Grifonin-1: A Small HIV-1 Entry Inhibitor Derived from the Algal Lectin, Griffithsin

Background: Griffithsin, a 121-residue protein isolated from a red algal Griffithsia sp., binds high mannose N-linked glycans of virus surface glycoproteins with extremely high affinity, a property that allows it to prevent the entry of primary isolates and laboratory strains of T- and M-tropic HIV-1. We used the sequence of a portion of griffithsin's sequence as a design template to create smaller peptides with antiviral and carbohydrate-binding properties. Methodology/Results: The new peptides derived from a trio of homologous β-sheet repeats that comprise the motifs responsible for its biological activity. Our most active antiviral peptide, grifonin-1 (GRFN-1), had an EC50 of 190.8±11.0 nM in in vitro TZM-bl assays and an EC50 of 546.6±66.1 nM in p24gag antigen release assays. GRFN-1 showed considerable structural plasticity, assuming different conformations in solvents that differed in polarity and hydrophobicity. Higher concentrations of GRFN-1 formed oligomers, based on intermolecular β-sheet interactions. Like its parent protein, GRFN-1 bound viral glycoproteins gp41 and gp120 via the N-linked glycans on their surface. Conclusion: Its substantial antiviral activity and low toxicity in vitro suggest that GRFN-1 and/or its derivatives may have therapeutic potential as topical and/or systemic agents directed against HIV-1

Comprehensive assessment of sequence variation within the copy number variable defensin cluster on 8p23 by target enriched in-depth 454 sequencing

<p>Abstract</p> <p>Background</p> <p>In highly copy number variable (CNV) regions such as the human defensin gene locus, comprehensive assessment of sequence variations is challenging. PCR approaches are practically restricted to tiny fractions, and next-generation sequencing (NGS) approaches of whole individual genomes e.g. by the 1000 Genomes Project is confined by an affordable sequence depth. Combining target enrichment with NGS may represent a feasible approach.</p> <p>Results</p> <p>As a proof of principle, we enriched a ~850 kb section comprising the CNV defensin gene cluster DEFB, the invariable DEFA part and 11 control regions from two genomes by sequence capture and sequenced it by 454 technology. 6,651 differences to the human reference genome were found. Comparison to HapMap genotypes revealed sensitivities and specificities in the range of 94% to 99% for the identification of variations.</p> <p>Using error probabilities for rigorous filtering revealed 2,886 unique single nucleotide variations (SNVs) including 358 putative novel ones. DEFB CN determinations by haplotype ratios were in agreement with alternative methods.</p> <p>Conclusion</p> <p>Although currently labor extensive and having high costs, target enriched NGS provides a powerful tool for the comprehensive assessment of SNVs in highly polymorphic CNV regions of individual genomes. Furthermore, it reveals considerable amounts of putative novel variations and simultaneously allows CN estimation.</p

Histidine-Rich Glycoprotein Protects from Systemic Candida Infection

Fungi, such as Candida spp., are commonly found on the skin and at mucosal surfaces. Yet, they rarely cause invasive infections in immunocompetent individuals, an observation reflecting the ability of our innate immune system to control potentially invasive microbes found at biological boundaries. Antimicrobial proteins and peptides are becoming increasingly recognized as important effectors of innate immunity. This is illustrated further by the present investigation, demonstrating a novel antifungal role of histidine-rich glycoprotein (HRG), an abundant and multimodular plasma protein. HRG bound to Candida cells, and induced breaks in the cell walls of the organisms. Correspondingly, HRG preferentially lysed ergosterol-containing liposomes but not cholesterol-containing ones, indicating a specificity for fungal versus other types of eukaryotic membranes. Both antifungal and membrane-rupturing activities of HRG were enhanced at low pH, and mapped to the histidine-rich region of the protein. Ex vivo, HRG-containing plasma as well as fibrin clots exerted antifungal effects. In vivo, Hrg−/− mice were susceptible to infection by C. albicans, in contrast to wild-type mice, which were highly resistant to infection. The results demonstrate a key and previously unknown antifungal role of HRG in innate immunity

Transgressive phenotypes and generalist pollination in the floral evolution of Nicotiana polyploids

This is the author's proofThe file attached is the Accepted/final draft post-refereeing version of the article. 6 month embargo now lapsed

The Formulated Microbicide RC-101 Was Safe and Antivirally Active Following Intravaginal Application in Pigtailed Macaques

Background: RC-101 is a congener of the antiretroviral peptide retrocyclin, which we and others have reported is active against clinical HIV-1 isolates from all major clades, does not hemagglutinate, and is non-toxic and non-inflammatory in cervicovaginal cell culture. Herein, film-formulated RC-101 was assessed for its antiviral activity in vitro, safety in vivo, retention in the cervix and vagina, and ability to remain active against HIV-1 and SHIV after intravaginal application in macaques. Methodology/Principal Findings: RC-101 was formulated as a quick-dissolving film (2000 μg/film), retained complete activity in vitro as compared to unformulated peptide, and was applied intravaginally in six pigtailed macaques daily for four days. At one and four days following the final application, the presence of RC-101 was assessed in peripheral blood, cervicovaginal lavage, cytobrushed cervicovaginal cells, and biopsied cervical and vaginal tissues by quantitative western blots. One day following the last film application, cervical biopsies from RC-101-exposed and placebo-controlled macaques were collected and were subjected to challenge with RT-SHIV in an ex vivo organ culture model. RC-101 peptide was detected primarily in the cytobrush and biopsied cervical and vaginal tissues, with little to no peptide detected in lavage samples, suggesting that the peptide was associated with the cervicovaginal epithelia. RC-101 remained in the tissues and cytobrush samples up to four days post-application, yet was not detected in any sera or plasma samples. RC-101, extracted from cytobrushes obtained one day post-application, remained active against HIV-1 BaL. Importantly, cervical biopsies from RC-101-treated animals reduced RT-SHIV replication in ex vivo organ culture as compared to placebo-treated animals. Conclusions/Significance:Formulated RC-101 was stable in vivo and was retained in the mucosa. The presence of antivirally active RC-101 after five days in vivo suggests that RC-101 would be an important molecule to develop further as a topical microbicide to prevent HIV-1 transmission. © 2010 Cole et al

The Tetraspanin Protein CD37 Regulates IgA Responses and Anti-Fungal Immunity

Immunoglobulin A (IgA) secretion by plasma cells in the immune system is critical for protecting the host from environmental and microbial infections. However, the molecular mechanisms underlying the generation of IgA+ plasma cells remain poorly understood. Here, we report that the B cell–expressed tetraspanin CD37 inhibits IgA immune responses in vivo. CD37-deficient (CD37−/−) mice exhibit a 15-fold increased level of IgA in serum and significantly elevated numbers of IgA+ plasma cells in spleen, mucosal-associated lymphoid tissue, as well as bone marrow. Analyses of bone marrow chimeric mice revealed that CD37–deficiency on B cells was directly responsible for the increased IgA production. We identified high local interleukin-6 (IL-6) production in germinal centers of CD37−/− mice after immunization. Notably, neutralizing IL-6 in vivo reversed the increased IgA response in CD37−/− mice. To demonstrate the importance of CD37—which can associate with the pattern-recognition receptor dectin-1—in immunity to infection, CD37−/− mice were exposed to Candida albicans. We report that CD37−/− mice are evidently better protected from infection than wild-type (WT) mice, which was accompanied by increased IL-6 levels and C. albicans–specific IgA antibodies. Importantly, adoptive transfer of CD37−/− serum mediated protection in WT mice and the underlying mechanism involved direct neutralization of fungal cells by IgA. Taken together, tetraspanin protein CD37 inhibits IgA responses and regulates the anti-fungal immune response

Effects of Aberrant Pax6 Gene Dosage on Mouse Corneal Pathophysiology and Corneal Epithelial Homeostasis

Background: Altered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6(+/-) heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6(+/Sey-Neu) (Pax6(+/-)) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77(Tg/-) transgenics, which over-express Pax6 and model human PAX6 duplication. Methodology/Principal Findings: We used electron microscopy to investigate ocular defects in Pax6(+/-) heterozygotes (low Pax6 levels) and PAX77(Tg/-) transgenics (high Pax6 levels). As well as the well-documented epithelial defects, aberrant Pax6 dosage had profound effects on the corneal stroma and endothelium in both genotypes, including cellular vacuolation, similar to that reported for human macular corneal dystrophy. We used mosaic expression of an X-linked LacZ transgene in X-inactivation mosaic female (XLacZ(Tg/-)) mice to investigate corneal epithelial maintenance by LESC clones in Pax6(+/-) and PAX77(Tg/-) mosaic mice. PAX77(Tg/-) mosaics, over-expressing Pax6, produced normal corneal epithelial radial striped patterns (despite other corneal defects), suggesting that centripetal cell movement was unaffected. Moderately disrupted patterns in Pax6(+/-) mosaics were corrected by introducing the PAX77 transgene (in Pax6(+/-), PAX77(Tg/-) mosaics). Pax6(Leca4/+), XLacZ(Tg/-) mosaic mice (heterozygous for the Pax6(Leca4) missense mutation) showed more severely disrupted mosaic patterns. Corrected corneal epithelial stripe numbers (an indirect estimate of active LESC clone numbers) declined with age (between 15 and 30 weeks) in wild-type XLacZ(Tg/-) mosaics. In contrast, corrected stripe numbers were already low at 15 weeks in Pax6(+/-) and PAX77(Tg/-) mosaic corneas, suggesting Pax6 under-and over-expression both affect LESC clones. Conclusions/Significance: Pax6(+/-) and PAX77(Tg/-) genotypes have only relatively minor effects on LESC clone numbers but cause more severe corneal endothelial and stromal defects. This should prompt further investigations of the pathophysiology underlying human aniridia and ARK

Rare coding variants in ten genes confer substantial risk for schizophrenia

Rare coding variation has historically provided the most direct connections between gene function and disease pathogenesis. By meta-analysing the whole exomes of 24,248 schizophrenia cases and 97,322 controls, we implicate ultra-rare coding variants (URVs) in 10 genes as conferring substantial risk for schizophrenia (odds ratios of 3–50, P < 2.14 × 10−6) and 32 genes at a false discovery rate of <5%. These genes have the greatest expression in central nervous system neurons and have diverse molecular functions that include the formation, structure and function of the synapse. The associations of the NMDA (N-methyl-d-aspartate) receptor subunit GRIN2A and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor subunit GRIA3 provide support for dysfunction of the glutamatergic system as a mechanistic hypothesis in the pathogenesis of schizophrenia. We observe an overlap of rare variant risk among schizophrenia, autism spectrum disorders1, epilepsy and severe neurodevelopmental disorders2, although different mutation types are implicated in some shared genes. Most genes described here, however, are not implicated in neurodevelopment. We demonstrate that genes prioritized from common variant analyses of schizophrenia are enriched in rare variant risk3, suggesting that common and rare genetic risk factors converge at least partially on the same underlying pathogenic biological processes. Even after excluding significantly associated genes, schizophrenia cases still carry a substantial excess of URVs, which indicates that more risk genes await discovery using this approach

Analysing the eosinophil cationic protein - a clue to the function of the eosinophil granulocyte

Eosinophil granulocytes reside in respiratory mucosa including lungs, in the gastro-intestinal tract, and in lymphocyte associated organs, the thymus, lymph nodes and the spleen. In parasitic infections, atopic diseases such as atopic dermatitis and asthma, the numbers of the circulating eosinophils are frequently elevated. In conditions such as Hypereosinophilic Syndrome (HES) circulating eosinophil levels are even further raised. Although, eosinophils were identified more than hundred years ago, their roles in homeostasis and in disease still remain unclear. The most prominent feature of the eosinophils are their large secondary granules, each containing four basic proteins, the best known being the eosinophil cationic protein (ECP). This protein has been developed as a marker for eosinophilic disease and quantified in biological fluids including serum, bronchoalveolar lavage and nasal secretions. Elevated ECP levels are found in T helper lymphocyte type 2 (atopic) diseases such as allergic asthma and allergic rhinitis but also occasionally in other diseases such as bacterial sinusitis. ECP is a ribonuclease which has been attributed with cytotoxic, neurotoxic, fibrosis promoting and immune-regulatory functions. ECP regulates mucosal and immune cells and may directly act against helminth, bacterial and viral infections. The levels of ECP measured in disease in combination with the catalogue of known functions of the protein and its polymorphisms presented here will build a foundation for further speculations of the role of ECP, and ultimately the role of the eosinophil