Сократительные белки сосудистых гладкомышечных клеток – универсальные маркеры сосудов микроциркуляторного русла

Highlights. The use of vascular smooth muscle cell markers, e.g. smooth muscle myosin heavy chain (SM-MHC) and alpha smooth muscle actin (α-SMA) for immunodetection of adventitial and perivascular microvessels (vasa vasorum) is preferrable over endothelial markers (CD31 and VE-cadherin) as it allows to define vascular geometry regardless of sectioning artifacts and provides ideal signal-to-noise ratio.Aside from elastic laminae which discriminate arterioles from venules and capillaries, we were unable to confirm any specific markers of arterial, venous, and capillary differentiation, although KLF2 and PROX1 transcription factors indicated venous specification and HEY1 suggested capillary identity in rat aortas.Aim. To develop an optimal approach to detection of microvessels and to evaluate the techniques for the differential immunostaining of arterioles, venules, and capillaries in human saphenous veins and rat aortas.Methods. Saphenous veins excised during the coronary artery bypass graft surgery were used for the study. Serial cryosections were analyzed by means of haematoxylin and eosin and Russell-Movat’s pentachrome stainings and by immunofluorescent staining for endothelial cell markers (CD31 and VE-cadherin), vascular smooth muscle cell markers (SM-MHC and α-SMA), mechanosensitive transcription factors (KLF2 and KLF4), transcription factors of arterial specification (HES1, HEY1, ERG), transcription factors and markers of venous identity (NR2F2, NRP2), and transcription factors and markers of lymphatic lineage (PROX1, LYVE1, VEGFR3). Samples were visualized by light and confocal microscopy.Results. In comparison with endothelial cell markers (CD31 and VE-cadherin), vascular smooth muscle cell markers (SM-MHC and α-SMA) permitted objective evaluation of vascular geometry and maximized signal-to-noise ratio irrespective of specific marker, microvessel specification or antibody used. Autofluorescence and specific histological  pattern  of  elastic  membranes  at  Russell-Movat’s  pentachrome staining allowed to discriminate arterioles from venules and capillaries. Albeit immunostaining of rat aortas found specific markers of venous endothelial cells (KLF2 and PROX1) and capillary endothelial cells (HEY1), these findings have not been confirmed in saphenous veins. We were unable to find specific markers of human venules and capillaries among the saphenous vein vasa vasorum despite an extensive screening of multiple markers.Conclusion. Immunodetection of microvessels (e.g., vasa vasorum) should be performed by using vascular smooth muscle cell markers (SM-MHC and α-SMA) rather than endothelial cell markers (CD31 and VE-cadherin). Lack of specific markers to discern microvessels of different lineages suggests Russell-Movat’s pentachrome staining as an optimal option for the machine learning of neural networks to analyse the microvessels including vasa vasorum.Основные положения. Показано, что использование маркеров сосудистых гладкомышечных клеток, к примеру тяжелых цепей миозина гладких мышц (SM-MHC) и альфа-актина гладких мышц (α-SMA), для иммунодетекции сосудов микроциркуляторного русла сосудистой стенки (vasa vasorum) предпочтительнее применения эндотелиальных маркеров (CD31 и VE-кадгерин), поскольку позволяет определять сосудистую геометрию вне зависимости от артефактов пробоподготовки и обеспечивает идеальное соотношение «сигнал – шум».За исключением эластических мембран, позволяющих дифференцировать артериолы от венул и капилляров, в тканях сосудов взрослого человека не выявлено специфичных маркеров артериальной, венозной и капиллярной дифференцировки, хотя в аортах крыс маркерами венозной дифференцировки служили транскрипционные факторы KLF2 и PROX1, а маркером капиллярной дифференцировки – транскрипционный фактор HEY1.Цель. Разработать оптимальный способ детекции сосудов микроциркуляторного русла и оценить подходы к дифференциальному окрашиванию артериол, венул и капилляров на примере сети vasa vasorum большой подкожной вены (БПВ) человека и аорты крысы.Материалы и методы. Для исследования использовали сегменты БПВ, извлеченные в процессе коронарного шунтирования, а также аорты нормолипидемических и нормотензивных крыс Wistar. Серийные криосрезы анализировали при помощи окрашивания гематоксилином и эозином, пентахромного окрашивания по Расселлу – Мовату, иммунофлюоресцентного окрашивания на маркеры эндотелиальных клеток (ЭК) (CD31 и VE-кадгерин), сосудистых гладкомышечных клеток (СГМК) (SM-MHC и α-SMA), механочувствительных транскрипционных факторов (KLF2 и KLF4), транскрипционные факторы артериальной дифференцировки (HES1, HEY1, ERG), транскрипционные факторы и маркеры венозной дифференцировки (NR2F2, NRP2), транскрипционные факторы и маркеры лимфатической дифференцировки (PROX1, LYVE1, VEGFR3), а также иммуногистохимического окрашивания на маркеры венозной дифференцировки NR2F2. Готовые микропрепараты анализировали посредством световой и конфокальной микроскопии.Результаты. В  сравнении  с  маркерами  ЭК  (CD31  и VE-кадгерином)  маркеры  СГМК (SM-MHC и α-SMA) позволяли осуществлять объективную визуализацию и оценку сосудистой геометрии и обеспечивали максимальное соотношение «сигнал – шум» независимо от вида маркера, типа сосуда или антитела. Аутофлюоресценция и специфичный гистологический паттерн эластических мембран в ходе окрашивания по Расселлу – Мовату позволяли дифференцировать артериолы от остальных сосудов микроциркуляторного русла. Несмотря на то что иммуноокрашивание аорт крыс показало специфичные маркеры венозного (KLF2 и PROX1) и капиллярного (HEY1) эндотелия, данные находки не нашли подтверждения при иммуноокрашивании БПВ человека. Таким образом, специфичных маркеров венул и капилляров в тканях взрослого человека не идентифицировано, несмотря на анализ широкой палитры классических маркеров различных эндотелиальных дифферонов.Заключение. Для иммунодетекции сосудов микроциркуляторного русла целесообразно использовать маркеры СГМК (SM-MHC и α-SMA), а не маркеры ЭК (CD31 и VE-кадгерин). Отсутствие специфичных маркеров дифференцировки микрососудов (в частности vasa vasorum) позволяет предположить пентахромное окрашивание по Расселлу – Мовату в качестве оптимальной опции для машинного обучения нейронных сетей с целью анализа сосудов микроциркуляторного русла

Analysis of GH1, GHR and PRL gene polymorphisms for estimation of the genetic diversity of Buryat and Altai cattle breeds

Small and unique Buryat and Altai cattle breeds of TuranoMongolian origin are well adapted to harsh conditions of the continental climate to be their habitat. However, the population-genetic structure of the breeds has been poorly studied. This paper presents the results of analysis of polymorphisms GH1 (AC_000176.1: BTA 19, exon 5, rs41923484, g.2141C>G, L127V), GHR (AC_000177.1: BTA 20, exon 10, rs109300983, g.257A>G, S555G) and PRL (AC_000180.1: BTA 23, exon 3, g.35108342A>G) in the samples of Buryat cattle breed of Russia, China and Mongolia, and indigenous Altai cattle breed (Russia) that belong to TuranoMongolian cattle. The Russian sample of Buryat breed was differentiated from the Mongolian sample based on pairwise G-test and FST values for the PRL-RsaI polymorphism and from the Chinese sample – based on pairwise G-test values for the GH1-AluI polymorphism. All the three samples of Buryat breed clearly differed from the sample of Altai breed based on pairwise G-test and FST values for the GHR-AluI polymorphism as well as on the base of FST values for the joint polymorphism of the three genes. Nei’s genetic distances calculated from the three gene polymorphisms also confirmed the difference between the two breeds. The results of AMOVA demonstrated that GHR gene variability (16 %) gave the largest contribution to the differentiation that was confirmed by FST values (0.12–0.27). The STRUCTURE software enabled us to reveal four clusters, with a specific ratio for each sample, in the Chinese and Mongolian samples of Buryat breed, and in the sample of Altai breed, while the Russian sample of Buryat breed had only three clusters. The differences within the breed level were determined based on the GH1-AluI and PRL-RsaI polymorphisms, while at the inter-breed level – based on the GHR-AluI polymorphism. Linkage disequilibrium analysis demonstrated significant linkage of the following pairs of genes in the Buryat breed: GH1-GHR, GH1-PRL, GHR-PRL

Biochemical and Molecular Phylogenetic Study of Agriculturally Useful Association of a Nitrogen-Fixing Cyanobacterium and Nodule Sinorhizobium with Medicago sativa L.

Seed inoculation with bacterial consortium was found to increase legume yield, providing a higher growth than the standard nitrogen treatment methods. Alfalfa plants were inoculated by mono- and binary compositions of nitrogen-fixing microorganisms. Their physiological and biochemical properties were estimated. Inoculation by microbial consortium of Sinorhizobium meliloti T17 together with a new cyanobacterial isolate Nostoc PTV was more efficient than the single-rhizobium strain inoculation. This treatment provides an intensification of the processes of biological nitrogen fixation by rhizobia bacteria in the root nodules and an intensification of plant photosynthesis. Inoculation by bacterial consortium stimulates growth of plant mass and rhizogenesis and leads to increased productivity of alfalfa and to improving the amino acid composition of plant leaves. The full nucleotide sequence of the rRNA gene cluster and partial sequence of the dinitrogenase reductase (nifH) gene of Nostoc PTV were deposited to GenBank (JQ259185.1, JQ259186.1). Comparison of these gene sequences of Nostoc PTV with all sequences present at the GenBank shows that this cyanobacterial strain does not have 100% identity with any organisms investigated previously. Phylogenetic analysis showed that this cyanobacterium clustered with high credibility values with Nostoc muscorum

Biochemical and Molecular Phylogenetic Study of Agriculturally Useful Association of a Nitrogen-Fixing Cyanobacterium and Nodule Sinorhizobium with Medicago sativa L.

Seed inoculation with bacterial consortium was found to increase legume yield, providing a higher growth than the standard nitrogen treatment methods. Alfalfa plants were inoculated by mono- and binary compositions of nitrogen-fixing microorganisms. Their physiological and biochemical properties were estimated. Inoculation by microbial consortium of Sinorhizobium meliloti T17 together with a new cyanobacterial isolate Nostoc PTV was more efficient than the single-rhizobium strain inoculation. This treatment provides an intensification of the processes of biological nitrogen fixation by rhizobia bacteria in the root nodules and an intensification of plant photosynthesis. Inoculation by bacterial consortium stimulates growth of plant mass and rhizogenesis and leads to increased productivity of alfalfa and to improving the amino acid composition of plant leaves. The full nucleotide sequence of the rRNA gene cluster and partial sequence of the dinitrogenase reductase (nifH) gene of Nostoc PTV were deposited to GenBank (JQ259185.1, JQ259186.1). Comparison of these gene sequences of Nostoc PTV with all sequences present at the GenBank shows that this cyanobacterial strain does not have 100% identity with any organisms investigated previously. Phylogenetic analysis showed that this cyanobacterium clustered with high credibility values with Nostoc muscorum