Exploration of Antibiofilm and In Vivo Wound Healing Activity of p-Cymene-Loaded Gellan/PVA Nanofibers.

Wound dressings with outstanding biocompatibility, antimicrobial, and tissue regeneration activities are essential to manage emerging recalcitrant antifungal infections to speed up healing. In this study, we have engineered p-cymene-loaded gellan/PVA nanofibers using electrospinning. Morphological and physicochemical properties of the nanofibers were characterized using a multitude of techniques to validate the successful integration of p-cymene (p-cym). The fabricated nanomaterials exhibited strong antibiofilm activity against Candida albicans and Candida glabrata compared to pure p-cymene. In vitro biocompatibility assay demonstrated that nanofibers did not possess any cytotoxicity to the NIH3T3 cell lines. In vivo, full-thickness excision wound healing study showed that the nanofibers were able to heal skin lesions faster than the conventional clotrimazole gel in 24 days without forming any scar. These findings unraveled p-cymene-loaded gellan gum (GA)/poly(vinyl alcohol) (PVA) nanofibers as an effective biomaterial for cutaneous tissue regeneration.

Eucalyptol/ ß-cyclodextrin inclusion complex loaded gellan/PVA nanofibers as antifungal drug delivery system.

Fungal infections pose a serious threat to humankind due to the toxicity of conventional antifungal therapy and continuous emerging incidence of multidrug resistance. Essential oils fascinated researchers because of their broad antimicrobial activity and minimal cytotoxicity. However, hydrophobic, volatile and low water solubility of essential oils hinder their applications in pharmaceutical industries. Therefore, in this study we have loaded eucalyptol/ ß-cyclodextrin inclusion complex to gellan/polyvinyl alcohol nanofibers (EPNF) to eradicate Candida albicans and Candida glabrata biofilms. The electrospun nanofibers characterized by various physicochemical techniques and it was observed that EPNF possess highly hydrophilic surface property that facilitate rapid drug release. EPNF inhibited approximately 70% biofilm of C. albicans and C. glabrata. Time kill results depicted that eucalyptol (EPTL) encapsulation in the nanofibers prolonged its antifungal activity than the pure EPTL. Electron microscopy studies revealed that EPNF disrupted the cell surface of Candida. Collectively the current study suggested nanofiber encapsulation enhanced antibiofilm activity of eucalyptol and these nanoscale systems can serve as an alternative therapeutic strategy to treat fungal infections. Further, the developed nanofibrous materials can be applied as cost effective coating agent for biomedical implants.

Eugenol-acacia gum-based bifunctional nanofibers as a potent antifungal transdermal substitute.

Aim: Fungal biofilms interfere with the wound healing processes. Henceforth, the study aims to fabricate a biomaterial-based nano-scaffold with the dual functionalities of wound healing and antibiofilm activity. Methods: Nanofibers comprising acacia gum, polyvinyl alcohol and inclusion complex of eugenol in ß-cyclodextrin (EG-NF) were synthesized using electrospinning. Antibiofilm studies were performed on Candida species, and the wound-healing activity was evaluated through an in vivo excision wound rat model. Results: The EG-NF potentially eradicated the mature biofilm of Candida species and their clinical isolates. Further, EG-NF also enhanced the re-epithelization and speed of wound healing in in vivo rat experiments. Conclusion: The study established the bifunctional applications of eugenol nanofibers as a transdermal substitute with antifungal potency.

Dissecting the Therapeutic Potency of Antimicrobial Peptides Against Microbial Biofilms.

Microbial resistance to conventional therapeutics has become a significant threat to human society. Biofilms serve as the major virulence factor for the microorganisms by resisting the antibiotics and host innate immune system. Antimicrobial peptides (AMPs) have emerged as a potential alternative to conventional therapeutics due to their exceptional anti-biofilm and broad-spectrum antimicrobial property. Researchers have applied bioinformatics, genetic engineering, tissue culture, and drug delivery approaches to enhance the production and therapeutic efficacy of antimicrobial peptides. This review comprehensively describes the various aspects of AMPs with particular focus on their anti-biofilm potential. Other detailed information highlighted in this review includes different classes of AMPs, their mode of action, and anti-biofilm activity both alone and in synergy with other AMPs or conventional antibiotics. Further, challenges and opportunities of AMPs based drug delivery systems such as nano-formulations, polymeric micelles, and vesicles are also summarized.

Insights into interplay of immunopathophysiological events and molecular mechanistic cascades in psoriasis and its associated comorbidities.

Psoriasis is an inflammatory skin disease with complex pathogenesis and multiple etiological factors. Besides the essential role of autoreactive T cells and constellation of cytokines, the discovery of IL-23/Th17 axis as a central signaling pathway has unraveled the mechanism of accelerated inflammation in psoriasis. This has provided insights into psoriasis pathogenesis and revolutionized the development of effective biological therapies. Moreover, genome-wide association studies have identified several candidate genes and susceptibility loci associated with this disease. Although involvement of cellular innate and adaptive immune responses and dysregulation of immune cells have been implicated in psoriasis initiation and maintenance, there is still a lack of unifying mechanism for understanding the pathogenesis of this disease. Emerging evidence suggests that psoriasis is a high-mortality disease with additional burden of comorbidities, which adversely affects the treatment response and overall quality of life of patients. Furthermore, changing trends of psoriasis-associated comorbidities and shared patterns of genetic susceptibility, risk factors and pathophysiological mechanisms manifest psoriasis as a multifactorial systemic disease. This review highlights the recent progress in understanding the crucial role of different immune cells, proinflammatory cytokines and microRNAs in psoriasis pathogenesis. In addition, we comprehensively discuss the involvement of various complex signaling pathways and their interplay with immune cell markers to comprehend the underlying pathophysiological mechanism, which may lead to exploration of new therapeutic targets and development of novel treatment strategies to reduce the disastrous nature of psoriasis and associated comorbidities.

Cinnamaldehyde incorporated gellan/PVA electrospun nanofibers for eradicating Candida biofilm.

Immunocompromised patients encounter fungal infections more frequently than healthy individuals. Conventional drugs associated health risk and resistance, portrayed fungal infections as a global health problem. This issue needs to be answered immediately by designing a novel anti-fungal therapeutic agent. Phytoactive molecules based therapeutics are most suitable candidate due to their low cytotoxicity and minimal side effects to the host. In this study, cinnamaldehyde (CA), an FDA approved phytoactive molecule present in cinnamon essential oil was incorporated into gellan (GA)/poly vinyl alcohol (PVA) based electrospun nanofibers to resolve the issues like low water solubility, high volatility and irritant effect associated with CA and also to enhance its therapeutic applications. The drug encapsulation, morphology and physical properties of the synthesized CA nanofibers were evaluated by FESEM, AFM, TGA, FTIR and static water contact angle analysis. The average diameters of CA encapsulated GA/PVA nanofibers and GA/PVA nanofibers were recorded to be 278.5 ± 57.8 nm and 204.03 ± 39.14 nm, respectively. These nanofibers were evaluated for their anti-biofilm activity against Candida using XTT (2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium salt) reduction assay. Data demonstrated that CA encapsulated GA/PVA nanofibers can effectively eradicate 89.29% and 50.45% of Candida glabrata and Candida albicans biofilm respectively. CA encapsulated nanofibers exhibited brilliant antimicrobial property against Staphylococcus aureus and Pseudomonas aeruginosa. The cytotoxicity assay demonstrated that nanofibers loaded with CA have anticancer properties as it reduces cell viability of breast cancer cells (MCF-7) by 27.7%. These CA loaded GA/PVA (CA-GA/PVA) nanofibers could be used as novel wound dressing material and coatings on biomedical implants to eradicate biofilm.

Computational studies on receptor-ligand interactions between novel buffalo (Bubalus bubalis) nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants and muramyl dipeptide (MDP).

Nucleotide binding and oligomerization domain 2 (NOD2), a member of intracellular NOD-like receptors (NLRs) family, recognizes the bacterial peptidoglycan, muramyl dipeptide (MDP) and initiates host immune response. The precise ligand recognition mechanism of NOD2 has remained elusive, although studies have suggested leucine rich repeat (LRR) region of NOD2 as the possible binding site of MDP. In this study, we identified multiple transcripts of NOD2 gene in buffalo (buNOD2) and at least five LRR variants (buNOD2-LRRW (wild type), buNOD2-LRRV1-V4) were found to be expressed in buffalo peripheral blood mononuclear cells. The newly identified buNOD2 transcripts were shorter in lengths as a result of exon-skipping and frame-shift mutations. Among the variants, buNOD2-LRRW, V1, and V3 were expressed more frequently in the animals studied. A comparative receptor-ligand interaction study through modeling of variants, docking, and molecular dynamics simulation revealed that the binding affinity of buNOD2-LRRW towards MDP was greater than that of the shorter variants. The absence of a LRR segment in the buNOD2 variants had probably affected their affinity toward MDP. Notwithstanding a high homology among the variants, the amino acid residues that interact with MDP were located on different LRR motifs. The binding free energy calculation revealed that the amino acids Arg850(LRR4) and Glu932(LRR7) of buNOD2-LRRW, Lys810(LRR3) of buNOD2-LRRV1, and Lys830(LRR3) of buNOD2-LRRV3 largely contributed towards MDP recognition. The knowledge of MDP recognition and binding modes on buNOD2 variants could be useful to understand the regulation of NOD-mediated immune response as well as to develop next generation anti-inflammatory compounds.

Diversity, Antimicrobial Action and Structure-Activity Relationship of Buffalo Cathelicidins.

Cathelicidins are an ancient class of antimicrobial peptides (AMPs) with broad spectrum bactericidal activities. In this study, we investigated the diversity and biological activity of cathelicidins of buffalo, a species known for its disease resistance. A series of new homologs of cathelicidin4 (CATHL4), which were structurally diverse in their antimicrobial domain, was identified in buffalo. AMPs of newly identified buffalo CATHL4s (buCATHL4s) displayed potent antimicrobial activity against selected Gram positive (G+) and Gram negative (G-) bacteria. These peptides were prompt to disrupt the membrane integrity of bacteria and induced specific changes such as blebing, budding, and pore like structure formation on bacterial membrane. The peptides assumed different secondary structure conformations in aqueous and membrane-mimicking environments. Simulation studies suggested that the amphipathic design of buCATHL4 was crucial for water permeation following membrane disruption. A great diversity, broad-spectrum antimicrobial action, and ability to induce an inflammatory response indicated the pleiotropic role of cathelicidins in innate immunity of buffalo. This study suggests short buffalo cathelicidin peptides with potent bactericidal properties and low cytotoxicity have potential translational applications for the development of novel antibiotics and antimicrobial peptidomimetics.

Comparative genomic analysis of buffalo (Bubalus bubalis) NOD1 and NOD2 receptors and their functional role in in-vitro cellular immune response.

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo--a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.

Insight into buffalo (Bubalus bubalis) RIG1 and MDA5 receptors: a comparative study on dsRNA recognition and in-vitro antiviral response.

RIG1 and MDA5 have emerged as important intracellular innate pattern recognition receptors that recognize viral RNA and mediate cellular signals controlling Type I interferon (IFN-I) response. Buffalo RIG1 and MDA5 genes were investigated to understand the mechanism of receptor induced antiviral response. Sequence analysis revealed that RIG1 and MDA5 maintain a domain arrangement that is common in mammals. Critical binding site residues of the receptors are evolutionary conserved among mammals. Molecular dynamics simulations suggested that RIG1 and MDA5 follow a similar, if not identical, dsRNA binding pattern that has been previously reported in human. Moreover, binding free energy calculation revealed that MDA5 had a greater affinity towards dsRNA compared to RIG1. Constitutive expressions of RLR genes were ubiquitous in different tissues without being specific to immune organs. Poly I:C stimulation induced elevated expressions of IFN-ß and IFN-stimulated genes (ISGs) through interferon regulatory factors (IRFs) mediated pathway in buffalo foetal fibroblast cells. The present study provides crucial insights into the structure and function of RIG1 and MDA5 receptors in buffalo.