Cortisol Biosynthesis in the Human Ocular Surface Innate Immune Response

Innate immune responses have a critical role in regulating sight-threatening ocular surface (OcS) inflammation. While glucocorticoids (GCs) are frequently used to limit tissue damage, the role of intracrine GC (cortisol) bioavailability via 11-beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in OcS defense, remains unresolved. We found that primary human corneal epithelial cells (PHCEC), fibroblasts (PHKF) and allogeneic macrophages (M1, GM-CSF; M2, M-CSF) were capable of generating cortisol (M1>PHKF>M2>PHCEC) but in corneal cells, this was independent of Toll-like receptor (TLR) activation. While PolyI∶C induced maximal cytokine and chemokine production from both PHCEC (IFNγ, CCL2, CCL3, and (CCL4), IL6, CXCL10, CCL5, TNFα) and PHKF (CCL2, IL-6, CXCL10, CCL5), only PHKF cytokines were inhibited by GCs. Both Poly I∶C and LPS challenged-corneal cells induced M1 chemotaxis (greatest LPS-PHKF (250%), but down-regulated M1 11β-HSD1 activity (30 and 40% respectively). These data were supported by clinical studies demonstrating reduced human tear film cortisol∶cortisone ratios (a biomarker of local 11β-HSD1 activity) in pseudomonas keratitis (1∶2.9) versus healthy controls (1∶1.3; p<0.05). This contrasted with putative TLR3-mediated OcS disease (Stevens-Johnson Syndrome, Mucous membrane pemphigoid) where an increase in cortisol∶cortisone ratio was observed (113.8∶1; p<0.05). In summary, cortisol biosynthesis in human corneal cells is independent of TLR activation and is likely to afford immunoprotection under physiological conditions. Contribution to ocular mucosal innate responses is dependent on the aetiology of immunological challenge.     

Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

TnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a C. perfringens plc virR double mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, P_attCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.     

Molecular modeling of cornulin (CRNN) for docking with phytocompounds from Justicia adhatoda L.

Cornulin (CRNN) is linked with tumour progression. Therefore, it is of interest to document data on the molecular modeling of cornulin (CRNN) for docking with phytocompounds (Pyrazinamide, Anisotine, Vasicinone, Vasicoline) from Justicia adhatoda L. Thus, we document the optimal binding features of these compounds with the cornulin model for further consideration.     

Molecular docking analysis of SARS-CoV-2 linked RNA dependent RNA polymerase (RdRp) with compounds from Plectranthus amboinicus

It is of interest to document the moelcular docking analysis of SARS-CoV-2 linked RNA dependent RNA polymerase (RdRp) with compounds from Plectranthus amboinicus. Hence, we report the binding features of rutin, Luteolin, Salvianolic acid A, Rosmarinic acid and p-Coumaric acid with the target protein SARS-CoV-2 linked RNA dependent RNA polymerase (RdRp) for further consideration.     

ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H(2)O(2) have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H(2)O(2) in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H(2)O(2) production. Stomatal closure induced by H(2)O(2) is inhibited by the removal of NO with NO scavenger, and both ABA and H(2)O(2) stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H(2)O(2) synthesis nor does NO treatment induce H(2)O(2) production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H(2)O(2) and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H(2)O(2) synthesis. In the NR double mutant nia1, nia2 both ABA and H(2)O(2) fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H(2)O(2) are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H(2)O(2) production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H(2)O(2) and NO-induced stomatal closure.     

Discovery of N-substituted pyridinones as potent and selective inhibitors of p38 kinase

The identification and evolution of a series of potent and selective p38 inhibitors is described. p38 inhibitors based on a N-benzyl pyridinone high-throughput screening hit were prepared and their SAR explored. Their design was guided by ligand bound co-crystals of p38α. These efforts resulted in the identification of 12r and 19 as orally active inhibitors of p38 with significant efficacy in both acute and chronic models of inflammation.     

Lamellipodin, an Ena/VASP ligand, is implicated in the regulation of lamellipodial dynamics

Lamellipodial protrusion is regulated by Ena/VASP proteins. We identified Lamellipodin (Lpd) as an Ena/VASP binding protein. Both proteins colocalize at the tips of lamellipodia and filopodia. Lpd is recruited to EPEC and Vaccinia, pathogens that exploit the actin cytoskeleton for their own motility. Lpd contains a PH domain that binds specifically to PI(3,4)P2, an asymmetrically localized signal in chemotactic cells. Lpd's PH domain can localize to ruffles in PDGF-treated fibroblasts. Lpd overexpression increases lamellipodial protrusion velocity, an effect observed when Ena/VASP proteins are overexpressed or artificially targeted to the plasma membrane. Conversely, knockdown of Lpd expression impairs lamellipodia formation, reduces velocity of residual lamellipodial protrusion, and decreases F-actin content. These phenotypes are more severe than loss of Ena/VASP, suggesting that Lpd regulates other effectors of the actin cytoskeleton in addition to Ena/VASP.     

Molecular mapping of wilt resistance genes in chickpea

Fusarium wilt is a widespread and serious chickpea disease caused by the soil-borne fungus Fusarium oxysporum f.sp. ciceri (Foc). We evaluated an F₉ recombinant inbred line population of chickpea for resistance to three Foc races (1, 2 and 3) in pot culture experiments and identified flanking and tightly linked DNA markers for the resistance genes. The simple sequence repeat markers H3A12 and TA110 flanked the Foc1 locus at 3.9 and 2.1 cM, respectively, while Foc2 was mapped 0.2 cM from TA96 and 2.7 cM from H3A12. The H1B06y and TA194 markers flanked the Foc3 locus at 0.2 and 0.7 cM, respectively. These markers were also validated using 16 diverse chickpea genotypes. Identification of tightly linked flanking markers for wilt resistance genes will be useful for their exploitation in breeding programs and to understand the mechanism of resistance and evolution of the genes. S. J. M. Gowda and P. Radhika contributed equally to this study.     

Short-term exposure of multipotent stromal cells to low oxygen increases their expression of CX3CR1 and CXCR4 and their engraftment in vivo

The ability of stem/progenitor cells to migrate and engraft into host tissues is key to their potential use in gene and cell therapy. Among the cells of interest are the adherent cells from bone marrow, referred to as mesenchymal stem cells or multipotent stromal cells (MSC). Since the bone marrow environment is hypoxic, with oxygen tensions ranging from 1% to 7%, we decided to test whether hypoxia can upregulate chemokine receptors and enhance the ability of human MSCs to engraft in vivo. Short-term exposure of MSCs to 1% oxygen increased expression of the chemokine receptors CX3CR1and CXCR4, both as mRNA and as protein. After 1-day exposure to low oxygen, MSCs increased in vitro migration in response to the fractalkine and SDF-1alpha in a dose dependent manner. Blocking antibodies for the chemokine receptors significantly decreased the migration. Xenotypic grafting into early chick embryos demonstrated cells from hypoxic cultures engrafted more efficiently than cells from normoxic cultures and generated a variety of cell types in host tissues. The results suggest that short-term culture of MSCs under hypoxic conditions may provide a general method of enhancing their engraftment in vivo into a variety of tissues.