Allele-Specific Behavior of Molecular Networks: Understanding Small-Molecule Drug Response in Yeast

The study of systems genetics is changing the way the genetic and molecular basis of phenotypic variation, such as disease susceptibility and drug response, is being analyzed. Moreover, systems genetics aids in the translation of insights from systems biology into genetics. The use of systems genetics enables greater attention to be focused on the potential impact of genetic perturbations on the molecular states of networks that in turn affects complex traits. In this study, we developed models to detect allele-specific perturbations on interactions, in which a genetic locus with alternative alleles exerted a differing influence on an interaction. We utilized the models to investigate the dynamic behavior of an integrated molecular network undergoing genetic perturbations in yeast. Our results revealed the complexity of regulatory relationships between genetic loci and networks, in which different genetic loci perturb specific network modules. In addition, significant within-module functional coherence was found. We then used the network perturbation model to elucidate the underlying molecular mechanisms of individual differences in response to 100 diverse small molecule drugs. As a result, we identified sub-networks in the integrated network that responded to variations in DNA associated with response to diverse compounds and were significantly enriched for known drug targets. Literature mining results provided strong independent evidence for the effectiveness of these genetic perturbing networks in the elucidation of small-molecule responses in yeast.     

STK39 Polymorphism Is Associated with Essential Hypertension: A Systematic Review and Meta-Analysis

Background

A recent genome-wide association study identified STK39as a candidate gene for blood pressure (BP) in Europeans. Subsequently, several studies have attempted to replicate the association across different ethnic populations. However, the results have been inconsistent.

Objective and Methods

We performed a meta-analysis to elucidate the association between the STK39 rs3754777 polymorphism (or proxy) and hypertension. Published literature from PubMed and Embase databases were retrieved and pooled odds ratio (OR) with 95% confidence interval (CI) was calculated using fixed- or random-effects model.

Results

Using appropriate inclusion/exclusion criteria, we identified 10 studies that included 21, 863 hypertensive cases and 24, 480 controls from different ethnicities. The meta-analysis showed a significant association of STK39 rs3754777 variant with hypertension (OR = 1.10, 95%CI = 1.06–1.15, p = 7.95×10⁻⁶). Further subgroup analysis by ethnicity suggested that the association was significant in Europeans (OR = 1.08, 95% CI = 1.03–1.14, p = 0.002) and in East Asians (OR = 1.16, 95%CI = 1.07–1.25, p = 4.34×10⁻⁴), but not in Africans (OR = 1.01, 95%CI 0.80–1.27, p = 0.932). We further confirmed the positive association by sensitivity analysis. No publication bias was detected (Begg’s test, p = 0.721; Egger’s test, p = 0.744).

Conclusions

The present meta-analysis confirms the significant association of STK39 polymorphism with susceptibility to hypertension in Europeans and East Asians. Future studies should include gene–gene and gene–environment interactions to investigate the identified association.     

Homeostatic Properties and Phenotypic Maturation of Murine CD4+ Pre-Thymic Emigrants in the Thymus

After a tightly regulated developmental program in the thymus, “mature” single positive (SP) thymocytes leave the thymus and enter the periphery. These newly arrived recent thymic emigrants (RTEs) are phenotypically and functionally immature, and will complete a dynamic maturation in the peripheral lymphoid organs before being licensed to be resident naïve T cells. To study the early events occurring in the RTE maturation process, we identified the phenotype of CD4⁺ pre-RTEs, a population of CD4⁺ SP thymocytes that have acquired the thymus egress capability. Compared to peripheral naïve T cells, CD4⁺ pre-RTEs displayed superior survival capability in lymphoreplete mice and faster proliferation under lymphopenic condition. The differences in Bcl2/Bim expression and/or heightened IL-7 signaling pathway may account for the pre-RTEs’ better responsiveness to homeostatic signals. Qa2, the expression of which indicates the phenotypic maturation of SPs and RTEs, was found to be upregulated in CD4⁺ pre-RTEs in thymic perivascular space. Migratory dendritic cells that surround this region contribute to Qa2 expression in pre-RTEs. The dendritic cell-driven Qa2 induction of CD4⁺ pre-RTEs is independent of MHC class II and Aire molecules.     

Molecular and Clinical Characteristics of Clonal Complex 59 Methicillin-Resistant Staphylococcus aureus Infections in Mainland China

Detailed molecular analyses of Clonal Complex 59 (CC59) methicillin-resistant Staphylococcus aureus (MRSA) isolates from children in seven major cities across Mainland China were examined. A total of 110 CC59 isolates from invasive and non-invasive diseases were analyzed by multilocus sequence typing (MLST), Staphylococcus cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) typing and pulsed-field gel electrophoresis (PFGE). Antibiotics susceptibilities, carriage of plasmids and 42 virulence genes and the expression of virulence factors were examined. ST59 (101/110, 91.8%) was the predominant sequence type (ST), while single locus variants (SLVs) belonging to ST338 (8/110, 7.3%) and ST375 (1/110, 0.9%) were obtained. Three SCCmec types were found, namely type III (2.7%), type IV (74.5%) and type V (22.7%). Seven spa types including t437, which accounted for 87.3%, were determined. Thirteen PFGE types were obtained. PFGE types A and B were the major types totally accounting for 81.8%. The dominant clone was ST59-t437-IVa (65.5%), followed by ST59-t437-V (14.5%). The positive rate of luks-PV and lukF-PV PVL encoding (pvl) gene was 55.5%. Plasmids were detected in 83.6% (92/110) of the strains. The plasmid size ranging from 23.4 kb to 50 kb was most prevalent which accounted for 83.7% (77/92). A significantly lower expression of hla was found in ST59-t437-IVa compared with ST59-t437-V. Among the 110 cases, 61.8% of the patients were less than 1 year old. A total of 90 cases (81.8%) were community-associated (CA) infections whereas 20 cases (18.2%) were hospital-associated (HA) infections. Out of the 110 patients, 36.4% (40/110) were diagnosed with invasive infectious diseases in which ST59-t437-IVa accounted for 67.5% (27/40). In brief, ST59-t437-IVa was proved as the dominant clone in CC59 MRSA strains. The carriage rate of pvl gene was high. CC59 MRSA could result in CA and HA infections. The majortiy of MRSA infection children were in young age.     

Shikonin Kills Glioma Cells through Necroptosis Mediated by RIP-1

Background and Purpose

Shikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms.

Methods

Shikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting.

Results

Shikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression.

Conclusions

We demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.     

镉吸附细菌的分离及其对土壤镉的固定

[目的] 探究镉吸附细菌是否能够高效固定土壤有效镉（Cd）,为土壤有效Cd的微生物固定提供理论依据。[方法] 利用含Cd²⁺牛肉膏蛋白胨液体培养基对细菌进行Cd的耐受性测试筛选出镉抗性强的菌株;通过16S rRNA基因相似性及系统进化分析鉴定耐镉细菌,将菌细胞加入含CdCl₂溶液中进行Cd²⁺吸附效率测定;通过土培模拟实验,测定土壤pH、碱解氮、有效磷、速效钾、有机质、CEC、有效Cd及微生物数量来分析镉吸附细菌对镉污染土壤的影响。[结果] 从德阳鱼腥草根际土壤中分离获得的57株细菌对Cd²⁺表现出不同程度的抗性,并从中筛选出3株耐Cd优势细菌普罗威登斯菌属（Providencia）DY8、芽孢杆菌属（Bacillus）DY3和芽孢杆菌属（Bacillus）DY1-4。其对溶液中的Cd²⁺表现出较好的吸附作用,吸附效率随着Cd²⁺浓度升高而降低。DY8、DY3、DY1-4能使镉污染土壤中有效Cd含量分别降低72.11%、68.55%、62.32%,同时显著提高镉污染土壤中碱解氮、有效磷的含量。[结论] Cd污染农田土壤中含有丰富的耐Cd微生物资源,Cd吸附细菌能降低土壤中有效Cd的含量,且能有效改善土壤养分条件。     

侵染中华蜜蜂6日龄幼虫的蜜蜂球囊菌的微小RNA差异表达谱及调控网络

【目的】蜜蜂球囊菌(Ascosphaeraapis,简称球囊菌)是专性侵染蜜蜂幼虫的致死性真菌病原。MicroRNA(miRNA)作为一类重要的基因表达调控因子,能够广泛参与真菌及其宿主的相互作用过程。本研究通过比较分析球囊菌孢子(AaCK)和侵染中华蜜蜂(Apis cerana cerana,简称中蜂) 6日龄幼虫肠道内的球囊菌(AaT)的smallRNA(sRNA)组学数据对球囊菌的差异表达miRNA(differentiallyexpressed miRNA,DEmiRNA)、靶mRNA及二者间的调控网络进行全面解析,旨在揭示miRNA介导的球囊菌对中蜂幼虫的侵染机制。【方法】对于球囊菌侵染的中蜂6日龄幼虫肠道的small RNA-seq (sRNA-seq)数据,利用BLAST工具连续比对东方蜜蜂(Apiscerana)和球囊菌的参考基因组筛滤得到AaT的sRNA组学数据。分别将AaCK和AaT的sRNA组学数据比对miRBase数据库,对球囊菌侵染宿主前后miRNA的数量和结构特征进行分析。联用RNAhybrid+svm_light、Miranda和TargetScan软件预测AaCK vs AaT比较组中DEmiRNA的靶mRNA,进而利用相关生物信息学软件对上述靶mRNA进行GO分类和KEGG代谢通路富集分析。通过Cytoscape软件对DEmiRNA-mRNA调控网络进行可视化。利用Stem-loop RT-PCR、RT-qPCR和分子克隆验证测序结果的可靠性。【结果】在AaCK和AaT中分别鉴定到380和387个miRNA。结构特征分析结果显示,AaCK和AaT的mi RNA皆集中分布在18–25 nt,且首位碱基主要偏向于U。AaCKvsAaT比较组共有270个DEmiRNA,包含155个上调miRNA和115个下调miRNA,分别靶向结合6091和6145个mRNA。GO分类结果显示,上述靶mRNA主要涉及代谢进程、细胞进程、应激反应等15个生物学进程;细胞、细胞组分、细胞器等12个细胞组分;催化活性、结合、转运子活性等11个分子功能。KEGG代谢通路富集分析结果显示,上述靶mRNA富集在123条代谢通路,参与对氨基酸代谢、碳水化合物代谢以及核苷酸代谢等物质代谢,氧化磷酸化、硫代谢、氮代谢等能量代谢,以及MAPK和Hippo等信号通路的调控。球囊菌DEmiRNA与靶mRNA之间存在复杂的调控关系,其中miR-29-x、miR-250-x、miR-4968-y、miR-11200-x、novel-m0023-5p、novel-m0130-5p和novel-m0135-5p等DEmiRNA可靶向结合与球囊菌的半胱氨酸蛋白酶、DNA甲基化转移酶以及几丁质酶相关的mRNA;此外,miR-7-x、miR-9-z、miR-319-y和miR-5951-y等同时参与调控MAPK信号通路;进一步分析发现,miR-250-x同时参与对DNA甲基化转移酶、MAPK信号通路及其他酶类合成与代谢途径的调控,并可能参与球囊菌与中蜂6日龄幼虫之间的跨界调控。通过Stem-loopRT-PCR和RT-qPCR验证了4个DEmiRNA的差异表达,并利用分子克隆和Sanger测序证实miR-7-x的序列与测序结果一致。【结论】本研究解析了侵染中蜂6日龄幼虫的球囊菌的miRNA差异表达谱及DEmiRNA的调控网络,揭示了球囊菌DEmiRNA可能通过调控病原的物质和能量代谢、增殖、毒力、信号通路及相关mRNA参与对中蜂幼虫的侵染过程。miR-7-x、miR-250-x、novel-m0023-5p等关键DEmiRNA有望作为白垩病治疗的新型分子靶点。     

烟草青枯病劳尔氏菌与拮抗菌对根系分泌物的竞争作用

[目的]研究青枯病病原菌与拮抗菌的营养特性及其对烟草根系分泌物的响应差异,对提高拮抗菌定殖能力、有效生物防控烟草青枯病具有非常重要的意义。[方法]本研究通过筛选与鉴定贵州烟区青枯病病原菌株及拮抗菌株,通过Biolog表型芯片技术分别检测病原菌与拮抗菌的特征性碳、氮源,利用气质联用(GC-MS)检测烟草主栽品种K326根系分泌物的主要物质,在此基础上进行病原菌与拮抗菌对其利用能力、利用强度以及共培养的研究。[结果]经鉴定,分离、筛选到的病原菌株和拮抗菌株分别为青枯劳尔氏菌(Ralstonia solawacearum)和枯草芽孢杆菌(Bacillus subtilis);在含量为0.01μg/mL以上的根系分泌物中,12种物质的含量从高到低排序为:果胶>葡萄糖>木糖>阿拉伯糖>半乳糖>核糖>蔗糖>苯甲酸>果糖=D-甘露醇>棕榈酸>富马酸,果胶含量最高且明显高于其他物质;拮抗菌(LX4)对碳源利用能力高于病原菌(Rs)的碳源有阿拉伯糖、木糖和核糖,分别是病原菌利用能力的1.22、1.95和2.17倍;前12 h拮抗菌利用果糖强度高于病原菌,不同碳源共培养24 h后LX4对gfp-Rs(绿色荧光蛋白标记后的青枯病病原菌)抑制率为18.34%(阿拉伯糖)、53.23%(木糖)、63.53%(核糖)和52.09%(果糖)。[结论]拮抗菌对烟草根系分泌物的利用不及病原菌,但在特定碳源条件下拮抗菌能够利用根系分泌物中的某些碳源产生某种拮抗物质抑制病原菌,拮抗菌与病原菌之间同时存在利用性竞争和干扰性竞争关系,研究结果为进一步研究烟草青枯病的生物防控提供了新的理论依据。     

相容溶质四氢嘧啶与羟基四氢嘧啶的代谢调控研究进展

四氢嘧啶(Ectoine)及其衍生物羟基四氢嘧啶(5-hydroxyectoine,5-HE)是嗜盐微生物胞内合成的一类能够抵抗外界高盐胁迫的相容溶质,具有细胞、细胞膜、蛋白质和核酸的保护作用,可抵抗高盐、高温、冷冻和干燥等极端环境因素的刺激,从而倍受关注。本文对不同类型微生物Ectoine/5-HE(Ects)生物合成代谢、分解代谢以及吸收/转运系统涉及的调控机制进行综述,以期为Ects合成产量的提升与高效积聚策略的优化,提供一定的理论参考依据。