Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab

<p>Abstract</p> <p>Background</p> <p>Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking.</p> <p>Results</p> <p>Using degenerative primers and the rapid amplification of cDNA ends (RACE) procedure, we cloned the Duplex-Specific Nuclease (DSN) gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 – 7.5) and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact.</p> <p>Conclusion</p> <p>We describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate specificity of DSN should prove valuable in certain molecular biology applications.</p

Entanglement Measures for Single- and Multi-Reference Correlation Effects

Electron correlation effects are essential for an accurate ab initio description of molecules. A quantitative a priori knowledge of the single- or multi-reference nature of electronic structures as well as of the dominant contributions to the correlation energy can facilitate the decision regarding the optimum quantum chemical method of choice. We propose concepts from quantum information theory as orbital entanglement measures that allow us to evaluate the single- and multi-reference character of any molecular structure in a given orbital basis set. By studying these measures we can detect possible artifacts of small active spaces.Comment: 14 pages, 4 figure

Step-Wise Computational Synthesis of Fullerene C60 derivatives. 1.Fluorinated Fullerenes C60F2k

The reactions of fullerene C60 with atomic fluorine have been studied by unrestricted broken spin-symmetry Hartree-Fock (UBS HF) approach implemented in semiempirical codes based on AM1 technique. The calculations were focused on a sequential addition of fluorine atom to the fullerene cage following indication of the cage atom highest chemical susceptibility that is calculated at each step. The effectively-non-paired-electron concept of the fullerene atoms chemical susceptibility lays the foundation of the suggested computational synthesis. The obtained results are analyzed from energetic, symmetry, and the composition abundance viewpoints. A good fitting of the data to experimental findings proves a creative role of the suggested synthesis methodology.Comment: 33 pages, 11 figures, 2 tables, 2 chart

Continuous symmetry of C60 fullerene and its derivatives

Conventionally, the Ih symmetry of fullerene C60 is accepted which is supported by numerous calculations. However, this conclusion results from the consideration of the molecule electron system, of its odd electrons in particular, in a close-shell approximation without taking the electron spin into account. Passing to the open-shell approximation has lead to both the energy and the symmetry lowering up to Ci. Seemingly contradicting to a high-symmetry pattern of experimental recording, particularly concerning the molecule electronic spectra, the finding is considered in the current paper from the continuous symmetry viewpoint. Exploiting both continuous symmetry measure and continuous symmetry content, was shown that formal Ci symmetry of the molecule is by 99.99% Ih. A similar continuous symmetry analysis of the fullerene monoderivatives gives a reasonable explanation of a large variety of their optical spectra patterns within the framework of the same C1 formal symmetry exhibiting a strong stability of the C60 skeleton.Comment: 11 pages. 5 figures. 6 table

Global hybrids from the semiclassical atom theory satisfying the local density linear response

We propose global hybrid approximations of the exchange-correlation (XC) energy functional which reproduce well the modified fourth-order gradient expansion of the exchange energy in the semiclassical limit of many-electron neutral atoms and recover the full local density approximation (LDA) linear response. These XC functionals represent the hybrid versions of the APBE functional [Phys. Rev. Lett. 106, 186406, (2011)] yet employing an additional correlation functional which uses the localization concept of the correlation energy density to improve the compatibility with the Hartree-Fock exchange as well as the coupling-constant-resolved XC potential energy. Broad energetical and structural testings, including thermochemistry and geometry, transition metal complexes, non-covalent interactions, gold clusters and small gold-molecule interfaces, as well as an analysis of the hybrid parameters, show that our construction is quite robust. In particular, our testing shows that the resulting hybrid, including 20\% of Hartree-Fock exchange and named hAPBE, performs remarkably well for a broad palette of systems and properties, being generally better than popular hybrids (PBE0 and B3LYP). Semi-empirical dispersion corrections are also provided.Comment: 12 pages, 4 figure

СЛУЧАЙ РАЗВИТИЯ ИНТРАМУРАЛЬНОЙ ГЕМАТОМЫ ЛЕВОГО ПРЕДСЕРДИЯ ПОСЛЕ ПРОВЕДЕНИЯ ЧРЕСКОЖНОГО КОРОНАРНОГО ВМЕШАТЕЛЬСТВА

Left atrial Intramural hematoma (LAIH) is rare complication cardiac invasive procedures. The article analyses the mechanism of formation of this pathology. The case of the LAIH after percutaneous coronary intervention (PCI) is presented. The patient complained of intensive chest pain. When performing ECHO, LAIH was found. LAIH occupied a large part of the cavity left atrium. The conservative supervision of the patient was chosen despite the compession of the left inferior pulmonary vein and symptoms of the acute heart failure. The successful symptomatic treatment was conducted. Taking into consideration the recent PCI dual antiplatelet therapy was not interrupted. LAIH gradually regressed. Five weeks after the desease began, the patient was discharged from the hospital. Nine months after almost full lysis of LAIH was observed. The discussion section is devoted to the issues of diagnosis, the choice of medical tactics, the necessity of surgical treatment and the stop of antitrombotic theatment.Интрамуральная гематома левого предсердия (ИГЛП) – редко встречающееся осложнение вмешательств на сердце. В статье рассматриваются механизмы формирования этой патологии. Представлен случай развития ИГЛП после проведения чрескожного коронарного вмешательства (ЧКВ). По окончании процедуры больной пожаловался на интенсивные боли в груди. При проведении ЭхоКГ выявлена ИГЛП, занимающая большую часть полости левого предсердия (ЛП). Несмотря на компрессию левой нижней легочной вены и симптомы острой сердечной недостаточности, была выбрана консервативная тактика ведения, проводилось успешное симптоматическое лечение. Учитывая недавнее ЧКВ, двойная дезагрегантная терапия не прерывалась. ИГЛП постепенно уменьшалась в размерах. Через пять недель от начала заболевания пациент был выписан из стационара, через девять месяцев наблюдался практически полный лизис ИГЛП. Раздел обсуждения посвящен вопросам диагностики, выбора лечебной тактики, целесообразности проведения хирургического лечения и отмены антитромботической терапии

Особенности характеристик миокарда пациентов с острым инфарктом миокарда с подъемом сегмента ST и сахарным диабетом по данным магнитно-резонансной томографии сердца с контрастированием

Introduction. The presence of diabetes mellitus in patients with acute infarction significantly worsens short- and long-term prognosis, but the features of the course of the infarction in this category of patients have not been fully studied.Objective. To compare clinical, functional and structural myocardial characteristics of patients with acute ST-segment elevation myocardial infarction with and without diabetes mellitus.Materials and methods. The study included 91 patients with revascularized ST-segment elevation myocardial infarction (41 patients with diabetes mellitus). All patients underwent cardiac MRI with contrast, including myocardial T1 mapping, tissue analysis of left ventricular myocardium with determination of infarct zone, heterogeneous zone, edema zone, pre- and post-contrast T1 values, extracellular volume values of healthy myocardium and infarct zone.Results. The presence of diabetes in patients with AMI was associated with increased EDV LV: 153 ± 38 and 181 ± 58 ml (p = 0.007), increased ESV LV: 76 ml [54–93] and 87 ml [71–122] (p = 0.035). Infarct size was significantly larger in patients with diabetes than in those without diabetes: 37 ± 15 g (95% CI: 33–41) and 47 ± 21 g (95% CI: 40–53), p = 0.017. Patients with acute infarction and diabetes mellitus had higher extracellular volume values in both healthy myocardium: 24% [22–27] and 28% [24–30], p = 0.002, and in the infarct zone: 48% [40–58] and 58% [50–61], p = 0.016.Conclusions. In patients with ST-segment elevation myocardial infarction with diabetes mellitus, the infarct size, the edema area, and the value of the extracellular volume fraction both in the infarct area and in the preserved myocardium were larger than in patients without diabetes, which may underlie the development of myocardial dysfunction and further progression of heart failure.Введение. Наличие сахарного диабета (СД) у пациентов с острым инфарктом миокарда (ОИМ) существенно ухудшает краткосрочный и долгосрочный прогноз, однако особенности течения инфаркта у данной категории пациентов до конца не изучены.Цель. Сравнить клинические, функциональные и структурные характеристики миокарда пациентов с ОИМ с подъемом сегмента ST с СД и без СД.Материалы и методы. В исследование включен 91 пациент с реваскуляризированным острым инфарктом миокарда с подъемом сегмента ST (41 пациент с СД, 50 пациентов без СД). Всем пациентам выполнена МРТ сердца с контрастированием, в том числе Т1-картирование миокарда, проведен тканевой анализ миокарда левого желудочка с определением зоны инфаркта, гетерогенной зоны, зоны отека, значений до- и постконтрастного Т1, фракции внеклеточного объема здорового миокарда и инфарктной зоны.Результаты. Наличие СД у пациентов с ОИМ ассоциировалось с увеличенным конечным диастолическим объемом левого желудочка: 153 ± 38 и 181 ± 58 мл (р = 0,007), увеличенным конечным систолическим объемом левого желудочка: 76 мл [54–93] и 87 мл [71– 122] (р = 0,035). Размер инфаркта у пациентов с СД был достоверно больше, чем у пациентов без СД: 37 ± 15 г (95% ДИ 33–41) и 47 ± 21 г (95% ДИ 40–53), р = 0,017. У пациентов с ОИМ и СД фракция внеклеточного объема была выше и в здоровом миокарде: 24% [22–27] и 28% [24–30], р = 0,002, и в инфарктной зоне: 48% [40–58] и 58% [50–61], р = 0,016.Заключение. У пациентов с инфарктом миокарда с подъемом сегмента ST и СД размер инфаркта, зона отека, а также значение фракции внеклеточного объема как в зоне инфаркта, так и в сохранном миокарде были больше, чем у пациентов без СД, что может лежать в основе развития дисфункции миокарда и дальнейшего прогрессирования сердечной недостаточности

Диагностическая эффективность перфузионной компьютерной томографии миокарда с чреспищеводной электрокардиостимуляцией у больных с исходным диагнозом «острый коронарный синдром»

Introduction. Computed tomography angiography (CTA) is widely used to detect atherosclerotic changes in coronary arteries (CA). However, the method is limited by the impossibility to perform functional assessment of detected stenoses. Perfusion computed tomography of the myocardium (PCT) can be used for this purpose.Aim of the study. To assess diagnostic accuracy of PCT of the myocardium with transesophageal electrocardiostimulation (TEES) and stress-echocardiography (stress-echoCG) with veloergometry (VEM) in detection of transient ischemia in patients with initial diagnosis of “acute coronary syndrome” (ACS) with borderline stenoses (50–75%) in CA in relation to measurements of fractional flow reserve (FFR).Materials and Methods. The study included 30 patients with the initial diagnosis of ACS with borderline (50–75%) stenoses in CA according to CTA or coronary angiography (CAG). Subsequently, they underwent myocardial PCT with 320-row detector with TEES, as well as stress-echoCG with VEM. Invasive FFR measurement was used as a reference method. FFR value &lt; 0.8 indicated hemodynamic significance of stenosis. Myocardial perfusion was assessed visually. Regional myocardial contractility of the left ventricle was assessed by stress-echoCG.Results. All patients were examined according to the stated protocol. PCT with TEES revealed FFR-significant stenoses with sensitivity, specificity, prognostic value of positive result and prognostic value of negative result 56, 93, 90, 65% respectively, stress-echoCG with VEM 62, 93, 91, 68% respectively.Conclusion. Myocardial PCT with TEES enables to detect perfusion defects associated with transient ischemia, and the diagnostic accuracy of the method in comparison with FFR is comparable with the already well-proven stress-echoCG. The use of PCT with TEES in combination with CTA can be considered as a promising diagnostic tool in patients without known coronary anatomy and with suspected ACS.Введение. Компьютерная томографическая ангиография (КТА) широко используется для выявления атеросклеротических изменений в коронарных артериях (КА). Однако метод ограничен невозможностью проведения функциональной оценки выявленных стенозов. С этой целью может использоваться перфузионная компьютерная томография миокарда (ПКТ).Цель исследования. Оценить диагностическую точность методов ПКТ миокарда с чреспищеводной электрокардиостимуляцией (ЧПЭС) и стресс-эхокардиографии (стресс-ЭхоКГ) с велоэргометрией (ВЭМ) в выявлении преходящей ишемии у больных с исходным диагнозом «острый коронарный синдром» (ОКС) с пограничными стенозами (50–75%) в КА по отношению к измерениям фракционного резерва кровотока (ФРК).Материалы и методы. В исследование были включены 30 больных с исходным диагнозом ОКС с пограничными (50–75%) стенозами в КА по данным КТА или коронарной ангиографии (КАГ). Впоследствии им были выполнены ПКТ миокарда на томографе с 320-рядным детектором с ЧПЭС, а также стресс-ЭхоКГ с ВЭМ. В качестве референтного метода использовалось инвазивное измерение ФРК. Значение показателя ФРК &lt; 0,8 указывало на гемодинамическую значимость стеноза. Перфузия миокарда оценивалась визуально. При проведении стресс-ЭхоКГ оценивалась региональная сократимость миокарда левого желудочка.Результаты. Все больные обследованы согласно заявленному протоколу. ПКТ с ЧПЭС выявила ФРК-значимые стенозы с чувствительностью, специфичностью, прогностической ценностью положительного результата и прогностической ценностью отрицательного результата 56, 93, 90, 65% соответственно, стресс-ЭхоКГ с ВЭМ 62, 93, 91, 68% соответственно.Выводы. ПКТ миокарда с ЧПЭС позволяет выявить дефекты перфузии, ассоциированные с преходящей ишемией, а диагностическая точность метода при сравнении с ФРК сопоставима с уже хорошо зарекомендовавшей себя стресс-ЭхоКГ. Использование ПКТ с ЧПЭС в комбинации с КТА можно рассматривать как перспективный диагностический инструмент у больных без известной коронарной анатомии и с подозрением на ОКС