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Tag Archives: STING protein

Hepatitis B Research Review: March

Welcome to the Hepatitis B Research Review! This monthly blog shares recent scientific findings with members of Baruch S. Blumberg Institute (BSBI) labs and the hepatitis B (HBV) community. Technical articles concerning HBV, Hepatocellular Carcinoma, and STING protein will be highlighted as well as scientific breakthroughs in cancer, immunology, and virology. For each article, a brief synopsis reporting key points is provided as the BSBI does not enjoy the luxury of a library subscription. The hope is to disseminate relevant articles across our labs and the hep B community.

Summary: This month, researchers at Fudan University in Shanghai, China have identified activation of the cGAS/STING pathway by extracellular DNA as a mediator of radiation-induced liver disease. At the Pennsylvania State University College of Medicine in Hershey, PA, HBV researchers have elucidated the role of the host kinase protein CDK2 in phosphorylating the HBV core protein, leading to new cccDNA formation. Researchers from the University of Charlottesville in Virginia have characterized the “apoptotic metabolite secretome”, a select group of molecules released from cells undergoing apoptosis. 

 DNA sensing and associated type 1 interferon signaling contributes to progression of radiation-induced liver injury – Cellular & Molecular Immunology

This paper from Fudan University in Shanghai, China reveals the role of the cGAS/STING pathway in radiation-induced liver disease (RILD). Either radiation therapy (RT) or accidental exposure to ionizing radiation may cause RILD. RT is used to treat various cancers, including hepatocellular carcinoma (HCC). The dose of radiation used when treating HCC and gastrointestinal malignancies is limited by the risk of RILD as the liver is a highly radiosensitive organ. RILD is associated with a high mortality in patients with HCC and typically occurs within four months of receiving RT. RILD is characterized by hepatic injury due to the deposition of fibrin into the central veins and sinusoids of the liver. While the exact mechanism of RILD development is not well understood, it has been shown that hepatic nonparenchymal cells (NPCs) such as Kupffer cells, sinusoidal endothelial cells, and hepatic stellate cells play an important role. NPCs are cells in the liver that are not hepatocytes; they consist of immune cells, endothelial cells, pericytes, and other cell types. The cGAS/STING pathway is a component of the innate immune system in cells responsible for sensing double-stranded DNA (dsDNA) in the cytoplasm and subsequently initiating the expression and secretion of type 1 interferons (IFN-I). This publication identifies the cGAS/STING-mediated production of IFN-I by NPCs as a key mediator of RILD. The authors propose that RT induces massive hepatocyte apoptosis, resulting in a large amount of ectopic dsDNA which is then taken up by liver NPCs, resulting in the activation of cGAS and subsequently STING. In order to determine this, the group exposed wild-type (WT), cGAS knockout, and STING knockout mice to 30Gy of radiation. While livers of WT mice subjected to radiation showed increased steatosis (retention of lipids), mice lacking either cGAS or STING showed less at 48 hours as measured by histological staining. The knockout mice also showed reduced apoptosis in liver tissue at 48 hours as measured by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay of histological sections. Additionally, histological staining of mouse liver tissues six weeks after radiation showed that the knockout mice had less veno-occlusive inflammation, an indicator of RILD. Next, the group showed that hepatocytes extracted from mice 24 hours following irradiation secrete much more dsDNA in vitro than NPCs extracted from the same liver. Furthermore, levels of cGAS, STING, IFN-α, IFN-β, and TLR9 mRNA transcripts were found to increase dramatically in liver NPCs but not in hepatocytes following radiation as measured by RT-qPCR. Additionally, expression levels of cGAS/STING-related genes TBK1, IRF3, ISG15, JAK1, TYK2, AKT1, AGBL5, TRIM32, RSAD2, and TTL4 were all increased in liver NPCs but not in hepatocytes following radiation. The group then showed that DNase treatment of mice during and after RT prevented increased expression levels of cGAS, STING, IFN-α, and IFN-β mRNAs. This result indicates that extracellular DNA is a trigger for RT-induced IFN-I secretion. Finally, the group showed that knockout of the IFNα and IFNβ receptors in mice reduced the amount of liver steatosis and apoptosis caused by RT. Additionally, blockade of IFN-I signaling with an interferon alpha and beta receptor subunit 1 (IFNAR1)-specific antibody did not negatively affect the tumor-reducing properties of RT in a mouse HCC model. This paper indicates that cGAS/STING-signaling in liver NPCs is a major cause of RILD. Extracellular DNA from hepatocytes killed during RT is taken up by NPCs where it activates cGAS/STING signaling to produce IFN-I. This finding could help scientists and clinicians devise ways to prevent RILD in patients undergoing RT for HCC or other cancers. Perhaps short-term immune modulators may be used in tandem with RT to prevent an excessive response of the innate immune system. 

Role of Hepatitis B Virus Capsid Phosphorylation in Nucleocapsid Disassembly and Covalently Closed Circular DNA Formation – PLOS Pathogens

This paper from Dr. Jianming Hu’s laboratory at the Pennsylvania State University College of Medicine in Hershey, PA outlines the role of phosphorylation of the HBV core protein (HBc) in the HBV life cycle. HBV has a relaxed circular (RC) DNA genome which it delivers to the nucleus of hepatocytes. In the nucleus, the RC DNA is converted into covalently closed circular (CCC) DNA which is the viral transcriptional template for all HBV mRNA species including pregenomic RNA (pgRNA). Along with the viral reverse transcriptase (RT), pgRNA is packaged by HBc into newly formed nucleocapsids (NC) where it is reverse-transcribed to form RC DNA resulting in mature NCs. Mature NCs may either be enveloped and secreted as infectious virions or uncoat within the cell and further contribute to CCC DNA formation. Because CCC DNA is the reservoir of HBV in infected hepatocytes, its eradication is highly sought after and is required to achieve a true cure for the virus. This publication reports a model wherein HBc phosphorylation by the host protein cyclin-dependent kinase 2 (CDK2) facilitates the uncoating of newly formed NCs and their subsequent formation of CCC DNA. Previously, this group has found that CDK2 is a host kinase which is incorporated into HBV NCs. CDK2 is a highly conserved kinase (phosphorylating protein) which is essential during the G1, S, and G2 phases of the cell cycle.  First, the group identified two S-P (serine-proline) motifs on the globular N-terminal domain (NTD) of HBc, S44 and S49 which are potential CDK2 substrates that are on the interior surface of assembled NCs. In order to mimic constitutive phosphorylation or to block phosphorylation of the serine residues, they were mutated to glutamic acid residues (N2E) or alanine residues (N2A) respectively. The phospho-mimetic mutant N2E showed decreased levels of pgRNA packaging into NCs as measured by native agarose gel electrophoresis (NAGE) and Southern blot following transfection of the constructs into HepG2 cells. After release from NCs into the nucleus, the RC DNA HBV genome takes the form of protein free (PF) RC DNA lacking the RT protein, prior to forming CCC DNA. The phospho-mimetic N2E mutant yielded more PF-RC DNA and CCC DNA than wild type (WT) HBV and conversely, the phospho-null N2A mutant yielded less of both species than WT HBV. These results show that while NCs phosphorylated at both S44 and S49 are less efficient at packaging pgRNA, they are more likely to uncoat and release their genomes into the nucleus. Next, PhoenixBio (PXB) primary human hepatocytes harvested from human-liver chimeric mice were infected with HBV and treated with two CDK2 small molecule inhibitors. PF DNA was then extracted from the cells and analyzed via Southern blot. Both CDK2 inhibitors dramatically reduced the level of CCC DNA formation as compared to the mock control. This result indicates that CDK2 activity within NCs modulates their stability causing them to uncoat and deliver their genomes to the nucleus as opposed to being exported as virions. This publication sheds light on the exact stages of HBc phosphorylation and how they affect CCC DNA formation. This work is important because understanding the molecular mechanisms of CCC DNA formation will help in the development HBV antivirals. Small molecules which interfere with specific stages of HBc phosphorylation and dephosphorylation may prove efficacious in preventing CCC DNA formation in individuals chronically infected with HBV.            

 ​Metabolites released from apoptotic cells act as tissue messengers – Nature

This paper from the University of Charlottesville in Virginia investigates the “apoptotic metabolite secretome” and its effect on neighboring cells. Apoptosis is a highly regulated form of programmed cell death (PCD) which accounts for approximately 90% of homeostatic cell turnover. Metabolites are small molecules that are the intermediates or end products of metabolism. Here, a panel of conserved apoptotic metabolites was identified in the supernatants of apoptotic cells using advanced spectroscopy techniques (spectroscopy-based metabolomics). Six metabolites were found to be secreted across a variety of cell types in response to various apoptosis inducers. These six metabolites are: adenosine monophosphate (AMP), guanosine 5′-monophosphate (GMP), creatine, spermidine, glycerol-3-phosphate (G3P), and adenosine triphosphate (ATP). These metabolites were all found in the supernatants of Jurkat cells (acute T cell leukemia) following exposure to UV irradiation as well as following treatment with anti-Fas antibody. These metabolites were also released from primary mouse bone-marrow-derived macrophages (BMDMs) treated with anthrax and primary mouse thymocytes treated with anti-Fas antibody. Additionally, lung and colon cancer cell lines A549 and HCT116 released four of these metabolites (ATP, spermadine, G3P, and creatine) when subjected to the BH3-mimetic ABT-737 (induces mitochondrial outer membrane permeabilization) as measured using commercial kits. Secretion of these metabolites was prevented by pretreatment of cells with the pan-caspase inhibitor zVAD, indicating apoptosis as the mechanism of release. The metabolites alanine, pyruvate, and creatinine were retained within apoptotic cells, showing that metabolite release was organized and not due to nonspecific rupture of apoptotic bodies. Because only specific metabolites were released during apoptosis, the group hypothesized that the opening of plasma membrane channels may determine the apoptotic secretome. Pannexin 1 (PANX1) is a membrane channel activated by caspase 3 and 7 cleavage during apoptosis. Previously, this group has demonstrated that PANX1 activation is responsible for the secretion of ATP and UTP from apoptotic cells, which function as “find me” signals to recruit phagocytes to perform efferocytosis. In order to determine the role of PANX1 activation in the apoptotic secretome, prior to UV irradiation, PANX1 was inhibited in Jurkat cells using two methods: pharmacological inhibition with the drugs trovafloxacin (Trovan) or spironolactone and generation of a cell line bearing a dominant-negative PANX1 mutation at the caspase cleavage site. Jurkat cells with inhibited or nonfunctional PANX1 showed less secretion of 25 metabolites released from UV-treated Jurkat cells as measured by spectroscopy-based metabolomics. Spermidine, GMP, AMP, and G3P were all secreted dependent upon PANX1 activation. Next, to test whether metabolic activity within the dying cell affects its secretome, the group chose to focus on the release of spermidine. Spermidine released from apoptotic cells naturally reduces local inflammation and counteracts autoimmunity. Interestingly, while spermidine was heavily secreted from apoptotic cells, its precursor molecule putrescine was not released at all. As the starting product of spermidine synthesis is arginine, the isotope carbon-13 (13C)-containing argenine was administered to Jurkat cells one minute prior to UV irradiation. Apoptotic cells showed 40% and 25% more incorporation of 13C label into putrescine and spermidine respectively than live cells at one hour post-UV. This indicates that in addition to the caspase-dependent opening of membrane channels, apoptotic cells also maintain or even upregulate certain metabolic pathways to contribute to the apoptotic secretome. Next, in order to test the effect of the apoptotic secretome on neighboring cells, supernatant from apoptotic Jurkat cells was administered to LR73 cells (phagocytic, Chinese hamster ovary). RNA-sequencing analysis of the LR73 cells after four hours in the apoptotic supernatant revealed altered transcription of programs linked to cytoskeletal rearrangements, inflammation, wound healing or tissue repair, antiapoptotic functions, metabolism and the regulation of cell size within the phagocyte. Finally, the group used two concoctions of PANX1-dependent metabolites to treat mouse models of inflammatory arthritis and lung-transplant rejection. Treatment with the metabolite mixtures resulted in significantly reduced inflammation and better clinical outcomes in both inflammatory disease models. This publication shows that apoptotic cells affect their microenvironment by secreting anti-inflammatory metabolites. It also demonstrates that apoptosis may be harnessed to ameliorate inflammatory diseases. Once fully elucidated, other forms of PCD may also prove useful in treating other diseases such as cancer and viral infections.  

Meet our guest blogger, David Schad, B.Sc., Junior Research Fellow at the Baruch S. Blumberg Institute studying programmed cell death such as  apoptosis and necroptosis in the context of hepatitis B infection under the direction of PI Dr. Roshan Thapa. David also mentors high school students from local area schools as part of an after-school program in the new teaching lab at the PA Biotech Center. His passion is learning, teaching and collaborating with others to conduct research to better understand nature.

Hepatitis B Research Review: February


Welcome to the Hepatitis B Research Review! This monthly blog shares recent scientific findings with members of Baruch S. Blumberg Institute (BSBI) labs and the hepatitis B (HBV) community. Technical articles concerning HBV, Hepatocellular Carcinoma, and STING protein will be highlighted as well as scientific breakthroughs in cancer, immunology, and virology. For each article, a brief synopsis reporting key points is provided as the BSBI does not enjoy the luxury of a library subscription. The hope is to disseminate relevant articles across our labs and the hep B community. 

 Summary: This month, researchers in Beijing, China have reported that a therapeutic vaccine composed of polylactic acid microparticles loaded with HBV surface antigen and the mouse STING agonist DMXAA showed efficacy in clearing HBV infection in a mouse model. Researchers from Wuhan, China have reported that SOX2, a transcription factor important for cell proliferation is also a host restriction factor for HBV infection. Also, researchers from the University of Boulder in conjunction with Dr. James Chen’s lab in Dallas have reported the synthesis of two potent cGAS inhibitors.

The incorporation of cationic property and immunopotentiator in poly (lactic acid) microparticles promoted the immune response against chronic hepatitis B – Journal of Controlled Release

This paper from the Chinese Academy of Sciences in Beijing, introduces a microparticle vaccine which may be used to treat chronic HBV infection (CHB). The 1μm diameter microparticle is made from polylactic acid (PLA), which is a biodegradable polymer typically synthesized from plant starch. The microparticle also contains didodecyldimethylammonium bromide (DDAB) which is a double-chain cationic surfactant. This group has previously shown that DDAB may be used as a carrier for the HBV surface protein (HBsAg). DDAB also gives the microparticle a positive charge, which accelerates its phagocytosis into antigen-presenting cells (APCs) and facilitates its escape from lysosomal degradation once in the cell. Additionally, the group loaded microparticles with the mouse STING agonist  5,6-dimethylxanthenone-4-acetic acid (DMXAA). The microparticles were refereed to as DDAB-PLA (DP) and DDAB-PLA-DMXAA (DP-D) respectively. Both types of microparticle were saturated with HBV surface antigen (HBsAg). The microparticles were first tested on mouse bone marrow dendritic cells (BMDCs). Administration of microparticles caused less than a 20% reduction of cell viability in these cultures. BMDCs treated with DP-D microparticles had at least ten-fold more expression of IRF-7 and IFN-β mRNA as measured by RT-qPCR than those treated with HBsAg or DP microparticles alone. Surprisingly, the DP-D microparticle-treated cells also had about twice the expression of these genes compared to the positive control HBsAg + DMXAA, which contained ten times more DMXAA than the microparticles. This indicates that the DP-D microparticles induced the STING pathway with high efficiency due to their bioavailability. Next, the group found that DP-D microparticles induced the highest level of chemokine expression (measured via RT-qPCR) and immune cell recruitment (measured via flow cytometry) at the site of injection in inoculated mice compared with HBsAg alone, HBsAg with aluminum salts (traditional vaccine adjuvant), and DP microparticles. This result shows that the DP-D microparticles induced both an innate immune response and an adaptive immune response in mice. Further, the group showed that BMDCs treated with DP-D microparticles had a high level of maturation, expressing CD40, CD86, and MHCII molecules on their surface as measured by flow cytometry. Finally, the group administered the HBsAg-primed microparticles to mice infected with recombinant HBV (rAAV-1.3HBV virus, serotype ayw). Mice treated with both types of microparticles showed a higher cytokine response as well as a higher titer of anti-HBsAg antibody as measured by ELISA. Mice treated with the DP-D microparticles had the most profound immune cell activation and  fastest clearance of serum HBsAg. The microparticle vaccine introduced in this publication is promising because it is highly efficient in delivering antigen to immune cells. The microparticles are unique in that they contain a small molecule STING agonist inside. This design is clever because this vaccine stimulates the innate immune system by activating STING and the adaptive immune system by displaying HBsAg to APCs. This promotes HBV clearance in a multifaceted approach: immune cells produce cytokines through the STING pathway, T cells recognize and destroy infected cells, and B cells secrete anti-HBsAg antibodies to neutralize newly formed viruses. This publication highlights the versatility of biodegradable microparticle technology in designing unique approaches to combat infection. Micro- and nanoparticle delivery systems represent a promising avenue for future drugs to combat HBV and other viruses.

SOX2 Represses Hepatitis B Virus Replication by Binding to the Viral EnhII/Cp and Inhibiting the Promoter Activation – Viruses
This paper from Wuhan University in China identifies the protein sex determining region Y box 2 (SOX2) as a host factor that restricts HBV replication. SOX2 is a transcription factor critical for cell proliferation and the tumorigenecity of solid tumors. In 2006, expression of SOX2 along with three other transcription factors was shown to convert somatic cells into induced pluripotent stem cells. Overexpression of SOX2 indicates poor prognosis in patients undergoing resection of HCC. In HCC cells, SOX2 has also been found to induce the expression of programmed death ligand-1 (PD-L1), leading to the tumor’s evasion of the host immune system. Previously, it has been demonstrated that HBV infection induces increased expression of SOX2 in hepatocytes. This study demonstrates that SOX2 inhibits HBV replication by binding to the Enhancer II (EnhII) and Core Promoter (Cp) regions of the HBV genome. By binding to the EnhII/Cp region, SOX2 disrupts the transcription of the mRNA species precore, core, polymerase, and pgRNA. This reduction of mRNA transcription results in reduced levels of core-associated DNA, HBV surface antigen (HBsAg), and HBV e antigen (HBeAg). To learn this, the group co-transfected both HepG2 and Huh7 cells with a fixed concentration of  HBV 1.3-mer plasmid DNA alongside variable concentrations of Flag-tagged SOX2 in pcDNA3.1 plasmid DNA. Cells transfected with higher concentrations of SOX2 plasmid DNA showed reduced levels of HBV mRNAs (3.5, 2.4, and 2.1 kb) via Northern blotting. SOX2-transfected cells also showed reduced levels of HBV core-associated DNA via qPCR as well as reduced levels of both HBsAg and HBeAg via ELISA. Next, in order to learn  if SOX2 interacts directly with an HBV promoter, a dual-luciferase reporter assay was implemented. Here, four vectors were used, each containing one of the HBV enhancer and/or promoter sequences (preS1, preS2, EnhⅡ/Cp, and EnhⅠ/Xp) upstream of a firefly luciferase reporter. Each of these firefly luciferase reporter vectors were co-transfected into HepG2 cells alongside variable concentrations of SOX2 plasmid DNA. A plasmid encoding Renilla luciferase was also included at a constant concentration in each transfection as a control for transfection efficiency. While firefly luciferase has an emission of 625 nm (red), Renilla luciferase has an emission of 525 nm (green). Therefore, levels of red fluorescence were used to measure the activity of the HBV enhancer/promoter sequences and levels of green fluorescence were utilized as a control for transfection efficiency. Co-transfection with SOX2 significantly diminished the luciferase activity of the EnhII/Cp reporter only and in a dose-responsive manner, indicating its interaction with that region of the HBV genome. Further, using HBV-producing HepAD38 cells, chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) was used to isolate SOX2 protein and then determine what DNA sequence it was bound to. The EnhII/Cp sequence was found to be highly enriched on SOX2 protein. In order to determine which part of the SOX2 protein is required for binding to the EnhII/Cp region, truncated forms of SOX2 were generated in the pcDNA3.1 plasmid. Using the assays described above, it was found that only SOX2 mutants lacking the high mobility group (HMG) domain were unable to bind to the EnhII/Cp region and suppress HBV products. Interestingly, it was found that SOX2 mutants lacking the transcription activation (TA) domain were still able to bind to the EnhII/Cp region. Further, it was demonstrated by Western blot of subcellular fractions and immunofluorescence that SOX2 mutants lacking the HMG domain were unable to enter the nucleus. Finally, studies were performed in an in vivo BALB/c mouse model. Mice were given a hydrodynamic injection of an adeno-associated viral vector conferring HBV (pAAV-HBV1.3) alongside pcDNA3.1 plasmid DNA conferring SOX, SOX2 lacking HMG domain ( SOXΔHMG), or empty vector. Levels of HBsAG and HBeAg in the blood at days two and four were reduced only in mice given the full length SOX2 plasmid. Additionally, mice given the full length SOX2 plasmid had a reduction of 3.5kb HBV mRNA in liver tissues as measured by qPCR and a lower abundance of HBV core antigen (HBcAg) in liver tissues as measured by immunohistochemical staining. This study shows that SOX2 protein, previously shown to be upregulated by HBV, plays an anti-HBV role in the liver. SOX2 is therefore a new host restriction factor of HBV replication. SOX2 may be one protein which contributes to HBV-induced hepatocarcinogenesis, given its role in promoting the transcription of genes involved in cell proliferation. In the future, SOX2 may be utilized for its anti-HBV activity or targeted for the treatment of HCC.

 Discovery of Small Molecule Cyclic GMP-AMP Synthase Inhibitors – The Journal of Organic Chemistry

This paper from the University of Colorado Boulder introduces the development of novel small molecule inhibitors of the protein cyclic GMP-AMP synthase (cGAS). This publication is in conjunction with Dr. James Chen’s laboratory at the University of Texas Southwestern Medical Center in Dallas, Texas. Dr. Chen’s lab discovered cGAS in 2012. cGAS is a cytosolic, double-stranded DNA (dsDNA)-sensing protein. It belongs to the nucleotidyltransferase family of enzymes which transfer nucleoside monophosphates, the substituents of nucleic acids. When cGAS recognizes dsDNA, it synthesizes the cyclic dinucleotide cyclic GMP-AMP (cGAMP). cGAMP acts as a second messenger and activates the stimulator of interferon genes protein (STING). Once activated, STING triggers TBK1- and IKK-mediated activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). In the nucleus, IRF3 and NF-kB induce the expression of type I interferons and other inflammatory cytokines. cGAS is essential for detecting foreign pathogens which contain dsDNA and triggering an innate immune response to clear them. However, excessive or dysfunctional cGAS activity may lead to chronic inflammation and/or autoimmunity. Pharmacologic inhibition of cGAS may provide treatments for diseases including Aicardi-Goutiés syndrome (AGS), lupus erythematosus, and cancer. Current small molecule inhibitors of cGAS are limited by poor specificity and/or cellular activity. In this study, a high throughput virtual screen (HTVS) was utilized to screen about 1.75 million drug-like compounds for activity against the dimer-forming and DNA-binding faces of mouse cGAS (mcGAS). mcGAS was utilized for the in silico screen because the human cGAS (hcGAS)-DNA complex was only recently published. From this virtual screen, ten compounds were further investigated, leading to the selection of one lead compound. This lead was further optimized for greater potency through chemical modifications resulting in the analogues CU-32 and CU-76. The IC50 of both compounds is below 1µM. To test these compounds’ selectivity for cGAS, human monocyte cells THP-1 were either transfected with  interferon-stimulatory DNA (ISD) or infected with Sendai virus (SeV). ISD is a 45-basepair DNA known to activate cGAS, while SeV is a single-stranded RNA (ssRNA) virus known to activate the RIG-I-MAVS pathway; both stimuli are known to result in IRF3 activation and dimerization. Following treatment with both compounds, Western blot of the cells was conducted probing for the formation of IRF3 dimers. In ISD-treated cells, CU-32 and CU-76 inhibited the formation of IRF3 dimers in a dose responsive manner. Neither compound had any effect on IRF3 dimer formation in SeV-infected cells. This result indicates that these inhibitors are selective to cGAS. Using in silico molecular docking studies, the group speculates that these compounds disrupt the interface of the cGAS dimer, allosterically inhibiting dimerization. The discovery of novel cGAS inhibitors is exciting and important for multiple reasons. These compounds, if made commercially available will allow for improved experimentation investigating the cGAS/STING pathway. If these compounds or their derivatives are found to be safe and effective in humans, they may be promising candidates for the treatment of autoimmune disorders or cancer.


Meet our guest blogger, David Schad, B.Sc., Junior Research Fellow at the Baruch S. Blumberg Institute studying programmed cell death such as apoptosis and necroptosis in the context of hepatitis B infection under the direction of PI Dr. Roshan Thapa. David also mentors high school students from local area schools as part of an after-school program in the new teaching lab at the PA Biotech Center. His passion is learning, teaching and collaborating with others to conduct research to better understand nature.