In silico identification and structural features of six new genes similar to MATER specifically expressed in the oocyte

In the present work, we have used the in silico subtraction methodology to identify six new mouse genes similar to NALP5/MATER, whose ESTs were represented almost exclusively in egg libraries. Five genes were selected for RT-PCR and/or in situ hybridization. These experiments confirmed their oocyte restricted expression. Five of these genes are localized on mouse chromosome 7, as is NALP5/MATER; among them, three are localized in a 300 kb cluster.     

Human acidic mammalian chitinase as a novel target for anti-asthma drug design using in silico screening

Human acidic mammalian chitinase (hAMCase) was recently shown to be involved in the development of asthma, suggesting a possible application for hAMCase inhibitors as novel therapeutic agents for asthma. We therefore initiated drug discovery research into hAMCase using a combination of in silico methodologies and a hAMCase assay system. We first selected 23 candidate hAMCase inhibitors from a database of four million compounds using a multistep screening system combining Tripos Topomer Search technology, a docking calculation and two-dimensional molecular similarity analysis. We then measured hAMCase inhibitory activity of the selected compounds and identified seven compounds with IC₅₀ values ?100 μM. A model describing the binding modes of these hit compounds to hAMCase was constructed, and we discuss the structure–activity relationships of the compounds we identified, suggested by the model and the actual inhibitory activities of the compounds.     

Modeling the interaction of gametes and embryos with the maternal genital tract: From in vivo to in silico

Understanding the complex interaction between gametes or embryos and the maternal genital tract requires the use of experimental models. The selection of the right model is an important task to undertake, and despite many new developments in this area, an ideal model system has not yet been developed. In this review article, we focus on how the most appropriate model species and model system can be selected, each with its particular advantages and disadvantages. Selection criteria need to be based on the evaluation of the aim of the experiment, the tools that are available to the scientist, and the ethics that are involved in working with particular animal species and model systems. Society and politics direct scientists to “Refine, Reduce, and Replace” the use of experimental animals, which means that the use of in vivo models is increasingly being discouraged. An in vivo model allows experimentation in the full biological environment of a living organism. In contrast with in vivo models, in vitro models are less complex and are abstracts of in vivo systems, leading often to results that are different from the in vivo situation. If an investigator could understand all the components of a complex biological system and re-create them as individual smaller models in a computer, he or she could create in silico models that would completely represent the complexity of in vivo models. We predict that in the future, in silico modeling will be the natural departure from in vivo, in situ, and in vitro modeling approaches. In addition to numerous advantages that this modeling approach can bring to studying maternal interaction with gametes and embryo, it is perhaps the only true alternative method to animal experimentation.     

Genomics of steroid hormones: in silico analysis of nucleotide sequence variants (polymorphisms) of the enzymes involved in the biosynthesis and metabolism of steroid hormones

Alterations of steroid hormone biosynthesis and metabolism are suspected to be involved in the pathogenesis of several diseases. Several polymorphisms of the enzymes involved in these processes have already been described and some could be associated with certain diseases. We attempted to examine the sequence variants of these genes in order to find novel variants by an in silico analysis. We analyzed the known human nucleotide sequences of the enzymes p450 side-chain cleavage enzyme, steroid 17-alpha-hydroxylase/17,20-lyase, 3-beta-hydroxysteroid dehydrogenase types 1 and 2, 21-hydroxylase, 11-beta-hydroxylase, aldosterone synthase, aromatase, 11-beta-hydroxysteroid dehydrogenase types 1 and 2, steroid 5-alpha-reductase types 1 and 2, steroid 5-beta-reductase, dehydroepiandrosterone sulfotransferase, 17-beta-hydroxysteroid dehydrogenase types 1–3. The analysis was performed using the National Center for Biotechnology Information Database by the search tool blastn. We found numerous sequence variants in both coding and non-coding sequences. The majority of these sequence variants have already been described, nevertheless, some appear as novel variants. Some of these may also have functional significance. We hypothesize over the possible significance of these findings and briefly review the available literature.     

Chromenylchalcones showing cytotoxicity on human colon cancer cell lines and in silico docking with aurora kinases

Due to toxicity problems, various plant-derived compounds have been screened to find the chemotherapeutic agents. As anticancer therapeutic agents, chalcones have advantages such as poor interaction with DNA and low risk of mutagenesity. Chromenones show anticancer activities too. Therefore, hybrids of chalcone and chromenone may be potent chemotherapeutic agents. We prepared 16 synthetic chromenylchalcones and applied a clonogenic long-term survival assay method for them on HCT116 human colorectal cancer cell lines. One of chromenylchalcones tested here, chromenylchalcone 11, showed IC₅₀ of 93.1 nM which can be competed with the IC₅₀ values of well-known flavonoids such as catechin gallate and epicatechin gallate. Further biological experiments including cell cycle analysis, apoptosis assay, Western blot analysis, and immunofluorescent microscopy were carried out for this compound. In addition, in vitro kinases binding assay performed to explain its molecular mechanism demonstrated the compound inhibited aurora kinases. The binding modes between chromenylchalcone 11 and aurora kinases were elucidated using in silico docking experiments. These findings could be used for designing cancer therapeutic or preventive plant-derived chromenylchalcone agents.     

葡萄基因组中KUP蛋白的生物信息学分析

植物钾(K+)转运体蛋白在K+的跨膜运输中起重要作用,进而维持植物体正常生长和代谢活动.本研究中,通过隐马尔科夫模型(HMM)和葡萄蛋白质库搜索,共找到18个葡萄钾转运体蛋白(VvKUPs).利用生物信息学方法,我们对葡萄家族12条KUP蛋白序列的系统发生和KUP基因组定位进行分析,然后对其氨基酸组成成分、理化性质以及二级结构进行预测和分析,同时还分析了葡萄与拟南芥、水稻和杨树的KUP基因家族之间的联系.基因组定位结果发现其分布在至少9条染色体上.二级结构预测结果发现不同成员间氨基酸数目、氨基酸序列间的疏水性存在一定的差异;α-螺旋和无规则卷曲为主要二级结构组成部分.基因结构分析表明,KUP基因家族成员分别含有7～10个内含子.葡萄KUP蛋白的亚细胞定位分析表明VvKUP主要定位于膜结构上.电子表达图谱分析结果表明:12条KUP基因有对应的EST序列,其中的11条KUP有相应的电子表达谱,并主要在花、果实、花序和花蕾等组织部位表达.     

Discrimination of intact mycobacteria at the strain level: a combined MALDI-TOF MS and biostatistical analysis

New methodologies for surveillance and identification of Mycobacterium tuberculosis are required to stem the spread of disease worldwide. In addition, the ability to discriminate mycobacteria at the strain level may be important to contact or source case investigations. To this end, we are developing MALDI-TOF MS methods for the identification of M. tuberculosis in culture. In this report, we describe the application of MALDI-TOF MS, as well as statistical analysis including linear discriminant and random forest analysis, to 16 medically relevant strains from four species of mycobacteria, M. tuberculosis, M. avium, M. intracellulare, and M. kansasii. Although species discrimination can be accomplished on the basis of unique m/z values observed in the MS fingerprint spectrum, discrimination at the strain level is predicted on the relative abundance of shared m/z values among strains within a species. For the 16 mycobacterial strains investigated in the present study, it is possible to unambiguously identify strains within a species on the basis of MALDI-TOF MS data. The error rate for classification of individual strains using linear discriminant analysis was 0.053 using 37 m/z variables, whereas the error rate for classification of individual strains using random forest analysis was 0.023 using only 18 m/z variables. In addition, using random forest analysis of MALDI-TOF MS data, it was possible to correctly classify bacterial strains as either M. tuberculosis or non-tuberculous with 100% accuracy.     

Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria

New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409-419 of the human c-myc proto-oncogene. Using the M. tuberculosis Ag85B as a reporter of gene expression, we demonstrate that the vectors permit the specific detection of recombinant products expressed in the host species M. bovis BCG. BCG over-expressing Ag85B was a potent inducer of Ag85B-specific T cells in immunised mice, indicating that the C-terminal c-myc tag did not alter the characteristics of the recombinant protein. The versatility of the epitope-tagging vectors was demonstrated by the efficient secretion and detection of recombinant products in BCG. The vectors described in this study will facilitate the expression of foreign proteins in mycobacterial host systems.     

普通烟草NtODB基因的电子克隆、表达及生物信息学分析

ODB基因在植物同源重组依赖性的DNA双键断裂修复过程中起重要作用,对植物诱变育种具有潜在的应用价值.克隆烟草NtODB基因并分析其表达特征为丰富ODB基因在同源重组DNA修复过程中的作用提供证据.为得到烟草NtODB基因序列,采用电子克隆技术获得该基因cDNA序列并克隆验证.进一步使用生物信息学方法分析该基因表达特征...     

In silico analysis of the large and small subunits of cereal RuBisCO as precursors of cryptic bioactive peptides

Ribulose-1,5-bisphosphate carboxylase (RuBisCO) is a plant metabolic enzyme and the most abundant protein on earth, but remains largely underutilized in the food system. Bioinformatics analysis was conducted to evaluate the prospects of RuBisCO from widely cultivated cereals (rice, barley, wheat, oat, sorghum, corn) as sources of bioactive peptides, and results were compared to commonly consumed proteins. The large and small RuBisCO subunits were found to contain several bioactive peptides with biological functions relevant to the management and treatment of diabetes, cardiovascular and neurodegenerative diseases, specifically inhibition of angiotensin converting enzyme and dipeptidyl peptidase-IV, antioxidative property and activation of ubiquitin-mediated proteolysis. Due to high sequence homology, there was negligible difference in occurrence frequency of bioactive peptides within large RuBisCO subunits unlike small subunits, which produced more diverse profiles of bioactive peptides among the cereals. The cereal RuBisCO displayed similar or better prospects as other food proteins except milk proteins, thereby providing cheaper and sustainable precursors of bioactive peptides. Simulated enzymatic hydrolysis of RuBisCO subunits indicated that thermolysin and papain had preferred cleavage patterns for releasing the cryptic peptides compared to gastrointestinal proteases. These findings will contribute towards utilization of RuBisCO as alternative sources of peptide-based nutraceuticals for human health promotion.     

A comprehensive in silico analysis of huntingtin and its interactome

A polyglutamine expansion of the N-terminal region of huntingtin (Htt) causes Huntington’s disease, a severe neurodegenerative disorder. Htt huge multidomain structure, the presence of disordered regions, and the lack of sequence homologs of known structure, so far prevented structural studies of Htt, making the study of its structure-function relationships very difficult. In this work, the presence and location of five Htt ordered domains (named from Hunt1 to Hunt5) has been detected and the structure of these domains has been predicted for the first time using a combined threading/ab initio modeling approach. This work has led to the identification of a previously undetected HEAT repeats region in the Hunt3 domain. Furthermore, a putative function has been assigned to four out of the five domains. Hunt1 and Hunt5, displaying structural similarity with the regulatory subunit A of protein phosphatase 2A, are predicted to play a role in regulating the phosphorylation status of cellular proteins. Hunt2 and Hunt3 are predicted to be homologs of two yeast importins and to mediate vescicles transport and protein trafficking. Finally, a comprehensive analysis of the Htt interactome has been carried out and is discussed to provide a global picture of the Htt’s structure–function relationships.     

In silico structural homology modeling of nif A protein of rhizobial strains in selective legume plants

Symbiosis is a complex genetic regulatory biological evolution which is highly specific pertaining to plant species and microbial strains. Biological nitrogen fixation in legumes is a functional combination of nodulation by nod genes and regulation by nif, fix genes. Three rhizobial strains (Rhizobium leguminosarum, Bradyrhizobium japonicum, and Mesorhizobium ciceri) that we considered for in silico analysis of nif A are proved to be the best isolates with respect to N₂ fixing for ground nut, chick pea and soya bean (in vitro) out of 47 forest soil samples. An attempt has been made to understand the structural characteristics and variations of nif genes that may reveal the factors influencing the nitrogen fixation. The primary, secondary and tertiary structure of nif A protein was analyzed by using multiple bioinformatics tools such as chou-Fasman, GOR, ExPasy ProtParam tools, Prosa -web. Literature shows that the homology modeling of nif A protein have not been explored yet which insisted the immediate development for better understanding of nif A structure and its influence on biological nitrogen fixation. In the present predicted 3D structure, the nif A protein was analyzed by three different software tools (Phyre2, Swiss model, Modeller) and validated accordingly which can be considered as an acceptable model. However further in silico studies are suggested to determine the specific factors responsible for nitrogen fixing in the present three rhizobial strains.     

埃及伊蚊、冈比亚按蚊和致倦库蚊polyUBQ基因的电子克隆及其序列分析

针对具有选择性蛋白质降解功能的泛素在调控昆虫生长发育过程中的重要作用,探讨埃及伊蚊、冈比亚按蚊和致倦库蚊基因组中polyUBQ基因的有关生物信息。采用电子克隆的方法钓取3种蚊虫基因组中polyUBQ基因序列,分析其特征、分子进化关系、遗传多态性和密码子偏爱性。结果显示,成功获取埃及伊蚊、冈比亚按蚊和致倦库蚊polyUBQ基因序列,命名为Aa-polyUBQ(GenBank登录号:AAGE02005963)、Ag-polyUBQ(ABKP02009650)和Cq-polyUBQ(AAWU01023041),分别编码1 065个、218个和533个氨基酸残基,各具14个、3个和7个泛素单体,Aa-polyUBQ、Ag-polyUBQ和Cq-polyUBQ蛋白二级结构主要元件是延伸带和无规则卷曲,Leu、Ile和Lys是构成3种蛋白的主要氨基酸,亚细胞主要定位于细胞质和细胞核,无前导肽、信号肽和跨膜结构域,除Ag-polyUBQ蛋白外均呈碱性;3种基因序列的同源性较高(83.8%-88.2%)且遗传距离较近(0.129-0.187);检出135个多态位点,共生成3个单倍型,单倍型多样性(Hd=1.000)、平均核苷...     

Carotenoid biosynthetic genes in Brassica rapa: comparative genomic analysis,phylogenetic analysis,and expression profiling

Background

Carotenoids are isoprenoid compounds synthesized by all photosynthetic organisms. Despite much research on carotenoid biosynthesis in the model plant Arabidopsis thaliana, there is a lack of information on the carotenoid pathway in Brassica rapa. To better understand its carotenoid biosynthetic pathway, we performed a systematic analysis of carotenoid biosynthetic genes at the genome level in B. rapa.

Results

We identified 67 carotenoid biosynthetic genes in B. rapa, which were orthologs of the 47 carotenoid genes in A. thaliana. A high level of synteny was observed for carotenoid biosynthetic genes between A. thaliana and B. rapa. Out of 47 carotenoid biosynthetic genes in A. thaliana, 46 were successfully mapped to the 10 B. rapa chromosomes, and most of the genes retained more than one copy in B. rapa. The gene expansion was caused by the whole-genome triplication (WGT) event experienced by Brassica species. An expression analysis of the carotenoid biosynthetic genes suggested that their expression levels differed in root, stem, leaf, flower, callus, and silique tissues. Additionally, the paralogs of each carotenoid biosynthetic gene, which were generated from the WGT in B. rapa, showed significantly different expression levels among tissues, suggesting differentiated functions for these multi-copy genes in the carotenoid pathway.

Conclusions

This first systematic study of carotenoid biosynthetic genes in B. rapa provides insights into the carotenoid metabolic mechanisms of Brassica crops. In addition, a better understanding of carotenoid biosynthetic genes in B. rapa will contribute to the development of conventional and transgenic B. rapa cultivars with enriched carotenoid levels in the future.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1655-5) contains supplementary material, which is available to authorized users.     

甘蔗几丁质酶基因的电子克隆与生物信息学分析

用电子克隆方法获得甘蔗几丁质酶基因SCCHI1,采用生物信息学方法,对该基因编码蛋白从氨基酸组成、理化性质、跨膜结构域、疏水性/亲水性、亚细胞定位、高级结构及功能域等方面进行了预测和分析。结果表明:SCCHI1基因全长1236bp,包含一个完整的990bp的ORF,编码329个氨基酸。SCCHI1基因属于糖苷水解酶19家族,含有N-端信号肽、交连区、催化区,与高粱等其它植物的几丁质酶基因具有高度的相似性。为SCCHI1基因的分子克隆、功能鉴定和应用提供参考。