Predicting and Designing Epitope Ensemble Vaccines against HTLV-1.

The infection mechanism and pathogenicity of Human T-lymphotropic virus 1 (HTLV-1) are ambiguously known for hundreds of years. Our knowledge about this virus is recently emerging. The purpose of the study is to design a vaccine targeting the envelope glycoprotein, GP62, an outer membrane protein of HTLV-1 that has an increased number of epitope binding sites. Data collection, clustering and multiple sequence alignment of HTLV-1 glycoprotein B, variability analysis of envelope Glycoprotein GP62 of HTLV-1, population protection coverage, HLA-epitope binding prediction, and B-cell epitope prediction were performed to predict an effective vaccine. Among all the predicted peptides, ALQTGITLV and VPSSSTPL epitopes interact with three MHC alleles. The summative population protection coverage worldwide by these epitopes as vaccine candidates was found nearly 70%. The docking analysis revealed that ALQTGITLV and VPSSSTPL epitopes interact strongly with the epitope-binding groove of HLA-A*02:03, and HLA-B*35:01, respectively, as this HLA molecule was found common with which every predicted epitope interacts. Molecular dynamics simulations of the docked complexes show they form stable complexes. So, these potential epitopes might pave the way for vaccine development against HTLV-1.

Polymodal Mechanism for TWIK-Related K+ Channel Inhibition by Local Anesthetic.

BACKGROUND: Local anesthetics cause reversible block of pain and robustly inhibit TWIK-related K channel (TREK-1) currents. Before local anesthesia onset, injection of local anesthetics can cause unwanted transient pain. TREK-1 is an anesthetic-sensitive potassium channel that when inhibited produces pain. A disordered C-terminal loop of TREK-1 is thought to contribute to anesthetic sensitivity, but the molecular basis for TREK-1 inhibition by local anesthetics is unknown. Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA) required for TREK-1 activation and also binds to the channel's C terminus. METHODS: Here, we use biophysical and cellular techniques to characterize direct and indirect lipid-mediated mechanism for TREK-1 inhibition (respectively). We characterized direct binding of local anesthetic to TREK-1 by reconstituting the purified channel into artificial membranes and measuring ion flux. We characterized indirect PA-mediated inhibition of TREK-1 by monitoring lipid production in live whole cells using a fluorescent PLD2 product release assay and ion channel current using live whole-cell patch-clamp electrophysiology. We monitored anesthetic-induced nanoscale translocation of PLD2 to TREK-1 channels with super-resolution direct stochastic reconstruction microscopy (dSTORM). RESULTS: We find local anesthetics tetracaine, lidocaine, and bupivacaine directly bind to and inhibit PLD2 enzymatic activity. The lack of PLD2 activity indirectly inhibited TREK-1 currents. Select local anesthetics also partially blocked the open pore of TREK-1 through direct binding. The amount of pore block was variable with tetracaine greater than bupivacaine and lidocaine exhibiting a minor effect. Local anesthetics also disrupt lipid rafts, a mechanism that would normally activate PLD2 were it not for their direct inhibition of enzyme catalysis. CONCLUSIONS: We propose a mechanism of TREK-1 inhibition comprised of (1) primarily indirect PLD2-dependent inhibition of lipid catalysis and (2) limited direct inhibition for select local anesthetics through partial open pore block. The inhibition through PLD2 explains how the C terminus can regulate the channel despite being devoid of structure and putative binding sites for local anesthetics.

Detection of CXCR2 cytokine receptor surface expression using immunofluorescence.

The interleukin-8 (IL-8, CXCL8) chemokine, also known as the neutrophil chemotactic factor, is a cytokine that plays a key role in inflammatory response, cell proliferation, migration, and survival. IL-8 expression is increased not only in inflammatory disorders, but also in many types of cancer, including prostate cancer. IL-8 acts as a ligand for the C-X-C chemokine receptor 2 (CXCR2) protein present on the cell plasma membrane. Binding of the IL-8 ligand to the CXCR2 receptor results in an intracellular signaling pathway mediated by GTP binding proteins coupled to the receptor itself. Knowledge of the CXCR2 expression levels facilitates the understanding of the role and function of IL-8. In this chapter, we describe a protocol that uses the immunofluorescence method and confocal microscopy to analyze the CXCR2 surface expression in human prostate cancer cells. However, this protocol is easily adaptable to analyze the surface expression of other cytokine receptors in different cell types.