clinical response of melanoma to the drug."}
+{"text": "The IGF/mTOR pathway, which is modulated by nutrients, growth factors, energy status and cellular stress regulates aging in various organisms. SIRT1 is a NAD+ dependent deacetylase that is known to regulate caloric restriction mediated longevity in model organisms, and has also been linked to the insulin/IGF signaling pathway. Here we investigated the potential regulation of mTOR signaling by SIRT1 in response to nutrients and cellular stress. We demonstrate that SIRT1 deficiency results in elevated mTOR signaling, which is not abolished by stress conditions. The SIRT1 activator resveratrol reduces, whereas SIRT1 inhibitor nicotinamide enhances mTOR activity in a SIRT1 dependent manner. Furthermore, we demonstrate that SIRT1 interacts with TSC2, a component of the mTOR inhibitory-complex upstream to mTORC1, and regulates mTOR signaling in a TSC2 dependent manner. These results demonstrate that SIRT1 negatively regulates mTOR signaling potentially through the TSC1/2 complex. The mTOR complex 1 (mTORC1) consists of mTOR, regulatory associated protein of mTOR (Raptor), LST8/G-protein \u03b2-subunit like protein (mLST8/G\u03b2L) and PRAS40. mTORC1 is stimulated by growth promoting conditions and inhibited by low nutrient levels, growth factor deprivation, reductive stress and the specific inhibitor of mTORC1, Rapamycin. Upstream to mTORC1 is the TSC1-TSC2 inhibitory complex, which functions as a GTPase activating protein (GAP) for the GTPase Rheb, an upstream activator of mTOR. The TSC1-TSC2 complex inactivates Rheb to inhibit mTOR signaling The mammalian Drosophila homologs dTSC1 or dTSC2 or mutation in dTOR or its downstream target dS6K, leads to longevity phenotype in DrosophilaThe mTOR pathway has been implicated in longevity in model organisms such as yeast, worms and flies. Over-expression of the The two best characterized substrates of mTORC1 are p70-S6 Kinase 1 (S6K1) and 4E-BP1, the eukaryotic initiation factor 4E (eIF4E) binding protein 1. Activation of mTOR results in phosphorylation of S6K1 and 4EBP1, which increases protein synthesis and ribosome biogenesis. Thus activation of mTOR results in an increase in cell size and mass Clearly mTOR and SIRT1 regulate many common effectors critical to the longevity signaling pathways in lower organisms and mice. However, no direct link has yet been established between these two important regulators. Here we investigated the potential functional interrelationship between these two proteins in regulating the stress response. Our results demonstrate that SIRT1 indeed regulates mTOR signaling, potentially through TSC2.We investigated the activity of mTOR pathway in SIRT1 deficient mouse embryonic fibroblasts (MEFs) by analyzing the phosphorylation of mTOR and its substrates S6K1 and 4EBP1. Phosphorylation of S6, the downstream target of S6K1 was also analyzed. As shown in \u2212/\u2212 and WT mouse tissue. As indicated by higher levels of phospho-S6 and phospho-4EBP1 , under stress (-leucine) or growth (insulin) conditions and mTOR signaling measured by examining phosphorylation levels of S6 and 4EBP1 . ResveraTo determine if SIRT1 modulates the expression of the critical proteins involved in regulation of the mTOR pathway, protein expression analysis of TSC1, TSC2, Raptor and Rheb in SIRT1 depleted cells under various stress conditions was performed. As shown in To determine if SIRT1 regulates the mTOR signaling pathway upstream or downstream from mTORC1, we examined whether rapamycin, a specific mTOR inhibitor that inhibits mTOR complex 1 (mTORC1) activity, could inhibit the upregulated mTOR activity in the SIRT1 depleted cells. Treatment of the matched-control or SIRT1 depleted HeLa cells with rapamycin significantly abrogated the phophorylation of S6K1, S6 and 4EBP1 , suggestmTOR is downregulated by the upstream TSC1/TSC2 complex. TSC2 null cells show upregulated mTOR signaling whereas over-expression of TSC1 and TSC2 result in mTOR inhibition Since the above results suggested that SIRT1 acts upstream of mTORC1 to downregulate mTOR signaling similar to TSC2, we investigated if the SIRT1-mediated down-regulation of mTOR signaling was TSC2 dependent. We treated WT and TSC2 null MEFs with the SIRT1 activator resveratrol (RES), and SIRT1 inhibitor nicotinamide (NAM). As expected, only the TSC2 null MEFs showed an increased S6 phosphorylation. In contrast, NAM treatment induced S6 phosphorylation in WT MEFs , suggestResveratrol has been shown to activate the AMP-activated kinase (AMPK) pathway independent of SIRT1 To confirm that the observed effects of resveratrol were indeed mediated through SIRT1, we investigated the effect of resveratrol on WT versus SIRT1 null MEFs. Resveratrol treatment strongly inhibited S6 phosphorylation in WT MEFs, but with reduced efficacy in SIRT1 null MEFs, suggesting that the effect of resveratrol on mTOR signaling is mediated partly through SIRT1 . We obsein vivo. Immunoprecipitation of endogenous TSC2 resulted in coimmunoprecipitation of endogenous SIRT1  were maintained under standard cell culture conditions. WT and SIRT1 null mouse embryonic fibroblasts For cell size determination, cells were stained for F-actin using rhodamine labeled phalloidin stain. The nucleus was stained with DAPI. Actin staining was visualized by fluorescent microscopy at 40X magnification.Whole cell extracts of WT and SIRT1 null MEFs and HeLa cells were made using NP-40 lysis buffer with standard protease inhibitors (Sigma Aldrich protease inhibitor cocktail) followed by western blot analysis for phosphorylation of the mTOR substrates using phospho-protein-antibody  for various mTOR substrate proteins. All treatments; -leucine, -glucose, -serum, tunicamycin (2 \u00b5g/ml) were given for 1hr before making extracts for western blot analysis.HeLa cells were immunostained for SIRT1 using SIRT1 monoclonal antibody (Upstate). The nucleus was stained with DAPI. Localization of SIRT1 was measured by fluorescent microcopy at 40X. Subcellular fractionation of HeLa, 293T, Jurkat cells and MEFs was performed using Pierce fractionation kit.For co-immunoprecipitation, whole cell extracts were made in NP-40 lysis buffer with standard protease inhibitors. All treatments were given for 1hr prior to making the cell lysates. Approximately 2-3 million cells were used. The immunoprecipitation was performed on 500 \u00b5g protein extract and 5% (25ug) of the immunoprecipitates were used as the input. SIRT1 antibody from upstate and TSC2 antibody from cell signaling technology was used for western blotting.Figure S1F-actin staining of TSC2+/+ and TSC2 \u2212/\u2212 MEFs: Actin was stained using rhodamine-phalloidin stain. Nucleus was stained with DAPI. Red: F-actin, Blue: nucleus.(0.51 MB TIF)Click here for additional data file.Figure S2AMPK activity in Control and SIRT1-RNAi HeLa cells: cell lysates from untreated or leucine starved cells were analysed by Western blot analysis using phospho-AMPK antibody . Tubulin is shown as loading control.(0.08 MB TIF)Click here for additional data file."}
+{"text": "The und-/- mice . Endothe-/- mice .       +-dependent histone deacetylase sirtuin-1 , whose activation has been shown to                        exert numerous beneficial effects in many aspects including life-span expansion,                        inhibition of endothelial senescence, inflammation, oxidative stress, and                        beneficial regulation of carbohydrate and lipid metabolism [It is of note that at early to middle                        stages of atherosclerosis and advanced age, eNOS protein level in the                        vasculature is not decreased and even upregulated ,7. Much tabolism .       -/-ApoEmice [+/-/ApoE-/-SIRT1                        mice to determine the role of endogenous SIRT1 on atherogenesis and endothelial                        function. They show in a recently published study reduced atherosclerotic                        lesion formation and inhibition of foam cell formation in +/-/ApoE-/-SIRT1                        mice as compared to control -/-ApoE mice [+/-/ApoE-/-SIRT1                        mice and endothelial expression of ICAM-1 and VCAM-1 upon in vivo                        challenge with lipopolysaccharide to induce systemic inflammation. The                        inhibitory role of SIRT1 in endothelial inflammatory responses is further                        confirmed in cultured human aortic endothelial cells in response to TNF-\u03b1. An increase in reactive oxygen species (ROS) in cells in which SIRT1                        is silenced by siRNA has been also demonstrated. The results support the                        findings by various studies demonstrating anti-inflammatory and anti-oxidative                        effects of SIRT1 in endothelial cells. Surprisingly, no difference in                        endothelium-dependent relaxations and eNOS-S1177 phosphorylation, an activating                        mechanism of the enzyme between +/-/ApoE-/-SIRT1                        and -/-ApoE mice is observed. It seems that there is                        discrepancy between this study and the study by Zhang, et al. [SIRT1                        transgenic mice. The discrepancy may be explained by different experimental                        conditions between the two studies. In the study by Zhang, endothelial function                        is investigated in wild type and endothelial specific SIRT1 transgenic                        mice fed high-fat-diet (HFD) for a relatively longer period i.e. 6 months,                        while the atherosclerotic burden, unfortunately not the endothelial function,                        is studied in -/-ApoE and -/-ApoE                        endothelial SIRT1 transgenic mice on HFD for 10 weeks, a protocol which                        is similar to that used in the study by Stein. It seems that the food utilized                        in the two studies is also different. Moreover, the protection of endothelial                        function observed in the study by Zhang could be due to an over-expression of                        the transgene SIRT1 which may exert much stronger effect than single SIRT1                        allele deletion model used in the study by Stein. Another possible explanation                        could be that the whole body single allele deletion of SIRT1 may readily                        affect other cell functions such as monocytes/macrophages as demonstrated by                        the authors in the same series of study [SIRT1                        in their mouse model in the future experiments. Nevertheless, the study by                        Stein and colleagues certainly further supports the hypothesis that                        SIRT1 may be a promising therapeutic target to prevent or treat                        atherosclerosis.                                Most recent studies demonstrate that SIRT1 interacts                        directly with eNOS, causes deacetylation of the enzyme at lysines 496 and 506                        and posttranslationally enhances eNOS activity , 11. TheE-/-mice . The   E-/-mice  take fur-/- mice , confirm, et al.  who repoof study , but may"}
+{"text": "Ethanol-induced folate deficiency is due to effects of ethanol on folate metabolism and absorption. We have already shown by using different methods that ethanol interferes with reabsorption of folate from the proximal tubule. In this study, we have used the folate analogue, the fluorescein methotrexate (FL-MTX), in order to evaluate effects of ethanol on FL-MTX uptake by the human proximal tubular (HPT) cells by using a confocal microscope and fluoroskan microplate reader. Since endothelins (ETs) play a major role in a number of diseases and also in the damage induced by a variety of chemicals, we have used endothelin-B (ET-B) and protein kinase-C (PKC) inhibitors to evaluate the role of endothelin in ethanol-mediated FL-MTX uptake by using fluoroskan microplate reader. Confocal microscope and fluoroskan studies reveal that cellular absorption of FL-MTX is concentration-dependent. Moreover, ethanol concentration has an impact on FL-MTX uptake. Fluoroskan studies reveal that the ethanol-induced decrease in FL-MTX uptake is reversed by adding the ET-B receptor antagonist (RES-701-1) or PKC-selective inhibitor (BIM). Thus, we can conclude that ethanol may act via ET and ET in turn may act via ET-B receptor and the PKC signaling pathway to impair FL-MTX transport.  Folate is a water-soluble vitamin that occurs in natural food sources as reduced methylated or formylated tetrahydrofolate. Folic acid is a synthetic analogue with no specialized metabolic activity . Folate Renal tubular reabsorption of filtered folate is essential for the conservation and normal homeostasis of this important vitamin . AlthougEpidemiologic studies on humans and experimental studies on animals show that alcohol causes cancer through different mechanisms, including through mutagenesis by acetaldehyde, through perturbation of estrogen metabolism and response, and through inducing oxidative damage and/or by affecting folate and one-carbon metabolism pathways . EthanolIn this study, we used the folate analogue, the fluorescein methotrexate (FL-MTX), as a model compound of our study to evaluate the effect of ethanol on FL-MTX uptake by the HPT cells by using confocal microscope and fluoroskan microplate reader. Fluorescein methotrexate (F-MTX), a fluorescent derivative of MTX, was synthesized by coupling the carboxyl of the glutamate moiety of MTX through a diaminopentane spacer to fluorescein isothiocyanate . We usedFL-MTX, BIM  were purchased from Molecular Probes  and RES-701-1 (ET-B receptor antagonist) was purchased from American Peptide Company . We purchased all other chemicals used in this study from Sigma Chemical Co , Collaborative Biomedical Research  and GIBCO . 2 flasks  in a serum-free mixture of Dulbecco's modified Eagle's medium-Ham's F-12 medium (50 : 50 by volume) with the following additions per L: selenium (5\u2009\u03bcg), insulin (5\u2009mg), transferrin (5\u2009\u03bcg), hydrocortisone (36\u2009\u03bcg), epidermal growth factor (10\u2009\u03bcg), triiodothyronine (4\u2009ng), and 2\u2009mmol glutamine. When the HPT cells were grown to confluency in tissue culture flasks, the cells were subcultured onto 24-well plates . We used the cells when they attained confluency for this study.HPT cells from normal human kidney cortex tissue (from kidneys unable to be used in transplantation) were isolated by the enzyme dissociation method using a DNAase-collagenase mixture as described previously . Isolate\u03bcM of FL-MTX and (b) 4\u2009\u03bcM of FL-MTX and different concentrations  of ethanol for 30\u201345\u2009minutes of incubation at 37\u00b0C. The cells were subjected to confocal microscopy.HPT cells in the 24 well plates were washed twice with room temperature buffer (PBS). The cells were treated with (a) 1.5\u2009mL of PBS with 0.5,1,2, and 4\u2009The cells in the 24 well plates were directly observed under Bio Rad Radiance 2000 Confocal microscope and viewed through a 40X CF infinity plan fluor objective. Excitation was provided by the 488\u2009nm line of an argon laser. A 515/30\u2009nm filter and a 545/40\u2009nm dichroic filter were used. The trapped signal was sent inside the Photo Multiplier Tubes which capture light using total internal reflection. A few cells in the wells were selected and the images were viewed. Bio-Rad's Lasersharp-I allows 32-bit image acquisition, display and 3D volume rendering, and image analysis.2, MgCl2, D-glucose, and NaHCO3) and then treated with (a) 1.5\u2009mL of the above incubation buffer containing 1, 2, 4, 8, and 16\u2009\u03bcM FL-MTX, (b) 1.5\u2009mL of incubation buffer containing 10\u2009\u03bcM FL-MTX and different concentrations  of ethanol, and (c) 1.5\u2009mL of incubation buffer containing 10\u2009\u03bcM FL-MTX, 500\u2009mg/dL ethanol and 10\u2009\u03bcM ET-B receptor antagonist (RES-701-1) or 2.5\u2009\u03bcM PKC selective inhibitor (BIM), respectively, for 2\u2009hours of incubation at 37\u00b0C. The cells were washed with incubation buffer and then the fluorescence intensity was read in fluoroskan reader with an appropriate filter.HPT cells in the 24 well plates were washed twice with room temperature pH 7.4 incubation buffer . All statistical analysis was carried out using SAS 9.1 . One way analysis of variance with Duncan's multiple range tests was performed to identify the statistical significance. A P- value of less than .05 was considered statistically significant.Data are expressed as mean \u00b1 SEM (P < .001)  .The cellf FL-MTX . When thP < .001), , see . We usedP < .001) by adding ET-B receptor antagonist (RES-701-1) or PKC selective inhibitor (BIM) , or 2.5\u2009\u03bcM PKC-selective inhibitor (BIM) for this study. From this experiment, we can suggest that ethanol may act via ET and ET in turn may act via ET-B receptor and PKC signaling pathway to impair FL-MTX transport. We standardized the concentrations of RES-710-1 and BIM in this study based on various experiments.Fluoroskan studies revealed that ethanol-induced decrease in FL-MTX uptake was reversed and it is significant (or (BIM) . We stanFor folate uptake and other toxicological studies, we are traditionally growing HPT cells, which formed a useful tool to understand various mechanisms. HPT cells which were fully grown and confluent in 24 well plates were used for this study. The cells form confluent monolayers on plastic as well as on porous membrane inserts and produce domes (hemicysts) in culture were indicating retention of ion transport properties and integrity of cell to cell junctions. Before conducting the present experiment, we also characterized these cells histochemically by using our recently developed methods for gamma glutamyl transpeptidase (GGT) and glucose-6-phosphatase (G-6-P) (data not shown). The cells were positive for GGT and G-6-P. The cells, thus, retained the membrane integrity and function like that of the intact proximal tubule and hence we used these cells to develop confocal and fluoroskan methods to understand the mechanism of FL-MTX uptake studies. We developed our confocal method in such a way so as to demonstrate the images in 24 well plates directly. When we treated the HPT cells with different concentrations of FL-MTX and viewed by using the confocal microscope, we observed that cellular uptake of FL-MTX is concentration-dependant. Fluoroskan method also showed that cellular uptake of FL-MTX was concentration dependant. In general, transport on the \u201cclassical\u201d organic anion system in renal proximal tubule is specific and active. Assaraf et al.  demonstr It is quite clear from both the confocal and fluoroskan methods that ethanol reduced FL-MTX uptake in a concentration dependant manner. Since the confocal methods were quite comparable with our fluoroskan methods, we concluded that our protocol was quite clear for both the methods. Miller  observedChronic ethanol decreases endothelin-stimulated glucose uptake in rat adipocytes . Folate By using the folate analogue, FL-MTX, we developed this method to evaluate the mechanism of ethanol mediated FL-MTX transport. Our uptake methods by using confocal microscope and fluoroskan microplate reader showed that ethanol-impaired FL-MTX uptake was mediated through endothelin signaling. Our HPT cells in 24 well plates provided a good model for this study. On the therapeutic point of view, ET receptor antagonists may prevent ethanol impaired FL-MTX or folate uptake."}
+{"text": "Our                        recent study defined a new role for SIRT1 as a regulator of hepatic lipid                        metabolism. In the liver a major target of this sirtuin is the PPAR\u03b1/PGC-1\u03b1 signaling                        axis. Ablation of SIRT1 in the liver results in disrupted fatty acid                        oxidation, increased cellular stress, and elevations in proinflammatory                        cytokines. However, contrary to previous studies, we observed no changes in                        glucose production in the absence of SIRT1, despite impaired PGC-1\u03b1 signaling.                        These findings point toward the involvement of other players in                        SIRT1-regulated hepatic metabolism. Here we discuss our findings, and                        comment on\u00a0some of the controversy surrounding this protein in the                        current literature.                 The food we eat has long been linked to the rate we                        age. Selective pressures in times of food abundance and scarcity have                        influenced our very genetic makeup, instilling in our genome genes believed to                        control the delicate balance between metabolism and aging. However, this                        balance has been disrupted in western societies with developments in                        agriculture and technologies that have promoted the intake of high-calorie                        diets and sedentary lifestyles. We are witnessing an alarming increase in the                        rate of metabolic syndrome, which consists of a collection of abnormalities                        including obesity, type 2 diabetes, dyslipidemia, fatty liver, and a                        pro-inflammatory and prothrombotic state ,2 Curren+-dependent                        protein deacetylases and/or ADP ribosyltransferases that                        target histones, transcription factors, and co-regulators to adapt gene                        expression in response to the cellular energy state [The sirtuin family of proteins appears to                        be at the crossroads between nutritional status and longevity. Sirtuins are highly conserved NADgy state . Many megy state .       The liver is a central metabolic organ in charge of                        regulating nutrient homeostasis in fed and fasting conditions. It controls key                        aspects of lipid and glucose metabolism in response to nutritional and hormonal                        signals . Tight rin vivo, primarily due to the complicated developmental defects                        in the SIRT1 whole-body knockout mouse [bona fide SIRT1 substrate. Further studies are necessary to elucidate weather                        SIRT1 indeed deacetylates PPAR\u03b1, thereby affecting its                        activity.                                Although SIRT1 is an important regulator                        of metabolism, the tissue-specific and systemic roles of SIRT1 are difficult to                        dissect ut mouse ,16. In sut mouse . Microarut mouse ,18. It ut mouse  (Figure A major focus of our study was to characterize how                        disruptions in PPAR\u03b1 signaling affect the physiology of SIRT1 LKO mice .       Several of the metabolic abnormalities we observed in                        the SIRT1 LKO mice , howeverUsing a similar                        hepatic-specific knockout mouse model, Chen et al. observed a reduction in                        weight gain and liver fat accumulation in LKO mice when fed a western-style                        diet. Additionally, their mice were protected from the physiological impacts of                        a western diet with lower blood glucose and insulin levels. Similar to our                        study, their group observed minor physiological differences in LKO mice fed a                        chow diet. In wake of these findings, Chen et al. proposed that SIRT1 activity                        in the liver is directly proportional to calorie intake, and that excess                        calories and/or SIRT1 activators may result in elevated synthesis of fat and                        cholesterol. One possible factor contributing to the discrepancy between our                        observations and those of Chen et al. may be the difference in age of animals                        at which the feeding was initiated and data were collected. In our study, mice                        were six-week old when high-fat diet feeding was initiated, whereas four-month                        old mice were utilized in the study carried                        out by Chen et al. The varied responses of SIRT1 LKO mice to a western-style                        diet at different ages raises the possibility that hepatic SIRT1 may                        selectively regulate alternative metabolic pathways at multiple stages of                        development. An inducible SIRT1 knockout model will be helpful to dissect                        age-dependent effects of SIRT1.  Moreover, since the liver is such a dynamic                        metabolic organ, small variations in dietetic components and genetic                        backgrounds may also contribute to the inconsistency between these two                        studies.                                Another surprising phenotype observed in the SIRT1 LKO                        mice is their normal gluconeogenesis in response to a 16-h fasting . The   In conclusion, while our study defines a                        new role for SIRT1 as a key regulator of hepatic lipid metabolism, it also adds                        fuel to the fire of controversy surrounding this protein as a central player in                        mammalian energy homeostasis. It appears that in the liver, a major target of                        this sirtuin is the PPAR\u03b1/PGC-1\u03b1 signaling axis. Ablation of                        SIRT1 in the liver creates disruptions in fatty acid oxidation, increased                        cellular stress, and elevations in proinflammatory cytokines. What remains to                        be determined is the precise role SIRT1 plays in regulating gluconeogenesis and                        cholesterol metabolism in the liver and how this, in turn, affects systemic                        metabolism. Our findings and others suggest that activation of SIRT1 may                        provide a therapeutic strategy for treatment of metabolic syndrome."}
+{"text": "The new trends in immunochemistry related to the replacement ofradioisotopic labels with non-radioactive labels are presented. Immunoenzymatic, fluorescent and chemiluminescent techniques are described in terms of their basic principles and their most common applications. The advantages of computer-controlled calibration are also discussed."}
+{"text": "Neocarzinostatin (NCS) was bound covalently to human/mouse chimeric Fab fragments of MAb A7 (chA7Fab) directed against human pancreatic carcinoma. The anti-tumour effect of chA7Fab-NCS was tested in a nude mouse model on pancreatic carcinoma and compared with A7-NCS or NCS alone. The anti-tumour effect of chA7Fab-NCS increased in a dose-dependent manner and was significantly greater than either A7-NCS or NCS. Tumour growth was completely suppressed after the administration of chA7Fab-NCS. An enzyme-linked immunosorbent assay with rabbit anti-mouse immunoglobulin was performed to examine the antigenicity of chA7Fab. ChA7Fab had less reactivity with rabbit anti-mouse immunoglobulin than either whole antibody A7 or murine Fab fragments of A7. Thus, chA7Fab-NCS can inhibit human pancreatic cancer growth in an animal and may be useful for targeting chemotherapy to pancreatic cancer in humans."}
+{"text": "The third-generation aromatase inhibitors (AIS) are largely replacing tamoxifen in the adjuvant treatment of early-stage breast cancer in postmenopausal women with hormone receptor\u2013positive tumours. To date, multiple trials have been conducted comparing tamoxifen treatment with an AI, and all have demonstrated improved disease-free survival with AI treatment. Trials have included direct 5-year comparisons between tamoxifen and an AI, switching to an AI within 5 years after initial tamoxifen treatment, or extending treatment with an AI after 5 years of completed tamoxifen treatment. Some of these trials have been completed; others are ongoing; and head-to-head trial comparisons of individual AIS are also in progress. The present article summarizes the data obtained from various clinical trials of hormonal therapy for early breast cancer. It also reviews recent data so as to shed light on the current status of these therapies. The focus is on the efficacy of treatment with an AI. Toxicity is discussed in the second article in this supplement. The efficacy of tamoxifen as adjuvant therapy for women with estrogen receptor (ER)\u2013positive early breast cancer has been clearly demonstrated. Adjuvant tamoxifen treatment has been associated with a 31% reduction in the annual breast cancer mortality rate among hormone receptor\u2013positive women with early breast cancer Clinically meaningful endpoints are distinct measurements or analyses that reflect how a patient feels, functions, or survives Although OS is the most objective measure of efficacy, its evaluation requires prolonged follow-up, and consequently, the use of surrogate endpoints has become acceptable. In addition, OS measurements are affected by the development of effective salvage treatments for patients with metastatic disease, and they therefore may not be a pure efficacy endpoint in the evaluation of adjuvant therapy. The most widely used surrogate endpoints in oncology are DFS or event-free survival, the latter being the time from the beginning of treatment until a patient experiences a recurrence, a new primary cancer, or death. For some cancers, DFS is now recognized as an acceptable endpoint for evaluating therapies in situ (DCIS and LCIS) within CLBC and others not Early trials of tamoxifen for the adjuvant treatment of breast cancer used OS and DFS as primary efficacy endpoints. However, these early trials were comparing tamoxifen with placebo, rather than with an active drug. The more recent AI trials have used DFS as the primary endpoint, with TTDR and RFS as surrogate endpoints of clinical benefit . AlthougThe use of DFS as a surrogate endpoint for OS is most appropriate in settings in which recurrence of the disease is responsible for a major component of mortality in the treated population, which is the case for most solid tumours p = 0.3); however, DFS was 83% and 77% respectively (p < 0.00001) in the two cohorts With respect to early breast cancer, OS alone was often used as the primary endpoint in early trials of adjuvant therapy. However, in interim analyses of some studies, it became evident that OS benefit would not be apparent and that other measures such as DFS also had to be considered. For example, in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 randomized trial involving 2644 ER-positive node-negative women, no significant advantage in OS was found after 4 years of follow-up  Women with breast cancer may have tumours with receptors for hormones such as estrogen or progesterone. Circulating estrogen binds to its cognate receptor in women with ER-positive breast tumours, which induces cell proliferation The risk of breast cancer recurrence is highest within the first 5 years of primary chemotherapy et al.Several trials with AIS for adjuvant endocrine treatment in postmenopausal women with early-stage breast cancer are either underway or have been completed. In addition to trials for upfront AI treatment, there have also been trials to evaluate switching from tamoxifen to an AI, or extending treatment with an AI after tamoxifen for 5 years  of a number of AI trials showed that, with AIS, breast cancer recurrences are significantly less frequent than they are with tamoxifen n = 3125), 20 mg tamoxifen alone (n = 3116), or a combination of the two (n = 3125). After the first analysis at 33 months, the combination arm was dropped when no benefit was demonstrated as compared with tamoxifen alone p = 0.005], an improvement in TTR , and a reduced occurrence of CLBC  as compared with women on tamoxifen The ATAC trial enrolled 9366 women from 21 countries, including 635 from Canada, all of whom were postmenopausal with early-stage breast cancer p = 0.0001). That finding suggests a carryover effect for anastrozole, whereby women continue to benefit from treatment with anastrozole after stopping treatment. Although fracture rates overall were higher in women on anastrozole during the treatment period, the incidence was the same for both groups after treatment had been completed Follow-up of the ATAC trial beyond 5 years of treatment and data obtained at 100 months showed significant improvement in DFS, TTR, TTDR, and CLBC in women on anastrozole, although no difference in OS was noted p = 0.003), defined as the time from randomization to the first local, regional, or distant recurrence; a new invasive cancer in the contralateral breast; a second, non-breast cancer; or death without a preceding cancer event p = 0.007), and the 5-year DFS estimates for letrozole and tamoxifen were 84% and 81% respectively. A CLBC occurred in 0.6% of women on letrozole as compared with 1.1% of women on tamoxifen, and a 1.2% absolute decrease in distant recurrence was observed among women on letrozole. Fewer gynecologic and thromboembolic events, but more bone fractures, arthralgia, and low-grade hypercholesterolemia were experienced by women on letrozole as compared with those on tamoxifen The BIG 1\u201398/IBCSG 18\u201398 study enrolled 8010 hormone receptor\u2013positive postmenopausal women worldwide, including 20 from Canada p = 0.08); however, whether this trend is real or confounded by the crossover of patients from tamoxifen to letrozole is difficult to assess Updated results at a median follow-up of 76 months (reported at the 2008 San Antonio Breast Cancer Symposium) continued to support improved survival for patients treated with letrozole The Tamoxifen Exemestane Adjuvant Multinational (TEAM) study was initiated in 2001 in 9 countries to examine the efficacy of exemestane versus tamoxifen in 9775 hormone-sensitive women with early breast cancer www.clinicaltrials.gov/ct/show/NCT00066573 for details) and has completed accrual. Results of these trials should yield useful information about the individual effects and relative efficacy of the AIS.Two key head-to-head trials of AIS as adjuvant treatment for women with early breast cancer are underway. The Femara versus Anastrozole Clinical Evaluation (FACE) is a phase III, open-label, randomized, multicentre trial designed to test whether anastrozole or letrozole has superior efficacy as adjuvant treatment for 5 years in postmenopausal women with hormone receptor\u2013positive, node-positive disease p = 0.01). Gastrointestinal complaints, musculoskeletal disorders, fractures, disorders of lipid metabolism, and hyperglycemia were more common in women on anastrozole. Women on tamoxifen developed more venous disorders and gynecologic changes . Notably, however, the trial was unable to fulfil the sample size calculations because of competing trials, and the results are therefore based on the patients who were able to be recruited to the trial.The Italian Tamoxifen Arimidex (ITA) trial has been evaluating a switch to anastrozole after 2\u20133 years of tamoxifen p \u2265 0.001), representing a 40% reduction in the risk of an event in women switched to anastrozole after being on tamoxifen for 2 years. Although more fractures and bone pain were recorded in the anastrozole group, fewer thromboses occurred Two additional trials, the Arimidex\u2013Nolvadex (ARNO 95) study and the Austrian Breast and Colorectal Cancer Study Group (ABCSG) trial 8, which together involve 3224 women with ER-positive early-stage breast cancer, are also examining the effect of continuation on tamoxifen after 2 years, as compared with a switch to anastrozole for a further 3 years p < 0.0001), event-free survival (p < 0.0001), distant recurrence-free survival (p = 0.002), and OS (p = 0.04) A meta-analysis of the three aforementioned trials confirmed that, among patients who switched to anastrozole as compared with those who remained on tamoxifen, fewer disease recurrences and deaths were observed, resulting in significant improvements in DFS , sequential trial strategies analyze events from the start of treatment. Sequential and switch trials investigate the same intervention, but they are conducted in different patient groups and are therefore expected to provide different results. The ABCSG 8 trial was recently completed, and the sequencing strategy continued to show improved benefit as compared with tamoxifen alone for 5 years The IES is a double-blind randomized trial that compares a switch to exemestane in postmenopausal women with early breast cancer who have been on tamoxifen for 2\u20133 years with continuation of tamoxifen alone for a total of 5 years The most recent analysis for the BIG 1\u201398/IBCSG 18\u201398 trial, in addition to reporting the monotherapy analysis with either letrozole or tamoxifen alone, also included an analysis on sequencing to letrozole for 3 years following 2 years on tamoxifen and vice versa The EBCTG recently carried out a meta-analysis of the ABCSG 8, ARNO 95, IES/BIG 2\u201397, and ITA trials and showed that AIS are associated with significantly fewer breast cancer recurrences, even in the switch setting, and furthermore, a statistically significant improvement in mortality rates p = 0.4) were observed in the group on extended treatment, but the risk of endometrial cancer was also observed to be doubled. Of interest, an earlier study found no benefit for extended treatment with tamoxifen in women with node-negative cancer p = 0.07) in those who continued to receive it.Several trials have examined the efficacy of adjuvant treatment extended beyond the standard 5-year duration. These have included extended treatment on tamoxifen or an AI beyond 5 years, or sequential treatment with an AI in women who were disease-free after 5 years on tamoxifen. The Adjuvant Tamoxifen, Longer Against Shorter (ATLAS) randomized trial is comparing 10 years with 5 years of tamoxifen in 11,500 women (59% ER-positive). A lower recurrence rate has been observed among women continuing on tamoxifen at a mean of 4.2 years following randomization p < 0.001) The double-blind MA.17 trial from the NCI C Clinical Trials Group is looking at the effect of either letrozole or placebo in women who have already undergone standard 5-year treatment with tamoxifen n = 856) were randomly assigned to receive either 3 years of anastrozole or no further treatment. At a median follow-up of 62.3 months, women who received anastrozole (n = 387) had a statistically significantly reduced risk of recurrence  as compared with women who received no further treatment (p = 0.031) The ABCSG trial 6a is similar to MA.17: It is examining the efficacy of 3 years of anastrozole treatment in women who have completed 5 years of tamoxifen treatment, with or without the first-generation AI aminoglutethimide for the first 2 years of therapy The NSABP B-33 trial was designed to examine whether exemestane treatment (versus placebo) prolongs DFS following 5 years of tamoxifen treatment The addition of 5 years of tamoxifen in premenopausal women with hormone receptor\u2013positive disease is associated with a 40% reduction in recurrence p = 0.01) at a median follow-up of 48 months, and fewer patients on that combination experienced bone metastases Treatment with AI alone has not been recommended in premenopausal women with functioning ovaries following chemotherapy. In these women, though AIs may reduce estrogen production in peripheral tissue to some extent, ovarian estrogen production is maintained or increased, which may lead to unfavourable effects on breast cancer risk of recurrence The IBCSG is currently conducting three trials in European and North American premenopausal women Several trials are being conducted to determine the optimal duration of AI treatment for postmenopausal women with early breast cancer. The MA.17R trial was initiated in 2004  to compare DFS in subjects who receive 5 years of letrozole or placebo after having received approximately 5 years (4.5\u20136 years) of aromatase inhibitor therapy , including those who have received 5 years of adjuvant letrozole as part of the MA.17 trial. In the ABCSG-16 Secondary Adjuvant Long-Term Study in the Arimidex trial, women who have been free of recurrence after approximately 5 years of endocrine therapy will receive either 2 years or 5 years of extended adjuvant therapy with anastrozole With ongoing studies looking into the benefits of an additional 5 years of treatment with an AI, some physicians question whether patients will be willing to take these medications for this length of time. To address that concern, a survey was recently conducted among patients with early breast cancer and the physicians who treat them. Physicians were asked to indicate the minimum incremental benefit that they believed would justify 5 additional years of AI therapy. Most indicated that they would prescribe an AI for a further 5 years only if it produced an additional 1%\u20132% benefit in OS. When patients with early breast cancer on an AI therapy were asked the same question, one third responded that they would continue AI therapy for a further 5 years even if it offered a less-than-1% incremental benefit in OS. These results suggest that physicians may be more risk-averse than are their patients; the results are also encouraging in that they show that most patients tolerate their AI medication well enough to consider a further 5 years of therapy for a very small potential benefit de novo) resistance refers to a lack of response at initial exposure to the drug; acquired resistance develops during therapy in patients who are initially responsive. Host factors, treatment type, and the biology of the tumour can all have a role to play in the development of resistance.Because adjuvant endocrine therapy is generally a long-term treatment, the development of resistance and other issues affecting drug efficacy must be considered because these events will have an effect on the benefits of treatment. Drug resistance may be intrinsic or acquired. Intrinsic  are at increased risk of recurrence, while those with high-activity variants have a more favourable outcome neu oncogene encodes a transmembrane tyrosine kinase receptor with extensive homology to the epidermal growth factor receptor and that overexpression of this gene may be associated with tamoxifen resistance Tamoxifen is converted to anti-estrogenic metabolites that are thought to be more potent than tamoxifen itself. One of the active metabolites, endoxifen, is produced through the action of the cytochrome P450 enzyme CYP2D6, and women who have particular allelic variants of the genes encoding this enzyme are poor metabolizers of tamoxifen neu status and AI efficacy, neoadjuvant letrozole has been found to be equally effective in HER2/neu\u2013positive and HER2/neu\u2013negative tumours neu-positive tumours, showed that the prognosis of those patients was less favourable with both letrozole and tamoxifen therapy Because the AIs inhibit the action of aromatase, the degree of expression of this enzyme\u2014and differences in the affinity of drug binding\u2014could have a bearing on drug efficacy The estrogen receptor is a member of the nuclear receptor family of ligand-activated transcription factors. Upon entering a cell, estrogen binds to the receptor, which then undergoes conformational changes and binds to elements upstream of estrogen-dependent genes and alters their transcription either by up- or downregulation Acquired resistance to the AIs has been demonstrated in tissue-culture cell lines in which cells acquire an adaptive hypersensitivity to estrogen through upregulation of estrogen receptors and increased crosstalk between various growth factor receptor signalling pathways Women on tamoxifen experiencing excessive menopausal symptoms such as hot flashes may be prescribed a serotonin reuptake inhibitor (SRI), and these agents have been shown to inhibit CYP2D6 activity. Venlafaxine has been found to be a weak inhibitor of CYP2D6; paroxetine is a potent inhibitor Data so far have unequivocally shown that, as compared with tamoxifen, the AIs offer superior benefit in DFS, and adjuvant AI treatment is accepted as the standard of care in early breast cancer. However, issues for the physician include which AI to use and when to start it. Further, questions such as duration of treatment and the optimal treatment strategy remain to be answered. The data so far point to AI treatment up front, because the risk of recurrence is highest within the first 2\u20133 years after surgery"}
+{"text": "Since the development of the third-generation aromatase inhibitors (AIs), anastrozole, letrozole and exemestane, these agents have been the subject of intensive research to determine their optimal use in advanced breast cancer. Not only have they replaced progestins in second-line therapy and challenged the role of tamoxifen in first-line, but there is also evidence for a lack of cross-resistance between the steroidal and nonsteroidal AIs, meaning that they may be used in sequence to obtain prolonged clinical benefit. Many questions remain, however, as to the best sequence of the two types of AIs and of the other available agents, including tamoxifen and fulvestrant, in different patient groups. What is the optimal sequence for hormonal therapy in breast cancer? Until recently, the antioestrogen tamoxifen was the only agent approved for adjuvant therapy. At the time of relapse, postmenopausal patients with tamoxifen-resistant disease (progressing during adjuvant tamoxifen or within 1 year from its discontinuation) were generally offered an aromatase inhibitor (AI), followed by a progestin  when they further progressed. This paradigm needs to be reconsidered in the light of recent developments following the early results of the Arimidex, Tamoxifen Alone or in Combination (ATAC) study , with anFurther results of ongoing adjuvant trials are eagerly awaited, but it is likely that in the future an increasing number of patients who present with advanced breast cancer will already have received an AI (with or without tamoxifen) as part of their adjuvant therapy. The remaining patients will have had tamoxifen only, and a few will be hormone-therapy na\u00efve. After taking into account previous treatments, the choice of an appropriate hormonal therapy should be based on efficacy and tolerability data of the available agents.P<0.001) compared with continuing on tamoxifen  0.83), but not for combination therapy compared with tamoxifen (HR 1.02). In women with oestrogen-receptor positive tumours, the HR was 0.78 in favour of anastrozole compared with tamoxifen or combination therapy. The recently published Intergroup Exemestane Study did not explore combined treatments, but its results support the sequential use of tamoxifen and AIs. Switching to exemestane after 2\u20133 years of tamoxifen therapy resulted in a significant reduction in disease-free survival events ? The improved efficacy (time to progression) and side effect profile of AIs compared with tamoxifen  and nonsteroidal (e.g. anastrozole and letrozole) agents are illustrated in Although the pharmacological and molecular bases for their lack of cross-resistance need to be investigated further, the possibility of successfully using steroidal and nonsteroidal AIs in sequence is supported by clinical studies  and musculoskeletal events. In addition, effects have been reported on bone density, lipid levels and thromboembolic events with hormonal treatments. In the ATAC trial, there was a significantly lower incidence of hot flushes, vaginal bleeding and discharge and venous thromboembolism with anastrozole than tamoxifen, but anastrozole was associated with more musculoskeletal symptoms and fractures  MIG-8 trial was designed to assess treatment with different AI sequences in postmenopausal patients with oestrogen receptor-positive or -unknown advanced breast cancer. This is a prospective, nonrandomised study where patients not previously treated with AIs received exemestane first, and were crossed over to a nonsteroidal AI (anastrozole or letrozole) at the time of progression. Patients with previous exposure to AIs received the alternative AI after entering the study  who had received the steroidal AI formestane and were treated with anastrozole (n=12) are considered, 78% achieved further stable disease on anastrozole . At the first data report, clinical benefit was obtained by 25% among 24 women in this group (n=20). The clinical benefit rate was 55%, with a median duration of 15 months.formestane followed by exemestane or anastrozole (SAINT);vice versa ;exemestane followed by anastrozole or nonsteroidal AIs followed by fulvestrant or exemestane in patients failing nonsteroidal AIs (SOFEA).There are several large trials currently in progress or planned which should throw more light on whether there is an optimal sequence for AI therapy. These include trials of:Trials in progress include the NCIC MA27 and the SOFEA trials. NCIC MA27 is a randomised phase III trial comparing exemestane (with or without celecoxib) with anastrozole (with or without celecoxib) for 5 years in preventing cancer recurrence in postmenopausal women following surgery for primary breast cancer. The study aims to recruit 6800 women. The SOFEA trial is comparing fulvestrant with or without anastrozole versus exemestane in 750 patients failing nonsteroidal AIs.The impact of new agents, such as fulvestrant, on drug sequencing will also need to be considered. Preliminary results from studies of fulvestrant after progression on tamoxifen and anastrozole reported a clinical benefit rate of 34\u201350%. Another study has examined fulvestrant in women who had progressed on tamoxifen and an AI (vice versa). In other words, antiaromatase drugs of the two types can be used at different times to prolong disease control, before changing to other, less tolerable treatments (such as progestins or chemotherapy). The traditional sequence of hormone therapy, which is to say, adjuvant tamoxifen followed by an AI as first-line therapy at relapse and then progestins as second line could therefore change to adjuvant tamoxifen followed by an AI as first-line therapy at relapse and then another AI as second line and finally, progestins as third-line therapy. The above sequence is likely to change further based on the results of recent and ongoing adjuvant trials , which may result in an increased number of patients exposed to AIs in the adjuvant setting. The choice of first- and subsequent-line hormone therapy for advanced disease will be based on the treatment history of the patient, on the (limited) data reported above on noncross resistance, and on a balance of the expected benefits and toxicities with the agent of choice.Current evidence suggests that there is clinical utility in using antiaromatase agents in sequence (steroidal inactivators after nonsteroidal inhibitors, or"}
+{"text": "A number of trials have studied the value of aromatase inhibitors (AIs) for the adjuvant treatment of early hormone-responsive postmenopausal breast cancer. Three different AIs have been used and they have been compared as initial treatment  or after 2\u20133 years of tamoxifen , in both cases against a standard arm of 5 years of tamoxifen. In addition, two trials have evaluated AIs against no treatment after 5 years of tamoxifen. In all circumstances, the AIs have demonstrated superior efficacy. However, no results are currently available for the key question, that is \u2013 is it better to start initially with an AI or use it sequentially after 2 years of tamoxifen? Here, we review the trial results and present two models, which address this issue. The models clearly show that early treatment with an AI is superior to using it after 5 years of tamoxifen. They also favour an upfront strategy to sequencing after 2 years of tamoxifen, but in this case the differences are small and model-dependent. A key question is whether AIs have substantially better efficacy than tamoxifen for ER-positive\u2013PgR-negative tumours, where the data are currently contradictory. A mechanism explaining why greater efficacy might be so is proposed. Further results from ongoing trials will be needed to resolve this issue. Several trials have shown that the aromatase inhibitors (AIs) achieve a lower recurrence rate than tamoxifen when used as adjuvant treatment for post-menopausal women with hormone-receptor-positive breast cancer. Interpretation of these results is complicated by the fact that three different AIs have been used, and also that they have been offered at different times in the treatment programme. Three types of schedule can be identified: initial treatment; sequencing treatment after 2\u20133 years of tamoxifen; and extended treatment after 5 years of tamoxifen. Larger reductions in the hazard ratio for recurrence have been reported in the sequencing and extended treatment regimens, but a proper evaluation of the overall impact on recurrence needs to account for the higher recurrence rates that occurred in the period before switching to an AI.Here, we briefly review the design and findings of these trials and use the results to develop models, which can be used to explore the long-term (10 year) impact of different approaches. The two types of model used are: (i) a surface model, which uses the available data in the most straightforward way to predict outcomes; and a so-called \u2018deep\u2019 model which tries to explain the current data by an underlying mechanistic model. The goal of this later model is to try to explain and understand at a biologic level what has been observed. If correct, it may provide more accurate long-term predictions of recurrence rates. The model also identifies some key questions that should be amenable to study in the current trials.Several end points could be used in evaluating the different strategies. Recurrence is the most sensitive endpoint for evaluating efficacy, and includes distant recurrence, local recurrence and contralateral tumours. This is preferable to disease-free survival (DFS), which would also include intercurrent deaths without recurrence. These latter events rightfully belong in the safety or overall analysis, but are not relevant for efficacy, where they dilute real differences in efficacy with events unrelated to breast cancer. Using only distant recurrences would provide a better surrogate for death from breast cancer, but their use requires longer follow-up, and it is too early to use this end point at this stage.The use of recurrence rates gives equal weight to early and late recurrences. It is clear that an early recurrence is more detrimental than a late one, and integrating the recurrence curves to give the per cent of \u2018time lost to recurrence\u2019 is probably the best summary measure of efficacy in this context. Thus a recurrence at year 1 would lose 90% of the first 10 years to recurrence, but a recurrence at year 9 would only lose 10%. For a 10-year window, the actual number of years lost is just the percentage multiplied by 10, and this gives a useful measure for comparing strategies. We will evaluate both measures, but focus on this latter outcome measure below.One key question is how long the effect will be maintained after treatment is stopped. The EBCTCG overview has shown that the effect of tamoxifen on recurrence is maintained for about 5 years after stopping 5 years of treatment . The onlCarryover is a consequence of curing a proportion of patients, and not simply holding tumours in check. If treatment is curative, then all future recurrences are prevented, so the hazard ratio will remain below unity even after treatment has stopped. This is expected for chemotherapy, but the long-term results from the overview strongly suggest that tamoxifen is also curing a proportion of patients, and that its action is more than cytostatic. A reasonable assumption is that the early superiority of AIs over tamoxifen will result in a higher cure rate and that the reduction in recurrence rates will also be maintained for 5 years after the treatment ceases. This assumption is used in our base case model. However, given the importance of this parameter and the paucity of data, models with a 2-year carryover are also evaluated.\u22121 provides an excellent fit to these data . They found a hazard ratio of 0.64 for extended treatment  after 5 years of tamoxifen . AlthougFor these trials, the treatment period extends beyond 5 years, so a fairer comparison would be against initial AI or sequencing strategies of the same duration. However, by assuming a 5-year carryover effect, this will have little impact on the results for the first 10 years.vs 17.3%), leading to 1 less recurrence for every 18 women treated. The average time lost to recurrence is reduced by 3.1% or 3.7 months. The model predicts a similar recurrence rate at 10 years for the strategy of switching to an AI after 2 years compared as to initial use . Waiting 5 years before commencing an AI is not an effective strategy; although for women who have already received 5 years of tamoxifen, it is more effective than stopping treatment.The results predicted from this model are shown in tial use ; but, ben=880) and 0.83 for PgR+ patients (n=3834) (n=564) was again very large (HR=0.42)  . Data onvs 20.2 vs 34.0%) and total time lost to recurrence (8.5 vs 12.3 vs 18.4%), leading to gains of 4.6 and 11.9 months, respectively, when compared with sequencing after 2 years of tamoxifen or use of tamoxifen only.If one uses a hazard ratio of 0.43 for anastrozole for ER+/PgR\u2212 patients, as reported in both the ATAC and ABCSG8/ARNO95 studies (vs 13.2%) and time lost to recurrence , but is twice as high on tamoxifen (4%\u2009year\u22121), but unchanged for an AI (2%\u2009year\u22121), when the current phenotype is ER+/PgR\u2212. This leads to the results shown in In the ATAC trial, PgR-negative patients had a particularly poor outcome if treated with tamoxifen. This is in keeping with other studies in which PgR negativity is a poor prognostic variable in tamoxifen-treated patients (In particular, this model suggests that it is better to use an AI first since the effect of using tamoxifen is not to prime patients, but to shift a proportion of them to a phenotype which will progress more rapidly if left on tamoxifen.vs a sequencing strategy. The deep model nicely explains the findings of the ATAC and ABCSG8/ARNO95 trials (It must be remembered that these predictions are model-based and will require empirical confirmation. The difference in averaged hazard ratios between the upfront and switching strategies is not statistically significant when a heterogeneity test is employed. In addition to the uncertainties in the estimates of the individual hazard ratios, a more important source of uncertainty is the accuracy of the structural model for extrapolation beyond currently available follow-up times, and the lack of direct comparisons of an AI upfront 5 trials  results If PgR is predictive, then initial use of an AI is clearly indicated for PgR-negative tumours. For PgR-positive tumours, there is more uncertainty, but initial use of an AI, for say 3 years, may still be better than sequencing it after 2 years of tamoxifen, as this is the period with highest recurrence rates. It also avoids the side effects associated with tamoxifen. Trials comparing these two approaches would be highly desirable.Direct evidence for a phenotype shift, especially in tamoxifen-treated patients may be obtainable by comparing recurrent disease with the primary cancer or monitoring PgR status in peripheral blood as has been done for HER-2 (Recently Our assumptions on carryover do not have a major effect for the first 10 years of follow-up, except for the extended treatment strategies, which would appear more favourable if there was no carryover. Of course, the duration of treatment is longer here, and a fairer comparison would be against upfront or sequencing strategies with the same duration of treatment. If this were done, the model again predicts that an AI upfront strategy is optimal. Optimal duration of treatment with an AI is a key outstanding question for which there are currently no data. Hopefully this will be addressed in future trials.vs sequencing after tamoxifen. The BIG 1\u201398 trial is scheduled to provide such data in 2008 for letrozole and the TEAM study will have data for exemestane, but this is likely to mature even later.Currently, there are also no data for the key comparison of an AI upfront These gaps in our knowledge provide the rationale for the modelling we have done, but again we emphasize that models should be used as interim measures to guide current thinking and practice. They will be superseded by the actual trial data once it becomes available.Acceptability and side effects are also part of an overall evaluation. Overall, the AIs are better tolerated than tamoxifen, with fewer hot flushes, gynaecologic symptoms, and thromboembolic events, but more joint symptoms and fractures . For mos"}
+{"text": "Postmenopausal patients with hormone-sensitive early breast cancer are typically treated with adjuvant endocrine therapy, which significantly reduces the risk of recurrence. Because treatment is of a long duration, side effects from adjuvant therapy can be problematic. The aromatase inhibitors (AIS) are replacing tamoxifen as first-line treatment agents for early breast cancer. Here, we present the side-effect data associated with AIS in relation to bone, gynecologic, and cardiovascular health and to arthralgia and myalgia. Although AIS have been shown to decrease bone density, increase arthralgia, and affect vaginal health, these adverse events are usually manageable, and several strategies can be followed to improve quality of life in women on AI treatment. To optimize adherence to therapy. It is important that these issues are addressed so that women can benefit from treatment. The selective estrogen receptor (SERM) antagonist tamoxifen and, more recently, the aromatase inhibitors (AIS) anastrozole, exemestane, and letrozole have all been used as adjuvant endocrine therapy agents for the treatment of hormone receptor\u2013positive early breast cancer. Because the presence of circulating estrogen is associated with breast cancer recurrence Long-term treatment with endocrine agents raises issues both for patients and for physicians in balancing the potential benefits of reducing recurrence with the possible side effects. The SERMS and AIS reduce estrogen in different ways. The SERMS bind to the estrogen receptor on tumour cells, thus blocking the ability of the cells to proliferate; however, the SERMS also have estrogenic effects on certain tissues such as bone The effects of tamoxifen are well documented and include gynecologic symptoms, an increased risk of endometrial cancer, and an increased risk of deep-vein thrombosis For example, to prevent potential bias in event reporting in the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial, the largest and longest trial evaluating an AI, investigators were required to ask patients at every visit whether they had suffered adverse effects rather to use pre-specified checklists Typically, bone health may deteriorate in menopausal women, because a decline in estrogen concentration accelerates bone loss Not surprisingly, given the profound estrogen depletion effect of AIS, clinical trials have demonstrated an increased risk of fractures with AI treatment as compared with treatment with tamoxifen . In thosp = 0.0001) decrease in hip and lumbar spine BMD The ATAC trial included a bone subprotocol involving 81 and 86 women on anastrozole and tamoxifen monotherapy respectively, who either had normal BMD or osteopenia at baseline. At 5 years, no women with normal BMD on either treatment became osteoporotic, but anastrozole was associated with a statistically significant , 234 women on anastrozole concurrently treated with the bisphosphonate risedronate showed an increase in BMD and a decrease in bone turnover markers at 12 months as compared with results in women on anastrozole alone In the Zometa\u2013Femara Adjuvant Synergy Trial, concomitant administration of zoledronic acid in 522 women started on letrozole (\u201cupfront group\u201d) resulted in significantly higher lumbar spine and hip BMD at 24 months than was seen in the 538 women who were given the bisphosphonate only if their T-score fell to \u22122 or lower during therapy, or if spinal fracture was evident at 36 months (8.2% vs. 4.7% respectively) In premenopausal women, treatment with goserelin plus tamoxifen or anastrozole for 3 years was associated with significant bone loss, and bone was still compromised after 5 years, particularly in women who had been on anastrozole. However, 4 mg zoledronic acid given every 6 months for 3 years completely inhibited bone loss and led to improved BMD at 5 years Bone loss should be a consideration in women on AI treatment, and regular screening is recommended. The American Society of Clinical Oncology (ASOC) recommended in 2003 that all women at high risk of osteoporosis, including women on AIS, undergo baseline assessment of BMD and annual monitoring while on treatment for breast cancer Joint and musculoskeletal pain increase with age in women, reaching a peak during menopause and postmenopause, suggesting that symptoms may be related to estrogen depletion If pain occurs, it seems to manifest shortly after the initiation of treatment. For example, in the ATAC trial, arthralgia was most common in the first 6 months after the start of treatment A substudy of the ATAC trial revealed that risk factors for developing joint symptoms were prior use of hormone replacement therapy, hormone receptor positivity, obesity, prior chemotherapy, and treatment with anastrozole, leading to significant symptom increases of 12.3%, 8.2%, 6.8%, 5.9%, and 5.7% respectively An association between persistent musculoskeletal pain and vitamin D hypovitaminosis has been reported The prevalence of arthralgia in patients on AIS can be significant. In a small study of 56 patients who were not in clinical trials but who were receiving AIS, arthralgia or bone pain or both were reported in 61% and resulted in discontinuation of the drug in 20% Physical examination and patient history are required to rule out pain from osteoarthritis, rheumatoid arthritis, sleep disturbance, and so on.Articular pain should be distinguished from nonarticular pain, and inflammatory from non-inflammatory pain.The most common sites of arthralgia are the knees, hands, wrists, and shoulders.The most appropriate intervention for pain management may be a combination of lifestyle changes\u2014for example, exercise, and calcium and vitamin D supplements\u2014in conjunction with pharmacologic interventions.Drug therapy includes the use of acetaminophen, nonsteroidal anti-inflammatory drugs, opiates, glucosamine, and topical medications such as capsaicin or methylsalicylate.Patients with severe pain should be referred to a rheumatologist.Pain from bone metastases should be ruled out by history and physical examination or, where appropriate, imaging studies.A drug holiday of 3\u20134 weeks can also be helpful in confirming the cause of the pain. Anecdotally, rechallenge with the same AI is often well tolerated, but if symptoms significantly affect quality of life or impair activities of daily living, a switch to another endocrine agent can be considered.As compared with placebo, tamoxifen treatment is associated with an increased incidence of vaginal bleeding, endometrial polyps, endometrial thickening, and ovarian cysts. Prolonged use is associated with significant gynecologic complications, including a doubled to tripled risk of endometrial cancer As compared with women on tamoxifen, women on letrozole in the BIG 1-98 study also suffered significantly less vaginal bleeding and fewer hot flashes . HoweverAs compared with the 60-month analysis Estrogen levels in women on AI treatment are so low that their response to estrogen may be heightened, and the effect of this heightened response on breast cancer recurrence is unknown. In a prospective study, 7 postmenopausal women on AI treatment who were started on a vaginal estrogen tablet for severe symptoms of atrophic vaginitis showed significantly raised serum estradiol levels within 2 weeks Lifestyle modifications such as the use of layered clothing\u2014or even acupuncture, meditation, and biofeedback\u2014have been suggested for alleviating hot flashes in women on adjuvant hormone therapy et al.Management of sexual dysfunction in postmenopausal women on AI therapy has recently been reviewed by Derzko A sexual history, physical examination, and hormonal evaluation should be carried out.Therapeutic interventions should be tailored to address each area of distress.Non-hormonal treatments are the first-line recommendation for urogenital atrophy, vaginitis, and dyspareunia.Vaginal dryness may be treated with lubricants.Low-dose local vaginal estrogen therapy may be considered for highly symptomatic patients who are unresponsive to non-hormonal therapy, but this approach is controversial.Mortality in breast cancer survivors occurs not only because of cancer recurrence, but also because of cardiovascular (CV) disease (death from other causes has not been found to be significantly elevated) In the ATAC trial, no difference was observed in the occurrence of ischemic CV events between women on tamoxifen and those on anastrozole, and the most common event was angina p < 0.001, A similar situation has held for letrozole: conflicting data on lipid levels have been seen. In the BIG 1-98 trial, analysis at 51 months showed hypercholesterolemia (predominantly grades 1 and 2) in 51% of women on letrozole as compared with 25% of women on tamoxifen  lower incidence of thromboembolic events was observed with letrozole as compared with tamoxifen in the BIG 1-98 trial p = 0.02); however, this increase was considered to be statistically nonsignificant, because the cut-off for significance was p = 0.01 It is important to remember that lipid levels are surrogate endpoints and do not necessarily have an effect on CV disease. Despite the favourable effect of tamoxifen on lipid profiles, a meta-analysis of tamoxifen trials showed no effect on the incidence of myocardial infarction (MI), though death from MI was decreased in women likely to have hyperlipidemia and coronary artery disease p < 0.001) of thromboembolic events was observed in women on letrozole The SABRE study, which was designed to assess the efficacy of concurrent administration of risedronate with anastrozole on bone health, also monitored lipid levels. After 12 months, LDL was decreased, HDL was increased, and no change was observed in total cholesterol both in women on anastrozole alone and in those concurrently treated with the bisphosphonate No current evidence suggests that the AIS have a particular adverse effect on CV health. As noted in the clinical trials, differences in lipid levels, as compared with levels in women taking tamoxifen, may have more to do with the protective effect of tamoxifen on lipid levels than with an adverse effect of the AIS Many of the side effects associated with the AIS stem from their accelerated effect on estrogen depletion, but in general, these agents are well tolerated. So far, no current evidence shows that AIS might be associated with a serious adverse event such as the increased incidence of endometrial cancer noted with tamoxifen treatment. However, although the AIS have been shown to have a negative effect on bone density and arthralgia, these effects are manageable, and interventions can be introduced to alleviate symptoms and prevent unwanted sequelae. Possible side effects should be discussed with the patient, and patient well-being should be monitored for the duration of treatment. Further, patients with known risk factors such as osteopenia, joint pain, or cardiovascular symptoms should be identified at the start of treatment, and appropriate interventions should be applied such that AI treatment can be continued without worsening of symptoms. The goal is to maintain patients on treatment for the allotted length of time while ensuring a comfortable quality of life."}
+{"text": "Despite the perception of many oncologists that tamoxifen is an inferior drug, and should be substituted by an aromatase inhibitor in post-menopausal women, the current evidence strongly supports the view that AIs should be used 2\u20133 years after tamoxifen to achieve the maximal overall survival (OS) advantage. The last year has been an interesting time for oncologists interested in the adjuvant hormonal treatment of post-menopausal women with receptor-positive early breast cancer. Three important new pieces of clinical research were presented at the 2008 San Antonio Breast Cancer Symposium: a meta-analysis of the Aromatase Inhibitor (AI) trials , and extension trials, where 5 years of tamoxifen treatment is followed by 2 to 3 years of an aromatase inhibitor. Each of these AI trials has clearly shown a disease-free survival (DFS) advantage for the AI. These DFS outcomes were so convincing that by 2005 the ASCO Technology Assessment Panel had ruled that optimal adjuvant hormonal therapy should include an AI, \u2018but it remains unclear if initial treatment with an aromatase inhibitor is superior to a planned cross over from tamoxifen\u2019 . In the There is increasing concern that some of the most influential AI trials have been unable to demonstrate an overall survival (OS) advantage of AI\u2019s compared with tamoxifen particularly when used up front. For example Seruga and Tannock recently reviewed the evidence and concluded that \u2018there is no evidence for superiority of AIs over tamoxifen when used as initial treatment\u2019 . They weHere, we develop the case put by Seruga and Tannock in the light of fresh evidence, focusing particularly on the case for a switching strategy. Most of the trials included have been updated since the meta-analysis, doubling the median follow up time. We also think that it is legitimate to consider trials that used exemestane, as many oncologists believe that there is little or no clinical difference between steroidal and non-steroidal AIs. We will also consider the statistical pitfalls that confront investigators as they grapple with the problem of crossover. This has affected almost all of the AI trials and has confused their reporting.The first AI trial to report was the ATAC study, a substitution trial that compares 5 years of anastrozole to 5 years of tamoxifen . The ATATwo explanations have been offered to explain the lack of an OS benefit in the ATAC trial. First, longer follow up is needed. Second, about two-thirds of the patients enrolled into the ATAC study were node negative, and it has been suggested that their relatively good baseline prognosis makes it harder to demonstrate a significant absolute mortality difference. It is equally possible, however, that the complete substitution of tamoxifen by an AI is not the best way to use these new drugs. Perhaps there are some patients who need tamoxifen for their cure.For patients in their first consultation after surgery for breast cancer, there is one question, which comes above all others: How can I reduce my risk of death? If we look at all of the AI data with this single question in mind, we can see that mortality benefits are only seen in a subset of trials. We have called these the \u2018OS-positive trials\u2019 , censored or retrospective. ITT analysis ignores the cross over and keeps patients in their originally assigned treatment groups, regardless of whether they are actually taking tamoxifen or not. This is a statistically rigorous approach, but it may contribute to an underestimation of the benefits of AIs. The ITT method therefore may be less informative and less persuasive for changing clinical practice. Alternatively, censored data can be used. Using the Kaplan\u2013Meier method, patients are silently removed from the data set (censored) at the moment that they withdraw themselves from the control arm of the trial to take an AI. The third approach is the retrospective analysis in which patients are analysed by what drug they received rather than how they were initially assigned.Although censored or retrospective analysis seems to deal with the problem of cross over, these approaches become problematic if there is any association between a woman's desire to leave the control arm of a trial and her risk of relapse or death. In fact, such an association is quite likely. For example, one can easily imagine that a younger woman at higher risk might take more interest in breast cancer research, including the emerging results from the ATAC trial, and have a lower threshold for asking to come out of trial and cross over to the AI arm, whereas older women with lower risk disease and other comorbidites might be content to remain on the original assigned treatment.There is now strong evidence that exactly this kind of crossover bias does exist. When the MA17 trial reported a DFS advantage for letrozole  at 76 months. This trial also included two switching arms, but OS data has not yet been reported for this part of the trial, so this is not included in the table. These latest results from BIG 1\u201398 reinforce the pattern previously described: OS positivity is not seen when AIs are given up front. We predict that an overall survival difference will eventually be seen in this trial and this will favour a switching strategy.The larger of the two trials to report at San Antonio 2008 was the BIG 1\u201398 study (The Austrian Breast Cancer Study Group (ABCSG8) trial was also reported at San Antonio and is one of the most interesting of the adjuvant AI trials because it shows a clear OS benefit with shorter follow up than ATAC in a patient population that is similar. The crossover rate from the tamoxifen arm in this trial was less than 6%, so the use of a censored method may be legitimate and, the survival advantage remains significant even if data are subjected to an ITT analysis . ABCSG8 undermines the arguments that have been used to explain away the lack of a mortality benefit in ATAC. OS positivity was clearly seen at 72 months, which is 2 years earlier than the ATAC follow up. Furthermore, the node positivity rates of the patients in the ABCSG trial are similar to those of ATAC. The mortality benefits from ABCSG8 suggest that if we treat 60 patients for 6 years, one life will be saved. It is now the fourth OS-positive AI trial. All four of the OS-positive AI trials have been switching trials. Or in other words, mortality benefits are only seen in trials, which give AIs after an initial period of tamoxifen.In our view, oncologists should always remember the question that comes before all others. How can an individual patient reduce her risk of death? The available evidence for receptor-positive post-menopausal women strongly supports the use of an approach in which all patients are exposed to 2\u20133 years of tamoxifen and 2\u20133 years of an aromatase inhibitor. All OS-positive trials use such an approach and this is the only treatment strategy for which a mortality benefit could be seen in the AI meta-analysis. Such an approach also limits the patient's exposure to either class of drug. As these two classes of drug have different safety profiles  this additional limited exposure to either agent is likely to mitigate the risk of serious complications.Recent trial data support the view that AIs should be used after 2\u20133 years of tamoxifen to achieve an OS advantage and add weight to scepticism surrounding up front use of these drugs , a strat"}
+{"text": "The third-generation aromatase inhibitors (AIs) anastrozole, exemestane and letrozole have largely replaced tamoxifen as the preferred treatment for hormone receptor \u2013 positive breast cancer in postmenopausal women. Approximately 185,000 new cases of invasive breast cancer are diagnosed yearly, and at least half of these women are both postmenopausal and eligible for adjuvant therapy with AIs. In addition, AIs are currently being tested as primary prevention therapy in large randomised trials involving tens of thousands of women at increased risk for breast cancer. Given the volume of use, internists will increasingly see postmenopausal women who are taking or considering treatment with AIs. Physicians need to be able to: (i) briefly discuss the pros and cons of using a selective estrogen receptor modulator such as tamoxifen or raloxifene vs. an AI for risk reduction and (ii) recognise and manage AI-associated adverse events. The primary purpose of this review is to help internists with these two tasks.Expert opinion based on review of literature on relevant clinical trials.Both tamoxifen and AIs are effective for the adjuvant and neoadjuvant treatment of postmenopausal breast cancer; the optimal choice of drug is dependent on the characteristics of the patient and tumour. Adverse events with both drug classes are manageable. Adverse events associated with tamoxifen include increased risk of uterine cancers and thromboembolic events vs. an increased incidence of vaginal dryness, loss of libido, musculoskeletal pain and bone mineral density loss with AIs. Promising studies of AIs in the breast cancer prevention setting are ongoing. Estrogen promotes the growth and survival of normal and cancerous breast epithelial cells by binding and activating the estrogen receptor (ER). The activated receptor in turn binds to gene promoters in the nucleus and activates many other genes responsible for cell division, inhibition of cell death, new blood vessel formation and protease activity. An increase in the proportion of cells that express ER is found at both the earliest stages of breast precancer and in approximately 70% of breast cancers . There aAromatase inhibitors are not without adverse effects, which primarily stem from profound estrogen depletion. Many women will turn to their internists for advice about whether to take these drugs, as well as help in preventing and managing adverse events. The purpose of this article is to provide primary care physicians with a basic understanding of AIs to help facilitate these interactions.Aromatase inhibitors and inactivators interfere with the body's ability to produce estrogen from androgens by suppressing aromatase enzyme activity. Before menopause, ovarian aromatase is responsible for the majority of circulating estrogen and is exquisitely sensitive to changes in luteinising hormone (LH). Following menopause, aromatase in fat and muscle may be responsible for much of the circulating estrogen. Aromatase in highly estrogen-sensitive tissues, such as the breast, uterus, vagina, bone, brain, heart and blood vessels, provides local estrogen in an autocrine fashion . The aroThree generations of AIs have been developed  3\u20138). E\u20138. E3\u20138)For women with newly diagnosed hormone receptor positive ER+ cancers requiring systemic adjuvant therapy, 5 years of tamoxifen reduces the relative odds of recurrence by 40% and relative risk of death from breast cancer by 34% . At 15 yThere is some evidence suggesting a worse outcome with tamoxifen for women with ER-positive tumours that lack progesterone receptor (PgR), and/or exhibit overexpression of growth factor receptors such as human epidermal growth factor receptors 1and 2 (EGFR and HER-2/neu) ,12. The Treatment with AIs produce frequent and durable responses in postmenopausal women previously treated with tamoxifen or endocrine ablative surgery, and AIs are more effective than tamoxifen in producing responses and delaying progression in first-line treatment of metastatic disease . A recenThe third-generation AIs are currently the preferred first-line treatment for metastatic hormone receptor-positive tumours and have all been approved by the US Food and Drug Administration for adjuvant use in postmenopausal women before or after surgery for ER-positive and/or PgR-positive breast cancer . AlthougSystemic treatment administered before definitive surgery is termed neoadjuvant therapy and is often used in women who have clinically involved nodes or a tumour that is \u2265 3 cm. Under these circumstances the chance of occult metastatic disease is high, and the chance of breast conservation with a cosmetically acceptable outcome is low. Neoadjuvant treatment both increases the chance of breast conservation and promotes timely treatment of occult metastases. Pathological response to neoadjuvant chemotherapy is an important prognostic factor. Women with a pathological complete response in breast and lymph nodes to neoadjuvant chemotherapy have as much as a 95%, 5-year distant, disease-free survival (DFS) . AlthougNeoadjuvant trials with antihormone therapy have generally shown that the chance of breast conservation is higher with AIs than tamoxifen and may be higher for AIs than for chemotherapy in women with hormone receptor positive tumours \u201322. In aClinical trials of AIs as adjuvant therapy have followed one of four approaches: (i) a head-to-head comparison of tamoxifen vs. an AI; (ii) extended adjuvant therapy following initial adjuvant therapy (5 years of an AI after 5 years of tamoxifen); (iii) switching to an AI for 2\u20133 years after 2\u20133 years of tamoxifen and (iv) combination therapy using both an AI and tamoxifen simultaneously. All AI approaches except the simultaneous combination of an AI and tamoxifen are associated with fewer breast cancer-related events than tamoxifen alone.The Anastrozole, Tamoxifen Alone or in Combination (ATAC) trial randomised more than 9000 women to 5 years of tamoxifen, anastrozole or both agents in combination. The combination treatment did not show a benefit and is not discussed further. Sixty-one per cent of women had no disease detected in their lymph nodes (referred to as node negative) at diagnosis. After 5 years of treatment, there was a significant improvement in DFS in the group of women treated with anastrozole alone regardless of tumour size, nodal status or use of adjuvant chemotherapy before the randomisation. There was a significant interaction with hormone receptor status: women who had ER-positive but PgR-negative tumours were likely to have a superior outcome with anastrozole, whereas women with tumours that were positive for both receptors did just as well with tamoxifen as with anastrozole. The absolute improvement in DFS with 5 years of anastrozole, compared with 5 years of tamoxifen, was 2.5% (p = 0.005). The incidence of contralateral breast cancer was reduced by 53% in women with hormone receptor-positive tumours. No overall survival benefit or significant reduction in deaths from breast cancer was demonstrated for anastrozole in this study. However, there appears to be an emerging survival benefit for women with ER-positive tumours who also had evidence of tumour cells in their draining lymph nodes (referred to as node positive) ,25.In the Breast International Group's Femara-Tamoxifen trial, also known as BIG 1\u201398, 5 years of adjuvant letrozole was compared with 5 years of tamoxifen in postmenopausal women with ER-positive and/or PgR-positive breast cancer. Eventually, this trial was modified with the addition of two treatment groups in which women were either switched from tamoxifen to letrozole or from letrozole to tamoxifen after the initial 2 years of treatment . Approxin = 9786). We are awaiting the efficacy results of this trial.The ongoing Tamoxifen Exemestane Adjuvant Multi-institutional (TEAM) trial compares exemestane with tamoxifen as first-line adjuvant treatment. The TEAM trial is designed to compare DFS in patients treated with exemestane vs. tamoxifen at 2.75 years, and to compare DFS in patients treated with 5 years of up-front exemestane vs. tamoxifen for 2.5\u20133 years followed by 2\u20132.5 years of exemestane. Enrolment was completed in January 2006  would improve DFS compared with placebo. At a median follow-up of 2.4 years from the time of randomisation, letrozole improved DFS, compared with placebo, by a relative value of 43% and an absolute value of 6%. This was significant regardless of nodal status . The triThe switching strategy was designed to: (i) combine the apparent superior efficacy of AIs with tamoxifen's favourable effects on bone and (ii) expose tumour cells to anti-hormonal therapies with two different mechanisms of action. Several adjuvant trials were designed in which, after 2\u20133 years of adjuvant tamoxifen, women were randomised to continue taking tamoxifen for another 2\u20133 years or switch to an AI. One such trial, the Intergroup Exemestane Study (IES), randomised 4742 postmenopausal women after 2\u20133 years of tamoxifen to exemestane 25 mg/day or to continued tamoxifen of sufficient duration to complete a 5-year course of adjuvant therapy . Fifty-oIn other switching trials, such as the Italian Tamoxifen Arimidex (ITA) trial and the Austrian Breast and Colorectal Study Group 8 (ABCSG 8)/Arimidex-Nolvadex (ARNO 95) combined analysis, switching to anastrozole after 2 years of tamoxifen was compared with continued tamoxifen treatment. A 39% relative improvement in DFS (p = 0.049) and 52% improvement in overall survival were seen at a median follow-up of 30 months in the ABCSG 8/ARNO 95. Improvement in DFS was observed for ITA ,35.In summary, all the adjuvant trials in postmenopausal women \u2013 whether they involved initial head-to-head comparison with tamoxifen , switching to an AI after 2\u20133 years of tamoxifen , or administering 5 years of an AI after 5 years of tamoxifen \u2013 show improvement in DFS favouring the AI. An overall survival benefit is emerging in at least two of the switching trials in women randomised to 2\u20133 years of an AI following 2\u20133 years of tamoxifen vs. continuing on tamoxifen ,35. No sThere is no clear advantage to one AI vs. another at the present time. Oncologists often select an AI depending on the type of adjuvant strategy they wish to employ. Several head-to-head trials comparing one AI to another in the adjuvant setting are ongoing. These include trials of anastrozole vs. exemestane and anastrozole vs. letrozole.Responses have been observed in premenopausal women with concomitant goserelin and AI treatment following tamoxifen failure ,37. ThisThe adverse event profile for AIs differs from that of tamoxifen. There is no increase in uterine cancers or thromboembolic events as is observed with tamoxifen, but with the exception of hot flushes. Women taking AIs are more likely to complain of symptoms related to estrogen deprivation. Women taking AIs are also more likely to report musculoskeletal adverse events than women taking tamoxifen. These are considered in detail below.Use of AIs is associated with a higher frequency of vaginal dryness, loss of libido and painful intercourse than is tamoxifen. There are fewer instances of vaginal bleeding and endometrial cancer with AIs than with tamoxifen ,33,38. AStudies of tamoxifen in postmenopausal women have shown reduction in bone turnover markers and an increase in bone density and the opposite effects with AIs \u201344. ThesIn adjuvant studies, all three third-generation AIs \u2013 anastrozole, letrozole and exemestane \u2013 have shown an increased risk of bone fracture compared with tamoxifen. The absolute differences, while statistically significant in the ATAC trial of anastrozole vs. tamoxifen and the BIG 1\u201398 trial of letrozole vs. tamoxifen, were only 1\u20134%. Most fractures were in the spine and not the hip ,48. The Bisphosphonates can be used to prevent the bone mineral loss observed with AIs. This strategy was successfully used in the Zometa-Femara Adjuvant Synergy trials, and the Austrian Breast and Colorectal Cancer Study Group trial 12, in which an intravenous bisphosphonate, zoledronic acid, was administered every 6 months for the duration of AI therapy ,50. VitaIn randomised studies, arthralgias/myalgias have been reported significantly more frequently in women randomised to AIs than in those randomised to tamoxifen or placebo. The absolute frequency varies tremendously from trial to trial (5.4\u201337% for AIs vs. 3.6\u201326% for tamoxifen or placebo), which in turn probably reflects the method used to record the symptoms. The incidence of arthralgias and myalgias appear to be about two-thirds higher with an AI than with tamoxifen or placebo but usually improves with time . Two smaAromatase inhibitors do not increase the risk of deep venous thrombosis; this differs from tamoxifen, for which the risk of deep venous thrombosis and pulmonary embolism is increased approximately twofold ,58. FurtAromatase inhibitors in adjuvant trials have been associated with an increase in ischaemic cardiovascular events and a numeric, but not statistically significant increase in cardiac deaths when compared with tamoxifen ,30,31, bBecause the proportional differences in cardiac deaths observed in women randomised to AI vs. tamoxifen are < 1%, a potential increase in cardiovascular events is not likely to be a major concern for women undergoing cancer therapy with an AI. However, enthusiasm for AI use in the primary prevention setting will be limited if AIs are found to be associated with a higher number of cardiac events compared with placebo or tamoxifen.As AI use becomes more common, internists will undoubtedly be asked by their patients for help with management and prevention of adverse events, although the relative risks and benefits of AIs vs. other hormonal therapy will hopefully have been discussed by the patient's oncologist.For vasomotor symptoms, non-hormonal methods such as selective serotonin reuptake inhibitors (SSRIs), gabapentin or clonidine should be tried first . In doseVaginal dryness that is not ameliorated with lubricants may be treated with poorly absorbed vaginal estrogens, such as oestradiol vaginal rings or tablets. However, a small study showed a significant increase in serum estrogen levels following use of these preparations . A weak Tamoxifen fails to prevent ER-negative breast cancer, and one-third or more of ER-positive breast cancers \u201370. The Of serious concern for prevention is the potential for increase in risk of bone fracture and cardiovascular disease related to long-term estrogen depletion with AIs. However, arthralgias, fatigue, dyspareunia, reduced libido and hot flushes may result in poor uptake and/or compliance. Ongoing phase III prevention trials will define the incidence of these adverse events relative to placebo in a healthy population, and potential solutions to avoid some of these problems in the prevention setting are already being explored.One small study indicates that bone mineral loss after AIs is primarily limited to women with insufficient 25-OH vitamin D levels . Given tStatins could be used along with AIs to improve both lipid profiles and endothelial function. There is also a suggestion that long-term use of a lipophilic statin might reduce breast cancer risk , but resApproximately one-quarter of perimenopausal and postmenopausal women take hormone replacement therapy for some period of time during menopause or menopause transition . AlthougPreclinical studies indicate that AIs might be effective in reducing the risk of breast cancer in hormonally intact animals under circumstances in which breast aromatase is up-regulated . In studEven with an initial response to treatment, for women with metastatic disease, resistance eventually develops to AIs and clinical regrowth of tumour is observed. In most cases, the resistant cancer continues to be ER positive. There are several mechanisms of resistance demonstrated in animal models. These include: (i) development of hypersensitivity of the ER to very low levels of estrogen; (ii) up-regulation of growth factor receptors and/or associated signalling pathways ) ,93. ReduThe third-generation AIs are now preferred therapy for postmenopausal women with hormone receptor-positive tumours in both the early and metastatic settings. Switching from adjuvant tamoxifen to an AI (exemestane or anastrozole) after 2\u20133 years of tamoxifen has shown superior DFS and overall survival compared with continuing on tamoxifen. Using anastrozole or letrozole instead of adjuvant tamoxifen as initial therapy (with or without prior adjuvant chemotherapy) has also shown superior DFS. Finally, for women completing 5 years of tamoxifen, extended adjuvant antihormonal therapy with letrozole has shown a reduced recurrence rate, particularly for node-positive patients. American Society of Clinical Oncology guidelines recommend that an AI be included in a woman's adjuvant regimen if she has ER-positive and/or PgR-positive breast cancer. The decision to use AI as initial endocrine therapy, as opposed to switching to an AI after 2\u20133 years of tamoxifen therapy, is likely to be guided by the tumour characteristics. Patients who have ER-positive tumours with unfavourable characteristics, such as HER-2 positivity, PgR negativity or nodal positivity, are likely to be selected for immediate AI therapy. However, patients with ER-positive tumours without unfavourable characteristics are likely to be selected for tamoxifen treatment for 2\u20133 years before taking an AI for 2\u20133 years. Several ongoing clinical trials are examining the use of AIs in women at an elevated risk of developing breast cancer. Critical to the ultimate success of AIs in both the adjuvant and preventive settings will be management of adverse events, particularly bone mineral density loss, arthralgias and gynaecological sequelae."}
+{"text": "It was aimed to review the literature and make a meta-analysis of the trials on both upfront, switching, and sequencing anastrozole in the adjuvant treatment of early breast cancer.The PubMed, ClinicalTrials.gov and Cochrane databases were systematically reviewed for randomized-controlled trials comparing anastrozole with tamoxifen in the adjuvant treatment of early breast cancer.P < 0.0001) for patients treated with anastrozole compared with tamoxifen. In the second analysis in which only ITA, ABCSG 8, and ARNO 95 trials were included and ATAC  was excluded, combined hazard rate for EFS was 0.64 (95%CI: 0.52\u20130.79) (P < 0.0001). In the third analysis including hazard rate for recurrence-free survival (excluding non-disease related deaths) of estrogen receptor-positive patients for ATAC trial and hazard rate for EFS of all patients for the rest of the trials, combined hazard rate was 0.73 (95%CI: 0.65\u20130.81) (P < 0.0001).The combined hazard rate of 4 trials for event-free survival (EFS) was 0.77 (95%CI: 0.70\u20130.85) (Anastrozole appears to have superior efficacy than tamoxifen in the adjuvant hormonal treatment of early breast cancer. Until further clinical evidence comes up, aromatase inhibitors should be the initial hormonal therapy in postmenopausal early breast cancer patients and switching should only be considered for patients who are currently receiving tamoxifen. The standard treatment of early breast cancer is surgery, with or without radiotherapy and chemotherapy, followed by hormonal therapy for women with hormone receptor-positive tumors. Although 5 years of tamoxifen has been the mainstay of adjuvant hormonal therapy for more than 20 years , tamoxifAlthough there are several studies on the advantages of anastrozole over tamoxifen as adjuvant hormonal therapy of hormone-sensitive early breast cancer, the timing and duration of anastrozole treatment remains in question. The Arimidex, Tamoxifen Alone or in Combination (ATAC) trial used an \"upfront approach\" where anastrozole was started as the first-line hormonal therapy ,5,9, wheTo address the overall impact of all three approaches, I sytematically reviewed the literature and did a meta-analysis of randomized trials on upfront, switching, and sequencing schedules of anastrozole adjuvant treatment in early breast cancer.Four clinical trials were included in this meta-analysis: ATAC , ItalianSystematic literature searches of PubMed, ClinicalTrials.gov and Cochrane databases were performed by using the keywords anastrozole (or Arimidex) and breast cancer. There was no date restriction, but the research was restricted to trials published in the English language. I reviewed the titles and abstracts of all the articles retrieved by this search. I considered randomized controlled trials to be eligible if they compared anastrozole with another agent in the adjuvant treatment of early breast cancer, with no restriction of study phase and regardless of doses or schedules. I didn't include dose escalation studies or single arm studies. When there were multiple records related to the same study, I extracted end point data from the report with the longest follow-up.There were 3 recent meta-analyses, 2 on advanced disease ,15 and 1The conduct and reporting of meta-analysis was performed in accordance with the Quality of Reporting of Meta-analyses (QUOROM) statement .I extracted the following data from each eligible trial: authors' names, journal, date of publication, number of patients randomized to each arm (ITT population) and analyzed per arm, median age of patients, menopausal and hormone receptor status, and treatment schedule and doses. I also recorded if there was a proper mode of randomization and allocation method described in the publication, median follow-up, median survival, drop-out rates and adverse events per arm, and definitions of EFS, DFS, and recurrence free survival (RFS). Hazard rates (HR) and confidence intervals (CI) for these end points were also extracted from the publications.Quality of trials were assessed based on the reporting of method of randomization and allocation concealment, comparability of baseline treatment groups, reasons for drop-outs and if there was any evidence of differential drop-outs between treatment arms.P > 0.10). For our primary analysis EFS rates from ABCSG 8, ARNO 95 and ITA trials were combined but RFS rates were used from ATAC trial because EFS rates weren't reported and RFS was the most similar end point to other trials in terms of exclusion of recurrence-free deaths. A group of sensitivity analyses were made by excluding ATAC trial from our analyses and also by analysing only hormone receptor-positive patient groups.HR from Cox proportional models reported in the original publications comparing anastrozole and comparator treatment was combined. The natural logarithms of the HR were combined using general variance models, and utilizing inverse variance weights. BetweenPublication bias could not be assessed due to small number of studies included in the analysis. All analyses were performed using STATA version 9 .I identifed 635 potential studies on anastrozole treatment in breast cancer during initial literature search Figure. . Each stDemographic and baseline cancer characteristics of patients and treatment regimens were similar across ITA, ABCSG 8, and ARNO 95 trials. In all these 3 trials, nearly all of the patients were estrogen receptor-positive and had received 2\u20133 years of tamoxifen before randomization to study groups. For both ITA and ARNO 95 trials, patients who had completed 2\u20133 years of adjuvant tamoxifen treatment and were relapse-free were randomized to either continue tamoxifen (20 or 30 mg/day) or switch to anastrozole 1 mg/day) for the remainder of their treatment . For ABCSG 8 trial, anastrozole vs. tamoxifen was prospectively sequenced after 2 years of tamoxifen treatment for newly diagnosed patients. One year after switching or sequencing, patients were clinically evaluated every 3 months in the ABCSG 8 and ITA trials; and then every 6 months or year for all 3 trials. All three studies assessed local and distant recurrence, contralateral breast cancers, and all deaths (deaths following recurrence and deaths without recurrence). The patient characteristics and treatment regimen were different for ATAC trial than the other 3 trials. ATAC trial compared 5 years of anastrozole with tamoxifen alone, or combination of the two drugs, as adjuvant therapy in postmenopausal women with localized breast cancer after surgery and chemotherapy (if applicable). Patients did not receive any hormonal treatment before randomization. Hormone receptor status for the 14% of study population was unknown at the time of randomization. It has the longest median follow-up time and highest sample size of all 4 trials    and combined HR was found to be 0.73 (95%CI: 0.65\u20130.81), P < 0.0001)  when all 4 trials were included in the analysis, 0.64 (0.52\u20130.79) when ATAC the only upfront trial was excluded, and 0.73 (0.65\u20130.81) when HR for RFS of estrogen receptor-positive patients in ATAC trial was included in the analysis.In the trials comparing anastrozole and tamoxifen, anastrozole was either used as initial therapy (upfront approach) ,5,9 or aIt is also very important to interpret the results of switching and sequencing trials properly since the same problem of \"attrition of susceptibles\" appears in this comparison, too. Although study designs look similar, there is a remarkable difference between the two approaches. In the switching approach, adjuvant therapy is switched to anastrozole in patients already under tamoxifen treatment for several years -8. But iP = 0.0009), despite the higher HR observed in ARNO 95 (HR = 0.66 [0.44\u20130.99]) [P = 0.068]) ) .Since different treatment strategies of anastrozole have not been compared in a single clinical study, statistical modeling methods were used to define the best strategy -30. Two P < 0.0001) over those who remained on tamoxifen ; amoxifen . Due to 2\u20130.71; aThis meta-analysis is important in terms of combining the results of upfront, switching, and sequencing approaches for anastrozole adjuvant treatment in early breast cancer. Our results show that anastrozole treatment improves EFS significantly, when compared to tamoxifen irrespective of the treatment approach used.The main limitation of our meta-analysis is the low number of trials included in the analysis. To make a definitive conclusion on the optimal strategy of anastrozole adjuvant treatment in early breast cancer, trials comparing different treatment approaches \u2013 upfront, switching, sequencing, and extended treatment \u2013 would be highly desirable.As a conclusion, anastrozole appears to have superior efficacy than tamoxifen in the adjuvant hormonal treatment of early breast cancer. The question of giving anastrozole upfront or after several years of tamoxifen treatment remains open, as it is not possible to make any direct comparisons. However, it appears unlikely that giving tamoxifen, an agent that has inferior activity both in the adjuvant and metastatic setting, as first line treatment would be more effective than starting with anastrozole. This concept is also supported by several mathematical models. It is thought that, until further clinical evidence comes up, aromatase inhibitors should be the initial hormonal therapy in postmenopausal early breast cancer patients. Switching should only be considered for patients who are currently receiving tamoxifen. With the current trends, this patient population should be expected to decrease in size during the years to come, with the rare exception of premenopausal patients who become postmenopausal during tamoxifen treatment. The introduction of tamoxifen or another estrogen antagonist after the completion of aromatase inhibitor therapy may be an interesting research topic.ABCSG: Austrian Breast and Colorectal Cancer Study Group; ARNO: Arimidex-Nolvadex; ATAC: Arimidex, Tamoxifen Alone or in Combination; CI: Confidence interval; DFS: Disease-free survival; EFS: Event-free survival; HR: Hazard rates; ITA: Italian Tamoxifen Anastrozole; ITT: Intent-to-treat; QUOROM: Quality of Reporting of Meta-analyses ; RFS: Recurrence free survival.The authors declare that they have no competing interests.AA conceived of the study, and participated in its design and coordination of the statistical analysis. FT participated in the coordination and improvement of the study. All authors read and approved the final manuscript."}
+{"text": "Over the last few decades, extensive efforts have been made worldwide to develop nanomedicine delivery systems, especially via oral route for antidiabetic drugs. Absorption of insulin is hindered by epithelial cells of gastrointestinal tract, acidic gastric pH and digestive enzymes.Recent reports have identified and explained the beneficial role of several structural molecules like mucoadhesive polymers  and other copolymers for the efficient transport and release of insulin to its receptors.Insulin nanomedicines based on alginate-dextran sulfate core with a chitosan-polyethylene glycol-albumin shell reduced glycaemia in a dose dependent manner. Orally available exendin-4 formulations exerted their effects in a time dependent manner. Insulin nanoparticles formed by using alginate and dextran sulfate nucleating around calcium and binding to poloxamer, stabilized by chitosan, and subsequently coated with albumin showed a threefold increase of the hypoglycemic effect in comparison to free insulin in animal models. Solid lipid nanoparticles showed an enhancement of the bioavailability of repaglinide (RG) within optimized solid lipid nanoparticle formulations when compared with RG alone.Nanoparticles represent multiparticulate delivery systems designed to obtain prolonged or controlled drug delivery and to improve bioavailability as well as stability. Nanoparticles can also offer advantages like limiting fluctuations within therapeutic range, reducing side effects, protecting drugs from degradation, decreasing dosing frequency, and improving patient compliance and convenience Type I diabetes is a fast growing epidemic which in 2011 was estimated to affect over 350 million people worldwide, and its prevalence is expected to increase to approximately 550 million by 2030 . Thus, tNanomedicines have numerous advantages for the oral drug delivery route. Several disease related drugs are successfully encapsulated in nanomedicines in order to improve bioavailability, bioactivity and control delivery. Especially, these particles are of small size (within the micro or nano range) and capable of encapsulating peptide drugs such as insulin . Also, nCurrently, multiple daily subcutaneous injections of insulin are the standard treatment for insulin-dependent diabetic patients. Nevertheless, clinical studies showed that a significant percentage of patients failed to attain lasting glycemic control on insulin treatment , 10. WelLassman-Vague and Raccah  reviewedOral formulations have some potential advantages and face several common problems, particularly for peptides and proteins: poor stability in the gastric fluid, low solubility/bioavailability and the mucus barrier can prevent drug penetration and absorption. Nanoparticle formulations are being developed to encapsulate and protect drugs and release them in a controlled manner to overcome these limitations , 18. NanThe absorption mechanisms of orally delivered drug loaded nanoparticles have attracted less attention than their design. The design of new nanoparticles for oral administration usually focuses on overcoming the different barriers in the gastrointestinal tract. The nanoparticles must resist the harsh gastrointestinal environment, e.g the low pH in the stomach and the degradative enzymes. However, the major barrier to their absorption remains the intestinal mucosa . In ordeThe concept of nanomedicine named in 2004 by the European Science Foundation, represents a new area in the field of drug delivery research concerning drug delivery vehicles . RecentlA number of reviews exist on nanomedicine based oral insulin delivery systems , 24. TheReis et al.  developeIn one study, insulin nanoparticles were formed by using alginate and dextran sulfate nucleating around calcium and binding to poloxamer, stabilized by chitosan, and subsequently coated with albumin and evaluated in streptozotocin-induced Wistar diabetic rats. Pharmacodynamic and pharmacokinetic parameters were evaluated at a dose of 50 IU/kg nanoencapsulated insulin, and the 13% oral bioavailability represented a threefold increase in comparison to free insulin. Therefore, the nanoparticles facilitated the oral delivery of insulin, and potentially that of other therapeutic proteins . Rawat e-3, i.e. a log Pmembr of -3. For comparison, the log octanol/water partition coefficient (log Poct) for molecules assumed to be transported by the paracellular route (e.g. mannitol) is also in the order of -3. Then it is assumed that the surface area of the luminal cell membrane of the intestinal epithelium is 1000-fold larger than that of the paracellular space. The larger surface area of the cell membrane will compensate for the difference in partitioning between the cell membrane and the extracellular fluid. As a result, it could be thought that the hydrophilic drug is transported in equal amounts by the paracellular and transcellular routes. However, in reality, the tight junctions which gate the entrance to the paracellular pathway restrict the paracellular transport of drugs even further  of the paracellular pathway , 39. InsInsulin receptors have been identified in the basolateral membranes of dog intestinal mucosa, in the mouse intestinal cells and in the membrane of Caco-2 cells . KendzieThis review mainly focused on the nanomedicines and their transport mechanism of antidiabetic drugs. Nanoparticles are multiparticulate delivery systems designed to obtain prolonged or controlled drug delivery and to improve bioavailability as well as stability. Nanoparticles can also offer advantages like limiting fluctuations within therapeutic range, reducing side effects, protecting drugs from degradation, decreasing dosing frequency, and improving patient compliance and convenience . From th"}
+{"text": "Color is one of the most powerful aspects of a psychological counseling environment. Little scientific research has been conducted on color design and much of the existing literature is based on observational studies. Using design of experiments and response surface methodology, this paper proposes an optimal color design approach for transforming patients\u2019 perception into color elements. Six indices, pleasant-unpleasant, interesting-uninteresting, exciting-boring, relaxing-distressing, safe-fearful, and active-inactive, were used to assess patients\u2019 impression. A total of 75 patients participated, including 42 for Experiment 1 and 33 for Experiment 2. 27 representative color samples were designed in Experiment 1, and the color sample  was the most preferred one. In Experiment 2, this color sample was set as the \u2018central point\u2019, and three color attributes were optimized to maximize the patients\u2019 satisfaction. The experimental results show that the proposed method can get the optimal solution for color design of a counseling room. Color, one of the most powerful aspects of the environment, has been reported to promote human adaptation to the environment and enhance spatial form The visual environment is a vital element influencing hospital staff morale and productivity; studies have even reported that an enhanced visual environment have produced improved faster recovery rates by as much as 10%. In fact, these improvements have been attributed to particular elements of the visual environment; they include the use of appropriate color in interior design. In hospital design, color can have an impact on peoples\u2019 perceptions and responses to the environment and also affect patient recovery rates, improving the quality and overall experience of patients, staff and visitors. Color is also powerful tools for coding, navigation and wayfinding, color can also promote a sense of well-being and independence.There are numerous studies in the field of psychology which demonstrate the relationship between human behavior and color. However, color studies in the environmental design field are almost non-existent. Nourse and Welch Scientific research means reviewing a concept through observation in order to test a theory or hypothesis in order to explain a phenomenon. The presupposed theory and hypotheses are tested by systematic observations to make a general explanation for the natural phenomena. Experiments are useful to explore the phenomena because they involve testing hypotheses and investigating cause-and-effect relationships Based on previous studies, the consistent trend is that blue and green are the most preferred colors. However, the majority of color preference studies failed to control the confounding variables such as color attributes All studies were approved by Institutional Review Board (IRB) at the Zhongshan Hospital, and all participants provided written informed consent.Sherman, Shepley, and Varni Drawing on the model presented by Sherman, Shepley, and Varni A total of 75 patients with normal or corrected-to-normal vision participated, including 42 for Experiment 1 and 33 for Experiment 2.The color samples were rendered using the software Photoshop on a Core i3 computer (HP ProBook 6450b) equipped with an AMD Radeon HD 6370 graphics adaptor. It was displayed on a large rear projection screen . The projection allowed for stereoscopic viewing by use of two projectors with a resolution of 1,920\u00d71,080 pixels each and a color depth of 32 bits. The refresh rate of the display was 60 Hz for each eye. The participant was seated at a distance of 2.0 m from the projection screen.Design Expert software was utilized to conduct the experimental design and analysis. The software is performed on a Pentium 4 PC with a 2.4 GHz CPU and 512 MB RAM.The color system of Commission International de l\u2019Eclairage (CIE) is chosen because its color space is more symmetrical than other color systems, such as Munsell color system and Ostwald color system In 1976, CIE recommended two standard color difference formulae: CIELAB and CIELUV for the colorant and lighting industries, respectively. Each of these two formulae also provides a uniform color space for representing colors in a perceptually even fashion.The CIELAB space has been up to now the most widely used for presenting colors. We employed the Affective Appraisal Scale With 3 attributes of color, 3 values of Lightness , 3 values of a , and 3 values of b  are selected to generate 27 (\u200a=\u200a3\u00d73\u00d73) representative color samples (as shown in The next step to improve color parameters was to apply a response surface method (RSM) using a central composite design (CCD), which were referred to NIST/SEMATECH. The work which initially generated interest in the package of techniques was a paper by Box and Wilson The design ranges for L, a, and b are , , and  respectively. Design Expert was utilized to conduct the experimental design and analysis. The design, consisting of 15 experimental runs , is summarized in RSM is a collection of experimental strategies, mathematical methods, and statistical inference that enables an experimenter to make efficient empirical exploration of the system of interest Because the functional relationship between the response and inputs is unknown and perhaps complicated, the first step in RSM is usually to start with the first-order designs.In regions of higher curvature, especially near the optimum, second-order models are commonly used:To determine the parameters that optimize the multiple responses, the desirability function approach is utilized. The desirability function approach proposed by Derringer and Suich In Experiment 1, results are listed in In Experiment 2, analyses of variance for response variables and significant factor estimations using least squares analysis were generated by the Design Expert program. Beside the numerical analysis, Design Expert also generated a graphical presentation to investigate the fitted model. For example, the contour plot for 6 responses is shown in Six responses are to be minimized. The contour plot of total desirability is shown in An important impetus for the growing international awareness of healthcare facility design has been mounting scientific evidence that certain environmental design strategies can promote improved outcomes.Rubin and her colleagues The theoretical foundation of this research was based on the Conceptual Model of the Impact of Physical Environments on Patients\u2019 Outcome proposed by Sherman, Shepley, and Varni Experiments showed that blue and green are the preferred colors, and our results are also confirmed by other research. Hemphill There are some possible explanations for the results about the patients\u2019 preference for partial black color. On the one hand, the patients saw black color as more pleasant and relaxing than white color, and perhaps the environment\u2019s atmosphere may induce their intimate behavior. Furthermore, the relatively less pleasant atmosphere from white color may possibly inhibit their intimate behavior. On the other hand, the favorable impression of the psychologist might mediate the patients\u2019 response. In the conditions with black color, the patients might disclose themselves more frequently, because they regarded the psychologist as more favorable than the conditions with white color.In addition, psychologists should be aware of the physiological effects of color choices. For example, although cool colors may be desirable because they may decrease blood pressure and pulse rate, research indicates that the may also contribute to feelings of sadness and fatigue. Color theory suggests that psychologists should consider choosing wall/ceiling colors that are visually pleasing to them because many psychologists occupy the office on a daily basis. Balanced against the desire to please oneself is the desire to create a counseling space in which patients with different color preferences can all be comfortable.In a recent review of research on color in environmental design, Pressly For example, experiments that use factorial designs can be very helpful in exploring whether the effect of one or more environmental components varies under differing levels of another component. We reviewed an investigation with such a design that found that the influence of counselor self-disclosure on perceptions of counselor credibility differed depending on whether there was a physical barrier between counselor and client To apply the findings to actual counseling room design, we have to consider some points.First, the patients were selected randomly in psychological clinic of general hospital. Thus, it is difficult to judge whether the differences of color would have the similar effects to patients who have serious depression and high anxiety. Consequently, further studies and observations that focused on persons who really come to therapy or counseling should be needed to test the effects of color in counseling settings.Second, this study focused on the physical variables that would have impression on patients. Therefore, our findings may not always bring about the same outcomes as this study in actual counseling in which the number of people and types of conversation are varied. In actual counseling, therapists and counselors conduct various treatments, such as group counseling and family therapy. It is implicated that the patterns of the effects might change with the types of communication. Thus, the application of color to various types of communication should be examined more precisely in additional research.Finally, this study highlighted the effects of color in a counseling room. Besides this factor, there may also be more effective physical issues to conduct counseling for patients. Further explorations of physical variables may stimulate or inhibit development of other physical factors and relationships among them.From the practical point of view, the knowledge about environmental factors in counseling such as effects of color helps psychologists to practice their therapy more effectively. Like some researchers This paper has carried out research to apply experimental design and optimization theory to develop a methodology for color design of counseling room. The results enable us to understand patients\u2019 preference in the color design and to develop method based upon optimization theory for creating color design more scientifically and effectively."}
+{"text": "Commission Internationale de l\u2019Eclairage (CIE) in 1931, along with its subsequent improvements, represents a family of light mixture models that has served well for many decades for stimulus specification and reproduction when highly controlled color standards are important. Still, with regard to color appearance many perceptual and cognitive factors are known to contribute to color similarity, and, in general, to all cognitive judgments of color. Using experimentally obtained odd-one-out triad similarity judgments from 52 observers, we demonstrate that CIE-based models can explain a good portion  of the color similarity data. Color difference quantified by CIELAB \u0394E explained behavior at levels of 81% , 79% (across red colors), and 66% (across blue colors). We show that the unexplained variation cannot be ascribed to inter- or intra-individual variations among the observers, and points to the presence of additional factors shared by the majority of responders. Based on this, we create a quantitative model of a lexicographic semiorder type, which shows how different perceptual and cognitive influences can trade-off when making color similarity judgments. We show that by incorporating additional influences related to categorical and lightness and saturation factors, the model explains more of the triad similarity behavior, namely, 91% , 90% (reds), and 87% (blues). We conclude that distance in a CIE model is but the first of several layers in a hierarchy of higher-order cognitive influences that shape color triad choices. We further discuss additional mitigating influences outside the scope of CIE modeling, which can be incorporated in this framework, including well-known influences from language, stimulus set effects, and color preference bias. We also discuss universal and cultural aspects of the model as well as non-uniformity of the color space with respect to different cultural biases.The system for colorimetry adopted by the  Humans constantly experience environmental color and interact with color appearance in everyday life. Because the perceptual processing of color arose as an adaptation through biological and cultural evolution, scientific analyses of human color processing must involve many factors, including physical properties of color stimuli, the processing of physical reflectance features by the human visual system, and higher-order cognitive processing of color in human visual cortex. On the other hand, modern scientific study of human interactions with color stimuli grew largely out of early engineering and technological applications where color specification and reproduction in industry are of crucial importance. As a result, a key emphasis in the early days of developing photograph and display technology was to provide methods to precisely define color appearance, and to do so in ways that were adequate for reproducing color across different formats .Commission Internationale de l\u2019Eclairage (or CIE) which led to a 1922 report on colorimetry by the Optical Society of America, and subsequently the formalization of the CIE 1931 XYZ color specification and the 1931 CIE 2-degree standard observer color space and human color matching functions, as well as a series of subsequent standard observer color space refinements A consequence of this applied emphasis was the founding of the international authority on light, illumination and color, established in 1913, known as the Given its originally intended use, it is amazing that CIE systems have served so well as models of complex color appearance computations. Still, it is well recognized that other perceptual and cognitive factors also contribute to color similarity, and, in general, to all cognitive judgments of color. We know, for example, that individual variation in visual processing features result in nonuniform color perception across observers In this paper we investigate whether CIE models are a good predictor of human choice behavior in color similarity judgments. We use results from a color triad task. Color triad tasks require observers to choose from three-color samples the singleton that does not form a natural grouping, or \u201cbelong with\u201d, the other two of samples provided. Such tasks have been used with the aim of understanding biological, psychophysical and cognitive constraints that guide the human responders in their choices Let us represent each color triad as a triangle in a three-dimensional CIE space. The likelihood for a given stimulus in one such triad to be chosen as an odd-one-out is, in the simplest null-model, dictated by the geometry of the triangle Intra-individual variation arises as a degree of test-retest inconsistencies of the observers, which are random chance events, and can be a reflection of individual behavior, such as sloppiness in completing the test. (2) Inter-individual variation can reflect (a) inhomogeneities of the observers, such as the presence of a small number of dichromats, (b) individual cognitive factors such as personal color preferences or linguistic influences relevant in subsets of participants. (3) Systematic variation, or patterns of deviation from the CIE model that are frequent in a large portion of the participants, which can arise from (a) inconsistencies in the CIE description of perceptual space, or (b) systematic cognitive factors such as variation in the assignment of shared color meaning or \u201cconventions\u201d. Inter- and intra-individual variations have previously been quantified and compared There are three possible sources of variation that can contribute to mismatches between the data and the CIE model. (1) lexicographic semi-order modeling In order to improve the null-model, we propose that in addition to the distance considerations provided by the representation of colors in CIE space, there are other perceptual-cognitive factors that contribute to individual odd-one-out color similarity choices. Some of these factors may be widely used (such as a warm-cool distinction described below), whereas others may be more dependent on pragmatic uses of color or even culturally specific uses that vary across groups of individuals. As shown in analyses that follow, such factors can trade-off perceptually salient features for pragmatic features in a manner that resembles choice behavior interpreted using A\u2019 and \u2018not A\u2019. Such distinctions can be based on idiosyncracies of individual perceptual salience One identifiable factor underlying choice behavior for color similarity that goes beyond the CIE distance model is a factor we refer to as \u201ccategorical considerations\u201d. Categorical considerations permit the natural partitioning of the color stimuli studied into subsets \u2018Here categorical considerations are given a quantitative implementation. We propose that the probability to be chosen defined by category , or if tcategory , then ala*b* plane of CIELAB (1976) space catW, was obtained as part of the optimization problem :To summarize, the most comprehensive model (3) can explain 91% of the data for the global, 90% for the red, and 87% for the blue condition.From the perspective of cognitive psychologists interested in understanding human color experience, the CIE models are perfect tools for empirical stimulus specification, but they are not ideal as models of the many ways humans experience and interact with color. Our results show that CIE color differences alone are insufficient to describe color similarity when strong sources of cognitive influence on color judgment are at play. This is because CIE formalizations were not designed to predict a variety of behavioral outcomes that arise from human color appearance processing. The formal geometric properties of CIE space do not very accurately predict human judgments of, for example, color similarity, color preference or color difference in complex scenes. The results presented here suggest that a family of models that use CIE distances in conjunction with other known perceptual and cognitive factors is a promising approach to using CIE formalisms as a basis for the quantitative modeling of cognitive color relations.The present results confirm the suggestion e.g.,  that thelexicographic semi-order modeling of choice behavior Our model can be considered a hierarchical model in the following sense. It includes the null model (the CIE distance model), which most of the time comprises the largest contribution to the choice probability. Other factors, although present, in the majority of cases do not have enough weight to modify the prediction given by the CIE distance model. In some cases, however, the CIE prediction is \u201cweak\u201d, which happens for example in the case when the triad is a nearly equilateral triangle. In this case, other linguistic or cognitive layers, such as category criteria or lightness-saturation baises, become the major basis for color similarity choices, and can determine the color choice behavior. These findings and this hierarchical modeling approach are related to When faced with complex multidimensional alternatives \u2026 it is extremely difficult to utilize properly all the available information. Instead \u2026 people employ various approximation methods that enable them to process the relevant information in making a decision. The particular approximation scheme depends on the nature of the alternatives, as well as on the ways they are presented \u2026 The lexicographic semiorder is one such approximation \u2026 \u201d  a new and highly plausible approach to modeling the perceptual-cognitive-pragmatic aspects of stimuli that naturally play a role in color similarity judgments, and (ii) is an appropriate and useful extension of the formalisms presented by lexicographic semiorder modeling.It should be noted that in this paper we emphasize a modeling approach that is based on the most widely used index of color difference: CIELAB \u0394E. We examined alternative models based on other more recently introduced CIE color difference equations  that have been developed by CIE scientists in the last decade. Although these more recent color appearance models (CAMs) strive to advance color difference estimation beyond CIELAB-based formulas, they remain under active development and are not considered final models, as they continue to evolve, and continue as topics of ongoing testing, debate, and discussion Finally, the CAMs are dramatically more complex computationally, to the point of bordering on being impractical for some applications. This is likely the reason they have not been embraced widely as tools in the empirical research literatures that study behaviors linked to cognitive and perceptual color relations. These two reasons underlie our choice to emphasize and build on the accepted, widely used, color difference index CIELAB \u0394E. However, once CAM development is finalized and vetted by CIE experts, a potentially useful pursuit might be to generalize our hierarchical approach by using a CAM as the starting point for computing distances \u2013 this is entirely compatible with the aims of the present paper, but is necessarily a task for future modeling research.Our analyses show that different warm-cool category and lightness-saturation emphases are found across global, red and blue triad choices . What ismarked/unmarked color relations \u2018A\u2019/\u2018not A\u2019 partition in the color space on which color similarity judgments can be evaluated.Note that categorical considerations modeled here could originate from any number of sources. They could arise from normal biological bases If limited appropriateness of categorical considerations across color space is possible, then it is not surprising that the present data shows warm-cool category considerations improve the fit to the data for global and red conditions, while they explain less of the data for the case of the blue condition. Similarly, the criteria of lightness and saturation seem to play a role in triad choice data for the blue condition but not for the red condition or the global condition. Such patterns are plausible because stimulus sets for the three conditions were not sampled to be subjectively equal on all possible dimensions. Thus, cognitive and perceptual salience may be differently represented by samples comprising the three stimulus sets .One advantage of CIE modeling that keeps formalizations tractable is that a standard observer approach to color modeling is used. This standard observer simplification does, however, limit the potential to address systematic variation in observer groups that can be linked to, for example, shared knowledge about color, which uniquely contributes to color similarity relations in observers with specific linguistic or cultural affiliations. In these cases where culturally relevant information is known, it would be beneficial to include such information as a factor relevant in color similarity judgments. Extending the modeling capabilities of the CIE in this way is one aim of the present hierarchical modeling approach.universal in this framework are the identifiable perceptual and cognitively salient features of chosen triad stimuli , and what is cultural are the alternative approximations that additionally contribute to triad choices . The universal-cultural contrast differs here from that typically seen in the literature \u2013 that is, although the two forms of influence arise from very different sources, they nevertheless can both contribute to odd-one-out color similarity judgments.This article considers data only from native-language speakers of English. But it is certainly true that category influences on color triad choices will differ for non-English speakers. For example, one would expect variation in lightness-saturation influences on color triad choices across ethnolinguistic groups piros and v\u00f6r\u00f6s) \u00f6t\u00e9t, respectively) to modify basic color terms much more frequently than that seen in native English color naming. This suggests that there are differences of lightness dimension salience between native-language Hungarian and English color naming xanh) is used for denoting all \u201cgreens\u201d and \u201cblues\u201d and the linguistic differentiation of green appearances from blue appearances is only achieved by modifying terms that specify which xanh appearance is \u201cgreen\u201d (or xanh l\u00e1 c\u00e2y or \u201cxanh like the leaves\u201d) and which xanh is \u201cblue\u201d (or xanh nu'\u00f3'c bi or \u201cxanh like the ocean\u201d) Examples of how category influences can differ across language groups are readily available. Russian has two distinct linguistic categories (or two \u201cbasic color terms\u201d) for colors that English denotes under the lexical category \u201cblue\u201d linguistic markedness could influence which criteria color similarity is based on, and these would be expected to vary across ethno-linguistic groups and might even arise due to subgroups within a linguistic culture, such as color experts  compared to individuals in the same ethno-linguistic culture with little color expertise.Thus, linguistic and cultural factors could set which criteria are most attended to in color similarity judgments compared across cultures. Indeed, many forms of Data from 52 native English speakers  are analyzed (see Text S1Methods.(PDF)Click here for additional data file."}
+{"text": "Similarly, 94% identification success for the yellow fields was obtained depending on the values of the parameter y of CIE XYZ color space. With the designed system, three kinds of apples  were investigated by classifying them into four groups with respect to two parameters, color and size. Finally, 99% success rate was achieved in the analyses conducted for 595 apples.This study was focused on the multicolor space which provides a better specification of the color and size of the apple in an image. In the study, a real-time machine vision system classifying apples into four categories with respect to color and size was designed. In the analysis, different color spaces were used. As a result, 97% identification success for the red fields of the apple was obtained depending on the values of the parameter \u201c Turkey has the third place in the world after China and the USA with a 2.782.370-ton apple production . The appRecently, with the improvement of vision processing technology, machine vision has started to be used in industrial food machines which enable the classification of different types of fruits in terms of their size, color, and defects with help of high-definition cameras. Machine vision systems or vision processing techniques have been used by researchers on different types of fruits , such asLeemans and Destain obtained the RGB images of Jonagold variety of apples via two CCD cameras placed in different angles. By using the images obtained, they were able to classify the apples into two quality groups with a 73% success rate by the segmentation of the apple surface . Li et aXYZ, CIE L*a*b*, and CIE L*C*h* can be counted among device independent color spaces.Color is the main information source for object classification, inspection, and sorting. Color space is the mathematical representation of colors. Color spaces are formed so that they represent all colors. Color spaces are designed three dimensionally. The most frequently used color spaces are RGB (on computer monitors), YIQ, YUV or YCbCr (in video systems), and CMYK (in color printers). Since none of these color spaces are related to brightness, saturation, and intuitive perception of the color, there are additional color spaces such as HSV and HSI . Color sL*a*b* system is suggested as the best color space for quantification in foods with curved surfaces [There have been several benefits of applying color space transformation that have been identified by studies on skin detection. First, the codification process is sharpened to enhance the distinctiveness between skin and nonskin classes through a certain color space transformation. Another benefit is the realization of illumination invariance, as varying illumination has its own set of challenges regarding skin detection. Finally, color space transformation presumably is able to categorize shades of different skin tones together . So, HSVsurfaces  and whensurfaces .L*a*b*, and CIE XYZ color space) component with thresholding. Different color space transformations were investigated to find the best color space in the classification of apples.There is a lot of studies in the literature on the classification of apple. In this study, with the difference of other studies in the literature, a conveyor system was developed to obtain data from different angles of each apple. The apple image was separated from the background by HSV color space component with thresholding. Apple size and color were obtained by multicolor space  resolution 25\u2009fps (frame per second) Complementary Metal Oxide Semiconductor (CMOS) camera with a manually adjustable 6\u2009mm focus length which is mounted on camera, C-mount lens, a lighting system, 600\u2009mm \u00d7 670\u2009mm \u00d7 300\u2009mm white painted diffusely illuminated tunnel with four fluorescent lamps, a conveyor belt on which fruits are placed, and automatic sorting unit. The setup of the system is shown in Figures Image acquisition device used for this research is a 1.3 mega pixel  close to 97%.In practice, the points to be taken into consideration in the design of lighting systems are color rendering, high index, close to daylight (color temperature 5000\u2009K\u22126500\u2009K), choice of lamp whose level of light is appropriate, and proper positioning of the lamp so that all surfaces of the object receive equal levels of light. In the lighting system, 4 Philips TL-D 90 Graphica 18W/950 models are used. The features of this lamp are a color temperature of 5300\u2009K (approximately D55) and a color-rendering index  color space. Unfortunately, RGB color space is not consistently uniform, and the proximity of colors does not indicate color similarity . In addiM \u00d7 N \u00d7 3 array of color space pixels, where each color pixel is a triplet corresponding to the red, green, and blue components of an RGB image at a specific spatial location [An RGB color image is an location .R,\u2009\u2009G, and\u2009\u2009B\u2009\u2009values of RGB color space are the sum of respective sensitivity functions and the incoming light;S(\u03bb)\u2009\u2009is the light spectrum, R(\u03bb), G(\u03bb), and B(\u03bb) are the sensitivity functions for the R, G, and B sensors, respectively [ectively .H (hue) distinguishes among the perceived colors, such as red, yellow, green, and blue.\u2009\u2009V  represents the brightness of a color and S (saturation) refers to how much the amount of white light mixed with a hue is [HSV color space is quite closer than the RGB system to the way in which human experiences and describes color sensations . HSV sepa hue is . ConsideR, G, and B values [HSV color space values are calculated according to B values (2)H={\u03b8,XYZ color space takes the human eye as a basis, it forms the base for all color management systems and includes all perceivable colors. This color space is determined by CIE in 1931 according to standard illuminant  and standard observer . The reason for this is that the most significant feature of CIE XYZ color space is being device independent.Since CIE \u03bb of CIE color system [S(\u03bb) is the relative spectral power of an illuminant, x(\u03bb), y(\u03bb), and z(\u03bb) are color-matching functions, P(\u03bb) is the spectral reflectance at wavelength \u03bb, and k is a normalizing factor given by 100/\u222bS(\u03bb)y(\u03bb)d(\u03bb).Tristimulus values at wavelength r system  are(3)XL*a*b* and CIE L*u*v*) in order to provide the structural equity space. There is a strong relation between these color spaces and a human's visual perception. The components are \u201cL\u201d (light), the rate from green to red \u201ca\u201d, and that from blue to yellow \u201cb\u201d.In 1976, CIE suggested two color spaces XYZ and CIE L*a*b* with D55-compliant. The sorting process is carried out by collecting the thresholding results of HSV color space \u201cS\u201d component, CIE XYZ color space \u201cY\u201d component, and CIE L*a*b* \u201ca\u201d component in one mask. Consider the following:f is the image obtained after the background has been removed, with z being the original image and T being the threshold. The threshold values based on image. The threshold values are defined by examining the histograms of the color space components. These values are fixed and added to the software after testing different apple images.Apple area was separated from the background with thresholding. The RGB image is transformed into HSV with the \u201crgb2hsv\u201d routine of the image processing toolbox of Matlab (Mathworks 2011) before applying thresholding. In addition, the RGB image is transformed to CIE x- and y-axes in the masked area.The next process after the process of separation of the background is the determination of the color and size of the apple. Size of the apple is calculated according the pixel numbers it occupies in Y\u201d component of CIE XYZ color space shows the yellow regions in the apple picture. The thresholding of \u201ca\u201d component of CIE L*a*b* color space shows the red regions in the apple picture. The information about the color and the type of the apple is gathered according to the area the yellow and red regions take up on the surface of the apple. The conversion of RGB apple picture to CIE XYZ and CIE L*a*b* color spaces can be applied with the help of built-in functions of Matlab. Yet, the conversion time of Matlab built-in function is too much for a real-time system. However, only one component of these color spaces is used.The color of the apple is determined according to the intensity and dispersion of the colors in the specified area. For this procedure, thresholding is used for red and yellow regions in the image. The thresholding of \u201cXYZ and CIE L*a*b* color spaces changes depending on the illuminant character of the lamp. For this reason, a function is written considering that the color temperature of the fluorescent lamp is 5300\u2009K (approximately D55).The conversion from RGB color space to CIE R, G, and B values are converted to linear and nominal values as follows:R\u2032 = R/255\u2009G\u2032 = G/255, B\u2032 = B/255, \u2009R\u2032, G\u2032, B\u2032 \u2264 0.04045 If \u2009Rs = R\u2032/12.92;\u2009\u2009Gs = G\u2032/12.92;\u2009\u2009Bs = B\u2032/12.92 ElseConverting RGB to CIE XYZ is performed with the help of a 3 \u00d7 3 matrix. XYZD65 color space is given below [Firstly, nonlinear XYZ source data differs from the illuminant target RGB space (destination). Humans have a visual ability, called chromatic adaptation, to acknowledge and discount the color of the illumination and approximately recall an object's color. Chromatic adaptation is most easily understood by viewing a white object under various illuminations. Several chromatic adaptation transformations exist in literature, most of which are based on the von Kries model [If the source differs from the illuminant of the destination, chromatic adaptations are used to decide whether the es model , 27.XYZD65 color space acquired according to D65 reference white is converted to CIE XYZD55 color space according to the D55 reference white used in our lighting system. Consider the following:XWS,\u2009\u2009YWS, and\u2009\u2009ZWS\u2009\u2009are source reference white;\u2009\u2009XWD,\u2009\u2009YWD, and\u2009\u2009ZWD\u2009\u2009are the destination reference white;\u2009\u2009\u03c1,\u2009\u2009\u03b3, and\u2009\u2009\u03b2\u2009\u2009are cone response domain. Accordig to conditions of D65 luminance, source reference values are\u2009\u2009XWS,\u2009\u2009YWS,\u2009\u2009ZWS = 0.9505, 1.0000, 1.0890 respectively. Accordig to conditions of D55 luminance, destinations reference values are\u2009\u2009XWD,\u2009\u2009YWD,\u2009\u2009ZWD = 0.95682, 1.0000, 0.92149 respectively. In calculations we assumed the 2\u00b0 standard colorimetric observer. The conversion matrix acquired via chromatic adaptation matrix (MA) isBy using von Kries transforms, Using the designed system, ten images were obtained for the test purpose. On each image, there is one yellow and one red apple. The experimental results for the average and standard deviation values of the parameters of different color spaces for the color pixels of red and yellow apples and the background are given in Ra) that is close to 97%. These RGB images are converted to the CIE XYZD65 color space according to illumination condition and 2\u00b0 standard observers. It is converted to the CIE XYZD55 color space by taking into consideration the D55 illumination conditions in the systems using the von Kries transforms matrix. The CIE L*a*b* color space and components are calculated from the CIE XYZD55 color space by using the aforementioned formulas.The apple images are obtained with special fluorescent lamps which have a color temperature of 5300\u2009K (approximately D55) and a color-rendering index . Therefore, the threshold value was specified as 15. The Y component of CIE XYZ was used to expose the yellow regions by prioritizing the luminance. The threshold value was specified as 0.5 in accordance with the normalized values. Further the pixels smaller threshold values were deleted. The results show that the identification success rates for the pixels in red and yellow regions are 97% and 94%, respectively. They also show that the parameter \u201cS\u201d of the HSV color space is a useful color space to distinguish the background and to remove the apple masks.After examining the histograms of the pixels along with the color parameter values , the parThe size and color information is collected from the pictures acquired by the machine vision system after the place of the apple is masked in the picture. The components of color spaces used to achieve this are shown in S\u201d component  and \u201ca\u201d component (of CIE L*a*b* color space), yellow and red regions are determined. Hence, apple type is obtained according to these color information.These results show that the apple size and color were basically extracted. Multicolor space is used to separate the apple from the background and to effectively determine its color and size. The determination of the position of the apple in the image is achieved considering all the thresholding results Figures  of the \u201component  in HSV component  in CIE Xomponent  in CIE Lx- and y-axes in the masked area, and the pixel length (0.312\u2009mm)  and size (regular-large) are taken into consideration in the experimental results. Furthermore, the size of the apple is calculated by multiplying pixel number of the area (the apple covers) in .312\u2009mm) .L*a*b* conversion took on average of 34.2\u2009ms with Matlab's built-in rgb2lab function, while the same conversion took on average of 13.4\u2009ms with the new function (D55-compliant). This result is seen in Moreover, for device independent color space conversions, a custom, D55-compliant function is developed and runs faster than the Matlab built-in counterpart. The RGB to CIE a\u201d component with red color sensitive to the CIE L*a*b* space. Comparing histograms obtained by two different functions, similarities are seen between aggregation curves of pixels, despite the different positions of histograms (These two functions (D55-compliant and Matlab built-in) are implemented on RGB original image  in orderstograms . So the stograms .As a result, 595 apples including three kinds, \u201cStarking, Jonagold, and Golden\u201d, were examined by the system . They weSpeed is yet another critical aspect to consider. With the use of a custom and fast D55-compliant program, 95 apples are classified in less than a minute, but the processing time to classify apples with the developed software according to two parameters like size and color is approximately 260\u2009ms. Therefore, the speed of automatic apple classification system can be further increased with the improvement of the sorting unit.Some important outputs drawn from the study on the apple classification system offered are as follows. In apple classification systems, there have been some important things as the follows: (i) what kind of apples is classified, (ii) which properties are considered for the classification, and (iii) what amount of apples are classified at a given time. Having such information indicates how much the system is practicable.Examining these kinds of studies existing in the literature, it is seen that classifications were performed with respect to mainly two parameters like the segmentation of the surface images and the surface quality. In this study, classification of apples are practically realized considering the parameters, color, and size. For this, software based color space, instead of cameras which are sensitive to the different wavelengths and filters, is utilized to have the color and size information of apples. The different color spaces were compared and the most suitable ones were selected. In the determination of the color spaces, the cameras and lighting equipment used in the system have a great importance.In this study, in accordance with the studies in the literature, a system which places under the camera and also which lets the camera make rotary motion was offered. With such a system, multiple photos can be taken and most of the surface (of the apple) can be viewed. Otherwise, naturally processing time increases because more photos are taken. To overcome this problem, only one photo was considered to get information about the color and size of the apples.In this experimental study, the system works on only one line. If the number of the lines is increased, more apples can be classified by the system. Since the separator of the system works in a short distance, it can not keep up with the processing rate of the software. Though the classification rate of the software for an apple is 260\u2009ms, which corresponds to 240 apples in one minute, the number of apples in a minute, in fact, remains 96. This problem can be solved easily by using a handle belt conveyor."}
+{"text": "The population of influenza virus consists of a huge variety of variants, called quasispecies, due to error-prone replication. Previously, we reported that progeny virions of influenza virus become infected to adjacent cells via cell-to-cell transmission pathway in the presence of oseltamivir. During cell-to-cell transmission, viruses become infected to adjacent cells at high multiplicity since progeny virions are enriched on plasma membrane between infected cells and their adjacent cells. Co-infection with viral variants may rescue recessive mutations with each other. Thus, it is assumed that the cell-to-cell transmission causes expansion of virus quasispecies. Here, we have demonstrated that temperature-sensitive mutations remain in progeny viruses even at non-permissive temperature by co-infection in the presence of oseltamivir. This is possibly due to a multiplex infection through the cell-to-cell transmission by the addition of oseltamivir. Further, by the addition of oseltamivir, the number of missense mutation introduced by error-prone replication in segment 8 encoding NS1 was increased in a passage-dependent manner. The number of missense mutation in segment 5 encoding NP was not changed significantly, whereas silent mutation was increased. Taken together, we propose that oseltamivir expands influenza virus quasispecies via cell-to-cell transmission, and may facilitate the viral evolution and adaptation. RNA viruses exist as genetically diverse populations, termed quasispecies, due to error-prone replication by viral RNA-dependent RNA polymerases1245671012Influenza virus is one of the most serious zoonotic pathogens causing seasonal epidemics and periodic pandemics among human beings around the world. The genome of influenza virus consists of eight-segmented and single-stranded RNAs. The segmented structure of the virus genome allows genetic reassortment when more than two genetically different viruses co-infect a single cell. The viral envelope consists of a lipid bilayer derived from cellular plasma membrane with two viral spike proteins, hemagglutinin (HA) and neuraminidase (NA). Influenza virus infection is initiated by the attachment of HA on virus particles to terminal sialic acid residues in glycoconjugates17Viruses are released as cell-free virions from an infected cell and become infected to distant cells and tissues. In contrast, the virus transmission mechanism from an infected cell to adjacent cells without viral diffusion into the extracellular environment, termed cell-to-cell transmission, has been reported in several viruses202223ts) mutations remain in progeny viruses even at non-permissive temperature by co-infection in the presence of oseltamivir. This is possibly due to a multiplex infection through the cell-to-cell transmission by the addition of oseltamivir. Further, Next-Generation sequencing analyses revealed that spontaneous mutations introduced by error-prone replication were accumulated in the virus population by oseltamivir treatment. In conclusion, we propose that oseltamivir expands the virus quasispecies via cell-to-cell transmission, and cell-to-cell transmission may contribute to the evolution of influenza virus.Here, we showed that temperature-sensitive  as helper virus. Thus, it is suggested that high multiplicity of infection is important for the expansion of virus quasispecies. Recently, we found that progeny influenza virions become infected to the adjacent cells via cell-to-cell transmission in the presence of oseltamivir possibly with high multiplicity of infectionts) mutant viruses, ts1 and ts53. The viral transcription of ts1 is defective at non-permissive temperature due to a mutational change in PB2 polymerase subunit at the amino acid position of 417 from Asp to Asn25ts53 PA polymerase subunit results in the defective virus genome replication at non-permissive temperature2728ts1 and ts53 during co-infection. To perform co-infection experiments using ts1 and ts53 efficiently, 0.1% confluent Madin-Darby canine kidney (MDCK) cells were co-infected with ts1 and ts53 at each multiplicity of infection (MOI) of 5 /ml of wild-type virus possibly due to segment reassortment. The virus titer determined at 34\u00b0C was 1.3 \u00d7 106\u2005PFU/ml including both ts mutant viruses and wild-type virus. Thus, the population rate of ts virus was calculated to be 91%  of 5 . In ordeeratures . It is rutations . The virue assay . Co-infeo be 91% . At non-Stu I as reported previouslyts53 virus has a substitution mutation from U to C at the nucleotide position of 701 in segment 3 encoding PA, while the sequence of segment 3 of ts1 virus is the same as that of wild-type virus. The digested DNA fragments containing 220 and 199 base pairs were derived from ts53 and wild-type viruses, respectively. As expected, the virus genome derived from ts53 was clearly found even at non-permissive temperature in the presence of oseltamivir 26ts mutants at MOI of 0.01\u2005PFU/cell, and incubated at 37\u00b0C and 34\u00b0C, respectively. After incubation for 48\u2005h, the culture fluid was harvested and centrifuged at 1,700 \u00d7 g for 10\u2005min. The virus suspension was stored at \u221280\u00b0C until use.MDCK cells were maintained in minimal essential medium (MEM) (Sigma) containing 10% fetal bovine serum. Influenza virus A/WSN/33 and temperature sensitive mutants  or non-permissive temperature (39.5\u00b0C) in the absence or presence of 50\u2005\u03bcg/ml of oseltamivir phosphate, cells were washed with serum-free MEM. Then, maintenance medium (MEM containing vitamins and 0.1% BSA) was added with or without 50\u2005\u03bcg/ml of oseltamivir phosphate and cultured at either 34\u00b0C or 39.5\u00b0C. At 60\u2005hpi, the culture supernatant was collected, and then its virus titer was determined by plaque assays under both 34\u00b0C and 39.5\u00b0C. Viruses grown in the presence of oseltamivir were harvested from the cell surface by treatment with 30\u2005mU/ml of bacterial NA derived from Clostridiun perfringens (SIGMA).To examine the transmission of influenza virus from individual co-infected cells to adjacent cells, we carried out co-infection of ts53 virus has a substitution mutation from U to C at the nucleotide position of 701 in segment 3 encoding PA, while the sequence of segment 3 of ts1 virus is the same as that of wild-type virus. To discriminate the genome of ts1 (wild-type) and that of ts53, total RNA was reverse-transcribed by SuperScript III (Invitrogen) with PA-895-rev (5\u2032-TTAATTTTAAGGCATCCATCAGCAGG-3\u2032), which is complementary to positive-sense RNA of the segment 3. The cDNA was amplified by PCR using primers, PA-895-rev and PA-695-cut (5\u2032-TCTCCCGCCAAACTTCTCAGGCC-3\u2032) partially corresponding to positive-sense RNA of segment 3 between nucleotide sequence positions 678 to 700 except for nucleotide positions 696 and 697. Since segment 3 of ts53 has a substitution mutation from U to C at the nucleotide position of 701, PCR products derived from wild-type could be digested by Stu I but not that from ts53. After PCR reactions, PCR products were digested with Stu I and separated on native-PAGE. Large and small fragments derived from ts53 and wild-type viruses were 220 and 199 base pairs, respectively4 for 1\u2005h, sequential dehydrations with ethanol in a stepwise manner were carried out followed by propylene oxide treatment, and embedded in Poly/Bed 812 (Polysciences). Ultrathin sections were examined with a JEM-1300 (JEOL) operated at 80\u2005kV.MDCK cells were infected with virus at MOI of 10, and cultured in the absence or presence of 50\u2005\u03bcg/ml of oseltamivir phosphate. After incubation at 37\u00b0C for 16\u2005h, cells were subsequently fixed with 2.5% of glutaraldehyde. After further fixation with 1% OsOAmplicon generation and NGS were carried out as previously reportedThe output from the GSJunior includes sequence results (FASTA) and quality score for every sequence position in a read. It is known that average quality score of a read is inversely proportional to the number of errors in that readSupplementary Information"}
+{"text": "Carassius auratus gibelio physiology after a single oral administration. In this study, Carassius auratus gibelio was subjected to oral gavage with various concentrations of trichlorfon . The trichlorfon concentration in the plasma and liver tissue was quantified using liquid chromatography-tandem mass spectrometry at different time points. At the beginning of oral exposure, the uptake of trichlorfon in the plasma and liver tissue was fast, and trichlorfon was rapidly eliminated to a low level within 24 h. In addition, acetylcholinesterase, superoxide dismutase, catalase, and glutathione-S-transferase activities in the plasma and liver tissue changed significantly after trichlorfon exposure. Additionally, vacuolar degeneration, necrosis, and congestion of the central vein were observed in the liver after trichlorfon exposure, as assessed by hematoxylin and eosin staining. Our results suggested that trichlorfon could accumulate and induce hematotoxicity and hepatotoxicity in the plasma and liver tissue, the toxicity induced by trichlorfon might result in physiological disturbances in fish.Trichlorfon is a most widely used organophosphate insecticide in aquaculture, many successful results have been reported for bath treatments of trichlorfon to control parasites. However, immersion treatments of large stocks with trichlorfon has caused serious environmental pollution. In contrast, oral administration treatment has advantages on reducing environmental pollution and having little effect in non-targeted species. The aim of this study was to investigate the effect of trichlorfon on  Organophosphate (OP) pesticides are used worldwide to control parasites in agriculture, industry, and households . The OP Ergasilus sp., Lernea sp., Dactylogyrus sp., Trichodinas sp. in freshwater fish , which causes accumulation of acetylcholine (ACh) at nerve synapses and disrupts nerve function . Superoxe damage . The inhe damage . Trichloe damage . With execreased . DespiteCarassius auratus gibelio physiology after an oral administration. Hence, the metabolism kinetics of trichlorfon in Carassius auratus gibelio plasma and liver tissues was examined. Furthermore, the AChE, GST, SOD, and CAT activities were determined in the plasma and liver tissue, and liver histopathology was measured to explore the toxic effect of trichlorfon.The aim of this study was to investigate the effect of trichlorfon on Carassius auratus gibelio; approximately 10 cm in body length) were obtained from the Wujiang National Farm of Chinese Four Family Carps, Jiangsu Province, China. Initially, the fish were reared at 22 \u00b1 2\u00b0C in 400 L aerated tanks for 1 week before the experiment and fed twice daily (in the morning and late in the afternoon) at a ratio of 5% of their total biomass. After 7 days of acclimation, fish were starved for 2 days before the administration of the drug. All experiments were performed according to the guidance of the Care and Use of Laboratory Animals in China. This study was approved by the Committee on the Ethics of Animal Experiments of Shanghai Ocean University, China.Trichlorfon (>90% pure) was purchased from Shanghai Biochemical Reagent, Shanghai, China. Healthy silver crucian carp (All the fish were divided into four groups (40 fish per group). The conditions were identical among the tanks and the fish were randomly distributed into the different tanks. To prepare the trichlorfon dosing solution, trichlorfon was dissolved in 5 mL of ethanol and mixed with water to achieve a final concentration of 2 g/L. The fish were given 2 g/L of the trichlorfon solution via gavage using a stomach tube, with final trichlorfon doses of 0.5 g/kg, 1 g/kg, or 2 g/kg. As controls, fish were given ethanol mixed with water at the same dosage. After oral administration, each fish was placed in an observation tank for 5 min to check for possible drug regurgitation; regurgitating fish were excluded from the analysis.g for 10 min in 4\u00b0C, and the plasma supernatant was collected for the later experiments. Hepatocyte tissues were washed with ice-cold saline (0.85% NaCl), homogenized in (1:9 w/v) ice-cold 0.1 M pH 7.4 phosphate buffer using a glass homogenizer, and the homogenate was centrifuged for 20 min (10000 \u00d7 g) at 4\u00b0C. The supernatant was used as the enzyme source to assess the enzyme activities. All the preparations were frozen at -80\u00b0C until analysis.Three fish of each group were anesthetized with 2-phenoxyethanol (2 mL/L) before handling, and sampled at 1, 2, 3, 4, 8, 12, 24, 48, 72, and 96 h after the oral drug administration. For the enzymatic analysis, blood was taken from the caudal vein, incubated at 37\u00b0C for 1 h, stored at 4\u00b0C for 12 h, and centrifuged at 2000 \u00d7 g for 5 min, and the clear supernatant was collected and transferred into a new 50 mL centrifuge tube. The extraction procedure was repeated twice with 10 mL of the ethyl acetate. The supernatants were combined and concentrated to 5 mL under a stream of nitrogen at 40\u00b0C. The residues were extracted using Copure Alumina SPE , and dissolved in 5 mL of ethyl acetate. Finally, the extract was evaporated to near dryness under a stream of nitrogen at 40\u00b0C, dissolved in 1.0 mL of the mobile phase . The extract was filtered using a 0.22-\u03bcm nylon filter for liquid chromatography-tandem mass spectrometry (LC\u2013MS/MS) analysis. All samples were analyzed in duplicate.One milliliter of blood was taken from the caudal vein at 1, 2, 3, 4, 8, 12, 24, 48, 72, and 96 h after oral drug administration and mixed with 14 mL of ethyl acetate. Then, 2 g of hepatocyte tissues were homogenized in centrifuge tube, and 15 mL of ethyl acetate was added. The slurry was vortexed for 1 min. The samples were centrifuged at 2,292 \u00d7 Samples were analyzed on an LTQ-Orbitrap XL instrument . The parameters were as follows: Mobile phase A: water containing 0.1% of formic acid (v/v); Mobile phase B: methanol; injection volume 2 \u03bcL; mobile phase flow rate 0.2 mL/min; mobile phase gradients: 35% B (0\u20136.00 min), 95% B (6.01\u201310.00 min), and 35% B (10.01\u201314.00 min); electrospray positive ionization (ESI+); gas flow was set at 10 L/min; gas temperature was set at 350\u00b0C; nebulizer pressure 25 psi; capillary voltage 3500 V, gas temperature 350\u00b0C; nebulizer pressure 50 psi; capillary voltage 5500 V. The multiple reaction monitoring (MRM) mode was positive, and the optimized MS/MS transitions were m/z 259.1>221.1.mg protein in hepatocytes and per ml in plasma.Acetylcholinesterase activity was quantified according to the colorimetric technique as previously described . All sam2O2 and the resulting decrease in absorbance at 405 nm over a 3 min period at 25\u00b0C. One unit of CAT activity was defined as the amount of enzyme required to consume 1 mol of H2O2 in 1 s and was expressed as U/mg of protein in hepatocytes and U/mL in plasma.The activities of SOD, CAT, and GST were quantified using commercial kits  according to the manufacturer\u2019s instructions. The total protein concentration of the samples was determined at 595 nm using the Bradford method. GST activity was assayed by measuring the formation of GSH (Glutathione) and the 1-chloro-2,4-dinitrobenzene (CDNB) conjugate. The specific activity of GST was expressed as U/mg of protein in hepatocytes and U/mL in plasma. SOD activity was measured at 550 nm, one unit of SOD activity was defined as the amount of enzyme required to inhibit the oxidation reaction by 50% and was expressed as U/mg of protein in hepatocytes and U/mL in plasma. CAT activity was determined by assaying the hydrolysis of HFor histopathological examination, three fish from each group per replicate for histopathological studies were removed after treatment periods of 24 h. Hepatocyte tissues were dissected and fixed in Bouin\u2019s fluid for 24 h, and washed with tap water. They were processed through a graded ethanol series, cleared in xylene, and embedded in paraffin. Paraffin samples were sectioned at a thickness of 5 \u03bcm, and stained with hematoxylin and eosin (HE) for light microscopic observation. The sections were examined under a light microscope .post hoc test). All the results are presented as mean \u00b1 SD of three independent experiments. The results of one-way ANOVAs are denoted as \u2217 for P < 0.05 and \u2217\u2217 for P < 0.01.Data was tested for normality and homogeneity of variance before statistical analysis. All data analyses were carried out using IBM SPSS Statistics Version 20. One-way analysis of variance (ANOVA) was used to test for differences, followed by the Tukey test  in plasma were 9.793, 11.715, and 13.208, and the Cmax in liver were 20.495, 24.912, and 45.058 . Additionally, the high dose (2 g/kg) resulted in a shorter time (3.56 h) to peak level compared with the lower dose (5.37 h for 0.5 g/kg) in plasma, and in liver tissue, the shortest time to peak level was 2.65 h at a dose of 1 g/kg (Table 1).The concentration-time profiles of plasma and liver tissue after an oral dose of 0.5 g/kg, 1 g/kg, and 2 g/kg of trichlorfon are shown in Figure 2A, a significant decrease of AChE activity was observed in all tested samples after oral administration of trichlorfon in the plasma, and this reduction was concentration and time-dependent; a higher dose of trichlorfon resulted in lower AChE activity. In liver tissues, AChE activity was significantly reduced at 12 h after oral administration (P < 0.01), and upregulated at 24 and 48 h post oral administration, followed by a gradual decrease at 72 and 96 h .Acetylcholinesterase activity in the plasma and liver tissue was monitored after a single dose of 0.5 g/kg, 1 g/kg, or 2 g/kg of trichlorfon. As shown in P < 0.01); at 1 g/kg, the activity of GST was significantly downregulated at 48, 72, and 96 h (P < 0.01); at 2 g/kg, the activity of GST gradually decreased in a time-dependent manner . However, in liver tissues, significant decreases in GST activity was observed at all tested samples after oral administration, and this reduction was concentration and time-dependent.To evaluate the toxicity of trichlorfon, GST activity was detected after a single dose of 0.5 g/kg, 1 g/kg, or 2 g/kg of trichlorfon in the plasma and liver tissues. At a dose of 0.5 g/kg, the activity of GST only decreased at 96 h post oral administration in the plasma , and significant increased of CAT activity was only found in dosing of 2 g/kg at 48 h (P < 0.05). However, CAT activity induction decreased with exposure time, and CAT was significantly suppressed at 72 and 96 h post oral administration . SOD activity increased at 12, 24, and 48 h at all tested concentrations of trichlorfon, and was inhibited at doses of 1 g/kg and 2 g/kg at 96 h post oral administration .Catalase and SOD activities in the plasma and liver tissues post oral administration are presented in P < 0.01), followed by a gradual decrease at 72 and 96 h (P < 0.01); at 1 g/kg, CAT activity was reduced at 24 h (P < 0.05), followed by a gradual decrease at 48, 72, and 96 h (P < 0.01); and at 2 g/kg, CAT activity was reduced from 12 h (P < 0.01) . At a dose of 0.5 g/kg, SOD activity was reduced at 24 h (P < 0.05), followed by a gradual decrease at 48, 72, and 96 h (P < 0.05). At doses of 1 g/kg and 2 g/kg, SOD activity was reduced after 12 h (P < 0.01) .In liver tissues, CAT activity was inhibited in a dose-dependent manner. At a dose of 0.5 g/kg, CAT activity was reduced at 48 h . However, at 1 g/kg, vacuolar degeneration, necrosis, and congestion of the central vein were observed in the liver tissues . Similar phenomena were observed in the liver tissues of the 2 g/kg group . These results of HE staining in liver tissues suggested that the orally administered of trichlorfon induced hepatic injury.A histopathological examination was carried out to determine the extent of hepatotoxicity as a consequence of trichlorfon treatment in liver tissue. As illustrated in Carassius auratus gibelio were examined after a single oral administration of trichlorfon. At the beginning of oral exposure, the uptake of trichlorfon in the plasma and liver tissue was fast, after which trichlorfon was rapidly eliminated to a low level within 24 h. In addition, the AChE, SOD, CAT, and GST activities in the plasma and liver tissues changed significantly after trichlorfon exposure, although the exact mechanisms underlying the toxicity responses are not clear. Finally, we detected the effect of trichlorfon on liver tissue using HE staining.It is reported that trichlorfon causes many physiological changes in experimental animals . In thisFigure 2). The persistent suppression of AChE can inhibit fish activities  (Acetylcholinesterase is mainly responsible for the digestion of the neurotransmitter ACh in the synaptic cleft . There awimming) . ACh perwimming) . ContinuCyprinus carpio exposed to bispyribac-sodium under field conditions . Downregulation of GST activity highlighted the fact that GST probably plays crucial physiological roles during trichlorfon detoxification in Carassius auratus gibelio.Glutathione S-transferase is an important member of a supergene family that is involved in protecting against the deleterious effects of xenobiotics and oxidative stress . In a prnditions . Similare Apollo . Our stuCarassius auratus gibelio following trichlorfon treatment might have resulted from increased formation of reactive oxygen, which could stimulate CAT and SOD activities. However, the CAT activity was significantly inhibited at 72 and 96 h, and SOD was decreased at doses of 1 g/kg and 2 g/kg at 96 h. This trend  of anti-oxidase levels has also been observed in zebrafish hepatocytes exposed to beta-cypermethrin, and could be a signal of an overwhelmed antioxidant capacity caused by long-term exposure . These results were similar to those obtained in tilapia, which indicated that low concentrations of trichlorfon did not cause significant alterations in the tissue (Histopathological assays have been widely used as indices for the investigations of pesticide toxicity . OP peste tissue . Howevere tissue . BesidesCarassius auratus gibelio. The exposure concentration played an important role in the bioconcentration of trichlorfon, and its increase could significantly affect the uptake rates, Cmax and Tmax. AChE, GST, SOD, and CAT activity were significantly inhibited following trichlorfon treatment in liver tissues. AChE and GST activity in plasma were inhibited by trichlorfon, and SOD and CAT activity were increased by trichlorfon. Additionally, high oral concentration of trichlorfon induced histopathological changes in liver tissues, including vacuolar degeneration, necrosis, and congestion of the central vein. Considering the toxicity of trichlorfon on Carassius auratus gibelio, there should be a serious apprehension about the potential danger of this pesticide on fish. Therefore, further work should be performed to determine the precise physiological action and molecular mechanism of trichlorfon in fish.Overall, by combining analysis of the metabolism and toxicity of trichlorfon in the plasma and liver post oral exposure, we investigated the hematotoxicity and hepatotoxicity of trichlorfon in LL designed and led the study. MZ and JL performed the experiments. JL drafted the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "There are an exceedingly large number of sequence variants discovered through whole genome sequencing in most populations, including cattle. Deciphering which of these affect complex traits is a major challenge. In this study we hypothesize that variants in some functional classes, such as splice site regions, coding regions, DNA methylated regions and long noncoding RNA will explain more variance in complex traits than others. Two variance component approaches were used to test this hypothesis \u2013 the first determines if variants in a functional class capture a greater proportion of the variance, than expected by chance, the second uses the proportion of variance explained when variants in all annotations are fitted simultaneously.Our data set consisted of 28.3 million imputed whole genome sequence variants in 16,581 dairy cattle with records for 6 complex trait phenotypes, including production and fertility. We found that sequence variants in splice site regions and synonymous classes captured the greatest proportion of the variance, explaining up to 50% of the variance across all traits. We also found sequence variants in target sites for DNA methylation (genomic regions that are found be highly methylated in bovine placentas), captured a significant proportion of the variance. Per sequence variant, splice site variants explain the highest proportion of variance in this study. The proportion of variance captured by the missense predicted deleterious (from SIFT) and missense tolerated classes was relatively small.The results demonstrate using functional annotations to filter whole genome sequence variants into more informative subsets could be useful for prioritization of the variants that are more likely to be associated with complex traits. In addition to variants found in splice sites and protein coding genes regulatory variants and those found in DNA methylated regions, explained considerable variation in milk production and fertility traits. In our analysis synonymous variants captured a significant proportion of the variance, which raises the possible explanation that synonymous mutations might have some effects, or more likely that these variants are miss-annotated, or alternatively the results reflect imperfect imputation of the actual causative variants.The online version of this article (10.1186/s12864-018-4617-x) contains supplementary material, which is available to authorized users. The genetic component of complex trait variation, for many traits, is due to large numbers of mutations which individually explain a small portion of genetic variance \u20133. GWAS Whole genome sequencing (WGS) is providing solutions to some of the limitations of GWAS and genomic prediction with SNP genotyping arrays. In human and mouse genomes, the number of discovered genetic variants from WGS is well into the millions  and thisOne strategy is to attempt to filter the large number of variants to a subset that is more likely to have effects , 16. ForOur hypothesis was to variants in some functional classes will explain more genetic variation, than expected by chance, and more variation than some other classes. Sequence variants from the 1000 Bull Genomes Project were annotated into 20 functional classes, including, but not limited too; target sites for DNA methylation regions , and 25% were located within intragenic regions (protein coding genic regions). 23% of the variants were located within introns . The number of annotated variants in each class Table\u00a0 were fouGenotypes for the 28.3 million sequence variants were imputed into 16,581 dairy cattle, including cows and bulls, from the Holstein and Jersey breeds, with phenotypes for milk production and fertility traits  as a reference. Accuracy was high when the minor allele frequency (MAF)\u2009>\u20090.1, however with low MAF the accuracy of imputation dropped rapidly.The accuracy of imputing sequence data was assessed for chromosome 14  and miRNA target site classes, just over 35% of variants have a MAF between 0.1\u20130.5 while in the splice site region class more than 60% of the variants have a MAF between 0.1\u20130.5. The highest in this study.There is a chance that variants that have very low allele frequencies could be sequence errors . So, theGenomic relationship matrices (GRM) were constructed for each annotation class from the genotypes of all variants in the classes according to the method by Yang et al. . To deteWe next tested whether the variants in each annotation class explained more variance than expected by chance, give the number of variants in that class. This was done by obtaining the variance explained, using the GRM for each class, in a restricted maximum likelihood (REML) analysis using ASReml 4.1   to measure how much variance each variant capture in a class. To perform this analysis, GCTA  was usedThe synonymous, DNA methylated regions and upstream classes capture the highest proportion of variance across most traits, Fig.\u00a0Out of the intragenic classes, the synonymous class captures the highest proportion of the variance for all traits, followed by the splice site class; in which the average proportion of the variance captured across traits was 8%. For variants in the missense deleterious and missense tolerated classes, the proportion of variance captured was, unexpectedly, almost nil. In the previous variance component analysis, comparable results were observed, except for fat percent Fig.\u00a0. These rAs the number of variants in each class varies greatly, we investigate how much variance is explained (on average) by each individual variant for all classes, Fig.\u00a0Out of the regulatory classes, the DNA methylated class explained most of the variance per sequence variant in fat percent and protein percent. While for variants in the miRNA target sites class, we observe a modest proportion of the variance explained per variant, particularly for fat, milk and protein. The variance explained per sequence variant in the lncRNA and TFBS was also relatively modest, particularly for the traits fat, protein percent and fertility, which explain slightly more variance than that in lncRNA. The upstream and downstream classes, on the other hand, capture very little of the variance per sequence variant Fig.\u00a0 which doAs with the previous analysis, a MAF threshold of 0.001 was used to filter out sequencing and imputation errors. The above analyses were also performed using an MAF threshold of 0.000000001 and 0.01, and those results can be found in Additional\u00a0files\u00a0Cow and bull phenotypic records were available for the traits milk volume, fat volume, protein volume, fat percent, protein percent and fertility. The bull dataset includes records from thousands of cows to obtain the daughter trait deviations, the majority of which are not present in the cow dataset. Therefore, the cow and bull datasets are very close to independent, and we can use them to cross-validate the results (given we expect to see few sex specific differences). As with the previous analysis, the GRM for all functional classes was fitted simultaneously in the model, only this time, the REML was calculated using phenotypic records strictly from either cow or from bulls. Both the proportion of variance captured and the variance explained per sequence variant for each class was recorded for the cow and bull phenotypic records.Overall both sexes follow very similar trends in the proportion of variance captured by each class Fig.\u00a0 and b, aWhole genome sequence variants were annotated into 13 functional classes, then genotypes for these variants were imputed into a large population of dairy cattle with milk and fertility phenotypes, to test the hypothesis that variants in some annotation classes will explain more variance than others for these traits. We find for most traits, the asRNA, splice site regions, 5\u2019 UTR, miRNA target sites and TFBS classes explained more variation than expected by chance, given the number of variants in these classes. The second variance component approach fitted the GRM constructed from variants in each class simultaneously. With this approach it was observed that variants in DNA methylation target regions (at least in bovine placenta), synonymous and upstream classes captured a considerable proportion of the variance. On a per sequence variant basis, variants in the splice site regions had the highest proportion of variance explained per sequence variant for most traits. Finally, we assessed bull and cow phenotypes in separate analyses to investigate any sex differences; finding that there was little evidence for this.Many authors propose that variants found in coding regions (intragenic variants) have the greatest potential to be functionally important and more likely to contribute to trait variation . Out of The most unexpected results in this study is the little to no variance captured by the missense deleterious and tolerated classes Fig.\u00a0. Only foAnother explanation is due to the potentially larger number of sequence and imputation errors present in the missense classes, given the low MAF for this class of variant. As shown in Fig.\u00a0The synonymous class, surprisingly, captured a much greater proportion of the variance than the missense deleterious class did for all traits. There are a number of possible explanations for this observation. Some studies have found that synonymous mutations are enriched for trait associated variants , 17, witHuman studies find that phenotypic variation for complex traits occurs in regulatory regions and highly conserved regions , 22. OurThe lncRNA and asRNA classes are known to have important regulatory functions in the cell  and mutaThe study by Das et al.  is one oA limitation in the variance component analysis 2, when all GRM across all classes were fitted simultaneously, we observed that different complex traits are affected by different annotation classes. One example of this is the trait fertility, which seems to be significantly affected by variants in the upstream class Fig.\u00a0. These rOne important limitation that impacts this study has to do with the state of annotation of the bovine genome. Compared to human and mouse genomes, the current annotation state of the bovine genome is relatively poorly characterized . This leThis study has shown that by using variance component analyses, sequence variants annotated in certain classes explain more of the variance than expected by chance, given the number of variants in the class. In addition to this, variants annotated in some of these classes explain substantially more trait variance on a per sequence variant basis . Many regulatory classes, particularly sites that have been observed to be methylated in some cases, lncRNA, miRNA target sites and TFBS captured modest to large proportions of the variance. The synonymous and splice site variants captured some of the highest proportions of the variance out of the protein coding classes. Further, the splice site class captured the greatest proportion of variance, per SNP, for most traits. We propose that splice site variants, and RNA splicing, should be of greater focus in future work to understand the associations these variants have in complex dairy traits. A limitation in the current study was the accuracy of imputation, particularly of variants with low MAF, and it is important to recognize this may have had an impact on our results, particularly for those annotation classes with many low MAF variants, such as the missense class.Bos Taurus sequences from a range of dairy and beef breeds (including 311 Holstein and 61 Jersey bulls). A total of 28.3 million sequence variants were available for this study.Sequence genotypes from real and imputed 800\u00a0K SNP Chip array genotypes  were imputed using Fimpute software  into fulSequence variants were annotated into the following classes; intergenic, intragenic, exon, intron, CDS, 5\u00a0kb upstream of a transcription start site, 5\u00a0kb downstream of a gene, frame-shift variants, splice site region variants and stop codons classes by querying the Ensembl variant database version 77 . Splice The classes missense deleterious, missense tolerated, synonymous, 3\u2019 UTR, 5\u2019 UTR were annotated in a previous study using the NGS-SNP tool . We querMicroRNA predicted target sites were obtained from the MicroCosm target site database . TFBS weThe DNA methylated regions were obtained from the paper by Su et al.  that prePatterns of DNA methylation have been reported to remain static between tissues and throughout the life of a cell , howeverLong noncoding RNA were obtained from the study by Koufariotis et al.  and fromThe variance component analysis was performed to determine if SNP in a functional class explain more variance than the variance explained by the same number of randomly selected variants from a random permutation test. The genome relationship was calculated for each functional class of SNP according to the Yang et al. method .To measure the similarities between each GRM for all functional class of SNP , the Euclidean distance was calculated using the following formula:m and p is the corresponding GRM for each class.Where To calculate the proportion of variance explained by each functional class of SNP, a REML analysis was performed by fitting the following model to the datay denotes a vector of the dairy phenotypic records obtained from the following study [b is a vector of fixed effects that includes the breed and sex, x is the design matrix that allocates the records to the fixed effects, z is design matrix that allocates records to breeding values and g denotes a vector of random breeding values obtain from the following formula:Where ng study , and theng study , b is a 2) from the above models.Where g is the GRM for the functional classes of SNP, and sion 4.1  was usedn SNP, where n corresponds to the number of variants found in the functional class while selecting for variants that had similar allele frequencies as the functional class they represent. For each class, the same number of variants was randomly selected from the sequence variant dataset and the GRM was calculated from the random set. ASReml 4.1 [The random permutation test involves calculating the variance explained from a randomly chosen set of Reml 4.1  was usedTo calculate the significance of the enrichment or depletion of a class Table\u00a0, the perh2 denotes the actual variance explained (heritability) for a functional class of SNP and the rh2 represents the variance explained from the random permutation test for each functional class, which is the average heritability obtained by the 5 iterations of the random permutation test.Where In the previous analysis, classes that have very large numbers of variants will capture most of the heritability for each trait, regardless if the variance explained is from the actual functional class being tested, or from the random permutation test set. This makes it particularly difficult to determine the true additional variance explained. To get around this, the total proportion of genetic variance a functional class explains was determined by fitting the GRM from all functional classes simultaneously in the model. The tool Plink  was usedh2 represents the variance captured by each functional class, divided by the total sum of all the variance captured for each trait and multiplied by 100.Where Further, to calculate the variances explained on a per sequence variant basis the following formula was applied to each class:h2 represents the variance captured by the class (heritability) and is divided by n, the total number of variants found in the class. This result was multiplied by 100 to get a percentage of the variances explained by the class and then divided the result by 10\u22124 so that the results can be visually represented.Where Additional file 1:Minor Allele Frequencies: Full matrix of the MAF for all classes. (XLSX 13\u00a0kb)Additional file 2:Euclidean matrix with distance values showing the similarities between functional class GRM. The more similar the GRM is between two classes the lower the Euclidean distance measure is. This is also represented by the color green. The more dissimilar the GRM is between two classes the higher the Euclidean distance measure is. Represented by the red color. (XLSX 10\u00a0kb)Additional file 3:The heritability for each trait along with the permutated heritability obtained from the permutation test using the same number but randomly chosen SNPs (which was replicated 5 times and significance is determined as greater or less than the average of the proportion of variance explained by the randomly chosen SNP\u2009\u00b1\u20092\u2009\u00d7\u2009S.E). The heritability percent difference is simply the difference between the class heritability and the permutated heritability multiplied by 100. (XLSX 13\u00a0kb)Additional file 4:Variance Component Analysis 2: performed using variants with MAF thresholds of 0.000000001 and 0.1. For each functional class is the proportion of variance captured by each class, along with the total proportion of variance captured for each trait represented as a percentage, in brackets. (XLSX 11\u00a0kb)Additional file 5:Variance Component Analysis 2: performed using variants with MAF thresholds of 0.000000001 and 0.1. For each functional class is the variance explained per sequence variant along with the variance component. (XLSX 10\u00a0kb)"}
+{"text": "Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus mosquitoes and determined infection rates, dissemination rates, and transmission rates. We found the highest vector competence for the imported Zika virus in Ae. aegypti mosquitoes, some susceptibility of Ae. albopictus mosquitoes, but no transmission ability for Cx. quinquefasciatus mosquitoes. Considering that, in China, Ae. albopictus mosquitoes are widely distributed but Ae. aegypti mosquito distribution is limited, Ae. albopictus mosquitoes are a potential primary vector for Zika virus and should be targeted in vector control strategies.In China, the prevention and control of Zika virus disease has been a public health threat since the first imported case was reported in February 2016. To determine the vector competence of potential vector mosquito species, we experimentally infected  However, vector competence  differs among mosquitoes of different species and among virus strains. Ae. aegypti mosquitoes collected from Singapore are susceptible and could potentially transmit Zika virus after 5 days of infection; however, no Zika virus genome has been detected in saliva of Ae. aegypti mosquitoes in Senegal after 15 days of infection   and Guangzhou (in 1993) in Guangdong Province. In 2005, the China Center for Disease Control and Prevention collected Ae. aegypti mosquitoes from the city of Haikou in Hainan Province. All mosquitoes were maintained under standard insectary conditions of 27 \u00b1 1\u00b0C, 70%\u201380% relative humidity, and a light:dark cycle of 16 h:8 h. To obtain enough individuals for the experiments, we collected eggs from mosquitoes of all 3 species and hatched them in dechlorinated water in stainless steel trays. The larvae (150\u2013200/L water) were reared and fed daily with yeast and turtle food. Pupae were put into 250-mL cups and placed in the microcosm (20 cm \u00d7 20 cm \u00d7 35 cm cage covered with nylon mesh) until they emerged. Adults were kept in the microcosms and given 10% glucose solution ad libitum.The Guangdong Provincial Center for Disease Control and Prevention collected ,10 copies/\u03bcL) was passaged once through C6/36 cells before the mosquitoes were infected. The fresh virus suspension (5.45 \u00b1 0.38 log10 copies/\u03bcL) was used to prepare the blood meal.Zika virus (GenBank accession no. KU820899.2), provided by the Guangdong Provincial Center for Disease Control and Prevention, was originally isolated from a patient in China in February 2016 and classified as the Asian lineage  with fresh virus suspension at a ratio of 1:2. The blood meal was warmed to 37\u00b0C and transferred into a Hemotek blood reservoir unit . Mosquitoes were then fed by using the Hemotek blood feeding system. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the virus concentration (copy level) in the blood meal before and after feeding. After 30 min of exposure to the infectious blood meal, mosquitoes were anesthetized with diethyl ether. Fully engorged females were transferred to 250-mL paper cups covered with net (10\u201315 mosquitoes/cup). The infected mosquitoes were provided with 10% glucose and maintained in an HP400GS incubator  at 28\u00b0C, 80% relative humidity, and a light:dark cycle of 16 h:8 h. The experiments were conducted according to standard procedures in a Biosafety Level 2 laboratory.We transferred 5\u20137-day-old female Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus mosquitoes, we selected 18\u201330 mosquitoes at postinfection days (dpi) 0 , 4, 7, 10, and 14. Each mosquito was placed in 50 \u03bcL TRIzol  and homogenized in a tissue grinder . Total RNA was extracted according to the manufacturer\u2019s protocol of TRIzol reagent and dissolved in 20 \u03bcL RNase-free water.To determine Zika virus infections in Zika virus cDNA synthesis reaction was performed by using the GoScript Reverse Transcription System . Total RNA and 10 \u03bcM Zika virus reverse primer (3\u2032 untranslated region: 5\u2032-ACCATTCCATTTTCTGGC-3\u2032) were incubated, and cDNA was synthesized according to the procedure.https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome), which was selective for 296-bp nucleotide . The following PCR conditions were used: 94\u00b0C for 3 min, followed by 35 cycles of 94\u00b0C for 30 s, 60\u00b0C for 30 s, 72\u00b0C for 30 s, and 72\u00b0C for 7 min. The PCR products were examined by use of 1% agarose gel electrophoresis. The target fragment was cloned into pMD18-T vector  and sequenced.The nonstructural protein 1 (NS1) primers of Zika virus were designed by NCBI/Primer-BLAST . A standard curve  was generated from a range of serial 10-fold dilutions of the plasmid (6.23 \u00d7 102\u20136.23 \u00d7 107 copies/\u03bcL).The viral genome in the Zika virus\u2013positive mosquitoes was determined by using absolute qRT-PCR. First, we constructed the standard. A 141-bp fragment across capsid and propeptide regions of Zika virus was amplified by specific primers  and cloned into pMD18-T vector. After sequencing, the recombinant plasmid was linearized by Each 20 \u03bcL of qRT-PCR was amplified by a 7500 Real-Time PCR System  under the following conditions: 1 cycle at 50\u00b0C for 2 min, 95\u00b0C for 2 min; 40 cycles at 95\u00b0C for 15 s, 60\u00b0C for 15 s, and 72\u00b0C for 1 min. Zika virus RNA copies from each sample were quantified by comparing cycle threshold value with the standard curve. The efficiency of this qRT-PCR system was evaluated by using blank control, uninfected C6/36 cells, C6/36 cells infected with Zika virus or DENV, and mosquitoes infected with Zika virus or DENV; the result displayed that its minimum detecting amount is 6.23 copies/\u03bcL of Zika virus and that the specificity is 100%.Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus mosquitoes, we infected another batch of mosquitoes with Zika virus and then dissected the midgut, head, and salivary glands of each mosquito at dpi 0, 4, 7, 10, and 14 by using 18\u201330 mosquitoes per time point. The legs and wings of mosquitoes were removed and placed into cold phosphate-buffered saline. Each tissue was dissected and washed 3 times in phosphate-buffered saline and transferred to 50 \u03bcL TRIzol . Logistic regression was used to compare the infection, dissemination, and transmission rates for different mosquito species at the same time or for the same species of mosquito at different times. p value significance was corrected by Bonferroni adjustments. The Zika virus RNA copy levels were log-transformed and then compared among mosquitoes of different species at the same time or among mosquitoes of the same species at different times by using post hoc Tukey honest significant difference tests.Ae. aegypti and Ae. albopictus mosquitoes at dpi 4, 7, 10, and 14   and Ae. albopictus mosquitoes  (Cx. quinquefasciatus mosquitoes (After the 414 mosquitoes (138 of each of the 3 species) had ingested the infectious blood meal (dpi 0), RT-PCR indicated that all were Zika virus positive , panel A, and 14 , panel A p>0.05) , panel A p<0.01) , panel Asquitoes , panel AAe. aegypti and Ae. albopictus mosquitoes was an increase with time after infection, but that for Cx. quinquefasciatus mosquitoes was a decrease , then increased gradually. For Ae. albopictus mosquitoes, the trend for copy levels of Zika virus was similar to that for Ae. aegypti mosquitoes, but levels were slightly lower before dpi 7 (p<0.05). However, the copy levels were the same for mosquitoes of the 2 species at dpi 10 and 14 (p>0.05). For Cx. quinquefasciatus mosquitoes, the virus copy levels were low before dpi 7 and totally diminished afterward  . Compared with the infection rates for Ae. aegypti and Ae. albopictus mosquitoes, that for Cx. quinquefasciatus mosquitoes was significantly lower at dpi 4  and 7 , and no midguts were positive for Zika virus at dpi 10 and 14  were infected by Zika virus, and the midguts were measured; the overall infection rates were 89.86% for squitoes . At dpi  p>0.05) , panel A0 and 14 , panel AAe. aegypti mosquitoes started from dpi 4 and increased rapidly up to 100% after dpi 7   and 7\u201310 for Ae. albopictus  mosquitoes. Overall, Zika virus infection was disseminated in 73.39% of midgut-infected Ae. aegypti mosquitoes but only 42.15% of midgut-infected Ae. albopictus mosquitoes  , panel Bsquitoes . Zika viAe. aegypti mosquitoes, the detection of Zika virus in salivary glands was consistent with that in heads  was lower at dpi 14 than that for Ae. aegypti mosquitoes at the same time   for Ae. albopictus mosquitoes. Zika virus was detected in the salivary glands of 78 (62.90%) midgut-infected Ae. aegypti and 29 (23.97%) midgut-infected Ae. albopictus mosquitoes. Furthermore, the population transmission rates of Zika virus were 56.52% for Ae. aegypti, 21.01% for Ae. albopictus, and 0% for Cx. quinquefasciatus mosquitoes  , panel C10) in midguts of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus mosquitoes did not differ significantly at dpi 0 (p>0.05). For Ae. aegypti mosquitoes, the Zika virus copies (log10) of midguts at dpi 4 were rapidly raised to 5.96 \u00b1 0.92, which was higher than that at dpi 0 (5.00 \u00b1 0.34) (p<0.05). Levels then increased continuously over time and reached 6.82 \u00b1 0.47 at dpi 14 . After that, the growth of Zika virus became rapid and the Zika virus copies (log10) at dpi 14 reached 7.20 \u00b1 0.48, which exceeded that in Ae. aegypti mosquitoes (p<0.05)  , panel Anfection , panel A10) in heads of Ae. aegypti mosquitoes continually increased from dpi 4 (4.97 \u00b1 0.45) to 14 (6.19 \u00b1 0.46) but remained stable for Ae. albopictus mosquitoes from dpi 7 (4.82 \u00b1 0.43) to 10 (4.82 \u00b1 0.64) and then reached 6.95 \u00b1 0.81 at dpi 14  (Ae. aegypti mosquitoes. Compared with the Zika virus copies (log10) at dpi 0 (0), the value (5.54 \u00b1 0.52) was apparently higher at dpi 14 (p<0.05)  was higher than that at dpi 10 (4.92 \u00b1 0.85). Furthermore, the number of virus copies in Ae. albopictus mosquitoes was higher than that in Ae. aegypti mosquitoes at dpi 14 (p<0.05)  , panel B(p<0.05) , panel C(p<0.05) , panel CAe. aegypti and Ae. albopictus mosquitoes in China possess the ability to transmit Zika virus, whereas Cx. quinquefasciatus mosquitoes were not able to transmit the virus under our laboratory conditions.Because of the absence of vaccines and specific treatment, the major approach to prevention and control of Zika virus disease is vector control (Ae. aegypti mosquitoes could serve as vectors to spread Zika virus in China and that Ae. aegypti mosquitoes were better vectors than Ae. albopictus mosquitoes because transmission rate was higher and extrinsic incubation period was shorter for the former. The strong vector competence of Ae. aegypti mosquitoes could be associated with Zika virus rapid reproduction in the midgut during dpi 0\u20134, which enabled the viral particles to easily overcome the midgut barrier and be released into the hemolymph cavity and invade the salivary gland (Ae. aegypti mosquitoes from Singapore and Italy (,Ae. aegypti mosquitoes is very limited in southern China, ranging from latitude 22\u00b0N to 25\u00b0N (Ae. aegypti mosquitoes for Zika virus required the authorities in China to pay close attention to local epidemics of Zika virus in these regions.Our results demonstrate that Ae. albopictus and Ae. aegypti mosquitoes were similar, but the replication of Zika virus in midgut was slower for Ae. albopictus mosquitoes. The dissemination and transmission of Asian genotype Zika virus by Ae. albopictus mosquitoes in China started on 7 and 10 dpi, respectively, which indicated lower vector competence than that for Ae. albopictus mosquitoes from Singapore infected with East African genotype Zika virus from Uganda but higher than that for Ae. albopictus mosquitoes from the Americas infected with Asian genotype Zika virus from New Caledonia (,Ae. albopictus than for Ae. aegypti mosquitoes, Ae. albopictus mosquitoes are widely distributed in China, especially in Guangdong Province, where dengue was often epidemic (Ae. albopictus mosquito density and survival time has increased with urbanization (,Ae. albopictus mosquitoes can potentially become the primary vector for Zika virus in China and need attention in the vector control strategy.Under the same experimental conditions, the whole-mosquito infection rates and midgut infection rates for Cx. quinquefasciatus are common blood-sucking mosquitoes in China, especially in southern cities, and are the vector of Western equine encephalitis virus (Cx. quinquefasciatus mosquitoes had ingested the virus, but the infection rate and Zika virus copies gradually decreased and no virus was detected in any tissues after dpi 7. The few positive midgut samples before dpi 7 could have resulted from an undigested blood meal because Cx. quinquefasciatus mosquitoes are larger and might take more blood than Aedes mosquitoes. Our results illustrate that Cx. quinquefasciatus mosquitoes in China are not able to transmit Zika virus, a finding that is consistent with the Zika virus susceptibility of Cx. pipiens mosquitoes from Iowa, USA, and Cx. quinquefasciatus mosquitoes from Rio de Janeiro, Brazil (,Cx. p. quinquefasciatus mosquitoes are potential Zika virus vectors in China (In our study, Zika virus from C6/36 cells or infected mosquitoes was sensitively and specifically identified by qRT-PCR. We used qRT-PCR to detect virus copies because the Zika virus strain isolated from the patient who imported the virus into China can infect C6/36, Aag2, and Vero cells but did not show obvious cytopathic effect, which could be associated with the patient\u2019s mild clinical signs. Furthermore, previous research proved that the viral copies calculated by qPCR were consistent with the PFU detected by plaque assay (Ae. aegypti and Ae. albopictus mosquitoes are susceptible to Zika virus, whereas Cx. quinquefasciatus mosquitoes are not able to transmit the imported Zika virus. Comparatively, the vector competence of Ae. albopictus mosquitoes is inferior to that of Ae. aegypti mosquitoes, but considering their wide distribution, Ae. albopictus mosquitoes might become the primary vector for Zika virus in China. These updated findings can be used for Zika virus disease prevention and vector control strategy.In conclusion, our findings indicate that in China,"}
+{"text": "Aedes caspius and Aedes albopictus mosquitoes in Spain for the transmission of Zika virus. Whereas Ae. albopictus mosquitoes were a competent vector, Ae. caspius mosquitoes were unable to transmit Zika virus. We also identified high levels of vertical transmission of Zika virus in Ae. albopictus mosquitoes.We assessed the vector competence of  Flaviviridae primarily transmitted by Aedes aegypti mosquitoes, but other Aedes species mosquitoes could be competent vectors  and F2 generation of Ae. albopictus mosquitoes  to Zika virus through infectious blood meals. We used F8 generation of colonized populations of Ae. aegypti mosquitoes  as a control population and Zika virus strains CAM  and PR , passaged 4 times on Vero cells and 2 times on C6/36 cells. We propagated on C6/36 cells for 4 days, and freshly harvested supernatant was mixed 1:1 with sheep blood  and 2.5% sucrose (We determined vector competence at different days postinfection (dpi) by exposing F0 generation of Ae. albopictus and Ae. aegypti female mosquitoes infectious blood meals containing either the CAM or PR strain at a final concentration of 7.6 log10 PFU/mL. Infection rates were determined by screening mosquitoes\u2019 bodies, dissemination rates by screening legs, and transmission rates by screening saliva, at 3 different time points  using Zika-specific quantitative reverse transcription PCR including negative controls in each reaction  for each trial  (To determine the ability of http://www.jmp.com).We performed generalized linear models with binomial error distribution and logit link function to assess the effect of mosquito species, virus strains, and dpi on the infection, dissemination, and transmission rates. We also considered the interactions between virus strain and dpi and between virus strain and mosquito species. We determined differences in mean viral titers between mosquito species, virus strains, and dpi in mosquito body, legs, and saliva using Kruskal-Wallis tests. Analyses were run in JMP version 9  but was similar between mosquito species and Zika strains. Transmission rate also increased with time, and mosquitoes infected with Zika CAM showed a higher transmission rate than those infected with Zika PR. Transmission rate did not differ between Ae. albopictus and Ae. aegypti mosquitoes  and higher titers for Zika PR compared with Zika CAM . Mean viral titers in legs were similar for both Zika strains , but were higher in Ae. aegypti relative to Ae. albopictus mosquitoes . Mean viral titers did not differ in saliva secretions between mosquito species  or Zika strains . We detected Zika virus infection in Ae. caspius mosquitoes at 7, 14, and 21 dpi, but detected no virus dissemination or transmission at any point  were positive for Zika virus, with a filial infection rate of 72.2 (95% CI 27.6\u2013156.1) and mean viral load of 2.5 log10 PFU/mL. This value equates to a ratio of 1:14.Infection rate was higher in squitoes ,2. Mean ny point . Five laAe. albopictus mosquitoes in Spain are competent vectors of Zika virus at levels similar to Ae. aegypti mosquitoes. We detected Zika CAM in saliva earlier than Zika PR, which suggests that genetically variable strains may have different transmission potential (Ae. albopictus mosquitoes from Spain and Italy (Ae. albopictus mosquitoes from Spain could transmit Zika virus at 7 dpi, 4 days earlier than mosquitoes in Italy (Ae. caspius mosquitoes, it is unable to escape the midgut and be effectively transmitted (Our results suggest Ae. albopictus mosquitoes in Spain at substantially higher rates than found in Ae. albopictus mosquitoes from New York and Italy (Ae. albopictus mosquitoes in Spain.Zika virus is vertically transmitted by the population of Ae. albopictus mosquitoes increase the risk for Zika virus transmission in Spain. The high number of imported Zika virus cases and the rapid spread of Ae. albopictus mosquitoes contribute to the risk for autochthonous transmission of Zika virus. The risk for transmission by Ae. caspius mosquitoes, however, may be considered extremely low.Our results confirm that populations of Aedes caspius and Ae. albopictus mosquitoes for Zika virus, Spain.Additional information about vector competence of"}
+{"text": "Inhalants are often abused\u00a0due to their ability to\u00a0acutely induce feelings of euphoria. Difluoroethane is a toxic lipophilic hydrocarbon that crosses the blood-brain barrier and inhibits the central nervous system. Studies have shown the cardiac, renal, and respiratory effects it has when abused; however, our literature review yielded no previous report of acute psychosis after difluoroethane inhalation. In order to prevent poor outcomes by missed diagnosis, we present a case of difluoroethane-induced acute psychosis.\u00a0 In the United States, there is a nine percent lifetime prevalence of inhalant abuse\u00a0. InhalanA 36-year-old female\u00a0patient was brought to the ED of the local community hospital after she was found unconscious and unresponsive on a grocery store\u2019s bathroom floor.\u00a0She later admitted to huffing \u201cdusters.\u201d She had consumed an unknown number of computer cleaner canisters to get high. She presented with auditory and visual hallucinations.\u00a0She reported delusions that she would give birth to Jesus Christ. She was also experiencing visual hallucinations of blurred images. She also reported hearing the voice of \u201cJesse,\u201d a former boyfriend. She endorsed depressive symptoms with sad mood, anhedonia, fatigue, and feelings of hopelessness. Despite being psychotic and depressed, she had\u00a0no cognitive impairment, suicidal ideations, or aggressive behavior. Her medical history includes anxiety, hypothyroidism, polycystic ovarian syndrome, endometriosis, and celiac disease.\u00a0She is prescribed clonazepam 1 mg TID, levothyroxine 50 mcg QD, loratadine 1 tab QD, glucophage 500 mg BID, and lisinopril 5 mg QD by her primary care physician. Urine toxicology screen at presentation was positive for benzodiazepine consistent with home medications and tetrahydrocannabinol\u00a0consistent with a self-reported recreational use of marijuana. Notably, the patient endorsed a several year history of daily\u00a0marijuana use of small quantity with no previous psychotic symptoms or adverse psychiatric effects. Due to the new onset of psychosis and melancholic depressive symptoms, the patient was admitted to the inpatient behavioral health unit.Treatment with olanzapine 20 mg QHS was initiated on the night of her arrival to the behavioral health unit, and on the fifth day she no longer displayed psychotic delusions or hallucinations. Olanzapine was preferred by the physician as the patient displayed insomnia and agitation, and also for its available formulations both PO and IM as needed to manage her psychosis. She tolerated\u00a0the medication well and\u00a0noted improvements of both her depressive and psychotic symptoms. She engaged appropriately in individual and group therapy sessions. After a five day hospital stay, the patient was transferred to an inpatient substance abuse program.\u00a0The Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition DSM-5) identifies inhalant use disorder as \u201ca problematic pattern of use of a hydrocarbon-based inhalant substance leading to clinically significant impairment or distress\u201d with the requirement of meeting two of 12 specific qualifiers relating largely to impairment in daily function\u00a0 identifi. This caAcute adverse effects of difluoroethane inhalation include loss of consciousness, frostbite at mucosal surfaces, rhabdomyolysis, and global myocardial hypokinesis. At the cardiac myocyte, fluorinated hydrocarbons alter potassium and calcium currents, leading to dysrhythmias and prolonged atrial refractory time\u00a0. These eInhalant-induced psychotic disorder is defined in the DSM-5 as psychosis with evidence from \u201chistory, physical examination, or laboratory findings that the deficits are etiologically related to the effects of inhalant substances\u201d\u00a0-9. It isOur patient\u2019s urine toxicology screen tested positive for cannabis, and studies have shown cannabis to induce acute psychosis. Recent literature notes that chronic cannabis use is related to psychosis and schizophrenia later in life\u00a0. No trueThis novel presentation suggests the need for further exploration into diagnosis and treatment of hydrocarbon intoxication. The standard toxicology screen done today does not confirm inhalant abuse; therefore, it is important to consider this as a source when a patient presents with altered mental status and acute psychotic symptoms. Difluoroethane abusing patients are acutely at risk for cardiac failure, and early identification can be life-saving. Thus, clinical suspicion for difluoroethane intoxication should be maintained for patients presenting with unexplained psychosis so as not to miss a crucial and time-sensitive diagnosis.This case report highlights the need to consider difluoroethane inhalation for patients presenting to the ED with acute psychosis unexplained by prior diagnosed psychotic disorder and a negative urine toxicology screen. 1,1-Difluoroethane abuse has been associated in publication with mortality by causing impairment leading to trauma, anhedonia resulting in suicide, and cardiac compromise.\u00a0Appropriate clinical suspicion can allow timely diagnosis which might\u00a0facilitate proper workup for critical adverse effects including myocardial hypokinesis and dysrhythmias, thus preventing delays in treatment."}
+{"text": "Extraskeletal Ewing sarcoma (EES) is an uncommon tumor with low prevalence in the head and neck region. Herein, we report a 13-year-old boy with EES in the temporal region, which was managed by surgery and chemotherapy. The histological characteristics and the clinical manifestations of the lesion and our surgical approach will be discussed as well. Ewing sarcoma (ES) is an invasive malignant tumor characterized by small round blue cells. It is often found in long bones and the pelvis during childhood . ExtraskTumors that primarily occur in the head and neck region are often found in the nasal cavity, oral cavity, paranasal sinuses or the soft tissue of the neck ,5. Hereiclinic of our institute complaining of a swelling in his right temporal region developed one month earlier. Clinical examination revealed a palpable swelling above the zygomatic arch and behind the lateral orbital rim, which became more prominent when the patient clenched his teeth.The patient had no pain, trismus or tenderness. Redness was not seen either. Neurological assessment was unremarkable. The patient did not report any weight loss. Magnetic resonance imaging (MRI) revealed a hyperintense well-defined mass in the right temporal fossa measuring 4 cm \u00d7 2.5 cm .An open biopsy was scheduled for the patient. We approached the lesion by making an incision over the temporal region and above the zygomatic arch. The lesion was located medial to the temporalis muscle. It contained a cystic fluid which was aspirated. The lesion did not have any adhesion to the surrounding tissues and was easily dissected.Hematoxylin and eosin (H&E) staining revealed atypical cells with round to oval nuclei and prominent nucleoli. Immunohistochemical (IHC) staining revealed positive CD99 , S100 F, and NSEThe patient was referred to an oncology clinic where he underwent a whole body positron emission tomography (PET) scan which showed no metastasis or primary origin. The patient underwent 14 sessions of chemotherapy with doxorubicin, etoposide, ifosfamide, cyclophosphamide, mesna, actinomycin D, and vincristine. He has been followed up for 3 years so far with no evidence of recurrence or metastasis and has an excellent lifestyle.EES is a rare tumor accounting for 1.1% of soft tissue malignancies. It usually affects males between the ages of 15 and 30 years and has a high recurrence rate . Its cliLocalized EES is treated by surgical excision in combination with adjuvant or neoadjuvant chemotherapy. In the present case, the surgical margins were free from the tumor, and the patient was referred to an oncology clinic and received 14 sessions of chemotherapy. Fortunately, he had good prognostic factors such as the absence of metastases, tumor size less than 10 cm in diameter, and clear surgical margins.Due to fallacious characteristics of these lesions, one must care when facing such clinical and radiologic manifestations, especially when they present at unusual sites. Clinical features tend to be nonspecific and may mimic common benign conditions.Our presented case highlights the significance of the inclusion of EES in the list of differential diagnoses of facial soft tissue lesions. IHC staining is highly applicable for such cases. After reaching a diagnosis of EES, an invasive multimodal treatment is imperative."}
+{"text": "A joint space-time array for communication signals is constructed in this paper to settle the contradiction between the performance of angle estimation and the array aperture. It introduces Doppler information caused by platform motion into the signal processing to obtain favorable performance with limited array aperture. We analyze the theoretical performance in the aspects of distinguishable source number, spatial resolution and Cram\u00e9r-Rao bound (CRB), respectively. Both theoretical analysis and simulation results demonstrate that the proposed space-time array can give rise to a significant enhancement in achievable array performance. Direction of arrival (DOA) estimation is an important branch of array signal processing. The performance of DOA estimators mainly depends on the aperture of the array. In the modern electromagnetic environment, antenna arrays may be deployed on moving platforms  to enhance their maneuverability . HoweverVarious methods have been proposed to solve this problem, for instance, high order cumulant , cyclostIn other studies, some compressive sensing (CS)-based DOA estimation algorithms take the moving array condition into consideration. Reference  formulatAlternatively, the synthetic aperture (SA) technique, which utilizes Doppler information to improve DOA performance, has received increasing attention in moving array systems. This technique was firstly used in cooperative scenarios for radar imaging ,11 and tTo the best of our knowledge, methods intended for communication signals with moving array have not received much attention. On the basis of the existing SA techniques, we propose a space-time array model for moving arrays in this paper. Its performance is thoroughly analyzed for comparison with a conventional array. First, the distinguished source number is derived under different conditions. Then we utilize the Euclidean norm to analyze the array spatial resolution enhancement. Finally, the Cram\u00e9r-Rao bound (CRB) is computed in order to evaluate the estimation accuracy. The theoretical results are further discussed so as to cope with the parameter selection problem for space-time arrays. In contrast to conventional arrays, the proposed space-time array introduces Doppler information into the DOA estimation and manifests superior performance. Numeric simulations are also provided to demonstrate its effectiveness.The paper is organized as follows: d , and the platform moves along its baseline at a constant velocity Assume that a uniform linear array is equipped on a moving platform. The array is composed of Q far-field plane waves impinge on the array from directions f0, which is known to the observer. c is the velocity of the electromagnetic wave. Because of movement of the array, a Doppler shift We assume that m-th sensor at time t as:q-th source with power Regarding the first sensor as a reference, we can express the observation signal of the Since From Equation (2), we can find that the time domain samples constitute a geometric sequence like a uniform linear array manifold, so we consider creating a manifold structure in the time domain which is similar to the space domain for obtaining the space-time array.Supposing that p-th segment, the observed signal of the m-th sensor at the j-th sample moment can be expressed as:p-th segment. Besides, the phase shift of every sample moment during the same segment can make up a vector as follows:Then in the m-th sensor in the p-th segment, we can represent q-th source. m-th sensor. We define The vector To express Equation (6) in matrix form, we define The modified model transforms temporal samples into virtual sensors, which is equivalent to changing an This section presents the performance analysis of the space-time array. The comparison with a conventional array is implemented to demonstrate its advantages in the aspects of distinguishable source number, spatial resolution and CRB.The number of sources that an array can specify is mainly dependent on its sensor number and the rank of the source sample-correlation matrix. As the equivalent sensor number of a space-time array is enlarged, the distinguishable source number is supposed to increase. We provide a detailed analysis below.Wax and Ziskind have deduced relevant conclusions in . Their wlts from , we can For space-time arrays, the condition is slightly more complicated. The maximum of ondition :(9)Qmax\u2208MJ, but a range from sT(1/ff). Equation (4) indicates that the equivalent element spacing of We note that the distinguishable source number of he space-time array is increased, which coincides with our expectation. However, its maximum is not equal to the virtual sensor number Resolution is one of the important indexes for measuring the array performance. The spatial resolution of an array is closely correlated with the changing rate of the steering vector. To intuitively compare the resolution of different arrays, a scalar  ability :(10)\u03b7(\u03b8)Here we suppose that Proof.\u00a0See We can observe that Equation (13) indicates that the space-time array can improve the spatial resolution in the scenario assumed in this paper. Moreover, we can find that In this section, we derive the DOA estimation CRB of conventional and space-time array model. Then a comparison is provided to provide some in-depth conclusions about the performance.The derivation of CRB generally relies on random signal (Gaussian) model or nonrandom  signal model. In this paper, we consider that the sources are nonrandom.We define  work of , the valThe CRB of space-time array model can also be obtained in a similar way. From Equations (6) and (7), we know that the dimension of the space-time observation signal To be convenient to make contrast between the CRB of two models, q-th signal and its value is In Equations (16) and (17), Proof.\u00a0See It is worth noting that Obviously, when Remark\u00a01.As concluded above, Remark\u00a03.The condition Remark\u00a03.Expressions of resolution and CRB have similar forms. Remark\u00a04.In this section, some computer simulations are presented to verify our deduction about space-time arrays and demonstrate its performance. In the figures of this section, the curves with postfix \u2018conventional\u2019 and \u2018ST\u2019 denote the performance curves of a conventional array and a space-time array, respectively. We consider a uniformly linear array with nine sensors. The array is equipped on an airplane and moves along its baseline at a constant velocity of 200 m/s.First, we resort to the MUSIC algorithm to check the resolving ability of the space-time array (the specific operations of the algorithm can be seen in To further demonstrate the spatial resolution enhancement, we plot the probability of the target resolution curves for different values of Next, we verify the deduction about CRB. Like In This paper constructs a joint space-time array model based on a moving platform. The proposed space-time array combines spatial information with temporal information and obtains better performance than a conventional array, which makes it applicable in those conditions assumed in this paper. The theoretical analysis demonstrates that the space-time array improves the distinguishable source number, spatial resolution and estimation accuracy. Numerical simulations validate our deduction and illustrate the reliability of the space-time array performance. Nevertheless, our method requires that the duration of the time segment be less than the coherence time of the signal. When the signal bandwidth is relatively wide, the performance of proposed space-time array will degrade severely. Thus, our future work will focus on developing methods for more general environment."}
+{"text": "Few studies have compared the surgical outcomes of different surgical procedures currently used to treat refractory colonic slow-transit constipation (STC), despite the increase in the number of cases. This study aimed to analyse the long-term surgical outcomes of subtotal colectomy with antiperistaltic caecorectal anastomosis (SC-ACRA) vs total colectomy with ileorectal anastomosis (TC-IRA) for severe STC.n\u2009=\u200935) or SC-ACRA (n\u2009=\u200920) for severe STC at our institution. The post-operative functional outcomes between the two groups were compared.Between January 2005 and January 2015, we retrospectively collected clinical data of 55 patients who underwent TC-IRA (P\u2009=\u20090.655), sex (P\u2009=\u20090.234), period of constipation (P\u2009=\u20090.105) and defecation frequency (P\u2009=\u20090.698) between the TC-IRA and SC-ACRA groups. During a median follow-up period of 72\u2009months , there were no significant differences between the TC-IRA and SC-ACRA groups regarding the median number of bowel movements per day , Cleveland Clinic Florida Constipation Score , Cleveland Clinic Incontinence Score  and Gastrointestinal Quality of Life Index . Moreover, there was no significant difference in the incidence of post-operative complications between the two groups .There were no significant differences in age (Our findings indicate that both TC-IRA and SC-ACRA are effective treatments for severe STC, with similar long-term outcomes. Colonicslow-transit constipation (STC) is defined as reduced colonic motility and stool frequency, usually accompanied by abdominal bloating; it is one of the most common types of constipation encountered by colorectal surgeons . CurrentAlthough more conservative approaches have recently been reported, ablative procedures continue to play effective roles in treating STC . Many STet al.  and the National Natural Science Foundation of China [No. 81500505 to W.C.L.] and the Natural Science Foundation of Hubei Province [No. 2015CFB636 to W.C.L.]."}
+{"text": "Clinical observations have shown that patients with chronic neuropathic pain or itch exhibit symptoms of increased anxiety, depression and cognitive impairment. Such patients need corrective therapy with antidepressants, antipsychotics or anticonvulsants. It is known that some psychotropic drugs are also effective for the treatment of neuropathic pain and pruritus syndromes due to interaction with the secondary molecular targets. Our own clinical studies have identified antipruritic and/or analgesic efficacy of the following compounds: tianeptine , citalopram (selective serotonin reuptake inhibitor), mianserin (tetracyclic antidepressant), carbamazepine (anticonvulsant), trazodone (serotonin antagonist and reuptake inhibitor), and chlorprothixene (antipsychotic). Venlafaxine (serotonin-norepinephrine reuptake inhibitor) is known to have an analgesic effect too. The mechanism of such effect of these drugs is not fully understood. Herein we review and correlate the literature data on analgesic/antipruritic activity with pharmacological profile of these compounds. Chronic pain accompanies many pathological conditions, such as diabetes, chronic renal failure, and cancer ,2,3. TheAt least half of patients suffering from chronic pain and itching are diagnosed with mental disorders such as depression and/or anxiety ,7. At thGenerally, the detection of analgesic and especially antipruritic effects of psychotropics is random, there are no certain structure criteria for determining their efficacy. In our previous studies, using molecular modelling methods, we showed that the tricyclic and tetracyclic psychotropic drugs that have the V-like conformation of their heterocyclic group , which are involved in the pain signal pathways [According to the data reported in literature, some TCAs are blockers of the pathways ,17. In 2pathways . We hypopathways . TrappinHowever, there were mismatches, too. Our own clinical studies at First Pavlov State Medical University have identified antipruritic and/or analgesic efficacy of the following compounds : tianeptRegarding analgesic and antipruritic activity of these psychotropics, certainly, there are some limitations in their effectiveness. Firstly, we studied their effect in patients having chronic itch and pain  associated with chronic renal failure (CRF) and chronic haemodialysis (CH), as well as in patients with diffuse itching accompanied by depression. It is not necessary that the compounds be helpful for other types of pain and pruritus. Secondly, the drugs were not effective for all persons. Some of them were beneficial for patients of a certain sex; some were ineffective for several subjects for unknown reason. Thirdly, the number of patients of First Pavlov State Medical University that we could observe was limited. Summarising the above, we suppose it is necessary to expand the frontiers of using of psychotropic drugs for treatment of chronic pain and pruritus.In the group of the compounds described above, there are no representatives of TCAs, gabapentinoids or serotonin-norepinephrine reuptake inhibitors (SNRI), which are also widely used for chronic pain management. That is why we have added venlafaxine, a SNRI, to the list of the drugs reviewed . We haveThe purpose of this review is to analyse and correlate the literature data and the results of our own research on analgesic/antipruritic activity and pharmacological profile of the compounds chosen. The information obtained could help to identify relationships between the pharmacological profiles of the drugs and the peculiarities of their analgesic/antipruritic efficacy, to identify \u201cblank spots\u201d and to determine the future direction for the development of effective personalised therapy in patients with pain/itch and mood disorders.In the first stage, we collated currently available information about the main receptors that are considered targets for analgesic and antipruritic therapy.The glutamatergic system plays an important role in the perception of sensory information of various modalities. Glutamatergic synapses participate in mediation of nerve impulses associated with the transmission of olfactory, vestibular, visual, tactile and pain signals. Glutamate receptors are present in all structures and regions of the central nervous system (CNS) that are responsible for responding to pain stimulation. The main participants in the transmission of pain impulse by the family of glutamate receptors are NMDAR and \u03b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) .AMPARs are more involved in acute pain processes . At the Investigation of the analgesic effect of NMDAR antagonists is ongoing, and a targeted search for potential blockers among various classes of drugs is being conducted. A randomised, double-blind, placebo-controlled study of l-4-chlorokynurenine, the precursor of 7-chlorokynurenic acid, which is in turn a well-known antagonist of the glycine site of NMDAR, was performed on healthy volunteers . The resAccording to their activation mechanism, sodium channels are divided into voltage-dependent (VGSCs or Nav) and ligand-dependent . VoltageRecent clinical and experimental studies indicate that Nav1.7 plays a crucial role in the hereditary mechanisms of neuropathic pain: mutations in the SCN9A gene were found to be associated with hereditary pain syndromes. The mutations associated with congenital pain sensitivity (CIP) lead to incomplete functioning of the Nav1.7 channels and to the inability to experience pain . Given tTo make the rational design of Nav inhibitors more powerful, the molecular mechanisms of Nav inhibition have been studied extensively. For example, in the paper , a modelThe term \u201ccalcium channels\u201d often means the voltage-dependent type of the channels, which is more common, but it should be mentioned that there are ligand-dependent calcium channels as well . AccordiN-type calcium channels are the most attractive target for analgesic drugs . These c50) equal to 4 \u00b5M, but does not block L- and N-type varieties. The antinociceptive effect of this compound was shown in rodents with formalin-induced inflammatory pain. In addition to the synthesis and testing of new drugs that can inhibit T-type VGCCs, such compounds are sought in nutraceuticals, which are food components having therapeutic properties. Thus, in the work [Analysis of the data reported in the literature in recent years revealed no clear breakthroughs in the development of new analgesic drugs among calcium channel blockers. Recently, the attention of researchers has focused on T-type calcium channels. T-type VGCCs are found in the primary afferent neurons of the spinal ganglia and in the free nerve endings. They contribute to the initiation of action potential in these sites by reducing the activation threshold . Intensithe work  it was sDespite successful in vitro experiments and preclinical testing of new calcium channel blockers, the results of their clinical trials are controversial. For example, a multicentre, double-blind, controlled and randomised trial with ethosuximide, an anticonvulsant and a T-type channel blocker, was performed recently in 114 patients with non-diabetic peripheral neuropathic pain . EarlierAnother well-known participant of nociceptive transmission and, therefore, a possible target for the analgesics, is the A-type gamma-aminobutyric acid receptor (GABAAR). It is a ligand-dependent ion (chlorine) channel in chemical synapses of the nervous system that inhibits the transmission of nervous excitation and is controlled by gamma-aminobutyric acid (GABA) . The strThere is evidence indicating that neuropathic pain can be controlled by modulating the GABAAR. It was shown that spinal administration of the GABAAR antagonist bicculine causes tactile allodynia and thermal hyperalgesia in rats . In contInteraction with the GABAAR is described not only for experimental drugs, but also for several clinically approved psychotropic drugs. Electrophysiological experiments in rat hippocampal slices showed that the antidepressants imipramine, fluoxetine and trazodone influenced the interaction of GABAAR with its antagonist pentylenetetrazole . The autThe role of the GABAAR in the transmission of different types of pain is currently being studied intensively. For example, the authors of  investigIn the paper , the \u03b16 The authors of  investigAnd finally, using cryo-electron microscopy, Masiulis and co-authors recently obtained the 3D-structures of the full-length human \u03b11\u03b23\u03b32L GABAA receptor and its complexes with the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA, and the classical benzodiazepines alprazolam and diazepam . The obtOpioid receptors were identified in the early 70s of the 20th century as the receptors that bind morphine-like substances . Opioid Currently, the efforts of researchers are aimed at the development of the opioid receptor agonists that would cause the maximum analgesic effect with minimal side effects ,105. TiwCurrently, the efforts of researchers are aimed at the development of the opioid receptor agonists that would cause the maximum analgesic effect with minimal side effects ,105. TiwIn the work , the effIn the paper , the anaIn the work , the anaAn interesting solution to the problem of side effects of opioid receptor agonists was proposed in . The autCannabinoid receptors are a class of cellular receptors belonging to the GPCR superfamily . The recTripterygium wilfordii and used in traditional Chinese medicine for inflammatory disorders. In mice, inflammatory and neuropathic pain was induced by injection of carrageenan and by SNI, respectively. The intraperitoneal injection of celastrol relieved the swelling and allodynia caused by carrageenan. In the animals with neuropathic pain, celastrol reduced mechanical hyperalgesia. The anti-pain effect of celastrol was abolished by the introduction of an antagonist of CB2. The authors concluded that the analgesic effect of celastrol is related to its ability to activate CB2, and the cannabinoid system can be a good target for anti-pain therapy. In the paper [The analgesic effect of cannabinoid receptor modulators is being actively studied in animal models. The authors of  investighe paper , diabetehe paper .Despite a large body of evidence having demonstrated the antinociceptive action of CB1 receptor agonists, opposite results were obtained in the paper . AnalgesThere were attempts of clinical trials of CB1 and CB2 modulators for the treatment of neuropathic pain. In a double-blind, placebo-controlled study in cancer patients suffering from neuropathic pain caused by chemotherapy, the efficacy of the cannabinoid-containing drug nabiximols was shown to relieve pain symptoms . It is iThereby, modulation of chronic pain through the cannabinoid system is probably more complicated than direct activation or inhibition of cannabinoid receptors.The 5-HT7 receptors are one of the subtypes of serotonin receptors belonging to the family of GPCRs on the cell membrane. It is activated by the neurotransmitter serotonin . The 5-HData have been accumulated indicating that the 5-HT7 receptors are involved in the transmission of the pain signal. Brenchat and co-authors caused mechanical hypersensitivity in mice using capsaicin . They reStudy of the analgesic effect of 5-HT7 receptor agonists is ongoing. It was found that the selective agonists of 5-HT7 receptor LP-44 and LP-211 reduced orofacial pain caused by the introduction of formalin in mice . Since cThe 5-HT7 receptors are of great interest also due to existing data on the relationships between the nervous and humoral systems. Neurotransmitters, such as dopamine and serotonin, regulate the secretion of various cytokines by stimulating the corresponding receptors on the surface of immunocompetent cells, including the peripheral ones. It was shown that in patients with mental disorders, the content of proinflammatory cytokines is increased in the cerebrospinal fluid. Cytokines are known to cause hyperalgesia, reduce pain threshold, sensitise afferent nociceptive neurons, and increase the frequency of discharges in nociceptive A\u03b4- and C-fibers. All these factors contribute to central sensitisation, which is displayed by secondary hyperalgesia and/or allodynia . For thiThe sodium-calcium exchanger (NCX) is a cell membrane bi-directional transporter. Under normal conditions, it removes calcium out of the cell (direct mode) but can also pump calcium into the cell (reverse mode) under special conditions, such as an increased concentration of intracellular sodium and/or membrane depolarisation ,131.The authors of the work  showed tIt was shown that many ion channel blockers are partial inhibitors of the NCX, for example, amiodarone , dronedarone , bepridil , sibenzolin (sodium channels blocker) . At the At present, the NCX is considered to be one of the possible targets of therapeutic effect in the development of new methods of pain therapy . The advIn addition to the targets for analgesic drugs, potential targets for antipruritic compounds are also of great interest. Histamine is one of the most well-known pruritus mediators ,139. TheHowever, it is known that antihistamines are ineffective in many cases of chronic itching, which indicates the existence of a histamine-independent mechanism . TherefoTRP channels are a group of ion channels located mainly in the plasma membrane of many types of animal and human cells. There are seven subfamilies of TRP channels: TRPC , TRPV (Vanilloid), TRPM (Melastatin), TRPA (Ankyrin), TRPP (Polycystin), TRPML (Mucolipin), TRPN . Most of these channels mediate various sensations, such as the sensation of pain, heat, warm or cold, different types of taste, pressure and vision. The channels have relatively non-selective permeability for cations, including sodium, calcium and magnesium ions. TRP channels are activated and regulated through various stimuli and synthesised in almost all body systems .A large amount of data has been accumulated on the involvement of TRP channels in pain pathways and on studies of modulators of the receptors for the management of neuropathic pain syndromes. This information has been fully presented in recent reviews ,152. CurIn our review, we couldn\u2019t cover all possible participants of the pain/itch signal transmission. There are many other macromolecules that are considered to be targets of analgesic/antipruritic therapy: adenosine receptors , P2X7 reAt the next stage of our review, we searched the data on analgesic and antipruritic efficacy of the psychotropic drugs studied in animal models and in clinical trials. Furthermore, the interaction of the drugs with the listed above targets for analgesic and antipruritic therapy was also considered. The information found is summarised in Kim and co-authors studied the effect of tianeptine in a rat model of inflammatory pain caused by intraplantar formalin injections . The paiIn the work , mechaniOne more group of Korean scientists studied the effects of tianeptine on mechanical allodynia in rats . TactileThe authors of the work  studied Thus, tianeptine was effective for reducing the symptoms of neuropathic pain in several experiments on rodents . As for Analysis of the literature did not reveal published work on the clinical trials of tianeptine as an analgesic and/or an antipruritic drug. As for our own data, at the Center for Haemodialysis in the First Pavlov State Medical University of St. Petersburg, we studied the effect of antidepressants tianeptine, citalopram and mianserin on chronic pain and itching in patients with chronic renal failure (CRF) who were on chronic haemodialysis (CH) . The stuThus, in our clinical studies tianeptine has been shown to be effective in reducing neuropathic pain and itching in patients with CRF. As an analgesic, the drug was equally effective for both men and women, and as an antipruritic agent, it turned out to be more effective for men . In our Our own research has shown that tianeptine blocks neither NMDAR nor AMPAR . NeverthIn the paper , with thLin and co-authors showed involvement of the 5-HT7 receptors in the antinociceptive effect of tianeptine . The rodSeveral ex vivo experiments showed that tianeptine does not bind at clinically relevant concentrations to calcium channels, glutamate receptors, GABA(B) receptors, and histamine receptors . GABAAR Published data does not show the effect of tianeptine on voltage-dependent sodium channels, cannabinoid receptors, the NCX, TRP channels. The information on tianeptine pharmacological profile is summarised in The authors of the work  studied The opposite result is described in . The autThe drug was effective for reducing the symptoms of neuropathic pain in some experiments in rodents . As for The authors of the work  performeN\u00f8rregaard and co-authors  in a douIn 1996, the authors of  investigThe authors of the work  also invAragona and co-authors  investigIn the work , the effThe authors of  studied In our study  and not at all effective as an AMPAR inhibitor (IC50 > 300 \u03bcM) [The analysis of the data reported in the literature has not revealed any studies on anti-glutamatergic effect of citalopram. In our study, we showed in isolated rat brain neurons using the patch-clamp technique that citalopram is almost ineffective as an inhibitor of NMDARs (IC 300 \u03bcM) .50 = 100 \u00b5M. The researchers concluded that the analgesic effect of citalopram and other SSRIs may be partially related to the blockade of sodium channels.The authors of  using th50 = 64.5 \u03bcM. Moreover, citalopram did not compete with the L-type calcium channel blocker diltiazem for binding to the receptor, which points to different binding sites of these drugs.With the help of electrophysiological approaches, the ability of citalopram to inhibit L-type calcium channels was shown in isolated ventilary cardiomyocytes of guinea pig  and in rIn the review , it is rIn the study , rats weHedlund and co-authors  studied Using radioligand binding assays, it was shown that R-citalopram and citalopram possess moderate affinities for histamine H1 receptors  .We have not found published data on the effect of citalopram on the NCX or TRP channels. The information on citalopram pharmacological profile is summarised in The authors of  in theirIn the work , the tanIn 2015, \u00dc\u00e7el and co-authors studied the effects of mianserin on neuropathic pain induced by diabetes in rats using several experimental models . The effIn the work , the autThus, in experiments in rodents, the drug showed its antinociceptive effect in some models of neuropathic pain . The effIn 1989, Langemark and co-authors studied the effect of mianserin and TCA clomipramine on chronic idiopathic pain in a group of 182 patients . During In the paper , the autIn 1993, in a double-blind placebo-controlled study, the authors of  tested tTwo groups of patients took part in the study : (1) patAnalysis of the literature did not reveal published work on the clinical trials of mianserin as an antipruritic drug.In our studies  the exp in a groTo summarise, the efficacy of mianserin for chronic pain is uncertain. Most studies indicate a lack of analgesic effect or a slight effect on certain pain under certain conditions, e.g., tension headache in patients suffering from depression. Thus, our results seem to contradict those reported in other works. We suppose that this could be explained by sex difference; our trial demonstrated high efficacy of mianserin in men and a low effect in women. The other studies did not distinguish the effect of mianserin in males and females, but there were more women than men in the studied groups in all of them ,195,196.In the work , the inh50 = 43 \u03bcM [Using the electrophysiological methods (5 Hz train protocol), a group of Hungarian scientists revealed that mianserin can inhibit the voltage-dependent sodium channels Nav1.2 in human HEK-293 cells with IC = 43 \u03bcM . The sam = 43 \u03bcM .Becker and co-authors studied the muscle relaxant effect of several antidepressants, including mianserin, in rat aortic rings . AccordiIn the work , the effOlianas and co-authors  studied The interaction of mianserin with the opioid system has been shown in vivo as well. In the study , the antCalliphora vicina. The effects of various antidepressants on the receptors were evaluated by measuring the transepithelial potential. It was determined that mianserin was the most effective antagonist of the 5-HT7 receptors among the studied antidepressants.In the study , by measUsing radioligand binding assays, it was shown that mianserin has extremely high affinity for histamine H1 receptor ,208, andNo published data was found either on the effect of mianserin or on cannabinoid receptors, the sodium-calcium exchanger, or TRP channels. Information related to the mianserin pharmacological profile is summarised in The analgesic activity of carbamazepine has been known since the mid-20th century, and continues to be studied. In rats, carbamazepine has been shown to reduce inflammatory hyperalgesia caused by the introduction of brewer\u2019s yeast .In the study , thermalIn another research investigation , using tThe authors of , by admiIn the work , neuropaIn the work , mechaniThus, in experiments in rodents, the drug showed its analgesic effect in quite a few models of neuropathic pain . As for In neuropathic pain treatment in humans, carbamazepine is used primarily as a first-line drug for trigeminal neuralgia. The first clinical placebo-controlled study proving the effectiveness of carbamazepine for the treatment of pain symptoms in trigeminal neuralgia was conducted in 1966 in 70 patients .In the study , 143 patThe last clinical study on carbamazepine and trigeminal neuralgia was presented in 1995 . The autCarbamazepine is also widely used in the treatment of diabetic neuropathy. In a small group of patients (40 patients), a double-blind placebo-controlled study showed the nociceptive efficacy of carbamazepine in patients with the disease . A recent study  involvedAntipruritic efficacy of carbamazepine was also studied. The work  describeIn our small trial, 11 patients were studied with chronic kidney disease on haemodialysis taking a course of carbamazepine. Status of the patients was estimated before and after 4 weeks from the beginning of the therapy. Before the medical treatment pains were localised in upper and lower extremities, vertebral region, and coxal bones; durations of pain paroxysm were from 2\u22123 h to 20 h per a day; the average pain intensity in the group was 4.98 \u00b1 2.37 (VAS); SCORAD index was 18.0 \u00b1 7.5. After carbamazepine treatment, the respective indexes were: VAS\u20141.64 \u00b1 0.88; SCORAD index\u20145.1 \u00b1 2.0 .Thus, among the seven psychotropics analysed, carbamazepine was the most studied analgesic drug, mainly in trigeminal neuralgia and diabetic neuropathy . MoreoveThe mechanisms of interaction of carbamazepine with the glutamatergic system are not fully understood. Some studies have found that carbamazepine affects the signalling pathways that involve NMDARs ,226,227.The ability of carbamazepine to block voltage-dependent sodium channels has been shown in many papers at the end of the last century ,230,231.In the work  in guine50 = 24.5 nM. At the same time, carbamazepine affected only the GABAARs containing gamma subunits and not the recombinant receptor containing only alpha and beta subunits. Moreover, benzodiazepines did not affect the effect of carbamazepine, which suggests different binding sites for carbamazepine and benzodiazepines.In the paper , it was There is no data in the literature about the direct effect of carbamazepine on opioid receptors. However, it has been shown that opioid receptor ligands and carbamazepine can enhance the effects of each other. Thus, in the work , epileptIn the work  in a strOl\u00e1h and co-authors, using mouse fibroblast cell culture expressing rat TRPV1 channels, studied the uptake of radioactive calcium caused by the effect of vanilloids on TRPV1 channels and the effect of various psychotropic compounds on this uptake . It turnWe could not find published data on the effect of carbamazepine on 5-HT7 receptors, the NCX, or histamine receptors. The information on carbamazepine pharmacological profile is summarised in The authors of  showed tOkuda and co-authors induced neuropathic pain in rats by sciatic nerve ligation . Then thThe authors of  tested tThus, trazodone demonstrated analgesic efficacy in several experiments in rodents . As for Wilson and co-authors, in their study , found oThe authors of the work  revealedSimilar results were obtained in the work  devoted Analysis of the literature did not reveal published work on the clinical trials of trazodone as an antipruritic drug.In our small study, the antipruritic effect of trazodone was checked in 17 patients with anxiety and depressive disorders, which also included diffuse itching in the clinical picture. The examined patients had no pathology of kidney or diabetes. Two patients left therapy because of stomach ache. In the remaining 15 patients, the reduction in the severity of pruritus was gradual and reached its maximum (up to 70%) in 2/3 of patients after 6\u201310 weeks of therapy. Thus, for the first time, we showed the antipruritic efficacy of trazodone in 2/3 of patients suffering from the syndrome of diffuse itching: the reduction was up to 70% .Thus, in several clinical studies, trazodone has shown its effectiveness in reducing chronic pain  and diffuse itching . HoweverOur electrophysiological measurements showed that the anti-glutamate activity of trazodone is very weak .Using the whole-cell patch-clamp method, the authors of  studied 50 value of 11.07 \u00b1 0.09 \u03bcM and inhibited the Ca2+-specific inward current peak amplitude in a concentration-dependent manner with IC50 value of 19.05 \u00b1 0.13 \u03bcM.The opposite result was obtained in . The res50 value was 23\u201345 \u03bcM) [Kraus and co-authors, using he whole-cell patch-clamp method in rat brain slice, showed that trazodone induced inhibition of T-type calcium-dependent calcium channels Cav 3.1, Cav 3.2 and Cav 3.3 in clinically relevant concentrations (IC3\u201345 \u03bcM) . The expIn the study , the ratIn the work , the molThe same study reported that the serotonergic system was also involved in the antinociceptive effect of trazodone; the antinociceptive effect of the drug was inhibited by metergoline, a non-selective serotonin receptor antagonist .The interaction of trazodone with histamine H1 receptors of the human brain frontal cortex was studied using the radioligand binding techniques. The results of the study showed that trazodone could be a potent receptor antagonist with an equilibrium dissociation constant of 350 \u00b1 60 nM .No effects of trazodone on cannabinoid receptors, the NCX, and TRP channels have been reported in the literature. The information on trazodone pharmacological profile is summarised in Surprisingly, the analysis of the data reported in the literature did not reveal any studies on the analgesic and antipruritic effects of chlorprothixene in animal models. However, chlorprothixene is used in many experiments as a component of anaesthesia for animals ,252.The paper  describeThe clinical trial described in  involvedIn 1978, the effect of chlorprothixene on postherpetic neuralgia was tested in two double-blinded studies . The firAccording to the results obtained in , patientSince the last study was published in 1981, it seems that chlorprothixene as an analgesic drug has been forgotten. As for itching, analysis of the literature did not reveal any published work on the clinical trials of chlorprothixene as an antipruritic drug.Our study showed the effectiveness of chlorprothixene for the treatment of chronic pruritus . The stuIn conclusion, chlorprothixene has shown its effectiveness in reducing chronic pain and itching in several clinical studies . There w50 = 2.5 \u00b1 0.6 \u00b5M and a Hill coefficient equal to 1.3 \u00b1 0.2 at \u221280 mV holding voltage in the absence of magnesium in extracellular solution , a group of Hungarian scientists demonstrated that chlorprothixene can inhibit voltage-dependent sodium channels Nav1.2 in human HEK-293 cells with IC = 43 \u03bcM .The papers ,258 descThe authors of  studied It was shown in human histamine receptors expressed in the CHO cells that chlorprothixene is an effective antagonist of H1 receptors with an inhibition constant Ki = 4 nM .There is indirect evidence that chlorprothixene can interact with TRP channels. Capsaicin is often used in experiments for potentiation of mechanical and thermal allodynia and hyperalgesia. Capsaicin binds to TRPV1 channels, activates them, sodium and calcium ions pass through the channels, and a nerve impulse is triggered. Resiniferatoxin is a capsaicin analogue with even greater activity against TRPV1 channels. Acs and co-authors studied the binding of radioactively labelled resiniferatoxin to TRPV1 channels and the effect of TCAs and phenothiazine-like antipsychotics on this binding . ChlorprAccording to the literature, the effect of chlorprothixene on AMPA- receptors, calcium channels, opioid receptors, cannabinoid receptors, and the NCX has not been studied. The information on chlorprothixene pharmacological profile is summarised in In the study  the antiThe authors of  studied In , the dosIn the study , the antIntriguing results were obtained by the authors of  in theirRowbotham and co-authors  tested tRegarding inflammatory pain, Piletz and co-authors reported concave significant associations between pro-inflammatory biomarkers (tumour necrosis factor (TNF)-\u03b1, interleukin (IL)-1\u03b2, and monocyte chemotactic protein-1) and plasma levels of venlafaxine in patients with a major depressive disorder .Lee et al. in 2010 investigated the effect of venlafaxine with a dosage of 75 mg/day in males with functional non-cardiac chest pain . The stuA comparative, double-blind clinical trial with venlafaxine, carbamazepine, and pregabalin was made by Razazian and co-authors . The patThe authors of both ,274 noteA comprehensive Cochrane review of venlafaxine efficacy in neuropathic pain was published by Gallagher and colleagues in 2015 . The autThe authors of  tested tThus, in several clinical studies, venlafaxine has shown its effectiveness in reducing chronic pain (predominantly in diabetic neuropathy). As for itching, we could not find any published trials on the antipruritic effect of venlafaxine. The following adverse effects of the drug were reported: nausea, somnolence, headache, insomnia, sexual dysfunction, and dizziness ,274,276.With the help of extrinsic fluorescence quenching, it was demonstrated that venlafaxine is able to bind to the extracellular S1S2 domain of the NR1-1b subunit of the NMDA receptor, but not in clinically relevant concentrations . It was 50 of 8 \u03bcM. Moreover, according to these results, venlafaxine binds to the resting state of the channel, while most tricyclic antidepressants bind to the inactivated state.Using the patch-clamp technique, the authors of  showed iIn the paper , the antAccording to , venlafaThe effect of venlafaxine on TRP channels has not been studied yet. However, the authors of  showed wWe did not find any published data on the effect of venlafaxine on calcium channels, GABAAR, the NCX or cannabinoid receptors. The information regarding the venlafaxine pharmacological profile is summarised in Having compared the data on the analgesic/antipruritic efficacy of drugs  and thei50 = 100 \u03bcM). On the other hand, it provides slight pain relief in diabetic neuropathy. We don\u2019t believe that it can be excluded that modulation of sodium current by citalopram contributes to its antinociceptive effect in diabetic neuropathy.The voltage-dependent sodium channels involved in nociception  are predominantly expressed in the PNS: in the dorsal root ganglion (DRG) and the trigeminal ganglion . Their rAs was expected, the drugs modulating opioid and cannabinoid receptors have been proven to be effective as supraspinal analgesia . SeveralAnalysis of the information collected leads us to suggest that the interaction of mianserin with the \u03ba-opioid receptors makes a considerable contribution to the analgesic effect. According to the data reported in the literature, the distribution, function and pharmacology of these receptors depend on sex ,291, andIn connection with these observations, it is important to mention that a large amount of evidence has been accumulated so far indicating that pain pathways are different for men and for women, and sex should be a key factor in personalised prescriptions .It is interesting that almost all compounds  are inhibitors of histamine receptors, despite their different structures. The interaction of carbamazepine with histamine receptors has not been studied, so it is not excluded. Perhaps the interaction of these drugs with histamine receptors explains their antipruritic effect.However, there are contradictions in this assumption. The anxiolytic effect of the histamine H1 receptor antagonist pyrilamine was studied in animal models . It was Experiments with animal models have shown that tianeptine, citalopram, carbamazepine, trazodone and venlafaxine are effective against inflammatory pain caused by formalin, carrageenan and brewer\u2019s yeast . ProinflOur analysis has revealed a huge number of \u201cblank spots\u201d in the mechanisms of the analgesic and, especially, the antipruritic effects of psychotropic drugs. The interaction of psychotropics with TRP channels, which are key players in transmitting a pruriceptive signal, has not been investigated adequately. The antipruritic effect of the drugs has not been studied in animal models at all. Also, we have found no explanation for the fact that, in our clinical studies, mianserin, carbamazepine and tianeptine suppress neuropathic itching in men more effectively than in women . PerhapsThe mechanisms of neuropathic pain and the analgesic effect of psychotropic drugs have been studied much better than pruritus. However, there are still many questions; most of all, in our opinion, regarding the role of calcium ions. First of all, there is no clear data for some drugs with respect to their ability to inhibit calcium channels. Secondly, the contribution of the sodium-calcium exchanger to the pharmacological profile of psychotropic drugs has not been studied at all. Thirdly, our review describing just seven compounds could not reveal any certain relationships between the type of their analgesic activity and their ability to modulate calcium flux through the calcium channels. This question requires further investigation.Having analysed the clinical data of the analgesic and antipruritic efficacy of these seven drugs, we found that almost no-one divides patients into groups by sex and/or age, although such studies are important for effective therapy development. We were practically the only ones who tried to analyse the efficacy of drugs separately in men and women, and such studies on large groups of patients are desirable.Chronic pain/itching and mood disorders accompany each other frequently. Much information has been obtained so far indicating that the molecular mechanisms of these pathologies overlap. Enhancement and ordering of our knowledge of the analgesic/antipruritic action of psychotropic drugs is an actual problem. The information will help to develop the personalised medicine and reduce the number of prescribed drugs and side effects. There is no doubt that work on this topic requires the continuation."}
+{"text": "Talaromyces helicus, a soil isolate, and of the model fungus Neurospora crassa through parallel microchannels. The distributions of growth velocity obtained for each strain were compared with measurements obtained in macroscopic solid culture. For the two strains used in the study, confinement caused the growth velocity to drop in comparison with unconfined experiments. In addition, N. crassa was also limited in its growth by the nutrient supply, while the microfluidic culture conditions seemed better suited for T. helicus. Qualitative observations of fungi growing in microfluidic chambers without lateral confinement also revealed that side walls influence the branching behaviour of hyphae. This study is one of the first to consider the confinement degree within soil microporosities as a key factor of fungal growth, and to address its effect, along with physicochemical parameters, on soil colonization, notably for bioremediation purposes.Soil fungi have the ability to form large mycelial networks. They rely on the resources available in the soil to produce biomass and are able to degrade complex biomolecules. Some of them can even degrade recalcitrant organic pollutants and are considered as promising candidates for soil bioremediation strategies. However, the success of this approach depends on the ability of fungi to colonize the soil matrix, where they encounter spatial and temporal variations of confinement, humidity and nutrient concentration. In this paper, we present a study of fungal growth at the scale of single hyphae in a microfluidic device, allowing fine control of nutrient and water supply. Time-lapse microscopy allowed simultaneous monitoring of the growth of dozens of hyphae of  They arTalaromyces helicus, has been selected as particularly efficient for the biodegradation of PAH both in mineral medium and in historically contaminated industrial soils [In this context, a soil-borne fungal strain, al soils . In orde\u22121 in cm-wide macropores [Natural soils are porous media with high heterogeneity in pore size and distribution and can be subjected to variations in water saturation and infiltration rates depending on weather conditions. As a result, average infiltration rates through a given volume of soil can vary in the range of a few millimetres to a few tens of centimetres per hour, while local flow velocities vary greatly depending on pore size and saturation, reaching up to 50 cm scropores . TransfeBacillus subtilis on the fungus Coprinopsis cinerea [Neurospora crassa [Fusarium fujikuroi [Candida albicans in filamentous form, geometric constraints have been shown to affect hyphal growth and morphology [Microfluidic tools have been proposed to study the interactions between microorganisms and their microenvironment, specifically in soils ,7. Numer cinerea , investia crassa  or imageujikuroi . By usinrphology .Most of the studies of fungal growth in microfluidic devices were performed in static conditions, i.e. in a resting fluid or gel culture medium. In such a system, the dead volume in the chamber is small and the growing microorganisms are likely to quickly consume the nutrients and oxygen initially present in the device. For animal cell culture, dynamic cell culture systems have been developed to avoid this risk of starvation ,15.In this study, we propose a microfluidic device to monitor the growth of individual hyphae experiencing lateral confinement in microchannels. This device is suited for microscopic observations and nutrient perfusion. Observations can be performed in static or dynamic conditions. The objective is to use these results to help discriminate between phenomena that result from the geometrical constraints and those that are merely a consequence of starvation.Talaromyces helicus and Neurospora crassa was studied with and without the implementation of a medium perfusion in the microfluidic chamber and compared with macroscopic growth on solid medium. We also studied more qualitatively the branching events that took place in the microchannels and the perfusion chamber. Apical extension velocity and branching are two key features that influence the ability for a fungus to colonize a soil.The growth of the two fungal strains 2.2.1.Talaromyces helicus from our laboratory collection and previously isolated from an industrial contaminated soil was used for this study. The model species Neurospora crassa was obtained from the BCCM/MUCL Agro-food & Environmental Fungal Collection (strain MUCL 041473). Strains were maintained on MYEA medium composed of malt extract 20 g l\u22121, yeast extract 2 g l\u22121 and agar 15 g l\u22121, at 22\u00b0C with a 12 h\u201312 h light\u2013dark cycle, and transplanted onto fresh medium every 10 days.A strain of the filamentous fungus 2.2.a, featuring two chambers connected through 50 parallel microchannels was patterned onto a silicon plate by photolithography, yielding a positive master . Each channel is 10 \u03bcm wide and 5.8 \u03bcm deep, in order to accommodate a single hypha, as illustrated by figure 1b,c. Microchannels are 0.5 or 1 mm long, which allows to observe full microchannels in the microscope field, using either a \u00d720 or a. The negatively patterned PDMS slab was bound to a glass microscope slide after surface oxidization in a plasma chamber (Harrick) for 60 s.A design, represented in figure 12.3.\u22121 fluorescein solution at 9 \u03bcl min\u22121 through the distal chamber using a peristaltic pump (Ismatec). The intensity of fluorescence in the chambers was monitored by fluorescence microscopy over 3 h using the fluorescein isothiocyanate (FITC) filter. Average fluorescence intensity at five different y positions in the perfusion chamber was measured for each time point. The mean grey value was measured using ImageJ software for each image over a 130 \u00d7 600 \u03bcm rectangular area as close as possible to the channel openings (but not including them).In order to characterize solute transport in the device, a chip featuring 1 mm long channels was filled with distilled water, then perfused with a 50 mg l2.4.et al. [\u22121, NaH2PO4 . 2H2O 1.54 g l\u22121, Na2HPO4 8 mg l\u22121, MgSO4 . 7H2O 0.25 g l\u22121, NH4NO3 1 g l\u22121, ZnSO4 . 7H2O 1 mg l\u22121, MnCl2 . H2O 0.1 mg l\u22121, FeSO4 . 7H2O 1 mg l\u22121, CuSO4 . 5H2O 0.5 mg l\u22121, CaCl2 . 2H2O 0.1 mg l\u22121, MoO3 0.2 mg l\u22121 and glucose 20 g l\u22121. All chips were filled with sterile MMG prior to seeding with mycelium. A 2 mm diameter mycelium plug was collected from a solid-medium culture of T. helicus or N. crassa at the edge of the growing colony and transferred to the inoculation inlet of the chip. The inlet was then closed with a PDMS plug to prevent drying and the system was placed in a sealed Petri dish in a water-saturated atmosphere. The mycelium was allowed to grow at 22\u00b0C with a 12 h\u201312 h light\u2013dark cycle, until hyphae reached the opening of the channels. After pre-incubation, systems were placed on the stage of the microscope. Systems used in dynamic conditions were connected to a peristaltic pump (Ismatec) and the distal chamber was perfused with MMG at 1.8 \u03bcl min\u22121, using a PTFE tubing (0.56 \u03bcm I.D. and 1.07 \u03bcm O.D.) directly plugged in the PDMS and connected to the silicon peristaltic tubing . The experimental set-up is pictured in electronic supplementary material, figure B1. Systems used in static conditions were not perfused. Hyphal growth was monitored in parallel in multiple microchannels over 10 h. The experiment was repeated three times for each strain, with and without perfusion with MMG. Growth was measured in a total of 280 channels from 15 separate chips, 6 in static conditions and 9 in dynamic conditions.Mineral medium supplemented with glucose (MMG) at pH 5.5, as described by Fayeulle et al. , was use2.5.N. crassa and T. helicus were each inoculated in three Petri dishes on MYEA and incubated at 22\u00b0C. Pictures of the N. crassa colonies were taken every 6 h for 48 h. For T. helicus, pictures were taken every day for 4 days, and then every 2 or 3 days until colonies reached the edge of the dish. All image analysis was performed in ImageJ. Colony size on each image was measured manually due to the low contrast. For each image, the maximum and minimum diameters of the colony were measured, and the mean of both values was calculated. Microscopic observations were made using an inverted microscope (Leica DMI-8) equipped with a motorized stage, and images of five fields for each chip were taken at a 10 \u00d7 magnification (or 4 \u00d7 for the fluorescein perfusion) with a camera (Leica DFC 3000G), every 10 min for T. helicus and every 5 min for N. crassa, for at least 10 h. In each channel, the distance between the channel entrance and the tip of the longest growing hypha was measured. Channels presenting obvious signs of drying or of PDMS peeling off from the glass slide were excluded from analysis, as well as those already filled with a hypha longer than half the length of the channel at the beginning of the observations. Time-averaged elongation velocities vm were calculated asti and tf denote the times at which the observation of the hypha started and stopped, and x(t) represents the position of the hyphal tip at the time t. The maximum observation time tf \u2212 ti is 10 h. For hyphae reaching the extremity of the channel earlier than 10 h, the last picture with a visible hyphal tip was taken into account. Spatio-temporal diagrams were generated using the built-in KymoGraph plugin to visualize elongation velocity variations. Image sequences of the two-dimensional growth of branched hyphae were manually thresholded, then analysed using the plugin Fractal Analysis. The Box Counting method was used to obtain the fractal dimension of each image in the sequence.In order to measure colony growth rates on a solid medium, 2.6.t-test was performed to compare results obtained in different experimental conditions. The confidence interval (99% or 95%) is indicated with the p-value in the legend.All results are presented as mean \u00b1 standard deviation. Whenever relevant, an unpaired Student\u2019s 3.3.1.\u22121 fluorescein solution at a flow rate \u22121, after 10 min of perfusion. In the inoculation chamber, the concentration remained well below this level, even after 150 min of perfusion. Figure 2a reveals that the concentration in the inoculation chamber started to increase fast after only 10 min. This characteristic time is shorter than the characteristic time \u03c4D for the diffusive transport of fluorescein  and N. crassa (b), in both static and dynamic conditions. While time-averaged velocities appeared to be consistent within a single chip, they varied significantly from one chip to the other, as indicated by electronic supplementary material, figure B2. This can be explained by inconsistent inoculum size, amount of nutrient-rich agar added with the inoculum and metabolic state: from one chip to the next, the amount of biomass and of nutrients inoculated in the chip was slightly variable. In spite of this variability, a clear difference in elongation velocities was found between the two strains. Without perfusion, vm was 190 \u00b1 46 \u03bcm h\u22121 on average for N. crassa and 47 \u00b1 19 \u03bcm h\u22121 for T. helicus.For both \u22121, lower than in the previous section, was chosen in order to minimize shear stress on hyphae and medium consumption: with chamber dimensions of w = 3 mm by h = 124 \u03bcm, the average velocity vf of the fluid in the perfusion chamber was about 80 \u03bcm s\u22121. The associated Reynolds number is defined as Re = hvf/\u03bd, with \u22122. The maximal velocity in the microchannels at the beginning of the experiments is vm. However, there was no noticeable influence of the channel position along y on the hyphal growth velocity vm, as illustrated by For experiments in dynamic conditions, a flow rate of 1.8 \u03bcl mina) shows, there was no significant difference for T. helicus between chips incubated in static conditions and those with a circulation of medium . In dynamic conditions, mycelial growth in N. crassa was again faster than in T. helicus. Also, in contrast to T. helicus, growth velocities were significantly different for N. crassa between static and dynamic conditions: hyphae grew at 190 \u00b1 46 \u03bcm h\u22121 on average without circulation of medium and 300 \u00b1 128 \u03bcm h\u22121 with circulation.As the distribution of elongation velocities displayed in 3.3.a. This graphical representation allows for rapid visualization of variations in elongation velocity. On such a diagram, growth at a constant rate appears as a straight line cutting diagonally from the top left to the bottom right of the figure, acceleration as a convex curve and deceleration as a concave curve. Hyphae sometimes stopped for a while and then resumed their growth. Pauses in T. helicus growth were usually associated with branching events, while in N. crassa, they were mostly observed when the cross-section of a microchannel was locally reduced due to a microfabrication defect. No further investigation of the temporal variations of N. crassa growth was performed, and we focused on T. helicus, whose thinner hyphae are much less sensitive to the irregularities in channel shape. In the example shown in figure 5a, T. helicus first grew at a constant rate, then accelerated. Accelerating hyphae are scattered along the system and not clustered close to (or far from) its entrance, and their average velocity is the same as the rest of the population. Figure 5b reveals that growth of T. helicus in static conditions tended to be mostly constant or decelerating, while velocity profiles noted in chips with perfusion were either constant or accelerating. The presence of a flow in the perfusion chamber thus appeared to influence the growth of T. helicus, although no difference between static and dynamic conditions was noticeable in the distributions of elongation velocity.Time-lapse images acquired during confined growth were used to build spatio-temporal diagrams such as the one displayed in figure 53.4.N. crassa and T. helicus were inoculated at the centre of Petri dishes and left to grow until they reached the edge of the dish. For both strains, the colony sizes were consistent between replicates and followed a similar trend: a short lag phase followed by a linear growth phase (a). The slope of these curves yields the radial growth velocity of the colony vc, for which an almost 10-fold difference was found between the two strains. The average diameter of T. helicus colonies after 7 days was 33.4 \u00b1 0.1 mm, which is consistent with values found in the literature (25\u201333 mm in 7 days) [vc of 1040 \u03bcm h\u22121 for N. crassa is consistent with values from the literature [\u22121 reported for unconstrained hyphal elongation rate of N. crassa [figure 6b, but the velocity ratio between micro- and macro-scale vm/vc is smaller for N. crassa. Error bars represent the standard deviation, which is quite large due to the number of measurements. Although error bars overlap in static conditions, Student\u2019s t-test indicates a significant difference between the two strains for both static and dynamic conditions.th phase a. The sl 7 days) . The colterature ,18, and . crassa . Growth 3.5.a. The tip velocity of single hyphae is not sufficient to predict the capacity of a fungal strain to colonize a soil, as a given total hyphal length can be associated with a diversity of mycelial morphologies [FD characterizing mycelial growth on a surface is a number between 1 and 2. Automated image analysis can be performed as long as the mycelium is not too dense, either in a dilute suspension of mycelial fragments [Figure 7b shows how pictures obtained by time-lapse microscopy were post-treated to extract the mycelial outline. Figure 7c presents the time evolution of fractal dimension. As hyphae started to grow out of the microchannels, the fractal dimension was close to 1, characteristic of linear growth, and increased over 10 hours to reach a value between 1.4 and 1.5. The surface occupied by the mycelium increased over time, as indicated by figure 7d.As they reach the perfusion chamber, hyphae start to deviate from the microchannel axis, as illustrated in figure 7hologies . Fractalragments  at the eragments  or at thragments . Figure 4.4.1.b results from these convective flows in the microchannels. We showed that convection could accelerate fluorescein transport, in comparison with pure diffusive transport. In experiments involving fungi, however, the presence of hyphae in the microchannels modifies their hydraulic resistance. When all the microchannels contain hyphae, their permeability is expected to decrease dramatically. As microchannels become more resistive, convective flows between the two chambers slow down, or even stop, and no longer fuel the concentration gradient in the inoculation chamber. This means that when microchannels are occupied by hyphae, the main effect of the flow is to replenish the perfusion chamber with fresh medium, which feeds fungal growth mostly by diffusion. If the concentration of nutrients in the medium is large enough, then the growth conditions encountered by hyphae in all microchannels are comparable. This is consistent with the fact that the hyphal elongation velocity does not depend on the y-position of the channel  [\u22129 m2 s\u22121) [N. crassa growth. The growth of N. crassa over a given length requires more energy than that of T. helicus: because its hyphae are thicker, the amount of biomass corresponding to this length is larger. Also, N. crassa grows faster than T. helicus, which is another reason why its nutrient consumption rate is expected to be larger than that of T. helicus. It thus seems that the growth of N. crassa is limited by the flux of nutrients diffusing from the perfusion chamber in static and probably also in dynamic conditions. In contrast, nutrient availability does not appear to significantly affect the growth of T. helicus in these conditions, since enhancing nutrient supply through perfusion does not modify its elongation velocity. Lack of space due to the geometric constraints and limited access to oxygen may thus be the main limiting factors for this last strain. The relatively slow growth of T. helicus seems to be associated with a moderate consumption of nutrients and its needs are met with the initial amount of nutrients in the chamber.Apart from water, fungal growth requires nutrients such as glucose, ammonium, aminoacids, phosphates and trace elements, and gases such as oxygen, carbon dioxide and ammonia \u201330. In r. crassa . The dif m2 s\u22121)  is 100 \u00d7 m2 s\u22121) , diffusi4.2.N. crassa is more affected than T. helicus by nutrient deprivation in microfluidic conditions, but it does not explain why the elongation velocity of both strains is so small in comparison with the growth velocity measured at the scale of the colony. It should be noted that MYEA, which is used for macroscopic scale experiments, is a complex medium as opposed to the MMG medium infused in the microsystems and thus provides nutrients in a different chemical form. However, in the case of T. helicus, elongation velocity is not affected by nutrient supplementation through a perfusion, which suggests that the availability of essential nutrients is not the main factor limiting its growth.Differences in nutrient consumption rate between the two strains might explain why N. crassa, thigmotropism has been proposed to be due to a putative calcium channel protein responding to mechanical stresses at the apex and triggering changes in the polarity machinery at the hyphal tip [Spatial limitations in the microfluidic chips may also explain this slower growth. It could be due to the detection of the microchannel surfaces by hyphal membranes, since several types of mechanosensitive receptors control fungal morphogenesis including hyphal growth . Notablyphal tip .T. helicus than with N. crassa. The second hypha generally grew more slowly than the first one, but the amount by which it was slowed down differed depending on the strain: the velocity decreased by 16% in T. helicus and 69% in N. crassa. This again illustrates the fact that N. crassa, being more confined in the channel than T. helicus, is also more sensitive to an additional confinement.The presence of a hypha in a channel did not automatically prevent new hyphae coming from the inoculation chamber to colonize the channel. Simultaneous growth of multiple hyphae happened more frequently with T. helicus, branching events were observed in the straight channels and usually associated with a brief pause or a decrease in elongation rate. This arrest of mycelial growth prior to branching events was also observed in the unconfined growth of single hyphae [N. crassa during its growth in the microchannels. Measurements of the branching distance of N. crassa in tortuous microsystems are found in the literature: it decreases from 219 \u03bcm on an agar plate to 43 \u03bcm in a microfluidic network of 12 \u00d7 10 \u03bcm channels and even to 23 \u03bcm in complex maze-like structures [N. crassa is also capable of adjusting its branching distance to the straight geometry of our channels, suppressing branching over lengths exceeding its unconfined branching distance. In soil, this could allow the fungus to rapidly follow cracks in rocks. To the best of our knowledge, no values of the fractal dimension characterizing the growth of T. helicus are available in the literature, but our experimental values are consistent with those reported for other strains.For e hyphae . We did ructures . This smd is remarkable. Indeed, if all the hyphae in the branched network were to grow with the same velocity as the initially straight hyphae, surface coverage should increase in a faster, nonlinear manner. But we observe that, as the network density increases, some hyphae stop completely while others keep growing. This is likely to cause, on average, a decrease in the apical velocity. Note that the thresholding method used to analyse our images does not allow us to distinguish between intersecting and bifurcating hyphae, which prevents us from tracking the elongation velocity of individual hyphae over time, but we can still evaluate the instantaneous tip velocity of several apices in the same field of view. This decrease in average apical velocity seems to almost equally compensate the increase in the number of hyphae. This arrested growth could be seen as an illustration of a quorum sensing phenomenon between hyphae, which is poorly understood and studied in fungi, but has been shown to exist in N. crassa and to control fungal growth in other species [The quasi-linear shape of the curve in figure 7 species . More pr species . A seemi5.T. helicus, while the model strain N. crassa was limited in its growth by the low nutrient supply in confined conditions. This suggests that N. crassa, although widely used in the literature due to its being easy to handle in the lab, is more sensitive to confinement. Experimental results obtained in unconfined conditions should therefore be handled with care, as they may not always be transferable to predict fungal behaviour in soil. The distance of each channel from the perfusion inlet had no effect on elongation velocities, suggesting that all channels provide equivalent conditions for hyphal growth. This also suggests that the presence of fungi in a soil significantly alters its permeability and thus water and nutrient transport. Given the high variability in elongation velocities for the same strain in the same conditions, this device proved useful to study the behaviour of individual hyphae with a high number of replicates. For both strains considered, hyphal elongation in microfluidic channels was always slower than unconstrained radial colony growth on agar. We showed that this reduced velocity can be explained in part by the limited amount of nutrients available in microsystems, but that confinement also has an effect on fungal behaviour. When growing in microchannels, the branching distance of N. crassa increased beyond its unconfined value. Qualitative observations of mutual interactions between hyphae in confined one- and two-dimensional geometries suggest that hyphal growth inhibition may also contribute to the reduction in growth velocity observed at the microscale. In the future, we hope that a more systematic study of growth velocities across scales will allow to extract experimental values for the parameters involved in models of fungal growth in silico, in order to better predict the behaviour of fungi in soils [The transport of solutes through microchannels was characterized in the microfluidic device in the absence of fungus, showing that stable concentrations in the culture chambers can be achieved for several hours. The culture of two filamentous fungal strains was achieved in the microfluidic chambers, in presence or absence of a perfusion, and time-averaged elongation velocities were measured. The size of the channels connecting the two chambers is comparable to that of small inter-aggregate spaces or large intra-aggregate pores, which is a relevant scale, in soils, for both water transport and soil\u2013microbe interactions . Our sysin soils ."}
+{"text": "The Electronic Health Record (EHR) has become an integral component of healthcare delivery. Survey based studies have estimated that physicians spend 4\u20136 hours of their workday devoted to EHR. Our study was designed to use computer software to objectively obtain time spent on EHR.We recorded EHR time for 248 physiciansover 2 time intervals. EHR active use was defined as more than 15 keystrokes, or 3 mouse clicks, or 1700 \"mouse miles\" per minute. We recorded total time and % of work hours spent on EHR, and differences in those based on seniority. Physicians reported duty hours using a standardized toolkit.Physicians spent 3.8 (\u00b12) hours on EHR daily, which accounted for 37% (\u00b117%), 41% (\u00b114%), and 45% (\u00b112%) of their day for all clinicians, residents, and interns, respectively. With the progression of training, there was a reduction in EHR time . During the first academic quarter, clinicians spent 38% (\u00b1 8%) of time on chart review, 17% (\u00b1 7%) on orders, 28% (\u00b111%) on documentation (i.e. writing notes) and 17% (\u00b17%) on other activities (i.e. physician hand-off and medication reconciliation). This pattern remained unchanged during the fourth quarter.Physicians spend close to 40% of their work day on EHR, with interns spending the most time. There is a significant reduction in time spent on EHR with training and greater experience, although the overall amount of time spent on EHR remained high. Electronic health record (EHR) has had a marked increase in penetrance in the United States health care since the passing of the Affordable Care Act (ACA) . NumerouAlthough EHRpromisesa number of advantages, there are also many challenges which include a slow learning curve, high costs and difficulty with Health Information Exchangebetween systems . StudiesDespite the high prevalence of EHR use in the United States, there are few studies documenting the precise time physicians spend on EHR. Studies that have been performed have relied largely on self-reporting of work hours \u201315. Our Our study was conducted at NewYork-Presbyterian Brooklyn Methodist Hospital, a 651-bed tertiary care center. The study period extended from June 2016 to October 2016 and was approved by the institutional review board at New York Presbyterian- Brooklyn Methodist Hospital with waived consent process. We sampled 2 two week periods. One was early in the academic year (first academic quarter) and one, at the very end of it\u2014fourth academic quarter. During these periods, the average hospital census remained above 95%. We used data from patients admitted to the medicine service including patients admitted to a units witha higher level of care unitWe thought that using \u201cencounters\u201d will be a better way to capture initial and subsequent daily patient-physician interactions and corresponding progress notes. We also performed a sub-study evaluating attending physicians with the following parameters: specialty, teaching position, number of patient encounters, and total time spent on EHR. Time per patient was calculated dividing total EHR time by the number of patient encounters. We used average time to better characterize different patient populations and have higher internal validity.\u00ae . EHR active use was defined by more than 15 keystrokes, or 3 mouse clicks, or 1700 \"mouse miles per minute\u201d. The accepted definition of the mouse mile is a measure of distance between two points travelled by mouse, with 1 mouse mile = 1 pixel [EHR time was determined by computerized software using CERNERDuring the study, patients completed a five-question survey . Three oAll statistical tests were two-tailed and p <0.05 was regarded as significant. STATA 14.2  was used to perform the statistical analysis. Continuous data were expressed as mean\u00b1standard deviation (SD), or in cases where the distribution is not normal, as median and the 25th and 75th percentiles. Comparisons of continuous data between groups were made using one-way or two-way analysis of variance or the Wilcoxon Rank Sum test as appropriate. The chi square test was used to make between group comparisons of discrete data. We used analysis of covariance for PGY level to access correlations between time on EHR with patient, or physician satisfaction. Number of hours a day was calculated accounting for days at work .During the study, a total of 248 clinicians were included in the analysis: In the first academic quarter there were 130 clinicians\u2014102 residents , 24 fellows, and 4 attending hospitalists. In the fourth academic quarter there were 118 clinicians\u201495 residents , 19 fellows and 4 attending hospitalists. In the sub-study, we analyzed data for 188 attending from 12 different medicine sub-specialties.In the first academic quarter, the average time spent by clinicians on EHR was 3.8 (\u00b12) hours a day, or 37% (\u00b117%), of their total work hours. When analyzing utilization of EHR based on the level of training; residents, fellows, and hospitalists spent 41% (\u00b114%), 23% (\u00b119%), and 37% (\u00b119%) of their works hours on EHR, respectively. During the fourth academic quarter, clinicians were spending less time on EHR averaging 2.9 hours a day or 30% of their time (p<0.01) . This reWhen analyzed by resident seniority, during the first academic quarter, PGY-1\u2019s spent 45% (\u00b112%) of their total work hours using EHR, while PGY-2\u2019s and PGY-3\u2019s spent 42% (\u00b114%) and 34% (\u00b116%), respectively. Comparing first and fourth academic quarters, it was noted that senior residents (PGY-2&3) were spending less time on EHR during the fourth academic quarter (p<0.04) with a trend toward less time on EHR among interns (p = 0.07). Throughout the year, PGY-1\u2019s and PGY-2\u2019s were spending more time on EHR than PGY-3\u2019s  , Fig 3.During the first academic quarter, clinicians spent 38% (\u00b1 8%) of time on chart review, 17% (\u00b1 7%) on orders, 28% (\u00b111%) on documentation (i.e. writing notes) and 17% (\u00b17%)on other activities (i.e. physician hand-off and medication reconciliation). There are no significant differences in time allocation on EHR based on seniority or academic quarter .Among the attending included in the sub-study, 108 (57%) were private attendings, with Internal Medicine  being the most represented specialtythe median number of patients seen per attending during the 2-week sample period was 75  with median time spent per patient on EHR being 21  minutes. There was no apparent difference in time spent per patient by faculty versus private attending  minutes vs. 21  minutes, respectively, p = 0.42). Among medicine sub-specialties the greatest number of patients were seen by Endocrinologists 101  spending 30  minutes on EHR per patient . For comin-patients were approached to participate in a 5 question survey and 167 (87%) completed it. The mean HCAHPS score was 10.9 (\u00b11.9), with 104 (62%) patients reporting a perfect score. There was no correlation between time spent by the treating resident on EHR and patient\u2019s HCAHPS score . Based on the patient survey, residents spent 10 (\u00b18) minutes in person with each patient per day. There was a trend towards a direct association between time spent on EHR and time spent with each patient . The patients reported a mean satisfaction of 83(\u00b123) with relation to the amount of time spent at the bedside by a resident, when asked on a scale of 0\u2013100, with 100 being the most satisfied. There was a direct association between patient satisfaction and resident time at the bedside reported by patients . The results of the Oxford Happiness Survey indicated residents to be \u201csomewhat happy\u201d with a mean score of 3.8 (\u00b10.8), however, we did not find a significant correlation between amount of time spent on EHR and resident\u2019s overall happiness .One hundred ninety two alert and awake Our analysis demonstrated that clinicians spent 3.7 hours per day, or 37% of their work day on EHR. There was a marked reduction in EHR time withboth clinician and resident seniority. Despite this improvement, the total time spent on EHR remained exceedingly high amongst even the most experienced physicians. We did not see a correlation between time spent on EHR by physicians with patient or physician satisfaction.We found thatresidents spent more time on EHR than in direct patient contact per encounter\u2013approximately 37 min compared with 10 minutes, respectively. This objectively demonstrates a shift away from the bedside and a greater emphasis on EHR, demonstrating the demands of modern medicine on physicians at every level of training. An important observation of our analysis is that EHR proficiency improves with progression of training, both in regards to seniority and familiarity of EHR. This may in part be attributed to suboptimal initial EHR training. EHR training at our institution is composed of a single 2-hour session incorporated as part of the hospital orientation followed by self-learning. Continued regular training in EHRwould help counter deficiencies in initial EHR orientations. Furthermore, updates to dated EHR systems may also be of use. Further research comparing different EHR interfaces, in addition to best practices for EHR training are needed.Our study is inline with a recent report by Young et al., evaluating multiple family medicine residency programs . This stThe significance of an increasing shift towards EHR is a growing paradigm that cannot be understated, particularly in the current era of healthcare when there is increasing scrutiny on documentation and a ceiling on the number of hours that can be worked by house staff , 14, 18.One limitation of our study was that we chose to only monitor our hospitalist service for attending work hours. This decision was made as their work hours are the most clearly defined with a schedule of working alternating weeks with 12 hour shifts daily. While we observed data in regards to time spent on EHR by faculty and private attendings, it was more challenging to clearly define their work hours.In conclusion, we found that physicians spend almost 40% of their work day on EHR with Interns spending the most time. Although there is a significant reduction in time spent on EHR with greater training and exposure to EHR, the overall amount of time spent on EHR remains high. We did not identify any factors linked with either physician\u2019s or patient\u2019s satisfaction.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file."}
+{"text": "To investigate how scaling affects the penetration of microorganisms into dentinal tubules, how pulpal cells seeded into the pulp cavity respond to bacterial challenge, and how penetration and inflammatory response may depend on the bacterial composition.Streptococcus gordonii and Actinomyces oris or S. gordonii and Porphyromonas gingivalis for 10 weeks. Bacterial invasion was assessed in a depth of 1 mm to the root surface. Human pulpal cells were seeded into the cavities to assess the expression of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and matrix metalloproteinase-3 (MMP-3) by real-time polymerase chain reaction and immunoassay.Root canals of 102 extracted human teeth underwent shaping and cleaning. Half of the teeth were subjected to scaling and root planing, the other half remained untreated. Teeth were exposed to either A. oris/S. gordonii, and 50% versus 25% when exposed to P. gingivalis/S. gordonii (p = 0.043). Scaling had no impact on IL-8 and MMP-3 expression in pulpal cells. P. gingivalis/S. gordonii caused higher levels of IL-8, MCP-1, and MMP-3 than A. oris/S. gordonii .The percentage of teeth with bacteria detected in dentine was higher when teeth received scaling than when they were untreated: 66.6% versus 44.4% when exposed to P. gingivalis may affect the immune response in pulpal cells.Scaling supports the penetration of bacteria into the dentine of extracted human teeth. Root surface debridement with hand instruments may facilitate bacterial penetration. Other kinds of mechanical instrumentation in this experimental setting should be investigated. The tooth\u2019s pulpal tissue together with the surrounding periodontal apparatus form one biologic unit, a continuum where alterations in one compartment may have an impact on the others. Therefore, also therapeutic interventions in one compartment potentially can exert broader effects. Mechanical instrumentation, the standard treatment of periodontitis, aims to eliminate hard and soft bacterial deposits on the root surface and results in improved clinical outcomes such as reduced bleeding on probing (BoP) and decreased probing pocket depth (PPD) , 2. HoweAs a matter of fact, subgingival mechanical instrumentation during active  and supportive periodontal therapy results in the removal of more or less cementum which can eventually lead to exposure of dentinal tubules, pulp injury, or hypersensitivity . Root suActinomyces sp. is abundant in deep dentine lesions [Candida albicans, it is possible to colonize the dentinal tubules system as well as the periodontal pocket [Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, or Tannerella forsythia could be detected [Periodontal disease and instrumentation of the root surface might influence pulpal tissues due to their close anatomical relationship: primarily through exposed dentinal tubules, lateral or accessory canals, and via the apical foramen. Once dentine tubules become exposed, microbial inflammation is potentially able to spread bi-directionally. As such, it is not surprising that the microbiota infecting the periodontium and the root canal systems are highly similar. Disease transmission and microbial similarity has been demonstrated by many studies \u20139 and is lesions  and is f lesions . Also fol pocket . Despitel pocket , 16. In detected .On the other side, periodontal inflammation can affect the vitality of the pulp. In periodontally compromised teeth without restorations and caries lesions, histological signs of pathology were found adjacent to accessory canals in 79% of teeth . FurtherStreptococcus gordonii with Actinomyces oris as well as S. gordonii with P. gingivalis), and (iii) differences between the two bacterial compositions.To date, evidence on bacterial challenges on the root surface after mechanical instrumentation and their impact on pulpal cells is scarce. The present study used a 10-week biofilm cultivation to determine (i) the impact of mechanical instrumentation on the penetration depth of bacteria from the\u00a0root surface into dentine and (ii) the cellular response of pulpal cells seeded into the pulpal cavum of extracted scaled and native teeth that were exposed to two-species bacterial mixtures  was necessary as the teeth were categorized as \u201cirreversibly anonymised.\u201d Pulpal chambers and root canals were instrumented 1 mm short of the apical foramen using endodontic files and ProTaper Next instruments up to 40.06 . During instrumentation, the teeth were irrigated with 3 ml of NaOCl solution . To remove remnants of the disinfecting solution, the teeth were stored in 0.9% w/v NaCl solution for two months with exchange of the solution every third day. Half of the teeth then received scaling and root planing with Gracey curettes  for 4 min by a periodontist. The apices of the teeth were closed with composite  and Syntac system  in order to prevent apical leakage. Predetermined breaking lines in the longitudinal axis were produced with a diamond bur in half of the scaled and half of the native teeth. Figure S. gordonii ATCC 10558, A. oris MG1, and P. gingivalis ATCC 33277. Strains were cultivated on trypticase soy agar [A. oris MG1 and P. gingivalis ATCC 33277 in anaerobic conditions). Thereafter, microorganisms were suspended in 0.9% w/v NaCl solution (OD600 nm = 1). Then, suspensions of S. gordonii were mixed 1:1 either with A. oris or with P. gingivalis, before adding to the nutrient broth  with 10 \u03bcg/ml \u03b2-NAD ) brain heart infusion broth Oxoid). Extracted teeth had been incubated in the nutrient media with bacteria for 10 weeks. For half of the teeth, S. gordonii and A. oris were used for the other half S. gordonii and P. gingivalis. The boxes containing the teeth were opened in a laminar air flow cabinet and the roots were inoculated with one of the two bacterial suspensions. The boxes were incubated in an anaerobic chamber at 37 \u00b0C for 10 weeks. Every week, new bacteria were added and bacterial growth in nutrient media was checked. Exchange of the nutrient medium was performed every 3 days.The following microbial strains were used: soy agar  plates  were harvested from the extracted donor teeth from patients who had given informed and written consent. As before, the volunteers were informed about the use of their extracted teeth in research and according to the guidelines, no previous approval from the Cantonal Ethical Committee KEK was necessary as the teeth were categorized as \u201cirreversibly anonymised.\u201d Cells growing out from dental pulp tissue were incubated in cell culture medium  with 10% fetal bovine serum . As fibroblasts within the dental pulp cell population show a high proliferation rate, it can be assumed that the dental pulp cell population predominantly consists of fibroblasts. A total of three strains of pulp cells were established by explant cultures and fewer than 10 passages were used for the experiments. Cells were maintained in a humidified atmosphere of 5% CO2 and sputtered with a 10-nm layer of gold. Microscopical analysis was performed using a scanning electron microscope  at an acceleration voltage of 8 kV.For scanning electron microscopy , the teeFor assessing the penetration depth of bacteria into dentin, the teeth were longitudinally split in two halves. With a sterile rose bur, dentine specimens were collected out in a distance of about 1 mm to the root surface. The debris were placed into an Eppendorf tube filled with 100 \u03bcL of Wilkins-Chalgren broth with NAD and distributed on TSA plates with an inoculating loop. After anaerobic incubation for 7 days, bacterial growth was recorded.mRNA expression of interleukin (IL)-8, monocyte chemoattractant protein 1 (MCP-1 = CCL2), and matrix metallo-protease (MMP)-3 were quantified in pulpal cells. RNA extraction was performed using the innuPREP Mini kit  and cDNA was generated from 100 ng total RNA using the GoScriptTM Reverse Transcription System  according to the manufacturer\u2019s instructions. Thereafter, real-time PCR using GoTaq\u00ae qPCR Master Mix (Promega) with respective primers . Significance was set at p < 0.05.Statistical analysis was based on the Kruskal-Wallis test and Mann-Whitney From the 102 teeth at the beginning, only 91 could be included in the final analysis. In part, leakages occurred and these teeth had to be removed from final analysis.A. oris/S. gordonii as well as P. gingivalis/S. gordonii, scaled teeth showed a trend for a higher percentage of teeth with bacteria infected dentine compared to native teeth .Both bacterial mixtures were able to penetrate into dentine. For eth Fig. . In the A. oris/S. gordonii  , there was a higher level of IL-8, MCP-1, and MMP-3 mRNA expression when the teeth were exposed to 37) Fig. .Fig. 5InIn this in vitro study, we assessed the impact of mechanical instrumentation first\u00a0on the penetration of bacteria into dentinal tubules and second on cellular responses of pulpal cells. The intent was to simulate the clinical exposure of root dentin either scaled or native to bacterial biofilm. For this purpose, the extracted adolescent premolar and upper third molar teeth were incubated in a two-species bacterial biofilm for 10 weeks. The major findings are that the instrumentation of the teeth affected the penetration of bacteria into dentine and furthermore that the expression of tested inflammatory mediators of pulpal cells was influenced by the bacterial species however not by instrumentation.S. gordonii, P. gingivalis, and A. oris. Interestingly, when P. gingivalis and A. oris were co-cultured with S. gordonii, penetration until the outer third has been observed for the combination with A. oris but not P. gingivalis. [A. oris/S. gordonii than for P. gingivalis/S. gordonii, but the results failed statistical significance. Others showed that the recognition of streptococcal cell wall-anchored polypeptides of the antigen I/II family accounts for the ability of P. gingivalis to invade dentinal tubules when in co-culture with S. gordonii but not with Streptococcus mutans [Particular emphasis in this study was set on the transmission of bacteria through dentine and the assumption that\u00a0dentinal tubules opened by scaling and root planing are more patent for bacteria and their toxins. In periodontally diseased teeth, it has been shown that most bacteria are located in the outer 300 \u03bcm of dentinal tubules . Here,\u00a0tgivalis. . In the s mutans . This inStreptococcus sanguinis [SEM images confirmed the penetration of bacteria into the dentinal tubules from the root surfaces. Further, there was a large number\u00a0of bacteria visible in irregularities at the root surface after scaling. Scaling increases in vitro surface roughness of root surfaces which is accompanied by higher adhesion of anguinis . Smootheanguinis .P. gingivalis/S. gordonii induced a significantly higher mRNA expression of all inflammatory mediators than A. oris/S. gordonii did. This effect might be related to gingipains. It can be assumed that gingipains can penetrate into the pulpal chamber. Already Bergenholtz and Lindhe suggested a pathway of communication between dentinal tubules, the periodontium, and the pulp [Although histology has shown differences in pulpal tissue of teeth with moderate-to-severe chronic periodontitis , we founthe pulp . In an\u00a0athe pulp , 31.P. gingivalis resulted in an increase of IL-8 and MCP-1 mRNA expression [Gingipains have been shown to regulate the expression of MMPs and of tissue inhibitors of metalloproteinases (TIMPs) in oral fibroblasts and epithelial cells thus contributing to tissue destruction . The Argpression . Both ILpression . For MMPpression , expresspression .Some limitations need to be considered. First, leakage at the apexes or accessory canals might have concealed possible differences among groups. Second, a healthy pulp is a complex tissue consisting of a panoply of cells. Third, the non-vital extracted teeth present different physical characteristics such as for example the redox potential and this might have severe consequences of bacterial transmission through dentinal tubules. Fourth, pulpal cells were not seeded into the pulpal chambers until the end of 10 weeks of biofilm incubation. And finally, it must be considered that in the clinical situation of a vital tooth, various defense mechanisms against a possible penetration of bacterial toxins or bacteria are present. This involves a physiological pulpal tissue pressure resulting in an outward flow through the dentinal tubules. When bacterial toxins reach the dental pulp, an inflammatory process occurs; this will initiate the deposition of tertiary dentine in the tubules and increase the above\u00a0mentioned tissue pressure. Thus, the clinical implication of these findings might be taken with caution and requires future research.Our study provides novel insights into the effects of root instrumentation on pulpal cell response when exposed to bacterial biofilm. Our experiments further indicated that penetration of bacteria through the dentine was more observed in the scaled group than in the group with the native teeth. Since the teeth were endodontically instrumented, we assume that in root canal\u2013treated teeth, similar penetration patterns might be observed. SEM images showed a more pronounced colonization of bacteria at the surfaces of the scaled teeth, in particular in irregularities. Therefore, it has to be pointed out that a smooth root surface might be crucial in preventing massive bacterial adherence. In this respect, other kinds of mechanical instrumentation in this experimental setting should be investigated.Escherichia coli lipopolysaccharide (LPS) into root cementum was detected neither in the periodontally healthy nor in the diseased teeth [Previously, cementum of periodontally diseased root surfaces was believed to harbor bacteria and bacterial endotoxins, and therefore the removal of the \u201cdiseased cementum\u201d was considered mandatory to allow periodontal healing , 37. Lated teeth . The bined teeth . ConsequFrom a clinical point of view, the available data indicate that mechanical instrumentation of diseased root surfaces can be performed less aggressively with respect to cementum removal. We recently showed that compared to hand instrumentation, the application of ultrasonication and air-polishing with erythritol prevents from substance loss while providing a smooth surface with nearly no residual biofilm . It mighRoot surface debridement with hand instruments may remove root cementum, which in turn facilitates bacterial penetration from the periodontal pocket and the oral cavity into the dentinal tubules system especially in the root canal\u2013treated teeth where a cellular defense to bacterial penetration is lacking. In vital teeth, periodontal pathogens may influence pulpal response."}
+{"text": "Building a human-like car-following model that can accurately simulate drivers\u2019 car-following behaviors is helpful to the development of driving assistance systems and autonomous driving. Recent studies have shown the advantages of applying reinforcement learning methods in car-following modeling. However, a problem has remained where it is difficult to manually determine the reward function. This paper proposes a novel car-following model based on generative adversarial imitation learning. The proposed model can learn the strategy from drivers\u2019 demonstrations without specifying the reward. Gated recurrent units was incorporated in the actor-critic network to enable the model to use historical information. Drivers\u2019 car-following data collected by a test vehicle equipped with a millimeter-wave radar and controller area network acquisition card was used. The participants were divided into two driving styles by K-means with time-headway and time-headway when braking used as input features. Adopting five-fold cross-validation for model evaluation, the results show that the proposed model can reproduce drivers\u2019 car-following trajectories and driving styles more accurately than the intelligent driver model and the recurrent neural network-based model, with the lowest average spacing error (19.40%) and speed validation error (5.57%), as well as the lowest Kullback-Leibler divergences of the two indicators used for driving style clustering. With the process of urbanization and the rapid growth in the number of vehicles, car following has become the most common driving behavior in daily driving. It is of great significance to build a model that can accurately simulate drivers\u2019 car-following behaviors. Car-following models have been widely applied in microscopic traffic simulation, the tests of driving assistant systems, and other fields ,2,3. In Research on car-following modeling has lasted for more than half a century. The modeling methods can be divided into two types: theoretical-driven methods and data-driven methods . The forThe data-driven methods can directly use data to learn drivers\u2019 control strategies and have fewer assumptions on driving behavior. Data-driven models such as neural networks have stronger expression ability and can be easily updated. Studies have shown that the data-driven car-following models have better generalization ability than the theoretical-driven models ,11. In tRL assumes that human behavior maximizes the cumulative long-term rewards . When thThe traditional IRL method often uses a linear representation of reward function, which may not reflect drivers\u2019 nonlinear intrinsic preferences . BesidesThe rest of the paper is organized as follows: The development of theoretical-driven car-following models can be dated back to the 1960s. Pipes  and Chan\u03b2 is the acceleration parameter. t. Among the mentioned models, IDM is one of the most widely used car-following models, which has the formulations as presented in Equations (1) and (2). In this paper, IDM was calibrated and compared with the proposed model.The earlier study using behavior cloning (BC) methods to model drivers\u2019 car-following behaviors was conducted by Jia et al. . The autS, A, T, R, \u03b3}, where S denotes the state space, A denotes the action space, T denotes the transition matrix, R denotes the reward function, and \u03b3 denotes the discount factor. RL assumes an agent exists in the predefined environment. At every time-step, the agent observes a state and takes an action following its policy, gets to the next state according to the transition matrix, and it then receives a reward.In the setting of RL, the Markov-decision process (MDP) is used to model a sequential decision-making problem. An MDP can be defined as a tuple {To solve an MDP using RL, the reward function must be specified, then the policy can be obtained by applying value-based or policy-based algorithms . As for t, the state includes the speed of the following vehicle denoted as t is the simulation time interval which was set as 0.1 s in the present study, and In this study, the MDP for modeling car-following is defined as follows: at a certain time-step The goal of IRL is to learn the reward function given a set of expert demonstrations. Many approaches have been proposed to solve this problem . The fraWith the above formulation of trajectory distribution, the reward function represented by a linear representation or by a neural network can be learned by maximizing the log-likelihood of demonstrators\u2019 trajectories. However, the Max-Ent-IRL typically requires expensive computational power as it needs to solve the forward problem of finding an optimal policy with respect to the current reward , making Since the basic ideas of IRL and generative adversarial nets (GANs) proposed by Goodfellow et al.  have a lThe structure of the proposed model denoted as GAIL-GRU ,39 is shAn actor-critic structure, which is the core component of the generator, was adopted as an agent to interact with the car-following environment. As can be seen in The proximal policy optimization (PPO) algorithm proposed in  was usedith state-action pair, and The discriminator is an FCN with two hidden layers as depicted in The discriminator also provides the reward signals for the car-following environment. The state-action reward Algorithm 1: Algorithm for modeling drivers\u2019 car-following behaviorsInput: The collected state-action pairs of drivers denoted as D\u2003Output:\u20031: The strategy of drivers denoted as \u20032: Algorithm begins:\u20033: Randomly initialize the parameters of the discriminator and the actor-critic networks as \u20034: For N, repeat the following steps\u20035: Using the policy \u20036: Update the parameters of the discriminator for three times from \u20037: Use the updated discriminator parameterized by \u20038: Algorithm endThe proposed algorithm is showed presented in Algorithm 1 below. The machine learning package PyTorch  was used for implementing the proposed model. The Adam optimizer  was usedData from two field tests conducted in Huzhou City in Zhejiang Province and Xi\u2019an city in Shaanxi Province in China were used in this study. The driving route included urban roads and expressways, as shown in A total of 42 drivers participated in the experiment. All of the subjects were male, the average age was 40.3 years old and the standard deviation was 5.1. The participants\u2019 driving experiences ranged from 2 to 30 years with the average being 15.2 years and the standard deviation was 7.5. During the test, all of the subjects were asked to drive the test vehicle according to their personal driving styles, and they were only told the starting and ending positions. It took 90\u2013110 min for the subjects to complete the entire test.The test data were collected by a test vehicle equipped with a variety of sensors as exhibited in In this study, the rules for determining a car-following period implemented in a previous study were adopted . The detTwo steps were taken to process the extracted data. First, all of the data were processed by a moving average filter to eliminate the noise in the data , where tBecause the proposed model is based on RL, it assumes that the agent that should be modeled has certain kinds of preferences or rewards. Every individual driver may have different preferences during car-following. However, in the literature of driving-behavior research, it is common to cluster drivers into different driving styles such as aggressive or conservative. Drivers belonging to one group may have similar preferences. So, in this study, instead of using the car-following data of each participant to train the proposed model, K-means was initially applied to cluster the participants into different driving styles. The data from the different groups were then used for model training.t = 6.748, p < 0.001) and mean TH when braking  than the aggressive group, which consisted of 26 drivers.There are many indicators that can be used to characterize the driving style of drivers in car-following. Except for basic kinematic indicators, such as spacing, speed, or relative speed, time-based variables such as time headway (TH) and time-to-collision (TTC) have also been accepted for characterizing driving styles ,48. In tThe initial state ith time; and ith time. As suggested in , this stKullback-Leibler (KL) divergence is a measure of the difference between two distributions that can be calculated by Equation (14). When the value of the KL divergence is smaller, the two distributions become more similar. The car-following models should produce a similar distribution of representative features with the drivers\u2019 demonstrations. Because this study used mean TH and mean TH when braking to divide the drivers into two different driving styles, the similarity between the distribution of the simulated mean TH and mean TH when braking and the empirical distributions were measured by calculating the KL divergence to evaluate the performance of car-following models in reproducing drivers\u2019 driving styles.k was determined to be 5 in this study due to the scale of the dataset. The dataset of aggressive and conservative drivers was equally divided into 5 groups. As illustrated in K-Fold cross-validation was adopted in this study to evaluate the generalization ability of car-following models. Specifically, As mentioned in The parameters of IDM were calibrated with the objective of minimizing the RMSPE of spacing as Equation (15). A large penalty of the crash was also added in the objective function, as no collision was observed in the collected data. In this paper, the population size and the maximum iteration times of the genetic algorithm (GA) are set to 100. In order to reduce the influence of the randomness of GA on the calibration of IDM, the GA algorithm is repeated 12 times, and the final result is the combination of parameters with the minimum error.In accordance with another study , an RNN The performances of the proposed model GAIL-GRU, the theoretical-driven model IDM, and the recent behavior-cloning model RNN for replicating the car-following trajectories of the drivers with different driving styles on the training sets and test sets are presented in The three models trained by the same data from the aggressive dataset were used for simulation to illustrate and compare the characters of the three models in reproducing drivers\u2019 car-following trajectories. The simulation results of spacing for three car-following periods randomly selected from the dataset are presented in The present study demonstrated that the proposed GAIL-GRU model can replicate human-like car-following behaviors and driving styles more accurately than the theoretical-driven model IDM and the behavior-cloning model RNN. Compared with IDM, the proposed model used the actor neural network which consists of two hidden layers including a GRU network and a fully connected network, to fit drivers\u2019 car-following policies, while IDM only contains six parameters to be calibrated. Significantly more parameters were contained in the proposed model, accounting for a better fitting ability and generalization ability compared to the IDM.Compared with RNN, the proposed model also used a kind of RNN to represent drivers\u2019 strategies, and the length of the input was also set to be 1 s. However, the RNN model has a higher error for replicating car-following trajectories. From the simulation results of spacing for three car-following periods, it can be seen that the RNN experienced the problem of cascading errors, while the proposed model did not have this problem. The reason for this is that the RNN only learns the state-action relationships during training. When the input for RNN is not included in the training data, the output of RNN may deviate from drivers\u2019 actual control actions. The proposed model is based on RL, the strategy output by the proposed model has the objective of maximizing the accumulative rewards, which award behavior similar to the demonstrator. Therefore, the strategy obtained by the proposed model can be extended to unseen states.Comparing RNN with IDM, the error of the BC model is higher than the theoretical-driven model, which seems to be inconsistent with previous studies ,11. HoweThe proposed model borrows the idea of GANs. In the fields of computer vision, GANs have been proven to be able to generate life-like images to the original image in recent years. In this study, the proposed model is demonstrated to be able to generate car-following strategies and driving styles closest to the drivers. It can be seen that the training process of the proposed model is very similar to that of GANs. The essence of the training process is the process of mutual confrontation between the generator and the discriminator. The training of the discriminator makes the discriminator distinguish the category of the samples more accurately. However, the training of the generator produces samples more similar to drivers\u2019 actual behaviors, so as to cheat the discriminator. After training, the samples generated by the generator can be perfectly close to the distribution of the real samples, while the discriminator cannot correctly tell the difference between the generated samples from the actual samples. The proposed model can output the strategy that is identical to the drivers\u2019 actual behaviors.This study also had some important limitations. First, the car-following behaviors modeled in this study could only represent a small group of drivers. A broader sample is needed in future studies. Secondly, this study did not consider the influence of vehicles in adjacent lanes on drivers\u2019 car-following behaviors. These factors will be fully considered in future research to obtain a more accurate description of drivers\u2019 behaviors."}
+{"text": "Using multiple hMSC donors, we showed that this process was robust and yielded hMSCs that maintained expansion, phenotypic characteristic, and functional properties. The developed process in a vertical-wheel suspension bioreactor can be scaled to the levels needed to meet commercial demand of hMSCs.Human mesenchymal stem/stromal cells (hMSCs) have been investigated and proven to be a well-tolerated, safe therapy for a variety of indications, as shown by over 900 registered hMSC-based clinical trials. To meet the commercial demand for clinical manufacturing of hMSCs, production requires a scale that can achieve a lot size of ~100B cells, which requires innovative manufacturing technologies such as 3D bioreactors. A robust suspension bioreactor process that can be scaled-up to the relevant scale is therefore crucial. In this study, we developed a fed-batch, microcarrier-based bioreactor process, which enhances media productivity and drives a cost-effective and less labor-intensive hMSC expansion process. We determined parameter settings for various stages of the culture: inoculation, bioreactor culture, and harvest. Addition of a bioreactor feed, using a fed-batch approach, was necessary to replenish the mitogenic factors that were depleted from the media within the first 3 days of culture. Our study resulted in an optimized hMSC culture protocol that consistently achieved hMSC densities between 2 \u00d7 10 Human mesenchymal stem/stromal cells (hMSCs) have been investigated and proven to be a well-tolerated, safe therapy for a variety of diseases , as indiTo accommodate hMSC expansion in suspension bioreactors, it is critical to choose a bioreactor platform that allows cell attachment to the microcarriers, homogeneously suspends the cells and microcarriers, as well as distributes gas and nutrients while imposing low shear, as hMSCs are known to be sensitive to shear stress ,8,9. In In clinical hMSC manufacturing, there has been movement away from serum-containing processes due to higher risk of graft rejection, immunoreactions, and virus contamination associated with animal-derived products ,20,21. TAnother paradigm for consideration is perfusion, in which media is continuously circulated through the culture, therefore simultaneously supplying nutrients and removing waste from the culture. While perfusion offers a continuous process and can achieve high cell densities, it utilizes large quantities of media and consequently induces high media costs and logistics/supply chain issues . Thus, pWe established this fed-batch process in hMSC expansion in the vertical-wheel bioreactors, where a fed-batch process showed a higher cell yield and therefore enhanced media productivity compared to a batch process A,B. AddiWith the fed-batch paradigm in place, further optimization of this MSC bioreactor culture process is central to maximizing yields and recovering healthy, functional cells at harvest. To achieve that, optimization studies need to be performed in the small-scale bioreactors prior to scaling up to a full production scale. Various stages of a XF bioreactor culture need to be studied: cell and microcarrier inoculation, seeding strategies, culture, feed, and harvest protocol D. Each o2 in Corning\u00ae CellBIND\u00ae culture flasks , in the accompanying High Performance XF Media Kit (RoosterBio). Cells were cultured at 37 \u00b0C with 5% CO2. After 4 days, cells were dissociated from the flasks with TrypLE\u2122 Select Enzyme , quenched with medium, then counted using the NucleoCounter\u00ae NC-200\u2122 . Cell suspension was then spun down at 300\u00d7 g for 5 min and resuspended in growth medium at a density of 1 \u00d7 106 cells/mL for cell inoculation in the following steps.hBM-MSC Working Cell Banks (WCB) from five adult donors  were used in this study. These cells had been isolated and previously expanded under XF conditions . Cells were thawed and seeded at a density of 3000 cells/cm\u00ae Low Concentration Synthemax\u2122 II microcarriers (Corning) inside PBS-0.1 single-use Vertical-Wheel\u2122 bioreactors . To seed the cells onto the microcarriers, a 1\u20136.3 mL cell suspension was inoculated into the bioreactor containing 30 mL of medium and 0.75\u20132.5 g of microcarriers, resulting in cell seeding density of 11,000\u201370,000 cells/mL, and areal density of 2222\u20139333 cells/cm2 of microcarrier surface. To facilitate attachment, the cells were incubated for 20 min at static condition, then gently shaken to re-distribute the cells and the microcarriers, and finally incubated for 20 more minutes to further cell attachment to microcarriers. Our data showed that an average of 84% cell attachment could be achieved using this seeding method , was added at 2% of the working volume and agitation speed was increased to 30 rpm.At the end of the cell attachment duration, fresh culture medium was added to a total volume of 90 mL. The PBS-0.1 vessel was placed on its controller base in a 5% CODaily sampling of the bioreactor was performed to quantify cell numbers as well as the metabolite and waste profile to assess the kinetics of cell growth and media consumption. A 3 mL sample was taken daily while the cells/microcarriers were in suspension at 30 rpm agitation. The cell/microcarrier sample were left to sediment, the supernatant was isolated for medium analysis, and an equal volume of TrypLE was added to dissociate the cells from the microcarriers. Following dissociation, the number of cells was counted. The medium supernatant was analyzed using the BioProfile FLEX2 Analyzer  to monitor the metabolite and waste concentration.Visualization of cell attachment and cell/microcarrier aggregation was performed by obtaining a 1 mL sample from the bioreactor and visualizing it under light microscopy .Following hMSC expansion in bioreactors for 5\u20136 days, in-vessel harvest was performed. While bioreactor sampling provides the kinetics during culture, the most accurate quantification of total cell yield after the culture duration is obtained through complete in-vessel harvest, minimizing any potential errors from non-representative sampling when large cell-microcarrier aggregates are present in the culture. After agitation is stopped and the cells/microcarriers settle to the bottom of the bioreactor, the medium was removed and the remaining cells/microcarrier solution were washed with PBS. TrypLE was added to the bioreactor at half of the working volume, and agitation was initiated (20\u2013100 rpm for 20\u201340 min) in a 37 \u00b0C incubator. Cells were then separated from the microcarriers using a 100-\u03bcm cell strainer, quenched with an equal volume of medium, and then counted to obtain the final harvest yield. Values are presented as concentrations (cells/mL) to emphasize the scalable process that was being developed.Evaluation of hMSC properties was conducted on the cells harvested from the bioreactor and compared to the properties of cells that were expanded on 2D planar flasks, which served as a control. Cell populations were evaluated for expression of MSC surface markers and differentiation potential . Functional properties of MSCs were also evaluated, such as immunomodulatory function and angiogenic cytokine secretion. To determine the health of the hMSCs harvested from the bioreactor, cell expansion was quantified by culturing the cells harvested from the bioreactor in CellBIND flasks for 5 days. Following a 5-day culture, cells were harvested and the fold expansion was quantified. Expansion capability of the cells harvested from the bioreactor is an important measure of cell quality/health and is an important validation of the bioreactor process following the final cell yield.\u00ae Adipogenesis Differentiation Kit media (Gibco) for 7\u201314 days, then fixed and stained with Oil Red O Solution (Sigma-Aldrich). For osteogenic differentiation, cells were cultured in STEMPRO\u00ae Osteogenesis Differentiation Kit media (Gibco) for 14 days, then fixed and stained with 2% Alizarin Red Stain . For chondrogenic differentiation, cell pellets were generated in ultra-low attachment plates and cultured in STEMPRO\u00ae Chondrogenesis Differentiation Kit media (Gibco) for 21 days, then fixed, sectioned, and stained with Alcian Blue. All differentiated samples were imaged following staining and compared with matched undifferentiated samples. Qualitative evaluation of hMSC differentiation down adipo-, osteo-, and chondrogenic lineages were performed on the cells harvested from the bioreactor and compared to those from the 2D flask controls. For adipogenic differentiation, harvested cells were cultured in STEMPROCells dissociated from the microcarriers and harvested from the bioreactors were analyzed for the expression of surface markers (CD73(+), CD90(+), CD105(+), CD166(+), CD14(\u2212), CD34(\u2212), CD45(\u2212)) using flow cytometry and compared to those from 2D flask controls. Induction of indoleamine 2,3-dioxygenase (IDO) activity by exposure of hMSCs to the pro-inflamatory cytokine interferon-gamma (IFN-\u03b3) is central to the immunosuppressive function of hMSCs ,35,36. hMSCs have been shown to secrete cytokines which help promote tissue angiogenesis ,38,39, tn = 1 bioreactor per group in these kinetic experiments therefore still allowed for the determination of the effects of the tested experimental parameters. This approach allowed for the breadth of experiments presented here. We previously quantified bioreactor-to-bioreactor variability within an experiment, and observed the following coefficient of variation values: CV < 6% for final harvest density and CV < 2% for post-bioreactor cell expansion . Due to 6 cells (1\u00d7) to three times the microcarriers and cell number at 3.75 g and 6.3 \u00d7 106 cells (3\u00d7). At 1.25 g, the microcarriers provide 450 cm2 surface area in 90 mL medium, which is the recommended surface area to media volume ratio by the manufacturer. The cell density for the 1\u00d7 condition is 23,333 cells/mL (4667 cells/cm2). The effect of media exchanges on the group with the highest cell number and microcarriers (at a concentration that far exceed the manufacturer\u2019s recommendations) was also investigated , 2.1 \u00d7 106 (4667 cells/cm2 seeding density), and 4.2 \u00d7 106 cells (9333 cells/cm2 seeding density). Seeding of 2.1 \u00d7 106 and 4.2 \u00d7 106 cells resulted in similar yield of 4 \u00d7 105 cells/mL, while bioreactor seeding with 1 \u00d7 106 cells resulted in the lowest yield (6 cells) reached a growth plateau after 3 days despite the addition of a bioreactor feed on Day 3  as the cell inoculation density in subsequent studies, as after 5 days of culture the cell yield is maximized, and cell health is maintained.The effects of cell seeding parameters, i.e., cell numbers and microcarrier surface area, were each investigated independently. First, various cell numbers were seeded into the bioreactor with 1.25 g microcarriers: 1 \u00d7 106 cells and 90 mL, the effect of seeding density was then investigated. The amount of microcarriers were varied, providing a range of available surface area for cell growth: 0.75 g (270 cm2), 1.25 g (450 cm2), and 2.5 g (900 cm2), which corresponds to seeding density of 7777 cells/cm2, 4667 cells/cm2, and 2333 cells/cm2, respectively. The growth kinetics and final cell yield after a 5-day culture were similar among the three conditions (2) showed markedly reduced expansion compared to the other conditions with 1.25 g (4667 cells/cm2) and 2.5 g microcarriers (2333 cells/cm2), as well as the 2D control  on Day 3 of bioreactor culture showed no effect on cell growth  is sufficient through headspace gassing, sparging is often necessary to increase medium oxygenation to large-scale cultures with higher densities . As biorFollowing 5-day culture, hMSCs were harvested from the bioreactor. To date, the effect of harvest parameters on hMSC health/characteristics has been mainly focused on the choice of dissociation buffer and its effect on harvest yield and cell viability ,58,59. HFollowing cell detachment from the microcarriers, the enzymatic reaction was quenched by the addition of medium. Downstream processing of a large-scale bioreactor process, especially volume reduction and wash, can often take up to 4 h of processing time ; therefoQuantification of the effects of various bioreactor process parameters on the cell yield and post-bioreactor health resulted in an optimized protocol for hMSC expansion in a vertical-wheel suspension bioreactor A. This fFollowing bioreactor culture, it is important to verify the cell health, quality, and functionality. The cell health following bioreactor culture was verified by quantifying the subsequent 2D expansion of the cells harvested from the bioreactor. For three donors, this cell expansion at P5 was comparable to each donor\u2019s respective 2D control at similar population doubling level (PDL) C. In addWe observed that cells harvested from the bioreactor maintained their CQAs of hMSC surface marker expression of CD14, CD34, CD45, CD73, CD90, CD105 and CD166 D as well5\u20136 \u00d7 105 cells/mL, which was within the reported range of hMSC yield in various microcarrier-based bioreactor systems [4\u20134.8 \u00d7 105 cells/mL density, with most of the studies being performed under serum-containing conditions in a 7\u201314 day bioreactor process [5\u20133 \u00d7 105 cells/mL were achieved after 14 days [Our developed bioreactor process yielded a range of harvest densities of 2 \u00d7 10 systems . Previou process . In xeno 14 days . In compWith this developed fed-batch microcarrier hMSC culture process, scaleup is the necessary next step to meet the demands for commercial manufacturing. In addition to verifying and further optimizing these parameters at larger scales, there are other considerations in large-scale production of hMSCs in bioreactors, such as the availability of platforms for downstream processing ,64. For 2 microcarriers surface area per mL of media and cell seeding density of 23,333 cells/mL resulted in an optimized yield and post-harvest cell health. The addition of a bioreactor feed on the third day of culture was critical to maintain the cell expansion by replenishing the mitogenic factors that were depleted from the medium. Our study resulted in an optimized hMSC culture protocol in a vertical-wheel suspension bioreactor. Our process is robust and works in multiple donors, and yields hMSCs with maintained expansion capability, phenotypic and functional properties that are comparable to traditional 2D flask cultures. The evaluated parameters were all investigated due to their relevance for larger scale production of hMSCs and parameter settings were selected both on biological response as well as system scalability to move towards production of hMSCs in larger bioreactors (>80 L). We intend to continue to scale up this process to development, pilot, and production scale bioreactors, which are critical steps to generating the cell numbers necessary for clinical therapies.We developed a fed-batch, microcarrier-based bioreactor process, which enhances media productivity and drives a cost-effective and less labor-intensive hMSC expansion process. In this study, we determined parameters for various stages of the bioreactor culture: cell number and microcarrier concentration at inoculation, agitation speed during expansion, feed timing, culture length, as well as agitation speed and duration during the harvest process. We found that bioreactor inoculation at 5 cm"}
+{"text": "However, howand to what extent the high env diversity relates to immuneprotection remains unclear. In this study, we compared immune protections and responses ofthree groups of horses stimulated by the high-diversity vaccine EIAV_HD, a singlemolecular clone of the vaccine EIAV_LD with low envdiversity, as well as a constructed vaccine strain EIAV_MD with moderate env diversity. The disparity of virus-host interactions betweenthree env diversity-varied groups (5 horses in each group)was evaluated using clinical manifestation, pathological scores, and env-specific antibody. We found the highest titres of env antibodies (Abs) or neutralizing Abs (nAbs) in the EIAV_HD group, followedby the EIAV_MD group, and the lowest titres in the EIAV_LD group (P<0.05). The occurrence of disease/death was different between EIAV_HDgroup (1/0), EIAV_MD (2/2), and EIAV_LD group (4/2). A similar env diversity-related linear relationship was observed in the clinicalmanifestations and pathological changes. This diversity-dependent disparity in changesbetween the three groups was more distinct after immunosuppression, suggesting thatenv diversity plays an important role in protection underlow host immunocompetence. In summary, inoculation with vaccines with higher geneticdiversity could present broader and more efficient protection. Our findings stronglysuggest that an abundance of Env antigens are required for efficient protection againstlentiviruses.Lentiviruses harbour high genetic variability for efficient evasion from host immunity.An attenuated equine infectious anaemia (EIA) vaccine was developed decades ago in Chinaand presented remarkably robust protection against EIA. The vaccine was recently proven tohave high genomic diversity, particular in  Thebackground signal was determined using the sera from the healthy control.Virus-specific humoral immunity was assessed using escribed . The avieviously. Here thescribed . The act-\u03b3, GrzB andIL-2) in blood plasma were determined using a direct-ELISA method according to themanufacturer\u2019s instructions . The background signal was determinedusing the serum from the healthy group. Three independent experiments were performed foreach treatment.The concentrations of three key virus-related cytokines  using the QIAamp DNA Blood Mini Kit . Theproviral DNA was amplified by PCR. Each PCR product was size-fractionated and recoveredfor sequencing (ligated with proprietary adaptors using T4 RNA ligase) according to themanufacturer\u2019s instructions. The deep-sequencing experiment was performed at the BeijingGenomics Institute , as a commercial service. Three different strains weresequenced separately using an Illumina Hiseq 2500 platform . An average depth of 150,000\u2013210,000 reads for each nucleotide was generated.Sequence assembly and mapping were conducted on the 3500xL Genetic Analyzer.Isolation of nucleic acids and deep sequencing was performed using Illumina Hiseq. Inbrief, proviral DNA was extracted from eMDM cells 4 per\u03bcl/blood were considered to have thrombocytopenia. Pathological assessments were performedon five autopsied organs by a professional pathologist. Autopsied specimens were embeddedin paraffin for 24\u2005h, then sliced and stained with haematoxylin and eosin (H&E) formorphological review. The five autopsied organs were lung, kidney, spleen, lymph nodes andliver. The severity of the pathological changes in each organ was categorized and scoredindependently between immunocompetent horses and immunosuppressed horses. For each organ,\u201c0\u201d indicated a normal organ, \u201c1\u201d indicated mild pathological changes, \u201c2\u201d indicatedmoderate pathological changes and \u201c3\u201d indicated severe pathological changes. A total offour professional pathologists scored each organ, by reviewing three randomly captured(\u00d710 magnification) images independently under double-blind conditions. For each organ,the resultant score was the score assigned consistently by at least three pathologists. Ifno such consensus was reached, the organ was reassessed and scored by a panel of more thanfive pathologists.Clinical manifestations of disease in horses were individually assessed. Horse rectaltemperature was recorded daily. Episodic fever was identified as being a temperature over39\u00b0C. Horses with a platelet count in peripheral blood of less than 1\u2009\u00d7\u200910env Abs titre, env Absavidity and nAbs against the vaccine strain in the three different env diversity-varied groups of horses were compared using a linearmixed-effects model and adjusting for time. The group inoculated with the EIAV stainharbouring env moderate-diversity was taken as a reference.The package \u201clmerTest\u201d in R (Version 3.6.0) was used to carry out the estimation.Multivariate ANOVA inference was employed to test for significance between the differentcharacteristics of the three env diversity-varied EIAVstrains. Results were considered significant or very significant when P-values were less than 0.05 or 0.01, respectively.The in vitro during the developing attenuation ofvirulent strain [env diversity were constructed,including an attenuated vaccine EIAV_HD, a recombinant strain EIAV_MD, and a monoclonalinfectious clone EIAV_LD . In EIAV_MD, three highly variable regions (V6/V7/V8) were seen. Only asingle highly variable region (V2) was found in EIAV_LD .Furthermore, a clear parallel was found between the increased diversity and the increasedpassaging t strain . Here, tLN40 at 24 weeks post-inoculation. The surviving horses were subjected toa follow-up treatment of dexamethasone to mimic an immunosuppression stage. The timelineof the whole experiment can be broken down into three stages: a 24-week stagepost-inoculation and prior to challenge; a 4-week stage post-challenge but prior toimmunosuppression, and an 8-week stage post immunosuppression. The immunity of the hostcan therefore be considered as immunocompetent (week 0\u201324) followed with immunosuppression(week 28\u201330). The rectal temperature was monitored and the number of platelets inperipheral blood was assessed throughout the experiment (2 to 4\u2009\u00d7\u2009103 copies/ml). There was insignificant variationin the viral load at different time points in sera from horses inoculated with the threeenv diversity-varied EIAV strains . Afterchallenge, all the horses in the control group, 2 horses from the EIAV_LD group, and 2horses from the EIAV_MD group developed acute disease that resulted in death. One horse inthe EIAV_LD group developed acute EIA, with concurrent fever and marked decline inplatelets, but did not die. The viral load in all the horses that developed clinicalsymptoms reached high levels (>1\u2009\u00d7\u2009107 copies/ml). However, none of thehorses inoculated with the high-diversity vaccine developed disease post-challenge andprior to immunosuppression, indicating that the EIAV_HD provided complete protection.Moreover, the horses without any clinical EIA symptoms harboured a low viral load(2\u2009\u00d7\u2009102\u20134\u2009\u00d7\u2009103 copies/ml) . The three mock-inoculated(sham control) horses displayed acute EIAs and died within 28 days post-challenge, and allhad viral loads above 1\u2009\u00d7\u2009107 copies/ml . Fifteen horses were randomly divided into three groups and inoculated with indicatedvaccine candidates, with an independent control group of three horses inoculated withsaline buffer, as indicated in the schematic diagram . All theenv diversity, 60% of horses were protected. Similarly increasing speed of avidity or nAbs were seen in allthree groups inoculated with diversity-varied strains. However, the magnitude of aviditydisplayed a diversity-dependent gradient. Additionally, the diversity-non-dependentcontrol, the titre and avidity of gag-specific Abs showed nodiscrepancy in three groups. Only at 4 weeks post inoculation were the titres of gag (p26) significantly greater in EIAV_LD than in the othergroups . Using a linear mixed effects model with adjustment for time, thisdisparity can be clearly demonstrated . In the group inoculated withEIAV_LD, only the env antibody avidity was significantlylower than the reference group, indicating that EIAV_LD could arouse similar humoralimmunity to EIAV_MD strain. We also analysed correlations between env diversity and nAbs to the challenge strain and several immune cytokines pre /post-challenge. Although the nAbs against the challenge strainwere found to be low during the observation period, a milder disparity was also observedbetween the three groups pre and post-challenge (Table S1). Moreover, a significantcorrelation could be seen between SNPs and nAbs for the challenge strain and IFN-\u03b3 priorand post-challenge using a Pearson method. (P\u2009<\u20090.05)(Table S2). A significant correlation could be seen between SNPs and nAbs against thechallenge strain and IFN-\u03b3 prior and post-challenge (P\u2009<\u20090.05) (Table S2). Only a mild correlation between SNPs and IL-2 was seenpost-challenge. The enhanced nAbs and IFN-\u03b3 observed might be elicited by viral strainswith increased diversity. In summary, higher diversity of the EIAV strain elicited greaterhumoral immunity even for IFN-\u03b3 than in lower diversity EIAV strains. The humoral responses to EIAV in all vaccinated horses were evaluated by detection andanalysis of the titre of nstrated .Taking We next examined the pathological changes in tissues from horses that had either diedfrom the disease or had been euthanized after challenge with a virulent viral strain. Atthe immunocompetent stage, four horses, two from EIAV_MD group (No.13 and NO.16) and twofrom EIAV_LD group (No.9 and No. 21), developed acute EIA and died post-challenge. Thepathological scores given to the organs of these horses after autopsy are shown in env diversity-varied groups separately. In general,pathological changes were much milder in specimens from immunosuppressed horses than thosefrom immunocompetent horses. For example, the changes seen in the lungs included sparse orabundant oedema with congested capillaries and dilated alveoli with widened alveolarsepta. In the spleen, horses underwent detectable structure disarrangement with mildfibrosis. In the lymphoid tissue, mild-to-moderate chronic adaptive changes, includinghyaline degeneration and interstitial fibrosis, were seen in organs , EIAV_MD(a recombinant strain with moderate env diversity) and EIAV_LD(an infectious clone with low env diversity). Statisticalanalysis has revealed a linear correlation between the extent of env-diversity and protection against challenging EIAV infection. Theimmunosuppression of hosts inoculated with diversity-varied stains has further \u201chighlighted\u201dthe existing linear correlation between env-diversity andprotection. We supposed that the suppression of host immunity could enhance the viralreplication, which was validated by the replicating curve prior and post-challenge.Combining the results of three env diversity-varied inoculatedgroups prior to challenge and post-challenge as well as prior to immunosuppression andpost-immunosuppression, it is clear that env diversity playsan important role in immune protection against EIAV. To the best of our knowledge, this isthe first study demonstrating how env diversity-variedlentiviral vaccines protected hosts against viruses, leaving behind decades of Gag beingused as a primary target immunogen [We hypothesized that a diversity-dependent mechanism should exist during virus-hostinteractions. We used a well-characterized EIAV attenuated vaccine able to provide effectiveimmune protection against a heterogenous virulence strain, and have constructed adiversity-dependent platform to verify our hypothesis. Our findings have confirmed ourhypothesis that the mmunogen .env diversity-dependentprotection, env-specific Abs were periodically measured andcompared between groups. As expected, there was a linear correlation between the titre,avidity of env-specific Abs or nAbs against vaccine, and themagnitude of env-diversity of the three inoculated strains.These results suggest that increased env-diversity has aprotective mechanism as it enhances humoral immunity through both eliciting and maturingenv-specific Abs. Notably, the titre of nAb in HD group ismuch higher than the other groups. Unexpectedly, we observed a markedly low titre of nAbs inEIAV_MD group, similar to that of the EIAV_LD group. By reviewing the deep sequencingdatabase and searching for differences between strains, we found that a hypervariable regionV3, which is correlated with neutralization antibody response [env-diversity couldalso play a role in autologous immune escape, which has been substantially documented inHIV-1 [env, it is nevertheless of great importance that wecontinue to attempt this, in order to be able to target the hypervariable regions and widerepitopes from the diverse quasispecies that may exist in viral stocks [To better understand the mechanism of response ,38, is ainHIV-1 ,39. Despl stocks ,41. Herein vitro andin vivo studies, the data presented here using EIAV env diversity-varied viruses clearly demonstrate the effects ofenv diversity on vaccine efficacy. Moreover, the complexityof the env diversity was able to increase during thevirus-host interactions. Numerous HIV vaccines developed with enhanced diversity haveconferred higher protection compared to the low diverse controls [env-specific Abs precursor pool. In addition, we found that thesignificance of env diversity for vaccine efficacy does notpreclude a role for gag diversity in a protective vaccine.Therefore, additional studies are still required for specific validation, and there is stilla long way to go before potent lentivirus vaccines can be reliably developed. We believethat our observations using env diversity-varied strains couldprovide useful information for the design of vaccine regimens for the other members of thelentivirus family.In general, combining results from both our controls ,22,42,43Click here for additional data file."}
+{"text": "Lepidagathis hyalina Nees is an ethnomedicinally potential Asian herb, locally used to treat cardiovascular diseases and coughs. The study was intended to evaluate qualitative and quantitative investigation to ensure numerous pharmacological properties of methanol extracts of L. hyalina Ness root (MELHR). MELHR manifested strong radical scavenging activity in the reducing power and DPPH  assays, and phenol and flavonoid in the quantitative assays. In the study of the thrombolytic assay, MELHR showed moderate explicit percentage of clot lysis (29.39\u2009\u00b1\u20091.40%) with moderate (135.35\u2009\u00b5g/mL) toxic properties. The in vitro anti-inflammatory activity was evaluated by the inhibition of hypotonicity-induced RBC hemolysis, whereas the plant extract exhibited a significant (pp\u2009\u02c2\u20090.005) dose-dependent inhibition and the highest inhibition was found 55.01\u2009\u00b1\u20093.22% at 1000\u2009\u00b5g/mL concentration. Moreover, the MELHR also showed significant (p\u2009<\u20090.005) dose-dependent potentiality on protein denaturation which is considered as antiarthritic activity, and the peak inhibition was found significant (71.97\u2009\u00b1\u20092.71%) at 1000\u2009\u00b5g/mL concentration. MELHR also exhibited the dose-dependent and statistically significant anthelmintic potential on aquarium worm (Tubifex tubifex). So, the present investigation suggests that L. hyalina could be the best choice for the management of cardiovascular, inflammation, arthritis, and anthelmintic diseases. Further investigation should be necessary to determine behind the mechanism of bioactivity and therapeutic potential of this plant. Free radicals are major concern for biological evolution and also have certain beneficial effects on some species . An imbaArthritis becomes a common cause of disability in developed countries nowadays, which is attributed to pain, restricted joint movement, and synovial membrane inflammation , 13. FibHelminthiasis (worm infestation) is a macroparasitic ailment in which parasitic worms such as nematodes, trematodes, cestodes, and other parasitic worms infest the human and animal body and cause a variety of disorders like pneumonia, malnutrition, eosinophilia, anemia, etc. It exacerbates global economic and social difficulties, particularly in tropical areas . AccordiAllium sativum (garlic), and Hydrastis canadensis are good options of antimicrobial activities with lesser side effects than their synthetic counterparts like streptomycin and Aureomycin  16-\u03b1-hydroxy-olean-12-en (13)-28-oic acid)) identified in the leaves of this plant has antibacterial and antifungal properties, and also the stem part of this plant manifested several pharmacological properties which are reported as antioxidant, thrombolytic, antidepressant, and anxiolytic activity [in vitro tests on Lepidagathis hyalina Ness.tivities , 31. Thitivities , 32. Accactivity , 32. To Sodium carbonate, ferric chloride, folin-ciocalteu reagent (FCR), trichloroacetic acid, methanol, potassium ferrocyanide, potassium acetate, aluminum chloride, hydrochloric acid, and sulfuric acid were purchased from Merck . Diclofenac sodium and levamisole were brought from ACME Laboratories Ltd. . Sodium acetate, quercetin, and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) were collected from Sigma Chemical Co. . Lyophilized streptokinase vial (1500000 IU) and vincristine sulfate  were gained from Beacon Pharmaceutical Ltd. . Ultraviolet-Vis spectrophotometer  was applied to take absorbance for this experiment. Specified reference-tagged chemicals were used in this research project which was an analytical grade reagent.Lepidagathis hyalina Ness were collected as fresh condition from the Golden Temple Hill area, Bandarban, Chittagong, Bangladesh. After collection, the plant taxonomy was authenticated by Prof. Dr. Sheikh Bokhtear Uddin , and also identified and confirmed by Prof. Dr. Abu Sayeed .The roots of L. hyalina were washed and stored in shade at low temperature for two weeks and after that ground into coarse powder through a blender machine. The blended powder of root was put in an impermeable container with a sufficient volume of methanol at room temperature for 10\u201314 days, and the mixed solution shook vigorously. A rotary evaporator was applied to filter the mixed solution at 40\u201350\u00b0C. By this process, a deep green sticky semisolid was formed and kept in a refrigerator until further study. All of the research design and protocols have been approved and authenticated by the P&D committee (Pharm-P&D17/08\u2032'-19), Department of Pharmacy, International Islamic University Chittagong, Chittagong, Bangladesh.The roots of MELHS was experimented for preliminary qualitative phytochemical analysis through the standard determination method of phytochemicals  .L. hyalina Ness roots (MELHR) was examined through the method described by Tayab et al. [Free radical scavenging assay of methanol extract of b et al. . The DPPHere, Ac \u2009=\u2009absorbance of the control and As \u2009=\u2009absorbance of the sample.\u00b5g/mL) was made and then mixed with 2.5\u2009ml of 0.2\u2009M phosphate buffer (pH 6.6) and 1% potassium ferricyanide. At 50\u00b0C temperature, the mixed solution was incubated for reaction completion. About 2.5\u2009ml of 10% trichloroacetic acid was added and centrifuged the mixer for 10 mins at 3000\u2009rpm. The formed supernatant solution was dispelled after centrifuging period. Then, a half milliliter of 0.1% ferric chloride and 2.5 milliliter distilled water were summed up and then investigated the absorbance at 700\u2009nm on a UV spectrophotometer. The blank and reference standard that applied in this experiment was phosphate buffer and ascorbic acid, respectively.The method of Sarkar et al. was conducted to evaluate the reducing power assay of MELHR . 1 milliThe total phenolic content of MELHR was measured by the following method, where gallic acid is used as standard . 1\u2009ml of\u03bcl aluminum chloride (10%), 200\u2009\u03bcl of 1\u2009M potassium acetate, and 5.6 milliliters of distilled water were added to each test tube one after another. The final mixture was placed in incubation for 30 minutes to facilitate the reaction. Then, the sample, standard, and a blank were placed into UV machine, and absorbance was measured at 420\u2009nm. Total flavonoid content was shown as mg of quercetin equivalent/gm of dried extract.The content of total flavonoid content of MELHR was measured by aluminum chloride colorimetric method using quercetin as standard . 1\u2009mL of\u00b5/mL). Vincristine sulfate was used in this experiment as a positive control as the preceding method in serial concentrations between 0.125\u2009\u00b5g/mL to 10\u2009\u00b5g/mL. In each experimental vial, ten alive nauplii were added and incubated at room temperature under the light. After incubation, an amplifying glass was applied to calculate the living nauplii in each vial and record the number. The mortality percentage of nauplii was figured out according to the equation:N0\u2009=\u2009number of nauplii taken and N1\u2009=\u2009number of nauplii alive.The cytotoxic properties of MELHR were investigated by the method of Alam et al. with slight modification . ArtemiaAlsever solution  was taken and equally mixed with fresh whole blood that was taken from some healthy volunteers. Then, the blood solution was centrifuged at 3000\u2009g for 10 mins. After that, isosaline was subjected to wash the packed cells, then made 10% v/v solution, and kept at 4\u00b0C before use in this experiment.\u00b5g/mL) of crude extract and diclofenac sodium were taken, and then, add 0.5\u2009mL of stock erythrocyte (RBC) suspension, 1\u2009mL of 10\u2009mM sodium phosphate buffer (pH 7.4), and 2\u2009mL of hypotonic solution (50\u2009mM sodium chloride), respectively. 0.5\u2009mL of stock erythrocyte (RBC) suspension and the hypotonic-buffered solution were mixed and considered as a control sample solution. The different mixer that was made in this experiment was incubated for half an hour at 37\u00b0C temperature and then centrifuged for 20 mins at 3000\u2009g. After centrifugation, the supernatant solution was subjected to calculate the absorbance at 560\u2009nm on a UV spectrophotometer. The percentage of inhibition was counted through the equation as follows:1\u2009=\u2009optical density of the hypotonic-buffered saline solution and OD2\u2009=\u2009optical density of test sample in a hypotonic solution.The anti-inflammatory effects of MELHR were inspected on hemolysis of human red blood cells (HRBCs) induced by a hypotonic solution and were evaluated using the described method with slight modification . Differe\u00b5g/mL) of MELHR and diclofenac sodium were taken. 1N HCl was applied to adjust the pH 6.3 of the solutions and then incubated first for 20\u2009mins at 37\u00b0C temperature. After that, all solutions were kept in an incubator for half an hour again at 57\u00b0C. After the incubation process, all the solutions were cooled, and then add 2.5\u2009milliliters of phosphate buffer. The UV-visible spectrophotometer was subjected to measure the absorbance of the solutions at 416\u2009nm. The control manifests 100% protein denaturation. The following equation was used to measure the percentage inhibition of protein denaturation:AC\u2009=\u2009absorbance of the control and AS\u2009=\u2009absorbance of the sample.The antiarthritic activity of MELHR was investigated by the method of the protein denaturation technique . The reain vitro thrombolytic assay was performed using streptokinase vial in the same method described by Hasnat et al. [\u00b5l of (10\u2009mg/ml) methanol extract was added to the preweighed clot. 100\u2009\u00b5l streptokinase was added to the positive control group and 100\u2009\u00b5l of distilled water in the negative control group. All the tubes were further incubated for 90\u2009min at 37\u00b0C for clot lysis. After removing the fluid, the weight of the clot was further measured and the difference in weight was calculated. The percentage clot lysis was computed using the formula:The t et al. . 0.5\u2009ml Tubifex tubifex was subjected to find out anthelmintic potential because it has anatomical similarity and belongs to the same group of intestinal worms. The sludge worm used in this experiment was collected from the aquarium shop of Chittagong. The experiment was divided into several groups; whereas the negative control group consists of only distilled water, the positive control group consisted of the standard drug levamisole (1\u2009mg/mL), and the test group consisted of different concentrations  of crude extracts, respectively. In this investigation, 10 to 12 worms were placed in each Petri dish in five different groups. Then, 3\u2009mL of the different concentrations of all groups was added to the Petri dish. The initial time, paralysis time, and the death time of the worms were observed and recorded carefully, in which paralysis time and death time of worms were considered as the evaluation of the anthelmintic activity of this experiment. When the worm's movement could not be observed after shaking vigorously, the paralysis time and the death time considered by the confirmation of worm's movement could not be observed either after shaking or when dipped in slightly warm water.The anthelmintic activity of crude extracts was measured by the method described by Ajaiyeoba et al. with some minor modifications . In this\u2217p\u2009<\u20090.05, \u2217\u2217p\u2009<\u20090.01, and \u2217\u2217\u2217p\u2009<\u20090.001 were considered as statistically significant. The one-way ANOVA was measured by following Dunnett's test compared to the negative control. All the assays were conducted as triplicate and repeated three times each for the consistency of the result and statistical function.GraphPad Prism version 7.00  was used to analyze the experimental results. The data were presented as mean\u2009\u00b1\u2009SEM (standard error mean), in which L. hyalina Nees root (MELHR) manifested the presence of carbohydrates, quinones, alkaloids, reducing sugar, phenols, polyphenols, flavonoids, triterpenoids, coumarins, and cardial glycosides. The yield of this experiment is summarized in The qualitative phytochemical experiment of methanolic extracts of \u00b5g/ml concentration, while standard ascorbic acid demonstrates a 97.49% effect at the same concentration. Here, the potentiality of scavenging properties was increased compared with the increased concentration. The IC50 values of MELHR and ascorbic acid were 189.01% and 20.59%, respectively, which was estimated via linear regression formula.The antioxidant activity of MELHR was investigated by DPPH free radical scavenging. The crude extract manifests potential antioxidant properties which were presented in \u00b5g/ml concentration, whereas the standard has shown 1.88 absorbance at the same concentrations.Reducing power is related to antioxidant properties in which the components containing reducing power can decrease the oxidized intermediated lipid peroxidation process. The reducing power properties of MELHR and ascorbic acid are summarized in y\u2009=\u20090.0102x\u2009\u2212\u20090.0637; for phenol assay, it was y\u2009=\u20090.0039x\u2009+\u20090.033).The total flavonoid and phenol content of the crude extract was estimated quantitatively. The result is displayed in 50 value of MELHR was 135.35\u2009\u00b5g/mL which was demonstrated via linear regression equation (y\u2009=\u20090.0691x\u2009+\u200940.647) and the average percentage of mortality was 63.33%.The potential of cytotoxicity of crude extract was evaluated via brine shrimp cytotoxic assay. The fatality result of plant extract was assessed in in vitro anti-inflammatory potential of MELHR is assessed in p\u2009\u02c2\u20090.05) increased the anti-inflammatory properties. The peak percentage of inhibition of hemolysis by hypotonic solution and heat-induced hemolysis of crude extract was found 55.01\u2009\u00b1\u20093.22 at 1000\u2009\u00b5g/mL concentration, whereas diclofenac Na manifested 82.46\u2009\u00b1\u20091.92 at the same concentration.The \u00b5g/mL, respectively.The antiarthritic activity of MELHR on protein denaturation is presented in p\u2009\u02c2\u20090.005) moderate (29.39\u2009\u00b1\u20091.40%) clot lysis properties compared with both positive and negative controls.The thrombolytic property of MELHR is shown in Tubifex tubifex worms which finding summarized in \u00b5g/mL concentrations the extract manifested significant paralysis time 13.2\u2009\u00b1\u20090.842, 7.37\u2009\u00b1\u20090.684, and 4.5\u2009\u00b1\u2009065\u2009min; and death time 36.16\u2009\u00b1\u20093.096, 27.3\u2009\u00b1\u20092.197, and 15.53\u2009\u00b1\u20091.88\u2009min, respectively, whereas the standard drug levamisole showed paralysis and death time 3.17\u2009\u00b1\u20090.189 and 6.5\u2009\u00b1\u20090.384\u2009min, respectively, at 1\u2009\u00b5g/mL. The result indicated that the effect of anthelmintic was directly proportional to the concentrations of crude extract.The anthelmintic activity of MELHR was investigated on Lepidagathis hyalina roots. Phytochemical screening of the methanolic extract of L. hyalina roots confirmed the presence of a wide range of chemical entities such as carbohydrates, quinones, alkaloids, reducing sugar, phenols, polyphenols, flavonoids, triterpenoids, coumarins, and cardial glycoside that can be associated with several pharmacological activities given by the plant extract.Despite the availability of modern medicines, medicinal plants in developing countries are becoming increasingly popular as primary healthcare products because of their low cost and low side effects and toxicity , 41. In 50 values of crude extract were found 189.01%, which by comparison with ascorbic acid stand as 20.59%. Then, the connection of methanol extracts between TPC, TFC, reducing power, and DPPH was analyzed, indicating that MELHR has strong antioxidant potentiality [50 at low concentration with a quick response indicates that the plant extract is quite potent to give cytotoxic activity [The numerous artificial antioxidants have been replaced with plant-based polyphenols and flavonoids in recent years. Nature source antioxidants are obviously safer to consume than artificial antioxidants, which are suspected of harmful effects on health , 42. Thentiality . Howeverntiality . LC50 atactivity .in vitro antiarthritic activity of MELHR was summarized in terms of inhibition of the protein denaturation method. The study demonstrated that both the standard drug and the plant extract increase inhibition level gradually with the increase in doses, but diclofenac Na was observed to give better results than plant extract indicating a better antiarthritic effect.Since the membrane of erythrocytes is regarded to be analogous to the lysosomal membrane, the erythrocyte membrane stabilization test is considered to be an efficient tool for the anti-inflammatory drug screening process. The extracellular release of lysosomal contents by activated neutrophils is an important phenomenon in the pathophysiology of inflammation that is why membrane stabilization tests play a crucial role to assess inflammation , 45. Botp\u2009<\u20090.05) reduced the percentage of clot lysis (29.35%) as moderate scale compared with standard. A significant difference between the percentage values of clot lysis of positive and treatment groups indicates the efficiency of plant extract to exert effective thrombolytic activity. In a previous study, carbon tetrachloride extract and dichloromethane soluble extract have shown an almost similar level of activity  against streptokinase standard [Thrombosis occurs as a result of hypercoagulation of the blood, damage to blood vessels, and blockage of blood flow within blood vessels. It is a life-threatening vascular complication of arthritis, myocardial infarction, pulmonary embolism, cerebrovascular ischemia, stroke, and venous embolism. Furthermore, venous thrombosis has been identified as the second greatest cause of cancer-related mortality . The plastandard .As part of a study to assess anthelmintic action, it was discovered that the crude extract causes dose-dependent paralysis, ranging from loss of mobility to death. The plant extract's paralysis and death time were compared to that of the standard medication levamisole. The anthelmintic activity of the test extract steadily increased, although at a higher concentration than levamisole. The fact that the plant extract includes a combination of components, but levamisole is a single component utilized as a medicine, might be the explanation. It was obtained from the previous studies that alkaloid, phenol, tannin, and terpenoids play an important role to give anthelmintic activity \u201352.L. hyalina Nees could be considered as a potential source for discovering the secondary metabolites which could be used for numerous pharmacological applications, and further studies are required to reveal the mechanism behind its potentiality.The complied pharmacological results indicated that this plant has a strong potentiality for the treatment of several diseases like arthritis, inflammation, cancer, etc. In our investigation, MELHR has been proved to have promising radical scavenging properties. Additionally, MELHR possesses significant arthritis, anti-inflammatory, thrombolytic, and anthelmintic activity with moderate toxic effects. This potentiality of MELHR could be due to the several bioactive compounds that are found in phytochemical analysis. Overall,"}
+{"text": "The Myc family of transcription factors are involved in the development and progression of numerous cancers, including prostate cancer (PCa). Under the pressure of androgen receptor (AR)-directed therapies resistance can occur, leading to the lethal form of PCa known as neuroendocrine prostate cancer (NEPC), characterized among other features by N-Myc overexpression. There are no clinically approved treatments for NEPC, translating into poor patient prognosis and survival. Therefore, there is a pressing need to develop novel therapeutic avenues to treat NEPC patients. In this study, we investigate the N-Myc-Max DNA binding domain (DBD) as a potential target for small molecule inhibitors and utilize computer-aided drug design (CADD) approaches to discover prospective hits. Through further exploration and optimization, a compound, VPC-70619, was identified with notable anti-N-Myc potency and strong antiproliferative activity against numerous N-Myc expressing cell lines, including those representing NEPC. The Myc protein family are transcription factors regulating communication networks within cells by binding to DNA and modulating the expression of numerous genes involved in cell growth and division. Dysregulation of Myc, especially N-Myc, is strongly associated with the development of the most resistant malignancies, including neuroendocrine prostate cancer (NEPC) ,2,3,4 foMyc has always been considered a difficult target and often classified as undruggable, due to its intrinsically disordered nature and the lack of distinct binding pockets on its surface ,9,10,11.For example, compound EN4 was recently designed as a covalent ligand targeting the Cys171 residue within the intrinsically disordered region of Myc . By bindGiven the high structural and functional similarity between the c-Myc and N-Myc oncoproteins ,20, we uPreviously, we identified a potentially druggable pocket where the Myc bHLHLZ domain interacts with its homologous domain from the Max protein, forming a stable helical configuration which binds specifically to DNA E-boxes at enhancers and promoters of Myc target genes . By screThis similarity search yielded the hit compound VPC-70127, which is characterized by the presence of a 2-nitro-4-(trifluoromethyl)phenyl and a pyrazine connected via a hydrazide linker. Although VPC-70127 showed strong Myc inhibitory activity in our primary transcriptional assay, initial assessment demonstrated that VPC-70127 only weakly disrupts DNA binding to Myc-Max complex. Similarly, although VPC-70511 showed promising inhibitory activity in the transcription assay in LNCaP cells, further testing in a counter-screen using Myc-negative HO15.19, revealed that VPC-70511 had a highly cytotoxic profile. Thus, we pursued another hit, VPC-70551, which contains a similar hydrazide linker connecting two substituted phenyl rings together. The 2-nitro-4-(trifluoromethyl)phenyl was replaced by 4-cyano-2-(trifluoromethyl)phenyl and the pyrazine was replaced by a (trifluoromethyl)phenyl, as shown in To confirm the new scaffold\u2019s specificity toward N-Myc, we performed a round of substructure searches using N\u2019-phenylbenzohydrazide as a query against the ZINC15 database and selected 34 analogues for experimental validation . We thenThe docking pose of VPC-70551 inside N-Myc-Max/DBD complex revealed that the compound forms an H-bond with the backbone of Max\u2019s Asp215 through its hydrazide linker, and a pair of weaker H-bond with sidechain Lys419 and Arg214 of N-Myc through its cyano group. The 4-cyano-2-(trifluoromethyl)phenyl group is involved in numerous hydrophobic contacts with N-Myc residues Leu397, Phe401, and Lys419, and with Max residues Arg212, Arg215 Ile218, Lys219, Phe222, and Arg239. These contacts anchor VPC-70551 into its position in the DBD pocket c. A compTo determine the optimal substitutions on the free phenyl ring, we used the active scaffold for a second substructure search against the ZINC15 database to identify a set of 181 analogues for further validation . Bulky and large chemical groups were deemed unfavorable at the meta-substitutions  as they would displace the scaffold completely from its proposed binding position and shift the compound to the solvent-exposed areas.. Although the sulfonyl linkers are interacting with the Max protein, they cannot compensate for the unfavorable position of the ligands\u2019 groups in the solvent-exposed area of the pocket. This lack of activity is observed in most compounds containing sulfur groups at para and meta positions . Interestingly, three compounds  with sulfur or large linkers were active, and all three had fluorine groups.We then tested another series of substitutions with sulfonyl linkers at the R3-position. Combinations of different groups with the linker, such as methyl, ethyl, amine, or methylaniline with the sulfonyl linker, did not help their inhibitory activity We did not observe significant inhibitory activity in compounds with an oxy linker in their R3-extended group , or compounds with para\u2013OH or meta \u2013OH substitutions . We observed a few exceptions: VPC-70654, 70663, 70732 and 70792. Both VPC-70732 and 70792 had oxy linkers in their substitution group, while VPC-70663 had a R3-nitro group, and they still maintained good transcription inhibition activity.Interestingly, most of the active compounds contained at least one regular halogenic substitution. Sixteen analogues contained an R3\u2013Cl substitution combined with other groups at the meta- or ortho-positions. Combinations of R3\u2013Cl with other halogens/halogenic group at the meta position demonstrated good inhibition at both concentrations tested , while small halogens at ortho position abolished inhibitory activity . At the meta positions, methyl groups (VPC-70617) or sulfonyl groups (VPC-70721) did not impart any inhibitory activity at 5 \u00b5M, while a methoxy group (VPC-70685) or cyano group (VPC-70662) recovered the activity. Interestingly, activity was maintained when R3\u2013Cl was combined with ortho \u2013OH groups (VPC-70633) or large halogenic groups . We tried compounds with triple substitutions centered on the R3\u2013Cl as well. Although compounds with ortho-halogens at R3\u2013Cl were previously inactive, adding a third substitution recovered the inhibition activity . The third substitution added often came from a compound considered active; thus, the third substitution could be inconsequential and only two substitution groups could be necessary for inhibitory activity.3 . The effects are diminished when the substitutions are combined with methyl-ending groups . The activity is completely abolished when the groups are added at the ortho position  or when combined with bulky chemical substitutions, such as sulfonamides . The positioning of the substitutions seems to influence the inhibition activity as an R1\u2013F, R3\u2013F, and R4\u2013F grouping nullified activity (VPC-70772), while an R1\u2013F, R2\u2013F, and R3\u2013F did not (VPC-70715).Similar to the R3\u2013Cl, we observed strong increase in inhibition activity when the R3-position is substituted for another halogen group such as \u2013F, \u2013Br, and \u2013CF3 had no activity; when replaced by meta\u2013Cl, the compounds had 100% transcription inhibition activity at 5 \u00b5M. Similarly, for VPC-70672, removing the methyl group at the R3 position restored activity (VPC-70653).Based on the above observations, we wanted to determine if the activity is maintained or increased by halogen substitutions at the meta- or ortho-positions only. The activity of compounds with halogen substitutions at the R1 or R5 position (ortho) often declined considerably or was lost completely . However, compounds such as VPC-70698, 70768, 70769, and 70776 have ortho-halogen substitutions and still showed high transcription inhibition activity. In general, compounds with meta-substitutions  reported a better inhibition activity when compared to ortho-substitutions, although some had no activity (VPC-70684). If the substitution at the R3 position is a methyl-like group, the compounds had a lower transcription inhibition or no activity at all . We observed that too many halogenic groups hampered activity (VPC-70656). Compounds with methyl substitutions were often just inactive (VPC-70666), no matter which position they were: VPC-70651 with meta\u2013CH3 substitutions demonstrated very strong transcription inhibition . We observed that \u2013Cl led to a strong effect that could compensate for other substitutions that were deemed inactive in other compounds. This is the case for VPC-70619 which substituted the meta \u2013OH group of VPC-70673, for a \u2013Cl, restoring the inhibitory activity at 5 \u00b5M.Compounds with simultaneous halogen substitutions with at least one \u2013Cl at the meta-positions of the scaffold are shown to be significantly more active. Combining \u2013Cl with either \u2013Cl, \u2013Br, and \u2013CFBased on the above SAR considerations we have concluded that due to the presence of the strong electronegative \u2013Cl group, mainly at the R3 position and to a lesser extent \u2013F and \u2013Br, there is a strong case to have at least one halogenic group or a functional group with significant electronegativity as a substituent on the free phenyl ring. We stipulated that halogenic groups might have a crucial role as a hydrophobic contact to anchor the ligand to Max. Consequently, we carried out further viability assays to determine whether the reported activity in -Cl compounds emerged from toxicity effects.We shortlisted 54 active compounds requiring further testing in cell viability assays to determine their inhibition effect on N-Myc-dependant cellular growth. The shortlisted compounds included all derivatives of the N\u2019-[4-Cyano-2-(trifluoromethyl)phenyl]benzohydrazide scaffold which were active at both 10 \u00b5M and 5 \u00b5M in our LNCaP-NMYC transcription inhibition assays. The compounds are mainly characterized by substitutions at the para- and meta-positions, with at least one of the substituted groups being a halogenic or a strong electronegative group.The 54 compounds were tested at 10 \u00b5M for their inhibition of the IMR32 human neuroblastoma cell line, in which the MYCN gene is amplified and actively expressed . HO15.1950 of 4.3 \u00b5M in the luciferase transcription inhibition assay, and presented a stable microsomal half-life of 2310 min, compared to 3 min for 10074-G5  was effective in the IMR32 cell lines with an inhibition higher than 50%. At 10 \u00b5M, VPC-70619 had an inhibition of 99.4% in IMR32 cells while having a minimal effect of 14% in HO15.19 cells, thus showing potential selective activity against N-Myc expressing cells. We reported that VPC-70619 shows good inhibition of NEPC in the in vitro model NCI-H660 cell line. Furthermore, VPC-70619 had an IC10074-G5 .Based on a promising activity profile of VPC-70619, we studied its binding pose in greater details. We observed that VPC-70619 has a similar docking profile to VPC-70551 thanks to their common shared scaffold. The compound maintained most of the same interactions, including engagement to the side chains of Lys419 of N-Myc. Additionally, side chains Lys219, Asp216, Arg212, and Arg215 act as clamps to anchor the phenyl ring into a stable conformation in the pocket through hydrophobic interactions . AlthougAs seen in the docking poses, the 3-chloro-4-cyano benzohydrazide portion of VPC-70619 overlaps significantly with the DNA backbone a. These We first performed a microscale thermophoresis (MST) assay to validate the direct binding of VPC-70619 to the recombinant N-Myc-Max complex and to calculate the binding affinity. We evaluated the effect of increasing concentrations of 70619 on the movement of a fluorescent labelled N-Myc-Max complex through a temperature gradient induced by an infrared laser. VPC-70619 demonstrated a dose-dependent shift of thermophoresis; however, due to the limited solubility of VPC-70619 in the assay buffer, we could only estimate a dissociation constant above 130 \u03bcM b.To confirm that 70619 could disrupt the binding of N-Myc-Max DBD to DNA, we performed a bio-layer interferometry (BLI) assay where we immobilized biotinylated E-box sequence on streptavidin sensors, and we evaluated recombinant N-Myc-Max binding to the DNA in the presence of increasing concentrations of VPC-70619. We found that the compound was effective at blocking N-Myc-Max binding to DNA in a dose-dependent manner c. We finmax) occurred at 480 min for PO and 60 min for IP, with peak concentrations (Cmax) of 2600 ng/mL and 6220 ng/mL, respectively, following administration in 75:25 propylene glycol (PG)/poly-(ethylene glycol) 400 (PEG) formulation at 10 mg/kg dosing. Plasma concentrations of 70551 decreased slowly, with a half-life (T1/2) of 332 min for IP administration. The clearance of 70551 for PO delivery was extremely slow, and we could not determine the T1/2 in the experimental conditions used for this assay. No obvious adverse effects of 70551 were observed in the tested animals. Regarding 70619, it presented much higher bioavailability, with a Cmax of 29,500 ng/mL and 24,000 ng/mL for IP and PO, respectively, following administration in a 20:80 Kolliphor EL formulation. Plasma concentrations decreased slowly, with a reported terminal half-life of 330 min for IP, while PO had a T1/2 of 427 min. No signs of toxicity were observed in any control or treated animals. Therefore, VPC-70619 was deemed to have appropriate efficacy in vivo, and a better pharmacokinetic profile due to higher bioavailability for both oral and intraperitoneal administration.In order to characterize VPC-70619 as a drug candidate against N-myc, its pharmacokinetic (PK) characteristics were evaluated in Balb/c mice following intraperitoneal (IP) and peroral (PO) administrations. As presented in While N-Myc is not significantly expressed in adult tissues, it was reported to be amplified and overexpressed in NEPC tumors . The preIn our previous studies, we described a novel anti-Myc compound series targeting the proposed Myc-Max heterodimer DBD pocket , and repTo improve the potency in the series, we explored the enhanced N-Myc scaffold by searching for new compounds with substitutions on the empty phenyl ring. Extensive SAR analysis revealed that successful inhibition relies on the presence of an electronegative group such as halogens at the para- or meta position of the phenyl ring. The halogen substitutions, combined with another small group, maintained and enhanced activity or specificity. Docking simulations show that the halogens at the para or meta positions interact with the N-Myc-Max DBD site through a network of hydrophobic contacts, while halogen substitutions at the ortho position do not impart any activity as they are not able to interact with the pocket or are displaced by the compound completely when bound. Similarly, large, extended, and bulky substitution groups are detrimental to the scaffold\u2019s anti-N-Myc activity, as they often displace the compound from the pocket in our docking simulations. We tested multiple series of ligands, including sulfonyl, methyl, ethyl, and oxy linkers, and the majority of them did not return significant improvement over the parental compound, VPC-70551. We stipulate that they could not enhance the proposed interaction network that was already in place, or could not compensate for the unfavorable solvation of the ligands\u2019 exposed groups. Our binding and SAR model provides valuable insight into the proposed N-Myc-Max DBD pocket, and suggests that active compounds must bind tightly to both N-Myc and Max and minimize solvent-exposed groups.50 of 7.0 \u03bcM. Importantly, 70619 was effective at inhibiting growth in N-Myc-specific cells (IMR32 and NCI-H660) while presenting minimal cytotoxicity in the Myc-negative HO15.19 cell lines. The compound was shown to bind to recombinant N-Myc-Max complex and to block DNA binding without disrupting the N-Myc-Max heterodimer itself. However, VPC-70619 presented somewhat weak affinity to N-Myc-Max; this might be due to the nature of the proposed pocket, and thus further optimization of the molecule is needed to ensure the success of our structural determination of the N-Myc-Max interaction with the identified inhibitors. Nonetheless, we propose that 70619\u2032s mode of action in inhibiting N-myc cell proliferation is via the blocking of the N-Myc-Max heterocomplex from binding to target genes.Using cell-based screening, we could exclude molecules presenting off-target effects or toxicity. We identified a potent compound, VPC-70619, characterized by the same 4-cyano-2-(trifluoromethyl)phenyl-benzohydrazide scaffold which anchors the compound into the hydrophobic core of the N-Myc-Max DBD pocket, as well as a 2-chlorobenzonitrile group which clashes with the DNA E-box binding to N-Myc-Max. Interestingly, 70619 has 15-fold increased microsomal stability, with a half-life of 2310 min compared to 140 min for VPC-70551. The lead compound, 70619, inhibits cell proliferation levels in the N-Myc-driven NCI-H660, with a low micromolar ICBased on the proposed SAR model, including a halogenic group in our series is highly beneficial to the reported anti-N-Myc activity. As previously highlighted, their inclusion as hydrophobic moieties could therefore be important as molecular interaction with weak to medium strength , specifiThus, we propose compound VPC-70619 as a single agent that could be effective with tolerable toxic effects within a specific therapeutic window, or as a chemotherapy agent which can be part of a combined oncogenic treatment regimen ,32. PharTo identify potentially improved derivatives, high-similarity 3D structural searches were performed on the ZINC15 database  using thThe Protein Preparation Wizard tool within Maestro 9.3 from Schr\u00f6dinger LLC   was usedDocking analysis of all compounds obtained from similarity searches or medicinal chemistry synthesis was performed with the Glide docking program from Schr\u00f6dinger LLC . A dockiLNCaP-NMYC cells were a generous gift from David Rickman at Weill Cornell Medicine , and were cultured in RPMI supplemented with 10% fetal bovine serum (FBS). HO15.19 cells cultured in DMEM supplemented with 10% FBS were a generous gift from John Sidivy at Brown University . NCI-H660 cells were purchased from the American Type Culture Collection  and cultured in RPMI supplemented 5% FBS, 1% Insulin-Transferrin-Selenium  , 10nM b-estradiol  , 10 nM hydrocortisone (Sigma H0888) and 1% matrigel. IMR32 cells were purchased from ATCC and were cultured in Eagle\u2019s Minimum Essential Medium (EMEM) supplemented with 10% FBS.LNCaP-NMYC cells were transfected using TransIT-2020 transfection reagents per the manufacturer\u2019s protocol. Cells were plated at a density of 10,000 cells per well of a 96-well plate. Following 24 h treatment with 5 \u00b5M or 10 \u00b5M of the compounds, the Myc reporter activity was measured using the Cignal Myc Reporter Assay Kit from Qiagen (#336841)  per the manufacturer\u2019s instructions.50 was measured with the TECAN InfiniteM200 plate reader .Viability of N-Myc positive (IMR32and NCI-H660) and negative (HO15.19) cells were determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega cat. G7570) . Cells were seeded into a 96-well white plate with a clear bottom: 10,000 cells per well for the IMR32 cells cultured in EMEM supplemented with 10% FBS, 2000 HO15.19 cells per well in Eagle\u2019s minimum essential medium (EMEM) supplemented with 10% FBS, and 2000 cells per well for the NCI-H660 neuroendocrine cells cultured in RPMI supplemented 5% FBS, 1% Insulin-Transferrin-Selenium , 10nM b-estradiol , 10nM hydrocortisone (Sigma H0888) and 1% matrigel. Following 24 h incubation, the cells were treated with serial dilutions of the derivatives along with the respective parental compound starting at 25 \u03bcM for 72 h. Cell Titer Glo was added to each well at a ratio of 1:1 and incubated on a shaker at RT for 2 min. Luminescence and ICThe bHLHLZ domain of N-Myc (amino acids 309\u2013394) and Max (amino acids 12\u201393) protein sequences were cloned into pETDuet with an N-terminal 6\u00d7His. pETDuet-His-NMyc-Max was transformed and expressed in E. coli BL21 and induced with 0.5 mM isopropyl-\u03b2-D-thiogalactopyranoside (IPTG) at 16 \u00b0C overnight. The cultures were lysed by sonication and combined for purification by immobilized metal ion chromatography (IMAC) with nickel-nitrilotriacetic acid (Ni-NTA) resin. The bound proteins were eluted from the column with 20 mM Tris, pH = 8, 500 mM NaCl, 5% glycerol, 300 mM imidazole, 5 mM \u03b2Me, and 0.1 mM PMSF. Size exclusion chromatography with Superdex 75 was used to increase the purity of the N-Myc-Max protein sample. The purified protein complex was used in subsequent BLI and MST experiments.Biotinylated-duplex DNA  was bound to the super-streptavidin (SSA) ForteBio Sensor in the assay buffer . The protein solution was mixed with the serially diluted compound (70619) to a final concentration of 0.1 mg/mL of protein and 200 \u03bcM, 100 \u03bcM, and 50 \u03bcM for the compound. Sample containing N-Myc-Max protein alone was used as a control.5 cells per well of a 6-well plate. After incubation for 24 h the cells were treated with 10 \u03bcM of either 10074-G5 or 70619 for 72 h. The coverslips were fixed with methanol:acetone (3:1) and incubated with primary antibodies n-Myc Antibody (NMYC-1)   and MAX Antibody (C-17) (Santa Cruz sc-197)  overnight at 4 \u00b0C. PLA was conducted using the Duolink In Situ Detection Reagents Red kit (Sigma DUO92008) according to the manufacturer\u2019s protocol. The coverslips were then mounted on slides with VECTASHIELD mounting media with DAPI, and images were taken on ZEISS 780 confocal microscope at 40\u00d7 magnification.LNCaP-NMYC cells were seeded on coverslips at a density of 10Bienta Enamine Biology Services  conducted PK studies according to the Enamine PK study protocols and Institutional Animal Care and Use Guidelines. The PK studies were reviewed and approved by the Bienta Animal Care and Use Committee (BACUC) under the project identification codes PK-VPC-112618 (approved 17 December 2018) and PK-VPC-101119 (approved 21 October 2019), for VPC-70551 and VPC-70916, respectively. Evaluation of 70619 and 70551 was conducted in male Balb/c mice through intraperitoneal (IP) and peroral (PO) administration. Measurements were taken at six time points: 5, 15, 60, 120, 240, and 360 min for IP; 15, 30, 60, 120, 240, and 480 min for PO. Four mice were included in each treatment group for each time point; one mouse each for IP and PO was included as a control. Initial target administration for both IP and PO was 10 mg/kg dose level, 2 mg/mL dose concentration, and 5 mL/kg dose volume. Concentration of the compounds was evaluated in the plasma samples using an LC-MS/MS system.g and incubated at 4 \u00b0C for 10 min. Samples were then loaded onto a LC-MS 96-well plate for analysis.A microsomal stability assay was performed using the Corning Gentest NADPH Regenerating System  . Mouse liver microsomes (MLM)   and compounds were diluted to a final concentration of 0.15 mg/mL and 1 \u03bcM respectively. These were prepared into a 500 \u03bcL solution with 100 mM potassium phosphate buffer (pH = 7.4). Imipramine was used as a control. NADPH solutions A and B were mixed in a 5:1 ratio, and 24 \u03bcL of the NADPH was added to each of the MLM/compound samples at 30 s intervals. At time points 10, 20, and 45 min, 100 \u03bcL of reaction was added to the stopping solution. Samples were centrifuged at 18,000\u00d7 An MST assay was conducted to determine the binding affinity of 70619 to the purified N-Myc-Max protein complex. N-Myc-Max protein samples were labeled with the red fluorescent dye NT647 using the Monolith NT Protein labeling kit RED-NHS amine-reactive ; 70619 was serial diluted with 100% DMSO and mixed with the labeled fluorescent N-Myc-Max  in the assay reaction buffer  starting at 600 \u03bcM, with a final concentration of DMSO of 5%. The protein/inhibitor solution was mixed and incubated at room temperature in the dark for 5 min before being filled into the capillaries. MST assays were performed with 20% LED/excitation power and medium MST power using premium capillaries for Monolith NT.115. Analysis of the data for Kd estimation was calculated with the MO Affinity Analysis software from Nanotemper .In this study, we determined that N\u2019-[4-Cyano-2-(trifluoromethyl)phenyl]benzohydrazide derivatives developed by CADD modelling synergized with experimental validation are potent and selective N-Myc inhibitors. We further reported that the previously-identified binding site on the N-Myc-Max/DBD complex represents a viable drug target for inhibition with small molecules. The lead compound VPC-70619, identified here from multiple rounds of structural similarity searches and molecular docking, demonstrated strong anti-proliferative activity against a neuroendocrine cell line. While we identified a potential therapeutic window that could situate VPC-70619 as a potential candidate in anti-Myc treatments, supplemental optimization studies will be required to determine its full potential for future clinical applications. We identified novel derivatives through extensive SAR studies of structural analogues, providing valuable insight into N-Myc-Max DBD site topology. Finally, we have proposed that interfering directly with the ability of N-Myc-Max to interact with DNA E-boxes could be a viable mechanism for the design of potent small molecules to treat NEPC patients.All compounds reported in this manuscript can be made available to other researchers after standard Material Transfer Agreement (MTA) implementation with the University of British Columbia.U.S. Patent Application Number: 17/250,810Filed 5 March 2021 based on PCT Application No. PCT/CA2019/051243Title: MYC-MAX INHIBITOR COMPOUND THERAPEUTICS FOR CANCER TREATMENT, METHODS AND USES ASSOCIATED THEREWITHInventors: TCHERKASSOV, A.; RENNIE, P. S.; BAN, F.; LEBLANC, E. J. J.; CARABET, L. A.; LALLOUS, N.; SINGH K.;, MORIN, H.; and TON, A."}
+{"text": "Miscanthus \u00d7 giganteus is a promising high-yielding perennial plant to meet growing bioenergy demands; however, the degree to which the soil microbiome affects its nitrogen cycling and subsequently, biomass yield remains unclear. In this study, we hypothesize that contributions of metabolically active soil microbial membership may be underestimated with DNA-based approaches. We assessed the response of the soil microbiome to nitrogen availability in terms of both DNA and RNA soil microbial communities from the Long-term Assessment of Miscanthus Productivity and Sustainability (LAMPS) field trial. DNA and RNA were extracted from 271 samples, and 16S small subunit (SSU) rRNA amplicon sequencing was performed to characterize microbial community structure. Significant differences were observed in the resulting soil microbiomes and were best explained by the sequencing library of origin, either DNA or RNA. Similar numbers of membership were detected in DNA and RNA microbial communities, with more than 90% of membership shared. However, the profile of dominant membership within DNA and RNA differed, with varying proportions of Actinobacteria and Proteobacteria and Firmicutes and Proteobacteria. Only RNA microbial communities showed seasonal responses to nitrogen fertilization, and these differences were associated with nitrogen-cycling bacteria. The relative abundance of bacteria associated with nitrogen cycling was 7-fold higher in RNA than in DNA, and genes associated with denitrifying bacteria were significantly enriched in RNA, suggesting that these bacteria may be underestimated with DNA-only approaches. Our findings indicate that RNA-based SSU characterization can be a significant and complementing resource for understanding the role of soil microbiomes in bioenergy crop production.IMPORTANCEMiscanthus \u00d7 giganteus is a promising candidate for bioeconomy cropping systems; however, it remains unclear how the soil microbiome supplies nitrogen to this low-input crop. DNA-based techniques are used to provide community characterization, but may miss important metabolically active taxa. By analyzing both DNA- and actively transcribed RNA-based microbial communities, we found that nitrogen cycling taxa in the soil microbiome may be underestimated using only DNA-based approaches. Accurately understanding the role of microbes and how they cycle nutrients is important for the development of sustainable bioenergy crops, and RNA-based approaches are recommended as a complement to DNA approaches to better understand the microbial, plant, and management interactions. Miscanthus \u00d7 giganteus (Greef et Deu.) is a promising perennial grass bioenergy crop because of its ability to produce large amounts of biomass with little fertilizer compared with hay or grain crops (\u2013M. \u00d7 giganteus has been observed to be up to three times higher than switchgrass (Panicum virgatum L. cv. Cave-in-Rock), similar to willow , three times higher than reed canary grass , and two times higher than triticale   Beauv) and reed canary grass  in crops \u20134. The p grass 7\u2013, in addi grass 7\u2013, 12. Thergy crop , 13\u201316.M. \u00d7 giganteus have been evaluated. Previously, M. \u00d7 giganteus has been observed to decrease in productivity at low temperatures  profiles and clone libraries, and found that RNA-based methods could identify enriched metabolically active membership  and fertilization (representing different N availability) on changes in microbial community membership and structure. We hypothesize that microbiome responses (as indicated by DNA and RNA) to M. \u00d7 giganteus management will differ and, specifically, that metabolically active (RNA) microbial communities will show a more rapid and sensitive response to fertilization than total (DNA) microbial communities. To test these hypotheses, soil samples were collected from the LAMPS site, a replicated chronosequence field previously used to investigate the effects of stand age and nitrogen fertilizer on M. \u00d7 giganteus and corn (Zea mays L.)  , 52. DNAM \u00d7 giganteus were compared. The origin of the sequencing library, either DNA or RNA, was found to have the greatest influence on the separation of the microbial community . These differences between DNA and RNA communities were also observed for each sampling day (pPERMANOVA = 0.001). Their influence was 3.9 times higher than that of stand age  and 15 times higher than that of N fertilization amount . DNA and RNA microbial communities were observed to separate into clear clusters using constrained analysis of principal coordinates (CAP) along the first axis of the CAP  and homogeneity  of DNA and RNA communities were also observed to be significantly different.The DNA and RNA 16S rRNA amplicons from soil samples representing three ages and three fertilization rates of Shannon < 0.001, pChao1 < 0.001, Alpha diversity of soil microbial communities was compared using the Shannon index, which evaluates both microbial richness and evenness, and Chao1, which evaluates the abundance of observed species. Both alpha diversity indices showed significant differences between DNA and RNA microbial communities, with higher alpha diversity observed in DNA microbial communities , where a total of 39,898 and 32,171 ASVs were identified in DNA and RNA, respectively. We compared the ASVs between DNA and RNA samples and found that 17,779 ASVs were shared between DNA and RNA microbial communities ; 22,119 and 14,392 ASVs were unique in DNA and RNA, respectively. Unique ASVs were generally low abundance (average\u2009<\u20090.000003%) and low prevalence (average\u2009<\u20090.022%) in their respective libraries . ASVs thASVs commonly identified between DNA and RNA libraries were further classified based on their enrichment in DNA or RNA, specifically using the ratio of RNA:DNA relative abundances. The RNA:DNA ratio of shared ASVs ranged from 0.0023 to 1,300. The majority of shared ASVs (58%) were more enriched in RNA relative to DNA . For ASVActinobacteria (26%) and Proteobacteria (33%) in DNA and mainly Proteobacteria (49%) in RNA  in RNA . While DActinobacteria and Bacteroidetes were more enriched in DNA , while Firmicutes and Proteobacteria were more enriched in the RNA microbial community . Differentiating ASVs unique in DNA included sequences associated with Actinobacteria, Gemmatimonadetes, Latescibacteria, and Parcubacteria. In contrast, sequences associated with Firmicutes were unique in RNA.We evaluated whether the phyla observed to be significantly different between DNA and RNA were comprised of ASVs unique to DNA or RNA or shared between the two methods . ASV shared by DNA and RNA microbial communities showed more pronounced variations in microbial community structures differences. Previously, the response of the soil microbial community at this site to plant stand age, fertilization history, and time since fertilization was studied based on DNA . In thisM. \u00d7 giganteus included were 2-, 3-, and 4-years-old, and the microbial community of each stand age was significantly different based on both DNA and RNA microbial communities (ppairwisePERMANOVA < 0.05). Similar patterns were observed for the response to N fertilization amount in both libraries, and both DNA and RNA microbial communities were found to have different microbial community compositions for three varying N fertilization amount (ppairwisePERMANOVA < 0.05). Pairwise comparison of DNA and RNA based on sampling day or the time since fertilization resulted in no significant differences observed in DNA, but significant differences between pre-fertilization (10\u2009days before fertilization) and 69\u2009days since fertilization in RNA .Next, pairwise comparisons of DNA and RNA microbial communities between stand ages were performed . The stand ages of Kruskal-Wallis < 0.05). The dominant phyla differed between DNA and RNA (Acidobacteria (>17%), Actinobacteria (>24%), and Proteobacteria (>32%) dominant in DNA, and Firmicutes (>12%) and Proteobacteria (>43%) in RNA. We subsequently selected these phyla to evaluate genera level differences between DNA and RNA methods.Stand age was consistently observed to explain the most variation between experimental factors, regardless of DNA or RNA methods . We next and RNA , with AcActinobacteria, Proteobacteria, and Firmicutes, and 308, 316, and 337 genera in 2-, 3-, and 4-year-old M. \u00d7 giganteus, respectively, showed significant differences between the DNA and RNA microbial communities. We selected the genera with greater than 0.1% relative abundance and compared differences between taxonomic profiles in DNA and RNA . Sequences associated with Bacillus, Clostridium, Paenibacillus, Sporosarcina of Firmicutes and Bradyrhizobium, Methyloversatilis, Nitrosomonas, Nitrosospira, and Steroidobacter of Proteobacteria were more enriched in RNA than in DNA. On the other hand, Gaiella and Solirubrobacter of Actinobacteria were more enriched in DNA.A total of 569 genera were detected among Actinobacteria, Firmicutes, Gemmatimonadetes, Hydrogenedentes, Latescibacteria, Nitrospirae, and Proteobacteria) showed significant differences between N fertilization amount differences compared with four phyla in DNA . Actinobacteria (>26%) was more enriched in DNA microbial communities , and Firmicutes (>12%) and Proteobacteria (>47%) were significantly more enriched in RNA microbial communities .Differences in response to fertilization were also observed between DNA and RNA microbial communities. Both DNA- and RNA-based methods identified that soil microbial communities showed different responses to N fertilization amount . Sequences associated with Bacillus, Clostridium, Paenibacillus, Sporosarcina of Firmicutes and Bradyrhizobium, Methyloversatilis, and Nitrosomonas of Proteobacteria more enriched in RNA than DNA. On the other hand, Gaiella from Actinobacteria and Sphingomonas of Proteobacteria were more abundant in DNA.Genus-level analysis was performed on the Acidobacteria, Armatimonadetes, Firmicutes, and Planctomycetes were increased compared with before fertilization, and the relative abundances of Actinobacteria, Bacteroidetes, Chloroflexi, and Latescibacteria decreased.In comparing pre- and postfertilization soil samples, differences in soil microbial communities were observed only in RNA libraries . AdditioFirmicutes, in which relative abundance was increased 10-fold, and Planctomycetes also increased by about 1.7-fold. These phyla are notable because they are known to contain known nitrogen cycling bacteria  in the RNA microbial community.Overall, the total relative abundance of these genera comprised 1.18% and 8.51% in the DNA and RNA microbial communities, respectively . The larM. \u00d7 giganteus from 2-, 3-, and 4-year-old stand ages, where generally RNA showed enrichment of taxa associated nitrogen cycling functions. In all stand ages, DNA was not significantly different pre- and postfertilization within a season. RNA did show seasonal differences, with trends varying depending on stand age .The taxa that showed distinct responses in RNA compared with DNA were classified by their known nitrogen cycling functions . Only taxa associated with denitrification in the RNA microbial communities showed a significant difference . This reFirmicutes showed the highest SSU gene copy number of 6.00, followed by Deinococcus-Thermus, Chlamydiae, and Actinobacteria. Planctomycetes, Proteobacteria, and Verrucomicrobia were enriched both DNA and RNA microbial communities, but the average number of SSU gene copies was higher in the taxa of RNA microbial communities.We further evaluated SSU copy numbers in genes associated with denitrifying phylum that were enriched after fertilization in the RNA microbial communities. Specially, we wanted to understand the potential impact of SSU gene copy numbers on biasing the observed enrichment of these taxa. Overall, we observed differences in the denitrifying phylum enriched in RNA and DNA libraries. In RNA and DNA, the enriched phyla have an average of 2.00 and 1.23 SSU gene copies, respectively . Among the bacterial membership that were enriched only in RNA microbial communities, M. \u00d7 giganteus soil microbiomes from DNA and RNA extractions, we found that the most significant factor in explaining variation between microbiomes was its sequencing library of origin, even more so than experimental factors of stand age, N fertilization amount, or sampling day (Table\u00a0S1). DNA and RNA microbiomes also had significantly different alpha diversity, with increased diversity and less variation observed in DNA relative to RNA. These results are consistent with what is known about DNA and RNA. DNA represents the potential genes or membership that may be active and thus is expected to represent more diverse membership with the potential to become metabolically active. RNA, which is actively transcribed, represents growing members, and its higher variability is consistent with its dynamic responses. Previous studies have shown that the RNA microbial community may also have lower alpha diversity because it does not contain the sequences of dormant or dead cells and also has greater variability in response to the environment , suggesting that both methods identify the similar presence of membership. The abundance of these shared membership, however, could be significantly different between DNA and RNA, and most of the shared membership were more enriched in RNA. Based on the assumption that taxa observed in both methods are the most reliable, it is likely that DNA-based methods are underestimating the relative abundance of membership. Further, these differences between DNA and RNA methods contributed to differences in estimated alpha diversity and varying observations of the microbial community response to plant host stand age and fertilization.M. \u00d7 giganteus soil microbial communities between the two library methods was in response to nitrogen fertilization. Only RNA microbial communities showed differences pre- and postfertilization and only at day 69. RNA is able to show more rapid changes in response to changes in environmental conditions than DNA , and there have been no published studies indicating that these are influenced by nucleic acid storage approaches.Finally, it has previously been observed that RNA preservation methods and chemicals can bias the detection of specific microbial membership. Generally, proaches \u201372. The In summary, we found that DNA and RNA methods for characterizing the general response of microbial communities varied. While our results support that these variations originate from biological differences, we also acknowledge that they may be influenced by some combination of known  and unknown biases that would benefit from further research. With relevance to developing sustainable bioenergy crops and understanding the role of microbes in nutrient cycling, RNA appears to better capture the response of taxa known to be involved in nitrogen cycling and is also more sensitive to seasonal shifts in microbiomes. To better link microbial communities to ecosystem processes, we need to move toward characterizing the functional response of microbial communities. Due to costs, the first step in this characterization is often phylogenetic characterization of SSU genes based on DNA. Our results indicate that this method alone may bias against the composition results of the relevant microbial membership.Notably, the integration of RNA-based methods into an experiment adds significant costs, requiring materials to quickly preserve samples for RNA extraction and typically more time for extraction and library preparation. RNA used for SSU characterization can be a complement to DNA-based studies, as it leverages the advantages and throughput of indicator gene amplification while not being as expensive as metatranscriptomics strategies. Based on our results, we recommend that DNA can be used for the initial and broad characterization of community membership. The use of RNA for SSU characterization could be used to complement DNA characterization when experimental questions have been developed. In the context of our experiment, DNA-based analyses were used to validate that there was a significant response to stand age and fertilization. RNA-based analyses were more helpful in identifying the specific taxa that respond to fertilization. With these specific taxa now identified, future research will be focused on functional characterization, guided by the result of this study . More broadly, in our understanding of microbial ecology, increasing numbers of studies are identifying the environments or gradients for which microbial communities are changing. In future work, it will be necessary to emphasize which taxa or what functions are changing, and our results indicate that RNA-based SSU characterization may be a substantial resource.\u00b0 N, 93.743\u00b0 W). This staggered-start experiment was planted with M. \u00d7 giganteus  at a density of \u223c11 plants m\u22122 in replicated blocks (n\u2009=\u20094) in 2015, 2016, and 2017 as described previously . Soil samples included in this analysis were obtained in triplicate from 60 experimental plots at each time point and analyzed independently. Samples for DNA extraction were stored on dry ice immediately after being taken as described previously  and 806R (5\u2032-GGACTACNVGGGTWTCTAAT-3\u2032)  following the standard protocol in this kit and liquid handling in Eppendorf epMotion 5075 (Eppendorf North America). The extracted RNA was transcribed into cDNA according to a standard protocol using iScript cDNA Synthesis Kit  for sequencing analysis. The resulting DNA and RNA were analyzed for quantity using an Invitrogen Qubit 4 Fluorometer . DNA and RNA sample concentrations above 10\u2009ng \u03bcLTAAT-3\u2032) , 77. BacDADA2 package (version 1.13.1) in R (version 4.1.0) was used to perform quality control of sequencing libraries and to determine the abundance of ASV. The quality filtering parameters for all sequences were the same as previously described for DNA amplicons (vegan package (version 2.5\u20137). Multivariate homogeneity of group dispersions, calculating the average distance of members to the centroid of the group, was used to analyze the dispersion of each sample using betadisper function from the vegan package (version 2.5\u20137). Significant differences in alpha diversity and homogeneity between DNA and RNA microbial communities were evaluated using the Kruskal-Wallis test with Dunn\u2019s post hoc test. Permutational multivariate analysis of variance (PERMANOVA) was performed with the adonis function of the vegan package using the Bray-Curtis dissimilarity matrix (version 2.5\u20137). PERMANOVA was performed to identify significant differences between centroids of each microbial community, and the R2 statistic represents the proportion of the variance for the separation of the microbial community that was explained by experimental and field environmental factors . PERMANOVA was performed using the \u201cstrata\u201d argument for the planted block, which was identified as one of the major factors to structure the microbial composition in the previous study, to better identify the effects of stand age and N fertilization amount, fertilization history, and time since fertilization. This analysis restricted permutations to the data set within each block and was used to quantify variations between and within treatments (adonis function of the vegan package (version 2.5\u20137). The level of significance in the statistical analysis was defined as P\u2009<\u20090.05. The rRNA operon copy number database  was used to evaluate SSU gene copy numbers  Sequence Read Archive"}
+{"text": "EMPFINDLICHER IM DUNKELROTEN LICHT 1  and NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED GENE 2  delayed both the phase and period of its circadian rhythms. The fact that variation in period and phase are separated in tomato provides an optimal tool to study how these factors affect the perception of photoperiod.Many biological processes follow circadian rhythmicity and are controlled by the circadian clock. Predictable environmental changes such as seasonal variation in photoperiod can modulate circadian rhythms, allowing organisms to adjust the timing of their biological processes to the time of the year. In some crops such as rice, barley or soybean, mutations in circadian clock genes have altered photoperiod sensitivity, enhancing their cultivability in specific seasons and latitudes. However, how changes in circadian rhythms interact with the perception of photoperiod in crops remain poorly studied. In tomato, the appearance\u00a0during domestication of mutations in EID1 and LNK2 and show that they recreate the changes in phase and period that occurred during its domestication. We perform transcriptomic profiling of these near isogenic lines under two different photoperiods, and observe that EID1, but not LNK2, has a large effect on how the tomato transcriptome responds to photoperiod. This large effect of EID1 is likely a consequence of the global phase shift elicited by this gene in tomato's circadian rhythms.Here we develop tomato near isogenic lines carrying combinations of wild alleles of Our study shows that changes in phase that occurred during tomato domestication determine photoperiod perception in this species, while changes in period have little effect.The online version contains supplementary material available at 10.1186/s12870-022-03565-1. Synchronization with the environment is crucial for survival. In order to efficiently anticipate predictable environmental changes linked to diurnal oscillations, all living organisms have developed endogenous timekeeping mechanisms named circadian clocks . In planThe circadian clock in plants is best studied in Arabidopsis, where it is organized in interlocked transcriptional feedback loops . In thisBecause circadian rhythms can be modulated by the environment, their variation could be beneficial for adaptation to certain settings, and opens the possibility to selective pressures on the circadian clock. For example, plants that live near the equator where the duration of the day is constant along the year, could evolve different mechanisms to time their biological processes than plants at higher or lower latitudes that experience strong seasonal variation. Indeed, it has been found that plants adapted to higher latitudes naturally present longer free running circadian periods \u201316. MoreEID1 and LNK2 [EID1 appeared early during domestication causing a\u2009~\u20092\u00a0h phase delay of its circadian rhythms [LNK2 presents a large deletion that increases the period of its circadian rhythms [EID1 and LNK2 show signatures of positive selection and their mutations are fixed in cultivated tomatoes but do not exists in its closest wild ancestor S. pimpinellifolium, suggesting that phase and period changes have been beneficial for its domestication [EID1 and LNK2 allowed tomato to expand its cultivation range to higher latitudes [Tomato is an interesting case because domestication significantly decelerated circadian rhythms through selection of knock-out alleles of two genes not identified as a source of variation in any other crop, and LNK2 , 27. EIDand LNK2 , that taand LNK2 . In toma rhythms , 27. LNK rhythms \u201333. In ctication , 27. Becatitudes , 27.EID1 and LNK2. We first confirm the validity of these lines and then study their transcriptional responses to variation in photoperiod and to the allelic configurations of EID1 and LNK2. Our study contributes to understanding how variation in circadian rhythms affects the molecular state of tomato under different photoperiods.Here we study the interaction between photoperiod and the mutations that delayed circadian rhythms in tomato. For this, we generate a set of tomato near isogenic lines that segregate for wild functional alleles of S. pimpinellifolium (accession TO937) in a cultivated S. lycopersicum (accession Moneymaker) background [S. pimpinellifolium introgressions at the positions of EID1 and LNK2. Whole genome short read sequencing of this line revealed the presence of 5 introgressions in chromosomes 1, 4, 5 (\u00d7\u20092) and 9 , the introgression in chromosome 9  or both introgressions . In addition, all lines selected carried an introgression at the bottom chromosome 4 that includes 208 genes and had been previously detected as a segregation distorter during the generation of the near isogenic line population [In order to investigate how mutations in EID1 and LNK2 interact with each other and how they affect the perception of photoperiod in tomato we generated near isogenic lines (NILs) that contain wild alleles of both genes in a cultivated tomato background. For this we performed several rounds of backcrossing in a set of previously developed NILs containing introgressions from the wild tomato ckground . We obtapulation .S. pimpinellifolium alleles of LNK2 and EID1 in these lines by characterizing its circadian rhythms in three independent experiments. As expected, wild LNK2 alleles decreased the long circadian period observed in cultivated tomato but had no significant effect on the phase of leaf movements  of circadian rhythms  and short day (SD) conditions, and we collected leaf samples from three biological replicates two hours after lights on . This time of the day was chosen to coincide with the highest expression of EID1 (ZT0) and LNK2 (ZT4) in a published time course experiment in tomato [We performed RNA-seq on the near isogenic lines carrying wild alleles of n tomato  .We obtained an average of 34.8 million read pairs per sample (minimum of 19.8 million and maximum of 43.8), that were aligned to the tomato reference genome sequence with an average success rate of 92.9%. Principal component analysis  in each photoperiod separately. Figure\u00a0We first investigated if wild alleles of LNK2 affected only 297 transcripts in short days and 254 in long days, suggesting that EID1 has a more important role shaping gene expression than LNK2. GO terms of biological processes associated to the transcripts affected by LNK2 are translation or photosynthesis (Table S2). We were surprised by the mild effect of LNK2\u2009+\u2009on expression, since LNK proteins in Arabidopsis are involved in transcriptional initiation of clock genes in the PSEUDO RESPONSE REGULATOR (PRR) family such as PRR5 and TOC1, and affects the expression of members of the REVEILLE (RVE) family such as CCA1 or LHY [RVE7, PRR5 or PRR7 had clear homologs in tomato, some others had been lost (CCA1 / LHY) or had undergone duplication (RVE6 and TOC1). To increase the likelihood of functional homology we considered as homologs only tomato transcripts whose expression oscillated during the diel cycle in a published RNA-seq time-course experiment [In contrast to the large effect found for EID1, the addition of wild alleles of 1 or LHY \u201333, 35. periment .RVE genes in tomato, wild alleles of LNK2 significantly decreased the expression of the homolog of RVE1 and RVE2 in short days, but had no effect on the homolog of CCA1 and LHY, as it does in Arabidopsis . Finally, wild alleles of EID1 increased the expression of PRR3 and PRR7 in short days. Interestingly, most of the significant differences of expression observed were photoperiod-specific, suggesting a complex seasonal effect in the regulatory function of LNK2 and EID1. In summary, although some transcripts in the PRR and RVE families in tomato are affected by allelic variation in LNK2 and EID1, we cannot conclude that the mechanism of action of their proteins in tomato are similar to those previously defined in Arabidopsis.Among the LNK2 and EID1 alleles with photoperiod in tomato we analyzed photoperiod sensitivity in each NIL. Fifty percent of the characterized transcriptome  was significantly affected by photoperiod in at least one genotype . The largest set is formed by 3671 transcripts responding to photoperiod in all genotypes, followed by 1866 transcripts affected exclusively in the EID1\u2009+\u2009genotype, 978 transcripts deregulated simultaneously in LE\u2009+\u2009and EID1\u2009+\u2009, and 855 differentially expressed in LE\u2009+\u2009, 4, 5 (two introgressions) and 9 (the region of EID1) , only the introgression in chromosome 9 containing EID1 (EID1\u2009+) or introgressions only in chromosomes 1 and 9 . In addition, all plants screened contained S. pimpinellifolium alleles on the distal region of chromosome 4 due to the presence of a segregation distortion locus identified during the generation of the population [Tomato near isogenic lines LNK2\u2009+\u2009, EID1\u2009+\u2009and LNK\u2009+\u2009/EID1\u2009+\u2009 were generated from a set of introgression lines derived from a cross between Monforte , 42. Intpulation , 42.S. pimpinellifolium introgressions in the region of LNK2 and EID1 (see above) was sequenced for whole genome genotyping. To do this, DNA from leaf tissue was extracted using the DNeasy Plant extraction kit from Qiagen following manufacturer's instructions. A single sequencing library was constructed using the standard Illumina method, and sequenced in an Illumina NovaSeq 6000 system, yielding 361,774,304 pairs of 150\u00a0bp reads. In order to assign alleles in the F1 hybrid to cultivated and wild tomato we obtained publicly available short reads from S. pimpinellifolium accession LA1589 and S. lycopersicum cv. Moneymaker [http://broadinstitute.github.io/picard) and indels were realigned using GATK IndelRealigner [S. pimpinellifolium alleles in the F1 hybrid was scored at 3,396,011 biallelic positions where Moneymaker and LA1589 were homozygous and different from each other, and the hybrid was heterozygous.A single F1 individual resulting from the cross of two lines carrying reduced neymaker , 44. Reaneymaker . Previouneymaker , duplicaealigner . We callealigner . PresencThree different experiments to measure leaf movements were conducted following a protocol that has been described in detail elsewhere . BrieflyEID1 and LNK2. Three biological replicates were collected from each genotype and condition, and total RNA was extracted with the RNeasy Plant Mini Kit from Qiagen. Libraries were prepared according to the Illumina TruSeq RNA protocol and sequenced in two lanes of a Novaseq 6000 system, yielding 834,954,102 150\u00a0bp read pairs .RNA-seq was conducted in two consecutive experiments in the same controlled environment chamber set first to LD  and then to SD . We collected leaf samples from 14-day old plants 2\u00a0h after dawn (ZT2), coinciding with the maximum peak of expression of S. lycopersicum cv Moneymaker in each photoperiod separately, and contained a unique variable with 8 factors grouping the genotype and condition for each sample . The second model was conceived to test the effect of photoperiod in each genotype and included the three variables \"photoperiod\", \"genotype at EID1\" and \"genotype at LNK2\", as well as all its possible interactions (photoperiod\u2009+\u2009EID1\u2009+\u2009LNK2\u2009+\u2009photoperiod:EID1\u2009+\u2009photoperiod:LNK2\u2009+\u2009EID1:LNK2\u2009+\u2009photoperiod:EID1:LNK2). From this model we extracted the effect of photoperiod in each genotype. In both models we considered as differentially expressed those transcripts with a q-value lower than 0.01. A dataset with normalized values for each sample used in all downstream analyses and graphical representations was generated using the vst function in the DEseq2 R package [Reads for each biological replicate were aligned independently to the tomato genome reference sequence v4.0 using hisat2 v2.1.0 , allowin package .For functional analysis of differentially expressed genes, GO terms in the ITAG4.1 genome annotation were assigned to the 23,226 transcripts that presented more than 10 reads in all samples. Overrepresentation analysis was performed with the GOseq R package based on the Wallenius non-central hyper-geometric distribution . GO cateTo obtain the homologs of PRRs and RVE genes in tomato, we compared the protein sequences from the following Arabidopsis TAIR10 ids: AT1G01060.1 (LHY), AT1G18330.2 (RVE7), AT2G46830.1 (CCA1), AT3G09600.1, (RVE8), AT5G02840.1 (RVE4), AT5G17300.1 (RVE1), AT5G37260.1 (RVE2) AT5G52660.2 (RVE6), AT2G46790.1 (PRR9), AT4G18020.1 (APRR2), AT5G02810.1 (PRR7), AT5G24470.1 (PRR5), AT5G60100.2 (PRR3) and AT5G61380.1 (TOC1) onto the tomato protein sequences from annotation ITAG4.1 using standalone BLAST\u2009+\u2009. For theS. lycopersicum var. M82 and S. pennellii seedlings were obtained from the SRA database . Although the original experiment consisted in duplicate samples every 4\u00a0h during one diel cycle and two circadian cycles, only reads corresponding to the diel cycle were used, corresponding to samples from time-points 12, 16, 20, 24, 28 and 32 . Short reads from these 24 libraries were aligned to the tomato genome reference sequence v4.0 using hisat2 v2.1.0 with default parameters [S. lycopersicum and S. pennellii using the meta2d function in the R package MetaCycle with parameters minper\u2009=\u200920, maxper\u2009=\u200928, cycMethod\u2009=\u2009\"ARS\", adjustPhase\u2009=\u2009\"predictedPer\", combinePvalue\u2009=\u2009\"fisher\", ARSmle\u2009=\u2009\"auto\" and ARSdefaultPer\u2009=\u200924 [p value\u2009<\u20090.05 for the cycling test, an estimated period between 22 and 26\u00a0h both in S. lycopersicum and S. pennellii, and did not present amplitudes or mean expression values more than 2.5 standard deviations away from the mean  The red line indicates the frequency of heterozygous SNPs in 1000-SNP windows along the 12 tomato chromosomes. (B) Zoom-in view in the chromosomal regions where the heterozygous line presented introgressions from S. pimpinellifolium. Red lines indicate the frequency of heterozygous SNPs in 100-SNP windows. The location of LNK2 and EID1 is indicated with a vertical line. The location of genes in each region is indicated with black dots along the x axis. Figure S2. Circadian parameters in the near isogenic lines generated. Three independent experiments are shown in columns and Period, Phase, Amplitude and Relative Amplitude Error in rows. The wild species S. pimpinellifolium was not included in the first experiment. Different letters in each boxplot indicate significant differences.\u00a0Figure S3. Expression oscillation of LNK2 and EID1 in tomato. Data was obtained from RNA-seq data published in M\u00fcller et al 2016. Plants were grown in 12:12 light/dark and 20:18 \u00b0C temperature cycles and leaf samples collected from 7-day old seedlings every 4 hours. Read counts on each gene are normalized by gene length and sample size.\u00a0Figure S4. Principal component analysis of expression values from the RNA-seq experiment in the near isogenic lines segregating for wild alleles of EID1 and LNK2. Only transcripts with more than 10 reads across all samples were included in the analysis.\u00a0Figure S5. Phase and percent of differentially expressed genes among cycling genes in tomato. (a) Phase distribution of the 6017 transcripts whose expression oscillates during the diel cycle in S. lycopersicum and S. pennellii. (b) Percentage of transcripts in (a) whose expression was significantly altered by photoperiod in our experiment.\u00a0Figure S6. Phylogenetic tree from protein sequence alignments for genes belonging to the LNK family in tomato and Arabidopsis. For LNK2, the sequence from the wild tomato species S. pennellii is used because of the large deletion present in this gene in cultivated tomato. Arabidopsis, cultivated tomato and S. pennellii protein names are highlighted in gray, red and green respectively. Tomato proteins whose transcript oscillates during the diel cycle are marked with an orange dot.\u00a0Additional file 2:Table S1. primers used to generate near isogenic lines with wild alleles of EID1 and LNK2. Table S2. GO enrichment analysis. Table S3. List of transcripts whose expression oscillate with ~24h cycles in tomato and their phase estimates."}
+{"text": "This study presents a new storm surge hazard potential index (SSHPI) for estimating tropical cyclone (TC) induced peak surge levels at a coast. The SSHPI incorporates parameters that are often readily available at real-time: intensity in 10-min maximum wind speed, radius of 50-kt wind, translation speed, coastal geometry, and bathymetry information. The inclusion of translation speed and coastal geometry information lead to improvements of the SSHPI to other existing surge indices. A retrospective analysis of SSHPI using data from 1978\u20132019 in Japan suggests that this index captures historical events reasonably well. In particular, it explains\u2009~\u200966% of the observed variance and\u2009~\u200974% for those induced by TCs whose landfall intensity was larger than 79-kt. The performance of SSHPI is not sensitive to the type of coastal geometry (open coasts or semi-enclosed bays). Such a prediction methodology can decrease numerical computation requirements, improve public awareness of surge hazards, and may also be useful for communicating surge risk. As the Earth\u2019s climate warms because of human activities, a more severe, widespread storm surge hazard is projected with high confidence due to both the rising sea-level and the possible increase in TC intensity2. Furthermore, coastal development results in high population density in low-lying cities roughly five times (241 people/km2) than the global mean (47 people/km2)3. The storm surge threat has never been greater and such concern is exemplified by several recent extreme surge events, such as Hurricane Katrina (2005) that generated a peak surge of 8 m and made it one of the costliest ($149 billion) natural disasters in the United States (US) history4. More recently, an 8 m storm surge due to Typhoon Haiyan (2013) killed 6300 people and left 1061 missing in the Philippines5. Precise and timely forecasts informing effective warnings are imperative to mitigate the risks to life and property posed by TCs and its associated storm surges8.Storm surge associated with tropical cyclones has a long history of causing catastrophic damage and many deaths along low-elevation (< 10 m) coastal zones. Based on a 2003 study, storm surge may be responsible for as many as 2.6 million deaths worldwide during the past 200 years9. Other TC prone countries such as Japan10, Bangladesh11, Australia12 also warn their coastal inhabitants employing a wind intensity-based scale, similar to SSHWS. However, these scales are defined based on wind-induced structural damage and do not account for other crucial factors that influence surge generation13. Thus, SSHWS has been widely criticized as an inappropriate estimate of storm surge potential16.One argument is that although TCs are often weakening during the landfall time frame, the storm surge potential may still be increasing at the same time. Thus, a lower category TC  can sometimes inflict a significant storm surge. In the 2003\u20132008 period, hurricanes have generated three of the largest five surges occurring in the US within the past 65\u00a0years. Yet none of these hurricanes registered higher than a category 3 hurricane at landfall15. Hurricane Sandy (2012) highlighted the hazard posed by a weakening TC. Sandy approached the United States coast as a category 3 hurricane, before weakening and making landfall as a post-tropical storm. It generated\u2009~\u20092.7\u00a0m storm surge and resulted in more than 60 direct deaths from drowning17. Another recent example is TC Amphan (2020) which approached India\u2013Bangladesh coast as a category 5 hurricane. Although, Amphan made landfall as a category 1 hurricane, it resulted in\u2009~\u20092.75\u00a0m storm surge and claimed hundreds of lives18. Considering storm surge is an extremely life-threatening hazard, there is an obvious need for an alternative means of more effectively characterizing TC surge potential.When quantifying and communicating natural disasters such as TC, practitioners and scientists have often employed categorization-based statistical approaches for ease of public understanding and usefulness. For instance, the Saffir\u2013Simpson hurricane wind scale (SSHWS) has been using for nearly five decades by the National Hurricane Center (NHC) in the US to categorize TC strength. Coastal inhabitants in the US thus have learned to assess the danger of TCs using SSHWS, e.g., evacuation intent increases linearly with SSHWS category19 found that the greatest storm surge events would occur when a hurricane makes landfall to the north of Tampa Bay (US), resulting in maximum winds at the mouth  of the bay. Irish, Resio, and Ratcliff15 evaluated the relationship between hurricane size (radius to maximum wind speed: Rmax) and maximum storm surge over idealized continental shelf slopes. Their results demonstrated that storm surges tend to increase with hurricane size and that this relationship becomes increasingly pronounced for shallow coastal waters. Sebastian et al.20 found that storm surge behavior is highly sensitive to the local wind direction and landfall location. These findings support the conclusion of our recent work21, in which we showed that storm surge characteristics in a semi-enclosed bay, such as Tokyo bay in Japan, is largely sensitive to the landfall location, local wind direction, and storm size.Numerical simulation-based surge modeling also shows that wind speed is not the only storm parameter that markedly influences surge extent. Weisberg and Zheng22 performed numerical experiments and found that a fast-moving TC (>\u200948.2\u00a0km/h) tended to intensify the storm surge. This tendency was also reported by Rego and Li23, who used Hurricane Rita (2005) as a reference storm; they demonstrated that faster propagation speed resulted in a greater surge but decreased the potential of the largest flood area along the open coasts of the Louisiana-Texas shelf. However, Peng, Xie, and Pietrafesa24 found that both the surge height and inundation areas over the Croatan-Albemarle-Pamlico Estuary decreased as hurricane forward speed increased. Using 42\u00a0years of tidal records and landfall TC best tracks in Japan, Islam and Takagi25, showed that fast-moving TCs tended to amplify the storm surge along open coastlines but reduced the surge at semi-enclosed bays (vice-versa). Several recent studies27 suggested that the forward speed of TCs has decreased significantly both at the global and regional scales. It indicates that coastal areas have experienced a longer influence of time by TCs. Therefore, it is of great interest to incorporate TC forward speed and coastal geometry information in the surge index.Other factors, including TC forward speed and coastal geometry are also found to be influential to surge generation in many previous studies. For example, Jelesnianski28 and coastal geometry (surge estimates for bays and open coasts separately). Another common limitation is that some of them can only be applied to NHC\u2019s responsible area because they use TC structure variables , Rmax) that are only available in the Atlantic hurricane forecasting database29 . Our simplified approach provides an instantaneous overall estimate of a TC\u2019s peak surge potential, which can supplement computationally expensive TC surge model forecasts. By providing a potential peak surge, the proposed index can be used to inform the public and emergency responders as a means of quantifying surge hazard effectively, similar to the role the SSHWS plays for wind hazard during a TC event.Table 33 was one of the first who criticized the SSHWS and suggested a non-dimensional relationship for estimating surge damage potential ) based on TC intensity (Vmax) and radius of hurricane force wind (R33):After the devastating damages incurred by Hurricane Katrina in 2005, KanthaVref and Rref are climatological reference constants: 33 m/s and 96.6 km. This scale has the advantage of yielding a continuous scale and does not saturate at the higher end as SSHWS does at category 5. Larger values of HSI indicate more severe surge damage potential. The HSI has quadratic dependence to Vmax because wind momentum input at the water surface is proportional to V2max. The reason for the linear dependence of HSI on the storm radius is because the storm surge impact is most often confined to a broad but roughly linear strip along the coastline33. Another underlying assumption that is not directly discussed in Kantha33 but may fit with Eq.  for the US coasts considering the integrated kinetic energy of the hurricane wind field:IKETS is the integrated kinetic energy for marine winds greater than tropical storm force (18 m/s). The larger value of SDP indicates more severe surge damage potential but with an upper bound limit at 614.Following Kantha\u2019s34 addressed the relative importance of water depth variation across the continental shelves adjacent to the US and proposed an improved dimensionless and continuous surge scale (SS) is of the formLater, Irish and Resio\u2206p is the hurricane central pressure difference, defined as the nominal atmospheric pressure around a hurricane minus the central pressure of that hurricane and directly proportional to the V2max. L30m is the horizontal distance (km) between the shoreline and the 30\u00a0m depth contour and L30m. \u03a8x is the dimensionless storm size function to adjust that ratio. L30m used in the scale is because Irish and Resio34 found that surge generation tended to be confined between the shore and the 30-m depth contour for the five representative shelf profiles in the US. Chavas et al.37 has also reported similar results where they suggested L30m is an optimal characteristic length scale for storm surge generation in the US coasts. Irish and Resio34 argued that Eq.\u00a0 for estimating surge potential, it was only validated with limited observed peak open coast surge values with sample size (n)\u2009=\u200929, leaving no index-based method for bays or estuaries. Kantha38 argued that it is better to consider TC forward speed in the Eq.  and TC forward speed information (S) introduced in Islam and Takagi25:Here we propose a dimensionless and continuous storm surge hazard potential index (SSHPI) Eq.\u00a0. The matR50 is a measure of radius to 50-kt (26 m/s) winds ), S is the forward speed (km/h), a is the characteristic coastal geometry25: for open coast, a = 1 and for semi-enclosed bay, a = \u2212 1. As TC\u2019s wind field can be highly asymmetrical, making it difficult to determine the actual aerial coverage of the wind field with a specific speed39, we use the arithmetic average of the longest and shortest R50. The reason of using R50 instead of R33 is because the former one is recorded consistently in HURDAT2 (Atlantic Hurricane Database), JMA, and JTWC (Joint Typhoon Warning Center) forecast/best track data sets for all named storms and is available for all landfalling TCs since 2004, which would reduce challenges that arise when applying an index on a global or a regional scale. Furthermore, the relationship between R50 and storm surge forecasting has been discussed in many prior studies41.Specifically, Vref, Rref, and Sref are reference constants and are defined as 50-kt, 95\u00a0nm , and 35\u00a0km/h , respectively. L* is chosen to be 40\u00a0km to make SSHPI roughly equal in magnitude to the peak storm surge height. These reference values are used for normalization that prevents the index values from being biased toward extreme values. The structure of Eq. (S\u2009=\u20095\u00a0km/h) would result in very low SSHPI numbers (using Eq.\u00a0(R50\u2009=\u2009170\u00a0nm) would also result in very high SSHPI numbers (vice-versa). Although such TCs are infrequent in Japan but can sometimes occur elsewhere. The form of Eq.\u00a04 without upper and lower bound of TC size and forward speed would probably not be a good representative for the surge hazard poses by such unusual TCs. Therefore, we limit 0.5 \u2264 S and R50 are exceptionally large or small. This upper and lower bound of TC size and forward speed will prevent discrete jumps in SSHPI numbers.It is noted that a stationary or very slow-moving TC  on the 43. Also, the time scale for mass redistribution (to generate a sea surface slope) within the shallow and geometrically complex estuaries is on the order of hours and longer than along the open coasts19. Thereby, with cross-shore wind stress components, a slower TC has more time to interact with the seawater and pushes more into shallow areas of a bay. Consequently, surge height begins to fully develop (or mature), causing a large sea-level gradient between the upper and lower bay. Thus, we set a\u2009=\u2009\u2212 1 for semi-enclosed bay and by construction, large , but slow-moving  and intense  TCs will generate greater storm surge potential.The linear dependence of SSHPI on TC forward speed is twofold. First, in semi-enclosed bays, the effective cross-shore shallow area over which TC winds act is larger; the contribution of wind stress tends to be more pronounced in the bay than the open coastlines due to a shallower depth44. This mechanism could be partially explained by Proudman\u2019s linear theory45, which showed that storm surges could be amplified when the TC translation speed was similar to the propagation speed of the long wave  storms will generate larger SSHPI. It should be noted that we did not directly consider inverse barometer effect (IBE) and the influence of TC approach angle, waves, and astronomical tide to keep SSHPI simple. Thus, SSHPI will tend to underestimate/overestimate total surge height somewhat for some TC events. The limitation is particularly relevant in open coasts, where wave set-up and IBE are often the dominant drivers behind storm surge and coastal flooding48. Furthermore, a very steep coast  would result in a low SSHPI number  TCs that made landfall in Japan; (b) TCs that incurred a minimum of 40 cm of storm surge, and (c) TCs that had intensity, size (R50), and forward speed information  were available. The choice of 40 cm is to evaluate storm surge index accuracy for more severe storm surge events.We use JMA best track data archives from 1978 to 2019. The best track data acquired during the pre-satellite era  contain inhomogeneities and large uncertainties in the data qualityBased on the above three criteria, 51 TCs were selected for analysis  instead of R50. Note that JMA provides two types of wind radius information: the longest and shortest radius42. The translation speed at time T is calculated with the TC central positions at T and T \u2013 6\u00a0h. For cases in which translation speed, intensity, and R50 data was unavailable immediately before TC landfall time, those data were obtained via linear interpolation of the available data at two neighboring positions . The bathymetry data over the target area was obtained from the Japan Coast Guard51. GIS environment was used to measure the closest horizontal distance between each selected tide station and 30\u00a0m depth contour (L30m)  located on open coastline or in a bay; (b) JMA predicted astronomical tide data53 were available; (c) elevation of the observation reference plane and the astronomical tide table reference plane were available; (d) fell right side of a selected TC track and located within the range of R50 ; and (e) no data were missing when a TC traversed the station. Eleven stations  were selected as representative observatories for storm surges on open coastlines and seven  were selected as representative observatories for storm surges in semi-enclosed bays. Figure\u00a0The storm surge heights recorded at 18 JMA-operated tidal stations55 which comprehensively account for coastal surge dynamics.The SSHPI is positive proportional to the peak surge height. Fig. Vmax with \u2206p as an intensity metric in Eq.\u00a0 as discussed in previous studies58. Correlation corresponding to the R30, was also calculated to explore whether any other definition of size metric would provide a better correlation than R50. The correlation statistic did not vary markedly  from the statistic presented in Fig.\u00a0Vmax vs. MSLP and R50 vs. R30 and thus, the alternative parameters (MSLP and R30) could potentially be used at a basin for a period where Vmax and R50 are unavailable.In the sensitivity test where we replaced c in Eq.\u00a0. The resNext, in an attempt to make sure that the significant storm surge events did not skew the Pearson correlation analysis, we performed additional correlation analysis for SSHPI but excluding the three highest storm surge events caused by Typhoon Mireille (1991), Typhoon Flo (1990), and Typhoon Jebi (2018). While the correlation coefficient decreases slightly, from 0.81 to 0.75 (p\u2009<\u20090.01), it remains significant and strong.A least squares-fit between surge height and SSHPI Fig.\u00a0a gives a\u03beest). The scatter plot . Fig. \u03beest are close to the observations. Root-mean square error (RMSE) in the estimated surge is \u00b118.09 cm, smaller than JMA\u2019s numerical storm surge prediction model errors47 (\u00b150 cm). It needs to be noted that JMA applies TC wind and pressure field to the numerical storm surge model as external forcing. Their TC model diagnoses wind and pressure fields using the necessary input of forecast values, including the TC center location, minimum pressure at the center, Vmax, R50 (if present), and the radius of 1000 hPa46. The difference in RMSE between SSHPI and JMA numerical model predicted surges is primarily because meteorological inputs for current SSHPI analysis are from best track data (post-processed), while JMA evaluated their surge model applying forecasted products during the period of 2015-201747. Nonetheless, operational forecasts of TC have been improving gradually, the performance of SSHPI showed in this study may not be affected significantly in forecast settings (considering SSHPI inputs are from TC forecasted advisories). The performance of the Eq.  for TCs in Japan. However, we attempted to apply SSHPI for the US significant surge cases 59, and the bias score (BS)59 to assess the accuracy of the SSHPI quantitatively. Their mathematical forms are:Next, we use the probability of detection (POD)All three scores Fig.  suggests\u03c3), RMSE) and correlation analyses were conducted using the versions of SSHPI that do not include all of the predictors, i.e., reduced versions of SSHPI (SSHPI (intensity); SSHPI ; SSHPI ). Figure \u03c3 = 12.7 cm) than the observations (\u03c3 = 31.06 cm). Gradual improvements are apparent as the TC parameters (size and forward speed information) are added to the surge indices and they have R = 0.50 (p < .01) and R = 0.55 (p < .01), respectively. It is worth noting that the Pearson correlation statistic for SSHPI  shows significant improvement over all three of the surge indices and the differences are significant at the 5% level. Comparing to other indices, SSHPI has similar variability (\u03c3 = 25.19 cm) as the observations has, the highest correlation, and the least RMSE (\u00b118.09 cm). A similar result can also be confirmed utilizing principal component analysis.To better understand the dependency of the SSHPI on its predictors, statistical measures  is located on a steep-slope coast facing the open sea, where the combined influence of wave-set up and IBE is large. Overall, the estimated major storm surge events agree well with the recorded surge data, although RMSE increases to \u00b138.7 cm for this set of surge events than the RMSE (\u00b118.09 cm) for all storm surge cases.We next examine the accuracy of SSHPI in predicting major storm surge events using two criteria: (a) estimating the largest ten storm surge events (using Eqs. Vmax) definition of major storm surge events, Fig.\u00a0Vmax\u2009=\u200995-kt), the rest were simply categorized as category 1 hurricanes (64-kt\u2009\u2264\u2009Vmax\u2009\u2264\u200982-kt) during the landfall time frame in SSHWS scale.If we use TC intensity-based  for quantifying/categorizing TC induced surge events in Japan. When applied retrospectively, it explains ~66% of the observed variance. A fundamental difference between the SSHPI and existing indices is that it considers coastal geometry and storm forward motion speed in surge estimates. Using reduced-versions of SSHPI, we found that while surge estimation derived from using only intensity and storm size information provides less information on the overall surge hazard than does full form of SSHPI.SSHPI utilizes the most common and readily available TC meteorological parameters, coastal geometry, and bathymetry information and thus can, hopefully, be applied to surge extremes produced by TCs in other basins . However, in that case, the index values and its associated hazard potential showed in this study likely have to be revised, as the surge hazard potential may be different in other basins. For instance, SSHPI values larger than 4.0 are representative of extremely dangerous for Japan, however, it may not necessarily constitute similar surge hazard potential in areas characterized by a large continental shelf or shallow ocean waters.SSHPI can predict hazardous surge events for Japan well and perform better for surge events caused by TCs with landfall intensity larger than 79-kt. It also has the advantage of providing an instantaneous measure of a TC\u2019s surge potential without heavy computational effort. Thus, it could be of utility to the general public for pre-TC measures and post-TC analysis. SSHPI values could be utilized in countries at risk of storm surge but have no access to advanced surge models. Furthermore, it can explain temporal variations in surge events on global, regional, and local scales as it considers climatological variables of a TC.67 associated with TC forecasted track information get smaller. The empirical equation (Eq.\u00a017. Ultimately, we would like to use SSHPI as SSHWS which provides public an intuitive understanding of the surge damage. Thus, the next step of this work includes connecting SSHPI to the losses, i.e., the actual risk, exploring effective communication methods through categorization, graphical and verbal techniques, which requires interdisciplinary efforts and collaboration between operational agencies and academics. Lastly, a global database of SSHPI and surge records would largely increase the usage of the SSHPI.It should be noted that SSHPI proposed in this study is largely dependent on the quality of the forecasted track information. The correlation statistics for SSHPI shown in this study will improve as the uncertaintiestion Eq.\u00a0 used in Supplementary Information."}
+{"text": "Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to joint destruction and bone erosion. Even if many treatments were developed with success in the last decades, some patients fail to respond, and disease chronicity is still a burden. Mechanisms involved in such resistance may include molecular changes in stromal cells. Other explanations can come from observations of tenosynovial giant cell tumor (TGCT), first considered as an inflammatory arthritis, but with unusual neoplastic features. TGCT leads to synovium hypertrophy and hyperplasia with hemosiderin deposition. It affects young adults, resulting in secondary osteoarthritis and increased morbidity. TGCT shows clinical, histological and genetic similarities with RA but affecting a single joint. However, the monoclonality of some synoviocytes, the presence of translocations and rare metastases also suggest a neoplastic disease, with some features common with sarcoma. TGCT is more probably in an intermediate situation between an inflammatory and a neoplastic process, with a main involvement of the proinflammatory cytokine CSF-1/CSF1R signaling axis. The key treatment option is surgery. New treatments, derived from the RA and sarcoma fields, are emerging. The tyrosine kinase inhibitor pexidartinib was recently FDA-approved as the first drug for severe TGCT where surgery is not an option. Options directly targeting the excessive proliferation of synoviocytes are at a preclinical stage. Rheumatoid arthritis (RA) is an inflammatory disease that primarily affects the joints. The aggressive synovitis observed is the consequence of synoviocyte hyperplasia, neoangiogenesis, and local infiltration by immune cells that produce pro-inflammatory cytokines , 2. DespTGCT is a rare disease characterized by massive proliferation of the synovium of joints, tendon sheaths, and bursa and affects mainly young adults. TGCT can be localized or diffuse, intra or extra-articular and leads to joint pain and swelling with bloody effusion or to a painful soft-tissue mass , 5. Key The purpose of this review is to discuss the position of TGCT between RA and sarcoma on different aspects. The first part will describe the clinical aspects. Then, histopathological lesions and genetic alterations will be analyzed. The final part will outline current treatments of TGCT and the new promising strategies arising from both RA and sarcoma management, with the importance of a multidisciplinary approach.TGCT is a locally aggressive synovial disease with inflammatory pattern sharing common aspects with RA. However, TGCT appears also like a neoplastic process because of some similarities with sarcoma. In this part, similarities and differences of the clinical manifestations of these three conditions will be developed Figure\u00a01TGCT is characterized by an overgrowth of the synovium of joints, less frequently of tendon sheaths, or bursa. It affects mostly young adults with a modest female predominance . Two anaAs these symptoms are rather unspecific, there is often a delay in diagnosis. The key exam is MRI that defines lesion extension and bone involvement. MRI signal intensity depends on lesion characteristics . Conventional radiographs are less contributive and show rather late joint damage. Although not systematic, pre-operative biopsy can help to confirm diagnosis showing synovial hypertrophy and hyperplasia with hemosiderin deposition .Diffuse TGCT has a higher recurrence rate than localized TGCT, with a more rapid joint destruction, number of surgical events, and secondary osteoarthritis (OA). Therefore, the resulting quality of life is decreased in TGCT patients compared with the general population .RA, one of the most prevalent joint diseases, can rarely start as a mono-articular arthritis. It can constitute, to some extent, a differential diagnosis for TGCT. This atypical presentation can delay RA diagnosis. In terms of epidemiology, RA affects much more women than TGCT, and TGCT begins earlier than RA. The role of estrogen on the immune system is systemic and may account, partially, to the different sex ratio observed between RA and TGCT. Indeed, RA is associated with systemic inflammation contrary to TGCT which is restricted to a single-joint. TGCT can follow traumatisms and occurs earlier , 17, 18.Another differential diagnosis of TGCT is sarcoma. Sarcomas are a heterogeneous group of rare mesenchymal malignancies with over 150 subtypes , 21. Theper se does not origin from synovium. Its occurrence within a joint is very rare and rather affects soft tissues around large joints, especially the knee. Compared with TGCT, patients with sarcoma tend to be younger and of male gender, lesions are usually large, with a small effusion, and calcifications  is the most frequent intra-articular sarcoma , 24. Theications .To conclude, TGCT shares common features with both RA and sarcoma. Clinical manifestations, laboratory tests and MRI help to differentiate these diseases, but biopsy remains the most powerful tool.Considering that TGCT is at the interface of RA and sarcoma, its pathophysiology is first described, and then compared with each other disease Figure\u00a01+ monocytes and are capable of lacunar resorption, while CD14- monocytes support osteoclast formation and express osteoclastogenic factors  ] . CSF1 re(CSF-1)] . Cytokin(CSF-1)] , 28. In (CSF-1)] , 29, 30.(CSF-1)] .RA synovitis is associated with hyperplasia of synoviocytes, neoangiogenesis, and local infiltration by immune cells . Macroph+  that involves CSF1/M-CSF1 gene, on 1p13, and collagen 6A3 gene, on 2q35. This translocation is present in the majority of TGCT. The resulting fusion protein leads to CSF1 (M-CSF) overexpression that increases and attracts non-neoplastic monocyte-like inflammatory cells that express CSFR1/M-CSFR. However, the translocation is present only in two to 16% of intralesional cells meaning that most cells are non-neoplastic ones and recruited solely through the local overexpression, and production of CSF1. Recently, the loss or the replacement of the negative regulatory elements in the 3\u2019end of CSF1 was described with FISH and RNAseq methods as even more frequent than COL6A3-CSF1 fusion. Overall, these alterations lead to the formation of a tumor mass with CSF-1/CSF-1R as a central mechanism of tumorigenesis , 41, 42.In vitro studies in RA synovial cells showed that trisomy 7 leads to synovial hyperproliferation , especially HLA-DRB1*01 and HLA-DRB1*04. These variants are associated with an increased risk of developing RA. Some of RA susceptibility genes are associated with increased severity , and 2% of myxoid liposarcomas . Even ifSarcomas can be classified according to cytogenetic changes and the complexity of their karyotypes. Approximately half of sarcomas are regarded as \u201csarcomas with complex genomics\u201d. Whereas the other half may be referred to as \u201csarcomas with simple genomics\u201d, characterized by recurrent genetic alterations . Other molecular alterations include mutation of KIT proto-oncogene receptor tyrosine kinase. This mutation occurs in some sarcomas and this has been the basis for the use of tyrosine kinase inhibitors (TKI) .To conclude this part, similarities and differences in genetic alterations that have been described between TGCT, RA and sarcoma are summarized in For many years, the key and only treatment was surgical excision, but without any consensus on the most appropriate type of surgery. These methods were also used in RA . The higSurgical procedures include different types of techniques: arthroscopic or open surgical excision with partial, or complete synovectomy. Usually, arthroscopic synovectomy is used for localized forms while open total synovectomy is common for dTGCT, but there is no clear consensus . LocalizArthroscopy is a minimally invasive procedure with a particularly low risk of complications. Compared with open surgery, arthroscopy allows faster rehabilitation, less postoperative pain, can be performed as a day care but can be technically demanding .Arthroscopic synovectomy appears as the best method to treat localized TGCT with low rate of recurrences (0-6%) , 55. TheOpen synovectomy is an open surgery that can be performed in all joints. This technique shows advantages as the easiest to perform but also drawbacks with an increased patient morbidity, a longer rehabilitation, and an overall risk of post-operative complications higher than arthroscopy .This method is generally chosen for dTGCT where recurrence rate is more important. Such open procedure is supposed to allow a more complete synovectomy. For the knee, a two-stage approach (anterior and posterior) appears as a safe treatment option with an average recurrence rate lower than for arthroscopy . There aOpen synovectomy and arthroscopy are often associated to control dTGCT. This combined synovectomy results in a better outcome for diffuse knee TGCT.Arthroplasty is not a first-line treatment of TGCT but remains necessary when patients have many recurrences or refractory disease leading to OA. Indeed, chronic joint inflammation and bleeding combined with the multiple surgeries lead to secondary OA. This requires joint replacement at a relatively young age . Total aTwo forms of radiation therapy have been used for TGCT: external beam or intra-articular radiotherapies. These two approaches improved local disease control and were mainly considered as adjuvant therapies but are rarely used today .External beam radiation (EBR) has two main indications in TGCT with inoperable disease or as an adjuvant treatment when recurrence occurred, or if surgery was incomplete. Better results are described when EBR is used just after synovectomy with a low recurrence rate. The benefit of EBR is more important when EBR is performed within three months after surgical knee synovectomy and not used to treat a recurrence that has already developed. No major adverse effects have been reported. This radiation therapy is not recommended for hand and foot lesions .Radiosynovectomy, also called isotopic synoviorthesis, consists in the intra-articular injection of radioactive products. The anti-inflammatory action consists of synoviocyte radio-lesion and necrosis with no risk of extra-articular migration and degradation of the surrounding tissues. This technique has been used a lot in RA before the use of biotherapies . As for These local treatments are often far from always active to control diffuse and recurrent TGCT. However, they are suited to local forms of the disease.As for the clinical presentation and pathogenesis, new treatments are emerging, often from the cancer and RA fields. As discussed above, surgical procedures do not prevent the risk of recurrence in diffuse TGCT, where systemic therapies have a growing interest. Other local therapies are discussed as perspectives.CSF-1 overexpression by TGCT clones has led to the option to target the CSF1/CSF1R-signaling axis . Two strTKI are widely used in oncology, notably in sarcomas. Imatinib that blocks CSF1R activation was the first to be used in TGCT, with induction of a complete response in a case of recurrent elbow TGCT . A relapvs. 0% (0/59) in the placebo group. Results were then confirmed with an overall response increased to 53% at 22 month-median follow-up. However, 44% (27/61) of patients treated with pexidartinib presented grade 3 or 4 adverse events vs. 12% (7/59) in the placebo group with a higher incidence of hepatic adverse events  and cadmium (Cd) share similar characteristics and transporters but their actions on the immune system is often opposite. Zn is necessary for a normal immune response whereas Cd has deleterious effects on cells , 79. Therthritis . Cd intrrthritis .in situ targeting of synoviocyte hyperproliferation and inflammation would be of interest. A model was developed in RA with the induction of synoviocyte apoptosis through gene targeting. The pro-apoptotic gene PUMA combined with an new adenovirus-baculovirus complex vector (in vivo model of RA (Regarding TGCT pathophysiology, the x vector  has showel of RA . TherefoTGCT is a cause of disability in young adults because of its high level of recurrence and of bone destruction, aggravated by recurrent bleeding. Its pathophysiology is now better understood. Genetic and chromosomal aberrations induce synovial hypertrophy and hyperplasia. TGCT shares common features with RA and sarcoma but with differences.The multiple presentations of the disease are the basis for treatment strategies. Usual treatments rely on surgical procedures, but limited results are affected by the high recurrence rate. New treatments are developed and inspired by those of RA and sarcoma. Systemic therapies targeting the CSF1/CSFR1 axis show promising results and pexidartinib is now FDA-approved for TGCT but managing liver toxicity is critical. Local treatments targeting synoviocytes are also under development. Overall, a multidisciplinary approach is essential for TGCT management. A such approach could also serve to understand better unresolved mechanisms of resistance in RA.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.MR and HF: writing and figures. PM: concept and proof reading. All authors contributed to the article and approved the submitted version.This work has been supported by the OPeRa IHU program, and the Institut Universitaire de France. PM is a senior member of the Institut Universitaire de France. MR is supported by the Ecole de l\u2019INSERM Liliane Bettencourt Programme.PM has a patent for the use of PUMA gene therapy in arthritis.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "ATP7B (c.3207C>A), SORL1 (c.352G>A), SETX (c.2385_2387delAAA), and FOXP1 (c.1762G>A) genes. The functional analysis revealed that the deletion in the SETX gene changed the splicing pattern, which was accompanied by lower SETX mRNA levels in the patient\u2019s fibroblasts, suggesting loss-of-function as the underlying mechanism. In addition, the patient\u2019s fibroblasts demonstrated altered mitochondrial architecture with decreased connectivity, compared to the control individuals. This is the first association of the CBS-PNFA phenotype with the most common ATP7B pathogenic variant p.H1069Q, previously linked to Wilson\u2019s disease, and early onset Parkinson\u2019s disease. This study expands the complex clinical spectrum related to variants in well-known disease genes, such as ATP7B, SORL1, SETX, and FOXP1, corroborating the hypothesis of oligogenic inheritance. To date, the FOXP1 gene has been linked exclusively to neurodevelopmental speech disorders, while our study highlights its possible relevance for adult-onset progressive apraxia of speech, which guarantees further study.Our aim was to analyze the phenotypic-genetic correlations in a patient diagnosed with early onset corticobasal syndrome with progressive non-fluent aphasia (CBS-PNFA), characterized by predominant apraxia of speech, accompanied by prominent right-sided upper-limb limb-kinetic apraxia, alien limb phenomenon, synkinesis, myoclonus, mild cortical sensory loss, and right-sided hemispatial neglect. Whole-exome sequencing (WES) identified rare single heterozygous variants in  MAPT, PGRN, LRKK2, PSEN1, or C9orf72 genes ..37].C9orf72 expansion was excluded [APOE \u03b52, \u03b52 genotype.We focused on the rare variants with the highest putative impact on the disease phenotype . The preexcluded . Among cSETX variant c.2385_2387delAAA in exon 10 activates a cryptic donor site, which may lead to splicing alteration , a known NMD target [DSCR1 levels increased upon cycloheximide treatment, showing robust NMD inhibition , characterized by excessive copper accumulation in various organs, such as the brain, liver, and cornea, leading to a wide spectrum of symptoms from hepatic to neuro-psychiatric [The chiatric ,46,47,48ATP7B gene [ATP7B pathogenic variant H1069Q in exon 14 accounts for 72% of WD cases in the Polish population [ATP7B mutations (Hzgs) demonstrate clinical symptoms, and biochemical and/or brain abnormalities [ATP7B Hzgs, such as early or late-onset Parkinson\u2019s disease (EOPD or LOPD) , while recessive mutations are associated with ataxia-oculomotor apraxia 2 (AOA2) characterized by cerebellar ataxia, oculomotor apraxia, and axonal sensorimotor neuropathy [SETX mutations have been detected also in Charcot Marie Tooth (CMT), distal hereditary motor neuropathy (dHMN) [The utophagy ,58. The uropathy ,59. SETXy (dHMN) ,61, chily (dHMN) , and Alzy (dHMN) . None ofSETX mRNA levels in the patient\u2019s fibroblasts suggesting loss-of-function as the underlying mechanism  gene encodes a transmembrane protein, named sortilin-related receptor (SORLA), involved in endo-lysosomal processes, amyloid precursor protein (APP) sorting and in the degradation of amyloid-beta (Ab) peptide, responsible for Alzheimer\u2019s disease (AD) pathology [SORL1 variants have been associated with AD [SORL1 is considered a cross-disease gene.The athology ,69. Init with AD ,71,72, w with AD ,73,74,75SORL1 rare variants resulted in altered maturation and cellular trafficking of the SORLA protein [A recent functional study revealed that many  protein . The V11 protein . The S12 protein . It is tFOXP1 (Forehead Box P1) gene encodes a transcription factor regulating tissue and cell-type-specific gene transcription during development and adulthood [FOXP1 syndrome present with considerable delays in language milestones [The dulthood . Pathogedulthood . Childrelestones . Expresslestones  , a motor speech disorder with effortful speech, impaired sequencing of articulatory gestures, and sound-based errors, distinct from both dysarthria and aphasia, is very likely to co-occur with non-fluent aphasia. It may be both neurodevelopmental (childhood-onset) or progressive. In most cases, progressive AOS accompanies PNFA or CBS.Heterogenous criteria are used in clinical practice to diagnose AOS . In the ATP7B heterozygous mutation carriers dysarthria has been reported [In WD, mixed dysarthria is often observed , but dysreported . In our reported . Only inDetailed clinical case descriptions with very long follow-ups and neuropathology data are unfortunately rarely reported. Of note, Tetzloff et al. provided a 10-year-long observation of the PPAOS case with autopsy-confirmed CBD . In thisFOXP1 findings, it remains to be established if FOXP1 abnormalities may influence the onset or pattern of progression of progressive AOS in adults.In the context of ATP7B, SETX, SORL1, and FOXP1, have been associated with mitochondrial dysfunction [Finally, we provide evidence of an altered mitochondrial network in the patient (less branched mitochondria with decreased connectivity) compared to control individuals. As mentioned before, all four genes, function ,42,43,44The patient\u2019s clinical diagnosis was not supported by the analysis of WD metabolic markers, cerebrospinal fluid (CSF) biomarkers, dopamine transporter SPECT , or amylAs DNA from the patient\u2019s family members was not available, the co-segregation of the identified variants with the disease phenotype in the family has not been established.The large-scale sequencing techniques have the potential to facilitate a diagnosis for intractable dementia cases with heterogenous clinical manifestations. Our results are in line with those of Ciani et al., who found that 50% of sporadic patients from the FTLD spectrum showed at least one rare missense variant in AD, PD, ALS, and LBD-associated genes . Our resATP7B mutation. While parkinsonian phenotypes have been previously linked to ATP7B heterozygotes, we also described other variants of potential functional impacts in well-known disease-related genes, such as SETX, SORL1, and FOXP1. Of note, these three latter genes have been associated with speech and/or language deficits. The dissection of the molecular mechanisms underlying possible complex interactions among these genes would be a great challenge for the future.To our knowledge, this is the first reported case of the CBS-NAV phenotype with a single heterozygous"}
+{"text": "Plasma treatment of polymeric materials is a cost-effective and efficient technique to modify the surface and change the constituent unit configuration. This research investigates the effects of argon DC glow discharge plasma on pure and DR1 dye-loaded polyamide 6 polymer films and stabilization of dye on the surface. Plasma breaks some bonds and activates the surface through creating reactive structures such as free radical sites on the surface and increases tertiary amides on the surface of polymer. Besides, this process alters surface topographical characteristics and conformation of azobenzene dye which are effective on the durability of the dye on the surface. Plasma causes interactions of the dye with the polymer and immobilizes the dye on the polymer. On the other hand, these interactions lead to changes in the dye's optical and geometric isomeric activity and stability. This work studies the chemical and morphological changes of polyamide 6 by plasma with AFM and spectroscopic methods. Furthermore, the aging of nylon 6 films loaded with DR1 dye is measured, and the conformational changes of the dye are investigated. Plasma stabilizes the dye on the polymer surface through making changes of chemical and physical properties on the surface components. Polyamides are the first types of globally mass-produced thermoplastic engineering plastics, produced commercially in two forms of films and fibers in the industry2. Polyamide 6, as a type of polyamide, is a significant polymer in the industry and is classified in the category of semicrystalline polymers3. This polymer is a synthetic polymer extensively used in electronic devices, machine industry, and military equipment5. Polyamide 6 has favorable properties, such as suitable chemical and aging resistance6, appropriate electrical and thermal resistance7, excellent mechanical properties, and low contact friction8. It has both hydrophilic parts resulting from carbonyl and amide groups and hydrophobic parts due to the presence of ethylene sequence in its structure9.Polymers have a variety of applications due to their different physical and chemical properties10. In these materials, surface properties sometimes limit their uses in different applications. As a result, improving the surface properties of polymers has great importance in applications, such as coating or printing11. There are different methods of surface modification for changing the surface properties while maintaining the bulk profile of material, like chemical modification12, radiation13, and thermal treatment14, which is crucial for various applications. Disadvantages of the mentioned methods include using hazardous chemicals, high energy consumption, and expensive materials and equipment.In general, polymers usually have low surface energy because of the deficiency of polar groups in their structure, leading to low wettability and adhesion17. Cold plasma is a quasi-neutral medium in which particles are in a thermal non-equilibrium state, and heavier particles possess lower temperatures than electrons18. The cold plasma treatment of materials is a suitable method to alter the surface characteristics of a polymer without any damage to its bulk20. A variety of energetic particles in plasma media, including ions, electrons, radicals, metastable species, and photons in the energy range of ultraviolet radiation, affect the surface22. The depth of modification via the plasma technique for the surface is from the order of 10\u00a0nm, without influencing the bulk characteristics21. The interactions between the plasma and the polymer surface lead to results, such as chain scission23, etching, polymerization, formation of new covalent bonding and functional groups, crosslinking24, generation of polar groups25, and elimination of surface contamination26. Thus, the plasma can change the chemical, physical, mechanical, and adhesion properties of the surface of polymers28. In addition, the plasma affects properties, such as printability, wettability, and biocompatibility of the surface29.Therefore, methods including not using chemical material for surface treatment and modification are more popular and practical nowadays. Plasma treatment of material is an environmentally friendly, clean, dry, chemical-free, and economical method to alter the surface properties30. Dye immobilization on the surface of material is an important subject that depends on surface physical and chemical properties32. Disperse Red 1 (DR1) dye is a pseudo-stilbene type azobenzene with push\u2013pull characteristic through its electron donor and acceptor substituents33. Azobenzene is a molecular switching system with two phenyl rings linked by (\u2013N=N\u2013) that provides reversible photoisomerization under ultraviolet light irradiation34. Azobenzene molecules are found in two configurations of E (trans) and Z (cis) isomers35. More stable trans isomer converts to cis conformation during exposure to UV light36. The molecule is transferred to cis state with about 50\u00a0kJ\u00a0mol\u22121 more energy, and the cis isomer changes to trans after thermal relaxation37. Photoisomerization of trans azobenzene causes geometrical structure change and brings the aromatic rings closer to each other38. Azobenzenes have a wide range of applications, such as optical data storage39, sensors40, nonlinear optics41, nanomachines42, drug delivery, holographic43, optical and solvatochromic applications, and so on45, due to their photoswitching, molecular motion, electronic, and optical properties. As a result, the dye must be immobilized on the surface of material for better efficiency in various applications40. Also, polymers containing azo dyes have extensive optical applications through their low cost and desirable optical response and have the potential to be used in optoelectronic devices47.Dyeing of materials is done in different ways, and dyeing after manufacture is one of the economical methods to reduce the consumption of dyes. But the main problem in this method is low color stability on the surface due to environmental stress conditions like heat, sunlight, and humidityIn this work, pure and dye-loaded polymer films were exposed to glow discharge plasma to apply plasma-induced changes on the surface of prepared samples. Fourier-transform infrared spectroscopy (FT-IR) and atomic force microscopy (AFM) were employed to evaluate the chemical and morphological changes of the polymers\u2019 surface as the result of plasma treatment. The accelerated aging process was used to estimate dye fastness on the surface of the polymer samples. UV\u2013Vis spectroscopy was applied to measure and compare the effects of plasma on dye fastness of the polymer films and dye conformational composition before and after plasma treatment.After preparing the thin films of pure and DR1 dye-doped polyamide 6, each sample was placed in the plasma reactor for treatment with the argon plasma. FT-IR spectra of the thin layer samples show that the argon plasma treatment of the films, for 300\u00a0s, changes the chemical structure of thin polymeric layers. Given that the glow discharge plasma affects the surface, it can be said that these chemical changes have occurred on the surface of polyamide 6. Figure\u00a0\u22121 belong to C-H (CH2) symmetric and asymmetric stretching vibrations. The amide group's presence and nitrogen non-bonding electrons conjugation with carbonyl compound in the amide group lead to the reduction of absorption frequency and wavenumber in the carbonyl band because of the resonance effect48. So, the nearby peaks at 1635\u00a0cm\u22121 are related to the primary amide by overlapping of C=O stretching bands and N\u2013H bending vibrations. The peak located at 1578\u00a0cm\u22121 refers to the secondary amide in the polymer structure, and the peak at 1457\u00a0cm\u22121 is attributed to the other forms of N\u2013H vibrations. In addition, the observed band in 1383\u00a0cm\u22121 corresponds to the tertiary amide group. The wide band in 699\u00a0cm\u22121 is ascribed to N\u2013H wagging vibrations.The FT-IR spectra show that the absorption bands in the range of 2850\u20133000\u00a0cmDR1 dye-doped polyamide 6 polymer thin film was exposed to plasma for different treatment times to further investigate plasma effects. Then UV\u2013Vis spectra of the sample for each time interval of plasma treatment were recorded and compared with the sample without plasma exposure. Figure\u00a0Changes in the chemical structure of the surface and the presence of different materials with polar properties cause changes in the absorption wavelength by the dye\u2019s species. The obtained data from the deconvoluted lines demonstrates that the plasma environment converts several trans isomers to cis form. As can be seen from Table Due to the surface treatment nature of cold discharge plasma, it seems that the study of the changes induced by plasma on the surface of DR1 dye-doped polyamide 6 polymer can provide interesting information. An atomic force microscope (AFM) was used to analyze the surface topography of the dye-doped polymer films. The results obtained from the AFM in Fig.\u00a0\u20133\u00a0M) to adsorb more dye on the surface of the sample. The sample was exposed to plasma for various time periods to study and compare the plasma influence on the surface components. UV\u2013Vis spectra were performed for the sample for each time interval of plasma treatment and compared with the state before plasma exposure to investigate the other effects of plasma. Figure\u00a0In this part, DR1 dye-doped polymer film was dipped in DR1 dye solution  are formed on the surface of the polymer after exposure to ambient air that increase the amount of some polar materials on the surface, like C=O and C\u2013O bonds53, which act as an intermediary to adsorb the dye into the polymer surface. Oxygenated compounds can help the dye adhere to the polymer surface by creating a hydrogen bond.The increase in the peak intensity of tertiary amides in the doped polymer state is more than pure polymer film. In addition, the peak related to 1635\u00a0cmAccording to Fig.\u00a054. The introduced peaks in the UV\u2013Vis absorption spectrum of the DR1 dye, as a pseudo-stilbene type dye, contributing in both isomeric states of this type of dyes, are so close to each other and overlapped55. The absorption spectra can be used to estimate the cis to trans isomers ratio before and after plasma treatment through calculation of the area under the deconvoluted curves. In this work, the absorption spectra were fitted by two Gaussian peaks to extract and compare the amount of cis and trans isomeric species using the OriginPro software. Figure\u00a0The absorption spectra obtained from the DR1 dye-doped into polyamide 6 polymer film, before and after plasma treatment for different times, were used to determine the alteration of dye conformation due to argon plasma treatment. Azobenzene dyes have two main peaks for each conformation in the UV\u2013Vis spectra arising from \u03c0\u2009\u2192\u2009\u03c0* and n\u2009\u2192\u2009\u03c0* transitions. The intensity of absorption band of \u03c0\u2009\u2192\u2009\u03c0* transition in the trans species is higher than the cis states, but the \u03c0\u2009\u2192\u2009\u03c0* and n\u2009\u2192\u2009\u03c0* transitions are slightly shifted further in the wavelength range for the cis modeThe area under the dependent fitted curves in the absorption spectra, due to plasma treatment in different time intervals, confirms an increase in the population of cis isomeric species compared to trans types, see Table a roughness as the average deviation from the surface baseline is obviously enhanced. Also, AFM results show that the Rq roughness are increased after glow discharge plasma treatment of the dye-doped polymer surface. According to Fig.\u00a0As shown in Table 56. Obviously, there are more empty spaces for dye isomerization on the surface of the polymer than the bulk. It is clear that more effective photons in the plasma environment collide with the dye molecules on the surface directly. The plasma changes the conformation of many dye molecules on the surface due to more free space than dye molecules in the bulk of the material. Some of the reasons for the stabilization of the cis conformation are because of locking the cis isomer due to dye alignment and spatial limitation57. On the other hand, plasma can change surface morphology and chemistry, and prepare the surface conditions to stable cis isomers physically. Increasing the number of pores and polar groups on the surface of the polymer by plasma contributes to stabilizing the dye in the cis state. Numerous subjects impress on the dye inversion, such as substituent, environmental polarity and temperature, and the other environmental conditions58. Creating new bonds between the dye and the polymer and new substituent of dye affect the stability of the cis isomer state. Plasma also affects the substituent of the dye and alters various chemical and physical characteristics of the surface. By creating bonds between the dye and the polymer, the plasma causes changes in the optical behavior and stability of the dye, in particular isomeric states. Additionally, in this study, the DC field applied for plasma creates some components with partial dipoles oriented in the field\u2019s direction on the surface. This leads to a regular polarization that brings about alignment, stability, and adhesion of the dye to the surface of the polymer, which is helpful for various nonlinear optical applications. In addition, the higher energy level of the cis isomer leads to more significant reactions with the polymer surface and causes the dye to become immobilized on the polymer surface. Therefore, it is obvious that the plasma increases the adhesion and durability of the dye on the surface. Polymers have good surface and optical properties, and it is easy to make thin films of polymeric material. Even though inorganic materials are usually used to store information due to greater stability of isomeric state of the dye41, plasma treatment suggests a new approach for stabilizing the cis isomer by a strong influence on the polymer surface and dye to create a better polymer environment for data storage and optical applications.According to Fig.\u00a0By comparing the UV\u2013Vis spectra of Fig.\u00a0The effects of argon DC glow discharge plasma on dyeing of polyamide 6 polymer surface were investigated. The results confirm that plasma-modified dye-loaded film suffers less dye leaching because of physical and chemical changes of the surface content. Plasma activates the surface of polyamide 6 polymer and creates radical groups through breaking some specific bonds on the surface. The subsequent reactions create covalent bonds leading to the increase of tertiary amides and immobilization of dye on the surface of polymer. Besides, plasma treatment of polymeric films increases electro-negativity and surface polarity by introducing polar groups that reinforce the dye\u2019s durability on the polymer surface. Plasma process introduces a new and effective method for converting trans to cis isomer states of DR1 dye with a higher conversion rate in nylon 6 polymer film. The higher energy of cis species enhances the reactivity of the dye with the polymer surface and increases the possibility of dye reactions with the surface. Plasma has significant effect on the conformational conversion and stability of the dye molecules present on the surface because of changes in substituent and polarity of the surface. The results demonstrate that the glow discharge plasma increases the interactive surface of the polymer by enhancing roughness and creating fine and porous structures on the surface, which improves the adhesion of the dye to the surface of the polymer. On the other hand, the surface roughness positively affects the stability of the cis isomer of the dye during plasma modification.Finally, plasma modification of the polymer surface can be an appropriate technique for dye immobilization on the surface. This technique increases color fastness on the surface and prepares it for use in different applicable substances like halochromic and holographic materials. Moreover, using plasma can multiply the capacity of dye-based optical memories or data storage systems and increase their stability. Also, increasing the amount of cis isomers of dye on the polymeric surface prevents degradation of the polymer through UV radiation absorption on the surface.2)5CO\u2013]n, used in this work, was purchased from Sigma-Aldrich with a density of 1.084\u00a0g/mL at the temperature of 25\u00a0\u00b0C and the glass transition temperature of 47\u00a0\u00b0C. DR1 dye, formic acid (HCOOH), and ethanol (C2H6O) as solvents and dimethylformamide (DMF) were obtained from Merck. The water used in this work was distilled water.Polyamide 6 polymer, with the linear formula of [\u2013NH analysis was performed on a Nanosurf Mobile S instrument.70, an FT-IR spectrophotometer, was used to record the vibration spectra over the wavenumber range of 400\u20134000\u00a0cmAn accelerated aging process was chosen to examine the immobilization of the dye on the surface of the polymer. The color fastness was compared between the plasma-treated and untreated dye-loaded polymer films during the aging test. The films were placed in an aging oven  exposed to an ultraviolet light at the temperature of 45\u00a0\u00b0C and humidity of 87%. The ultraviolet light source was a 500\u00a0W mercury-vapor lamp. It was used for various time intervals at a distance of 22\u00a0cm from all samples\u2019 surfaces at equal angles, and humidity of 87% was obtained by evaporating distilled water from the floor of the chamber. After the aging process at different times, the UV\u2013Vis absorption spectra were recorded to determine the color retention on the surface of the samples.The polymer solution was prepared by dissolving polyamide 6 polymer in formic acid at a concentration of 0.5\u00a0W/W% at room temperature. The nylon 6 films were cast on glass slides by the spin-coating method. Generally, three sets of samples were prepared; the first set includes pure polyamide 6 polymer films to investigate the chemical changes and the amount of dye fastness on the surface of pure polymer films immersed in dye solution before and after plasma treatment. The second set of films was cast from the polyamide 6 solution doped with DR1 dye at a concentration of 2\u00a0W/W% to study physical and chemical changes of surface content. For the last group, the prepared doped polymer films were dipped in the solution of DR1 dye in ethanol solvent with a concentration of 10\u00a0mg/mL in ambient temperature for optimized time of 90\u00a0s, so the films adsorb more dye homogeneously. These samples were used for conformational studies. It should be noted that the prepared samples were dried at a temperature of 30\u00a0\u00b0C in an oven. The films were exposed to a plasma environment, and the surface changes were compared with the plasma-free state. The experiments were repeated three times for each batch of samples.\u20131\u00a0Torr. Then the power supply connected to the electrodes introduced a glow discharge plasma in the space between the two electrodes by applying the DC bias voltage of 1.1\u00a0kV and discharge current of 0.15 A with the power density of 660 mW/cm3. After plasma modification of the samples, the chamber pressure reaches the atmospheric pressure. Figures S4 and S5 in supplementary information indicate respectively the image and scheme of argon DC glow discharge plasma instrument treating a sample.The chamber of glow discharge plasma in this work was a Pyrex tube with a length of 500\u00a0mm with two aluminum electrodes attached to the two ends of this cylindrical tube. The polymer films were placed in the positive column zone of the glow discharge plasma for various time durations. The applied gas was argon (with a purity of 99.99%), and the gas pressure in the plasma chamber was decreased with a rotary vacuum pump (Alcatel) joint. After purging the chamber with argon gas, the target gas pressure was fixed at 2\u2009\u00d7\u200910The absorption spectra were fitted by two Gaussian peaks to extract and compare the amount of cis and trans isomeric species using the OriginPro 2018 software. The area under each deconvoluted curve indicates the population of any isomeric state. The raw data of UV\u2013Vis spectra were smoothed by the OriginPro software using Savitzky-Golay method with 32 points of window.59.A batch of dye-doped samples immersed in dye solution, were washed with aqueous 50% DMF at the temperature of 45\u00a0\u00b0C for 40\u00a0min after plasma treatment and compared with untreated washed samples with the same conditions. The color strength  of each sample was determined by reflectance measurement according to Kubelka\u2013Munk equationSupplementary Information."}
+{"text": "Magnetic bone cement not only supports bone metastasis defects but can also achieve magnetic hyperthermia to eliminate tumor cells around the bone defect. However, the physicochemical properties of the bone cement matrix will change if the weight ratio of the magnetic nanoparticles in the cement is too high. We mixed 1 weight percent Zn Skeletal-related events are caused by bone metastases, including pain, pathologic fracture, and spinal cord compression, which are the main reasons for the decline of quality of life among advanced oncology patients ,2. With The spine is the predilection site of bone metastases, and 90% of spinal tumors are metastatic. At present, percutaneous vertebroplasty with bone cement is the main minimally invasive surgery for the treatment of spinal bone metastases. Bone cement is a crucial filling material in the treatment of bone metastases; however, the antitumor effect of conventional bone cement is limited.The addition of chemotherapy drugs to bone cement struggles to achieve a constant antitumor effect due to the \u201csudden release effect\u201d , and the0.3Fe2.7O4 nanoparticles using the one-pot method, as the nanoparticles have high heating efficiency and good biocompatibility [0.3Fe2.7O4 nanoparticles to polymethyl methacrylate (PMMA) to prepare magnetic bone cement (M-PMMA), which minimizes the affection to the physicochemical properties, and satisfies the hyperthermia requirement in the AMF. The in vitro and in vivo performance of M-PMMA was also evaluated, and 1 wt% Zn0.3Fe2.7O4 PMMA-based bone cement was able to suppress bone metastases and provide a promising clinical transformation.In this study, we synthesized 22 nm Zntibility . We then0.3Fe2.7O4 nanoparticles (200 mg) were dissolved in a methacrylic acid monomer , and 20 g PMMA powder was divided into 10 bottles of 2 g PMMA powder in a sterile environment. Two grams of PMMA powder and 1 mL of MMA containing Zn0.3Fe2.7O4 nanoparticles were mixed and injected into the bone marrow cavity of the tibia to fill the bone defect, and several minutes later, the mixture was solidified.Zinc (II) acetylacetonate (0.3 mmol), iron (III) acetylacetonate (2.7 mmol), sodium oleate (2 mmol), and oleic acid (4 mL) were mixed with benzyl ether (20 mL) under a gentle flow of argon. The mixture solution was stirred by a magnetic stirring apparatus in an argon atmosphere and heated to 393 K for 1 h. The mixture was further heated to reflux temperature (\u2248573 K) under an argon blanket and maintained at the reflux temperature for 1 h. After cooling down to 300 K by removing the heat mantle, the NPs were collected by centrifugation. The size of the nanoparticles was regulated by a heating rate from 393 to 573 K. The morphology and size of the nanoparticles were characterized using a high-resolution (120 kV) transmission electron microscope . PMMA bone cement was purchased from the Ningbo Hicron Biotechnology Limited Company . Zn0.3Fe2.7O4 bone cement samples were prepared using the method mentioned above. The samples were dissolved by a mixture of nitrohydrochloric acid (HCL: HNO3 = 3:1), the iron in the mixture was measured by inductively coupled plasma mass spectrometry (ICP-MS), and the mass percent of nanoparticles in the bone cement was calculated using the chemical formula of Zn0.3Fe2.7O4.Three 200 mg solidified PMMA-Zn0.3Fe2.7O4 bone cement samples were prepared and adhered to conducting resin. After metal spraying, surface characterization was captured by FE-SEM, and the element distribution was observed by energy disperse spectroscopy (EDS).The SEM images were captured using a field emission electron microscope  operating at 10 kV. The solidified PMMA-Zn0.3Fe2.7O4 bone cement was prepared, and the bone cement pastes were injected into a cylindrical column mold at the dough stage and compacted with metal clips. Compressive strength test samples (12 mm in length and 6 mm in diameter) were prepared and dried for 24 h at room temperature. The bone cement\u2019s pastes were poured into square molds at the dough stage and compacted by metal clips. Bending strength test samples  were prepared and dried for 24 h at room temperature. All compressive and bending strength samples were tested using an electronic universal testing machine .The PMMA-Zn2 at 37 \u00b0C. According to the guidelines of ISO5833, 2 g ball-shaped solidified PMMA-Zn0.3Fe2.7O4 bone cement was immersed in 1 mL of culture medium for 24 h in a humidified atmosphere of 5% CO2 at 37 \u00b0C to prepare the leaching solution. The PMMA leaching solution was prepared as described above. The two types of 100% leaching solutions were diluted to acquire a 50% leaching solution. The 50% and 100% PMMA-Zn0.3Fe2.7O4 and PMMA leaching solutions were incubated with MEF for 24 h, 48 h, and 72 h. The cell viabilities were evaluated by Cell Counting Kit-8 assay , and the absorbance was measured at 450 nm with a multimode plate reader . Cell viability was expressed as the percentage of viable cells compared with controls (cells incubated with pure culture medium).Mouse embryonic fibroblast (MEF) cells were purchased from the American Type Culture Collection . Cells were all cultured in a DMEM/HIGH GLUCOSE  medium supplemented with 10% fetal bovine serum and antibiotics (100 \u00b5g/mL penicillin and 100 \u00b5g/mL streptomycin) in a humidified atmosphere of 5% CO0.3Fe2.7O4 bone cement solidified samples (5 mm in diameter and 10 mm in length) were prepared. One hole (1 mm in diameter and 5 mm in depth) was drilled from the center of the sample basal circle. The PMMA samples were prepared using the same method described above. The cylinder-shaped samples were put into a 2.5 mL Eppendorf tube and covered with polystyrene foam to avoid thermal dissipation. A fiber optic probe  was inserted into the previously drilled hole. The temperatures in the core of the samples were documented every second after switching on the AMF  with a frequency of 430 kHz.PMMA-Zn3) by ophthalmic scissors, then collected by centrifugation and re-suspended by PBS. The VX2 tumor mass suspension was added to a 10 mL syringe.New Zealand white rabbits were supplied by the laboratory animal center of the Chinese PLA General Hospital. The rabbits were approximately 20\u201324 weeks old and weighed 2.5\u20133.0 kg. All animal experiments were approved by the animal welfare ethics committee of the Chinese PLA General Hospital. The VX2 solid tumor mass was purchased from Tongpai Biological Technology Limited Company . After fast thawing in warm water (37 \u00b0C), the cryopreserved tumor mass was sheared into a small piece (1 mmAfter successful anesthetization with ketamine (10 mg/kg) and lumianning (5 mg/kg), the right hind leg skin of a rabbit was prepared, and the VX2 tumor mass suspension was injected into the muscles. Three weeks later, the tumor tissue of the tumor-bearing rabbit was harvested and sliced into small pieces to prepare the bone metastases model.3) were put into the bone marrow cavity of the tibia through the bone hole, and the hole was sealed by bone wax. The rabbits were fed normally 6 h after the operation.The rabbits were successfully anesthetized with ketamine (10 mg/kg) and lumianning (5 mg/kg), the right hind leg skin was prepared, and the puncture point was located 1 cm under the tibial medial plateau. After local anesthesia with 1% lidocaine, a 3 mm diameter puncture needle was penetrated into the tibia plateau, several fresh VX2 tumor masses  were included in the M-PMMA + AMF group; rabbits injected with M-PMMA without AMF, the M-PMMA group; and rabbits without M-PMMA and AMF, the control group. One week after the model establishment operation, all animal models were confirmed by X-ray examination . After the tumor-bearing rabbit was punctured vertically with a vertebroplasty puncture needle 3 cm into the tibial plateau of the affected limb, 1 mL of the contents of the bone marrow cavity was withdrawn to prevent spillage during the bone cement injection. Approximately 0.5\u20130.8 mL of magnetic bone cement was injected into the bone marrow cavity along the bone hole, and the M-PMMA injection was completed and treated in AMF  for 30 min.Magnetic hyperthermia began at 24 h post-M-PMMA injection. The rabbits in the M-PMMA + AMF group were administered magnetic hyperthermia treatment after one day, and all rabbits were treated seven times. Magnetothermal therapy uses medical tape to affix the optical fiber approximately 1 cm below the tibial plateau, and the affected limb is placed inside the magnetothermal therapy coil. The magnetic thermotherapy apparatus was turned on, and the magnetic field strength was adjusted to raise the temperature to 43 \u00b0C. The X-ray examination was carried out post-M-PMMA injection and at the end of magnetic hyperthermia. A chest CT scan  was also administered at the end of the magnetic hyperthermia. The weight and cross-section diameter of the tumor-bearing hind legs in each group were measured, and the appearance of the tumor-bearing hind legs was also observed. When the rabbits died, the tumor-bearing hind legs were harvested and scanned using micro-CT ; bone mass was measured using auxiliary software. The harvested tumor-bearing hind legs were decalcified and embedded in paraffin, the samples were sliced by microtome  and stained by hematoxylin and eosin, and the pathological sections were observed by microscope .\u00ae . A p value of less than 0.05 was considered statistically significant.The results are expressed as the mean \u00b1 standard deviation for as many continuous variables as possible. A normal distribution was confirmed, and significant differences were calculated using a one-way ANOVA. Non-normally distributed data were analyzed using the Kruskal\u2013Wallis test, followed by the Mann\u2013Whitney U-test to find significant differences between groups. Statistical analyses were performed using GraphPad Prism0.3Fe2.7O4 nanoparticles and PMMA-Zn0.3Fe2.7O4 bone cement is shown in 0.3Fe2.7O4 nanoparticles mean size was 21.8 \u00b1 2.0 nm and polyhedral in shape. TEM images  monomer. The ratio of nanoparticles to the MMA monomer was 20 mg to 1 mL, and 2 g of PMMA powder  mapping (The solidified magnetic bone cement is shown in  mapping a\u2013c show p < 0.05). The mechanical strength of PMMA was enhanced after adding the Zn0.3Fe2.7O4 nanoparticles. The mechanical properties of M-PMMA meet the criteria listed in the ISO 5833:2002(E) guidelines, which require that the compressive and bending strength must be higher than 70 MPa and 50 MPa.p > 0.05). Consequently, PMMA-Zn0.3Fe2.7O4 bone cement has no toxic effects on MEF cells, which means that it is biocompatible.As shown in 0.3Fe2.7O4 and pure PMMA in the AMF of 4\u201327 kA/m at a frequency of 430 kHz. As illustrated in 0.3Fe2.7O4 barely increased in the low magnetic field strength (4\u20137 kA/m), when the field strength reached 10 kA/m, the temperature increased gradually. When the field strength was switched to 27 kA/m, the change in surface temperature increased to 60 \u00b0C within 100 s.p > 0.05). One week after the model was established, the tumor-bearing hind legs began to swell. As shown in p > 0.05), but at the beginning of the second week, the difference became significant (p < 0.05). The condition of the rabbits worsened following lung metastases. Food intake decreased rapidly at three weeks post-model establishment in the M-PMMA and control groups, while such conditions appeared later in the PMMA + AMF group. Although all the tumor-bearing rabbits died in our experiment, the rabbits in the M-PMMA + AMF group lived longer. p < 0.05), and the survival time of the M-PMMA + AMF group was longer than the other two groups.We further evaluated bone resorption using X-ray and micro-CT scans. The X-ray image was first roughly examined using the morphologic changes of the tumor-bearing tibias. As shown in p < 0.05). Magnetic hyperthermia induced by PMMA-Zn0.3Fe2.7O4 bone cement can inhibit bone resorption.In addition, the incidence of pathologic fractures was 10% in the M-PMMA and control groups, which did not occur in the M-PMMA + AMF group. Furthermore, skeletal morphologies and bone resorption were evaluated by micro-CT after magnetic hyperthermia. Although the cortical bone thickness of tumor-bearing tibias in the M-PMMA + AMF group a1,b1,c1 0.3Fe2.7O4 with good biocompatibility and high heating efficiency into a PMMA matrix to prepare M-PMMA, which minimized the affection for physicochemical properties and satisfied the hyperthermia requirement in the AMF. Magnetic bone cement is composed of magnetic nanoparticles and a bone cement matrix. The magnetic nanoparticles, the core of magnetic bone cement, directly determine the heating efficiency of AMF. We chose Zn0.3Fe2.7O4 nanoparticles as the threads in magnetic bone cement because of their high heating efficiency and good biocompatibility, which have been reported previously [We mixed 1 wt% Zneviously .0.3Fe2.7O4 nanoparticles were synthesized using a one-pot approach [4 being antiferromagnetic and Zn2+ ions occupying only the A-site of the spinal lattice. Consequently, Ms, and anisotropy can be tuned by changing the Zn:Fe ratio, and Ms can be increased by properly doping zinc elements in ferrite [0.3Fe2.7O4 nanoparticles have the highest SAR in a series of zinc-doped ferrite nanoparticles [Znapproach . General ferrite . Zn0.3Fearticles ,11. Prevarticles . Meanwhiarticles .The design and synthesis of magnetic bone cement to treat bone metastases has been reported ,17. HoweProviding mechanical support and filling bone defects are the two basic functions of bone cement. Furthermore, mechanical support is especially important in the treatment of spinal osteolytic metastases because the spine supports the whole trunk. If the spine is unstable, axial pain and nerve symptoms will appear. In this work, the mechanical strength is improved by incorporating magnetic nanoparticles into the bone cement matrix, and the results are consistent with previous studies ,20, wher0.3Fe2.7O4 nanoparticles (Zn0.3Fe2.7O4/SiO2) is better than naked Zn0.3Fe2.7O4 nanoparticles [0.3Fe2.7O4 nanoparticles as threads.Biocompatibility is essential for the biomedical application of bone cement . Generalarticles . Due to \u22121 s\u22121 [0.3Fe2.7O4 nanoparticles as threads due to their appropriate hyperthermia performance in a low-strength field.Hyperthermia occurs between 43 \u00b0C to 45 \u00b0C, while ablation temperatures are above 60 \u00b0C . When th\u22121 s\u22121 , which i0.3Fe2.7O4 with high heating efficiency and good biocompatibility can be used for the magnetic hyperthermia treatment of bone metastases. In this work, the VX2 bone tumor was extremely similar to the bone metastases in the clinic. Most rabbits in the M-PMMA and control groups presented with lung metastases three weeks after model establishment. When the tumor cells metastasized to the lungs, the food intake of the rabbits decreased rapidly, and the body weight simultaneously decreased. Hence, an MRI scan was conducted before M-PMMA injection to observe whether the model establishment was successful and to determine the time of injection. Two weeks after model establishment, the tumor-bearing hind legs in the M-PMMA and control groups swelled rapidly, and areas of necrosis and cavitation occurred within the tumor. However, the tumor-bearing hind leg in the M-PMMA + AMF group swelled slowly. Accordingly, the corresponding time-weight and time-circumference curves revealed that magnetic hyperthermia can suppress bone metastases locally.Immunodeficient animal models are widely used in tumor research. Bone tumor cells are percutaneously injected into the flank of nude mice, and the model is established several days later. Although such bone tumor animal models are convenient to observe and measure , they ma0.3Fe2.7O4 bone cement exposed to AMF. All the rabbits died from visceral metastases in our experiment, which indicates that magnetic hyperthermia locally suppresses bone tumors and that bone metastases need multidisciplinary treatment. In addition, we found that the therapeutic effects were closely related to treatment time. Bone metastases are more difficult to inhibit if tumor cells invade the surrounding tissues. This result is consistent with oncology treatment guidelines, which require early detection and treatment [0.3Fe2.7O4-meditated magnetic hyperthermia could not only locally suppress bone tumors but also inhibit osteoclastic bone resorption.Bone metastasis patients will deteriorate if tumor cells metastasize to the visceral organs in the clinic . Bone mereatment . We reve0.3Fe2.7O4 nanoparticles with high heat efficiency and good biocompatibility. These nanoparticles were then incorporated with PMMA bone cement to develop a magnetic bone cement with 1 wt% Zn0.3Fe2.7O4. The PMMA-Zn0.3Fe2.7O4 bone cement not only provides reliable mechanical support but also high heat efficiency. In vitro studies have revealed the biocompatibility of the cement. In vivo investigations indicate that PMMA-Zn0.3Fe2.7O4-meditated magnetic hyperthermia can suppress bone metastases locally and inhibit osteolytic bone resorption. Our studies demonstrate that with heating emission ability improvement, the nanoparticles incorporated into the bone cement matrix will be further reduced, and the effect on physicochemical properties will be minimized. PMMA-Zn0.3Fe2.7O4 bone cement may be appropriate for clinical use.In summary, we synthesized Zn"}
+{"text": "Lecanoromycetes, the largest extant clade of LFSs. All LFSs possess a robust CAZyme arsenal including enzymes acting on cellulose and hemicellulose, confirmed by experimental assays. However, the number of genes and predicted functions of CAZymes vary widely, with some fungal symbionts possessing arsenals on par with well-known saprotrophic fungi. These results suggest that stable fungal association with a phototroph does not in itself result in fungal CAZyme loss, and lends support to long-standing hypotheses that some lichens may augment fixed CO2 with carbon from external sources.Lichen symbioses are thought to be stabilized by the transfer of fixed carbon from a photosynthesizing symbiont to a fungus. In other fungal symbioses, carbohydrate subsidies correlate with reductions in plant cell wall-degrading enzymes, but whether this is true of lichen fungal symbionts (LFSs) is unknown. Here, we predict genes encoding carbohydrate-active enzymes (CAZymes) and sugar transporters in 46 genomes from the  Lichen symbioses are thought to be stabilized by the transfer of fixed carbon from a photosynthesizing symbiont to a fungus. Here, Resl et al. show that, contrary to other fungal symbioses, fungal association with a phototroph in lichens does not result in loss of fungal enzymes for plant cell-wall degradation. Almost a hundred years later, Smith and colleagues revealed these transferred photosynthates to be polyols and glucose in the case of algae and cyanobacteria, respectively2. They and others traced the transfer of algal fixed carbon into fungal cells, where they found it to be converted into mannitol and arabitol4. The fungal-photosymbiont relationship is widely interpreted as conferring net independence from external carbohydrates on the resulting lichen thallus. Accordingly, lichen fungal symbionts have been classified as biotrophs5, and the symbiotic outcome, the lichen thallus, as a \u201cphotosynthetic carbon autotroph\u201d6 or \u201ccomposite autotroph\u201d7.Stable fungal associations with algae and/or cyanobacteria, usually referred to as lichens, feature prominently in the history of the discovery and study of symbiosis. In describing the pairing of fungi with microbial photosymbionts for the first time, the Swiss botanist Simon Schwendener proposed that lichen fungal symbionts derive nutrition from \u201cassimilates\u201d of their photosynthesizing partners9, reflecting a common pattern of compensated trait loss in symbioses10. So what happened to CAZymes in lichen fungal symbionts? Multiple lines of indirect evidence have emerged over the last 40 years to suggest the retention of diverse CAZymes in lichen fungal symbionts (LFSs) or their secondary evolutionary derivates, especially specific plant cell wall-degrading enzymes (PCWDEs). First, molecular phylogenetic studies have shown multiple independent origins of putative saprotroph lineages from lichen fungal symbiont ancestors, both ancient11 and recent14. How these newly evolved lineages acquired the carbohydrate breakdown arsenal they would presumably need for life without an alga has not been explained. Second, some fungi near the symbiont-to-saprotroph transition have been shown to switch between the two lifestyles facultatively, so-called \u201coptional lichens\u201d16; in these cases, the fungus appears not to be obligately dependent on the alga for nutrition. Third, many lichen symbioses exhibit anomalous \u201csubstrate specificity\u201d, i.e., they are restricted to specific organic substrates and unable to colonize others, suggesting lack of nutritional autonomy19. Fourth, lichen fungi are capable of growing axenically in vitro on a variety of sugars other than sugar alcohols, including crystalline cellulose and sucrose (reviewed by Fahselt20). Finally, enzymes involved in breakdown of lichen-exogenous polymers, including cellulose and lignin, have been isolated from lichens in nature . These phenomena could be dismissed as exceptions, but their distribution across the fungal symbiont tree hints at deeper underlying fungal capabilities, which if combined with phototroph symbiosis could lead to a kind of \u201chybrid\u201d lifestyle, in which carbohydrates are obtained from multiple sources. The possibility of multiple carbon sources for lichens was even suggested by Schwendener himself in his original 1869 paper, in which he predicted that two tracks of nutrient acquisition would ultimately be proven: one for lichens that have minimal substrate contact, which he predicted to depend mostly on algal assimilates; and one for lichens that closely hug organic substrates such as tree bark or wood1.Fungi are assimilative heterotrophs and thus require a robust machinery of enzymes for scavenging and transporting extracellular nutrients, including carbohydrates. In arbuscular mycorrhizal and ectomycorrhizal fungi, the stable supply of glucose from plants is thought to have led to erosion or loss in many families of carbohydrate-active enzymes (CAZymes)22, it has become possible to infer CAZymes for species for which a genome, but no experimental evidence, is currently available. Comparative genomic overviews of CAZyme repertoires are now available for many symbiotic fungi9, but no survey exists of comparative CAZyme arsenals in LFSs.Unlike for many fungi, phenotypic carbohydrate use profiles have seldom, if ever, been developed for LFSs. This is in part due to the recalcitrance of most LFSs to culturing and their extreme slow growth, if culturing is successful. Knowledge gaps around unculturable or slow-growing fungi are common, but have been offset in recent years by genome sequencing. Coupled with the development of widely available databases such as CAZy23), we hypothesized that LFSs would exhibit functional losses in CAZymes coinciding with the beginning of stable association with phototroph symbionts, similar to what has been found for PCWDEs in arbuscular- and ectomycorrhizal fungi25. To test this, however, we would need to map CAZymes across a well-sampled phylogeny and reconstruct the ancestral states of common ancestors, many of which are considerably older than the reconstructed origins of e.g., ectomycorrhizal fungi26. Here we map the occurrence of genes encoding CAZymes at two levels: across representative species of the Leotiomyceta group of Ascomycota, including the origin of the Lecanoromycetes, the largest extant lineage of LFSs; and among major groups within Lecanoromycetes, including species representing different ecological substrate specificities  as well as major morphological outcomes of the lichens they occur in . Our survey of 46 lecanoromycete genomes, 29 of which we sequenced for this project, reveals a complex pattern of retention and loss that lends support to Schwendener\u2019s hypothesis of hidden saprotrophy in some lichens and is not unequivocally consistent with CAZyme erosion upon acquisition of phototroph symbionts.Given the common assumption that lichen symbiont complementarity confers collective autotrophy on the symbiosis, and past results inferring they evolved from saprotrophic or non-lichen biotrophic ancestors  selected genomes should be representative of a range of CAZyme repertoires, already mapped by Miyauchi et al.9, amongst others; and 2) they should draw primarily from Leotiomyceta, the group that includes the sibling classes of Lecanoromycetes. Because the few published lecanoromycete genomes currently over-represent fungal symbionts of generalist lichens in one taxonomic group within the subclass Lecanoromycetidae\u2014the order Lecanorales\u2014we generated 29 new genomes for this study, with emphasis on capturing both a diversity of substratum specializations as well as diverse lineages of the subclass Ostropomycetidae (sampling explained in greater detail in Methods). Eighteen of the genomes were obtained as metagenome-assembled genomes . To these we assigned activity on the common plant cell wall substrates cellulose, hemicellulose, lignin and pectin following\u00a0Miyauchi et al.9.We assembled a data set of 83 fungal genomes, including 46 from the class Lecanoromycetes , which are involved in glycosylation and the synthesis of polysaccharides, differ little across all analyzed genomes and do not exhibit any significant reduction in Lecanoromycetes, suggesting that a core synthetic machinery remains largely unchanged across the evolution of the sampled Ascomycota Fig.\u00a0. While sAll lecanoromycete genomes possessed genes encoding enzymes predicted to act on plant cell wall compounds, including cellulose and hemicellulose  and lignin AA1, AA2 and AA5). Depending on the symbiont configuration, these organic polymers may be produced by the phototroph partner and/or be exogenous to the lichen symbiosis (see Discussion). Principal components analyses (PCA) of CAZyme family numbers in the sampled genomes reveals differences in the amount of variation in CAZyme composition among lecanoromycete and other sampled ascomycete genomes ; acetylxylan is a major component of hemicellulose. An analysis of predicted GH43 gene sequences and their subcellular location compared to those of characterized enzymes shows that most characterized GH43s are in Ostropomycetidae , but as with GH5s, the majority of sequences are not close to any characterized CAZymes , and several are phylogenetically close to experimentally characterized enzymes from EC 1.10.3.2, which included laccase, p-diphenol:oxygen oxidoreductase and ferroxidase . All three PCWDE groups are reduced already at the split between Eurotiomycetes and Lecanoromycetes  after aligning sequences from all Xylographa species recovered in GH5 Subfamily 5  and barley \u03b2-glucan  but not xylan   Fig.\u00a0. Both exogs Fig.\u00a0. In contive Fig.\u00a0.Fig. 4EnAspergillus and Penicillium. Furthermore, LFSs associated with the same genus of algal phototroph, Trebouxia, can retain multiple genes coding for pectin degradation as well as cellodextrin transporters , or they can largely lack these genes . This implies that association with Trebouxia does not in itself result in gene loss, and the exact nature of the fungal-phototroph interaction, and other aspects of symbiosis biology, may need to be reconsidered. Understanding when and where in the fungal mycelium the CAZymes are deployed will be critical to determining their biological significance.Our analysis of CAZyme and sugar transporter genes paints a picture of a lecanoromycete arsenal that is larger, more diverse and more consistently present than expected. Our data show a reduction in mean numbers of lecanoromycete degradative CAZyme genes relative to other sampled Ascomycota, but also reveal significant differences within Lecanoromycetes. Genes for the breakdown of cellulose, hemicellulose and pectin are disproportionately enriched in the subclass Ostropomycetidae, and some genomes, notably in the OG clade, possess overall CAZyme numbers and functionality similar to those of well-known model saprotrophs such as 32. Cellulases and other CAZymes have been postulated to be involved in haustorial penetration of the algal cell wall34 and degradation of algal cell walls in fresh lichen growth tips35. They could also play a role in digestion of dead algal cell walls, especially if algal populations turn over during the lifespan of the thallus, as has long been suspected36. Transcriptomic studies of isolates, co-cultures and natural lichens could provide evidence of this. In two studies that reported CAZyme differential expression in symbiont coculture experiments, one detected upregulation of multifunctional GHs that could also be involved in fungal cell wall modification (GH2 and GH12), but none of the core lecanoromycete cellulases or hemicellulases we report here37, while another reported upregulation of gene models assigned to GH5, GH43 and GH4538 . Most common algal symbionts are thought to contain cellulose in their cell walls but no pectin39; it is unclear if any lichen algal symbiont possesses pectin in its cell walls.The largest differences in CAZyme gene numbers among the sampled lecanoromycete genomes represent PCWDEs. This raises a central question: what are the targets of LFS PCWDEs? The two most obvious candidates are the phototroph itself, on the one hand, and lichen-exogenous polysaccharides, such as wood and tree bark, or even non-plant polymers, on the other. The first possibility \u2014 targeting of the alga \u2014 echoes suggestions in studies of ectomycorrhizal fungi, where PCWDEs have been postulated to play a role in \u201ccell softening\u201d or \u201cremodeling\u201d of root tissues in their vascular plant symbionts upon contact initiation41 and absence42 of cellulose-containing algal photosymbionts. Cellulase production furthermore has been reported to vary depending on the species of tree it is growing on43. Some lichens have also been experimentally shown to possess an enzymatic and extracellular redox cycling toolbox consistent with lignin modification45. The second line of evidence relates to the ecological substratum use and relationship to known saprotrophs. The enrichment of PCWDEs we found tracks closely with the secondary origin of non-lichen saprotrophs and, among lichens, those with wood-obligate ecology. Several secondary origins of putative fungal saprotrophs occur within the Lecanoromycetes, some of which include fungi involved in \u201coptional lichens\u201d, in which the fungus can occur either as an LFS or saprotroph46. The most speciose of these saprotroph groups, the Ostropales, arose within our \u201cOG clade\u201d that possesses the largest PCWDE enrichment of all sampled lecanoromycete genomes. We also found cellulase gene family expansions in LFSs of wood-obligate lichens such as Ptychographa and Xylographa, and our experimental evidence for cellulase activity derives from one of these genomes (Xylographa bjoerkii).The second possibility, that LFS PCWDEs target lichen-exogenous polysaccharides, is supported by two lines of evidence. The first is experimental. Both cellulases and polygalacturonases, which are active on pectins, have been detected in incubated whole lichens both in the presence47. Determining where and when these genes are expressed in nature will require consideration of the life cycle and spatial extent of LFS mycelia, both of which are still poorly understood. The mycelia of sexually reproducing LFSs go through an aposymbiotic stage of unknown length, during which it has been suggested they may be saprotrophic48. Lichens at high latitudes or under snowpack may go through seasonal fluctuations in fixed carbon input, which could be augmented by other sources21. Even after symbiosis is established, parts of the mycelium can be free of phototroph cells, and exhibit deviations in carbohydrates that suggest other metabolic processes are in effect than in the phototroph-associated mycelium49. Many lichens include a phototroph-free \u201cprothallus\u201d, in which fungal hyphae radiate beyond the zone of phototroph cells50. Others possess a \u201chypothallus\u201d, a phototroph-free cushion of mycelium in direct contact with the substrate49. Macrolichens are often anchored onto their substrates by phototroph-free \u201choldfasts\u201d, \u201crhizines\u201d or mycelial pegs which have traditionally been interpreted as having an exclusively structural, stabilizing function, but can extend as mycelial networks into xylem51 or living moss mats42. Parts of the mycelium with phototroph cells may also differ based on position relative to growth tips, which are thought to include larger proportions of living cells52, and entire strata can be phototroph-free. We currently lack data on the role CAZymes could play in nutrient scavenging or cell wall remodeling in these phototroph-free sectors of the symbiont mycelium.Enzymatic targeting of algal cell walls or lichen-exogenous polymers are not mutually exclusive possibilities. Nor do all exogenous substrates necessarily have to be plant cell walls, as lichen symbionts also interact with a wide range of other fungi, notably fungal parasites and bacteria53, as well as a variety of metal-containing oxidoreductases including laccase and heme peroxidases that are induced by starvation and treatment with soluble cellulose and lignin breakdown products44. Specifically, experimental work has shown that LsaPOX, a putative heme peroxidase from the fungal symbiont of the lichen Leptogium saturninum, can metabolize the \u03b2-O-4 lignin model dimer adlerol into veratraldehyde, a reaction also performed by white-rot fungi45  \u2014 with CAZyme arsenals as large or larger than those of many saprotrophs \u2014 shows that LFSs do not necessarily lack CAZyme arsenals. Despite their minority representation in our data set, CAZyme-rich LFSs may in fact be numerous in nature: the Ostropales and Gyalectales which make up the OG clade include over 3200 named LFS species, almost 17% of named LFSs54, in addition to approximately 500 named non-LFS fungi, mostly saprotrophs. For phototroph acquisition to have no evolutionary consequences for the CAZyme arsenal, symbiont-derived photosynthates would likely have to provide a function other than as a substrate for growth and respiration. In fact this has already long been postulated, in the form of a role for polyols as compatible solutes55. Some have even suggested this may be the main role for transferred photosynthates57. A further indication that photosynthates are not solely nutritional subsidies could be the retention of invertases in Lecanoromycetes. In ectomycorrhizal fungi, the loss of invertases is thought to limit their ability to access plant sucrose and reinforce their dependence on plant-derived glucose9. Though the orthologs we found did not exhibit high similarity to characterized sequences, the prediction of an invertase is consistent with the ability to culture LFSs on sucrose58 and experimental evidence of invertases in lichen fungi59.Did stable phototroph association coincide with the loss of PCWDEs in LFSs? Looking only at the reductions in mean CAZyme gene numbers, the answer would appear to be positive, but the occurrence of LFSs in the OG clade  and Arthonia (Arthoniomycetes). Both genomes echo the lack of predicted cellobiose transporters found within numerous Lecanoromycetes, but differ in their predicted pectin degradation capacity, with Arthonia possessing gene numbers similar to those found in the Ostropomycetidae. Arthonia also possesses higher numbers of non-CAZyme peroxidases, similar to the OG clade. If the OG clade CAZyme arsenals are related to saprotrophy, similar CAZyme arsenals could be expected in lichen fungal symbionts e.g., from the Dothideomycetes, which have long been postulated to have dual lifestyles as mutualists and saprotrophs60. Within Lecanoromycetes, the most striking apparent functional losses, both in terms of CAZyme genes as well as in cellodextrin and cellobiose transporters, occurred outside of the Ostropomycetidae, especially in the subclass Lecanoromycetidae, which largely lacks these two types of transporters. LFSs in Lecanoromycetidae, like those in Ostropomycetidae, are considered obligate symbionts of algae or cyanobacteria. They share many of the same algal symbionts, especially Trebouxia, and have no known lifecycle differences. However, they differ in general thallus architecture. Ostropomycetidae almost exclusively form crustose thalli in which thallus-to-substrate surface area contact is maximized. Lecanoromycetidae, and to some extent Acarosporomycetidae and Umbilicariomycetidae, include LFSs involved both in crustose thalli as well as so-called macrolichens, in which the thallus often becomes greatly expanded into leaf-, hair- or shrub-like forms, in which surface contact is minimized. Lecanoromycetidae macrolichens include some of the largest lichens by biomass, and in theory these would require more carbon, not less. So what additional adaptational processes could drive CAZyme loss?Lecanoromycetes, the focus of our study, are the largest extant group of LFSs. A fuller picture of CAZyme arsenal retention during lichen symbioses and especially in pairing with specific phototroph partners will emerge as more genomic data become available from other fungal classes. Two additional LFS genomes were included in the outgroups of our dataset, 61. The exact consequence of this is unknown, but intracellular haustoria are characteristic of pathogenic fungi and may require a greater variety of CAZymes to penetrate the algal cell wall. In the second, macrolichens owe their architecture to a well-developed exopolysaccharide gel, termed cortex, which forms a rigid structural scaffold considered a prerequisite to macrolichen formation62. The polysaccharide composition of this layer varies widely across lichen symbioses63 and is poorly characterized and mapped across the phylogenetic tree. Additional evolution in cortex layering happened in many lichens involving Lecanoromycetidae64, though in how many remains unclear. The cortex, which is hydrophilic, also mediates water retention63 and has been shown to operate like a sponge for passive uptake of dissolved nutrients65 and glucose66. One of the largest epiphytic macrolichens has been experimentally demonstrated to take up tree-derived glucose67. Whether this is specifically facilitated by the cortex remains to be tested, but as the cortex mediates the passage of environmental molecules for many macrolichens, it seems likely. If capture of simple sugars is found to be a general function of the cortex in different environments, it could be expected to have significant evolutionary consequences for maintenance of CAZyme genes used for more costly carbohydrates.Two basic biological traits surrounding the crust-to-macrolichen transition deserve more attention with respect to their potential effect on sugar uptake. In the first, the type of fungal-algal contact differs, from intracellular haustoria in many crust lichens to so-called intraparietal haustoria, which do not breach the cell wall, in some crusts and all macrolichens2 fixation. The finding that some LFSs have CAZyme arsenals on par with saprotrophs lends support to Schwendener\u2019s hypothesis that different types of lichens exist: those with fungal symbionts that depend mostly on their phototroph, and those with fungal symbionts\u00a0that augment their carbon assimilation from external sources1. The longstanding assumption that LFSs solely utilize fixed carbon must now be weighed against competing hypotheses, including A) that some or many LFSs build their mycelia from non-algal carbon sources, including absorbed monosaccharides and complex polysaccharides, before or during symbiosis; and B) that many different models of carbon acquisition\u2014fixed, seasonal, facultative, scavenged and/or absorbed\u2014may exist under the umbrella of what we currently call lichen symbiosis.The existence of a robust carbohydrate breakdown machinery across a large swathe of LFS evolution calls into question the assumption, underlying decades of ecophysiological work on lichens, that the lichen carbon economy is exclusively the sum of algal COExtended Materials and Methods as well as Supplementary Figures and Tables are available as Supplementary Information. All used software is listed in Supplementary Table\u00a0Cladonia, Evernia, Lobaria, Peltigera, Pseudevernia, Pseudocyphellaria, Ramalina, Sticta, Umbilicaria); 2) LFS lineages involved in crust-forming lichens for which carbon acquisition from the substrate has been postulated, including wood specialists , bark specialists , specialists of decaying plant matter , mineral soil (Dibaeis), rock  and a lichen-on-lichen \u201cparasite\u201d (Lambiella); 3) LFS lineages from crust-forming lichens that behave as ecological generalists ; and 4) a lecanoromycete saprotroph (Agyrium) and endophyte (Cyanodermella68). Our sampling simultaneously represents a cross-section of lecanoromycete evolution, with 24 genomes from the subclass Ostropomycetidae, 17 from the subclass Lecanoromycetidae, and four and one each from the species-poor subclasses Acarosporomycetidae and Umbilicariomycetidae, respectively. Ten genomes were derived from cultured samples and 18 are metagenome-assembled genomes (MAGs) assembled and binned according to Tagirdzhanova et al.69. Culture-derived genomes differed little from MAGs in estimated completeness and gene numbers  LFSs of macrolichens  and Pfam (v33.1) as well as eggnog-scanner searches against EggNOG (v4.5.1) databases.https://github.com/reslp/phylociraptor). We ran BUSCO 3.0.2 on each genome assembly to identify single copy-orthologous, combined amino-acid sequences of each BUSCO gene from all genomes. Only genes which were present in >50% of genomes were aligned using mafft 7.464 and trimmed using trimal 1.4.1. We calculated single-gene trees using iqtree 2.0.7 and a species tree using ASTRAL 5.7.1. We created a concatenated alignment from all alignments, estimated the best substitution model for each and calculated a tree based on a partitioned analysis of the concatenated alignment using IQ-Tree 2.0.7. We used the concatenated phylogeny to generate an ultrametric tree using r8s 1.81. This tree was used for subsequent analyses. We used custom python and R scripts to plot phylogenomic trees.Phylogenomic trees were calculated using the phylociraptor pipeline (25 (Supplementary Table\u00a0http://peroxibase.toulouse.inra.fr/) using Orthofinder 2.5.2.We selected sets of CAZyme families involved in (hemi-)cellulose, pectin and lignin degradation based on previous studiesWe reconstructed the ancestral size of each CAZyme family using CAZyme counts from our genome annotations and our ultrametric phylogenomic tree in R. We used anc.ML from phytools (v0.7\u201370) under an Ornstein-Uhlenbeck model of trait evolution. We then visualize ancestral states with custom R scripts.We calculated phylogenetically corrected PCAs in R as implemented in the phytools function phyl.pca using maximum-likelihood optimization and regular PCAs using the prcomp function . We ran CAFE twenty times while accounting for different gene-birth rates and error-models and summarized the results using custom R and python scripts.CAZyme families known to contain important degradative enzymes  were further characterized using Saccharis v1 . Based on sequence similarity, we selected two candidate cellulase sequences from Xylographa bjoerkii for testing cellulolytic activity.Sequences assigned to GH5 subfamily 5, with confirmed cellulolytic activity, from obligately wood inhabiting LFS Enzyme activity of the two candidate cellulases A and B was tested by combining in a 1.5\u2009mL microcentrifuge tube 50\u2009\u00b5L enzyme; 100\u2009\u00b5L buffer A at either pH 4, 5, or 6; and 50\u2009\u00b5L either AZCL-HE-cellulose, AZCL-\u03b2-glucan, or AZCL-xylan. Tubes were incubated at 4, 20, 37, 50, or 60\u2009\u00b0C for 48\u2009h. To measure activity, samples were centrifuged at 13,000\u2009rpm for 5\u2009min to settle any debris, then 100\u2009\u00b5L supernatant was removed to a 96-well flat-bottom plate. Absorbance at 595\u2009nm was measured by plate reader and blanked with a sample containing water instead of enzyme.http://pfam.xfam.org/family/sugar_tr; accessed August 5, 2021) combined with characterized sugar transporter sequences from the PF00083 seed set. Additionally, we added characterized cellodextrin , S8AIR7 ) and sugar alcohol transporters . We used the presence of characterized sequences in the inferred orthogroups to identify orthologs in each genome included in this study. The number of orthologs of different transporters per genome were visualized using custom R scripts.To identify putative sugar- and sugar-alcohol transporters we used Orthofinder 2.5.2 on all sequences from all genomes with Pfam annotation PF00083  against the predicted proteins in all 83 genomes studied here. We then extracted all predicted protein sequences which had a diamond hit to any of the downloaded sequences. Similar to the identification of sugar transporters we used Orthofinder 2.5.2 to classify putative class II peroxidases present in our genomes based on the presence of downloaded genes in individual orthogroups. The number of orthologs per genome for different peroxidases were visualized using custom R scripts.Further information on research design is available in the\u00a0Supplementary InformationReporting SummaryPeer Review FileDescription of Additional Supplementary FilesSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3"}
+{"text": "All human infants acquire language, but their brains do not know which language/s to prepare for. This observation suggests that there are fundamental components of the speech signal that contribute to building a language system, and fundamental neural processing mechanisms that use these components, which are shared across languages. Equally, disorders of language acquisition are found across all languages, with the most prevalent being developmental language disorder , where oral language comprehension and production is atypical, and developmental dyslexia , where written language acquisition is atypical. Recent advances in auditory neuroscience, along with advances in modelling the speech signal from an amplitude modulation  perspective, have increased our understanding of both language acquisition and these developmental disorders. Speech rhythm patterns turn out to be fundamental to both sensory and neural linguistic processing. The rhythmic routines typical of childcare in many cultures, the parental practice of singing lullabies to infants, and the ubiquitous presence of BabyTalk (infant-directed speech) all enhance the fundamental AM components that contribute to building a linguistic brain. All human cultures have also invented music. A fundamental feature of both language and music is rhythmic structure. In music, it is more obvious to the listener that there are different levels of rhythmic patterning nested within the overall beat rate; however, this is also the case in human languages. In essence, speech involves patterns of strong and weak beats that recur periodically in hierarchical structures. This patterning is referred to as metre or prosody and syllable stress. Recent computational modelling suggests that the infant brain initially relies on this rhythmic patterning when building a language system . In this. 2Over three decades ago, cross-language behavioural research showed that newborn infants could discriminate languages from different rhythm classes (stress-timed versus syllable-timed), and it was proposed that rhythm discrimination could be a cross-language precursor of language acquisition . Around More recently, experimental studies have explored musical behaviours in newborn infants. These studies have revealed sensitivity to different musical beat structures at birth, with systematic rhythmic movement to music measurable by five months of age ,7. IndeeIn contrast to the phoneme-dominant view, a purely acoustic analysis of the speech signal reveals that \u2018phonemes\u2019 are an abstraction from the signal itself, and are perceptual categories imposed by the literate listener . If infa. 3a). Children with DD also have robust and profound difficulties hearing both speech and non-speech acoustic rhythm [Disorders of language acquisition carry severe developmental costs. The two most prevalent disorders are DD and DLD . Children with DD appear to acquire spoken language with ease, but show difficulties once tuition in reading begins. Despite apparently normal hearing and normal intellectual functioning, they fail to develop age-appropriate reading skills, and these difficulties persist even with intensive remediation . A wealtfigure 2c rhythm .Figure Children with DLD have persistent difficulties with learning oral language that cannot be explained by a known condition such as sensori-neural hearing loss, and are typically characterized as having difficulties with the accurate processing and production of grammatical structures in speech . For exab, using a phrase from the popular children's story book \u2018Room on the Broom\u2019, by Julia Donaldson. Accordingly, accurate sensory learning and neural representation of these acoustic rhythmic hierarchies may be crucial to developing syntax and grammar. Children with DLD also have robust and profound difficulties in hearing acoustic rhythm.Again, new psychoacoustic understandings may throw light on these developmental patterns. All human languages use prosodic phrasing (tightly integrated hierarchies of metre and syntax) to highlight the grammatical structure of language . This isfigure 2et al. [The investigation of rhythm perception and production in children with DLD and DD using matched experimental tasks is revealing. The simplest example of a rhythmic behaviour is tapping or clapping in time with a regular beat. Corriveau & Goswami  asked chet al.  showed iet al. .et al. [et al. [et al. [On the perceptual side, regarding DLD Cumming et al.  reported [et al.  reported [et al. ,39 reporUsing younger controls when studying children with atypical development is important experimentally, to help equate for the effects of learning on the developing brain. In principle, younger language-age-matched or reading-level-matched children provide a way of controlling for the effects of experiencing oral or written language. These younger children have reached the same developmental level of oral or written language learning as the children with DLD or DD. Further, rhythm is a multi-modal percept. Visual rhythm cues given by head and cheek movements synchronized to the act of articulation, as well as motor knowledge of how to prepare the articulators to speak, all converge with acoustic rhythmic information to aid language comprehension . Speech . 4c). The speech spectrogram depicts the raw sound wave (shown in figure 3b) in terms of changes in frequency (pitch) over time. The speech envelope contains a range of AM patterns at different temporal rates which are found across the frequencies depicted in the spectrogram, as do many other biological sounds like wind and rain . The doanguages . The abia) reveals the core spectro-temporal modulation patterning nested in the speech envelope in each speech genre. To achieve this, the speech signal is filtered into the same bands imposed by the human cochlea, and modulation structure is then estimated using a principal components analysis approach. This reveals three core AM bands in both IDS and CDS spanning five core pitch bandings. Modulation structure is computed using key modulation statistics such as AM maxima (peaks in bands) and oscillatory phase relationships (rhythmic synchronicity) between different bands of AMs. This \u2018spectral-amplitude modulation phase hierarchy\u2019 (S-AMPH) modelling approach reveals that rhythmic patterning in IDS and CDS is represented in the phase relations of slower AM bands in the amplitude envelope, particularly those corresponding temporally to \u2018brain waves\u2019 in the delta (\u223c2 Hz) and theta (\u223c5 Hz) bands in electroencephalography (EEG). EEG records brain electrical activity, measuring the intrinsic rhythms of cell signalling in the brain, and the same rhythms  are found across the cortex. The acoustic AM phase synchronization for AM bands centred on approximately 2 Hz and approximately 5 Hz in IDS is significantly stronger in IDS than in speech directed to adults, and these AM bands are automatically encoded by brain waves in the infant brain [The potential role of the acoustic structure of the amplitude envelope in children's linguistic learning can be investigated by separating the AM characteristics of speech from the frequency modulation (FM) characteristics. This is achieved by acoustic engineering methods for decomposing the amplitude envelope (demodulation) . The AM figure 3nt brain . Furthernt brain ,48. Intent brain ,48. The nt brain ,49. Anotnt brain . As a synt brain . Not allnt brain . These rnt brain  (most Ennt brain  (to divint brain . If the nt brain ). Cross-. 5et al. [In the last 20 years, the potential role of amplitude envelope perception in the \u2018phonological deficit\u2019 characterizing dyslexia across languages has been investigated intensively by myself and my colleagues . et al. ; see [66et al. ,68.Other studies have assessed sensitivity to AM directly by using temporal modulation transfer functions (TMTFs). TMTFs assess the minimum depth of AM (variation in intensity) that can be detected by a listener. Studies with French DD children demonstrate reduced discrimination of AM at a range of temporal rates, with the largest difference at 4 Hz ,70. Englb), as these are also AM hierarchies [Amplitude rise time discrimination is also impaired in children with DLD, although to date only English-speaking children have been tested \u201374. Althfigure 2rarchies . The perrarchies . Johnson. 6neural function regarding encoding the speech signal. Speech encoding relies in part on matching the rhythmic changes in electrical brain potentials in large cortical cell networks  to temporally matching rhythmic energy modulations (AMs) in speech. The functional organization of the bands of neuroelectric oscillations responsive to speech inputs (primarily the brain rhythms at delta (0.5\u20134 Hz), theta (4\u20138 Hz), beta (12\u201320 Hz) and gamma (35+ Hz)) is also hierarchical [automatic alignment of neuroelectric oscillations to the AM information hierarchy in speech [Recent data from auditory neuroscience studies with adults show that rise times/auditory edges also have an important archical , and thearchical . Rise tiarchical ,79 . Succesarchical . Accordi), beta 1\u201320 Hz anboth younger reading-level controls and chronological age-matched controls, even when sentence recognition accuracy was matched across groups [et al. [Studies of this speech\u2013brain alignment in children with DD suggest that the alignment process is disrupted in dyslexia, particularly in the delta band (0.5\u20134 Hz). When listening to noise-vocoded sentences, the accuracy of speech\u2013brain oscillatory alignment in the delta band (0\u20132 Hz) was found to be significantly reduced for DD children compared to s groups . Despite [et al. . Childre [et al. ,85. This [et al. ). NevertRegarding rhythm production, if children with DD are trained to tap to every second pulse of a 2.4 Hz metronome beat so that their synchronization performance is no less variable than control children, their brains still show a phase difference at 2.4 Hz when EEG is recorded . This phrising phase of the delta oscillation, which would be expected if sensory performance is enhanced at the oscillatory peak (more neurons are active at the peak), the DD participants showed no relationship between oscillatory phase and behaviour. This suggests that reaction time in the DD adults was not governed by oscillatory phase in delta, and hence that they were using other sensory strategies to succeed in the task. Finally, although cross-language neural data are sparse, the sentence listening task has also been used with Spanish children with DD [EEG studies with highly remediated adults with DD have also been conducted. In a study when a rhythmic tone stream was delivered at 2 Hz, phase entrainment was significantly reduced in DD compared to control adults . The par with DD . The aut with DD . This is with DD . This is. 7To capture these diverse data from psychoacoustic, neural, behavioural and speech modelling studies, a \u2018temporal sampling\u2019 (TS) framework for language acquisition can be proposed ,19,22,28surfing the sound waves. For a successful surfer, the temporal accuracy and prediction of this alignment is key to catching the peak of the wave. TS theory proposes that the brains of typically developing infant surfers make accurate judgements about the peaks of these waves, but that the brains of infants at family risk for DD and DLD do not. This alignment process is automatic; it is part of language acquisition for every baby, but for infants with less accurate rise time discrimination, the automatic alignment is out of time at one or more temporal rates . Oral language processing is atypical in both DD and DLD because these automatic brain processes are working slightly differently, although this is easier to demonstrate experimentally in perception than in production. Oral timing studies with DD adults enable the expected speech production differences to be calibrated [A possible theoretical instantiation of TS theory is provided as librated ; to datelibrated , and manIndeed, although children with DD appear to comprehend and produce spoken language without notable differences from other children, closer investigation shows that this is not the case. Their performance in phonological awareness tasks and their word finding difficulties provide experimental examples . PerceivBy contrast, children with DLD clearly do not comprehend and produce language as other children do. Their difficulties in perceiving some of the energy changes in the speech signal \u201374 lead Finally, it may be possible to adapt the speech signal in light of our discoveries about the AM hierarchy, so that the amplitude envelope cues in speech that are perceived poorly by children with DD and DLD are synthetically amplified or exaggerated. Envelope enhancement algorithms are already being applied in DD, with measurable benefits for hearing speech-in-noise ,94. A cl"}
+{"text": "Obstructive sleep apnea (OSA) and obesity are associated with stress system activation involving the hypothalamic-pituitary-adrenal (HPA) axis in adults, but these effects in childhood and adolescence remain unclear. We examined diurnal salivary cortisol as a measurement of the HPA axis function in obese adolescents with and without OSA and the relationships between cortisol levels, body weight, and parameters of polysomnography (PSG).After PSG, saliva samples were collected from obese participants (with and without OSA) and lean participants four times over a 24-h period, namely, at 7:00 h (m-sCort), 13:00 h (a-sCort), 19:00 h (e-sCort), and 23:00 h (n-sCort). An enzyme-linked immunosorbent assay (ELISA) was used to measure salivary cortisol levels. The mean values of cortisol levels and fixed-time point diurnal cortisol slope (DCS) were calculated and compared among the three study groups. Correlations between parameters were analyzed using Spearman's correlation coefficients.2) and time with SpO2 <90%. Moreover, in the obese OSA group, DCS was significantly flatter than in the other two groups. The a-sCort in obese non-OSA participants was significantly higher than that in the lean control group and, surprisingly, was positively correlated with the apnea/hypopnea index. Additionally, m-sCort was related to body weight.Obese OSA participants had significantly higher e-sCort and n-sCort levels than both obese non-OSA participants and lean controls. However, m-sCort and a-sCort in these patients had a pronounced upward trend. M-sCort was significantly correlated with both the lowest oxygen saturation (SpOThis study provided further evidence for alterations in diurnal cortisol production in obese adolescents, which may indicate a chronically stressed HPA axis. However, there were significant differences in salivary cortisol parameters between participants with and without OSA. Furthermore, patients with OSA had more associations between time-point cortisol levels and OSA-related indices. Nonetheless, this research is a pilot study, and further investigations are necessary. Sleep is an important part of our lives. It is essential for all functions of an organism . Altered sleep is potentially harmful to human health and may result in perturbations in cardiovascular, metabolic, neurocognitive, immune, and endocrine functions. It is known that diurnal changes in human physiology, including hormone production, are driven by the endogenous circadian timing system (ECTS), which is composed of the suprachiasmatic nucleus of the hypothalamus and circadian oscillators in peripheral tissues. ECTS enables a consolidated period of wakefulness during the day as well as consolidated sleep during the night . Under aNotably, cortisol is an important neuroendocrine biomarker, which is present in all bodily fluids, including urine, serum, and saliva. Although measurements of serum cortisol have traditionally been used as an indicator of the HPA axis activity in clinical and scientific studies, even a small amount of stress, such as venipuncture, can increase hormone levels . MoreoveObstructive sleep apnea (OSA) and obesity are associated with the activation of the HPA axis. OSA is a common sleep disorder characterized by snoring, obstruction of the upper airways during sleep, intermittent nocturnal hypoxia, and sleep fragmentation, resulting in significant health consequences. OSA affects at least 2% of adolescents globally . Among pThe aim of our study was to evaluate diurnal salivary cortisol as a measure of the HPA axis activity, in obese adolescents with and without OSA, and determine the relationships between cortisol levels, body weight, and the parameters of polysomnography (PSG). We hypothesized that the circadian rhythm of salivary cortisol is different in these patients. Furthermore, we expected that obese adolescents with OSA would show HPA axis hyperactivity and have more significant correlations between PSG indices and circadian cortisol levels compared with those of obese patients without OSA.In this study, we recruited adolescents who were referred to the Clinic of the Scientific Center for Family Health and Human Reproduction Problems between September 2018 and December 2020 for overweight and obesity management and normal-weight age-matched controls from the community. We screened 99 adolescents (72 boys with obesity and 27 lean peers) against the inclusion and exclusion criteria and enrolled 84 participants (63 obese boys and 21 lean boys) in this cross-sectional study. Of those, four adolescents were lost at follow-up. Our final sample for the future study and analysis included 80 adolescents comprised of 60 obese adolescents and 20 lean control participants.Eligible participants met the following criteria: male sex, age 15\u201317 years, body mass index (BMI) \u226595th percentile for obese patients and BMI in the range between 5th and 85th percentile for lean participants, no intake of sleep-promoting pills, the performance of usual activities, no stress for at least 1 week before the measurement period, and signed informed consent. Exclusion criteria included neuromuscular diseases and craniofacial anomalies, positive airway pressure therapy (PAP), and unwillingness to participate in the study. Basic characteristics were obtained for all subjects, and a clinical examination was performed.The participants completed the Adolescent Sleep Habits Survey, which was adapted for Russian schoolchildren , to asseAll subjects completed the salivary cortisol portion and PSG of this study. The assignment of obese subjects to subgroups was based on the PSG results: 33 adolescents were included in the OSA obese group and 27 adolescents without OSA were age-matched as obese controls. OSA was identified if the apnea/hypopnea index (AHI) was \u22652 number/h . All 20 2). BMI-for-age percentiles were computed using the Centers for Disease Control and Prevention (CDC) reference values (2\u201320 years)  . Obesity2). Sleep stages and associated events were scored according to the American Academy of Sleep Medicine (AASM) scoring rules : electroencephalogram, oculogram, submental and tibial electromyogram, electrocardiogram, chest and abdominal respiratory movements, oronasal airflow, body position, and blood oxygen saturation , 13:00, 19:00, and 23:00 h . Continuous variables comparisons between the obese OSA group, obese controls, and lean controls were performed using analysis of variance (ANOVA) or the non-parametric Mann\u2013Whitney (M\u2013W) All procedures performed in this study involving human participants were in accordance with the ethical standards of the national research committee and with the 1964 Helsinki Declaration and its later amendments. This study was reviewed and approved by the Committee on Biomedical Ethics of the Scientific Center for Family Health and Human Reproduction Problems . All adolescents and their parents or legal guardians provided written informed consent to participate in this study.p < 0.0001 and 0.003, respectively) but did not differ between obese groups (the obese with OSA group and obese controls). According to the sleep self-assessment, OSA symptoms had a significant proportion of obese respondents with OSA than obese without OSA adolescents and more than the lean control group (there were two persons with daytime sleepiness). Slightly more than half of the obese respondents with OSA snored. Obese respondents without OSA also snored, but significantly less than the obese participants with OSA . Lean adolescents were self-confessed \u201cnonsnorers.\u201d Breathing pauses during sleep were self-reported by only 11 apneic subjects. There was a significant difference between the obese with OSA group and obese controls , as well as between obese participants and lean controls  with respect to daytime sleepiness.The baseline characteristics of the study participants are shown in p = 0.001), lower average and minimal SpO2 , higher RAI (p = 0.003), more time spent in superficial sleep (p = 0.01), and less time spent in both deep (p = 0.06), and REM sleep (p = 0.03) than the obese without OSA group and lean controls. Almost all PSG parameters in the obese non-OSA and lean controls were not significantly different, except for the snore and the number of respiratory arousals .Polysomnography data of the three groups are summarized in p = 0.215, respectively), although this measurement had a pronounced upward trend. However, e-sCort and n-sCort in obese with OSA participants were significantly higher compared with those in the lean control group  as well as to those with obesity but without OSA . There were also significant differences between a-sCort levels in the obese with OSA group and the lean control group . The a-sCort in obese non-OSA participants was significantly higher than that in lean controls  but was similar to that in the obese with OSA group.As shown in p = 031, respectively), which is clearly identified in An interesting component of the circadian rhythm of cortisol is the diurnal cortisol slope (DCS). This measurement is the degree of change in cortisol concentrations from morning to evening during wakefulness. In our study, we calculated fixed-time point slopes, in which samples were gathered . We usedSpearman's rank correlation analysis was performed to assess correlations of m-sCort, a-sCor, e-sCort, and n-sCort levels with BMI and sleep indices in obese adolescents with and without OSA. The results are shown in Rs =0.476; p = 0.046) in obese non-OSA participants, and with the lowest SpO2 , and time with SpO2 <90%  in obese patients with OSA. Interestingly, a-sCort was significantly positively correlated with AHI in the obese non-OSA group. Of note, salivary evening cortisol, as well as night cortisol, levels were significantly correlated with PSG parameters such as SWS  for both AHI and N1\u2013N2  in obese OSA adolescents. Finally, circadian salivary cortisol levels did not correlate significantly with the other studied PSG characteristics such as RAI, TST, and REM sleep in any of the groups.Salivary morning cortisol was found to correlate significantly with BMI-SDS  but negative correlations with lowest SpO2 (m-sCort) in obese OSA youth. These data suggest that circadian cortisol levels can be predictors of OSA severity, and this requires further research.Similar to the results in adult studies, we found highly contradictory data regarding the circadian cortisol rhythm in children and youth. Some characteristics are known to influence cortisol concentrations  and may explain the discrepancies in cortisol levels observed in these studies. Malakasioti et al. , Park etThe attempts to establish a direct connection between obesity/metabolic syndrome (MS) and cortisol excretion brought contradictory results , 32. So,Notably, adolescence  is a time of important physical, hormonal, and psychological changes , and daiIt is known that flattened slopes of cortisol secretion, which exhibits suppressed morning peak levels or failures to reach sufficiently low levels by evening, are indicative of the HPA axis dysregulation . They mavice versa in adolescence and to evaluate the effect of PAP therapy on diurnal cortisol levels in this population.The data from the pilot study provided more pronounced diurnal cortisol alternations in apneic obese male adolescents than in those without OSA. Furthermore, patients with OSA had more associations between time-point cortisol levels and OSA-related indices. Our preliminary findings are consistent with previous reports of HPA axis hyperactivity in adolescents with OSA. Reported modulations of circadian cortisol rhythmicity associated with OSA and obesity may underlie some of the above-mentioned negative health outcomes. At present, evaluation of circadian secretion of free cortisol appears to be promising for the detection of subtle alterations of the HPA axis in conditions, such as obesity and OSA, in daily clinical practice, whereas dynamic tests following stimulation with different substances and psychological stress challenges or suppression with inhibiting agents of the HPA axis at different levels requires the use of specific clinical settings and special patient preparation. Salivary cortisol offers a suitable and easily obtainable measure for assessing diurnal cortisol alternations in children and adolescents and can therefore aid in further advancing understanding in this area, as well as to enhance diagnostic, prognostic, and treatment possibilities in obesity and OSA. Further studies are necessary to elucidate how cortisol metabolism is involved in the pathogenesis of both obesity-induced OSA and This was a pilot study; therefore, it had several limitations. The major limitation was the small sample size, potentially limiting our power to uncover more associations and predictors in the study groups. We believed that increasing the sample size in our future research will allow us to strengthen the study because we can divide participants according to both OSA and obesity severity and consider a general linear model to evaluate the possible effect of OSA severity, as well as obesity severity, and the comorbidity of OSA with obesity on circadian cortisol concentration. A second limitation of this study was that only men were assessed, which eliminated the gender effect, but the study results can only be applied to male adolescents, which also requires future research to study the associations between cortisol and OSA, body weight, and sex. The last limitation was using BMI-for-age percentiles but not a full assessment of body composition to classify subjects in the study, potentially increasing classification errors. We can revise the methodology in our future research.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Committee on Biomedical Ethics of the Scientific Center for Family Health and Human Reproduction Problems. Written informed consent to participate in this study was provided by the participants and their legal guardian/next of kin.IM and LR: conceptualization and design. IM and LS: methodology. OB and SB: investigation and formal analysis. OB, IM, and LS: data curation. OB: writing original draft. OB, IM, SB, LS, and LR: writing\u2013review and editing. LR: supervision. All authors agree to be accountable for the content of the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "No correlation was present with any severity outcomes in diabetic patients without AH . All of the New-onset Diabetes patients  had AH, and 12 had severe COVID-19; additionally, five patients were admitted to the ICU and three patients died. However, severity outcomes did not reach statistical correlation significance in this group. In nondiabetic patients with AH , there was a statistically significant association with the need for oxygen therapy (p = 0.001), invasive mechanical ventilation (p = 0.01), and ICU admission (p = 0.03). Our results support data regarding the impact of AH on severity outcomes. It also suggests an effect of AH on the prognosis of COVID-19 inpatients, regardless of the presence of pre-existing diabetes or new-onset diabetes. We reinforce the importance to assess at admission glycemia in all patients admitted with COVID-19.Diabetes mellitus (DM) has emerged as a major risk factor for COVID-19 severity and SARS-CoV-2 infection can worsen glycemic control and may precipitate new-onset diabetes. At-admission hyperglycemia (AH) is a known predictor for worse outcomes in many diseases and seems to have a similar effect in COVID-19 patients. In this study, we aimed to assess the impact of AH regardless of pre-existing diabetes mellitus and new-onset diabetes diagnosis in the clinical severity of COVID-19 inpatients in the first months of the pandemic. A retrospective monocentric study on 374 COVID-19 inpatients  was developed to assess associations between AH  and severity outcomes  and mortality) in patients with and without diabetes. Considering diabetic patients with AH  there was a correlation with COVID-19 severity ( The first cases of pneumonia caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) were identified in December 2019. After the rapid spread of this virus, on 11 March 2020, World Health Organization (WHO) declared COVID-19 a global pandemic .Several health conditions have been associated with a worse prognosis of COVID-19; diabetes mellitus had emerged as a major comorbidity for COVID-19 severity ,5. In onSARS-CoV-2 infection may worsen glycemic control and may precipitate new-onset diabetes. The at-admission glycemia is a known predictor of critical illness and worse outcomes in many diseases; the same seems to apply to COVID-19 patients ,10,11,12CORONADO study (Coronavirus SARS-CoV-2 and diabetes Outcomes) was a French nationwide multicenter observational study and one of the first to investigate the relationship between diabetes and COVID-19. They found an association between at-admission plasma glucose levels and the severity of COVID-19 .Hyperglycemia occurs secondarily to the physiological response to stress hormones released during acute illness and also to metabolic dysregulations that impair insulin signaling . HyperglThe present study was developed in a central Portuguese hospital dedicated to COVID-19, during the first months of the pandemic. In this study, we try to clarify the importance of hyperglycemia as a potential prognostic factor for COVID-19 inpatients, both with and without diabetes.We conducted a retrospective study on COVID-19 adult patients admitted to a medical ward  of Centro Hospitalar Universit\u00e1rio Lisboa Central (CHULC), a tertiary hospital, from 1 March 2020 to 31 October 2020. During the referred period, 374 patients were admitted to the ward.All patients had been tested for SARS-CoV-2 by real-time polymerase chain reaction at admission in the emergency room. The medical care provided to each patient was selected for their clinical context, considering the clinical judgment and protocols provided by the Portuguese Directorate General of Health. This study has not influenced the type of care provided to the patients.We extracted patients\u2019 demographic and clinical data from electronic medical records: age, sex, gender, previous diabetes diagnosis, and blood glucose level at admission , HbA1c measured during hospitalization, disease severity, and clinical outcomes. The disease severity was classified according to the national standards .The exclusion criteria in this study were: (1) Patients who were not confirmed by a positive result of SARS-CoV-2 detection in respiratory specimens by the reverse transcriptase polymerase chain reaction assay; (2) Patients with COVID-19 diagnosed only during hospital staying and not in the emergency room (3) Patients younger than 18 years old; (4) Patients without blood glucose measurement in the first 24 h after admission.Considering these exclusion criteria and since we had no data from admission blood glucose in 14 patients (4 patients with previous DM and 10 patients without this diagnosis), only the 360 patients with admission blood glucose evaluated were considered in the present study.The clinical outcomes analyzed included clinical severity, the need for oxygen therapy, Invasive Mechanical Ventilation (IMV), admission to the ICU, and mortality.To analyze and compare clinical severity and outcomes, we considered three groups of patients : patientThe Ethical Committee from Centro Hospitalar Universit\u00e1rio Lisboa Central (Comiss\u00e3o de \u00c9tica para a Sa\u00fade) approved this study  and waived patients\u2019 Informed Consent due to the retrospective characteristics of the present study and anonymous data used.The case definitions of confirmed human infection with SARS-CoV-2 were under interim guidance from the WHO directives. We considered the presence of previous diabetes when the diagnosis of the disease and/or antidiabetic therapy was referred to in the patient\u2019s medical record. New-onset diabetes was assumed in cases without a previous diagnosis and a measured HbA1c equal to or higher than 6.5% at any time during hospitalization. Absolute hyperglycemia at hospital admission was defined as any blood glucose level greater than 140 mg/dL, within the first 24 h after hospital admission or in the emergency department presentation, according to the American Diabetes Association guidelines and the joint recommendations of the Portuguese Society of Diabetology and the Portuguese Society of Internal Medicine on the management and treatment of hyperglycemia in hospital inpatients ,19. The p < 0.05 (2-tailed) were considered results statistically significant. Data analysis was performed using IBM SPSS\u00ae Statistics, version 23.Descriptive statistics included means and standard deviations or median and interquartile range (IQR) for continuous variables  and frequencies and percentages for the categorical ones. Normal distribution was checked using the Shapiro-Wilk test or skewness and kurtosis. Categorical variables were compared with the \u03c72 method and continuous variables were compared using the Spearman correlation test. The association of AH with different outcomes was quantified as odds ratios (OR) that were adjusted using multiple logistic regression analyses with 95% Cis. Results with During the period considered, 374 patients with COVID-19 were admitted to the ward, but admission blood glucose was evaluated in only 360 patients admitted. AH occurred in 119 patients (33.1%). In this subgroup of patients, 57.1% (n = 68) had diabetes mellitus previously diagnosed and 12.6% (n = 15) had new-onset diabetes with two of them presenting with diabetic ketoacidosis.The demographic and clinical characteristics of each subgroup are shown in p < 0.001). In hyperglycemic patients with diabetes, the median HbA1c was 7.5% .HbA1c was measured in 116 patients . The median HbA1c was 7.5% (IQR 6.2\u20139.3) in patients with diabetes and 5.9% (IQR 5.6\u20136.5%) in patients without diabetes  and 15.8% (n = 39) of patients without the diagnosis. All of the fifteen patients with new-onset diabetes had hyperglycemia when admitted to the hospital. Admission blood glucose was higher in the groups of diabetic patients with at-admission hyperglycemia and the group with new-onset diabetes, with a median value of 211 mg/dL (IQR 168-269.8) and 203 (IQR 156\u2013291) respectively.In all groups, the majority of patients were Caucasian. Patients with new onset diabetes were younger, with a median age of 58 years old (IQR 43\u201378). At-admission blood glucose and HbA1c were higher in the group of diabetic patients with AH.Clinical outcomes were analyzed according to the presence of AH or AN  and accop < 0.001), to be submitted to IMV (p < 0.001), to be admitted to the ICU (p = 0.002), or to present more severe presentations of the disease (p < 0.001). The mortality rate among patients was 18.3%, however, the presence of AH did not show a statistical correlation with this outcome.Compared with those without, patients presenting at-admission hyperglycemia were more likely to need oxygen therapy  of the respective column. In this table, we cannot include the patients without admission blood glucose measured (n = 14). p = 0.001). The likelihood for patients to be submitted to IMV was far greater in those presenting with AH in the group with a previous diabetes diagnosis as well as in those without diabetes . The same was observed for ICU admission with a higher probability of that need among patients presenting with AH in those two groups: 9.4% vs. 70.6% (p = 0.026) in the group with a previous diabetes diagnosis and 30.6% vs. 69.4% (p = 0.03) in those without diabetes. According to the presence or absence of AH, no significant difference was found in the risk of death in any of the groups considered.The presence of AH was a predictive factor for the need for oxygen therapy in patients without diabetes .We found AH to be a risk factor for higher severity of the disease among patients with a previous diagnosis of diabetes: critical disease in 30.9% of patients (n = 21) severe disease in 36.8% (n = 25), moderate disease in 9.6% (n = 6), mild disease in 14.7% (n = 10), and 8.8% (n = 6) being asymptomatic , invasive mechanical ventilation  and ICU admission .The results of this study support the hypothesis that there is a direct impact of AH on the clinical outcomes of COVID-19, namely clinical severity, need for respiratory support (oxygen supply or IMV) or ICU admission. For most clinical outcomes, the impact of AH occurs regardless of a previous diagnosis of diabetes. In some cases, the impact of AH on clinical outcomes can be stronger than the presence of diabetes per se. Thus, our results support previous data regarding the impact of AH on clinical outcomes of SARS-CoV-2 infection.Before this global pandemic, substantial evidence was already suggesting that patients with diabetes or AH could have worse outcomes in community-acquired pneumonia. Some recent studies showed similar results concerning COVID-19 ,20. HoweShi Q et al., found a higher proportion of ICU admission (17.6%) and more fatal cases (20.3%) of COVID-19 in patients with diabetes.  A retrosIn a large retrospective study, with COVID-19 inpatients from hospitals in Hubei province, it was observed that patients with diabetes needed more frequent oxygen therapy (76.9% versus 61.2%) and IMV (3.6% versus 0.7%) compared to those without diabetes. A higher mortality rate in patients with diabetes (7.8% versus 2.7%) was also observed . HoweverThere is evidence also suggesting an increased risk for severe illness and mortality in critically and noncritically ill patients with COVID-19 who present with hyperglycemia  ,23,24,25A pooled analysis and meta-summary of literature described that SARS-CoV-2 infected patients who presented with raised blood glucose levels had an approximately threefold increased risk of mortality . In a muOur study has also revealed that patients with AH had a higher risk of having a severe or critical clinical presentation compared with normoglycemic patients. The same pattern of comparison was verified for other clinical outcomes such as oxygen therapy (74.8% vs. 54.4%), IMV (28.6% vs. 11.6%) or ICU admission (30.3% vs. 14.9%) ,20,27. CWe have observed that the presence of AH was a risk factor for worse outcomes in patients regardless of the presence or absence of a previous diagnosis of diabetes. However, opposite to other studies, we did not find those statistical associations in patients with new-onset diabetes maybe because of the sample size of this subgroup (15 patients) ,29.Our investigation revealed a higher proportion of patients with no previous diagnosis of diabetes but with AH who needed oxygen supply, IMV, and ICU admission. Concerning patients with a previous diagnosis of diabetes, AH showed to be a risk factor for the need for IMV, ICU admission, and a COVID-19 clinical severity. These findings suggest that stress hyperglycemia could play a role in the prognosis of patients hospitalized with COVID-19 regardless of the previous diagnosis of diabetes.In addition to glycemia measured in the first 24 h after hospital admission, some studies investigated the association between other glycemic variables, such as fasting blood glucose, HbA1c or glycemic fluctuations during the hospital stay, with health outcomes related to COVID-19 ,32,33,34Zhang et al., studied the outcomes of 166 COVID-19 inpatients in one hospital in Wuhan. After adjustment for confounding variables, the odds ratio for composite outcomes  was 5.47 (95% CI 1.56\u201319.82) for those with secondary hyperglycemia  and 2.61 (95% CI 0.86\u20137.88) for those with diabetes  .A Chinese study with 548 COVID-19 inpatients, (18% with diabetes) suggested that patients who had higher mean levels of glucose during their first week of hospitalization were more likely to have a more prolonged hospital stay and greater risks for severe pneumonia, acute respiratory distress syndrome (ARDS) and death . SimilarFrom a pathophysiologic perspective, the release of stress hormones is known to be the main component of the general adaptation to critical illness. However, stress hormone response results in persistent insulin resistance and hyperglycemia and this directly favors SARS-CoV-2 replication in human monocytes . AccordiOur study was conducted in one of the most important hospitals in Portugal managing COVID-19 inpatients during the peak of the pandemic. The results reinforce the strong association found between hyperglycemia and in-hospital worse outcomes in patients with COVID-19, regardless of the presence of previous diabetes. It also reinforces the importance to evaluate blood glucose levels in all patients and to strengthen blood glucose control in patients with diabetes to reduce the severity of SARS-CoV-2 infection . The AmeSome studies have adopted a higher cut-off to consider hyperglycemia to study the impact on clinical outcomes in patients with diabetes. In our study, we used the same definition of AH in patients with and without diabetes, as suggested by ADA. Once we considered and evaluated only the effect of AH, we excluded the potential influence of steroid-induced hyperglycemia. Different criteria have been used to stratify COVID-19 clinical severity; in this study we considered national clinical standards to apply the classification. However, in other studies, different criteria do not seem to change the interpretation of results .As shown in the Methods, we conducted the present retrospective study on all COVID-19 patients admitted to a medical ward between 1 March 2020 and 31 October 2020. This means that we included in our study only patients with COVID-19 who did not receive any vaccination. We believe that studying a wider group during a longer period of time was not the ideal approach for the aims of the present study, and other confounders would have been introduced, including the different schemes of vaccinations. Indeed, one of the strengths of the present study is the inclusion of patients during the first hit of SARS-CoV-2 infection, which can reveal better the impact of this coronavirus on clinical outcomes. We also need to mention that there was a delay in the preparation of the article and subsequent publication due to the pending approval by the local Ethics Committee.There are some limitations that should be mentioned. First, a relatively small sample size possibly limits the statistical power of analyses, and it is possible that several observed trends would reach a statistical significance in a larger sample. Comorbidities such as hypertension, cardiovascular disease, chronic kidney, liver disease, and the presence of chronic diabetic complications were not taken into consideration for the results of our study. Since the vaccines were still not available in the referred period, we cannot exclude the impact of this potential confounder.At-admission glycemia is an easy and inexpensive parameter to assess in all inpatients infected with SARS-CoV-2 . It can"}
+{"text": "Higher educational attainment is observationally associated with lower risk of Alzheimer\u2019s disease. However, the biological mechanisms underpinning this association remain unclear. The protective effect of education on Alzheimer\u2019s disease may be mediated via increased brain reserve. We used two-sample Mendelian randomization to explore putative causal relationships between educational attainment, structural brain reserve as proxied by MRI phenotypes and Alzheimer\u2019s disease.n = 1 131 881), late-onset Alzheimer\u2019s disease  and 15 measures of grey or white matter macro- or micro-structure derived from structural or diffusion MRI (nmax = 33 211). We conducted univariable Mendelian randomization analyses to investigate bidirectional associations between (i) educational attainment and Alzheimer\u2019s disease; (ii) educational attainment and imaging-derived phenotypes; and (iii) imaging-derived phenotypes and Alzheimer\u2019s disease. Multivariable Mendelian randomization was used to assess whether brain structure phenotypes mediated the effect of education on Alzheimer\u2019s disease risk.Summary statistics were obtained from genome-wide association studies of educational attainment . There were positive associations between genetically predicted educational attainment and four cortical metrics : surface area 0.30 ; volume 0.29 ; intrinsic curvature 0.18 ; local gyrification index 0.21 ]; and inverse associations with cortical intracellular volume fraction [\u22120.09 ] and white matter hyperintensities volume [\u22120.14 ]. Genetically proxied levels of surface area, cortical volume and intrinsic curvature were positively associated with educational attainment . We found no evidence of associations between genetically predicted imaging-derived phenotypes and Alzheimer\u2019s disease. The inverse association of genetically predicted educational attainment with Alzheimer\u2019s disease did not attenuate after adjusting for imaging-derived phenotypes in multivariable analyses.Our results provide support for a protective causal effect of educational attainment on Alzheimer\u2019s disease risk, as well as potential bidirectional causal relationships between education and brain macro- and micro-structure. However, we did not find evidence that these structural markers affect risk of Alzheimer\u2019s disease. The protective effect of education on Alzheimer\u2019s disease may be mediated via other measures of brain reserve not included in the present study, or by alternative mechanisms. et al. provide evidence that education leads to changes in brain structure: greater brain volume, surface area and curvature. However, the protective effects of education against Alzheimer's disease do not appear to be mediated by these effects on brain structure.Seyedsalehi  A potential mechanism by which higher educational attainment (EA) protects against risk of Alzheimer\u2019s is through increasing or maintaining the underlying \u2018brain reserve\u2019 of the individual.5 Brain reserve refers to individual differences in the anatomical and structural characteristics of the brain that enable some individuals to preserve their cognitive and functional status despite neuropathology.9\u201311 The progression of amyloid-\u03b2 and tau pathology in Alzheimer\u2019s disease is associated with several structural alterations in the brain, including progressive cortical thinning,12\u201315 widespread grey matter atrophy in cortical and subcortical regions17 and damage to the integrity and organization of white matter tracts.18\u201321 Many of these structural characteristics can be measured in vivo using structural or diffusion MRI, and thus their pre-morbid levels may serve as a useful proxy for the structural basis of brain reserve capacity.23 Consistent with the brain reserve hypothesis, observational neuroimaging studies of cognitively intact older adults have provided evidence for an association of education with several MRI measures of brain macro- and micro-structure that are implicated in Alzheimer\u2019s disease. For instance, higher education levels have been associated with increased whole-brain and regional grey matter volume,24\u201326 cortical thickness27\u201331 and increased surface areas of sub-regions of the hippocampus and amygdala that are vulnerable to Alzheimer\u2019s disease pathology.32 Similar findings have been reported for the association between education levels and white matter micro-structure in cognitively intact elderly, with more highly educated individuals showing increased white matter tract integrity in several brain regions that are characteristic sites of Alzheimer\u2019s pathology.33Despite a large body of observational epidemiological evidence supporting education as a protective factor for Alzheimer\u2019s disease,34 For instance, the association of education with brain structural metrics may be confounded by early-life factors that are common predictors of both EA and brain structure, such as childhood socioeconomic status,36 birthweight,37\u201341 childhood cognitive ability42 and maternal smoking during pregnancy.43 Confounding may also affect the relationship between MRI markers of structural brain reserve and Alzheimer\u2019s risk. Several of the established risk factors for Alzheimer\u2019s disease and other dementias  have been shown to be associated with various aspects of brain macro- and micro-structure in community-dwelling populations.44\u201355 Therefore, it may be that these factors, rather than poor pre-morbid structural brain health, underlie the increase in Alzheimer\u2019s disease risk. In addition, even if the observed associations reflect causal relationships, the direction of causality (i.e. from the exposure to the outcome or vice versa) cannot be definitively established using observational methods.34 For instance, the observational association between education and brain structure could arise as a result of longer duration of education causing a change in brain structural metrics, or alternatively, individuals with better structural brain health  seeking longer durations of education (or a combination of both). Reverse causation could also affect the association between MRI markers of brain reserve and Alzheimer\u2019s disease. Specifically, while greater structural brain reserve (as proxied by MRI metrics) may confer protection against advancing neuropathology and risk of an Alzheimer\u2019s diagnosis, it is also possible that changes in these structural markers occur in response to pre-clinical Alzheimer\u2019s disease .Given the observed associations between education and structural markers of brain reserve, it is plausible to hypothesize that changes in brain structure may mediate the protective effect of education on Alzheimer\u2019s disease risk, through determining the underlying brain reserve of the individual. However, interpreting these observational associations as evidence of causal relationships between education, brain structure and Alzheimer\u2019s risk relies on several untestable and potentially implausible assumptions, including the absence of residual and/or unmeasured confounding and reverse causation.56\u201358 can be a useful approach for assessing putative causal relationships between education, brain structure and Alzheimer\u2019s disease, as it addresses some of the common limitations of classical epidemiological studies (e.g. confounding and reverse causation). By using genetic variants that are specifically associated with a putative exposure as instrumental variables, MR can be used to make inferences about the causal effect of an exposure on an outcome.60 Due to the random assortment of alleles at conception, the distribution of genetic variants that are associated with a particular exposure is largely independent of factors that confound exposure\u2013outcome associations in conventional observational analyses.61\u201363 Therefore, estimates from MR are less affected by environmental confounders, and can provide more reliable insights into causal relationships between risk factors and disease outcomes than classical epidemiological studies. In addition, given that the genotype of an individual is determined at conception and cannot be modified by subsequent disease outcomes, the direction of causation will always be from the genetic variant to the trait of interest, eliminating the potential for reverse causation.34 Therefore, MR can be particularly useful for obtaining reliable causal inferences in retrospective settings (e.g. in case-control studies) where genetic variants are measured after the occurrence of the disease outcome.64 For instance, while MRI markers of structural brain reserve cannot be reliably measured in individuals with Alzheimer\u2019s disease (as the disease is likely to distort their measurement in cases), genetic variants associated with brain structure cannot be modified by the disease event and can therefore be used as a reliable proxy for pre-morbid brain structure in individuals with Alzheimer\u2019s disease.Mendelian randomization (MR)65\u201369 However, the mechanisms underlying the protective effect of education remain relatively unexplored. Recent advances in genotyping and multi-modal neuroimaging technologies have facilitated the collection of these data in large-scale prospective cohort studies,70 enabling the discovery of genetic variants associated with brain structure using considerably larger sample sizes than was previously possible.71\u201374 The present study aimed to expand on previous work by using MR to investigate putative causal relationships between EA, brain structure as measured by MRI and risk of Alzheimer\u2019s disease. In addition, we aimed to explore whether there was any evidence for the hypothesis that brain structural alterations lie on the causal pathway from education to Alzheimer\u2019s disease (i.e. the brain reserve hypothesis). We proxied structural brain reserve using imaging markers of grey and white matter macro- and micro-structure derived from structural or diffusion MRI (see Methods for the description and choice of these imaging-derived phenotypes). Our primary objectives were: (i) to replicate, using the latest genome-wide association study (GWAS) data, the previous MR findings supporting a protective causal effect of EA on Alzheimer\u2019s disease risk; (ii) to assess whether EA has a causal effect on brain macro- and/or micro-structure; (iii) to assess whether brain macro- and/or micro-structure phenotypes causally affect risk of Alzheimer\u2019s disease; and (iv) to assess whether the protective effect of EA on Alzheimer\u2019s disease risk is mediated via changes to brain macro- and/or micro-structure, using multivariable MR (an extension to univariable MR which can be used to investigate mediating relationships).76 A directed acyclic graph illustrating the putative causal effects explored in this study is presented in 77In recent years, a growing number of MR investigations have found evidence for an inverse association of genetically predicted EA with Alzheimer\u2019s disease risk.79 To minimize the possibility of confounding due to population stratification, all data sources were restricted to individuals of European ancestry. The GWAS used to obtain the summary statistics for each phenotype are listed in The present study is based on a two-sample MR analysis strategy, an extension of MR in which the effects of the genetic instruments on the exposure and the outcome are obtained from two separate datasets.et al.80 EA was defined as the number of years of schooling (YOS) completed, measured at an age of at least 30 years. Genetic variant association estimates with Alzheimer\u2019s disease were taken from the largest available GWAS meta-analysis of clinically diagnosed late-onset Alzheimer\u2019s disease (onset age > 65 years), as conducted by the International Genomics of Alzheimer\u2019s Project.81 Summary statistics for the imaging-derived phenotypes were obtained from three separate data sources based on the early-2020 release of combined genetic and multi-modal brain imaging data from the UK Biobank.83 Although several thousand imaging-derived phenotypes can be generated from UK Biobank MRI data, many are highly inter-correlated, and analysis of all imaging-derived phenotypes could lead to spurious results or challenges in controlling for multiple comparisons. Our strategy was to focus on 15 imaging-derived phenotypes  or total brain volume, as these were all global measures and would be highly correlated with whole-brain volume. However, for the localized phenotypes , we used genetic association estimates that were corrected for ICV. All genetic association estimates were taken from GWAS conducted on the combined discovery and replication cohorts.Summary-level genetic association estimates with EA were obtained from a GWAS meta-analysis of \u223c1.1 million individuals by Lee enotypes , most of release . GeneticP < 5 \u00d7 10\u22128) as instrumental variables. The resulting instruments were pruned to near-independence using a linkage disequilibrium (LD) threshold of r2 < 0.001 over 10 000 kilobase pairs. We used the LD reference panel for the European super-population in the 1000 Genomes Project reference dataset, which was restricted to bi-allelic SNPs with minor allele frequency >0.01. We extracted the following summary-level data from each exposure and outcome GWAS: SNP rs number, effect and other alleles, effect allele frequency, sample size, number of cases/controls (if applicable), standardized \u03b2-coefficients, standard errors and P-values. Where an instrument SNP was not available in the outcome dataset, we used LDlink88 to identify a proxy SNP in LD with the target SNP (r2 > 0.8). The exposure and outcome GWAS datasets were then harmonized to ensure that the genetic variant association estimates corresponded to the effect of the same allele. We used allele frequency information to infer the orientation of alleles in the exposure and outcome GWAS. Palindromic SNPs with minor allele frequency >0.42 were dropped from the analysis.89 Details of the SNPs used as instruments in each MR analysis (including proxies for SNPs not available in the outcome datasets) are available in the 90 (https://sb452.shinyapps.io/power/) to estimate the minimum causal effect that we had 80% power to detect at a multiple-testing-corrected significance threshold (see the \u2018Correction for multiple testing\u2019 section later). Power calculations are presented in For each MR analysis, we selected all single nucleotide polymorphisms (SNPs) associated with the exposure of interest at a genome-wide significance threshold  MR92 with multiplicative random-effects as the primary analysis method, as it provides the most efficient combination of the variant-specific ratio estimates91\u201393 and accounts for heterogeneity in the causal estimates obtained from individual variants.94We performed univariable MR analyses to estimate each of the following total causal effects: (i) EA on Alzheimer\u2019s disease; (ii) EA on each of the 15 imaging-derived phenotypes; and (iii) imaging-derived phenotypes on Alzheimer\u2019s disease. To examine the direction of association and investigate the possibility of reverse causation, we additionally estimated each of these causal effects in the direction opposite to that initially hypothesized .et al.68 Given the correlation structure between the various MRI metrics considered, applying a simple Bonferroni correction could be overly conservative. We therefore used a principal component analysis approach (https://github.com/hagax8/independent_tests) to estimate the number of independent hypotheses tested from the matrix of squared phenotypic correlations between the imaging-derived phenotypes /2 (=78), and the square root of T (=8.83) was used to establish the final number of individual imaging-derived phenotypes to correct for. Bidirectional associations between EA and imaging-derived phenotypes and between imaging-derived phenotypes and Alzheimer\u2019s disease were therefore considered significant at a multiple-testing corrected threshold of P < 0.006 (=0.05/8.83). For testing bidirectional causal relationships between EA and Alzheimer\u2019s disease, we set statistical significance at P < 0.05.To account for the number of MR analyses performed to test for bidirectional effects of education on brain structure and brain structure on Alzheimer\u2019s disease, we applied the multiple-testing correction approach described by Lord enotypes . We firs95 Given that each of the objectives of interest were distinct , we applied the multiple-testing correction to the number of hypotheses within each objective.Note that the purpose of the multiple-testing correction was to control the family-wise error rate within each objective (i)\u2013(iv), rather than across the total number of univariable MR analyses performed (i.e. 62). Controlling the family-wise error rate across all 62 tests would be appropriate if we were interested in the global null hypothesis .96 Weighted Median MR,97 MR-Egger98 and the MR pleiotropy residual sum and outlier (MR-PRESSO) method.99 These four robust methods were selected since they each produce a valid estimate of the causal effect of the exposure on the outcome under different assumptions  to be included as instruments in the analysis, and can be used to dissect the total causal effect of the exposure on the outcome into an indirect effect via the mediator, and a direct effect of the exposure on the outcome not via the mediator  effect of the exposure on the outcome can then be estimated by subtracting the direct effect from the total effect.108 This broadly mirrors the difference-in-coefficients method used in traditional non-instrumental variable regression-based approaches to mediation.109We performed multivariable MR analyses108 For each multivariable MR analysis, we selected all SNPs associated with the primary exposure (EA) or the mediator (imaging-derived phenotype of interest) as instrumental variables. The pooled set of SNPs were clumped to pairwise LD (r2 < 0.001) over 10 000 kilobase pairs, on the basis of the lowest P-value for association with any of the two traits. The extraction of summary-level data from the Alzheimer\u2019s disease GWAS, harmonization of the exposure and outcome datasets, and identification of proxies for missing SNPs, followed the same procedure as univariable MR, as described before.Using this approach, we estimated the direct causal effect of EA on Alzheimer\u2019s disease risk, while adjusting for each of the 15 imaging-derived phenotypes in turn. A difference between the estimate of the total causal effect of EA on Alzheimer\u2019s disease (from univariable MR) and the direct causal effect estimate (from the multivariable MR model including an imaging-derived phenotype) would indicate a mediating role of that brain structure phenotype on the causal pathway from education to Alzheimer\u2019s disease.110 We used the \u2018TwoSampleMR\u2019 package89 (v.0.5.6) for data extraction and harmonization, the \u2018ieugwasr\u2019 package (https://mrcieu.github.io/ieugwasr/index.html) for clumping and the \u2018LDlink\u2019 package88 (v.1.1.2.9) for extracting LD proxies. All MR analyses were carried out using the \u2018MendelianRandomization\u2019111 (v.0.5.1) and \u2018MRPRESSO\u201999 (v.1.0) packages.All statistical analyses were performed in R software .The present study is a secondary analysis of publicly available data. Ethical approval was granted for each of the original GWAS studies, the details of which can be found in the respective publications.https://portal.ide-cam.org.uk. Individual-level imaging and genetic data used for the in-house GWAS analyses may be requested through the UK Biobank (https://www.ukbiobank.ac.uk/), and the code used in generating the imaging phenotypes is available on GitHub (https://github.com/ucam-department-of-psychiatry/UKB). Summary-level genetic association estimates with all other phenotypes were obtained from publicly available published GWAS, and can be accessed from the respective publications. R code for reproducing all MR analyses can be found on https://github.com/as2970/EA_brain_AD_MR.Summary statistics from the in-house GWAS of cortical macro- and micro-structure are available at There was strong evidence for an inverse association between genetically proxied EA and Alzheimer\u2019s disease across all MR methods . The resQ statistic provided no evidence that there was more heterogeneity in the variant-specific causal estimates than expected due to chance .In the opposite direction, as expected, there was no association between genetically predicted Alzheimer\u2019s disease and years of education. The IVW estimate (representing the SD change in YOS per doubling the odds of Alzheimer\u2019s disease) was 0.00  and the total volume of white matter hyperintensities . The results of the pleiotropy robust methods were broadly consistent with the IVW analyses  were positively associated with EA . The IVWQ statistic, we found evidence of heterogeneity in all three analyses , 95% CI \u22120.03, \u22120.01], increased white matter mean diffusivity  and reduced hippocampal volume in both hemispheres . For left and right hippocampal volume, all robust methods provided similar estimates to the IVW analysis  more broadly. We have focused on EA as the exposure of interest as it is a potentially modifiable factor, and has been suggested to be \u2018the most consistent, robust and durable method yet to be identified for raising intelligence levels\u2019.117 Indeed, a large meta-analysis of quasi-experimental studies estimated every additional year of education to be associated with an increase of \u223c1\u20135 standardized intelligence quotient points,117 a result that was consistent across the lifespan and across broad categories of cognitive ability. If, as previous multivariable MR evidence suggests, intelligence lies on the causal pathway from education to Alzheimer\u2019s disease, increasing the number of YOS could still be an effective public health strategy for reducing the incidence of Alzheimer\u2019s disease, even if the protective effect of higher EA is entirely mediated via increased intelligence levels. Findings from a quasi-experimental study suggest that increasing the school-leaving age in the UK in 1972 has led to improvements in intelligence quotient as well as a range of health and well-being outcomes, including reduced risks of diabetes and all-cause mortality.37 Our results indicate that education policy reforms that target the length of compulsory schooling are also likely to be effective for the primary prevention of Alzheimer\u2019s disease at the population level.69Our finding of a protective causal effect of education on Alzheimer\u2019s disease risk is consistent with the large body of observational epidemiological literature.32 as well as evidence for positive genetic correlations between education and MRI markers of cortical macro-structure.118 To investigate the direction of causation in these associations, we performed sensitivity analyses using Steiger filtering where we excluded any SNPs that explained more variance in the outcome than in the exposure. Although the magnitude of the effect size attenuated in all analyses following the removal of these SNPs, all causal effect estimates remained statistically significant. We note that when a bidirectional MR analysis suggests causal effects in both directions, it is difficult to settle the question of whether these truly represent reciprocal causality or may reflect shared aetiology, whereby the genetic variants used as instrumental variables in both analyses predict a common cause of both traits. Thus, while all our sensitivity analyses suggest distinct causal effects in both directions between EA and these macro-structural markers, the shared aetiology explanation is also possible. We also found evidence for causal effects of EA on local gyrification index and on the total volume of white matter hyperintensities. However, when we removed variants that explained more variance in the outcome than in the exposure, these effects were no longer statistically significant, suggesting that they may be explained by reverse causation or horizontal pleiotropy. As white matter hyperintensities mainly develop in elderly individuals,87 reverse causation is unlikely in this case and the association with EA probably reflects horizontal pleiotropy.We found evidence for bidirectional causal relationships between EA and three global macro-structural markers of cortical morphology, namely, volume, surface area and intrinsic curvature. These findings are in agreement with the existing observational neuroimaging literature on the association between EA and brain morphology,119 Grey matter volume shows marked changes across the lifespan, increasing substantially along with cortical surface expansion in early childhood121 and decreasing through adulthood with reductions in cortical thickness and subcortical volumes.122\u2013124 ICV, on the other hand, increases until mid-to-late adolescence, with little if any changes observed after,125 and may therefore serve as a proxy for maximal neuroanatomical volume.119 If education was associated with enhanced brain maturation in childhood/adolescence, we would expect years of education to be strongly related to ICV. If, however, education had a neuroprotective effect in ageing, we would expect a specific association between higher EA and grey matter volume in adulthood/ageing, after controlling for ICV. The study found consistent positive associations between education and ICV in both development and adulthood, but there was no evidence that education was related to ICV-adjusted grey matter volumes in ageing cohorts,119 indicating a primarily developmental effect of education that is established in early life rather than a protective effect against neurodegeneration in ageing.119 This finding is also compatible with recent observational evidence on the association between education and rates of cognitive/brain ageing over the course of adulthood and old age. For instance, a longitudinal neuroimaging study of >2000 individuals found no evidence of an association between levels of education and longitudinal rates of volume loss in atrophy-prone cortical regions or in the hippocampus.29 Similarly, a recent review that examined the effects of EA on cognitive ageing126 concluded that while there was strong evidence of a positive association between education and level of cognitive functioning, associations between education and the rate of ageing-associated cognitive decline were negligible. Overall, these findings are more consistent with the account that the effects of education on brain structure and cognitive function are established primarily during neurodevelopment in childhood/adolescence, and are largely preserved into old age rather than being grounded in neuroprotective effects on cognitive/brain ageing.126 In other words, individuals with higher EA might have an initial advantage in terms of cognitive/brain reserve that persists through their adulthood/ageing. It is this advantage, rather than attenuated longitudinal changes in brain structure and cognition, which reduces the risk of being diagnosed with Alzheimer\u2019s disease in more highly educated individuals (i.e. an intercept effect of education across the lifespan rather than an effect on the slope/rate of change).126The observed causal effect of EA on these macro-structural metrics may arise from the beneficial effects of education on neurodevelopment in childhood/adolescence, and/or from its protective effects on neurodegeneration in adulthood/ageing. As we have used GWAS of brain structure measured cross-sectionally in middle to old age, we cannot prioritize either of the two hypotheses in the current study. However, there are several observational studies that have attempted to distinguish between the effects of education on neurodevelopment and neurodegeneration. For instance, a recent study addressed this question by examining the association of EA with total grey matter volume and ICV in both developmental and adult cohorts.32 We only present analyses in the main paper based on cross-sectional brain imaging data, and it is possible that analyses based on longitudinal changes may differ\u2014see 127 Cognitive reserve is a dynamic and active process of adaptation, and is typically measured using behavioural testing or functional neuroimaging modalities (e.g. resting state or task-related networks of brain activation that moderate the effect of Alzheimer\u2019s pathology on cognition).128 Although the concepts of cognitive and brain reserve are not mutually exclusive and are hypothesized to operate synergistically in moderating the effect of Alzheimer\u2019s pathology on clinical outcome,22 it is possible that the protective effect of education on Alzheimer\u2019s disease risk is largely mediated via increased cognitive reserve.128 Therefore, the use of functional, rather than structural MRI, or cognitive/behavioural phenotypes might be better suited to capturing such mediating effects. Additionally, some of the putative benefits of education on Alzheimer\u2019s risk could be mediated primarily through mechanisms minimally related to brain MRI metrics, such as reduced smoking and improved vascular health5 or via other brain processes (e.g. microscopic or intracellular or vascular) that cannot be captured well by our MRI metrics.Across the 15 cross-sectional imaging-derived phenotypes examined, we did not find any evidence of a causal effect of brain structure on Alzheimer\u2019s disease risk or for a mediating role of these brain structure phenotypes on the causal pathway from EA to Alzheimer\u2019s disease. However, we cannot, on the basis of these findings, rule out the possibility that changes in brain structure mediate the protective effect of education on Alzheimer\u2019s risk through determining the underlying brain reserve of the individual. It is possible that capturing the neural basis of brain reserve requires more granular and specific MRI metrics than the ones used in the current study. We reasoned that any putative protective effects of education on Alzheimer\u2019s risk would be unlikely to be manifest only in a highly regionally specific manner, and hence decided to primarily study global rather than localized imaging phenotypes. Nevertheless, the use of global imaging metrics in the current study may have masked any significant regionally specific causal effects of brain macro- or micro-structure on Alzheimer\u2019s disease risk.APOC1 gene region (rs12721046), which has been associated with cerebral amyloid deposition as measured by PET imaging in a recent GWAS.129 The APOC1 gene encodes a member of the apolipoprotein C1 family, which plays a central role in the regulation of lipid levels and metabolism.130 It is located in a cluster on chromosome 19, \u223c5 kilobase pairs downstream from the APOE gene,130 where common genetic variation is known to have a relatively large effect on Alzheimer\u2019s disease risk.132 A recent fine-mapping study of the APOE and surrounding regions using whole-genome sequencing data identified a cluster of risk variants (including rs12721046) in the APOC1 region as potential causal variants for Alzheimer\u2019s disease.133 This cluster of variants were found to confer increased risk of Alzheimer\u2019s independent of the APOE-\u03b54 genotype.133 In all four analyses, the precision of the variant-specific causal estimate for rs12721046 was substantially higher than the remaining instruments, causing this variant to dominate the IVW estimate. When this variant was removed from the analysis, the overall causal effect estimate was attenuated towards the null and was no longer significant. Since all causal effects were only evidenced by the rs12721046 variant, they may represent horizontally pleiotropic pathways from this variant to brain structure. Therefore, we cannot draw definitive conclusions regarding the effect of genetic liability to Alzheimer\u2019s disease on brain structure.In the reverse direction, we found evidence for an association of genetically predicted Alzheimer\u2019s disease with cortical orientation dispersion index, mean diffusivity in the white matter tracts and bilateral hippocampal volumes. However, these results were entirely driven by a single intron variant in the 22 Given the age range of the UK Biobank imaging cohort (45\u201382 years),73 the possibility that some individuals in the imaging GWAS harbour pre-clinical Alzheimer\u2019s disease cannot be excluded. Using both self-reported diagnoses and International Classification of Diseases134 codes, we found that only 0.6% of participants in the UK Biobank have a current Alzheimer\u2019s diagnosis. Therefore, it is unlikely that a large proportion of the imaging GWAS sample had current or imminent Alzheimer\u2019s disease at the time of scanning. Nevertheless, with increasing duration of follow-up, a substantial proportion of these participants may go on to develop Alzheimer\u2019s or other dementias.Our findings should be interpreted in the context of some limitations. First, although we have used imaging-derived phenotypes as markers of structural brain reserve, we cannot determine the extent to which these phenotypes capture pre-morbid brain reserve, as opposed to neuropathological changes secondary to disease (e.g. atrophy secondary to Alzheimer\u2019s disease).135 Although this bias could be avoided by using three non-overlapping datasets from the same underlying population for genetic discovery and the estimation of variant-exposure and variant-outcome associations, restricting our analyses to non-overlapping datasets for the phenotypes examined would have substantially reduced the sample sizes. Therefore, a compromise was necessary to balance the risk of bias against imprecision of the causal estimates.64Second, causal effect estimates in our analyses might be affected by Winner\u2019s curse bias, which occurs when the same dataset is used to select the genetic variants as instrumental variables and to estimate their association with the exposure.91 with the magnitude of bias being a linear function of the degree of overlap between the two samples.136 However, brain imaging is a recent addition to the UK Biobank data collection protocol, and <10% of the UK Biobank cohort contribute to the imaging sample used in the present study. Even if all participants from the UK Biobank imaging sample were included in the EA GWAS, the percentage overlap between the exposure and outcome datasets in the current study would range from 2.8 to 2.9% for the education \u2192 brain structure analyses, and from 4.1 to 4.3% for the brain structure \u2192 education analyses, suggesting that the extent of sample overlap in the current study is minimal. Therefore, it is unlikely that the use of completely non-overlapping samples would have changed the direction or magnitude of our results.Third, although there were no overlapping samples in the analyses of education and brain structure on Alzheimer\u2019s disease, 39% of participants in the EA GWAS and all of those in the brain imaging GWAS were recruited from the UK Biobank. Overlap between the exposure and outcome datasets in two-sample MR can exacerbate bias due to weak instruments,137 In MR, both the exposure and the outcome are causally downstream of the genetic variants and any confounders of the exposure\u2013outcome association . Therefore, if selection into the study sample is a function of the exposure or the outcome, this can introduce an association between the instrumental variable and the confounder138 leading to a violation of the instrumental variable assumptions.139 In the analyses assessing the causal effect of EA on imaging-derived phenotypes, selection into the imaging sample is likely to have been a function of the exposure, as participants in the UK Biobank have higher education levels compared to the UK general population.141 Selection bias could have also occurred in the analyses assessing the causal effect of brain structure on Alzheimer\u2019s disease, as recruitment into the imaging sample may have been a function of the outcome. Since UK Biobank participants are healthier than the general population,142 and the imaging cohort is a subset of those participants who have volunteered to undergo extensive additional assessments,143 it is reasonable to assume that those recruited into the imaging cohort were less likely to have Alzheimer\u2019s disease at the time of scanning compared to the general population (or develop Alzheimer\u2019s disease in the future). However, simulation studies have shown that the impact of selection bias on MR estimates and Type I error rates is only severe when the collider has a particularly large effect on selection,139 which is unlikely to be the case in the examples discussed previously. Therefore, we believe it is unlikely that our findings can be explained solely by selection bias.Fourth, our findings may have been affected by selection bias in the UK Biobank imaging cohort, which occurs when selection into the study sample depends on a collider.142 They have also been shown to have a lower prevalence of obesity, smoking and daily alcohol consumption compared with the UK general population, as well as lower age-adjusted rates of total cancer incidence and all-cause mortality.142 These differences are consistent with the well-established \u2018healthy volunteer effect\u2019,144 which may limit the generalizability of our findings.Fifth, genetic association estimates with several phenotypes were taken from GWAS based on the UK Biobank cohort, which is not representative of the general population with regards to several sociodemographic, lifestyle and health-related characteristics. For instance, UK Biobank participants are more likely to be female, older and living in less socioeconomically deprived areas compared with non-participants.Sixth, to minimize the possibility of confounding due to population stratification, we limited our analyses to participants of European ancestry. Therefore, it remains unclear whether our findings will extrapolate to other populations.145 these are meta-analyses of MRI data from different imaging cohorts. The use of uniform genotyping and neuroimaging protocols in the UK Biobank and the application of consistent genetic and phenotypic quality control pipelines to the data used in the current study is likely to have resulted in lower measurement error and enhanced power, despite the slightly smaller sample size. Nevertheless, statistical power calculations indicated that our analyses may have been underpowered to detect particularly subtle causal effects of brain structure on Alzheimer\u2019s risk (73 As data from more participants become available, replication of these findings using larger and better-powered GWAS of imaging-derived phenotypes will enable us to draw more definitive conclusions regarding putative causal relationships between education, brain structure and Alzheimer\u2019s disease.Finally, the GWAS data sources used for brain imaging phenotypes had relatively small sample sizes. Although larger GWAS of brain structure are available,r\u2019s risk . The imaIn conclusion, our findings add to the extensive body of observational epidemiological literature, as well as evidence from a growing number of MR investigations, providing support for a causally protective role of increased EA on risk of Alzheimer\u2019s disease. In addition, our results support potential bidirectional causal associations between education and several aspects of cortical macro- and micro-structure. However, we found no evidence that these structural alterations have downstream effects on risk of Alzheimer\u2019s or that they lie on the causal pathway from education to Alzheimer\u2019s disease. The observed protective effect of increased EA on risk of Alzheimer\u2019s may be mediated via other structural brain changes not captured by these particular MRI phenotypes or by alternative biological mechanisms.awac392_Supplementary_DataClick here for additional data file."}
+{"text": "Person-centredness is a cornerstone to a palliative approach to care. However, there is a risk that a person-centred perspective is lost in how a palliative approach is evaluated. We explored the extent to which evaluations of a palliative approach are consistent with its person-centred ethical stance. Using a narrative review approach, we critically reflected on how the experiences, priorities and concerns of patients and family are represented, or not represented, in evaluations of a palliative approach. We were guided by the following questions: (1) What types of outcomes and indicators are commonly used to evaluate a palliative approach? (2) Whose perspectives are represented in current evaluations of a palliative approach? And (3) What are the foci of evaluation in this body of research? We observed that the evaluations of a palliative approach are commonly based on indicators of its implementation and predominantly reflect the perspectives of healthcare providers and healthcare systems, rather than patients or family. Although evaluations focused on healthcare providers and systems are important for integrating a palliative approach, there is concern that the essence of person-centredness is lost when the perspectives of patients and families about their healthcare needs, outcomes and experiences are not consistently measured as the ultimate goal of care. There is a need for more emphasis on evaluation practices that value person-centred outcomes, in addition to outcomes oriented to the needs of healthcare providers and systems. The World Health Organization defines palliative care as an approach that improves the quality of life of patients and their families facing the problems associated with life-threatening illness. This is accomplished through the prevention and relief of suffering by means of early identification and comprehensive assessment and treatment of pain and other problems \u2013 physical, psychosocial and spiritual. The WHO further states: \u2018Palliative care is a crucial part of integrated, people-centred health services\u2019.3 Although a palliative approach applies along the entire trajectory of life-limiting illness, from experiencing early symptoms to dying and grief, it is distinct from specialized palliative care. A palliative approach is conceptualized as an upstream orientation towards the needs of people who have life-limiting conditions and their families: it adapts the principles, knowledge and skills of palliative care to all life-limiting illnesses and conditions, and it integrates them into non-specialist palliative care settings.Although palliative care was defined as an approach for all those facing life-limiting illness, the initial populations receiving palliative care were predominantly persons living with malignant disease. Driven by the need to develop accessible high-quality end-of-life care for all who stand to benefit from it, the idea of a more widespread palliative approach to care has been constructed. From the person\u2019s perspective, this person-centred approach involves being listened to, being informed and having a say in one\u2019s care.6 From the perspective of palliative care providers, a person-centred approach has been described as \u2018respectful of, and responsive to, the needs, preferences and values of the person receiving care, their family, and other caregivers\u2019. Communication and the relationship between the person and the healthcare provider, characterized as a partnership, are central to person-centred care.9 Within the healthcare system, person-centred care can be viewed as an ethical stance focused on caring for the individual as a person and promoting \u2018just institutions\u2019 as reflected in the services, organization and evaluation of care.11A palliative approach often melds chronic disease management with a person-centred  approach focused on quality of life for the person and not just the medical care of the disease. there has been a concurrent and increasing need to evaluate its implementation and impact. However, competing agendas influence the need to evaluate a palliative approach at micro-, meso- and macro-levels of the health system. This has led to the development of multiple standardized indicators, metrics and tools for evaluation. At the micro-level, evaluations are predominantly concerned with quality of life, desires and needs of patients and family members as the basis for shared decision-making and care planning. Meso-level considerations emphasize organizational priorities focused on monitoring, evaluating and improving the implementation of a palliative approach in care delivery. At the macro-level, the emphasis is on evaluation of population health considerations including regulation, adherence to policies and measurement of cost-efficiency. As a result of these competing agendas, there is a risk for the person-centred perspective to be lost in evaluations of a palliative approach.Although much work has focused on introducing and integrating a palliative approach into healthcare systems,all people receiving care as well as indicators of care delivery and healthcare system performance. Our research was motivated by the desire to identify to what extent person-centredness is actually represented in how a palliative approach is being evaluated and to critically reflect on these potentially competing perspectives.Given that a palliative approach is to be person-centred, yet integrated within healthcare systems, it is important that evaluations reflect the diverse perspectives of To explore the extent to which evaluations of a palliative approach are consistent with its person-centred ethical stance, we conducted a review of evaluation practices reported in empirical literature. We used the following questions to guide our analysis:(1) What types of outcomes and indicators are being commonly used to evaluate a palliative approach?(2) Whose perspectives are represented in current evaluations of a palliative approach?(3) What are the foci of evaluation in this body of research?The answers to these questions led to our critical reflection on how the experiences, priorities and concerns of patients and family are represented, or not represented, in the outcomes and indicators used to evaluate a palliative approach. We identified relevant manuscripts from our original review on a palliative approach (which only included articles from prior to 2012) and conducted an updated search (2012\u20132019) of the same library databases using the terms \u2018palliative approach\u2019 or \u2018palliative care approach\u2019. The resulting 528 articles were screened to identify those that discuss outcomes or indicators used to evaluate a palliative approach. Consistent with our conceptualization of a palliative approach, we did not include articles that focused only on: (a) the final days or hours of life, (b) the treatment of a particular symptom or (c) specialized palliative care. We included 88 studies focused on adult populations, which were written in English, and those that reported on a primary study . Guided by the above three research questions, we captured information about (a) the outcomes or indicators used to evaluate a palliative approach, (b) the data sources for evaluation  and (c) the intervention or initiative being evaluated. We employed a constant comparative analytical approach to clarify, expand on, refine and organize the initial conceptual groupings we observed, followed by a critical reflective process to ask interpretive questions of what we saw in that body of literature.Considering our objective towards critical reflection rather than systematic documentation, we employed a narrative review approach focused on constructing a \u2018scholarly summary along with interpretation and critique\u2019.Overall, the 88 articles revealed 3 overarching categories of different indicators and outcomes: (a) healthcare provider outcomes regarding interventions to prepare them for delivering a palliative approach, (b) indicators (including process indicators) of a palliative approach implementation and (c) outcomes reflecting the results of implementation see . Most arn\u2009=\u200971), the largest category being the healthcare providers\u2019 experiences of care (n\u2009=\u200947). To illustrate, in one study district nurses were interviewed about \u2018how they perceived their role in supporting cancer patients with palliative care needs at home\u2019 . Another study used questionnaires in residential care to measure \u2018nurse assistants experiences of care provision and caring climate\u2019 , and yet another study used focus groups, interviews and a survey of medical and nursing staff to evaluate transitions to palliative care . Further, 38 articles included implementation outcomes focused on aspects of care provision, such as: advance care planning, identification of people who would benefit from a palliative approach, goals of care conversations and symptom assessment interventions. Several articles (11) evaluated workplace culture (e.g. relationships and communication between care providers) as an indicator of whether a palliative approach was being successfully implemented. Relatively fewer articles (7) focused on adherence to healthcare system policies or guidelines for implementing a palliative approach. Additionally, 33 articles focused on preparing healthcare providers to deliver a palliative approach, including evaluations of healthcare provider attitudes or confidence (21), knowledge (20) and the provider perspectives on their experiences with educational programmes (15).A palliative approach was most commonly evaluated based on indicators of its implementation . This includes 40 articles that evaluated patient- and family-centred health and quality of life outcomes, and 25 that evaluated healthcare system outcomes. Patient health (in particular symptoms and functioning) and quality of life were evaluated as outcomes of a palliative approach in only 26 of the articles , and 7 articles evaluated health and quality of life of family members. Experience of care (as perceived by patients or family) was evaluated in 26 of the articles. For example, one study protocol involved use of the Schedule for the Evaluation of Individual Quality of Life, the Palliative care outcome Scale-Symptoms-MS, and the Core Palliative care Outcomes Scale to evaluate impact of a home-based palliative approach on overall quality of life, symptoms, and \u2018emotional, psychological and spiritual needs, and provision of information and support\u2019.process of delivering a palliative approach and less emphasis on outcome measures for evaluating the results of a palliative approach. In addition, delivery is more often evaluated from the healthcare providers\u2019 point of view; many articles focus on healthcare providers\u2019 care experiences or elements of care provision, whereas patient- and family-centred outcomes are less prominently measured. Given that person-centredness is a core value embedded within a palliative approach to care, important questions arise, such as: Why are patient- and family-centred outcomes not more routinely evaluated? How are patients\u2019 and families\u2019 voices accounted for in evaluations of a palliative approach? Of the articles that did describe outcomes, what are the motivations for measuring system outcomes, including service use and cost? To what extent are we using these system outcomes to advance a person-centred perspective? Although the WHO sees palliative care as integral to a people-centred integrated healthcare system, these reflections highlight the need for more attention to measuring outcomes and indicators from a person-centred point of view.Overall, the above observations indicate an emphasis on indicators regarding the Regarding the data sources of the 88 studies, 12 studies utilized administrative health data, 24 utilized chart review data, and 64 utilized data obtained from healthcare providers. Conversely, only 30 studies included data that were reported directly by patients or family, whereas the remaining 58 studies included data only from sources  for each of the palliative approach outcomes or indicators see . Althoug20 These results lead to important questions about the representation of patients and families in evaluations of a palliative approach. Why are patients and families not more routinely included in studies focused on their healthcare experiences and outcomes? Whose voices are not well-represented because of these evaluative practices? Clearly, there is a critical need to develop strategies and means to ensure inclusion of diverse patients and family perspectives.The findings from this narrative literature review show that a palliative approach has been predominantly evaluated using data from providers and healthcare systems. Although measures of person-centredness can be derived from various data sources, it is nonetheless important to ensure that, where possible, measures of how patients and families are doing (i.e. their outcomes) and how they are experiencing the care they received are obtained directly from them.n\u2009=\u200934), followed by staff education or community of practice initiatives (n\u2009=\u200924), and policies related to the implementation of a palliative approach (n\u2009=\u200912). Relatively few studies focused on evaluating specific palliative approach interventions (see  with eight studies focusing on GSF in Primary Care and three on GSF in Nursing Homes. The remaining 23 studies described models for care delivery organized around a particular condition (e.g. chronic obstructive pulmonary disease or multiple sclerosis), or a particular practice setting, such as rural nursing or long-term care.Studies evaluating models of care were most frequent (ions see . For exan\u2009=\u200920), such as their attitudes, knowledge and confidence, in addition to indicators of palliative approach implementation (n\u2009=\u200918), healthcare system outcomes (n\u2009=\u20094) and patient- and family-centred outcomes (n\u2009=\u20098). By comparison, most of the 34 studies focusing on models of care included evaluations based on indicators of palliative care implementation (n\u2009=\u200929), in addition to patient- and family-centred outcomes (n\u2009=\u200916), healthcare system outcomes (n\u2009=\u200914) and, to some extent, HCP provider outcomes  the attributes of centredness (what it is), (2) how centredness is translated into practice (how it is done) and (3) evaluation of the effects (possible ways of measuring the effects of centredness). Although care processes, including communication and shared decision-making, are central to a palliative approach, challenges arise when these are reduced to standardized guidelines and checklists that are not individualized. For example, implementation of a palliative approach has focused on criteria for identifying people who need a palliative approach and the use of practice guidelines for discussions such as goals of care. However, person-centredness requires flexible approaches that tailor the application of such standardized guidelines and tools to the person\u2019s preferences and capabilities to engage in care planning and decision-making. Process evaluation of a palliative approach therefore requires evidence from patients and families of the extent to which such tailoring is being addressed in educational interventions and the implementation of guidelines and tools.The second theme of centredness, translation into practice, draws attention to the interactions and relationships between the person, the family and the healthcare providers.et al., such evaluation should focus on health-related effects ; person self-evaluations ; family participation and experiences; and organizational effects . While the importance of improving our measurements of quality of life and healthcare experiences of patients and families is widely acknowledged, person centredness further challenges us to shift the prevailing focus on measuring what matters most on average to findings ways of conducting evaluations that capture uniqueness \u2013 diverse healthcare needs, outcomes and experiences.In addition to evaluations focused on palliative approach implementation, evaluations of centredness are needed to determine whether the implementation is having the desired impacts. According to Feldthusen 22 Guided by the centredness attributes of \u2018being unique\u2019, \u2018being heard\u2019 and \u2018shared responsibility\u2019, we need to shift our focus on measuring what typical patients might want on average (nomothetic point of view) towards revealing diverse perspectives of particular patients (idiographic point of view). This motivation becomes increasingly relevant and important as our systems are being challenged to attend to matters of equity in health care \u2013 extending our offerings of the typical to meet the needs of the minority, marginalized and far too often underserved among our patient populations.Collectively, the themes and attributes of centredness point to the need to integrate commonalities and individual differences in our evaluations of a palliative approach. Whereas the predominant emphasis in healthcare system evaluation practices has been on nomothetic measurements that focus on population averages, idiographic assessments and measurements of individual differences are needed to understand how, whether and for whom a palliative approach is achieving desired person-centred outcomes.We believe that health system evaluation practices privilege healthcare provider and system perspectives and indirect indicators rather than tackling the complex and inherently messy question of whether patients and families experience care as individualized, person-centred and relevant to their unique and diverse needs. This may have led to an unintended consequence regarding the generation of a body of knowledge that seems to further support conventional evaluation practices rather than posing the fundamental question upon which a palliative approach is grounded: How will we know if the care we provided is the best possible care for all people who need a palliative approach? How do we ensure that our evaluations lead to interpretations, actions and decisions that are aligned with a person-centred orientation? How do we prevent the unintended consequences of not representing the needs, experiences and outcomes of marginalized and underserved populations? Given that healthcare professionals are collectively committed to person-centred care, we need to construct alternative  ways to ask these profoundly complex questions.System-oriented evaluation metrics delude us into thinking we have neatly packaged and convenient answers. While there may be many good reasons to continue to collect them, we must not forget the larger purpose for which we are doing this work\u2014the crucial objectives of health care. There may never be a neatly packaged answer to the question of how we would know if we were providing the right care for this patient and family in their season of need. But that does not mean we should ever stop trying to ask it.Click here for additional data file.Supplemental material, sj-docx-1-pcr-10.1177_26323524231193041 for Voices lost: where is the person in evaluating a palliative approach to care? by Richard Sawatzky, Pat Porterfield, Erin Donald, Carolyn Tayler, Kelli Stajduhar and Sally Thorne in Palliative Care and Social Practice"}
+{"text": "Exogenous lipoid pneumonia (ELP) is a rare disease caused by the inhalation of oily materials in the alveoli with the pathological characterization by the presence of laden\u2010lipid macrophages in the respiratory specimens. At present, the treatment norm for ELP has not well defined, and so the aim of this study is to evaluate the effect of bronchoalveolar lavage in combination with glucocorticoids on children with ELP.We retrospectively reviewed 17 children with a confirmed history of exogenous oily materials aspiration, admitted to the First Affiliated Hospital of Guangzhou Medical University from June 2012 to December 2021. Clinical features, blood investigations, tomographic evaluations, therapeutic bronchoalveolar lavage and glucocorticoids use were carried out at the beginning of therapy and throughout a follow\u2010up period.The included children are the median age of 2\u2009years. Fever, dypnea and tachypnea were the most common symptoms. The most common radiological features were airspace consolidations . Chest CT scans showed areas of consolidation with air bronchogram , poorly defined centrilobular nodules , areas of ground\u2010glass attenuation  and \u2018crazy\u2010paving\u2019 pattern  in the both lower, right middle lung lobes. Neutrophil percentage of peripheral blood and bronchoalveolar lavage fluid exhibited a significantly higher than the normal range. After treatment with multiple bronchoalveolar lavages and local administration of budesonide during the hospital stay, taken by oral prednisolone (1\u2009~\u20092\u2009mg/kg) after discharge, all of children became asymptomatic and presented normal radiological imagings in the follow\u2010up period.The most frequently findings in the CT scan of ELP were consolidations and ground\u2010glass attenuation in the both lower and right middle lung lobes. Multiple bronchoalveolar lavages in combination with oral prednisolone for children who had a confirmed history of exogenous oily substances ingestion were an efficient and safe for the clearance of oily materials from the lung and the prevention of fibrosis. This strategy contributed to reducing the damage of ELP in children patients. The most common radiological features were airspace consolidations in children with acute exogenous lipoid pneumonia. Bronchoalveolar lavage with glucocorticoids was an efficient and safe for with acute exogenous lipoid pneumonia. Medical records were retrospectively reviewed by using a standardized form to collect demographic data, clinical manifestations, laboratory test results and radiological findings. All patients with established ELP were evaluated by the senior attending paediatrician at the inpatient units on the basis of signs, symptoms and laboratory/imaging findings during hospital stay. The exclusion criterion was suspected with a history of or no evidence of exogenous substances inhalation. A total of 17 children patients was included in the final analysis. CT scans of all children found diffuse pulmonary lesion at admission. Supporative measures, such as oxygen therapy, empirical antibiotic therapy, local or systematic corticosteriods use to slow the inflammatory response, were performed in children with clinical symptoms or severe lung damage. The number of bronchoscopic lung lavage was carried out in terms of the severity of clinical symptoms/signs and radiological findings.2.2We collected data on demographics , symptoms/signs, hospital stay, blood investigations , radiological imagings, bronchoalveolar lavage fluid analysis (lymphocyte subset percentage and neutrophil/lymphocyte/macrophage percentage), source of inhaled oil substance, comorbidity, number of therapeutic bronchoalveolar lavage and clinical course. Clinical course was classed into four categories as follows: complete or partial resolution; no charge; progression and lost to follow\u2010up. Bronchoscopic data on lymphocyte subset was determined by flow cytometry.2.3High\u2010resolution CT (HRCT) scans of all children were evaluated for the presence and distribution of the following findings: air\u2010space consolidation, ground glass attenuation, crazy\u2010paving pattern, and poorly defined centrilobular nodules.2.42 test or Fisher's exact test, respectively. P\u2009\u2264\u20090.05 was considered statistically significant. Statistical analysis was carried out by using IBM SPSS Statistics for Windows, verision 25.0  software.Data were expressed as medians with interquartile ranges for continuous variables and as numbers with percentages for categorical variables. Among two groups, continuous and categorical variables were compared by using the Mann\u2013Whitney U test and \u03c733.1n\u2009=\u200914), fever (n\u2009=\u200914) and tachypnea (n\u2009=\u20097). Clinical visit data were available for the 17 children who finally were asymptomatic and presented normal radiological imagings. One patient taking sewing machine oil for 2\u2009days had a severe pneumothorax but a good clinical evolution. Corticosteroid and preventive antibiotics use were administered in 12 of the 17 children. Bronchoscopic lavage using normal saline was performed in all children. Characteristics of the patients are listed in the table\u00a0In our study, a total of 17 patients were included, with median age of 2\u2009years , in two of which had severe malnutrition and in one of which had obesity, the remaining 14 patients had no risk factors. The most frequent symptoms were cough , peripheral blood investigations, bronchoscopic lavage fluid (BALF) analysis and CT features are presented in Figure\u00a0In most cases, the lesions were multilobular; both lungs were equally involved; the right lung was more extensively involved than the left lung. On CT scan, the most common radiological features in the patients with ELP were bilateral areas of air\u2010space consolidation 15, 93.75%) and poorly defined centrilobular nodules . The children had no pleural effusion, lymph\u2010node enlargement or any other chest abnormality. The presence of ground\u2010glass attenuation was observed in 11 cases and \u2018crazy\u2010paving\u2019 pattern  in 6 cases  is a rare condition that can be difficult to recognize and results from accumulation of oily materials in the alveoli that are aspirated from vegetable, mineral and animal origins, characterized by lipid\u2010laden macrophages in the sputum or bronchoscopy lavage.To precisely review the effect of bronchoalveolar lavage on acute ELP, we only included patients with a confirmed history of aspiration, clinical symptoms and CT scan images.BAL is a successful method recommended in the therapy of pulmonary alveolar proteinosis,Regardless of location, the inflammatory response can destroy the alveolar walls and the interstitium, and the resultant fibrosis can occasionally progress to end\u2010stage lung disease.There are also several limitations in the present study. First, this was a respective study conducted in only one hospital so that many confounding factors and various bias could not be avoided due to the inherent nature of the retrospective study. Second, the radiological findings of all children varied greatly from CT scan images at initial presentation to X\u2010ray films during the follow\u2010up period; therefore, we did not get quantitative data to compare. Finally, the number of children was insufficient to reach a credible and robust conclusion. In the future, larger multicenter prospective cohorts are needed to elucidate the risks and benefits of bronchoscopic lavage and glucocorticoids on children with ELP.In conclusion, diagnosis of acute ELP is often difficult. Causative agents of ELP can vary depending on differences in culture and life style of the affected countries and regions. Diagnosis of acute ELP should be based on confirmed exposure to lipid substances and clinical\u2010radiological features. Sufficient exposure data are necessary to reach a diagnosis of acute ELP and to differentiate it from pneumoconiosis.All of authors declare that they have no conflict of interest.SY and SZW were involved in collecting the medical records and drafted the first manuscript. JXX and YNL were engaged in data extraction and reviewing the quality of included studies with the aid of ZHH, XWC and QYX. Statistical analysis was carried by YNL and ZHH. All authors reviewed this article and contributed the revisions. DHC and CYL are the guarantor for this article.The study was approved by the institutional ethic review committee, which waived the requirement for written informed consent because of the nature of the retrospective study."}
+{"text": "Giara of Gesturi  provides a significant case for the study of their relations with visibility and movement. Context-oriented GIS models based on viewsheds and least-cost paths have been devised as targeted tools. The results show a certain correlation between nuraghi and potential movement on the slopes, thanks to the selection of plateau morphologies such as outward crests. Anyway, nuraghi do not stand exactly at the most accessible points of the plateau. Nuraghi offered ample visual control, especially at large distances, but not specifically over the closest accessible ways. This suggests that the function of nuraghi is somehow connected to defense and visibility, but it is not explained directly by local territorial control: a role as landmarks and multifaceted monuments has likely to be envisioned.The built landscape of Nuragic Sardinia is an exceptional case for geostatistical analysis, allowing for a discussion of long-held assumptions and ideas. The function of nuraghi (ca. 1700\u20131100 BCE), the most prominent settled monument of the Sardinian Bronze Age, has been addressed via a multiplicity of landscape approaches, mainly relying on intuitive assessments of their spatial properties: nuraghi were assumed as means of territorial control. The series of nuraghi crowning the mesa plateau named  The potential of the archaeology of Sardinia in shedding light over the processes of Mediterranean deep history has been increasingly remarked by recent research . SardiniIn this sense, we guess Sardinia, particularly during Nuragic times of the Bronze and Early Iron Ages . A specific paragraph will be devoted to the discussion of the different observed uses of GIS in the study of Nuragic landscapes .the Giara di Gesturi (hereafter quoted as: the Giara) a wide mesa-type basaltic plateau located in South-Central Sardinia, with a characteristic distribution of nuraghi and villages of the Bronze and Iron Ages .In this study we select the area around the Giara and its surroundings .A series of quantitative GIS-based models, aimed at the assessment and quantification of commonly accepted ideas regarding the spatial properties of nuraghi, will be devised and employed in order to explore context-specific patterns of movement and visibility in Specifically, this will entail:the Giara plateau (mobility/accessibility);the building of a generalized movement model, based on the generation of least-cost paths with randomized origin, directed towards the use of viewsheds, centered on the existing nuraghi, as a means to investigate the degree of visual control over the reconstructed movement pattern (visibility).the Giara nuraghi with those associated with points randomly generated in analogous positions, in order to test for their significance.The results will allow us to compare the mobility/accessibility vs. visibility properties associated with the Giara can be better conceptualized. These results can also provide a conceptual framework to tackle spatial trends across the island in a more sound way than the still dominant intuitive and descriptive interpretive proposals of settlement patterns, even when using GIS as a tool. We hope in this way to contribute to strengthen the use and evaluation of formal modelling .The definition and name of the Nuragic culture stem from its most recognizable monuments\u2013the nuraghi\u2013which still constitute an integral and highly visible part of the contemporary landscape of the island , 10\u201312. Nuraghi are widely spread in the different areas of the island, with an estimated number ranging around 7000 units, or even more , 32, 51;14C dates, alternatively to the Sardinian Middle Bronze Age 1 (MBA1 ca. 1750/1700-1600 BCE) or MBA 2 (ca. 1600\u20131500 BCE) Reviewers' comments:Reviewer's Responses to Questions <font color=\"black\"> Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0PartlyReviewer #2:\u00a0Yes********** <font color=\"black\"> 2. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 4. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 5. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The authors contribute to the research on the function and context of the Sardinian nuraghi from a landscape perspective, using GIS-based methods  and viewsheds to understand the possible role of these monuments for territorial control of the surrounding landscape. Movement through the landscape and towards the Giara of Gesturi plateau in southern Sardinia, which provides the setting for this case-study, is assessed with LCPA, CSA is used to assess accessibility and defensibility, while viewsheds address monument visibility as well as the possibility to control the surroundings. The study shows that contrary to former assumptions, the role of nuraghi in the cultural landscape of the Giara plateau  appears to have been much more complex than simply allowing for the control of natural access and transit routes. The authors highlight the significance of the nuraghi\u2019s visibility, especially by remotely moving people. They could not directly connect their results to advantages for a defensive/military use of the monuments , and surprisingly, their results might in some cases even contradict any interest in movement control. Despite the naturally small spatial extent of the study, it should be an important contribution to the ongoing research of the still barely understood nuragic towers, which will help to evaluate results from upcoming landscape analyses in other regions with nuraghi on the island.The manuscript certainly delivers a well-defined approach, the execution is properly described and the questions asked are archaeologically relevant. Nonetheless, I still have some serious issues with the manuscript. The authors appear to stress a false dichotomy between what they call \u201cintuitive\u201d archaeology and apparently \u201csuperior\u201d quantitative approaches and models, instead of highlighting the importance of combined methodologies for a meaningful examination of the archaeological records. This would be especially rewarding for the study of the Sardinian nuragic period, where scientific analyses and sound landscape analyses are still in their beginnings. However, the complexity of the contexts and materials require a multi-faceted research, including the \u201ctraditional\u201d archaeological methods.While the original study appears to be sound (with the below mentioned major reservations), the English language is sometimes unclear and needs revision. I advise the authors work with a copyeditor to improve the flow and readability of the text. The fact that the research aims at an English speaking audience does not relativize the publications by Sardinian and other authors, especially since there are relevant papers available in English.The figures are generally good quality, readable, and illustrate the findings of the authors appropriately. The same is true for the supplementary materials.This supposed dichotomy affects the drawn conclusions from the nonetheless most interesting results from the case-study of nuraghi on the Giara plateau, where some obvious interpretative approaches, for example concerning the symbolic importance of the monuments, are largely ignored. Finally, this biased vision leads to partly inappropriate attacks, beyond justified criticism, against Sardinian scholars who are proficient in the field but hold divergent views on the applicability of particular modelling approaches. Instead of convincing arguments for the chosen methodology and its undisputed contribution to understanding the role and function of nuraghi as monuments that shape the Bronze Age landscape of the island, the authors use polemics  and selective quotations as well as a providing a selective bibliography. The latter has unfortunately become almost standard amongst some researchers studying nuragic Sardinia, however it has definitely to be considered bad scientific practice. The expedient discussion of relevant questions will be obstructed as long as disparate groups of scholars refuse to exchange arguments and evaluate the actual contributions of the respective \u201cother\u201d groups. The results and conclusions clearly show that there would be the potential to integrate the proposed landscape analysis with the knowledge and approaches that have been provided by the Sardinian scholars \u201cunder attack\u201d. I will substantiate my criticism by summarizing each section of the manuscript:\u2022 The Abstract is well presented and provides a good overview of the aims, results and methods of this study.\u2022 The Introduction needs thorough revision: Firstly, an uncritical citation of Kristiansen\u2019s \u201cThird science Revolution\u201d (lines 43-44) seems utterly unacceptable after the weighty criticism from paleo-geneticists and archaeologists alike, together with the expectable appropriation of Kristiansen\u2019s assumptions by the far right. This indispensable criticism is summed up in World Archaeology, vol. 51, no. 4 (2019), especially in the editorial, in the contributions of Hakenbeck and Frieman & Hofmann, as well as by Martin Furholt (2018 & 2021). Furthermore, the aDNA data from nuragic Sardinia is far from comprehensive, and the ca. 15 or so samples from this period presented by Marcus et al. 2020 are rather to be seen as the basis of a yet to establish dataset, thus no assumptions should be based on this small glimpse on Sardinian paleo-genetics. The authors themselves admit that this \u201cIt is likely that this image (\u2026) is somehow deceptive\u201d (l. 46-47), so why claiming that the island \u201chas emerged as one of the most conservative places in Europe\u201d before there is sufficient information on this (l. 44-45)? Next, using Webster (1996) as a main reference for Nuragic Sardinia and adapting his chronology seems odd. This ignores relevant work by Sardinian scholars who know the record first hand as well as the established chronology by Lo Schiavo & Perra (2018). In the last paragraph, the scope of the research is adequately summarized.\u2022 \u00a72 From Monuments to Nuragic Landscapes aims at providing an overview of Nuragic archaeology but unfortunately fails to do so by using the partly outdated and heavily debated work of Gary Webster as the main source. A more differentiated view and the inclusion of relevant, also contradicting, viewpoints of scholars working in Nuragic archaeology would be necessary to complete this contextualization. The random collection on papers  refers to sometimes outdated research or papers dealing with particularly narrow topics and single sites, thus leaving Webster\u2019s works the only actual overviews. Not even one of the general works of G. Lilliu on Nuragic Sardinia is included. Much work has been done in Sicily since Leighton\u2019s \u201cSicily before history\u201d and the archaeological record of Corsica has been boosted by recent comprehensive, high-quality research. The author\u2019s statements (lines 112-114) can hardly be acceptable by scholars working on these islands. For the contextualization and connections between them and Sardinia, there are relevant, recent publications for example by Lo Schiavo, Peche-Quilichini, Araque Gonzalez, Fundoni and Miletti, to name but a few, of which the authors do not seem to be aware of. The bibliography on Giant\u2019s tombs as well as on the sanctuaries is deficient and partly outdated. The marginally mentioned interpretations of social relations in Nuragic Sardinia (lines 119-121), restricted to G. Webster and a paper by M. Perra that is not even centred on this issue , ignoring the contradictory but relevant ideas of Tronchetti, Russu and Araque Gonzalez, without revealing the author\u2019s ideas on the topic, must either be perceived as listless or uninformed. However, the nuraghi are almost always discussed in their social contexts, and consequently the latter should be considered in more than merely a single line. The symbolic aspects of the nuraghi (lines 168-173) are referred to with outdated literature and do not mention the most relevant tome \u201cSimbolo di un Simbolo\u201d by Campus & Leonelli (2012) or new approaches by Araque Gonzalez (2021). Regarding the latter, there is no short but poignant discussion on the construction process, its possible organization and its relevance for nuragic society. The confirmed uses of nuraghi, as illustrated for example by the results from excavations at nuraghe Arrubiu at Orroli, are also being ignored. The overall omission of citations of relevant work by archaeologists who disagree with Webster\u2019s and other British scholar\u2019s interpretations is conspicuous. This paragraph needs thorough revision to provide a serious contextualization of the monuments.\u2022 \u00a73 Theoretical Issues and Landscape Approaches, sums up former landscape studies centred around the nuraghi and highlights the relevance of the author\u2019s research. This is generally done in a comprehensive way, but it is evident that again, Sardinian scholars and their statement that rigid and abstract models are not always useful for the unique and complex nuragic landscape undergo heavy, sometimes polemic criticism (lines 208-232). The approach of the Spanish school of Granada is also criticized (lines 233-246). Finally, what is not clear to me is in how far the case study of nuraghi should be relevant for the interpretation of \u201cMediterranean societies\u201d who did not build nuraghi. However, the evaluation of monument construction within prehistoric societies as a social event and collective action pointing against or towards hierarchizing processes would be relevant. This point needs clarification. The following explanation and discussion of the use of GIS (lines 256-298) is well informed and competent. However, in the second part of this section inadequate polemics and the invention of the term (Nuragic scholars\u2019 empiricism) should be nuanced and brought forward in the form of fair, transparent criticism, including quotes and ideas and their deconstruction (lines 299-319). This is a serious problem I have with this manuscript. The promoted approach by the authors seems sound and I agree fully on its usefulness (lines 320-333).\u2022 \u00a74 Study area: The Giara of Gesturi is well laid out and provides all relevant information. Figures 1 and 2 are reversed and their order has to be corrected.\u2022 \u00a75 Methods and \u00a76 GIS Analysis follow a clear structure and the approach (a basic GIS landscape approach) is well defined. A quote is needed in line 605  and the link to bibliography online in line 500 is dead (or mistaken?). Quotes are needed for each specific criticism, otherwise it remains a blurred \u201copponent\u201d that is targeted.\u2022 \u00a77 Results is also well structured and provides interesting insights, for example that secluded location was more important than access and movement control (lines 660-661) and that visibility over the closest 100 m radius around the nuraghi is scarce (lines 668-669), which both contradict a military use, the latter would be quite tragic in case of a siege. In the case that actually visibility per se was more important than visual control, as it is stated (lines 672-674 and 691-695), it must be considered  that this hints towards a strong symbolic meaning and function of the nuraghi, beyond its obvious usefulness as landmark (line 697).\u2022 \u00a78 Discussion and conclusions presents the intriguing results and the author\u2019s (very cautious) interpretation. Although they did not find clear indicators of a defensive (military) use of the monuments (lines 748-750), they still do not want to exclude it, but they do not discuss the issue. I would suggest to explain why all these contradictions would still favour such an explanation of the monuments, except for the fact that Lilliu and Webster attributed a defensive function to nuraghi . Again citations are missing, for example in line 710 (\u201cas postulated in the literature\u201d \u2013 which?). the authors hint at a possible spatial relationship between nuraghi and the traditional pathways to the plateau \u2013 and here they seem to indicate that the denigrated \u201cempirical2 archaeologists who have postulated this relationship were not all wrong. However, they do not mention this coincidence between both approaches. Unfortunately, they do not discuss the symbolic implications that the focus on visibility has . The authors interpretation of their findings unfortunately remains vague regarding their results (lines 763-768). I liked the transparency given by the authors by stating that their analysis remains preliminary and not fully contextual. They show that future work in landscape archaeology will be essential to clarify aspects of the nuraghi, however they come up with the mentioned false dichotomy again and (lines 778-782) and fail to acknowledge the potential of a combination of all knowledge. Some sentences could easily be erased from the manuscript because their only aim seems to be directly discrediting particular scholars (lines 784-786).I recommend publication after revising these major concerns. If the authors would nuance their criticism, summarize the actual positions of the researchers they rightfully want to criticize (with proper quotes) and provide concrete arguments, the paper would improve significantly. I strongly recommend them to re-think their own position and consider if their landscape approach would not be enhanced by combining it with the results from Sardinian field archaeologists and with theoretical approaches by researchers whom they ignored for now, and joint forces might finally shed more light on the still mysterious nuraghi and their functions and meanings. A bolder statement on the interpretation of the focus on visibility by remotely moving people would also be desirable, if the authors would like to provide one.Reviewer #2:\u00a0The proposed contribution is of an excellent level, quite original, methodologically impeccable. The bibliography used is good and exhaustive. The work undoubtedly deserves publication.However, there are some observations that can improve the contribution:- In paragraph 2.1, as far as the Nuragic civilization is concerned, the authors speak only of the Bronze Age, while it would be appropriate to speak also of the I\u00b0 Iron Age, as the authors do in other paragraphs.- The term \"classical\", used in the text to define the most recent nuraghes with rooms with a \"tholos\" vault, should, in our opinion, be used in parentheses, so as not to give the reader who is not an expert in Nuragic civilization the doubt that they are monuments belonging to the Greek and Roman classical age.- A paleo-geomorphological analysis of the investigated area is missing. In fact, over time, some areas may have undergone substantial changes; in fact, on the edges and slopes of the plateaus, we often witness landslides and mudslides. Even considering the importance attributed to the Scalas, i.e. the natural accesses to the plateau, a mention of this aspect would be useful, also to take it into account in the analyses.- Equally, it would be interesting to at least mention paleoenvironmental aspects. Are there archaeozoological studies or pedological and pollen analyzes for the investigated area?- The method of retrieving data is not well specified: the authors talk about the analysis of aerial photos, but in studies of this type the direct analysis of the monuments and possibly a systematic survey of the entire territory would also have been appropriate. Was it made? Why was it not considered appropriate to do so?- As also mentioned by the authors, we do not have a precise chronological location of the monuments considered by the analyses, as very few sites have been the object of archaeological excavations. This is important, because the type of analysis presented would preferably require having architectures of the same phase as objects. It would therefore be necessary to better explain the reasons for the choices made and how the authors think they have solved the problem.********** <font color=\"black\"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #1:\u00a0NoReviewer #2:\u00a0No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at\u00a0figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,\u00a0 15 Jun 2023Response to reviewersThe Authors wish to express their appreciation of the remarks and observations by the Reviewers, which have addressed important issues, and have pointed to the necessity to be more clear and definite in the expression of their proper point of view. It is a pleasure to see that both Reviewers have accepted the GIS quantitative approach that we presented, and that each of them has recognized that the crucial parts of the analysis which is the core of the study are well organized and rather convincing (\u00a7 4-5-6-7); the observations, mainly by Reviewer #2 about paleo-geomorphology, paleo-environment, data retrieval, and chronological detail will be addressed properly, and these observations contribute to the clarification of our arguments, we guess.At the same point, particularly Reviewer #1, expressed some more criticism about our submitted paper, which we guess can solve, also by considering in an appropriate mode some wider literature that has been indicated by Reviewer #1. Criticism by Reviewer #1 is wide, and in fact, while approving the rigor of the statistical analysis and the availability of data, this Reviewer only \u201cpartly\u201d considers the manuscript sound and that data support the conclusions.We have therefore introduced what we deem are appropriate changes to the paper, and we present a response for all the remarks by both Reviewers. We hope this will make clear our views, while reflecting our full adhesion to the peers\u2019 blind review principle.Reviewer #1General responseBefore answering to each observation by the Reviewer, we think it is appropriate to make some general remarks to the provided criticism: in fact, we can identify three major arguments inside the critical observations to our submitted paper by Reviewer #1.1. The most relevant one, in our view, regards our observation that a dichotomy seems to exist in Sardinian studies between what we call \u201ctraditional\u201d and \u201cquantitative\u201d approaches to the interpretation of the landscape patterns in Nuragic archaeology. The Reviewer observes that we would be unfair in stressing these positions, and this could reach the level of polemics, and include selective referencing, or even aims at discrediting specific scholars. We would furthermore exclude the possibilities for a synthesis, and more integrated approaches, caring both the empirical/traditional views and the model-using/quantitative perspectives. We think we can clarify this issue.With regards to the dichotomy between \u201ctraditional\u201d and \u201cquantitative\u201d approaches in Sardinian landscape archaeology, we argue that it actually exists: in fact, many archaeological syntheses on Nuragic landscapes do not take into account the results of the now numerous quantitative and formal approaches to the subject. Starting from this observation, which can find evidence in the quotations and in some precise observations by some authors, particularly in years 2000s , it is not the aim of this paper, nor of its Authors, to perpetrate this divide. Instead, the Authors believe that traditional approaches have really provided a vast and compelling body of knowledge, a discussion of which is unavoidable even when adopting quantitative approaches, and we state this point all over the paper, by referring since the beginning to A. Taramelli\u2019s views, and by using the empirical syntheses, such as the works in Campus et al. 2008, Depalmas and Usai 2015 as the basis for the construction of the questions to be checked by geo-statistical tools. We therefore aim to build on traditional approaches, interpretations and ideas, rather than reject them outrightly. An outright rejection would not have resulted in the need for a translation process as the Authors have discussed (line 329). In other words, we think it is definitely right to provide an actual evaluation of the contributions derived from traditional approaches; we also think that such an evaluation we made in the submitted paper (lines 756-768) has resulted in partial confirmation and equally partial rejection: we consider this result a testimony of the strength of traditional arguments, but also of the need for their formal verification and refinement. At the same time, we think we can dilute some of the apparent criticism which could be dictated in the submitted text by the need to express clearly the positions of some scholars, in order to make clear the Authors\u2019 constructive attitude.Therefore, in our updated manuscript, some of the criticism has been nuanced and our appreciation for traditional work better highlighted. We also agree that our citations in the general parts could be seen as partial, but in our intention this had been caused by the need to give only examples of works rather than a comprehensive list. We have corrected this approach, which was not in any way meant to favor any particular interpretation, making clear what we think are different views of the situation. 2. Reviewer #1 considers outdated our reference to Gary Webster\u2019s views in terms of chronology and Nuragic society, and excessive our reliance on his overviews. We would also omit the views on Sardinia by other scholars disagreeing with Webster and \u201cother British scholars\u201d; finally, the quotation of Robert Leighton\u2019s \u201cSicily Before History\u201d (1998) would be outdated, and also the reference to Corsica would downscale the recent improvement in knowledge about the region.We accept the criticism about the rather dated literature, but at the same time we wish to stress that we wanted to refer to the  handbooks and syntheses available on the prehistoric and protohistoric sequence of the areas, particularly for Sardinia and Sicily, as the context of the paper would -in our view- require a more general look for potential readers interested in the methodological framework, but not expert in the details of the areas; by the way, we consider the views by Gary Webster  as certainly partial, and expression of single scholars\u2019 views, but not out of scientific quality: when appropriate, we guessed we had introduced a balanced series of quotations for different opinions. As it seems not to be the case, we reviewed our quotations and integrated them, thus hoping to have removed this negative impression.3. A third relevant criticism by Reviewer #1 is what would be the Authors\u2019 interpretive position toward the symbolic and social meaning of the nuraghe as a monument. We thought indeed that a complete discussion of the social and symbolic factors implied by the construction processes of nuraghi and by the possible implications of their being a monumental preseence in the land had to be discussed in a differently targeted paper, but the criticism fosters us to be more clear and detailed. We hope to have solved this problem as well, by adding to the text some more observations on the issue, as reported afterwards.Furthermore, as for the observation by Reviewer #1 (in the Review text and not in the answer to the bulleted question about intelligibility and standard English) that English language could be made more fluent and readable, we regret for the possible inadequacy of our style, but we can assure that the manuscript has already been proof-read by an (American-)English speaking scholar, who has now confirmed the opinion on the suitability of the text.We now list the specific actions required by Reviewer #1, reporting and commenting our changes and response. We are particularly happy that the -sometimes harsh- criticism by this reviewer didn\u2019t affect \u00a76 \u2013 GIS analyses nor  \u00a77 \u2013 Results.IntroductionREVIEWER\u2019S OBSERVATION 1Submitted text lines 43-47: The Reviewer criticizes our \u201cuncritical citation of Kristiansen\u2019s \u201cThird Science Revolution\u201d\u201d, counter-referring to some papers, namely published in World Archaeology 51/4 (2019) and elsewhere by M. Furholt , which express prudence and \u201cweighty criticism\u201d.The Reviewer further writes that, as the sample of ca. 15 Nuragic Sardinians presented by Marcus et al. 2020  is small, it is \u201crather to be seen as the basis of a yet to establish dataset, thus no assumptions should be based on this small glimpse on Sardinian paleo-genetics.\u201dANSWER 1We fully understand the Reviewer\u2019s position on aDNA studies, and we agree with the prudence that is called for, in front of the small number of samples. As for the papers expressing reserve about the use of aDNA data , we know them and particularly appreciate the thorough scrutiny that Martin Furholt gave of the Yamnaya migration hypothesis. We furthermore would also recommend to take care of the tools employed for tracing aDNA data, such as the Admixture approach , NATURE COMMUNICATIONS (2018) 9:3258).In any case, there are two papers on aDNA, so far, regularly published in scientific journals, which independently stress the same conclusion, i.e. that human remains found in Sardinia -after Early Neolithic times- show a major contribution from external aDNA only after Nuragic/Bronze Age times, that is, there is an intense genetic and demographic continuity in the island. They are:- the quoted Marcus et al. 2020, with 16 samples of Nuragic age, and comprehensively 48 well-dated individuals from Neolithic to Nuragic age;- the not quoted (but now quoted: see below) Fernandes et al. (2020), NATURE ECOLOGY & EVOLUTION 4: 334-345, adding ca. 15 samples dated to the Bronze age and comprehensively 30 from Neolithic to Bronze age .Therefore, the long-stated even modern DNA peculiarity of Sardinian inhabitants , Science 341 (6145): 565-569; Sikora et al. (2014), PLoS Genet 10(5): e1004353), seems to have a relation with the island\u2019s relative isolation, which is affirmed by different samplings of aDNA, and traced until the Iron Age/Phoenician contact. Also recent syntheses, albeit expressing doubts about the supposed Early Neolithic input of Anatolian/Aegean aDNA, repeat the same considerations , Annals of Human Biology 48/3: 203-212).ACTIONS 1Summing up:a) we deem that a quotation of the present scientific agreement about a significant genetic/population/demographic continuity fron Pre-Nuragic to Nuragic times is appropriate for our paper\u2019s introduction, and we maintain it;b) We downscale a bit the quotation of Kristiansen\u2019s \u201cThird Science Revolution\u201d, by postponing it in the text, and by raising some righteous care;c) As our paper is not a DNA (neither ancient nor modern) paper, a thorough discussion such as in Cal\u00f2 et al. (2021) is out of scope and would deflect the paper from its main goal. Therefore, we only quote Marcus et al. (2020) and we add Fernandes et al. (2020);d) We still claim, as observed by Reviewer #1 that \u201cIt is likely that this image of Sardinia as a separated enclave is somehow deceptive \u2026\u201d.REVIEWER\u2019S OBSERVATION 2Submitted text lines 51-53: the Reviewer states that we would ignore \u201crelevant work by Sardinian scholars who know the record first hand as well as the established chronology by Lo Schiavo & Perra (2018).\u201dANSWER AND ACTIONS 2We do agree that the general reference to only Webster 1996 is outdated; we have explained above  why we had decided to introduce this reference. We maintain it anyway, but we add 2 references (6-7 in text) to chronologies established by Sardinian and other scholars .Chapter 2GENERAL OBSERVATION \u00a72: the Reviewer remarks that, in this paragraph, the \u201coverview of Nuragic archaeology [...] unfortunately fails to do so by using the partly outdated and heavily debated work of Gary Webster as the main source\u201d.ANSWER AND ACTIONS, GENERAL \u00a72: We have explained above  why we decided to use Webster as a formal reference (both Webster 1996 and Webster 2015). Indeed, it is our opinion that \u00a72 does not refer only, nor mainly, to Webster\u2019s views, but that it depends on our own direct experience in the study of Nuragic Sardinia, as well as much on a paper that we quote, but don\u2019t want to overstress, also to avoid self-referncing, namely Vanzetti et al. 2013. As the Reviewer has perceived this feeling, of dependence on a single (relevant) scholar, we introduced more quotations (such as in NEW MANUSCRIPT lines 96-99) and expressed in many points of the text the different positions.\u00a7 2.1REVIEWER\u2019S OBSERVATION 3Submitted text\u2019s lines 97-101; 118-119The Reviewer considers the bibliography on Giants\u2019 tombs and sanctuaries \u201cdeficient and partly outdated\u201d.ANSWER AND ACTIONS 3The bibliography on Giants\u2019 tombs and sanctuaries has been integrated with further and up-to-date references. REVIEWER\u2019S OBSERVATION 4Submitted text\u2019s lines 109-110The Reviewer considers the references presented here as a \u201crandom collection on papers  refer[ring] to sometimes outdated research or papers dealing with particularly narrow topics and single sites, thus leaving Webster\u2019s works the only actual overviews\u201d.ANSWER AND ACTIONS 4While we do agree that the idea of somehow \u201crandom collection\u201d is real, as it was intention of the Authors to give an idea of the wide range of papers and views that Nuragic impressive archaeology has fostered, we don\u2019t want to give any impression that Webster\u2019s works are the only existing overviews. For this reason, we widened the series of quoted papers, and we inserted some basic Sardinian literature, such as, e.g. Lilliu\u2019s founding syntheses.REVIEWER\u2019S OBSERVATION 5Submitted text\u2019s line 114The Reviewer remarks that \u201cMuch work has been done in Sicily since Leighton\u2019s \u201cSicily before history\u201d and the archaeological record of Corsica has been boosted by recent comprehensive, high-quality research.\u201dANSWER AND ACTIONS 5The Autors agree with the Reviewer, but argue that, in the frame of the submitted paper, a further discussion of Sicily and Corsica would be out of scope, and therefore referencing had been selective and synthetic . Anyway, the two cases are different:As for Sicily, the mass of work published after the syntheses by Sebastiano Tusa (1983 and 1999) and Robert Leighton has not resulted in any synthetic overview, and therefore one has to refer still to these publications. We added the quotation of Tusa\u2019s synthesis, as Tusa 1999 and of a recent Conference on the Prehistory of Sicily. Sicily, as stated also by our research in Cannatello, is characterized by a notable internal variability of cultural manifestations and monumental landscapes, both at a synchronic and diachronic dimension.As for Corsica, the new works have provided some synthetic overviews, of which we are aware, but it is clearly fieldwork by Peche Quilichini (and somehow Matteo Milletti) that has provided the best new results. In any case, Corsica monumentality of tower-like buildings is not widespread all over the island, and is characterized by a considerable variability. As the reference to Corsica has also the function, in the paper\u2019s frame, to trace the divergent trajectories of Sardinia and Corsica- in other terms why we can discuss of Sardinia (and of a specific region inside it), without necessarily debating Corse monumentality - we consider the best option to add a single paper by Peche Quilichini, precisely discussing this issue (Peche Quilichini 2021). We don\u2019t quote here Vanzetti et al. 2013, which had evaluated the situation of Corsica and other major islands of the Mediterranean, as we consider it already slightly outdated.REVIEWER\u2019S OBSERVATION 6The Reviewer argues: \u201cThe marginally mentioned interpretations of social relations in Nuragic Sardinia (lines 119-121), restricted to G. Webster and a paper by M. Perra that is not even centred on this issue , ignoring the contradictory but relevant ideas of Tronchetti, Russu and Araque Gonzalez, without revealing the author\u2019s ideas on the topic, must either be perceived as listless or uninformed. However, the nuraghi are almost always discussed in their social contexts, and consequently the latter should be considered in more than merely a single line.\u201dANSWER AND ACTIONS 6We do agree with the Reviewer that the discussion of Nuragic social relations was compressed, but at the same time we remark that this derived from our precise knowledge of the debate, as this debate is so varied that even a whole paper could not include all Authors\u2019 opinions. We still think that hierarchic and heterarchic are the main poles (and the best examples) between the scholars\u2019 view of Nuragic society dwindle, but we added the anarchic and cooperative CONTROLLI LEI SE VA BENE view that has been recently expressed by Araque Gonzalez . As we agree that an ampler discussion is more appropriate, we have further expanded, as requested, the discussion on different social interpretations of Nuragic Sardinia. We have anyway avoided to insert a deeper discussion on FBA-EIA social dynamics, since such discussion would lie somewhat outside the scope of the paper, which is more centered on MBA-RBA landscapes. \u00a7 2.2REVIEWER\u2019S OBSERVATION 7This observation by the Reviewer is linked to what expressed above, as the argument discussed at General response 3, encompassing also other points in the submitted paper. \u201cThe symbolic aspects of the nuraghi (lines 168-173) are referred to with outdated literature and do not mention the most relevant tome \u201cSimbolo di un Simbolo\u201d by Campus & Leonelli (2012) or new approaches by Araque Gonzalez (2021). Regarding the latter, there is no short but poignant discussion on the construction process, its possible organization and its relevance for nuragic society. The confirmed uses of nuraghi, as illustrated for example by the results from excavations at nuraghe Arrubiu at Orroli, are also being ignored.\u201dANSWER AND ACTIONS 7We accept the observation, and we give now a more comprehensive overview of symbolic interpretations of nuraghi, updating the references and basically including the Reviewer\u2019s proposals.As noted above (Answer and actions 6) for the social reconstruction, we avoided a deeper review of symbolic meanings of nuraghi in FBA-EIA (and beyond), when scholars generally underline a symbolic shift in many archaeological contexts inside the nuraghi, because such discussion would lie somewhat outside the scope of the paper, which is more centered on MBA-RBA landscapes.Chapter 3\u00a7 3.1REVIEWER\u2019S OBSERVATION 8The Reviewer identifies, in our critique, a polemical vein, stating the following: \u201cSardinian scholars and their statement that rigid and abstract models are not always useful for the unique and complex nuragic landscape undergo heavy, sometimes polemic criticism (lines 208-232). The approach of the Spanish school of Granada is also criticized (lines 233-246).\u201dANSWER AND ACTIONS 8In the first part of the paragraph, we have abandoned some of the observations that Reviewer #1 identified as polemical in our critique: while the main points of our observations remain intact, the merits of the traditional approaches have also been better underlined. Our critique of the Spanish school of Granada has been slightly nuanced, by rephrasing some sentences. At the same time, some of its shortcomings had already been underscored by the same Spanish authors, whose critique we had also quoted. REVIEWER\u2019S OBSERVATION 9The Reviewer states: \u201cwhat is not clear to me is in how far the case study of nuraghi should be relevant for the interpretation of \u201cMediterranean societies\u201d who did not build nuraghi.\u201dANSWER AND ACTIONS 9The Authors do not believe that the analysis of Nuragic societies is immediately informative of any other processes happening in other Mediterranean contexts; at the same time, the high richness of monumental and spatial data about the Nuragic world seems to have the potential to become and represent a rather complete comparative model, even if chronological details have some degree of uncertainty. It is in this sense that we consider it relevant for the study of Mediterranean societies in general. We hope to have made this point clearer in the new manuscript. \u00a7 3.2REVIEWER\u2019S OBSERVATION 10The Reviewer, similarly to the previous observation, states that \u201cin the second part of this section inadequate polemics and the invention of the term (Nuragic scholars\u2019 empiricism) should be nuanced and brought forward in the form of fair, transparent criticism, including quotes and ideas and their deconstruction (lines 299-319).\u201dANSWER AND ACTIONS 10This section has been extensively re-written, in order to better specify the Authors\u2019 point of view and to provide a more detailed and nuanced presentation of our criticism towards traditional landscape approaches. First of all, relevant examples of work towards which our criticism is addressed are provided, with a careful description (in a necessarily synthetic way); secondly, rather than focusing on some of what we do perceive as limitations of traditional approaches, we have stressed how current ideas and formal approaches could actually be integrated. This was the original idea of the paper, and we think now we made sure that our criticism did not obscure this original intent. Chapter 4REVIEWER\u2019S OBSERVATION 11\u201cFigures 1 and 2 are reversed and their order has to be corrected.\u201dACTION 11The order of figures 1 and 2 has been reversed in the uploaded documents. Chapter 5REVIEWER\u2019S OBSERVATION 12\u201cA quote is needed in line 605  and the link to bibliography online in line 500 is dead (or mistaken?). Quotes are needed for each specific criticism, otherwise it remains a blurred \u201copponent\u201d that is targeted.\u201dACTION 12Quotes have been added specifically where requested and more generally in cases where they better needed reiteration. The dead link has been substituted with a working one. Chapter 7REVIEWER\u2019S OBSERVATION 13The Reviewer suggests that a more in-depth discussion of the possible symbolic meaning of nuraghi should be put forward, by stating that \u201cIn the case that actually visibility per se was more important than visual control, as it is stated (lines 672-674 and 691-695), it must be considered  that this hints towards a strong symbolic meaning and function of the nuraghi, beyond its obvious usefulness as landmark (line 697).\u201dANSWER AND ACTION 13We have chosen to further discuss the possible symbolic role of nuraghi in the following Discussions section of the paper (Chapter 8). This new discussion partly corresponds to the General response #3 discussed above.Chapter 8REVIEWER\u2019S OBSERVATION 14The Reviewer observes that our position towards the militaristic view of nuraghi is not entirely clear and justified by the data, by stating that \u201cAlthough they did not find clear indicators of a defensive (military) use of the monuments (lines 748-750), they still do not want to exclude it, but they do not discuss the issue. I would suggest to explain why all these contradictions would still favour such an explanation of the monuments, except for the fact that Lilliu and Webster attributed a defensive function to nuraghi .\u201dANSWER AND ACTIONS 14With regards to our interpretation of the nuraghi in a militarist sense, we have made our position clearer. We think that to a certain degree nuraghi show spatial features that could -broadly speaking- be used for site protection purposes . However, as already noted, the visual control lacks the specificity that one would expect and that has been hypothesized by the Authors who favor these interpretations. In fact, both the classic \u201cmilitarist\u201d interpretation and the one seeing nuraghi as means of \u201cterritorial control\u201d are significiantly weakened in the light of our results, but some degree of \u201cdefensiveness\u201d appears to be actually enhanced, especially with regards of the secluded position of the monuments. This detected degree of defensivess  is still not enough, in our view, to make nuraghi monuments optimised for militar defense or even territorial control. Instead, as already stated, other goals should be considered; conversely, one should consider also the possibility to be seen from faraway: in this respect, we have broadened our discussion of the possible symbolic value of the visual aspects of Nuragic towers, while also rephrasing and expanding our general point of view.REVIEWER\u2019S OBSERVATION 15\u201cAgain citations are missing, for example in line 710 (\u201cas postulated in the literature\u201d \u2013 which?).\u201dACTION 15Quotes for \u201cas postulated in literature\u201d have been added. REVIEWER\u2019S OBSERVATION 16According to the reviewer, we \u201cseem to indicate that the denigrated \u201cempirical\u201d archaeologists who have postulated this relationship  were not all wrong.\u201d and thence we would fail to \u201cmention this coincidence between both approaches\u201d, that is our and previous approaches.ANSWER AND ACTIONS 16Our text stated \u201cAt the same time, as postulated in the literature, proximity between nuraghi and access routes has been detected by our model, but not in the sense of a strict association between a nuraghe and the route terminus\u201d. The coincidence was therefore mentioned, with the caveat that such association could have been obtained in a spatially different way. We have made slight modifications to our wording and sentences, but we still feel that the overall meaning and intention remain intact, with an important difference between the results of the two different approaches.REVIEWER\u2019S OBSERVATION 17\u201cIf the authors would nuance their criticism, summarize the actual positions of the researchers they rightfully want to criticize (with proper quotes) and provide concrete arguments, the paper would improve significantly. I strongly recommend them to re-think their own position and consider if their landscape approach would not be enhanced by combining it with the results from Sardinian field archaeologists and with theoretical approaches by researchers whom they ignored for now, and joint forces might finally shed more light on the still mysterious nuraghi and their functions and meanings.\u201dANSWER AND ACTIONS 17In the second-last paragraph, rather than remarking some of the shortfalls of traditional approaches, as the reviewer highlights and criticizes, we have opted to better stress the contribution of these empirically-based scholars, both in general and specifically for the construction of the arguments discussed (as well as for the results suggested) by our paper, where traditional interpretations have served as a necessary basis for further testing. A critical reappraisal of valid and solid hypotheses is the core goal of our contribution; we feel that this was in part  demonstrated by the translation process (line 382 of the new manuscript) from traditional ideas in spatial and quantitative models. REVIEWER\u2019S OBSERVATION 18\u201cSome sentences could easily be erased from the manuscript because their only aim seems to be directly discrediting particular scholars (lines 784-786).\u201dANSWER AND ACTIONS 18While firmly rejecting such an accusation, we hope that it originated from an unfortunate wording. The sentence has been changed accordingly. Reviewer #2\u00a7 2.1REVIEWER OBSERVATION 1In paragraph 2.1, as far as the Nuragic civilization is concerned, the authors speak only of the Bronze Age, while it would be appropriate to speak also of the I\u00b0 Iron Age, as the authors do in other paragraphs.ACTION 1References to Early Iron Age have been added where appropriate.\u00a7 2.2REVIEWER OBSERVATION 2The term \"classical\", used in the text to define the most recent nuraghes with rooms with a \"tholos\" vault, should, in our opinion, be used in parentheses, so as not to give the reader who is not an expert in Nuragic civilization the doubt that they are monuments belonging to the Greek and Roman classical age.ACTION 2The term \u201cclassical\u201d has been substituted with \u201ctypical\u201d.\u00a7 4.1REVIEWER OBSERVATION 3A paleo-geomorphological analysis of the investigated area is missing. In fact, over time, some areas may have undergone substantial changes; in fact, on the edges and slopes of the plateaus, we often witness landslides and mudslides. Even considering the importance attributed to the Scalas, i.e. the natural accesses to the plateau, a mention of this aspect would be useful, also to take it into account in the analyses.ANSWER AND ACTIONS 3Further observations on the partly unstable geomorphological nature of the Giara plateau have been added. However, there is no specific study allowing for a paleogeomorphological reconstuction, which should in turn be taken in consideration by future research. REVIEWER OBSERVATION 4Equally, it would be interesting to at least mention paleoenvironmental aspects. Are there archaeozoological studies or pedological and pollen analyzes for the investigated area?ANSWER AND ACTIONS 4There are no specific archaeozoological, palynological and pedological studies for the Giara of Gesturi. The closest available data comes from the Pranu \u2018e Muru plateau, but a direct comparison between the two contexts could be misleading. These observations have been added to the manuscript.\u00a7 4.2REVIEWER OBSERVATION 5The method of retrieving data is not well specified: the authors talk about the analysis of aerial photos, but in studies of this type the direct analysis of the monuments and possibly a systematic survey of the entire territory would also have been appropriate. Was it made? Why was it not considered appropriate to do so?ANSWER AND ACTIONS 5A more complete description of the data retrieval methods has been provided, together with a better justification of the survey methods here adopted, as an effective ground check of all the nuraghi of the area has been performed by one of the Authors (D.S.). REVIEWER OBSERVATION 6As also mentioned by the authors, we do not have a precise chronological location of the monuments considered by the analyses, as very few sites have been the object of archaeological excavations. This is important, because the type of analysis presented would preferably require having architectures of the same phase as objects. It would therefore be necessary to better explain the reasons for the choices made and how the authors think they have solved the problem.ANSWER AND ACTIONS 6As requested, we have expanded our discussion on the issue of chronology: we do agree that a better chronological resolution would be preferable, but we think that the analysis still remains useful even in its absence (further details in the manuscript). The only caveat is that our results should be taken as the average of long term dynamics, the details of which are still to be assessed.AttachmentResponse to reviewers - Climbing the Giara.odtSubmitted filename: Click here for additional data file. 10 Jul 2023Climbing the Giara: A quantitative reassessment of movement and visibility in the Nuragic landscape of the Gesturi plateau PONE-D-23-01219R1Dear Dr. Vanzetti,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Peter F. Biehl, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to Questions <font color=\"black\"> Comments to the Author </font> 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #1:\u00a0All comments have been addressedReviewer #2:\u00a0All comments have been addressed********** <font color=\"black\"> 2. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 3. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 5. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 6. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0I am very pleased to see that the authors addressed all comments, provided sound answers to each criticism and used the rationale behind them to significantly enhance this paper.My admittedly harsh criticism of what I considered polemic statements in the first version has been adressed and is now minimalized by mostly clearer wording and better contextualization. The term \"nuragic scholar's empiricism\" is in my opinion still a bit difficult and lines 299-319 seem still slightly polemic, however this might (hopefully) spark a fruitful discussion with the adressed scholars.I really appreciate the extended interesting discussion on the scalas (lines 369-386) as wel as the more profound discussion of results. The bibliography and English language have both been significantly impoved. Personally, i would not let stand Kristiansen on aDNA uncommented or without citing his critics, but this is the author's decision.I look forward to seeing this important and pioneering contribution to nuragic archaeology in print soon.Reviewer #2:\u00a0The authors responded satisfactorily to the suggestions and questions. The requested improvements have been made in the text. For me the text is now publishable.********** <font color=\"black\"> 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #1:\u00a0NoReviewer #2:\u00a0No********** 26 Jul 2023PONE-D-23-01219R1 the Giara: a quantitative reassessment of movement and visibility in the Nuragic landscape of the Gesturi plateau  Climbing Dear Dr. Vanzetti:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Peter F. Biehl Academic EditorPLOS ONE"}
+{"text": "P\u2009<\u20090.001 for trend). These associations were similar when adjusting for religious upbringing and when cases occurring within the first two years of follow-up were excluded from the analysis. The association was somewhat attenuated when religious practices were removed from the model as covariates, though it remained statistically significant  (P\u2009<\u20090.029 for trend). This longitudinal study provides evidence for the first time that low religiosity in adulthood may be a strong risk factor for developing PD.Parkinson\u2019s disease (PD) is associated with low religiosity cross-sectionally. Whether low religiosity might be associated with an increased risk for developing PD is unknown. This study investigated whether low religiosity in adulthood is associated with increased risk for developing PD. A population-based prospective cohort study was conducted. Participants from the English Longitudinal Study of Aging and the Midlife in the United States study who were free from PD at baseline (2004\u20132011) and completed questionnaires on self-reported religiosity, were included in a pooled analysis. Incident PD was based on self-report. Multivariable logistic regression was used to estimate odds ratios (OR) for developing PD according to baseline religiosity, with adjustment for sociodemographic characteristics, health and lifestyle factors and engagement in religious practices. Among 9,796 participants in the pooled dataset, 74 (0.8%) cases of incident PD were identified during a median follow-up of 8.1\u00a0years. In the fully adjusted model, compared with participants who considered religion very important in their lives at baseline, it was found that\u00a0participants who considered religion \u201cnot at all important\u201d in their lives had a tenfold risk of developing PD during follow-up . Moreover, there was a dose\u2013response relationship between decreasing religiosity and increasing PD risk ( The prevalence of Parkinson\u2019s disease (PD) has increased substantially during the last three decades  from July 2010 through to July 2019, and the Midlife in the United States (MIDUS) study from January 1995 through to June 2014. ELSA is an ongoing population-based cohort study which began in 2002, that has now included over 18,000 predominantly middle-aged and older adults who are representative of the English population  and in MIDUS participants have been followed up every 9\u201310\u00a0years since the baseline survey (\u201cMIDUS 1\u201d). ELSA Wave 5 (2010\u20132011) and MIDUS 2 (2004\u20132006) were considered as the baseline for this analysis, since these were the first waves where information on religiosity, spirituality and PD were assessed. Follow-up for ELSA totaled 9\u00a0years and follow-up for MIDUS totaled 10\u00a0years.n\u2009=\u200911,644). Of these participants, those with missing data for any sociodemographic covariates (n\u2009=\u2009166), or who did not take part in at least the first follow-up after baseline (n\u2009=\u20091682), were excluded. This yielded a total sample of 9796 participants, including 7124 (72.7%) participants from ELSA and 2672 (27.3%) participants from MIDUS.To be included in this analysis, participants must have been free from PD at baseline and have completed all relevant questions on religion and spirituality  \u201cReligion very important\u201d, (b) \u201cSpirituality very important but not religion\u201d, and (c) \u201cNeither spirituality nor religion very important\u201d  were self-reported at baseline in both cohorts. Religious affiliations were recoded into three groups: (1) Christian religion, (2) non-Christian religion, and (3) no religion. Frequency of service attendance was recoded as: (1) more than weekly, (2) weekly, (3) monthly, (4) less than monthly, and (5) never. Frequency of private religious practices were recoded as: (1) daily,\u00a0(2) often, (3) rarely, and (4) never.Information on religious upbringing was assessed at MIDUS 1 (1995\u20131996), with the following question \u201cHow important was religion in your home when you were growing up?\u201d. The options included: (1) very important, (2) somewhat important, (3) not very important, and (4) not at all important.In a secondary analysis, participants in MIDUS were recategorised based on changes in religiosity prior to baseline. This was estimated by subtracting their scores for religiosity measured at baseline (MIDUS 2), from their scores for religiosity measured 10\u00a0years earlier (MIDUS 1). Participants could therefore be categorised into three groups based on pre-baseline changes in religiosity: (a) religiosity increased during the 10\u00a0years preceding baseline, (b) religiosity decreased during the 10\u00a0years preceding baseline, and (c) religiosity unchanged during the 10\u00a0years preceding baseline.During the 10-year follow-up period, participants were asked at each interview (ELSA) or survey (MIDUS), to report whether they had ever been diagnosed with PD by a medical professional. Incident PD was defined as self-reported PD at the most recent interview/survey.The following covariates, measured at baseline, were obtained by self-report in both cohorts: age in years (continuous), ethnicity , marital status , educational qualifications , smoking status , alcohol consumption , diabetes (yes/no), hypertension, (yes/no), mental health conditions , self-rated health  and physical activity levels . In addition, information on cognitive impairment  and severe mental disorders  were available in ELSA. Information on head injuries  was available in MIDUS. Missing values indicators were used for participants with missing information for these covariates.The association of baseline religiosity with incident PD during follow-up was assessed using logistic regression to determine odds ratios (OR) with 95% confidence intervals (CI). The group that reported religion as being very important in their lives served as the reference. Religiosity was also entered as a single multilevel variable to test for a linear trend across groups.Pooled and cohort-specific analyses were conducted with adjustment for possible confounders. Model 1 was minimally adjusted for age, sex and either cohort (pooled dataset), geographical region (ELSA), or sample (MIDUS). Model 2 additionally adjusted for ethnicity, education, marital status, smoking status, alcohol consumption, self-rated health, physical activity levels, diabetes, hypertension, mental health conditions, frequency of private religious practices, and frequency of religious service attendance.Several sensitivity analyses were performed to confirm the robustness of the findings. Within the pooled dataset, the analyses were repeated after: (1) excluding participants who did not profess a religious affiliation at baseline, (2) restricting the analysis to participants with complete covariate data, and (3) removing religious practices as covariates in the regression model. In ELSA, the analyses were repeated after: (1) introducing a lag time of approximately 2\u00a0years, including only PD cases identified after the first visit, (2) excluding individuals with cognitive impairment or severe mental disorders at baseline, and (3) shortening the follow-up to the first 4 years (two visits) after baseline. In MIDUS, the analyses were repeated after: (1) adjusting for religious upbringing, (2) adjusting for a history of serious head injury at baseline, and (3) recategorising participants based on a combination of religiosity and spirituality.In addition, a secondary analysis was carried out in MIDUS, which related changes in religiosity during the 10-years preceding baseline, with the subsequent risk of incident PD during follow-up.P\u2009<\u20090.05. All analyses were performed using SPSS version 28 .Statistical testing was performed two-sided at The baseline characteristics of the 7,124 participants in ELSA and 2,672 participants in MIDUS are presented in Tables Overall, there were lower levels of religiosity in ELSA than in MIDUS. In ELSA, the largest group comprised participants who considered religion not at all important in their daily lives (32.7%) whilst the smallest group comprised participants who considered religion very important in their daily lives (17.1%). Whereas, in MIDUS, the largest group comprised participants who considered religion very important in their lives (40.9%) and the smallest group comprised participants who considered religion not at all important in their lives (8.7%).P\u2009<\u20090.001 for trend). These associations were also significant in both cohort-specific analyses  were identified during a median follow-up of 8.1\u00a0years . In the fully adjusted logistic regression model Table , comparees Table .Table 3BP\u2009<\u20090.001 for trend). Compared with religiously affiliated individuals who reported religion being very important in their lives at baseline, religiously affiliated individuals who reported religion being not at all important in their lives had a greater than tenfold risk of developing PD . Complete case analysis (n\u2009=\u20099684) revealed a similar association . The associations were somewhat attenuated when removing religious service attendance , private religious practices , or both religious service attendance and private religious practices  from the regression model, though they remained statistically significant. Furthermore, there was still a linear trend across the groups when all religious practices were removed from the model (P\u2009<\u20090.029 for trend).Within the pooled dataset, lower baseline religiosity was associated with a higher risk of developing PD even when restricting the analysis to participants who professed a religious affiliation , and after excluding individuals with cognitive impairment or severe mental disorders at baseline . The association was considerably strengthened after shortening the follow-up to the first 4 years after baseline .In MIDUS, the association was strengthened after adjusting for religious upbringing  and was unchanged after adjusting for a history of serious head injury . After recategorising participants based on a combination of religiosity and spirituality, participants who considered spirituality very important in their lives but not religion  and participants who considered neither spirituality nor religion very important in their lives , were both at higher risk of developing PD compared to participants who considered religion very important.After recategorising participants in MIDUS based on changes in religiosity during the 10-years preceding baseline, compared with participants whose level of religiosity was unchanged during this period, participants whose level of religiosity decreased had a higher risk of developing PD , whereas participants whose level of religiosity increased had a lower risk of developing PD .Using prospective data from two population-based cohort studies in England and the USA, the current study shows for the first time that low religiosity in adulthood may be associated with an increased risk for developing PD, accounting for a wide range of potential confounders.The findings of this longitudinal study are consistent with previous cross-sectional studies, which showed a robust association between PD and low religiosity . Accordingly, adjusting for religious practices might have made the association more apparent\u2014by isolating the effects of intrinsic religiosity on PD. Intriguingly, this theory may be in line with a recent cross-sectional study, which showed that newly diagnosed people with PD had lower intrinsic religiosity than age-and sex- matched healthy controls, despite the two groups being similar for frequency of religious practices  were included as covariates in this analysis. On the surface, this would seem to imply that religious practices were harmful, i.e., participants with higher religiosity had a lower risk of developing PD This study has several strengths, including the prospective design, long follow-up period, use of two large and well-documented population-representative cohorts, inclusion of a wide range of potential confounders, measurement of religiosity at two different time periods in two different continents and employment of a variety of sensitivity analyses. Furthermore, the participants were not selected on the basis of religiosity or PD diagnosis. Several limitations also warrant discussion. Following previous published studies (Kamel et al., This study provides evidence for the first time that low religiosity in adulthood may be associated with an increased risk for developing PD; especially among individuals who have a spiritual, but not religious, outlook on life. If replicated by other researchers, these findings could prove important in understanding global trends in the incidence of PD (GBD 2016 Parkinson's Disease Collaborators,"}