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The placenta is essential for reproductive success. The murine placenta includes polyploid giant cells that are crucial for its function. Polyploidy occurs broadly in nature but its regulators and significance in the placenta are unknown. We have discovered that many murine placental cell types are polyploid and have identified factors that license polyploidy using single-cell RNA sequencing. Myc is a key regulator of polyploidy and placental development, and is required for multiple rounds of DNA replication, likely via endocycles, in trophoblast giant cells. Furthermore, MYC supports the expression of DNA replication and nucleotide biosynthesis genes along with ribosomal RNA. Increased DNA damage and senescence occur in trophoblast giant cells without Myc, accompanied by senescence in the neighboring maternal decidua. These data reveal Myc is essential for polyploidy to support normal placental development, thereby preventing premature senescence. Our study, combined with available literature, suggests that Myc is an evolutionarily conserved regulator of polyploidy.
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Placenta , Trofoblastos , Animales , Femenino , Ratones , Embarazo , Placenta/metabolismo , Placentación , Poliploidía , Trofoblastos/metabolismoRESUMEN
BACKGROUND & AIMS: Vacuole membrane protein 1 (VMP1) is an endoplasmic reticulum (ER) transmembrane protein that regulates the formation of autophagosomes and lipid droplets. Recent evidence suggests that VMP1 plays a critical role in lipoprotein secretion in zebra fish and cultured cells. However, the pathophysiological roles and mechanisms by which VMP1 regulates lipoprotein secretion and lipid accumulation in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are unknown. METHODS: Liver-specific and hepatocyte-specific Vmp1 knockout mice as well as Vmp1 knock-in mice were generated by crossing Vmp1flox or Vmp1KI mice with albumin-Cre mice or by injecting AAV8-TBG-cre, respectively. Lipid and energy metabolism in these mice were characterized by metabolomic and transcriptome analyses. Mice with hepatic overexpression of VMP1 who were fed a NASH diet were also characterized. RESULTS: Hepatocyte-specific deletion of Vmp1 severely impaired VLDL secretion resulting in massive hepatic steatosis, hepatocyte death, inflammation and fibrosis, which are hallmarks of NASH. Mechanistically, loss of Vmp1 led to decreased hepatic levels of phosphatidylcholine and phosphatidylethanolamine as well as to changes in phospholipid composition. Deletion of Vmp1 in mouse liver also led to the accumulation of neutral lipids in the ER bilayer and impaired mitochondrial beta-oxidation. Overexpression of VMP1 ameliorated steatosis in diet-induced NASH by improving VLDL secretion. Importantly, we also showed that decreased liver VMP1 is associated with NAFLD/NASH in humans. CONCLUSIONS: Our results provide novel insights on the role of VMP1 in regulating hepatic phospholipid synthesis and lipoprotein secretion in the pathogenesis of NAFLD/NASH. LAY SUMMARY: Non-alcoholic fatty liver disease and its more severe form, non-alcoholic steatohepatitis, are associated with a build-up of fat in the liver (steatosis). However, the exact mechanisms that underly steatosis in patients are not completely understood. Herein, the authors identified that the lack of a protein called VMP1 impairs the secretion and metabolism of fats in the liver and could therefore contribute to the development and progression of non-alcoholic fatty liver disease.
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Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Lipoproteínas/metabolismo , Hígado/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Fosfolípidos/metabolismoRESUMEN
Increased hepatic ischemia-reperfusion (IR) injury in steatotic livers is a major reason for rejecting the use of fatty livers for liver transplantation. Necroptosis is implicated in the pathogenesis of fatty liver diseases. Necroptosis is regulated by three key proteins: receptor-interacting serine/threonine-protein kinase (RIPK)-1, RIPK3, and mixed-lineage kinase domain-like protein (MLKL). Here, we found that marked steatosis of the liver was induced when a Western diet was given in mice; steatosis was associated with the inhibition of hepatic proteasome activities and with increased levels of key necroptosis-related proteins. Mice fed a Western diet had more severe liver injury, as demonstrated by increases in serum alanine aminotransferase and necrotic areas of liver, after IR than did mice fed a control diet. Although hepatic steatosis was not different between Mlkl knockout mice and wild-type mice, Mlkl knockout mice had decreased hepatic neutrophil infiltration and inflammation and were protected from hepatic IR injury, irrespective of diet. Intriguingly, Ripk3 knockout or Ripk3 kinase-dead knock-in mice were protected against IR injury at the late phase but not the early phase, irrespective of diet. Overall, our findings indicate that liver steatosis exacerbates hepatic IR injury via increased MLKL-mediated necroptosis. Targeting MLKL-mediated necroptosis may help to improve outcomes in steatotic liver transplantation.
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Hígado Graso/metabolismo , Necroptosis , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Daño por Reperfusión/metabolismo , Animales , Hígado Graso/genética , Hígado Graso/patología , Ratones , Ratones Noqueados , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Daño por Reperfusión/genética , Daño por Reperfusión/patologíaRESUMEN
Autophagy is a lysosomal degradation pathway that degrades cytoplasmic proteins and organelles. Absence of autophagy in hepatocytes has been linked to promoting liver injury and tumorigenesis; however, the mechanisms behind why a lack of autophagy induces these complications are not fully understood. The role of mammalian target of rapamycin (mTOR) in impaired autophagy-induced liver pathogenesis and tumorigenesis was investigated by using liver-specific autophagy related 5 knockout (L-ATG5 KO) mice, L-ATG5/mTOR, and L-ATG5/Raptor double knockout (DKO) mice. We found that deletion of mTOR or Raptor in L-ATG5 KO mice at 2 months of age attenuated hepatomegaly, cell death, and inflammation but not fibrosis. Surprisingly, at 6 months of age, L-ATG5/mTOR DKO and L-ATG5/Raptor DKO mice also had increased hepatic inflammation, fibrosis, and liver injury, similar to the L-ATG5 KO mice. Moreover, more than 50% of L-ATG5/mTOR DKO and L-ATG5/Raptor DKO mice already developed spontaneous tumors, but none of the L-ATG5 KO mice had developed any tumors at 6 months of age. At 9 months of age, all L-ATG5/mTOR DKO and L-ATG5/Raptor DKO had developed liver tumors. Mechanistically, L-ATG5/mTOR DKO and L-ATG5/Raptor DKO mice had decreased levels of hepatic ubiquitinated proteins and persistent nuclear erythroid 2 p45-related factor 2 activation but had increased Akt activation compared with L-ATG5 KO mice. Conclusion: Loss of mTOR signaling attenuates the liver pathogenesis in mice with impaired hepatic autophagy but paradoxically promotes tumorigenesis in mice at a relatively young age. Therefore, the balance of mTOR is critical in regulating the liver pathogenesis and tumorigenesis in mice with impaired hepatic autophagy.
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Proteína 5 Relacionada con la Autofagia/fisiología , Autofagia/fisiología , Neoplasias Hepáticas/etiología , Serina-Treonina Quinasas TOR/fisiología , Animales , Carcinogénesis , Hepatomegalia/etiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteína Reguladora Asociada a mTOR/fisiologíaRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem worldwide, ranging from nonalcoholic fatty liver (NAFL, steatosis without hepatocellular injury) to the more aggressive nonalcoholic steatohepatitis (NASH, steatosis with ballooning, inflammation, or fibrosis). Although many studies have greatly contributed to the elucidation of NAFLD pathogenesis, the disease progression from NAFL to NASH remains incompletely understood. Nuclear receptor small heterodimer partner (Nr0b2, SHP) is a transcriptional regulator critical for the regulation of bile acid, glucose, and lipid metabolism. Here, we show that SHP levels are decreased in the livers of patients with NASH and in diet-induced mouse NASH. Exposing primary mouse hepatocytes to palmitic acid and lipopolysaccharide in vitro, we demonstrated that the suppression of Shp expression in hepatocytes is due to c-Jun N-terminal kinase (JNK) activation, which stimulates c-Jun-mediated transcriptional repression of Shp Interestingly, in vivo induction of hepatocyte-specific SHP in steatotic mouse liver ameliorated NASH progression by attenuating liver inflammation and fibrosis, but not steatosis. Moreover, a key mechanism linking the anti-inflammatory role of hepatocyte-specific SHP expression to inflammation involved SHP-induced suppression of NF-κB p65-mediated induction of chemokine (C-C motif) ligand 2 (CCL2), which activates macrophage proinflammatory polarization and migration. In summary, our results indicate that a JNK/SHP/NF-κB/CCL2 regulatory network controls communications between hepatocytes and macrophages and contributes to the disease progression from NAFL to NASH. Our findings may benefit the development of new management or prevention strategies for NASH.
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Modelos Animales de Enfermedad , Inflamación/prevención & control , Cirrosis Hepática/prevención & control , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/metabolismo , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
Oomycete pathogens cause serious damage to a wide spectrum of plants. Although host pathogen recognition via pathogen effectors and cognate plant resistance proteins is well established, the genetic basis of host factors that mediate plant susceptibility to oomycete pathogens is relatively unexplored. Here, we report on RTP1, a nodulin-related MtN21 family gene in Arabidopsis that mediates susceptibility to Phytophthora parasitica. RTP1 was identified by screening a T-DNA insertion mutant population and encoded an endoplasmic reticulum (ER)-localized protein. Overexpression of RTP1 rendered Arabidopsis more susceptible, whereas RNA silencing of RTP1 led to enhanced resistance to P. parasitica. Moreover, an RTP1 mutant, rtp1-1, displayed localized cell death, increased reactive oxygen species (ROS) production and accelerated PR1 expression, compared to the wild-type Col-0, in response to P. parasitica infection. rtp1-1 showed a similar disease response to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, including increased disease resistance, cell death and ROS production. Furthermore, rpt1-1 exhibited resistance to the fungal pathogen Golovinomyces cichoracearum, but not to the necrotrophic pathogen Botrytis cinerea. Taken together, these results suggest that RTP1 negatively regulates plant resistance to biotrophic pathogens, possibly by regulating ROS production, cell death progression and PR1 expression.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiología , Resistencia a la Enfermedad , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Enfermedades de las Plantas/microbiología , Arabidopsis/genética , Botrytis/fisiología , Muerte Celular , Resistencia a la Enfermedad/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Proteínas Fluorescentes Verdes/metabolismo , Mutación/genética , Phytophthora/fisiología , Raíces de Plantas/microbiología , Pseudomonas syringae/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Fracciones Subcelulares/metabolismo , Transformación GenéticaRESUMEN
Perioperative outcomes research using anesthesia information management systems (AIMS) is an emerging research method that has not been comprehensively reviewed. This review reports an initial analysis of the use of AIMS for perioperative patient outcomes research from articles published between January 1980 and January 2013. Perioperative patient outcomes research based on AIMS has increased greatly since 2001. Although risk stratification studies involving large study populations were most commonly reported, AIMS were also used to report a rare life-threatening anesthesia-related complication, malignant hyperthermia. Use of AIMS for perioperative outcomes research can address clinically relevant topics that traditional research methods have been unable to adequately address, mainly because of the innate challenges presented by perioperative anesthesia practice. It is expected that perioperative effectiveness and outcomes research using large AIMS databases will be more widely embraced in the future to generate useful evidence and knowledge to improve anesthesia care.
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Anestesia/métodos , Anestesiología/métodos , Gestión de la Información en Salud/estadística & datos numéricos , Hipertermia Maligna/terapia , Atención Perioperativa/estadística & datos numéricos , Complicaciones Posoperatorias/terapia , Bases de Datos Factuales , Humanos , Resultado del TratamientoRESUMEN
Background and Purpose: The rapid spread of coronavirus disease 2019 (COVID-19) worldwide prompted global fear and panic among all populations. The Fear of COVID-19 Scale was developed to assess the degree of fear. This review summarizes and synthesizes the revised cross-cultural validation studies of the Revised Fear of COVID-19 Scale to explore the properties of the scale and fear characteristics among different populations. Methods: A systematic literature search was conducted on the electronic databases of PubMed and Scopus. The review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis framework. Results: The review demonstrated that the Revised Fear of COVID-19 Scale has robust psychometric properties and fear impacts mental health across all study populations. The characteristics of the fear of COVID-19 are associated with specific variables. Conclusion: The Revised Fear of COVID-19 Scale can be used as a screening tool to assess levels of COVID-19 fear among different populations globally.
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Bats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size. In vivo studies of bats can be challenging for several reasons such as ability to locate and capture them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation. Yet, cells in frozen tissue are usually damaged and represent low integrity and viability. As a result, isolating primary cells from frozen tissues poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this the first protocol specially focused on fibroblasts isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines. Basic Protocol 1: Bat wing biopsy collection and preservation Support Protocol 1: Blood collection from bat- venipuncture Basic Protocol 2: Isolation of primary fibroblasts from adult bat frozen wing biopsy Support Protocol 2: Maintenance of primary fibroblasts Support Protocol 3: Cell banking and thawing of primary fibroblasts Support Protocol 4: Growth curve and doubling time Support Protocol 5: Lentiviral transduction of bat primary fibroblasts Basic Protocol 3: Bat stable fibroblasts cell lines development Support Protocol 6: Bat fibroblasts validation by immunofluorescence staining Support Protocol 7: Chromosome counting.
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The tetra fish species Astyanax mexicanus comprises two morphotypes: cavefish that live in caves and surface fish that inhabit rivers and lakes. Because cavefish have adapted to the nutrient-poor conditions in their habitat whereas the surface fish populations can be used as a proxy for the ancestral condition, this species has become a powerful model system for understanding genetic variation underlying metabolic adaptation. The liver plays a critical role in glucose and fat metabolism in the body and hence is an important tissue for studying altered metabolism in health and disease. Cavefish morphs of A. mexicanus have been shown to develop fatty livers and exhibit massive differences in gene expression and chromatin architecture. Primary cell lines from various tissues have become invaluable tools for biochemical, toxicology, and cell biology experiments, as well as genetic and genomic analyses. To enhance the utility of the model system by enabling an expanded set of biochemical and in vitro experiments, we developed protocols for the isolation and maintenance of primary liver cells from A. mexicanus surface fish and cavefish. We also describe methods that can be used for primary cell characterization, including cloning, characterization of cell growth pattern, and lentivirus transduction. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Primary culture of liver cells Support Protocol 1: Maintenance of A. mexicanus primary liver cells Support Protocol 2: Banking of A. mexicanus primary liver cells Support Protocol 3: Recovery of A. mexicanus primary liver cells Support Protocol 4: Primary liver cell cloning Support Protocol 5: Characterization of A. mexicanus primary liver cell growth pattern Basic Protocol 2: Lentiviral transduction of A. mexicanus primary liver cells.
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Characidae , Animales , Characidae/genética , Genoma , Adaptación Fisiológica , HígadoRESUMEN
Exosomes are small extracellular vesicles (sEVs) of ~30-150 nm in diameter that have the same topology as the cell, are enriched in selected exosome cargo proteins, and play important roles in health and disease. To address large unanswered questions regarding exosome biology in vivo, we created the exomap1 transgenic mouse model. In response to Cre recombinase, exomap1 mice express HsCD81mNG, a fusion protein between human CD81, the most highly enriched exosome protein yet described, and the bright green fluorescent protein mNeonGreen. As expected, cell type-specific expression of Cre induced the cell type-specific expression of HsCD81mNG in diverse cell types, correctly localized HsCD81mNG to the plasma membrane, and selectively loaded HsCD81mNG into secreted vesicles that have the size (~80 nm), topology (outside out), and content (presence of mouse exosome markers) of exosomes. Furthermore, mouse cells expressing HsCD81mNG released HsCD81mNG-marked exosomes into blood and other biofluids. Using high-resolution, single-exosome analysis by quantitative single molecule localization microscopy, we show here that that hepatocytes contribute ~15% of the blood exosome population whereas neurons contribute <1% of blood exosomes. These estimates of cell type-specific contributions to blood EV population are consistent with the porosity of liver sinusoidal endothelial cells to particles of ~50-300 nm in diameter, as well as with the impermeability of blood-brain and blood-neuron barriers to particles >5 nm in size. Taken together, these results establish the exomap1 mouse as a useful tool for in vivo studies of exosome biology, and for mapping cell type-specific contributions to biofluid exosome populations. In addition, our data confirm that CD81 is a highly-specific marker for exosomes and is not enriched in the larger microvesicle class of EVs.
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Oomycetes represent a unique group of plant pathogens that are phylogenetically distant from true fungi and cause significant crop losses and environmental damage. Understanding of the genetic basis of host plant susceptibility facilitates the development of novel disease resistance strategies. In this study, we report the identification of an Arabidopsis thaliana T-DNA mutant with enhanced resistance to Phytophthora parasitica with an insertion in the Raf-like mitogen-activated protein kinase kinase kinase gene Raf36. We generated additional raf36 mutants by CRISPR/Cas9 technology as well as Raf36 complementation and overexpression transformants, with consistent results of infection assays showing that Raf36 mediates Arabidopsis susceptibility to P. parasitica. Using a virus-induced gene silencing assay, we silenced Raf36 homologous genes in Nicotiana benthamiana and demonstrated by infection assays the conserved immune function of Raf36. Mutagenesis analyses indicated that the kinase activity of Raf36 is important for its immune function and interaction with MKK2, a MAPK kinase. By generating and analysing mkk2 mutants and MKK2 complementation and overexpression transformants, we found that MKK2 is a positive immune regulator in the response to P. parasitica infection. Furthermore, infection assay on mkk2 raf36 double mutant plants indicated that MKK2 is required for the raf36-conferred resistance to P. parasitica. Taken together, we identified a Raf-like kinase Raf36 as a novel plant susceptibility factor that functions upstream of MKK2 and directly targets it to negatively regulate plant resistance to P. parasitica.
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Proteínas de Arabidopsis , Arabidopsis , Phytophthora , Quinasas raf/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Phytophthora/fisiología , Enfermedades de las Plantas/microbiologíaRESUMEN
Investigating the function of delicate mammalian eyes often requires chemical fixation, histological sectioning, immunohistochemistry (IHC) and in situ hybridization (ISH). One of the long-standing challenges in the ocular histology field is the limited success of maintaining intact morphology via cryo- or paraffin procedures. Although our latest protocol significantly improved the morphology of mouse eyeball sections, the window technique is time-consuming and requires extensive practice to avoid damage while making windows. In this study, we present a novel glyoxal fixative that is suitable for a freeze-substitution approach to improve both morphology and molecular target preservation of mouse eyes. The method prevents morphology distortion in all tested eyeballs. Therefore, it suits a variety of research needs from morphological examination to investigation of single-molecule RNA expression, using hematoxylin and eosin (H&E) stain, IHC, and ISH assays on either frozen (cryo) or paraffin-infiltrated tissue sections. In addition, this method can be easily performed in many histology laboratories.
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Glioxal , Parafina , Animales , Ratones , Fijadores/farmacología , Glioxal/farmacología , Solventes , Hibridación in Situ , MamíferosRESUMEN
Exercise is critical for improving metabolic health and putatively maintains or enhances mitochondrial quality control in metabolic tissues. Although previous work has shown that exercise elicits hepatic mitochondrial biogenesis, it is unknown if acute exercise activates hepatic mitophagy, the selective degradation of damaged or low-functioning mitochondria. We tested if an acute bout of treadmill running increased hepatic mitophagic flux both right after and 2-h postexercise in 15- to 24-wk-old C57BL/6J female mice. Acute exercise did not significantly increase markers of autophagic flux, however, mitophagic flux was activated 2-h post-treadmill running as measured by accumulation of both LC3-II and p62 in isolated mitochondria in the presence of leupeptin, an inhibitor of autophagosome degradation. Furthermore, mitochondrial-associated ubiquitin, which recruits the autophagy receptor protein p62, was also significantly increased at 2 h. Further examination via Western blot and proteomics analysis revealed that acute exercise elicits a time-dependent, dynamic activation of mitophagy pathways. Moreover, the results suggest that exercise-induced hepatic mitophagy is likely mediated by both polyubiquitination and receptor-mediated signaling pathways. Overall, we provide evidence that acute exercise activates hepatic mitophagic flux while also revealing specific receptor-mediated proteins by which exercise maintains mitochondrial quality control in the liver.NEW & NOTEWORTHY This study provides evidence that acute exercise activates hepatic mitophagic flux and mitochondrial polyubiquitination while additionally revealing specific receptor-mediated proteins by which exercise maintains mitochondrial quality control in the liver.
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Mitocondrias , Mitofagia , Animales , Autofagia , Femenino , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitofagia/fisiologíaRESUMEN
Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases. Many methods have been developed for EV characterization. However, current methods for EV analysis all have different limitations. Thus, developing efficient and effective methods for EV isolation and characterization remains one of the crucial steps for this cutting-edge research field as it matures. Here, we provide a detailed protocol outlining a single-particle interferometric reflectance imaging sensor (SP-IRIS), as a method that is capable of detecting and characterizing EVs from unpurified biological sources and purified EVs by other methodologies. This advanced technique can be used for multi-level and comprehensive measurements for the analysis of EV size, EV count, EV phenotype, and biomarker colocalization.
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Vesículas Extracelulares , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Interferometría , Dinámica Poblacional , Proteínas/metabolismoRESUMEN
Alcohol-related liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are the primary causes of chronic liver disease in western countries. Liver transplantation is currently one of the most efficient approaches to save patients with liver failure, which is often associated with hepatic ischemia-reperfusion (IR) injury. IR injury is exacerbated by hepatic steatosis, yet the mechanism remains elusive. Necroptosis is a form of regulated cell death mediated by receptor-interacting protein kinase 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL) protein, which has been implicated in the pathogenesis of ALD and NAFLD. Though necroptosis plays an important role in IR injury of high fat diet - induced steatotic livers, the role of necroptosis in IR injury of ethanol - induced steototic livers has not been investigated. In the present study, we used chronic plus binge alcohol (Gao-binge) feeding followed by IR surgery to investigate IR liver injury with ethanol-associated steatosis. We found that the levels of key necroptotic proteins MLKL and RIP3 increased in alcohol-fed mouse livers. Moreover, we observed increased liver injury after IR in control diet-fed mice, which was further exacerbated by alcohol feeding based on serum alanine aminotransferase (ALT) levels and TUNEL staining of necrotic cells. Hepatic neutrophil infiltration also increased in alcohol-fed mice after IR surgery. However, deletion of Mlkl did not protect against IR liver injury in alcohol-fed mice compared with matched wild-type mice. In conclusion, alcoholic steatosis promotes IR injury, which seems to be independent of MLKL-mediated necroptosis.
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Enfermedad del Hígado Graso no Alcohólico , Proteínas Quinasas , Daño por Reperfusión , Animales , Humanos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Necroptosis , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Daño por Reperfusión/metabolismoRESUMEN
Nonacholic fatty liver disease, or hepatic steatosis, is the most common liver disorder affecting the western world and currently has no pharmacologic cure. Thus, many investigations have focused on alternative strategies to treat or prevent hepatic steatosis. Our laboratory has shown that chronic heat treatment (HT) mitigates glucose intolerance, insulin resistance, and hepatic steatosis in rodent models of obesity. Here, we investigate the direct bioenergetic mechanism(s) surrounding the metabolic effects of HT on hepatic mitochondria. Utilizing mitochondrial proteomics and respiratory function assays, we show that one bout of acute HT (42°C for 20 min) in male C57Bl/6J mice (n = 6/group) triggers a hepatic mitochondrial heat shock response resulting in acute reductions in respiratory capacity, degradation of key mitochondrial enzymes, and induction of mitophagy via mitochondrial ubiquitination. We also show that chronic bouts of HT and recurrent activation of the heat shock response enhances mitochondrial quality and respiratory function via compensatory adaptations in mitochondrial organization, gene expression, and transport even during 4 weeks of high-fat feeding (n = 6/group). Finally, utilizing a liver-specific heat shock protein 72 (HSP72) knockout model, we are the first to show that HSP72, a protein putatively driving the HT metabolic response, does not play a significant role in the hepatic mitochondrial adaptation to acute or chronic HT. However, HSP72 is required for the reductions in blood glucose observed with chronic HT. Our data are the first to suggest that chronic HT (1) improves hepatic mitochondrial respiratory efficiency via mitochondrial remodeling and (2) reduces blood glucose in a hepatic HSP72-dependent manner.
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Resistencia a la Insulina , Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Masculino , Glucemia/metabolismo , Mitocondrias , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Resistencia a la Insulina/genéticaRESUMEN
Alzheimer's disease (AD) is the most common neurodegenerative disease, yet there are no disease-modifying treatments available and there is no cure. It is becoming apparent that metabolic and vascular conditions such as type 2 diabetes (T2D) and hypertension promote the development and accumulation of Alzheimer's disease-related dementia pathologies. To this end, aerobic exercise, which is a common lifestyle intervention for both metabolic disease and hypertension, is shown to improve brain health during both healthy aging and dementia. However, noncompliance or other barriers to exercise response are common in exercise treatment paradigms. In addition, reduced intracellular proteostasis and mitochondrial function could contribute to the etiology of AD. Specifically, compromised chaperone systems [i.e., heat shock protein (HSP) systems] can contribute to protein aggregates (i.e., ß-amyloid plaques and neurofibrillary tangles) and reduced mitochondrial quality control (i.e., mitophagy). Therefore, novel therapies that target whole body metabolism, the vasculature, and chaperone systems (like HSPs) are needed to effectively treat AD. This review focuses on the role of heat therapy in the treatment and prevention of AD. Heat therapy has been independently shown to reduce whole body insulin resistance, improve vascular function, activate interorgan cross talk via endocytic vesicles, and activate HSPs to improve mitochondrial function and proteostasis in a variety of tissues. Thus, heat therapy could offer immense clinical benefit to patients suffering from AD. Importantly, future studies in patients are needed to determine the safety and efficacy of heat therapy in preventing AD.
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Enfermedad de Alzheimer , Diabetes Mellitus Tipo 2 , Enfermedades Neurodegenerativas , Enfermedad de Alzheimer/terapia , Encéfalo , Cognición , HumanosRESUMEN
Phytophthora species are destructive plant pathogens that cause significant crop losses worldwide. To understand plant susceptibility to oomycete pathogens and to explore novel disease resistance strategies, we employed the Arabidopsis thaliana-Phytophthora parasitica model pathosystem and screened for A. thaliana T-DNA insertion mutant lines resistant to P. parasitica. This led to the identification of the resistant mutant 267-31, which carries two T-DNA insertion sites in the promoter region of the ethylene-responsive factor 19 gene (ERF019). Quantitative reverse transcription PCR (RT-qPCR) assays showed that the expression of ERF019 was induced during P. parasitica infection in the wild type, which was suppressed in the 267-31 mutant. Additional erf019 mutants were generated using CRISPR/Cas9 technology and were confirmed to have increased resistance to P. parasitica. In contrast, ERF019 overexpression lines were more susceptible. Transient overexpression assays in Nicotiana benthamiana showed that the nuclear localization of ERF019 is crucial for its susceptible function. RT-qPCR analyses showed that the expression of marker genes for multiple defence pathways was significantly up-regulated in the mutant compared with the wild type during infection. Flg22-induced hydrogen peroxide accumulation and reactive oxygen species burst were impaired in ERF019 overexpression lines, and flg22-induced MAPK activation was enhanced in erf019 mutants. Moreover, transient overexpression of ERF019 strongly suppressed INF-triggered cell death in N. benthamiana. These results reveal the importance of ERF019 in mediating plant susceptibility to P. parasitica through suppression of pathogen-associated molecular pattern-triggered immunity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Unión al ADN/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Phytophthora/fisiología , Enfermedades de las Plantas/inmunología , Factores de Transcripción/metabolismo , Arabidopsis/inmunología , Arabidopsis/parasitología , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/genética , Resistencia a la Enfermedad , Susceptibilidad a Enfermedades , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/parasitología , Inmunidad de la Planta , Factores de Transcripción/genéticaRESUMEN
Exosomes are small membrane vesicles (ranging from 30 nm to 150 nm), secreted by different cell types upon fusion of multivesicular bodies (MVB) to the cell plasma membrane under a variety of normal and pathological conditions. Through transferring their cargos such as proteins, lipids and nucleic acids from donor cells to recipient cells, exosomes play a crucial role in cell-to-cell communication. Due to their presence in most body fluids (such as blood, breast milk, saliva, urine, bile, pancreatic juice, cerebrospinal and peritoneal fluids), and their role in carrying bioactive molecules from the cells of origin, exosomes have attracted great interest in their diagnostic and prognostic value for various diseases and therapeutic approaches. Although a large body of literature has documented the importance of exosomes over the past decade, there is no article systematically summarizing protein markers of exosome from different resources and the antibodies that are suited to characterize exosomes. In this review, we briefly summarize the exosome marker proteins, exosomal biomarkers for different diseases, and the antibodies suitable for different bio-resources exosomes characterization.