Comparing methods for metabolic network analysis and an application to metabolic engineering

Bioinformatics tools have facilitated the reconstruction and analysis of cellular metabolism of various organisms based on information encoded in their genomes. Characterization of cellular metabolism is useful to understand the phenotypic capabilities of these organisms. It has been done quantitatively through the analysis of pathway operations. There are several in silico approaches for analyzing metabolic networks, including structural and stoichiometric analysis, metabolic flux analysis, metabolic control analysis, and several kinetic modeling based analyses. They can serve as a virtual laboratory to give insights into basic principles of cellular functions. This article summarizes the progress and advances in software and algorithm development for metabolic network analysis, along with their applications relevant to cellular physiology, and metabolic engineering with an emphasis on microbial strain optimization. Moreover, it provides a detailed comparative analysis of existing approaches under different categories.     

Construction and elementary mode analysis of a metabolic model for Shewanella oneidensis MR-1

A stoichiometric model describing the central metabolism of Shewanella oneidensis MR-1 wild-type and derivative strains was developed and used in elementary mode analysis (EMA). Shewanella oneidensis MR-1 can anaerobically respire a diverse pool of electron acceptors, and may be applied in several biotechnology settings, including bioremediation of toxic metals, electricity generation in microbial fuel cells, and whole-cell biocatalysis. The metabolic model presented here was adapted and verified by comparing the growth phenotypes of 13 single- and 1 double-knockout strains, while considering respiration via aerobic, anaerobic fumarate, and anaerobic metal reduction (Mtr) pathways, and utilizing acetate, n-acetylglucosamine (NAG), or lactate as carbon sources. The gene ppc, which encodes phosphoenolpyruvate carboxylase (Ppc), was determined to be necessary for aerobic growth on NAG and lactate, while not essential for growth on acetate. This suggests that Ppc is the only active anaplerotic enzyme when cultivated on lactate and NAG. The application of regulatory and substrate limitations to EMA has enabled creation of metabolic models that better reflect biological conditions, and significantly reduce the solution space for each condition, facilitating rapid strain optimization. This wild-type model can be easily adapted to include utilization of different carbon sources or secretion of different metabolic products, and allows the prediction of single- and multiple-knockout strains that are expected to operate under defined conditions with increased efficiency when compared to wild type cells.     

Musculoskeletal determinants of pelvic sucker function in hawaiian stream gobiid fishes: Interspecific comparisons and allometric scaling

Gobiid fishes possess a distinctive ventral sucker, formed from fusion of the pelvic fins. This sucker is used to adhere to a wide range of substrates including, in some species, the vertical cliffs of waterfalls that are climbed during upstream migrations. Previous studies of waterfall‐climbing goby species have found that pressure differentials and adhesive forces generated by the sucker increase with positive allometry as fish grow in size, despite isometry or negative allometry of sucker area. To produce such scaling patterns for pressure differential and adhesive force, waterfall‐climbing gobies might exhibit allometry for other muscular or skeletal components of the pelvic sucker that contribute to its adhesive function. In this study, we used anatomical dissections and modeling to evaluate the potential for allometric growth in the cross‐sectional area, effective mechanical advantage (EMA), and force generating capacity of major protractor and retractor muscles of the pelvic sucker (m. protractor ischii and m. retractor ischii) that help to expand the sealed volume of the sucker to produce pressure differentials and adhesive force. We compared patterns for three Hawaiian gobiid species: a nonclimber (Stenogobius hawaiiensis), an ontogenetically limited climber (Awaous guamensis), and a proficient climber (Sicyopterus stimpsoni). Scaling patterns were relatively similar for all three species, typically exhibiting isometric or negatively allometric scaling for the muscles and lever systems examined. Although these scaling patterns do not help to explain the positive allometry of pressure differentials and adhesive force as climbing gobies grow, the best climber among the species we compared, S. stimpsoni, does exhibit the highest calculated estimates of EMA, muscular input force, and output force for pelvic sucker retraction at any body size, potentially facilitating its adhesive ability. J. Morphol. 2013. © 2013 Wiley Periodicals, Inc.     

基于EMA-qPCR的茄科青枯菌活体检测技术的建立

【目的】利用特异性核酸染料叠氮溴乙锭(Ethidium monoazide bromide, EMA)与实时荧光定量PCR技术相结合, 建立一种能有效区分青枯菌死活细胞的检测方法。【方法】样品DNA制备前经EMA渗透预处理, 再进行实时荧光定量PCR特异扩增菌体DNA。【结果】终浓度为2.0 mg/L的EMA能有效排除1.0×107 CFU/mL灭活青枯菌细胞DNA的扩增, 对活细胞和不可培养状态(Viable but non-culturable, VBNC)活菌的DNA扩增均没有影响。当每个定量PCR反应体系中的活细胞在5.0×100?5.0×104 CFU范围内时, 扩增Ct值与定量PCR反应体系中活细胞CFU对数值呈良好的负相关性(R2=0.992 5)。比较EMA-qPCR法和平板计数法对经过不同温度短期保存的青枯菌检测结果发现, 待检样品可在24 °C与4 °C冷藏条件下短期保存。【结论】本研究建立的EMA-qPCR方法能有效检测青枯菌VBNC细胞和有效区分死活菌, 避免或减少青枯菌PCR检测的假阳性和假阴性。     

History of gene therapy

Two decades after the initial gene therapy trials and more than 1700 approved clinical trials worldwide we not only have gained much new information and knowledge regarding gene therapy in general, but also learned to understand the concern that has persisted in society. Despite the setbacks gene therapy has faced, success stories have increasingly emerged. Examples for these are the positive recommendation for a gene therapy product (Glybera) by the EMA for approval in the European Union and the positive trials for the treatment of ADA deficiency, SCID-X1 and adrenoleukodystrophy. Nevertheless, our knowledge continues to grow and during the course of time more safety data has become available that helps us to develop better gene therapy approaches. Also, with the increased understanding of molecular medicine, we have been able to develop more specific and efficient gene transfer vectors which are now producing clinical results.     

基于能源代谢分析的南昌市能源消费碳排放综合生态效率研究

有关能源消费的代谢机理及生态效率研究是当前生态经济研究领域中的重点和难点,也是政府决策部门、相关行业及人群关注的热点。基于能源代谢分析理念,构建了基于相对变量的城市能源消费碳排放综合生态效率度量模型,对2000—2013年南昌市能源消费结构、碳排放量及其生态效率进行了测算与分析。结果表明:(1)南昌市在2000—2013年,能源消费量和碳排放量整体上呈"N"型曲线上升的一致性变化特征,主要经历了快速增长、短暂下降、恢复平稳增长3个阶段,且煤炭是南昌市能源消费和碳排放的主要来源,短时期内难以改变;(2)南昌市的能源消费效率和碳排放效率整体上在不断优化;(3)南昌市能源消费碳排放综合生态效率经历了较大的波动变化,整体上的生态效率分值并不高;仍需要加大节能减排力度,全面优化南昌市能源消费结构。     

Waiving in vivo studies for monoclonal antibody biosimilar development: National and global challenges

Biosimilars are biological medicinal products that contain a version of the active substance of an already authorised original biological medicinal product (the innovator or reference product). The first approved biosimilar medicines were small proteins, and more recently biosimilar versions of innovator monoclonal antibody (mAb) drugs have entered development as patents on these more complex proteins expire. In September 2013, the first biosimilar mAb, infliximab, was authorised in Europe. In March 2015, the first biosimilar (Zarxio?, filgrastim-sndz, Sandoz) was approved by the US Food and Drug Administration; however, to date no mAb biosimilars have been approved in the US. There are currently major differences between how biosimilars are regulated in different parts of the world, leading to substantial variability in the amount of in vivo nonclinical toxicity testing required to support clinical development and marketing of biosimilars. There are approximately 30 national and international guidelines on biosimilar development and this number is growing. The European Union's guidance describes an approach that enables biosimilars to enter clinical trials based on robust in vitro data alone; in contrast, the World Health Organization's guidance is interpreted globally to mean in vivo toxicity studies are mandatory.

We reviewed our own experience working in the global regulatory environment, surveyed current practice, determined drivers for nonclinical in vivo studies with biosimilar mAbs and shared data on practice and study design for 25 marketed and as yet unmarketed biosimilar mAbs that have been in development in the past 5y. These data showed a variety of nonclinical in vivo approaches, and also demonstrated the practical challenges faced in obtaining regulatory approval for clinical trials based on in vitro data alone. The majority of reasons for carrying out nonclinical in vivo studies were not based on scientific rationale, and therefore the authors have made recommendations for a data-driven approach to the toxicological assessment of mAb biosimilars that minimises unnecessary use of animals and can be used across all regions of the world.     

Urine as a source for clinical proteome analysis: From discovery to clinical application

The success of clinical proteome analysis should be assessed based on the clinical impact following implementation of findings. Although there have been several technological advancements in mass spectrometry in the last years, these have not resulted in similar advancements in clinical proteomics. In addition, application of proteomic biomarkers in clinical diagnostics and practical improvement in the disease management is extremely rare. In this review, we discuss the relevant issues associated with identification of robust biomarkers of clinical value. Urine appears to be an ideal source of biomarkers, for theoretical, methodological, and practical reasons. Therefore, this review is focused on the search for biomarkers in urine within the last decade. Urine can be used for non-invasive assessment of a variety of diseases including those affecting the urogenital tract and also other pathologies such as cardiovascular disease or appendicitis. We also discuss the importance of data validation, an essential step in translating biomarkers into the clinical practice. Furthermore, we examine several examples of apparently successful proteomic biomarker discovery studies and their implications for disease diagnosis, prognosis, and therapy evaluation. We also discuss some current challenges in this field and reflect on future research prospects. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Rapid detection of viable bacteria by nested polymerase chain reaction via long DNA amplification after ethidium monoazide treatment

In assays to determine whether viable cells of Enterobacteriaceae are present in pasteurized milk, the typical ethidium monoazide (EMA) polymerase chain reaction (PCR) targets a short stretch of DNA. This process often triggers false-positive results owing to the high level of dead cells of Enterobacteriaceae that had initially contaminated the sample. We have developed a novel, direct, real-time PCR that does not require DNA isolation (DQ-PCR) to detect low levels of cells of Enterobacteriaceae regardless of live and dead cells first. We confirmed that the DQ-PCR targeting a long DNA (the 16S ribosomal RNA [rRNA] gene, amplified length of 1514 bp) following EMA treatment is a promising tool to detect live bacteria of all genera owing to the complete suppression of background signal from high levels of dead bacteria in pasteurized milk. However, when identifying viable bacteria in pasteurized milk, commercial PCR primers designed for detecting long stretches of DNA are generally not available. Thus, we treated samples with EMA and then carried out an initial round of PCR of a long stretch of DNA (16S gene, 1514 bp). We then performed another round of PCR, a novel nested PCR to generate short products using commercial primers. This procedure resulted in the rapid detection of low levels of viable cells of Enterobacteriaceae.