Complementary Metagenomic Approaches Improve Reconstruction of Microbial Diversity in a Forest Soil.

Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this "uncultivated majority" remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages.IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism

A Genetic Screen Identifies New Steps In Oocyte Maturation That Enhance Proteostasis In The Immortal Germ Lineage

Somatic cells age and die, but the germ-cell lineage is immortal. In Caenorhabditis elegans, germline immortality involves proteostasis renewal at the beginning of each new generation, when oocyte maturation signals from sperm trigger the clearance of carbonylated proteins and protein aggregates. Here, we explore the cell biology of this proteostasis renewal in the context of a whole-genome RNAi screen. Oocyte maturation signals are known to trigger protein-aggregate removal via lysosome acidification. Our findings suggest that lysosomes are acidified as a consequence of changes in endoplasmic reticulum activity that permit assembly of the lysosomal V-ATPase, which in turn allows lysosomes to clear the aggregates via microautophagy. We define two functions for mitochondria, both of which appear to be independent of ATP generation. Many genes from the screen also regulate lysosome acidification and age-dependent protein aggregation in the soma, suggesting a fundamental mechanistic link between proteostasis renewal in the germline and somatic longevity

Infrared photoinduced electrochemiluminescence microscopy of single cells

Electrochemiluminescence (ECL) is evolving rapidly from a purely analytical technique into a powerful microscopy. Herein, we report the imaging of single cells by photoinduced ECL (PECL; λem = 620 nm) stimulated by an incident near-infrared light (λexc = 1050 nm). The cells were grown on a metal–insulator–semiconductor (MIS) n-Si/SiOx/Ir photoanode that exhibited stable and bright PECL emission. The large anti-Stokes shift allowed for the recording of well-resolved images of cells with high sensitivity. PECL microscopy is demonstrated at a remarkably low onset potential of 0.8 V; this contrasts with classic ECL, which is blind at this potential. Two imaging modes are reported: (i) photoinduced positive ECL (PECL+), showing the cell membranes labeled with the [Ru(bpy)3]2+ complex; and (ii) photoinduced shadow label-free ECL (PECL−) of cell morphology, with the luminophore in the solution. Finally, by adding a new dimension with the near-infrared light stimulus, PECL microscopy should find promising applications to image and study single photoactive nanoparticles and biological entities

Synchrotron X-ray diffraction experiments with a prototype hybrid pixel detector

International audienceA prototype X-ray pixel area detector (XPAD3.1) has been used for X-ray diffraction experiments with synchrotron radiation. The characteristics of this detector are very attractive in terms of fast readout time, high dynamic range and high signal-to-noise ratio. The prototype XPAD3.1 enabled various diffraction experiments to be performed at different energies, sample-to-detector distances and detector angles with respect to the direct beam, yet it was necessary to perform corrections on the diffraction images according to the type of experiment. This paper is focused on calibration and correction procedures to obtain high-quality scientific results specifically developed in the context of three different experiments, namely mechanical characterization of nanostructured multilayers, elastic-plastic deformation of duplex steel and growth of carbon nanotubes

Complementary Metagenomic Approaches Improve Reconstruction of Microbial Diversity in a Forest Soil

Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this “uncultivated majority” remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes. Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages. IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism

Peculiar effective elastic anisotropy of nanometric multilayers studied by surface Brillouin scattering

We show in this paper by using a two-scale transition model that the elastic anisotropy of a thin film specimen can be tuned by appropriate stacking design. The anisotropic behaviour is illustrated for two monophase thin films, namely W which is perfectly elastically isotropic and Au which is strongly elastically anisotropic, and for a nanometric W/Au multilayers. The experimental measurements show that the model capture the elastic anisotropy rather well even for a nanometric multilayer stacking (period of 12 nm) and that the elastic anisotropy of W/Au multilayer is more pronounced than the ones of the two components for a fraction of 50%. This enhanced anisotropy is discussed in view of the multilayer microstructur

Controlled biaxial deformation of nanostructured W/Cu thin films studied by X-ray diffraction

The deformation behaviour of 150. nm thick W/Cu nanocomposite deposited on polyimide substrates has been analysed under equi-biaxial tensile testing coupled to X-ray diffraction technique. The experiments were carried out using a biaxial device that has been developed for the DiffAbs beamline of SOLEIL synchrotron source. Finite element analysis has been performed to study the strain distribution into the cruciform shape substrate and define the homogeneous deformed volume. X-ray measured elastic strains in tungsten sub-layers could be carried out for both principal directions. The strain field was determined to be almost equi-biaxial as expected and compared to finite element calculations

A low proportion of HBeAg among HBsAg-positive pregnant women with known HIV status could suggest low perinatal transmission of HBV in Cameroon

<p>Abstract</p> <p>Background</p> <p>Transmission of hepatitis B virus (HBV) from HBV-positive mothers to their infants is common and usually occurs when the mother is hepatitis B e antigen (HBeAg) positive and/or has a high HBV DNA load. In this study, we determined the prevalence of hepatitis B surface antigen (HBsAg) and HBeAg among pregnant women with known HIV status.</p> <p>Findings</p> <p>A total of 650 pregnant women with a mean age of 26.2 years including 301 HIV-positives and 349 HIV-negatives were screened for HBsAg (Monolisa AgHBs Plus Biorad, France). Among the HBsAg-positives, HBeAg and anti-HBe were tested (Monolisa Ag HBe Plus Biorad, France). Overall, 51 (7.85%) were positive for HBsAg. The prevalence of HBsAg was not statistically different between HIV-positive and HIV-negative pregnant women [28/301 (9.3%) vs 23/349 (6.59%); p = 0.2]. None of the 45 HBsAg-positive samples was reactive for HBeAg.</p> <p>Conclusions</p> <p>Our study indicates a high prevalence of HBsAg with very low proportion of HBeAg in Cameroonian pregnant women. Since perinatal transmission of HBV is mostly effective when the mother is also HBeAg-positive, our data could suggest that perinatal transmissions play a minor role in HBV prevalence in Cameroon. In line with previous African studies, these findings further suggests that horizontal transmission could be the most common mechanism of HBV infections in Cameroon.</p

IκBβ acts to inhibit and activate gene expression during the inflammatory response

The activation of pro-inflammatory gene programs by nuclear factor-κB (NF-κB) is primarily regulated through cytoplasmic sequestration of NF-κB by the inhibitor of κB (IκB) family of proteins1. IκBβ, a major isoform of IκB, can sequester NF-κB in the cytoplasm2, although its biological role remains unclear. Although cells lacking IκBβ have been reported3, 4, in vivo studies have been limited and suggested redundancy between IκBα and IκBβ5. Like IκBα, IκBβ is also inducibly degraded; however, upon stimulation by lipopolysaccharide (LPS), it is degraded slowly and re-synthesized as a hypophosphorylated form that can be detected in the nucleus6, 7, 8, 9, 10, 11. The crystal structure of IκBβ bound to p65 suggested this complex might bind DNA12. In vitro, hypophosphorylated IκBβ can bind DNA with p65 and c-Rel, and the DNA-bound NF-κB:IκBβ complexes are resistant to IκBα, suggesting hypophosphorylated, nuclear IκBβ may prolong the expression of certain genes9, 10, 11. Here we report that in vivo IκBβ serves both to inhibit and facilitate the inflammatory response. IκBβ degradation releases NF-κB dimers which upregulate pro-inflammatory target genes such as tumour necrosis factor-α (TNF-α). Surprisingly, absence of IκBβ results in a dramatic reduction of TNF-α in response to LPS even though activation of NF-κB is normal. The inhibition of TNF-α messenger RNA (mRNA) expression correlates with the absence of nuclear, hypophosphorylated-IκBβ bound to p65:c-Rel heterodimers at a specific κB site on the TNF-α promoter. Therefore IκBβ acts through p65:c-Rel dimers to maintain prolonged expression of TNF-α. As a result, IκBβ^(−/−) mice are resistant to LPS-induced septic shock and collagen-induced arthritis. Blocking IκBβ might be a promising new strategy for selectively inhibiting the chronic phase of TNF-α production during the inflammatory response

Synchrotron X-ray diffraction experiments with a prototype hybrid pixel detector

A prototype X-ray pixel area detector (XPAD3.1) has been used for X-ray diffraction experiments with synchrotron radiation. The characteristics of this detector are very attractive in terms of fast readout time, high dynamic range and high signal-to-noise ratio. The prototype XPAD3.1 enabled various diffraction experiments to be performed at different energies, sample-to-detector distances and detector angles with respect to the direct beam, yet it was necessary to perform corrections on the diffraction images according to the type of experiment. This paper is focused on calibration and correction procedures to obtain high-quality scientific results specifically developed in the context of three different experiments, namely mechanical characterization of nanostructured multilayers, elastic-plastic deformation of duplex steel and growth of carbon nanotubes