Ensemble approach to predict specificity determinants: benchmarking and validation

<p>Abstract</p> <p>Background</p> <p>It is extremely important and challenging to identify the sites that are responsible for functional specification or diversification in protein families. In this study, a rigorous comparative benchmarking protocol was employed to provide a reliable evaluation of methods which predict the specificity determining sites. Subsequently, three best performing methods were applied to identify new potential specificity determining sites through ensemble approach and common agreement of their prediction results.</p> <p>Results</p> <p>It was shown that the analysis of structural characteristics of predicted specificity determining sites might provide the means to validate their prediction accuracy. For example, we found that for smaller distances it holds true that the more reliable the prediction method is, the closer predicted specificity determining sites are to each other and to the ligand.</p> <p>Conclusion</p> <p>We observed certain similarities of structural features between predicted and actual subsites which might point to their functional relevance. We speculate that majority of the identified potential specificity determining sites might be indirectly involved in specific interactions and could be ideal target for mutagenesis experiments.</p

Movement Protein Pns6 of Rice dwarf phytoreovirus Has Both ATPase and RNA Binding Activities

Cell-to-cell movement is essential for plant viruses to systemically infect host plants. Plant viruses encode movement proteins (MP) to facilitate such movement. Unlike the well-characterized MPs of DNA viruses and single-stranded RNA (ssRNA) viruses, knowledge of the functional mechanisms of MPs encoded by double-stranded RNA (dsRNA) viruses is very limited. In particular, many studied MPs of DNA and ssRNA viruses bind non-specifically ssRNAs, leading to models in which ribonucleoprotein complexes (RNPs) move from cell to cell. Thus, it will be of special interest to determine whether MPs of dsRNA viruses interact with genomic dsRNAs or their derivative sRNAs. To this end, we studied the biochemical functions of MP Pns6 of Rice dwarf phytoreovirus (RDV), a member of Phytoreovirus that contains a 12-segmented dsRNA genome. We report here that Pns6 binds both dsRNAs and ssRNAs. Intriguingly, Pns6 exhibits non-sequence specificity for dsRNA but shows preference for ssRNA sequences derived from the conserved genomic 5′- and 3′- terminal consensus sequences of RDV. Furthermore, Pns6 exhibits magnesium-dependent ATPase activities. Mutagenesis identified the RNA binding and ATPase activity sites of Pns6 at the N- and C-termini, respectively. Our results uncovered the novel property of a viral MP in differentially recognizing dsRNA and ssRNA and establish a biochemical basis to enable further studies on the mechanisms of dsRNA viral MP functions

The FGGY carbohydrate kinase family : insights into the evolution of functional specificities

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS Computational Biology 7 (2011): e1002318, doi:10.1371/journal.pcbi.1002318.Function diversification in large protein families is a major mechanism driving expansion of cellular networks, providing organisms with new metabolic capabilities and thus adding to their evolutionary success. However, our understanding of the evolutionary mechanisms of functional diversity in such families is very limited, which, among many other reasons, is due to the lack of functionally well-characterized sets of proteins. Here, using the FGGY carbohydrate kinase family as an example, we built a confidently annotated reference set (CARS) of proteins by propagating experimentally verified functional assignments to a limited number of homologous proteins that are supported by their genomic and functional contexts. Then, we analyzed, on both the phylogenetic and the molecular levels, the evolution of different functional specificities in this family. The results show that the different functions (substrate specificities) encoded by FGGY kinases have emerged only once in the evolutionary history following an apparently simple divergent evolutionary model. At the same time, on the molecular level, one isofunctional group (L-ribulokinase, AraB) evolved at least two independent solutions that employed distinct specificity-determining residues for the recognition of a same substrate (L-ribulose). Our analysis provides a detailed model of the evolution of the FGGY kinase family. It also shows that only combined molecular and phylogenetic approaches can help reconstruct a full picture of functional diversifications in such diverse families.This study was funded by NIH and DOE grants

Expression of Linear and Novel Circular Forms of an INK4/ARF-Associated Non-Coding RNA Correlates with Atherosclerosis Risk

Human genome-wide association studies have linked single nucleotide polymorphisms (SNPs) on chromosome 9p21.3 near the INK4/ARF (CDKN2a/b) locus with susceptibility to atherosclerotic vascular disease (ASVD). Although this locus encodes three well-characterized tumor suppressors, p16INK4a, p15INK4b, and ARF, the SNPs most strongly associated with ASVD are ∼120 kb from the nearest coding gene within a long non-coding RNA (ncRNA) known as ANRIL (CDKN2BAS). While individuals homozygous for the atherosclerotic risk allele show decreased expression of ANRIL and the coding INK4/ARF transcripts, the mechanism by which such distant genetic variants influence INK4/ARF expression is unknown. Here, using rapid amplification of cDNA ends (RACE) and analysis of next-generation RNA sequencing datasets, we determined the structure and abundance of multiple ANRIL species. Each of these species was present at very low copy numbers in primary and cultured cells; however, only the expression of ANRIL isoforms containing exons proximal to the INK4/ARF locus correlated with the ASVD risk alleles. Surprisingly, RACE also identified transcripts containing non-colinear ANRIL exonic sequences, whose expression also correlated with genotype and INK4/ARF expression. These non-polyadenylated RNAs resisted RNAse R digestion and could be PCR amplified using outward-facing primers, suggesting they represent circular RNA structures that could arise from by-products of mRNA splicing. Next-generation DNA sequencing and splice prediction algorithms identified polymorphisms within the ASVD risk interval that may regulate ANRIL splicing and circular ANRIL (cANRIL) production. These results identify novel circular RNA products emanating from the ANRIL locus and suggest causal variants at 9p21.3 regulate INK4/ARF expression and ASVD risk by modulating ANRIL expression and/or structure

Computing Highly Correlated Positions Using Mutual Information and Graph Theory for G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are a superfamily of seven transmembrane-spanning proteins involved in a wide array of physiological functions and are the most common targets of pharmaceuticals. This study aims to identify a cohort or clique of positions that share high mutual information. Using a multiple sequence alignment of the transmembrane (TM) domains, we calculated the mutual information between all inter-TM pairs of aligned positions and ranked the pairs by mutual information. A mutual information graph was constructed with vertices that corresponded to TM positions and edges between vertices were drawn if the mutual information exceeded a threshold of statistical significance. Positions with high degree (i.e. had significant mutual information with a large number of other positions) were found to line a well defined inter-TM ligand binding cavity for class A as well as class C GPCRs. Although the natural ligands of class C receptors bind to their extracellular N-terminal domains, the possibility of modulating their activity through ligands that bind to their helical bundle has been reported. Such positions were not found for class B GPCRs, in agreement with the observation that there are not known ligands that bind within their TM helical bundle. All identified key positions formed a clique within the MI graph of interest. For a subset of class A receptors we also considered the alignment of a portion of the second extracellular loop, and found that the two positions adjacent to the conserved Cys that bridges the loop with the TM3 qualified as key positions. Our algorithm may be useful for localizing topologically conserved regions in other protein families

Biological properties of swine vesicular disease virus strain 2348 Italy/2008

Swine vesicular disease (SVD) is a viral infectious disease, which, if acute, is manifested by the clinical pattern similar to a number of vesicular diseases including foot-and-mouth disease. In case of subclinical disease, there are no evident clinical signs, therefore the diagnosis is problematic, and there can be the risk of the disease introduction into the Russian Federation with the infected pigs. The key measure for the prevention of SVD introduction involves control diagnostic testing of all animals imported in the country that makes it necessary to keep updated the currently used methods and tools for the disease laboratory diagnosis. The paper demonstrates data on experimental infection of pigs with SVDV strain 2348 Italy/2008 that belongs to the most recent one of the four known phylogenetic groups. The virus was kindly provided by the World Reference Laboratory for Foot-and-Mouth Disease (Pirbright, Great Britain), and it was adapted to the monolayer continuous cell cultures of porcine origin (IB-RS-2 and PGSK-30). The pigs were intradermally infected with concentrated cultured virus at a dose of 109 TCID50. The infected animals demonstrated clinical signs typical for the acute disease. There was evidence that the virus was not transmitted to the intact animal in case husbandry conditions were met that allowed to avoid the infection transmission by the fecal-oral and contact mechanisms. As a result of the experiment, reference sera were collected at different time intervals post infection and their activity was determined using virus microneutralization test in cell culture and ELISA. Aphthae collected from the infected animals were deposited into the Strain collection of the Reference Laboratory for Foot-and-Mouth Disease, FGBI “ARRIAH”

Validation of the technique aimed at the determination of FMD antibodies in liquid phase blocking sandwich Elisa

The paper describes the process of the validation of the technique aimed at the determination of FMD antibodies in liquid phase blocking sandwich ELISA for FMD types A, 0 and Asial. The results were analyzed and the following main validation characteristics were determined: sensitivity, specificity, accuracy, consistency, predictability of positive and negative results, reproducibility and intermediate precision under the conditions of repeatability and reproducibility. The results of the ELISA procedure validation were in compliance with the acceptability criteria

Вибір матеріалу та технологія виготовлення заготовок із алюмінієвого сплаву АК6

Purpose. Justification of the material and heat treatment method of aluminum alloy for the manufacturing of parts, type «plate» based on the results of microstructure and mechanical properties research; development of technological process of blanks operation of AK6 aluminum alloy. Methodology. Powdered alloy based on aluminum type AK6 was the research material. Finished forgings with the size 2520×1520×65 mm were obtained as a result of the preparation and forging of the blanks. After mechanical treatment of the blanks they were exposed to thermal processing and milling. Structure of the metal was examined under light microscope MIM-8M. Brinell hardness was used as the strength alloy characteristic. Findings. Influence analysis of alloy elements on the structure of deformable aluminum alloys was carried out. Research of influence of heat treatment modes on structure and properties of the AK6 alloy were performed. The improved technological process, which made it possible to obtain the item with the improved structure and properties and lower costs is offered. Originality. The samples of AK6 powdered alloy on fire resistance were tested. It is established that under heating of an example in the oxidative flame, it does not ignite to a temperature of 705 °C. The cause of high fire resistance of AK6 alloy samples was found, it is connected with the presence in the material the evenly distributed, small oxide inclusions and amorphous oxide film on the surface. Practical value. Hard conditions of work (corrosion in marine and industrial atmosphere, static and shock loads, cyclic temperature) allow the use of the item in various designs.Цель. В работе необходимо осуществить обоснования материала и способа термической обработки алюминиевого сплава для изготовления детали типа «плита» на основе результатов исследования микроструктуры и механических свойств; разработку технологического процесса изготовления заготовок из алюминиевого сплава АК6. Методика. Материалом для исследования был порошковый сплав на основе алюминия типа АК6. Готовые поковки размером 2 520×1 520×65 мм получали в результате подготовки заготовки и её ковки. После механической обработки заготовки подвергались термической обработке и фрезерованию. Структуру металла исследовали под световым микроскопом МИМ-8М. В качестве характеристики прочности сплава была использована твердость по Бринеллю. Результаты. Проведен анализ влияния легирующих элементов на структуру деформируемых алюминиевых сплавов. Выполнены исследования влияния режимов термической обработки на структуру и свойства сплава АК6. Предложен усовершенствованный технологический процесс, который позволил получить деталь с улучшенной структурой и свойствами, а также меньшей себе-стоимостью. Научная новизна. Проведены испытания образцов порошкового сплава АК6 на огнестой-кость. Установлено, что при нагревании образца в окислительном пламени он не воспламеняется до темпе-ратуры 705 °C. Выяснена причина высокой огнестойкости образцов сплава АК6, что связано с наличием в материале равномерно распределенных, мелких оксидных включений и аморфной оксидной пленки на поверхности. Практическая значимость. Жесткие условия труда (коррозия в морской и промышленной атмосфере, статические и ударные нагрузки, циклические температуры) позволяют использовать деталь в различных конструкциях.Мета. У роботі необхідно здійснити обґрунтування матеріалу та способу термічної обробки алюмінієвого сплаву для виготовлення деталі типу «плита» на основі результатів дослідження мікроструктури та механічних властивостей; розробити технологічний процес виготовлення заготовок із алюмінієвого сплаву АК6. Методика. Матеріалом для дослідження був порошковий сплав на основі алюмінію типу АК6. Готові поковки розміром 2 520×1 520×65 мм отримували в результаті підготовки заготовки та її кування. Після механічної обробки заготовки піддавалися термічній обробці та фрезеруванню. Структуру металу досліджували під світловим мікроскопом МІМ-8М. В якості характеристики міцності сплаву була використана твердість за Брінеллем. Результати. Проведено аналіз впливу легуючих елементів на структуру деформованих алюмінієвих сплавів. Виконано дослідження впливу режимів термічної обробки на структуру та властивості сплаву АК6. Запропоновано удосконалений технологічний процес, який дозволив отримати деталь із поліпшеною структурою та властивостями, а також меншою собівартістю. Наукова новизна. Проведено випробування зразків порошкового сплаву АК6 на вогнестійкість. Встановлено, що при нагріванні зразка в окислювальному полум’ї він не запалюється до температури 705 °C. Встановлено причину високої вогнестійкості зразків сплаву АК6, що пов’язано з наявністю в матеріалі рівномірно розподілених, дрібних оксидних включень та аморфної оксидної плівки на поверхні. Практична значимість. Жорсткі умови праці (корозія в морській та промисловій атмосфері, статичні та ударні навантаження, циклічні температури) дозволяють використовувати деталь у різноманітних конструкціях

EFFECTS OF METABOLIC DRUG ELTACINE ON CLINICAL, FUNCTIONAL AND BIOCHEMICAL INDICES IN PATIENTS WITH CHRONIC HEART FAILURE

Aim. To study clinical efficacy of a new domestic metabolic drug Eltacine in patients with chronic heart failure (CHF).Material and methods. 134 patients with CHF of I-III functional classes were randomized in two parallel groups of patients receiving Eltacine or placebo additionally to standard therapy. Common clinical and laboratory investigations were used as well as 6-minute-walking test and Echocardiography. Besides Holter monitoring with determination of heart rate variability, peroxidal oxidation of lipids (POL) and cell anti-oxidant protection were implemented.Results. Eltacine increased in tolerance to physical burden, improved cardiac haemodynamics, parameters of POL and cell anti-oxidant protection, improved the patient quality of life.Conclusion. The efficacy and safety of Eltacine as metabolic and antioxidant therapy was shown in patients with CHF.</p

ЦИРКУЛИРУЮЩАЯ РЕКОМБИНАНТНАЯ ФОРМА ВИРУСА ГЕПАТИТА С RF2k/1b: ПРОБЛЕМЫ ДИАГНОСТИКИ И ТЕРАПИИ

Study objective: To find out the causes of nonstandard results of hepatitis C virus genotyping using the test system Abbott RealTime HCV Genotype II. Materials and methods: 19 plasma samples from HCV/HIV patients showing nonstandard genotyping results were studied by sequencing the NS5B and 5’UTR/core regions of viral genomes. Genotyping kits manufactured by Vector-Best and Interlabservice were additionally used. Results: Patients were found to be infected with RF2k/1b HCV. RF2k/1b HCV was found in 6% of the co-infected HCV/HIV patients. Conclusion: The routinely used test systems targeted to only one fragment of HCV genome, i.e. to 5’UTR/core, erroneously identify the RF2k/1b recombinant as genotype 2. Data on antiviral therapy for RF2k/1b HCV suggest that therapeutic regimens recommended for genotype 2 HCV are inadequate for RF2k/1b cases.Цель исследования: установить причину «нестандартных» результатов при генотипировании вируса гепатита С (ВГС) с применением тест-системы «Abbott RealTime HCV Genotype II». Материалы и методы исследования: 19 образцов плазмы крови коинфицированных ВГС/ВИЧ пациентов с «нестандартными» результатами генотипирования ВГС были исследованы путем секвенирования областей генома ВГС NS5B и 5’UTR/core, а также с применением генотипирующих наборов производства «Вектор-Бест» и «Интерлабсервис». Результаты: установлено, что обследованные пациенты инфицированы RF2k/1b ВГС. Среди коинфицированных ВИЧ/ВГС доля инфицированных RF2k/1b составляет 6%. Заключение: широко применяемые в рутинной практике тест-системы, мишенью которых является только один фрагмент генома вируса 5’UTR/core, ошибочно идентифицируют рекомбинант ВГС RF2k/1b как генотип 2. Данные о результатах противовирусной терапии у пациентов, инфицированных RF2k/1b, говорят о недостаточной эффективности схем, рекомендованных для лечения вируса генотипа 2