RESUMO
The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNASerCGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain.
Assuntos
Cromossomos Artificiais de Levedura , Genoma Fúngico , Saccharomyces cerevisiae , Sequência de Bases , Cromossomos/genética , Saccharomyces cerevisiae/genética , Biologia SintéticaRESUMO
The production of pore-forming toxins that disrupt the plasma membrane of host cells is a common virulence strategy for bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA)1-3. It is unclear, however, whether host species possess innate immune mechanisms that can neutralize pore-forming toxins during infection. We previously showed that the autophagy protein ATG16L1 is necessary for protection against MRSA strains encoding α-toxin4-a pore-forming toxin that binds the metalloprotease ADAM10 on the surface of a broad range of target cells and tissues2,5,6. Autophagy typically involves the targeting of cytosolic material to the lysosome for degradation. Here we demonstrate that ATG16L1 and other ATG proteins mediate protection against α-toxin through the release of ADAM10 on exosomes-extracellular vesicles of endosomal origin. Bacterial DNA and CpG DNA induce the secretion of ADAM10-bearing exosomes from human cells as well as in mice. Transferred exosomes protect host cells in vitro by serving as scavengers that can bind multiple toxins, and improve the survival of mice infected with MRSA in vivo. These findings indicate that ATG proteins mediate a previously unknown form of defence in response to infection, facilitating the release of exosomes that serve as decoys for bacterially produced toxins.
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Proteínas Relacionadas à Autofagia/metabolismo , Toxinas Bacterianas/metabolismo , Exossomos/metabolismo , Células A549 , Proteína ADAM10/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , DNA Bacteriano/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/ultraestrutura , Feminino , Células HEK293 , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Staphylococcus aureus Resistente à Meticilina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções Estafilocócicas/mortalidadeRESUMO
The bacterial microbiota promotes the life cycle of the intestine-dwelling whipworm Trichuris by mediating hatching of parasite eggs ingested by the mammalian host. Despite the enormous disease burden associated with Trichuris colonization, the mechanisms underlying this transkingdom interaction have been obscure. Here, we used a multiscale microscopy approach to define the structural events associated with bacteria-mediated hatching of eggs for the murine model parasite Trichuris muris. Through the combination of scanning electron microscopy (SEM) and serial block face SEM (SBFSEM), we visualized the outer surface morphology of the shell and generated 3D structures of the egg and larva during the hatching process. These images revealed that exposure to hatching-inducing bacteria catalyzed asymmetric degradation of the polar plugs prior to exit by the larva. Unrelated bacteria induced similar loss of electron density and dissolution of the structural integrity of the plugs. Egg hatching was most efficient when high densities of bacteria were bound to the poles. Consistent with the ability of taxonomically distant bacteria to induce hatching, additional results suggest chitinase released from larva within the eggs degrade the plugs from the inside instead of enzymes produced by bacteria in the external environment. These findings define at ultrastructure resolution the evolutionary adaptation of a parasite for the microbe-rich environment of the mammalian gut.
Assuntos
Microbiota , Trichuris , Camundongos , Animais , Microscopia Eletrônica de Varredura , Bactérias , Larva , Óvulo , MamíferosRESUMO
Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19). Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchoalveolar lavage fluid (BALF) from critically ill COVID-19 patients was associated with reduced intensive care unit (ICU) and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.
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Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19 , Humanos , Peptidil Dipeptidase A/metabolismo , Receptores Virais , SARS-CoV-2RESUMO
The most distal portion of the ventricular conduction system (VCS) contains cardiac Purkinje cells (PCs), which are essential for synchronous activation of the ventricular myocardium. Contactin-2 (CNTN2), a member of the immunoglobulin superfamily of cell adhesion molecules (IgSF-CAMs), was previously identified as a marker of the VCS. Through differential transcriptional profiling, we discovered two additional highly enriched IgSF-CAMs in the VCS: NCAM-1 and ALCAM. Immunofluorescence staining showed dynamic expression patterns for each IgSF-CAM during embryonic and early postnatal stages, but ultimately all three proteins became highly enriched in mature PCs. Mice deficient in NCAM-1, but not CNTN2 or ALCAM, exhibited defects in PC gene expression and VCS patterning, as well as cardiac conduction disease. Moreover, using ST8sia2 and ST8sia4 knockout mice, we show that inhibition of post-translational modification of NCAM-1 by polysialic acid leads to disrupted trafficking of sarcolemmal intercalated disc proteins to junctional membranes and abnormal expansion of the extracellular space between apposing PCs. Taken together, our data provide insights into the complex developmental biology of the ventricular conduction system.
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Ventrículos do Coração/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurogênese/fisiologia , Molécula de Adesão de Leucócito Ativado , Animais , Moléculas de Adesão Celular/metabolismo , Contactina 2/metabolismo , Expressão Gênica , Coração , Sistema de Condução Cardíaco/metabolismo , Camundongos , Camundongos Knockout , Ácidos Siálicos , SialiltransferasesRESUMO
IMPORTANCE: Generation of virus-host protein-protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention.
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Proteases Semelhantes à Papaína de Coronavírus , Interações entre Hospedeiro e Microrganismos , Glicoproteínas de Membrana , Proteínas de Membrana , Proteínas de Membrana Transportadoras , SARS-CoV-2 , Replicação Viral , Humanos , COVID-19/virologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/metabolismoRESUMO
Glioblastoma multiforme (GBM) is the most aggressive brain cancer with a poor prognosis. Therefore, the correlative molecular markers and molecular mechanisms should be explored to assess the occurrence and treatment of glioma.WB and qPCR assays were used to detect the expression of CXCL5 in human GBM tissues. The relationship between CXCL5 expression and clinicopathological features was evaluated using logistic regression analysis, Wilcoxon symbolic rank test, and Kruskal-Wallis test. Univariate, multivariate Cox regression and Kaplan-Meier methods were used to assess CXCL5 and other prognostic factors of GBM. Gene set enrichment analysis (GSEA) was used to identify pathways associated with CXCL5. The correlation between CXCL5 and tumor immunoinfiltration was investigated using single sample gene set enrichment analysis (ssGSEA) of TCGA data. Cell experiments and mouse subcutaneous transplanted tumor models were used to evaluate the role of CXCL5 in GBM. WB, qPCR, immunofluorescence, and immunohistochemical assays showed that CXCL5 expression was increased in human GBM tissues. Furthermore, high CXCL5 expression was closely related to poor disease-specific survival and overall survival of GBM patients. The ssGSEA suggested that CXCL5 is closely related to the cell cycle and immune response through PPAR signaling pathway. GSEA also showed that CXCL5 expression was positively correlated with macrophage cell infiltration level and negatively correlated with cytotoxic cell infiltration level. CXCL5 may be associated with the prognosis and immunoinfiltration of GBM.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Animais , Camundongos , Humanos , Glioblastoma/patologia , Prognóstico , Processos Neoplásicos , Neoplasias Encefálicas/metabolismo , Transdução de Sinais , Quimiocina CXCL5/genéticaRESUMO
Colorectal cancer (CRC) is a significant health problem with elevated mortality rates, prompting intense exploration of its complex molecular mechanisms and innovative therapeutic avenues. Resveratrol (RSV), recognised for its anticancer effects through SIRT1 activation, is a promising candidate for CRC treatment. This study focuses on elucidating RSV's role in CRC progression, particularly its effect on autophagy-related apoptosis. Using bioinformatics, protein imprinting and immunohistochemistry, we established a direct correlation between FOXQ1 and adverse CRC prognosis. Comprehensive in vitro experiments confirmed RSV's ability to promote autophagy-related apoptosis in CRC cells. Plasmids for SIRT1 modulation were used to investigate underlying mechanisms. Molecular docking, glutathione-S-transferase pull-down experiments and immunoprecipitation highlighted RSV's direct activation of SIRT1, resulting in the inhibition of FOXQ1 expression. Downstream interventions identified ATG16L as a crucial autophagic target. In vivo and in vitro studies validated RSV's potential for CRC therapy through the SIRT1/FOXQ1/ATG16L pathway. This study establishes RSV's capacity to enhance autophagy-related cell apoptosis in CRC, positioning RSV as a prospective therapeutic agent for CRC within the SIRT1/FOXQ1/ATG16L pathway.
Assuntos
Apoptose , Autofagia , Neoplasias Colorretais , Fatores de Transcrição Forkhead , Resveratrol , Sirtuína 1 , Humanos , Resveratrol/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Sirtuína 1/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Nus , Masculino , Simulação de Acoplamento Molecular , Feminino , Progressão da Doença , Camundongos Endogâmicos BALB CRESUMO
In this study, simple-structured wavelength sensors were developed by depositing two back-to-back Au/MAPbI3/Au photodetectors on an MAPbI3 single crystal. This sensor could quantitatively distinguish wavelengths. Further device analysis showed that both photodetectors possess entirely disparate optoelectronic properties. Consequently, the as-developed wavelength sensor could accurately distinguish incident-light wavelengths ranging from 265 to 860 nm with a resolution of less than 1.5 nm based on the relation between the photocurrent ratios of both photodetectors and the incident light wavelengths. Notably, a high resolution and wide detection range are among the optimum reported values for such sensors and enable full-color imaging. Furthermore, technology computer-aided design (TCAD) simulations showed that a mechanism involved in distinguishing wavelengths is attributed to the wavelength-dependent photon generation rate in MAPbI3 single crystals. The high-performance MAPbI3 wavelength sensor can potentially drive the research progress of perovskites in wavelength recognition and full-color imaging.
RESUMO
RATIONALE: The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined. OBJECTIVE: To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria. METHODS AND RESULTS: Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na+/Ca2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding NaV1.5) and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A. CONCLUSIONS: We describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.
Assuntos
Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular , Feminino , Humanos , Cinética , Masculino , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Mitocôndrias Cardíacas/ultraestrutura , Proteínas Mitocondriais/genética , Miócitos Cardíacos/ultraestrutura , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Imagem Individual de Molécula , Trocador de Sódio e Cálcio/genética , Superóxidos/metabolismoRESUMO
Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 µmâ s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.
Assuntos
Microscopia/métodos , Microsporídios/patogenicidade , Organelas/imunologia , Organelas/ultraestrutura , Esporos Fúngicos/imunologia , Esporos Fúngicos/ultraestrutura , Proteínas Fúngicas/metabolismo , Microsporídios/imunologia , Microsporídios/ultraestrutura , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Signaling in the ancestral branch of the unfolded protein response (UPR) is initiated by unconventional splicing of HAC1/XBP1 mRNA during endoplasmic reticulum (ER) stress. In mammals, IRE1α has been known to cleave the XBP1 intron. However, the enzyme responsible for ligation of two XBP1 exons remains unknown. Using an XBP1 splicing-based synthetic circuit, we identify RtcB as the primary UPR RNA ligase. In RtcB knockout cells, XBP1 mRNA splicing is defective during ER stress. Genetic rescue and in vitro splicing show that the RNA ligase activity of RtcB is directly required for the splicing of XBP1 mRNA. Taken together, these data demonstrate that RtcB is the long-sought RNA ligase that catalyzes unconventional RNA splicing during the mammalian UPR.
Assuntos
Proteínas/fisiologia , Splicing de RNA/genética , Resposta a Proteínas não Dobradas , Aminoacil-tRNA Sintetases , Animais , Sequência de Bases , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Endorribonucleases/metabolismo , Técnicas de Inativação de Genes , Camundongos , Dados de Sequência Molecular , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/química , Proteínas/genética , Interferência de RNA , Fatores de Transcrição de Fator Regulador X , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteína 1 de Ligação a X-BoxRESUMO
Type I and III interferons induce expression of the "myxovirus resistance proteins" MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles ("biomolecular condensates"). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40-50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: â¼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20-30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.
Assuntos
Antivirais/uso terapêutico , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Filamentos Intermediários/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Infecções por Rhabdoviridae/prevenção & controle , Animais , Núcleo Celular/genética , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Proteínas de Resistência a Myxovirus/genética , Infecções por Rhabdoviridae/metabolismo , Infecções por Rhabdoviridae/virologia , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
This work reports the design of a wavelength sensor composed of two identical perovskite (FA0.85 Cs0.15 PbI3 ) photodetectors (PDs) that are capable of discriminating incident wavelength in a quantitative way. Due to strong wavelength-dependent absorption coefficient, the penetration depth of the photons in the FA0.85 Cs0.15 PbI3 nanofilms increases with the increasing wavelength, leading to a gradual decrease of photo-generated current for PD1, but an increase of photocurrent in PD2, according to the theoretical simulation of Technology Computer Aided Design. This special evolution of photo-generated current as a function of wavelength facilitates the quantitative determination of the wavelength since the current ratio of both PDs monotonously decreases with the increase of wavelength from 265 to 810 nm. The average absolute error and the average relative error are estimated to be 7.6 nm and 1.68%, respectively, which are much better than other semiconductors materials-based wavelength sensors previously reported. It is believed that the present perovskite film-based wavelength sensor will have potential application in the future color/spectrum optoelectronic devices.
RESUMO
In OA chondrocytes, there is diminished mitochondrial production of ATP and diminished extracellular adenosine resulting in diminished adenosine A2A receptor (A2AR) stimulation and altered chondrocyte homeostasis which contributes to the pathogenesis of OA. We tested the hypothesis that A2AR stimulation maintains or enhances mitochondrial function in chondrocytes. The effect of A2AR signaling on mitochondrial health and function was determined in primary murine chondrocytes, a human chondrocytic cell line (T/C-28a2), primary human chondrocytes, and a murine model of OA by transmission electron microscopy analysis, mitochondrial stress testing, confocal live imaging for mitochondrial inner membrane polarity, and immunohistochemistry. In primary murine chondrocytes from A2AR-/- null mice, which develop spontaneous OA by 16 weeks, there is mitochondrial swelling, dysfunction, and reduced mitochondrial content with increased reactive oxygen species (ROS) burden and diminished mitophagy, as compared to chondrocytes from WT animals. IL-1-stimulated T/C-28a2 cells treated with an A2AR agonist had reduced ROS burden with increased mitochondrial dynamic stability and function, findings which were recapitulated in primary human chondrocytes. In an obesity-induced OA mouse model, there was a marked increase in mitochondrial oxidized material which was markedly improved after intraarticular injections of liposomal A2AR agonist. These results are consistent with the hypothesis that A2AR ligation is mitoprotective in OA.
Assuntos
Condrócitos/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Osteoartrite/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor A2A de Adenosina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosina/uso terapêutico , Agonistas do Receptor A2 de Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/uso terapêutico , Animais , Linhagem Celular , Células Cultivadas , Criança , Condrócitos/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dinâmica Mitocondrial , Osteoartrite/tratamento farmacológico , Fenetilaminas/farmacologia , Fenetilaminas/uso terapêutico , Receptor A2A de Adenosina/genéticaRESUMO
INTRODUCTION AND HYPOTHESIS: The aim was to investigate the effectiveness and safety of electroacupuncture (EA) in women with balanced mixed urinary incontinence (MUI) compared with PFMT plus solifenacin. METHODS: This is a secondary analysis of a randomized non-inferiority clinical trial. Seventy-nine patients with balanced MUI were randomly assigned to receive either 12-week EA with 24-week follow-up or 36-week PFMT and solifenacin. Primary outcome was the proportion of participants with ≥50% reduction of mean 24-h incontinence episode frequency (IFE) through weeks 1-12 from baseline. Analysis was performed in an intention-to-treat population using a generalized linear model with a binomial distribution, adjusted for imbalances in baseline variables, and a two-sided p value of less than 0.05 was considered significant. RESULTS: A total of 34 participants in the EA group and 45 participants in the PFMT plus solifenacin group were included in the intention-to-treat analysis of primary outcome. Through weeks 1-12, the proportion of participants with ≥50% reduction of mean 24-h IEF was 32.4% in the EA group, and 37.2% in the PFMT plus solifenacin group, with a mean difference of -2.82% (95%CI: -23.88 to 18.23, p=0.79), revealing non-inferiority. No significant difference held true for all the secondary outcomes. Six adverse events occurred in the EA group and 22 in the PEMT plus solifenacin group. CONCLUSIONS: The effect of EA is similar to PFMT plus solifenacin in relieving the symptoms of both SUI and UUI and increasing participants' quality of life but with better safety. The effects of EA may sustain 24 weeks after treatment.
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Eletroacupuntura , Incontinência Urinária por Estresse , Incontinência Urinária , Feminino , Humanos , Diafragma da Pelve , Qualidade de Vida , Resultado do Tratamento , Incontinência Urinária/terapia , Incontinência Urinária de Urgência/tratamento farmacológicoRESUMO
BACKGROUND: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+]i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. CONCLUSIONS: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.
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Displasia Arritmogênica Ventricular Direita/metabolismo , Conexina 43/metabolismo , Desmossomos/metabolismo , Miócitos Cardíacos/fisiologia , Placofilinas/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Células Cultivadas , Modelos Animais de Doenças , Homeostase , Humanos , Camundongos , Camundongos Knockout , Mutação/genética , Placofilinas/genéticaRESUMO
Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles (e.g., stress granules and P-bodies) in the mammalian cell cytoplasm and nucleus. In contrast to the long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA) associated with the endoplasmic reticulum (ER) and Golgi apparatus, we report that MxA formed membraneless metastable (shape-changing) condensates in the cytoplasm. In our studies, we used the same cell lines and methods as those used by previous investigators but concluded that wild-type MxA formed variably sized spherical or irregular bodies, filaments, and even a reticulum distinct from that of ER/Golgi membranes. Moreover, in Huh7 cells, MxA structures associated with a novel cytoplasmic reticular meshwork of intermediate filaments. In live-cell assays, 1,6-hexanediol treatment led to rapid disassembly of green fluorescent protein (GFP)-MxA structures; FRAP revealed a relative stiffness with a mobile fraction of 0.24 ± 0.02 within condensates, consistent with a higher-order MxA network structure. Remarkably, in intact cells, GFP-MxA condensates reversibly disassembled/reassembled within minutes of sequential decrease/increase, respectively, in tonicity of extracellular medium, even in low-salt buffers adjusted only with sucrose. Condensates formed from IFN-α-induced endogenous MxA also displayed tonicity-driven disassembly/reassembly. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with spherical GFP-MxA condensates in cells showing an antiviral effect. These observations prompt comparisons with the extensive literature on interactions between viruses and stress granules/P-bodies. Overall, the new data correct a long-standing misinterpretation in the MxA literature and provide evidence for membraneless MxA biomolecular condensates in the uninfected cell cytoplasm.IMPORTANCE There is a long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA), which displays antiviral activity against several RNA and DNA viruses, associates with the endoplasmic reticulum (ER) and Golgi apparatus. We provide data to correct this misinterpretation and further report that MxA forms membraneless metastable (shape-changing) condensates in the cytoplasm consisting of variably sized spherical or irregular bodies, filaments, and even a reticulum. Remarkably, MxA condensates showed the unique property of rapid (within 1 to 3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions. Moreover, GFP-MxA condensates included the VSV nucleocapsid (N) protein, a protein previously shown to form liquid-like condensates. Since intracellular edema and ionic changes are hallmarks of cytopathic effects of a viral infection, the tonicity-driven regulation of MxA condensates may reflect a mechanism for modulation of MxA function during viral infection.
Assuntos
Citoplasma/virologia , Proteínas de Resistência a Myxovirus/metabolismo , Linhagem Celular , Efeito Citopatogênico Viral/fisiologia , Citoplasma/metabolismo , Humanos , Orthomyxoviridae/metabolismo , Proteínas/metabolismo , Vírus da Estomatite Vesicular Indiana/metabolismo , Viroses/metabolismo , Vírus/metabolismoRESUMO
In this study, a highly sensitive and self-driven near-infrared (NIR) light photodetector based on PdSe2 /pyramid Si heterojunction arrays, which are fabricated through simple selenization of predeposited Pd nanofilm on black Si, is demonstrated. The as-fabricated hybrid device exhibits excellent photoresponse performance in terms of a large on/off ratio of 1.6 × 105 , a responsivity of 456 mA W-1 , and a high specific detectivity of up to 9.97 × 1013 Jones under 980 nm illumination at zero bias. Such a relatively high sensitivity can be ascribed to the light trapping effect of the pyramid microstructure, which is confirmed by numerical modeling based on finite-difference time domain. On the other hand, thanks to the broad optical absorption properties of PdSe2 , the as-fabricated device also exhibits obvious sensitivity to other NIR illuminations with wavelengths of 1300, 1550, and 1650 nm, which is beyond the photoresponse range of Si-based devices. It is also found that the PdSe2 /pyramid Si heterojunction device can also function as an NIR light sensor, which can readily record both "tree" and "house" images produced by 980 and 1300 nm illumination, respectively.
RESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1005625.].