Comparative analysis of the DNA isolated from thyme leaves using different methods

Background. The base for a molecular analysis is DNA of high quality. For DNA isolation, different kits or classical methods are used. For mass analysis, isolation with kits is a very expensive process. So, the objective of our investigation was to find a cheap method for high-quality DNA isolation from leaves of various thyme cultivars.Materials and methods. Leaves cut from thyme accessions (Thymus mastichina L. cv. ‘Svetliachok’, T. striatus Vahl. cv. ‘Jubileiniy’, T. vulgaris L. cv. ‘Fantasia’, and T. vulgaris cv. ‘Jalos’.) maintained ex situ in the collection of the Nikita Botanical Gardens were used as the material for the analysis. Light microscopy was used to study leaf anatomy and localize essential oil on leaf cross sections. Essential oil was extracted on Ginsberg devices, and phenolic content was measured with The Folin–Ciocâlteu reagent (FCR). Commercial kits (DiamondDNATM, PureLink® Plant Total DNA Purification Kit) and classical methods (CTAB, CTAB with 2% polyvinylpyrrolidone) were used for DNA isolation. DNA quality was evaluated spectrophotometrically, with electrophoresis (horizontal, automated system Agilent 4200 TapeStation) and PCR.Results. The analysis showed that the leaf blade mesophyll of four thyme cultivars had inclusions with essential oil. The content of essential oil and phenolic compounds was measured biochemically. Since the plants were characterized by the presence of secondary metabolites, DNA was isolated by different methods. Spectrophotometry demonstrated that the classical CTAB method and CTAB with 2% PVP provided the best results. Using an automated electrophoresis system, the presence of high-molecularweight DNA (more than 52000 bp) in significant amounts was detected in the samples isolated with DiamondDNATM kit and CTAB + 2% PVP.Conclusion. Among the tested kits and methods, CTAB + 2% PVP provided thyme DNA suitable for PCR and, presumably, for genome library preparation. The low cost of reagents for this technique makes it applicable for future mass analysis of plant material

Синтез цис- та транс-3-(4-гідроксифеніл)циклобутанкарбонових кислот та дослідження їх похідних як лігандів рецептора GPR-40

Aim. To synthesize cis- and trans-isomers of 3-(4-hydroxyphenyl)cyclobutanecarboxylic acid and evaluate the biological activity of their derivatives against GPR-40.Results and discussion. Cis- and trans-isomers of 3-(4-hydroxyphenyl)cyclobutanecarboxylic acid were synthesized. The derivatives of this compound were tested as GPR-40 agonists and exhibited the micromolar activity.Experimental part. The methyl ester of 3-(4-hydroxyphenyl)cyclobutanecarboxylic acid was obtained as a mixture of cis/trans-isomers in 3 steps starting from a commercially available 3-oxocyclobutanecarboxylic acid. Further transformation of this compound into isomerically pure 3-(4-hydroxyphenyl)cyclobutanecarboxylic acids was achieved in five steps based on the chromatographic separation of diastereomeric amide derivatives. New GPR-40 ligands were obtained by O-alkylation of a phenolic oxygen atom of the corresponding carboxylic acid methyl ester. The biological activity of the agonists synthesized was studied using a fluorometric bioassay and the engineered Chinese hamster ovary (CHO) stable cell line expressing the human GPR-40.Conclusions. An effective synthetic approach to 3-(4-hydroxyphenyl)cyclobutanecarboxylic acid allowing to isolate two single cis/trans-stereoisomers of this compound has been developed. In order to demonstrate the possibility for the bioisosteric replacement of the ethylene moiety in the structures of free fatty acid receptor (FFAR) agonists by the cyclobutane ring, four new GPR-40 ligands possessing the micromolar activity have been synthesized.Received: 22.08.2020 Revised: 11.10.2020 Accepted: 17.10.2020Мета. Синтезувати цис- та транс-ізомери 3-(4-гідроксифеніл)циклобутанкарбонової кислоти та виявити біологічну активність їх похідних щодо рецептора GPR-40.Результати та їх обговорення. Було синтезовано цис- та транс-ізомери 3-(4-гідроксифеніл)циклобутанкарбонової кислоти, а також їх похідні, що виявили мікромолярну активність як агоністи рецептора GPR-40.Експериментальна частина. Метиловий естер 3-(4-гідроксифеніл)циклобутанкарбонової кислоти було одержано у вигляді суміші цис- та транс-ізомерів у три стадії, виходячи з комерційно доступної 3-оксоциклобутанкарбонової кислоти. Подальше перетворення цієї речовини на ізомерно чисті 3-(4-гідроксифеніл)циклобутанкарбонові кислоти було досягнуто у п’ять стадій, що ґрунтувалося на хроматографічному розділенні діастереомерних амідних похідних. Нові ліганди рецептора GPR-40 одержано шляхом О-алкілування за фенольним атомом оксигену відповідного метилового естеру кислоти. Біологічну активність синтезованих агоністів досліджено на стабільній лінії клітин яєчників китайського хом’яка (CHO), яка експресує людський рецептор GPR-40, за допомогою флуориметричного біотесту.Висновки. Розроблено ефективний підхід до синтезу 3-(4-гідроксифеніл)циклобутанкарбонової кислоти, що дозволяє одержати її у вигляді двох індивідуальних цис/транс-стереоізомерів. Для демонстрації можливості біоізостерної заміни етиленової ланки в структурі агоністів рецепторів вільних жирних кислот (FFAR) на циклобутанове кільце було також синтезовано чотири нові ліганди GPR-40, що мали мікромолярну активність.Received: 22.08.2020 Revised: 11.10.2020 Accepted: 17.10.202

Varespladib and cardiovascular events in patients with an acute coronary syndrome: the VISTA-16 randomized clinical trial

IMPORTANCE: Secretory phospholipase A2(sPLA2) generates bioactive phospholipid products implicated in atherosclerosis. The sPLA2inhibitor varespladib has favorable effects on lipid and inflammatory markers; however, its effect on cardiovascular outcomes is unknown. OBJECTIVE: To determine the effects of sPLA2inhibition with varespladib on cardiovascular outcomes. DESIGN, SETTING, AND PARTICIPANTS: A double-blind, randomized, multicenter trial at 362 academic and community hospitals in Europe, Australia, New Zealand, India, and North America of 5145 patients randomized within 96 hours of presentation of an acute coronary syndrome (ACS) to either varespladib (n = 2572) or placebo (n = 2573) with enrollment between June 1, 2010, and March 7, 2012 (study termination on March 9, 2012). INTERVENTIONS: Participants were randomized to receive varespladib (500 mg) or placebo daily for 16 weeks, in addition to atorvastatin and other established therapies. MAIN OUTCOMES AND MEASURES: The primary efficacy measurewas a composite of cardiovascular mortality, nonfatal myocardial infarction (MI), nonfatal stroke, or unstable angina with evidence of ischemia requiring hospitalization at 16 weeks. Six-month survival status was also evaluated. RESULTS: At a prespecified interim analysis, including 212 primary end point events, the independent data and safety monitoring board recommended termination of the trial for futility and possible harm. The primary end point occurred in 136 patients (6.1%) treated with varespladib compared with 109 patients (5.1%) treated with placebo (hazard ratio [HR], 1.25; 95%CI, 0.97-1.61; log-rank P = .08). Varespladib was associated with a greater risk of MI (78 [3.4%] vs 47 [2.2%]; HR, 1.66; 95%CI, 1.16-2.39; log-rank P = .005). The composite secondary end point of cardiovascular mortality, MI, and stroke was observed in 107 patients (4.6%) in the varespladib group and 79 patients (3.8%) in the placebo group (HR, 1.36; 95% CI, 1.02-1.82; P = .04). CONCLUSIONS AND RELEVANCE: In patients with recent ACS, varespladib did not reduce the risk of recurrent cardiovascular events and significantly increased the risk of MI. The sPLA2inhibition with varespladib may be harmful and is not a useful strategy to reduce adverse cardiovascular outcomes after ACS. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01130246. Copyright 2014 American Medical Association. All rights reserved

Genetic aspects of infertility and miscarriage -a review of literature

Abstract We present current data on the disturbance of reproductive function in men and women according to genetic causes. The purpose of this paper was to discuss the influence of heredity on sexual differentiation, gametogenesis, early stages of embryogenesis and implantation

Component composition of essential oil in the North American <i>Pinus</i> L. species introduced to the Southern Coast of Crimea

Background. Studying essential oils in conifers is of great scientific and practical interest due to their high bactericidal properties. Their volatiles play an important role in combating pathogenic microflora and removing harmful microorganisms from the air, thus benefiting human health. Conifers are highly effective as part of parklands and urban landscaping.Materials and methods. Pinus radiata D. Don, P. sabiniana Douglas and P. coulteri D. Don grown on the Southern Coast of Crimea were studied. Essential oil was extracted from pine needles by hydrodistillation on Ginsberg devices, and its component composition was analyzed using gas–liquid chromatography on a 6890N system with a 5973N mass selective detector.Results. Among the studied species, P. radiata manifested high essential oil content in needles: 0.15% on the wet weight basis (0.36%, dry weight). Under the conditions of the southern coast of Crimea, the major essential oil components in P. radiata were β-pinene (29.5% of the total essential oil), α-pinene (21.2%) and limonene (12.4%); in P. sabiniana, phenylethyl butyrate (20.5%), limonene (15.2%) and α-pinene (13.7%); in P. coulteri, β-pinene (11.6%), δ-cadinene (11.0%) and α-pinene (10.6%). In the essential oil of P. radiata monoterpenes dominated (74.9%); in P. sabiniana, monoterpenes (38.7%) and their derivatives − alcohols (25.3%) and esters (20.5%); in P. coulteri, sesquiterpenes (38.2%) and monoterpenes (28.8%).Conclusion. The essential oils of P. radiata and P. sabiniana under different climate conditions contained mostly monoterpenes (β-pinene in P. radiata, and α-pinene in P. sabiniana) and their derivatives. The component composition of P. coulteri essential oil was the most variable, with a general tendency towards the predominance of sesquiterpenes and diterpenes; the ratio between those groups and the qualitative composition of sesquiterpenes both varied

Anatomy, ploidy level, and essential oil composition of <i>Hyssopus officinalis</i> ʻNikitskiy Beliyʼ <i>in vitro</i> and <i>ex situ</i>

Background. Clonal micropropagation is a biotechnological method for plant multiplication. The existing data on the structure of organs in vitro, genetic stability, and essential oil composition are limited for Hyssopus officinalis L., so this study was aimead at investigating these aspects under a short period of in vitro culturing.Materials and methods. Plants of Hyssopus officinalis ʻNikitskiy Beliyʼ cultivated ex situ, in vitro and ex vitro were analyzed. Conventional methods were applied to study plant anatomy, ploidy level, and relative DNA content, as well as to extract and analyze essential oil. Statistical analysis was performed using the Past 4.03 software.Results. According to the results obtained, with 6-BAP introduced into MS nutrient medium in optimal concentrations (0.3– 0.5 mg/L), the general in vitro structure of leaf blades in the developed microshoots was similar to those in ex situ plants, while the qualitative and quantitative changes observed were induced by the effect of specific culturing conditions and plant rejuvenation. The analysis of the ploidy level and relative DNA content in the nuclei isolated from the leaf tissue cells of the microshoots ex vitro after adaptation revealed no changes compared to the ex situ leaf parameters. The mass fraction of essential oil and its component composition in the mother plants and ex vitro regenerants were similar.Conclusion. Cultivation of Hyssopus officinalis ʻNikitskiy Beliyʼ microshoots on MS nutrient medium with 6-BAP optimal concentrations promotes morphogenesis without significant deviations in the ploidy level, relative DNA content, essential oil yield, or its component composition. The developed protocol for clonal micropropagation of Hyssopus officinalis ʻNikitskiy Beliyʼ provides clones identical to the ex situ plants

Development Calculation Methods of Technological Parameters for Receipt Deformed Semi-Finished Products of Gold Based Alloys of Probe 585

Разработаны алгоритмы, программное обеспечение и методика расчета деформационных режимов и энергосиловых параметров холодной сортовой прокатки и волочения прутков и проволоки из сплавов золота 585-й пробы. Получена формула для расчета контактной площади при прокатке в калибрах, близких по форме к ромбическим. Представлены результаты анализа заводских режимов и предложены новые маршруты прокатки и волочения, которые прошли промышленную апробацию с получением опытных партий ювелирных цепей.Developed algorithms, software, and calculation methods of deformation regimes and power parameters for cold bar rolling and drawing of rods and wires of gold alloys 585 standard. Obtain a formula for the calculation of the contact area during rolling in calibers similar in form to the rhombic. Presented the results of analysis factory regimes and proposed new routes of rolling and drawing, which have passed tested to obtain commercial pilot batches of jewelry chains

Cyclobutyl-Containing Rigid Analogues of Threonine: Synthesis and Physical Chemical Properties

Hitherto unknown <i>cis</i>- and <i>trans</i>-1-amino-3-hydroxy-3-methylcyclobutanecarboxylic acids were synthesized in multigram scale. The obtained compounds can be considered as achiral conformationally restricted analogues of threonine with fixed spatial orientation of functional groups. p<i>K</i>_a values are noticeably different for both amino acids. According to the X-ray data the cyclobutane rings in both compounds are almost planar (the corresponding torsion angles are below 7°)

Development Calculation Methods of Technological Parameters for Receipt Deformed Semi-Finished Products of Gold Based Alloys of Probe 585

Разработаны алгоритмы, программное обеспечение и методика расчета деформационных режимов и энергосиловых параметров холодной сортовой прокатки и волочения прутков и проволоки из сплавов золота 585-й пробы. Получена формула для расчета контактной площади при прокатке в калибрах, близких по форме к ромбическим. Представлены результаты анализа заводских режимов и предложены новые маршруты прокатки и волочения, которые прошли промышленную апробацию с получением опытных партий ювелирных цепей.Developed algorithms, software, and calculation methods of deformation regimes and power parameters for cold bar rolling and drawing of rods and wires of gold alloys 585 standard. Obtain a formula for the calculation of the contact area during rolling in calibers similar in form to the rhombic. Presented the results of analysis factory regimes and proposed new routes of rolling and drawing, which have passed tested to obtain commercial pilot batches of jewelry chains