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+{"text": "It is a high priority that health care providers have effective communication skills. It has been well documented that the doctor-patient relationship is central to the delivery of high quality medical care, and it has been shown to affect patient satisfaction, to decrease the use of pain killers, to shorten hospital stays, to improve recovery from surgery and a variety of other biological, psychological and social outcomes. This study sought to quantify the current knowledge of interns in Iran about communication skills.A cross-sectional study using a self-report questionnaire was conducted among interns. Data analysis was based on 223 questionnaires. The internal consistency of the items was 0.8979.Overall, knowledge levels were unsatisfactory. Results indicated that interns had a limited knowledge of communication skills, including identification of communication skills. In addition, there was a significant difference between the mean scores of interns on breaking bad news and sex education. The confidence of males about their communication skills was significantly higher than for females. Analysis of the total scores by age and sex showed that there was a statistically significant main effect for sex and the interaction with age was statistically significant. Free response comments of the interns are also discussed.It is argued that there is a real need for integrating a communication skills course, which is linked to the various different ethnic and religious backgrounds of interns, into Iranian medical curricula. Some recommendations are made and the limitations of the study are discussed. The expectations of the public have been dramatically increased and the majority of them are familiar with their rights in the health care system. As a consequence, it is a high priority that health care providers have effective communication skills. It has been well documented that the doctor-patient relationship is central to the delivery of high quality medical care. It has been shown to affect patient satisfaction, to decrease the use of pain killers, to shorten hospital stays, to improve recovery from surgery and a variety of other biological, psychological and social outcomes -4. Lack However, little is known about the importance of communication skills in the practice and training of doctors in Iran, where the culture differs greatly from that of the West. Sensitivity to religious matters is particularly important in Iranian doctor-patient relationships where Islam is more than a religion; it is a way of life. It controls politics, local laws, behaviour and many other aspects of daily life. It gives guidance in all spheres of human activity from birth to death. Therefore doctors coming into contact with religious patients need to be aware that there are numerous potential barriers to good communication .A major criticism of current medical training in Iran is that communication skills have not been embedded in the curriculum of Iranian medical students, despite the richness and variety of evidence from elsewhere concerning the importance of communication skills. Concerns over poor doctor-patient communication amongst Iranian doctors led to an exploration of the current situation . In thisA quantitative survey was performed at Tehran University of Medical Science (TUMS). A cross-sectional study was conducted using a questionnaire administered to 235 interns. Anonymity was maintained throughout. The subjects received the self-administered questionnaire with a covering letter explaining the project and the subject's rights. 12 subjects did not return the questionnaire and an additional 7 subjects did not give their age and one person did not give his/her sex. Therefore data analysis was based on 223 questionnaires, but covariate-based analysis on fewer. The subjects were asked to complete the questionnaire without referring to source books.The questionnaire consisted of three sections. The first section asked students to give personal details including the demographic items age and gender .The choice of items was based on the communication skills an intern will need. All items were verified and subjected to content validation by three major experts in communication skills. These experts were given copies of the CSKS and the purpose and objectives of the study. They then evaluated the CSKS on an individual basis. Comparisons were made between these evaluations and the authors then made some minor changes within the CSKS. The CSKS had a high internal consistency  is 10 to 100, with 10 indicating low knowledge. Analysis of the total scores produced a mean score of 51.30 [95 per cent confidence interval (CI) 49.05\u201353.55]. The subjects' performance on the CSKS suggests a knowledge deficit in communication skills. The mean scores for males and females were respectively 53.6 and 48.2 (P = 0.02). The vast majority of interns (78.1%) had not studied a paper on communication skills. When asked whether they had formally attended communication skills courses, 91.4% of interns reported \"no\". Of the few interns who reported \"yes\", these interns specified courses such as CPR, injections and semiology . There was a statistically significant main effect for sex  and the interaction effect [F  = 4.06, p = 0.04) did reach statistical significance. However the effect size was small (eta squared = 0.02). The young male interns were more confident than average, while the young female interns were less confident.Free responses included the following comments:Nobody has trained us about communication skills. Our knowledge in respect of communication skills is very poor. Your items show that we are very far behind other countries. Our universities are not as advanced as other universities'.''I feel we are not familiar with the ABC of communication skills'.'A good guide to communication skills needed'.'I feel communication skills would be an excellent course since it gives us an idea of how we can handle bad news'.'Attending doctors are not totally familiar with the aims and use of communication skills in the clinical setting'.'All our courses only focus on biological issues rather than psychosocial issues'There were a number of limitations to this study.1. The CSKS has not been normed for a population of interns.2. Criterion-related validity of the CSKS was not determined, although content validity was established on the instrument.3. Since it is a self-assessed questionnaire, these may be problems with bias, such as prestige bias.The very high response rate (95%) of this questionnaire may have reflected general interest, or may have resulted from the advantages of self-assessment which itself may improve performance. The results on the CSKS show that basic knowledge of interns in Iran about communication skills is limited. Researchers have reported similar findings in other countries which reveal a deficit in the knowledge of doctors about communication skills . The impThe vast majority of research studies have been conducted on the outcome of communication skills in the practice and training of doctors in western countries. Even here, despite doctors trained in communication skills and the advocacy of the use of a patient-oriented approach, some evidence suggests that there are difficulties in practice ,20. HoweThe results of the study show that there were significant differences between males and females with regard to their reported knowledge of the main communication skills. Women were less confident of their skills. The deficit may partly be an artefact of an inadvertent prestige bias of the male students. The deficit is particularly notable in sex education. There are three possible explanations for this. Firstly, in general, Iranian female interns are very shy to ask patients about sexual issues. Therefore they may feel that sex education skills have no implications for their practice and hence pay less attention to sex education training. Secondly, it may be a systematic error in female respondents, i.e. they may be shy to discuss their knowledge about sex education skills rather than lack knowledge. Thirdly, in the past, sex education was regarded as a taboo in Iran and was not available in schools, especially for girls . This peThe results on the CSKS suggest that there are areas of weakness in the communication skills confidence of interns, particularly in breaking bad news. While it is well recognised that delivering bad news is a difficult task that requires skills and sensitivity , both feThere is a significant difference between the mean score of the interns on breaking bad news. The female interns have reported lower confidence than the male interns. The deficit could be an inadvertent prestige bias of the male students. However, to our knowledge, there is no evidence that underpin such finding. Although Orlander et al's work  demonstrGiven the poor levels of confidence about communication skills, particularly sex education skills, revealed in this study, it is concluded that educational programmes are necessary. In sex education skills training, given the complex interplay of cultural and religious beliefs in Iran, particular attention must be paid to multicultural and religious issues. Therefore, further work is needed on gender education and stereotypes in sex education; learning styles; the 'hidden curriculum'; and how far medical schools make organisational and administrative arrangements on the basis of gender and the implication for female and male interns.The enthusiastic response to the questionnaires may suggest that medicine is accepting the need for developing communication skills within the medical curriculum. Medical education in Iran must respond to this challenge.Finally, our findings may be somewhat limited in generalisability because they are derived from only one medical school in Iran. Self-assessment data may suffer from biases such as prestige bias. Despite these caveats, the authors believe the data to be an accurate reflection of current practice in Iran, based on the Iranian authors training experiences, and consistency with previous accounts.Whilst the approach to this research has been shaped by a government-recognised health need, the authors recognise the need for, and welcome, further examination of these findings from multiple perspectives, especially with regards to ethnicity and social issues. Since not enough attention has been focused on individuals as makers of health as a service rather than customers of health care services, it is strongly recommended, therefore, that medical students be trained in the context of psychosocial issues that may influence health behaviour, as has been indicated by one of the participants. It is particularly important that this type of approach be incorporated into the curricula of medical training. This may assist in transferring from the disease-oriented to the patient-oriented approach and ultimately lead to patients understanding more and taking greater responsibility for their own health.The author(s) declare that they have no competing interests.MT and ST carried out the conception, design, initial analysis and interpretation of the data. MT drafted the paper. ODL was involved in revising the draft critically, revising the statistical analysis and gave final approval of the version to be published. AAZ contributed to the collection of data and the reviewing of the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "The first world wide symposium on the topic of gender-specific medicine provided the latest research on differences in sex and/or gender in medicine and medical care. The presentations ranged beyond the topic of reproduction to encompass the entire human organism. This report critically reviews three issues that emerged during the Conference: gender mainstreaming, the concept of sex/gender differences and the issue of men's health. It suggests that the interdisciplinary concept of gender-specific medicine has to be mirrored by the integration of social and cultural studies into medical research and practice. Gender & Medicine, 3, Supplement A, 2006, or on the website [This three-day conference, the first world-wide symposium on the topic of gender-specific medicine, attracted a wide range of distinguished researchers and clinicians. The focus of the Conference was to report on the latest research on sex and/or gender difference and its impact on physiological function, treatment procedure and patient outcomes. The presentations ranged beyond the topic of reproduction \u2013 the most obvious field of sex difference \u2013 to encompass the entire human organism, with the scheduled 92 papers and 98 poster presentations covering diverse medical sub-specialities. Individual papers can be accessed as abstracts in the journal  website . This reThe concept of gender mainstreaming was originally established by OSAGI in 1997, and was later also adopted by the WHO. Gender mainstreaming calls for equitable attention to male and female examples in clinical research, treatment, education, and indeed the whole health care system. It aims to ensure recognition and understanding of gender in social policy processes, and thereby counteract gender inequality . Gender The last day of the conference saw a vigorous discussion on the dilemma of how to integrate gender mainstreaming into medical research and education, which is made particularly difficult as medical schools have taught gender blindness until now. Staff from institutions that already teach gender-specific medicine, including Columbia University (USA), Charit\u00e9 Universit\u00e4tsmedizin (Berlin), Karolinska University Hospital (Stockholm) and Chiba Prefectoral Institute of Public Health (Japan), all offered their individual strategies for resolving this problem. Clinical research and hard data were needed to persuade colleagues to integrate gender-specific medicine into other medical sub-specialties. It was agreed that the transition from research to the application of such knowledge in patient care was difficult. While clinical researchers were preoccupied with identifying sex/gender differences in biomedicine, health care practitioners voiced their need for practical guidelines on how to institute gender-specific medicine in their offices. Some promising attempts to establish guidelines were presented, such as: at Monash University , where gender mainstreaming has been introduced to medical education; at the Office of Research on Women's Health, NIH  Institute, which has scheduled web-based training course on gender/sex medicine for 2006; and at the Charit\u00e9, which also will commence teaching a European graduate course in gender-specific medicine this year. Finally, the establishment of an international society for gender-specific medicine was proposed.The way in which most papers dealt with the concepts of gender and sex is problematic. In contrast to the specificity of the medical data that was presented, particularity was often abandoned in relation to sex and gender, and the two concepts conflated. Or, as was the case for many papers, 'sex'  and 'gender'  were presented (unwittingly or explicitly) as binary oppositions. Since at least the 1970s, mainly Western feminist and social theorists have criticised an oppositional conceptualisation of sex as opposed to gender, and nature as opposed to nurture, where the first term is privileged. Such a pattern of conceptualisation tended to define traits as solely biological  and indOnly a few papers presented an integrated concept of sex/gender; for example, a paper on the role of male and female health factors in stress, or a paper on the social and cultural factors imbedded in the clinical presentation of chronic pain ,9. Only Despite the conference title, only a few papers and poster presentations dealt explicitly with sex/gender difference in men. This may be a product of the Western feminist movement's campaign for the establishment of 'women's health' as a separate branch of medicine, to compensate for the limitations of the gender-neutral \u2013 male-orientated \u2013 norm in medicine. The legacy of this has been a perception of gender-specific medicine as 'women's medicine' with a political agenda. Of course, further research in the specific fields of women's health remains necessary, particularly as research for women is still restricted in scope due to concerns that clinical trials might affect women's fertility. However, there is a multitude of reasons that men's health should be a valid field of research, as was highlighted in 1999 by the first World Congress on Men's Health in Vienna . The papThe Conference was not the first to examine the neglected question of sex and gender difference in medical research and treatment. Yet by presenting a wide range of the latest international medical research, the Conference drew attention to the capacity of this as yet little-understood field of research, in time, to reshape medical understanding. A further conference is scheduled in 2007, and the organisers intend a stronger integration of the social sciences as a separate discussion stream, and more medical studies on men. This is commendable, for as this Conference has demonstrated, it is clear that the interdisciplinary concept of gender-specific medicine calls for the integration of social and cultural studies into medical research and practice. While the American Institute of Medicine of the National Academies in 2001 stated that 'every cell has a sex' - the emphasis here was on the sexual genotype and chromosomal differences  - Dr JohOSAGI Office of the Special Advisor on Gender Issues and Advancement of Women, United NationsWHO World Health OrganisationNIH National Institute of HealthThe author(s) declare that they have no competing interests."}
+{"text": "The illustrated case is a 55-year-old asymptomatic multiparous post-menopausal female who was diagnosed to have microscopic hematuria on serial urine cytology examinations . There were 5-10 isomorphic erythrocytes per high-power field, without any proteinuria or pus cells in any examination. There was no history of diabetes, hypertension or any major illness in the past. The complete blood cell count, serum biochemistry and coagulation profile were within normal limits. Ultrasound examination did not reveal any definite structural abnormality in the urinary system. The multi-detector computed tomography examination, however, showed significant dilatation of the left ovarian vein with formation of pelvic varices in the broad ligament  and b. Tet al., suggest the abnormal angle of the origin and course of the proximal part of SMA to be responsible for this phenomenon.[Nutcracker phenomenon was first described by de Schepper in 1972, as compression of the LRV, usually in the fork between the aorta and superior mesenteric artery.2 The abnoenomenon. The roleenomenon. Aberrantenomenon.et al., described the role of LRV diameter and peak flow velocity measurements on B-mode ultrasound and Doppler respectively.[Kim ectively. The LRV ectively. The B-moThe patients with nutcracker phenomenon are usually young and thin. The nutcracker phenomenon may be an important cause of pediatric varicocele. In children, the role of Doppler US for the diagnosis of nutcracker phenomenon is affected by the fact that the LRV sampling area is smaller. Apart from this, the Doppler angle is larger in children than in adults. The high reno-caval pressure gradient may lead to retrograde blood flow from the LRV into the testicular vein. Doppler spectral analysis and venography of the LRV are useful in children with varicocele to look for the presence of underlying nutcracker phenomena. Compress9The MDCT provides an important noninvasive alternative to diagnose this condition based on LRV dimensions, reflux of contrast medium from LRV into left ovarian vein and delineation of collaterals. The LRV dimensions are measured as antero-posterior dimension in the axial planes at two different points. The first measurement is done for the lateral segment of LRV at a point where the left ovarian vein drains into the LRV. The second measurement is done for the aorto-mesenteric segment of LRV at the narrowest point where the LRV passes between the aorta and the superior mesenteric artery. The dimensions are expressed as LRV ratio which is the ratio of measurements in the lateral segment and aorto-mesenteric segments of LRV. Reflux iThe treatment of NS significantly depends on the patient symptoms and urine microscopy findings. The patients with mild hematuria can be offered conservative treatment with close follow-up. The illu21In the absence of structural causes, LRV hypertension or \u2018nutcracker syndrome\u2019 should always be ruled out in a case of macroscopic or microscopic hematuria. The phase-contrast urine microscopy shows isomorphic erythrocytes. B-mode ultrasound including Doppler assessment and MDCT are the primary imaging modalities. Invasive venography may be required for further evaluation. It is, however, important to differentiate asymptomatic dilatation of LRV (nutcracker phenomenon) and \u2018nutcracker syndrome\u2019 presenting with gross or microscopic hematuria, orthostatic proteinuria, varicocele and hypertension. The diagnosis of LRV hypertension should be made with caution in multiparous women. As illustrated in the present case of \u2018hematuria of unknown origin\u2019, the MDCT diagnosis of LRV hypertension is feasible in clinical practice if due attention is paid to finer imaging details and the index of suspicion is high."}
+{"text": "We recently set standards for gender-specific medicine training as an integrated part of the GP training curriculum. This paper describes the programme and evaluation of this training.The programme is designed for GP registrars throughout the 3-year GP training. The modules emphasize interaction, application, and clinically integrated learning and teaching methods in peer groups. In 2005 - 2008, after completion of each tutorial, GP registrars were asked to fill in a questionnaire on a 5-point Likert scale to assess the programme's methods and content. GP registrars were also asked to identify two learning points related to the programme.The teaching programme consists of five 3-hour modules that include gender themes related to and frequently seen by GPs such as in doctor-patient communication and cardiovascular disease. GP registrars evaluated the training course positively. The written learning points suggest that GP registrars have increased their awareness of why attention to gender-specific information is relevant.In summary, gender-specific medicine training has been successfully integrated into an existing GP training curriculum. The modules and teaching methods are transferable to other training institutes for postgraduate training. The evaluation of the teaching programme shows a positive impact on GP registrars' gender awareness. Gender-specific medicine (GSM) studies the relationship between gender and health. It is concerned with the promotion of equal opportunity and fair treatment of men and women and aims to redress current gender disparities or gender bias in the healthcare system. Both genders will benefit when GPs deliver healthcare based on education on the role of sex and gender in health and illness. Various studies have revealed the importance of considering sex and gender issues when providing patient care in general practice. For instance, gender has implications in the presentation of chronic obstructive pulmonary disease (COPD). Women with COPD show higher levels of anxiety and depression and worse symptom-related quality of life than their male counterparts,3 A healIn the last decade, the field of gender-specific medicine has focussed on training educational professionals, and on reforms aimed to include gender aspects in the curricula of medical schools, and in health research. -12 AlthoThe influence of gender of physician and patient occurs on all levels of medical encounters and it often comes about unintentionally. Education on knowledge about gender-related processes will likely not be enough to prevent gender disparities in patient care. It is also necessary to address physicians' attitudes and preconceived notions about roles of men and women. The complexity of teaching the subject gender necessitates to go beyond biomedical factors and to include the social context of men and women. That acknowledgement implies that a full integration of a training programme about gender issues may be a necessary condition to getting change and acceptance among GP registrars. One of the methods medical educators can use to ensure future GPs' competency in gender issues is to offer training over time, with reflection on the action, and integrated into clinical practice.  InteractRecently, the department of Women's Studies Medicine and the Institute of Postgraduate Training in General Practice at Radboud University Nijmegen Medical Centre launched an initiative for the development of an interactive, long-term training programme in gender-specific medicine. Our goal as teachers is to move GP registrars along a gender sensitivity scale and to (a) enable GP registrars to understand basic concepts of gender, (b) sensitize GP registrars to gender as determinant of health, and (c) ensure that GP registrars attain a reasonable understanding of why gender-specific medicine is relevant. These goals are in line with recent consensus statements which underscore the significance of gender as a key determinant of health and advocate gender equity,20We developed and evaluated an integrated, interactive training programme to teach GP registrars about gender-specific medicine. In this paper we describe and evaluate this training programme.The training programme was developed by four GPs with expertise in and a commitment to gender issues. They synthesized and outlined the training programme in close collaboration with the department of Women's Studies Medicine at Radboud University Medical Centre. The group worked on the specific areas to be covered and educational methods to be used. The experts have been guided by the following questions:1. Is the topic relevant for and frequently seen by GPs?2. Does the topic have gender-specific aspects that impact practice?3. Is there underlying evidence for the gender-specific aspects?They selected cross-disciplinary topics: cardiovascular disease, depression/anxiety disorders, urinary incontinence, addiction to alcohol or benzodiazepines, domestic and sexual violence. They also included communicative aspects in the training programme. In addition, for each topic they defined a set of objectives, and translated the relevant information and evidence from literature into educational material based on the principles of problem-based learning. Our framework for gender needs assessment is partially based on gender concepts written by Phillips After the course, GP registrars will be able to:1. consider the ways in which gender aspects and differences have an impact on health and illness, and an understanding of how gender-related issues may bias the provision of healthcare.2. consider gender aspects and gender differences in epidemiology, presentation, diagnostic management, and treatment strategies in primary care.3. understand the impact of doctors' gender in relationships and communication, and model reflective practice around gender issues.Teaching and learning in general practice takes place primarily at work. The key to productive learning in postgraduate training is to focus on the experiences of GP registrars in their practice and to connect theoretical education to these experiences The tutoReflection on gender issues supports GP registrars to understand complex situations by considering them in a larger context, and to identify their particular needs. ,26 RefleWe work in small-group sessions with 10 to 15 GP registrars who are familiar with each other to facilitate comfort. We used a mix of video-consultations, paper cases, role plays with simulated patients, and reading gender-related articles or narratives. We choose different methods of learning over time as a variety of educational experiences can be more stimulating.Our GP-supervisors have special skills and specific expertise in teaching gender-specific medicine. They know available resources on gender-specific medicine. Furthermore, they are familiar with resistance and know how to cope with defensive postures of GP registrars when they touch gender issues.The training programme consists of five 3-hours tutorials. An introductory tutorial discusses the intent of the course including the basic concepts of gender, and four specific tutorials address clinical topics in general practice. The sequence used within the tutorials proceeds from an introduction of the topic and an icebreaker exercise, followed by reflection upon experiences and stimulation of self-assessment, to an interactive assignment with a plenary discussion. Typically, the tutorial ends with an overview of the knowledge acquired.Tutorial one and two of our training programme are followed in first year , tutorial three in second year , and tutorial four and five in third year  of GP training. The key features of each tutorial are shortly outlined hereafter and the main factors are presented in table gender and socialization, introduces the concepts of gender and sex. The purpose is to initiate a gender issue perspective into GP registrars' medical encounters. For example, gender differences in life experiences and the influences of family, peers, and media on gender roles. Factors of gender-related attitudes and themes with regard to doctor-patient encounters are discussed to help facilitate a heightened level of gender awareness.Tutorial one, gender and communication, focuses on eliciting the influence of gender on doctor and patient communication and how stereotyped expectations of men and women can affect doctor-patient relationships. Gender differences are addressed that can cause misunderstanding and that can hamper communication between dyads of men and women. An overview of potential gender-related pitfalls in doctor-patient communication is given.Tutorial two,(a) gender in depression and anxiety disorders, and (b) abuse of alcohol or benzodiazepines. In this tutorial we address one's own beliefs, norms and values with regard to gender that can influence the provision of care to others. Also we focus and clarify the differences of social expectations for appropriate behaviours of men as compared to women as is the case for alcohol consumption.Tutorial three consists of two parts of one and a half hour each: gender differences in cardiovascular disease (CVD) and urinary incontinence (UI). Here, we explain the persistent gender differences in cardiovascular disease and the potential biases in the care for patients with CVD such as the stereotypical conceptualisation of CVD as a male disease. GP registrars are taught the importance to reflect on their own and others interpretations, reactions, and conduct in patient care with regard to coronary risk factors in men and women. We address gender differences in patients' beliefs with urinary incontinence for example despite incontinence in men being less severe they experience more distress than women.Tutorial four deals with recognizing and responding to sexual abuse, addresses sexual violence, a serious and widespread problem for women with a number of social and gender-related barriers that make it hard for GPs to identify such abuse. For example doctor's availability for abused women differs by gender as female doctors tend to restrict their availability due to distress it brought about and male doctors because of time constraints.Tutorial five, p < 0.05) was assessed with the use of Chi square test. Similarly, the learning points were evaluated after each tutorial. They were coded and analysed according to the three objectives of the course by the first author (PD).After each tutorial, GP registrars were asked to indicate their level of agreement with initially 5 and later 7 statements to evaluate the course. Each statement was designed to assess the quality of and their opinion on the learning and teaching methods, the perceived relevance for practice, and the usefulness of the applied knowledge. GP registrars' participation was voluntary. We did not assess demographic features with the exception of their sex. We used Likert scales where 1 = totally disagree, and 5 = totally agree. Data were analysed in the SSPS 16.0. Answers were dichotomized so that a response of 1, 2 or 3 suggested a rejection of the program and a response of 4 or 5 implied acceptance of the programme. Significance . 32,8% of the GP registrars were male (n = 145), 64,7% were female (n = 286). 11 GP registrars did not disclose their sex .2 = 2.42; ns). Gender-specific information provided in the programme was highly beneficial to their practice . There were no significant gender differences between the evaluations of the programme but female GP registrars valued the programme consistently higher than male GP registrars.The methodology and the supervisors of the GSM programme were well received by the GP registrars. GP registrars' evaluation of the learning methods was in average positive. Overall, more than 80% of the GP registrars positively rated the learning methods used in the programme, the supervisor's role and the approach of the topic. Also, GP registrars appraised gender issues as significant for their learning programme . General comments included an increasing awareness of gender issues in general practice or gender-related disease presentation. The learning points dealing with gender bias were about diagnostic management, current lack of gender knowledge and dealing with delicate situations as sexual violence. The theme \"understanding of gender bias in healthcare\" was almost equal mentioned by male and female GP registrars . Also, both male GP registrars as well as female GP registrars  described learning points concerning differences between male and female patients and doctors in communication and working patterns. There were remarks about traditional gender roles of doctors and patients in their consultation e.g. in the perception of disease presentation.GP registrars noted 743 learning points on 442 readable evaluation forms. Three main themes were identified in the GP registrars' learning points: Although the evaluation did not confirm negative attitudes about the gender-specific programme on a regular basis, GP registrars did report negative comments on the evaluation forms. For example, some male as well as female GP registrars were of the opinion that the teacher occasionally focussed too much on the backlog of women. Both male and female GP registrars made negative comments on the evaluation forms from time to time that fit into all the types of resistance against gender issues .This paper describes the development and pilot-evaluation of a teaching programme in gender-specific medicine for GP training. We present a successful implementation of a mandatory training programme, extended over time, and integrated in the full three-year period of the Dutch GP training. Both male and female GP registrars evaluated the training in gender-specific medicine positively and were satisfied with the education. GP residents considered the education relevant for their learning and daily practice. Based on these outcomes, it demonstrates the feasibility and usefulness of incorporating gender-specific medicine in the curriculum of GP training on a regular basis.To the best of our knowledge, this is the first programme for on-going teaching and learning of GSM in GP-training. Much of the literature on learning and teaching GSM has been descriptive in nature, providing objectives, goals and theoretical models but not offering mandatory training ,28 A laceither women or men in teaching gender issues in medical education[Our GSM teaching programme is aimed at all GP registrars and it intends to offer a suitable framework to address gender-specific medicine. Therefore the teaching programme provided a comprehensive overview of medical conditions pertinent to both men and women in primary care. Usually it is education,34 We foeducation Nevertheeducation,37 IntroWe feel that the success of our teaching programme is also based on the light the programme sheds on the complex way in which men and women are advantaged and disadvantaged by biological and social factors in health issues and the successful clarification of it by the programme. The programme captured the specific gender-related processes and pathways that leads to health outcomes and emphasized strongly on gender socialization. It went beyond the biomedical framework and discussed gender-related life-experiences concerning health and disease. ,39 We exThe training programme as presented seems to work well but we have to point out some limitations. Our evaluation is not rigorous partly because of the low response rate. The assessment and ideas of the GP registrars who did not complete the survey, their assessment of the programme, and indeed their ideas about gender, may differ from those of the GP registrars who did complete the survey. Also, the evaluation did not include a sound assessment of the acquired knowledge and skills. This limits the scope of the evaluation of the programme. Demographic data of non-responders are not available, so comparisons between responders and nonresponders could not be performed. Furthermore, some of our GP registrars who were undergraduate students at Radboud University Nijmegen Medical Centre already have had the opportunity to learn about GSM by following courses in the undergraduate curriculum. Those GP registrars may therefore be somewhat more aware of gender-related issues than GP registrars who were undergraduate students at universities elsewhere.The next step in incorporating GSM in medical education is to take new initiatives to teach also GP trainers and to include gender-related issues in GP registrars' examinations. GP trainers entrusted with the educational supervision of GP registrars have a special responsibility for inculcating the principles of good medical practice including gender-specific medicine. If the GP trainer does not share these new insights, it is hard to implement change for GP registrars. ,43 Also,Teaching and learning gender-specific medicine is feasible in GP training. GP registrars found gender-specific medicine important and interested to learn. We recommend and encourage stakeholders of medical schools to provide learning opportunities in GSM to GP registrars.The authors declare no financial or non-financial competing interests other than they are members of department of primary care (Nijmegen) and social medicine (Maastricht) and wish to ensure a sustainable supply of general practitioners.TLJ developed the design of the study and the training programme. PD carried out the analysis and wrote the first draft of the manuscript. All authors were involved in critically revising the manuscript, and have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "During the last decades research has reported unmotivated differences in the treatment of women and men in various areas of clinical and academic medicine. There is an ongoing discussion on how to avoid such gender bias. We developed a three-step-theoretical model to understand how gender bias in medicine can occur and be understood. In this paper we present the model and discuss its usefulness in the efforts to avoid gender bias. In the model gender bias is analysed in relation to assumptions concerning difference/sameness and equity/inequity between women and men. Our model illustrates that gender bias in medicine can arise from assuming sameness and/or equity between women and men when there are genuine differences to consider in biology and disease, as well as in life conditions and experiences. However, gender bias can also arise from assuming differences when there are none, when and if dichotomous stereotypes about women and men are understood as valid. This conceptual thinking can be useful for discussing and avoiding gender bias in clinical work, medical education, career opportunities and documents such as research programs and health care policies. Too meet the various forms of gender bias, different facts and measures are needed. Knowledge about biological differences between women and men will not reduce bias caused by gendered stereotypes or by unawareness of health problems and discrimination associated with gender inequity. Such bias reflects unawareness of gendered attitudes and will not change by facts only. We suggest consciousness-rising activities and continuous reflections on gender attitudes among students, teachers, researchers and decision-makers. The concept of gender has been used in the social and humanistic sciences since the 1960's. It was originally introduced to designate how different societies and cultures interpret biological sex -5. The pIn medical research today, the term gender is often used wrongly as being synonymous with biological sex . SometimIn clinical medicine studies have shown unjustified differences in the investigation and treatment of male and female patients; measures that are not evidence-based. Most research on this subject has been about coronary heart disease ,16, but In addition to gender bias in the investigation and treatment of patients gender bias occurs in medical education, professional careers, and in medical research as well. Analyses of medical textbooks, curricula, education material and examination questions have revealed stereotypical gender patterns and even open patriarchal views -32. In aAs a contribution to the ongoing discussion on how to prevent gender bias, the aim of this article is to describe a three-step theoretical model on how gender bias in medicine can occur and be understood, and to discuss the usefulness of the model in the work to detect and understand gender bias.In our model we use results from three of our empirical gender studies conducted in Sweden as examples. Below these studies are briefly described.1. Physician teachers' knowledge and attitudes are important for the addressing of gender issues in medical education. At the Ume\u00e5 Medical School in Sweden, we surveyed 303 (29% women) physician teachers' attitudes towards gender issues via a questionnaire. They were asked to rate their degree of agreement with five statements about the importance of gender in consultation, in tutoring students, and in contact with colleagues, with staff and in research. Women assessed gender more important in all these professional relationships than men did ,41. Ther2. In Sweden there are no recommendations to treat irritable bowel syndrome (IBS) differently for women and men patients. Still, in a paper case about IBS in a national exam for 289 Swedish residents there were different suggestions about investigations and treatment depending on whether the patient was described as a woman or a man. For example, more x-rays of the colon were suggested for men, while more tranquilizers and advice about life style were suggested for women .3. In a research assessment study, Swedish physicians, especially female physicians, upgraded the scientific accuracy of a fictive qualitative research abstract if the author was said to be a woman . These rThe results of our three empirical gender bias studies brought our thoughts to the long history of feminist theories on the meaning of gender difference and sameness and how it interacts with gender equity and inequity ,46. In tThe concepts of sameness/difference and/or equity/inequity are common in gender theory and research, but there is an ongoing discussion, sometimes rather confusing, about how to define, interpret, and apply them.On an ontological level, the question about sameness/difference is whether men and women are seen as essentially different or not. A question on the practical level, illustrating the ontological dimensions of sameness and difference, would be: Should women have the same rights as men and have access to all sections of society, for example to become surgeons, because they are human beings just as men are, with individual and divergent skills and interests, or because they represent other values than men and therefore have something to add? This question also illustrates that 'sameness', as used in the context sameness/difference, corresponds to diversity within gender.On the epistemological level there has been a discussion whether sameness/difference are fruitful analytical tools for gender researchers. Difference has been associated with essentialism, and sameness with the risk of reproducing the male norm since 'same' is easily understood as 'same as a man'. A practical view has been to use the concepts as means to bring about knowledge that can be used for change, not as goals. This is the way we use them in our analysis of gender bias in medicine.The discussion on sameness/difference has sometimes been confused when the term 'sameness' has been exchanged by 'equality'. The opposite of equality is inequality and not difference. The terms equality/inequality relate to whether or not individuals, irrespective of if they are a man or a women, have the same value and are free to develop their personal abilities without (gendered) limitations ,47. It iIn our analysis in this paper we were also inspired by the work by Ruiz and Verbrugge in 1997 , where aIn our model we develop the theories of gender as sameness/difference and/or equity/inequity in three steps as a way to understand gender bias in medicine. We illustrate our analysis by providing typical examples from the studies above. All quotations in step 1 and step 2 in the model are comments from physician teachers in study 1. Each quotation is identified by gender, age and specialty group of the informant. The specific specialty is not presented for anonymity reasons. Two crossing axes are the basis of the model  arise where divergent assumptions about women and men can be identified figure .\"Men and women doctors can function equally well at our ward. Still, for me, it is easier to talk to male colleagues because we share views on how to investigate and treat patients in most cases. There are fewer irrational elements than in contact with female colleagues.\" I. Women and men are on equal footing but different: \"I like to think that we are all equal human beings and can understand each other.\" II. Women and men are on equal footing and the same: \"It is a shame that women, who are more sensitive and suffer more from pain, are often paid no attention to, and even humiliated, during consultations for pain.\" III. Women and men are different and there is inequity emanating from downgrading of women, women's duties and 'female' characteristics:\"I think I would be questioned more in my work by the patients if I were a woman. They never take me for a nurse\" IV. Women and men are much the same but there is inequity between them, emanating from differences in position, leading to gendered experiences and life conditions:The divergent assumptions about women and men described in step 1 can lead to different approaches to the subject of gender figure .\"At my workplace all patients are treated in the best way possible and regardless of gender. And, competence, not gender, is what counts at our department.\" I and II. Emphasizing equity and denying that gender is a basis for inequities: \"In research about differences between women and men there is a tendency to generalize from mean values and forget the distribution within the groups. Are men and women really that different?\" II and IV. Emphasising diversity among women and among men and questioning gendered dichotomies: \"Female students are sometimes neglected and often underestimate themselves. This indicates that when tutoring you have to instruct female students 'how to fly' and male students 'how to land.\"' III and IV. Emphasizing inequity and working for change: \"Men and women can never really understand each other, they express themselves altogether differently.\" I and III. Emphasizing differences and accepting stereotype dichotomies about women and men as realities: The approaches to the subject of gender described in step 2 all run the risk of being manifested as gender bias figure .-In fields I and II equity between men and women is presumed. There is a blindness to gender differences in position, influence, life conditions and experiences, which are factors of importance for health and illness and also for the opportunity to make a career. This can lead to upgrading of men and downgrading of women. Perceiving that men and women are treated the same and have equal opportunities, as many teachers did in the open-ended answers , opens u-In fields II and IV sameness between and diversity within each gender is emphasized. Differences are apprehended as originating in power asymmetry and/or norms. The bias near at hand is to overlook differences connected with biology and disease, for example, differences between men and women in their reaction to drugs, in standard values for blood tests, or in symptoms of coronary disease . This in-In fields III and IV differences in position and power between men and women are acknowledged. In order to obtain equity, there is a risk of upgrading women in an inappropriate way. The women physicians who upgraded female authors in the abstract assessment study  demonstr-In fields I and III there is an unawareness of the influence of gendered norms and doing-gender processes. There is a risk that differences are assumed where there are none, due to un-reflected dichotomous thinking about women and men. The reasoning of the residents in the paper case study about IBS leading to a different outcome for male and female cases  , exempliResearch focusing on biological differences between women and men has produced useful research during the last decades very much on biological differences and has produced a lot of useful research on the subject. However, as illustrated in fields I and III, there is always the risk that small and insignificant differences are exaggerated or that differences are understood strictly in biological terms. Moreover, once knowledge of gender differences in a condition has been established, this might in fact cause gender-biased assessments of individual patients in clinical practice. The differences seen in research between men and women on the group level are often smaller than the differences between individual men in the male group and individual women in the female group. If that fact is disregarded stereotyped generalizations are quickly made. We have labelled this mechanism 'knowledge-mediated gender bias' .This model based on assumptions of difference/sameness and equity/inequity between women and men can be useful in understanding the processes leading to gender bias in medicine. Gender bias can be comprehended as originating in unawareness of gendered norms and doing-gender processes and/or of differences between women and men regarding positions in society, life conditions, life experiences and biology. Consequently, gender bias in medicine can arise from assuming sameness but also of equity between women and men when there are genuine differences to consider in biology and disease, as well as in life conditions, experiences and power. However, it can also arise from assuming differences when there are none, when or if dichotomous perceptions about women and men are understood as valid.As is illustrated in our model no assumptions about women and men and no approaches to the subject of gender are free from the risk of gender bias. There can be a delicate balance between identifying which differences between women and men are valid and need to be considered to avoid injustices, and which assumed differences are stereotypical ideas leading to bias.Our analysis on gender bias is an extension of the one made by Ruiz and Verbrugge in 1997: \"Like a polarised lens gender bias can arise from two views-one assuming equality where there are genuine differences and the other assuming differences where none may exist\" . In the Nowadays studying the biological differences between women and men is a growing research field  This calls for consciousness-rising activities like role-plays and case discussions from a gender perspective in educational programs for students as well as for medical faculty, physicians and medical decision-makers and department chairs. Continuous reflections on gender attitudes should be encouraged in such programs.However, these types of bias are associated with attitudes about gender and will not change by facts only. Quite often there is an unawareness of gender attitudes and a belief in objectivity and neutrality among physicians . One of The conceptual thinking about gender bias derived from the model can provide ideas and instruments to use for physicians, teachers and researchers in order to handle unawareness of and resistance to gender issues in medicine and to prevent gender bias. We will give some examples:In clinical situations it is important to evaluate if the investigation and treatment would have been different had the patient been of the opposite sex. If so, is the difference evidence-based or an expression of gender bias? It is also advisable to address gender questions now and then at sittings and rounds at hospital wards and to use the model to perform local follow-ups on investigations and outcome of different diseases and disorders in relation to gender.In medical education gender issues should be introduced at an early time at medical school and also be part of the educational programs within each specialty. The students get a better understanding of sex and gender and gender bias by being introduced to the theoretical model. It can be used as a basis and starting-point in discussions about diseases where we know that gender bias has been reported for example in coronary heart disease and in depressions. Specific work-shops and interactive discussions with actor patients can be used to address gender attitudes.As has been discussed by Bickel et al many young women entering medicine and most men conclude that gender equity is achieved by now . They arThe conceptual ideas of the model can also be helpful when assessing and evaluating a variety of documents such as research programs, research applications or health care and health promotion policies. If researchers, members of research councils and decision-makers get acquainted with the model, they would ask questions like: Which (implicit) assumptions about women and men underlie the document and what type of bias can these assumptions bring about? The same type of questions could be asked concerning individuals, including oneself. How does the individual perceive sameness/difference and equity/inequity between women and men? Which type of gender bias does he/she run the risk of because of these assumptions?The model can also be applied to and used as a tool to analyse and understand other hierarchical systems than gender, for example class and ethnicity.Our model illustrates that gender bias in medicine can arise from assuming sameness and/or equity between women and men when there are genuine differences to consider in biology and disease, as well as in life conditions and experiences. Gender bias can also arise from assuming differences when there are none, when and if dichotomous stereotypes about women and men are understood as valid. This conceptual thinking can be useful for discovering and preventing gender bias in clinical work, medical education, career opportunities and documents such as research programs and health care policies. The strength of the model is the combined focus on knowledge and awareness \u2013 are men and women the same/different and are we aware of the gender order? The model can be expanded to other categories of diversity and opens up for future research and other consciousness-rising activities.The authors declare that they have no competing interests.All three authors contributed to the conception, design, and analysis of this article. GR drafted the article and KH and EEJ revised the article critically for important intellectual content. All authors read and approved the final manuscript."}
+{"text": "The CTLs showed cytotoxic activity against not only the peptide-pulsed target cells but also HLA-A24 colorectal tumour cell lines that endogenously overexpressed HER2 / neu. The antigen-specificity was confirmed by cold target inhibition assay using HE1-pulsed target cells. In summary, HER2 / neu peptide, RWGLLLALL, may contribute to the induction of antitumour immunity with the peptide-based immunotherapy for the colorectal carcinomas. \u00a9 2001 Cancer Research Campaign http://www.bjcancer.comHER2 / neu is a potential antigen candidate for immunotherapy because of its correlation to a poor prognosis and high expressions in many kinds of epithelial tumours. Especially in the colorectal carcinomas, the higher expression of HER2 / neu is recognized in metastatic regions as well as in primary sites. Several CTL epitopes restricted by HLA-A2.1 and -A3 were identified so far, however epitopes restricted by HLA-A24, that is one of the most common allele in Japanese and Caucasians, have not been identified. In this paper, we showed identification of a CTL epitope peptide of HER2 / neu restricted by HLA-A24. HLA-A24 binding peptides selected by an analysis based on HLA-A24 binding motifs were determined for their binding affinities to HLA-A24 molecules. The peptide with a sequence of RWGLLLALL (position 8\u201316) named HE1 showed the highest affinity. We induced CTLs from CD8"}
+{"text": "At a crossroads, global public health surveillance exists in a fragmented state. Slow to detect, register, confirm, and analyze cases of public health significance, provide feedback, and communicate timely and useful information to stakeholders, global surveillance is neither maximally effective nor optimally efficient. Stakeholders lack a globa surveillance consensus policy and strategy; officials face inadequate training and scarce resources.Three movements now set the stage for transformation of surveillance: 1) adoption by Member States of the World Health Organization (WHO) of the revised International Health Regulations (IHR[2005]); 2) maturation of information sciences and the penetration of information technologies to distal parts of the globe; and 3) consensus that the security and public health communities have overlapping interests and a mutual benefit in supporting public health functions. For these to enhance surveillance competencies, eight prerequisites should be in place: politics, policies, priorities, perspectives, procedures, practices, preparation, and payers.To achieve comprehensive, global surveillance, disparities in technical, logistic, governance, and financial capacities must be addressed. Challenges to closing these gaps include the lack of trust and transparency; perceived benefit at various levels; global governance to address data power and control; and specified financial support from globa partners.universal, global access to interoperable public health information when it\u2019s needed, where it\u2019s needed. This vision mitigates the tension between two fundamental human rights: first, the right to privacy, confidentiality, and security of personal health information combined with the right of sovereign, national entities to the ownership and stewardship of public health information; and second, the right of individuals to access real-time public health information that might impact their lives.We propose an end-state perspective for comprehensive, effective and efficient global, multiple-hazard public health surveillance and describe a way forward to achieve it. This end-state is st century.The vision can be accomplished through an interoperable, global public health grid. Adopting guiding principles, the global community should circumscribe the overlapping interest, shared vision, and mutual benefit between the security and public health communities and define the boundaries. A global forum needs to be established to guide the consensus governance required for public health information sharing in the 21 At a crossroads and fragmented, global public health surveillance is defined as the ongoing systematic collection, analysis, and interpretation of health data, essential to the planning, implementation, and evaluation of public health practice, closely integrated to the dissemination of these data to those who need to know and linked to prevention and control ,2. GlobaWhile the promise and intuitive added value of integrating public health surveillance highlighted in earlier reports  still reNew movements have now come to the fore to expedite tomorrow\u2019s digital, paperless public health surveillance workplace and promote comprehensive surveillance. There is strategic merit in conceiving a vision of the end state for comprehensive, global, multiple-hazard public health surveillance; one that acknowledges the challenges and identifies steps to overcome them. We propose here a perspective for comprehensive, effective and efficient global, multiple-hazard public health surveillance and describe a way forward to achieve it.Three important movements now set the stage to achieve comprehensive, effective and efficient global, multiple-hazard public health surveillance:1. The adoption of the revised International Health Regulations [IHR(2005)] by all World Health Organization (WHO) Member States, which includes national obligations to achieve a set of core surveillance and response capacities to prevent the international spread of disease ;2. The maturation of information sciences  capabilities and the remarkable penetration of information technologies (IT) to the most distant parts of the globe ; and3. A consensus that both the security and public health communities have overlapping interests and mutual benefits to collaborate in supporting the development of essential public health functions, especially public health surveillance .These movements offer the practical opportunity to empower, enhance, and enjoin global public health surveillance, as never before.The IHR(2005) constitutes the WHO\u2019s legal and operational framework for activities around prevention and control of the international spread of disease, regardless of origin or intent . The adoption of IHR(2005) by WHO Member States challenges them in a new and urgent way to assess and strengthen core surveillance capacities . This agst century global public health surveillance digital (paperless) workplace. By identifying and defining standards and making them easier to apply, PHI adds value to efforts already performed by public health practitioners at all health levels. This added value derives from the inherent ability of PHI to facilitate digital communication in a more robust, efficient, and standards-based manner.Information sciences and IT both used in a public health setting  are a means to an end; the end being the achievement of effective and efficient public health surveillance. Concerned with effective and efficient collection, collation, transmission, analyses, visualization, storage, and retrieval of electronic data, the scientific discipline of PHI has emerged to leverage the inherent merits found in IT and computer science technology. But this merger yields more than the sum of its individual parts\u2014it has the potential to enhance the transformation to a 21Over the past ten years, many public health processes have been improved by PHI solutions . These iPHI also offers paper-to-digital conversion techniques and tools that empower and enable epidemiologists and surveillance practitioners to work better, faster, and cheaper  by provist century\u201d, addresses the interface of health and security [The third movement aligns the overlapping, mutual interests of the security and public health communities around the domain of public health surveillance. It forces policy discussion, increases focus, as well as provides sources to drive progress. The WHO\u2019s 2007 World Health Report, \u201cA safer future: global public health in the 21security . The folAdditionally, World Health Assembly Resolutions 54.14 and 55.16, respectively, requested the WHO to \u201cprovide technical support to Member States for developing intervention programmes that prevent epidemics and respond to communicable disease threats and emergencies, particularly with regard to epidemiologic investigations, laboratory diagnoses and community and clinical management of cases\u201d and \u201cto continue, in consultation with relevant intergovernmental agencies and other inter national organizations, to strengthen global surveillance of infectious diseases, water quality, and food safety, and related activities such as the revision of the International Health Regulations and development of WHO\u2019s food safety strategy, by coordinating information gathering on potential health risks and disease outbreaks, data verification, analysis and dissemination, by providing support to laboratory networks, and by making a strong contribution to any international humanitarian response, as required.\u201d ,19.The WHO plays a role in the international response to accidental or deliberate use of biological and chemical agents or radio-nuclear materials that affect health. They have a vision for international public health security, ready to respond collectively to the threat of epidemics and other public health emergencies, both natural and man-made. Correspondingly, WHO has adopted mechanisms for supporting countries and strengthening the international response .Among global public health stakeholders there now seems to exist both the political will and an acknowledged, if yet undefined, overlapping interest and mutual benefit to achieving comprehensive, effective and efficient global, multiple-hazard public health surveillance. There are areas where security and public health interests are seen to be at odds. However, some countries may not wish to share public health information with other countries that may be used for the security or economic advantage of the other nation. These perceptions have been a challenge to global public health surveillance in recent years and may be considered a key challenge going forward.In order for these three movements to empower and enhance surveillance competencies and lead to the end-state perspective described here, eight prerequisites or conditions should be in place Figure . They inMany challenges (gaps and impediments) exist between the current and end-state described here. They can be disaggregated into technical, logistic, governance, and financial domains. Within the technical domain, single (silo) categorical, disease-specific surveillance systems create the situation where public health practitioners cannot determine relationships between health conditions or co-morbidities . For example, new tuberculosis (TB) cases could be missed because HIV/AIDS data are not cross-matched with the TB registry. In this situation, surveillance becomes ineffective, because it is incomplete; events that represent a public health threat or that could inform about a potential public health threat are missed. Many times disease-specifi c silos are created and sustained by single funding streams and corresponding program obligations and priorities. They are often then maintained by program corporate cultures. Secondly, appropriate public health information is often not collected because what needs to be measured is often not known ,21. In tThere is a critical gap in global governance, under which all Member States would agree to function. Countries now collect and communicate public health information within and outside their natural border. The amount and type of information and willingness to collaborate varies from region to region. While there is a justifiable need to share important public health information that might impact neighboring states , there still exists some uncertainty about what types of public health information are appropriate, how they should be communicated, and how quickly they should be shared.Challenges (or impediments) to bridge the gaps include both the lack of trust and perceived benefit at various levels, the lack of a global governance model to address power and control of public health information, and the lack of focused financial support from global partners.st century involves empowering and enabling existing public health surveillance systems to interoperate . Surveillance systems should also be enjoined (or integrated \u2013 meaning streamlining data collection among systems to reduce redundancy of the data collected where it makes sense to do so). These activities will lead to universal global access to interoperable public health information when it\u2019s needed, where it\u2019s needed. In the process sense, interoperable and integrated public health information means achieving effective and efficient public health business practices and workflow empowered and enabled to be better, faster, and cheaper by IT.Enhancing global public health surveillance in the 21Operationally, comprehensive, global public health surveillance means one sign-on access to authorized and necessary public health information. Public health information includes other information \u2013 when combined with health-related information \u2013 that provides a picture of population or community health. Consumers should have one-stop shopping for public health information; and there should be one source for integration of public health information for all users. This also means one common set of standards for \u201cbringing together\u201d or interoperating existing or new data streams. Most importantly, one size does not fit all.Demographic, clinical, laboratory and other information about patients with diseases of public health significance should only have to be entered once, saving time and resources. The local, district, national, or inter national health authorities should be able to access real-time health outcome data and perform analyses or take timely and appropriate public health action based on that information. It should be stressed that keeping electronic health information private, confidential, and secure while automatically and immediately electronically communicating public health information to local public health authorities to satisfy mandatory public health reporting purposes is critical. Different reporting systems may be in existence depending on the types of data and information being reported, purpose and urgency of relaying the information, and where the data/information is being reported.This perspective embraces and mitigates the tension between two fundamental human rights. The first is the human right to privacy, confidentiality, and security of personal health information. This includes any informa-tion about individuals demonstrated to be related to health  and the right of sovereign, national entities to the ownership and authority over their citizens\u2019 public health information. The second is the human right of individuals to have real-time access to public health information that might impact their lives.th century replicated database model, a federated model of public health information sharing allows multiple participants to share data without having to give up ownership, thus accommodating universal access to public health information [Data management requires much more than investment in technology; it involves how data are created, stored, moved, used, and retired. As opposed to the 20ormation . A federhttp://cdc.confex.com/cdc/phin2009/webprogram/Paper21091.html)  their actions . Neither of us have financial relationships  likely to undermine our credibility. We have no conflicts for other reasons, such as personal relationships, academic competition, and intellectual passion.As a guest editor, Dr. McNabb will avoid selecting external peer reviewers with obvious potential conflicts of interest\u2014for example, those who work in the same department or institution as any of the authors. He has no personal, professional, or financial involvement in any of the issues he might judge.We both contributed to the conceptualization, writing, and editing of this manuscript. We will both defend its findings."}
+{"text": "IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.The Map kinase Activating Death Domain containing protein (MADD) isoform of the  IG20 gene, is essential for cancer cell survival and confers resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. TRAIL normally binds to death receptors-4 (DR4) and -5 (DR5) on cancer cells resulting in DR oligomerization and subsequent recruitment of the Fas associated Death Domain containing protein (FADD) and procaspase-8 to DRs Map kinase Activating Death Domain containing protein (MADD), a splice variant of the TRAIL is unique in that it generally does not adversely affect normal cells or tissues Our earlier studies have shown that MADD is one such anti-apoptotic proteinTo determine if MADD is expressed differentially we stained breast cancer tissue microarrays using a MADD reactive antibody IG20 isoforms, and MADD alone in MCF-7 and T47D cells, which expressed only MADD and Differentially Expressed in Normal and Neoplastic tissues Splicing Variant (DENN-SV) isoforms  . Our preisoforms . In contisoforms . Unlike vs 16E induced apoptosis) and MDA-MB-231  cells (vs 16E induced apoptosis)  cells  A\u2013B, butoptosis) . Down moRelative to control cells and cells treated with 16E-shRNA, cells expressing 13L-shRNA showed a significant reduction in their numbers . To diffvs. 16E induced apoptosis; P<0.001 in MCF-7, and P<0.05 in MDA-MB-231). Furthermore, combining MADD knockdown with TRAIL treatment resulted in enhanced apoptosis in both cell types (vs. untreated cells). MADD knockdown combined with doxorubicin treatment significantly increased apoptosis in MCF-7 cells  for up to 72 hours and determined DR expression on their surface. We did not find any increase in the expression of decoy receptors in both cell lines (data not shown). As shown in To determine if increased activation of the extrinsic apoptotic pathway contributed to increased death in cells devoid of MADD expression, we blocked the extrinsic pathway (i.e. prevent capase-8 activation) by expressing a dominant negative form of FADD (DN-FADD) in MCF-7 cells. As seen in MADD knockdown resulted in DR5 associated caspase-8 activation , which was further enhanced when MADD knockdown was coupled with TRAIL or doxorubicin treatment , while cIG20 gene, is significantly over expressed in breast tissues containing DCIS and invasive breast cancer, as well as in the three evaluated breast cancer cell lines. We confirmed the ability to down regulate MADD expression using shRNA, which resulted in spontaneous apoptosis that was further augmented by treatment with TRAIL or doxorubicin, a standard chemotherapeutic agent used in advanced invasive breast cancer patients. In the setting of MADD knockdown, TRAIL induced apoptosis was as effective as that induced by doxorubicin.We have previously shown enhanced levels of expression of MADD in multiple cancer tissues IG20 splice variants using exon-specific shRNAs have shown that the MADD isoform of the IG20 gene is required and sufficient for cancer cell survival Previous studies from our laboratory that employed selective knockdown of L, which is a TRAIL resistance factor L binds to FADD and prevents the recruitment of procaspase-8 and thus confers resistance to TRAIL-induced apoptosis While MCF-7 and MDA-MB-231 showed significant levels of spontaneous apoptosis upon MADD knockdown, apoptosis noted in T47D cells, although higher, did not reach statistical significance . The T47Because TRAIL induces apoptosis in cancer cells but not in their normal counterparts, it is an attractive candidate for cancer therapy. TRAIL and several agonistic antibodies that specifically bind to TRAIL receptors are currently being tested in clinical trials. A recent study demonstrated that the majority of breast cancer cell lines are very sensitive to TRAIL-induced apoptosis, suggesting that it could be used to treat recalcitrant breast cancers MCF-7 cells were less sensitive to TRAIL-induced apoptosis at every concentration tested, whereas the more aggressive MDA-MB-231 cells were relatively more sensitive, which positively correlated with the dose of TRAIL used (data not shown). In order to test a possible synergistic effect between MADD knockdown and TRAIL treatment, we deliberately treated cells with a suboptimal dose of TRAIL and were able to induce much higher levels of apoptosis in both MDA-MB-231 and MCF-7 cells with MADD knockdown .All known pro-survival functions of MADD have been associated with the extrinsic apoptosis pathway involving the death receptors. However, the impact MADD knockdown might have on doxorubicin induced apoptosis, which primarily activates the intrinsic apoptosis pathway, has not been investigated. We had shown previously that over-expression of the pro-apoptotic isoform, IG20pa which can act as a dominant negative MADD rendered HeLa and PA-1 cancer cells more susceptible to chemotherapeutic drugs and \u03b3-radiation- induced apoptosis We found that upon treatment with a low dose of doxorubicin, the cell surface levels of DR4 and DR5 were increased in MCF-7 cells, while DR5 was increased in MDA-MB-231 cells . This suMADD knockdown reduces the threshold for apoptosis as indicated by enhanced spontaneous death In summary, our results show that in addition to the enhancement of TRAIL-induced apoptosis, MADD knockdown could synergize the apoptotic effects of doxorubicin in breast cancer cells. These findings may have implications for developing novel strategies to treat breast cancer, overcome TRAIL resistance, and enhance conventional chemotherapies.IG20 (SVRRRIYDNPYFEPQYGFPPEEDEDEQGESYTPRFSQHVSGNR), was generated as described beforePhycoerythrin (PE) conjugated anti-DR4 (DJR1 clone), anti-DR5 (DJR2-4 clone) anti-DcR1 (Phycoerythrin (PE) conjugated DJR3 clone), anti-DcR2 (DJR4-1 clone), anti-TRAIL (clone RIK 2), and an IgG isotype controlused for FACS analyses were obtained from eBioscience. Anti-DR5and caspase-8 (1C12) antibodies used in western blots were obtained from Cell Signaling. Human TRAILwas obtained from Peprotech Inc. Doxorubicinwas obtained from Sigma-Aldrich. An antibody (13L antibody) that specifically binds to a peptide encoded by exon 13L of in situ (DCIS) specimens and 86 malignant breast tumors were provided by Dr. Wiley. All the human tissue sections were processed is from existing de-identified tissue samples only, and thus, this research qualified as a minimal risk to patients and their privacy, and approval for the use of these specimens with a exemption of consent was granted by the Institutional Review Board of the Office for the Protection of Research Subjects at University of Illinois at Chicago .Tissue microarrays (TMA) containing 44 benign breast lesions, 39 ductal carcinoma TMAs were incubated at 60\u00b0C for 1 hour, de-waxed, and immersed in alcohol. Sections were covered with a citrate-based antigen unmasking solution kits (Vector Laboratories) and subjected to microwave treatment for 10 minutes. Subsequently, the sections were incubated in 0.3% hydrogen peroxide for 30 minutes to inactivate endogenous peroxidase activity. Prior to application of the primary antibody, nonspecific interactions were blocked for 10 min using a blocking serum. Sections were incubated at 4\u00b0C overnight with an anti-MADD antibody at a 1\u2236800 dilution. The subsequent steps used the Vectastain Universal Quick kit (Vector Laboratories) and followed the manufacturer's instructions. Peroxidase staining was revealed with 3, 3-diaminobenzidine and then the sections were counterstained with Vector Hematoxilin QS. Sections were dehydrated, cleared and mounted in Vecta Mount mounting medium. Negative control staining was performed for each TMA slide without the primary antibody. Slides were scored for the intensity of staining in a semi-quantitative fashion independently by two pathologists: negative (0), weak (1+), intermediate (2+), or strong (3+).2.293T cells and MDA-MB-231 cells were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum(Invitrogen), 2 mM L-glutamine(Invitrogen), 100 units/ml penicillin-G, and 100 \u00b5g/ml streptomycin(Invitrogen). MCF-7 cells were cultured in Minimal Essential medium (Invitrogen) supplemented with 5% fetal bovine serum, 100units/ml penicillin-G, 100 \u00b5g/ml streptomycin and 5.86% sodium bicarbonate(Invitrogen). Both MCF-7 and MDA-MB-231 cells were obtained from Dr. Jonna Frasor . T-47D cell line was obtained from Dr. Andrei Gartel . All these cell lines were original purchased from ATCC . These cells were maintained in RPMI (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml of penicillin-G and 100 \u00b5g/ml of streptomycin. All cell lines were maintained at 37\u00b0C in a humidified atmosphere with 5% CO5) were placed into 60 cm culture dishes with cover glasses, and cultured overnight. The cells were fixed with acetone and permeabilized with 0.01% Triton X-100 followed by blocking with 1% BSA for 30 min at RT, Cells grown on cover slips were treated overnight with the rabbit anti-IG20 13L antibody at 4\u00b0C. Normal rabbit serum(Invitrogen) was used as a control. Subsequently, cells were stained with a biotinylated anti-rabbit antibody  and FTIC labeled streptavidin (Santa Cruz). The image was visualized and captured using LSM 510 META confocal microscope.MCF-7, MDA-MB-231 and T47D cells , and 1 \u00b5g of RNA was used for reverse transcription\u2013polymerase chain reaction (RT\u2013PCR) using the Super-Script One-Step RT\u2013PCR system (Invitrogen). cDNAs were synthesized at 50\u00b0C for 30 minutes followed by incubation at 94\u00b0C for 2 minutes. Subsequently, 35 cycles of PCR were carried out with denaturation at 95\u00b0C for 30 seconds, annealing at 55\u00b0C for 30 seconds and extension at 72\u00b0C for 1 minute; followed by a final incubation at 72\u00b0C for 7 minutes. The sequences of F2-B2 and GAPDH primers have been previously reported 4) and MDA-MB-231 (2\u00d7104) cells were plated in 6-well plates and transduced with lentiviruses. Seventy-two hours post-transduction, cells were stained with 100 nM tetramethylrhodamine methyl ester (TMRM) (Molecular Probes-Invitrogen) for 15 minutes at 37\u00b0C. Cells were collected, washed with cold PBS and analyzed using a BD FACS Calibur (BD Biosciences). Only GFP-positive (shRNA-expressing) cells indicating lentivirus expression were included in the analysis.Cells positive for TMRM have intact mitochondria, and a reduction in TMRM staining served as a reliable marker for apoptosis. The MCF-7 (3.5\u00d7104) and MDA-MB-231 (1\u00d7104) cells were plated into 6-well plates. Twenty-four hours later, cells were treated with different shRNA-expressing lentiviruses for 4 hours followed by the addition of fresh warm medium. The medium was replaced every 3 days. When control wells were confluent, they were fixed in ice-cold methanol and stained with crystal violet to assess viability and colony formation.MCF-7 (2\u00d7104) and MDA-MB-231 (1\u00d7104) cells were plated into 6-well plates. Twenty-four hours later, cells were treated with different shRNA-expressing lentiviruses for 4 hours and then replenished with fresh warm medium. Cells were counted 24 hours post-transduction and counted again on days 3 and 5. Cell death was determined by Trypan Blue (Invitrogen) exclusion.MCF-7 , washed once with PBS containing 0.5% bovine serum albumin, and allowed to stand in the same buffer for 10 minutes at 4\u00b0C. PE-conjugated antibodies were used to stain samples for 30 minutes at 4\u00b0C. A mouse IgG antibody was used as an isotype control. Cells were washed with PBS and analyzed using a BD FACS Calibur (BD Biosciences).Cells were lysed for 30 min at 4\u00b0C in a lysis buffer  containing a protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor (Sigma-Aldrich). Protein concentrations were determined using Bradford assay (Bio-Rad). Protein samples (50 ug per lane) were separated by SDS-PAGE using a 10% gel and transferred to nitrocellulose membranes, blocked with 5% skim milk and probed using corresponding primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. The protein bands were visualized by enhanced chemiluminescence.One-way Anova, the Tukey-Kramer test  or the student's t-test (for the remaining figures) was used to compare groups as indicated. A P-value of 0.05 was considered significant. Analysis was performed utilizing GraphPad Prism 5 statistical software and Microsoft Excel Software (version 2003). Results are expressed as mean \u00b1 SE.Figure S1MADD knockdown leads to decreased breast cancer cell survival. Upon MADD knockdown MCF-7 (A) and MDA-MB-231 (B) cells were plated in 6-well plates, transduced with indicated viruses and live cells were counted at indicated days after transduction using trypan blue. Data shown are representative of three different experiments. MCF-7 (C) and MDA-MB-231 (D) cells were cultured until control wells were fully confluent. At that time the cells were fixed with ice cold methanol and stained with crystal violet.(TIF)Click here for additional data file.Figure S2Doxorubicin treatment over time results in enhanced cell surface expression of death receptors. At each time point , MCF-7  and MDA-MB-231  cells were collected and stained with PE conjugated antibodies to DR4  and DR5  or an IgG isotype negative control antibody and analyzed by FACS, P<0.05 vs. un-treated group. Data are shown as the fold change in the absolute fluorescence intensity at various time points compared to zero time point. Summarized data from three independent experiments are shown.(TIF)Click here for additional data file."}
+{"text": "We show that liver regeneration is accompanied by large triglyceride and protein increases without changes in total phospholipids both in E2F2+/+ and E2F2\u2212/\u2212 mice. Remarkably, we found that the phenotype of quiescent liver tissue from E2F2\u2212/\u2212 mice resembles the phenotype of proliferating E2F2+/+ liver tissue, characterized by a decreased phosphatidylcholine to phosphatidylethanolamine ratio and a reprogramming of genes involved in generation of choline and ethanolamine derivatives. The diversity of fatty acids in total lipid, triglycerides and phospholipids was essentially preserved on E2F2 loss both in proliferating and non-proliferating liver tissue, although notable exceptions in inflammation-related fatty acids of defined phospholipid classes were detected. Overall, our results indicate that E2F2 activity sustains the hepatic homeostasis of major membrane glycerolipid components while it is dispensable for storage glycerolipid balance.Increasing evidence links metabolic signals to cell proliferation, but the molecular wiring that connects the two core machineries remains largely unknown. E2Fs are master regulators of cellular proliferation. We have recently shown that E2F2 activity facilitates the completion of liver regeneration after partial hepatectomy (PH) by regulating the expression of genes required for S-phase entry. Our study also revealed that E2F2 determines the duration of hepatectomy-induced hepatic steatosis. A transcriptomic analysis of normal adult liver identified \u201clipid metabolism regulation\u201d as a major E2F2 functional target, suggesting that E2F2 has a role in lipid homeostasis. Here we use wild-type (E2F2 The mammalian liver is a lipidostat that plays a central role in whole body lipid metabolism. Healthy livers regenerate efficiently after partial hepatectomy (PH). Successful regeneration requires replenishing all of the various epithelial and stromal cell types that compose the liver and a complex matrix remodeling to restore tissue homeostasis. Following resection of 70% of adult liver, 90-95% of the remaining hepatocytes leave their quiescent state and quasi-synchronously reenter the cell-cycle to begin regeneration E2F2 gene in hepatocytes led to a reduced rate of S-phase entry and to delayed liver regeneration, along with prolonged hepatectomy-induced steatosis. By contrast, other members of the E2F family (E2F1 and E2F4) are dispensable for this function 1Many cell-cycle regulators are known to contribute to liver regeneration -/- regenerating liver -/- hepatocytes, and that E2F2-regulated transcripts in the quiescent tissue could impact lipid homeostasis during regeneration.Transcriptomic studies have revealed that E2F factors not only regulate the expression of genes involved in cell-cycle control. Genes involved in differentiation, apoptosis, autophagy or metabolism are also regulated by E2Fs The role of E2F transcription factors in the regulation of bioenergetics and metabolism is being increasingly recognized in vivo. Using E2F2+/+ and E2F2-/- mice, we have studied two metabolically distinctive contexts: (i) quiescent tissue, when E2F2 transcriptional activity is presumably repressed and metabolic requirements are basal, and (ii) proliferating 48-h post-PH tissue, when the high metabolic demand that follows resection of 70% of the liver challenges E2F2 activity E2F2 gene activity is necessary to sustain normal PL composition in the adult mouse liver, affecting particularly the phosphatidylcholine (PC) to phosphatidylethanolamine (PE) ratio and the content of defined FA in glycerolipid and sphingolipid classes.Lipid homeostasis is a dynamic phenomenon in which import, synthesis, traffic, degradation and exportation are tightly regulated in order to maintain a specific lipid composition optimal for their individual functions. In the current work, we have investigated whether E2F2 contributes to sustain liver PL and FA compositions +/+  and E2F2-/- mice used in the described experiments are progeny of the first-generation backcross of the E2F2 null allele onto the C57Bl6 genetic background (mixed C57B16\u2236129Sv strain). All the mice were housed and bred in our mouse colony at the University of the Basque Country animal facility, and were genotyped as previously described -/- mice. We used three cohorts of 8 animals per genotype making a total of 24 E2F2-/- mice and their respective littermate controls (n\u200a=\u200a24) for the microarray analysis  hepatectomy was performed under isofluorane anesthesia according to the Higgins and Anderson method, as recently described -/- mice cohorts used here. Liver mass on day 2 doubled in the two groups, rising from \u223c30% to ca. 68% of the initial pre-PH hepatic index, which was also similar in the two experimental groups .Confirming previous observations 2 and stored at \u221280\u00b0C for mRNA analysis.Animal experiments were approved by the University of the Basque Country ethical committee CEEA in accordance with the guidelines of European Research Council for animal care and use. Liver aliquots were immediately homogenized for lipid analysis or frozen in liquid N2 until analysis.Livers (200 mg) were homogenized in ice-cold phosphate-buffered saline  immediately after surgery (0-h) or euthanasia (48-h) using a Polytron homogenizer . Protein content in homogenates was analyzed using a bicinconinic acid-based commercial reagent (Bio-Rad). Lipids were exhaustively extracted from homogenates as described before TAG was quantified by commercial kits used in clinical settings , according to the manufacturer's instructions. Phosphorus content of PL classes was determined after isolation by thin-layer-chromatography using chloroform\u2236methanol\u2236acetic acid\u2236water  as the solvent system in saturated chambers 2 in the presence of anhydrous methanol containing 0.5 N H2SO4 in Teflon lined, screw capped tubes 2 (30 ml/min) was the carrier gas. The FA peaks were detected with flame ionization detectors, operated in the dual-differential mode, and quantified by electronic integration (Varian Workstation).FA composition of lipids was determined by gas-chromatography of their FA methyl ester derivatives prepared by keeping the lipid samples overnight at 45\u00b0C under NTotal RNA was extracted from liver tissue (100 mg) using TRIzol reagent (Invitrogen), purified , DNase I-treated (Invitrogen) and re-purified according to the manufacturers' instructions. The purity and concentration of RNA was determined with a NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies). The 260/280 nm absorbance ratio of samples was in the 1.8\u20132.1 range. RNA integrity was further assessed by running samples in 1% agarose gel electrophoresis. cDNA was synthesized from 3.6 \u00b5g total RNA using the SuperScript III First-Strand Synthesis System for reverse transcription PCR kit (Invitrogen) according to the manufacturer's recommendations. For quantitative real-time PCR analyses of target mRNA levels TaqMan Gene Expression Assays (Applied Biosystems) were used. References of TaqMan assays were the following: Mm00839436_m1 for phosphatidylethanolamine N-methyltransferase ; Mm00772290_m1 for stearoyl-CoA desaturase 1 ; Mm01208299_g1 for fatty acid desaturase 2 ; Mm00522694_m1 for choline phosphotransferase 1 ; Mm01200029_g1 for choline/ethanolamine phosphotransferase 1 ; Mm02601848_g1 for phosphatidylethanolamine binding protein 1 ; and Mm00448878_m1 for serine palmitoyltransferase, long chain base subunit 2 . The relative quantities of each gene were determined by the \u0394\u0394Ct method. Normalization was performed using normalization factors computed by GeNorm for pyruvate carboxylase , transferrin receptor  and vascular endotelial zinc finger containing factor 1  mRNAs, as detailed previously t-test for unpaired data for comparisons between two individual data groups or for paired data for comparisons of FA composition values. A P\u22640.05 value was considered statistically significant unless otherwise stated.Lipid composition results are presented as means \u00b1 SD. Statistical analyses were performed using GraphPad Prism v5.02 for Windows . Significance was determined by the Student's +/+ and E2F2-/- mice liver transcriptomes that we had gathered in a previous study www.ebi.ac.uk/arrayexpress) under accession number E-MEXP-1413. The microarray analysis produced an unexpectedly large number of deregulated codes (2915), 59.5% of which were upregulated and 40.5% of which were downregulated in the liver of E2F2-/- compared with WT mice (P\u22640.01) (P\u22640.05) and Benjamini-Hochberg (P\u22640.01) multiple testing corrections  and peroxisomal  FA oxidation; FA synthesis  and precursor use of FA ; glycerolipid metabolism ; sphingolipid metabolism , as well as genes involved in steroid metabolism and bile secretion  and plasma lipid transport and metabolism . These genetic alterations argue that mice lacking E2F2 could display abnormalities in hepatic lipid balance. These findings prompted us to further investigate the role of E2F2 in liver lipid homeostasis in vivo, and to examine if the PL and FA composition of quiescent and regenerating (48-h) liver tissue was dependent on E2F2 gene activity.To better understand the connection between E2F2 activity and lipid metabolism, we re-evaluated microarray raw data of quiescent E2F2(P\u22640.01) . Gene Onrections . Remarkarections . We foun-/- mice, suggesting that the shift from PL to TAG as the dominant lipid component in the tissue ongoing regeneration is E2F2-independent.Mouse liver total TAG, PL and protein content (mg per g of liver) were assessed in quiescent state and after PH . Regener-/- mice. PC and PE were the major PL classes in quiescent WT liver, and accounted for \u223c52% and \u223c22% of PL content, respectively. Remarkably, we found that PC levels were lower (30%) and that PE levels were significantly higher (69%) in quiescent E2F2-/- liver tissue relative to WT counterparts. Consequently, the hepatic PC/PE ratio in quiescent tissue was much lower in E2F2-/- mice (0.987) compared to E2F2+/+ mice (2.375), indicating that E2F2 gene activity contributes to sustain the homeostasis of the two major membrane phospholipid classes when cells do not proliferate. Likewise, the ratio of choline-containing PL (PC+LPC+SM) versus amino-containing PL (PE+PS) was lower in E2F2-/- mice liver (2.138) compared to WT (1.002). 48 hours after partial hepatectomy, PC and PE percentages  but also constrains the normal response of the liver to regeneration-associated signals that control PC and PE homeostasis. By contrast to major PL classes, neither E2F2 deficiency nor regeneration signals affected significantly the relative abundance of the minor amino-phospholipid phosphatidylserine (PS), the choline-phospholipids lysophosphatidylcholine (LPC) and sphingomyelin (SM), phosphatidylinositol (PI), cardiolipin  or the intermediate phosphatidic acid (PA).To evaluate whether E2F2 activity has an effect on PL distribution we determined the relative proportion of each PL class by measuring the phosphorous content of the major PL classes after thin-layer-chromatography separation, both in quiescent and regenerating livers . Liver Pcentages  as well -/- mouse liver samples (-/- mice and the regeneration induced changes were E2F2-independent. The most abundant FA in the liver of the mouse strains used here were 18\u22361 (oleic acid) of the n-9 family, 18\u22362 (linoleic acid) and 20\u22364  of the n-6 family, 22\u22366  of the n-3 family, and the saturated fatty acids (SFA) 16\u22360  and 18\u22360 (stearic acid). At 48-h post-PH, SFA, mostly 18\u22360, decreased  and the monounsaturated fatty acids  increased , whereas total n-6 polyunsaturated fatty acids (PUFA) did not change. Interestingly, the marked decrease in the pro-inflammatory AA  was compensated by the increase in its precursor linoleic acid . Anti-inflammatory n-3 PUFA decreased  due to DHA reduction. Remarkably, in regenerating liver, C18 fatty acids represented nearly 65% of the fatty acids, and the proportion of products to precursor 20\u22365+22\u22366/18\u22363n-3 and 20\u22364/18\u22362n-6 dropped to one fourth. Since the overall changes were similar in the two genotypes, it is possible that repression of elongase activity and activation of \u03949 desaturase activity during regeneration be independent of E2F2 activity. Only two of the parameters measured at 48-h post-PH were indeed sensitive to E2F2 loss: the rise in 18\u22362n-6, which was 50% higher in the absence of E2F2, and the 20% decrease in unsaturation index, which was not apparent in E2F2-/- mice.To test whether E2F2 gene is required for normal esterification of FA into lipids, and to maintain homeostasis of liver lipids, we determined the FA composition of total lipids 48 hours after PH and compared to the pre-PH composition in WT and E2F2 samples . The gloThe differences in the FA composition of total lipid in liver regeneration described above were consistent with previous reports on proliferating cells -/- compared to WT. The singular FA composition of hepatic SM was slightly different in mutant mice, with exchange of very-long-chain saturated 24\u22360 and 23\u22360 for 18\u22360 and 18\u22361n-9. Our results indicate that, besides the abnormal low proportion of PC to PE in quiescence, constitutive E2F2 deletion in mice leads to reorganization of the molecular species of hepatic PC, PE, PS and SM. Since these changes are sustained during normal proliferation of liver cells, loss of E2F2 could have a potential impact on multiple membrane-dependent cell processes.The FA composition of PL classes was remarkably similar before and 48 hours after PH, and E2F2 deletion caused similar changes in quiescent and regenerating tissue -driven methylation of PE Chka, Pcyt1a and Chpt1. The Kennedy pathway and the Lands cycle operate in PE synthesis using analogous reactions to PC synthesis. A third PE source is mitochondrial PS decarboxylation, while subsequent base-exchange reactions catalyzed by PS synthase between PC (Ptdss1) or PE (Ptdss2) and serine generate PS. PS synthase 2 can also work in reverse producing PE from PS -/-  pathway for m PS -/- , we founce liver , suggestce liver  whereas /- liver . It is png liver  support -/- mice.A characteristic feature of liver regeneration is the accumulation of LDs in the tissue de novo pathways Scd1, Fads2) and elongases (Elovl6/Fasn) are essential in supporting life-sustaining cellular processes. The global changes during regeneration are similar in the two strains  under accession code E-MEXP-1413. We used six RNA pools, three pools of E2F2+/+  (n\u200a=\u200a24) and three pools of E2F2-/- (n\u200a=\u200a24) mice genotypes. Each pool contained quiescent liver samples from 8 animals. Affymetrix chips and microarray data analysis were performed exactly as described in Ref. 5. TIGR MultiExperiment Viewer Mev version 4.1  was used for the statistical analysis of the microarray data. To identify the genes whose expression levels changed significantly in E2F2-/- compared with WT mice we used Welch's t-test with a P\u22640.01 significance level cut-off. After curation, 2915 sequences were found to elicit significant expression change in quiescent liver upon E2F2 loss; of them 1735 (59.5%) were upregulated and 1180 (40.5%) were downregulated.(XLSX)Click here for additional data file.Table S2-/- liver relative to wild-type controls.Overrepresented Gene Ontology functional categories in quiescent E2F2aRefers to the number of genes included in each overrepresented functional category. bRefers to the percentage of genes detected in each functional category relative to the total number of genes included in that particular category. Functional classification of deregulated genes was made using FatiGO+, a public domain web tool for finding significant associations of Gene Ontology (GO) terms within groups of genes. Statistical significance was determined by Fischer's exact test and P-values were adjusted applying Benjamini-Hochberg (P\u22640.01) and Bonferroni (P\u22640.05) multiple testing correction.(DOCX)Click here for additional data file.Table S3E2F2 deletion deregulates the expression of a set of genes involved in lipid metabolism in quiescent liver. Genes (208) were extracted from the identified sequences listed in (DOCX)Click here for additional data file."}
+{"text": "Mouse models have served a valuable role in deciphering various facets of Salivary Gland (SG) biology, from normal developmental programs to diseased states. To facilitate such studies, gene expression profiling maps have been generated for various stages of SG organogenesis. However these prior studies fall short of capturing the transcriptional complexity due to the limited scope of gene-centric microarray-based technology. Compared to microarray, RNA-sequencing (RNA-seq) offers unbiased detection of novel transcripts, broader dynamic range and high specificity and sensitivity for detection of genes, transcripts, and differential gene expression. Although RNA-seq data, particularly under the auspices of the ENCODE project, have covered a large number of biological specimens, studies on the SG have been lacking.To better appreciate the wide spectrum of gene expression profiles, we isolated RNA from mouse submandibular salivary glands at different embryonic and adult stages. In parallel, we processed RNA-seq data for 24 organs and tissues obtained from the mouse ENCODE consortium and calculated the average gene expression values. To identify molecular players and pathways likely to be relevant for SG biology, we performed functional gene enrichment analysis, network construction and hierarchal clustering of the RNA-seq datasets obtained from different stages of SG development and maturation, and other mouse organs and tissues. Our bioinformatics-based data analysis not only reaffirmed known modulators of SG morphogenesis but revealed novel transcription factors and signaling pathways unique to mouse SG biology and function. Finally we demonstrated that the unique SG gene signature obtained from our mouse studies is also well conserved and can demarcate features of the human SG transcriptome that is different from other tissues.Our RNA-seq based Atlas has revealed a high-resolution cartographic view of the dynamic transcriptomic landscape of the mouse SG at various stages. These RNA-seq datasets will complement pre-existing microarray based datasets, including the Salivary Gland Molecular Anatomy Project by offering a broader systems-biology based perspective rather than the classical gene-centric view. Ultimately such resources will be valuable in providing a useful toolkit to better understand how the diverse cell population of the SG are organized and controlled during development and differentiation.The online version of this article (doi:10.1186/s12864-016-3228-7) contains supplementary material, which is available to authorized users. Salivary gland (SG) morphogenesis requires the complex coordination of cells to orchestrate a number of dynamic cellular processes including cell specification, lineage commitment, cell migration, proliferation and differentiation, all culminating in the formation of this specialized gland \u20133. A netFor many years now, mouse genetic models have been widely utilized to study various facets of salivary gland biology including branching morphogenesis, cleft formation, organ development and differentiation. While these studies have been instrumental in identifying some of the individual genes and signaling pathways necessary for proper salivary gland function, a limited number of studies have focused on the global examination of salivary gland gene expression in mouse , 8\u201310. IWhile microarray technology have been the application of choice in the past for transcriptome analysis, recent advancements have seen the supplanting of microarrays by genomic methods driven by next-generation sequencing (NGS) approaches like RNA-sequencing (RNA-seq) . CompareIn the present study, we have performed RNA-seq to generate a comprehensive gene expression profile of the mouse submandibular gland at various time points of development during embryogenesis and maturation in adult. To the best of our knowledge this is the first reported RNA-seq based study to examine the transcriptome of the mouse submandibular gland. An extensive bioinformatics analysis of our datasets has revealed interesting gene regulatory networks and maps that are enriched for and define the various stages of salivary glands. Finally we have leveraged the ENCODE, FANTOM, Human Protein Atlas as well as other published gene expression datasets to identify a salivary gland specific gene signature that is to a large extent conserved between mouse and human. Collectively our study not only validates existing literature but also provides a wealth of genomic resources that can be harnessed for the discovery of new genes and biologically important pathways in the salivary gland and for formulating testable hypotheses.Mus musculus (mm9 build) using Tophat2 . We subsequently performed between-sample normalization using the DESeq median normalization method and calculated fragments per kilobase of transcripts per million (FPKM) mapped reads thereby giving us measurements of relative expression of genes within and between biological samples.In order to better define the dynamic changes in global gene expression levels and to identify new tissue-specific and tissue-enriched regulators, we performed RNA-seq based expression profiling of the mouse submandibular salivary gland at various key stages of embryonic development in addition to post-natal and adult tissues. Towards this end, we isolated total RNA from the mouse submandibular gland dissected from E14.5\u00a0day old embryos (Pseudoglandular stage - representing the onset of branching morphogenesis), E16.5  and E18.5  [In order to better analyze and appreciate the overall gene expression patterns between the various developmental and adult time points, we utilized principal component analysis (PCA), a statistical technique that reduces and summarizes large datasets while illustrating relationships between samples based on co-variance of the data being examined , 15. Usip-value of less than 0.1. We also considered genes that were expressed at \u22651 FPKM in at least one biological replicate. Using this criterion, we identified a total of 3601 differentially expressed genes (DEGs) between the embryonic and neonatal stages with 1597 genes found to be up-regulated and 2004 genes showing downregulation , neonatal (P5) and adult (4wk and 12wk). For this analysis, we selected genes that showed at least a two-fold change in expression between each time point while showing an adjusted K-Means clustering on the standardized log2 transformed FPKM values for each of the samples. This analysis led to the identification of 8 different gene clusters that corresponded to genes enriched in a specific developmental time point as well as genes enriched in grouped time points, which we refer to as Embryo, Neonatal and Adult Enriched clusters  showed an FPKM of 1 or greater in at least one of the sample replicates across all samples and b) were comparatively differentially expressed, with a log2-fold change of 1 or greater, at one time point compared to other time points across all 6 datasets. Based on the above criteria we identified a total of 1924 genes that were specifically enriched, across all datasets as demonstrated in Fig.\u00a0ers Fig.\u00a0. Interesers Fig.\u00a0.Fig. 2ClGene Ontology (GO) analysis of the developmental stage-specific gene profile identified overrepresentation of genes associated with specific functions, indicating the existence of distinct biological processes occurring during embryonic, neonatal and adult stages of salivary gland development. A closer examination of genes that were uniquely expressed during embryogenesis revealed specific enrichment of biological pathways associated with organ, embryonic and vasculature development, cell differentiation, and transcription - all of these biological processes are associated with organ morphogenesis Fig.\u00a0. InteresSince our analysis of the broad gene-enriched profile of embryonic, neonatal and adult salivary gland revealed interesting facets of its biology, we wondered if further in-depth parsing of the datasets could provide more detailed insights into salivary gland maturation, particularly in the embryonic stages. Hence, we next evaluated the transcriptomic activities from E14.5 to E18.5 to identify genes and pathways that may be critical during the early stages of salivary gland organogenesis and development. Of the 1924 comparatively differentially expressed developmental specific genes we identified, 1064 of them were specifically enriched during embryogenesis  project (Illumina-based RNA-seq) and the FANTOM5 consortium ). To make proper comparisons, we only chose representative human tissue samples for which the corresponding data was available for the mouse tissues. Upon examination of the Human Protein Atlas (HPA) samples, we found that of the 126 human genes that were homologous to the mouse, 45 genes (~36%) showed enrichment in the human salivary gland as compared to the 16 other tissues analyzed , from dissected mouse C57BL/6 submandibular glands at indicated time points. For each RNA sample, cDNA libraries were prepared using the TrueSeq RNA Sample Preparation Kit (Illumina) and were then 50\u00a0bp single-end sequenced on an Illumina HiSeq 2500. Quality control metrics were performed on raw sequencing reads using the FASTQC v0.4.3 application. Reads were mapped to the Mus musculus genome (mm9 build) with TopHat2  v2.0.13,p-value held to 0.1. Additionally we considered only genes with values of at least one FPKM in at least one biological replicate. The second analysis involved the creation of the salivary gland developmental gene profile. In this analysis, all time-points were considered separate and only true biological replicates were used. In order to create a highly stringent list of time-point specific genes the following approach was used: 1) Contrasts were created between one time-point and every other subsequent time-point 2) DEGs for each of these analyses were called using the aforementioned criteria and 3) The intersection of these DEGs were reported for the individual time-point, thereby creating a stringent list of genes that were consistently differentially expressed in either the positive or negative direction. This process was iterated until all time-points were analyzed identically. The union of genes identified in the aforementioned analyses were taken and reported as the gene profile for the developing salivary gland. K-Means clustering was used to capture the patterns of gene expression as development proceeds in the salivary gland. Gene FPKM values were averaged across biological replicates and subsequently standardized to their Z-Scores. These values were used as inputs to the K-Means clustering algorithm  analysis was performed using DESeq2. The developmental stage salivary gland count data was imported as one matrix and was subsequently divided into two separate DEG analyses. The first analysis treated each of the embryo time-points as biological replicates as well as the two separate adult stages when making contrasts as part of the DESeq2 standard protocol. Genes were considered differentially expressed if the log2 Fold Change between samples was at least 1, with the adjusted uster3.0 ) using kCounts data from the MouseENCODE data plus the whole adult skin and the 12\u00a0week old salivary gland (12wk SG) experiments were imported and normalized as one matrix. In order to identify gland specific genes, DESeq2 was used to create contrasts between the 12wk SG and each of the 24 other tissues. DEGs were reported for each contrast according to the aforementioned criteria and the intersection of the genes from each analyses that were considered differentially expressed in the positive direction  were reported as the tissue specific salivary gland gene signature.All analyses and figures were made using R v3.2.3. The heatmap program from the NMF  v0.20.6 In order to examine if the same genes identified in the tissue specific salivary gland signature would be enriched in human salivary gland compared to all other tissues, processed RNA-seq experiments generated by the Human Protein Atlas  were dowThe R-package CAGEr(v1.12.0) was used to download raw FANTOM5 Cap Analysis of Gene Expression (CAGE) data taken from human tissue samples. Only samples which matched those used in the mouse specific analysis were selected. Raw tag counts were read in for each sample and TSS locations were estimated using the clusterCTSS function, which will cluster neighboring TSSs if they are closer than 20\u00a0bp and remove potential TSS sites if there are less than 2 tag counts. Promoter widths were calculated by calculating the cumulative distribution of CAGE tag signal across the clustered TSS locations, followed by determining the position of the Upper and Lower quantile locations of the cumulative CAGE signal, whereupon the distance between these quantiles represents the promoter width. Consensus promoter locations across all samples were determined in order to best estimate gene expression. The consensusCluster functions was used to aggregate tag clusters using the promoter widths calculated previously, whereupon promoter locations were merged if located within 100\u00a0bp of each other and TSS locations with extremely low overall tag counts were eliminated. This provided a raw count matrix for all determined TSS locations, which represented overall gene expression. Nearest gene annotation was applied for each TSS location and promoter tags were aggregated based on gene identity. This gene-level tag count matrix was then used as input for DESeq2 differential gene expression analysis pipeline. This final count matrix was log2 transformed, DESeq2 median normalized and filtered for generation of the mouse tissue specific gene signature.p-value and gene-sets were selected to highlight unique features of the enrichment analysis. These features were graphically represented using the EnrichmentMap [The KEGG enrichment analysis shown in Additional file hmentMap  tool, whhmentMap , using dhmentMap  add-on f"}
+{"text": "Similarly, combination of DOX with RES and DID in HT-29 decreased the IC50\u2019s of DOX from 0.88\u2009\u00b1\u20090.03\u2009\u03bcM to 0.47\u2009\u00b1\u20090.02\u2009\u03bcM and 0.29\u2009\u00b1\u20090.04\u2009\u03bcM, respectively. The expressions of p53 and Bax genes were markedly elevated in HCT 116 cells after exposure to DOX/DID. In HT-29 cells, the expression of Bcl-XL gene was significantly decreased after exposure to DOX/DID. In addition, combination of DOX with RES significantly increased the expression of Bax gene in HCT 116 cells. RES treatment induced significant S-phase arrest in DOX-treated HCT 116 cells, while DID induced G2/M- and S-phase arrest in HCT 116 and HT-29, respectively. Both RES and DID significantly enhanced the intracellular entrapment of DOX due to blocking the efflux activity of p-glycoprotein pump. In conclusion, RES and DID sensitize colorectal cancer cells to DOX via facilitating apoptosis and enhancing intracellular entrapment of DOX.Doxorubicin (DOX) has limited efficacy in colorectal cancer due to multi-drug resistance. Resveratrol (RES) and didox (DID) are polyhydroxyphenols with potential chemosensitizing effects. Herein, we assessed the chemomodulatory effects of RES and DID to DOX in colorectal cancer cells. Equitoxic combination of DOX with RES and DID in HCT 116 reduced the IC Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females with an estimated 1.4 million cases and 693.000 deaths occurring in 2012, accounting for 8% of all cancer related deathsMDRl gene; and is considered a member of the ATP-binding cassette (ABC) transporter superfamily. It is energy-dependent transporter pump that removes xenobiotics such as, DOX outward from cells and confer drug resistance in tumor cells7The anthracycline, doxorubicin (DOX), is a widely used chemotherapy due to its efficacy in fighting wide range of cancers such as carcinomas, sarcomas and hematological neoplasias3in vivo and in vitro studies showed that RES possesses potential anti-tumor activity against several malignancies242526MDR1 geneCompounds of natural origin are very rich source for leads with potential anticancer properties as well as chemomodulatory effects such as, P-gp inhibitors791011121314161819202122Didox (DID) is a synthetic polyphenolic compound which shares important biochemical targets with RES323328363738MDR1 gene.DID and RES might be potential successful adjuvant candidates for combination with DOX3350 of 0.96\u2009\u00b1\u20090.02\u2009\u03bcM  decreased from 0.96\u2009\u00b1\u20090.02\u2009\u03bcM to 0.52\u2009\u00b1\u20090.05\u2009\u03bcM and 0.4\u2009\u00b1\u20090.06\u2009\u03bcM, respectively  decreased from 0.88\u2009\u00b1\u20090.03\u2009\u03bcM to 0.47\u2009\u00b1\u20090.02\u2009\u03bcM and 0.29\u2009\u00b1\u20090.04\u2009\u03bcM, respectively  at concentration of 0.3\u2009\u03bcM . Cellula\u20090.03\u2009\u03bcM . Also, R\u20090.03\u2009\u03bcM . Both REell line . IC50\u2019s ectively   increased the relative Bcl2 gene expression  . In addiIn HT-29, DOX significantly increased cells in G0/G1-phase from 69.6\u2009\u00b1\u20091.0% to 82.3\u2009\u00b1\u20091.0%, while RES significantly decreased cell population in G0/G1-phase to 44.3\u2009\u00b1\u20090.7%. DID did not induce any significant change in G0/G1-phase cell population. Combination of DOX with RES and DID significantly decreased G0/G1 cell population to 77.0\u2009\u00b1\u20092.1% and 74.2\u2009\u00b1\u20090.4%, respectively compared to DOX treatment alone (82.3\u2009\u00b1\u20091.0%) . DOX aloMDR1 gene and highly abundant in colorectal cancer cells (MDR1 gene is highly expressed in HCT 116 cells and weakly expressed in HT-29 cellsP-glycoprotein (P-gp) is a plasma membrane efflux pump coded by To further investigate the sub-molecular interaction between RES and DID with P-gp molecules, ATP consumption assay was carried out on purified human recombinant P-gp molecules attached to ATPase enzyme. VRP  and sodium vanadate (ATPase inhibitor) were used as positive controls; and are supposed to increase and decrease ATP consumptions, respectively compared to non-treated control (NTC). RES significantly decreased remaining ATP molecules by 29% compared to 31.5% for VRP . InteresMDR1 gene was measured by real time PCR. In both HCT 116 and HT-29, none of the treatments under investigation showed any significant change in MDR1 gene expression  is involved in multidrug resistance of CRC42per se did not show any promising cytotoxicity in CRC cells under investigation (HCT 116 and HT-29). In the current work as well as in our previous publication, RES showed weak cytotoxicity against HCT 116 cells (IC50 17.5 \u03bcM)et al.According to our data, DID 2, p53 and Bcl-XL) were further investigated. According to our observations, in HCT 116 cell line, RES enhanced DOX-induced Bax gene expression which might explain the significant increase in active caspase-32, Bax and p53 gene expression. Yet, p53 promotes apoptosis through down-regulating the antiapoptotic Bcl2 and up-regulating the apoptotic Bax protein expression at transcriptional level2 gene expression  was generously gifted from Professor Howard L. Elford, Molecules for Health Inc., Richmond, VA, USA. Doxorubicin (DOX), resveratrol (RES), and sulpharodamine (SRB) were purchased from Sigma Chemical Co. . RPMI-1640 media, fetal bovine serum and other cell culture materials were purchased from Lonza Group Ltd. . Other reagents were of the highest analytical grade.\u00aeCCL-247) and HT-29 (ATCC\u00aeHTB-38), were obtained from the Vaccera . Cells were maintained in RPMI-1640 supplemented with streptomycin (100\u2009\u03bcg/mL); penicillin (100\u2009units/mL) and heat-inactivated fetal bovine serum (10% v/v) in a humidified, 5% (v/v) CO2 atmosphere at 37\u2009\u00b0C.Human colorectal cell lines, HCT 116 The cytotoxicity of DOX, RES and DID were tested against HCT 116 and HT-29 cells by SRB assay as previously describedThe dose response curves of compounds were analyzed using  model .d is the drug concentration that produces 50% reduction of the maximum inhibition rate and m is a Hill-type coefficient. IC50 is defined as the drug concentration required to reduce absorbance to 50% of that of the control where R is the residual unaffected fraction (the resistance fraction); [D] is the drug concentration used; KCombination index (CI) was calculated as previously describedThe nature of drug interaction is defined as synergism if CI\u2009>\u20090.8; antagonism if CI\u2009<\u20091.2; and additive if CI ranges from 0.8\u20131.2.2, Bax, Bcl-XL, p53 and MDR1 following treatment of cells with DOX, RES, DID and their combination, total RNA extraction from cells was performed using RNeasy Mini Kit\u00ae . Reverse transcription was undertaken to construct cDNA library from different treatments using High-capacity cDNA Reverse Transcription Kit . The archived cDNA libraries were then subjected to quantitative real time PCR reactions using cyber green fluorophore . Primer sequences were as follow: Bcl2 forward primer GGG-TAC-GAT-AAC-CGG-GAG-AT and reverse primer CTG-AAG-AGC-TCC-TCC-ACC-AC; Bax forward primer TCT-GAC-GGC-AAC-TTCAAC-TG and reverse primer TGG-GTG-TCC-CAA-AGT-AGG-AG; Bcl-XL forward primer GGC GGA TTT GAA TCT CTT TCT C and reverse primer TTA TAA TAG GGA TGG GCT CAA CC; p53 forward primer CCT-CAC-CAT-CAT-CAC-ACT-GG and reverse primer CTG-AGT-CAGGCC-CTT-CTG-TC; MDR1 forward primer GCT-GGG-AAG-ATC-GCT-ACT-GA and reverse primer GGT-ACC-TGC-AAA-CTC-TGA-GCA. GAPDH was used as reference housekeeping gene with forward primer TGC-ACC-ACC-AAC-TGC-TTAG and reverse primer GAT-GCA-GGG-ATG-ATG-TTCmdr1 Gene expression was normalized using the expression level of corresponding GAPDH gene. The rest of gene expressions in different treatments (single or combined) were expressed relative to its corresponding normalized level in control untreated cells (cells exposed to drug free media) and control treatment gene expression level was expressed as dotted line.To assess the gene expression of Bcl\u00ae caspase-3 ELISA kit . Briefly, the cells were exposed to the predetermined IC50\u2019s of test compounds (single or combined treatments) or drug free media (control group) for 24\u2009h. Cells were harvested and washed with PBS, then incubated with the biotin-ZVKD-fmk inhibitor for 1\u2009hour. Cells were transferred into the wells of a microplate pre-coated with a monoclonal antibody specific for caspase-3. Following a wash to remove any unbound substances, streptavidin conjugated to horseradish peroxidase was added to the wells and bound to the biotin on the inhibitor. Following a wash to remove any unbound Streptavidin-HRP, a substrate solution was added to the wells. The enzyme reaction yields a blue product that turned yellow when a Stop Solution was added. The optical density of each well was determined within 30\u2009minutes, using a microplate reader set to 450\u2009nm with a wavelength correction at 540\u2009nm or 570\u2009nm. The concentrations of active caspase-3 were calculated from a standard curve of constructed with known concentrations of active caspase-3. Caspase concentration was expressed as ng/mg protein. Proteins were determined spectrophotometrically by the method of Bradford using purified bovine serum albumin as a standard.To assess the effect of RES, DID, DOX and their combination on apoptosis, the active caspase-3 level was measured by using Quantikine50\u2019s of test compounds (single or combination) for 24\u2009h and compared to control cells (treated with drug free media). Cell cycle analysis was performed using the CycleTEST\u2122 PLUS DNA Reagent Kit . Control cells with known DNA content (PBMCs) were used as a reference point for determining the DI (DNA Index) for the test samples. Cells were stained with propodium iodide following the procedure provided by the kit and then run on the DNA cytometer. Cell cycle distribution was calculated using CELLQUEST software .To determine the effect of DOX, RES, DID and their combinations on the cell cycle distribution in HCT 116 and HT-29 cell lines; cells were exposed to the predetermined IC5 cells/well. Cells were exposed to DOX (10\u2009\u03bcM), DOX (10\u2009\u03bcM) and RES (10\u2009\u03bcM), DOX (10\u2009\u03bcM) and DID (10\u2009\u03bcM) or DOX (10\u2009\u03bcM) and VRP (10\u2009\u03bcM) for 2\u2009h at 37\u2009\u00b0C and subsequently extracellular DOX containing media was washed trice with ice cold PBS. Intracellular DOX were extracted after cell lysis by incubation with SDS (2% w/v), saturated aqueous solution of ZnSO4 (100\u2009\u03bcl), Acetonitril (500\u2009\u03bcl) and acetone (500\u2009\u03bcl) for 30\u2009min at 37\u2009\u00b0C. After centrifugation, supernatant was collected and DOX concentration was measured spectroflourometrically at \u03bbex/em 482/550\u2009nm28To assess the effect of RES and DID on the efflux pumping activity of P-gp in tumor cell lines, the P-gp substrate, DOX, was used as a probe and verapamil (VRP) was used as standard p-gp blocker. Briefly, exponentially growing cells were plated in 6-well plates in plating density of 103VO4, VRP, RES, or DID. Recombinant human P-gp membrane fraction was incubated with the reaction mixture at 37\u2009\u00b0C for about 5\u2009minutes, and then the reaction was initiated by adding 10\u2009\u03bcl of 25\u2009mM MgATP and incubating for 40\u2009minutes at 37\u2009\u00b0C. An identical reaction mixture without drug treatment (NTC) was assayed in parallel. The reaction was stopped and the remaining un-consumed ATP was detected using luciferase firefly luminescent signal and ATP standard curve was plotted. The remaining concentrations of ATP were expressed as (p. mole/\u03bcg P-gp molecules). Sodium vanadate (Na3VO4) was used as selective inhibitor of P-gp related ATPase enzyme, and samples treated with Na3VO4 have greater luminescent signal than un-treated samples (NTC). In addition, VRP inhibits pgp molecules via covalent binding and competing with other substrates, hence, stimulating ATPase activity resulting in more consumption of ATP molecules compared to NTC.To reveal the mechanism of P-gp-inhibition induced by RES and DID, assessment of P-gp attached ATPase activity was performed using Pgp-Glo\u2122 Assay Systems . Briefly, in 96-well plates, Pgp-Glo\u2122 Assay Buffer was mixed with Na\u00ae for windows, version 17.0.0. p\u2009<\u20090.05 was taken as a cut off value for significance.Data are presented as mean\u2009\u00b1\u2009SD. Analysis of variance (ANOVA) with LSD post hoc test was used for testing the significance using SPSSHow to cite this article: Khaleel, S. A. et al. Didox and resveratrol sensitize colorectal cancer cells to doxorubicin via activating apoptosis and ameliorating P-glycoprotein activity. Sci. Rep.6, 36855; doi: 10.1038/srep36855 (2016).Publisher\u2019s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "Correlation coefficients were significant for the PCL parameters product (0.73), area (0.71), T2*-time (-0.67), FA (0.65) and ADC (0.55). Our initial results suggest that HS type 1, HS type 2 and no-HS subtypes can be distinguished from each other using ex vivo UHF MRI based on T2-weighted morphologic images and the assessment of the parameter product. Upon clinical translation, UHF-MRI may provide a promising technique for the preoperative differentiation of HS subtypes in patients.The aim of the present study is to differentiate subtypes of hippocampal sclerosis (HS) using ex vivo ultra-high field magnetic resonance imaging (MRI). Included were 14 surgically resected hippocampi of patients with medically intractable temporal lobe epilepsy. The resected hippocampi were histologically categorized into subtypes of hippocampal sclerosis (HS type 1 (n = 10), HS type 2 (n = 2) and no-HS (n = 2)) and subsequently scanned on a preclinical 7T MRI acquiring T2-weighted morphology, relaxometry and diffusion tensor imaging. On the morphological images, the pyramidal cell layer (PCL) of the hippocampus was segmented and the following parameters were derived: T2 signal intensity, T1-, T2- and T2*-relaxation times, apparent diffusion coefficient (ADC), fractional anisotropy (FA) and mean diffusivity (MD). Furthermore, the area of the PCL was determined, as well as the parameter product which refers to the widths of the PCL parallel and perpendicular to the stratum moleculare. Spearman correlation coefficient was used to demonstrate relationships between MR-parameters and type of sclerosis. In comparison to no-HS specimens, the PCL was significantly narrower in HS type 1 and HS type 2 hippocampi. This narrowing affected the entire cornu ammonis sector (CA) 1 in HS type 1, while it was limited to the upper half of CA1 in direction to CA2 in HS type 2. The parameter product median increased from 0.43 to 1.67 and 2.91 mm Hippocampal sclerosis (HS) is observed in up to 70% of patients with mesial temporal lobe epilepsy (TLE) . Up to oAccording to the International League against Epilepsia (ILAE), HS can be histologically divided into four subtypes based on specific patterns of neuronal cell loss in the pyramidal cell layer (PCL) . DiffereMagnetic resonance imaging (MRI) is crucial for diagnosing hippocampal sclerosis in TLE patients. Typical features of HS are reduced hippocampal volume, increased signal intensity of the PCL on T2-weighted and T2-fluid attenuation inversion recovery (FLAIR) images and loss of internal architecture ,14. FurtWhile current clinical protocol focuses on differentiating HS from no-HS, little is known about the MR characteristics of different HS subtypes. One reason for this is the limited ability of clinical 1.5T and 3.0T MRI scanners to image the internal architecture of the hippocampus due to spatial resolution and tissue contrast \u201314. As aTo evaluate the potential of UHF MRI, we chose an ex vivo UHF MRI setting by making use of strong magnetic fields, fast and powerful gradients, long scan times and the absence of motion, thus allowing for micrometer-scale resolutions, strong tissue contrasts and an increased signal-to-noise ratio in multiparametric studies. The present study therefore aims to address the morphology, relaxometry and diffusion metrics of surgically resected HS type 1, HS type 2 and no-HS hippocampi. The question we focus on in this ex vivo study is whether HS characteristics can be visualized and quantified by morphological and multiparametric UHF MRI.This study includes 14 patients diagnosed with medically intractable TLE who underwent surgery for the relief of seizures at University Hospital Erlangen after imaging and electroclinical characteristics had shown evidence of hippocampal involvement in seizure generation.Directly after surgery, resected hippocampi were fixed in a 4% paraformaldehyde buffered solution, washed with phosphate buffered saline (PBS) and submitted for neuropathological examination. The specimens were diagnosed with HS type 1 (n = 10), HS type 2 (n = 2) and no-HS (n = 2) according to ILAE guidelines. All procedures were approved by the local ethics committee . Participant\u2019s written informed consent was obtained.2), slice thickness (300 \u03bcm) and image center were identical for all sequences. Morphological images were acquired using a 3D T2-weighted turbo spin echo sequence /echo time (TE): 8520/95 ms, matrix: 704x704, averages (av): 4, acquisition time (TA): 207 min per slice). Relaxometry was acquired using a 3D fast low angle shot-sequence  for T1-times. A 2D spin-echo sequence  was used for T2-times and a 2D-gradient echo sequence  for T2*-times, each with 3 averages, a matrix of 576x576 and acquisition times of 5, 210 and 14 minutes per slice, respectively. Diffusion tensor imaging (DTI) was performed with six b-values  in 265 directions using a 2D echo planar imaging sequence .For imaging, hippocampal specimens were fixed in a standardized procedure for 24 h in paraformaldehyde and afterwards embedded in 1.5% agarose. Imaging war performed on a preclinical 7T MRI  using a volume resonator radiofrequency coil . For technical reasons, three samples  were imaged with a surface coil. The imaging protocol included T2-weighted morphology, relaxometry and diffusion tensor imaging (DTI). The field of view .Image analysis was performed using an Osirix Dicom Viewer  in conjunction with Chimaera's segmentation tool . The pyramidal cell layer (PCL) was segmented on the first slice of the T2-weighted morphological image stack on which the hippocampus was homogeneously visible. The manually drawn segmentation covered the four cornu ammonis sectors  of the PCL. The CA1/subiculum border was determined according to Mueller et al  by drawiStatistical analysis was performed using statistic software R . Spearma2), product (mm2), T2 signal intensity (a.u.), T1-time (ms), T2-time (ms), T2*-time (ms), FA (a.u.), ADC (*10\u22126 mm2/s) and MD (*10\u22126 mm2/s).The study results can be divided into a qualitative analysis, based on morphologic T2-weighted MR images, and a quantitative part, including the parameters area  to 42.3 (HS type 2) to 40.3 ms (no-HS), with significant differences between HS type 1 and no-HS. FA increased for HS type 1, HS type 2 and no-HS, while ADC and MD show decreasing tendencies. Spearman Rank correlation coefficients demonstrated significant relationship between type of sclerosis and the MR-parameters product (0.73), area (0.71), T2* (-0.67), FA (0.56) and ADC (-0.55). All other parameters including T2Si (-0.45), T1-time (0.15), T2-time (0.15) and MD (-0.46) did not reach significance. The results of all quantitative parameters assessed in this study are summarized in Quantitative parameters were determined for each hippocampus when segmenting the pyramidal cell layer. The following results were obtained by grouping the specimens according to their neuropathologic diagnosis: The median value of the parameter product increases from 0.43 to 1.67 and 2.91 mmIn patients suffering from medically intractable TLE, the surgical outcome and prognosis for post-operative seizure freedom are associated with different subtypes of hippocampal sclerosis that can currently only be diagnosed and categorized by histological analysis. As a first step towards a preoperative subtype differentiation using non-invasive imaging methods, we investigated the morphological and quantitative characteristics of surgically resected HS type 1, HS type 2 and no-HS hippocampi by multiparametric ex vivo UHF MRI.Our results suggest that HS type 1, HS type 2 and no-HS gliosis only hippocampi can be distinguished from each other based on specific morphological patterns of the PCL, which we were able to depict in high-resolution T2-weighted MR-images acquired with long repetition times and the resulting strong tissue contrasts. These morphological differences can be quantitatively captured by the parameter product, which we have introduced and defined in the present study. Furthermore, the morphological parameters product and area, T2*-time, FA and ADC yielded significant correlation coefficients depending on the type of sclerosis.The median values of T2*-time in the PCL increased from HS type 1 to HS type 2 and no-HS hippocampi. This tendency was also observed in a recent in vivo study by Santyr et al 20, who found R2* (= 1/T2*) to be significantly lower in the entire hippocampus of HS patients when compared to controls. However, the authors did not find a significant decrease of R2* for patients with HS in comparison to patients diagnosed with no-HS [As T2* relaxation results from susceptibilities among tissues, this increase could correlate with sclerosis-induced tissue homogenization in the PCL, similarly to an increase in the T2 signal and T2 relaxation time. It has been formerly suggested that the later reflect gliosis \u201323 and a2) and that only126 diffusion directions were investigated, whereas the DTI sequence used in this study included six b-values and 256 diffusion directions.Our finding of FA values is clearly lower in the PCL of HS type 1 when compared with HS type 2 and no-HS hippocampi, which may reflect stronger neuronal cell loss. This result concurs with clinical studies that found FA to be decreased in patients with HS in comparison to patients without HS \u201317. HoweIn terms of a histological interpretation, an increase of ADC-values in stronger pathologies may reflect a loss of neurons, respectively the shrinkage of neuronal bodies, which may lead to less restricted water diffusion and therefore higher diffusivity .T2 signal intensity, as well as T1 and T2 relaxation times, did not show significant correlation with the type of sclerosis investigated in our study. T2 relaxation time in particular has been shown to be increased in patients with TLE, in vivo as well as ex vivo . For thiOur study has several limitations: The sample size of our specimens, especially for HS type 2 and no-HS hippocampi is very small. A higher number of HS type 2 and no-HS specimens or even HS type 3 hippocampi would increase the power of our study, however, as these are very rare forms of HS, no further specimens were available at our institution. Therefore, our results must be interpreted as preliminary and need to be confirmed in a larger patient collective. Furthermore, according to ILAE guidelines, the classification into different HS subtypes is based on distinct scores for neuronal cell loss in the individual subfields of the PCL (CA1-CA4 and the dentate gyrus). It therefore remains to be elucidated as to how far our observation that different HS subtypes express different atrophy patterns in the PCL can be correlated to histological data.When performing ex vivo MRI, the impact of fixation on MR-parameters has to be considered. Formalin fixation has been reported to cause a strong decrease of all relaxation times \u201331. In aOur data may provide a reference for the adaptation of in vivo imaging protocols on ultra-high field scanners. A first in vivo feasibility study of 7T MRI of hippocampal sclerosis by Stefanits et al  demonstrOur initial results suggest that HS type 1, HS type 2 and no-HS subtypes can be qualitatively and quantitatively distinguished from each other using ex vivo UHF MRI based on T2-weighted morphological images and the assessment of the parameter product. T2*-relaxation time and diffusion metrics may additionally contribute to a differentiation of HS subtypes.In terms of a potential clinical translation, UHF MRI may be a promising technique to determine HS subtypes preoperatively in patients.S1 Table(PDF)Click here for additional data file."}
+{"text": "Novel features of the longitudinal instability of a single electron bunch circulating in a low-emittance electron storage ring are discussed. Measurements and numerical simulations, performed both in time and frequency domain, show a non-monotonic increase of the electron beam energy spread as a function of single bunch current, characterized by the presence of local minima and maxima, where a local minimum of the energy spread is interpreted as a higher-order microwave instability threshold. It is also shown that thresholds related to the same zero-intensity bunch length depend linearly on the accelerating radio frequency voltage. The observed intensity-dependent features of the energy spread, confirmed by measurements with two independent diagnostics methods, i.e. horizontal beam profile measurements by a synchrotron light monitor and photon energy spectrum measurements of undulator radiation, are given a theoretical interpretation by applying a novel eigenvalue analysis based on the linearized Vlasov equation. While these and the new generation of storage ring light sources such as the ESRF-EBS4, APS-U5, ALS-U6 and Sirius7 significantly reduce the horizontal emittance, they do not offer any improvement of another important part of the full 6-dimensional electron beam emittance, namely, the energy spread. The electron beam energy spread can limit the peak harmonic flux from undulators, particularly at the high harmonics that are extensively used in medium-energy storage rings. In addition, the energy spread affects the angular divergence of the emitted radiation, and may ultimately limit the x-ray brightness in future ultra-low-emittance storage ring based light sources. Therefore, it is important to have a detailed understanding of what determines the electron beam energy spread in storage ring facilities under a variety of operating conditions.Successful construction, commissioning and operation of several ultra-low emittance storage rings have had a decisive impact on the development direction of future synchrotron light source facilities. For example, PETRA-III, NSLS-II, and, recently, MAX-IV, have all demonstrated reliable operation with a horizontal electron beam emittance equal to or smaller than 1 nm-rad8. As the beam current increases, the intra-bunch particle interaction via short-range wakefields induced by the beam in a vacuum chamber modifies the longitudinal particle distribution as a function of the beam intensity . The energy spread is not changed by\u00a0the collective effects if the beam intensity is low enough, but the longitudinal microwave instability occurs above a certain threshold current and results in the energy spread growth and in the beam brightness degradation. This instability typically manifests itself as a high-frequency perturbation of the beam that increases both the energy spread and bunch length until a new quasi-equilibrium state is established. One of the goals of a storage ring design is to keep the instability threshold above the operating current.The energy spread of a low-current electron beam in a storage ring is determined by the equilibrium between radiation damping and quantum fluctuations11. Our measurements show that the energy spread growth is not monotonic with the beam current, but rather is characterized by a number of local minima\u00a0and maxima. In addition, spectral analysis of the beam motion show that as the energy spread goes through a local minimum, the oscillation frequency of the perturbation changes almost discontinuously by a value comparable to the synchrotron frequency. Thus, we find evidence for the higher-order instability thresholds, and observe this general phenomenology over a wide range of accelerating radio frequency (RF) voltages and electron bunch lengths. We then reproduce the energy spread growth and spectral features with the simulation code \u00a0SPACE12, and finally compare the measurements and simulations to a recently developed theory extending Sacherer\u2019s14 insights to interpret these microwave instability thresholds in terms of a succession of classical mode coupling. We find that the predictions based on the mode coupling theory are quite successful and appear to provide a useful way for better understanding of the microwave instability over a wide range of beam parameters.In this paper, we study the onset of the microwave instability and its behavior well above the threshold current using precise measurements at the NSLS-II storage ring\u03c3\u03b4 as a function of single-bunch current I0 in the NSLS-II storage ring, where \u03b4\u2009=\u2009(E\u2009\u2212\u2009E0)/E0 is the relative energy deviation with respect to the reference energy E0, and I0\u2009=\u2009Q/T0, where Q is the bunch charge and T0 is the revolution period. Two independent diagnostic methods have been applied to measure the current-dependent energy spread \u03c3\u03b4(I0)16. The first method is based on measurements of horizontal beam profile using a synchrotron light monitor (SLM) installed in a low-dispersion area17, where the energy spread \u03c3\u03b4 is related to the horizontal beam size \u03c3x as18:\u03b5x is the horizontal emittance; \u03b2x and \u03b7x are, respectively, the horizontal beta function and dispersion in the bending magnet, in which the beam generates light detected by the SLM. In our case, \u03b2x\u2009=\u20092.77 m and \u03b7x\u2009=\u20090.13\u2009m. The second method is based on the measurement of photon energy spectrum from the in-vacuum undulator (IVU) of the Soft Matter Interfaces (SMI) X-ray beamline19.The main results of this paper hinge upon experimental data for the energy spread 21. This study of longitudinal beam dynamics in the spectral domain allows us to characterize the current-dependent behavior of the synchrotron frequency and its harmonics above the first threshold of microwave instability.In addition to the energy spread measurements, spectra of the beam motion were also measured using a stripline, which is a part of NSLS-II bunch-by-bunch transverse feedback systemE0\u2009=\u20093\u2009GeV storage ring with the revolution period T0\u2009=\u20092.6\u2009\u03bcs and the momentum compaction \u03b1c\u2009=\u20093.7\u2009\u00d7\u200910\u22124. The storage ring is equipped with three damping wigglers (DWs) designed to control the\u00a0horizontal beam emittance and energy spread. If the magnet gaps of the DWs are open, the\u00a0horizontal emittance and energy spread are determined by the \u201cbare\u201d magnet lattice (BL): \u03b5x\u2009=\u20092\u2009nm, \u03c3\u03b4\u2009=\u20090.05%. For user operations, the gaps of all three DWs are closed to achieve the design emittance \u03b5x\u2009=\u20090.9\u2009nm22 and the energy spread \u03c3\u03b4\u2009=\u20090.087%. We denote this magnet lattice as 3DW. For the 3DW lattice with RF voltage VRF\u2009=\u20093\u2009MV, the bunch length is \u03c3z0\u2009=\u20094.6\u2009mm and the synchrotron tune is \u03bds0\u2009=\u20098.67\u2009\u00d7\u200910\u22123 in the limit of I0\u2009\u2192\u20090. The reduction of the\u00a0horizontal beam emittance by DWs and corresponding changes in the energy spread and beam lifetime have been confirmed experimentally2.NSLS-II is a \u03c3\u03b4 dependence on the number of DWs is illustrated by the different low-current values of the measured \u03c3x in Fig.\u00a0VRF\u2009=\u20092.6\u2009MV in both cases. Using Eq.  of the 7th harmonic of the SMI IVU radiation spectra. Both sets of data closely agree over the whole range of I0, and the measured growth of both \u03c3x and the IVU FWHM are characterized by the presence of local minima and maxima at certain values of the beam current. This non-monotonic behavior is of particular interest, and we will use the measurements, simulations, and theory to show that they arise when different modes of oscillation become unstable as the\u00a0beam current increases.The sing Eq.  to extrath harmonic of radiation from 23\u2009mm period\u00a0and 2.8\u2009m long IVU of the SMI beamline demonstrated about 16% larger harmonic width than this was predicted by simulations. This systematic effect is attributed to a possible small misalignment of this undulator with respect to electron beam23 and a detector effect. Measurements with another undulator, 20\u2009mm period\u00a0and 3\u2009m long IVU of Coherent Hard X-ray (CHX) scattering beamline, performed in parallel with the measurements at the SMI, were in better agreement with simulations (within 1%), however, these measurements were done using a slower detection system, that required exposure times exceeding the electron beam lifetime at high currents per bunch. Therefore, only the SMI data were included in this paper. We note that the single-electron spectral width of the 7th harmonic of the SMI IVU at 8.1\u2009keV photon energy is about 9.5\u2009eV, which contributes only a few percent to the measured harmonic width .At the nominal NSLS-II operation mode (3DW), the spectral measurements of the 7\u03c3x dependence on I0 for the 3DW lattice and various RF voltages. The energy spread is derived from the SLM measurements of \u03c3x(I0) using Eq.\u00a0\u03c3\u03b4(0)\u2009=\u20090.087% in the low-current limit. This value was also confirmed experimentally by measurements of decoherence of betatron oscillation24. Below a (RF voltage-dependent) threshold current, which determines the first threshold of microwave instability, the energy spread is approximately constant and equal to its unperturbed value, as shown in Fig.\u00a0\u03c3\u03b4 drift below I0\u2009=\u20090.5 mA is a systematic error caused by the diagnostic setup\u00a0of SLM for \u03c3x measurements. Above the threshold current, the microwave instability induces an energy spread increase that may be significant even at low beam intensity. The energy spread increase above the first microwave instability threshold is modulated by a number of local maxima and minima of \u03c3\u03b4. At any RF voltage, the difference between the peaks and valleys becomes smaller as I0 increases, and we will refer to the currents at which \u03c3\u03b4 reaches a local minimum as higher-order thresholds . It is interesting to note that for a given RF voltage these higher-order thresholds approximately make a straight line, and that the slope VRF.In Fig.\u00a0\u03c3\u03b4 were observed in the \u03c3\u03b4 are naturally grouped according to the measured bunch length, so that the points with the same color in Fig.\u00a0Figure\u00a0To interpret theoretically the measurements of the intensity dependence of the energy spread we distinguish three different bunch intensity regimes: 1) the low-intensity, or single-particle dynamics regime, where intensity-dependent effects are negligible; 2) the medium-intensity, or bunch-lengthening regime, where the dynamics is characterized by a stationary distribution with a constant energy spread and intensity-dependent bunch length; 3) the high-intensity, or microwave instability regime, where both energy spread and bunch length exhibit a time and intensity-dependent behavior.\u03b2x and \u03b7x and on the equilibrium values of the energy spread and emittance, the latter two of which are determined by the single-particle process of incoherent emission of synchrotron radiation. The time evolution of the electron phase space density in the low-intensity regime is governed by the Fokker-Planck equation25, which consists of a Hamiltonian part determined by the lattice configuration, and by a non-Hamiltonian part that describes the damping and diffusion due to the emission of synchrotron radiation. In the second, medium-intensity regime, the dynamics is changed by the addition of a longitudinal wakefield that is produced by the electromagnetic interaction of the electron bunch with its surroundings. The medium-intensity dynamics is similar to that of the low-intensity regime, with the same equilibrium energy spread but with a longitudinal profile that is modified by the wakefield.In the first, low-intensity regime, the measured horizontal beam size depends upon the lattice functions The microwave instability threshold current delimits the transition from the second regime to the third, high-intensity regime. Above the threshold current the energy spread increases with current, and the collective dynamics is governed by the time-dependent Vlasov-Fokker-Planck equation. Our goal is to characterize the transition to, and dynamical behavior within, the third regime.26q and p are normalized with respect to the bunch length \u03c3z and energy spread \u03c3\u03b4 respectively, i.e. z is the longitudinal distance with respect to the reference particle. In Eq.  is the longitudinal wakefield and 12 using a longitudinal wakefield W|| that was obtained numerically using the code GdfidL27. For the calculation of W|| the point-source Green\u2019s function was approximated by a Gaussian distribution of length W|| includes the resistive wall and geometric changes in vacuum chamber components of NSLS-II.Here entclass1pt{minima\u03c3\u03b4 correspond to the currents at which the electron beam becomes less perturbed, with smaller fluctuations of energy spread about its new and inflated near-equilibrium \u03c3\u03b4. As shown in Figs.\u00a0Numerical simulations of the Vlasov-Fokker-Planck equation using space code are compared with SLM measurements. Figure\u00a028, which interprets the longitudinal mode coupling as the physical mechanism responsible for the onset of the microwave instability.To analyze the evolution of the energy spread above the first threshold and to investigate its modes of oscillation, we discuss now a theoretical framework based on the linearized Vlasov equation0, with perturbation \u03a81 assumed to have an harmonic time dependence with complex frequency \u03a9:The linearized Vlasov equation is obtained by neglecting the right hand side of Eq. , which i29. In addition, by assuming that above the Ith the energy spread increases to quench the instability, we can extend our analysis to the high intensity, microwave instability regime. The quenching mechanism can be understood as due to the additional Landau damping provided by the increase in energy spread.The approximation of the equilibrium density with an intensity-dependent two-dimensional Gaussian distribution allows us to approximately take into account the effect of bunch lengthening and incoherent synchrotron frequency shift without the construction of the action-angle variables associated with the self-consistent equilibrium as was done in\u03c3z, synchrotron frequency \u03c9s and energy spread \u03c3\u03b4 on the bunch intensity I0. In this case, by linearizing about the Gaussian equilibrium density \u03a80 one can derive the following integral equation for the density bunching \u00a028:me is the electron rest mass and c is the velocity of light. The symmetric kernel In(x) byIn what follows we omit the explicit dependence of the bunch length Z|| is the Fourier Transform of the longitudinal wakefield W||.In Eq.  Z|| is t31, but their work effectively neglected the RF focusing so that the synchrotron frequency \u03c9s played no explicit role, which is in sharp contrast to Eq.\u00a0 to c to c5) tr of Eq.  this impI0 at VRF\u2009=\u20093\u2009MV combining the measured, numerically simulated, and semi-analytically calculated data. The stable modes of oscillation calculated analytically with Eqs.\u00a0(VRF\u2009=\u20093\u2009MV. Based on the applied theory, the appearance of an unstable mode at To confirm these conclusions, Fig.\u00a0ith Eqs.\u00a0,5 are reWe studied dynamic properties of the energy spread above the microwave instability threshold, in regimes characterized by states of quasi-equilibrium and by the presence of one or several stable modes of oscillation. The beam dynamics has been studied experimentally by high-precision measurements performed at the NSLS-II storage ring with different machine settings. The measurements carried out with three distinct diagnostic methods, show very clear and reproducible intensity-dependent patterns, thus allowing a theoretical interpretation. To this end, we performed numerical simulations of the Vlasov-Fokker-Plank equation and compared the results with a novel eigenvalue analysis based on the linearized Vlasov equation. This analysis, with the assumption that two stable oscillation modes merge together at the instability threshold, allows us to interpret the microwave instability as a classical mode coupling. Moreover, by assuming that the energy spread above threshold increases to provide sufficient Landau damping to suppress the growth of unstable modes, the full spectral analysis is reduced to the determination of solely stable modes of oscillation. The measurements discussed in this paper, together with the methods used for their interpretation, offer an opportunity to predict and further characterize the microwave instability threshold, and represent an effort to enhance the understanding of the dynamic evolution of the energy spread above the threshold which may further enable controlled operation of electron storage rings at high current."}
+{"text": "A post-conflict vaccination campaign, Central African Republic. Bull World Health Organ. 2018 Aug 1;96(8):540\u201347. on page 544,"}
+{"text": "Animal models and, in particular, mice models, are important tools to investigate the pathogenesis of respiratory diseases and to test potential new therapeutic drugs. Lung function measurement is a key step in such investigation. In mice, it is usually performed using forced oscillation technique (FOT), negative pressure-driven forced expiratory (NPFE) and pressure-volume (PV) curve maneuvers. However, these techniques require a tracheostomy, which therefore only allows end-point measurements. Orotracheal intubation has been reported to be feasible and to give reproducible lung function measurements, but the agreement between intubation and tracheostomy generated-data remains to be tested.Using the Flexivent system, we measured lung function parameters , forced expiratory volume in the first 0.1\u2009s (FEV0.1), compliance (Crs) of the respiratory system, compliance (C) measured using PV loop and an estimate of inspiratory capacity (A)) in healthy intubated BALB/cJ mice and C57BL/6\u2009J mice and compared the results with similar measurements performed in the same mice subsequently tracheostomized after intubation, by means of paired comparison method, correlation and Bland-Altman analysis. The feasibility of repetitive lung function measurements by intubation was also tested.We identified parameters that are accurately evaluated in intubated animals . Repetitive lung function measurements were obtained in C57BL/6\u2009J mice.This subset of lung function parameters in orotracheally intubated mice is reliable, thereby allowing relevant longitudinal studies.Supplementary information accompanies this paper at (10.1186/s12931-019-1177-9). Chronic airway diseases e.g. chronic obstructive pulmonary disease (COPD) are devastating diseases characterized by impaired respiratory function. For instance, parameters derived from the forced expiratory maneuvers, such as the forced expiratory volume in the first second FEV1) to the forced vital capacity (FVC) ratio are widely used to characterize functional obstruction  to the f\u20134, mice Similar to spirometry in cooperative humans, equipment for lung function testing such as the Buxco or the Flexivent system has been developed for mice. Inflating mouse lungs to a given pressure followed by connection of animal\u2019s airways to a negative pressure reservoir allows to trigger a forced expiratory maneuver and thus to measure FEV0.1 (forced expiratory volume in the first 0.1\u2009s), FVC , and the surrogate of the human FEV1/FVC ratio, so-called FEV0.1/FVC ratio . Single However, monitoring lung function in mice, as above described, requires a tracheostomy, a procedure that has hampered longitudinal studies in animal models of chronic respiratory diseases. Orotracheal intubation could overcome this problem although the small size of the mouse, presenting numerous advantages such as cheap housing and cost minimizing for testing expensive drugs, is challenging for this technique . HoweverIn two inbred mice strains that are routinely studied , we thus aimed at comparing lung function measurements obtained using orotracheal intubation with the same data obtained using tracheostomy. We evaluated the agreement between the two different methods by means of intra-class correlation and Bland-Altman analyses. We also tested the feasibility of repetitive lung function measurements by intubation.Male BALB/cJ mice  and male C57BL/6\u2009J mice  were obtained from Janvier . The mice were housed in a conventional animal facility with free access to food and water. All animal studies were performed according to European and French directives about vertebrate animals protection use for animal experiments. Agreement was obtained from French authorities (number A33\u2013063-907) and all the protocols used were approved by the local ethics committee .2O. Forced oscillation measurements were performed using the single-FOT maneuver (\u201cSnapshot-150 perturbation\u201d) and the broadband FOT maneuver (\u201cQuick Prime-3 perturbation\u201d). The single FOT measurements were fitted to single compartment model to determine respiratory system resistance (Rrs) and respiratory system compliance (Crs). The multi-frequency FOT measurements were fitted to constant phase model to obtain newtonian resistance (Rn), tissue damping (G) and tissue elastance (H). PV loops were also generated to obtain compliance (C) of the respiratory system, an estimate of inspiratory capacity (A), curvature of the upper portion of the deflation limb of the PV curve (K) and the area enclosed by the PV loop (Area). The negative pressure-driven forced expiratory (NPFE) maneuver was then performed by inflating the mouse lungs to a pressure of 30\u2009cm H2O over 1\u2009s, hold this pressure for 2\u2009s before connecting the animal\u2019s airways to the negative pressure reservoir (\u2212\u200950\u2009cm H2O) for 2\u2009s. The forced expired volume over 0.1\u2009s (FEV0.1), forced vital capacity (FVC) and the peak expiratory flow (PEF) were calculated directly from the flow-volume loop generated during lung deflation. In every mouse, each maneuver was repeated until 2 acceptable measurements (coefficient of determination \u22650.95) were recorded. The median of at least 2 acceptable measurements was then calculated.Mice were anesthetized with an intraperitoneal injection of 125\u2009mg/kg ketamine and 10\u2009mg/kg xylazine . The same anesthetic protocol was used for intubation and tracheostomy. Orotracheal intubation was performed using an 18G, 30\u2009mm intravenous plastic catheter previously cut and tapered at 20\u2009mm , without suture sealing the wall of the trachea around the intubation catheter. Tracheostomy was subsequently performed using either the 18G metal cannula or the intubation catheter, with a suture sealing the wall of the trachea around the cannula. The animal was then connected to the small animal ventilator  and mechanically ventilated at a respiratory rate of 150 breaths/min, a tidal volume of 10\u2009mL/kg and a PEEP set at 3 cmHThe order of the experiment was randomized for C57BL/6\u2009J and BALB/cJ mice. The Flexivent was re-calibrated for the different cannulas used in intubated and tracheostomized mice if necessary. Lung function measurements were performed first in intubated mice and immediately after in the same tracheotomized animal, either using different cannulas or using the same cannula . The measurements performed in tracheostomized mouse were compared with those previously recorded in the same intubated mouse. To test the impact of previous intubation on lung function measured on tracheotomized C57BL/6\u2009J mice, other lung function measurements were performed on either solely intubated mice or solely tracheotomized animals. The measurements performed in tracheostomized mice were compared with those obtained in intubated mice. Mice were then sacrificed through pentobarbital injection of 500\u2009mg/kg.The study protocol is shown Fig.\u00a0P\u2009<\u20090.05, was analyzed using paired or unpaired t tests for variables with parametric distribution, or using Wilcoxon or Mann\u2013Whitney tests for variables with nonparametric distribution. The linear relationship between measurements obtained in intubated and tracheostomized mice was evaluated by Pearson correlation. Bland-Altman plots were built with Prism 6 software. The relationship between the difference and the mean of the two methods  was evaluated by Spearman correlation.The statistical analysis was performed with Prism 6 software . Statistical significance, defined as 2O.s/mL. This value was close to that of 18G metal cannula used for tracheostomy (0.27 cmH2O.s/mL). One of the 15 BALB/cJ mice (7%) and 5 of the 15 C57BL/6\u2009J mice (33%) died during the intubation procedure. One of the 15 BALB/cJ mice (7%) was successfully intubated, but showed aberrant lung function data using intubation as well as tracheostomy. On average, 23\u2009min and 26\u2009min separated the measurements performed following intubation from those following tracheostomy in C57BL/6\u2009J and BALB/cJ mice, respectively. No sign of respiratory drive was observed during the measurements. Overall, lung function was successfully measured in 13 of the 15 BALB/cJ mice (87%) and 10 of the 15 of the C57BL/6\u2009J mice (67%) in response to NPFE maneuver, in 14 of the 15 BALB/cJ mice (93%) and 10 of the 15 of the C57BL/6\u2009J mice (67%) using FOT single compartment model and PV loop maneuver, and in 12 of the 15 BALB/cJ mice (80%) and 9 of the 15 of the C57BL/6\u2009J mice (60%) using FOT constant phase model. We then compared the lung function data obtained using intubation and subsequently tracheostomy in the same mice of each strains.Because the metal cannula used for tracheostomy was traumatic when used for orotracheal intubation, we tested other intubation catheters, and finally chose the cannula that offered the best compromise between a resistance close to that of the tracheostomy cannula and sufficient flexibility to enable orotracheal intubation. To facilitate intubation, the 18G catheter was narrowed by slightly heating the distal end of the tube. The resistance of the tube, measured during calibration of the FlexiVent system was 0.32 cmHRegarding BALB/cJ mice, the expiratory flow-volume curve was downward shifted especially at the onset of expiration in intubated compared with tracheostomized mice Fig.\u00a0a. As a cTo evaluate the possibility that lung function measured in tracheotomized C57BL/6\u2009J mice could have been affected by previous intubation, we compared lung function obtained in animals that have been only intubated or tracheostomized. We observed a downward shift of the expiratory flow-volume curve for intubated C57BL/6\u2009J animals compared with tracheostomized mice  in intubated mice compared to tracheostomized animals  was higher than that required in tracheotomized mice , this only represented a significant change in BALBc/J mice. The analysis of CODcp did not show any difference in intubated mice compared to tracheostomized animals , C was strongly positively correlated with Crs in both strains, whatever the measurement method, orotracheal intubation or tracheostomy , we also performed another set of experiments using the same cannula. Because the metal cannula used for tracheostomy was traumatic when used for orotracheal intubation, we chose the intubation catheter for both procedures. Two of the 22 BALB/cJ mice (9%) died during the intubation procedure; 3 of the 22 BALB/cJ mice (14%) and 5 of the 22 C57BL/6\u2009J mice (23%) could not be intubated. One of the 22 BALB/cJ mice (4%) was successfully intubated, but showed aberrant lung function data using intubation as well as tracheostomy. Overall, lung function was successfully measured in 16 of the 22 BALB/cJ mice (73%) and 16 of the 22 of the C57BL/6\u2009J mice (73%) in response to NPFE maneuver, in 16 of the 22 BALB/cJ mice (73%) and 17 of the 22 of the C57BL/6\u2009J mice (77%) using FOT single compartment model and PV loop maneuver, and in 8 of the 22 BALB/cJ mice (36%) and 15 of the 22 of the C57BL/6\u2009J mice (68%) using FOT constant phase model.In both strains of mice, the flow-volume curves obtained in intubated mice were similar to those measured in tracheostomized animals Fig.\u00a0a-b. ExceIn intubated C57BL/6\u2009J mice, but not in BALB/cJ mice, Rn derived from the impedance curves was significantly increased compared to tracheostomized C57BL/6\u2009J mice Fig.\u00a0a-b. C anAltogether, with the exception of Crs and C for C57BL/6\u2009J mice, the second set of experiments allowed us to identify exactly the same subsets of parameters that are accurately evaluated in intubated BALB/cJ animals: forced vital capacity (FVC), forced expiratory volume in the first 0.1\u2009s (FEV0.1), compliance (Crs) of the respiratory system, compliance (C) measured using PV loop, and an estimate of inspiratory capacity (A).To test the possibility of repeated intubations, we performed sequential lung function measurements using intubation in C57BL/6\u2009J mice with 19\u2009days interval , that were accurately evaluated in intubated BALB/cJ animals. The measurements of Crs and C in intubated C57BL/6\u2009J mice should be used carefully in this specific strain. Indeed, their use was validated by our first comparison study using different cannulas, but not the second one with the same catheters. However, the existence of a proportional bias does not prevent the use of intubation to measure C before and after an experimental challenge, on the condition that only relative changes are mentioned.Some apparent differences between both strains can be observed. In particular, the expiratory flow-volume curve measured in intubated BALB/cJ mice was altered compared to that obtained in tracheostomized mice, while both curves were very similar in C57BL/6\u2009J mice. Regarding Rn, as expected, taking into account that it evaluates \u201cproximal\u201d resistance, Rn was smaller in tracheostomized BALB/cJ whereas G and H were similar in intubated and tracheostomized BALB/cJ. A similar result was not found in C57BL/6\u2009J since Rn was not different in intubated and tracheostomized mice. This may be a consequence of a scattering of the data in a smaller C57BL/6\u2009J group than in the BALB/cJ group. Moreover, calculation of the threshold frequency that discriminates the central compartment alone (at high frequency) from the combination central and distal compartments (at low frequency) showed that it was different in both strains giving some support to JH Bates et al. conclusion  stating We have also investigated the possibility of repeated lung function measurements. As already shown by De Vleeschauwer et al, all mice lost weight after the first intubation . The larper se, but intubation with a cannula compatible with lung function measurement, which must offer the best compromise between a resistance close to that of the tracheostomy cannula, sufficient flexibility to enable orotracheal intubation and a diameter close to that of the trachea to avoid air leakage. Nevertheless, it can be anticipated that mortality should be lowered as the experience of the operator increases.The technique of intubation had side-effects with an increase in mortality associated to the intubation procedure. Different methods of orotracheal intubation have been proposed , 23\u201331. In conclusion, we have identified a subset lung function parameters that could accurately be evaluated in intubated mice . Repetitive lung function measurements by orotracheal intubation are feasible and seem to be reproducible in C57BL/6\u2009J mice. This suggests that lung function can be reliably assessed using orotracheal intubation in mice and therefore may be used for longitudinal studies in model of chronic respiratory diseases. The ability of lung function measurement using orotracheal intubation to detect functional and pathological changes in mice remains to be tested in various pathological models. However, we strongly believe that repeated lung function measurement in intubated mice would have several advantages in longitudinal studies with limited numbers of expensive animals.Additional file 1: Table S1. Association between parameters measured using tracheostomy and orotracheal intubation. Table S2. Agreement between the measurements of parameters by tracheostomy and orotracheal intubation. Table S3. Association between the difference and the average of the two methods for the measurements using tracheostomy and orotracheal intubation. Table S4. Association between parameters measured using tracheostomy and orotracheal intubation, both using the intubation cannula. Table S5. Agreement between the measurements of parameters by tracheostomy and orotracheal intubation, both using the intubation cannula. Table S6. Association between the difference and the average of the two methods for the measurements using tracheostomy and orotracheal intubation, both using the intubation cannula. Table S7. Association between parameters measured using orotracheal intubation at day 0 et day 19. Figure S1. Evaluation of peak expiratory flow and the FEV0.1/FVC ratio measurements obtained in mice using orotracheal intubation and tracheostomy. Figure S2. Evaluation of lung function measurement in C57BL/6\u2009J mice that have been either intubated or tracheostomized. Figure S. Evaluation of quality of the single compartment, contant phase and Salazar-Knowles models fit. Figure S4. Evaluation of the curvature of the upper portion of the deflation limb of the Pressure-Volume loop and the area enclosed by the same curve, obtained in mice using orotracheal intubation and tracheostomy. Figure S5. Relationships between the respiratory system compliance (Crs) and the compliance (C) measured in intubated and tracheostomized BALB/cJ (A) and C57BL/6\u2009J mice (B). Figure S6. Evaluation of peak expiratory flow and the FEV0.1/FVC ratio measurements obtained in mice using orotracheal intubation and tracheostomy, both using the intubation cannula. Figure S7. Evaluation of the curvature of the upper portion of the deflation limb of the Pressure-Volume loop and the area enclosed by the same curve, obtained in mice using orotracheal intubation and tracheostomy, both using the intubation cannula. (DOCX 757 kb)"}
+{"text": "Transgender women (TGW) in the U.S. experience high rates of stigma, depression, and elevated rates of suicide. This study examined correlates of suicidal ideation and estimated the conditional indirect effects of perceived stigma and psychosocial mediators on suicidal ideation.N\u00a0=\u200992) were recruited through snowball sampling in Atlanta, Georgia. Structured interviews were conducted. Suicidal ideation was assessed by combining two variables that measured suicidal thoughts. Logistic regression models were performed to identify the potential risk and protective factors for\u00a0suicidal ideation. We examined hypothesized psychosocial factors, including anxiety, depression, psychosocial impact of gender minority status, and substance use behaviors as potential mediators for the relationship between perceived stigma and suicidal ideation. All models were controlled for age, race, education, and homelessness.Using a cross-sectional study design, TGW (N\u00a0=\u200930) of the study participants. In multivariable analysis, suicidal ideation was associated with sexual abuse , anxiety , family verbal abuse , stranger verbal abuse , and psychosocial impact of gender minority status . Partner support was found to be the protective factor for suicidal ideation . In the mediation analysis, the psychosocial impact of gender minority status mediated the relationship between perceived stigma and suicidal ideation. The estimated conditional indirect effect was 0.46, (95% CI\u2009=\u20090.12\u20131.11).Suicidal ideation was reported by 33% (Interventions that aim to reduce suicidal behaviors among TGW should address stigma, psychosocial impact of gender minority status, and different forms of violence and abuse. Transgender is an umbrella term for individuals\u00a0whose gender identity or gender expression differs from what is typically associated with the sex that\u00a0they were assigned at birth. The transgender community includes individuals, who were assigned male at birth and identify as female, who were assigned female at birth and identify as male, and who identify their gender as outside the binary categories of male or female \u20134. In thAmong cisgender populations, that is, people whose gender identity and gender expression align with their assigned sex at birth , researcThe stigma and discrimination experienced due to their gender identity may be associated with several adverse health outcomes among TGW . Stigma Minority stress theory states that sexual and gender minorities experience stressors, such as discrimination and stigma, that lead to increased levels of stress that\u00a0can, in turn, deplete psychological resources  and lead to poor overall mental and physical health outcomes . In\u00a0thisThere is evidence that\u00a0psychological and psychosocial factors, such as substance use, depression, and anxiety, may explain the association between perceived stigma and suicidal ideation . SubstanEvidence for the prevalence and correlates of suicidal ideation among TGW is found in the literature , 25\u201327, n\u00a0=\u200992) between the\u00a0ages of\u00a018 and 65\u2009years who\u00a0reside in Atlanta, GA. Venues that\u00a0serve TGW and word\u2013of\u2013mouth recommendations\u00a0from transgender advocates provided the primary methods of recruitment. These venues offer\u00a0HIV prevention and care, housing, and counseling services to the TGW. The study was known as the Transgender Atlanta Personal Survey. Transgender advocates notified the study project director when they located a woman who was\u00a0willing to be screened for study participation. In addition, the project was advertised through formal and informal communication channels via advocacy groups and Lesbian, Gay, Bisexual, and Transgender (LGBT) service organizations. The project director used\u00a0print materials to\u00a0provide their\u00a0contact information. Data were collected from August 2014 through June 2015.In this cross-sectional study, multiple community\u2013based outreach strategies were used to recruit a sample of TGW (TGW were screened to determine eligibility. The inclusion criteria\u00a0were: (1) 18 to 65\u2009years of age, (2) male sex assigned at birth, and (3)\u00a0self\u2013identifying as either female or transgender. All participants who were screened,\u00a0except one individual who identified as \u201cother\u201d were eligible and consented to participate in the study. After providing written informed consent, women engaged in a face\u2013to\u2013face structured interview with a trained graduate research assistant. The training involved cultural-competency and the\u00a0use of\u00a0non-judgmental statements. Interview responses were recorded on a portable electronic tablet, using Qualtrics\u00a9 software . The Institutional Review Board of Georgia State University approved study protocols following a full board review.The survey assessed sociodemographic characteristics, a broad range of theoretical contextual factors, and self-reported HIV status. In addtion, we\u00a0assessed the prevalence of several trauma exposures, such as \u201cever experienced physical abuse by an intimate partner,\u201d \u201cever being a victim of sexual abuse,\u201d \u201cever experienced childhood sexual abuse,\u201d and psychosocial factors.Perceived stigma was assessed by using four items adapted for TGW from the original scale developed for gay individuals , 45. TheThe psychosocial impact of gender minority status was assessed using three items from a 4-item subscale developed by Sjoberg and colleagues . The 4-iSuicidal ideation was assessed by combining two items that measured suicidal thoughts. The\u00a0items were: (1) \u201cIn the past 12\u2009months, have you considered attempting suicide?\u201d for which the\u00a0response options were Yes/No; and (2) \u201cI have thought about suicide because of my gender status,\u201d for which\u00a0response options were provided on a 5-point Likert scale, ranging from 1 = strongly disagree to 5 =\u00a0strongly agree. We dichotomized Item 2 by collapsing the\u00a0responses of\u00a04 (agree) and 5 (strongly agree) as \u201cYes\u201d and all other responses as \u201cNo.\u201d Then, we created a new variable, \u201csuicidal ideation,\u201d for\u00a0the participants who responded \u201cYes\u201d to\u00a0either of the two items; these participants were considered\u00a0as experiencing suicidal ideation and other participants, as not experiencing suicidal ideation.Depression was measured using six items from the Brief Symptom Inventory . This suAnxiety was measured using the 3-item subscale from the Brief Symptom Inventory. The\u00a0items were: (1) \u201cExperienced nervousness or shakiness inside\u201d; (2) \u201cFeeling tense or keyed up\u201d; and (3) \u201cFeeling so restless you couldn\u2019t sit still.\u201d Response options for all items were on a 5-point Likert scale, ranging from 1 = not at all to 5 =extremely. For the\u00a0anxiety score, we calculated\u00a0the mean of the three items.Excessive drinking was measured by three items: (1) \u201cIn the past 30\u2009days, on how many days did you drink any alcohol?\u201d; (2) \u201cOn the days when you drank alcohol in the past 30\u2009days, about how many drinks did you have on average?\u201d; and\u00a0(3) \u201cIn the past 30\u2009days, how many times did you have 5 or more alcoholic drinks in one sitting?\u201d Based on the Dietary Guidelines for Americans, 2015\u20132020 , particiNon-injection drug use was measured by one item: \u201cIn the past 12\u2009months, have you used any non-injection drugs, other than those prescribed for you?\u201d Response options were Yes/No.Injection drug use was measured by one item: \u201cHave you ever in your life shot up or injected any drugs other than those prescribed for you? By drugs, I am referring to drugs such as heroin, meth \u2013 not hormones or silicone? By shooting up, we mean anytime you might have used drugs with a needle, either by mainlining, skin popping, or muscling.\u201d Response options were Yes/No.Intimate partner violence\u00a0is the experience of physical and emotional violence by a romantic or sexual partner in one's\u00a0lifetime. This variable was measured by three items: (1) \u201cIn your lifetime, have you ever been physically abused by a romantic or sexual partner? By physical abuse, we mean a range of behaviors, from slapping, pushing, or shoving, to severe acts, such as being beaten, burned, or choked\u201d; (2) \u201cIn your lifetime, have you ever been emotionally abused by a romantic or sexual partner? By l emotional\u00a0abuse, we mean name-calling, or humiliating you, or trying to monitor and control or threaten you\u201d; and (3) \u201cHave you ever been physically abused or beaten by a romantic or sexual partner because of your gender identity or presentation?\u201d Response options were Yes/No.Sexual abuse is\u00a0the experience of forced oral/anal sex in one's\u00a0lifetime. This variable was measured by three items: (1) \u201cIn your lifetime, has someone ever made you perform oral sex?\u201d; (2) \u201cIn your lifetime, has someone ever made you receive anal sex? By receiving anal sex, we mean they put their penis in your anus (you were the bottom)\u201d; and\u00a0(3) \u201cIn your lifetime, has someone ever made you perform anal sex? By performing anal sex, we mean they made you put your\u00a0penis in their anus (you were the top)\u201d. Response options were Yes/No.Child sexual abuse was measured by one item: \u201cAs a child (less than 16\u2009years old), were you ever sexually abused?\u201d Response options were Yes/No/I do not remember. HIV status was measured by one item, \u201cWhat was the result of your most recent HIV test?\u201d for which the\u00a0response options were \u201cnegative\u201d/ \u201cpositive\u201d/ \u201cI do not know my status\u201d.Descriptive statistics were computed among TGW who reported suicidal ideation and those\u00a0who reported no suicidal ideation. To estimate bivariate associations, TGW with suicidal ideation were compared to TGW without suicidal ideation, using chi-square analyses and Fisher\u2019s exact test for categorical variables . Wilcoxon rank-sum tests were used for continuous variables . Control variables were selected based on the literature and the presence of statistically significant differences in our bivariate analyses . Age, race, and education were statistically significantly different between both groups, and homelessness was associated with suicidal ideation among TGW in prior studies. To estimate the association of substance abuse behaviors, violence, abuse, HIV status, and other psychosocial factors with suicidal ideation, we conducted separate multivariable logistic regression models, adjusting for age, race, education, and homelessness.path c); the effect of X on M is indicated by path a; the effect of M on Y controlling for X, is indicated by path b; and the direct effect of X on Y, controlling for M, is path c\u2019. The indirect effect is the product of path a and path b, which is path ab. The equation (c\u2009=\u2009c\u2019\u2009+\u2009ab), which indicates the total effect is equal to direct and indirect effects, does not\u00a0hold true due to the use of logistic regression. By standardizing the coefficients expressed on a\u00a0log-odds metric (multiplied by the standard deviation of the predictor variable and divided by standard deviation of outcome variable), however,\u00a0c would be approximately equal to c\u2019\u2009+\u2009ab [path a, path b, path c\u2019, and path c were generated. For testing the dichotomous mediators  we used the INDIRECT macro [N=\u20095000) was used to construct confidence intervals\u00a0(CIs) for the indirect effect (path ab) to determine statistically significant mediators. The Statistical Package for Social Sciences (SPSS), version 25.0, , was used for all analyses.We also examined the impact of perceived stigma on suicidal ideation through mediation analyses. A mediator is a variable that explains, or accounts for, the effect of the independent variable on the dependent variable\u00a0, 50. To  c\u2019\u2009+\u2009ab . Using P c\u2019\u2009+\u2009ab , we testN=\u200992), the prevalence of suicidal ideation was 33% (n=\u200930). The average\u00a0age of participants was 35\u2009years, 51% were homeless, 60% of the participants who are aware of their HIV status reported as HIV positive, 50% had more than a\u00a0high-school education, and 84% identified as Black or African American , . The psychosocial impact of gender minority status also\u00a0was significantly associated with suicidal ideation (path b) , and there was a significant indirect effect (path ab) of the psychosocial impact of gender minority status on the association between perceived stigma and suicidal ideation . The other psychosocial mediator variables, anxiety, depression, excessive drinking, injection drug use, and non-injection drug use, were not\u00a0significant mediators. The mediation effect size was estimated by calculating the ratio of (path ab/path c) [In the mediation analyses Table\u00a0, results/path c) . The psyIn this\u00a0study, we sought to determine the correlates for suicidal ideation among TGW and examined the mediation pathways that\u00a0explain the underlying relationships. In our sample, the prevalence of suicidal ideation was 33%, within the range reported by other studies , 53. We Using the postulates of minority stress theory and the\u00a0psychological mediation framework, we investigated whether certain\u00a0psychosocial factors explained the associations between perceived stigma and suicidal ideation. We found that the psychosocial impact of gender minority status was a statistically significant mediator in our sample, indicating that the effect of perceived stigma on suicidal ideation may be explained by this pathway. The other psychosocial mediators examined, depression, anxiety, and substance use behaviors, were not statistically significant, which is contrary to the\u00a0findings\u00a0of previous research .The risk factors that we found to be significantly associated with suicidal ideation are in keeping\u00a0with those of\u00a0other studies that reported a\u00a0lack of or low social support , 13, 54,As would be expected, interpersonal factors, such as experiencing sexual abuse; psychosocial factors, such as anxiety and depression; and trans-specific factors, such as perceived stigma, family and stranger verbal abuse due to gender identity; and the psychosocial impact of gender minority status predicted the likelihood of suicidal ideation among TGW. We also found that partner support was a protective factor among TGW, similar to other studies that found a reduced risk of suicidal ideation among the TGW with higher levels of social support , indicatWe found that\u00a0the construct, the psychosocial impact of gender minority status, which measures the psychosocial distress experienced by TGW related to their unique gender identity, was significantly associated with suicidal ideation. The psychosocial impact of gender minority\u00a0status is different from the other general forms of distress, such as anxiety, psychological distress, and depression , and repperceptions of the prejudice and discrimination in their community. In this context, perceived stigma could be viewed as a proxy for distal external and objective stressors. We found that perceived stigma nonetheless had an impact on psychological processes among the TGW in our study. The implication of this conceptualization is that perceptions of the societal anti-transgender attitudes and structural level anti-transgender polices are important to assess even if they are considered as proxies for distal objective measures of stressors and that,\u00a0for some TGW, perceptions are important to their mental health. TGW who perceive transgender-related stigma in our study, experienced increased psychosocial effects that could eventually be\u00a0manifested in behaviors such as suicidal ideation.We also found that the psychosocial impact of gender minority status was a statistically significant mediator that partially explains the relationship between perceived stigma and suicidal ideation. This finding was similar to the studies that: (a) reported that\u00a0internalized trans-negativity  mediated the relationship between distal stressors  and suicidal ideation , and (b)Although\u00a0this study fills an important gap in the literature, several limitations exist. First, our study is a cross-sectional study, and we cannot infer causation between predictor variables and suicidal ideation. In particular, the lack of temporality limits the interpretation of the findings from the mediation model. Future studies should investigate these associations among TGW in a longitudinal framework. In addition, convenience sampling was utilized to recruit participants, and\u00a0most of the participants were referred through the community-based organization\u00a0that provides support services to TGW. Therefore, our sample may not be representative of TGW who live\u00a0in Atlanta. Due to the small sample size, this study may not have adequate statistical power to detect some significant associations. Finally, although culturally competent interviewers were utilized, social desirability bias may have affected\u00a0some of the sensitive responses from participants. Recall bias also\u00a0may have affected the participants\u2019 responses, particularly with regard to\u00a0questions about early childhood.TGW are disproportionately affected by suicidal ideation and are in urgent need of tailored and effective interventions to ameliorate their mental health concerns. Interventions that increase social inclusion may be particularly beneficial . In one"}
+{"text": "The tropane and granatane alkaloids belong to the larger pyrroline and piperidine classes of plant alkaloids, respectively. Their core structures share common moieties and their scattered distribution among angiosperms suggest that their biosynthesis may share common ancestry in some orders, while they may be independently derived in others. Tropane and granatane alkaloid diversity arises from the myriad modifications occurring to their core ring structures. Throughout much of human history, humans have cultivated tropane- and granatane-producing plants for their medicinal properties. This manuscript will discuss the diversity of their biological and ecological roles as well as what is known about the structural genes and enzymes responsible for their biosynthesis. In addition, modern approaches to producing some pharmaceutically important tropanes via metabolic engineering endeavors are discussed. Plants are sessile organisms and thus evolved natural products or \u201cspecialized metabolites\u201d as a chemical response to both biotic and abiotic forces. Specialized metabolites are used by plants to defend themselves and communicate with other plants and organisms in their environments. Whilst the chemical diversity of plant specialized metabolites is vast, with total numbers thought to exceed over 200,000 structures, common themes of structure and function are the result of repeated and convergent evolution of both their biosynthesis and biological roles . MoreoveErythroxylum coca, a species notable for the production of the tropane alkaloid cocaine (1), was used in Peruvian households at least 8000 years ago -octane core structure of TAs is found in over 200 alkaloids [N-methyl-9-azabicyclo[3.3.1]-nonane, the core scaffold of GAs, appears in considerably fewer alkaloid metabolites (Tropane (TA) and granatane (GA) alkaloids are structural homologues, sharing similar chemical compositions and core scaffolds. Despite their similarities, TAs and GAs show different distribution patterns across the plant kingdom. The bicyclo3..1-octanelkaloids ,6  and other TAs serves as a structural analog of acetylcholine. TAs have been observed to attach to and inhibit muscarinic acetylcholine receptors .,51.50,51Examples of scattered distributions of alkaloid classes can be observed throughout the plant kingdom. For example, the legumes (Fabaceae) contain several alkaloid secondary metabolites that are not found in all members within the family . In someDiversity among tropane and granatane alkaloids can also be seen at the biochemical level. Most data available regarding enzymes involved in TA biosynthesis comes from species within the Solanaceae family. Despite recent advancements, some critical steps in TA biosynthesis remain ambiguous. Additionally, recent advances in high throughput sequencing technologies, plant genomics, and biochemical methods have aided the progress of elucidating TA biosynthesis in other non-model species, specifically within the Erythroxylaceae. Lastly, even though the biosynthesis of GAs is predicted to be similar to TAs, plausibly a whole new set of enzymes were recruited for the biosynthesis of GAs. This would mean that untapped gene and enzyme diversity is present in the GA and TA biosynthetic pathways. It is therefore important to elucidate the biosynthesis of these alkaloids to help further understand specific parts of their mechanisms and their metabolic processes.10) and arginine (11) are the amino acids predicted to be the starting substrates of TA biosynthesis  may be a starting amino acid for incorporation into the tropane moiety. Other studies using D. metel and D. stramonium have also shown the incorporation of proline (12) into TA compounds, such as scopolamine (3) and tropine (13) . Fe. Fe10) aine (13) . Biosyntine (13) . Consequ10) is first methylated at the \u03b3-N position. A proposed alternative route includes the decarboxylation of ornithine (10) to form the polyamine putrescine (14) as the first step -1,5,10-triazadecane fed to Nicotiana glutinosa plants revealed that N-methylspermidine can also be incorporated into the pyrrolidine ring of nicotine -4-(N-methyl-2-pyrrolidinyl)-3-oxobutanoate -4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate to the leaves of E. coca -cinnamic acid and the N-acetylcysteamine thioester of -trans-cinnamic acid , which in reality corresponds to pelletierine (5). The name isopelletierine is the optically inactive racemate of pelletierine (5) [N-methylpelletierine (7) is 1-[(2R)-1-methyl-2piperdinyl]-2-propanone. Pseudopelletierine (6), also referred to as granatanone, contains a bicyclic core and can be referred to as  nonan-3-one]. Additionally, the presence of anabasine (8), in Nicotiana species would by extension mean that selected members of the Solanaceae can be classified as granatane producing members. Furthermore, the solanaceous species W. somifera produces anabasine (8), which could also be classified as a GA. In addition, several Sedum species (family Crassulaceae) produce the compounds pelletierine (5), N-methylpelletierine (7), and pseudopelletierine (6). All of the species discussed above are members of the order Solanales and are more closely related to one another than they are to other granatane producing angiosperms  . The coriosperms . This ma25) is the starting substrate for the entry into granatane biosynthesis. In several cases, different forms of labeled lysine were found incorporated into the core granatane structure  and [1.5-14C 3H2] cadaverine yields incorporation of the labels into N-methylpelletierine (7) and pseudopelletierine (6)  cadaverine, Leistner et al. (1990) have predicted that there should be a lysine decarboxylase enzyme that also contains oxidase activity such that the only cadaverine (26) intermediate that exists is always enzyme bound -4-(l-methyl-2-pyrrolidinyl)-3-oxobutanoate to D. stramonium plants -lysine into anabasine (8) [26) would be achieved with the help of a P. granatum cadaverine N-methyltransferase (CMT). CMTs have not been isolated and characterized in other species, but this enzyme is predicted to be related to putrescine N-methyltransferases (PMT) and spermidine synthases (SPDS). PMT cDNA sequences have been found in Nicotiana species by the sequencing of large genomic libraries [N-methylcadaverine (27) in P. granatum and Sedum species could be performed with the aid of an enzyme similar to the methylputrescine oxygenase (MPO) present in the biosynthesis of nicotine in N. tabacum. While a copper dependent oxidase is used for tropane alkaloid biosynthesis, it is not possible to rule out alternative enzymes such as polyamine oxidases that require FAD as a cofactor [25) described in Hypothesis III would be performed by a lysine methyltransferase (LMT). The responsible enzyme may be related to the lysine methyltransferases ubiquitously present in eukaryotic primary metabolism for gene access regulation to chromatin [The enzymes responsible for carrying out the biochemical reactions described above are based on an extension of similar reactions carried out in tropane producing species. The decarboxylation of lysine (sine (8) . The metibraries ,126. Thecofactor . The methromatin .20) [20) was a true intermediate of the biosynthesis of TAs. However, it is most likely that cuscohygrine is a dimerized product of hygrine (20) which in turn is a breakdown product of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoyl-CoA (34). If this compound is present as a free acid under physiological conditions a \u03b2-ketoester is formed, \u03b2-ketoesters very often spontaneously decarboxylate [9) is the dimerization product of pelletierine (5). This also supports the presence of an oxobutanoate intermediate.In both granatane and tropane alkaloid producing species, dimerized versions of intermediates within their respective pathways have been found. For example, cuscohygrine is the dimerized form of hygrine (20) . Originaboxylate . In the 3). The World Health Organization (WHO) includes these important pharmaceutical compounds on their list of essential drugs [3), however their low yield of 16% does not make this method economically feasible [3) in hairy root cultures is achieving industrial level yields [There has been an increasing interest in the biosynthesis of tropane alkaloids (TAs), especially to up-regulate the production of valued compounds, such as atropine and scopolamine  is the most popular choice for increasing yields via metabolic engineering. Past methods such as genetic breeding, polyploid breeding and radiation breeding have failed to yield a higher content of scopolamine (3) in A. belladonna [in planta. A common focal point centers on what is considered the first and last rate-limiting enzymes in the TA pathway, putrescine N-methyltransferase (PMT) and hyoscyamine 6\u03b2-hydroxylase (H6H). The overexpression of only one PMT gene in transgenic hairy root cultures of A. belladonna did not change the total TA content [PMT gene in D. metel was overexpressed, the TA content was significantly increased by almost four times that of the control [4) to scopolamine (3) [H6H gene resulted in an increase in the biosynthesis of scopolamine (3) in the transformed TA producing plant, A. belladonna. Another successful use of H6H was in transgenic Hyoscyamus muticus hairy root cultures, where scopolamine (3) levels increased to over 100 times that of the controls [H6H in transgenic A. belladonna plants resulted in the leaf and stem alkaloid contents to be exclusively scopolamine (3) [Due to its high demand in medicine, scopolamine (lladonna . Researc content . If the  control . H6H hasmine (3) . The ovecontrols . Furthermine (3) .PMT and H6H were overexpressed simultaneously in transgenic H. niger root cultures, scopolamine (3) biosynthesis increased to levels over nine times more than the wild type [NtPMT and HnH6H in A. belladonna significantly increased scopolamine (3) content of secondary roots when compared to wild-type plants [Metabolic engineering endeavors are becoming more complex and are moving away from only modifying one gene at a time. When both ild type . In an ie plants . More stD. metel produces important medicinal tropanes and is used by researchers because of its tractable hairy root culture system. Agrobacterium rhizogenes can transform plant roots to hairy roots by utilizing the Ri T-DNA plasmid it carries. Hairy roots that have been induced by A. rhizogenes have high growth rates, are genetically stable, and produce copious amounts of lateral roots [Staphylococcus aureus and Bacillus cereus as biotic elicitors and silver nitrate and nanosilver as abiotic elicitors on the hairy root cultures of D. metel to see their effects on biomass and atropine production. When live bacteria are present in transformed root cultures, there is a considerable influence on secondary metabolite accumulation [D. metel infected by B. cereus and S. aureus was reduced more than half when compared to the control. The authors hypothesize that this may be a cause of atropine secretion into the culture medium that was then converted into scopolamine (3) [3) was not analyzed in the spent media. The living bacteria can cause various influences on roots, affecting enzymes in the TA pathway to produce alkaloids in D. metel roots [al roots . Increasal roots . Shakeramulation . Contrarmine (3) . Howeverel roots . Althougel roots .2) content in Anisodus acutangulus hairy root cultures [4) content in D. stramonium hairy root cultures [Other attempts to engineer higher TA contents in plants include those that use abiotic elicitors. A summary of recent metabolic engineering studies using elicitors can be seen on cultures . In addicultures . Silver cultures ,149. As cultures ,149,150.cultures . D. metel and observed an increase in the transformed hairy root biomass of approximately 16%. However, only half of the expected atropine accumulation was observed in these treatments. Although secretion of atropine was not measured in this study, subsequent reports corroborated this hypothesis by finding a three-fold increase in alkaloids in the spent culture media following silver nitrate treatment [Artemisia annua and D. metel hairy roots increasing tropanes by at least 2.4 fold [14) must be present in abundance during TA production for a high yield of the final alkaloid. Currently there are only a few studies attempting to engineer the TA pathway in microorganisms. Qian et al. engineered a strain of Escherichia coli capable of efficiently producing putrescine (14) [14) degradation, uptake and utilization were also deleted. Stress to cells by the overproduction of putrescine (14) was handled by the deletion of RpoS, a stress responsive RNA polymerase sigma factor. To increase the conversion of ornithine (10) to putrescine (14), overexpression of ornithine biosynthetic enzymes and ornithine decarboxylase (ODC) was also necessary. The final metabolically engineered E. coli strain produced 1.68 g/L of putrescine (14) and high cell density cultures (HCDCs) produced 24.2 g/L of putrescine (14). This would be the first step for engineering alkaloid biosynthesis that relies on putrescine (14) in microorganisms. In a follow-up study, Qian et al. (2011) performed similar manipulations to produce a strain of E. coli capable of producing the polyamine cadaverine (26). Introduction of an l-lysine decarboxylase in addition to overexpressing dapA, the gene encoding the enzyme dihydrodipicolinate synthase successfully resulted in the new strain producing as much as 9.61 g/L cadaverine (26) from renewable resources [Shakeran et al. (2015) used silver nitrate as an abiotic elicitor in reatment . Still, reatment ,154. Nan2.4 fold . Initialine (14) . Howeveresources . If metaE. coca in the Erythroxylaceae were used to study TA biosynthesis [1) production. The jasmonic acid-isoleucine (JA-Ile) analogue coronalon and salicylic acid (SA) were also used as elicitors to observed their effects on calli metabolism. All three culture media growing calli accumulated cocaine (1). The medium used to grow calli also significantly affected natural product metabolism. The only treatments that yielded higher amounts of cocaine (1) were dependent upon culture media, not upon elicitor treatment. For example, Anderson rhododendron medium (ARM) produced cocaine (1) an order of a magnitude greater than both Gamborg B5 (GB5) and modified Murashige-Tucker medium (MMT), but lower levels of hydroxycinnamate-quinate esters such as chlorogenic acid (CGA) were detected. Interestingly the elicitors coronalon and salicylic acid did not yield any increase in TA production suggesting that TAs, at least in E. coca, may not be regulated by common plant defense hormones.Using plant in vitro cell culture or tissue culture is an important tool when studying the regulation and biosynthesis of secondary metabolites. Large quantities of plant material can be produced under controlled and sterile conditions. In tissue cultures of solanaceous plant species, using elicitors mimicking stress hormones can increase important secondary metabolite production . Recentlynthesis . VariousRecent advances in genomics, transcriptomic and metabolomic technologies are poised to illuminate the biosynthetic foundations of TAs and GAs. Future research on the biosynthesis of TAs and GAs will affect multiple fields of research. First, enzymes involved in TA and GA biosynthesis will expand our fundamental knowledge of chemistry and enzymology. Second, elucidation of the genes and enzymes underlying TA and GA biosynthesis will expand the molecular tools available to synthetic biologists. With these additional tools, scientists can look to not only produce TAs and GAs in heterologous hosts but also to engineer novel molecules . Lastly,"}
+{"text": "Diffusive losses of nitrogen and phosphorus from agricultural areas have detrimental effects on freshwater and marine ecosystems. Mitigation measures treating drainage water before it enters streams hold a high potential for reducing nitrogen and phosphorus losses from agricultural areas. To achieve a better understanding of the opportunities and challenges characterising current and new drainage mitigation measures in oceanic and continental climates, we reviewed the nitrate and total phosphorus removal efficiency of: (i) free water surface constructed wetlands, (ii) denitrifying bioreactors, (iii) controlled drainage, (iv) saturated buffer zones and (v) integrated buffer zones. Our data analysis showed that the load of nitrate was substantially reduced by all five drainage mitigation measures, while they mainly acted as sinks of total phosphorus, but occasionally, also as sources. The various factors influencing performance, such as design, runoff characteristics and hydrology, differed in the studies, resulting in large variation in the reported removal efficiencies.The online version of this article (10.1007/s13280-020-01345-5) contains supplementary material, which is available to authorized users. The high intensive agricultural production dominating parts of the world, such as Western Europe and North America, is one of the main causes of eutrophication resulting in water quality problems and ecosystem degradation worldwide , denitrifying bioreactors (DBR) and controlled drainage (CD) and the two emergent technologies saturated buffer zones (SBZ) and integrated buffer zones (IBZ) Fig.\u00a0. To obtaIn FWS, drainage water typically passes one or more deep basins or channels and shallow vegetated zones (berms) before reaching the outlet and eventually the stream  before it reaches the outlet . The\u00a0relevant studies was selected\u00a0considering the following criteria:The inlet water had to originate from drainage systems transporting water from agricultural fields, and must not be mixed with water from other sources such as streams.Based on the K\u00f6ppen-Geiger climate classification system, the sites had to be located in oceanic  or continental  climates Fig.\u00a0, where t2.The study had to be a field study with sites exposed to ambient temperature and with a surface area larger than 10\u00a0mThe study had to include a mass balance for either nitrate\u2014N, total phosphorus (TP) or total suspended solids (TSS) for at least one drainage season, whose length depended on the climate region.To find relevant studies for our review, a search of published studies was conducted via ISI Web of Science for 1900\u20132019 employing four different search strings, which are described in the Supplementary Material (Table S2). Absolute removal was reported in various units , and we therefore identified and used the most commonly reported unit, which meant that\u00a0recalculation of removal efficiencies were\u00a0necessary in some studies.If two studies were conducted at the same study site within overlapping monitoring periods, the study with the longest time series was selected. Not all extracted data could be separated into years or seasons, implying that standard deviation (\u03c3) for nitrate removal was not available for nine sites and for TP removal for one site; still, these sites were included in the calculation of the arithmetic mean  and by the Shapiro\u2013Wilk test, and where the assumptions were not fulfilled this is mentioned in the result section. Meta-analysis was only conducted for a mitigation measure if sufficient data were available, i.e. data from more than two sites originating from different studies. The meta-analysis was performed in R software 3.6.1  and the between-study variance (T2). The DerSimonian and Laird (DL) method was applied to estimate T2, and the Hartung-Knapp method was used to adjust the confidence intervals (CI), producing more conservative results, as recommended by Borenstein (K\u2009<\u200920). To evaluate whether the use of the overall summary effect was appropriate, the degree of consistency of the effect sizes was assessed using forest plot, funnel plot and multiple statistical measures. The observed variation (Q) was tested to investigate if the true effect varied between studies and if application of the random effect model was appropriate (Borenstein I2) gave an indication of what proportion of the variation was real, and reflected the extent of overlapping CIs. However, care must be taken, as in the case of an I2 close to zero, it can either be ascribed to that all variance is due to sampling error within the studies, though it can also be \u00a0caused by very imprecise studies with substantial difference between effect sizes. Thus, large I2 values can either indicate the possible existence of different subgroups or that the analysis contain highly precise studies with very small differences between the effect sizes. The estimate of the absolute variance, T2, was used as an indication of dispersion, and it was compared with \u03c32. The standard deviation of the effect size (T) was also reported. In the funnel plot, the removal efficiencies were plotted against the SE, thus asymmetry or other shapes in the funnel plot might indicate bias related to publication bias, heterogeneity or sampling error. Funnel plots were only inspected if the analysis contained more than ten studies, as recommend by Borenstein  and large sample size, as these were regarded as more precise. The summary effect was calculated based on the effect sizes and their weight, using a\u00a0random effect\u00a0model, which allow the true mean to vary between studies, as the selected studies differed in design, materials and methods. To account for this variability, the weighting factor assigned to each effect size incorporated both the within-study variance . According to our risk of bias assessment, the risk of bias was low in 35% of the studies. Insufficient monitoring of the water balance was the main reason that many studies were categorised as having \u2018moderate to high\u2019 risk of bias (Table\u00a02 per regulation well). The hydraulic loading rate (HLR) to the systems differed substantially, as expected, being highest for DBR and lowest for CD due to the difference in size. Treatment of drainage water is a relatively new concept, as illustrated by that the oldest facilities were two 20-year-old FWS and the second oldest a 10-year-old DBR. The youngest and least studied measure was IBZ.The initial search yielded 8126 studies in total, and after evaluating the inclusion criteria, we had a master bibliography of 42 articles containing 84 sites distributed across eleven countries (Table\u00a0I2\u2009=\u200996%), and T2 (260%) was higher than \u03c32 (70%). The subset analysis of data with either low risk of bias or sampling periods longer than two years/drainage seasons showed the average removal ranged between 40 and 44%, and CI and PI were slightly more narrow than for the full dataset  (Table\u00a0\u22122 year\u22121 (CI: 29 to 91\u00a0g\u00a0N m\u22122 year\u22121).The weighted average obtained by meta-analysis showed that FWS significantly reduced nitrate loading by 41% within a range from \u2212\u00a08 to 63% Fig.\u00a0. However2 (226%) was much lower than \u03c32 (838%). The subset data analysis for TP removal reported a slightly higher removal (35%) than the initial data; however, \u03c32 was still very high as the included studies reported both removal and release of TP (Table\u00a0\u22122 year\u22121 (\u2212\u00a01.16 to 2.52\u00a0g P m\u22122 year\u22121)  Fig.\u00a0. As expeI2 was high (99%), as some of the studies were very precise, but showed different removal efficiency. Average T2 (436%) was much higher than \u03c32 (169%). The subset analysis of data with either low risk of bias or sampling periods longer than two years/drainage seasons reported lower removal efficiency (35%), and CI and PI were slightly narrower for studies with N\u2009>\u20092 (Table\u00a0T2 and higher \u03c32. The arithmetic mean efficiency was 44% (CI: 35 to 53%), while the absolute nitrate removal per DBR volume amounted, on average, to 715\u00a0g\u00a0N m\u22123 year\u22121 (CI: 292 to 760\u00a0g\u00a0N m\u22123 year\u22121), ranging from 66 to 2033\u00a0g\u00a0N m\u22123 year\u22121 (Table\u00a0\u22122 year\u22121 (CI: 333 to 855\u00a0g\u00a0N m\u22122 year\u22121).The weighted average calculated by meta-analysis showed a significant reduction of the annual nitrate loading by DBR of 40% within a range from 6 to 79%   and the other net removal (28% or 6\u00a0g P m\u22122 year\u22121)  Table\u00a0.I2\u2009=\u200979%), yet T2 was only slightly higher than \u03c32. The removal efficiency of studies including sampling periods longer than two years/drainage seasons was more or less similar to the result of the full data analysis (Table\u00a0\u22122 year\u22121 (1.16 to 1.24\u00a0g\u00a0N m\u22122 year\u22121), corresponding to 12\u00a0kg\u00a0N ha\u22121 year\u22121 (CI: 8 to 16\u00a0kg\u00a0N ha\u22121 year\u22121). The relative nitrate reduction correlated well with the relative reduction of drainage flow (R\u2009=\u20090.80 (Pearson), p < 0.0001, K\u2009=\u200919), and exclusion of studies with sub-irrigation, a practice implying an additional water supply, improved this correlation  , while T2 (406%) and \u03c32 (403%) were more or less identical, suggesting that the studies were similar enough to justify combination. The arithmetic mean was 29% (CI 10 to 48%) (Table\u00a0\u22122 year\u22121 (0.01 to 0.05\u00a0g P m\u22122 year\u22121) or 0.30\u00a0kg P ha\u22121 year\u22121 (0.10 to 0.49\u00a0kg P ha\u22121 year\u22121) (Table\u00a0R\u2009=\u20090.87 (Pearson), p < 0.01, K = 6)  according to the meta-analysis Fig.\u00a0. The rem\u22122 year\u22121 (CI: 9 to 37\u00a0g\u00a0N m\u22122 year\u22121). There were no available data on TP balances for SBZ in the articles selected for this review. For IBZ, the annual nitrate removal efficiency, calculated as the arithmetic mean, was 26% (CI: 20 to 32%) (Table\u00a0\u22122 year\u22121 (71 to 209\u00a0g\u00a0N m\u22122 year\u22121). The removal efficiency of TP was 48% (CI: 40 to 56%), while the absolute TP removal per IBZ area was 2.4\u00a0g P m\u22122 year\u22121 (CI: 1.4 to 3.5\u00a0g P m\u22122 year\u22121).Removal efficiencies could not be aggregated using meta-analysis for the emergent technologies, SBZ and IBZ, as, until now, only one study containing multiple sites has been published for each practice Table\u00a0. The ann%) Table\u00a0. The absS1). The loading rate of nutrients are highly site specific, as it is determined by the concentration of nutrients in the water and by HLR, which is highly variable from site to site. For example, for DBR, the specific loading rate of nitrate per DBR area differed substantially between sites . Furthermore, the HLR varies from year to year, although this variation can be accounted for to some extent by monitoring over multiple years. In this review, it was demonstrated by that study sites monitored for multiple years (N\u2009>\u20092) had higher \u03c32, and thus incorporated more variation. Absolute removal was reported relative to mitigation measures surface area in our review, however, another possibility would be to report absolute removal per catchment area, however, the estimate of catchment areas are often very uncertain, adding more uncertainty to the removal estimate. The HLR also influence relative removal, where the removal efficiency tends to increase with decreasing HLR , though temperature is at least as important. The design of mitigation measures is commonly guided by DMMCAR, as a rough estimate of HLR and temperature; for instance, in New Zealand a guideline predicts that a DMMCAR of 5% will yield an approximate nitrate reduction of 50\u2009\u00b1\u200915% .Removal efficiency was quantified in both absolute and relative values in our review. However, care should be taken when comparing values\u00a0from different sites, as the absolute removal efficiency depended heavily on the nutrient loading to the system . This implies that the estimates depend especially on the frequency of nutrient sampling and the water flow monitoring strategy. Estimates of TP retention might be more uncertain than those of nitrate\u00a0as TP concentrations in tile drainage water tend to\u00a0change quickly over time, especially at high flow, which can be difficult to capture , which was not surprising, as their review included a broad range of created and restored wetlands treating both agricultural runoff, riverine water, secondary and tertiary domestic wastewater and urban stormwater. Our review of FWS showed that they did not always remove TP, as four out of 15 FWS sites acted as a source of P. This net release of P might be due to mobilisation of dissolved reactive P (DRP) from the sediment or the size of the FWS being too small to adequately decelerate the flow , age monitoring schemes and run off characteristics, factors that all affected the removal efficiency. At one site, nitrate release was reported, which was most likely due to the lack of monitoring of one of the inlets  between the studied sites, not least regarding nitrate loading rates. Many of the study sites were experimental facilities or pilot studies, implying that they were\u00a0established to investigate and identify factors influencing performance. For example, among the studies included in our review, the low removal efficiency could be ascribed to short-circuiting within the system  also prevail.Besides suitability to the landscape, implementation strategies are often guided by cost efficiency. Cost efficiency, including capital and operational cost of the drainage systems, has been calculated earlier Supplementary material 2 (XLSX 93 kb)Below is the link to the electronic supplementary material."}
+{"text": "In cultures amended with 4 \u00b5M vitamin B12 and 13C labelled CF, formation of 13CO2 was detected. Known organohalide-respiring bacteria and reductive dehalogenase genes were neither detected using quantitative PCR nor metagenomic analysis of the enrichment cultures. Rather, members of the order Clostridiales, known to co-metabolically transform CF to DCM and CO2, were detected. Accordingly, metagenome-assembled genomes of Clostridiales encoded enzymatic repertoires for the Wood-Ljungdahl pathway and cobalamin biosynthesis, which are known to be involved in fortuitous and nonspecific CF transformation. This study indicates that hypersaline lake microbiomes may act as a filter to reduce CF emission to the atmosphere.Chloroform (CF) is an environmental contaminant that can be naturally formed in various environments ranging from forest soils to salt lakes. Here we investigated CF removal potential in sediments obtained from hypersaline lakes in Western Australia. Reductive dechlorination of CF to dichloromethane (DCM) was observed in enrichment cultures derived from sediments of Lake Strawbridge, which has been reported as a natural source of CF. No CF removal was observed in abiotic control cultures without artificial electron donors, indicating biotic CF dechlorination in the enrichment cultures. Increasing vitamin B Until the 1970s, halogenated organic compounds, organohalogens, were believed to originate exclusively from anthropogenic sources . A remarAcetobacterium woodii  and archaea [(8.6 \u00b1 0.25) \u00d7 107 copies/g dry sediment] among all the sediment samples from the two lakes  in top and bottom layer sediments of Lake Strawbridge  increased from 5%\u201316%  and 3%\u20137%  in the bottom layer sediments to ~67% and ~18%, respectively, in the initial sediment microcosm and subsequent transfer enrichment cultures : 86.91%).A total of 50\u201364 million reads were obtained from sequencing of each enrichment culture with and without vitamin B12 . Six nea12  were rec12 A. More t12 . Taxonomp-value < 0.5, p-value = 0.452) and MAG1, MAG3, and MAG4 to novel families . The remaining MAGs likely belong to novel genera based on MiGA classification (rdh) were neither detected in the MAGs nor in the unbinned contigs (acs gene cluster encoding carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex, the signature enzyme that connects the carbonyl- and methyl- branch of the WLP  citrate or DTT, acterium ,36 and Dlobacter  were nei contigs B, or theronments , and theronments . Our finClostridiales were abundantly present in the sediment microcosms and enrichment cultures /cob(II)alamins, which can catalyze reductive dechlorination of CF [2, which we observed in our culture when 4 \u00b5M of vitamin B12 was added, is not clear. Increasing vitamin B12 from 0.4 to 4 \u00b5M shifted the dominant CF transformation pathway from reductive dechlorination (to DCM) to CF oxidation to CO2 (12-dependent WLP enzyme(s) in CF hydrolysis to CO [2 by CO dehydrogenase  pathways. Accordingly, we identified all genes for cobalamin biosynthesis and transport in the Clostridiales MAGs  CF transformation and contribute to local halogen cycling and reducing VOX emissions to the atmosphere. Hypersaline lakes are among the major sources for VOX production and emission on Earth . This st"}
+{"text": "Home self-monitoring of blood pressure is widely used in primary care to assist in the diagnosis of hypertension, as well as to improve clinical outcomes and support adherence to medication. The National Institute for Health and Care Excellence (NICE) care pathways for hypertension recommend specific guidelines, although they lack detail on supporting patients to self-monitor.To elicit primary care practitioners\u2019 experiences of managing patients\u2019 home blood pressure self-monitoring, across surgeries located in different socioeconomic areas.A qualitative focus group study was conducted with a total of 21 primary care professionals.Participants were GPs and practice nurses (PNs), purposively recruited from surgeries in areas of low and high deprivation, according to the English indices of multiple deprivation. Six vignettes were developed featuring data from interviews with people who self-monitor and these were used in five focus groups. Results were thematically analysed.Themes derived in the thematic analysis largely reflected topics covered by the vignettes. These included: advice on purchase of a device; supporting home monitoring; mitigating patient anxiety experienced as a result of home monitoring; valuing patients\u2019 data; and effect of socioeconomic factors.The work provides an account of methods used by primary care practitioners in the management of home blood pressure self-monitoring, where guidance may be lacking and primary care practitioners act on their own judgement. Findings complement recent policy documentation, which recognises the need to adopt new ways of working to empower patients , but lacks detail on how this should be done. Policy documents, including the NHS Long Term plan, advocate for the increased use of technology in healthcare pathways across the NHS. Blood pressure self-monitoring has been conducted by patients in their own homes for many years, and thus provides a case from which lessons may be learnt on a variety of aspects of management of patient-owned technologies mobilised in primary care. The study provides an account of methods used by primary care practitioners in the management of home blood pressure self-monitoring, where guidance may be lacking and primary care practitioners act on their own judgement.1, and is associated with improved clinical outcomes in hypertension, when accompanied by appropriate interventions.4 Since 2011,5 NICE has advocated home blood pressure monitoring as one method of assisting the diagnosis of hypertension, and continues to issue guidance on the use of this method in the updated (2019) guidelines.6 Now, home self-monitoring of blood pressure is widely used in primary care to assist in the diagnosis of hypertension, although adherence to NICE guidelines is known to vary.7 Furthermore, the guidelines do not provide detail on supporting patients to self-monitor, for example, no detail is given regarding advice on purchase or use of a monitor.6The self-monitoring of blood pressure at home was introduced in the 1930s8 There is a need to explore the management of self-monitoring in the everyday work of primary care, where practitioners act on their own discretion and where practice is likely to vary. Smolen and colleagues have developed a conceptual model of hypertension management in general practice, identifying three main strategies used by providers to support patients: education (about the condition and its consequences); relationship building (relating to continuity of care); and use of self-management tools (including home monitors).9 Their study and other prior work focuses largely on managing care once a patient has obtained a monitor, excluding guidance provided prior to this point.9 Some work has additionally explored the potential of new technologies  for measuring blood pressure in primary care,10 although not considering this within the context of current everyday practice. In terms of guidance, patients are sometimes unconfident in the use of home monitors, and may seek information online.4 Healthcare assistants may be involved in providing support and guidance to patients who self-monitor, although research into their role is limited.11Qualitative work exploring blood pressure self-monitoring has, to date, mostly been conducted within the confines of randomised controlled trials, where self-monitoring has been directed as part of a research protocol.Tracking Ourselves? Project12 has explored everyday experiences of self-monitoring, through interviews with people who had acquired a device either on recommendation of a clinician or out of their own interest. Finding consistent references to primary care in many of these interviews, the study sought to further explore the relationship between primary care professionals and patients who self-monitor blood pressure by conducting focus groups with GPs and PNs. Discussions focused on routine daily practice and professional experience.Funded by a 3-year Leverhulme Trust research grant, the The study aimed to elicit primary care practitioners\u2019 views and experiences of managing patients\u2019 home blood pressure self-monitoring.Primary care health professionals were recruited with assistance from the National Institute for Health Research\u2019s (NIHR) Clinical Research Network (CRN) in Yorkshire and Humber, UK. GP practices and participants were contacted via two CRN clusters (groups of research-active GP practices). Six practices and one research group were approached, although two practices were unable to participate. The authors purposively recruited from surgeries in both higher and lower socioeconomic areas, to explore differences in views and practice across primary care practitioners working in these areas. Healthcare professionals were eligible to take part if they had experience working as a GP or PN in general practice. Written informed consent was obtained from all participants.13In total, five focus groups were conducted, with a total of 21 health professionals, from two practices in higher socioeconomic areas, two practices in lower socioeconomic areas, and one group formed of practitioners from a cluster based in lower socioeconomic areas. The English Indices of Deprivation 2015 was used to determine socioeconomic status.14 Vignettes have been used in surveys,17 interviews18 and focus groups,19 and their content is usually hypothetical.Vignettes were developed to structure the group discussions. Vignette methodology involves the use of short sections of text describing a person or situation to prompt a discussion between the researcher(s) and the participant(s).The research discussed in this article is part of a larger project exploring how and why people self-monitor, involving interviews with 84 people who monitor their own blood pressure and/or body mass index (BMI). The authors drew on analysis from these interviews to design vignettes that would stimulate discussion among healthcare professionals, selecting excerpts that provided good illustrations of common experience. The vignettes focused on multiple points of care: diagnosis; obtaining a monitor; use and storage of blood pressure data in clinic; self-management of medication; and patient anxiety. Vignettes were refined through piloting with local clinical and academic staff. In the focus groups, participants considered the scenarios presented in the vignettes and reflected on their own experiences.Of the five focus groups, four took place in rooms at the GP surgeries where participants were employed, and one was held in a university building at the research meeting of one of the clusters. Each focus group involved discussion of the same six vignettes.After taking informed consent, the researchers read out each vignette before asking an opening question to prompt discussion. Each 1-hour focus group then followed a semi-structured approach, using a question guide prepared in advance (see supplementary files). Two researchers were present at each focus group, except for one focus group (FG5) where only one researcher was able to attend. Both researchers took notes when present. Sessions were audio-recorded.19 JA led the analysis, while KW and CW analysed a subsample of the data (one focus group) and discussed the development of initial themes with JA. Final themes were derived through an iterative analysis of all focus group transcripts, in line with Braun and Clarke\u2019s methodology.19 In the quotes below, participants are labelled by their focus group number (\u2018FG#\u2019), then by their role in general practice (GP or PN), then by their participant number within each focus group, for example, \u2018FG3GP2\u2019. The letter in brackets dictates the participant\u2019s sex.Audio-recordings were transcribed verbatim and analysed using thematic analysis.Five focus groups were conducted, with a total of 21 general practice clinicians. These consisted of 14 GPs and seven PNs. Focus groups ranged in size from two to eight participants. Of the 21 participants, 14 were female (66%). Years of experience in general practice ranged from 1\u201324 years, with a median of 8 years. Participant demographics are presented in Supplementary Table S1 and Supplementary Table S2.Themes mostly reflected the focus of the vignettes that were developed; however, some discussions on particular vignettes cut across themes, and vice versa. For example, quotes in the theme on valuing patients\u2019 self-monitoring data occurred in reference to both Frank and Emily\u2019s vignettes. The final themes were: \u2018advice on purchase of a device\u2019; \u2018supporting home monitoring\u2019; \u2018mitigating patient anxiety experienced as a result of home monitoring\u2019; \u2018valuing patients\u2019 self-monitoring data\u2019; and \u2018effect of socioeconomic factors\u2019. Each of these is presented below with excerpts from the focus groups to demonstrate the main findings.'I think it's morally questionable' [FG1GP6{F}]), and discussed how they would advise patients on what to buy and where to source it from.One vignette described how an interviewee had been given advice by her GP on which blood pressure monitor she should buy on Amazon. Many participants were shocked that this had occurred For others, it was considered unethical to make any recommendations at all:'We\u2019re not meant to recommend a pharmacist to have their medications sent to, are we? So you sort of do feel a bit that you can\u2019t over-recommend things.' (FG3GP2[F])Several participants suggested they would recommend their patients to consult the British Hypertension Society\u2019s list of recommended devices:'I think you can get British Hypertension Society, you know, approved blood pressure monitors, so [I would recommend] one of them.' (FG1PN2[F])The theme on supporting home monitoring included two subthemes. First, this concerned methods that practitioners had used to educate patients on the use of a home blood pressure monitor. These included sending links to NHS Choices (now referred to as the NHS website) via SMS, and referring patients to YouTube, as well as setting up appointments with healthcare assistants for face-to-face instruction on how to use a monitor:'There\u2019s lots of really good stuff on YouTube from very reputable sources [...] they can go back and look at it again, rather than me explaining it and you think, \u201coh, I've done a great job there\u201d, and off they go and they're like, \u201cwhat do I do with this?\u201d' (FG1GP5[M])'I use SMS. So, we\u2019re on SystmOne and it\u2019s set up so you can send people SMS and I quite often send them a link to NHS Choices.' (FG5GP2[F])Second, participants discussed how patients had brought their blood pressure monitor to the surgery in order to test its accuracy against the surgery monitor. They related their experience to Frank\u2019s vignette, which described how Frank had been advised by his GP to measure his blood pressure at home, and it was no longer being measured in the clinic:'I think one thing I would want to know is that the machine he was using had been validated, so the first time I would get him to come and see the healthcare assistant, say, \u201cbring your machine in, we\u2019ll check it on yours, check it on ours and make sure it's the same and then off you go and maybe we would do that every 12 months.\u201d' (FG1GP3[M])However, there were mixed views on the value of patients bringing their monitor for calibration, with some GPs finding it unnecessary given the accuracy of blood pressure monitors overall. Some GPs were unaware that this was being conducted by colleagues in other roles:'I think I sigh when somebody brings a blood pressure monitor in to me, inwardly sigh and think, \u201coh really?\u201d [...] You have to take a ball park I think really with it, [...] if I did ten blood pressures with my monitor consecutively, they\u2019re all going to be different.' (FG4GP1[M])'I don\u2019t think we\u2019ve ever done that, have we? [...]' (FG5GP2[F])\u2018Some do.' (FG5PN1[F])\u2018Do they?' (FG5GP1[M])Several participants saw it as the healthcare assistant\u2019s role to support home monitoring and take blood pressures in the surgery where required, allowing GPs to concentrate on the analysis of data and decision making around care:'A lot of these problems that have been identified are the sort of things that with a well-trained healthcare assistant they could sort that out.' (FG1PN1[F])One vignette focused on the case of a patient (Gary) who had been advised to monitor his blood pressure less often because he had health anxiety. The participants were unanimous in backing the decision of his GP, and recognised patients like this in their own practice:'Gary may need some other type of help to manage his health anxiety, but monitoring his blood pressure all the time, in my opinion, is not going to do him any favours.' (FG4GP1[M])One way of mitigating the anxiety caused by self-monitoring was to share guidelines on how often to monitor with the patient, as a way to reassure them that it was safe not to monitor on a frequent basis:'There\u2019s fairly clear guidelines on how frequently people should have blood pressure checks, which I might, kind of, share with him as a way of reassuring him.' (FG4GP2[M])Other practitioners used the offer of ambulatory monitoring as a way to take pressure off patients and reduce their level of anxiety:'It\u2019s on for 24 hours and then you can forget about it, he\u2019s not got the added anxiety of, \u201coh, I\u2019ve got to take my blood pressure\u201d and, \u201coh, what\u2019s it going to be and am I doing it right?\u201d' (FG2PN1[F])Participants also advocated emphasising the importance of a longer-term approach to blood pressure with more anxious patients to mitigate concern:'It\u2019s important and we need to do it properly, but we need to do it as a routine matter, this is not something that you need to get worried or stressed about and it\u2019s something we need to sort out over months and years.' (FG2GP1[M])Another vignette presented the case of Emily, who had remarked that her GP had not recorded the blood pressure readings she took with her to the surgery. Participants discussed the importance of making obvious to the patient why they are collecting data, recognising that patients may be unclear about how their data is used:'She\u2019s [Emily] [...] not sure whether or not the doctor is taking it seriously or really cares. I hope my patients don\u2019t think that, I think I tell them the conclusion I\u2019ve drawn.' (FG2GP1[M])Some reflected that this may be a blind spot in the way they work:'I think this touches on a really important thing though and I think I'm really bad at this actually [...] I don't value the work and the time they have put into producing this information [...] it's not going to encourage them to carry on doing it.' (FG1PN2[F])Some followed particular ways of working in relation to blood pressure records that patients had brought, but were unsure whether the data were processed in line with those policies:'So, people just tend to bring in the sheets we give them, and I do give those to the receptionists to scan in. I think we scan them in. I\u2019ve never checked.' (FG5GP1[M])The authors purposively sampled from practices in areas of higher and lower deprivation according to the English Indices of Deprivation. In practices in more deprived areas, clinicians reported that they might recommend their patients to ask family and friends if they could borrow a blood pressure monitor, or lend them one from the surgery, rather than advising patients to buy their own:'Income is an issue. This is a fairly deprived practice. So, sometimes you end up having conversations about, has your mum got one, do you know someone who\u2019s got one, as well.' (FG5GP2[F])One of the vignettes described the case of Bob, a tiler who reported \u2018confessing\u2019 to his GP that he had taken less medication than instructed. Reacting to this vignette, participants in higher socioeconomic areas commented that they did not find their patients to be deferential or to express guilt about querying instructions from healthcare professionals, and expressed their assumption that they were more used to patients directing their own monitoring or medication:'It\u2019s a very, very middle class area, so they ask a lot more questions.' (FG3GP1[F])'It\u2019s very educated.' (FG3GP2[F])'A lot less deferential.' (FG3GP1[F])'So our patient demographic is [...] skewed towards the higher socioeconomic end of the spectrum, I guess, so we\u2019ve got loads of professionals and, you know, medics and lawyers and teachers [...] there\u2019s lots of educated people [...] I encounter quite a few people who want to be quite autonomous in how they do these things and I\u2019m quite happy for them to do that.' (FG4GP2[M])This study has explored primary care practitioners\u2019 experiences and practices in supporting blood pressure self-monitoring in the management of hypertension. The findings demonstrate how clinicians guide patients to make choices about purchasing healthcare technologies, and make clear the need for balance between maintaining professional impartiality and providing patients with guidance to ensure they purchase appropriate devices. Participants indicated pharmacies as reputable vendors of approved blood pressure monitors, and lists of approved devices as resources to which they refer their patients. On educating patients about their devices, several participants stated that they use email or SMS to refer patients to online materials, including videos on YouTube, when these originate from reputable sources. They also refer patients to healthcare assistants to learn how to use blood pressure monitors.The findings highlight a recognition among primary care professionals of the anxiety patients may experience as a result of home monitoring, and a variety of approaches to mitigate it. These included use of ambulatory monitors to reduce outputs visible to patients that may cause them concern, or providing patients with guidance on monitoring frequency. Participants suggested they were aware of the need to be open with patients about how their self-generated data would be used. This openness was recognised as a way to encourage continued monitoring, and to communicate to patients that primary care professionals value the data that patients provide.Cost was highlighted as a barrier to some patients purchasing monitors and suggestions were given for overcoming this barrier, for example, patients might borrow a monitor from friends or family, or use one owned by the surgery. The results demonstrate too the possibility of variations in patients\u2019 attitudes to discussing monitoring and blood pressure medication with their primary care practitioner.18 The use of vignettes more generally provided a useful framework within which to promote discussion, as indicated by Keane and colleagues.14The use of vignettes developed from interviews with people who self-monitor provided authenticity to the study materials. In contrast to traditional vignette methodology, this approach may reduce research participants\u2019 sense that the research is contrived.The study is limited in that practitioners were all recruited from the same geographical region in one area of the UK. However, by conducting focus groups with a total of 21 primary care practitioners across both GP and PN roles, a wide variety of practices and experiences of managing patient use of blood pressure monitors at home have been elicited. Inclusion of healthcare assistants and practice managers in the focus groups may have shed more light on how self-monitoring fits within the wider structure of primary care. Use of vignettes may have led participant conversations, although it is considered this was necessary in order to stimulate discussion of the topics examined in the research.The thematic analysis was limited in that it was principally completed by one researcher (JA); however, the analysis of one of the transcripts by three researchers, and the subsequent discussion of themes arising, ensured that themes were robust.9 The findings provide detail in the ways that these provider actions are currently undertaken, highlighting variation in practice occurring among surgeries. For example, the findings indicate that healthcare assistants are increasingly providing support for home monitoring, which GPs and PNs may not have time to provide. Healthcare assistants may be more likely to understand local patient culture and provide a sense of continuity of care,11 which contributes to aspects of relationship building. Their role in supporting home monitoring could, therefore, bring benefits to patient care, beyond time-saving for GPs.Smolen and colleagues\u2019 conceptual model of providers\u2019 approach to hypertension management details three particular provider actions for successful blood pressure management: relationship building; self-management tools; and patient education.8 the results show that there are important considerations to be made around whether and how patients obtain a monitor in the first place. Primary care professionals were found to advise patients differently on where to purchase a blood pressure monitor, including from pharmacies and online, and patients on low incomes were given other suggestions about how they might obtain a monitor. Thus, while Smolen and colleagues highlight the importance of self-management tools,9 the participants in the present study gave examples of how they guide patients to obtain these tools, taking account of differences across socioeconomic backgrounds.While prior research in this area has primarily focused on care occurring after a patient has obtained a home blood pressure monitor,4 the results demonstrate how primary care professionals advise patients to seek out online materials from sources, including YouTube, professional associations and NHS Choices (now referred to as the NHS website), and may send them direct links to online information. Thus, the study demonstrates the kinds of resources primary care professionals draw on and the way that these are disseminated to patients in the third part of Smolen et al\u2019s conceptual model, patient education.While patients are known to turn to the internet to seek guidance for home monitoring of their own volition,'not a good idea for everyone' and that 'some people feel more anxious when taking their own blood pressure',20 although some research suggests that anxiety does not increase in those self-monitoring blood pressure.21 Here, the authors add that primary care professionals share guidelines on the frequency of monitoring and encourage patients to take a longer term view of blood pressure in order to mitigate patient concerns and worry.The findings also highlight how primary care professionals experience patient anxiety around self-monitoring, including through the use of ambulatory monitors. Anxiety around self-monitoring is recognised in guidance for patients with hypertension from the British Heart Foundation, which suggests that self-monitoring is 22 The findings show too that clinicians are finding workarounds to enable patients to obtain blood pressure monitors in more deprived areas. The NHS Long Term Plan promises greater funding for poorer areas,23 which could provide self-monitoring technologies to those who cannot afford them, without the need for such workarounds.This article has explored the experience of blood pressure self-monitoring in primary care. It has been demonstrated how pharmacists are called on to supply and support the use of blood pressure monitors by patients with hypertension, supporting suggestions in the Topol Review that the role of the pharmacist will need to evolve to include the supply and support of multiple healthcare technologies as the use of these increases.6 currently advocates that practitioners provide guidance and education to their patients on blood pressure monitoring, but lacks detail on how this should be conducted. The five aspects of managing self-monitoring that have been highlighted may also be useful when considering how primary care practitioners can best support self-monitoring by patients with conditions other than hypertension. The importance of the role of the healthcare assistant in helping patients to manage a self-monitoring practice is emphasised. It is also highlighted that there is a need to ensure patients are clear about how their data is used to encourage continued monitoring.The results provide a view of how primary care practitioners currently manage the use of home blood pressure monitoring, and provide examples of practice that may be informative for those working in this area. In particular, a need to consider five aspects of patient self-monitoring of blood pressure  is highlighted. These findings are of particular importance since NICE guidanceFurther research is required to understand views of pharmacists and healthcare assistants on spending more of their time supporting patients with self-monitoring technologies, particularly as the number of self-monitoring technologies implemented in primary care for different conditions increases. Further work could also examine the potential of a standardised NHS care pathway for self-monitoring, which could include detail on how devices should be obtained, how their accuracy can be checked, target readings, when readings should be taken, and which healthcare professional should be consulted once readings are collated."}
+{"text": "This work examines the nature of self-employment arrangements of older adults in the United States. Many people engage in self-employment - in the 2016 Health and Retirement Study (HRS), 20 percent of respondents working for pay reported being self-employed - yet there exists a dearth of data on these arrangements. This lack of data prevents consideration of important questions relevant to employment, inequality, and policy. Who works in different types of self-employment? What resources facilitate some individuals obtaining higher quality self-employment arrangements? To what extent does the income from different types of arrangements keep people out of poverty? Are different types of arrangements associated with individuals being happier and having more job satisfaction? This work leverages novel restricted-access data collected in the HRS in 2016 on the employer names and locations for individuals reporting self-employment along with respondent narratives on industry and type of work to classify self-employment reports into three entrepreneurial roles  across 14 different types of work. Using the breadth of information collected in the HRS and linkage to administrative records, this work then presents differences in characteristics, such as demographics, income, wealth, savings, health insurance coverage, home ownership, health status, and expectations of working longer, associated with different classifications of self-employment. Exploring these questions provides unique insights into the changing nature of work and the transition to retirement relevant to policy considerations across the health, insurance, and retirement income dimensions, among others."}
+{"text": "Adipocytes can directly interact with and activate AT-resident invariant natural killer T (iNKT) cells through CD1d-dependent presentation of lipid antigens, which is associated with anti-inflammatory cytokine production in lean AT . Whether alterations in the microenvironment, i.e., increased free fatty acids concentrations or altered cytokine/adipokine profiles as observed in obesity, directly affect adipocyte-iNKT cell communication and subsequent cytokine output is currently unknown. Here we show that the cytokine output of adipocyte-iNKT cell interplay is skewed by a lipid-rich microenvironment. Incubation of mature 3T3-L1 adipocytes with a mixture of saturated and unsaturated fatty acids specifically reduced insulin sensitivity and increased lipolysis. Reduced activation of the CD1d-invariant T-Cell Receptor (TCR) signaling axis was observed in Jurkat reporter cells expressing the invariant NKT TCR, while co-culture assays with a iNKT hybridoma cell line (DN32.D3) skewed the cytokine output toward reduced IL-4 secretion and increased IFN\u03b3 secretion. Importantly, co-culture assays of mature 3T3-L1 adipocytes with primary iNKT cells isolated from visceral AT showed a similar shift in cytokine output. Collectively, these data indicate that iNKT cells display considerable plasticity with respect to their cytokine output, which can be skewed toward a more pro-inflammatory profile  Insulin resistance is one of the hallmarks of type II diabetes mellitus T2DM) pathogenesis, with both overlapping and unique molecular mechanisms affecting different metabolic organs, including muscle, liver and adipose tissue (AT) , 2. AT hDM pathogOne of the AT-resident immune cell types that decrease dramatically with obesity both in mouse models and in humans are the invariant natural killer (iNKT) cells . iNTo study adipocyte-iNKT cell communication directly, i.e., without interference of other cell types, we and others have developed and characterized various co-culture assays, combining mouse or human (pre)adipocyte cell lines or primary adipocytes, with either reporter cells expressing the iNKT TCR , iNKT hyHere we investigated the effects of FFA, as well as other obesity-associated components of the AT microenvironment, on the direct communication between adipocyte, and iNKT cells. First, we developed and characterized a cell culture model using mature murine 3T3-L1 adipocytes cell line treated with a commercially available and chemically-defined lipid mixture to mimic a high lipid microenvironment. Impaired insulin signaling and increased lipolysis was observed, without overall disruption of adipocyte-specific gene expression. Interestingly, while iNKT hybridoma cells, and primary iNKT cells produced both IL-4 and IFN\u03b3 under basal conditions, their cytokine output was skewed when adipocytes were pre-treated with the lipid mixture toward a low IL-4, high IFN\u03b3 profile. Taken together, these data indicate that iNKT cells display considerable plasticity with respect to their cytokine output, which can be skewed by microenvironmental factors like FFA.Dexamethasone (Sigma), 3-isobutyl-1-methylxanthine (IBMX)(Sigma), insulin , Lipid mix 1 (sigma L0288), albumin conjugated linoleic (sigma L9530), and oleic acid (sigma O3008), sodium palmitate (sigma P9767), myristic acid (sigma M3128), stearic acid (sigma S4751), cholesterol , \u03b1Galactosylceramide KRN7000 , IFN\u03b3 (sigma SRP3058), TNF\u03b1 (sigma H8916), Pam3Cys , LPS (sigma L4516), rabbit-anti-AKT  , rabbit-REP\u2212iNKT\u2212\u03b22M_KO reporter cell lines (The murine 3T3-L1 cell line (ZenBio) was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine serum (Invitrogen), penicillin and streptomycin . For differentiation of 3T3-L1 cells to adipocytes, the cells were grown to confluence and after 2 days (day 0) stimulated with culture medium containing dexamethasone (250 nM), 3-isobutyl-1-methylxanthine (500 \u03bcM), and insulin (170 nM) for 2 days. On day 2, the medium was changed for culture medium containing insulin (170 nM) and maintained for 4\u20136 days. The murine iNKT cell hybridoma line DN32.D3 was cultured in RPMI-1640 medium (Sigma Aldrich) supplemented with 10% fetal bovine serum, penicillin and streptomycin (both 100 \u03bcg/ml), MEM Non-Essential Amino Acids Solution , HEPES , Glutamine  and \u03b2MeOH . The JE6-1ll lines  were culStimulation of 3T3-L1 adipocytes was done post differentiation for 3 days with lipid mix and individual fatty acids, and for 24 h with the residual obesogenic stimuli. One Millimolar of Palmitic, stearic and myristic acid was conjugated to fatty acid free BSA in a 6:1 ratio. Lipids were dissolved in 150 mM NaCl at 70\u00b0C while stirring. Before added to BSA 150 mM NaCl solution at 37\u00b0C while stirring. After adjusting pH to 7, 4 aliquots where stored at \u221220\u00b0C before further use (protocol adapted from Seahorse Bioscience).3T3-L1 cells were grown in a six well format and differentiated accordingly. After stimulation cells were washed with ice cold PBS, scraped and lysed in 300 \u03bcl ice cold RIPA buffer  and NaF acting as phosphorylase inhibitor) for 15\u2032 at 4\u00b0C. After centrifugation on max speed at 4\u00b0C in table top centrifuge, supernatant was collected, supplemented with Laemmli Sample Buffer (LSB). All western blot samples were boiled for 5 min at 95\u00b0C before use. Samples were subjected to SDS-PAGE and transferred to PVDF membrane (Milipore). Blocking was done with 5% Skim Milk TBST. Primary antibody staining was performed overnight at 4\u00b0C . Secondary antibody staining was performed for 1 h at RT . After staining, membranes were washed for 1 h with TBST before being treated with ECL western blot substrate solution (Pierce). Protein expression was measured with LAS4000 ImageQuant.Secreted Glycerol was measured using the Sigma-aldrich Glycerol Assay Kit (#MAK117-1KT) with 50 \u03bcl of media.Intracellular Triglycerides were measured using the kit Stanbio\u2122 Triglycerides LiquiColor\u2122 (#SB2200-225). Cells were washed with PBS and lysed in 50 \u03bcl cold PBS via syringe pull technique.REP\u2212iNKT\u2212\u03b22M_KO reporter cells (50.000 cells/well) were added to the 3T3-L1 adipocytes and co-cultured for 24 h. Medium from DN32.D3 co-cultures (200 \u03bcl) was used to determine secreted IL-4 and IFN\u03b3 via the Invitrogen\u2122 eBioscience\u2122 Mouse IL-4 ELISA Ready-SET-Go!\u2122 Kit (#501128931) and the IFN\u03b3 ELISA kit (BD-bioscience).3T3-L1 wild type (Zenbio) were plated in a 96-well format and differentiated according to the protocol mentioned above. Mature 3T3-L1 Cells were then treated with lipid mix or individual fatty acids for 4 days, or 24 h with residual obesogenic stimulants. After stimulation, either DN32.D3 iNKT hybridoma cells (50.000 cells/well) or JE6-1REP\u2212iNKT\u2212\u03b22M_KO reporter cells (JE6-1er cells  were colRNA was extracted using TRIzol reagent (Invitrogen). cDNA was synthesized using the superscript first strand synthesis system (Invitrogen) according to manufacturer's protocol. Gene expression levels were determined by quantitative real time PCR with the MyIq cycler (Bio-Rad) using SYBR-green (Bio-Rad) and normalized to 36B4 expression. Primers for quantitative RT-PCR are described in https://www.ivd-utrecht.nl/en/). Spleens and epididymal white AT (eWAT) were dissected from male C57BL/6J mice aged between 11 and 14 weeks. Spleens were disintegrated through a 70 \u03bcM cell strainer into 50 ml cold PBS and centrifuged for 10 min at 400 g. After discarding the supernatant, the pellet was resuspended in 5 ml of 10x Red Blood Cell (RBC) Lysis Buffer  for 5 min at room temperature. Forty five milliliter of cold PBS was added and cells centrifuged for 10 min at 400 g. After discarding the supernatant, the pellet was resuspended in 10 ml of cold PBS and filtered through a 70 \u03bcM cell strainer. Cells were retained on ice.Tissue collection was performed on animals sacrificed for other purposes and therefore exempted form ethical approval by the local Animal Welfare Body Utrecht, a body of Utrecht University and the University Medical Center Utrecht . Per 1 g adipose, 1 ml of 10 mg/ml collagenase  was added and incubated at 37\u00b0C for 15\u201320 min, with vigorous shaking every 5 min until AT was digested completely. Digested AT was then washed through a 100-\u03bcm cell filter with 20 ml of cold digestion buffer. Following centrifugation (500 g for 10 min) supernatant was removed and the cell pellet was resuspended in cold PBS. Cells were retained on ice.Before processing blood vessels and lymph nodes were removed from the eWAT which was then minced in 10 ml cold digestion buffer . Purified cells were then used in co-culture as described above.The increased concentration of circulating FFA observed in obesity contributes to the development of insulin resistance in adipose tissue , 2. To mMcp1 was expressed in both undifferentiated and differentiated cells, and unaffected by the lipid mixture (HSL and PNPLA2 (encoding the ATGL protein) were more highly expressed in differentiated cells compared to undifferentiated 3T3-L1 cells, but no significant change in expression was observed upon lipid mixture treatment. The Plin gene, encoding Perilipin1, was the only gene tested here to show a significant decrease in expression. As also observed for insulin signaling , inflammation, and lipid storage by RT-qPCR . Express mixture . Further mixture , we analignaling  and lipoignaling , none ofignaling . To veriignaling .in vitro studies on adipocyte-iNKT cell communication.Taken together, these data indicate that treatment of mature 3T3-L1 adipocytes with a chemically-defined lipid mixture results in a robust insulin resistance phenotype with increased lipolysis, without causing an overall disruption of cellular functionality, as the cells were clearly functional in terms of lipid metabolism and no dramatic changes in various key genes were observed. These observed characteristics support this cellular model as suitable for subsequent experimental approaches, including REP\u2212iNKT\u2212\u03b22M_KO reporter cells , or that could potentially play in role in this based on findings in other lipid APC . In addition, we analyzed 3 genes implicated in the biosynthesis of potential endogenous lipid antigens in adipocytes (Ugcg) and other lipid APC's . In agreement with previous reports , Niemann Pick type C2 (Npc2), \u03b1-galactosidase (Gla), were also not altered dramatically  in IL-4 (p = 0.0086) in IFN\u03b3 secretion  lipid antigens (In the present study we compared several co-culture systems to address the delicate cross-talk between adipose and iNKT cells in a lipid-rich environment. Under our experimental conditions, we obtained similar results from both DN32.D3 hybridoma cells and primary iNKT cells form eWAT, suggesting that DN32.D3 hybridoma cells represent an appropriate model for iNKT cells in the context of eWAT . In our er cells  reflecteread-out ; our datantigens .In contrast to iNKT cells from eWAT, we have also shown in the present study that splenic iNKT cells do not exhibit an IL-4/IFN\u03b3 preference in co-culture assays upon lipid mixture treatment. Splenic iNKT cells have been previously reported to have an IL-4 and IFN\u03b3 cytokine secretion capacity , 43, we in vivo, and ultimately into whole body energy homeostasis will be the topic of future studies. Indeed, very recent in vivo studies by LaMarche et al. already underscored the microenvironmental impact on iNKT cell output (Immune cell plasticity refers to immune cells which have a flexible, context-dependent inflammatory phenotype. This flexibility allows for rapid response to stimulus without being permanently defined by it , 48, 50.l output .All datasets generated for this study are included in the article/Ethical review and approval was not required for the animal study because it was exempted by the local animal ethics committee.RE, IM, AB, AM, and EK designed the experiments. RE, IM, AB, and AM performed experiments and analyzed the data. RE and IM drafted the manuscript. RE, IM, and EK edited and revised the manuscript. All authors contributed to the article and approved the submitted version.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": "Alzheimer\u2019s disease (AD) is a progressive neurodegenerative disorder leading to the most common form of dementia in elderly people. Modifiable dietary and lifestyle factors could either accelerate or ameliorate the aging process and the risk of developing AD and other age-related morbidities. Emerging evidence also reports a potential link between oral and gut microbiota alterations and AD. Dietary polyphenols, in particular wine polyphenols, are a major diver of oral and gut microbiota composition and function. Consequently, wine polyphenols health effects, mediated as a function of the individual\u2019s oral and gut microbiome are considered one of the recent greatest challenges in the field of neurodegenerative diseases as a promising strategy to prevent or slow down AD progression. This review highlights current knowledge on the link of oral and intestinal microbiome and the interaction between wine polyphenols and microbiota in the context of AD. Furthermore, the extent to which mechanisms bacteria and polyphenols and its microbial metabolites exert their action on communication pathways between the brain and the microbiota, as well as the impact of the molecular mediators to these interactions on AD patients, are described. The healthy human brain contains eighty-six thousand million neurons and many more glial cells. Each neuron can contact thousands or even tens of thousands of others. They send messages between different parts of the brain, and from the brain to the muscles and organs of the body. Alzheimer\u2019s disease (AD) disrupts this communication among neurons, resulting in loss of function and cell death . AccumulAll types of dementias, including AD, have in common a series of stereotyped changes including brain inflammation, loss of synaptic plasticity, and many biochemical and metabolic changes that eventually lead to neuronal death. AD is diagnosed by the presence of lesions in the brain often referred as senile plaques, mainly composed of amyloid-\u03b2 (A\u03b2) peptides, and neurofibrillary tangles, also as the result of protein accumulation . In AD, Increasing age is the most important non-modifiable risk factor for dementias and, in particular, for AD  , dementiIn high-income countries, such as the USA, the \u2018National Plan to Address Alzheimer\u2019s Disease\u2019 has been running for years as a national strategy, including prevention trials, and infrastructure supporting open access to big data, in order to address the scientific needs for prevention and treatment of AD by 2025 . As a reThe causes of AD are not completely understood, but it is believed they include a combination of genetic, environmental, and lifestyle factors . Since iOther than the above mentioned unmodifiable factors, a number of acquired factors have been identified as probable risk factors that could lead to AD . Among tRegarding AD, the influence of the gut microbiome in the bidirectional crosstalk between gut and the brain, known as the gut-brain axis, constitutes a research field of growing interest . ParticuConsidering the limited efficacy of current therapies, medical or psychological, for neurologic disorders (such as AD), the discovery of new mediators and moderators of these disorders, such as the oral and gut microbiome as well as the interactions with the diet and the lifestyle may open new diagnostic, preventive and nutritional and pharmaceutical therapeutic avenues for mental conditions and, particularly, for AD.In this paper, we have summarized the current knowledge regarding the influence of diet  on human microbiota and its potential impact on AD. Three main sections structure the content in a sequential and concatenated way. As with other chronic diseases related to aging, the need of preventive measures has become apparent, and most current approaches to tackle cognitive decline (the earliest manifestation of dementia) rely on lifestyle changes. Although both genetic and lifestyle factors play a role in determining individual risk of AD and dementia in general , recent Smoking has been associated with an increased risk of all-cause dementia , AD  and vascular dementia  . Albeit Diet is a modifiable environmental factor that has been associated with many non-communicable diseases with connections to dementia, such as diabetes and cardiovascular disease . A largehttps://dietamediterranea.com/en/fundacion): (i) use olive oil as your main fat source; (ii) eat plenty of plant products such as fruits, vegetables, legumes and nuts; (iii) bread and other grain products  should be a part of the everyday diet; (iv) fresh and locally low-processed products are preferred; (v) consume dairy products on a daily basis, mainly yogurt and cheese; (vi) red meat should be consumed in moderation and if possible as a part of stews and other recipes; (vii) consume fish abundantly and eggs in moderation; (viii) fresh fruit should be your everyday dessert and, sweets, cakes and dairy desserts should be consumed only on occasion; (ix) water is the beverage of choice, wine should be taken in moderations and with meals; and (x) be physically active every day.Owing to the complex biological interactions between different components of the diet, it has been proposed that the use of a whole-diet approach, through the study of dietary patterns rather than individual nutrients or food groups, might help to understand the role of diet in AD and related dementia . At presIn this line, the MIND (Mediterranean\u2013Dietary Approaches to Stop Hypertension (DASH) Intervention for Neurodegenerative Delay) diet incorporates the DASH (Dietary Approaches to Stop Hypertension) diet, which has been shown to lower high blood pressure , a risk While excessive alcohol use is an established risk factor for multiple chronic diseases , the linAlthough the long-term high consumption of alcoholic beverages has been associated with an increased prevalence of cancer, cardiovascular diseases, liver cirrhosis, dementia and depression ,65, modev/v [The first results suggesting a protective effect of moderate consumption of red wine in AD were published in the late 90s using data from a prospective cohort of elder participants in the Bordeaux area . Later, v/v . In contrast to other alcoholic beverages, such as spirits, for which increased risks have been reported , the proGemella, Granulicatella, Streptococcus and Veillonella genera), Bacteroidetes (strongly represented by Prevotella), Proteobacteria (Neisseria and Haemophilus genera), Actinobacteria , and Fusobacteria (genus Fusobacterium). The role of these oral microorganisms includes digestion of food, resistance against pathogens, maintenance of homeostasis, and the modulation of the immune system, contributing to oral and general well-being. However, they are also responsible for a variety of oral diseases [Millions of human microbiomes have been sequenced . Achievidiseases ,76. MoreClostridium, Enterococcus, Lactobacillus, and Ruminococcus genera), Bacteroidetes (including Bacteroides and Prevotella genera) and Actinobacteria, which represent approximately 90% of the microbiota. Other subdominant or minor phyla include Proteobacteria, Fusobacteria, and Verrucomicrobia [The human gut microbiome is mainly constituted by representatives of bacteria, but also include archaea, lower and higher eukarya and viruses. The intestinal ecosystem brings together the best environmental parameters for bacterial development. That is why it is the area of our body where the highest density of microorganisms with great complexity are housed; more than 1000 microbial species and all of these bacteria encode a microbial gene pool, exceeding the size of the human genome, known as the microbiome. These inhabitants of the human body are separated in different phyla. Among them, adult human gut microbiota is mainly represented by the phyla Firmicutes \u2014are able to invade and colonize the host cells, and disrupt host immune system manipulating cytokine networks, involved in bacteria adhesion, and inactivating protease inhibitors. Subsequently, P. gingivalis was able to initiate microglial cell activation and promote the synthesis of innate immune inflammatory proteins [P. gingivalis infections increased earlier occurrence of age-related granules in APOE\u2212/\u2212 mice following inflammation-mediated tissue injury, accompanied by loss of cerebral BBB integrity [In controls ,32. Serucontrols . Moreoveproteins . Furtherntegrity . n = 158), all cognitively normal, were studied in a longitudinal neurological program at the University of Kentucky. Raised baseline antibody levels, specific for the oral anaerobes F. nucleatum and Prevotella intermedia, correlated with cognitive deficits in subjects 10 years later [Going one step further, other studies showed the potential of the detection of periodontal pathogens as an AD predictive tool. Biological resilient adults , Eubacterium  and Clostridium sp SY8519, R.hominis and F.prausnitzzi and greater abundance of O. splanchnicus, Odoribacter sp, K. pneumoniae, B. fragilis, E. lenta and Desulfovibrio genus (D. fairfieldensis) [Clostridium, Eubacterium, and Butyrivibrio genera, are able to produce butyrate in the gut lumen at mM levels [P. gingivalis, leading to dysbiosis [P. gingivalis, similarly to periodontitis, may influence intestinal dysbiosis. Apart from P. gingivalis, other periodontopathogens including A. actinomycetemcomitans, can also disseminate to the colon [The evidence on the role of the gut microbiota on AD includes direct actions of bacteria as well as indirect actions or aging-related processes ,92. In lShigella . Other sldensis) . In manyM levels . Butyratysbiosis . In parthe colon . TherefoHafnia alvei, Pseudomonas aeruginosa, Morganella morganii, Pseudomonas putida, Citrobacter koseri, and Klebsiella pneumonia [As far as the intestinal level is concerned, studies have also shown that gut microbiota regulates the function of different body systems through interactions between the gut and distant organs, including the so-called \u2018gut-brain axis\u2019, via immune, endocrine and neural routes that are not fully understood ,100. Proneumonia . The comneumonia .Clostridioides difficile infections, may be a promising treatment option for several neurological dysfunctions [Finally, preliminary literature suggests that fecal microbiota transplantation, considered currently, as the most effective gut microbiota intervention for recurrent unctions . In factunctions  showed sOver the last 20 years, dietary polyphenolic compounds have received much attention because of their potential biological activities in many chronic diseases such as cardiovascular diseases, diabetes, obesity, and other inflammation-related diseases and lifestyle-related cancer ,114. TheRed wine is one of the richest sources of polyphenols in the diet. In particular, red wine provides a unique and very diverse combination of phenolic structures including flavonols, flavan-3-ols and anthocyanins, among the flavonoid compounds, and hydroxybenzoic and hydroxycinamic acids, phenolic alcohols and stilbenes, among the non-flavonoids. Taking into account that the influence of wine polyphenols on human microbiota is becoming widely recognized, and many new studies about microbiota and AD have been reported over last few years, we want to place a special emphasis on studies that comprehensively explore, from an integral perspective, the connection between modulation of oral and intestinal microbiota by wine polyphenols, and the derived consequences in AD .P. gingivalis can occur resulting in documented translocation to a variety of tissues and organs. P. gingivalis may access the brain and spread via different pathways including direct infection and damage to endothelial cells protecting the BBB, infection of monocytes followed by brain recruitment and/or infection and spreading through cranial nerves. Is has been suggested that after entering the brain, P. gingivalis spreads slowly over many years in mice from neuron to neuron along anatomically connected pathways [P. gingivalis brain colonization and neurodegeneration in AD. The dissemination of oral microorganisms to the brain is controlled by antimicrobial peptides, as part of the innate immune system. Indeed, in the last decade, several studies have explored the role of these antimicrobial peptides as potential biomarkers for AD; in particular, salivary lactoferrin discriminates between patients with mild cognitive impairment and AD from control subjects [F. nucleatum, P. gingivalis and A. actynomycetemcomitans, in experiments in oral subgingival biofilm models. This effect was independent of the presence of ethanol [P. gingivalis adherence, some phenolic metabolites with antimicrobial and immunomodulatory actions, such as caffeic and p-coumaric acids, at concentrations naturally found in wine, inhibited the adhesion of periodontal pathogens to oral cells as shown recently [Streptococcus dentisani resulted in a synergistic anti-adhesive effect against AD causative bacteria in oral fibroblasts with evident anti-inflammatory activity against cytokine production, all together preventing the progression of periodontal disease and promoting host-microbe homeostasis [P. gingivalis virulence. Some polyphenols and flavonoids present in wine and tea are known to inhibit gingipain activity and interfere with biofilm formation by P. gingivalis in gingival cells models [P. gingivalis by reducing the expression of virulence factor genes such as fimbriae (Type II and IV) and proteinases (kgp and rgpA) [Increased data link oral microbes with AD. Different hypothesis reveal how increased brain microbial burden may directly exacerbate A\u03b2 deposition, inflammation, and AD progression. Once the oral cavity is infected, transient bacteremia of pathways . Elderlysubjects . Anothersubjects ,121. Tre ethanol . A critirecently . The comeostasis . Secretes models . Resverand rgpA) .After pass through the oral cavity, polyphenols suffer the action of microbiota in the gastrointestinal tract. Polyphenols can reach the colon in high proportions (90\u201395%), where they can be transformed into an inventory of bioavailable microbial metabolites by the resident microbiota, which are considered to be responsible for the health promoting effects that are attributed to the parent compounds. As result of this bi-directional relationship between polyphenols and the microbiome of the human gut, polyphenols can also modulate the composition of an individual\u2019s microbiome.Enterococcus, Prevotella, Bacteroides and Bifidobacterium, at genera level; and in Blautia coccoides, Eubacterium rectale group and B. uniformis and Eggerthella bacterial species [Faecalibacterium prausnitzii and Akkermansia sp., butyrate producers with brain anti-inflammatory properties [Regarding wine polyphenols-gut microbiome interactions, only a few intervention studies have investigated its impact in host and mental health . Intere species ,128,129. species , red win species . These b species . Modulat species . Interesoperties ,134, andoperties ,137,138,operties , hence, operties ,140.Despite these valuable findings, more robust studies with larger populations and from both taxonomic and functionality approaches are needed to confirm and complete such results in causal AD patients. Besides, an individual variability in metabolite production has been reported, depending on the specific chemical structure of the polyphenol and differences/variations in gut microbiota. Consequently, different gut microbiota-responsive phenotypes to wine polyphenol interventions have been reported ,138. TheThe interactions and metabolic pathways of wine microbial polyphenols have been widely documented, however, most studies were carried out in in vitro colonic and cell models or animal models ,143. An As there is a crucial need for the development of new strategies capable to prevent, delay the onset or treat brain dysfunction and associated cognitive decline, different studies have focused on biological activity of individual phenolic compounds and derived microbial metabolites . Interacn = 20), and, even though only 1% of resveratrol reached central nervous system, AD biomarker changes were reported, including a significantly less pronounced decline in cerebrospinal fluid and plasma amyloid-beta levels , suggesting that resveratrol had indeed engaged its target in the brain [n = 37), Moran et al. [Regarding AD prevention and treatment, it is essential that bioavailability issues be addressed for neuroprotection to be relevant in a clinical study scenario. Different clinical studies have been carried out that explored the benefits of resveratrol for treating individuals having AD . Wine polyphenols could exert their effects in the context of AD through different mechanisms: (i) direct actions on the brain; (ii) through their ability to modulate gut microbiota composition and functionality and, lastly, (iii) through the actions and properties of the metabolites produced in the gut. More studies including a deep investigation of all these mechanisms are needed. Considering that metabolic changes are most likely the result of polyphenol-microbiota interactions, an integrated microbiome-metabolome study is suggested to better understand neuroprotective effects or red wine.Giving the rapid increase in the proportion of older adults worldwide, it is of crucial importance to identify modifiable risk factors that may prevent or delay the onset of cognitive impairment and extend years of healthy life. The identification and implementation of effective dietary strategies early in the adult life, could help to improve cognition and mental health. This systematic review has tried to clarify the connections between diet, and wine in particular, and human microbiome at the frame of the AD. New challenges in the role of wine polyphenols in the prevention of functional decline and AD have been tried to be identified.Accumulated evidence suggests that microorganisms are implicated in AD pathogenesis. In the cascade of events preceding AD, oral cavity and gastrointestinal microbiome seem to play a role and different bacteria have shown an important contribution to stimulate A\u03b2 aggregation and neuro-inflammation. Although AD is a complex disease and microbial infection may not be the sole cause, this new paradigm may provide novel targets for the prevention and treatment of this devastating disease.P. gingivalis) into the brain producing inflammatory mediators, so causing neuro-inflammation and possibly acting as primary agents for AD. Then strategies to prevent and/or treat periodontitis might be a possible treatment/beneficial option in countering AD progression. An adequate oral hygiene habits are the main strategy used to prevent the onset of these disorders. Dietary patterns and, in particular diets including red wine polyphenols, modulate the composition and integrity of the oral microbiota suggesting plausible benefits in the prevention of periodontal diseases. Although no published studies linking directly the modulation of oral microbes by wine polyphenols (or from other dietary sources) and AD were found, their multiple observed mechanisms of action, including antimicrobial, anti-adherent ability, inmunomodulatory effects and inhibition of virulence gene expression need to be evaluated to understand whether this orally-driven disruption will reduce P. gingivalis infection in the brain and slow or prevent further neurodegeneration and accumulation of pathology in AD patients. However, this research field is still in need of further in vitro and especially in vivo well-conducted studies for further support of these beneficial effects.Studies in oral microbiomes seems to be critical in our understanding of AD and of how to prevent it through diet and lifestyle. The mechanism for the relationship between periodontitis and cognitive decline is still unclear but there is accumulated evidence to support a role for systemic infection and inflammation, direct entry of bacteria  as well as by microbial stimuli by specific intestinal bacteria. However, caution needs to be taken when interpreting the results of these studies and extrapolating them to humans, since mostly cellular and animal models have been performed. Further intervention trials are warranted to increase understanding of the impact of wine-microbiota interaction on features on mental health and AD risk. In addition, it will be important to extend these findings in larger-scale studies with adequate geographic separation. This will allow to accurately analysis of the influence of other potential confounding variables.Cohesive research is needed through the precise and quantified integration of data from wine consumption, omics and imaging technologies, advanced models and multi-organ-on-a-chip models to mechanistic studies that offer many possibilities to address the major research challenges arising from the complexity of interactions between wine/diet polyphenols and AD preventive outcomes, as well as to advance in the development of microbiome-informed predictive tools and biomarkers of the health status and early disease detection."}
+{"text": "LEP and their associations with clinicopathological features including survival outcomes of OC patients. The protein expression of LEP was evaluated in 208 samples using both tissue microarray and immunohistochemistry techniques. The methylation profiles of LEP were measured in 63 formalin-fixed, paraffin-embedded tumor tissues by quantitative polymerase chain reaction using a MethyLight assay. Our results showed a significant association of LEP protein overexpression with several clinicopathological variables, mainly tumor subtype, LVI, age of menarche, tumor size and stage (p < 0.04). Kaplan\u2013Meier analysis (using low expression versus high expression as a discriminator) indicated that LEP protein overexpression is a powerful positive prognosticator of both OC recurrence (DFS) and disease-specific survival (DSS) in our OC cohort . This implies that patients with high LEP expression profiles live longer with less recurrence rates. Methylation analysis results demonstrated a clear association between no/low LEP protein expression pattern (38%) and LEP promoter CpG island hypermethylation (43%). Results of this study suggest that LEP is a powerful prognosticator of OC recurrence and DSS. LEP expression in OC seems to be regulated by its promoter hypermethylation through gene partial/total silencing. Further multi-institutional studies using larger cohorts are required to demystify the intricate molecular functions of this leptin-driven effects in OC pathophysiology and to accurately assess its theranostic potential and validate its prognostic/predictive power in OC onset, progression towards more effective and personalized management of OC patients.Ovarian cancer (OC) is the deadliest among all gynecological cancers. Epidemiological studies showed that obesity might influence many cancers including OC. One of the key factors that may link obesity and OC is leptin (LEP), known as an adipokine with pleiotropic effects on body homeostasis. This study aims to investigate the expression pattern of LEP, assess the methylation profiles of  Ovarian cancer (OC) is the fifth most lethal malignancy among women and the deadliest of all gynecological cancers . In SaudOb/LEP gene located on ch7q32.1, and binds to its receptors (Ob-R/LEPR) that are found on several tissues, mainly the brain and hypothalamus [LEP is regulated by food intake, hormones, metabolites, and cytokines, and its secretion correlates with fat mass and hormones like estrogen, progesterone, and insulin levels [Epidemiological studies showed that obesity is a risk factor for many cancers including OC . Every 5thalamus . LEP, knthalamus ,14,15,16n levels ,17. LEP importance resides in maintaining the immunometabolism balance between weight and energy. This occurs by signaling satiety to the hypothalamus subsequently reducing dietary intake and fat storage while modulating energy expenditure and carbohydrate metabolism, preventing further weight gain. Noteworthy, most obese individuals are not leptin deficient but rather leptin \u201cresistant\u201d. Consequently, they have elevated levels of circulating leptin leading the hyperleptinemia [LEP) has an established role in angiogenesis, ovulation, and fertilization [LEP (G2548A) within the 5\u2032 promoter region. This mutation has been associated with variations in BMI, the concentrations of LEP in the plasma [ptinemia ,19. Hypeptinemia ,24,25,26lization ,28,29. Me plasma ,31,32,33e plasma . LEP dysregulation and OC development, however, the exact mechanism and prognostic value of leptin remain unclear. In this study, we assessed the expression levels of LEP in OC tissues and its correlation with the methylation status of the LEP promoter region. Finally, we assessed its prognostic value in our cohort. The involvement of the LEP in many diseases, as previously mentioned, has also been reported in various types of cancer. Previous data reported overexpression of LEP protein in renal carcinoma  and brea3. Also, nearly half of the cohort was diagnosed with the serous histological subtype.As shown in Immunohistochemical (IHC) analysis was performed in tissue microarray (TMA) slides to assess the expression pattern of LEP protein. Analyzed OC tissue samples showed diffuse cytoplasmic localization of LEP expression. According to the suggested cut-off point of evaluation (low expression vs. high expression), 24% of our OC tissue samples showed low  cytoplasmic expression of LEP protein as compared to 76% of high cytoplasmic expression pattern .p = 0.001), LVI (p = 0.001), age of menarche (p = 0.01), tumor size (p = 0.01) and stage (p = 0.04). The association of LEP expression pattern with the OC patients\u2019 clinicopathological features was assessed using (low expression versus high expression) as the best discriminator . LEP prop = 0.01 and p = 0.002, respectively). In addition, LEP protein overexpression was a powerful positive prognosticator of both OC recurrence and DSS in our cohort. Interestingly, more than half of our patients\u2019 cohort with high LEP expression were alive  and Disease-Specific Survival (DSS) data using Kaplan\u2013Meier analysis and (low expression versus high expression) as a discriminator indicated that both DFS and DSS were significantly associated with LEP protein expression in OC patients  of our OC patient cohort. Interestingly, our data showed that LEP methylation profiles were mainly correlated with histological subtype (p = 0.001), tumor site (p = 0.04), menopausal status (p = 0.09), and patients\u2019 age (p = 0.06). No association was noted between LEP methylation in OC patients and BMI and tumor stage (p > 0.05)  . LEP methylation profiles .For the survival outcomes, Kaplan\u2013Meier survival analysis revealed that there was no association between either DSS or DFS with LEP in the process of tumorigenesis and metastasis is mediated through regulating the EMT, cell adhesion and proteolysis of the ECM components [Ovarian cancer is a disease marked by the absence of early specific signs and silent clinical symptoms making its diagnosis at an early stage challenging, thus leading to higher mortality rates, being the highest deadly cancer among all gynecological tumors ,41,42,43mponents . The invLEP expression and OC, however, the exact mechanism of this gene and its potential prognostic value remain unclear. Furthermore, several studies have investigated the protein expression pattern of LEP in tissues of many cancer types such as breast [LEP promoter methylation status, and its potential prognostic value. Our IHC results showed a cytoplasmic (p < 0.04) (p > 0.05) [These findings suggested possible links between s breast ,60 and cs breast , but onls breast . In the oplasmic  expressi < 0.04) . Compare > 0.05) . Similar > 0.05) ,66,67,68 > 0.05) ,69. Like > 0.05) ,39,40.p = 0.01; p = 0.002; Interestingly, Kaplan\u2013Meier survival analysis of our cohort showed a strong prognostic power of LEP protein expression. Indeed, patients with LEP overexpression had a strong trend toward better prognosis marked by lower recurrence  of OC patients. Interestingly, our data showed that LEP methylation profiles were correlated mainly with histological subtype, menopausal status, and patients\u2019 age (p > 0.05), but no significant associations were found with BMI and tumor stage (p > 0.05)  .LEP promoter methylation status and patients\u2019 survival outcomes (DFS and DSS) (data not shown), our results demonstrated a clear association between LEP promoter hypermethylation profiles and a no/low LEP protein expression pattern. In reality, 43% of LEP promoter hypermethylation in our cohort is approximately matching with the proportion of OC patients with no/low LEP protein expression (38%) of OC patients  were documented in several studies [Therefore, these concordances between IHC and methylation analysis results confirm our hypothesis that ilencing ,78. Our  studies ,78. Our  studies ,82,83,84LEP and its receptor LEPR, the epigenetic events (DNA CpG islands methylation events as shown in this study) affecting the LEP gene, the LEPR (Ob-R) polymorphism , and the soluble LEPR isoform (Ob-Re)) should be considered to better understand the downstream LEP signal transduction pathways to define more accurately both the LEP prognostic and predictive value, and to refine the cohort of cancer patients that will have better outcomes [LEP gene promoter methylation, protein expression, and the obesity-related metabolic events highlight the magnitude and complexity of LEP molecular functions in both health or disease as discussed earlier  tissue collected in the Departments of Pathology and Gynecology, King Abdulaziz University Hospital (KAUH) between 1995 and 2014. It has been identified primarily by the Tumor Node Metastasis (TNM) classification system using histopathological features. The tissue samples and the associated clinical data were collected after all the necessary ethical approvals were obtained in compliance with the guidelines of the King Abdulaziz University Hospital Ethical Committee (Ref. number: KAU-189-14). The patients\u2019 clinicopathological characteristics , follow-up data, and survival are summarized in TMA was generated in-house from the archived FFPE tissue blocks. After sections preparation, the slides were stained with hematoxylin and eosin (H&E). Five TMA recipient paraffin blocks used in this study were produced. The TMA contained duplicate specimens of the OC patient and duplicate specimens of the placenta as control tissue. Immunohistochemical staining of 5 TMA slides derived from 208 patients diagnosed with OC in KAUH, Jeddah was performed using an automated titration run staining system , which allows manual addition of antibodies. The rabbit polyclonal antibody against LEP  was used at the dilution of 1:50.LEP protein staining was evaluated using a standard x40 magnification light microscope, blinded by tumor grade, point, or clinical outcome details. A four category  scoring system was adopted for cell cytoplasmic staining: (0) means no expression, no detectable stain in <10% of the membrane; (1+) mild yet detectable discontinuous stain present in 10\u201339% of the membranes; (2+) moderate, clearly positive discontinuous stain present in 40\u201390% of the membranes; and (3+) strong continuous membrane stain. With both the staining intensity and the fraction of positively stained cells considered using the following formula, the staining index of the membranous expression pattern was calculated based on the following equation: 0 and 3 ,90. The \u00ae  software packages . Frequency tables were analyzed using the Chi-square test, with Fischer\u2019s exact test to assess the significance of the correlation between the categorical variables. Univariate survival analysis for the outcome measure  was based on the Kaplan\u2013Meier method, with log-rank (Mantel\u2013Cox) comparison test. In all tests, the values p < 0.05 were considered as statistically significant.Statistical analyses were performed using the SPSSXylene  was used for paraffin dissolution to extract DNA from FFPE rolls followed by washing with 100% ethanol . After the deparaffinization step, the DNA was extracted from tissues using the QIAamp DNA FFPE Tissue Kit (Qiagen) as directed by the manufacturer. The concentrations of all the DNA samples were measured using the Thermo Scientific NanoDrop 2000 Spectrophotometer.LEP promoter region was performed using (Qiagen EpiTect \u00ae Bisulfite Conversion). Briefly, NaH2SO4 was incubated with 0.5 \u03bcg of the extracted DNA. The unmethylated cytosine residues were converted into uracil residues, while the methylated ones remained as it is (cytosine). For MethyLight assay, EpiTect \u00ae MethyLight PCR Kit  and EpiTect \u00ae Control DNA and Control DNA Package  were used. Specially designed primers and fluorescent dual-labeled TaqMan probe were produced  were used for DNA amplification. After that, we proceeded with a qRT-PCR machine using StepOnePlus \u2122 -Real-time PCR system  by placing the qPCR master mix with the DNA samples. As detailed in the manufacturer\u2019s manual and stated elsewhere , bisulfi\u00ae  software packages . To identify the statistically significant correlation between hypermethylation events and clinicopathological features of our cohort, we used ANOVA followed by Fisher\u2019s exact test. Furthermore, DSS and DFS were calculated by univariate Kaplan\u2013Meier analysis, and equality of the survival functions was determined by log-rank (Mantel-Cox) test, withal, p-values < 0.05 were considered statistically significant.Statistical analyses were performed using the SPSS"}
+{"text": "Governments have a responsibility to provide equal opportunities for sport and physical activity to all people of population. Chinese governments have issued many policies, such as \u201cexhibition in the south, expansion in the West and East\u201d of ice and snow sports to promote and stimulate the participation of the broad masses of the people. As a high-cost sport, the participants of ice and snow sports are usually socially elite groups. This study investigated the participation of cultural elite groups in ice and snow sports and investigated the social mobilization effect of ice and snow sports participation promotion policies by using binary regression and sequential regression models. The research shows that there are two different stages of one-time and continuous participation in the development of ice and snow sports in China. The one-time participation of ordinary people in ice and snow sports is mainly in response to the social mobilization of government policies. At the same time, it is positively correlated with site restrictions and knowledge of ice and snow sports. In the continuous participation group, gender, income, perception of ice and snow culture, and convenience near the site were highly positively correlated with consumption level. According to the results, low- and middle income people are less likely to participate in these activities because of their income. Therefore, this policy can increase inequalities. Increasing physical activity levels is one of key component of the UN's 2030 Sustainable Development Goals. Investing in population based policies to promote sport and physical activity opportunities including walking, cycling, active recreation, and play can contribute directly to achieving many of the 2030 Sustainable Development Goals. So, policy actions on sport and physical activity can have multiplicative health, social and economic benefits, and will directly contribute to achieving SDGs . GovernmChinese government, with its policies, seeks to increase level of physical activity. One of these policies is increasing people's participation in ice and snow sports. To realize the vision of \u201c300 million people on ice and snow\u201d, Chinese governments at all levels and their functional departments have issued policies to promote the participation of ice and snow sports. In 2016, the State General Administration of Sports, the National Development and Reform Commission, the Ministry of Education and the National Tourism Administration jointly issued notices on the development plan of ice and snow sports (2016\u20132025). In November of the same year, the State General Administration of sports issued the plan for the promotion and popularization of mass winter sports (2016\u20132020). In 2019, the general office of the CPC Central Committee and the general office of the State Council issued the notice the opinions on taking the 2022 Beijing Winter Olympic Games as an opportunity to vigorously develop ice and snow sports can clearly see that China has made great efforts to develop ice and snow sports with the help of Beijing's hosting of the Winter Olympic Games . Second, to avoid repeated answers, each IP address could only answer the questionnaire once during the network survey. Third, questionnaires with an answer time <1 min were eliminated, and some questionnaires that obviously did not answer carefully were eliminated through manual browsing in the later stage. A total of 326 samples were collected. After excluding missing values and outliers, 324 valid questionnaires were obtained, and 215 samples were selected for this study.In particular, the data of this study are the survey conducted by the research team when informing the respondents of the purpose, which does not involve any ethics and privacy, and can be applied to the study of this project.This paper uses questionnaire survey to collect data. In this paper, Stata 16.0 software was used to analyze the data, and logit and ologit models were used for estimation. Among them, the logit model is one of the discrete choice models. In this paper, a binary logit regression model is selected for the analysis of cultural elite participation. In the dependent variable, two values 1 and 0  are taken, that is, participation is yes or no. Ologit has become a sequential regression model. This paper converts the participation times into sequential data for analysis of the annual participation times of cultural elites and studies the factors that increase their continuous participation times through the sequential model.In terms of variable selection, we not only select according to the traditional sociological analysis variables but also design according to the preset research ideas and determine the result variables and independent variables in combination with the existing literature research, as follows:In this study, two outcome variables, whether they have participated in ice and snow sports and the number of times they participate in ice and snow sports every year, were used to evaluate the social mobilization effect of 300 million people participating in ice and snow sports. The option of whether to participate in ice and snow sports is set to yes or no, and the values are 1 and 0 according to the two classification variables. Now, the options for the number of times to participate in ice and snow sports each year are set to 0, 1, 2, 3, and 4 times or more, and the values are assigned as 0, 1, 2, 3, and 4 according to the continuity variable.The purpose of this study is to explore the social mobilization effect of 300 million people participating in ice and snow sports based on an empirical study of cultural elites. Therefore, the sociological characteristics of the population and social sports factors are selected as independent variables.The sociological characteristics of the population mainly include gender, education and income. The first is the gender characteristics. Traditional ethical values still affect the value standards of Chinese women. It can be basically predicted from life experience that men prefer to participate in sports activities than women. In terms of gender, men = 1 and women = 0. The second is the characteristics of education. The scope of this study is the cultural elite. The selected category of cultural elites is personnel with a graduate degree or above. After converting the degree into the variable of years of education, it is 18. Income inequality has an important impact on the consumption behavior and social attitude of social members. The total income of residents includes personal wage income, business income, property income and transfer income. The income variable in this paper is: what was your annual income in the previous year? The options are set as below 100,000 yuan, 100,000\u2013200,000 yuan, 200,000\u2013300,000 yuan, 300,000\u2013500,000 yuan and above 500,000 yuan. The values are assigned as 0, 1, 2, 3, and 4 according to the continuity variable.The social sports participation factors of the crowd mainly select the cultural habits and the convenience of the venue. Bourdieu once pointed out that people's interests and hobbies exist in a certain \u201cfield and habits\u201d. This study selects \u201clike watching ice and snow competitions\u201d and \u201cunderstanding ice and snow sports\u201d as explanatory variables. Among them, the options of \u201clike watching ice and snow competitions\u201d are set as: very dislike, dislike, general, like and very like, and are assigned as 0, 1, 2, 3, and 4 according to the continuity variable; The option of \u201cunderstanding ice and snow sports\u201d is set to very little understanding, little understanding, general, understanding and very understanding, and is assigned as 0, 1, 2, 3, and 4 according to the continuity variable. As the carrier of ice and snow sports, the convenience of ice and snow sports venues (pavilions) affects people's enthusiasm to participate in ice and snow sports activities. Therefore, this study takes ice and snow sports venues (pavilions) as an important independent variable. Therefore, \u201cIs it convenient for you to go to the ice and snow venues near your residence?\u201d To explain the convenience of ice and snow venues, this option is set to very inconvenient, inconvenient, general, convenient and very convenient. It is assigned as 0, 1, 2, 3, and 4 according to the continuity variable.The three key independent variables of sports participation are consumption perception, policy cognition and participation times. First, due to the particularity of venues, climate and equipment, the consumption of ice and snow sports is higher than that of general sports. We choose\u201d do you think the consumption of ice and snow sports are expensive?\u201d to explain the consumption perception of ice and snow sports. The options are set to very expensive, relatively expensive, general, not too expensive and very cheap and are assigned as 0, 1, 2, 3, and 4 according to the continuity variable. Second, the social mobilization effect of ice and snow sports policy should first be the awareness of the policy. The research uses the question \u201chave you heard of '300 million people participating in ice and snow sports\u201d, the options are set to no and yes, and the values are 0 and 1 according to the two classification variables. At the same time, the question \u201cwhen did you first participate in ice and snow sports?\u201d is used to respond. The previous value is 02015, and the first time after 2015 is 1. Third, to investigate the continuity of the effect of social mobilization, this study sets the variable of \u201cthe number of times of participating in ice and snow sports every year\u201d, which is set as 0, 1, 2, 3, and 4 times and is assigned as 0, 1, 2, 3, and 4 according to the continuity variable. The descriptive statistics of the above variables are shown in According to the descriptive statistics of the variables in The social mobilization effect of the policy of \u201c300 million people participating in ice and snow sports\u201d is first reflected in the degree of response. From the contingency table of policy response , of the Whether the social mobilization effect of cultural elites participating in ice and snow sports in the year is sustainable needs to be further discussed. According to The first dependent variable of this study is the dichotomous variable of whether they have participated in ice and snow sports. Therefore, dichotomous linear regression (logit) is used to analyze the impact of cultural elites' participation in ice and snow sports. Gender, income, and the understanding of ice and snow sports had no significant impact on the participation of cultural elites in ice and snow sports.In terms of cultural factors, the number of cultural elites who like watching ice and snow competitions will increase by 1.97 times (e0.722 = 1.97) compared to those who do not like watching ice and snow competitions.In terms of venue convenience, the convenience of ice and snow venues (pavilions) near the residence will increase the participation probability of cultural elite groups by 1.75 times (e0.674 = 1.75).In terms of consumption perception, whether consumption is expensive has a positive correlation with participation in ice and snow, but there is no significant difference.In terms of policy response, those who have heard of \u201c300 million people participating in ice and snow sports\u201d are 4.72 times more likely to participate in ice and snow sports than those who have not heard of it (e1.73 = 4.72).The estimated results of the regression equation in The second dependent variable of this study is the continuous variable of the annual number of cultural elites participating in ice and snow sports. Therefore, ordered linear regression (ologit) is used to analyze the impact of cultural elites participating in ice and snow sports. The gender difference was significant, and the number of men participating in ice and snow sports was more significant than that of women. In model 6, with the strongest explanatory power, the possibility of increasing the number of men participating in ice and snow sports is 4.1 times (e1.51 = 4.1); even in model 4, with the lowest probability, it is 2.4 times (e0.882 = 2.4).Four of the six models showed that there was a positive relationship between high income and the number of participants .Interest and watching ice and snow competitions had a significant positive correlation with the number of participants participating in ice and snow sports .There was no significant relationship between understanding that 300 million people have participated in ice and snow sports and the number of participants .Venue convenience and consumer price had an impact on the number of participants .The model estimation results in In this study, it was seen that the interest in watching ice and snow sports, the convenience of the venue, and hearing that 300 million people are participating in ice and snow sports have a stable impact on the participation of cultural elites in ice and snow sports. This reveals that the popularization of Chinese, venues and government calls has played a positive role in promoting the development of ice and snow sports. The number of cultural elites who like watching ice and snow competitions have increased, which shows that the dissemination of ice and snow events attracts people to participate in ice and snow sports. Holding high-level ice and snow events will help guide people's initiative and enthusiasm to participate in ice and snow sports, which is also a common problem for sports events to promote sports participation . Also, CIn current study it was seen that gender had no significant impact on whether cultural elites participate in ice and snow sports. Wang et al. has been shown that pleasure and increasing ice and snow experience are the key points for female consumers to show off their ice and snow experience by ice and snow sports tourism life . At the In terms of consumption perception, whether consumption is expensive had a positive correlation with participation in ice and snow, but there was no significant difference. This may be because for most one-time participants, occasional participation will not affect the decline of their quality of life. As Liu and Liu found in his research on the characteristics of sports consumption view of urban residents of different sizes, the sports consumption view mainly involves three aspects, such as \u201cmarket environment\u201d, \u201cindividual conditions\u201d and \u201csocial reference\u201d , and is Factors affecting the number of participants were also examined in this study. This study showed that the number of men participating in ice and snow sports was more significant than that of women. This indicated that gender inequality was in sustainable ice and snow sports participation. The reason for this inequality may be that the technical threshold of ice and snow sports is relatively high, there are certain risks in the process of participation, and it is related to the solidified female characteristics in China's social consciousness .Income also affects the number of participation. This may be because the high consumption of ice and snow sports leads to the lack of economic ability of low-income groups to support their participation in ice and snow sports many times; alternatively, the higher the income is, the higher the cognitive participation of ice and snow sports .The effect of interest on the number of participants indicated that watching ice and snow competitions had a significant positive correlation with the number of participants participating in ice and snow sports, which is not only consistent with the estimation results of the participation model but also confirms the research conclusion of Pan and Hai; that is, psychological internal drive can positively promote the behavior of sports tourism participants .The current study showed that understanding ice and snow sports and policy responses cannot increase the number of cultural elites participating. This indicated that in the current Chinese society, the call of the government only plays a role in promoting participation, and economic strength and ice and snow cultural knowledge are still needed in sustainable participation.The variable venue convenience and consumer price had a significant impact on the increase the annual number of cultural elites participating in ice and snow sports, and the degree of impact was also close. From another dimension, ice and snow infrastructure is an important factor in the development of China's ice and snow sports industry .Increasing the physical activity level, as one of key component of the UN's 2030 Sustainable Development Goals, can have multiplicative health, social and economic benefits . HoweverIn general, this paper systematically analyses the factors of Chinese cultural elites participating in ice and snow sports. The main discus are as follows: first, Chinese cultural elites actively responded to the call of the Chinese government and significantly increased their participation in ice and snow sports after 2015; second, the participation of Chinese cultural elites in ice and snow sports is mostly one-time, which is related to the government's call, site restrictions and knowledge of ice and snow sports; third, the continuous participation of Chinese cultural elites in ice and snow sports is related to gender, income, ice and snow culture, venue convenience and consumption level; fourth, the government's call can only increase the participation rate, but the social promotion of ice and snow sports needs to be strengthened for sustainable participation. Therefore, to better realize the strategy of \u201cexhibition in the south, expansion in the west and eastward\u201d of ice and snow sports, we also need to strengthen the policy call, put forward targeted publicity strategies for the public, and improve the possibility and sustainability of the public's participation in ice and snow; cultivate ice and snow culture and build ice and snow culture according to local conditions; stimulate the ice and snow market, provide ice and snow venues and formulate sports consumption compensation mechanisms according to the consumption characteristics of ice and snow sports.There were some deficiencies and limitations in this study. First, the survey sample of this study is targeted at the cultural elite group, which leads to group bias in the results. Therefore, in the future, we will consider adding category attributes to conduct personnel category analysis to more accurately analyze the consumption of ice and snow sports. Second, this study mainly uses the basic variables commonly used in sociological research. In the follow-up research, variables can be further added, and the promotion and participation factors and obstacles can be introduced into the regression model to explore the promotion and hindrance mechanism.Ice and snow sports are usually expensive. According to the results, low- and middle-income people are less likely to participate in these activities because of their income. Therefore, this policy can increase inequalities.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.PL drafting the work and revising it critically for important intellectual content. WL contributed to the initial drafting of the manuscript. Both authors contributed to the article and approved the submitted version.This work was supported by the National Social Science Foundation of China (Grant No. 19BTY041).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": "Background: The metabolic syndrome (MetS) is prevalent in Arabian populations. Several small-scale studies have been performed to investigate the genetic basis of MetS. This systematic review and meta-analysis aimed to examine whether candidate gene polymorphisms are associated with MetS susceptibility among ethnic groups of the Arabian world and to suggest possible directions for future research regarding genetic markers and MetS.Methods: A search was conducted for peer-reviewed articles that examined the genetic association of MetS in Arabian populations in the following databases: Medline, Embase, Scopus, Direct Science, Web of Science, ProQuest, and Google Scholar until March 31, 2021. Articles were eligible if they were case-control studies, which investigated MetS as a dichotomous outcome (MetS vs no MetS). To assess the quality of the studies, the Q-Genie tool  was used. A non-central chi2 (random-effect) distribution was used to determine the heterogeneity (H) of Q and I  statistics.Results: Our search strategy identified 36 studies that met our inclusion criteria. In most cases, studies were excluded due to a lack of statistical information such as odds ratios, confidence intervals, and p-values. According to the Q-Genie tool, 12 studies scored poorly (a score of\u226435), 13 studies scored moderately ( >35 and\u226445), and 12 studies had good quality ( >45 or higher). The most frequently studied genes were FTO and VDR (both included in four studies). Three SNPs indicated increased risk for MetS after calculating the pooled odds ratios: FTO-rs9939609 ; LEP-rs7799039 ; and SERPINA12-rs2236242 . Meta-analysis studies showed no significant heterogeneity.Conclusion: There were many sources of heterogeneity in the study settings. Most of the studies had low to moderate quality because of sample size and power issues, not considering all potential sources of bias, and not providing details about genotyping methods and results. As most studies were small-scale, aimed to replicate findings from other populations, we did not find any unique genetic association between MetS and Arabian populations. The metabolic syndrome (MetS), which is characterized by insulin resistance, hypertension, obesity, and dyslipidemia , is a sihttps://www.ebi.ac.uk/gwas/search?query=metabolic%20syndrome) revealed 18 publications on MetS but 193 publications when the search term diabetes was used. Using data from 291,107 individuals from the United Kingdom Biobank, Lind and his colleagues identified 93 independent loci associated with MetS, of which 80 were previously unidentified , Embase, Scopus, Direct Science, Web of Science, ProQuest, and Google Scholar, from database inception to March 31, 2021, using these search terms and filters:\u2022 \u201cArab\u201d OR \u201cArab ancestry\u201d of \u201cArab ethnicity\u201d OR one of the 22 Arab countries: \u201cAlgeria\u201d OR \u201cBahrain\u201d OR \u201cComoros\u201d OR \u201cDjibouti\u201d OR \u201cEgypt\u201d OR \u201cUnited Arab Emirate\u201d OR \u201cIraq\u201d OR \u201cJordan\u201d OR \u201cKuwait\u201d OR \u201cLebanon\u201d OR \u201cLibya\u201d OR \u201cMauritania\u201d OR \u201cMorocco\u201d OR \u201cOman\u201d OR \u201cPalestine\u201d OR \u201cQatar\u201d OR \u201cSaudi\u201d OR \u201cSaudi Arabia\u201d OR \u201cSomalia\u201d OR \u201cSudan\u201d OR \u201cSyria\u201d OR \u201cTunisia\u201d OR \u201cYemen. \u201d\u2022 AND \u201cMetabolic syndrome\u201d OR \u201cMetS\u201d OR \u201cSyndrome X\u201d OR \u201cDysmetabolic syndrome\u201d OR \u201cInsulin resistance syndrome. \u201d\u2022 AND \u201cGenetic\u201d OR \u201cGenetics\u201d OR \u201cSNP\u201d OR \u201cSingle nucleotide polymorphism\u201d OR \u201cGenetic polymorphism\u201d OR \u201cGenetic variant\u201d OR \u201cGenetic variation\u201d OR \u201cGenetic polymorphism\u201d OR \u201cAllele\u201d OR \u201cGenetic locus. \u201dA sample search strategy for Medline (via OvidSP) can be found in the Eligibility: The eligibility criteria comprised peer-reviewed articles on genetic association with MetS in Arabian populations . In this context, we defined the Arabian population as those who are associated with the Arab League, as shown in the following table. Articles eligible for consideration included case-control studies examining MetS as a dichotomous outcome (MetS versus no MetS), and published in English. The exclusion criteria were studies that did not meet the inclusion criteria, grey literature, duplicate publications, and studies without detailed results, including odds ratios, confidence intervals, and p values. We also excluded case report review papers and family association studies. To reduce the risk of selection and methodological bias, studies that reported one of the standard definitions of MetS  were included in the meta-analysis  after screening the titles and abstracts to determine their eligibility. Any disagreements were resolved by a fourth researcher (WO). Further, detailed data were extracted from standardized data extraction forms, including author affiliations, study designs and settings, study population characteristics, MetS classification criteria, number of cases and controls, obesity determination and measurements, genetic markers, tested alleles, and association analysis methods and outcomes.Quality of studies: To assess the quality of the studies, the Q-Genie  tool was applied, which has a scoring system covering eleven different elements, as previously reported . In Stata software, we used the \u201cmetan\u201d command. The heterogeneity (H) was determined using the non-central chi  and Egger\u2019s test were not considered because the number of studies used in the meta-analysis was small ( <10 studies), and the test had a relatively low statistical power to determine whether the observed asymmetry was caused by chance.tral chi  (common-l chi (I  statistihi (I (I  > 50%). (I (I (\u03c4 ) in addi and Tunisia (10 studies) (Table 1). The majority of studies adopted the IDF definition of MetS over other definitions like the AHA/NHLBI (Our search strategy . In mostHA/NHLBI  and the HA/NHLBI  (SupplemHA/NHLBI .Table 1) that selected genes based on previous literature reports or their predicted functions in relation to MetS development. There was considerable variability in the number of genes selected, ranging from one to six. The most commonly studied genes were FTO and VDR , rs4420638 (APOE), rs7799039 (LEP), and rs2236242 (SERPINA12). As all of these indicated a significant heterogeneity [I ; LEP-rs7799039 ; and SERPINA12-rs2236242 , All studies were gene-candidate studies /haplotype blocks and the type and frequency of monomorphic/polymorphic SNPs. Many studies have investigated the genetics of MetS, but few have examined MetS as a binary outcome. In light of the high prevalence of MetS in Arab populations and its components , the curIn the narrative synthesis, 36 studies were included after several were excluded due to insufficient methodology or statistical analysis information. All of the studies were candidate gene studies, and none of the studies utilized a genome-wide or sequencing approach. Moreover, there was considerable variation in the tested markers and genes, with most studies evaluating a single gene  to severTable 1). For example, while most of the studies focused on diverse populations, a few included only females in their research  are shown in FTO gene encodes alpha-ketoglutarate-dependent dioxygenase, which regulates food intake and energy expenditure  and how they were handled appropriately.1) The protocol of this review has not been pre-registered for this , which may introduce potential bias per Cochrane guidelines.2) There have been no comprehensive studies or genome-wide studies conducted on the association of MetS in any Arab population. Additionally, several studies have been conducted to examine some individual components of MetS rather than MetS as a whole. We recommend establishing a regional consortium that can address this problem and unify the definition of MetS, the study settings, and the analysis plans.3) There was an underrepresentation of studies from the low- and middle-income regions of the Arab world, especially those that are subject to war and political instability .4) There were several studies with poor design or analysis of the results. In addition, several studies were excluded due to a lack of results detail, such as a lack of odds ratios or confidence intervals. Therefore, we recommend that MetS researchers in the region carefully design their studies, consider potential bias sources, and report results in detail.PPAR\u03b32 gene or rs2228570 in VDR gene), making it difficult to conclude these studies. Therefore, we recommend using common gene or SNP markers nomenclature systems, such as the HUGO Gene Nomenclature for genes and the bSNP Reference SNP (rs or RefSNP) number for genetic markers.5) In this systematic review, a significant concern was using different names for the same marker  Lastly, as reported in Beshyah and colleagues in 2018 , we obseWe summarized the current literature on the genetic associations of MetS in Arab populations in this review. We found that three markers, FTO-rs9939609, LEP-rs7799039, and SERPINA12-rs2236242, contributed significantly to the pooled risk for MetS development. As a result of considerable variations in study settings and numerous sources of heterogeneity, most of these studies are rated as of low to moderate quality."}
+{"text": "Our results showed that Sal B significantly ameliorated hyperlipidemia, hyperglycemia, hyperinsulinemia, and insulin resistance in db/db mice. Furthermore, it significantly alleviated diabetes-induced vascular endothelial dysfunction according to histopathology analysis. In diabetic thoracic aorta, HG- and CCCP-induced HUVECs, Sal B distinctly increased Bcl-2 expression and reduced BAX, Beclin1, Parkin and Pink1 expression, thereby protecting endothelial cells from apoptosis and mitophagy. Moreover, Sal B markedly enhanced migration, mitochondrial activity and intracellular Ca2+ levels both in HG- and CCCP-induced HUVECs. Collectively, Sal B exhibited a potential to improve diabetes-induced endothelial and mitochondrial dysfunction through down-regulating apoptosis and mitophagy of endothelial cells.Endothelial dysfunction is a critical mediator in the pathogenesis of vascular complications of diabetes. Herein, this study was conducted to investigate the therapeutic effects of Salvianolic acid B  on diabetes-induced endothelial dysfunction and the underlying mechanisms. Diabetic models were established both in db/db mice and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs). Moreover, HUVECs were exposed to carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to induce endothelial cell damage. Following Sal B treatment, pathological changes of thoracic aorta were investigated by hematoxylin and eosin, alcian blue (AB), elastic fiber, Masson, and reticular fiber staining. BCL2-associated X (BAX), B-cell lymphoma-2 (Bcl-2), Beclin1, Parkin and PTEN Induced Kinase 1 (Pink1) expression was detected by Western blot, immunohistochemistry, and immunofluorescence in thoracic aorta, HG- and CCCP-induced HUVECs. Cell scratch test, MitoTracker Red CMXRos staining and Flou-4 AM staining were separately presented to detect migration, mitochondrial activity and intracellular CaAbbreviations: DM: diabetes mellitus; T2DM: type 2 diabetes mellitus; Sal B: Salvianolic acid B; HG: high glucose; FBG: fasting blood glucose; TC: total cholesterol; TG: triglycerides; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; FINS: fasting insulin; HOMA-IR: homeostasis model assessment insulin resistance; QUICKI: quantitative insulin-sensitivity check index; H&E: hematoxylin and eosin; HUVECs: human umbilical vein endothelial cells; IHC: immunohistochemistry; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; FCM: flow cytometry; CCK-8: cell counting kit-8 Diabetes mellitus (DM) is gradually becoming a global public health concern . Type 2 In diabetic patients, endothelial cells exhibit mitochondrial damage, mitochondrial dysfunction, and reduced oxidative ability . MitochoSalvianolic acid B  is a natural bioactive antioxidant derived from Radix Salvia miltiorrhiza . It has http://www.cavens.com.cn/). These animals were raised at 22\u00a0\u00b1\u00a02\u00b0C in an atmosphere of 60\u00a0\u00b1\u00a05% relative humidity and a 12\u00a0h light/dark cycle. The animal experimental procedures strictly followed the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. This project gained the approval of the Animal Care and Use Committee of the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine (GK2019010). Diabetes was confirmed if fasting blood glucose level >16.7 mM. The mice were randomly separately into three groups (15 mice/group): control group (db/m mice); model group (db/db mice) and Sal B group (db/db mice). All mice were fed a commercial diet and allowed free access to water and food. Sal B was purchased from Med Chem Express Company , with a purity 99.93% by high performance liquid chromatography (HPLC). For the Sal B group, db/db mice were orally treated with 50 mg/kg Sal B each day for 6 weeks. The dose of Sal B was determined according to a published study [7-week-old male diabetic C57BL/KsJ db/db mice (35\u201340\u00a0g) and age-matched non-diabetic C57BL/KsJ db/m mice with 7-week-old and 16\u201318\u00a0g were purchased from Changzhou cavens experimental animal Co., Ltd  was tested through Glucose Assay Kit . Then, whole blood was harvested and serum was isolated for biochemical measurement. Through automatic biochemical analyzer, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were detected in serum specimens. Fasting insulin (FINS) was examined Insulin Assay Kit . Homeostasis model assessment insulin resistance (HOMA-IR) was calculated following the formula: HOMA-IR\u00a0=\u00a0FBG (mmol/L) \u00d7 FINS (mIU/L)/22.5 .Thoracic aorta tissues were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 5\u00a0\u00b5m. The sections were deparaffinized to water, followed by being stained with hematoxylin and eosin , alcian blue , elastic fiber , Masson  as well as reticular fiber . For H&E staining, the sections were stained by hematoxylin lasting 5 min and differentiated by hydrochloric acid ethanol at room temperature for 3 s. After returning to blue by rinsing with tap water for 30\u00a0min, the sections were counter-stained with eosin staining solution for 2\u00a0min. For AB staining, the slices were stained by alcian blue lasting 20\u00a0min. Afterward, the sections were oxidized with 0.5% periodic acid aqueous solution for 10\u00a0min, and stained with Schiff reagent for 15\u00a0min. For elastic fiber staining, the sections were incubated with Lugol iodine solution lasting 5 min and treated by sodium thiosulfate lasting 5\u00a0min. Subsequently, the sections were stained with aldehyde fuchsin staining solution for 10\u00a0min. For Masson staining, the sections were stained with Weigert\u2019s iron hematoxylin for 5\u00a0min. After a few seconds of differentiation with 1% hydrochloric acid ethanol, the sections were washed with tap water for 30\u00a0min and turned blue. The slices were stained by ponceau acid fuchsin solution lasting 10\u00a0min. Following being treated by phosphomolybdic acid aqueous solution lasting 5\u00a0min, the slices were counterstained by aniline blue solution lasting 5\u00a0min as well as treated by 1% glacial acetic acid for 1\u00a0min. For reticular fiber staining, the sections were oxidized with 1% potassium permanganate lasting 5\u00a0min as well as treated by 2% oxalic acid lasting 2\u00a0min and 1% uranium nitrate for 10\u00a0sec, followed by being immersed in ammonia silver solution for 1\u00a0min. After washing with 95% alcohol, the sections were developed in the developer for 1\u00a0min. Subsequently, the sections were tinted with 0.2% gold chloride for 1\u00a0min and treated with 5% sodium sulfite for 1\u00a0min. After dehydration with gradient ethanol, the sections were mounted with transparent xylene, followed by neutral gum. Finally, the sections were investigated under a light microscope .2 at 37\u00b0C. Diabetes cell model was constructed by 30\u00a0mM high-glucose (HG)-induced HUVECs. 0.1% DMSO was utilized for improving the solubility and bioavailability of Sal B. HUVECs were treated with 0, 5, 10, 30, 50, 80 and 100\u00a0\u03bcM Sal B for 48\u00a0h in HG-pre-treated HUVECs. Furthermore, HUVECs were treated with 50\u00a0nM oxidative phosphorylation uncoupler carbonyl cyanide m-chlorophenyl hydrazone  that was dissolved by 0.1% DMSO.Human umbilical vein endothelial cells  were grown in Dulbecco\u2019s modified Eagle\u2019s medium  plus 10% fetal bovine serum (FBS), 100\u00a0U/ml penicillin and 100\u00a0U/ml streptomycin. HUVECs were grown in an environment of 5% COThoracic aorta tissues or HUVECs were lysed through lysis buffer (20\u00a0mM Tris-hydrochloric acid (pH\u00a0=\u00a07.5), 137\u00a0mM sodium chloride, 0.5% ethyl phenyl polyethylene glycol, 0.5\u00a0mM dithiothreitol, complete protease inhibitor cocktail, and phosphatase inhibitor). Bradford protein concentration determination kits were applied for determining the concentration of total protein. The protein was separated by 10% SDS-PAGE and transferred to PVDF membrane . The membrane was incubated with primary antibody , anti-B-cell lymphoma-2 , anti-Beclin1 , anti-Parkin , anti-PTEN Induced Kinase 1  and anti-\u03b2-actin ) overnight at 4\u00b0C. Afterward, the membrane was incubated by horseradish peroxidase-conjugated secondary antibody  lasting 1\u00a0h at room temperature. The protein band was developed with enhanced chemiluminescence kits , followed by quantification through Image-Pro Plus 6.0 software .5 cells/well), followed by glass coverslips for 24\u00a0h. Thoracic aorta tissues or HUVECs were fixed with 4% paraformaldehyde for 40\u00a0min on the ice. Afterward, thoracic aorta tissues or HUVECs were permeabilized by 0.1% Triton X-100 for 20\u00a0min and blocked by 5% BSA for 30\u00a0min. For immunohistochemistry, the sections were incubated with primary antibody lasting 90\u00a0min at 37\u00b0C. Primary antibodies included anti-BAX , anti-Bcl-2 , anti-Beclin1 , anti-Parkin  and anti-Pink1 . Then, the sections were incubated by secondary antibodies  for 30\u00a0min. The sections were stained by diaminobenzidine solution in the dark for 15\u00a0min, followed by being counterstained by hematoxylin for 10\u00a0min. For immunofluorescence, the sections were incubated by primary antibody , anti-Bcl-2 , anti-Beclin1 , anti-Parkin  and anti-Pink1 ) overnight at 4\u00b0C. Then, the sections were incubated by Alexa Fluor\u00ae 488 Conjugate  or Alexa Fluor\u00ae 594 Conjugate  secondary antibodies for 1\u00a0h at 37\u00b0C, followed by being stained with DAPI . Images were acquired utilizing a light microscope  or a fluorescence microscope .HUVECs were planted onto a 6-well plate . HUVECs were digested by trypsin and centrifugated for 10\u00a0min at 4\u00b0C. In the dark at 4\u00b0C, the cell suspension was stained by 5\u00a0\u00b5l Annexin V-FITC lasting 15\u00a0min as well as 10\u00a0\u00b5l PI lasting 5\u00a0min. Afterward, HUVECs were instantly detected through flow cytometer .3 cells/well). Then, 10\u00a0\u03bcl CCK-8 solution was added to each well. HUVECs continued to incubate lasting 4 h at 37\u00b0C. Each test was set to 3 duplicate holes and repeated 3 times. The absorbance values at 450\u00a0nm were determined with a microplate reader.CCK-8  was applied to detect proliferation of HUVECs. HUVECs were added to a 96-well plate (2\u00a0\u00d7\u00a0106 cells/well). After 24\u00a0h, HUVECs were grown to complete confluence and scratched by 10\u00a0\u00b5l pipette tips. Afterward, HUVECs were cultured by serum-free medium for 24\u00a0h. Images were acquired under a light microscope at 0 and 24\u00a0h. Cell scratch was assessed through measurement of the wound distance utilizing Image-Pro Plus 6.0 software.HUVECs were seeded onto a 6-well plate . Mitochondrial fluorescence intensity was quantified utilizing Image-Pro Plus 6.0 software.2+ levels. HUVECs were seeded onto a 6-well plate. In the dark, HUVECs were incubated by 2\u00a0\u03bcmol/L Fluo-4 AM for 30\u00a0min. Being washed twice with PBS, results were acquired under a fluorescence microscope . The fluorescence intensity was quantified with Image-Pro Plus 6.0 software.Fluo-4 AM fluorescent probe  was utilized to detect intracellular CaData are displayed as the mean \u00b1 standard deviation of \u22653 independent assays. Statistical analysis was presented with GraphPad Prism software (version 8.0.1). One-way analysis of variance followed by Tukey's test was applied for multi-group comparison. P <\u00a00.05 indicated a statistical significance.This study aimed to investigate the therapeutic effects of Sal B on diabetes-induced vascular endothelial dysfunction both in db/db mice and HG- and CCCP-induced HUVECs, and explored the underlying molecular mechanisms.Here, db/db mice were used for establishing T2DM model. Following treatment of 50 mg/kg Sal B for 6 weeks, we measured serum lipid profiles including TC, TG, LDL-C and HDL-C by biochemical analysis. Our results showed that db/db mice displayed significantly increased serum TC, TG and LDL-C levels as well as reduced serum HDL-C levels than control mice . HoweverH&E staining showed that compared with the control group, the arterial wall was significantly thickened, the arterial media and adventitia space was widened, and infiltrating cells displayed significant increase in the model group ). As shoThe effects of Sal B on apoptosis were observed in thoracic aorta of db/db mice through detection of apoptosis markers BAX and Bcl-2. Western blot showed that db/db mice presented significantly elevated expression of BAX as well as significantly reduced expression of Bcl-2 in thoracic aorta . HoweverWe further observed the effects of Sal B on HG-induced HUVECs. By FCM and CCK-8, 20\u00a0\u03bcM was identified as the optimal dose of Sal B in HUVECs . We obse2+ levels. Both in HG- and CCCP-induced HUVECs, intracellular Ca2+ levels were markedly weakened compared to controls . Sal The role of Sal B on apoptosis and mitophagy was further evaluated in CCCP-induced HUVECs. As shown in Western blot results, BAX presented increased expression and Bcl-2 exhibited reduced expression in CCCP-induced HUVECs than controls \u2013c). NeveDiabetes may increase the risk of development of cardiovascular diseases . ChronicThis study established diabetic db/db mice characterized by hyperlipidemia, hyperglycemia, hyperinsulinemia, and insulin resistance. Treatment of 50 mg/kg Sal B for 6 weeks significantly ameliorated hyperlipidemia implied by reduced serum TC, TG and LDL-C levels and increased serum HDL-C levels, hyperglycemia implied by decreased FBG levels, hyperinsulinemia implied by lowered FINS levels, and insulin resistance implied by reduced HOMA-IR and QUICKI values. It has been found that Sal B may ameliorate glucose metabolism and insulin sensitivity in cirrhotic rats . HyperglHG is the main link between diabetes and relevant vascular complications . Thus, w2+ levels both in HG- and CCCP-induced HUVECs. Furthermore, Sal B treatment enhanced migration and mitochondrial activity in HG- and CCCP-induced HUVECs. Taken together, our findings revealed that Sal B treatment may protect against diabetes-induced endothelial and mitochondrial dysfunction.By treating the cells with the uncoupling agent CCCP, mitochondria can be depolarized to simulate the mitochondrial damage state under experimental conditions ,36. CCCP2+ levels both in HG- and CCCP-induced HUVECs. Hence, Sal B possessed the potential as a promising therapeutic agent against diabetic endothelial and mitochondrial dysfunction, which was involved in the down-regulation of apoptosis and mitophagy of endothelial cells.Collectively, this study demonstrated that Sal B protected against hyperglycemia-induced endothelial dysfunction. In thoracic aorta of db/db mice, HG- and CCCP-induced HUVECs, Sal B alleviated apoptosis and mitophagy of endothelial cells. Moreover, Sal B markedly improved migration, mitochondrial activity and intracellular Ca"}
+{"text": "This study aims to develop a risk model to predict esophageal fistula in esophageal cancer (EC) patients by learning from both clinical data and computerized tomography (CT) radiomic features.In this retrospective study, computerized tomography (CT) images and clinical data of 186 esophageal fistula patients and 372 controls  were collected. All patients had esophageal cancer and did not receive esophageal surgery. 70% patients were assigned into training set randomly and 30% into validation set. We firstly use a novel attentional convolutional neural network for radiographic descriptor extraction from nine views of planes of contextual CT, segmented tumor and neighboring structures. Then clinical factors including general, diagnostic, pathologic, therapeutic and hematological parameters are fed into neural network for high-level latent representation. The radiographic descriptors and latent clinical factor representations are finally associated by a fully connected layer for patient level risk prediction using SoftMax classifier.512 deep radiographic features and 32 clinical features were extracted. The integrative deep learning model achieved C-index of 0.901, sensitivity of 0.835, and specificity of 0.918 on validation set with superior performance than non-integrative model using CT imaging alone (C-index = 0.857) or clinical data alone (C-index = 0.780).The integration of radiomic descriptors from CT and clinical data significantly improved the esophageal fistula prediction. We suggest that this model has the potential to support individualized stratification and treatment planning for EC patients. EC is the 8th most common tumor worldwide , and neaPrevious studies on esophageal fistula mostly focused on clinical parameters, using the logistics regression analysis to establish predictive models \u20137. Such Deep learning methods can identify non-linear relationships between different types of parameters, and have been explored in large data analysis  and mediIn this study, we developed a deep learning model of esophageal fistula for EC patients. Our model automatically extracted the information in the CT imaging and integrated the clinical features. In addition, the attention map was drawn to visualize the neural network based on CT images.This retrospective study was approved by the local review board. For this type of study, formal informed consent was not required, and all collected data was kept confidential and anonymous. EC patients who developed esophageal fistula in Shandong Cancer Hospital from July 2014 to August 2019 were retrospectively enrolled as the case group. Patients who were clearly described with esophageal fistula or perforation in CT, esophagogram or endoscopy systems were collected. Because anastomotic fistula is a special type of esophageal fistula closely related to surgical methods and surgical techniques, our study did not involve anastomotic fistula after esophagus surgery. We only study the esophageal fistula caused by tumor itself and treatment. The inclusion criteria included: 1) patients diagnosed as EC pathologically with the World Health Organization (WHO) criteria; 2) availability of general, diagnostic and therapeutic data; 3) availability of contrast-enhanced CT imaging before treatment; 4) diagnosed as esophageal fistula by either endoscopy, CT or contrast radiography of the upper gastrointestinal tract. Exclusion criteria were: 1) patients treated by esophageal surgery; 2) the fistula developed due to medical injure or trauma; 3) concomitant with another carcinoma. By such, there are 186 eligible patients. At the same time, we collected a control group of 372 patients, 1:2 matched with the case group by the diagnosis time of EC, sex, marriage, and race. Patients in the control group followed the same inclusion and exclusion criteria as above but didn\u2019t develop esophageal fistula. The included patients were divided into training set (n = 390) and validation set (n = 168) randomly. Specifically, We applied the method of simple randomization to separate the whole dataset into training and validation sets using random numbers generated by the computer.We collected data from medical records using a standardized questionnaire about general, diagnostic, therapeutic and esophageal fistula data. Specifically, general parameters include gender, age at initial diagnosis, Eastern Cooperative Oncology Group performance status (ECOG PS) score, Body Mass Index (BMI), history of smoking, history of drinking, history of hypertension, history of diabetes, history of coronary heart disease and eating obstruction. Diagnostic parameters include tumor stage (T4), node stage (N2-3), stage, tumor site, longitudinal length of lesions, pathological and general type. Therapeutic parameters consist of chemotherapy, radiotherapy, target therapy and serum albumin and cholesterol. Esophageal fistula parameters include fistula type and therapy of fistula. The details are given in All patients underwent esophagoscopy, esophagogram and contrast-enhanced CT scan of neck, chest, and abdomen before treatment. We collected pre-treatment CT imaging and diagnostic CT of esophageal fistula. Intravenous contrast enhancement was used for all patients. The CT-scans were acquired by SOMATOM Definition AS  using a tube voltage of 120 kVp, a tube current of 200 mAs, a detector of 64\u00d70.625 mm and a beam pitch of 1.5. Esophageal tumor boundaries on all 558 pre-treatment CT imaging were manually delineated with reference to esophagoscopy, barium meal or PET-CT in mediastinal window twice using 3D-Slicer by two experienced radiologists separately to reduce the deviation. For patients with satellite tumors, only the primary tumor or the tumor that caused esophageal fistula was appreciated.3. A 200\u00d7200\u00d7200 mm3 cube was defined as located at the center of manually segmented tumor volume. We used its transverse, sagittal, coronal and six diagonal planes as nine views . The inputs of the network were nine views of panels where there are patches of contextual CT, segmented tumor and neighboring structures in each view. To extract nine views and patches, CT images were firstly resampled to a voxel size of 1\u00d71\u00d71 mmne views Figure\u00a01Clinical records were fed into a neural network for high-level representation extraction. Finally, the radiographic features and clinal factor representation are associated with a fully connected layer for patient-level risk prediction using SoftMax classifier.The performance of the proposed risk prediction model was validated by comparing it with the risk prediction model using CT images alone and clinical records data alone.sensitivity = TP/ (TP+FN), specificity = TN/ (TN+FP).Evaluation measures included C-index, sensitivity, and specificity. Given true positive (TP), false negative (FN), true negative (TN), and false positive (FP) numbers, sensitivity and specificity are obtained as 691 patients developed esophageal fistula during the study period. 413 had complete pre-treatment CT imaging. All perforations were developed at the location where the tumor invaded the esophagus. After excluding 227 patients with postoperative anastomotic fistulas who had surgical operations, 186 patients were finally enrolled in the case group. 372 controls never received esophageal operation matching the case cases. The detailed workflow is given in Among all 558 eligible patients, 468 (83.9%) are male and 90 (16.1%) are female. The median age is 61 (range 41-85) in the case group and 64 (range 37-89) in the control group separately. Patients with squamous carcinoma predominated account for 93.2% where most of them had stage III EC (52.2%) with T3 (63.6%) or N1 (40.1%) disease. Before developing perforation, the proportions of patients who received chemotherapy or radiotherapy were 71.5% and 54.3% respectively, while 45.7% of patients received both of them, and 12.4% received concurrent chemoradiotherapy. Besides, 37 (19.9%) patients developed esophageal fistula before treatment. The median interval time from baseline CT to the diagnosis of esophageal fistula was 5 days (3-9 days). The interval time between the development of esophageal fistula and the diagnosis of esophageal cancer ranged from 3 to 1401 days with a median value of 72 days. The median survival time after esophageal fistula is 2.9 months.In the case group, 90 patients (48.4%) had fistula formation to the trachea or bronchus, 91 patients (48.9%) had fistula formation to the mediastinum, and two patients (1.1%) and one patient (0.5%) had fistula formation to the pleural cavity and the arteria, respectively. Two patients developed two kinds of fistula simultaneously. After the development of fistula, most patients received nutritional support. Meanwhile, some of the patients accepted nutrient canal (34.9%), esophageal stent (31.7%), gastrostomy (7.5%), and radical resection (0.5%). Conservative treatment represents only intravenous nutrition, without nutrition tubes or gastrostomy. Of all 558 patients, no patient was placed with stent before treatment or received intraluminal radiotherapy. The esophageal fistula characteristics are listed in In univariate logistic regression analysis, there are significant differences between patients with and without fistula in age, ECOG PS score, serum albumin, T4 stage, N stage, stage, longitudinal length of lesions, general type, and treatment-related parameters. All significant factors were further included in the multiple regression analysis. Age, ECOG PS score, serum albumin, T4 stage, N stage, general type, chemotherapy, total dose of radiotherapy, and radiotherapy range (metastatic lymph nodes) are independent risk factors for esophageal fistula. The detailed results are shown in The detailed architecture of AMM-CNN is given in To improve the learning effectiveness, data augmentation was performed, including pixel shifting and rotation for the training set. As there were imbalanced positive and negative cases, shifting operations of -10, -5, 0, + 5, +10 pixels along the x and y-axis and rotations of -10, +10 degrees were performed for positive cases, resulting in 9750 positive samples. For negative training cases, 9360 negative samples were obtained after shifting operations of -5, 0, + 5, +10 along x and -5, 0, + 5 along y-axis, and rotations of -10, +10 degrees.Combining clinical features and CT imaging, deep learning achieved a C-index of 0.921 in the internal validation and 0.901 in the external validation, which outperformed CT imaging alone  and clinical data alone . The sensitivity was 0.835, and specificity was 0.918. The integrative model produced higher predictive performance than models using single modality data. For the clinical characteristics, the C-index obtained by deep learning is 0.780, which is better than the traditional logistics regression model .To study the interpretability of the model, we draw the attention map to explain the focus of the neural network on CT images. As shown in Esophageal fistula is a fatal complication of EC. Therefore, a risk prediction model integrating CT imaging and clinical features is worth investigation. In this study, we used the deep learning method to comprehensively analyze the influence of various parameters on the esophageal fistula, including clinical parameters such as stage, treatment, and CT imaging. Deep learning models can directly learn patient characteristics from raw data or imaging without feature selection or design . Therefovs 0.857, 0.780). Because deep learning algorithms can integrate clinical parameters and CT images well. Deep learning is very suitable for the analysis of multi-domain parameters, such as the fusion of histopathological images and genomic data , the Innovation Project of Shandong Academy of Medical Sciences (2019-04), and the Academic Promotion Program of Shandong First Medical University (2019ZL002).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": "Small extracellular vesicles (sEVs) with genetic information secreted by cells play a crucial role in the cellular microenvironment. In this study, our purpose is to explore the characteristics of the small extracellular vesicles of human adipose-derived mesenchymal stromal cells (hADMSC-sEVs) and studied the role of hADMSC-sEVs in improving the survival rate of grafted fat.In the present study, we used the transmission electron microscopy, nano-tracking analysis, nanoflow surface protein analysis, and zeta potential value to identify sEVs. SEVs\u2019 trajectory was traced dynamically to verify whether hADMSC-sEVs can be internalized into human umbilical vein endothelial cells (HUVECs) in vitro at different times. The angiogenic property of hADMSC-sEVs was observed by measuring the volume, weight, and histological analysis of the grafted fats in nude mouse models.Our research showed that the hADMSC-sEVs were sEVs with double-layer membrane structure and the diameter of which is within 30\u2013150\u2009nm. hADMSC-sEVs exert biological influence mainly through internalization into cells. Compared with the control group, the hADMSC-sEVs group had a significantly higher survival rate of grafted fat, morphological integrity, and a lower degree of inflammation and fibrosis. And immunohistochemistry showed that hADMSC-sEVs significantly increased the neovascularisation and the expression of CD34, VEGFR2, and Ki-67 in the graft tissue.As a potential nanomaterial, hADMSC-sEVs have been explored in the field of cell-free application of stem cell technology. hADMSC-sEVs promoted the survival of grafted fats by promoting the formation of new blood vessels, which is another promising progress in the field of regenerative medicine. We believe that hADMSC-sEVs will have a broad application prospect in the field of regenerative medicine in the future.The online version contains supplementary material available at 10.1186/s13287-021-02319-4. Small extracellular vesicles (sEVs) are 30\u2013150\u2009nm membranous vesicles actively released by cells . In partIn clinical, autologous fat grafting is playing an increasingly important role in the esthetic and reconstructive field for its extensive utility for soft tissue rejuvenation, volume augmentation, and body contouring. In contrast, the unpredictable fat resorption and low survival rates limit its further application and development , 10. MouIn this context, we extracted sEVs from the fourth passage of hADMSCs and identified the characteristics of hADMSCs and hADMSC-sEVs. Then, we explore the mechanism of hADMSC-sEVs\u2019 exerting influence in this biological progress. We chose a nude mice fat grafting model to identify whether hADMSC-sEVs could potentially promote angiogenesis after fat grafting and studied the underlying mechanism of hADMSC-sEVs\u2019 effect in improving the retention of fat graft.All animal protocols were implemented under the Animal Ethical Committee of Fujian Medical University\u2019s supervision and approval. Eighteen male nude mice (6\u2009weeks of age) were raised in the Experimental Animal Center of Fujian Medical University. Animals were kept in cages individually after grafting and maintained under ambient temperature.2. The seeding density of cells was 5\u2009\u00d7\u2009105/25\u2009cm2. The fourth passage of hADMSCs, cultivated until around 80% confluency, was used in future experiments. hADMSCs from different donors were cultivated or kept separately. The collected hADMSCs were observed by inverted microscope  and characterized by osteogenic and adipogenic induction and flow cytometry .We obtained lipoaspirates from three healthy female patients who underwent liposuction of the thigh at the Department of Plastic Surgery, Fujian Medical University Union Hospital. The donors ranged in age between 25 and 45\u2009years. All participants signed the informed consent. This study was approved by the Ethics Committee of Union hospital of Fujian Medicine University and performed following the principles described in the Declaration of Helsinki. hADMSCs were isolated as previously described . BrieflyWe chose the fourth-passage hADMSCs, which were in the state of logarithmic growth. When the cells grow to more than 80% confluence, we added adipogenesis induction media  and changed the medium every 3\u2009days. Oil Red Assay kit  was for lipid droplets staining according to the manufacturer\u2019s specifications, and the results were observed under a microscope after 2\u2009weeks.Similarly, we used the fourth-passage hADMSCs which proliferated in logarithmically and added osteogenic induction media  after the cells grew to more than 80% confluence. Accordingly, we changed the medium every 3\u2009days and used an alkaline phosphatase calcium cobalt staining kit  for staining cells after 3\u2009weeks according to the manufacturer\u2019s instructions, and the results were observed under the microscope.The fourth passage of hADMSCs was selected for flow cytometry analysis for phenotypic identification of mesenchymal stromal cells. CD29, CD90, CD31, and CD45 along with related isotype controls  were used for hADMSCs\u2019 immunofluorescence staining. Flow cytometry was performed by using the BD Accuri C6 System .g for 10\u2009min, 2000\u00d7g for 10\u2009min, and 10,000\u00d7g for 30\u2009min). The supernatant removed the sediment was then filtered through a 0.22-\u03bcm filter  to remove the large extracellular vesicles further and ultracentrifuged at 100,000\u00d7g for 70\u2009min by using the High-Speed Refrigerated Centrifuge . The supernatant was discarded, and the precipitation was resuspended with PBS. Finally, the suspension was ultracentrifuged at 100,000\u00d7g for 70\u2009min again, and sEVs were obtained after precipitation collection. The obtained sEVs concentration was measured with bicinchoninic acid (BCA) protein detection kit  and stored at \u2212\u200980\u2009\u00b0C for further use. All centrifugations are operated at 4\u2009\u00b0C.hADMSC-sEVs were collected and purified according to the following processes. We selected the fourth-passage hADMSCs to extract sEVs. After cells\u2019 confluency reaching 70\u201380%, hADMSCs\u2019 culture medium was replaced with serum-free low glucose DMEM for 48\u2009h to collect cells\u2019 supernatant. To isolate and remove cell particles, dead cells, and cell debris of the obtained supernatant, we performed a series of differential centrifugal precipitation  . Compared with polystyrene beads (RI\u2009=\u20091.59), in the Nano-FCM system, monodisperse silica nanoparticles (RI\u2009=\u20091.46) are employed as the reference to calibrate the size of EVs. In the nanoFCM system, the detection efficiency is 100%. Particle concentration can be determined via single-particle enumeration, which defines the particle concentration of the number of particles collected in a given period. Finally, the size, distribution, and total concentration of EVs were calculated by NTA software.Zeta potentials of sEVs were measured three times using a Nano laser particle size analyzer . Data were collected and analyzed using Anton Paar Kalliope software.EVs were imaged by transmission electron microscopy (TEM) to verify their morphology. The sample with a volume of 5\u2009\u03bcl (366\u2009\u03bcg/ml) was prepared and dropped on the sealing film. Covered with a copper mesh and stood for 20\u2009min so that the copper mesh fully absorbed sEVs. The copper mesh with sEVs adsorbed was transferred to 4% paraformaldehyde for fixation for 5\u2009min. Then using 50\u2009\u03bcl of 2% uranyl acetate to stain negatively with copper mesh for 5\u2009min, and then copper mesh was dried at room temperature for 30\u2009min. Finally, using FEI transmission electron microscopy  for imaging at 100\u2009kV.Take 20\u2009\u03bcl sEVs samples and freeze-dry them in a freeze dryer for 16\u2009h. Then, the lyophilized sEVs powder was evenly dispersed on the conductive tape of the sample holder, and the sample holder was placed in the gold evaporation chamber for ion sputtering gold plating. Finally, the shape and quantity of sEVs were observed under a high-low vacuum scanning electron microscope (SEM) , and the images were taken and recorded.g ultracentrifuging for 70\u2009min. Carefully remove the supernatant, resuspend it in 50\u2009\u03bcL of pre-cooled PBS, and detect the protein index results with a NanoFCM instrument .Taking 30\u2009\u03bcL of sEVs diluent and adding FITC Mouse Anti-Human CD9, CD81  and FITC Mouse IgG  to mix, then incubating at 37\u2009\u00b0C for 30\u2009min in the dark, adding 1\u2009ml of pre-cooled PBS, 110,000\u00d7g for 1\u2009h at 4\u00b0. HUVECs  and hADMSC-sEVs labeled with PKH67 were incubated in 37\u2009\u00b0C with serum-free medium for 6\u2009h and 12\u2009h. Meanwhile, HUVECs and PKH67 dye were incubated in 37\u2009\u00b0C for 6\u2009h and 12\u2009h as control. After fixation with 4% paraformaldehyde (PFA) for 30\u2009min and staining with 4, 6-diamino-2-phenylindoles , the samples were observed under a fluorescence microscope .The hADMSC-sEVs were labeled with a PKH67 dye  for 4\u2009min. Then, Bovine Serum Albumin (BSA) was used to terminate staining. The excess dye after labeling was removed by ultracentrifugation at 100,000The grafted fat paraffin-embedded sections were stained with hematoxylin, eosin, and immunohistochemistry. The sections were primarily incubated with rabbit anti-human and mouse CD34 , VEGFR2 , and Ki-67  separately, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Finally, the staining color was developed using the DAB Detection Kit . Histologic parameters were examined under a light microscope. HE staining was observed in 5 low magnification field analysis methods for the assessment of fat graft integrity, as evidenced by the presence of intact and nucleated adipocytes and the presence of cysts and vacuoles. Each parameter was graded by two observers independently on a semiquantitative scale ranging from 0 to 5 .10 particles/ml) hADMSC-sEVs solution (hADMSC-sEVs group) or 0.1\u2009ml PBS (control group) was injected subcutaneously into the back of nude mice (the fat particles was from the other patient who had liposuction of thighs). The mice were sacrificed at 1, 2, and 3\u2009months after fat grafting. Grafted fat samples were harvested and measured by the weight, volume, hematoxylin-eosin (HE), and immunochemistry staining. The weights of fat grafts were determined by a balance and the volumes were measured by the liquid overflow method. The analysis of HE and IHC staning has been discussed above.Six-week-old, male nude mice were obtained from the Laboratory Animal Center of Fujian medicine University . The experimental protocol was approved by the Animal Ethical Committee of Fujian Medical University (Permit Number: FJMU IACUC 2019-0131). The mice were randomly assigned to hADMSC-sEVs or control groups (six mice in each group). A mixture of 0.4\u2009ml of fat particles and 0.1\u2009ml . Results were presented as the mean\u2009\u00b1\u2009standard deviation (SD). The two-tailed Student\u2019s Under light microscope, a characteristic morphology of slender spindle-like cells of fourth-passage hADMSCs was observed Fig.\u00a0a. Mature8) to 1\u2009\u00d7\u2009106 cells and the ratio of protein amount (\u03bcg) to 1\u2009\u00d7\u2009106 cells through the measurement of NTA and previous BCA , which was confirmed by the better quantitative results of weight and volume of grafted fat were better in the hADMSC-sEVs group compared to the control group, indicating a protective effect of hADMSC-sEVs on grafted fat survival. For micro-evaluation, we analyzed the HE staining, which revealed that the grafted fat in the hADMSC-sEVs groups exhibited better survival and morphologic integrity compared to the control group, as shown in Fig.\u00a0P\u2009<\u20090.05)  can be obtained by using this device. Studies have shown that compared with the traditional 2D culture, the total amount of sEVs in the 3D culture system has increased 19.4 times , 47. MorIn terms of sEVs extraction, the mainstream sEVs extraction methods mainly include ultracentrifugation (UC), density gradient centrifugation (DGC), exclusion chromatography (SEC), ultrafiltration (UF), Immune capture (IC) and polymer precipitation (Precip) . Among tThe present study had several limitations. First, the downstream molecules in the VEGF/VEGFR2 signaling pathway remain to be defined in our further investigation. Second, the theory of graft retention or endogenous adipose repassage is still being defined. Consequently, further studies are warranted to address this issue.Small extracellular vesicles, as a novel kind of nanoparticle without nuclear structure, do not show apparent side effects, such as immunogenicity or tumorigenicity when applied in animal models. Studies have found that EVs can replicate the function of the cells which they are derived. Our research has proved that hADMSC-sEVs play a considerable role in fat grafting nude mouse model. hADMSC-sEVs can promote neovascularization and increase the retention of grafted fat, whose mechanism may be explained by VEGF/VEGFR2 signal transduction. These findings indicate that hADMSC-sEVs can be regarded as a potential treatment option for fat transplantation. As a new type of nanomaterial, we need further and more in-depth studies to promote hADMSC-sEVs to apply in a broader range of diseases.Additional file 1. Supplemntal material (enlarged figures) hADMSC-sEVs promoted neovascularization in the nude mice fat grafting model (rows meaned different staining and lines represents different groups and months)."}
+{"text": "Syphilis has long been known as \u201cthe great imitator\u201d, mimicking a wide variety of diseases, and often its diagnosis is delayed or missed. It remains an important public health issue that continues to occur at high rates among patients with HIV. We report a case of a 52-year-old man who presented with a constellation of unusual symptoms highlighting that syphilis should be included in the differential diagnosis in patients with HIV presenting with abnormal liver enzymes, rash, proteinuria, conjunctivitis, and/or sexual risk factors. The rate of syphilis continues to rise in the United States, despite effective antitreponemal therapy, essentially due to the increasing incidence of primary and secondary syphilis among men who have sex with men (MSM) . Clinicag benzathine injections x 3 doses. The patient had significant improvement in his symptoms and labs normalized after 1 month. RPR Titer came back at 16 after 3 months.A 52 year-old man (MSM) with a history of Gilbert's disease, HIV for 18 years well controlled on emtricitabine-rilpivirine-tenofovir alafenamide (Odefsey) presented to his primary care physician with a constellation of unusual symptoms of low-grade fevers, fatigue, muscle aches, joint stiffness, bilateral red eyes with tearing, rash, and dark urine for 2 months. The patient stated he initially noticed low-grade fevers followed by fatigue, muscle aches, and joint stiffness that responded to as-needed pain medications. He scheduled an urgent visit when he started noticing bilateral red eyes with tearing but no visual disturbances, a rash that started on his right leg with no associated pain or itching, and dark urine gradually worsening over the last week. The patient had no other significant medical history, alcohol intake, illicit drug use, herbal supplement intake, or family history of autoimmune disorders. The patient worked outside, often had contact with ticks, and frequently picked ticks off his body when he was out in the woods. The patient did not recall having a bull eye rash at the time. The patient was sexually active and did have unprotected sex with a single partner since the last sexually transmitted infections screen 3 months ago. The patient was otherwise healthy. On examination, the patient was hemodynamically stable, afebrile, and appeared well, with a nickel and dime lesion on his right leg . His bloThe incidence of syphilis among patients with HIV, especially MSM, has been rising in the United States, and several outbreaks have been reported . SyphiliTo conclude, this case emphasizes the importance of early recognition of rare presenting characteristics of syphilis and the importance of a high index of suspicion, especially when evaluating proteinuria, glomerulonephritis, hepatitis, rash, or conjunctivitis in patients with HIV."}
+{"text": "In recent years, protecting important objects by simulating animal camouflage has been widely employed in many fields. Therefore, camouflaged object detection (COD) technology has emerged. COD is more difficult to achieve than traditional object detection techniques due to the high degree of fusion of objects camouflaged with the background. In this paper, we strive to more accurately and efficiently identify camouflaged objects. Inspired by the use of magnifiers to search for hidden objects in pictures, we propose a COD network that simulates the observation effect of a magnifier called the MAGnifier Network (MAGNet). Specifically, our MAGNet contains two parallel modules: the ergodic magnification module (EMM) and the attention focus module (AFM). The EMM is designed to mimic the process of a magnifier enlarging an image, and AFM is used to simulate the observation process in which human attention is highly focused on a particular region. The two sets of output camouflaged object maps were merged to simulate the observation of an object by a magnifier. In addition, a weighted key point area perception loss function, which is more applicable to COD, was designed based on two modules to give greater attention to the camouflaged object. Extensive experiments demonstrate that compared with 19 cutting-edge detection models, MAGNet can achieve the best comprehensive effect on eight evaluation metrics in the public COD dataset. Additionally, compared to other COD methods, MAGNet has lower computational complexity and faster segmentation. We also validated the model\u2019s generalization ability on a military camouflaged object dataset constructed in-house. Finally, we experimentally explored some extended applications of COD. In nature, animals evolve according to the principle of survival of the fittest. They may be able to camouflage their shape or retain shape characteristics similar to those of their habitat to avoid being hunted by predators or to ambush prey better . CurrentIn addition to its military application, camouflaged object detection (COD) can be applied in industrial detection , medicaThe task of COD is to detect objects that have similar patterns  to their surroundings. However, studies on COD are lacking. For example, in military fields, military camouflaged objects are often identified by means of infrared-, polarization-, and hyperspectral-based imaging and other technologies ,10,11. Hhttps://github.com/jiangxinhao2020/Magnet_eval (accessed on 1 December 2022)). Now, with the development of deep learning technology, some scholars have applied segmentation to the detection of camouflaged objects, providing new ideas for the detection of camouflaged objects in visible wavelengths [Before the rapid development of deep learning, researchers commonly used traditional digital image processing methods, such as spectral transforms , sparse elengths . HoweverThe originality of this study is that, instead of rigidly solving the problem from the perspective of deep learning, we took our inspiration from life observations and designed a segmentation network suitable for camouflaged objects by simulating the magnifying glass observation effect on a target. This is called the MAGnifier Network (MAGNet). MAGNet differs from other COD methods in that it has a clearer structure and can achieve a better segmentation performance with lower computational complexity. We apply the concept of observation with a magnifier to the COD problem and propose a novel camouflaged object segmentation network called MAGNet with a clear structure. MAGNet can achieve higher segmentation accuracy with lower computational complexity.We design a parallel structure with the ergodic magnification module (EMM) and attention focus module (AFM) to simulate the magnifier functions. We propose a weighted key point area perception loss function to improve the focus of the camouflaged object, thus improving segmentation performance.We perform extensive experiments using public COD benchmark datasets and a camouflaged military object dataset constructed in-house. MAGNet has the best comprehensive effect in eight evaluation metrics in comparison with 19 cutting-edge detection models, and it can enable real-time segmentation. Finally, we experimentally explore several potential applications of camouflaged object segmentation.In summary, the major contributions of this paper are threefold:This paper is organized as follows. Similar previous research is introduced in In recent years, scene understanding technologies for use in autonomous driving , virtualIn contrast to camouflaged objects, salient objects are the most noticeable objects in an image. The research of salient object detection (SOD) can promote image understanding , stereo Because of the similarity between a camouflaged object and the background, the boundary between the foreground and the background is very difficult to distinguish; therefore, the production of a camouflaged object dataset is very time-consuming . CurrentA magnifier can help an observer quickly find a camouflaged object in an image. This is because the magnifying effect of the magnifier makes it easier for the observer to spot the center, key points, and minuscule details of the camouflaged object. Inspired by the magnifier, we applied the magnifier observation effect to the COD problem and designed the EMM and the AFM. The EMM is designed to mimic the process of a magnifier enlarging an image, mainly using the designed central excitation module to excite the center and magnify the receptive field. Additionally, AFM is used to simulate the human visual system, and its channel-spatial attention module can simulate the effect of a human focusing on observing objects in the magnifier\u2019s field of view. Finally, we design a more applicable weighted key point area perception loss function for camouflaged object segmentation, which directs more attention to the camouflaged object in the region by weighting. The network structure of MAGNet is shown in We input a camouflaged object image into this network. MAGNet first extracts multi-scale feature maps through a Res2Net-50 backbone , and ResAs shown in The CEM is used to traverse the feature maps of the different scales of output from the last three layers of the backbone to expand the receptive field and intensify the central point and key points.The MFFM is designed to fully integrate the multi-scale feature maps after the CEM to realize the efficient utilization of high-level and low-level features.We find that when observers use a magnifier to observe an object, they observe the central area of the magnifying glass more carefully than the edge areas. With the human visual receptive field mechanism, an observer is more attracted to the center of an object. Then, we use the magnifier to traverse the whole picture until the center of the magnifier coincides with the center of the object. Several studies  discuss To simulate the visual magnification and central excitation of the magnifier, we design a simple and efficient CEM, as shown in Specifically, the CEM consists of four branches, and the input feature maps are simultaneously fed into all four branches. The four branches first use a 1 \u00d7 1 convolution to change the number of output channels. Then, to achieve efficient multi-scale visual amplification, three of the branches use 3 \u00d7 3, 5 \u00d7 5, and 7 \u00d7 7 DConvs with an expansion factor of 2. After the three sets of output feature maps were connected, a 3 \u00d7 3 convolutional layer was used for fusion between channels. The fourth branch is the residual connection module, which aims to retain part of the original features to reduce the feature loss due to convolution. The two sets of features are connected to obtain a centrally excited feature map. The multi-scale centrally excited feature maps obtained from the last three layers of backbone input to the CEM have the same number of channels to ensure a balanced utilization of information at each scale.The connection of three sets of DConvs can increase the importance of the central features while increasing the receptive field, thus achieving a central excitation of the input. The visualization of the feature map output from the CEM is shown on the right in The function of the MFFM is to fully integrate the feature maps after central excitation of different scales, thereby outputting a camouflaged object map that contains abundant high- and low-level features. The MFFM structure diagram is shown in The front-end fusion method of the module adopts the Hadamard product (\u2299). The Hadamard product calculation method is a pixel-by-pixel multiplication, which can better achieve feature crossover, eliminating the difference between the two groups of features and improving the feature fusion capabilities.The back end of the module is fused by adding the channels, which can fuse the features of each layer to increase the feature dimension but does not increase the internal feature information, making full use of the semantic information of the high-level and low-level features.out. Algorithm 1 is the pseudocode of the MFFM:Algorithm 1: MFFM AlgorithmInput: CEM2, CEM3, CEM4. \u2003\u2003\u2003CEM4_1 = CEM4\u2003\u2003\u2003CEM3_1 = CBR (UP (CEM4))\u2299CEM3\u2003\u2003\u2003CEM3_2 = Concat ))\u2003\u2003\u2003CEM2_1 = CBR (UP (CEM3))\u2299CEM2\u2003\u2003\u2003CEM2_2 = CBR (UP (CEM3_1))\u2299CEM2_1\u2003\u2003\u2003CEM2_3 = Concat ))out = CBR (CEM2_3)\u2003\u2003\u2003FOutput: Fout.The module output map is denoted as FAFM has two steps. First, through upsampling and convolution operations, the three sets of feature maps output by the backbone are processed into feature maps of the same size with the same number of channels. Then, the maps are input into the channel-spatial attention module (CSAM) to simulate the effect of human attention focused on observing objects in the magnifier field of view.Attention mechanisms in deep learning can simulate the human visual attention mechanism, where the goal is to obtain more important information . AttentiAlgorithm 2: CSAM AlgorithmInput: L2, L3, L4.\u2003\u2003\u2003# 1. Feature Maps Concat\u2003\u2003\u2003X_original = Concat\u2003\u2003\u2003# 2. Spatial Attention\u2003\u2003\u2003For i = 2, 3, 4: \u2003\u2003\u2003xsa_i = SAmodule (Li)\u2003\u2003\u2003# 3. Channel Attention\u2003\u2003\u2003xca_i = CAmodule(Li)\u2003\u2003\u2003Xsa = Concat \u2003\u2003\u2003Xsa = Softmax (Xsa)\u2003\u2003\u2003# 4. Fusion Attention Maps\u2003\u2003\u2003Xca = Concat \u2003\u2003\u2003Xout = X_original \u2299 Xca \u2299 XsaOutput: Xout.As illustrated in Feature Maps Concat: Superimposing the feature maps of the same size with the same number of channels in the latter three layers of the backbone after processing can achieve the average utilization of feature maps of each scale and fully fuse the semantic information of high- and low-level features. Therefore, the feature maps of the three different layers were input into the channel attention and spatial attention mechanism branches to generate a channel attention map and a spatial attention map, respectively.Channel Attention: The squeeze-and-excitation (SE) module is the most commonly used method of channel attention [ttention . It can ttention , which cFinally, the new and old feature maps were multiplied pixel by pixel by a Hadamard convolution to generate a channel attention map with embedded location and direction information.Spatial Attention: The spatial attention mechanism is particularly important for finding special targets and can retain important local information. For the input feature map Finally, we connect three sets of spatial attention maps and use softmax to normalize again.Fusion Channel and Spatial Attention Maps: We use the Hadamard product for the fusion of attention maps, that is, the pixel-by-pixel multiplication method, which can better enhance feature information to obtain a more accurate feature map.Finally, the feature maps output by the EMM and AFM are transformed into a single-channel camouflaged object map through an upsampling operation. The two feature maps are fused by pixel-by-pixel addition.The binary cross entropy (BCE) loss function and the intersection over union (IOU) loss function are the most common  functionP is the prediction map, GT is the ground truth map, H and W are the picture length and width, respectively, and h and w are the sizes of the regions around the pixel points in the GT map, and Combining the designed pair of focusing and amplifying modules, we propose a weighted key point area perception loss based on the BCE loss and IOU loss  h and w should not be smaller than the maximum perceptual field of 32 \u00d7 32 for a single pixel; (2) should be as small as possible; and (3) should be set to an odd number. In this section, we selected 23 \u00d7 23, 33 \u00d7 33, and 43 \u00d7 43 for comparison experiments. In h and w are set to 33 \u00d7 33, they are more conducive to efficient training and can achieve the best performance.Therefore, experiments prove that when From the comparison with the latest methods in In addition, we explore other extended applications similar to COD. This paper is dedicated to achieving more accurate detection of camouflaged objects. By simulating the search function of a magnifier, we propose a new network based on the observed effect of a magnifier named MAGNet. We designed two bionic modules that can be processed in parallel and presented a more applicable weighted key-point- area perception loss that allows the network to exploit important information about an object further, and thus, achieving an accurate search for camouflaged objects. The results demonstrate the accuracy advantages of MAGNet for COD through quantitative and qualitative evaluation of challenging public datasets and an in-house-built dataset. MAGNet also offers lower computational complexity and faster segmentation than other COD methods. Additionally, MAGNet has potential value for applications in other fields . In the future, we will continue to explore the accurate recognition of low-detectability objects."}
+{"text": "School dropout is a significant concern universally. This paper investigates the incorporation of spatial dependency in estimating the topographical effect of school dropout rates in India. This study utilizes the secondary data on primary, upper primary, and secondary school dropout rates of the different districts of India available at the Unified District Information System for Education plus (UDISE+) for the year 2020 to contemplate the impact of these dropouts from one region to different regions in molding with promotion rate and repetition rate. The Global Moran\u2019s I, Univariate and Bivariate Local Indicators of Spatial Association, and spatial models are utilized to investigate the geographical variability and to find the possible relationship between dropout rates and the school-level factors at the district level. The outcomes provide clear spatial clustering and precisely highlight the hot zone dropout regions with high repetition and low promotion rates. Based on this study\u2019s results, educational administrators can make evidence-based decisions to reduce dropout rates in hot zones of various regions of India. Furthermore, futuristic studies focusing on linking spatial hot zones with causal factors will add consistent data in assisting policymakers in taking necessary measures to develop a sound education management system. Education is a foundation for human progress toward creating a healthy society. Its effects are significant in the development of individuals and the whole country. India\u2019s school education vision 2030 intends to qualitatively improve the nation\u2019s current educational system and provide high-quality education to all children of the school-attending age group, whose numbers are estimated to climb from 25 crores in 2010 to 30 crores in 2030. Though the school enrolment rates have increased in the past few years, the percentage of students who drops out of school has either remained the same or increased. As per UDISE+ 2020 report, the dropout rate in the secondary level (17%) is still high compared to the primary (1.8%) and upper primary level (1.5%). This percentage of school dropouts adds a quantitative inclusion burden to India\u2019s vision of establishing education goals [The Dissimilarity in the primary education curriculum and school infrastructure has been noted to play a key role in primary education outcomes. As a result, the government executed a new policy named \"universalization of elementary education\" (UEE) in 2001. UEE focuses on three major elements: universalization, which guarantees that all students between the ages of 6 and 14 have direct exposure to a school; enrolment universalization, which guarantees that all students in the aforesaid age are enrolled in school; and retention universalization, which guarantees that students who started primary school progress till they complete the upper primary level .In the continuum of the retention universalization goal, research on factors influencing school dropout generally concentrates on child, family, school and community-related factors \u20138. Only To improve the efficiency of the school management framework, this approach incorporating geospatial technology will assist policy-makers and researchers in better understanding the existing status. Additionally, this would support the Sarva Shiksha Abhiyan and National Education Policy in providing direction for formulating preventive measures to decrease dropouts. The spatial representations of education policies across districts can help to guarantee that every student in India completes their schooling at any cost to enhance their quality of life. Policy-makers and government representatives can also utilize findings from this study to improve the current policies, fostering India\u2019s school education and assisting in developing, testing, and implementing cost-effective strategies in hotspot regions to reduce dropouts in India.https://dashboard.udiseplus.gov.in. The reports have been published under the Department of School Education and Literacy (DoSEL), Ministry of Education, Government of India. This report is based on data that schools with active UDISE+ codes in a reference year voluntarily uploaded using a data collection format (DCF) specifically created for this report. The State/UT government of the school\u2019s location assigns the UDISE+ code for institutions. The District Education Officer (DEO) at the district level ensures that the information entered into the DCFs is accurate. This report offers essential information on several factors, such as the number of schools, teachers, and students who were enrolled, promoted, and dropped out, in terms of counts and percentages. This data source is the input for the analysis [The UDISE+ of India recently released data for the year 2020, accessible on the UDISE+ website analysis .Following the conceptual frameworks of earlier studies \u201314, the https://www.kaggle.com/datasets/raghulgandhi/indian-district-map-726. Initially, the number of districts reported in UDISE+ was 733. However, a few districts in Arunachal Pradesh, Delhi, Karnataka, Manipur, Tamil Nadu, and West Bengal were merged for analysis purposes using their boundary, and the number of districts considered for the analysis was 726 given in The primary researcher downloaded India\u2019s district-level base map from kaggle at Wmn denotes the standardised weight matrix connecting observation m and n; and S0 denotes the total of all geographical weights.Wmn denotes the standardised weight matrix connecting observation m and n; and S0 denotes the sum of all geographical weights.To examine the geographic distribution of dropouts in primary, upper primary, and secondary levels in India, several quartile maps at the district-level were created. To investigate the geographical correlation and grouping of districts, Moran\u2019s I, LISA cluster map, and significance maps were created. To calculate the distance in space between every potential pair of observable units in the dataset. The Queens\u2019 contiguity approach has been used to produce the spatial weight matrix of order one . Since tA positive spatial autocorrelation suggests that spots with identical data points are strongly connected in the area, while a negative spatial autocorrelation shows that strongly connected spots are more distinct. The values of Moran\u2019s I typically range from , with positive measures indicating the geographical grouping of comparable measures and negative measures indicating the spatial grouping of different measures. In the absence of any spatial autocorrelation, a measure of 0 indicates a random geographical distribution. The association of nearby values around a particular geographic region is measured by univariate LISA . It estaIi:The following formula provides the measure Four different types of autocorrelations were identified based on the Moran\u2019s scatter plots and are referred to as:Districts with high measures and identical neighbouring districts are known as \u201chot spots\u201d (High-High).Districts with low measures and identical neighbouring districts are known as \u201ccold spots\u201d (Low-Low).Districts that are high in measures but have low-measure neighbouring districts (High-Low) and districts that are low in measures but have high-measure neighbouring districts (Low-High) are known as \u201cSpatial Outliers\u201d.In the same way, bivariate LISA were also calculated to examine the relationship between the repetition rate and promotion rate of both boys and girls of regions with different dropout rates.LISA cluster and significance map were produced in the Geo-Da by utilizing the LISA tools. The map shows the districts with significant Moran\u2019s I value categorized in terms of spatial autocorrelation, where hotspots are defined by red, coldspots by deep blue, and spatial outliers by light blue and light red. To investigate the possible relationships between the dropouts and predictors, we carried out statistical regressions. We first used the ordinary least square (OLS) model, then we calculated the spatial autocorrelation in the OLS regression residuals to check the spatial heterogeneity caused by spatial dependency. As soon as we determined that the Moran\u2019s I statistic for each of the outcomes was statistically significant, we calculated the spatial lag model (SLM) and spatial error model (SEM) to obtain unbiased measures of the correlations between the predictors and dropout while addressing the geographical heterogeneity that existed in the data. A standard SLM assumes that the data points are spatially dependent and lag to one another in the nearby regions, In contrast, the SEM assumes that the disturbance terms are correlated with nearby geographical units. The best model was then determined by comparing the Akaike Information Criterion (AIC) and Schwartz Criterion (BIC) values, and we observed that SEM provided the better fit for this particular study.The fundamental formula for OLS is as follows:SLM, if the response variable (Y), is correlated to the weighted mean of the observations in its surrounding regions, where \u03c1 is the auto-regressive parameter, thenSEM, if residuals reveal spatial dependency, the subsequent model effectively manages the spatial effect.\u03b6 is the i.i.d. disturbance term. By increasing the relevant likelihood functions, both SEM and SLM are estimated [Here, \u03bb denotes the auto-regressive parameter; stimated . QGIS deThe spatial distribution of districts\u2019 primary, upper primary and secondary dropout rates is depicted in The estimated values of the Moran\u2019s I statistic shows primary, upper primary and secondary dropout rates across the districts have spatial autocorrelation. The LISA cluster map in The estimated values of bivariate LISA shown in The estimated coefficients of OLS regression were 0.103(<0.05) for boys\u2019 promotion rate, -0.113(<0.05) for girls\u2019 promotion rate, 0.604(<0.05) for boys\u2019 dropout rate, 0.418(<0.05) for girls dropout rate were statistically significant to primary dropout. After making spatial modifications using the spatial model, it was observed that the pattern of the relationship between predictors and primary dropouts remained the same. We found SEM  as the best fit since it has the lowest AIC and BIC values when comparing SLM  and OLS .The estimated coefficients of OLS regression were -0.255(0.001) for boys\u2019 promotion rate, 0.245(0.001) for girls\u2019 promotion rate, -0.345(0.001) for boys\u2019 repetition rate, 0.327(0.001) for girls\u2019 repetition rate, 0.229(0.001) for boys dropout rate, 0.785(0.001) for girls dropout rate were statistically significant to upper primary dropout. After making spatial modifications using the spatial model, it was observed that the pattern of the relationship between predictors and upper primary dropouts remained the same. The corresponding AIC and BIC values of OLS , SLM  and SEM  are obtained and SEM is considered to be the best fit.The estimated coefficients of OLS regression were 0.059(0.001) for boys\u2019 promotion rate, -0.075(0.001) for girls\u2019 promotion rate, -0.101(0.001) for boys\u2019 repetition rate, 0.099(0.001) for girls repetition rate, 0.624(0.001) for boys dropout rate, 0.377(0.001) for girls dropout rate were highly significant to secondary dropout. After making spatial modifications using the spatial model, it was observed that the pattern of the relationship between predictors and secondary dropouts remained the same. We found SEM  as the best fit since it has the lowest AIC and BIC values when comparing OLS  and SLM .School graduation denotes promotion from primary to secondary. Hence high promotion rate yields a collective benefit in aligning towards the retention universalization policy of UEE, propelling the whole education system processing towards India\u2019s vision for 2030. Our study, based on geospatial techniques, reveals that School dropouts are one of the essential criteria for increasing a country\u2019s overall school graduation percentage. Hence, dropping out is an issue that has to be solved \u201325. In aThe current study intends to apply the existing method for assessing the geographical neighborhood factors that influence dropout incidence and to evaluate significant findings on these factors in India. We emphasize that spatial statistics can give valuable information for analyzing dropout distribution and point out its importance.With the use of the district-level information from the UDISE+ India of 2020, a LISA cluster map is generated. Based on the thematic maps, we observed that district dropout rates were not simply random. In particular, several districts in Arunachal Pradesh, Assam, Bihar, Gujarat, Jammu & Kashmir, Jharkhand, Madhya Pradesh, Manipur, Meghalaya, Mizoram, Nagaland, Odisha, Tamil Nadu, Tripura, and Uttar Pradesh are considered to be hotspots for dropouts in the primary, upper primary and secondary levels because these clusters of neighborhoods have a low rate of promotion and a high rate of repetition.Further investigation on hotspots reveals a correlation between the dropouts and the promotion rate and repetition rate of boys and girls. Although previous studies have linked child and family factors to a higher risk of dropout , the curDespite being focused on Indian districts, this study\u2019s methodology may also be employed at the block level. Understanding the spatial features and clusters of districts with greater block-level dropout rates is more required than merely identifying the districts with the highest dropout rates. In this regard, geospatial studies can assist in identifying block-level dropout hotspots to help make decisions that possess the capacity in changing district-level dropout trends.In this study, we emphasized the importance of spatial analysis of district-level dropout rates using quartile, univariate-bivariate LISA, and spatial autoregressive models. It is possible to find the district-level variations of dropout rates using quartile maps. Clustered LISA maps displayed districts with comparable high or low dropout rates close to one another. This identification of dropout hotspots or coldspots can increase the effectiveness of contextualized interventions . LISA maAlthough this study contributes some conceptual and methodological insights, it has certain limitations. Firstly, GIS-based research will always have difficulty with data availability, and this study also faced the same. Second, this study analyzed the pattern of district-level dropout rates and their influencing school-level characteristics across districts within a district-level framework. This analysis might be taken further and applied to block levels to determine the intra-district variation in student dropout rates, which could help to determine school-level factors influencing the dropout variation within districts. Third, though this study examined the regions in India with the highest dropout rates, our study only focused on spatial factors and not the causative framework. Fourth, we exclusively considered the characteristics at the school level specific to the promotion-repetition rates and excluded other factors related to child, family and community. This study is the first attempt to use spatial analysis for dropout data at the district level in India, therefore this limitation offers an opportunity for future work to expand the knowledge about how certain factors might be geographically clustered and examine regional patterns.Despite its limitations, this study highlights the conceptual findings to advance our understanding of how dropout rates are influenced by geographical locations and methodological strategies for using GIS tools to investigate educational sector challenges. The inference that dropout rates in India are geographically grouped in specific districts in the north-eastern and central states is thematically supported by the evidence. These factors are not deterministic, although high dropout rates are correlated with low promotion rates and high repetition rates.This study recommends and supports the use of GIS approaches that are suitable for concerns and challenges in education. Such techniques can enhance conceptual knowledge and practical implications by identifying spatially effective remedies. These conceptual and methodological contributions may motivate the researchers to explore the methodology for real-world problems including strategic planning, policy-making, and decision-making in the education sector.Although school enrolment in India has seen a substantial increase over a few years, there is still no decrease in dropout rates. This study provides clear evidence that school dropout is a persisting issue in India affecting educational attainment progress. The results of our research indicate geospatial district-level variations in primary, upper primary, and secondary dropouts.In addition to highlighting the geographical variations in dropout rates, this study examines the association between dropouts and promotion-repetition rates. In particular, it shows a strong negative relationship between promotion rates and school dropouts, and a positive relationship between the repetition rates and the dropouts. Based on the findings, the study recommends that India\u2019s education policy must target the hotspot districts with high dropouts rate.Extending the study to analyze and correlate all causal factors in hot spot districts will throw light on creating navigable pathways for comprehensive intervention strategies. In addition, the maps and tables provided in our study will provide a substantial base for policymakers to initiate conversations based on highlighted hotspots. This kind of GIS-based study can assist the first step in developing a strategy to enhance the nation\u2019s educational infrastructure and, in turn, its economic situation.Though the government has been consistently making efforts to improve the educational standards, the north-eastern and central districts in India need to be in a better position to provide standard education due to the high number of hotspots in this region. In the continuum to this conclusion, this study calls for rigorous preventive measures to reduce dropouts in achieving quality education. with the ultimate goal of achieving global education standards that will enable our future generations to strive and prosper more successfully on the planet.S1 Dataset(ZIP)Click here for additional data file.S1 Appendix(CSV)Click here for additional data file."}
+{"text": "Alopecia is a treatable disorder that usually occurs due to high levels of 5\u2010alpha dihydrotestosterone in hair follicles. To enhance the storage capacity of hair follicles and alleviate the inherent characteristics of dutasteride, 5\u2010alpha\u00a0reductase\u00a0inhibitor, a prolonged\u2010release nanocarrier was synthesised, and its influence on rat abdomen's skin was investigated. Results showed the lower ratio of S/Co (higher ethanol concentration) increased the hydrodynamic nanocarriers' particle size due to thermodynamic disturbance and Ostwald ripening. In contrast, an increase in surfactant through a decrease in interfacial tension resulted in smaller nanocarriers of 32.4\u00a0nm. Moreover, an increase in viscosity had an inverse correlation with the nanoemulsions' particle size. Nanocarriers containing ethanol showed less entrapment efficacy, perhaps due to the rapid dissolution of dutasteride into ethanol during nanoemulsification, while, based on Stokes' equation, the addition of ethanol resulted in smaller particle size and stability of the system. Skin permeation analysis using Franz diffusion cells showed nanocarriers could pass through the skin and release dutasteride for 6\u00a0days. In conclusion, the optimum concentration of ingredients is decisive in guaranteeing the ideal particle size, stability, and skin permeation of nanocarriers. The Present dutasteride nanocarrier would promise a prolonged and sustained\u2010release drug delivery system for Alopecia therapy. Alopecia is a treatable disorder that, over time, gets to be chronically influencing a noteworthy part of the population. To enhance the storage capacity of hair follicles and alleviate the inherent characteristics of dutasteride, a prolonged\u2010release nanocarrier was synthesised that could pass through the skin and release dutasteride for more than six days. The synthesised dutasteride nanocarrier would promise a prolonged and sustained\u2010release drug delivery system for Alopecia therapy. Hair still has this function along with its aesthetic function. Skewering body hair is a remnant of the period when a hairy man pulled his hair together to increase insulation. Research shows that humans lost the hair that covered most of their bodies 3.3 million years ago . The humVarious factors can affect hair growth and cause permanent or temporary hair loss. Hair loss and thinning have been an issue for men and women throughout history. Finding the cause and treatment of hair loss has become a controversial topic. Scalp hair retains heat and keeps the body warm, protecting the head from sunlight and cold and heat. Hair follicles are divided into two categories depending on androgen and hormones. The follicles on the skull are usually hormone\u2010dependent. About 3\u00a0mm of hair is in the hypodermis. Usually, every 1\u20134 ends of the hair in the skull and 1\u20132 fluffy hairs form a follicular unit surrounded by hair straightening muscle ) , 35. EntC is the total amount of dutasteride, and TC is the free amount of dutasteride, which was detected only in the supernatant media. The assay was performed in triplicate, and the values provided are the mean\u00a0\u00b1\u00a0SD of three independent experiments.2.62. The abdomen skin of the rat was shaved and cut. Then, the fat tissue was removed from the skin pieces, washed with Phosphate buffer saline, and assessed for accuracy. Dutasteride nanoemulsion was poured on the skin in the donor section. A sample of 100\u00a0\u03bcl was extracted from the section under the skin in the Franz diffusion cell at predetermined time intervals of 20\u00a0min, 1\u00a0h, 2\u00a0h, 4h, 24\u00a0h, 48\u00a0h, and 6\u00a0days. The concentration of released dutasteride was measured using a UV\u2010Vis spectrophotometer. However, a volume equal to the receptor phase was immediately applied to the Franz diffusion cell to maintain a constant volume.Drug permission analysis on rat skin using a Franz diffusion cell is one of the gold methods to evaluate drug permission through the skin , 24, 25.2.7p\u2010value of less than 0.05 was considered statistically significant.Graph pad InStat software (V3) was applied to calculate and analyse the dutasteride nanoemulsion's particle size, EE%, and skin permeation. All triplicate experiments were repeated three times. Experiments were performed as mean\u00a0\u00b1\u00a0SD. A 33.1The maximum wavelength of dutasteride was determined using\u00a0a UV\u2010Visible spectrophotometer. Based on the spectra obtained through the spectrophotometer, the maximum absorption wavelength of dutasteride was determined to be 240\u00a0nm.R2\u00a0=\u00a00.9955 was calculated, and the concentration equation was obtained in absorption wavelength (y\u00a0=\u00a00.0218x\u00a0+\u00a00.373). Samples that had adsorption above 2 were excluded from the study. The linear regression with the R2\u00a0=\u00a00.9955 indicates good linearity, ranging from 62 to 3\u00a0\u03bcg/ml . However, soybean oil significantly increased the dissolution of dutasteride than sesame oil (p\u00a0<\u00a00.05) and mineral oil (p\u00a0<\u00a00.01) . Moreover, the dutasteride nanoemulsion of E had a significantly larger hydrodynamic particle size than B (P\u00a0<\u00a00.01) and C (P\u00a0<\u00a00.001). SPAN analysis showed a smaller SPAN for C and E dutasteride nanoemulsions than for D nanoemulsion . Although the pH of nanoemulsion E (5.43\u00a0\u00b1\u00a00.07) was significantly higher than D nanoemulsion (p\u00a0<\u00a00.05), there were no significant differences between the pH of C and E nanoemulsions (p\u00a0>\u00a00.05).pH measurement data showed that there were no significant differences between the pH of C (5.29\u00a0\u00b1\u00a00.04) and D (5.25\u00a0\u00b1\u00a00.05) nanoemulsions (3.5p\u00a0>\u00a00.05). At the same time, both of them showed significantly higher viscosity than E nanoemulsion (p\u00a0<\u00a00.001) .The EE% was measured using the ultrafiltration method and showed that EE% of C, D, and E nanoemulsions were 45.07%\u00a0\u00b1\u00a00.31, 69.95%\u00a0\u00b1\u00a00.48, and 99.9%\u00a0\u00b1\u00a00.1. There was a significant difference between the EE% of all nanoemulsions (3.7To select an ideal nanoemulsion, one of the most important characteristics is the thermodynamic stability of the nanoemulsion. In other words, thermodynamically unstable nanoemulsions were removed from the study. Following thermodynamic stability analysis of freeze\u2010thaw, centrifugation, and heating\u2010cooling, A dutasteride emulsion got biphasic at the centrifugation stage and was removed from the study. Although B, C, and D dutasteride emulsions were monophasic, their appearance was changed. Therefore, they were removed from the study. E dutasteride nanoemulsion was stable and went to further analysis Table\u00a0.3.8Based on thermodynamic stability findings, E nanoemulsion was selected for the ex vivo study of permeation analysis. Drug release and skin permeation analysis were evaluated using Franz diffusion cells. The dutasteride nanoemulsion's release profile showed a sustained dutasteride release at 24\u00a0h and continued for over 6\u00a0days. The figure shows that approximately 76% of dutasteride was released over 6\u00a0days Figure\u00a0 and b.4R2\u00a0=\u00a00.9715, indicating a linear relationship between concentration and adsorption at 240\u00a0nm. Moreover, soybean oil is more solubilised dutasteride than other oils. Changes in synthesis parameters alter hydrodynamic particle size and other nanocarriers' characteristics, such as pH, thermodynamic stability, viscosity, and EE%.Hair loss or Alopecia is a treatable disorder that, over time, gets to chronically influencing a noteworthy part of the populace to decrease their self\u2010esteem. It is worth mentioning that the wealthy ancient Egyptians shaved their hair, and shaving was considered a symbol of wealth. Nevertheless, cultures have changed; now, thick hair symbolises beauty in many parts of the world. However, re\u2010styling hair and eyebrows may symbolise beauty in the following centuries. Nevertheless, in the present century, based on the criteria of beauty, let us look for a solution to this disorder. The present drug delivery system and formulations are not efficient enough to deliver the drug to the hair follicles and cannot release the drug in the hair follicles for a long time. Formulating and synthesising a drug delivery system based on nanocarriers would be beneficial and a gold strategy for a prolonged and sustained release drug delivery system in hair follicles. In the present study, a prolonged\u2010release nanocarrier of dutasteride was synthesised, characterised, and its influence on rat abdomen's skin was investigated. The concentration curve standard showed an As shown in the formulations of A and B, the lower ratio of S/Co made a larger particle size of dutasteride nanocarriers. When the ratio of surfactant to ethanol was increased from 1: 1\u20132: 1, particle size decreased from approximately 5230\u20134340\u00a0nm. Ethanol had enhanced hydrodynamic particle size; however, the increase of Smic also was critical in B nanocarriers. There are some controversial data related to ethanol and particle size. Tavakol et\u00a0al.  and FerrAnother point relates to the increased surfactant and S/Co in the C nanocarrier. An increased surfactant through decreased surface tension results in smaller nanocarriers of approximately 32.4\u00a0nm. An increase in oil concentration from 5% to 10% did not significantly change hydrodynamic particle size and the pH of C and D nanocarriers. It seems that an increase of surfactant from 30% to 40% in A and C nanocarriers was sufficient to cover the surface of nanocarriers and decrease surface tension resulting in particle size of nanocarriers. The best formulations for hydrodynamic particle size were C, D, and E.Data related to the viscosity of nanoemulsion showed that the increase of oil did not significantly change the viscosity of C and D nanocarriers and also did not significantly change the particle sizes. However, D nanocarriers with higher non\u2010significant viscosity than C nanocarriers had smaller particle sizes. Moreover, E dutasteride nanocarriers with significantly lower viscosity had larger hydrodynamic particle sizes. It is demonstrated that an increase in viscosity is inverse to the particle size of nanoemulsions , 38.Based on the data derived from the EE% and particle size, and thermodynamic stability, it was conferred that larger nanocarriers of E nanocarriers had higher EE% than smaller nanocarriers. E nanoemulsion did not have ethanol and showed the highest EE%. It should be said that ethanol dissolves the drug and probably solubilise more drug in the nanocarrier; therefore, in the absence of ethanol, more drug can be kept in the nanocarrier, and its EE% has increased. Other nanocarriers of C and D showed less EE%, perhaps due to the rapid dissolution of dutasteride into ethanol during nanoemulsification; as a result, the accumulation of the drug inside the emulsion nanoparticles decreased and led to the drug leaving the internal phase, and the presence of the dutasteride in the dispersed phase.The stability of nanocarriers was eventually owing to the electrostatic repulsion derived from deprotonated moieties. The optimum concentration of oil, surfactant, and co\u2010surfactant is decisive in guaranteeing the stability of nanoemulsion. Larger particles of A and B were thermodynamically unstable and could not pass the thermodynamic stability phase. Although based on Stokes' equation , the add5Dutasteride nanoemulsion was prepared using soybean oil, tween 80, and span 80, ethanol as a co\u2010surfactant, and different ratios of Smic and S/Co. Results showed that the lower ratio of S/Co made a larger particle size of dutasteride nanocarriers. Ethanol had enhanced hydrodynamic particle size. Particle size increases at higher ethanol concentration due to thermodynamic disturbance and Ostwald ripening, while particle size decreases due to the decreasing interfacial surface. An increased surfactant through decreased surface tension results in smaller nanocarriers of approximately 32.4\u00a0nm. In our study, an increase of Smic to 40% and S/Co to 3: 1 were decisive in the particle size of dutasteride nanocarriers. Dutasteride nanocarriers with significantly lower viscosity had larger hydrodynamic particle sizes. It is demonstrated that the increase in viscosity has an inverse correlation with the particle size of nanoemulsions. Other nanocarriers of C and D showed less EE%, perhaps due to the rapid dissolution of dutasteride into ethanol during nanoemulsification; as a result, the accumulation of the drug inside the emulsion nanoparticles decreased and led to the drug leaving the internal phase, and the presence of the dutasteride in the dispersed phase. The optimum concentration of oil, surfactant, and co\u2010surfactant is decisive in guaranteeing the stability of nanoemulsion. Although based on Stokes' equation, the addition of ethanol probably through the reduction of interfacial tensions in nanoemulsion results in smaller particle size and stability of the system. Skin permeation analysis using Franz diffusion cells showed sustained release of dutasteride in 6\u00a0days. The first point is related to the permeation of dutasteride through the skin, showing the efficacy of dutasteride nanoemulsion in hair loss therapy; another critical point is that only 34% of dutasteride was released from the skin within 24\u00a0h. Nanoemulsion did not show a burst release during the first day, and 76% of dutasteride was released in 6\u00a0days.Mehri Memar Bashi Aval and Saeedeh Saeedi synthesised the nanocarriers and characterised them, Elham Hoveizi wrote the manuscript, Reza Mombeiny and Mostafa Kazemi characterised the nanocarriers and performed Ex\u2010vivo study permeation analysis, Shima Tavakol got the grant, designed and supervised the research, and wrote the paper.The authors declare no conflict of interest."}
+{"text": "Community Health Workers (CHWs) often share cultural, geographic, or other lived experiences with patients and provide health education and support. Use of CHWs and telehealth approaches are promising strategies for addressing the needs of patients with metabolic syndrome (MetS). This narrative review analyzed how these approaches were integrated into programs expanding care access for patients with MetS. Searching PubMed, PSYCInfo, Embase, Web of Science, and Google Scholar resulted in 1,630+ abstracts screened and 12 articles meeting inclusion criteria. These studies examined implementation of tele-mentoring approaches (n=4), patient group classes via videoconferencing (n=2), or individual telehealth consultations facilitated by CHWs (n=7), with some programs including multiple intervention types. This review included adults ranging from 37-79 years old. Most studies focused on late mid-life (ages 50-64). Because health behaviors in midlife have important implications for MetS and related health concerns in later life, it is important to consider midlife interventions. Using the RE-AIM framework, we evaluated studies on five dimensions: reach, effectiveness, adoption, implementation, and maintenance. Reach and implementation indicators suggest reducing barriers to engagement  allows for higher participation and program completion rates. Measures of MetS-related behavioral outcomes were heterogeneous across study designs, making overall effectiveness difficult to determine. Adjusting time spent with patients according to health literacy and clinical needs is a strategy CHW programs use to provide equitable, cost-effective care. Programmatic considerations for implementing programs that include both CHWs and telehealth are discussed, with special consideration for what works in late middle age and in older adulthood."}
+{"text": "IC50 values against cancer cells in vitro. In this study, two Coprinopsis cinerea galectins, CGL1 and CGL2, were heterologously expressed, and their biochemistry properties and anticancer effects were evaluated. The purified galectins were thermostable at neutral pH conditions. They both existed as tetramers and shared a high affinity towards lactose. CGL1 and CGL2 strongly inhibited the cell viability of many cancer cell lines, including three colorectal cancer cells, in a dose-dependent manner by inducing mitochondria-mediated caspase-dependent apoptosis. Furthermore, CGL1 exhibited higher apoptosis-inducing ability and cytotoxicity than CGL2. In vivo cell viability experiments based on two xenograft mouse models showed that CGL1 had a more substantial inhibitory effect than CGL2 on HCT116 tumor growth (p < 0.0001), whereas only CGL1 inhibited DLD1 tumor growth (p < 0.01). This is the first study to evaluate the anti-colorectal cancer effect of mushroom lectins in vivo, and our results showed that CGL1 is a potent agent for colorectal cancer treatment.Mushroom galectins are promising anticancer agents for their low  Lectins are a family of carbohydrate epitopes-binding proteins of non-immune origin with at least one specific and reverse carbohydrate-recognition domain. In nature, lectins distribute widely among plants, animals, viruses, bacteria, fungi, and humans , with 82Pholiota adipose , in which width (W) is defined as the smaller of the two measurements and length (L) is defined as the larger of the two measurements [To obtain the subcutaneous xenografts of human colorectal tumors in the mice, HCT116 and DLD1 cells were harvested during exponential growth. Two million HCT116 or DLD1 cells in PBS were suspended in a 1:1 (urements .https://swissmodel.expasy.org (accessed on 14 October 2021) using C. cinerea CGL2 (PDB code: 1UL9) as the template. The structure was further visualized using the Pymol software . The interaction between CGL1 and the substrate lactose was analyzed by Autodock 4.2 [The three-dimensional structure of CGL1 was modeled with the Swiss Model . p < 0.05 was considered statistically significant.All experimental data were presented as mean \u00b1 standard deviation (SD). Statistical significance was evaluated by one-way ANOVA followed by Student\u2019s In conclusion, CGL1 and CGL2 are thermally stable and remain at maximum activities at neutral and alkaline conditions. CGL1 is a tetramer similar to CGL2. They share identical binding residues, motif-based hydrogen bond networks, and binding constants when interacting with lactose. Their application potential in colorectal cancers is explored in vitro by cell cytotoxicity and apoptosis assays and in vivo using the subcutaneous colorectal tumor models. Our results suggest that CGL1 exhibits more substantial effects in colorectal cancer inhibition than CGL2 and is a promising agent for colorectal cancer treatment."}
+{"text": "Pimpinella L. is one of the large genera in the Apiaceae family. In a previous study, the molecular phylogenies of Pimpinella were explored using nuclear ribosomal DNA internal transcribed spacers (ITS) and several chloroplast DNA segments. There have been few studies conducted on chloroplast genomes in Pimpinella, which has limited systematic understanding of this genus. We assembled the complete chloroplast genomes of nine Pimpinella species from China using data generated from next generation sequencing (NGS). The chloroplast (cp) DNA used were standard double-stranded molecules, ranging from 146,432 base pairs (bp)  to 165,666 bp (P. purpurea) in length. The circular DNA contained a large single-copy (LSC) region, small single-copy (SSC) region, and pair of inverted repeats (IRs). The cp DNA of the nine species contained 82\u201393 protein-coding genes, 36\u201337 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes, respectively. Four species  exhibited striking distinctions in genome size, gene number, IR boundary, and sequence identity. We confirmed the non-monophyly of the Pimpinella species on the basis of the nine newly identified plastomes. The distant relationship between the above-mentioned four Pimpinella species and Pimpinelleae was indicated with high support values. Our study provides a foundation for future in-depth phylogenetic and taxonomic studies of genus Pimpinella. Pimpinella L.  is comprised of approximately 150 species that are mainly distributed throughout Europe, Africa, and Asia, with 39 species and two varieties found in China  are frequently blurred. The published molecular phylogenies of Pimpinella were inferred using the nuclear ribosomal internal transcribed spacer (ITS) region and a few plastid markers   . The genan clade .Pimpinella species from China was conducted using data from the ITS and two cpDNA intron sequences  gene from Pimpinella diversifolia DC. (GenBank accession number MT561033) as the seed file, the raw reads of genome-related chloroplasts were assembled. After aligning with congeneric species in pairs, the plastome sequences were annotated using Geneious Prime 2019.2.3 (ON321877 \u2013ON321885).Utilizing NOVOPlasty 3.7  with the2019.2.3 , supplemhttps://sourceforge.net/projects/codonw/), the relative synonymous codon usage (RSCU) values of each species were determined. These RSCU values were presented on a heatmap created using TBtools v1.086  regions were determined using the \u2018Repeat Finder\u2019 plugin in Geneious Prime . Utilizis v1.086 . With ths v1.086 , RNA edihttps://webblast.ipk-gatersleben.de/misa/) (REPuter  was utile/misa/) , simple https://irscope.shinyapps.io/irapp/) to display the structure of the nine plastomes, and adjusted manually if necessary. Utilizing the MAFFT  and the Kimura 2-parameter model , one LSC region, and one SSC region , seven tRNA genes , and four rRNA genes .The complete cp genome of the nine C region . The plaP. smithii and P. valleculosa contained 127 genes: 82 protein-coding genes, 36 tRNA genes, and eight rRNA genes. Among the 127 genes, 99 were unique and 14 genes were duplicated in the IR region, including four protein-coding genes , six tRNA genes  and four rRNA genes .Moreover, the plastid genomes of P. rhomboidea consisted of 131 genes, including 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Among these, 95 genes were unique and 18 genes were duplicated in the IR region, including seven protein-coding genes , seven tRNA genes  and four rRNA genes .The plastid genomes of P. purpurea consisted of 139 genes, including 93 protein-coding genes, 37 tRNA genes and eight rRNA genes. Among these, 87 genes were unique and 26 genes were duplicated in the IR region, including 15 protein-coding genes , seven tRNA genes , and four rRNA genes .The plastid genomes of Pimpinella plastomes. The total sequence sizes of these genes for codon analysis ranged from 63,423\u201363,516 bp. These protein sequences encoded 21,141\u201321,172 codons . All of the RNA editing sites belonged to the same conversion type (cytosine to uracil (C-U)). Among these, the majority (41\u201345) were located in the 2nd codon position, and the remainder (13\u201317) were in the 1st position.In an analysis of the RNA editing sites, a total of 516 RNA editing sites were identified. The smallest number of editing sites was 56 for leculosa . In all Pimpinella plastomes. Moreover, the repeats with lengths of 30-40 bp accounted for the greatest proportion  coincided. The LSC/IRb border of P. smithii and P. valleculosa, located in the ycf2 gene, and the trnL gene were 900 bp and 374 bp away from the IRa/LSC border, respectively. The LSC/IRb border of P. rhomboidea, located in the rps19 gene, and the trnH gene was five bp away from the IRa/LSC border. The LSC/IRb border of P. purpurea, located in the rps11 gene, and the rpl36 gene had a distance of 136 bp from the IRa/LSC border.The comparison of IR/SC borders among the nine plastomes are shown in P. candolleana and P. purpurea, while the lowest was 0.0007 between P. candolleana and P. diversifolia.Genetic distance analysis results are shown in Pimpinella species and related taxa in the Apioideae was well resolved with high support values   .Pimpinella is a relatively large member of the Apiaceae family and understanding the phylogeny and taxonomy of this genus is important. In this study, the Pimpinella plastomes were determined to be all quadripartite in the genome structure. We determined it had several distinct characteristics. First, the overall sizes of chloroplast genomes varied from 146,432 bp  to 165,666 bp (P. purpurea). Second, the numbers of the unique genes encoded by plastomes ranged from 87 (P. purpurea) to 99 . Among the closely-related species, the plastomes rarely fluctuated in size and gene content. For example, the plastome sizes of nine Chamaesium species reported  (GenBank accession No. Z00044). Most notably, the large contractions of the IR regions in P. smithii (\u223c7.2 kb) and P. valleculosa (\u223c7.6 kb) made their plastome sizes significantly smaller than the newly identified plastomes. Subsequently, in P. smithii and P. valleculosa, rps19, rpl2, rpl23, and ycf2 had only one copy. The huge expansion (\u223c5.7 kb) of the IR regions in P. purpurea with a length of 31,049 bp made it the largest of the nine newly-obtained Pimpinella plastomes. At the LSC/IRb border, its IR expanded \u223c6.1 kb compared with P. diversifolia, and resulted in many genes  contained within the IR region. This kind of large IR expansion  was not usual, and also happened to Crithmum maritimum (Apiaceae) (Apiaceae is a family that is famous for its unique fruit characteristics. reported  ranged ftructure . In mosttructure . IR cont tobacco  was suggpiaceae) . The exppiaceae) , althougPimpinella plastomes was confirmed and also observed in other genera in Apiaceae (Ligusticum species in Apiaceae (Pimpinella chloroplast genomes were discovered to be mononucleotides (A/T), similar to many other angiosperms.The preference for codons ending with A/T in the Apiaceae . The conApiaceae . Short rApiaceae . A largerps16 intron and rpl16 intron) sequences   to combine these two species into Tongoloa. The greatly divergent plastomes of P. purpurea and P. rhomboidea, compared to the other Pimpinella plastomes examined, may be related to potential chloroplast capture events. \u2018Capture\u2019 cases  . Four spe\u2019 cases  have beee\u2019 cases . It is ne\u2019 cases . Althouge\u2019 cases , the implogenies . These tPimpinella and allied taxa may have a more complicated phylogeny, and future in-depth studies based on the chloroplast genomes are necessary.Previous studies  have shoPimpinella genus. The circular DNA of nine newly obtained plastomes contained a LSC region, SSC region, and pair of IRs. The plastomes ranged from 146,432 bp  to 165,666 bp (P. purpurea) in length. The plastid genomes of P. candolleana, P. diversifolia, P. rubescens, P. scaberula, and P. thellungiana were comprised of 130 genes, including 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. However, P. smithii and P. valleculosa contained only 127 genes. Moreover, the plastome of P. rhomboidea consisted of 131 genes. Most intriguingly, the plastid genome of P. purpurea consisted of 139 genes, including 93 protein-coding genes, accompanied by 15 protein-coding genes, and seven tRNA genes  duplicated. Phylogenetic analysis revealed that P. candolleana, P. diversifolia, P. rubescens, P. scaberula, and P. thellungiana were clustered in Pimpinelleae; P. smithii and P. valleculosa were clustered with Selineae members; and P. rhomboidea and P. purpurea in the East-Asian clade were relatively distant from their congeners. This study provides new evidence that chloroplast genomes might be useful when reconstructing the phylogeny of Pimpinella with a larger species sampling, which will be helpful for solving taxonomy problems in the genus.This study is the first attempt to comprehensively examine plastome features and infer phylogeny using plastome data for the 10.7717/peerj.14773/supp-1Table S1Click here for additional data file.10.7717/peerj.14773/supp-2Table S2Click here for additional data file.10.7717/peerj.14773/supp-3Table S3Click here for additional data file.10.7717/peerj.14773/supp-4Supplemental Information 1Click here for additional data file."}
+{"text": "Vibrio cholerae, persists as a devastating acute diarrheal disease. Despite availability of information on socio-cultural, agent and hosts risk factors, the disease continues to claim lives of people in Tanzania. The present study explores spatial patterns of cholera cases during a 2015\u201316 outbreak in Mwanza, Tanzania using a geographical information system (GIS) to identify concentrations of cholera cases. This cross-sectional study was conducted in Ilemela and Nyamagana Districts, Mwanza City. The two-phase data collection included: 1) retrospectively reviewing and capturing 852 suspected cholera cases from clinical files during the outbreak between August, 2015, and April, 2016, and 2) mapping of residence of suspected and confirmed cholera cases using global positioning systems (GPS). A majority of cholera patients were from Ilemela District , were males  and their median age was 27 (19\u201336) years. Of the 452 (55.1%) laboratory tests, 352 (77.9%) were confirmed to have Vibrio cholerae infection. Seven patients (0.80%) died. Cholera cases clustered in certain areas of Mwanza City. Sangabuye, Bugogwa and Igoma Wards had the largest number of confirmed cholera cases, while Luchelele Ward had no reported cholera cases. Concentrations may reflect health-seeking behavior as much as disease distribution. Topographical terrain, untreated water, physical and built environment, and health-seeking behaviors play a role in cholera epidemic in Mwanza City. The spatial analysis suggests patterns of health-seeking behavior more than patterns of disease. Maps similar to those generated in this study would be an important future resource for identifying an impending cholera outbreak in real-time to coordinate community members, community leaders and health personnel for guiding targeted education, outreach, and interventions.Cholera, which is caused by  Vibrio cholerae, persists as a devastating acute diarrheal disease and public health challenge in low and middle-income countries [LMICs]  , 2. Worl [LMICs] , 3. The  [LMICs] , and the [LMICs] . Again,  [LMICs] . Mwanza, [LMICs] . However [LMICs] , 9.Environmental, socio-cultural and economic factors contribute to primary and secondary cholera transmission \u201314. AreaCholera pathogenesis and associated risk factors that pre-dispose human population to cholera are well documented. Even so, cholera outbreaks continue reemerging year after year at an alarming rate , contribIn Tanzania, once a person is suspected of having cholera at a health facility, s/he is isolated in a special room for treatment and observation. If a sudden increase in the number of cholera cases that are linked by time and place occurs, health authorities declare an outbreak. When a cholera outbreak is declared, suspected cholera cases  are referred to isolated camps as a containment measure to have designated locations for cholera care and to minimize spread of the disease. Patients are assessed, treated, and discharged once they do not show any indication of the disease. Transfer to the camp is considered mandatory and all people suspected of having cholera are expected to comply. In this regard, the current study focused on understanding the spatial distribution of suspected cholera cases in Ilemela and Nyamagana municipals.Geographic information systems (GIS) is frequently applied as a tool for exploring spatial patterns and relationships in public health, including examining cholera . StudiesThis cross-sectional analytical study was carried out in Ilemela and Nyamagana municipals that make-up Mwanza City located along the southern shores of Lake Victoria in northern Tanzania . Each ofAll patients with suspected and/or confirmed with cholera of all age groups and sex who reported to any of the five cholera camps from August 2015, through April 2016, were included in the study (n = 852). Patients\u2019 files with no specifications of residence were excluded , as were those with a residence outside of Mwanza City .The data collection occurred in two phases. Phase I focused on the retrospective collection of secondary data from patients\u2019 files from each of the camps from August 2015, through April 2016. The data included: patients\u2019 age, sex, occupation, area of residence, water sources, duration spent at the camp, time from symptoms to transfer to the camp, laboratory testing, and outcome.Phase II mapped the residences of cholera cases during fieldwork conducted May-June, 2016. Because no addresses or street indexing exists in this setting, capturing the locational data involved visiting specific areas of residence for the cholera cases and capturing the global positioning coordinates (GPS) of the household. Identification of the respective patient\u2019s household location involved a series of steps. First, the name of the street from where the patient came was recorded from the cholera camp register. Next, having identified the name of the street, one of the research assistants would list all the names from the respective street. Then, the list of names was submitted to the government office where the chairperson of the street in support of the ward leaders identified the households listed from the camp. Lastly, the location of each household was captured using GPS. Data/information collected was anonymously stored and secured on a password protected computer and files and were only accessible to the research team. Each entry was assigned an anonymous code for further protection.ArcGIS Desktop 10.3 Reviewers' comments:Reviewer's Responses to Questions <font color=\"black\"> Comments to the Authorpublication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented. </font> 1. Does this manuscript meet PLOS Global Public Health\u2019s Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 2. Has the statistical analysis been performed appropriately and rigorously? </font> Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 3. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 4. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 5. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The manuscript titled Spatial distribution of suspected and confirmed cholera cases in Mwanza City,Northern Tanzania by Monica Madullu, MPHDeborah S.K. Thomas, PhDElias C. Nyanza, MPH, PhDJeremiah SeniSospatro E. Ngallaba, MDSophia KiluviaMoses AsoriJoseph Kangmennaang,has been an interesting read. Cholera still remains a health issue in some parts of LMICs. The emphasis on use of GIS for mapping cholera incidence and possible use in making health decisions by government that could reduce cholera in those areas in the manuscript is welcomed.However, the authors should take note of the following observations;1. Introduction: Line 54, rephrasing of sentence needed. \"Just one decade later in 2015-16, again? more than.....\"2. Method: more details on the phase 11 study will be appreciated. For example where all the 852 participants mapped from their residences to the various camps? if so, that means you had access to their home addresses. Did the individuals give consent to this information and use of same?3. Results: Kindly give more details on distribution of cholera camps across the two districts surveyed. Reconcile details about mortality in line 144-149 where 6 and 7 were mentioned. If they are different, clearly specify.4. Discussion: Line 192....corroborate studies? or with? line 208, \"10% attended more than 24 hours from onset of symptoms\" I think you need to check that again. same as line 221-223, it may need rephrasingLine 229, statement regarding cholera in Northern part near lake Victoria should be added to section where water source closeness in relation to cholera incidence was mentioned (196-200) for better organization. same as line 233, well and tap water user has been mentioned before (203), merge bothline 255-256...rephrase sentence.5. Reference: check 1,5 and 7. when referencing website, date accessed is usually included30, add journal or website6: Table: kindly recheck Table 4, what p-value is considered significant from the analysis you did? this was not mentioned in the methods. from the confidence intervals in the results, p values of 0.10 are not significant with the single asterisk. If you think otherwise, kindly provide more information to the conclusion in method-data analysis section.& Supporting Information: I successfully downloaded the file and opened it, but could not see data on the file. kindly check again.Reviewer #2:\u00a0This paper has the potential to be published because it provides epidemiological insights on the cholera outbreak in a specific region in Tanzania. However, in the introduction I miss why the mapping of the disease was important. Also I am missing strong implications and recommendations that the local authorities can implement to resolve or prevent any future cholera outbreaks.- Please rewrite the final part of the introduction sentence. I am missing a problem statement that will naturally lead to the aim and the objectives of this research paper and why this research was necessary to be conducted- Methods: I don\u2019t understand why the wards are mentioned. Please elaborate.- L84- 92 should be in the introduction section.- L95 rename to eligibility criterea- Separate subheading for data analysis- Please create subheadings to make reading the results easier.- Please create a separate subheading for limitations and strengths in the discussion section********** <font color=\"black\"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.Do you want your identity to be public for this peer review? If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Privacy Policy. </font> For information about this choice, including consent withdrawal, please see our Reviewer #1:\u00a0NoReviewer #2:\u00a0No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  29 Oct 2022AttachmentResponse to Reviewers_October 29 2022.docxSubmitted filename: Click here for additional data file. 28 Nov 2022PGPH-D-22-01133R1Spatial distribution of suspected and confirmed cholera cases in Mwanza City, Northern TanzaniaPLOS Global Public HealthDear Dr. Nyanza,Thank you for submitting your manuscript to PLOS Global Public Health. After careful consideration, we feel that it has merit but does not fully meet PLOS Global Public Health\u2019s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.globalpubhealth@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgph/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Dec 28 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.We look forward to receiving your revised manuscript.Kind regards,David Musoke, PhDAcademic EditorPLOS Global Public HealthJournal Requirements:1. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article\u2019s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments (if provided):May the authors address the minor comment on Table 4.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions <font color=\"black\"> Comments to the Author </font> 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #1:\u00a0All comments have been addressedReviewer #2:\u00a0All comments have been addressed********** <font color=\"black\"> 2. Does this manuscript meet PLOS Global Public Health\u2019s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented. </font> Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 3. Has the statistical analysis been performed appropriately and rigorously? </font> Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 4. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 5. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 6. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0Authors have effected most observations seen in the original manuscript. However, Table 4 still need review. The notes under the table in the manuscript with track changes and that without track changes are different. the notes with manuscript without tract changes have two asterisks with same interpretation. kindly recheck.Reviewer #2:\u00a0All issues were addressed and the content is ready for publication.********** <font color=\"black\"> 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.Do you want your identity to be public for this peer review? If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Privacy Policy. </font> For information about this choice, including consent withdrawal, please see our Reviewer #1:\u00a0NoYes:\u00a0dr. C. ZemouriReviewer #2:\u00a0**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  28 Nov 2022AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 7 Dec 2022Spatial distribution of suspected and confirmed cholera cases in Mwanza City, Northern TanzaniaPGPH-D-22-01133R2Dear Dr. Nyanza,We are pleased to inform you that your manuscript 'Spatial distribution of suspected and confirmed cholera cases in Mwanza City, Northern Tanzania' has been provisionally accepted for publication in PLOS Global Public Health.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.globalpubhealth@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they'll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Global Public Health.Best regards,David Musoke, PhDAcademic EditorPLOS Global Public Health***********************************************************Congratulations on the acceptance of your manuscript.Reviewer Comments :"}
+{"text": "A variety of anticancer therapeutic targets have been identified over the decades. Nevertheless, the complexity of biological regulation dictates the necessity of knowledge about mechanisms specific to a particular tumor type. Using the DepMap CRISPR/Cas9 knockout database, we performed a comprehensive search for genes critical for tumor survival. Both established and novel markers of tumor viability were identified, many of which are transcriptional regulators. Our results substantiate new therapeutic strategies applicable to individual tumors.The identification of mechanisms that underlie the biology of individual tumors is aimed at the development of personalized treatment strategies. Herein, we performed a comprehensive search of genes (termed Supertargets) vital for tumors of particular tissue origin. In so doing, we used the DepMap database portal that encompasses a broad panel of cell lines with individual genes knocked out by CRISPR/Cas9 technology. For each of the 27 tumor types, we revealed the top five genes whose deletion was lethal in the particular case, indicating both known and unknown Supertargets. Most importantly, the majority of Supertargets (41%) were represented by DNA-binding transcription factors. RNAseq data analysis demonstrated that a subset of Supertargets was deregulated in clinical tumor samples but not in the respective non-malignant tissues. These results point to transcriptional mechanisms as key regulators of cell survival in specific tumors. Targeted inactivation of these factors emerges as a straightforward approach to optimize therapeutic regimens. Genetic factors of tumor progression are distinct for individual cancer types. In each case, there is a unique set of regulatory circuits  whose dysfunction contributes to the biology of specific malignancies. Commonly, conventional chemotherapeutics are indiscriminately toxic to many cell types including non-malignant counterparts. Ideally, for each tumor type, it is necessary to select the unique targets whose inactivation would preferentially affect the particular tumor ,2.https://depmap.org/portal/, accessed on 15\u201330 April 2022 and on 17\u201319 April 2023). This database contains a comprehensive set of genes individually knocked out by CRISPR/Cas9 technology in a broad panel of human-transformed cell lines. Results are represented as the \u2018gene effect\u2019 score indicating the probability of dependency of each cell line on the gene of interest. Strong negative values mark the cases where a given gene is critically important for cell proliferation/viability [A large-scale analysis of transformed cell lines has been demonstrated to be promising for the identification of genes essential for tumor cell proliferation/survival ,5,6,7,8.In this study, we used the DepMap panel to search for genes whose knockout most specifically affected the viability of the particular tumor type in comparison with other tumors. We coined the term \u2018Supertargets\u2019 for these critical genes. In total, we analyzed cell lines derived from 27 tumor types . The top five genes within each tumor type were identified on the basis of efficacy and selectivity for cell viability inhibition. Next, using the TNMplot database portal, we analyzed the expression of identified genes and uncovered the transcripts deregulated in tumor vs. normal samples. The majority of identified Supertargets have been previously implicated in tumor biology and/or served as therapeutic targets, thereby validating the adequacy of the approach. Importantly, our cohort of Supertargets also contains poorly studied genes with unknown roles. These genes deserve future in-depth investigation as potential new targets for anticancer therapy.https://depmap.org/portal/, accessed on 15\u201330 April 2022 and on 17\u201319 April 2023) analysis of the dependency of tumor cell lines on individual genes was conducted using the CRISPR  and RNAi ; accessed on 17\u201319 April 2023). Gene effect difference was calculated as T-statistic scores by DepMap portal using scipy.stats.ttest_ind . Gene expression analysis in cell lines was carried out by DepMap Expression Public 22Q4 release, accessed on 10\u201313 April 2023.The DepMap website , which contains transcriptome data from The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) repositories [http://geneontology.org/, accessed on 20 January 2023). Annotation version and release date: GO Ontology database DOI: 10.5281/zenodo.6799722 (released 1 July 2022). Analysis type: PANTHER Overrepresentation Test (released 13 October 2022); test type: Fisher\u2019s exact [Gene expression in tumor samples and the respective normal tissues were evaluated with Mann\u2013Whitney test using the TNMplot database . DNA-binding domains of transcription factors were determined using Homo sapiens Comprehensive Model Collection . The survival analysis was carried out using the Pan-Cancer datasets of the online tool www.kmplot.com (accessed on 20 April 2023).The GOplot analysis was carried out using the TNMplot database , is insufficient for a majority of cancer cell lines, although MYB inactivation has a strong inhibitory effect in hematopoietic tumors.In the DepMap project, a cell line is considered dependent if the probability of dependency is <0.5; a score of 0 is equivalent to a non-essential gene whereas a score of \u22121 corresponds to the median of all commonly essential genes. The difference in average \u2018gene effect\u2019 values between the selected group and the rest of the cell lines can be counted as a T-statistic score . The lowest T-statistic scores correspond to genes whose knockout affects cell viability in the particular tumor type. We selected the top five genes (referred to as Supertargets) with the lowest T-statistic scores for 27 cancer types using the DepMap portal. Since T-statistics scores depend on the number of cell lines for the respective tumor type, in each cohort we analyzed the top five genes independently.To further validate the proposed approach, we focused on AML and ALL, the malignancies with well-characterized therapeutic targets ,13. IndeMYB gene encoding the MYB transcription factor is deregulated in hematological malignancies including AML [The ding AML  and ALL ding AML ,16. In oding AML . In the ding AML . Severalding AML  for a deLMO2 in acute T-cell leukemia; to date, LMO2 function has been characterized mostly in this tumor type [LMO2 knockout , LIM domain only 2 (LMO2), Janus kinase 2 (JAK2), and growth factor independence 1 (GFI1) proteins A. The PUmor type . Howeverknockout . FurtherLEF1 (lymphoid enhancer binding factor 1), RUNX1 (RUNX family transcription factor 1), and EBF1 (EBF transcription factor 1) genes, identified as ALL Supertargets, are key hematopoietic transcription regulators that play central roles in differentiation and survival of lymphocyte progenitors [ATP6V0A2 (ATP6V0A2 gene encodes the component of the proton channel of vacuolar ATPase (V-ATPase). A recent study indicated that the V-ATPase complex, in addition to its main function in generating the electrochemical proton gradient across the membranes, is involved in Notch/Wnt-dependent tumor progression [The genitors   and ALL (FC median = 113.25). The expression of other genes also increased (FC ranged from 1.45 to 8.59). One exception was the SPI1 mRNA decrease in AML , son of sevenless homolog 1 (SOS1), and signal transducer and activator of transcription 5B (STAT5B), are the components of JAK/STAT signaling pathway critical in BCR-ABL1-positive neoplasms [All CML cell lines in the DepMap panel contain the Philadelphia chromosome that leads to the formation of BCR-ABL1 fusion proteins. As expected, at the top of CML Supertargets are the c scores A. Furtheeoplasms ,32,33,34IKZF1) [BATF3) [The most important genes in Hodgkin\u2019s lymphoma HL; B encode IKZF1)  and the  [BATF3) .MEF2B), EBF transcription factor 1 (EBF1), BCL6 transcriptional repressor (BCL6), and paired box 5 (PAX5)) encode well-studied transcription factors with significant roles in B-cell malignancies (reviewed in [SH3GL1 gene that encodes the ubiquitously expressed endophilin-A2 implicated in endocytosis [In non-Hodgkin\u2019s lymphoma (NHL), four out of five Supertargets (myocyte enhancer factor 2B (iewed in ) , a component of the quality control system of ubiquitin-dependent degradation of misfolded proteins. HERPUD1 is implicated in ovarian [SH3GL1 for NHL and HERPUD1 for MM.In multiple myeloma (MM), four Supertarget genes encode transcription factors such as interferon regulatory factor 4 (IRF4), PR/SET domain 1 (PRDM1), POU class 2 homeobox associating factor 1 (POU2AF1), and myocyte enhancer factor 2C MEF2C, B. Produc ovarian  and live ovarian  cancers,The search of Supertargets was performed for 21 solid tumor types presented in the DepMap panel. p < 0.01, Mann\u2013Whitney test). The most dramatically elevated transcripts of the top five genes were observed in neuroblastoma (FC ranging from 218 to 4085). Additionally, in breast cancer and osteosarcoma, all the top five Supertargets were up-regulated. In contrast, SMARCA2 (a subunit of the SWI/SNF chromatin remodeling complex) and DDX5 (RNA helicase)  B were signancies B. In then tumors . The PAXd in RCC .MYCN is a molecular hallmark of this tumor, thereby justifying the significance of the other four markers. Indeed, MYCN, ISL1, HAND2, and PHOX2B, together with GATA3 and TBX2, comprise the core regulatory circuit, an autoregulatory transcriptional loop that maintains the malignant phenotype of MYCN-positive neuroblastoma [We next focused on the tumor types in which the expression of all Supertargets was significantly increased in clinical samples: neuroblastoma, breast cancer, and osteosarcoma . In neurblastoma . One mayblastoma . In lineblastoma . Thus, pSPDEF (ranked 1) contains the ETS DNA binding domain, a subtype of the bHLH domain; functions of this gene product are vital for normal development as well as for the survival of breast cancer cells [FOXA1, ESR1, and GATA3, are among the most studied prognostic markers and therapeutic targets in breast cancer  gene, its overexpression has been recently shown to drive genome evolution in breast carcinomas [Breast cancer was also the disease with substantially increased transcription of each of the five Supertargets . SPDEF . As rcinomas .SMARCAL1, IRS1, SUB1, HMGA2, and FANCM were identified as Supertargets  threshold; 73.7% Supertarget genes were expressed in >50% tumor cell lines if we set a higher threshold log2(TPM + 2) to cut very low expressing genes . Thus, tMYOG and MYOD1 transcripts were 50-fold higher. Thus, while transcription of Supertargets is not tumor type-specific, its relative expression is generally elevated.However, the expression of certain Supertargets may vary between cell lines of different tissue origins. We estimated the relative expression of Supertargets by measuring the ratio between the median expression of Supertargets in cell lines of the specific tumor type (for which the Supertarget was identified) and its median expression in the total cohort of tumor cells. We found that 83% of Supertargets were expressed higher in the particular tumor type compared to their average median expression. Moreover, the expression of 35% Supertargets was at least 2-fold higher; strikingly, the amounts of http://geneontology.org/, accessed on 20 January 2023, revealed that 92 out of 124 (73%) unique Supertargets were present in the nucleus  and 58 (46%) were associated with chromosomes . Supertargets were involved in different signaling pathways; the principal enrichment of observed vs. expected values was revealed for JAK/STAT , interleukin , insulin/IGF pathway mitogen-activated protein kinase kinase/MAP kinase cascade , and p53 pathway feedback loops 2   .p-value = 6.36 \u00d7 10\u221232, FDR = 6.94 \u00d7 10\u221230; The analysis of molecular functions by the GOplot tool indicated an overwhelming enrichment of transcription factors among Supertargets A. AccordIn accordance with the predicted tumor specificity, the GOplot analysis of biological pathways revealed the \u2018lymphocyte and T-cell differentiation\u2019 and \u2018regulation of hemopoiesis\u2019 groups as the most enriched Supertargets of blood tumors, and the \u2018cell fate commitment\u2019 and \u2018gland, muscle, and mesenchyme development\u2019 groups for solid tumors .Homo sapiens Comprehensive Model Collection  classifhttps://kmplot.com, accessed on 15 February 2022). Out of 27 tumor types in DepMap, the KMplot database contains data on 12 types. We analyzed the overall survival (OS) of patients based on 60 (12 \u00d7 5) Supertargets. As a result, 17 genes demonstrated significant (p < 0.01) differences in life expectancy depending on the high or low expression of individual Supertargets    B and the cancer) C hazard Besides the CRISPR analysis, DepMap includes RNAi data. However, these parameters cannot be compared directly, since CRISPR and RNAi contain different sets of tumor lineages. Additionally, the knockdown efficiency by RNAi differs significantly for different genes. We estimated the effect of RNAi knockdown for top5 CRISPR identified Supertargets using data on five blood malignancies  and for five solid tumors . In the solid tumors, the five selected cancer types are those having the lowest T-statistic values by the CRISPR analysis . Overallp < 0.0005 set by DepMap. If the gene fit this criterion, it was considered a Supertarget and was given an \u201cRNAi KD\u201d score of \u201c1\u201d; otherwise, the score was \u201c0\u201d.To take into account the RNAi efficiency, the genes were divided into two subgroups using the DepMap database criterion: 25 genes with high prediction accuracy and 25 with low prediction accuracy. Next, we determined whether each gene in each group can be considered a Supertarget by RNAi analysis for CRISPR-determined tumor type. For each gene, the statistical significance of the negative gene effect difference was assessed using the confidence threshold p < 0.0005. Thus, in the case of efficient RNAi, the genes act as Supertargets upon knockdown.NFIA gene are better understood in brain development [The Supertargets identified herein included the previously characterized markers as well as proteins whose function has not been attributed to the respective tumor. To analyze the latter group, we performed a PubMed search of manuscripts with a text combination of the gene and the respective tumor type. We found that 24 out of 135 Supertargets have not been linked to the respective tumors . Among telopment , althougelopment . RegardiIn the present study, we used the DepMap database to search for genes that can serve as therapeutic targets for individual tumor types. For each of the total 27 tumor types, we identified the top five genes whose CRISPR/Cas9 mediated knockout was lethal. A similar inhibitory effect was observed through the analysis of RNAi DepMap data. Importantly, we found 24 new Supertarget genes whose functions have not been investigated previously in the respective tumor.Selectivity of Supertargets for an individual tumor can arise from two mechanisms. In the first scenario, the expression of Supertargets is cell type-specific. Second, the expression of Supertargets is ubiquitous, but its knockout affects cell type-specific cascades. Using RNAseq data available at the DepMap portal, we showed that, although transcription of Supertargets is not confined to a particular tumor type, the relative abundance of specific transcripts is higher in those cells in which the Supertarget gene was identified. Furthermore, using the TNMplot portal, we demonstrated that the expression of >50% of identified genes was significantly increased in matched tumor vs. non-malignant clinical samples. The most dramatic transcriptional burden was observed in neuroblastoma samples (a 200\u20134000-fold increase in steady-state levels of five Supertarget mRNAs).Interestingly, genes coding for transcription factors with various DNA-binding domains are the predominant group within Supertargets (41%). Our data substantiate the key role of transcription factors in tumor biology ,64. For From this viewpoint, three newly identified DNA binding Supertargets, NFIA, ZBTB18, and TEAD3, are of particular interest. They are required for the proliferation of cervical, rhabdomyosarcoma, and urinary tract tumors, respectively. In addition, several new Supertargets encoding non-DNA binding transcriptional regulators were identified for osteosarcoma , cervix (histone chaperone SPTY2D1), and upper aerodigestive tract . In additional, several other non-DNA binding transcriptional regulators are present among all Supertargets: a component of the cohesion complex STAG1 (Ewing sarcoma), SMARCA2 (lung) and SMARCAL1 (osteosarcoma). SMARCA2, a.k.a. BRM, is an ATPase subunit of SWI/SNF remodeling complexes ,75, wherAmong the discovered Supertargets are the genes for which the oncogenic driver mutations have been found. Individual mutations can cause tumor hypersensitivity to deletion of the mutated gene; for example, GOF mutations of the EZH2 methyltransferase gene in lymphoid tumor cells . A similHERPUD1 expression has been reported for ovarian [HERPUD1 gene knockout is most crucial in MM. Likewise, TRPM7 has been implicated in breast, pancreatic and gastric malignancies . NevertRPP25L as a novel Supertarget. Currently, there is only one report that attributed the RPP25L gene product to RNase P/MRP complexes involved in tRNA processing [WDR88 gene encoding a protein with six WD40 repeat domains; however, information about its functions is lacking.Several new Supertargets remain poorly characterized; therefore, little is known about their functional properties. In glioma cells, this study identified ocessing . In uterFinally, we provide initial evidence in support of the clinical relevance of Supertargets. Although the dataset for analysis is currently limited to 12 tumor types, a subgroup of Supertargets showed promising value in OS prognosis. However, it is premature to indiscriminately use Supertargets as prognostic markers because of data shortage and the complexities in interpretation. Indeed, the role of an individual Supertarget should be considered with regard to the specific context, largely the tissue origin of the tumor. One would expect that the patterns of gene expression significantly vary in the course of tumor development and the response to treatment. Therefore, the differential ranking and representation of individual Supertargets should be kept in mind as a prerequisite for evaluating their practical usefulness.Our comprehensive search of critically vital genes, termed Supertargets, in the panel of tumors of various origins identified both established and yet unknown mechanisms. Importantly, the majority of proteins encoded by Supertargets appeared to be transcriptional regulators such as DNA binders and chromatin modifiers. This finding justifies the relevance of chemical tools that target transcription factors for treatment optimization. The development of selective instruments that combat the specific transcription regulators would help elucidate the functional roles of the identified genes. In turn, these tools would serve as prototypes of specific inhibitors for personalized therapies."}
+{"text": "Currently, only 1 in 4 children in the U.S. engage in the recommended amount of physical activity (PA) and disparities in PA participation increase as income inequities increase. Moreover, leading health organizations have identified rural health as a critical area of need for programming, research, and policy. Thus, there is a critical need for the development and testing of evidence-based PA interventions that have the potential to be scalable to improve health disparities in children from under-resourced rural backgrounds. As such, the present study utilizes human-centered design, a technique that puts community stakeholders at the center of the intervention development process, to increase our specific understanding about how the PA-based needs of children from rural communities manifest themselves in context, at the level of detail needed to make intervention design decisions. The present study connects the first two stages of the NIH Stage Model for Behavioral Intervention Development with a promising conceptual foundation and potentially sustainable college student mentor implementation strategy.We will conduct a three-phase study utilizing human-centered community-based participatory research (CBPR) in three aims: (Aim 1) conduct a CBPR needs assessment with middle school students, parents, and teachers/administrators to identify perceptions, attributes, barriers, and facilitators of PA that are responsive to the community context and preferences; (Aim 2) co-design with children and adults to develop a prototype multi-level PA intervention protocol called Hoosier Sport; (Aim 3) assess Hoosier Sport\u2019s trial- and intervention-related feasibility indicators. The conceptual foundation of this study is built on three complementary theoretical elements: (1) Basic Psychological Needs mini-theory within Self-Determination Theory; (2) the Biopsychosocial Model; and (3) the multilevel Research Framework from the National Institute on Minority Health and Health Disparities.Our CBPR protocol takes a human-centered approach to integrating the first two stages of the NIH Stage Model with a potentially sustainable college student mentor implementation strategy. This multidisciplinary approach can be used by researchers pursuing multilevel PA-based intervention development for children. Cardiovascular disease (CVD) is the leading cause of death in the United States (US) and disproportionately impacts people from rural areas and lower socioeconomic backgrounds . While tWhile the health consequences of physical inactivity affect all children, those from rural areas are disproportionately affected compared to urban children , 14. ChiAs rural populations continue to bear disproportionate burdens of disease and adverse health conditions, there has been growing support for developing and piloting of novel community-derived multilevel interventions . MultileThe powerful influence that college students can have on role modeling and supporting the behaviors of children is well recognized . For insa systematic approach that holds empathy at its core and encourages its practitioners to return repeatedly to the context, emotions, needs, and desires of the key stakeholders they are developing their solutions for\u201d . Our primary hypotheses are that Hoosier Sport will be feasible as defined by multiple trial- and intervention-related feasibility indicators   . This foHoosier Sport. Aim 3 is to assess Hoosier Sport\u2019s trial- and intervention-related feasibility indicators in a sample of 6th grade middle school students. The present study defines PA in line with the Centers for Disease Control and Prevention as any bodily movement that is produced by the contraction of skeletal muscle and that substantially increases energy expenditure  needs assessment with middle school students, parents, and teachers/administrators to identify perceptions, attributes, barriers, and facilitators of PA that are responsive to the community context and preferences. Aim 2 is to co-design with children and adults to develop a prototype multi-level PA intervention protocol called enditure . We seleenditure , 11 withThe conceptual foundation of this study is built on three complementary theoretical elements : (1) Basic Psychological Needs mini-theory within Self-Determination Theory (SDT) ; (2) theThe first theoretical element of the present study, basic psychological needs, posits that increasing autonomy, competence, and relatedness will increase child well-being , 40\u201342. The second theoretical element of the present study, the Biopsychosocial Model, allows for recognition of the interconnectedness between biological, psychological, and social factors in shaping an individual\u2019s health and well-being . The BioLastly, the study also aligns with the NIMHD Research Framework that provides a system for targeting multiple levels of influence  . The PA-Aim 1 (Needs Assessment): Conduct a community-based participatory research (CBPR) needs assessment with middle school students, parents, and teachers/administrators to identify perceptions, attributes, barriers, and facilitators of PA that are responsive to the community context and preferences.The study\u2019s first phase is a CBPR needs assessment (NIH Stage 0) to identify the community\u2019s physical activity-related needs, goals, opportunities, and assets. Despite knowledge of PA-based needs from a population level, we need to develop specific understanding of how the needs manifest themselves in context at the level of detail needed to make intervention design decisions. To conduct the PA-related needs assessment, we will survey children, parents, and teachers/administrators using a multi-level survey design targeting individual, interpersonal, and community levels of influence. This CBPR needs assessment will serve as a starting point for examining the PA context  in the current school partner and inform future CBPR needs assessments with additional school partners.n\u2009=\u200940 students, n\u2009=\u200940 parents, and n\u2009=\u200915 teachers/administrators . The proposed sample size was selected to be feasible while having a large enough sample to have approximately normal distributions in our outcomes based on the central limit theorem . Data collection will include a survey sample of  theorem . InclusiAfter receiving consent from parents, we will obtain assent from children before they participate in the study to ensure children fully understand the assent document information, including the purpose of the study, study requirements, and potential risks or benefits. Parental consent will be collected remotely through an informed consent document distributed through Qualtrics survey software. Child assent and survey administration will be conducted through Qualtrics and occur in-person to increase compliance and understanding. The survey measures will include demographics, the Physical Activity Questionnaire for Children (PAQ-C) , ExpandeMeasures section for additional details and see For the adult survey, we will obtain consent and administer the survey remotely. Similar to the child survey, the adult surveys will include demographics, questions from the EFNEP Food and Physical Activity Behaviors Questionnaire , BPNES , 44, andno\u201d activity being a 1 and \u201c7 times or more\u201d being a 5. In children, the PAQ-C has demonstrated good internal consistency, acceptable validity, and an adequate Cronbach\u2019s alpha coefficient of 0.72\u20130.88  will be used to assess self-reported physical activity behaviors in children , 52. The.72\u20130.88 , 51. Forover the last 7\u2009days\u201d and \u201cyesterday.\u201d Of the original 30 questions on the questionnaire, the research team selected eight questions for children and 10 questions for adults to help ensure the survey will be feasible in terms of respondent burden. Response options allow participants to select how often they consume various food and drink options. The EFNEP began in 1969, serves all states and U.S. territories, and reaches 450,000 low-income youth and 200,000 low-income adults each year (Questions from the Expanded Food and Nutrition Education Program (EFNEP) Food and Physical Activity Behaviors Questionnaire will be used to assess dietary intake. Questions covered nutritional behaviors \u201cach year , 55. Theach year .I do not agree at all\u201d to \u201cI completely agree.\u201d Four items assessed autonomy, four for competence, and three for relatedness . The BPNES measures psychological needs satisfaction in an exercise context based on autonomy, competence, and relatedness , 44, 57.The adult survey will include questions addressing the PSE level of influence. Questions will assess adults\u2019 interest in PA, nutrition, positive behavioral programming, and perceptions of current school PA policies and interest in new school PA policies. PA environmental questions were informed by past research on perceived environmental variables that may influence PA . As PA bFor descriptive statistics, we will compute frequencies and percentages for each categorical variable and calculate means and standard deviations for continuous variables. Quantitative analyses will be performed in R 4.0.3 . In collHoosier Sport.Aim 2 (Participatory Co-design): Co-design with children and adults to develop a prototype multi-level PA intervention protocol, called Hoosier Sport intervention protocol (NIH Stage 1A) to understand the unique PA-based needs of youth from primarily low-SES rural backgrounds by targeting individual, interpersonal, and school levels of influence. We will conduct a 5-step participatory co-design protocol that includes the following 5 session sequence: (1) problem identification; (2) solution generation; (3) solution evaluation; (4) operationalization; and (5) prototype evaluation. The participatory co-design process in our study context is designed to empower children and adults  to provide input into the prototype Hoosier Sport PA intervention protocol. Based on preliminary school stakeholder input and previous PA intervention literature  leaterature ; (3) socterature ; (4) empterature ; (5) PA terature . School gramming .n\u2009=\u20095 individuals, which aligns with the standard range of participants needed for a participatory design  and one group of children, each with y design . The oddThe two design teams will complete a series of five co-design sessions across 3 months, with approximately 2-weeks between sessions. The child group will begin the process, and in parallel, the adult group will have alternating sessions between the child sessions . The adult group will co-design with the study team to review and revise the child-developed prototype while striving to maintain as many child-derived components as possible. This parallel and alternating co-design process will allow children to have a sense of autonomy in the process to include important concepts to them  while allowing the adults to refine the intervention protocol to increase the likelihood of feasibility and practicality.The sessions will be facilitated by an experienced research team member with training in facilitating group coaching and discussions. The research team will develop open-ended questions to guide each session that are aligned with each session\u2019s goals. For instance, in session 1, the design session agenda focuses on understanding challenges with children\u2019s PA-related behaviors. The design process is an iterative process where we begin by coming to a common understanding of the challenges with PA-related behaviors, then collaboratively develop numerous divergent solution ideas for (1) sport/PA participation; (2) leadership development; (3) social support for PA; (4) empowering education; and (5) PA take-home equipment & activities. Then, we progressively move toward a detailed and high-fidelity intervention protocol. During each session, the facilitator will encourage discussion, interpretation, and respectful debate among design team members while ensuring progress. PA-based needs, goals, opportunities, and assets identified in Aim 1 will be integrated throughout the design session discussions by providing survey results to co-designers during session agenda development and finalization.Hoosier Sport protocol feasibility, acceptability, and appropriateness on the Feasibility of Intervention Measure (FIM), Acceptability of Intervention Measure (AIM), and Intervention Appropriateness Measure (IAM)  , each adHoosier Sport will be feasible, acceptable, and appropriate  method . The RITHoosier Sport\u2019s trial- and intervention-related feasibility indicators.Aim 3 (Pilot Testing): Assess Hoosier Sport intervention with 6th grade students from one rural middle school twice per week for 8-weeks during physical education class (NIH Stage 1B). We will assess recommended trial- and intervention-related feasibility measures for pilot/feasibility studies  to guide intervention revisions and retesting Hoosier Sport the following semester. Hoosier Sport college student mentors will work alongside the research team to deliver the intervention . The college student mentors will be upper division undergraduate and graduate students enrolled in public health majors. To enhance intervention fidelity and as a part of the academic course, college student mentors will participate in 4-weeks of training classes prior to being deployed to the middle school, where they will receive course credit for hours spent delivering the intervention. Hypotheses for Aim 3 include the following: (3a) achieve full enrollment (n\u2009=\u200920); (3b) 85% retention at the end of the intervention; (3c) 75% attendance rate; (3d) mean score of \u226516 (a \u201cgood\u201d score) on the FIM; (3e) mean score of \u226516 (a \u201cgood\u201d score) on the AIM; (3f) mean scores of \u226516 (a \u201cgood\u201d score) on the IAM; (3\u2009g) 80% fidelity with intervention procedures.The third phase of the study is to assess intervention feasibility by testing the  studies . As expl studies , 70), bl studies . After tHoosier Sport college student mentors will be recruited through a service-learning course developed by the research team, titled \u201cIntroduction to Youth Sport Development,\u201d and housed within the Department of Kinesiology at the Indiana University School of Public Health-Bloomington. This course includes topics such as effective mentoring techniques, communication strategies, and administering safety protocols. College student mentor models have been used to engage youth in PA, attain knowledge, and apply healthy behaviors, transferrable life skills, and academic enrichment  join the study. The research team will confirm eligibility via email/phone. Consent from parents to have their child(ren) join will populate a list of children who are eligible to be approached for assent. The research team will then host a study information and recruitment session for children at the school site. At this session, child participants will be provided with study information and a Qualtrics-based assent survey in appropriate and understandable terms for 6th grade students. Children will be provided with an opportunity to ask questions and informed that they can discontinue participation at any time during the study.Enrolled participants will be mailed their initialized Axivity AX3 accelerometer , 70 and In line with recently published NIH-funded pilot/feasibility research , we willHoosier Sport (consent from parent/guardian and assent from child) and (2) the number of college students successfully enrolled into the \u201cIntroduction to Sport-Based Youth Development\u201d course.Recruitment capability will be determined based on (1) the number of children successfully enrolled into Retention will be measured based on (1) the number of children who participate in the post-intervention data collection event and (2) the number of college students who successfully completed their service-learning hours at the middle school. A make-up post-intervention event will be scheduled for child participants who miss the post-intervention data collection event.to what extent was the intervention delivered as planned? (2) in what ways, if any, did the college student mentors adjust the program? (3) what were the reasons for any adjustments?Three groups of stakeholders  will assess treatment fidelity using self-report measures at two time points (mid- and post-intervention) to explore whether the intervention was delivered accurately, consistently, and with quality. Assessment of fidelity will be guided by three questions from past school-based PA implementation research : (1) to Hoosier Sport will rate the feasibility, acceptability, and appropriateness of the intervention using the FIM, AIM, and IAM, respectively (each described in Aim 2).Children who participate in Accelerometer compliance will be assessed for each of the two PA data collections by determining the number of days accelerometers collected data compared to the target.Cost of the intervention will be monitored throughout the study period and determined in comparison to the prospective study budget.Data analysisFor analysis, we will check the completeness and distributions of all variables. Normalizing transformations will be applied as needed for non-normally distributed variables. Internal consistencies of scaled scores are assessed with Cronbach\u2019s alpha. Analyses of primary outcomes  and exploratory outcomes  will be descriptive with means and standard deviations (SD).Health interventions incorporating evidence and engaging key community members in the planning process generate more effective outcomes , 75, 76.trial-related feasibility was reported in many studies ; however, important intervention-related feasibility indicators were not widely reported   . Additioateness) .Hoosier Sport intervention poised for refinement or expansion in a Stage 2/3 efficacy clinical trial, powered to test changes in physical activity, with secondary outcomes of other cardiovascular disease risk factors . Findings will also help inform other academic institutions practicing CBPR and aiming to partner with local schools. Ultimately, Hoosier Sport should be feasible and adaptable to a range of school contexts that could benefit immediately from partnerships with major academic institutions with the college student service-learning workforce to deliver programming at scale.In sum, successfully completing the aims will lead to a feasible The studies involving human participants were reviewed and approved by Indiana University Institutional Review Board. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.SG and KK designed the initial study protocol and drafted the manuscript. SG, PF, VM, CC, KP, TE, AG, JE, NW, and KK contributed to the conceptualization and design of the study protocol. All authors contributed to the article and approved the submitted version.This work was supported by the SNAP-Ed grant program within the Division of Nutrition and Physical Activity at the Indiana Department of Health, as well as the Indiana University Office of the Vice Provost of Research, and the Indiana University Center for Innovative Teaching and Learning.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": "CanChild), and to identify and share key ingredients for developing partnerships between organizations and academic institutions. A companion study is underway to examine partnership experiences with CanChild from the partners\u2019 perspective. Four CanChild researchers and two co-facilitators participated in a collaborative auto-ethnography approach to share experiences with organizational research partnerships and to reflect, interpret, and synthesize common themes and lessons learned. The researchers and facilitators met virtually via Zoom for 105\u00a0min. Researchers were asked to discuss the following: the formation of their organizational partnerships; if/how partnerships evolved over time; if/how partnerships were sustained; and lessons learned about benefits and challenges to building research partnerships with organizations. The meeting was recorded, transcribed verbatim, and analyzed by the facilitators to identify and synthesize common experiences and reflections. Multiple rounds of asynchronous reflection and feedback supported refinement of the final set of analytic themes. Researchers agreed that partnerships with organizations should be formed through a mutual interest, and that partnerships evolved by branching to include new organizations and researchers, while also involving trainees. Researchers identified the importance of defining roles and responsibilities of key individuals within each partnering group to sustain the partnership. Lessons learned from organizational partnerships included reciprocity between the partnering organization and academic institution, leveraging small pockets of funds to sustain a partnership over time, and building a strong rapport with individuals in a partnership. This commentary summarized lessons-learned and provided recommendations for researchers and organizations to consider when forming, growing, and sustaining research partnerships over time.There is an increased interest from both researchers and knowledge users to partner in research to generate meaningful research ideas, implement research projects, and disseminate research findings. There is accumulating research evidence to suggest the benefits of engaging children/youth with disabilities and their parents/families in research partnerships; however, less is known about the benefits of, and challenges to, engaging organizations as partners in research. The purpose of this commentary is to reflect on successful organizational partnership experiences from the perspectives of researchers at an internationally-recognized childhood disability research centre ( Researchers and people who use research findings are partnering to create research projects and share results. There are examples of children with disabilities and their families participating in research partnerships, but less is known about the involvement of healthcare organizations and community organizations as research partners. The purpose of this article is to share successful examples of partnership between organizations and a childhood disability research centre from the perspective of researchers. Four researchers and two facilitators met to reflect on their experiences with organizational research partnerships. They met online for 105\u00a0min using Zoom software. The researchers were asked to talk about how their partnerships with organizations were formed, how they grew over time, and how they were maintained. The meeting was recorded, and the facilitators took the researchers\u2019 experiences and summarized them into common messages. Everyone then read the summary on their own and added their ideas. This happened three different times until everyone agreed on one set of ideas. The researchers agreed that partnerships with organizations should be formed through common goals, that they should grow to include new partners and junior researchers, and that clear roles and responsibilities were needed to keep the partnership going. The experiences shared in this article are valuable to other researchers and organizations that are interested in forming research partnerships. Increasingly, health care organizations, funding bodies, policy-makers, and professional organizations are interested in partnering with diverse knowledge users in research. Many well established research approaches or methodologies include aspects of partnership, such as integrated knowledge translation (iKT), collaborative research, community-engaged research, community-based participatory research, or co-production of knowledge . In CanaThere is emerging evidence about involving children/youth with disabilities and/or their families as partners in childhood disability research, including a systematic review of 22 articles that identified benefits and challenges of including children and young people with disabilities as partners in research . BenefitCanChild Centre for Childhood Disability Research (hereafter referred to as CanChild) was created in 1989 and is located at McMaster University in Hamilton, Ontario, Canada. It is a research centre comprised of local, national, and international students, researchers, and partners who collaborate in conducting childhood disability research. CanChild researchers from different academic institutions have historically engaged in research partnerships with diverse knowledge users, ranging from individual children and youth with disabilities and/or their families, to organizational stakeholders such as service providers, program leadership, and/or policy makers. CanChild researchers have contributed to best practice guidelines for engaging various stakeholder groups in research and knowledge translation [nslation , 11 and nslation .CanChild from the perspectives of researchers and identify key ingredients for fostering research partnerships between organizational and academic institutions. While our group has studied research partnership trajectories with children, youth, and families [CanChild from the partners\u2019 perspectives and is expected to provide further guidance.The purpose of this paper is to examine successful organizational partnership experiences at families , 13, we CanChild researchers took part in a collaborative auto-ethnography approach to gather descriptions of their experiences with organizational research partnerships and to reflect, interpret, and generate common themes and lessons learned. Collaborative auto-ethnography is a form of qualitative research in which authors use self-reflection and writing to explore anecdotal and personal experiences and connect these autobiographical stories to wider meanings and understandings [CanChild in which approximately 15 trainees, local and international researchers discussed how best to advance \u201cIntentional Collaboration\u201d. These discussions identified organizational partnerships as a strength at CanChild and a need to further develop, learn from, and share examples from well-established organizational partnerships. Stage 2 included selection of the example teams and projects and their participation in a focus group . Stage 3 included three rounds of asynchronous reflection and feedback on the interim analytic themes.tandings . It \u201cbritandings . Collabotandings . ResearcResearchers in the present study included those with previously published and ongoing research partnerships with organizations \u201318. We sTo facilitate discussions of these experiences, researchers were asked about how their organizational partnerships were formed; if (and how) the partnerships evolved over time; if (and how) the partnerships were sustained over time; lesson(s) learned about the benefits and challenges to building their research partnerships; and recommendations for other childhood disability researchers and scientists interested in partnering with organizations. Researchers were provided with these questions prior to meeting in a virtual setting to prepare their thoughts.Four researchers and two co-facilitators met in a virtual setting using Zoom teleconference software  for 105\u00a0min. Each researcher first offered their reflections to each of the above questions and were afforded chances to ask each other follow-up questions to identify commonalities and differences in their experiences. The virtual meeting was recorded and transcribed verbatim. PGM and KP as co-facilitators, reviewed the transcriptions, collaboratively synthesized these experiences, and drafted the key ideas into manuscript form. Three iterations of the findings and manuscript were reviewed and revised by all researchers who participated in the videoconference to ensure that the information presented reflected their experiences, selected their most powerful examples, considered the diversity in exemplars provided, and communicated asynchronously using the comment and reply functions in the written document.CanChild, shared lessons learned, and provides recommendations for future research in this area. This article aims to present a collective voice of researcher experiences with organizational partnerships, while acknowledging the individual reflections of each researcher on each project or study. As collaborative auto-ethnography is a qualitative research design, we present our discussions and recommendations with the intent of providing our collective reflections and examples that may offer guidance for researchers who wish to conduct research in partnership with organizations [The discussion that follows describes a synthesized reflection of different organizational partnership experiences from researchers within izations .CanChild researchers who took part agreed that it was valuable for organizations and researchers to initiate partnerships through a shared interest in a topic or research question. A common starting point was perceived to be mutual interest from both the organizational and research institutions in developing a solution for a clinical challenge. For example, in P4C, a leader in a healthcare organization responsible for delivering school-based occupational therapy (OT) services, approached researchers to investigate service delivery issues . Likewise, the Readiness Support Project was initially started at KidsAbility  becauseCanChild researchers across multiple institutions and onboard trainees in a new academic institution and geographic locale. Trainees were able to advance partnership growth when they brought new partnership ideas to the researcher or when the researchers extended their existing partnerships to include trainees [CanChild led to extended partnerships between researchers; for example, the Principal Investigators of the P4C and Readiness Support Projects supported each other\u2019s growth through introduction to new study partners and opportunities. This underscored the value of a (CanChild) research centre\u2019s infrastructure to further partnerships.Researchers reflected on the opportunities for growing partnerships through linking in new partners and onboarding trainees. In PROSPECT, there was early opportunity to grow their new organizational partnership while partnering to solve a clinical challenge in a specific geographical locale. The researcher (MK) received initial funding as they were relocating to a new institution and their partner organization was initiating adoption of statewide changes impacting their current workflow. As a result of these major changes, they chose to \u201cfail forward\u201d together by experimenting with how to best conduct research together  and negotiating the parameters of their partnership . They created a new research group to recruit and retain organizational partners and co-author products, and they created norms and mechanisms to partner with trainees , 21. An CanChild colleagues, and/or external community organization colleagues, allowing the research to expand with increased reach and impact.Individuals within organizational partnerships were identified by researchers as key players in the initiation and sustainability of partnerships. Layers within the partnership indicated the importance of multiple levels of engagement between researchers and organizational partners, and the roles and responsibilities of individuals within each partnering group. These roles and responsibilities included representation from each partner, and how every partner would like to be involved in each stage of the project, to ensure ongoing engagement and facilitation towards a shared end goal. For example, to support continued engagement, the P4C project developed and implemented a terms of reference guide to provide information about who is involved in the partnership, their mandate, and their roles and responsibilities. Similarly, PROSPECT implemented a dissemination guide at the start of each phase of work, to map each partner to their credited roles for contributing to research products. Across researchers, each partnership that was described grew over time, with new connections made with trainees, other \u201cWhat do we offer to our partners in addition to thinking about what they offer back to us?\u201d (MP)The researchers identified three important lessons learned and recommendations for forming, evolving, and maintaining partnerships. Three illustrative quotes were selected and agreed upon by the researchers that named and captured the essence of these lessons. These are presented below.\u201cBuild habits for being a good steward of smaller pockets of funds\u201d (MK)A reciprocal relationship between an academic institution and a partnering organization is fundamental for establishing a foundation from which partnerships can strengthen and evolve . In our \u201cPeople value the relationships and how they feel engaging in the partnership\u201d (WC and LD)It is important to recognize and understand, early on, that it takes resources to build and sustain partnerships over time. We therefore acknowledge the importance of fully delivering on smaller initial investments in the partnership , and establishing habits for negotiating risk while producing on project deliverables with \u2018shoestring\u2019 budgets, as they can reinforce creativity and accountability in the partnership as needed to seek and secure larger investments and rewards. A literature review on creating successful partnerships identified critical success factors for partnerships, which included sufficient funds, staffing personnel, materials and time, and skilled leadership . NotwithRegardless of whether a program of research engages in a partnership with a single organization or multiple organizations, it is paramount to appreciate the quality and the authenticity that individuals and groups bring to the partnership. Researchers emphasized the importance of investing time and effort in establishing personal relationships with partners to make the partnership truly authentic. Building a rapport with partners that is authentic can lead to multiple partnerships in the future, with mutual benefits . In P4C,CanChild. Therefore, this paper may be limited in scope as it was focused on providing information about organizational partnerships in a childhood disability context from researchers to other researchers and organizations. It is important to acknowledge that organizational partners might not perceive the same experiences, benefits, and lessons learned as the researchers involved in this study. Therefore, our companion study will include and compare the experiences of childhood disability research partners.Although the purpose of this paper was to share successful organizational partnership experiences from the perspectives of researchers, we recognize the importance of also examining the partners\u2019 experience and recommendations. Our team is currently conducting a study with youth, parents, clinicians, and organizational leaders to examine their experiences of partnering with In summary, researchers shared experiences of establishing partnerships with organizations from a childhood disability research centre. Through a collaborative auto-ethnography approach and subsequent reflection, we shared lessons-learned and provided recommendations that other researchers can utilize when forming, evolving, and maintaining partnerships with organizations. The key ingredients of successful organizational partnerships were identified to be reciprocity, infrastructure, and relationship-building. Future work in this area should understand factors that contribute to the longevity of organizational partnerships and to understand the first-hand experiences of organizational partners."}
+{"text": "Optimal antithrombotic therapy depicts a challenge to clinicians treating atrial fibrillation (AF) patients who are undergoing percutaneous coronary intervention (PCI). Theoretically, these patients would require a combination therapy of oral anticoagulant and dual antiplatelet therapy (DAPT) with aspirin and a P2Y12 inhibitor, known as triple antithrombotic therapy (TAT). However, TAT is known to carry a significant risk of bleeding. The purpose of the present paper is to provide a focused review of the evidence about the safety of TAT as well as to address contemporary directions regarding antithrombotic therapy following PCI in patients with AF who received a drug-eluting stent.Novel oral anticoagulant studies consistently demonstrated a better safety profile when compared to Vitamin K antagonist (warfarin), especially in AF patients who have other indications of DAPT after PCI. Evidence from several studies showed that the use of TAT in AF patients undergoing stent implantation or PCI has no significant clinical benefit with more risk of major bleeding when compared to DAT. Therefore, the current recommendations for AF have taken into account the mounting evidence of antithrombotic treatment after PCI in AF patients, which has caused a major shift away from the TAT strategy toward DAT over time.Cardiologists face challenges in determining the best antithrombotic treatment for AF patients after PCI with DES implantation. Growing data suggest that TAT is associated with considerable bleeding and worse safety, without significant effectiveness. Hence, TAT is strictly applied for individuals with significant thrombotic risk and low bleeding risk, and for a limited duration. This paper highlights the safety concerns of TAT and current trends in antithrombotic therapy after PCI in patients with AF and DES. Patients undergoing percutaneous coronary intervention (PCI) occasionally have or acquire accompanying indications for anticoagulation. These can include atrial fibrillation (AF), deep vein thrombosis (DVT), and mechanical valve replacements. Among these, AF is the most frequent concomitant indication for anticoagulant therapy . AF is t2DS2-VASc score\u2009\u2265\u20092  and\u2009\u2265\u20093  for reducing stroke risk  appeared in 11.1% of the DAT cohort versus 17.6% of the TAT group (p\u2009=\u20090.025) [Until recently, there has been little evidence regarding the safety of triple therapy (TAT) in AF patients after PCI or DES. To investigate whether TAT is the optimum antithrombotic strategy in these patients, a number of randomized clinical trials (RCTs) have been conducted. The WOEST trial  was the first RCT to highlight this issue and it was a tiebreaker between interventionalist and electrophysiologist. While interventionalists advocate for antiplatelet therapy to avoid stents thrombosis, electrophysiologists point to the importance of anticoagulants for stroke prevention in AF, with neither therapy adequate on its own . WOEST c=\u20090.025) . Howeverp\u2009<\u20090.001) than in the warfarin population  at the 1-year follow-up. The diversity of ethnicity was better than in previous trials.; however, the study still lacked good numbers from the East Asian population. This may be important due to the increased incidence of clopidogrel resistance, especially in the Asian population due to the prevalence of genetic polymorphisms [Four RCTs compared TAT (with Vit K antagonist) versus DAT  plus multiple adjusted doses of NOACs, rivaroxaban 15\u00a0mg o.d. (PIONEER AF-PCI), dabigatran 110\u00a0mg or 150\u00a0mg b.i.d. (RE-DUAL PCI), apixaban 5\u00a0mg b.i.d. (AUGUSTUS), and edoxaban 60\u00a0mg o.d. (ENTRUST-AF PCI) in AF patients undergoing PCI , and 5.5% in DAPT (150\u00a0mg dabigatran) when compared with TAT (p\u2009=\u20090.002). Furthermore, the safety regarding stent thrombosis between the regimens was almost similar, with just a 1.1% increase in stent thrombosis in the DAT group [p\u2009=\u20090.15), while in the 150\u00a0mg dabigatran cohort, it was exactly the same as in the TAT group (0.9% vs. 0.9%) (HR\u2009=\u20090.99 (95% CI 0.35\u20132.81), p\u2009=\u20090.98) [In the RE-DUAL PCI trial , a total of 2725 daily patients with AF who had PCI were randomly allocated (1:1:1) to obtain TAT (warfarin plus a P2Y12 inhibitor and aspirin for a period of 1\u20133 months), dual antithrombotic therapy (DAT) which includes dabigatran (dose of 110\u00a0mg twice daily) plus a P2Y12 inhibitor, or DAT with dabigatran (150\u00a0mg twice daily) plus a P2Y12 inhibitor. The most important findings of this study were a significant decrease (11.5%) of major bleeding  in DAPT (110\u00a0mg dabigatran) when compared with TAT (AT group . Neverth\u2009=\u20090.98) .p\u2009=\u20090\u00b7001 only for non-inferiority), without any significant difference in the ischemic outcomes. This was the first trial to compare DAT with edoxaban against TAT (warfarin-based) in AF patients following PCI [p\u2009=\u20090.12) of NOAC but only non-inferiority (p\u2009=\u20090.001) for bleeding events, compared with a warfarin-based strategy.The ENTRUST-AF PCI trial enrolled 1506 individuals with AF and recent PCI who were randomized into two groups including TAT (warfarin and clopidogrel 75\u00a0mg daily for 1 year and aspirin 100\u00a0mg q.d. for 1\u201312 months) and edoxaban 60\u00a0mg daily plus clopidogrel 75\u00a0mg daily for 12 months. This trial adds to accumulating evidence that TAT is linked to higher major bleeding rates (20%), compared to 17% in the edoxaban arm (wing PCI . Howeverp\u2009<\u20090.001 for non-inferiority and superiority test). Patients who received a placebo had lower bleeding event rates with only 9% as compared with 16.1% of those who received aspirin (p\u2009<\u20090.001). For the secondary outcomes, the apixaban group had lower mortalities and hospitalizations than the warfarin group , and a comparable ischemic event (including stent thrombosis) in both groups. Regarding the aspirin versus placebo groups, there were similar numbers of deaths, hospitalizations, and ischemic events between the two groups [More recently, the AUGUSTUS trial included 4614 patients to compare two antithrombotic regimens (apixaban vs. VKA/warfarin). In addition, this study compared aspirin against a placebo. This broad objective came after the uncertainty of whether the low rate of bleeding observed on DAT in previous trials was the result of NOAC usage or due to withholding aspirin. Major or clinically significant bleeding occurred in only 10.5% of the individuals who received apixaban, while in 14.7% of those who had warfarin . However, this observation does not fully explain why it is higher during the first month. This leads to the consideration of other possible significant factors such as stent under-expansion and whether intracoronary imaging  was done to ensure stent optimization would add more clarity to the result. Additionally, some of these trials had a mixed population of ACS patients who were treated by PCI and others who had medical therapy alone. Therefore, further work is required to explore the mechanisms behind early stent thrombosis and to highlight other important clinical questions Figs.  and 9.FiThere are some strategies cardiologists can use to mitigate bleeding risk when managing such patients. First comes an appropriate assessment of the bleeding and ischemic risk using validated risk scores, such as the CHA2DS2-VASc and the HAS-BLED score. Second is the use of DAT with NOACs and clopidogrel instead of using triple therapy whenever possible . BecauseIn summary, the complicated relationship between AF, PCI, and antithrombotic therapy poses a significant challenge for clinicians aiming to find an optimal approach that effectively prevents thromboembolic events while minimizing the potential for bleeding complications. As the most frequent sustained arrhythmia that also raises the risk of thrombotic event for patients, making adequate antithrombotic therapy are essential to prevent strokes and other ischemic consequences. The advent of NOACs beyond the traditional warfarin therapy has improved safety profile. The comprehensive analysis of pivotal trials, such as WOEST, PIONEER AF-PCI, RE-DUAL PCI, ENTRUST-AF PCI, and AUGUSTUS, has yielded valuable insights into the relative advantages and disadvantages of various antithrombotic regimens. TAT was once the standard approach following PCI, but the weight of evidence has progressively shifted toward DAT with NOACs and clopidogrel, with the goal of achieving a balance between reducing bleeding risk and maintaining ischemic protection. The disparity in hemorrhage rates and ischemic outcomes between these trials highlights the need for individualized treatment decisions that take into account the unique clinical profiles of individual patients. Guidelines have evolved in response to these findings, guiding clinicians toward refined strategies that favor DAT over TAT, while emphasizing the role of risk assessment scores (CHADSVASC and HAS-BLED) and lowering the duration of aspirin as possible in high bleeding patients, and the use of protective strategies such as proton pump inhibitors to further mitigate bleeding risks. Nevertheless, it is noteworthy that the primary endpoints of these trials have predominantly concentrated on bleeding safety, possibly overshadowing rarer yet critical outcomes such as stent thrombosis, MI, and stroke. Additionally, emerging questions surrounding early stent thrombosis, use of lower NOAC doses need further investigation to enhance therapeutic precision. In this dynamic landscape of AF management following PCI, clinical judgment must align with evidence-based guidance, considering patient characteristics, ischemic risk, and bleeding vulnerability. The synthesis of research, guidelines, and clinical expertise will continue to improve the delicate art of managing anticoagulant strategies that protect against thromboembolic events while maintaining the delicate balance of bleeding risks, toward more tailored therapeutic approaches and improved patient outcomes."
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