Seroprevalence of Helicobacter pylori Infection in Patients with Connective Tissue Diseases

To assess the possibility that Helicobacter pylori might be an etiologic agent, titers of anti-H. pylori IgG in sera of patients with connective tissue diseases [rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), polymyositis or dermatomyositis (PM/DM), progressive systemic sclerosis (PSS), mixed connective tissue disease (MCTD) and Sjögren's syndrome (SjS)] were compared with those of non-patient (healthy) volunteers and of patients with chronic pulmonary diseases (CPD) by ELISA using an extract of sonicated H. pylori as the antigen. Among patients with connective tissue diseases, those with SLE and RA had anti-H. pylori titers as low as healthy volunteers. Patients with SjS had much higher average titers than patients with CPD (P<0.05). We previously reported that levels of myeloid calcium-binding protein (MRP8 and MRP14) were elevated in the serum of patients with connective tissue diseases. No correlation was found between serum levels of anti-H. pylori IgG and of MRP, a novel marker of inflammation. Furthermore, sera with high IgG titers were selected, and their reactivity with the H. pylori antigen were analyzed by Western blotting. H. pylori antigens with a variety of molecular masses were immunostained with sera from patients and from healthy volunteers, but a 16-kDa antigen was only immunostained by reaction with the sera of patients with MCTD and SjS, although the number of test samples was small.     

Individual Limb Muscle Bundles Are Formed through Progressive Steps Orchestrated by Adjacent Connective Tissue Cells during Primary Myogenesis

1. Download : Download high-res image (152KB)
2. Download : Download full-size image

     

结缔组织生长因子小干扰RNA的构建及其干扰效果检测

目的：构建结缔组织生长因子（CTGF）基因的小干扰RNA（siRNA）,并检测其对CTGF基因表达的干扰效果。方法：根据CTGF基因序列,设计2条合理的CTGF-siRNA,并将其克隆到siRNA载体pSliencer2．1-U6neo中,转化大肠杆菌DH5a,挑取阳性菌落进行酶切和测序鉴定;将构建成功的CTGF-siRNA重组质粒与带nJAG标签的CTGF表达载体共同转染人胚肾293T细胞,Western印迹检测siRNA的干扰效果。结果：构建了2个CTGFpsiRNA重组质粒,2条siRNA都有干扰作用,其中一条的干扰效果可达75％以上。结论：构建的CTGF-siRNA可为进一步研究CTGF的功能提供参考。     

Mutation Spectrum of Six Genes in Chinese Phenylketonuria Patients Obtained through Next-Generation Sequencing

Background

The identification of gene variants plays an important role in the diagnosis of genetic diseases.

Methodology/Principal Findings

To develop a rapid method for the diagnosis of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficiency, we designed a multiplex, PCR-based primer panel to amplify all the exons and flanking regions (50 bp average) of six PKU-associated genes (PAH, PTS, GCH1, QDPR, PCBD1 and GFRP). The Ion Torrent Personal Genome Machine (PGM) System was used to detect mutations in all the exons of these six genes. We tested 93 DNA samples from blood specimens from 35 patients and their parents (32 families) and 26 healthy adults. Using strict bioinformatic criteria, this sequencing data provided, on average, 99.14% coverage of the 39 exons at more than 70-fold mean depth of coverage. We found 23 previously documented variants in the PAH gene and six novel mutations in the PAH and PTS genes. A detailed analysis of the mutation spectrum of these patients is described in this study.

Conclusions/Significance

These results were confirmed by Sanger sequencing. In conclusion, benchtop next-generation sequencing technology can be used to detect mutations in monogenic diseases and can detect both point mutations and indels with high sensitivity, fidelity and throughput at a lower cost than conventional methods in clinical applications.     

抗结缔组织生长因子特异性小干扰RNA的设计、合成及筛选

目的 设计和合成针对抗肝纤维化关键基因抗结缔组织生长因子（connective tissue growth factor,CTGF）的特异性小干扰RNA（siRNA）,并筛选高效的CTGF siRNA抗肝纤维化序列.方法 参照siRNA设计原则,应用RNA在线设计软件,设计3段RNA干扰候选序列,分别转染正常干细胞株（L-02）,以转染非特异性siRNA（与CTGF mRNA无同源性）作为对照,应用Western 印迹检测L-02细胞CTGF蛋白质表达.结果 与对照相比,转染siRNA的L-02细胞CTGF蛋白表达明显下调,且siCTGF-1、siCTGF-2、siCTGF-3各片段干扰率分别为44.91 %、93.99 %、81.34%,其中以siCTGF-2干扰率最高,效果最明显.转染非特异性siRNA的L-02细胞CTGF蛋白表达无明显变化（P＜0.05）.结论 不同的CTGF siRNA对L-02细胞CTGF表达水平具有不同的干扰效能,成功合成能高效阻抑CTGF表达的CTGF siRNA,为进一步探索更特异、更有效的抗肝纤维化基因治疗这一新途径打下坚实基础.     

结缔组织生长因子对血管生成和细胞功能的调节

结缔组织生长因子(CTGF)是CCN家族的主要成员,是研究较多的CCN蛋白之一。CTGF能促进细胞的增殖、存活、迁移和黏附,调节血管生成分子(bFGF,VEGF)以及一些影响胞外基质完整性和稳定性的分子(胶原、MMPs和TIMPs)的活性。CTGF可以在多个控制位点采取直接或间接的机制调控细胞功能和血管生成。结合新的发现和新的视点,阐述CTGF对细胞功能和血管生成的调控作用以及与肿瘤生长的关系。     

Use of Human Fibroblasts Grown on Microcarriers for Formation of Connective Tissue Equivalent

Living equivalents of tissues, specifically those produced on the basis of fibroblasts and collagen gel, are widely used for repair of organ and tissues defects. In clinical practice, it is more convenient to use the fibroblasts grown on microcarriers or such a connective tissue equivalent when the fibroblasts on microcarriers are embedded in collagen gel. We studied the properties of a connective tissue equivalent produced by embedding the fibroblasts grown on microcarriers in collagen gel for its prospective use in clinical practice. According to our results, the optimal time of use of the living tissue equivalent amounts to three–four days after embedding of fibroblasts on microcarriers in gel. At that time, contraction only begins, which facilitates manipulations with the gel.     

Comparison of Next-Generation Sequencing and Polymerase Chain Reaction for Personalized Treatment-Related Genomic Status in Patients with Metastatic Colorectal Cancer

Personalized treatments based on the genetic profiles of tumors can simultaneously optimize efficacy and minimize toxicity, which is beneficial for improving patient outcomes. This study aimed to integrate gene alterations associated with predictive and prognostic outcomes in patients with metastatic colorectal cancer (mCRC) with polymerase chain reaction (PCR) and in-house next-generation sequencing (NGS) to detect KRAS, NRAS, and BRAF mutations. In the present study, 41 patients with mCRC were assessed between August 2017 and June 2019 at a single institution. The overall concordance between NGS and PCR results for detecting KRAS, NRAS, and BRAF mutations was considerably high (87.8–92.7%), with only 15 discrepant results between PCR and NGS. Our companion diagnostic test analyzes KRAS, NRAS, and BRAF as a panel of CRC molecular targets; therefore, it has the advantages of requiring fewer specimens and being more time and cost efficient than conventional testing for separate analyses, allowing for the simultaneous analysis of multiple genes.     

Diversity of extracellular matrix morphology in vertebrate skeletal muscle

Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.     

An in vitro model to evaluate the properties of matrices produced by fibroblasts from osteogenesis imperfecta and Ehlers-Danlos Syndrome patients

Aim of the studyOsteogenesis imperfecta and Ehlers Danlos syndrome are hereditary disorders caused primarily by defective collagen regulation. Osteogenesis imperfecta patients were divided to haploinsufficient and dominant negative depending on the effect of COL1A1 and COL1A2 mutations whereas Ehlers Danlos syndrome patients had a mutation in PLOD1. Although collagen abnormalities have been extensively studied in monolayer cultures, there are no reports about 3D in vitro models which may reflect more accurately the dynamic cell environment. This is the first study presenting the structural and mechanical characterization of a 3D cell-secreted model using primary patient fibroblasts.Materials and methodsFibroblasts from patients with osteogenesis imperfecta and Ehlers Danlos syndrome were cultured with ascorbic acid for 5 weeks. The effect of mutations on cytosolic and secreted collagen was tested by electrophoresis following incubation with radiolabeled ¹⁴C proline. Extracellular matrix was studied in terms of collagen fiber orientation, stiffness, as well as glycosaminoglycan and collagen content.Results and conclusionsOsteogenesis imperfecta patients with haploinsufficient mutations had higher percentage of anisotropic collagen fibers alignment compared to other patient groups; all patients had a lower percentage of anisotropic samples compared to healthy controls. This correlated with higher average stiffness in the control group. Glycosaminoglycan content was lower in the control and haploinsufficient groups. In cells with PLOD1 mutations, there were no differences in PLOD2 expression. This proof of concept study was able to show differences in collagen fiber orientation between different patient groups which can potentially pave the way towards the development of 3D models aiming at improved investigation of disease mechanisms.     

介导大鼠结缔组织生长因子基因沉默的慢病毒载体转移质粒的构建及鉴定

目的：构建介导大鼠结缔组织生长因子（CTGF）基因沉默的慢病毒载体转移质粒pGCL-CTGF,为进一步包装慢病毒载体奠定基础。方法：以大鼠CTGF基因为靶基因,根据RNA干扰（RNAi）序列设计原则,设计4对有小发夹结构的RNAi靶点序列,退火形成双链DNA,双酶切后定向克隆到慢病毒载体转移质粒pGCL-GFP中,构建4个含靶基因片段的重组慢病毒载体转移质粒pGCL-CTGF,并对质粒进行PCR及测序鉴定。结果：CTGF的短发夹RNA（shRNA）片段被成功克隆到慢病毒载体转移质粒pGCL-GFP中,4个插入序列与设计的靶基因片段完全一致。结论：构建了能够表达4个含CTGF靶基因片段的慢病毒载体转移质粒,为进一步包装介导CTGF基因沉默的慢病毒载体奠定了基础。     

快速直视筛选结缔组织生长因子特异RNA干扰序列

利用一种快速简便经济直接的筛选特异RNA干扰序列的方法,筛选结缔组织生长因子（CTGF）基因特异RNA干扰序列.设计合成分别含EcoRⅠ和HindⅢ酶切位点的引物,以pTM1-CTGF为模板PCR获得CTGF基因,定向克隆入pSOS-HUS质粒得到质粒pSOS-HUS-CTGF. 质粒pSOS-HUS-CTGF经过SfiⅠ酶切后分别与5条CTGF干扰序列和1条随机对照序列克隆获得pSOS-CTGFi1,pSOS-CTGFi2,pSOS-CTGFi3,pSOS-CTGFi4,pSOS-CTGFi5,pSOS-CTGFicontrol.质粒转入HEK293细胞中,于第1、3、5、7 d倒置荧光显微镜照相,IPP图像分析软件测定光强度值.5种CTGF特异RNA干扰载体的相对光强度分别为0.24%、41.47%、2.93%、20.40%和28.85%. 特异性序列site 1和site 3的沉默效果最好. pSOS-HUS系统采用双向启动子,通过绿色荧光蛋白下调水平来衡量干扰序列的沉默效果具有可靠直观重复性好的优点.利用该方法成功筛选了高沉默效果的CTGF特异RNA干扰序列.     

Using Next-Generation Sequencing and Bioinformatic Methods to Predict New Genes That May Be Regulated by CD47 in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the world, and the incidence and death rate of OSCC in men is twice that of women. CD47 is a ubiquitous cell surface transmembrane protein, also known as integrin-related protein (IAP). Previous studies have pointed out that CD47 can inhibit the growth of OSCC, but the detailed mechanism is not clear. This study aimed to explore the effect of CD47 gene expression profiles in OSCC. The OSCC cell lines, OECM-1 and OC-2, overexpressed CD47, and the expression profiles of mRNAs were analyzed through next-generation sequencing (NGS) with a bioinformatic approach. A total of 14 differentially expressed genes (DEGs) were listed. In addition, ingenuity pathway analysis (IPA) was used to analyze the molecular function (MF), biological process (BP), and cellular component (CC) network signaling. The human protein atlas (HPA) database was used to analyze gene expression and the survivability of human cancer. The results found that HSPA5, HYOU1, and PDIA4 were involved in the IPA network and when highly expressed, mediated the survivability of cancer. In addition, HSPA5 was positively and significantly correlated with CD47 expression (p < 0.0001) and induced by CD47-overexpression in the OECM-1 and OC-2 OSCC cancer cell lines. These findings provide important insights into possible new diagnostic strategies, including unfolded protein for OSCC-targeting CD47.     

High-Throughput Discovery of Mutations in Tef Semi-Dwarfing Genes by Next-Generation Sequencing Analysis

Tef (Eragrostis tef) is a major cereal crop in Ethiopia. Lodging is the primary constraint to increasing productivity in this allotetraploid species, accounting for losses of ∼15–45% in yield each year. As a first step toward identifying semi-dwarf varieties that might have improved lodging resistance, an ∼6× fosmid library was constructed and used to identify both homeologues of the dw3 semi-dwarfing gene of Sorghum bicolor. An EMS mutagenized population, consisting of ∼21,210 tef plants, was planted and leaf materials were collected into 23 superpools. Two dwarfing candidate genes, homeologues of dw3 of sorghum and rht1 of wheat, were sequenced directly from each superpool with 454 technology, and 120 candidate mutations were identified. Out of 10 candidates tested, six independent mutations were validated by Sanger sequencing, including two predicted detrimental mutations in both dw3 homeologues with a potential to improve lodging resistance in tef through further breeding. This study demonstrates that high-throughput sequencing can identify potentially valuable mutations in under-studied plant species like tef and has provided mutant lines that can now be combined and tested in breeding programs for improved lodging resistance.     

产蛋下降综合征病毒核酸J片段的确证和序列测定

产蛋下降综合征病毒核酸Ｊ片段的确证和序列测定张兹钧,胡木枝（拱北动植物检疫局,珠海５１９０２０）金由辛（中国科学院上海生物化学研究所分子生物学国家重点实验室,上海２０００３１）关键词产蛋下降综合征;Ｊ片段;ＤＮＡ片段产蛋下降综合征（ＥｇｇＤｒｏＰＳｙ...     

调节性T 细胞在发热结缔组织病患者外周血中的表达及临床意义

目的:研究Treg细胞在发热CTD患者外周血表达对结核感染的诊断价值。方法:对103例发热CTD患者进行T-SPOT.TB试验,将39例阳性者设为实验组-1,进行抗结核治疗,将64例阳性者设为实验组-2,另选取40例健康者作为对照组,检测三组外周血CD4+CD25+Treg细胞、Foxp3基因、IL-10、TGF-β的表达。结果:实验组CD4+CD25+Foxp3 Treg细胞占CD4+T比例高于对照组(P0.05),实验组-1治疗前外周血CD4+CD25+Foxp3 Treg细胞占CD4+T比例高于实验组-1治疗后、实验组-2(P0.05);实验组TGF-β表达量低于对照组(P0.05),实验组-1治疗前低于实验组-1治疗后及实验组-2(P0.05);实验组-1治疗前IL-10表达量低于实验组-1治疗后、实验组-2及对照组(P0.05)。结论:CD4+CD25+Foxp3 Treg细胞在发热CTD伴有结核感染患者外周血中的表达升高,其变化可作为结核感染诊断的辅助性指标。