RESUMO
G-protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate activated GPCRs, thereby priming them for desensitization1. Although it is unclear how GRKs recognize these receptors2-4, a conserved region at the GRK N terminus is essential for this process5-8. Here we report a series of cryo-electron microscopy single-particle reconstructions of light-activated rhodopsin (Rho*) bound to rhodopsin kinase (GRK1), wherein the N terminus of GRK1 forms a helix that docks into the open cytoplasmic cleft of Rho*. The helix also packs against the GRK1 kinase domain and stabilizes it in an active configuration. The complex is further stabilized by electrostatic interactions between basic residues that are conserved in most GPCRs and acidic residues that are conserved in GRKs. We did not observe any density for the regulator of G-protein signalling homology domain of GRK1 or the C terminus of rhodopsin. Crosslinking with mass spectrometry analysis confirmed these results and revealed dynamic behaviour in receptor-bound GRK1 that would allow the phosphorylation of multiple sites in the receptor tail. We have identified GRK1 residues whose mutation augments kinase activity and crosslinking with Rho*, as well as residues that are involved in activation by acidic phospholipids. From these data, we present a general model for how a small family of protein kinases can recognize and be activated by hundreds of different GPCRs.
Assuntos
Receptor Quinase 1 Acoplada a Proteína G/química , Rodopsina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Microscopia Crioeletrônica , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Alphaviruses can cause severe human arthritis and encephalitis. During virus infection, structural changes of viral glycoproteins in the acidified endosome trigger virus-host membrane fusion for delivery of the capsid core and RNA genome into the cytosol to initiate virus translation and replication. However, mechanisms by which E1 and E2 glycoproteins rearrange in this process remain unknown. Here, we investigate prefusion cryoelectron microscopy (cryo-EM) structures of eastern equine encephalitis virus (EEEV) under acidic conditions. With models fitted into the low-pH cryo-EM maps, we suggest that E2 dissociates from E1, accompanied by a rotation (â¼60°) of the E2-B domain (E2-B) to expose E1 fusion loops. Cryo-EM reconstructions of EEEV bound to a protective antibody at acidic and neutral pH suggest that stabilization of E2-B prevents dissociation of E2 from E1. These findings reveal conformational changes of the glycoprotein spikes in the acidified host endosome. Stabilization of E2-B may provide a strategy for antiviral agent development.
Assuntos
Vírus da Encefalite Equina do Leste , Proteínas do Envelope Viral , Antivirais/química , Antivirais/farmacologia , Microscopia Crioeletrônica , Vírus da Encefalite Equina do Leste/química , Concentração de Íons de Hidrogênio , Conformação Proteica , Estabilidade Proteica/efeitos dos fármacos , Proteínas do Envelope Viral/químicaRESUMO
Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio1284-1959, in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Proteínas Serina-Treonina Quinases/química , Fatores de Troca de Nucleotídeo Guanina Rho/química , Animais , Microscopia Crioeletrônica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Neoplasias Uveais , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Eastern equine encephalitis virus (EEEV), a mosquito-borne icosahedral alphavirus found mainly in North America, causes human and equine neurotropic infections. EEEV neurovirulence is influenced by the interaction of the viral envelope protein E2 with heparan sulfate (HS) proteoglycans from the host's plasma membrane during virus entry. Here, we present a 5.8-Å cryoelectron microscopy (cryo-EM) structure of EEEV complexed with the HS analog heparin. "Peripheral" HS binding sites were found to be associated with the base of each of the E2 glycoproteins that form the 60 quasi-threefold spikes (q3) and the 20 sites associated with the icosahedral threefold axes (i3). In addition, there is one HS site at the vertex of each q3 and i3 spike (the "axial" sites). Both the axial and peripheral sites are surrounded by basic residues, suggesting an electrostatic mechanism for HS binding. These residues are highly conserved among EEEV strains, and therefore a change in these residues might be linked to EEEV neurovirulence.
Assuntos
Desenho de Fármacos , Vírus da Encefalite Equina do Leste/ultraestrutura , Encefalomielite Equina/tratamento farmacológico , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/ultraestrutura , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Sulfatos de Condroitina/farmacologia , Microscopia Crioeletrônica , Vírus da Encefalite Equina do Leste/metabolismo , Encefalomielite Equina/virologia , Proteoglicanas de Heparan Sulfato/análogos & derivados , Heparina/metabolismo , Humanos , Mesocricetus , Estrutura Molecular , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/ultraestrutura , Ligação Viral/efeitos dos fármacosRESUMO
Metabolic associated fatty liver disease (MAFLD) is one of the most common causes of chronic liver disease worldwide. To date, there is no FDA-approved treatment, so there is an urgent need to determine its pathophysiology and underlying molecular mechanisms. Autophagy is a lysosomal degradation pathway that removes damaged organelles and misfolded proteins after cell injury through endoplasmic reticulum stress or starvation, which inhibits apoptosis and promotes cell survival. Recent studies have shown that autophagy plays an important role in removing lipid droplets from hepatocytes. Autophagy has also been reported to inhibit the production of pro-inflammatory cytokines and provide energy for the hepatic stellate cells activation during liver fibrosis. Thyroid hormone, irisin, melatonin, hydrogen sulfide, sulforaphane, DA-1241, vacuole membrane protein 1, nuclear factor erythroid 2-related factor 2, sodium-glucose co-transporter type-2 inhibitors, immunity-related GTPase M, and autophagy-related gene 7 have been reported to ameliorate MAFLD via autophagic induction. Lipid receptor CD36, SARS-CoV-2 Spike protein and leucine aminopeptidase 3 play a negative role in the autophagic function. This review summarizes recent advances in the role of autophagy in MAFLD. Autophagy modulates major pathological changes, including hepatic lipid metabolism, inflammation, and fibrosis, suggesting the potential of modulating autophagy for the treatment of MAFLD.
Assuntos
Autofagia , Hepatopatias , Hepatopatia Gordurosa não Alcoólica , Humanos , Fígado/metabolismo , Hepatopatias/metabolismo , Hepatopatias/fisiopatologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/fisiopatologiaRESUMO
Background: Hypoxia-inducible factor-1α (HIF-1α), heat shock protein-72 (HSP-72), hemeoxygenase-1 (HO-1), and matrix metalloproteinase-9 (MMP-9) have been identified as potential therapeutic targets in the brain for cerebral ischemia. To elucidate their underlying mechanisms, we first aimed to ascertain whether these proteins participate in the pathogenesis of heat-induced ischemic damage to the hypothalamus of rats. Second, we investigated whether hypobaric hypoxia preconditioning (HHP) attenuates heat-induced hypothalamic ischemic/hypoxic injury by modulating these proteins in situ. Methods: Anesthetized rats treated with or without HHP were subjected to heat stress. Hypothalamic ischemic/hypoxic damage was evaluated by measuring hypothalamic levels of cerebral blood flow (CBF), partial oxygen pressure (PO2), and hypothalamic temperature via an implanted probe. Hypothalamic apoptotic neurons were counted by measuring the number of NeuN/caspase-3/DAPI triple-stained cells. Hypothalamic protein expression of HIF-1α, HSP-72, HO-1, and MMP-9 was determined biochemically. Results: Before the start of the thermal experiments, rats were subjected to 5 hours of HHP (0.66 ATA or 18.3% O2) daily for 5 consecutive days per week for 2 weeks, which led to significant loss of body weight, reduced brown adipose tissue (BAT) wet weight and decreased body temperature. The animals were then subjected to thermal studies. Twenty minutes after heat stress, heat-exposed rats not treated with HHP displayed significantly higher core and hypothalamic temperatures, hypothalamic MMP-9 levels, and numbers of hypothalamic apoptotic neurons but significantly lower mean blood pressure, hypothalamic blood flow, and PO2 values than control rats not exposed to heat. In heat-exposed rats, HHP significantly increased the hypothalamic levels of HIF-1α, HSP-72, and HO-1 but significantly alleviated body and hypothalamic hyperthermia, hypotension, hypothalamic ischemia, hypoxia, neuronal apoptosis and degeneration. Conclusions: HHP may protect against hypothalamic ischemic/hypoxic injury and overexpression of MMP-9 by upregulating the hypothalamic expression of HIF-1α, HSP-72, and HO-1 in rats subjected to heatstroke.
Assuntos
Isquemia Encefálica/terapia , Golpe de Calor/terapia , Hipotálamo/patologia , Hipóxia/fisiopatologia , Metaloproteinase 9 da Matriz/metabolismo , Animais , Apoptose , Isquemia Encefálica/etiologia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Modelos Animais de Doenças , Golpe de Calor/complicações , Golpe de Calor/patologia , Golpe de Calor/fisiopatologia , Humanos , Hipotálamo/citologia , Hipotálamo/fisiopatologia , Masculino , Neurônios/patologia , RatosRESUMO
To develop accurate prognostic models is one of the biggest challenges in "omics"-based cancer research. Here, we propose a novel computational method for identifying dysregulated gene subnetworks as biomarkers to predict cancer recurrence. Applying our method to the DNA methylome of endometrial cancer patients, we identified a subnetwork consisting of differentially methylated (DM) genes, and non-differentially methylated genes, termed Epigenetic Connectors (EC), that are topologically important for connecting the DM genes in a protein-protein interaction network. The ECs are statistically significantly enriched in well-known tumorgenesis and metastasis pathways, and include known epigenetic regulators. Importantly, combining the DMs and ECs as features using a novel random walk procedure, we constructed a support vector machine classifier that significantly improved the prediction accuracy of cancer recurrence and outperformed several alternative methods, demonstrating the effectiveness of our network-based approach.
Assuntos
Algoritmos , Biomarcadores Tumorais , Metilação de DNA , Neoplasias do Endométrio , Recidiva Local de Neoplasia , Ilhas de CpG , DNA de Neoplasias , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Epigenômica , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Genéticos , Prognóstico , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNARESUMO
The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF-ATP) revealed that the phosphate-binding loop (amino acids 97-105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.
Assuntos
Carboxiliases/metabolismo , Ácido Mevalônico/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Carboxiliases/fisiologia , Cristalografia por Raios X/métodos , Enterococcus faecalis/enzimologia , Enterococcus faecalis/metabolismo , Hemiterpenos/biossíntese , Cinética , Ácido Mevalônico/metabolismo , Compostos Organofosforados , Especificidade por SubstratoRESUMO
BACKGROUND: The novel concept of continuous intraoperative neuromonitoring (Cont-IONM) through stimulation of the vagal nerve has been used in thyroidectomies to prevent imminent injury of the recurrent laryngeal nerve (RLN). This article reports on this technology and the results of using transoral Cont-IONM in natural orifice transluminal endoscopic surgery for thyroid disease. METHODS: Cont-IONM of the RLN was achieved through automatic cyclical stimulation of the vagal nerve using a C2 monitor and delta stimulating electrode. During the operation, three vestibular incisions were made, and the stimulating electrode was transorally inserted, with its cable line lying outside the trocar. The vagal nerve was gently dissected, looped, and then enveloped by the electrode cuff. Electromyography (EMG) of the vocalis muscle was performed, and the alarm was set to activate when the EMG amplitude reduced by 50% and latency was prolonged by 10%. Demographic data and outcome variables, including incremental time required to achieve Cont-IONM, were obtained. RESULTS: A total of 20 patients (28 nerves at risk) undergoing a transoral endoscopic thyroidectomy vestibular approach were enrolled in this study. All Cont-IONM procedures were successfully completed. In all patients, the stimulation was set at 0.7 milliamps every 1 s, and Cont-IONM use was unassociated with any untoward neural, cardiovascular, or gastrointestinal sequelae. On average, the ipsilateral Cont-IONM procedure required 10.33 ± 2.57 min to complete. Except for one instance, no significant problems occurred with electrode displacement. In one patient, a combined EMG event occurred, which improved after releasing the thyroid retractor, and the patient had no vocal cord paralysis postoperatively. CONCLUSION: Cont-IONM is feasible and safe to use during transoral endoscopic thyroidectomies and may assist in the early detection of adverse EMG changes, thereby preventing paralysis of the RLNs.
Assuntos
Monitorização Neurofisiológica Intraoperatória , Cirurgia Endoscópica por Orifício Natural , Traumatismos do Nervo Laríngeo Recorrente/prevenção & controle , Tireoidectomia/métodos , Paralisia das Pregas Vocais/prevenção & controle , Adulto , Eletromiografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/prevenção & controle , Doenças da Glândula Tireoide/cirurgia , Adulto JovemRESUMO
Identifying prostate cancer-driving transcription factors (TFs) in addition to the androgen receptor promises to improve our ability to effectively diagnose and treat this disease. We employed an integrative genomics analysis of master TFs CREB1 and FoxA1 in androgen-dependent prostate cancer (ADPC) and castration-resistant prostate cancer (CRPC) cell lines, primary prostate cancer tissues and circulating tumor cells (CTCs) to investigate their role in defining prostate cancer gene expression profiles. Combining genome-wide binding site and gene expression profiles we define CREB1 as a critical driver of pro-survival, cell cycle and metabolic transcription programs. We show that CREB1 and FoxA1 co-localize and mutually influence each other's binding to define disease-driving transcription profiles associated with advanced prostate cancer. Gene expression analysis in human prostate cancer samples found that CREB1/FoxA1 target gene panels predict prostate cancer recurrence. Finally, we showed that this signaling pathway is sensitive to compounds that inhibit the transcription co-regulatory factor MED1. These findings not only reveal a novel, global transcriptional co-regulatory function of CREB1 and FoxA1, but also suggest CREB1/FoxA1 signaling is a targetable driver of prostate cancer progression and serves as a biomarker of poor clinical outcomes.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Fator 3-alfa Nuclear de Hepatócito/fisiologia , Recidiva Local de Neoplasia/metabolismo , Neoplasias da Próstata/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Sítios de Ligação , Biomarcadores Tumorais , Linhagem Celular Tumoral , Sequência Consenso , Intervalo Livre de Doença , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Subunidade 1 do Complexo Mediador/metabolismo , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/mortalidade , Prognóstico , Modelos de Riscos Proporcionais , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Transcrição GênicaRESUMO
China's disaster management system contains no law-based presidential disaster declarations; however, the national leader's instructions (pishi in Chinese) play a similar role to disaster declarations, which increase the intensity of disaster relief. This raises the question of what affects presidential disaster instructions within an authoritarian regime. This research shows that China's disaster politics depend on a crisis threshold system for operation and that the public and social features of disasters are at the core of this system. China's political cycle has no significant impact on disaster politics. A change in the emergency management system has a significant bearing on presidential disaster instructions, reflecting the strong influence of the concept of rule of law and benefiting the sustainable development of the emergency management system. In terms of disaster politics research, unlocking the black box of China's disaster politics and increasing the number of comparative political studies will benefit the development of empirical and theoretical study.
Assuntos
Planejamento em Desastres , Política , China , HumanosRESUMO
Ossifying fibromyxoid tumor (OFMT) is an uncommon mesenchymal neoplasm of uncertain differentiation and intermediate malignant potential. Recurrent gene fusions involving either PHF1 or BCOR have been found in 85% of OFMT, including typical and malignant examples. As a subset of OFMT still lack known genetic abnormalities, we identified two OFMTs negative for PHF1 and BCOR rearrangements, which were subjected to transcriptome analysis for fusion discovery. The RNA sequencing found a novel CREBBP-BCORL1 fusion candidate in an axillary mass of a 51 year-old male and a KDM2A-WWTR1 in a thigh mass of a 36 year-old male. The gene fusions were validated by RT-PCR and FISH in the index cases and then screened by FISH on 4 additional OFMTs lacking known fusions. An identical CREBBP-BCORL1 fusion was found in an elbow tumor from a 30 year-old male. Both OFMTs with CREBBP-BCORL1 fusions had areas of typical OFMT morphology, exhibiting uniform round to epithelioid cells arranged in cords or nesting pattern in a fibromyxoid stroma. The OFMT with KDM2A-WWTR1 fusion involved dermis and superficial subcutis, being composed of ovoid cells in a fibromyxoid background with hyalinized giant rosettes. The S100 immunoreactivity ranged from very focal to absent. Similar to other known fusion genes in OFMT, BCORL1, CREBBP and KDM2A are also involved in histone modification. In summary, we expand the spectrum of molecular abnormalities in OFMT with 2 novel fusions, CREBBP-BCORL1 and KDM2A-WWTR1, further implicating the epigenetic deregulation as the leading pathogenetic mechanism in OFMT. © 2016 Wiley Periodicals, Inc.
Assuntos
Biomarcadores Tumorais/genética , Fibroma Ossificante/genética , Fibroma/genética , Proteínas de Fusão Oncogênica/genética , Neoplasias de Tecidos Moles/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Proteína de Ligação a CREB/genética , Proteínas F-Box/genética , Fibroma/patologia , Fibroma Ossificante/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Repressoras/genética , Neoplasias de Tecidos Moles/patologia , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
An increasing number of congenital and infantile sarcomas displaying a primitive, monomorphic spindle cell phenotype have been characterized to harbor recurrent gene fusions, including infantile fibrosarcoma and congenital spindle cell rhabdomyosarcoma. Here, we report an unusual spindle cell sarcoma presenting as a large and infiltrative pelvic soft tissue mass in a 4-month-old girl, which revealed a novel TFG-MET gene fusion by whole transcriptome RNA sequencing. The tumor resembled the morphology of an infantile fibrosarcoma with both fascicular and patternless growth, however, it expressed strong S100 protein immunoreactivity, while lacking SOX10 staining and retaining H3K27me3 expression. Although this immunoprofile suggested partial neural/neuroectodermal differentiation, overall features were unusual and did not fit into any known tumor types (cellular schwannoma, MPNST), raising the possibility of a novel pathologic entity. The TFG-MET gene fusion expands the genetic spectrum implicated in the pathogenesis of congenital spindle cell sarcomas, with yet another example of kinase oncogenic activation through chromosomal translocation. The discovery of this new fusion is significant since the resulting MET activation can potentially be inhibited by targeted therapy, as MET inhibitors are presently available in clinical trials.
Assuntos
Proteínas de Fusão Oncogênica/genética , Proteínas/genética , Proteínas Proto-Oncogênicas c-met/genética , Sarcoma/genética , Neoplasias de Tecidos Moles/genética , Feminino , Histonas/genética , Histonas/metabolismo , Humanos , Lactente , Proteínas de Fusão Oncogênica/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Sarcoma/diagnóstico por imagem , Sarcoma/patologia , Neoplasias de Tecidos Moles/diagnóstico por imagem , Neoplasias de Tecidos Moles/patologiaRESUMO
CIC rearrangements have been reported in two-thirds of EWSR1-negative small blue round cell tumors (SBRCTs). However, a number of SBRCTs remain unclassified despite exhaustive analysis. Fourteen SBRCTs lacking driver genetic events by RNA sequencing (RNAseq) analysis were collected. Unsupervised hierarchical clustering was performed using samples from our RNAseq database, including 13 SBRCTs with non-CIC genetic abnormalities and 2 CIC-rearranged angiosarcomas among others. Remarkably, all 14 study cases showed high mRNA levels of ETV1/4/5, and by unsupervised clustering most grouped into a distinct cluster, separate from other tumors. Based on these results indicating a close relationship with CIC-rearranged tumors, we manually inspected CIC reads in RNAseq data. FISH for CIC and DUX4 abnormalities and immunohistochemical stains for ETV4 were also performed. In the control group, only 2 CIC-rearranged angiosarcomas had high ETV1/4/5 expression. Upon manual inspection of CIC traces, 7 of 14 cases showed CIC-DUX4 fusion reads, 2 cases had DUX4-CIC reads, while the remaining 5 were negative. FISH showed CIC break-apart in 7 cases, including 5 cases lacking CIC-DUX4 or DUX4-CIC fusion reads on RNAseq manual inspection. However, no CIC abnormalities were detected by FISH in 6 cases with CIC-DUX4 or DUX4-CIC reads. ETV4 immunoreactivity was positive in 7 of 11 cases. Our results highlight the underperformance of FISH and RNAseq methods in diagnosing SBRCTs with CIC gene abnormalities. The downstream ETV1/4/5 transcriptional up-regulation appears highly sensitive and specific and can be used as a reliable molecular signature and diagnostic method for CIC fusion positive SBRCTs.
Assuntos
Algoritmos , Proteínas de Ligação a DNA/genética , Rearranjo Gênico , Hibridização in Situ Fluorescente , Proteínas Repressoras/genética , Sarcoma de Ewing/diagnóstico , Análise de Sequência de RNA , Fatores de Transcrição/genética , Transcrição Gênica , Regulação para Cima , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sarcoma de Ewing/genética , Adulto JovemRESUMO
BACKGROUND: Vibrio vulnificus is a marine bacterial species that causes opportunistic infections manifested by serious skin lesions and fulminant septicemia in humans. We have previously shown that the multifunctional autoprocessing repeats in toxin (MARTXVv1) of a biotype 1 V. vulnificus strain promotes survival of this organism in the host by preventing it from engulfment by the phagocytes. The purpose of this study was to further explore how MARTXVv1 inhibits phagocytosis of this microorganism by the macrophage. METHODS: We compared between a wild-type V. vulnificus strain and its MARTXVv1-deficient mutant for a variety of phagocytosis-related responses, including morphological change and activation of signaling molecules, they induced in the macrophage. We also characterized a set of MARTXVv1 domain-deletion mutants to define the regions associated with antiphagocytosis activity. RESULTS: The RAW 264.7 cells and mouse peritoneal exudate macrophages underwent cell rounding accompanied by F-actin disorganization in the presence of MARTXVv1. In addition, phosphorylation of some F-actin rearrangement-associated signaling molecules, including Lyn, Fgr and Hck of the Src family kinases (SFKs), focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), phosphoinositide 3-kinase (PI3K) and Akt, but not p38, was decreased. By using specific inhibitors, we found that these kinases were all involved in the phagocytosis of MARTXVv1-deficient mutant in an order of SFKs-FAK/Pyk2-PI3K-Akt. Deletion of the effector domains in the central region of MARTXVv1 could lead to reduced cytotoxicity, depending on the region and size of deletion, but did not affect the antiphagocytosis activity and ability to cause rounding of macrophage. Reduced phosphorylation of Akt was closely associated with inhibition of phagocytosis by the wild-type strain and MARTXVv1 domain-deletion mutants, and expression of the constitutively active Akt, myr-Akt, enhanced the engulfment of these strains by macrophage. CONCLUSIONS: MARTXVv1 could inactivate the SFKs-FAK/Pyk2-PI3K-Akt signaling pathway in the macrophages. This might lead to impaired phagocytosis of the V. vulnificus-infected macrophage. The majority of the central region of MARTXVv1 is not associated with the antiphagocytosis activity.
Assuntos
Toxinas Bacterianas/imunologia , Fagocitose/imunologia , Vibrioses/microbiologia , Vibrio vulnificus/imunologia , Vibrio vulnificus/patogenicidade , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Citotoxinas/imunologia , Citotoxinas/metabolismo , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos BALB C , Vibrioses/patologia , Vibrio vulnificus/genéticaRESUMO
BACKGROUND: An attenuated mutant (designated NY303) of Vibrio vulnificus, which causes serious wound infection and septicemia in humans, was isolated fortuitously from a clinical strain YJ016. This mutant was defective in cytotoxicity, migration on soft agar and virulence in the mouse. The purpose of this study was to map the mutation in this attenuated mutant and further explore how the gene thus identified is involved in virulence. METHODS: The whole genome sequence of mutant NY303 determined by next-generation sequencing was compared with that of strain YJ016 to map the mutations. By isolating and characterizing the specific gene-knockout mutants, the gene associated with the phenotype of mutant NY303 was identified. This gene encodes a global regulator, Lrp. A mutant, YH01, deficient in Lrp was isolated and examined in vitro, in vivo and ex vivo to find the affected virulence mechanisms. The target genes of Lrp were further identified by comparing the transcriptomes, which were determined by RNA-seq, of strain YJ016 and mutant YH01. The promoters bound by Lrp were identified by genome footprinting-sequencing, and those related with virulence were further examined by electrophoretic mobility shift assay. RESULTS: A mutation in lrp was shown to be associated with the reduced cytotoxicity, chemotaxis and virulence of mutant NY303. Mutant YH01 exhibited a phenotype resembling that of mutant NY303, and was defective in colonization in the mouse and growth in mouse serum, but not the antiphagocytosis ability. 596 and 95 genes were down- and up-regulated, respectively, in mutant YH01. Many of the genes involved in secretion of the MARTX cytotoxin, chemotaxis and iron-acquisition were down-regulated in mutant YH01. The lrp gene, which was shown to be negatively autoregulated, and 7 down-regulated virulence-associated genes were bound by Lrp in their promoters. A 14-bp consensus sequence, mkCrTTkwAyTsTG, putatively recognized by Lrp was identified in the promoters of these genes. CONCLUSIONS: Lrp is a global regulator involved in regulation of cytotoxicity, chemotaxis and iron-acquisition in V. vulnificus. Down-regulation of many of the genes associated with these properties may be responsible, at least partly, for loss of virulence in mutant NY303.
Assuntos
Proteínas de Bactérias/genética , Regulação para Baixo , Proteína Reguladora de Resposta a Leucina/genética , Mutação , Vibrio vulnificus/genética , Vibrio vulnificus/patogenicidade , Virulência/genética , Animais , Proteínas de Bactérias/metabolismo , Proteína Reguladora de Resposta a Leucina/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Doenças dos Roedores/microbiologia , Vibrioses/microbiologia , Vibrio vulnificus/fisiologiaRESUMO
The bone destruction disease including osteoporosis and rheumatoid arthritis are caused by the imbalance between osteoblastogenesis and osteoclastogenesis. Inhibition of the NF-κB pathway was responsible for decreased osteoclastogenesis. Recently many studies indicated that niclosamide, the FDA approved an antihelminth drug, inhibits prostate and breast cancer cells growth by targeting NF-κB signaling pathways. This study evaluated the effects of niclosamide on osteoclast and osteoblast differentiation and function in vitro. In RANKL-induced murine osteoclast precursor cell RAW264.7 and M-CSF/RANKL-stimulated primary murine bone marrow-derived macrophages (BMM), niclosamide dose-dependently inhibited the formation of TRAP-positive multinucleated osteoclasts and resorption pits formation between 0.5uM and 1uM. In addition, niclosamide suppressed the expression of nuclear factor of activated T cells c1 (NFATc1) and osteoclast differentiated-related genes in M-CSF/ RANKL-stimulated BMM by interference with TRAF-6, Erk1/2, JNK and NF-κB activation pathways. However, the cytotoxic effects of niclosamide obviously appeared at the effective concentrations for inhibiting osteoclastogenesis (0.5-1uM) with increase of apoptosis through caspase-3 activation in osteoblast precursor cell line, MC3T3-E1. Niclosamide also inhibited ALP activity, bone mineralization and osteoblast differentiation-related genes expression in MC3T3-E1. Therefore, our findings suggest the new standpoint that niclosamide's effects on bones must be considered before applying it in any therapeutic treatment.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Niclosamida/efeitos adversos , Osteogênese/efeitos dos fármacos , Animais , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Camundongos , Niclosamida/farmacologia , Niclosamida/uso terapêutico , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteogênese/genética , Ligante RANK/genética , Ligante RANK/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacosRESUMO
SMARCB1 inactivation occurs in a variety of tumors, being caused by various genetic mechanisms. Since SMARCB1 and EWSR1 genes are located close to each other on chromosome 22, larger SMARCB1 deletions may encompass the EWSR1 locus. Herein, we report four cases with SMARCB1-deletions showing concurrent EWSR1 gene abnormalities by FISH, which lead initially to misinterpretations as EWSR1-rearranged tumors. Our study group included various morphologies: a poorly differentiated chordoma, an extrarenal rhabdoid tumor, a myoepithelial carcinoma, and a proximal-type epithelioid sarcoma. All cases showed loss of SMARCB1 (INI1) by immunohistochemistry (IHC) and displayed characteristic histologic features for the diagnoses. The SMARCB1 FISH revealed homozygous or heterozygous deletions in three and one case, respectively. The co-hybridized EWSR1 probes demonstrated either unbalanced split signals or heterozygous deletion in two cases each. The former suggested bona fide rearrangement, while the latter resembled an unbalanced translocation. However, all the FISH patterns were quite complex and distinct from the simple and uniform split signals seen in typical EWSR1 rearrangements. We conclude that in the context of 22q11-12 regional alterations present in SMARCB1-deleted tumors, simultaneous EWSR1 involvement may be misinterpreted as equivalent to EWSR1 rearrangement. A detailed clinicopathologic correlation and supplementing the EWSR1 FISH assay with complementary methodology is mandatory for correct diagnosis. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Cordoma/genética , Mioepitelioma/genética , Proteínas de Ligação a RNA/genética , Tumor Rabdoide/genética , Proteína SMARCB1/genética , Sarcoma/genética , Adolescente , Adulto , Proteínas de Ligação a Calmodulina/isolamento & purificação , Pré-Escolar , Cordoma/diagnóstico , Cordoma/patologia , Cromossomos Humanos Par 22/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Mioepitelioma/diagnóstico , Mioepitelioma/patologia , Proteína EWS de Ligação a RNA , Proteínas de Ligação a RNA/isolamento & purificação , Tumor Rabdoide/diagnóstico , Tumor Rabdoide/patologia , Proteína SMARCB1/isolamento & purificação , Sarcoma/diagnóstico , Sarcoma/patologiaRESUMO
OBJECTIVE: Prior studies highlighted that patients on chronic medications who used complementary and alternative medicines (CAMs) did not share this information with their healthcare providers. Furthermore, there might be potential adverse interactions between CAMs and antiepileptic drug (AED) therapy. However, there are no studies that investigate the effect of religion or race on CAM use in a multiethnic community. Therefore, we aimed to investigate CAM usage among pediatric patients with epilepsy (PPE) and identify predictors of CAM usage in our multiethnic setting. METHODS: This was a cross-sectional study where caregivers of recruited at an outpatient epilepsy clinic between September 2013 and July 2014 completed a self-administered survey. Those who declined participation, lacked understanding of English or Mandarin, or missed the clinic appointments were excluded. Those caring for more than one patient participated only once. RESULTS: Of the 195 surveys collected, 178 were used for analysis. The PPE used an average of 1.50 AEDs (range: 0-4). Forty-nine (27.5%) caregivers reported giving CAMs to their PPE. Commonly used CAMs were multivitamins (44.4%), traditional herbs (42.2%), and acupuncture (17.4%). Multivitamins were mostly given by Christian/Catholic caregivers while Buddhist caregivers mostly gave traditional herbs or acupuncture. The majority of the CAM users were Chinese. Univariate analysis showed that caregivers with secondary school (high school) education were 3.52 times more likely to use CAMs compared to those with primary school (elementary school) education. CONCLUSIONS: While some caregivers gave CAMs to their PPE, they had various misconceptions and did not discuss the usage with their healthcare providers. We propose that the predictors identified in this study can help to identify potential CAM users. Healthcare providers should routinely probe about the usage of CAMs by PPE, and thereafter, the suitability of CAM use could be evaluated. The caregivers will benefit from open discussion with healthcare professionals on the safe use of AEDs with or without CAMs.
Assuntos
Terapias Complementares/estatística & dados numéricos , Epilepsia/terapia , Adolescente , Adulto , Anticonvulsivantes/uso terapêutico , Cuidadores , Criança , Pré-Escolar , Estudos Transversais , Medicamentos de Ervas Chinesas/uso terapêutico , Etnicidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Singapura/epidemiologia , Inquéritos e Questionários , Vitaminas/uso terapêuticoRESUMO
A series of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium derivatives was synthesized and evaluated for osteoclast inhibition using a TRAP-staining assay. Among them, two compounds, LCCY-13 and LCCY-15, dose-dependently suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. Moreover, the cytotoxicity assay on RAW264.7 cells suggested that the inhibition of osteoclastic bone resorption by these compounds was not a result of their cytotoxicity. Further, the inhibitory activities of compounds LCCY-13 and LCCY-15 were further confirmed by including specific inhibition of NFATc1 expression levels in nuclei using an immunofluorescent analysis. In addition, LCCY-13 and LCCY-15 also significantly attenuated the bone resorption activity of osteoclasts according to a pit formation assay. Thus, a new class of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium compounds might be considered as an essential lead structure for the further development of anti-resorptive agents.