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BACKGROUND: Chronic obstructive pulmonary disease (COPD) has the highest increased risk due to household air pollution arising from biomass fuel burning. However, knowledge on COPD patho-mechanisms is mainly limited to tobacco smoke exposure. In this study, a repeated direct wood smoke (WS) exposure was performed using normal- (bro-ALI) and chronic bronchitis-like bronchial (bro-ALI-CB), and alveolar (alv-ALI) lung mucosa models at air-liquid interface (ALI) to assess broad toxicological end points. METHODS: The bro-ALI and bro-ALI-CB models were developed using human primary bronchial epithelial cells and the alv-ALI model was developed using a representative type-II pneumocyte cell line. The lung models were exposed to WS (10 min/exposure; 5-exposures over 3-days; n = 6-7 independent experiments). Sham exposed samples served as control. WS composition was analyzed following passive sampling. Cytotoxicity, total cellular reactive oxygen species (ROS) and stress responsive NFkB were assessed by flow cytometry. WS exposure induced changes in gene expression were evaluated by RNA-seq (p ≤ 0.01) followed by pathway enrichment analysis. Secreted levels of proinflammatory cytokines were assessed in the basal media. Non-parametric statistical analysis was performed. RESULTS: 147 unique compounds were annotated in WS of which 42 compounds have inhalation toxicity (9 very high). WS exposure resulted in significantly increased ROS in bro-ALI (11.2%) and bro-ALI-CB (25.7%) along with correspondingly increased NFkB levels (bro-ALI: 35.6%; bro-ALI-CB: 18.1%). A total of 1262 (817-up and 445-down), 329 (141-up and 188-down), and 102 (33-up and 69-down) genes were differentially regulated in the WS-exposed bro-ALI, bro-ALI-CB, and alv-ALI models respectively. The enriched pathways included the terms acute phase response, mitochondrial dysfunction, inflammation, oxidative stress, NFkB, ROS, xenobiotic metabolism of AHR, and chronic respiratory disorder. The enrichment of the 'cilium' related genes was predominant in the WS-exposed bro-ALI (180-up and 7-down). The pathways primary ciliary dyskinesia, ciliopathy, and ciliary movement were enriched in both WS-exposed bro-ALI and bro-ALI-CB. Interleukin-6 and tumor necrosis factor-α were reduced (p < 0.05) in WS-exposed bro-ALI and bro-ALI-CB. CONCLUSION: Findings of this study indicate differential response to WS-exposure in different lung regions and in chronic bronchitis, a condition commonly associated with COPD. Further, the data suggests ciliopathy as a candidate pathway in relation to WS-exposure.
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Bronquitis Crónica , Ciliopatías , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Bronquitis Crónica/inducido químicamente , Bronquitis Crónica/metabolismo , Humo/efectos adversos , Madera/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Membrana Mucosa , Productos de TabacoRESUMEN
BACKGROUND: Fibroblast-to-myofibroblast conversion is a major driver of tissue remodelling in organ fibrosis. Distinct lineages of fibroblasts support homeostatic tissue niche functions, yet their specific activation states and phenotypic trajectories during injury and repair have remained unclear. METHODS: We combined spatial transcriptomics, multiplexed immunostainings, longitudinal single-cell RNA-sequencing and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. Our findings were validated in idiopathic pulmonary fibrosis patient tissues in situ as well as in cell differentiation and invasion assays using patient lung fibroblasts. Cell differentiation and invasion assays established a function of SFRP1 in regulating human lung fibroblast invasion in response to transforming growth factor (TGF)ß1. MEASUREMENTS AND MAIN RESULTS: We discovered a transitional fibroblast state characterised by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1 + cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage-specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1 + transitional fibroblasts and Cthrc1 + myofibroblasts. TGFß1 downregulated SFRP1 in noninvasive transitional cells and induced their switch to an invasive CTHRC1+ myofibroblast identity. Finally, using loss-of-function studies we showed that SFRP1 modulates TGFß1-induced fibroblast invasion and RHOA pathway activity. CONCLUSIONS: Our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies SFRP1 as a modulator of TGFß1-driven fibroblast phenotypes in fibrogenesis. These findings are relevant in the context of therapeutic interventions that aim at limiting or reversing fibroblast foci formation.
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Fibrosis Pulmonar Idiopática , Miofibroblastos , Ratones , Animales , Humanos , Miofibroblastos/metabolismo , Fibroblastos/metabolismo , Pulmón/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Diferenciación Celular , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
INTRODUCTION: Environmental pollutants injure the mucociliary elevator, thereby provoking disease progression in chronic obstructive pulmonary disease (COPD). Epithelial resilience mechanisms to environmental nanoparticles in health and disease are poorly characterised. METHODS: We delineated the impact of prevalent pollutants such as carbon and zinc oxide nanoparticles, on cellular function and progeny in primary human bronchial epithelial cells (pHBECs) from end-stage COPD (COPD-IV, n=4), early disease (COPD-II, n=3) and pulmonary healthy individuals (n=4). After nanoparticle exposure of pHBECs at air-liquid interface, cell cultures were characterised by functional assays, transcriptome and protein analysis, complemented by single-cell analysis in serial samples of pHBEC cultures focusing on basal cell differentiation. RESULTS: COPD-IV was characterised by a prosecretory phenotype (twofold increase in MUC5AC+) at the expense of the multiciliated epithelium (threefold reduction in Ac-Tub+), resulting in an increased resilience towards particle-induced cell damage (fivefold reduction in transepithelial electrical resistance), as exemplified by environmentally abundant doses of zinc oxide nanoparticles. Exposure of COPD-II cultures to cigarette smoke extract provoked the COPD-IV characteristic, prosecretory phenotype. Time-resolved single-cell transcriptomics revealed an underlying COPD-IV unique basal cell state characterised by a twofold increase in KRT5+ (P=0.018) and LAMB3+ (P=0.050) expression, as well as a significant activation of Wnt-specific (P=0.014) and Notch-specific (P=0.021) genes, especially in precursors of suprabasal and secretory cells. CONCLUSION: We identified COPD stage-specific gene alterations in basal cells that affect the cellular composition of the bronchial elevator and may control disease-specific epithelial resilience mechanisms in response to environmental nanoparticles. The identified phenomena likely inform treatment and prevention strategies.
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Células Epiteliales , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Enfermedad Pulmonar Obstructiva Crónica/etiología , Células Epiteliales/metabolismo , Masculino , Persona de Mediana Edad , Células Cultivadas , Bronquios/patología , Femenino , Anciano , Óxido de Zinc , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Cilios , Nanopartículas , Diferenciación CelularRESUMEN
Obesity is a global health problem characterized by excessive fat accumulation, driven by adipogenesis and lipid accumulation. Long non-coding RNAs (lncRNAs) have recently been implicated in regulating adipogenesis and adipose tissue function. Mouse lncRNA U90926 was previously identified as a repressor of in vitro adipogenesis in 3T3-L1 preadipocytes. Consequently, we hypothesized that, in vivo, U90926 may repress adipogenesis, and hence its deletion would increase weight gain and adiposity. We tested the hypothesis by applying U90926-deficient (U9-KO) mice to a high-throughput phenotyping pipeline. Compared with WT, U9-KO mice showed no major differences across a wide range of behavioral, neurological, and other physiological parameters. In mice fed a standard diet, we have found no differences in obesity-related phenotypes, including weight gain, fat mass, and plasma concentrations of glucose, insulin, triglycerides, and free fatty acids, in U9-KO mice compared to WT. U90926 deficiency lacked a major effect on white adipose tissue morphology and gene expression profile. Furthermore, in mice fed a high-fat diet, we found increased expression of U90926 in adipose tissue stromal vascular cell fraction, yet observed no effect of U90926 deficiency on weight gain, fat mass, adipogenesis marker expression, and immune cell infiltration into the adipose tissue. These data suggest that the U90926 lacks an essential role in obesity-related phenotypes and adipose tissue biology in vivo.
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ARN Largo no Codificante , Ratones , Animales , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Adipocitos/metabolismo , Obesidad/genética , Obesidad/metabolismo , Adipogénesis/genética , Aumento de Peso , Dieta Alta en Grasa/efectos adversos , Fenotipo , Ratones Endogámicos C57BLRESUMEN
CONTEXT: Exercise training is known to improve glucose tolerance and reverse insulin resistance in people with obesity. However, some individuals fail to improve or even decline in their clinical traits following exercise intervention. OBJECTIVE: This study focused on gene expression and DNA methylation signatures in skeletal muscle of low (LRE) and high responders (RES) to 8 weeks of supervised endurance training. METHODS: We performed skeletal muscle gene expression and DNA methylation analyses in LRE and RES before and after exercise intervention. Additionally, we applied the least absolute shrinkage and selection operator (LASSO) approach to identify predictive marker genes of exercise outcome. RESULTS: We show that the two groups differ markedly already before the intervention. RES were characterized by lower expression of genes involved in DNA replication and repair, and higher expression of extracellular matrix (ECM) components. The LASSO approach identified several novel candidates (eg, ZCWPW2, FOXRED1, STK40) that have not been previously described in the context of obesity and exercise response. Following the intervention, LRE reacted with expression changes of genes related to inflammation and apoptosis, RES with genes related to mitochondrial function. LRE exhibited significantly higher expression of ECM components compared to RES, suggesting improper remodeling and potential negative effects on insulin sensitivity. Between 45% and 70% of differences in gene expression could be linked to differences in DNA methylation. CONCLUSION: Together, our data offer an insight into molecular mechanisms underlying differences in response to exercise and provide potential novel markers for the success of intervention.
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Nanoparticles (NPs) released from engineered materials or combustion processes as well as persistent herpesvirus infection are omnipresent and are associated with chronic lung diseases. Previously, we showed that pulmonary exposure of a single dose of soot-like carbonaceous NPs (CNPs) or fiber-shaped double-walled carbon nanotubes (DWCNTs) induced an increase of lytic virus protein expression in mouse lungs latently infected with murine γ-herpesvirus 68 (MHV-68), with a similar pattern to acute infection suggesting virus reactivation. Here we investigate the effects of a more relevant repeated NP exposure on lung disease development as well as herpesvirus reactivation mechanistically and suggest an avenue for therapeutic prevention. In the MHV-68 mouse model, progressive lung inflammation and emphysema-like injury were detected 1 week after repetitive CNP and DWCNT exposure. NPs reactivated the latent herpesvirus mainly in CD11b+ macrophages in the lungs. In vitro, in persistently MHV-68 infected bone marrow-derived macrophages, ERK1/2, JNK, and p38 MAPK were rapidly activated after CNP and DWCNT exposure, followed by viral gene expression and increased viral titer but without generating a pro-inflammatory signature. Pharmacological inhibition of p38 activation abrogated CNP- but not DWCNT-triggered virus reactivation in vitro, and inhibitor pretreatment of latently infected mice attenuated CNP-exposure-induced pulmonary MHV-68 reactivation. Our findings suggest a crucial contribution of particle-exposure-triggered herpesvirus reactivation for nanomaterial exposure or air pollution related lung emphysema development, and pharmacological p38 inhibition might serve as a protective target to alleviate air pollution related chronic lung disease exacerbations. Because of the required precondition of latent infection described here, the use of single hit models might have severe limitations when assessing the respiratory toxicity of nanoparticle exposure.
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Enfisema , Nanopartículas , Nanotubos de Carbono , Neumonía , Animales , Ratones , Pulmón , Neumonía/inducido químicamente , Nanopartículas/toxicidadRESUMEN
Metformin-induced glycolysis and lactate production can lead to acidosis as a life-threatening side effect, but slight increases in blood lactate levels in a physiological range were also reported in metformin-treated patients. However, how metformin increases systemic lactate concentrations is only partly understood. Because human skeletal muscle has a high capacity to produce lactate, the aim was to elucidate the dose-dependent regulation of metformin-induced lactate production and the potential contribution of skeletal muscle to blood lactate levels under metformin treatment. This was examined by using metformin treatment (16-776 µM) of primary human myotubes and by 17 days of metformin treatment in humans. As from 78 µM, metformin induced lactate production and secretion and glucose consumption. Investigating the cellular redox state by mitochondrial respirometry, we found metformin to inhibit the respiratory chain complex I (776 µM, P < 0.01) along with decreasing the [NAD+]:[NADH] ratio (776 µM, P < 0.001). RNA sequencing and phospho-immunoblot data indicate inhibition of pyruvate oxidation mediated through phosphorylation of the pyruvate dehydrogenase (PDH) complex (39 µM, P < 0.01). On the other hand, in human skeletal muscle, phosphorylation of PDH was not altered by metformin. Nonetheless, blood lactate levels were increased under metformin treatment (P < 0.05). In conclusion, the findings suggest that metformin-induced inhibition of pyruvate oxidation combined with altered cellular redox state shifts the equilibrium of the lactate dehydrogenase (LDH) reaction leading to a dose-dependent lactate production in primary human myotubes.NEW & NOTEWORTHY Metformin shifts the equilibrium of lactate dehydrogenase (LDH) reaction by low dose-induced phosphorylation of pyruvate dehydrogenase (PDH) resulting in inhibition of pyruvate oxidation and high dose-induced increase in NADH, which explains the dose-dependent lactate production of differentiated human skeletal muscle cells.
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Ácido Láctico , Metformina , Humanos , Ácido Láctico/metabolismo , Metformina/farmacología , NAD/metabolismo , Oxidación-Reducción , Fibras Musculares Esqueléticas/metabolismo , Piruvatos , Oxidorreductasas/metabolismo , Lactato Deshidrogenasas/metabolismoRESUMEN
OBJECTIVES: Better disease management can be achieved with earlier detection through robust, sensitive, and easily accessible biomarkers. The aim of the current study was to identify novel epigenetic biomarkers determining the risk of type 2 diabetes (T2D). METHODS: Livers of 10-week-old female New Zealand Obese (NZO) mice, slightly differing in their degree of hyperglycemia and liver fat content and thereby in their diabetes susceptibility were used for expression and methylation profiling. We screened for differences in hepatic expression and DNA methylation in diabetes-prone and -resistant mice, and verified a candidate (HAMP) in human livers and blood cells. Hamp expression was manipulated in primary hepatocytes and insulin-stimulated pAKT was detected. Luciferase reporter assays were conducted in a murine liver cell line to test the impact of DNA methylation on promoter activity. RESULTS: In livers of NZO mice, the overlap of methylome and transcriptome analyses revealed a potential transcriptional dysregulation of 12 hepatokines. The strongest effect with a 52% decreased expression in livers of diabetes-prone mice was detected for the Hamp gene, mediated by elevated DNA methylation of two CpG sites located in the promoter. Hamp encodes the iron-regulatory hormone hepcidin, which had a lower abundance in the livers of mice prone to developing diabetes. Suppression of Hamp reduces the levels of pAKT in insulin-treated hepatocytes. In liver biopsies of obese insulin-resistant women, HAMP expression was significantly downregulated along with increased DNA methylation of a homologous CpG site. In blood cells of incident T2D cases from the prospective EPIC-Potsdam cohort, higher DNA methylation of two CpG sites was related to increased risk of incident diabetes. CONCLUSIONS: We identified epigenetic changes in the HAMP gene which may be used as an early marker preceding T2D.
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Diabetes Mellitus Tipo 2 , Hepcidinas , Humanos , Femenino , Ratones , Animales , Hepcidinas/genética , Hepcidinas/metabolismo , Metilación de ADN , Diabetes Mellitus Tipo 2/metabolismo , Estudios Prospectivos , Insulina/metabolismo , Obesidad/genética , Biomarcadores/metabolismo , Células Sanguíneas/metabolismoRESUMEN
Inherited disorders of mitochondrial metabolism, including isolated methylmalonic aciduria, present unique challenges to energetic homeostasis by disrupting energy-producing pathways. To better understand global responses to energy shortage, we investigated a hemizygous mouse model of methylmalonyl-CoA mutase (Mmut)-type methylmalonic aciduria. We found Mmut mutant mice to have reduced appetite, energy expenditure and body mass compared with littermate controls, along with a relative reduction in lean mass but increase in fat mass. Brown adipose tissue showed a process of whitening, in line with lower body surface temperature and lesser ability to cope with cold challenge. Mutant mice had dysregulated plasma glucose, delayed glucose clearance and a lesser ability to regulate energy sources when switching from the fed to fasted state, while liver investigations indicated metabolite accumulation and altered expression of peroxisome proliferator-activated receptor and Fgf21-controlled pathways. Together, these shed light on the mechanisms and adaptations behind energy imbalance in methylmalonic aciduria and provide insight into metabolic responses to chronic energy shortage, which may have important implications for disease understanding and patient management.
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Errores Innatos del Metabolismo de los Aminoácidos , Ratones , Animales , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Metabolismo Energético/genética , Hígado/metabolismoRESUMEN
BACKGROUND: Exercise exerts many health benefits by directly inducing molecular alterations in physically utilized skeletal muscle. Molecular adaptations of subcutaneous adipose tissue (SCAT) might also contribute to the prevention of metabolic diseases. AIM: To characterize the response of human SCAT based on changes in transcripts and mitochondrial respiration to acute and repeated bouts of exercise in comparison to skeletal muscle. METHODS: Sedentary participants (27 ± 4 yrs) with overweight or obesity underwent 8-week supervised endurance exercise 3×1h/week at 80% VO2peak. Before, 60 min after the first and last exercise bout and 5 days post intervention, biopsies were taken for transcriptomic analyses and high-resolution respirometry (n = 14, 8 female/6 male). RESULTS: In SCAT, we found 37 acutely regulated transcripts (FC > 1.2, FDR < 10%) after the first exercise bout compared to 394, respectively, in skeletal muscle. Regulation of only 5 transcripts overlapped between tissues highlighting their differential response. Upstream and enrichment analyses revealed reduced transcripts of lipid uptake, storage and lipogenesis directly after exercise in SCAT and point to ß-adrenergic regulation as potential major driver. The data also suggest an exercise-induced modulation of the circadian clock in SCAT. Neither term was associated with transcriptomic changes in skeletal muscle. No evidence for beigeing/browning was found in SCAT along with unchanged respiration. CONCLUSIONS: Adipose tissue responds completely distinct from adaptations of skeletal muscle to exercise. The acute and repeated reduction in transcripts of lipid storage and lipogenesis, interconnected with a modulated circadian rhythm, can counteract metabolic syndrome progression toward diabetes.
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Tejido Adiposo , Ejercicio Físico , Músculo Esquelético , Femenino , Humanos , Masculino , Tejido Adiposo/metabolismo , Ejercicio Físico/fisiología , Músculo Esquelético/metabolismo , Transcriptoma , Adulto Joven , Adulto , Terapia por Ejercicio , Sobrepeso/terapia , Obesidad/terapia , Resultado del TratamientoRESUMEN
Since their initial description by Elie Metchnikoff, phagocytes have sparked interest in a variety of biologic disciplines. These important cells perform central functions in tissue repair and immune activation as well as tolerance. Myeloid cells can be immunoinhibitory, particularly in the tumor microenvironment, where their presence is generally associated with poor patient prognosis. These cells are highly adaptable and plastic, and can be modulated to perform desired functions such as antitumor activity, if key programming molecules can be identified. Human clear cell renal cell carcinoma (ccRCC) is considered immunogenic; yet checkpoint blockades that target T cell dysfunction have shown limited clinical efficacy, suggesting additional layers of immunoinhibition. We previously described "enriched-in-renal cell carcinoma" (erc) DCs that were often found in tight contact with dysfunctional T cells. Using transcriptional profiling and flow cytometry, we describe here that ercDCs represent a mosaic cell type within the macrophage continuum co-expressing M1 and M2 markers. The polarization state reflects tissue-specific signals that are characteristic of RCC and renal tissue homeostasis. ErcDCs are tissue-resident with increasing prevalence related to tumor grade. Accordingly, a high ercDC score predicted poor patient survival. Within the profile, therapeutic targets (VSIG4, NRP1, GPNMB) were identified with promise to improve immunotherapy.
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Productos Biológicos , Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/patología , Macrófagos/metabolismo , Células Dendríticas , Plásticos/metabolismo , Productos Biológicos/metabolismo , Microambiente Tumoral , Glicoproteínas de Membrana/metabolismoRESUMEN
Heated tobacco products (HTP) are novel nicotine delivery products with limited toxicological data. HTP uses heating instead of combustion to generate aerosol (HTP-smoke). Physiologically relevant human bronchial and alveolar lung mucosa models developed at air-liquid interface were exposed to HTP-smoke to assess broad toxicological response (n = 6-7; ISO puffing regimen; compared to sham; non-parametric statistical analysis; significance: p < 0.05). Elevated levels of total cellular reactive oxygen species, stress responsive nuclear factor kappa-B, and DNA damage markers [8-hydroxy-2'-deoxyguanosine, phosphorylated histone H2AX, cleaved poly-(ADP-Ribose) polymerase] were detected in HTP-smoke exposed bronchial and/or alveolar models. RNA sequencing detected differential regulation of 724 genes in the bronchial- and 121 genes in the alveolar model following HTP-smoke exposure (cut off: p ≤ 0.01; fold change: ≥ 2). Common enriched pathways included estrogen biosynthesis, ferroptosis, superoxide radical degradation, xenobiotics, and α-tocopherol degradation. Secreted levels of interleukin (IL)1êµ and IL8 increased in the bronchial model whereas in the alveolar model, interferon-γ and IL4 increased and IL13 decreased following HTP-smoke exposure. Increased lipid peroxidation was detected in HTP-smoke exposed bronchial and alveolar models which was inhibited by ferrostatin-1. The findings form a basis to perform independent risk assessment studies on different flavours of HTP using different puffing topography and corresponding chemical characterization.
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Productos de Tabaco , 8-Hidroxi-2'-Desoxicoguanosina , Adenosina Difosfato Ribosa , Aerosoles/análisis , Estrógenos , Histonas , Humanos , Interferón gamma , Interleucina-13 , Interleucina-4 , Interleucina-8 , Membrana Mucosa/química , Nicotina/análisis , Especies Reactivas de Oxígeno , Humo/análisis , Superóxidos/análisis , Nicotiana , Productos de Tabaco/análisis , alfa-TocoferolRESUMEN
According to the current consensus, murine neural stem cells (NSCs) apically contacting the lateral ventricle generate differentiated progenitors by rare asymmetric divisions or by relocating to the basal side of the ventricular-subventricular zone (V-SVZ). Both processes will ultimately lead to the generation of adult-born olfactory bulb (OB) interneurons. In contrast to this view, we here find that adult-born OB interneurons largely derive from an additional NSC-type resident in the basal V-SVZ. Despite being both capable of self-renewal and long-term quiescence, apical and basal NSCs differ in Nestin expression, primary cilia extension and frequency of cell division. The expression of Notch-related genes also differs between the two NSC groups, and Notch activation is greatest in apical NSCs. Apical downregulation of Notch-effector Hes1 decreases Notch activation while increasing proliferation across the niche and neurogenesis from apical NSCs. Underscoring their different roles in neurogenesis, lactation-dependent increase in neurogenesis is paralleled by extra activation of basal but not apical NSCs. Thus, basal NSCs support OB neurogenesis, whereas apical NSCs impart Notch-mediated lateral inhibition across the V-SVZ.
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Ventrículos Laterales , Células-Madre Neurales , Animales , Diferenciación Celular/genética , Femenino , Ventrículos Laterales/metabolismo , Ratones , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Bulbo Olfatorio/metabolismoRESUMEN
Glucotoxic metabolites and pathways play a crucial role in diabetic complications, and new treatment options which improve glucotoxicity are highly warranted. In this study, we analyzed bezafibrate (BEZ) treated, streptozotocin (STZ) injected mice, which showed an improved glucose metabolism compared to untreated STZ animals. In order to identify key molecules and pathways which participate in the beneficial effects of BEZ, we studied plasma, skeletal muscle, white adipose tissue (WAT) and liver samples using non-targeted metabolomics (NMR spectroscopy), targeted metabolomics (mass spectrometry), microarrays and mitochondrial enzyme activity measurements, with a particular focus on the liver. The analysis of muscle and WAT demonstrated that STZ treatment elevated inflammatory pathways and reduced insulin signaling and lipid pathways, whereas BEZ decreased inflammatory pathways and increased insulin signaling and lipid pathways, which can partly explain the beneficial effects of BEZ on glucose metabolism. Furthermore, lysophosphatidylcholine levels were lower in the liver and skeletal muscle of STZ mice, which were reverted in BEZ-treated animals. BEZ also improved circulating and hepatic glucose levels as well as lipid profiles. In the liver, BEZ treatment reduced elevated fumarate levels in STZ mice, which was probably due to a decreased expression of urea cycle genes. Since fumarate has been shown to participate in glucotoxic pathways, our data suggests that BEZ treatment attenuates the urea cycle in the liver, decreases fumarate levels and, in turn, ameliorates glucotoxicity and reduces insulin resistance in STZ mice.
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The oligodendrocyte progenitors (OPCs) are at the front of the glial reaction to the traumatic brain injury. However, regulatory pathways steering the OPC reaction as well as the role of reactive OPCs remain largely unknown. Here, we compared a long-lasting, exacerbated reaction of OPCs to the adult zebrafish brain injury with a timely restricted OPC activation to identify the specific molecular mechanisms regulating OPC reactivity and their contribution to regeneration. We demonstrated that the influx of the cerebrospinal fluid into the brain parenchyma after injury simultaneously activates the toll-like receptor 2 (Tlr2) and the chemokine receptor 3 (Cxcr3) innate immunity pathways, leading to increased OPC proliferation and thereby exacerbated glial reactivity. These pathways were critical for long-lasting OPC accumulation even after the ablation of microglia and infiltrating monocytes. Importantly, interference with the Tlr1/2 and Cxcr3 pathways after injury alleviated reactive gliosis, increased new neuron recruitment, and improved tissue restoration.
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Células Precursoras de Oligodendrocitos , Animales , Encéfalo , Gliosis/metabolismo , Inmunidad Innata , Células Precursoras de Oligodendrocitos/metabolismo , Pez CebraRESUMEN
Physical training improves insulin sensitivity and can prevent type 2 diabetes (T2D). However, approximately 20% of individuals lack a beneficial outcome in glycemic control. TGF-ß, identified as a possible upstream regulator involved in this low response, is also a potent regulator of microRNAs (miRNAs). The aim of this study was to elucidate the potential impact of TGF-ß-driven miRNAs on individual exercise response. Non-targeted long and sncRNA sequencing analyses of TGF-ß1-treated human skeletal muscle cells corroborated the effects of TGF-ß1 on muscle cell differentiation, the induction of extracellular matrix components, and identified several TGF-ß1-regulated miRNAs. qPCR validated a potent upregulation of miR-143-3p/145-5p and miR-181a2-5p by TGF-ß1 in both human myoblasts and differentiated myotubes. Healthy subjects who were overweight or obese participated in a supervised 8-week endurance training intervention (n = 40) and were categorized as responder or low responder in glycemic control based on fold change ISIMats (≥+1.1 or <+1.1, respectively). In skeletal muscle biopsies of low responders, TGF-ß signaling and miR-143/145 cluster levels were induced by training at much higher rates than among responders. Target-mining revealed HDACs, MYHs, and insulin signaling components INSR and IRS1 as potential miR-143/145 cluster targets. All these targets were down-regulated in TGF-ß1-treated myotubes. Transfection of miR-143-3p/145-5p mimics in differentiated myotubes validated MYH1, MYH4, and IRS1 as miR-143/145 cluster targets. Elevated TGF-ß signaling and miR-143/145 cluster induction in skeletal muscle of low responders might obstruct improvements in insulin sensitivity by training in two ways: by a negative impact of miR-143-3p on muscle cell fusion and myofiber functionality and by directly impairing insulin signaling via a reduction in INSR by TGF-ß and finetuned IRS1 suppression by miR-143-3p.
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Diabetes Mellitus Tipo 2/genética , Ejercicio Físico/genética , Proteínas Sustrato del Receptor de Insulina/genética , Insulina/sangre , MicroARNs/genética , Factor de Crecimiento Transformador beta1/genética , Adulto , Diferenciación Celular/genética , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/fisiopatología , Diabetes Mellitus Tipo 2/terapia , Ejercicio Físico/fisiología , Femenino , Regulación de la Expresión Génica/genética , Humanos , Insulina/genética , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/fisiopatología , Mioblastos/metabolismo , Acondicionamiento Físico Humano , Transducción de Señal/genéticaRESUMEN
Fibrogenic processes instigate fatal chronic diseases leading to organ failure and death. Underlying biological processes involve induced massive deposition of extracellular matrix (ECM) by aberrant fibroblasts. We subjected diseased primary human lung fibroblasts to an advanced three-dimensional phenotypic high-content assay and screened a repurposing drug library of small molecules for inhibiting ECM deposition. Fibrotic Pattern Detection by Artificial Intelligence identified tranilast as an effective inhibitor. Structure-activity relationship studies confirmed N-(2-butoxyphenyl)-3-(phenyl)acrylamides (N23Ps) as a novel and highly potent compound class. N23Ps suppressed myofibroblast transdifferentiation, ECM deposition, cellular contractility, and altered cell shapes, thus advocating a unique mode of action. Mechanistically, transcriptomics identified SMURF2 as a potential therapeutic target network. Antifibrotic activity of N23Ps was verified by proteomics in a human ex vivo tissue fibrosis disease model, suppressing profibrotic markers SERPINE1 and CXCL8. Conclusively, N23Ps are a novel class of highly potent compounds inhibiting organ fibrosis in patients.
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BACKGROUND: The SARS-CoV-2 spike protein mediates attachment of the virus to the host cell receptor and fusion between the virus and the cell membrane. The S1 subunit of the spike glycoprotein (S1 protein) contains the angiotensin converting enzyme 2 (ACE2) receptor binding domain. The SARS-CoV-2 variants of concern contain mutations in the S1 subunit. The spike protein is the primary target of neutralizing antibodies generated following infection, and constitutes the viral component of mRNA-based COVID-19 vaccines. METHODS: Therefore, in this work we assessed the effect of exposure (24 h) to 10 nM SARS-CoV-2 recombinant S1 protein on physiologically relevant human bronchial (bro) and alveolar (alv) lung mucosa models cultured at air-liquid interface (ALI) (n = 6 per exposure condition). Corresponding sham exposed samples served as a control. The bro-ALI model was developed using primary bronchial epithelial cells and the alv-ALI model using representative type II pneumocytes (NCI-H441). RESULTS: Exposure to S1 protein induced the surface expression of ACE2, toll like receptor (TLR) 2, and TLR4 in both bro-ALI and alv-ALI models. Transcript expression analysis identified 117 (bro-ALI) and 97 (alv-ALI) differentially regulated genes (p ≤ 0.01). Pathway analysis revealed enrichment of canonical pathways such as interferon (IFN) signaling, influenza, coronavirus, and anti-viral response in the bro-ALI. Secreted levels of interleukin (IL) 4 and IL12 were significantly (p < 0.05) increased, whereas IL6 decreased in the bro-ALI. In the case of alv-ALI, enriched terms involving p53, APRIL (a proliferation-inducing ligand) tight junction, integrin kinase, and IL1 signaling were identified. These terms are associated with lung fibrosis. Further, significantly (p < 0.05) increased levels of secreted pro-inflammatory cytokines IFNγ, IL1êµ, IL2, IL4, IL6, IL8, IL10, IL13, and tumor necrosis factor alpha were detected in alv-ALI, whereas IL12 was decreased. Altered levels of these cytokines are also associated with lung fibrotic response. CONCLUSIONS: In conclusion, we observed a typical anti-viral response in the bronchial model and a pro-fibrotic response in the alveolar model. The bro-ALI and alv-ALI models may serve as an easy and robust platform for assessing the pathogenicity of SARS-CoV-2 variants of concern at different lung regions.
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Pulmón/metabolismo , Mucosa Respiratoria/metabolismo , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Bronquios/metabolismo , Citocinas/metabolismo , Perfilación de la Expresión Génica , Humanos , Modelos Biológicos , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
OBJECTIVE: Liver mitochondria adapt to high-calorie intake. We investigated how exercise alters the early compensatory response of mitochondria, thus preventing fatty liver disease as a long-term consequence of overnutrition. METHODS: We compared the effects of a steatogenic high-energy diet (HED) for six weeks on mitochondrial metabolism of sedentary and treadmill-trained C57BL/6N mice. We applied multi-OMICs analyses to study the alterations in the proteome, transcriptome, and lipids in isolated mitochondria of liver and skeletal muscle as well as in whole tissue and examined the functional consequences by high-resolution respirometry. RESULTS: HED increased the respiratory capacity of isolated liver mitochondria, both in sedentary and in trained mice. However, proteomics analysis of the mitochondria and transcriptomics indicated that training modified the adaptation of the hepatic metabolism to HED on the level of respiratory complex I, glucose oxidation, pyruvate and acetyl-CoA metabolism, and lipogenesis. Training also counteracted the HED-induced glucose intolerance, the increase in fasting insulin, and in liver fat by lowering diacylglycerol species and c-Jun N-terminal kinase (JNK) phosphorylation in the livers of trained HED-fed mice, two mechanisms that can reverse hepatic insulin resistance. In skeletal muscle, the combination of HED and training improved the oxidative capacity to a greater extent than training alone by increasing respiration of isolated mitochondria and total mitochondrial protein content. CONCLUSION: We provide a comprehensive insight into the early adaptations of mitochondria in the liver and skeletal muscle to HED and endurance training. Our results suggest that exercise disconnects the HED-induced increase in mitochondrial substrate oxidation from pyruvate and acetyl-CoA-driven lipid synthesis. This could contribute to the prevention of deleterious long-term effects of high fat and sugar intake on hepatic mitochondrial function and insulin sensitivity.