Horizontal transmission of epizootic ulcerative syndrome (EUS)-associated virus in the snakehead Ophicephalus striatus under simulated natural conditions

Natural transmission of the epizootic ulcerative syndrome (EUS) was conducted on na?ve snakeheads Ophicephalus striatus (also known as Channa striata) kept (A) in aquifer water, (B) in lakewater, (C) cohabiting with EUS snakeheads in lakewater, and (D) cohabiting with apparently healthy snakeheads in lakewater during the 1994 to 1995 EUS season. The results showed that EUS-like lesions developed in 6 to 14 d among na?ve snakeheads cohabiting with EUS snakeheads and with apparently healthy snakeheads in lakewater (Treatments C and D). Among na?ve fish exposed to lakewater (Treatment B), similar lesions developed in 16 to 21 d, while na?ve fish in aquifer water (Treatment A) did not develop EUS-like lesions. EUS signs began as Grade I (slight) lesions that gradually progressed to Grades III-IV (severe) 3 to 5 d from lesion onset, similar to the naturally affected EUS fish. The virus was recovered from some but not all naturally EUS-affected snakeheads, snakeheads with healing lesions and apparently healthy snakeheads, but not from na?ve snakeheads. The results provide evidence of a waterborne horizontal transmission of the EUS-associated virus. This is the first report of a successful horizontal transmission of the EUS-associated virus from apparently healthy snakeheads to na?ve fish under natural conditions and of virus recovery in tissue culture from naturally exposed experimental fish.     

Benzochromenes from the roots of Bourreria pulchra

Two new benzochromenes, (6,6-dimethyl-2-methoxy-6H-benzo[c]chromen-9-yl)methanol (1), and 2-methoxy-6,6-dimethyl-6H-benzo[c]chromen-9-carbaldehyde (2), together with several already known metabolites, were isolated from the root extract of Bourreria pulchra (Boraginaceae). The structures of 1 and 2 were established on the basis of their spectroscopic data. Both were assayed for in vitro antiprotozoan activity, and especially 1 was found to possess significant activity against Leishmania mexicana and Trypanosoma cruzi parasites (IC₅₀ 4.6 μg/mL and 7.5 μg/mL, respectively).     

Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.     

Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum

Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans.     

Small-molecule cyclic alpha V beta 3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion

Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial alpha V beta 3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block alpha V beta 3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA (alpha-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind alpha V beta 3. An inactive peptide, EMD 135981 (cR beta-ADFV) was used as control. Cyclization and the introduction of D-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind alpha V beta 3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either alpha V beta 3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic alpha V beta 3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.     

Potent and selective cyclohexyl-derived imidazopyrazine insulin-like growth factor 1 receptor inhibitors with in vivo efficacy

Preclinical and emerging clinical evidence suggests that inhibiting insulin-like growth factor 1 receptor (IGF-1R) signaling may offer a promising therapeutic strategy for the treatment of several types of cancer. This Letter describes the medicinal chemistry effort towards a series of 8-amino-imidazo[1,5-a]pyrazine derived inhibitors of IGF-1R which features a substituted quinoline moiety at the C1 position and a cyclohexyl linking moiety at the C3 position. Lead optimization efforts which included the optimization of structure-activity relationships and drug metabolism and pharmacokinetic properties led to the identification of compound 9m, a potent, selective and orally bioavailable inhibitor of IGF-1R with in vivo efficacy in an IGF-driven mouse xenograft model.     

SPL7013 Gel (VivaGel?) Retains Potent HIV-1 and HSV-2 Inhibitory Activity following Vaginal Administration in Humans

SPL7013 Gel (VivaGel^®) is a microbicide in development for prevention of HIV and HSV. This clinical study assessed retention and duration of antiviral activity following vaginal administration of 3% SPL7013 Gel in healthy women. Participants received 5 single doses of product with ≥5 days between doses. A cervicovaginal fluid (CVF) sample was collected using a SoftCup™ pre-dose, and immediately, or 1, 3, 12 or 24 h post-dose. HIV-1 and HSV-2 antiviral activities of CVF samples were determined in cell culture assays. Antiviral activity in the presence of seminal plasma was also tested. Mass and concentration of SPL7013 in CVF samples was determined. Safety was assessed by reporting of adverse events. Statistical analysis was performed using the Wilcoxon signed-rank test with Bonferroni adjustment; p≤0.003 was significant. Eleven participants completed the study. Inhibition of HIV-1 and HSV-2 by pre-dose CVF samples was negligible. CVF samples obtained immediately after dosing almost completely inhibited (median, interquartile range) HIV-1 [96% (95,97)] and HSV-2 [86% (85,94)], and activity was maintained in all women at 3 h (HIV-1 [96% (95,98), p = 0.9]; HSV-2 [94% (91,97), p = 0.005]). At 24 h, >90% of initial HIV-1 and HSV-2 inhibition was maintained in 6/11 women. SPL7013 was recovered in CVF samples obtained at baseline (46% of 105 mg dose). At 3 and 24 h, 22 mg and 4 mg SPL7013, respectively, were recovered. More than 70% inhibition of HIV-1 and HSV-2 was observed if there was >0.5 mg SPL7013 in CVF samples. High levels of antiviral activity were retained in the presence of seminal plasma. VivaGel was well tolerated with no signs or symptoms of vaginal, vulvar or cervical irritation reported. Potent antiviral activity was observed against HIV-1 and HSV-2 immediately following vaginal administration of VivaGel, with activity maintained for at least 3 h post-dose. The data provide evidence of antiviral activity in a clinical setting, and suggest VivaGel could be administered up to 3 h before coitus.

Trial Registration

The study is registered at ClinicalTrials.gov under identifier: {"type":"clinical-trial","attrs":{"text":"NCT00740584","term_id":"NCT00740584"}}NCT00740584     

Tagging genes with cassette-exchange sites

In an effort to make transgenesis more flexible and reproducible, we developed a system based on novel 5′ and 3′ ‘gene trap’ vectors containing heterospecific Flp recognition target sites and the corresponding ‘exchange’ vectors allowing the insertion of any DNA sequence of interest into the trapped locus. Flp-recombinase-mediated cassette exchange was demonstrated to be highly efficient in our system, even in the absence of locus-specific selection. The feasibility of constructing a library of ES cell clones using our gene trap vectors was tested and a thousand insertion sites were characterized, following electroporation in ES cells, by RACE–PCR and sequencing. We validated the system in vivo for two trapped loci in transgenic mice and demonstrated that the reporter transgenes inserted into the trapped loci have an expression pattern identical to the endogenous genes. We believe that this system will facilitate in vivo studies of gene function and large-scale generation of mouse models of human diseases, caused by not only loss but also gain of function alleles.