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OBJECTIVE: Our study aimed to investigate the association between maternal pre-pregnancy body mass index (BMI), gestational weight gain (GWG), and impaired pelvic floor muscle (PFM) morphology and function during the early postpartum period. METHODS: This retrospective cohort study was conducted at Shanghai First Maternity and Infant Hospital from December 2020 to December 2022. A total of 1118 primiparous women with singleton pregnancies who underwent vaginal deliveries and participated in postpartum PFM assessments were included. Maternal pre-pregnancy BMI and GWG were considered as exposures. PFM morphology and function impairment were the primary outcomes. PFM morphology impairment, defined as levator ani muscle avulsion, was assessed using transperineal ultrasound. PFM function impairment, manifested as diminished PFM fiber strength, was assessed through vaginal manometry. Multivariable logistic regression analysis was employed to calculate adjusted odds ratios (aOR) with 95% confidence intervals (CI). Restricted cubic spline models were used to validate and visualize the relationship. RESULTS: Women with lower pre-pregnancy BMI were at an increased risk of levator ani muscle avulsion (aOR = 1.73, 95% CI: 1.10-2.70, P = 0.017), particularly when combined with excessive GWG during pregnancy (aOR = 3.20, 95% CI: 1.15-8.97, P = 0.027). Lower pre-pregnancy BMI was also identified as an independent predictor of PFM weakness (aOR = 1.53, 95% CI: 1.08-2.16, P = 0.017 for type I fiber injuries). Notably, regardless of the avulsion status, both underweight and overweight/obese women faced an elevated risk of reduced PFM strength (aOR = 1.74, 95% CI: 1.17-2.59, P = 0.006 for underweight women with type I fiber injuries; aOR = 1.67, 95% CI: 1.06-2.64, P = 0.027; and aOR = 1.73, 95% CI: 1.09-2.76, P = 0.021 for overweight/obese women with type I and type II fibers injuries, respectively). CONCLUSIONS: Both lower and higher pre-pregnancy BMI, as well as excessive GWG, were strongly associated with PFM impairments. These findings highlighted the critical importance of comprehensive weight management throughout pregnancy to effectively promote women's pelvic health.
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Transverse mode switchable ultrashort optical pulses with narrow bandwidths can create potential for exploring what we believe are new physical effects. We demonstrate the generation of transverse mode switchable ultrashort pulses with narrow bandwidths in an all-fiber mode-locked laser by exploring a mode-selective photonic lantern (MSPL). The laser cavity serves not only as a ring resonator but also as an intrinsic spectral filter. For mode-locking with the LP01, LP11a, and LP11b modes, the bandwidths are 3.0â nm, 86.7 pm and 101.7 pm, respectively. The narrowband pulses with higher-order modes are generated by an intrinsic spectral filter due to the spectral-domain intermodal interference. Mode-locked pulses with a signal-to-noise ratio better than 60â dB for LP01, LP11a, and LP11b modes are independently generated, i.e., transverse mode switchable by changing the input port of the MSPL. The mode-locked wavelength can be tuned for the LP11a mode and LP11b mode by adjusting the state of polarization. Furthermore, our experimental results also show that, the slope efficiency of LP11a and LP11b modes can be improved, by the use of LP11 mode pump scheme. We anticipate that, narrowband pulses with complex mode profiles can be generated by simultaneously phase-locked transverse and longitudinal modes.
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To understand the effects of a high-fat diet (HFD) on lung cancer progression and biomarkers, we here used an inducible mutant epidermal growth factor receptor (EGFR)-driven lung cancer transgenic mouse model fed a regular diet (RD) or HFD. The HFD lung cancer (LC-HFD) group exhibited significant tumor formation and deterioration, such as higher EGFR activity and proliferation marker expression, compared with the RD lung cancer (LC-RD) group. Transcriptomic analysis of the lung tissues revealed that the significantly changed genes in the LC-HFD group were highly enriched in immune-related signaling pathways, suggesting that an HFD alters the immune microenvironment to promote tumor growth. Cytokine and adipokine arrays combined with a comprehensive analysis using meta-database software indicated upregulation of C-reactive protein (CRP) in the LC-HFD group, which presented with increased lung cancer proliferation and metastasis; this was confirmed experimentally. Our results imply that an HFD can turn the tumor growth environment into an immune-related pro-tumorigenic microenvironment and demonstrate that CRP has a role in promoting lung cancer development in this microenvironment.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Ratones , Animales , Proteína C-Reactiva , Dieta Alta en Grasa , Ratones Transgénicos , Adenocarcinoma del Pulmón/genética , Neoplasias Pulmonares/genética , Receptores ErbB/genética , Microambiente TumoralRESUMEN
Located in the frontline against the largest population of microbiota, the intestinal mucosa of mammals has evolved to become an effective immune system. γδ T cells, a unique T cell subpopulation, are rare in circulation blood and lymphoid tissues, but rich in the intestinal mucosa, particularly in the epithelium. Via rapid production of cytokines and growth factors, intestinal γδ T cells are key contributors to epithelial homeostasis and immune surveillance of infection. Intriguingly, recent studies have revealed that the intestinal γδ T cells may play novel exciting functions ranging from epithelial plasticity and remodeling in response to carbohydrate diets to the recovery of ischemic stroke. In this review article, we update regulatory molecules newly defined in lymphopoiesis of the intestinal γδ T cells and their novel functions locally in the intestinal mucosa, such as epithelial remodeling, and distantly in pathological setting, e.g., ischemic brain injury repair, psychosocial stress responses, and fracture repair. The challenges and potential revenues in intestinal γδ T cell studies are discussed.
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Linfocitos Intraepiteliales , Receptores de Antígenos de Linfocitos T gamma-delta , Animales , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Mucosa Intestinal , Epitelio/metabolismo , Tejido Linfoide/metabolismo , Linfocitos Intraepiteliales/metabolismo , Mamíferos/metabolismoRESUMEN
Long-form collapsin response mediator protein-1 (LCRMP-1) belongs to the CRMP family which comprises brain-enriched proteins responsible for axon guidance. However, its role in spermatogenesis remains unclear. Here we find that LCRMP-1 is abundantly expressed in the testis. To characterize its physiological function, we generate LCRMP-1-deficient mice (Lcrmp-1-/-). These mice exhibit aberrant spermiation with apoptotic spermatids, oligospermia, and accumulation of immature testicular cells, contributing to reduced fertility. In the seminiferous epithelial cycle, LCRMP-1 expression pattern varies in a stage-dependent manner. LCRMP-1 is highly expressed in spermatids during spermatogenesis and especially localized to the spermiation machinery during spermiation. Mechanistically, LCRMP-1 deficiency causes disorganized F-actin due to unbalanced signaling of F-actin dynamics through upregulated PI3K-Akt-mTOR signaling. In conclusion, LCRMP-1 maintains spermatogenesis homeostasis by modulating cytoskeleton remodeling for spermatozoa release.
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Actinas , Proteínas del Tejido Nervioso , Espermátides , Animales , Masculino , Ratones , Actinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Espermátides/metabolismo , Espermatogénesis/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
With an aging population and the numerous health impacts associated with old age, the identification of anti-aging drugs has become an important new research direction. Although mitochondria have been recognized to affect aging, anti-aging drugs specifically targeting the mitochondria are less well characterized. In this study, diphenyleneiodonium (DPI) was identified as a potential senomorphic drug that functions by promoting mitochondrial fission. DPI significantly reduced the number of senescence-associated ß-galactosidase (SA-ß-gal) positive cells and increased the number of proliferating Ki-67 positive cells in BrdU or irradiation stress-induced senescent NIH3T3 cells or IMR90 cells and mouse embryonic fibroblasts (MEFs) replicative senescent cells. Cell cycle arrest genes and senescence-associated secretory phenotype (SASP) factors were downregulated with DPI treatment. In addition, the oxygen consumption rate (OCR) of mitochondrial respiration showed that DPI significantly reduced senescence-associated hyper OCR. Mechanistically, DPI promoted mitochondrial fission by enhancing AMPK/MFF phosphorylation and DRP1 mitochondrial translocation. Inhibition of DRP1 by Mdivi-1 abolished DPI-induced mitochondrial fission and the anti-senescence phenotype. Importantly, Eighty-eight-week-old mice treated with DPI had significantly reduced numbers of SA-ß-gal positive cells and reduced expression of cell cycle arrest genes and SASP factors in their livers and kidneys. Pathological and functional assays showed DPI treatment not only reduced liver fibrosis and immune cell infiltration but also improved aged-related physical impairments in aged mice. Taken together, our study identified a potential anti-aging compound that exerts its effects through modulation of mitochondrial morphology.
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Proteínas Quinasas Activadas por AMP , Dinaminas , Animales , Ratones , Dinaminas/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Senoterapéuticos , Dinámicas Mitocondriales , Células 3T3 NIH , Proteínas Mitocondriales/metabolismo , Fibroblastos/metabolismoRESUMEN
Metamaterials, from concept to application level, is currently a high-trending topic. Due to the strict requirements of the simultaneous reasonable structural design and stability of materials, the construction of a high-performance metamaterial for extreme environments is still difficult. Here, combining metamaterial design with materials optimization, we propose a completely different strategy and synthesize a type of monomeric ceramic meta-atom to construct metamaterials. Based on a geometric design with multiple degrees of freedom and dielectric properties, hybrid anapole modes with impedance matching can be produced, experimentally inducing nearly perfect absorption with high temperature stability (high tolerable temperature of approximately 1300 °C, with almost zero temperature drift) in microwave/millimeter-wave bands. We surpass the oxidation temperature limitation of 800 °C in conventional plasmonic absorbers, and provide an unprecedented direction for the further development of integrated high-performance metamaterial wireless sensors responding to extreme environmental scenarios, which will also lead to a new direction of specific ceramic research toward device physics.
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FIP200, also known as RB1CC1, is a protein that assembles the autophagy initiation complex. Its post-translational modifications and degradation mechanisms are unclear. Upon autophagy activation, we find that FIP200 is methylated at lysine1133 (K1133) by methyltransferase SETD2. We identify the E3 ligase Trim21 to be responsible for FIP200 ubiquitination by targeting K1133, resulting in FIP200 degradation through the ubiquitin-proteasome system. SETD2-induced methylation blocks Trim21-mediated ubiquitination and degradation, preserving autophagy activity. SETD2 and Trim21 orchestrate FIP200 protein stability to achieve dynamic and precise control of autophagy flux.
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Autofagia , Ubiquitina , Metilación , Autofagia/fisiología , Proteínas Relacionadas con la Autofagia/metabolismo , Ubiquitinación , Ubiquitina/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMEN
Heat shock protein (HSP) 40 has emerged as a key factor in both innate and adaptive immunity, whereas the role of HLJ1, a molecular chaperone in HSP40 family, in modulating endotoxin-induced sepsis severity is still unclear. During lipopolysaccharide (LPS)-induced endotoxic shock, HLJ1 knockout mice shows reduced organ injury and IFN-γ (interferon-γ)-dependent mortality. Using single-cell RNA sequencing, we characterize mouse liver nonparenchymal cell populations under LPS stimulation, and show that HLJ1 deletion affected IFN-γ-related gene signatures in distinct immune cell clusters. In CLP models, HLJ1 deletion reduces IFN-γ expression and sepsis mortality rate when mice are treated with antibiotics. HLJ1 deficiency also leads to reduced serum levels of IL-12 in LPS-treated mice, contributing to dampened production of IFN-γ in natural killer cells but not CD4+ or CD8+ T cells, and subsequently to improved survival rate. Adoptive transfer of HLJ1-deleted macrophages into LPS-treated mice results in reduced IL-12 and IFN-γ levels and protects the mice from IFN-γ-dependent mortality. In the context of molecular mechanisms, HLJ1 is an LPS-inducible protein in macrophages and converts misfolded IL-12p35 homodimers to monomers, which maintains bioactive IL-12p70 heterodimerization and secretion. This study suggests HLJ1 causes IFN-γ-dependent septic lethality by promoting IL-12 heterodimerization, and targeting HLJ1 has therapeutic potential in inflammatory diseases involving activated IL-12/IFN-γ axis.
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Proteínas del Choque Térmico HSP40/metabolismo , Interleucina-12 , Sepsis , Animales , Linfocitos T CD8-positivos/metabolismo , Endotoxinas/toxicidad , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Sepsis/inducido químicamenteRESUMEN
HLJ1 (also called DNAJB4) is a member of the DNAJ/Hsp40 family and plays an important role in regulating protein folding and activity. However, there is little information about the role of HLJ1 in the regulation of physiological function. In this study, we investigated the role of HLJ1 in blood coagulation using wild-type C57BL/6 mice and HLJ1-null (HLJ1-/-) mice. Western blot analysis and immunohistochemistry were used to assess the expression and distribution of HLJ1 protein, respectively. The tail bleeding assay was applied to assess the bleeding time and blood loss. A coagulation test was used for measuring the activity of extrinsic, intrinsic and common coagulation pathways. Thromboelastography was used to measure the coagulation parameters in the progression of blood clot formation. The results showed that HLJ1 was detectable in plasma and bone marrow. The distribution of HLJ1 was co-localized with CD41, the marker of platelets and megakaryocytes. However, genetic deletion of HLJ1 did not alter blood loss and the activity of extrinsic and intrinsic coagulation pathways, as well as blood clot formation, compared to wild-type mice. Collectively, these findings suggest that, although HLJ1 appears in megakaryocytes and platelets, it may not play a role in the function of blood coagulation under normal physiological conditions.
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Coagulación Sanguínea/genética , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Animales , Biomarcadores/metabolismo , Plaquetas/metabolismo , Eliminación de Gen , Masculino , Megacariocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Glicoproteína IIb de Membrana Plaquetaria/genéticaRESUMEN
BACKGROUND: The nuclear receptor Rev-erbα/ß, a key component of the circadian clock, emerges as a drug target for heart diseases, but the function of cardiac Rev-erb has not been studied in vivo. Circadian disruption is implicated in heart diseases, but it is unknown whether cardiac molecular clock dysfunction is associated with the progression of any naturally occurring human heart diseases. Obesity paradox refers to the seemingly protective role of obesity for heart failure, but the mechanism is unclear. METHODS: We generated mouse lines with cardiac-specific Rev-erbα/ß knockout (KO), characterized cardiac phenotype, conducted multi-omics (RNA-sequencing, chromatin immunoprecipitation sequencing, proteomics, and metabolomics) analyses, and performed dietary and pharmacological rescue experiments to assess the time-of-the-day effects. We compared the temporal pattern of cardiac clock gene expression with the cardiac dilation severity in failing human hearts. RESULTS: KO mice display progressive dilated cardiomyopathy and lethal heart failure. Inducible ablation of Rev-erbα/ß in adult hearts causes similar phenotypes. Impaired fatty acid oxidation in the KO myocardium, in particular, in the light cycle, precedes contractile dysfunctions with a reciprocal overreliance on carbohydrate utilization, in particular, in the dark cycle. Increasing dietary lipid or sugar supply in the dark cycle does not affect cardiac dysfunctions in KO mice. However, obesity coupled with systemic insulin resistance paradoxically ameliorates cardiac dysfunctions in KO mice, associated with rescued expression of lipid oxidation genes only in the light cycle in phase with increased fatty acid availability from adipose lipolysis. Inhibition of glycolysis in the light cycle and lipid oxidation in the dark cycle, but not vice versa, ameliorate cardiac dysfunctions in KO mice. Altered temporal patterns of cardiac Rev-erb gene expression correlate with the cardiac dilation severity in human hearts with dilated cardiomyopathy. CONCLUSIONS: The study delineates temporal coordination between clock-mediated anticipation and nutrient-induced response in myocardial metabolism at multi-omics levels. The obesity paradox is attributable to increased cardiac lipid supply from adipose lipolysis in the fasting cycle due to systemic insulin resistance and adiposity. Cardiac molecular chronotypes may be involved in human dilated cardiomyopathy. Myocardial bioenergetics downstream of Rev-erb may be a chronotherapy target in treating heart failure and dilated cardiomyopathy.
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Ritmo Circadiano/fisiología , Miocardio/patología , Obesidad/fisiopatología , Animales , Relojes Circadianos , Cardiopatías , Humanos , Ratones , Ratones NoqueadosRESUMEN
Fissure sealant is a recommended preventive measure on permanent molars, but its efficacy on primary molars in preschool children is still in doubt. Sodium fluoride varnish (NaFV) enhances remineralization and is effective in preventing caries in smooth surfaces, but limited information is available regarding its use on occlusal surfaces of primary molars. The present study aimed to compare the efficacy of glass ionomer sealant (GIS) versus topical application of 5% NaFV in preventing and arresting occlusal caries in primary second molars among preschool children. One calibrated examiner examined children aged 3-4 years for visible plaque index (VPI), decayed-missing-filled primary surface (dmfs) index, and extent of carious lesions by the International Caries Detection and Assessment System (ICDAS). Subjects with 1 or more primary second molars that were sound or with incipient lesions (ICDAS 0-1), with distinct visual enamel changes (ICDAS 2), or with localized enamel breakdown (ICDAS 3) were recruited. The children were randomly assigned to either NaFV group - application of 5% NaFV at 3-monthly intervals or GIS group - 1 single placement of GIS. A parental questionnaire was used to collect information on the children's sociodemographic background and oral health-related habits. Two blinded examiners conducted clinical examinations after 6 and 12 months to evaluate the development of occlusal caries and the retention of GIS. Negative binomial regression using generalized estimating equations was employed to adjust for the clustering effect and evaluate the influence of selected variables on the development of occlusal caries into dentin at 12 months. A total of 323 children with 1,159 primary second molars received the respective preventive measures at baseline. At 12 months, 280 children with 989 molars were evaluated. Caries had developed or progressed into dentin in 7.8% and 8.0% of the molars in the NaFV and GIS groups, respectively, with no significant difference between the groups (p = 0.913). The overall retention rates of GIS were 24.6% and 13.5% at 6 and 12 months, respectively. Regression analyses showed only baseline caries experience, and an ICDAS code had a significant association with occlusal caries progression. Quarterly NaFV application and single GIS placement showed similar effectiveness in the prevention of occlusal caries development among preschool children.
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Caries Dental , Fluoruros Tópicos , Resinas Acrílicas , Preescolar , Caries Dental/prevención & control , Susceptibilidad a Caries Dentarias , Fluoruros , Fluoruros Tópicos/uso terapéutico , Humanos , Diente Molar , Selladores de Fosas y Fisuras/uso terapéutico , Dióxido de SilicioRESUMEN
BACKGROUND: COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus is transmitted through respiratory droplets and by physical contact from contaminated surfaces to the mucosa. Its route of transmission has caused a significant challenge in medical and dental healthcare. OBJECTIVE: This article aims to review the literature and information available on the provision of paediatric dental treatment during and post-pandemic and to provide specific recommendations on the safe provision of paediatric dental care. RESULTS: Children infected by SARS-CoV-2 have no or milder COVID-19 symptoms and are potential vectors in spreading the disease. Routine dental treatment is suspended in many countries because of the increased risk of cross-infection in dental practices. Only emergency dental care is provided for urgent conditions. It is necessary to gradually reinstate regular dental care to paediatric patients and maintain their good oral health. To control the disease transmission and maintain the oral health of the population, minimally intervention techniques that minimise or eliminate aerosol generation, plus comprehensive oral health preventive measures should be practised to safeguard safety at dental practices in this unprecedented time. CONCLUSIONS: Robust infection control guidelines should be implemented in dental clinics to minimise the risk of infection and to ensure the safety of patients and staff during the pandemic. Three levels of preventive care should be practised to prevent oral diseases and improve children's oral health in this COVID-19 era. Treatment should be prioritized to patients in urgent needs and aerosol-generating procedures should be minimized.
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Ventriculoatrial (VA) shunt is one of the most commonly used solutions for hydrocephalus. In recent years, the number of VA shunt has decreased worldwide, given the perceived technical challenges and the potentially serious complications associated with VA shunt. However, VA shunt remains as a viable treatment option for hydrocephalus in selected patients. Novel placement strategies and monitoring methods have been developed to reduce complications following VA shunt. In this article, we reported that seven consecutive cases who received a VA shunt. VA shunts were applied in seven hydrocephalic patients who experienced previous ventriculoperitoneal (VP) shunt failures or had contraindications to abdominal catheter placement. The insertion of VA shunt catheters was guided with the aid of intraoperative electromagnetic neuronavigation and electrocardiographic technique. There were three female and four male patients with a mean age of 46 years (range 22-68 years) received VA shunts under the guidance of electromagnetic neuronavigation and electrocardiographic method intraoperatively. In all cases, postoperative cranial CT scans and chest radiography demonstrated appropriate positioning of the catheter tips. And no postoperative complications occurred during the follow-up period of 3-26 months. VA shunts are potential favorable alternatives for patients who cannot tolerate VP shunts. It is noteworthy that VA is not free of complications. Intraoperative application of electromagnetic neuronavigation and electrocardiographic technique contributes to safe and optimal catheter placement of VA shunts.
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Lesiones Traumáticas del Encéfalo/cirugía , Ventrículos Cerebrales/cirugía , Manejo de la Enfermedad , Atrios Cardíacos/cirugía , Hidrocefalia/cirugía , Neuronavegación/métodos , Adulto , Anciano , Lesiones Traumáticas del Encéfalo/diagnóstico por imagen , Ventrículos Cerebrales/diagnóstico por imagen , Estudios de Factibilidad , Femenino , Atrios Cardíacos/diagnóstico por imagen , Humanos , Hidrocefalia/diagnóstico por imagen , Monitorización Neurofisiológica Intraoperatoria/métodos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del Tratamiento , Adulto JovenRESUMEN
Diastolic dysfunction is condition of a stiff ventricle and a function of aging. It causes significant cardiovascular mortality and morbidity, and in fact, three million Americans are currently suffering from this condition. To date, all the pharmacological clinical trials have been negative. The lack of success in attenuating/ameliorating diastolic dysfunction stems from lack of duplication of myriads of clinical manifestation in pre-clinical settings. Here we report, a novel genetically engineered mice which may represents a preclinical model of human diastolic dysfunction to some extent. Topoisomerase 2 beta (Top2b) is an important enzyme in transcriptional activation of some inducible genes through transient double-stranded DNA breakage events around promoter regions. We created a conditional, tissue-specific, inducible Top2b knockout mice in the heart. Serendipitously, echocardiographic parameters and more invasive analysis of left ventricular function with pressure-volume loops show features of diastolic dysfunction. This was also confirmed histologically. At the cellular level, the Top2b knockdown showed morphological changes and molecular signaling akin to human diastolic dysfunction. Reverse phase protein analysis showed activation of p53 and inhibition of, Akt, as the possible mediators of diastolic dysfunction. Finally, activation of p53 and inhibition of Akt were confirmed in myocardial biopsy samples obtained from human diastolic dysfunctional hearts. Thus, we report for the first time, a Top2b downregulated preclinical mice model for diastolic dysfunction which demonstrates that Akt and p53 are the possible mediators of the pathology, hence representing novel and viable targets for future therapeutic interventions in diastolic dysfunction.
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The Rho family of GTPases consists of 20 members including RhoE. Here, we discover the existence of a short isoform of RhoE designated as RhoEα, the first Rho GTPase isoform generated from alternative translation. Translation of this new isoform is initiated from an alternative start site downstream of and in-frame with the coding region of the canonical RhoE. RhoEα exhibits a similar subcellular distribution while its protein stability is higher than RhoE. RhoEα contains binding capability to RhoE effectors ROCK1, p190RhoGAP and Syx. The distinct transcriptomes of cells with the expression of RhoE and RhoEα, respectively, are demonstrated. The data propose distinctive and overlapping biological functions of RhoEα compared to RhoE. In conclusion, this study reveals a new Rho GTPase isoform generated from alternative translation. The discovery provides a new scope of understanding the versatile functions of small GTPases and underlines the complexity and diverse roles of small GTPases.
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Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Animales , Secuencia de Bases , Línea Celular , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Masculino , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Biosíntesis de Proteínas , Isoformas de Proteínas , Proteínas de Unión al GTP rho/químicaRESUMEN
Exosomes are secreted extracellular vesicles with lipid bilayer membranes. They are emerging as a new category of messengers that facilitate cross-talk between cells, tissues, and organs. Thus, a critical demand arises for the development of a sensitive and non-invasive tracking system for endogenous exosomes. We have generated a genetic mouse model that meets this goal. The Nano-luciferase (NanoLuc) reporter was fused with the exosome surface marker CD63 for exosome labeling. The cardiomyocyte-specific αMHC promoter followed by the loxP-STOP-loxP cassette was engineered for temporal and spatial labeling of exosomes originated from cardiomyocytes. The transgenic mouse was bred with a tamoxifen-inducible Cre mouse (Rosa26Cre-ERT2) to achieve inducible expression of CD63NanoLuc reporter. The specific labeling and tissue distribution of endogenous exosomes released from cardiomyocytes were demonstrated by luciferase assay and non-invasive bioluminescent live imaging. This endogenous exosome tracking mouse provides a useful tool for a range of research applications.
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Exosomas/metabolismo , Fibroblastos/metabolismo , Luciferasas/metabolismo , Miocitos Cardíacos/metabolismo , Tetraspanina 30/metabolismo , Animales , Animales Modificados Genéticamente , Células Cultivadas , Exosomas/ultraestructura , Fibroblastos/ultraestructura , Integrasas/genética , Integrasas/metabolismo , Luciferasas/genética , Mediciones Luminiscentes , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/ultraestructura , Cadenas Pesadas de Miosina/genética , Regiones Promotoras Genéticas , ARN no Traducido/genética , Proteínas Recombinantes de Fusión/metabolismo , Tetraspanina 30/genética , Factores de Tiempo , Distribución TisularRESUMEN
Polyubiquitination encompasses complex topologies through various linkage types to deliver diverse cellular signals, which has been recognized as a sophisticated ubiquitin code. Accurate comparison of polyubiquitination signals is critical for revealing the dynamic cellular ubiquitination-regulated events. Western blotting (WB) is the most widely used biochemical method to quantify proteins and posttranslational modifications under diverse physiological conditions. The accuracy and sensitivity of the WB mainly depend on the quality and specificity of the antibody. In this study, we found that the antiubiquitin antibodies exhibited different affinities to the eight linkage types of ubiquitin chains, with the highest sensitivity for the K63-linked chain, lower efficiency for M1 and K48, and very low affinity for the other types of chains. Herein, we introduced the tandem hybrid ubiquitin-binding domain (ThUBD)-based far-Western blotting (TUF-WB) to visualize the signal of synthetic ubiquitin chains or ubiquitinated conjugates on a solid membrane by utilizing the unbiased affinity of ThUBD to all types of ubiquitin linkages. As compared to antiubiquitin antibody detection, TUF-WB can accurately quantify the signal intensity to the mass amounts of all eight ubiquitin chains. Meanwhile, the sensitivity of this method in detecting complex ubiquitinated samples was 4-5-fold higher than those of antibodies. Consequently, TUF-WB allows accurate quantification of polyubiquitination signal on the membrane with great sensitivity and wider dynamic range.
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Far-Western Blotting/métodos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitinación , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Escherichia coli/química , Células HEK293 , Humanos , Proteínas de la Membrana/química , Dominios Proteicos , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Inflammatory response after myocardial infarction (MI) is essential for cardiac healing, whereas excessive and prolonged inflammation extends the infarction and promotes adverse cardiac remodeling. Understanding the mechanistic insight of these tightly controlled inflammatory processes has a significant impact on post-MI recovery and therapy. Here, we uncover the critical role of small GTPase RhoE in post-MI recovery and its clinical implication. METHODS: Three genetic mouse lines are used: global RhoE knockout, cardiomyocyte-specific RhoE heterozygous, and cardiomyocyte-specific RhoE overexpression mice. A set of molecular signaling experiments, including bimolecular fluorescence complementation, immunoprecipitation, electrophoretic mobility shift assay, and mRNA microarray analysis, were conducted. Permanent ligation of the left anterior descending artery was performed, followed by the assessments of cardiac function, inflammation, and survival in the first week after MI. Finally, we examined the correlation of the expression levels of RhoE in MI patient heart and patient prognosis. RESULTS: RhoE deficiency turns on a group of proinflammatory gene expressions in mouse heart. Mice with cardiomyocyte-specific haploinsufficiency exhibit excessive inflammatory response with deleterious cardiac function after MI. A profound increase in nuclear factor-κB activity is detected in the mutant heart and the isolated cardiomyocytes. We further find that the expression of RhoE is upregulated in response to MI. Mechanistically, RhoE interacts with p65 and p50 individually in cytosol and blocks their nuclear translocation. RhoE also occupies the dimerization domain of p65 and subsequently disrupts the heterodimerization between p65 and p50. Cardiac RhoE overexpression inhibits nuclear factor-κB activity, restrains post-MI inflammation, and improves cardiac function and survival. Consistently, we find that the expression level of RhoE is elevated in the heart of patients with MI and that the patients with a higher expression level of RhoE exhibit a better prognosis in cardiac function recovery. CONCLUSIONS: The study uncovers RhoE as a new fine-tuning factor modulating MI-induced inflammation and promoting injured heart recovery. RhoE may serve as a new potential biomarker for the assessment of MI patient prognosis. Manipulation of RhoE could be as a potential therapeutic approach for MI and other inflammatory diseases.