Increased DNA typing success for feces and feathers of capercaillie (Tetrao urogallus) and black grouse (Tetrao tetrix)

Noninvasive sampling, for example, of droppings or feathers, is a promising approach for molecular genetic studies on endangered and elusive animal species. Yet, such specimens are known for containing only minute amounts of DNA, resulting in lower typing success rates relative to analyses on fresh tissues such as muscle or blood. Furthermore, artefactual signals as well as contamination are more likely to occur when DNA is limited. To increase the reliability of DNA typing from noninvasive samples, optimized DNA extraction and polymerase chain reaction protocols were developed, taking advantage of developments in the forensic field aiming at successful molecular genetic analysis of DNA templates being low in quality and quantity. In the framework of an extensive monitoring project on population dynamics of capercaillie and black grouse in the Tyrolean Alps, feces samples and molted feathers from both species were collected. On a subset comprising about 200 specimens of either species, eight polymorphic short tandem repeat (STR) markers were analyzed to test these improved protocols. Besides optimizing DNA yields, both lowered sample consumption and reduced hands‐on time were achieved, and the rates of informative profiles amounted to 90.7% for capercaillie and 92.4% for black grouse. Similarly, high success rates had not been achieved in earlier studies and demonstrate the benefit of the improved methodology, which should be easily adaptable for use on animal species other than those studied here. The STR genotypes were not only powerful enough to discriminate among unrelated birds but also appeared fit for telling apart closely related animals, as indicated by Pi and Pi_sib values. The software package allelematch aided analysis of genotypes featuring possible dropout and drop‐in effects. Finally, a comparison between molecular genetic and morphology‐based species‐of‐origin determination revealed a high degree of concordance.     

An ER clamp for endosome fission

Endosomes are known to undergo budding and fission reactions that separate regions destined for lysosomal degradation from carriers to be recycled to the plasma membrane. A recent paper (Rowland et al, 2014 ) shows that contact sites between endosomes and the endoplasmic reticulum (ER) define the position and timing for fission. This uncovers an unanticipated role for the ER in controlling endosomal sorting and maturation.     

Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants

Background

Obesity is a major health problem. Although heritability is substantial, genetic mechanisms predisposing to obesity are not very well understood. We have performed a genome wide association study (GWA) for early onset (extreme) obesity.

Methodology/Principal Findings

a) GWA (Genome-Wide Human SNP Array 5.0 comprising 440,794 single nucleotide polymorphisms) for early onset extreme obesity based on 487 extremely obese young German individuals and 442 healthy lean German controls; b) confirmatory analyses on 644 independent families with at least one obese offspring and both parents. We aimed to identify and subsequently confirm the 15 SNPs (minor allele frequency ≥10%) with the lowest p-values of the GWA by four genetic models: additive, recessive, dominant and allelic. Six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) within one linkage disequilibrium (LD) block including the GWA SNP rendering the lowest p-value (rs1121980; log-additive model: nominal p = 1.13×10⁻⁷, corrected p = 0.0494; odds ratio (OR)_CT 1.67, 95% confidence interval (CI) 1.22–2.27; OR_TT 2.76, 95% CI 1.88–4.03) belonged to the 15 SNPs showing the strongest evidence for association with obesity. For confirmation we genotyped 11 of these in the 644 independent families (of the six FTO SNPs we chose only two representing the LD bock). For both FTO SNPs the initial association was confirmed (both Bonferroni corrected p<0.01). However, none of the nine non-FTO SNPs revealed significant transmission disequilibrium.

Conclusions/Significance

Our GWA for extreme early onset obesity substantiates that variation in FTO strongly contributes to early onset obesity. This is a further proof of concept for GWA to detect genes relevant for highly complex phenotypes. We concurrently show that nine additional SNPs with initially low p-values in the GWA were not confirmed in our family study, thus suggesting that of the best 15 SNPs in the GWA only the FTO SNPs represent true positive findings.     

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.     

Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM₆), octasaccharide (HM₈), and decasaccharide (HM₁₀), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM₈ and HM₁₀ are significantly more potent than HM₆ in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM₈ relative to HM₆ in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM₈ to bind and dimerize FGF2. Notably FGF2·HM₈ exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.     

Pitfalls in the sample preparation and analysis of N-acylethanolamines

N-acylethanolamines (NAEs) are a group of lipid mediators synthesized in response to a number of physiological and pathological stimuli. Because of the low tissue concentrations of NAEs, analyses often include liquid extraction followed by solid-phase extraction and subsequent quantitation by LC/MS or GC/MS. Reported levels of NAEs vary considerably, however, and often no explanation is given for these discrepancies. Brought on by difficulties encountered during method development, the effects of using four different brands of silica-containing solid phase extraction (SPE) columns and five different brands of chloroform for sample preparation were investigated. Considerable variation in the retention and recoveries of seven NAEs and 2-arachidonoylglycerol existed between the SPE columns. Furthermore, it was found that some chloroforms contained quantifiable amounts of N-palmitoylethanolamine and N-stearoylethanolamine. Finally, it was found that use of one of the chloroforms resulted in a loss of N-oleoylethanolamine from solution due to addition of chlorine to the ω-9 bond. The identity of this reaction product was confirmed by LC-MS/MS and NMR. It is recommended that these aspects of sample preparation and analysis should be thoroughly validated during method development and the relevant information on specific brands used be reported in future communications in order to better estimate the validity of reported quantitative data.     

Ionenverhältnisse und die Besiedlung hyperhaliner Gewässer,besonders durch Insekten

Zusammenfassung Die Besiedlung hyperhaliner Gewässer ist nur den Tieren möglich, die eine ausgeprägte Osmoregulation haben. In einem kurzen Überblick über die wichtigsten Ionenwirkungen wird die Bedeutung der einzelnen Ionen auf die Osmoregulation dargelegt. Dabei wird vor allem auf die Wirkung der gemeinsamen Einwirkung mehrerer Ionen hingewiesen. Im ökologischen Teil wird auf die Besiedlungsbedingungen hyperhaliner Gewässer und die im Freiland beobachtete Verteilung der Organismen in Gewässern mit unbalanziertem Ionenmilieu eingegangen. Um die an derartige Ionenverhältnisse gebundenen Tiere und ihre Lebensbedingungen zu charakterisieren wird der Ausdruck Kairie vorgeschlagen und diskutiert, der eine Erweiterung des Rapie-Begriffes vonPora darstellt. Ein stenokairischer Organismus stellt sehr enge Bedingungen an die ionale Zusammensetzung des Aussenmilieus, ein eurykairischer Organismus vermag Gewässer verschiedenster ionaler Zusammensetzung zu besiedeln.     

A global analysis of peptide fragmentation variability

Understanding the fragmentation process in MS/MS experiments is vital when trying to validate the results of such experiments, and one way of improving our understanding is to analyze existing data. We here present our findings from an analysis of a large and diverse data set of MS/MS-based peptide identifications, in which each peptide has been identified from multiple spectra, recorded on two commonly used types of electrospray instruments. By analyzing these data we were able to study fragmentation variability on three levels: (i) variation in detection rates and intensities for fragment ions from the same peptide sequence measured multiple times on a single instrument; (ii) consistency of rank-based fragmentation patterns; and (iii) a set of general observations on fragment ion occurrence in MS/MS experiments, regardless of sequence. Our results confirm that substantial variation can be found at all levels, even when high-quality identifications are used and the experimental conditions as well as the peptide sequences are kept constant. Finally, we discuss the observed variability in light of ongoing efforts to create spectral libraries and predictive software for target selection in targeted proteomics.