Tom Zielinski has worked at Bellbrook Labs for over 8 years where he is the Manager of Biochemical assays. Over 20 years, he has been an R&D Scientist who has helped develop numerous cellular and biochemical assays for enzymes in diverse classes such as GPCRs, kinases, methyltransferases and glyosyltransferases.

Glycosyltransferase enzymes participate in diverse metabolic and regulatory roles by catalyzing the transfer of sugars to protein, lipid and carbohydrate acceptors as well as to other endogenous and xenobiotic molecules. Of more than 200 human glycosyltransferases (GTs), there are over 20 distinct polypeptide N-acetylgalactosaminyltransferases (GALNTs) that catalyze the initial step of O-glycosylation by transferring GalNac to Thr or Ser residues on multiple targets including mucins. Abnormal post-translational glycosylation of mucin is a driver of cancer-associated changes diversely affecting growth and survival of cancer cells and their ability to invade and metastasize. GALNT3 overexpression and dysregulation has been directly linked to multiple cancers including gastric, pancreatic, ovarian, lung and others making it a compelling target for drug discovery. Development of an HTS workflow for GALNT3 is described here. Recombinant First, GALNT3 enzyme activity robustness was first evaluated tested and optimized in the Trasncreener UDP2 Assay with the donor and acceptor substrates UDP-GalNac and Mucin 10 (153-165) EA2 peptide. Second, in order to perform a pilot screen, optimal GALNT3 concentration and Km values for both substrates were determined. Thirdly, A pilot screen was run using the TR-FRET-based assay with a with a diverse, pre-screened filtered 8,000 compound OPS orthogonally pooled compound set library from LCGC from the Lankenau Institute for Medical Research (LIMR) Chemical Genomics Center which allowed screening of 8,000 compounds in duplicate in just five 384-well plates. Hits were confirmed followed by dose-response curves of all potential hits measurements with the primary screening assay and then further validated with a FP based Transcreener UDP2 Assay second assay format. Finally, two confirmed hits were further evaluated for the longevity of target engagement by doing jump dilution performing rapid dilution experiments to assess measure dissociation rates residence time and reversibility.

Seung Yeon Chung completed Bachelor of Science at the age of 22 years from Suwon University. I am a graduate student in the master course of molecular biology under the supervision of Professor H. A. Kang at Department of Life Science, Chung-Ang University in Seoul, Korea.

The human fungal pathogen Cryptococcus neoformans causes life-threatening meningitis in immunocompromised patients. The virulence of C. neoformans depends on production of a polysaccharide capsule and deposition of melanin in the cell wall along with an ability to secrete various proteins. Secreted phospholipase B1 (PLB1) with high levels of N-linked glycosylation is one of fungal virulence determinants. Several mannoproteins such as chitin deacetylase 2(MP98) are also known as key antigens stimulating T-cell response. Here, we report secretory expression analysis of PLB1 and MP98 by constructing His-tagged and GPI anchorless PLB1 (PLB1-HisGPI-) and MP98 (MP98-HisGPI-) proteins and expressing them in the wild-type and several glycosylation mutant strains of C. neoformans. The apparent molecular weights of PLB1-HisGPI- and MP98-HisGPI- were decreased in the mutant strains such as alg3Δ, mnn2Δ and och1Δ strains respectively which have defects in biosynthesis of N-linked glycans. Unexpectedly, the molecular weight of MP98-HisGPI- secreted from the O-glycosylation mutants was identical to that secreted from the wild type suggesting that ser/thr-rich regions in C-terminus of MP98 might not be O-glycosylated. Moreover, we observed that MP98 was secreted as more heavily glycosylated forms in synthetic complete medium compared to the cultivation with rich medium. The purified mannoproteins with different degree of glycosylation would be valuable tools to investigate the role of glycans assembled on mannoproteins in the interaction of C. neoformans with host cells.

Eun Jung Thak has completed Bachelor of Science at the age of 24 years from Chung-Ang University. He is now a Post graduate student in the Masters course of Molecular Biology under the supervision of Professor H A Kang at Department of Life Science, Chung-Ang University in Seoul, Korea.

Environmental stresses are sensed by transmembrane sensors, the WSC family, Mid2p and Mtl1p in Saccharomyces cerevisiae. These surface sensor proteins are highly O-mannosylated and involved in the cell wall integrity signaling pathway. In this study, we identified two putative cell wall sensors WSC1 (CNAG_03328) and WML1 (WSC/Mtl2-Like, CNAG_01255) in the opportunistic human fungal pathogen Cryptococcus neoformans by in silico analysis. The overall protein sequence similarities between C. neoformans proteins to S. cerevisiae sensor proteins are quite low. CnWsc1p shows 16.8% and 21.3% identities with S. cerevisiae Wsc1p and S. pombe Wsc1p respectively. CnWml1p is related to SpWsc1p and ScMtl1p with 19.3% and 16.7% identities respectively. We observed that the CnWsc1p and CnWml1p were expressed as high molecular weight (MW) forms much larger than the predicted size in the wild-type strain. Noticeably, CnWsc1p with low MW was accumulated in the Cnktr3Δ mutant who has a defect in O-glycosylation. Sub-cellular fractionation experiments showed the enriched expression of CnWsc1p and CnWml1p in the cell wall/membrane fraction indicating their expression at cell surface. Furthermore, whereas no detectable defects were shown in the Cnwcs1Δ and Cnwml1Δ single mutants, the Cnwcs1wml1Δ double mutant displayed apparent cell growth defects under osmotic stresses. These results suggest that C. neoformans Wsc1p and Wml1p are O-mannosylated surface proteins with important roles in responding to osmotic stresses.

Ajeesh Kumar K K has completed his Post graduation in Biochemistry at the age of 22 years from Bharathiar University affiliated college. He has more than three years experience in biochemistry research field and mainly working on proteoglycans from shark cartilage. He is currently working as a Senior Research Fellow in Central Institute of Fisheries Technology, Cochin in India and has one national journal publication. He attended two national and international seminars and presented poster and paper.

Proteoglycans were extracted from deep sea shark Echinorhinus brucus cartilage and their anti-proliferative activity against MCF-7 cell lines was evaluated. Cytotoxicity effect was determined by assessing the cytotoxic effect of the proteoglycans by MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide). The following assays were carried out to find out apoptosis mechanism was the cause for cytotoxicity, the concentration of caspases 3 and 9, double fluorescent staining with acridine orange/ethedium bromide and DNA laddering assay. Gene expression assay for BAX, BCL-2 and p53 has been done to find out the expression of mentioned genes products to confirm apoptosis pathway. Different concentrations such as 5, 10, 25, 50 and 100 µg/ml were used for assessing anti-proliferative effect. Significant cytotoxic effect of 75% cell death was observed for 100 µg/ml treated sample. Significant raise in activity of caspase 3 and 9 was found for 100 µg/ml treated sample i.e., 3.92 and 4.35 (expression fold compared with control as 1) respectively. DNA laddering effect was also observed for the 100 µg/ml treated sample. Maximum cell death determined by double fluorescent staining was observed for 100 µg/ml treated sample. A significant raise in the fold expression was observed for BCL-2 and p53 of 4.5 and 3.55 respectively (band intensity was expressed as relative absorbance unit); whereas, low BAX expression of 0.42 was noticed for 100 µg/ml treated sample. Significant anti-proliferative effect was observed for optimal concentration of 100 µg/ml treated sample and mechanism behind cell death was found to be apoptosis as inferred from the above results.

Mehrdad Larry is pursuing his graduation and he will be MD-MPH from Tehran University of Medical Sciences next year. He has been the first place in The Scientific and Laboratory Examination in Isfahan province two times. He earned the second place in National Entrance Exam for Universities in Iran. Currently, he is working on oxidative stress in patients with diabetic nephropathy at Endocrinology and Metabolism Research Center (EMRC) at Vali-Asr Hospital affiliated with Tehran University of Medical Sciences. He has published 6 papers in reputed journals so far.

Complications of diabetes mellitus are associated with non-enzymatic glycation and oxidative stress which damages proteins. Sufficient data supports that such inflammatory process affects advanced glycation end products (AGE) and advanced oxidation protein products (AOPP). These glycooxidation mechanisms have been recently stated to play an important role in progression of vascular complications in diabetes. To date, the exact relationships between glycation and oxidation pathways have not been discovered. Our study assesses the effect of albuminuria progression in patients with Type 2 diabetes on AGE and AOPP levels and their relationship according to the albuminuria. We designed a matched case-control study of 38 microalbuminuric patients with Type 2 diabetes and 38 AGE and body mass index (BMI) matched normoalbuminuric patients with Type 2 diabetes. Serum concentrations of AGE and AOPP were measured. There were no significant difference between both AGE and AOPP comparing normoalbuminuric and microalbuminuric patients. Multiple logistic regression analysis showed a significant correlation between AGE and AOPP in normoalbuminuric patients (r = 0.375, p-value<0.05) which remained significant after adjustment for age, BMI, systolic and diastolic blood pressure (r=0.493, p-value<0.05); while such correlation was not observed in microalbuminuric patients by far. (r=0.08) Our finding suggests that disappearance of the relationship between glycation and oxidation marker molecules in diabetic nephropathy progression happens. Such disturbance in the balance between these molecules may be able to reveal the pathogenesis of biochemical processes which leads to vascular damage in complications of diabetes.