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1.
Sci Rep ; 11(1): 19817, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615949

RESUMO

Recent studies have focused their attention on conjunctivitis as one of the symptoms of coronavirus disease 2019 (COVID-19). Therefore, tear samples were taken from COVID-19 patients and the presence of SARS-CoV-2 was evidenced using Real Time reverse transcription polymerase chain reaction. The main aim of this study was to analyze mRNA expression in the tears of patients with COVID-19 compared with healthy subjects using Next Generation Sequencing (NGS). The functional evaluation of the transcriptome highlighted 25 genes that differ statistically between healthy individuals and patients affected by COVID-19. In particular, the NGS analysis identified the presence of several genes involved in B cell signaling and keratinization. In particular, the genes involved in B cell signaling were downregulated in the tears of COVID-19 patients, while those involved in keratinization were upregulated. The results indicated that SARS-CoV-2 may induce a process of ocular keratinization and a defective B cell response.


Assuntos
COVID-19/genética , Oftalmopatias/virologia , Lágrimas/metabolismo , Transcriptoma , Idoso , Linfócitos B/metabolismo , COVID-19/patologia , COVID-19/virologia , Oftalmopatias/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Queratinas/metabolismo , Masculino , SARS-CoV-2/isolamento & purificação , Análise de Sequência de RNA/métodos , Pele/metabolismo , Pele/patologia , Pele/virologia , Lágrimas/virologia
2.
Sci Rep ; 11(1): 17863, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504224

RESUMO

Colorectal carcinoma (CRC) is associated with significant morbidity and mortality worldwide. Cytokeratins (CKs) are widely expressed in various types of carcinomas, whereas in CRC it is usually CK7 - and CK20 + . A subset of CRCs is CK7 + . This study aims to determine the prevalence of CK7 expression in CRC and its impact on overall survival. We analyzed 300 randomly selected surgically treated CRC cases using paraffin embedded tumor tissue samples and evaluated CK7 and CK20 expression using the tissue microarray method. Tumors with positivity > 10% and > 25% of tumor cells were considered CK7 and CK20 positive, respectively. Expression of both CKs and several clinical-pathological variables (stage, grade, laterality, mismatch-repair/MMR status) were evaluated using patient follow up data (Kaplan-Meier analysis of cancer-specific survival (CSS)). Significant results include shorter CSS (restricted mean 4.98 vs. 7.74 years, P = 0.007) and 5-year survival (29.4% vs. 64.6%, P = 0.0221) in CK7 + tumors compared to CK7 - tumors, respectively; without significant association with grade, stage or right-sided location. These results were significant in a multivariate analysis. CK20 + tumors are more frequently MMR-proficient and left-sided. MMR-deficient tumors are more frequently right-sided and had longer survival. CK7 expression, right-sided location (rmean CSS 6.83 vs. 8.0 years, P = 0.043), MMR-proficiency (rmean CSS 7.41 vs. 9.32 years, P = 0.012), and UICC stages III + IV (rmean CSS 6.03 vs. 8.92 years, P < 0.001) of the tumor correlated with negative prognostic outcomes, whereas the most significant results concern stage and CK7 positivity. The result concerning negative prognostic role of CK7 differs from those obtained by several previous studies focused on this topic.


Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Queratina-7/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Diagnóstico Diferencial , Feminino , Humanos , Queratinas/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico
3.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360875

RESUMO

Single prostate stem cells can generate stem and progenitor cells to form prostaspheres in 3D culture. Using a prostasphere-based label retention assay, we recently identified keratin 13 (KRT13)-enriched prostate stem cells at single-cell resolution, distinguishing them from daughter progenitors. Herein, we characterized the epithelial cell lineage hierarchy in prostaspheres using single-cell RNA-seq analysis. Keratin profiling revealed three clusters of label-retaining prostate stem cells; cluster I represents quiescent stem cells (PSCA, CD36, SPINK1, and KRT13/23/80/78/4 enriched), while clusters II and III represent active stem and bipotent progenitor cells (KRT16/17/6 enriched). Gene set enrichment analysis revealed enrichment of stem and cancer-related pathways in cluster I. In non-label-retaining daughter progenitor cells, three clusters were identified; cluster IV represents basal progenitors (KRT5/14/6/16 enriched), while clusters V and VI represent early and late-stage luminal progenitors, respectively (KRT8/18/10 enriched). Furthermore, MetaCore analysis showed enrichment of the "cytoskeleton remodeling-keratin filaments" pathway in cancer stem-like cells from human prostate cancer specimens. Along with common keratins (KRT13/23/80/78/4) in normal stem cells, unique keratins (KRT10/19/6C/16) were enriched in cancer stem-like cells. Clarification of these keratin profiles in human prostate stem cell lineage hierarchy and cancer stem-like cells can facilitate the identification and therapeutic targeting of prostate cancer stem-like cells.


Assuntos
Queratinas/metabolismo , Células-Tronco Neoplásicas , Neoplasias da Próstata , RNA/metabolismo , Adulto , Células Cultivadas , Humanos , Masculino , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Cultura Primária de Células , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Análise de Célula Única , Adulto Jovem
4.
Nat Struct Mol Biol ; 28(8): 681-693, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373646

RESUMO

The synaptonemal complex (SC) is a supramolecular protein assembly that mediates synapsis between homologous chromosomes during meiosis. SC elongation along the chromosome length (up to 24 µm) depends on its midline α-fibrous component SYCE2-TEX12. Here, we report X-ray crystal structures of human SYCE2-TEX12 as an individual building block and on assembly within a fibrous lattice. We combine these structures with mutagenesis, biophysics and electron microscopy to reveal the hierarchical mechanism of SYCE2-TEX12 fiber assembly. SYCE2-TEX12's building blocks are 2:2 coiled coils that dimerize into 4:4 hetero-oligomers and interact end-to-end and laterally to form 10-nm fibers that intertwine within 40-nm bundled micrometer-long fibers that define the SC's midline structure. This assembly mechanism bears striking resemblance with intermediate filament proteins vimentin, lamin and keratin. Thus, SYCE2-TEX12 exhibits behavior typical of cytoskeletal proteins to provide an α-fibrous SC backbone that structurally underpins synaptic elongation along meiotic chromosomes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Pareamento Cromossômico/fisiologia , Complexo Sinaptonêmico/metabolismo , Cristalografia por Raios X , Proteínas do Citoesqueleto/metabolismo , Humanos , Queratinas/metabolismo , Laminas/metabolismo , Meiose/fisiologia , Estrutura Quaternária de Proteína , Vimentina/metabolismo
5.
Exp Eye Res ; 211: 108720, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34389315

RESUMO

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin ß4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.


Assuntos
Córnea/citologia , Epitélio Corneano/citologia , Limbo da Córnea/citologia , Técnicas de Cultura de Órgãos/métodos , Células-Tronco/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Separação Celular , Células Cultivadas , Colágeno/metabolismo , Córnea/metabolismo , Epitélio Corneano/metabolismo , Humanos , Queratinas/metabolismo , Limbo da Córnea/metabolismo , Pessoa de Meia-Idade , Células-Tronco/metabolismo , Fatores de Tempo , Doadores de Tecidos , Preservação de Tecido/métodos , Vimentina/metabolismo
6.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207662

RESUMO

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory-Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62- total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-ß-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.


Assuntos
Queratinas/metabolismo , Agregados Proteicos , Proteína Sequestossoma-1/metabolismo , Animais , Células CHO , Cricetulus , Humanos , Queratinas/genética , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Sequestossoma-1/genética
7.
Braz J Biol ; 83: e246389, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34320050

RESUMO

Poultry industry is expanding rapidly and producing million tons of feather waste annually. Massive production of keratinaceous byproducts in the form of industrial wastes throughout the world necessitates its justified utilization. Chemical treatment of keratin waste is proclaimed as an eco-destructive approach by various researchers since it generates secondary pollutants. Keratinase released by a variety of microbes (bacteria and fungi) can be used for the effective treatment of keratin waste. Microbial degradation of keratin waste is an emerging and eco-friendly approach and offers dual benefits, i.e., treatment of recalcitrant pollutant (keratin) and procurement of a commercially important enzyme (keratinase). This study involves the isolation, characterization, and potential utility of fungal species for the degradation of chicken-feather waste through submerged and solid-state fermentation. The isolated fungus was identified and characterized as Aspergillus (A.) flavus. In a trial of 30 days, it was appeared that 74 and 8% feather weight was reduced through sub-merged and solid-state fermentation, respectively by A. flavus. The pH of the growth media in submerged fermentation was changed from 4.8 to 8.35. The exploited application of keratinolytic microbes is, therefore, recommended for the treatment of keratinaceous wastes to achieve dual benefits of remediation.


Assuntos
Galinhas , Plumas , Animais , Fermentação , Fungos , Resíduos Industriais , Queratinas/metabolismo
8.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070506

RESUMO

Concentration of hyaluronic acid (HA) in the lungs increases in idiopathic pulmonary fibrosis (IPF). HA is involved in the organization of fibrin, fibronectin, and collagen. HA has been proposed to be a biomarker of fibrosis and a potential target for antifibrotic therapy. Hyaluronidase (HD) breaks down HA into fragments, but is a subject of rapid hydrolysis. A conjugate of poloxamer hyaluronidase (pHD) was prepared using protein immobilization with ionizing radiation. In a model of bleomycin-induced pulmonary fibrosis, pHD decreased the level of tissue IL-1ß and TGF-ß, prevented the infiltration of the lung parenchyma by CD16+ cells, and reduced perivascular and peribronchial inflammation. Simultaneously, a decrease in the concentrations of HA, hydroxyproline, collagen 1, total soluble collagen, and the area of connective tissue in the lungs was observed. The effects of pHD were significantly stronger compared to native HD which can be attributed to the higher stability of pHD. Additional spiperone administration increased the anti-inflammatory and antifibrotic effects of pHD and accelerated the regeneration of the damaged lung. The potentiating effects of spiperone can be explained by the disruption of the dopamine-induced mobilization and migration of fibroblast progenitor cells into the lungs and differentiation of lung mesenchymal stem cells (MSC) into cells of stromal lines. Thus, a combination of pHD and spiperone may represent a promising approach for the treatment of IPF and lung regeneration.


Assuntos
Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/farmacologia , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/efeitos dos fármacos , Espiperona/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Hialuronoglucosaminidase/administração & dosagem , Hialuronoglucosaminidase/farmacocinética , Hidroxiprolina/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/enzimologia , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Queratinas/metabolismo , Pulmão/enzimologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Poloxâmero/química , Receptores de IgG/metabolismo , Espiperona/administração & dosagem , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
9.
Int J Mol Sci ; 22(10)2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-34063352

RESUMO

PubMed searches reveal much literature regarding lipids in barrier function of skin and less literature on lipids in barrier function of the oral mucosa. In terrestrial mammals, birds, and reptiles, the skin's permeability barrier is provided by ceramides, fatty acids, and cholesterol in the outermost layers of the epidermis, the stratum corneum. This layer consists of about 10-20 layers of cornified cells embedded in a lipid matrix. It effectively prevents loss of water and electrolytes from the underlying tissue, and it limits the penetration of potentially harmful substances from the environment. In the oral cavity, the regions of the gingiva and hard palate are covered by keratinized epithelia that much resemble the epidermis. The oral stratum corneum contains a lipid mixture similar to that in the epidermal stratum corneum but in lower amounts and is accordingly more permeable. The superficial regions of the nonkeratinized oral epithelia also provide a permeability barrier. These epithelial regions do contain ceramides, cholesterol, and free fatty acids, which may underlie barrier function. The oral epithelial permeability barriers primarily protect the underlying tissue by preventing the penetration of potentially toxic substances, including microbial products. Transdermal drug delivery, buccal absorption, and lipid-related disease are discussed.


Assuntos
Lipídeos/fisiologia , Membrana Mucosa/fisiologia , Dermatopatias/patologia , Pele , Administração Cutânea , Humanos , Queratinas/química , Queratinas/metabolismo , Mucosa Bucal/fisiologia , Permeabilidade , Pele/química , Pele/citologia , Dermatopatias/etiologia
10.
Am J Clin Pathol ; 156(5): 846-852, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34125164

RESUMO

OBJECTIVES: The microcystic, elongated, and fragmented (MELF) pattern of myoinvasion in endometrial carcinoma (EC) is associated with an increased risk of lymph node metastasis. Our aim is to assess the role of cytokeratin immunohistochemical (IHC) stains in detecting sentinel nodal metastasis in MELF pattern tumors. METHODS: We recovered 19 MELF pattern EC hysterectomies with lymphadenectomy from our files. Negative nodes were subjected to cytokeratin AE1/AE3 IHC. Ten additional cases with sentinel lymph node (SLN) biopsies primarily assessed by IHC were also analyzed. RESULTS: Of the 19 cases of EC, 6 had positive lymph nodes based on H&E-stained sections at the time of their initial diagnosis. With the addition of IHC stains, 8 previously negative cases were found to have node metastases, and 3 of these were SLNs. Among the 10 cases primarily assessed by IHC, 5 had malignant cells in their SLNs. CONCLUSIONS: Cytokeratin IHC staining detected malignant cells in 9 of 16 cases with SLNs in our sample of women with MELF pattern of myoinvasion. Immunohistochemical stains should be routinely performed on SLNs from all MELF-positive cases to detect occult lymph node metastases and isolated tumor cells.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma Endometrioide/patologia , Neoplasias do Endométrio/patologia , Queratinas/análise , Metástase Linfática/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Queratinas/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias/métodos , Estudos Retrospectivos , Linfonodo Sentinela/patologia , Biópsia de Linfonodo Sentinela
11.
Sci Rep ; 11(1): 12007, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099743

RESUMO

Microbial secondary metabolites from extreme environments like hydrothermal vents are a promising source for industrial applications. In our study the protease gene from Bacillus cereus obtained from shallow marine hydrothermal vents in the East China Sea was cloned, expressed and purified. The protein sequence of 38 kDa protease SLSP-k was retrieved from mass spectrometry and identified as a subtilisin serine proteinase. The novel SLSP-k is a monomeric protein with 38 amino acid signal peptides being active over wide pH (7-11) and temperature (40-80 °C) ranges, with maximal hydrolytic activities at pH 10 and at 50 °C temperature. The hydrolytic activity is stimulated by Ca2+, Co2+, Mn2+, and DTT. It is inhibited by Fe2+, Cd2+, Cu2+, EDTA, and PMSF. The SLSP-k is stable in anionic, non-anionic detergents, and solvents. The ability to degrade keratin in chicken feather and hair indicates that this enzyme is suitable for the degradation of poultry waste without the loss of nutritionally essential amino acids which otherwise are lost in hydrothermal processing. Therefore, the proteinase is efficient in environmental friendly bioconversion of animal waste into fertilizers or value added products such as secondary animal feedstuffs.


Assuntos
Bacillus cereus/enzimologia , Proteínas de Bactérias/metabolismo , Queratinas/metabolismo , Serina Proteases/metabolismo , Subtilisinas/metabolismo , Animais , Organismos Aquáticos , Bacillus cereus/química , Bacillus cereus/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Braquiúros/microbiologia , Galinhas , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Plumas/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Fontes Hidrotermais/microbiologia , Modelos Moleculares , Oceano Pacífico , Conformação Proteica , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serina Proteases/química , Serina Proteases/genética , Serina Proteases/isolamento & purificação , Especificidade por Substrato , Subtilisinas/química , Subtilisinas/genética , Subtilisinas/isolamento & purificação
12.
Toxins (Basel) ; 13(3)2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33808971

RESUMO

Mammalian animal toxicity of ochratoxin A (OTA) has focused largely in the past half-century on pigs because of initial recognition of it as a principal cause of intermittent growth suppression and renal disease caused by mouldy feed. Subsequent classical toxicology has used laboratory rodents because renal pathology in pigs raised questions concerning possible involvement in the human idiopathic bilateral renal atrophy of Balkan endemic nephropathy for which OTA was a focus of attention for human nephropathy through 1980s and into 2000s. Emphasis on human nephropathy has more recently concerned the plant metabolite aristolochic acid. Recognition that agricultural management can often minimise food and feed-stuff spoilage by OTA-producing Aspergilli and Penicillia has moderated some of the risks for animals. Legislation for human food safety combined with sophisticated analysis generally provides safety in the developed world. Chronic experimental exposure of male rats, in the absence of clinical dis-ease, specifically causes renal cancer. The possibility of this as a unique model for the human has generated considerable experimental evidence which may be more directly relevant for carcinogenesis in the complex kidney than that obtained from biochemical toxicities in vitro. Nevertheless, there does not appear to be any case of human renal or urinary tract cancer for which there is verified etiological proof for causation by OTA, contrary to much claim in the literature. To contribute to such debate, histopathology review of OTA/rat renal cancers, augmented where appropriate by immune profiles, has been completed for all remaining tumours in our research archive. Overall consistency of positivity for vimentin, is matched with occasional positives either for CD10 or the cytokeratin MNF 116. The current situation is discussed. Suggestion that OTA could cause human testicular cancer has also been challenged as unsupported by any experimental findings in rats, where the Leydig cell tumour immune profile does not match that of human germ cell neoplasms.


Assuntos
Testes de Carcinogenicidade , Neoplasias Renais/induzido quimicamente , Ocratoxinas/toxicidade , Neoplasias Testiculares/induzido quimicamente , Animais , Exposição Dietética , Humanos , Queratinas/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Neprilisina/metabolismo , Ratos Endogâmicos F344 , Medição de Risco , Fatores de Risco , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia , Fatores de Tempo , Vimentina/metabolismo
13.
Indian J Pathol Microbiol ; 64(2): 323-328, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33851627

RESUMO

Background: Primary cutaneous amyloidosis (PCA) comprises several forms of localized cutaneous amyloidosis characterized by amyloid deposits occurring at or near dermal-epidermal junctions. Immunohistochemical studies have shown the expression of cytokeratin (CK) suggesting that it has an epidermal origin. Objectives: To study the clinicopathological features of PCA and expression of CK5/6 and correlate it with Congo red stain. Materials and Methods: A total of 30 histologically proven cases of PCA were studied. Congo red staining and immunohistochemical expression of CK5/6 were analyzed. Statistical Analysis: : The qualitative data has been expressed as proportions and the quantitative data has been expressed as mean ± SD. All data was analyzed using the Statistical Package for Social Sciences (SPSS) software version 22. Results: Deposits of amyloid in papillary dermis were seen in all 30 cases. Mild focal basal cell vacuolar degeneration and apoptotic bodies in epidermis were seen in six cases. The presence of pigment cells in dermis were seen in 26 cases. CK5/6 showed weak/mild immunopositivity in nine cases, moderate in 20 cases, and strong in one case. Conclusion: The presence of dermal melanophages interspersed within eosinophilic deposits gives a clue to the diagnosis. Congo red stain highlights the deposits and visualization under polarized light gives apple green birefringence which is diagnostic of amyloid. Staining of amyloid deposits by CK5/6 proves that the amyloid is of keratinocyte origin. There was 100% sensitivity with Congo red and CK5/6. Thus, CK5/6 can be used as an adjunct tool to Congo red stain in the diagnosis of primary cutaneous amyloidosis.


Assuntos
Amiloide/metabolismo , Amiloidose Familiar/diagnóstico , Amiloidose Familiar/patologia , Epiderme/patologia , Queratinas/metabolismo , Dermatopatias Genéticas/diagnóstico , Dermatopatias Genéticas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Vermelho Congo , Derme/patologia , Feminino , Humanos , Imuno-Histoquímica , Queratinócitos/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos Retrospectivos , Coloração e Rotulagem , Adulto Jovem
14.
Int J Mol Sci ; 22(4)2021 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-33669958

RESUMO

The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.


Assuntos
Citoesqueleto de Actina/metabolismo , Hemidesmossomos/metabolismo , Queratinas/metabolismo , Movimento Celular , Adesões Focais/metabolismo , Células HaCaT , Humanos , Microtúbulos/metabolismo , Modelos Biológicos
15.
Proc Natl Acad Sci U S A ; 118(13)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33762306

RESUMO

High levels of the intermediate filament protein keratin 17 (K17) are associated with poor prognoses for several human carcinomas. Studies in mouse models have shown that K17 expression is positively associated with growth, survival, and inflammation in skin and that lack of K17 delays onset of tumorigenesis. K17 occurs in the nucleus of human and mouse tumor keratinocytes where it impacts chromatin architecture, gene expression, and cell proliferation. We report here that K17 is induced following DNA damage and promotes keratinocyte survival. The presence of nuclear K17 is required at an early stage of the double-stranded break (DSB) arm of the DNA damage and repair (DDR) cascade, consistent with its ability to associate with key DDR effectors, including γ-H2A.X, 53BP1, and DNA-PKcs. Mice lacking K17 or with attenuated K17 nuclear import showed curtailed initiation in a two-step skin carcinogenesis paradigm. The impact of nuclear-localized K17 on DDR and cell survival provides a basis for the link between K17 induction and poor clinical outcomes for several human carcinomas.


Assuntos
Carcinoma/genética , Reparo do DNA , Queratina-17/metabolismo , Queratinas/metabolismo , Neoplasias Experimentais/genética , 9,10-Dimetil-1,2-benzantraceno/administração & dosagem , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Transporte Ativo do Núcleo Celular , Animais , Carcinogênese/induzido quimicamente , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma/induzido quimicamente , Carcinoma/patologia , Núcleo Celular/metabolismo , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Feminino , Técnicas de Inativação de Genes , Células HeLa , Humanos , Microscopia Intravital , Queratina-17/genética , Queratinócitos , Queratinas/genética , Masculino , Camundongos Knockout , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/patologia , Imagem com Lapso de Tempo
16.
J Mycol Med ; 31(2): 101133, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33774386

RESUMO

A total of 70 feathers samples of Emu (Dromaius novaehollandiae) were collected from 7 Emu farms situated at two districts (Raigad and Thane) of Maharashtra (India) and screened for resident keratinophilic fungi. Among them, 44 isolates were recovered and identified by evaluating characteristic macro- and micro-morphological features. Further gene products corresponding to the ITS1-5.8S-ITS2 rDNA region from all isolates were amplified and sequenced. Homology search was performed using BLAST program against non-redundant nucleotide database, and significantly matched DNA sequences deposited to the NCBI Gene Bank for reference purposes. Eight identified fungal species belongs to 7 different genera named as Aphanoascus terreus Ac_MW577456 (21.43%), Microsporum gypseum Ac_MW580920 (14.29%), Ctenomyces serratus Ac_MW577459 (10.0%), Uncinocarpus orissi Ac_MW577461 (5.17%), Aphanoascus verrucosus Ac_MW577458 (4.29%), Gymnascella dankaliensis Ac_MW577460 (2.86%), Gymnoascoideus petalosporus Ac_MW577462 (2.86%) and Arthroderma tuberculatum Ac_MW577457 (1.43%).


Assuntos
Dromaiidae/microbiologia , Plumas/microbiologia , Fungos/classificação , Fungos/genética , Queratinas/metabolismo , Animais , DNA Ribossômico/genética , Dromaiidae/anatomia & histologia , Fazendas , Fungos/isolamento & purificação , Índia , Microbiologia do Solo
17.
PLoS Negl Trop Dis ; 15(2): e0009136, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33630844

RESUMO

Mycobacterium ulcerans is the causative agent of the chronic, necrotizing skin disease Buruli ulcer. Modes of transmission and molecular mechanisms involved in the establishment of M. ulcerans infections are poorly understood. Interactions with host glycans are often crucial in bacterial pathogenesis and the 22 kDa M. ulcerans protein MUL_3720 has a putative role in host cell attachment. It has a predicted N-terminal lectin domain and a C-terminal peptidoglycan-binding domain and is highly expressed on the surface of the bacilli. Here we report the glycan-binding repertoire of whole, fixed M. ulcerans bacteria and of purified, recombinant MUL_3720. On an array comprising 368 diverse biologically relevant glycan structures, M. ulcerans cells showed binding to 64 glycan structures, representing several distinct classes of glycans, including sulfated structures. MUL_3720 bound only to glycans containing sulfated galactose and GalNAc, such as glycans known to be associated with keratins isolated from human skin. Surface plasmon resonance studies demonstrated that both whole, fixed M. ulcerans cells and MUL_3720 show high affinity interactions with both glycans and human skin keratin extracts. This MUL_3720-mediated interaction with glycans associated with human skin keratin may contribute to the pathobiology of Buruli ulcer.


Assuntos
Queratinas/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium ulcerans , Polissacarídeos/metabolismo , Pele/microbiologia , Úlcera de Buruli/microbiologia , Células Epiteliais , Sulfatos
18.
Mycoses ; 64(6): 624-633, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33586267

RESUMO

BACKGROUND: Trichophyton schoenleinii is an anthropophilic dermatophyte that causes tinea favosa. Nowadays, it remains an important pathogen in some regions of the world, mainly epidemic in Africa and West Asia. Despite the medical importance of T. schoenleinii infections, a high-quality reference genome for T. schoenleinii is still unavailable, neither its transcriptomic profile. OBJECTIVES: The aim of the current study was to improve understanding of the underlying pathogenic mechanism of T. schoenleinii, and to define the candidate pathogenic genes of T. schoenleinii. METHODS: Comprehensive genomic analysis of T. schoenleinii was carried out by Illumina and PacBio sequencing platforms. Transcriptome profiles of T. schoenleinii cultured in vitro in two media containing either keratin or soy protein were determined using RNA sequencing (RNA-seq) technology. RESULTS: Here, we present the first draft genome sequence of T. schoenleinii strain T2s, which consists of 11 scaffolds containing 7474 predicted genes. Transcriptome analysis showed that genes involved in keratin hydrolysis have higher expression in T. schoenleinii grown in keratin medium, including genes encoding proteases, cysteine dioxygenase and acetamidase. Other genes with higher expression include genes encoding the components of the pH-responsive signal transduction pathways and transcription factors, many of which may play a role in pathogenicity. CONCLUSION: In summary, this study provides new insights into the pathogenic mechanism of T. schoenleinii and highlights candidate genes for further development of novel targets in disease diagnosis and treatment of tinea favosa.


Assuntos
Genoma Fúngico , Trichophyton/genética , Virulência/genética , Arthrodermataceae/genética , Arthrodermataceae/isolamento & purificação , Perfilação da Expressão Gênica , Genes Fúngicos , Humanos , Queratinas/metabolismo , Tinha Favosa/microbiologia , Trichophyton/metabolismo
19.
PLoS One ; 16(2): e0247238, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33596250

RESUMO

The TSA Opal multiplex immunohistochemistry (mIHC) protocol (PerkinElmer) has been used to characterize immune infiltration in human cancers. This technique allows multiple biomarkers to be simultaneously stained in a single tissue section, which helps to elucidate the spatial relationship among individual cell types. We developed and optimized two improved mIHC protocols for a 7-color panel containing 6 biomarkers (CD3, CD8, CD163, PD-L1, FoxP3, and cytokeratin (CK)) and DAPI. The only difference between these two protocols was the staining sequence of those 6 biomarkers as the first sequence is PD-L1/CD163/CD8/CK/CD3/FoxP3/DAPI and the second sequence is FoxP3/CD163/CD8/CK/CD3/PD-L1/DAPI. By comparing PD-L1/FoxP3 staining in mIHC and singleplex PD-L1/FoxP3 staining on the adjacent slide, we demonstrated that the staining sequence does not affect the staining intensity of individual biomarkers as long as a proper antigen retrieval method was used. Our study suggests that use of an antigen retrieval buffer with higher pH value (such as Tris-EDTA pH9.0) than that of the stripping buffers (such as citrate buffer pH6.0) is helpful when using this advanced mIHC method to develop panels with multiple biomarkers. Otherwise, individual biomarkers may exhibit different intensities when the staining sequence is changed. By using this protocol, we characterized immune infiltration and PD-L1 expression in head and neck squamous cell carcinoma (HNSCC), breast cancer (BCa), and non-small cell lung cancer (NSCLC) specimens. We observed a statistically significant increase in CD3+ cell populations within the stroma of NSCLC as compared to BCa and increased PD-L1+ tumor cells in HNSCC as opposed to BCa.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias da Mama/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias Pulmonares/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores Tumorais/metabolismo , Complexo CD3/metabolismo , Antígenos CD8/metabolismo , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imuno-Histoquímica , Indóis/química , Queratinas/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Receptores de Superfície Celular/metabolismo
20.
Sci Rep ; 11(1): 4392, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33623133

RESUMO

Corneal haze post refractive surgery is prevented by mitomycin c (MMC) treatment though it can lead to corneal endothelial damage, persistent epithelial defects and necrosis of cells. Suberanilohydroxamic acid (SAHA) however has been proposed to prevent corneal haze without any adverse effects. For clinical application we have investigated the short and long term outcome of cells exposed to SAHA. Human donor cornea, cultured limbal epithelial cells, corneal rims and lenticules were incubated with SAHA and MMC. The cells/tissue was then analyzed by RT-qPCR, immunofluorescence and western blot for markers of apoptosis and fibrosis. The results reveal that short term exposure of SAHA and SAHA + MMC reduced apoptosis levels and increased αSMA expression compared to those treated with MMC. Epithelial cells derived from cultured corneal rim that were incubated with the MMC, SAHA or MMC + SAHA revealed enhanced apoptosis, reduced levels of CK3/CK12, ∆NP63 and COL4A compared to other treatments. In SAHA treated lenticules TGFß induced fibrosis was reduced. The results imply that MMC treatment for corneal haze has both short term and long term adverse effects on cells and the cellular properties. However, a combinatorial treatment of SAHA + MMC prevents expression of corneal fibrotic markers without causing any adverse effect on cellular properties.


Assuntos
Epitélio Corneano/efeitos dos fármacos , Mitomicina/farmacologia , Vorinostat/farmacologia , Adulto , Apoptose , Células Cultivadas , Colágeno Tipo IV/metabolismo , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Feminino , Fibrose , Humanos , Queratinas/metabolismo , Masculino , Pessoa de Meia-Idade , Mitomicina/efeitos adversos , Vorinostat/efeitos adversos
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