Mannose-containing oligosaccharides of non-specific human secretory immunoglobulin A mediate inhibition of Vibrio cholerae biofilm formation

The role of antigen-specific secretory IgA (SIgA) has been studied extensively, whereas there is a limited body of evidence regarding the contribution of non-specific SIgA to innate immune defenses against invading pathogens. In this study, we evaluated the effects of non-specific SIgA against infection with Vibrio cholerae O139 strain MO10 and biofilm formation. Seven day old infant mice deficient in IgA (IgA(-/-) mice) displayed significantly greater intestinal MO10 burden at 24 hr post-challenge when compared to IgA(+/+) pups. Importantly, cross-fostering of IgA(-/-) pups with IgA(+/+) nursing dams reversed the greater susceptibility to MO10 infection, suggesting a role for non-specific SIgA in protection against the infection. Since biofilm formation is associated with virulence of MO10, we further examined the role of human non-specific SIgA on this virulence phenotype of the pathogen. Human non-specific SIgA, in a dose-dependent fashion, significantly reduced the biofilm formation by MO10 without affecting the viability of the bacterium. Such an inhibitory effect was not induced by human serum IgA, IgG, or IgM, suggesting a role for the oligosaccharide-rich secretory component (SC) of SIgA. This was supported by the demonstration that SIgA treated with endoglycosidase H, to cleave the high-mannose containing terminal chitobiose residues, did not induce a reduction in biofilm formation by MO10. Furthermore, the addition of free mannose per se, across a wide dose range, induced significant reduction in MO10 biofilm formation. Collectively, these results suggest that mannose containing oligosaccharides within human non-specific secretory IgA can alter important virulence phenotypes of Vibrio cholerae such as biofilm formation, without affecting viability of the microorganism. Such effects may contribute significantly to innate immune defenses against invading pathogens in vivo in the gastrointestinal tract.     

Osmosis in Cortical Collecting Tubules : ADH-Independent Osmotic Flow Rectification

The present experiments were designed to evaluate the effects of varying the osmolality of luminal solutions on the antidiuretic hormone (ADH)-independent water and solute permeability properties of isolated rabbit cortical collecting tubules. In the absence of ADH, the osmotic water permeability coefficient (cm s^–1) P_f^l→b, computed from volume flows from hypotonic lumen to isotonic bath, was 20 ± 4 x 10^–4 (SEM); the value of P_f^b→l in the absence of ADH, computed from volume flows from isotonic bath to hypertonic lumen, was 88 ± 15 x 10^–4 cm s^–1. We also measured apparent urea permeability coefficients (cm s^–1) from ¹⁴C-urea fluxes from lumen to bath (P_DDurea^l→b) and from bath to lumen (P_DDurea^b→l). For hypotonic luminal solutions and isotonic bathing solutions, P_DDurea^l→b was 0.045 ± 0.004 x 10^–4 and was unaffected by ADH. The ADH-independent values of P_DDurea^l→b and P_urea^b→l were, respectively, 0.216 ± 0.022 x 10^–4 cm s^–1 and 0.033 ± 0.002 x 10^–4 cm s^–1 for isotonic bathing solutions and luminal solutions made hypertonic with urea, i.e., there was an absolute increase in urea permeability and asymmetry of urea fluxes. Significantly, P_DDurea^l→b did not rise when luminal hypertonicity was produced by sucrose; and, bathing fluid hypertonicity did not alter tubular permeability to water or to urea. We interpret these data to indicate that luminal hypertonicity increased the leakiness of tight junctions to water and urea but not sucrose. Since the value of P_f^b→l in the absence of ADH, when tight junctions were open to urea, was approximately half of the value of P_f^l→b in the presence of ADH, when tight junctions were closed to urea, we conclude that tight junctions are negligible paracellular shunts for lumen to bath osmosis with ADH. These findings, together with those in the preceding paper, are discussed in terms of a solubility-diffusion model for water permeation in which ADH increases water solubility in luminal plasma membranes.     

Tools to analyze protein farnesylation in cells

Farnesylation of proteins is catalyzed by protein farnesyl transferase (FTase) and is obligatory for the function of the oncoprotein Ras and a variety of other physiologically important proteins. The rapid and selective detection of cellular protein farnesylation status is crucial to understanding both the function of farnesylated proteins and FTase inhibitors. The unnatural FPP analogue 8-anilinogeranyl diphosphate (AGPP, 3b) is an effective alternative substrate for mammalian FTase. Using antibodies specific for the anilinogeranyl moiety, we show that the alcohol precursor (AGOH, 5b) of AGPP is incorporated into cellular proteins in an FTase dependent manner competitive with endogenous pools of FPP. Continuous treatment of HEK-293 cells with 100 microM AGOH for up to 3 days is neither cytotoxic or cytostatic. Antibodies to detect the unnatural anilinogeranyl group were raised against bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) bioconjugates of the activated hapten N-hydroxyphthalimido-succinyl-(S-anilinogeranyl)-L-cysteine methyl ester 9a. Polyclonal antisera containing anti-anilinogeranyl antibodies were generated by immunization of rabbits and a monoclonal anti-anilinogeranyl antibody was raised in mice. ELISA and western blotting of anilinogeranyl modified proteins were used to show the selectivity and measure the titer of the antibodies. The unnatural FPP analogue and corresponding antibodies provide a simple and rapid method for monitoring FTase activity in cells and detection of cellular proteins modified by AGOH 5a.     

Vaccine-induced immunity in baboons by using DNA and replication-incompetent adenovirus type 5 vectors expressing a human immunodeficiency virus type 1 gag gene

The cellular immunogenicity of formulated plasmid DNA and replication-defective human adenovirus serotype 5 (Ad5) vaccine vectors expressing a codon-optimized human immunodeficiency virus type 1 gag gene was examined in baboons. The Ad5 vaccine was capable of inducing consistently strong, long-lived CD8(+)-biased T-cell responses and in vitro cytotoxic activities. The DNA vaccine-elicited immune responses were weaker than those elicited by the Ad5 vaccine and highly variable; formulation with chemical adjuvants led to moderate increases in the levels of Gag-specific T cells. Increasing the DNA-primed responses with booster doses of either Ad5 or modified vaccinia virus Ankara vaccines suggests a difference in the relative levels of cytotoxic and helper responses. The implications of these results are discussed.     

Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane

Background  

MicroRNAs (miRNAs) are small regulatory RNAs, some of which are conserved in diverse plant genomes. Therefore, computational identification and further experimental validation of miRNAs from non-model organisms is both feasible and instrumental for addressing miRNA-based gene regulation and evolution. Sugarcane (Saccharum spp.) is an important biofuel crop with publicly available expressed sequence tag and genomic survey sequence databases, but little is known about miRNAs and their targets in this highly polyploid species.     

Health effects in fish from the polluted Orlando Dam and Klipspruit wetland system,Soweto, South Africa

Pollution of aquatic ecosystems often results in adverse environmental disturbances, including physiological and/ or histomorphological changes in fish. The health of Clarias gariepinus sampled from two polluted water bodies, Orlando Dam and a pond in the Klipspruit wetland catchment, Soweto, was investigated in 2015–2016. Limited fish health-related data is available for this highly impacted freshwater ecosystem. Fish were collected between April 2015 and February 2016. A necropsy and a histological assessment were done on various target organs of each fish. Water and sediment samples were analysed for selected organic and inorganic compounds. Macroscopic assessment revealed abnormally shaped urogenital papillae, morphological alterations of the gonads, as well as discoloration of liver tissue. These observations were supported by microscopic evidence of hepatic histological alterations in fish from Orlando Dam, as well as the presence of both female and male gonadal tissue (intersex) in 13.6% and 50% of fish from the wetland pond and the Orlando Dam, respectively. Water analyses showed high levels of faecal coliform bacteria and metal concentrations and sediment analyses showed detectable levels of potential endocrine disrupting chemicals.     

Secretagogue response of goblet cells and columnar cells in human colonic crypts

Crypts of Lieberkühn were isolated fromhuman colon, and differential interference contrast microscopydistinguished goblet and columnar cells. Activation with carbachol(CCh, 100 µM) or histamine (10 µM) released contents from gobletgranules. Stimulation with prostaglandinE₂(PGE₂, 5 µM) or adenosine (10 µM) did not release goblet granules but caused the apical margin ofcolumnar cells to recede. Goblet volume was lost during stimulationwith CCh or histamine (~160 fl/cell), but not withPGE₂ or adenosine. Three-quartersof goblet cells were responsive to CCh but released only 30% of gobletvolume. Half-time for goblet volume release was 3.7 min.PGE₂ stimulated a prolonged fluidsecretion that attained a rate of ~350 pl/min. Columnar cells lost~50% of apical volume during maximalPGE₂ stimulation, with a half-timeof 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. Theseresults support separate regulation for mucus secretions from gobletcells and from columnar cells, with control mechanisms restrictingtotal release of mucus stores.

     

Frequency and clinical patterns of stroke in Iran - Systematic and critical review

Background  

Cerebrovascular disease is the second commonest cause of death, and over a third of stroke deaths occur in developing countries. To fulfil the current gap on data, this systematic review is focused on the frequency of stroke, risk factors, stroke types and mortality in Iran.