Collagen-binding Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) of Gram-positive Bacteria Inhibit Complement Activation via the Classical Pathway

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r₂C1s₂ was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.     

Conformational stability of triazolyl functionalized collagen triple helices

Functionalized collagen is attractive for the development of synthetic biomaterials. Herein we present the functionalization of azidoproline containing collagen model peptides with various alkynes using click chemistry. The influence on the stability of the collagen triple helix of the stereochemistry of the introduced triazolyl prolines (4R or 4S), the position of their incorporation (Xaa or Yaa) and the substituents attached to them are shown. The results provide a useful guide for the optimal functionalization of collagen using click chemistry.     

Bioavailable affinity label for collagen prolyl 4-hydroxylase

Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification catalyzed by the nonheme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report that 4-oxo-5,6-epoxyhexanoate, a mimic of the α-ketoglutarate co-substrate, inactivates human P4H. The inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caenorhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H. Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treatment of a variety of fibrotic diseases.     

抑制瘢痕细胞中串珠素表达对Ⅰ型胶原蛋白生成量的影响

目的：硬膜外瘢痕,又叫硬膜外纤维化,是指在硬膜外腔的手术涉及范围内形成的瘢痕组织或纤维化,是机体对创伤的修复反应。瘢痕的粘连和收缩会牵拉硬膜和神经根,限制其活动,被瘢痕包绕的神经根受到非正常的牵拉和挤压,神经纤维的轴浆运输、动脉血供、静脉回流受阻,神经根和背侧神经节对机械压迫很敏感,会产生一系列症状,如疼痛、麻木及下肢肌力降低等。近年来,对硬膜外瘢痕防治的研究大多是椎板切除术后如何通过物理或化学屏障来减少术后因瘢痕粘连导致的并发症。但对通过瘢痕形成过程中抑制其主要构成成分的生成来减轻椎板切除术后硬膜外瘢痕形成的相关研究还较少。通过减少椎板切除术后硬膜外瘢痕主要成分Ⅰ型胶原蛋白的生成来实现抑制椎板切除术后硬膜外瘢痕的形成。方法：选用30只250克两月鼠龄的SD雄性大鼠随机按1、2、3、4、5、6周分为6组,行后路4、5腰椎全椎板切除术。术后1、2、3、4、5、6周时每周取一组大鼠全锥板切除术后硬膜后方瘢痕组织,分别行病理切片HE染色,组织块贴壁法细胞培养。筛选第三周瘢痕组织培养的成纤维细胞进行慢病毒干扰串珠素表达并设对照组,通过Western-blot、RT-PCR分析Ⅰ型胶原蛋白生成量与对照组的差别并进行统计学分析。结果：慢病毒干扰小组Ⅰ型胶原蛋白生成量较对照组及纯病毒组明显减少（RT-PCR F=509.331,q A,B=-43.371,P〈0.01,q A,C=-46.133,P〈0.01,Western-Blot F=337.578;q A,B=-112.433,P〈0.01,q A,C=-89.227,P〈0.01）。结论：干扰串珠素表达能有效减少术后硬膜外瘢痕成纤维细胞生成Ⅰ型胶原蛋白,对抑制椎板切除术后硬膜外瘢痕形成应有一定作用。通过慢病毒介导的shRNA干扰成纤维细胞中的串珠素后,其生成的Ⅰ型胶原蛋白量与对照组相比较差异有统计学意义（P〈0.05）,这说明通过抑制瘢痕成纤维细胞的串珠素表达能够有效减少Ⅰ型胶原蛋白的生成。这种方法不论从Ⅰ型胶原蛋白是瘢痕主要构成成分方面,还是Ⅰ型胶原蛋白在瘢痕生成过程中分泌胶原中占得比例增多导致机体由胎儿期的无瘢痕愈合转化至成体的瘢痕愈合这个方面来将,理论上都能够做到有效地抑制、减少硬膜外瘢痕的形成,因此通过干扰硬膜外成纤维细胞串珠素表达从而达到抑制硬膜外瘢痕的形成这一理论是可行的,为进一步进行椎板切除术后抑制硬膜外瘢痕形成的体内试验奠定了理论基础。     

Controlled formaldehyde fixation of fibronectin layers for expansion of mesenchymal stem cells

Extracellular cell matrices deposited by cells stimulate cell proliferation. However, their generation is cumbersome and time consuming. We show here that controlled fixation of fibronectin layers after coating culture vessels significantly enhances expansion of murine and human mesenchymal stem cells (MSCs) and, to a lesser extent, primary fibroblasts. In contrast, fibronection fixation did not stimulate proliferation of established cancer cell lines. Fixed vitronectin or collagen IV layers also enhanced proliferation of murine MSCs. Thus, controlled formaldehyde fixation of layers formed by fibronectin or some other extracellular matrix components represents a simple and reproducible way to enhance proliferation of primary cells.     

胃癌患者Sox2蛋白和CTHRC-1蛋白的表达与肿瘤发展密切相关

本研究选取病理科收集的自2016年1月至2016年12月的90例术后胃癌组织标本(胃癌组)、45例癌旁组织标本(对照组),采用免疫组化染色法检测两组标本中的Y框蛋白(Sox2)、胶原三股螺旋重叠蛋白-1表达,并分析其与肿瘤TNM分期、淋巴结转移、浸润深度、分化程度的关系,采用Spearman秩相关检验检测两种蛋白的相互关系,试图探讨胃癌患者癌组织中性别决定区Y框蛋白(Sox2)、胶原三股螺旋重叠蛋白-1(CTHRC-1)的表达及其相关性。研究结果表明:胃癌组的Sox2蛋白阳性表达率63.33%显著低于对照组的93.33%(p<0.05),胃癌组的CTHRC-1蛋白阳性表达率78.89%显著高于对照组的24.44%(p<0.05);胃癌组的Sox2蛋白阳性表达与胃癌的分化程度、发生淋巴结转移具有相关性(p<0.05);胃癌组的CTHRC-1蛋白阳性表达与胃癌的淋巴结转移、TNM分期、肿瘤浸润深度具有显著的相关性(p<0.05);胃癌组的Sox2蛋白与CTHRC-1蛋白呈显著的负相关表达(Spearman相关系数r=-0.322, p=0.002<0.05)。本研究的初步结论表明:胃癌组织中Sox2蛋白低表达和CTHRC-1蛋白高表达,并且与肿瘤发展密切相关,且二者表达呈负相关。     

Transforming growth factor-beta1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation

Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-beta1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-beta1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-beta1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-beta1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression.     

How does the annelid Alvinella pompejana deal with an extreme hydrothermal environment?

Alvinella pompejana, the so-called Pompeii worm (Desbruyères and Laubier, 1980), is found exclusively in association to high temperature venting, at the surface of hydrothermal chimneys of the East Pacific Rise. The main characteristics of this emblematic species is its tolerance to high temperature but its ability to colonize extremely hot substrates has been the subject of much controversy. In the last decade, new tools allowing in situ and in vivo investigation have been determinant in the understanding of the strategies and adaptations required to colonize such an extremely hot environment. New data relative to the characterization of the animal habitat conditions, on one hand, to the molecular adaptations of this organism and the colonization processes by this species, on the other hand, are now available. Advanced methods and tools, that have fostered the physico-chemical characterization of vent habitats in recent years, are first reviewed. Factors controlling the physico-chemical variability of vent habitats and the threats A. pompejana might effectively face are discussed. The exceptional thermotolerance of this species and the maximum temperature it could sustain are then considered in the light of molecular data relative to its collagen stability. Life history traits as well as biological controls on tube micro-habitat conditions are discussed on the basis of new in situ and in vivo experiments and characterization. Finally, the current knowledge and opened questions related to the molecular adaptations to chemical stresses are briefly stated. The ability of Alvinella pompejana to colonize these substrates is far from being fully understood, but the exceptional properties of its extracellular biopolymers and the behavior of the worm can be now considered as major clues in the colonization process. Alvinella pompejana could thus stand at the limits authorized for its biological machinery in a highly dynamic environment where temperature can readily reach lethal values, but where temperature regulation by the animal itself would prevent exposure to deleterious thermal spikes. The dynamic system associating this pioneer species and its associated microflora might be viewed as a key to the subsequent colonization of these environments by less tolerant species, highlighting A. pompejana as a new type of ecosystem bioengineer.     

Fathoming Aspergillus oryzae metabolomes in formulated growth matrices

The stochasticity of Aspergillus oryzae (Trivially: the koji mold) pan-metabolomes commensurate with its ubiquitously distributed landscapes, i.e. growth matrices have been seemed uncharted since its food fermentative systems are mostly being investigated. In this review, we explicitly have discussed the likely tendencies of A. oryzae metabolomes pertaining to its growth milieu formulated with substrate matrices of varying nature, composition, texture, and associated physicochemical parameters. We envisaged typical food matrices, namely, meju, koji, and moromi as the semi-natural cultivation models toward delineating the metabolomic patterns of the koji mold, which synergistically influences the organoleptic and functional properties of the end products. Further, we highlighted how tailored conditions in sub-natural growth matrices, i.e. synthetic cultivation media blends, inducers, and growth surfaces, may influence A. oryzae metabolomes and targeted phenotypes. In general, the sequential or synchronous growth of A. oryzae on formulated matrices results in a number of metabolic tradeoffs with its immediate microenvironment influencing its adaptive and regulatory metabolomes. In broader context, evaluating the metabolic plasticity of A. oryzae relative to the tractable variables in formulated growth matrices might help approximate its growth and metabolism in the more complex natural matrices and environs. These approaches may considerably help in the design and manipulation of hybrid cultivation systems towards the efficient harnessing of commercial molds.     

Effect of haemin on enzyme activity and cytotoxin production by Bacteriodes gingivalis W50

Abstract Bacteriodes gingivalis W50 was grown in a chemostat at pH 7.5 under haemin-limited and haemin excess conditions in order to provide cells with a known high or low inefectivity and virulence for mice, respectively. The activities of enzymes and formation of cytotoxic fermentation products by these cells were compared. No significant differences in chymotrypsin activity or in the ability to degrade hyaluronic acid were found; neither were there any significant differences in the production of butyrate, propionate or succinate. At pH 7.5, trypsin activity was 3.5-fold higher in cells grown under haemin excess conditions whereas collagenolytic activity was nearly 3-fold higher in haemin-limited cells. Although collagenolytic activity may be important in tissue damage, a high ratio of trypsin to collagen breakdown activities was associated with virulent cells grown under an excess of haemin.