Detection of methylated septin 9 in tissue and plasma of colorectal patients with neoplasia and the relationship to the amount of circulating cell-free DNA

BACKGROUND: Determination of methylated Septin 9 (mSEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, the relationship between methylated DNA in plasma and colon tissue of the same subjects has not been reported. METHODS: Plasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 patients with adenoma and 34 patients with CRC. Following bisulfite conversion of DNA a commercial RT-PCR assay was used to determine the total amount of DNA in each sample and the fraction of mSEPT9 DNA. The Septin-9 protein was assessed using immunohistochemistry. RESULTS: The percent of methylated reference (PMR) values for SEPT9 above a PMR threshold of 1% were detected in 4.2% (1/24) of NED, 100% (26/26) of adenoma and 97.1% (33/34) of CRC tissues. PMR differences between NED vs. adenoma and NED vs. CRC comparisons were significant (p<0.001). In matching plasma samples using a PMR cut-off level of 0.01%, SEPT9 methylation was 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC. Significant PMR differences were observed between NED vs. CRC (p<0.01) and adenoma vs. CRC (p<0.01). Significant differences (p<0.01) were found in the amount of cfDNA (circulating cell-free DNA) between NED and CRC, and a modest correlation was observed between mSEPT9 concentration and cfDNA of cancer (R2 = 0.48). The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with abundant expression in normals, and diminished expression in adenomas and tumors. CONCLUSIONS: Methylated SEPT9 was detected in all tissue samples. In plasma samples, elevated mSEPT9 values were detected in CRC, but not in adenomas. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Additional parameters including the amount of cfDNA in plasma appear to also play a role

КОНСЕРВИРОВАННЫЕ ОВОЩНЫЕ ПРОДУКТЫ ДЛЯ СОЦИАЛЬНОГО ПИТАНИЯ

The developed technology, regulatory and technical documentation allowed to produce 3-5-kg packages of sterilized vegetables: potatoes, beets, carrots and cabbage, which are in great demand in the social nutrition (the army, schools, hospitals). The use of packaging for products of composite materials allows to reduce the amount of required storage space.Разработанная технология и нормативно-техническая документация позволяют выпускать 3-5-килограммовые упаковки стерилизованных овощей: картофеля, свеклы, моркови и капусты, которые пользуются большим спросом в социальном питании (армия, учебные заведения, больницы). Использование для продуктов тары из комбинированных материалов позволяет уменьшить объемы необходимых складских помещений

ФЕРМЕНТАТИВНАЯ АКТИВНОСТЬ БАКТЕРИЙ РОДА ARTHROBACTER И ИХ ГЕНОТИПИЧЕСКАЯ ИДЕНТИФИКАЦИЯ

Two-stage screening enabled one to define the composition of enzyme complexes produced by bacteria of genus Arthrobacter in media with specific substrates and activity of individual constituents involved in the t ransformation of milk proteins, lipids and carbohydrates. It was found that enzyme complexes of Arthrobacter strains BIM B-2239, BIM B-2240, BIM B-2241 and BIM B-2242 showed the most balanced activity of lipase, protease, β-galactosidase and glucose(xylose)isomerase components. Following the nucleotide sequence analysis of 16S rRNA gene the examined cultures were identified as Arthrobacter sulfonivorans. 16S rRNA gene sequences over 1400 bp in size were deposited in GenBank database.Detailed investigation of conditions favoring the efficient hydrolysis of proteins and lipids by proteases and lipases produced by Arthrobacter sulfonivorans strains BIM B-2239, BIM B-2240, BIM B-2241, BIM B-2242 and the transformation of milk lactose and derived hydrolysis products mediated by β-galactosidases and glucose(xylose)isomerases will allow one to select microbial strain to develop the biotechnology of manufacturing hypoallergenic feed additive with prebiotic activity from dairy substrates.В результате двухступенчатого скрининга определены состав ферментных комплексов, продуцируемых бактериями рода Arthrobacter в средах с о с пецифическим субстратом, и а ктивность и х о тдельных компонентов, участвующих в трансформации белков, жиров и углеводов молока. Установлено, что наиболее сбалансированными по активности одновременно липаз, протеаз, β-галактозидаз и глюкозо(ксилозо)изомераз являются ферментные комплексы штаммов БИМ В-2239, БИМ В-2240, БИМ В-2241 и БИМ В-2242 Arthrobacter sp.Методом анализа нуклеотидных последовательностей генов 16S рРНК указанные бактериальные культуры идентифицированы как Arthrobacter sulfonivorans. Последовательности их генов 16S рРНК протяженностью более 1400 п. о. депонированы в базе данных GenBank.Детальное исследование условий эффективного гидролиза белков и жиров с участием продуцируемых штаммами БИМ В-2239, БИМ В-2240, БИМ В-2241 и БИМ В-2242 Arthrobacter sp. липаз и протеаз, а также трансформации лактозы молока и продуктов ее гидролиза с участием β-галактозидаз и глюкозо(ксилозо)изомераз позволит отобрать штамм-продуцент для разработки биотехнологии получения из молочного сырья гипоаллергенной биологически активной кормовой добавки пребиотического действия

Выделение, характеристика и молекулярно-генетическая идентификация нового штамма бактерий Paenibacillus species

Bacterial variant PS-K-17 was isolated from wheat grain contaminated by polysaccharide-producing microbiota for further characterization. It was found that the isolate grown on agar slants and in submerged culture on media with specific substrates synthesized beta-galactosidase, amylase, protease, pectinase, cellulase, beta-glucanase, lipase (esterase), alginase, extracellular polysaccharides, and pigments, probably carotenoids. Based on cultural-morphological and physiological-biochemical properties and phylogenetic analysis of nucleotide sequences of 16S rRNA gene (access code MF443394 in GenBank) the bacterial culture was identified as Paenibacillus species PS-K-17. The studied isolate forms one phylogenetic branch with type strains Paenibacillus nicotianae (98.3 %), Paenibacillus hordei (98.2 %), Paenibacillus kyungheensis (97.9 %), holding wherein a separate position. Strain Paenibacillus sp. PS-K-17 may find use in biotechnology as a producer of extracellular polysaccharides and enzymes splitting plant polymeric substances as well as a component of microbial consortium-ingredient of a new complex feed additive.Из зерна пшеницы, контаминированного образующей полисахарид микрофлорой, выделена и охарактеризована культура бактерий ПС-К-17. Установлено, что изолят на агаризованных средах и в глубинной культуре со специфическими субстратами синтезирует бета-галактозидазу, амилазу, протеазу, пектиназу, целлюлазу, бета- глюканазу, липазу (эстеразу), альгиназу, а также внеклеточные полисахариды и пигменты, возможно, каротиноиды. На основании культурально-морфологических и физиолого-биохимических особенностей, а также филогенетического анализа нуклеотидных последовательностей гена 16S рРНК (код доступа MF443394 в GenBank) бактериальная культура идентифицирована как Paenibacillus species ПС-К-17. Исследуемый изолят образует одну филогенетическую ветвь с типовыми штаммами Paenibacillus nicotianae (98,3 %), Paenibacillus hordei (98,2 %), Paenibacillus kyungheensis (97,9 %), в пределах которой занимает обособленное положение. Штамм Paenibacillus sp. ПС-К-17 может найти применение в биотехнологии как продуцент внеклеточных полисахаридов и ферментов, расщепляющих растительные полимеры, а также как компонент микробного консорциума в составе новой кормовой добавки комплексного действия

Поиск протеомных маркеров рака молочной железы в составе суммарных экзосом крови

To improve early detection of cancer, search for tumor markers in biological fluids is of great importance. A significant portion of exosomes is associated with the surface of blood cells, however, the protein spectrum of such exosomes has not been previously studied. The use of total blood exosomes (plasma exosomes and exosomes associated with the surface of blood cells) can not only significantly increase the specificity and sensitivity of existing methods, but also suggest new tumor markers for liquid biopsy.Objective. Search for candidate protein tumor markers of breast cancer by comparing 2D-proteomic maps of total blood exosomes of healthy females (HFs) and breast cancer patients (BCPs).Methods. Exosomes were isolated from plasma and total blood of HFs and BCPs by ultrafiltration followed by ultracentrifugation and were characterized using transmission electron microscopy (TEM) and immunocytochemistry. Protein concentration in exosomes was determined using the NanoOrange Protein Quantitation kit (Invitrogen) commercial kit. Proteomes of exosomes were studied using 2d electrophoresis followed by protein identification by mass spectrometry.Results. Highly purified samples of vesicles from plasma and total blood of no more than 100 nm in size were obtained, on the surface of which markers specific for exosomes were detected by monoclonal antibodies CD9. A comparative analysis of the proteomic maps of exosomal proteins of the HFs and BCPs obtained by 2D-electrophoresis allowed us to establish significant differences in the expression level and protein set in normal and pathological conditions. The 11 proteomic markers of breast cancer were identified by the peptide fingerprint method, of which LRG и у-chain FGB were detected in the composition of exosomes for the first time (according to the Exocarta database). Using MALDI-TOF mass spectrometry, 99 proteins were identified in the exosome preparations of the blood of HFs and BCPs, of which 35% were detected in the composition of the exosomes for the first time (according to the Exocarta database). 17 (53%) proteins associated with breast cancer were detected in total blood exosomes of cancer patients.Conclusion. The results obtained indicate that total blood exosomes are a promising source of diagnostic material for the search for proteomic markers of breast cancer. The identified proteomic tumor markers require further validation.Актуальность. Актуальной задачей повышения эффективности ранней диагностики онкологических заболеваний является поиск высокоспецифичных опухолевых маркеров в биологических жидкостях организма. Значительная часть экзосом ассоциирована с поверхностью форменных элементов крови, однако белковый спектр таких экзосом ранее не исследовался. использование суммарных экзосом крови (экзосом плазмы и экзосом, ассоциированных с поверхностью клеток крови) может не только значительно повысить специфичность и чувствительность существующих методов, но и выявить новые онкомаркеры для жидкостной биопсии.Цель работы - поиск кандидатных белковых онкомаркеров рака молочной железы (РМЖ) путем сравнения 2D-протеомных карт суммарных экзосом крови здоровых женщин и больных РМЖ.Материал и методы. Экзосомы выделены из крови здоровых женщин и больных РМЖ методом ультрафильтрации с последующим ультрацентрифугированием и охарактеризованы при помощи трансмиссионной электронной микроскопии и иммуноцитохимии. Концентрацию белка в экзосомах определяли при помощи коммерческого набора NanoOrange Protein Quantitation kit (Invitrogen). Протеом экзосом исследован с помощью 2D-электрофореза с последующей идентификацией белков методом масс-спектрометрии.Результаты. Получены высокоочищенные препараты микровезикул суммарной крови размером не более 100 нм, на поверхности которых иммуноцитохимически были выявлены специфические для экзосом маркеры (CD63, CD9 и CD24). Сравнительный анализ протеомных карт экзосомальных белков здоровых женщин и больных РМЖ, полученных с помощью 2D-электрофореза, позволил установить значимые различия в уровне экспрессии и наборе белков в норме и патологии. Методом пептидного фингерпринта идентифицировано 11 перспективных протеомных маркеров РМЖ, из них LRG и у-цепь FGB выявлены в составе экзосом впервые (согласно базе Exocarta). Методом MALDI-TOF масс-спектрометрии идентифицировано 99 белков в препаратах экзосом крови здоровых женщин и больных РМЖ, из них 35 % выявлены в составе экзосом впервые (согласно базе Exocarta). В составе суммарных экзосом крови онкологических больных выявлено 17 (53 %) белков, ассоциированных с РМЖ.Заключение. Полученные результаты свидетельствуют о том, что суммарные экзосомы крови являются перспективным источником диагностического материала для поиска протеомных маркеров РМЖ. идентифицированные протеомные онкомаркеры в их составе требуют дальнейшей валидации

Liquid biopsies come of age: towards implementation of circulating tumour DNA

Improvements in genomic and molecular methods are expanding the range of potential applications for circulating tumour DNA (ctDNA), both in a research setting and as a ‘liquid biopsy’ for cancer management. Proof-of-principle studies have demonstrated the translational potential of ctDNA for prognostication, molecular profiling and monitoring. The field is now in an exciting transitional period in which ctDNA analysis is beginning to be applied clinically, although there is still much to learn about the biology of cell-free DNA. This is an opportune time to appraise potential approaches to ctDNA analysis, and to consider their applications in personalized oncology and in cancer research.We would like to acknowledge the support of The University of Cambridge, Cancer Research UK (grant numbers A11906, A20240, A15601) (to N.R., J.D.B.), the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n. 337905 (to N.R.), the Cambridge Experimental Cancer Medicine Centre, and Hutchison Whampoa Limited (to N.R.), AstraZeneca (to R.B., S.P.), the Cambridge Experimental Cancer Medicine Centre (ECMC) (to R.B., S.P.), and NIHR Biomedical Research Centre (BRC) (to R.B., S.P.). J.G.C. acknowledges clinical fellowship support from SEOM

Search for breast cancer proteomic markers in total blood exosomes

To improve early detection of cancer, search for tumor markers in biological fluids is of great importance. A significant portion of exosomes is associated with the surface of blood cells, however, the protein spectrum of such exosomes has not been previously studied. The use of total blood exosomes (plasma exosomes and exosomes associated with the surface of blood cells) can not only significantly increase the specificity and sensitivity of existing methods, but also suggest new tumor markers for liquid biopsy.Objective. Search for candidate protein tumor markers of breast cancer by comparing 2D-proteomic maps of total blood exosomes of healthy females (HFs) and breast cancer patients (BCPs).Methods. Exosomes were isolated from plasma and total blood of HFs and BCPs by ultrafiltration followed by ultracentrifugation and were characterized using transmission electron microscopy (TEM) and immunocytochemistry. Protein concentration in exosomes was determined using the NanoOrange Protein Quantitation kit (Invitrogen) commercial kit. Proteomes of exosomes were studied using 2d electrophoresis followed by protein identification by mass spectrometry.Results. Highly purified samples of vesicles from plasma and total blood of no more than 100 nm in size were obtained, on the surface of which markers specific for exosomes were detected by monoclonal antibodies CD9. A comparative analysis of the proteomic maps of exosomal proteins of the HFs and BCPs obtained by 2D-electrophoresis allowed us to establish significant differences in the expression level and protein set in normal and pathological conditions. The 11 proteomic markers of breast cancer were identified by the peptide fingerprint method, of which LRG и у-chain FGB were detected in the composition of exosomes for the first time (according to the Exocarta database). Using MALDI-TOF mass spectrometry, 99 proteins were identified in the exosome preparations of the blood of HFs and BCPs, of which 35% were detected in the composition of the exosomes for the first time (according to the Exocarta database). 17 (53%) proteins associated with breast cancer were detected in total blood exosomes of cancer patients.Conclusion. The results obtained indicate that total blood exosomes are a promising source of diagnostic material for the search for proteomic markers of breast cancer. The identified proteomic tumor markers require further validation