Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer

Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal alpha-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

PMCID: PMC3534021This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Association study of genetic variation in DNA repair pathway genes and risk of basal cell carcinoma

DNA repair plays a critical role in protecting the genome from ultraviolet radiation and maintaining the genomic integrity of cells. Genetic variants in DNA repair-related genes can influence an individual's DNA repair capacity, which may be related to the risk of developing basal cell carcinoma (BCC). We comprehensively assessed the associations of 2,965 independent single-nucleotide polymorphisms (SNPs) across 165 DNA repair pathway genes with BCC risk in a genome-wide association meta-analysis totaling 17,187 BCC cases and 287,054 controls from two data sets. After multiple testing corrections, we identified three SNPs (rs2805831 upstream of XPA: OR = 0.93, P = 1.35 × 10-6 ; rs659857 in exon of MUS81: OR = 1.06, P = 3.09 × 10-6 and rs57343616 in 3' UTR of NABP2: OR = 1.11, P = 6.47 × 10-6 ) as significantly associated with BCC risk in meta-analysis, and all of them were nominally significant in both data sets. Furthermore, rs659857 [T] was significantly associated with decreased expression of MUS81 mRNA in the expression quantitative trait locus (eQTL) analysis. Our findings suggest that the inherited common variation in three DNA repair genes-XPA, MUS81 and NABP2-may be involved in the development of BCC. To our knowledge, our study is the first report thoroughly examining the effects of SNPs across DNA repair pathway genes on BCC risk based on a genome-wide association meta-analysis

Genomic and environmental selection patterns in two distinct lettuce crop-wild hybrid crosses

Genomic selection patterns and hybrid performance influence the chance that crop (trans)genes can spread to wild relatives. We measured fitness(-related) traits in two different field environments employing two different crop–wild crosses of lettuce. We performed quantitative trait loci (QTL) analyses and estimated the fitness distribution of early- and late-generation hybrids. We detected consistent results across field sites and crosses for a fitness QTL at linkage group 7, where a selective advantage was conferred by the wild allele. Two fitness QTL were detected on linkage group 5 and 6, which were unique to one of the crop–wild crosses. Average hybrid fitness was lower than the fitness of the wild parent, but several hybrid lineages outperformed the wild parent, especially in a novel habitat for the wild type. In early-generation hybrids, this may partly be due to heterosis effects, whereas in late-generation hybrids transgressive segregation played a major role. The study of genomic selection patterns can identify crop genomic regions under negative selection across multiple environments and cultivar–wild crosses that might be applicable in transgene mitigation strategies. At the same time, results were cultivar-specific, so that a case-by-case environmental risk assessment is still necessary, decreasing its general applicability

Right and left ventricles: as inseparable as the twin brothers ‘Castor and Pollux’

NA (Editorial

Problem gambling: a suitable case for social work?

Problem gambling attracts little attention from health and social care agencies in the UK. Prevalence surveys suggest that 0.6% of the population are problem gamblers and it is suggested that for each of these individuals, 10–17 other people, including children and other family members, are affected. Problem gambling is linked to many individual and social problems including: depression, suicide, significant debt, bankruptcy, family conflict, domestic violence, neglect and maltreatment of children and offending. This makes the issue central to social work territory. Yet, the training of social workers in the UK has consistently neglected issues of addictive behaviour. Whilst some attention has been paid in recent years to substance abuse issues, there has remained a silence in relation to gambling problems. Social workers provide more help for problems relating to addictions than other helping professions. There is good evidence that treatment, and early intervention for gambling problems, including psycho-social and public health approaches, can be very effective. This paper argues that problem gambling should be moved onto the radar of the social work profession, via inclusion on qualifying and post-qualifying training programmes and via research and dissemination of good practice via institutions such as the Social Care Institute for Excellence (SCIE). Keywords: problem gambling; addictive behaviour; socia

Roux-en-Y Gastric Bypass Increases Glycemic Variability and Time in Hypoglycemia in Patients With Obesity and Prediabetes or Type 2 Diabetes: A Prospective Cohort Study

OBJECTIVE: Roux-en-Y gastric bypass (RYGB) is an established treatment for type 2 diabetes and obesity. The study objective was to establish RYGB's effects on glycemic variability (GV) and hypoglycemia. RESEARCH DESIGN AND METHODS: This was a prospective observational study of 10 participants with obesity and prediabetes or type 2 diabetes who underwent RYGB. Patients were studied before RYGB (Pre) and 1 month, 1 year, and 2 years postsurgery with continuous glucose measurement (CGM). A mixed-meal test (MMT) was conducted at Pre, 1 month, and 1 year. RESULTS: After RYGB, mean CGM decreased (at 1 month, 1 year, and 2 years), and GV increased (at 1 year and 2 years). Five of the 10 participants had a percent time in range (%TIR) <3.0 mmol/L (54 mg/dL) greater than the international consensus target of 1% at 1 or 2 years. Peak glucagon-like peptide-1 (GLP-1) and glucagon area under the curve during MMT were positively and negatively associated, respectively, with contemporaneous %TIR <3.0 mmol/L. CONCLUSIONS: Patients undergoing RYGB are at risk for development of postbariatric hypoglycemia due to a combination of reduced mean glucose, increased GV, and increased GLP-1 response

Improvements in sperm motility following low or high intensity dietary interventions in men with obesity

Introduction: Obesity increases risks of male infertility, but bariatric surgery does not improve semen quality. Recent uncontrolled studies suggest that a low-energy diet (LED) improves semen quality. Further evaluation within a randomized, controlled setting is warranted. Methods: Men with obesity (18-60 years) with normal sperm concentration (normal count) (n = 24) or oligozoospermia (n = 43) were randomized 1:1 to either 800 kcal/day LED for 16 weeks or control, brief dietary intervention (BDI) with 16 weeks’ observation. Semen parameters were compared at baseline and 16 weeks. Results: Mean age of men with normal count was 39.4 ± 6.4 in BDI and 40.2 ± 9.6 years in the LED group. Mean age of men with oligozoospermia was 39.5 ± 7.5 in BDI and 37.7 ± 6.6 years in the LED group. LED caused more weight loss than BDI in men with normal count (14.4 vs 6.3 kg; P < .001) and men with oligozoospermia (17.6 vs 1.8 kg; P < .001). Compared with baseline, in men with normal count total motility (TM) increased 48 ± 17% to 60 ± 10% (P < .05) after LED, and 52 ± 8% to 61 ± 6% (P < .0001) after BDI; progressive motility (PM) increased 41 ± 16% to 53 ± 10% (P < .05) after LED, and 45 ± 8% to 54 ± 65% (P < .001) after BDI. In men with oligozoospermia compared with baseline, TM increased 35% [26] to 52% [16] (P < .05) after LED, and 43% [28] to 50% [23] (P = .0587) after BDI; PM increased 29% [23] to 46% [18] (P < .05) after LED, and 33% [25] to 44% [25] (P < .05) after BDI. No differences in postintervention TM or PM were observed between LED and BDI groups in men with normal count or oligozoospermia. Conclusion: LED or BDI may be sufficient to improve sperm motility in men with obesity. The effects of paternal dietary intervention on fertility outcomes requires investigation

A Novel Approach to Standard Techniques in the Assessment and Quantification of the Interventricular Systolic Relationship

<p>Abstract</p> <p>Background</p> <p>Blood flow between the right and left ventricles is subject to the continuity equation and systolic ventricular interdependence. Quantification of this relationship might aid in understanding inter-ventricular function. The purpose of this study was to evaluate and quantify ventricular interdependence by directly comparing right and left ventricular systolic function though echocardiographic surrogates of right and left ventricular systolic function such as MAPSE, TAPSE, RV TVI and LV TVI.</p> <p>Methods</p> <p>This study prospectively evaluated 51 healthy participants (mean age, 41 ± 17 years) by resting echocardiography. In addition to standard measurements, tricuspid annular plane of systolic excursion, (TAPSE), mitral annular plane of systolic excursion (MAPSE), and the peak annulus systolic velocity of the right ventricular (RVs) and left ventricular (LVs) free walls were measured by M-mode and pulsed wave Doppler tissue echocardiography and further evaluated for variance across age, gender, and body surface area.</p> <p>Results</p> <p>TAPSE (22.1 ± 2.9 mm) was over 54.5% greater than MAPSE (14.3 ± 2.6 mm) and RVs was 64.4% greater than LVs. The LV to RV systolic relationship measured by MAPSE/TAPSE and LVs/RVs ratios were 0.66 ± 0.14 and 0.76 ± 0.21 respectively. These values were not significantly affected by age, gender or body surface area (BSA).</p> <p>Conclusion</p> <p>MAPSE/TAPSE and LVs/RVs ratios appear stable across age, gender, and BSA potentially making them good surrogates of systolic ventricular relationship and interdependence.</p