In Silico Design of an Oseltamivir Derivative with Increased Affinity against Wild-Type and Mutant Variants of Neuraminidase and Hemagglutinin of Influenza A H1N1 Virus

Antiviral resistance has turned into a world concern nowadays. Influenza A H1N1 emerged as a problem at the world level due to the neuraminidase (NA) mutations. The NA mutants conferred resistance to oseltamivir and zanamivir. Several efforts were conducted to develop better anti-influenza A H1N1 drugs. Our research group combined in silico methods to create a compound derived from oseltamivir to be tested in vitro against influenza A H1N1. Here we show the results of a new compound derived from oseltamivir but with specific chemical modifications, with significant affinity either on NA (in silico and in vitro assays) or HA (in silico) from influenza A H1N1 strain. We include docking and molecular dynamics (MD) simulations of the oseltamivir derivative at the binding site onto NA and HA of influenza A H1N1. Additionally, the biological experimental results show that oseltamivir derivative decreases the lytic-plaque formation on viral susceptibility assays, and it does not show cytotoxicity. Finally, oseltamivir derivative assayed on viral NA showed a concentration-dependent inhibition behavior at nM, depicting a high affinity of the compound for the enzyme, corroborated with the MD simulations results, placing our designed oseltamivir derivative as a potential antiviral against influenza A H1N1.     

基于N蛋白单克隆抗体的小反刍兽疫病毒抗体检测胶体金试纸条的研制

本研究旨在建立一种简便、快捷、可直观检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)抗体的检测方法。将pET-32a-N重组质粒转化至大肠杆菌(Escherichia coli) Rosetta(DE3)感受态细胞中进行诱导表达,以纯化的PPRVN蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,间接酶联免疫吸附试验(enzyme-linked immunosorbent assays, ELISA)筛选及亚克隆,获得了抗PPRV N蛋白的单克隆抗体。将PPRV N蛋白分别作为金标抗原及检测线(T线)包被抗原、单克隆抗体作为质控线(C线)包被抗体,组装成检测PPRVN蛋白抗体的胶体金免疫层析试纸条。结果显示：成功获得1株能稳定分泌抗N蛋白抗体的杂交瘤细胞株,命名为1F1;间接ELISA检测1F1腹水效价为1:128000;亚类鉴定结果为IgG1,轻链为kappa链。Westernblotting结果显示,1F1能与PPRV N蛋白特异性结合;间接免疫荧光(indirect immunofluorescent ass...     

CPI-17-mediated contraction of vascular smooth muscle is essential for the development of hypertension in obese mice

Several factors have been implicated in obesity-related hypertension, but the genesis of the hypertension is largely unknown. In this study, we found a significantly upregulated expression of CPI-17(C-kinasepotentiated protein phosphatase 1 inhibitor of 17 kDa) and protein kinase C(PKC) isoforms in the vascular smooth muscles of high-fat diet(HFD)-fed obese mice. The obese wild-type mice showed a significant elevation of blood pressure and enhanced calcium-sensitized contraction of vascular smooth muscles. However, the obese CPI-17-deficient mice showed a normotensive blood pressure, and the calcium-sensitized contraction was consistently reduced. In addition, the mutant muscle displayed an abolished responsive force to a PKC activator and a 30%-50% reduction in both the initial peak force and sustained force in response to various G protein-coupled receptor(GPCR) agonists. Our observations showed that CPI-17-mediated calcium sensitization is mediated through a GPCR/PKC/CPI-17/MLCP/RLC signaling pathway. We therefore propose that the upregulation of CPI-17-mediated calcium-sensitized vasocontraction by obesity contributes to the development of obesity-related hypertension.     

Examination of relationships between stable isotopes and cortisol concentrations along the length of phocid whiskers

Alaskan seals are found in remote and sometimes inaccessible locations, making it difficult to collect time‐series information. This study explores a novel method to examine temporal changes in diet and physiological status of ringed (Pusa hispida), spotted (Phoca largha), and harbor (Phoca vitulina) seals using cortisol concentrations and δ¹⁵N and δ¹³C stable isotopes (SIs) measured in serial sections of whiskers. As whiskers grow, whisker tissue is deposited sequentially making these measurements temporally aligned. Whisker cortisol presented in a distinct pattern with elevated concentrations at the root section followed by a curvilinear decline moving toward the tip of most whiskers. Comparing SIs at the root to the rest of the whiskers, δ¹³C values were slightly lower in ringed and harbor seal whiskers and δ¹⁵N values were slightly higher in harbor seal whiskers. The data were modeled controlling for the observed trends in cortisol concentrations and further associations between cortisol concentrations and SIs were detected in spotted and harbor seal whiskers. Additional research examining the source and stability of whisker cortisol is warranted. However, the methods presented here demonstrate that whiskers could prove valuable to gather long‐term and naturally aligned dietary and physiological information.     

Cyanobacterial bioactive metabolites—A review of their chemistry and biology

Cyanobacterial blooms occur when algal densities exceed baseline population concentrations. Cyanobacteria can produce a large number of secondary metabolites. Odorous metabolites affect the smell and flavor of aquatic animals, whereas bioactive metabolites cause a range of lethal and sub-lethal effects in plants, invertebrates, and vertebrates, including humans. Herein, the bioactivity, chemistry, origin, and biosynthesis of these cyanobacterial secondary metabolites were reviewed. With recent revision of cyanobacterial taxonomy by Anagnostidis and Komárek as part of the Süβwasserflora von Mitteleuropa volumes 19(1–3), names of many cyanobacteria that produce bioactive compounds have changed, thereby confusing readers. The original and new nomenclature are included in this review to clarify the origins of cyanobacterial bioactive compounds.Due to structural similarity, the 157 known bioactive classes produced by cyanobacteria have been condensed to 55 classes. This review will provide a basis for more formal procedures to adopt a logical naming system. This review is needed for efficient management of water resources to understand, identify, and manage cyanobacterial harmful algal bloom impacts.     

Incipient forebrain boundaries traced by differential gene expression and fate mapping in the chick neural plate

We correlated available fate maps for the avian neural plate at stages HH4 and HH8 with the progress of local molecular specification, aiming to determine when the molecular specification maps of the primary longitudinal and transversal domains of the anterior forebrain agree with the fate mapped data. To this end, we examined selected gene expression patterns as they normally evolved in whole mounts and sections between HH4 and HH8 (or HH10/11 in some cases), performed novel fate-mapping experiments within the anterior forebrain at HH4 and examined the results at HH8, and correlated grafts with expression of selected gene markers. The data provided new details to the HH4 fate map, and disclosed some genes (e.g., Six3 and Ganf) whose expression domains initially are very extensive and subsequently retract rostralwards. Apart from anteroposterior dynamics, some genes soon became downregulated at the prospective forebrain floor plate, or allowed to identify an early roof plate domain (dorsoventral pattern). Peculiarities of the telencephalon (initial specification and differentiation of pallium versus subpallium) are contemplated. The basic anterior forebrain subdivisions seem to acquire correlated specification and fate mapping patterns around stage HH8.     

人源H5N1禽流感病毒血凝素分子遗传变异特征分析

从GenBank获取已公布的人及相应国家禽感染的H5N1禽流感病毒HA核酸和蛋白序列,用生物软件ClustalX1.83和MEGA4.0对HA基因序列和蛋白分析,构建HA核苷酸遗传进化树.结果表明,HA蛋白上再次出现既能与人又能与禽受体特异性结合的QNG;东南亚和东亚人感染毒株亲缘关系密切,西亚、南亚和非洲人感染毒株遗传距离近;进化树组成和HA蛋白关键位点氨基酸的变异,揭示多数国家的人感染的毒株与当地禽感染毒株高度同源,具有地域性特征,部分国家之间存在着病毒扩散现象.     

Diet segregation between two colonies of little penguins Eudyptula minor in southeast Australia

We studied foraging segregation between two different sized colonies of little penguins Eudyptula minor with overlapping foraging areas in pre‐laying and incubation. We used stomach contents and stable isotope measurements of nitrogen (δ¹⁵N) and carbon (δ¹³C) in blood to examine differences in trophic position, prey‐size and nutritional values between the two colonies. Diet of little penguins at St Kilda (small colony) relied heavily on anchovy while at Phillip Island (large colony), the diet was more diverse and anchovies were larger than those consumed by St Kilda penguins. Higher δ¹⁵N values at St Kilda, differences in δ¹³C values and the prey composition provided further evidence of diet segregation between colonies. Penguins from each colony took anchovies from different cohorts and probably different stocks, although these sites are only 70 km apart. Differences in diet were not reflected in protein levels in the blood of penguins, suggesting that variation in prey between colonies was not related to differences in nutritional value of the diet. Anchovy is currently the only available prey to penguins throughout the year and its absence could have a negative impact on penguin food supply, particularly at St Kilda where the diet is dominated by this species. While it is difficult to establish whether diet segregation is caused by inter‐ or intra‐colony competition or spatial differences in foraging areas, we have shown that colonies with broadly overlapping foraging ranges could have significant differences in trophic position, diet composition and prey size while maintaining a diet of similar nutritional value.     

Regulation of c-Src activity by the expression of wild-type v-Src and its kinase-dead double Y416F-K295N mutant

Active, wild-type v-Src and its kinase-dead double Y416F-K295N mutant were expressed in hamster fibroblasts. Expression of the active v-Src induced activation of endogenous c-Src and increased general protein-tyrosine phosphorylation in the infected cells. Expression of the kinase-dead mutant induced hypophosphorylation of Tyr416 of the endogenous c-Src. The inactivation of c-Src was reversible, as confirmed by in vitro kinase activity of c-Src immunoprecipitated from the kinase-dead v-Src-expressing cells. Both activation and inactivation of c-Src may be explained by direct interaction of the v-Src and c-Src that may either facilitate transphosphorylation of the regulatory Tyr416 in the activation loop, or prevent it by formation of transient dead-end complexes of the Y416F-K295N mutant with c-Src. The interaction was also indicated by co-localization of v- and c-Src proteins in immunofluorescent images of the infected cells. These results suggest that dimerization of Src plays an important role in the regulation of Src tyrosine kinase activity.     

Sheep Grazing Decreases Organic Carbon and Nitrogen Pools in the Patagonian Steppe: Combination of Direct and Indirect Effects

We explored the net effects of grazing on soil C and N pools in a Patagonian shrub–grass steppe (temperate South America). Net effects result from the combination of direct impacts of grazing on biogeochemical characteristics of microsites with indirect effects on relative cover of vegetated and unvegetated microsites. Within five independent areas, we sampled surface soils in sites subjected to three grazing intensities: (1) ungrazed sites inside grazing exclosures, (2) moderately grazed sites adjacent to them, and (3) intensely grazed sites within the same paddock. Grazing significantly reduced soil C and N pools, although this pattern was clearest in intensely grazed sites. This net effect was due to the combination of a direct reduction of soil N content in bare soil patches, and indirect effects mediated by the increase of the cover of bare soil microsites, with lower C and N content than either grass or shrub microsites. This increase in bare soil cover was accompanied by a reduction in cover of preferred grass species and standing dead material. Finally, stable isotope signatures varied significantly among grazed and ungrazed sites, with δ¹⁵N and δ¹³C significantly depleted in intensely grazed sites, suggesting reduced mineralization with increased grazing intensity. In the Patagonian steppe, grazing appears to exert a negative effect on soil C and N cycles; sound management practices must incorporate the importance of species shifts within life form, and the critical role of standing dead material in maintaining soil C and N stocks and biogeochemical processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Author Contributions  RAG designed study, performed research, analyzed data, wrote the paper; ATA designed study, wrote the paper; CGGM designed study, performed research, analyzed data; MGP performed research; OES designed study; RBJ designed study, contributed new methods.