YRKL sequence of influenza virus M1 functions as the L domain motif and interacts with VPS28 and Cdc42

Earlier studies have shown that the C-terminal half of helix 6 (H6) of the influenza A virus matrix protein (M1) containing the YRKL sequence is involved in virus budding (E. K.-W. Hui, S. Barman, T. Y. Yang, and D. P. Nayak, J. Virol. 77:7078-7092, 2003). In this report, we show that the YRKL sequence is the L domain motif of influenza virus. Like other L domains, YRKL can be inserted at different locations on the mutant M1 protein and can restore virus budding in a position-independent manner. Although YRKL is a part of the nuclear localization signal (NLS), the function of YRKL was independent of the NLS activity and the NLS function of M1 was not required for influenza virus replication. Some mutations in YRKL and the adjacent region caused a reduction in the virus titer by blocking virus release, and some affected virus morphology, producing elongated particles. Coimmunoprecipitation and Western blotting analyses showed that VPS28, a component of the ESCRT-I complex, and Cdc42, a member of the Rho family GTP-binding proteins, interacted with the M1 protein via the YRKL motif. In addition, depletion of VPS28 and Cdc42 by small interfering RNA resulted in reduction of influenza virus production. Moreover, overexpression of dominant-negative Cdc42 inhibited influenza virus replication, whereas a constitutively active Cdc42 mutant enhanced virus production in infected cells. These results indicated that VPS28, a component of ESCRT-I, and Cdc42, a small G protein, are associated with the M1 protein and involved in the influenza virus life cycle.     

Maximum Likelihood Estimation of Long‐Term HIV Dynamic Models and Antiviral Response

Summary HIV dynamics studies, based on differential equations, have significantly improved the knowledge on HIV infection. While first studies used simplified short‐term dynamic models, recent works considered more complex long‐term models combined with a global analysis of whole patient data based on nonlinear mixed models, increasing the accuracy of the HIV dynamic analysis. However statistical issues remain, given the complexity of the problem. We proposed to use the SAEM (stochastic approximation expectation‐maximization) algorithm, a powerful maximum likelihood estimation algorithm, to analyze simultaneously the HIV viral load decrease and the CD4 increase in patients using a long‐term HIV dynamic system. We applied the proposed methodology to the prospective COPHAR2–ANRS 111 trial. Very satisfactory results were obtained with a model with latent CD4 cells defined with five differential equations. One parameter was fixed, the 10 remaining parameters (eight with between‐patient variability) of this model were well estimated. We showed that the efficacy of nelfinavir was reduced compared to indinavir and lopinavir.     

Plasma-based approach to measure target engagement for liver-targeting stearoyl-CoA desaturase 1 inhibitors

A positive correlation between stearoyl-CoA desaturase (SCD)1 expression and metabolic diseases has been reported in rodents and humans. These findings indicate that SCD1 is a promising therapeutic target for the chronic treatment of diabetes and dyslipidemia. The SCD1 enzyme is expressed at high levels in several human tissues and is required for the biosynthesis of monounsaturated fatty acids, which are involved in many biological processes. Liver-targeted SCD inhibitors were designed to pharmacologically manipulate SCD1 activity in the liver to avoid adverse events due to systemic inhibition. This article describes the development of a plasma-based SCD assay to assess the level of SCD inhibition, which is defined in this article as target engagement. Essentially, animals are dosed with an exogenous deuterated tracer (d7-stearic acid) as substrate, and the converted d7-oleic acid product is measured to monitor SCD1 inhibition. This study reveals that this plasma-based assay correlates with liver SCD1 inhibition and can thus have clinical utility.     

Philometrids of the southern flounder Paralichthys lethostigma: a multidimensional approach to determine their diversity

Two species of philometrid nematode, Philometra overstreeti and Philometroides paralichthydis, infect the southern flounder, Paralichthys lethostigma. Individuals of P. overstreeti are located between the teeth and inside the bony part of the branchial arches of the fish. Individuals of P. paralichthydis are associated with the bones of the buccal cavity and among muscles that control the dorsal and anal fins. Sequencing of part of the cytochrome oxidase I gene revealed 4 distinct genetic clades, each corresponding exactly to the 4 respective locations of the parasites in the host, suggesting the need for taxonomic revision. We hypothesized that each clade represented a separate species and, because the worms are morphologically indistinguishable, compared population level parameters of the clades comprising each currently recognized species. For each currently recognized species, the presence of worms from 1 clade was negatively correlated with the presence of worms from the other. Results also indicated significant differences between the clades in prevalences relative to both biotic and abiotic factors. Results clearly indicated major differences in the ecology of the philometrids constituting each clade. Taken as a whole, molecular and ecological data support the contention that the 4 genetic clades are likely 4 distinct species.     

Identification of a molecular activator for insulin receptor with potent anti-diabetic effects

Insulin exerts its actions through the insulin receptor (IR) and plays an essential role in diabetes. The inconvenient daily injection and undesirable side-effects associated with insulin injection demand novel drugs for the diseases. To search for bioactive insulin mimetics, we developed an in vitro screening assay using phospho-IR ELISA. After screening the small molecule chemical libraries, we have obtained a compound (5,8-diacetyloxy-2,3-dichloro-1,4-naphthoquinone) that provokes IR activation by directly binding to the receptor kinase domain to trigger its kinase activity at micromolar concentrations. This compound selectively activates IR but not other receptors and sensitizes insulin's action. Moreover, it elevates glucose uptake in adipocytes and has oral hypoglycemic effect in wild-type C57BL/6J mice and db/db and ob/ob mice without demonstrable toxicity. Hence, this promising compound mimics the biological functions of insulin and is useful for further drug development for diabetes treatment.     

Substituted aminopyridines as potent and selective phosphodiesterase-4 inhibitors

The synthesis and the biological evaluation of new potent phosphodiesterase type 4 (PDE4) inhibitors are presented. This new series was elaborated by replacement of the metabolically resistant phenyl hexafluorocarbinol of L-791,943 (1) by a substituted aminopyridine residue. The structure-activity relationship of N-substitution on 3 led to the identification of (-)-3n which exhibited a good PDE4 inhibitor activity (HWB-TNFalpha=0.12 microM) and an improved pharmacokinetic profile over L-791,943 (rat t(1/2)=2 h). (-)-3n was well tolerated in ferret with an emetic threshold of 30 mg/kg (po) and was found to be active in the ovalbumin-induced bronchoconstriction model in guinea pig (54%, 0.1 mg/kg, ip) as well as the ascaris-induced bronchoconstriction model in sheep (64%/97%, early/late, 0.5 mg/kg, iv).     

The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.     

Genome-Wide Investigation of DNA Methylation Marks Associated with FV Leiden Mutation

In order to investigate whether DNA methylation marks could contribute to the incomplete penetrance of the FV Leiden mutation, a major genetic risk factor for venous thrombosis (VT), we measured genome-wide DNA methylation levels in peripheral blood samples of 98 VT patients carrying the mutation and 251 VT patients without the mutation using the dedicated Illumina HumanMethylation450 array. The genome-wide analysis of 388,120 CpG probes identified three sites mapping to the SLC19A2 locus whose DNA methylation levels differed significantly (p<3 10⁻⁸) between carriers and non-carriers. The three sites replicated (p<2 10⁻⁷) in an independent sample of 214 individuals from five large families ascertained on VT and FV Leiden mutation among which 53 were carriers and 161 were non-carriers of the mutation. In both studies, these three CpG sites were also associated (2.33 10⁻¹¹<p<3.02 10⁻⁴) with biomarkers of the Protein C pathway known to be influenced by the FV Leiden mutation. A comprehensive linkage disequilibrium (LD) analysis of the whole locus revealed that the original associations were due to LD between the FV Leiden mutation and a block of single nucleotide polymorphisms (SNP) located in SLC19A2. After adjusting for this block of SNPs, the FV Leiden mutation was no longer associated with any CpG site (p>0.05). In conclusion, our work clearly illustrates some promises and pitfalls of DNA methylation investigations on peripheral blood DNA in large epidemiological cohorts. DNA methylation levels at SLC19A2 are influenced by SNPs in LD with FV Leiden, but these DNA methylation marks do not explain the incomplete penetrance of the FV Leiden mutation.