Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia

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Comparison of SSCP and DHPLC for the detection of LDLR mutations in a New Zealand cohort

Familial hypercholesterolaemia (FH) is a common inherited disorder, associated with premature vascular disease. FH may be caused by many different mutations in the low density lipoprotein receptor (LDLR) gene, about 700 mutations have been described, most of which occur rarely and often only in single families. Although particular mutations are prevalent in certain ethnic groups, countries with heterogeneous population bases (such as NZ) may carry a wide variety of mutations; making a gene screening approach the appropriate first step for a mutation detection programme. We have compared SSCP with DHPLC to assess their effectiveness as methods for LDLR mutation detection. Although five novel LDLR mutations were detected by SSCP in patients with FH, DHPLC was more sensitive, with eight novel mutations detected. Six of these mutations (T392M, R419G, Y421N, 1206-1207delCT, 1872delC, and 1943delC) were clustered in exons 9 and 13 of the EGF precursor homology domain, one (679-680delAC) in the ligand binding domain (exon 4) and the eighth (P774H) in the membrane-spanning domain (exon 16). Twenty five mutations were identified in 35 patients in total. Of these, we were able to detect only 64% of mutations by SSCP even though all variants were detected by DHPLC. All patients are heterozygous for the mutations, which is consistent with the clinical phenotypes.     

抗纤维化细胞因子对TGF-β1基因启动转录活性的影响

转化生长因子β1(transforming growth factor-β1)是一类现已明确的致纤维化细胞因子,具有广泛的生物学效应,对细胞外基质(ECM)基因表达、降解、细胞增殖分化、凋亡及免疫功能都具有重要调节作用。前期我们研究发现,TGF-β1启动调控序列中单核苷酸多态性(single nucleotide polymorphism,SNP)位点-509C>T与肝纤维化进展及血浆中TGF-β1浓度有明显的相关性。本研究旨在探讨抗纤维化细胞因子(IL-10、HGF、IFN-γ)对含TGF-β1基因-509C>T启动调控序列活性的影响。我们以特定-509C>T基因型患者DNA为模板,用PCR方法扩增得到一对长度为2.14kb(-1328~+812)含有-509C>T变异的TGF-β1上游基因片段,并将其与不含启动子的pCAT3-enhancer报告基因载体重组,构建重组体phT-GF2.14C和phTGF2.14T。用脂质体转染法将两种重组体分别转染至正常人肝脏细胞中,分别用IL-10(4ng/ml)、HGF(10ng/ml)、IFN-γ(20ng/ml)干预转染后细胞。ELISA法测定转染细胞的报告基因CAT活性。结果表明:肝细胞转染重组体phTGF2.14C细胞的CAT活性明显高于转染重组体phTGF2.14T(t=12.5882,P=0.0002)。IFN-γ对TGF-β1基因启动子phTGF2.14C、phTGF2.14T均具有显著抑制作用。细胞因子IL-10和HGF对其调控作用不显著。TGF-β1基因-509C>T中C等位基因可明显增强TGF-β1基因上游启动调控序列的转录活性,IFN-γ作为一种抗纤维化细胞因子在基因转录水平对含有两种等位基因-509C>T的TGF-β1基因上游启动调控序列均具有抑制作用。而作为抗纤维化细胞因子的IL-10与HGF在2.14Kb(-1328~+812)区域内对TGF-β1基因上游启动调控序列的作用不显著。     

A method for exceptionally low noise single channel recordings

Summary We present a method whereby, with integrating electronics, quartz patch electrodes and a novel use of silicone oil, background noise levels as low as .083 pA RMS in a 5 kHz bandwidth (4-pole Butterworth filter) have been achieved in single channel patch clamp recordings. These approaches result in much higher signal to noise ratios for single channel recording than have previously been reported and should allow many investigators to significantly reduce noise at a constant bandwidth or to increase their recording bandwidths by several kHz.     

卵巢辐射损伤对大鼠腰椎骨密度和生物力学的影响

目的探讨大鼠卵巢辐射损伤后腰椎骨密度、微结构和生物力学的改变。方法手术暴露大鼠双侧卵巢并应用50Gy的γ射线局部照射,术后90d取大鼠腰椎,DEXA测定骨密度,扫描电镜显示微结构,并行压缩实验检测腰椎最大载荷。结果与假手术组相比,卵巢辐射组大鼠的腰椎骨密度显著减少(P<0.05),骨微结构破坏,生物力学性能下降(P<0.05)。结论卵巢辐射损可导致大鼠腰椎的骨质疏松样改变。     

低能量激光血管内照射治疗脑梗死的临床观察

目的:探讨采用低能量半导体激光血管内照射疗法(intravascular laser irradiation on blood,ILIB)治疗脑梗死的效果及机制.方法:随机选取80例脑梗死患者分为治疗组与对照组,两组各40例,两组患者接受同样的药物治疗,治疗组在上述治疗的基础上,于发病1 d～3 d内,加用半导体激光血管内照射治疗.采用国产半导体激光治疗仪,波长650nm,功率1.5mW～2.0mW.患者平卧,消毒后用静脉留置针穿刺上肢正中静脉或贵要静脉,成功后留置外套管,通过管脑导入激光剂,开启半导体激光治疗仪进行照射治疗.结果:治疗组有效率92.5%,与对照组差异有显著性意义(p＜0.01),治疗组患者临床症状和体征治疗后明显改善.结论:该疗法有调节脂质代谢,改善血液流变学性质,恢复神经传导功能等功效,进而提高脑梗死的治疗效果.     

Broadening the phenotypic spectrum of Pearson syndrome: Five new cases and a review of the literature

Pearson syndrome (PS) is a multisystem mitochondrial respiratory chain disorder typically characterized by sideroblastic anemia and exocrine pancreatic insufficiency. PS is caused by a single large‐scale mitochondrial DNA (mtDNA) deletion. PS classically presents in the first year of life and may be fatal in infancy. Children who survive PS may progress to develop Kearns–Sayre syndrome later in life. The full phenotypic spectrum and prognosis of the condition continue to evolve. Here we report five new patients with PS with unique clinical presentations, including four patients with onset later than previously reported in the literature, and one patient with prenatal onset of symptoms. The timing and unique features of these presentations support an expanded phenotypic spectrum of single large‐scale mtDNA deletion syndromes (SLSMDS) and reinforce the importance of including SLSMDS in the differential for children with complex multisystem presentations.     

High-throughput single strand conformation polymorphism mutation detection by automated capillary array electrophoresis: validation of the method

Capillary array electrophoresis (CAE) is a novel technique, which allows for high throughput analysis of DNA fragments. When screening for mutations in whole populations or large patient groups it is necessary to have robust and well-characterized setups for high throughput analysis. For large-scale mutation screening, we have developed procedures for single strand conformation polymorphism (SSCP) assays using CAE (CAE-SSCP) whereby we may increase both the sensitivity and the throughput compared to conventional SSCP analysis. In this study we have validated CAE-SSCP by 1) comparing detection by slab-gel based SSCP with CAE-SSCP of mutations in the MYH7, MYL2, and MYL3 genes encoding sarcomere proteins from patients suffering from hypertrophic cardiomyopathy; and 2) by constructing a series of 185 mutants having substitution mutations, as well as insertion/deletion mutations, or some combinations of these, in different sequence contexts in four exons and different positions relative to the end of the amplicon (three from the KCNQ1 gene, encoding a cardiac potassium channel, and one from the TNNI3 gene encoding cardiac troponin I). The method identified 181 out of 185 mutations (98%), and the data suggest that the position of mutation in the fragment had no effect on the sensitivity. Analysis of the specificity of the method showed that only very few mutants could not be distinguished from each other and there were no false positives.     

应用复合诱导突变分离PCR法检测mtDNA的单核苷酸多态性

目的应用复合诱导突变分离PCR(multiplexed mutagenically separated PCR,MS-PCR)技术、银染分型,建立线粒体DNA(mitochondrial DNA,mtDNA)编码区单核苷酸多态(single nucleotide polymorphism,SNP)分型系统,探讨其应用价值。并调查了成都汉族群体mtDNA编码区4个SNP基因座等位基因频率和单倍型分布情况。方法根据SNP基因座(C12705T、A8701G、G8584A、C10400T)设计两条片段相差4个碱基的等位基因特异性引物和一条公共引物,4个SNP基因座复合扩增,PCR产物经聚丙烯酰胺凝胶电泳、银染显带后确定样本的基因型。结果不同SNP基因座为长度不同的单一谱带,其分型结果与直接测序一致。在成都汉族160名无关个体中,4个SNP基因座C12705T、A8701G、G8584A、C10400T等位基因频率分别为0.3813/0.6187、0.4813/0·5187、0.8250/0.1750、0.4938/0.5062;共检出6种单倍型,单倍型的基因多样性为0.7137。结论建立的MMS-PCR银染分型系统是一种简单、快速、准确、有效的SNP分型方法,对建立mtDNA编码区SNP数据库,研究群体遗传学、进化学和进行法医学个人识别和亲子鉴定有重要意义。     

Surface markers of human natural killer cells as analyzed in a modified single cell cytotoxicity assay on poly-L-lysine coated cover slips

A modified single cell cytotoxicity assay using poly-L-lysine coated cover slips (PLL-SCCA) was employed to study the frequency and surface marker profile of human peripheral blood lymphocytes (PBL) with NK reactivity against K 562 target cells. When compared with the previously described agarose single cell cytotoxicity assay (A-SCCA) identical results were obtained. For 13 donors tested 18.1 +/- 4.4% of the PBL formed conjugates with K 562 and 2.7 +/- 1.6% displayed NK reactivity. In contrast to the A-SCCA, the PLL-modified assay permits direct identification of both conjugate forming (TBC) and cytolytic PBL (NK) by means of surface markers. Indirect immunofluorescence studies with monoclonal anti-PBL antibodies revealed that neither the plating procedures nor the incubation conditions employed affected the expression of the antigens recognized by these reagents. This method of directly identifying NK cells showed that OKM1+ cells were enriched among the NK cells as compared to PBL and TBC (55% vs. 23% and 43%, respectively). In contrast, the OKT3+ or Leu1+ fraction of the NK cells was reduced as compared to PBL and TBC. However, using this method of identification at the effector cell level, a substantial proportion of the NK cells were OKT3+ or Leu1+ (57% or 58% respectively, 7 donors). Approximately 25% of the NK cells were Leu2a+ and 30% were Leu3a+, respectively. However, the size of the Leu3a+ fraction varied considerably with individual donors and the size of this fraction appeared to be inversely related to that of the donors NK pool.