- Email: [email protected]
Genetics of multiple sclerosis: Association analysis of GWAS-identified and microRNA-linked common genetic variations
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Abstracts / Journal of Biotechnology 208 (2015) S5–S120
at about 1.5 cm apart. A quantity of 0.2 ml solution was inoculated in each hole. Fifteen days after inoculation, micro-tomography computed images of longitudinal sections of the infected tibia revealed a change in the morphology of the cancellous bone just between the two infected holes. A connecting path made of modified cancellous bone was observed between the holes. This change of bone morphology is due to the uni-directional expansion of the two bacterial population sites. This result suggests that bacteria are capable to communicate not only within a packed population (the so called quorum sensing), but also at a distance and in a relatively dense environment such as the bone. This communication is needed for their growth, virulence and formation of biofilms. Acknowledgements: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS–UEFISCDI, project number PN-II-ID-PCE-2011-3-0953. http://dx.doi.org/10.1016/j.jbiotec.2015.06.293 Study upon the haematological parameters in minipig models Ioan Hut¸u 1 , Irina Patras 1,∗ , Esra Elena Foditsh 2 , Rainhold Zimmermann 2 , Calin Mircu 1 1 Faculty of Veterinary Medicine, Banat University of Agricultural Sciences and Veterinary Medicine “King Mihai I of Romania” Timisoara, Romania 2 Spinal Cord Injury and Tissue Regeneration Centre, Paracelsus Medical University, Austria
E-mail address: iri [email protected] (I. Patras). Minipig models are used frequently in many fields of biomedical research due to the (i) social acceptance, (ii) existence of a large number of similarities between the swine’s and human’s physiology and (iii) easy training and handling. To observe and compare the haematology dynamics during the follow-up period between four groups of minipigs, considering the type of intervention applied. The experiment was conducted on 21 Gottingen Minipig, adult females which underwent different surgical procedures, as follows: thoracic vertebrae laminectomy, laparoscopic intervention or both – representing the spinal cord injury model (SCI) and control group (healthy individuals). 64 blood samples were processed using an automatic Haematology analyser IDEXX model Procyte DxTM with veterinary software for 25 parameters. The results obtained was classified as low, normal or high. Comparing the obtained values with other parameters and results of clinical examination we conclude that the classification of the equipment not necessary meets the normal status of Göttingen minipigs, as reflected in the software interpretation. Therefore, we used the reference values provided by the supplier of biological material – Elegaard Göttingen Minipigs® . Kruskal Wallis statistical analysis performed on blood counts shows that surgical interventions and neuromodulation had an impact on mean corpuscular volume (p < 0.033), white blood cell count (neutrophils p < 0.004 and basophils) and percentage of the distribution in differential leucocyte count (p < 0.008) to lymphocytes (p < 0.002), monocytes (p < 0.020) and basophils (p < 0.008). http://dx.doi.org/10.1016/j.jbiotec.2015.06.294
Evaluating the clinical and anatomopatological investigative techniques of liver neoplastic processes in dogs Cristina Alice Vulpe 1,∗ , Vasile Vulpe 2 , Leonardo Meomartino 3 , Sorin Aurelian Pasca 2 , Ionel Bondoc 2 , Ionel Papuc 1 1 Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania 2 Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Iasi, Romania 3 Faculty of Veterinary Medicine, University Federico II, Naples, Italy
E-mail address: [email protected] (C.A. Vulpe). Primary hepatic tumours in dogs are rare. On the other hand, metastases are frequent and their main center of proliferation is the liver. It’s common knowledge that patients (dogs) are brought in for a clinical investigation in advanced stages of neoplastic conditions. This occurs mostly because of the extraordinary capacity of the liver to make up for the lost functions. The clinical examination is the first step in diagnosing a dog suffering from a liver tumour. Clinical signs vary according to the size of the neoplasm and the age of the patient. The imaging examination guides the clinician towards a diagnosis; the ultrasound is a defining step, even more so if it used with a puncture. A radiologic examination attests the occurrence of lung metastases. The etiologic diagnosis is set in virtue of an anatomopathological examination. The main objective of the paper consists of evaluating the relevancy of the utilised methods in establishing the diagnosis of liver tumours in dogs. These studies have led to the following etiologic RESULTS: 8 carcinomas; 1 cholangiocarcinoma; 5 adenocarcinomas; 2 lymphosarcomas; 1 multiple myeloma. http://dx.doi.org/10.1016/j.jbiotec.2015.06.295 Genetics of multiple sclerosis: Association analysis of GWAS-identified and microRNA-linked common genetic variations Vitalina V. Bashinskaya ∗ , Olga G. Kulakova, Ivan S. Kiselev, Natalia M. Baulina, Alexey N. Boyko, Ekaterina Yu. Tsareva, Olga O. Favorova Pirogov Russian National Research Medical University, Moscow, Russia E-mail address: [email protected] (V.V. Bashinskaya). Multiple sclerosis (MS) is a chronic autoimmune polygenic disease of the central nervous system. The results of genome-wide association studies (GWASs) only partially explain MS etiology. Among other factors underlying MS susceptibility, the role of microRNAs is suggested pursuant to GWAS and expression studies. Here we aimed to analyze in Russians possible associations with MS of several GWAS-identified loci, the majority of them being proven miRNA targets, and with SNPs located in miRNA genes. The selected loci are CD58, IL7RA, IL2RA, CD6, TNFRSF1A, CLEC16A, IRF8, STAT3, MIR499A, and MIR146A. 509 MS patients and 276 healthy volunteers from Moscow were enrolled in the study. The carriage of individual alleles/genotypes and their combinations was compared in unstratified case/control samples and after gender stratification of MS patients and controls using APSampler software, which is based on Bayesian MCMC method and provides validation by means of Fisher’s exact test.
Abstracts / Journal of Biotechnology 208 (2015) S5–S120
We validated associations of CLEC16A and IL2RA with MS. Besides, CLEC16A and IRF8 were associated with MS in women, and IL7RA and CD58 – in men. With multi-locus association analysis the protective biallelic combinations (TNFRSF1A*T + CLEC16A*A), (TNFRSF1A*T + IRF8*A), and (IRF8*A + MIR499*A) were identified in women. Our data show involvement of immune response genes and genes, linked to miRNA system, in MS pathogenesis. This study was supported with Russian Scientific Foundation (grant 14-14-00605). http://dx.doi.org/10.1016/j.jbiotec.2015.06.296 Examination of pleural effusion in cats – 7 cases Alexandru Gabriel Neagu ∗ , Niculae Tudor, Poliana Tudor, Constantin Vlagioiu Faculty of Veterinary Medicine, University of Agronomical Sciences and Veterinary Medicine, Bucharest, Romania E-mail address: [email protected] (A.G. Neagu). Pleural effusions represent one cause of dyspnea in companion animals and can have multiple causes. Clinical signs associated with pleural effusions are varied, depending on the amount of the effusion and how quickly the fluid accumulates. Pleural effusions may be represented either by transsudat, modified transsudat or exudates, but the radiographic examination does not determine the type of the effusion, therefore further investigations are needed. In the study were enrolled seven cats aged between 3 years and 14 years who presented respiratory disorders on physical examination. Radiographic examination performed in the lateral view incidence, ventro/dorsal and dorso/ventral revealed the presence of pleural effusion. Thoracocentesis was performed unilateral or bilateral then the pleural fluid was collected and examined macroscopically and then microscopically. Cytological examinations established that pleural effusions were represented by: hemorrhagic effusion (2); neoplastic effusion (2); septic exudates (3). Pleural effusions are a secondary phenomenon, which requires further investigation to establish the underlying cause. The cytological examination provides important clues for the diagnosis. http://dx.doi.org/10.1016/j.jbiotec.2015.06.297 Seroprevalence of Anaplasma phagocytophilum in dogs from Constant¸a county Daniela Enache 1,∗ , Mirela Imre 2 , Marius Stelian Ilie 2 , Dragomir Coprean 3 1 Ovidius University, Department of Biology, Faculty of Natural and Agricultural Sciences, 900470 Constant¸a, Romania 2 Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I of Romania” from Timis¸oara, Department of Parasitology and Parasitic Diseases, 300645 Timis¸oara, Romania 3 Academy of Romanian Scientists, Ovidius University, Department of Biology, Faculty of Natural and Agricultural Sciences, 900470 Constant¸a, Romania
E-mail address: [email protected] (D. Enache). Canine anaplsmosis is a tick-borne disease of dogs who progresses with serious clinical manifestations which are dependent of the pathogens location (granulocytes, platelets). The pathogens who
S95
cause the diseases are from Anaplasma genera (Anaplasma phagocytohilum, Anaplasma platys). Anaplasma phagocytophilum has an important significance for both animals and humans health. The goal of our study was to etablish the status of Anaplasma phagocytophilum infection in dogs from South–Eastern part of Romania. Serum samples from 257 dogs living in 35 localities from Constant¸a county, were assayed through Diff-Quik stained blood smears and IFAT. Our results showed that the seroprevalence of antibodies of Anaplasma phagocytophilum was 6.22%. Overall, the seroprevalence of Anaplasma phagocytophilum infection in dogs from our study is enough to worry us and to be considered by all veterinarians from Constant¸a county when they will evaluate a dog. In the present study no significant differences were detected (p > 0.05) between the prevalence and age, gender and breed of the dogs. http://dx.doi.org/10.1016/j.jbiotec.2015.06.298 The in vitro investigation of cytotoxic and apoptotic effects vulpinic acid on normal and cancer cells O˘guzhan Karaosmano˘glu 1 , Hülya Sivas 1,∗ , Turgay Tay 2 , Ays¸en Özdemir Türk 1 1
Anadolu University, Faculty of Sciences, Department of Biology, Eskisehir, Turkey 2 Anadolu University, Faculty of Sciences, Department of Chemistry, Eskisehir, Turkey E-mail address: [email protected] (H. Sivas). Vulpinic acid is one of the important seconder metabolites in lichens which are known to have many biological properties. Here, the cytotoxic and apoptotic effects of vulpinic acid on a human hepatocellular carcinoma cell line HepG2 and an embryonic rat fibroblast cell line F2408 have been investigated. HepG2 and F2408 cells were routinely cultured in DMEM supplemented with 10% foetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, in monolayer culture, and incubated at 37 ◦ C in a humidified atmosphere of 95% air and 5% CO2 . The cells were harvested by means of trypsinization (1% trypsin/EDTA). Cytotoxicity of vulpinic acid was determined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction and neutral red uptake methods. Apoptotic morphology of cells was observed after acridine orange/ethidium bromide (AO/EB) double staining under a fluoresan microscope. As a result, vulpinic acid has significantly cytotoxic effect on both cell lines in a time and dose dependent manner. The value of IC50 was around 450–500 M for F2408 and 300–400 M for HepG2, after 48 h. The treatment of cells with 200 M vulpinic acid significantly increased apoptotic cell ratio after 48 h. These results show that vulpinic acid inhibits the normal and the hepatocellular carcinoma cell growth and activates apoptosis. This research was supported financially by Scientific Research Project Commission of Anadolu University via contract 1205F080. http://dx.doi.org/10.1016/j.jbiotec.2015.06.299
Abstracts / Journal of Biotechnology 208 (2015) S5–S120
at about 1.5 cm apart. A quantity of 0.2 ml solution was inoculated in each hole. Fifteen days after inoculation, micro-tomography computed images of longitudinal sections of the infected tibia revealed a change in the morphology of the cancellous bone just between the two infected holes. A connecting path made of modified cancellous bone was observed between the holes. This change of bone morphology is due to the uni-directional expansion of the two bacterial population sites. This result suggests that bacteria are capable to communicate not only within a packed population (the so called quorum sensing), but also at a distance and in a relatively dense environment such as the bone. This communication is needed for their growth, virulence and formation of biofilms. Acknowledgements: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS–UEFISCDI, project number PN-II-ID-PCE-2011-3-0953. http://dx.doi.org/10.1016/j.jbiotec.2015.06.293 Study upon the haematological parameters in minipig models Ioan Hut¸u 1 , Irina Patras 1,∗ , Esra Elena Foditsh 2 , Rainhold Zimmermann 2 , Calin Mircu 1 1 Faculty of Veterinary Medicine, Banat University of Agricultural Sciences and Veterinary Medicine “King Mihai I of Romania” Timisoara, Romania 2 Spinal Cord Injury and Tissue Regeneration Centre, Paracelsus Medical University, Austria
E-mail address: iri [email protected] (I. Patras). Minipig models are used frequently in many fields of biomedical research due to the (i) social acceptance, (ii) existence of a large number of similarities between the swine’s and human’s physiology and (iii) easy training and handling. To observe and compare the haematology dynamics during the follow-up period between four groups of minipigs, considering the type of intervention applied. The experiment was conducted on 21 Gottingen Minipig, adult females which underwent different surgical procedures, as follows: thoracic vertebrae laminectomy, laparoscopic intervention or both – representing the spinal cord injury model (SCI) and control group (healthy individuals). 64 blood samples were processed using an automatic Haematology analyser IDEXX model Procyte DxTM with veterinary software for 25 parameters. The results obtained was classified as low, normal or high. Comparing the obtained values with other parameters and results of clinical examination we conclude that the classification of the equipment not necessary meets the normal status of Göttingen minipigs, as reflected in the software interpretation. Therefore, we used the reference values provided by the supplier of biological material – Elegaard Göttingen Minipigs® . Kruskal Wallis statistical analysis performed on blood counts shows that surgical interventions and neuromodulation had an impact on mean corpuscular volume (p < 0.033), white blood cell count (neutrophils p < 0.004 and basophils) and percentage of the distribution in differential leucocyte count (p < 0.008) to lymphocytes (p < 0.002), monocytes (p < 0.020) and basophils (p < 0.008). http://dx.doi.org/10.1016/j.jbiotec.2015.06.294
Evaluating the clinical and anatomopatological investigative techniques of liver neoplastic processes in dogs Cristina Alice Vulpe 1,∗ , Vasile Vulpe 2 , Leonardo Meomartino 3 , Sorin Aurelian Pasca 2 , Ionel Bondoc 2 , Ionel Papuc 1 1 Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania 2 Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Iasi, Romania 3 Faculty of Veterinary Medicine, University Federico II, Naples, Italy
E-mail address: [email protected] (C.A. Vulpe). Primary hepatic tumours in dogs are rare. On the other hand, metastases are frequent and their main center of proliferation is the liver. It’s common knowledge that patients (dogs) are brought in for a clinical investigation in advanced stages of neoplastic conditions. This occurs mostly because of the extraordinary capacity of the liver to make up for the lost functions. The clinical examination is the first step in diagnosing a dog suffering from a liver tumour. Clinical signs vary according to the size of the neoplasm and the age of the patient. The imaging examination guides the clinician towards a diagnosis; the ultrasound is a defining step, even more so if it used with a puncture. A radiologic examination attests the occurrence of lung metastases. The etiologic diagnosis is set in virtue of an anatomopathological examination. The main objective of the paper consists of evaluating the relevancy of the utilised methods in establishing the diagnosis of liver tumours in dogs. These studies have led to the following etiologic RESULTS: 8 carcinomas; 1 cholangiocarcinoma; 5 adenocarcinomas; 2 lymphosarcomas; 1 multiple myeloma. http://dx.doi.org/10.1016/j.jbiotec.2015.06.295 Genetics of multiple sclerosis: Association analysis of GWAS-identified and microRNA-linked common genetic variations Vitalina V. Bashinskaya ∗ , Olga G. Kulakova, Ivan S. Kiselev, Natalia M. Baulina, Alexey N. Boyko, Ekaterina Yu. Tsareva, Olga O. Favorova Pirogov Russian National Research Medical University, Moscow, Russia E-mail address: [email protected] (V.V. Bashinskaya). Multiple sclerosis (MS) is a chronic autoimmune polygenic disease of the central nervous system. The results of genome-wide association studies (GWASs) only partially explain MS etiology. Among other factors underlying MS susceptibility, the role of microRNAs is suggested pursuant to GWAS and expression studies. Here we aimed to analyze in Russians possible associations with MS of several GWAS-identified loci, the majority of them being proven miRNA targets, and with SNPs located in miRNA genes. The selected loci are CD58, IL7RA, IL2RA, CD6, TNFRSF1A, CLEC16A, IRF8, STAT3, MIR499A, and MIR146A. 509 MS patients and 276 healthy volunteers from Moscow were enrolled in the study. The carriage of individual alleles/genotypes and their combinations was compared in unstratified case/control samples and after gender stratification of MS patients and controls using APSampler software, which is based on Bayesian MCMC method and provides validation by means of Fisher’s exact test.
Abstracts / Journal of Biotechnology 208 (2015) S5–S120
We validated associations of CLEC16A and IL2RA with MS. Besides, CLEC16A and IRF8 were associated with MS in women, and IL7RA and CD58 – in men. With multi-locus association analysis the protective biallelic combinations (TNFRSF1A*T + CLEC16A*A), (TNFRSF1A*T + IRF8*A), and (IRF8*A + MIR499*A) were identified in women. Our data show involvement of immune response genes and genes, linked to miRNA system, in MS pathogenesis. This study was supported with Russian Scientific Foundation (grant 14-14-00605). http://dx.doi.org/10.1016/j.jbiotec.2015.06.296 Examination of pleural effusion in cats – 7 cases Alexandru Gabriel Neagu ∗ , Niculae Tudor, Poliana Tudor, Constantin Vlagioiu Faculty of Veterinary Medicine, University of Agronomical Sciences and Veterinary Medicine, Bucharest, Romania E-mail address: [email protected] (A.G. Neagu). Pleural effusions represent one cause of dyspnea in companion animals and can have multiple causes. Clinical signs associated with pleural effusions are varied, depending on the amount of the effusion and how quickly the fluid accumulates. Pleural effusions may be represented either by transsudat, modified transsudat or exudates, but the radiographic examination does not determine the type of the effusion, therefore further investigations are needed. In the study were enrolled seven cats aged between 3 years and 14 years who presented respiratory disorders on physical examination. Radiographic examination performed in the lateral view incidence, ventro/dorsal and dorso/ventral revealed the presence of pleural effusion. Thoracocentesis was performed unilateral or bilateral then the pleural fluid was collected and examined macroscopically and then microscopically. Cytological examinations established that pleural effusions were represented by: hemorrhagic effusion (2); neoplastic effusion (2); septic exudates (3). Pleural effusions are a secondary phenomenon, which requires further investigation to establish the underlying cause. The cytological examination provides important clues for the diagnosis. http://dx.doi.org/10.1016/j.jbiotec.2015.06.297 Seroprevalence of Anaplasma phagocytophilum in dogs from Constant¸a county Daniela Enache 1,∗ , Mirela Imre 2 , Marius Stelian Ilie 2 , Dragomir Coprean 3 1 Ovidius University, Department of Biology, Faculty of Natural and Agricultural Sciences, 900470 Constant¸a, Romania 2 Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I of Romania” from Timis¸oara, Department of Parasitology and Parasitic Diseases, 300645 Timis¸oara, Romania 3 Academy of Romanian Scientists, Ovidius University, Department of Biology, Faculty of Natural and Agricultural Sciences, 900470 Constant¸a, Romania
E-mail address: [email protected] (D. Enache). Canine anaplsmosis is a tick-borne disease of dogs who progresses with serious clinical manifestations which are dependent of the pathogens location (granulocytes, platelets). The pathogens who
S95
cause the diseases are from Anaplasma genera (Anaplasma phagocytohilum, Anaplasma platys). Anaplasma phagocytophilum has an important significance for both animals and humans health. The goal of our study was to etablish the status of Anaplasma phagocytophilum infection in dogs from South–Eastern part of Romania. Serum samples from 257 dogs living in 35 localities from Constant¸a county, were assayed through Diff-Quik stained blood smears and IFAT. Our results showed that the seroprevalence of antibodies of Anaplasma phagocytophilum was 6.22%. Overall, the seroprevalence of Anaplasma phagocytophilum infection in dogs from our study is enough to worry us and to be considered by all veterinarians from Constant¸a county when they will evaluate a dog. In the present study no significant differences were detected (p > 0.05) between the prevalence and age, gender and breed of the dogs. http://dx.doi.org/10.1016/j.jbiotec.2015.06.298 The in vitro investigation of cytotoxic and apoptotic effects vulpinic acid on normal and cancer cells O˘guzhan Karaosmano˘glu 1 , Hülya Sivas 1,∗ , Turgay Tay 2 , Ays¸en Özdemir Türk 1 1
Anadolu University, Faculty of Sciences, Department of Biology, Eskisehir, Turkey 2 Anadolu University, Faculty of Sciences, Department of Chemistry, Eskisehir, Turkey E-mail address: [email protected] (H. Sivas). Vulpinic acid is one of the important seconder metabolites in lichens which are known to have many biological properties. Here, the cytotoxic and apoptotic effects of vulpinic acid on a human hepatocellular carcinoma cell line HepG2 and an embryonic rat fibroblast cell line F2408 have been investigated. HepG2 and F2408 cells were routinely cultured in DMEM supplemented with 10% foetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, in monolayer culture, and incubated at 37 ◦ C in a humidified atmosphere of 95% air and 5% CO2 . The cells were harvested by means of trypsinization (1% trypsin/EDTA). Cytotoxicity of vulpinic acid was determined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction and neutral red uptake methods. Apoptotic morphology of cells was observed after acridine orange/ethidium bromide (AO/EB) double staining under a fluoresan microscope. As a result, vulpinic acid has significantly cytotoxic effect on both cell lines in a time and dose dependent manner. The value of IC50 was around 450–500 M for F2408 and 300–400 M for HepG2, after 48 h. The treatment of cells with 200 M vulpinic acid significantly increased apoptotic cell ratio after 48 h. These results show that vulpinic acid inhibits the normal and the hepatocellular carcinoma cell growth and activates apoptosis. This research was supported financially by Scientific Research Project Commission of Anadolu University via contract 1205F080. http://dx.doi.org/10.1016/j.jbiotec.2015.06.299