RESUMO
Cryogenic electron microscopy (cryo-EM) has become one of the most powerful techniques to reveal the atomic structures and working mechanisms of biological macromolecules. New designs of the cryo-EM grids-aimed at preserving thin, uniform vitrified ice and improving protein adsorption-have been considered a promising approach to achieving higher resolution with the minimal amount of materials and data. Here, we describe a method for preparing graphene cryo-EM grids with up to 99% monolayer graphene coverage that allows for more than 70% grid squares for effective data acquisition with improved image quality and protein density. Using our graphene grids, we have achieved 2.6-Å resolution for streptavidin, with a molecular weight of 52 kDa, from 11,000 particles. Our graphene grids increase the density of examined soluble, membrane, and lipoproteins by at least 5-fold, affording the opportunity for structural investigation of challenging proteins which cannot be produced in large quantity. In addition, our method employs only simple tools that most structural biology laboratories can access. Moreover, this approach supports customized grid designs targeting specific proteins, owing to its broad compatibility with a variety of nanomaterials.
RESUMO
Synthesis and implementation of highly active, stable, and affordable electrocatalysts for the oxygen evolution reaction (OER) is a major challenge in developing energy efficient and economically viable energy conversion devices such as electrolyzers, rechargeable metal-air batteries, and regenerative fuel cells. The current benchmark electrocatalyst for OER is based on iridium oxide (IrOx) due to its superior performance and excellent stability. However, large scale applications using IrOx are impractical due to its low abundance and high cost. Herein, we report a highly active hafnium-modified iridium oxide (IrHfxOy) electrocatalyst for OER. The IrHfxOy electrocatalyst demonstrated ten times higher activity in alkaline conditions (pH = 11) and four times higher activity in acid conditions (pH = 1) than a IrOx electrocatalyst. The highest intrinsic mass activity of the IrHfxOy catalyst in acid conditions was calculated as 6950 A gIrOx-1 at an overpotential (η) of 0.3 V. Combined studies utilizing operando surface enhanced Raman spectroscopy (SERS) and DFT calculations revealed that the active sites for OER are the Ir-O species for both IrOx and IrHfxOy catalysts. The presence of Hf sites leads to more negative charge states on nearby O sites, shortening of the bond lengths of Ir-O, and lowers free energies for OER intermediates that accelerate the OER process.
RESUMO
BACKGROUND: Plasmacytoid urothelial carcinoma (PUC) is a rare, aggressive histologic variant of urothelial cancer characterised by a diffuse growth pattern and CDH1 mutation. We studied the efficacy of preoperative platinum-based chemotherapy in nonmetastatic PUC and immune checkpoint inhibitors (ICIs) in advanced PUC. METHODS: Cases of nonmetastatic PUC and advanced PUC treated with ICIs at our institution were identified. Outcomes were compared to those of a published cohort of patients with urothelial carcinoma not otherwise specified. RESULTS: We identified 81 patients with nonmetastatic PUC. Of the patients with localised disease who underwent neoadjuvant chemotherapy, pathologic complete response and downstaging rates were 12 and 21%, respectively. Pathologic downstaging was not associated with significant improvement in clinical outcomes. Up to 18% of localised disease and 28% of locally advanced cases had unresectable disease at the time of surgery. ICI-treated advanced PUC (N = 21) had progression-free and overall survival of 4.5 and 10.5 months, respectively, and a 38% response rate. FGFR3 and DNA damage response gene alterations were observed in 3 and 15% of cases, respectively. CONCLUSIONS: PUC is associated with high disease burden and poor chemosensitivity. Increased awareness and recognition of this disease variant will allow for new treatment strategies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Inibidores de Checkpoint Imunológico/uso terapêutico , Mutação , Terapia Neoadjuvante/mortalidade , Neoplasias da Bexiga Urinária/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Adulto JovemRESUMO
Corticosteroids are highly prescribed and effective anti-inflammatory drugs but the burden of side effects with chronic use significantly detracts from patient quality of life, particularly in children. Developing safer steroids amenable to long-term use is an important goal for treatment of chronic inflammatory diseases such as Duchenne muscular dystrophy (DMD). We have developed vamorolone (VBP15), a first-in-class dissociative glucocorticoid receptor (GR) ligand that shows the anti-inflammatory efficacy of corticosteroids without key steroid side effects in animal models. miRNAs are increasingly recognized as key regulators of inflammatory responses. To define effects of prednisolone and vamorolone on the muscle miRNAome, we performed a preclinical discovery study in the mdx mouse model of DMD. miRNAs associated with inflammation were highly elevated in mdx muscle. Both vamorolone and prednisolone returned these toward wild-type levels (miR-142-5p, miR-142-3p, miR-146a, miR-301a, miR-324-3p, miR-455-5p, miR-455-3p, miR-497, miR-652). Effects of vamorolone were largely limited to reduction of proinflammatory miRNAs. In contrast, prednisolone activated a separate group of miRNAs associated with steroid side effects and a noncoding RNA cluster homologous to human chromosome 14q32. Effects were validated for inflammatory miRNAs in a second, independent preclinical study. For the anti-inflammatory miRNA signature, bioinformatic analyses showed all of these miRNAs are directly regulated by, or in turn activate, the inflammatory transcription factor NF-κB. Moving forward miR-146a and miR-142 are of particular interest as biomarkers or novel drug targets. These data validate NF-κB signaling as a target of dissociative GR-ligand efficacy in vivo and provide new insight into miRNA signaling in chronic inflammation.
Assuntos
Inflamação/genética , MicroRNAs/genética , Músculos/metabolismo , Prednisona/farmacologia , Pregnadienodiois/farmacologia , Animais , Sequência de Bases , Doença Crônica , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , MicroRNAs/metabolismo , Modelos Biológicos , Músculos/efeitos dos fármacos , Músculos/patologia , Distrofia Muscular de Duchenne/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Receptores de Glucocorticoides/metabolismo , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
BACKGROUND: The current study compared the incidence of vascular thromboembolic events (VTEs) in patients with metastatic or unresectable urothelial carcinoma (UC) who were treated with gemcitabine and carboplatin (GCb); gemcitabine, carboplatin, and bevacizumab (GCbBev); or gemcitabine and cisplatin (GCis). METHODS: Patients with UC who were treated with GCbBev on protocol were analyzed prospectively and 2 contemporary control cohorts receiving GCb or GCis were evaluated retrospectively. VTE was defined as either venous or arterial (myocardial infarctions or cerebral vascular accidents) thrombosis. VTEs were considered to be related to treatment if they occurred during treatment or within 4 weeks of the completion of treatment. Associations with chemotherapy regimen were tested using either the Fisher exact test or Kruskal-Wallis test. Clinical factors associated with VTEs were analyzed using conditional logistic regression stratified by treatment regimen. RESULTS: Among 198 patients, VTEs occurred in 13 of 51 patients treated with GCbBev (26%), 22 of 92 patients treated with GCb (24%), and 8 of 55 patients treated with GCis (15%). Patient characteristics were significantly different between the treatment cohorts in terms of age, prior cystectomy, tumor location near pelvic vessels, Khorana risk group, and receipt of antiplatelet therapy. The incidence of VTE and type of VTE (arterial vs venous) did not differ by type of chemotherapy. Prior cystectomy was associated with an increased risk of VTE (odds ratio, 2.2; 95% confidence interval, 1.0-4.9 [P = .047]). CONCLUSIONS: The incidence of VTE in Cis-treated patients was similar to prior reports. However, the VTE rate in Cb-treated patients was > 20%, a figure not previously defined in patients with UC and higher than expected. This high incidence of both Cis-related and Cb-related VTEs warrants greater awareness by treating physicians and deserves further study. Cancer 2016;122:712-721. © 2015 American Cancer Society.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células de Transição/tratamento farmacológico , Infarto do Miocárdio/epidemiologia , Acidente Vascular Cerebral/epidemiologia , Neoplasias Urológicas/tratamento farmacológico , Tromboembolia Venosa/epidemiologia , Idoso , Bevacizumab/administração & dosagem , Carboplatina/administração & dosagem , Carcinoma de Células de Transição/patologia , Carcinoma de Células de Transição/secundário , Estudos de Casos e Controles , Cisplatino/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Feminino , Humanos , Incidência , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Pelve Renal/patologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos Retrospectivos , Neoplasias Ureterais/tratamento farmacológico , Neoplasias Ureterais/patologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Neoplasias Urológicas/patologia , GencitabinaRESUMO
Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by the absence of dystrophin protein. One current DMD therapeutic strategy, exon skipping, produces a truncated dystrophin isoform using phosphorodiamidate morpholino oligomers (PMOs). However, the potential of exon skipping therapeutics has not been fully realized as increases in dystrophin protein have been minimal in clinical trials. Here, we investigate how miR-146a-5p, which is highly elevated in dystrophic muscle, impacts dystrophin protein levels. We find inflammation strongly induces miR-146a in dystrophic, but not wild-type myotubes. Bioinformatics analysis reveals that the dystrophin 3' UTR harbors a miR-146a binding site, and subsequent luciferase assays demonstrate miR-146a binding inhibits dystrophin translation. In dystrophin-null mdx52 mice, co-injection of miR-146a reduces dystrophin restoration by an exon 51 skipping PMO. To directly investigate how miR-146a impacts therapeutic dystrophin rescue, we generated mdx52 with body-wide miR-146a deletion (146aX). Administration of an exon skipping PMO via intramuscular or intravenous injection markedly increases dystrophin protein levels in 146aX vs. mdx52 muscles while skipped dystrophin transcript levels are unchanged supporting a post-transcriptional mechanism of action. Together, these data show that miR-146a expression opposes therapeutic dystrophin restoration, suggesting miR-146a inhibition warrants further research as a potential DMD exon skipping co-therapy.
RESUMO
Absence of dystrophin results in muscular weakness, chronic inflammation and cardiomyopathy in Duchenne muscular dystrophy (DMD). Pharmacological corticosteroids are the DMD standard of care; however, they have harsh side effects and unclear molecular benefits. It is uncertain whether signaling by physiological corticosteroids and their receptors plays a modifying role in the natural etiology of DMD. Here, we knocked out the glucocorticoid receptor (GR, encoded by Nr3c1) specifically in myofibers and cardiomyocytes within wild-type and mdx52 mice to dissect its role in muscular dystrophy. Double-knockout mice showed significantly worse phenotypes than mdx52 littermate controls in measures of grip strength, hang time, inflammatory pathology and gene expression. In the heart, GR deletion acted additively with dystrophin loss to exacerbate cardiomyopathy, resulting in enlarged hearts, pathological gene expression and systolic dysfunction, consistent with imbalanced mineralocorticoid signaling. The results show that physiological GR functions provide a protective role during muscular dystrophy, directly contrasting its degenerative role in other disease states. These data provide new insights into corticosteroids in disease pathophysiology and establish a new model to investigate cell-autonomous roles of nuclear receptors and mechanisms of pharmacological corticosteroids.
Assuntos
Cardiomiopatias , Distrofia Muscular de Duchenne , Receptores de Glucocorticoides , Animais , Camundongos , Cardiomiopatias/patologia , Cardiomiopatias/metabolismo , Distrofina/metabolismo , Distrofina/genética , Distrofina/deficiência , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/patologia , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/metabolismo , Miocárdio/patologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/efeitos dos fármacos , Fenótipo , Receptores de Glucocorticoides/metabolismoRESUMO
There is no approved therapy for Becker muscular dystrophy (BMD), a genetic muscle disease caused by in-frame dystrophin deletions. We previously developed the dissociative corticosteroid vamorolone for treatment of the allelic, dystrophin-null disease Duchenne muscular dystrophy. We hypothesize vamorolone can treat BMD by safely reducing inflammatory signaling in muscle and through a novel mechanism of increasing dystrophin protein via suppression of dystrophin-targeting miRNAs. Here, we test this in the bmx mouse model of BMD. Daily oral treatment with vamorolone or prednisolone improves bmx grip strength and hang time phenotypes. Both drugs reduce myofiber size and decrease the percentage of centrally nucleated fibers. Vamorolone shows improved safety versus prednisolone by avoiding or reducing key side effects to behavior and growth. Intriguingly, vamorolone increases dystrophin protein in both heart and skeletal muscle. These data indicate that vamorolone, nearing approval for Duchenne, shows efficacy in bmx mice and therefore warrants clinical investigation in BMD.
RESUMO
Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by the absence of dystrophin protein. One current DMD therapeutic strategy, exon skipping, produces a truncated dystrophin isoform using phosphorodiamidate morpholino oligomers (PMOs). However, the potential of exon skipping therapeutics has not been fully realized as increases in dystrophin protein have been minimal in clinical trials. Here, we investigate how miR-146a-5p, which is highly elevated in dystrophic muscle, impacts dystrophin protein levels. We find inflammation strongly induces miR-146a in dystrophic, but not wild-type myotubes. Bioinformatics analysis reveals that the dystrophin 3'UTR harbors a miR-146a binding site, and subsequent luciferase assays demonstrate miR-146a binding inhibits dystrophin translation. In dystrophin-null mdx52 mice, co-injection of miR-146a reduces dystrophin restoration by an exon 51 skipping PMO. To directly investigate how miR-146a impacts therapeutic dystrophin rescue, we generated mdx52 with body-wide miR-146a deletion (146aX). Administration of an exon skipping PMO via intramuscular or intravenous injection markedly increases dystrophin protein levels in 146aX versus mdx52 muscles; skipped dystrophin transcript levels are unchanged, suggesting a post-transcriptional mechanism-of-action. Together, these data show that miR-146a expression opposes therapeutic dystrophin restoration, suggesting miR-146a inhibition warrants further research as a potential DMD exon skipping co-therapy.
RESUMO
BACKGROUND: Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype. METHODS: We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000 bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography. RESULTS: Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P < 0.0001), wire hang (P = 0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P = 0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P < 0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P = 0.0017) and centrally located nuclei indicating degeneration/regeneration (P < 0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P < 0.0001), collagen deposition (+1.4-fold, P = 0.0217), and sarcolemmal damage measured by intracellular IgM (P = 0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes. CONCLUSIONS: The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.
Assuntos
Distrofina , Distrofia Muscular de Duchenne , Animais , Humanos , Camundongos , Distrofina/genética , Distrofina/metabolismo , Éxons/genética , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Oligonucleotídeos Antissenso , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Modelos Animais de DoençasRESUMO
Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies -Eteplirsen, Golodirsen, Viltolarsen, and Casimersen -for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (â¼44âd vs. â¼24âd, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.
Assuntos
Distroglicanas/metabolismo , Distrofina/metabolismo , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/uso terapêutico , Animais , Éxons , Camundongos , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/metabolismoRESUMO
Epidemiological and preclinical observations have suggested a role for one or more products of the mevalonate/cholesterol biosynthesis pathway in the progression of prostate cancer. In this study, we used ezetimibe (Zetia), a specific, FDA-approved, cholesterol uptake-blocking drug, in combination with either a hyper- or hypocholesterolemic diet, to show that elevated circulating cholesterol levels promote, whereas a reduction in circulating cholesterol levels retard, the growth of human prostate cancer xenograft tumors in mice. Circulating cholesterol levels also modified tumor angiogenesis; higher cholesterol levels increased microvessel density and other indicators of vascularity. Consistent with these data, the reduction of cholesterol levels also increased the levels of the angiogenesis inhibitor thrombospondin-1 in the xenografts. Our results thus suggest that hypercholesterolemia directly accelerates the growth of prostate carcinomas, and that the pharmacological reduction of serum cholesterol levels may retard prostate cancer growth by inhibiting tumor angiogenesis.
Assuntos
Antineoplásicos/uso terapêutico , Azetidinas/uso terapêutico , Neovascularização Patológica/prevenção & controle , Neoplasias da Próstata/patologia , Animais , Anticolesterolemiantes/uso terapêutico , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Colesterol/farmacologia , Ezetimiba , Hemoglobinas/metabolismo , Humanos , Masculino , Camundongos , Microcirculação/efeitos dos fármacos , Transplante de Neoplasias , Neoplasias da Próstata/tratamento farmacológico , Transplante HeterólogoRESUMO
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate important intracellular biological processes. In myasthenia gravis (MG), a disease-specific pattern of elevated circulating miRNAs has been found, and proposed as potential biomarkers. These elevated miRNAs include miR-150-5p, miR-21-5p, and miR-30e-5p in acetylcholine receptor antibody seropositive (AChR+) MG and miR-151a-3p, miR-423-5p, let-7a-5p, and let-7f-5p in muscle-specific tyrosine kinase antibody seropositive (MuSK+) MG. In this study, we examined the regulation of each of these miRNAs using chromatin immunoprecipitation sequencing (ChIP-seq) data from the Encyclopedia of DNA Elements (ENCODE) to gain insight into the transcription factor pathways that drive their expression in MG. Our aim was to look at the transcription factors that regulate miRNAs and then validate some of those in vivo with cell lines that have sufficient expression of these transcription factors This analysis revealed several transcription factor families that regulate MG-specific miRNAs including the Forkhead box or the FOXO proteins (FoxA1, FoxA2, FoxM1, FoxP2), AP-1, interferon regulatory factors (IRF1, IRF3, IRF4), and signal transducer and activator of transcription proteins (Stat1, Stat3, Stat5a). We also found binding sites for nuclear factor of activated T-cells (NFATC1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), early growth response factor (EGR1), and the estrogen receptor 1 (ESR1). AChR+ MG miRNAs showed a stronger overall regulation by the FOXO transcription factors, and of this group, miR-21-5p, let-7a, and let 7f were found to possess ESR1 binding sites. Using a murine macrophage cell line, we found activation of NF-κB -mediated inflammation by LPS induced expression of miR-21-5p, miR-30e-5p, miR-423-5p, let-7a, and let-7f. Pre-treatment of cells with the anti-inflammatory drugs prednisone or deflazacort attenuated induction of inflammation-induced miRNAs. Interestingly, the activation of inflammation induced packaging of the AChR+-specific miRNAs miR-21-5p and miR-30e-5p into exosomes, suggesting a possible mechanism for the elevation of these miRNAs in MG patient serum. In conclusion, our study summarizes the regulatory transcription factors that drive expression of AChR+ and MuSK+ MG-associated miRNAs. Our findings of elevated miR-21-5p and miR-30e-5p expression in immune cells upon inflammatory stimulation and the suppressive effect of corticosteroids strengthens the putative role of these miRNAs in the MG autoimmune response.
Assuntos
MicroRNA Circulante/genética , MicroRNA Circulante/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Fatores Reguladores de Interferon/metabolismo , Miastenia Gravis/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Transcrição STAT/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos/imunologia , Estudos de Coortes , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Células RAW 264.7 , RNA Mensageiro/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologia , Transdução de Sinais/genética , Linfócitos T/metabolismoRESUMO
OBJECTIVE: Muscle inflammation is a feature in myositis and Duchenne muscular dystrophy (DMD). Autoimmune mechanisms are thought to contribute to muscle weakness in patients with myositis. However, a lack of correlation between the extent of inflammatory cell infiltration and muscle weakness indicates that nonimmune pathologic mechanisms may play a role. The present study focused on 2 microRNA (miRNA) sets previously identified as being elevated in the muscle of patients with DMD-an "inflammatory" miRNA set that is dampened with glucocorticoids, and a "dystrophin-targeting" miRNA set that inhibits dystrophin translation-to test the hypothesis that these miRNAs are similarly dysregulated in the muscle of patients with myositis, and could contribute to muscle weakness and disease severity. METHODS: A major histocompatibility complex class I-transgenic mouse model of myositis was utilized to study gene and miRNA expression and histologic features in the muscle tissue, with the findings validated in human muscle biopsy tissue from 6 patients with myositis. Mice were classified as having mild or severe myositis based on transgene expression, body weight, histologic disease severity, and muscle strength/weakness. RESULTS: In mice with severe myositis, muscle tissue showed mononuclear cell infiltration along with elevated expression of type I interferon and NF-κB-regulated genes, including Tlr7 (3.8-fold increase, P < 0.05). Furthermore, mice with severe myositis showed elevated expression of inflammatory miRNAs (miR-146a, miR-142-3p, miR-142-5p, miR-455-3p, and miR-455-5p; ~3-40-fold increase, P < 0.05) and dystrophin-targeting miRNAs (miR-146a, miR-146b, miR-31, and miR-223; ~3-38-fold increase, P < 0.05). Bioinformatics analyses of chromatin immunoprecipitation sequencing (ChIP-seq) data identified at least one NF-κB consensus element within the promoter/enhancer regions of these miRNAs. Western blotting and immunofluorescence analyses of the muscle tissue from mice with severe myositis demonstrated reduced levels of dystrophin. In addition, elevated levels of NF-κB-regulated genes, TLR7, and miRNAs along with reduced dystrophin levels were observed in muscle biopsy tissue from patients with histologically severe myositis. CONCLUSION: These data demonstrate that an acquired dystrophin deficiency may occur through NF-κB-regulated miRNAs in myositis, thereby suggesting a unifying theme in which muscle injury, inflammation, and weakness are perpetuated both in myositis and in DMD.
Assuntos
Distrofina/metabolismo , MicroRNAs/genética , Debilidade Muscular/genética , Músculo Esquelético/metabolismo , Miosite/genética , Animais , Sequenciamento de Cromatina por Imunoprecipitação , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Camundongos , Camundongos Transgênicos , MicroRNAs/metabolismo , Debilidade Muscular/metabolismo , Miosite/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Índice de Gravidade de Doença , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismoRESUMO
Duchenne muscular dystrophy (DMD) is a chronic muscle disease characterized by poor myogenesis and replacement of muscle by extracellular matrix. Despite the shared genetic basis, severity of these deficits varies among patients. One source of these variations is the genetic modifier that leads to increased TGF-ß activity. While anti-TGF-ß therapies are being developed to target muscle fibrosis, their effect on the myogenic deficit is underexplored. Our analysis of in vivo myogenesis in mild (C57BL/10ScSn-mdx/J and C57BL/6J-mdxΔ52) and severe DBA/2J-mdx (D2-mdx) dystrophic models reveals no defects in developmental myogenesis in these mice. However, muscle damage at the onset of disease pathology, or by experimental injury, drives up TGF-ß activity in the severe, but not in the mild, dystrophic models. Increased TGF-ß activity is accompanied by increased accumulation of fibroadipogenic progenitors (FAPs) leading to fibro-calcification of muscle, together with failure of regenerative myogenesis. Inhibition of TGF-ß signaling reduces muscle degeneration by blocking FAP accumulation without rescuing regenerative myogenesis. These findings provide in vivo evidence of early-stage deficit in regenerative myogenesis in D2-mdx mice and implicates TGF-ß as a major component of a pathogenic positive feedback loop in this model, identifying this feedback loop as a therapeutic target.
Assuntos
Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Desenvolvimento Muscular/fisiologia , Regeneração/fisiologiaRESUMO
BACKGROUND: We sought to identify microRNAs (miRNAs) associated with response to anti-TNF-α or glucocorticoids in children with inflammatory bowel disease (IBD) to generate candidate pharmacodynamic and monitoring biomarkers. METHODS: Clinical response was assessed by Pediatric Crohn's Disease Activity Index and Pediatric Ulcerative Colitis Activity Index. Quantitative real-time polymerase chain reaction via Taqman Low-Density Array cards were used to identify miRNAs in a discovery cohort of responders (nâ =â 11) and nonresponders (nâ =â 8). Seven serum miRNAs associated with clinical response to treatment, along with 4 previously identified (miR-146a, miR-146b, miR-320a, miR-486), were selected for further study. Candidates were assessed in a validation cohort of serum samples from IBD patients pre- and post-treatment and from healthy controls. Expression of miRNA was also analyzed in inflamed mucosal biopsies from IBD patients and non-IBD controls. RESULTS: Discovery cohort analysis identified 7 miRNAs associated with therapeutic response: 5 that decreased (miR-126, miR-454, miR-26b, miR-26a, let-7c) and 2 that increased (miR-636, miR-193b). In the validation cohort, 7 of 11 candidate miRNAs changed in the same direction with response to anti-TNF-α therapies, glucocorticoids, or both. In mucosal biopsies, 7 out of 11 miRNAs were significantly increased in IBD vs healthy controls. CONCLUSIONS: Five candidate miRNAs associated with clinical response and mucosal inflammation in pediatric IBD patients were identified (miR-126, let-7c, miR-146a, miR-146b, and miR-320a). These miRNAs may be further developed as pharmacodynamic and response monitoring biomarkers for use in clinical care and trials.
Assuntos
Colite Ulcerativa/sangue , Doença de Crohn/sangue , Monitoramento de Medicamentos/métodos , MicroRNAs/sangue , Inibidores do Fator de Necrose Tumoral/farmacocinética , Adolescente , Biomarcadores/sangue , Biópsia , Criança , Pré-Escolar , Estudos de Coortes , Colite Ulcerativa/tratamento farmacológico , Doença de Crohn/tratamento farmacológico , Feminino , Humanos , Mucosa Intestinal/patologia , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
INTRODUCTION: The aim of this study was to determine drug delivery/toxicity, and pathologic/surgical outcomes of patients with muscle-invasive bladder cancer (MIBC) receiving neoadjuvant gemcitabine-cisplatin (GC) plus radical cystectomy-pelvic lymph node dissection (RC-PLND). PATIENTS AND METHODS: Chemotherapy and surgical/pathologic outcomes were retrospectively analyzed with 5-year survival follow-up at a referral center. Post-neoadjuvant chemotherapy (NAC) pathologic endpoints included complete response (pT0N0), residual non-MIBC (pTa/Tis/T1N0), and ≥ MIBC (≥ pT2 and/or N+). Associations of pathologic/surgical findings with overall survival (OS), disease-free survival (DFS), and surgical management with RC-PLND were analyzed (Cox regression). RESULTS: Clinical T2a-T4aN0M0 MIBC patients (n = 154) from January 2000-October 2012 received GC plus RC-PLND. Patients (n = 117; 76%) received GC × 4 and 136 (88%) GC × 3. Five-year OS was 61% (95% confidence interval [CI], 53-71). Median number of resected lymph nodes (LNs) was 19. Down-staging was observed as follows: pT0N0: 21%; pTa/Tis/T1N0: 25%, with similar 5-year OS (85% and 89%, respectively). Five-year OS for < pT2 versus ≥ pT2 residual disease was 87% (95% CI, 78%-98%) versus 38% (95% CI, 27%-53%); P < .001. Post-NAC stage ≥ pT2 (HR, 6.79; 95% CI, 2.63-17.53; P < .001), positive LN (HR, 3.64; 95% CI, 1.84-7.19; P < .001), and positive margins (HR, 4.15; 95% CI, 1.68-10.25; P = .002) were associated with increased risk of all-cause death (multivariable analysis). An HR of 0.97 (95% CI, 0.94-1.00) was observed for each additional node removed, but this effect was not statistically significant (P = .056). CONCLUSIONS: Neoadjuvant GC achieves meaningful pathologic responses. Patients with ≥ pT2 residual disease, positive margins, or positive LN post-chemotherapy have inferior survival.
Assuntos
Cistectomia , Neoplasias da Bexiga Urinária , Cisplatino , Desoxicitidina/análogos & derivados , Humanos , Excisão de Linfonodo , Músculos , Terapia Neoadjuvante , Estudos Retrospectivos , Resultado do Tratamento , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/cirurgia , GencitabinaRESUMO
The development of therapeutics for muscle diseases such as facioscapulohumeral dystrophy (FSHD) is impeded by a lack of objective, minimally invasive biomarkers. Here we identify circulating miRNAs and proteins that are dysregulated in early-onset FSHD patients to develop blood-based molecular biomarkers. Plasma samples from clinically characterized individuals with early-onset FSHD provide a discovery group and are compared to healthy control volunteers. Low-density quantitative polymerase chain reaction (PCR)-based arrays identify 19 candidate miRNAs, while mass spectrometry proteomic analysis identifies 13 candidate proteins. Bioinformatic analysis of chromatin immunoprecipitation (ChIP)-seq data shows that the FSHD-dysregulated DUX4 transcription factor binds to regulatory regions of several candidate miRNAs. This panel of miRNAs also shows ChIP signatures consistent with regulation by additional transcription factors which are up-regulated in FSHD (FOS, EGR1, MYC, and YY1). Validation studies in a separate group of patients with FSHD show consistent up-regulation of miR-100, miR-103, miR-146b, miR-29b, miR-34a, miR-454, miR-505, and miR-576. An increase in the expression of S100A8 protein, an inflammatory regulatory factor and subunit of calprotectin, is validated by Enzyme-Linked Immunosorbent Assay (ELISA). Bioinformatic analyses of proteomics and miRNA data further support a model of calprotectin and toll-like receptor 4 (TLR4) pathway dysregulation in FSHD. Moving forward, this panel of miRNAs, along with S100A8 and calprotectin, merit further investigation as monitoring and pharmacodynamic biomarkers for FSHD.
RESUMO
Cardiomyopathy is a leading cause of death for Duchenne muscular dystrophy. Here, we find that the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) can share common ligands but play distinct roles in dystrophic heart and skeletal muscle pathophysiology. Comparisons of their ligand structures indicate that the Δ9,11 modification of the first-in-class drug vamorolone enables it to avoid interaction with a conserved receptor residue (N770/N564), which would otherwise activate transcription factor properties of both receptors. Reporter assays show that vamorolone and eplerenone are MR antagonists, whereas prednisolone is an MR agonist. Macrophages, cardiomyocytes, and CRISPR knockout myoblasts show vamorolone is also a dissociative GR ligand that inhibits inflammation with improved safety over prednisone and GR-specific deflazacort. In mice, hyperaldosteronism activates MR-driven hypertension and kidney phenotypes. We find that genetic dystrophin loss provides a second hit for MR-mediated cardiomyopathy in Duchenne muscular dystrophy model mice, as aldosterone worsens fibrosis, mass and dysfunction phenotypes. Vamorolone successfully prevents MR-activated phenotypes, whereas prednisolone activates negative MR and GR effects. In conclusion, vamorolone targets dual nuclear receptors to treat inflammation and cardiomyopathy with improved safety.