The spermidine analogue GC7 (N1-guanyl-1,7-diamineoheptane) induces autophagy through a mechanism not involving the hypusination of eIF5A

The exogenous administration of spermidine promotes longevity in many model organisms. It has been proposed that this anti-age activity of spermidine is related to this polyamine's ability to promote autophagy. Since spermidine is the substrate for the eIF5A post-translational modification by hypusination, we asked ourselves whether mature eIF5A may represent the link between spermidine and autophagy induction. To test this hypothesis, we inhibited the conversion of native eIF5A by a pharmacological approach, using the N1-guanyl-1,7-diamineoheptane (GC7), a spermidine analogue which competitively and reversibly inhibits deoxyhypusine synthase (DHS). In addition, we also employed genetic approaches by ablating both the eIF5A protein itself and DHS, the rate limiting enzyme catalyzing the conversion of lysine to hypusine. Collectively the data presented in this study demonstrate that the mature eIF5A (hypusinated form) is not involved in the autophagic pathway and that the inhibitor of DHS, GC7, produces off-target effect(s) resulting in marked induction of basal autophagy. These data are relevant in light of the fact that GC7 is considered a potent and selective inhibitor of DHS and is a potential candidate drug for cancer, diabetes and HIV therapy

Quantification of the Chemical Chaperone 4-Phenylbutyric Acid (4-PBA) in Cell Culture Media via LC-HRMS: Applications in Fields of Neurodegeneration and Cancer

In recent years, 4-phenylbutyric acid (4-PBA), an FDA-approved drug, has increasingly been used as a nonspecific chemical chaperone in vitro and in vitro, but its pharmacodynamics is still not clear. In this context, we developed and validated a Liquid Chromatography–High Resolution Mass Spectrometry (LC-HRMS) method to quantify 4-PBA in NeuroBasal-A and Dulbecco’s Modified Eagle widely used cell culture media. Samples were injected on a Luna® 3 µm PFP(2) 100 Å (100 × 2.0 mm) column maintained at 40 °C. Water and methanol both with 0.1% formic acid served as mobile phases in a step gradient mode. The mass acquisition was performed by selected ion monitoring (SIM) in negative mode for a total run time of 10.5 min at a flow rate of 0.300 mL/min. The analogue 4-(4-Nitrophenyl)-Butyric Acid served as internal standard. Validation parameters were verified according to FDA and EMA guidelines. The quantification ranges from 0.38–24 µM. Inter and intraday RSDs (Relative Standard Deviations) were within 15%. The developed LC-HRMS method allowed the estimation of 4-PBA absorption and adsorption kinetics in vitro in two experimental systems: (i) 4-PBA improvement of protein synthesis in an Alzheimer’s disease astrocytic cell model; and (ii) 4-PBA reduction of endoplasmic reticulum stress in thapsigargin-treated melanoma cell lines. © 2023 by the authors

Inhibition of the histone methyltransferase ezh2 enhances protumor monocyte recruitment in human mesothelioma spheroids

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a long latency period and dismal prognosis. Recently, tazemetostat (EPZ\u20106438), an inhibitor of the histone methyltransferase EZH2, has entered clinical trials due to the antiproliferative effects reported on MPM cells. However, the direct and indirect effects of epigenetic reprogramming on the tumor microenvironment are hitherto unexplored. To investigate the impact of tumor\u2010associated macrophages (TAMs) on MPM cell responsiveness to tazemetostat, we developed a three-dimensional MPM spheroid model that recapitulates in vitro, both monocytes\u2019 recruitment in tumors and their functional differentiation toward a TAM\u2010like phenotype (Mo\u2010TAMs). Along with an increased expression of genes for monocyte chemoattractants, inhibitory immune checkpoints, immunosuppressive and M2\u2010like molecules, Mo\u2010TAMs promote tumor cell proliferation and spreading. Prolonged treatment of MPM spheroids with tazemetostat enhances both the recruitment of Mo\u2010TAMs and the expression of their protumor phenotype. Therefore, Mo\u2010TAMs profoundly suppress the antiproliferative effects due to EZH2 inhibition in MPM cells. Overall, our findings indicate that TAMs are a driving force for MPM growth, progression, and resistance to tazemetostat; therefore, strategies of TAM depletion might be evaluated to improve the therapeutic efficacy of pharmacological inhibition of EZH2

High levels of trim5a are associated with xenophagy in hiv‐1‐infected long‐term nonprogressors

Autophagy is a lysosomal‐dependent degradative mechanism essential in maintaining cellular homeostasis, but it is also considered an ancient form of innate eukaryotic fighting against invading microorganisms. Mounting evidence has shown that HIV‐1 is a critical target of autoph-agy that plays a role in HIV‐1 replication and disease progression. In a special subset of HIV‐1‐infected patients that spontaneously and durably maintain extremely low viral replication, namely, long‐term nonprogressors (LTNP), the resistance to HIV‐1‐induced pathogenesis is ac-companied, in vivo, by a significant increase in the autophagic activity in peripheral blood mon-onuclear cells. Recently, a new player in the battle of autophagy against HIV‐1 has been identified, namely, tripartite motif protein 5α (TRIM5α). In vitro data demonstrated that TRIM5α directly recognizes HIV‐1 and targets it for autophagic destruction, thus protecting cells against HIV‐1 in-fection. In this paper, we analyzed the involvement of this factor in the control of HIV‐1 infection through autophagy, in vivo, in LTNP. The results obtained showed significantly higher levels of TRIM5α expression in cells from LTNP with respect to HIV‐1‐infected normal progressor patients. Interestingly, the colocalization of TRIM5α and HIV‐1 proteins in autophagic vacuoles in LTNP cells suggested the participation of TRIM5α in the autophagy containment of HIV‐1 in LTNP. Al-together, our results point to a protective role of TRIM5α in the successful control of the chronic viral infection in HIV‐1‐controllers through the autophagy mechanism. In our opinion, these findings could be relevant in fighting against HIV‐1 disease, because autophagy inducers might be employed in combination with antiretroviral drugs

Design, Realization, and Characterization of Advanced Adhesives for Joining Ultra-Stable C/C Based Components

The aim of this work is to develop high-performance adhesives to join carbon fiber reinforced composites (C/C) for use in aerospace applications; in order to guarantee sound mechanical strength, a low coefficient of thermal expansion, and ease of application on large components. Several different adhesive formulations, based on phenolic or cyanate-ester resins (charged with the maximum experimentally feasible amount of carbon-based fillers), are developed and tested. The measurements of the lap shear strength at room temperature of the C/C joined by means of one phenolic and one cyanate ester-based resin demonstrates that these formulations are the most suitable for the given application. A complete characterization, by means of viscosimetry, dilatometry, and thermal gravimetric analysis, coupled with gas analysis by means of mass spectroscopy, confirms that the phenolic-based formulation is the most promising joining material. A nano-indenter is used to obtain its Young modulus and hardness, both inside the joint and as a bulk cured adhesive

Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD

This work is licensed under a Creative Commons Attribution-NonCommercialNoDerivs 3.0 Unported License.-- et al.The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD.We acknowledge the following grant support: SAF2012-33283 (MINECO, Spain), Comunidad de Madrid S2010/BMD-2423, EFSD and Amylin Paul Langerhans Grant and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, ISCIII, Barcelona, Spain) to AMV.; SAF2010-16037, SAF2013-43713-R (MINECO) and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD, ISCIII) to PMS. RD12/0042/0019 (ISCIII) and S2010/BMD-2478 (Comunidad de Madrid) to LB, PI 13/01299 and Fundación Mutua Madrileña 2012 to C G-M and AIRC IG-2012 to GMF.Peer Reviewe

Targeting homeostatic mechanisms of endoplasmic reticulum stress to increase susceptibility of cancer cells to fenretinide-induced apoptosis: the role of stress proteins ERdj5 and ERp57

Endoplasmic reticulum (ER) malfunction, leading to ER stress, can be a consequence of genome instability and hypoxic tissue environments. Cancer cells survive by acquiring or enhancing survival mechanisms to counter the effects of ER stress and these homeostatic responses may be new therapeutic targets. Understanding the links between ER stress and apoptosis may be approached using drugs specifically to target ER stress responses in cancer cells. The retinoid analogue fenretinide [N-(4-hydroxyphenyl) retinamide] is a new cancer preventive and chemotherapeutic drug, that induces apoptosis of some cancer cell types via oxidative stress, accompanied by induction of an ER stress-related transcription factor, GADD153. The aim of this study was to test the hypothesis that fenretinide induces ER stress in neuroectodermal tumour cells, and to elucidate the role of ER stress responses in fenretinide-induced apoptosis. The ER stress genes ERdj5, ERp57, GRP78, calreticulin and calnexin were induced in neuroectodermal tumour cells by fenretinide. In contrast to the apoptosis-inducing chemotherapeutic drugs vincristine and temozolomide, fenretinide induced the phosphorylation of eIF2α, expression of ATF4 and splicing of XBP-1 mRNA, events that define ER stress. In these respects, fenretinide displayed properties similar to the ER stress inducer thapsigargin. ER stress responses were inhibited by antioxidant treatment. Knockdown of ERp57 or ERdj5 by RNA interference in these cells increased the apoptotic response to fenretinide. These data suggest that downregulating homeostatic ER stress responses may enhance apoptosis induced by oxidative stress-inducing drugs acting through the ER stress pathway. Therefore, ER-resident proteins such as ERdj5 and ERp57 may represent novel chemotherapeutic targets

Microinjection Manipulation Resulted in the Increased Apoptosis of Spermatocytes in Testes from Intracytoplasmic Sperm Injection (ICSI) Derived Mice

The invention of intracytoplasmic sperm injection (ICSI) has possibly been the most important development in reproductive medicine, one that has given hope to thousands of infertile couples worldwide. However, concerns remain regarding the safety of this method since it is a more invasive procedure than in vitro fertilization (IVF), since a spermatozoon is injected into the oocyte cytoplasm. Using mice derived from IVF technology as a control, we assessed the influence of invasive microinjection in the process of transferring sperm into oocyte cytoplasm in ICSI procedure on the development and physiologic function of resultant offspring. Our results demonstrated that mice produced from ICSI and IVF had no significant difference in phenotypic indices including body weight, forelimb physiology, and learning and memory ability. However, increased spermatocyte apoptosis was observed in the testis of adult ICSI mice, when compared with IVF mice. And, decreased testis weight and marked damage of spermatogenic epithelia were found in aged ICSI mice. Furthermore, proteomic analysis verified that most of the differentiated proteins in testes between adult ICSI and IVF mice were those involved in regulation of apoptosis pathways. Our results demonstrated that the microinjection manipulation used in the ICSI procedure might pose potential risks to the fertility of male offspring. The changed expression of a series of proteins relating to apoptosis or proliferation might contribute to it. Further studies are necessary to better understand all the risks of ICSI

Biomarkers of Inflammation and Lung recovery in ECMO patients with Persistent Pulmonary Hypertension of the Newborn (PPHN): A feasibility study

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Objectives: Extracorporeal membrane oxygenation is a treatment for Persistent Pulmonary Hypertension of the Newborn with high mortality. Hypothesis: the extracorporeal membrane oxygenation circuit results in inflammatory responses that mitigate against successful weaning. Design: Single-center prospective observational feasibility study. Setting: PICU. Patients: Twenty-four neonates requiring extracorporeal membrane oxygenation support for Persistent Pulmonary Hypertension of the Newborn. Interventions: None. Measurements and Main Results: The reference outcome was death or more than 7 days of extracorporeal membrane oxygenation support. Other outcomes included serial measures of plasma-free hemoglobin and markers of its metabolism, leucocyte, platelet and endothelial activation, and biomarkers of inflammation. Of 24 participants recruited between February 2016 and June 2017, 10 died or required prolonged extracorporeal membrane oxygenation support. These patients were sicker at baseline with higher levels of plasma-free hemoglobin within 12 hours of cannulation (geometric mean ratio, 1.92; 95% CIs, 1.00–3.67; p = 0.050) but not thereafter, versus those requiring less than 7 days extracorporeal membrane oxygenation. Serum haptoglobin concentrations were significantly elevated in both groups. Patients who died or required prolonged extracorporeal membrane oxygenation support demonstrated elevated levels of platelet-leucocyte aggregation, but decreased concentrations of mediators of the inflammatory response: interleukin-8, C-reactive protein, and tumor necrosis factor α. Conclusions: Clinical status at baseline and not levels of plasma-free hemoglobin or the systemic inflammatory response may determine the requirement for prolonged extracorporeal membrane oxygenation support in neonates