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+{"text": "Medical education is increasingly being conducted in community-based teaching sites at diverse locations, making it difficult to provide a consistent curriculum. We conducted a randomized trial to assess whether students who viewed digital lectures would perform as well on a measure of cognitive knowledge as students who viewed live lectures. Students' perceptions of the digital lecture format and their opinion as whether a digital lecture format could serve as an adequate replacement for live lectures was also assessed.Students were randomized to either attend a lecture series at our main campus or view digital versions of the same lectures at community-based teaching sites. Both groups completed the same examination based on the lectures, and the group viewing the digital lectures completed a feedback form on the digital format.There were no differences in performance as measured by means or average rank. Despite technical problems, the students who viewed the digital lectures overwhelmingly felt the digital lectures could replace live lectures.This study provides preliminary evidence digital lectures can be a viable alternative to live lectures as a means of delivering didactic presentations in a community-based setting. Medical education is increasingly being conducted in community-based teaching sites outside of the traditional academic medical setting , At the Our institution has a community integrated structure where medical students spend the clinical portion of their training in one of six community campuses spread throughout the State of Michigan. Although this structure has many advantages, it is difficult to provide a consistent educational experience for the students. To help address this challenge, we implemented an all-day lecture series held at one of the community campuses two weeks before the end of the internal medicine clerkship.The students and faculty presenters from other campuses traveled from their home campus to the campus hosting the lecture series. End-of-clerkship feedback from the students has indicated the lecture series is both valuable and well received. Traveling to the host community, however, was inconvenient and time consuming for both students and faculty presenters. In addition, it is not practical for students at our rural medicine campus to attend due to the distance (approximately 400 miles) from the other communities.There is evidence that delivering the audio from a lecture in combination with the presenter's slides can be an effective means of delivering lectures at remote sites, and may even be as effective as traditional lectures,4. We saStudents taking the third-year required internal medicine clerkship during the 2003\u20132004 academic year at our institution were offered the opportunity to participate in the study. Those agreeing to participate were randomized into to one of two arms of the study. The random assignment of students to the two arms of the study was done within each community to control for the potential of community differences. The control group traveled to the host community campus and attended the live lectures with their colleagues who chose not to participate in the study. The experimental group stayed at their home campus on the same day and completed a parallel set of CD-ROM-based multimedia modules made from digital recordings of the previous year's lectures. They completed these digital lectures in computer laboratories in either the community campus office or within one of the teaching hospitals.The series included six lectures covering asthma, coronary artery disease (CAD), acute renal failure, liver disease, thyroid disease, and antibiotic pharmacology. The Clerkship Education Committee chose these topics based on their perceived importance and consistency with the Society for General Internal Medicine/Clerkship Directors of Internal Medicine (SGIM/CDIM) Curriculum Guide.Between the 2002\u20132003 academic year when the lectures were taped and the 2003\u20132004 academic year when the study was conducted, the clerkship faculty decided to revise the lecture series to be more case-based though the topics were kept the same. Two of the lectures, CAD and renal failure, were not modified and kept as consistent as possible with the previous year in order to conduct the study. Though there might have been minor inconsistencies between the digital and live versions of these two lectures, the same faculty member presented each lecture during both the 2002\u20132003 academic year when the lectures were taped and the 2003\u20132004 academic year when presented live. The two lecturers also attempted to keep the live lectures as consistent as possible with the digital lectures and used the same slides that they had used the previous year. While the format of the other four lectures changed, the material covered and instructional objectives remained consistent.At the end of the live lecture series, students were asked to complete a short examination that included four to five questions based on each of the six lectures. These questions were written by the presenters of the lectures and designed to assess student mastery of the lectures' key objectives. The students were informed that the purpose of the examination was to provide them with feedback on the mastery of the material and the presenters with feedback on the effectiveness of the lectures, and would not impact on their clerkship grade. After the students completed the exam, they were given a copy that included the correct answers and a short explanation for the correct answer. The exam forms contained no student identifiers, but students in the control group were asked to indicate on the examination form that they had agreed to participate in the study so they could be differentiated from the students who had chosen not to participate in the study.Students in the experimental arm of the study completed the same examination in their home community after they had completed the digital lectures. They were also asked to complete a short feedback form asking whether they had any technical problems using the modules, to rate their agreement with what the researchers felt to be three potential advantages and three potential disadvantages of the modules, and whether they felt the modules could serve as a suitable replacement for live lectures.The specific questions are listed below.Advantages of the Modules\u2022 Convenience of viewing the presentations when you choose.\u2022 Avoiding having to travel to another community for an all day lecture series.\u2022 Ability to keep copies of these presentations for use in the future.Disadvantages of these modules\u2022 Inability to ask questions of the presenter\u2022 Lack of group interaction/discussion of a topic\u2022 Just not like being in the room with the presenter. They included digitized video and audio from the taped presentation inserted as a window in the PowerPoint\u00ae slides from the presentation. As students displayed each of the slides, they were able to observe the presenter in the multimedia window discussing the slide that was being viewed.The CD-ROM modules were created using a technique developed by the first author. A manual outlining how to develop these modules is available from Nine of the items on the exam focused on the material in the CAD and acute renal failure lectures, where the lecturers presented the lectures in the same format as they had used in the previous year when the lectures were taped. The remaining 20 examination items covered material in the other four lectures. Group differences were tested for statistical significance by both an independent sample t-test for means and a Mann-Whitney test for ranks. A Levene's test for equality of variance between the two groups was also performed. These analyses were conducted separately for the subset of items covering the material in the CAD/acute renal failure lectures and the other four lectures. A power analysis was conducted to assess the magnitude of the difference between the groups that would likely be detectable given the number of students participating in the study. The coefficient alpha reliability of the exam was also estimated. The data were presented descriptively using means, standard deviations and mean ranks within the control and experimental groups. All analyses were conducted using the Statistical Package for the Social Sciences version 11. Approval for the project was obtained from the University Committee for Research Involving Human Subjects within our institution.A total of 96 students completed the internal medicine clerkship during the 2003\u20132004 academic year. As described below 56 of the students were eligible to participate in the study. A total of 29 students or 52% of the eligible students agreed to participate in the study. Complete data were available for 12 students who attended the live lectures and 17 students who completed the digital lectures.During the first rotation, there were some technical problems in a demonstration of the digital lectures. The net result was that very few students chose to participate during that rotation. During the second and third rotations, approximately two-thirds of the students agreed to participate. There were also 20 students who were ineligible to participate. These included six students from the rural medicine program at Marquette who do not participate in the live lecture series due to the distance from the other communities. Additionally, some of the communities conducted a fourth rotation of the internal medicine clerkship due to space limitations in the three regular rotations and a live version of the lecture series was not given for the students in the fourth rotation. These 20 students all completed the CD-ROM modules but because they could not be randomized between the live and digital formats, they were not able to participate in the study.Differences in the sample sizes for the two groups were due to some of the students in the live lecture group failing to mark that they were participating the study. During the second clerkship rotation, the proctor inadvertently failed to remind the students participating in the study to mark this information on their examinations when the exams were handed out.A power analysis indicated that with the number of subjects in the study, it would be possible to detect differences of nine tenths of a standard deviation with a power of 80%, p < 0.05 for a one-tailed t-test.Table The Levene's test for equality of variance between the control and experimental groups was statistically significant (p = 0.03) for the CAD and renal failure items. The variation among the scores of students who observed the CD-ROMs was almost twice as large as for students who observed the live lectures. There was no statistically significant difference among the groups for the variance of the items from the other four lectures.The coefficient alpha reliability for the items covering CAD/renal failure and the items covering the other four lectures were 0.33 and 0.66 for the 9 and 20 item scales respectively and was 0.70 for the combined 29-item exam.The 17 students who completed the digital lectures also completed a short feedback form on their experiences and impressions of the digital lecture format. These data are presented in Table There were no statistically significant differences found between students who viewed the live and CD-ROM based lectures. The observed mean scores in the two groups were in fact almost identical. Unfortunately, the small sample size limits the power of the study and confidence in which we can assert that digital lectures can be as effective as live lectures in increasing students' knowledge. The study does suggest that it is unlikely that there are large differences in the performance of students who view CD-ROM based lectures as opposed to live lectures and adds to the growing body of literature concerning the effectiveness of technology for implementing distance learning in medical education.There was a statistically significant difference in variances among the two groups for the items covering the CAD/renal failure lectures. The standard deviation in the scores was twice as large for the students who completed the digital modules. The differences in the dispersion are also evident in the range of values in each group. It is not clear why there was more variation in the scores among the students who completed the digital modules. It may be related to the technical problems encountered by many of the students in accessing the modules, though one would expect this would have resulting in extending the lower tail of the distribution but not the upper tail. It may have also in part reflected the impact of discussions that occurred during the live lectures that may have reduced the variability among the students in their responses to the examination.Despite the fact that almost all of the students experienced some technical difficulties using the modules, they all agreed and most strongly agreed the modules could serve as an adequate replacement for live lectures. They were particularly appreciative of not having to travel to another community to attend didactic presentations and having the flexibility of viewing the modules at their convenience. Of the three potential disadvantages of the format that were listed on the feedback form, they felt their inability to ask a question of the presenter was the most important. In the future we are considering using a web-based bulletin board system as a means of allowing students to ask questions of the presenter.The number of students with technical difficulties viewing the modules was surprising. We had tested the modules on a variety of different computers with very few problems. In a few cases, the CD-ROMs we distributed apparently had not been copied correctly. Additionally, we switched the video formats from MPEG to Windows media files. We assumed there would be less compatibility problems with the Windows media files given that this is a format developed by Microsoft. Unfortunately, we later found out the Windows media files require software that was not shipped with earlier versions of Windows. We expect this was a significant cause of the technical problems the students experienced.We are now using a commercial software package which greatly simplifies the process of creating the digital lectures and allows them to be distributed over the Web as well as via CD-ROM requires no special software minimizing the compatibility issues. Students who completed the fourth rotation of the clerkship and did not participate in the study were provided with the new version of the modules. Only one of the 12 students indicated they had technical problems accessing the lectures off the CD-ROM disks on which the modules were distributed and that student was able to access the modules via the Web. Such combined Web and CD-ROM distance learning formats have been shown to be effective in a number of educational settings ,7.There were several important limitations in the study. First is the very small sample size which limited the power of the study for detecting differences in the performance of the students completing the live and CD-ROM based lectures. It also increased the likelihood the two groups of students were not equivalent. Since there were no student identifiers on the exams, it was not possible to compare the characteristics of the two groups. The outcome measure was a locally developed test. While the items were written by the presenters and based on the major objectives in their lectures, there was no assessment of validity other than content validity. It is also not clear the extent the findings of this study can be generalized to other digital lecture formats.Although the data collected in this study were limited, it provides some evidence that digital lectures are both well received by students and can provide a satisfactory substitute for live lectures from a performance standpoint.The author(s) declare that they have no competing interests.DJS designed the study, developed the feedback instrument on for the digital modules conducted the statistical analyses and wrote the first draft of the paper. GSF, HSF and KK developed and gave presentations, wrote questions for the knowledge examination and edited and helped revise the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Objective. To prospectively determine the rate of unplanned extubations and contributing factors and determine whether a targeted intervention program would be successful in decreasing the rate of unplanned extubations. Design. Prospective, observational study. Setting. A 10-bed Pediatric Intensive Care Unit (PICU). Patients. All intubated pediatric patients during two time periods: September 1, 2000\u2013March 31, 2001 and November 1, 2001\u2013April 30, 2002. Interventions. After determining the rate and causes of unplanned extubation, a program was developed consisting of education and a formalized endotracheal tube taping policy. Data were then collected after implementation of the program. Measurements and Main Results. Prior to the implementation of the program, there were 10 (14.7%) unplanned extubations for a rate of 6.4 unplanned extubations per 100 ventilated days. Of the ten unplanned extubations, reintubation was required in 2 (20%). Inadequate sedation, poor taping, and improper position of the endotracheal tube were the items most frequently cited as causing an unplanned extubation. Following the program, there were two (3.4%) unplanned extubations for 1.0 unplanned extubations per 100 ventilated days. Neither patient required reintubation. There were no significant differences (P > .05) in age, weight, endotracheal tube size, or duration of intubation in the two time periods. However, there was a significant decrease in both the number (P = .03) and the rate (P = .04) of unplanned extubations after the implementation of the quality improvement program. Conclusions. The rate of unplanned extubation in a PICU can be decreased with a quality improvement program that targets the institution's specific needs. Improving quality of care in the pediatric intensive care unit (PICU) is an imperative in healthcare. Quality improvement efforts are focused on care processes with the goal of eliminating errors and adverse events. This process begins with the identification of a problem and its causative factors. Then, a plan is implemented to eliminate these factors. The results are analyzed to ascertain whether the plan has decreased the identified problem.The use of endotracheal intubation is routine in the care of critically ill children . ExtubatUnplanned extubation exposes the patient to morbidity and mortality over and above those associated with the patient's underlying disease , 7. KuraIt is more common to require reintubation after an unplanned extubation than after a planned extubation . In addiOur impression was that there was a high rate of unplanned extubations in our PICU. As a quality improvement effort, we prospectively determined the unplanned extubation rate in the PICU as well as the contributing factors. Based on these data, we developed a targeted intervention program hypothesizing that it would be able to decrease unplanned extubations.The Institutional Review Board waived the need for informed consent. The study included all intubated patients in a 10-bed PICU located in a general county teaching hospital. The PICU is staffed by board certified pediatric critical care attendants, pediatric critical care fellows as well as pediatric and emergency department residents and interns. At night, care is provided by a senior pediatric resident (PGY 2 or 3) and an intern. The fellow and attendant are available by phone and return to the hospital if needed. Nurse-to-patient ratios are 1 : 1 or 1 : 2 depending on acuity. Nurses administer sedatives and neuromuscular blocking agents as ordered by the physicians. The choice of the particular agent and the dose is based upon the patient's clinical requirements. Sedation protocols are not utilized in the PICU. Physical restraints may be used with a physician order.  After intubation, the endotracheal tube is secured with tape, a chest radiograph is performed, and the position of the endotracheal tube is adjusted, if indicated. For patients who are intubated before admission to the PICU, a chest X-ray is obtained as soon as possible after arrival and the tube is adjusted if needed. Although there is no standardized protocol for obtaining radiographs on intubated patients, they are often done on a daily basis. At the time of initiation of the project, there was no standardized policy for taping of the endotracheal tube. For the purpose of this study, an extubation was considered to be unplanned when the displacement or removal of the endotracheal tube occurred at a time other than that chosen for a planned extubation. Reintubation was defined as the replacement of the endotracheal tube within 24 hours, regardless of whether the extubation was planned or unplanned. Since both 8- and 12-hour shifts are utilized in the PICU, time periods were arbitrarily defined as 0600\u20131200, 1201\u20131800, 1801\u20130000, and 0001\u20130559.Data collected included patient's age, weight, diagnosis, indication for intubation, size of endotracheal tube, and date and time of intubation and extubation. The data were collected by the physician responsible for the patient's care while intubated. For patients who were intubated prior to arrival to the PICU, the time of admission to the PICU was documented as the time of intubation. If a patient was transferred to an outside institution or expired, the time of transfer or death was documented as the time of extubation. These were considered planned extubations. If the extubation was unplanned, the presumed cause was documented by the data collector. Any questions about the cause of the unplanned extubation were discussed with the study investigator who made the final determination. If the patient required reintubation, a new data sheet was started. Each intubation was considered a separate event. Data were collected during two time periods. Data gathered during the first time period, September 1, 2000 through March 31, 2001, were analyzed, and the rate and causes of unplanned extubation were determined. A small task force comprised of physician, nursing staff, and respiratory therapy staff was formed to identify specific areas for intervention. An intervention program was developed and subsequently implemented. The program was comprised of an education component including a didactic session to improve knowledge about sedation for the intubated patient and the complications of an unplanned extubation. In addition, an endotracheal tube taping policy was developed. This mandated that the endotracheal tube was to be secured by painting the endotracheal tube, upper lip, and cheek with skin adhesive. One piece of pink tape was used to secure the endotracheal tube by placing one end on the right cheek and drawing it across the top lip, pressing firmly for good adhesion. The tape was then wrapped around the tube for a minimum of two revolutions in a clockwise direction. Excess tape was secured to the right cheek. Using a second piece of tape on the left cheek, the procedure was repeated wrapping the tape in a spiral fashion up the tube and back down again. Excess tape was secured to the left cheek. The security of application was tested by gently pulling the endotracheal tube up and away from the patient's face. The tape on the endotracheal tube was required to be completely changed at least every 48 hours or when loose, grossly contaminated, or needed to be repositioned. A detailed procedure was written and a brief computer video was made to illustrate the proper procedure. All nurses and respiratory care practitioners were required to view the video. Competency was demonstrated by correctly performing the proper taping of the endotracheal tube and stating the policy requirements. Prior to this program, unplanned extubations were viewed as a routine part of PICU care. After the implementation of this program, a zero tolerance attitude towards unplanned extubations was adopted.The effects were evaluated in a second data collection period, November 1, 2001 through April 30, 2002. There were no changes in the use of noninvasive ventilation modalities during the two time periods. Although extensive education about sedation of the intubated patient took place, no sedation protocols were instituted, and the medications continued to be prescribed by physicians. Nursing staff could administer sedative drugs only with a physician order.P < .05.In order to standardize the number of intubated days, the unplanned extubation rate per 100 ventilated days was calculated. Ventilator days were calculated using the difference between the times of intubation and extubation in hours and minutes. Ventilated days were only counted for those patients with an endotracheal tube; ventilator days for patients with a tracheostomy were not collected or used in the calculations. Data from the first and second periods were analyzed using Mann-Whitney Utest, Chi-squaretest, or Fisher's exact test, as appropriate. Rates of unplanned extubation per 100 ventilated days for each month of the study were also determined. Data are presented as medians , except as noted. Statistical significance was defined at During the initial period, there were 68 intubations in 62 patients . PatientOf the 10 unplanned extubations in the initial part of the study, five happened between 0600\u20131200, two between 1201\u20131800, two between 1801\u20130000, and one between 0001\u20130559. In the second time interval, one occurred in the 1801\u20130000 time period and the other occured between 0001\u20130559.Inadequate patient sedation, poor taping where the endotracheal tube is not properly secured to the face or \u201cslips\u201d through the tape, improper position of the endotracheal tube either above the clavicles or at or below the carina, and unknown were the items most frequently cited as leading to an unplanned extubation . Based oThe program was instituted in September 2001 and training was completed in October 2001. Following the intervention program, there were 59 intubations in 59 patients . The patP > .05). There was no difference (P > .05) in the use of cuffed endotracheal tubes in the first time period (32% of patients) compared with that in the second period (42%). In addition, there were no changes in personnel or assignments in the two periods. However, there was a difference in the reasons for intubation between the two groups for respiratory failure and apnea. When comparing the two time periods, age, weight, endotracheal tube size, and duration of intubation were similar (n = 2), there were insufficient data to perform process control [P = .03) and the rate (P = .04) of unplanned extubations after the implementation of the quality improvement program. The ratio of the incidence rate of unplanned extubations before and after the intervention program was 0.15 with a 95% confidence interval of 0.04\u20130.59. This indicates that the postintervention rate is not greater than 59% and not less than 4% of what it was in the preintervention period.There was no apparent increase or decrease in the monthly rate of unplanned extubations prior to the institution of the intervention program . Due to  control . There wThe ultimate goal of every intervention is to improve the health and quality of life in all patients. The objective of this study was to improve the quality of care in our PICU by reducing unplanned extubations. In order to accomplish this, we used the plan (P) do (D) study (S) act (A) model . PDSA isThe first stage of PDSA is the planning (P) stage in which there is analysis of the intended area of improvement. In this case, it was determined that the rate of unplanned extubations in the PICU was well above national benchmark standards.At the same time that we determined the rate of unplanned extubations in, we also examined possible causes. Several factors that contribute to an unplanned extubation have been previously identified , 10, 13.In the study (S) phase, we recollected data to determine whether the changes achieved the desired results. Using the targeted intervention program, we were able to reduce the unplanned extubation rate from 6.4 to 1.0 unplanned extubations, per 100 ventilated days. When examining the time of day in which the unplanned extubations occurred, the intervention reduced unplanned extubations in all time periods. Nonetheless, even after education about sedation of the intubated pediatric patient, inadequate sedation continued to be a contributing factor in unplanned extubations. Clearly, improved sedation contributed to the decrease in unplanned extubations but since both unplanned extubations in the second time period were attributed to inadequate sedation, the education program was not completely effective.  Assessing the level of sedation in the intubated pediatric patient is difficult. Sedation assessment scales such as the Ramsay scale, modified Ramsay sedation protocol, and the COMFORT scale have been used in the assessment of sedation in intubated children as well as for guiding medication administration , 14\u201317. Because our interventions were successful, we acted (A) on them by adopting them on a permanent basis. Nonetheless, we must be careful in attributing our success to our interventions. Some would argue that with the small sample size, the improvement in rate of unplanned extubation was due to the Hawthorne effect  where peIdeally, the statistical process control method would have been used to investigate trends in the rate of unplanned extubation prior to the implementation of the program. However, since there were only ten unplanned extubations in the first time period and two in the second period, this method could not be utilized. Nonetheless, there was no indication that the rate of unplanned extubations had begun to decrease prior to the implementation of the program .The time periods chosen for the study were similar in both groups. They were carefully selected due to the seasonality of pediatric diseases such as respiratory syncytial virus. The six-month period when there were no data collection was to allow for this seasonality. The age, weight, size of endotracheal tube, and duration of intubation were not different in the groups. Although there were differences in the reasons for intubation in the two groups, the differences likely would have biased the results towards a higher rate of unplanned extubation in the postintervention group since the patients intubated for respiratory failure would likely have more secretions and be more ill than those intubated for apnea. The similarity in the two groups leads us to believe that the decrease in the rate of unplanned extubation was due to our interventions and not due to differences in patient groups.In conclusion, we demonstrated that the rate of unplanned extubation in a PICU can be decreased with a targeted intervention program tailored for the specific problems. This illustrates that efforts directed at improving quality of care should be based on the issues operative at that institution. By doing so, providers will be able to decrease the rate of unplanned extubations in their PICU."}
+{"text": "Tumours of the central nervous system are the second most common group of childhood cancers in 0\u201314\u00a0year olds  and represent a major diagnostic group in 15\u201324\u00a0year olds. The pilot case\u2013control study aimed to establish methodologies for a future comprehensive aetiological investigation among children and young adults.Eligible cases were newly diagnosed with an intracranial tumour of neuroepithelial tissue aged 0\u201324\u00a0years. The pilot recruited patients through Leeds and Manchester Principal Treatment Centres. Controls were drawn from general practice lists. Controls were frequency matched by age and gender.We interviewed 49 cases and 78 controls comprising 85% of the target sample size. Response rates were 52% for cases and 32% for controls. Completion of the questionnaire was successful, with a very small proportion of missing data being reported (5-10%). The age distribution of cases and controls was similar with around three-quarters of interviewed subjects aged 0\u201314. Half of cases and almost two-thirds of controls reported using a mobile phone with the majority starting between 10\u201314\u00a0years of age. Prevalence of breastfeeding was lower in cases than controls , whilst cases were more likely to be delivered by caesarean section . Cases were significantly more likely to have a birthweight >\u20093.5\u00a0kg compared to controls. Cases were also more likely to come from a family with 3 or more siblings than controls . The majority of participants (>80%) were in favour of taking either blood or saliva to aid molecular epidemiological research.Successful methods were established for identifying and recruiting a high proportion of case subjects, exploiting strong links with the clinical teams at the treatment centres. Control procedures proved more difficult to implement. However, working closely with national clinical and professional research networks will enable improved control identification and recruitment, with good prospects for collecting biological samples in the future. Tumours of the central nervous system (CNS) are the second most common group of childhood cancers comprising a quarter of all malignancies in patients aged 0\u201314\u00a0years with approximately 350 children diagnosed each year in the UK . SurvivaCNS tumours presenting in the young differ notably from those in older adults in terms of the cellular origins, pathological subtypes and anatomic site. The most common subtypes in young people are astrocytic tumours (50%) and embryonal tumours including medulloblastoma (25%) . Apart fThe only established environmental risk factor for CNS tumours is ionising radiation -8. ExposPre-school nurseries have a high prevalence and diversity of infectious disease (e.g. ) and attAs a forerunner to a population-based case control study of neuroepithelial CNS tumours in children, teenagers and young adults we aimed to undertake a pilot study involving a multidisciplinary team comprising paediatric and adolescent oncologists, research nurses, and epidemiologists. The aims were to 1) establish procedures for optimal case and control ascertainment, 2) pilot a questionnaire and study materials, 3) optimise the collection and storage of biological samples 4) develop a protocol and grant application for the full study.Eligible cases were those children and young people who were aged 0\u201324\u00a0years at diagnosis presenting with a primary intracranial tumour of neuroepithelial tissue as defined by WHO classification [Controls from the Leeds centre were randomly selected from general practice (GP) lists to identify a population-based sample and provide access to medical records. As part of the feasibility process, controls were frequency matched according to the age (0\u201324\u00a0years) and sex distribution of the case sample. GP practices were selected whose population demographic  reflected those of the larger geographical area. Once approval was obtained, a study administrator based themselves in the practice and randomly selected a list of eligible participants. Study invitation letters were distributed on behalf of the person\u2019s GP. Where a control refused to take part, replacement controls were used and the socio-demographic breakdown of response rates monitored to assess the representation of the participants. From the Manchester centre, three friend controls who fulfilled the required age and sex were selected and interviewed. Numerous GP practices were approached but despite extensive efforts and involvement with the Primary Care Research Network (PCRN), a group which supports clinical research studies involving primary care services in England, we were unable to recruit any practices (see Results).The pilot study set out to recruit and interview 25 cases and 50 controls per centre .Exposure prevalence was assessed through information collected from face-to-face interviews. The interview proforma was designed to be compatible with a large parallel international case\u2013control study covering the Nordic countries , a copy Ethical approval for the study was granted by the North West Research Ethics Committee in July 2007 (reference number 07/MRE08/46) and informed consent obtained for every participant. The study conformed to the principles embodied in the Declaration of Helsinki. The recruitment periods were September 2007 to March 2009 in Leeds and June 2008 to June 2010 in Manchester.Conditional logistic regression stratified by age (5-year age groups) and sex was undertaken to derive odds ratios (OR) and 95% CI. Adjustment for deprivation was carried out using the Townsend score of the child\u2019s address at diagnosis by the use of Townsend score quintiles based on the UK population distribution. In view of the limited sample size and power and range of possible aetiological factors involved in the development of CNS tumours in children and young people, we undertook a careful regression analysis including a small number of risk factors which were deemed important based on the epidemiological literature . A full list of risk factors collected from the interviews is provided in the Additional file Aggregating the data from across both centres yielded 49 cases and 78 controls who were interviewed. Overall, although both centres experienced some problems in terms of recruitment, across both centres we recruited 85% of the target sample size. The flowchart in Figure\u00a0Table\u00a0Response rates were 52% and 32% for cases and controls respectively out of those who were eligible for the study Figure\u00a0. We founFrom Manchester deprivation scores were available from cases who did not take part and it was found that there was no significant difference in Townsend score between the two groups. In the Leeds area deprivation was available for interviewed and non-interviewed controls and when comparing deprivation quintile there was found to be a significant trend of reducing participation with increasing quintiles of deprivation .Half of cases and almost two-thirds of controls indicated that they had used a mobile phone Table\u00a0. The majLogistic regression modelling for selected birth related and environmental factors Table\u00a0 showed sOf the 12 cases and 25 controls who responded about their willingness to provide a blood or saliva sample to carry out future biological research, all cases and controls said they would agree to provide saliva whilst 89% of cases and 81% of controls would agree to provide a blood sample.Through this pilot study, we have demonstrated that by working closely as a multidisciplinary team, recruitment of participants diagnosed with brain tumours is feasible as part of a \u2018case-control\u2019 design to address aetiological questions, despite the huge challenges facing these young people shortly after diagnosis.In terms of addressing the aims of the study, we developed successful methods for identifying and recruiting a high proportion of case subjects by exploiting our strong links with local clinicians and research nurses. Control procedures proved more difficult; nonetheless, this pilot study was informative and we propose the following recommendations to facilitate the design and recruitment of future UK case\u2013control investigations involving childhood and young adult cancer:1. Close collaboration with primary care and the National Institute for Health Research (NIHR) Comprehensive Clinical Research Network (CCRN), a body which oversees all clinical NHS-based research in England and which supports widening research participation to improve patient benefit across all clinical domains. This will help to optimise recruitment for both cases and controls.2. Engagement with the PCRN and National Cancer Research Institute (NCRI) Primary Care Clinical Studies Group (CSG) to identify general practices which are familiar with research studies, the latter a professional group helping to develop major primary care oncology research studies in the UK.3. Collaboration with the relevant Childhood Cancer and Leukaemia Group (CCLG) sub-group, e.g. the CNS sub-group, a national group of professionals dedicated to improving the delivery of care for young people with cancer.4. Collection of saliva samples for the purposes of molecular or genetic epidemiology.Both Leeds and Manchester experienced a number of problems relating to recruitment of controls via GP practices. In Leeds, procedures for identifying controls were resource intensive leading to a much lower than anticipated recruitment rate of 31%. The delayed start in Manchester meant adhering to new NHS structures and despite full ethical and Research and Development/Caldicott Guardian approval for the control recruitment protocol and acceptance of the study onto the NIHR/PCRN portfolio, little progress was made in identifying GP practices willing to participate in the research. Exhaustive efforts over a long period of time were made by the Manchester staff to engage with general practices both through the PCRN and directly to practices with little success.We believe that the new NHS General Medical Services contract for General Practice, which was implemented in 2004 and allocated certain resources to GPs based on how well they manage patient care , may have influenced the willingness of GP practices throughout Manchester to participate. We have since taken advice from the national NCRI Primary Care CSG to help develop control recruitment procedures for future research by ensuring that we work closely with the regional CCRN. We are also exploring alternative sources of control subjects such as child health records through our existing links with primary care.Completion of the questionnaire was a success, with a very small proportion of missing data being reported. The collection of biological samples would be an integral part of future epidemiological research in this field. We explored the possibility of exploiting the national CCLG tumour bank samples in conjunction with case control research projects. The Brain Tumour sub-group of the CCLG was fully supportive and indicated willingness to collaborate with future studies. All tumour and blood samples collected by the CCLG adhere to specific protocols which would be closely mirrored in future studies. Although we did not collect biological material, we did however ask participants about their willingness to provide biological samples and there was a clear consensus in favour of taking either blood or saliva to aid molecular epidemiological research. Participants also stated that saliva samples would be more readily donated than blood, particularly from younger controls.Although our pilot study had limited statistical power, findings agreed with previous aetiological work in showing an increased risk associated with birthweight greater than 3500\u00a0g . HoweverFeedback from participants has provided us with key information with which to revise the study questionnaires and recruitment procedures to ensure that participation rates in future case\u2013control studies can be maximised. It has provided a valuable insight into questionnaire design and recruitment procedures, particularly in terms of overcoming problems associated with the identification of suitable healthy control subjects.In summary, this pilot has provided us with all the elements necessary to produce a full protocol for a future UK study, including extensive documentation on all aspects of recruiting and approaching case and control subjects and their families. Findings from this pilot will provide essential information for refining the methods for a future large, multi-centre case\u2013control study.Written informed consent was obtained from the patient or their guardian/parent/next of kin for the publication of this report.The authors declare that they have no competing interest.JB and PM devised the study; DM and JA organised the interviews and gathered the data for the study from participants; SF and RF carried out the statistical analysis; RF drafted the manuscript; all authors provided critical comments and approved the contents of the paper prior to submission. All authors read and approved the final manuscript.Family questionnaire.Click here for file"}
+{"text": "Objective: Interest in flaps based on the subscapular vascular system has decreased because of the need for intraoperative patient repositioning and the inability to employ a simultaneous 2-team approach. The aims of this study are to review our experience using dorsal decubitus patient positioning for subscapular-based flap harvest and to demonstrate the effectiveness and safety of this approach. Methods: A retrospective review of all subscapular-based flap cases performed by the senior author at 2 hospital centers from 1995 to 2010 was conducted. Variables studied included indications for reconstruction, flap characteristics, and postoperative complications. A longitudinal roll placed between the scapulae as well as an optional perpendicularly placed shoulder roll are used to achieve dorsal decubitus patient positioning. Results: One hundred five flaps were performed during the study period, and dorsal decubitus positioning was used in all cases. Eighty-four flaps were free and 21 were pedicled. Indications for reconstruction included cancer resection (n = 58), trauma (n = 32), infection (n = 9), and others (n = 6). A simultaneous 2-team approach was carried out in 70 cases. Major complications included 9 cases of arterial or venous thrombosis/insufficiency, 2 of which resulted in total flap failure. Intraoperative conversion to lateral decubitus positioning was never required. Conclusions: Dorsal decubitus harvesting for subscapular-based flaps is a practical and effective technique that enables a simultaneous 2-team approach in complex reconstructive cases. Previous limitations of these highly versatile flaps, such as the need for intraoperative patient repositioning, can thus be avoided. This approach is employed for all subscapular-based flap reconstructions performed by the senior author. In reconstructive surgery, the subscapular vascular system is an important anatomical crossroad that provides a wide variety of flaps: the latissimus dorsi flap (LDF), scapular or parascapular fasciocutaneous flaps, serratus muscle flap, scapular bone flap, and thoracodorsal artery perforator (TAP) flap. The LDF, harvested in either muscular or musculocutaneous forms, remains the most commonly used flap of the subscapular system.,5,7-9First described in 1906 by Iginio Tansini to permanently cover deficits of the anterior thorax following radical mastectomies,3-14The LDF has very predictable vascularization with a robust pedicle measuring up to 8 cm in length12Over the last few decades, interest in flaps based on the subscapular vascular system has decreased. We believe this trend is largely due to inconveniences related to the traditional lateral decubitus harvesting position with which intraoperative position changes are necessary and simultaneous 2-team operations are often impractical. The aims of this study are to review our experience using an alternative dorsal decubitus patient position for raising a wide range of subscapular-based flaps and to demonstrate the effectiveness, safety, and potential limitations of this approach.A retrospective review of all subscapular-based flap cases performed by the senior author at 2 hospital centers between 1995 and 2010 was conducted. All patient charts were reviewed to determine the underlying etiology and specific location of each reconstructed tissue deficit. Whether a 1- or 2-team approach was used in each case was also documented. Other variables studied included age; sex; mode of tissue transfer; flap composition; and size/quantity of the cutaneous, muscular, and osseous components. All incidences of complications were documented to determine the overall success rate of these procedures. Complications were classified as either minor or major. Major complications were defined as those cases requiring operative intervention. Only patients having undergone a minimum of 1-year postoperative follow-up were included in this study.With the patient standing, the usual key landmarks are identified: the inferior border of the scapula, the posterior midline corresponding to the spinous processes, and the superior border of the iliac crest. The anterior border of the muscle is then delineated while the patient places his or her hands on the hips and pushes downward toward the pelvis. Once the patient is fully anesthetized, a first cushion is placed under the spine along the longitudinal axis of the body and, if deemed necessary to optimize exposure, an optional second cushion is positioned perpendicularly just under the scapular belt, thereby forming a \u201cT-Roll.\u201d Positioning the patient in this manner permits full access to all components of the subscapular vascular system. Prior to incision, the anterior border of the LD muscle is always verified via digital palpation as it can be displaced posteriorly in this position; the anterior border is then injected with a diluted solution of 500 mL of normal saline and 1 mg of epinephrine 1:1000. The initial skin incision is then made according to the composition of the flap to be harvested. If only a muscular component is envisioned, the incision is made just anteriorly to the demarcated border of the LD muscle; otherwise, it is made with respect to the area of the planned skin paddle.In the case of LD musculocutaneous flaps, subcutaneous dissection is performed until the anterior border of the LD is identified; in cases not requiring a cutaneous component, a direct incision is made to the deep fascia and suprafascial dissection is performed as needed. Once the anterior border of the LD is reached, the areolar plane distinguishing it from the serratus muscle is identified; the LD's natural adhesions to the serratus muscle slips are then separated. At this stage, it is useful to suture the skin of the thoracic wall onto itself; doing so optimizes exposure and allows the assistant to exert countertraction on the serratus fascia as pedicle dissection is carried out into the axillary region in the plane between the serratus anterior muscle and the LD. The first vascular branch encountered is the serratus branch and it is normally ligated, unless a serratus muscle component is planned.In cases where a chimeric osseous flap is planned, care is taken to preserve the angular branch; when present, the angular artery may originate from either the thoracodorsal artery or the serratus branch. Once the branches of the subscapular system are identified, the minor perforating arteries are progressively ligatured allowing additional length for advancing the pedicle as necessary. Axillary exposure is maximized with an assistant holding the ipsilateral arm opposite the site of the dissection, thereby facilitating pedicle isolation. Dissection proceeds and the thoracodorsal nerve and circumflex scapular branch are sequentially met. We prefer to isolate the pedicle at the axillary junction; this not only maximizes the vessel length and, in the case of pedicled reconstructions, the arc of rotation, but enables us to preserve the circumflex scapular branch which may permit the addition of an osseous component (if the angular branch is either absent or severed) or scapular/parascapular fasciocutaneous component .As referred to earlier, the various branches of the subscapular vascular system may be preserved and utilized to add serratus muscle, scapular bone, or scapular/parascapular fasciocutaneous elements. The harvesting of the aforementioned flap components may take place in a sequential fashion as the subscapular pedicle dissection proceeds from caudal to cephalad. Once the serratus arterial branch is met, the surgeon may opt to preserve it to include up to 3 or 4 serratus digitations as part of a chimeric flap; in such cases, the serratus branch is dissected under loupe magnification and the secondary vascular branches preserved up to their entrances into their respective muscle slips. The serratus digitations forming the flap may subsequently be released anterior to posterior from their attachments to the thoracic cage with the help of a periosteal elevator. The angular artery, which is the next branch encountered as the dissection proceeds cephalad, may be followed to the inferior angle of the scapula to raise an osseous flap segment. The scapular attachment of the teres major muscle is cut and detached, thereby exposing the bone's lateral edge and associated distal angular artery. The infraspinatus muscle is then incised according to the dimension of underlying scapular bone desired. The osteotomy is performed with an oscillating saw; importantly, the inferior angle of the scapula should be preserved. The circumflex scapular artery branch is particularly useful for adding a distinct fasciocutaneous component in chimeric flaps. The branch is followed into the triangular space bordered by the teres minor above, teres major below, and long head of the triceps laterally. A corresponding scapular or parascapular skin paddle is designed and harvested. In the case of chimeric flap harvest, this fasciocutaneous component may then be passed retrograde through the triangular space to join the other flap component(s) derived from the subscapular vascular system.Finally, once all the necessary flap components have been raised, the pedicle  may be divided from its axillary origins with sharp dissecting scissors prior to transposition, if applicable. The thoracodorsal nerve is preserved up to the division of the flap so as to minimize the traction transferred to the pedicle. Closure of the donor site is performed following placement of closed suction drains into superficial and deep planes; a third suction drain is placed at the site of raised scapular bone if applicable. A running horizontal mattress suture with 2-0 Vicryl is done on the free anterior margin of residual LD muscle to establish good hemostasis; the muscle edge is then sutured back onto the lateral thoracic wall. Final closure is subsequently completed in 2 planes .2 (range: 21-460 cm2). Multiple skin paddles were harvested in 29 patients. In cases involving a scapular bone segment, mean osseous flap length was 9.8 cm (range: 5-14 cm). Overall, 24 of the flaps in this series were chimeric and contained at least 2 separate tissue components based on their respective arterial pedicles which were all derived from the subscapular system. A 2-team approach was employed in 70 cases (66.7%) with cancer resection or preparation of the recipient site being performed concurrently with flap harvest , with a mean age of 47.6 years (range: 14-78). Eighty-four flaps were free and 21 were pedicled. Indications for reconstruction included cancer resection, soft tissue loss secondary to trauma, postinfectious deficits, soft tissue loss secondary to burn, upper extremity functional loss, facial paralysis, and recurrent fistula . The com harvest . IntraopOverall, 1.9% of patients had minor complications and 8.6% had major complications giving a total rate of 10.5%. Minor complications were restricted to the donor site and included 1 seroma and 1 hematoma. There were 9 cases of arterial (n = 5) and venous (n = 4) thrombosis or insufficiency. Six of these flaps presented with some degree of necrosis; 4 of these were partially salvageable and exhibited only partial flap loss, while 2 of the cases resulted in complete flap loss for an overall failure rate of 1.9% .Over the last 100 years, myocutaneous LDFs and other derivatives of the subscapular system have had a growing range of applications. The significant quantities of available tissue as well as their robust vascular pedicles make these flaps highly useful tools in a wide variety of reconstructive scenarios. However, through innovations in microsurgical technique and evolving 3-dimensional anatomic descriptions of the body's vascular territories, many reconstructive surgeons have moved away from subscapular-based flaps in favor of free tissue transfer from alternate donor sites.,One explanation for the decreasing interest in flaps from the subscapular region is the need to place the patient in the lateral decubitus position to facilitate harvesting. Lee and Mun42 and when indicated, scapular bone in complex chimeric flaps, demonstrates the effectiveness of this technique in obtaining sufficient exposure. Using our proposed positioning technique, the surgeon is enabled to harvest the required tissue in an amount equal to that achievable in the traditional lateral decubitus position. Importantly, conversion to the lateral decubitus position was never required in our series; for this reason, we strongly believe that this dorsal decubitus approach is applicable to the full spectrum of subscapular-based flaps. Moreover, the TAP flap can also be raised with the patient placed in this position.In our study, we present our experience using the dorsal decubitus position for subscapular-based flap harvest in complex reconstructive cases. To the best of our knowledge, our team is the first to publish and describe this novel solution to the current logistical limitations of subscapular-based flaps. The positioning for this technique is extremely simple, requiring only the installation of 1 or 2 cushions along with full prepping of the ipsilateral upper extremity to allow intraoperative traction as described earlier. Our ability to harvest cutaneous paddles measuring up to 460 cmThe low total failure rate of 1.9% observed in this study demonstrates the safety of this technique. Overall, 70 combined procedures were performed allowing 2 surgical teams to work simultaneously on separate sites, therefore optimizing operating room time. Since the dorsal decubitus position obviates the need for intraoperative position changes, it may further decrease the procedure length; this benefit also applies to cases that are carried out by a single surgical team. Manipulation of the initial sterile field is also avoided thereby limiting the risk of field contamination. Consequently, patient morbidity may be potentially lessened not only due to decreased time under general anesthesia but by minimizing the chances of postoperative infection. In addition, our technique has possible economic implications as a result of reduced operating room\u2013associated costs. Further studies being planned by our team that prospectively compare harvest times and complication rates between subscapular-based flaps raised in the dorsal decubitus and lateral decubitus positions will serve to shed further light on the proposed benefits of this technique.Flaps based on the subscapular vascular system, whether pedicled or free, are reliable and versatile tools that provide excellent coverage for the reconstruction of a wide range of tissue deficits in various anatomical locations. Harvesting these flaps in the dorsal decubitus position, as described by our team, provides the reconstructive surgeon with an effective and safe method of achieving flaps that match those obtainable in the conventional lateral decubitus position, regardless of the required volume or tissue composition. Furthermore, a 2-team approach is rendered possible in almost any operative scenario. In cases where a subscapular-based flap is deemed the most suitable option for reconstruction, the application of the described dorsal decubitus technique may serve to eliminate many of the limitations associated with the conventional harvesting method."}
+{"text": "MGA271 is an Fc optimized humanized IgG1 monoclonal antibody that binds to B7-H3 (CD276), a member of the B7 family, currently undergoing Phase I testing. The Fc domain is engineered for enhanced binding to the activating Fc\u03b3R, CD16A, and decreased binding to the inhibitory Fc\u03b3R, CD32B. B7-H3 has limited expression in normal tissue and high expression in multiple tumors including melanoma (M), squamous cell cancer of the head and neck (SCCHN) and non-small cell lung cancer (NSCLC). The correlation between B7-H3 overexpression and poor prognosis in certain cancers suggests a role for B7-H3 in tumor escape. Despite the clinical success of agents including anti-CTLA-4, anti-PD-1 and anti-PD-L1 antibodies, the majority of patients with M, SCCHN or NSCLC progress nonetheless, and substantial unmet need exists for these patients. The underlying hypotheses for combining MGA-271 (anti-B7-H3) with pembrolizumab (anti-PD-1) are: 1) combining immune-modulating agents may mediate additive or synergistic antitumor activity (e.g. anti-CTLA-4+anti-PD-1), and can do so where neither single agent has pronounced antitumor activity (e.g. anti-PD-1+anti-LAG-3), 2) coordinate engagement of both innate and adaptive immunity, 3) both agents may enhance the immune response against tumors via modulation of T cell immunosuppression, and 4) limited expression of B7-H3 on normal tissues may help focus an immune attack on tumors, limiting the risk of immune-related adverse events (irAEs) resulting from the disruption of self-tolerance, allowing MGA271 to be combined more readily with other immune-modulating agents, including anti-CTLA-4 and anti-PD-1 antibodies. /PD-1 /PD-L1.This US, multi-center, open-label trial (NCT02475213) enrolls patients with advanced B7-H3-expressing SCCHN, M, or squamous NSCLC. Progression on previous checkpoint inhibitor is allowed. Following a 3+3 +3 dose escalation scheme, successive cohorts of patients will receive escalating doses of weekly IV MGA271 beginning at 3 mg/kg, and a fixed dose of IV pembrolizumab (2 mg/kg) administered every three weeks. Both study drugs will be administered starting on Day 1 of the study and given for up to one year. A three-cohort expansion phase will open at the established MTD with 16 patients each with M, SCCHN and NSCLC. The primary objective of this study is to determine the safety, dose-limiting toxicity and maximum tolerated dose of the MGA271/pembrolizumab combination. Secondary objectives include evaluation of pharmacokinetics, pharmacodynamics and preliminary anti-tumor activity of this combination. This novel study will provide the first clinical assessment of coordinated targeting of both the B7-H3 and PD-1/PD-L1 axes in patients with advanced cancer.ClinicalTrials.gov identifier NCT02475213."}
+{"text": "The diagnosis of IgE-mediated cow\u2019s milk allergy is often based on anamnesis, and on specific IgE (sIgE) levels and/or Skin Prick Tests (SPT), which have both a good sensitivity but a low specificity, often causing positive results in non-allergic subjects. Thus, oral food challenge is still the gold standard test for diagnosis, though being expensive, time-consuming and possibly at risk for severe allergic reactions.The aim of the present study was to perform a systematic review of the studies that have so far analyzed the positive predictive values for sIgE and SPT in the diagnosis of allergy to fresh and baked cow\u2019s milk according to age, and to identify possible cut-offs that may be useful in clinical practice.A comprehensive search on Medline via PubMed and Scopus was performed August 2017. Studies were included if they investigated possible sIgE and/or SPT cut-off values for cow\u2019s milk allergy diagnosis in pediatric patients. The quality of the studies was evaluated according to QUADAS-2 criteria.The search produced 471 results on Scopus, and 2233 on PubMed. Thirty-one papers were included in the review and grouped according to patients\u2019 age, allergen type and cooking degree of the milk used for the oral food challenge.A/L or when SPT with commercial extract are above 6\u00a0mm or Prick by Prick (PbP) with fresh cow\u2019s milk are above 8\u00a0mm. Any cut-offs are proposed for single cow\u2019s milk proteins and for baked milk allergy in children younger than 2\u00a0years. In Children \u2265\u20092\u00a0years of age it is hard to define practical cut-offs for allergy to fresh and baked cow\u2019s milk. Cut-offs identified are heterogeneous.In children <\u20092\u00a0years, CMA diagnosis seems to be highly likely when sIgE to CM extract are \u2265\u20095 KUNone of the cut-offs proposed in the literature can be used to definitely confirm cow\u2019s milk allergy diagnosis, either to fresh pasteurized or to baked milk. However, in children <\u20092\u00a0years, cut-offs for specific IgE or SPT seem to be more homogeneous and may be proposed. Cow\u2019s milk (CM) is one of the first causes of food allergy in the first years of life  and of fIt has been shown that, the greater the food-sIgE levels and the SPTs wheal size, the higher the chances that patients react during an OFC . This isThe aim of this study was to compare, in children with suspected CMA, the levels of sIgEs and the wheal sizes of SPTs for CM or its three main allergenic molecules , \u03b2-lactoglobulin (\u03b2LG), and casein) with the Reference Standard (RS) test, OFC, in order to identify any validated cut-off value. We analyzed available data from a methodological point of view and tried to provide practical clinical indications for the diagnosis of CMA in children. At the best of our knowledge, such a classification has never been considered in previous studies \u20138.We included studies in which authors looked for a cut-off value for SPTs or sIgEs levels for the diagnosis of CMA in children. In most cases, diagnosis was based on the results of the OFC. Studies were also considered whenever a clear relationship between CM exposure and allergic reaction was highlighted and sIgE or SPTs were carried out.Studies were excluded if information was not specific enough for CMA, or if the Authors identified the optimal cut-off only (meaning a cut-off based on the best combination between sensibility and specificity), which does not allow to adequately select patients at high risk of reacting to the OFC.We included children with suspected CMA.We searched for cut-off values for CMA diagnosis using CM extract, \u03b1LA, \u03b2LG, casein, for sIgE or SPT, and using fresh milk for PbP.On August 2017, we performed a comprehensive search on Medline via PubMed and Scopus, by using the strings \u201csIgE\u201d or \u201cspecific IgE\u201d or \u201cSPT\u201d or \u201cskin prick test\u201d and \u201cmilk allergy\u201d or \u201cmilk hypersensitivity\u201d. Search was not restricted by publication type or language or study design. If any relevant paper was identified afterwards, we included it as well , 10, 11.We checked reference of all included studies and reviews, for additional references as well.For each string, two authors independently screened titles and abstracts to consider for inclusion all potential identified studies. Full texts were searched as well, to identify studies for inclusion. We resolved disagreements through discussion or, if required, by consultation with a third person. Data extraction from reports was in duplicate and in case of doubts we directly contacted the authors to obtain and confirm data. Studies were all widely discussed in detail and evaluated by the authors in a standardized and independent manner.We recorded the selection process to complete a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram Fig.\u00a0.Fig. 1FlThe methodological quality of the included studies was evaluated according to criteria proposed by QUADAS-2 . In ordesIgEs levels or SPT wheal size;< 2\u00a0years;>2\u00a0years;any age;patients\u2019 age, enrolling children:CM: fresh pasteurized CM (or CM formula in children <12\u00a0months of age);baked milk: extensively heated CM .the cooking degree of CM administered during the OFC:The search identified 2233 articles of potential interest on Medline, and 471 articles on SCOPUS. After the selection process, a total of 31 articles were included in this systematic review. Of these, 22 referred to the cut-off for sIgE and 13 for SPT cut-offs (4 proposed cut-offs for both)  and 7/13 (53.8%), respectively were prospective , 13\u201326, Most studies analyzed the role of sIgE and SPTs for CM in patients allergic to fresh pasteurized milk , 27\u201339. According to QUADAS-2 evaluation: a) for sIgE studies: patients\u2019 selection was considered at low risk for both bias and applicability in 8 studies, index test choice was at low risk for bias and applicability in all the studies, reference standard in 10 and flow and timing only in 5 Fig.\u00a0; b for SA/L.Table\u00a0A/L [A/L [A/L [A/L [A/L; \u03b2LG: 0.35\u20139.91 KUA/L; casein: 0.78\u20136.6 KUA/L) [A/L cut-off for CM extract with a 97.5% specificity [As for sIgE against CM, considering those studies including only children younger than 2\u00a0years, two prospective studies with a good QUADAS-2 evaluation and with significant patients\u2019 numbers showed quite similar cut-offs for a 95% positive predictive value (PPV) (\u22653.5 KUA/L  and \u22655 KA/L [A/L ), even iA/L [A/L  and 50 KA/L [A/L . Four paA/L [A/L , 21, 33 6 KUA/L) , 19, 21.Four studies evaluated PPV of SPTs through commercial extracts Table\u00a0. StudiesTwo papers evaluated a possible cut-off using PbP for CMA in children younger than 2\u00a0years , 37 and Three studies analyzed the diagnostic efficacy of sIgE against CM or its allergenic proteins in patients allergic to baked milk Table\u00a0 9, 24, , 24, 40.Three studies investigated a possible cut-off for SPTs using milk extracts or its proteins to diagnose allergy to milk that was extensively cooked in a grain matrix (muffins). Only two of these studies reached a conclusion Table  5, 20, , 20, 36.Over the last years, several studies have looked for cut-offs for sIgE or SPTs able to predict CMA without the need to perform an OFC.patients\u2019 age. Most of the studies on fresh pasteurized CMA diagnosis included children aged from few months to several years. Only the paper from Chung  enrolledtype of allergen. Several studies showed that SPT mean wheal diameter is usually different between commercial extracts and fresh food , 41;cooking degree of the milk. It is well known that CM proteins are modified by exposure to high temperatures, which not only modify the conformational epitopes, but partly the sequential ones as well. Heating is one of the most common technological treatment applied to milk processing and it may have different effects on the binding of IgE to proteins. Mild treatments are not sufficient to reduce the allergenicity of milk as it has been shown for pasteurized milk, which is able to elicit allergic responses in milk allergic patients . On the To find more homogeneous cut-offs, we grouped the studies according to:different statistical methods (e.g. PPV or specificity). Two different kinds of cut-offs values are proposed in literature, both for SPT and for sIgEs: those based on a high PPV (95% PPV) and those based on a high specificity (95% specificity). The first ones, being based on the predictive value, depend on several factors, above all on the prevalence of allergy in the studied population, background history, sex, etc., and are applicable in allergy centers where it is assumed that the diagnostic criteria and the prevalence of food allergy are similar to those found in those studies providing the values. On the contrary, cut-off values based on 95% specificity do not change with the prevalence of the disease in the population and give us the possibility to better select children to test with OFC, given the high risk of a positive challenge. These two kinds of cut-off values may produce different results even in the same study population .variations in the chosen level of predictive value in different studies (e.g. 90% vs. 95%) may substantially change the proposed cut-offs.methodological quality ; d) differences in patients\u2019 selection or in the definition of a positive OFC  or unknown issues could explain these differences. As for baked milk allergy, there are only a few studies investigating cut off values for both specific IgE and SPT, and they showed a low methodological value. However, using CM extract, cut-offs seem to be higher if compared with those for fresh pasteurized milk. A single prospective study with a low risk of bias and applicability showed a 100% PPV for wheal diameter cut-off value of 7\u00a0mm when fresh CMA patients were pricked with baked cake for predicting baked CMA [in children \u22652\u00a0years of age, it is hard to define practical cut-offs for CMA. The cut-offs proposed for SPT with commercial extracts or fresh milk are heterogeneous, probably because most of the studies included children of any age and with no differentiation in age groups. A large variability in cut-offs has been recorded for single CM proteins as well, especially for sIgE levels, even when selecting methodologically valid studies using the same statistical methods. For example, two DBPCFC prospective studies , 16, witaked CMA .Given the large variability of the proposed cut-offs, it is hard to propose practical clinical indications. However:A/L or when SPT with commercial extract are above 6\u00a0mm or PbP with CM are above 8\u00a0mm, the real need for a diagnostic confirmation of CMA through an OFC should be carefully evaluated.No proposed cut-off can be used to definitely confirm a diagnosis of CMA, either with fresh pasteurized or with baked milk. Cut-offs may be affected by many factors, and especially PPV cut-offs may be considered as useful only in the same allergy unit in which they were detected, and may be extrapolated to other centers only if they have similar allergy prevalence. However, with these limits, in children <\u20092\u00a0years, when sIgE against CM are above 5 KU"}
+{"text": "To this aim, we exploited the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) data set, selected a subpopulation of 591 patients with an overlapping clinical history and analyzed treatment outcome according to dosage \u221220 or 40\u2009mg per day of citalopram. We found that sociodemographic characteristics affected treatment response in the same direction in the two dose groups, but these effects reached statistical significance only in the 40\u2009mg per day dose group. In the latter, higher improvement rate was associated with having a working employment status (P=0.0219), longer education (P=0.0053), high income (P=0.01) or a private insurance (P=0.0031), and the higher remission rate was associated with having a working employment status (P=0.0326) or longer education (P=0.0484). Moreover, the magnitude of the effect of the sociodemographic characteristics on mood, measured as the percent of patients showing a positive outcome when exposed to favorable living conditions, was much greater\u2014up to 37-fold\u2014in the 40 compared to the 20\u2009mg per day dose group. Overall, our results indicate that citalopram amplifies the influence of the living conditions on mood in a dose-dependent manner. These findings provide a potential explanation for the variable efficacy of SSRIs and might lead to the development of personalized strategies aimed at enhancing their efficacy.Selective serotonin reuptake inhibitors (SSRIs), the most commonly prescribed antidepressant drugs, have a variable and incomplete efficacy. In order to better understand SSRI action, we explored the hypothesis that SSRIs do not affect mood  A new hypothesis, the undirected susceptibility to change hypothesis, predicts that SSRI treatment does not drive changes in mood per se but, by increasing brain plasticity, creates a window of opportunity for a change in mood that is driven by the quality of the living conditions.2 In particular, the serotonin increase induced by SSRIs enhances brain plasticity and thus renders the individual more susceptible to the influence of the living conditions. The main consequence is the lack of a univocal outcome of SSRI administration: in a favorable environment treatment leads to a reduction of symptoms; by contrast, in a stressful environment it may lead to a worse prognosis. As a further consequence, SSRIs are expected to amplify the influence of living conditions on mood in a dose-dependent manner. Such hypothesis is supported by preclinical data5 showing that fluoxetine treatment leads to an improvement of the depression-like phenotype when administered in an enriched condition, while it leads to a worsening when administered in a stressful condition. In addition, SSRI treatment consequences on selected end points, such as vulnerability to obesity, have been shown to be dependent on the quality of the environment.7 Finally, a number of clinical studies have shown that the environment moderates the effects of antidepressant treatment.13 However, the approaches used so far in these studies do not allow for assessing the effect of SSRIs on the susceptibility to the influence of the living conditions.Antidepressant drugs are the current standard treatment for major depressive disorder (MDD) and, among these, selective reuptake inhibitors (SSRIs) are the most commonly prescribed. However, their efficacy is variable and incomplete: 60\u201370% of depressed patients do not experience remission and 30\u201340% do not show a significant response.14 Our prediction was that the 40\u2009mg per day dose, compared to the 20\u2009mg per day dose, would amplify the influence of sociodemographic characteristics on patients' mood, since it should increase plasticity to a higher degree and thus lead to greater susceptibility to the environment. We therefore predicted citalopram to affect susceptibility to the living conditions in a dose-dependent manner. In addition, since we hypothesized that the environment drives the change in MDD induced by SSRIs, we predicted that patients living in conditions associated to a high quality of life should show a more effective response to treatment.The main aim of the present study was to test whether SSRI treatment amplifies the influence of the living conditions on mood in a dose-dependent manner. We thus exploited the data collected in the framework of the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study. We considered a subpopulation of 591 patients having similar MDD severity and overlapping treatment history, and analyzed the efficacy of treatment between weeks 4 and 6 according to the dose received\u2014either 20 or 40\u2009mg per day of citalopram. Longer or different treatment periods or other patients' groups could not be considered without losing data validity because of the limitations imposed by the STAR*D trial design. The socioeconomic characteristics included in the analysis are proxy of the quality of the patient's life environment and widely considered reliable indicators of socioeconomic status.15 In brief, STAR*D was a multisite, prospective, randomized, multistep clinical trial conducted in the United States of America aimed to determine which of several treatments would be most effective for outpatients with nonpsychotic MDD.16 The study was conducted at 18 primary care and 23 psychiatric care centers and enrolled 4041 nonpsychotic MDD patients, aged 18\u201375, with a baseline score \u2a7e14, on the 17-item Hamilton Depression Rating Scale (HAM-D17).This paper is based on the STAR*D  study data. The design details of STAR*D are described accurately elsewhere.http://www.ids-qids.org/.MDD symptom severity in the STAR*D clinical trial was measured longitudinally using the 16-item Quick Inventory of Depressive Symptomatology (QIDS). The QIDS is a briefer version of the more commonly used 30-item Inventory of Depressive Symptomatology\u2014IDS. The QIDS is available in the clinician and self-rated version and has been designed to assess the severity of depressive symptoms through the evaluation of all the criterion symptom domains designated by the American Psychiatry Association Diagnostic and Statistical Manual of Mental Disorders\u20144th edition (DSM-IV) to diagnose a major depressive episode. Each item was scored on a scale from 0 to 3 points: 0 indicating no problem and 3 indicating severe problem. Total score ranges from 0, that is, not depressed, to 27, that is, most depressed. For further details on the validity, reliability, generalizability, scoring and interpretation, see 17 According to the Guidelines for discontinuing participants from the randomized treatment study, patient drop out could be due to a number of reasons including participant request and decision by clinicians to discontinue the study is in the best interest of the participant.At Level 1, all participants were treated with citalopram, a SSRI, for a minimum of 8 weeks and were encouraged to complete 12 weeks to maximize benefit. All participants started treatment with a dose of 20\u2009mg per day citalopram, with clinical visits at 2, 4, 6, 9 and 12 weeks. To ensure satisfactory dosing for an appropriate period of time, treatment was conducted using measurement-based care. This included flexible dosing recommendations based on symptom and side effect at each treatment visit.17Dose adjustments were decided according to the QIDS-Clinician-rate (QIDS-C16) score: QIDS-C16\u2a7d5, continue current dose; QIDS-C16=6\u20138, continue or increase current dose according to the clinician assessment; QIDS-C16\u2a7e9, increase current dose. In addition, if the reduction in baseline symptom severity was found to be <20% at week 4, the initial dose (20\u2009mg per day) was raised to 40\u2009mg per day, assuming tolerable side effects. At week 4, participants with intolerable side effects could move to the next treatment level.In the present study, data from level 1 of STAR*D were considered. Only patients having similar MDD severity and overlapping treatment history were included in the analysis. In particular, we selected patients treated with a dose of 20\u2009mg per day for the first 4 weeks following enrollment, showing a QIDS-SR16 score equal to 6\u201310 (mild depression) on week 4 and whose information concerning the QIDS score on week 6 was available . Longer 14 sex, race, marital status, employment status, insurance status, education, income, experience of traumatic events and drug abuse. In addition, we analyzed the following descriptors of the onset and progression of the psychopathology: age at onset of first major depressive episode (MDE), number of MDE, difference in QIDS-SR16 score between enrollment and week 4. Education was shown as years of schooling completed; we considered two levels of education: <college (<16 years) and \u2a7ecollege (\u2a7e16 years). Income was analyzed subdividing the examined population into three monthly income classes: the low-income group was set at \u2a7d$1000, while middle and high income groups at $1000\u2013$2500 and >$2500, respectively. Employment status was analyzed considering only two conditions: employed  and unemployed (unemployed and retired). For marital status we considered: never married, married (married or cohabiting) and no more married . Finally, we considered only two ethnicities, Caucasian (white) and non-Caucasian .The efficacy of the two dosing regimens was analyzed in relation to a number of sociodemographic characteristics considered as proxy of the socioeconomic status and the quality of the living environment of the patient:MDD symptom severity was measured using the QIDS-SR16. Remission was defined as a QIDS-SR16 score \u2a7d5. In order to determine the effects of citalopram treatment, according to sociodemographic characteristics, we considered three variables: (i) percent of patients showing an improvement, measured as a reduction \u2a7e1 in QIDS-SR16 score between week 4 and 6; (ii) percent of patients achieving remission, measured as the attainment of a QIDS-SR16 score \u2a7d5 at week 6; and (iii) variation in the QIDS-SR16 score, measured between weeks 4 and 6.18 For this reason, we additionally computed the percent of improvement or remission in the favorable  and in the unfavorable  sociodemographic condition, and the RR of unfavorable versus favorable condition, with the corresponding 95% confidence interval. Finally, the preventive fraction (1\u2212RR) was computed to estimate the effect size and thus the magnitude of influence of the living conditions on the outcome. As in our study the outcome measured is a beneficial one, that is improvement or remission, values of 1\u2212RR>0 indicate that the unfavorable condition decreases the probability of the beneficial outcome compared to the favorable condition, and vice versa. The variation in the QIDS-SR16 score between weeks 4 and 6 was analyzed with analyses of variance including sociodemographic characteristics as between-subject factors. Separate analyses of variance were performed for each sociodemographic characteristic within the two treatment doses. Post hoc comparisons were performed using the Tukey's test. The variation in percent of improvement, stationary and worsening according to sociodemographic characteristics has been analyzed independently in the two dose groups with X2-test.Of the 4041 participants, a sample of 591 patients was identified according to the selection criteria . SummaryOf 591 patients who comprised the evaluable sample, 357 (60.4%) were treated with 20\u2009mg of citalopram per day and 234 (39.6%) with 40\u2009mg of citalopram per day between weeks 4 and 6. Females comprised two-thirds of the sample and minority representation was robust. The two dose groups did not show any meaningful clinical difference before receiving different citalopram doses . At enroAccording to our hypothesis, the two citalopram dosages amplified the influence of sociodemographic characteristics on the percent of patients showing an improvement in a dose-dependent manner. In the 20\u2009mg per day dose group, patients' response was not significantly affected by sociodemographic characteristics. By contrast, in the 40\u2009mg per day dose group, sociodemographic characteristics were associated to significantly different outcomes. In the latter group, a higher rate of improvement was associated with having a working employment status, more than 16 years of education, high income and a private insurance . In ordeAs for the rate of improvement, the two citalopram dosages produced a different amplification of the influence of the sociodemographic characteristics on the percent of patients achieving remission. In particular, in the 40\u2009mg per day group, a significantly higher rate of remission was found to be associated with being employed and having longer education . The magP=0.0218), having a private insurance =4.427, P=0.0132), a high income =3.629, P=0.0281) or more years of education =11.344, P=0.0009) was associated with a significant larger reduction of QIDS-SR16 score  and having a private versus a public insurance (P<0.05).The variation in the QIDS-SR16 score produced results in line with the previous ones, as it significantly differed according to sociodemographic variables only in the 40\u2009mg per day dose group. In particular, in this group, being of Caucasian ethnicity =5.334, 16 score . Post ho2 which predicts that, as SSRI increases the susceptibility to the environment, treatment outcome is more profoundly affected by the quality of the living conditions in patients receiving high dosages of SSRIs.The results of the present study show that citalopram amplifies the influence of the living conditions on mood in a dose-dependent manner as sociodemographic characteristics modify treatment response in the same direction in the two dose groups, but in the 40\u2009mg per day dose group the effect is much larger and reaches statistical significance. In addition, the magnitude of the influence of the living conditions on mood is much greater\u2014up to 37-fold\u2014in the 40 compared to the 20\u2009mg per day dose group. These results support the undirected susceptibility to change hypothesis,19The STAR*D clinical trial provides a unique opportunity to investigate the role of citalopram as amplifier of the influence of the living conditions on mood. It has allowed to consider a subpopulation of patients with similar MDD severity and overlapping treatment history in order to analyze the amplification of the influence of the sociodemographic features induced by different dosages of citalopram. Given the clear ethical limitations to perform a clinical trial aimed at directly measuring the effects of the SSRIs in amplifying the beneficial, but especially the detrimental effects of the environment in patients, the STAR*D data set is the best alternative to test the undirected susceptibility to change hypothesis. In addition, the STAR*D clinical trial is the largest ecologically valid 'real world' study of outpatients with nonpsychotic major depressive disorder to date.Sociodemographic characteristics modified treatment outcome in the same direction in the two dose groups, but these changes did not reach statistical significance in the 20\u2009mg per day dose group. By contrast, in the 40\u2009mg per day dose group, each one of five sociodemographic characteristics\u2014income, education, ethnicity, insurance and employment\u2014significantly affected treatment outcome. In addition, the magnitude of the effect of the sociodemographic characteristics on mood, measured as the percent of patients showing a positive outcome when exposed to a favorable environment, was dose-dependent. In particular, the influence of the living conditions was much greater in the 40 than in the 20\u2009mg per day dose group: improvement rate went from a minimum of fivefold for employment status to a maximum of 37-fold for education while remission rate went from twofold for education to eightfold for income.20 the present results indicate not only that citalopram amplifies the influence of the living conditions on mood, but also that the quality of the living environment drives the change in mood. In particular, in the 40\u2009mg per day dose group, where this change reaches statistical significance, improvement and remission were shown at significantly higher rates by patients living in conditions associated with a high quality of life, such as having a working employment status, more than 16 years of education and a high income. The exception concerns insurance where those individuals having public insurance showed the worse outcome, even compared to those having no insurance.21 This is concordant with previous studies reporting, for instance, that having public insurance predicts the highest attrition.17 In addition, higher rates of improvement and remission were associated with being of Caucasian ethnicity. The sociodemographic characteristics here found to affect treatment outcome have been previously shown to both determine rates of major depression morbidity and mortality22 and affect SSRI outcome.1 According to our hypothesis,2 even a worsening of symptomatology could be predicted when citalopram treatment is administered in an adverse environment. However, only a very limited worsening of the QIDS-SR score was expected in patients receiving the treatment while living in an unfavorable condition . This discordant picture can be coherently interpreted in light of our results and the undirected susceptibility to change hypothesis, positing that high serotonin levels lead to increased plasticity and thus to high susceptibility to change, which may promote either an improvement or a worsening, according to the quality of the environment.2 It is worth noting that the effects of citalopram described in the present paper may represent only part of the action of SSRIs on mood as the main target of these drugs, that is, the serotoninergic system, has a high molecular complexity and is involved in a wide range of physiological functions.Taking into account the role of SSRIs as amplifier of the influence of living conditions on mood\u2014and, consequently, the quality of the environment as a moderator of SSRI treatment outcome25The major limitation of the present study is the short time frame (2-week period) over which data of the STAR*D clinical trial have been extracted. This period had to be chosen because it is the only one allowing to consider patients with overlapping clinical history. Longer or different treatment periods or other patient groups would have not allowed to keep data validity. By contrast, the fact that coherent significant results have been found in such a short time frame suggests that the described phenomenon is robust. Further limitations include open treatment design, the use of a single antidepressant agent  and the lack of placebo control. Although data analyses did not include correction for multiple comparisons, the overall consistence of the results support their reliability. It is worth noting that depression treatment disparities experienced by different ethnicities may result from stigma, clinician failure to engage with the patient, poor patient activation, treatment adherence and other factors, including biological differences.26 or taking them in charge through appropriate specialized services, as it is unlikely that people can rapidly and effectively change their living milieu. The cost of this approach is limited as no new psychoactive molecules need to be developed, while the benefits for the patients could be substantial. Finally, the undirected susceptibility to change hypothesis may contribute building a new theoretical framework capable to integrate the 'chemical imbalance theory' with other hypotheses acknowledging the importance of social\u2013psychological factors in MDD, as both approaches are needed to explain the mechanisms underlying the recovery from the disease.In conclusion, acknowledging the role of SSRIs as amplifier of the influence of the living conditions on mood represents a critical step in developing a personalized medicine approach aimed at better matching patients with treatment and avoiding potential harmful consequences. The control of the patients' living environment could be achieved by training patients to cope with harsh conditions, for instance, through cognitive behavioral therapy,"}
+{"text": "We propose a probabilistic method, CancerLocator, which exploits the diagnostic potential of cell-free DNA by determining not only the presence but also the location of tumors. CancerLocator simultaneously infers the proportions and the tissue-of-origin of tumor-derived cell-free DNA in a blood sample using genome-wide DNA methylation data. CancerLocator outperforms two established multi-class classification methods on simulations and real data, even with the low proportion of tumor-derived DNA in the cell-free DNA scenarios. CancerLocator also achieves promising results on patient plasma samples with low DNA methylation sequencing coverage.The online version of this article (doi:10.1186/s13059-017-1191-5) contains supplementary material, which is available to authorized users. Cancer cells often display aberrant DNA methylation patterns, such as hypermethylation of the promoter regions of tumor suppressor genes and pervasive hypomethylation of intergenic regions \u20135. ThereIn recent years, several studies have reported plasma methylation biomarkers for different types of cancers \u201315. UsuaSeveral approaches have recently been proposed for non-invasive universal cancer detection. These methods do not rely on detecting biomarkers specific to certain tumor types. Instead, they utilize properties of ctDNA that are common to various cancer types, such as copy number aberration (CNA) \u201319, pervIn summary, no existing cfDNA-based method can simultaneously detect cancer and predict its tissue of origin. We are therefore proposing a novel method, CancerLocator, that simultaneously infers the proportion and tissue of origin of ctDNA in a blood sample using genome-wide DNA methylation data. As shown in Fig.\u00a0We first evaluated our method on simulation data with known ctDNA fractions. The results show that CancerLocator can achieve a Pearson\u2019s correlation coefficient (PCC) of 0.975 between the predicted and true proportions of ctDNA, and an error rate of 0.078 for the classification of non-cancer and tumor types. Moreover, our method far outperforms two well-established multi-class classification methods in both simulations and using real data, especially when the proportion of tumor-derived DNAs in the cfDNAs is lower than 50% . We note that CancerLocator achieved promising results on patient plasma samples, including around two-thirds of cancer samples collected from early-stage cancer patients.A flowchart of CancerLocator is illustrated in Fig.\u00a0K =14,429 CpG clusters (features), on average\u03b1t, \u03b2t). The index t\u2009=\u20090 represents normal plasma, while t\u2009=\u20091, \u2026, T represents a tumor type.In the first step, we select CpG clusters (our procedure for grouping CpG sites into CpG clusters is described in the \u201cMethods\u201d section) as features if their methylation range (MR) is sufficiently large. MR is defined as the range of average methylation levels observed in healthy plasma and different solid tumor tissues. We selected In the second step, we use the selected features and their beta distributions to deconvolute a patient\u2019s plasma cfDNA into the normal plasma cfDNA distribution and, possibly, a solid tumor DNA distribution. We have designed a probabilistic method that can simultaneously infer the burden and the tissue of origin of the ctDNA. Intuitively, if the likelihood of presence for any tumor type is not substantially higher than the likelihood that the observed distribution is the normal background, the patient is predicted to not have cancer. Otherwise, the patient is predicted to have the tumor type that is associated with the highest likelihood.\u03b8 and tumor type t can be formulated as a maximum-likelihood estimation (MLE) problem, where the likelihood function is expressed as the product of the likelihoods of each CpG cluster, assuming that all of the K selected CpG clusters are independent of each other. This is expressed as:xk denotes the methylation level of CpG site k in a cancer patient\u2019s cfDNA. In principle, xk is a linear combination of the DNA methylation levels in normal plasma and solid tumor type t with fraction \u03b8. The normal and tumor components of the methylation are denoted by vk and uk, respectively v\u2009+\u2009\u03b8u . As mentioned earlier, since v and u follow the Beta distributions Beta and Beta, respectively, x follows the distribution \u03c8, which is calculated as the convolution of two Beta distributions Beta and Beta.Inferring the ctDNA burden entclass1pt{minimaxk of CpG cluster k can be derived from two numbers, nk and mk, denoting the total number of cytosines and the number of methylated cytosines mapped to CpG cluster k. We can model mk and nk together as a binomial distribution mk\u2009~\u2009Binomial, and rewrite the likelihood function as:Because cfDNA has low abundance in plasma, its methylation is usually measured by sequencing-based methods. Therefore, the methylation level Detailed formulas and our optimization method are given in the \u201cMethods\u201d section.For a comprehensive performance evaluation, we compare our method with two popular multi-class classification methods, i.e., random forest (RF) and support vector machine (SVM), on two types of data: simulation data with known ctDNA burden and real data with known clinical information but unknown ctDNA burden. The evaluations on simulation data and real data are complementary in assessing the predictive power of the methods.\u03b8 values). This strategy can make the simulated methylation data keep the potential correlations of methylation values between CpG clusters in real data. In addition, to make the simulated data more realistic, we add tumor CNA events at pre-defined probabilities . The procedure for these simulations is described in the \u201cMethods\u201d section. The results described below are on the simulation dataset with 30% CNA events\u2014simulation data with other CNA event rates yield similar results , at a variety of ctDNA burdens (\u03b8) increases with the true \u03b8, implying that the burden estimation becomes less precise when patients are in mid- or late cancer stages. This result could be partially explained by the fact that tumor heterogeneity may be higher in late stage tumor samples, which introduces the complexity of ctDNA burden prediction. However, this increased variance does not hurt the performance of the cancer detection because the predicted \u03b8 is still much higher than the normal background. Indeed, as demonstrated in Fig.\u00a0We first assessed CancerLocator for ctDNA burden predictions. Overall, the predicted and true proportions of ctDNA are highly consistent, with a Pearson\u2019s correlation coefficient of 0.975 and a root mean squared error of 0.074, respectively. As shown in Fig.\u00a0\u03b8, \u03b8\u2009+\u200910%], where \u03b8\u2009=\u20090, 10, 20, 30, 40, 50, 60, 70, 80, and 90%. For a six-class classification problem , we adopt the error rate measure for assessing the classification performance (see \u201cMethods\u201d). The results are shown in Fig.\u00a0\u03b8 \u2208  largely outperforms RF and SVM , which are only slightly better than random guesses (0.833). For the second lowest ctDNA burdens \u03b8 \u2208 , while RF and SVM still have very poor performance . The two competing methods do not perform well until the ctDNA burdens are greater than 50%, which is mainly seen in plasma samples of late-stage cancer patients. The superior performance of CancerLocator on low to moderate ctDNA fractions indicates that without considering the mixture nature of cfDNAs in plasma, existing popular classification methods always fail to distinguish normal plasma samples and cancer patients\u2019 plasma samples. This result highlights the advantage of our method for cancer diagnosis.We then compared the performance of CancerLocator to that of two popular multi-class classification methods  ten times, the predictions of each of the three methods in ten runs were summarized into a confusion matrix, as shown in Table\u00a0The results in Table\u00a0To understand the relationship between estimated ctDNA burdens and tumor types in real data, we plotted their relationships in Fig.\u00a0We also note that CancerLocator correctly predicted seven out of eight chronic hepatitis B virus (HBV) samples to be non-cancer samples. In addition, our method successfully predicted the only one sample with benign lung tumor as non-cancer in all ten runs, with the predicted ctDNA burden always being 0.0%. These results demonstrate that CancerLocator can go beyond distinguishing healthy samples from cancer samples and handle more sophisticated scenarios, such as differentiating HBV carriers or benign tumor patients from cancer patients.Blood-based cancer diagnosis, unlike traditional diagnosis based on tissue biopsy, has the potential to diagnose tumors from many organs. The proposed CancerLocator aims to exploit this potential of cfDNA by not only diagnosing the presence of tumors, but also predicting the tissue of origin. Although three very recent studies have investigated the inference of tissue of origin \u201323, thes\u03bb (defined in the \u201cMethods\u201d section and computed based on the likelihood) used to differentiate cancer or non-cancer samples is specifically determined for this set of plasma samples for the best performance. When data on more plasma samples become available, this cutoff could be determined by the training data to be robust to most testing scenarios. Finally, we note that we identified markers by comparing methylation profiles of normal plasma cfDNAs and tumor DNAs. This procedure may introduce markers that are tissue-specific but not tumor-specific. This effect can be largely reduced by first using paired samples (tumor sample and the matched adjacent non-tumor sample) to identify tumor-specific markers, then further narrowing down to those markers that show differentiating signals from normal plasma cfDNAs. We foresee the increased power by such identified biomarkers when sufficient paired samples become available.In this work, we used DNA methylation microarrays of solid tumor tissues to train the model due to the scarcity of whole-genome bisulfite sequencing data (WGBS) in the public domain. Since DNA methylation arrays focus only on promoter regions, they may miss important signature regions of cancer. Therefore, we expect that the growing amount of WGBS data will significantly empower the proposed approach by revealing better and higher resolution signatures. Owing to the limited number of plasma samples, the results of this study are evaluated only on three cancer types . However, our new approach has the potential to perform well on all cancer types with well-circulated originating organs. Also, due to the limited plasma samples, the cutoff of the prediction score In this section, we describe: 1) how the data are processed (including methylation microarray and sequencing data); 2) the implementation of CancerLocator; 3) how the simulation data are generated while taking into account copy number aberrations; 4) how the training and testing data are split; and 5) what measures we use to evaluate performance.We collect a large set of public methylation data of solid tumors and plasma cfDNA samples taken from both healthy people and cancer patients. The majority of tumor methylation profiles in TCGA were assayed using the Infinium HumanMethylation450 microarray. We collect those data for solid tumors with >100 samples from five different organs: 681 samples of breast (BRCA), 290 samples of colon (COAD), 522 samples of kidney (including 300 KIRC and 156 KIRP samples), 169 samples of liver (LIHC), and 809 samples of lung (including 450 LUAD and 359 LUSC samples) cancer.The public methylation data of plasma cfDNA samples are from Chan et al.  and Sun The blood samples of eight lung cancer patients and one benign lung tumor patient were collected. The demographic and clinical features of the patients profiled are presented in Additional file Blood samples were centrifuged at 1600\u2009\u00d7\u2009g for 10\u00a0minutes and then the plasma was transferred into new microtubes and centrifuged at 16,000\u2009\u00d7\u2009g for another 10\u00a0minutes. The plasma was collected and stored at \u221280\u00a0\u00b0C. cfDNA was extracted from 5\u00a0ml plasma using the Qiagen QIAamp Circulating Nucleic Acids Kit and quantified using a Qubit 3.0 Fluoromter (Thermo Fisher Scientific). Bisulfite conversion of cfDNA was performed using a EZ-DNA-Methylation-GOLD kit (Zymo Research). After that, an Accel-NGS Methy-Seq DNA library kit (Swift Bioscience) was used to prepare the sequencing libraries. The DNA libraries were then sequenced with 150-bp paired-end reads.The Infinium HumanMethylation450 microarray data from TCGA measure all solid tumor samples at ~450,000 CpGs. Since our testing sample  compriseThe microarray data (level 3 in TCGA database) provide the methylation levels of individual CpG sites. We define the methylation level of a CpG cluster as the average methylation level of all CpG sites in the cluster. A cluster\u2019s methylation level is marked as \u201cnot available\u201d (NA) if more than half of its CpG sites do not have methylation measurements.Bismark  is emploFor each CpG cluster, we used the methylation range (MR) to indicate a feature\u2019s differential power between classes. We first obtained the average methylation level of all samples from each class , then defined MR as the range of this set of mean values . The higher the MR of a cluster is, the more differential power it has. Finally, we selected those CpG clusters whose MRs were no lower than a threshold.k \u2208 {1, 2, \u22ef, K}, the methylation level xk of the plasma cfDNA from a given patient can be approximated as a mixture of vk and uk, which are the methylation levels of the normal plasma sample and the solid tumor tissue, respectively. Let \u03b8 \u2208  denote the proportion of tumor-derived DNAs in plasma cfDNA. Then xk can be expressed as the weighted sum of vk and uk, i.e., xk\u2009=\u2009(1\u2009\u2212\u2009\u03b8)vk\u2009+\u2009\u03b8uk.The cfDNA in the plasma of cancer patients can be regarded as a mixture of normal background DNA and tumor-released DNA. Formally, for each CpG cluster T possible tumor types. Let t \u2208 {0, 1, 2, \u22ef, T} be the variable representing either normal plasma (t\u2009=\u20090) or a tumor type (1\u2009\u2264\u2009t\u2009\u2264\u2009T). For each CpG cluster k, we model its methylation level in a sample of type t as a Beta distribution: vk\u2009~\u2009Beta for normal plasma samples (t =0) and uk\u2009~\u2009Beta for solid tumor samples of type t \u2208 {1, \u22ef, T}, where \u03b1k0 and \u03b2k0 (\u03b1kt and \u03b2kt) are the parameters of the beta model of methylation levels of CpG cluster k in normal plasma (solid tumor) samples. As illustrated in step 1 of Fig.\u00a0We assume that an individual carries at most one type of tumor among the vk and uk), as shown in Fig.\u00a0xk can be modeled by a derived distribution with the given ctDNA burden \u03b8 and source tumor type t. This model is denoted as the probability density function \u03c8, which is calculated by the convolution of Beta and Beta. It is formally expressed as:fBeta is the probability mass function of the Beta distribution.By integrating the two Beta distributions (xk) of a CpG cluster k can be characterized by the numbers of methylated and unmethylated cytosines in the reads. Let M\u2009=\u2009 and N\u2009=\u2009 be the number of methylated cytosines and the total number of cytosines mapped to all CpG sites, respectively, where the index runs over all K CpG clusters. For each CpG cluster k, mk can be modeled by a binomial distribution: mk\u2009~\u2009Binomial. By integrating the mixture model of xk in Eq.\u00a0k which has the inputs from the model parameters  and the sequence measurements of plasma samples :fBinomial is the probability density function of the binomial distribution.Due to its low abundance in plasma, the methylation profile of cfDNA is usually measured by sequencing-based methods, and the methylation levels , the more precise the estimation. After obtaining the solution  because \u03b8\u2009=\u20090 indicates a normal plasma sample. The larger the prediction score \u03bb, the higher the chance that the patient has a cancer tumor of type \u03bb is greater than a threshold, the patient is predicted as having cancer with the ctDNA burden Since the integrals in Eqs.\u00a0We simulate the methylation sequencing data of a patient\u2019s plasma cfDNAs using the previously described probabilistic models: (i) a mixture model that treats the cfDNA as a mixture of normal plasma cfDNA and DNAs released from primary tumor sites; and (ii) a binomial model for the methylated cytosine count of plasma cfDNA sequencing data. In addition, to make the simulation data more realistic, we incorporate CNAs and read depth bias. The procedure for simulating plasma cfDNA methylation sequencing data is detailed in the following sections.K CpG clusters; (ii) the total number of cytosines (Z) on the sequencing reads that are aligned to any CpG cluster; (iii) the range of \u03b8 : ; (iv) the collections of normal plasma samples  and solid tumor samples (denoted as POOLtumor); and (v) bk, the background probability for a CpG dinucleotide to be aligned to CpG cluster k, satisfying \u2211k\u2009=\u20091Kbk\u2009=\u20091. The last input reflects the read-depth bias introduced during the sequencing process and read alignment and the density of CpG sites in the clusters. Refer to Additional file bk.Inputs include: (i) the genomic regions of all M\u2009=\u2009 and N\u2009=\u2009. The elements mk and nk are the number of methylated cytosines and the total number of cytosines in the reads mapped to CpG cluster k, respectively.Output comprises a simulated methylation sequencing profile of a plasma sample, represented by the integer vectors \u03b8 from the distribution \u03b8\u2009~\u2009Uniform.Generate a random ctDNA fraction ck for each CpG cluster k, from the categorical distribution ck\u2009~\u2009Cat. Here, pc denotes the probability of observing copy number c \u2208 {0, 1, 2, 3, 4, 5} in the sequencing data. The probabilities pc satisfy three criteria: (i) their sum is equal to one, c\u2009=\u200905c\u2009\u2217\u2009pc\u2009=\u20092; and (iii) extreme CNAs are less likely to occur. In this work, we predefine p0 = 0.005, p1 = 0.16, p2 = 0.7, p3 = 0.105, p4 = 0.025, p5 = 0.005. Note that the sum of all these probabilities except p2 (30% in this case) is the probability of any given CpG cluster having a CNA event. We have tried other probability configurations for the simulation with more (50%) or fewer (10%) CNA events and obtained similar results  when simulating a normal plasma sample.Generate a random integer copy number normal whose methylation profile is denoted by , and randomly select a solid tumor from POOLtumor whose methylation level profile is denoted by . Note that we also randomly select two normal plasma samples from POOLnormal in order to simulate a new normal plasma sample.Randomly select a normal plasma sample from POOLxk of plasma cfDNA at CpG cluster k. This is the adjusted linear combination of vk and uk after incorporating the copy number ck generated in step 2. That is, xk = (1 \u2212 \u03b8k')vk + \u03b8k'uk, where \u03b8k' is the adjusted value of \u03b8 given by \u03b8k' describes the actual ctDNA fraction after considering the copy number ck of the ctDNA.Calculate the methylation level nk, representing the total number of cytosines in CpG cluster k, from the Poisson distribution nk ~ Poisson(ZBk). Bk is the adjusted CpG dinucleotide bias bk, given by ck generated in step 2.Generate a random number mk from the binomial distribution mk ~ Binomial.Generate a random number Due to the limited number of normal plasma samples, we also simulated new normal plasma samples by mixing two normal plasma samples at different mixture ratios. The procedure is the same as above except that step 2 is ignored by fixing all copy numbers as two because there are no CNA events in the normal plasma samples.\u03bb, which is set as 0.023 to generate the predictions on the real plasma samples. For consistency with the real data experiments, we apply the same strategies to simulation data experiments and calculate the error rate averaged over ten runs.All TCGA solid tumor tissues and plasma samples are divided into non-overlapping sets for three tasks: (i) learning discriminating features; (ii) simulation experiments; and (iii) testing on the real data. Specifically, as shown in Fig.\u00a0The error rate and accuracy are the most popular and established multi-class classification performance measures \u201329. They"}
+{"text": "MIC-A, TNF-\u03b1 genes) identified as associated to BD because of their LD with HLA-B*51. In fact, the HLA-B*51 is inherited as part of extended HLA haplotypes which are well preserved in patients with BD. Sardinian population is highly differentiated from other Mediterranean populations because of a distinctive genetic structure with very highly preserved HLA haplotypes.Beh\u00e7et\u2019s disease (BD) is a polygenic immune-mediated disorder characterized by a close association with the HLA-B*51 allele. The HLA region has a strong linkage disequilibrium (LD) and carries several genetic variants (e.g. AIF-1) gene variants among BD patients and healthy controls from Sardinia. Six  AIF-1 single nucleotide polymorphisms (SNPs) and related extended haplotypes have been investigated as well as their LD within the HLA region and with HLA-B*51. Overall, 64 BD patients, 43 HLA-B*51 positive healthy controls (HC) and 70 random HC were enrolled in the study.In order to identify other genes of susceptibility to BD within the HLA region we investigated the distribution of human Allograft Inflammatory Factor-1 (pc = 0.0021) in BD patients (40.6%) than in HC (9.8%). The rs2259571TAIF-1 variant had a significantly reduced phenotypic, but not allelic frequency in BD patients  compared to healthy population (91.3%). That was likely due to the LD between HLA-B*51 and rs2259571G (pc = 9E-5), even though the rs2259571G distribution did not significantly differ between BD patients and HC.HLA-B*51 was the only allele with significantly higher frequency (AIF-1 SNPs haplotypes was observed between BD patients and HC and between HLA-B*51 positive BD patients and HLA-B*51 positive HC. Taken together, these results suggest that AIF-1 gene is not associated with susceptibility to BD in Sardinia.No significant difference in distribution of  B*15, B*57, A*26), the TNF-\u03b1 and the MHC Class I chain-related gene A (MIC-A) have been associated to an increased risk of BD [Beh\u00e7et\u2019s disease (BD) is a chronic vasculitis characterized by recurrent oral ulcers, genital ulcers, ocular and skin manifestations with involvement of arteries and veins of all sizes. BD clusters in an area between latitudes 30\u00b0 N and 45\u00b0 N spanning from the far Eastern Asia to the Mediterranean basin ,2. Such sk of BD \u201312.We previously pointed out that , in SardAIF-1) is a 143 amino acid, 17\u2009kDa, cytoplasmic calcium-binding protein, encoded within the HLA class III region on chromosome 6p21 which is densely clustered with genes involved in the inflammatory responses including TNF-\u03b1. Because its pro-inflammatory role, AIF-1 is involved in various inflammatory pathological processes such as allograft rejection, autoimmune diseases, inflammatory central nervous system injury. Several single-nucleotide polymorphisms (SNPs) have been identified in the AIF-1 gene as associated with autoimmune diseases [TNF-\u03b1 gene promoter and the HLA-B locus, and its pro-inflammatory activity we deemed interesting to study AIF-1 as a candidate gene for BD susceptibility.Human Allograft Inflammatory Factor-1 .AIF-1 gene . The amount of DNA was determined using the Qubit fluorometric quantitation that comprises the Qubit 3.0 Fluorometer and sensitive, specific Qubit quantitation DNA assay (Thermo Fisher Scientific). All patients and controls were genotyped for 6 different SNPs of the F-1 gene  by the rAIF-1 SNPs in the extended HLA haplotypes.Patients and controls were also typed for HLA-A, B, C, DRB1, DQA1 and DQB1 using commercial kits  in order to identify a different distribution of the It is well known that choosing preliminary candidate SNPs is critical for candidate gene association studies. The chosen SNPs were based on previously described associations in various immune-mediated diseases ,18, as wAIF-1 polymorphic alleles and disease associations in healthy controls (HC) versus BD patients, chi-square test or two-tailed Fisher\u2019s exact test, for low frequency, were performed using MedCalc software . The strength of association was estimated by calculating the odds ratio (OR) with 95% Confidence Interval (95% CI). Under the assumption of independence, a value of p<0.05 was considered statistically significant where Bonferroni correction was applied for multiple comparisons to all novel associations, with a correction factor derived from the number of alleles examined; pc indicates where the Bonferroni correction was applied. The LD among the 6 SNPs of the AIF-1 gene and between single SNPs and HLA-B*51 was calculated using the HaploView 4.2 software.Hardy\u2013Weinberg equilibrium (HWE) was tested using the Chi-square test. To assess differences in the proportions of pc = 0.0021; OR = 6.2; 95%CI 2.5 to 15.8) in BD patients (40.6%) than in HC (9.8%). No other HLA class I and II alleles were independently associated with BD.HLA-B*51 phenotype frequency was significantly higher  did not show different allelic and phenotypic distribution between patients and HC. Only the rs2259571 SNP had a significantly decreased phenotypic, but not allelic, frequency of the rs2259571T variant in BD patients  compared to healthy population (91.3%) without a significantly different phenotypic distribution of the rs2259571G variant between BD patients and HC despite its LD with the HLA-B*51 (pc = 9E-5). Noteworthy, the rs2259571T phenotypic frequency distribution did not significantly differ between HLA-B*51 positive BD patients (56.5%) and HLA-B*51 positive HC (62.8%).Five out of 6 AIF-1 SNPs haplotypes, no significantly different haplotype distribution between BD patients and HC was detected (AIF-1 SNP haplotypes. As effect of the LD between rs2259571G and HLA-B*51 the GGGCA was found at a higher frequency in HLA-B*51 positive subjects (56.3%) and the GGTCA was most frequently carried by HLA-B*51 negative subjects (52.9%) irrespective of the disease status.Analysing the distribution of detected . The GGTpc = 0.065 OR 0.06 95%CI 0.01\u20130.54) according to, but not fully confirming, the previous observation of a lack of association between B*51-DR*4 and BD susceptibility in Sardinians (13). No significant difference in the distribution of AIF-1 single or combined SNPs was observed between the B*51-DR*11 and B*51-DR*4 extended haplotypes  of HLA-B*51 positive BD patients and in 14/37 (37.8%) of HLA-B*51 positive HC (p = 0.231); and B*51-DR*4 in 1/26 (3.8%) of HLA-B*51 positive BD patients and in 14/37 (37.8%) of HLA-B*51 positive HC , chemokines, inducible nitric oxide synthase and promotes inflammatory cell proliferation and migration [AIF-1 in rheumatoid arthritis and systemic sclerosis has been investigated and the rs2269475 SNP was found associated with an increased risk of developing both diseases [AIF-1 in BD susceptibility, we did not find any suggestion for this in our study population.Because of its position within the HLA class III region, between the HLA-B and HLA-DR loci, and its pro-inflammatory effect, the igration . Moreoveigration ,27. The diseases ,29. AlthAIF-1 in BD. With the aim to elucidate the genetic basis of BD we set a candidate gene case-control association study and we tested six different AIF-1 SNPs. Major strengths are represented by the peculiar genetic background of Sardinians and by the enrolment of two different control populations allowing to identify a different distribution of AIF-1 in patients and in controls but also in HLA-B*51 carriers versus other subjects. A major limitation is related to sample size, therefore caution is advised when interpreting results as they may be related to the small size of the population under study.To the best of our knowledge, this was the first study investigating the role of AIF-1 expression or change in protein structure may predispose to the development of BD in Sardinian patients. Further, larger studies are required to confirm our findings in other populations.In conclusion, the present study does not support the hypothesis that a genetically determined regulation of S1 TableGenotyping results for AIF1 are reported here.(XLSX)Click here for additional data file."}
+{"text": "Purpose: Despite the wide adoption of tumor molecular profiling, there is a dearth of evidence linking molecular biomarkers for treatment selection to prediction of treatment outcomes in patients with metastatic pancreatic cancer. We initiated a pilot study to test the feasibility of designing a larger phase II trial of molecularly tailored treatment for metastatic pancreatic cancer.Methods: Our study aimed to assess the feasibility of following a treatment algorithm based on the expression of three published predictive markers of response to chemotherapy: ribonucleotide reductase catalytic subunit M1 (for gemcitabine); excision repair cross-complementation group 1 (for platinum agents); and thymidylate synthase (for 5-fluorouracil) in patients with untreated, metastatic pancreatic cancer. Results of the tumor biopsy analysis were used to assign patients to one of seven doublet regimens. Key secondary objectives included response rate (RR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS).Results: Between December 2012 and March 2015, 30 patients were enrolled into the study. Ten patients failed screening primarily due to inadequate tumor tissue availability. Of the remaining 20 patients, 19 were assigned into 6 different chemotherapy doublets, and achieved an RR of 28%, with a DCR rate of 78%. The median PFS and OS were 5.78 and 8.21 months, respectively.Conclusions: The incorporation of biomarkers into a treatment algorithm is feasible and resulted in a PFS and OS similar to other doublet therapies for patients with metastatic pancreatic cancer. Based on the results from this pilot study, a larger phase II randomized trial of molecularly targeted therapy versus physicians' choice of standard of care has been initiated in the second-line setting (NCT02967770). Surgical resection is currently the only potentially curative treatment option, but unfortunately, only 9% and 29% of patients have operable or localized, nonmetastatic disease, respectively. The vast majority of patients are diagnosed with metastatic disease on initial presentation, and these patients have a reported median survival of only 8\u201311 months.2 Modern day chemotherapy combinations have since improved outcomes over single agent gemcitabine, thereby becoming the new standards of care. In 2011, the combination of 5-fluorouracil (5FU), oxaliplatin, and irinotecan (FOLFIRINOX) was shown to improve median overall survival (OS) to 11.1 months compared with gemcitabine, which demonstrated a median OS of just 6.8 months in patients with a good performance status (ECOG 0-1).3 In 2013, the combination of gemcitabine and nab-paclitaxel was also shown to be better than single agent gemcitabine. The median OS of gemcitabine and nab-paclitaxel was 8.5 months, which was statistically superior to the 6.7-month survival seen with gemcitabine.6 These chemotherapy regimens in current clinical practice were evaluated empirically in nonbiomarker-enriched patient populations, and there are no accompanying predictive tools to guide their use in patients. Given the short-lived benefit from chemotherapy, it would be a worthwhile endeavor to select patients who are most likely to benefit from a given treatment while sparing treatment-related side effects for those who are less likely to benefit. Although the predictive strength of any single biomarker is debatable,7 the utility of a composite of multiple biomarkers in patient selection to match them with best available treatment option(s) has not been widely explored.Chemotherapy continues to be the cornerstone of treatment for pancreatic cancer since the approval of gemcitabine in the frontline setting.8 In addition, the expression of excision repair cross-complementation group 1 (ERCC1) and thymidylate synthase (TS) may be markers for platinum resistance10 and 5FU resistance,12 respectively. We have recently performed a review of the value of these predictive biomarkers across disease types, and our findings suggest that there is value at a minimum to exploring the utilization of these biomarkers in clinical trials for patients with pancreatic and other cancer types.13 Examples of the incidence of high or low expression of RRM1, ERCC1, and TS have been previously published. For example, for RRM1, Valsecchi et al. detailed that, of 93 patients assessed, RRM1 expression by immunohistochemistry (IHC) was low in 61 (65%) and high in 32 patients (35%).14 Other smaller data sets reveal high expression of RRM1 to be observed in 34\u2009\u2212\u200950% of patient samples.15\u201317 For ERCC1, Valsecchi et al. detailed that, of 94 patients assessed, ERCC1 expression by IHC was low in 41 (44%) and high in 53 patients (56%).14 Fareed et al. and Hwang et al. detailed that high levels of ERCC1 expression were observed in \u223c50% of patient samples.19 Finally, for TS, Hu et al. evaluated pancreatic tumor tissue from 132 resected patients and determined that TS expression was high in 83 of 132 (63%) and low in 49 of 132 patients (37%).20 Formentini et al. revealed that TS expression in 130 pancreatic cancer patients was low in 56% and high in 43% of patients.21Despite the availability of molecular profiling, there is scarcity of high-quality prospective data evaluating the efficacy of linking molecular profiles to specific treatment choices in patients with pancreatic adenocarcinoma. Studies have explored the predictive role of some biomarkers to specific chemotherapeutic agents. One such biomarker is ribonucleotide reductase catalytic subunit M1 (RRM1) expression, which is a potential marker for gemcitabine resistance.We designed a pilot study to explore the feasibility of incorporating the use of predictive biomarkers RRM1, ERCC1, and TS to select therapeutic \u201cdoublet\u201d chemotherapy for patients with metastatic pancreatic cancer. As per protocol, the stated primary objective was \u201cto determine the estimates of outcomes necessary to plan and conduct subsequent studies with molecularly tailored therapy, for patients with metastatic pancreatic cancer.\u201d More practically stated, the primary objective was essentially to assess the feasibility of testing these markers in newly diagnosed patients, and incorporating the test results into the treatment-decision process. Our key secondary end-points, including disease control rate (DCR), time to progression, and OS, were preliminary assessed to determine the feasibility and efficacy of our \u201cmolecularly tailored therapy\u201d selections.Eligible patients were \u226518 years of age, with cytologically/histologically confirmed metastatic pancreatic adenocarcinoma that was amenable to biopsy to obtain sufficient tissue for molecular profiling. Patients must have not received prior systemic therapy for metastatic disease, have measurable disease by RECIST version 1.1, an Eastern Cooperative Oncology Group (ECOG) performance status of 0\u20132, and acceptable liver, renal, and hematologic laboratory values. The study protocol (NCT01888978) was approved by the local institutional review board, and all patients provided written informed consent.This was an open-label pilot study designed to assess the feasibility of following a simple algorithm to treat patients with metastatic pancreatic cancer with a chemotherapy regimen based on three published predictive markers of response/resistance to chemotherapy. As per protocol, the primary objective was \u201cto determine the estimates of outcomes necessary to plan and conduct subsequent studies with molecularly tailored therapy, for patients with metastatic pancreatic cancer.\u201d Biopsies of accessible metastatic lesions were performed by an interventional radiologist under standard biopsy procedures. The specimen was sent to Caris Life Sciences (Caris) for molecular analysis to be checked for expression of, at minimum, RRM1, ERCC1, and TS, although a broader panel was typically assessed. IHC was used to measure the expression of the markers, as per standard operating procedures internal to Caris. For each IHC, the staining intensity and the percent of positive cells was provided by Caris, and the cutoff of \u201chigh\u201d versus \u201clow\u201d was determined by Caris, using their internal database. These cutoff values did not change significantly in the course of this study. Patients with inadequate biopsy specimens who were unwilling to undergo a second biopsy were considered screen failures. Patients were assigned a treatment doublet based on expression of RRM1, ERCC1, and TS, using the algorithm depicted below .2 gemcitabine on day 1 and 100\u2009mg/m2 oxaliplatin on day 2, both given every 14 days,22\u201324 and was selected for patients with low RRM1 and ERCC1 expression. Gem-5FU consisted of the combination of 1000\u2009mg/m2 gemcitabine and 2000\u2009mg/m2 5FU, given as a 24-h slow infusion, both administered on days 1, 8, and 15 of a 28-day cycle,25\u201327 and was selected for patients with low RRM1, high ERCC1, and low TS expression. Gem-Abrax consisted of the combination of 1000\u2009mg/m2 gemcitabine and 125\u2009mg/m2 nab-paclitaxel, both given on days 1, 8, and 15 of a 28-day cycle,5 and was selected for patients with low RRM1, high ERCC1, and high TS expression. FOLFOX consisted of the combination of 85\u2009mg/m2 oxaliplatin on day 1, 400\u2009mg/m2 5FU on day 1, 400\u2009mg/m2 leucovorin on day 1, and 2400\u2009mg/m2 5FU over 46\u2009h, all administered every 14 days,28\u201330 and was selected for patients with high RRM1, low ERCC1, and low TS expression. Ox-Tax consisted of the combination of 100\u2009mg/m2 oxaliplatin on day 1 and 65\u2009mg/m2 docetaxel on day 1, both given every 3 weeks with growth factor support,31 and was selected for patients with high RRM1, low ERCC1, and high TS expression. 5FU/leucovorin and irinotecan (FOLFIRI) consisted of the combination of 180\u2009mg/m2 irinotecan on day 1, 400\u2009mg/m2 5FU on day 1, leucovorin 400\u2009mg/m2 on day 1, and 5FU 2400\u2009mg/m2 over 46\u2009h, all administered every 14 days,32\u201335 and was selected for patients with high RRM1, high ERCC1, and low TS expression. Finally, Tax-Iri consisted of the combination of 35\u2009mg/m2 docetaxel and 50\u2009mg/m2 irinotecan, both given weekly for 4 of 6 weeks,37 and was selected for patients with high RRM1, high ERCC1, and high TS expression. Dose modifications for adverse events were detailed in the protocol for each regimen. Treatment in the assigned subgroup was continued until disease progression or patient intolerance occurred, and all patients were followed until death.Patients were treated with one of seven possible chemotherapy doublets, based on their tumor molecular profile . Gem-Ox N, median, range for continuous variables; and N, percent for categorical variables). Kaplan\u2013Meier methodology was used to estimate the progression-free survival (PFS) and OS. Median PFS and OS were presented with their 95% confidence intervals. SAS 9.3  was used for the analysis.As a pilot study, no specific statistical hypotheses were tested or were utilized in determining the \u201cestimates of outcomes necessary to plan and conduct subsequent studies.\u201d Rather, descriptive summary statistics were used for patient demographics as well as toxicities  had enough tissue available for tumor molecular profiling and consequent treatment assignment. One patient was a screen failure after biopsy due to biliary obstruction. The remaining 19 patients were able to initiate molecularly tailored therapy. The median time from biopsy to treatment initiation was 32.5 days (range: 14\u201368). The most common tumor molecular profile was low RRM1 and ERCC1 (44.4%), and these patients were treated with a doublet of gemcitabine and oxaliplatin. The second-most commonly seen profile was high RRM1, high ERCC1, and low TS (22.2%), and these patients were treated with a doublet of FOLFIRI.Patients continued therapy with protocol-defined dose modifications for adverse events until disease progression or intolerance. At the time of submission of this article, all patients had completed follow-up for PFS and OS, with the exception of one patient who moved out of the country and was lost to follow-up after 3 months.The CA 19-9 best response is shown in Grade 3 adverse events reported among the patients treated with molecularly tailored regimens included nausea/vomiting (10%), anemia (10%), thrombocytopenia (10%), venous thromboembolism (5%), peripheral neuropathy (5%), and febrile neutropenia (5%). There were no Grade 4 adverse events reported  did not receive any further treatment, of whom seven (88%) died within 3 months of failure of the frontline therapy. Twelve patients (60%) underwent second-line therapy at the discretion of the treating physician, of whom four patients (33%) received gemcitabine and nab-paclitaxel, three patients (25%) received FOLFOX/XELOX, two patients (16.7%) received FOLFIRI, two patients (16.7%) pursued further clinical trials , and one patient (8%) received single agent 5FU. Patients who had disease progression after frontline treatment but were able to receive second-line therapy had a median OS with further treatment of 5.4 (range: 0.7\u201313.4) months.5 These successes indicate that optimal utilization of cytotoxic agents, including gemcitabine, oxaliplatin, irinotecan, 5FU, and taxanes , can improve patient outcomes. Unfortunately, the success of the various cytotoxic chemotherapy regimens is often diluted when tested in large, unselected patient populations.39 The inability to translate the significant benefits seen in some patients to the pancreatic cancer population at large is because, in common practice, promising chemotherapy combinations are never administered to select patients who are most likely to respond based on their tumor's molecular profile. However, more data have emerged recently40 defining molecular subgroups of patients with pancreatic cancer, who, if appropriately selected, could benefit from currently available therapies. This understanding makes a persuasive argument to enhance patient outcomes by adapting predictive tumor biomarkers that will provide a realistic platform for physicians to select treatment with better precision from an array of available agents and regimens for their patients. We initiated a pilot trial to assess the feasibility of tailoring established and FDA-approved cytotoxic chemotherapies for pancreatic cancer patients through molecular analyses of their tumors.Although we have greatly improved our understanding of the molecular etiology of pancreatic adenocarcinoma, this has not translated into clinically meaningful treatment options for patients. In fact, no personalized nor even targeted therapy has resulted in clinically meaningful improvements in patient outcomes. By contrast, novel cytotoxic chemotherapy regimens such as FOLFIRINOX and gemcitabine plus nab-paclitaxel have led to an increased OS, while maintaining a reasonable quality of life.Recognizing the low probability of a single-tumor biomarker to be predictive of chemotherapy benefit in general, we utilized an algorithm of several published markers with the intent of improving clinical outcomes compared with contemporary, nonbiomarker-enriched treatment strategies.8 The overexpression of ERCC1 can lead to resistance to platinum drugs.10 The expression of TS can predict resistance to 5FU.12 Based on the tumor biomarker profile, the algorithm outlined in In this study, patients underwent fresh tumor biopsies and the tumors were assessed for three specific published predictive markers of response to chemotherapy. The first such marker is RRM1, which can predict resistance to gemcitabine.Treatment offered to patients based on their molecular profiles was very well tolerated, although it is difficult to compare any specific adverse event to larger studies due to the small number of patients. This trial demonstrated the feasibility of obtaining and analyzing fresh tumor biopsies to guide treatment selection. However, there was a 40% screen failure rate, indicating the constraints of making treatment decisions based on the molecular profile of tumors in previously untreated patients who are likely to have significant disease-related symptoms and may not be able to afford waiting for tumor profiling results before a treatment decision can be made and therapy is initiated.38Moreover, our data suggest a potential benefit to molecularly tailored therapy, with the majority of patients achieving a 6-month DCR of 78%, with partial response and stable disease seen in 28% and 50% of patients, respectively. The majority (55%) of patients had >50% reduction in their tumor marker (CA 19-9). The median PFS of 5.78 months (95% CI 5.39\u201315.72) and the median OS of 8.21 months (95% CI 7.16\u201315.72) were similar to other published doublet therapy results.41\u201348 Although the sample was small, our study's findings are in line with those previously described.41\u201348A significant portion (40%) of the study population was not able to pursue further treatment for their disease. This underscores the need for effective frontline therapy to potentially benefit these patients, a significant proportion of whom will never be able to pursue treatment in the second-line setting once the frontline regimen fails. These patients may potentially benefit from molecularly tailored treatment strategies, which may help identify regimens that are likely/unlikely to be effective. Contrary to the treatment outcomes in the frontline setting, the effectiveness of chemotherapy beyond the frontline setting sharply declines in patients with metastatic pancreatic carcinoma. Second-line trials historically have shown very low response rates (less than 30%) and OS of 4\u20136 months.The incorporation of biomarkers to guide the selection of chemotherapy is feasible and resulted in a similar PFS and OS compared with other standard therapies for patients with metastatic pancreatic cancer. To investigate the benefit of using this approach, a randomized trial versus standard of care has been developed in the second-line setting, taking into consideration the high rate of screen failures due to inadequate tissue sampling (NCT02967770)."}
+{"text": "Following publication of the original article , a typesThe publisher apologizes to the authors and readers for the inconvenience."}
+{"text": "The publisher apologizes to the readers and authors for the inconvenience.In the original publication of article , \u201820 \u00d7 1The original article has been corrected."}
+{"text": "We sought to explore these relationships using grass carp (Ctenopharyngodon idellus), which often suffers functional disorder of liver and gallbladder. We studied fluctuations of BAs in the gall and changes of microbial communities in the gut in response to seven different diets: five different BS, chelating BS agent, and control. The BS comprised two primary BS [sodium taurochololate (TCAS) and sodium taurochenodeoxycholate (TCDCAS)], sodium tauroursodeoxycholate (TUDCAS), and two secondary BS [sodium taurodeoxycholate (TDCAS) and sodium taurolithocholate (TLCAS)]. Supplementation of primary BS caused a more significant fluctuation of biliary BAs than secondary BS, and TCAS caused a more prominent increase than TCDCAS and TUDCAS. For the gut microbiota, primary BS tended to increase their diversity and induce community succession, secondary BS resulted in a higher firmicutes/bacteroidetes ratio, while TUDCAS had no significant effects. Changes of the gut microbiota triggered by different types of BS caused alteration in BAs biotransformation. Two-obesity-associated families, Lachnospiraceae and Ruminococcaceae were positively correlated with biliary cholic acid (CA), taurochenodeoxycholic acid (TCDCA), and deoxycholic acid (DCA). As both primary and secondary BS resulted in increased synthesis of toxic secondary Bas by the gut microbiota, future studies should pay closer attention to gut microbiota when considering BA treatment.Lipid metabolism can influence host\u2019s health. There is increasing evidence for interplay between two key regulating factors in lipid metabolism: bile acids (BAs) and gut microbiota. However, very little is known about how types of different diet-supplemented bile salts (BS) influence this interaction  Sodium tauroursodeoxycholate (TUDCAS) was purchased from Huaaobio Company . TUDCAS eschews classification in this aspect, or more precisely, its classification as primary or secondary BS is host dependant. For example, UDCA is considered to be a secondary BA in humans , but a pn \u2248 600) were obtained from the Wuhu Fish Hatchery . Prior to the experiment, fish were reared in 1000-L plastic tanks for 2 weeks to acclimate them to the experimental conditions. After the acclimatization period, 315 fish specimens of similar size (average weight: 40 \u00b1 5 g) were randomly distributed into 21 aquariums (125 L of water each) at the stocking density of 15 fish per tank. Each aquarium was randomly assigned a diet, and each dietary treatment was conducted in triplicate . Fish were fed to apparent satiation three times daily  for 6 weeks. During the feeding trial, fish were maintained under the natural photoperiod conditions, water temperature ranged from 22 to 23\u00b0C, pH fluctuated between 7.2 and 7.4, and water replacement rate was adjusted to keep the total ammonia nitrogen and nitrites below 0.2 and 0.005 mg/L, respectively. At the end of the feeding trial, three fish specimens from each diet replicate (adding up to nine specimens per each treatment) were anesthetized in diluted tricaine methanesulfonate  at the concentration of 100 mg/L. The fish were then euthanized, dissected, and gallbladder and hindgut content of each fish collected and stored at -80\u00b0C for the downstream use. All the samplings were done between 5 and 6 h after feeding in order to avoid the temporal variation of gut microbiota  was conducted to test for significant differences between groups in terms of overall biliary BAs composition using Bray-Curtis distance using the R Vegan package (BAs were extracted according to the method described by  package .DNA was extracted from hindgut contents of the 56 samples for which biliary BAs were measured successfully (see previous section) using QIAamp Fast DNA Stool Mini Kit . NanoDrop 2000 Spectrophotometer  was used to check the concentration and quality of the extracted DNA. Extracted DNA was diluted to 10 ng/\u03bcL and stored at -80\u00b0C for downstream use.Universal primers 515F (5\u2032-GTGYCAGCMGCCGCGGTA-3\u2032) and 806R (5\u2032-GGACTACHVGGGTWTCTAAT-3\u2032), with a 12 nt unique barcode at 5\u2032-end of 515F, were used to amplify the hypervariable V4 region of the bacterial 16S rRNA gene . Each sa1  algorithm. Principal coordinate analysis (PCoA) was used to visualize similarities between groupings with weighted_unifrac distance . PERMANOt-tests.Group differences in relative abundance at the genus taxonomic level were tested using the nonparametric Kruskal\u2013Wallis test correcting for multiple comparisons with the Benjamini\u2013Hochberg false discovery rate (FDR) procedure. Pairwise comparisons between each experimental group and Ctrl group were tested using t-tests.Metagenomic content of samples was inferred from 16S rRNA gene sequence data using PICRUSt 1.0 and KEGG database, which includes 6909 bacterial genes  annotated in the reference genomes . Stamp vr).Biliary BAs and microbiota taxa from 56 samples were analyzed and used to construct molecular ecological networks between different biliary BAs and top 50 microbial taxa at the genus level. Networks were constructed using MENA pipeline with default parameters, based on the random matrix theory (RMT) method with the RMT cutoff value set at 0.5 . ModulesPRJNA416986).The obtained raw 16S rRNA sequences were deposited in the NCBI/EBI/DDBJ Sequence Read Archive . The sum of the concentrations of all measured biliary BAs  varied greatly among the six experimental treatments. Compared with the Ctrl group, the concentration of total BAs in groups fed with primary BS (TCAS and TCDCAS) and TUDCAS showed significant increase, the groups fed with secondary BS (TDCAS and TLCAS) and chelating agent (Chol group) did not show significant increase . The concentration of total BAs was ranked as follows: TCA fed group  > TCDCA fed group  > TUDCA fed group  > TDCA fed group  > Chol group  > TLCA fed group  > Ctrl group . In detail, TCA fed group exhibited an increased concentration of seven measured BAs, with extremely high levels of DCA , TCDCA , CA , and a moderate increase in concentrations of UDCA , CDCA , TCA , and TUDCA ; TCDCA fed group exhibited a moderate increase in concentrations of CA , TUDCA , and CDCA ; TUDCA fed group showed a moderate increase in concentrations of CA , TUDCA , UDCA , CDCA , and TCA ; TDCA fed group showed increased levels of CA , CDCA , and UDCA . Finally, Chol group exhibited elevated levels of CA , CDCA , and TCA .BAs in the gallbladder of grass carp were dominated by CA (1.0\u2013120.1 ng/\u03bcl over groups) and TUDCA , followed by medium levels of CDCA (0.1\u20134.1 ng/\u03bcl over groups), TCDCA (0\u20139.3 ng/\u03bcl over groups), and DCA (0\u20139.1 ng/\u03bcl over groups), and low levels of TCA (0\u20130.4 ng/\u03bcl over groups), TDCA (0\u20130.6 ng/\u03bcl over groups), UDCA (0\u20130.03 ng/\u03bcl over groups), LCA (0\u20130.006 ng/\u03bcl over groups), and TLCA (0\u20130.0001 ng/\u03bcl over groups) ; TCDCA fed group was significantly different from all groups except TUDCA fed group; the two secondary BS groups (TDCA fed and TLCA fed groups) were not significantly different from the Ctrl group . Chol group showed significant differences with all other groups .The result of PERMANOVA test showed that biliary BAs of the TCA fed group were significantly different from all other groups  did not significantly alter biliary BAs; The chelating agent slightly promoted the concentration of biliary CA, CDCA and TCA.Cetobacterium, followed by Bacteroides (10.2%) and Citrobacter (5.6%). The composition of gut microbiota varied greatly among different groups. At the phylum level , Fusobacteria ranged from 4.1% in TCA fed group to 88.5% in Chol group. Proteobacteria, Bacteroidetes, and Firmicutes decreased from 48.5, 38.2, and 8.7% in the TCA fed group to 5.4, 4.1, and 0.8% in the Chol group, respectively. Compared with the Ctrl group, both TCA fed and TCDCA fed groups exhibited increased Bacteroidetes, Firmicutes, and Proteobacteria, and decreased Fusobacteria. Only two of these, Firmicutes and Proteobacteria, were increased in the TLCA fed group. However, in TUDCA fed group and Chol group, Proteobacteria were decreased, while Fusobacteria were increased . The median value of Firmicutes/Bacteroidetes (F/B ratio) ranged from 0.108 in the Ctrl group to 5.594 in the TLCA fed group . It was significantly increased in TCA fed, TLCA fed, and TDCA fed groups in comparison to the Ctrl group .Gut microbiota of the 56 fish samples were sequenced. After the initial quality filtering, chimera checking, and OTU picking, 624 non-singleton OTUs were identified at the 97% similarity level. At the genus level, roughly 57.4% of the total reads were annotated as P < 0.05 in all cases); Chao1 index was significantly higher in TCDCA fed and TLCA fed groups (P < 0.05 in both cases), PD_whole_tree index was lower in the Chol group (P = 0.023). No significant difference in any of the diversity parameters was detected for the TUDCA fed group .In comparison to the Ctrl group, Shannon and Simpson indices of two primary BS groups (TCA fed and TCDCA fed groups) and one secondary BS group (TLCA fed groups) were significantly higher , where the majority of samples from TCA fed, TCDCA fed, and TLCA fed groups clustered together. PCoA and PERMANOVA analyses with weighted_unifrac distance showed  that TCA fed group was separated from the remaining groups ; TCDCA fed group was mixed with the TLCA fed group , and separated from the remaining groups: TCA fed group , TUDCA fed group , Chol , and Ctrl . TUDCA fed group, TDCA fed group, Chol, and Ctrl groups could not be set apart from each other. In comparison to the Ctrl group, two primary BS groups (TCA fed and TCDCA fed groups) were significantly different , whereas TUDCA fed group, Chol, and two secondary BA groups (TLCA fed and TDCA fed group) were not significantly different. Correlation analysis showed that Cetobacterium , Bacteroides , and Lachnospiraceae  contributed largely to the PCoA1 coordinate, whereas Delftia , Enterobacteriaceae , and Bacteroides  contributed largely to the PCoA2 coordinate (Supplementary Table S4).For beta diversity, cluster analysis indicated that all samples were divided into two clades  significantly decreased proportion of the Fusobacteria while promoted proportion of the Proteobacteria, and they also increased alpha diversity and induced community succession of the gut microbiota; Secondary BS (TLCAS and TDCAS) resulted in a higher F/B ratio, TLCAS also increased proportion of the Proteobacteria and Firmicutes. TUDCAS had no significant effect either on structure or F/B ratio of the gut microbiota.P < 0.05 in all cases, Supplementary Table S5; Figure 3). In comparison to the Ctrl group, and with the exception of TDCA fed group, the remaining groups exhibited varying degrees of decrease in Fusobacteriaceae  and Cetobacterium. More specifically, 15 taxa, including Clostridiaceae, Ruminococcaceae, and Citrobacter, were significantly changed in the TCA fed group; 5 genera, including Lactococcus and Coprococcus, were significantly changed in the TCDCA fed group; 10 taxa, including Ruminococcaceae and Citrobacter, were significantly changed in the TUDCA fed group; 8 taxa, including Brevibacillus, Clostridiaceae, and Meiothermus, were significantly changed in the TLCA fed group; 2 taxons, including Clavibacter and Fusobacteriaceae, were significantly changed in the TDCA fed group; and 8 taxa, including Rhodospirillaceae and Acinetobacter, were significantly changed in the Chol group .In the Kruskal\u2013Wallis test of taxonomic abundance at the genus level, 32 bacterial genera exhibited significant differences among different groups . Specifically, our results revealed a marked higher abundance in genes involved in the biosynthesis of secondary BAs (P = 0.000). Another notable observation was the significantly lower abundance of genes involved in fatty acid elongation pathway in mitochondria in all treatment groups (P = 0.000). Furthermore, genes in pathways associated with the biosynthesis of steroids (P = 0.011) and unsaturated fatty acids (P = 0.000), as well as metabolism of sphingolipids (P = 0.001) and alpha-linolenic acid (P = 0.000), were more abundant in TCA fed, TCDCA fed, and TLCA fed groups, and less abundant in the remaining three groups. Contrarily, genes in linoleic acid metabolism (P = 0.000) and glycerolipid metabolism (P = 0.000) were less abundant in the TCA fed, TCDCA fed, and TLCA fed groups, and more abundant in the remaining three groups. As regards, the microbial genes involved in biomodification of BAs, the abundance of BSH was highly significantly increased in the TCA fed group (P = 0.01), and the abundance of 7\u03b1-HSDH (P = 0.020), 3\u03b1-HSDH (P = 0.053), and 3\u03b2-HSDH (P = 0.056) increased in the TLCA fed group .The PICRUST prediction revealed that most KEGG pathways associated with microbial lipid metabolism were altered in the fish fed BS-supplemented diets .The network inferred through MENA analysis showed that eight biliary BAs and 43 microbial taxa were involved in the interplay between biliary BAs and gut microbiota. These BAs were CA, CDCA, TCDCA, DCA, TDCA, LCA, UDCA, and TUDCA. Microbial taxa included 10 taxa from Firmicutes, 6 taxa from Bacteroides, 20 taxa from Proteobacteria, 4 taxa from Fusobacteria, 1 taxon from Thermomicrobia, 1 taxon from Actinobacteria, and 1 taxon from Verrucomicrobia. In the Firmicutes, Lachnospiraceae, Ruminococcaceae, Clostridiaceae, and Citrobacter, were strongly correlated with the concentration of biliary CA, with r = 0.85, 0.60, and 0.77, respectively . These three taxa also exhibited strong correlations with the concentrations of DCA and TCDCA. Bacteroides and Coprococcus genera exhibited moderate correlations with the concentrations of CA , DCA , and TCDCA . The concentration of CDCA was correlated with the abundance of Lachnospiraceae , Coprococcus , Ruminococcaceae , and Citrobacter ; LCA with Lachnospiraceae ; TCA with Anoxybacillus , Lysobacter , Cupriavidus , Rhodospirillaceae , Brevibacillus , and Lactococcus . Beside these interactions, we also found some weak correlations: Clostridiaceae was weakly related to the concentration of LCA , CA , and DCA . A negative relationship between Cetobacterium and the concentration of biliary CA , DCA , and TCDCA  was also detected.Among the microbial taxa directly correlated with BAs, Lachnospiraceae, Ruminococcaceae, and in vivo experiments focusing on both the fluctuation of biliary BAs and gut microbiota responses to different BS supplemented in diet remain exceedingly rare. Although impacts of different BS dietary supplementations on regulation of BA synthesis have been studied in rats . This result was consistent evidence that the level of free CA with was higher than conjugated TCA in TCA fed group and corroborated that the gut microbiota in grass carp intestine took part in deconjugation of the BAs. The same evidence was also found in TCDCA fed group where the level of free CDCA was higher than conjugated TCDCA and a higher level of the BSHs in the gut microbiota accompanied. Otherwise, the higher abundance of 7\u03b1-HSDHs and 7\u03b1-HSDH in TLCA fed group suggested an altered effect of its gut microbiota on BA oxidation and epimerization  and biosynthesis of secondary BAs. In turn, this change in the rate of BA biotransformation might regulate the biosynthesis of primary BAs in liver . We founAge , diet T, and metSupplementary Figure S1). TCAS, TCDCAS, and TLCAS significantly increased the proportion of Proteobacteria as well as its member taxon, Pseudoalteromonadaceae . The interaction network among biliary BAs and gut microbiota showed that CA and TCDCA were positively related to the proportion of most Proteobacteria taxa . Fish bacterial diseases are usually caused by the Proteobacteria genera, such as Pseudomonas  distant groups of animals. Our results also revealed the distinct effects of different dietary BAs on gut microbiota. TUDCAS had no significant effect on composition of the gut microbiota in grass carp. The changes of the gut microbiota resulted in alteration in BAs biotransformation and increased synthesis of toxic secondary BAs. These results suggested that UDCA should be more appropriate for treatment of grass carp hepatobiliary disease because it had lesser side effects.S-GW and G-TW were the main contributors of the manuscript. S-GW and G-TW contributed to the study design, the acquisition of the funding, the oversight of the study, the interpretation of the data, and the editing of the manuscript. FX was responsible for the design, the data collection, the analysis, the interpretation of data, and the writing of the manuscript. S-GW and JZ contributed to the collection, the analysis, and the interpretation of the data. IJ was responsible for the quality control, the interpretation of the data, and language editing of the manuscript. HZ was responsible for the study coordination and quality control, and the editing of the manuscript. W-XL and ML contributed to the fieldwork data collection and the drafting 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": "As genome-wide association studies (GWAS) have grown in size, the number of genetic variants that have been associated per disease has correspondingly increased. Despite this increase in the number of single-nucleotide polymorphisms (SNPs) identified per disease, their biological interpretation has in many cases remained elusive. To address this, we have combined GWAS results with orthogonal sources of evidence, namely the current knowledge of molecular pathways; real-world clinical data from six million patients; RNA expression across tissues from Alzheimer\u2019s disease (AD) patients, and purpose-built rodent models for experimental validation. In more detail, first we show that when examined at a pathway level, analysis of all GWAS studies groups AD in a cluster with disorders of immunity and inflammation. Using clinical data, we show that the degree of comorbidity of these diseases with AD correlates with the strength of their genetic association with molecular participants in the Janus kinases/signal transducer and activator of transcription (JAK-STAT) pathway. Using four independent RNA expression datasets we then find evidence for the altered regulation of JAK-STAT pathway genes in AD. Finally, we use both in vitro and in vivo rodent models to demonstrate that A\u03b2 induces gene expression of the key drivers of this pathway, providing experimental evidence to validate these data-driven observations. These results therefore nominate JAK-STAT anomalies as a prominent aetiopathological event in AD and hence a potential target for therapeutic development, and moreover demonstrate a de novo multi-modal approach to derive information from rapidly increasing genomic datasets. CLU encoding clusterin, is involved in processes as diverse as complement signaling, protein binding and chaperoning, and cell survival [As genome wide association studies (GWAS) have grown in size, often now numbering tens of thousands of research participants, the number of genes identified as contributing to disease susceptibility have correspondingly grown. This is as true for Alzheimer\u2019s Disease (AD) as it is for many other disorders, and bioinformatic and pathway analyses of this large number of susceptibility genes is providing a highly efficient method of proposing and prioritising underlying biological pathways for further study ,2. In sosurvival . In the https://www.ebi.ac.uk/gwas/) together with a co-morbidity study from real-world data to identify shared pathological processes. We then tested the resulting pathway in observational and empirically derived genome wide expression datasets from human and rodent studies, and finally validated the results in empirical studies in rat models in vitro and in vivo. The results, demonstrating a role for JAK-STAT signaling in AD, are in line with the known contribution of inflammatory processes to the disease, but they further nominate a specific target for therapy and provide a possible approach to interpretation of GWAS data for other disease areas. In an effort to address this limitation, we reasoned that it should be possible to hone pathway analysis by utilising orthogonal datasets. Specifically, we hypothesised that pathways are more relevant to disease aetiopathogenesis if diseases that shared pathways also shared morbidity. Put another way, if two or more diseases are more commonly found to co-occur rather than by chance, and if those comorbid diseases also share molecular pathways, one would predict that those shared pathways are more likely to play a role in aetiopathogenesis. In order to test this reasoning, we combined pathway analysis of the GWAS associations from all diseases \u2019 , \u2018A\u2019 as the set of genes associated with disease \u2018A\u2019, and \u2018B\u2019 as the set of genes associated with disease \u2018B\u2019, the first sample \u2018SGiven that the results from the analysis described above suggested an intersection between AD and inflammatory diseases and hence confirming known associations, as discussed in the results and discussion sections, we subsequently focused on this overlap. For each inflammatory disease sampled in the GWAS-catalogue that had statistical power for subsequent analysis (see below), we calculated a so-called pathway load for each KEGG pathway. This is a numeric value that represents the proportion of susceptibility genes that a given disease has on a given KEGG pathway. Given disease \u2018A\u2019 and pathway \u2018p\u2019, the pathway load is equal to the number of susceptibility genes that disease \u2018A\u2019 has on pathway \u2018p\u2019, divided by the total number of times that any associated gene of disease \u2018A\u2019 is annotated as belonging to any KEGG pathway. Formally, if we denote \u2018m\u2019as the number of genes of disease \u2018A\u2019 that belong to pathway \u2018p\u2019, the pathway load is: We divide by https://www.cdc.gov/nchs/nhds/, [http://www.icpsr.umich.edu/icpsrweb/ICPSR/studies/24281), or in the National Archive of Computerized Data on Aging  recordsg  AddNeuroMed (ANM), a longitudinal multi-centre cohort study with blood samples from 105 AD cases, 125 Mild Cognitive Impairment (MCI) individuals and 114 controls ,10; 2) tp values we report correspond to the RNA-expression variable per gene. To test for statistically significant differences in RNA-expression in these four datasets for a given pathway, we use a per gene general linear model (GLM) with a binomial link function, which models AD status (two levels per person\u2013either AD patient or control) as a function of RNA-expression while controlling for a number of covariates . The 2 atmosphere. Neuronal cultures were treated 7\u20139 days post-plating. In order to explore JAK-STAT signaling in vitro, primary neuronal cultures were generated from Sprague Dawley E18 rat embryos by papain dissociation according to the manufacturer\u2019s instructions  and cultured as previously described . Briefly2O added. A\u03b2 was incubated for 24 h at 37 \u00b0C and further diluted to 100 \u03bcM in PBS followed by 18 h incubation at 37 \u00b0C. Rat primary neuronal cultures were treated with 3 \u00b5M A\u03b2 for either 4 h or 24 h. These time points were selected based on the commonly observed time progression of changes in RNA after A\u03b2 treatment.A\u03b21\u201342 peptide was purchased from Dr. David Teplow  and was resuspended in 100% 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP) at a final concentration of 1 mM. For complete solubilisation the peptide was homogenized using a Teflon plugged 250 \u03bcL Hamilton syringe. HFIP was removed by evaporation in a SpeedVac , A\u03b21\u201342 resuspended at a concentration of 5 mM in dimethylsulfoxide (DMSO) and sonicated for 10 min. Oligomers were prepared as previously described : A\u03b21\u201342 We further tested whether the observations made in vitro were also found in in vivo models. Male wild type Wistar rats  were subjected to bilateral injections of 50 \u00b5M A\u03b21-42 or PBS (n = 5 per group) into the lateral ventricles . Brains were collected 3 h post injection. Entorhinal cortex was subdissected, frozen in liquid nitrogen and processed for RNA extraction. Frozen tissues were thawed and homogenised in Trizol and total RNA extracted according to the manufacturer\u2019s instructions . \u2212\u0394\u0394CT method as described by Livak and Schmittgen [Total RNA from entorhinal cortex (1\u2009\u03bcg) was reverse transcribed using random hexamers and a Taqman RT kit  according to the manufacturer\u2019s instructions. PCR primers were designed using the Universal Probe Library package  and used in SYBR Green-based PCR reactions performed on a StepOnePlusTM (96 well) thermal cycler . Relative quantification of gene expression between samples was determined using the 2hmittgen . InternaPrimer sequences: Jak1(NM_053466.1): Forward: 5\u2032-ccaccgggacatttcact-3\u2032; Reverse: 5\u2032-ttgtgggaaacctgtctcatc-3\u2032; Jak2 (NM_031514.1): Forward: 5\u2032-ggagagtatgttgccgaagaa-3\u2032; Reverse: 3\u2032-atattatgatacacaggcgtaatacca-3\u2032; Jak3 (NM_012855.2): Forward: 5\u2032-ggccaaagtcccatcttct-3\u2032; Reverse: 5\u2032-gaagctccacacgtcagattg-3\u2032; Tyk2 : Forward: 5\u2032-tgccatcttgctctcaacc-3\u2032; Reverse: 5\u2032-gtgagggatacagttcttgaagc-3\u2032.\u2212\u0394\u0394CT calculations performed. The fold change in the target genes  were calculated for each sample and the mean calculated. Statistical significance was determined by Student\u2019s t-test. Data are represented as normalised fold increases over control samples. Values are given as mean \u00b1 s.e.m (standard error of the mean).All samples were run in triplicate from three independent experiments. The mean crossing threshold (CT) values for both the target and internal control genes in each sample were determined and the 2All animal studies described in this manuscript were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986. We further certify that the research  was conducted according to the requirements of POL-GSKF-410 and associated relevant SOPs, and that all related documentation is stored in an approved HBSM database. Human biological samples were sourced ethically and their research use was in accord with the terms of the informed consentsGiven that recent large scale association studies suggest genes related to AD risk are involved in many different biological pathways, only some of which would have been predicted in advance, we wondered whether some of these pathway associations would be shared with other diseases. To address this question systematically, we first obtained from the GWAS catalogue  a list oUsing this data of genes associated with diseases and the KEGG pathways that those genes appear in, we then generated a gene-gene matrix, plotting for each gene associated with each disease the numbers of KEGG pathways shared with genes associated with each other disease in the dataset. Not surprisingly, we find many genes participate in many different pathways. This is illustrated for three disorders in p value < 0.05). Fourteen of these diseases were disorders commonly classified as diseases of immunity, while the other four were AD, age-related macular degeneration (ARMD), T1DM and Hypothyroidism , while \u2018JAK-STAT\u2019(hsa04630) did survive  suggesting that the shared GWAS genes on this pathway might contribute to the observed co-morbidity between AD and these disorders of immunity Having demonstrated that AD, together with ARMD, hypothyroidism and T1DM, share multiple KEGG pathways with disorders of immunity, we then explored which of these pathways was responsible for this overlap. In order to do this systematically, we first determined the strength of association of each disease with each KEGG pathway, calculating the proportion of susceptibility genes that a given disease has for each KEGG pathway (\u2018pathway load\u2019). Then, using the US National Hospital Discharge Survey (NHDS), a dataset including diagnostic information from more than six million patients, we calculated the co-morbidity between disorders of immunity and late onset AD on discharge from in-patient care between 1979 and 2006. Finally, for each individual pathway we then calculated the degree of correlation between pathway load and comorbidity with AD. Most of the KEGG pathways showed no significant correlation, with only three exceptions, indicating possible meaningful shared pathways contributing to disease comorbidity see . Of thesIf the KEGG pathway is responsible for the observed co-morbidity between AD and disorders of immunity, then we reasoned that altered JAK-STAT signaling would be a feature of AD. In order to explore this, we utilised gene expression datasets from blood from human cohort studies (Datasets 1 and 2), from brain from post-mortem human studies (Dataset 3) and from a rodent in vitro model relevant to AD (Dataset 4) with robust proof of concept using empirical studies with gene-knockdown, in animal models and in post-mortem human brain. p < 0.05 after Bonferroni correction, see p value 5 \u00d7 10\u22129). In DCR, six of the 47 JAK-STAT genes were significantly altered (p < 0.05), also revealing significance at the pathway level . p values obtained in ANM were significantly correlated  in the AD population when compared with the control population. We then examined JAK-STAT genes in the MCI population compared to unaffected controls in both cohort datasets, finding 10 out of 47 genes dysregulated in both studies, with binomial test being also significant .We first examined expression of genes from the JAK-STAT pathway (KEGG ID hsa04630) in blood from patients with AD compared to unaffected controls, reasoning that post-mortem brain might have more late-stage, secondary changes of inflammation, possibly less relevant to aetiopathological pathways. We used two cohorts, one a multinational longitudinal study , and another, a single centre longitudinal study utilising the exact same protocol , and 37 of which survived multiple comparison correction . A binomial exact test reveals that this proportion of significant genes in the JAK-STAT pathway is larger than expected by chance . With respect to the five genes that were significant in ANM (Dataset 1) and that were also sampled in DCR (Dataset 2), TYK2 , IFNAR2  and PIAS1  were also significantly altered in Dataset 3 . Binomial testing showed that this proportion of dysregulated genes was higher than expected by chance alone . Of the four genes that were significant in ANM (Dataset 1) and were also sampled in all other datasets , Akt1 , Ifnar2 (0.007) and Pias1 (0.016) were also dysregulated in Dataset 4  and maintained up to 24h (p = 0.0063). Jak1 and Jak2 mRNA levels were only significantly increased at 24h with p values of p = 0.024 and p = 0.015, respectively. Jak3 expression was modulated by A\u03b2 at any time point (p > 0.05). We then further validated these bioinformatics results using empirical studies, measuring the key drivers of the pathway, Jak1, Jak2, Jak3 and Tyk2, in an in vitro rat model of neuronal toxicity. Previously, we and many others have demonstrated that rodent neurons exposed to amyloid peptides are susceptible of A\u03b2-induced toxicity and other phenotypes, including tau phosphorylation and synaptic alterations ,21,22,23p value 0.001), Jak2 (0.0071) and Tyk3 (0.0032) were significantly upregulated (p < 0.05) in the brains of these rats but not in control animals . Five male rats were subjected to bilateral intracerebroventricular injection of 50 \u00b5M A\u03b21-42 or equal volumes of phosphate buffered saline (PBS). After 3h following injection, animals were sacrificed, and brains harvested for RNA extraction. We measured the mRNA levels for Jak1, Jak2, Jak3 and Tyk2 in entorhinal cortex from A\u03b2-injected rats or PBS-sham controls by real time PCR. 3h after A\u03b2 injection, the levels of Jak1  and Type 1 Diabetes mellitus (T1DM). The association with ARMD is particularly interesting as it has previously been found to be a risk factor for AD ,25, becafactor H ,29,30. Tfactor H , our datHowever, the most extensive association between shared pathways and disease we find is with disorders of immunity and inflammation. The role of inflammation in AD has been apparent for many years. This evidence is very extensive and comes from many directions . It inclAs in any \u2018big data\u2019 approach, there are limitations both to the datasets available and to our use of them. First, in using the GWAS catalogue as a primary data-source, we limit ourselves to disease-gene associations where a significant number of genes have been identified. We do this in order to provide sufficient power for analysis, but acknowledge that the limit of 25 susceptibility genes to enable a disease to enter analysis is both arbitrary and dependent on the size and numbers of studies that happen to have been conducted to date. Almost certainly, we miss information as a consequence of data limitation. Secondly, by segregating genes into pathways we attempt to overcome the intrinsic limitation of GWAS studies, in that biology is mechanistically enacted at the level of pathway and not gene, let alone SNP. Given the large number of SNPs and genes in the human genome, two diseases may have no elements measured in GWAS, or even sequencing studies, in common and yet share an overlapping disease pathogenesis. Measuring association not with SNP but with multiple SNPs across a gene (\u2018gene-wide association\u2019) is one attempt to overcome the limitation; here we go one step beyond this with a pathway-wide association approach. However, in attempting to derive such information from the GWAS data, we are severely limited by current understanding of biological pathways, which is rudimentary at best. This limitation is bound to hinder our derivation of knowledge from information in this context. Thirdly, in seeking to identify diseases comorbid with AD, we have crudely utilised a dataset of concurrent diagnoses, taking no account of some of the confounds or other concerns of conventional epidemiology. Indeed we cannot be sure whether the co-morbidity we observe is due to the disease itself or the drugs used to treat the disease. However, we note that similar claims-level analyses of real-world clinical data have recently proven valuable in studying genetic and environmental factors shared amongst diseases .We accept the limitations of our approach described above. However, in mitigation of these potential limitations, the datasets we examine are huge; namely, all genetic studies with all genes and all diseases in the first phase, and a dataset of over six million people in the second. Furthermore, we would suggest that some confounds are less critical in the analysis we present here. For example, in studies of risk and protection then clearly understanding direction of effect\u2013whether it is the disease or the treatment that affects risk\u2013is fundamental. However, it becomes less important, potentially irrelevant, where we are determining simply whether the same processes are involved, as both disease and treatment will at some level and in some cases affect the same molecular pathways, which are the axis of our analysis. Finally, despite the limitations of deriving knowledge from data using this approach, the fact that the findings replicate in observational molecular studies in man and in experimental studies in rodents offers strong support to the results.The JAK-STAT signaling pathway, nominated as a potential target for therapy through data-driven genomics and real-world data in this study, is a key regulator of the response to mediators of inflammation, including cytokines, chemokines  and micrIn summary, a combined, sequential analysis of GWAS data agnostic to disease type, combined with real-world data of co-incidence of AD with other diseases, nominated JAK-STAT signaling amongst other pathways as a possible underlying pathogenetic mechanism shared across multiple diseases. Remarkably, these diseases\u2013inflammatory disorders, ARMD and Diabetes had previously been implicated as risk factors for AD. Adding to the weight of evidence for JAK-STAT signaling in AD, we subsequently found altered gene expression of the pathway in multiple human and rodent datasets and in empirical studies of A\u03b2 exposure in rodents, both in vitro and in vivo. These data are not the first to nominate JAK-STAT signaling for therapeutic intervention , as expe"}
+{"text": "This work provides new insights for the safe disposal of Cd-enriched wastewater and for improving the economic viability of Cd-contaminated resources by recovering a value-added photocatalyst.Biochar is widely used for the adsorptive removal of Cd from water and soil, but the Cd-enriched biochar produced carries a risk of secondary pollution. In this work, biochar derived from rice straw was used to adsorb Cd from plating wastewater. The Cd-enriched biochar showed a saturated adsorption capacity of about 63.5 mg/g and could be recycled and used in a mesoporous carbon-supported CdS (CdS@C) photocatalyst after pyrolysis carbonization and a hydrothermal reaction. The results demonstrated that the as-prepared CdS@C photocatalyst contained mixed cubic and hexagonal CdS phases, with a considerably lower band gap (2.1 eV) than pure CdS (2.6 eV). CdS@C exhibited an enhanced photocatalytic performance for the degradation of organic dyes under visible light irradiation compared with pure CdS due to its excellent light-harvesting capacity and efficient electron-hole separation. Moreover, the continuous formation of active species (h Cadmium (Cd) is extremely toxic to plants and poses a serious threat to humans and animals when it enters the food chain  , rhodamine B , methylene blue , acid red 11 , and 5,5\u2032\u2013dimethyl\u20131\u2013pyrroline\u2013N\u2013oxide  were commercially purchased. Deionized water was used to prepare reaction and stock solutions. The rice straw biomass was picked from the College of Life Sciences, Fujian Agriculture and Forestry University  was obtained through centrifugation, followed by washing with deionized water and drying at 60\u00b0C for 24 h.2/O2  mixed atmosphere, Cd-Biochar was oxidized at 650\u00b0C for 0.5 h, which increased the cadmium content in the biochar from 2.5 to 5%. This was dispersed into a 5 mM Na2S solution in a Teflon-lined stainless steel pot, heated in a muffle furnace at 180\u00b0C for 72 h, and then cooled to ambient temperature. The CdS@C composite photocatalyst was obtained after washing with deionized water and filtering  of the photocatalyst was calculated by the Kubelka-Munk (K-M) formula:Scanning electron microscopy (SEM) images were acquired using a JSM6700-F  operating at 10 kV to compare the surface morphology of biochar before and after synthesis. Inductively coupled plasma mass spectrometry (ICP-MS) and elemental analysis (EA) were used to determine the contents of C, H, S, and Cd in the material. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images were used to determine the crystal morphology and phase of the materials using a JEM-2010 at 200 kV. X-ray powder diffraction (XRD) patterns were used to determine the composition and molecular structure of the materials; these patterns were obtained using the Ultima IV , operating at a 40-kV tube voltage and a 40-mA tube current. X-ray photoelectron spectroscopy (XPS) was carried out to analyze the valence states of elements using an ESCALAB 250  with an Al/Mg double anode target as the radiation source . The C 1s peak at 284.7 eV was used to calibrate the energy scale of the XPS spectra. The diffuse reflectance spectra (DRS) were recorded from 200 to 800 nm on an UV2550 UV-Vis spectromater  using a BaSOA is the absorbance, hv is 1,240/wavelength, and K is a constant. The electron spin resonance (ESR) spectra were obtained of active substances produced by visible light irradiation using a Bruker A300 spectrometer .where The role of RhB in photocatalytic degradation was studied. In a general catalysis procedure, photocatalyst (20.0 mg) was dispersed in 100 mL of a photocatalytic reactor with a 50 mL RhB solution . The samples were magnetically stirred at 800 rpm for 30 min in the dark. A xenon lamp  equipped with cut-off filters at 420 and 780 nm was used as the light source. After visible light irradiation for a certain period of time, the photocatalytic absorbance experiments were monitored by a 554-nm ultraviolet-visible spectrophotometer. The efficiency of photocatalysis was calculated by the following formula:R is the degradation rate, C0 is the characteristic absorbance of RhB before photocatalysis, and C is the characteristic absorbance of RhB after photocatalysis  during photocatalytic reactions using the capture agent DMPO. Hydroxyl radicals (\u2022OH) generated during the photocatalytic reaction could react with terephthalic acid (TA) to produce highly fluorescent 2-hydroxyterephthalic acid (TAOH), and the fluorescence was detected by fluorescence spectroscopy.The current vs. time (I-t) curves were obtained using a Shanghai Chenhua CHI600E electrochemical workstation. First, 5 mg of a sample was ultrasonically dispersed in a 5% naphthene solution. About 5 \u03bcl of the suspension was then uniformly smeared on an ITO glass substrate (1 cm \u00d7 1 cm) and allowed to dry at room temperature. The coating process was repeated 10 times to form a thin film on the ITO glass substrate. The change in the instantaneous current with time was used to evaluate whether the photocatalyst had a photoelectric effect. An electron spin resonance (ESR) spectrometer (Bruker A300) was used to detect the presence of superoxide radical  atmosphere at 650\u00b0C before the sulfuration  and 412.3 eV (Cd 3d3/2), which was consistent with the characteristic peaks of Cd2+ in CdS   to evaluate its photocatalytic activity. As shown in Previous works have demonstrated that mixed-phase CdS composites supported on porous materials have strong photocatalytic activities . As shown in 2\u2022\u2212 were detected after light irradiation of CdS@C, whereas the ESR signal of DMPO\u2013O2\u2022\u2212 remained silent under dark conditions  during the photocatalysis of CdS@C.This work presents the first report of the synthesis of a CdS@C composite photocatalyst by upcycling Cd from plating wastewater. The photocatalytic performance of the composite catalyst obtained was evaluated by using it in the photodegradation of organic dyes. CdS@C contained a mixture of cubic and hexagonal CdS and exhibited a considerably higher light-harvesting capacity and electron-hole separation efficiency. CdS@C displayed enhanced photocatalytic efficiency toward the degradation of organic dyes compared with pure CdS. Further investigation of the photocatalytic mechanism suggested that the photodegradation of organic dyes was largely attributable to the continuous formation of catalytically active species (The datasets generated for this study are available on request to the corresponding author.ZC, S-GZ, and R-ZX designed the research and co-wrote the paper. R-ZX, X-GY, Z-WC, and J-XL conducted experiments and characterized the materials. RH and Z-XC conducted the catalysis.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": "Oxidative stress is identified as a major inducer of retinal pigment epithelium (RPE) cell dysregulation and is associated with age-related macular degeneration (AMD). The protection of RPE disorders plays an essential role in the pathological progress of retinal degeneration diseases. The pharmacological functions of fucoxanthin, a characteristic carotenoid, including anti-inflammatory and antioxidant properties, may ameliorate an outstanding bioactivity against premature senescence and cellular dysfunction. This study demonstrates that fucoxanthin protects RPE cells from oxidative stress-induced premature senescence and decreased photoreceptor cell loss in a sodium iodate-induced AMD animal model. Similarly, oxidative stress induced by hydrogen peroxide, nuclear phosphorylated histone (\u03b3H2AX) deposition and premature senescence-associated \u03b2-galactosidase staining were inhibited by fucoxanthin pretreatment in a human RPE cell line, ARPE-19 cells. Results reveal that fucoxanthin treatment significantly inhibited reactive oxygen species (ROS) generation, reduced malondialdehyde (MDA) concentrations and increased the mitochondrial metabolic rate in oxidative stress-induced RPE cell damage. Moreover, atrophy of apical microvilli was inhibited in cells treated with fucoxanthin after oxidative stress. During aging, the RPE undergoes well-characterized pathological changes, including amyloid beta (A\u03b2) deposition, beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) expression and tight junction disruption, which were also reduced in fucoxanthin-treated groups by immunofluorescence. Altogether, pretreatment with fucoxanthin may protect against premature senescence and cellular dysfunction in retinal cells by oxidative stress in experimental AMD animal and human RPE cell models. While aging causes a decline in the ability to respond and adapt to the accumulative impact of different exposures, age-related disease develops when cellular dysfunction from compromised cytoprotective pathways is severe enough to cause tissue destruction . Aging iAge-related macular degeneration (AMD) is a leading cause of blindness affected by oxidative stress among the elderly, characterized by RPE degeneration. Histological changes in RPE cells, including atrophy of apical microvilli and disruption of cellular junctions, occur before the loss of photoreceptor cells . Sodium The RPE is derived from the outer layer of the optic cup that grows out from the forebrain. Therefore, AMD, a devastating neurodegenerative disease, has many pathological characteristics that are common to Alzheimer\u2019s disease, including amyloid beta (A\u03b2) accumulation, which has also been demonstrated to be associated with drusen in eyes ,12. ElevGenerally, oxidative stress is characterized by increased levels of ROS, resulting in its damage. ROS, such as hydrogen peroxide, that extensively attack DNA, cellular junctions and other cellular organelles play an important physiological and pathophysiological role in controlling various cellular functions such as cellular differentiation, proliferation, senescence and death. Further, ROS are important signaling molecules that play an essential role in the progression of inflammation. Malondialdehyde (MDA) expression and nuclear phosphorylated histone (\u03b3H2AX) deposition were used as markers of lipid peroxidation and DNA strand damage. Administration of antioxidants targeting ROS generation and reducing ROS-induced cell damage of the RPE may prevent AMD progression ,21. CataHijikia fusiformis, Laminaria japonica and Sargassum fulvellum [Fucoxanthin  is an orulvellum , and resulvellum ,26,27,28ulvellum ,30. Due p < 0.05) and 10 mg/kg fucoxanthin (p < 0.01)  B.To examine inhibitory prosenescent properties of fucoxanthin in vivo, we pretreated the animals with fucoxanthin  for seven days before sodium iodate-induced retinal degeneration and examined retinal senescence and histological changes. \u03b2-Galactosidase , a lysosomal hydrolytic enzyme with the physiological function of catalyzing the hydrolysis of glycosidic bonds which transform lactose into galactose, is known to be characteristic of senescent cells. The intense blue deposits of senescence-associated \u03b2-galactosidase  staining were observed in sodium iodate-induced experimental animals C, comparTo investigate the cytoprotective effect of fucoxanthin and hydrogen peroxide in human ARPE-19 cells, the cells were pretreated with 1, 5 or 10 \u03bcM fucoxanthin for 48 h and with 500 \u03bcM hydrogen peroxide for an additional 48 h. Hydrogen peroxide increased the ROS level in the ARPE-19 cells and fucoxanthin exhibited an inhibitory effect on oxidative stress-induced ROS production. Compared with the ROS generation detected in the hydrogen peroxide-exposed groups, drastically decreased ROS generation was observed in the group treated with 5 and 10 \u03bcM fucoxanthin A. MoreovHydrogen peroxide is a potent senescence inducer and plays an important role in the induction of senescence. It is well established that hydrogen peroxide triggers potent senescence and DNA damage by various signaling cascades through oxidative stress. To investigate whether fucoxanthin protects against oxidative stress-induced cellular senescence and DNA damage, ARPE-19 cells were pretreated with fucoxanthin for 24 h and then exposed to 500 \u03bcM hydrogen peroxide for another 24 h. The cellular senescence was determined with galactosidase activity. The proportion of intense blue deposits after SA-b-Gal staining cells exhibited a statistically significant increase in the ARPE-19 cultures treated with 500 \u03bcM hydrogen peroxide for 24 h alone B, comparMoreover, there are well-known molecular triggers for the senescence response, including the DNA damage response, so we also examined the protective effect of fucoxanthin from DNA damage in ARPE-19 cells after oxidative stress. Immunofluorescence staining for \u03b3H2AX, the substitute marker of the DNA damage reaction, revealed that oxidative stress commanded to nuclear cH2AX deposition, compared with the significant increase in nuclear \u03b3H2AX observed in the hydrogen peroxide-treated groups D\u2013F, whicTo test the cell structure protective effects of fucoxanthin, scanning electron microscopy (SEM) was used to investigate the ultrastructure morphological changes. The intact cell junction of cultured cells was observed by electron microscopy. The RPE operates specialized metabolic and transport functions critical for homeostasis, thereby forming a part of the blood\u2013retina barrier. The apical surface of RPE cells radiates long and thin microvilli that establish a complex of close structures A. The ceAs the abovementioned experiment observed the protective effect of fucoxanthin on microvilli formation following exposure to hydrogen peroxide, further study was performed to examine the morphogenesis of the cytoskeleton and cell junction. The effect of fucoxanthin on the fluorescence staining of tight junction protein zonular occludens (ZO-1) and F-actin in ARPE-19 exposure to hydrogen peroxide was investigated by immunofluorescence microscopy. The results show that the expression continuous around the ZO-1 cells and the filamentous structure of actin were observed in the ARPE-19 monolayer culture A\u2013D. WithBACE1 is a transmembrane protease responsible for the \u03b2-site cleavage of the amyloid precursor protein to produce A\u03b2, and A\u03b2 is an important component of plaques in neurological disease and drusen deposits in AMD. To test the protective effect of fucoxanthin on the AMD cellular model, fucoxanthin was added to ARPE-19 cells exposed to hydrogen peroxide. To further confirm the involvement of AMD, the expression of its various drusen-related proteins including A\u03b21-42 and BACE1 was examined by immunofluorescence assays. Hydrogen peroxide exposure up-regulated cellular expressions of A\u03b21-42 and BACE1 E\u2013H compaThis study demonstrated that fucoxanthin has a cytoprotective effect on retinal cell degeneration in a dose\u2013response fashion in experimental animal and cultured cell models. Treatment with fucoxanthin substantially inhibited the DNA strand damage marker, and nuclear \u03b3H2AX deposition and premature SA \u03b2-galactosidase staining were observed following fucoxanthin pretreatment. An administration of fucoxanthin significantly inhibited ROS generation, reduced MDA concentrations and increased the mitochondrial metabolic rate in oxidative stress-induced RPE cell damage. Moreover, atrophy of apical microvilli, A\u03b2 deposition, Beta-secretase 1 (BACE1) expression and tight junction disruption were also reduced and inhibited in cells treated with fucoxanthin after oxidative stress.AMD is a complex eye disease and is classified into wet or dry forms. Wet AMD is characterized by the sprouting of new vessels from choriocapillaris through Bruch\u2019s membrane. Drusen and RPE alterations are the hallmark of dry AMD . OxidatiMitochondrial dysfunction and oxidative damage are appreciably improved with senescence and aging-related diseases. During aging, increased ROS disrupt mitochondrial DND, lipids and structure and limit energy production in AMD ,38,39. RThe actin cytoskeleton is a highly dynamic structure that participates in the morphogenesis of apical microvilli. Due to phototoxicity, the daily renewal of the outer segment of the photoreceptor is ensheathed by microvilli arising from the surface of the pigment epithelial cells and is intensely important to the survival of photoreceptors ,44. DisrThe A\u03b2s, from the amyloid \u03b2 precursor protein cleaved by BACE1, are associated with the pathogenesis of Alzheimer\u2019s disease . A\u03b2 is aHealthy 4\u20135-week-old male Sprague-Dawley rats  weighing 200\u2013300 g were used. All care and treatments of experimental animal studies were approved and monitored by the Mackay Medical College Institutional Animal Care Committee (IACUC-A1070033) in accordance with institutional animal ethical guidelines. Standard diet and tap water were provided ad libitum.The sodium iodate-induced AMD animal model has been widely used for studying retinal degeneration diseases and drug treatment effects. To induce retinal generation, experimental methods followed a previously described protocol  with sli2. The next day, \u03b2-galactosidase staining was identified by development of blue color using microscopy.\u03b2-Galactosidase activity is known to be characteristic of senescent cells and is used as a biomarker for senescent and aging cells. To evaluate the protective effect of fucoxanthin on cellular senescence, galactosidase activity was analyzed. For senescence assay, experimental samples were performed according to the protocol of the SA b-Gal Staining Kit . Tissue and cultured cell samples were incubated overnight at 37 \u00b0C with 5% COTissues or cells were harvested and incubated in PBS containing 10 mM general oxidative stress Indicator CM-H2DCFDA  for 30 min to 1 h at 37 \u00b0C in the dark to allow loading of dye into the cells. This test compound is nonfluorescent when chemically reduced, but after intracellular esterases and oxidation occur within the cell, it becomes fluorescent. The intracellular production of ROS was monitored by a microplate reader with excitation at 490 nm to obtain the absorbance value. The results were expressed as percentage of change and the blank control group was taken as 100%.g at 4 \u00b0C. Later, the absorbance of the supernatant obtained was measured spectrophotometrically at 532 nm according to the manufacturer, and the analyzed data were expressed as malondialdehyde equivalents (nmol/mg tissue protein). These analyzed results were expressed as percentage of change and the blank control group was taken as 100%.The thiobarbituric acid (TBA) reactive substances of product MDA assay (Sigma-Aldrich) was conducted for an index of lipid peroxidation and oxidative stress of retinal tissues. The method involved heating up the assay mixture comprising tissue homogenates. MDA-TBA adduct was prepared by adding TBA solution into each vial containing the standard and the sample was incubated at 95 \u00b0C for 60 min. After cooling to room temperature in an ice bath for 20 min, the reaction mixture was centrifuged for 10 min at 16,000\u00d7 2 and 37 \u00b0C in a humidified incubator. When cells reached confluency, cells were pretreated with various concentrations of fucoxanthin for 3 days and replaced every 24 h for the duration of the experiment. After a brief wash with medium, cells were incubated with 500 \u03bcM hydrogen peroxide in culture media for oxidative stress. All experiments were performed in triplicate.Human RPE cell line ARPE-19 cells  were grown in DMEM/F12 media with 10% fetal bovine serum (FBS) and standard antibiotics  at 5% COMTT assay (Thermo Fisher Scientific) was used to evaluate cellular metabolic activity as an indicator of cell viability and cytotoxicity. After the experimental incubation period, cells were washed once and then incubated with 0.5 mg/mL MTT labeling reagent at 37 \u00b0C for 4 h. Solubilization solution was added to solubilize the produced purple formazan crystals (MTT metabolic product). The formazan was then solubilized, and its concentration was determined by optical density at absorbance of 570 nm using a microplate reader.For DNA strand damage assay, cells were fixed with 4% paraformaldehyde in PBS for 30 min at 25\u201327 \u00b0C and then anti-\u03b3-H2AX  at 4 \u00b0C overnight. After washing, the cells were incubated with a fluorochrome-conjugated secondary antibody at 27 \u00b0C for 1 h. After additional rinsing three times in PBS (10 min each), the cells were stained with DAPI  nuclear probe  at RT for 2 min. After drying and fixation, the samples were visualized with a fluorescence microscope.Briefly, experimental cells were fixed in 2.5% paraformaldehyde and 2.5% glutaraldehyde in 0.125 M cacodylate buffer (pH 7.4) with 2 mM CaCl2. Upon postfixing with 2% osmium tetroxide in 0.1 M cacodylate buffer, experimental cells were dehydrated through a graded series of ethanol\u2013water mixtures and then dried by the critical point method. After drying, the sample was sputter coated with gold, and cells were examined on a JEOL 100CX transmission electron microscope.Human RPE cell line ARPE-19 cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeated in 0.05% Triton-X 100 for 15 min and blocked with 4% FBS in PBS for 30 min. Anti-ZO-1 antibody  was used to determine the expression of junctional proteins. Anti-Amyloid \u03b2 42 (A\u03b242) (Abcam) and anti-BACE1 against \u03b2-secretase  antibodies were used to determine the expression of amyloid \u03b2 peptides. Hoechst 33,342 or DAPI  was used to stain nucleic acids for the nuclear staining. Images on slides were taken using a fluorescence microscope system.t-test was evaluated to compare between any two groups. One-way analysis of variance (ANOVA) followed by Dunnett\u2019s or Bonferroni\u2019s multiple comparison test was evaluated to analyze the parametric value groups. Statistically significant differences between groups were established when p-values were less than 0.05.Statistical data were analyzed using the SPSS program for Windows software . Means and standard deviations (SD) were presented for all experimental values in this study. The Kolmogorov\u2013Smirnov normality test was performed to verify the normal distribution of the data. The Mann\u2013Whitney test was used to analyze the non-parametric values. Student\u2019s"}
+{"text": "Endometrial cancer is a common gynecological cancer with annually increasing incidence worldwide. However, the biomarkers that provide prognosis and progression for this disease remain elusive.Two eligible human endometrial cancer datasets (GSE17025 and GSE25405) were selected for the study. A total of 520 differentially expressed mRNAs and 30 differentially expressed miRNAs were identified. These mRNAs were mainly enriched in cell cycle, skeletal system development, vasculature development, oocyte maturation, and oocyte meiosis signalling pathways. A total of 160 pairs of differentially expressed miRNAs and mRNAs, including 22 differentially expressed miRNAs and 71 overlapping differentially expressed mRNAs, were validated in endometrial cancer samples using starBase v2.0 project. The prognosis analysis revealed that Cyclin E1  was significantly linked to a worse overall survival in endometrial cancer patients.The hub genes and differentially expressed miRNAs identified in this study might be used as prognostic biomarkers for endometrial cancer and molecular targets for its treatment. Endometrial cancer (EC), that is, uterine corpus endometrial carcinoma (UCEC), originates from the epithelial malignant tumours in endometrium. With an increase in obesity and an aging population, the incidence and mortality rates of EC are increasing in developed countries . AccordiCurrently, there are no known reliable diagnostic and prognostic biomarkers for EC. Cancer antigen 125 (CA125), being most frequently used as a biomarker for ovarian cancer, has some diagnostic/prognostic value in EC . HoweverDue to these factors reduce the clinical value of the existing biomarkers in the progress and prognosis of EC, it is crucial to discover new reliable biomarkers as well as to unravel the underlying molecular mechanisms of the EC progression.http://bioinfogp.cnb.csic.es/tools/venny/index.html)  Affymetrix Human Genome U133 Plus 2.0 Array platform [The mRNA and miRNA expression data of the GSE17025 and GSE25405 datasets were respectively downloaded from the GEO database (platform . The miRwww.cancergenome.nih.gov) by the tool named SangerBox . The mRNA-seq and miRNA-seq datasets contained 544 EC tissue samples, 35 NE tissue samples, and 539 EC tissue samples and 33 NE tissue samples, respectively.The mRNA-seq and miRNA-seq data of patients with UCEC were downloaded from TCGA  was obtained by correcting P-value using the \u2018Benjamini-Hochberg\u2019 method, adj.P-value <\u20090.05 and |log2 fold change (FC)| >\u20091 were set as the threshold value [The Limma package (version 3.36.5) in R/Bioconductor was used to identify differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) between EC and NE tissue samples . The adjld value . The orihttp://david.ncifcrf.gov) facilitates users to perform biological analysis from data collection [The Database for Annotation, Visualization and Integrated Discovery  and visualized using Cytoscape (version 3.7.1) [PPI network of DEGs was constructed using STRING database  , 60. Then 3.7.1) . The funhttp://c1.accurascience.com/miRecords/), which includes 11 different miRNA target genes predicted databases [The target genes for miRNAs (TG-miRNAs) were predicted by employing miRecords (atabases . A TG-mihttp://starbase.sysu.edu.cn/) to verify the association between these potentially valuable differentially expressed target genes and DEMs in patients with EC [The intersection of TG-miRNAs and DEGs were considered to be potentially valuable differentially expressed target genes. Pearson correlation analysis was then used in starBase ( with EC . These shttp://www.oncolnc.org/) [The overall survival of patients with EC with regard to hub genes was calculated using Kaplan-Meier analysis in OncoLnc (nc.org/) . The patAdditional file 1. Node-degree of interaction analysis of the 82 hub genes (Degree of interaction \u226510).Additional file 2. Correlation between differentially expressed miRNAs and target genes in patients with endometrial cancer (Data source: starBase v2.0 project)."}
+{"text": "Spontaneous isolated superior mesenteric artery dissection (SISMAD) is a rare potentially fatal disease. We present a case of cocaine-related SISMAD in a patient with abdominal pain. A 38-year-old African American male with hypertension and alcohol, cocaine, and tobacco abuse presented with abdominal pain and recent cocaine use. A CT angiogram revealed SISMAD; he was treated with conservative management. Cocaine and SISMAD share similar pathophysiologic mechanisms pertaining to vascular smooth muscle cell apoptosis and increased shear stress at fixed vascular positions. Our report emphasizes the need to consider cocaine abuse in SISMAD pathophysiology, risk stratification, and treatment algorithms in future studies. Initially reported by Bauersfeld in 1947, spontaneous isolated superior mesenteric artery dissection (SISMAD) is a rare but potentially fatal arterial disease with an incidence of 0.06% from a cohort of 6666 autopsies  reflectiA 38-year-old African American male with a history of hypertension and alcohol, cocaine, and tobacco use presented with recurrent abdominal pain. Three days prior, he was admitted with abdominal pain and hypertensive urgency. Cocaine and cannabinoids were found in his urine. A computed tomography (CT) scan of the abdomen with IV contrast demonstrated atherosclerosis of the aortoiliac system. With conservative management, the patient was diagnosed with cocaine-induced vasospasm and was discharged after two days.The following day, he returned with recurrence of his abdominal pain and reported continued cocaine use. He described his symptoms as crampy in nature localizing to the upper right and left quadrants. He denied any associated nausea, vomiting, diarrhea, melena, or hematochezia. On arrival, his blood pressure was 190/120\u2009mmHg. His abdomen was soft and flat with diffuse abdominal tenderness to soft and deep palpation with no guarding, organomegaly, rebound tenderness, or peritoneal signs.Results of laboratory tests revealed a serum lactic acid of 2.7\u2009mmol/L. A CT angiogram (CTA) of the abdomen and pelvis revealed a SISMAD within the proximal-to-mid SMA with a 3\u2009cm thrombosis of the proximal false lumen and distal false lumen patency; 70% stenosis of the proximal SMA was noted secondary to this process . There wSISMAD is rare with <1000 reported cases , 3. ClinFive classifications have been proposed by Sakamoto et al., Yun et al., Zerbib et al., Luan et al., and Li et al. . Yun's cAlthough the precise pathogenesis of SISMAD remains uncharacterized, it is suggested to be related to arterial wall dysfunction involving atherosclerosis, vasculitis, cystic medial necrosis, segmental arteriolar mediolysis, fibromuscular dysplasia, and connective tissue diseases , 8, 9. HIn our case, we postulated that our patient's chronic cocaine abuse along with other risk factors  has likely resulted in gradual SMA wall dysfunction forming a nidus for subsequent dissection. His recent acute cocaine use may have produced a significant shear stress elevation leading to dissection. Therefore, with the aforementioned mechanisms, our patient's cocaine use played a significant role in his disease development.No prospectively randomized clinical trial has been published regarding treatment guidelines of SISMAD. Treatment options range from conservative management with or without antithrombotic therapy (anticoagulation or antiplatelet), endovascular SMA stenting, or open surgery. In a recent meta-analysis of 25 selected studies, conservative medical treatment was successful in 397 of 447 of cases, irrespective of anticoagulation or antiplatelet therapy, and 50 underwent conversion to endovascular therapy or open surgery . Park et"}
+{"text": "A significant decrease in 2D cell growth was observed at high GSH-NS concentrations, with the formulation with a low disulfide-bond content, GSH-NS B, being more cytotoxic than the formulation with a high disulfide-bond content, GSH-NS D. The cell growth decrease induced by GSH-NS was owing to G1 cell cycle arrest. Moreover, a significant down-regulation of mRNA expression of the cyclin genes CDK1, CDK2, and CDK4 and up-regulation of mRNA expression of the cyclin inhibitor genes CDKN1A and CDKN2A were observed. On the other hand, a significant decrease in MCS growth was also observed at high GSH-NS concentrations, but not influenced by the nanosponge disulfide-bond content, with the MCS IC50 values being significantly higher than those obtained on 2D cell cultures. GSH-NSs are suitable nanocarries as they provoke limited cellular effects, as cell cycle arrest only occurred at concentrations significantly higher than those used for drug delivery.This study aims to evaluate the bioeffects of glutathione-responsive \u03b2-cyclodextrin-based nanosponges (GSH-NSs) on two- (2D) and three-dimensional (3D) cell cultures. The bioeffects of two types of GSH-NS formulations, with low (GSH-NS B) and high (GSH-NS D) disulfide-bond content, were evaluated on 2D colorectal (HCT116 and HT-29) and prostatic (DU-145 and PC3) cancer cell cultures. In particular, the cellular uptake of GSH-NS was evaluated, as their effects on cell growth, mitochondrial activity, membrane integrity, cell cycle distribution, mRNA expression, and reactive oxygen species production. The effect of GSH-NSs on cell growth was also evaluated on multicellular spheroids (MCS) and a comparison of the GSH-NS cell growth inhibitory activity, in terms of inhibition concentration (IC) The ideal nanoparticle-based drug delivery system assures the safe delivery and selective action of a drug to a target site. Indeed, nanomaterials can add further functionality to the conjugated/loaded drug and, taking advantage of their unique size, are able to play a crucial therapeutic role. This has triggered an increased interest in nanopharmaceuticals ,2 and thNanosponges are hyper-cross-linked cyclodextrin polymers generally obtained from \u03b1, \u03b2, and \u03b3 cyclodextrins, containing suitable amounts of linear dextrin cross-linked with a proper cross-linking agent. A cage-like structure is obtained via the cross-linking of cyclodextrins, thus creating nanochannels in the polymer matrix that can be modulated employing different types of cross-linking agents and/or varying the amount used ,14. It iNanosponges offer several features, such as sustained and controlled release, improvement of aqueous solubility, bioavailability, and stability of the hosted molecules, which could be advantageously exploited for drug delivery ,17,18. IInterestingly, nanosponge-based drug delivery systems can be tuned to form \u2018stimuli-responsive\u2019 nanocarriers that modify their structure in response to external changes, such as pH or redox potential ,26,27. TTrotta et al. have developed a next generation of nanosponges that are bioresponsive to GSH external concentration . This beThe fact that GSH-NS may well represent an efficient stimuli-responsive drug delivery system for anticancer drugs prompted in-depth study into their biological effect per se on cell growth reported herein. As preliminary cellular evaluations of nanocarriers are usually carried out on 2D cell cultures, previously, the effects of cyclodextrin-based nanosponges have been widely tested in 2D cell monolayer cultures. However, 3D cell cultures, such as multicellular spheroids (MCS), have various in vivo tissue characteristics including the production of an extracellular matrix ,40. ThisGSH-NSs have a size of about 200 nm and a negative surface charge, in agreement with our previous papers ,37,38. T1 and IC50 values of GSH-NS, which were determined at 24, 48, and 72 h of exposure.The basal level of reduced glutathione was measured in each of the different cell lines, which were grouped according to cancer type. The results show that HT-29 A and PC-50 values according to disulfide-bond content and exposure time. These data highlight the remarkable cytotoxicity difference between the two nanosponge formulations in colorectal cancer cell lines, as lower IC50 values were observed for the nanosponge formulation with the lower disulfide bridge content (GSH-NS B) at 24, 48, and 72 h  and cytotoxic (IC50) concentrations of fluorescent GSH-NS was analyzed by flow cytometry and fluorescence microscope imaging after 24 h exposure. HCT116, HT-29, DU145, and PC-3 cell monolayers were exposed to 6-coumarin loaded GSH-NS B or 6-coumarin loaded GSH-NS D for 24 h. Significant dose-dependent differences in nanosponge cellular uptake were observed  in the culture supernatant, we measured the LDH leakage of HCT116, HT-29, DU145, and PC-3 cells after 24, 48, and 72 h of incubation with an experimental medium containing different GSH-NS B or GSH-NS D concentrations . No significant increase in LDH leakage percentage over untreated control cells was observed under any of the test conditions (data not shown). The lack of apparent plasma membrane-damaged cells in the LDH assay would appear to contrast significantly with the decrease in cell growth observed by WST-1 cell proliferation assay , which w50 values of each cell line after 24 h of GSH-NS B or GSH-NS D incubation  was performed, as they participate in cell cycle regulation, especially during the Ged cells B,D,F,H. ed cells B,D,F,H. h higher A\u2013F and lh higher C,D,G,H.50 values at 24 h, the dichlorofluorescein-diacetate (DCFH-DA) assay did not indicate a significant intracellular ROS increase at 1, 12 (data not shown), and 24 h  and cytotoxic (IC50) concentrations of fluorescent GSH-NS was confirmed by fluorescence microscope imaging after 24 h of exposure , cellular aggregates organized in a specific cell-to-cell and cell\u2013matrix interaction, closer to in vivo features . Dose re0 values A,G. The exposure B,C,H,I.50 values in both cell lines  and DU145 MCS , which are compared to GSH-NS B and GSH-NS D treated HCT116 MCS  (p < 0.05) and DU145 MCS  (p < 0.05).GSH-NS B and D gave similar ICll lines A,G and all lines A. Interell lines A,G. Thisll lines A,C. More16 cells G, sugges16 cells A,C. FiguUnderstanding the effect that nanoparticles have on cells is crucial to predict their in vivo toxicity and avoid any undesirable nanoparticle activities. Although there are numerous in vitro cytotoxicity assays that can be applied for the general screening of nanoparticles ,43, it iGSH plays a key role in cellular defense against oxidative stress  and its \u03b2-cyclodextrin toxicology has been evaluated in in vitro and in vivo studies that have reported it as non-toxic and well tolerated even at very high doses . Previou1) and cytotoxic (IC50) GSH-NS concentrations were determined by a 2D cell assay, which measured mitochondrial activity. A decrease in cell growth with significantly different IC1 or IC50 values was observed when the two nanosponge formulations were compared in all cell lines, except in DU145 cell line, where no statically significant difference was observed.As no reports have been published on the effects at a cellular level of GSH-NS as such, it was decided to study the effect of GSH-NS per se on HCT116, HT-29, DU145, and PC-3 cancer cell lines with various GSH content in a concentration range that is about fifty times higher than that used in the above mentioned studies to ensure the use of cytotoxic concentrations. HCT116 and DU145 cells showed the highest GSH values in colorectal and prostatic cancer cell lines, respectively; previous research studies have shown that DU145 cells have the highest GSH content . Non-toxN-acetyl cysteine or buthionine sulfoximine [DU145 cell line was the most sensitive to the GSH-NS D cytotoxic effect among all cell lines tested. Notably, DU145 cells are more resistant to electrophilic toxicity than other cells owing to their high levels of redox-sensitive transcription factor, nuclear factor erythroid 2-related factor-2 (Nrf2), which activates cytoprotective pathways against oxidative injury, such as GSH synthesis ,55. As tfoximine  could af50 would appear to indicate that prostatic cancer cell lines are more sensitive to GSH-NS cytotoxic effects.Our study shows that colorectal cancer cells, in particular HCT116 cells, have the most pronounced GSH-NS B and D cellular uptake. This difference in nanosponge cellular uptake in this cell line may be owing to differing uptake mechanisms, as cell surface thiols have been reported to affect disulfide-conjugated peptide cell entry . Indeed,0/G1 phase in all cell lines at 24 h IC50 values. Thus, to further investigate this cell cycle arrest, we analyzed a panel of genes that are involved in cell cycle regulation. Notably, the results show significant mRNA over-expression in the cell cycle progression regulators at G1, CDKN1A, and CDKN2A, which code for p21 and p16 that inhibit the cyclin-CDK2 and -CDK4 complexes, respectively. Apart from this, the mRNA expression of CDC25A, CDK1, CDK2, and CDK4 was either unaffected or down-regulated in all cell lines. These data demonstrate that GSH-NS inhibition of cell proliferation is essentially owing to G1 cell cycle arrest, in agreement with previous reports by Choi et al. [Worthy of note is that cell cycle analyses revealed a significant cell cycle arrest in the Gi et al. . Interes50 values were significantly lower in the 2D cultures than in the 3D cultures, especially after 24 h incubation, where similar values were reached only after 72 h of incubation. For example, IC50 was twofold higher after 24 h in 3D cultures for HCT116 and three-fold higher in DU145 than in their respective monolayers. On the other hand, IC1 concentrations were significantly lower in HCT116 MCS than in HCT116 cell monolayers, whereas IC1 was quite similar both in DU145 spheroids and monolayers.Lastly, the investigation of nanosponges effects on MCS growth was carried out. The results were interesting as differences in the 2D study were observed. There were no significant differences between the two GSH-NS formulations in HCT116 and DU145 MCS, whereas there was a significant difference in the 2D HCT-116 culture. Indeed, IC1 cell cycle arrest, without membrane damage or oxidative stress generation at significantly higher concentrations about fifty times those used for the delivery of anticancer drugs.GSH-NS cytotoxicity might appear to be linked to disulfide-bond content in 2D cell monolayers as the formulation with the higher disulfide-bond content, GSH-NS D, had the lowest cytotoxic effect in all cell lines, except for the DU145 cell line. On the other hand, GSH-NS cytotoxicity was not influenced by the disulfide-bond content in MCS and the most pronounced cell growth decrease was observed in the colorectal cancer cell line, HCT116, after 24 h of exposure to GSH-NS. Tissue-like morphology and phenotypic change may be identified as the major factors in diminishing toxicity on MCS. This means that in vitro 3D cell culture models could act as an intermediate stage and bridge the gap between in vitro 2D and in vivo studies, which would extend current cellular level cytotoxicity to the tissue level and improve the predictive power of in vitro nanoparticle toxicology . FinallyGlutathione-responsive \u03b2-cyclodextrin-based nanosponges (GSH-NSs) were synthetized according to the method developed by Trotta et al. .Briefly, GSH-NSs were obtained using a one-step synthetic route by reacting \u03b2-cyclodextrin and pyromellitic dianhydride, in the presence of 2-hydroxyethyl disulfide to insert disulfide bridges in the NS nanostructure . Varying\u00ae, IKA, Konigswinter, Germany) for 5 min at 24,000 rpm. The sample was then homogenized on a high-pressure homogenizer  for 90 min at a back pressure of 500 bar to further reduce the size of the nanosponges. The aqueous nanosponge nanosuspension was subsequently purified by dialysis  to eliminate potential synthesis residues. The nanosuspension was stored at +4 \u00b0C and used for all experiments.GSH-NS nanosuspensions were prepared following the preparation protocol previously reported ,38. A weFluorescent GSH-responsive nanosponges were obtained by adding 6-coumarin (0.1 mg/mL) to the aforementioned aqueous GSH-NS nanosuspensions (previously described) (10 mg/mL) under stirring for 24 h at room temperature in the dark.v/v). For zeta potential determination, the samples were placed in an electrophoretic cell where an electric field of approximately 15 V/cm was applied. Three batches were analyzed for each NS type and each measured value was the average of ten repetitions. Nanosponge morphology was evaluated by transmission electron microscopy (TEM)  after the diluted aqueous nanosponge nanosuspensions were sprayed onto a Form war-coated copper grid and air-dried.The two types of GSH-NS (GSH-NS B and D), either blank or 6-coumarin loaded, were characterized in vitro to measure their physico-chemical parameters. The average diameters, polydispersity indices, and zeta potential values were determined by photon correlation spectroscopy (PCS) and electrophoretic mobility using a 90 Plus Instrument  at a fixed angle of 90\u00b0 and a temperature of +25 \u00b0C. The analyses were performed on diluted GSH-NS samples (1:30 v/v) heat-inactivated fetal calf serum  in a humidified atmosphere of 5% CO2 air at 37 \u00b0C. At 85% confluence, cells were harvested with 0.25% trypsin and sub-cultured into 75 cm2 flasks, 6-well plates or 96-well plates according to need. Cells were allowed to attach to the surface for 24 h prior to treatment. GSH-NS B and D were then suspended in a cell culture medium and diluted to the appropriate concentrations. After treatment, the cells were harvested to determine cytotoxicity, cell cycle distribution and ROS production. The cells that were not exposed to GSH-NS were used as control conditions for each experiment.Human colorectal cancer cell lines, HCT116 and HT-29 , and human prostatic carcinoma cell lines, DU145 and PC-3 (ICLC), were cultured in McCoy\u2019s 5A Medium and RPMI-1640 Medium, respectively. These media were supplemented with 2 mM L-glutamine, 100 UI/mL penicillin, 100 \u00b5g/mL streptomycin, and 10%  in HT-29, HCT116, DU154, and PC-3 cells were assayed by the Glutathione Assay Kit , according to manufacturer\u2019s instructions. The protein concentration (\u03bcg/mL) was quantified by the Qubit fluorometer  and the Quant-IT Protein Assay Kit . Calibration was performed by the application of a two-point standard curve, according to the manufacturer\u2019s instructions.Briefly, reduced glutathione (GSH) reacts with 5,5\u2032-dithiobis(2-nitrobenzoic acid) (DTNB) in a recycling assay and produces glutathione disulfide (GSSG) and the 1,3,5-trinitrobenzene (TNB) anion, which can be detected by absorbance. In turn, the enzyme glutathione reductase then reduces GSSG, which release GSH that can react with another DTNB molecule. Therefore, the rate of TNB production is measured rather than a single determination of how much DTNB react with GSH, as it is proportional to the initial amount of GSH . The pla3 HT-29, 1.5 \u00d7 103 HCT116, 5.0 \u00d7 102 DU145, and 1.2 \u00d7 103 PC-3 cells were seeded in 100 \u00b5L of growth medium in replicates (n = 8) in 96-well culture plates; the seeding density of each cell line was chosen according to the best proliferation rate. The medium was removed after 24 h and the cells were incubated with in an experimental medium containing differing GSH-NS B or GSH-NS D concentrations . At 24, 48, and 72 h, WST-1 reagent (10 \u00b5L) was added and the plates were incubated at 37 \u00b0C in 5% CO2 for 1.5 h. Well absorbance was measured at 450 and 620 nm (reference wavelength) on a microplate reader Asys UV 340.The effect that GSH-NS B and D had on HCT116, HT-29, DU145, and PC-3 cell growth was evaluated by WST-1 cell proliferation assay . Briefly, 2.0 \u00d7 1050), defined as the dose of compound that inhibited 50% of cell growth, was interpolated from the growth curves, as was the inhibition concentration 1% (IC1), defined as the dose of compound that inhibited 1% of cell growth. Thus, to compare the effects of GSH-NS on the different cell lines, the IC1 and IC50 values obtained were used to carry out the following experiments.Cell proliferation data were expressed as a percentage of control, that is, untreated cells. At 24, 48, and 72 h, the inhibition concentration 50%  and cytotoxic (IC50) concentrations of either fluorescent GSH-NS B or fluorescent GSH-NS D at 24 h. After a 24 h incubation, the cells were washed three times with phosphate-buffered saline PBS, suspended in 250 \u00b5L PBS, and run on the flow cytometer with 488 nm excitation. Intracellular fluorescence was expressed as integrated mean fluorescence intensity (iMFI), which was the product of the frequency of 6-coumarin-loaded GSH-NS positive cells and the mean fluorescence intensity.Coumarin 6-loaded GSH-NS cellular uptake was assessed by cytofluorimetric analysis using a C6 flow cytometer  and imaging analysis using a DMI4000B fluorescence microscopy . For flow cytometry analysis, 5.0 \u00d7 104 cells/coverslip for 48 h of incubation. The coumarin 6-loaded nanosponges were then added at the respective IC50 values for GSH-NS B and D, and incubated for 24 h. The cells were incubated with 1 \u00b5g/mL of 4\u2032,6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining 30 min before the programmed stop time. After the cells were washed with PBS, the cells on the coverslip were mounted on a glass slide, observed under a fluorescence microscope, and photographed.Microscopy observation was carried out after glass coverslips were placed in 24-well plates and the cells seeded at a density of 5.0 \u00d7 103, 1.5 \u00d7 103, 5.0 \u00d7 102, and 1.2 \u00d7 103 cells/100 \u00b5L culture medium, respectively. Twenty-four hours after the seeding, 100 \u00b5L of different concentrations  of GSH-NS B or GSH-NS D was added to the wells. The plates were then incubated for 24, 48, and 72 h, at 37 \u00b0C, in a humidified atmosphere of 5% CO2 air. Cell-free culture media were then collected and incubated with the same volume of reaction mixture for 30 min. LDH activity was measured at 490 nm on a microplate reader Asys UV 340. The background control was obtained by measuring the LDH activity of the assay medium, the untreated control by measuring the LDH activity of untreated cells, and the positive control by measuring the maximum releasable LDH activity after the treatment with the lysis buffer. The LDH leakage percentage was calculated as follows: LDH leakage (%) = /(positive control\u2212untreated control) \u00d7 100, and is the mean of three independent wells.Lactate dehydrogenase (LDH) is an enzyme that is widely present in cytosol and catalyzes the conversion of lactate to pyruvic acid. If plasma membrane integrity is disrupted, the LDH leaks into culture media, increasing its extracellular level, and the amount of LDH release is proportional to the number of damaged cells . The LDH50 of GSH-NS B or GSH-NS D. The occurrence of the so-called sub-G0/G1 peak, which is a distinct cell population characterized by subdiploid DNA fluorescence and might correlate with the internucleosomal DNA fragmentation typical of apoptosis , was also evaluated. Briefly, 1 \u00d7 106 HCT116, 1 \u00d7 106 HT-29, 1 \u00d7 106 DU145, and 1 \u00d7 106 PC-3 cells were incubated with 2 \u00b5M of the live cell staining Vybrant Dye Cycle Green (Invitrogen) for 30 min at 37 \u00b0C. The samples were run on a flow cytometer with 488 nm excitation to measure Vybrant Dye Cycle Green staining and data analysis was performed by FCS Express software version 4 .Cell cycle distribution was evaluated 24 h after cell treatment with the respective IC50. Briefly, the cells were collected in RNA Cell Protection Reagent  and stored at \u221280 \u00b0C. Total RNA was obtained by the RNeasy Plus Mini Kit . Total RNA concentration (\u00b5g/mL) was determined using the fluorometer Qubit (Invitrogen) and the Quant-IT RNA Assay Kit (Invitrogen). Calibration was carried out by applying a two points standard curve, according to the manufacturer\u2019s instructions. RNA sample integrity was determined by the Total RNA 6000 Nano Kit  using the Agilent 2100 Bioanalyzer .Total RNA was isolated from the HCT116, HT-29, DU145, and PC-3 cells, 24 h after incubation with the respective GSH-NS B or GSH-NS D ICReal-time RT-PCR analysis was carried out using 1 \u00b5g of total RNA, which was reverse transcribed in a 20 \u00b5L cDNA reaction volume using the QuantiTect Reverse Transcription Kit . Each 10 \u00b5L real-time RT-PCR reaction was obtained using 12.5 ng of cDNA, according to the manufacturer\u2019s instructions. Quantitative RT-PCR was performed by the SsoFast EvaGreen  and the QuantiTect Primer Assay  was used as the gene-specific primer pair for the studied gene panel .RRN18S) was used to normalize mRNA data and real-time RT-PCR was performed by the MiniOpticon Real Time PCR system . The PCR protocol conditions were as follows: a HotStarTaq DNA polymerase activation step at +95 \u00b0C for 30 s, followed by 40 cycles at +95 \u00b0C for 5 s and +55 \u00b0C for 10 s. All runs were performed on at least three independent cDNA preparations per sample and all samples were run in duplicate. At least two non-template controls were included in each PCR run. Quantification data analyses were performed by the Bio-Rad CFX Manager software version 1.6 , according to the manufacturer\u2019s instructions. These analyses were performed in compliance with MIQE guidelines  [The transcript of the reference gene 18S ribosomal RNA (riments) .1 and IC50 at 24 h, HT-29, HCT116, DU145, and PC-3 cells were washed twice with PBS in six-well plates and incubated with 10 \u00b5M DCFH at 37 \u00b0C in the dark for 30 min. The cells were then washed with PBS, trypsinized, collected in 500 \u00b5L of PBS, and analyzed. ROS production was expressed as iMFI ratio, that is, the difference between the iMFI of treated and untreated cells over the iMFI of untreated cells (iMFI is the product of the frequency of ROS-producing cells and the median fluorescence intensity).The production of intracellular reactive oxygen species (ROS) was measured by flow cytometry using dichlorofluorescein-diacetate (DCFH-DA)  as the oxidation-sensitive probe. Briefly, after 1, 12, and 24 h cell exposure to the respective GSH-NS B or GSH-NS D IC\u00ae 96-well Hanging Drop Plate . On day 8 of the HCT116 and DU145 spheroid culture, 15 \u00b5L of different GSH-NS B or GSH-NS D concentrations  was added to each cell hanging drop and MCS growth was analyzed at 24, 48, and 72 h after nanosponge incubation. Noteworthy is the fact that we had to use a different concentration range for 3D cell growth assay  to obtain the dose-response data necessary to calculate the IC50 values from the one used in the 2D cell growth assay . Phase contrast photographs were taken by the DMI4000B microscope  and the diameter of each MCS was measured by Leica Application Suite Software (Leica) and the volume (V) was calculated using the equation V = 4/3\u03c0r3. Coumarin 6-loaded nanosponge uptake by MCS at the respective IC50 at 24 h for GSH-NS B or GSH-NS D was analyzed by fluorescence microscopy using a DMI4000B microscope (Leica).Cell suspensions (250-cell spheroids) 40 \u00b5L were dispensed into the access hole at each cell culture site to form a hanging drop on a Perfecta3D1) and for a 50% inhibition of cell growth (IC50) for each nanosponge formulation. Statistical analyses were performed on Prism software version 6  using a Student\u2019s t-test and one-way analysis of variance (ANOVA) to calculate the threshold of significance as appropriate. Statistical significance was set at p < 0.05.The results are expressed as the average value \u00b1 standard deviation (St.Dev) of three independent experiments. Median-effect analysis was performed by CalcuSyn software version 2.11  to calculate the values of the concentration required to cause a 1% inhibition of cell growth (IC"}
+{"text": "HCV vaccine development is stymied by the high genetic diversity of the virus and the variability of the envelope glycoproteins. One strategy to overcome this is to identify conserved, functionally important regions\u2014such as the epitopes of broadly neutralizing antibodies (bNAbs)\u2014and use these as a basis for structure-based vaccine design. Here, we report an anti-idiotype approach that has generated an antibody that mimics a highly conserved neutralizing epitope on HCV E2. Crucially, a mutagenesis screen was used to identify the antibody, designated B2.1\u2009A, whose binding characteristics to the bNAb AP33 closely resemble those of the original antigen. Protein crystallography confirmed that B2.1\u2009A is a structural mimic of the AP33 epitope. When used as an immunogen B2.1\u2009A induced antibodies that recognized the same epitope and E2 residues as AP33 and most importantly protected against HCV challenge in a mouse model. Effective drugs are now available to treat chronic infections4 but we still lack a prophylactic vaccine to prevent primary infection.There is an urgent need for a vaccine to prevent infection with hepatitis C virus (HCV). Over 70\u2009million people are chronically infected with the virus6. Successful clearance of virus correlates with a broad, strong T-cell response and rapid induction of neutralizing antibodies during the early phase of infection8. Importantly, the rate of spontaneous clearance in individuals who have previously cleared the virus increases to about 80% upon reinfection, showing that a protective immune response has been induced, indicating that vaccination is a realistic strategy10.A minority of newly-infected individuals (10\u201340%), clear HCV infection, but the majority develop a chronic infection11. Another obstacle is the presence of immunodominant, hypervariable regions within the envelope glycoproteins13 which direct the immune response towards non-neutralizing antibody production. The challenge is to create a vaccine that directs the immune response to conserved, functionally important regions, which typically are poorly immunogenic15.The high genetic diversity of the virus\u2014which exists as seven distinct genotypes and over 60 subtypes\u2014makes it difficult to create a single vaccine that will protect against all infections17. Most broadly-neutralizing antibodies (bNAbs) bind to the E2 glycoprotein and block its interaction with the CD81 receptor18. AP33 is one of several bNAbs that bind to a highly conserved region of E2 (residues 412\u2013423)20. AP33 potently neutralizes all genotypes of HCV in vitro23. It protected mice from HCV infection when passively administered24. HCV1, a bNAb which binds to the same epitope, prevented HCV infection in chimpanzees25 and reduced allograft rejection after liver transplantation in human trials27. A molecule able to elicit antibodies similar to AP33/HCV1 would therefore be an attractive vaccine candidate.Much work has been done to identify and characterize antibodies that can neutralize a broad range of HCV isolates by binding to conserved sites of vulnerability on the virus412\u2013423. We and others tried this, as soon as E2412\u2013423 was identified as the linear epitope of several bNAbs, but immunization with the peptide coupled to KLH never resulted in any antibodies that recognized E228. The failure of the peptide to elicit an anti-E2 response was not surprising, because a peptide is flexible and does not necessarily adopt the same shape as a conformationally constrained section of protein, despite having the same amino acid sequence. Structural studies were then carried out to determine the shape adopted by the peptide when complexed with the bNAbs, with a view to constraining it in a conformation that would elicit the desired antibodies. X-ray crystallography revealed that the peptide, and, by implication, the E2412\u2013423 epitope, adopts three distinct conformations: the most potent bNAbs AP33 and HCV1 bind to a \u03b2-hairpin structure31, bNAb 3/11 binds to a double-turn structure while bNAbs HC33.1, HC33.4 and HC33.8 bind to a more open, single-turn structure33. We attempted to stabilize the \u03b2-hairpin conformation recognized by AP33 by making a cyclic peptide immunogen34. Immunization with the cyclic peptide did elicit antibodies, but further analysis showed that these antibodies bound a different conformation of the E2 peptide, they had low affinity for E2 and they failed to neutralize HCV.The most obvious such molecule would be a peptide corresponding to E236. Initial exposure to an antigen induces the production of antibodies against it, termed Ab1. The unique paratope is recognized as a set of idiotypic epitopes, or idiotopes, by the immune system. Anti-idiotype (anti-Id) antibodies generated against the Ab1 are termed Ab2. A subset of these, called Ab2\u00df, complement the paratope of the Ab1 precisely enough to effectively mimic the structure of its epitope on the original antigen. An Ab2\u00df antibody can be used as a surrogate antigen to elicit Ab3 antibodies, and a subset of these, termed Ab1\u2032, bind to the original antigen37. There are several other classes of anti-Id antibodies that do not mimic the antigen, such as Ab2\u03b3, which recognize part of the Ab1 paratope. Distinguishing Ab2\u00df from Ab2\u03b3 can be difficult, because both these classes inhibit the binding of Ab1 to antigen. Numerous studies have demonstrated that Ab2\u00df anti-Id antibodies can function as vaccines to elicit a protective immune response against infectious pathogens and against tumor-associated antigens41. Traditional vaccine approaches have failed for highly variable viruses such as HCV and HIV. Both fields are now focusing on the conserved epitopes of bNAbs as leads for vaccine design45. Here we have used the anti-Id approach to make a molecular structural mimic of the E2412\u2013423 epitope on HCV E2. We applied a mutagenic strategy to identify a specific Ab2\u00df, B2.1\u2009A, which requires the same binding residues for interaction with AP33 as the viral antigen. We demonstrate that immunization with B2.1\u2009A induces Ab1\u2032 antibodies that protect against viral challenge.Here we describe an alternative strategy to create a molecule that will generate AP33-like antibodies. We use AP33 as a template to make a structural mimic of its antigen-binding site (paratope), by exploiting the ability of the immune system to generate antibodies that are specific for the paratope of another antibody. Jernes\u2019 idiotype network theory postulates that the immune system generates a cascade of antibodies that interact with each other46 showed that the AP33 epitope is conserved in 93% of HCV E2 sequences  bind to biotinylated AP33 and (ii) block binding of AP33 to HCV E2 glycoprotein. These anti-idiotype antibodies are rare; in total, we screened approximately 5000 primary hybridoma cell clones from 19 fusion events to obtain 122 that secreted anti-idiotype (Ab2) antibodies. All the Ab2 antibodies were able to inhibit the binding of MAb AP33 to E2 therefore it is likely that they recognize determinants within, or close to, the AP33 antigen-binding site Fig. . They hae32 Fig. .Fig. 1Ab412\u2013423 epitope. Nevertheless, sequencing was useful in that it identified duplicates, which were excluded from further analysis. Ultimately, there were 18 unique anti-Id clones. All the Ab2s only bound to intact AP33 and not AP33 light chain or a hybrid antibody comprising AP33 heavy chain and an irrelevant \u03ba light chain, suggesting that perhaps they were all Ab2\u00dfs48.Rather than test all the Ab2s in mice, molecular techniques were used to identify Ab2\u00df antibodies. Nucleotide sequencing showed that there was no similarity between the Ab2 complementarity determining regions (CDRs) and the E229  by one or more mutations, indicating that they recognized determinants within the AP33 antigen-binding pocket. In contrast, the binding of some Ab2s was not affected by any mutation, indicating that they recognized determinants outside the antigen-binding pocket. The Ab2s could be ranked according to how closely their binding profile resembled that of E2, and this analysis revealed that only one Ab2, designated B2.1\u2009A, was affected by exactly the same mutations as E2  of B2.1\u2009A, and the structure determined to a resolution of 1.8\u2009\u00c5. The structure unambiguously shows the positions of all the amino acid side-chains and of water molecules at the interface between the two antibodies. The asymmetric unit of this Ab1-Ab2 complex was composed of one molecule of AP33 Fab and one molecule of B2.1\u2009A scFv.To verify that B2.1\u2009A is a genuine mimic of the E2ie AP33 in complex with a peptide corresponding to its E2 epitope ) shows that B2.1\u2009A docks into the AP33 antigen-binding site  and B2.1\u2009A Fab. The binding kinetics of the two molecules were very similar  for B2.1\u2009A Fab and E2661 were 9.73 and 16.0\u2009\u00d7\u2009104\u2009M\u22121 s\u22121, respectively, and their respective dissociation rate constants (kd) were 3.64 and 5.42\u2009\u00d7\u200910\u22124\u2009s\u22121. Thus B2.1\u2009A Fab had slightly slower rates of association and dissociation than E2661, resulting in almost identical equilibrium dissociation constants (KD) of 3.76\u2009nM for B2.1\u2009A Fab and 3.4\u2009nM for E2661. These measurements show that AP33 binds to B2.1\u2009A as strongly as it binds to its authentic antigen, E2.Surface Plasmon Resonance (SPR) was used to directly compare the binding affinity for AP33 of soluble E2 that lacks the transmembrane domain  than that obtained by boosting with B2.1\u2009A Fab-KLH, but the difference between the two boosting regimens is not statistically significant  strongly inhibited binding of AP33 to B2.1\u2009A Fig. , showingant Fig. . We and 412\u2013423 epitope, whereas an identical peptide containing a W420R mutation had no effect  at 2\u2009\u00b5g/ml and with an IC50 about twice that of AP33  virus is poorly neutralized by AP33 whereas Gt2a (BiCre-JC1) is much more sensitive  cells were grown in TC-100 medium (GIBCO) supplemented with penicillin/streptomycin and 10% FCS. Hi Five\u2122 Trichoplusia ni cells (BTI-TN-5B1-4) were grown in Express Five\u2122 serum-free medium supplemented with 16\u2009mM L-glutamine. Mammalian cells were incubated at 37\u2009\u00b0C in 5% CO2, insect cells at 28\u2009\u00b0C.Human epithelial kidney cells (HEK)-293T (ATCC CRL-1573), Sp2/0-Ag 14 myeloma cells (ATCC CRL-1581), human hepatoma Huh-7, Huh7.5, and Huh7-J20 were grown in Dulbecco\u2019s modified Eagle\u2019s medium (GIBCO) supplemented with 10% fetal calf serum (FCS), non-essential amino acids (GIBCO), and penicillin/streptomycin. AP33 and B2.1\u2009A hybridoma cells were grown in the same medium supplemented with 10% ultra-low IgG FCS in CELLine CL350 bioreactors (Thermofisher) according to the manufacturer\u2019s instructions. Baby hamster kidney (BHK-21) cells (ATCC CCL-10) were grown in Glasgow minimal essential Eagle\u2019s medium supplemented with 10% newborn calf serum, 4% tryptose phosphate broth, and penicillin/streptomycin. 59. The anti-idiotype murine MAb B2.1\u2009A was generated as described herein. MAbs were purified from hybridoma supernatant on HiTrap protein G columns using an \u00c4kta Purifier . Fab fragments of AP33 and B2.1\u2009A were made by digesting the respective IgGs for 7\u2009h with immobilized papain, followed by purification using a Nab protein A Plus column (Thermofisher). AP33 Fab was further purified for crystallization by anion exchange on Mono Q 5/50 GL  in 20\u2009mM Tris pH 8.5, using a gradient of 0\u2013300\u2009mM NaCl. Biotinylation was carried out using the ImmunoprobeTM biotinylation kit . B2.1\u2009A Fab was purified for surface plasmon resonance (SPR) by size exclusion chromatography (SEC) on Superdex 200 GL .The anti-E2 murine MAbs AP33 and ALP98, and the rat MAb 3/11 have been described previously661 was cloned by limiting dilution, amplified, and used to infect Hi Five\u2122 cells at a multiplicity of infection (moi) of 1. After 3 days\u2019 incubation the supernatant medium was harvested, filtered through a 0.22\u2009\u00b5m membrane, and dialyzed first against 20\u2009mM sodium phosphate pH 6.8, 150\u2009mM NaCl and subsequently against 20\u2009mM sodium phosphate pH 8.0, 300\u2009mM NaCl. Ultrapure imidazole was added to a concentration of 20\u2009mM, and the supernatant loaded onto a HisTrap HP Ni Sepharose column using an \u00c4kta Purifier . E2661 was eluted with a gradient of 40\u2013200\u2009mM imidazole in 20\u2009mM sodium phosphate pH 8.0, 300\u2009mM NaCl, to separate monomeric molecules from dimers and multimers. Monomeric E2661 was purified to homogeneity by size exclusion chromatography (SEC) on a Superdex 200 Increase 10/300 GL column .The sequence encoding amino acid residues (aa) 384\u2013661 of HCV genotype 1a strain H77c (GenBank accession no. AF009606), plus an N-terminal 6-histidine tag, was inserted into the pAcGP67-A transfer vector (BD Biosciences). Sf9 cells were co-transfected with the transfer vector and Baculogold\u2122 linearized baculovirus DNA, to make recombinant baculovirus (rBac) expressing the ectodomain of E2 as a secreted, soluble protein. The rBac E2Balb/c mice were immunized with AP33 conjugated to Keyhole Limpet Hemocyanin (KLH). Purified AP33 IgG was conjugated to KLH, in a ratio of 2:1 (wt/wt), using 0.2% glutaraldehyde, and then dialyzed into phosphate-buffered saline (PBS). A primary subcutaneous (s/c) injection of 15\u2009\u00b5g AP33-KLH emulsified in Complete Freund\u2019s Adjuvant (CFA) was followed by three s/c injections of 15\u2009\u00b5g in Incomplete Freund\u2019s Adjuvant (IFA) at two-weekly intervals. A week after the last injection, serum samples were tested for Ab2 antibodies. The mice received a final intraperitoneal injection of 150\u2009\u00b5g AP33-KLH in PBS and five days later their spleen cells were fused with Sp2/0-Ag 14 mouse myeloma cells using 50% PEG. After 12 days of selection in HAT medium, hybridoma colonies were tested for Ab2 production, and serially diluted to obtain single-cell clones. Ab2 antibodies were purified by protein G affinity for further characterization.Microtitre plates were coated with goat anti-mouse IgG (H\u2009+\u2009L chains) 40\u2009mg/ml (Jackson Immuno Research) at a dilution of 1:20000 in PBS. Mouse IgG was captured from the hybridoma supernatant during a 1\u2009h incubation at RT. Biotinylated AP33 (b-AP33) was added at 0.1\u2009\u03bcg/ml and incubated at RT for 1\u2009h. Bound b-AP33 was detected with streptavidin-conjugated to horse radish peroxidase (HRP)  followed by 3,3\u2032,5,5\u2032-Tetramethylbenzidine (TMB) substrate. Absorbance was measured at 450\u2009nm.TM protease inhibitor cocktail (Roche). Nuclei were pelleted by centrifugation at 15,000\u2009\u00d7\u2009g and the cytoplasmic extract containing E2660 stored in aliquots at \u221220\u2009\u00b0C. Microtitre plates coated with 2.5\u2009\u00b5g/ml Galanthus nivalis agglutinin (GNA) were used to capture E2 glycoproteins from cell lysate. Hybridoma supernatant medium or purified Ab2s were mixed with b-AP33, added to the plates and incubated for 1\u2009h at RT. Bound b-AP33 was detected with streptavidin-conjugated to horse radish peroxidase (HRP)  followed by 3,3\u2032,5,5\u2032-Tetramethylbenzidine (TMB) substrate. Absorbance was measured at 450\u2009nm. A decreased signal indicated blocking by Ab2 antibodies of b-AP33\u2013E2 interaction. To test whether Ab2s were specific for AP33, b-AP33 was replaced by b-3/11.A modified GNA-capture ELISA was used to detect antibodies that compete with AP33 for binding to E2. Soluble genotype 1a E2 was made by infecting BHK cells at 5 PFU/cell with recombinant vaccinia virus expressing aa 384\u2013660 of HCV genotype 1a strain H77c . Four days after infection the cells were harvested, washed in PBS and resuspended in lysis buffer . Captured chimeric MAbs were detected with anti-human IgG HRP , followed by TMB. Equal amounts of WT and mutant AP33 MAbs were then captured onto microtitre plates coated with anti-human IgG antibody. Ab2 MAbs were added at 1\u2009\u00b5g/ml and incubated for 1\u2009h at RT. Bound Ab2 MAbs were detected with anti-mouse IgG HRP  followed by TMB. Absorbance was measured at 450\u2009nm.Wild type (WT) and mutant AP33 MAbs were produced by transient transfection of HEK cells with expression vectors encoding the appropriate antibody heavy and light chain combinations as previously describedMicrotitre plates were coated with 1\u2009\u00b5g/ml B2.1\u2009A. Dilutions of sera were mixed with b-AP33, added to the plates, and incubated for 1\u2009h at RT. Bound b-AP33 was detected with streptavidin-HRP followed by TMB. Absorbance was measured at 450\u2009nm. A decreased signal indicated blocking of b-AP33\u2013B2.1\u2009A interaction by competing serum antibodies.661 (genotype 1a strain H77c). Dilutions of MAbs or sera were added and incubated for 1\u2009h at RT. To test for peptide inhibition, MAbs or sera were pre-incubated with peptides for 30\u2009mins at RT. Bound antibodies were detected with anti-mouse IgG HRP followed by TMB. Absorbance was measured at 450\u2009nm. Titer was defined as the reciprocal of the highest dilution of immune serum that gave a signal at least three times higher than the average signal of pre-immune sera at the same dilution.Microtitre plates were coated with 1\u2009\u00b5g/ml of purified E2Microtitre plates were coated with 5\u2009\u00b5g/ml of AP33 IgG. Serial dilutions of WT and mutant MBP-B2.1\u2009A scFv were added and incubated for 1\u2009h at RT. Binding of MBP-B2.1\u2009A scFv was detected with anti-MBP-HRP  followed by TMB. Absorbance was measured at 450\u2009nm.In all ELISAs, Immulon 2HB microtitre plates were used. The plates were washed three times between each step in PBS plus 0.05% Tween 20 (PBST). PBST was used as a diluent at every step except coating, which was done in PBS. After coating, plates were blocked with 2% skimmed milk in PBST.411\u2013424 peptide (IQLINTNGSWHINS) conjugated to KLH. Serum samples were taken 7\u201310 days after the last injection, or after each injection.Balb/c or Rosa 26-Fluc mice were immunized with Ab2 IgG, or IgG fragments, conjugated to KLH as above. A primary s/c injection of 50\u2009\u00b5g Ab2-KLH emulsified in CFA was followed by up to five s/c booster injections of 50\u2009\u00b5g antigen in IFA at two-weekly intervals. In some experiments, the antigen used for alternate booster injections was E2To generate Ab1\u2032 IgG for passive transfer into the human chimeric liver mouse model (see below), Rosa 26-Fluc mice were mock-immunized or immunized with 50\u2009\u03bcg B2.1\u2009A Fab coupled to KLH. Sera was collected and pooled. The IgG was purified on a HiTrap Protein G column using an \u00c4KTA\u2122 Pure chromatography system . Fractions were pooled and dialyzed into PBS.Escherichia coli expression vector derived from the pMAL-c2x vector (NEB). The construct encodes a maltose-binding protein (MBP)-B2.1\u2009A scFv fusion protein, which carries a 6-histidine sequence at its N-terminus and a tobacco etch virus (TEV) protease cleavage site at the C-terminus of MBP. Briefly, the fusion protein was expressed in E. coli strain Rosetta-gami 2(DE3)pLysS (EMD Millipore) and the protein purified from cell extract using HisTrap and MBPTrap columns . The protein was then digested with TEV protease and re-applied to the HisTrap column. The unbound fraction containing B2.1\u2009A scFv was further purified on a 16/60 Sephacryl S100  in PBS.The genes encoding the variable heavy (vH) and variable light (vL) chain were generated by reverse transcription-polymerase chain reaction from total RNA of the B2.1A-secreting hybridomas cells. The vH and vL segments were linked via a nucleotide sequence encoding a flexible linker (GGSGGSGGGGSGGGGSGGGAS) and this cassette placed downstream from a Igk-leader sequence in a modified mammalian expression vector pDisplay (Life Technologies) to generate a single-chain variable fragment (scFv) expression construct. The vH-linker-vL cassette was subsequently sub-cloned into pLOU3, an A Crystal Gryphon Liquid Handling System (Art Robbins Instruments) was used to set up co-crystallization trials using the sitting drop method. B2.1\u2009A scFv was buffer exchanged into crystallization buffer  and combined with AP33 Fab in a 1:1 molar ratio at a final concentration of 5\u2009mg/mLTwo datasets were obtained from a crystal grown under condition D4  PEG 8000) of the PEGs suite (Qiagen). One low-resolution set (3\u2009\u00c5) was collected on a Rigaku-MSC Micromax-007 X-ray generator and Saturn 944+ CCD detector at 100\u2009K in-house. The second high-resolution dataset (1.85\u2009\u00c5) was collected from the same crystal at the Diamond Synchrotron facility, IO3 beamline on a PILATUS 3 6\u2009M detector.60. Two ensembles were used for the search structure: the AP33 structure (pdb 4gaj) and an in-silico model of B2.1\u2009A created by the SWISS-MODEL server. Three cycles of rigid body refinement and several restrained refinement cycles were performed in Phenix61. This low-resolution structure was used to model the high-resolution dataset with the same space group selected with Pointless62. Again this model was refined using rigid body refinement in Phenix then alternating cycles of restrained refinement with TLS parameters and water refinement with model inspection and manual refinement on Coot63. Contact in the CCP4 suite and the PDBePISA server64 were used to analyze inter- and intra-molecular interactions. Finally, MolProbity65 was used for model validation which showed that 98% of residues were in favored regions of the Ramachandran plot, with 0.15% in outlier regions, and a Clashscore of 4.86. Pymol was used to generate all images.Molecular replacement using Phaser was used to determine the structure from the low-resolution dataset6SXI.The coordinates and structure factors are deposited in the Protein Data Bank under accession code 661 or B2.1\u2009A Fab) were injected over the chip at 30\u2009\u00b5l/min in buffer , at concentrations ranging from 0.6 to 80\u2009nM. The sensor surface was regenerated between each cycle with two 60\u2009s injections of 10\u2009mM glycine pH 1.5. Each sensorgram was corrected for nonspecific binding by subtraction of the signal obtained from the negative control flow cell. The sensorgrams were analyzed using Biacore X100 Evaluation software (BIAcore) by applying a 1:1 binding model to obtain kinetic affinity constants.SPR experiments were performed on a Biacore X100 instrument at 25\u2009\u00b0C. The proteins used were purified to homogeneity by affinity chromatography followed by SEC. AP33 IgG was immobilized on a CM5 chip by standard amine coupling to a level of approximately 400 resonance units (RU). A second flow cell on the same chip was activated and deactivated in the absence of antibody as a negative control. Analytes (E266 and the SNP-IN tool (http://korkinlab.org/snpintool/) were used to predict single point mutations that may enhance the affinity of AP33 and B2.1\u2009A. Site-directed mutagenesis was used to generate mutations in the B2.1\u2009A ScFv background. The proteins were expressed in E.coli and purified using the His-tag on a BioSprint 15 workstation (Qiagen).The BeAtMuSiC program67 were linearized with XbaI then transcribed using T7 RiboMAXTM Express Large Scale RNA Production System (Promega) and purified using RNeasy kit (Qiagen). RNA was electroporated into Huh7.5 cells and infectious virus was harvested every 3\u2009h from 24\u2009h to 96\u2009h post-electroporation then concentrated using a MilliporeSigma\u2122 Amicon\u2122 Bioseparations stirred cells (Merck). Virus titers were determined in a focus-forming unit assay by serial dilution on Huh7.5 cells. Infected cells were stained for the NS5A viral protein as described previously68.Plasmids encoding the BiCre-Con1 and BiCre-JC1 genomes22. Briefly, cells were seeded at a density of 4\u2009\u00d7\u2009103 cells per well into a 96-well plate and incubated at 37\u2009\u00b0C overnight prior to infection. Virus was preincubated for 1\u2009h at 37\u2009\u00b0C for with AP33 prior to infection at a multiplicity of infection (MOI) of 0.1.AP33 antibody neutralization assays were performed in the SEAP-reporter cell line Huh7-J20 as described previously69 between 8\u201312 weeks old of age were transplanted with 0.5\u2009\u00d7\u2009106 of cryopreserved adult human hepatocytes . Hepatocytes were injected by intrasplenic injection during isoflurane anesthesia using an insulin syringe. The peritoneum was sutured using 4.0 VICRYL sutures  and skin was closed using MikRon Autoclip surgical clips . To facilitate the engraftment of adult human hepatocytes, mice were cycled off and on with the drug nitisinone  according to weight loss and health state. Nitisinone was provided in the drinking water to FNRG mice for maintenance from birth and was retired after surgery. Nitisinone was added again after 10\u201315% body weight loss was reached. Once the body weight loss was recovered, mice were provided with 10% Nitisinone solution for 2 days before animals were put back on plain drinking water.Female FNRG mice70. Mice were bled from the tail vein and serum was serially diluted before use.Human albumin ELISA was used to quantify the level of human hepatocyte engraftment after surgery following a previously published protocol4 TCID50 of BicreCon1 or BiCreJc1 in PBS. To evaluate the level of infection mice were bled through the tail vein at day 7, 21, and 35 post-infection.FNRG mice highly engrafted with human adult hepatocytes according to serum human albumin secretion  were selected to test the protective capacity of B2.1\u2009A antibodies against BiCreJc1 (genotype 2a) and BicreCon1 (genotype 1b) HCV viruses. Mice were intraperitoneally injected 3 times with 80 \u03bcg per mouse of IgG from mice immunized with B2.1\u2009A Fab, or from mock-immunized mice. The first injection was 24\u2009h and the second was 1\u2009h prior HCV infection, the third was 24\u2009h post-HCV infection. Mice were intravenously infected with 10All animal work with infectious agents was conducted in BSL3 facilities in accordance with local rules at Imperial College London, UK. All animal research described in this study was approved by appropriate local Ethics Committee and carried out under United Kingdom Home Office Licenses in accordance with the approved guidelines and under the Animals (Scientific Procedures) Act 1986 (ASPA).Further information on research design is available in the Supplementary FileReporting Summary"}
+{"text": "Several endpoints were measured at several days. Brain hemorrhage and platelet depletion were observed in RI and CI mice. Brain hemorrhage severity was significantly higher in CI mice than in RI mice. Ghrelin therapy with pegylated G-CSF reduced the severity in brains of both RI and CI mice. RI and CI did not alter PARP and NF-\u03baB but did significantly reduce PGC-1\u03b1 and ghrelin receptors; the therapy, however, was able to partially recover ghrelin receptors. RI and CI significantly increased IL-6, KC, Eotaxin, G-CSF, MIP-2, MCP-1, MIP-1\u03b1, but significantly decreased IL-2, IL-9, IL-10, MIG, IFN-\u03b3, and PDGF-bb; the therapy inhibited these changes. RI and CI significantly reduced platelet numbers, cellular ATP levels, NRF1/2, and AKT phosphorylation. The therapy significantly mitigated these CI-induced changes and reduced p53-mdm2 mediated caspase-3 activation. Our data are the first to support the view that Ghrelin therapy with pegylated G-CSF is potentially a novel therapy for treating brain hemorrhage after RI and CI.Medical treatment becomes challenging when complicated injuries arise from secondary reactive metabolic and inflammatory products induced by initial acute ionizing radiation injury (RI) or when combined with subsequent trauma insult(s) (CI). With such detrimental effects on many organs, CI exacerbates the severity of primary injuries and decreases survival. Previously, in a novel study, we reported that ghrelin therapy significantly improved survival after CI. This study aimed to investigate whether brain hemorrhage induced by RI and CI could be inhibited by ghrelin therapy with pegylated G-CSF . B6D2F1 female mice were exposed to 9.5 Gy  These combined radiation injuries (CIs) were observed at Hiroshima and Nagasaki, Japan, where 60\u201370% of victims received thermal burns concurrent with radiation injury.2 At the Chernobyl reactor meltdown, 10% of 237 victims exposed to radiation received thermal burns as well.3 Yet nowadays, public health concerns relating to radiation exposure are on the rise due to advanced development and proliferation of nuclear technologies, radiation and nuclear medicine, and nuclear weapon systems. The growing risk of radiation incidents from terrorist acts with mass casualties thereby warrants increased caution, attention, and study.Vast volumes of literature report that ionizing radiation (IR) produces detrimental, and potentially devastating, effects to cells, organs and systems in humans.12It is generally believed that radiation at any given dose affects biological systems, with each biological system succumbing to acute radiation syndrome (ARS) at certain specific \u201cthreshold\u201d doses. The most radiosensitive organ is the first to show sickness\u2013namely, bone marrow, where damage occurs within hours after total-body irradiation. Consequently, hematopoietic-ARS (H-ARS) results: low bone marrow cellularity and circulatory blood cell depletion present first; spleen shrinkage, followed by splenomegaly, results next; and by day 7, gastrointestinal tract (GI)-ARS arises. Ultimately, impairment of sensitive tissues that sustain crucial immunochemical and metabolic homeostasis, breach of biological barriers, and post-irradiation sepsis leads to multiple organ failure (MOF).15 burns,17 sepsis,18 and/or hemorrhage19 aggravate ARS, particularly in CI. Similar observations were found in humans exposed to ionizing radiation and burn trauma.20 Moreover, radiation suppresses progenitor cells in wounded tissues and bone marrow, thereby leading to complications in tissue renewal, neovascularization for wound healing, as well as remodeling of microvascular beds.22 Therefore, it is essential to develop and identify countermeasure agents or combinations for managing CI. We previously investigated and reported beneficial effects of Ghrelin (a growth hormone-like peptide containing 28 amino acids), specifically amelioration of hematopoietic syndrome of ARS and recovery from CI-associated trauma in mice,23 including increased survival, mitigation of bodyweight loss, wound healing acceleration, as well as increased hematocrit values, neutrophil counts, lymphocyte counts, platelet counts, and bone-marrow cellularity.23 These results were the first to suggest that Ghrelin therapy effectively improved survival not only by attenuating CI-induced leukocytopenia, thrombocytopenia and bone-marrow damage but also by accelerating wound healing rate.23Animal studies from literature clearly indicate that wounds,24 In that report, when mice were exposed to 15% total skin surface burn following 9.5 Gy 60Cobalt-\u03b3 photon radiation, extracranial hemorrhage and intracranial hemorrhage were found. Extracranial hemorrhage was observed in the olfactory lobe, mid-brain, and cerebellum. The latter displayed bleeding that was distributed widely. Histological examination showed subdural and intraparenchymal bleeding in the cerebral cortex and cerebellar cortex. Platelet depletion concurrently occurred, suggesting a correlation between platelet counts and brain hemorrhage.24 Radiation in combination with wound trauma causes cellular ATP depletion in the ileum, pancreas, brain, spleen, kidney, lung, and liver.25 In that report, we found that combined radiation with wound trauma induced cellular ATP reduction by inhibiting pyruvate dehydrogenase and activating pyruvate dehydrogenase kinase 1. A similar result was found in mice after hemorrhage.26Our laboratory recently reported that brain hemorrhage was observed on days 13\u201316 after irradiation in an experimental animal model of radiation combined with burn trauma.27 It was not clear whether CI alters AKT activation. Nevertheless, AKT and MAPK are known to be associated with apoptosis. Caspase-3 is a critical protease in caspase-dependent apoptosis.29 Pegylated G-CSF (Neulasta\u00ae) was approved by FDA in 2016 for hematopoietic syndrome of ARS.30 Pegylated G-CSF, which has been shown to significantly increase survival, modified hematological profiles after irradiation in our experimental animal model.32 Whether RI and CI would result in different severities of brain hemorrhage remained unclear. Furthermore, whether Ghrelin combined with pegylated G-CSF enabling to inhibit brain hemorrhage also remained unknown. In this report, we aim to investigate these two questions. Because CI is evident to amplify hematopoietic ARS and gastrointestinal ARS,4 we hypothesized that 1) CI results in greater brain hemorrhage than radiation alone, and 2) treatment with Ghrelin in the presence of pegylated G-CSF is effective in mitigating brain hemorrhage from RI and CI. Data presented in this report demonstrate that increases in brain hemorrhage incidents are associated with RI and CI and CI induced more lesions than RI. The increases are mitigated by Ghrelin therapy with pegylated G-CSF, thus proving the main hypotheses.It is evident that CI increases MAPK activation.All procedures involving animals were reviewed and approved by the AFRRI Institutional Animal Care and Use Committee. Euthanasia was carried out in accordance with the recommendations and guidance of the American Veterinary Medical Association. Research was conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).33 also used female mice for this reason. As such, we continued to conduct this study with female mice so that data collected could be compared with previous ones.B6D2F1/J female mice  obtained from Jackson Laboratory  were maintained in a facility accredited by AAALAC in plastic microisolator cages with hardwood chip bedding and allowed to acclimate to their surroundings for at least 7 days prior to initiation of the study. Male mice were not used in this study because of potential problems associated with male mouse aggression, such as fight wounds which were not desirable during the experimental period. Previous combined injury studiesad libitum. Animal holding rooms were maintained at 22\u00b0C\u00b12\u00b0C with 50%\u00b120% relative humidity using at least 10\u201315 air changes/h of 100% conditioned fresh air. A 12-h 0600 (light) to 1800 (dark) full-spectrum lighting cycle was used. Mouse tails were tattooed for individual identification during acclimation. B6D2F1/J female mice were randomly divided into 8 groups:sham+vehicle+vehicle,wound+vehicle+vehicle,radiation+vehicle+vehicle,radiation+wound+vehicle+vehicle,sham+Ghrelin+pegylated G-CSF,wound+Ghrelin+pegylated G-CSF,Radiation+ Ghrelin+pegylated G-CSF, andRadiation+wound+Ghrelin+pegylated G-CSF.These mice were maintained in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International in plastic microisolator cages on hardwood chip bedding. Commercial rodent chow  and acidified tap water (pH=2.5\u20132.8) were provided Each group received topical gentamicin cream and was administered with oral levofloxacin. The sham-irradiated animals  were treated in the same manner but not exposed to the radiation source.33 whole-body bilateral 60Co \u03b3-photon radiation, delivered at a dose rate of 0.4 Gy/min, as described previously.23 The dose of 9.5 Gy is the dose to cause 50% population death over 30 days postradiation, abbreviated LD50/30. The field was uniform within \u00b12%. The exposure time for each radiation was determined from the mapping data; corrections for the 60Co decay and the small difference in the mass energy absorption coefficients for water and soft tissue were applied. The accuracy of the actual dose delivery was verified with an ionization chamber adjacent to the mouse rack, which had been calibrated in terms of dose to the midline soft tissue of mice.Mice were given 9.5 Gy23 All mice subjected to the skin injury were given 0.5 mL sterile 0.9% NaCl intraperitoneally (i.p.), which contained 150mg/kg of acetaminophen  immediately after skin injury to alleviate pain. Four hours later, mice were given a second dose of 150mg/kg of acetaminophen. Skin-wounded mice without radiation exposure received only one dose of 150mg/kg of acetaminophen immediately after skin injury.Skin surface injuries were performed on the shaved dorsal surface of mice. Animals receiving skin wounds were anesthetized by isoflurane inhalation. A 15% total-body-surface-area skin wound was performed within 1h after irradiation.23Ghrelin was purchased from Phoenix Pharmaceutical . Three doses of 113\u03bcg/kg were administered subcutaneously (s.c.) in a volume of 0.2 mL 24h, 2d, and 3d after RI or CI. The vehicle given to control mice was sterile 0.9% sodium chloride solution for injection, USP, based on the survival data published previously.32 in a volume of 0.2ml 24 h, 8d, and 15d after RI or CI, i.e., 25\u03bcg/25-g mouse. Neulasta\u00ae was supplied in 0.6mL prefilled syringes for s.c. injection. Each syringe contains 6mg Peg-G-CSF in a sterile, clear, colorless, preservative-free solution containing 0.35mg acetate, 0.02mg polysorbate 20, 0.02mg sodium, and 30mg sorbitol in water for injection, USP. The vehicle mouse received 0.2ml of vehicle containing 0.35mg acetate, 0.02mg polysorbate 20, 0.02mg sodium, and 30mg sorbitol in 0.6mL water.32Pegylated G-CSF  is a polyethylene glycol pharmaceutical-formulated-grade drug, also known as pegfilgrastim, that was purchased from the AmerisourceBergen Corporation . A dose of 1000\u03bcg/kg was administered by s.c. injectionsGentamicin sulfate cream, 0.1% , was applied daily for 10 days to the skin injuries on days 1\u201310. Levofloxacin (LVX), , 100mg/kg in 0.2mL/mouse, was administered p. o. daily for 14 days beginning on day 3.Blood samples were collected in EDTA tubes after Sham, wound, RI and CI and assessed with the ADVIA 2120 Hematology System . Differential analysis was conducted using the peroxidase method and the light scattering techniques recommended by the manufacturer.Mouse craniums and/or the extracted brains were kept in 10% neutral buffered formalin as above until processing by routine methods for histopathologic examinations. The formalin-fixed tissues were embedded in paraffin, cut into 5-\u03bcm sections, stained with hematoxylin and eosin, and examined by light microscopy. Histologic lesions were graded by number of hemorrhage lesions.2 during the 30-day monitoring period for collecting their blood and brains. Their entire brains from surviving mice and moribund mice were collected. Because the hemorrhagic lesions were dominant in small brain, the small brain was used for further biochemical studies. The small brains were mixed with Na+ Hanks\u2019 solution containing 10\u03bcl/ml protease inhibitor cocktail, 10mM phosphatase 2 inhibitor, 10mM phosphatase 3 inhibitor, 10mM DTT, 5mM EDTA and 10mM PMSF, homogenized using Bullet Blender Homogenizer Storm  for 4 min at speed 10 and centrifuged at 9,000 xg for 10 min . Supernatant fluids were conserved for protein determination and stored at \u221280\u00b0C until use.Surviving mice were anesthetized by isoflurane followed by vertebrate dislocation on day 30 after sham, wound, RI and CI for blood collection and brain collection. Mice with moribundity were euthanized by CO\u2122 23 Cytokine Assay kit and 9 Cytokine kit  following the manufacturer\u2019s protocol. Data were analyzed using the LuminexH 100\u2122 System  and quantified using MiraiBio MasterPlexH CT and QT Software . Data were expressed as pg/mg protein in tissues.Cytokine concentrations in small brain lysates were analyzed using the Bio-Plex+ Hanks\u2019 buffer containing 1% sodium dodecyl sulfate (SDS) and 1% 2-mercaptoethanol were resolved on SDS-polyacrylamide slab gels . After electrophoresis, proteins were blotted onto a polyvinylidene difuoride (PVDF) membrane  using a Tran-Blot Turbo System and the manufacturer\u2019s protocol . The blot was then incubated for 90min at room temperature with 5% non-fat dried milk in tris-buffered saline-0.5% tween20 (TBST) at room temperature. After blocking, the blot was incubated with a selected antibody against PGC-1\u03b1, NRF1, NRF2, Mfn1, Total C1\u20135 Oxpho Rodent WB Antibody Cocktail , Drp1 , PARP , Ghrelin receptors, GAPDH , NF-\u03baBp65, NF-\u03baBp50, AKT, p-AKT, ERK1/2, p-ERK1/2, JNK. P-JNK, p38, p-p38 , and IgG  at a final concentration of 1\u03bcg/ml in TBST-5% milk. The blot was washed 3 times (10 min each) in TBST before incubating for 60 min at room temperature with a 1000X dilution of species-specific IgG peroxidase conjugate  in TBST. The blot was washed 6 times (5 min each) in TBST before detection of the peroxidase activity using the Enhanced Chemiluminescence kit . IgG and GAPDH levels were not altered by radiation and used as a control for protein loading. Protein bands of interest were quantitated using the ImageJ program and normalized to either IgG or GAPDH levels. Data were expressed as intensity ratio to IgG or GAPDH levels.Total protein in the small brain lysates was determined with Bio-Rad reagent . Samples with 20\u03bcg of protein in NaCellular ATP levels were determined using the ATP Bioluminescence Assay Kit HS II . Luminescence was measured with a TD-20/20 luminometer . Data were normalized to total protein and cellular ATP levels were expressed as fmol/\u03bcg protein.Data were expressed as mean\u00b1s.e.m. For each western blot and assay, the data were compared using the ANOVA, Tukey post hoc test, and student\u2019s t-test with a significance level of 5%.50/30) and has been used for previous publications on testing drug efficacy.33Radiation at 9.5 Gy was used to investigate the brain hemorrhage after RI and CI. This radiation dose is a lethal dose causing 50% population death within 30 days postirradiation -\u03b3 coactivator-1\u03b1 (PGC-1\u03b1) is shown to downregulate NF-\u03baB levels.34 Therefore, we measured PGC-1\u03b1 and NF-\u03baB in the cerebellum through Western blotting analysis. RI and CI significantly reduced PGC-1\u03b1 protein levels in cerebellum samples of mice treated with vehicle -\u03baB activation in ileum and skin on days 1\u20137. vehicle . RI and  vehicle \u2013c.35 It was of interest to find out whether RI and CI altered ghrelin receptors. Indeed, RI and CI decreased these receptors. Ghrelin therapy with pegylated G-CSF was able to recover these reductions in samples of CI mice . Ghrelin therapy with pegylated G-CSF mitigated this depletion and significantly elevated platelet counts back to 567\u00b186 (p<0.05). No platelet counts were available in CI mice treated with the combinational therapy.RI and CI are known to induce platelet depletion.46 RI concurrently induces massive release of numerous reactive factors, coagulopathy, suppression of vascular growth factors, and vascular remodeling and complicates the endothelial injury-associated peripheral perfusion.48 The microvascular barriers  sustain circulatory homeostasis. Therefore, the impact of endothelium impairment becomes long-lasting from an acute phase to a delayed phase, and thereafter, to a prolonged phase.48 These effects of interstitial hemorrhage, cell hypoxia, and cell necrosis are life-threatening and represent a great challenge; not only in the development of countermeasures against radiological/nuclear accidents, but also can complicate outcomes in radiation therapy.50In this report, we provide evidence that in B6D2F1/J mice, brain hemorrhage is associated with RI- and CI-induced moribundity. CI induced brain hemorrhage earlier and more severely than RI. Ghrelin therapy with pegylated G-CSF after RI and CI mitigated sickness, moribundity and impact of brain hemorrhage. Either total or partial body radiation exposure results in damage of microvascular networks, which is one of the most important outcomes of acute radiation sickness.13 The discrepancy is due to (1) different tissues analyzed and (2) the tissue collection at different time points after RI and CI. In this report, the brain of RI and CI mice were collected from moribund mice euthanized on days 13\u201321 and days 12\u201317, respectively. RI and CI reduced ghrelin receptors, PGC-1\u03b1 and PARP in the cerebellum. Ghrelin administration with pegylated G-CSF partially recovered the receptors but not PGC-1\u03b1 and PARP, suggesting that the therapy effect is specific. RI and CI increased proinflammatory cytokines and chemokines in the cerebellum. Ghrelin therapy with pegylated G-CSF effectively inhibited RI-induced proinflammatory cytokines and chemokines  is inactivated and pyruvate dehydrogenase kinase (PDK) is activated. In this report, we showed that RI and CI significantly reduced ATP production, with CI leading to more drastic reductions than RI in cerebellum. These reductions were mediated by decreased levels of NRF1/2. Like ATP, RI and CI also reduced NRF1/2, with CI leading to more drastic reductions than RI . The possibility of Ghrelin therapy with pegylated G-CSF modifies microRNAs that are associated with thrombopoiesis also cannot be excluded and should be further explored.It is evident that radiation combined with burn trauma increases miR-690 and miR-223 in serum.In summary, RI and CI significantly increased brain hemorrhage. CI induced more hemorrhage lesions than RI. These lesions were mitigated by Ghrelin treatment with pegylated G-CSF. RI and CI decreased ghrelin receptors, increased proinflammatory cytokines/chemokines, and decreased anti-inflammatory cytokines/chemokines in the small brain. Ghrelin therapy with pegylated G-CSF remarkably inhibited proinflammatory cytokines/chemokines in RI mice and elevated anti-inflammatory cytokines/chemokines in CI mice. RI and CI inhibited cellular ATP amounts by decreasing NRF1/2 and complex 1-V proteins. Ghrelin therapy with pegylated G-CSF recovered ATP, NRF1/2 and complex III in CI mice. In RI mice, the combinational therapy mitigated RI-induced platelet depletions, which may contribute to inhibition of brain hemorrhage. These results suggest that Ghrelin treatment with pegylated G-CSF is potentially useful for treating brain hemorrhage.We demonstrate that ionizing radiation followed by skin wounds induces cerebro-vascular impairment, intracranial hemorrhage, ghrelin receptor reduction, cytokine/chemokine increases, cellular ATP reduction, and platelet depletion. The results suggest that ATP reduction and platelet depletion highly likely contribute to the onset of brain hemorrhage, at least in part; thereby, this intracranial hemorrhage partly leads to ultimate mortality. In RI mice, Ghrelin therapy with pegylated G-CSF significantly mitigated platelet depletion, proinflammatory cytokines/chemokines, and ERK1/2 and JNK activation . In CI m"}
+{"text": "The process of metastasis is one of the most destructive characteristics of cancer, yet it is still poorly understood. Formation of metastasis comprises several distinct steps: cancer cells first detach from the primary tumor and then enter the bloodstream where they circulate freely in the body and eventually adhere to vessel walls in distant organs where they can form secondary tumors. A recent study discovered that a co-treatment of two common drugs that interfere with cellular metabolism, metformin and 2-deoxy-D-glucose, induced cellular changes resembling those observed in metastasis: the drugs induced detachment of certain breast cancer cells and their proliferation in the floating state. In this study, we investigated if this treatment also induces other changes that are related to metastasis, i.e., if the detached cells are softer and if they are more prone to adhesion than control cells. The results of our in vitro experiments showed that this was indeed the case and thus indicate possible relations between metabolism and metastatic potential. While the results of this study cannot be directly projected to cancers in vivo, they present new observations that can be important for the analysis of cancer cell detachment and anchorage-independent growth.Metastatic cancer cells can overcome detachment-induced cell death and can proliferate in anchorage-independent conditions. A recent study revealed that a co-treatment with two drugs that interfere with cell metabolism, metformin and 2-deoxy-D-glucose, promotes detachment of viable MDA-MB-231 breast cancer cells. In the present study, we analyzed if these detached viable MDA-MB-231 cells also exhibit other features related to cancer metastatic potential, i.e., if they are softer and more prone to adhere to epithelial cells. The cell mechanics of attached cells and floating cells were analyzed by optical tweezers and cell deformability cytometry, respectively. The adhesion was assessed on a confluent monolayer of HUVEC cells, with MDA-MB-231 cells either in static conditions or in a microfluidic flow. Additionally, to test if adhesion was affected by the state of the epithelial glycocalyx, HUVEC cells were treated with neuraminidase and tunicamycin. It was found that the treated MDA-MB-231 cells were more prone to adhere to HUVEC cells and that they were softer than the control, both in the floating state and after re-seeding to a substrate. The changes in the HUVEC glycocalyx, however, did not increase the adhesion potential of MDA-MB-231. One of the most lethal features of various cancer types is the formation of metastasis, a process where cancer cells spread from the primary tumor to other organs or tissues to form secondary tumors. The metastatic process involves several distinct steps, starting with detachment from the primary tumor and entry into the bloodstream by intravasation ,2,3,4,5.Normal epithelial cells are anchorage dependent, and when they detach from the extracellular matrix, they enter a particular type of cell death called anoikis . HoweverIn the next step of the metastatic process, the adhesion of CTCs to endothelial cells, it is crucial that both cell types express adequate ligands and receptors . An impoAs shown by Bizjak et al. , co-treaHUVEC (ATCC) were grown in serum-reduced minimum essential medium (MEM)  supplemented with 5% fetal bovine serum (Life Technologies) and MDA-MB-231 cells (ATCC) in RPMI medium  supplemented with 10% fetal bovine serum (Life Technologies) and 4.5 g/L glucose. Antibiotics (streptomycin-penicillin) (Life Technologies) were added to both media. To obtain floating MDA-MB-231 cells, they were first grown in a standard T-25 cell culture flask. The control MDA-MB-231 cells were detached by trypsinization 48 h after seeding and then harvested from the solution. The floating met2DG-treated MDA-MB-231 cells were treated with 5mM metformin and 0.6 mM 2-deoxy-D-glucose after seeding for 48 h and then harvested from the solution .For the experiments, HUVEC cells were treated one day after seeding with neuraminidase (Sigma-Aldrich) at 1 U/mL for 24 h and with tunicamycin (Sigma-Aldrich) at 5 \u00b5g/mL for 48 h. The concentrations and duration of treatments used were chosen based on the literature data and our preliminary experiments.4 cells/mL. The cell viability was tested on control, neuraminidase and tunicamycin treated HUVEC cells. The cells were plated in 96-well microtiter plates (TPP) at a concentration of 3 \u00d7 10H-tetrazolium) test. To each well, 20 \u03bcL of MTS  was added to the cell culture medium. After 2 h, the absorbance at 490 nm was measured using a Bio-Tek microplate reader . The absorption corresponded to the amount of the soluble formazan product that was formed, which is directly proportional to the number of viable cells. The viability was calculated as the ratio between absorbance at 490 nm of the treated and control cells. The cell viability was determined using the MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-24 cells/mL) into a custom-built experimental chamber with an uncoated, plasma-treated glass bottom. One day after seeding, the cells were left untreated or were treated with neuraminidase or tunicamycin. The assay was performed 48 h after seeding for control and neuraminidase treatments and 72 h for tunicamycin treatment. Floating MDA-MB-231 cells were obtained by trypsinization (control), met2DG treatment or by harvesting of cells cultured on polyHEMA (Sigma-Aldrich) coated flasks for 48 h. All MDA-MB-231 cells were stained after detachment with 0.5 \u00b5M calcein acetoxymethyl ester  for 15 min in the incubator. Thereafter, 1 \u00d7 104 cells/mL of stained MDA-MB-231 cells were seeded on the confluent monolayer of HUVEC cells and incubated for 20 min. Then the cells in the experimental chambers were washed thoroughly, so all weak and unattached cells were washed away. The samples were monitored under Eclipse Ti inverted microscope  with epi-fluorescence, and 20 random fields of views were recorded. The adhered cells were then counted with the help of the ImageJ Cell Count Plugin [HUVEC cells were seeded (3 \u00d7 10t Plugin .4 cells/mL) into an uncoated flow chamber (ibidi \u00b5-slide I0.2 Luer). As in the static adhesion assay, the cells were whether left untreated or treated with neuraminidase or tunicamycin. For the adhesion assay, floating MDA-MB-231 cells (105 cell/mL) stained with 0.5 \u00b5M calcein AM were flown over the monolayers of HUVEC cells for 15 min. The flow was kept constant using a neMESYS syringe pump  at 0.5 \u00b5L/sec. Thereafter the cells were rinsed with the growth buffer at 1 \u00b5L/sec for 10 min, so all weakly attached and unattached MDA-MB-231 cells were washed away. The samples were examined under the Eclipse Ti inverted microscope (Nikon) with epi-fluorescence. Nine fields of view from the central area of the \u00b5-slide at 4 different distances  from the microchannel entrance were imaged. The cells in the respective fields of views were then counted in the same way as in 2.3.HUVEC cells were seeded  were measured 72 h after seeding. For the measurements of re-seeded cells, control and met2DG-treated floating MDA-MB-231 cells were seeded into the chamber at the concentration of 3 \u00d7 104 cells/mL. The cells were left to attach to the substrate for one hour before the measurements were conducted. For the measurements of HUVEC cells, they were seeded into the chamber at a low concentration (1.5 \u00d7 104 cells/mL) so that they did not form a confluent layer even after 24 or 48 h of treatment with neuraminidase or tunicamycin.The stiffness of cells was measured with optical tweezers in a custom-build PDMS experimental chamber adhered to an uncoated glass bottom. Adhered MDA-MB-231 cells were seeded into the chamber at the concentration of 1.5 \u00d7 10Cell stiffness measurements were performed on an Eclipse Ti inverted microscope (Nikon) equipped with laser tweezers , as described in Zemljic Jokhadar et al. . In brieThe experimental chamber with cells was mounted on the microscope, and silica beads with a diameter of 5.06 \u00b5m  were added into the sample, where they were trapped by optical tweezers. The beads were then positioned near the selected cell. The bead center was approximately 5 \u00b5m above the surface, which was achieved with the piezo microscope stage . By moving the piezo stage with a constant velocity of 1 \u00b5m/s, the cell was pushed into the bead. The bead position was monitored in real-time with a digital camera  at 50 fps and by using a custom-written Matlab program . The piezo stage was automatically retracted after the bead was pushed for 0.5 \u00b5m from the center of the optical trap. The force was calculated fromThe cell stiffness was determined as the slope of the linear part of the force\u2013deformation curve . The ind6 cells/mL. The cell suspensions were loaded into 1 mL syringe, which was fixed to the Nemesys pump  and connected to the Flic30 microfluidic chip . The flow rate of the cell suspension was set to 0.25 \u00b5L/s, and the flow rate of the buffer was set to 0.75 \u00b5L/s [The method for deformability cytometry was adopted from Otto et al. . Detache.75 \u00b5L/s . Before A of the cells by counting pixels inside the automatically detected contours and the perimeter l as contour length. Following the definition from Otto et al. [We processed the acquired images with custom-written image recognition software . The automatic shape detection employed background subtraction, Gaussian blur, automatic Otsu thresholding and was filtered according to the expected size and solidity of the obtained shapes. We determined the size (projected cell surface area) o et al. , the def4 cells/mL. One day after seeding, cells in uncoated wells were left untreated or were treated with met2DG for 48 h. Forty-eight hours after seeding, the suspension from uncoated and polyHEMA coated wells and 72 h from wells with met2DG treated MDA-MB-231 cells was collected and centrifuged. The supernatant was stored at \u221280 \u00b0C and then used for ELISA analyses. For the detection of cytokine, we used immune-enzyme test Human TNF alpha Uncoated ELISA  following the manufacturer\u2019s instructions.MDA-MB-231 cells were seeded in uncoated or polyHEMA coated wells of a 6-well plate (TPP) at 5 \u00d7 10HUVEC cells were grown and treated with neuraminidase or tunicamycin as in other experiments. After the treatments, we incubated the cells with 4 \u00b5g/mL of wheat germ agglutinin, Alexafluor 488 conjugate  in the growth media for 15 min. Then we gently washed the cells and examined the samples under the Nikon ECLIPSE TE2000-E microscope  in the confocal mode (Nikon C1). HUVEC cells were grown and treated with neuraminidase or tunicamycin as in other experiments. HUVEC cells were seeded in 24-well culture plates for 24 h and then treated for 24 h with neuraminidase or for 48 h with tunicamycin.After the treatments, we incubated the cells with 4 \u00b5g/mL of wheat germ agglutinin, Alexafluor 488 conjugate  in the growth media for 15 min. Cells were washed with physiological saline and harvested by trypsinization. After trypsinization, cells were resuspended in cold PBS and measured with Attune N \u00d7 T flow cytometer (Thermo Fisher Scientific). Three independent biological repeats were performed.For detached cells, MDA-MB-231 cells were plated on 6-well plates at 160,000 cells per well for cells treated with 5 mM metformin + 0.6 mM 2DG for 48. For cells grown on poly-HEMA, MDA-MB-213 cells were seeded in complete RPMI medium with 1 g/L glucose on poly-HEMA coated 6-well plates at 215,000 per well for 72 h. After treatment, detached cells population were collected, and attached cells were detached with trypsin. All cell populations were spun down and resuspended in Seahorse XF RPMI 1640-based Seahorse XF Glycolytic Rate Assay medium . The Seahorse Cell Energy Phenotype Assay (Agilent) was performed following manufacturer instructions, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using Seahorse Analyser XFp .p-values. For comparison of mean values between the treated and the control cells, the Student\u2019s t-test was used. In both cases, p-values < 0.05 deemed significant.For comparison of median values between the treated and the control cells, the Mann\u2013Whitney\u2013Wilcoxon test  was used to calculate the An important step in the metastatic cascade is the adhesion of CTCs to endothelial cells. We, therefore, analyzed if floating MDA-MB-231 cells that were detached by met2DG treatment are more prone to adhere to normal HUVEC monolayer than the control MDA-MB-231 cells that were detached by the standard trypsinization protocol. The test was conducted in static conditions, as well as in a microfluidic system that mimics the shear flow in the vasculature. In the static conditions, floating MDA-MB-231 cells were placed over a HUVEC monolayer in the experimental chamber for 20 min and then thoroughly washed. In the microfluidic system, floating MDA-MB-231 cells were flown over a HUVEC monolayer in a microfluidic system for 15 min. The results showed that met2DG-treated MDA-MB-231 cells adhere significantly more to the HUVEC cells then control MDA-MB-231 cells in both the static conditions a and undWe analyzed the effects of met2DG treatment on stiffness of MDA-MB-231 cells in three different conditions with two different techniques. To test if met2DG treatment alone can alter cellular stiffness, we used optical tweezers to measure the stiffness of the attached control and met2DG-treated MDA-MB-231 cells. Second, we collected floating cells obtained after 48 h of met2DG treatment and control cells detached by trypsinization and performed deformability cytometry, where deformations of individual floating cells were measured as they were pumped at high velocity through a constricted channel. To test if trypsinization has an effect on cell stiffness, we also included cells grown in flasks coated with polyHEMA. Third, we re-seeded the floating cells and probed them again with optical tweezers. Both techniques are single-cell-based, but probing with optical tweezers measures cell stiffness at small deformations, i.e., at the level of the cell membrane, while microfluidic cell deformability assay measures deformations of whole cells. In the first case, we did not measure any significant difference in cell stiffness between control and met2DG-treated adhered MDA-MB-231 cells a. On theAs metformin and 2DG are metabolic inhibitors, we determined the cell energy phenotype of the floating met2DG-treated cells using Seahorse Analyser XFp . MDA-MB-We observed that Met2DG-treated cells had significantly altered cell energy phenotype, with almost completely suppressed mitochondrial respiration a compareIn the second part of our study, we examined the role of epithelial GLX in the adhesion of MDA-MB-231 cells to HUVEC cells. HUVEC GLX was treated by tunicamycin, which blocks N-glycosylation, and neuraminidase, which degrades the sialic parts of GLX. As the adhesion can be affected by cell stiffness, we also measured the stiffness of treated HUVEC cells with optical tweezers.We first evaluated the effects of treatments on the amount of GLX on HUVEC cells using confocal microscopy and flow cytometry by staining the cells with fluorescent WGA, which is a nonspecific GLX stain. The representative images obtained by confocal microscopy are shown in To quantify these results, we also analyzed the WGA-stained HUVEC cells with flow cytometry. After the treatments, the cells were harvested by trypsinization and introduced into the flow cytometer. The measured mean fluorescence intensity is presented in In the next step, we analyzed if the impaired HUVEC GLX influences the adhesion of MDA-MB-231 cells. The measurements were again conducted in static conditions and under a microfluidic flow. Control MDA-MB-231 cells (detached by trypsinization) were compared to the MDA-MB-231 cells detached by met2DG treatment . For allFinally, we tested if the treatments of HUVEC cells affected their stiffness. Again we employed the measurements with optical tweezers. While tunicamycin-treated cells showed no marked changes, the neuraminidase-treated cells were significantly stiffer than the control .Cancer cells can detach from the primary tumor, bypass anoikis and attach to the matrix of distant organs in the body, where they can form secondary tumors ,4,13. ThMDA-MB-231 human breast cancer cell line is commonly used to model late-stage breast cancer and has metastatic properties . A studySince deregulation of anoikis is linked to metastatic ability, our goal was to investigate if this subpopulation of met2DG-treated, viable, floating MDA-MB-231 cells has mechanical characteristics that are related to increased metastatic potential; specifically, we analyzed their stiffness and ability to adhere to endothelial monolayer. Additionally, we analyzed the effects of two drugs that disrupt epithelial GLX on MDA-MB-231 adhesion. In the first part of the study, we compared the adhesion of normal and met2DG-treated MDA-MB-231 cells to normal endothelial HUVEC cells. Since the adhesion of CTC to vessel walls is most prominent in post-capillary venules, where only low shear stress is present ,26, we cSeveral studies had shown that cancerous cells exhibit different mechanical properties than normal cells of the same tissue origin. For example, highly invasive malignant MDA-MB-231 cells are softer than benign human mammary gland cell line MCF-10 or MCF-7, which is a non-invasive malignant breast cancer cell line ,29,30. TIn parallel, we observed significantly altered cell energy phenotype of the floating met2DG-treated compared to control  MDA-MB-231 cells, with almost completely blocked mitochondrial respiration . On the GLX covers the endothelium, and in normal conditions, it forms a selective barrier between the endothelium and its environment by preventing adverse ligands the access to adhesion receptors. Therefore, in the second part of our study, we focused on the role of the endothelial GLX in the adhesion of MDA-MB-231 cells. We treated the HUVEC cells with two substances that affect the GLX on different levels. The endothelial GLX is composed of different glycoproteins, soluble proteins and residual chains, among others such as sialic acid, which are attached to core proteins . Sialic Flow-cytometric analysis of HUVEC cells labeled with fluorescent WGA confirmed that both treatments reduced the amount of GLX . InteresTreatments of HUVEC cells with neuraminidase and tunicamycin did not result in an increase in the adhesion of MDA-MB-231 cells . In factWe found that MDA-MB-231 cells that detach in vitro due to a combined treatment of two cell metabolism-interfering drugs, metformin and 2DG, are more prone to adhere to endothelial HUVEC cells than control MDA-MB-231 cells. They are also softer (more deformable) than the control cells both in suspension and after they re-attach to the glass substrate. While these features could be related to a higher metastatic potential, further studies are needed to confirm this connection in vivo."}
+{"text": "Breast cancer is one of the major causes of deaths due to cancer, especially in women. The crucial barrier for breast cancer treatment is resistance to radiation therapy, one of the important local regional therapies. We previously established and characterized radio-resistant MDA-MB-231 breast cancer cells (RT-R-MDA-MB-231 cells) that harbor a high expression of cancer stem cells (CSCs) and the EMT phenotype. In this study, we performed antibody array analysis to identify the hub signaling mechanism for the radiation resistance of RT-R-MDA-MB-231 cells by comparing parental MDA-MB-231 (p-MDA-MB-231) and RT-R-MDA-MB-231 cells. Antibody array analysis unveiled that the MAPK1 protein was the most upregulated protein in RT-R-MDA-MB-231 cells compared to in p-MDA-MB-231 cells. The pathway enrichment analysis also revealed the presence of MAPK1 in almost all enriched pathways. Thus, we used an MEK/ERK inhibitor, PD98059, to block the MEK/ERK pathway and to identify the role of MAPK1 in the radio-resistance of RT-R-MDA-MB-231 cells. MEK/ERK inhibition induced cell death in both p-MDA-MB-231 and RT-R-MDA-MB-231 cells, but the death mechanism for each cell was different; p-MDA-MB-231 cells underwent apoptosis, showing cell shrinkage and PARP-1 cleavage, while RT-R-MDA-MB-231 cells underwent necroptosis, showing mitochondrial dissipation, nuclear swelling, and an increase in the expressions of CypA and AIF. In addition, MEK/ERK inhibition reversed the radio-resistance of RT-R-MDA-MB-231 cells and suppressed the increased expression of CSC markers (CD44 and OCT3/4) and the EMT phenotype (\u03b2-catenin and N-cadherin/E-cadherin). Taken together, this study suggests that activated ERK signaling is one of the major hub signals related to the radio-resistance of MDA-MB-231 breast cancer cells. Breast cancer is one of the major causes of death due to cancer worldwide, especially in women . For breRadio-resistance is a process in which the tumor cells or tissues adapt to radio therapy-induced damage  and survMAPK pathways are key signaling pathways involved in the regulation of normal cell proliferation, survival, and differentiation. Aberrant regulation of the MAPK pathways contributes to the development of cancer; particularly, the extracellular signal-regulated kinase (ERK) is crucially involved in cancer cell proliferation, survival, and metastasis . ERK conRadio-resistant MDA-MB-231 breast cancer cells (RT-R-MDA-MB-231 cells) are reported to have a high proliferation rate, metastatic activity, and adhesion to endothelial cells compared with the parental MDA-MB-231 (p-MDA-MB-231) breast cancer cell line. RT-R-MDA-MB-231 cells harbor increased expressions of cancer stem cell (CSC) markers and the epithelial\u2013mesenchymal transition (EMT) phenotype . We hypoThis study was designed to decipher the proteomic differences between p-MDA-MB-231 and RT-R-MDA-MB-231 cells, enabling us to corroborate our findings at the molecular level. In addition, we also aimed to investigate the importance of the key altered signaling in the reversal of radio-resistance and the regulation of the CSC and EMT phenotypes that are strongly associated with radio-resistance.To determine the key altered expressions of proteins involved in the radio-resistance of RT-R-MDA-MB 231 cells, we performed and analyzed antibody microarrays to assess the difference in protein expressions between p-MDA-MB-231 and RT-R-MDA-MB-231 cells. The internal normalization ratio (INR) was kept as INR > 1.0 and INR < 1.0. With respect to this value, we selected around 10 upregulated proteins and 16 downregulated proteins, which are specified in Among the 16 downregulated proteins, caspase 3 was the most downregulated in the RT-R-MDA-MB-231 cells, which is suggested as one of the mechanisms for RT resistance . Figure Gene ontology (GO) enrichment analysis of differentially expressed proteins was carried out with the use of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. The KEGG pathway analysis showed that the most significant pathway involved in the RT-R-MDA-MB-231 cells was the MAPK1 signaling pathway B. GO enrTo investigate the inhibitory effect of a MEK/ERK inhibitor, PD98059, in RT-R-MDA-MB-231 cells, we performed cell viability assays in p-MDA-MB-231 cells and RT-R-MDA-MB-231 cells. Morphological analysis A revealeTo explore the radio-sensitivity of both p-MDA-MB-231 and RT-R-MDA-MB-231 cells, we performed a colony formation assay. This revealed that RT-R-MDA-MB-231 cells were resistant to radiation (RT) until 4 Gy, whereas p-MDA-MB-231 cells were sensitive to RT treatment A,B. The \u00ae Red staining is used to show the live time status of mitochondria [In chondria . The staTo molecularly confirm the difference in cell death between p-MDA-MB-231 and RT-R-MDA-MB-231 cells, we performed Western blot analysis. It was reported that CSC markers and EMT phenotypes were highly expressed in RT-R-MDA-MB-231 cells compared to the p-MDA-MB-231 cells, and that their high expression was closely related to radio-resistance . Here, wThe total proteins of p-MDA-MB-231 and RT-R-MDA-MB-231 cells were isolated with a radioimmunoprecipitation assay (RIPA) buffer, which contained 0.1% NP-40 and 0.1% sodium dodecyl sulfate in phosphate-buffered saline (PBS) containing a protease inhibitor cocktail . The expression profiling of proteins was analyzed by a Signaling Explorer Antibody Array .https://www.genome.jp/kegg/pathway.html, accessed on 20 October 2018) was employed. The selected genes were investigated for potential protein\u2013protein interactions using STRING  database version: 10.5 . For the display of protein interactions, selected proteins were uploaded into the STRING database and assessed using Cytoscape Software version Cytoscape_v3.7.1 . To access the interaction of the experimental data and to provide unambiguous comprehensive coverage, the online tool string was used.The obtained proteins from the antibody array analysis were further submitted to DAVID . DAVID is an online tool that provides a biological understanding between two or more data sets of genes, and it can also be used to determine gene ontology (GO) in terms of biological processes and cellular processes. To determine the pathways involved in the identified genes, KEGG (Kyoto Encyclopedia of Genes and Genomes) (2 incubator. The cells were grown with 80% confluence and were treated with a MEK/ERK inhibitor (PD98059) dissolved in DMSO or DMSO alone.RT-MDA-MB-231 cells were established as previously described . Briefly5 cells/well, they were treated with the MEK/ERK inhibitor, and they were maintained for 24 and 48 h at 37 \u00b0C in a 5% CO2 incubator. After incubation, 50 \u00b5L of MTT (0.5 mg in 1\u00d7 PBS) was added to each well and incubated for about 2 h at 37 \u00b0C in a 5% CO2 incubator. The media were removed and the formazan crystals that formed in the live cells were dissolved with the 500 \u00b5L of DMSO. The solubilized formazan crystals were transferred to 96-well plates and the absorbance was read by an enzyme-linked immunosorbent assay (ELISA) reader at 540 nm. The cell viability was quantified in percentage, while vehicle-treated control cells were set at 100%.We used a calorimetric assay, MTT -2,5-diphenyltetrazolium bromide), to analyze the cell viability. The cells were seeded in 24-well plates with a confluence of 1 \u00d7 103 cells/well) were seeded in six-well plates, treated with the indicated doses of the MEK/ERK inhibitor, and maintained at 37 \u00b0C in a 5% CO2 incubator. The cells were irradiated with a given concentration, and the media were discarded after 24 h and replaced with fresh complete media every 2\u20133 days. After 14 days, the medium was discarded and the cells were washed with 1\u00d7 PBS thrice. The colonies were fixed with absolute methanol for 10 min, stained with Giemsa staining solution, and then maintained at room temperature. The number of colonies was counted using ImageJ software.P-MDA-MB-231 or RT-R MDA-MB-231 cells  was added to the 500 \u00b5L of 1\u00d7 PBS, which was incubated for 30 min at 37 \u00b0C with a 5% CO2 incubator. After incubation, the cells were washed with 1\u00d7 PBS and were fixed with 90% glycerol in 1\u00d7 PBS. The cells were viewed under a fluorescent microscope .For the nuclear morphological changes, DAPI staining was performed. The cells were seeded in 12-well plates at a density of 1 \u00d7 106 cells/plate. The cells were treated with the MEK/ERK inhibitor or DMSO as a vehicle control and were maintained for 48 h at 37 \u00b0C with a 5% CO2 incubator. After 48 h, the cells were harvested and transferred to 15 mL falcon tubes, and they were centrifuged for 5 min at 2000 rpm. The supernatant was discarded, and the tubes were centrifuged again to remove the residual supernatant. After complete removal of the supernatant, 500 \u00b5L of the 2X sample buffer containing 100 mM of Tris-Cl (pH 6.8), 4% (w/v) sodium dodecyl sulphate (SDS), 0.2% (w/v) bromophenol blue, and 200 mM of dithiothreitol was added. The protein lysates were collected in 1.5 mL Eppendorf tubes and kept at 100 \u00b0C for 10 min. The protein concentration was determined by the Bradford assay. In addition, 30 \u00b5g of the proteins was resolved in 8\u201312% SDS-PAGE and was transferred to a polyvinylidene difluoride membrane. After transfer, the membranes were blocked with 3% skimmed milk in Tris-buffered saline containing 1% Tween 20 (TBST) buffer for 30 min at room temperature, and they were incubated at 4 \u00b0C overnight with antibodies against actin , ERK , p-ERK , CypA , pro-caspase 9 , pro-caspase 3 , AIF , PARP-1 , CD44 , \u03b2-catenin , Oct 3/4 , E-cadherin , and N-cadherin . After overnight incubation in primary antibodies, the membranes were washed with TBST buffer thrice for about 10 min per wash. Then, the membranes were incubated in horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature with 1:2000 dilution. The membranes were later washed with TBST buffer three times (10 min/wash) and were developed with ECL (electrochemiluminescence) solutions .P-MDA-MB-231 and RT-MDA-MB-231 cells were seeded in 100 mm plates with a cell density of 2 \u00d7 10p-value <0.05 was considered statistically significant.All experiments were performed at least in triplicate, and all analyses were performed with the use of GraphPad Prism 7 software . One-way ANOVA followed by the Newman\u2013Keuls post hoc test was performed to compare various treatment groups. The data were presented as mean \u00b1 standard deviation (SD). A Radiation therapy is one of the common and essential parts of breast cancer treatment. Around half of the cancer patients go through radiation therapy at some point in their treatment . IonizinThis study was designed to find the hub signaling involved in the RT resistance of RT-R-MDA-MB-231 cells and to investigate the importance of the hub signaling in the reversal of radio-resistance and the regulation of the CSC and EMT phenotype that is highly associated with radio-resistance. We found that ERK signaling was highly activated in RT-R-MDA-MB-231 cells compared to in p-MDA-MB-231 cells and that ERK signaling was essential for the survival of both p-MDA-MB-231 and RT-R-MDA-MB-231 cells. In addition, the RT resistance of RT-R-MDA-MB-231 cells was reversed by the inhibition of ERK signaling . FurtherBefore concluding, we should discuss some questions. The first question would be whether activated ERK signaling is the main mechanism for the radio-resistance of MDA-MB-231 cells. In The second point to discuss would be the relationship between ERK signaling and EMT, as well as the CSC phenotype of RT-R-MDA-MB-231 cells, because it is mentioned that several other signaling pathways such as JAK/STAT, Hedgehog, Wnt, Notch, PI3K/PTEN, and nuclear factor-\u03baB (NF-\u03baB) signaling pathways, compared to ERK signaling, are closely related to CSC properties ,54,55, aThe third point to discuss is why the phenotype of cell death induced by ERK inhibition differed between p-MDA-MB-231 cells and RT-R-MDA-MB-231 cells, while ERK inhibition induced cell death and suppressed the increased expression of CSC markers and the EMT phenotype of both p-MDA-MB-231 cells and RT-R-MDA-MB-231 cells. We speculate that the reason could be that RT-R-MDA-MB-231 cells exhibit a decreased activity of caspase. It has been reported that the cancer cells harboring caspase defects frequently undergo necroptosis or necrosis instead of apoptosis when the death signal appears . InitialThe fourth point to discuss would be the role of the other upregulated and downregulated proteins in RT-R-MDA-MB-231 cells. Although we could not discuss here all of the 26 proteins, recent studies have suggested that the inhibition of CLK1 also decreases cell proliferation . CLK1 anThe fifth point to discuss would be the mechanisms driving the upregulation of ERK signaling in RT-R-MDA-MB-231 cells. As we know that radiotherapy works by damaging the DNA of cancer cells, our first thought was that the upregulation of ERK signaling would be related to some of the mutations in the Ras-Raf-MEK-ERK pathway. Therefore, we performed whole genome sequencing, but there was no additional mutation of ERFR, PI3K/Akt, Ras, Raf, MEK, or ERK molecules of MDA-MB-231 cells (data not shown). In this paper, we inhibited ERK signaling with PD98059, a non-adenosine triphosphate competitive MAPK (MEK) inhibitor . TherefoThe weakness of this study is that we performed the experiment with only one cell line. It is in question whether the main mechanism of the radio-resistance of RT-R-MDA-MB-231 cells can be applied to all radiation-resistant breast cancer cell lines or can be generalized to triple-negative breast cancer cells. In addition, even regarding the radio-resistance of RT-R-MDA-MB 231 cells, other signaling pathways are also suggested as a key signaling pathway involved in the resistance. Similar to the signaling involved in CSC, the signaling involved in the radio-resistance of RT-R-MDA-MB 231 cells could also be complexed. However, aberrantly upregulated ERK signaling contributes to cancer cell proliferation, survival, and metastasis , and manIn summary, we found that ERK signaling was highly activated in RT-R-MDA-MB-231 cells compared to p-MDA-MB-231 cells. The activated ERK signaling was associated with an increased cancer stemness and EMT phenotype. In addition, the RT resistance of RT-R-MDA-MB-231 cells was reversed by the inhibition of ERK signaling. Furthermore, the inhibition of ERK suppressed the CSC marker proteins. With all of these findings, we conclude that activated ERK signaling is one of the major hub signals related to the acquisition of radio-resistant MDA-MB-231 cells. This study suggests a distinct and advantageous therapeutic value of the targeting of the ERK signaling pathway in MDA-MB-231 cells. Further research is also warranted regarding ERK signaling on the radio-resistance of breast cancer, especially on TNBC."}
+{"text": "Results: Our final model results show that browsing on Instagram was associated with lower levels of body appreciation, fully mediated by upward social comparison with social media influencers, not close or distant peers. Commenting on others\u2019 looks and posting own content were not associated with body dissatisfaction. Being an adolescent female (compared to a young woman) and having a higher BMI were associated with worse body appreciation. Conclusions: Our findings highlight the need for public health interventions to raise awareness about the posting practices of social media influencers and to strengthen a positive body image among young females susceptible to social comparison processes.Background: Instagram is one of the most popular social media platforms among young females. Idealized body images shared on the platform have been associated with lower levels of body satisfaction in this population, likely due to social comparison processes. In the present study, we tested a mediation model linking Instagram use  to body dissatisfaction , mediated by upward social comparison with close peers, distant peers, and social media influencers. Methods: We applied structural equation modeling to self-report cross-sectional data collected from 291 female adolescents and young women (M In September 2021, the Wall Street Journal published the Facebook Files, uncovering that the platform owner conducted their own research and found that Instagram is making body issues worse for one in three teenage girls, mainly because Instagram contributes to unhealthy social comparison among teens . On InstThe current study was designed to investigate the relationship between Instagram use and body dissatisfaction among female adolescents and young women. In particular, the study considered (1) different types of Instagram use , (2) the mechanism through upward social comparison, and (3) the role of close and distant peers as well as social media influencers as comparison targets.Instagram includes a plethora of (seemingly) authentic pictures, and many of them represent body ideals. Physical appearance, in fact, plays an important role on Instagram, and studies have found that adolescents and young people experience distress, are dissatisfied with their bodies, and feel the pressure to look perfect on social media , especiaBrowsing, commenting, and posting on Instagram is likely not directly linked to body dissatisfaction, but, as also evidenced in the Facebook Files, through social comparison among teens. According to the Tripartite Influence Model of Body Dissatisfaction and Eating Disturbance , young pupward social comparison processes [Past research suggested that particular social media environments such as Instagram may contribute to feelings of inadequacy as a result of rocesses ,27. Fromrocesses . People rocesses . The phrrocesses . In an erocesses  found throcesses  found throcesses . In factrocesses . Eventuarocesses  found throcesses . Consideclose and distant peers. In fact, previous research found that exposure to thin and attractive distant peers had a more detrimental effect than the exposure to close peers, mainly because of the plethora of idealized images of distant peers to which young women were exposed on social media [Many images and stories on Instagram are posted by peers. A qualitative study by Kenny et al.  demonstral media . To follAlong with peers, Instagram allows users to be directly in contact with people they typically do not know personally, such as social media influencers. Influencers are those people who have been able to establish a strong online presence, which is constantly strengthened through the use of regular posts and their ability to create communities on their social media accounts . One exaAlthough social media, and Instagram in particular, are frequently blamed for disseminating idealized body images, voluntarily or involuntarily , there iAll investigated relationships are summarized in the theoretical mediation model in The data for this study were collected through a survey in spring 2019. To be eligible to the study, participants had to be female, less than 30 years of age, and fluent in either Italian or English. Participants were recruited in two different ways: young women aged 18 to 28 were invited to respond to an online survey through posts and snowball sampling on Instagram and Facebook over the course of 30 days. To avoid targeting underage females on social media platforms, female adolescents aged 15 to 17 were asked to fill out a paper-and-pencil survey at a large collaborating high school in Northern Italy. We opted for paper-and-pencil approach since a PC with an Internet connection was not available at school for all adolescent participants and for the dedicated time slot for data collection. The participants were informed beforehand about the aim of the study. Study participation was voluntary, and data were collected in an anonymous form. The Ethics Committee of the university where the research was carried out as well as the headmaster of the collaborating high school approved the study design.Instagram use was assessed using three items. Browsing was assessed with \u201cHow often do you look at the looks of others\u2019 in their photos and stories on Instagram?\u201d , commenting with \u201cHow often do you comment on the looks of others in their photos and stories on Instagram?\u201d , and posting with \u201cHow often do you share your photos and stories on Instagram?\u201d .Upward social comparison was measured with three items adapted from Fardouly and Vartanian . The iteThe Body Dissatisfaction Scale developed by Mutale et al.  was usedThe Body Appreciation Scale  was usedCovariates included participants\u2019 age, which was collapsed into 1 = \u201cadolescents\u201d and 0 = \u201cyoung adults\u201d. This variable simultaneously captured the two different assessment modes . Furthermore, the analysis considered participants\u2019 body mass index (BMI), calculated from self-reported height and weight. For participants up to 19 years of age, we calculated age- and gender-specific z-scores using the World Health Organization (WHO) growth reference . For oldSPSS was used to conduct the preliminary statistical analyses and impute missing data. Missing data were imputed using an expectation-maximization algorithm on a dataset including only participants with less than two missing data points on the variables included in the final mediation models. Descriptive statistics were calculated for all measures to assess normal distribution and detect outliers. Skewness and kurtosis values in the absolute range of two were considered acceptable. Scale reliability was assessed with Cronbach\u2019s alpha. Bivariate correlations among all the variables were conducted. The lavaan package  in R 4444 was usn = 291; 82.2%). The average age of the final sample was 19.8 (SD = 4.57). Given the bimodal distribution of age, the variable was collapsed into adolescents  and young adults . The dichotomous age variable further reflected the two different data collection procedures . The large majority of participants were Italian . The interquartile range of age-specific z-scores for BMI was 1.19. According to the growth standards of the WHO [n = 3) of females in the final sample were underweight, 83% (n = 242) were normal weight, 13% (n = 38) were overweight, and 3% (n = 8) were obese. Concerning weekly Instagram use, the majority declared spending more than five hours per week using the platform , followed by three to five hours  and one to two hours . Regarding the different types of Instagram use, more than half of the participants reported often or always browsing through others\u2019 looks in photos or stories , while only a minority commented on others\u2019 looks on a daily basis . Approximately one in four females posted her own photos or stories on Instagram .Survey data were obtained from 354 females. The analytical sample only included females with an Instagram account and two or fewer missing data points on the variables included in the final model  = 121.762, p \u2264 0.001; CFI = 0.963; RMSEA = 0.066; SRMR = 0.044). The final measure for the lack of body appreciation thus included eleven items with factor loadings ranging from 0.282 to 0.891  revealed that the two items \u201cI am attentive to my body\u2019s needs\u201d and \u201cI engage in healthy behaviors to take care of my body\u201d as part of the (lack of) Body Appreciation Scale were indicators of a distinct concept. They were thus eliminated, which resulted in good model fit (\u03c7.891 see . Given tBivariate correlations  did not 2 (12) = 23.374, p = 0.025; CFI = 0.963; RMSEA = 0.057; SRMR = 0.042). Among the three different types of Instagram use , browsing through other people\u2019s looks on Instagram turned out to be significantly and positively associated with upward comparison when social media influencers were the comparison target. The model did not include significant associations between browsing on Instagram and upward comparison with close or distant peers, and neither did it include significant associations between commenting or posting on Instagram and upward comparison with close peers, distant peers or influencers. In turn, controlling for being an adolescent and BMI (measured with age-specific z-scores), upward comparison with the three different comparison targets was significantly and positively associated with a lack of body appreciation, while it was not related to perceived body discrepancy. Being a female adolescent (compared to a young woman) and higher BMI were related to worse body appreciation. Furthermore, higher BMI, but not being an adolescent, was associated with higher levels of body image discrepancy. Eventually, the indirect association between browsing on Instagram and lack of body appreciation, through upward comparison with influencers, was positive and significant . The coefficients of all direct paths and correlations among endogenous variables are summarized in The tested mediation model showed good model fit , though multi-item indicators should be preferred because of their greater stability. Fifth and last, we did not consider additional psychological characteristics such as self-esteem or negative affect [The present study comes with some limitations. First, a cross-sectional design was used, which does not allow for drawing conclusions on the causal mechanisms between the investigated variables. A longitudinal or experimental design would overcome this limitation. Yet, our hypothesized mediation model was based on prior research on the topic, both cross-sectional and longitudinal or experimental. Furthermore, we tested an alternative mediation model reversing the hypothesized paths between Instagram use and body dissatisfaction, which led to a significant deterioration of all fit indices. Second, our sampling method limits the generalizability of the findings to a larger female population and the final sample size was comparably small. A follow-up power analysis with the semPower package  revealede affect ,53 as moBoost Body Confidence and Social Media Savvy intervention [In conclusion, the present study found that the relationship between browsing through the looks of others on Instagram and body dissatisfaction, measured by the lack of body appreciation, is fully mediated by upward appearance comparison with social media influencers. Thus, the exposure to idealized pictures and stories of this comparison target is associated with detrimental outcomes in female adolescents and young women. The findings of our study highlight the need for public health interventions to raise awareness about the posting practices of social media influencers and to strengthen a positive body image, with special attention to particularly vulnerable girls. Prior interventions to promote a positive body image among women proved to be effective . Howeverrvention , should rvention  can helprvention  as well rvention , can be"}
+{"text": "In a Cox regression analysis after adjusting for all covariates, with advantaged and disadvantaged neighborhoods classified according to individual household income, the adjusted HR for patients living in a disadvantaged area was higher compared to patients living in an advantaged area in patients with middle income, compared to the reference group (a high income within an advantaged neighborhood) . The adjusted HR for patients with low income who lived in a disadvantaged location was greater than for patients who lived in an advantaged area . Conclusions: Individual SES has a greater impact on all-cause mortality among diabetic patients when they live in a low-income neighborhood.Background: Neighborhood environmental factors along with individual factors are beginning to make a mark as factors which influence individual health outcomes. The goal of this study is to look at the combined impact of individual and neighborhood socioeconomic status on all-cause mortality in diabetic patients who have just been diagnosed. Methods: The Korean National Health Insurance (2002\u20132013) was employed in this cohort research, which used a stratified random sample. During the years 2003\u20132006, a total of 15,882 individuals who were newly diagnosed with diabetes and using oral disease-controlling medication were included in the study. Individual income and neighborhood deprivation index were used to examine the combined effect on all-cause mortality. The frailty model was performed using Cox\u2019s proportional hazard regression. Results: During the study period, 28.3 percent ( Diabetes mellitus is a typical chronic disease, and disease burden due to this disease is considered a major public health challenge in developed countries. According to the statistics on the cause of death, the number of deaths due to diabetes in 2020 was 8456 per 100,000 people, accounting for 2.8% of the total cause of death, and ranked sixth. In the same year, the number of deaths from heart disease reached 32,347 per 100,000 people, and the number of deaths from cerebrovascular disease reached 21,860 per 100,000 people ,10,11,12In the past, the focus has been on individual characteristics as risk factors for health ,19,20,21In previous studies on health inequality, research used a multilevel analysis methodology ,31 that Several recent studies have highlighted the distinct effects of individual and neighborhood-level socioeconomic status (SES) on several aspects of diabetes care, including treatment approach, care quality, and mortality. However, results from research that examined neighborhood-level characteristics and their impact on individual health have been mixed. Many people feel that community competition has an independent impact on the health of all citizens ,37,38,39The first aim of this study was to investigate a possible association between individual-level SES and neighborhood-level SES, and mortality in patients with newly diagnosed diabetes. The second aim of this study was to examine if individual and neighborhood SES have a combined influence on diabetes patient mortality.The Korean National Health Insurance (KNHI) claims database for 2002\u20132013 and the 2005 Korea Census were utilized in this investigation. The National Health Insurance Corporation obtains data from cohorts that are representative of the population of the country. These records contain information on 1,025,340 individuals who were chosen from a stratified random sample based on age, gender, area, health insurance type, income quintiles, and individual total medical expenses in 2002. The database contains reimbursement information for each medical treatment, including basic demographic patient information, a clinic or hospital identity, an illness code, expenses incurred, results of health screenings, past/family health history, health habits, and death information. We studied the relationship between combined individual and neighborhood socioeconomic level and mortality in newly diagnosed diabetes patients in a cohort study. The Institutional Review Board of Yonsei University\u2019s Graduate School of Public Health granted this project ethical approval . Because the study was based on routinely available administrative and claims data, informed permission was not required.A total of 55,157 diabetics were included in the KNHI enrollee database. Between 2003 and 2006, 26,156 people with newly diagnosed diabetes  were chosen. A lack of diabetes claims in 2002\u20132005, a first diabetes claim in 2003\u20132006, and the absence of diabetes in the health history prior to the year of diagnosis were all used to confirm new diagnoses. The subjects were followed for a minimum of seven years and a maximum of ten; 10,274 of the 26,156 individuals were eliminated because 483 were under the age of 20 and 9791 patients did not follow their hypoglycemic prescription. These exclusion criteria were required in order to identify true diabetic patients. A total of 15,882 people were included in the final study sample .In this study, the primary outcome was all-cause mortality. The survival time from diagnosis to death, or study end-date, was the outcome variable, and mortality was defined as all-cause mortality as determined from death certificate data in the national death registry. Ischemic heart disease (ICD-10 code I20\u2013I25), cerebrovascular disease (ICD-10 code I60\u2013I69), and diabetes (ICD-10 code E10\u2013E14) were defined as diabetes-related mortality.As a proxy variable, the average monthly insurance premium for household income was utilized. In Korea, there are two types of health insurance: National health insurance and medical aid. Medical aid is available to anyone with a household income of less than Korea Won (KRW) 600 per month based on a single family. People with household income of more than KRW 600 per month can apply for basic livelihood security recipient, and if somebody is eligible for basic livelihood security recipient, they are automatically entitled to medical aid . People To quantify deprivation at the neighborhood level, a summary measure was utilized. Using census data from 2005, the modified Carstairs index  was usedAge , sex, residential area , Charlson comorbidity index  , number p-value for mortality among areas were 0.022 and 0.004, respectively. Scaled Schoenfeld residuals were used to evaluate the proportional hazards assumptions, and no violations were discovered. SAS 9.3 software was used for all statistical analyses.The chi-square test was used to obtain descriptive statistics for all variables, including frequencies and percentages for categorical variables. The Kaplan\u2013Meier product limit technique was used to assess survival probability for all-cause mortality, using log rank tests stratified by socioeconomic level. Survival analysis was performed using a Cox proportional hazard model by frailty model. This frailty model could evaluate whether there is intra-cluster homogeneity of the outcome between individual socioeconomic status and neighborhood deprivation through the integration of random effect . When thn = 1714) vs. 14.2%(n = 637) for those with high household income and lived in an advantaged neighborhood, 17.8% (n = 2031) vs. 17.9% (n = 803) for those with high household income and lived in a disadvantaged neighborhood, 22.2% (n = 2533) vs. 20.1% (n = 901) for those with middle household income and lived in an advantaged neighborhood, 29.8% (n = 3396) vs. 28.5% (n = 1281) for those with middle household income and lived in a disadvantaged neighborhood, 6.1% (n = 694) vs. 8.2% (n = 370) for those with low household income and lived in an advantaged neighborhood, and 9.0% (n = 1021) vs. 11.2% (501) for those who with low household income and lived in a disadvantaged neighborhood.During the research period, 4493 (28.3%) of the 15,882 eligible individuals died; 11,389 (71.7%) survived . Betweenp-value 0.0001 by log-rank test; Individuals with high income, middle income, and low income had 9.2, 8.5, and 8.3 mean years of survival, respectively (p-value 0.0001 by log-rank test; Individuals with low income in a disadvantaged location had an average of 8.3 mean years of survival, whereas those with low income in an advantaged area had an average of 7.8 mean years of survival (After adjusting for all factors, The significance of individual and neighborhood socioeconomic variables in patients with diabetes was investigated in this study, which used a comparative and longitudinal methodology. Patients with diabetes who lived in a poor neighborhood had a greater risk of all-cause death than those who lived in an advantaged location, even if they had the same amount of individual income. Additionally, in patients dwelling in the same disadvantaged area or advantaged area, the lower earning individuals had increased risk of all-cause mortality, even after controlling for individual and neighborhood characteristics. In diabetes-related mortality, the risk was high only in individuals with low-income and living within an advantaged area compared to those having high-income and living within a disadvantaged area.In previous studies, diabetes was more prevalent in low socioeconomic groups ,59. HoweThe second argument is that more direct psychological routes resulting from inequality, such as despondency, lack of control, or loss of esteem, have an impact on individual health ,63,64,65The third point, perspectives on the role of socioeconomic position, suggests reasons why the wealthy in more affluent areas may be healthier. Residing in affluent areas would bolster the capacity of the comparatively rich to exploit their expertise, money, power, status, and social connections . The weaThe fourth explanation is that a lack of a safe environment limits exercise opportunities, making it more difficult to maintain a healthy lifestyle . FurtherIn addition to these four possible explanations, when examining other perspectives related to mortality with diabetes and calculating the CCI score, hypertension and dyslipidemia were not considered and treated as separate risk factors. This is because diabetic patients with high blood pressure or dyslipidemia can develop stroke and cardiovascular diseases, and act as a mediating factor leading to death from these conditions. Similarly, CCI reflected the presence of cancer and the other chronic diseases, which may have contributed to the death.In the case of Korea, the proportion of government or compulsory insurance funds in current healthcare was 61% in 2019, lower than the average of 74.1% in OECD countries. This means that the burden of medical expenses on households is greater than that of OECD countries. Therefore, there is a need for a policy that increases access to healthcare by reducing the burden on individual low economic levels by strengthening insurance coverage for medical expenses. In addition, at the regional level, health promotion education and related projects are needed to maintain and improve a healthy life, and at the same time, it is considered necessary to expand infrastructure such as sports facilities and good healthcare facilities.Our research has significant limitations. To begin, we only included high-risk diabetic populations. Our findings cannot be applied to the general population in the absence of diabetes. Second, because this study employed data from a claims database, lifestyle and educational characteristics that impact mortality could not be taken into account. Third, we did not consider modifying the research participants\u2019 neighborhood deprivation status if they relocated inside the study region. Furthermore, although geographic accessibility, transportation to healthcare resources, extreme temperature and natural environment could have a major impact on mortality, we did not consider these variables. Thus, there may be a problem with unmeasured confounding bias. Additionally, we did not reflect the changes over time for CCI or the other risk factors affecting mortality, which may be a limitation of our study. Finally, we used the modified Carstairs index that was used in previous studies, although we did not confirm the reliability and validity of this index. Finally, multicollinearity between health insurance type and household income exists in this analysis because participants in the medical aid group are definitely in the low household income group. This may induce an inconsistent direction of HR between the crude HR and the adjusted HR of medical aid.Despite its problems, our research offers some advantages. To our knowledge, this research was the first investigation of the link between individual and neighborhood SES and mortality, and it was a prospective design with a comparatively large sample size, resulting in strong statistical power for detecting the impacts of neighborhood deprivation. Second, utilizing nationwide representative cohort data, a representative sample of diabetic patients was studied. Third, we tried to make our research sample more homogeneous by enrolling individuals who had just been diagnosed with diabetes.Deprivation in one\u2019s neighborhood leads to all-cause death. Individual and community level variables accumulate weight against people, putting those with both individual- and neighborhood-level risk factors at the highest risk. These findings bring severe clinical and public-health problems, indicating that in the creation of healthcare policy, both individual and neighborhood-level approaches are critical."}
+{"text": "Cancer is a leading cause of death and disability worldwide. Epigenetic deregulation is one of the most critical mechanisms in carcinogenesis and can be classified into effects on DNA methylation and histone modification. MicroRNAs are small noncoding RNAs involved in fine-tuning their target genes after transcription. Various microRNAs control the expression of histone modifiers and are involved in a variety of cancers. Therefore, overexpression or downregulation of microRNAs can alter cell fate and cause malignancies. In this review, we discuss the role of microRNAs in regulating the histone modification machinery in various cancers, with a focus on the histone-modifying enzymes such as acetylases, deacetylases, methyltransferases, demethylases, kinases, phosphatases, desumoylases, ubiquitinases, and deubiquitinases. Understanding of microRNA-related aberrations underlying histone modifiers in pathogenesis of different cancers can help identify novel therapeutic targets or early detection approaches that allow better management of patients or monitoring of treatment response. Epigenetics is defined as stable and heritable alterations in gene expression and cellular function without changes to the original DNA sequence and can still be passed on from generation to generation . This te3) on cytosine residues of CpG islands, especially those located in the gene promoter region [DNA methylation is defined as the addition of methyl groups  are small noncoding RNAs with a length of approximately 22 nucleotides . In the Cancer development is a multistep process, and genetic alterations in every step are manifested by significant dysregulation of proteins involved in cell cycle regulation, which may have been triggered by miRNAs , 12. miRThis review will discuss histone modifications and the microRNA-mediated regulation of the histone modification machinery in cancer.Histones are lysine\u2013arginine abundant proteins involved in chromosome condensation, consisting of four core types  located in the bead of the nucleosome, along with two linker histones (H1/H5). The amino and carboxy termini of these proteins may undergo modifications, such as methylation, acetylation, phosphorylation, sumoylation, ubiquitination, and ADP-ribosylation, which are pivotal for transcriptional regulation. The addition of acetyl group on lysine residues of the H3 and H4 classes of histones results in a lightly packed chromosome structure and transcription activation . Lysine Given that histone modification affects gene transcription and appears early in tumorigenesis, considerable research has been carried out on the role of these alterations in malignancies. H4K16 hypoacetylation has been identified in breast, colon, lung, and liver cancers as well as in medulloblastomas , 18. ThiAll of these modifications are involved in malignancy induction by revising tumor suppressors or oncogene expression. H3 and H4 hypoacetylation and hypermethylation lead to p21WAF1 tumor suppressor inactivation . Loss ofFurthermore, dysregulation of histone-modifying enzymes in cancer and their distinct expression profile in tumor cells compared to normal cells have been identified in some studies . For examiRNAs are involved in the regulation of biological processes such as development, growth, differentiation, proliferation, and apoptosis . AlteratOnco MiRs (oncomir) switch tumor-linked operations, such as unlimited cell growth, transformation, and metastasis. miR-21 is an oncomir known to be elevated in many cancers and drives cell proliferation. A sort of H3K4 demethylase, known as RBP2, can decrease miR-21 levels followed by decreased H3K4 trimethylation of its promoter and could act as a novel treatment in chronic myeloid leukemia cells . OverexpmiR-29 is known as a tumor suppressor gene because of its function in preventing cell growth and proliferation. H3K27 trimethylation is accomplished by recruiting YY1 and Ezh2. This change is related to the miR-29 promoter and could repress its expression in skeletal muscle cells. Aberrant downregulation of miR-29 by raised H3K27me3 is found in rhabdomyosarcoma . FurtherThe epigenetic profile in numerous cancers is altered by the action of miRNAs , 50. TheThere are numerous studies demonstrating miRNA-mediated regulation of different histone acetyltransferases including EP300, PCAF, TIP60, and hCLOCK . The polHDACs are classified into four classes including classes I\u2013IV . There hThe upregulation of HDAC1 results in uncontrolled growth and cisplatin-resistant in ovarian cancer cells. miR-34a suppresses this process by targeting HDAC1 . MoreoveAlterations in HDAC4 expression occur via several miRNAs and vary based on cancer type. Amodio et al. found that miR-29b/HDAC4 serves as an epigenetic loop in multiple myeloma and the induction of miR-29b expression could repress HDAC4 and result in cell survival and reduced malignancy in myeloma . HoweverSIRT is a family of histone deacetylase compromised seven proteins and divided into four classes. Class I includes SIRT1, SIRT2, and SIRT3. Class II consists of SIRT4. SIRT5 belongs to class III, and SIRT6 and SIRT7 are class IV members . Being eThere are two major types of histone methyltranferases termed histone lysine N-methyltransferases and histone arginine N-methyltransferases. The following sections discuss the miRNA-mediated regulation of both types.Lysine methyletransferases (KMTs) are divided into groups based on the site of methyl group addition . In this(1) Suv39H1, Suv39H2, SETDB1, and G9A/EHMT2 (H3K9). miR-125a-5p, a recognized prognostic factor in gastric cancer, regulates SUV39H1 (KMT1A) and prevents angiogenesis [ogenesis . The dowogenesis . In addiogenesis . miR-675ogenesis . Low levogenesis . Wu et aogenesis .(2) KMT2A (MLL1). KMT2A is a direct target of hsa-miR-22-3p, and hsa-miR-22-3p upregulation has been found in the metastatic form of prostatic cancer [c cancer .(3) NSD1 and ASH1L (H3K36). miR-142 could inhibit ASH1L (KMT2H), and its downregulation leads to increased levels of ASH1L in leukemia [leukemia . Moreoveleukemia . KMT3B (leukemia .(4) SMYD3 (H4K5). miR\u2010124 acts as a SMYD3 (KMT3E) expression modifier, and its decreased levels that result in cellular invasive criteria have been shown in intrahepatic cholangiocarcinoma cells [ma cells . miR-346ma cells .(5) hDOT1L (H3K79). DOT1L (KMT4) is blocked by miR-133b. miR-133b is a tumor suppressor, and low levels cause chemoresistance in colorectal cancers [ cancers .(6) SET8 (H4K20). SET8 is a histone methyltransferase that adds one methyl group on lysine 20 of H4. Its expression can be switched by miR-502, and it has oncogenic effects and contributes to cell growth and migration. This oncogene expression can be increased due to reduced levels of miR-502 in many malignancies including breast cancer, ovarian cancer, small-cell lung cancer, colorectal cancer, non-Hodgkin's lymphoma, esophageal squamous cell carcinoma, clear cell renal cell carcinoma, and hepatocellular carcinoma [arcinoma , 103. Foarcinoma , 124. Moarcinoma .(7) EZH1 and EZH2 (H3K27). Elevation of KMT6B (EZH1) caused by miR-17-5p downregulation is related to erlotinib resistance in NSCLC [in NSCLC . miR-765in NSCLC . Additioin NSCLC . In a stin NSCLC .Multiple microRNAs can alter EZH2 expression. Effects of miR-101 on EZH2 have been observed in various cancers , and dowProtein arginine methyltransferases (PRMTs) are involved in histone post-translational methylation , and mulThere are multiple histone demethylase enzymes divided into lysine and arginine methyltransferase groups. Histone lysine demethylases are classified into KDM1-8 families . miRNA-mLSD1 is a histone demethylase, controlled by miR-137 in a negative feedback loop in endometrial cancer . miR-302FBXL10 is a direct target of miR-146b, and downregulation of this miRNA in later stages of epithelial ovarian cancer has been linked to FBXL10 increase that, in turn, induces metastasis. However, in the early stage of the disease, miR-146b reduction results in overexpression of cyclin D1 and cell proliferation . Hong anJMJD1A is a direct target of miR-627, and its low expression is related to growth and differentiation inhibition . LMP1 an\u03b1-positive breast cancers and cell lines. miR-491-5p upregulation results in cell cycle arrest and attenuates growth through inhibition of JMJD2B in the same cancer [JMJD2B can be regulated by miR-491-5p, and its overexpression via miR-491-5p downregulation has been observed in ERe cancer . miR-491e cancer . Yong ete cancer .Studies have shown that JARID1B levels are ameliorated by miR\u2010137 . Thus, imiR-221 can target PHF2, and its upregulation along with PHF2 decrease has been shown in hepatocellular carcinoma cells .\u03b2-catenin pathway activation has been found in NSCLC [JMJD6 is a miR-770 direct target, and its overexpression due to miR-770 downregulation followed by WNT/in NSCLC . Furtherin NSCLC .There are several clusters of kinases classified by kinase domain sequences similarity, biological function, and other criteria . miRNA-mReduction of miR-517a is found in bladder cancer cell lines. It has a tumor-suppressive effect and can target RPS6KA4/MSK2 . The PRKJAK2 is one of the most important kinases involved in histone regulation and the key member of the JAK2/STAT3 signaling pathway recruited in inflammation and apoptosis . The levLIMK2 plays an oncogenic role in bladder cancer and can be decreased by miR\u2010135a .miR-23 can target NEK6, the enzyme that negatively regulates p53. The natural substance berberine plays a role in hepatocellular carcinoma treatment by activating this signal . In anot(1) CAMK . DAPK3 is a p53-activating kinase and a direct target of miR-1307, which is overexpressed in chemoresistant ovarian cancer cell lines [ll lines . CHEK2 cll lines . miR-182ll lines . CHEK1 ill lines . Howeverll lines . miR-497ll lines . miR-145ll lines , 202. mill lines . p53 indll lines . miR-451ll lines . A similll lines . miR-101ll lines , 209.(2) STE (STK4 and PAK2). miR-18a elevation motivates prostate cancer development through STK4 suppression [pression . PAK2 ispression \u2013214. Morpression . In addipression , and miRpression .(3) CMGC . miR-589-5p limits MAP3K8 expression and causes suppression of CD90+ cancer stem cells in hepatocellular carcinoma [arcinoma . miR-144arcinoma , 181. miarcinoma . CDK3 isarcinoma . miR-873arcinoma . miR-446arcinoma .The main groups of protein phosphatases are sorted considering the structural fold of the catalytic domain , and miRPPM1D has been found to be modulated by miR-499a-5p and miR-499a-5p downregulation followed by PPM1D upregulation in osteosarcoma .PPP2CA is a direct target of miR\u2010155, and its overexpression leads to PPP2CA low levels in colon cancer . miR-650The apoptosis activator miR-101 can repress EYA1 and is diminished in breast cancer . miR-562\u03b2-catenin [DUSP1 is controlled by various miRNAs. In osteosarcoma, miR-34a targets DUSP1, and its repression is related to elevated levels of Bax and E-cadherin, along with diminished levels of Bcl-2, cyclin E, cyclin D1, and -catenin . Upregul-catenin . Various-catenin , 238.SENP1 is one of the most important desumoylation proteins modulated by various miRNAs . SENP1 cRBX1, RNF8, HUWE1, and UHRF1 are histone ubiquitinating enzymes involved in different malignancies. Below, we summarize the miRNA-mediated effects on the regulation of these enzymes . RBX1 ha\u03b2\u2010catenin signaling target genes (Axin2 and MYC) and Sirt1/JAK/STAT3 signaling modulation [USP3, USP7, and USP22 are three deubiquitination enzymes studied in cancers, and these are controlled by various miRNAs . USP3 thdulation \u2013261. miRdulation . In gastdulation . Additiodulation .With due attention to the high cancer mortality rate, early diagnosis and initiation of appropriate therapeutics are urgently needed. To further these objectives, it is crucial to increase our understanding of the mechanisms and pathways involved in malignancy progression and improvement. The histone-modifying enzymes that catalyze the remodeling of chromatin structures play a major role in cancer biology. In this review, we discussed miRNAs that interact with a complex array of histone modifiers and reviewed the effects of their aberrant expression in various cancers. These alterations impact the fluctuation of multiple cancer cell properties such as drug sensitivity, drug resistance, proliferation, apoptosis, and malignancy trajectories. Hence, recognition of these small noncoding RNAs is imperative for the early diagnosis of cancer and may lead to the identification of new biomarker tests to facilitate earlier diagnosis and treatment than is currently possible for the best outcomes."}
+{"text": "Background: Apolipoprotein A5 (ApoA5), an important modulator of plasma and hepatic triglyceride metabolism, has been found to be downregulated by metformin to improve non-alcoholic fatty liver disease. Meanwhile, exercise has been recommended as a therapeutic strategy for non-alcoholic steatohepatitis (NASH). However, no study has yet determined whether exercise affects hepatic ApoA5 expression or the inhibition of ApoA5 to toll-like receptor 4 (TLR4). We herein examined the effects of exercise on hepatic ApoA5 expression and the relevance of ApoA5 and TLR4-mediated pathway in mice with high-fat diet (HFD)-induced NASH.Methods: Male C57BL/6J mice were built NASH model with high-fat diet for 12 weeks, and following mice were subjected to exercise for 12 weeks on a treadmill. Microscopy and enzyme-linked immunosorbent assay were used to measure histological analysis of liver and hepatic lipids, respectively. Quantitative real-time PCR and western blot were used to determined mRNA and protein levels of ApoA5 and TLR4-mediated nuclear factor kappa B (NF-\u03baB) pathway components, respectively. ApoA5 overexpression plasmids transfected into mice to investigate the relevance of ApoA5 and TLR4.Results: 12 weeks of exercise remarkably alleviated HFD-induced hepatic lipid accumulation, inflammation, and fibrosis, as well as reduced serum lipopolysaccharide (LPS), hepatic TLR4, myeloid differentiation factor 88 (MyD88), and NF-\u03baBp65 expression. Importantly, exercise did not reduce ApoA5 expression but instead enhanced its ability to suppress TLR4-mediated NF-\u03baB pathway components by decreasing circulating LPS in our experiments involving transfection of ApoA5 overexpression plasmids and LPS interventions.Conclusion: The results demonstrated that exercise improved HFD-induced NASH by triggering the inhibitory effects of ApoA5 on the TLR4-mediated NF-\u03baB pathway. Non-alcoholic steatohepatitis (NASH) is a severe manifestation of non-alcoholic fatty liver disease (NAFLD) and has been considered the main cause of liver failure, cirrhosis, and cancer . The lipAPOA5 transgenic mice and hepatoma cells transfected with ApoA5 expression plasmids exhibited increased hepatic TG accumulation. Furthermore, recent data have indicated that hepatic ApoA5 mRNA and protein is overexpressed in patients and mice with NAFLD. The hepatic steatosis and other phenotypes of NAFLD may be alleviate with decrease of ApoA5 mRNA expression or with down-regulation of ApoA5 involving signaling pathway (APOA5 (\u2212/\u2212) mice fed high fat diet manifest greater hepatic steatosis, and ApoA5 overexpression prevented ectopic lipid accumulation rather than increasing it. These findings implicated that ApoA5 may be as a potential therapeutic target for NASH, whereas the mechanisms need to be clarified. Additionally, ApoA5 acts as a predictor for remnant liver growth after preoperative portal vein embolization and liver surgery (Apolipoprotein A5 (ApoA5), a member of the apolipoprotein family specifically expressed in the liver , has bee pathway . But the surgery . Evidenc surgery . However surgery .Lipopolysaccharide is part of the outer membranes of gram-negative bacteria, with its circulating concentrations significantly increasing in mice with high-fat diet (HFD)-induced NASH . Howevern = 24), a high-fat diet group , and a high-fat diet plus exercise group . The LFD group received a low-fat diet containing 10% kcal from fat , whereas the HFD and HFD + EXE groups were fed a HFD containing 60% kcal from fat  for 24 weeks, and after 12 weeks of HFD feeding, mice in HFD + EXE group were subjected to exercise training for 12 weeks. A day after the final training session, mice were killed under anesthesia  for collection of serum and liver.Male C57BL/6J mice aged 6 weeks were purchased from the Experimental Animal Center of Guangdong Province  and acclimated for 1 week. The mice were housed on a 12-h light\u2013dark cycle at 22\u201324\u00b0C and were provided free access to food and water. All animal care and lab experimental procedures were conducted in accordance with the Chinese Guidelines for Animal Welfare and Experimental Protocols and were approved by the Animal Experiment Administration Committee of Guangzhou Sport University (2020DWLL-005). All mice were randomly divided into three groups: low-fat diet control group  , and 5 mHepatic TG and TC levels were measured using commercial kits , according to the manufacturer\u2019s instructions.Fresh liver tissues were fixed with 4% paraformaldehyde solution for 24 h, embedded in paraffin, and sliced into 4-\u03bcm sections for hematoxylin-eosin (H&E) staining and Sirius Red staining. The NAFLD activity score (NAS) was calculated according to the guidance provided by the Pathology Committee of the NASH Clinical Research Network : steatosSerum lipopolysaccharide (LPS) levels was measured using ELISA kits (CUSABIO Technology LLC.), according to the manufacturer\u2019s instructions.n = 4/group) and injected with an ApoA5 overexpression plasmid , negative control empty vector , and normal saline through the tail vein, respectively. The mice were killed, after which serum and liver samples were harvested and stored for analysis after treatment for 3 days. The ApoA5 overexpression plasmid (pEGF-N1-ApoA5) and negative control empty vector (pEGF-N1 vector) were designed and purchased from Heyuan Biotechnology .To analyze the effects of ApoA5 on TLR4-mediated signaling pathway, LFD mice were randomly divided into three groups (n = 4/group) that subsequently received intraperitoneal injections of normal saline, 5 \u03bcg/kg\u22c5wt LPS and 10 \u03bcg/kg\u22c5wt LPS, respectively. After the mice were killed, serum and liver samples were harvested and stored for analysis after treatment for 12 h. LPS  was purchased from Sigma .To investigate the ability of ApoA5 to inhibit TLR4-mediated NF-\u03baB pathway within a certain LPS concentration, HFD + EXE mice were randomly divided into three groups . Equal amounts of total protein were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. These membranes were then blocked and incubated with primary antibodies against ApoA5 , TLR4, MyD88, NF-\u03baBp65, and \u03b2-actin . Membranes were incubated for 1 h with the following secondary antibodies: goat anti-mouse IgG-HRP and mouse anti-rabbit IgG-HRP . Signal detection was performed using SuperSignal Dura Substrate , after which immunoblot signals were quantified using Quantity One software.P value of \u22640.05 indicating statistical significance.All data were expressed as mean \u00b1 standard error of the mean. Statistical significance was evaluated using one-way analysis of variance with the Bonferroni test for multiple comparisons. All analyses were performed using GraphPad Prism 5.0, with a Non-alcoholic steatohepatitis is characterized by hepatic steatosis, inflammation, and fibrosis . Mice wiCompared to the LFD group, the HFD group exhibited significantly higher circulating LPS concentrations (609.42 \u00b1 42.21 vs. 71.11 \u00b1 14.15 ng/mL), which sharply declined after exercise (230.88 \u00b1 35.03 vs. 609.42 \u00b1 42.21 ng/mL) . AlthougTo investigate whether ApoA5 could inhibit the TLR4-mediated signaling pathway, we assessed the expression of TLR4, MyD88, and NF-\u03baBp65 after transfection with the ApoA5 overexpression plasmid (pEGF-N1-ApoA5) in LFD mice. Accordingly, LFD mice transfected with pEGF-N1-ApoA5 demonstrated remarkably higher mRNA and protein expression of ApoA5 and distinctly lower mRNA and protein expression of TLR4, MyD88, and NF-\u03baBp65 compared to untransfected mice , indicatHowever, the inhibitory effects of ApoA5 on TLR4 were limited by circulating LPS concentrations. HFD + EXE mice had lower LPS concentrations, which were obviously enhanced after injection of 5 and 10 \u03bcg/kg\u22c5wt of LPS, respectively . HFD + Ede novo synthesis of free fatty acids, all of which have an effect on NASH .YY and LZ performed study concept and design. YY, LY, XL, and CF performed development of methodology and writing, review, and revision of the manuscript. YY, LY, and SL provided acquisition, analysis and interpretation of data, and statistical analysis. LY provided technical and material support. NC performed supplementary experiments and data analysis. All authors read and approved the final 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.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": "This Special Issue is dedicated toward the understanding of the physicochemical properties and structure changes of food products during processing. Processing food is necessary to not only extend product shelf life but also lead to changes in the physicochemical properties and structure of foods, which can be desirable or undesirable. Processing operations include dehydration, thermal treatment, encapsulation, and extrusion. The physicochemical properties of food are mainly responsible for the final quality of the product. Moreover, the measurement of these properties is important for design and quality control during the processing of the food.The physicochemical and structural changes in food during processing depend on the type of food being processed (solid or fluid) or its constituents. There are numerous physicochemical properties of food, for example, hydration properties , rheological fluid behavior, mechanical properties, optical properties , and thermal properties. However, each type of food needs to be characterized by studying certain specific physicochemical properties. The choice of the most appropriate physicochemical properties in each case is very important in food research.Physical and chemical changes in each constituent and ingredient result from processing operations and often lead to physical, sensory, and nutritional changes in food, and therefore, in the quality.In the present article collection, there are 14 articles of high scientific value. Several of them have evaluated the extrusion process to obtain new snacks or ingredients ,3; fermeLactobacillus spicheri DSM 15429 strain on textural, volatile profile, and sensorial properties of gluten-free muffins in order to obtain baked goods with improved quality characteristics. Lactobacillus spicheri is a novel strain isolated from industrial rice sourdough but unexploited for bakery products manufacturing. The results showed that Lactobacillus spicheri DSM 15429 was able to grow in the rice flour, influencing the texture and the volatile profile of gluten-free muffins as well as their sensory characteristics. Both textural parameters and volatiles recorded significant differences compared to muffins obtained with a spontaneously fermented rice sourdough. The hardness and cohesiveness decreased, while the springiness and resilience of gluten-free muffins improved their values. The volatile profile of gluten-free muffins was significantly improved by utilization of the rice sourdough fermented with Lactobacilus spicheri DSM 15429. 3-methylbutanal, 2-methylbutanal, acetophenone, and limonene were the main volatile derivatives responsible for the aroma and odor scores of the sensory analysis.Gluten-free products available on the market have a low textural quality associated with the high crumbly structure, low flavor, aroma, poor mouthfeel, lesser appearance in comparison with the conventional final baked products. The aim of Chis et al.\u2019s work  was to ap < 0.05). Additionally, Kaleda et al. [The interest in plant-based products is growing in Western countries, mostly due to health and environmental issues that arise from the consumption and production of animal-based food products. Many vegan products today are made from soy, but the drawbacks include the challenges of cultivating soy in colder climates, such as northern Europe. Therefore, the study of Zahari et al.  investiga et al.  studied Espert et al.  investigThe physico-chemical and microstructural changes of \u201cRojo Brillante\u201d persimmons in two maturity stages were evaluated during air drying in Vilhena et al.\u2019s article . AuthorsThe role of roasting in cold brew coffee chemistry is poorly understood. The brewing temperature influences the extraction processes and may have varying effects across the roast spectrum. To understand the relationship between brew temperature and roast temperature, the group of Rao prepared hot and cold brew coffees from Arabica Colombian coffee beans roasted to light, medium, and dark levels .The study of O\u2019Donoghue et al.  studied The Special Issue also contains interesting works, such as the article published by Gaspare et al. , which sSchmid et al.  analyzedThe work of Arilla et al.  aimed toFinally, the rheological properties of twelve different licorice root extracts were evaluated using a rotational viscometer as a function of soluble solids content (15\u201345 \u00b0Bx) and temperature (30\u201370 \u00b0C) by Nasiri et al. .Concluding the presentation of this Special Issue, we would like to thank the abovementioned research teams for their contributions to the present article collection, which provide excellent examples of the multidisciplinary character of the research on physicochemical properties and structure changes of food products during processing."}
+{"text": "DUSP6, MDM2, and EIF2S3 were consistently selected as CRC-associated factors with high significance in all logistic models. CPEB4 became an insignificant factor only when combined with the clusters for cell cycle processes and for transcription. The CPEB4/DUSP6 complex was a prerequisite for the significance of MMD, whereas EXT2, RNF4, ZNF264, WEE1, and MCM4 were affected by more than two clusters. Intricate networks among MMD, RAS signaling factors , and translation factors  were also revealed. Our results suggest that limited G1/S transition, uncontrolled DNA replication, and the cap-independent initiation of translation may be dominant and concurrent scenarios in circulating tumor cells derived from colorectal cancer. This gene-function-based cluster approach is simple and useful for revealing intricate CRC-associated gene expression networks. These findings may provide clues to the metastatic mechanisms of circulating tumor cells in patients with colorectal cancer.Colorectal cancer (CRC) is a complex disease characterized by dynamically deregulated gene expression and crosstalk between signaling pathways. In this study, a new approach based on gene-function-based clusters was introduced to explore the CRC-associated networks of gene expression. Each cluster contained genes involved in coordinated regulatory activity, such as RAS signaling, the cell cycle process, transcription, or translation. A retrospective case\u2013control study was conducted with the inclusion of 119 patients with histologically confirmed colorectal cancer and 308 controls. The quantitative expression data of 15 genes were obtained from the peripheral blood samples of all participants to investigate cluster\u2013gene and gene\u2013gene interactions.  Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer-related deaths worldwide, with an estimated 1,880,725 new cases and 915,880 deaths in 2020 [Escherichia coli, yeast, and mammalian cells [Cancer cells can disseminate from the primary tumor in both the early and late stages of the disease. These rare circulating tumor cells (CTCs) are thought to be highly correlated with distal metastasis, recurrence, and poor clinical outcomes ,4. Whilean cells . In addian cells . Althougan cells ,17, littIn this study, we introduced the concept of a gene-function-based cluster, which is defined as a group of genes with coordinated biological functions or subcellular locations. The expression data of 15 genes obtained from CTC-containing blood samples were used, as viable CTCs seem to be appropriate for studying the cellular mechanisms of metastasis ,6. BasedWe used a retrospective case\u2013control study to identify the interactions between colorectal cancer-associated genes with the inclusion of 119 cases and 308 non-cancer controls. One hundred and nineteen patients with histologically confirmed colorectal cancer (CRC) were enrolled (2006\u20132009) in a prospective investigational protocol, which was approved by the Institutional Review Board at Cheng Hsin General Hospital .The non-cancer control group included 308 volunteers who visited our institution for routine health examinations between November 2005 and November 2010. There was no evidence of any clinically detectable cancer diseases at the time of the blood sample collection. The follow-up period of the controls ranged from 4.8 to 9.9 years. Twenty-six controls (8.4%) were censored, and the health statuses of 282 controls were followed up as of September 2015. Of 282 control subjects, 9 (3.2%) were diagnosed with cancer during the follow-up period. The cancer types of these controls included bladder cancer (1), breast cancer (2), ovarian cancer (1), hepatoma (1), urothelial cell carcinoma of the renal pelvis (1), B-cell lymphoma over the bilateral adrenal gland (1), B-cell lymphoma of the stomach (1), and prostate cancer (1).In addition, among the participants, 111 patients and 227 controls were the same as those used in a previous study by Huang et al. [TM , and TaqMan Master Mix  were used for analysis [Blood samples (6\u20138 mL) were collected for the isolation of the mononuclear cell fraction containing tumor cells, followed by total RNA extraction and cDNA synthesis. The relative expression levels (mRNA) of 15 investigated genes in isolated cells of the study sample were measured using quantitative real-time PCR according to a previous report . Pre-desanalysis ,11.\u00ae 9.4 Language Reference: Concepts, 6th Ed., SAS Institute, Cary, NC, USA).All statistical analyses were performed using SAS  with the Logistic procedure  and status (case or control) while controlling for sex and age.jth sample. Then, the derived estimate is defined as Because the number of cases (N = 119) was smaller than that of the controls (N = 308), we randomly selected 500 subsamples from the controls with a sample size of 119 to represent heterogeneous populations. A logistic regression model was built for each subsample along with the cases. The derived estimates of the coefficients were summarized as the average of the estimates of the coefficients for 500 samples. Let 2 and bse2.The odds ratios (ORs) and corresponding confidence intervals (CIs) were estimated by exponentiating the derived estimates and the corresponding confidence intervals of the coefficients. The derived standard error (SE) of the derived estimates for the CI includes two parts. The first part is the average of the standard error (wse) of the estimates of the coefficients for the 500 subsamples. The second part is the variation between the samples and is given by the standard deviation (bse) of the estimates of the coefficients between 500 subsamples. The final SE is equal to the square root of the sum of wseA hierarchical analysis procedure was designed to investigate the possible interactions between genes and gene clusters . There wIn STEP-1, single-gene modeling was conducted for each investigated gene, and the PS and OR at ground status were obtained.MMD because its coding protein is involved in the dynamics of lysosomal membranes. The TLA cluster had three translation factors, EIF2S3, EXT2, and CPEB4. Four factors associated with the regulation of the cell cycle process, MCM4, MDM2, WEE1, and POLDIP2, were grouped into the CY cluster. The TRf cluster contained two general transcription factors, ZNF264 and RNF4, while the TRm cluster had two factors for the regulation of immune-associated transcription, IRF4 and STAT2. Finally, the SN cluster included three genes involved in RAS signaling, GRB2, NF1, and DUSP6.In STEP-2, (1) 15 investigated genes were grouped into 6 clusters, named LY, TLA, CY, TRf, TRm, and SN, respectively, according to the biological function or subcellular location of each gene as follows: The LY cluster contained (2) Six single-cluster models were constructed to obtain basal cluster-PS (B-PS) and cluster-OR (B-OR) for each cluster-grouped gene, which is written as \u201cgene name/cluster name\u201d.In STEP-3, a series of multiple-cluster analyses was performed by constructing two-, three-, and four-cluster models. For each model, a primary cluster  and one cluster listed in square brackets () were included in the analysis. Each multiple-cluster model was constructed with the sequential inclusion of clusters in the following order: LY, TLA, CY, TRf, TRm, and SN. For example, the LY/TLA model (model ID: M15) consists of the primary cluster, LY, and the first cluster (TLA) listed in square brackets. Five primary cluster sets were used for the three-cluster models: LY/SN, TLA/SN, LY/TLA, LY/CY, and TLA/CY. Three primary cluster sets were used to construct the four-cluster models: LY/TLA/SN, LY/CY/SN, and TLA/CY/SN.In STEP-4, gene\u2013gene analysis was performed by constructing two- and three-gene models through the inclusion of two and three genes, respectively, which were grouped in the LY, TLA, and SN clusters.https://string-db.org/cgi/input?sessionId=bVqn1xBuFseC&input_page_active_form=single_identifier (accessed on 13 December 2022) and https://string-db.org/cgi/input?sessionId=bVqn1xBuFseC&input_page_active_form=multiple_sequences, respectively (accessed on 17 December 2022).STRING  Database version 11.5 was used to analyze protein\u2013protein interaction networks. Two options for the \u201cSearch\u201d domain, \u201cSingle Protein by Name/Identifier\u201d and \u201cMultiple Proteins by Names/Identifiers\u201d, were input with the names of the 15 investigated genes, using the links p = 0.039) between CRC cases and non-cancer controls using the chi-square test, but not in age (p = 0.644) or smoking status (p = 0.157). In addition, there were more female smokers (17.64%) than male smokers (2.52%) in the case group, of whom 12 did not have corresponding information.This retrospective case\u2013control study was conducted using blood samples from 119 patients with colorectal cancer (CRC) and 308 non-cancer controls. The characteristics of the study sample are presented in Female smokers in the 36\u201365 age subgroup accounted for approximately 60% of the total female smokers in the case group. Smoking status was not included as a confounding factor in the logistic models because of two considerations: (1) Smoking status was not significantly different between case and control groups. (2) Detailed measurements of cigarette smoking, especially pack-years, were not collected, which might have biased the estimates.The clinico-pathological characteristics of CRC cases are listed in A single-gene model using logistic regression was implemented for each investigated gene to obtain the percent of significance (PS) and odds ratio (OR) for the ground status  in the single-cluster analysis, whereas STAT2 and GRB2 lost their significance upon their inclusion in TRm and SN clusters, respectively.The fifteen investigated genes were grouped into six clusters, named LY, TLA, CY, TRf, TRm, and SN, respectively, according to their biological functions or subcellular locations, as mentioned in the section on the analysis procedure . The greMultiple-cluster analyses were sequentially performed to identify cluster\u2013gene interactions by constructing two-, three, and four-cluster models , STEP-3.EIF2S3/TLA and EXT2/TLA, and three risk factors (OR > 1), CPEB4/TLA, MDM2/CY, and DUSP6/SN (Fifteen two-cluster models were conducted. Five cluster-grouped genes were represented with consistently high significance (PS > 50), and no or negligible effects through inclusion of the second cluster were observed. There were two protective factors (OR < 1), DUSP6/SN .In total, 18 cluster\u2013gene effects were considered valid interactions according to changes in the PS of cluster-grouped genes under any of the following conditions : (a) theEXT2/TLA was positively increased by four other single clusters, namely, CY, TRf, TRm, and SN clusters. CPEB4/TLA was influenced by the CY cluster because of its reduced PS. Furthermore, the PS values of two genes grouped in the CY cluster, MCM4 and WEE1, were negatively affected by the TLA or SN clusters. MCM4/CY lost its significance when controlling for the TLA cluster, while WEE1/CY became an insignificant factor because of the strong negative effect of both TLA and SN clusters. Additionally, ZNF264/TRf was negatively affected by four other clusters, with the CY cluster exhibiting the strongest influence. RNF4/TRf became a significant factor through an increase in PS when controlling for CY, TRm, or SN clusters. Furthermore, the TRf cluster had a moderate positive effect on the PS of STAT2/TRm. Finally, the interaction between the CY cluster and NF1/SN was observed.The PS of EIF2S3/TLA, EXT2/TLA, MDM2/CY, and DUSP6/SN were commonly represented as CRC-associated with high significance (PS = 77.6\u2013100). High expression of MDM2/CY and DUSP6/SN was significantly associated with a higher risk of colorectal cancer with OR ranges of 5.95\u201310.62 and 2.14\u20133.82, respectively. EIF2S3/TLA and EXT2/TLA were protective factors with OR ranges of 0.17\u20130.35 and 0.11\u20130.44, respectively , except for the presence of the CY/TRf combination . With respect to EXT2/TLA, the positive effect derived from the CY cluster  or TRf cluster  disappeared in the TLA/CY/TRf model .First, the CY/TRf combination had an intrinsic influence on two genes grouped in the TLA cluster: MMD/LY , LY/TLA/SN set for STAT2/TRm (M21), and LY/TLA set for GRB2/SN  in the CY cluster, the findings of the TLA-derived negative effect on their PS were mostly similar to those obtained from the two-cluster analysis. MCM4/CY and WEE1/CY lost significance (PS < 50) upon the inclusion of the TLA cluster in the three- and four-cluster models ; however, the LY/TLA/SN set suppressed the PS of ZNF264/TRf (M20). In addition, the TLA cluster eliminated the SN-derived positive influence on the PS of RNF4/TRf (M6 vs. M11).Fifth, MMD, the only gene in the LY cluster, became a significant factor for LY/TLA/SN-containing models. According to this finding, we constructed 11 two-gene models and 15 three-gene models  to 99.8 (M59), 79.8 (M58), and 52.4 (M33), respectively. Moreover, the PS of NF1 was positively affected by EIF2S3 (M53) or CPEB4 (M48), but not by the EIF2S3/CPEB4 combination (M49). However, this EIF2S3\u2013NF1 interaction was impeded by the addition of MMD (M45 versus M53). In addition, the significance of EXT2 was increased in the presence of GRB2 (M56), DUSP6 (M62), CPEB4/DUSP6 (M65), CPEB4/GRB2 (M59), or CPEB4/NF1 (M50). Finally, MMD was identified as a significant CRC-associated factor in the MMD/CPEB4/DUSP6 model (M36).The complex interactions between seven genes are schematically presented in The search results for interaction networks of \u201cSingle Protein\u201d based on the STRING database showed that no interactions were the same as those identified in this study . FurtherCancer cells are characterized by the deregulation of cell signaling ,17, whicDUSP6 and the upstream regulator GRB2 of RAS/ERK signaling [WEE1 [MCM4, MDM2, and POLDIP2. Furthermore, EIF2S3 and CPEB4 together had noticeable effects on EXT2 using the TLA model. In addition, the existence of the IRF4\u2013STAT2 interaction is revealed by the TRm model, and this agrees with the search results obtained from the STRING database. Based on the current knowledge, IRF4 is involved in STAT3-oncogenic signaling [STAT2 is reported to be associated with STAT1 and interferon regulatory factor 9 (IRF9) to form IFN-stimulated gene factor 3 (ISGF3) [Single-cluster models uncover interactions between genes involved in different steps of coordinated cellular processes: The SN model reveals interactions between the downstream ERK1/2 inactivator ignaling ,20, wherng [WEE1  is stronignaling , whereas (ISGF3) .EXT2/TLA is positively affected by four different clusters, EXT2 presumably plays a central role in the invasive character of colorectal cancer with respect to extracellular matrix (ECM) assembly [ZNF264/TRf was suppressed by four different clusters, whereas the CY cluster exerted the strongest effect. This finding is concordant with a report on the involvement of ZNF264-based transcriptional activity during the cell cycle process, especially replication stress and genomic instability in cancer [RNF4/TRf by the CY cluster may indicate that RNF4 likely participates in the DNA damage response and the maintenance of chromosomal integrity during the cell cycle through the SUMO-targeted ubiquitin ligase (STUbL) pathway [RNF4/TRf is acceptable, since the impactful factor ERK1/2 inactivator DUSP6/SN [MCM4/CY and WEE1/CY, in which strong and intricate modulations derived from SN and TLA clusters were observed. It is presumed that factors involved in RAS signaling and translation closely coordinate or counteract each other to regulate the cell cycle process.The complexities of cluster\u2013gene interactions indicate gene regulations across different pathways in circulating tumor cells. First, as assembly ,25,26. Sn cancer . Thirdly pathway . Based oDUSP6/SN ,20 may dGRB2/SN only represents a significant factor for the coexistence of the LY/TLA combination but without the CY cluster. In contrast, the CY cluster was crucial for the significance of NF1/SN. Thus, NF1 might be required for spindle organization and chromosome segregation in circulating tumor-derived cells, as in neuron cells [ZNF264/TRf was partially reversed in the TLA/SN combination model, whereas the TLA-derived effect was enhanced by the addition of the LY cluster. Moreover, the SN-derived positive effect on the significance of RNF4/TRf was completely suppressed by the TLA/SN combination. Finally, our results suggest that CPEB4 may be involved not only in the translational regulation of mitosis as reported [CPEB4/TLA. Furthermore, CPEB4/TLA unexpectedly became an insignificant factor in the presence of the CY/TRf combination.Interaction networks between LY, TLA, and SN clusters are supposedly associated with the regulation of the cell cycle process. For instance, on cells , rather on cells . In addireported  but alsoMMD and factors involved in RAS signaling and translation. The presence of the CPEB4/DUSP6 combination is a prerequisite for the significance of MMD, which supports the reported five-gene model for colorectal cancer [MMD expression may indicate a low amount of active ERK1/2 if MMD is regulated by LPS stimulation in macrophages, as reported by Liu et al. [MMD expression and DUSP6 overexpression significantly impedes the transduction of mitogenic signals. It is presumed that some circulating tumor cells are likely to exhibit reversible G0-G1 arrest and have high metastatic potential, as reported for dormant cancer cells in the circulation [GRB2 and DUSP6, may be involved in the invasiveness of circulating tumor cells through intricate interactions with the suppressor gene EXT2. Finally, we verified the presumption that translation factors may participate in the regulation of cell proliferation via the MAPK/ERK signaling pathway, since EIF2S3 and CPEB4 showed strong but opposite effects on NF1. Our results for EIF2S3 expression in colorectal cancer-derived cells are in accordance with findings in acute myeloid leukemia [Two-gene and three-gene models confirmed intricate interactions between l cancer ,8. Loweru et al. . Based oculation . Moreoveculation  and chemculation ,35,36. Ileukemia .DUSP6, MDM2, and EIF2S3 were consistently selected as significant factors associated with colorectal cancer in all logistic models and were not modulated by any other genes or clusters. These results suggest that limited G1/S transition, uncontrolled DNA replication, and the cap-independent initiation of translation may be dominant and concurrent scenarios in circulating tumor cells from colorectal cancer. The primary strength of this study is the approach based on gene-function-based clusters for the identification of complex interactions between factors involved in mitogenic signaling, cell cycle processes, transcription, and translation in cells with high metastatic potential. Moreover, the cluster\u2013gene interactions identified in this study are novel. Most of the interactions between proteins encoded by the 15 investigated genes have been neither published nor included in the STRING database. In addition, these results provide clues for the varied gene-specific associations with colorectal cancer in univariate and multivariate analyses. Thus, the approach based on gene-function-based clusters is expected to be useful for the identification of rational gene signatures for clinical diagnostic and prognostic utilities, as well as for the validation of drug targets.Overall, Our study had several limitations. First, the findings of cluster\u2013gene and gene\u2013gene interactions might not be associated with colorectal cancer, since patients with other cancer types were not included in the investigation. Second, the subjects in the control group were only confirmed to be cancer-free, without knowing other health conditions, such as inflammatory bowel diseases; thus, interference with the identified interactions in this study could not be excluded. Third, the appropriateness of the grouping of genes is uncertain because the cellular functions of some proteins encoded by the investigated genes are not fully understood. For instance, proteins exert oncogenic activity only after translocation from the cytosol to the nucleus. Furthermore, we lack the knowledge of proteins that may function as double-edged swords. Therefore, the grouping of gene clusters in this study may provide a partial scope for crosstalk between regulatory pathways in cancer cells. Fourth, only age and sex were controlled for in the logistic models, whereas other confounding variables, such as measurements of cigarette smoking, body mass index (BMI), and other lifestyle risk factors, were not fully collected during the inclusion period. Fifth, the small sample size and the proportion of patients with different clinical stages of the disease may have influenced the results.Future research should include different grouping principles of genes to identify more novel interactions. Moreover, how these factors, when located in different subcellular compartments, interact with each other requires further investigation. In addition, the examination of the specificity of cluster\u2013gene and gene\u2013gene interactions for colorectal cancer is required through investigations of other cancer types. Multiple drug targets could be potentially conceived to develop advanced therapeutic agents in precision medicine.It was concluded that combined cluster-based and gene\u2013gene analyses can be used to explore the crosstalk between cellular activities and rationally represent parallel scenarios in colorectal cancer-derived cells. Multiple gene-based signatures can provide a better overview of the characteristics of circulating tumor cells isolated from patients with colorectal cancer and further dynamic and personalized information for prognoses and therapeutic responses."}
+{"text": "COVID-19 infection is less severe among children than among adults; however, some patients require hospitalization and even critical care. Using data from the French national medico-administrative database, we estimated the risk factors for critical care unit (CCU) admissions among pediatric COVID-19 hospitalizations, the number and characteristics of the cases during the successive waves from January 2020 to August 2021 and described death cases.We included all children (age < 18) hospitalized with COVID-19 between January 1st, 2020, and August 31st, 2021. Follow-up was until September 30th, 2021 (discharge or death). Contiguous hospital stays were gathered in \u201ccare sequences.\u201d Four epidemic waves were considered . We excluded asymptomatic COVID-19 cases, post-COVID-19 diseases, and 1-day-long sequences (except death cases). Risk factors for CCU admission were assessed with a univariable and a multivariable logistic regression model in the entire sample and stratified by age, whether younger than 2.We included 7,485 patients, of whom 1988 (26.6%) were admitted to the CCU. Risk factors for admission to the CCU were being younger than 7 days [OR: 3.71 95% CI (2.56\u20135.39)], being between 2 and 9 years old [1.19 (1.00\u20131.41)], pediatric multisystem inflammatory syndrome (PIMS) [7.17 (5.97\u20138.6)] and respiratory forms [1.26 (1.12\u20131.41)], and having at least one underlying condition [2.66 (2.36\u20133.01)]. Among hospitalized children younger than 2 years old, prematurity was a risk factor for CCU admission [1.89 (1.47\u20132.43)]. The CCU admission rate gradually decreased over the waves (from 31.0 to 17.8%). There were 32 (0.4%) deaths, of which the median age was 6 years (IQR: 177 days\u201315.5 years).Some children need to be more particularly protected from a severe evolution: newborns younger than 7 days old, children aged from 2 to 13 years who are more at risk of PIMS forms and patients with at least one underlying medical condition. Two years after the first cases, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to spread around the world with the regular emergence of new variants of concern. Rapid dissemination of the delta variant B.1.617.2) in the summer of 2021 and then of the omicron variant B.1.1.529) in the winter of 2021\u20132022 were associated with an increase in children hospitalizations, which worries the pediatric community in several affected countries .617.2 in. Althoug29 in theEven if morbidity has been much lower for children than for adults, the pediatric population has not been completely spared \u201314. ChilSome studies have already looked at pediatric hospitalizations and risk factors for severity, but none has provided a review of hospitalizations nationwide over a long period and taken into account contiguous hospital stays. Thus, the aim of our national observational study were since the outbreak of COVID-19  to determine the risk factors associated with pediatric critical care unit (CCU) admissions. The secondary aims were to describe the number and characteristics of pediatric hospitalizations in all French hospitals and the clinical characteristics of children who died of COVID-19.Programme de M\u00e9dicalisation des Syst\u00e8mes d\u2019Information\u201d (PMSI) database , with an inscription on the Health Data Hub public register (N\u00b0 F20201117130456). Patients were not involved, as we used pseudonymized discharge data.We included data from all children (age < 18) admitted to French hospitals with COVID-19 between January 1st, 2020, and August 31st, 2021. Patients were followed up until discharge or death until September 30th, 2021. Hospital stays for COVID-19 were identified according to national guidelines . Acute Csoins intensifs\u201d), and step-down units . The secondary outcome was in-hospital death.The primary outcome was the requirement for hospitalization in CCUs. The CCUs included intensive care units, intermediate care units , and vasoactive drugs were defined based on the act coded using the Common French Classification of Medical Acts .The ICD-10 codes used to specify underlying chronic conditions were defined by a team of physicians experienced in medical information and a pediatrician and are listed in For continuous variables, data were described by their median and interquartile ranges [IQRs]. Categorical variables were described as numbers of patients and percentages. Age was also divided into age groups: 0\u20137 days, 8\u201389 days, 90 days-1 year, 2\u20139 years, 10\u201313 years, and 14\u201317 years. For global chronological description, the time unit used was the month of the sequence\u2019s starting date. Four periods, corresponding to 4 different epidemic waves, were considered from January 1st to August 11th 2020, from August 12th 2020 to January 1st 2021, from January 2nd to July 4th 2021 and from July 5th to August 31st 2021.Qualitative variables were compared between groups using Chi-square tests, and quantitative variables were compared using ANOVA. Risk factors for CCU admission were assessed with a univariable and a multivariable logistic regression model in the entire sample and stratified by age, whether younger than 2. Multivariable models included all the variables with at least 10 patients per modality.P-values lower than 0.05 were considered statistically significant.The analyses were performed on the secure platform of the ATIH. Data extraction and preparation were carried out on January 11th 2022 with SAS Enterprise Guide version 8.3. We identified 11,414 patients younger than 18 years who were hospitalized with SARS-CoV-2 infection in France between March 2020 and August 2021. We excluded 1,848 (16.2%) patients who were discharged alive after a length of stay less than 1 day, 1,972 (17.3%) patients with asymptomatic infections and 109 (1.0%) hospitalizations related to post-COVID symptoms. The characteristics of the excluded patients are given in n = 2,392) ; 32.0% were under 3 months old  . Most ch= 2,392) . No unden = 3,294). The PIMS rate was 10.8% (n = 806) for all admitted children, with a maximum rate of 44.0% for the 7-year-old children (n = 453) of all PIMS. The proportion of PIMS and respiratory forms of COVID-19 hospitalized by age is shown in Respiratory forms represented 44.0% of hospitalizations  children required ventilatory support . Seven cAs prematurity was assessed only for children younger than 2 years old, the analysis of risk factors for CCU admission was stratified according to the age of children (<> 2 years old). Among hospitalized children younger than 2 years old, most underlying conditions identified were significantly associated with CCU admissions except for Down syndrome, chronic lung disease excluding asthma, and neurologic and metabolic disease . PrematuAmong those older than 2 years old, metabolic disease [2.97 (2.15\u20134.08)], sickle cell disease [2.70 (1.97\u20133.70)], asthma [2.10 (1.64\u20132.69)], cardiovascular disease [2.10 (1.45\u20133.03)], obesity [2.06 (1.36\u20133.13)] and neurologic disease [1.84 (1.40\u20132.43)] significantly increased the risk of CCU admission . In thisn = 19, 59.4%), followed by chronic lung disease , metabolic disease  and immune deficiency . Five of those who died had a cardiovascular disease (15.6%), and one was obese (3.1%). None had Down syndrome, sickle cell disease, or a chronic renal or digestive disease. Among the children younger than 2 who died (n = 13), 5 were premature. Regarding the distribution over time, 14 deaths occurred during the first wave (until August 2020), 5 during the second wave, 7 during the third wave and 6 during the fourth wave.Among the children in the study, 32 (0.4%) died during their hospital stay . The medp < 0.001).Symptomatic COVID-19 admissions among children in French hospitals peaked in March 2020 (first wave), October 2020 (second wave), April 2021 (third wave) and August 2021 (fourth wave), with 478, 648, 713, and 923 monthly admissions, respectively . The perThe national PMSI database enabled the exhaustive analysis of 7,485 pediatric hospitalizations for symptomatic SARS-CoV-2 infection from January 2020 to September 2021 in France. This study, gathering the largest number of symptomatic children hospitalized over the longest period ever assessed, strengthens knowledge of pediatric COVID-19 and highlights risk factors for severe disease. More than one-fourth of these children required admission to the CCU, and risk factors identified by multivariate analyses were (i) being younger than 7 days old or from 2 to 9 years old and (ii) PIMS or \u201crespiratory form.\u201d Most underlying conditions were significantly associated with CCU admissions. Unlike adults, Down syndrome and diabetes were not significantly associated (The rate of admissions to the CCU (26.6%) was in the upper range of those reported in similar pediatric studies between 4.1 and 30.1% , 13, 22.The analysis of CCU admission revealed an increased risk for patients younger than 7 days old. The fragility of this age group is presumably partly linked to particular neonatal contexts  . Indeed,The analysis of underlying respiratory conditions did not reveal any excess risk of CCU associated with chronic respiratory diseases other than asthma, and the involvement of these comorbidities in severe forms of infection is not yet clearly documented for all of them . Among pIn pediatrics, the youngest children, including new-borns and those with comorbidities, are the most vulnerable to respiratory infections , 32, andA limitation of the PMSI national database is the possible variation in the quality of coding. We may be confident in the quality of the collected data because national COVID-19 coding guidelines have been published shortly after the beginning of the pandemic. Moreover, strict national rules with regular checks carried out by the payer may limit the risk of coding errors. The PMSI database does have many advantages, such as being exhaustive and national, including all the data from public and private hospitals. In addition, this study allowed us to gather continuous stays within different hospitals so that the risk of overestimating the number of hospitalizations is limited. We included all patients presenting any symptoms of COVID-19 regardless of the severity during their stay if an ICD-10 code was present in their records. If the patients became symptomatic after admission, then an ICD-10 code of symptomatic COVID-19 was present in the file and the patient was considered.In conclusion, the SARS-CoV-2 pandemic among children is not comparable to the adults\u2019 in terms of hospitalization and mortality rates. Nevertheless, childhood morbidity is not negligible, as evidenced by the number of hospitalizations for symptomatic forms in France, the high rate of CCU admission and the number of deaths. We are able to confirm that some children are particularly at risk of evolving toward a severe infection: new-borns younger than 7 days old, children aged from 2 to 13 years who are more at risk of PIMS, and patients with at least one underlying medical condition. Repercussions on the child\u2019s overall health are also a constant concern of pediatricians and should of course guide the long-term management of the pandemic.support@atih.sante.fr. Aggregated data can be provided upon request to the authors.Raw data are available upon request to the Agence technique de l\u2019information sur l\u2019hospitalisation (ATIH): Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants\u2019 legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.BP, AR, FB-F, PT, and ST involved in the methodology, formal analysis, investigation, data curation, writing the original draft, reviewing and editing the manuscript, designing of tables and graphs, and verified the underlying data. ABo, ABr, HC, VG, ML, XL, JM, EO, and FS involved in data provision and reviewing and editing the manuscript. All authors had full access to all data in the study and accept responsibility for the decision to submit for publication."}
+{"text": "Equus caballus Papillomavirus Type9 (EcPV9) was thus far only reported in the semen of a stallion with penile lesions in Australia. This study reports for the first time the presence of EcPV9 in asymptomatic Italian horses. From July 2020 to January 2022, genital brush samples were collected from 209 horses with no apparent signs of neoplastic disease and no PV-associated lesions, clinically examined at the Didactic Veterinary University Hospital (OVUD) of Perugia and at the Veterinary University Hospital (OVU) of Turin. Brushes were submitted to real-time PCR targeting the EcPV9-L1 region. The first amplification targeted a region of ~116 bp, followed by the amplification and sequencing of ~533 bp of the positive samples. EcPV9-L1 DNA was found in eleven horses (5.3%), all female and mainly English Thoroughbred. Co-infection with EcPV2-L1 was found in 7 out of the 11 EcPV9-L1 positive horses (63.6%). This study contributes to the description of the prevalence of exposure or infection of EcPVs in the horse population in Italy, for which data are still limited. In this regard, here we provide a phylogenetic analysis and the completely reconstructed viral genomes of two Italian EcPV type 9 isolates, as well as four EcPV type 2 obtained from co-infected animals.Papillomavirus (PV) infections may be related to anogenital lesions and cancer development in humans and several other animal species. To date, 11 different PVs have been reported in horses. Among them, a newly described PV named  Papillomaviruses (PVs) are a group of highly host-adapted, small, non-enveloped DNA viruses. They show a specific tropism for cutaneous and mucosal keratinocytes, and infection is generally thought to require epithelial wounding or micro-wounding to allow access of the virus to the basal lamina ,2,3. In Bos taurus papillomavirus 1 (BPV1), 2 (BPV2), and 13 (BPV13), Equus caballus papillomaviruses 1\u20138 (EcPV1\u20138), Equus asinus papillomaviruses 1\u20132 (EaPV1\u20132) [As regards Equidae, to date, 13 PVs have been documented to infect horses and donkeys: EaPV1\u20132) . In addiEaPV1\u20132) . Equids Several studies provided evidence for an active involvement of PV infection for cancer development also in horses ,21,22. IIn this study, we describe for the first time the presence of EcPV9 in Italian horses, and we provide the complete genome reconstructed from two different animals. Phylogenetic analyses were performed to compare these EcPV9 Italian isolates with the other ones available in the public databases, confirming their relationships with the EcPV9 reported in the semen of a thoroughbred stallion in Australia.From July 2020 to January 2022, genital brush samples were collected from horses during routine clinical examination at the Didactic Veterinary University Hospital (OVUD) of Perugia and at the Veterinary University Hospital (OVU) of Turin for medical reasons unrelated to the study, with the following inclusion criteria: (i) no apparent sign of neoplastic disease; (ii) no PVs associated lesions. No restrictions were set for breed, age and sex.Penile and vulvar swabs were collected through sampling with sterile cytobrushes . Vulvar swabs were taken from the vaginal vestibulum of mares, while penile swabs were obtained from stallions and geldings by gently rubbing the glans mucosa, close to fossa glandis.The brushes were stored in 2 mL tubes containing 800 \u00b5L of DNA/RNA Shield Stabilization Solution , then kept at \u221220 \u00b0C until processing.L1 detection was assessed in 100 ng DNA samples by using primers and related specific probes for the amplification of a target region of ~116 bp . Sequences were aligned using the SeqMan software  to obtain a consensus sequence and compared with available sequences retrieved from the National Center for Biotechnology Information (NCBI) database through the BLAST tool.DNA extraction was carried out using a QIAamp DNA Mini Kit , according to manufacturer\u2019s instruction. For each sample, total DNA was extracted from 200 \u00b5L of DNA/RNA Shield Stabilization Solution  in which cytobrushes were previously kept. Samples were eluted in 100 \u00b5L of elution buffer , and DNA was quantified by Qubit fluorimeter . EcPV9- ~116 bp ; equine  ~116 bp  were useL1 presence was also assessed for EcPV9-L1 positive samples, by following the previously described protocol. Primers and related specific probes used for EcPV2-L1 detection are reported in EcPV2-L1 sequencing in positive samples. The amplification was obtained through Platinum\u2122 Taq DNA Polymerase  using the primer pairs reported in \u00ae Taq DNA Polymerase, 1.25 \u03bcL of 50 mM MgCl2 in a final volume of 25 \u03bcL. Thermocycling parameters consisted of an initial denaturation step  followed by 40 cycles of denaturation , annealing  and extension , amplifications were run on a CFX 9600 . The PCR products were separated on 2% agarose gels, the bands were excised from the gel and purified using the NucleoSpin\u00ae Gel and PCR Clean-up . Purified DNA was used for sequencing of both strands with the Big Dye Terminator v.1.1 cycle sequencing kit . The consensus sequence was determined by the alignment of forward and reverse strand using SeqMan . The consensus sequences were compared by Blast analyses with the only EcPV9 sequence deposited in GenBank. A multiple sequence alignment including the newly generated L1 sequences and sequences from other EcPVs was built with BioEdit v.7.0.5.2 using CLUSTALW [A fragment of ~533 bp was targeted for the EcPV9-CLUSTALW . NucleotWe carried out an NGS-based identification of the EcPV9 genome sequence for the samples where enough DNA was available. In detail, total DNA was extracted from five horse samples listed in Equus caballus genome  was used as reference in a filtering step performed using BWA v 0.7.12 aligner and samtools v1.13 [http://tree.bio.ed.ac.uk/software/figtree, accessed on 22 June 2022).To keep only the non-horse reads, the ls v1.13 ,30. For ls v1.13  was usedls v1.13  for aligls v1.13 . A phylols v1.13 , to buills v1.13 . The treL1 (dependent variable) and 4 classes of age  and 2 of breed (English Thoroughbred vs. the others) (independent variable). Statistical analysis was then restricted to the mares: the OR was assessed through logistic regression using as dependent variables the positivity/negativity to EcPV9-L1, artificial insemination/natural service, pluriparous/maiden, while breed and age were considered as independent variables. Finally, a third logistic regression model was fit to assess the OR between being pregnant vs. being empty (dependent variable) and the positivity/negativity to EcPV9-L1 and fertility/ipofertility (independent variables).According to submission information, horses were classified by age as \u201cyoung\u201d (<8 years) or \u201cadult\u201d (\u22658 years). Breed and origin information was obtained from medical records. Microsoft Excel (2016) software was used for descriptive statistics analysis such as mean \u00b1 1 standard deviation and median calculations of age, male and female, and proportion of positivity. Moreover, STATA16.1  software was used to fit a logistic regression model assessing the association, expressed as odds ratio (OR) between the positivity or negativity to EcPV9-A total of 209 horses  were sampled in this study during clinical examination at OVUD (Perugia) and OVU (Turin). The age ranged from 6 months to 24 years, with a mean of 10.2 (\u00b15.2 SD) years and a median of 10 years. Overall, 31.1% of sampled animals were <8 years old (young) and 65.6% \u22658 years old (adult). The age was not known for seven animals. Moreover, following the four categories of age division, 41 animals were <6 yy (very young), 34 of 6\u2013<9 yy (young), 62 of 9\u2013<13 yy (adult) and 65 of \u226513 yy (elderly). The sampled animals belonged to different breeds: 89 Italian Standard Breed (42.6%), 72 English Thoroughbred (34.4%), 12 Italian Saddle (5.7%), 9 Arabian Thoroughbred (4.3%), 4 Quarter Horse (1.9%), 4 Shire (1.9%), 3 Belgian (1.4%), 2 Hannover (1%), 2 Maremman (1%), 2 Anglo Arabian (1%), 2 ponies (1%), 1 Sardinian Anglo Arabian (0.5%), 1 Appaloosa (0.5%) and 1 Argentine Criollo (0.5%). Moreover, the breed was not known for five horses (2.4%). Sampled animals came from various Italian regions: 128 (61.2%) were from Piedmont, 31 (14.8%) from Umbria, 13 (6.2%) from Lazio, 9 (4.3%) from Tuscany, 8 (3.8%) from Emilia-Romagna, 5 (2.4%) from Marche and Lombardy, 3 (1.4%) from Sardinia, 2 (1%) from Campania, 1 (0.5%) from Sicily and France, while for 3 (1.4%), the geographical origin was unknown.L1 DNA was found in 5.3% (11 out of 209) of the examined horses. These animals were subjected to clinical examination.Overall, EcPV9-L1 DNA, seven of them (63.6%) also showed the presence of EcPV2-L1  compared to group 2 (5.9%) and 4 (1.6%), while no positive subjects were found in group 1 . The ageL1 and artificial insemination vs. natural service. No significant association was found nor between positivity/negativity to EcPV9 and fertility/hypofertility, neither between positivity/negativity to EcPV9 and being pregnant/being empty. Moreover, all positive mares were followed by a veterinarian until March 2022. No signals of PV-related lesions were detected. However, seven animals out nine showed reproductive problems during follow-up  were Italian Trotter, 62 (39.2%) English Thoroughbred, 4 (2.5%) Shire, 2 (1.3%) Arabian Thoroughbred and Italian Saddle. Moreover, it was not possible to know the breed of one horse. Their age ranged between 3 and 21 years, with an average of 10.4 (\u00b14.4 SD) years and a median of 10 years. English Thoroughbred and Shire were subjected to natural service, whereas Italian Trotter, Italian Saddle and Arabian Thoroughbred to artificial insemination. Among the sampled mares, 119 were pluriparous and 28 maiden. It was not possible to obtain this information for 11 mares. As mentioned in ollow-up .L1 sequences of 533 bp were obtained for all eleven positive samples. The quality of the sequences was checked manually using Sequencing Analysis v. 5.2 (Applied Biosystems). The sequences were then used for the alignment of the forward and reverse sequences of each sample in order to obtain a consensus sequence for phylogenetic analysis.EcPV9-All nucleotide sequences obtained were identical to each other and showed a 100% similarity to EcPV9 strain SW (MN117918.1). A 405-nucleotide alignment including the newly generated sequences, other EcPV sequences, and human PV1 as outgroup, was used for phylogenetic analysis. The phylogenetic tree confirmed that sequences identified in this study cluster with EcPV9 within the Dyoiota genus .The total number of raw reads obtained from Illumina sequencing, together with the number of trimmed and filtered reads, is reported in The completely reconstructed sequences, either related of EcPV2 or EcPV9, were further validated by mapping the raw reads with BWA, calculating the breadth and depth coverage with samtools  and visuThe phylogenetic relationship among the newly reconstructed EcPV isolates is in line with their papillomavirus type : as expeL1, phylogenetic analysis and viral genome reconstruction by NGS.This study represents the first investigation on EcPV9 genoprevalence in horses worldwide and contributes to the description of EcPVs epidemiology in Italy, a viral infection for which data are still limited ,37. It aL1 is the most conserved and is thus widely used for PV detection and identification by sequencing. Moreover, after the infection, the viral genome can be integrated into the host DNA or maintained as multiple episomes that replicate concomitantly to the host cells [As concerns the molecular characterization, PV genes are grouped into early (E) and late (L), depending on their expression phase during the infection. Among late PV genes, the st cells . Viral ist cells ,3.In this study, the full genome of two EcPV9 was obtained; thus, we can speculate that in these two animals, the viral genome was not integrated into the host genome. The role of this new virus in EcPV-related lesions and its possible involvement in cancer will need to be further investigated. In humans, to date, over 200 types of HPVs have been characterized. However, the majority are classified as \u201clow-risk\u201d (lrHPV) types, and about 12 types as \u201chigh risk\u201d oncogenic PVs  ; more spConcerning EcPV9, as mentioned, it was thus far only detected in the semen of a Thoroughbred stallion with a penile lesion in Australia . In the The sequences of EcPV9 isolated in this study differ from the Australian sequences only for three SNPs. This suggests higher conservation of this type of EcPV compared to EcPV2 where even isolates from the same country differ among each other. Among HPV types, sequence diversity can be found, where intratype variant lineages differ by 1\u201310%, and sublineages by 0.5\u20131%. Since PVs are double-stranded DNA viruses, they can use their host proofreading DNA polymerase for their replication, thus avoiding high mutation rates. For this reason, mutations in HPV genomes have been acquired slowly, defining HPV types whose infection cycle has adapted to different host cells. The nucleotide polymorphisms of PV intratype variants derives from random mutations occurring within viral types generated .Our findings further prove the broad diversity of PV types in horses and highlight the need for further investigation of the clinical significance of this newly described virus. The clinical impact of the nine EcPVs affecting the equine species is still not well characterized. In addition, there is increasing evidence for the involvement of PVs in the development of canine, ovine and feline SCCs ,56,57,58Our results show that in horses, as in human cases, co-infections may be present and that infections may be asymptomatic . MoreoveThis paper provides for the first time the genoprevalence of EcPV9 in Italian horses. Our results suggest four important conclusions: 1\u2014in horses, as in humans, many infections are asymptomatic and probably resolve spontaneously; 2\u2014Thoroughbred are more susceptible to the infection and show an OR of 26.4 to be positive with respect to other breeds used as a comparison variable; 3\u2014in horses, as in humans, sexual transmission is a plausible model; 4\u2014a very high genetic conservation of EcPV9 compared to other EcPVs was observed."}
+{"text": "Do pulse oximetry discrepancies, hidden hypoxemia, and clinical outcomesdiffer among racial and ethnic subgroups?In this cross-sectional study of 5 databases with 87\u2009971 patients,significant disparities in pulse oximetry accuracy across racial and ethnicsubgroups  were found,with higher rates of hidden hypoxemia associated with mortality, futureorgan dysfunction, and abnormal laboratory test results.In this study, discrepancies in pulse oximetry accuracy among racial andethnic subgroups were associated with higher rates of hidden hypoxemia,mortality, and organ dysfunction. This cross-sectional study examines racial and ethnic discrepancies betweenoxygen saturation measured by pulse oximetry and arterial blood gas and theirassociations with clinical outcomes. o2), when compared with arterial oxygensaturation (Sao2) measured by arterial blood gas (ABG),may differentially affect patients according to race and ethnicity. However,the association of these disparities with health outcomes is unknown.Discrepancies in oxygen saturation measured by pulse oximetry databases  as well as Emory Healthcare (2014-2021)and Grady Memorial (2014-2020) databases, spanning 215 hospitals and 382ICUs. From 141\u2009600 hospital encounters with recorded ABG measurements,87\u2009971 participants with first ABG measurements and anSpo2 Edit 1 <sc                        ></sc>\u226588% but Sao2 <88%).Patients with hidden hypoxemia , clinical outcomes , organ dysfunction by scores , and laboratory values (lactate and creatininelevels) before and 24 hours after the ABG measurement.Outcomes, stratified by race and ethnicity, were Sao2-Sao2 pairs from87\u2009971 patient encounters  were analyzed, with 4859 (5.5%) having hidden hypoxemia. Hiddenhypoxemia was observed in all subgroups with varying incidence  and wasassociated with greater organ dysfunction 24 hours after the ABGmeasurement, as evidenced by higher mean (SE) SOFA scores (7.2 [0.1] vs 6.29[0.02]) and higher in-hospital mortality .Furthermore, patients with hidden hypoxemia had higher mean (SE) lactatelevels before (3.15 [0.09] mg/dL vs 2.66 [0.02] mg/dL) and 24 hours after(2.83 [0.14] mg/dL vs 2.27 [0.02] mg/dL) the ABG test, with less lactateclearance (\u22120.54 [0.12] mg/dL vs \u22120.79 [0.03] mg/dL).The first Spo2 level in patients who self-identified asBlack, followed by Hispanic, Asian, and White. Patients with and withouthidden hypoxemia were demographically and clinically similar at baseline ABGmeasurement by SOFA scores, but those with hidden hypoxemia subsequentlyexperienced higher organ dysfunction scores and higher in-hospitalmortality.In this study, there was greater variability in oxygen saturation levels fora given Sp They described occult hypoxemia asan Sao2 of less than 88% when the Spo2 wasbetween 92% and 96%. In both cohorts, the incidence of hidden hypoxemia was almost 3times higher among patients self-reported as Black vs White.1Recently, reports of systemic racial bias in which oxygen saturation measured bypulse oximeter  only two-thirds of the time.2Pulse oximetry is a useful tool to monitor blood oxygen saturation without obtainingan invasive arterial blood gas (ABG) measurement. The US Food and DrugAdministration (FDA) requires root mean square accuracy within 2% for values between70% and 100%, implying that an adequate pulse oximeter returns anSp7 This study used 5 large EHR data sets ofcritically ill patients to further evaluate the incidence and clinical outcomes ofhidden hypoxemia across racial and ethnic groups.Studies have highlighted the inaccuracy of pulse oximetry in critically ill patients;however, smaller sample sizes hindered in-depth analysis of race, ethnicity, andoutcomes (eTable 1 in the STROBE) reporting guideline.8 Data in Medical Information Mart forIntensive Care III (MIMIC-III), MIMIC-IV, and Electronic Intensive CareUnit\u2013Clinical Research Database (eICU-CRD) had been previously deidentifiedand did not require a waiver for informed consent. The Medical Information Mart forIntensive Care (MIMIC) database is a collaboration between the Beth Israel DeaconessMedical Center and the Laboratory for Computational Physiology at the MassachusettsInstitute of Technology. The database contains granular, deidentified ICU data fromthe Beth Israel Deaconess Medical Center. The data have been generated from morethan 70 intensive care unit beds with medical, surgical, cardiac, and neurologicalpatients. We used the latest data version, MIMIC-III (version 1.4), which containsdeidentified data associated with 53 423 ICU admissions.9 Physionet approved the use of MIMIC forthis study. Emory University approved the use of the Emory and Grady databases forresearch, with a complete HIPAA and informed consent waiver.This study followed the Strengthening the Reporting of Observational Studies inEpidemiology  and MIMIC databases were used. The Sequential Organ FailureAssessment (SOFA) scores  and Grady Memorial Hospital . Emory Healthcare and Grady patient dataspanned 2014 to 2021 and 2014 to 2020, respectively. SOFA scores and itscomponents were not available for the Emory and Grady databases.o2 values were extracted from the EHR.Data analysis was conducted with R version 3.6.3  and Python version 3.6 (Python Software Foundation). All ABG andSpo2 range of 88% to 100% was selected as an intervalin which patients may have hypoxemia but falsely be considered as havingarterial blood oxygenation in the reference range according toSao2. Each ABG-measured Sao2 wasmatched with the closest Spo2 value recorded within theprevious 5 minutes. To eliminate repeated measurements and limitconfounding, only the first ABG measurement from each hospital encounter wasused. Spo2 measurements of less than 88% were notexamined because of low prevalence in the EHR.An SpIn each EHR data set, race and ethnicity were defined using self-reporteddemographic data, including administrative entries with additionalidentifiers. All patients with race and ethnicity information who could notbe classified as Asian, Black, Hispanic, or White were excluded. Patientswere stratified by age, sex, race and ethnicity, and CVSOFA score. If any ofthese characteristics were missing, a patient was excluded from thecorresponding subgroup analysis but included in the overall analysis.For each racial and ethnic group, the frequency of ABG measurement wascharacterized using 2 complementary analyses. First, encounters with ABGmeasurements were compared with encounters without ABGs measurements todetermine the likelihood of receiving an ABG measurement during an encounter byrace and ethnicity. Second, to characterize the rate of ABG collection acrossencounters with at least 1 ABG, the total number of ABGs normalized by thelength of stay (in days) was calculated. Given possible confounding by illnessseverity, the second estimates were stratified by CVSOFA score at the time ofABG.o2-Sao2 pairs werecharacterized by modified Bland-Altman plots. We used \u03c72 teststo compare the distribution of categorical variables  between any 2groups, while Mann-Whitney nonparametric tests were used for continuous andordinal variables . Notably, differences in thedistribution of numeric clinical end points  were evaluated viabootstrapping (100 iterations), followed by a Mann-Whitney nonparametric test.Differences between stratified odds differences  were tested with the Breslow-Day test.Differences in Spo2 Edit 2 <sc></sc>of Edit 3 <sc                    ></sc>88% or greater despite an Sao2 of less than 88%.Patients with and without hidden hypoxemia were compared at the time of ABGmeasurement by baseline demographic characteristics  and by organfailure scores . The long-term association of hiddenhypoxemia with clinical outcomes was analyzed by estimating differences inlength of stay and in-hospital mortality. The short-term association of hiddenhypoxemia with organ dysfunction was examined using SOFA and CVSOFA scoresmeasured 24 hours after the baseline ABG measurement. Associations betweenhidden hypoxemia, RSOFA, and in-hospital mortality were also examined. Theconsequences of hidden hypoxemia were also evaluated by comparing the last serumlactate and serum creatinine levels in a 7-day window before the ABG measurementwith the first value in a 7-day window starting 24 hours after the ABGmeasurement. Values were compared at each time in addition to the differencebetween the before and after values.Hidden hypoxemia was defined as an SpMultivariate logistic regression was used for assessing binary end points , multivariate ordinal regression for numerical end points, and multivariate linear models for continuous endpoints , using analysis of variance to test forthe impact of hidden hypoxemia while adjusting for other covariates   and Python version 3.6 (Python Software Foundation).Statistical significance was set at o2-Sao2 pairs from 87\u2009971patient encounters were analyzed among 4 race/ethnicity subgroups , with 4859 (5.5%) having hidden hypoxemia. In total, 141\u2009600patients, with 679\u2009909 ABGs and 5\u2009435\u2009144Spo2-Sao2 pairs within 30 minutes ofeach other were identified. Patient characteristics are presented in o2measurements to those within the 5 minutes preceding the ABG measurement resulted in268\u2009904 Spo2-Sao2 pairs; furtherselecting the first ABG in an encounter led to 87\u2009971Spo2-Sao2 pairs and 0.4% (369 encounters), respectively. Given that there were no SOFA scoredata from Emory and Grady, RSOFA, CVSOFA, and SOFA scores had 52.7%(46\u2009381 encounters), 52.9% (46\u2009538 encounters), and 52.7%(46\u2009381 encounters) missingness, respectively. Missingness forSpThere were differences in the likelihood of receiving an ABG measurement during ahospital encounter that varied by race and ethnicity, with the White subgroupmost likely to receive an initial ABG measurement despite similar RSOFA andCVSOFA scores: White patients, 85\u2009872 of 1\u2009532\u2009492 encounters(5.6%); Asian patients, 3249 of 95\u2009813 (3.4%); Black patients,49\u2009053 of 1\u2009781\u2009868 (2.8%); and Hispanic patients, 3426 of179\u2009617 (1.9%)  . There was avarying incidence of hidden hypoxemia among racial and ethnic group indescending order: Black, 1785 [6.8%]; Hispanic, 160 [6.0%]; Asian, 92 [4.8%];White, 2822 [4.9%] (P\u2009<\u2009.001)  .Across all racial and ethnic groups statistically significant, althoughclinically small, differences in baseline organ dysfunction  were present at the time of the first ABG measurements betweenpatients with and without hidden hypoxemia . FurtherP\u2009<\u2009.001). This associationpersisted even when adjusted for age, sex, SOFA score. However, there was nodifferences in length of stay for patients with and without hidden hypoxemiawhen considering survivors only  and serum lactate level  before the ABGmeasurement than patients without hypoxemia. Patients with hidden hypoxemiacontinued to maintain significantly higher mean (SE) serum creatinine and lactate  values after the ABG measurements.When comparing values before and after the ABG, serum lactate demonstrated asmaller mean (SE) decrease among patients with hidden hypoxemia overall and in all racial and ethnicgroups except Asian patients. However, the difference in serum creatininelevels before and after the ABG measurement was not consistent across raceand ethnicity and hidden hypoxemia status.Across all racial and ethnic subgroups, patients with hidden hypoxemia had asignificantly higher mean (SE) serum creatinine level  , Hispanic (97%), and Asian (95%) patients. The risk of hidden hypoxemia atan SpO2 of 93% to 96% is 6.5% in White patients, increasing to 6.6%for Hispanic patients and up to 10.9% for Black and Asian patients (a 68% higherrelative risk). In conjunction with eTable 8 in the o2 target by selecting the highestSpo2 with a fixed risk of hidden hypoxemia. To ensure arisk of hidden hypoxemia of less than 10%, Spo2 should begreater than 93% among Asian patients , 96% amongBlack patients ; 92% among Hispanic patients , and 93% among White patients   .o2 <88%, butSpo2 \u226588%) by race and ethnicity inhospitalized patients\u2019 first ABG measurements in their hospitalization across5 large US databases. Racial and ethnic disparities in the incidence of hiddenhypoxemia in the hospital are worrying because low oxygen saturation levels, whenundetected, can lead to complications in the short and long term.To our knowledge, this study is the first to characterize the prevalence of hiddenhypoxemia , andincreased laboratory findings , even whenadjusting for covariates, such as age, sex, and SOFA score. These continue to holdtrue when restricted to ABGs with carboxyhemoglobin and methemoglobin levels lessthan 2%   .As this analysis was restricted to the first ABG measurement in a hospitalization,the clinician could not have been aware of hidden hypoxemia prior to the ABG test.It is therefore unknowable how long a patient was truly hypoxemic before their ABGmeasurement, although the clinician would be aware of hypoxemia once they had theABG results. Despite similar organ dysfunction scores at the time of the ABGmeasurement, patients with hidden hypoxemia had greater laboratory abnormalities before the ABG measurement that persistedfor at least 24 hours, suggesting that these patients may have more severe illness.This study was not designed to assess causality; it is both plausible that thepatient\u2019s illness could be causative of hidden hypoxemia (and thus be a markerof dysfunction) and that hidden hypoxemia for an unknown (perhaps prolonged)duration resulted in worse organ dysfunction.14 and, in healthy patients, brief cognitive impairment withoutsustained long-term cognitive changes.15 However, hypoxia has been associated with increasedoxidative stress, reactive-oxygen species, angiogenesis, hypoxia-inducible factors,and systemic and vascular inflammation with endothelial dysfunction.18 Critically ill patients may have impaired tolerance ofthese changes,17 andhypoxia can result in kidney injury and lactic acidosis.21The effects of hypoxia can be organized by the duration a patient experienceshypoxia. Brief, acute episodes of hypoxia have been associated withelectrocardiogram changes22However, this sampling does not reflect the United States 2010 census of Asian (5.9%of population), Black (13.4%), Hispanic (18.5%), and White (60.1%)individuals.24 Going forward, populationdifferences will only increase in relevance as the United States becomes moreracially and ethnically diverse.25 Furthermore, although older studies questioned the accuracyof pulse oximetry in critical illness, sample sizes were too small to examine theissue of skin color and race and ethnicity in meaningful detail  is toowide at low blood oxygenation levels, and third, pulse oximeters are insufficientlytested across different racial and ethnic groups prior to approval by the FDA. Asthe results of this study demonstrate, this combination has unintended negativehealth outcomes. By providing a data-driven approach to identify hidden hypoxemiausing pulse oximetry, this study is a step toward greater health care equity.26 If anything, greater ABG sampling ismerely a stopgap to cover the use of imperfect medical technology. The importantmessage is that health care devices, like predictive algorithms and medications,must be designed more inclusively to achieve comparable measurement accuracyirrespective of race and ethnicity. As noted in previous studies,28 pulse oximetry devices are not reliably accurate and do notcapture blood oxygenation readings equally across different skin colors. In themeantime, prudent clinicians should note the Spo2reading at the time the ABG is drawn to accurately identify anySpo2-to-Sao2discrepancy once the ABG result is reported.While a short-term solution to hidden hypoxemia may be to more frequently sample ABGvalues, such a strategy is invasive and inefficient.It is important to be cognizant of the patient population in which pulse oximetersused in critical care are validated. There is a need for more transparency in thelabeling of all patient care devices, including the detailed characteristics ofgroups on which they were evaluated. To further achieve more equitable healthoutcomes, we call for reinforced testing and recalibration of health caredevices\u2014across all target patient populations.o2-Spo2 pairscombine measurements that are not always collected simultaneously. Analysis wasthus restricted to Spo2 values recorded in the 5minutes preceding the ABG test. Shock and critical illness, in conjunction withother comorbid conditions , mayfurther affect pulse oximetry accuracy and need further characterization forproper adjustment for confounding beyond CVSOFA score.This retrospective EHR analysis has inherent limitations that were systematicallyaddressed. First,Sao2 signal quality or pulse oximeterbrand, leading to unclear knowledge of Spo2accuracy and homogeneity.There was high missingness, especially in SOFA scores and laboratory values, thatwas accounted for with missing flags during regression analysis; SOFA scoreswere only calculated for eICU-CRD, MIMIC-III, and MIMIC-IV data and were notcalculated for Emory or Grady data. Multiple imputation methods could improverobustness. Additionally, the EHR data did not recordSpThere may be a selection bias in acquiring ABG measurements. Given similar SOFAscores at the time of testing, White patients were significantly more likely toreceive the criterion-standard test. It is plausible that there was selectionbias with underdetection of hidden hypoxemia among Asian, Black, and Hispanicpatients. The disparities in clinical outcomes may be underestimated if more ABGtests were performed in these racial and ethnic subgroups. Additionally, theseretrospective analyses reflect associations; future studies should be designedto assess causality.o2 measurements (vs trueSao2 measurements) were associated withincreased incidence of hidden hypoxemia (Sao2 Edit 4 <sc                ></sc><88% despite Spo2 \u226588%). Althoughdemographically and clinically similar to patients without hypoxemia at baseline ABGmeasurement, those with hidden hypoxemia had higher rates of organ dysfunction 24hours later and higher in-hospital mortality. Validation of all health technologies,including pulse oximetry, must be performed across a wider range of patientpopulations to avoid perpetuating harm from miscalibration.22In this study, all racial and ethnic subgroups experienced high variability inarterial oxygen saturation for fixed pulse oximetry levels, with a greaterdiscrepancy in patients self-reporting as Asian, Black, and Hispanic than White.Small but statistically significant differences in the bias ofSp"}
+{"text": "Resection of lung cancer with chest wall involvement is an invasive procedure.We report a case of pulmonary adenocarcinoma with chest wall involvement that was resected through video-assisted thoracoscopic segmentectomy and combined en bloc resection of the chest wall (2nd to 4th ribs). Surgical stress was decreased by reducing the extent of lung parenchymal resection and applying a video-assisted technique with an additional posterior paravertebral incision.A thoracoscopic surgical approach involving incisions in areas requiring resection of the proximal, lateral, and posterior sides of the involved ribs can be applied to tumors invading the chest wall. Video-assisted thoracoscopic surgery (VATS) is a minimally invasive approach for early-stage non-small-cell lung cancer (NSCLC). Minimally invasive approaches reduce surgical stress, consequently shortening the length of hospitalization and ensuring a better patient quality of life . HoweverA 69-year-old woman experiencing back pain on her left side was referred to our hospital. Chest computed tomography (CT) revealed a 4.5\u2009\u00d7\u20093.8\u00a0cm mass in the apicoposterior segment of the left upper lobe, invading the third rib Fig.\u00a0. MediastAlthough a four-incision approach was selected based on our standard surgical technique, the access port site was modified to allow resection of the anterior parts of the 2nd to 4th ribs (Fig.\u00a0Chest wall reconstruction was not performed because the resected portion was completely covered by the scapula even upon elevation of the left arm. The operative time and blood loss were 370\u00a0min and 282 mL, respectively.The postoperative course was uneventful and the patient was discharged on the 5th postoperative day. The final pathological finding was pT3N0M0 solid adenocarcinoma with invasion of only the third rib Fig.\u00a0G. AdjuvaLobectomy with combined resection is the standard surgical procedure for NSCLC showing invasion of the adjacent organs. Minimally invasive techniques for lung cancer with chest wall invasion have been reported to reduce surgical stress \u20137. CerfoVATS segmentectomy is performed to reduce invasiveness and avoid overall complications. Consequently, the patient quality of life is improved and the hospital stay is decreased . In patiAs described by Dal Agnol et al., reconstruction of the chest wall was not performed because the tumor was located posteriorly and above the 5th rib; thus, the chest wall defect was covered and protected by the scapula and overlying muscles (In conclusion, VATS pulmonary resection combined with chest wall resection can be achieved through the placement of incisions that bridge the resected ribs. The VATS approach is useful for visual and palpatory recognition of the surgical margins."}
+{"text": "Non-invasive magnetic imaging techniques are necessary to assist magnetic nanoparticles in biomedical applications, mainly detecting their distribution inside the body. In Alternating Current Biosusceptometry (ACB), the magnetic nanoparticle's magnetization response under an oscillating magnetic field, which is applied through an excitation coil, is detected with a balanced detection coil system.2 with a spatial resolution of 2.0\u00a0cm and sensitivity in the milligram scale. A correlation coefficient between quantitative reconstructed and nominal magnetic nanoparticle distributions above 0.6 was found for all measurements.We built a Multi-Channel ACB system (MC-ACB) containing nineteen pick-up coils and obtained 2D quantitative images of magnetic nanoparticle distributions by solving an inverse problem. We reconstructed the magnetic nanoparticles spatial distributions in a field of view of 14\u2009\u00d7\u200914 cmBesides other interesting features such as sufficient large field of view dimension for mice and rat studies, portability, and the ability to assess the quantitative magnetic nanoparticles distributions in real-time, the MC-ACB system is a promising tool for quantitative imaging of magnetic nanoparticles distributions in real-time, offering an affordable setup for easy access in clinical or laboratory environments. Magnetic nanoparticles (MNPs) present great versatility due to their inherent magnetic properties and reduced size, enabling many biomedical applications . Reliabl\u22121 and real-time for a field of view (FOV) of 4.5\u00a0cm, respectively.Alternatively, AC susceptibility devices were applied for quantitative imaging of MNPs detecting their AC susceptibility. Ficko and collaborators introduced three MNPs imaging methodologies based on AC susceptibility measurements, in which the authors highlighted the low-cost instrumentation of the approaches . The sysIn this way, the ACB system applies an alternating magnetic field to magnetize the MNPs and detect their dynamic magnetic response through a pair of coils in a gradiometric configuration . The sinIn this paper, we present the progress of improving the ACB system as a measurement tool to provide a quantitative assessment of the spatial distribution of MNPs. To this end, we established a feasible system that could be useful for real-time MNPs quantification in rodents. We increased the FOV, implemented the appropriate forward model, and solved the inverse problem for quantitative MNPs imaging. Different MNP amounts inserted in gypsum cubes were used to reconstruct the particle distribution and estimate the MC-ACB system\u2019s spatial resolution and sensitivity.The MC-ACB system consists of one pair of excitation and nineteen pairs of pick-up coils. The pick-up coils are connected in a first-order gradiometric configuration to reduce the excitation field and the environmental noise, thereby increasing the system\u2019s sensitivity. A baseline of 13.7\u00a0cm separates the coils within a pair to suppress mutual interferences. The system was developed with the excitation coils having a radius of 6.24\u00a0cm and 135 turns (AWG 20); and the pick-up coils with a radius of 0.83\u00a0cm and 1200 turns (AWG 32), arranged in a honeycomb configuration as represented in Fig.\u00a0Measurements were carried out using 19 lock-in amplifiers  and an audio power amplifier  to apply a 2 mT magnetic field at 10\u00a0kHz in the excitation coils, which provides an inhomogeneous alternating magnetic field to magnetize the MNPs in the linear susceptibility range. The MNPs response, induced into the pick-up coils, was readout as a voltage by lock-in amplifiers. The voltage amplitude detected by the lock-in amplifier is our raw data, and it was acquired at a 20\u00a0Hz sampling rate using an A/D acquisition board  and LabView software.Stability and noise experiments were performed in the MC-ACB system at the Berlin Magnetically Shielded Room 2 (BMSR-2) of the Physikalisch-Technischen Bundesanstalt (PTB). The BMSR-2 is an eight-layered magnetically shielded room in which seven layers are Mu-metal to shield low-frequency magnetic fields while an additional layer of very high conductivity aluminum works as an eddy current barrier to attenuate high-frequency fields.The MC-ACB was operated in its work function. The SR830 was employed to generate an electrical signal of 0.7\u00a0V at a frequency of 10\u00a0kHz and amplified by power amplifiers (\u2212\u20093\u00a0dB). Both MC-ACB signals inside and outside the BSMR-2 were acquired continuously for 10\u00a0min at a 20\u00a0Hz sampling rate using the A/D board previously described. The measurement was performed using no magnetic material.We also evaluated the system's response to different magnetic nanoparticle conjugations with varying synthesis protocols  and different sizes .The mathematical approach of both the forward and inverse problems for quantitative 2D ACB imaging has been described by Biasotti et al. . Brieflyp-th pick-up coil and the v-th voxel, where The separation of geometric parameters and material properties of the MNP from the MNP mass inside the mentclass2pt{minimwhere We solved Eq.\u00a0 by apply imaging , 26 and where S of about 90 Am2/kg iron (H\u2009>\u2009800 kA/m) and a hydrodynamic diameter dhyd of about 130\u00a0nm. For our measurements, the MNPs were immobilized in gypsum at different concentrations to produce 10 cubic phantoms with dimensions of 1.2\u2009\u00d7\u20091.2\u2009\u00d7\u20091.2 cm3 and an iron mass ranging from 0.05 to 4.8\u00a0mg [We employed Perimag MNPs . The MNP stock suspension has an iron concentration c(Fe)\u2009=\u20099.5\u00a0mg/ml, with the MNPs exhibiting a saturation magnetization Mo 4.8\u00a0mg .L, which was measured as the point spread function (PSF) at each possible voxel position in the FOV.Firstly, we established the forward model to discretize our FOV in We performed measurements using the reference gypsum cube with a Fe content of 1.04\u00a0mg, positioned at a vertical distance of 0.3\u00a0cm from the pick-up coils\u2019 surface. As the A/D board acquires signals at a rate of 20\u00a0Hz, we previously determined that the acquisition signal with immobile material would be given by the average of the signals acquired over 10\u00a0s.2 grid at a step of 1.0\u00a0cm. Therefore, the selected FOV is centered according to the central detector coil with a volume of voxels of The cube was moved by a 2D CNC (Computed Numeric Control) in a 14\u2009\u00d7\u200914 cmWe assessed how the system would reconstruct separated cubes at a certain distance to estimate the system's sensitivity and spatial resolution.We positioned two gypsum cubes with a total MNP content of 4.80 and 3.06\u00a0mg along the FOV\u2019s central line, distanced at 3, 2, and 1\u00a0cm. The vertical distance between both cubes and the pick-up coils\u2019 surface was 0.3\u00a0cm. We determined the spatial resolution as the minimum distance the system can resolve two cubes . Then, we diluted the sample until no signal was detected. Figure\u00a0L+ the truncated singular values of the sensitivity matrix L as parameters. As a result, the maximum number of voxels containing MNPs, which can be simultaneously reconstructed, is determined by the number of non-zero singular values above a truncation threshold. Therefore, the obtained normalized singular values for the MC-ACB system using a voxel of 3 are shown in Fig.\u00a0L for the multi-channel ACB system is of dimensions matrix We determined the inverse solution's stability by the sensitivity matrices' singular values. The TSVD method calculates from the pseudo-inverse matrix Regarding the arrangement of the detection coils of the MC-ACB system (circular) and the rectangular FOV used, we simulated a sensitivity map of both proposed geometry and a squared array with 25 pick-up coils. It is worth pointing out that the coils simulation has the same length, number of turns, and electronic parameters in this simulation.Figure\u00a0Although rectangular geometries of coils can increase the effective area of detection and enable to detect in the corner of FOV, there are problems associated with less homogeneity along the FOV due to radial asymmetry. Some works showed that the spatial inhomogeneity around the FOV is associated with the worst inverse problem solutions in biomagnetic measurements. Furthermore, the exciting rectangular coil provides less sensitivity in the central area of FOV. We used circular geometries to avoid these problems. We emphasize that the method proposed in this work is a standard model for ACB real-time quantitative imaging, in which there is the possibility to optimize the electronic parameters, number, and geometries of the coils to enable different applications in future works.The main advantage of using rectangular FOV is the high computational performance in reconstructing images, once the presence of non-responsive voxels in the reconstructed distribution does not show worst results when compared to a hexagonal FOV without these voxels.To estimate the system's spatial resolution, we investigated how the ACB system resolves two cubes depending on their distance. Figure\u00a0mentclass2pt{minimL, in which the voxel's position were shifted in the direction out of center cubes. However, even with the MNP distribution partially occupying some voxels, we found a The quantitative reconstruction of the assembled 2D MNP distribution is presented in Fig.\u00a03 subdivided into 196 voxels of 3 with a reasonable spatial resolution of about 2\u00a0cm.In a first step, we introduced MC-ACB to map the MNPs biodistribution without gaining any quantitative information . Here, wThrough the MC-ACB, it is possible to reconstruct a more significant number of voxels, making the system an interesting tool in animal experiments since another magnetic system can reconstruct a limited number of voxels . Even thOn the other hand, the MC-ACB system can be implemented in any environment without the extra cost of shielding and maintenance. We also recognize that the first introduced ACB quantitative reconstruction of MNPs distributions employing a single-channel system yielded a more stable inverse problem due to the increased number of equations in the sensitivity matrix and reconstructed the MNPs distributions with higher spatial resolution due to the sensor\u2019s dimensions. However, the scanning time process of approximately 10\u00a0min required by this setup limits the methodology to perform real-time measurements of a rat, while the multi-channel works in real-time at a 20\u00a0Hz sampling rate, enabling longer measurement times. Also, future improvements regarding the instrumentation and electronics as the lock-in amplifiers may increase the system portability and even reduce the costs, facilitating the use of the system in any environment. Although the MC-ACB presents high temporal resolution and an increased FOV, the current sensitivity limitation influences the stability of the inverse problem, reducing its performance in reconstructions and quantification. In this context, we decided to use the MNPs concentration at the mg/mL level to ensure that the ACB would have enough sensitivity to detect the samples. Besides, the literature reports showed in vivo studies involving the ACB in which the MNPs concentration ranged from 10 to 70\u00a0mg/ml , 23, 28.e.g., multiple excitations coils, magnetic field gradients, and frequency decode) to improve the inverse problem\u2019s stability [Despite the advantages of the methodology proposed, the current state of the art of MC-ACB presents considerable technical limitations compared to other quantitative MNP imaging modalities. MRX and MPI apply distinct spatial encoding strategies (tability , 32. To tability , 33, it tability , 33. A stability  and multThis work described a significant advance regarding the ACB methodology, which will enable further application studies involving MNPs applications and biomedical engineering and in pharmaceutics, gastroenterology, and physiology.In this study, we presented an MC-ACB system with nineteen pick-up coils and its employment to perform quantitative imaging of MNP distributions in a Our group has extensive knowledge in drug delivery studies and gastroenterology\u00a0assessments in animals and humans using the ACB methodology, focusing on assessing solid dosage forms in the through images , 35\u201338. Furthermore, the MC-ACB system is a promising tool for quantitative imaging of MNP distributions in real-time, offering an affordable setup for easy access in clinical or laboratory environments."}
+{"text": "Current clinical rating scales in frontotemporal dementia (FTD) often do not incorporate neuropsychiatric features and may therefore inadequately measure disease stage.832 participants from the Genetic FTD Initiative (GENFI) were recruited: 522 mutation carriers and 310 mutation-negative controls. The standardised GENFI clinical questionnaire assessed the frequency and severity of 14 neuropsychiatric symptoms: visual, auditory, and tactile hallucinations, delusions, depression, anxiety, irritability/lability, agitation/aggression, euphoria/elation, aberrant motor behaviour, hypersexuality, hyperreligiosity, impaired sleep, and altered sense of humour. A principal component analysis (PCA) was performed to identify key groupings of neuropsychiatric and behavioural items in order to create a new neuropsychiatric module that could be used as an addition to the Clinical Dementia Rating (CDR) plus National Alzheimer\u2019s Coordinating Center Behaviour and Language Domains (NACC FTLD) rating scale.C9orf72, 40.8% GRN, 46.6% MAPT) compared with 24.5% of controls. Anxiety and depression were the most common in all genetic groups but fluctuated longitudinally and loaded separately in the PCA. Hallucinations and delusions loaded together, with the remaining neuropsychiatric symptoms loading with the core behavioural features of FTD. These results suggest using a single \u2018psychosis\u2019 neuropsychiatric module consisting of hallucinations and delusions. Adding this to the CDR plus NACC FTLD, called the CDR plus NACC FTLD-N, leads to a number of participants being scored more severely, including those who were previously considered asymptomatic now being scored as prodromal.Overall, 46.4% of mutation carriers had neuropsychiatric symptoms (51.6% Neuropsychiatric symptoms occur in mutation carriers at all disease stages across all three genetic groups. However, only psychosis features provided additional staging benefit to the CDR plus NACC FTLD. Inclusion of these features brings us closer to optimising the rating scale for use in trials. Neuropsychiatric features are common in sporadic and genetic forms of frontotemporal dementia (FTD), particularly the behavioural variant.Neuropsychiatric symptoms occur early on in FTD, before individuals become symptomatic. There are three main groups: affective symptoms (anxiety and depression), \u2018psychosis\u2019 symptoms  and other neuropsychiatric symptoms that formed a group with the core behavioural criteria of FTD. The affective symptoms were common in controls as well as mutation carriers and fluctuated longitudinally, so were excluded from the scale. The psychosis symptoms therefore formed the neuropsychiatric component that was added to the Clinical Dementia Rating plus National Alzheimer\u2019s Coordinating Center Behaviour and Language Domains, resulting in individuals being scored more severely.Future revisions of FTD clinical rating scales should include neuropsychiatric symptoms to capture the entire spectrum of disease. This will in turn optimise selection of individuals into therapeutic trials.Frontotemporal dementia (FTD) is a heterogeneous neurodegenerative disorder that commonly presents with either personality change  or speech and language difficulties . The core behavioural symptoms recognised in the diagnostic criteria of bvFTD are disinhibition, apathy, loss of sympathy or empathy, ritualistic-compulsive behaviour and appetite change.GRN), chromosome 9 open reading frame 72 (C9orf72) and microtubule-associated protein tau (MAPT).While studies have highlighted the presence of neuropsychiatric symptoms in sporadic forms of FTD,This study, therefore, aims to understand how best to include neuropsychiatric symptoms as an additional module to the CDR plus NACC FTLD, a scale commonly used in current clinical trials of FTD. In particular, the study aims to investigate whether neuropsychiatric symptoms are best assessed as part of, or separately to, the core behavioural symptoms that are currently included in the behaviour module of the CDR plus NACC FTLD. Better understanding of an individual\u2019s disease stage and their progression by incorporating neuropsychiatric symptoms into the scale will enhance therapeutic trial design and improve identification of treatment response.Participants were recruited from the fifth data freeze of the Genetic FTD Initiative (GENFI) study between 20 January 2012 and 30 May 2019, including sites in the UK, Canada, Belgium, France, Germany, Italy, the Netherlands, Portugal, Spain and Sweden.C9orf72, 213 GRN and 88 MAPT mutation carriers. Of these 522 mutation carriers, the CDR plus NACC FTLD global score was 0 in 55.7%, 0.5 in 15.7% and \u22651 in 28.5%. Participants were also separately judged by a clinician whether they were felt to be symptomatic. In this group, 109 had bvFTD (20.9% of the mutation carriers studied),The standardised GENFI clinical assessment included a clinical history and neurological examination, neuropsychometric assessment and the CDR plus NACC FTLD.Neuropsychiatric symptoms were assessed using the GENFI neuropsychiatric symptom scale .4 This c10.1136/jnnp-2022-330152.supp1Supplementary dataWe also wanted to explore the overlap of these neuropsychiatric symptoms with the core behavioural symptoms described in the international consensus criteria for bvFTD.2 test (sex), and age- and sex-adjusted linear regressions to compare the Mini-Mental State Examination (MMSE) and CDR plus NACC FTLD. Frequency and severity of individual neuropsychiatric symptoms were compared between groups using ordinal logistic regressions adjusting for age and sex, except for the comparisons of disease group versus controls for severity  and frequency (when the controls scored zero) where linear regressions adjusting for age and sex were used, with 95% bias-corrected bootstrapped confidence intervals with 2000 repetitions.All statistical analyses were performed using Stata/MP V.16.1 unless otherwise specified. All graphs were produced using GraphPad Prism V.9 except for the Sankey diagrams which were made using SankeyMATIC. Demographics were compared between groups using either linear regression (age and education) or a \u03c7C9orf72, GRN, MAPT). A further PCA included behavioural as well as neuropsychiatric symptoms in the analyses was also performed. Items within a component with loadings closest to +1.00\u2009or \u22121.00 were interpreted as loading strongly onto that factor, while those nearest to zero were considered as loading weakly.Principal component analysis (PCA) was performed in R V.4.1.2As mood symptoms are common in the general population and can fluctuate with time, both with and without treatment , we decided to additionally perform a subanalysis of depression and anxiety and how these features change in frequency and severity over time. Descriptive differences were reported for each time point over three visits.Finally, we investigated the addition of a neuropsychiatric module to the CDR plus NACC FTLD rating scale. We compared this new scale (termed CDR plus NACC FTLD-N) with the original CDR plus NACC FTLD. We also developed a new version of the behavioural module based on the PCA findings and compared this (termed CDR plus NACC FTLD-N-B+) with the original CDR plus NACC FTLD.C9orf72 mutation carriers had, on average, significantly fewer years of education than controls . C9orf72 and GRN mutation carriers were significantly older than controls (both p<0.001) and MAPT mutation carriers (p<0.001\u2009and p=0.001 respectively), while the C9orf72 group contained more males than the GRN group   .C9orf72 compared with the GRN group .The MMSE and CDR plus NACC FTLD Sum of Boxes scores were significantly different to controls in each genetic group overall . There wC9orf72 group, 40.8% of the GRN group and 46.6% of the MAPT group. In comparison, 24.5% of controls were reported as showing symptoms. Stratifying by CDR plus NACC FTLD, neuropsychiatric symptoms occurred in 18.0% of mutation carriers with a global score of 0 , 70.7% of mutation carriers with a global score of 0.5  and 89.3% of mutation carriers with a global score\u22651   .When looking at the individual symptoms in the combined mutation carrier group, anxiety was the most frequent and severe neuropsychiatric symptom, followed by depression, impaired sleep and irritability/lability. Stratifying this group by CDR plus NACC FTLD, all of the neuropsychiatric symptoms were significantly more impaired  than controls when the global score was \u22651 : labilitC9orf72 mutation carriers with the majority of symptoms also significantly more frequent (and in most cases more severe) in the symptomatic GRN and MAPT groups  vs 7.7%, 0.10 (0.36) in GRN mutation carriers and 8.0%, 0.06 (0.22) in MAPT mutation carriers; auditory hallucinations 22.2%, 0.31 (0.71) vs 7.7%, 0.06 (0.21) and 0.0%, 0.00 (0.00); tactile hallucinations 9.7%, 0.14 (0.53) vs 0.0%, 0.00 (0.00) and 4.0%, 0.02 (0.10); delusions 36.1%, 0.51 (0.81) vs 17.3%, 0.13 (0.36) and 12.0%, 0.16 (0.47). In contrast, altered sense of humour was more frequent and severe in symptomatic MAPT mutation carriers compared with the other two groups  vs 40.3%, 0.52 (0.79) in C9orf72 and 42.3%, 0.47 (0.70) in GRN). When the CDR plus NACC FTLD was 0.5 anxiety , depression , impaired sleep  and irritability/lability  were the most frequent symptoms. However, visual hallucinations were more common in all three groups  and agitation/aggression (in C9orf72 and GRN), euphoria/elation (in C9orf72 and MAPT), aberrant motor behaviour (in GRN), hypersexuality (in C9orf72) and altered sense of humour (in C9orf72) all occurred more frequently than in controls. Only euphoria/elation was more frequent (in the C9orf72 group) at a CDR plus NACC FTLD of 0 with no other symptoms more frequent or severe than controls  T groups . Comparicontrols .PCA of the neuropsychiatric symptoms loaded on to four main components, which cumulatively explained 28%, 49%, 69% and 81% of the variation in the data respectively. Component one showed loading mainly of the non-psychosis and non-affective  symptoms, component two strongly loaded visual and auditory hallucinations and delusions , component three loaded depression, anxiety and impaired sleep , and component four loaded hyperreligiosity . PCA of When adding the core behavioural symptoms to the PCA, a similar result was found with four components, but in this analysis all the core behavioural symptoms loaded with the other \u2018behavioural\u2019  symptoms from the neuropsychiatric symptom scale  .In the PCA, depression and anxiety consistently loaded together, often with symptoms likely to be secondary to these affective disorders such as impaired sleep and irritability/lability. In order to further consider whether affective symptoms should be included or excluded from any rating scale the longitudinal change was assessed. Within the combined mutation carrier group, and stratifying by CDR plus NACC FTLD, 55.6% had depression (mean severity 0.7 (0.8)) and 51.9% had anxiety (0.7 (0.9)) at visit one in the symptomatic (\u22651) group, while at visit two 37.0% had depression (mean severity 0.4 (0.7)) and 44.4% had anxiety (0.6 (0.8)) and at visit three 40.7% had depression (mean severity 0.5 (0.7)) and 40.7% had anxiety (0.8 (0.8)). For the prodromal (0.5) group, 25.0% had depression (mean severity 0.2 (0.4)) and 25.0% had anxiety (0.2 (0.4)) at visit one, while at visit two 12.5% had depression (mean severity 0.3 (0.7)) and 31.3% had anxiety (0.3 (0.6)), and at visit three 25.0% had depression (mean severity 0.3 (0.5)) and 25.0% had anxiety (0.3 (0.6)). Finally, for the asymptomatic (0) group 7.8% had depression (mean severity 0.1 (0.3)) and 10.3% had anxiety (0.1 (0.4)) at visit one, while at visit two 12.9% had depression (mean severity 0.1 (0.4)) and 15.5% had anxiety (0.1 (0.4)), and at visit three 12.1% had depression (mean severity 0.1 (0.3)) and 12.1% had anxiety (0.1 (0.3)). In light of the above results, we first investigated adding a neuropsychiatric module to the CDR plus NACC FTLD rating scale consisting only of the psychosis symptoms , excluding both affective symptoms and the other symptoms which loaded with the core behavioural features. We termed this the CDR plus NACC FTLD-N. This scale was positively correlated with the original CDR plus NACC FTLD . The new scale led to a number of participants (0.6%) now being considered prodromal who had previously been asymptomatic on the CDR plus NACC FTLD, as well as symptomatic participants with a bvFTD diagnosis now being considered more severely affected .C9orf72 mutation carriers  incorporating the non-psychosis and non-affective symptoms included in the GENFI neuropsychiatric symptoms scale and (2) generating an Algorithm-based Behaviour score from each of the individual behavioural symptoms  resulted in people previously being considered as asymptomatic now being considered prodromal, and in general people being rated as more severe, highlighting the importance of including psychotic symptoms in clinical rating scales of FTD.Neuropsychiatric features were present in almost half the cohort studied, more commonly in those who were judged as symptomatic, but also occurring to a lesser extent prodromally  and in some people at an asymptomatic stage . Anxiety, depression and impaired sleep were the most common symptoms in mutation carriers. The presence of affective symptoms has been previously reported in symptomatic FTD, for example, a systematic review suggested a prevalence of 7%\u201369% of depression in FTD in general, and of 19%\u201363% of anxiety in bvFTD; the frequency in our study of 40.3% (depression) and 49.7% (anxiety) fits well within this range. However, such symptoms were also common in our controls with 12.9%, 15.5% and 13.2% reporting symptoms of depression, anxiety and impaired sleep, respectively. The prevalence of these conditions is around 5% for depression, 7% for anxiety and up to 30% for sleep problems in European countries, suggesting potentially higher rates in our cohort of controls compared with the general population .C9orf72 expansion carriers had the highest frequency of neuropsychiatric symptoms, followed by MAPT then GRN mutation carriers. Many studies have shown a close link between C9orf72 expansion carriers and the presence of neuropsychiatric symptoms, both early on as a presenting feature and during the course of the disease.C9orf72 mutations are the most common genetic cause of ALS,Of the mutation groups studied, C9orf72 than GRN and MAPT mutation carriers. This is consistent with prior studies showing they occur commonly in C9orf72 mutation carriers,C9orf72 mutation carriers in the presymptomatic period, increasing in frequency when entering the symptomatic phase.MAPT mutation carriers, although were still present, suggesting that these symptoms are not pathognomonic of a particular genetic subtype.Although less common than affective symptoms, \u2018psychosis\u2019 features, that is, hallucinations and delusions occur in up to a quarter of symptomatic patients, and more frequently in MAPT mutation carriers compared with the other groups was altered sense of humour. This has been poorly studied but has been associated with temporal lobe atrophy and MAPT-related FTD in one prior study.The only symptom that was more frequent in PCA identified three main groups of symptoms as hypothesised. Importantly, this suggests that one cannot use a single neuropsychiatric score within a rating scale that incorporates all of the psychotic, affective and other neuropsychiatric symptoms. Combined with the depression/anxiety subanalysis, the results from this study suggest that a single neuropsychiatric score consisting of features of psychosis  will be necessary in any future clinical rating scale.Depression and anxiety not only formed a separate group to other neuropsychiatric symptoms, but also fluctuated longitudinally. These two factors combined with the relatively common occurrence in non-carriers support the argument for excluding depression and anxiety from global CDR scoring. There are multiple potential reasons for this fluctuation: the presence of depression and/or anxiety may coincide with life events including recovery with therapy or medication, a change in insight as the disease progresses, biological processes associated with the disease process, heightened anxiety/depression as individuals approach the age at onset of their relatives and subsequent fall in these symptoms if they pass this age, and a disease-related increase in anxiety as mutation carriers get older and more symptomatic from other affected domains.Behavioural symptoms within the consensus diagnostic criteria for bvFTDInterestingly, the symptom of hyperreligiosity was distinct from the other three groups. This symptom has been described in association with temporal lobe deficits, particularly of the right hemisphere, and can be seen in right temporal lobe epilepsyOverall, the PCAs and depression/anxiety subanalyses within the individual genetic mutation groups mirrored the findings when all mutation carriers were studied together, supporting the use of the same approach for all FTD individuals regardless of the genetic mutation, that is, a neuropsychiatric module consisting of \u2018psychotic\u2019 features, consideration of the \u2018other\u2019 neuropsychiatric features when scoring the behavioural domain, and exclusion of affective symptoms from the scale.Inclusion of the neuropsychiatric score into the CDR plus NACC FTLD (CDR plus NACC FTLD-N) led to more people being considered at higher disease stages than the original CDR, including some people being considered prodromal who were previously considered asymptomatic. In people clinically judged to be symptomatic the change in stage was mainly seen in those with bvFTD and minimal change in those with other clinical phenotypes, consistent with prior studies showing that while neuropsychiatric symptoms are seen in PPA and parkinsonian syndromes they are less common.We also investigated a second addition to the CDR plus NACC FTLD, incorporating a version of the behavioural score that required consideration of multiple different symptoms including not only the core bvFTD symptoms but also the \u2018other\u2019 symptoms from the neuropsychiatric feature list. This also led to a general increase in disease stage severity, suggesting that perhaps clinicians are not considering the whole spectrum of behavioural features in FTD when scoring the behavioural module in the CDR plus NACC FTLD.It is likely that these two additions to the CDR plus NACC FTLD lead to improved accuracy of rating disease stage in FTD and bring us closer to optimising the rating scale for use in therapeutic trials where precise scoring of an individual\u2019s disease severity is important so that treatment responses can be more representative of their real-life experience with FTD.Although this study represents one of the largest familial FTD cohorts, numbers become smaller in each group as they are stratified, particularly in longitudinal analyses, such as performed for depression and anxiety.We had included symptoms in the GENFI neuropsychiatric symptom scale that had been previously reported in patients with FTD. However, reports of other symptoms such as anhedoniaWhen considering depression and anxiety we did not record the effect of therapeutic interventions on symptom severity such as antidepressants or psychological therapies. This did not allow us to measure their effect on the longer-term fluctuation in symptoms, and future studies should address this limitation.All of the neuropsychiatric symptoms were recorded as being present or absent through the use of the GENFI symptom scale. While this is helpful for our current focus on improving clinical rating scales, it is important to note that this is distinct from making formal clinical diagnoses according to standard criteria of depression, anxiety, psychosis, etc, and therefore, may not be a true representation of the prevalence. Nonetheless, there are also potential reasons why neuropsychiatric symptoms may be underrepresented in GENFI as participants with active affective or psychotic disorders may not be able to (or want to) take part in an ongoing observational research study.Lastly, although each rater received training in use of the neuropsychiatric symptom scales, now that its use is established it will be important in future studies to formally assess both intra- and inter-rater reliability.In summary, neuropsychiatric symptoms commonly occur in individuals with FTD in all the main genetic mutation groups, including in those classed as \u2018asymptomatic\u2019. The failure of the current CDR plus NACC FTLD scale to account for psychotic symptoms runs the risk of adopting inaccurate tools to identify the disease staging and treatment response in individuals enrolled in therapeutic studies. This study suggests an initial step to rectifying this issue."}
+{"text": "Sargassum graminifolium fucoidans (SGFs) also show marvelous immunoregulatory effects. In the present study, two fractions, SGF\u22121 and SGF\u22122, were purified from SGFs by DEAE-Sepharose Fast Flow and Sephacryl S-400 HR column chromatography. We investigated the in vivo immune regulatory activity of SGF\u22122 and explored the immune activation of SGF\u22122 fecal fermentation products with in vitro fecal fermentation combined with a Caco-2/RAW264.7 co-culture system. In vivo results exhibited that SGF\u22122 could elevate the thymus/spleen indices, CD8+ splenic T lymphocyte subpopulations, and CD4+ Foxp3+ splenic Tregs. The 16S high-throughput sequencing results showed that SGF\u22122 administration significantly increased the relative abundance of Lactobacillus, Alloprevotella, Ruminococcus, and Akkermansia. In addition, it was found that SGF\u22122 fermented by feces could significantly improve the phagocytosis, NO, and cytokine  production of macrophages in the co-culture system. These results indicated that SGFs have the potential to modulate immunity and promote health by affecting the gut microbiota.Fucoidan, brown seaweed-derived dietary fibers (DFs), can be considered a promising candidate for modulating immune responses. Due to its structural complexity and diversity, it is unclear whether  DFs and and 1 \u2192 -linked \u03b1Sargassum graminifolium, belonging to the genus Sargassum, is an edible marine brown alga widely distributed along the southeast coast of China. Previously, there were few reports to evaluate the polysaccharide bioactivity of S. graminifolium. These few studies indicated that crude polysaccharides from S. graminifolium exhibited antioxidant properties and a protective effect on ethylene glycol-induced kidney damage [S. graminifolium might relieve the symptoms of food allergy in OVA-induced mice by regulating gut microbiota [S. graminifolium and its underlying mechanisms need to be investigated. In addition, the previous studies mainly evaluated the biological activity of crude polysaccharides from S. graminifolium without evaluating the biological function of their purification fractions. Therefore, this study aimed to further isolate and purify crude polysaccharides and evaluate the immunomodulatory activity of its purified fractions in vivo.y damage ,6. Our rcrobiota . HoweverIn recent years, an in vitro fecal fermentation model has been widely established to effectively simulate the process of decomposing and utilizing polysaccharides by intestinal microorganisms ,9. This S. graminifolium was further purified by DEAE-Sepharose Fast Flow chromatography. As shown in \u22121), C-H stretching vibration , and C=O asymmetric stretching vibration (a stretching peak near 1620 cm\u22121) [\u22121. An obvious 1224 cm\u22121 absorption peak, which was assigned to the sulfate group, was present in SGF\u22122, while a small sulfate group peak at 1249 cm\u22121 was observed in SGF\u22121, which is consistent with the previous results of high sulfate content in SGF\u22122 (31.81%) and low sulfate content in SGF\u22121 (5.00%). Although two fractions, SGF\u22121 and SGF\u22122, were purified from the fucoidan of S. graminifolium, only the immunomodulatory activity of fraction SGF\u22122 was further explored in the following experiments due to the low yield found in SGF\u22121.The crude fucoidan extracted from 20 cm\u22121) ; howeverp < 0.05).The effects of SGF\u22122 on body, spleen, and thymus weights are presented in n = 6, with 70,599 \u00b1 2851 reads/sample), 420,325 reads from the FL group , and 421,350 reads from the FH group . The species accumulation curve can be used to estimate the adequacy of the sample quantity and determine the species richness. A rank abundance curve is an effective tool to investigate species evenness and abundance. If the curve is smoother, the species composition of the sample will be more uniform. As shown in Accumulating data have demonstrated that human intestinal flora is important in modulating the nutrition, metabolism, and immunity of its host ,13. FollBacteroidetes and Firmicutes in the control group were the main dominant bacterial communities, accounting for 55.90% and 36.47%, respectively. In the FL and FH groups, the intestinal microflora was still dominated by Bacteroidetes and Firmicutes; Bacteroidetes increased slightly, whereas Firmicutes showed a mild decrease after being treated with SGF\u22122, but there was no significant difference. It has been reported that Firmicutes and Bacteroidetes contain many glycoside hydrolases that can degrade non-digestible polysaccharides [Bacteroidetes compared to Firmicutes; these pathways and glycoside hydrolases play an important role in fermenting glycans into SCFAs [Alistipes, Lactobacillus, Alloprevotella, Odoribacter, Bacteroides, and Ruminiclostridium were the dominant bacteria (Lactobacillus significantly increased in the FL group (p < 0.01) and FH group (p < 0.05), and the relative abundance of Alloprevotella also significantly increased in the FH group (p < 0.01) -producers [Akkermansia, whose abundance was negatively correlated with obesity, diabetes, and cardiovascular diseases [Akkermansia has also been illustrated to restore the percentage of Tregs [Akkermansia to regulate immunity. As such, in both the FL and FH groups, the treatment significantly increased the relative abundance of Akkermansia, so it is reasonable to attribute the benefits of SGF\u22122 in part to its regulation of this gut microbe.To better understand the structural response of the intestinal flora before and after SGF\u22122 administration, the taxonomic compositions of the three groups were then compared. At the phylum level, the relative abundance of the three groups is shown in charides . In addito SCFAs . At the bacteria B. Compar < 0.01) C. Moreove groups D. After roducers . SCFAs  method, and the results are shown in + gated from CD3+ T cells was significantly increased after the SGF\u22122 treatment (FH group), while CD4+ gated from CD3+ T cells was significantly decreased after the SGF\u22122 treatment (FL and FH groups). The DCs in the mouse splenocytes were defined by CD11c+ MHC-II+, and the flow cytometry result showed that there were no significant differences in the percentage of CD11c+MHC-II+ DCs among the three groups , the percentage of CD4+Foxp3+ Tregs in the splenocytes of the SGF\u22122-treated mice (FL and FH) significantly increased to 47.6% and 45.7%, respectively  cells and T cells, promote DC maturation, and then promote pro-inflammatory cytokine production [+CD8+ T cells and CD4+ Foxp3+ Tregs, which to some extent verified the immunostimulatory activity of SGF\u22122. The critical roles of CD4+Foxp3+ Tregs in immune regulation and the fact that SCFAs can promote the differentiation of T cells into Tregs have been discussed above. Here, we demonstrated that SGF\u22122 treatment could promote the production of Tregs, suggesting that fucoidans induce Treg differentiation by increasing the production of SCFAs by gut microbiota. However, the conclusion that SGF\u22122 treatment can increase SCFA levels still needs to be verified by detecting the levels of SCFAs in mouse serum. In addition, SCFAs can enhance the production of IL-10 by DCs through G-coupled receptors to promote the differentiation of Tregs [+MHC-II+) were observed after SGF\u22122 treatment.The immunomodulatory effects of polysaccharides can be achieved by activating effector cells including macrophages, lymphocytes, and dendritic cells . Fucoidaoduction ,25. Our of Tregs . HoweverIn recent years, a series of connections have emerged between host physiology, the composition of the gut microbiota, and DFs. The main view is that the combination of plant fiber with fiber-digesting probiotics could increase the components of immune regulation activity and further modulate the host physiology by transmitting signals to immune cells through pattern recognition receptors (PRRs) of IECs . TherefoAfter being treated with SGF\u22122 fecal fermentation products for 24 h, the CCK8 assay was used to examine the cytotoxic effects of RAW264.7 macrophages in the basal chamber. As shown in p < 0.05). Considering that the addition of SGF\u22122 fermented by feces in the co-culture system did not significantly affect the macrophage cell viability, it was eventually revealed that SGF\u22122 fermented by feces may partially enhance the phagocytic activity of RAW264.7 cells in the basal chamber.Phagocytic activity is a main indicator of the functional activation of macrophages. Hence, the phagocytic activity of RAW264.7 macrophages in the basal chamber was determined after being treated with SGF\u22122 fecal fermentation products for 24 h. As shown in Nizamuddinia zanardinii could stimulate macrophages to secrete NO [Phagocyte nitric oxide (NO), which is the hallmark of macrophage activation, can effectively prevent the invasion of pathogens. Hence, the detection of NO content in the culture medium can be used as a reliable method to evaluate the activation of macrophages . To evalcrete NO . The reaBased on the above results, the cytokine levels  in the co-culture model were detected by specific ELISA experiments . As shown in + Tregs [Macrophages play critical roles in immune regulation by producing inflammatory factors and pro-inflammatory cytokines. Previous studies have shown that fucoidan can stimulate macrophages to secrete NO, TNF-\u03b1, and IL-6 ,29. In t+ Tregs . This reS. graminifolium and then evaluated the immune activity of SGF\u22122 in vivo and in vitro. Up to now, the immune activity of purified fractions from S. graminifolium has been poorly documented. By including the activity evaluation of a purified fraction of SGF\u22122, it will be possible to interpret the data more soundly and hopefully to the reproducibility of studies. Our work adds an additional level of complexity in interpreting the mechanisms by which DFs contribute to host health. It is generally considered that DFs can be metabolized by gut microorganisms into beneficial metabolites including short-chain fatty acids (SCFAs) [In the present study, we included a detailed study on the isolation and purification of polysaccharides from  (SCFAs) . This co (SCFAs) . These c (SCFAs) . In the 3, NaCl, KH2PO4, K2HPO4, CaCl2\u00b76H2O, MgSO4\u00b77H2O, and Tween-80 were obtained from Sinopharm Chemical Reagent Co., Ltd. . Methanol, ethyl acetate, acetone, and dichloromethane were acquired from Tianjing Chemical Reagents Co. .DEAE-Sepharose Fast Flow and Sephacryl S-400 HR were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. . Detoxi-Gel endotoxin removing gel was obtained from Thermo Fisher Scientific . Dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS), vitamin K1, resazurin, and standard monosaccharides were acquired from Sigma-Aldrich . Yeast extract, peptone, hemin, L-cysteine, bile salts, NaHCHTrypsin-EDTA, fetal bovine serum (FBS), and Dulbecco\u2019s Modified Eagle\u2019s medium (DMEM) were obtained from Gibico BRL . ELISA kits of IL-6, TNF-\u03b1, and IL-10 were acquired from R&D Systems . The spleen lysis buffer kit was purchased from Sorlabio Co. . Griess reagent, BCA, and CCK-8 kit were acquired from Beyotime Biotech . (FITC)-conjugated-CD8, APC-conjugated CD3, FITC-conjugated MHC-II, APC-conjugated CD11c, PerCP-Cy5.5-conjugated CD4, and Alexa Fluor 647 Foxp3 were acquired from Biolegend .2 method was used to precipitate alginic acid. Thereafter, the polysaccharide was deproteinized five times by the Sevag method. Then, the crude fucoidan solution was purified and isolated by an anion exchange column (DEAE-Sepharose Fast Flow) with a gradient elution of 0.5 mol/L of NaCl and 1.0 mol/L of NaCl, respectively . The eluents under the same elution peak were combined and desalinated using dialysis bags (1000 Da) according to the elution curve plotted by the phenol-sulfuric acid method [2O, flow rate: 1.5 mL/min, 3.0 mL/tube). Similarly, the eluents were pooled under the same elution peak. After lyophilization, two yellow powders were obtained, named SGF\u22121 and SGF\u22122. The contamination of endotoxin in the SGF\u22121 and SGF\u22122 was removed by Detoxi Endotoxin Removing Gel.The crude fucoidans were prepared by referring to our previous studies , and thed method . A Sepha2-gelatin method [The sugar content, sulfate content, and protein content of the SGF\u22121 and SGF\u22122 were determined by the phenol-sulfuric acid method, the BaCln method , and then method , respectn method . High-pen method . A FTIR n = 8): the control group , the FL group (gavaged with 125 mg/kg b. w. of SGF\u22122), and the FH group (gavaged with 250 mg/kg b. w. of SGF\u22122). The mice were gavaged with SGF\u22122 dissolved in physiological saline for 28 consecutive days. Then, the mice were killed using the cervical dislocation method on the last day, and each group of mice was dissected and weighed for body weight and immune-related organ weight (spleen and thymus). The thymus and spleen indexes were calculated by the following formula: thymus/spleen index (mg/g) = weight of thymus (spleen)/body weight.All the following experimental procedures were approved by the relevant laws and conducted at the Experimental Animal Center of Huaqiao University of School of Medicine . Male BALB/c mice (25 \u00b1 2 g) were purchased from Minhou Wu\u2019s experimental Animal Trading Co., Ltd. , acclimatized in an environment with a stable temperature of 22 \u00b1 2 \u00b0C and provided with sufficient standard rodent water and chow for at least two weeks. Then, three experimental groups were set up as follows  to detect and amplify the V3\u2013V4 regions of 16S rRNA using the general primer pair. Sequences with 97% clustering similarity were assigned to the same operation taxonomic unit (OUT). Subsequently, \u03b1-diversity analysis, species taxonomy analysis, and different species screening were performed.w/v, 20%). The mixed fecal solution was then filtered with gauze, and the insoluble particles were removed by centrifugation . The remaining supernatants were collected for in vitro SGF\u22122 fermentation. Sterilized Erlenmeyer flasks were filled with 9 mL of sterilized basal medium (pH 7.6) and rinsed with oxygen-free N2 to maintain anaerobic conditions. The basal medium included yeast extract (2.0 g/L), peptone (2.0 g/L), hemin (0.02 g/L), L-cysteine (0.5 g/L), bile salts (0.5 g/L), NaHCH3 (2.0 g/L), NaCl (0.1 g/L), KH2PO4 (0.04 g/L), K2HPO4 (0.04 g/L), CaCl2\u00b76H2O (0.01 g/L), MgSO4\u00b77H2O (0.01 g/L), vitamin K1 (0.01 g/L), resazurin (0.01 g/L), and Tween-80 (2.0 mL/L). The pH of the fermentation medium was adjusted to 7.6 and sterilized at 121 \u00b0C for 20 min. The fecal slurry (1.0 mL) and 50 mg of SGF\u22122 were inoculated into the appropriate fermentation flask. In addition, fermentation flasks with or without LPS were included as the experimental group and positive control, respectively. After fermentation , samples were collected and centrifuged to remove the precipitation . The remaining supernatants were then passed through a 0.22 \u03bcm filter membrane and stored at \u221220 \u00b0C before use.In vitro fermentation was performed using mice fecal microbiota in an anaerobic culture system . Fresh fv/v) FBS, 100 \u03bcg/ml of streptomycin, and 100 units/ml of penicillin in a humidified atmosphere of 5% CO2 at 37 \u00b0C.A human intestinal epithelial Caco-2 cell line and murine macrophage RAW264.7, obtained from the Chinese Academy of Sciences Cell Bank , were suspended in DMEM supplemented with 10%  value of the Caco\u22122 cells was above 300 \u03a9\u00b7cm2, 1 \u00d7 105 RAW264.7 cells were inoculated in the basal chamber of the Transwell. After co-culture for 24 h, the LPS or SGF\u22122 fermentation supernatant (supernatants were diluted with culture medium at the ratio of 1:1000) was added to the apical chamber of the Transwell. In the control group, the fermentation supernatant without SGF\u22122 was added to the apical chamber of the Transwell. After co-culture for another 24 h, the culture medium or RAW264.7 macrophages in the basal chamber were collected for subsequent experiments.The Caco-2/RAW264.7 co-culture systems were performed on 24-well Transwell plates . The Caco-2 cells were inoculated at 1 \u00d7 10After treatment, a cell counting kit (CCK-8) was used to measure the cell viability of RAW264.7 in the basal chamber. Briefly, an amount of CCK-8 solution (10 \u03bcL per 100 \u03bcL) was added to each well and incubated for another 2 h. Finally, the absorbance at 450 nm was read using a TECAN microplate reader.After the treatment, the corresponding ELISA kits were used to quantify the levels of cytokines  in the basal culture medium. Briefly, 50 \u03bcL of sample or standard was added to the appropriate wells, and then 50 \u03bcL of the antibody cocktail was added to each well. The plate was incubated on a plate shaker for 1 h at room temperature. Then, each well was washed with wash buffer three times; 100 \u03bcL of TMB development solution was added to incubate for 10 min; and then 100 \u03bcL stop solution was added to each well. Finally, the absorbance at 450 nm was read. The cytokine levels were calculated based on the standard curve.The Griess method was used to determine the NO production in the basal culture medium. Briefly, 50 \u03bcL of sample or standard was added to the appropriate wells, and then 50 \u03bcL of Griess reagent I and 50 \u03bcL of Griess reagent II were sequentially added to each well. Finally, the absorbance at 540 nm was read. The concentration of NO in the sample was calculated based on the standard curve.v/v) containing 1% acetic acid (v/v) for 10 min. Finally, a TECAN microplate reader was used to assess the optical density at 540 nm.The phagocytosis of RAW264.7 macrophages was measured by the neutral red uptake . After tp < 0.05 (two-sided) was regarded as a statistically significant difference. All the experimental data were presented by the mean \u00b1 SD.GraphPad Prism 8.01 was used for mapping and data analysis. One-way ANOVA and Tukey tests were used to analyze the significant differences. S. graminifolium on the immune modulatory effects and gut microbiota, which could promote the development of S. graminifolium pharmaceuticals.In general, two fractions SGF\u22121 and SGF\u22122 were obtained from the SGFs: SGF\u22121 was a polysaccharide with a 113 kDa average molecular weight, 74.72% total sugar content, and 5.00% sulfate content; SGF\u22122 was a polysaccharide with a 258.07 kDa average molecular weight, 55.79% of total sugar content, and 31.81% of sulfate content. The high-yield SGF\u22122 was further investigated for its in vivo and in vitro immunomodulatory activities. The in vivo experiments indicated that SGF\u22122 can increase the spleen and thymus indices, the subsets of T lymphocytes, and Tregs in mice. The 16S high-throughput sequencing of mice intestinal contents showed that oral administration of SGF\u22122 did not significantly change the diversity of gut microbiota, but did change the composition of intestinal microorganisms, especially the proportion of probiotics. Moreover, SGF\u22122 fermented by feces improved the phagocytic ability and promoted the NO and cytokine production of RAW264.7 macrophages in the co-culture system. Overall, the present study verifies the effects of fucoidan from"}
+{"text": "Spontaneous reporting is the most used method to monitor post-marketing safety information. Although patient involvement in spontaneous reporting has increased overtime, little is known about factors associated with patients\u2019 adverse drug reaction (ADR) reporting.To identify and assess the sociodemographic characteristics, attitudes and knowledge that influence spontaneous reporting and the reasons associated with ADR underreporting by patients.A systematic review was conducted according to PRISMA guidelines. A search on the MEDLINE and EMBASE scientific databases was performed to retrieve studies published between 1 January 2006 and 1 November 2022. Studies were included if they addressed knowledge and attitudes associated with ADR underreporting.A total of 2512 citations were identified, of which 13 studies were included. Sociodemographic characteristics were frequently identified with ADR reporting in 6 studies, being age (3/13) and level of education (3/13) the most often reported. Older age groups (2/13) and individuals with higher level of education (3/13) were more likely to report ADRs. Underreporting was shown to be motivated by reasons related to knowledge, attitudes, and excuses. Ignorance (10/13), complacency (6/13), and lethargy (6/13) were the most frequent reasons for not reporting.This study highlighted the scarcity of research conducted with the aim of assessing ADR underreporting by patients. Knowledge, attitudes, and excuses were commonly observed in the decision to report ADRs. These motives are characteristics that can be changed; hence strategies must be designed to raise awareness, continually educate, and empower this population to change the paradigm of underreporting.The online version contains supplementary material available at 10.1007/s11096-023-01592-y. Approaches to increasing ADR reporting should consider the sociodemographic characteristics of patients, including the level of education.Strategies to decrease ignorance, complacency, and lethargy seem to be necessary to address the lack of reporting culture of ADR by patients.The continuous implementation of awareness campaigns and educational programs tailored to different knowledge levels, could increase the engagement, and encourage the active participation of patients.More research is required to inform the public, enhance ADR reporting and to provide current evidence on the effectiveness of pharmacovigilance interventions in reporting practice.Adverse drug reactions (ADRs) are a significant and worldwide public health problem, being a frequent cause of increased mortality \u20135, morbiAlthough in the early years of pharmacovigilance, reporting ADRs was restricted to healthcare professionals (HCPs), HCPs and patients can\u00a0now report suspected ADRs to spontaneous reporting systems , 13. In It is acknowledged that the type of information reported by patients and HCPs is different . PatientADRs are significantly underreported worldwide, with estimates that more than 94% are not reported by HCPs . UnderreLittle is known about factors associated with ADR reporting by patients; however, studies have been emerging in recent\u00a0years. Identifying the main barriers is crucial to understand gaps and to design specific strategies that can positively impact the quantity and quality of ADR reports and ultimately increase the safety of medicines. One systematic review was performed in 2006 regarding patient reporting of suspected ADRs, however none of the included studies concerned spontaneous reporting . More reThe purpose of this systematic review was to identify and assess the sociodemographic characteristics, attitudes and knowledge that influence spontaneous reporting, and the reasons associated with underreporting of ADRs by patients.This systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines  and a reA search of MEDLINE PubMed and EMBASE scientific databases was performed to retrieve articles published between 1 January 2006 and 1 November 2022, with final search conducted on 2 November 2022. The search strategy used was: (attitud* OR knowledge* OR barrier* OR facilitators*) AND (Adverse Drug Reaction Reporting Systems[MesH] OR Drug-Related Side Effects and Adverse Reactions[MeSH]) AND report* . The references cited by the included studies were examined by manual search.Studies were considered eligible for inclusion if: (i) the target population were patients and/or consumers; (ii) written in English, French, Portuguese, or Spanish; (iii) aimed to assess factors  associated with ADR underreporting; (iv) addressed ADR reporting through spontaneous reporting. The studies included could be either observational or interventional as they comprised original data. However, in interventional studies only baseline data were collected.Conference abstracts/proceedings, reviews, editorials, letters to the editor, comments, theses/dissertations, systematic reviews and/or meta-analysis were excluded. Studies in which the target population were HCPs and/or students, focused on a specific pathology or treatment, and performed through intensive monitoring schemes or clinical trials were also excluded. For studies with no access, the authors were contacted. Finally, studies that identified attitudes and knowledge but did not directly associate underreporting reasons were excluded.All articles retrieved were independently screened by 2 reviewers (CC and PA or CC and DR), who conducted full-text analysis to assess suitability for inclusion. For\u00a0any divergent decisions, a third reviewer  acted as referee to reach consensus.Critical assessment of the quality and risk of bias of the included studies were assessed using the Appraisal tool for Cross-Sectional Studies (AXIS) . This toData from the eligible studies were extracted based on Lopez-Gonzalez et al. . ReasonsThe extraction was performed by 2 authors (CC and PA or CC and DR) independently and included: author (year of publication), country, study design, setting, study population, response rate, sample size, data source, personal characteristics associated to reporting and reasons for not reporting ADRs. In case of inconsistencies, a third reviewer  made the final decision. If further information was needed the corresponding authors were contacted.. either extracted directly from the study (one specific question for each reason) or calculated using the mean/median when more than one question within the same study was associated to the same reason category mentioned above).The characteristics of the included studies were assessed through a descriptive analysis with each reason of underreporting reported numerically as a percentage  and EMBASE n\u2009=\u2009376). After screening titles and abstracts, 113 duplicate studies were removed, and 2286 citations potentially met the inclusion criteria. Since 9 studies were excluded due to inaccessibility of full-text, a total of 253 articles underwent to full-text analysis. This resulted in\u00a0thirteen studies 6. After  meeting The general characteristics of included studies are presented in Table Five studies were conducted in Africa \u201344, 50, Four studies \u201347 were The sample size ranged from 15 to 4981 subjects and the response rate from 6.0 to 100%, being above 50.0%\u00a0in 8 of the studies. The study with the highest number of recruited subjects (n\u2009=\u20094981) had the lowest response rate (6.0%) .Six studies were conducted through direct interviews, which were face-to-face \u201345 or teLikert scale was the most often used (n\u2009=\u20095) , 45\u201347. Six studies , 41\u201344 mThe two most prevalent factors associated with the act of reporting were the level of education (n\u2009=\u20093) and age (n\u2009=\u20093). Age did not reach consensus since 1 study  revealedvs rural areas) [The remainder reported factors were sex , 45, regl areas) , with hil areas)  and highl areas)  were assl areas) .. unsupportive physician, diminishing ADR importance, lack of guidance, feeling it has to be reported to a physician and not another HCP); (v) lack of feedback or action taken by the authorities regarding previously submitted reports; and (vi) being abroad. Two studies did not present descriptive statistics for their assessment [The reasons that influenced the reporting of ADRs by patients were restricted to four categories. Ignorance  \u201346, 49 asessment , 44.This review showed that sociodemographic characteristics were commonly observed with ADR reporting whereas knowledge, attitudes, and excuses were frequently identified with ADR underreporting by patients. This review also highlighted the scarcity of research conducted with the aim of assessing ADR underreporting by patients. Over the last 15\u00a0years, most of the studies were conducted in African countries, where the pharmacovigilance framework is weak and relatively new . The indOur review has some limitations. Firstly, a search for studies in grey literature was not carried out. However, we believe that our results would not be affected since studies with this type of objective are usually associated with academic institutions where the incentive and ambition for publication is higher. We decided not to include conference abstracts/proceedings and editorials as these materials often reflect preliminary analysis, and it is less likely that methods and results are described with the necessary detail for the purpose of our study. Moreover, the percentages associated with each barrier might be subject to bias as means/medians were calculated if two or more statements were grouped in the same category within the same study. Finally, we acknowledge that our systematic review is limited by the details that authors have reported. However, an assessment of quality was performed for all included studies. Despite these acknowledged limitations, we believe that our main findings are relevant, and the data collected are comprehensive to assess the factors associated with underreporting of ADRs, thereby adding information to the body of evidence.Regarding sociodemographic characteristics, the age factor was not unanimous. One study showed that the younger population is more likely to report ADRs , as theyReasons related to ignorance were widely reported as obstacles to ADR reporting. These reasons may be associated with the fact that they have\u00a0never heard about pharmacovigilance and may not\u00a0recognize its importance. This ignorance may also be described as poor knowledge dissemination given that\u00a0reporting systems might be more aligned with HCPs than patients .Complacency was another reported reason as patients might believe that an\u00a0ADR is not serious enough, or that\u00a0it is not necessary to report. Complacency and ignorance could be highly related to the belief that marketed drugs are well-documented and safe. Lethargy justifies statements about forgetfulness, procrastination, lack of time or interest to report, and difficulty filling out the report. Simplifying reporting, promoting easier access, and boosting strategies that demonstrate that it is neither burdensome nor a bureaucratic process can overcome this barrier.Overall, previous studies conducted among HCPs showed a wider spectrum of reasons for underreporting: fear, indifference, diffidence, ignorance, complacency, insecurity, unavailability of the reporting form, lethargy, ambition, and financial reimbursement , 59\u201365. As in our review, patient-HCP communication problems have been identified\u00a0in other studies , 66\u201368 a. on-line platforms, mobile apps), which do not have associated costs or time limitations [A previous systematic review identified additional reasons for not reporting such as postal mailing costs or the limitation of restricting reporting to telephone during working hours . Howeveritations . Of noteitations .The lack of interventions for patients was highlighted in a systematic review  which coSeveral altruistic and personal motives that encourage patients to report ADRs have been described, such as contributing to increase safety knowledge, the need to be heard and to prevent harm to other patients , 57, 66.From the methodological point of view, only 6 studies of our systematic review were considered of high quality and low susceptibility to bias, which emphasizes the need for improvement when conducting future studies. Most of the included studies used a convenience sample and representativeness may be lacking, leading to potential selection bias. Most studies did not present a denominator and although some studies\u00a0attempted to address non-responder\u00a0rates, none was completely\u00a0successful.There is an urgent need to develop and conduct high quality, intervention based\u00a0studies in this population to optimize ADR reporting and to provide evidence of effectiveness. Different communication, educational and promotional strategies targeted to patients should be designed and implemented to overcome the potentially modifiable barriers. Training and dissemination related to online platforms with clear, simple, and interactive content could be an efficient option to overcome these barriers.This review highlighted the scarcity of studies conducted with the aim of assessing the influencing factors of underreporting ADRs by patients.Sociodemographic characteristics appear to influence spontaneous reporting, with age and level of education being the most frequently reported. Patients\u2019 attitudes observed linked to\u00a0underreporting included ignorance, complacency, lethargy. These can be improved through the implementation of awareness campaigns and theoretical/practical educational programs tailored to different knowledge levels. Increasing engagement and encouraging active participation of patients is needed to reverse the current paradigm of underreporting.Supplementary file1 (DOCX 32 KB)Below is the link to the electronic supplementary material."}
+{"text": "Scientific Reports 10.1038/s41598-021-83872-z, published online 01 March 2021Correction to: The original version of this Article contained an error in the Data availability section, where the link to the raw data in the Sequence Read Archive (NCBI-SRA) was incorrect.https://www.ncbi.nlm.nih.gov/sra). The detailed scripts for all analyses are available at:\u00a0https://github.com/gboris/AD_RNA-Seq.All data supporting the results in found within the Manuscript and accompanying Figures or in Supplementary Tables\u00a01\u20136. All summary data generated in this study are included in this published article (and supplementary information files). The raw data has been submitted to the Sequence Read Archive . The detailed scripts for all analyses are available at: https://github.com/gboris/AD_RNA-Seq.All data supporting the results in found within the Manuscript and accompanying Figures or in Supplementary Tables 1\u20136. All summary data generated in this study are included in this published article (and supplementary information files). The raw data has been submitted to the Sequence Read Archive (NCBI-SRA; The original Article has been corrected."}
+{"text": "Although positive changes were found during both lockdown periods, they were less pronounced throughout the winter lockdown. Further studies are needed to elucidate the real impact of these changes in the post-COVID period.The present study aims to assess the diet quality, the relationship between diet quality and lifestyle, and the association of diet quality with body mass index and students\u2019 field of study during COVID-19 lockdown periods (spring and winter) in 2020. Datasets were collected via an anonymous online self-reported questionnaire distributed during two time periods using social media. A total of 1939 Croatian students (82.4% women and 17.6% men) completed the questionnaire. Diet quality was assessed using the pro-healthy diet index (pHDI) and non-healthy diet index (nHDI). An increase in diet quality was noted during both lockdown periods but was lower during the winter lockdown. Cooking for oneself was associated with a high level of pHDI, while ordering or buying ready-to-eat food was linked to a low level of pHDI. Additionally, a decrease in screen time and increased physical activity was associated with high pHDI values. Furthermore, during both lockdown periods, students with a BMI above 30 kg/m In response to the outbreak and rapid spread of COVID-19 (coronavirus disease 2019), including the increasing death rate, the World Health Organization declared the COVID-19 pandemic on 12 March 2020 [The relaxation of the global lockdown, introduced on 19 March 2020 , began iThe COVID-19 pandemic brings upon a potential deterioration of the global obesity pandemic we have been battling for decades . An incrWith universities\u2019 closure, the entire teaching program was moved online. In order to fulfill their academic obligations, students were forced to spend more time in front of the screen, which was also noted in the population of adolescents in Croatia . SpendinOn the other side, the COVID-19 lockdown was also associated with some beneficial changes. An increase in cooking frequency and the time and effort spent on food preparation were recorded and positively associated with higher diet quality during COVID-19 ,13,14. AIn this study, three parameters were accurately investigated: (i) diet quality, (ii) the relationship between diet quality and lifestyle, and (iii) the association of diet quality with body mass index and students\u2019 field of study. We assessed these before the lockdown, and during the spring and winter of 2020\u2014lockdowns in the Republic of Croatia.This research was conducted as a part of the UNI-COVID project, which included Polish, Spanish, Portuguese, Ukrainian, Turkish, and Croatian students with no exclusion criteria. This project is an observational cross-sectional study that used a structured questionnaire developed using the Google Forms tool . StudentThis research is in line with the ethical standards of the institutional and national committee and the Helsinki Declaration. Students agreed to participate in the research via the digital form for informed consent. Since this study does not belong to a medical experiment, it was exempt from ethical approval from the Poznan University of Medical Sciences Bioethics Committee according to Polish laws and Good Clinical Practice (GCP) regulations (decision number: 527/20). The questionnaire consisted of questions taken from a KomPAN questionnaire .The questionnaire was taken on two occasions. It was first implemented during the spring lockdown 2020\u2014it was available for fulfillment from 18 May to 7 June 2020. In that period, responses were collected from 751 students . It was conducted for the second time during the winter lockdown in 2020, and 1188 students  completed the questionnaire from 14 to 22 December 2020. Sociodemographic characteristics of students included age, gender, marital status, residence, level of education, and the general situation in the household.Students answered questions about dietary and lifestyle behaviors before introducing lockdowns and whether those behaviors changed during the lockdowns. Lifestyle behaviors included screen time, sleeping time, cooking habits, food ordering frequency, the daily number of meals, their time of consumption, and the level of physical activity.Questionnaire part B, on the frequency of consumption of certain foods, was used to assess the quality of nutrition before the introduction of lockdowns. The structure of questionnaire part B is briefly described by Pfeifer et al. .Students marked the frequency of consumption of certain foods by choosing one of six frequency categories\u2014from never to several times a day. They also assessed the change in the frequency of consumption for 22 food groups by choosing for each group: \u201cmore\u201d, \u201cless\u201d, or \u201cequal\u201d, in relation to consumption before the introduction of lockdowns.Each of the six categories of food consumption frequencies was recalculated to the daily frequency as follows: (1) never \u2192 0 times a day, (2) 1\u20133 times a month \u2192 0.06 times a day, (3) once a week \u2192 0.14 times a day, (4) several times a week \u2192 0.5 times a day, (5) once a day \u2192 1 time a day and (6) several times a day \u2192 2 times a day.Two indices were used to assess the diet quality\u2014pHDI  and nHDI . For the period before the lockdowns, indices were calculated as a sum of recalculated daily frequencies of respectable corresponding food groups as described in the previous papers ,18.(a)pHDI: lower tertile (<23.9%), middle tertile (23.9\u201338.3%), and upper tertile (\u226538.3%).(b)nHDI: lower tertile (<8.2%), middle tertile (8.2\u201314.4%), and upper tertile (\u226514.4%).The pHDI and nHDI were expressed as the scored and total possible points ratio. The indices were categorized using an a posteriori approach. Three levels were defined according to the tertiary distribution:(a)pHDI: lower tertile (<27.6%), middle tertile (27.6\u201342.1%), and upper tertile (\u226543.1%).(b)nHDI: lower tertile (<8.9%), middle tertile (8.9\u201316.8%), and upper tertile (\u226516.8%) .During the lockdown period, students assessed the change in their consumption frequency for each food group. For a student who increased their consumption of a certain food group, the number of points rose to the points for the corresponding frequency category above the one marked for the period before the lockdowns. Respectively, for a student who decreased their intake, a corresponding number of points was awarded for the frequency category below the one indicated for the period before the lockdowns were introduced. The sum for both indices was calculated and categorized the same way as before the measures. For each index, three levels were defined according to the tertiary distribution:t-test and \u03c72-test for categorical variables. A box plot diagram was used to show the correlation between pHDI, nHDI values, and body mass index groups , and to show the pHDI and nHDI of individual scientific fields of study. The sample size was calculated as described by Pfeifer et al. [All of the data obtained by the questionnaire were qualitative, except for students\u2019 body height, body weight, and age. Qualitative data for the frequency of certain food groups\u2019 consumption were converted and coded into quantitative data (pHDI and nHDI). The F-test was used to check the equivalence of the variances between the two datasets, while the statistical significance of the difference was tested by the Student\u2019s Furthermore, descriptive statistics were used to show the general characteristics of students depending on the levels of pHDI during the lockdowns and the changes in dietary and lifestyle behaviors during the lockdowns. For the data relating to the time before the lockdowns, descriptive statistics were used depending on the levels of pHDI before the introduction of the lockdowns. MS Office Excel 2016 and SPSS v. 17  were used for all of the above data processing.The distribution by age, gender, and body mass index (BMI) of students during the spring (response rate 0.5%) and winter lockdowns (response rate 0.8%) is shown in p < 0.001) compared to pHDI values before the lockdown. A similar change was observed by repeating the questionnaire in winter 2020\u2014a statistically significant increase (p < 0.001) in pHDI values during the lockdown. Additionally, a statistically significant difference was noted between pHDI values during the spring and winter lockdowns\u2014pHDI values were higher during the spring lockdown.p < 0.001) compared to the periods before the lockdowns were observed during both lockdowns  (p < 0.05).The \u03c7Although not statistically significant, women were more likely than men to have a high level of pHDI\u201438.6 vs. 29.1% during the spring and 28.7 vs. 21.5% during the winter lockdown. Furthermore, during spring and winter lockdowns, students in cities with more than 100,000 inhabitants, compared to other residences, most often had a medium level of pHDI, while students residing in villages or cities with less than 20,000 inhabitants most often had a high level of pHDI. During both lockdowns, students under the age of 20 most often had a low level, those aged 25\u201330 years had a medium level, and students with a master\u2019s degree had a high level of pHDI .2 (4.07 \u00b1 2.18). Although the mean pHDI for the group with BMI values from 18.5 to 24.9 kg/m2 was lower than for the group with lower BMI, the median pHDI for the normal weight group was 4.56, while the mean pHDI for malnourished students was 4.34. On the other hand, both mean values and medians of nHDI increased with increasing BMI\u2014the lowest nHDI was noted for students with a BMI of less than 18.5 kg/m2 (1.39 \u00b1 1.03), while the highest nHDI was detected in obese students (2.52 \u00b1 1.81).During the spring lockdown, a declining trend in diet quality with an increase in body mass index was observed . The groThe dependency between diet quality and body mass index during the winter lockdown shows equal relation for nHDI but not for pHDI. Students with the lowest BMI have the lowest nHDI (1.66 \u00b1 1.19), while students with the highest BMI have the highest nHDI (2.14 \u00b1 1.47). On the other hand, pHDI values were similar in all BMI groups, with the result approximately 4 .Students\u2019 characteristics  according to their fields of study are presented in Depending on the field of study, the mean values of pHDI and nHDI differ . The meap < 0.05) were confirmed in the values of pHDI [During the two round of lockdowns, significant differences  difference between the groups of certain lifestyle characteristics, both before the spring and winter lockdowns. Thus, 42.4 and 37.8% of students who cooked for themselves before the spring and winter lockdowns, respectively, had a high level of pHDI, while 41.8 and 41.0% of students who mainly ordered or bought ready-to-eat food before the spring and winter lockdowns, respectively, had a low pHDI level. Students who predominantly ordered food had the lowest propensity for high pHDI. Furthermore, prior to the introduction of lockdowns, students who had high physical activity at work and in their free time predominantly had a high level of pHDI. Of the total number of highly active students, 53.1 and 53.6% had the high pHDI before the spring lockdown, while the analogous percentages before the winter lockdown were 43.9 and 42.8%. On the other hand, students with low physical activity most often had a low level of pHDI  in pHDI level between students with different places of residence and working status before the introduction of winter but not before spring lockdown . Additionally, students who did not have a habit of consuming meals at regular times during the spring and winter lockdowns predominantly had a low pHDI level, while those who consumed all meals at normal times were more likely to have a high level of pHDI during the lockdowns .In contrast to the period during the winter lockdown, students who increased the frequency of ordering food had a predominantly low pHDI, while students who reduced or maintained the same frequency during the spring lockdown tended to have a high level of pHDI. The same trend during the spring lockdown was also observed for body weight change\u2014students who increased their body weight were more likely to have low pHDI. Regarding the change in cooking habits, students who started cooking for themselves during the spring lockdown had high pHDI .p > 0.05). During the winter lockdown, both students who increased and those who decreased the number of meals consumed were more likely to have low pHDI, while during the spring lockdown, those who reduced the number of meals predominantly had a high level of pHDI.Furthermore, students who lost their jobs during the lockdowns showed a predominantly high level of pHDI during both winter and spring lockdowns. An increase in screen time showed an association with a low level of pHDI during the winter lockdown, while students who reduced screen time tended to have higher pHDI values\u2014results during the spring lockdown showed the opposite effect  on the affiliation to a certain level of pHDI during the spring lockdown. The trend was similar\u2014students who started cooking for themselves during the measures were more likely to have high pHDI values. On the other hand, students who mostly ordered or bought ready-made food had a low level of pHDI, 41.8 and 41.0%, before the spring and winter lockdown, respectively. In contrast to the positive effect of cooking, ordering or buying ready-made food negatively affects diet quality. Moreover, it is associated with the consumption of low nutrient-density foods and a higher intake of saturated fats and sweets [The increased cooking frequency was highlighted as a positive change  and studd sweets ,31. Acco2 had the lowest pHDI and the highest nHDI compared to other students during the spring lockdown. However, it should be borne in mind that these observations could happen only during lockdowns and might not be permanent. Higher BMI values are associated with lower diet quality, lower levels of physical activity, and a higher frequency of overeating during the spring lockdown [2, where the mean value increased. An increase in nHDI values was also noted, again except for the group of students with obesity where, compared to the spring, the mean value decreased during the winter lockdown. These results can be explained by long-term stress and anxiety, which are associated with higher consumption of energy-rich and nutritionally poor foods [Individuals with excessive body weight and obesity are at greater risk of developing more severe clinical outcomes of COVID-19 disease . People lockdown . Additioor foods . For exaor foods . On the The pHDI and nHDI values varied according to the students\u2019 field of study, so students from biotechnical sciences and biomedicine and health had the highest pHDI values. In contrast, those from technical sciences had the lowest. Students from biotechnical sciences also had one of the lowest nHDI values, while technical and social sciences students had the highest nHDI values. In a paper by Mu\u00f1oz-Rodr\u00edguez et al., students from the field of biomedicine had a better diet quality in contrast to non-biomedical students . Similarp < 0.05). Students who consumed all meals at usual times tended to have a high level of pHDI. At the same time, irregular consumption was associated with a low level of pHDI.Although research shows an inconsistent association between mealtimes and diet quality , meal coThe daily rhythm and adequate amount of sleep appear to be related to the functioning of the immune system at an optimal level . Before Spending more time in front of the screen is associated with poorer diet quality and reduced physical activity ,11,46. COn the other hand, increasing physical activity levels could likely reduce the severity of COVID-19 disease symptoms . Before P\u00e9rez-Rodrigo et al.  observedSince the students\u2019 diet quality increased during both lockdowns, it is essential for future studies to identify all of the contributors to this phenomenon and find a way to maintain them in the post-COVID period. Additionally, it was recorded that higher diet quality was associated with higher cooking frequency and lower food ordering practice\u2014therefore, it should consider the implementation of students\u2019 cooking workshops as a potent tool to increase diet quality among the student population. In addition, students from biotechnical science and the field of biomedicine and health recorded the highest diet quality\u2014since these subpopulations of students have the greatest knowledge about nutrition, the introduction of basic education regarding nutrition in syllabi of other study fields may be a way to go for providing better student nutrition.Using an online questionnaire as a tool, in a short time we collected the information of many students without breaking the regulations and recommendations of social distancing. However, some limitations should be acknowledged. A KomPAN questionnaire, from which most questions were taken to create an online questionnaire used in this study, has not been validated in Croatia\u2014but it was used in the study since it best fits the target population, and it allowed the comparison with the results of other countries included in the UNI-COVID project. During spring and winter lockdowns, students who decided to take part in the study were predominantly females , untrained, and could not ask for explanations if they had any doubts. This might have resulted in under/overestimation of actual food proportion intakes. The students answered questions retrospectively about their diet and lifestyle before the lockdown, which is also a limitation due to memory recollection. Additionally, the self-reported weight and height could have led to bias when BMI was calculated. Transferring qualitative data to quantitative for the assessment of diet quality during lockdowns and restricting the variation in the HDI options could also lead to a potential calculation error. However, it is possible to express nHDI and pHDI values in a scale ranging from 0 to 100 ,50, inst2. Regarding the association of diet quality and field of study, students from biotechnical sciences had the highest cumulative diet quality\u2014the largest difference between the mean value of pHDI and nHDI. In second place were students whose studies belong to the field of biomedicine and health.The diet quality of students in Croatia generally increased during the spring and winter lockdown. Higher diet quality, during both winter and spring lockdowns, was associated with a decrease in food ordering frequency, a lesser increase in body weight, and an increase in physical activity either at work or in leisure time. As the student\u2019s BMI increased, the diet quality, shown as pHDI, decreased, and the lowest mean pHDI was noted for students with a BMI above 30 kg/m"
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