Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 50
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Forensic Sci ; 65(2): 406-420, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31670846

RESUMO

Recent reports have demonstrated that genetically variant peptides derived from human hair shaft proteins can be used to differentiate individuals of different biogeographic origins. We report a method involving direct extraction of hair shaft proteins more sensitive than previously published methods regarding GVP detection. It involves one step for protein extraction and was found to provide reproducible results. A detailed proteomic analysis of this data is presented that led to the following four results: (i) A peptide spectral library was created and made available for download. It contains all identified peptides from this work, including GVPs that, when appropriately expanded with diverse hair-derived peptides, can provide a routine, reliable, and sensitive means of analyzing hair digests; (ii) an analysis of artifact peptides arising from side reactions is also made using a new method for finding unexpected modifications; (iii) detailed analysis of the gel-based method employed clearly shows the high degree of cross-linking or protein association involved in hair digestion, with major GVPs eluting over a wide range of high molecular weights while others apparently arise from distinct non-cross-linked proteins; and (v) finally, we show that some of the specific GVP identifications depend on the sample preparation method.


Assuntos
Cabelo/metabolismo , Queratinas Específicas do Cabelo/metabolismo , Peptídeos/metabolismo , Proteoma/metabolismo , Artefatos , Cromatografia Líquida , Bases de Dados de Proteínas , Medicina Legal , Humanos , Masculino , Espectrometria de Massas , Proteômica , Reprodutibilidade dos Testes
2.
Nutrients ; 11(12)2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31847069

RESUMO

Hair disorders may considerably impact the social and psychological well-being of an individual. Recent advances in the understanding the biology of hair have encouraged the research and development of novel and safer natural hair growth agents. In this context, we have previously demonstrated-at both preclinical and clinical level-that an Annurca apple-based dietary supplement (AMS), acting as a nutraceutical, is endowed with an intense hair-inductive activity (trichogenicity), at once increasing hair tropism and keratin content. Herein, in the framework of preclinical investigations, new experiments in primary human models of follicular keratinocytes and dermal papilla cells have been performed to give an insight around AMS biological effects on specific hair keratins expression. As well as confirming the biocompatibility and the antioxidant proprieties of our nutraceutical formulation, we have proven an engagement of trichokeratins production underlying its biological effects on human follicular cells. Annurca apples are particularly rich in oligomeric procyanidins, natural polyphenols belonging to the broader class of bioflavonoids believed to exert many beneficial health effects. To our knowledge, none of the current available remedies for hair loss has hitherto shown to stimulate the production of hair keratins so clearly.


Assuntos
Folículo Piloso , Queratinas Específicas do Cabelo , Malus , Extratos Vegetais/farmacologia , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Suplementos Nutricionais , Flavonoides , Folículo Piloso/citologia , Folículo Piloso/efeitos dos fármacos , Folículo Piloso/metabolismo , Humanos , Queratinas Específicas do Cabelo/análise , Queratinas Específicas do Cabelo/metabolismo , Modelos Biológicos
3.
PLoS One ; 14(10): e0218642, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31577805

RESUMO

PURPOSE: Development of a supervised machine-learning model capable of predicting clinically relevant molecular subtypes of pancreatic ductal adenocarcinoma (PDAC) from diffusion-weighted-imaging-derived radiomic features. METHODS: The retrospective observational study assessed 55 surgical PDAC patients. Molecular subtypes were defined by immunohistochemical staining of KRT81. Tumors were manually segmented and 1606 radiomic features were extracted with PyRadiomics. A gradient-boosted-tree algorithm was trained on 70% of the patients (N = 28) and tested on 30% (N = 17) to predict KRT81+ vs. KRT81- tumor subtypes. A gradient-boosted survival regression model was fit to the disease-free and overall survival data. Chemotherapy response and survival were assessed stratified by subtype and radiomic signature. Radiomic feature importance was ranked. RESULTS: The mean±STDEV sensitivity, specificity and ROC-AUC were 0.90±0.07, 0.92±0.11, and 0.93±0.07, respectively. The mean±STDEV concordance indices between the disease-free and overall survival predicted by the model based on the radiomic parameters and actual patient survival were 0.76±0.05 and 0.71±0.06, respectively. Patients with a KRT81+ subtype experienced significantly diminished median overall survival compared to KRT81- patients (7.0 vs. 22.6 months, HR 4.03, log-rank-test P = <0.001) and a significantly improved response to gemcitabine-based chemotherapy over FOLFIRINOX (10.14 vs. 3.8 months median overall survival, HR 2.33, P = 0.037) compared to KRT81- patients, who responded significantly better to FOLFIRINOX over gemcitabine-based treatment (30.8 vs. 13.4 months median overall survival, HR 2.41, P = 0.027). Entropy was ranked as the most important radiomic feature. CONCLUSIONS: The machine-learning based analysis of radiomic features enables the prediction of subtypes of PDAC, which are highly relevant for disease-free and overall patient survival and response to chemotherapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma Ductal Pancreático , Desoxicitidina/análogos & derivados , Queratinas Específicas do Cabelo/metabolismo , Queratinas Tipo II/metabolismo , Aprendizado de Máquina , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas , Adulto , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/terapia , Desoxicitidina/administração & dosagem , Intervalo Livre de Doença , Feminino , Fluoruracila/administração & dosagem , Humanos , Irinotecano/administração & dosagem , Leucovorina/administração & dosagem , Masculino , Pessoa de Meia-Idade , Oxaliplatina/administração & dosagem , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Estudos Retrospectivos , Sensibilidade e Especificidade , Taxa de Sobrevida
4.
Clin Cancer Res ; 24(2): 351-359, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29101303

RESUMO

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is associated with a dismal prognosis and poor therapeutic response to current chemotherapy regimens in unselected patient populations. Recently, it has been shown that PDAC may be stratified into functionally and therapeutically relevant molecular subgroups and that some of these subtypes can be recapitulated by IHC for KRT81 [quasi-mesenchymal (QM)/squamous/basal-like] and HNF1A (non-QM, overlap with exocrine/ADEX subtype).Experimental Design: We validated the different outcome of the HNF1A/KRT81 PDAC subtypes in two independent cohorts of surgically treated patients and examined the treatment response to chemotherapy in a third cohort of unresectable patients. The first two cohorts included 262 and 130 patients, respectively, and the third independent cohort comprised advanced-stage PDAC patients who were treated with either FOLFIRINOX (64 patients) or gemcitabine (61 patients).Results: In both cohorts with resected PDAC, the HNF1A-positive subtype showed the best, the KRT81-positive subtype the worst, and the double-negative subtype an intermediate survival (P < 0.013 and P < 0.009, respectively). In the chemotherapy cohort, the survival difference between the double-negative and the HNF1A-positive subtype was lost, whereas the dismal prognosis of KRT81-positive PDAC patients was retained (P < 0.021). Patients with a KRT81-positive subtype did not benefit from FOLFIRINOX therapy, whereas those with HNF1A-positive tumors responded better compared with gemcitabine-based treatment (P < 0.038).Conclusions: IHC stratification recapitulating molecular subtypes of PDAC using HNF1A and KRT81 is associated with significantly different outcomes and responses to chemotherapy. These results may pave the way toward future pretherapeutic biomarker-based stratification of PDAC patients. Clin Cancer Res; 24(2); 351-9. ©2017 AACR.


Assuntos
Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Queratinas Específicas do Cabelo/metabolismo , Queratinas Tipo II/metabolismo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/terapia , Estudos de Coortes , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Estadiamento de Neoplasias , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/terapia , Prognóstico , Resultado do Tratamento
6.
Biotechniques ; 63(3): 131-134, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28911317

RESUMO

Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). While Tet- BMP4-KRT31-Luc iPS cells could be maintained as stable iPS cells, the cells differentiated to produce luciferase luminescence in the presence of all-trans retinoic acid (RA) and doxycycline (Dox), and addition of a hair differentiation factor significantly increased luciferase fluorescence. Thus, this cell line may provide a reliable cell-based screening system to evaluate drug candidates for hair differentiation activity.


Assuntos
Alopecia/terapia , Diferenciação Celular , Engenharia Celular/métodos , Cabelo/citologia , Cabelo/crescimento & desenvolvimento , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Linhagem Celular , Doxiciclina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Células-Tronco Pluripotentes Induzidas/metabolismo , Queratinas Específicas do Cabelo/genética , Queratinas Específicas do Cabelo/metabolismo , Queratinas Tipo I/genética , Queratinas Tipo I/metabolismo , Luciferases/metabolismo , Substâncias Luminescentes/metabolismo , Camundongos , Regiões Promotoras Genéticas , Tetraciclina/farmacologia , Tretinoína/farmacologia
7.
DNA Cell Biol ; 36(7): 552-564, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28509589

RESUMO

As an important commercial trait for sheep, curly fleece has a great economic impact on production costs and efficiency in sheep industry. To identify genes that are important for curly fleece formation in mammals, a suppression subtractive hybridization analysis was performed on the shoulder skin tissues exposed to two different growth stages of Chinese Tan sheep with different phenotypes (curly fleece and noncurling fleece). BLAST analysis identified 67 differentially expressed genes, of which 31 were expressed lower and 36 were expressed higher in lambs than in adult sheep. Differential expressions of seven randomly selected genes were verified using quantitative real-time polymerase chain reaction (qRT-PCR). KRT71 gene was selected for further study due to its high correlation with the curly hair phenotype in various mammal species. Semi-qPCR showed distinctively high expression of KRT71 in skin tissues. Moreover, qPCR result showed a significantly higher expression of KRT71 in curly fleece than noncurling Tan sheep. The luciferase assay and electrophoresis mobility shift assay showed that there were transcription factor binding sites in the promoter region of KRT71 related to the differential expression of KRT71 at the two growth stages of Tan sheep. Online bioinformation tools predicted MFZ1 as a transcriptional factor that regulates the expression of KRT71. These studies on KRT71 gene revealed some mechanisms underlying the relationship between the KRT71 gene and the curly fleece phenotype of Tan sheep.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Cabelo/metabolismo , Queratinas Específicas do Cabelo/genética , Característica Quantitativa Herdável , Fatores de Transcrição/genética , Fatores Etários , Animais , Sítios de Ligação , Perfilação da Expressão Gênica , Biblioteca Gênica , Ontologia Genética , Genes Reporter , Cabelo/anatomia & histologia , Cabelo/crescimento & desenvolvimento , Humanos , Queratinas Específicas do Cabelo/metabolismo , Luciferases/genética , Luciferases/metabolismo , Anotação de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Carneiro Doméstico , Técnicas de Hibridização Subtrativa , Fatores de Transcrição/metabolismo
8.
Proteomics ; 17(11)2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28445005

RESUMO

Rhizochalinin (Rhiz) is a novel marine natural sphingolipid-like compound, which shows promising in vitro and in vivo activity in human castration-resistant prostate cancer. In the present study, a global proteome screening approach was applied to investigate molecular targets and biological processes affected by Rhiz in castration-resistant prostate cancer. Bioinformatical analysis of the data predicted an antimigratory effect of Rhiz on cancer cells. Validation of proteins involved in the cancer-associated processes, including cell migration and invasion, revealed downregulation of specific isoforms of stathmin and LASP1, as well as upregulation of Grp75, keratin 81, and precursor IL-1ß by Rhiz. Functional analyses confirmed an antimigratory effect of Rhiz in PC-3 cells. Additionally, predicted ERK1/2 activation was confirmed by Western blotting analysis, and revealed prosurvival effects in Rhiz-treated prostate cancer cells indicating a potential mechanism of resistance. A combination of Rhiz with MEK/ERK inhibitors PD98059 (non-ATP competitive MEK1 inhibitor) and FR180204 (ATP-competitive ERK1/2 inhibitor) resulted in synergistic effects. This work provides further insights into the molecular mechanisms underlying Rhiz bioactivity. Furthermore, our research is exemplary for the ability of proteomics to predict drug targets and mode of action of natural anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Álcoois Graxos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Proteoma/análise , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Interleucina-1beta/metabolismo , Queratinas Específicas do Cabelo/metabolismo , Queratinas Tipo II/metabolismo , Proteínas com Domínio LIM/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Proteínas de Membrana/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Proteômica/métodos , Rhizoctonia/química , Estatmina/metabolismo
9.
Oncol Rep ; 37(5): 2964-2970, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28405679

RESUMO

Keratins are fibrous proteins. Hair keratins constitute hard structures such as the hair and nails, and cytokeratins have been used as markers of breast carcinoma. However, the expression and function of full-size hair keratin genes have not been previously demonstrated in breast cancer. We investigated the expression of the hair keratin, KRT81, and its function in human breast cancer and normal mammary epithelial cells. Western blotting showed full size 55-kDa KRT81 expression in the human breast cancer cell lines, MCF7, SKBR3 and MDA-MB-231, normal human mammary epithelial cells (HMEC), and non-neoplastic cells (MCF10A). Reverse transcription-polymerase chain reaction revealed that the full size KRT81, including its 5' region is expressed in breast cells. Immunohistochemical and immunofluorescence analyses showed that KRT81 was located in the cytoplasm. To investigate the function of KRT81, we knocked down KRT81 by siRNA in MCF10A cells. Microarray analysis revealed that the expression of genes related to invasion such as matrix metallopeptidase (MMP)9 was decreased. In KRT81-knockdown MDA-MB231 cells, zymography revealed a decrease in MMP9 activity, while scratch and invasion assays revealed that KRT81-knockdown decreased cell migration and invasion abilities. This is the first study showing that full size KRT81 is expressed in normal breast epithelial cells and breast cancer cells. Moreover, our results indicate that KRT81 contributes to the migration and invasion of breast cancer cells.


Assuntos
Neoplasias da Mama/metabolismo , Queratinas Específicas do Cabelo/genética , Queratinas Específicas do Cabelo/metabolismo , Queratinas Tipo II/genética , Queratinas Tipo II/metabolismo , Glândulas Mamárias Humanas/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Citoplasma/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos/métodos
10.
Int J Biol Macromol ; 101: 805-814, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28315768

RESUMO

We selected 1235 decapeptides from human hair proteins encoded by human genes of keratins and keratin associated proteins. The peptides were linked to glass arrays and screened for their affinity towards a solution of human hair extracted keratin fraction. Based on the physicochemical properties of the peptides, ten variables were studied: content of different types of amino acid side chains (cysteine, hydrophobic, polar, basic, acidic, aromatic rings, amide, alcohol side chains), isoelectric point, and net charge. We found differences statistically significant on the binding affinity of peptides based on their content of cysteine, hydrophobic and polar amino acids, mainly containing alcohols. These results point to the formation of hydrophobic interactions and disulfide bonds between small peptides and human hair keratins as the main driving forces for the interaction of possible cosmetic peptides, namely designed to strength human hair. As so, our results enlighten the nature of the interaction of keratin based materials with human hair, which are claimed to enhance hair fiber strength, and enable a more directed and sustained hair care peptide design.


Assuntos
Queratinas Específicas do Cabelo/metabolismo , Fragmentos de Peptídeos/metabolismo , Humanos , Queratinas Específicas do Cabelo/química , Análise Serial de Proteínas , Ligação Proteica
11.
Anat Sci Int ; 92(2): 248-261, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26880085

RESUMO

Hardening of the human hair shaft during cornification results from the bonding of keratins and keratin-associated proteins. In situ hybridization and light immunocytochemical studies have shown the general distribution of different keratins and some associated proteins but not determined their ultrastructural localization. I report here the localization of hair keratins, two high-sulfur keratin-associated proteins and sulfhydryl oxidase has been studied under the transmission electron microscope in the cornification zone of the human hair. The ultrastructural study on keratin distribution in general confirms previous light microscopic studies. Sulfur-rich KAP1 is mainly cortical but the labeling disappears in fully cornified cortical cells while a diffuse labeling is also present in differentiating cuticle cells. Sulfur-rich K26 immunolocalization is only detected in the exocuticle and endocuticle. Sparse labeling for sulfhydryl oxidase occurs in differentiating cortical cells but is weak and uneven in cuticle cells and absent in medulla and inner root sheath. Labeling disappears in the upper fully cornified cortex and cuticle. The observations indicate that sulfhydryl oxidase and keratin associated proteins are initially produced in the cytoplasm among keratin bundles accumulating in cortical and cuticle cells but these proteins undergo changes during the following cornification that alter the epitopes tagged by the antibodies.


Assuntos
Folículo Piloso/ultraestrutura , Cabelo/ultraestrutura , Queratinas Específicas do Cabelo/ultraestrutura , Oxirredutases/ultraestrutura , Diferenciação Celular , Cabelo/metabolismo , Folículo Piloso/metabolismo , Humanos , Queratinas Específicas do Cabelo/metabolismo , Oxirredutases/metabolismo
12.
PLoS One ; 11(4): e0153936, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27100288

RESUMO

The keratin-associated proteins (KAPs) are the structural proteins of hair fibers and are thought to play an important role in determining the physical properties of hair fibers. These proteins are activated in a striking sequential and spatial pattern in the keratinocytes of hair fibers. Thus, it is important to elucidate the mechanism that underlies the specific transcriptional activity of these genes. In this study, sheep KRTAP 3-3 and KRTAP11-1 genes were found to be highly expressed in wool follicles in a tissue-specific manner. Subsequently, the promoter regions of the two genes that contained the 5' flanking/5' untranslated regions and the coding regions were cloned. Using an in vivo transgenic approach, we found that the promoter regions from the two genes exhibited transcriptional activity in hair fibers. A much stronger and more uniformly expressed green fluorescent signal was observed in the KRTAP11-1-ZsGreen1 transgenic mice. In situ hybridization revealed the symmetrical expression of sheep KRTAP11-1 in the entire wool cortex. Consistently, immunohistochemical analysis demonstrated that the pattern of ZsGreen1 expression in the hair cortex of transgenic mice matches that of the endogenous KRTAP11-1 gene, indicating that the cloned promoter region contains elements that are sufficient to govern the wool cortex-specific transcription of KRTAP11-1. Furthermore, regulatory regions in the 5' upstream sequence of the sheep KRTAP11-1 gene that may regulate the observed hair keratinocyte specificity were identified using in vivo reporter assays.


Assuntos
Biomarcadores/metabolismo , Regulação da Expressão Gênica , Cabelo/metabolismo , Queratinas Específicas do Cabelo/genética , Regiões Promotoras Genéticas/genética , Animais , Clonagem Molecular , Feminino , Perfilação da Expressão Gênica , Técnicas Imunoenzimáticas , Hibridização In Situ , Queratinas Específicas do Cabelo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Filogenia , Ovinos
13.
Nat Med ; 22(3): 278-87, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26855150

RESUMO

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers--HNF1A and KRT81--that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/genética , Citocromo P-450 CYP3A/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Queratinas Específicas do Cabelo/metabolismo , Queratinas Tipo II/metabolismo , Neoplasias Pancreáticas/genética , Idoso , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Dasatinibe/uso terapêutico , Cloridrato de Erlotinib/uso terapêutico , Feminino , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Transplante de Neoplasias , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Receptor de Pregnano X , Prognóstico , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Esteroides/metabolismo , Regulação para Cima
14.
Matrix Biol ; 52-54: 260-265, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26709044

RESUMO

Dental enamel is the hardest tissue in the human body, and although it starts as a tissue rich in proteins, by the time of eruption of the tooth in the oral cavity only a small fraction of the protein remains. While this organic matrix of enamel represents less than 1% by weight it plays essential roles in improving both toughness and resilience to chemical attacks. Despite the fact that the first studies of the enamel matrix began in the 19th century, its exact composition and mechanisms of its function remain poorly understood. It was proposed that keratin or a keratin-like primitive epithelial component exists in mature enamel, however due to the extreme insolubility of its organic matrix the presence of keratins there was never clearly established. We have recently identified expression of a number of hair keratins in ameloblasts, the enamel secreting cells, and demonstrated their incorporation into mature enamel. Mutation in epithelial hair keratin KRT75 leads to a skin condition called pseudofollicularis barbae. Carriers of this mutation have an altered enamel structure and mechanical properties. Importantly, these individuals have a much higher prevalence of caries. To the best of our knowledge, this is the first study showing a direct link between a mutation in a protein-coding region of a gene and increased caries rates. In this paper we present an overview of the evidence of keratin-like material in enamel that has accumulated over the last 150years. Furthermore, we propose potential mechanisms of action of KTR75 in enamel and highlight the clinical implications of the link between mutations in KRT75 and caries. Finally, we discuss the potential use of keratins for enamel repair.


Assuntos
Cárie Dentária/epidemiologia , Esmalte Dentário/química , Queratinas Específicas do Cabelo/genética , Queratinas Específicas do Cabelo/metabolismo , Queratinas Tipo II/genética , Queratinas Tipo II/metabolismo , Animais , Cárie Dentária/genética , Cárie Dentária/metabolismo , Esmalte Dentário/metabolismo , Esmalte Dentário/patologia , Doenças do Cabelo/complicações , Doenças do Cabelo/genética , Humanos , Mutação
15.
PLoS One ; 10(8): e0137233, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26317522

RESUMO

Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines) have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2) has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD) of 1.8 × 10(-4) M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals) were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10(-7) M), within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS) shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10(-5) M). BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks), suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5), below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the development of better, more clinically relevant BMP-2-conjugated systems for bone repair and regeneration.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Ouro/metabolismo , Queratinas Específicas do Cabelo/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Proteína Morfogenética Óssea 2/química , Ouro/química , Cabelo/química , Humanos , Concentração de Íons de Hidrogênio , Queratinas Específicas do Cabelo/química , Ligação Proteica , Eletricidade Estática , Propriedades de Superfície
16.
J Allergy Clin Immunol ; 136(5): 1277-87, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26316095

RESUMO

BACKGROUND: Alopecia areata (AA) is a common T cell-mediated disorder with limited therapeutics. A molecular profile of cytokine pathways in AA tissues is lacking. Although studies have focused on TH1/IFN-γ responses, several observations support a shared genetic background between AA and atopy. OBJECTIVE: We sought to define the AA scalp transcriptome and associated biomarkers with comparisons with atopic dermatitis (AD) and psoriasis. METHODS: We performed microarray and RT-PCR profiling of 27 lesional and 17 nonlesional scalp samples from patients with AA for comparison with normal scalp samples (n = 6). AA gene expression was also compared with samples from patients with lesional or nonlesional AD and those with psoriasis. A fold change of greater than 1.5 and a false discovery rate of less than 0.05 were used for differentially expressed genes (DEGs). RESULTS: We established the AA transcriptomes (lesional vs nonlesional: 734 DEGs [297 upregulated and 437 downregulated]; lesional vs normal: 4230 DEGs [1980 upregulated and 2250 downregulated]), including many upregulated immune and downregulated hair keratin genes. Equally impressive as upregulation in TH1/interferon markers (IFNG and CXCL10/CXCL9) were those noted in TH2 (IL13, CCL18, CCL26, thymic stromal lymphopoietin, and periostin), TH9/IL-9, IL-23 (p40 and p19), and IL-16 mediators (all P < .05). There were no increases in TH17/TH22 markers. Hair keratin (KRT) expressions (ie, KRT86 and KRT85) were significantly suppressed in lesional skin. Greater scalp involvement (>25%) was associated with greater immune and keratin dysregulation and larger abnormalities in nonlesional scalp samples (ie, CXCL10 and KRT85). CONCLUSIONS: Our data associate the AA signature with TH2, TH1, IL-23, and IL-9/TH9 cytokine activation, suggesting consideration of anti-TH2, anti-TH1, and anti-IL-23 targeting strategies. Similar to psoriasis and AD, clinical trials with selective antagonists are required to dissect key pathogenic pathways.


Assuntos
Alopecia em Áreas/imunologia , Dermatite Atópica/imunologia , Psoríase/imunologia , Células Th1/imunologia , Células Th2/imunologia , Adulto , Biomarcadores/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Feminino , Humanos , Interleucina-23/metabolismo , Queratinas Específicas do Cabelo/genética , Queratinas Específicas do Cabelo/metabolismo , Ativação Linfocitária , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Transcriptoma , Adulto Jovem
17.
J Cutan Pathol ; 42(5): 361-7, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25727336

RESUMO

Neoplasms originating from nail matrix keratinocytes are very rare. Onychomatricoma and onychocytic matricoma are benign tumors arising from nail matrix keratinocytes. Only one case of onychocytic carcinoma, the malignant counterpart of onychocytic matricoma, has been reported in the literature. Herein, we describe a case of invasive onychocytic carcinoma. Two biopsy specimens of the tumor, obtained at early and invasive stages, were examined histopathologically. The first biopsy specimen showed a retiform proliferation of eosinophilic and basophilic cells in the nail matrix. The second biopsy specimen showed a retiform basophilic cell proliferation with focal keratinization. Similar to normal nail matrix keratinocytes, the proliferating basophilic cells failed to express cytokeratin (CK)1, CK6 and CK10. Focal expression of hair-specific keratins, including K31, K85 and K86, was observed. On the basis of these findings, the tumor was identified as an invasive malignant tumor originating from nail matrix keratinocytes.


Assuntos
Carcinoma/patologia , Dedos/patologia , Doenças da Unha/patologia , Unhas/patologia , Invasividade Neoplásica/patologia , Neoplasias Cutâneas/patologia , Carcinoma/metabolismo , Feminino , Humanos , Queratinócitos/patologia , Queratinas Específicas do Cabelo/metabolismo , Pessoa de Meia-Idade , Doenças da Unha/metabolismo , Unhas/metabolismo , Neoplasias Cutâneas/metabolismo
18.
Ann Oncol ; 26(6): 1142-8, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25716425

RESUMO

BACKGROUND: MicroRNAs (miRNAs) have a key role in carcinogenesis through negative regulation of their target genes. Therefore, genetic variations in miRNAs or their target sites may affect miRNA-mRNA interactions, thereby result in altered expression of target genes. This study was conducted to investigate the associations between single-nucleotide polymorphisms (SNP) located in the miRNA target sites (poly-miRTSs) and survival of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS: Using public SNP database and miRNA target sites prediction program, 354 poly-miRTSs were selected for genotyping. Among these, 154 SNPs applicable to Sequenom's MassARRAY platform were investigated in 357 patients. A replication study was carried out on an independent patient population (n = 479). Renilla luciferase assay and reverse transcription-polymerase chain reaction were conducted to examine functional relevance of potentially functional poly-miRTSs. RESULTS: Of the 154 SNPs analyzed in a discovery set, 14 SNPs were significantly associated with survival outcomes. Among these, KRT81 rs3660G>C was found to be associated with survival outcomes in the validation cohort. In the combined analysis, patients with the rs3660 GC + CC genotype had a significantly better overall survival compared with those with GG genotype [adjusted hazard ratio (aHR) for OS, 0.65; 95% confidence interval (CI) 0.50-0.85; P = 0.001]. An increased expression of the reporter gene for the C allele of rs3660 compared with the G allele was observed by luciferase assay. Consistently, the C allele was associated with higher relative expression level of KRT81 in tumor tissues. CONCLUSION: The rs3660G>C affects KRT81 expression and thus influences survival in early-stage NSCLC. The analysis of the rs3660G>C polymorphism may be useful to identify patients at high risk of a poor disease outcome.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Queratinas Específicas do Cabelo/genética , Queratinas Tipo II/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Regiões 3' não Traduzidas , Idoso , Sítios de Ligação , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Biologia Computacional , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Predisposição Genética para Doença , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Queratinas Específicas do Cabelo/metabolismo , Queratinas Tipo II/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fenótipo , Modelos de Riscos Proporcionais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Risco , Fatores de Tempo , Transfecção
19.
BMB Rep ; 48(1): 19-24, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24667173

RESUMO

A mouse homozygous for the spontaneous mutation uncovered (Uncv) has a hairless phenotype. A 309-bp non-frameshift deletion mutation in the N-terminal cytoplasmic domain of iRhom2 was identified in Uncv mice (iRhom2(Uncv)) using target region sequencing. The detailed molecular basis for how the iRhom2 mutation causes the hairless phenotype observed in the homozygous iRhom2(Uncv) mouse remains unknown. To identify differentially expressed proteins in the skin of wild-type and homozygous iRhom2(Uncv) littermates at postnatal day 5, proteomic approaches, including two-dimensional gel electrophoresis and mass spectrometry were used. Twelve proteins were differentially expressed in the skin in a comparison between wild-type and homozygous iRhom2(Uncv) mice. A selection of the proteomic results were tested and verified using qRT-PCR, western blot and immunohistochemistry. These data indicate that differentially expressed proteins, especially KRT73, MEMO1 and Coro-1, might participate in the mechanism by which iRhom2 regulates the development of murine skin.


Assuntos
Proteínas de Transporte/genética , Pele/metabolismo , Animais , Proteínas de Transporte/metabolismo , Regulação para Baixo , Eletroforese em Gel Bidimensional , Genótipo , Homozigoto , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratinas Específicas do Cabelo/genética , Queratinas Específicas do Cabelo/metabolismo , Camundongos , Camundongos Pelados , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutação , Fenótipo , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Regulação para Cima
20.
Brain Pathol ; 25(1): 1-10, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24716541

RESUMO

Adamantinomatous craniopharyngiomas (adaCP) cause hypothalamic pituitary dysfunction. Elucidation of pathomechanisms underlying tumor progression is essential for the development of targeted chemotherapeutic treatment options. In order to study the mechanisms of tumor outgrowth, we implanted human primary adaCP tissue from three different surgical specimens stereotactically into the brain of immunodeficient mice (n = 20). Three months after tumor inoculation, magnetic resonance imaging and histology confirmed tumor engraftment in all 20 mice (100%) that obtained tissue transplants. The lesions invaded adjoining brain tissue with micro finger-shaped protrusions. Immunohistochemical comparison of the primary tumor and xenotransplants revealed a similar amount of proliferation (Mib-1) and cytokeratin expression pattern (KL-1). Whole tumor reconstruction using serial sections confirmed whirl-like cell clusters with nuclear ß-catenin accumulations at the tumor brain border. These whirls were surrounded by a belt of Claudin-1 expressing cells, showed an activated epidermal growth factor receptor (EGFR) and distinct CD133 as well as p21(WAF1/Cip1) positivity, indicating a tumor stem cell phenotype. Consistent with our previous in vitro studies, intracranial xenotransplants of adaCP confirmed cells with nuclear ß-catenin and activated EGFR being the driving force of tumor outgrowth. This model provides the possibility to study in vivo tumor cell migration and to test novel treatment regimens targeting this tumor stem cell niche.


Assuntos
Neoplasias Encefálicas/fisiopatologia , Encéfalo/fisiopatologia , Craniofaringioma/fisiopatologia , Adolescente , Adulto , Animais , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Claudina-1/metabolismo , Craniofaringioma/patologia , Progressão da Doença , Receptores ErbB/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Queratinas Específicas do Cabelo/metabolismo , Imagem por Ressonância Magnética , Masculino , Camundongos Mutantes , Pessoa de Meia-Idade , Transplante de Neoplasias , Ubiquitina-Proteína Ligases/metabolismo , beta Catenina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA