Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method

A new algorithm was developed for the estimation of the metabolic flux distribution based on GC-MS data of proteinogenic amino acids. By using a sensitive GC-MS protocol as well as by combining the global search algorithm such as the genetic algorithm with the local search algorithm such as the Levenberg-Marquardt algorithm, not only the distribution of the net fluxes in the entire network, but also certain exchange fluxes which contribute significantly to the isotopomer distribution could be quantified. This mass isotopomer analysis could identify the biochemical changes involved in the regulation where acetate or glucose was used as a main carbon source. The metabolic flux analysis clearly revealed that when the specific growth rate increased, only a slight change in flux distribution was observed for acetate metabolism, indicating that subtle regulation mechanism exists in certain key junctions of this network system. Different from acetate metabolism, when glucose was used as a carbon source, as the growth rate increased, a significant increase in relative pentose phosphate pathway (PPP) flux was observed for Escherichia coli K12 at the expense of the citric acid cycle, suggesting that when growing on glucose, the flux catalyzed by isocitrate dehydrogenase could not fully fulfill the NADPH demand for cell growth, causing the oxidative PPP to be utilized to a larger extent so as to complement the NADPH demand. The GC-MS protocol as well as the new algorithm demonstrated here proved to be a powerful tool for characterizing metabolic regulation and can be utilized for strain improvement and bioprocess optimization.     

TSNAdb: A Database for Tumor-specific Neoantigens from Immunogenomics Data Analysis

Tumor-specific neoantigens have attracted much attention since they can be used as biomarkers to predict therapeutic effects of immune checkpoint blockade therapy and as potential targets for cancer immunotherapy. In this study, we developed a comprehensive tumor-specific neoantigen database (TSNAdb v1.0), based on pan-cancer immunogenomic analyses of somatic mutation data and human leukocyte antigen (HLA) allele information for 16 tumor types with 7748 tumor samples from The Cancer Genome Atlas (TCGA) and The Cancer Immunome Atlas (TCIA). We predicted binding affinities between mutant/wild-type peptides and HLA class I molecules by NetMHCpan v2.8/v4.0, and presented detailed information of 3,707,562/1,146,961 potential neoantigens generated by somatic mutations of all tumor samples. Moreover, we employed recurrent mutations in combination with highly frequent HLA alleles to predict potential shared neoantigens across tumor patients, which would facilitate the discovery of putative targets for neoantigen-based cancer immunotherapy. TSNAdb is freely available at http://biopharm.zju.edu.cn/tsnadb.     

PRIP (Phospholipase C-related but Catalytically Inactive Protein) Inhibits Exocytosis by Direct Interactions with Syntaxin 1 and SNAP-25 through Its C2 Domain

Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K⁺-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation.     

Predicting protein-protein interactions by combing various sequence- derived features into the general form of Chou's Pseudo amino acid composition

Knowledge of protein-protein interactions (PPIs) plays an important role in constructing protein interaction networks and understanding the general machineries of biological systems. In this study, a new method is proposed to predict PPIs using a comprehensive set of 930 features based only on sequence information, these features measure the interactions between residues a certain distant apart in the protein sequences from different aspects. To achieve better performance, the principal component analysis (PCA) is first employed to obtain an optimized feature subset. Then, the resulting 67-dimensional feature vectors are fed to Support Vector Machine (SVM). Experimental results on Drosophila melanogaster and Helicobater pylori datasets show that our method is very promising to predict PPIs and may at least be a useful supplement tool to existing methods.     

PSMD: An extensive database for pan‐species microsatellite investigation and marker development

Microsatellites are widely distributed throughout nearly all genomes which have been extensively exploited as powerful genetic markers for diverse applications due to their high polymorphisms. Their length variations are involved in gene regulation and implicated in numerous genetic diseases even in cancers. Although much effort has been devoted in microsatellite database construction, the existing microsatellite databases still had some drawbacks, such as limited number of species, unfriendly export format, missing marker development, lack of compound microsatellites and absence of gene annotation, which seriously restricted researchers to perform downstream analysis. In order to overcome the above limitations, we developed PSMD (Pan‐Species Microsatellite Database, http://big.cdu.edu.cn/psmd/ ) as a web‐based database to facilitate researchers to easily identify microsatellites, exploit reliable molecular markers and compare microsatellite distribution pattern on genome‐wide scale. In current release, PSMD comprises 678,106,741 perfect microsatellites and 43,848,943 compound microsatellites from 18,408 organisms, which covered almost all species with available genomic data. In addition to interactive browse interface, PSMD also offers a flexible filter function for users to quickly gain desired microsatellites from large data sets. PSMD allows users to export GFF3 formatted file and CSV formatted statistical file for downstream analysis. We also implemented an online tool for analysing occurrence of microsatellites with user‐defined parameters. Furthermore, Primer3 was embedded to help users to design high‐quality primers with customizable settings. To our knowledge, PSMD is the most extensive resource which is likely to be adopted by scientists engaged in biological, medical, environmental and agricultural research.     

Predictive value of circulating miR-328 and miR-134 for acute myocardial infarction

MicroRNA (miRNAs) is demonstrated to be present in the blood of humans and has been increasingly suggested as a novel biomarker for various pathological processes in the heart, including myocardial infarction, myocardial remodeling and progression to heart failure. In this study, we aim to evaluate the diagnostic and prognostic value of circulating miR-328 and miR-134 in patients with acute myocardial infarction (AMI). Circulating levels of miR-328 and miR-134 were detected by quantitative real-time PCR in plasma samples from 359 AMI patients and 30 healthy volunteers. Concentrations of high-sensitivity cardiac troponin T (hs-cTnT) were measured using electrochemiluminescence-based methods. MiRNAs were assessed for discrimination of a clinical diagnosis of AMI and for association with primary clinical endpoint defined as a composite of cardiogenic death and development of heart failure within 6 months after infarction. Results showed that levels of plasma miR-328 and miR-134 were significantly higher in AMI patients than in healthy controls. Receiver operating characteristic curve analyses showed significant diagnostic value of miR-328 and miR-134 for AMI. However, neither of them was superior to hs-cTnT for the diagnosis. Additionally, increased miRNA levels were strongly associated with increased risk of mortality or heart failure within 6 months for miR-328 (OR 7.35, 95 % confidence interval 1.07–17.83, P < 0.001) and miR-134 (OR 2.28, 95 % confidence interval 1.03–11.32 P < 0.001). In conclusion, circulating miR-328 and miR-134 could be potential indicators for AMI, and the miRNA levels are associated with increased risk of mortality or development of heart failure.     

奇异果甜蛋白及其基因工程

奇异果甜蛋白(thaumatin)是迄今为止最甜的物质之一,对其研究具有很重要的意义。奇异果甜蛋白的生化性质基本清楚,基因序列和氨基酸序列都已测定。它的甜味可能是由奇异果甜蛋白上特定基团和受体结合引起的。对奇异果甜蛋白的生理功能知之甚少。近二十年来,在奇异果甜蛋白的基因工程上取得了一定进展,但仍然存在许多困难。 Abstract:Thaumatin is one of the sweetest substances known to date,it is important to study the thaumatin.The biochemical properties of thaumatin have been clarified clearly.Thaumatin had been isolated and sequenced.The mechanism of the sweetness of thaumatin may be due to the combination of some special groups and the receptors.The exact function of thaumatin is still not clear.Although gene engineering of thaumatin has been carried out for 20 years,there are still some difficulties to be solved for using in the market.     

Inhibition of severe acute respiratory syndrome virus replication by small interfering RNAs in mammalian cells

Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease that spread worldwide in early 2003. The cause was determined as a novel coronavirus (CoV), SARS-associated CoV (SARS-CoV), with a single-stranded, plus-sense RNA. To date, no effective specific treatment has been identified. To exploit the possibility of using RNA interference as a therapeutic approach to fight the disease, plasmid-mediated small interfering RNAs (siRNAs) were generated to target the SARS-CoV genome. The expression of siRNAs from two plasmids, which specifically target the viral RNA polymerase, effectively blocked the cytopathic effects of SARS-CoV on Vero cells. These two plasmids also inhibited viral replication as shown by titer assays and by an examination of viral RNA and protein levels. Thus, our results demonstrated the feasibility of developing siRNAs as effective anti-SARS drugs.     

THE ULTRASTRUCTURE OF THE ENAMEL IN THE GIANT PANDA OF PLEISTOCENE

<正> Enamel ultrastructures in the molar teeth of the giant panda, including Ailuropoda microta of the Early Pleistocene, Ailuropoda melanoleuca bacont of the Middle and Late Pleistocene and a living form, Ailuropoda rnelanoleuca, have been investigated by scanning electron microscopy. Transverse and longitudinal sections of enamel were made in order co evaluate shape, size and arrangement of the prisms. The sections were etched then with 0.074 M H_3PO_4 for 30-60 sec. Our investigations have shown certain features of the enamel which allow us to recognize differences among Ailuropoda on the basis of examination of large areas of the enamel. The results are summarized below.     

Identification, characterization, and localization of a novel kidney polycystin-1-polycystin-2 complex

The functions of the two proteins defective in autosomal dominant polycystic kidney disease, polycystin-1 and polycystin-2, have not been fully clarified, but it has been hypothesized that they may heterodimerize to form a "polycystin complex" involved in cell adhesion. In this paper, we demonstrate for the first time the existence of a native polycystin complex in mouse kidney tubular cells transgenic for PKD1, non-transgenic kidney cells, and normal adult human kidney. Polycystin-1 is heavily N-glycosylated, and several glycosylated forms of polycystin-1 differing in their sensitivity to endoglycosidase H (Endo H) were found; in contrast, native polycystin-2 was fully Endo H-sensitive. Using highly specific antibodies to both proteins, we show that polycystin-2 associates selectively with two species of full-length polycystin-1, one Endo H-sensitive and the other Endo H-resistant; importantly, the latter could be further enriched in plasma membrane fractions and co-immunoprecipitated with polycystin-2. Finally, a subpopulation of this complex co-localized to the lateral cell borders of PKD1 transgenic kidney cells. These results demonstrate that polycystin-1 and polycystin-2 interact in vivo to form a stable heterodimeric complex and suggest that disruption of this complex is likely to be of primary relevance to the pathogenesis of cyst formation in autosomal dominant polycystic kidney disease.