Recombining overlapping BACs into a single larger BAC

BACKGROUND: BAC clones containing entire mammalian genes including all the transcribed region and long range controlling elements are very useful for functional analysis. Sequenced BACs are available for most of the human and mouse genomes and in many cases these contain intact genes. However, large genes often span more than one BAC, and single BACs covering the entire region of interest are not available. Here we describe a system for linking two or more overlapping BACs into a single clone by homologous recombination. RESULTS: The method was used to link a 61-kb insert carrying the final 5 exons of the human CFTR gene onto a 160-kb BAC carrying the first 22 exons. Two rounds of homologous recombination were carried out in the EL350 strain of bacteria which can be induced for the Red genes. In the first round, the inserts of the two overlapping BACs were subcloned into modified BAC vectors using homologous recombination. In the second round, the BAC to be added was linearised with the very rare-cutting enzyme I-PpoI and electroporated into recombination efficient EL350 bacteria carrying the other BAC. Recombined BACs were identified by antibiotic selection and PCR screening and 10% of clones contained the correctly recombined 220-kb BAC. CONCLUSION: The system can be used to link the inserts from any overlapping BAC or PAC clones. The original orientation of the inserts is not important and desired regions of the inserts can be selected. The size limit for the fragments recombined may be larger than the 61 kb used here and multiple BACs in a contig could be combined by alternating use of the two pBACLink vectors. This system should be of use to many investigators wishing to carry out functional analysis on large mammalian genes which are not available in single BAC clones

Cryonic Suspension and the Law

Three central problems which adversely affect the intriguing use, development, and perfection of the cryonic suspension of individuals are analyzed: the extent to which a physician may be guilty of malpractice in assisting with a suspension - owing to present weaknesses in defining death and coordinate criminal liability attaching theretofor murder; the need for a recognition of suspension; and the present effect of the law\u27s anachronistic treatment of estate devolution upon a cryon - or one undergoing suspension. To meet these difficulties, a partnership is proposed between law and medicine which would respond to challenges to this type of new biology in measured anticipation of the future consequences, rather than with a passive spirit of resignation to things to come

Cryonic Suspension and the Law

Three central problems which adversely affect the intriguing use, development, and perfection of the cryonic suspension of individuals are analyzed: the extent to which a physician may be guilty of malpractice in assisting with a suspension - owing to present weaknesses in defining death and coordinate criminal liability attaching theretofor murder; the need for a recognition of suspension; and the present effect of the law\u27s anachronistic treatment of estate devolution upon a cryon - or one undergoing suspension. To meet these difficulties, a partnership is proposed between law and medicine which would respond to challenges to this type of new biology in measured anticipation of the future consequences, rather than with a passive spirit of resignation to things to come

Associations between retirement reasons, chronic pain, athletic identity, and depressive symptoms among former professional footballers

Background: Retirement from professional sport has been recognised as a major psychological stressor, and there is a need to identify factors that increase the risk of mental health problems after career termination. The current study examined associations between career-ending injury, chronic pain, athletic identity and depressive symptomology in retired professional footballers. Methods: A cross-sectional study was performed with 307 retired male footballers who had played within a professional United Kingdom league. Participants completed measures of depressive symptoms (Short Depression-Happiness Scale), chronic pain (Pain Intensity Numerical Rating Scale), and athletic identity (Athletic Identity Measurement Scale), and reported their reasons for retirement. Results: A total of 48 participants (16%) met the cut-off score for possible cases of clinically-relevant depression. These participants were more recently retired, and had higher athletic identity than those without depressive symptoms. Former players with depressive symptoms were more likely to cite injury as a retirement reason, and report higher levels of ongoing injury-related pain. Multivariate logistic regression revealed that presence of depressive symptoms was independently associated with retirement through injury (OR = 3.44; 95% CI = 1.39, 8.51), higher pain levels (OR = 1.38; 95% CI = 1.02, 1.86), and increased athletic identity (OR = 1.28; 95% CI = 1.14, 1.44). Conclusions: Career-ending injury is strongly associated with higher odds of depressive symptomology during retirement, while experiencing chronic pain, and maintaining a high sense of athletic identity, are additional potential contributors

A new spreadsheet method for the analysis of bivariate flow cytometric data

BACKGROUND: A useful application of flow cytometry is the investigation of cell receptor-ligand interactions. However such analyses are often compromised due to problems interpreting changes in ligand binding where the receptor expression is not constant. Commonly, problems are encountered due to cell treatments resulting in altered receptor expression levels, or when cell lines expressing a transfected receptor with variable expression are being compared. To overcome this limitation we have developed a Microsoft Excel spreadsheet that aims to automatically and effectively simplify flow cytometric data and perform statistical tests in order to provide a clearer graphical representation of results. RESULTS: To demonstrate the use and advantages of this new spreadsheet method we have investigated the binding of the transmembrane adhesion receptor CD44 to its ligand hyaluronan. In the first example, phorbol ester treatment of cells results in both increased CD44 expression and increased hyaluronan binding. By applying the spreadsheet method we effectively demonstrate that this increased ligand binding results from receptor activation. In the second example we have compared AKR1 cells transfected either with wild type CD44 (WT CD44) or a mutant with a truncated cytoplasmic domain (CD44-T). These two populations do not have equivalent receptor expression levels but by using the spreadsheet method hyaluronan binding could be compared without the need to generate single cell clones or FACS sorting the cells for matching CD44 expression. By this method it was demonstrated that hyaluronan binding requires a threshold expression of CD44 and that this threshold is higher for CD44-T. However, at high CD44-T expression, binding was equivalent to WT CD44 indicating that the cytoplasmic domain has a role in presenting the receptor at the cell surface in a form required for efficient hyaluronan binding rather than modulating receptor activity. CONCLUSION: Using the attached spreadsheets and instructions, a simple post-acquisition method for analysing bivariate flow cytometry data is provided. This method constitutes a straightforward improvement over the standard graphical output of flow cytometric data and has the significant advantage that ligand binding can be compared between cell populations irrespective of receptor expression levels

Sociodemographic inequalities in student achievement: An intersectional multilevel analysis of individual heterogeneity and discriminatory accuracy (MAIHDA) with application to students in London, England

Sociodemographic inequalities in student achievement are a persistent concern for education systems and are increasingly recognized to be intersectional. Intersectionality considers the multidimensional nature of disadvantage, appreciating the interlocking social determinants which shape individual experience. Intersectional multilevel analysis of individual heterogeneity and discriminatory accuracy (MAIHDA) is a new approach developed in population health but with limited application in educational research. In this study, we introduce and apply this approach to study sociodemographic inequalities in student achievement across two cohorts of students in London, England. We define 144 intersectional strata arising from combinations of student age, gender, free school meal status, special educational needs, and ethnicity. We find substantial strata-level variation in achievement composed primarily by additive rather than interactive effects with results stubbornly consistent across the cohorts. We conclude that policymakers should pay greater attention to multiply marginalized students and intersectional MAIHDA provides a useful approach to study their experiences.Comment: 63 pages (main text 39 pages), 10 figures, 10 table

Determining the role for uric acid in non-alcoholic steatohepatitis development and the utility of urate metabolites in diagnosis:An opinion review

There has long been a recognised association between non-alcoholic fatty liver disease (NAFLD) and the composite aspects of the metabolic syndrome. Part of this association highlighted the supposed co-existence of elevated uric acid levels in those with NAFLD. There is interest in exploitation of this as a putative diagnostic and prognostic biomarker in NAFLD. Given the increased economic and health burden associated with the NAFLD epidemic, improved methods of population-based, minimally-invasive methods and biomarkers are clearly highly sought and necessary. In this opinion review we review the proposed role of uric acid in the pathogenesis of NAFLD and its potential utilisation in the diagnosis and monitoring of the disease process

Understanding the role of Epstein-Barr virus in T- and NK-cell disorders

Epstein-Barr virus (EBV) is associated with B- and epithelial cell malignancies. It is also associated with lymphoproliferations and malignancies of T- and natural killer (NK) cells. The global impact of these conditions is significant, and although rare, they are aggressive and are often resistant to treatment. Diagnosis is often delayed, and evidence-based treatment strategies are limited due to their rarity. Viral gene expression in extranodal T- and NK-cell lymphoma (ENKTL), chronic active Epstein-Barr virus (CAEBV) and haemophagocytic lymphohistiocytosis (HLH) is limited. The viral latent membrane proteins LMP1, LMP2A and LMP2B-TR-TR have growth-transforming properties in B- and epithelial cells. Their effects on cellular gene expression in primary NK cells included pathways involved in cell cycle and stress responses. LMP1 and LMP2B-TR expression by ENKTL and CAEBV cell lines is associated with increased survival in the absence of relevant growth factors, but also with increased susceptibility to apoptosis. This cannot be fully explained by variation in the expression of proteins involved in the intrinsic apoptotic pathway. Finally, we describe PrimeFlow RNA, a new protocol for identification of the EBV-infected lymphocyte subset. Importantly, this technique means that we can begin to identify druggable targets on the EBV-infected cells directly from patient blood samples

The "acutely sick" African child: applying new statistical methods to delineate mortality risks and identify ways to improve management

The admission burden to paediatric wards in African hospitals is very high, and many children have life-threatening complications of common infectious diseases including malaria. In this setting the Fluid Expansion As Supportive Therapy (FEAST) trial unexpectedly showed that fluid resuscitation (giving a bolus of saline or albumin) to febrile children with impaired perfusion was harmful. This PhD used data from the FEAST trial to answer several important questions: 1) how can children at the highest risk of mortality be identified and prioritised in these low-income settings; 2) were there surrogate markers in bedside measures recorded over admission that could explain some of the detrimental impact of the bolus; and 3) did the bolus increase mortality risk immediately after administration or was there a different mechanism of action? Prognostic factors for mortality were identified in the FEAST data and a bedside risk score developed. The score was validated in data collected on children admitted to a rural district hospital in Kenya and compared to other risk scores. The heterogeneity of the effect of bolus over levels of different measures, including malaria parasitaemia, was explored. The proportion of treatment effect explained by measures recorded over time was calculated. No one measure was shown to be a suitable surrogate marker, but the impact of the bolus varied across levels of oxygen saturation, and across levels of base excess in those with malaria at baseline. Further insight into the mechanism by which the bolus had detrimental impact on the children in the FEAST trial was sought by modelling the mortality risk over time. The maximum mortality risk occurred in both arms within the first 2 hours but the risk in the bolus arm was slower to decrease, showing that children were recovering more slowly in the bolus arms compared to the no bolus arm