Ascertaining the biochemical function of an essential pectin methylesterase in the gut microbe Bacteroides thetaiotaomicron

Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II–derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/β-hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed β-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella. Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.     

Engineering Strontium Binding Affinity in an EF-hand Motif: A Quantum Chemical and Molecular Dynamics Study

Abstract

Proteins with the ability to specifically bind strontium would potentially be of great use in the field of nuclear waste management. Unfortunately, no such peptides or proteins are known—indeed, it is uncertain whether they exist under natural conditions due to low environmental concentrations of strontium. To investigate the possibility of devising such molecules, one of us (CV), in a previous experimental study [J. Biol. Inorg. Chem. 8, 33440 (2003)], proposed starting from an EF-hand motif of the protein calmodulin and mutating some residues to change the motif's specificity for calcium into one for strontium. In this paper, which represents a theoretical complement to the experimental work, we analyzed small-molecule crystallographic structures and performed quantum chemical calculations to identify possible mutations. We then constructed seven mutant sequences of the EF-hand motif and analyzed their dynamical and binding behaviors using molecular dynamics simulations and free-energy calculations (using the MM/PBSA method). As a result of these analyzes we were able to isolate some characteristics that could lead to mutant peptides with enhanced strontium affinity.     

Aedes aegypti Mosquitoes Exhibit Decreased Repellency by DEET following Previous Exposure

DEET (N,N-Diethyl-m-toluamide) is one of the most widely used mosquito repellents. Although DEET has been shown to be extremely effective, recent studies have revealed that certain individual insects are unaffected by its presence. A genetic basis for this has been shown in Aedes aegypti mosquitoes and the fruit fly Drosophila melanogaster, but, for the triatomine bug, Rhodnius prolixus, a decrease in response to DEET occurred shortly after previous exposure, indicating that non-genetic factors may also be involved in DEET “insensitivity”. In this study, we examined host-seeking behaviour and electrophysiological responses of A. aegypti after pre-exposure to DEET. We found that three hours after pre-exposure the mosquitoes showed behavioural insensitivity, and electroantennography revealed this correlated with the olfactory receptor neurons responding less to DEET. The change in behaviour as a result of pre-exposure to DEET has implications for the use of repellents and the ability of mosquitoes to overcome them.     

Temporal Variation in Physiological Biomarkers in Black Flying-Foxes (<Emphasis Type="Italic">Pteropus alecto</Emphasis>), Australia

Bats of the genus Pteropus (Pteropodidae) are recognised as the natural host of multiple emerging pathogenic viruses of animal and human health significance, including henipaviruses, lyssaviruses and ebolaviruses. Some studies have suggested that physiological and ecological factors may be associated with Hendra virus infection in flying-foxes in Australia; however, it is essential to understand the normal range and seasonal variability of physiological biomarkers before seeking physiological associations with infection status. We aimed to measure a suite of physiological biomarkers in P. alecto over time to identify any seasonal fluctuations and to examine possible associations with life-cycle and environmental stressors. We sampled 839 adult P. alecto in the Australian state of Queensland over a 12-month period. The adjusted population means of every assessed hematologic and biochemical parameter were within the reported reference range on every sampling occasion. However, within this range, we identified significant temporal variation in these parameters, in urinary parameters and body condition, which primarily reflected the normal annual life cycle. We found no evident effect of remarkable physiological demands or nutritional stress, and no indication of clinical disease driving any parameter values outside the normal species reference range. Our findings identify underlying temporal physiological changes at the population level that inform epidemiological studies and assessment of putative physiological risk factors driving Hendra virus infection in P. alecto. More broadly, the findings add to the knowledge of Pteropus populations in terms of their relative resistance and resilience to emerging infectious disease.     

Analysis of the Maize dicer-like1 Mutant,fuzzy tassel,Implicates MicroRNAs in Anther Maturation and Dehiscence

Sexual reproduction in plants requires development of haploid gametophytes from somatic tissues. Pollen is the male gametophyte and develops within the stamen; defects in the somatic tissues of the stamen and in the male gametophyte itself can result in male sterility. The maize fuzzy tassel (fzt) mutant has a mutation in dicer-like1 (dcl1), which encodes a key enzyme required for microRNA (miRNA) biogenesis. Many miRNAs are reduced in fzt, and fzt mutants exhibit a broad range of developmental defects, including male sterility. To gain further insight into the roles of miRNAs in maize stamen development, we conducted a detailed analysis of the male sterility defects in fzt mutants. Early development was normal in fzt mutant anthers, however fzt anthers arrested in late stages of anther maturation and did not dehisce. A minority of locules in fzt anthers also exhibited anther wall defects. At maturity, very little pollen in fzt anthers was viable or able to germinate. Normal pollen is tricellular at maturity; pollen from fzt anthers included a mixture of unicellular, bicellular, and tricellular pollen. Pollen from normal anthers is loaded with starch before dehiscence, however pollen from fzt anthers failed to accumulate starch. Our results indicate an absolute requirement for miRNAs in the final stages of anther and pollen maturation in maize. Anther wall defects also suggest that miRNAs have key roles earlier in anther development. We discuss candidate miRNAs and pathways that might underlie fzt anther defects, and also note that male sterility in fzt resembles water deficit-induced male sterility, highlighting a possible link between development and stress responses in plants.     

Metaheuristic approaches in biopharmaceutical process development data analysis

Bioprocess and Biosystems Engineering - There is a growing interest in mining and handling of big data, which has been rapidly accumulating in the repositories of bioprocess industries....     

Composition and Variation of the Human Milk Microbiota Are Influenced by Maternal and Early-Life Factors

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Synthesis of triazole-linked pseudo-starch fragments

Rapid assembly of starch fragment analogues was achieved using 'click chemistry'. Specifically, a pentadecasaccharide and two hexadecasaccharide mimics containing two parallel maltoheptaosyl chains linked via [1,2,3]-triazoles to glucose or maltose core were synthesised using Cu(I)-catalyzed [3+2] dipolar cycloaddition of azidosaccharides and 4,6-di-O-propargylated methyl alpha-d-glucopyranoside and 6,6'- and 4',6'-di-O-propargylated p-methoxyphenyl beta-maltoside.     

Mutations in DIIS5 and the DIIS4-S5 linker of Drosophila melanogaster sodium channel define binding domains for pyrethroids and DDT

Mutations in the DIIS4-S5 linker and DIIS5 have identified hotspots of pyrethroid and DDT interaction with the Drosophila para sodium channel. Wild-type and mutant channels were expressed in Xenopus oocytes and subjected to voltage-clamp analysis. Substitutions L914I, M918T, L925I, T929I and C933A decreased deltamethrin potency, M918T, L925I and T929I decreased permethrin potency and T929I, L925I and I936V decreased fenfluthrin potency. DDT potency was unaffected by M918T, but abolished by T929I and reduced by L925I, L932F and I936V, suggesting that DIIS5 contains at least part of the DDT binding domain. The data support a computer model of pyrethroid and DDT binding.