Functional Diversity of Carbohydrate-Active Enzymes Enabling a Bacterium to Ferment Plant Biomass

Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.     

Very High Surface Capacity Observed Using Si Negative Electrodes Embedded in Copper Foam as 3D Current Collectors

Cu foam is evaluated as a replacement for metal foil current collectors to create 3D composite electrodes with the objective to produce Si‐based anodes with high loadings. The electrodes are prepared by casting the slurry into the porosity of the foam. With such a design, the loading and the surface capacity can reach values as high as 10 mg cm^?2 and 10 mAh cm^?2. Compared to the common 2D design, the 3D copper framework shows a great advantage in the cycle life (more than 400 cycles at a Si loading of 10 mg cm^?2 with commercial micrometric particles) and power performance. The thinness of the composite coating on the foam walls favors a better preservation of the electronic wiring upon cycling and fast lithium ion diffusion. A higher coulombic efficiency in half cells with lithium metal as the counter electrode is achieved by using carbon nanofibers (CNF) rather than carbon black (CB). The possibility to reach, in practice, higher surface capacity could allow a significant increase in both the volumetric and gravimetric energy densities by 23% and 19%, respectively, for the Cu foam‐silicon//LiFePO₄ stack compared to the graphite/LiFePO₄ stack of traditional design.     

Inter-Annual Variability of Fledgling Sex Ratio in King Penguins

As the number of breeding pairs depends on the adult sex ratio in a monogamous species with biparental care, investigating sex-ratio variability in natural populations is essential to understand population dynamics. Using 10 years of data (2000–2009) in a seasonally monogamous seabird, the king penguin (Aptenodytes patagonicus), we investigated the annual sex ratio at fledging, and the potential environmental causes for its variation. Over more than 4000 birds, the annual sex ratio at fledging was highly variable (ranging from 44.4% to 58.3% of males), and on average slightly biased towards males (51.6%). Yearly variation in sex-ratio bias was neither related to density within the colony, nor to global or local oceanographic conditions known to affect both the productivity and accessibility of penguin foraging areas. However, rising sea surface temperature coincided with an increase in fledging sex-ratio variability. Fledging sex ratio was also correlated with difference in body condition between male and female fledglings. When more males were produced in a given year, their body condition was higher (and reciprocally), suggesting that parents might adopt a sex-biased allocation strategy depending on yearly environmental conditions and/or that the effect of environmental parameters on chick condition and survival may be sex-dependent. The initial bias in sex ratio observed at the juvenile stage tended to return to 1∶1 equilibrium upon first breeding attempts, as would be expected from Fisher’s classic theory of offspring sex-ratio variation.     

Constitutive Nuclear Localization of an Alternatively Spliced Sirtuin-2 Isoform

Sirtuin-2 (SIRT2), the cytoplasmic member of the sirtuin family, has been implicated in the deacetylation of nuclear proteins. Although the enzyme has been reported to be located to the nucleus during G₂/M phase, its spectrum of targets suggests functions in the nucleus throughout the cell cycle. While a nucleocytoplasmic shuttling mechanism has been proposed for SIRT2, recent studies have indicated the presence of a constitutively nuclear isoform. Here we report the identification of a novel splice variant (isoform 5) of SIRT2 that lacks a nuclear export signal and encodes a predominantly nuclear isoform. This novel isoform 5 fails to show deacetylase activity using several assays, both in vitro and in vivo, and we are led to conclude that this isoform is catalytically inactive. Nevertheless, it retains the ability to interact with p300, a known interaction partner. Moreover, changes in intrinsic tryptophan fluorescence upon denaturation indicate that the protein is properly folded. These data, together with computational analyses, confirm the structural integrity of the catalytic domain. Our results suggest an activity-independent nuclear function of the novel isoform.     

Recombinant Antigens Expressed in Pichia pastoris for the Diagnosis of Sleeping Sickness Caused by Trypanosoma brucei gambiense

Background

Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.

Methodology/Principal Findings

We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.

Conclusions/Significance

The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.     

Selection Signatures in Worldwide Sheep Populations

The diversity of populations in domestic species offers great opportunities to study genome response to selection. The recently published Sheep HapMap dataset is a great example of characterization of the world wide genetic diversity in sheep. In this study, we re-analyzed the Sheep HapMap dataset to identify selection signatures in worldwide sheep populations. Compared to previous analyses, we made use of statistical methods that (i) take account of the hierarchical structure of sheep populations, (ii) make use of linkage disequilibrium information and (iii) focus specifically on either recent or older selection signatures. We show that this allows pinpointing several new selection signatures in the sheep genome and distinguishing those related to modern breeding objectives and to earlier post-domestication constraints. The newly identified regions, together with the ones previously identified, reveal the extensive genome response to selection on morphology, color and adaptation to new environments.     

Cellular Source and Mechanisms of High Transcriptome Complexity in the Mammalian Testis

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Deorphanization and Characterization of Human Olfactory Receptors in Heterologous Cells

Olfaction plays an indispensable role in human and animals in self and environmental recognition, as well as intra‐ and interspecific communication. Following the discovery of a family of olfactory receptors (ORs) by Buck and Axel in 1991, it has been established that the sense of smell begins with the molecular recognition of a chemical odorant by one or more ORs expressed in the olfactory sensory neurons. Therefore, characterization of the molecular interactions between odorant molecules and ORs is a key step in the elucidation of the general properties of the olfactory system and in the development of applications, i.e., design of new odorants, search for blockers, etc. The process putted in place at ChemCom to improve the expression of ORs at the cytoplasmic membrane of the HEK293 cell and assays enabling large‐scale deorphanization, and to characterize the interaction between chemical odorants and ORs is described. The family of human ORs includes ca. 400 putatively functional ORs which are GPCRs (G protein‐coupled receptors); to date over 100 human ORs have been deorphanized.     

Cytokeratin analysis of breast and leukemia tumor cell lines by flow cytometry

Using four human tumor cell lines, MCF-7 and T47-D from breast tumors, MOLT-4 and K-562 from leukemia, flow cytometric DNA analysis of pure and mixed cell population was performed using monoclonal antibodies to cytokeratin to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratins. Surprisingly, on pure or mixed K-562 cells, we found positive labeling with KL1, CK8, and CK18 antibodies (results confirmed by immunocytology). This preliminary study has allowed a DNA analysis on epithelial cells of human breast tumors.     

Combinatorial engineering to enhance thermostability of amylosucrase

Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50 degrees C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B' domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50 degrees C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30 degrees C.