In-silico and in-vitro investigation of STAT3-PIM1 heterodimeric complex: Its mechanism and inhibition by curcumin for cancer therapeutics.

The functional activity among STAT3 and PIM1, are key signaling events for cancer cell function. Curcumin, a diarylheptanoid isolated from turmeric, effectively inhibits STAT3 signaling. Selectively, we attempted to address interactions of STAT3, PIM1 and Curcumin for therapeutic intervention using in silico and in vitro experimental approaches. Firstly, protein-protein interactions (PPI) between STAT3-PIM1 by molecular docking studies reflected salt bridges among Arg279 (STAT3)-Glu140 (PIM1) and Arg282 (STAT3)-Asp100 (PIM1), with a binding affinity of -38.6 kcal/mol. Secondly, molecular dynamics simulations of heterodimeric STAT3-PIM1 complex with curcumin revealed binding of curcumin on PIM-1 interface of the complex through hydrogen bonds (Asp155) and hydrophobic interactions (Leu13, Phe18, Val21, etc.) with a binding energy of -7.3 kcal/mol. These PPIs were confirmed in vitro by immunoprecipitation assays in MDA-MB-231 cells. Corroborating our results, expression levels of STAT3 and PIM1 decreased after curcumin treatment. We observed that PIM1 interacts with STAT3 and these functional interactions are disrupted by curcumin. The calculated band energy gap of heterodimeric STAT3-PIM1-Curcumin complex was of 9.621 kcal/mol. The present study revealed the role of curcumin in STAT3/PIM1 signaling and its binding affinity to the complex for design of advanced cancer therapeutics.

The steroidal lactone withaferin A impedes T-cell motility by inhibiting the kinase ZAP70 and subsequent kinome signaling.

The steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera. It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found that WFA dose-dependently (within the concentration range of 0.3-1.25 µM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhesion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome analysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, suggesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and impedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologies.

GSK3ß Interacts With CRMP2 and Notch1 and Controls T-Cell Motility.

The trafficking of T-cells through peripheral tissues and into afferent lymphatic vessels is essential for immune surveillance and an adaptive immune response. Glycogen synthase kinase 3ß (GSK3ß) is a serine/threonine kinase and regulates numerous cell/tissue-specific functions, including cell survival, metabolism, and differentiation. Here, we report a crucial involvement of GSK3ß in T-cell motility. Inhibition of GSK3ß by CHIR-99021 or siRNA-mediated knockdown augmented the migratory behavior of human T-lymphocytes stimulated via an engagement of the T-cell integrin LFA-1 with its ligand ICAM-1. Proteomics and protein network analysis revealed ongoing interactions among GSK3ß, the surface receptor Notch1 and the cytoskeletal regulator CRMP2. LFA-1 stimulation in T-cells reduced Notch1-dependent GSK3ß activity by inducing phosphorylation at Ser9 and its nuclear translocation accompanied by the cleaved Notch1 intracellular domain and decreased GSK3ß-CRMP2 association. LFA-1-induced or pharmacologic inhibition of GSK3ß in T-cells diminished CRMP2 phosphorylation at Thr514. Although substantial amounts of CRMP2 were localized to the microtubule-organizing center in resting T-cells, this colocalization of CRMP2 was lost following LFA-1 stimulation. Moreover, the migratory advantage conferred by GSK3ß inhibition in T-cells by CHIR-99021 was lost when CRMP2 expression was knocked-down by siRNA-induced gene silencing. We therefore conclude that GSK3ß controls T-cell motility through interactions with CRMP2 and Notch1, which has important implications in adaptive immunity, T-cell mediated diseases and LFA-1-targeted therapies.

Targeted Gene Silencing in Malignant Hematolymphoid Cells Using GapmeR.

Delivery of conventional antisense oligonucleotides or small interfering RNA (siRNA) molecules into hematolymphoid cells for targeted gene silencing has been proven to be difficult. Here, we describe a simple protocol to knockdown specific gene(s) in malignant hematolymphoid cells using "GapmeR." This protocol could be applicable to a wide range of cell-types and thus solves an important problem for researchers working with cell lines or primary cells derived from patients with hematolymphoid malignancies.

Combination Therapy Using Inhalable GapmeR and Recombinant ACE2 for COVID-19.

Here we report our perspective on applying GapmeR technology in combination with recombinant angiotensin-converting enzyme 2 (ACE2) in the treatment of COVID-19 patients. GapmeR is a cell-permeating antisense single-stranded DNA molecule that can be designed to specifically target intracellular severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Once internalized into host cells, such as lung alveolar cells, GapmeR molecules can bind to the viral RNA. This RNA/DNA hybrid will then be degraded by the RNase H enzyme abundantly present in the host cells. GapmeRs can be delivered to COVID-19 patients through inhalation or via nebulization. SARS-CoV-2-targeted GapmeR can also be given to frontline healthcare workers as a prophylactic protection. The recombinant ACE2 protein, the efficacy of which is being evaluated in clinical trials, will bind to the spike (S) glycoprotein of extracellular SARS-CoV-2 and potentially block viral infectivity. We propose that combining inhalable SARS-CoV-2-targeted GapmeRs with recombinant ACE2 could provide a viable and rapidly implementable more effective therapeutic approach for eradicating SARS-CoV-2 and save millions of lives.

Multifunctional Antimicrobial Nanofiber Dressings Containing ε-Polylysine for the Eradication of Bacterial Bioburden and Promotion of Wound Healing in Critically Colonized Wounds.

Bacterial colonization of acute and chronic wounds is often associated with delayed wound healing and prolonged hospitalization. The rise of multi-drug resistant bacteria and the poor biocompatibility of topical antimicrobials warrant safe and effective antimicrobials. Antimicrobial agents that target microbial membranes without interfering with the mammalian cell proliferation and migration hold great promise in the treatment of traumatic wounds. This article reports the utility of superhydrophilic electrospun gelatin nanofiber dressings (NFDs) containing a broad-spectrum antimicrobial polymer, ε-polylysine (εPL), crosslinked by polydopamine (pDA) for treating second-degree burns. In a porcine model of partial thickness burns, NFDs promoted wound closure and reduced hypertrophic scarring compared to untreated burns. Analysis of NFDs in contact with the burns indicated that the dressings trap early colonizers and elicit bactericidal activity, thus creating a sterile wound bed for fibroblasts migration and re-epithelialization. In support of these observations, in porcine models of Pseudomonas aeruginosa and Staphylococcus aureus colonized partial thickness burns, NFDs decreased bacterial bioburden and promoted wound closure and re-epithelialization. NFDs displayed superior clinical outcome than standard-of-care silver dressings. The excellent biocompatibility and antimicrobial efficacy of the newly developed dressings in pre-clinical models demonstrate its potential for clinical use to manage infected wounds without compromising tissue regeneration.

SnAP reagents for the synthesis of selenomorpholines and 1,4-selenazepanes and their biological evaluation.

Herein, we disclose the first set of unique selenium-containing SnAP reagents for the direct synthesis of C-substituted selenomorpholines and 1,4-selenazepanes, including their amino acid derivatives from commercially available aldehydes under mild conditions. These elusive N-unprotected heterocycles are not accessible by classical routes. Biological evaluation of these compounds revealed promising activities against clinically relevant fungal strains.

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. Here, we demonstrate that GGL27, a TFPI-1-derived novel cationic host defence peptide, facilitates the delivery of nucleic acid cargo into the cytosol of a range of mammalian cells. The GGL27 peptide is non-cytotoxic and is internalized in a broad range of mammalian cell-types, including transformed cell lines and primary cells. GGL27 spontaneously forms complexes with nucleic acids of variable sizes, protects them from nuclease degradation, and delivers cargo effectively. Together, our observations demonstrate the versatile cell-penetrating property of GGL27, providing an excellent template for developing a simple, non-toxic peptide-based cytosolic delivery tool for wide use in biomedical research.

Protective Action of Linear Polyethylenimine against Staphylococcus aureus Colonization and Exaggerated Inflammation in Vitro and in Vivo.

Increased evolution of multidrug resistant pathogens necessitates the development of multifunctional antimicrobials. There is a perceived need for developing new antimicrobials that can interfere with acute inflammation after bacterial infections. Here, we investigated the therapeutic potential of linear polyethylenimine (LPEI) in vitro and in vivo. The minimum inhibitory concentration of LPEI ranged from 8 to 32 µg/mL and elicited rapid bactericidal activity against clinical isolates of meticillin-resistant Staphylococcus aureus (MRSA). The polymer was biocompatible for human cultured ocular and dermal cells. Prophylactic addition of LPEI inhibited the bacterial colonization of human primary dermal fibroblasts (hDFs). In a scratch wound cell migration assay, LPEI attenuated the migration inhibitory effects of bacterial secretions. The polymer neutralized the cytokine release by hDFs exposed to bacterial secretions, possibly by blocking their accessibility to host cell receptors. Topical instillation of LPEI (1 mg/mL) was noncytotoxic and did not affect the re-epithelialization of injured porcine cornea. In a prophylactic in vivo model of S. aureus keratitis, LPEI was superior to gatifloxacin in terms of reducing stimulation of cytokines, corneal edema, and overall severity of the infection. These observations demonstrate therapeutic potential of LPEI for antimicrobial prophylaxis.

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.

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 αLß2 integrin receptor LFA-1, which interacts primarily with its cognate ligand ICAM-1 expressed on the endothelium. This interaction triggers a plethora of downstream signaling pathways, which are not fully understood. Thus, in order to dissect the signal transduction processes at molecular levels and phenotypic changes in migrating T-cells, a laboratory model mimicking T-cell motility is important. Here, we describe a simple and highly reproducible in vitro model to study T-cell migration.

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. Here, we describe the use of novel cell-permeating antisense molecules, called "GapmeR", to knockdown specific gene(s) in human primary T-cells.

Profiling Activity of Cellular Kinases in Migrating T-Cells.

T-Lymphocyte kinases are important checkpoints that control T-cell motility by regulating a diverse range of signal transduction pathways. The distinct configuration of kinase events in T-cell could be used to fingerprint the status of T-cells. However, only small fraction human kinases have been characterized so far and little is known about the dynamics of the kinome in motile T-cells. Although several direct and indirect strategies exist to characterize cellular kinase activities, such as RNA interference, antibody arrays, enzyme kinetics, and mass spectrometry, this chapter focuses on an alternative multiplex phosphopeptide array-based methodology, which allows the kinome-wide identification of hyper-activated kinases involved in the regulation of T-cell migration.

A Protocol to Study T-Cell Signaling in an Immune Synapse by Microscopy.

The immune synapse is a complex cellular structure that enables cell-cell communications between immune cells, mainly at the interface of an effector T-cell and an antigen-presenting cell (APC) that expresses the appropriate peptide-MHC complexes. With progressive technological advances, there has been increasing interest in understanding molecular events that take place in motile T-lymphocytes at the immune synapse. Here, we provide an optimized method to induce the formation of an immune synapse between a T-cell and an APC in vitro. The experimental protocol described herein would be useful in characterizing the role of cell surface molecules and downstream signaling pathways in the context of cell-to-cell communications between T-cells and APCs.

Computational Analysis of Protein-Protein Interactions in Motile T-Cells.

Analysis of protein-protein interactions is important for better understanding of molecular mechanisms involved in immune regulation and has potential for elaborating avenues for drug discovery targeting T-cell motility. Currently, only a small fraction of protein-protein interactions have been characterized in T-lymphocytes although there are several detection methods available. In this regard, computational approaches garner importance, with the continued explosion of genomic and proteomic data, for handling protein modeling and protein-protein interactions in large scale. Here, we describe a computational method to identify protein-protein interactions based on in silico protein design.

Antimicrobial Activity and Cell Selectivity of Synthetic and Biosynthetic Cationic Polymers.

The mammalian and microbial cell selectivity of synthetic and biosynthetic cationic polymers has been investigated. Among the polymers with peptide backbones, polymers containing amino side chains display greater antimicrobial activity than those with guanidine side chains, whereas ethylenimines display superior activity over allylamines. The biosynthetic polymer ε-polylysine (εPL) is noncytotoxic to primary human dermal fibroblasts at concentrations of up to 2,000 µg/ml, suggesting that the presence of an isopeptide backbone has greater cell selectivity than the presence of α-peptide backbones. Both εPL and linear polyethylenimine (LPEI) exhibit bactericidal properties by depolarizing the cytoplasmic membrane and disrupt preformed biofilms. εPL displays broad-spectrum antimicrobial properties against antibiotic-resistant Gram-negative and Gram-positive strains and fungi. εPL elicits rapid bactericidal activity against both Gram-negative and Gram-positive bacteria, and its biocompatibility index is superior to those of cationic antiseptic agents and LPEI. εPL does not interfere with the wound closure of injured rabbit corneas. In a rabbit model of bacterial keratitis, the topical application of εPL (0.3%, wt/vol) decreases the bacterial burden and severity of infections caused by Pseudomonas aeruginosa and Staphylococcus aureus strains. In vivo imaging studies confirm that εPL-treated corneas appeared transparent and nonedematous compared to untreated infected corneas. Taken together, our results highlight the potential of εPL in resolving topical microbial infections.

Bio-inspired crosslinking and matrix-drug interactions for advanced wound dressings with long-term antimicrobial activity.

There is a growing demand for durable advanced wound dressings for the management of persistent infections after deep burn injuries. Herein, we demonstrated the preparation of durable antimicrobial nanofiber mats, by taking advantage of strong interfacial interactions between polyhydroxy antibiotics (with varying number of OH groups) and gelatin and their in-situ crosslinking with polydopamine (pDA) using ammonium carbonate diffusion method. Polydopamine crosslinking did not interfere with the antimicrobial efficacy of the loaded antibiotics. Interestingly, incorporation of antibiotics containing more number of alcoholic OH groups (NOH ≥ 5) delayed the release kinetics with complete retention of antimicrobial activity for an extended period of time (20 days). The antimicrobials-loaded mats displayed superior mechanical and thermal properties than gelatin or pDA-crosslinked gelatin mats. Mats containing polyhydroxy antifungals showed enhanced aqueous stability and retained nanofibrous morphology under aqueous environment for more than 4 weeks. This approach can be expanded to produce mats with broad spectrum antimicrobial properties by incorporating the combination of antibacterial and antifungal drugs. Direct electrospinning of vancomycin-loaded electrospun nanofibers onto a bandage gauze and subsequent crosslinking produced non-adherent durable advanced wound dressings that could be easily applied to the injured sites and readily detached after treatment. In a partial thickness burn injury model in piglets, the drug-loaded mats displayed comparable wound closure to commercially available silver-based dressings. This prototype wound dressing designed for easy handling and with long-lasting antimicrobial properties represents an effective option for treating life-threatening microbial infections due to thermal injuries.

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. Here, we show that GapmeR internalizes into human primary T-cells through macropinocytosis. Internalized GapmeR molecules can associate with SNX5-positive macropinosomes in T-cells, as detected by super-resolution microscopy. Utilizing the intrinsic self-internalizing capability of GapmeR, we demonstrate significant and specific depletion (>70%) of the expression of 5 different endogenous proteins with varying molecular weights (18 kDa Stathmin, 80 kDa PKCε, 180 kDa CD11a, 220 kDa Talin1 and 450 kDa CG-NAP/AKAP450) in human primary and cultured T-cells. Further functional analysis confirms CG-NAP and Stathmin as regulators of T-cell motility. Thus, in addition to screening, identifying or verifying critical roles of various proteins in T-cell functioning, this study provides novel opportunities to silence individual or multiple genes in a subset of purified human primary T-cells that would be exploited as future therapeutics.

Latent Oxidative Polymerization of Catecholamines as Potential Cross-linkers for Biocompatible and Multifunctional Biopolymer Scaffolds.

Electrospinning of naturally occurring biopolymers for biological applications requires postspinning cross-linking for endurance in protease-rich microenvironments and prevention of rapid dissolution. The most commonly used cross-linkers often generate cytotoxic byproducts, which necessitate high concentrations or time-consuming procedures. Herein, we report the addition of "safe" catecholamine cross-linkers to collagen or gelatin dope solutions followed by electrospinning yielded junction-containing nanofibrous mats. Subsequent in situ oxidative polymerization of the catecholamines increased the density of soldered junctions and maintained the porous nanofiber architecture. This protocol imparted photoluminescence to the biopolymers, a smooth noncytotoxic coating, and good mechanical/structural stability in aqueous solutions. The utility of our approach was demonstrated by the preparation of durable antimicrobial wound dressings and mineralized osteoconductive scaffolds via peptide antibiotics and calcium chloride (CaCl2) incorporation into the dope solutions. The mineralized composite mats consist of amorphous calcium carbonate that enhanced the osteoblasts cell proliferation, differentiation, and expression of important osteogenic marker proteins. In proof-of-concept experiments, antibiotic-loaded mats displayed superior antimicrobial properties relative to silver (Ag)-based dressings, and accelerated wound healing in a porcine deep dermal burn injury model.

Insight into membrane selectivity of linear and branched polyethylenimines and their potential as biocides for advanced wound dressings.

UNLABELLED: We report here structure-property relationship between linear and branched polyethylene imines by examining their antimicrobial activities against wide range of pathogens. Both the polymers target the cytoplasmic membrane of bacteria and yeasts, eliciting rapid microbicidal properties. Using multiscale molecular dynamic simulations, we showed that, in both fully or partially protonated forms LPEI discriminates between mammalian and bacterial model membranes whereas BPEI lacks selectivity for both the model membranes. Simulation results suggest that LPEI forms weak complex with the zwitterionic lipids whereas the side chain amino groups of BPEI sequester the zwitterionic lipids by forming tight complex. Consistent with these observations, label-free cell impedance measurements, cell viability assays and high content analysis indicate that BPEI is cytotoxic to human epithelial and fibroblasts cells. Crosslinking of BPEI onto electrospun gelatin mats attenuate the cytotoxicity for fibroblasts while retaining the antimicrobial activity against Gram-positive and yeasts strains. PEI crosslinked gelatin mats elicit bactericidal activity by contact-mediated killing and durable to leaching for 7days. The potent antimicrobial activity combined with enhanced selectivity of the crosslinked ES gelatin mats would expand the arsenel of biocides in the management of superficial skin infections. The contact-mediated microbicidal properties may avert antimicrobial resistance and expand the diversity of applications to prevent microbial contamination. STATEMENT OF SIGNIFICANCE: Current commercially available advanced wound dressings are either impregnated with metallic silver or silver salts which have side effects or may not avert antimicrobial resistance. In this article, we have used multidisciplinary approach comprising of computational, chemical and biological methods to understand the antimicrobial properties and biocompatibility of linear (LPEI) and branched (BPEI) polyethylenimines. We then applied this knowledge to develop dual purpose wound dressings containing these polymers, which encourages healing while maintain antimicrobial activity. In addition, the approach can be expanded to rationalize the antimicrobial vs. cytotoxicity of other cationic polymers and the method of crosslinking would enhance their potentials as biocides for advanced materials.