Identification of Medicinal Aconitum Species Based on ITS2 Sequences and Analysis of Genetic Relationship / 中国实验方剂学杂志

ObjectiveThe internal transcribed spacer （ITS） 2 region of ribosomal gene， a DNA barcode， was employed to identify 12 medicinal Aconitum species and the genetic relationship among the species was analyzed. MethodA total of 30 samples of the 12 species were collected. The DNA was extracted with spin column plant genomic DNA kit and the universal primers of ITS2 sequence were used for polymerase chain reaction （PCR） amplification， followed by electrophoresis detection and bi-directional sequencing. The yielded sequences were aligned and spliced by CodonCode Aligner 17.0 and sequence variation was analyzed by MEGA 7.0. The secondary structure was predicted by ITS2 Database and the neighbor-joining （NJ） method was applied to generate the phylogenetic tree. ResultThe ITS2 sequences of the 12 species were 220-221 bp， with the average guanine and cytosine （GC） content of 64.09%， 140 variable sites， 137 informative sites， and 81 conservative sites. The intraspecific genetic distance （K2P） was smaller than the interspecific genetic distance. According to the secondary structures of ITS2 sequences and NJ cluster analysis， A. scaposum， A. sinomontanum， and A. barbatum had close genetic relationship， while the rest nine showed close kinship， particularly A. soongaricum and A. yinschanicum. ConclusionITS2 sequence is of great value for the molecular identification and genetic relationship determination of Aconitum， which provides a new method for the study of ethnomedicine.

3-State Protein Secondary Structure Prediction based on SCOPe Classes

Abstract Improving the accuracy of protein secondary structure prediction has been an important task in bioinformatics since it is not only the starting point in obtaining tertiary structure in hierarchical modeling but also enhances sequence analysis and sequence-structure threading to help determine structure and function. Herein we present a model based on DSPRED classifier, a hybrid method composed of dynamic Bayesian networks and a support vector machine to predict 3-state secondary structure information of proteins. We used the SCOPe (Structural Classification of Proteins-extended) database to train and test the model. The results show that DSPRED reached a Q3 accuracy rate of 82.36% when trained and tested using proteins from all SCOPe classes. We compared our method with the popular PSIPRED on the SCOPe test datasets and found that our method outperformed PSIPRED.

Molecular authentication of the medicinal species of Rhizoma et Radix Heraclei, Radix Angelicae Sinensis, Radix Angelicae Pubescentis and Rhizoma et Radix Notopterygii by integrating ITS2 and its secondary structure / 药学学报

This study is to identify Chinese medicinal materials Rhizoma et Radix Heraclei, Angelicae Sinensis, Radix Angelicae Pubescentis and Rhizoma et Radix Notopterygii based on ITS2 and its secondary structure. Total 26 ITS sequences of 7 species were downloaded from GenBank, the ITS2 sequences were annotated by HMMer method. The NJ phylogenetic tree was built by MEGA software, the intraspecific and interspecific K2P genetic distance were analyzed by MEGA as well. The ITS2 secondary structures of all taxa were predicted by ITS2 database. Sequence matrix of primary structure and secondary structure was aligned by 4Sale software. And the profile neighbor joining (PNJ) phylogenetic tree was constructed via the the ProfDistS software based on the distance method. The results show that, the average interspecific genetic distance was far greater than the average intraspecific genetic distance, an obvious barcoding gap was noted among all taxa; NJ tree showed that all species were clustered into seperate branches; each species had different secondary structures; the PNJ tree showed higher resolution than NJ tree. Therefore, ITS2 is suggested to be used as a barcode for distinguishing the original plants of Rhizoma et Radix Heraclei, Radix Angelicae Sinensis, Radix Angelicae Pubescentis and Rhizoma et Radix Notopterygii in this study, this provides some scientific basis for classification and accurate identification of these Chinese medicinal materials.

Identification of Astragalus and its adulterants based on ITS2sequence and secondary structure / 药学学报

To effectively identify the Astragalus and its adulterants based on ITS2 sequence and secondary structure, in this study, 32 portions of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Beg.) Hsiao and Astragalus membranaceus (Fisch.) Bge. collected were conducted ITS2 sequence amplification and bidirectional sequencing, whose results were then spliced by CExpress software remove the 5.8S and 28S sequences at both ends to obtain a complete ITS2 sequence. In addition, 3 ITS2 sequences for each of the adulterants of Astragalus, respectively, Oxytropis coerulea, Caragana sinica, Hedysarum polybotrys, Althaea rosea were downloaded from GenBank. The intra-specific and inter-specific genetic distances were calculated by the software MEGA7 to analyze the difference of each sequence; the Neighbor-joining (NJ) method was used to construct the phylogenetic tree based on ITS2 sequence (primary structure) as well as joint ITS2 sequence and its secondary structure. The results showed that the average ITS2 sequence length of both A. mongolicus and A. membranaceus was 216 bp, and their average GC content was 50.00% and 50.46%, respectively. The similarity of ITS2 sequence length and GC content between the two kind of Astragalus and Oxytropis coerulea was the highest, while the ITS2 sequence length and GC content of Althaea rosea showed great differences with those of Astragalus. The inter-specific distance between Astragalus and Oxytropis coerulea was the smallest, while that between the medicinal Astragalus and Hedysarum polybotrys, Caragana sinica as well as Althaea rosea was great. The phylogenetic trees constructed based on the ITS2 sequence (primary structure) and joint ITS2 sequence and its secondary structure showed that the topological relations of the two phylogenetic trees were basically the same, and both could effectively identify the Astragalus and its adulterants. What’s more, the addition of secondary structure information made end branch of the phylogenetic tree become more in its construction, and the distinguish ability and approval rating were also improved, which further reflected the genetic relationship of Astragalus and its adulterants. This provides some scientific basis for classification and accurate identification of Astragalus and its adulterants.

Identification of Bupleurum Seeds by ITS2 Genetic Barcoding and Seed Morphology / 中国实验方剂学杂志

Objective:To accurately identify Bupleurum seeds by traditional morphological identification method combined with DNA barcoding technique. Method:A total of 41 seed samples on the market were collected and 75 ribosomal DNA internal transcribed spacer 2 (ITS2) sequences of 15 varieties were downloaded from the GenBank database as experimental materials. The seeds were measured and observed by stereomicroscope and vernier caliper, and their 1 000-grain weights were calculated. Genomic DNA was extracted from the seeds and used as a template, and ITS2 sequences were amplified using polymerase chain reaction (PCR) and bidirectional sequencing. Species identification was conducted based on BLAST method, neighbor-joining (NJ) phylogenetic tree method, Kimura two-parameter model (K2P) genetic distance method, and secondary structure of ITS2 sequence. Result:There were slight differences in the length, width, cross-section, and 1 000-grain weight among Bupleurum seeds from different origins. The ITS2 sequences of B. chinense seeds had 2 intraspecific variable sites and 3 haplotypes, the maximum intraspecific genetic distance (0.009) was far smaller than the minimum interspecific genetic distance (0.032). B. chinense and B. scorzonerifolium in the NJ phylogenetic tree were clustered into independent branches with good monophyletic property. The secondary structure of ITS2 sequences could make up for the shortcomings of NJ tree in identifying variants. The collected 41 seeds included 30 B. chinense seeds, 3 B. scorzonerifolium seeds, 5 B. falcatum seeds, 2 B. marginatum var. stenophyllum seeds, and 1 B. smithii var. parvifolium seeds. Conclusion:The B. chinense seeds on the market have problems of diverse sources and chaotic origins. Based on the combination of ITS2 gentic barcoding and seed morphological identification, the Bupleurum seeds can be accurately identified, which provides scientific bases for establishing the quality standard of Bupleurum seeds, standardizing the cultivation of B. chinense, and solving the quality problems of B. chinense from the source, and provides a reference for the accurate identification of other medicinal plant seeds or seed medicinal materials.

Molecular Identification of Atractylodes Plants Based on ITS2 Sequence / 中国实验方剂学杂志

Objective: To identify the genetic relationship of cultivated and wild Atractylodes and its closely related species by using the Internal Transcribed Spacer 2(ITS2)barcode,in order to explore the cultivation origin of A. coreana from Northeast China. Method: Genomic DNAs were extracted from 40 samples of Atractylodes and its closely related species from different cultivated habitats,and 7 samples of wild A. coreana. were also extracted. The ITS2 sequences of these samples were amplified, and bidirectional sequencing was conducted by polymerase chain reaction(PCR). Totally 47 ITS2 sequences were aligned by using MEGA 5.0,5.8S and 28S sequences were removed to obtain the complete ITS2 sequence and build neighbor-joining (NJ) tree. Result: The lengths of ITS2 sequences of all samples were 232 bp. The NJ tree and the secondary structures of ITS2 showed that various varieties could be distinguished obviously except A. chinesis and A. coreana,which showed a good monophyly. The NJ tree showed that cultivated and wild A. coreana can also get together very well. Conclusion: As a DNA barcode,ITS2 sequences can be used to stably and accurately distinguish various varieties of Atractylodes. The relationship between A. chinesis and A. coreana is very close. A. coreana can be considered as a variant of A. coreana in North China. It is recommended to incorporate A. coreana into A. chinesis. The large-scale cultivation of A. coreana may originate from local wild population in Liaoning province,and the provenance may come from Xiuyan and other places in Liaoning province.

Analysis of Zingiberaceae in E'mei Area Using ITS2 Sequences / 中国实验方剂学杂志

Objective: To evaluate the use of ITS2 sequences as DNA barcode to identify the Zingiberaceae medicinal plants from E'mei area. Method: The genomic DNAs were extracted from 43 Zingiberaceae medicinal plant samples from Sichuan E'mei area. The ITS2 sequences of these samples were amplified and bidirectionally sequenced by PCR. 40 ITS2 sequences were downloaded from the GenBank,and then the interspecific and intraspecific genetic distances were calculated and analyzed by using MEGA 6.0 to construct Neighbor-joining (NJ) tree; TAXON DNA software was also used to analyze intraspecific and interspecific variations and barcoding gaps. The differences in secondary structure of the ITS2 sequences were predicted and compared. Result: The minimum interspecific distance in Zingiberaceae samples was greater than the maximum intra specific distance,with obvious barcoding gap. The NJ tree showed that the samples were clustered into five different branches,Alpinia,Curcuma,Globba,Hedychium,and Zingiber respectively,and further cluster into sub-branches. Significant differences were also present in the secondary structures of ITS2 between different samples. Conclusion: ITS2 sequences as DNA barcode can be used to conduct accurate and rapid identification of the Zingiberaceae plants and clearly figure out the phylogenetic relationship among them,providing guidance for the study of the distribution of medicinal plants of this genus,as well as theoretical basis for the quality control,medication safety and rational development of Zingiberaceae medicinal plants in E'mei area.

Identification of Chrysanthemum indicum and its adulterants based on ITS2 barcode / 中国中药杂志

DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum based on ITS2 sequences. The total DNA was extracted from 22 collected samples,and the ITS2 sequence was amplified by PCR and sequenced,and the information of ITS2 sequence was obtained. Another 14 items of the same family or the same genus were downloaded from Gen Bank.We aligned all 36 sequences,calculated the intraspecific and interspecific distances,and constructed Neighbor Joining( NJ) phylogenetic tree,using MEGA 7. 0. The difference of the secondary structure between the ITS2 sequences was compared. The results showed that the genetic distance of Ch. indicum and Ch. morifolium was overlapped,but the maximum intraspecific distance was far less than the minimum interspecific distance between and among Ch. indicum and other species,with an obvious barcoding gap. The NJ tree showed that Ch. indicum and Ch. morifolium shared a clade,and most of Ch. morifolium with some Ch. indicum were shared a subclade,while Inula lineariifolia,Sinosenecio oldhamianus and Senecio scandens belonged to one clade separately. ITS2 secondary structures for I. lineariifolia,S. oldhamianus and S. scandens were significantly different enough to identify completely but Ch. indicum and Ch. morifolium shared two secondary structures of A and B. It was proved that Ch. indicum was one of the evolutionary sources of Ch.morifolium. Therefore ITS2 sequence as DNA barcode can identify Ch. indicum and its adulterants accurately and quickly. The study provides an important basis for Ch. indicum for the identification of germplasm resources and the safety of clinical medication.

Effects of dihydromyricetin on biological properties of Bloom syndrome helicase / 中国药理学通报

; Aim To explore the anti-tumor mechanism of dihydromyricetin (DMY), a kind of flavonoid compound with anti-inflammatory and anti-tumor effects, via studying the effect of DMY on biological activities of Bloom helicase. Methods The effect of DMY on the biological activities of BLM helicase was studied by ultraviolet spectrum (UV), circular dichroism (CD), fluorescence polarization and free phosphorus detection. Results The results of CD and UV showed that DMY could bind to a site of the BLM helicase. In the concentration of DMY in 0 ~ 25 μmol . L 1 range, DMY showed a positive correlation with the interference ability of BLM helicase secondary structure with the increase of concentration, while in the concentration of DMY in 25 ~ 75 μmol . L 1 range, DMY showed a negative correlation. Fluorescence polarization and free phosphorus detection experiments showed that DMY could bind to BLM helicase, thus inhibiting the helicase activity of BLM helicase. Conclusions DMY can competitively bind to the DNA binding site of BLM helicase and change the spatial structure of BLM helicase, inhibiting the binding of BLM helicase to DNA and the biological activity of BLM helicase accordingly.

ITS2 sequence analysis and identification of medicinal Artemisia plants / 中国中药杂志

Artemisia hedinii occupies an important position in the Tibetan medicine. Plants in Artemisia vary a lot and are widely distributed in the Qinghai-Tibet Plateau, many plants in Artemisia look similar, making traditional identification methods laborious. In this article, ITS2 sequences were used as DNA barcoding to identify four kinds of confusable Tibetan medicine plants in Artemisia, aiming to establish a rapid and accurate identification methods. Twenty-one samples in Artemisia were collected from the Qinghai-Tibet Plateau, ITS2 sequence PCR amplification and sequencing were conducted after the extraction of DNA. Another 11 sequence downloaded from Genbank were added to the analysis. Genetic distance calculation and analysis, building Neighbor Joining (NJ) phylogenetic tree were conducted by MEGA 6.0, also comparison of secondary structures of ITS2 sequences among samples. A. hedinii, A. annua, A. dubia and A. argyi shared close genetic distance, but the maximum distance between the four species was much greater than the minimum distance within each species, NJ tree showed that the four species went to four separate branches, differences among secondary structures of ITS2 sequences also made it clear to identify these medical plants. It could be an accurate and rapid method for identification and recognition, as well as the evolutionary relationships between the species by using ITS2 sequence as DNA barcode for plants of Tibetan Artemisia. The study provides theoretical basis for quality control, medication safety and rational exploitation.

Application of phylogenetic information of ITS2 secondary structure in DNA barcoding of Solanum medicinal plant / 中国中药杂志

Internal transcript spacer 2 (ITS2) is one of the broadly used standard core barcodes and also the only nuclear barcode in identification of Chinese traditional medicine. Although the DNA barcode method based on ITS2 is popular and has been used in Chinese Pharmacopoeia, its low discriminatory efficiency is still a problem to its extensive application. Therefore, further study is still necessary to explore its phylogenetic information for medicinal plants identification. In cells, ITS2 activity is based on its secondary structure. The secondary structures are particularly useful in phylogenetic analysis because they include information not found in the primary sequence. In this study ITS2 secondary structure of 40 samples from 26 species were predicted and used to explore their utility in addressing the identification problems of Chinese traditional medicine in Solanum. The secondary structures were predicted and aligned, and their consensus models were generated using the three different software of LocARNA, MASTR and PicXAA-R. RNAstat software was used to transform the secondary structures into 28 symbol code data for maximum parsimony (MP) analysis. The results showed that the phylogenetic information increased 88.57% after ITS2 secondary structure information has been added, and then the support values above 50%, 75% and 90% in the tree increased 19.05%, 66.67% and 66.67%, respectively, indicating that the identification of Solanum medical plants has been well resolved. Thus, our analysis suggests that ITS2 secondary structure information should be incorporated into the current DNA barcoding analysis as a beneficial supplement of phylogenetic information.

Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae

Background: Antithrombin III (ATIII) is a protein that inhibits abnormal blood clots (or coagulation) by breaking down thrombin and factor Xa. ATIII helps to keep a healthy balance between hemorrhage and coagulation. The present work demonstrated the production, purification and characterization of recombinant human antithrombin (rhAT) from yeast Saccharomyces cerevisiae BY4741 was demonstrated. After expression of rhAT by S. cerevisiae, the biomass and rhAT concentration were analyzed through fed-batch fermentation process. Results: In fed-batch fermentation, the biomass (maximum cell dry weight of 11.2 g/L) and rhAT concentration (312 mg/L) of the expressed rhAT were achieved at 84 h of cultivation time. The maximum cell lysis efficiency (99.89%) was found at 8 s sonication pulse and 7 mL lysis buffer volume. The rhAT protein solution was concentrated and partially purified using cross-flow filtration with the recovery yield and purity of 95 and 94%, respectively. The concentrated solution was further purified by the single step ion exchange chromatography with the recovery yield and purity of 55 and >98%, respectively. The purified rhAT was characterized by various analytical techniques, such as RP-HPLC, FT-IR, CD, SDS-PAGE, western blotting, and Liquid chromatography mass spectrometry (LC-MS) analysis. The biological activity of rhAT was analyzed as heparin cofactor to meet the therapeutic grade applications. Conclusions: The simple, cost-effective and economically viable nature of the process used in the present study for the production of rhAT will be highly beneficial for the healthcare sector. This may also be used to produce other value-added therapeutic recombinant proteins expressed in S. cerevisiae, with greater effectiveness and ease.

Optimization of extraction technology of two immune active proteins from Astragalus membranaceus var. mongholicus / 中草药

Objective: To optimize the extraction technology of immune active glycoproteins AmPR10-16kD and HQGP-2 from Astragalus membranaceus var. mongholicus (AMM). Methods: The optimized extraction temperature conditions were investigated by circular dichroism of water-soluble protein involving in AmPR10-16kD and HQGP-2 with secondary structure from AMM. The optimized extraction technology was investigated using single factor test and orthogonal test with gray value of water-soluble protein AmPR10-16kD and HQGP-2 as the index which was determined by Image of gel graphical analysis software. In this study, the effects of temperature, solid-liquid ratio, time, solvent, granularity, and times on gray value were investigated, for which the inhibitory effect of water-soluble protein was determined as an evidence by CCK-8 method, and the content of water-soluble protein is determined as an evidence by BCA method. Results: The optimized extraction technique for proteins AmPR10-16kD and HQGP-2 in AMM was established, that was 5.0 g powder of AMM over the No.4 sieve, olvent Tris-HCl, solid-liquid ratio 1:16 and 60 min for extraction at the temperature of 40 ℃ and being mixed under 100 r/min. The water-soluble protein extract rate in the orthogonal test analysis was 65 mg/g, of which inhibitory effect was 90.90% at a concentration of 90 μg/mL. Conclusion: The optimal extraction conditions could accurately reflect the relative amounts of AmPR10-16kD and HQGP-2 maximum extraction rate, providing a stable, reasonable, and feasible extraction process for further study of the bioactive substance of AMM.

Effect and mechanism of danshensu on hepatitis B virus reverse transcriptase and antigen expression / 中国中药杂志

MTT assay was used in this study to investigate the inhibitory effect of danshensu on the activity of 2.2.15 cells among human hepatoma cell line (HepG2); indirect fluorescence labeling method was used to measure the changes of reactive oxygen levels in the cells; ELISA method was used to determine hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels in cellular supernatants; HBV DNA level was measured with fluorogenic quantitative PCR method. The inhibitory effect of danshensu on HBV RT(hepatitis B virus reverse transcriptase) was studied by using enzyme inhibition dynamics, and the effect of danshensu on secondary structure of HBV reverse transcriptase was monitored by using circular dichroism. The results showed that danshensu had a good inhibitory effect on the growth of HepG2.2.15 cells, with a half inhibitory concentration (IC₅₀) of (15.35±2.43) μmol•L⁻¹; danshensu could significantly inhibit HBsAg and HBeAg expressions, and showed an inhibitory effect on HBV DNA replication. In addition, danshensu was an effective inhibitor for HBV reverse transcriptase [IC₅₀ (21.32±2.43) μmol•L⁻¹]. The fluorescence labeling results showed that the reactive oxygen levels in the cells were increased with the increase of danshensu concentration. Circular dichroism analysis showed that danshensu could induce partial change of conformation of HBV reverse transcriptase and gradually increased α-helical content. These results indicated that danshensu could make the structure of the enzyme become closer by binding to HBV reverse transcriptase, which was not conducive to the formation of the active center, so it could finally decrease the activity of HBV reverse transcriptase. Such decrease in enzyme activity would directly affect the HBV DNA replication, and combined with the decrease of the antigen levels, the effect of danshensu on HBV was increased.

Compiling Multicopy Single-Stranded DNA Sequences from Bacterial Genome Sequences

A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.

CARACTERIZACIÓN DE LA ESTRUCTURA SECUNDARIA DE SUBTIPOS DE LA HISTONA H1 POR DICROÍSMO CIRCULAR / CHARACTERIZATION OF THE SECONDARY STRUCTURE OF HISTONE H1 SUBTYPES BY CIRCULAR DICHROISM (CD)

Introducción: Las histonas H1 modulan la estructura y la función de la cromatina. Las células somáticas de mamífero contienen los subtipos H1º, H1a, H1b, H1c, H1d y H1e; en células germinales de testículo y en ovocito, se encuentran respectivamente H1t y H1oo. Su estructura está conformada por un dominio central globular flanqueado por los dominios N-Terminal (DNT) y C-Terminal (DCT). Objetivo: Caracterizar la estructura secundaria de subtipos de la histona H1 mediante dicroísmo circular (DC). Materiales y Métodos: La histona H1 total se extrajo de núcleos de cerebro de rata por cromatografía de intercambio catiónico; la H1º se purificó por filtración en gel y las H1a, H1b, H1c y H1e por cromatografía líquida de alta resolución de fase reversa (RF-HPLC). Los espectros de DC se realizaron en tampón fosfato 10 mM; tampón fosfato 10 mM, 20% TFE (trifluoroetanol); tampón fosfato 10 mM, 40% TFE; tampón fosfato 10 mM, 60% TFE; tampón fosfato 10 mM, 150 mM NaCl y tampón fosfato 10 mM, 1 M NaCl. El análisis de los espectros se realizó con el programa Standard Analysis. Resultados: El porcentaje de hélice-alfa se calculó por diferentes métodos matemáticos teniendo en cuenta elipticidad molar a 193 nm y a 222 nm; con programa de deconvolución K2D y con relaciones cualitativas R1 y R2. El TFE induce la estructura en hélice-alfa en cada uno de los subtipos, mientras que NaCl no induce ningún cambio importante. Conclusión: Los subtipos con mayor contenido de hélice-alfa son H1a y H1c. Las diferencias observadas en el porcentaje de hélice-alfa entre los diferentes subtipos puede ser importante para su diferenciación funcional.

H1 histones modulate the structure and function of chromatin. Mammalian somatic cells contain H1º, H1a, H1b, H1c, H1d and H1e subtypes; H1t and H1oo are found in testicular germ cells and oocyte, respectively. Its structure consists of a globular core domain flanked by N-terminal (DNT) and C-terminal (DCT) domains. Objective: To characterize the secondary structure of histone H1 subtypes through circular dichroism (CD). Materials and Methods: Total histone H1 was extracted for rat brain nuclei by cation exchange chromatography; histone H1º was purified by gel filtration and the histones H1a, H1b, H1c and H1e were purified by reversed phase high performance liquid chromatography (RP-HPLC). CD spectra were performed in 10 mM phosphate buffer; 10 mM, 20% TFE phosphate buffer (trifluoroethanol); 10 mM, 40% TFE; phosphate buffer 10 mM, 60% TFE; phosphate buffer 10 mM, 150 mM NaCl and phosphate buffer 10 mm, 1 M NaCl. The analysis of the spectra was performed with JASCO Standard Analysis. Results: The percentage of alpha-helix was calculated using different mathematical methods, taking into account the molar ellipticity at 193 nm, and 222 nm, with K2D deconvolution program and the R1 and R2 qualitative relationships. The results indicate that TFE induced the alpha-helix structure in each of the subtypes, whereas NaCl did not induce any significant change. Conclusion: H1a and H1c are subtypes with highest content of alpha-helix. The observed differences in the percentage of alpha-helix between different subtypes may be important for their functional differentiation.

FASTR: A novel data format for concomitant representation of RNA sequence and secondary structure information.

Given the importance of RNA secondary structures in defining their biological role, it would be convenient for researchers seeking RNA data if both sequence and structural information pertaining to RNA molecules are made available together. Current nucleotide data repositories archive only RNA sequence data. Furthermore, storage formats which can frugally represent RNA sequence as well as structure data in a single file, are currently unavailable. This article proposes a novel storage format, ‘FASTR’, for concomitant representation of RNA sequence and structure. The storage efficiency of the proposed FASTR format has been evaluated using RNA data from various microorganisms. Results indicate that the size of FASTR formatted files (containing both RNA sequence as well as structure information) are equivalent to that of FASTA-format files, which contain only RNA sequence information. RNA secondary structure is typically represented using a combination of a string of nucleotide characters along with the corresponding dot-bracket notation indicating structural attributes. ‘FASTR’ – the novel storage format proposed in the present study enables a frugal representation of both RNA sequence and structural information in the form of a single string. In spite of having a relatively smaller storage footprint, the resultant ‘fastr’ string(s) retain all sequence as well as secondary structural information that could be stored using a dot-bracket notation. An implementation of the ‘FASTR’ methodology is available for download at http://metagenomics.atc.tcs.com/compression/fastr.

Structural and functional analysis of chitinase gene family in wheat (Triticum aestivum).

Chitinases are the hydrolytic enzymes which protect plants against pathogen attack. However, the precise role of chitinases in disease resistance has not been explored in wheat. In the present study, in silico approach, including secondary structure analysis, detailed signature pattern study, cis-acting regulatory elements survey, evolutionary trends and three-dimensional molecular modeling was used for different chitinase classes of wheat (Triticum aestivum). Homology modeling of class I, II, IV and 3 chitinase proteins was performed using the template crystal structure. The model structures were further refined by molecular mechanics methods using different tools, such as Procheck, ProSA and Verify3D. Secondary structure studies revealed greater percentage of residues forming α helix conformation with specific signature pattern, similar to casein kinase II phosphorylation site, amidation site, N-myristoylation (N-MYR) site and protein kinase C phoshorylation site. The expression profile suggested that wheat chitinase gene was highly expressed in cell culture and callus. We found that wheat chitinases showed more functional similarity with rice and barley. The results provide insight into the evolution of the chitinase family, constituting a diverse array of pathogenesis-related proteins. The study also provides insight into the possible binding sites of chitinase proteins and may further enhance our knowledge of fungal resistance mechanism in plants.

Spontaneous clearance of chronic hepatitis C infection is associated with an internal ribosomal entry site IIId stem loop structure variant.

Aim: To investigate if any mutations in hepatitis C virus (HCV) internal ribosome entry site (IRES) can inhibit the translation of viral polyprotein. Materials and Methods: A 26-year-old male patient infected with HCV 10 years ago was followed up. After 9 years of chronic infection. The patient had managed to resolve the infection for a period of 9 months, after which the patient experienced a viral recurrence characterized by high viral load and diverse HCV quasispecies. The IRES structures of the viral strains that disappeared were comparable with those that are currently active using structural mutational analysis. Results: A novo mutational position 254 combined with a rarely observed mutation at position 253 in the stem of the IIId subdomain were observed and the new conformation had an octa-apical loop (AGUGUUGG) and a shift in the 3 ` GU from the loop to the stem. Conclusions: These mutations were found to be highly deleterious, and they affected the direct binding of the IIId loop to the 40S ribosomal subunit with a subsequent inhibition of translation of viral polyprotein and clearance of the virus.

Prediction of the secondary structure and B-cell epitopes of Mycoplasma suis ORF5 protein / 中国人兽共患病学报

The objective of the study is to predict the spatial structure and B‐cell epitopes of Mycoplasma suis ORF5 pro‐tein .The secondary structure ,hydrophilicity ,flexible region ,antigenic index and surface probability were analyzed and predic‐ted by the Protean module in DNAStar software and B Cell Epitope Prediction Tools of IDEB ,then B‐cell epitopes were predic‐ted by aggregate analysis .Results showed that the secondary structure of Mycoplasma suis ORF5 protein was relatively regu‐lar ,in which the potential B cell antigenic epitopes were located at GGVDGGRD ,GMRLPEDSR ,and EGHPDLESAR .The methods of prediction of the secondary structure and B‐cell epitopes of Mycoplasma suis ORF5 protein may provide a new method for the study of M .suis immunogenicity ,and provides a new idea for the study on immunogenicity of pathogenic micro‐organisms .