Dynamics of Membrane Tethers Reveal Novel Aspects of Cytoskeleton-Membrane Interactions in Axons

Mechanical properties of cell membranes are known to be significantly influenced by the underlying cortical cytoskeleton. The technique of pulling membrane tethers from cells is one of the most effective ways of studying the membrane mechanics and the membrane-cortex interaction. In this article, we show that axon membranes make an interesting system to explore as they exhibit both free membrane-like behavior where the tether-membrane junction is movable on the surface of the axons (unlike many other cell membranes) as well as cell-like behavior where there are transient and spontaneous eruptions in the tether force that vanish when F-actin is depolymerized. We analyze the passive and spontaneous responses of axonal membrane tethers and propose theoretical models to explain the observed behavior.     

Mutational approach for N2-fixing and P-solubilizing mutant strains of Klebsiella pneumoniae RSN19 by microwave mutagenesis

Nitrogen (N) fixing Klebsiella pneumoniae RSN19 has high inorganic phosphorus (P) solubilizing capability, but its N₂-fixing capability is limited. In order to acquire a P-solubilizing mutant strain with high efficiency N-fixing capability, different microwave irradiation intensities and durations were tested on RSN19 in an attempt to produce mutants with improved N₂-fixation and P-solubilization capabilities. The effect of microwave irradiation power and time were studied and the microwave mutagenesis parameters were optimized. Nitrogenase activity was tested on the mutant strains by acetylene reduction method; and their P-solubilizing capability and genetic stability were determined. The results indicated that the best conditions for microwave mutagenesis that produced better performed mutant strains were 250W, 36 s. Under these conditions a maximum positive mutation rate of 1.66% was obtained, resulting in five genetically stable strains with promoted nitrogenase activity which was designated as RSM-219, RSM-206, RSM-224, RSM-225 and RSM-275. Subculture tests showed that RSM-219 and RSM-206 were genetically stable mutant strains with higher nitrogenase activity and phosphate solubilizing capabilities than the original strain. Both RSM-219 and RSM-206 performed better than the original strain under N-free conditions when supplied with calcium phosphate only, and produced greater increases in the biomass of alfalfa seedlings.     

Stable and orally bio-available pro-drugs of CPS11

Stable and orally bio-available pro-drugs of CPS11 were synthesized. They are active on human umbilical vein endothelial cell proliferation assay and tube formation assay. The therapeutic efficacy and safety of 4 as a single agent or combined with Taxol in the treatment of MX-1 human breast cancer xenograft were evaluated. Compound 4 as a single agent failed to produce an anti-tumor activity, while it significantly enhanced antitumor potency of Taxol.     

微滴培养技术的新应用

微滴培养技术在卵母细胞培养和胚胎早期发育研究中有广泛的应用.近期研究发现这项技术又有新的应用范围.有科学家发现将该技术应用于胚胎干细胞可以实现高效传代,在精原干细胞培养和精子发生相关过程的研究方面也可取得很好效果,同时,对早期人类胚胎干细胞的分离过程的监控和对精原干细胞生长过程的观察也相对容易.这些研究结果显示,微滴培养技术在这些新领域的研究与常规培养方法相比具有独特的优势.回顾微滴培养技术的主要发展过程,重点探讨微滴培养技术的最新应用及其优点.     

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

EGFP (enhanced green fluorescent protein) tagged to either the N (amino)-terminus [EGFP/hERG (human ether-a-go-go-related gene)] or C (carboxyl)-terminus (hERG/EGFP) of hERG channel is used to study mutant channel protein trafficking for several years. However, it has been reported that the process can alter hERG channel properties. The aim of the study was to determine whether EGFP tagged to N-terminus of hERG channels would alter the cellular localizations and the electrophysiological properties of hERG channels compared with untagged hERG channels. The hERG channels tagged with or without EGFP were transiently expressed in HEK (human embryonic kidney) 293 cells using a lipofectamine method. HEK 293 cells expressing pCDNA3-hERG or pEGFP-hERG were double immunolabelled with anti-hERG and anti-calnexin (an ER marker protein) followed with FITC- and TRITC (tetramethylrhodamine β-isothiocyanate)-labelled secondary antibodies, respectively. Confocal laser scanning microscope was used to observe the cellular localization of EGFP-tagged hERG channels and untagged hERG channels. Patch-clamp technique was used to record whole cell currents. We found that the EGFP/hERG fusion protein and untagged hERG channels were both expressed not only on the cell surface membrane but also in the cytoplasm of HEK293 cells. The EGFP/hERG appeared to influence the hERG channel gating properties, including reduction of the peak tail current density, more rapid inactivation process, faster recovery from inactivation and faster deactivation kinetics compared with untagged hERG channels. Our results suggest that the EGFP/hERG channel alter the electrophysiological properties of hERG channel, but it does not seem to alter the cellular location of hERG channels. Thus, EGFP tagging to N-terminus might be used for research of subcellular location of hERG channels but not for the channel electrophysiological properties.     

Seroprevalence of pandemic (H1N1) 2009 in pregnant women in China: an observational study

Background

We investigated the seropositive rates and persistence of antibody against pandemic (H1N1) 2009 virus (pH1N1) in pregnant women and voluntary blood donors after the second wave of the pandemic in Nanjing, China.

Methodology/Principal Findings

Serum samples of unvaccinated pregnant women (n = 720) and voluntary blood donors (n = 320) were collected after the second wave of 2009 pandemic in Nanjing. All samples were tested against pH1N1 strain (A/California/7/2009) with hemagglutination inhibition assay. A significant decline in seropositive rates, from above 50% to about 20%, was observed in pregnant women and voluntary blood donors fifteen weeks after the second wave of the pandemic. A quarter of the samples were tested against a seasonal H1N1 strain (A/Brisbane/59/2007). The antibody titers against pH1N1 strain were found to correlate positively with those against seasonal H1N1 strain. The correlation was modest but statistically significant.

Conclusions and Significance

The high seropositive rates in both pregnant women and voluntary blood donors suggested that the pH1N1 virus had widely spread in these two populations. Immunity derived from natural infection seemed not to be persistent well.     

Genetic characteristics of 2009 pandemic H1N1 influenza a viruses isolated from Mainland China

A total of 100 HIN1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang,Hubei and Guangdong between June and November 2009,were provided by local CDC laboratories.After MDCK cell culture,57 Influenza A Pandemic (H1N1) viruses were isolated and submitted for whole genome sequencing.A total of 39 HA sequences,52 NA sequences,36 PB2 sequences,31 PB1 sequences,40 PA sequences,48 NP sequences,51 MP sequences and 36 NS sequences were obtained,including 20 whole genome sequences.Sequence comparison revealed they shared a high degree of homology (96％～99％) with known epidemic strains (A/Califomia/04/2009(H1N1).Phylogenetic analysis showed that although the sequences were highly conserved,they clustered into a small number of groups with only a few distinct strains.Site analysis revealed three substitutions at loop 220 (221-228) of the HA receptor binding site in the 39 HA sequences:A/Hubei/86/2009 PKVRDQEG→PKVRDQEA,A/Zhejiang/08/2009 PKVRDQEG→PKVRDQER,A/Hubei/75/2009PKVRDQEG→PKVRDQGG,the A/Hubei/75/2009 was isolated from an acute case,while the other two were from patients with mild symptoms.Other key sites such as 119,274,292 and 294 amino acids of NA protein,627 of PB2 protein were conserved.Meanwhile,all the M2 protein sequences possessed the Ser32Asn mutation,suggesting that these viruses were resistant to adamantanes.Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.     

The persistent circulation of enterovirus 71 in People's Republic of China: causing emerging nationwide epidemics since 2008

Emerging epidemics of hand-foot-and-mouth disease (HFMD) associated with enterovirus 71 (EV71) has become a serious concern in mainland China. It caused 126 and 353 fatalities in 2008 and 2009, respectively. The epidemiologic and pathogenic data of the outbreak collected from national laboratory network and notifiable disease surveillance system. To understand the virological evolution of this emerging outbreak, 326 VP1 gene sequences of EV71 detected in China from 1987 to 2009 were collected for genetic analyses. Evidence from both traditional and molecular epidemiology confirmed that the recent HFMD outbreak was an emerging one caused by EV71 of subgenotype C4. This emerging HFMD outbreak is associated with EV71 of subgenotype C4, circulating persistently in mainland China since 1998, but not attributed to the importation of new genotype. Originating from 1992, subgenotype C4 has been the predominant genotype since 1998 in mainland China, with an evolutionary rate of 4.6∼4.8×10⁻³ nucleotide substitutions/site/year. The phylogenetic analysis revealed that the majority of the virus during this epidemic was the most recent descendant of subgenotype C4 (clade C4a). It suggests that the evolution might be one of the potential reasons for this native virus to cause the emerging outbreak in China. However, strong negative selective pressure on VP1 protein of EV71 suggested that immune escape might not be the evolving strategy of EV71, predicting a light future for vaccine development. Nonetheless, long-term antigenic and genetic surveillance is still necessary for further understanding.