Comparative activity and functional ecology of permafrost soils and lithic niches in a hyper‐arid polar desert

Permafrost in the high elevation McMurdo Dry Valleys of Antarctica ranks among the driest and coldest on Earth. Permafrost soils appear to be largely inhospitable to active microbial life, but sandstone lithic microhabitats contain a trophically simple but functional cryptoendolithic community. We used metagenomic sequencing and activity assays to examine the functional capacity of permafrost soils and cryptoendolithic communities in University Valley, one of the most extreme regions in the Dry Valleys. We found metagenomic evidence that cryptoendolithic microorganisms are adapted to the harsh environment and capable of metabolic activity at in situ temperatures, possessing a suite of stress response and nutrient cycling genes to fix carbon under the fluctuating conditions that the sandstone rock would experience during the summer months. We additionally identified genes involved in microbial competition and cooperation within the cryptoendolithic habitat. In contrast, permafrost soils have a lower richness of stress response genes, and instead the metagenome is enriched in genes involved with dormancy and sporulation. The permafrost soils also have a large presence of phage genes and genes involved in the recycling of cellular material. Our results underlie two different habitability conditions under extreme cold and dryness: the permafrost soil which is enriched in traits which emphasize survival and dormancy, rather than growth and activity; and the cryptoendolithic environment that selects for organisms capable of growth under extremely oligotrophic, arid and cold conditions. This study represents the first metagenomic interrogation of Antarctic permafrost and polar cryptoendolithic microbial communities.     

IL-27 induces the production of IgG1 by human B cells

It has been reported that IL-27 specifically induces the production of IgG2a by mouse B cells and inhibits IL-4-induced IgG1 synthesis. Here, we show that human na?ve cord blood expresses a functional IL-27 receptor, consisting of the TCCR and gp130 subunits, although at lower levels as compared to na?ve and memory splenic B cells. IL-27 does not induce proliferative responses and does not increase IgG1 production by CD19(+)CD27(+) memory B cells. However, it induces a low, but significant production of IgG1 by na?ve CD19(+)CD27(-)IgD(+)IgG(-) spleen and cord blood B cells, activated via CD40, whereas it has no effect on the production of the other IgG subclasses. In addition, IL-27 induces the differentiation of a population of B cells that express high levels of CD38, in association with a down-regulation of surface IgD expression, and that are surface IgG(+/int), CD20(low), CD27(high), indicating that IL-27 promotes isotype switching and plasma cell differentiation of naive B cells. However, as compared to the effects of IL-21 and IL-10, both switch factors for human IgG1 and IgG3, those of IL-27 are modest and regulate exclusively the production of IgG1. Finally, although IL-27 has no effect on IL-4 and anti-CD40-induced Cepsilon germline promoter activity, it up-regulates IL-4-induced IgE production by naive B cells. These results point to a partial redundancy of switch factors regulating the production of IgG1 in humans, and furthermore indicate the existence of a common regulation of the human IgG1and murine IgG2a isotypes by IL-27.     

Microbial gut evaluation in an angolan paediatric population with sickle cell disease

Sickle cell disease (SCD) is one of the most common genetic conditions worldwide. It can contribute up to 90% of under‐5 mortality in sub‐Saharan Africa. Clinical manifestations are very heterogeneous, and the intestinal microbiome appears to be crucial in the modulation of inflammation, cell adhesion and induction of aged neutrophils, the main interveners of recurrent vaso‐occlusive crisis. Enterocyte injury, increased permeability, altered microbial composition and bacterial overgrowth have all been documented as microbial and pathophysiologic changes in the gut microbiome of SCD patients in recent studies. Our aim was to sequence the bacterial 16S rRNA gene in order to characterize the gut microbiome of Angolan children with SCA and healthy siblings as a control. A total of 72 stool samples were obtained from children between 3 and 14 years old. Our data showed that the two groups exhibit some notable differences in microbiota relative abundance at different classification levels. Children with SCA have a higher number of the phylum Actinobacteria. As for the genus level, Clostridium cluster XI bacteria was more prevalent in the SCA children, whereas the siblings had a higher abundance of Blautia, Aestuariispira, Campylobacter, Helicobacter, Polaribacter and Anaerorhabdus. In this study, we have presented the first microbiota analysis in an Angolan paediatric population with SCD and provided a detailed view of the microbial differences between patients and healthy controls. There is still much to learn before fully relying on the therapeutic approaches for gut modulation, which is why more research in this field is crucial to making this a reality.     

The 20S proteasome α5 subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the lettuce mosaic potyvirus HcPro protein

In plants, the ubiquitin/26S proteasome system (UPS) plays a central role in protein degradation and is involved in many steps of defence mechanisms, regardless of the types of pathogen targeted. In addition to its proteolytic activities, the UPS ribonuclease (RNase) activity, previously detected in 20S proteasome preparations from cauliflower and sunflower (Helianthus annuus), has been shown to specifically target plant viral RNAs in vitro. In this study, we show that recombinant Arabidopsis thaliana proteasomal α(5) subunit expressed in Escherichia coli harbours an RNase activity that degrades Tobacco mosaic virus (TMV, Tobamovirus)- and Lettuce mosaic virus (LMV, Potyvirus)-derived RNAs in vitro. The analysis of mutated forms of the α(5) subunit demonstrated that mutation of a glutamic acid at position 110 affects RNase activity. Furthermore, it was demonstrated, using a bimolecular fluorescence complement assay, that the multifunctional helper component proteinase (HcPro) of LMV, already known to interfere with the 20S proteasome RNase activity in vitro, can interact in vivo with the recombinant α(5) subunit. Further experiments demonstrated that, in LMV-infected lettuce cells, α(5) is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α(5) , showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thaliana α(5) the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.     

Increase in epidermal growth factor receptor and its messenger ribonucleic acid levels with differentiation of human trophoblast cells in culture

Epidermal growth factor receptor (EGFR) expression was studied during the differentiation of human trophoblast cells in culture. In vitro, intravillous mononuclear cytotrophoblasts aggregate and fuse within 24 h to form a syncytium. This morphological differentiation was associated with a significant twofold increase in specific ¹²⁵I-EGF binding capacity (P < 0.01). Scatchard analyses showed an apparent rise in the number of high-affinity binding sites (0.33 ± 0.04 and 0.63 ± 0.07 pmol/mg protein at 24 and 48 h, respectively), with no change in their affinity (1.34 and 1.42 × 10^?10 mol/L). Affinity labeling of ¹²⁵I-EGF in cultured trophoblast cells followed by SDS-PAGE and autoradiography revealed a band of 175 KDa corresponding to EGFR, the intensity of which increased with the time in culture. EGF-dependent phosphorylation of membrane proteins from cultured trophoblast cells revealed major phosphorylated proteins of 170 KDa (EGFR) and 35 KDa, which were both increased at 48 h, indicating a rise in EGFR-kinase activity during syncytium formation. Northern blot analysis of EGFR-mRNA, followed by hybridization with a ³²P-cDNA probe for EGFR, revealed an increase in EGFR gene expression in syncytiotrophoblasts, as compared to cytotrophoblasts. Thus, the increase in bioactive EGFR observed during the differentiation of trophoblast cells was due to an increase in their synthesis. Cultured trophoblast cells are therefore a good model of spontaneous up-regulation of EGFR expression with cell differentiation. © 1993 Wiley-Liss, Inc.     

P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains

P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.