RESUMEN
RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-ß2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-ß1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-ß2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-ß2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.
Asunto(s)
Transporte Activo de Núcleo Celular , Priones/química , Proteínas de Unión al ARN/química , Receptores Citoplasmáticos y Nucleares/química , Adulto , Anciano , Animales , Citoplasma/química , Proteínas de Unión al ADN/química , Drosophila melanogaster , Femenino , Proteínas Fluorescentes Verdes/química , Células HEK293 , Células HeLa , Homeostasis , Humanos , Carioferinas/química , Masculino , Persona de Mediana Edad , Chaperonas Moleculares/química , Mutación , Enfermedades Neurodegenerativas/patología , Dominios Proteicos , Proteína EWS de Unión a ARN/química , Factores Asociados con la Proteína de Unión a TATA/química , beta Carioferinas/químicaRESUMEN
Cytoplasmic FUS aggregates are a pathological hallmark in a subset of patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). A key step that is disrupted in these patients is nuclear import of FUS mediated by the import receptor Transportin/Karyopherin-ß2. In ALS-FUS patients, this is caused by mutations in the nuclear localization signal (NLS) of FUS that weaken Transportin binding. In FTD-FUS patients, Transportin is aggregated, and post-translational arginine methylation, which regulates the FUS-Transportin interaction, is lost. Here, we show that Transportin and arginine methylation have a crucial function beyond nuclear import-namely to suppress RGG/RG-driven phase separation and stress granule association of FUS. ALS-associated FUS-NLS mutations weaken the chaperone activity of Transportin and loss of FUS arginine methylation, as seen in FTD-FUS, promote phase separation, and stress granule partitioning of FUS. Our findings reveal two regulatory mechanisms of liquid-phase homeostasis that are disrupted in FUS-associated neurodegeneration.
Asunto(s)
Arginina/química , Proteína FUS de Unión a ARN/química , beta Carioferinas/química , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Citoplasma/metabolismo , Metilación de ADN , ADN Complementario/metabolismo , Densitometría , Degeneración Lobar Frontotemporal/metabolismo , Células HeLa , Homeostasis , Humanos , Carioferinas/química , Espectroscopía de Resonancia Magnética , Metilación , Chaperonas Moleculares/química , Mutación , Enfermedades Neurodegenerativas/metabolismo , Unión Proteica , Dominios ProteicosRESUMEN
Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-ß2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-ß2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-ß2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-ß2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-ß2 may act analogously to control condensates in diverse cellular contexts.
Asunto(s)
Transporte Activo de Núcleo Celular , Señales de Localización Nuclear , Proteína FUS de Unión a ARN/química , beta Carioferinas/química , Sitios de Unión , Degeneración Lobar Frontotemporal/metabolismo , Humanos , Carioferinas/metabolismo , Luz , Extracción Líquido-Líquido , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética , Mutación , Nefelometría y Turbidimetría , Unión Proteica , Dominios Proteicos , ARN/química , Dispersión de Radiación , TemperaturaRESUMEN
FUS is a nuclear RNA-binding protein, and its cytoplasmic aggregation is a pathogenic signature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It remains unknown how the FUS-RNA interactions contribute to phase separation and whether its phase behavior is affected by ALS-linked mutations. Here we demonstrate that wild-type FUS binds single-stranded RNA stoichiometrically in a length-dependent manner and that multimers induce highly dynamic interactions with RNA, giving rise to small and fluid condensates. In contrast, mutations in arginine display a severely altered conformation, static binding to RNA, and formation of large condensates, signifying the role of arginine in driving proper RNA interaction. Glycine mutations undergo rapid loss of fluidity, emphasizing the role of glycine in promoting fluidity. Strikingly, the nuclear import receptor Karyopherin-ß2 reverses the mutant defects and recovers the wild-type FUS behavior. We reveal two distinct mechanisms underpinning potentially disparate pathogenic pathways of ALS-linked FUS mutants.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genética , Mutación/genética , Proteína FUS de Unión a ARN/genética , ARN/genética , Transporte Activo de Núcleo Celular/genética , Glicina/genética , HumanosRESUMEN
Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11ß2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.
Asunto(s)
Cilios/efectos de los fármacos , Cilios/metabolismo , Oxiesteroles/farmacología , Animales , Línea Celular , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Células 3T3 NIH , Transducción de Señal/efectos de los fármacosRESUMEN
Mucus obstruction is a central feature in the cystic fibrosis (CF) airways. A genome-wide association study (GWAS) of lung disease by the CF Gene Modifier Consortium (CFGMC) identified a significant locus containing two mucin genes, MUC20 and MUC4. Expression quantitative trait locus (eQTL) analysis using human nasal epithelia (HNE) from 94 CF-affected Canadians in the CFGMC demonstrated MUC4 eQTLs that mirrored the lung association pattern in the region, suggesting that MUC4 expression may mediate CF lung disease. Complications arose, however, with colocalization testing using existing methods: the locus is complex and the associated SNPs span a 0.2 Mb region with high linkage disequilibrium (LD) and evidence of allelic heterogeneity. We previously developed the Simple Sum (SS), a powerful colocalization test in regions with allelic heterogeneity, but SS assumed eQTLs to be present to achieve type I error control. Here we propose a two-stage SS (SS2) colocalization test that avoids a priori eQTL assumptions, accounts for multiple hypothesis testing and the composite null hypothesis, and enables meta-analysis. We compare SS2 to published approaches through simulation and demonstrate type I error control for all settings with the greatest power in the presence of high LD and allelic heterogeneity. Applying SS2 to the MUC20/MUC4 CF lung disease locus with eQTLs from CF HNE revealed significant colocalization with MUC4 (p = 1.31 × 10-5) rather than with MUC20. The SS2 is a powerful method to inform the responsible gene(s) at a locus and guide future functional studies. SS2 has been implemented in the application LocusFocus.
Asunto(s)
Sistemas de Transporte de Aminoácidos/genética , Fibrosis Quística/genética , Modelos Estadísticos , Mucina 4/genética , Mucinas/genética , Sitios de Carácter Cuantitativo , Alelos , Sistemas de Transporte de Aminoácidos/metabolismo , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Heterogeneidad Genética , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Desequilibrio de Ligamiento , Pulmón/metabolismo , Pulmón/patología , Mucina 4/metabolismo , Mucinas/metabolismo , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Polimorfismo de Nucleótido SimpleRESUMEN
The acute respiratory virus infection can induce uncontrolled inflammatory responses, such as cytokine storm and viral pneumonia, which are the major causes of death in clinical cases. Cyclophilin A (CypA) is mainly distributed in the cytoplasm of resting cells and released into the extracellular space in response to inflammatory stimuli. Extracellular CypA (eCypA) is upregulated and promotes inflammatory response in severe COVID-19 patients. However, how eCypA promotes virus-induced inflammatory response remains elusive. Here, we observe that eCypA is induced by influenza A and B viruses and SARS-CoV-2 in cells, mice, or patients. Anti-CypA mAb reduces pro-inflammatory cytokines production, leukocytes infiltration, and lung injury in virus-infected mice. Mechanistically, eCypA binding to integrin ß2 triggers integrin activation, thereby facilitating leukocyte trafficking and cytokines production via the focal adhesion kinase (FAK)/GTPase and FAK/ERK/P65 pathways, respectively. These functions are suppressed by the anti-CypA mAb that specifically blocks eCypA-integrin ß2 interaction. Overall, our findings reveal that eCypA-integrin ß2 signaling mediates virus-induced inflammatory response, indicating that eCypA is a potential target for antibody therapy against viral pneumonia.
Asunto(s)
COVID-19 , Ciclofilina A , Ciclofilina A/metabolismo , Animales , Humanos , Ratones , COVID-19/metabolismo , COVID-19/virología , COVID-19/inmunología , Antígenos CD18/metabolismo , SARS-CoV-2 , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Neumonía Viral/metabolismo , Neumonía Viral/inmunología , Citocinas/metabolismo , Anticuerpos Monoclonales/farmacología , Transducción de Señal , Virus de la Influenza A , Modelos Animales de EnfermedadRESUMEN
Widespread release of norepinephrine (NE) throughout the forebrain fosters learning and memory via adrenergic receptor (AR) signaling, but the molecular mechanisms are largely unknown. The ß2 AR and its downstream effectors, the trimeric stimulatory Gs-protein, adenylyl cyclase (AC), and the cAMP-dependent protein kinase A (PKA), form a unique signaling complex with the L-type Ca2+ channel (LTCC) CaV1.2. Phosphorylation of CaV1.2 by PKA on Ser1928 is required for the upregulation of Ca2+ influx on ß2 AR stimulation and long-term potentiation induced by prolonged theta-tetanus (PTT-LTP) but not LTP induced by two 1-s-long 100-Hz tetani. However, the function of Ser1928 phosphorylation in vivo is unknown. Here, we show that S1928A knock-in (KI) mice of both sexes, which lack PTT-LTP, express deficiencies during initial consolidation of spatial memory. Especially striking is the effect of this mutation on cognitive flexibility as tested by reversal learning. Mechanistically, long-term depression (LTD) has been implicated in reversal learning. It is abrogated in male and female S1928A knock-in mice and by ß2 AR antagonists and peptides that displace ß2 AR from CaV1.2. This work identifies CaV1.2 as a critical molecular locus that regulates synaptic plasticity, spatial memory and its reversal, and LTD.SIGNIFICANCE STATEMENT We show that phosphorylation of the Ca2+ channel CaV1.2 on Ser1928 is important for consolidation of spatial memory and especially its reversal, and long-term depression (LTD). Identification of Ser1928 as critical for LTD and reversal learning supports the model that LTD underlies flexibility of reference memory.
Asunto(s)
Plasticidad Neuronal , Memoria Espacial , Ratones , Masculino , Femenino , Animales , Plasticidad Neuronal/fisiología , Potenciación a Largo Plazo/fisiología , Transducción de Señal , Fosforilación , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Hipocampo/fisiologíaRESUMEN
Primary angle-closure glaucoma (PACG) is the leading cause of irreversible blindness in the world. Angle closure induced by pupil block and secondary iris synechia is the fundamental pathology of the PACG. The molecular mechanisms of angle closure have not yet been clearly illustrated. This study was designed to investigate the protein difference in the aqueous humour and explore new biomarker of the PACG. Aqueous humour (AH) was collected from patients with acute primary angle closure (APAC) and cataract (n = 10 in APAC group) and patients with cataract only (n = 10 in control group). Samples were pooled and measured using label-free proteome technology. Then, the differentially expressed proteins (DEPs) were verified by ELISA using independent AH samples (n = 20 each group). More than 400 proteins were revealed in both groups through proteomics. Comparing the two groups, there were 91DEPs. These proteins participate in biological activities such as inflammation, fibrosis, nerve growth and degeneration and metabolism. We found that the expression of transforming growth factor-ß2 and matrilin2 was downregulated in the APAC group. The two proteins are related to inflammation and extracellular matrix formation, which might be involved in angle closure. This study characterized DEPs in AH of the APAC and found a downregulated protein matrilin2.
Asunto(s)
Humor Acuoso , Catarata , Humanos , Enfermedad Aguda , Humor Acuoso/metabolismo , Catarata/metabolismo , Ensayo de Inmunoadsorción Enzimática , Inflamación/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Proteínas Matrilinas/metabolismoRESUMEN
BACKGROUND: Herein, we report results from a genome-wide study conducted to identify protein quantitative trait loci (pQTL) for circulating angiogenic and inflammatory protein markers in patients with metastatic colorectal cancer (mCRC). The study was conducted using genotype, protein marker, and baseline clinical and demographic data from CALGB/SWOG 80405 (Alliance), a randomized phase III study designed to assess outcomes of adding VEGF or EGFR inhibitors to systemic chemotherapy in mCRC patients. Germline DNA derived from blood was genotyped on whole-genome array platforms. The abundance of protein markers was quantified using a multiplex enzyme-linked immunosorbent assay from plasma derived from peripheral venous blood collected at baseline. A robust rank-based method was used to assess the statistical significance of each variant and protein pair against a strict genome-wide level. A given pQTL was tested for validation in two external datasets of prostate (CALGB 90401) and pancreatic cancer (CALGB 80303) patients. Bioinformatics analyses were conducted to further establish biological bases for these findings. RESULTS: The final analysis was carried out based on data from 540,021 common typed genetic variants and 23 protein markers from 869 genetically estimated European patients with mCRC. Correcting for multiple testing, the analysis discovered a novel cis-pQTL in LINC02869, a long non-coding RNA gene, for circulating TGF-ß2 levels (rs11118119; AAF = 0.11; P-value < 1.4e-14). This finding was validated in a cohort of 538 prostate cancer patients from CALGB 90401 (AAF = 0.10, P-value < 3.3e-25). The analysis also validated a cis-pQTL we had previously reported for VEGF-A in advanced pancreatic cancer, and additionally identified trans-pQTLs for VEGF-R3, and cis-pQTLs for CD73. CONCLUSIONS: This study has provided evidence of a novel cis germline genetic variant that regulates circulating TGF-ß2 levels in plasma of patients with advanced mCRC and prostate cancer. Moreover, the validation of previously identified pQTLs for VEGF-A, CD73, and VEGF-R3, potentiates the validity of these associations.
Asunto(s)
Neoplasias Colorrectales , ARN Largo no Codificante , Factor de Crecimiento Transformador beta2 , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/patología , Masculino , Femenino , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/sangre , ARN Largo no Codificante/sangre , ARN Largo no Codificante/genética , Sitios de Carácter Cuantitativo , Persona de Mediana Edad , Metástasis de la Neoplasia , Anciano , Polimorfismo de Nucleótido Simple , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Estudio de Asociación del Genoma CompletoRESUMEN
Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.
Asunto(s)
Discapacidades del Desarrollo/genética , Proteínas de Drosophila/genética , Enfermedades Hereditarias del Ojo/genética , Discapacidad Intelectual/genética , Carioferinas/genética , Anomalías Musculoesqueléticas/genética , beta Carioferinas/genética , Proteína de Unión al GTP ran/genética , Alelos , Secuencia de Aminoácidos , Animales , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Enfermedades Hereditarias del Ojo/metabolismo , Enfermedades Hereditarias del Ojo/patología , Femenino , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Genoma Humano , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Carioferinas/antagonistas & inhibidores , Carioferinas/metabolismo , Masculino , Anomalías Musculoesqueléticas/metabolismo , Anomalías Musculoesqueléticas/patología , Mutación , Neuronas/metabolismo , Neuronas/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Secuenciación Completa del Genoma , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/metabolismoRESUMEN
Hypertrophic scar (HS) is a common fibroproliferative disease caused by abnormal wound healing after deep skin injury. However, the existing approaches have unsatisfactory therapeutic effects, which promote the exploration of newer and more effective strategies. MiRNA-modified functional exosomes delivered by dissolvable microneedle arrays (DMNAs) are expected to provide new hope for HS treatment. In this study, a miRNA, miR-141-3p, which is downregulated in skin scar tissues and in hypertrophic scar fibroblasts (HSFs), is identified. MiR-141-3p mimics inhibit the proliferation, migration, and myofibroblast transdifferentiation of HSFs in vitro by targeting TGF-ß2 to suppress the TGF-ß2/Smad pathway. Subsequently, the engineered exosomes encapsulating miR-141-3p (miR-141-3pOE -Exos) are isolated from adipose-derived mesenchymal stem cells transfected with Lv-miR-141-3p. MiR-141-3pOE -Exos show the same inhibitive effects as miR-141-3p mimics on the pathological behaviors of HSFs in vitro. The DMNAs for sustained release of miR-141-3pOE -Exos are further fabricated in vivo. MiR-141OE -Exos@DMNAs effectively decrease the thickness of HS and improve fibroblast distribution and collagen fiber arrangement, and downregulate the expression of α-SMA, COL-1, FN, TGF-ß2, and p-Smad2/3 in the HS tissue. Overall, a promising, effective, and convenient exosome@DMNA-based miRNA delivery strategy for HS treatment is provided.
Asunto(s)
Cicatriz Hipertrófica , Exosomas , MicroARNs , Humanos , Cicatriz Hipertrófica/terapia , Cicatriz Hipertrófica/genética , Cicatriz Hipertrófica/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Exosomas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Fibroblastos/metabolismo , Proliferación Celular/genéticaRESUMEN
Unprovoked thrombosis (thrombosis occurring without an established environmental factor favouring the episode) is a classic feature of APS. In the general population, provoked venous thromboembolism (VTE) is clearly defined and has clinical and therapeutic differences compared with unprovoked VTE. Whether provoked VTE in the context of APS may lead to a limited treatment duration is not well established. Therefore, careful clinical and laboratory evaluation is needed to identify patients eligible for a limited duration of anticoagulation treatment after provoked VTE. Given the uncertainties of available data, the risks and benefits of treatment decisions should be clearly explained. Decisions should be shared by both the patient and physician. Cardiovascular risk factors are common in patients with APS with arterial thrombosis. There are insufficient data suggesting that cardiovascular risk factor control would allow the cessation of anticoagulation. In most instances, arterial thrombosis will require prolonged anticoagulants. A careful analysis of clinical characteristics and laboratory evaluation, particularly the aPL antibody profile, is needed to make decisions on a case-by-case basis.
Asunto(s)
Síndrome Antifosfolípido , Trombosis , Tromboembolia Venosa , Humanos , Síndrome Antifosfolípido/complicaciones , Tromboembolia Venosa/tratamiento farmacológico , beta 2 Glicoproteína I , Trombosis/etiología , Anticoagulantes/uso terapéuticoRESUMEN
ß-defensin 2 (BD2) is a small cationic peptide that exerts a critical role in host defense against bacterial infections. Here, we aimed to investigate the role of BD2 in protecting against acute urinary tract infection (AUTI) caused by Escherichia coli (UPEC). Here, LPS-induced human urinary bladder epithelial cell (HCV-29) model and UPEC-induced mice model were used for assessing AUTI. Visceral organ lesions of mice following treatment was assessed by HE staining. Cell viability was determined by CCK-8 assay. Permeability in HCV-29 cells was analyzed in Transwell assay. Expression of inflammatory factors (IL-1ß, IL-6, and TNF-α) was measured by ELISA assay. The levels of BD2, ß-catenin and tight-junction proteins (ZO-1, Occludin, and Claudin-1) were detected by RT-qPCR, western blotting, immunofluorescence or immunohistochemistry. Our results showed that BD2 was lowly expressed and ß-catenin showed the reverse trend in response to bacterial infection in vitro and in vivo. BD2 overexpression alleviated the decreased cell viability, increased cell permeability, upregulation of inflammatory factors, downregulation of tight-junction protein and high ß-catenin expression in LPS-induced HCV-29 cells, which may contribute to the negative regulation of ß-catenin expression. Furthermore, BD2 overexpression attenuated the bacterial infection of tissues, high levels of inflammatory factors and ß-catenin, and low levels of tight-junction proteins in mice stimulated with UPEC. This study showed that BD2 played a crucial role in protecting against AUTI caused by gram-negative bacteria through suppressing ß-catenin expression. Targeting BD2 may provide a potential therapeutic approach for the prevention and treatment of AUTI.
Asunto(s)
Infecciones por Escherichia coli , Hepatitis C , Infecciones Urinarias , beta-Defensinas , Animales , Humanos , Ratones , beta Catenina/genética , beta Catenina/metabolismo , beta-Defensinas/genética , beta-Defensinas/metabolismo , Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Lipopolisacáridos/toxicidad , Proteínas de Uniones Estrechas/metabolismoRESUMEN
Transforming growth factor-ß2 (TGF-ß2) induced fibrogenic changes in human trabecular meshwork (HTM) cells have been implicated in trabecular meshwork (TM) damage and intraocular pressure (IOP) elevation in primary open-angle glaucoma (POAG) patients. Silibinin (SIL) exhibited anti-fibrotic properties in various organs and tissues. This study aimed to assess the effects of SIL on the TGF-ß2-treated HTM cells and to elucidate the underlying mechanisms. Our study found that SIL effectively inhibited HTM cell proliferation, attenuated TGF-ß2-induced cell migration, and mitigated TGF-ß2-induced reorganization of both actin and vimentin filaments. Moreover, SIL suppressed the expressions of fibronectin (FN), collagen type I alpha 1 chain (COL1A1), and alpha-smooth muscle actin (α-SMA) in the TGF-ß2-treated HTM cells. RNA sequencing indicated that SIL interfered with the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, also known as AKT) signaling pathway, extracellular matrix (ECM)-receptor interaction, and focal adhesion in the TGF-ß2-treated HTM cells. Western blotting demonstrated SIL inhibited the activation of Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) and the downstream PI3K/AKT signaling pathways induced by TGF-ß2, potentially contributing to its inhibitory effects on ECM protein production in the TGF-ß2-treated HTM cells. Our study demonstrated the ability of SIL to inhibit TGF-ß2-induced fibrogenic changes in HTM cells. SIL could be a potential IOP-lowering agent by reducing the fibrotic changes in the TM tissue of POAG patients, which warrants further investigation through additional animal and clinical studies.
Asunto(s)
Movimiento Celular , Proliferación Celular , Transducción de Señal , Silibina , Malla Trabecular , Humanos , Antioxidantes/farmacología , Western Blotting , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Fibrosis , Glaucoma de Ángulo Abierto/metabolismo , Glaucoma de Ángulo Abierto/tratamiento farmacológico , Glaucoma de Ángulo Abierto/patología , Janus Quinasa 2/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Silibina/farmacología , Silimarina/farmacología , Factor de Transcripción STAT3/metabolismo , Malla Trabecular/efectos de los fármacos , Malla Trabecular/metabolismo , Malla Trabecular/patología , Factor de Crecimiento Transformador beta2/farmacología , Factor de Crecimiento Transformador beta2/metabolismoRESUMEN
Vibrio parahaemolyticus possesses two distinct type VI secretion systems (T6SS), namely T6SS1 and T6SS2. T6SS1 is predominantly responsible for adhesion to Caco-2 and HeLa cells and for the antibacterial activity of V. parahaemolyticus, while T6SS2 mainly contributes to HeLa cell adhesion. However, it remains unclear whether the T6SS systems have other physiological roles in V. parahaemolyticus. In this study, we demonstrated that the deletion of icmF2, a structural gene of T6SS2, reduced the biofilm formation capacity of V. parahaemolyticus under low salt conditions, which was also influenced by the incubation time. Nonetheless, the deletion of icmF2 did not affect the biofilm formation capacity in marine-like growth conditions, nor did it impact the flagella-driven swimming and swarming motility of V. parahaemolyticus. IcmF2 was found to promote the production of the main components of the biofilm matrix, including extracellular DNA (eDNA) and extracellular proteins, and cyclic di-GMP (c-di-GMP) in V. parahaemolyticus. Additionally, IcmF2 positively influenced the transcription of cpsA, mfpA, and several genes involved in c-di-GMP metabolism, including scrJ, scrL, vopY, tpdA, gefA, and scrG. Conversely, the transcription of scrA was negatively impacted by IcmF2. Therefore, IcmF2-dependent biofilm formation was mediated through its effects on the production of eDNA, extracellular proteins, and c-di-GMP, as well as its impact on the transcription of cpsA, mfpA, and genes associated with c-di-GMP metabolism. This study confirmed new physiological roles for IcmF2 in promoting biofilm formation and c-di-GMP production in V. parahaemolyticus.
Asunto(s)
Proteínas Bacterianas , Biopelículas , GMP Cíclico , Sistemas de Secreción Tipo VI , Vibrio parahaemolyticus , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/fisiología , Vibrio parahaemolyticus/metabolismo , Biopelículas/crecimiento & desarrollo , Sistemas de Secreción Tipo VI/genética , Sistemas de Secreción Tipo VI/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Humanos , Regulación Bacteriana de la Expresión Génica , Células HeLaRESUMEN
OBJECTIVES: Recently published 2023 ACR/EULAR APS classification criteria emphasize the importance of quantifying single-, double-, and triple-antiphospholipid antibody positivity, distinguishing between IgG and IgM isotypes, and delineating moderate/high levels of anticardiolipin (aCL) and anti-ß2 glycoprotein I (anti-ß2GPI) antibodies. We aimed to establish clinically important moderate/high thresholds for aCL and anti-ß2GPI IgG/IgM chemiluminescent immunoassays (CLIA), in particular QUANTA Flash, comparable to our in-house ELISAs used for over two decades, and to evaluate their diagnostic performance. METHODS: QUANTA Flash CLIA and in-house ELISAs were used to measure aCL and anti-ß2GPI IgG/IgM. Moderate thresholds for QUANTA Flash CLIA were determined using a non-parametric approach, calculating a 99th percentile on serum samples from 139 blood donors, and by mirroring the diagnostic performance of in-house ELISA on 159 patient samples. RESULTS: Thresholds for QUANTA Flash CLIA achieving diagnostic performance equivalent to in-house ELISAs were 40â¯CU for moderate and 80â¯CU for high levels for aCL and anti-ß2GPI IgG and IgM. The assays showed good qualitative agreement, ranging from 76.10 to 91.19â¯%. When considering in-house ELISA results, 14 out of 80 (17.5â¯%) patients did not fulfill the new ACR/EULAR laboratory classification criteria, while 27 out of 80 (33.8â¯%) did not when considering QUANTA Flash CLIA results. CONCLUSIONS: We determined moderate and high thresholds for aCL and anti-ß2GPI IgG and IgM detected with QUANTA Flash CLIA, aligning with long-established in-house ELISA thresholds. These thresholds are crucial for seamlessly integrating of the new 2023 ACR/EULAR classification criteria into future observational clinical studies and trials.
RESUMEN
BACKGROUND: Psoriasis, a chronic inflammatory skin disease, is increasingly effectively managed with the targeted immunotherapy; however, long-term immunotherapy carries health risks, and loss of response. Therefore, we need to develop the alternative treatment strategies. Mesenchymal stem/stromal cell (M.S.C.) exosomes stand out for their remarkable immunomodulatory properties, gaining widespread recognition. This study investigated whether M.S.C. exosomes can reduce psoriasis-induced hyperplasia by inducing Transforming Growth Factor beta 2 (TGF-beta2) signaling. METHODOLOGY: Exosomes were isolated from M.S.C.s by ultracentrifugation. Then, scanning electron microscopy was used for the morphology of exosomes. To ascertain the exosome concentration, the Bradford test was used. To ascertain the cellular toxicity of exosomes in Human Umbilical Vein Endothelial Cells ( H.U.V.E.C), an MTT experiment was then conducted. Real-time PCR was used to quantify TGF beta2 expression levels, whereas an ELISA immunosorbent assay was used to determine the protein concentration of TGF beta2. RESULTS: In this study, the exosomes of 15-30 nm in size that were uniform, and cup-shaped were isolated. Moreover, the IC50 value for this Treatment was calculated to be 181.750 µg/ml. The concentration of TGF-ß2 gene in the target cells significantly increased following Treatment with the exosomes. Furthermore, the expression level of the studied gene significantly increased due to the Treatment. CONCLUSION: Upregulating the expression of TGF-ß2 in psoriatic cells via TGF-ß2 signaling is one way exosomes can help reduce hyperplasia.
Asunto(s)
Exosomas , Células Endoteliales de la Vena Umbilical Humana , Hiperplasia , Células Madre Mesenquimatosas , Psoriasis , Factor de Crecimiento Transformador beta2 , Exosomas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Psoriasis/metabolismo , Humanos , Factor de Crecimiento Transformador beta2/metabolismo , Hiperplasia/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Transducción de Señal , AnimalesRESUMEN
Bacteria belonging to the genus Algoriphagus have been isolated from various sources, such as Antarctic sea ice, seawater, and sediment, and some strains are known to produce orange to red pigments. However, the pigment composition and biosynthetic genes have not been fully elucidated. A new red-pigmented Algoriphagus sp. strain, oki45, was isolated from the surface of seaweed collected from Senaga-Jima Island, Okinawa, Japan. Genome comparison revealed oki45's average nucleotide identity of less than 95% to its closely related species, Algoriphagus confluentis NBRC 111222 T and Algoriphagus taiwanensis JCM 19755 T. Comprehensive chemical analyses of oki45's pigments, including 1H and 13C nuclear magnetic resonance and circular dichroism spectroscopy, revealed that the pigments were mixtures of monocyclic carotenoids, (3S)-flexixanthin ((3S)-3,1'-dihydroxy-3',4'-didehydro-1',2'-dihydro-ß,ψ-caroten-4-one) and (2R,3S)-2-hydroxyflexixanthin ((2R,3S)-2,3,1'-trihydroxy-3',4'-didehydro-1',2'-dihydro-ß,ψ-caroten-4-one); in particular, the latter compound was new and not previously reported. Both monocyclic carotenoids were also found in A. confluentis NBRC 111222 T and A. taiwanensis JCM 19755 T. Further genome comparisons of carotenoid biosynthetic genes revealed the presence of eight genes (crtE, crtB, crtI, cruF, crtD, crtYcd, crtW, and crtZ) for flexixanthin biosynthesis. In addition, a crtG homolog gene encoding 2,2'-ß-hydroxylase was found in the genome of the strains oki45, A. confluentis NBRC 111222 T, and A. taiwanensis JCM 19755 T, suggesting that the gene is involved in 2-hydroxyflexixanthin synthesis via 2-hydroxylation of flexixanthin. These findings expand our knowledge of monocyclic carotenoid biosynthesis in Algoriphagus bacteria. KEY POINTS: ⢠Algoriphagus sp. strain oki45 was isolated from seaweed collected in Okinawa, Japan. ⢠A novel monocyclic carotenoid 2-hydroxyflexixanthin was identified from strain oki45. ⢠Nine genes for 2-hydroxyflexixanthin biosynthesis were found in strain oki45 genome.
Asunto(s)
Carotenoides , Agua de Mar , Agua de Mar/microbiología , Bacterias/genética , Oxigenasas de Función Mixta/genética , Familia de Multigenes , Filogenia , Ácidos Grasos/química , ARN Ribosómico 16S/genética , ADN Bacteriano/genética , Análisis de Secuencia de ADN , Técnicas de Tipificación BacterianaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic inflammation, lung tissue fibrotic changes and impaired lung function. Pulmonary fibrosis 's pathological process is thought to be influenced by macrophage-associated phenotypes. IPF treatment requires specific targets that target macrophage polarization. Cytokine-like 1(CYTL1) is a secreted protein with multiple biological functions first discovered in CD34+ haematopoietic cells. However, its possible effects on IPF progression remain unclear. This study investigated the role of CYTL1 in IPF progression in a bleomycin-induced lung injury and fibrosis model. In bleomycin-induced mice, CYTL1 is highly expressed. Moreover, CYTL1 ablation alleviates lung injury and fibrosis in vivo. Further, downregulating CYTL1 reduces macrophage M2 polarization. Mechanically, CYTL1 regulates transforming growth factor ß (TGF-ß)/connective tissue growth factor (CCN2) axis and inhibition of TGF-ß pathway alleviates bleomycin-induced lung injury and fibrosis. In conclusion, highly expressed CYTL1 inhibits macrophage M2 polarization by regulating TGF-ß/CCN2 expression, alleviating bleomycin-induced lung injury and fibrosis. CYTL1 could, therefore, serve as a promising IPF target.