- Email: [email protected]
In vitro cytotoxicity of gold ions and gold nanoparticles on human bone marrow-derived mesenchymal stem cells
S100
Abstracts / Journal of Biotechnology 208 (2015) S5–S120
(CCHFv and TBEv) and bacterial (Francisella tularensis and Borrelia burgdorferi s.l.) agents transmitted by ticks”. Lyophilised strains were rehydrated in nutrient broth, cultured in Francisella tularensis specific medium (CHAB-PACCV) and passed on nutrient medium. Microbiological diagnosis was confirmed by immunoassay (Tularemia biothreat Alert kit, Tetracore) and molecular tests – Real Time PCR (TaqMan Francisella tularensis detection kit, Applied Biosystems) for the 2 analysed genes. The three identification methods have confirmed the presence of Francisella tularensis strains in lyophilised samples analysed. Acknowledgements: This work was funded by MEN-UEFISCDI PN II “Partnerships in priority areas” program, Grant No. 295/2014. http://dx.doi.org/10.1016/j.jbiotec.2015.06.312 Studies on the effect of antigonadotropics and inhibitors of neoplastic proliferations in polypeptide pineal hormone dependent tumors Raluca Elena Negreanu 1,∗ , Razvan Adrian Negreanu 2 , Iulia Cringanu 3 , Aneta Pop 4 , Dan Cringanu 1 1
Department of Oncology and Pet Pathology, Faculty of Veterinary Science, Bucharest, Romania 2 Department of Medical Oncology, Coltea Clinical Hospital, Bucharest, Romania 3 Department of Zootechnics, University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania 4 Department of Biochemistry and Molecular Biology, Faculty of Veterinary Science, Bucharest, Romania E-mail address: [email protected] (R.E. Negreanu). Protocols for treatment of cancer patients are in constant change. Our role as physicians and researchers is to discover new therapies that combine maximum effect with minimal toxicity. This led us to consider a gland with multiple functional implications in metabolic and breeding processes, the pineal gland. We performed selective extraction of polypeptide pineal hormones both from fresh urine of prepubescent pig males and from the pineal gland, and tested on a number of canine patients (dogs), males, age ranging from 7 to 11 years old, with prostate malignancies. Our studies have highlighted, through electronic transmission microscopy and scanning, the pineal peptide hormone synthesis and release in the cerebrospinal fluid and the blood in the capillaries of the epiphysis’ structure. Opo therapeutic extracts obtained by us using lectins from the seeds of Rhaphanus sativus (black radish) were used as parenteral therapy for a series of hormone-dependent neoplastic diseases, thus using the pharmaco-dynamic mechanisms of hormone replacement therapy with antigonadotropic effect. Highlighting the gonadotropic and also the anti tumor effects of polypeptides pineal hormone dependent prostate tumor therapy, represents a potentially valuable addition to the classical adjuvant chemotherapy treatment protocols that may be immunosuppressive and even toxic for the patient. http://dx.doi.org/10.1016/j.jbiotec.2015.06.313
Immunohistochemical method for identification of Lawsonia intracellularis infection in pigs Anca Sofiana Surpat (Hulea) 1,∗ , Viorel Herman 1 , Iosif Marincu 2 , Narcisa Mederle 3 , Ovidiu Alexandru Mederle 4 1 Department of Infectious Diseases, Banat’s University of Agricultural Sciences and Veterinary Medicine Timisoara, Romania 2 Department of Infectious Diseases, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania 3 Department of Parasitology, Banat’s University of Agricultural Sciences and Veterinary Medicine Timisoara, Romania 4 Department of Microscopic Morphology, Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania
E-mail address: [email protected] (A.S. Surpat (Hulea)). Intracellular infection by Lawsonia intracellularis, the etiological agent of proliferative enteritis, occurs all over the world, in different types of production systems that affect young pigs. The aim of this study was to identify the infection with Lawsonia intracellularis in young pigs by the immunohistochemistry. 123 samples of ileum of young pigs with specific intestinal adenomatosis lesions were studied. We used the working system NovoLink Max Polymer detection with antibody Lawsonia intracelularis (Novocastra, Newcastle UponTyne, and UK). We performed imunohistochemical method using DakoCytomation Autostainer. We used chromogen 3.3-diamino-benzidine and Lille haematoxylin for counterstain. Microscopical identification was performed by Nikon Eclipse E 600 microscope and the images were captured by the system LUCIA G. Immunohistochemical method demonstrated a high sensitivity and specificity. We identified by this method the etiological agent of proliferative enteritis in 114 (92.68%) intestinal samples studied. Immunohistochemistry is a method for accurate diagnosis of infection with Lawsonia intracelularis in porcine proliferative enteritis outbreaks. http://dx.doi.org/10.1016/j.jbiotec.2015.06.314 In vitro cytotoxicity of gold ions and gold nanoparticles on human bone marrow-derived mesenchymal stem cells Gamze Tan 1,∗ , Melek Yaman 3 , Nihan Örüklü 3 , Necdet Sa˘glam 2 , Emin Ümit Ba˘grıac¸ık 3 1 Department of Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey 2 Institute of Science, Nanotechnology and Nanomedicine Division, Hacettepe University, Ankara 06800 Turkey 3 Department of Immunology, Faculty of Medicine, Gazi University, Ankara, Turkey
E-mail address: [email protected] (G. Tan). The combination of human bone marrow-derived mesenchymal stem cells (MSCs) and gold nanoparticles (AuNPs) may have a promising potential to develop new therapeutic methodologies. The purpose of this study was to understand the cytotoxic effects of AuNPs and Au ions on MSCs. AuNPs were synthesized by reducing gold salts with citrate. AuNPs were characterized by TEM and DLS. Characterization of
Abstracts / Journal of Biotechnology 208 (2015) S5–S120
MSCs markers was determined by flow cytometry. Cellular cytotoxicity was assessed by MTT assay. Viability of MSCs at 24, 48 and 72 h was evaluated following treatments of AuNPs or Au ions at various concentrations ranging from 0.01 to 5 nM. ANOVA was used for statistical analysis. Viabilities of MSCs treated with AuNPs or Au ions were compared with untreated control groups. AuNPs at 5 nM caused relatively higher toxicity compared to AuNPs at 0.01 nM. We observed no significant (p < 0.01) difference between treated and non-treated groups for Au ions. Presence of human thyroid stimulating hormone in cell cultures did not affect cytotoxicity that was caused by AuNPs. In conclusion, next generation AuNPs should be prepared in non-toxic forms for use in biological applications. Supported by Grant No: TUBITAK-SBAG-112S451. http://dx.doi.org/10.1016/j.jbiotec.2015.06.315 Conditions of the bacterial cellulose mechanical treatment during the isolation of cellulose nanocrystals Elena Alexandrovna Kotina ∗ , Nikolay Alexandrovich Pestov, Victor Vasilievich Revin Ogarev Mordovia State University, Saransk, Russia E-mail address: [email protected] (E.A. Kotina). The cellulose is considered a perspective material for nanotechnologies, due to that crystal sites of cellulose possess very high mechanical strength. Isolation of cellulose nanocrystals from bacterial cellulose (BC) demands carrying out milling of the dried bacterial cellulose. Powdering of BC will reduce the degree of its crystallinity. In this regard the research on the effect of intensity of mechanical treatment on the degree of cellulose crystallinity was carried out. Dried BC was milled by the ball mill MTI SFM-1 at 750 rpm within 2–16 min. The size analysis of the received particles showed that milling within 2 min led to receiving particles having a size of 235–250 nm. The further increasing of the milling time led to receiving particles having a bigger size, which can occur due to the aggregation of separate particles of BC. Milling during 2 min decreased the crystallinity index of BC by 32%. Increasing of the milling time to 4 and 6 min led to additional stage-by-stage decreasing of the crystallinity index by 15 and 13% respectively. Milling during 8 min decreased the crystallinity index of BC by 65%. Milling during 16 min did not result in further decreasing of the crystallinity index. The work was supported by the Ministry of Education and Science of the Russian Federation, base section, and grant No. 2913. http://dx.doi.org/10.1016/j.jbiotec.2015.06.316
S101
Toxic effects of gold nanoparticles and gold ions in human umbilical cord derived mesenchymal stem cells Gamze Tan 1,∗ , Necdet Sa˘glam 2 , Melek Yaman 3 , Nihan Örüklü 3 , Emin Ümit Ba˘grıac¸ık 3 1 Department of Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey 2 Institute of Science, Nanotechnology and Nanomedicine Division, Hacettepe University, Ankara 06800, Turkey 3 Department of Immunology, Faculty of Medicine, Gazi University, Ankara, Turkey
E-mail address: [email protected] (G. Tan). We investigated whether gold nanoparticles (AuNPs) and gold ions have toxic effects in human umbilical cord-derived mesenchymal stem cells (UCMSCs) that were isolated in our laboratory as well as UCMSCs that were purchased from the American Type Culture Collection (ATCC). Surface markers of UCMSCs were determined by flow cytometry. Various methods of cellular cytotoxicity were studied. UCMSCs were treated with either AuNPs or gold ions for 24 h, 48 h, and 72 h. Cell viability was measured by MTT assay. Oxidative stress was assessed by Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Apoptosis was also tested. We found that under in vitro culture conditions, AuNPs at 1 nM and 5 nM decreased viability of human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) in all time points tested whereas gold ions at the same concentrations did not affect viability UCBMSCs at significant levels. Presence of human thyroid stimulating hormone (TSH) in cell cultures did not change the results regarding toxicity. We concluded that AuNPs should be evaluated carefully in biological applications. Supported by Grant No: TUBITAK-SBAG-112S451. http://dx.doi.org/10.1016/j.jbiotec.2015.06.317 Molecular fingerprinting of Romanian resident Bison bonasus in order to avoid consanguinisation Calin Mircu, Oana Maria Boldura ∗ , Gheorghe Bonca, Camelia Tulcan, Sorina Popescu, Ioan Hut¸u Banat University of Agricultural Sciences and Veterinary Medicine “King Mihai I of Romania”, Timisoara, Romania E-mail address: oana [email protected] (O.M. Boldura). Genomic DNA isolated from blood of 14 sampled individuals of Bison bonasus was used in order to obtain a molecular fingerprints and a dendrogram exhibiting the phylogenetic relationship among the individuals. Two types of molecular marker systems: Inter simple sequence repeats (ISSR) and directed amplification of minisatellite region DNA (DAMD) were used with primers for PCR method and development of the DNA fingerprint. The degree of polymorphism was either absent or very low especially in the case of ISSR marker system. Because of their superiority in reveling individual genetic polymorphism, DAMD molecular markers are recommended as first choice for similar studies. Based on molecular data a common binary matrix was developed. Using IBM SPSS Statistic version 22 a dissimilarity matrix and a dendrogram were generated. The obtained DNA fingerprint is a cost effective, accurate and fast method that can be further used to establish the optimal partners for mating in order to avoid sanguinisation and the parenting relation among members of the herd and offsprings. Our data
Abstracts / Journal of Biotechnology 208 (2015) S5–S120
(CCHFv and TBEv) and bacterial (Francisella tularensis and Borrelia burgdorferi s.l.) agents transmitted by ticks”. Lyophilised strains were rehydrated in nutrient broth, cultured in Francisella tularensis specific medium (CHAB-PACCV) and passed on nutrient medium. Microbiological diagnosis was confirmed by immunoassay (Tularemia biothreat Alert kit, Tetracore) and molecular tests – Real Time PCR (TaqMan Francisella tularensis detection kit, Applied Biosystems) for the 2 analysed genes. The three identification methods have confirmed the presence of Francisella tularensis strains in lyophilised samples analysed. Acknowledgements: This work was funded by MEN-UEFISCDI PN II “Partnerships in priority areas” program, Grant No. 295/2014. http://dx.doi.org/10.1016/j.jbiotec.2015.06.312 Studies on the effect of antigonadotropics and inhibitors of neoplastic proliferations in polypeptide pineal hormone dependent tumors Raluca Elena Negreanu 1,∗ , Razvan Adrian Negreanu 2 , Iulia Cringanu 3 , Aneta Pop 4 , Dan Cringanu 1 1
Department of Oncology and Pet Pathology, Faculty of Veterinary Science, Bucharest, Romania 2 Department of Medical Oncology, Coltea Clinical Hospital, Bucharest, Romania 3 Department of Zootechnics, University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania 4 Department of Biochemistry and Molecular Biology, Faculty of Veterinary Science, Bucharest, Romania E-mail address: [email protected] (R.E. Negreanu). Protocols for treatment of cancer patients are in constant change. Our role as physicians and researchers is to discover new therapies that combine maximum effect with minimal toxicity. This led us to consider a gland with multiple functional implications in metabolic and breeding processes, the pineal gland. We performed selective extraction of polypeptide pineal hormones both from fresh urine of prepubescent pig males and from the pineal gland, and tested on a number of canine patients (dogs), males, age ranging from 7 to 11 years old, with prostate malignancies. Our studies have highlighted, through electronic transmission microscopy and scanning, the pineal peptide hormone synthesis and release in the cerebrospinal fluid and the blood in the capillaries of the epiphysis’ structure. Opo therapeutic extracts obtained by us using lectins from the seeds of Rhaphanus sativus (black radish) were used as parenteral therapy for a series of hormone-dependent neoplastic diseases, thus using the pharmaco-dynamic mechanisms of hormone replacement therapy with antigonadotropic effect. Highlighting the gonadotropic and also the anti tumor effects of polypeptides pineal hormone dependent prostate tumor therapy, represents a potentially valuable addition to the classical adjuvant chemotherapy treatment protocols that may be immunosuppressive and even toxic for the patient. http://dx.doi.org/10.1016/j.jbiotec.2015.06.313
Immunohistochemical method for identification of Lawsonia intracellularis infection in pigs Anca Sofiana Surpat (Hulea) 1,∗ , Viorel Herman 1 , Iosif Marincu 2 , Narcisa Mederle 3 , Ovidiu Alexandru Mederle 4 1 Department of Infectious Diseases, Banat’s University of Agricultural Sciences and Veterinary Medicine Timisoara, Romania 2 Department of Infectious Diseases, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania 3 Department of Parasitology, Banat’s University of Agricultural Sciences and Veterinary Medicine Timisoara, Romania 4 Department of Microscopic Morphology, Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania
E-mail address: [email protected] (A.S. Surpat (Hulea)). Intracellular infection by Lawsonia intracellularis, the etiological agent of proliferative enteritis, occurs all over the world, in different types of production systems that affect young pigs. The aim of this study was to identify the infection with Lawsonia intracellularis in young pigs by the immunohistochemistry. 123 samples of ileum of young pigs with specific intestinal adenomatosis lesions were studied. We used the working system NovoLink Max Polymer detection with antibody Lawsonia intracelularis (Novocastra, Newcastle UponTyne, and UK). We performed imunohistochemical method using DakoCytomation Autostainer. We used chromogen 3.3-diamino-benzidine and Lille haematoxylin for counterstain. Microscopical identification was performed by Nikon Eclipse E 600 microscope and the images were captured by the system LUCIA G. Immunohistochemical method demonstrated a high sensitivity and specificity. We identified by this method the etiological agent of proliferative enteritis in 114 (92.68%) intestinal samples studied. Immunohistochemistry is a method for accurate diagnosis of infection with Lawsonia intracelularis in porcine proliferative enteritis outbreaks. http://dx.doi.org/10.1016/j.jbiotec.2015.06.314 In vitro cytotoxicity of gold ions and gold nanoparticles on human bone marrow-derived mesenchymal stem cells Gamze Tan 1,∗ , Melek Yaman 3 , Nihan Örüklü 3 , Necdet Sa˘glam 2 , Emin Ümit Ba˘grıac¸ık 3 1 Department of Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey 2 Institute of Science, Nanotechnology and Nanomedicine Division, Hacettepe University, Ankara 06800 Turkey 3 Department of Immunology, Faculty of Medicine, Gazi University, Ankara, Turkey
E-mail address: [email protected] (G. Tan). The combination of human bone marrow-derived mesenchymal stem cells (MSCs) and gold nanoparticles (AuNPs) may have a promising potential to develop new therapeutic methodologies. The purpose of this study was to understand the cytotoxic effects of AuNPs and Au ions on MSCs. AuNPs were synthesized by reducing gold salts with citrate. AuNPs were characterized by TEM and DLS. Characterization of
Abstracts / Journal of Biotechnology 208 (2015) S5–S120
MSCs markers was determined by flow cytometry. Cellular cytotoxicity was assessed by MTT assay. Viability of MSCs at 24, 48 and 72 h was evaluated following treatments of AuNPs or Au ions at various concentrations ranging from 0.01 to 5 nM. ANOVA was used for statistical analysis. Viabilities of MSCs treated with AuNPs or Au ions were compared with untreated control groups. AuNPs at 5 nM caused relatively higher toxicity compared to AuNPs at 0.01 nM. We observed no significant (p < 0.01) difference between treated and non-treated groups for Au ions. Presence of human thyroid stimulating hormone in cell cultures did not affect cytotoxicity that was caused by AuNPs. In conclusion, next generation AuNPs should be prepared in non-toxic forms for use in biological applications. Supported by Grant No: TUBITAK-SBAG-112S451. http://dx.doi.org/10.1016/j.jbiotec.2015.06.315 Conditions of the bacterial cellulose mechanical treatment during the isolation of cellulose nanocrystals Elena Alexandrovna Kotina ∗ , Nikolay Alexandrovich Pestov, Victor Vasilievich Revin Ogarev Mordovia State University, Saransk, Russia E-mail address: [email protected] (E.A. Kotina). The cellulose is considered a perspective material for nanotechnologies, due to that crystal sites of cellulose possess very high mechanical strength. Isolation of cellulose nanocrystals from bacterial cellulose (BC) demands carrying out milling of the dried bacterial cellulose. Powdering of BC will reduce the degree of its crystallinity. In this regard the research on the effect of intensity of mechanical treatment on the degree of cellulose crystallinity was carried out. Dried BC was milled by the ball mill MTI SFM-1 at 750 rpm within 2–16 min. The size analysis of the received particles showed that milling within 2 min led to receiving particles having a size of 235–250 nm. The further increasing of the milling time led to receiving particles having a bigger size, which can occur due to the aggregation of separate particles of BC. Milling during 2 min decreased the crystallinity index of BC by 32%. Increasing of the milling time to 4 and 6 min led to additional stage-by-stage decreasing of the crystallinity index by 15 and 13% respectively. Milling during 8 min decreased the crystallinity index of BC by 65%. Milling during 16 min did not result in further decreasing of the crystallinity index. The work was supported by the Ministry of Education and Science of the Russian Federation, base section, and grant No. 2913. http://dx.doi.org/10.1016/j.jbiotec.2015.06.316
S101
Toxic effects of gold nanoparticles and gold ions in human umbilical cord derived mesenchymal stem cells Gamze Tan 1,∗ , Necdet Sa˘glam 2 , Melek Yaman 3 , Nihan Örüklü 3 , Emin Ümit Ba˘grıac¸ık 3 1 Department of Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey 2 Institute of Science, Nanotechnology and Nanomedicine Division, Hacettepe University, Ankara 06800, Turkey 3 Department of Immunology, Faculty of Medicine, Gazi University, Ankara, Turkey
E-mail address: [email protected] (G. Tan). We investigated whether gold nanoparticles (AuNPs) and gold ions have toxic effects in human umbilical cord-derived mesenchymal stem cells (UCMSCs) that were isolated in our laboratory as well as UCMSCs that were purchased from the American Type Culture Collection (ATCC). Surface markers of UCMSCs were determined by flow cytometry. Various methods of cellular cytotoxicity were studied. UCMSCs were treated with either AuNPs or gold ions for 24 h, 48 h, and 72 h. Cell viability was measured by MTT assay. Oxidative stress was assessed by Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Apoptosis was also tested. We found that under in vitro culture conditions, AuNPs at 1 nM and 5 nM decreased viability of human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) in all time points tested whereas gold ions at the same concentrations did not affect viability UCBMSCs at significant levels. Presence of human thyroid stimulating hormone (TSH) in cell cultures did not change the results regarding toxicity. We concluded that AuNPs should be evaluated carefully in biological applications. Supported by Grant No: TUBITAK-SBAG-112S451. http://dx.doi.org/10.1016/j.jbiotec.2015.06.317 Molecular fingerprinting of Romanian resident Bison bonasus in order to avoid consanguinisation Calin Mircu, Oana Maria Boldura ∗ , Gheorghe Bonca, Camelia Tulcan, Sorina Popescu, Ioan Hut¸u Banat University of Agricultural Sciences and Veterinary Medicine “King Mihai I of Romania”, Timisoara, Romania E-mail address: oana [email protected] (O.M. Boldura). Genomic DNA isolated from blood of 14 sampled individuals of Bison bonasus was used in order to obtain a molecular fingerprints and a dendrogram exhibiting the phylogenetic relationship among the individuals. Two types of molecular marker systems: Inter simple sequence repeats (ISSR) and directed amplification of minisatellite region DNA (DAMD) were used with primers for PCR method and development of the DNA fingerprint. The degree of polymorphism was either absent or very low especially in the case of ISSR marker system. Because of their superiority in reveling individual genetic polymorphism, DAMD molecular markers are recommended as first choice for similar studies. Based on molecular data a common binary matrix was developed. Using IBM SPSS Statistic version 22 a dissimilarity matrix and a dendrogram were generated. The obtained DNA fingerprint is a cost effective, accurate and fast method that can be further used to establish the optimal partners for mating in order to avoid sanguinisation and the parenting relation among members of the herd and offsprings. Our data