Structural characterization of a hypothetical protein: a potential agent involved in trimethylamine metabolism in Catenulispora acidiphila

Catenulispora acidiphila is a newly identified lineage of actinomycetes that produces antimicrobial activities and represents a promising source of novel antibiotics and secondary metabolites. Among the discovered protein coding genes, 68 % were assigned a putative function, while the remaining 32 % are genes encoding “hypothetical” proteins. Caci_0382 is one of the “hypothetical” proteins that has very few homologs. Sequence analysis shows that the protein belongs to the NTF2-like protein family. The structure of Caci_0382 demonstrates that it shares the same fold and has a similar active site as limonene-1,2-epoxide hydrolase, which suggests that it may have a related function. Using a fluorescence thermal shift assay, we identified stabilizing compounds that suggest potential natural ligands of Caci_0382. Using this information, we determined the crystal structure in complex with trimethylamine to provide a better understanding of the function of this uncharacterized protein. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10969-014-9176-z) contains supplementary material, which is available to authorized users

Structural and biochemical studies of novel Aldo-keto Reductases (AKRs) for the biocatalytic conversion of 3-hydroxybutanal to 1,3-butanediol

The non-natural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases. This pathway requires an aldo-keto reductase (AKR) selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one pot synthesis. In this work, we screened over 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 from Pseudomonas aeruginosa showed the highest activity and was selected for comparative studies with STM2406 from Salmonella typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as cofactor, reduced a broad range of aldehydes, and showed low activity toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for activity against 3-HB and aromatic aldehydes, which include the residues of the substrate binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced both activity and affinity of this protein toward 3-HB resulting in a seven-fold increase in kcat/Km. Our work provided further insights into the molecular mechanisms of substrate selectivity of AKRs and rational design of these enzymes towards new substrates. Importance In this study, we identified several aldo-keto reductases with significant activity in the reduction of 3-hydroxybutanal to 1,3-BDO, an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate binding residues including the two structural determinants necessary for high activity in the biosynthesis of 1,3-BDO. This work expands our understanding of the molecular mechanisms of substrate selectivity of AKRs and the potential for protein engineering of these enzymes for applications in the biocatalytic production of 1,3-BDO and other valuable chemicals

ВОЗМОЖНОСТИ АРТРОПЛАСТИКИ ТАЗОБЕДРЕННОГО СУСТАВА НА ФОНЕ СУБКОМПЕНСИРОВАННОГО КОСТНОГО МЕТАБОЛИЗМА

The results of clinic and X – ray monitoring after total hip replacement of patients with disturbed bone metabolism had been analyzed. Zoledronic acid effectiveness combined with calcium and Vit D was studied. Parameters of functional activity, life quality and DEXA monitoring had been researched. Thus we can conclude that short time treatment is effective for life quality and functional activity.Проанализированы результаты клинического и лучевого мониторинга у пациентов, перенесших артропластику тазобедренного сустава, на фоне субкомпенсированного костного метаболизма. Изучена эффективность золедроновой кислоты в сочетании с препаратами кальция и активными метаболитами витамина Д3. Изучены объективные параметры функциональной активности, субъективное восприятие пациентами качества жизни, а также количественные параметры лучевого мониторинга. Отмечено, что короткие курсы антирезорбтивной терапии эффективно влияют на параметры функциональной активности и качества жизни пациентов

АРТРОПЛАСТИКА ТАЗОБЕДРЕННОГО СУСТАВА ПРИ ДЕСТРУКТИВНО-ДИСТРОФИЧЕСКИХ ПОРАЖЕНИЯХ

Introduction. Hip arthroplasty is considered to be the most effective method providing social and household reintegration for destructive-dystrophic lesions. Goal of the study is to improve the results of surgical treatment using the technology of arthroplasty in patients with decompensated forms of destructive-dystrophic hip joint lesions. Material and methods. It was an open prospective, randomized study carried out on the base of traumatology and ortopaedics department of the Bashkir State Medical University including 710 patients with decompensated destructivedystrophic hip joint lesions. All patients (n=710, of which 6.1% were female) depending on the diacritic approach were divided into groups: the control group included (n=406) patients with hip joint trauma who were provided a conventional range of diagnostic and treatment procedures; the experimental group (n=304) included patients who had a range of diagnostics and treatment of destructive-dystrophic lesions of hip joints based on assessment of the connective tissue phase state. Results. Biometric indicators of stance and walking phase firmly improved in all groups 3 years after arthroplasty (p<0.05). Comparative analysis of arthroplasty results in the research groups showed effectiveness of the developed concept to surgically treat destructive and dystrophic lesions of hip joint on the basis of the system approach in the form of improved parameters of the components of the local and systemic level by 2.38% and 2.3% for osteoarthrosis (p<0.05), by 1.61% and 1.84% for aseptic necrosis of femoral head (p>0.05), by 5.62% and 4.37% for post-traumatic damage of hip joint (p<0.05). Conclusion. Analysis of short-term and long-term results of arthroplasty showed high efficiency of the developed concept of surgical treatment and monitoring of connective tissue phase state at destructive-dystrophic lesions of hip joints based on the system approach in the form of reduced pain syndrome, improvement of functional possibilities and patients life quality.Введение. Эндопротезирование тазобедренного сустава (ТБС) считается наиболее эффективным методом, обеспечивающим социальную и бытовую реинтеграцию при деструктивно-дистрофических поражениях.Цель исследования: улучшить результаты хирургического лечения по технологии артропластики пациентов с декомпенсированными формами деструктивно-дистрофических поражений ТБС.Материалы и методы. На клинических базах кафедры травматологии и ортопедии Башкирского государственного медицинского университета проведено открытое проспективное рандомизированное исследование, включающее 710 пациентов с декомпенсированными деструктивно-дистрофическими поражениями ТБС. Все пациенты (n=710, из них 61,97% женщин), в зависимости от диагностических подходов, были разделены на группы: контрольную группу составили (n=406) пациенты с поражением ТБС, которым проводился традиционный комплекс диагностических и лечебных процедур; исследуемая группа (n=304) включала пациентов, которым проводились комплексная диагностика и лечение деструктивно-дистрофических поражений ТБС на основе оценки фазового состояния соединительной ткани.Результаты. Показатели биометрии фаз опоры и ходьбы достоверно улучшились во всех группах через три года после артропластики (р<0,05). Сравнительный анализ результатов артропластики в группах показал эффективность разработанной концепции хирургического лечения при поражениях ТБС на основе системного подхода в виде улучшения показателей компонент локального и системного уровня на 2,38 и 2,3% при остеоартрозе (р<0,05), на 1,61 и 1,84% при асептическом некрозе головки бедра (р>0,05), на 5,62 и 4,37% при посттравматических поражениях ТБС (р<0,05).Заключение. Анализ ближайших и отдаленных результатов артропластики показал высокую эффективность разработанной концепции хирургического лечения и мониторинга фазового состояния соединительной ткани при деструктивно-дистрофических поражениях ТБС на основе системного подхода в виде уменьшения болевого синдрома, улучшения функциональных возможностей и качества жизни пациентов.

СРАВНИТЕЛЬНЫЙ АНАЛИЗ ХИРУРГИЧЕСКОГО ЛЕЧЕНИЯ НЕСТАБИЛЬНЫХ ПЕРЕЛОМОВ КЛЮЧИЦЫ

Introduction. Study of different treatments effectiveness of these injuries allows to distinguish the rational side of medical factors, which directly reflect the key pathogenetic mechanisms. A substantial portion of the currently used methods of clavicle fractures surgical treatment allow one to achieve rapid recovery of structural and functional patterns of the patient in most cases.The purpose of the study is to improve the results of surgical treatment of patients with fractures of the clavicle in the middle third. Material and methods. The study included 104 patients with fractures of the clavicle in the middle third.In the main group (48 patients) the clavicle lockable rod of the original design was used for osteosynthesis, in the comparison group (56 patients) plate osteosynthesis was performed.Results and discussion. The proposed technology of operative treatment of clavicle fractures in the middle third using the original hip compression-locking rod must improve the results of treatment in this group of patients. The developed algorithm of diagnostics, treatment and rehabilitation of patients with clavicle fractures allows to improve the results of treatment, provides domestic and social reintegratie patients. Conclusion. Analysis of the clinical studies results have revealed a reliable advantage of the results on a scale DASH and a lower frequency of complications in the main group (p<0.05).Введение. Изучение эффективности различных способов лечения данных повреждений позволяет выделить рациональные стороны лечебных факторов, что прямо отражает ключевые патогенетические механизмы. При этом значительная часть используемых в настоящее время методик оперативного лечения переломов ключицы позволяют достичь быстрого восстановления структурно-функциональных стереотипов пациента в большинстве случаев. Цель исследования: улучшение результатов хирургического лечения пациентов с переломами ключицы в средней трети.Материал и методы. В исследование вошли 104 пациента с переломами ключицы в средней трети. В основной группе (48 пациентов) для остеосинтеза ключицы использовался блокируемый стержень оригинальной конструкции, в группе сравнения (56 пациентов) проводился накостный остеосинтез. Оценка результатов проводилась по шкале DASH через 3, 6 и 12 месяцев после остеосинтеза. Результаты и их обсуждение. Предложенная технология оперативного лечения переломов ключицы в средней трети с использованием оригинального компрессирующего блокируемого стержня повысила результативность лечения данной группы пациентов. Разработанный алгоритм диагностики, лечения и реабилитации пострадавших с переломами ключицы позволяет улучшить результаты лечения, обеспечивает бытовую и социальную реинтеграцию пациентов.Заключение. Анализ результатов клинического исследования выявил достоверное преимущество результатов по шкале DASH и меньшую частоту осложнений в основной группе (p<0,05)

Surgical treatment of unstable fractures of the clavicle

Clavicle fractures are one of the most common types of injuries and range from 2.6 to 4% of all skeletal injuries. The aim of the study was to improve the results of surgical treatment of patients with fractures of the clavicle in the middle third. Material and methods. The study included 104 patients with fractures of the clavicle in the middle third. In the main group (48 patients) for osteosynthesis of the clavicle, a lockable rod of the original design was used, in the comparison group (56 patients) bone osteosynthesis was performed. Evaluation of the results was carried out on the DASH scale 3,6 and 12 months after osteosynthesis. Results: the proposed technology of operative treatment of clavicle fractures in the middle third with the use of an original compression blocking rod to improve the results of treatment of this group of patients. The developed algorithm for diagnosing, treating and rehabilitating victims with fractures of the collarbone allows to improve the results of treatment, provides for household and social reintegration of patients. Analysis of the results of the clinical trial revealed a significant advantage of the results on the DASH scale and a lower incidence of complications in the main group (p <0.05).Переломы ключицы относятся к одному из наиболее часто встречающихся видов травм и составляют от 2,6 до 4% среди всех повреждений скелета. Цель исследования: улучшение результатов хирургического лечения пациентов с переломами ключицы в средней трети. Материал и методы. В исследование вошли 104 пациента с переломами ключицы в средней трети. В основной группе (48 пациентов) для остеосинтеза ключицы использовался блокируемый стержень оригинальной конструкции, в группе сравнения (56 пациентов) проводился накостный остеосинтез. Оценка результатов проводилась по шкале DASH через 3,6 и 12 месяцев после остеосинтеза. Результаты: предложенная технология оперативного лечения переломов ключицы в средней трети с использованием оригинального компрессирующего блокируемого стержня улучшить результаты лечения данной группы пациентов. Разработанный алгоритм диагностики, лечения и еабилитации пострадавших с переломами ключицы позволяет улучшить результаты лечения, обеспечивает бытовую и социальную реинтгеграцию пациентов. Анализ результатов клинического исследования выявил достоверное преимущество результатов по шкале DASH и меньшую частоту осложнений в основной группе (р<0,05)

2′-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5′-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2′-O position of the first nucleotide. To investigate the conformational changes of the complex during 2′-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2′-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target

Targeting Human Central Nervous System Protein Kinases: An Isoform Selective p38αMAPK Inhibitor that Attenuates Disease Progression in Alzheimer\u27s Disease Mouse Models

The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150\u27s exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior

Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction

Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction

Circular Permutation in the Ω-Loop of TEM-1 β-Lactamase Results in Improved Activity and Altered Substrate Specificity

Generating diverse protein libraries that contain improved variants at a sufficiently high frequency is critical for improving the properties of proteins using directed evolution. Many studies have illustrated how random mutagenesis, cassette mutagenesis, DNA shuffling and similar approaches are effective diversity generating methods for directed evolution. Very few studies have explored random circular permutation, the intramolecular relocation of the N- and C-termini of a protein, as a diversity-generating step for directed evolution. We subjected a library of random circular permutations of TEM-1 β-lactamase to selections on increasing concentrations of a variety of β-lactam antibiotics including cefotaxime. We identified two circularly permuted variants that conferred elevated resistance to cefotaxime but decreased resistance to other antibiotics. These variants were circularly permuted in the Ω-loop proximal to the active site. Remarkably, one variant was circularly permuted such that the key catalytic residue Glu166 was located at the N-terminus of the mature protein