Crossing the threshold: an amphibian assemblage highly infected with Batrachochytrium dendrobatidis in the southern Brazilian Atlantic forest

The fungal infection caused by Batrachochytrium dendrobatidis (Bd) in amphibians is known to be lethal when infection intensity values exceed loads of 10,000 zoospores per individual. We investigated Bd infection intensity in 100 anurans of southern Brazil. Almost half of the individuals were infected and the intensity ranged from four to about 156,000 zoospore genomic equivalents. We found no clinical signs of chytridiomycosis and no evidence of mortality. However, we observed a reduction in the number of infected individuals with loads above 10,000 zoospores. This fact could be considered indirect evidence that individuals with high loads are removed from the population.     

The role of pigment based plumage traits in resolving conflicts

The role of melanin ‘badges of status’, in male–male competition has been well‐studied, in contrast, carotenoid based plumage has largely been examined in the context of female mate choice. Recent work has shown that carotenoid signals can also function in male–male competition, although the functions of the two types of signals is currently unclear. Here, we examine the relationships between colouration, dominance and aggression in the crimson finch Neochmia phaeton, a species where males have both conspicuous red carotenoid plumage and a black melanin patch. We examined the importance of carotenoid and melanin based signals in three contexts: 1) among free‐living birds interacting at a feeding station: we found that neither colour signal influenced the outcome of interactions; 2) in staged dyadic contest in captivity: we found that coloration from carotenoid pigments was positively related to the probability of winning a contest, while the size of the melanin plumage patch was not related to winning; and 3) in staged dyadic contests where male plumage colour had been masked: we found that the number of interactions required to determine dominance increased. While the underlying natural plumage colour was still important in these contests, birds with more intense carotenoid colouration were now more likely to lose. These results confirm carotenoid‐based signalling in male–male contests. However this signal is used in conjunction with other factors such as self‐assessment and body condition. Contrary to traditional expectations, the black melanin patch was not found to be important in this context.     

Evolution of ASPM coding variation in apes and associations with brain structure in chimpanzees

Studying genetic mechanisms underlying primate brain morphology can provide insight into the evolution of human brain structure and cognition. In humans, loss‐of‐function mutations in the gene coding for ASPM (Abnormal Spindle Microtubule Assembly) have been associated with primary microcephaly, which is defined by a significantly reduced brain volume, intellectual disability and delayed development. However, less is known about the effects of common ASPM variation in humans and other primates. In this study, we characterized the degree of coding variation at ASPM in a large sample of chimpanzees (N = 241), and examined potential associations between genotype and various measures of brain morphology. We identified and genotyped five non‐synonymous polymorphisms in exons 3 (V588G), 18 (Q2772K, K2796E, C2811Y) and 27 (I3427V). Using T1‐weighted magnetic resonance imaging of brains, we measured total brain volume, cerebral gray and white matter volume, cerebral ventricular volume, and cortical surface area in the same chimpanzees. We found a potential association between ASPM V588G genotype and cerebral ventricular volume but not with the other measures. Additionally, we found that chimpanzee, bonobo, and human lineages each independently show a signature of accelerated ASPM protein evolution. Overall, our results suggest the potential effects of ASPM variation on cerebral cortical development, and emphasize the need for further functional studies. These results are the first evidence suggesting ASPM variation might play a role in shaping natural variation in brain structure in nonhuman primates.     

Structure elucidation of arabinoxylan isomers by normal phase HPLC-MALDI-TOF/TOF-MS/MS

Normal phase-high performance liquid chromatography (NP-HPLC) coupled to matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry is evaluated for the detailed structural characterization of various isomers of arabinoxylan (AX) oligosaccharides produced from endo-beta-(1-->4)-xylanase (endoxylanase) digestion of wheat AX. The fragmentation characteristics of these oligosaccharides upon MALDI-TOF/TOF high-energy collision induced dissociation (CID) were investigated using purified AX oligosaccharide standards labeled at the reducing end with 2-aminobenzoic acid (2-AA). A variety of cross-ring cleavages and 'elimination' ions in the fragment ion spectra provided extensive structural information, including Araf substitution patterns along the xylan backbone and comprehensive linkage assignment. The off-line coupling of this MALDI-CID technique to capillary normal phase HPLC enabled the separation and identification of isomeric oligosaccharides (DP 4-8) produced by endoxylanase digestion of AX. Furthermore, this technique was used to characterize structurally different isomeric AX oligosaccharides produced by endoxylanase enzymes with different substrate specificities.     

Tbx20 regulation of endocardial cushion cell proliferation and extracellular matrix gene expression

While recent work has implicated Tbx20 in myocardial maturation and proliferation, the role of Tbx20 in heart valve development remains relatively unknown. Tbx20 expression was manipulated in primary avian endocardial cells in order to elucidate its function in developing endocardial cushions. Tbx20 gain of function was achieved with a Tbx20-adenovirus, and endogenous Tbx20 expression was inhibited with Tbx20-specific siRNA in cultured endocardial cushion cells. With Tbx20 gain of function, the expression of chondroitin sulfate proteoglycans (CSPG), including aggrecan and versican, was decreased, while the expression of the matrix metalloproteinases (MMP) mmp9 and mmp13 was increased. Consistent results were observed with Tbx20 loss of function, where the expression of CSPG genes increased and MMP genes decreased. In addition, cushion mesenchyme proliferation increased with infection of a Tbx20-adenovirus and decreased with transfection of Tbx20-specfic siRNA. Furthermore, BMP2 treatment resulted in increased Tbx20 expression in endocardial cushion cells, and loss of Tbx20 led to increased Tbx2 and decreased N-myc gene expression. Taken together, these data support a role for Tbx20 in repressing extracellular matrix remodeling and promoting cell proliferation in mesenchymal valve precursor populations in endocardial cushions during embryonic development.     

O-fucosylation is required for ADAMTS13 secretion

ADAMTS13 is a plasma metalloproteinase that cleaves von Willebrand factor to smaller, less thrombogenic forms. Deficiency of ADAMTS13 activity in plasma leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains eight thrombospondin type 1 repeats (TSR), seven of which contain a consensus sequence for the direct addition of fucose to the hydroxyl group of serine or threonine. Mass spectral analysis of tryptic peptides derived from human ADAMTS13 indicate that at least six of the TSRs are modified with an O-fucose disaccharide. Analysis of [(3)H]fucose metabolically incorporated into ADAMTS13 demonstrated that the disaccharide has the structure glucose-beta1,3-fucose. Mutation of the modified serine to alanine in TSR2, TSR5, TSR7, and TSR8 reduced the secretion of ADAMTS13. Mutation of more than one site dramatically reduced secretion regardless of the sites mutated. When the expression of protein O-fucosyltransferase 2 (POFUT2), the enzyme that transfers fucose to serines in TSRs, was reduced using siRNA, the secretion of ADAMTS13 decreased. A similar outcome was observed when ADAMTS13 was expressed in a cell line unable to synthesize the donor for fucose addition, GDP-fucose. Although overexpression of POFUT2 did not affect the secretion of wild-type ADAMTS13, it did increase the secretion of the ADAMTS13 TSR1,2 double mutant but not that of ADAMTS13 TSR1-8 mutant. Together these findings indicate that O-fucosylation is functionally significant for secretion of ADAMTS13.     

Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts

Metabolic profiling, metabolomic and metabonomic studies mainly involve the multicomponent analysis of biological fluids, tissue and cell extracts using NMR spectroscopy and/or mass spectrometry (MS). We summarize the main NMR spectroscopic applications in modern metabolic research, and provide detailed protocols for biofluid (urine, serum/plasma) and tissue sample collection and preparation, including the extraction of polar and lipophilic metabolites from tissues. 1H NMR spectroscopic techniques such as standard 1D spectroscopy, relaxation-edited, diffusion-edited and 2D J-resolved pulse sequences are widely used at the analysis stage to monitor different groups of metabolites and are described here. They are often followed by more detailed statistical analysis or additional 2D NMR analysis for biomarker discovery. The standard acquisition time per sample is 4-5 min for a simple 1D spectrum, and both preparation and analysis can be automated to allow application to high-throughput screening for clinical diagnostic and toxicological studies, as well as molecular phenotyping and functional genomics.