- Email: [email protected]
Comparative study on nutritional management in CKD
Abstracts / Journal of Biotechnology 256S (2017) S44–S116
Performance characteristics and comparison of DxN Veris system versus Qiagen PCR kit for Hepatitis B virus quantification Murat Sayan 1,∗ , Murat Sayan 2,∗ , Ayse Arıkan 3 , Ayse Arıkan 4 , Tamer Sanlidag 2 , Tamer Sanlidag 5 1 Kocaeli University, Faculty of Medicine, Clinical Laboratory, PCR Unit, Kocaeli, Turkey 2 Near East University, Research Center of Experimental Health Sciences, Nicosia, Cyprus 3 Near East University, Faculty of Medicine, Department of Medical Microbiology, Nicosia, Cyprus 4 Kyrenia University, Faculty of Medicine, Department of Medical Microbiology, Kyrenia, Cyprus 5 Celal Bayar University, Faculty of Medicine, Department of Medical Microbiology, Manisa, Turkey
E-mail address: [email protected] (M. Sayan). The Beckman Coulter DxN VERIS System is a fully automated, random access, closed molecular diagnostic instrument for viral load quantification of hepatitis B virus (HBV). The purpose of this study was to compare the performance of the VERIS HBV Assay (Beckman Coulter DxN Veris HBV kit, U.S.A.) with our routine system (Artus HBV Qiagen kit, Germany) in clinical samples. A total of 67 HBV plasma samples from Kocaeli city, with the HBV DNA levels ranging from negative to >106 IU/ml, were analyzed by both using Qiagen kit and fully automated VERIS instrument that incorporates the extraction, purification, quantification, and results interpretation of infectious disease nucleic acid targets. For assay analysis, Bland Altman Data Plotting and Passing-Bablok regression analysis methods were used. According to the Bland Altman Plot (Veris combined Qiagen Log IU/ml), the specificity resulted 95% LoA (Cl 95% = −1.55–0.61), correlation 0.97 with standard deviation (SD) 0.55 and a mean SD = −0.47.HBV Passing-Bablok regression analysis indicates that Veris combined = −1.179 + 1.153 Qiagen Log IU/ml with correlation 0.97. The Veris DxN HBV system exhibited high sensitivity, specifity, linearity and reproducibility. In addition to that characteristics, being low cost and easy to use, make this system new solution for the detection of HBV infection. http://dx.doi.org/10.1016/j.jbiotec.2017.06.1103 Preparation of bacterial cellulose films with regenerative effect Marina Yurievna Yazykova Department BBB, Samara NR University, Samara, Russia E-mail address: [email protected]. A wound is defined as a disruption in the continuity of the epithelial lining of the skin or mucosa resulting from physical or thermal damage. With the advancement in technology, currently, different types of wound dressing materials are available for all types of wounds. But the selection of a material for a particular wound is important to achieve faster healing. In this work, an attempt has been made to create regenerative wound dressing based on bacterial cellulose and human cord blood platelet lysate. Umbilical cord blood was used to isolate platelets. The tubes containing platelet concentrate were frozen at −80 ◦ C and thawed at +37 ◦ C. Then the samples were centrifuged at 3000 rpm during 20 min. Obtained this way supernatant was used for evaluation of protein
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profile. Protein profile of platelet lysate was measured in 5 samples using Maxis Impakt mass-spectrometer. Sample preparation included separation of platelet lysate proteins by one-dimensional Laemmli electrophoresis (SDS-PAGE) and further protein trypsinolysis in gel. According to mass-spectrometry all samples contained vitronectin and thrombospondin known to participate in regenerative processes. Bacterial cellulose was exposed in platelet lysate and applied on post-surgical suture of experimental animal. Significant regenerative effect was achieved on 3rd day. http://dx.doi.org/10.1016/j.jbiotec.2017.06.1104 miRNA expression and polymorphism of miRNA genes in the myocardial infarction Natalia Baulina 1,∗ , Ivan Kiselev 2 , German Osmak 2 , Natalia Matveeva 2 , Vitalina Bashinskaya 1 , Olga Kulakova 2 , Boris Titov 1 , Nino Kukava 1 , Roman Shakhnovich 1 , Olga Favorova 1 1 Russian Cardiology Scientific and Production Center, Moscow, Russia 2 Pirogov Russian National Research Medical University, Moscow, Russia
E-mail address: [email protected] (N. Baulina). Myocardial infarction (MI) is the most severe type of coronary artery disease and one of the leading causes of death worldwide. In spite of progress in cardiovascular genomics, data for MI are still limited and contradictory. The differential miRNA expression as well as genome variability in miRNA genes may contribute to MI development. We performed high-throughput miRNA sequencing (Miseq, Illumina) in peripheral blood mononuclear cells and plasma of MI patients and controls. A total of 8 plasma miRNAs were differentially expressed (p < 0.05), of which miR-375 and miR26a downregulation remained significant after FDR correction (p < 0.05). Significantly down-regulated miRNAs were also identified using RT-qPCR in plasma of MI patients. 325 MI patients and 185 individuals free of cardiovascular diseases (all Russian ethnicity) were genotyped for single nucleotide polymorphisms in miRNA genes: MIR196A2 rs11614913, MIR499A rs3746444, MIR146A rs2910164, and MIR423 rs6505162. Association analysis of miRNA genes’ variants with MI was performed using the APSampler algorithm. Rs11614913*C carriage in MIR196A2 gene was significantly associated with MI. According to our data, miRNAs are active players in MI development, and their further investigation may contribute to the solution of the problem of missing heritability for MI. This study was supported with Russian Scientific Foundation (grant 16-14-10251). http://dx.doi.org/10.1016/j.jbiotec.2017.06.1105 Comparative study on nutritional management in CKD Violeta Elena Simion 1,∗ , Monica Parvu 1 , Elena Mitranescu 2 , Laurentiu Tudor 2 1
Faculty of Veterinary Medicine, Spiru Haret University, Bucharest, Romania 2 Faculty of Veterinary Medicine, U.A.S.V.M. Bucharest, Romania E-mail address: [email protected] (V.E. Simion).
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Abstracts / Journal of Biotechnology 256S (2017) S44–S116
Chronic kidney disease (CKD), one of the most common diseases in older cats, requires an adequate nutritional management to increase the survival rate in this disease that affects a high percentage of animals. Two groups, each composed of 6 cats, were selected from CKD cases at the veterinary clinic. The first group consumed homemade food that contained meat/organs, seeds, cereals and celery (Apium graveolens) juice and the second group consumed the commercial food recommended for this condition. The results obtained revealed that cats in the group fed with home prepared food a Creatinine initial values between 4.6–6.1 mg/dL and 2.2–3.2 mg/dL, while the cats in the second group had similar results from 4.3–6.5 mg/dL to 2.2–2.6 mg/dL at the end of the monitoring. For BUN, the results of the first group went from 115–211 mg/dL initial values to 56–70 mg/dL and for the second group, fed with commercial food, from 90–168 mg/dL to 63–79 mg/dL at the end of the monitoring. The slight difference obtained at BUN was due to the inclusion of celery juice in the diet that decreases BUN levels while the rest of the ingredients provide important sources of antioxidants and anti-inflammatory substances for CKD diagnosed cats. http://dx.doi.org/10.1016/j.jbiotec.2017.06.1106 Investigation on some selected vector-borne pathogens and evaluation of co-infections in clinically suspected dogs, in Southern Romania Roxana Georgiana Anghel ∗ , Valentina Alina Simion, Ioan Liviu Mitrea, Mariana Ionita University of Agronomical Sciences and Veterinary Medicine of Bucharest, Faculty of Veterinary Medicine, Romania E-mail address: [email protected] (R.G. Anghel). In the last decades, the importance of vector borne diseases (VBDs) increased, as arthropod-transmitted diseases are both common to human and animals. The present study brings an update on the current situation in Romania regarding the prevalence of canine vector borne diseases (CVBDs) to increase the awareness on the occurrence and importance of CVBDs. Blood samples were collected (March 2015–March 2017) from 436 dogs, showing clinical signs compatible for VBDs, from Bucharest area (Southern Romania) and were analyzed by an Elisa test and blood smears. The results revealed a seroprevalence for Dirofilaria immitis of 22.48%, followed by Anaplasma spp. (2.06%), Ehrlichia canis/ewingii (1.83%) and Borrelia burgdorferi (0.46%). Mixed infections, with D. immitis + Anaplasma spp. (3 dogs), D. immitis + E. canis (2 dogs), E. canis + B. burgdorferi (1 dog) and E. canis + Anaplasma spp. (1 dog) were detected. The presence of large piroplasms (Babesia spp.) was registered in 30.50% of dogs. Co-infections D. immitis + Babesia spp. were also registered (6 dogs). These findings show a high exposure of dogs to vector borne pathogens and emphasize potential high risks for the both animal and human health. http://dx.doi.org/10.1016/j.jbiotec.2017.06.1107
Insights on Trichinella-infection in domestic pigs and hunted game, on 2011–2015 period, in Romania Cristian Vasile Buzatu ∗ , Isabela Madalina Nicorescu, Ioan Liviu Mitrea, Mariana Ionita Department of Parasitology and Parasitic Diseases & Animal Biology, Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest, Romania E-mail address: [email protected] (C.V. Buzatu). Trichinellosis is a major food-borne parasitic-zoonosis acquired by eating raw or undercooked meat with infective Trichinellalarvae. Pork and hunted game are the main sources of outbreaks of human trichinellosis in Romania. This study aimed to provide new insights on Trichinella-infections in animals (pigs and game) correlated with the human trichinellosis’ incidence in Romania. For this, reported data for 2011–2015 period were analyzed, including: over 800,000 pigs raised under non-controlled housing conditions and 53,000 hunted game (wild boar and bears), which were tested for Trichinella using standard procedures. The results showed a mean percentage of Trichinella-positive pigs raised in households (0.1%) higher than the UE mean (<0.01%). The mean incidence in game (0.98%) was significant high, with the highest in bears (22.27%), whereas, in wild boars was lower (0.86%). For the same period, a positive significant correlation (Spearman rank correlation coefficient of 0.46, P-value < 0.01) between the level of infection in pigs and wild boar and the number of human cases was registered. However, an important decrease of Trichinella prevalence was observed in 2015, for both, animals (pigs-0.06% and game-0.62%) and humans (0.28/100,000). Nonetheless, the pressure of Trichinella infection is still present in Romania, with potential high risks for the human health. http://dx.doi.org/10.1016/j.jbiotec.2017.06.1108 Expression and purification of breast cancer marker HER2 Aigerim Turgimbayeva ∗ , Sailau Abeldenov, Nurgul Sarina, Erlan Ramanculov, Saule Eskendirova, Bekbolat Khassenov National Center for Biotechnology, Astana, Kazakhstan E-mail address: [email protected] (A. Turgimbayeva). Human epidermal growth factor receptor 2 (HER2) transmits signals in healthy cells to regulate cell proliferation and survival. As many as 20–30% breast cancer patients have elevated levels of HER2 expression. The aim of this work was to obtain monoclonal anti-HER2 antibodies for cancer diagnostics. The recombinant HER2 antigen was tagged at N-terminus with octa-histidine tag and overexpressed in mammalian cells. Protein yield was low: 100 g from 8 × 107 cells. The procedure for obtaining HER2 from eukaryotic cells was too labor-consuming for practical application, therefore alternative expression systems in yeast and bacteria were tested. We found E. coli expression system to be the most efficient: the extracellular domain of HER2 (Her2-ECD) was purified from soluble fraction of BL21 (DE3) strain by immobilized metal affinity chromatography to yield 5 mg of protein per liter of bacterial culture. BALB/c mice were successfully immunized by Her2-ECD to produce monoclonal antibodies by hybridoma technology. Obtained
Performance characteristics and comparison of DxN Veris system versus Qiagen PCR kit for Hepatitis B virus quantification Murat Sayan 1,∗ , Murat Sayan 2,∗ , Ayse Arıkan 3 , Ayse Arıkan 4 , Tamer Sanlidag 2 , Tamer Sanlidag 5 1 Kocaeli University, Faculty of Medicine, Clinical Laboratory, PCR Unit, Kocaeli, Turkey 2 Near East University, Research Center of Experimental Health Sciences, Nicosia, Cyprus 3 Near East University, Faculty of Medicine, Department of Medical Microbiology, Nicosia, Cyprus 4 Kyrenia University, Faculty of Medicine, Department of Medical Microbiology, Kyrenia, Cyprus 5 Celal Bayar University, Faculty of Medicine, Department of Medical Microbiology, Manisa, Turkey
E-mail address: [email protected] (M. Sayan). The Beckman Coulter DxN VERIS System is a fully automated, random access, closed molecular diagnostic instrument for viral load quantification of hepatitis B virus (HBV). The purpose of this study was to compare the performance of the VERIS HBV Assay (Beckman Coulter DxN Veris HBV kit, U.S.A.) with our routine system (Artus HBV Qiagen kit, Germany) in clinical samples. A total of 67 HBV plasma samples from Kocaeli city, with the HBV DNA levels ranging from negative to >106 IU/ml, were analyzed by both using Qiagen kit and fully automated VERIS instrument that incorporates the extraction, purification, quantification, and results interpretation of infectious disease nucleic acid targets. For assay analysis, Bland Altman Data Plotting and Passing-Bablok regression analysis methods were used. According to the Bland Altman Plot (Veris combined Qiagen Log IU/ml), the specificity resulted 95% LoA (Cl 95% = −1.55–0.61), correlation 0.97 with standard deviation (SD) 0.55 and a mean SD = −0.47.HBV Passing-Bablok regression analysis indicates that Veris combined = −1.179 + 1.153 Qiagen Log IU/ml with correlation 0.97. The Veris DxN HBV system exhibited high sensitivity, specifity, linearity and reproducibility. In addition to that characteristics, being low cost and easy to use, make this system new solution for the detection of HBV infection. http://dx.doi.org/10.1016/j.jbiotec.2017.06.1103 Preparation of bacterial cellulose films with regenerative effect Marina Yurievna Yazykova Department BBB, Samara NR University, Samara, Russia E-mail address: [email protected]. A wound is defined as a disruption in the continuity of the epithelial lining of the skin or mucosa resulting from physical or thermal damage. With the advancement in technology, currently, different types of wound dressing materials are available for all types of wounds. But the selection of a material for a particular wound is important to achieve faster healing. In this work, an attempt has been made to create regenerative wound dressing based on bacterial cellulose and human cord blood platelet lysate. Umbilical cord blood was used to isolate platelets. The tubes containing platelet concentrate were frozen at −80 ◦ C and thawed at +37 ◦ C. Then the samples were centrifuged at 3000 rpm during 20 min. Obtained this way supernatant was used for evaluation of protein
S89
profile. Protein profile of platelet lysate was measured in 5 samples using Maxis Impakt mass-spectrometer. Sample preparation included separation of platelet lysate proteins by one-dimensional Laemmli electrophoresis (SDS-PAGE) and further protein trypsinolysis in gel. According to mass-spectrometry all samples contained vitronectin and thrombospondin known to participate in regenerative processes. Bacterial cellulose was exposed in platelet lysate and applied on post-surgical suture of experimental animal. Significant regenerative effect was achieved on 3rd day. http://dx.doi.org/10.1016/j.jbiotec.2017.06.1104 miRNA expression and polymorphism of miRNA genes in the myocardial infarction Natalia Baulina 1,∗ , Ivan Kiselev 2 , German Osmak 2 , Natalia Matveeva 2 , Vitalina Bashinskaya 1 , Olga Kulakova 2 , Boris Titov 1 , Nino Kukava 1 , Roman Shakhnovich 1 , Olga Favorova 1 1 Russian Cardiology Scientific and Production Center, Moscow, Russia 2 Pirogov Russian National Research Medical University, Moscow, Russia
E-mail address: [email protected] (N. Baulina). Myocardial infarction (MI) is the most severe type of coronary artery disease and one of the leading causes of death worldwide. In spite of progress in cardiovascular genomics, data for MI are still limited and contradictory. The differential miRNA expression as well as genome variability in miRNA genes may contribute to MI development. We performed high-throughput miRNA sequencing (Miseq, Illumina) in peripheral blood mononuclear cells and plasma of MI patients and controls. A total of 8 plasma miRNAs were differentially expressed (p < 0.05), of which miR-375 and miR26a downregulation remained significant after FDR correction (p < 0.05). Significantly down-regulated miRNAs were also identified using RT-qPCR in plasma of MI patients. 325 MI patients and 185 individuals free of cardiovascular diseases (all Russian ethnicity) were genotyped for single nucleotide polymorphisms in miRNA genes: MIR196A2 rs11614913, MIR499A rs3746444, MIR146A rs2910164, and MIR423 rs6505162. Association analysis of miRNA genes’ variants with MI was performed using the APSampler algorithm. Rs11614913*C carriage in MIR196A2 gene was significantly associated with MI. According to our data, miRNAs are active players in MI development, and their further investigation may contribute to the solution of the problem of missing heritability for MI. This study was supported with Russian Scientific Foundation (grant 16-14-10251). http://dx.doi.org/10.1016/j.jbiotec.2017.06.1105 Comparative study on nutritional management in CKD Violeta Elena Simion 1,∗ , Monica Parvu 1 , Elena Mitranescu 2 , Laurentiu Tudor 2 1
Faculty of Veterinary Medicine, Spiru Haret University, Bucharest, Romania 2 Faculty of Veterinary Medicine, U.A.S.V.M. Bucharest, Romania E-mail address: [email protected] (V.E. Simion).
S90
Abstracts / Journal of Biotechnology 256S (2017) S44–S116
Chronic kidney disease (CKD), one of the most common diseases in older cats, requires an adequate nutritional management to increase the survival rate in this disease that affects a high percentage of animals. Two groups, each composed of 6 cats, were selected from CKD cases at the veterinary clinic. The first group consumed homemade food that contained meat/organs, seeds, cereals and celery (Apium graveolens) juice and the second group consumed the commercial food recommended for this condition. The results obtained revealed that cats in the group fed with home prepared food a Creatinine initial values between 4.6–6.1 mg/dL and 2.2–3.2 mg/dL, while the cats in the second group had similar results from 4.3–6.5 mg/dL to 2.2–2.6 mg/dL at the end of the monitoring. For BUN, the results of the first group went from 115–211 mg/dL initial values to 56–70 mg/dL and for the second group, fed with commercial food, from 90–168 mg/dL to 63–79 mg/dL at the end of the monitoring. The slight difference obtained at BUN was due to the inclusion of celery juice in the diet that decreases BUN levels while the rest of the ingredients provide important sources of antioxidants and anti-inflammatory substances for CKD diagnosed cats. http://dx.doi.org/10.1016/j.jbiotec.2017.06.1106 Investigation on some selected vector-borne pathogens and evaluation of co-infections in clinically suspected dogs, in Southern Romania Roxana Georgiana Anghel ∗ , Valentina Alina Simion, Ioan Liviu Mitrea, Mariana Ionita University of Agronomical Sciences and Veterinary Medicine of Bucharest, Faculty of Veterinary Medicine, Romania E-mail address: [email protected] (R.G. Anghel). In the last decades, the importance of vector borne diseases (VBDs) increased, as arthropod-transmitted diseases are both common to human and animals. The present study brings an update on the current situation in Romania regarding the prevalence of canine vector borne diseases (CVBDs) to increase the awareness on the occurrence and importance of CVBDs. Blood samples were collected (March 2015–March 2017) from 436 dogs, showing clinical signs compatible for VBDs, from Bucharest area (Southern Romania) and were analyzed by an Elisa test and blood smears. The results revealed a seroprevalence for Dirofilaria immitis of 22.48%, followed by Anaplasma spp. (2.06%), Ehrlichia canis/ewingii (1.83%) and Borrelia burgdorferi (0.46%). Mixed infections, with D. immitis + Anaplasma spp. (3 dogs), D. immitis + E. canis (2 dogs), E. canis + B. burgdorferi (1 dog) and E. canis + Anaplasma spp. (1 dog) were detected. The presence of large piroplasms (Babesia spp.) was registered in 30.50% of dogs. Co-infections D. immitis + Babesia spp. were also registered (6 dogs). These findings show a high exposure of dogs to vector borne pathogens and emphasize potential high risks for the both animal and human health. http://dx.doi.org/10.1016/j.jbiotec.2017.06.1107
Insights on Trichinella-infection in domestic pigs and hunted game, on 2011–2015 period, in Romania Cristian Vasile Buzatu ∗ , Isabela Madalina Nicorescu, Ioan Liviu Mitrea, Mariana Ionita Department of Parasitology and Parasitic Diseases & Animal Biology, Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest, Romania E-mail address: [email protected] (C.V. Buzatu). Trichinellosis is a major food-borne parasitic-zoonosis acquired by eating raw or undercooked meat with infective Trichinellalarvae. Pork and hunted game are the main sources of outbreaks of human trichinellosis in Romania. This study aimed to provide new insights on Trichinella-infections in animals (pigs and game) correlated with the human trichinellosis’ incidence in Romania. For this, reported data for 2011–2015 period were analyzed, including: over 800,000 pigs raised under non-controlled housing conditions and 53,000 hunted game (wild boar and bears), which were tested for Trichinella using standard procedures. The results showed a mean percentage of Trichinella-positive pigs raised in households (0.1%) higher than the UE mean (<0.01%). The mean incidence in game (0.98%) was significant high, with the highest in bears (22.27%), whereas, in wild boars was lower (0.86%). For the same period, a positive significant correlation (Spearman rank correlation coefficient of 0.46, P-value < 0.01) between the level of infection in pigs and wild boar and the number of human cases was registered. However, an important decrease of Trichinella prevalence was observed in 2015, for both, animals (pigs-0.06% and game-0.62%) and humans (0.28/100,000). Nonetheless, the pressure of Trichinella infection is still present in Romania, with potential high risks for the human health. http://dx.doi.org/10.1016/j.jbiotec.2017.06.1108 Expression and purification of breast cancer marker HER2 Aigerim Turgimbayeva ∗ , Sailau Abeldenov, Nurgul Sarina, Erlan Ramanculov, Saule Eskendirova, Bekbolat Khassenov National Center for Biotechnology, Astana, Kazakhstan E-mail address: [email protected] (A. Turgimbayeva). Human epidermal growth factor receptor 2 (HER2) transmits signals in healthy cells to regulate cell proliferation and survival. As many as 20–30% breast cancer patients have elevated levels of HER2 expression. The aim of this work was to obtain monoclonal anti-HER2 antibodies for cancer diagnostics. The recombinant HER2 antigen was tagged at N-terminus with octa-histidine tag and overexpressed in mammalian cells. Protein yield was low: 100 g from 8 × 107 cells. The procedure for obtaining HER2 from eukaryotic cells was too labor-consuming for practical application, therefore alternative expression systems in yeast and bacteria were tested. We found E. coli expression system to be the most efficient: the extracellular domain of HER2 (Her2-ECD) was purified from soluble fraction of BL21 (DE3) strain by immobilized metal affinity chromatography to yield 5 mg of protein per liter of bacterial culture. BALB/c mice were successfully immunized by Her2-ECD to produce monoclonal antibodies by hybridoma technology. Obtained