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1.
Exp Parasitol ; 217: 107956, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32659234

RESUMO

The species name Cryptosporidium bollandi n. sp. is proposed for Cryptosporidium piscine genotype 2 based on morphological, biological and molecular characterisation. Phylogenetic analyses of 18S rRNA (18S) sequences revealed that C. bollandi n. sp. was most closely related to piscine genotype 4 (5.1% genetic distance) and exhibited genetic distances of 10.0%, 12.2% and 25.2% from Cryptosporidium molnari, Cryptosporidium huwi and Cryptosporidium scophthtalmi, respectively. At the actin locus, C. bollandi n. sp. was again most closely related to piscine genotype 4 (6.8% genetic distance) and exhibited 15.5% (C. molnari), 18.4% (C. huwi), 22.9% (C. scophthalmi) and up to 27.5% genetic distance from other Cryptosporidium spp. (Cryptosporidium felis). Phylogenetic analysis of concatenated 18S and actin sequences showed that C. bollandi n. sp. exhibited 12.9% (C. molnari) to 21.1% (C. canis) genetic distance from all other Cryptosporidium spp. Genetic data as well as previous histological analysis clearly supports the validity of C. bollandi n. sp. as a separate species.


Assuntos
Ciclídeos/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium/fisiologia , Doenças dos Peixes/parasitologia , Actinas/química , Actinas/genética , Animais , Sequência de Bases , Evolução Biológica , Criptosporidiose/epidemiologia , Cryptosporidium/classificação , Cryptosporidium/genética , Cryptosporidium/ultraestrutura , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Doenças dos Peixes/epidemiologia , Pesqueiros , Genótipo , Funções Verossimilhança , Microscopia Eletrônica de Transmissão/veterinária , Filogenia , Reação em Cadeia da Polimerase/veterinária , Prevalência , RNA Ribossômico 18S/química , Washington/epidemiologia , Austrália Ocidental/epidemiologia
2.
Prostate ; 80(13): 1087-1096, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32609927

RESUMO

BACKGROUND: Prostate cancer is the second most common cancer worldwide. Tumor microenvironment is composed of activated fibroblasts, the so called carcinoma-associated fibroblasts (CAFs). They express high levels of α-smooth muscle actin (α-SMA) and type I collagen (COL1), and support proliferation and migration of tumor epithelial cells. Extracorporeal shock waves (ESWs), acoustic waves, are effective in the treatment of hypertrophic scars, due to their ability to modulate fibrosis. Based on this rationale, the study evaluated the effects of ESWs on CAF activation and the influence of ESW-treated CAFs on the growth and migration of epithelial prostatic carcinoma cells. METHODS: Primary cultures of CAFs (n = 10) were prepared from tumors of patients undergoing surgery for high-risk prostate carcinoma. CAFs were treated with ESWs (energy levels: 0.32 mJ/mm2 , 1000 pulses; 0.59 mJ/mm2 , 250 pulses). After treatment, the messenger RNA and protein levels of the stromal activation markers α-SMA and COL1 were determined. Subsequently, two different stabilized cell lines (PC3 and DU145) of androgen-resistant prostate cancer were treated with the conditioned media produced by ESW-treated CAFs. At different times, viability and migration of PC3 and DU145 cells were evaluated. Viability was also assessed by coculture system using CAFs and PC3 or DU145 cells. RESULTS: ESWs reduced gene expression and protein level of α-SMA and COL1 in CAFs. The treatment of PC3 and DU145 with conditioned media of ESW-treated CAFs determined a reduction of their growth and invasive potential. Coculture systems between ESW-treated CAFs and PC3 or DU145 cells confirmed the epithelial cell number reduction. CONCLUSIONS: This in vitro study demonstrates for the first time that ESWs are able to modulate the activation of prostate CAFs in favor of a less "reactive" stroma, with consequent slowing of the growth and migration of prostate cancer epithelial cells. However, only further studies to be performed in vivo will confirm the possibility of using this new therapy in patients with prostate cancer.


Assuntos
Tratamento por Ondas de Choque Extracorpóreas/métodos , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/terapia , Células Estromais/patologia , Actinas/genética , Actinas/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Progressão da Doença , Humanos , Masculino , Células PC-3 , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Estromais/metabolismo
3.
Gene ; 757: 144939, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32640306

RESUMO

Osteoarthritis (OA) is a chronic degenerative change with high incidence and leads to a lower quality of life and a larger socioeconomic burden. This study aimed to explore potential crucial genes and pathways associated with OA that can be used as potential biomarkers forearly treatment. Single-cell gene expression profile of 1464 chondrocytes and 192 fibroblasts in OA were downloaded from the public database (GSE104782 and GSE109449) for subsequent analysis. A total of eight clusters in chondrocytes and three clusters in fibroblasts of OA were identified using the Seurat pipeline and the "SingleR" package for cell-type annotation. Moreover, 44 common marker-genes between fibroblastic-like chondrocytes and fibroblasts were identified and the focal adhesions pathway was further identified as a significant potential mechanism of OA via functional enrichment analysis. Further, the reverse transcription quantitative real-time PCR (RT-qPCR) experiments at tissue's and cellular level confirmed that two key marker-genes (COL6A3 and ACTG1) might participate in the progression of OA. Summarily, we inferred that chondrocytes in OA might up-regulate the expression of COL6A3 and ACTG1 to complete fibroblasts transformation through the focal adhesion pathway. These findings are expected to gain a further insight into the development of OA fibrosis process and provide a promising target for treatment for early OA.


Assuntos
Actinas/genética , Colágeno Tipo VI/genética , Osteoartrite/genética , Actinas/metabolismo , Idoso , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo VI/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Osteoartrite/patologia , Mapas de Interação de Proteínas , RNA-Seq , Análise de Célula Única , Transcriptoma , Regulação para Cima
4.
Science ; 368(6496): 1205-1210, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32527825

RESUMO

Cell migration is driven by local membrane protrusion through directed polymerization of F-actin at the front. However, F-actin next to the plasma membrane also tethers the membrane and thus resists outgoing protrusions. Here, we developed a fluorescent reporter to monitor changes in the density of membrane-proximal F-actin (MPA) during membrane protrusion and cell migration. Unlike the total F-actin concentration, which was high in the front of migrating cells, MPA density was low in the front and high in the back. Back-to-front MPA density gradients were controlled by higher cofilin-mediated turnover of F-actin in the front. Furthermore, nascent membrane protrusions selectively extended outward from areas where MPA density was reduced. Thus, locally low MPA density directs local membrane protrusions and stabilizes cell polarization during cell migration.


Assuntos
Actinas/metabolismo , Movimento Celular , Extensões da Superfície Celular , Actinas/química , Actinas/genética , Membrana Celular , Polaridade Celular , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos
5.
Arch Insect Biochem Physiol ; 104(4): e21685, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32350927

RESUMO

The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is one of the most notorious and difficult to control pests of potato and other solanaceous crops in North America. This insect has evolved a remarkable ability to detoxify both plant and synthetic toxins, allowing it to feed on solanaceous plants containing toxic alkaloids and to develop resistance to synthetic chemicals used for its control. RNA interference (RNAi) is a natural mechanism that evolved as an immune response to double-stranded RNA (dsRNA) viruses where dsRNA triggers silencing of target gene expression. RNAi is being developed as a method to control CPB. Here, we evaluated four CPB-specific genes to identify targets for RNAi-mediated control of this insect. Out of the four dsRNAs evaluated in CPB larvae and adults, dsIAP (dsRNA targeting inhibitor of apoptosis, iap gene) performed better than dsActin, dsHSP70, and dsDynamin in inducing larval mortality. However, in adults, the mortality induced by dsActin is significantly higher than the mortality induced by dsIAP, dsHSP70, and dsDynamin. Interestingly, a combination of dsIAP and dsActin performed better than either dsIAP or dsActin alone by inducing feeding inhibition in 24 hr and mortality in 48 hr in larvae. When the dsIAP and dsActin were expressed in the Escherichia coli HT115 strain and applied as a heat-killed bacterial spray on potato plants, it protected the plants from CPB damage. These studies show that the combination of dsIAP and dsActin shows promise as an insecticide to control CPB.


Assuntos
Besouros/genética , Proteínas Inibidoras de Apoptose/genética , Interferência de RNA , Actinas/genética , Animais , Besouros/efeitos dos fármacos , Besouros/crescimento & desenvolvimento , Escherichia coli , Controle de Insetos/métodos , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , RNA de Cadeia Dupla , Solanum tuberosum
6.
Nat Cell Biol ; 22(6): 674-688, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32451441

RESUMO

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Comunicação Celular , Drosophila melanogaster/metabolismo , Dinaminas/metabolismo , Endocitose , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/genética , Sequência de Aminoácidos , Animais , Drosophila melanogaster/genética , Dinaminas/genética , Feminino , Guanosina Trifosfato/metabolismo , Masculino , Mioblastos/citologia , Mioblastos/metabolismo , Ligação Proteica , Homologia de Sequência
7.
Environ Toxicol ; 35(9): 1007-1014, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32441858

RESUMO

Arecoline, a component of betel nuts, is a known carcinogen that causes oral cancers among those who chew betel nuts. Betel nut chewing is also associated with an increased risk of chronic kidney disease (CKD), but the role of arecoline in this association is unclear. This in vitro study investigates the effects of arecoline on cultured human kidney (HK2) cells. We observed that arecoline had no effect on cell viability but increased cell migration in a dose-dependent manner. Western blot analysis showed that arecoline treatment caused a dose-dependent decrease in E-cadherin expression and dose-dependent increases in N-cadherin, vimentin, α-SMA, and collagen expression; reverse transcriptase-polymerase chain reaction analysis revealed dose-dependent increases in α-SMA and collagen mRNA. Arecoline treatment upregulated the expression of phosphorylated extracellular signal-regulated kinase through epithelial mesenchymal transition and renal fibrosis in HK2 cells. These findings demonstrate that arecoline plays a role in inducing the epithelial mesenchymal transition and fibrogenesis in renal tubule cells and suggest that arecoline promotes the progression of CKD.


Assuntos
Areca/toxicidade , Arecolina/toxicidade , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Actinas/genética , Actinas/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Areca/química , Caderinas/genética , Caderinas/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Transição Epitelial-Mesenquimal/genética , Fibrose , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Sistema de Sinalização das MAP Quinases/genética , Fosforilação , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Regulação para Cima
8.
PLoS Pathog ; 16(5): e1008489, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365140

RESUMO

Remodelling of the actin cytoskeleton in response to external stimuli is obligatory for many cellular processes in the amoebic cell. A rapid and local rearrangement of the actin cytoskeleton is required for the development of the cellular protrusions during phagocytosis, trogocytosis, migration, and invasion. Here, we demonstrated that EhC2B, a C2 domain-containing protein, is an actin modulator. EhC2B was first identified as an effector of EhRab21 from E. histolytica. In vitro interaction studies including GST pull-down, fluorescence-based assay and ITC also corroborated with our observation. In the amoebic trophozoites, EhC2B accumulates at the pseudopods and the tips of phagocytic cups. FRAP based studies confirmed the recruitment and dynamics of EhC2B at the phagocytic cup. Moreover, we have shown the role of EhC2B in erythrophagocytosis. It is well known that calcium-dependent signal transduction is essential for the cytoskeletal dynamics during phagocytosis in the amoebic parasite. Using liposome pelleting assay, we demonstrated that EhC2B preferentially binds to the phosphatidylserine in the presence of calcium. The EhC2B mutants defective in calcium or lipid-binding failed to localise beneath the plasma membrane. The cells overexpressing these mutants have also shown a significant reduction in erythrophagocytosis. The role of EhC2B in erythrophagocytosis and pseudopod formation was also validated by siRNA-based gene knockdown approach. Finally, with the help of in vitro nucleation assay using fluorescence spectroscopy and total internal reflection fluorescence microscopy, we have established that EhC2B is an actin nucleator. Collectively, based on the results from the study, we propose that EhC2B acts like a molecular bridge which promotes membrane deformation via its actin nucleation activity during the progression of the phagocytic cup in a calcium-dependent manner.


Assuntos
Actinas/metabolismo , Citofagocitose , Entamoeba histolytica/metabolismo , Eritrócitos , Proteínas de Protozoários/metabolismo , Pseudópodes/metabolismo , Actinas/genética , Domínios C2 , Entamoeba histolytica/genética , Humanos , Proteínas de Protozoários/genética , Pseudópodes/genética
9.
Exp Parasitol ; 215: 107918, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32464220

RESUMO

According to the World Health Organization, lymphatic filariasis (LF), a mosquito-borne neglected tropical disease (NTD), should be eliminated as a public health concern by the end of 2020. To this end, the goals of the Global Programme to Eliminate Lymphatic Filariasis (GPELF) include interrupting transmission through mass drug administration (MDA). After two decades, several countries have implemented MDA and are now ready to confirm whether transmission has been interrupted. The method for detecting the parasites in mosquito vectors known as xenomonitoring is a non-invasive tool for assessing the current transmission status of the filarial nematode Wuchereria bancrofti (which is responsible for 90% of cases) by their vectors. There are several methods available for detection of the worm in mosquito samples, such as dissection or polymerase chain reaction (PCR). However, most of these techniques still produce a considerable number of false-negative results. The present study describes a new duplex PCR protocol, which is an improvement on the traditional PCR methodology, enhanced by introducing the actin gene as an endogenous control gene. After adjusting the mosquito pool size, DNA extraction, and WbCx PCR duplex design, we achieved a reliable and sensitive molecular xenomonitoring protocol. This assay was able to eliminate 5% of false negative samples and detected less than one Wb larvae. This high sensitivity is particularly valuable after MDA, when prevalence declines. This new method could reduce the number of false-negative samples, which will enable us to improve our ability to generate accurate results and aid the monitoring strategies used by LF elimination programmes.


Assuntos
Culex/parasitologia , Filariose Linfática/transmissão , Mosquitos Vetores/parasitologia , Reação em Cadeia da Polimerase Multiplex/métodos , Wuchereria bancrofti/fisiologia , Actinas/genética , Animais , Sequência de Bases , Eletroforese em Gel de Ágar , Filariose Linfática/sangue , Filariose Linfática/tratamento farmacológico , Filariose Linfática/prevenção & controle , Feminino , Humanos , Doenças Negligenciadas/parasitologia , Sensibilidade e Especificidade , Wuchereria bancrofti/genética
10.
PLoS One ; 15(4): e0231905, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315372

RESUMO

Myocardial fibrosis is often associated with cardiac hypertrophy; indeed, fibrosis is one of the most critical factors affecting prognosis. We aimed to identify the molecules involved in promoting fibrosis under hypertrophic stimuli. We previously established a rat model of cardiac hypertrophy by pulmonary artery banding, in which approximately half of the animals developed fibrosis in the right ventricle. Here, we first comprehensively analyzed mRNA expression in the right ventricle with or without fibrosis in pulmonary artery banding model rats by DNA microarray analysis (GSE141650 at NCBI GEO). The expression levels of 19 genes were up-regulated more than 1.5-fold in fibrotic hearts compared with non-fibrotic hearts. Among them, fibrosis growth factor (FGF) 23 showed one of the biggest increases in expression. Real-time PCR analysis also revealed that, among the FGF receptor (FGFR) family, FGFR1 was highly expressed in fibrotic hearts. We then found that FGF23 was expressed predominantly in cardiomyocytes, while FGFR1 was predominantly expressed in fibroblasts in the rat ventricle. Next, we added FGF23 and transforming growth factor (TGF)-ß1 (10-50 ng/mL of each) to isolated fibroblasts from normal adult rat ventricles and cultured them for three days. While FGF23 itself did not directly affect the expression levels of any fibrosis-related mRNAs, FGF23 enhanced the effect of TGF-ß1 on increasing the expression levels of α-smooth muscle actin (α-SMA) mRNA. This increase in xx-SMA mRNA levels due to the combination of TGF-ß1 and FGF23 was attenuated by the inhibition of FGFR1 or the knockdown of FGFR1 in fibroblasts. Thus, FGF23 synergistically promoted the activation of fibroblasts with TGF-ß1, transforming fibroblasts into myofibroblasts via FGFR1. Thus, we identified FGF23 as a paracrine factor secreted from cardiomyocytes to promote cardiac fibrosis under conditions in which TGF-ß1 is activated. FGF23 could be a possible target to prevent fibrosis following myocardial hypertrophy.


Assuntos
Fatores de Crescimento de Fibroblastos/farmacologia , Cardiopatias/patologia , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacos , Actinas/genética , Actinas/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibrose , Cardiopatias/metabolismo , Masculino , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Pirróis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo
12.
PLoS One ; 15(4): e0230818, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315311

RESUMO

The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.


Assuntos
Força Muscular/genética , Músculo Esquelético/fisiologia , Atrofia Muscular/genética , Miostatina/genética , Actinas/genética , Animais , Extremidades/fisiologia , Fêmur/fisiologia , Expressão Gênica/genética , Guanidinoacetato N-Metiltransferase/genética , Imunoglobulina G/genética , Camundongos , Camundongos Endogâmicos BALB C , Força Muscular/fisiologia , Atrofia Muscular/fisiopatologia , Voo Espacial/métodos , Ausência de Peso
13.
Proc Natl Acad Sci U S A ; 117(18): 9922-9931, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32312818

RESUMO

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare -/- mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.


Assuntos
Cílios/genética , Distrofias de Cones e Bastonetes/genética , Proteínas do Olho/genética , Segmento Externo da Célula Bastonete/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/genética , Animais , Cílios/patologia , Distrofias de Cones e Bastonetes/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Segmento Externo da Célula Bastonete/patologia
14.
Proc Natl Acad Sci U S A ; 117(18): 10055-10066, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32312822

RESUMO

Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such "early activation" genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.


Assuntos
Transtorno do Espectro Autista/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Hipocampo/patologia , Actinas/genética , Trifosfato de Adenosina/genética , Animais , Transtorno do Espectro Autista/patologia , Comportamento Animal/fisiologia , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Pareamento Cromossômico/genética , Pareamento Cromossômico/fisiologia , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Dendritos/genética , Dendritos/fisiologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mutação/genética , Neurônios/metabolismo , Neurônios/patologia , Fatores de Transcrição/genética
15.
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
16.
Nat Commun ; 11(1): 1943, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327648

RESUMO

Kidney fibrosis is a highly deleterious process and a final manifestation of chronic kidney disease. Alpha-(α)-synuclein (SNCA) is an actin-binding neuronal protein with various functions within the brain; however, its role in other tissues is unknown. Here, we describe the expression of SNCA in renal epithelial cells and demonstrate its decrease in renal tubules of murine and human fibrotic kidneys, as well as its downregulation in renal proximal tubular epithelial cells (RPTECs) after TGF-ß1 treatment. shRNA-mediated knockdown of SNCA in RPTECs results in de novo expression of vimentin and α-SMA, while SNCA overexpression represses TGF-ß1-induced mesenchymal markers. Conditional gene silencing of SNCA in RPTECs leads to an exacerbated tubulointerstitial fibrosis (TIF) in two unrelated in vivo fibrotic models, which is associated with an increased activation of MAPK-p38 and PI3K-Akt pathways. Our study provides an evidence that disruption of SNCA signaling in RPTECs contributes to the pathogenesis of renal TIF by facilitating partial epithelial-to-mesenchymal transition and extracellular matrix accumulation.


Assuntos
Nefropatias/patologia , Rim/patologia , alfa-Sinucleína/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Rim/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Vimentina/genética , Vimentina/metabolismo , alfa-Sinucleína/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
Anticancer Res ; 40(3): 1239-1245, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32132020

RESUMO

BACKGROUND/AIM: Since the first description of five pericytomas with the t(7;12)/ACTB-GLI1 fusion gene, only three new tumors were studied by both cytogenetics and molecular techniques. We report here genetic data on another case of this rare tumor. MATERIALS AND METHODS: Cytogenetic, fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), and Sanger sequencing analyses were performed. RESULTS: The pericytoma carried two structurally rearranged chromosomes: der(7)t(7;12)(p22;q13) and der(12)t(1;12)(q12;q13). In FISH experiments with a break-apart probe for GLI1, the distal part of the probe hybridized to der(7) whereas the proximal part to der(12). RT-PCR and Sanger sequencing detected an ACTB-GLI1 fragment in which exon 2 of ACTB was fused to exon 6 of GLI1. CONCLUSION: The ACTB-GLI1 fusion gene was mapped at der(7)t(7;12)(p22;q13) and coded for a putative ACTB-GLI1 protein in which the first 41 amino acid (aa) of ACTB replaced the first 177 aa of GLI1.


Assuntos
Actinas/genética , Proteínas de Fusão Oncogênica/genética , Pericitos/patologia , Neoplasias de Tecidos Moles/genética , Proteína GLI1 em Dedos de Zinco/genética , Idoso de 80 Anos ou mais , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 7 , Feminino , Humanos , Neoplasias de Tecidos Moles/patologia , Coxa da Perna/patologia , Translocação Genética
18.
PLoS Pathog ; 16(3): e1008437, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176741

RESUMO

Magnaporthe oryzae causes rice blast disease, but little is known about the dynamic restructuring of the actin cytoskeleton during its polarized tip growth and pathogenesis. Here, we used super-resolution live-cell imaging to investigate the dynamic organization of the actin cytoskeleton in M. oryzae during hyphal tip growth and pathogenesis. We observed a dense actin network at the apical region of the hyphae and actin filaments originating from the Spitzenkörper (Spk, the organizing center for hyphal growth and development) that formed branched actin bundles radiating to the cell membrane. The actin cross-linking protein Fimbrin (MoFim1) helps organize this actin distribution. MoFim1 localizes to the actin at the subapical collar, the actin bundles, and actin at the Spk. Knockout of MoFim1 resulted in impaired Spk maintenance and reduced actin bundle formation, preventing polar growth, vesicle transport, and the expansion of hyphae in plant cells. Finally, transgenic rice (Oryza sativa) expressing RNA hairpins targeting MoFim1 exhibited improved resistance to M. oryzae infection, indicating that MoFim1 represents an excellent candidate for M. oryzae control. These results reveal the dynamics of actin assembly in M. oryzae during hyphal tip development and pathogenesis, and they suggest a mechanism in which MoFim1 organizes such actin networks.


Assuntos
Actinas , Proteínas Fúngicas , Hifas , Magnaporthe , Glicoproteínas de Membrana , Proteínas dos Microfilamentos , Oryza/microbiologia , Doenças das Plantas/microbiologia , Actinas/genética , Actinas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hifas/genética , Hifas/crescimento & desenvolvimento , Magnaporthe/genética , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-32159970

RESUMO

Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40-60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-ß-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/genética , Proteínas Serina-Treonina Quinases/genética , Transativadores/genética , Fatores de Transcrição/genética , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Comunicação Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
20.
J Neurosci ; 40(15): 2993-3007, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32139584

RESUMO

During differentiation, oligodendrocyte precursor cells (OPCs) extend a network of processes that make contact with axons and initiate myelination. Recent studies revealed that actin polymerization is required for initiation of myelination whereas actin depolymerization promotes myelin wrapping. Here, we used primary OPCs in culture isolated from neonatal rat cortices of both sexes and young male and female mice with oligodendrocyte-specific deletion of mechanistic target of rapamycin (mTOR) to demonstrate that mTOR regulates expression of specific cytoskeletal targets and actin reorganization in oligodendrocytes during developmental myelination. Loss or inhibition of mTOR reduced expression of profilin2 and ARPC3, actin polymerizing factors, and elevated levels of active cofilin, which mediates actin depolymerization. The deficits in actin polymerization were revealed in reduced phalloidin and deficits in oligodendrocyte cellular branching complexity at the peak of morphologic differentiation and a delay in initiation of myelination. We further show a critical role for mTOR in expression and localization of myelin basic protein (Mbp) mRNA and MBP protein to the cellular processes where it is necessary at the myelin membrane for axon wrapping. Mbp mRNA transport deficits were confirmed by single molecule RNA FISH. Moreover, expression of the kinesin family member 1B, an Mbp mRNA transport protein, was reduced in CC1+ cells in the mTOR cKO and in mTOR inhibited oligodendrocytes undergoing differentiation in vitro These data support the conclusion that mTOR regulates both initiation of myelination and axon wrapping by targeting cytoskeletal reorganization and MBP localization to oligodendrocyte processes.SIGNIFICANCE STATEMENT Myelination is essential for normal CNS development and adult axon preservation and function. The mechanistic target of rapamycin (mTOR) signaling pathway has been implicated in promoting CNS myelination; however, there is a gap in our understanding of the mechanisms by which mTOR promotes developmental myelination through regulating specific downstream targets. Here, we present evidence that mTOR promotes the initiation of myelination through regulating specific cytoskeletal targets and cellular process expansion by oligodendrocyte precursor cells as well as expression and cellular localization of myelin basic protein.


Assuntos
Citoesqueleto/genética , Bainha de Mielina/genética , Oligodendroglia , Serina-Treonina Quinases TOR/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Axônios , Diferenciação Celular/genética , Cinesina/genética , Cinesina/metabolismo , Camundongos , Camundongos Knockout , Proteína Básica da Mielina/genética , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Oligodendroglia/ultraestrutura , Ratos , Ratos Sprague-Dawley , Células-Tronco , Serina-Treonina Quinases TOR/genética , Peixe-Zebra
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