Fabricating a Partially Fluorinated Hybrid Cation-Exchange Membrane for Long Durable Performance of Vanadium Redox Flow Batteries.

The long-term durability of vanadium redox flow batteries (VRFBs) depends on the stability and performance of the membrane separator. We have architected a hybrid membrane by uniform dispersion of MIL-101(Cr) (Cr-MOF) in a partially fluorinated polymer grafted with sulfonic acid groups (PHP@AMPSCr-MOF(1.0)). The single cell VRFB performance of the PHP@AMPSCr-MOF(1.0) membrane was studied in comparison with the Cr-MOF incorporated Nafion membrane (NafionCr-MOF(1.0)) and showed an excellent result with 97.5% Coulombic efficiency (CE) at 150 mA/cm2 without any significant deterioration in the charge-discharge process for 1500 cycles (over 650 h). Meanwhile, the CE value of the NafionCr-MOF membrane (94.5%) deteriorated after 800 cycles (about 360 h) under similar conditions. The high VRFB performance of the PHP@AMPSCr-MOF(1.0) membrane has been attributed to the synergized properties and good interactions between Cr-MOF and partially fluorinated polymer matrix responsible for the creation of hydrophilic proton-conducting channels to achieve high selectivity. Furthermore, the cost-effective polymer and thus membranes may open new windows for practical applications in other energy devices such as fuel cells, electrolysis, and water treatment.

A rationally designed synthetic antimicrobial peptide against Pseudomonas-associated corneal keratitis: Structure-function correlation.

Contact lens wearers are at an increased risk of developing Pseudomonas-associated corneal keratitis, which can lead to a host of serious ocular complications. Despite the use of topical antibiotics, ocular infections remain a major clinical problem, and a strategy to avoid Pseudomonas-associated microbial keratitis is urgently required. The hybrid peptide VR18 (VARGWGRKCPLFGKNKSR) was designed to have enhanced antimicrobial properties in the fight against Pseudomonas-induced microbial keratitis, including contact lens-related keratitis. In this paper, VR18's modes of action against Pseudomonas membranes were shown by live cell Raman spectroscopy, live cell NMR, live-cell fluorescence microscopy and measures taken using sparsely tethered bilayer lipid membrane bacterial models to be via a bacterial-specific membrane disruption mechanism. The high affinity and selectivity of the peptide were then demonstrated using in vivo, in vitro and ex vivo models of Pseudomonas infection. The extensive data presented in this work suggests that topical employment of the VR18 peptide would be a potent therapeutic agent for the prevention or remedy of Pseudomonas-associated microbial keratitis.

Streptococcus pneumoniae exerts oxidative stress, subverts antioxidant signaling and autophagy in human corneal epithelial cells that is alleviated by tert-Butylhydroquinone.

Streptococcus pneumoniae is one of the leading causes of bacterial keratitis in the developing world and globally. In the current study, we have determined oxidative stress as pathogenesis of S. pneumoniae infection in corneal tissues and human corneal epithelial cells (HCEC) and explored host immune response of HCEC towards S. pneumoniae. We also determined whether treatment with tert-Butylhydroquinone (tBHQ), a Nrf2 inducer, could alleviate oxidative stress and reduce bacterial cytotoxicity in these cells. Oxidative stress was determined in corneal tissues of patients and HCEC by immunohistochemistry and immunofluorescence analysis, respectively. The expression of antioxidant genes, cytokines and antimicrobial peptides was determined by quantitative PCR. Infection of HCEC by S. pneumoniae was determined by colony-forming units. The autophagy and cell death were determined by fluorescence microscopy. The phosphorylation of signaling proteins was evaluated by immunoblot analysis. S. pneumoniae induced oxidative stress during corneal infections and inhibited antioxidant signaling pathways and immune responses like autophagy. tBHQ aided in restoring Nrf2 activation, reduced reactive oxygen species generation and prevented cytotoxicity and cell death in S. pneumoniae-infected HCEC. tBHQ also induced autophagy in a Nrf2-dependent manner and reduced bacterial survival in HCEC. Increased expression of antimicrobial peptides by tBHQ might have contributed to a reduction of bacterial load and cytotoxicity, as exemplified in LL-37 depleted corneal epithelial cells exposed to S. pneumoniae compared to control siRNA-transfected cells. tBHQ mediates alleviation of oxidative stress induced by S. pneumoniae by activating Nrf2-mediated antioxidant signaling in corneal epithelial cells. tBHQ also enhances expression of antimicrobial peptides in corneal cells and aids in inhibition of bacterial survival and cytotoxicity of HCEC.

Use of Induced Pluripotent Stem Cells to Build Isogenic Systems and Investigate Type 1 Diabetes.

Type 1 diabetes (T1D) is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of ß-cells by autoreactive CD8+ T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individual's collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and ß-cells. Additionally, we also engineered T cell avatars from the same donor to provide an in vitro platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g. IFIH1, PTPN22, SH2B3, TYK2) in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.

Alternative Therapeutic Interventions: Antimicrobial Peptides and Small Molecules to Treat Microbial Keratitis.

Microbial keratitis is a leading cause of blindness worldwide and results in unilateral vision loss in an estimated 2 million people per year. Bacteria and fungus are two main etiological agents that cause corneal ulcers. Although antibiotics and antifungals are commonly used to treat corneal infections, a clear trend with increasing resistance to these antimicrobials is emerging at rapid pace. Extensive research has been carried out to determine alternative therapeutic interventions, and antimicrobial peptides (AMPs) are increasingly recognized for their clinical potential in treating infections. Small molecules targeted against virulence factors of the pathogens and natural compounds are also explored to meet the challenges and growing demand for therapeutic agents. Here we review the potential of AMPs, small molecules, and natural compounds as alternative therapeutic interventions for the treatment of corneal infections to combat antimicrobial resistance. Additionally, we have also discussed about the different formats of drug delivery systems for optimal administration of drugs to treat microbial keratitis.

Recombinant IL-22 promotes protection in a murine model of Aspergillus flavus keratitis and mediates host immune responses in human corneal epithelial cells.

Aspergillus flavus is a leading cause of corneal infections in India and worldwide, resulting in severe visual impairment. We studied the host immune response towards A. flavus in immortalised human corneal epithelial cells (HCEC) and found increased expression of Toll-like receptors, antimicrobial peptides and proinflammatory cytokines like IL-6 and IL-8. Differential expressions of antimicrobial peptides were determined in corneal scrapings from A. flavus keratitis patients with significantly increased expression of LL-37, S100A12 and RNase 7. Increased levels of IL-22 expression were observed both in patients with A. flavus keratitis and in experimental mice model of corneal infections along with IL-17, IL-23 and IL-18. IL-22 is an important mediator of inflammation during microbial infections, and acts primarily on fibroblasts and epithelial cells. We observed constitutive expression of IL-22 receptors in HCEC, and IL-22 mediated activation of NF-κB, MAPK pathways and STAT3, along with increased expression of antimicrobial peptides in these cells. IL-22 also efficiently lessened cell deaths in corneal epithelial cells during A. flavus infection in vitro. Furthermore, recombinant IL-22 reduced fungal burden and corneal opacity in an experimental murine model of A. flavus keratitis.

Attenuation of Pseudomonas aeruginosa infection by INP0341, a salicylidene acylhydrazide, in a murine model of keratitis.

PSEUDOMONAS AERUGINOSA: is an opportunistic pathogen and a major cause of corneal infections worldwide. The bacterium secretes several toxins through its type III secretion system (T3SS) to subvert host immune responses. In addition, it is armed with intrinsic as well as acquired antibiotic resistance mechanisms that make treatment a significant challenge and new therapeutic interventions are needed. Type III secretion inhibitors have been studied as an alternative or in accompaniment to traditional antibiotics to inhibit virulence of bacteria. In this study, INP0341, a T3SS inhibitor, inhibited cytotoxicity by P. aeruginosa toward human corneal epithelial cells (HCEC) at 100 µM without affecting bacterial growth in the liquid media. An increased expression of antimicrobial peptides and reactive oxygen species generation was also observed in cells exposed to P. aeruginosa in the presence of INP0341. Furthermore, INP0341 efficiently attenuated corneal infection by P. aeruginosa in an experimental model of murine keratitis as evident from corneal opacity, clinical score and bacterial load. Thus, INP0341 appears to be a promising candidate to treat corneal infection caused by P. aeruginosa and can be further considered as an alternative therapeutic intervention.

Differential Expression of Antimicrobial Peptides in Streptococcus pneumoniae Keratitis and STAT3-Dependent Expression of LL-37 by Streptococcus pneumoniae in Human Corneal Epithelial Cells.

Streptococcus pneumoniae is the leading cause of bacterial keratitis in the developing world with a growing trend of acquiring resistance against various antibiotics. In the current study, we determined the expression of different antimicrobial peptides (AMPs) in response to S. pneumoniae in patients, as well as in primary and immortalized human corneal epithelial cells. We further focused on LL-37 and determined its expression in human cornea infected with S. pneumoniae and studied the killing ability of LL-37 against S. pneumoniae. The expression of AMPs was determined by quantitative PCR and the phosphorylation of signaling proteins was evaluated by immunoblot analysis. LL-37 expression was also determined by immunofluorescence and Western blot method and the killing ability of LL-37 against S. pneumoniae was determined by colony-forming units. Differential expression of antimicrobial peptides was observed in patients with S. pneumoniae keratitis. Although S. pneumoniae induced expression of the AMPs in human corneal epithelial cells (HCEC), it did not induce AMP expression in U937, a human monocyte cell line. S. pneumoniae also caused activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB)and mitogen activated protein kinase (MAPK) pathways in corneal epithelial cells. LL-37 was found to be effective against both laboratory and clinical strains of S. pneumoniae. LL-37 induction by S. pneumoniae in human corneal epithelial cells was mediated by signal transducer and activator of transcription 3 (STAT3) activation, and inhibition of STAT3 activation significantly reduced LL-37 expression. Our study determines an extensive profile of AMPs expressed in the human cornea during S. pneumoniae infection, and suggests the potential of LL-37 to be developed as an alternative therapeutic intervention to fight increasing antibiotic resistance among bacteria.

Differential expression of antimicrobial peptides in corneal infection and regulation of antimicrobial peptides and reactive oxygen species by type III secretion system of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen and is the major cause of corneal infection worldwide that secret several virulent toxins through its type III secretion system (T3SS). In defense against pathogenic insults, epithelial cells and macrophages express antimicrobial peptides (AMPs) that are essential components of host immune response. In this study, we have determined the expression of several AMPs in patients with P. aeruginosa corneal infection. We also used an in vitro model of infection using human corneal epithelial cells and macrophages to determine the gene expression of AMPs and cellular response to wild-type and T3SS mutant P. aeruginosa. We found differential expression of several AMPs in patient samples and also found that P. aeruginosa repress AMP expression in both epithelial cells and macrophages by its T3SS in vitro. It dampens AMP expression by causing delay in NF-κB, p38 and ERK activation and inhibits reactive oxygen species generation in these cells by its T3SS. Our study show the profile of AMPs expressed during P. aeruginosa keratitis and suggest the pivotal role of the T3SS in epithelial cells and macrophages during P. aeruginosa infection.