scRNA-seq profiling of human granulocytes reveals expansion of developmentally flexible neutrophil precursors with mixed neutrophil and eosinophil properties in asthma.

Neutrophils and eosinophils share common hematopoietic precursors and usually diverge into distinct lineages with unique markers before being released from their hematopoietic site, which is the bone marrow (BM). However, previous studies identified an immature Ly6g(+) Il-5Rα(+) neutrophil population in mouse BM, expressing both neutrophil and eosinophil markers suggesting hematopoietic flexibility. Moreover, others have reported neutrophil populations expressing eosinophil-specific cell surface markers in tissues and altered disease states, confusing the field regarding eosinophil origins, function, and classification. Despite these reports, it is still unclear whether hematopoietic flexibility exists in human granulocytes. To answer this, we utilized single-cell RNA sequencing (scRNA-seq) and CITE-seq to profile human BM and circulating neutrophils and eosinophils at different stages of differentiation and determine whether neutrophil plasticity plays role in asthmatic inflammation. We show that immature metamyelocyte neutrophils in humans expand during severe asthmatic inflammation and express both neutrophil and eosinophil markers. We also show an increase in tri-lobed eosinophils with mixed neutrophil and eosinophil markers in allergic asthma and that IL-5 promotes differentiation of immature blood neutrophils into tri-lobed eosinophilic phenotypes suggesting a mechanism of emergency granulopoiesis to promote myeloid inflammatory or remodeling response in patients with chronic asthma. By providing insights into unexpectedly flexible granulocyte biology and demonstrating emergency hematopoiesis in asthma, our results highlight the importance of granulocyte plasticity in eosinophil development and allergic diseases.

Molecular regulation of neutrophil swarming in health and disease: Lessons from the phagocyte oxidase.

Neutrophil swarming is a complex coordinated process in which neutrophils sensing pathogen or damage signals are rapidly recruited to sites of infections or injuries. This process involves cooperation between neutrophils where autocrine and paracrine positive-feedback loops, mediated by receptor/ligand pairs including lipid chemoattractants and chemokines, amplify localized recruitment of neutrophils. This review will provide an overview of key pathways involved in neutrophil swarming and then discuss the cell intrinsic and systemic mechanisms by which NADPH oxidase 2 (NOX2) regulates swarming, including modulation of calcium signaling, inflammatory mediators, and the mobilization and production of neutrophils. We will also discuss mechanisms by which altered neutrophil swarming in disease may contribute to deficient control of infections and/or exuberant inflammation. Deeper understanding of underlying mechanisms controlling neutrophil swarming and how neutrophil cooperative behavior can be perturbed in the setting of disease may help to guide development of tools for diagnosis and precision medicine.

NADPH oxidase 2 limits amplification of IL-1ß-G-CSF axis and an immature neutrophil subset in murine lung inflammation.

The leukocyte NADPH oxidase 2 (NOX2) regulates inflammation independent of its antimicrobial activity. Inherited defects in NOX2 lead to chronic granulomatous disease (CGD), associated with recurrent bacterial and fungal infections, often with excessive neutrophilic inflammation that results in significant inflammatory burden and tissue damage. We previously showed that excessive leukotriene B4 (LTB4) production by NOX2-deficient mouse neutrophils was a key driver of elevated lung neutrophil infiltration in the initial response to pulmonary challenge with the model fungal particle zymosan. We now identify interleukin-1ß (IL-1ß) and downstream granulocyte colony-stimulating factor (G-CSF) as critical amplifying signals that augment and sustain neutrophil accrual in CGD mice. Neutrophils, delivered into the lung via LTB4, were the primary source of IL-1ß within the airways, and their increased numbers in CGD lungs led to significantly elevated local and plasma G-CSF. Elevated G-CSF simultaneously promoted increased granulopoiesis and mobilized the release of higher numbers of an immature CD101- neutrophil subset from the marrow, which trafficked to the lung and acquired a significantly more proinflammatory transcriptome in CGD mice compared with wild-type mice. Thus, neutrophil-produced IL-1ß and downstream G-CSF act sequentially but nonredundantly with LTB4 to deploy neutrophils and amplify inflammation in CGD mice after inhalation of zymosan. NOX2 plays a critical role in dampening multiple components of a feed-forward pipeline for neutrophil recruitment, and these findings highlight NOX2 as a key regulator of neutrophil number, subsets, and function at inflamed sites.

Neutrophil and Macrophage NADPH Oxidase 2 Differentially Control Responses to Inflammation and to Aspergillus fumigatus in Mice.

Aspergillus fumigatus is an important opportunistic fungal pathogen and causes invasive pulmonary aspergillosis in conditions with compromised innate antifungal immunity, including chronic granulomatous disease, which results from inherited deficiency of the superoxide-generating leukocyte NADPH oxidase 2 (NOX2). Derivative oxidants have both antimicrobial and immunoregulatory activity and, in the context of A. fumigatus, contribute to both fungal killing and dampening inflammation induced by fungal cell walls. As the relative roles of macrophage versus neutrophil NOX2 in the host response to A. fumigatus are incompletely understood, we studied mice with conditional deletion of NOX2. When NOX2 was absent in alveolar macrophages as a result of LysM-Cre-mediated deletion, germination of inhaled A. fumigatus conidia was increased. Reducing NOX2 activity specifically in neutrophils via S100a8 (MRP8)-Cre also increased fungal burden, which was inversely proportional to the level of neutrophil NOX2 activity. Moreover, diminished NOX2 in neutrophils synergized with corticosteroid immunosuppression to impair lung clearance of A. fumigatus. Neutrophil-specific reduction in NOX2 activity also enhanced acute inflammation induced by inhaled sterile fungal cell walls. These results advance understanding into cell-specific roles of NOX2 in the host response to A. fumigatus. We show that alveolar macrophage NOX2 is a nonredundant effector that limits germination of inhaled A. fumigatus conidia. In contrast, reducing NOX2 activity only in neutrophils is sufficient to enhance inflammation to fungal cell walls as well as to promote invasive A. fumigatus. This may be relevant in clinical settings with acquired defects in NOX2 activity due to underlying conditions, which overlap risk factors for invasive aspergillosis.

Macrophage NOX2 NADPH oxidase maintains alveolar homeostasis in mice.

The leukocyte NADPH oxidase 2 (NOX2) plays a key role in pathogen killing and immunoregulation. Genetic defects in NOX2 result in chronic granulomatous disease (CGD), associated with microbial infections and inflammatory disorders, often involving the lung. Alveolar macrophages (AMs) are the predominant immune cell in the airways at steady state, and limiting their activation is important, given the constant exposure to inhaled materials, yet the importance of NOX2 in this process is not well understood. In this study, we showed a previously undescribed role for NOX2 in maintaining lung homeostasis by suppressing AM activation, in CGD mice or mice with selective loss of NOX2 preferentially in macrophages. AMs lacking NOX2 had increased cytokine responses to Toll-like receptor-2 (TLR2) and TLR4 stimulation ex vivo. Moreover, between 4 and 12 week of age, mice with global NOX2 deletion developed an activated CD11bhigh subset of AMs with epigenetic and transcriptional profiles reflecting immune activation compared with WT AMs. The presence of CD11bhigh AMs in CGD mice correlated with an increased number of alveolar neutrophils and proinflammatory cytokines at steady state and increased lung inflammation after insults. Moreover, deletion of NOX2 preferentially in macrophages was sufficient for mice to develop an activated CD11bhigh AM subset and accompanying proinflammatory sequelae. In addition, we showed that the altered resident macrophage transcriptional profile in the absence of NOX2 is tissue specific, as those changes were not seen in resident peritoneal macrophages. Thus, these data demonstrate that the absence of NOX2 in alveolar macrophages leads to their proinflammatory remodeling and dysregulates alveolar homeostasis.

Thermostable and organic solvent-tolerant acid pectinase from Aspergillus terreus FP6: purification, characterization and evaluation of its phytopigment extraction potential.

The present study discusses the purification, characterization and application of pectinase from Aspergillus terreus FP6 in fruit pigment extraction. By the four-step purification involving precipitation, dialysis, ion-exchange chromatography, gel filtration chromatography, a 20.85-fold purification of the enzyme to homogeneity was achieved. The apparent molecular mass of the pectinase was 47 kDa, as found by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme was recorded at pH 6.0 and 50 °C. The enzyme retained 80.3% and 79.1% residual activity, respectively at pH 6.0 and 50 °C for 90 min. The pectinase was best functional in the presence of toluene and retained its activity for 30 min. Cu2+ and Co2+ acted as enzyme activators, while Ca2+, ß-mercaptoethanol, dimethyl sulfoxide and ethylenediaminetetraacetic acid proved to be the inhibitors. The K m and V max values of the pectinase with pectin as substrate were 0.002 mM and 27.39 U/mL, respectively thus indicating the high enzyme affinity towards the substrate. After 30-min treatment of the grape skin with the partially purified enzyme, microscopic observation revealed that a short time of the enzymatic treatment resulted in substantial loss of pigment and shrinkage of the grape skin cells thereby highlighting the high efficiency of the pectinase. The current study implies that the A. terreus FP6 pectinase may be applied as a bio-agent in the food and beverage industries and has the potential to replace harmful solvents by promoting a greener approach to extract plant pigments.

Co-substrate-mediated utilization of high concentration of phenol by Aspergillus niger FP7 and reduction of its phytotoxicity on Vigna radiata L.

Phenol and its derivatives behave as mutagens, teratogens and carcinogens inducing adverse physiological effects and are considered environmental hazards. The present study focuses on high concentration phenol utilization by Aspergillus niger FP7 under various physicochemical parameters. The soil remediation potential of the culture for reducing phenol toxicity against Vigna radiata L. seed germination was also evaluated along with the extent of phenol utilization using high-performance liquid chromatography. Aspergillus niger FP7 showed phenol tolerance up to 1000 mg/l, beyond which there was a sharp reduction in phenol utilization. Supplementation of the mineral salt medium with glucose and peptone and application of a 100 rpm agitation rate enhanced phenol utilization (up to 88.3%). Phenol utilization efficiency decreased (up to 29.6%) when cadmium and mercury salts were present, but the same improved (59.4-75.5%) by the incorporation of cobalt, copper and zinc salts. Vigna radiata L. seeds sown in the non-augmented soil revealed a 3.27% germination index, and with fungal augmentation, the germination index improved (97.3%). The non-augmented soil demonstrated 3.1% phenol utilization, while for the augmented soil, the utilization was 79.3%. Based on the phytotoxicity study and chromatographic analysis, it could be inferred that Aspergillus niger FP7 significantly enhanced phenol utilization in soil. In the future, Aspergillus niger FP7 could be of potential use in bioremediation of sites polluted with high concentrations of phenol.

Fibrinolytic protease from Bacillus cereus S46: Purification, characterization, and evaluation of its in vitro thrombolytic potential.

Intravascular thrombosis is a prime cause of cardiac complications worldwide. Microbial fibrinolytic proteases are of clinical significance in thrombosis treatment. The present study discusses the purification and characterization of a protease from Bacillus cereus S46, ascertaining its in vitro thrombolytic activity against a blood clot. By the three-step purification involving precipitation, dialysis, and diethylaminoethyl-cellulose ion-exchange chromatography, a 12.37-fold purification of the enzyme to homogeneity was achieved. The apparent molecular mass of the protease was 30 kDa, as found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme was observed at pH 8.0 and 40°C. The enzyme retained an 82.19% residual activity at pH 8.0 and 40°C for 1 h. The Km and Vmax values of the protease with casein were 0.0027 mM and 9.712 µmol/min, respectively. In an in vitro assay, the purified protease resulted in 97.02% lysis of the blood clot. The fibrinolytic potential of the enzyme, together with its characteristics of being active and stable under near-physiological conditions, may suggest its application as a therapeutic agent.

Bitter Melon Enhances Natural Killer-Mediated Toxicity against Head and Neck Cancer Cells.

Natural killer (NK) cells are one of the major components of innate immunity, with the ability to mediate antitumor activity. Understanding the role of NK-cell-mediated tumor killing in controlling of solid tumor growth is still in the developmental stage. We have shown recently that bitter melon extract (BME) modulates the regulatory T cell (Treg) population in head and neck squamous cell carcinoma (HNSCC). However, the role of BME in NK-cell modulation against HNSCC remains unknown. In this study, we investigated whether BME can enhance the NK-cell killing activity against HNSCC cells. Our results indicated that treatment of human NK-cell line (NK3.3) with BME enhances ability to kill HNSCC cells. BME increases granzyme B accumulation and translocation/accumulation of CD107a/LAMP1 in NK3.3 cells exposed to BME. Furthermore, an increase in cell surface expression of CD16 and NKp30 in BME-treated NK3.3 cells was observed when cocultured with HNSCC cells. Collectively, our results demonstrated for the first time that BME augments NK-cell-mediated HNSCC killing activity, implicating an immunomodulatory role of BME. Cancer Prev Res; 10(6); 337-44. ©2017 AACR.

Degradation of benzo[a]pyrene by Pleurotus ostreatus PO-3 in the presence of defined fungal and bacterial co-cultures.

Benzo[a]pyrene, a high molecular weight polycyclic aromatic hydrocarbon possesses carcinogenic, teratogenic, and mutagenic properties. The present study focuses on benzo[a]pyrene degradation by Pleurotus ostreatus PO-3, characterization and identification of metabolites produced and the extent of degradation in the presence of axenic culture of P. ostreatus PO-3 and defined co-cultures of the basidiomycete with bacteria and non-basidiomycete fungi. Thin-layer chromatography revealed that P. ostreatus PO-3 transformed benzo[a]pyrene to polar metabolites. Following degradation, appearance of numerous peaks in the mass spectrum indicated that benzo[a]pyrene degradation was a result of the metabolic activity of P. ostreatus PO-3. A degradation product corresponding to the m/z 284.2 was detected which could possibly be BaP-quinone, resulting from the oxidation of benzo[a]pyrene. Compared to the axenic culture of P. ostreatus PO-3 (64.3%), co-cultures of P. ostreatus PO-3 and Penicillium chrysogenum MTCC 787 and P. ostreatus PO-3 and Pseudomonas aeruginosa MTCC 1688 could degrade 86.1 and 75.1% of benzo[a]pyrene, respectively. Thus it could be inferred from the present investigation that the combined catabolic activities of P. ostreatus PO-3 with bacteria and non-basidiomycete fungi can produce synergistic effects to enhance BaP degradation. The increase in the generation of polar metabolites as degradation products from the recalcitrant parent compound advocates the potential application of P. ostreatus PO-3 in benzo[a]pyrene bioremediation.

Involvement of c-Fos in the Promotion of Cancer Stem-like Cell Properties in Head and Neck Squamous Cell Carcinoma.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Although improvements in surgical techniques, chemotherapy and radiation delivery, and supportive care have improved quality of life for patients with HNSCC, regional and distant recurrence remain common. Recent evidence suggests that cancer stem-like cells (CSC) play a significant role in recurrence and chemoresistance. We previously observed that c-Fos was highly upregulated in the HNSCC sphere-forming cells. Consequences of c-Fos upregulation for the biology of HNSCC-CSCs are poorly understood. In this study, we investigated the role of c-Fos in renewal of stemness of HNSCC and tumor growth.Experimental Design and Results: We generated stable HNSCC cell lines ectopically expressing the c-Fos gene. Exogenous expression of c-Fos in nontumorigenic MDA1386Tu cells makes these cells tumorigenic in nude mice. Furthermore, subcutaneous transplantation of c-Fos-overexpressing Cal27 cells (tumorigenic) into immunocompromised mice enhanced tumor growth as compared with parental cells. Mechanistic investigations demonstrated that c-Fos overexpression enhanced the epithelial-mesenchymal transition (EMT) state and expression of CSC markers (Nanog, c-Myc, Sox2, and Notch1). Ectopic expression of c-Fos in HNSCC cells also displays increased sphere formation. We further observed that overexpression of c-Fos increased the expression of pERK and cyclin D1 in HNSCC cells.Conclusions: Together, our results strongly suggest a novel role of c-Fos as a regulator of EMT and cancer stem cell reprogramming in HNSCC cells, which may hold potential as a CSC-directed therapeutic approach to improve HNSCC treatment. Clin Cancer Res; 23(12); 3120-8. ©2016 AACR.

Coconut oil induced production of a surfactant-compatible lipase from Aspergillus tamarii under submerged fermentation.

Filamentous fungi are efficient producers of lipases. The present study focuses on identification of a potent lipolytic fungus and enhancement of lipase production through optimization of nutritional and cultural conditions under submerged fermentation. Molecular characterization of the fungus by 18S rDNA sequencing revealed its identity as Aspergillus tamarii with 98% homology. Maximum lipase production was noted in mineral salts medium supplemented with coconut oil (2.5%, v/v). A combination of ammonium chloride (2%, w/v) and tryptone (2%, w/v) facilitated maximum lipase production at pH 5 of the production medium. A carbon: nitrogen ratio of 1:4 led to significant (p < 0.00008) increase in the enzyme production in the presence of surfactant cetyltrimethylammonium bromide (0.5%, w/v). Maximum lipase activity (2,32,500 ± 192 U/ml/min) was recorded after 7 days of incubation at 25 °C on a rotary shaker at 120 rpm. A 9.8-fold increase in lipase activity was recorded after optimization of the process parameters. Addition of crude lipase enhanced the oil stain removal activity of a commercially available detergent by 2.2-fold. The current findings suggest the potentiality of this fungal lipase to be used in detergent formulation.

Anti-inflammatory activity of cyclo-oxygenase2 inhibitory anionic protein fraction from Lamellidens marginalis (Lamarck).

Aqueous extract of freshwater mussel, Lamellidens marginalis is known to possess potent antioxidant and anti-inflammatory activity. Here, we have made an attempt to purify anti-inflammatory protein from Lamellidens marginalis extract (LME). Aqueous LME was prepared, and total protein was precipitated by 60% ammonium sulfate followed by purification through ion exchange chromatography. Isolated fractions were studied for anti-inflammatory activity in in vitro and in vivo experimental models. Active fractions were characterized by SDS PAGE and HPLC. Protein recovered from ammonium sulfate precipitation showed four distinct peaks in diethyl-aminoethyl cellulose ion exchange chromatography when eluted with stepwise salt gradient. Protein fraction eluted in 0.5 M sodium chloride solution showed maximum specific activity and anti-inflammatory activity in acute model and adjuvant induced chronic inflammation model. This fraction also showed cyclo-oxygenase 2 (COX2) enzyme inhibitory activity in in-vitro system. In SDS-PAGE 0.5 M NaCl fraction showed multiple bands after Coomassie brilliant blue staining and three distinct peaks in HPLC. In this study, we identified an anti-inflammatory protein fraction with high anionic property which could be attributed to inhibition of COX2 enzyme activity.

Anti-miR-203 suppresses ER-positive breast cancer growth and stemness by targeting SOCS3.

Breast cancer is a major public health problem worldwide in women and existing treatments are not adequately effective for this deadly disease. microRNAs (miRNAs) regulate the expression of many target genes and play pivotal roles in the development, as well as in the suppression of many cancers including breast cancer. We previously observed that miR-203 was highly upregulated in breast cancer tissues and in ER-positive breast cancer cell lines. In our present study, we observed that anti-miR-203 suppresses breast cancer cell proliferation in vitro. Orthotopic implantation of miR-203 depleted MCF-7 cells into nude mice displays smaller tumor growth as compared to control MCF-7 cells. Furthermore, miR-203 expression is significantly higher in ER-positive breast cancer patients as compared to ER-negative patients. We identified suppressor of cytokine signaling 3 (SOCS3) as a direct target of miR-203. Here we observed that miR-203 expression is inversely correlated with SOCS3 expression in ER-positive breast cancer samples. Additionally, we found that anti-miR-203 suppressed the expression of pStat3, pERK and c-Myc in MCF-7 and ZR-75-1 cells. We also demonstrated that anti-miR-203 decreased mammospheres formation and expression of stem cell markers in MCF-7 and ZR-75-1 cells. Taken together, our data suggest that anti-miR-203 has potential as a novel therapeutic strategy in ER-positive breast cancer treatment.

Immunomodulatory role of bitter melon extract in inhibition of head and neck squamous cell carcinoma growth.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer and leading cause of cancer related mortality worldwide. Despite the advancement in treatment procedures the overall survival rate of patients has not considerably enhanced in the past few decades. Therefore, new strategies to achieve a favorable response for the improvement in the prognosis of HNSCC are urgently needed. In this study, we examined the role of bitter melon extract (BME) in HNSCC tumor microenvironment. Mouse head and neck cancer (SCCVII) cells were subcutaneously injected into the flanks of syngeneic mice. We observed that oral gavage of BME significantly inhibits the tumor growth in mice as compared to control group. Further study suggested that BME inhibits cell proliferation as evident from low expression of proliferating cell nuclear antigen (PCNA) and c-Myc in the tumors of BME fed mice as compared to that of control group. We next investigated the role of BME as an immunomodulator in HNSCC model. Forkhead box protein P3+ (FoxP3+) T cells suppress tumor immunity. Our data suggested that BME treatment decreases the infiltrating regulatory T (Treg) cells by inhibiting FoxP3+ populations in the tumors and in spleens. Additionally, BME treatment reduces Th17 cell population in the tumor. However, BME treatment did not alter Th1 and Th2 cell populations. Together, our findings offer a new insight into how bitter melon extract inhibits head and neck tumor growth by modulating cell proliferation and Treg populations, with implications for how to control tumor-infiltrating lymphocytes and tumor progression.

Serum miR-30e and miR-223 as Novel Noninvasive Biomarkers for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers and is the third leading cause of all cancer-related death. Limited noninvasive biomarkers are available for HCC detection. Early detection is the key in improving the survival of HCC patients. In this study, we tested the hypothesis that serum miRNAs can be used as a potential biomarker for HCC. Quantitative RT-PCR for miRNA analysis was performed using 70 serum samples. Receiver operating characteristic analysis was performed to measure the prognostic power of the miRNAs. The miRNA expression level was also measured from liver biopsy samples. Our study revealed that two miRNAs, miR-30e and miR-223, were expressed at significantly lower levels (P < 0.003) in the sera of HCC patients compared with healthy volunteers. Furthermore, expression of these miRNAs was compared between sera from chronic liver disease and sera from HCC patients. miR-30e and miR-223 expression was significantly lower in HCC sera compared with sera from chronic liver disease patients. Both miRNA expression levels were lower in HCC liver biopsy specimens compared with normal liver RNA. Taken together, our results suggested that serum miR-30e and miR-223 are useful biomarkers of HCC, irrespective of etiology, and deserve further study for their diagnostic value.

Biosynthesis and characterization of a thermostable, alkali-tolerant chitinase from Bacillus pumilus JUBCH08 displaying antagonism against phytopathogenic Fusarium oxysporum.

The present investigation highlights the process parameters influencing the submerged fermentation of chitinase by Bacillus pumilus JUBCH08, purification and characterization of the enzyme and determination of antagonistic activity of the bacterium against Fusarium oxysporum. Medium supplemented with 0.5 % chitin and peptone, at initial pH 8.0, when incubated at 35 °C for 72 h favored highest chitinase production. The enzyme was purified 25.1-fold to homogeneity. The chitinase was found to be thermostable and alkali-tolerant with maximum activity at pH 8.0 and 70 °C for 1 h. The molecular weight of chitinase was found to be 64 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Mg2+, Co2+, Ca2+ and Mn2+ improved the chitinase activity. The K m and V max values of the enzyme were 0.13 mg/ml and 38.23 U/ml, respectively. When subjected to dual plate assay, the bacterium showed 45 % antagonism against F. oxysporum. Thus, it could be inferred that cultural conditions strongly affected the chitinase production by B. pumilus JUBCH08. The enzyme being thermostable and best functional under alkaline conditions could be useful for the feed industry and related biotechnological applications. Inhibition of F. oxysporum by the culture through lytic mechanism indicates its potentiality as a biocontrol agent.

Purification and characterization of a surfactant-compatible lipase from Aspergillus tamarii JGIF06 exhibiting energy-efficient removal of oil stains from polycotton fabric.

An extracellular lipase with 23,666.66 U/ml/min activity was produced by Aspergillus tamarii JGIF06 under submerged fermentation in mineral salt medium containing coconut oil (2.5 % v/v), tryptone (2 % w/v) and ammonium chloride (2 % w/v), with initial pH of 5 ± 0.2, incubated at 25 °C for 7 days on a rotary shaker at 120 rpm. A 7.9-fold increase in lipase-specific activity was recorded after purification by DEAE Sepharose ion exchange and Sephadex G200 column chromatography. The apparent molecular mass of this enzyme was revealed as 50 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The optimal lipase activity was recorded at pH 4 and 37 °C. The enzyme revealed broad specificity towards different vegetable oils. The K m and V max of the lipase on olive oil was found to be 330.4 mg and 53,690 U/ml/min, respectively. The lipase activity was stable in the presence of surfactants such as cetrimonium bromide, sodium dodecyl sulphate and Tween 80, and metal ions and reagents such as Ca2+, Ba2+ and 2-mercaptoethanol. However, the activity was greatly reduced in the presence of organic solvents such as chloroform. The stain removal potential of the crude lipase was determined on polycotton fabric pieces stained with peanut oil. Lipase added to cold water alone significantly enhanced the removal of stain by 152 %. The addition of lipase also improved the stain removal efficiency of a commercially available detergent in the presence of either cold (25 ± 2 °C) or hot (65 ± 2 °C) water. The current findings suggest the potentiality of this enzyme for energy-efficient biocatalytic application.

Production, characterization and Congo red dye decolourizing efficiency of a laccase from Pleurotus ostreatus MTCC 142 cultivated on co-substrates of paddy straw and corn husk.

A laccase produced by Pleurotus ostreatus MTCC 142 under solid-state fermentation using co-substrates of paddy straw and corn husk (1.5:1.5, g w/w) showed an activity of 2.54 U gds-1. Laccase activity was determined spectrophotometrically using 0.5 mM 2,2'- azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). Supplementation with fructose and potassium nitrate resulted in maximum enzyme production at initial pH 5.8 ± 0.2 and initial moisture content of 70%. A carbon: nitrogen ratio of 0.5:0.1 yielded highest laccase activity in the presence of surfactant Tween 20 (0.05%, w/v). Incorporation of vanillin (5 mM) and copper sulphate (10 mM) facilitated enhanced synthesis of laccase. A 4.8-fold increase in enzyme activity was recorded after optimization of nutritional parameters. The apparent molecular mass of this enzyme was revealed as 43 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The laccase showed optimal activity at pH 3 and 35 °C with 82.8% residual activity after 1 h of incubation. The K m and V max values on ABTS were found to be 0.52 mM and 9.33 U gds-1, respectively. The enzyme activity was enhanced by Cu2+ and remained unaffected with Ba2+, Mn2+, Pb2+, Mg2+, Ca2+ and Fe3+. However, pre-incubation of the enzyme with reagents like sodium azide, sodium lauryl sulphate and 2-mercaptoethanol demonstrated an inhibition of its activity. Addition of crude laccase to Congo red dye solution resulted in 36.84% decolourization after 20 h of incubation at 35 ± 2 °C. This study discusses the production and characterization of a laccase from P. ostreatus strain with potential for azo dye decolourization.

Brans-Dicke Theory with Λ>0: Black Holes and Large Scale Structures.

A step-by-step approach is followed to study cosmic structures in the context of Brans-Dicke theory with positive cosmological constant Λ and parameter ω. First, it is shown that regular stationary black-hole solutions not only have constant Brans-Dicke field ϕ, but can exist only for ω=∞, which forces the theory to coincide with the general relativity. Generalizations of the theory in order to evade this black-hole no-hair theorem are presented. It is also shown that in the absence of a stationary cosmological event horizon in the asymptotic region, a stationary black-hole horizon can support a nontrivial Brans-Dicke hair. Even more importantly, it is shown next that the presence of a stationary cosmological event horizon rules out any regular stationary solution, appropriate for the description of a star. Thus, to describe a star one has to assume that there is no such stationary horizon in the faraway asymptotic region. Under this implicit assumption generic spherical cosmic structures are studied perturbatively and it is shown that only for ω>0 or ω≲-5 their predicted maximum sizes are consistent with observations. We also point out how, many of the conclusions of this work differ qualitatively from the Λ=0 spacetimes.