Experimental infection of pigs porcine epidemic diarrhea virus

Було проведено зараження неонатальних безмолозивних поросят у першу добу їх життя суспензією вірусу ЕДС, отриманого раніше від поросят, хворих на ЕДС. Діагноз на ЕДС у поросят-донорів вірусу ЕДС був вставлений комплексним методом за клініко-епізоотологічними показниками і підтверджений ідентифікацією PEDV методом PCR-RT за допомогою тест-системи «EZ-RED / TGE / PDCoV MPX 1.0 Real time RT-PCR» фірми Tetracore (США) на ампліфікаторі CFX 96 Real-Time System фірми BIO RAD (США). Гомогенат тонкого кишечнику поросят-донорів PEDV, готували на блендері для PCR у вигляді густого смузі від 18 трупів тварин, заморожували при -18 °С без додавання кріоконсервантів і зберігали 359 діб. Перед зараженням поросят смузі розморожували і методом PCR-RT визначили концентрацію вірусу в геном-еквівалентах (Г.Е.) без кількісного встановлення життєздатних віріонів збудника. Для біопроби відбирали 20 аналогових неонатальних безмолозивних поросят, розділяли їх на 3 дослідні групи (1 група – 5 поросят, 2 група – 5 поросят і 3 група – 7 поросят) та одну контрольну (3 поросяти). Дослідних поросят per os заражали вірусвмісною суспензією, з концентрацією PEDV 1,03 × 106 Г.Е./см3. Доза для зараження першої групи склала 6 см3 (6,18 × 106 Г.Е./см3), для другої – 5 см3 (5,15 × 106 Г.Е./см3), для третьої – 4 см3 (4,12 × 106 Г.Е./см3) гомогенату. Четверта група – контрольна (не заражали). Всі поросята перебували в ідентичних умовах, які повністю відповідають фізіологічним потребам організму. З 17 заражених поросят лише 2 поросят заразилися PEDV. ЕДС було підтверджено лабораторними методами. При бактеріологічному дослідженні внутрішніх органів поросят, що вийшли з експерименту як дослідних, так і контрольної груп, був діагностований колібактеріоз. У контрольній групі була ізольована з серця і кишечнику непатогенна для білих мишей E. coli. Від поросят 1 і 2 дослідних груп була виділена непатогенна для білих мишей E. coli, тобто встановлений колібактеріоз; у 2 дослідній групі у одного поросяти виявили гемолітичну кишкову паличку; у 3 дослідній групі з внутрішніх органів поросят спільно з непатогенною для колишніх мишей кишкової палички ізолювали Klesiella spp., тобто діагностували мікс-інфекцію – колібактеріоз і клебсієльоз. З кишечнику всіх дослідних і контрольних поросят не виділили фізіологічно корисної індигенної мікрофлори – аерококів, молочно-кислих- і біфідобактерій, а висіяли гнильну факультативно-анаеробну спорову і неспорову мікрофлору.Было проведено заражение неонатальных безмолозивних поросят в первые сутки их жизни суспензией вируса ЭДС, полученного ранее от поросят больных ЭДС. Диагноз ЭДС у поросят-доноров вируса ЭДС был установлен комплексным методом по клинико-эпизоотологическим показателями и подтвержден идентификацией PEDV методом PCR-RT с помощью тест-системы «EZ-RED/TGE/PDCoV MPX 1.0 Real time RT-PCR» фирмы Tetracore (США) в амплификаторе CFX 96 Real-Time System фирмы BIO RAD (США). Гомогенат тонкого кишечника поросят-доноров PEDV, готовили на блендере для PCR в виде густого смузи от 18 трупов животных, замораживали при -18 °С без добавления криоконсервантов и хранили 359 суток. Перед заражением поросят смузи размораживали и методом PCR-RT определяли концентрацию вируса в геном-эквивалентах (Г.Е.) без количественного установления жизнеспособных вирионов возбудителя. Для биопробы отбирали 20 аналоговых неонатальных безмолозивних поросят, делили их на 3 опытные группы (1 группа – 5 поросят, 2 группа – 5 поросят и 3 группа – 7 поросят) и одну контрольную (3 поросенка). Опытных поросят per os заражали вирусосодержащей суспензией, с концентрацией PEDV 1,03×106 Г.Е./см3. Доза для заражения первой группы составила 6 см3 (6,18×106 Г.Е./см3), для второй – 5 см3 (5,15×106 Г.Е./см3), для третьей – 4 см3 (4,12×106 Г.Е./см3) гомогената. Четвертая группа – контрольная (не заражали). Все поросята находились в идентичных условиях, которые полностью соответствуют физиологическим потребностям организма. Из 17 зараженных поросят только 2 поросенка заразились PEDV, что было подтверждено лабораторными методами. При бактериологическом исследовании внутренних органов поросят, вышедших из эксперимента как опытных, так и контрольной групп, был диагностирован колибактериоз. В контрольной группе была изолирована из сердца и кишечника непатогенная для белых мышей E. coli. От поросят 1 и 2 опытных групп была выделена непатогенная для белых мышей E. coli, то есть установлен колибактериоз; во 2 опытной группе у одного поросенка обнаружили гемолитическую кишечную палочку; в 3 опытной группе из внутренних органов поросят совместно с непатогенной для белых мышей кишечной палочкой изолировали Klesiella spp., то есть диагностировали микс-инфекцию – колибактериоза и клебсиеллеза. Из кишечника всех опытных и контрольных поросят не выделили физиологически полезной индигенной микрофлоры – аэрококков, молочно кислых- и бифидобактерий, а высеяли гнилостную факультативно-анаэробную споровую и неспоровую микрофлору.There were infected neonatal piglets in the first days of their lives PED virus suspension derived from pigs previously PED patients. Diagnosis for PED in piglets donor virus PED was inserted complex method for clinical and epizootic performance and confirmed the identification PEDV by PCR-RT using the test system «EZ-RED/TGE/PDCoV MPX 1.0 Real time RT-PCR» company Tetracore (USA) Thermocyclers CFX 96 Real-Time System company BIO RAD (USA). Homogenate small intestine of pigs PEDV donor, prepared in a blender for PCR in a thick band of 18 animal carcasses, frozen at -18 °C without cryopreservation and kept 359 days. Before infecting pigs and strip defrost by RT-PCR identified the concentration of the virus genome equivalents (GE) without establishing viable virions quantitative pathogen. For Sample 20 selected analog neonatal piglets, divided them into 3 experimental groups (group 1 – 5 piglets, group 2 – 5 piglets and group 3 – 7 piglets) and one control (3 piglets). Research pigs infected per os virus-containing suspension with a concentration PEDV 1.03×106 GE/cm3. The dose for infection first group was 6 cm3 (6.18×106 GE/cm3), for the second – 5 cm3 (5,15 × 106 GE/cm3), for the third – 4 cm3 (4.12 GE×106/cm3) homogenate. The fourth group – control (not infected). All the pigs were in identical conditions that fully meet the physiological needs of the body. Of the 17 infected pigs only 2 was infected PEDV. PED was confirmed by laboratory methods. In bacteriological examination of internal organs of pigs that came out of a research experiment and control group were diagnosed colibacteriosis. In the control group was isolated from heart and intestinal non-pathogenic for white mice E. coli. From pigs 1 and 2 research groups has been allocated to white mice nonpathogenic E. coli, is set colibacteriosis; 2 experimental group found in one pig hemolytic E. coli; 3 experimental group from the internal organs of pigs in conjunction with non-pathogenic for mice intestinal former cane isolated Klesiella spp., is diagnosed with mixed infection (E. coli, Klesiella spp.). From the intestine of experimental and control pigs do not identified beneficial microflora – aerococcus, lactobacteria, bifidobacteria and cultured putrefactive anaerobic spore facultative and non spore microflora

Aberrant expression of selenium-containing glutathione peroxidases in clear cell renal cell carcinomas

Aim: To find putative diagnostic markers for clear cell renal cell carcinomas (ccRCC). Material and methods: Quantitative polymerase chain reaction (Q-PCR), bisulfite treatment, methylation-specific PCR, analysis on cBioPortal for Cancer Genomics. Results: We have found that expression of GPX 1, GPX3, and GPX4 genes was decreased in ccRCC. We have shown that the number of alanine (GCG) repeats at the amino terminus of the GPX1 protein is variable. It was reported earlier that an allele that possess 5 alanine repeats is associated with the increased cancer risk. According to the obtained data, the allele with the 5 alanine repeats was also present in a group of healthy donors. Moreover, the frequency of alleles with repeats was similar among ccRCC patients and healthy individuals. We found that decreased expression of GPXs genes was not associated with promoter methylation. To provide other explanation, an analysis on the gene copy number was performed. We have found the heterozygous deletions for GPX1 gene, amplification for GPX3 gene, and no change in gene copy number for GPX4. Conclusions: Our data support the hypothesis that GPX1, GPX3, and GPX4 genes may play a role in ccRCC cancerogenesis and therefore they might be considered as putative diagnostic markers for ccRCC. Key Words: clear cell renal cell carcinomas, selenium-containing GPXs, genetic and epigenetic regulation

Genetic risk factors for Parkinson’s disease in Ukraine

The paper focuses on the genetic risk factors for Parkinson’s disease (PD) such as polymorphisms in genes CYP1A1, GSTM1 and APOE. A total number of 516 people were examined. 300 persons were in the control group (mean age 67,0 ± 0,4 years; 200 males and 100 females) and 216 persons were patients with PD (mean age 65,0 ± 0,7 years, 116 males and 100 females). Whole blood samples collected from each person were genotyped using PCR-RFLP. Amplification and restriction results were assessed by conducting vertical agarose gel electrophoresis. The study analyzed marker с.2452C>A in the CYP1A1 gene. In the control group, allele C frequency was 0.79, and allele A frequency – 0.21. Genotype frequencies were: CC – 0.61, AC – 0.36, AA – 0.03. In the group of patients alleles C and A frequencies were 0.64 and 0.36 correspondingly. Genotype frequencies were: CC – 0.35, AC – 0.58, AA – 0.07. There was a significant difference between both groups in allele A frequency. It is considered that 0/0 genotype for the GSTM1 gene is a risk factor for PD. In the controls, +/+ and 0/0 genotypes frequencies were 0.67 and 0.33 correspondingly. In the group of patients +/+ genotype frequency was 0.55 and 0/0 genotype frequency – 0.45. The difference was statistically significant. In the control group genotype frequencies for the АРОЕ gene were 0.715 (Е3/Е3), 0.077 (Е3/Е4), 0.009 (Е4/Е4), 0.167 (Е2/Е3), 0.031 (Е2/Е4) and 0.000 (Е2/Е2). In the group of patients with PD they were 0.634 (Е3/Е3), 0.148 (Е3/Е4), 0.032 (Е4/Е4), 0.157 (Е2/Е3), 0.023 (Е2/Е4) and 0.000 (Е2/Е2). Е3/Е4 genotype frequency was significantly higher in the group of patients with PD than in the control group. Pathogenic allele с.2452C>A of the CYP1A1 gene is associated with increased risk of PD (OR = 1.72). 0/0 genotype carriers have higher risk to develop PD (OR = 1.72). Allele έ4 of the АРОЕ gene may be associated with increased risk of PD. Risk of the disease is higher in έ2 allele carriers (OR = 2.35) and έ4 allele carriers (OR=1.97). People with genotype Е4/Е4 have chances to be affected by PD 3.48 times higher (OR = 3.48). Associations revealed in the different human populations between genetic factors and PD may vary that is associated with the genetic heterogeneity and proportion of environmental factors which affect people. Despite the results are sometimes controversial, they can be helpful in developing DNA-tests for early diagnosis of PD

Dynamic changes in human-gut microbiome in relation to a placebo-controlled anthelminthic trial in Indonesia

Background: Microbiome studies suggest the presence of an interaction between the human gut microbiome and soil-transmitted helminth. Upon deworming, a complex interaction between the anthelminthic drug, helminths and microbiome composition might occur. To dissect this, we analyse the changes that take place in the gut bacteria profiles in samples from a double blind placebo controlled trial conducted in an area endemic for soil transmitted helminths in Indonesia. Methods: Either placebo or albendazole were given every three months for a period of one and a half years. Helminth infection was assessed before and at 3 months after the last treatment round. In 150 subjects, the bacteria were profiled using the 454 pyrosequencing. Statistical analysis was performed cross-sectionally at pre-treatment to assess the effect of infection, and at post-treatment to determine the effect of infection and treatment on microbiome composition using the Dirichlet-multinomial regression model. Results: At a phylum level, at pre-treatment, no difference was seen in microbiome composition in terms of relative abundance between helminth-infected and uninfected subjects and at post-treatment, no differences were found in microbiome composition between albendazole and placebo group. However, in subjects who remained infected, there was a significant difference in the microbiome composition of those who had received albendazole and placebo. This difference was largely attributed to alteration of Bacteroidetes. Albendazole was more effective against Ascaris lumbricoides and hookworms but not against Trichuris trichiura, thus in those who remained infected after receiving albendazole, the helminth composition was dominated by T. trichiura. Discussion: We found that overall, albendazole does not affect the microbiome composition. However, there is an interaction between treatment and helminths as in subjects who received albendazole and remained infected there was a significant alteration in Bacteroidetes. This helminth-albendazole interaction needs to be studied further to fully grasp the complexity of the effect of deworming on the microbiome. Trial registration: ISRCTN Registy, ISRCTN83830814

Endemic course of epidemic diarrhea of pigs in the stabilized focus of infection

Porcine epidemic diarrhea virus (PEDV) has been circulating in Ukraine since 2014 and induces an especially dangerous viral infection with a lethal diarrheal syndrome in newborn piglets, with the initial appearance at the focus of infection. The number of infected cases and lethality among diseased piglets of 1–5 days of age can reach 100%, which together with the forced anti-epizootic measures brings significant economic losses. PED can spread to all pigs, but the emergent quality of infectious pathology appears in newborn piglets. No effective and biologically safe means of specific antiviral prophylaxis, which substantially halts the epizootic process is registered, and etiopathogenetic therapy is not developed, therefore PED is an emergent infection which is difficult to control. Over time there appear stationary foci of infection, where evolutionary changes in relationships in the host-parasite system take place fairly rapidly, since pigs are prolific and fast maturing animals able to replace each generation up to three times each year. This leads to a significant variability in interpopulation relationships and the induction of biodiversity in the molecular mechanisms of adaptation and processing of the viral genome. Clinically, genetic modifications of local variants of PEDV – populations are manifested in the form of changes in epizootic peculiarities in the course of infectious pathology in different age groups of animals. Modifications of PEDV may be accompanied by a slight weakening of the intensity of the infectious process, a decrease in mortality and a decrease in the severity of the pathogenesis of diarrheal syndrome. At the same time, the age range of severe abdominal lesions expands from newborn piglets to fattening animals of older age groups of 28, 32, 70 days. Using a set of measures to combat the PED, including “reverse feeding” recycled infected biomaterial from convalescent pigs, eradication of the pathogen from the environment of the host macroorganisms through a total disinfection regime and strict compliance with veterinary and sanitary rules of animal husbandry provide temporary positive results, but in theory this approach is incorrect, since contamination of animals leads to the dispersal of the virus and the formation of endemic foci of infection. The persistence of the virus in convalescent organisms is not fixed, the external inanimate environment can only be a mechanical factor in transmission of the pathogen preserving the viability of PEDV over time. Stabilization of the epizootic foci of infection is possible due to three factors: a) dissemination of the virus in “reverse feeding”; b) preservation of the virus in the external environment as a result of poor-quality disinfection; c) occurrence of a non-immune element among the convalescent young gilts, who as a result of juvenile insufficiency of the immune system have a low titer accumulation of colostral antibodies to the virus received in the biomaterial through reverse feeding. Due to the lack of “lactogenic immunity”, neonatal pigs as biological indicators for the presence of PEDV in the environment begin reproducing the virus in the enterocytes and develop a typical diarrheal syndrome PED

ABERRANT EXPRESSION OF SELENIUM-CONTAINING GLUTATHIONE PEROXIDASES IN CLEAR CELL RENAL CELL CARCINOMAS

Aim: To find putative diagnostic markers for clear cell renal cell carcinomas (ccRCC). Material and methods: Quantitative polymerase chain reaction (Q-PCR), bisulfite treatment, methylation-specific PCR, analysis on cBioPortal for Cancer Genomics. Results: We have found that expression of GPX 1, GPX3, and GPX4 genes was decreased in ccRCC. We have shown that the number of alanine (GCG) repeats at the amino terminus of the GPX1 protein is variable. It was reported earlier that an allele that possess 5 alanine repeats is associated with the increased cancer risk. According to the obtained data, the allele with the 5 alanine repeats was also present in a group of healthy donors. Moreover, the frequency of alleles with repeats was similar among ccRCC patients and healthy individuals. We found that decreased expression of GPXs genes was not associated with promoter methylation. To provide other explanation, an analysis on the gene copy number was performed. We have found the heterozygous deletions for GPX1 gene, amplification for GPX3 gene, and no change in gene copy number for GPX4. Conclusions: Our data support the hypothesis that GPX1, GPX3, and GPX4 genes may play a role in ccRCC cancerogenesis and therefore they might be considered as putative diagnostic markers for ccRCC. Key Words: clear cell renal cell carcinomas, selenium-containing GPXs, genetic and epigenetic regulation

Raman dissipative soliton source of ultrashort pulses in NIR-III spectral window

We present a novel fiber source of ultrashort pulses at the wavelength of 1660 nm based on the technique of external cavity Raman dissipative soliton generation. The output energy of the generated 30 ps chirped pulses is in the range of 0.5–3.6 nJ with a slope efficiency of 57%. Numerical simulations are in excellent agreement with the experimental results and the shape of the compressed pulses. The compressed pulses consist of a central part with a duration of 300 fs and a weak pedestal. Our results clearly demonstrate the potential to extend the spectral range of the Raman-assisted technique for generating ultra-short pulses to new frequency regions, including biomedical windows. This paves the way for the development of new dissipative soliton sources in these bands

Communicative-functional Components of Discourse

The article considers the main types of semantic-sigmatic and communicative-functional transformations of lexical units against the background of changes in certain parameters of discourse and analyzes the fundamental motives for changing the load of lexical units in modern space. The relevance of this work is determined by modern transformations of linguistic study of different types of discourse, which occur on the basis of the study of communication and the search for universal foundations of linguistic interaction. Language is considered as a complex communicative and cognitive phenomenon that determines the state of intercultural communication. The components that directly affect the communication process and the success of the modern language formation must be identified. The aim of this study was to analyze the semantic-sigmatic and communicative-functional components of discourse and the theory of their understanding in modern communication. The theoretical and methodological basis of the study included the works of scientists in the field of modern methods of communication. The objective method was the analysis of documents (monographs, articles, statistics, scientific papers and textbooks on the selected topic)