Rab 5 is required for the cellular entry of dengue and West Nile viruses

The mechanisms of cellular entry of dengue and West Nile viruses are not well characterized. We show that both these viruses enter HeLa cells by clathrin-dependent endocytosis and require vacuolar acidic pH. Inhibition of the GTPase Rab 5 or 7, which regulates transport to early or late endosomes, respectively, demonstrated that Rab 5 was essential for survival of both dengue and West Nile virus. These data broaden our understanding of the pathways required for productive dengue and West Nile virus infection and may facilitate new strategies for combating disease.     

alpha-Glucosidase inhibitory pentacyclic triterpenes from the stem bark of Fagara tessmannii (Rutaceae)

In addition to fatty acids, a mixture of sterols (beta-sitosterol, stigmasterol, campesterol and stigmastanol), lupeol, arctigenin methylether, sesamin, vanillic acid (1), 2,6-dimethoxy-1,4-benzoquinone (2), betulinic acid and two pentacyclic triterpene acetates were isolated from Fagara tessmannii Engl. They were identified as 3beta-acetoxy-16beta-hydroxybetulinic acid (3a) and 3beta,16beta-diacetoxybetulinic acid (3b), and their structures were established using 1 and 2D NMR spectra and by comparison with published data. Two derivatives of the compounds were prepared. Some isolated compounds were evaluated for their antifungal and antibacterial activities. Compounds 1 and 3a showed significant inhibition of alpha-glucosidase.     

Role of huddling on the energetic of growth in a newborn altricial mammal

Huddling is considered as a social strategy to reduce thermal stress and promote growth in newborn altricial mammals. So far, the role of huddling on the allocation of saved energy has not been quantified nor have the related impacts on body temperature rhythms. To determine the energy partitioning of rabbit pups either raised alone or in groups of eight, four, or two individuals, when thermoregulatory inefficient (TI) and efficient (TE), we first investigated their total energy expenditure and body composition. We then monitored body temperature and activity rhythms to test whether huddling may impact these rhythms, centered on the suckling event. Pups in a group of eight utilized 40% less energy for thermogenesis when TI than did pups alone and 32% less energy when TE. Pups in groups of eight and four had significantly lower thermoregulatory costs in the TI period, whereas pups in groups of two, four, and eight had lower costs during the TE period. Huddling pups could therefore channel the energy saved into processes of growth and accrued more fat mass (on average 4.5 +/- 1.4 g) than isolated pups, which lost 0.7 g of fat. Pups in groups of four and eight had a body temperature significantly higher by 0.8 degrees C than pups in groups of two and one when TI, whereas no more differences were noted when the TE period was reached. Moreover, pups alone showed an endogenous circadian body temperature rhythm that differed when compared with that of huddling pups, with no rise before suckling. Thus huddling enables pups to invest the saved energy into growth and to regulate their body temperature to be more competitive during nursing, particularly at the early time when they are TI.     

Do ABO Blood Group Antigens Hamper the Therapeutic Efficacy of Mesenchymal Stromal Cells?

Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens – genetically governed antigenic determinants – at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome.We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products.We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals.     

Factors Affecting Date of Implantation,Parturition, and Den Entry Estimated from Activity and Body Temperature in Free-Ranging Brown Bears

Knowledge of factors influencing the timing of reproduction is important for animal conservation and management. Brown bears (Ursus arctos) are able to vary the birth date of their cubs in response to their fat stores, but little information is available about the timing of implantation and parturition in free-ranging brown bears. Body temperature and activity of pregnant brown bears is higher during the gestation period than during the rest of hibernation and drops at parturition. We compared mean daily body temperature and activity levels of pregnant and nonpregnant females during preimplantation, gestation, and lactation. Additionally we tested whether age, litter size, primiparity, environmental conditions, and the start of hibernation influence the timing of parturition. The mean date of implantation was 1 December (SD = 12), the mean date of parturition was 26 January (SD = 12), and the mean duration of the gestation period was 56 days (SD = 2). The body temperature of pregnant females was higher during the gestation and lactation periods than that of nonpregnant bears. The body temperature of pregnant females decreased during the gestation period. Activity recordings were also used to determine the date of parturition. The parturition dates calculated with activity and body temperature data did not differ significantly and were the same in 50% of the females. Older females started hibernation earlier. The start of hibernation was earlier during years with favorable environmental conditions. Dates of parturition were later during years with good environmental conditions which was unexpected. We suggest that free-ranging pregnant brown bears in areas with high levels of human activities at the beginning of the denning period, as in our study area, might prioritize investing energy in early denning than in early parturition during years with favorable environmental conditions, as a strategy to prevent disturbances caused by human.     

Temporal and Anatomical Host Resistance to Chronic Salmonella Infection Is Quantitatively Dictated by Nramp1 and Influenced by Host Genetic Background

The lysosomal membrane transporter, Nramp1, plays a key role in innate immunity and resistance to infection with intracellular pathogens such as non-typhoidal Salmonella (NTS). NTS-susceptible C57BL/6 (B6) mice, which express the mutant Nramp1^D169 allele, are unable to control acute infection with Salmonella enterica serovar Typhimurium following intraperitoneal or oral inoculation. Introducing functional Nramp1^G169 into the B6 host background, either by constructing a congenic strain carrying Nramp1^G169 from resistant A/J mice (Nramp-Cg) or overexpressing Nramp1^G169 from a transgene (Nramp-Tg), conferred equivalent protection against acute Salmonella infection. In contrast, the contributions of Nramp1 for controlling chronic infection are more complex, involving temporal and anatomical differences in Nramp1-dependent host responses. Nramp-Cg, Nramp-Tg and NTS-resistant 129×1/SvJ mice survived oral Salmonella infection equally well for the first 2–3 weeks, providing evidence that Nramp1 contributes to the initial control of NTS bacteremia preceding establishment of chronic Salmonella infection. By day 30, increased host Nramp1 expression (Tg>Cg) provided greater protection as indicated by decreased splenic bacterial colonization (Tg<Cg). However, despite controlling bacterial growth within MLN as effectively as 129×1/SvJ mice, Nramp-Cg and Nramp-Tg mice eventually succumbed to infection. These data indicate: 1) discrete, anatomically localized host resistance is conferred by Nramp1 expression in NTS-susceptible mice, 2) restriction of systemic bacterial growth in the spleens of NTS-susceptible mice is enhanced by Nramp1 expression and dose-dependent, and 3) host genes other than Nramp1 also contribute to the ability of NTS-resistant 129×1/SvJ mice to control bacterial replication during chronic infection.     

Expression pattern of AtABCC13/MRP11 reveals developmental,hormonal, and nutritional regulations

ATP binding cassette (ABC) proteins constitute one of the largest families of transporters. In Arabidopsis thaliana, over 100 genes encoding ABC transporters have been identified. Here, we described the expression pattern of AtABCC13/MRP11, a member of the multidrug resistance associated protein MRP/ABCC subfamily, previously uncharacterized. The histochemical analysis of transgenic Arabidopsis harboring AtABCC13 promoter-β-glucuronidase gene fusion shows that the AtABCC13 expression was specifically associated with vascular bundles. Moreover, AtABCC13 displayed a complex hormonal regulation. β-glucuronidase (GUS) fluorimetric assays revealed that the gene expression was induced by gibberellic acid and downregulated by naphthalene acetic acid, abscisic acid, and zeatin. Because AtABCC13 is also expressed during seed development and during germination, its expression was assessed upon exposure to various nutrients: nitrate, phosphate, and sucrose stimulated the AtABCC13 expression in seedlings, whereas their lack strongly reduced it.     

Activation of Peripheral Blood Mononuclear Cells by Dengue Virus Infection Depotentiates Balapiravir

In a recent clinical trial, balapiravir, a prodrug of a cytidine analog (R1479), failed to achieve efficacy (reducing viremia after treatment) in dengue patients, although the plasma trough concentration of R1479 remained above the 50% effective concentration (EC₅₀). Here, we report experimental evidence to explain the discrepancy between the in vitro and in vivo results and its implication for drug development. R1479 lost its potency by 125-fold when balapiravir was used to treat primary human peripheral blood mononuclear cells (PBMCs; one of the major cells targeted for viral replication) that were preinfected with dengue virus. The elevated EC₅₀ was greater than the plasma trough concentration of R1479 observed in dengue patients treated with balapiravir and could possibly explain the efficacy failure. Mechanistically, dengue virus infection triggered PBMCs to generate cytokines, which decreased their efficiency of conversion of R1479 to its triphosphate form (the active antiviral ingredient), resulting in decreased antiviral potency. In contrast to the cytidine-based compound R1479, the potency of an adenosine-based inhibitor of dengue virus (NITD008) was much less affected. Taken together, our results demonstrate that viral infection in patients before treatment could significantly affect the conversion of the prodrug to its active form; such an effect should be calculated when estimating the dose efficacious for humans.     

Molecular basis of oligoubiquitin-dependent internalization of membrane proteins in Mammalian cells

Ubiquitination induced down-regulation of cell surface proteins by internalization and lysosomal targeting plays a fundamental role in cell physiology and pathogenesis of diseases. The molecular basis of a single ubiquitin (Ub) as an autonomous endocytic signal, the widely accepted mechanism, however, remains elusive in higher eukaryotes. Using Ub containing reporter proteins without signalling abilities, we present evidence that only multiple Ub moieties, linked either covalently or assembled as oligomers with an intact interface for recognition by Ub-interacting motifs (UIMs), are recognized by the endocytic machinery in vivo and associate with a subset of Ub-binding clathrin adaptors in vitro. Genetic and pharmacological approaches show that internalization of plasma membrane proteins harbouring multiple Ub moieties is clathrin-dependent, but caveolin-independent. Functional assays demonstrate the cargo-dependent involvement of eps15/15R and epsin, UIM containing clathrin adaptors, in the endocytosis of model proteins, CD4 and the activated beta(2)-adrenergic receptor complex, containing polymeric or oligomeric Ub. These results provide a paradigm for the clathrin-mediated uptake of ubiquitinated membrane proteins in mammalian cells, requiring the assembly of multiple UIM-Ub interactions to overcome the low affinity binding of mono-Ub to UIM.