Design,synthesis and biological evaluation of N2,N4-disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine derivatives as HIV-1 NNRTIs

A series of N₂,N₄-disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine derivatives (PTTDs) was designed and synthesized by a facile route. The biological assay results showed that five most potent compounds displayed inhibitory activity against HIV-1 at low micromolar concentrations (EC₅₀ = 5.1–8.9 μM). Structure–activity relationship analysis indicated that N₂-(3-halogenated-benzyl) analogues were more potent than N₂-(unsubstituted-benzyl) analogues. The N₄-substitutions contributed to the antiviral activity in the following order: 2-/3-cyano substituted benzyl > 2-/3-halogenated benzyl > non-substituted benzyl > 4-halogenated benzyl. Docking studies of the representative compound revealed the binding conformation of these compounds and provided critical insights for the further development of PTTD analogues.     

Ambient temperature alters body size and gut microbiota of Xenopus tropicalis

Temperature is important to determine physiological status of ectotherms. However, it is still not fully understood how amphibians and their symbiotic microbiota acclimate to ambient temperature. In this study, we investigated the changes of gut microbiota of Xenopus tropicalis at different temperatures under controlled laboratory conditions. The results showed that microbial communities were distinct and shared only a small overlap among froglet guts, culture water and food samples.Furthermore, the dominant taxa harbored in the gut exhibited low relative abundance in water and food. It indicates that bacterial taxa selected by amphibian gut were generally of low abundance in the external environment. Temperature could affect betadiversity of gut microbiota in terms of phylogenetic distance, but it did not affect alpha diversity. The composition of gut microbiota was similar in warm and cool treatments. However, signature taxa in different temperature environments were identified. The relationships between temperature, gut microbiota and morphology traits of X. tropicalis revealed in this study help us to predict the consequences of environmental changes on ectothermic animals.     

Application of statistical design for the optimization of microbial community of synthetic domestic wastewater

A fractional factorial design (FFD) and a response surface methodology (RSM) were used to optimize the inoculum composition of six strains for treatment of synthetic domestic wastewater. The model predicted the highest overall specific substrate utilization rate (q) of 6.88 g TOC/(d-gVSS). The value is in accordance with the actual maximum q, and is 1.5 and 1.97 times greater than those without optimization for 4 and 6 strains respectively. Additionally, the shortest time to reach stationary phase (3.5 h) and highest maximum total organic carbon (TOC) removal efficiency (92%) were also achieved under the optimum condition. The results indicated that the FFD and RSM are powerful screening and optimizing tools for the microbial community. The experimental approaches enhance the overall specific rate of substrate utilization as well as other biodegradation parameters.     

Direct or Indirect Regulation of Calcium-Activated Chloride Channel by Calcium

Calcium-activated chloride channels (CaCCs) play fundamental roles in numerous physiological processes. Despite their physiological importance, the molecular identity of CaCCs has not been fully investigated until now. Recently, transmembrane 16A (TMEM16A) was demonstrated by three independent research groups to be a strong candidate for the CaCC molecular basis. To further investigate the electrophysiological characteristics, we constructed TMEM16A (abcd) stably transfected HEK293 cell lines and carried out whole-cell and excised inside–out patch-clamp experiments. The TMEM16A channel was Ca²⁺-dependent in both patch configurations. The TMEM16A current could be strongly inhibited by niflumic acid, and when Cl⁻ was substituted by gluconate ions, the current was reduced considerably. In inside–out configuration, TMEM16A channel was time-independent but voltage-dependent, in which the half-maximum activating free Ca²⁺ concentration was 63 nM at 80 mV. While in whole-cell configuration, the current was both time- and voltage-dependent. About the rectification feature, the TMEM16A current also showed distinct characteristics in the two patch configurations. In whole cells, the TMEM16A channel expressed outward rectification at low Ca²⁺ concentration but when the Ca²⁺ concentration was high it became linear. On the contrary, in inside–out configuration, it always expressed outward rectification. Comparing the different characteristics in the two configurations, some underlying mechanisms remain to be identified, which is discussed with respect to direct or indirect activation. There was irreversible rundown in this channel.     

Sequential interaction of actin-related proteins 2 and 3 (Arp2/3) complex with neural Wiscott-Aldrich syndrome protein (N-WASP) and cortactin during branched actin filament network formation

The WASP and cortactin families constitute two distinct classes of Arp2/3 modulators in mammalian cells. Physical and functional interactions among the Arp2/3 complex, VCA (a functional domain of N-WASP), and cortactin were examined under conditions that were with or without actin polymerization. In the absence of actin, cortactin binds significantly weaker to the Arp2/3 complex than VCA. At concentrations of VCA 20-fold lower than cortactin, the association of cortactin with the Arp2/3 complex was nearly abolished. Analysis of the cells infected with Shigella demonstrated that N-WASP located at the tip of the bacterium, whereas cortactin accumulated in the comet tail. Interestingly, cortactin promotes Arp2/3 complex-mediated actin polymerization and actin branching in the presence of VCA at a saturating concentration, and cortactin acquired 20 nm affinity for the Arp2/3 complex during actin polymerization. The interaction of VCA with the Arp2/3 complex was reduced in the presence of both cortactin and actin. Moreover, VCA reduced its affinity for Arp2/3 complex at branching sites that were stabilized by phalloidin. These data imply a novel mechanism for the de novo assembly of a branched actin network that involves a coordinated sequential interaction of N-WASP and cortactin with the Arp2/3 complex.     

Enhancement of polysialic acid yield by reducing initial phosphate and feeding ammonia water to <Emphasis Type="Italic">Escherichia coli</Emphasis> CCTCC M208088

Polysialic acid (PSA) is a capsular polysaccharide obtained from aerobic fermentation with Escherichia coli. To enhance PSA production and eliminate the influence of phosphate on the PSA purification process, a lower level of initial phosphate was adopted with pH control. The resulting PSA yield reached 4.1 g/L in fed-batch fermentation with 2.5 g/L K₂HPO₄ and E. coli strain CCTCC M208088. In addition, an ammonia water (NH₄OH) feeding strategy to control the pH at 6.4 was developed resulting in PSA production that reached as high as 5.2 g/L. NMR spectra confirmed the purified biopolymer as a α-2,8 linked PSA, identical to the published NMR spectra, with a molecular weight in the range of 16 ∼ 50 kDa.     

Delayed and Restricted Expression of UAS-Regulated GFP Gene in Early Transgenic Zebrafish Embryos by Using the GAL4/UAS System

A stable Tg(UAS:GFP) zebrafish line was generated and crossed with Tg(hsp70:GAL4) line, in which the GAL4 gene is under the control of an inducible zebrafish promoter derived from the heat shock 70 protein gene (hsp70). The dynamic green fluorescent protein (GFP) expression in early zebrafish embryos in the GAL4/UAS binary system was then investigated. We found that, at early developmental stages, expression of GFP effector gene was restricted and required a long recovery time to reach a detectable level. At later developmental stage (after 2 days postfertilization), GFP could be activated in multiple tissues in a shorter time, apparently due to a higher level of GAL4 messenger RNA induction. It appears that the type of tissues expressing GFP was dependent on whether they had been developed at the time of heat shock. Therefore, the delayed and restricted transgene expression should be taken into consideration when GAL4/UAS system is used to study transgene expression in early developmental stages.