Performance of high-throughput DNA quantification methods

BACKGROUND: The accuracy and precision of estimates of DNA concentration are critical factors for efficient use of DNA samples in high-throughput genotype and sequence analyses. We evaluated the performance of spectrophotometric (OD) DNA quantification, and compared it to two fluorometric quantification methods, the PicoGreen(® )assay (PG), and a novel real-time quantitative genomic PCR assay (QG) specific to a region at the human BRCA1 locus. Twenty-Two lymphoblastoid cell line DNA samples with an initial concentration of ~350 ng/uL were diluted to 20 ng/uL. DNA concentration was estimated by OD and further diluted to 5 ng/uL. The concentrations of multiple aliquots of the final dilution were measured by the OD, QG and PG methods. The effects of manual and robotic laboratory sample handling procedures on the estimates of DNA concentration were assessed using variance components analyses. RESULTS: The OD method was the DNA quantification method most concordant with the reference sample among the three methods evaluated. A large fraction of the total variance for all three methods (36.0–95.7%) was explained by sample-to-sample variation, whereas the amount of variance attributable to sample handling was small (0.8–17.5%). Residual error (3.2–59.4%), corresponding to un-modelled factors, contributed a greater extent to the total variation than the sample handling procedures. CONCLUSION: The application of a specific DNA quantification method to a particular molecular genetic laboratory protocol must take into account the accuracy and precision of the specific method, as well as the requirements of the experimental workflow with respect to sample volumes and throughput. While OD was the most concordant and precise DNA quantification method in this study, the information provided by the quantitative PCR assay regarding the suitability of DNA samples for PCR may be an essential factor for some protocols, despite the decreased concordance and precision of this method

Discovery and Differential Processing of HLA Class II-Restricted Minor Histocompatibility Antigen LB-PIP4K2A-1S and Its Allelic Variant by Asparagine Endopeptidase

Minor histocompatibility antigens are the main targets of donor-derived T-cells after allogeneic stem cell transplantation. Identification of these antigens and understanding their biology are a key requisite for more insight into how graft vs. leukemia effect and graft vs. host disease could be separated. We here identified four new HLA class II-restricted minor histocompatibility antigens using whole genome association scanning. For one of the new antigens, i.e., LB-PIP4K2A-1S, we measured strong T-cell recognition of the donor variant PIP4K2A-1N when pulsed as exogenous peptide, while the endogenously expressed variant in donor EBV-B cells was not recognized. We showed that lack of T-cell recognition was caused by intracellular cleavage by a protease named asparagine endopeptidase (AEP). Furthermore, microarray gene expression analysis showed that PIP4K2A and AEP are both ubiquitously expressed in a wide variety of healthy tissues, but that expression levels of AEP were lower in primary acute myeloid leukemia (AML). In line with that, we confirmed low activity of AEP in AML cells and demonstrated that HLA-DRB1*03:01 positive primary AML expressing LB-PIP4K2A-1S or its donor variant PIP4K2A-1N were both recognized by specific T-cells. In conclusion, LB-PIP4K2A-1S not only represents a novel minor histocompatibility antigen but also provides evidence that donor T-cells after allogeneic stem cell transplantation can target the autologous allelic variant as leukemia-associated antigen. Furthermore, it demonstrates that endopeptidases can play a role in cell type-specific intracellular processing and presentation of HLA class II-restricted antigens, which may be explored in future immunotherapy of AML

Evidence for the role of EPHX2 gene variants in anorexia nervosa.

Anorexia nervosa (AN) and related eating disorders are complex, multifactorial neuropsychiatric conditions with likely rare and common genetic and environmental determinants. To identify genetic variants associated with AN, we pursued a series of sequencing and genotyping studies focusing on the coding regions and upstream sequence of 152 candidate genes in a total of 1205 AN cases and 1948 controls. We identified individual variant associations in the Estrogen Receptor-ß (ESR2) gene, as well as a set of rare and common variants in the Epoxide Hydrolase 2 (EPHX2) gene, in an initial sequencing study of 261 early-onset severe AN cases and 73 controls (P=0.0004). The association of EPHX2 variants was further delineated in: (1) a pooling-based replication study involving an additional 500 AN patients and 500 controls (replication set P=0.00000016); (2) single-locus studies in a cohort of 386 previously genotyped broadly defined AN cases and 295 female population controls from the Bogalusa Heart Study (BHS) and a cohort of 58 individuals with self-reported eating disturbances and 851 controls (combined smallest single locus P<0.01). As EPHX2 is known to influence cholesterol metabolism, and AN is often associated with elevated cholesterol levels, we also investigated the association of EPHX2 variants and longitudinal body mass index (BMI) and cholesterol in BHS female and male subjects (N=229) and found evidence for a modifying effect of a subset of variants on the relationship between cholesterol and BMI (P<0.01). These findings suggest a novel association of gene variants within EPHX2 to susceptibility to AN and provide a foundation for future study of this important yet poorly understood condition

Long-term trends in anthropogenic land use in Siberia and the Russian Far East : a case study synthesis from Landsat

As globally important forested areas situated in a context of dramatic socio-economic changes, Siberia and the Russian Far East (RFE) are important regions to monitor for anthropogenic land-use trends. Therefore, we compiled decadal Landsat-derived land-cover and land-use data for eight dominantly rural case study sites in these regions and focused on trends associated with settlements, agriculture, logging, and roads 1975-2010. Several key spatial-temporal trends emerged from the integrated landscape-scale analyses. First, road building increased in all case study sites over the 35-year period, despite widespread socio-economic decline post-1990. Second, increase in settlements area was negligible over all sites. Third, increased road building, largely of minor roads, was especially high in more rugged and remote RFE case study sites not associated with greater agriculture extent or settlement densities. High demands for wood export coupled with the expansion of commercial timber harvest leases starting in the mid-1990s are likely among leading reasons for an increase in roads. Fourth, although fire was the dominant disturbance over all sites and dates combined, logging exerted a strong land-use pattern, serving as a reminder that considering local anthropogenic landscapes is important, especially in Siberia and the RFE, which represent almost 10% of the Earth's terrestrial land surface. The paper concludes by identifying remaining research needs regarding anthropogenic land use in the region: more frequent moderate spatial resolution imagery and greater access to more finely resolved statistical and other spatial data will enable further research. Social media abstract Landsat reveals long-term anthropogenic land-use trends in Siberia and Russian Far EastPeer reviewe

Pharmacotherapy for smoking cessation:effects by subgroup defined by genetically informed biomarkers

BACKGROUND: Smoking cessation therapies are not effective for all smokers, and researchers are interested in identifying those subgroups of individuals (e.g. based on genotype) who respond best to specific treatments. OBJECTIVES: To assess whether quit rates vary by genetically informed biomarkers within pharmacotherapy treatment arms and as compared with placebo. To assess the effects of pharmacotherapies for smoking cessation in subgroups of smokers defined by genotype for identified genome-wide significant polymorphisms. SEARCH METHODS: We searched the Cochrane Tobacco Addiction Group specialised register, clinical trial registries, and genetics databases for trials of pharmacotherapies for smoking cessation from inception until 16 August 2016. SELECTION CRITERIA: We included randomised controlled trials (RCTs) that recruited adult smokers and reported pharmacogenomic analyses from trials of smoking cessation pharmacotherapies versus controls. Eligible trials included those with data on a priori genome-wide significant (P andlt; 5 and#215; 10-8) single-nucleotide polymorphisms (SNPs), replicated non-SNPs, and/or the nicotine metabolite ratio (NMR), hereafter collectively described as biomarkers. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. The primary outcome was smoking abstinence at six months after treatment. The secondary outcome was abstinence at end of treatment (EOT). We conducted two types of meta-analyses- one in which we assessed smoking cessation of active treatment versus placebo within genotype groups, and another in which we compared smoking cessation across genotype groups within treatment arms. We carried out analyses separately in non-Hispanic whites (NHWs) and non-Hispanic blacks (NHBs). We assessed heterogeneity between genotype groups using Tand#178;, Iand#178;, and Cochrane Q statistics. MAIN RESULTS: Analyses included 18 trials including 9017 participants, of whom 6924 were NHW and 2093 NHB participants. Data were available for the following biomarkers: nine SNPs (rs1051730 (CHRNA3); rs16969968, rs588765, and rs2036527 (CHRNA5); rs3733829 and rs7937 (in EGLN2, near CYP2A6); rs1329650 and rs1028936 (LOC100188947); and rs215605 (PDE1C)), two variable number tandem repeats (VNTRs; DRD4 and SLC6A4), and the NMR. Included data produced a total of 40 active versus placebo comparisons, 16 active versus active comparisons, and 64 between-genotype comparisons within treatment arms.For those meta-analyses showing statistically significant heterogeneity between genotype groups, we found the quality of evidence (GRADE) to be generally moderate. We downgraded quality most often because of imprecision or risk of bias due to potential selection bias in genotyping trial participants. Comparisons of relative treatment effects by genotypeFor six-month abstinence, we found statistically significant heterogeneity between genotypes (rs16969968) for nicotine replacement therapy (NRT) versus placebo at six months for NHB participants (P = 0.03; n = 2 trials), but not for other biomarkers or treatment comparisons. Six-month abstinence was increased in the active NRT group as compared to placebo among participants with a GG genotype (risk ratio (RR) 1.47, 95% confidence interval (CI) 1.07 to 2.03), but not in the combined group of participants with a GA or AA genotype (RR 0.43, 95% CI 0.15 to 1.26; ratio of risk ratios (RRR) GG vs GA or AA of 3.51, 95% CI 1.19 to 10.3). Comparisons of treatment effects between genotype groups within pharmacotherapy randomisation armsFor those receiving active NRT, treatment was more effective in achieving six-month abstinence among individuals with a slow NMR than among those with a normal NMR among NHW and NHB combined participants (normal NMR vs slow NMR: RR 0.54, 95% CI 0.37 to 0.78; n = 2 trials). We found no such differences in treatment effects between genotypes at six months for any of the other biomarkers among individuals who received pharmacotherapy or placebo. AUTHORS' CONCLUSIONS: We did not identify widespread differential treatment effects of pharmacotherapy based on genotype. Some genotype groups within certain ethnic groups may benefit more from NRT or may benefit less from the combination of bupropion with NRT. The reader should interpret these results with caution because none of the statistically significant meta-analyses included more than two trials per genotype comparison, many confidence intervals were wide, and the quality of this evidence (GRADE) was generally moderate. Although we found evidence of superior NRT efficacy for NMR slow versus normal metabolisers, because of the lack of heterogeneity between NMR groups, we cannot conclude that NRT is more effective for slow metabolisers. Access to additional data from multiple trials is needed, particularly for comparisons of different pharmacotherapies.</p

Characterization of Imaging Luminance Measurement Devices (ILMDs)

CIE 244:2021This document describes the elements, function and characterization of imaging luminance measuring devices (ILMDs). Furthermore, the calibration of ILMDs is described and some guidelines for their use are provided. Using ILMDs the projection of the luminance distribution of a scene can be recorded and made available for further evaluation. In addition to a simple documentation of measurements, the geometrical assignment of the image points into the object coordinate system often allows more complex calculations by combining luminance, directional and, if necessary, solid angle information (e.g. for glare evaluation). In addition to the flexible evaluation option, it is possible to acquire a large number of measured values quickly and, if necessary, even synchronously. Furthermore, the type of evaluation can also be coupled to the image content, i.e. the image areas to be evaluated can be determined in the image either by their position within the image or by their luminance value

From Goya to Afghanistan. An essay on the ratio and ethics of medical war pictures

For centuries pictures of the dead and wounded have been part and parcel of war communications. Often the intentions were clear, ranging from medical instructions to anti-war protests. The public's response could coincide with or diverge from the publisher's intention. Following the invention of photography in the nineteenth century, and the subsequent claim of realism, the veracity of medical war images became more complex. Analysing and understanding such photographs have become an ethical obligation with democratic implications. We performed a multidisciplinary analysis of War Surgery (2008), a book containing harsh, full-colour photographs of mutilated soldiers from the Iraq and Afghanistan wars. Our analysis shows that, within the medical context, this book is a major step forward in medical war communication and documentation. In the military context the book can be conceived as an attempt to put matters right given the enormous sacrifice some individuals have suffered. For the public, the relationship between the 'reality' and 'truth' of such photographs is ambiguous, because only looking at the photographs without reading the medical context is limiting. If the observer is not familiar with medical practice, it is difficult for him to fully assess, signify and acknowledge the value and relevance of this book. We therefore assert the importance of the role of professionals and those in the humanities in particular in educating the public and initiating debate. © 2010 Taylor & Francis

Medicos, poultice wallahs and comrades in service: masculinity and military medicine in Britain during the First World War

The subject of British military medicine during the First World War has long been a fruitful one for historians of gender. From the bodily inspection of recruits and conscripts through the expanding roles of women as medical care providers to the physical and emotional aftermath of conflict experienced by men suffering from war-related wounds and illness, the medical history of the war has shed important light on how the war shaped British masculinities and femininities as cultural, subjective and embodied identities. Much of this literature has, however, focused on the gendered identities of female nurses and sick and wounded servicemen. Increasingly, however, more complex understandings of the ways in which medical caregiving in wartime shaped the gender identities of male caregivers are starting to emerge. This article explores some of these emerging understandings of the masculinity of male medical caregivers, and their relationship to the wider literature around the complex and sometimes contradictory relationship between warfare and medicine. It examines the ways in which the masculine identity of male medical caregivers from the ranks of the Royal Army Medical Corps, namely stretcher bearers and medical orderlies, was perceived and represented both by the men themselves and those they cared for. In doing so it argues that total war played a crucial role in shaping social and cultural perceptions of caregiving as a gendered practice. It also identifies particular tensions between continuity and change in social understandings of medical care as a gendered practice which would continue to shape twentieth-century British society in the war’s aftermath

Analysis of schizophrenia-related genes and electrophysiological measures reveals ZNF804A association with amplitude of P300b elicited by novel sounds

Several genes have recently been identified as risk factors for schizophrenia (SZ) by genome-wide association studies (GWAS), including ZNF804A which is thought to function in transcriptional regulation. However, the downstream pathophysiological changes that these genes confer remain to be elucidated. In 143 subjects (68 clinical high risk, first episode or chronic cases; 75 controls), we examined the association between 21 genetic markers previously identified by SZ GWAS or associated with putative intermediate phenotypes of SZ against three event-related potential (ERP) measures: mismatch negativity (MMN), amplitude of P300 during an auditory oddball task, and P300 amplitude during an auditory novelty oddball task. Controlling for age and sex, significant genetic association surpassing Bonferroni correction was detected between ZNF804A marker rs1344706 and P300 amplitude elicited by novel sounds (beta=4.38, P=1.03 × 10−4), which is thought to index orienting of attention to unexpected, salient stimuli. Subsequent analyses revealed that the association was driven by the control subjects (beta=6.35, P=9.08 × 10−5), and that the risk allele was correlated with higher novel P300b amplitude, in contrast to the significantly lower amplitude observed in cases compared to controls. Novel P300b amplitude was significantly correlated with a neurocognitive measure of auditory attention under interference conditions, suggesting a relationship between novel P300b amplitude and higher-order attentional processes. Our results suggest pleiotropic effects of ZNF804A on risk for SZ and neural mechanisms that are indexed by the novel P300b ERP component

SM-1 (APPR-1) RESEARCH AND DEVELOPMENT PROGRAM ACTIVITY BUILDUP PROGRAM. TASK I. Status Report for February to November 1958

Results are given of experimental work performed from February to November 1958 to determine the nature and extent of the build-up of radioactivity in the primary system of the SM-1 (APPR-1). The results of radiochemical and chemical analyses of primary water, circulating crud, and deposits removed from metal test specimens are presented. The relationships between build-up of activity and calendar hours, reactor operation, and crud levels are given. The methods and results of dose rate measurements made on the primary system components are reported. (auth