Autophagy drives fibroblast senescence through MTORC2 regulation

Sustained macroautophagy/autophagy favors the differentiation of fibroblasts into myofibroblasts. Cellular senescence, another means of responding to long-term cellular stress, has also been linked to myofibroblast differentiation and fibrosis. Here, we evaluate the relationship between senescence and myofibroblast differentiation in the context of sustained autophagy. We analyzed markers of cell cycle arrest/senescence in fibroblasts in vitro, where autophagy was triggered by serum starvation (SS). Autophagic fibroblasts expressed the senescence biomarkers CDKN1A/p21 and CDKN2A/p16 and exhibited increased senescenceassociated GLB1/beta-galactosidase activity. Inhibition of autophagy in serum-starved fibroblasts with 3-methyladenine, LY294002, or ATG7 (autophagy related 7) silencing prevented the expression of senescence-associated markers. Similarly, suppressing MTORC2 activation using rapamycin or by silencing RICTOR also prevented senescence hallmarks. Immunofluorescence microscopy showed that senescence and myofibroblast differentiation were induced in different cells, suggesting mutually exclusive activation of senescence and myofibroblast differentiation. Reactive oxygen species (ROS) are known inducers of senescence and exposing fibroblasts to ROS scavengers decreased ROS production during SS, inhibited autophagy, and significantly reduced the expression of senescence and myofibroblast differentiation markers. ROS scavengers also curbed the AKT1 phosphorylation at Ser473, an MTORC2 target, establishing the importance of ROS in fuelling MTORC2 activation. Inhibition of senescence by shRNA to TP53/p53 and shRNA CDKN2A/p16 increased myofibroblast differentiation, suggesting a negative feedback loop of senescence on autophagy-induced myofibroblast differentiation. Collectively, our results identify ROS as central inducers of MTORC2 activation during chronic autophagy, which in turn fuels senescence activation and myofibroblast differentiation in distinct cellular subpopulations

Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance.

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.Cancer Research U

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Impaired pituitary actions of thyrotropin-releasing hormone underlie central hypothyroidism in immunoglobulin superfamily, member 1 deficiency syndrome

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause central hypothyroidism. IGSF1 is a transmembrane glycoprotein of unknown function. It is expressed in thyroid-stimulating hormone (TSH) producing thyrotrope cells of the anterior pituitary gland. The protein is co-translationally cleaved into N- and C-terminal domains (NTD and CTD). The CTD is trafficked to the plasma membrane, whereas the NTD is retained in the endoplasmic reticulum (ER). Most intragenic IGSF1 mutations in patients map to the CTD. To better understand IGSF1 function, we used the CRISPR-Cas9 system to introduce a loss-of-function mutation into the IGSF1-CTD in mice. The modified allele harbors a 312 bp deletion, removing part of exon 18 and intron 18. The resulting mRNA is expressed, though at greatly reduced levels relative to wild-type, and contains a novel hybrid exon. This exon introduces frame-shift and a premature stop codon, which affects the trafficking of the CTD. Igsf1 deficient mice show normal serum TSH levels and normal numbers of TSH-expressing thyrotropes. However, expression of the TSH subunits, Tshb and Cga, and TSH protein content are reduced in these animals relative to wild-type littermates. Hypothalamic thyrotropin-releasing hormone (TRH) stimulates TSH synthesis and release. Importantly, pituitary TRH receptor (Trhr) mRNA levels are reduced in Igsf1 deficient males. When challenged with exogenous TRH, Igsf1 deficient mice release TSH, but to a significantly lesser extent than wild-type animals. The mice show similar impairments when endogenous TRH release is increased in response to reduced thyroid hormone feedback. Collectively, these results suggest that IGSF1 deficiency leads to central hypothyroidism via impairments in pituitary TRHR expression and/or downstream signaling.Les mutations causant une perte de fonction du gène membre 1 de la superfamille des immunoglobulines (IGSF1) qui se trouve sur le chromosome X, sont responsables d'un syndrome d'hypothyroïdie centrale. IGSF1 est une glycoprotéine transmembranaire ayant une fonction inconnue. Elle est exprimée dans les thyréotropes de l'hypophyse produisant la thyréostimuline (TSH). IGSF1 est clivées au moment de la traduction et se sépare en deux domaines: le domaine du terminal N (NTD) et du terminal C (CTD). Le CTD est transporté à la surface cellulaire tandis que le NTD est retenu dans le réticulum endoplasmique (ER). La majorité des mutations intra-géniques de IGSF1, découverte chez les patients, se trouvent dans la région qui encode le CTD. Pour mieux comprendre le rôle de IGSF1, nous avons utilisé le système CRISPR-Cas9 pour introduire une mutation causant une perte de fonction de IGSF1-CTD. L'allèle modifié contient une perte de 312 paires de bases qui engendre la perte d'une partie de l'exon 18 et de l'intron 18. L'ARN résultant est exprimé, malgré un niveau d'expression très bas comparativement aux souris de type sauvage, et contient un nouvel exon hybride. Cet exon introduit une mutation qui affecte le cadre the lecture de l'ARN, ce qui cause l'introduction d'un codon-stop prématuré, qui affecte le transport du CTD. Les souris déficientes en Igsf1 démontrent des niveaux normaux de TSH en circulation ainsi qu'un nombre normal de thyréotropes produisant du TSH. Par contre, l'expression des différentes parties de TSH, Tshb and Cga, ainsi que le niveau de protéine de TSH contenu dans l'hypophyse sont réduits chez ces souris comparativement aux souris de type sauvage dans la même cage. L'hormone thyréostimuline (TRH) de l'hypothalamus stimule la synthèse et la sécrétion de TSH. Intéressement, l'ARNm des récepteurs de TRH dans l'hypophyse (Trhr) est réduit chez les souris mâles déficientes en Igsf1. Quand ces souris sont stimulées avec une dose exogène de TRH, elles sécrètent du TSH, mais en quantité moindre que les souris de type sauvage. Les souris déficientes en Igsf1 répondent semblablement à une hausse des niveaux de TRH endogène dû à une réduction des niveaux d'hormones thyroïde. Ensemble, ces résultats suggèrent qu'une déficience en IGSF1 engendre le syndrome d'hypothyroïdie centrale via une réduction de l'expression ou de la cascade de TRHR au niveau de l'hypophyse

DNA Damage, Repair, and Cancer Metabolism

Although there has been a renewed interest in the field of cancer metabolism in the last decade, the link between metabolism and DNA damage/DNA repair in cancer has yet to be appreciably explored. In this review, we examine the evidence connecting DNA damage and repair mechanisms with cell metabolism through three principal links. (1) Regulation of methyl- and acetyl-group donors through different metabolic pathways can impact DNA folding and remodeling, an essential part of accurate double strand break repair. (2) Glutamine, aspartate, and other nutrients are essential for de novo nucleotide synthesis, which dictates the availability of the nucleotide pool, and thereby influences DNA repair and replication. (3) Reactive oxygen species, which can increase oxidative DNA damage and hence the load of the DNA-repair machinery, are regulated through different metabolic pathways. Interestingly, while metabolism affects DNA repair, DNA damage can also induce metabolic rewiring. Activation of the DNA damage response (DDR) triggers an increase in nucleotide synthesis and anabolic glucose metabolism, while also reducing glutamine anaplerosis. Furthermore, mutations in genes involved in the DDR and DNA repair also lead to metabolic rewiring. Links between cancer metabolism and DNA damage/DNA repair are increasingly apparent, yielding opportunities to investigate the mechanistic basis behind potential metabolic vulnerabilities of a substantial fraction of tumors

DNA Damage, Repair, and Cancer Metabolism

Although there has been a renewed interest in the field of cancer metabolism in the last decade, the link between metabolism and DNA damage/DNA repair in cancer has yet to be appreciably explored. In this review, we examine the evidence connecting DNA damage and repair mechanisms with cell metabolism through three principal links. (1) Regulation of methyl- and acetyl-group donors through different metabolic pathways can impact DNA folding and remodeling, an essential part of accurate double strand break repair. (2) Glutamine, aspartate, and other nutrients are essential for de novo nucleotide synthesis, which dictates the availability of the nucleotide pool, and thereby influences DNA repair and replication. (3) Reactive oxygen species, which can increase oxidative DNA damage and hence the load of the DNA-repair machinery, are regulated through different metabolic pathways. Interestingly, while metabolism affects DNA repair, DNA damage can also induce metabolic rewiring. Activation of the DNA damage response (DDR) triggers an increase in nucleotide synthesis and anabolic glucose metabolism, while also reducing glutamine anaplerosis. Furthermore, mutations in genes involved in the DDR and DNA repair also lead to metabolic rewiring. Links between cancer metabolism and DNA damage/DNA repair are increasingly apparent, yielding opportunities to investigate the mechanistic basis behind potential metabolic vulnerabilities of a substantial fraction of tumors

A Novel IGSF1 Mutation in a Boy With Short Stature and Hypercholesterolemia: A Case Report

IGSF1 deficiency is a recently discovered syndrome consisting of congenital central hypothyroidism (CeH) and macroorchidism. Here, we report on a patient presenting with short stature, who was found to carry a pathogenic mutation in the IGSF1 gene. A 14-year-old Israeli boy was referred to the Academic Medical Center in Amsterdam, The Netherlands, for follow-up on short stature ascribed to constitutional delay of growth and puberty, and familial hypercholesterolemia. Primary hypothyroidism had previously been excluded by a normal thyroid-stimulating hormone (TSH) concentration. However, in follow-up, plasma free thyroxine (FT4) concentrations were repeatedly low, and the patient was diagnosed with CeH. Because of coexistent relative macroorchidism, IGSF1 gene analysis was performed, revealing a mutation (c.2588C>G; p.Ser863Cys). The mutant IGSF1 protein was retained mainly in the endoplasmic reticulum and reached the plasma membrane with poor efficiency compared with wild-type protein. After starting thyroxine treatment, plasma cholesterol almost normalized. This case illustrates the necessity of measuring both FT4 and TSH when hypothyroidism is suspected, or needs to be ruled out. In addition, this case suggests that the presence of childhood hypercholesterolemia may be an indication of undiagnosed hypothyroidis

Mesoporous Silica Nanoparticles under Sintering Conditions: A Quantitative Study

Thin films made of mesoporous silica nanoparticles (MSNs) are finding new applications in catalysis, optics, as well as in biomedicine. The fabrication of MSNs thin films requires a precise control over the deposition and sintering of MSNs on flat substrates. In this study, MSNs of narrow size distribution (150 nm) are synthesized, and then assembled onto flat silicon substrates, by means of a dip-coating process. Using concentrated MSN colloidal solutions (19.5 mg mL^–1 SiO₂), withdrawal speed of 0.01 mm s^–1, and well-controlled atmospheric conditions (ambient temperature, ∼ 70% of relative humidity), monolayers are assembled under well-structured compact patterns. The thin films are sintered up to 900 °C, and the evolution of the MSNs size distributions are compared to those of their pore volumes and densities. Particle size distributions of the sintered thin films were precisely fitted using a model specifically developed for asymmetric particle size distributions. With increasing temperature, there is first evidence of intraparticle reorganization/relaxation followed by intraparticle sintering followed by interparticle sintering. This study is the first to quantify the impact of sintering on MSNs assembled as thin films