Molecular cloning, overexpression, characterization, and In silico modelling analysis of a novel GDSL autotransporter-dependent outer membrane lipase (OML) of Pseudomonas guariconensis.

The outer membrane lipase (oml) gene, encoding a novel autotransporter-dependent lipase from Pseudomonas guariconensis, was cloned and sequenced. The oml gene has an open reading frame of 1866 bp. It encodes the 621 amino acid autotransporter-dependent GDSL lipase (OML), which has the highest sequence similarity (64.08 %) with the EstA of Pseudomonas aeruginosa (PDB:3kvn.1. A). OML was expressed and purified, which showed a purified band of approximately 70 kDa. The purified enzyme showed maximum activity at pH 9 and 40 °C. Substrate specificity studies and kinetic study by Lineweaver-Burk plot of purified OML showed Km of 1.27 mM and Vmax of 333.33 U/mL with p-nitrophenyl palmitate. The purified enzyme showed good stability in the presence of hexane, methanol, and ethanol, while the presence of the metal ion Mg2+ showed maximum lipase activity. Bioinformatics analysis supported the in vitro findings by predicting enzyme substrate specificity towards long-chain fatty acids and fatty acids with shorter chain lengths. The stability of the interaction of the protein-ligand complex (OML-ricinoleic acid) was confirmed using MDS and castor oil bioconversion using purified OML was confirmed using High-Performance Liquid Chromatography (HPLC).

Inhibition of Listeria Monocytogenes HtrA Protease with Camostat, Gabexate and Nafamostat Mesylates and the Binding Mode of the Inhibitors.

In many bacteria, the High Temperature requirement A (HtrA) protein functions as a chaperone and protease. HtrA is an important factor in stress tolerance and plays a significant role in the virulence of several pathogenic bacteria. Camostat, gabexate and nafamostat mesylates are serine protease inhibitors and have recently shown a great impact in the inhibition studies of SARS-CoV2. In this study, the inhibition of Listeria monocytogenes HtrA (LmHtrA) protease activity was analysed using these three inhibitors. The cleavage assay, using human fibrinogen and casein as substrates, revealed that the three inhibitors effectively inhibit the protease activity of LmHtrA. The agar plate assay and spectrophotometric analysis concluded that the inhibition of nafamostat (IC50 value of 6.6 ± 0.4 µM) is more effective compared to the other two inhibitors. Previous studies revealed that at the active site of the protease, these inhibitors are hydrolysed and one of the hydrolysates is covalently bound to the active site serine. To understand the mode of binding of these inhibitors at the active site of LmHtrA, docking of the inhibitors followed by molecular dynamics simulations were carried out. Analysis of the LmHtrA-inhibitor complex structures revealed that the covalently bound inhibitor is unable to occupy the S1 pocket of the LmHtrA which is in contrast to the previously determined camostat and nafamostat complex structures. This observation provides the first glimpse of the substrate specificity of LmHtrA which is not known. The obtained results also suggest that the development of novel inhibitors of LmHtrA and its homologs with active site architecture similar to LmHtrA can be pursued with suitable modification of these inhibitors. To date, only a very few studies have been carried out on identifying the inhibitors of HtrA proteolytic activity.

Ocular surface sphingolipids associate with the refractory nature of vernal keratoconjunctivitis: newer insights in VKC pathogenesis.

BACKGROUND: The etiopathogenesis of vernal keratoconjunctivitis (VKC) is incompletely understood. Bioactive lipids play a key role in allergic disorders. This study focused on the sphingolipid metabolism on the ocular surface of VKC and to explore if it has a contributory role in the refractoriness of the disease. METHODS: Active VKC cases, (n=87) (classified as mild/moderate and severe/very severe based on the disease symptoms) and age-matched healthy controls (n=60) were recruited as part of a 2-year prospective study at a tertiary eye care centre in South India. Conjunctival imprint cytology was used to assess gene expression of enzymes of sphingolipids metabolism. Sphingolipids were estimated in the tears by LC-MS/MS analysis. In vitro study was done to assess IgE-induced alterations in sphingosine-1-phosphate (S1P) receptor expression and histone modification in cultured mast cells. RESULTS: Significantly altered gene expression of the sphingolipids enzymes and S1P receptor (SIP3R) were observed in conjunctival imprint cells of VKC cases. Pooled tears analysis revealed significantly lowered levels of S1P(d17:0), S1P(d20:1) (p<0.001) and S1P(d17:1) (p<0.01) specifically in severe/very severe VKC compared with both mild/moderate VKC and control. Cer(d18:/17:0) (p<0.001), ceramide-1-phosphate (C1P)(d18:1/8:0) (p<0.01) and C1P(d18:1/2.0 (p<0.05) were lowered in severe/very severe VKC compared with mild/moderate VKC. Cultured mast cells treated with IgE revealed significantly increased gene expression of S1P1 and 3 receptors and the protein expression of histone deacetylases (1, 6). CONCLUSION: Altered sphingolipid metabolism in the ocular surface results in low tear ceramide and sphingosine levels in severe/very severe VKC compared with the mild/moderate cases. The novel finding opens up fresh targets for intervention in these refractory cases.

In silico analysis of nitrilase-3 protein from Corynebacterium glutamicum for bioremediation of nitrile herbicides.

BACKGROUND: The nitrile compounds are produced either naturally or synthetically and are highly used in many manufacturing industries such as pharmaceuticals, pesticides, chemicals, and polymers. However, the extensive use and accumulation of these nitrile compounds have caused severe environmental pollution. Nitrilated herbicides are one such toxic substance that will persist in the soil for a long time. Therefore, effective measures must be taken to avoid its pollution to the environment. A variety of nitrile-converting bacterial species have the ability to convert these toxic substances into less toxic ones by using enzymatic processes. Among the bacterial groups, actinobacteria family members show good degradation capacity on these pollutants. The soil-dwelling Gram-positive industrial microbe Corynebacterium glutamicum is one such family member and its nitrile-degradation pathway is not well studied yet. In order to understand the effectiveness of using C. glutamicum for the degradation of such nitrile herbicides, an in silico approach has been done. In this perspective, this work focus on the structural analysis and molecular docking studies of C. glutamicum with nitrilated herbicides such as dichlobenil, bromoxynil, and chloroxynil. RESULTS: The bioinformatics analysis using different tools and software helped to confirm that the genome of C. glutamicum ATCC 13032 species have genes (cg 3093) codes for carbon-nitrogen hydrolase enzyme, which specifically act on non-peptide bond present in the nitrile compounds. The conserved domain analysis indicated that this protein sequence was nitrilase-3 and comes under the nitrilase superfamily. The multiple sequence alignment analysis confirmed that the conserved catalytic triad residues were 40E, 115K, and 151C, and the existence of nitrilase-3 protein in the genome of Corynebacterium sp. was evaluated by a phylogenetic tree. The analysis of physico-chemical properties revealed that alanine is the most abounded amino acid (10.20%) in the nitrilase-3 protein, and these properties influence the substrate specificity of aliphatic and aromatic nitrile compounds. The homology modelled protein showed better affinity towards nitrile herbicides such as 2,6-dichlorobenzamide (BAM) and 3,5-dichloro-4-hydroxy-benzamide (CIAM) with the affinity value of - 5.8 and - 5.7 kcal/mol respectively. CONCLUSIONS: The in silico studies manifested that C. glutamicum ATCC 13032 is one of the promising strains for the bioremediation of nitrilated herbicides contaminated soil.

Development of a U-bent plastic optical fiber biosensor with plasmonic labels for the detection of chikungunya non-structural protein 3.

This study presents a novel plasmonic fiber optic sandwich immunobiosensor for the detection of chikungunya, an infectious mosquito-borne disease with chronic musculoskeletal pain and acute febrile illness, by exploiting non-structural protein 3 (CHIKV-nsP3) as a biomarker. A plasmonic sandwich immunoassay for CHIKV-nsP3 was realized on the surface of a compact U-bent plastic optical fiber (POF, 0.5 mm core diameter) with gold nanoparticles (AuNPs) as labels. The high evanescent wave absorbance (EWA) sensitivity of the U-bent probes allows the absorption of the light passing through the fiber by the AuNP labels, upon the formation of a sandwich immunocomplex of CHIKV-nsP3 on the core surface of the U-bent probe region. A simple optical set-up with a low-cost green LED and a photodetector on either end of the U-bent probe gave rise to a detection limit of 0.52 ng mL-1 (8.6 pM), and a linear range of 1-104 ng mL-1 with a sensitivity of 0.1043A530 nm/log(CnsP3). In addition, the plasmonic POF biosensor shows strong specificity towards the CHIKV-nsP3 analyte in comparison with Pf-HRP2, HIgG, and dengue whole virus. The results illustrate the potential of plasmonic POF biosensors for direct and sensitive point-of-care detection of the chikungunya viral disease.

Patch Test Results of 276 Cases with Footwear Dermatitis - A Retrospective Study from a Tertiary Care Centre in South India.

BACKGROUND: Allergic contact dermatitis (ACD) is a major cause for foot wear dermatitis. Patch testing is the standard investigation for diagnosis of ACD. Identification of the causative allergen and avoidance of the same is the most important for patient management. AIMS: This study was conducted to find the common allergens in footwear, causing ACD, by retrospective analysis of the data of patients who had undergone patch testing with footwear series (FWS), approved by the Contact and Occupational Dermatoses Forum of India. MATERIALS AND METHODS: A total of 276 cases with footwear dermatitis who underwent patch test with FWS using Finn chamber method were studied. Statistical analysis was done using statistical package for social sciences (SPSS) version 24. Data was described using frequency and percentages. P value of less than 0.05 was considered significant. RESULTS: In this study 101 (36.5%) patients had positive patch test to at least one allergen. Among this, 43 (15.6%) were positive for single allergen only and 58 (21.01%) patients had positive patch test reactions to multiple allergens. The most common allergens with positive patch test were black rubber mix, mercapto benzo thiazole, and thiuram mix. Patients with either a positive or negative patch test had no statistically significant difference in the history of atopy. The limitations of this study include the lack of patch testing with the patient's own footwears and lack of follow-up after informing patients regarding allergen avoidance. CONCLUSIONS: Patch test must be done for all foot eczema cases for early identification of the causative allergen and also to provide suitable alternatives.

Lithium as an adjunct to radioactive iodine for the treatment of hyperthyroidism: a systematic review and meta-analysis.

BACKGROUND: Radioactive iodine (RAI) is commonly used in the treatment of hyperthyroidism but is not uniformly successful. Lithium increases thyroidal iodine retention without reducing iodide uptake, increasing the radiation dose to the thyroid when administered with RAI. Although these actions suggest that adjuvant lithium may increase the efficacy of RAI, its role as an adjunct to RAI remains contentious. OBJECTIVE: To evaluate the safety and efficacy of adding lithium to RAI to treat hyperthyroidism. METHODS: Relevant studies were identified by a search of Medline and the Cochrane Central Register of Controlled Trials. To be included, a study had to be a controlled trial comparing the effect of RAI alone to RAI with lithium in the treatment of hyperthyroidism. Relevant data were extracted and meta-analyses were performed. RESULTS: Of the 75 identified studies, 6 met the inclusion criteria; 4 of these studies were interventional and 2 were observational trials. Meta-analysis of the observational trials (N = 851), both of which were retrospective cohort studies, showed significant improvement in the primary outcome (i.e., cure rate) with adjunctive lithium (odds ratio [OR], 1.92; 95% confidence interval [CI], 1.24 to 2.96). The combined interventional trials (N = 485) also showed an improvement in cure rate, but the difference did not reach statistical significance (OR, 1.28; 95% CI, 0.85 to 1.91). Adjunctive lithium reduced time to cure and blunted thyroid hormone excursions after RAI. Lithium-related side effects were infrequent and usually mild. CONCLUSION: The observational trials demonstrated significant improvement in the cure rate of hyperthyroidism when lithium is added to RAI. The improvements shown in the interventional trials did not reach statistical significance due to the effect of a single, large negative trial.

Macrophage activation syndrome following Acinetobacter baumannii sepsis.

Macrophage activation syndrome (MAS) is a systemic disorder with a high mortality, commonly associated with rheumatological conditions, but which can also occur as a complication of several infections. Here we present a case of MAS following Acinetobacter baumannii sepsis. Early institution of therapy with prednisolone, cyclosporine, colistin, and polymyxin resulted in a prompt clinical recovery. There are very few reported cases of Acinetobacter-related MAS that have been successfully treated.