Two new sponge species (Demospongiae: Chalinidae and Suberitidae) isolated from hyperarid mangroves of Qatar with notes on their potential antibacterial bioactivity.

This study presents the taxonomic description of two new sponge species that are intimately associated with the hyperarid mangrove ecosystem of Qatar. The study includes a preliminary evaluation of the sponges' potential bioactivity against pathogens. Chalinula qatari sp. nov. is a fragile thinly encrusting sponge with a vivid maroon colour in life, often with oscular chimneys and commonly recorded on pneumatophores in the intertidal and shallow subtidal zone. Suberites luna sp. nov. is a massive globular-lobate sponge with a greenish-black colour externally and a yellowish orange colour internally, recorded on pneumatophores in the shallow subtidal zone, with large specimens near the seagrass ecosystem that surrounds the mangrove. For both species, a drug extraction protocol and an antibacterial experiment was performed. The extract of Suberites luna sp. nov. was found to be bioactive against recognized pathogens such as Staphylococcus epidermidis, Staphylococcus aureus and Enterococcus faecalis, but no bioactive activity was recorded for Chalinula qatari sp. nov. This study highlights the importance of increasing bioprospecting effort in hyperarid conditions and the importance of combining bioprospecting with taxonomic studies for the identification of novel marine drugs.

Human granzymes: related but far apart.

Granzymes (GZMs) are a class of serine protease, found in cytoplasmic granules of cytotoxic T cells and natural killer cells. The main function of these proteins has been recognized as wiping out viral infections via inducing the apoptosis. This review will highlight inter and intra species differences of GZMs in terms of their functional and structure. These futures may help to device a strategy for isolation of human specific GZMs, which are needed for understating of their role in immune system and devising an effective immune therapy.

A V(L) single-domain antibody library shows a high-propensity to yield non-aggregating binders.

A synthetic human V(L) phage display library, created by the randomization of all complementarity-determining regions (CDRs) in a V(L) scaffold, was panned against three test antigens to determine the propensity of the library to yield non-aggregating binders. A total of 22 binders were isolated against the test antigens and the majority (20) were monomeric. Thus, human V(L) repertoires provide an efficient source of non-aggregating binders and represent an attractive alternative to human V(H) repertoires, which are notorious for containing high proportions of aggregating species. Moreover, the solubility of V(L)s, in contrast to V(H)s, appears much less CDR dependent.

Orally administered P22 phage tailspike protein reduces salmonella colonization in chickens: prospects of a novel therapy against bacterial infections.

One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans.

Bacteriophage tailspike proteins as molecular probes for sensitive and selective bacterial detection.

We report the use of genetically engineered tailspike proteins (TSPs) from the P22 bacteriophage for the sensitive and selective detection of Salmonella enterica serovar Typhimurium. High yields of two mutant TSPs, one with an N-terminal cysteine (N-Cys) and another with a C-terminal cysteine (C-Cys), have been obtained using recombinant protein expression and purification in Escherichia coli. The mutant TSPs did not have the native endorhamnosidase enzymatic activity of intact P22 phage as well as wild type TSPs (wtTSPs). We have used the Cys-tag to immobilize these TSPs onto gold coated surfaces using thiol-chemistry. Our results demonstrate that the N-Cys configuration of TSPs gives a bacterial capture density of 25.87 ± 0.61 bacteria/100 µm(2) while the C-Cys configuration shows a density of 8.57 ± 0.19 bacteria/100 µm(2). This confirms that the appropriate orientation of the TSPs on the surface is important for efficient capture of the host bacteria. The bacterial capture density of the mutant N-Cys TSP was also 6-fold better than that obtained for intact P22 phage as well as wtTSPs. Bovine-serum albumin was used as a protective layer to prevent any non-specific binding of the bacteria onto the gold substrate. The recognition specificity was confirmed using 3 strains of E. coli which showed negligible binding. In addition, the host bacteria did not show any binding in the absence of the TSPs on the surface. We further show a selective real-time analytical detection of Salmonella by N-Cys mTSP-immobilized on gold coated SF-10 glass plates using surface plasmon resonance. The sensitivity of detection was found to be 10(3)cfu/ml of bacteria.

Deleterious CHEK2 1100delC and L303X mutants identified among 38 human breast cancer cell lines.

The CHEK2 protein plays a major role in the regulation of DNA damage response pathways. Mutations in the CHEK2 gene, in particular 1100delC, have been associated with increased cancer risks, but the precise function of CHEK2 mutations in carcinogenesis is not known. Human cancer cell lines with CHEK2 mutations are therefore of main interest. Here, we have sequenced 38 breast cancer cell lines for mutations in the CHEK2 gene and identified two cell lines with deleterious CHEK2 mutations. Cell line UACC812 has a nonsense truncating mutation in the CHEK2 kinase domain (L303X) and cell line SUM102PT has the well-known oncogenic CHEK2 1100delC founder mutation. Immunohistochemical analysis revealed that the two CHEK2 mutant cell lines expressed neither CHEK2 nor P-Thr(68) CHEK2 proteins, implying abrogation of normal CHEK2 DNA repair functions. Cell lines UACC812 and SUM102PT thus are the first human CHEK2 null cell lines reported and should therefore be a major help in further unraveling the function of CHEK2 mutations in carcinogenesis.

An association of autoantibody status and serum cytokine levels in type 1 diabetes.

At onset of type 1 diabetes, the islet autoantibody status of patients has been reported to predict progression of the disease. We therefore tested the hypothesis that the systemic immunoregulatory balance, as defined by levels of circulating cytokines and chemokines, is associated with islet autoantibody status. In 50 patients with recent-onset type 1 diabetes, antibodies to GAD and insulinoma-associated antigen 2 (IA-2) were analyzed by radioimmunoassay; cytoplasmic islet cell antibodies were determined by indirect immunofluorescence. Cytokine and chemokine concentrations were measured by rigidly evaluated double antibody enzyme-linked immunosorbent assay. Of four classically defined Th1/Th2 cytokines (gamma-interferon, interleukin [IL]-5, IL-10, IL-13), none showed an association with multiple autoantibody positivity. Of six mediators mainly produced by innate immunity cells, three were associated with multiple autoantibody status (IL-18 increased, MIF and MCP-1 decreased) and three were unaffected (IL-12, MIP-1beta, IP-10). GAD and/or IA-2 antibody titers negatively correlated with systemic concentrations of MIF, MIP-1beta, and IL-12. Combining the data of several cytokine and chemokine levels made it possible to predict islet antibody positivity in individual patients with 85% sensitivity and 94% specificity. These data suggest a close association of islet antibody status with systemic immunoregulation in type 1 diabetes.

Cholera toxin B pretreatment of macrophages and monocytes diminishes their proinflammatory responsiveness to lipopolysaccharide.

The cholera toxin B chain (CTB) has been reported to suppress T cell-dependent autoimmune diseases and to potentiate tolerance of the adaptive immune system. We have analyzed the effects of CTB on macrophages in vitro and have found that preincubation with CTB (10 microg/ml) suppresses the proinflammatory reaction to LPS challenge, as demonstrated by suppressed production of TNF-alpha, IL-6, IL-12(p70), and NO (p < 0.01) in cells of macrophage lines. Pre-exposure to CTB also suppresses LPS-induced TNF-alpha and IL-12(p70) formation in human PBMC. Both native and recombinant CTB exhibited suppressive activity, which was shared by intact cholera toxin. In cells of the human monocyte line Mono Mac 6, exposure to CTB failed to suppress the production of IL-10 in response to LPS. Control experiments excluded a role of possible contamination of CTB by endotoxin or intact cholera toxin. The suppression of TNF-alpha production occurred at the level of mRNA formation. Tolerance induction by CTB was dose and time dependent. The suppression of TNF-alpha and IL-6 production could be counteracted by the addition of Abs to IL-10 and TGF-beta. IFN-gamma also antagonized the actions of CTB on macrophages. In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred silently, i.e., in the absence of a measurable proinflammatory response. These findings identify immune-deviating properties of CTB at the level of innate immune cells and may be relevant to the use of CTB in modulating immune-mediated diseases.