Gasdermin D Hypermethylation Inhibits Pyroptosis And LPS-Induced IL-1β Release From NK92 Cells

INTRODUCTION: Although natural killer (NK) are major cells used to treat cancer patients, recent clinical trials showed that NK92 cells can be also used for the same purpose due to their high anti-tumor activity. Here, we examined whether these cells might be inflammatory due to the release of interleukin-1β (IL-1β), and whether the anti-inflammatory molecules dimethyl fumarate (DMF), or monomethyl fumarate (MMF) impair this activity. METHODS: NK92 cells were examined for the synthesis and release of IL-1β utilizing RT-PCR and ELISA assay, respectively. The expression of hydroxy-carboxylic acid receptors (HCA)1, HCA2 and HCA3 was detected by immunoblotting, flow cytometry, immunofluorescence and RT-PCR assays. The activation of caspase-1 and Gasdermin D (GSDMD) was evaluated by immunoblot assay. Pyroptosis was demonstrated by immunofluorescence imaging. Expression of DNA methyltransferases (DNMTs) mRNA was determined by whole transcriptome and immunoblot analyses. RESULTS: LPS-induced the release of IL-1β from NK92 cells, whereas DMF or MMF inhibited this induction. The effect of these drugs was due to inhibiting the conversion of procaspase-1 into active caspase-1. NK92 cells highly expressed GSDMD, a pyroptotic-mediated molecule. However, LPS induced the distribution of GSDMD into the cell membranes, corroborated with the presence of pyroptotic bodies, an activity that was inhibited by DMF or MMF. These molecule also inhibited the generation of GSDMD through DNMT-mediated hypermethylation of the promoter region of GSDMD gene. These results were supported by increased expression of DNMTs mRNA as determined by whole transcriptome analysis. DISCUSSION: Our results are the first to show that NK92 cells utilize GSDMD pathway to release IL-1β. Further, DMF and MMF which were previously shown to enhance NK cell cytotoxicity, also inhibit the inflammatory effects of these cells, making them most suitable for treating cancer patients

Enriched transcriptome analysis of laser capture microdissected populations of single cells to investigate intracellular heterogeneity in immunostained FFPE sections

To investigate intracellular heterogeneity, cell capture of particular cell populations followed by transcriptome analysis has been highly effective in freshly isolated tissues. However, this approach has been quite challenging in immunostained formalin-fixed paraffin-embedded (FFPE) sections. This study aimed at combining the standard pathology techniques, immunostaining and laser capture microdissection, with whole RNA-sequencing and bioinformatics analysis to characterize FFPE breast cancer cell populations with heterogeneous expression of progesterone receptor (PR). Immunocytochemical analysis revealed that 60% of MCF-7 cells admixture highly express PR. Immunocytochemistry-based targeted RNA-seq (ICC-RNAseq) and in silico functional analysis revealed that the PR-high cell population is associated with upregulation in transcripts implicated in immunomodulatory and inflammatory pathways (e.g. NF-κB and interferon signaling). In contrast, the PR-low cell population is associated with upregulation of genes involved in metabolism and mitochondrial processes as well as EGFR and MAPK signaling. These findings were cross-validated and confirmed in FACS-sorted PR high and PR-low MCF-7 cells and in MDA-MB-231 cells ectopically overexpressing PR. Significantly, ICC-RNAseq could be extended to analyze samples captured at specific spatio-temporal states to investigate gene expression profiles using diverse biomarkers. This would also facilitate our understanding of cell population-specific molecular events driving cancer and potentially other diseases

Heterogeneous treatment effects of therapeutic-dose heparin in patients hospitalized for COVID-19

Importance Randomized clinical trials (RCTs) of therapeutic-dose heparin in patients hospitalized with COVID-19 produced conflicting results, possibly due to heterogeneity of treatment effect (HTE) across individuals. Better understanding of HTE could facilitate individualized clinical decision-making. Objective To evaluate HTE of therapeutic-dose heparin for patients hospitalized for COVID-19 and to compare approaches to assessing HTE. Design, Setting, and Participants Exploratory analysis of a multiplatform adaptive RCT of therapeutic-dose heparin vs usual care pharmacologic thromboprophylaxis in 3320 patients hospitalized for COVID-19 enrolled in North America, South America, Europe, Asia, and Australia between April 2020 and January 2021. Heterogeneity of treatment effect was assessed 3 ways: using (1) conventional subgroup analyses of baseline characteristics, (2) a multivariable outcome prediction model (risk-based approach), and (3) a multivariable causal forest model (effect-based approach). Analyses primarily used bayesian statistics, consistent with the original trial. Exposures Participants were randomized to therapeutic-dose heparin or usual care pharmacologic thromboprophylaxis. Main Outcomes and Measures Organ support–free days, assigning a value of −1 to those who died in the hospital and the number of days free of cardiovascular or respiratory organ support up to day 21 for those who survived to hospital discharge; and hospital survival. Results Baseline demographic characteristics were similar between patients randomized to therapeutic-dose heparin or usual care (median age, 60 years; 38% female; 32% known non-White race; 45% Hispanic). In the overall multiplatform RCT population, therapeutic-dose heparin was not associated with an increase in organ support–free days (median value for the posterior distribution of the OR, 1.05; 95% credible interval, 0.91-1.22). In conventional subgroup analyses, the effect of therapeutic-dose heparin on organ support–free days differed between patients requiring organ support at baseline or not (median OR, 0.85 vs 1.30; posterior probability of difference in OR, 99.8%), between females and males (median OR, 0.87 vs 1.16; posterior probability of difference in OR, 96.4%), and between patients with lower body mass index (BMI 90% for all comparisons). In risk-based analysis, patients at lowest risk of poor outcome had the highest propensity for benefit from heparin (lowest risk decile: posterior probability of OR >1, 92%) while those at highest risk were most likely to be harmed (highest risk decile: posterior probability of OR <1, 87%). In effect-based analysis, a subset of patients identified at high risk of harm (P = .05 for difference in treatment effect) tended to have high BMI and were more likely to require organ support at baseline. Conclusions and Relevance Among patients hospitalized for COVID-19, the effect of therapeutic-dose heparin was heterogeneous. In all 3 approaches to assessing HTE, heparin was more likely to be beneficial in those who were less severely ill at presentation or had lower BMI and more likely to be harmful in sicker patients and those with higher BMI. The findings illustrate the importance of considering HTE in the design and analysis of RCTs. Trial Registration ClinicalTrials.gov Identifiers: NCT02735707, NCT04505774, NCT04359277, NCT0437258

Not Available

Not AvailablePolycyclic aromatic hydrocarbons (PAHs), including phenanthrene, are commonly found as pollutants in soils, estuarine, and sediments, as well as in terrestrial and other aquatic ecosystems. In this context, the phenanthrene-degrading bacteria were isolated and characterized in contaminated mangrove surface sediment, on the coast of Thane Creek, Mumbai, India by enrichment method, using phenanthrene as the sole source of carbon and energy. The phylogenetic diversity of the isolates were evaluated by 16S rRNA gene analysis and characterized as Bacillus mojavensis strain KSS001, Bacillus firmus strain KSS002, Bacillus flexus strain KSS003, Bacillus vietnamensis strain KSS004, and Bacillus amyloliquefaciens strain KSS005. Each isolate was grown on the phenanthrene up to 100 mg/L and the biodegradation ability was evidenced using a gas chromatography–flame ionization detector. Further, the mean value of phenanthrene degradation by 5 bacterial isolates after incubation in mineral salt medium for 7 days was 63% at 100 mg/L. The study reports that mangrove sediments of Thane Creek, Mumbai, contain a diverse population of phenanthrene-degrading bacteria that have the potential and capability to degrade PAHs contaminated sites, and are consequently recommended for bioremediation.Not Availabl