The association of a SNP upstream of INSIG2 with body mass index is reproduced in several but not all cohorts

A SNP upstream of the INSIG2 gene, rs7566605, was recently found to be associated with obesity as measured by body mass index (BMI) by Herbert and colleagues. The association between increased BMI and homozygosity for the minor allele was first observed in data from a genome-wide association scan of 86,604 SNPs in 923 related individuals from the Framingham Heart Study offspring cohort. The association was reproduced in four additional cohorts, but was not seen in a fifth cohort. To further assess the general reproducibility of this association, we genotyped rs7566605 in nine large cohorts from eight populations across multiple ethnicities (total n = 16,969). We tested this variant for association with BMI in each sample under a recessive model using family-based, population-based, and case-control designs. We observed a significant (p < 0.05) association in five cohorts but saw no association in three other cohorts. There was variability in the strength of association evidence across examination cycles in longitudinal data from unrelated individuals in the Framingham Heart Study Offspring cohort. A combined analysis revealed significant independent validation of this association in both unrelated (p = 0.046) and family-based (p = 0.004) samples. The estimated risk conferred by this allele is small, and could easily be masked by small sample size, population stratification, or other confounders. These validation studies suggest that the original association is less likely to be spurious, but the failure to observe an association in every data set suggests that the effect of SNP rs7566605 on BMI may be heterogeneous across population samples.     

The use of BMI and waist circumference as surrogates of body fat differs by ethnicity

Objective: To compare the prediction of percentage body fat using BMI and visceral adipose tissue (VAT) using waist circumference (WC) in individuals of Chinese, European, and South Asian origin. Research Methods and Procedures: Healthy men and women of Chinese, European, and South Asian origin (n = 627) between the ages of 30 and 65 years were recruited to ensure equal distribution of gender and representation across BMI ranges (18.5 to 24.9, 25 to 29.9, and ≥30 kg/m²). Participants were assessed for demographics, anthropometry, lifestyle, and regional adiposity. Percentage body fat and VAT were measured by DXA and computer tomography scan, respectively. Results: BMI and WC were highly correlated with total and regional measures of adiposity in each ethnic group. At any BMI, the percentage body fat of Chinese participants was similar to that of Europeans, but that of South Asians was greater by 3.9% (p < 0.001). Above a WC of 71.0 cm, the Chinese participants had an increasingly greater amount of VAT than the Europeans (p = 0.017 for interaction). South Asians had significantly more VAT than the Europeans at all but the most extreme WC (above 105 cm) (p < 0.05). Discussion: Compared with Europeans, percentage body fat was higher for a given BMI in South Asians, whereas VAT was higher for a given WC in both Chinese and South Asian men and women. These findings support the use of ethnic‐specific anthropometric targets.     

Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm

Background

Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete reprogramming of the male germ line could, in theory, result in both altered sperm DNA methylation and compromised spermatogenesis.

Methodology/Principal Finding

We determined concentration, motility and morphology of sperm in semen samples collected by male members of couples attending an infertility clinic. Using MethyLight and Illumina assays we measured methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm.

Conclusions

This is the first report of a broad epigenetic defect associated with abnormal semen parameters. Our results suggest that the underlying mechanism for these epigenetic changes may be improper erasure of DNA methylation during epigenetic reprogramming of the male germ line.     

Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine

Recent advances in redox proteomics have provided significant insight into the role of oxidative modifications in cellular signalling and metabolism. At present, these techniques rely heavily on Western blots to visualize the oxidative modification and corresponding two dimensional (2D) gels for detection of total protein levels, resulting in the duplication of efforts. A major limitation associated with this methodology includes problematic matching up of gels and blots due to the differences in processing and/or image acquisition. In this study, we present a new method which allows detection of protein oxidation and total protein on the same gel to improve matching in image analysis. Furthermore, the digested protein spots are compatible with standard MALDI mass spectrometry protein identification. The methodology highlighted here may be useful in facilitating the development of biomarkers, assessing potential therapeutic targets and elucidating new mechanisms of redox signalling in redox-related conditions.     

Fundamental characteristics of the immunoglobulin VH repertoire of chickens in comparison with those of humans, mice, and camelids

Examination of 1269 unique naive chicken V(H) sequences showed that the majority of positions in the framework (FW) regions were maintained as germline, with high mutation rates observed in the CDRs. Many FW mutations could be clearly related to the modulation of CDR structure or the V(H)-V(L) interface. CDRs 1 and 2 of the V(H) exhibited frequent mutation in solvent-exposed positions, but conservation of common structural residues also found in human CDRs at the same positions. In comparison with humans and mice, the chicken CDR3 repertoire was skewed toward longer sequences, was dominated by small amino acids (G/S/A/C/T), and had higher cysteine (chicken, 9.4%; human, 1.6%; and mouse, 0.25%) but lower tyrosine content (chicken, 9.2%; human, 16.8%; and mouse 26.4%). A strong correlation (R(2) = 0.97) was observed between increasing CDR3 length and higher cysteine content. This suggests that noncanonical disulfides are strongly favored in chickens, potentially increasing CDR stability and complexity in the topology of the combining site. The probable formation of disulfide bonds between CDR3 and CDR1, FW2, or CDR2 was also observed, as described in camelids. All features of the naive repertoire were fully replicated in the target-selected, phage-displayed repertoire. The isolation of a chicken Fab with four noncanonical cysteines in the V(H) that exhibits 64 nM (K(D)) binding affinity for its target proved these constituents to be part of the humoral response, not artifacts. This study supports the hypothesis that disulfide bond-constrained CDR3s are a structural diversification strategy in the restricted germline v-gene repertoire of chickens.     

Thioredoxin 1 is responsible for antibody disulfide reduction in CHO cell culture

During large-scale manufacturing of an IgG1 monoclonal antibody in Chinese hamster ovary (CHO) cells, reduction of the antibody's disulfide bonds was observed. We present evidence that mammalian thioredoxin 1 (TXN1) is the terminal enzyme responsible for this reduction event. We demonstrate a marked prevention of IgG1 disulfide bond reduction in a cell-density dependent manner by knocking down expression of TXN1 via lentivirus transduction of short hairpin RNA.     

Blk haploinsufficiency impairs the development, but enhances the functional responses, of MZ B cells

Blk was identified two decades ago as a B-cell-specific member of the Src family of tyrosine kinases. Recent studies, however, have discovered that Blk is expressed in many cell types outside of the B lineage, including early thymic precursors, interleukin-17-producing γδ T cells and pancreatic β-cells. In light of these recent discoveries, we performed a more comprehensive analysis of Blk expression patterns in hematopoietic cells and found that Blk is differentially expressed in mature B-cell subsets, with marginal zone (MZ) B cells expressing high levels, B1 B cells expressing intermediate-to-high levels and follicular (FO) B cells expressing low levels of Blk. To determine whether these differences in Blk expression levels reflected differential requirements for Blk in MZ, B1 and FO B-cell development, we analyzed the effects of reducing and eliminating Blk expression on B-cell development. We report that both Blk haploinsufficiency and Blk deficiency impaired the generation of MZ B cells. Moreover, although there were fewer MZ B cells in Blk(+/-) and Blk(-/-) mice as compared with Blk(+/+) mice, Blk-mutant MZ B cells were hyper-responsive to B-cell receptor stimulation, both in vitro and in vivo. Thus, this study has revealed a previously unappreciated role for Blk in the development and activation of MZ B cells.     

Functional characterization of a GJA1 frameshift mutation causing oculodentodigital dysplasia and palmoplantar keratoderma

A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms.     

Structure-activity relationships of triazolopyridine oxazole p38 inhibitors: identification of candidates for clinical development

The synthesis, structure-activity relationship, in vivo activity, and metabolic profile for a series of triazolopyridine-oxazole based p38 inhibitors are described. The deficiencies of the lead structure in the series, CP-808844, were overcome by changes to the C4 aryl group and the triazole side-chain culminating in the identification of several potential clinical candidates.     

Species coexistence, intransitivity, and topological variation in competitive tournaments

Competitive intransitivity occurs when species’ competitive abilities cannot be listed in a strict hierarchy, but rather form competitive loops, as in the game ‘Rock-Paper-Scissors’. Indices are useful for summarizing intransitivity in communities; however, as with most indices, a great deal of information is compressed into single number. So while recent ecological theory, experiments, and natural history observations demonstrate that competitive intransitivity can promote species coexistence, the consequence of variation in the ‘topology’ of competitive interactions that is not accounted for by intransitivity indices is much less well understood. We use a continuous analytical model and two complementary discrete lattice models (one spatially explicit, the other aspatial) to demonstrate that such variation does indeed greatly affect species coexistence. Specifically, we show that although intransitivity indices are good at capturing broad patterns of coexistence, communities with different levels of intransitivity can have equal coexistence, and communities with equal intransitivity can have different coexistence, due to underlying variation in competitive network topology.