Rational Substitution of ε-Lysine for α-Lysine Enhances the Cell and Membrane Selectivity of Pore-Forming Melittin.

Here, we present a rational approach that enhances the membrane selectivity of a prolific pore-forming peptide, melittin, based on experimental observations that the cationic polymer, ε-polylysine, disrupts bacterial membranes with greater affinity over mammalian cells when compared to poly-l-lysine and poly-d-lysine. We systematically replaced three α-lysine residues in melittin with ε-lysine residues and identified key residues that are important for cytotoxicity. We then assessed the antimicrobial properties of the modified peptides which carry two or three ε-lysyl residues.

CG-NAP/Kinase Interactions Fine-Tune T Cell Functions.

CG-NAP, also known as AKAP450, is an anchoring/adaptor protein that streamlines signal transduction in various cell types by localizing signaling proteins and enzymes with their substrates. Great efforts are being devoted to elucidating functional roles of this protein and associated macromolecular signaling complex. Increasing understanding of pathways involved in regulating T lymphocytes suggests that CG-NAP can facilitate dynamic interactions between kinases and their substrates and thus fine-tune T cell motility and effector functions.

A C-terminal peptide of TFPI-1 facilitates cytosolic delivery of nucleic acid cargo into mammalian cells.

Efficient intracellular nucleic acid delivery into mammalian cells remains a long-standing challenge owing to poor cell permeability and uptake of naked nucleic acids across the cell membrane and limited cargo stability. Conventional delivery methods have several drawbacks, such as cytotoxicity, limited cell-type applicability, low efficiency, hindrances that limit the potential of oligonucleotide delivery in functional genomics, therapeutics and diverse research applications. Thus, new approaches that are robust, safe, effective and valid across multiple cell types are much needed.

Combating Microbial Contamination with Robust Polymeric Nanofibers: Elemental Effect on the Mussel-Inspired Cross-Linking of Electrospun Gelatin.

Designing biocompatible nanofibrous mats capable of preventing microbial colonization from resident and nosocomial bacteria for an extended period remains an unmet clinical need. In the present work, we designed antibiotic free durable antimicrobial nanofiber mats by taking advantage of synergistic interactions between polydopamine (pDA) and metal ions with varying degree of antimicrobial properties (Ag, Mg, Ca, and Zn). Microscopic analysis showed successful pDA-mediated cross-linking of the gelatin nanofibers, which further improved by the inclusion of Ag, Mg, and Ca ions as supported by mechanical and thermal studies.

Enzyme-Linked Immunosorbent Assay for T-Cell Dependent Immunogenicity Assessment of Therapeutic Peptides.

Immunogenicity assessment of therapeutic peptides, proteins, oligonucleotides, and hybrid molecules, such as nucleopeptides, is a major aspect in understanding their safety and efficacy. Both T-cell independent and dependent immune reactions contribute to an immunogenic response against antigen, including secretion of cytokines and production of an antigen-specific antibody. Various assays exist for detecting and quantifying such immunogenic responses by human T-cells ex vivo or in mouse serum, which primarily include enzyme-linked immunosorbent assay (ELISA, direct and indirect), flow-cytometry and surface plasmon resonance (SPR).

Immunometabolomic Phenotyping of Motile T-Cells.

The immune system and its components defend our body against diverse pathogens and help in maintaining tissue homeostasis. Immune cells are highly dynamic in terms of their growth, migration, differentiation, and effector functions, and adopt diverse metabolic configurations to respond to varying immunological challenges. Growing body of evidence suggests that metabolic pathways fuel immune cells for their functioning, including T-cell migration to the site of infection.

GapmeR-Mediated Gene Silencing in Motile T-Cells.

Gene silencing is an important method to study gene functions in health and diseases. While there are various techniques that are applied to knockdown specific gene(s) of interest, they have certain limitations in application to T-lymphocytes. T-cells are "hard-to-transfect" cells and are recalcitrant to transfection reagents.

A Laboratory Model to Study T-Cell Motility.

Regulated migration of T-lymphocytes through high endothelial venules and secondary lymphoid organs is necessary for an adaptive immune response. Uncontrolled trafficking of T-cells is implicated in many pathological conditions, including autoimmune disorders, such as psoriasis and inflammatory bowel disease. T-cell migration is regulated mainly by the αLβ2 integrin receptor LFA-1, which interacts primarily with its cognate ligand ICAM-1 expressed on the endothelium.

Isolation of Human Peripheral Blood T-Lymphocytes.

Peripheral blood is the most common source of T-lymphocytes for in vitro culture. Here, we present a simple and standardized method for small- or large-scale isolation of viable T-lymphocytes and other mononuclear cells from fresh peripheral blood or buffy coat blood samples using the density gradient centrifugation. T-cells obtained using the protocol described here can be used for a variety of downstream analysis, including cellular, molecular, and functional assays.

Surface characteristics and antimicrobial properties of modified catheter surfaces by polypyrogallol and metal ions.

Catheter associated infections (CAIs) are the major cause of nosocomial infections leading to increased morbidity, mortality rates and economical loss. Though the antibiotic coated surface modified catheters are reported to be effective in preventing CAIs, presence of sub-lethal concentrations of antibiotics in long term instilled catheters poses a risk of development and spread of drug resistant microbial strains. Herein, we have developed an antibiotic-free alternative strategy to coat catheter surfaces using pyrogallol (PG) and metal ions (Ag/Mg).

Centrosome- and Golgi-Localized Protein Kinase N-Associated Protein Serves As a Docking Platform for Protein Kinase A Signaling and Microtubule Nucleation in Migrating T-Cells.

Centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP), also known as AKAP450, is a cytosolic scaffolding protein involved in the targeted positioning of multiple signaling molecules, which are critical for cellular functioning. Here, we show that CG-NAP is predominantly expressed in human primary T-lymphocytes, localizes in close proximity (<0.2 μm) with centrosomal and Golgi structures and serves as a docking platform for Protein Kinase A (PKA).

GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells.

Post-transcriptional gene silencing holds great promise in discovery research for addressing intricate biological questions and as therapeutics. While various gene silencing approaches, such as siRNA and CRISPR-Cas9 techniques, are available, these cannot be effectively applied to "hard-to-transfect" primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called "GapmeR", is an emerging new class of gene silencing molecule.

E50K-OPTN-induced retinal cell death involves the Rab GTPase-activating protein, TBC1D17 mediated block in autophagy.

The protein optineurin coded by OPTN gene is involved in several functions including regulation of endocytic trafficking, autophagy and signal transduction. Certain missense mutations in the gene OPTN cause normal tension glaucoma. A glaucoma-causing mutant of optineurin, E50K, induces death selectively in retinal cells.

A cataract-causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain.

Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.

M98K-OPTN induces transferrin receptor degradation and RAB12-mediated autophagic death in retinal ganglion cells.

Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN.

Optineurin mediates a negative regulation of Rab8 by the GTPase-activating protein TBC1D17.

Rab GTPases regulate various membrane trafficking pathways but the mechanisms by which GTPase-activating proteins recognise specific Rabs are not clear. Rab8 is involved in controlling several trafficking processes, including the trafficking of transferrin receptor from the early endosome to the recycling endosome. Here, we provide evidence to show that TBC1D17, a Rab GTPase-activating protein, through its catalytic activity, regulates Rab8-mediated endocytic trafficking of transferrin receptor.