Modulation of Extracellular ISG15 Signaling by Pathogens and Viral Effector Proteins.

ISG15 is a ubiquitin-like modifier that also functions extracellularly, signaling through the LFA-1 integrin to promote interferon (IFN)-γ release from natural killer (NK) and T cells. The signals that lead to the production of extracellular ISG15 and the relationship between its two core functions remain unclear. We show that both epithelial cells and lymphocytes can secrete ISG15, which then signals in either an autocrine or paracrine manner to LFA-1-expressing cells. Microbial pathogens and Toll-like receptor (TLR) agonists result in both IFN-ß-dependent and -independent secretion of ISG15, and residues required for ISG15 secretion are mapped. Intracellular ISGylation inhibits secretion, and viral effector proteins, influenza B NS1, and viral de-ISGylases, including SARS-CoV-2 PLpro, have opposing effects on secretion of ISG15. These results establish extracellular ISG15 as a cytokine-like protein that bridges early innate and IFN-γ-dependent immune responses, and indicate that pathogens have evolved to differentially inhibit the intracellular and extracellular functions of ISG15.

Tumor-Derived GM-CSF Promotes Granulocyte Immunosuppression in Mesothelioma Patients.

Purpose: The cross-talk between tumor cells, myeloid cells, and T cells can play a critical role in tumor pathogenesis and response to immunotherapies. Although the etiology of mesothelioma is well understood, the impact of mesothelioma tumor cells on the surrounding immune microenvironment is less well studied. In this study, the effect of the mesothelioma tumor microenvironment on circulating and infiltrating granulocytes and T cells is investigated.Experimental Design: Tumor tissues and peripheral blood from mesothelioma patients were evaluated for presence of granulocytes, which were then tested for their T-cell suppression potential. Different cocultures of granulocytes and/or mesothelioma tumor cells and/or T cells were set up to identify the mechanism of T-cell inhibition.Results: Analysis of human tumors showed that the mesothelioma microenvironment is enriched in infiltrating granulocytes, which inhibit T-cell proliferation and activation. Characterization of the whole blood at diagnosis identified similar, circulating, immunosuppressive CD11b+CD15+HLADR- granulocytes at increased frequency compared with healthy controls. Culture of healthy-donor granulocytes with human mesothelioma cells showed that GM-CSF upregulates NOX2 expression and the release of reactive oxygen species (ROS) from granulocytes, resulting in T-cell suppression. Immunohistochemistry and transcriptomic analysis revealed that a majority of mesothelioma tumors express GM-CSF and that higher GM-CSF expression correlated with clinical progression. Blockade of GM-CSF with neutralizing antibody, or ROS inhibition, restored T-cell proliferation, suggesting that targeting of GM-CSF could be of therapeutic benefit in these patients.Conclusions: Our study presents the mechanism behind the cross-talk between mesothelioma tumors and the immune microenvironment and indicates that targeting GM-CSF could be a novel treatment strategy to augment immunotherapy in patients with mesothelioma. Clin Cancer Res; 24(12); 2859-72. ©2018 AACR.

Streptonigrin Inhibits SENP1 and Reduces the Protein Level of Hypoxia-Inducible Factor 1α (HIF1α) in Cells.

Streptonigrin (CAS no. 3930-19-6) is a natural product shown to have antitumor activities in clinical trials conducted in the 1960s-1970s. However, its use in clinical studies eventually faded, and the molecular mechanisms of streptonigrin antitumor effects remain poorly defined. Despite its lack of current clinical use, efforts on its total synthesis have continued. Here, we show that streptonigrin binds and inhibits the SUMO-specific protease SENP1. NMR studies identified that streptonigrin binds to SENP1 on the surface where SUMO binds and disrupts SENP1-SUMO1 interaction. Site-directed mutations in combination with NMR chemical shift perturbation suggest key roles of aromatic π stacking interactions in binding streptonigrin. Treatment of cells with streptonigrin resulted in increased global SUMOylation levels and reduced level of hypoxia inducible factor alpha (HIF1α). These findings inform both the design of SENP1 targeting strategy and the modification of streptonigrin to improve its efficacy for possible future clinical use.

Efficacy of Anti-mesothelin Immunotoxin RG7787 plus Nab-Paclitaxel against Mesothelioma Patient-Derived Xenografts and Mesothelin as a Biomarker of Tumor Response.

Purpose: The purpose of this study was to evaluate the antitumor efficacy of the reduced immunogenicity anti-mesothelin immunotoxin RG7787 plus nab-paclitaxel against primary mesothelioma cell lines and tumor xenografts and the utility of mesothelin as a biomarker of tumor response.Experimental Design: Early-passage human malignant mesothelioma cell lines NCI-Meso16, NCI-Meso19, NCI-Meso21, and NCI-Meso29 were evaluated for sensitivity to RG7787 or nab-paclitaxel alone or in combination. In addition, the antitumor activity of RG7787 plus nab-paclitaxel was evaluated using NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumor xenografts in immunodeficient mice. Serum mesothelin was measured at different time points to determine whether its levels correlated with tumor response.Results: All four primary mesothelioma cell lines highly expressed mesothelin with 41 × 103 to 346 × 103 mesothelin sites per cell and were sensitive to RG7787, with IC50 ranging from 0.3 to 10 ng/mL. Except for NCI-Meso19, these cells were also sensitive to nab-paclitaxel, with IC50 of 10 to 25 ng/mL. In vitro, RG7787 plus nab-paclitaxel led to decreased cell viability compared with either agent alone. In NCI-Meso16 tumor xenografts, treatment with RG7787 plus nab-paclitaxel led to sustained complete tumor regressions. Similar antitumor efficacy was observed against NCI-Meso21 and NCI-Meso29 tumor xenografts. In all three tumor xenograft models, changes in human serum mesothelin correlated with response to therapy and were undetectable in mice with complete tumor regression with RG7787 and nab-paclitaxel.Conclusions: RG7787 plus nab-paclitaxel is very active against primary human mesothelioma cells in vitro as well as in vivo, with serum mesothelin levels correlating with tumor response. These results indicate that this combination could be useful for treating patients with mesothelioma. Clin Cancer Res; 23(6); 1564-74. ©2016 AACR.

Malignant Mesothelioma Effusions Are Infiltrated by CD3+ T Cells Highly Expressing PD-L1 and the PD-L1+ Tumor Cells within These Effusions Are Susceptible to ADCC by the Anti-PD-L1 Antibody Avelumab.

INTRODUCTION: The functional aspects of programmed death 1 (PD-1) and PD ligand 1 (PD-L1) immune checkpoints in malignant mesothelioma have not been studied. METHODS: Tumor samples from 65 patients with mesothelioma were evaluated for PD-L1 expression by immunohistochemistry, and its prognostic significance was examined. Malignant effusions from patients with pleural and peritoneal mesothelioma were evaluated for PD-1-positive and PD-L1-positive infiltrating lymphocytes and their role in inducing PD-L1 expression in tumor cells. Antibody-dependent cellular cytotoxicity (ADCC) of avelumab, a fully humanized immunoglobulin G1 anti PD-L1 antibody against primary mesothelioma cell lines, was evaluated in presence of autologous and allogeneic natural killer cells. RESULTS: Of 65 pleural and peritoneal mesothelioma tumors examined, 41 (63%) were PD-L1-positive, which was associated with slightly inferior overall survival compared to patients with PD-L1-negative tumors (median 23.0 versus 33.3 months, p = 0.35). The frequency of PD-L1 expression was similar in patients with pleural and peritoneal mesothelioma, with 62% and 64% of samples testing positive, respectively. In nine mesothelioma effusion samples evaluated, the fraction of cells expressing PD-L1 ranged from 12% to 83%. In seven patients with paired malignant effusion and peripheral blood mononuclear cell (PBMC) samples, PD-L1 expression was significantly higher on CD3-positive T cells present in malignant effusions as compared with PBMCs (p = 0.016). In addition, the numbers of CD14-positive PD-1-positive cells were increased in malignant effusions compared with PBMCs (p = 0.031). The lymphocytes present in malignant effusions recognized autologous tumor cells and induced interferon-γ-mediated PD-L1 expression on the tumor cell surface. Of the three primary mesothelioma cell lines tested, two were susceptible to avelumab-mediated ADCC in the presence of autologous natural killer cells. CONCLUSIONS: Most pleural as well as peritoneal mesotheliomas express PD-L1. Malignant effusions in this disease are characterized by the presence of tumor cells and CD3-positive T cells that highly express PD-L1. In addition, mesothelioma tumor cells are susceptible to ADCC by the anti-PD-L1 antibody avelumab.

Mechanistic insight into sono-enzymatic degradation of organic pollutants with kinetic and thermodynamic analysis.

In this paper, we have attempted to get a physical insight into process of sono-enzymatic treatment for degradation of recalcitrant organic pollutants. Decolourization of an azo dye has been used as model reaction with different experimental protocols that alter characteristics of ultrasound and cavitation phenomena in the system. Experimental data is analyzed to determine kinetic and thermodynamic parameters of decolorization process. The trends observed in kinetic and thermodynamic parameters of decolourization are essentially manifestations of the dominating mechanism of the decolorization of the textile dye (or nature of prevalent chemical reaction in the system), viz. either molecular reaction due to enzyme or radical reaction due to transient cavitation. The activation energy for sonochemical protocol is negative, which indicates instantaneity of the radical reactions. The frequency factor is also low, which is attributed to high instability of radicals. For enzymatic and sono-enzymatic protocols, activation energy is positive with higher frequency factor. Enthalpy change for sonochemical protocol is negative, while that for enzymatic and sono-enzymatic protocols is positive. The net entropy change for sonochemical protocol is more negative than enzymatic or sono-enzymatic protocol due to differences in prevalent chemical mechanism of dye decolorization. Due to inverse variations of frequency factor and activation energy, marginal rise in reaction kinetics is seen for sono-enzymatic protocol, as compared to enzymatic treatment alone. Due to inverse variations of enthalpy and entropy change, net Gibbs energy change in all experimental protocols shows little variation indicating synergism of the mechanism of ultrasound and enzyme.

Effect of fermentation parameters on bio-alcohols production from glycerol using immobilized Clostridium pasteurianum: an optimization study.

This article addresses the issue of effect of fermentation parameters for conversion of glycerol (in both pure and crude form) into three value-added products, namely, ethanol, butanol, and 1,3-propanediol (1,3-PDO), by immobilized Clostridium pasteurianum and thereby addresses the statistical optimization of this process. The analysis of effect of different process parameters such as agitation rate, fermentation temperature, medium pH, and initial glycerol concentration indicated that medium pH was the most critical factor for total alcohols production in case of pure glycerol as fermentation substrate. On the other hand, initial glycerol concentration was the most significant factor for fermentation with crude glycerol. An interesting observation was that the optimized set of fermentation parameters was found to be independent of the type of glycerol (either pure or crude) used. At optimum conditions of agitation rate (200 rpm), initial glycerol concentration (25 g/L), fermentation temperature (30°C), and medium pH (7.0), the total alcohols production was almost equal in anaerobic shake flasks and 2-L bioreactor. This essentially means that at optimum process parameters, the scale of operation does not affect the output of the process. The immobilized cells could be reused for multiple cycles for both pure and crude glycerol fermentation.

Sonoenzymatic decolourization of an azo dye employing immobilized horse radish peroxidase (HRP): a mechanistic study.

For degradation of biorefractory pollutants, enzymatic treatments and sonochemical treatments have shown high potential. A combined technique of sono-enzymatic treatment is of special interest as it has shown enhancement effect than the individual techniques. This work has attempted to give a mechanistic insight into the interaction of sonochemical and enzymatic treatments using immobilized horseradish peroxidase (HRP) enzyme on the decolourization of acid red dye (an azo dye). In order to segregate the effect of ultrasound and cavitation, experiments were conducted at elevated static pressure. The kinetic parameters of HRP, viz. Vmax and Km were marginally affected by immobilization. There was a minor change in pH optima and temperature optima for immobilized HRP (6.5, 25°C) from free HRP (7.0, 20-25°C). Though the specific activity of free enzyme (0.272U/mg) was found to be higher than the immobilized enzyme (0.104U/mg), immobilized enzyme exhibited higher stability (up to 3 cycles) and degradation potential than free enzyme in all experiments. The results revealed that the coupling of sonication and enzymatic treatment at high pressure in presence of polyethylene glycol (PEG) yielded the highest decolourization of acid red (61.2%). However, the total decolourization achieved with combined technique was lesser than the sum of individual techniques, indicating negative synergy between the sonochemical and enzymatic techniques.

Mechanistic investigation of ultrasonic enhancement of glycerol bioconversion by immobilized Clostridium pasteurianum on silica support.

Glycerol, the principal byproduct of biodiesel production, can be a valuable carbon source for bioconversion into diverse class of compounds. This article attempts to investigate the mechanistic aspects of ultrasound mediated bioconversion of glycerol to ethanol and 1,3-propanediol (1,3-PDO) by immobilized Clostridium pasteurianum cells on silica support. Our approach is of coupling experimental results with simulations of cavitation bubble dynamics and enzyme kinetics. In addition, the statistical analysis (ANOVA) of experimental results was also done. The glycerol uptake by cells was not affected by either immobilization or with ultrasonication. Nonetheless, both immobilization and ultrasonication were found to enhance glycerol consumption. The enhancement effect of ultrasound on glycerol consumption was most marked (175%) at the highest glycerol concentration of 25 g/L (271.7 mM). The highest glycerol consumption (32.4 mM) was seen for 10 g/L (108.7 mM) initial glycerol concentration. The immobilization of cells shifted the metabolic pathway almost completely towards 1,3-PDO. No formation of ethanol was seen with mechanical shaking, while traces of ethanol were detected with ultrasonication. On the basis of analysis of enzyme kinetics parameters, we attribute these results to increased substrate-enzyme affinity and decreased substrate inhibition for 1,3-PDO dehydrogenase in presence of ultrasound that resulted in preferential conversion of glycerol into 1,3-PDO.

Physical features of ultrasound assisted enzymatic degradation of recalcitrant organic pollutants.

This work has attempted to provide answer to the interaction of sonolysis and enzymatic treatment on degradation of recalcitrant dyes in a combined treatment. The model system comprises of two dyes, acid red and malachite green as model pollutants, along with horseradish peroxidase as a model enzyme and ultrasound of 20 kHz frequency. A dual approach of coupling experimental results with simulations of cavitation bubble dynamics has been adopted. Utilization of oxidation potential of horseradish peroxidase has been found to be a function of convection level in the medium. Cavitation phenomenon is found to have an adverse effect on enzyme action due to generation of high amplitude shock waves, which denature the enzyme. Degradation of dye at high static pressure increases due to absence of cavitation and high energy interaction (or collisions) between enzyme and dye molecules, which are beneficial towards enzymatic oxidation of the latter. High intensity convection generated by ultrasound also obviates need for an external shielding agent such as PEG that prevents attachment of the phenoxy radicals to enzyme that blocks the active sites of the enzyme.

Microbial conversion of glycerol: present status and future prospects.

Biodiesel has emerged as a potential alternate renewable liquid fuel in the past two decades. Total annual production of biodiesel stands at 6.96 million tons and 11.2 million tons in USA and Europe, respectively. In other countries, Asia and Latin America, biodiesel production has increased at unprecedented rate. Despite this, the economy of biodiesel is not attractive. An obvious solution for boosting the economy of the biodiesel industry is to look for markets for side products of the transesterification process of biodiesel synthesis. The main by-product is glycerol. However, this glycerol is contaminated with alkali/acid catalyst and alcohol, and thus, is not useful for conventional applications such as in toothpaste, drugs, paints and cosmetics. Conversion of this glycerol to value-added product is a viable solution for effective and economic utilization, which would also generate additional revenue for the biodiesel industry. Intensive research has taken place in area of conversion of glycerol to numerous products. The conventional catalytic route of glycerol transformation employs prohibitively harsh conditions of temperature and pressure, and thus, has slim potential for large-scale implementation. In addition, the selectivity of the process is rather small with formation of many undesired side products. The bioconversion processes, on the other hand, are highly selective although with slower kinetics. In this review, we have given an assessment and overview of the literature on bioconversion of glycerol. We have assessed as many as 23 products from glycerol bioconversion, and have reviewed the literature in terms of microorganism used, mode of fermentation, type of fermentor, yield and productivity of the process and recovery/purification of the products. The metabolic pathway of conversion of glycerol to various products has been discussed. We have also pondered over economic and engineering issues of large-scale implementation of process and have outlined the constraints and limitations of the process. We hope that this review will be a useful source of information for biochemists, biotechnologists, microbiologists and chemical engineers working in the area of glycerol bioconversion.

Effect of direct peroxide bleach application to bovine dentin on flexural strength and modulus in vitro.

OBJECTIVES: The objective of this study was to determine the effects of carbamide peroxide (CP) and hydrogen peroxide (HP) bleaching on the flexural strength (FS) and flexural modulus (FM) of dentin. METHODS: 2x2x20mm bovine dentin specimens were immersed in the bleaching agents to simulate overnight (10 or 15% CP, 6h daily, 2 weeks), exaggerated overnight (10% CP, 6h/day, 5 days/week, 2 months), daytime (6.5 or 7.5% HP, 1h daily, 3 weeks) and in-office (35% HP, 1h/day, 2 days/week, 3 weeks) treatment protocols. Distilled water (DW) and a placebo gel acted as control immersion materials. After immersion, the specimens were rinsed and stored in DW. Mechanical testing was performed 24h after the last treatment using an Instron Universal Testing Machine with a crosshead speed of 0.75 mm/min. The results were analyzed by ANOVA and Tukey's tests (p<0.05). RESULTS: There were significant reductions in the FS and FM of dentin after 2-week and 2-month exposures to CP. There were no significant differences in the FS or the FM of the dentin among the HP treatment and control groups. CONCLUSIONS: Direct in vitro application of CP bleaches caused significant decreases in dentin FS and FM. Similar decreases were not observed among the HP-treated dentin groups, which were exposed to shorter treatment times. Further research is needed to determine the effect of CP and HP on dentin in vivo.