Structural and biochemical studies of proteins implicated in Kaposi’s Sarcoma-associated Herpesvirus pathobiology

The Kaposi’s sarcoma-associated Herpes virus (KSHV) infects hundreds of millions of people world-wide contributing to the development of Kaposi’s sarcoma (the most common HIV-related cancer), Multicentric Castleman’s disease and Primary effusion lymphoma. The project focused on two key proteins: KSHV SOX and KSHV-vFLIP that operate during the lytic and latent phases of the KSHV life-cycle respectively. KSHV SOX is a virally encoded highly conserved alkaline exonuclease which plays a key role in the global and rapid degradation of host mRNA in a process termed host shutoff as well as the packaging of viral genomes into capsids following replication. Although published (Bagneries et al., 2011) and unpublished (Lee, 2015, Master’s thesis, UCL) crystal structures of SOX bound to DNA has given key insights into the mechanism of DNA recognition and cleavage, it was unclear how RNA could be recognised owing to the lack of canonical RNA binding motifs and therefore uncertain whether the mechanism of cleavage proposed for the exonucleolytic cleavage of DNA substrates would apply. A combination of structural and biochemical approaches were therefore used to address these key outstanding questions which are presented in this thesis. They include the first crystal structure of SOX-E244S mutant bound to the KSHV pre-miRNA K2-31 which was determined at 3.3 Å by molecular replacement. Analysis of this structure has revealed a distinct binding mode for RNA binding relative to DNA in which the “bridge” motif, spanning the C- and N-terminal lobes of the SOX molecule, has an essential role in substrate recognition. Despite these differences, endonucleolytic processing of RNA transcripts could still be achieved by the SN2 (biomolecular nucleophilic substitution) mechanism originally proposed for the exonucleolytic cleavage of DNA. The results of biochemical and biophysical assays performed on wild-type and mutant proteins defective in host shut off further revealed that the degradation of RNA is largely sequence non-specific, but requires structured elements such as stem loop or bulge motifs. They also revealed that impaired host shut off activity could be explained by impaired RNase activity against structured substrates for a subset of the mutants. The ability of compare SOX-DNA and SOX-RNA structures afforded by these studies lead to the identification of phytic acid as a potential inhibitor of both its DNase and RNase activities confirmed by the crystal structure of SOX-phytic acid complex refined to 2.3 Å. Here it is shown that phytic acid not only has the potential to physically block the association of DNA/RNA substrates but also inhibit nucleolytic cleavage by directly co-ordinating to a catalytically important magnesium ion. During latency, KSHV produces a limited number of proteins essential for its survival. One of these is the viral FLICE inhibitory proteins (vFLIPs) that directly activates the canonical and alternative NF-κB pathways resulting in increased cell proliferation, transformation, cytokine secretion, and protection against growth factor withdrawal-induced apoptosis. Previous studies have shown that KSHV-vFLIP forms a ternary complex with the transcription factor p100 and the kinase IKKα for persistent activation of the alternative pathway shown to have an important role in cellular transformation. Although the mechanism underlying this process is unclear, a physical interaction between KSHV-vFLIP and p100 has been implicated. Having successfully produced all three proteins in E. Coli and baculovirus expression systems, the nature of this complex was investigated using pull down assays and site-directed mutagenesis. From these results, it has been possible to deduce that KSHV-vFLIP interacts with residues 860-900 located at the C-terminal end of p100 and does not interact with the death domain of p100

Three-Dimensional Metal-Oxide Nanohelix Arrays Fabricated by Oblique Angle Deposition: Fabrication, Properties, and Applications

Three-dimensional (3D) nanostructured thin films have attracted great attention due to their novel physical, optical, and chemical properties, providing tremendous possibilities for future multifunctional systems and for exploring new physical phenomena. Among various techniques to fabricate 3D nanostructures, oblique angle deposition (OAD) is a very promising method for producing arrays of a variety of 3D nanostructures with excellent controllability, reproducibility, low cost, and compatibility with modern micro-electronic processes. This article presents a comprehensive overview of the principle of OAD, and unique structural and optical properties of OAD-fabricated thin films including excellent crystallinity, accurate tunability of refractive indices, and strong light scattering effect which can be utilized to remarkably enhance performances of various systems such as antireflection coatings, optical filters, photoelectrodes for solar-energy-harvesting cells, and sensing layers for various sensors.1114Ysciescopu

KSHV SOX mediated host shutoff: the molecular mechanism underlying mRNA transcript processing.

Onset of the lytic phase in the KSHV life cycle is accompanied by the rapid, global degradation of host (and viral) mRNA transcripts in a process termed host shutoff. Key to this destruction is the virally encoded alkaline exonuclease SOX. While SOX has been shown to possess an intrinsic RNase activity and a potential consensus sequence for endonucleolytic cleavage identified, the structures of the RNA substrates targeted remained unclear. Based on an analysis of three reported target transcripts, we were able to identify common structures and confirm that these are indeed degraded by SOX in vitro as well as predict the presence of such elements in the KSHV pre-microRNA transcript K12-2. From these studies, we were able to determine the crystal structure of SOX productively bound to a 31 nucleotide K12-2 fragment. This complex not only reveals the structural determinants required for RNA recognition and degradation but, together with biochemical and biophysical studies, reveals distinct roles for residues implicated in host shutoff. Our results further confirm that SOX and the host exoribonuclease Xrn1 act in concert to elicit the rapid degradation of mRNA substrates observed in vivo, and that the activities of the two ribonucleases are co-ordinated

Chemotherapeutic Candidate Inducing Immunological Death of Human Tumor Cell Lines

The immunological death induction by EY-6 on the human tumor cell lines was screened. Human colon carcinoma (HCT15, HCT116), gastric carcinoma (MKN74, SNU668), and myeloma (KMS20, KMS26, KMS34) cells were died by EY-6 treatment with dose-dependent manner. CRT expression, a typical marker for the immunological death, was increased on the EY-6-treated colorectal and gastric cancer cells. Interestingly, the effects on the myeloma cell lines were complicated showing cell line dependent differential modulation. Cytokine secretion from the EY-6 treated tumor cells were dose and cell-dependent. IFN-γ and IL-12 secretion was increased in the treated cells (200% to over 1000% of non-treated control), except HCT116, SNU668 and KMS26 cells which their secretion was declined by EY-6. Data suggest the potential of EY-6 as a new type of immuno-chemotherapeutics inducing tumor-specific cell death. Further studies are planned to confirm the efficacy of EY-6 including in vivo study

Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene

The maintenance of undifferentiated human pluripotent stem cells (hPSC) under xeno-free condition requires the use of human feeder cells or extracellular matrix (ECM) coating. However, human-derived sources may cause human pathogen contamination by viral or non-viral agents to the patients. Here we demonstrate feeder-free and xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which completely rule out the concern of human pathogen contamination. DAS-NG exhibited advanced biocompatibilities including surface nanoroughness, oxygen containing functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain pluripotency in vitro and in vivo, and especially cell adhesion-related gene expression profile was comparable to those of cultured on feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free culture method using DAS-NG will facilitate the generation of clinical-grade hPSC.ope

Immunotherapy of Malignant Melanoma with Tumor Lysate-Pulsed Autologous Monocyte-Derived Dendritic Cells

PURPOSE: Dendritic cell (DC) vaccination for melanoma was introduced because melanoma carries distinct tumor-associated antigens. The purpose of this study was to investigate the efficacy and safety of DC vaccination for melanoma in Korea. MATERIALS AND METHODS: Five patients with stage IV and one with stage II were enrolled. Autologous monocyte-derived DCs (MoDCs) were cultured and pulsed with tumor-lysate, keyhole limpet hemocyanin, and cytokine cocktail for mature antigen-loaded DC. DC vaccination was repeated four times at 2-week intervals and 2-4×10⁷ DC were injected each time. RESULTS: Reduced tumor volume was observed by PET-CT in three patients after DC vaccination. Delayed type hypersensitivity responses against tumor antigen were induced in five patients. Tumor antigen-specific IFN-γ-producing peripheral blood mononuclear cells were detected with enzyme-linked immunosorbent spot in two patients. However, the overall clinical outcome showed disease progression in all patients. CONCLUSION: In this study, DC vaccination using tumor antigen-loaded, mature MoDCs led to tumor regression in individual melanoma patients. Further standardization of DC vaccination protocol is required to determine which parameters lead to better anti-tumor responses and clinical outcomes.ope

Induced Endothelial Cell-Integrated Liver Assembloids Promote Hepatic Maturation and Therapeutic Effect on Cholestatic Liver Fibrosis

The transplantation of pluripotent stem cell (PSC)-derived liver organoids has been studied to solve the current donor shortage. However, the differentiation of unintended cell populations, difficulty in generating multi-lineage organoids, and tumorigenicity of PSC-derived organoids are challenges. However, direct conversion technology has allowed for the generation lineage-restricted induced stem cells from somatic cells bypassing the pluripotent state, thereby eliminating tumorigenic risks. Here, liver assembloids (iHEAs) were generated by integrating induced endothelial cells (iECs) into the liver organoids (iHLOs) generated with induced hepatic stem cells (iHepSCs). Liver assembloids showed enhanced functional maturity compared to iHLOs in vitro and improved therapeutic effects on cholestatic liver fibrosis animals in vivo. Mechanistically, FN1 expressed from iECs led to the upregulation of Itg alpha 5/beta 1 and Hnf4 alpha in iHEAs and were correlated to the decreased expression of genes related to hepatic stellate cell activation such as Lox and Spp1 in the cholestatic liver fibrosis animals. In conclusion, our study demonstrates the possibility of generating transplantable iHEAs with directly converted cells, and our results evidence that integrating iECs allows iHEAs to have enhanced hepatic maturation compared to iHLOs

Development and Verification of Time-Series Deep Learning for Drug-Induced Liver Injury Detection in Patients Taking Angiotensin II Receptor Blockers: A Multicenter Distributed Research Network Approach

Objectives The objective of this study was to develop and validate a multicenter-based, multi-model, time-series deep learning model for predicting drug-induced liver injury (DILI) in patients taking angiotensin receptor blockers (ARBs). The study leveraged a national-level multicenter approach, utilizing electronic health records (EHRs) from six hospitals in Korea. Methods A retrospective cohort analysis was conducted using EHRs from six hospitals in Korea, comprising a total of 10,852 patients whose data were converted to the Common Data Model. The study assessed the incidence rate of DILI among patients taking ARBs and compared it to a control group. Temporal patterns of important variables were analyzed using an interpretable time-series model. Results The overall incidence rate of DILI among patients taking ARBs was found to be 1.09%. The incidence rates varied for each specific ARB drug and institution, with valsartan having the highest rate (1.24%) and olmesartan having the lowest rate (0.83%). The DILI prediction models showed varying performance, measured by the average area under the receiver operating characteristic curve, with telmisartan (0.93), losartan (0.92), and irbesartan (0.90) exhibiting higher classification performance. The aggregated attention scores from the models highlighted the importance of variables such as hematocrit, albumin, prothrombin time, and lymphocytes in predicting DILI. Conclusions Implementing a multicenter-based time-series classification model provided evidence that could be valuable to clinicians regarding temporal patterns associated with DILI in ARB users. This information supports informed decisions regarding appropriate drug use and treatment strategies