Construction of an engineered strain capable of degrading two isomeric nitrophenols via a sacB- and gfp-based markerless integration system

In this study, a gfp-based novel markerless allelic exchange integration system was developed. By employing gfp gene and sacB gene as counter-selectable markers, an ortho-nitrophenol degradation operon (onpABC gene cluster) was successfully inserted into the chromosome of meta-nitrophenol utilizer Cupriavidus necator JMP134. Through two rounds of recombination, the engineered strain (strain JMP134-ONP) was directly selected from the plate by fluorescence screening and has the ability to degrade both ortho-nitrophenol and meta-nitrophenol, simultaneously. This relatively simple and efficient method can be used as an alternative strategy of allelic exchange insertion for the application of metabolic engineering in various bacterial strains, complementary to existing gene knock-in procedures.     

Down-Regulation of miRNA-30a Alleviates Cerebral Ischemic Injury Through Enhancing Beclin 1-Mediated Autophagy

The understanding of molecular mechanism underlying ischemia/reperfusion-induced neuronal death and neurological dysfunction may provide therapeutic targets for ischemic stroke. The up-regulated miRNA-30a among our previous identified 19 MicroRNAs (miRNAs) in mouse brain after 6 h middle cerebral artery occlusion (MCAO) could negatively regulate Beclin 1 messenger RNA (mRNA) resulting in decreased autophagic activity in tumor cells and cardiomyocytes, but its role in ischemic stroke is unclear. In this study, the effects of miRNA-30a on ischemic injury in N2A cells and cultured cortical neurons after oxygen glucose deprivation (OGD), and mouse brain with MCAO-induced ischemic stroke were evaluated. The results showed that miRNA-30a expression levels were up regulated in the brain of mice after 6 h MCAO without reperfusion, but significantly down regulated in the peri-infarct region of mice with 1 h MCAO/24 h reperfusion and in N2A cells after 1 h OGD/6–48 h reoxygenation. Both the conversion ratio of microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I and Beclin 1 protein level increased in N2A cells and cultured cortical neurons following 1 h OGD/24 h reoxygenation. The down-regulated miRNA-30a could attenuate 1 h OGD/24 h reoxygenation-induced ischemic injury in N2A cells and cultured cortical neurons through enhancing Beclin 1-mediated autophagy, as miRNA-30a recognized the 3′-untranslated region of beclin 1 mRNA to negatively regulate Beclin 1-protein level via promoting beclin 1 messenger RNA (mRNA) degradation, and Beclin 1 siRNA abolished anti-miR-30a-induced neuroprotection in 1 h OGD/24 h reoxygenation treated N2A cells. In addition, anti-miR-30a attenuated the neural cell loss and improved behavioral outcome of mice with ischemic stroke. These results suggested that down-regulation of miRNA-30a alleviates ischemic injury through enhancing beclin 1-mediated autophagy, providing a potential therapeutic target for ischemic stroke.     

Identification of differentially expressed genes in hepatopancreas of oriental river prawn, Macrobrachium nipponense exposed to environmental hypoxia

Hypoxia represents a major physiological challenge for prawn culture, and the hepatopancreas plays an important role in these processes. Here, we applied high-throughput sequencing technology to detect the gene expression profile of the hepatopancreas in Macrobrachium nipponense in response to hypoxia for 3 h and hypoxia for 24 h. Gene expression profiling identified 1925 genes that were significantly up- or down-regulated by dissolved oxygen availability. Functional categorization of the differentially expressed genes revealed that oxygen transport, electron transport chain, reactive oxygen species generation/scavenging, and immune response were the differentially regulated processes occurring during environmental hypoxia. Finally, quantitative real-time polymerase chain reaction using six genes independently verified the tag-mapped results. Immunohistochemistry analysis revealed, for the first time, hemocyanin protein expression as significant hypoxia-specific signature in prawns, which opens the way for in depth molecular studies of hypoxia exposure. The analysis of changes in hepatic gene expression in oriental river prawn provides a preliminary basis for a better understanding of the molecular response to hypoxia exposures.     

Biocontrol of Late Blight Disease (Phytophthora capsici) of Pepper and the Plant Growth Promotion by Paenibacillus ehimensis KWN38

For field application of a bacterial strain used to control Phythophthora capsici, we will need a biologically and economically efficient carrier medium. The known antagonist Paenibacillus ehimensisKWN38 was grown in a grass medium where it showed high antifungal and lytic enzyme activities. To demonstrate the potential of P. ehimensisKWN38 for biocontrol of late blight disease in pepper, pot trials were conducted by treating the 1‐month‐old plants with water (W), a selected grass medium (G3), G plus P. ehimensisKWN38 inoculation (G3P) or synthetic fungicide (F). The shoot dry weight in G3P was higher than that in W and F treatments at 15 days after zoospore infection (DZI). The root dry weight in G3P was also higher than that in W. The root mortality of G3 and W increased over 58 and 80% at 15 DZI, and some plants in those treatments wilted due to the failure of root physiology. The plants in G3P and F survived well because of their better root health conditions. Soil cellulase activity of G3P was consistently higher than that of W and F at earlier observation times (0, 2 and 6 DZI). The root β‐1,3‐glucanase activity of G3P promptly increased to maximum shortly after zoospore infection and reached the maximum value of 51.12 unit g^?1 of fresh weight at 2 DZI. All these results indicate that inoculation of P. ehimensisKWN38 to the root zone of potted pepper plants increases plant growth, root and soil enzyme activities and alleviates the root death caused by infection with P. capsici zoospores.     

利用双萤光素酶报告系统研究FoxM1转录活性调控

目的:构建细胞周期蛋白B1(Cyclin B1)启动子萤光素酶报告系统,用于转录因子FoxM1转录活性调控研究。方法:利用瞬时转染、实时荧光定量PCR及Western印迹,验证人胚肾HEK293细胞中FoxM1对Cyclin B1 mRNA的转录及蛋白表达的调节作用;利用PCR技术从人宫颈癌HeLa细胞基因组中钓取人源Cyclin B1的启动子序列,克隆至pGL3-Basic Vector中,构建含有Cyclin B1启动子的萤火虫萤光素酶报告质粒pGL3-Cyclin B1;通过瞬时转染、Western印迹及萤火虫/海肾萤光素酶双报告系统,研究c-Abl酪氨酸激酶对FoxM1转录活性的调控作用。结果:构建了Cyclin B1萤火虫萤光素酶报告系统,该报告系统可用于FoxM1转录活性研究;c-Abl酪氨酸激酶能够显著抑制FoxM1靶向Cyclin B1启动子的转录活性,且抑制作用依赖c-Abl的激酶活性。结论:pGL3-Cyclin B1萤光素酶报告系统可用于研究c-Abl酪氨酸激酶介导的FoxM1转录活性调节。     

TRANSPARENT TESTA2 regulates embryonic fatty acid biosynthesis by targeting FUSCA3 during the early developmental stage of Arabidopsis seeds

TRANSPARENT TESTA2 (TT2) regulates the biosynthesis of proanthocyanidins in the seed coat of Arabidopsis. We recently found that TT2 also participates in inhibition of fatty acid (FA) biosynthesis in the seed embryo. However, the mechanism by which TT2 suppresses the accumulation of seed FA remains unclear. In this study, we show that TT2 is expressed in embryos at an early developmental stage. TT2 is directly bound to the regulatory region of FUSCA3 (FUS3), and mediates the expression of numerous genes in the FA biosynthesis pathway. These genes include BCCP2, CAC2, MOD1 and KASII, which encode proteins involved in the initial steps of FA chain formation, FAD2 and FAD3, which are responsible for FA desaturation, and FAE1, which catalyzes very‐long‐chain FA elongation. Loss of function of TT2 results in reduced expression of GLABRA2 but does not cause a significant reduction in the mucilage attached to the seed coats, which competes with FA for photosynthates. TT2 is expressed in both maternal seed coats and embryonic tissues, but proanthocyanidins are only found in wild‐type seed coats and not in embryonic tissues. The amount of proanthocyanidins in the seed coat is negatively correlated with the amount of FAs in the embryo.     

机插配合控释掺混肥对水稻产量和土壤肥力的影响

在浙江黄泥田进行两年田间试验,研究了不同栽培与施肥方式结合对水稻产量和成熟期土壤养分、微生物生物量碳、氮以及脲酶、磷酸酶、蔗糖酶、过氧化氢酶活性的影响.结果表明: 机插配合控释掺混肥处理的水稻平均产量比手插配合常规化肥处理高16.3%,比直播配合控释掺混肥处理高27.0%,与手插配合控释掺混肥处理无显著差异;与手插配合常规化肥相比,机插配合控释掺混肥处理的土壤全氮、碱解氮、有效磷含量分别提高了21.6%、13.6%和41.6%,土壤微生物生物量碳提高了27.1%,土壤脲酶、蔗糖酶、过氧化氢酶活性分别提高了50.0%、22.2%和46.2%.表明机插配合控释掺混肥技术是一种值得推广的高效水稻轻简栽培方式.     

Functional Polymorphisms of Interferon-gamma Affect Pneumonia-Induced Sepsis

Objective

Sepsis is an inflammatory syndrome caused by infection, and both its incidence and mortality are high. Because interferon-gamma (IFN-γ) plays an important role in inflammation, this work assessed IFN-γ single nucleotide polymorphism (SNPs) that may be associated with sepsis.

Methods

A total of 196 patients with pneumonia-induced sepsis and 213 age- and sex-matched healthy volunteers participated in our study from July 2012 to July 2013 in Guangzhou, China. Patient clinical information was collected. Clinical pathology was assessed in subgroups defined based on clinical criteria, APACHE II (acute physiology and chronic health evaluation) and SOFA (sepsis-related organ failure assessment) scores and discharge rate. Four functional SNPs, −1616T/C (rs2069705), −764G/C (rs2069707), +874A/T (rs2430561) and +3234C/T (rs2069718), were genotyped by Snapshot in both sepsis patients and healthy controls. Pearson’s chi-square test or Fisher’s exact test were used to analyze the distribution of the SNPs, and the probability values (P values), odds ratios (OR) and 95% confidence intervals (CIs) were calculated.

Results

No mutations in the IFN-γ −764G/C SNP were detected among the participants in our study. The +874A/T and +3234C/T SNPs were in strong linkage disequilibrium (LD) (r² = 0.894). The −1616 TC+TT, +874 AT+AA genotype and the TAC haplotype were significantly associated with sepsis susceptibility, while the CTT haplotype was associated with protection against sepsis incidence. Genotype of −1616 TT wasn’t only protective against severity of sepsis, but also against higher APACHE II and SOFA scores as +874 AA and +3234 CC. The TAC haplotype was was protective against progression to severe sepsis either.

Conclusion

Our results suggest that functional IFN-γ SNPs and their haplotypes are associated with pneumonia-induced sepsis.