LUBAC-mediated M1 Ub regulates necroptosis by segregating the cellular distribution of active MLKL.

Plasma membrane accumulation of phosphorylated mixed lineage kinase domain-like (MLKL) is a hallmark of necroptosis, leading to membrane rupture and inflammatory cell death. Pro-death functions of MLKL are tightly controlled by several checkpoints, including phosphorylation. Endo- and exocytosis limit MLKL membrane accumulation and counteract necroptosis, but the exact mechanisms remain poorly understood. Here, we identify linear ubiquitin chain assembly complex (LUBAC)-mediated M1 poly-ubiquitination (poly-Ub) as novel checkpoint for necroptosis regulation downstream of activated MLKL in cells of human origin. Loss of LUBAC activity inhibits tumor necrosis factor α (TNFα)-mediated necroptosis, not by affecting necroptotic signaling, but by preventing membrane accumulation of activated MLKL. Finally, we confirm LUBAC-dependent activation of necroptosis in primary human pancreatic organoids. Our findings identify LUBAC as novel regulator of necroptosis which promotes MLKL membrane accumulation in human cells and pioneer primary human organoids to model necroptosis in near-physiological settings.

Spatially resolved transcriptomic profiles reveal unique defining molecular features of infiltrative 5ALA-metabolizing cells associated with glioblastoma recurrence.

BACKGROUND: Spatiotemporal heterogeneity originating from genomic and transcriptional variation was found to contribute to subtype switching in isocitrate dehydrogenase-1 wild-type glioblastoma (GBM) prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) enables intraoperative visualization of infiltrative tumors outside the magnetic resonance imaging contrast-enhanced regions. The cell population and functional status of tumor responsible for enhancing 5ALA-metabolism to fluorescence-active PpIX remain elusive. The close spatial proximity of 5ALA-metabolizing (5ALA +) cells to residual disease remaining post-surgery renders 5ALA + biology an early a priori proxy of GBM recurrence, which is poorly understood. METHODS: We performed spatially resolved bulk RNA profiling (SPRP) analysis of unsorted Core, Rim, Invasive margin tissue, and FACS-isolated 5ALA + /5ALA - cells from the invasive margin across IDH-wt GBM patients (N = 10) coupled with histological, radiographic, and two-photon excitation fluorescence microscopic analyses. Deconvolution of SPRP followed by functional analyses was performed using CIBEROSRTx and UCell enrichment algorithms, respectively. We further investigated the spatial architecture of 5ALA + enriched regions by analyzing spatial transcriptomics from an independent IDH-wt GBM cohort (N = 16). Lastly, we performed survival analysis using Cox Proportinal-Hazards model on large GBM cohorts. RESULTS: SPRP analysis integrated with single-cell and spatial transcriptomics uncovered that the GBM molecular subtype heterogeneity is likely to manifest regionally in a cell-type-specific manner. Infiltrative 5ALA + cell population(s) harboring transcriptionally concordant GBM and myeloid cells with mesenchymal subtype, -active wound response, and glycolytic metabolic signature, was shown to reside within the invasive margin spatially distinct from the tumor core. The spatial co-localization of the infiltrating MES GBM and myeloid cells within the 5ALA + region indicates PpIX fluorescence can effectively be utilized to resect the immune reactive zone beyond the tumor core. Finally, 5ALA + gene signatures were associated with poor survival and recurrence in GBM, signifying that the transition from primary to recurrent GBM is not discrete but rather a continuum whereby primary infiltrative 5ALA + remnant tumor cells more closely resemble the eventual recurrent GBM. CONCLUSIONS: Elucidating the unique molecular and cellular features of the 5ALA + population within tumor invasive margin opens up unique possibilities to develop more effective treatments to delay or block GBM recurrence, and warrants commencement of such treatments as early as possible post-surgical resection of the primary neoplasm.

A transcriptome study of p53-pathway related prognostic gene signature set in bladder cancer.

p53 pathway is important in tumorigenesis. However, no study has been performed to specifically investigate the role of p53 pathway genes in bladder cancer (BLCA). In this study, transcriptomics data of muscle invasive bladder cancer patients (n = 411) from The Cancer Genome Atlas (TCGA) were investigated. Using the hallmark p53 pathway gene set, the Non-Negative Matrix factorization (NMF) analysis identified two subtypes (C1 and C2). Clinical, survival, and immunological analysis were done to validate distinct characteristics of the subtypes. Pathway enrichment analysis showed the subtype C1 with poor prognosis having enrichment in genes of the immunity related pathways, where C2 subtype with better prognosis being enriched in genes of the steroid synthesis and drug metabolism pathways. A signature gene set consisting of MDGA2, GNLY, GGT2, UGT2B4, DLX1, and DSC1 was created followed by a risk model. Their expressions were analyzed in RNA extracted from the blood and matched tumor tissues of BLCA patients (n = 10). DSC1 had significant difference of expression (p = 0.005) between the blood and tumor tissues in our BLCA samples. Contrary to the usual normal bladder tissue to blood ratio, DLX1 expression was lower (p = 0.02734) in tumor tissues than in blood. Being the first research of p53 pathway related signature gene set in bladder cancer, this study potentially has a substantial impact on the development of biomarkers for BLCA.

Alteration of Proteotranscriptomic Landscape Reveals the Transcriptional Regulatory Circuits Controlling Key-Signaling Pathways and Metabolic Reprogramming During Tumor Evolution.

The proteotranscriptomic landscape depends on the transcription, mRNA-turnover, translation, and regulated-destruction of proteins. Gene-specific mRNA-to-protein correlation is the consequence of the dynamic interplays of the different regulatory processes of proteotranscriptomic landscape. So far, the critical impact of mRNA and protein stability on their subsequent correlation on a global scale remained unresolved. Whether the mRNA-to-protein correlations are constrained by their stability and conserved across mammalian species including human is unknown. Moreover, whether the stability-dependent correlation pattern is altered in the tumor has not been explored. To establish the quantitative relationship between stability and correlation between mRNA and protein levels, we performed a multi-omics data integration study across mammalian systems including diverse types of human tissues and cell lines in a genome-wide manner. The current study illuminated an important aspect of the mammalian proteotranscriptomic landscape by providing evidence that stability-constrained mRNA-to-protein correlation follows a hierarchical pattern that remains conserved across different tissues and mammalian species. By analyzing the tumor and non-tumor tissues, we further illustrated that mRNA-to-protein correlations deviate in tumor tissues. By gene-centric analysis, we harnessed the hierarchical correlation patterns to identify altered mRNA-to-protein correlation in tumors and characterized the tumor correlation-enhancing and -repressing genes. We elucidated the transcriptional regulatory circuits controlling the correlation-enhancing and -repressing genes that are associated with metabolic reprogramming and cancer-associated pathways in tumor tissue. By tightly controlling the mRNA-to-protein correlation of specific genes, the transcriptional regulatory circuits may enable the tumor cells to evolve in varying tumor microenvironment. The mRNA-to-protein correlation analysis thus can serve as a unique approach to identify the pathways prioritized by the tumor cells at different clinical stages. The component of transcriptional regulatory circuits identified by the current study can serve as potential candidates for stage-dependent anticancer therapy.

Integration of Online Omics-Data Resources for Cancer Research.

The manifestations of cancerous phenotypes necessitate alterations at different levels of information-flow from genome to proteome. The molecular alterations at different information processing levels serve as the basis for the cancer phenotype to emerge. To understand the underlying mechanisms that drive the acquisition of cancer hallmarks it is required to interrogate cancer cells using multiple levels of information flow represented by different omics - such as genomics, epigenomics, transcriptomics, and proteomics. The advantage of multi-omics data integration comes with a trade-off in the form of an added layer of complexity originating from inherently diverse types of omics-datasets that may pose a challenge to integrate the omics-data in a biologically meaningful manner. The plethora of cancer-specific online omics-data resources, if able to be integrated efficiently and systematically, may facilitate the generation of new biological insights for cancer research. In this review, we provide a comprehensive overview of the online single- and multi-omics resources that are dedicated to cancer. We catalog various online omics-data resources such as The Cancer Genome Atlas (TCGA) along with various TCGA-associated data portals and tools for multi-omics analysis and visualization, the International Cancer Genome Consortium (ICGC), Catalogue of Somatic Mutations in Cancer (COSMIC), The Pathology Atlas, Gene Expression Omnibus (GEO), and PRoteomics IDEntifications (PRIDE). By comparing the strengths and limitations of the respective online resources, we aim to highlight the current biological and technological challenges and possible strategies to overcome these challenges. We outline the available schemes for the integration of the multi-omics dimensions for stratifying cancer patients and biomarker prediction based on the integrated molecular-signatures of cancer. Finally, we propose the multi-omics driven systems-biology approaches to realize the potential of precision onco-medicine as the future of cancer research. We believe this systematic review will encourage scientists and clinicians worldwide to utilize the online resources to explore and integrate the available omics datasets that may provide a window of opportunity to generate new biological insights and contribute to the advancement of the field of cancer research.

Investigation of the efficacy and safety of eltrombopag to correct thrombocytopenia in moderate to severe dengue patients - a phase II randomized controlled clinical trial.

BACKGROUND: The dengue-infected patients with or without hemorrhagic manifestations, typically exhibit moderate to severe thrombocytopenia. A thrombopoietin receptor agonist - eltrombopag has been efficacious in correcting thrombocytopenia in patients with various pathological conditions including immune thrombocytopenia, chronic liver disease, and severe aplastic anemia. This study investigated the efficacy and safety of eltrombopag to correct dengue-mediated thrombocytopenia. METHODS: In this open-label, randomized controlled phase-II trial, patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) having platelet (PLT) count lower than 100 × 109/L without comorbidity, pregnancy, and liver abnormalities were enrolled in Dhaka Medical College Hospital, Better Life Hospital and AMZ hospital, Dhaka, Bangladesh. Between October 10, 2019, and December 30, 2019, 123 DF and DHF patients were assessed for eligibility to be enrolled in the trial. Fourteen patients were excluded as they failed to fulfill the inclusion criteria (N = 6) or refused to participate in the trial (N = 8). Finally, 109 patients were randomly assigned to either Group 1, (N = 36), Group 2 (N = 37), or Control-group (N = 36) in a 1:1:1 ratio. Two doses of eltrombopag - 25 mg/day and 50 mg/day were administered to Group-1 and Group-2 patients, respectively whereas the control-group patients received standard dengue treatment without eltrombopag. The management of all enrolled patients was according to WHO guidelines. The randomization procedure was performed by using a computerized system (STATA Inc.). CBC and immature platelet fraction (IPF) were monitored from Day-0 to Day-7. Absolute immature platelet count (A-IPC) was calculated from PLT count and IPF for each patient. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured on Day-0 and Day-4 and an Ultrasonogram (USG) of the abdomen was performed on Day-4 and Day-7 for each patient. The efficacy of eltrombopag as the primary outcome of the trial was investigated by the proportion of patients with recovered platelet count receiving eltrombopag with corrected platelet count (platelet count above the lower normal limit: 150 × 109/L) on Day-7 of the enrollment as compared to the Control-group. As the secondary outcomes, the reduction of bleeding tendency in response to eltrombopag as well as the safety of eltrombopag in dengue patients were assessed. The safety was evaluated in case of adverse events, liver function enzymes AST/ALT levels and USG. This trial is registered with the international clinical trial registry, number SLCTR/2019/037. RESULTS: A total of 101 patients including 77 DF and 24 DHF patients completed the trial as eight patients left the trial without completing the follow-up. Patients of the different groups were compared with respect to mean age (26±8, 30±10 and 30±9 years for, Group-1,-2 and Control-group, respectively) (p-value= 0.23) and basal PLT count (Group-1: 58±24 × 109; Group-2: 52±29 × 109 and control-group: 55±30 × 109) (p-value= 0.63). The mean PLT counts for Group-1 (332 × 109/L ± 92) and Group-2 (371 × 109/L ± 111) were significantly higher than control-group (194 × 109/L ± 96) on Day-7 (adjusted p-value= 1.15 × 10-06 for Group-1 vs. Control-group, and adjusted p-value= 1.82 × 10-08 for Group-2 vs. Control-group).). On Day-7, 91% of Group-1 (N = 30) and Group-2 (N = 32) patients who received eltrombopag achieved primary endpoint of PLT count above than lower normal limit (150 × 109/L) (Group-1: 91%, OR: 8.33, 95% CI: 2.11 to 32.80, p-value: 0.0024 and Group-2: 91%, OR: 8.89, 95% CI: 2.26 to 34.89, p-value: 0.0017) compared to 55% (N = 18) of control-group patients who did not receive eltrombopag. The bleeding manifestations for thirteen out of fourteen grade-II DHF patients were subsided within Day-7 who received eltrombopag, whereas four out of ten grade-II DHF patients with PLT counts lower than the lower normal limit in the control group showed intermittent bleeding symptoms throughout the trial period. Mean A-IPC but not IPF was significantly higher for eltrombopag-treated groups in comparison to the Control-group. The frequency of the most common adverse events (vomiting and diarrheal tendencies) was similar in the treated-and control-groups (N = 5, 15%, and N = 3, 9% for Group-1 and -2, respectively vs. N = 4, 12% in the Control-group). Ten (30%) patients of Group-1 and, fourteen (40%) patients of Group-2 showed increased AST (U/L) as opposed to nine patients (27%) in the Control-group. Increased ALT levels were observed for three (9%), nine (26%), and seven (21%) patients belonging to the Group-1, -2, and Control-group, respectively. PLT counts higher than the upper normal limit (450 × 109/L) on Day-7 were observed for seven patients who were administered the higher dose (50 mg/day) in contrast to the three patients receiving the lower dose (25 mg/day). USG reports did not show thrombosis events in any of the patients. INTERPRETATION: The trial revealed that the administration of eltrombopag in a short regimen for three days was efficacious to restore the PLT count in DF and DHF patients. The higher number of A-IPCs in eltrombopag treated patients underscored the possible mode of action of eltrombopag through stimulating megakaryopoiesis in dengue patients. The trial hints toward the positive effect of eltrombopag in the cessation of bleeding manifestation. Administration of the lower dose (25 mg/day) of eltrombopag was shown to be safer and equally efficacious to the higher dose (50 mg/day) in treating dengue-infected patients.

Microbiology, clinical spectrum and outcome of peritonitis in patients undergoing peritoneal dialysis in India: Results from a multicentric, observational study.

BACKGROUND: Peritoneal dialysis (PD)-related peritonitis is a major risk factor for drop out of patients on continuous ambulatory PD (CAPD) and automated PD (APD). Factors affecting PD-related peritonitis and centre-specific microbiological data are lacking in India. A multicentric prospective observational study was designed to overcome the gaps in the existing data regarding causative organism and outcome. METHODOLOGY: The present study was a prospective, uncontrolled, open-label; observational study conducted in 21 centres representing all the four geographical regions (North, South, East and West) of India between April 2010 and December 2011. RESULTS: A total of 244 patients on chronic PD with peritonitis were enrolled in the study (CAPD and APD), who met the inclusion criteria, from 21 centres covering the different geographical areas of India. Amongst the 85 samples that were culture positive, 38 (44.7%) were in the monsoon season followed by 23 (27.1%) in the post-monsoon, 18 (21.2%) during winter and 11 (12.9%) during summer. Maximum culture positivity (72.7%) was observed with automated culture technique. Microorganisms could be isolated in only 85 cases (35.3%) while the remaining samples were culture negative (156/241, 64.7% of samples). Organisms isolated were Gram-negative in 47.8%, Gram-positive in 36.7%, fungal in 13.3% and Mycobacterium tuberculosis in 2.2%. CONCLUSION: This large multicentre study of peritonitis offers insights into the aetiology and outcomes of infectious complications of chronic PD in India that are germane to clinical decision-making.