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A novel assay for identification of LGB genotypes in cattle
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Abstracts / Journal of Biotechnology 231S (2016) S4–S109
in the development of radioresistance during radiotherapy. In glioblastoma cell lines we found that silver nanoparticles-inducing radiosensitisation is associated with the DIDS-sensitive Clsw current activation involved in G2/M cell cycle phase accumulation upon ionizing radiation treatment. In contrast, silver nanoparticles do not affect the KCa3.1 current. Interesting, the radiosensitizer LY294002 reduces KCa3.1 current without promoting Clsw current activation. Altogether these data suggest the presence of distinct ionic mechanisms involved in radiosensitisation in glioblastoma cells. Combined treatment of silver nanoparticles and LY294002 could thus represent a new strategy against the development of radioresistance during glioblastoma radiotherapies. Further studies are needed to confirm these perspectives. http://dx.doi.org/10.1016/j.jbiotec.2016.05.350 A novel assay for identification of LGB genotypes in cattle Daniela Elena Ilie Research and Development Station for Bovine, Arad, Romania E-mail address: [email protected]. The development of novel assay for rapid detection of milk protein polymorphisms in cattle is important in the process of breeding and improving milk protein quality. We describe here the application of high-resolution melting analysis (HRM) to determine the main polymorphism of LGB (-lactoglobulin) gene in cattle. For rennet coagulation and the cheesemaking quality of milk, the LGB*B variant have been shown to be more favorable and the identification of this genetic variant is thus important. In the present study was developed a novel high-resolution melting assay to detect the main polymorphism in exon 4 of the LGB gene in cattle. We tested 3 different genotypes (AA, AB and BB) that had been previously analyzed by PCR-RFLP method and then examined the sensitivity of genetic variants detection using 185 bp amplicons and determination of single nucleotide polymorphisms (rs109625649) among Romanian Brown cattle by HRM curve analysis. Known LGB genetic variants in Romanian Brown cattle were readily detectable using HRM assay. The 185 bp amplicon was sensitive in detecting of all three genotypes. Considering its sensitivity and simplicity, the assay is a good fit for genetic selection as a rapid, inexpensive method to detect the LGB genetic variants in cattle. Acknowledgement: This work was published during the project: ADER 5.2.4./23.10.2015. http://dx.doi.org/10.1016/j.jbiotec.2016.05.351 Construction of Saccharomyces cerevisiae hybrid diploids for bioethanol production Bojan Zunar, Andrea Pranklin, Ana Loncar, Davor Nestic, Marina Svetec Miklenic, Anamarija Stafa, Bozidar Santek, Ivan Kresimir Svetec ∗ Faculty of Food Technology and Biotechnology, Department of Biochemical Engineering, Pierottijeva 6, 10000 Zagreb, Croatia E-mail address: [email protected] (I.K. Svetec). Bioethanol production from raw lignocelullosic material requires a strain of a microorganism which tolerates high ethanol concentrations and presence of growth inhibitors. Therefore, we constructed hybrid diploid Saccharomyces cerevisiae strains by mating two
haploids having desirable traits. One haploid was isolated from wine thus tolerating high ethanol concentration and the other has been shown to be resistant to several growth inhibitors. These haploids were mated and resulting homozygous and heterozygous (hybrid) diploids were tested for the ability to produce ethanol and to grow in the presence of several growth inhibitors frequently found in different lignocelullosic hydrolysates (acetic and levulinic acid, 2-furaldehyde). The results showed that some hybrid diploids were more resistant to tested inhibitors than parental haploid strains and homozygous diploids. It is also interesting that constructed strains showed different fermentation ability in CO2 production test, suggesting genetic variability of constructed hybrids. All together, these results suggest that the approach used in this study may help to construct new strains as well as to improve traits of biotechnologically interesting strains. http://dx.doi.org/10.1016/j.jbiotec.2016.05.352 Reproductive biotechnologies for the conservation of Romanian Grey Steppe genetic heritage Ada Cean 1,∗ , Daniela Ilie 1 , Oana Isabela Gavriliuc 2 , Virgil Paunescu 2 1
Research and Development Station for Bovine – Arad, Bodrogului 32, 310059 Arad, Romania 2 Victor Babes University of Medicine and Pharmacy, Timisoara, Romania E-mail address: [email protected] (A. Cean). Romanian Grey Steppe is an autochthonous cattle breed on the edge of extinction due to its low milk and meat productivity. Beyond the patrimonial and cultural importance, this breed has unique hereditary character and important genetic traits that should be preserved and used for improving productive longevity and fitness of highly-specialized breeds. As a result of a poor management of the in situ preservation of the breed, at this moment there are fewer than 100 individuals included in the national gene reserve, while the number is continuously decreasing. Our current work aims to test the efficiency of reproductive biotechnologies in preserving the Grey Steppe and to create a bank of cells and genetic material. The inventory we carried out in Western part of Romania, in 2013, counted 295 heads, from which only 7 were of Romanian variety, while the rest were of Hungarian descent. We recovered oocytes after in vivo maturation, which were subsequently vitrified using Open Pulled Straw technique and vitrification medium M199 with Hank’s, 20% FCS, 16% Ethylene glycol, 16% DMSO, 0.5 mol L−1 sucrose, then stored in liquid nitrogen as part of the genetic bank. We also performed in vitro embryo production with cryopreservation of the embryos by OPS technique. In view of our experience, we can conclude that Assisted Reproductive Technologies combined with biotechnologies can serve as an alternative method for preserving the Romanian Grey Steppe genetic heritage. Acknowledgment: The study was supported by a UEFISCDI, PN II PCCA under grant agreement number 120/ 2012 (CONSENS acronym). http://dx.doi.org/10.1016/j.jbiotec.2016.05.353
Abstracts / Journal of Biotechnology 231S (2016) S4–S109
in the development of radioresistance during radiotherapy. In glioblastoma cell lines we found that silver nanoparticles-inducing radiosensitisation is associated with the DIDS-sensitive Clsw current activation involved in G2/M cell cycle phase accumulation upon ionizing radiation treatment. In contrast, silver nanoparticles do not affect the KCa3.1 current. Interesting, the radiosensitizer LY294002 reduces KCa3.1 current without promoting Clsw current activation. Altogether these data suggest the presence of distinct ionic mechanisms involved in radiosensitisation in glioblastoma cells. Combined treatment of silver nanoparticles and LY294002 could thus represent a new strategy against the development of radioresistance during glioblastoma radiotherapies. Further studies are needed to confirm these perspectives. http://dx.doi.org/10.1016/j.jbiotec.2016.05.350 A novel assay for identification of LGB genotypes in cattle Daniela Elena Ilie Research and Development Station for Bovine, Arad, Romania E-mail address: [email protected]. The development of novel assay for rapid detection of milk protein polymorphisms in cattle is important in the process of breeding and improving milk protein quality. We describe here the application of high-resolution melting analysis (HRM) to determine the main polymorphism of LGB (-lactoglobulin) gene in cattle. For rennet coagulation and the cheesemaking quality of milk, the LGB*B variant have been shown to be more favorable and the identification of this genetic variant is thus important. In the present study was developed a novel high-resolution melting assay to detect the main polymorphism in exon 4 of the LGB gene in cattle. We tested 3 different genotypes (AA, AB and BB) that had been previously analyzed by PCR-RFLP method and then examined the sensitivity of genetic variants detection using 185 bp amplicons and determination of single nucleotide polymorphisms (rs109625649) among Romanian Brown cattle by HRM curve analysis. Known LGB genetic variants in Romanian Brown cattle were readily detectable using HRM assay. The 185 bp amplicon was sensitive in detecting of all three genotypes. Considering its sensitivity and simplicity, the assay is a good fit for genetic selection as a rapid, inexpensive method to detect the LGB genetic variants in cattle. Acknowledgement: This work was published during the project: ADER 5.2.4./23.10.2015. http://dx.doi.org/10.1016/j.jbiotec.2016.05.351 Construction of Saccharomyces cerevisiae hybrid diploids for bioethanol production Bojan Zunar, Andrea Pranklin, Ana Loncar, Davor Nestic, Marina Svetec Miklenic, Anamarija Stafa, Bozidar Santek, Ivan Kresimir Svetec ∗ Faculty of Food Technology and Biotechnology, Department of Biochemical Engineering, Pierottijeva 6, 10000 Zagreb, Croatia E-mail address: [email protected] (I.K. Svetec). Bioethanol production from raw lignocelullosic material requires a strain of a microorganism which tolerates high ethanol concentrations and presence of growth inhibitors. Therefore, we constructed hybrid diploid Saccharomyces cerevisiae strains by mating two
haploids having desirable traits. One haploid was isolated from wine thus tolerating high ethanol concentration and the other has been shown to be resistant to several growth inhibitors. These haploids were mated and resulting homozygous and heterozygous (hybrid) diploids were tested for the ability to produce ethanol and to grow in the presence of several growth inhibitors frequently found in different lignocelullosic hydrolysates (acetic and levulinic acid, 2-furaldehyde). The results showed that some hybrid diploids were more resistant to tested inhibitors than parental haploid strains and homozygous diploids. It is also interesting that constructed strains showed different fermentation ability in CO2 production test, suggesting genetic variability of constructed hybrids. All together, these results suggest that the approach used in this study may help to construct new strains as well as to improve traits of biotechnologically interesting strains. http://dx.doi.org/10.1016/j.jbiotec.2016.05.352 Reproductive biotechnologies for the conservation of Romanian Grey Steppe genetic heritage Ada Cean 1,∗ , Daniela Ilie 1 , Oana Isabela Gavriliuc 2 , Virgil Paunescu 2 1
Research and Development Station for Bovine – Arad, Bodrogului 32, 310059 Arad, Romania 2 Victor Babes University of Medicine and Pharmacy, Timisoara, Romania E-mail address: [email protected] (A. Cean). Romanian Grey Steppe is an autochthonous cattle breed on the edge of extinction due to its low milk and meat productivity. Beyond the patrimonial and cultural importance, this breed has unique hereditary character and important genetic traits that should be preserved and used for improving productive longevity and fitness of highly-specialized breeds. As a result of a poor management of the in situ preservation of the breed, at this moment there are fewer than 100 individuals included in the national gene reserve, while the number is continuously decreasing. Our current work aims to test the efficiency of reproductive biotechnologies in preserving the Grey Steppe and to create a bank of cells and genetic material. The inventory we carried out in Western part of Romania, in 2013, counted 295 heads, from which only 7 were of Romanian variety, while the rest were of Hungarian descent. We recovered oocytes after in vivo maturation, which were subsequently vitrified using Open Pulled Straw technique and vitrification medium M199 with Hank’s, 20% FCS, 16% Ethylene glycol, 16% DMSO, 0.5 mol L−1 sucrose, then stored in liquid nitrogen as part of the genetic bank. We also performed in vitro embryo production with cryopreservation of the embryos by OPS technique. In view of our experience, we can conclude that Assisted Reproductive Technologies combined with biotechnologies can serve as an alternative method for preserving the Romanian Grey Steppe genetic heritage. Acknowledgment: The study was supported by a UEFISCDI, PN II PCCA under grant agreement number 120/ 2012 (CONSENS acronym). http://dx.doi.org/10.1016/j.jbiotec.2016.05.353