Balancing Specificity and Promiscuity in Enzyme Evolution: Multidimensional Activity Transitions in the Alkaline Phosphatase Superfamily

Highly proficient, promiscuous enzymes can be springboards for functional evolution, able to avoid loss of function during adaptation by their capacity to promote multiple reactions. We employ systematic comparative study of structure, sequence and substrate specificity to track the evolution of specificity and reactivity between promiscuous members of clades of the alkaline phosphatase (AP) superfamily. Construction of a phylogenetic tree of protein sequences maps out the likely transition zone between arylsulfatases (ASs) and phosphonate monoester hydrolases (PMHs). Kinetic analysis shows that all enzymes characterized have four chemically distinct phospho- and sulfoesterase activities, with rate accelerations ranging from 1011-1017-fold for their primary and 109-1012-fold for their promiscuous reactions, suggesting that catalytic promiscuity is widespread in the AP-superfamily. This functional characterization and crystallography reveal a novel class of ASs that is so similar in sequence to known PMHs that it had not been recognized as having diverged in function. Based on analysis of snapshots of catalytic promiscuity ‘in transition’ we develop possible models that would allow functional evolution and determine scenarios for trade-off between multiple activities. For the new ASs we observe largely invariant substrate specificity that would facilitate the transition from ASs to PMHs via trade-off-free molecular exaptation, i.e. evolution without initial loss of primary activity and specificity toward the original substrate. This ability to bypass low activity generalists provides a molecular solution to avoid adaptive conflict.BBSR

Contrasting origin of B chromosomes in two cervids (Siberian roe deer and grey brocket deer) unravelled by chromosome-specific DNA sequencing

Abstract Background B chromosomes are dispensable and variable karyotypic elements found in some species of animals, plants and fungi. They often originate from duplications and translocations of host genomic regions or result from hybridization. In most species, little is known about their DNA content. Here we perform high-throughput sequencing and analysis of B chromosomes of roe deer and brocket deer, the only representatives of Cetartiodactyla known to have B chromosomes. Results In this study we developed an approach to identify genomic regions present on chromosomes by high-throughput sequencing of DNA generated from flow-sorted chromosomes using degenerate-oligonucleotide-primed PCR. Application of this method on small cattle autosomes revealed a previously described KIT gene region translocation associated with colour sidedness. Implementing this approach to B chromosomes from two cervid species, Siberian roe deer (Capreolus pygargus) and grey brocket deer (Mazama gouazoubira), revealed dramatically different genetic content: roe deer B chromosomes consisted of two duplicated genomic regions (a total of 1.42-1.98 Mbp) involving three genes, while grey brocket deer B chromosomes contained 26 duplicated regions (a total of 8.28-9.31 Mbp) with 34 complete and 21 partial genes, including KIT and RET protooncogenes, previously found on supernumerary chromosomes in canids. Sequence variation analysis of roe deer B chromosomes revealed a high frequency of mutations and increased heterozygosity due to either amplification within B chromosomes or divergence between different Bs. In contrast, grey brocket deer B chromosomes were found to be more homogeneous and resembled autosomes in patterns of sequence variation. Similar tendencies were observed in repetitive DNA composition. Conclusions Our data demonstrate independent origins of B chromosomes in the grey brocket and roe deer. We hypothesize that the B chromosomes of these two cervid species represent different stages of B chromosome sequences evolution: probably nascent and similar to autosomal copies in brocket deer, highly derived in roe deer. Based on the presence of the same orthologous protooncogenes in canids and brocket deer Bs we argue that genomic regions involved in B chromosome formation are not random. In addition, our approach is also applicable to the characterization of other evolutionary and clinical rearrangements

Contrasting origin of B chromosomes in two cervids (Siberian roe deer and grey brocket deer) unravelled by chromosome-specific DNA sequencing.

BACKGROUND: B chromosomes are dispensable and variable karyotypic elements found in some species of animals, plants and fungi. They often originate from duplications and translocations of host genomic regions or result from hybridization. In most species, little is known about their DNA content. Here we perform high-throughput sequencing and analysis of B chromosomes of roe deer and brocket deer, the only representatives of Cetartiodactyla known to have B chromosomes. RESULTS: In this study we developed an approach to identify genomic regions present on chromosomes by high-throughput sequencing of DNA generated from flow-sorted chromosomes using degenerate-oligonucleotide-primed PCR. Application of this method on small cattle autosomes revealed a previously described KIT gene region translocation associated with colour sidedness. Implementing this approach to B chromosomes from two cervid species, Siberian roe deer (Capreolus pygargus) and grey brocket deer (Mazama gouazoubira), revealed dramatically different genetic content: roe deer B chromosomes consisted of two duplicated genomic regions (a total of 1.42-1.98 Mbp) involving three genes, while grey brocket deer B chromosomes contained 26 duplicated regions (a total of 8.28-9.31 Mbp) with 34 complete and 21 partial genes, including KIT and RET protooncogenes, previously found on supernumerary chromosomes in canids. Sequence variation analysis of roe deer B chromosomes revealed a high frequency of mutations and increased heterozygosity due to either amplification within B chromosomes or divergence between different Bs. In contrast, grey brocket deer B chromosomes were found to be more homogeneous and resembled autosomes in patterns of sequence variation. Similar tendencies were observed in repetitive DNA composition. CONCLUSIONS: Our data demonstrate independent origins of B chromosomes in the grey brocket and roe deer. We hypothesize that the B chromosomes of these two cervid species represent different stages of B chromosome sequences evolution: probably nascent and similar to autosomal copies in brocket deer, highly derived in roe deer. Based on the presence of the same orthologous protooncogenes in canids and brocket deer Bs we argue that genomic regions involved in B chromosome formation are not random. In addition, our approach is also applicable to the characterization of other evolutionary and clinical rearrangements

Drug-Induced Atrial Fibrillation / Atrial Flutter

Drug-induced atrial fibrillation / flutter (DIAF) is a serious and potentially life-threatening complication of pharmacotherapy. Purpose of the work: systematization and analysis of scientific literature data on drugs, the use of which can cause the development of DIAF, as well as on epidemiology, pathophysiological mechanisms, risk factors, clinical picture, diagnosis and differential diagnosis, treatment and prevention of DIAF. Analysis of the literature has shown that many groups of drugs can cause the development of DIAF, with a greater frequency while taking anticancer drugs, drugs for the treatment of the cardiovascular, bronchopulmonary and central nervous systems. The mechanisms and main risk factors for the development of DIAF have not been finally established and are known only for certain drugs, therefore, this section requires further study. The main symptoms of DIAF are due to the severity of tachycardia and their influence on the parameters of central hemodynamics. For diagnosis, it is necessary to conduct an electrocardiogram (ECG) and Holter monitoring of an ECG and echocardiography. Differential diagnosis should be made with AF, which may be caused by other causes, as well as other rhythm and conduction disturbances. Successful treatment of DIAF is based on the principle of rapid recognition and immediate discontinuation of drugs (if possible), the use of which potentially caused the development of adverse drug reactions (ADR). The choice of management strategy: heart rate control or rhythm control, as well as the method of achievement (medication or non-medication), depends on the specific clinical situation. For the prevention of DIAF, it is necessary to instruct patients about possible symptoms and recommend self-monitoring of the pulse. It is important for practitioners to be wary of the risk of DIAF due to the variety of drugs that can potentially cause this ADR

Characterisation of Australian MRSA Strains ST75- and ST883-MRSA-IV and Analysis of Their Accessory Gene Regulator Locus

Background: Community-acquired methicillin-resistant Staphylococcus aureus have become a major problem in Australia. These strains have now been isolated throughout Australia including remote Indigenous communities that have had minimal exposure to healthcare facilities. Some of these strains, belonging to sequence types ST75 and ST883, have previously been reported to harbour highly divergent alleles of the housekeeping genes used in multilocus sequence typing. Methodology/Principal Findings: ST75-MRSA-IV and ST883-MRSA-IV isolates were characterised in detail. Morphological features as well as 16S sequences were identical to other S. aureus strains. Although a partial rnpB gene sequence was not identical to previously known S. aureus sequences, it was found to be more closely related to S. aureus than to other staphylococci. Isolates also were screened using diagnostic DNA microarrays. These isolates yielded hybridisation results atypical for S. aureus. Primer directed amplification assays failed to detect species markers (femA, katA, sbi, spa). However, arbitrarily primed amplification indicated the presence of unknown alleles of these genes. Isolates could not be assigned to capsule types 1, 5 or 8. The allelic group of the accessory gene regulator (agr) locus was not determinable. Sequencing of a region of agrB, agrC and agrD (approximately 2,100 bp) revealed a divergent sequence. However, this sequence is more related to S. aureus agr alleles I and IV than to agr sequences from other Staphylococcus species. The predicted autoinducing peptide (AIP) sequence of ST75 was identical to that of agr group I, while the predicted AIP sequence of ST883 was identical to agr group IV. Conclusions/Significance: The genetic properties of ST75/ST883-MRSA may be due to a series of evolutionary events in ancient insulated S. aureus strains including a convergent evolution leading to agr group I- or IV-like AIP sequences and a recent acquisition of SCCmec IV elements

COMPLICATED DIAGNOSIS: RETROPERITONEAL ALVEOCOCCOSIS IN THE GUISE OF ORMOND’S DISEASE

Diagnosis of parasitic diseases, in a type of their rarity and diversity, always presents considerable difficulties. Clinical observation of an alveococcosis with invasion in the retroperitoneal space with distribution on mediastinal fat proceeding with progressing retroperitoneal fibrosis with ureteral obstruction and hydronephrosis development, with an aorta lumen stenosis, with formation of SVC syndrome in the absence of changes in other parts of the body, first of all in the liver, in the absence of eosinophilia in the peripheral blood throughout all disease and lack of antibodies to echinococci in the blood serum, twice during observation determined by an immunofermental method is presented in this article

Влияние химической структуры карбоксилсодержащих полимеров на морфологию осадков и устойчивость дисперсий карбонатов

The influence of the molecular structure of carboxyl-containing polymers on their inhibitory and stabilizing effect in the precipitation processes was studied using a number of polymeric acids with different contents of carboxyl groups (X): polyacrylic acid (Mn = 5100 g-mol–1, X = 61.11 %), polymethacrylic acid (Mn = 5500 g-mol–1, X = 48.79 %), copolymer of methacrylic acid with turpentine terpene (α-pinene) (Mn = 350 g-mol–1, X = 14.54 %). It has been established that polyacid molecules slow down the process of crystal growth and recrystallization of the amorphous phase into more stable crystalline forms, which leads to a change in the morphology and structure of a carbonate precipitate. It has also been shown that polycarboxylic acids increase the colloidal stability of a mixed dispersion of carbonates and kaolin. The synergism of the stabilizing effect of mixtures of different hydrophobicity-polyacids was noted: in the presence of a mixture of polyacrylic acid with a copolymer of methacrylic acid with turpentine terpene, the stabilization effect increases 2 times compared to dispersion without additives and 1.4–1.8 times compared with individual components of the mixture. С целью изучения влияния молекулярно-структурного строения карбоксилсодержащих полимеров на их ингибирующее и стабилизирующее действие в процессах осадкообразования был исследован ряд полимерных кислот с различным содержанием карбоксильных групп (Х): полиакриловая кислота (Мn = 5100 г-моль–1, Х = 61,11 %), полиметакриловая кислота (Мn = 5500 г-моль–1, Х = 48,79 %), сополимер метакриловой кислоты с терпеном скипидара (α-пинен) (Мn = 3500 г-моль–1, Х = 14,54 %). Установлено, что введение поликислот замедляет процесс роста кристаллов и приводит к изменению морфологии и структуры осадка карбонатов в результате рекристаллизации аморфной фазы в более устойчивые кристаллические формы. Показано, что поликарбоновые кислоты повышают коллоидную стабильность смешанной дисперсии карбонатов и каолина. Отмечен синергизм стабилизирующего действия смесей поликислот с различной гидрофобностью: в присутствии смеси полиакриловой кислоты с сополимером метакриловой кислоты с терпеном скипидара стабилизационный эффект возрастает 2 раза по сравнению с дисперсией без добавок и в 1,4–1,8 раза по сравнению с индивидуальными компонентами смеси.

Genotyping of Capreolus pygargus Fossil DNA from Denisova Cave Reveals Phylogenetic Relationships between Ancient and Modern Populations

BACKGROUND: The extant roe deer (Capreolus Gray, 1821) includes two species: the European roe deer (C. capreolus) and the Siberian roe deer (C. pygargus) that are distinguished by morphological and karyotypical differences. The Siberian roe deer occupies a vast area of Asia and is considerably less studied than the European roe deer. Modern systematics of the Siberian roe deer remain controversial with 4 morphological subspecies. Roe deer fossilized bones are quite abundant in Denisova cave (Altai Mountains, South Siberia), where dozens of both extant and extinct mammalian species from modern Holocene to Middle Pleistocene have been retrieved. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed a 629 bp fragment of the mitochondrial control region from ancient bones of 10 Holocene and four Pleistocene Siberian roe deer from Denisova cave as well as 37 modern specimen belonging to populations from Altai, Tian Shan (Kyrgyzstan), Yakutia, Novosibirsk region and the Russian Far East. Genealogical reconstructions indicated that most Holocene haplotypes were probably ancestral for modern roe deer populations of Western Siberia and Tian Shan. One of the Pleistocene haplotypes was possibly ancestral for modern Yakutian populations, and two extinct Pleistocene haplotypes were close to modern roe deer from Tian Shan and Yakutia. Most modern geographical populations (except for West Siberian Plains) are heterogeneous and there is some tentative evidence for structure. However, we did not find any distinct phylogenetic signal characterizing particular subspecies in either modern or ancient samples. CONCLUSION/SIGNIFICANCE: Analysis of mitochondrial DNA from both ancient and modern samples of Siberian roe deer shed new light on understanding the evolutionary history of roe deer. Our data indicate that during the last 50,000 years multiple replacements of populations of the Siberian roe deer took place in the Altai Mountains correlating with climatic changes. The Siberian roe deer represent a complex and heterogeneous species with high migration rates and without evident subspecies structure. Low genetic diversity of the West Siberian Plain population indicates a recent bottleneck or founder effect

Анализ многолетнего опыта изучения инактивированных культуральных вакцин для профилактики f клещевого энцефалита отечественного и зарубежного производства по показателю качества -специфическая активность (иммуногенность)

The analysis of the use of the method for the evaluation of specific (immunogenic) activity of vaccines for preventing tick-borne encephalitis (TBE vaccines) against protective dilution (PD50) and the minimum immunizing dose (MID50) has been performed. The method was standardized and submitted to the regulatory documents for TBE vaccines authorized in the Russian Federation. When analyzing the results of the study of specific (immunogenic) activity of TBE vaccines (107 TBE vaccine batches by different manufacturers have been studied) it was confirmed that the choice of real lethal dose (RLD50) indicator of TBE virus (test strain «Absettarov») in the range of 100-3000 LD50, the reasonability of using BALB /c cell-line mice, the effectiveness of the national method of determining immunogenicity in terms of MlD50 for TBE vaccines authorized in the Russian Federation. The reasonability of using immunogenicity reference standard for TBE-OSO 42-28-48 to assess the reproducibility of the experiments, and the homogeneity of laboratory animals in terms of quality. Methods for determining TBE vaccine immunogenicity («specific activity (immunogenicity)» in terms of PD50 and MID50 is applicable both for Russian commercial TBE vaccines and for FSME-Immun vaccine, manufactured by «Baxter Vaccine AG», Austria.Проведен анализ применения метода оценки специфической (иммуногенной) активности современных вакцин клещевого энцефалита (вакцины КЭ) по протективному разведению (ПР50) и минимальной иммунизирующей дозе (МИД50). Метод стандартизован и внесен в нормативные документы на зарегистрированные в Российской Федерации вакцины КЭ. При анализе результатов изучения специфической (иммуногенной) активности вакцин КЭ (исследовано 107 серий вакцин КЭ различных производителей) подтверждены: выбор показателя реальной летальной дозы (РЛД50) вируса КЭ (тест-штамм «Абсеттаров») в пределах 100-3000 ЛД50, целесообразность применения линейных мышей BALB/с, эффективность применения отечественного метода определения иммуногенности по показателю МИД50 для вакцин КЭ, зарегистрированных в Российской Федерации. Подтверждена целесообразность применения стандартного образца иммуногенности вакцины КЭ - ОСО 42-28-48 для оценки сходимости результатов постановки экспериментов и однородности лабораторных животных по качеству. Метод определения иммуногенности вакцин КЭ (показатель качества «Специфическая активность (иммуногенность)» по показателям ПР50 и МИД50 применим как для российских коммерческих вакцин КЭ, так и для вакцины ФСМЕ-Иммун, производства компании «Бакстер вакцины АГ», Австрия