Оценка внутрисортового полиморфизма генов устойчивости к грибным болезням томата у сортов селекции Мичуринского ГАУ

Relevance. The studies are aimed at assessing the intravarietal allelic diversity of genes for resistance to fusariosis and cladosporiosis of tomato cultivars bred at the Michurinsky State Agrarian University using molecular markers.Methods. The biological objects of the study are tomato varieties bred at the Michurinsky State Agrarian University. A total of 10 varieties were analyzed. The assessment of intravarietal polymorphism was determined on a sample of 10 plants of each variety. DNA extraction was performed using a Quick-DNA Plant/Seed Miniprep Kit (Zymo Research, USA) for DNA extraction according to the manufacturer's protocol. The P7 DNA marker was used to identify the cladosporosis resistance gene Cf-19. The presence of the fusarium wilt resistance gene I-2 was determined using the marker I-2/5. Amplification results were visualized by electrophoresis in 2% agarose gel.Results. 10 plants of 10 varieties of Michurinsky GAU selection tomato were analyzed using molecular markers of genes for resistance to cladosporiosis and fusariosis. An assessment of intravarietal polymorphism was carried out. In most of the varieties studied, the genes analyzed are identified in a heterozygous state. Three varieties (Vivat, Carotinka, Krasavets) containing only alleles of susceptibility to the causative agent of the disease in the genotype I-2 identified by the gene. Individual samples were homozygous for the fusariosis resistance allele. Analysis of the gene for Cf-19 resistance to cladosporiosis showed that most of the varieties studied are heterozygous forms. In some varieties, all ten plants were heterozygous. These include Krasavets, Vivat, Orlik, Bui Tour. The remaining samples had different allelic compositions. The pathogen resistance allele has been identified in plants of the Nepryadva and Chernysh varieties. The dominant allele in the homozygous state was detected in the Japanese variety. At the same time, all the analyzed plants of this variety were monotonous.Conclusion. It has been established, but a significant part of the varieties of tomato selection of Michurinsky GAU is polymorphic in the genes for resistance to cladosporiosis and fusariosis. In this case, a significant part of the varieties can be used in selection during preliminary molecular analysis. According to the gene for resistance to fusariosis, the following varieties are identified sources of the resistance allele: Sokol, Nepryadva, Yaponchik, Orlik, Zolotnichok, Chernysh. In them, the gene is I-2 represented in a heterozygous state. Intraortic polymorphism was also noted for the Cf-19 gene. Most samples have two alleles. The exception is the Japanese variety, which is the dominant homozygote for this gene. Цель. Исследования направлены на оценку внутрисортового аллельного разнообразия генов устойчивости к фузариозу и кладоспориозу томата сортов селекции Мичуринского ГАУ с использованием молекулярных маркеров.Методология. Биологическими объектами исследования являются сорта томата селекции Мичуринского ГАУ. Всего проанализировано 10 сортов. Оценка внутрисортового полиморфизма определялась на выборке из 10 растений каждого сорта. Экстрагирование ДНК было проведено с использованием набора для экстрагирования ДНК Quick-DNA Plant/Seed Miniprep Kit (Zymo Research, USA) согласно протоколу производителя. Для идентификации гена устойчивости к кладоспоризу Cf19 использовали ДНК-маркер Р7. Наличие гена устойчивости к фузариозному увяданию I-2 определяли с помощью маркера I-2/5. Визуализацию результатов амплификации осуществляли с помощью электрофореза в 2% агарозном геле.Результаты. Было проанализировано по 10 растений 10 сортов томата селекции Мичуринского ГАУ с использованием молекулярных маркеров генов устойчивости к кладоспориозу и фузариозу. Проведена оценка внутрисортового полиморфизма. У большинства исследуемых сортов анализируемые гены идентифицированы в гетерозиготном состоянии. По гену I-2 выявлены три сорта (Виват, Каротинка, Красавец), содержащих в генотипе только аллели восприимчивости к возбудителю заболевания. Отмечены отдельные образцы гомозиготные по аллелю устойчивости к фузариозу. Анализ гена Cf-19 устойчивости к кладоспориозу показал, что большинство исследуемых сортов являются гетерозиготными формами. У части сортов все десять растений были гетерозиготны. К ним относятся Красавец, Виват, Орлик, Буй-Тур. Остальные образцы имели различный аллельный состав. Аллель устойчивости к возбудителю был идентифицирован у растений сорта Непрядва и Черныш. Доминантный аллель в гомозиготном состоянии выявлен у сорта Япончик. При этом все анализируемые растения этого сорта были однообразны.Заключение. Установлено, что значительная часть сортов томата селекции Мичуринского ГАУ является полиморфной по генам устойчивости к кладоспориозу и фузариозу. При этом значительная часть сортов может быть использована в селекции при проведении предварительного молекулярного анализа. По гену устойчивости к фузариозу выделены следующие сорта источники аллеля устойчивости: Сокол, Непрядва, Япончик, Орлик, Золотничок, Черныш. У них ген I-2 представлен в гетерозиготном состоянии. Внутрисортовой полиморфизм отмечен и для гена Cf-19. Большинство образцов имеют два аллеля. Исключение составляет сорт Япончик, который является доминантной гомозиготой по данному гену.

Создание новых форм томата с генами устойчивости к грибным болезням на основе маркерной селекции

Relevance. The presented studies are aimed at obtaining new forms of tomato with a complex of genes for resistance to fungal diseases in combination with a standard type of bush and dark coloring of fruits based on marker-mediated selection.Methodology. The biological objects of the study are varieties and hybrid forms of tomato from the collection of the Michurinsky SAU. Molecular genetic analysis was performed using the following methods. DNA extraction was carried out from young leaves using a kit for isolation of NC Sample NC manufactured by Agrodiagnostika LLC according to the manufacturer's protocol. Fermentas production kits were used for PCR. Identification of the cladosporosis resistance gene was Cf-19 performed using the DNA marker R7. The presence of a fusarious wilting resistance gene was determined by a I-2/5 marker. The amplification results were visualized by agarose gel electrophoresis.Results. During the research, a collection of varieties and hybrid forms of tomato of the Michurinsky GAU was analyzed in order to identify genes for resistance to cladosporiosis Cf-19 and fusarium wilt I-2. A total of 52 genotypes were analyzed. It was found that most samples (41 samples) are characterized by a heterozygous state of the Cf-19 gene. All indeterminant and semi-determinant forms had both alleles. Of the 23 determinant forms presented in the collection, 10 had only one allele corresponding to recessive homozygote. Among all analyzed tomato genotypes, no dominant homozygous forms were noted. The study of the collection revealed several alleles of the I-2 gene. In total, four fragments corresponding to various alleles were amplified. A total of 50 resistant genotypes have been identified in the collection. Two alleys of the I-2 gene (633/693 bp) were identified in 42 tomato samples. Four varieties are homozygous in one allele (633 bp), which determines resistance. Three varieties have a second resistance allele (566 bp). One genotype has only an allele defining susceptibility (693 bp). On the basis of molecular analysis, as well as an assessment of the type of bush and fetal color, initial forms were selected with subsequent hybridization. 67 hybrid tomato plants were obtained. Evaluation of the presence of resistance genes showed that most of the resulting hybrids are resistant to cladosporiosis and fuzariosis. This is due to the presence of dominant alleles of Cf-19 and I-2 genes in a heterozygous state. Among the resulting hybrids, plants with a bark type of bush were identified. A total of 13 such plants were obtained.Conclusion. Thus, the work carried out allowed to obtain hybrid forms of tomato combine the signs of resistance to two pathogens of fungal diseases and the stem type of the bush. These forms are planned to be used in further selection work.Цель. Исследования направлены на получение новых форм томата с комплексом генов устойчивости к грибным болезням в сочетании со штамбовым типом куста и темной окраской плодов на основе маркер-опосредованной селекции.Методы. Объект исследований – сорта и гибридные формы томата из коллекции Мичуринского ГАУ. Молекулярно-генетический анализ проводили с использованием следующих методов. Экстрагирование ДНК осуществляли из молодых листьев с применением набора для выделения НК «Проба НК» производства ООО «Агродиагностика» согласно протоколу производителя. Для проведения ПЦР использованы наборы производства компании Fermentas. Идентификацию гена устойчивости к кладоспоризу Cf-19 проводили с использованием ДНК-маркера Р7. Наличие гена устойчивости к фузариозному увяданию определяли с помощью маркера I-2/5. Визуализацию результатов амплификации осуществляли с помощью электрофореза в агарозном геле.Результаты. При проведении исследований была проанализирована коллекция сортов и гибридных форм томата Мичуринского ГАУ с целью идентификации генов устойчивости к кладоспориозу Cf-19 и фузариозному увяданию I-2. Всего проанализировано 52 генотипа. Установлено, что для большинства образцов (41 образец) характерно гетерозиготное состояние гена Cf-19. Все индетерминантные и полудетерминантные формы имели оба аллеля. Из 23 представленных в коллекции детерминантных форм у 10 отмечен только один аллель, соответствующий рецессивной гомозиготе. Среди всех анализируемых генотипов томата не отмечено доминантных гомозиготных форм. Изучение коллекции позволило выявить нескольких аллелей гена I-2. Всего амплифицировано четыре фрагмента, соответствующих различным аллелям. Всего устойчивых генотипов в коллекции выделено 50. У 42 образцов томата идентифицированы два аллея гена I-2 (633/693 п.н). Четыре сорта гомозиготны по одному аллелю (633 п.н.), обуславливающему устойчивость. Три сорта имеют второй аллель (566 п.н.) устойчивости. Один генотип имеет только аллель определяющий восприимчивость (693 п.н.).На основании молекулярного анализа, а также оценки типа куста и окраски плода был проведен отбор исходных форм с последующей гибридизацией. Получено 67 гибридных растений томата. Оценка наличия генов устойчивости показала, что большинство полученных гибридов являются устойчивыми к кладоспориозу и фузариозу. Это обусловлено наличием доминантных аллелей генов Cf-19 и I-2 в гетерозиготном состоянии. Среди полученных гибридов выделены растения со штамбовым типом куста. Всего таких растений получено 13. Проведенная работа позволила получить гибридные формы томата, сочетающие признаки устойчивости к двум возбудителям грибных болезней и штамбовый тип куста. Эти формы планируется использовать в дальнейшей селекционной работе

Desmoglein 3, via an Interaction with E-cadherin, Is Associated with Activation of Src

Desmoglein 3 (Dsg3), a desmosomal adhesion protein, is expressed in basal and immediate suprabasal layers of skin and across the entire stratified squamous epithelium of oral mucosa. However, increasing evidence suggests that the role of Dsg3 may involve more than just cell-cell adhesion.To determine possible additional roles of Dsg3 during epithelial cell adhesion we used overexpression of full-length human Dsg3 cDNA, and RNAi-mediated knockdown of this molecule in various epithelial cell types. Overexpression of Dsg3 resulted in a reduced level of E-cadherin but a colocalisation with the E-cadherin-catenin complex of the adherens junctions. Concomitantly these transfected cells exhibited marked migratory capacity and the formation of filopodial protrusions. These latter events are consistent with Src activation and, indeed, Src-specific inhibition reversed these phenotypes. Moreover Dsg3 knockdown, which also reversed the decreased level of E-cadherin, partially blocked Src phosphorylation.Our data are consistent with the possibility that Dsg3, as an up-stream regulator of Src activity, helps regulate adherens junction formation

Plakophilin-3 Is Required for Late Embryonic Amphibian Development, Exhibiting Roles in Ectodermal and Neural Tissues

The p120-catenin family has undergone a significant expansion during the evolution of vertebrates, resulting in varied functions that have yet to be discerned or fully characterized. Likewise, members of the plakophilins, a related catenin subfamily, are found throughout the cell with little known about their functions outside the desmosomal plaque. While the plakophilin-3 (Pkp3) knockout mouse resulted in skin defects, we find larger, including lethal effects following its depletion in Xenopus. Pkp3, unlike some other characterized catenins in amphibians, does not have significant maternal deposits of mRNA. However, during embryogenesis, two Pkp3 protein products whose temporal expression is partially complimentary become expressed. Only the smaller of these products is found in adult Xenopus tissues, with an expression pattern exhibiting distinctions as well as overlaps with those observed in mammalian studies. We determined that Xenopus Pkp3 depletion causes a skin fragility phenotype in keeping with the mouse knockout, but more novel, Xenopus tailbud embryos are hyposensitive to touch even in embryos lacking outward discernable phenotypes, and we additionally resolved disruptions in certain peripheral neural structures, altered establishment and migration of neural crest, and defects in ectodermal multiciliated cells. The use of two distinct morpholinos, as well as rescue approaches, indicated the specificity of these effects. Our results point to the requirement of Pkp3 in amphibian embryogenesis, with functional roles in a number of tissue types

Immunoglobulin, glucocorticoid, or combination therapy for multisystem inflammatory syndrome in children: a propensity-weighted cohort study

Background: Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory condition associated with SARS-CoV-2 infection, has emerged as a serious illness in children worldwide. Immunoglobulin or glucocorticoids, or both, are currently recommended treatments. Methods: The Best Available Treatment Study evaluated immunomodulatory treatments for MIS-C in an international observational cohort. Analysis of the first 614 patients was previously reported. In this propensity-weighted cohort study, clinical and outcome data from children with suspected or proven MIS-C were collected onto a web-based Research Electronic Data Capture database. After excluding neonates and incomplete or duplicate records, inverse probability weighting was used to compare primary treatments with intravenous immunoglobulin, intravenous immunoglobulin plus glucocorticoids, or glucocorticoids alone, using intravenous immunoglobulin as the reference treatment. Primary outcomes were a composite of inotropic or ventilator support from the second day after treatment initiation, or death, and time to improvement on an ordinal clinical severity scale. Secondary outcomes included treatment escalation, clinical deterioration, fever, and coronary artery aneurysm occurrence and resolution. This study is registered with the ISRCTN registry, ISRCTN69546370. Findings: We enrolled 2101 children (aged 0 months to 19 years) with clinically diagnosed MIS-C from 39 countries between June 14, 2020, and April 25, 2022, and, following exclusions, 2009 patients were included for analysis (median age 8·0 years [IQR 4·2–11·4], 1191 [59·3%] male and 818 [40·7%] female, and 825 [41·1%] White). 680 (33·8%) patients received primary treatment with intravenous immunoglobulin, 698 (34·7%) with intravenous immunoglobulin plus glucocorticoids, 487 (24·2%) with glucocorticoids alone; 59 (2·9%) patients received other combinations, including biologicals, and 85 (4·2%) patients received no immunomodulators. There were no significant differences between treatments for primary outcomes for the 1586 patients with complete baseline and outcome data that were considered for primary analysis. Adjusted odds ratios for ventilation, inotropic support, or death were 1·09 (95% CI 0·75–1·58; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids and 0·93 (0·58–1·47; corrected p value=1·00) for glucocorticoids alone, versus intravenous immunoglobulin alone. Adjusted average hazard ratios for time to improvement were 1·04 (95% CI 0·91–1·20; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids, and 0·84 (0·70–1·00; corrected p value=0·22) for glucocorticoids alone, versus intravenous immunoglobulin alone. Treatment escalation was less frequent for intravenous immunoglobulin plus glucocorticoids (OR 0·15 [95% CI 0·11–0·20]; p<0·0001) and glucocorticoids alone (0·68 [0·50–0·93]; p=0·014) versus intravenous immunoglobulin alone. Persistent fever (from day 2 onward) was less common with intravenous immunoglobulin plus glucocorticoids compared with either intravenous immunoglobulin alone (OR 0·50 [95% CI 0·38–0·67]; p<0·0001) or glucocorticoids alone (0·63 [0·45–0·88]; p=0·0058). Coronary artery aneurysm occurrence and resolution did not differ significantly between treatment groups. Interpretation: Recovery rates, including occurrence and resolution of coronary artery aneurysms, were similar for primary treatment with intravenous immunoglobulin when compared to glucocorticoids or intravenous immunoglobulin plus glucocorticoids. Initial treatment with glucocorticoids appears to be a safe alternative to immunoglobulin or combined therapy, and might be advantageous in view of the cost and limited availability of intravenous immunoglobulin in many countries. Funding: Imperial College London, the European Union's Horizon 2020, Wellcome Trust, the Medical Research Foundation, UK National Institute for Health and Care Research, and National Institutes of Health

P-cadherin expression in breast cancer: a review

P-cadherin is frequently over-expressed in high-grade invasive breast carcinomas and has been reported to be an enhancer of migration and invasion of breast cancer cells, being correlated with tumour aggressiveness. In addition, expression of P-cadherin is well established as an indicator of poor prognosis in human breast cancer, which has stimulated our interest in studying its role in this setting. This review describes the most important findings on P-cadherin expression and function in normal mammary tissue and breast cancer cells, emphasizing that further research is required to elucidate the role played by this protein in human mammary tumours

Immunoglobulin, glucocorticoid, or combination therapy for multisystem inflammatory syndrome in children: a propensity-weighted cohort study

Background Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory condition associated with SARS-CoV-2 infection, has emerged as a serious illness in children worldwide. Immunoglobulin or glucocorticoids, or both, are currently recommended treatments. Methods The Best Available Treatment Study evaluated immunomodulatory treatments for MIS-C in an international observational cohort. Analysis of the first 614 patients was previously reported. In this propensity-weighted cohort study, clinical and outcome data from children with suspected or proven MIS-C were collected onto a web-based Research Electronic Data Capture database. After excluding neonates and incomplete or duplicate records, inverse probability weighting was used to compare primary treatments with intravenous immunoglobulin, intravenous immunoglobulin plus glucocorticoids, or glucocorticoids alone, using intravenous immunoglobulin as the reference treatment. Primary outcomes were a composite of inotropic or ventilator support from the second day after treatment initiation, or death, and time to improvement on an ordinal clinical severity scale. Secondary outcomes included treatment escalation, clinical deterioration, fever, and coronary artery aneurysm occurrence and resolution. This study is registered with the ISRCTN registry, ISRCTN69546370. Findings We enrolled 2101 children (aged 0 months to 19 years) with clinically diagnosed MIS-C from 39 countries between June 14, 2020, and April 25, 2022, and, following exclusions, 2009 patients were included for analysis (median age 8·0 years [IQR 4·2–11·4], 1191 [59·3%] male and 818 [40·7%] female, and 825 [41·1%] White). 680 (33·8%) patients received primary treatment with intravenous immunoglobulin, 698 (34·7%) with intravenous immunoglobulin plus glucocorticoids, 487 (24·2%) with glucocorticoids alone; 59 (2·9%) patients received other combinations, including biologicals, and 85 (4·2%) patients received no immunomodulators. There were no significant differences between treatments for primary outcomes for the 1586 patients with complete baseline and outcome data that were considered for primary analysis. Adjusted odds ratios for ventilation, inotropic support, or death were 1·09 (95% CI 0·75–1·58; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids and 0·93 (0·58–1·47; corrected p value=1·00) for glucocorticoids alone, versus intravenous immunoglobulin alone. Adjusted average hazard ratios for time to improvement were 1·04 (95% CI 0·91–1·20; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids, and 0·84 (0·70–1·00; corrected p value=0·22) for glucocorticoids alone, versus intravenous immunoglobulin alone. Treatment escalation was less frequent for intravenous immunoglobulin plus glucocorticoids (OR 0·15 [95% CI 0·11–0·20]; p<0·0001) and glucocorticoids alone (0·68 [0·50–0·93]; p=0·014) versus intravenous immunoglobulin alone. Persistent fever (from day 2 onward) was less common with intravenous immunoglobulin plus glucocorticoids compared with either intravenous immunoglobulin alone (OR 0·50 [95% CI 0·38–0·67]; p<0·0001) or glucocorticoids alone (0·63 [0·45–0·88]; p=0·0058). Coronary artery aneurysm occurrence and resolution did not differ significantly between treatment groups. Interpretation Recovery rates, including occurrence and resolution of coronary artery aneurysms, were similar for primary treatment with intravenous immunoglobulin when compared to glucocorticoids or intravenous immunoglobulin plus glucocorticoids. Initial treatment with glucocorticoids appears to be a safe alternative to immunoglobulin or combined therapy, and might be advantageous in view of the cost and limited availability of intravenous immunoglobulin in many countries. Funding Imperial College London, the European Union's Horizon 2020, Wellcome Trust, the Medical Research Foundation, UK National Institute for Health and Care Research, and National Institutes of Health

Basic principles of enteral feeding in premature infants

То optimize the feeding of preterm infants is now one of the priority areas in neonatology and pediatrics because neonatal malnutrition has a negative impact on the health and development of a preterm infant, including on those at a later age. Current recommendations for the enteral feeding of preterm infants are based on the knowledge of their anatomic and physiological features and the need to prevent a menacing disease, such as necrotizing enterocolitis. The feeding of preterm infants at any gestational age with their mother's native breast milk is most optimal. Recommendations for breast milk enrichment are warranted for preterm infants with higher nutrient requirements. The paper presents the differential algorithms and regimens for the feeding of preterm infants with both breast milk and milk formulas depending on many factors, such as gestational age, birth weight, and clinical status

Evaluation of intravarietal polymorphism of tomato fungal disease resistance genes in Michurinsky State Agrarian University breeding varieties

Relevance. The studies are aimed at assessing the intravarietal allelic diversity of genes for resistance to fusariosis and cladosporiosis of tomato cultivars bred at the Michurinsky State Agrarian University using molecular markers.Methods. The biological objects of the study are tomato varieties bred at the Michurinsky State Agrarian University. A total of 10 varieties were analyzed. The assessment of intravarietal polymorphism was determined on a sample of 10 plants of each variety. DNA extraction was performed using a Quick-DNA Plant/Seed Miniprep Kit (Zymo Research, USA) for DNA extraction according to the manufacturer's protocol. The P7 DNA marker was used to identify the cladosporosis resistance gene Cf-19. The presence of the fusarium wilt resistance gene I-2 was determined using the marker I-2/5. Amplification results were visualized by electrophoresis in 2% agarose gel.Results. 10 plants of 10 varieties of Michurinsky GAU selection tomato were analyzed using molecular markers of genes for resistance to cladosporiosis and fusariosis. An assessment of intravarietal polymorphism was carried out. In most of the varieties studied, the genes analyzed are identified in a heterozygous state. Three varieties (Vivat, Carotinka, Krasavets) containing only alleles of susceptibility to the causative agent of the disease in the genotype I-2 identified by the gene. Individual samples were homozygous for the fusariosis resistance allele. Analysis of the gene for Cf-19 resistance to cladosporiosis showed that most of the varieties studied are heterozygous forms. In some varieties, all ten plants were heterozygous. These include Krasavets, Vivat, Orlik, Bui Tour. The remaining samples had different allelic compositions. The pathogen resistance allele has been identified in plants of the Nepryadva and Chernysh varieties. The dominant allele in the homozygous state was detected in the Japanese variety. At the same time, all the analyzed plants of this variety were monotonous.Conclusion. It has been established, but a significant part of the varieties of tomato selection of Michurinsky GAU is polymorphic in the genes for resistance to cladosporiosis and fusariosis. In this case, a significant part of the varieties can be used in selection during preliminary molecular analysis. According to the gene for resistance to fusariosis, the following varieties are identified sources of the resistance allele: Sokol, Nepryadva, Yaponchik, Orlik, Zolotnichok, Chernysh. In them, the gene is I-2 represented in a heterozygous state. Intraortic polymorphism was also noted for the Cf-19 gene. Most samples have two alleles. The exception is the Japanese variety, which is the dominant homozygote for this gene