The Smac mimetic BV6 cooperates with STING to induce necroptosis in apoptosis-resistant pancreatic carcinoma cells

Pancreatic cancer (PC) still remains a major cause of cancer-related death worldwide and alternative treatments are urgently required. A common problem of PC is the development of resistance against apoptosis that limits therapeutic success. Here we demonstrate that the prototypical Smac mimetic BV6 cooperates with the stimulator of interferon (IFN) genes (STING) ligand 2′,3′-cyclic guanosine monophosphate–adenosine monophosphate (2′3′-cGAMP) to trigger necroptosis in apoptosis-deficient PC cells. Pharmacological inhibition of key components of necroptosis signaling, such as receptor-interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), significantly rescues PC cells from 2′3′-cGAMP/BV6/zVAD.fmk-mediated cell death, suggesting the induction of necroptosis. Consistently, 2′3′-cGAMP/BV6 co-treatment promotes phosphorylation of MLKL. Furthermore, we show that 2′3′-cGAMP stimulates the production of type I IFNs, which cooperate with BV6 to trigger necroptosis in apoptosis-deficient settings. STING silencing via siRNA or CRISPR/Cas9-mediated gene knockout protects PC cells from 2′3′-cGAMP/BV6/zVAD.fmk-mediated cell death. Interestingly, we demonstrate that nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNFα), and IFN-regulatory factor 1 (IRF1) signaling are involved in triggering 2′3′-cGAMP/BV6/zVAD.fmk-induced necroptosis. In conclusion, we show that activated STING and BV6 act together to exert antitumor effects on PC cells with important implications for the design of new PC treatment concepts.Subject terms: Cancer, Cancer     

Inflammatory and coagulation biomarkers and mortality in patients with HIV infection

Background

In the Strategies for Management of Anti-Retroviral Therapy trial, all-cause mortality was higher for participants randomized to intermittent, CD4-guided antiretroviral treatment (ART) (drug conservation [DC]) than continuous ART (viral suppression [VS]).We hypothesized that increased HIV-RNA levels following ART interruption induced activation of tissue factor pathways, thrombosis, and fibrinolysis.

Methods and Findings

Stored samples were used to measure six biomarkers: high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), amyloid A, amyloid P, D-dimer, and prothrombin fragment 1+2. Two studies were conducted: (1) a nested case–control study for studying biomarker associations with mortality, and (2) a study to compare DC and VS participants for biomarker changes. For (1), markers were determined at study entry and before death (latest level) for 85 deaths and for two controls (n = 170) matched on country, age, sex, and date of randomization. Odds ratios (ORs) were estimated with logistic regression. For each biomarker, each of the three upper quartiles was compared to the lowest quartile. For (2), the biomarkers were assessed for 249 DC and 250 VS participants at study entry and 1 mo following randomization. Higher levels of hsCRP, IL-6, and D-dimer at study entry were significantly associated with an increased risk of all-cause mortality. Unadjusted ORs (highest versus lowest quartile) were 2.0 (95% confidence interval [CI], 1.0–4.1; p = 0.05), 8.3 (95% CI, 3.3–20.8; p < 0.0001), and 12.4 (95% CI, 4.2–37.0; p < 0.0001), respectively. Associations were significant after adjustment, when the DC and VS groups were analyzed separately, and when latest levels were assessed. IL-6 and D-dimer increased at 1 mo by 30% and 16% in the DC group and by 0% and 5% in the VS group (p < 0.0001 for treatment difference for both biomarkers); increases in the DC group were related to HIV-RNA levels at 1 mo (p < 0.0001). In an expanded case–control analysis (four controls per case), the OR (DC/VS) for mortality was reduced from 1.8 (95% CI, 1.1–3.1; p = 0.02) to 1.5 (95% CI, 0.8–2.8) and 1.4 (95% CI, 0.8–2.5) after adjustment for latest levels of IL-6 and D-dimer, respectively.

Conclusions

IL-6 and D-dimer were strongly related to all-cause mortality. Interrupting ART may further increase the risk of death by raising IL-6 and D-dimer levels. Therapies that reduce the inflammatory response to HIV and decrease IL-6 and D-dimer levels may warrant investigation. Trial Registration: ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT00027352","term_id":"NCT00027352"}}NCT00027352).     

Interleukin-17A mediates acquired immunity to pneumococcal colonization

Although anticapsular antibodies confer serotype-specific immunity to pneumococci, children increase their ability to clear colonization before these antibodies appear, suggesting involvement of other mechanisms. We previously reported that intranasal immunization of mice with pneumococci confers CD4+ T cell-dependent, antibody- and serotype-independent protection against colonization. Here we show that this immunity, rather than preventing initiation of carriage, accelerates clearance over several days, accompanied by neutrophilic infiltration of the nasopharyngeal mucosa. Adoptive transfer of immune CD4+ T cells was sufficient to confer immunity to na?ve RAG1(-/-) mice. A critical role of interleukin (IL)-17A was demonstrated: mice lacking interferon-gamma or IL-4 were protected, but not mice lacking IL-17A receptor or mice with neutrophil depletion. In vitro expression of IL-17A in response to pneumococci was assayed: lymphoid tissue from vaccinated mice expressed significantly more IL-17A than controls, and IL-17A expression from peripheral blood samples from immunized mice predicted protection in vivo. IL-17A was elicited by pneumococcal stimulation of tonsillar cells of children or adult blood but not cord blood. IL-17A increased pneumococcal killing by human neutrophils both in the absence and in the presence of antibodies and complement. We conclude that IL-17A mediates pneumococcal immunity in mice and probably in humans; its elicitation in vitro could help in the development of candidate pneumococcal vaccines.     

Long term repeated prescribed burning increases evenness in the basidiomycete laccase gene pool in forest soils

Repeated prescribed burning alters the biologically labile fraction of nutrients and carbon of soil organic matter (SOM). Using a long-term (30 years) repeated burning experiment where burning has been carried out at a 2- or 4-year frequency, we analysed the effect of prescribed burning on gross potential C turnover rates and phenol oxidase activity in relation to shifts in SOM composition as observed using Fourier-transform infrared spectroscopy. In tandem, we assessed the genetic diversity of basidiomycete laccases. While the overall effect of burning was a decline in phenol oxidase activity, Shannon diversity and evenness of laccases was significantly higher in burned sites. Co-correspondence analysis of SOM composition and laccase operational taxonomic unit frequency data also suggested a strong correlation. While this correlation could indicate that the observed increase in laccase genetic diversity due to burning is due to increased resource diversity, a temporal replacement of the most abundant members of the assembly by an otherwise dormant pool of fungi cannot be excluded. As such, our results fit the intermediate disturbance hypothesis. Effects were stronger in plots burned in 2-year rotations, suggesting that the 4-year burn frequency may be a more sustainable practice to ensure the long-term stability of C cycling in such ecosystems.     

Directed optimization of biocatalytic transglycosylation processes by the integration of genetic algorithms and fermentative approaches into a kinetic model

Many biocatalysts exhibit strict stereospecificity and regioselectivity. However, their thermodynamically controlled equilibria often limit yields in industrial production processes. Herein, we describe the synthesis of fructooligosaccharides from sucrose by various fructansucrases. We previously demonstrated that transfructosylation to diverse acceptors yields d-glucose and the fructose-containing product along with diverse by-products. To streamline this reaction, we developed a procedure that allows the enhanced transfructosylation of diverse acceptors by different fructansucrases. By diverting the released glucose from the reaction via metabolism by living cells we limited the back reaction and forced the consumption of sucrose. The basic conditions for the resulting fermentation process were optimized by a genetic algorithm and integrated into a kinetic model. This strategy allows the prediction of optimal reaction parameters for the production of desired target compounds.     

Assessment of iodine‐treated filter media for removal and inactivation of MS2 bacteriophage aerosols

Aims: To investigate the performance of an iodine‐releasing filter medium for use as a protective device against airborne pathogens. Methods and Results: The filter’s physical and viable removal efficiencies (VRE) were investigated with challenges of MS2 bacteriophage aerosols, and the infectivity of MS2 collected on the filter was analysed. To test a proposed inactivation mechanism, media containing thiosulfate or bovine serum albumin (BSA) were put in impingers to quench and consume I₂ released from the filter. In direct plating experiments, treated filters presented significantly higher VREs than did untreated filters; however, collection in excess BSA decreased VRE by half and in thiosulfate the apparent VRE decreased drastically. No significant difference in infectivity of retained viruses on treated and untreated filters was observed at the same environmental condition. Conclusions: Evidence presented herein for competition by dissolved I₂ in infectivity assays supports a mechanism of induced displacement and capture of I_2. It also requires that dissociation of iodine from the filter and capture of iodine by MS2 aerosols as they pass through the filter be factored in the design of the assessment methodology. The filter’s strong retention capability minimizes reaerosolization but also makes it difficult to discriminate the antimicrobial effect at the surface. Significance and Impact of the Study: This study shows the direct plating assay method to be sensitive to interference by iodine‐releasing materials. This requires reevaluation of earlier reports of VRE measurements.     

A large molecular size fraction of riverine high molecular weight dissolved organic matter (HMW DOM) stimulates growth of the harmful dinoflagellate Alexandrium minutum

An increase in the concentration of riverine dissolved organic matter (DOM) has been observed during the last decades, and this material can stimulate marine plankton in coastal waters with significant freshwater input. We studied the effect of two size fractions of riverine high molecular weight dissolved organic matter (HMW DOM), isolated with tangential ultrafiltration, on the harmful dinoflagellate Alexandrium minutum and a natural isolate of marine bacteria under laboratory conditions. Both A. minutum and bacteria grew significantly better with the low MW DOM compared to both the high MW DOM fraction and controls (no DOM additions). This experiment demonstrates that the harmful algae A. minutum and bacteria benefit from larger molecules of river HMW DOM, and highlights the potential of A. minutum to utilize organic nitrogen from large DOM molecules. This ability may enhance their likelihood of success in estuaries/costal waters with a humic rich freshwater input, especially when the relative amount of large molecules within DOM is more pronounced.     

Modeling system states in liver cells: Survival,apoptosis and their modifications in response to viral infection

Background  

The decision pro- or contra apoptosis is complex, involves a number of different inputs, and is central for the homeostasis of an individual cell as well as for the maintenance and regeneration of the complete organism.     

Proteome profile of functional mitochondria from human skeletal muscle using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS

Mitochondria can be isolated from skeletal muscle in a manner that preserves tightly coupled bioenergetic function in vitro. The purpose of this study was to characterize the composition of such preparations using a proteomics approach. Mitochondria isolated from human vastus lateralis biopsies were functional as evidenced by their response to carbohydrate and fat-derived fuels. Using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS, 823 unique proteins were detected, and 487 of these were assigned to the mitochondrion, including the newly characterized SIRT5, MitoNEET and RDH13. Proteins detected included 9 of the 13 mitochondrial DNA-encoded proteins and 86 of 104 electron transport chain (ETC) and ETC-related proteins. In addition, 59 of 78 proteins of the 55S mitoribosome, several TIM and TOM proteins and cell death proteins were present. This study presents an efficient method for future qualitative assessments of proteins from functional isolated mitochondria from small samples of healthy and diseased skeletal muscle.