安徽地区汉族心血管疾病患者SLCO1B1与ApoE基因多态性分布及其在他汀类药物临床个体化应用中的意义

目的：探讨脂质及药物代谢相关基因SLCO1B1和ApoE的基因多态性在安徽地区汉族心血管疾病患者中的分布，以评估他汀类药物个体化用药的效益/风险比。方法：利用PCR-荧光探针法技术检测2019年1月至2020年8月合肥市第二人民医院736例心血管疾病患者外周血基因组中SLCO1B1基因的rs2306283（388A>G）和rs4149056（521T>C）位点和ApoE基因的rs429358（388T>C）和rs7412（526C>T）位点的基因多态性分布特点，并与已报道的中国其他地区汉族心血管疾病患者的数据进行比较，分析不同地区间的基因型分布差异。结果：检测到安徽地区汉族心血管疾病患者中SLCO1B1基因型有6种，分别为*1a/*1a型（6.11%）、*1a/*1b型（29.08%）、*1b/*1b型（44.57%）、*1a/*15型（4.08%）、*1b/*15型（15.49%）、*15/*15型（0.68%），未检测到*1a/*5型、*5/*5型和*5/*15型；ApoE基因有6种表型，分别为E2/E2型（0.41%）、E2/E3型（11.96%）、E2/E4型（1.09%）、E3/E3型（67.66%）、E3/E4型（17.93%）、E4/E4型（0.95%）。两种基因的基因多态性频率分布满足Hardy-Weinberg遗传平衡，具有群体代表性。本研究人群中携带SLCO1B1正常肌病风险型的比例最高，约占79.76%；SLCO1B1中度肌病风险型和高度肌病风险型的人群比例较低，分别为19.57%和0.68%。ApoE大众类基因型比例最高，约占68.75%；ApoE保护类基因型及风险类基因型的人群比例分别为12.37%和18.88%。不同性别间SLCO1B1和ApoE基因表型患者差异无统计学意义。与华南地区心血管疾病患者相比，安徽地区ApoE基因多态性分布差异有统计学意义（P<0.05）。结论：安徽地区736例心血管疾病患者SLCO1B1和ApoE基因型分别以他汀药物剂量耐受性较高的正常肌病风险型和对他汀药物敏感的大众类基因型为主，服用他汀类药物诱发肌病的风险较低，降脂疗效较好；且两种基因的多态性分布均不受性别的影响，但ApoE基因多态性分布特征可能在地域上存在差异。因此，检测SLCO1B1和APOE基因多态性对于临床评估效益/风险比有重要的指导意义。     

In Vivo Simultaneous Analysis of Gene Expression by Dual-Color Luciferases in Caenorhabditis elegans

Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions.     

Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.     

Role of the HSP70 Co-Chaperone SIL1 in Health and Disease

Cell surface and secreted proteins provide essential functions for multicellular life. They enter the endoplasmic reticulum (ER) lumen co-translationally, where they mature and fold into their complex three-dimensional structures. The ER is populated with a host of molecular chaperones, associated co-factors, and enzymes that assist and stabilize folded states. Together, they ensure that nascent proteins mature properly or, if this process fails, target them for degradation. BiP, the ER HSP70 chaperone, interacts with unfolded client proteins in a nucleotide-dependent manner, which is tightly regulated by eight DnaJ-type proteins and two nucleotide exchange factors (NEFs), SIL1 and GRP170. Loss of SIL1′s function is the leading cause of Marinesco-Sjögren syndrome (MSS), an autosomal recessive, multisystem disorder. The development of animal models has provided insights into SIL1′s functions and MSS-associated pathologies. This review provides an in-depth update on the current understanding of the molecular mechanisms underlying SIL1′s NEF activity and its role in maintaining ER homeostasis and normal physiology. A precise understanding of the underlying molecular mechanisms associated with the loss of SIL1 may allow for the development of new pharmacological approaches to treat MSS.     

Alterations of Gut Microbiota by Overnutrition Impact Gluconeogenic Gene Expression and Insulin Signaling

A high-fat, Western-style diet is an important predisposing factor for the onset of type 2 diabetes and obesity. It causes changes in gut microbial profile, reduction of microbial diversity, and the impairment of the intestinal barrier, leading to increased serum lipopolysaccharide (endotoxin) levels. Elevated lipopolysaccharide (LPS) induces acetyltransferase P300 both in the nucleus and cytoplasm of liver hepatocytes through the activation of the IRE1-XBP1 pathway in the endoplasmic reticulum stress. In the nucleus, induced P300 acetylates CRTC2 to increase CRTC2 abundance and drives Foxo1 gene expression, resulting in increased expression of the rate-limiting gluconeogenic gene G6pc and Pck1 and abnormal liver glucose production. Furthermore, abnormal cytoplasm-appearing P300 acetylates IRS1 and IRS2 to disrupt insulin signaling, leading to the prevention of nuclear exclusion and degradation of FOXO1 proteins to further exacerbate the expression of G6pc and Pck1 genes and liver glucose production. Inhibition of P300 acetyltransferase activity by chemical inhibitors improved insulin signaling and alleviated hyperglycemia in obese mice. Thus, P300 acetyltransferase activity appears to be a therapeutic target for the treatment of type 2 diabetes and obesity.     

Genome-Wide Analyses of the Temperature-Responsive Genetic Loci of the Pectinolytic Plant Pathogenic Pectobacterium atrosepticum

Temperature is one of the critical factors affecting gene expression in bacteria. Despite the general interest in the link between bacterial phenotypes and environmental temperature, little is known about temperature-dependent gene expression in plant pathogenic Pectobacterium atrosepticum, a causative agent of potato blackleg and tuber soft rot worldwide. In this study, twenty-nine P. atrosepticum SCRI1043 thermoregulated genes were identified using Tn5-based transposon mutagenesis coupled with an inducible promotorless gusA gene as a reporter. From the pool of 29 genes, 14 were up-regulated at 18 °C, whereas 15 other genes were up-regulated at 28 °C. Among the thermoregulated loci, genes involved in primary bacterial metabolism, membrane-related proteins, fitness-corresponding factors, and several hypothetical proteins were found. The Tn5 mutants were tested for their pathogenicity in planta and for features that are likely to remain important for the pathogen to succeed in the (plant) environment. Five Tn5 mutants expressed visible phenotypes differentiating these mutants from the phenotype of the SCRI1043 wild-type strain. The gene disruptions in the Tn5 transposon mutants caused alterations in bacterial generation time, ability to form a biofilm, production of lipopolysaccharides, and virulence on potato tuber slices. The consequences of environmental temperature on the ability of P. atrosepticum to cause disease symptoms in potato are discussed.