Piceid Octanoate Protects Retinal Cells against Oxidative Damage by Regulating the Sirtuin 1/Poly-ADP-Ribose Polymerase 1 Axis In Vitro and in rd10 Mice.

Retinitis pigmentosa is a common cause of inherited blindness in adults, which in many cases is associated with an increase in the formation of reactive oxygen species (ROS) that induces DNA damage, triggering Poly-ADP-Ribose Polymerase 1 (PARP1) activation and leading to parthanatos-mediated cell death. Previous studies have shown that resveratrol (RSV) is a promising molecule that can mitigate PARP1 overactivity, but its low bioavailability is a limitation for medical use. This study examined the impact of a synthesized new acylated RSV prodrug, piceid octanoate (PIC-OCT), in the 661W cell line against H2O2 oxidative stress and in rd10 mice. PIC-OCT possesses a better ADME profile than RSV. In response to H2O2, 661W cells pretreated with PIC-OCT preserved cell viability in more than 38% of cells by significantly promoting SIRT1 nuclear translocation, preserving NAD+/NADH ratio, and suppressing intracellular ROS formation. These effects result from expressing antioxidant genes, maintaining mitochondrial function, reducing PARP1 nuclear expression, and preventing AIF nuclear translocation. In rd10 mice, PIC-OCT inhibited PAR-polymer formation, increased SIRT1 expression, significantly reduced TUNEL-positive cells in the retinal outer nuclear layer, preserved ERGs, and enhanced light chamber activity (all p values < 0.05). Our findings corroborate that PIC-OCT protects photoreceptors by modulating the SIRT1/PARP1 axis in models of retinal degeneration.

Impaired Plakophilin-2 in obesity breaks cell cycle dynamics to breed adipocyte senescence.

Plakophilin-2 (PKP2) is a key component of desmosomes, which, when defective, is known to promote the fibro-fatty infiltration of heart muscle. Less attention has been given to its role in adipose tissue. We report here that levels of PKP2 steadily increase during fat cell differentiation, and are compromised if adipocytes are exposed to a pro-inflammatory milieu. Accordingly, expression of PKP2 in subcutaneous adipose tissue diminishes in patients with obesity, and normalizes upon mild-to-intense weight loss. We further show defective PKP2 in adipocytes to break cell cycle dynamics and yield premature senescence, a key rheostat for stress-induced adipose tissue dysfunction. Conversely, restoring PKP2 in inflamed adipocytes rewires E2F signaling towards the re-activation of cell cycle and decreased senescence. Our findings connect the expression of PKP2 in fat cells to the physiopathology of obesity, as well as uncover a previously unknown defect in cell cycle and adipocyte senescence due to impaired PKP2.

Membrane Vesicles of Toxigenic Clostridioides difficile Affect the Metabolism of Liver HepG2 Cells.

Clostridioides difficile infection (CDI) appears to be associated with different liver diseases. C. difficile secretes membrane vesicles (MVs), which may be involved in the development of nonalcoholic fatty liver disease (NALFD) and drug-induced liver injury (DILI). In this study, we investigated the presence of C. difficile-derived MVs in patients with and without CDI, and analyzed their effects on pathways related to NAFLD and DILI in HepG2 cells. Fecal extracellular vesicles from CDI patients showed an increase of Clostridioides MVs. C. difficile-derived MVs that were internalized by HepG2 cells. Toxigenic C. difficile-derived MVs decreased mitochondrial membrane potential and increased intracellular ROS compared to non-toxigenic C. difficile-derived MVs. In addition, toxigenic C. difficile-derived MVs upregulated the expression of genes related to mitochondrial fission (FIS1 and DRP1), antioxidant status (GPX1), apoptosis (CASP3), glycolysis (HK2, PDK1, LDHA and PKM2) and ß-oxidation (CPT1A), as well as anti- and pro-inflammatory genes (IL-6 and IL-10). However, non-toxigenic C. difficile-derived MVs did not produce changes in the expression of these genes, except for CPT1A, which was also increased. In conclusion, the metabolic and mitochondrial changes produced by MVs obtained from toxigenic C. difficile present in CDI feces are common pathophysiological features observed in the NAFLD spectrum and DILI.

Pdx1 Is Transcriptionally Regulated by EGR-1 during Nitric Oxide-Induced Endoderm Differentiation of Mouse Embryonic Stem Cells.

The transcription factor, early growth response-1 (EGR-1), is involved in the regulation of cell differentiation, proliferation, and apoptosis in response to different stimuli. EGR-1 is described to be involved in pancreatic endoderm differentiation, but the regulatory mechanisms controlling its action are not fully elucidated. Our previous investigation reported that exposure of mouse embryonic stem cells (mESCs) to the chemical nitric oxide (NO) donor diethylenetriamine nitric oxide adduct (DETA-NO) induces the expression of early differentiation genes such as pancreatic and duodenal homeobox 1 (Pdx1). We have also evidenced that Pdx1 expression is associated with the release of polycomb repressive complex 2 (PRC2) and P300 from the Pdx1 promoter; these events were accompanied by epigenetic changes to histones and site-specific changes in the DNA methylation. Here, we investigate the role of EGR-1 on Pdx1 regulation in mESCs. This study reveals that EGR-1 plays a negative role in Pdx1 expression and shows that the binding capacity of EGR-1 to the Pdx1 promoter depends on the methylation level of its DNA binding site and its acetylation state. These results suggest that targeting EGR-1 at early differentiation stages might be relevant for directing pluripotent cells into Pdx1-dependent cell lineages.

The Role of Nitric Oxide in Stem Cell Biology.

Nitric oxide (NO) is a gaseous biomolecule endogenously synthesized with an essential role in embryonic development and several physiological functions, such as regulating mitochondrial respiration and modulation of the immune response. The dual role of NO in embryonic stem cells (ESCs) has been previously reported, preserving pluripotency and cell survival or inducing differentiation with a dose-dependent pattern. In this line, high doses of NO have been used in vitro cultures to induce focused differentiation toward different cell lineages being a key molecule in the regenerative medicine field. Moreover, optimal conditions to promote pluripotency in vitro are essential for their use in advanced therapies. In this sense, the molecular mechanisms underlying stemness regulation by NO have been studied intensively over the current years. Recently, we have reported the role of low NO as a hypoxia-like inducer in pluripotent stem cells (PSCs), which supports using this molecule to maintain pluripotency under normoxic conditions. In this review, we stress the role of NO levels on stem cells (SCs) fate as a new approach for potential cell therapy strategies. Furthermore, we highlight the recent uses of NO in regenerative medicine due to their properties regulating SCs biology.

Herbal and Dietary Supplements-Induced Liver Injury in Latin America: Experience From the LATINDILI Network.

BACKGROUND: Herbal and dietary supplements (HDS) consumption, a growing cause of hepatotoxicity, is a common practice among Latin-American populations. OBJECTIVES: To evaluate clinical, laboratory features and outcome in HDS-hepatotoxicity included in the Latin America-Drug Induced Liver Injury (LATINDILI) Network. METHODS: A total of 29 adjudicated cases of HDS hepatotoxicity reported to the LATINDILI Network from October 2011 through December 2019 were compared with 322 DILI cases due to conventional drugs and 16 due to anabolic steroids as well as with other series of HDS-hepatotoxicity. RESULTS: From 367 DILI cases, 8% were attributed to HDS. An increasing trend in HDS-hepatotoxicity was noted over time (p = .04). Camellia sinensis, Herbalife® products, and Garcinia cambogia, mostly used for weight loss, were the most frequently adjudicated causative agents. Mean age was 45 years (66% female). Median time to onset was 31 days. Patients presented typically with hepatocellular injury (83%) and jaundice (66%). Five cases (17%) developed acute liver failure. Compared to conventional medications and anabolic steroids, HDS hepatotoxicity cases had the highest levels of aspartate and alanine transaminase (p = .008 and p = .021, respectively), had more re-exposure events to the culprit HDS (14% vs 3% vs 0%; p = .026), and had more severe and fatal/liver transplantation outcomes (21% vs 12% vs 13%; p = .005). Compared to other DILI cohorts, less HDS hepatotoxicity cases in Latin America were hospitalized (41%). CONCLUSIONS: HDS-hepatotoxicity in Latin-America affects mainly young women, manifests mostly with hepatocellular injury and is associated with higher frequency of accidental re-exposure. HDS hepatotoxicity is more serious with a higher chance of death/liver transplantation than DILI related to conventional drugs.

Stemness of Human Pluripotent Cells: Hypoxia-Like Response Induced by Low Nitric Oxide.

The optimization of conditions to promote the stemness of pluripotent cells in vitro is instrumental for their use in advanced therapies. We show here that exposure of human iPSCs and human ESCs to low concentrations of the chemical NO donor DETA/NO leads to stabilization of hypoxia-inducible factors (HIF-1α and HIF-2α) under normoxia, with this effect being dependent on diminished Pro 402 hydroxylation and decreased degradation by the proteasome. Moreover, the master genes of pluripotency, NANOG and OCT-4, were upregulated. NO also induces a shift in the metabolic profile of PSCs, with an increased expression of hypoxia response genes in glycolysis. Furthermore, a reduction in the mitochondrial membrane potential with lower oxygen consumption and increased expression of mitochondrial fusion regulators, such as DRP1, was observed. The results reported here indicate that NO mimics hypoxia response in human PSCs and enhances their stemness properties when cultured under normoxic conditions.

Influence of Performance Status on the Effectiveness of Pembrolizumab Monotherapy in First-Line for Advanced Non-Small-Cell Lung Cancer: Results in a Real-World Population.

The KEYNOTE-024 clinical trial showed promising results for pembrolizumab in the first-line of treatment of advanced non-small-cell lung cancer (NSCLC). However, the profile of patients in real-world practice differs from those included in this clinical trial. Here, an observational single-center retrospective study was performed through a comparative analysis of clinical outcomes after pembrolizumab therapy according to the Eastern Cooperative Oncology Group Stage Performance Status (ECOG PS). Moreover, univariate and multivariate analyses were carried out to detect prognostic factors. In our cohort, 63.7% of patients had an ECOG PS of 0-1. Regarding response rate, 31.8% of patients had a partial response (PR), 19.3% had stable disease (SD) and 23.9% had progression disease. On the other hand, patients with ECOG PS ≥ 2 showed a significantly lower rate of PR and SD to pembrolizumab than patients with a PS of 0-1. The rate of response, median overall survival (OS) and progression-free survival (PFS) were significantly higher in patients with ECOG PS 0-1 than in those with ECOG PS ≥ 2. In the current study, we found ECOG PS as the only independent predictor of OS and PFS. Due to the ECOG PS scale being a subjective parameter, other tools are needed to identify treatment effectiveness to each patient.

Differential iNKT and T Cells Activation in Non-Alcoholic Fatty Liver Disease and Drug-Induced Liver Injury.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) and idiosyncratic drug-induced liver injury (DILI) could share molecular mechanisms involving the immune system. We aimed to identify activation immunological biomarkers in invariant natural killer T (iNKT) and CD4/CD8+ T cells in NAFLD and DILI. METHODS: We analyzed the activation profile (CD69, CD25, and HLA-DR) and natural killer group 2 member D (NKG2D) on iNKT cells, and CD4/CD8 T cells in peripheral blood mononuclear cells from NAFLD, with or without significant liver fibrosis, and DILI patients. RESULTS: There was an increase in iNKT cells in NAFLD patients compared to DILI or control subjects. Regarding the cellular activation profile, NAFLD with significant liver fibrosis (F ≥ 2) displayed higher levels of CD69+iNKT cells compared to NAFLD with none or mild liver fibrosis (F ≤ 1) and control patients. CD69+iNKT positively correlated with insulin resistance, aspartate aminotransferase (AST) level, liver fibrosis-4 index (FIB4) and AST to Platelet Ratio Index (APRI). DILI patients showed an increase in CD69+ and HLA-DR+ in both CD4+ and CD8+ T cells, detecting the most relevant difference in the case of CD69+CD8+ T cells. CONCLUSIONS: CD69+iNKT may be a biomarker to assess liver fibrosis progression in NAFLD. CD69+CD8+ T cells were identified as a potential distinctive biomarker for distinguishing DILI from NAFLD.

Combining metabolic profiling of plasma and faeces as a fingerprint of insulin resistance in obesity.

BACKGROUND & AIMS: Insulin resistance (IR) is one of the main risk factor for type 2 diabetes mellitus (T2DM). Nevertheless, its underlying pathophysiology is not completely established because IR is triggered by a complex interconnection of numerous factors impairing metabolism, promoting metabolome changes. METHODS: We used a metabolomics approach to identify plasma and faecal metabolites related to IR and obesity. We explored a cohort of 44 subjects at baseline, with 30 of them followed two years thereafter in a longitudinal study after an hypocaloric diet in the obese subjects. RESULTS: In all individuals as a whole, 11 plasma metabolites positively associated with BMI (acetoacetate, creatinine, glycerol, glycerol of lipids, VLDL, fatty esters, myo-inositol, phenylalanine, threonine, tyrosine and valine) and one negatively (phosphocholine), with similar associations at baseline and follow-up. Four of these metabolites (myo-inositol, valine, acetoacetate and phosphocholine) remained significant within obese and non-obese groups. Thirteen faecal metabolites positively associated with BMI at baseline and one negatively (glutamine). However, these correlations did not remain significant at follow-up. The correlations were not always consistent at baseline and at follow-up and the metabolites that showed significant correlations were different for the obese group compared with the control group. The percent change in plasma Δethanolamine, Δglucose, Δuracil and Δhypoxanthine were positively associated with ΔBMI. The percent change in plasma Δphosphocholine and of faecal Δhydroxyphenylacetate, and Δ2-hydroxyphenylacetate were associated with ΔHOMA-IR in those patients that lost weight. Faecal branched chain amino acids (BCAAs) in faeces were associated with IR, following a similar pattern to that described for plasma BCAAs. Choline derivates had an opposite behaviour. CONCLUSIONS: The integration of plasma and faecal metabolites represents a valuable fingerprint that could help in the identification of patients at risk for IR and in the design of novel therapeutic strategies to prevent IR and the development of overt T2DM in the context of obesity. The results are coherent with diet having a much greater impact on faecal metabolomic profile than on plasma metabolome.

Cytoskeletal transgelin 2 contributes to gender-dependent adipose tissue expandability and immune function.

During adipogenesis, preadipocytes' cytoskeleton reorganizes in parallel with lipid accumulation. Failure to do so may impact the ability of adipose tissue (AT) to shift between lipid storage and mobilization. Here, we identify cytoskeletal transgelin 2 (TAGLN2) as a protein expressed in AT and associated with obesity and inflammation, being normalized upon weight loss. TAGLN2 was primarily found in the adipose stromovascular cell fraction, but inflammation, TGF-ß, and estradiol also prompted increased expression in human adipocytes. Tagln2 knockdown revealed a key functional role, being required for proliferation and differentiation of fat cells, whereas transgenic mice overexpressing Tagln2 using the adipocyte protein 2 promoter disclosed remarkable sex-dependent variations, in which females displayed "healthy" obesity and hypertrophied adipocytes but preserved insulin sensitivity, and males exhibited physiologic changes suggestive of defective AT expandability, including increased number of small adipocytes, activation of immune cells, mitochondrial dysfunction, and impaired metabolism together with decreased insulin sensitivity. The metabolic relevance and sexual dimorphism of TAGLN2 was also outlined by genetic variants that may modulate its expression and are associated with obesity and the risk of ischemic heart disease in men. Collectively, current findings highlight the contribution of cytoskeletal TAGLN2 to the obese phenotype in a gender-dependent manner.-Ortega, F. J., Moreno-Navarrete, J. M., Mercader, J. M., Gómez-Serrano, M., García-Santos, E., Latorre, J., Lluch, A., Sabater, M., Caballano-Infantes, E., Guzmán, R., Macías-González, M., Buxo, M., Gironés, J., Vilallonga, R., Naon, D., Botas, P., Delgado, E., Corella, D., Burcelin, R., Frühbeck, G., Ricart, W., Simó, R., Castrillon-Rodríguez, I., Tinahones, F. J., Bosch, F., Vidal-Puig, A., Malagón, M. M., Peral, B., Zorzano, A., Fernández-Real, J. M. Cytoskeletal transgelin 2 contributes to gender-dependent adipose tissue expandability and immune function.

Glutamate interactions with obesity, insulin resistance, cognition and gut microbiota composition.

AIMS: To investigate the interactions among fecal and plasma glutamate levels, insulin resistance cognition and gut microbiota composition in obese and non-obese subjects. METHODS: Gut microbiota composition (shotgun) and plasma and fecal glutamate, glutamine and acetate (NMR) were analyzed in a pilot study of obese and non-obese subjects (n = 35). Neuropsychological tests [Trail making test A (TMT-A) and Trail making test B (TMT-B)] scores measured cognitive information about processing speed, mental flexibility and executive function. RESULTS: Trail-making test score was significantly altered in obese compared with non-obese subjects. Fecal glutamate and glutamate/glutamine ratio tended to be lower among obese subjects while fecal glutamate/acetate ratio was negatively associated with BMI and TMT-A scores. Plasma glutamate/acetate ratio was negatively associated with TMT-B. The relative abundance (RA) of some bacterial families influenced glutamate levels, given the positive association of fecal glutamate/glutamine ratio with Corynebacteriaceae, Coriobacteriaceae and Burkholderiaceae RA. In contrast, Streptococaceae RA, that was significantly higher in obese subjects, negatively correlated with fecal glutamate/glutamine ratio. To close the circle, Coriobacteriaceae/Streptococaceae ratio and Corynebacteriaceae/Streptococaceae ratio were associated both with TMT-A scores and fecal glutamate/glutamine ratio. CONCLUSIONS: Gut microbiota composition is associated with processing speed and mental flexibility in part through changes in fecal and plasma glutamate metabolism.

Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells.

Mitochondrial dysfunction and endoplasmic reticulum stress (ERS) are global processes that are interrelated and regulated by several stress factors. Nitric oxide (NO) is a multifunctional biomolecule with many varieties of physiological and pathological functions, such as the regulation of cytochrome c inhibition and activation of the immune response, ERS and DNA damage; these actions are dose-dependent. It has been reported that in embryonic stem cells, NO has a dual role, controlling differentiation, survival and pluripotency, but the molecular mechanisms by which it modulates these functions are not yet known. Low levels of NO maintain pluripotency and induce mitochondrial biogenesis. It is well established that NO disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux that induce ERS. Thus, at high concentrations, NO becomes a potential differentiation agent due to the relationship between ERS and the unfolded protein response in many differentiated cell lines. Nevertheless, many studies have demonstrated the need for physiological levels of NO for a proper ERS response. In this review, we stress the importance of the relationships between NO levels, ERS and mitochondrial dysfunction that control stem cell fate as a new approach to possible cell therapy strategies.

Nitric Oxide Prevents Mouse Embryonic Stem Cell Differentiation Through Regulation of Gene Expression, Cell Signaling, and Control of Cell Proliferation.

Nitric oxide (NO) delays mouse embryonic stem cell (mESC) differentiation by regulating genes linked to pluripotency and differentiation. Nevertheless, no profound study has been conducted on cell differentiation regulation by this molecule through signaling on essential biological functions. We sought to demonstrate that NO positively regulates the pluripotency transcriptional core, enforcing changes in the chromatin structure, in addition to regulating cell proliferation, and signaling pathways with key roles in stemness. Culturing mESCs with 2 µM of the NO donor diethylenetriamine/NO (DETA/NO) in the absence of leukemia inhibitory factor (LIF) induced significant changes in the expression of 16 genes of the pluripotency transcriptional core. Furthermore, treatment with DETA/NO resulted in a high occupancy of activating H3K4me3 at the Oct4 and Nanog promoters and repressive H3K9me3 and H3k27me3 at the Brachyury promoter. Additionally, the activation of signaling pathways involved in pluripotency, such as Gsk3-ß/ß-catenin, was observed, in addition to activation of PI3 K/Akt, which is consistent with the protection of mESCs from cell death. Finally, a decrease in cell proliferation coincides with cell cycle arrest in G2/M. Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that NO is necessary but not sufficient for the maintenance of pluripotency and the prevention of cell differentiation. J. Cell. Biochem. 117: 2078-2088, 2016. © 2016 Wiley Periodicals, Inc.

Role of nitric oxide in the maintenance of pluripotency and regulation of the hypoxia response in stem cells.

Stem cell pluripotency and differentiation are global processes regulated by several pathways that have been studied intensively over recent years. Nitric oxide (NO) is an important molecule that affects gene expression at the level of transcription and translation and regulates cell survival and proliferation in diverse cell types. In embryonic stem cells NO has a dual role, controlling differentiation and survival, but the molecular mechanisms by which it modulates these functions are not completely defined. NO is a physiological regulator of cell respiration through the inhibition of cytochrome c oxidase. Many researchers have been examining the role that NO plays in other aspects of metabolism such as the cellular bioenergetics state, the hypoxia response and the relationship of these areas to stem cell stemness.