The novel adipocytokine visfatin exerts direct cardioprotective effects

Visfatin is an adipocytokine capable of mimicking the glucose-lowering effects of insulin and activating the pro-survival kinases phosphatidylinositol-3-OH kinase (PI3K)-protein kinase B (Akt) and mitogen-activated protein kinase kinase 1 and 2 (MEK1/2)-extracellular signal-regulated kinase 1 and 2 (Erk 1/2). Experimental studies have demonstrated that the activation of these kinases confers cardioprotection through the inhibition of the mitochondrial permeability transition pore (mPTP). Whether visfatin is capable of exerting direct cardioprotective effects through these mechanisms is unknown and is the subject of the current study. Anaesthetized C57BL/6 male mice were subjected to in situ 30 min. of regional myocardial ischaemia and 120 min. of reperfusion. The administration of an intravenous bolus of visfatin (5 x 10(-6) micromol) at the time of myocardial reperfusion reduced the myocardial infarct size from 46.1+/-4.1% in control hearts to 27.3+/-4.0% (n>or= 6/group, P<0.05), an effect that was blocked by the PI3K inhibitor, wortmannin, and the MEK1/2 inhibitor, U0126 (48.8+/-5.5% and 45.9+/-8.4%, respectively, versus 27.3+/-4.0% with visfatin; n>or= 6/group, P<0.05). In murine ventricular cardiomyocytes subjected to 30 min. of hypoxia followed by 30 min. of reoxygenation, visfatin (100 ng/ml), administered at the time of reoxygenation, reduced the cell death from 65.2+/-4.6% in control to 49.2+/-3.7%(n>200 cells/group, P<0.05), an effect that was abrogated by wortmannin and U0126 (68.1+/-5.2% and 59.7+/-6.2%, respectively; n>200 cells/group, P>0.05). Finally, the treatment of murine ventricular cardiomyocytes with visfatin (100 ng/ml) delayed the opening of the mPTP induced by oxidative stress from 81.2+/-4 sec. in control to 120+/-7 sec. (n>20 cells/group, P<0.05) in a PI3K- and MEK1/2-dependent manner. We report that the adipocytokine, visfatin, is capable of reducing myocardial injury when administered at the time of myocardial reperfusion in both the in situ murine heart and the isolated murine cardiomyocytes. The mechanism appears to involve the PI3K and MEK1/2 pathways and the mPTP.     

Elevated incubation temperature improves later-life swimming endurance in juvenile Chinook salmon,Oncorhynchus tshawytscha

The effect of incubation and rearing temperature on muscle development and swimming endurance under a high-intensity swimming test was investigated in juvenile Chinook salmon (Oncorhynchus tshawytscha) in a hatchery experiment. After controlling for the effects of fork length (L_F) and parental identity, times to fatigue of fish were higher when fish were incubated or reared at warmer temperatures. Significant differences among combinations of pre- and post-emergence temperatures conformed to 15–15°C > 15–9°C > 9–9°C > 7–9°C > 7–7°C in 2011 when swimming tests were conducted at 300 accumulated temperature units post-emergence and 15–9°C > (7–9°C = 7–7°C) in 2012 when swimming tests were conducted at an L_F of c. 40 mm. The combination of pre- and post-emergence temperatures also affected the number and size of muscle fibres, with differences among temperature treatments in mean fibre cross-sectional area persisting after controlling for L_F and parental effects. Nonetheless, neither fibre number nor fibre size accounted for significant variation in swimming endurance. Thus, thermal carryover effects on swimming endurance were not mediated by thermal imprinting of muscle structure. This is the first study to test how temperature, body size and muscle structure interact to affect swimming endurance during early development in salmon.     

Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain

Sprouty (Spry) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor and epidermal growth factor (EGF) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. Data base searches lead to the identification and cloning of, to date, four mammalian sprouty genes. The primary sequences of the mammalian sprouty gene products share a well conserved cysteine-rich C-terminal domain with the Drosophila protein. The N-terminal regions, however, do not exhibit significant homology. This study aimed at determining the disposition of Spry proteins in intact cells before and after stimulation of the EGF receptor tyrosine kinase. Full-length or deletion mutants of Spry, tagged at the N termini with the FLAG-epitope, were expressed in COS-1 cells by transient transfection and analyzed by immunofluorescence microscopy before and after EGF stimulation of the cells. In unstimulated cells, the Spry proteins were distributed throughout the cytosol except for human Sprouty2 (hSpry2), which, although generally located in the cytosol, co-localized with microtubules. In all cases, the Spry proteins underwent rapid translocation to membrane ruffles following EGF stimulation. The optimal translocation domain was identified by deletion and immunofluorescence analysis to be a highly conserved 105-amino acid domain in the C-terminal half of the hSpry2 protein. The translocation of this conserved domain, based on hSpry2 data, was independent of the activation of phosphatidylinositol-3 kinase.     

Structural hybridization of pyrrolidine-based T-type calcium channel inhibitors and exploration of their analgesic effects in a neuropathic pain model

Highly effective and safe drugs for the treatment of neuropathic pain are urgently required and it was shown that blocking T-type calcium channels can be a promising strategy for drug development for neuropathic pain. We have developed pyrrolidine-based T-type calcium channel inhibitors by structural hybridization and subsequent assessment of in vitro activities against Ca_v3.1 and Ca_v3.2 channels. Profiling of in vitro ADME properties of compounds was also carried out. The representative compound 17h showed comparable in vivo efficacy to gabapentin in the SNL model, which indicates T-type calcium channel inhibitors can be developed as effective therapeutics for neuropathic pain.     

Expanding ART for treatment and prevention of HIV in South Africa: estimated cost and cost-effectiveness 2011-2050

Background

Antiretroviral Treatment (ART) significantly reduces HIV transmission. We conducted a cost-effectiveness analysis of the impact of expanded ART in South Africa.

Methods

We model a best case scenario of 90% annual HIV testing coverage in adults 15–49 years old and four ART eligibility scenarios: CD4 count <200 cells/mm³ (current practice), CD4 count <350, CD4 count <500, all CD4 levels. 2011–2050 outcomes include deaths, disability adjusted life years (DALYs), HIV infections, cost, and cost per DALY averted. Service and ART costs reflect South African data and international generic prices. ART reduces transmission by 92%. We conducted sensitivity analyses.

Results

Expanding ART to CD4 count <350 cells/mm³ prevents an estimated 265,000 (17%) and 1.3 million (15%) new HIV infections over 5 and 40 years, respectively. Cumulative deaths decline 15%, from 12.5 to 10.6 million; DALYs by 14% from 109 to 93 million over 40 years. Costs drop $504 million over 5 years and $3.9 billion over 40 years with breakeven by 2013. Compared with the current scenario, expanding to <500 prevents an additional 585,000 and 3 million new HIV infections over 5 and 40 years, respectively. Expanding to all CD4 levels decreases HIV infections by 3.3 million (45%) and costs by $10 billion over 40 years, with breakeven by 2023. By 2050, using higher ART and monitoring costs, all CD4 levels saves $0.6 billion versus current; other ART scenarios cost $9–194 per DALY averted. If ART reduces transmission by 99%, savings from all CD4 levels reach $17.5 billion. Sensitivity analyses suggest that poor retention and predominant acute phase transmission reduce DALYs averted by 26% and savings by 7%.

Conclusion

Increasing the provision of ART to <350 cells/mm3 may significantly reduce costs while reducing the HIV burden. Feasibility including HIV testing and ART uptake, retention, and adherence should be evaluated.     

ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA

Background

Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.

Methodology/Principal Findings

Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR^−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR^−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR^−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.

Conclusions

proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.     

An improved method of plant megabase DNA isolation in agarose microbeads suitable for physical mapping and YAC cloning

The isolation of high quality megabase DNA from plant cells that is susceptible to a variety of molecular reagents is a critical first step in the physical analysis of complex genomes. A method for the isolation of such DNA by encapsulating plant protoplasts in agarose microbeads is presented. In comparison with the conventional agarose plug method, microbeads provide a dramatic increase in the surface area yielding megabase DNA that can be treated essentially as an aqueous DNA solution. Examples of the utility of DNA prepared by this technique for physical mapping, partial restriction enzyme digestion and cloning of large inserts as YACs are presented.