RseB binding to the periplasmic domain of RseA modulates the RseA:sigmaE interaction in the cytoplasm and the availability of sigmaE.RNA polymerase.

The Escherichia coli sigmaE regulon has evolved to sense the presence of misfolded proteins in the bacterial envelope. Expression of periplasmic chaperones and folding catalysts is under the control of sigmaE RNA polymerase. The N-terminal domain of RseA sequesters sigmaE in the cytoplasmic membrane, preventing its association with core RNA polymerase. The C-terminal domain of RseA interacts with RseB, a periplasmic protein. The relative concentration of sigmaE:RseA:RseB is 2:5:1 and this ratio remains unaltered upon heat shock induction of the sigmaE regulon. Purification from crude cellular extracts yields cytoplasmic, soluble sigmaE RNA polymerase as well as membrane sequestered sigmaE.RseA and sigmaE.RseA.RseB. RseB binding to the C-terminal domain of RseA increases the affinity of RseA for sigmaE by 2- to 3-fold (Kd 50-100 nM). RseB binds also to the misfolded aggregates of MalE31, a variant of maltose binding protein that forms inclusion bodies in the periplasm. We discuss a model whereby the RseB-RiseA interaction represents a measure for misfolded polypeptides in the bacterial envelope, modulating the assembly of sigmaE RNA polymerase and the cellular heat shock response.     

Cbp1, a fungal virulence factor under positive selection,forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.     

Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, we show that Netrin-1 is expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. We demonstrate that alpha6beta4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of alpha6beta4 and alpha3beta1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells.     

Kosteletzkya virginica, a halophytic species with potential for agroecotechnology in Jiangsu Province, China

Kosteletzkya virginica (L.) Presl. is a perennial dicot halophytic species, that grows in brackish portions of coastal tidal marshes of the mid-Atlantic and southeastern United States. New saline mudflats have been increasing every year in Northern Jiangsu, China. In 1993, we introduced K. virginica (L.) into China from the Halophyte Biotechnology Center (University of Delaware, USA) as a potential species to improve the soil and develop ecologically sound saline agriculture. Our nearly 10 years of experimental research, both in a test garden and in the field, indicated that K. virginica adapts excellently to the heavy saline soils of Jiangsu Province, China. The average seed yield of K. virginica in 2001 at Yancheng was 638 kg/ha from 1-year old seedlings. There were significant variations among individuals from the unselected population of K. virginica on growth, quality, and seed yield traits. There is great potential to increase the seed yield if superior clones are selected. The seed yield of 35 selected individuals was six times greater than that of the average. Four growth traits of K. virginica were found to have a significant correlation with seed yield. However, there was no strong positive or negative correlation among seed quality traits. The mean seed weight and the germination ratio of the 35 selected individuals of K. virginica were 16.36±0.32 g and 76.11±1.82%, respectively. The percentage content of oil and crude protein in the seeds was 11.28±0.67 and 8.17±0.19%, respectively. The percentage of seed vigor of the selected individuals was 99%, as determined by soft X-ray radiography.     

Relationships between renal morphology and diet in 26 species of new world bats (suborder microchiroptera)

The renal morphology of 24 species of mormoopid and phyllostomid bats feeding on six different diets was examined to test evolutionary changes in several structural traits presumably led by dietary shifts from ancestral insectivorous diets. The kidneys of a fish-eating vespertilionid and an insect-eating emballonurid were also examined but not included in the phylogenetic comparison. The length, width, and breadth of the kidneys were used to calculate relative medullary thickness (RMT). Tissues were processed for stereological analysis, and the volumes of the kidney, nephron components, and vasculature were determined. RMT did not correlate with body mass in either animal-eating or plant-eating phyllostomid and mormoopid bats. The shift from insectivory to frugivory and nectarivory was accompanied by a reduction in RMT, a reduction in the percent of renal medulla, and an increase in the percent of renal cortex. No changes in these traits were observed in bats that shifted to carnivorous, omnivorous or sanguinivorous habits. No changes were observed in renal vasculature, in the percentage of cortical and medullary nephron components or of capillaries surrounding the nephrons in any feeding group. Vespertilionid and emballonurid species had similar values in all traits examined as compared to insectivorous phyllostomids and mormoopids. Our data suggest that diet does not influence a single area of the nephron, but rather the entire nephron such that the relative amounts of renal cortex and medulla are affected.     

Tuberculosis Exacerbates HIV-1 Infection through IL-10/STAT3-Dependent Tunneling Nanotube Formation in Macrophages

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Role of the sonchus yellow net virus N protein in formation of nuclear viroplasms

Sonchus yellow net virus is a plant nucleorhabdovirus whose nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins form large viroplasms in the nuclei of infected plants (C. R. F. Martins, J. A. Johnson, D. M. Lawrence, T. J. Choi, A. Pisi, S. L. Tobin, D. Lapidus, J. D. O. Wagner, S. Ruzin, K. McDonald, and A. O. Jackson, J. Virol. 72:5669-5679, 1998). When expressed alone, the N protein localizes to the nuclei of plant and yeast (Saccharomyces cerevisiae) cells and the P protein is distributed throughout the cells, but coexpression of N and P results in formation of subnuclear viroplasm-like foci (M. M. Goodin, J. Austin, R. Tobias, M. Fujita, C. Morales, and A. O. Jackson, J. Virol. 75:9393-9406, 2001; M. M. Goodin, R. G. Dietzgen, D. Schichnes, S. Ruzin, and A. O. Jackson, Plant J. 31:375-383, 2002). We now show that the N protein and various fluorescent derivatives form similar subnuclear foci in plant cells and that homologous interactions mediated by a helix-loop-helix region near the amino terminus are required for formation of the foci. Mutations within the helix-loop-helix region also interfere with N- and P-protein interactions that are required for N and P colocalization in the subnuclear foci. Affinity purification of N proteins harboring single mutations within the motif revealed that Tyr40 is critical for N-N and N-P interactions. Additional in vitro binding assays also indicated that the N protein binds to yeast and plant importin alpha homologues, whereas mutations in the carboxy-terminal nuclear localization signal abrogate importin alpha binding. The P protein did not bind to the importin alpha homologues, suggesting that the N and P proteins use different pathways for nuclear entry. Our results in toto support a model suggesting that during infection, the N and P proteins enter the nucleus independently, that viroplasm formation requires homologous N-protein interactions, and that P protein targeting to the viroplasm requires N-P protein interactions that occur after N and P protein import into the nucleus.     

Mapping ephemeral wetlands: manual digitisation and logistic regression modelling in Nelson Mandela Bay Municipality,South Africa

Until recently, little research has been conducted on the distribution and structure of ephemeral systems in semi-arid areas. This information is critical for appropriate wetland management and conservation. The Nelson Mandela Bay Municipality is a semi-arid area along the south-eastern coastline of the Eastern Cape Province of South Africa. The Municipality encapsulates a wide range of geological and geomorphological features as well as vegetation types within an area of some 1950 km², providing an ideal area for such research. The distribution and abundance of wetlands were defined, and a logistic regression (LR) model was used to establish whether this modelling technique is viable in semi-arid areas with highly variable rainfall patterns. Wetlands were delineated manually using geographical information systems, high-resolution aerial photographs and environmental data. More than 1700 wetland polygons were identified, with 80% of the systems being categorised as depressions, seeps and wetland flats. Unchannelled (8%) and channelled (7%) valley bottom wetlands and floodplain wetlands (5%) were also identified. The wetland database was then used to create a wetland occurrence probability model. There were 19 environmental variables used to develop the LR model, with eight variables used in the final model output. The predictive capacity of the model was good, with an area under curve value of 0.68 and an overall accuracy of 66%. This indicates that probabilistic wetland models are useful in highly variable environments with high numbers of small (<1 ha) wetlands. Such predictive models provide a tool to assist in improving the accuracy of land cover datasets in semi-arid areas, and can be used to inform management decisions on flood risk areas and key conservation zones. In addition, abiotic variables that are significant in the model output provide an indication of the factors influencing wetland functioning in the region.     

Genome‐wide association study identifies loci associated with liability to alcohol and drug dependence that is associated with variability in reward‐related ventral striatum activity in African‐ and European‐Americans

Genetic influences on alcohol and drug dependence partially overlap, however, specific loci underlying this overlap remain unclear. We conducted a genome‐wide association study (GWAS) of a phenotype representing alcohol or illicit drug dependence (ANYDEP) among 7291 European‐Americans (EA; 2927 cases) and 3132 African‐Americans (AA: 1315 cases) participating in the family‐based Collaborative Study on the Genetics of Alcoholism. ANYDEP was heritable (h ² in EA = 0.60, AA = 0.37). The AA GWAS identified three regions with genome‐wide significant (GWS; P < 5E‐08) single nucleotide polymorphisms (SNPs) on chromosomes 3 (rs34066662, rs58801820) and 13 (rs75168521, rs78886294), and an insertion‐deletion on chromosome 5 (chr5:141988181). No polymorphisms reached GWS in the EA. One GWS region (chromosome 1: rs1890881) emerged from a trans‐ancestral meta‐analysis (EA + AA) of ANYDEP, and was attributable to alcohol dependence in both samples. Four genes (AA: CRKL, DZIP3, SBK3; EA: P2RX6) and four sets of genes were significantly enriched within biological pathways for hemostasis and signal transduction. GWS signals did not replicate in two independent samples but there was weak evidence for association between rs1890881 and alcohol intake in the UK Biobank. Among 118 AA and 481 EA individuals from the Duke Neurogenetics Study, rs75168521 and rs1890881 genotypes were associated with variability in reward‐related ventral striatum activation. This study identified novel loci for substance dependence and provides preliminary evidence that these variants are also associated with individual differences in neural reward reactivity. Gene discovery efforts in non‐European samples with distinct patterns of substance use may lead to the identification of novel ancestry‐specific genetic markers of risk.     

Development and validation of a prototype 16S rRNA-based taxonomic microarray for Alphaproteobacteria

The microarray approach has been proposed for high throughput analysis of the microbial community by providing snapshots of the microbial diversity under different environmental conditions. For this purpose, a prototype of a 16S rRNA-based taxonomic microarray was developed and evaluated for assessing bacterial community diversity. The prototype microarray is composed of 122 probes that target bacteria at various taxonomic levels from phyla to species (mostly Alphaproteobacteria). The prototype microarray was first validated using bacteria in pure culture. Differences in the sequences of probes and potential target DNAs were quantified as weighted mismatches (WMM) in order to evaluate hybridization reliability. As a general feature, probes having a WMM > 2 with target DNA displayed only 2.8% false positives. The prototype microarray was subsequently tested with an environmental sample, which consisted of an Agrobacterium-related polymerase chain reaction amplicon from a maize rhizosphere bacterial community. Microarray results were compared to results obtained by cloning-sequencing with the same DNA. Microarray analysis enabled the detection of all 16S rRNA gene sequences found by cloning-sequencing. Sequences representing only 1.7% of the clone library were detected. In conclusion, this prototype 16S rRNA-based taxonomic microarray appears to be a promising tool for the analysis of Alphaproteobacteria in complex ecosystems.