Laccases are blue copper oxidases (E.C. 1.10.3.2 benzenediol: oxygen oxidoreductase) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. They are currently seen as highly interesting industrial enzymes because of their broad substrate specificity. A positive strain was isolated and characterized as nonspore forming Basidiomycetes Pleurotus sp. Laccase activity was determined using ABTS as substrate. Laccase was purified by ionexchange and gel filtration chromatography. The purified laccase was a monomer showed a molecular mass of 40 ± 1 kDa as estimated by SDS-PAGE and a 72-fold purification with a 22% yield. The optimal pH and temperature were 4.5 and 65°C, respectively. The K m and V max values are 250 (mM) and 0.33 (μmol/min), respectively, for ABTS as substrate. Metal ions like CuSO4, BaCl2, MgCl2, FeCl2, ZnCl2 have no effect on purified laccase whereas HgCl2 and MnCl2 moderately decrease enzyme activity. SDS and sodium azide inhibited enzyme activity, whereas Urea, PCMB, DTT, and mercaptoethanol have no effect on enzyme activity. The isolated laccase can be used in development of biosensor for detecting the phenolic compounds from the effluents of paper industries.

Bronchopulmonary dysplasia (BPD) is a multifactorial chronic lung disease of premature infants. BPD can be attributed to the dysregulation of normal lung development due to ventilation and oxygen toxicity, resulting in pathologic complications of impaired alveolarization and vascularization. MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression posttranscriptionally and are implicated in diverse biological processes and diseases. The objectives of this study are to identify the changed miRNAs and their target genes in neonatal rat lungs in response to hyperoxia exposure. Using miRNA microarray and real-time PCR analyses, we found downregulation of five miRNAs, miR-342, miR-335, miR-150, miR-126*, and miR-151*, and upregulation of two miRNAs, miR-21 and miR-34a. Some of these miRNAs had the highest expression during embryonic and early postnatal development. DNA microarray analysis yielded several genes with conserved binding sites for these altered miRNAs. Glycoprotein nonmetastatic melanoma protein b (GPNMB) was experimentally verified as a target of miR-150. In summary, we identified seven miRNAs that were changed in hyperoxia-exposed neonatal lungs. These results provide a basis for deciphering the mechanisms involved in the spatial and temporal regulation of proteins that contribute to the pathogenesis of BPD.

Long non-coding RNAs (lncRNAs) are a new arm of gene regulatory mechanism as discovered by sequencing techniques and follow-up functional studies. There are only few studies on lncRNAs as related to gene expression regulation and anti-viral activity during influenza virus infection. We sought to identify and characterize lncRNAs involved in influenza virus replication. Using RNA sequencing analysis, we found that 1,912 lncRNAs were significantly changed in human lung epithelial A549 cells infected with influenza A/Puerto Rico/8/34. Gene ontology analysis on neighboring genes of these lncRNAs revealed that the genes involved in type I interferon signaling and cellular response were highly enriched. Seven selected up-regulated lncRNAs (AC015849.2, RP-1-7H24.1, PSMB8-AS1, CTD-2639E6.9, PSOR1C3, AC007283.5 and RP11-670E13.5) were verified by real-time PCR. These lncRNAs were also induced by other two influenza H1N1 virus strains (A/WSN/1933 and A/Oklahoma/3052/09) and interferon β1. Repression of PSMB8 antisense RNA 1 (PSMB8-AS1) using CRISPR interference reduced viral mRNA and protein levels as well as the release of progeny influenza virus particles. Our study suggests that lncRNA PSMB8-AS1 could be a new host factor target for developing antiviral therapy against influenza virus infection.

A soil screened Bacillus flexus XJU-1 was induced to simultaneously produce alkaline amylase, alkaline lipase and alkaline protease at their optimum levels on a common medium under submerged fermentation. The basal cultivation medium consisted of 0.5% casein, 0.5% starch and 0.5% cottonseedoil as an inducer forprotease, amylase, and lipase, respectively. The casein also served as nitrogen source for all 3 enzymes. The starch was also found to act as carbon source additive for both lipase and protease. Maximum enzyme production occurred on fermentation medium with 1.5% casein, 1.5% soluble starch, 2% cottonseed oil, 2% inoculum size, initial pH of 11.0, incubation temperature of 37 °C and 1% soybean meal as a nitrogen source supplement. The analysis of time course study showed that 24 h was optimum incubation time for amylase whereas 48 h was the best time for both lipase and protease. After optimization, a 3.36-, 18.64-, and 27.33-fold increase in protease, amylase and lipase, respectively was recorded. The lipase was produced in higher amounts (37.72 U/mL) than amylase and protease about 1.27 and 5.85 times, respectively. As the 3 enzymes are used in detergent formulations, the bacterium can be commercially exploited to secrete the alkaline enzymes for use in detergent industry. This is the first report for concomitant production of 3 alkaline enzymes by a bacterium.

Pulmonary neutrophils are the initial inflammatory cells that are recruited during lung injury and are crucial for innate immunity. However, pathological recruitment of neutrophils results in lung injury. The objective of this study is to determine whether the novel neutrophil chemoattractant, soluble VCAM-1 (sVCAM-1), recruits pathological levels of neutrophils to injury sites and amplifies lung inflammation during acute lung injury. The mice with P2X7 receptor deficiency, or treated with a P2X7 receptor inhibitor or anti-VCAM-1 antibodies were subjected to a clinically relevant two-hit LPS and mechanical ventilation-induced acute lung injury. Neutrophil infiltration and lung inflammation were measured. Neutrophil chemotactic activities were determined by a chemotaxis assay. VCAM-1 shedding and signaling pathways were assessed in isolated lung epithelial cells. Antibody neutralization of sVCAM-1 or deficiency or antagonism of P2X7R reduced neutrophil infiltration and proinflammatory cytokine levels. The ligands for sVCAM-1 were increased during acute lung injury. sVCAM-1 had neutrophil chemotactic activities and activated alveolar macrophages. VCAM-1 is released into the alveolar airspace from alveolar epithelial type I cells through P2X7 receptor-mediated activation of the metalloproteinase ADAM-17. In conclusion,sVCAM-1 is a novel chemoattractant for neutrophils and an activator for alveolar macrophages. Targeting sVCAM-1 provides a therapeutic intervention that could block pathological neutrophil recruitment, without interfering with the physiological recruitment of neutrophils, thus avoiding the impairment of host defenses.

Proteases are one of the most important commercial enzymes used in various industrial domains such as detergent and leather industries. The alkaline proteases as well as other detergent-compatible enzymes such as lipases and amylases serve now as the key components in detergent formulations. They break down various stains during fabric washing. The search for detergent-compatible proteases with better properties is a continuous exercise. The current trend is to use detergent-compatible proteases that are stable over a wide temperature range. Although the proteases showing stability at elevated pH have the capacity to be used in detergent formulations, their usage can be significant if they are also stable and compatible with detergent and detergent ingredients, and also able to remove protein stains. Despite the existence of some reviews on alkaline proteases, there is no specification for the use of alkaline proteases as detergent additives. The present review describes the detergent-compatible proteases tested as detergent additives. An overview was provided for screening, optimization, purification, and properties of detergent compatible proteases, with an emphasis on the stability and compatibility of the alkaline proteases with the detergent and detergent compounds, as well as stain removal examination methods.

Wnt/β-catenin signaling is an essential pathway in cell cycle control. Dysregulation of the Wnt/β-catenin signaling pathway during viral infection has been reported. In this study, we examined the effect of modulating Wnt/β-catenin signaling during influenza virus infection. The activation of the Wnt/β-catenin pathway by Wnt3a increased influenza virus mRNA and virus production in in vitro in mouse lung epithelial E10 cells and mRNA expresson of influenza virus genes in vivo in the lungs of mice infected with influenza virus A/Puerto Rico/8/34. However, the inhibition of Wnt/β-catenin signaling by iCRT14 reduced virus titer and viral gene expression in human lung epithelial A549 cells and viral replication in primary mouse alveolar epithelial cells infected with different influenza virus strains. Knockdown of β-catenin also reduced viral protein expression and virus production. iCRT14 acts at the early stage of virus replication. Treatment with iCRT14 inhibited the expression of the viral genes (vRNA, cRNA and mRNA) evaluated in this study. The intraperitoneal administration of iCRT14 reduced viral load, improved clinical signs, and partially protected mice from influenza virus infection.

Glycoprotein nonmetastatic melanoma protein B (GPNMB), a transmembrane protein, has been reported to have an important role in tissue repair and angiogenesis. Recently, we have demonstrated that hyperoxia exposure down-regulates microRNA (miR)-150 expression and concurrent induction of its target gene, GPNMB, in neonatal rat lungs. This study aimed to test the hypothesis that soluble GPNMB (sGPNMB) promotes angiogenesis in the hyperoxic neonatal lungs. Wild-type (WT) or miR-150 knockout (KO) neonates, exposed to 95% O 2 for 3, 6, and 10 days, were evaluated for lung phenotypes, GPNMB protein expression in the lungs, and sGPNMB levels in the bronchoalveolar lavage. Angiogenic effects of sGPNMB were examined both in vitro and in vivo. After a 6-day exposure, similar analyses were performed in WT and miR-150 KO neonates during recovery at 7, 14, and 21 days. miR-150 KO neonates displayed an increased capillary network, decreased inflammation, and less alveolar damage compared with WT neonates after hyperoxia exposure. The early induction of GPNMB and sGPNMB were found in miR-150 KO neonates. The recombinant GPNMB, which contained a soluble portion of GPNMB, promoted endothelial tube formation in vitro and enhanced angiogenesis in vivo. The increased capillaries in the hyperoxic lungs of miR-150 KO neonates appeared dysmorphic. They were abnormally enlarged in size and occasionally laid at subepithelial regions in the alveoli. However, the lung architecture returned to normal during recovery, suggesting that abnormal vascularity during hyperoxia does not affect postnatal lung development. GPNMB plays an important role in angiogenesis during hyperoxia injury. Treatment with GPNMB may offer a novel therapeutic approach in reducing pathologic complications in bronchopulmonary dysplasia.