Ecophysiological properties of cultivable heterotrophic bacteria and yeasts dominating in phytocenoses of Galindez Island,maritime Antarctica

Antarctic plants are stable specific microenvironments for microbial colonization that are still less explored. In this study, we investigated cultivable heterotrophic bacteria and yeasts dominating in plant samples collected from different terrestrial biotopes near Ukrainian Antarctic Base on Galindez Island, maritime Antarctica. Phylogenetic analysis revealed affiliation of the bacterial isolates to genera Pseudomonas, Stenotrophomonas, Brevundimonas, Sporosarcina, Dermacoccus, Microbacterium, Rothia and Frondihabitans, and the yeast isolates to genera Rhodosporidium, Cryptococcus, Leucosporidiella, Candida and Exophiala. Some ecophysiological properties of isolated strains were determined that are important in response to different stresses such as psychro- and halotolerance, UV-resistance and production of hydrolytic enzymes. The majority of isolates (88 %) was found to be psychrotolerant; all are halotolerant. Significant differences in survival subsequent to UV-C radiation were observed among the isolates, as measured by culturable counts. For the bacterial isolates, lethal doses in the range 80–600 J m^?2 were determined, and for the yeast isolates—in the range 300–1,000 J m^?2. Dermacoccus profundi U9 and Candida davisiana U6 were found as most UV resistant among the bacterial and yeast isolates, respectively. Producers of caseinase, gelatinase, β-glucosidase, and cellulase were detected. To the best of our knowledge, this is the first report on isolation of UV resistant strain D. profundi, and Frondihabitans strain from Antarctica, and on detection of cellulase activity in Antarctic yeast strain C. davisiana. The results obtained contribute to clarifying adaptation strategies of Antarctic microbiota and its possible role in functional stability of Antarctic biocenoses. Stress tolerant strains were detected that are valuable for ecological and applied studies.     

The LMP2A protein of Epstein–Barr virus regulates phosphorylation of ITSN1 and Shb adaptors by tyrosine kinases

Latent Membrane Protein 2A (LMP2A) is an Epstein–Barr virus-encoded protein that is important for the maintenance of latent infection. Its activity affects cellular differentiation, migration, proliferation and B cell survival. LMP2A resembles a constitutively activated B cell antigen receptor and exploits host kinases to activate a set of downstream signaling pathways. In the current study we demonstrate the interaction of LMP2A with intersectin 1 (ITSN1), a key endocytic adaptor protein. This interaction occurs via both the N- and C-tails of LMP2A and is mediated by the SH3 domains of ITSN1. Additionally, we identified the Shb adaptor and the Syk kinase as novel binding ligands of ITSN1. The Shb adaptor interacts simultaneously with the phosphorylated tyrosines of LMP2A and the SH3 domains of ITSN1 and mediates indirect interaction of ITSN1 to LMP2A. Syk kinase promotes phosphorylation of both ITSN1 and Shb adaptors in LMP2A-expressing cells. In contrast to ITSN1, Shb phosphorylation depends additionally on Lyn kinase activity.Considering that Shb and ITSN1 are implicated in various receptor tyrosine kinase signaling, our results indicate that LMP2A can affect a number of signaling pathways by regulating the phosphorylation of the ITSN1 and Shb adaptors.     

Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design

An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens.     

Analyses in human urothelial cells identify methylation of miR-152, miR-200b and miR-10a genes as candidate bladder cancer biomarkers

Urinary miRNAs are discussed as potential biomarkers for bladder cancer. The majority of miRNAs, however, are downregulated, making it difficult to utilize reduced miRNA signals as reliable diagnostic tools. Because the downregulation of miRNAs is frequently associated with hypermethylation of the respective regulative sequences, we studied whether DNA hypermethylation might serve as an improved diagnostic tool compared to measuring downregulated miRNAs. miRNA expression arrays and individual qPCR were used to identify and confirm miRNAs that were downregulated in malignant urothelial cells (RT4, 5637 and J82) when compared to primary, non-malignant urothelial cells (HUEPC). DNA methylation was determined by customized PCR-arrays subsequent to methylation-sensitive DNA-restriction and by mass spectrometry. miRNA expression and DNA methylation were determined in untreated cells and in cultures treated with the demethylating agent 5-Aza-2′-deoxycytidine. miR-200b, miR-152 and miR-10a displayed differential expression and methylation among untreated cancer cell lines. In addition, reduced miRNA expression of miR-200b, miR-152, and miR-10a was associated with increased DNA methylation in malignant cells versus HUEPC. Finally, the demethylation approach revealed a causal relationship between both parameters for miR-152 in 5637 and also suggests a causal connection of both parameters for miR-200b in J82 and miR-10a in 5637. In conclusion, our studies in multiple bladder cancer cell lines and primary non-malignant urothelial cells suggest that hypermethylation of miR-152, miR-10a and miR-200b regulative DNA sequences might serve as epigenetic bladder cancer biomarkers.     

Bone morphogenetic protein-2 upregulates expression and function of voltage-gated K+ channels in human pulmonary artery smooth muscle cells

Activity of voltage-gated K(+) (K(V)) channels in pulmonary artery smooth muscle cells (PASMC) plays an important role in control of apoptosis and proliferation in addition to regulating membrane potential and pulmonary vascular tone. Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in normal human PASMC, whereas dysfunctional BMP signaling and downregulated K(V) channels are involved in pulmonary vascular medial hypertrophy associated with pulmonary hypertension. This study evaluated the effect of BMP-2 on K(V) channel function and expression in normal human PASMC. BMP-2 (100 nM for 18-24 h) significantly (>2-fold) upregulated mRNA expression of KCNA5, KCNA7, KCNA10, KCNC3, KCNC4, KCNF1, KCNG3, KCNS1, and KCNS3 but downregulated (at least 2-fold) KCNAB1, KCNA2, KCNG2, and KCNV2. The most dramatic change was the >10-fold downregulation of KCNG2 and KCNV2, two electrically silent gamma-subunits that form heterotetramers with functional K(V) channel alpha-subunits (e.g., KCNB1-2). Furthermore, the amplitude and current density of whole cell K(V) currents were significantly increased in PASMC treated with BMP-2. It has been demonstrated that K(+) currents generated by KCNB1 and KCNG1 (or KCNG2) or KCNB1 and KCNV2 heterotetramers are smaller than those generated by KCNB1 homotetramers, indicating that KCNG2 and KCNV2 (2 subunits that were markedly downregulated by BMP-2) are inhibitors of functional K(V) channels. These results suggest that BMP-2 divergently regulates mRNA expression of various K(V) channel alpha-, beta-, and gamma-subunits and significantly increases whole cell K(V) currents in human PASMC. Finally, we present evidence that attenuation of c-Myc expression by BMP-2 may be involved in BMP-2-mediated increase in K(V) channel activity and regulation of K(V) channel expression. The increased K(V) channel activity may be involved in the proapoptotic and/or antiproliferative effects of BMP-2 on PASMC.     

Normal and Friedreich ataxia cells express different isoforms of frataxin with complementary roles in iron-sulfur cluster assembly

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by insufficient expression of frataxin (FXN), a mitochondrial iron-binding protein required for Fe-S cluster assembly. The development of treatments to increase FXN levels in FRDA requires elucidation of the steps involved in the biogenesis of functional FXN. The FXN mRNA is translated to a precursor polypeptide that is transported to the mitochondrial matrix and processed to at least two forms, FXN(42-210) and FXN(81-210). Previous reports suggested that FXN(42-210) is a transient processing intermediate, whereas FXN(81-210) represents the mature protein. However, we find that both FXN(42-210) and FXN(81-210) are present in control cell lines and tissues at steady-state, and that FXN(42-210) is consistently more depleted than FXN(81-210) in samples from FRDA patients. Moreover, FXN(42-210) and FXN(81-210) have strikingly different biochemical properties. A shorter N terminus correlates with monomeric configuration, labile iron binding, and dynamic contacts with components of the Fe-S cluster biosynthetic machinery, i.e. the sulfur donor complex NFS1·ISD11 and the scaffold ISCU. Conversely, a longer N terminus correlates with the ability to oligomerize, store iron, and form stable contacts with NFS1·ISD11 and ISCU. Monomeric FXN(81-210) donates Fe(2+) for Fe-S cluster assembly on ISCU, whereas oligomeric FXN(42-210) donates either Fe(2+) or Fe(3+). These functionally distinct FXN isoforms seem capable to ensure incremental rates of Fe-S cluster synthesis from different mitochondrial iron pools. We suggest that the levels of both isoforms are relevant to FRDA pathophysiology and that the FXN(81-210)/FXN(42-210) molar ratio should provide a useful parameter to optimize FXN augmentation and replacement therapies.     

Crystal structure of glyceraldehyde-3-phosphate dehydrogenase from Plasmodium falciparum at 2.25 A resolution reveals intriguing extra electron density in the active site

The crystal structure of D-glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH) from the major malaria parasite Plasmodium falciparum is solved at 2.25 A resolution. The structure of PfGAPDH is of interest due to the dependence of the malaria parasite in infected human erythrocytes on the glycolytic pathway for its energy generation. Recent evidence suggests that PfGAPDH may also be required for other critical activities such as apical complex formation. The cofactor NAD(+) is bound to all four subunits of the tetrameric enzyme displaying excellent electron densities. In addition, in all four subunits a completely unexpected large island of extra electron density in the active site is observed, approaching closely the nicotinamide ribose of the NAD(+). This density is most likely the protease inhibitor AEBSF, found in maps from two different crystals. This putative AEBSF molecule is positioned in a crucial location and hence our structure, with expected and unexpected ligands bound, can be of assistance in lead development and design of novel antimalarials.     

Yeast strains with N-terminally truncated ribosomal protein S5: implications for the evolution,structure and function of the Rps5/Rps7 proteins

Ribosomal protein (rp)S5 belongs to the family of the highly conserved rp’s that contains rpS7 from prokaryotes and rpS5 from eukaryotes. Alignment of rpS5/rpS7 from metazoans (Homo sapiens), fungi (Saccharomyces cerevisiae) and bacteria (Escherichia coli) shows that the proteins contain a conserved central/C-terminal core region and possess variable N-terminal regions. Yeast rpS5 is 69 amino acids (aa) longer than the E. coli rpS7 protein; and human rpS5 is 48 aa longer than the rpS7, respectively. To investigate the function of the yeast rpS5 and in particular the role of its N-terminal region, we obtained and characterized yeast strains in which the wild-type yeast rpS5 was replaced by its truncated variants, lacking 13, 24, 30 and 46 N-terminal amino acids, respectively. All mutant yeast strains were viable and displayed only moderately reduced growth rates, with the exception of the strain lacking 46 N-terminal amino acids, which had a doubling time of about 3 h. Biochemical analysis of the mutant yeast strains suggests that the N-terminal part of the eukaryotic and, in particular, yeast rpS5 may impact the ability of 40S subunits to function properly in translation and affect the efficiency of initiation, specifically the recruitment of initiation factors eIF3 and eIF2.