Characterisation of Recombinant Human Erythropoietin Obtained from CHOpE—Erythropoietin Producing Strain of Chinese Hamster Ovary Cells

Development and implementation in clinical practice of recombinant human erythropoietins (rhEPOs) remain a priority task today. Additional studies were performed in order to obtain clinical trial authorisation for rhEPO tablets for oral use. The studies were aimed to demonstrate the suitability of the erythropoietin producer strain based on Chinese hamster ovary cells (CHOpE) for the production of rhEPO, and the compliance of the substance characteristics with the requirements for erythropoietin (EPO).The aim of the study was to characterise the rhEPO substance obtained from the CHOpE strain cells in accordance with the requirements for EPO.Materials and methods: the rhEPO substance was obtained by culturing the strain of Chinese hamster ovary cells—CHOpE. The expression construct of the producer strain was evaluated using methods for determination of nucleotide and amino acid sequences. The Sanger method was used to perform sequencing of the nucleotide sequence encoding the human EPO gene. The amino acid sequences of the rhEPO molecule C- and N-termini were determined by the Edman method. The copy number of the EPO gene in CHOpE cells was determined by real-time polymerase chain reaction. The properties of the rhEPO substance were evaluated in accordance with the requirements for EPO. Isoelectric focusing, peptide mapping, and polyacrylamide gel electrophoresis were used for identification of the rhEPO substance. The ratio of isoform composition was determined by capillary electrophoresis. Dimer impurities and high molecular weight related substances were determined by high-pressure liquid chromatography. The content of protein and residual nucleic acids was determined by spectrophotometry. The concentration of the rhEPO substance was assessed by enzyme immunoassay.The results of the study confirmed genetic stability of the CHOpE producer strain and demonstrated identity of N- and C-terminal amino acid sequences of the rhEPO molecule to those of the natural EPO. The CHOpE producer strain was used to obtain a rhEPO substance which is homogenous and does not contain impurities of EPO oligomeric forms. Dimers and high molecular weight related substances account for less than 0.5%. The rhEPO molecular weight ranges from 32 to 38 kDa, and the isoelectric point is within 2.8—4.15. The study identified the peaks of isoforms 1–8, the isoform composition of the rhEPO substance corresponds to that of EPO. It was determined that 1 mol of the substance contains 13.75 mols of sialic acids.Conclusions: the study confirmed the suitability of the CHOpE producer strain for the production of rhEPO. The obtained rhEPO substance meets requirements for EPO

Highly Effective xMAP Multiplex Assay for the Detection and Identification of Hemorrhagic Fever Agents, Including Ebola Virus

Developed has been the oligonucleotide liquid biochip based on xMAP technology, designed for the laboratory detection of particularly dangerous viral pathogens such as Ebola and Marburg filoviruses, and Machupo , Junin, and Lassa arenaviruses. The suggested approach allows for the detection of up to 100 viral genome equivalents in a sample. The sensitivity and specificity of oligonucleotide biochip is 100 % when the laboratory panels of positive and negative samples are used. These results indicate that the xMAP multiplexing for the detection and identification of tropical hemorrhagic fever agents, including Ebola virus, is not inferior to the conventional method such as real-time RT-PCR and can be applied for evaluation of viral load, and further on can easily be expanded for both the analysis of new viral agents and for the detection of critical mutations in viral genomes

Development of the Multiplex Real-Time PCR for Marburg, Ebola, and Lassa Viruses Identification

Presented are the data on the development and approbation of the method of Marburg, Ebola, and Lassa viruses identification based on real-time multiplex PCR with hybridization-fluorescent detection. This method is meant for the differential diagnostics of hemorrhagic fevers caused by these viruses. Displayed are the results of determination of multiplex PCR analytical sensitivity and specific activity

Participation of UGMU dental students in the faculty's volunteer movement

In 2017, the Happy Smile project was developed at the dental faculty of Ural State Medical University and its active implementation began, during which teachers and students conduct preventive measures in educational institutions shopping centers, and hospitals in the city. Studied the involvement of students in the implementation of this, and identified factors affecting the development of this activit

Epidemiology of hard tissues diseases of teeth among sportsmens in the Ural region

The article presents the results of a dental examination of 125 athletes involved in cyclical, speed-strength and team sports. The study revealed a high prevalence of dental diseases among athletes, which proves the need for the development of new methods of treatment and prophylactic and a differential approach of dental care to this population.В статье представлены результаты стоматологического обследования 125 спортсменов, занимающихся циклическими, скоростносиловыми и игровыми видами спорта. В ходе исследования была выявлена высокая распространенность стоматологических заболеваний среди спортсменов, что доказывает необходимость в разработке новых лечебнопрофилактических методов коррекции и дифференциального подхода к оказанию стоматологической помощи данному контингенту населения

Характеризация субстанции рекомбинантного эритропоэтина человека, полученного на основе штамма клеток яичника китайского хомячка CHOpE - продуцента эритропоэтина

Development and implementation in clinical practice of recombinant human erythropoietins (rhEPOs) remain a priority task today. Additional studies were performed in order to obtain clinical trial authorisation for rhEPO tablets for oral use. The studies were aimed to demonstrate the suitability of the erythropoietin producer strain based on Chinese hamster ovary cells (CHOpE) for the production of rhEPO, and the compliance of the substance characteristics with the requirements for erythropoietin (EPO).The aim of the study was to characterise the rhEPO substance obtained from the CHOpE strain cells in accordance with the requirements for EPO.Materials and methods: the rhEPO substance was obtained by culturing the strain of Chinese hamster ovary cells—CHOpE. The expression construct of the producer strain was evaluated using methods for determination of nucleotide and amino acid sequences. The Sanger method was used to perform sequencing of the nucleotide sequence encoding the human EPO gene. The amino acid sequences of the rhEPO molecule C- and N-termini were determined by the Edman method. The copy number of the EPO gene in CHOpE cells was determined by real-time polymerase chain reaction. The properties of the rhEPO substance were evaluated in accordance with the requirements for EPO. Isoelectric focusing, peptide mapping, and polyacrylamide gel electrophoresis were used for identification of the rhEPO substance. The ratio of isoform composition was determined by capillary electrophoresis. Dimer impurities and high molecular weight related substances were determined by high-pressure liquid chromatography. The content of protein and residual nucleic acids was determined by spectrophotometry. The concentration of the rhEPO substance was assessed by enzyme immunoassay.The results of the study confirmed genetic stability of the CHOpE producer strain and demonstrated identity of N- and C-terminal amino acid sequences of the rhEPO molecule to those of the natural EPO. The CHOpE producer strain was used to obtain a rhEPO substance which is homogenous and does not contain impurities of EPO oligomeric forms. Dimers and high molecular weight related substances account for less than 0.5%. The rhEPO molecular weight ranges from 32 to 38 kDa, and the isoelectric point is within 2.8—4.15. The study identified the peaks of isoforms 1–8, the isoform composition of the rhEPO substance corresponds to that of EPO. It was determined that 1 mol of the substance contains 13.75 mols of sialic acids.Conclusions: the study confirmed the suitability of the CHOpE producer strain for the production of rhEPO. The obtained rhEPO substance meets requirements for EPO. Разработка и внедрение в клиническую практику препаратов рекомбинантного эритропоэтина человека (рчЭПО) продолжают оставаться актуальными в настоящее время. Для получения разрешения на проведение клинических исследований таблетированной формы рчЭПО для перорального применения проведены дополнительные исследования для подтверждения пригодности штамма-продуцента CHOpE для производства рчЭПО и соответствия характеристик субстанции требованиям, предъявляемым к ЭПО.Цель работы: характеризация субстанции рчЭПО, полученной на основе клеток штамма продуцента СНОрЕ, в соответствии с требованиями, предъявляемыми к ЭПО.Материалы и методы: субстанцию рчЭПО получали при культивировании штамма клеток яичника китайского хомячка CHOpE. Экспрессирующую конструкцию штамма-продуцента оценивали с помощью методов определения нуклеотидной и аминокислотной последовательностей. Секвенирование нуклеотидной последовательности, кодирующей ген ЭПО человека, проводили по методу Сэнгера. Аминокислотную последовательность С- и N-концов молекулы рчЭПО определяли методом Эдмана. Копийность гена ЭПО в клетках СНОрЕ определяли методом полимеразной цепной реакции в реальном времени. Свойства субстанции рчЭПО изучали в соответствии с требованиями, предъявляемыми к ЭПО. Подлинность субстанции рчЭПО определяли с помощью методов изоэлектрического фокусирования, пептидного картирования и электрофореза в полиакриламидном геле. Для определения соотношения изоформного состава использовали метод капиллярного электрофореза. Для выявления примесей димеров и высокомолекулярных родственных веществ в субстанции применяли метод высокоэффективной жидкостной хроматографии высокого давления. Содержание белка и остаточных нуклеиновых кислот определяли спектрофотометрическим методом. Концентрацию субстанции рчЭПО оценивали методом иммуноферментного анализа.Результаты: подтверждена генетическая стабильность штамма-продуцента СHOpE, доказана идентичность аминокислотной последовательности N- и С-концов молекулы рчЭПО природному ЭПО. На основе штамма-продуцента СHOpE получена субстанция рчЭПО, которая является гомогенной и не содержит примесей олигомерных форм ЭПО. Димеры и высокомолекулярные родственные вещества составляют менее 0,5%. Молекулярная масса рчЭПО находится в диапазоне значений от 32 до 38 кДа, изоэлектрическая точка – от 2,8 до 4,15. Идентифицированы пики изоформ 1–8, изоформный состав субстанции рчЭПО соответствует ЭПО. Установлено, что в 1 моль субстанции содержится 13,75 моль сиаловых кислот.Выводы: подтверждена пригодность штамма-продуцента СНОрЕ для производства рчЭПО. Полученная субстанция рчЭПО соответствует требованиям, предъявляемым к ЭПО

The problem of life quolity in patients with herpetic lesion of the face and oral cavity mucosa

The quality of life in medicine includes a set of physical, psychological, emotional and social functioning of a person based on his subjective perception. The appearance of herpetic lesions in a functionally and aesthetically important zone has a significant effect on the quality of life of such patients and makes this problem not only medical, but also social. The aim of the study was to assess the effect of herpetic damage of the facial skin and oral mucosa on the quality of life of patients. The study involved 175 patients with herpes simplex of skin (B00.10, ICD-10, 1997), herpes simplex of lips (B00.11), herpetic gingivostomatitis (B00.2X) between September 2011 and July 2018. For quality assessment of patients, a specialized validated questionnaire for the quality of life "Oral health impact profile" (OHIP-49 RU) (Gileva OS, 2009) was used. In case of recurrence of herpes simplex of the face, lip, herpetic gingivostomatitis, the quality of life decreased by 46.6% (the quality of life index was 91.3 ± 8.1 points) on the vesicular and erosive stage; by 21.6% (42.4 ± 3.1, respectively) at the crust stage. During the remission, the quality of life in such patients does not reach its maximum and was only 96.6% (the quality of life index is 6.7 ± 0.8 points), which is associated with a number of restrictions in daily life.Качество жизни в медицине включает в себя совокупность физического, психологического, эмоционального и социального функционирования человека, основанного на его субъективном восприятии. Появление герпетических высыпаний в функционально и эстетически значимой зоне оказывает существенное влияние на качество жизни таких пациентов, а значит, делает данную проблему не только медицинской, но и социальной. Цель исследования – оценить влияние герпетического поражения кожи лица и слизистой оболочки рта на качество жизни пациентов. В исследовании приняли участие 175 пациентов с диагнозом простой герпес лица (B00.10, МКБ-10, 1997), простой герпес губы (В00.11), герпетический гингивостоматит (В00.2Х) в период с сентября 2011 по июль 2018 г. Для оценки качества жизни пациентов был использован специализированный валидированный опросник качества жизни «Профиль влияния стоматологического здоровья» (OHIP-49 RU) (Гилева О.С., 2009 г.). При возникновении рецидива простого герпеса лица, губы, герпетического гингивостоматита установлено снижение качества жизни на 46,6% (показатель качества жизни - 91,3±8,1 балла) на везикулярной и эрозивной стадии; на 21,6% (42,4±3,1 балла, соответственно) на стадии корки. В период ремиссии качество жизни у таких пациентов не достигает своего максимума и составляет лишь 96,6% (показатель качества жизни - 6,7±0,8 балла), что связано с рядом ограничений в повседневной жизни

Detection of tick-borne pathogens in wild birds and their ticks in Western Siberia and high level of their mismatch

Abstract: The Tomsk region located in the south of Western Siberia is one of the most high-risk areas for tick-borne diseases due to elevated incidence of tick-borne encephalitis and Lyme disease in humans. Wild birds may be considered as one of the reservoirs for tick-borne pathogens and hosts for infected ticks. A high mobility of wild birds leads to unpredictable possibilities for the dissemination of tick-borne pathogens into new geographical regions. The primary goal of this study was to evaluate the prevalence of tick-borne pathogens in wild birds and ticks that feed on them as well as to determine the role of different species of birds in maintaining the tickborne infectious foci. We analysed the samples of 443 wild birds (60 species) and 378 ticks belonging to the genus Ixodes Latraille, 1795 collected from the wild birds, for detecting occurrence of eight tick-borne pathogens, the namely tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and species of Borrelia, Rickettsia, Ehrlichia, Anaplasma, Bartonella and Babesia Starcovici, 1893, using RT-PCR/or PCR and enzyme immunoassay. One or more tick-borne infection markers were detected in 43 species of birds. All markers were detected in samples collected from fieldfare Turdus pilaris Linnaeus, Blyth’s reed warbler Acrocephalus dumetorum Blyth, common redstart Phoenicurus phoenicurus (Linnaeus), and common chaffinch Fringilla coelebs Linnaeus. Although all pathogens have been identified in birds and ticks, we found that in the majority of cases (75.5%), there were mismatches of pathogens in birds and ticks collected from them. Wild birds and their ticks may play an extremely important role in the dissemination of tick-borne pathogens into different geographical regions