Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

<p>Abstract</p> <p>Background</p> <p>Fumarase catalyzes the reversible hydration of fumarate to <smcaps>L</smcaps>-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of <smcaps>L</smcaps>-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications.</p> <p>Results</p> <p>A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from <it>Bacteroides </it>sp. 2_1_33B and <it>Parabacteroides distasonis </it>ATCC 8503 (26% identical and 43% similar). The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in <it>E. coli </it>BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form <smcaps>L</smcaps>-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg²⁺. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: <it>K</it>_m= 0.48 mM, <it>V</it>_max= 827 μM/min/mg, and <it>k</it>_cat/<it>K</it>_m= 1900 mM/s.</p> <p>Conclusions</p> <p>We isolated a novel fumarase gene, <it>fumF</it>, from a sequence-based screen of a plasmid metagenomic library from uncultivated marine microorganisms. The properties of FumF protein may be ideal for the industrial production of <smcaps>L</smcaps>-malate under higher temperature conditions. The identification of FumF underscores the potential of marine metagenome screening for novel biomolecules.</p

Thymosin alpha 1 in the prevention of infected pancreatic necrosis following acute necrotising pancreatitis (TRACE trial): protocol of a multicentre, randomised, double-blind, placebo-controlled, parallel-group trial

Introduction Infected pancreatic necrosis (IPN) and its related septic complications are the major causes of death in patients with acute necrotising pancreatitis (ANP). Therefore, the prevention of IPN is of great clinical value, and immunomodulatory therapy with thymosin alpha 1 may be beneficial. This study was designed to test the hypothesis that the administration of thymosin alpha 1 during the acute phase of ANP will result in a reduced incidence of IPN. Methods and analysis This is a randomised, multicentre, double-blind, placebo-controlled study. 520 eligible patients with ANP will be randomised in a 1:1 ratio to receive either the thymosin alpha 1 or the placebo using the same mode of administration. The primary endpoint is the incidence of IPN during the index admission. Most of the secondary endpoints will be registered within the index admission including in-hospital mortality, the incidence of new-onset organ failure and new-onset persistent organ failure (respiration, cardiovascular and renal), receipt of new organ support therapy, requirement for drainage or necrosectomy, bleeding requiring intervention, human leucocyte antigens-DR(HLA-DR) on day 0, day 7, day 14, and so on and adverse events. Considering the possibility of readmission, an additional follow-up will be arranged 90 days after enrolment, and IPN and death at day 90 will also be served as secondary outcomes. Ethics and dissemination This study was approved by the ethics committee of Jinling Hospital, Nanjing University (Number 2015NZKY-004-02). The thymosin alpha 1 in the prevention of infected pancreatic necrosis following acute necrotising pancreatitis(TRACE) trial was designed to test the effect of a new therapy focusing on the immune system in preventing secondary infection following ANP. The results of this trial will be disseminated in peer-reviewed journals and at scientific conferences. Trial registration number ClinicalTrials.gov Registry (NCT02473406)

A Novel Xenograft Model in Zebrafish for High-Resolution Investigating Dynamics of Neovascularization in Tumors

Tumor neovascularization is a highly complex process including multiple steps. Understanding this process, especially the initial stage, has been limited by the difficulties of real-time visualizing the neovascularization embedded in tumor tissues in living animal models. In the present study, we have established a xenograft model in zebrafish by implanting mammalian tumor cells into the perivitelline space of 48 hours old Tg(Flk1:EGFP) transgenic zebrafish embryos. With this model, we dynamically visualized the process of tumor neovascularization, with unprecedented high-resolution, including new sprouts from the host vessels and the origination from VEGFR2+ individual endothelial cells. Moreover, we quantified their contributions during the formation of vascular network in tumor. Real-time observations revealed that angiogenic sprouts in tumors preferred to connect each other to form endothelial loops, and more and more endothelial loops accumulated into the irregular and chaotic vascular network. The over-expression of VEGF165 in tumor cells significantly affected the vascularization in xenografts, not only the number and size of neo-vessels but the abnormalities of tumor vascular architecture. The specific inhibitor of VEGFR2, SU5416, significantly inhibited the vascularization and the growth of melanoma xenografts, but had little affects to normal vessels in zebrafish. Thus, this zebrafish/tumor xenograft model not only provides a unique window to investigate the earliest events of tumoral neoangiogenesis, but is sensitive to be used as an experimental platform to rapidly and visually evaluate functions of angiogenic-related genes. Finally, it also offers an efficient and cost-effective means for the rapid evaluation of anti-angiogenic chemicals

Control of a class of chaotic systems by a stochastic delay method

summary:A delay stochastic method is introduced to control a certain class of chaotic systems. With the Lyapunov method, a suitable kind of controllers with multiplicative noise is designed to stabilize the chaotic state to the equilibrium point. The method is simple and can be put into practice. Numerical simulations are provided to illustrate the effectiveness of the proposed controllable conditions

Variational Iteration Method for Singular Perturbation Initial Value Problems with Delays

The variational iteration method (VIM) is applied to solve singular perturbation initial value problems with delays (SPIVPDs). Some convergence results of VIM for solving SPIVPDs are given. The obtained sequence of iterates is based on the use of general Lagrange multipliers; the multipliers in the functionals can be identified by the variational theory. Moreover, the numerical examples show the efficiency of the method

Benchmarking the Memory Hierarchy of Modern GPUs

Part 2: Parallel and Multi-Core TechnologiesInternational audienceMemory access efficiency is a key factor for fully exploiting the computational power of Graphics Processing Units (GPUs). However, many details of the GPU memory hierarchy are not released by the vendors. We propose a novel fine-grained benchmarking approach and apply it on two popular GPUs, namely Fermi and Kepler, to expose the previously unknown characteristics of their memory hierarchies. Specifically, we investigate the structures of different cache systems, such as data cache, texture cache, and the translation lookaside buffer (TLB). We also investigate the impact of bank conflict on shared memory access latency. Our benchmarking results offer a better understanding on the mysterious GPU memory hierarchy, which can help in the software optimization and the modelling of GPU architectures. Our source code and experimental results are publicly available

Interventional Treatment of Iatrogenic False Aneurysm of Subclavian Artery Combine with Arteriovenous Malformation

Abstract: Subclavian artery pseudo-aneurysm is a rare disease, we report a case combine with arteriovenous malformation. She received failed covered stent implantation in subclavian artery before interventional embolization. And we makes a brief literature review on interventional therapy of false aneurysm of subclavian artery

Study on CO2 bubble growth and detachment in liquid steel induced by thermal decomposition reaction with physical modeling

The bubbles have been widely applied in industrial processing, including steelmaking. Recently, industrial trials of natively generated bubbles via decomposition reactions have been successfully adopted in steel refining and inclusion removal. The refining efficiency was proved to be outstanding due to the small bubble sizes and the subsequent satisfying dynamic conditions. However, the bubble behaviors, including growth and detachment, are still not thoroughly clarified due to the strict requirements for observation conditions. Therefore, the growth and detachment behavior of the bubbles generated by decomposition reactions were carefully observed with physical modeling and shadow imaging method. The detachment sizes of the bubbles were extracted accordingly. Based on the statistical data on the detachment bubble sizes, a traditional nozzle flow model was modified for the prediction of the bubble detachment size in decomposition reactions. This work provides the foundation for further studies on the interaction mechanism between the natively generated bubbles and the impurities in liquid steel in terms of inclusion removal