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1.
Saudi Pharm J ; 25(7): 1022-1031, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29158711

RESUMO

The process of assessment of drug efficacy produces multivariate data which are difficult to interpret. The interpretation and extraction of relevant data requires application of chemometric algorithms for multivariate data analysis. The aim of our study was evaluation of the efficacy of local treatment with chlorhexidine (CHX) in patients suffering from periodontal disease by chemometric algorithms for multivariate data analysis. Several algorithms were used: principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). The PCA models identified the examined variables as suitable for monitoring the periodontal disease progression at the same time revealing mutual relationship among them. The developed PLS-DA model successfully distinguished patients treated with CHX from non-treated patients. The OPLS-DA model revealed differences in the mechanism of action of the two widely applied treatments in periodontal disease, local administration of CHX and local administration of doxycycline (DOX). The approach presented in this study opens the possibility of application of chemometric algorithms for multivariate data analysis for assessment of treatment efficacy.

2.
Biochem Biophys Rep ; 32: 101364, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36237442

RESUMO

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of brain tumors, but the role of PRMT1 in medulloblastoma, the most common malignant pediatric brain tumor, remains unexplored. By examining the publicly available databases of pediatric brain tumor collection, we found that PRMT1 was predominantly expressed in medulloblastomas across all the pediatric brain tumors and that the high-level expression of PRMT1 correlated with poor survival of medulloblastoma patients. To determine the role of PRMT1 in medulloblastoma cells, we established an inducible knockdown system and demonstrated that PRMT1 depletion decreased medulloblastoma cell proliferation and induced cell apoptosis. Furthermore, the diamidine compounds, previously shown to exhibit selective PRMT1 inhibition, suppressed medulloblastoma cell viability in a dose-dependent manner. Finally, we observed induction of medulloblastoma cell apoptosis by the potent diamidine compounds at low micromolar concentrations. Together, our results suggest that PRMT1 could be an actionable therapeutic target in medulloblastoma.

3.
JID Innov ; 2(2): 100076, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35146482

RESUMO

Murine cancer cell lines are powerful research tools to complement studies in genetically engineered mouse models. We have established 21 melanoma cell lines from embryonic stem cell-genetically engineered mouse models driven by alleles that model the most frequent genetic alterations in human melanoma. In addition, these cell lines harbor regulatory alleles for the genomic integration of transgenes and the regulation of expression of such transgenes. In this study, we report a comprehensive characterization of these cell lines. Specifically, we validated melanocytic origin, driver allele recombination and expression, and activation of the oncogenic MAPK and protein kinase B pathways. We further tested tumor formation in syngeneic immunocompetent recipients as well as the functionality of the integrated Tet-ON system and recombination-mediated cassette exchange homing cassette. Finally, by deleting the transcription factor MAFG with an inducible CRISPR/Cas9 approach, we show the utility of the regulatory alleles for candidate gene modulation. These cell lines will be a valuable resource for studying melanoma biology and therapy.

4.
Biochem Biophys Rep ; 25: 100906, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33490652

RESUMO

Notch signaling is activated in the intestinal epithelial cells (IECs) of patients with inflammatory bowel disease (IBD), and contributes to mucosal regeneration. Our previous study indicated that TNF-α and Notch signaling may synergistically promote the expression of the intestinal stem cell (ISC) marker OLFM4 in human IECs. In the present study, we investigated the gene regulation and function of OLFM4 in human IEC lines. We confirmed that TNF-α and Notch synergistically upregulate the mRNA expression of OLFM4. Luciferase reporter assay showed that OLFM4 transcription is regulated by the synergy of TNF-α and Notch. At the protein level, synergy between TNF-α and Notch promoted cytoplasmic accumulation of OLFM4, which has potential anti-apoptotic properties in human IECs. Analysis of patient-derived tissues and organoids consistently showed cytoplasmic accumulation of OLFM4 in response to NF-κB and Notch activation. Cytoplasmic accumulation of OLFM4 in human IECs is tightly regulated by Notch and TNF-α in synergy. Such cytoplasmic accumulation of OLFM4 may have a cell-protective role in the inflamed mucosa of patients with IBD.

5.
Comput Struct Biotechnol J ; 17: 1171-1177, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31462973

RESUMO

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have been employed as a powerful versatile technology for programmable gene editing, transcriptional modulation, epigenetic modulation, and genome labeling, etc. Yet better control of their activity is important to accomplish greater precision and to reduce undesired outcomes such as off-target events. The use of small molecules to control CRISPR/Cas activity represents a promising direction. Here, we provide an updated review on multiple drug inducible CRISPR/Cas systems and discuss their distinct properties. We arbitrarily divided the emerging drug inducible CRISPR/Cas systems into two categories based on whether at transcription or protein level does chemical control occurs. The first category includes Tet-On/Off system and Cre-dependent system. The second category includes chemically induced proximity systems, intein splicing system, 4-Hydroxytamoxifen-Estrogen Receptor based nuclear localization systems, allosterically regulated Cas9 system, and destabilizing domain mediated protein degradation systems. Finally, the advantages and limitations of each system were summarized.

6.
Biochem Biophys Rep ; 14: 178-181, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29872750

RESUMO

Dedicator-of-cytokinesis (DOCK) proteins are a family of guanine-nucleotide exchange factors (GEF) for Rho GTPases. The DOCK-D homology subfamily comprises DOCK9, DOCK10, and DOCK11. DOCK9 and DOCK11 are GEFs for Cdc42 and induce filopodia, while DOCK10 is a dual GEF for Cdc42 and Rac1 and induces filopodia and ruffles. We provide data showing that DOCK9, the only one of the DOCK-D members that is not considered hematopoietic, is nevertheless expressed at high levels in T lymphocytes, as do DOCK10 and DOCK11, although unlike these, it is not expressed in B lymphocytes. To investigate DOCK9 function, we have created a stable HeLa clone with inducible expression of HA-DOCK9. Induction of expression of HA-DOCK9 produced loss of elongation and polygonal shape of HeLa cells. Regarding membrane protrusions, HA-DOCK9 prominently induced filopodia, but also an increase of membrane ruffles. The latter was consistent with an increase in the levels of activation of Rac1, suggesting that DOCK9 carries a secondary ability to induce ruffles through activation of Rac1.

7.
Cell Mol Gastroenterol Hepatol ; 5(3): 333-352, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29552622

RESUMO

BACKGROUND & AIMS: Aberrations in the esophageal proliferation-differentiation gradient are histologic hallmarks in eosinophilic esophagitis (EoE) and gastroesophageal reflux disease. A reliable protocol to grow 3-dimensional (3D) esophageal organoids is needed to study esophageal epithelial homeostasis under physiological and pathologic conditions. METHODS: We modified keratinocyte-serum free medium to grow 3D organoids from endoscopic esophageal biopsies, immortalized human esophageal epithelial cells, and murine esophagi. Morphologic and functional characterization of 3D organoids was performed following genetic and pharmacologic modifications or exposure to EoE-relevant cytokines. The Notch pathway was evaluated by transfection assays and by gene expression analyses in vitro and in biopsies. RESULTS: Both murine and human esophageal 3D organoids displayed an explicit proliferation-differentiation gradient. Notch inhibition accumulated undifferentiated basal keratinocytes with deregulated squamous cell differentiation in organoids. EoE patient-derived 3D organoids displayed normal epithelial structure ex vivo in the absence of the EoE inflammatory milieu. Stimulation of esophageal 3D organoids with EoE-relevant cytokines resulted in a phenocopy of Notch inhibition in organoid 3D structures with recapitulation of reactive epithelial changes in EoE biopsies, where Notch3 expression was significantly decreased in EoE compared with control subjects. CONCLUSIONS: Esophageal 3D organoids serve as a novel platform to investigate regulatory mechanisms in squamous epithelial homeostasis in the context of EoE and other diseases. Notch-mediated squamous cell differentiation is suppressed by cytokines known to be involved in EoE, suggesting that this may contribute to epithelial phenotypes associated with disease. Genetic and pharmacologic manipulations establish proof of concept for the utility of organoids for future studies and personalized medicine in EoE and other esophageal diseases.

8.
Cell Mol Gastroenterol Hepatol ; 3(3): 484-499, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28462385

RESUMO

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) occurs more frequently and aggressively in men than in women. Although sex hormones are believed to play a critical role in this disparity, the possible contribution of other factors largely is unknown. We aimed to investigate the role of serotonin on its contribution of sex discrepancy during HCC. METHODS: By using an inducible zebrafish HCC model through hepatocyte-specific transgenic krasV12 expression, differential rates of HCC in male and female fish were characterized by both pharmaceutical and genetic interventions. The findings were validated further in human liver disease samples. RESULTS: Accelerated HCC progression was observed in krasV12-expressing male zebrafish and male fish liver tumors were found to have higher hepatic stellate cell (HSC) density and activation. Serotonin, which is essential for HSC survival and activation, similarly were found to be synthesized and accumulated more robustly in males than in females. Serotonin-activated HSCs could promote HCC carcinogenesis and concurrently increase serotonin synthesis via transforming growth factor (Tgf)b1 expression, hence contributing to sex disparity in HCC. Analysis of liver disease patient samples showed similar male predominant serotonin accumulation and Tgfb1 expression. CONCLUSIONS: In both zebrafish HCC models and human liver disease samples, a predominant serotonin synthesis and accumulation in males resulted in higher HSC density and activation as well as Tgfb1 expression, thus accelerating HCC carcinogenesis in males.

9.
Data Brief ; 7: 1004-9, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27408912

RESUMO

This paper describes data related to a research article entitled "Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects" [1]. This paper includes data of the quantitative analysis of individual forms of respiratory chain complexes I, III and IV present in SURF1 knockout (SURF1 (-/-) ) and control (SURF1 (+/+) ) mouse fibroblasts and tissues and in fibroblasts of human control and patients with SURF1 gene mutation. Also it includes data demonstrating response of complex IV, cytochrome c oxidase (COX), to reversible inhibition of mitochondrial translation in SURF1 (-/-) mouse and SURF1 patient fibroblast cell lines.

10.
Regen Ther ; 3: 1-6, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31245465

RESUMO

T-box transcription factors play important roles in vertebrate mesoderm formation. Eomesodermin is involved in the initial step of the prospective mesodermal cells recruited near the primitive streak. Then T or Brachyury gene is responsible for general and axial mesodermal development. Tbx6, on the other hand, promotes paraxial mesodermal development while suppressing neural differentiation. Here, we studied differentiative properties of mouse ES cells (mESCs) with its Tbx6 expression regulated under the Tet-off system. mESCs were treated with noggin to promote neural differentiation. When Tbx6 was simultaneously turned on, later neural differentiation of these cells hardly occurred. Next, mESCs were subjected to formation of the embryoid bodies (EBs). When Tbx6 was turned on during EB formation, the rate of later cardiac troponin T (cTnT)-positive cells increased. If the cells were further treated with a wnt inhibitor KY02111 after EB formation, a synergistic increase of cTnT-positive cells occurred. Tbx6 expression in mESCs influenced the constituent ratio of the cardiac myosin light chain types, such that atrial species markedly increased over ventricular ones. These results are coincident with the function of Tbx6 in normal development, in that Tbx6 strongly suppressed neural differentiation while promoting cardiac development in a cooperative manner with wnt inhibition.

11.
Cell Cycle ; 14(8): 1188-96, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25695848

RESUMO

Direct lineage conversion is a promising approach for disease modeling and regenerative medicine. Cell divisions play a key role in reprogramming of somatic cells to pluripotency, however their role in direct lineage conversion is not clear. Here we used transdifferentiation of fibroblasts into neuronal cells by forced expression of defined transcription factors as a model system to study the role of cellular division in the direct conversion process. We have shown that conversion occurs in the presence of the cell cycle inhibitors aphidicolin or mimosine. Moreover, overexpression of the cell cycle activator cMyc negatively influences the process of direct conversion. Overall, our results suggest that cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells.


Assuntos
Divisão Celular , Fibroblastos/citologia , Neurônios/citologia , Animais , Afidicolina/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Transdiferenciação Celular/efeitos dos fármacos , Reprogramação Celular , Doxorrubicina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Mimosina/farmacologia , Neurônios/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Cell Cycle ; 14(5): 744-54, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25565400

RESUMO

Interstrand crosslinks induce DNA replication fork stalling that in turn activates the ATR-dependent checkpoint and DNA repair on nuclear chromatin. Mitomycin C (MC) and Decarbamoyl Mitomycin C (DMC) induce different types of DNA crosslinks with DMC being a more cytotoxic agent. We previously reported that the novel DMC induced ß-interstrand DNA crosslinks induce a p53-independent form of cell death. The p53-independent DMC cytotoxicity associates with the activation, and subsequent depletion, of Chk1. In this study we further dissect the novel DMC signal transduction pathway and asked how it influences chromatin-associated proteins. We found that treatment with DMC, but not MC, stimulated the disassociation of ATR from chromatin and re-localization of ATR to the cytoplasm. The chromatin eviction of ATR was coupled with the formation of nuclear Rad51 foci and the phosphorylation of Chk1. Furthermore, DMC but not MC, activated expression of gadd45α mRNA. Importantly, knocking down p53 via shRNA did not inhibit the DMC-induced disassociation of ATR from chromatin or reduce the activation of transcription of gadd45α. Our results suggest that DMC induces a p53-independent disassociation of ATR from chromatin that facilitates Chk1 checkpoint activation and Rad51 chromatin recruitment. Our findings provide evidence that ATR chromatin eviction in breast cancer cells is an area of study that should be focused on for inducing p53-independent cell death.


Assuntos
Cromatina/metabolismo , Mitomicinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , DNA/metabolismo , Dano ao DNA , Recombinação Homóloga/efeitos dos fármacos , Humanos , Células MCF-7 , Modelos Biológicos , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Quinases/metabolismo , Rad51 Recombinase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Autophagy ; 11(4): 643-52, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25919711

RESUMO

Tafazzin (TAZ) is a phospholipid transacylase that catalyzes the remodeling of cardiolipin, a mitochondrial phospholipid required for oxidative phosphorylation. Mutations of TAZ cause Barth syndrome, which is characterized by mitochondrial dysfunction and dilated cardiomyopathy, leading to premature death. However, the molecular mechanisms underlying the cause of mitochondrial dysfunction in Barth syndrome remain poorly understood. Here we investigated the role of TAZ in regulating mitochondrial function and mitophagy. Using primary mouse embryonic fibroblasts (MEFs) with doxycycline-inducible knockdown of Taz, we showed that TAZ deficiency in MEFs caused defective mitophagosome biogenesis, but not other autophagic processes. Consistent with a key role of mitophagy in mitochondria quality control, TAZ deficiency in MEFs also led to impaired oxidative phosphorylation and severe oxidative stress. Together, these findings provide key insights on mitochondrial dysfunction in Barth syndrome, suggesting that pharmacological restoration of mitophagy may provide a novel treatment for this lethal condition.


Assuntos
Autofagia/fisiologia , Cardiolipinas/metabolismo , Mitofagia/fisiologia , Fatores de Transcrição/metabolismo , Aciltransferases , Animais , Autofagia/genética , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Humanos , Camundongos , Mitocôndrias/genética , Mutação/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
14.
Cell Cycle ; 13(16): 2600-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25486200

RESUMO

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting carriers of premutation forms of the FMR1 gene, resulting in a progressive development of tremor, ataxia and neuropsychological problems. The disease is caused by an expanded CGG repeat in the FMR1 gene, leading to an RNA gain-of-function toxicity mechanism. In order to study the pathogenesis of FXTAS, new inducible transgenic mouse models have been developed that expresses either 11CGGs or 90CGGs at the RNA level under control of a Tet-On promoter. When bred to an hnRNP-rtTA driver line, doxycycline (dox) induced expression of the transgene could be found in almost all tissues. Dox exposure resulted in loss of weight and death within 5 d for the 90CGG RNA expressing mice. Immunohistochemical examination of tissues of these mice revealed steatosis and apoptosis in the liver. Decreased expression of GPX1 and increased expression of cytochrome C is found. These effects were not seen in mice expressing a normal sized 11CGG repeat. In conclusion, we were able to show in vivo that expression of an expanded CGG-repeat rather than overexpression of a normal CGG-repeat causes pathology. In addition, we have shown that expanded CGG RNA expression can cause mitochondrial dysfunction by regulating expression levels of several markers. Although FTXAS patients do not display liver abnormalities, our findings contribute to understanding of the molecular mechanisms underlying toxicity of CGG repeat RNA expression in an animal model. In addition, the dox inducible mouse lines offer new opportunities to study therapeutic interventions for FXTAS.


Assuntos
Ataxia/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Mitocôndrias/metabolismo , RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Tremor/metabolismo , Animais , Antibacterianos/metabolismo , Apoptose/efeitos dos fármacos , Ataxia/genética , Modelos Animais de Doenças , Doxiciclina/metabolismo , Fígado Gorduroso/patologia , Síndrome do Cromossomo X Frágil/genética , Fígado/metabolismo , Fígado/ultraestrutura , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Regiões Promotoras Genéticas , RNA/genética , Espécies Reativas de Oxigênio , Tremor/genética
15.
Mol Metab ; 3(4): 474-83, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24944907

RESUMO

We recently reported that local overexpression of VEGF-A in white adipose tissue (WAT) protects against diet-induced obesity and metabolic dysfunction. The observation that VEGF-A induces a "brown adipose tissue (BAT)-like" phenotype in WAT prompted us to further explore the direct function of VEGF-A in BAT. We utilized a doxycycline (Dox)-inducible, brown adipocyte-specific VEGF-A transgenic overexpression model to assess direct effects of VEGF-A in BAT in vivo. We observed that BAT-specific VEGF-A expression increases vascularization and up-regulates expression of both UCP1 and PGC-1α in BAT. As a result, the transgenic mice show increased thermogenesis during chronic cold exposure. In diet-induced obese mice, introducing VEGF-A locally in BAT rescues capillary rarefaction, ameliorates brown adipocyte dysfunction, and improves deleterious effects on glucose and lipid metabolism caused by a high-fat diet challenge. These results demonstrate a direct positive role of VEGF-A in the activation and expansion of BAT.

16.
Cell Cycle ; 13(16): 2554-63, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25486196

RESUMO

Defects in the completion of cell division by cytokinesis have long been proposed to foster carcinogenesis by engendering chromosome instability, but few tumor suppressor mechanisms controlling this process have so far been identified. Here, we identify a carboxyl (C)-terminal region of the high-mobility group protein HMG20b that is essential for cytokinesis, and report that it is inactivated by a cancer-associated mutation. We find that a C-terminal region of HMG20b spanning residues 173-317 is necessary and sufficient not only for its localization to cytokinetic structures, but also for its interaction with the tumor suppressor BRCA2, implicated in the abscission step of cytokinesis. Indeed, expression of this C-terminal HMG20b region suffices to restore cytokinesis in HMG20b-depleted cells. The non-conservative substitution of HMG20b residue Ala247 with Pro, reported in human lung cancer, disrupts these activities of HMG20b, impairing cytokinesis in a trans-dominant manner. Our findings provide fresh insight into the mechanism by which the HMG20b-BRCA2 complex controls mitotic cell division, and implicate heterozygous HMG20b mutations affecting cytokinesis regulation in the genesis of human cancers.


Assuntos
Proteína BRCA2/metabolismo , Citocinese/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Mutação , Alanina , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Prolina
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