Production of proline and protease with different organic wastes in bacteria (Production proline and protease with organic wastes) / Produção de prolina e protease com diferentes resíduos orgânicos em bactérias (produção de prolina e protease com resíduos orgânicos)

In this study, we investigated the proline and protease production of different bacteria in several organic waste materials. Our aim was to produce proline and protease economically in waste that is abundantly available while reducing its environmental impact. 5 ml of different organic waste materials (OWW: Olive waste water; N.B: Nutrient Broth; EW: Eggshell; PBS: PBS buffer; PLW: Peach leaf wastes; TCW: Turkish coffee wastes; TWW: Tea waste water; WCW: Waste cheese whey; WFO: Waste frying oil) were placed in 10 ml grow tubes, inoculated and incubated for 24 h. Phosphate-buffered saline and 10% solutions of different organic wastes were added. These cultures were subsequently incubated at 37°C for 24 h. Cells were harvested at 24 h for L-proline assay. 1 ml of culture was transferred by pipette into an Eppendorf tube and centrifuged at 14,000 rpm for 20 min at room temperature. Cellular debris was removed by centrifuge and the supernatant was used for proline activity assays. Protease activity was determined using a modified method with casein as the substrate. We found that proline and protease can easily be produced economically using Turkish coffee wastes (TCW), Waste cheese whey (WCW) and Olive waste water (OWW) organic waste. We believe that this study will result in similar research leading to the economical use of these waste materials thus reducing their impact on the environment.

Neste estudo, investigamos a produção de prolina e protease de diferentes bactérias em diversos resíduos orgânicos. Nosso objetivo era produzir prolina e protease economicamente em resíduos que estão disponíveis em abundância, reduzindo seu impacto ambiental. Cinco ml de diferentes materiais de resíduos orgânicos (OWW: resíduos de azeitona; NB: caldo nutriente; EW: casca de ovo; PBS: tampão PBS; PLW: resíduos de folhas de pêssego; TCW: resíduos de café turco; TWW: resíduos de chá; WCW: resíduos de queijo soro de leite; WFO: óleo de fritura residual) foram colocados em tubos de cultivo de 10 ml, inoculados e incubados por 24 horas. Adicionaram-se solução salina tamponada com fosfato e soluções a 10% de diferentes resíduos orgânicos. Essas culturas foram subsequentemente incubadas a 37° C durante 24 h. As células foram colhidas às 24 h para o ensaio de L-prolina. Um ml de cultura foi transferido por pipeta para um tubo Eppendorf e centrifugado a 14.000 rpm, por 20 min, em temperatura ambiente. Os detritos celulares foram removidos por centrifugação e o sobrenadante foi usado para ensaios de atividade de prolina. A atividade da protease foi determinada usando um método modificado com caseína como substrato. Descobrimos que a prolina e a protease podem ser facilmente produzidas economicamente, usando resíduos de café turco (TCW), resíduos de soro de queijo (WCW) e resíduos orgânicos de água de oliva (OWW). Acreditamos que este estudo resultará em pesquisas semelhantes, levando ao uso econômico desses materiais residuais, reduzindo, assim, seu impacto no meio ambiente.

The COP9 signalosome subunit 3 is necessary for early embryo survival by way of a stable protein deposit in mouse oocytes.

Investigations of genes required in early mammalian development are complicated by protein deposits of maternal products, which continue to operate after the gene locus has been disrupted. This leads to delayed phenotypic manifestations and underestimation of the number of genes known to be needed during the embryonic phase of cellular totipotency. Here we expose a critical role of the gene Cops3 by showing that it protects genome integrity during the 2-cell stage of mouse development, in contrast to the previous functional assignment at postimplantation. This new role is mediated by a substantial deposit of protein (94th percentile of the proteome), divided between an exceptionally stable cortical rim, which is prevalent in oocytes, and an ancillary deposit in the embryonic nuclei. Since protein abundance and stability defeat prospects of DNA- or RNA-based gene inactivation in oocytes, we harnessed a classical method next to an emerging method for protein inactivation: antigen masking (for functional inhibition) versus TRIM21-mediated proteasomal degradation, also known as 'Trim away' (for physical removal). Both resulted in 2-cell embryo lethality, unlike the embryos receiving anti-green fluorescent protein. Comparisons between COPS3 protein-targeted and non-targeted embryos revealed large-scale transcriptome differences, which were most evident for genes associated with biological functions critical for RNA metabolism and for the preservation of genome integrity. The gene expression abnormalities associated with COPS3 inactivation were confirmed in situ by the occurrence of DNA endoreduplication and DNA strand breaks in 2-cell embryos. These results recruit Cops3 to the small family of genes that are necessary for early embryo survival. Overall, assigning genes with roles in embryogenesis may be less safe than assumed, if the protein products of these genes accumulate in oocytes: the inactivation of a gene at the protein level can expose an earlier phenotype than that identified by genetic techniques such as conventional gene silencing.

PDIA6 promotes pancreatic cancer progression and immune escape through CSN5-mediated deubiquitination of ß-catenin and PD-L1.

Protein Disulfide Isomerase Family A Member 6 (PDIA6) is an endoplasmic reticulum protein that is capable of catalyzing protein folding and disulfide bond formation. Abnormally elevated expression of PDIA6 has been reported to predict poor outcomes in various cancers. Herein, gain-of- and loss-of-function experiments were performed to investigate how PDIA6 participated in the carcinogenesis of pancreatic cancer (PC). By analyzing the protein expression of PDIA6 in 28 paired PC and para carcinoma specimens, we first found that PDIA6 expression was higher in PC samples. Both the overall survival and disease-free survival rates of PC patients with higher PDIA6 expression were poorer than those with lower PDIA6 (n = 178). Furthermore, knockdown of PDIA6 impaired the malignancies of PC cells - suppressed cell proliferation, invasion, migration, cisplatin resistance, and xenografted tumor growth. PDIA6-silenced PC cells were more sensitive to cytotoxic natural killer (NK) cells. Overexpression of PDIA6 had opposite effects on PC cells. Interestingly, COP9 signalosome subunit 5 (CSN5), a regulator of E3 ubiquitin ligases known to promote deubiquitination of its downstream targets, was demonstrated to interact with PDIA6, and its expression was increased in PC cells overexpressing PDIA6. Additionally, PDIA6 overexpression promoted deubiquitination of ß-catenin and PD-L1 and subsequently upregulated their expression in PC cells. These alterations were partly reversed by CSN5 shRNA. Collectively, the above results demonstrate that PDIA6 contributes to PC progression, which may be associated with CSN5-regulated deubiquitination of ß-catenin and PD-L1. Our findings suggest PDIA6 as a potential target for the treatment of PC.

Zinc deficiency induces abnormal development of the myocardium by promoting SENP5 overexpression.

Gestational zinc deficiency is a cause of congenital heart disease in the fetus, and sentrin/small ubiquitin-like modifier (SUMO)-specific proteases (SENPs) as deSUMOylation enzymes play a crucial role in the development of cardiac structures. However, current studies of the regulation and function of SENP in zinc-deficient status during heart development remain limited. In this study, SUMO1 modification was found to gradually decrease during heart development, and the level of SENP5 exhibited a similar trend to SUMO1 conjugation. In addition, zinc deficiency resulted in cardiac dysplasia, increased cell apoptosis, decreased cell viability, and differentiation inhibition of hiPSC-CMs. In order to investigate the function of SENP5 in zinc deficiency, hiPSC-CMs were transfected with SENP5 small interfering RNA. The negative effects of zinc lacking conditions were reversed with depletion of SENP5. It was confirmed that zinc deficiency induced abnormal differentiation of hiPSCs and increased apoptosis of hiPSC-CMs by promoting SENP5 overexpression, which led to cardiac dysplasia. Thus, it was concluded that SENP5 regulates the SUMO1 deconjugation during heart development and zinc deficiency may reduce conjugated SUMO by promoting SENP5 overexpression, which induces abnormal development of the myocardium.

Relative contributions of non-essential Sec pathway components and cell envelope-associated proteases to high-level enzyme secretion by Bacillus subtilis.

BACKGROUND: Bacillus subtilis is an important industrial workhorse applied in the production of many different commercially relevant proteins, especially enzymes. Virtually all of these proteins are secreted via the general secretion (Sec) pathway. Studies from different laboratories have demonstrated essential or non-essential contributions of various Sec machinery components to protein secretion in B. subtilis. However, a systematic comparison of the impact of each individual Sec machinery component under conditions of high-level protein secretion was so far missing. RESULTS: In the present study, we have compared the contributions of non-essential Sec pathway components and cell envelope-associated proteases on the secretion efficiency of three proteins expressed at high level. This concerned the α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis, and the serine protease BPN' from Bacillus amyloliquefaciens. We compared the secretion capacity of mutant strains in shake flask cultures, and the respective secretion kinetics by pulse-chase labeling experiments. The results show that secDF, secG or rasP mutations severely affect AmyE, AmyL and BPN' secretion, but the actual effect size depends on the investigated protein. Additionally, the chaperone DnaK is important for BPN' secretion, while AmyE or AmyL secretion are not affected by a dnaK deletion. Further, we assessed the induction of secretion stress responses in mutant strains by examining AmyE- and AmyL-dependent induction of the quality control proteases HtrA and HtrB. Interestingly, the deletion of certain sip genes revealed a strong differential impact of particular signal peptidases on the magnitude of the secretion stress response. CONCLUSIONS: The results of the present study highlight the importance of SecDF, SecG and RasP for protein secretion and reveal unexpected differences in the induction of the secretion stress response in different mutant strains.

Amino Acid Supplementation Improves the Production of Extracellular Peptidases by Aspergillus Section Flavi and their Ionic Immobilization

Abstract Bioprocess studies have been highlighted due to the importance of physiological processes and industrial applications of enzymes. The potential of peptidase production from Aspergillus section Flavi using different amino acids as a supplemental nitrogen source was investigated. A production profile revealed that amino acids had positive effects on peptidase production when compared to the control without amino acids. Optimal production (100 U/mL) was obtained with Arginine amino acid in 96 h of fermentation. Extracellular peptidase from Aspergillus section Flavi was identified in submerged bioprocesses by in situ activity. Biochemical studies revealed that the maximum activities of the enzyme extract were obtained at pH 6.5 and a temperature of 55°C. The inhibition by EDTA and PMSF suggests the presence of more than one peptidase while the Ni2+ and Cu2+ had a negative influence on the enzyme activity. When the crude extract was reversibly immobilized on ionic supports, DEAE-Agarose and MANAE-Agarose the derivative showed different profiles of thermal and pH stabilities. Hence, this study revealed the basic properties and biochemical characteristics that allowed the production improvement of this class of enzyme. Moreover, with known properties stabilization and immobilization process is required to further explore its biotechnological capacities.

Pan-PIM kinase inhibitors enhance Lenalidomide's anti-myeloma activity via cereblon-IKZF1/3 cascade.

Multiple myeloma remains an incurable disease, and continued efforts are required to develop novel agents and novel drug combinations with more effective anti-myeloma activity. Here, we show that the pan-PIM kinase inhibitors SGI1776 and CX6258 exhibit significant anti-myeloma activity and that combining a pan-PIM kinase inhibitor with the immunomodulatory agent lenalidomide in an in vivo myeloma xenograft mouse model resulted in synergistic myeloma cell killing without additional hematologic or hepatic toxicities. Further investigations indicated that treatment with a pan-PIM kinase inhibitor promoted increased ubiquitination and subsequent degradation of IKZF1 and IKZF3, two transcription factors crucial for survival of myeloma cells. Combining a pan-PIM kinase inhibitor with lenalidomide led to more effective degradation of IKZF1 and IKZF3 in multiple myeloma cell lines as well as xenografts of myeloma tumors. We also demonstrated that treatment with a pan-PIM kinase inhibitor resulted in increased expression of cereblon, and that knockdown of cereblon via a shRNA lentivirus abolished the effects of PIM kinase inhibition on the degradation of IKZF1 and IKZF3 and myeloma cell apoptosis, demonstrating a central role of cereblon in pan-PIM kinase inhibitor-mediated down-regulation of IKZF1 and IKZF3 and myeloma cell killing. These data elucidate the mechanism of pan-PIM kinase inhibitor mediated anti-myeloma effect and the rationale for the synergy observed with lenalidomide co-treatment, and provide justification for a clinical trial of the combination of pan-PIM kinase inhibitors and lenalidomide for the treatment of multiple myeloma.

Malassezia pachydermatis from animals: Planktonic and biofilm antifungal susceptibility and its virulence arsenal.

The yeast Malassezia pachydermatis is a component of the microbiota of dogs and cats, however it can cause otitis and seborrheic dermatitis in these animals. The objective of this study was to determine the antifungal susceptibility, and evaluate virulence and pathogenicity of 25 M. pachydermatis strains from animals. Susceptibility to ketoconazole, fluconazole, itraconazole, voriconazole, terbinafine, and amphotericin B was evaluated by broth microdilution assay. In addition, biofilm-forming ability, protease, phospholipase, hemolysin and melanin production and adhesion to epithelial cells by this yeast species were assessed. Finally, strain pathogenicity was investigated using the nematode Caenorhabditis elegans. Concerning the planktonic susceptibility, minimum inhibitory concentrations varied from <0.03 to>64 µg/mL for azole derivatives, 1 to >16 µg/mL for amphotericin B and 0.03 to 0.25 µg/mL for terbinafine. All strains were classified as strong biofilm producers, and ketoconazole, fluconazole and amphotericin B presented the best inhibitory effect against mature biofilms. All fungal isolates produced proteases, whereas 14/25 strains were positive for phospholipase production. Hemolytic activity was not observed and 18/25 strains showed dark pigmentation in the presence of L-DOPA. Regarding adhesion to epithelial cells, a low adhesion rate was observed in 10/12 evaluated strains. C. elegans mortality rate reached 95.9% after 96 h of exposure of the worms to M. pachydermatis. This yeast species produces important virulence factors and presents high pathogenicity, corroborating its clinical importance.

SENP3 protects H9C2 cells from apoptosis triggered by H/R via STAT3 pathway.

OBJECTIVE: To investigate whether SENP3 protects H9C2 cells from apoptosis triggered by H/R through the signal transducer and activator of transcription 3 (STAT3) pathway. MATERIALS AND METHODS: Male C57BL mice were cultured and mouse models of myocardial I/RI were established. At the same time, cardiomyoblast H9C2 cell line of rat embryo was cultured. Reactive oxygen species (ROS) level was detected during H/R using 2',7'-dichlorofluorescein diacetate (DCFH) kit. Apoptotic cells were checked by flow cytometry. The expressions of p-JAK2, JAK2, STAT3, p-STAT3, cleaved-caspase3 (c-caspase3), and Bcl/Bax were detected using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: We revealed that SENP3 rose in mice of I/R group and in H9C2 cells following H/R with an increase in p-STAT3. Furthermore, increased expression of SENP3 was found to be dependent on the generation of ROS, as the SENP3 accumulation was inhibited by antioxidant (NAC). Inhibition of SENP3 suppressed the p-STAT3 expression, but promoted cell apoptosis, c-caspase3 expression, and Bcl/Bax ratio. Besides, SENP3 overexpression alleviated the cell apoptosis, which was abrogated by AG490. CONCLUSIONS: SENP3 could protect H9C2 against H/R through enhancing JAK2/STAT3 pathway.

In vivo expression of proteases and protease inhibitor, a serpin, by periodontal pathogens at teeth and implants.

Porphyromonas gingivalis and Tannerella forsythia secrete proteases, gingipains and KLIKK-proteases. In addition, T. forsythia produces a serpin (miropin) with broad inhibitory spectrum. The aim of this pilot study was to determine the level of expression of miropin and individual proteases in vivo in periodontal and peri-implant health and disease conditions. Biofilm and gingival crevicular fluid (GCF)/ peri-implant sulcular fluid (PISF) samples were taken from healthy tooth and implant sites (n = 10), gingivitis and mucositis sites (n = 12), and periodontitis and peri-implantitis sites (n = 10). Concentration of interleukin-8 (IL-8), IL-1ß and IL-10 in GCF was determined by enzyme-linked immunosorbent assay. Loads of P. gingivalis and T. forsythia and the presence of proteases and miropin genes were assessed in biofilm by quantitative PCR, whereas gene expression was estimated by quantitative RT-PCR. The presence of P. gingivalis and T. forsythia, as well as the level of IL-8 and IL-1ß, were associated with disease severity in the periodontal and peri-implant tissues. In biofilm samples harboring T. forsythia, genes encoding proteases were found to be present at 72.4% for karilysin and 100% for other KLIKK-protease genes and miropin. At the same time, detectable mRNA expression of individual genes ranged from 20.7% to 58.6% of samples (for forsylisin and miropsin-1, respectively). In comparison with the T. forsythia proteases, miropin and the gingipains were highly expressed. The level of expression of gingipains was associated with those of miropin and certain T. forsythia proteases around teeth but not implants. Cumulatively, KLIKK-proteases and especially miropin, might play a role in pathogenesis of both periodontal and peri-implant diseases.

In vitro evaluation of hydrolytic enzyme activity and biofilm formation of Candida parapsilosis species complex from a nosocomial environment

Abstract INTRODUCTION: Candida parapsilosis complex species, frequently found in hospital environments, have gained importance as etiological agents of candidemia. METHODS: Candida parapsilosis complex isolates from a nosocomial environment were identified and their hydrolitic enzyme activity and ability to form biofilm were characterized. RESULTS: Twenty-two C. parapsilosis sensu stricto isolates produced proteinase and three produced phospholipase. Most Candida metapsilosis isolates produced proteinase and one also produced phospholipase. All 29 isolates formed biofilms. CONCLUSIONS: The nosocomial environment may act as a reservoir for C. parapsilosis complex isolates with phenotypic features that could possibly lead to nosocomial infections and health complications in hospital patients.

Recent advances in understanding basophil-mediated Th2 immune responses.

Basophils, the least common granulocytes, represent only ~0.5% of peripheral blood leukocytes. Because of the small number and some similarity with mast cells, the functional significance of basophils remained questionable for a long time. Recent studies using newly-developed analytical tools have revealed crucial and non-redundant roles for basophils in various immune responses, particularly Th2 immunity including allergy and protective immunity against parasitic infections. In this review, we discuss the mechanisms how basophils mediate Th2 immune responses and the nature of basophil-derived factors involved in them. Activated basophils release serine proteases, mouse mast cell protease 8 (mMCP-8), and mMCP-11, that are preferentially expressed by basophils rather than mast cells in spite of their names. These proteases elicit microvascular hyperpermeability and leukocyte infiltration in affected tissues, leading to inflammation. Basophil-derived IL-4 also contributes to eosinophil infiltration while it acts on tissue-infiltrating inflammatory monocytes to promote their differentiation into M2 macrophages that in turn dampen inflammation. Although basophils produce little or no MHC class II (MHC-II) proteins, they can acquire peptide-MHC-II complexes from dendritic cells via trogocytosis and present them together with IL-4 to naive CD4 T cells, leading to Th2 cell differentiation. Thus, basophils contribute to Th2 immunity at various levels.

Bacterial and Fungal Proteolytic Enzymes: Production, Catalysis and Potential Applications.

Submerged and solid-state bioprocesses have been extensively explored worldwide and employed in a number of important studies dealing with microbial cultivation for the production of enzymes. The development of these production technologies has facilitated the generation of new enzyme-based products with applications in pharmaceuticals, food, bioactive peptides, and basic research studies, among others. The applicability of microorganisms in biotechnology is potentiated because of their various advantages, including large-scale production, short time of cultivation, and ease of handling. Currently, several studies are being conducted to search for new microbial peptidases with peculiar biochemical properties for industrial applications. Bioprospecting, being an important prerequisite for research and biotechnological development, is based on exploring the microbial diversity for enzyme production. Limited information is available on the production of specific proteolytic enzymes from bacterial and fungal species, especially on the subgroups threonine and glutamic peptidases, and the seventh catalytic type, nonhydrolytic asparagine peptide lyase. This gap in information motivated the present study about these unique biocatalysts. In this study, the biochemical and biotechnological aspects of the seven catalytic types of proteolytic enzymes, namely aspartyl, cysteine, serine, metallo, glutamic, and threonine peptidase, and asparagine peptide lyase, are summarized, with an emphasis on new studies, production, catalysis, and application of these enzymes.

Oxidized LDL-induced JAB1 influences NF-κB independent inflammatory signaling in human macrophages during foam cell formation.

BACKGROUND: Oxidized low-density lipoprotein (oxLDL) mediates the transformation of macrophages (MΦ) to cholesterol-rich foam cells and the release of pro-inflammatory cytokines during atherogenesis. JAB1 (Jun activation domain binding protein-1) is present in all stages of human plaques, involved in the Toll-like receptor-mediated activation of p38 mitogen-activated protein kinase (MAPK) and controls nuclear factor-kappa B (NF-κB) activation. Thus, we were interested in the role of JAB1 during foam cell formation of MΦ after oxLDL exposition. METHODS AND RESULTS: We found that JAB1 was present in CD68-immunoreactive (-ir) MΦ in atherosclerotic plaques of apolipoprotein E knockout (ApoE-/-) mice after a high cholesterol/fat diet. Furthermore, differentiated human U937 MΦ - incubated with oxLDL (4 h) to induce foam cell formation - showed a significant increase of JAB1 (50 µg/ml: 1.39 + 0.15-fold; 100 µg/ml: 1.80 + 0.26-fold; 200 µg/ml: 2.05 + 0.30-fold; p < 0.05) on the protein level compared to the control. Independent from JAB1 silencing, we found an increase of total cholesterol (TC), free cholesterol (FC) and cholesteryl ester (CE) after oxLDL exposition. However, siJAB1-MФ showed a reduction of tumor necrosis factor-alpha (TNF-α) (36%; p < 0.05 vs. non-transfected MФ) and interleukin (IL)-6 (30%; p < 0.05 vs. non-transfected MФ) mRNA expression, as well as TNF-α (46%; p < 0.05 vs. non-transfected MФ) and IL-6 (32%; p < 0.05 vs. non-transfected MФ) protein secretion after oxLDL exposition. In parallel with an upregulation of inflammatory cytokines (TNF-α, IL-6) after oxLDL exposition, we found a significant (p < 0.05) increase of 37% in p38 MAPK activation after 4 h oxLDL-treatment, independent from NF-kB signaling. In this context, we showed regional co-localization of JAB1 with p38 MAPK in atherosclerotic plaques of ApoE-/- mice. Moreover, we detected interaction of JAB1 with p38 MAPK in U937 cells. CONCLUSION: We demonstrate that oxLDL induces JAB1 expression and influences its cellular localization, whereby the p38 MAPK signaling pathway is modified with consequences for inflammation of human MΦ in foam cells and atherosclerotic lesions.

Pharmacological and genetic reappraisals of protease and oxidative stress pathways in a mouse model of obstructive lung diseases.

Protease-antiprotease imbalance and oxidative stress are considered to be major pathophysiological hallmarks of severe obstructive lung diseases including chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), but limited information is available on their direct roles in the regulation of pulmonary phenotypes. Here, we utilized ßENaC-transgenic (Tg) mice, the previously established mouse model of severe obstructive lung diseases, to produce lower-mortality but pathophysiologically highly useful mouse model by backcrossing the original line with C57/BL6J mice. C57/BL6J-ßENaC-Tg mice showed higher survival rates and key pulmonary abnormalities of COPD/CF, including mucous hypersecretion, inflammatory and emphysematous phenotypes and pulmonary dysfunction. DNA microarray analysis confirmed that protease- and oxidative stress-dependent pathways are activated in the lung tissue of C57/BL6J-ßENaC-Tg mice. Treatments of C57/BL6J-ßENaC-Tg mice with a serine protease inhibitor ONO-3403, a derivative of camostat methylate (CM), but not CM, and with an anti-oxidant N-acetylcystein significantly improved pulmonary emphysema and dysfunction. Moreover, depletion of a murine endogenous antioxidant vitamin C (VC), by genetic disruption of VC-synthesizing enzyme SMP30 in C57/BL6J-ßENaC-Tg mice, exaggerated pulmonary phenotypes. Thus, these assessments clarified that protease-antiprotease imbalance and oxidative stress are critical pathways that exacerbate the pulmonary phenotypes of C57/BL6J-ßENaC-Tg mice, consistent with the characteristics of human COPD/CF.

Simultaneous production of amylases and proteases by Bacillus subtilis in brewery wastes

ABSTRACT The simultaneous production of amylase (AA) and protease (PA) activity by Bacillus subtilis UO-01 in brewery wastes was studied by combining the response surface methodology with the kinetic study of the process. The optimum conditions (T = 36.0 °C and pH = 6.8) for high biomass production (0.92 g/L) were similar to the conditions (T = 36.8 °C and pH = 6.6) for high AA synthesis (9.26 EU/mL). However, the maximum PA level (9.77 EU/mL) was obtained at pH 7.1 and 37.8 °C. Under these conditions, a considerably high reduction (between 69.9 and 77.8%) of the initial chemical oxygen demand of the waste was achieved. In verification experiments under the optimized conditions for production of each enzyme, the AA and PA obtained after 15 h of incubation were, respectively, 9.35 and 9.87 EU/mL. By using the Luedeking and Piret model, both enzymes were classified as growth-associated metabolites. Protease production delay seemed to be related to the consumption of non-protein and protein nitrogen. These results indicate that the brewery waste could be successfully used for a high scale production of amylases and proteases at a low cost.

Chronic electronic cigarette exposure in mice induces features of COPD in a nicotine-dependent manner.

BACKGROUND: The use of electronic (e)-cigarettes is increasing rapidly, but their lung health effects are not established. Clinical studies examining the potential long-term impact of e-cigarette use on lung health will take decades. To address this gap in knowledge, this study investigated the effects of exposure to aerosolised nicotine-free and nicotine-containing e-cigarette fluid on mouse lungs and normal human airway epithelial cells. METHODS: Mice were exposed to aerosolised phosphate-buffered saline, nicotine-free or nicotine-containing e-cigarette solution, 1-hour daily for 4 months. Normal human bronchial epithelial (NHBE) cells cultured at an air-liquid interface were exposed to e-cigarette vapours or nicotine solutions using a Vitrocell smoke exposure robot. RESULTS: Inhalation of nicotine-containing e-cigarettes increased airway hyper-reactivity, distal airspace enlargement, mucin production, cytokine and protease expression. Exposure to nicotine-free e-cigarettes did not affect these lung parameters. NHBE cells exposed to nicotine-containing e-cigarette vapour showed impaired ciliary beat frequency, airway surface liquid volume, cystic fibrosis transmembrane regulator and ATP-stimulated K+ ion conductance and decreased expression of FOXJ1 and KCNMA1. Exposure of NHBE cells to nicotine for 5 days increased interleukin (IL)-6 and IL-8 secretion. CONCLUSIONS: Exposure to inhaled nicotine-containing e-cigarette fluids triggered effects normally associated with the development of COPD including cytokine expression, airway hyper-reactivity and lung tissue destruction. These effects were nicotine-dependent both in the mouse lung and in human airway cells, suggesting that inhaled nicotine contributes to airway and lung disease in addition to its addictive properties. Thus, these findings highlight the potential dangers of nicotine inhalation during e-cigarette use.

Thalidomide-based induction regimens are as effective as bortezomib-based regimens in elderly patients with multiple myeloma with cereblon expression.

Cereblon (CRBN) has been identified as a primary target of immunomodulatory drugs and is considered a biomarker for the prediction of outcomes after thalidomide- or lenalidomide-based treatments. In this study, we evaluated CRBN expression in bone marrow (BM) tissue at diagnosis and investigated the relationship between CRBN expression and treatment outcomes after thalidomide- or bortezomib-based front-line therapies in 89 elderly patients with multiple myeloma (MM). CRBN expression at the time of diagnosis was evaluated with immunohistochemical (IHC) staining for myeloma cells in paraffin wax-embedded BM tissue. CRBN-immunostained slides were scored by intensity and diffuseness, and a total score of >6 was defined as CRBN-positive (CRBN(+)). Thirty-eight patients (45.2 %) were CRBN(+). Among patients treated with thalidomide-based regimens, CRBN(+) patients showed a better treatment response than did CRBN-negative patients (35.0 vs. 11.8 % complete response rate, respectively; HR = 4.038, P = 0.137). During a median follow-up of 31.8 months, patients treated with bortezomib-based regimens had a longer time to progression (TTP) than did patients treated with thalidomide-based regimens (15.6 vs. 13.2 months, respectively; P = 0.047), but early mortality occurred frequently in patients treated with bortezomib-based regimens. Additionally, there was no significant difference in survival outcomes between thalidomide- and bortezomib-based regimens in CRBN(+) patients (median TTP, 13.8 vs. 15.6 months, respectively; P = 0.842 and median OS, 39.3 vs. 30.1 months, respectively; P = 0.074). These data suggest that thalidomide-based regimens are as effective as bortezomib-based regimens in elderly patients with MM who are CRBN(+). Thus, CRBN positivity, by IHC staining, may be useful in deciding appropriate treatment options in elderly patients with MM.

Suppression of charcoal rot in soybean by moderately halotolerant Pseudomonas aeruginosa GS-33 under saline conditions.

Charcoal rot severely limits the soybean crop yield under saline conditions. The present studies focus on biocontrol and plant growth promoting potential of phenazine producing moderately halotolerant Pseudomonas aeruginosa (GS-33) in soybean under saline soil conditions. A marine isolate; GS-33 was identified as P. aeruginosa based on polyphasic characterization. This strain showed potent in vitro biocontrol activity against charcoal rot causing fungus Macrophomina phaseolina. It was capable of producing phenazine-1-carboxylic acid even at elevated salt concentrations. Moreover, GS-33 possessed other biocontrol traits like production of siderophores, HCN and protease under saline conditions. Multiple traits for plant growth promotion such as synthesis of IAA, NH3 , and solubilization of phosphate were also exhibited by GS-33. Plant growth promoting and biocontrol control potentials of GS-33 were evaluated by pot assay under saline soil conditions. Higher biomass and chlorophyll content were observed in GS-33 treated seedlings. A greater reduction in charcoal rot caused by fungal pathogens under both normal and saline soil conditions in GS-33 treated seedlings was observed. In a nut shell, phenazine producing halotolerant strain GS-33 could mitigate saline soil conditions (abiotic stress) and infestation of M. phaseolina (biotic stress) in soybean.

Enhanced production and organic solvent stability of a protease from Brevibacillus laterosporus strain PAP04.

A bacterial strain (PAP04) isolated from cattle farm soil was shown to produce an extracellular, solvent-stable protease. Sequence analysis using 16S rRNA showed that this strain was highly homologous (99%) to Brevibacillus laterosporus. Growth conditions that optimize protease production in this strain were determined as maltose (carbon source), skim milk (nitrogen source), pH 7.0, 40°C temperature, and 48 h incubation. Overall, conditions were optimized to yield a 5.91-fold higher production of protease compared to standard conditions. Furthermore, the stability of the enzyme in organic solvents was assessed by incubation for 2 weeks in solutions containing 50% concentration of various organic solvents. The enzyme retained activity in all tested solvents except ethanol; however, the protease activity was stimulated in benzene (74%) followed by acetone (63%) and chloroform (54.8%). In addition, the plate assay and zymography results also confirmed the stability of the PAP04 protease in various organic solvents. The organic solvent stability of this protease at high (50%) concentrations of solvents makes it an alternative catalyst for peptide synthesis in non-aqueous media.