MYC multimers shield stalled replication forks from RNA polymerase.

Oncoproteins of the MYC family drive the development of numerous human tumours1. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II2,3. MYC proteins can also coordinate transcription with DNA replication4,5 and promote the repair of transcription-associated DNA damage6, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks7,8. MYC multimerization is triggered in a HUWE16 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.

Baseline and post-treatment biomarkers of resistance to anti-PD-1 therapy in acral and mucosal melanoma: an observational study.

BACKGROUND: Immunotherapies targeting programmed cell death-1 (PD-1) and its ligands have improved clinical outcomes for advanced melanoma. However, many tumors exhibit primary resistance or acquire secondary resistance after an initial positive response. The mechanisms of resistance are not well understood, and no validated predictive biomarkers are available. This exploratory study aimed to characterize baseline differences and molecular changes arising during treatment in acral and mucosal melanomas that exhibited primary or secondary resistance to anti-PD-1 monotherapy. METHODS: This was an observational retrospective study of 124 patients who had been treated for metastatic acral or mucosal melanoma with anti-PD-1 monotherapy. Tumor samples were collected at baseline (all patients) and post-treatment (resistant tumors only) and were assayed by immunohistochemistry, whole-exome sequencing, and RNA sequencing. RESULTS: At baseline, more non-progressor than resistant tumors exhibited expression of PD-L1, immune cell infiltration, and high tumor mutational burden (TMB); baseline PD-L1 expression was also more common in secondary-resistant than in primary-resistant tumors as well as in late versus early secondary-resistant tumors. Non-progressor tumors also had higher median baseline expression of an 18-gene T cell-inflamed gene expression profile (TcellinfGEP). Among resistant tumors, the proportion of PD-L1-positive melanomas and the expression of the TcellinfGEP mRNA signature increased during treatment, while the expression of mRNA signatures related to WNT and INFA1 signaling decreased. There was evidence for greater changes from baseline in secondary-resistant versus primary-resistant tumors for some markers, including expression of RAS-related and WNT-related mRNA signatures and density of CD11c+ and FOXP3+ T cells. Greater changes in CD11c+ cell density were observed in early compared with late secondary-resistant tumors. CONCLUSIONS: Our findings suggest that TcellinfGEP and PD-L1 expression, TMB, immune cell infiltration, and RAS and WNT signaling warrant further investigation as potential mechanisms and/or biomarkers of anti-PD-1 therapy resistance in acral and mucosal melanomas. Confirmation of these findings in larger populations is needed.

Effect of 17ß-estradiol on the daily pattern of ACE2, ADAM17, TMPRSS2 and estradiol receptor transcription in the lungs and colon of male rats.

Covid-19 progression shows sex-dependent features. It is hypothesized that a better Covid-19 survival rate in females can be attributed to the presence of higher 17ß-estradiol (E2) levels in women than in men. Virus SARS-CoV-2 is enabled to enter the cell with the use of angiotensin converting enzyme 2 (ACE2). The expression of several renin-angiotensin system components has been shown to exert a rhythmic pattern, and a role of the circadian system in their regulation has been implicated. Therefore, the aim of the study is to elucidate possible interference between E2 signalling and the circadian system in the regulation of the expression of ACE2 mRNA and functionally related molecules. E2 was administered at a dosage of 40 µg/kg/day for 7 days to male Wistar rats, and sampling of the lungs and colon was performed during a 24-h cycle. The daily pattern of expression of molecules facilitating SARS-CoV-2 entry into the cell, clock genes and E2 receptors was analysed. As a consequence of E2 administration, a rhythm in ACE2 and TMPRSS2 mRNA expression was observed in the lungs but not in the colon. ADAM17 mRNA expression showed a pronounced rhythmic pattern in both tissues that was not influenced by E2 treatment. ESR1 mRNA expression exerted a rhythmic pattern, which was diminished by E2 treatment. The influence of E2 administration on ESR2 and GPER1 mRNA expression was greater in the lungs than in the colon as a significant rhythm in ESR2 and GPER1 mRNA expression appeared only in the lungs after E2 treatment. E2 administration also increased the amplitude of bmal1 expression in the lungs, which implicates altered functioning of peripheral oscillators in response to E2 treatment. The daily pattern of components of the SARS-CoV-2 entrance pathway and their responsiveness to E2 should be considered in the timing of pharmacological therapy for Covid-19.

Concomitant Expression of IL-6 and TGF-ß Cytokines and their Receptors in Peripheral Blood of Patients with Multiple Sclerosis: The Effects of INFß Drugs.

BACKGROUND: Concomitant signals from IL-6 and TGF-ß have a central role in the Th17 cells development and differentiation, and these cells are the main promoters of demyelinating inflammation in the central nervous system (CNS) resulting in multiple sclerosis (MS). OBJECTIVES: To evaluate the simultaneous IL-6 and TGF-ß gene and their receptor protein expression in patients with Relapsing-Remitting (RR)-MS. MATERIALS AND METHODS: IL-6 and TGF-ß mRNA and their receptor expression on the surface of CD4+T cells were evaluated using real-time PCR (RT-PCR) and flow cytometry, respectively. RESULTS: The IL-6 mRNA expression in patients with RRMS was significantly higher than in the controls (p= 0.019). When patients who did not receive any other treatment were compared with the controls, the significant difference was substantial (p=0.006). The TGF-ß mRNA expression in patients was lower than in the controls (p = 0.03). However, in patients receiving IFNß, it increased compared with the other patients (p= 0.036). There was no difference in cytokine receptor expression between patients and the control group. CONCLUSION: Our data conclude an increase and decrease in mRNA expression levels of IL-6 and TGF-ß in patients with RRMS, respectively. Moreover, there were no significant differences in receptor expression of either cytokines. Based on our data the balance of TGF and IL-6 appears to have a positive impact on the disease control.

Novel Benzoxazoles Containing 4-Amino-Butanamide Moiety Inhibited LPS-Induced Inflammation by Modulating IL-6 or IL-1ß mRNA Expression.

LPS induces inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, and causes an inflammatory response. The development of small molecules that have suppressive effect on those inflammatory cytokines is a desirable strategy for the treatment of inflammatory diseases. We synthesized 12 novel compounds with 4-amino-N-(4-(benzo[d]oxazol-2-ylamino)phenyl)butanamide moiety and evaluated their biological activities. Among them, 4 compounds (compound 5d, 5c, 5f, 5m and synthetic intermediate 4d) showed potent inhibition activities on IL-1ß and IL-6 mRNA expression in vitro. Further, in vivo activity was evaluated with two compounds (5f and 4d) and mRNA levels of IL-1ß, IL-6, and TNF-α were significantly decreased without hepatotoxicity. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective for suppression of representative inflammatory cytokines.

A New Hybrid Protein Is a Novel Regulator for Experimental Colitis in Rats.

We newly developed a hybrid protein, tentatively named rMIKO-1, using gene technology. We herein investigated the effects of rMIKO-1 on activated macrophages and discussed its potential as a suppressor of experimental colitis. Fluorescent microscopy was used to observe the dynamic mobility of rMIKO-1 in macrophages. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, fluorescent immunochemical staining, flow cytometry, enzyme-linked immunosorbent assays, a polymerase chain reaction/quantitative polymerase chain reaction, and hematoxylin and eosin staining were conducted to assess the potential activity of rMIKO-1. A large amount of bleeding was observed in rats treated with 5% dextran sulfate sodium (DSS) alone on day 8 after treatment initiation, but not in those treated with 5% DSS plus rMIKO-1. In the in vitro assay, rMIKO-1 rapidly bound to macrophages, immediately entered cells by an unknown mechanism, and then migrated inside the nucleus. This result suggests that rMIKO-1 plays important immunological roles in the nucleus. Despite the activation of macrophages by lipopolysaccharide, the mRNA expression of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß, was significantly suppressed in macrophages preliminarily treated with rMIKO-1 for 1 h. Complexes of rMIKO-1 with nuclear factor-kappa B (NF-κB)/p65 and ß-actin formed in activated macrophages, which attenuated experimental colitis in rats. These results strongly suggest that rMIKO-1 negatively regulates excessively activated macrophages through the NF-κB/p65 signaling pathway. Therefore, rMIKO-1 is a novel suppressor of experimental colitis in rats through the negative regulation of activated macrophages.

Predicting functional circular RNA-based competitive endogenous RNA network in gastric carcinoma using novel bioinformatics analysis.

Gastric cancer (GC) remains one of the most prevalent types of malignancies worldwide, and also one of the most reported lethal tumor-related diseases. Circular RNAs (circRNAs) have been certified to be trapped in multiple aspects of GC pathogenesis. Yet, the mechanism of this regulation is mostly undefined. This research is designed to discover the vital circRNA-microRNA (miRNA)-messenger RNA (mRNA) regulatory network in GC. Expression profiles with diverse levels including circRNAs, miRNAs, and mRNAs were all determined using microarray public datasets from Gene Expression Ominous (GEO). The differential circRNAs expressions were recognized against the published robust rank aggregation algorithm. Besides, a circRNA-based competitive endogenous RNA (ceRNA) interaction network was visualized via Cytoscape software (version 3.8.0). Functional and pathway enrichment analysis associated with differentially expressed targeted mRNAs were conducted using Cytoscape and an online bioinformatics database. Furthermore, an interconnected protein-protein interaction association network which consisted of 51 mRNAs was predicted, and hub genes were screened using STRING and CytoHubba. Then, several hub genes were chosen to explore their expression associated with survival rate and clinical stage in GEPIA and Kaplan-Meier Plotter databases. Finally, a carefully designed circRNA-related ceRNA regulatory subnetwork including four circRNAs, six miRNAs, and eight key hub genes was structured using the online bioinformatics tool.

High FGFR1-4 mRNA Expression Levels Correlate with Response to Selective FGFR Inhibitors in Breast Cancer.

PURPOSE: FGFR1 amplification (FGFR1amp) is recurrent in metastatic breast cancer (MBC) and is associated with resistance to endocrine therapy and CDK4/6 inhibitors (CDK4/6is). Multi-tyrosine kinase inhibitors (MTKIs) and selective pan-FGFR inhibitors (FGFRis) are being developed for FGFR1amp breast cancer. High-level FGFR amplification and protein expression by IHC have identified breast cancer responders to FGFRis or MTKIs, respectively. EXPERIMENTAL DESIGN: Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient-derived xenografts (PDXs) harboring amplification in FGFR1/2/3/4 and in 10 patients receiving either an FGFRi/MTKI. mRNA levels were measured on FFPE tumor samples using two commercial strategies. Proliferation and angiogenesis were evaluated by detecting Ki-67 and CD31 in viable areas by immunofluorescence. RESULTS: High FGFR1-4 mRNA levels but not copy-number alteration (CNA) is associated with FGFRi response. Treatment with MTKIs showed higher response rates than with FGFRis (86% vs. 53%), regardless of the FGFR1-4 mRNA levels. FGFR-addicted PDXs exhibited an antiproliferative response to either FGFRis or MTKIs, and PDXs exclusively sensitive to MTKI exhibited an additional antiangiogenic response. Consistently, the clinical benefit of MTKIs was not associated with high FGFR1-4 mRNA levels and was observed in patients previously treated with antiangiogenic drugs. CONCLUSIONS: Tailored therapy with FGFRis in molecularly selected MBC based on high FGFR1-4 mRNA levels warrants prospective validation in patients with CDK4/6i-resistant luminal breast cancer and in patients with TNBC without targeted therapeutic options.

Regulations of expressions of rat/human sulfotransferases by anticancer drug, nolatrexed, and micronutrients.

Cancer is related to the cellular proliferative state. Increase in cell-cycle regulatory function augments cellular folate pool. This pathway is therapeutically targeted. A number of drugs influences this metabolism, that is, folic acid, folinic acid, nolatrexed, and methotrexate. Our previous study showed methotrexate influences on rat/human sulfotransferases. Present study explains the effect of nolatrexed (widely used in different cancers) and some micronutrients on the expressions of rat/human sulfotransferases. Female Sprague-Dawley rats were treated with nolatrexed (01-100 mg/kg) and rats of both sexes were treated to folic acid (100, 200, or 400 mg/kg) for 2-weeks and their aryl sulfotransferase-IV (AST-IV; ß-napthol sulfation) and sulfotransferase (STa; DHEA sulfation) activities, protein expression (western blot) and mRNA expression (RT-PCR) were tested. In human-cultured hepatocarcinoma (HepG2) cells nolatrexed (1 nM-1.2 mM) or folinic acid (10 nM-10 µM) were applied for 10 days. Folic acid (0-10 µM) was treated to HepG2 cells. PPST (phenol catalyzing), MPST (dopamine and monoamine), DHEAST (dehydroepiandrosterone and DHEA), and EST (estradiol sulfating) protein expressions (western-blot) were tested in HepG2 cells. Present results suggest that nolatrexed significantly increased sulfotransferases expressions in rat (protein, STa, F = 4.87, P < 0.05/mRNA, AST-IV, F = 6.702, P < 0.014; Student's t test, P < 0.01-0.05) and HepG2 cells. Folic acid increased sulfotransferases activity/protein in gender-dependant manner. Both folic and folinic acid increased several human sulfotransferases isoforms with varied level of significance (least or no increase at highest dose) in HepG2 cells pointing its dose-dependent multiphasic responses. The clinical importance of this study may be furthered in the verification of sulfation metabolism of several exogenous/endogenous molecules, drug-drug interaction and their influences on cancer pathophysiological processes. Further studies are necessary.

Clinical significance of FAT1 gene mutation and mRNA expression in patients with head and neck squamous cell carcinoma.

The FAT1 gene functions as a tumor suppressor or promoter and remains incompletely understood. We examined the clinical significance of FAT1 in head and neck squamous cell carcinoma (HNSCC) using four publicly available HNSCC cohorts and one HNSCC cohort enrolled at a tertiary medical center. We developed FAT1 signatures reflecting FAT1 mutations and mRNA expression using one cohort. Patients with HNSCC were classified into FAT1-associated low risk (FAT1-LR; n = 195) and FAT1-associated high risk (FAT1-HR; n = 371) subgroups. The five-year overall survival and recurrence-free survival rates were significantly lower in the FAT1-HR subgroup than in the FAT1-LR subgroup (P = 0.01 and 0.003, respectively). The clinical significance of FAT1 was validated using four independent cohorts. Cox proportional hazards models showed that the FAT1 signature was an independent prognostic factor for HNSCC patients. In addition, FAT1 signature was associated with the response to radiotherapy, advanced stage, and human papilloma virus (HPV) status in HNSCC patients. In conclusion, the FAT1 gene signature was associated with prognosis of HNSCC and may help to provide personalized treatments for HNSCC patients.

A Potential Probiotic for Diarrhea: Clostridium tyrobutyricum Protects Against LPS-Induced Epithelial Dysfunction via IL-22 Produced By Th17 Cells in the Ileum.

Probiotics are clinically used for diarrhea and inflammatory bowel diseases in both humans and animals. Previous studies have shown that Clostridium tyrobutyricum (Ct) protects against intestinal dysfunction, while its regulatory function in the gut needs further investigation and the related mechanisms are still not fully elucidated. This study aims to further verify the protective function of Ct and reveal its underlying mechanisms in alleviating diarrhea and intestinal inflammation. Ct inhibited LPS-induced diarrhea and intestinal inflammation in the ileum. IL-22 was identified and the protective role of Ct in the ileum presented an IL-22-dependent manner according to the transcriptomic analysis and in vivo interference mice experiments. The flow cytometric analysis of immune cells in the ileum showed that Ct enhanced the proportions of Th17 cells in response to LPS. The results of in situ hybridization further verified that Ct triggered Th17 cells to produce IL-22, which combined with IL-22RA1 expressed in the epithelial cells. Moreover, Ct was unable to enhance the levels of short-chain fatty acids (SCFAs) in the ileum, suggesting that the protective role of Ct in the ileum was independent of SCFAs. This study uncovered the role of Ct in alleviating diarrhea and inflammation with the mechanism of stimulating Th17 cells in the lamina propria to produce IL-22, highlighting its potential application as a probiotic for diarrhea and inflammation in the ileum.

Targeted Long-Read Sequencing Decodes the Transcriptional Atlas of the Founding RAS Gene Family Members.

The complicity of human RAS proteins in cancer is a well-documented fact, both due to the mutational hyperactivation of these GTPases and the overexpression of the genes encoding these proteins. Thus, it can be easily assumed that the study of RAS genes at the transcriptional and post-transcriptional level is of the utmost importance. Although previous research has shed some light on the basic mechanisms by which GTPases are involved in tumorigenesis, limited information is known regarding the transcriptional profile of the genes encoding these proteins. The present study highlights for the first time the wide spectrum of the mRNAs generated by the three most significant RAS genes (KRAS, NRAS and HRAS), providing an in-depth analysis of the splicing events and exon/intron boundaries. The implementation of a versatile, targeted nanopore-sequencing approach led to the identification of 39 novel RAS mRNA transcript variants and to the elucidation of their expression profiles in a broad panel of human cell lines. Although the present work unveiled multiple hidden aspects of the RAS gene family, further study is required to unravel the biological function of all the novel alternative transcript variants, as well as the putative protein isoforms.

LPS Stimulation Induces Small Heterodimer Partner Expression Through the AMPK-NRF2 Pathway in Large Yellow Croaker (Larimichthys crocea).

The mall heterodimer partner (SHP) plays an important regulatory role in mammal inflammation. The main objective of this study was to investigate the response of SHP to inflammatory stimulation and its underlying mechanism. The shp gene from large yellow croakers, was cloned, and this gene is mainly expressed in the liver and intestine. Lipopolysaccharide (LPS) stimulation induced the mRNA expression and protein level of SHP in macrophages of large yellow croakers. Overexpression of SHP significantly decreased mRNA expression of tnfα, il-1ß, il-6 and cox2 induced by LPS treatment in macrophages. LPS stimulation increased the phosphorylation level of Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) in macrophages. AMPK inhibitor treatment significantly decreased the expression of SHP induced by LPS while AMPK activator significantly increased the expression of SHP. The nuclear factor-erythroid 2-related factor 2 (NRF2) increased the promoter activity of SHP in large yellow croakers and the level of nuclear NRF2 was increased by LPS stimulation and AMPK activation. NRF2 inhibitor treatment significantly decreased mRNA expression of shp induced by LPS and AMPK activator. In conclusion, LPS can induce SHP expression by activating the AMPK-NRF2 pathway while SHP could negatively regulate LPS-induced inflammation in large yellow croakers. This study may be benefit to the development of immunology of marine fish and provide new ideas for inflammation-related diseases.

Graft-Versus-Host Disease Prevention by In Vitro-Generated Myeloid-Derived Suppressor Cells Is Exclusively Mediated by the CD11b+CD11c+ MDSC Subpopulation.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells that dampen overwhelming adaptive immune responses through multiple mechanisms and are recognized as an attractive novel immune intervention therapy for counteracting the destructive effects of graft- versus -host disease (GVHD) developing after allogeneic bone marrow transplantation (BMT). MDSCs can be produced in great numbers for cellular therapy, but they present a mixture of subsets whose functions in GVHD prevention are undefined. Here, we generated MDSCs in vitro from murine BM cells in the presence of GM-CSF and defined the integrin CD11c as a marker to subdivide MDSCs into two functional subgroups: CD11b+CD11c+ and CD11b+CD11c- MDSCs. Isolated CD11b+CD11c+ and CD11b+CD11c- MDSCs both inhibited alloantigen-stimulated T-cell proliferation in vitro, although CD11b+CD11c+ MDSCs were more efficient and expressed higher levels of different immunosuppressive molecules. Likewise, expression of surface markers such as MHC class II, CD80, CD86, or PD-L1 further delineated both subsets. Most importantly, only the adoptive transfer of CD11b+CD11c+ MDSCs into a single MHC class I-disparate allogeneic BMT model prevented GVHD development and strongly decreased disease-induced mortality, while CD11b+CD11c- MDSCs were totally ineffective. Surprisingly, allogeneic T-cell homing and expansion in lymphatic and GVHD target organs were not affected by cotransplanted CD11b+CD11c+ MDSCs indicating a clear contradiction between in vitro and in vivo functions of MDSCs. However, CD11b+CD11c+ MDSCs shifted immune responses towards type 2 immunity reflected by increased Th2-specific cytokine expression of allogeneic T cells. Induction of type 2 immunity was mandatory for GVHD prevention, since CD11b+CD11c+ MDSCs were ineffective if recipients were reconstituted with STAT6-deficient T cells unable to differentiate into Th2 cells. Most importantly, the beneficial graft- versus -tumor (GVT) effect was maintained in the presence of CD11b+CD11c+ MDSCs since syngeneic tumor cells were efficiently eradicated. Strong differences in the transcriptomic landscape of both subpopulations underlined their functional differences. Defining CD11b+CD11c+ MDSCs as the subset of in vitro-generated MDSCs able to inhibit GVHD development might help to increase efficiency of MDSC therapy and to further delineate relevant target molecules and signaling pathways responsible for GVHD prevention.

High Induction of IL-6 Secretion From hUCMSCs Optimize the Potential of hUCMSCs and TCZ as Therapy for COVID-19-Related ARDS.

Biological and cellular interleukin-6 (IL-6)-related therapies have been used to treat severe COVID-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure, which prompted further exploration of the role of IL-6 in human umbilical cord mesenchymal stem cell (hUCMSC) therapy. Peripheral blood mononuclear cells (PBMCs) were responders cocultured with hUCMSCs or exogenous IL-6. A PBMC suppression assay was used to analyze the anti-inflammatory effects via MTT assay. The IL-6 concentration in the supernatant was measured using ELISA. The correlation between the anti-inflammatory effect of hUCMSCs and IL-6 levels and the relevant roles of IL-6 and IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was further evaluated. We reported that hUCMSCs did not initially exert any inhibitory effect on PHA-stimulated proliferation; however, a potent inhibitory effect on PHA-stimulated proliferation was observed, and the IL-6 concentration reached approximately 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 inhibited PHA-stimulated inflammation but less so than hUCMSCs. The inhibitory effects of hUCMSCs on PHA-stimulated PBMCs disappeared after adding an IL-6 neutralizing antibody or pretreatment with tocilizumab (TCZ), an IL-6R antagonist. hUCMSCs exert excellent anti-inflammatory effects by inducing higher IL-6 levels, which is different from TCZ. High concentration of IL-6 cytokine secretion plays an important role in the anti-inflammatory effect of hUCMSC therapy. Initial hUCMSC therapy, followed by TCZ, seems to optimize the therapeutic potential to treat COVID-19-related acute respiratory distress syndrome (ARDS).

AGO2 expression levels and related genetic polymorphisms: influence in renal cell progression and aggressive phenotypes.

Aim: Renal cell carcinoma (RCC) is the most lethal urological cancer and up to 40% of patients submitted to surgery will relapse. Thus, the study aim was to analyze the associations of AGO2 SNPs with RCC patients' prognosis, and evaluate their effect on AGO2 mRNA levels. Materials & methods: The AGO2 rs4961280, rs3928672 and rs11996715 polymorphisms and the relative quantification of AGO2 mRNA levels were analyzed by real-time PCR. Results: We observed that AGO2 rs4961280 AC + AA genotypes carriers presented a higher cancer progression risk (odds ratio= 3.13, p < 0.001), a reduced progression-free survival (log rank test, p = 0.003) and an increased risk of an early relapse (hazard ratio= 2.26, p = 0.008). In fact, these patients also presented higher circulating levels of AGO2 mRNA (p = 0.043), with the high levels being associated with more aggressive tumors. Conclusion: The AGO2 rs4961280 AA/AC genotypes are unfavorable RCC prognostic biomarkers, with the AGO2 levels being a useful RCC aggressive phenotype biomarker.

Neutrophils Facilitate Prolonged Inflammasome Response in the DAMP-Rich Inflammatory Milieu.

Aberrant inflammasome activation contributes to various chronic inflammatory diseases; however, pyroptosis of inflammasome-active cells promptly terminates local inflammasome response. Molecular mechanisms underlying prolonged inflammasome signaling thus require further elucidation. Here, we report that neutrophil-specific resistance to pyroptosis and NLRP3 desensitization can facilitate sustained inflammasome response and interleukin-1ß secretion. Unlike macrophages, inflammasome-activated neutrophils did not undergo pyroptosis, indicated by using in vitro cell-based assay and in vivo mouse model. Intriguingly, danger-associated molecular patterns (DAMP)-rich milieu in the inflammatory region significantly abrogated NLRP3-activating potential of macrophages, but not of neutrophils. This macrophage-specific NLRP3 desensitization was associated with DAMP-induced mitochondrial depolarization that was not observed in neutrophils due to a lack of SARM1 expression. Indeed, valinomycin-induced compulsory mitochondrial depolarization in neutrophils restored inflammasome-dependent cell death and ATP-induced NLRP3 desensitization in neutrophils. Alongside prolonged inflammasome-activating potential, neutrophils predominantly secreted interleukin-1ß rather than other proinflammatory cytokines upon NLRP3 stimulation. Furthermore, inflammasome-activated neutrophils did not trigger efferocytosis-mediated M2 macrophage polarization essential for the initiation of inflammation resolution. Taken together, our results indicate that neutrophils can prolong inflammasome response via mitochondria-dependent resistance to NLRP3 desensitization and function as major interleukin-1ß-secreting cells in DAMP-rich inflammatory region.

Gene expression pattern of Kisspeptin and RFamide-related peptide (Rfrp) in the male Damaraland mole-rat hypothalamus.

Damaraland mole-rats (Fukomys damarensis) are cooperatively breeding, subterranean mammals, which exhibit high reproductive skew. Reproduction is monopolized by the dominant female of the group, while subordinates are anovulatory. Similarly, male subordinates within the colony show no sexual behaviour although they have functional gonads and do not differ from reproductive males in circulating levels of pituitary hormones and testosterone. However, reproductive status affects the neuroendocrine phenotype of males with breeders possessing increased mRNA expression of androgen and progesterone receptors compared to non-breeders in several forebrain regions implicated in the regulation of reproductive behaviour. The RFamide peptides kisspeptin and RFRP-3, encoded by the Kiss1 and Rfrp gene, are considered potent regulators of gonadotropin release. In females, reproductive inhibition is associated with reduced Kiss1 expression within the arcuate nucleus (ARC) and increased Rfrp expression in the anterior hypothalamus. To assess whether differential gene expression of Kiss1 and Rfrp underlies the difference in reproductive behaviour of males, we studied the expression of both genes by means of in situ hybridisation in wild-caught male Damaraland mole-rats with different reproductive status. The distribution of Kiss1 and Rfrp within the hypothalamus was found to be similar to females. Quantification of the Kiss1 hybridisation signal revealed no significant differences in relation to reproductive status. However, there was a significant positive correlation between testis mass and the number of Kiss1-expressing cells in the ARC and the mRNA content per cell, respectively. Analysis of the Rfrp hybridisation signal along the rostro-caudal extent of the hypothalamus revealed that non-reproductive males possessed an increased number of Rfrp-expressing cells at the level of the dorsomedial hypothalamic nucleus (DMH) than reproductive males. These data suggest the Kiss1 expression within the ARC is not associated with reproductive quiescence in subordinate males but instead, inhibitory effects may be mediated by Rfrp-expressing cells in the DMH.

Hypoxic Culture Maintains Cell Growth of the Primary Human Valve Interstitial Cells with Stemness.

The characterization of aortic valve interstitial cells (VICs) cultured under optimal conditions is essential for understanding the molecular mechanisms underlying aortic valve stenosis. Here, we propose 2% hypoxia as an optimum VIC culture condition. Leaflets harvested from patients with aortic valve regurgitation were digested using collagenase and VICs were cultured under the 2% hypoxic condition. A significant increase in VIC growth was observed in 2% hypoxia (hypo-VICs), compared to normoxia (normo-VICs). RNA-sequencing revealed that downregulation of oxidative stress-marker genes (such as superoxide dismutase) and upregulation of cell cycle accelerators (such as cyclins) occurred in hypo-VICs. Accumulation of reactive oxygen species was observed in normo-VICs, indicating that low oxygen tension can avoid oxidative stress with cell-cycle arrest. Further mRNA quantifications revealed significant upregulation of several mesenchymal and hematopoietic progenitor markers, including CD34, in hypo-VICs. The stemness of hypo-VICs was confirmed using osteoblast differentiation assays, indicating that hypoxic culture is beneficial for maintaining growth and stemness, as well as for avoiding senescence via oxidative stress. The availability of hypoxic culture was also demonstrated in the molecular screening using proteomics. Therefore, hypoxic culture can be helpful for the identification of therapeutic targets and the evaluation of VIC molecular functions in vitro.

Acute carbohydrate overfeeding: a redox model of insulin action and its impact on metabolic dysfunction in humans.

A role for fat overfeeding in metabolic dysfunction in humans is commonly implied in the literature. Comparatively less is known about acute carbohydrate overfeeding (COF). We tested the hypothesis that COF predisposes to oxidative stress by channeling electrons away from antioxidants to support energy storage. In a study of 24 healthy human subjects with and without obesity, COF was simulated by oral administration of excess carbohydrates; a two-step hyperinsulinemic clamp was used to evaluate insulin action. The distribution of electrons between oxidative and reductive pathways was evaluated by the changes in the reduction potentials (Eh) of cytoplasmic (lactate, pyruvate) and mitochondrial (ß-hydroxybutyrate, acetoacetate) redox couples. Antioxidant redox was measured by the ratio of reduced to oxidized glutathione. We used cross-correlation analysis to evaluate the relationships between the trajectories of Eh, insulin, glucose, and respiratory exchange during COF. DDIT3 and XBP1s/u mRNA were measured as markers of endoplasmic reticulum stress (ER stress) in adipose tissue before and after COF. Here, we show that acute COF is characterized by net transfer of electrons from mitochondria to cytoplasm. Circulating glutathione is oxidized in a manner that significantly cross-correlates with increasing insulin levels and precedes the decrease in cytoplasmic Eh. This effect is more pronounced in overweight individuals (OW). Markers of ER stress in subcutaneous fat are detectable in OW within 4 h. We conclude that acute COF contributes to metabolic dysfunction through insulin-dependent pathways that promote electron transfer to the cytoplasm and decrease antioxidant capacity. Characterization of redox during overfeeding is important for understanding the pathophysiology of obesity and type 2 diabetes.NEW & NOTEWORTHY Current principles assume that conversion of thermic energy to metabolically useful energy follows fixed rules. These principles ignore the possibility of variable proton uncoupling in mitochondria. Our study shows that the net balance of electron distribution between mitochondria and cytoplasm is influenced by insulin in a manner that reduces proton leakage during overfeeding. Characterization of the effects of insulin on redox balance is important for understanding obesity and insulin resistance.