Hansaem Lee has completed her PhD from Ulsan University, Republic of Korea and Post-doctoral studies from Mayo Clinic, Minnesota, USA. She is a Staff Scientist at division of Emerging Infectious Diseases and Vector Research, Korea National Institute of Health, Korea Centers for Disease Control and Prevention, Republic of Korea.

Isolating human B cell cells is inevitable to investigate the development of protective antibodies, as vaccine or therapeutics candidates, against emerging infectious viruses. Single B cell sorting is a powerful tool for define ant-viral B cells. Thus, the acquisition of abundant B cells is essential to synthesize the broad-ranged antibody cDNAs for the screening of the broadly neutralizing monoclonal antibodies against the infectious viruses. In this study, we test and compare the human B cell isolation rates among the three human B cell isolation tools, a fluorescence-activated cell sorting (FACS), EasySepâ„¢ human B cell enrichment kit (STEMCELL Technologies), and human CD19 MciroBeads (MACS Miltenyi Biotec). We found that the method using FACS was more suitable to obtain more human B cells than the other methods. In addition, using a CMV tetramer probe or MERS-CoV spike protein probes, the antibody genes against CMV and MERS-CoV were amplified by heavy chain and light chain RT-PCR and nested PCR successfully. These genes were also analyzed by IMGT, resulting that the antibody sequences were proper IgG genes. The results demonstrate that human B cell isolation using FACS are a reliable tool for assisting in the rapid development of prophylactic or therapeutic antibodies neutralizing new emerging infectious diseases.

Hye-Min Woo has completed her PhD from Kookmin University, Republic of Korea. She is a Senior Researcher of division of Emerging Infectious Diseases & Vector Research, Korea National Institute of Health, Korea Centers for Disease Control & Prevention, Republic of Korea.

Middle East Respiratory Syndrome (MERS) is a severe respiratory infection caused by MERS Coronavirus (MERS-CoV). After the outbreak of MERS occurred in the Republic of Korea in 2015, it has been required to develop the efficient and safe therapeutics against MERS-CoV infection. The spike glycoproteins (S) of MERS-CoV bind cellular receptors, and mediate infection of target cells. Therefore, S protein is one of the most promising antigen candidates for the development of neutralizing antibodies of a newly emerging MERS-CoV. In this study, we employed the peripheral blood mononuclear cells (PBMCs) of Korean MERS recovered patients to obtain the memory B cells bearing immunoglobulin Gs (IgGs) against MERS S protein. The cells were isolated by using FACS. The cDNAs of IgGs were synthesized from the sorted B cell clones. The variable regions of heavy and light chains were amplified by PCR, and recombinant plasmids were cloned with pFUSE vectors containing signal peptides and the constant region of human IgG. The anti-MERS-CoV S IgGs were expressed in 293F cells and purified by a protein G affinity chromatography. Characteristics of anti-MERS-CoV S IgGs were analyzed by the S protein affinity assay and the neutralizing assays using a MERS-CoV pseudovirus and MERS-CoV strains. The antibodies generated in this study are expected to be applied as therapeutic agents or tools for diagnosis.

Bob Snyder has over 30 years’ experience in Life Science Research and Commercialization. He has previously held executive positions at Abbott Laboratories, Merck & Co., Magneto Organics and Elsevier Science, before leading the spin-out of Proteios from the University of Washington. He has received his BS in Chemistry from Humboldt State University and a PhD in Physical Biochemistry from the University of California at Santa Barbara. He has received a National Research Council (NRC) Post-doctoral fellowship to conduct research at NASA's Space Bioprocessing Laboratory. He also received an MBA from the University of Chicago Graduate School of Business.

Problem: The use of recombinant proteins has increased greatly in recent years, with the advantages of employing a tag fused to the recombinant protein of interest to facilitate its purification, now widely recognized. The His-tag is the most frequently used affinity tag for protein enrichment. His-tagged proteins can be readily purified by using immobilized transition metal ions such as Ni2+ or Co2+ as affinity ligands. However, the technology is not without disadvantages: His-tags can interfere with the biological function of proteins to which they are fused; the high imidazole concentrations used for elution can inactivate certain proteins by stripping them of metal ions; and purification resins contain toxic transition metal ions raising the real possibility of heavy metal leaching from the column during purification. However, the main drawback of His-tagging is its cost, which has been estimated to be as high as $2/mg of purified protein. Orientation: In collaboration with the University of Washington, Proteios is developing a suite of protein purification kits based on the Car9â„¢-tagging technology developed in Prof. Baneyx's laboratory. Car9 is a hydrophilic dodecapetide that enables recombinant proteins to bind with high affinity to silica, when fused to their N- or C-termini. The interaction can be disrupted with lysine, forming the basis of an affinity purification system that provides simple, one-step operation with rapid (less than 15 minutes) purification of recombinant proteins at lab- and small-scale quantities. Findings: The Proteios kits will be five-to-ten times cheaper than current market options. The disposable, silica-based resin ProteoResinâ„¢ has been optimized for Car9 absorption and will not require lengthy and expensive cleaning/regeneration steps like Ni-NTA resins. The Car9 workflow, which has been validated and optimized, is being extended to include the low-cost cleavage of the N- or C-terminal Car9 tag using site- specific proteases.

Statement of the Problem: The yeast Pichia pastoris is a popular host for expressing and exporting recombinant proteins, such as human insulin and a hepatitis B vaccine protein, out of its cell. Secreted proteins are easier to purify and therefore are more useful than non-secreted proteins. However, Pichia pastoris has been known to secrete certain proteins efficiently while struggling to secrete others. Approach & Methodology: Our lab has created a strain with a mutated Bgs13 gene that is a super-secreter of multiple recombinant peptides. To understand why the Bgs13 strain displays enhanced secretion, cell wall assays were first performed using Congo red and Calcoflour white to determine if super-secretion is a result of defective cell walls. In addition, Bgs13 appears to be a homolog of the Saccharomyces cerevisiae protein kinase C (PKC1). Thus, we tested if super-secretion in our Bgs13 strain is a result of elevated or decreased protein kinase C activity compared to the wild type parent. Lastly, the localization of wild type Bgs13 and mutant Bgs13 proteins was compared by fusing each protein to EGFP and examining them with fluorescence microscopy analysis. Results & Significance: The mutant Bgs13 strain had a cell wall with apparent structural defects. Not only did the mutant Bgs13 protein have lower protein kinase C activity, but it also was localized to different parts of the P. pastoris cell compared to the wild type Bgs13 protein. By characterizing mutant and wild type Bgs13 proteins, the results will help us create strains with optimized secretion of many different recombinant proteins.

Statement of the Problem: The yeast Pichia pastoris is a popular host for expressing and exporting recombinant proteins, such as human insulin and a hepatitis B vaccine protein, out of its cell. Secreted proteins are easier to purify and therefore are more useful than non-secreted proteins. However, Pichia pastoris has been known to secrete certain proteins efficiently while struggling to secrete others. Approach & Methodology: Our lab has created a strain with a mutated Bgs13 gene that is a super-secreter of multiple recombinant peptides. To understand why the Bgs13 strain displays enhanced secretion, cell wall assays were first performed using Congo red and Calcoflour white to determine if super-secretion is a result of defective cell walls. In addition, Bgs13 appears to be a homolog of the Saccharomyces cerevisiae protein kinase C (PKC1). Thus, we tested if super-secretion in our Bgs13 strain is a result of elevated or decreased protein kinase C activity compared to the wild type parent. Lastly, the localization of wild type Bgs13 and mutant Bgs13 proteins was compared by fusing each protein to EGFP and examining them with fluorescence microscopy analysis. Results & Significance: The mutant Bgs13 strain had a cell wall with apparent structural defects. Not only did the mutant Bgs13 protein have lower protein kinase C activity, but it also was localized to different parts of the P. pastoris cell compared to the wild type Bgs13 protein. By characterizing mutant and wild type Bgs13 proteins, the results will help us create strains with optimized secretion of many different recombinant proteins.

Sandeep Mittan’s overall research goal is to design rational, molecularly-targeted therapies for diffuse large B-cell lymphoma (DLBCL), the most frequent type of B-cell Non-Hodgkin’s Lymphoma (B-NHL, 50000 cases/year). To do this, he plans to exploit functional genomics approaches to link DLBCL genetics and epigenetic lesions to pathway sensitivities and therapeutic principles. Ultimately, he wants to build a connectivity map between genetics and drugs, a compass to navigate personalized medicine approaches. We anticipate that our findings will not only identify new drug-gene relationships but also help to address fundamental questions in Germinal Center B-cell biology and shed new light on key pathways in mature B cells.

Statement of the problem: Diffuse Large B-Cell Lymphoma (DLBCL) is the most frequent B-cell Non-Hodgkin Lymphoma (B-NHL) which derives from Germinal Center (GC) B cells, and classified into GCB and ABC subtypes, which differ in their cell of origin, genetic alterations and responses to therapy, with ABC having inferior prognosis in response to current immune-chemotherapeutic regimen (R-CHOP). This standard first-line treatment is successful in ~70% of cases by 5-year survival, while the remaining ~30% of patients remains incurable. Bruton Kinase (BTK) inhibitor (Ibrutinib) and PI3K-delta inhibitors are providing novel targeted strategies but their impact on DLBCL is still limited to a fraction of cases, mostly in the ABC subtype. Methodology & Theoretical Orientation: To identify drugs that can be repositioned for selective efficacy against DLBCL subtypes (GCB- and ABC-DLBCL) compared to non-GC lymphomas, we screened focused libraries including FDA-approved drugs and other promising targeted compounds in advanced clinical testing and the results were validated in vivo using DLBCL xenografts. Findings: Dasatinib has broader activity than Ibrutinib, since it is active against all ABC-DLBCL Ibrutinib-sensitive cell lines along with ABC- and GCB-DLBCL cell lines. Notably, Dasatinib overcomes Ibrutinib-resistance caused by BTK C481S mutation, as Dasatinib is active against ABC-DLBCL lines (LY-10, HBL1 and TMD-8) rendered Ibrutinib-resistant by transduction of BTK C481S mutant. In these cell lines, Dasatinib is unable to suppress BTK-C481S auto-phosphorylation at Tyrosine 223, indicating that Dasatinib may act independently of BTK. As Dasatinib can suppress all three BCR-associated Src-Family Kinases (SFKs) LYN, FYN and BLK, we employed Dasatinib-resistant, gatekeeper mutants of each SFK, alone or in combination, to understand which kinase or combination of kinases needs to be suppressed for Dasatinib activity. Our results showed that FYN suppression is essential for Dasatinib activity in both DLBCL subtypes and PTEN disruption is the most significant predictor of Dasatinib resistance. PTEN is inactivated in most of resistant lines resulting in inability by Dasatinib to restrict PI3K-mediated AKT activation. Furthermore, these data were further validated by showing that 3 Dasatinib-sensitive DLBCL lines were made resistant by transduction of a constitutively active PIK3CA mutant (PIK3CA-H1047R) and the suppression of AKT activation by mTORC2 inhibitors can revert Dasatinib resistance. Conclusion & Significance: The present study identified the multi-kinase inhibitor Dasatinib as the most DLBCL-specific agent and provided a preclinical framework for Dasatinib-based targeted therapies for DLBCL.

Somer L Doody is a senior at Utah Valley University, finishing up double Bachelor of Science degrees in Biology and Biotechnology. She has worked on several projects in Molecular Biology and Protein Biochemistry through Innovabio, a contract research organization through Salt Lake Community College in Salt Lake City, Utah. The SKIP project is being done as one of her thesis projects for graduation.

Human SKIP (skeletal muscle and kidney-enriched inositol phosphatase) is a 51 kDa protein found in many tissues including brain, eye, and abundantly in the heart, kidney and skeletal muscle. SKIP exists in the endoplasmic reticulum under resting conditions and translocates to the membrane upon insulin signaling which stimulates protein complex formation involving the insulin receptor. SKIP displays sensitivity towards the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) with SKIP hydrolyzing the phosphate at position 5 of the inositol ring of PIP2. PI(4,5)P2 is predominantly located at the plasma membrane where it is involved in protein recruitment and insulin signaling. The purpose of this work is to obtain enzymatically active human SKIP. Previous experiments utilized a fusion protein from pLATE31 SKIP that contained a His-tag. This construct was difficult to purify and produced a low yield of inactive SKIP (as tested via Malachite Green detection of inorganic phosphate). We constructed a new GST-SKIP chimera produced in a heterologous expression system. A similar GST-SKIP species has been reported to have phosphatase activity towards short-chain diC8 (PI(4,5)P2. Glutathione affinity chromatography will be utilized to purify soluble GST-SKIP. The detection of the chimera will be conducted by western blot with an anti-GST probe. Fractions enriched with GST-SKIP will be tested for 5-phosphatase activity with both diC16 and diC8 PI(4,5)P2 substrates. Free phosphate detection in SKIP post-reaction mixtures will be carried out using Malachite Green Solution. Confirmation of SKIP activity will lead to removal of the GST-tag and further purification of the target protein to obtain a SKIP preparation for the study of phosphatidylinositol signaling pathways.