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1.
Plast Reconstr Surg ; 146(2): 165e-176e, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32740585

RESUMO

BACKGROUND: Trigger finger, or stenosing tenosynovitis, is one of the most common conditions affecting the hand, yet its pathophysiology remains poorly understood, and genetic association studies of trigger finger are lacking. The purpose of this study was to identify single-nucleotide polymorphisms associated with trigger finger through a genomewide approach. METHODS: The authors performed a case-control genomewide association study in the Partners HealthCare Biobank. Single-nucleotide polymorphism- and gene-based association analyses were carried out after quality control, imputation, and filtering. RESULTS: Among 942 trigger finger cases and 24,472 controls, the authors tested 7,846,471 single-nucleotide polymorphisms for association with trigger finger. In the single-nucleotide polymorphism-based analysis, a single locus on chromosome 13 corresponding to KLHL1 met the genomewide significance threshold (lead single-nucleotide polymorphism rs59988404; OR, 1.74; 95 percent CI, 1.47 to 2.07; p = 1.99 × 10). After mapping, gene-based analysis demonstrated a significant association with POLE2 (p = 7.53 × 10) on chromosome 14. Among trigger finger cases, rs59988404 genotype was significantly associated with the total number of trigger finger procedures performed (p = 0.026). CONCLUSIONS: In the first reported genomewide association study of trigger finger, the authors report significant associations of KLHL1 and POLE2 with risk of trigger finger. The authors' results may help to elucidate the pathophysiology of trigger finger and facilitate an individualized, precision-medicine treatment approach. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.


Assuntos
DNA Polimerase II/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Proteínas dos Microfilamentos/genética , Dedo em Gatilho/genética , Adulto , Estudos de Casos e Controles , Humanos , Polimorfismo de Nucleotídeo Único , Medicina de Precisão , Fatores de Risco , Dedo em Gatilho/terapia
2.
Nat Commun ; 11(1): 4187, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826901

RESUMO

EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico/fisiologia , Alinhamento de Sequência , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/genética
3.
Nat Commun ; 11(1): 3457, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651364

RESUMO

Glioblastoma is a deadly cancer, with no effective therapies. Better understanding and identification of selective targets are urgently needed. We found that advillin (AVIL) is overexpressed in all the glioblastomas we tested including glioblastoma stem/initiating cells, but hardly detectable in non-neoplastic astrocytes, neural stem cells or normal brain. Glioma patients with increased AVIL expression have a worse prognosis. Silencing AVIL nearly eradicated glioblastoma cells in culture, and dramatically inhibited in vivo xenografts in mice, but had no effect on normal control cells. Conversely, overexpressing AVIL promoted cell proliferation and migration, enabled fibroblasts to escape contact inhibition, and transformed immortalized astrocytes, supporting AVIL being a bona fide oncogene. We provide evidence that the tumorigenic effect of AVIL is partly mediated by FOXM1, which regulates LIN28B, whose expression also correlates with clinical prognosis. AVIL regulates the cytoskeleton through modulating F-actin, while mutants disrupting F-actin binding are defective in its tumorigenic capabilities.


Assuntos
Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas dos Microfilamentos/metabolismo , Animais , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Citoesqueleto/metabolismo , Imunofluorescência , Glioblastoma/genética , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo Real
4.
Acta Cir Bras ; 35(4): e202000406, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32578724

RESUMO

PURPOSE: To investigate the role of Rosmarinic acid (RA) in the prevention of traumatic brain injury and the immunohistochemical analysis of IBA-1 and GFAP expressions. METHODS: Healthy male rats were randomly divided into 3 groups consisting of 10 rats. Groups were as follows; control group, traumatic brain injury (TBI) group, and TBI+RA group. After traumatic brain injury, blood samples were taken from the animals and analyzed with various biochemical markers. And then IBA-1 and GFAP expressions were evaluated immunohistochemically. RESULTS: Significant results were obtained in all biochemical parameters between groups. Immunohistochemical sections showed IBA-1 not only in microglia and macrophage activity but also in degenerative neurons in blood vessel endothelial cells. However, GFAP reaction and post-traumatic rosmarinic acid administration showed positive expression in astrocytes with regular structure around the blood vessel. CONCLUSION: Rosmarinic acid in blood vessel endothelial cells showed that preserving the integrity of astrocytic structure in the blood brain barrier may be an important antioxidant.


Assuntos
Lesões Encefálicas Traumáticas/prevenção & controle , Proteínas de Ligação ao Cálcio/análise , Cinamatos/farmacologia , Craniotomia/métodos , Depsídeos/farmacologia , Proteína Glial Fibrilar Ácida/análise , Proteínas dos Microfilamentos/análise , Fármacos Neuroprotetores/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/cirurgia , Glutationa Peroxidase/análise , Imuno-Histoquímica , Masculino , Malondialdeído/análise , Distribuição Aleatória , Ratos Sprague-Dawley , Valores de Referência , Reprodutibilidade dos Testes
5.
PLoS One ; 15(4): e0232025, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353019

RESUMO

The actin cytoskeleton plays a central role in establishing cell polarity and shape during embryonic morphogenesis. Daam1, a member of the Formin family of actin cytoskeleton regulators, is a Dvl2-binding protein that functions in the Wnt/Planar Cell Polarity (PCP) pathway. To examine the role of the Daam proteins in mammalian development, we generated Daam-deficient mice by gene targeting and found that Daam1, but not Daam2, is necessary for fetal survival. Embryonic development of Daam1 mutants was delayed most likely due to functional defects in the labyrinthine layer of the placenta. Examination of Daam2 and Daam1/2 double mutants revealed that Daam1 and Daam2 are functionally redundant during placental development. Of note, neural tube closure defects (NTD), which are observed in several mammalian PCP mutants, are not observed in Wnt5a or Daam1 single mutants, but arise in Daam1;Wnt5a double mutants. These findings demonstrate a unique function for Daam genes in placental development and are consistent with a role for Daam1 in the Wnt/PCP pathway in mammals.


Assuntos
Proteínas dos Microfilamentos/genética , Placentação/genética , Proteínas rho de Ligação ao GTP/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/metabolismo , Polaridade Celular , Citoesqueleto/metabolismo , Desenvolvimento Embrionário , Feminino , Forminas/genética , Forminas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Placenta/embriologia , Gravidez , Via de Sinalização Wnt , Proteínas rho de Ligação ao GTP/metabolismo
6.
Mol Biol (Mosk) ; 54(2): 285-292, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32392198

RESUMO

The actin-binding proteins profilin, fascin, and ezrin were tested for involvement in metastasis of non-small cell lung cancer (NSCLC). The levels of the PFN1, FSCN1, and EZR mRNAs and respective proteins were determined by real-time PCR and Western blotting; tumor and adjacent normal lung tissue samples were obtained from 46 NSCLC patients. Patients with lymphatic metastasis had higher expression levels of the profilin, fascin, and ezrin mRNAs and the profilin and fascin proteins. Both mRNA and protein expression levels increased in patients with distant metastasis. The molecules may serve as predictors to evaluate the prognosis in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Neoplasias Pulmonares/patologia , Proteínas dos Microfilamentos/metabolismo , Profilinas/metabolismo , Linhagem Celular Tumoral , Humanos , Metástase Neoplásica , RNA Mensageiro
7.
Proc Natl Acad Sci U S A ; 117(22): 12324-12331, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32409605

RESUMO

Glioblastoma multiforme (GBM) is an aggressive cancer without currently effective therapies. Radiation and temozolomide (radio/TMZ) resistance are major contributors to cancer recurrence and failed GBM therapy. Heat shock proteins (HSPs), through regulation of extracellular matrix (ECM) remodeling and epithelial mesenchymal transition (EMT), provide mechanistic pathways contributing to the development of GBM and radio/TMZ-resistant GBM. The Friend leukemia integration 1 (Fli-1) signaling network has been implicated in oncogenesis in GBM, making it an appealing target for advancing novel therapeutics. Fli-1 is linked to oncogenic transformation with up-regulation in radio/TMZ-resistant GBM, transcriptionally regulating HSPB1. This link led us to search for targeted molecules that inhibit Fli-1. Expression screening for Fli-1 inhibitors identified lumefantrine, an antimalarial drug, as a probable Fli-1 inhibitor. Docking and isothermal calorimetry titration confirmed interaction between lumefantrine and Fli-1. Lumefantrine promoted growth suppression and apoptosis in vitro in parental and radio/TMZ-resistant GBM and inhibited tumor growth without toxicity in vivo in U87MG GBM and radio/TMZ-resistant GBM orthotopic tumor models. These data reveal that lumefantrine, an FDA-approved drug, represents a potential GBM therapeutic that functions through inhibition of the Fli-1/HSPB1/EMT/ECM remodeling protein networks.


Assuntos
Antimaláricos/administração & dosagem , Antineoplásicos Alquilantes/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Lumefantrina/administração & dosagem , Temozolomida/administração & dosagem , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Transativadores/genética , Transativadores/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(21): 11648-11657, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32398370

RESUMO

The intestinal mucosa exists in dynamic balance with trillions of luminal microbes. Disruption of the intestinal epithelial barrier, commonly observed in mucosal inflammation and diseases such as inflammatory bowel diseases (IBDs), is often associated with dysbiosis, particularly decreases in species producing short-chain fatty acids (SCFAs), such as butyrate. It remains unclear to what extent microbiota-derived factors contribute to the overall maintenance of intestinal homeostasis. Initial studies revealed that butyrate selectively promotes epithelial barrier function and wound healing. We aimed to define the specific mechanism(s) through which butyrate contributes to these epithelial responses. Guided by an unbiased profiling approach, we identified the dominant regulation of the actin-binding protein synaptopodin (SYNPO). Extensions of this work revealed a role for SYNPO in intestinal epithelial barrier function and wound healing. SYNPO was localized to the intestinal epithelial tight junction and within F-actin stress fibers where it is critical for barrier integrity and cell motility. Butyrate, but not other SCFAs, induced SYNPO in epithelial cell lines and murine colonic enteroids through mechanisms possibly involving histone deacetylase inhibition. Moreover, depletion of the microbiota abrogated expression of SYNPO in the mouse colon, which was rescued with butyrate repletion. Studies in Synpo-deficient mice demonstrated exacerbated disease susceptibility and increased intestinal permeability in a dextran sulfate sodium colitis model. These findings establish a critical role for the microbiota and their products, specifically butyrate, in the regulated expression of SYNPO for intestinal homeostasis and reveal a direct mechanistic link between microbiota-derived butyrate and barrier restoration.


Assuntos
Butiratos/metabolismo , Microbioma Gastrointestinal/fisiologia , Mucosa Intestinal/metabolismo , Proteínas dos Microfilamentos , Animais , Linhagem Celular , Homeostase/fisiologia , Humanos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Junções Íntimas/metabolismo
9.
PLoS Genet ; 16(5): e1008754, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365093

RESUMO

FSHD is characterized by the misexpression of DUX4 in skeletal muscle. Although DUX4 upregulation is thought to be the pathogenic cause of FSHD, DUX4 is lowly expressed in patient samples, and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we performed bulk RNA-seq on a 6-day differentiation time-course in primary FSHD2 patient myoblasts. We identify a set of 54 genes upregulated in FSHD2 cells, termed FSHD-induced genes. Using single-cell and single-nucleus RNA-seq on myoblasts and differentiated myotubes, respectively, we captured, for the first time, DUX4 expressed at the single-nucleus level in a native state. We identified two populations of FSHD myotube nuclei based on low or high enrichment of DUX4 and FSHD-induced genes ("FSHD-Lo" and "FSHD Hi", respectively). FSHD-Hi myotube nuclei coexpress multiple DUX4 target genes including DUXA, LEUTX and ZSCAN4, and also upregulate cell cycle-related genes with significant enrichment of E2F target genes and p53 signaling activation. We found more FSHD-Hi nuclei than DUX4-positive nuclei, and confirmed with in situ RNA/protein detection that DUX4 transcribed in only one or two nuclei is sufficient for DUX4 protein to activate target genes across multiple nuclei within the same myotube. DUXA (the DUX4 paralog) is more widely expressed than DUX4, and depletion of DUXA suppressed the expression of LEUTX and ZSCAN4 in late, but not early, differentiation. The results suggest that the DUXA can take over the role of DUX4 to maintain target gene expression. These results provide a possible explanation as to why it is easier to detect DUX4 target genes than DUX4 itself in patient cells and raise the possibility of a self-sustaining network of gene dysregulation triggered by the limited DUX4 expression.


Assuntos
Núcleo Celular/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular Facioescapuloumeral , RNA-Seq/métodos , Análise de Célula Única/métodos , Estudos de Casos e Controles , Diferenciação Celular , Núcleo Celular/química , Núcleo Celular/classificação , Núcleo Celular/patologia , Células Cultivadas , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/ultraestrutura , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Distrofia Muscular Facioescapuloumeral/patologia , Mioblastos/metabolismo , Mioblastos/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sequenciamento Completo do Exoma
10.
PLoS One ; 15(5): e0233152, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453755

RESUMO

Obesity is associated with significantly higher mortality rates, and excess adipose tissue is involved in respective pathologies. Here we established a human adipose tissue slice cultures (HATSC) model ex vivo. HATSC match the in vivo cell composition of human adipose tissue with, among others, mature adipocytes, mesenchymal stem cells as well as stroma tissue and immune cells. This is a new method, optimized for live imaging, to study adipose tissue and cell-based mechanisms of obesity in particular. HATSC survival was tested by means of conventional and immunofluorescence histological techniques, functional analyses and live imaging. Surgery-derived tissue was cut with a tissue chopper in 500 µm sections and transferred onto membranes building an air-liquid interface. HATSC were cultured in six-well plates filled with Dulbecco's Modified Eagle's Medium (DMEM), insulin, transferrin, and selenium, both with and without serum. After 0, 1, 7 and 14 days in vitro, slices were fixated and analyzed by morphology and Perilipin A for tissue viability. Immunofluorescent staining against IBA1, CD68 and Ki67 was performed to determine macrophage survival and proliferation. These experiments showed preservation of adipose tissue as well as survival and proliferation of monocytes and stroma tissue for at least 14 days in vitro even in the absence of serum. The physiological capabilities of adipocytes were functionally tested by insulin stimulation and measurement of Phospho-Akt on day 7 and 14 in vitro. Viability was further confirmed by live imaging using Calcein-AM (viable cells) and propidium iodide (apoptosis/necrosis). In conclusion, HATSC have been successfully established by preserving the monovacuolar form of adipocytes and surrounding macrophages and connective tissue. This model allows further analysis of mature human adipose tissue biology ex vivo.


Assuntos
Adipócitos , Tecido Adiposo , Modelos Biológicos , Obesidade , Técnicas de Cultura de Tecidos , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Adolescente , Adulto , Idoso , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Sobrevivência Celular , Feminino , Humanos , Antígeno Ki-67/metabolismo , Masculino , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Obesidade/metabolismo , Obesidade/patologia
11.
PLoS Pathog ; 16(5): e1008503, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365138

RESUMO

Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host's gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through "discreet-invasion". This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context.


Assuntos
Aderência Bacteriana , Mucosa Intestinal/microbiologia , Infecções por Salmonella , Salmonella typhimurium , Sistemas de Secreção Tipo I/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cães , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Infecções por Salmonella/genética , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Infecções por Salmonella/patologia , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Sistemas de Secreção Tipo I/genética
12.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(1): 67-72, 2020 Jan 30.
Artigo em Chinês | MEDLINE | ID: mdl-32376560

RESUMO

OBJECTIVE: The obtain purified recombinant asprosin and test its functions. METHODS: The recombinant plasmid of pET-22b-asprosin was constructed and transformed into competent E.coli BL (DE3) strain. After IPTG-induced expression, asprosin inclusion body was renatured by gradient urea and purified by Ni-NTA affinity chromatography column followed by removal of endotoxin to obtain recombinant asprosin for use in cells and animals experiments. C57 mice were injected intraperitoneally with the recombinant asprosin and blood glucose was detected using a blood glucose meter. Alamar Blue assay was used to evaluate of the effect of the recombinant asprosin on the viability of MIHA cells, and cellular glycogen content was detected using the anthrone method. RESULTS: At the absorbance at 600 nm of 0.8, induction of the recombinant host bacteria with 1 mmol/L IPTG at 37 ℃ for 4 h optimally induced the expression of asprosin inclusion body. After purification and endotoxin removal, the purity of the recombinant asprosin exceeded 95% with the content of endotoxin below 1 EU/mg. In C57 mice, intraperitoneal injection with recombinant asprosin significantly increased blood glucose level, which reached the peak level at 60 min following the injection (P=0.021) and recovered the normal level at 120 min (P=0.03). Treatment with the recombinant asprosin for 24 h did not cause obvious adverse effect on the viability of MIHA cells but significantly lowered glycogen content in the cells (P < 0.05). CONCLUSIONS: We successfully obtained recombinant asprosin using a prokaryotic expression system. The recombinant asprosin can decrease glycogen content in MIHA cells and increase blood glucose level in mice.


Assuntos
Corpos de Inclusão , Proteínas dos Microfilamentos/biossíntese , Fragmentos de Peptídeos/biossíntese , Hormônios Peptídicos/biossíntese , Animais , Glicemia/análise , Linhagem Celular , Escherichia coli , Glicogênio/análise , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos , Proteínas Recombinantes/biossíntese
13.
Nanotoxicology ; 14(6): 774-787, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32401088

RESUMO

Gastric carcinoma is one of the most lethal malignant tumors. As part of our long-term efforts on seeking effective diagnosis and therapeutic strategies of gastric cancer, we present herein novel ternary copper-based chalcogenide nanoplatform CuS-NiS2 nanomaterials with outstanding photothermal (PT)/photodynamic (PD) property that could effectively suppress human gastric cancer in vitro and in vivo without obvious side effects. We revealed that CuS-NiS2 induced reactive oxygen species (ROS) generation, leading to apoptosis through Bcl-2/Bax pathway of human gastric cancer cells under 808 nm near-infrared (NIR) irradiation. In addition, we also confirmed that the combination of CuS-NiS2 and 808 nm NIR laser treatment triggered necroptosis by regulating the novel pathway MLKL/CAPG of human gastric cancer cells. Moreover, the CuS-NiS2 exhibited excellent contrast enhancement according to magnetic resonance imaging (MRI). Taken together, we reported new ternary copper-based chalcogenide nanomaterials CuS-NiS2, which could be successfully applied for MRI-guided PT/PD therapy of gastric carcinoma through mitochondria-mediated apoptosis and MLKL/CAPG-mediated necroptosis.


Assuntos
Apoptose/efeitos dos fármacos , Cobre/uso terapêutico , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/efeitos dos fármacos , Nanoestruturas/uso terapêutico , Necroptose/efeitos dos fármacos , Níquel/uso terapêutico , Proteínas Nucleares/metabolismo , Fototerapia/métodos , Proteínas Quinases/metabolismo , Neoplasias Gástricas/terapia , Animais , Linhagem Celular Tumoral , Cobre/administração & dosagem , Humanos , Imagem por Ressonância Magnética , Masculino , Camundongos Nus , Mitocôndrias/metabolismo , Nanoestruturas/administração & dosagem , Níquel/administração & dosagem , Neoplasias Gástricas/diagnóstico por imagem , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
14.
J Anim Sci ; 98(4)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32249920

RESUMO

Smooth muscle cells (SMCs) play an important role in physiology and production in farm animals such as pigs. Here, we report the generation of a pig SMC line. Our original objective was to establish an enteroendocrine cell line from the pig ileum epithelium through lentiviral transduction of the Simian Virus (SV) 40 large T antigen. However, an initial expression analysis of marker genes in nine cell clones revealed that none of them were enteroendocrine cells or absorptive enterocytes, goblet cells, or Paneth cells, some of the major cell types existing in the ileum epithelium. A more detailed characterization of one clone named PIC7 by RNA-seq showed that these cells expressed many of the known smooth muscle-specific or -enriched genes, including smooth muscle actin alpha 2, calponin 1, calponin 3, myosin heavy chain 11, myosin light chain kinase, smoothelin, tenascin C, transgelin, tropomyosin 1, and tropomyosin 2. Both quantitative PCR and RNA-seq analyses showed that the PIC7 cells had a high expression of mRNA for smooth muscle actin gamma 2, also known as enteric smooth muscle actin. A Western blot analysis confirmed the expression of SV40 T antigen in the PIC7 cells. An immunohistochemical analysis demonstrated the expression of smooth muscle actin alpha 2 filaments in the PIC7 cells. A collagen gel contraction assay showed that the PIC7 cells were capable of both spontaneous contraction and contraction in response to serotonin stimulation. We conclude that the PIC7 cells are derived from an enteric SMC from the pig ileum. These cells may be a useful model for studying the cellular and molecular physiology of pig enteric SMCs. Because pigs are similar to humans in anatomy and physiology, the PIC7 cells may be also used as a model for human intestinal SMCs.


Assuntos
Suínos/fisiologia , Actinas/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Íleo/fisiologia , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso/fisiologia , Miócitos de Músculo Liso/fisiologia , Miosinas/genética , Especificidade de Órgãos , RNA Mensageiro/genética , Suínos/genética , Tenascina/genética , Tropomiosina/genética
15.
Medicine (Baltimore) ; 99(14): e19628, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32243390

RESUMO

BACKGROUND: Prostate cancer (PCa) is one of the leading causes of cancer-related death. In the present research, we adopted a comprehensive bioinformatics method to identify some biomarkers associated with the tumor progression and prognosis of PCa. METHODS: Differentially expressed genes (DEGs) analysis and weighted gene co-expression network analysis (WGCNA) were applied for exploring gene modules correlative with tumor progression and prognosis of PCa. Clinically Significant Modules were distinguished, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to Annotation, Visualization and Integrated Discovery (DAVID). Protein-protein interaction (PPI) networks were used in selecting potential hub genes. RNA-Seq data and clinical materials of prostate cancer from The Cancer Genome Atlas (TCGA) database were used for the identification and validation of hub genes. The significance of these genes was confirmed via survival analysis and immunohistochemistry. RESULTS: 2688 DEGs were filtered. Weighted gene co-expression network was constructed, and DEGs were divided into 6 modules. Two modules were selected as hub modules which were highly associated with the tumor grades. Functional enrichment analysis was performed on genes in hub modules. Thirteen hub genes in these hub modules were identified through PPT networks. Based on TCGA data, 4 of them (CCNB1, TTK, CNN1, and ACTG2) were correlated with prognosis. The protein levels of CCNB1, TTK, and ACTG2 had a degree of differences between tumor tissues and normal tissues. CONCLUSION: Four hub genes were identified as candidate biomarkers and potential therapeutic targets for further studies of exploring molecular mechanisms and individual therapy on PCa.


Assuntos
Biomarcadores Tumorais/análise , Redes Reguladoras de Genes/genética , Neoplasias da Próstata/genética , Actinas/análise , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ciclo Celular/análise , Biologia Computacional/métodos , Ciclina B1/análise , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Humanos , Masculino , Proteínas dos Microfilamentos/análise , Gradação de Tumores , Prognóstico , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/análise , Proteínas Tirosina Quinases/análise
16.
PLoS One ; 15(4): e0231597, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32287325

RESUMO

Resident microglia of the central nervous system are being increasingly recognized as key players in diseases such as neuropathic pain. Biochemical and behavioral studies in neuropathic pain rodent models have documented compelling evidence of the critical role of ATP mediated-P2X4R-brain-derived neurotrophic factor (BDNF) signaling pathway in the initiation and maintenance of pain hypersensitivity, a feature driving neuropathic pain-related behavior. The goal of this study was to develop and characterize an in vitro cell line model of activated microglia that can be subsequently utilized for screening neuropathic pain therapeutics. In the present study, we characterized the SIM-A9 microglia cell line for key molecules in the P2X4R-BDNF signaling axis using a combination of biochemical techniques and developed an ATP-activated SIM-A9 microglia model. We present three novel findings: first, SIM-A9 cells expressed P2X4R and BDNF proteins, second, ATP, but not LPS, was cytocompatible with SIM-A9 cells and third, exposure of cells to optimized ATP concentrations for defined periods increased intracellular expression of Iba1 and BDNF proteins. Increased Iba1 levels confirmed microglia activation and increased BDNF expression confirmed ATP-mediated stimulation of the P2X4R signaling pathway. We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.


Assuntos
Microglia/metabolismo , Neuralgia/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Técnicas de Cultura de Células/métodos , Lipopolissacarídeos/farmacologia , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Neuralgia/patologia , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/metabolismo
17.
Nat Commun ; 11(1): 1797, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286273

RESUMO

Mutations that inactivate negative translation regulators cause autism spectrum disorders (ASD), which predominantly affect males and exhibit social interaction and communication deficits and repetitive behaviors. However, the cells that cause ASD through elevated protein synthesis resulting from these mutations remain unknown. Here we employ conditional overexpression of translation initiation factor eIF4E to increase protein synthesis in specific brain cells. We show that exaggerated translation in microglia, but not neurons or astrocytes, leads to autism-like behaviors in male mice. Although microglial eIF4E overexpression elevates translation in both sexes, it only increases microglial density and size in males, accompanied by microglial shift from homeostatic to a functional state with enhanced phagocytic capacity but reduced motility and synapse engulfment. Consequently, cortical neurons in the mice have higher synapse density, neuroligins, and excitation-to-inhibition ratio compared to control mice. We propose that functional perturbation of male microglia is an important cause for sex-biased ASD.


Assuntos
Transtorno Autístico/metabolismo , Comportamento Animal , Microglia/metabolismo , Biossíntese de Proteínas , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular , Feminino , Perfilação da Expressão Gênica , Genótipo , Homeostase , Masculino , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Neurônios/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fagocitose , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/ultraestrutura , Comportamento Social , Sinapses/metabolismo
18.
Mutat Res ; 852: 503167, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32265040

RESUMO

Chronic kidney disease (CKD) is a multifactorial disorder with an important genetic component, and several studies have demonstrated potential associations with allelic variants. In addition, CKD patients are also characterized by high levels of genomic damage. Nevertheless, no studies have established relationships between DNA damage, or genomic instability present in CKD patients, and gene polymorphisms. To fill in this gap, the potential role of polymorphisms in genes involved in base excision repair (OGG1, rs1052133; MUTYH, rs3219489; XRCC1, rs25487), nucleotide excision repair (ERCC2/XPD, rs1799793, rs171140, rs13181; ERCC4, rs3136166); phase II metabolism (GSTP1, rs749174; GSTO1, rs2164624; GSTO2, rs156697), and antioxidant enzymes (SOD1, rs17880135, rs1041740, rs202446; SOD2, rs4880; CAT, rs1001179; GPX1, rs17080528; GPX3, rs870406: GPX4, rs713041) were inquired. In addition, some genes involved in CKD (AGT, rs5050; GLO1, rs386572987; SHROOM3, rs17319721) were also evaluated. The genomic damage, the genomic instability, and oxidative damage were evaluated by using the micronucleus and the comet assay in 589 donors (415 CKD patients and 174 controls). Our results showed significant associations between genomic damage and genes directly involved in DNA repair pathways (XRCC1, and ERCC2), and with genes encoding for antioxidant enzymes (SOD1 and GPX1). GSTO2, as a gene involved in phase II metabolism, and MUTYH showed also an association with genomic instability. Interestingly, the three genes associated with CKD (AGT, GLO1, and SHROOM3) showed associations with both the high levels of oxidatively damaged DNA and genomic instability. These results support our view that genomic instability can be considered a biomarker of the CKD status.


Assuntos
Angiotensinogênio/genética , Reparo do DNA , Instabilidade Genômica , Lactoilglutationa Liase/genética , Proteínas dos Microfilamentos/genética , Insuficiência Renal Crônica/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Angiotensinogênio/metabolismo , Estudos de Casos e Controles , Ensaio Cometa , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Lactoilglutationa Liase/metabolismo , Masculino , Proteínas dos Microfilamentos/metabolismo , Testes para Micronúcleos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/fisiopatologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo
19.
Gene ; 744: 144635, 2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-32244053

RESUMO

Spermatogenesis is a highly complex physiological process which contains spermatogonia proliferation, spermatocyte meiosis and spermatid morphogenesis. In the past decade, actin binding proteins and signaling pathways which are critical for regulating the actin cytoskeleton in testis had been found. In this review, we summarized 5 actin-binding proteins that have been proven to play important roles in the seminiferous epithelium. Lack of them perturbs spermatids polarity and the transport of spermatids. The loss of Arp2/3 complex, Formin1, Eps8, Palladin and Plastin3 cause sperm release failure suggesting their irreplaceable role in spermatogenesis. Actin regulation relies on multiple signal pathways. The PI3K/Akt signaling pathway positively regulate the mTOR pathway to promote actin reorganization in seminiferous epithelium. Conversely, TSC1/TSC2 complex, the upstream of mTOR, is activated by the LKB1/AMPK pathway to inhibit cell proliferation, differentiation and migration. The increasing researches focus on the function of actin binding proteins (ABPs), however, their collaborative regulation of actin patterns and potential regulatory signaling networks remains unclear. We reviewed ABPs that play important roles in mammalian spermatogenesis and signal pathways involved in the regulation of microfilaments. We suggest that more relevant studies should be performed in the future.


Assuntos
Citoesqueleto de Actina/metabolismo , Espermatogênese , Proteínas Quinases Ativadas por AMP/metabolismo , Actinas/metabolismo , Animais , Humanos , Masculino , Camundongos , Proteínas dos Microfilamentos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais
20.
Clin Implant Dent Relat Res ; 22(3): 424-450, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32319195

RESUMO

BACKGROUND AND OBJECTIVE: Although periimplantitis and periodontitis share similar features, particularly clinical features, they are two different diseases and should be analyzed separately. Thus far, few omics-level differences in periimplantitis and periodontitis have been reported. This study was aimed at exploring the differential effects of expression mRNAs, lncRNAs, and miRNAs in periodontitis and periimplantitis by high-throughput sequencing and competitive endogenous RNA (ceRNA) analysis. METHODS: Gingival tissues of healthy individuals (HI) and periimplantitis (PI) and periodontitis (P) patients were collected and used for genome-wide sequencing. The differentially expressed genes (DEGs) were screened and visualized by R software. The functions and pathways of DEGs were analyzed using Metascape, and the ceRNA network was constructed using the Cytoscape software. Finally, gene set enrichment analysis (GSEA) was used to predict the function of key nodes in ceRNA. RESULTS AND CONCLUSION: By constructing the regulated ceRNA network, six genes (FAM126B, SORL1, PRLR, CPEB2, RAP2C, and YOD1) and 16 miRNAs (hsa-miR-338-5p, hsa-miR-650, hsa-miR-9-5p, hsa-miR-1290, hsa-miR-544a, hsa-miR-3179, hsa-miR-1269a, hsa-miR-3679-5p, hsa-miR-149-5p, hsa-miR-615-3p, hsa-miR-33b-5p, hsa-miR-31-5p, hsa-miR-4639-5p, hsa-miR-204-5p, hsa-miR-5588-5p, and hsa-mir-196a-5p) were detected. Five long non-coding RNAs (lnc-CORO2B-1, lnc-MBL2-7, lnc-TRIM45-1, lnc-CHST10-2, and lnc-TNP1-6) were found to target these miRNAs in this ceRNA network. The ceRNA network based on transcriptome data revealed that FAM126B, SORL1, PRLR, CPEB2, RAP2C, and YOD1 were crucial proteins of differential effects in periodontitis and periimplantitis. The lncRNA-miRNA-mRNA interaction involved the regulation of the Hippo signaling pathway, Wnt signaling pathway, Toll-like receptor signaling pathway, NOD signaling pathway, oxidative stress, and innate immune process. These regulated pathways and biological processes may be factors contributing to the pathogenesis of periimplantitis being distinct from that of periodontitis.


Assuntos
Peri-Implantite , Periodontite , RNA Longo não Codificante , Endopeptidases , Humanos , Proteínas dos Microfilamentos , RNA Mensageiro , Proteínas de Ligação a RNA , Proteínas Repressoras , Tioléster Hidrolases , Transcriptoma , Proteínas ras
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