Breakdown of different peptides byPrevotella (Bacteroides) ruminicola and mixed microorganisms from the sheep rumen

Several di-, tri-, and oligopeptides were incubated individually in vitro with rumen fluid from two sheep receiving a mixed grass hay/concentrate diet and with washed cells ofPrevotella (formerlyBacteroides)ruminicola M384 andP. ruminicola B₁4. The rates of breakdown of most peptides were similar in the rumen fluid from the two sheep. Acidic and proline-containing peptides tended to be more slowly degraded than neutral or basic peptides. The dipeptide at the N-terminus of higher peptides was observed as an early product of hydrolysis, confirming that a dipeptidyl aminopeptidase type of activity was present. The relative rates of breakdown of dipeptides byP. ruminicola were different from that of rumen fluid, but the hydrolysis of higher peptides followed a similar pattern, and dipeptides from the N-terminus were detected as early products.     

Chemoselective Deprotection of Triethylsilyl Ethers

An efficient and selective method was developed for the deprotection of triethylsilyl (TES) ethers using formic acid in methanol (5–10%) or in methylene chloride 2–5%) with excellent yields. TES ethers are selectively deprotected to the corresponding alcohols in high yields using formic acid in methanol under mild reaction conditions. Other hydroxyl protecting groups like t-butyldimethylsilyl (TBDMS) remain unaffected.     

Hybridisation,paternal leakage and mitochondrial DNA linearization in three anomalous fish (Scombridae)

Using mitochondrial DNA for species identification and population studies assumes that the genome is maternally inherited, circular, located in the cytoplasm and lacks recombination. This study explores the mitochondrial genomes of three anomalous mackerel. Complete mitochondrial genome sequencing plus nuclear microsatellite genotyping of these fish identified them as Scomberomorus munroi (spotted mackerel). Unlike normal S. munroi, these three fish also contained different linear, mitochondrial genomes of Scomberomorus semifasciatus (grey mackerel). The results are best explained by hybridisation, paternal leakage and mitochondrial DNA linearization. This unusual observation may provide an explanation for mtDNA outliers in animal population studies.     

Flavodoxin cofactor binding induces structural changes that are required for protein–protein interactions with NADP+ oxidoreductase and pyruvate formate-lyase activating enzyme

Flavodoxin (Fld) conformational changes, thermal stability, and cofactor binding were studied using circular dichroism (CD), isothermal titration calorimetry (ITC), and limited proteolysis. Thermodynamics of apo and holo-Fld folding were examined to discern the features of this important electron transfer protein and to provide data on apo-Fld. With the exception of fluorescence and UV–vis binding experiments with its cofactor flavin mononucleotide (FMN), apo-Fld is almost completely uncharacterized in Escherichia coli. Fld is more structured when the FMN cofactor is bound; the association is tight and driven by enthalpy of binding. Surface plasmon resonance binding experiments were carried out under anaerobic conditions for both apo- and holo-Fld and demonstrate the importance of structure and conformation for the interaction with binding partners. Holo-Fld is capable of associating with NADP⁺-dependent flavodoxin oxidoreductase (FNR) and pyruvate formate-lyase activating enzyme (PFL-AE) whereas there is no detectable interaction between apo-Fld and either protein. Limited proteolysis experiments were analyzed by LC-MS to identify the regions in Fld that are involved in conformation changes upon cofactor binding. Docking software was used to model the Fld/PFL-AE complex to understand the interactions between these two proteins and gain insight into electron transfer reactions from Fld to PFL-AE.     

Climate change resilience of a globally important sea turtle nesting population

Few studies have looked into climate change resilience of populations of wild animals. We use a model higher vertebrate, the green sea turtle, as its life history is fundamentally affected by climatic conditions, including temperature‐dependent sex determination and obligate use of beaches subject to sea level rise (SLR). We use empirical data from a globally important population in West Africa to assess resistance to climate change within a quantitative framework. We project 200 years of primary sex ratios (1900–2100) and create a digital elevation model of the nesting beach to estimate impacts of projected SLR. Primary sex ratio is currently almost balanced, with 52% of hatchlings produced being female. Under IPCC models, we predict: (a) an increase in the proportion of females by 2100 to 76%–93%, but cooler temperatures, both at the end of the nesting season and in shaded areas, will guarantee male hatchling production; (b) IPCC SLR scenarios will lead to 33.4%–43.0% loss of the current nesting area; (c) climate change will contribute to population growth through population feminization, with 32%–64% more nesting females expected by 2120; (d) as incubation temperatures approach lethal levels, however, the population will cease growing and start to decline. Taken together with other factors (degree of foraging plasticity, rookery size and trajectory, and prevailing threats), this nesting population should resist climate change until 2100, and the availability of spatial and temporal microrefugia indicates potential for resilience to predicted impacts, through the evolution of nest site selection or changes in nesting phenology. This represents the most comprehensive assessment to date of climate change resilience of a marine reptile using the most up‐to‐date IPCC models, appraising the impacts of temperature and SLR, integrated with additional ecological and demographic parameters. We suggest this as a framework for other populations, species and taxa.     

Synthesis of chiral phosphoantigens and their activity in gamma delta T cell stimulation

Gammadelta T cells expressing Vgamma2Vdelta2 T cell receptors are activated by a broad range of phosphorus-containing small molecules, termed phosphoantigens, and are of interest in the context of the chemotherapy of B cell malignancies. Here, we report the synthesis of four pairs of chiral phosphoantigens: the bromohydrins of isopentenyl diphosphate (Phosphostim), the epoxides of isopentenyl diphosphate (EIPP); and the corresponding bromohydrin and epoxide analogs of but-3-enyl diphosphate. The ability of each compound to stimulate human Vgamma2Vdelta2 T cells was determined by TNF-alpha release and cell proliferation. In these assays, the (R)-bromohydrin diphosphates were, on average, about twice as active as the (S)-bromohydrin diphosphates. In contrast, the (S)-form of EIPP was about twice as active as (R)-EIPP. The activities of the epoxy but-3-enyl diphosphates were both very low. These results suggest that chiral phosphoantigens, as opposed to racemic mixtures, may have utility in immunotherapy.     

Model organisms: new insights into ion channel and transporter function. L-type calcium channels regulate epithelial fluid transport in Drosophila melanogaster

The neuropeptide CAP2b stimulates fluid transport obligatorily via calcium entry, nitric oxide, and cGMP in Drosophila melanogaster Malpighian (renal) tubules. We have shown by RT-PCR that the Drosophila L-type calcium channel alpha1-subunit genes Dmca1D and Dmca1A (nbA) are both expressed in tubules. CAP2b-stimulated fluid transport and cytosolic calcium concentration ([Ca2+]i) increases are inhibited by the L-type calcium channel blockers verapamil and nifedipine. cGMP-stimulated fluid transport is verapamil and nifedipine sensitive. Furthermore, cGMP induces a slow [Ca2+]i increase in tubule principal cells via verapamil- and nifedipine-sensitive calcium entry; RT-PCR shows that tubules express Drosophila cyclic nucleotide-gated channel (cng). Additionally, thapsigargin-induced [Ca2+]i increase is verapamil sensitive. Phenylalkylamines bind with differing affinities to the basolateral and apical surfaces of principal cells in the main segment; however, dihydropyridine binds apically in the tubule initial segment. Immunocytochemical evidence suggests localization of alpha1-subunits to both basolateral and apical surfaces of principal cells in the tubule main segment. We suggest roles for L-type calcium channels and cGMP-mediated calcium influx in both calcium signaling and fluid transport mechanisms in Drosophila.     

Common Variation at 1q24.1 (ALDH9A1) Is a Potential Risk Factor for Renal Cancer

So far six susceptibility loci for renal cell carcinoma (RCC) have been discovered by genome-wide association studies (GWAS). To identify additional RCC common risk loci, we performed a meta-analysis of published GWAS (totalling 2,215 cases and 8,566 controls of Western-European background) with imputation using 1000 Genomes Project and UK10K Project data as reference panels and followed up the most significant association signals [22 single nucleotide polymorphisms (SNPs) and 3 indels in eight genomic regions] in 383 cases and 2,189 controls from The Cancer Genome Atlas (TCGA). A combined analysis identified a promising susceptibility locus mapping to 1q24.1 marked by the imputed SNP rs3845536 (P _combined =2.30x10^-8). Specifically, the signal maps to intron 4 of the ALDH9A1 gene (aldehyde dehydrogenase 9 family, member A1). We further evaluated this potential signal in 2,461 cases and 5,081 controls from the International Agency for Research on Cancer (IARC) GWAS of RCC cases and controls from multiple European regions. In contrast to earlier findings no association was shown in the IARC series (P=0.94; P _combined =2.73x10^-5). While variation at 1q24.1 represents a potential risk locus for RCC, future replication analyses are required to substantiate our observation.