DNA probes to identify members of the Anopheles farauti complex

DNA probes have been constructed to distinguish between the members of the Anopheles farauti complex of mosquitoes known as species numbers 1, 2 and 3. Partial genomic libraries of the three known species were exposed to labelled total genomic DNA from each species. Colonies showing differential hybridization were selected for further testing. These probes were found which allow identification of the three known species: probe pAf1 (160 bp fragment) hybridizes to DNA from An. farauti nos. 1 and 2; probe pAf2 (95 bp fragment) hybridizes to DNA from An. farauti no. 2 only; and probe pAf3 (1.3 kb fragment) hybridizes strongly to DNA from An. farauti no. 3, less to no. 1 and faintly to no. 2. Increasing the stringency of hybridization reduced the cross-hybridization of probes pAf1 and pAf3. Only radioactively labelled probes were tested. Males and females and individuals from diverse habitats and localities showed the same species/probe hybridization characteristics. This technique allows faster identification of the sibling species than previous methods, and has the added advantage that it allows air-dried and alcohol stored specimens to be identified.     

Engineering Streptomyces tenebrarius to synthesize single component of carbamoyl tobramycin

Aims: To engineer Streptomyces tenebrarius for producing carbamoyl tobramycin as a main component. Methods and Results: The aprH‐M gene fragment (apramycin biosynthetic gene from GenBank) in S. tenebrarius Tt49 was knocked out by genetic engineering to form S. tenebrarius T106 (△aprH‐M). Compared to the wild‐type strain, mutant strain T106 (△aprH‐M) no longer produced apramycin, while mainly synthesize carbamoyl tobramycin. TLC and HPLC‐MS analyses indicated that the mutant strain significantly increased the production of carbamoyl tobramycin. Conclusions: The metabolic flow for the apramycin and its analogues biosynthesis was blocked by disrupting the aprH‐M gene clusters. The aprH‐M gene clusters might be essential for the biosynthesis of apramycin. The mutant strain T106 mainly synthesized carbamoyl tobramycin. Significance and Impact of Study: The mutant T106 mainly produces carbamoyl tobramycin without synthesizing apramycin, which will reduce cost of postextraction from fermentation products. Therefore, it has good prospects for industrial application.     

Contributions of basic neurochemistry towards a novel concept of epilepsy

Epilepsy is an ancient disorder which treatment over the centuries has been guided by preconceptions regarding its origin. The major improvements in epilepsy management came following the discovery of the EEG and the development of seizure suppressing agents. These advances in diagnosis and anticonvulsant therapy have further ingrained the conviction that epilepsy is a disease of neurons. Evidence presented here is intended to support a different point of view which suggests that the metabolic modifications in epileptogenic tissue denote subtle alterations in the anatomical and biochemical relationship between neurons and their glial envelopes. As a result the extracellular environment of these cells contain higher than normal levels of glutamic acid. This creates an unnatural functional connectivity between neurons so that they establish abnormal synchronous activity between them and become hyperexcitable due to the depolarizing milieu. To compensate for these biochemical changes it is suggested that some thought might be given to epilepsy management by metabolic manipulation. The measures should be directed specifically towards improving the ability of glia to remove glutamic acid from the extracellular milieu. Two obvious possibilities are to enhance glial glutamine synthesis and to improve the interstitial wash-out of glutamic acid in epileptogenic epicenters. Such a therapy would anticipate to gradually diminish seizure incidence and susceptibility without, however, having a direct action on convulsive episodes per se. The approach must be considered an adjunct to current epilepsy treatment and not a substitute for the use of anticonvulsants.Special Issue Dedicated to Dr. Abel Lajtha.     

Whole genome DNA methylation profiling of oral cancer in ethnic population of Meghalaya,North East India reveals novel genes

Oral Squamous Cell Carcinoma (OSCC) is a serious and one of the most common and highly aggressive malignancies. Epigenetic factors such as DNA methylation have been known to be implicated in a number of cancer etiologies. The main objective of this study was to investigate physiognomies of Promoter DNA methylation patterns associated with oral cancer epigenome with special reference to the ethnic population of Meghalaya, North East India. The present study identifies 27,205 CpG sites and 3811 regions that are differentially methylated in oral cancer when compared to matched normal. 45 genes were found to be differentially methylated within the promoter region, of which 38 were hypermethylated and 7 hypomethylated. 14 of the hypermethylated genes were found to be similar to that of the TCGA-HNSCC study some of which are TSGs and few novel genes which may serve as candidate methylation biomarkers for OSCC in this poorly characterized ethnic group.     

Anisakiasis: preparation of a stable antigen for indirect fluorescent antibody test

By coupling Anisakis larval hemoglobin with cyanogen bromide-activated Sepharose, a stable and sensitive antigen for the indirect fluorescent antibody test for anisakiasis was prepared. Using this antigen, cross-reaction with other helminth infections was minimized and the test was greatly simplified.Furthermore, the results obtained indicate that this technique may be useful not only in the sero-diagnosis of anisakiasis but may also be applicable to the diagnosis of other helminthic diseases.     

Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

Genetic Relationships Among Five Basic Genomes St,E,A,B and D in Triticeae Revealed by Genomic Southern and in situ Hybridization

The St and E are two important basic genomes in the perennial tribe Triticeae (Poaceae). They exist in many perennialspecies and are very closely related to the A, B and D genomes of bread wheat (Triticum aestivum L.). Genomic Southernhybridization and genomic in situ hybridization (GISH) were used to analyze the genomic relationships between the twogenomes (St and E) and the three basic genomes (A, B and D) of T. aestivum. The semi-quantitative analysis of the Southernhybridization suggested that both St and E genomes are most closely related to the D genome, then the A genome, andrelatively distant to the B genome. GISH analysis using St and E genomic DNA as probes further confirmed the conclusion.St and E are the two basic genomes of Thinopyrum ponticum (StStE~eE~bE~x) and Th. intermedium (StE~eE~b), two perennialspecies successfully used in wheat improvement. Therefore, this paper provides a possible answer as to why most of thespontaneous wheat-Thinopyrum translocations and substitutions usually happen in the D genome, some in the A genomeand rarely in the B genome. This would develop further use of alien species for wheat improvement, especially thosecontaining St or E in their genome components.     

中红侧沟茧蜂化学感受蛋白MmedCSP1的结合特征

化学感受蛋白是一类存在于昆虫化学感受器中的可溶性蛋白, 被认为与昆虫识别外界化学信息有关。本研究使用pGEX-4T-1表达载体在BL21 (DE3)异源表达系统中表达中红侧沟茧蜂Microplitis mediator化学感受蛋白MmedCSP1, 并通过亲和层析法纯化得到去表达标签的MmedCSP1;使用bis-ANS作为荧光配基, 在荧光分光光度计上研究它与50种气味标样的结合特征, 从而得到此类化学感受蛋白在中红侧沟茧蜂嗅觉识别中识别气味的种类。结果表明: MmedCSP1只能与水杨酸甲酯、戊烷、罗勒烯、β-紫罗兰酮、3, 4-二甲基苯甲醛、2-己酮和叶醇结合。但只有脂类化合物β-紫罗兰酮能在浓度为1 mmol/L下将bis-ANS从MmedCSP1中替换50%, β-紫罗兰酮与MmedCSP1的结合常数为16.89 μmol/L。这些结果提示MmedCSP1参与中红侧沟茧蜂对水杨酸甲酯、戊烷、罗勒烯、β-紫罗兰酮、3, 4-二甲基苯甲醛、2-己酮和叶醇等气味的识别过程, 且在不同气味中的识别过程中对于气味的运输能力有差异。