- Email: [email protected]
Biological activities of beta-glucans isolated from spent brewer's yeast
S14
Abstracts / Journal of Biotechnology 256S (2017) S5–S16
market of biotechnology including the incentives of the capital providers, who demand a required return of investment. The justification of the drug price can be based on the Discounted Cash Flow method. We propose an alternative policy approach for the evaluation of innovative drugs from a broader perspective by bridging concepts from health economics and business economic valuation. This approach may justify a drug price when the ICER exceeds the threshold.
viral strategies in cell targeting and nucleic acid delivery. The potential advantage of these biomolecular particles lies in complementary blend of viral efficiency and chemical particle tractability suitable for design of highly effective and safe gene therapy vehicles. This may be achieved by using standard molecular biology techniques as well as in vitro chemical modification in order to improve the bio-particles’ composition and mode of action.
http://dx.doi.org/10.1016/j.jbiotec.2017.06.048
Yeast whole genome growth analysis
New technologies in gynaecologic cytology and its effects on both Bethesda classification and screening policy
http://dx.doi.org/10.1016/j.jbiotec.2017.06.050
Zelimir Franjo Kurtanjek Department of Food Technology and Biotechnology, University of Zagreb, Croatia
Gamze Mocan
E-mail address: [email protected].
Department of Medical Pathology, Faculty of Medicine, Near East University, Nicosia, Cyprus
Cervical cancer is one of the most common cancers. The conventional Papanicolaou (Pap) smear has been mainstay of cervical cancer prevention for more than 75 years. Due to this success rate, it has been accepted as the most successful screening test. The success of Pap smear depends on the performance of the cytopathologist, control of the positive and negative reports and ancillary techniques. Image analysers (rescreening), archives, consultation and use of common terminology in reporting (Bethesda System 2014) also play an important role. The spectrum of cervical cytologic abnormalities ranges from equivocal changes to the pathognomonic nuclear and cytoplasmic effects of Human Papilloma Virus (HPV) infection to severe cytologic neoplastic changes. The new alternative technologies, as liquid-based cytology, molecular diagnostics and additional screening options provide further insight into the biology of HPV. Changes in histopathology terminology, approval and implementation of prophylactic HPV vaccines, updated guidelines for screening and clinical management, have led to increased accuracy in prevention, diagnosis and management.
Applied is “big data analytics” for inference of yeast whole genome expressions under chemostat limited nutrient growth conditions (glucose, ammonium, sulphate, phosphate, uracil, leucine). Expression profiles cover the range of specific growth rates from 0.005 to 0.35/h measured at steady states. Applied are linear and nonlinear models for inference of key genome expressions dependencies on the nutrient limitations and growth rate. The linear models are with sparse elastic algorithms of partial least squares, Fisher discrimination, and mixed multivariate distributions discrimination, all available in R environment. Variable importance (gene) are inferred by the nonlinear model of regularized decision tree forests. Gene expression cluster analysis reveals that most of gene expressions (about 80%) are linearly correlated with the specific growth rates independent of nutrient limitations. However, identified are specific gene pools with expressions dependent on limitations by ammonium, sulphate and phosphate. For example, from the proposed model, under glucose limitations the most important factor with positive effects are: MOB2 protein amino acid phosphorylation protein kinase activator activity, and RPL22B protein biosynthesis structural constituent of ribosome; while the most important suppression is for APM2 vesicle-mediated transport clathrin binding, RNA elongation from RNA polymerase, and CIT3 tricarboxylic acid cycle citrate (Si)-synthase activity.
http://dx.doi.org/10.1016/j.jbiotec.2017.06.049
http://dx.doi.org/10.1016/j.jbiotec.2017.06.051
Recent trends in application of virus-derived particles as nucleic acid delivery agents
Biological activities of beta-glucans isolated from spent brewer’s yeast
Igor Slivac ∗ , Kristina Radosevic, Visnja Gaurina Srcek
Vesna Zechner-Krpan ∗ , Lidija Sver, Vlatka Petravic Tominac
Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia
Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia
E-mail address: gm [email protected].
E-mail address: [email protected] (I. Slivac). Nucleic acid delivery comprises vector mediated transfer of genetic material into cells and favourable alternation of cellular function or structure. Considering the intolerance of cells toward exogenous genetic material, the vectors of interest have to act on different steps along the gene expression pathway in order to evoke the desired outcome. In the last 25 years of gene/cell therapy, viral vectors have dominated clinical trials due to their superior transduction efficiency acquired through evolution. Non-viral vectors involve a variety of (bio)chemical particles that are generally less efficient than viruses, but their application is comparatively less costly and with reduced adverse effects on patients and biosafety. A relatively novel category of non-viral transfection agents includes derivatives of viral peptides that owing to their origin can mimic
E-mail address: [email protected] (V. Zechner-Krpan). -d-Glucans (hereafter referred to as “-glucans”) are glucose polymers which belong to a group of physiologically active compounds called “biological response modifiers” (BRMs). Their immunostimulatory activity makes them suitable for application in human and veterinary medicine, in pharmaceutical and chemical industries as well as in production of food, feed and cosmetics. In the Western world, dietary supplements containing -glucans up to now have been mostly produced from baker’s yeast, Saccharomyces cerevisiae. Among various compounds that can be isolated from yeast, -glucans can be used in wound healing and treatment of various diseases, such as cancer, infectious diseases, hypercholesterolaemia and diabetes. Their antioxidative properties and synergistic
Abstracts / Journal of Biotechnology 256S (2017) S5–S16
effect with various drugs and treatments are also reported. In recent years, there has been an increasing interest in isolation and use of -glucan from brewer’s yeast, which is a byproduct of beer production. All samples of insoluble -glucans, isolated in our experiments from brewer’s yeast using three different procedures and dried using three different methods, showed biological activity. -Glucan preparations, obtained in modified drying conditions, showed noticeably higher ability of producing TNF-␣. Use of spent brewer’s yeast as a raw material for -glucan production is technologically and economically justified. http://dx.doi.org/10.1016/j.jbiotec.2017.06.052 Carbohydrate composition and site-occupancy determination in pituitary and recombinant preparations of human thyrotropin Paolo Bartolini 1,∗ , Maria Teresa Cp Ribela 1 , Renata Damiani 1 , Felipe D. Silva 1 , Eliana R. Lima 1 , João E. Oliveira 1 , Cibele N. Peroni 1 , Peter A. Torjesen 2 , Carlos R. Soares 1 1 Biotechnology Center, IPEN-CNEN/University of São Paulo, São Paulo, Brazil 2 Hormone Laboratory, Oslo University Hospital, Oslo, Norway
E-mail address: [email protected] (P. Bartolini). Human thyrotropin (hTSH) is a glycoprotein with three potential glycosylation sites: two in the ␣-subunit and one in the -subunit. Carbohydrate site-occupancy is frequently neglected in glycoprotein characterization, even if related to folding, trafficking, initiation of inflammation, host defence and congenital disorders of glycosylation (CDG). For the first-time N-glycoprofiling analysis was applied to site-occupancy determination of two native pituitary hTSH, in comparison with three CHO-derived preparations of hTSH, a widely used biopharmaceutical. A single methodology provided: (i) average N-glycan mass; (ii) mass fraction of each monosaccharide and of sulfate; (iii) percent carbohydrate. The results indicate that occupancy (65–87%) and carbohydrate mass (12–19%) can be 34–57% higher in recombinant hormones. The average glycan mass is 24% lower in pituitary hTSH and contains ∼3-fold fewer moles of galactose (P < 0.005) and sialic acid (P < 0.01). The number of moles of fucose per mole of hTSH was found 2.5-fold higher in the pituitary preparations. One of these native preparations, presenting the smallest glycan mass, lowest occupancy, GalNAc, sulfate, Gal and sialic acid contents, also presented the lowest in vivo bioactivity and circulatory half-life. This methodology, extremely important for comparing a recombinant biopharmaceutical to its native equivalent, can be applied to any physiologically or clinical relevant glycoprotein.
S15
Construction of novel Saccharomyces cerevisiae hybrid strains resistant to growth and fermentation inhibitors Anamarija Stafa 1,∗ , Andrea Pranklin 1 , Antonio Zandona 1 , Bojan Zunar 1 , Marina Svetec Miklenic 1 , Bozidar Santek 2 , Ivan Kresimir Svetec 1 1 Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia 2 Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia
E-mail address: [email protected] (A. Stafa). Bioethanol production from raw lignocellulosic material requires a producer strain which tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous (hybrid) diploid S. cerevisiae strains by mating two haploid isolates having those desirable traits. In comparison to parental haploids, hybrid diploids were more resistant to several inhibitors frequently found in different lignocellulosic hydrolysates, such as acetic and levulinic acid and 2-furaldehyde. Interestingly, hybrids showed different fermentation ability in a CO2 production test. Our results suggest that construction of intraspecies hybrids coupled with the use of different genetic engineering techniques is a promising approach for the improvement or development of new biotechnologically relevant strains of S. cerevisiae. Additionally, we also show that in two biotechnologically interesting natural isolates percentage of successful gene targeting (gene targeting fidelity) is much lower than in widely used laboratory strains. Furthermore, the most frequent off-targeted event was duplication of a targeted chromosome yielding aneuploids having two copies of targeted chromosome, one containing transformed and the other containing untransformed allele. These results warn us that it could be more difficult to make precise genetic modifications of natural S. cerevisiae strains than expected. http://dx.doi.org/10.1016/j.jbiotec.2017.06.054 Application of mathematical modelling in biorefinery processes design Tonci Rezic 1,∗ , Martina Andlar 1 , Roland Ludwig 2 , Bozidar Santek 1 1
http://dx.doi.org/10.1016/j.jbiotec.2017.06.053
Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia 2 Department of Food Sciences and Technology, University of Natural Resources and Life Sciences, Vienna, Austria E-mail address: [email protected] (T. Rezic). A biorefinery is a facility that integrates biomass conversion processes and equipment to produce bio-products from biomass. Biorefineries will have to employ the best possible processes (for biomass cultivation, harvesting, storage and transport, pretreatment, fermentation and bio-chemical conversion) to ensure efficient bio-based production. Nevertheless, numerous technical, strategic and commercial challenges have to be overcome to
Abstracts / Journal of Biotechnology 256S (2017) S5–S16
market of biotechnology including the incentives of the capital providers, who demand a required return of investment. The justification of the drug price can be based on the Discounted Cash Flow method. We propose an alternative policy approach for the evaluation of innovative drugs from a broader perspective by bridging concepts from health economics and business economic valuation. This approach may justify a drug price when the ICER exceeds the threshold.
viral strategies in cell targeting and nucleic acid delivery. The potential advantage of these biomolecular particles lies in complementary blend of viral efficiency and chemical particle tractability suitable for design of highly effective and safe gene therapy vehicles. This may be achieved by using standard molecular biology techniques as well as in vitro chemical modification in order to improve the bio-particles’ composition and mode of action.
http://dx.doi.org/10.1016/j.jbiotec.2017.06.048
Yeast whole genome growth analysis
New technologies in gynaecologic cytology and its effects on both Bethesda classification and screening policy
http://dx.doi.org/10.1016/j.jbiotec.2017.06.050
Zelimir Franjo Kurtanjek Department of Food Technology and Biotechnology, University of Zagreb, Croatia
Gamze Mocan
E-mail address: [email protected].
Department of Medical Pathology, Faculty of Medicine, Near East University, Nicosia, Cyprus
Cervical cancer is one of the most common cancers. The conventional Papanicolaou (Pap) smear has been mainstay of cervical cancer prevention for more than 75 years. Due to this success rate, it has been accepted as the most successful screening test. The success of Pap smear depends on the performance of the cytopathologist, control of the positive and negative reports and ancillary techniques. Image analysers (rescreening), archives, consultation and use of common terminology in reporting (Bethesda System 2014) also play an important role. The spectrum of cervical cytologic abnormalities ranges from equivocal changes to the pathognomonic nuclear and cytoplasmic effects of Human Papilloma Virus (HPV) infection to severe cytologic neoplastic changes. The new alternative technologies, as liquid-based cytology, molecular diagnostics and additional screening options provide further insight into the biology of HPV. Changes in histopathology terminology, approval and implementation of prophylactic HPV vaccines, updated guidelines for screening and clinical management, have led to increased accuracy in prevention, diagnosis and management.
Applied is “big data analytics” for inference of yeast whole genome expressions under chemostat limited nutrient growth conditions (glucose, ammonium, sulphate, phosphate, uracil, leucine). Expression profiles cover the range of specific growth rates from 0.005 to 0.35/h measured at steady states. Applied are linear and nonlinear models for inference of key genome expressions dependencies on the nutrient limitations and growth rate. The linear models are with sparse elastic algorithms of partial least squares, Fisher discrimination, and mixed multivariate distributions discrimination, all available in R environment. Variable importance (gene) are inferred by the nonlinear model of regularized decision tree forests. Gene expression cluster analysis reveals that most of gene expressions (about 80%) are linearly correlated with the specific growth rates independent of nutrient limitations. However, identified are specific gene pools with expressions dependent on limitations by ammonium, sulphate and phosphate. For example, from the proposed model, under glucose limitations the most important factor with positive effects are: MOB2 protein amino acid phosphorylation protein kinase activator activity, and RPL22B protein biosynthesis structural constituent of ribosome; while the most important suppression is for APM2 vesicle-mediated transport clathrin binding, RNA elongation from RNA polymerase, and CIT3 tricarboxylic acid cycle citrate (Si)-synthase activity.
http://dx.doi.org/10.1016/j.jbiotec.2017.06.049
http://dx.doi.org/10.1016/j.jbiotec.2017.06.051
Recent trends in application of virus-derived particles as nucleic acid delivery agents
Biological activities of beta-glucans isolated from spent brewer’s yeast
Igor Slivac ∗ , Kristina Radosevic, Visnja Gaurina Srcek
Vesna Zechner-Krpan ∗ , Lidija Sver, Vlatka Petravic Tominac
Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia
Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia
E-mail address: gm [email protected].
E-mail address: [email protected] (I. Slivac). Nucleic acid delivery comprises vector mediated transfer of genetic material into cells and favourable alternation of cellular function or structure. Considering the intolerance of cells toward exogenous genetic material, the vectors of interest have to act on different steps along the gene expression pathway in order to evoke the desired outcome. In the last 25 years of gene/cell therapy, viral vectors have dominated clinical trials due to their superior transduction efficiency acquired through evolution. Non-viral vectors involve a variety of (bio)chemical particles that are generally less efficient than viruses, but their application is comparatively less costly and with reduced adverse effects on patients and biosafety. A relatively novel category of non-viral transfection agents includes derivatives of viral peptides that owing to their origin can mimic
E-mail address: [email protected] (V. Zechner-Krpan). -d-Glucans (hereafter referred to as “-glucans”) are glucose polymers which belong to a group of physiologically active compounds called “biological response modifiers” (BRMs). Their immunostimulatory activity makes them suitable for application in human and veterinary medicine, in pharmaceutical and chemical industries as well as in production of food, feed and cosmetics. In the Western world, dietary supplements containing -glucans up to now have been mostly produced from baker’s yeast, Saccharomyces cerevisiae. Among various compounds that can be isolated from yeast, -glucans can be used in wound healing and treatment of various diseases, such as cancer, infectious diseases, hypercholesterolaemia and diabetes. Their antioxidative properties and synergistic
Abstracts / Journal of Biotechnology 256S (2017) S5–S16
effect with various drugs and treatments are also reported. In recent years, there has been an increasing interest in isolation and use of -glucan from brewer’s yeast, which is a byproduct of beer production. All samples of insoluble -glucans, isolated in our experiments from brewer’s yeast using three different procedures and dried using three different methods, showed biological activity. -Glucan preparations, obtained in modified drying conditions, showed noticeably higher ability of producing TNF-␣. Use of spent brewer’s yeast as a raw material for -glucan production is technologically and economically justified. http://dx.doi.org/10.1016/j.jbiotec.2017.06.052 Carbohydrate composition and site-occupancy determination in pituitary and recombinant preparations of human thyrotropin Paolo Bartolini 1,∗ , Maria Teresa Cp Ribela 1 , Renata Damiani 1 , Felipe D. Silva 1 , Eliana R. Lima 1 , João E. Oliveira 1 , Cibele N. Peroni 1 , Peter A. Torjesen 2 , Carlos R. Soares 1 1 Biotechnology Center, IPEN-CNEN/University of São Paulo, São Paulo, Brazil 2 Hormone Laboratory, Oslo University Hospital, Oslo, Norway
E-mail address: [email protected] (P. Bartolini). Human thyrotropin (hTSH) is a glycoprotein with three potential glycosylation sites: two in the ␣-subunit and one in the -subunit. Carbohydrate site-occupancy is frequently neglected in glycoprotein characterization, even if related to folding, trafficking, initiation of inflammation, host defence and congenital disorders of glycosylation (CDG). For the first-time N-glycoprofiling analysis was applied to site-occupancy determination of two native pituitary hTSH, in comparison with three CHO-derived preparations of hTSH, a widely used biopharmaceutical. A single methodology provided: (i) average N-glycan mass; (ii) mass fraction of each monosaccharide and of sulfate; (iii) percent carbohydrate. The results indicate that occupancy (65–87%) and carbohydrate mass (12–19%) can be 34–57% higher in recombinant hormones. The average glycan mass is 24% lower in pituitary hTSH and contains ∼3-fold fewer moles of galactose (P < 0.005) and sialic acid (P < 0.01). The number of moles of fucose per mole of hTSH was found 2.5-fold higher in the pituitary preparations. One of these native preparations, presenting the smallest glycan mass, lowest occupancy, GalNAc, sulfate, Gal and sialic acid contents, also presented the lowest in vivo bioactivity and circulatory half-life. This methodology, extremely important for comparing a recombinant biopharmaceutical to its native equivalent, can be applied to any physiologically or clinical relevant glycoprotein.
S15
Construction of novel Saccharomyces cerevisiae hybrid strains resistant to growth and fermentation inhibitors Anamarija Stafa 1,∗ , Andrea Pranklin 1 , Antonio Zandona 1 , Bojan Zunar 1 , Marina Svetec Miklenic 1 , Bozidar Santek 2 , Ivan Kresimir Svetec 1 1 Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia 2 Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia
E-mail address: [email protected] (A. Stafa). Bioethanol production from raw lignocellulosic material requires a producer strain which tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous (hybrid) diploid S. cerevisiae strains by mating two haploid isolates having those desirable traits. In comparison to parental haploids, hybrid diploids were more resistant to several inhibitors frequently found in different lignocellulosic hydrolysates, such as acetic and levulinic acid and 2-furaldehyde. Interestingly, hybrids showed different fermentation ability in a CO2 production test. Our results suggest that construction of intraspecies hybrids coupled with the use of different genetic engineering techniques is a promising approach for the improvement or development of new biotechnologically relevant strains of S. cerevisiae. Additionally, we also show that in two biotechnologically interesting natural isolates percentage of successful gene targeting (gene targeting fidelity) is much lower than in widely used laboratory strains. Furthermore, the most frequent off-targeted event was duplication of a targeted chromosome yielding aneuploids having two copies of targeted chromosome, one containing transformed and the other containing untransformed allele. These results warn us that it could be more difficult to make precise genetic modifications of natural S. cerevisiae strains than expected. http://dx.doi.org/10.1016/j.jbiotec.2017.06.054 Application of mathematical modelling in biorefinery processes design Tonci Rezic 1,∗ , Martina Andlar 1 , Roland Ludwig 2 , Bozidar Santek 1 1
http://dx.doi.org/10.1016/j.jbiotec.2017.06.053
Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia 2 Department of Food Sciences and Technology, University of Natural Resources and Life Sciences, Vienna, Austria E-mail address: [email protected] (T. Rezic). A biorefinery is a facility that integrates biomass conversion processes and equipment to produce bio-products from biomass. Biorefineries will have to employ the best possible processes (for biomass cultivation, harvesting, storage and transport, pretreatment, fermentation and bio-chemical conversion) to ensure efficient bio-based production. Nevertheless, numerous technical, strategic and commercial challenges have to be overcome to