Identification and characterization of a chondroitin synthase from Avibacterium paragallinarum

Applied Microbiology and Biotechnology - Avibacterium paragallinarum is a Gram-negative bacterium that causes infectious coryza in chicken. It was reported that the capsule polysaccharides...     

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.     

Oligomerization of Baculovirus LEF-11 Is Involved in Viral DNA Replication

We have previously reported that baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) late expression factor 11 (lef-11) is associated with viral DNA replication and have demonstrated that it potentially interacts with itself; however, whether LEF-11 forms oligomers and the impact of LEF-11 oligomerization on viral function have not been substantiated. In this study, we first demonstrated that LEF-11 is capable of forming oligomers. Additionally, a series of analyses using BmNPV LEF-11 truncation mutants indicated that two distinct domains control LEF-11 oligomerization (aa 42–61 and aa 72–101). LEF-11 truncation constructs were inserted into a lef-11-knockout BmNPV bacmid, which was used to demonstrate that truncated LEF-11 lacking either oligomerization domain abrogates viral DNA replication. Finally, site-directed mutagenesis was used to determine that the conserved hydrophobic residues Y58&I59 (representing Y58 and I59), I85 and L88&L89 (representing L88 and L89) are required for LEF-11 oligomerization and viral DNA replication. Collectively, these data indicate that BmNPV LEF-11 oligomerization influences viral DNA replication.     

奥利亚罗非鱼与鳜杂交的受精细胞学及胚胎发育研究

通过对奥利亚罗非鱼(♀)和鳜(♂)杂交的受精细胞学观察发现:异源精子入卵后产生星光,并核化成雄性原核,与雌原核互相靠近,接触最终完全融合为一个合子核,合子核继续分裂。分析比较了奥利亚罗非鱼,鳜以及奥鳜杂种胚胎发育情况,发现杂种胚胎的发育速度在囊胚早期与母本奥利亚罗非鱼基本一致,到囊胚后期速度有所减慢,但与鳜胚胎发育速度相差很大。这表明奥利亚罗非鱼和鳜虽属不同科间的远缘杂交,但有正常的受精细胞学程序和常规的细胞分裂(卵裂)方式。     

β-TCP支架体外构建组织工程骨及其流体力学研究

骨髓基质干细胞在β-tricalciumphosphate(β-TCP)支架上分别进行了1、2和4周的三维动态培养,对支架上不同时间和部位的细胞面积/微孔面积及支架动态培养的流体环境进行了研究.研究表明,第1周细胞在支架大部分孔道内粘附生长并出现一定区域的单细胞层和多细胞层,第2周部分区域的部分孔道已填满了细胞并出现多细胞层,第4周大部分孔道几乎填满了细胞,主管道内壁出现了较多的细胞生长.同时发现,支架上各个区域细胞粘附面积不等,部分区域无细胞存在,有的部位2周后细胞逐渐减少.为了研究支架各个位置细胞増殖与流速、剪切应力的关系,建立了支架随机孔道结构的流体分析模型,通过支架上流速和剪切应力分布探讨实验中细胞分布现象的机理.结合计算和实验发现,流体能流到的部位几乎都有细胞生长,细胞生长较快的部位速度大多集中在0.24～0.53mm/s,剪切力大多在0.0050～0.023Pa,主管道底部及靠近进口的部位可能存在由于过大的剪切力影响细胞生长的区域.上述结果在一定程度上反映了细胞-支架-流体三者在成骨转化过程中的作用,对指导体外灌注培养的流量确定、灌注工艺及骨转化动力学研究有重要的意义.     

Systematic Identification of H274Y Compensatory Mutations in Influenza A Virus Neuraminidase by High-Throughput Screening

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.     

Dynasore Protects Mitochondria and Improves Cardiac Lusitropy in Langendorff Perfused Mouse Heart

Background

Heart failure due to diastolic dysfunction exacts a major economic, morbidity and mortality burden in the United States. Therapeutic agents to improve diastolic dysfunction are limited. It was recently found that Dynamin related protein 1 (Drp1) mediates mitochondrial fission during ischemia/reperfusion (I/R) injury, whereas inhibition of Drp1 decreases myocardial infarct size. We hypothesized that Dynasore, a small noncompetitive dynamin GTPase inhibitor, could have beneficial effects on cardiac physiology during I/R injury.

Methods and Results

In Langendorff perfused mouse hearts subjected to I/R (30 minutes of global ischemia followed by 1 hour of reperfusion), pretreatment with 1 µM Dynasore prevented I/R induced elevation of left ventricular end diastolic pressure (LVEDP), indicating a significant and specific lusitropic effect. Dynasore also decreased cardiac troponin I efflux during reperfusion and reduced infarct size. In cultured adult mouse cardiomyocytes subjected to oxidative stress, Dynasore increased cardiomyocyte survival and viability identified by trypan blue exclusion assay and reduced cellular Adenosine triphosphate(ATP) depletion. Moreover, in cultured cells, Dynasore pretreatment protected mitochondrial fragmentation induced by oxidative stress.

Conclusion

Dynasore protects cardiac lusitropy and limits cell damage through a mechanism that maintains mitochondrial morphology and intracellular ATP in stressed cells. Mitochondrial protection through an agent such as Dynasore can have clinical benefit by positively influencing the energetics of diastolic dysfunction.     

Effect of Strongly Alkaline Electrolyzed Water on Silk Degumming and the Physical Properties of the Fibroin Fiber

Strongly alkaline electrolyzed water (SAEW) was prepared by electrolysis of tap water in a laboratory-made water electrolyzer. The pH of stored SAEW was stable for more than one month. The hardness of the electrolyzed water was 30% lower and the Na⁺ concentration was 18% higher than those of the tap water. Silkworm cocoon shells were boiled in pH 11.50 SAEW at a ratio of 1∶40∼80 (W/V) for 20 min and the sericin layers around the silk fibroin fibers were removed completely. The tensile properties and thermal decomposition temperature of a single filament of silk fibroin obtained by the SAEW method were almost the same as those for the fiber obtained by the neutral soap, and much higher than those for the fiber obtained by Na₂CO₃ degumming. The results demonstrate that SAEW is an environmentally friendly and pollution-free silk degumming agent that allows highly efficient, low cost recovery of sericin.     

Tumor Necrosis Factor Alpha rs1800629 Polymorphism and Risk of Cervical Lesions: A Meta-Analysis

Background

Tumor necrosis factor- alpha (TNF-α) is an inflammatory cytokine which may play important role on the immune response may control the progression of cervical lesions. There is a possible association between TNF-α rs1800629 G/A polymorphism and cervical lesions, but previous studies report conflicting results. We performed a meta-analysis to comprehensively assess the association between TNF-α rs1800629 polymorphism and cervical lesions risk.

Methods

Literature searches of Pubmed, Embase, Web of Science, and Wanfang databases were performed for all publications on the association between TNF-α rs1800629 polymorphism and cervical lesions through December 15, 2012. The pooled odds ratios (ORs) with their 95% confidence interval (95%CIs) were calculated to assess the strength of the association.

Results

Twenty individual case-control studies from 19 publications with a total of 4,146 cases and 4,731 controls were finally included into the meta-analysis. Overall, TNF-α rs1800629 polymorphism was significantly associated with increased risk of cervical lesions under two main genetic comparison models (For A versus G: OR 1.22, 95%CI 1.04–1.44, P = 0.017; for AA versus GG: OR 1.32, 95%CI 1.02–1.71, P = 0.034). Subgroup analysis by ethnicity further showed that there was a significant association between TNF-α rs1800629 polymorphism and increased risk of cervical lesions in Caucasians but not in Asians. Subgroup analysis by the types of cervical lesions showed that there was a significant association between TNF-α rs1800629 polymorphism and increased risk of cervical cancer (For A versus G: OR 1.24, 95%CI 1.05–1.47, P = 0.011; for AA versus GG: OR 1.31, 95%CI 1.01–1.70, P = 0.043; for AA/GA versus GG: OR 1.25, 95%CI 1.01–1.54, P = 0.039).

Conclusion

The meta-analysis suggests that TNF-α rs1800629 polymorphism is associated with increased risk of cervical lesions, especially in Caucasians.