Technical guide for genetic advancement of underdeveloped and intractable Clostridium

In recent years, the genus Clostridium has risen to the forefront of both medical biotechnology and industrial biotechnology owing to its potential in applications as diverse as anticancer therapy and production of commodity chemicals and biofuels. The prevalence of hyper-virulent strains of C. difficile within medical institutions has also led to a global epidemic that demands a more thorough understanding of clostridial genetics, physiology, and pathogenicity. Unfortunately, Clostridium suffers from a lack of sophisticated genetic tools and techniques which has hindered the biotechnological exploitation of this important bacterial genus. This review provides a comprehensive summary of biotechnological progress made in clostridial genetic tool development, while also aiming to serve as a technical guide for the advancement of underdeveloped clostridial strains, including recalcitrant species, novel environmental samples, and non-type strains. Relevant strain engineering techniques, from genome sequencing and establishment of a gene transfer methodology through to deployment of advanced genome editing procedures, are discussed in detail to provide a blueprint for future clostridial strain construction endeavors. It is expected that a more thorough and rounded-out genetic toolkit available for use in the clostridia will bring about the construction of superior bioprocessing strains and a more complete understanding of clostridial genetics, physiology, and pathogenicity.     

Finite element analysis of the biomechanical interaction between coronary sinus and proximal anchoring stent in coronary sinus annuloplasty

Recent clinical studies of the percutaneous transvenous mitral annuloplasty (PTMA) devices have shown a short-term reduction of mitral regurgitation after implantation. However, adverse events associated with the devices such as compression and perforation of vessel branches, device migration and fracture were reported. In this study, a finite element analysis was carried out to investigate the biomechanical interaction between the proximal anchor stent of a PTMA device and the coronary sinus (CS) vessel in three steps including: (i) the stent release and contact with the CS wall, (ii) the axial pull t the stent connector and (iii) the pressure inflation of the vessel wall. To investigate the impact of the material properties of tissues and stents on the interactive responses, the CS vessel was modelled with human and porcine material properties, and the proximal stent was modelled with two different Nitinol materials with one being stiffer than the other. The results indicated that the vessel wall stresses and contact forces imposed by the stents were much higher in the human model than the porcine model. However, the mechanical differences induced by the two stent types were relatively small. The softer stent exhibited a better fatigue safety factor when deployed in the human model than in the porcine model. These results underscored the importance of the CS tissue mechanical properties. Vessel wall stress and stent radial force obtained in the human model were higher than those obtained in the porcine model, which also brought up questions as to the validity of using the porcine model to assess device mechanical function. The quantification of these biomechanical interactions can offer scientific insight into the development and optimisation of the PTMA device design.     

Synthesis of some novel quinone diimine derivatives of benzo‐15‐crown‐5 for application in Hg2+ recognition

A series of novel fluoroionophore bearing derivatives of benzo‐15‐crown‐5 were synthesized by the amination of benzo‐15‐crown‐5 followed by condensation with different quinones in the presence of titanium tetrachloride (TiCl₄) and 1,4‐diazabicyclo‐[2.2.2]octane. The compounds were characterized by infrared, ¹H and ¹³C nuclear magnetic resonance, mass spectroscopy and elemental analysis. Absorption and fluorescence spectral characteristics of these compounds were studied. It was observed that the anthraquinone derivative was acting as an Hg²⁺ ion sensor. Copyright © 2013 John Wiley & Sons, Ltd.     

Detection of toxin translocation into the host cytosol by surface plasmon resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit(1). These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol(2-4). In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target(5). The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER(6-12). To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)(13-15). The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.     

Hexavalent Chromium Reduction and Accumulation by <Emphasis Type="Italic">Acinetobacter</Emphasis> AB1 Isolated from Fez Tanneries in Morocco

Hexavalent chromium reduction and accumulation by Acinetobacter AB1 isolated from Fez tanneries effluents were tested. The effects of some environmental factors such as pH, temperature, and exposure time on Cr(VI) reduction and resistance were investigated. We found that this strain was able to resist to concentrations as high as 400 mg/l of Cr(VI). Moreover, pH 10 and the temperature 30°C constitute favourable conditions to the growth and reduction of Acinetobacter AB1. Complete reduction of Cr(VI) was observed at low initial Cr(VI) concentrations of 50 mg/l after 72 h of incubation. Furthermore, Transmission electron microscope (TEM) analysis showed morphological changes in AB1 strain due 48H exposure to 100 mg/l chromate concentration and revealed circular electron dense (dark black point) inclusion within the cell cytoplasm suggesting chromium deposition within the cells.     

血栓弹力图指导支架内血栓后不稳定心绞痛治疗1 例及文献复习

目的:探讨在血栓弹力图监测血小板抑制率的情况下,调整氯吡格雷及阿司匹林用量,治疗冠心病、PCI术后支架内再发血栓患者的临床意义。方法:报告中国人民解放军总医院1例支架内亚急性血栓患者的临床资料并复习相关文献,对其临床表现、诊断、在血栓弹力图指导下的治疗进行分析。结果:1例支架内亚急性血栓患者经治疗病情好转出院,出院后继续调整氯吡格雷及阿司匹林用量,达到满意血小板抑制率,患者症状消失。结论:冠状动脉介入治疗后发生支架内血栓的患者,应用血栓弹力图指导氯吡格雷及阿司匹林用量,可达到令人满意的血小板抑制率,并防止出血情况发生。     

Amino-terminal domain stability mediates apolipoprotein E aggregation into neurotoxic fibrils

The three isoforms of apolipoprotein (apo) E are strongly associated with different risks for Alzheimer's disease: apoE4>apoE3>apoE2. Here, we show at physiological salt concentrations and pH that native tetramers of apoE form soluble aggregates in vitro that bind the amyloid dyes thioflavin T and Congo red. However, unlike classic amyloid fibrils, the aggregates adopt an irregular protofilament-like morphology and are seemingly highly alpha-helical. The aggregates formed at substantially different rates (apoE4>apoE3>apoE2) and were significantly more toxic to cultured neuronal cells than the tetramer. Since the three isoforms have large differences in conformational stability that can influence aggregation and amyloid pathways, we tested the effects of mutations that increased or decreased stability. Decreasing the conformational stability of the amino-terminal domain of apoE increased aggregation rates and vice versa. Our findings provide a new perspective for an isoform-specific pathogenic role for apoE aggregation in which differences in the conformational stability of the amino-terminal domain mediate neurodegeneration.     

Catalyst-induced growth with limited catalyst lifespan and competition

Spatially extended catalyst-induced growth processes are studied. This type of processes exists in all domains of biology, ranging from ecology (nutrients and growth), through immunology (antigens and lymphocytes) to molecular biology (signaling molecules initiating signaling cascades). The extinction-proliferation transition is considered for a system containing discrete catalysts (A) that induces the proliferation of a discrete reactant (B). The realization of this model on an infinite capacity d-dimensional discrete lattice for immortal catalysts has been previously considered (the AB model). It was shown that the adaptation of the reactants to the diffusive noise induced by stochastic fluctuations of catalyst density yields proliferation even if the average environmental conditions lead to extinction. This model is extended here to include more realistic situations, like finite lifespan of the catalysts and finite carrying capacity of the reactants. By using a combination of Monte Carlo simulation, percolation-theory-based estimations and an analytic perturbative analysis, the asymptotic behavior of these systems is studied. In both cases studied, it turns out that the overall survival of the reactant population at the long run is based on the size and shape of a typical single colony, related to the localized proliferation around spatio-temporal catalyst density fluctuations. If the density of these colonies (based on the lifetime of the spatial fluctuation and the carrying capacity of the medium) is large enough, i.e. above the percolation threshold, the reactant population survives even in (on average) hostile environment. This model provides a new insight on the population dynamics in chemical, biological and ecological systems.     

药物涂层球囊概述及其在外周动脉疾病治疗中的应用进展

经皮经腔血管成形术(PTA)已广泛用于外周动脉疾病(PAD)的治疗。然而,该技术存在血管壁弹性回缩和内膜增生等不足。PTA术后植入金属裸支架(BMS)虽然可以减少血管壁弹性回缩,但由此引起的支架内再狭窄(ISR)又成为治疗中的一个突出问题。药物洗脱支架(DES)被用来解决狭窄问题,但晚期支架内血栓形成(LST)、内皮化延迟和必须长期抗血小板治疗等问题也随之而来。在这样的背景下,药物涂层球囊(DCB)获得了快速发展。DCB作为非支架方案,可将所携载的活性药物转移至病变段血管壁,对ISR或原发病变均有较好的治疗效果。本文简要介绍了DCB的发展历史,并通过实验室研究、动物实验和临床试验,从机制上阐述涂层技术、涂层药物、赋形剂等对DCB功效和安全性的影响以及DCB在PAD治疗中的应用进展。