Biography:

Brian Miller is an Associate Professor of Biochemistry at the Florida State University, USA. He did his Ph.D. from the University of North Carolina, Chapel Hill in the year 2001. His research interest is protein structure, function and evolution.

Abstract:

Human glucokinase (GCK), the body’s primary glucose sensor and a major determinant of glucose homeostatic diseases, displays a unique form of allosteric-like behavior that is manifested as a cooperative kinetic response to glucose. The allosteric-like behavior of GCK is particularly intriguing since the enzyme is monomeric and contains only one glucose binding site. Recent work in our laboratory has shown that millisecond timescale order-disorder transitions within the enzyme’s small domain govern cooperativity. Here, we present the results of biophysical studies that elucidate the structural and dynamic origins of the time-dependent, “allokairic” properties of GCK. Using high-resolution nuclear magnetic resonance we identify two distinct mechanisms by which GCK can be activated, both of which result in hyperinsulinemia. The first activation mechanism alters the equilibrium distribution of GCK conformers in favor of a single-state, whereas the second mechanism alters the intrinsic dynamics of the enzyme without perturbing the relative distribution of states in the structural ensemble. Time-resolved fluorescence measurements map the dynamic conformational landscape of GCK and provide evidence for three distinct conformations of the enzyme in the absence of glucose. Together our findings provide a framework for understanding the origins of time-dependent changes in activity in other regulatory enzymes.

Biography:

Min-Kyu Kwak has his expertise in Metabolic Regulation driven by physiological roles of methylglyoxal biosynthesis/degradation enzymes. His model based on methylglyoxal production/detoxification hierarchy suggests different metabolic control systems between prokaryotes and eukaryotes. Because his findings provide a basis for understanding cell growth, viability, and differentiation to elevate the intracellular metabolites including methylglyoxal, glutathione, and reactive oxygen species, this should be of interest to scientists who are interested in the methylglyoxal metabolism and it’s regulating enzymes in cells. His research is also to elucidate the mechanism of energy transfer which is a fundamental mechanism in life. Energy transfer mechanisms are mediated by electrons and photons. He aims to elucidate the function and structure of intermediate products, enzymes and genes involved in cell change down to the level of electrons and photons; mechanisms of “polymorphic changes of Candida albicans, developmental processes of Dictyostelium discoideum, and sporulation of Bacillus subtilis regarding electron transfer.

Abstract:

Glutathione reductase maintains the glutathione level in a reduced state. As previously demonstrated, glutathione is required for cell growth/division and its biosynthesizing-enzyme deficiency causes methylglyoxal accumulation. However, experimental evidences for reciprocal relationships between Cph1-/Efg1-mediated signaling pathway regulation and methylglyoxal production exerted by glutathione reductase on yeast morphology remain unclear. Glutathione reductase (GLR1) disruption/overexpression was performed to investigate aspects of pathological/morphological alterations in Candida albicans. These assumptions were proved by observations of cellular susceptibility to oxidants and thiols, and measurements of methylglyoxal and glutathione content in hyphal-inducing conditions mainly through the activity of GLR1-overexpressing cells. Additionally, the transcriptional/translational levels of bio-energetic enzymes and dimorphism-regulating protein kinases were examined in the strain. The GLR1-deficient strain was non-viable when GLR1 expression under the control of a CaMAL2 promoter was conditionally repressed, despite partial rescue of growth by exogenous thiols. During filamentation, non-growing hyphal GLR1-overexpressing cells exhibited resistance against oxidants and cellular methylglyoxal was significantly decreased, which concomitantly increased expressions of genes encoding energy-generating enzymes, including fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alcohol dehydrogenase (ADH1), with remarkable repression of Efg1-signaling cascades. This is the first report that GLR1-triggered Efg1-mediated signal transduction repression strictly reduces dimorphic switching and virulence by maintaining the basal level of methylglyoxal following the enhanced gene expressions of glycolytic enzymes and ADH1. The Efg1 downregulatory mechanism by GLR1 expression has possibilities to involve in other complex network of signal pathways. Understanding how GLR1 overexpression affects multiple signaling pathways can help identify attractive targets for antifungal drugs.

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Biography:

Petra Borilova Linhartova has completed her Master’s degree at the Faculty of Science and PhD at the Faculty of Medicine Masaryk University Brno, Czech Republic. She is a team member in the project promoting excellence in basic research, Czech Science Foundation Centre of Orofacial development. She has published 12 papers in reputed journals and has been serving as a reviewer of international journals.

Abstract:

Dental caries is a complex chronic multifactorial disease representing a major oral health problem in the world. In its pathogenesis, metabolic activity of cariogenic bacteria and host enzymes involved in the immune response and dentine formation plays an important role. The advanced molecular genetics methods allow by combining the sophisticated cultivation techniques with genome-level studies to investigate more thoroughly how bacterial pathogens respond to environmental stimuli. In vitro study was focused on changes in metabolomes/transcriptomes of cariogenic bacteria (Streptococcus mutans, etc.) in dependence on external conditions, such as various substrates (carbohydrates, human/animal milk, and infant formula). Their cariogenic potential was evaluated by measuring acidity of the environment and biomass concentration. The composition of metabolites and gene expression profiles were monitored by modern biotechnology techniques (CE/HPLC/MS and NGS, respectively). Further, case-control association study comprising 803 Czech children (172 controls and 111/520 patients with dental caries in the primary/permanent dentition) was carried out. Candidate genes encoding matrix metalloproteinase-9 and -20 (MMPs), which are included in the development, remodeling and destruction of oral tissues, were selected for the analysis. Polymorphisms rs17576 and rs1784418 were determined by real-time PCR using TaqMan assays. Both SNPs were associated with severity of but not susceptibility to dental caries in the permanent dentition (P<0.05).Biotechnological and molecular genetics approaches offer new possibilities for the study of complex diseases etiopathogenesis, such as dental caries.

Biography:

Dejan Bezbradica obtained his PhD degree in the Department of Biochemical Engineering and Biotechnology of Faculty of Technology and Metallurgy in Belgrade in 2007. Since 2013, he is an Associate Professor in the Department of Biochemical Engineering and Biotechnology. During 2009, he was on sabbatical working in the Laboratory of Enzyme Engineering at Institute of Catalysis in Madrid. His scientific work covers following areas: Cell and enzyme immobilization, enzymatic synthesis in microaqueous media, application of membrane reactors in biocatalytic processes; microbial production and purification of industrial enzymes, kinetic modeling of bisubstrate enzymatic reactions, application of enzymes with transglycosylative activity in synthesis of bioactive compounds, chemical modification of enzymes and immobilization supports, and nanobiocatalysis. His recent research activities are focused on the development of food and feed products containing bioactive galactosides with prebiotic activities targeted for specific probiotic species.

Abstract:

Statement of Problem: Galacto-oligosaccharides (GOS) are group of β- galactoside compounds with significant market value due to their prebiotic properties utilized in infant nutrition products. Physiological activity is based on their short chain carbohydrate structure which makes them non-digestible by digestive enzymes, but digestible by beneficial probiotic bacteria with consequential property of selective promotion of their growth and improvement of overall health status. State of the art in current industrial GOS production based on transgalactosylation activity of β-galactosidases implies that attempts for further advance could be focused on: Fine-tuning of physiological properties by targeted control of enzymatic process toward obtaining GOS of desired structure and developing novel immobilized β-galactosidase preparations with improved affinity towards GOS synthesis.

Methodology & Theoretical Orientation: For evaluation of the effect of enzyme origin on degree of polymerization and type of β-linkages within obtained GOS compounds, transgalactosylation was performed with different β-galactosidases: from Aspergillus oryzae and Lactobacillus acidophilus. Elucidation of chemical structures in obtained GOS mixtures was performed using ion-mobility spectrometry−tandem mass spectrometry (IMS-MS/MS) one-step approach. Improvement in the field of β-galactosidase immobilization was attempted by producing novel nanobiocatalyst with functionalized nonporous fumed nano-silica (FNS) particles as immobilization support.

Conclusion & Significance: IMS-MS/MS analysis has shown that structure of obtained GOS is influenced by origin of β-galactosidase, since one from A. oryzae produced GOSs with β(1→6) and β(1→3) linkages, while enzyme from L. acidophilus produces GOSs with β(1→6) and β(1→4) linkages. Type of glycosidic linkages influences prebiotic properties of GOS, hence determination of linkage type will have great significance in enabling adequate selection of β-galactosidase for targeted prebiotic application. The immobilization on nano-supports indicated that the most adequate support is one functionalized with amino groups, which enabled several times higher transgalactosylation activities than conventionally immobilized β-galactosidase.

References:

Biography:

Ayse Ezgi Unlu has expertise on enzymes, enzymatic reactions, fermentation, protein synthesis, proteomics, enzymatic biopolymers and green solvents. The synthesis of Naproxen, a member of NSAIDs, was the subject of the master thesis using commercial lipase subjected to various pre-treatment strategies that enhanced the activity. Investigation of different parameters on the production of lipase by Candida rugosa and also proteomic analysis of the isoenzymes was another subject of interest. Two important antioxidant enzymes, catalase and superoxide dismutase production by Rhodotorula glutinis was studied comprehensively during PhD thesis. A post-doc research on the synthesis of flavonoids using green solvents was completed.

Abstract:

Catechin is a crucial member of flavonoids that show antioxidant properties both in vivo and in vitro. However, flavonoid monomers, like catechin, have some disadvantages such as low solubility and pro-oxidant activity. These drawbacks are reported to disappear in the polymerized form. The polymerization of catechin was reported using organic solvents to provide solubility in many studies. We present here the effect of natural deep eutectic solvent (NADES) as green solvents on laccase catalyzed polycatechin synthesis. The reaction media contained catechin (5 mg ml^-1), acetate buffer (pH=5) and betaine (B)-mannose (M) (5:2, molar amount) at mentioned amounts. The effect of B-M amount (5, 50-90%), laccase concentration (15.6-125 U) and temperature (25-40ºC) were investigated on polycatechin synthesis. The antioxidant activities of the polycatechins were tested in terms of superoxide radical scavenging activity and xanthine/xanthine oxidase activity. Size exclusion chromatography and HPLC analysis were used as analytical methods. According to the results, 5% B-M containing reaction media provided high molecular weight polycatechin that was comparable with acetone containing media. Therefore organic solvent content could be discarded from the reaction. However, handling of the reaction media and recovery of the product were challenging steps at increased NADES content. The conversion rate of catechin was found to increase with increasing laccase amount. Additionally, high laccase concentration (125 U) was found to provide high molecular weight and yield. On the other hand, temperature had no significant effect on polycatechin formation at tested range (25-40ºC). All polycatechins obtained were found to have increased superoxide radical scavenging activity and xanthine/xanthine oxidase inhibitory activity when compared to monomer catechin. This study showed that polycatechin synthesis pathway could be shifted to a green route using NADES.

References:

1. Unlu A E, Takac S (2012) Investigation of the simultaneous production of superoxide dismutase and catalase enzymes from Rhodotorula glutinis under different culture conditions. Artif. Cells Blood Substit. Immobil. Biotechnol. 40: 338-344.

Biography:

Mohammed has completed his PhD from the Department of Biochemistry, Faculty of Veterinary Medicine, Benha University, Egypt. He is a Faculty Member in the Department of Biochemistry, Faculty of Pharmacy, October 6 University, Egypt. He has published 6 papers in reputed journals.

Abstract:

Biochemical effect of tannic acid and curcumin on female mice experimentally induced Ehrlich ascites carcinoma (EAC) was investigated. This study was carried out on 220, 12-14 weeks old female mice and weighted 25-30 g. Mice were classified into two main large experiments. Experiment 1: Non-tumor bearing mice (NTB) Included 100 of animals and divided into four groups each one comprised 25 mice. Group 1: NTB- control saline treated. Group 2: NTB-treated with curcumin orally (350 mg/kg/day) for 6 weeks. Group 3: NTB-treated with tannic acid orally (160 mg/kg/day) for 6 weeks. Group 4: NTB-treated with curcumin and tannic acid orally at ratio (50%:50%) for 6 weeks. Experiment 2: Tumor bearing (TB) mice. Out of the total 120 animals, were divided into four groups each one comprised of 30 mice. Group 1: TBM-control saline treated. Group 2: TBM-treated with curcumin orally (350 mg/kg/day) for 6 weeks. Group 3: TBM-treated with tannic acid orally (160 mg/kg/day) for 6 weeks. Group 4: TBM-treated with curcumin and tannic acid orally at ratio (50%: 50%) for 6 weeks. Blood samples were collected from all animals groups after 2, 4 and 6 weeks from treatment. Serum were separated and processed directly for glucose, insulin, total cholesterol, triacylglycerol, total protein determination. The obtained results revealed that, a highly significant decrease in serum glucose, total cholesterol, total protein concentration, meanwhile, a highly significant increase in serum triacylglycerol concentration was also observed. But a non-significant decrease in serum insulin levels were observed in tumor bearing mice when compared with control. The results of this study indicated that curcumin, tannic acid and their combination treatment have potential benefits in cancer treatment.

Biography:

Rachel Chen received her PhD from California Institute of Technology in 1994 and subsequently worked as a Research Scientist in Bristol-Myers Squibb. She began her independent academic career in Virginia Commonwealth University and continued at Georgia Institute of Technology. Her research interfaces Biology, Chemistry, and Engineering with major focuses on applying molecular engineering tools in the synthesis of molecules that are not attainable with conventional means. She has published over 80 peer-reviewed papers and has been serving as an Associate Editor for Microbial Cell Factories and on Editorial Boards of Biotechnology and Bioengineering, AIMS Bioengineering and AIMS Microbiology.

Abstract:

As one of the four building blocks of life, sugar molecules permeate almost all aspects of life. The widespread occurrence of glycosylation and its broad impact in biological processes underscores the importance of studying glycosylation. To study glycans and probe their roles in a biological system significant amount of pure molecules are needed. Besides basic research, there are a wide range of opportunities of utilizing oligosaccharides, polysaccharides, and glycoproteins and other glyco-conjugates for diagnosis, vaccine development, as new drug entities, and many other medical applications. Unfortunately, these potential applications are all impeded by the lack of large scale synthesis technology for these molecules. Metabolic engineering, since its inception in late 80’s, has grown to be a field impactful in the synthesis of a variety of molecules of commercial and societal importance. Opportunities abound at the interface of glycosciences and metabolic engineering. In fact, all sugar moieties in biological components, small or big, free or bound, are important targets for metabolic engineering. Over the past decades, its use in the synthesis of sugar-containing molecules has gained significance. Glycosidic bond formation catalyzed by glycosyltransferase enzyme is in the center of the synthesis of most glycan structures in nature. Oligosaccharides, polysaccharides and glycoproteins share the commonality that requires glycosyltransferases in their synthesis, differing only in the nature of the acceptors. Therefore, from a metabolic engineering point of view, they share much of the synthesis challenges. These include the high energy demand due to the need for sugar nucleotides as precursors, the complexity of metabolic pathways and regulations involved, and the adequate supply of acceptors when and where the glycosyltransferases are most active. Represented by 2’-fucosyllactose, the success in bringing highly valuable oligosaccharides to commercial production demonstrates the power of metabolic engineering. On the other hand, given the enormous diversity and significant complexity of saccharide-containing structures, a handful of molecules attaining commercial success can only qualify as a promising beginning. In fact, the surface of the gigantic glyco-sphere has barely scratched. Providing scientists with hundreds and thousands of glycans in quantities sufficient to probe their structure and function relationships and supplying clinicians with selective compounds (such as Globo H and heparin in Kg quantities) for clinical studies in a cost effective manner are challenges before metabolic engineers and synthetic biologists. The inherent challenges in complex carbohydrates demands innovative metabolic engineering strategies beyond a simple extension of those used in successful examples. In this presentation, metabolic engineering challenges common to glycosyltransferase-catalyzed synthesis of oligosaccharides are analyzed and successful examples from Chen labs are showcased to emphasize the power of metabolic engineering as an enabling technology.

Reference:

1. Hyun-Dong Shin, Long Liu, Mi-Kyong Kim, Yong-IL Park, Rachel Chen (2016) Metabolic engineering of Agrobacterium spp. ATCC31749 for curdlan production from cellobiose. J. Ind. Microbiol. Biotechnol. 43: 1323-1331.

2. Anne Ruffing and Rachel Chen (2011) Citrate stimulates oligosaccharides synthesis in metabolically engineered Agrobacterium spp. Applied Biochemistry and Biotechnology 164(6):851-66.

3. Mao Z, Shin H D, Chen R (2009) A recombinant E. coli bioprocess for hyaluronan synthesis. Applied Microbiology and Biotechnology 84:63-9.

4. Anne Ruffing and Rachel Chen (2010) Metabolic engineering of Agrobacterium spp. strain ATCC31749 for production of an alpha-Gal epitope synthesis. Microbial Cell Factories 9:1.

5. Anne Ruffing, Zichao Mao, Rachel Chen, (2006) Metabolic engineering of Agrobacterium spp. for UDP-galactose regeneration and oligosaccharide synthesis. Metabolic Engineering 8:465-473.

Biography:

Valentina Citro is interested in developing Pharmacological Chaperones (PC) to cure rare diseases. She works on the identification of the mutations which can be responsive to chaperones and develop method for assays in vitro in two model systems: The Fabry disease, a lysosomal storage disorder and PMM2-CDG (CDG-Ia) disease, a disorder of glycosylation with no cure at present.

Abstract:

Pharmacological chaperones are useful for the treatment of enzymopathies arising from mutations that lower the free energy difference between an unfolded and a folded enzyme shifting the equilibrium towards the first form. The unfolded enzyme, although retaining the functional chemical groups needed for the biological activity, does not maintain them in the appropriate spatial disposition defined as native state. Improperly folded mutant enzymes are usually sensitive to proteolysis and are cleared by the protein quality control systems in the cytosol and endoplasmic reticulum. Activity can be rescued if the equilibrium is pushed back towards the native state. This can be obtained binding a pharmacological chaperone to the folded enzyme. In fact the binding energy of the ligand compensates for the loss in DeltaG of unfolding. Lysosomal alpha-galactosidase represents a good model system for the therapy with pharmacological chaperones. Lysosomal alpha-galactosidase catalyzes the removal of α-galactosyl residues from a glycosphingolipid, globotriaosylceramide. Mutations of lysosomal alpha-galactosidase cause Fabry disease. We use three methods to test the effect of pharmacological chaperones: 1) Thermal shift assay. This test takes advantage of an environmentally sensitive fluorescent dye which binds the enzyme when it reaches the melting temperature; 2) Urea induced unfolding coupled with limited proteolysis and Western blot detection. This test can be carried out on mutants in cell extracts; and 3) Administration of the pharmacological chaperone to cells expressing mutant enzymes. Open reading frames encoding mutated enzymes are introduced into vectors suitable for transient expression. Eukaryotic cells, COS7 or HEK293, are transfected and cultivated in the presence and in the absence of the drug. If the chaperone works and the mutant are stabilized, a larger amount of protein is detected by western blot and consequently a higher enzymatic activity measured.

References:

Biography:

Giuseppe Manfroni has graduated in Pharmaceutical Chemistry and Technology (2001) and received his PhD in Medicinal Chemistry (2006) from the University of Perugia (Italy). From 2006 to 2008, he worked as a Post-doctoral Researcher at the University of Perugia. From 2008 to date he is an Assistant Professor in the Department of Pharmaceutical Sciences and is a Lecturer in Pharmaceutical Analysis. He has spent short periods as a Visiting PhD Student at Rega Institute for Medical Research (Leuven, Belgium) and at the Molecular Modeling Laboratory (University of Perugia) under the supervision of Professor Johan Neyts and Professor Gabriele Cruciani, respectively. He is the author of 40 papers, and his research is mainly focused on Medicinal Chemistry of antiviral (HIV, HCV, and Flavivirus), antitumor, and anti-inflammatory (p38 inhibitors) agents. He is an expert on the synthesis of heterocyclic compounds and microwave assisted synthesis.

Abstract:

Among more than 70 related members of Flavivirus genus, Dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), Yellow fever virus (YFV), and Zika virus (ZV) are considered (re)-emerging pathogens that were originally endemic in the tropical regions but recently are spreading also in a wider geographic area. Indeed, there are several environmental, demographic, and ecological factors that promote the worldwide diffusion of known and/or novel flaviviruses. Flaviviruses can produce from mild flu-like symptoms to hemorrhagic fevers, hepatitis and neuropathies, such as encephalopathy, meningitis, and microcephaly in human embryos depending on the infective agents. Vaccines are available against YFV, JEV, TBEV, and more recently against DENV but the coverage is far from being complete. Moreover, the lack of an effective and specific therapy further worsens the scenario. The RNA-dependent RNA polymerase (RdRp) of the non-structural NS5 protein is one of the most favored target to find new potential anti- Flavivirus drugs. With the aim to find new inhibitors of the RdRp we undertook a research program exploiting, consecutively, two different approaches: i) A virtual screening carried out on the NS5 polymerase domain (DENV RdRp, 2j7U) followed by a biochemical validation on the isolate target, ii) a direct biochemical screening carried out on DENV NS5 polymerase with the intent to not exclude any potential hit compounds eventually missed during the in silico procedures. Both these approaches were realized using an in-house library of about 200, published and unpublished, compounds previously designed and synthesized as HCV NS5B inhibitors. To validate the potential of the identified hits an anti-viral activity against a panel of Flavivirus was evaluated. The two strategies led us to identify new RdRp inhibitors able to reduce the polymerase activity in the low micromolar range. In particular, the in silico procedure (i) was fruitful for the identification of a pyridobenzothiazole which was extensively characterized with biochemical and structural studies; the second approach (ii) led us to identify functionalized 2,1-benzothiaziens with promising anti-RdRp activity, not emerged as hit compounds during the in silico studies (Figure 1). Also in this case, a representative compound derived from a chemical optimization was better characterized in biochemical and virological assays. The strategy applied in this study led us to identify new promising inhibitors of the NS5 polymerase, worthy of further optimization with the final aim to discover anti-Flavivirus agents.

Biography:

Zeina Nasr passion is to understand the molecular aspect of tumor initiation and progression. Her research focuses on studying the effect of translation initiation dysregulation on cancer behavior. She has worked with several cell lines and transgenic mouse models and deciphered important pathways that contribute to cancer initiation and progression to metastasis. She built her experience by conducting research and teaching in academic institutions. She is currently interested in understanding the extra-ribosomal functions of ribosomal proteins and their effects on tumorigenesis.

Abstract:

Statement of the Problem: In addition to their role in ribosome biogenesis, ribosomal proteins (RPs) play important roles in DNA repair, proliferation, apoptosis and resistance to drugs and chemotherapy. Ribosomal protein S3 (RPS3), a DNA repair endonuclease, is known to be overexpressed in colon adenocarcinoma. In order to ensure their survival, cancer cells rely on aerobic glycolysis catalyzed by the enzyme lactate dehydrogenase (LDH). Our aim is to identify the role of RPS3 in colon cancer growth and metabolism.

Methodology & Theoretical Orientation: Human colon adenocarcinoma Caco-2 and normal colon NCM-640 cells were tested for the expression of RPS3 by Western blot. In order to inhibit RPS3 expression, cells were transfected with siRNA against RPS3 or a non-targeting siRNA (siNT) as a negative control. Upon RPS3 knockdown, cell behaviors were tested including proliferation and survival by trypan blue and WST-1 assays, and cell migration and invasion by the Boyden chamber assays. The glycolysis state of colon cancer cells was assessed by measuring LDH activity upon RPS3 knockdown using the LDH assay.

Findings: RPS3 was shown to be expressed in both Caco-2 and NCM-640 cells. RPS3 knockdown in Caco-2 significantly reduced cell proliferation, survival, migration and invasion compared to siNT-transfected cells. In NCM-640, RPS3 knockdown did not significantly affect cell proliferation and survival implying that RPS3 expression is selectively crucial for colon cancer cell growth. Interestingly, LDH activity was suppressed upon RPS3 knockdown, suggesting a decrease in glycolysis which explains in part the decrease in proliferation.

Conclusion & Significance: This is the first report that shows a role of RPS3 in regulating LDH activity therefore affecting the glycolytic state, the survival and proliferation of cancer cells. Our results also demonstrate that RPS3 is a selective molecular marker in colon cancer and a potential attractive target for colon cancer therapy.

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