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1.
Braz. j. biol ; 84: e257473, 2024. tab, graf, ilus
Artigo em Inglês | VETINDEX | ID: biblio-1374639

RESUMO

Feathers make up 7% of the total weight of adult chickens and keratin protein makes up 85% of the feathers. Today, the keratinase enzymes of some Bacillus strains are used to degrade and process raw keratin waste for animal and poultry feed. According to various studies, the probiotic properties of some spore-shaped Bacillus have also been proven. The study aimed to isolation of the keratinolytic Bacillus bacteria that they have probiotic properties for using in the livestock and poultry feed industry. We were able to isolate 8 strains of Bacillus licheniformis with kreatin degrading properties from the soil of Baharan chicken slaughterhouse (Qom city, Iran) applying heat shock, alcohol- and keratin-rich culture medium, and after microscopic and biochemical analysis, 16S rDNA gene was isolated. The measurement results of keratinase activity showed that the three strains of Bacillus licheniformis pvkr6, pvkr 15, and pvkr41 had the highest activity with 124.08, 101.1, and 100.18 U/ml. The results of probiotic properties evaluation also revealed that among all the isolates, only Bacillus licheniformis pvkr15 and Bacillus licheniformis PTCC 1595 (positive control) were γ-hemolytic strains. The percentage of surface hydrophobicity of the strains was obtained from 3.27 to 30.57. It was also shown that, on average, all the strains had acceptable susceptibility to the tested antibiotics except penicillin G. Bacillus licheniformis pvkr15 with highest keratinase activity (101.1U/ml) was considered an optional probiotics due to its abilities such as (biofilm formation, being safe cause of γ-hemolytic activity, high susceptibility to antibiotics such as streptomycin, gentamicin, cefixime, amoxicillin, tetracycline, vancomycin, erythromycin and having a moderate hydrophilic (hydrophobicity: 19.09%), high survivability in pH 2, 2.5 and 3, strong resistance to bile salts and moderate antagonistic activity against pathogenic bacterium like Proteus mirabilis and the ability to grow under anaerobic conditions). By using this strain, after hydrolysis of keratin protein in the feather structure, to replace part of the protein of livestock and poultry feed, not only is no need to separate bacteria from the feed, but also the strain play role of an useful and effective additive in animal growth.


As penas representam 7% do peso total das galinhas adultas e a proteína de queratina compõe 85% das penas. Hoje, as enzimas queratinase de algumas cepas de Bacillus são usadas para degradar e processar resíduos de queratina brutos para alimentação de animais e aves. De acordo com vários estudos, as propriedades probióticas de alguns Bacillus em forma de esporos também foram comprovadas. O estudo teve como objetivo o isolamento das bactérias queratinolíticas Bacillus que possuem propriedades probióticas para uso na indústria de ração animal e avícola. Conseguimos isolar 8 cepas de Bacillus licheniformis com propriedades degradantes de creatina do solo do abatedouro de frangos de Baharan (cidade de Qom, Irã) aplicando choque térmico, meio de cultura rico em álcool e queratina e, após análise microscópica e bioquímica, o gene 16S rDNA foi isolado. Os resultados da medição da atividade da queratinase mostraram que as três cepas de Bacillus licheniformis pvkr6, pvkr15 e pvkr41 tiveram a maior atividade com 124,08, 101,1 e 100,18 U/ml. Os resultados da avaliação das propriedades probióticas também revelaram que dentre todos os isolados apenas Bacillus licheniformis pvkr15 e Bacillus licheniformis PTCC 1595 (controle positivo) eram cepas γ-hemolíticas. A porcentagem de hidrofobicidade superficial das cepas foi obtida de 3,27 a 30,57. Também foi demonstrado que, em média, todas as cepas apresentaram suscetibilidade aceitável aos antibióticos testados, exceto penicilina G. Bacillus licheniformis pvkr15 com maior atividade de queratinase (101,1U/ml) foi considerado um probiótico opcional devido às suas habilidades como formação de biofilme, sendo causa segura de atividade γ-hemolítica, alta suscetibilidade a antibióticos como estreptomicina, gentamicina, cefixima, amoxicilina, tetraciclina, vancomicina, eritromicina e ter uma hidrofílica moderada (hidrofobicidade: 19,09%), alta capacidade de sobrevivência em pH 2, 2,5 e 3, forte resistência aos sais biliares e atividade antagonista moderada contra bactérias patogênicas como Proteus mirabilis e a capacidade de crescer em condições anaeróbicas. Ao utilizar esta cepa, após a hidrólise da proteína queratina na estrutura da pena, para substituir parte da proteína da ração de gado e aves, não só não há necessidade de separar as bactérias da ração, mas também a cepa desempenha um papel útil e eficaz aditivo no crescimento animal.


Assuntos
Animais , Solo , Resíduos , Probióticos , Bacillus licheniformis , Queratinas , Ração Animal
2.
Braz. J. Biol. ; 83: 1-8, 2023. graf
Artigo em Inglês | VETINDEX | ID: vti-765419

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.(AU)


A indústria avícola está se expandindo rapidamente e produzindo milhões de toneladas de resíduos de penas anualmente. A produção massiva de subprodutos queratinosos na forma de resíduos agrícolas e industriais em todo o mundo exige sua utilização justificada. O tratamento químico de resíduos de queratina é proclamado como uma abordagem ecodestrutiva por vários pesquisadores, uma vez que gera poluentes secundários. A queratinase liberada por uma variedade de micróbios (bactérias e fungos) pode ser usada para o tratamento eficaz de resíduos de queratina. A degradação microbiana de resíduos de queratina é uma abordagem emergente e ecológica e oferece benefícios duplos, ou seja, tratamento de poluente recalcitrante (queratina) e obtenção de uma enzima comercialmente importante (queratinase). Este estudo envolve o isolamento, caracterização e utilidade potencial de espécies de fungos para a degradação de resíduos de penas de frango por meio da fermentação submersa e em estado sólido. O fungo isolado foi identificado e caracterizado como Aspergillus (A.) flavus. Em um ensaio de 30 dias, constatou-se que 74% e 8% do peso das penas foram reduzidos por A. flavus, respectivamente, por meio da fermentação submersa e em estado sólido. O pH do meio de crescimento em fermentação submersa foi alterado de 4,8 para 8,35. A aplicação explorada de micróbios queratinolíticos é, portanto, recomendada para o tratamento de resíduos ceratinosos para obter benefícios duplos de remediação.(AU)


Assuntos
Aspergillus flavus/isolamento & purificação , Queratinas/análise , Queratinas/toxicidade , Biotransformação
3.
Braz. j. biol ; 83: e246389, 2023. graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1285638

RESUMO

Abstract 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.


Resumo A indústria avícola está se expandindo rapidamente e produzindo milhões de toneladas de resíduos de penas anualmente. A produção massiva de subprodutos queratinosos na forma de resíduos agrícolas e industriais em todo o mundo exige sua utilização justificada. O tratamento químico de resíduos de queratina é proclamado como uma abordagem ecodestrutiva por vários pesquisadores, uma vez que gera poluentes secundários. A queratinase liberada por uma variedade de micróbios (bactérias e fungos) pode ser usada para o tratamento eficaz de resíduos de queratina. A degradação microbiana de resíduos de queratina é uma abordagem emergente e ecológica e oferece benefícios duplos, ou seja, tratamento de poluente recalcitrante (queratina) e obtenção de uma enzima comercialmente importante (queratinase). Este estudo envolve o isolamento, caracterização e utilidade potencial de espécies de fungos para a degradação de resíduos de penas de frango por meio da fermentação submersa e em estado sólido. O fungo isolado foi identificado e caracterizado como Aspergillus (A.) flavus. Em um ensaio de 30 dias, constatou-se que 74% e 8% do peso das penas foram reduzidos por A. flavus, respectivamente, por meio da fermentação submersa e em estado sólido. O pH do meio de crescimento em fermentação submersa foi alterado de 4,8 para 8,35. A aplicação explorada de micróbios queratinolíticos é, portanto, recomendada para o tratamento de resíduos ceratinosos para obter benefícios duplos de remediação.


Assuntos
Animais , Galinhas , Plumas , Fermentação , Fungos , Resíduos Industriais , Queratinas/metabolismo
4.
Commun Biol ; 5(1): 1270, 2022 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-36402892

RESUMO

Here we show that intradermal injection of keratin promotes hair growth in mice, which results from extracellular interaction of keratin with hair forming cells. Extracellular application of keratin induces condensation of dermal papilla cells and the generation of a P-cadherin-expressing cell population (hair germ) from outer root sheath cells via keratin-mediated microenvironmental changes. Exogenous keratin-mediated hair growth is reflected by the finding that keratin exposure from transforming growth factor beta 2 (TGFß2)-induced apoptotic outer root sheath cells appears to be critical for dermal papilla cell condensation and P-cadherin-expressing hair germ formation. Immunodepletion or downregulation of keratin released from or expressed in TGFß2-induced apoptotic outer root sheath cells negatively influences dermal papilla cell condensation and hair germ formation. Our pilot study provides an evidence on initiating hair regeneration and insight into the biological function of keratin exposed from apoptotic epithelial cells in tissue regeneration and development.


Assuntos
Proteínas do Citoesqueleto , Queratinas , Camundongos , Animais , Projetos Piloto , Cabelo , Caderinas
5.
Nano Lett ; 22(22): 8835-8844, 2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36375092

RESUMO

Although keratins are robust in nature, hydrogels producing their extracts exhibit poor mechanical properties due to the complicated composition and ineffective self-assembly. Here we report a bioinspired strategy to fabricate robust keratin hydrogels based on mechanism study through recombinant proteins. Homotypic and heterotypic self-assembly of selected type I and type II keratins in different combinations was conducted to identify crucial domain structures for the process, their kinetics, and relationship with the mechanical strength of hydrogels. Segments with best performance were isolated and used to construct novel assembling units. The new design outperformed combinations of native proteins in mechanical properties and in biomedical applications such as controlled drug release and skin regeneration. Our approach not only elucidated the critical structural domains and underlying mechanisms for keratin self-assembly but also opens an avenue toward the rational design of robust keratin hydrogels for biomedical applications.


Assuntos
Hidrogéis , Queratinas , Hidrogéis/química , Queratinas/química , Queratinas/farmacologia , Pele , Liberação Controlada de Fármacos
6.
Artigo em Inglês | MEDLINE | ID: mdl-36360819

RESUMO

The high demand for keratinolytic enzymes and the modest presentation of fungal keratinase diversity studies in scientific sources cause a significant interest in identifying new fungal strains of keratinase producers, isolating new enzymes and studying their properties. Four out of the 32 cultures showed a promising target activity on protein-containing agar plates-Aspergillus amstelodami A6, A. clavatus VKPM F-1593, A. ochraceus 247, and Cladosporium sphaerospermum 1779. The highest values of keratinolytic activity were demonstrated by extracellular proteins synthesized by Aspergillus clavatus VKPM F-1593 cultivated under submerged conditions on a medium containing milled chicken feathers. The enzyme complex preparation was obtained by protein precipitation from the culture liquid with ammonium sulfate, subsequent dialysis, and lyophilization. The fraction of a pure enzyme with keratinolytic activity (pI 9.3) was isolated by separating the extracellular proteins of A. clavatus VKPM F-1593 via isoelectric focusing. The studied keratinase was an alkaline subtilisin-like non-glycosylated protease active over a wide pH range with optimum keratinolysis at pH 8 and 50 °C.


Assuntos
Plumas , Queratinas , Animais , Queratinas/metabolismo , Biodegradação Ambiental , Fungos/metabolismo , Concentração de Íons de Hidrogênio , Temperatura
7.
Cell Mol Life Sci ; 79(12): 591, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376494

RESUMO

Mechanoreceptors are implicated as functional afferents within mucosa of the airways and the recent discovery of mechanosensitive channels Piezo1 and Piezo2 has proved essential for cells of various mechanically sensitive tissues. However, the role for Piezo1/2 in vocal fold (VF) mucosal epithelia, a cell that withstands excessive biomechanical insult, remains unknown. The purpose of this study was to test the hypothesis that Piezo1 is required for VF mucosal repair pathways of epithelial cell injury. Utilizing a sonic hedgehog (shh) Cre line for epithelial-specific ablation of Piezo1/2 mechanoreceptors, we investigated 6wk adult VF mucosa following naphthalene exposure for repair strategies at 1, 3, 7 and 14 days post-injury (dpi). PIEZO1 localized to differentiated apical epithelia and was paramount for epithelial remodeling events. Injury to wildtype epithelium was most appreciated at 3 dpi. Shhcre/+; Piezo1loxP/loxP, Piezo2 loxP/+ mutant epithelium exhibited severe cell/nuclear defects compared to injured controls. Conditional ablation of Piezo1 and/or Piezo2 to uninjured VF epithelium did not result in abnormal phenotypes across P0, P15 and 6wk postnatal stages compared to heterozygote and control tissue. Results demonstrate a role for Piezo1-expressing VF epithelia in regulating self-renewal via effects on p63 transcription and YAP subcellular translocation-altering cytokeratin differentiation.


Assuntos
Proteínas Hedgehog , Queratinas , Proteínas Hedgehog/metabolismo , Queratinas/genética , Prega Vocal/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo
8.
Environ Sci Pollut Res Int ; 29(58): 86913-86932, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36271998

RESUMO

Since the dawn of century, tons of keratin bio-waste is generated by the poultry industry annually, and they end up causing environmental havoc. Keratins are highly flexible fibrous proteins which exist in α- and ß- forms and provide mechanical strength and stability to structural appendages. The finding of broad-spectrum protease, keratinase, from thermophilic bacteria and fungi, has provided an eco-friendly solution to hydrolyze the peptide bonds in highly recalcitrant keratinous substances such as nails, feathers, claws, and horns into valuable amino acids. Microorganisms produce these proteolytic enzymes by techniques of solid-state and submerged fermentation. However, solid-state fermentation is considered as a yielding approach for the production of thermostable keratinases. This review prioritized the molecular and biochemical properties of microbial keratinases, and the role of keratinases in bringing prodigious impact for the sustainable progress of the economy. It also emphasizes on the current development in keratinase production with the focus to improve the biochemical properties related to enzyme's catalytic activity and stability, and production of mutant and cloned microbial strains to improve the yield of keratinases. Recently, multitude molecular approaches have been employed to enhance enzyme's productivity, activity, and thermostability which makes them suitable for pharmaceutical industry and for the production of animal feed, organic fertilizers, biogas, clearing of animal hides, and detergent formulation. Hence, it can be surmised that microbial keratinolytic enzymes are the conceivable candidates for numerous commercial and industrial applications.


Assuntos
Queratinas , Peptídeo Hidrolases , Animais , Peptídeo Hidrolases/metabolismo , Queratinas/metabolismo , Biotecnologia/métodos , Plumas/metabolismo , Responsabilidade Social , Concentração de Íons de Hidrogênio
9.
Sci Rep ; 12(1): 16584, 2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36198710

RESUMO

Multiphoton fluorescence lifetime imaging microscopy (MPM-FLIM) is extensively proposed as a non-invasive optical method to study tissue metabolism. The approach is based on recording changes in the fluorescence lifetime attributed to metabolic co-enzymes, of which nicotinamide adenine dinucleotide (NADH) is of major importance. However, intrinsic tissue fluorescence is complex. Particularly when utilizing two-photon excitation, as conventionally employed in MPM. This increases the possibility for spectral crosstalk and incorrect assignment of the origin of the FLIM signal. Here we demonstrate that in keratinocytes, proteins such as keratin may interfere with the signal usually assigned to NADH in MPM-FLIM by contributing to the lifetime component at 1.5 ns. This is supported by a change in fluorescence lifetime distribution in KRT5- and KRT14-silenced cells. Altogether, our results suggest that the MPM-FLIM data originating from cellular autofluorescence is far more complex than previously suggested and that the contribution from other tissue constituents should not be neglected-changing the paradigm for data interpretation in this context.


Assuntos
NAD , Imagem Óptica , Queratinas , Microscopia de Fluorescência , Microscopia de Fluorescência por Excitação Multifotônica/métodos , NAD/metabolismo
10.
Oral Oncol ; 134: 106184, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36191479

RESUMO

Fanconi anemia (FA) patients frequently develop oral squamous cell carcinoma (OSCC). This cancer in FA patients is diagnosed within the first 3-4 decades of life, very often preceded by lesions that suffer a malignant transformation. In addition, they respond poorly to current treatments due to toxicity or multiple recurrences. Translational research on new chemopreventive agents and therapeutic strategies has been unsuccessful partly due to scarcity of disease models or failure to fully reproduce the disease. Here we report that Fanca gene knockout mice (Fanca-/-) frequently display pre-malignant lesions in the oral cavity. Moreover, when these animals were crossed with animals having conditional deletion of Trp53 gene in oral mucosa (K14cre;Trp53F2-10/F2-10), they spontaneously developed OSCC with high penetrance and a median latency of less than ten months. Tumors were well differentiated and expressed markers of squamous differentiation, such as keratins K5 and K10. In conclusion, Fanca and Trp53 genes cooperate to suppress oral cancer in mice, and Fanca-/-;K14cre;Trp53F2-10/F2-10 mice constitute the first animal model of spontaneous OSCC in FA.


Assuntos
Carcinoma de Células Escamosas , Anemia de Fanconi , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Modelos Animais de Doenças , Anemia de Fanconi/complicações , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Queratinas , Camundongos , Camundongos Knockout , Neoplasias Bucais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço
11.
Anticancer Res ; 42(11): 5315-5322, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36288876

RESUMO

BACKGROUND/AIM: Clear cell sarcoma of soft tissue (CCSST) and conventional malignant melanoma (MM) are rare and aggressive tumours with similarities in morphology and the expression of melanocytic markers. MATERIALS AND METHODS: We established two CCSST cell lines (FU-CCSST-1 and FU-CCSST-2) from soft tissues of the patella and supraclavicular. A MM cell line (FU-MM-1) was established from lymph node metastases of subungual malignant melanoma. RESULTS: FU-CCSST-2 cells were transplantable to immunodeficient mice. Immunohistochemical studies demonstrated tumour cells were negative for cytokeratin AE1/AE3 and positive for S100 protein, HMB45, Melan-A, CD146 and SOX10 in all specimens. FU-CCSST-1 and FU-CCSST-2 harboured t(12;22)(q13;q12) translocations with expression of the EWSR1/ATF1 fusion gene. FU-MM-1 demonstrated loss of the short arm of chromosome 9 and harboured wild-type BRAF (codon 469 and 600) and NRAS (codon 12, 13 and 61). CONCLUSION: We report the establishment and characterisation of CCSST and MM cell lines that may have utility in the study of pathogenic mechanisms and development of novel therapeutic reagents.


Assuntos
Melanoma , Sarcoma de Células Claras , Humanos , Camundongos , Animais , Sarcoma de Células Claras/patologia , Antígeno MART-1/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Antígeno CD146 , Melanoma/genética , Melanoma/metabolismo , Proteínas S100/metabolismo , Queratinas/metabolismo , Linhagem Celular
12.
J Cell Sci ; 135(20)2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-36285538

RESUMO

A large group of keratin genes (n=54 in the human genome) code for intermediate filament (IF)-forming proteins and show differential regulation in epithelial cells and tissues. Keratin expression can be highly informative about the type of epithelial tissue, differentiation status of constituent cells and biological context (e.g. normal versus diseased settings). The foundational principles underlying the use of keratin expression to gain insight about epithelial cells and tissues primarily originated in pioneering studies conducted in the 1980s. The recent emergence of single cell transcriptomics provides an opportunity to revisit these principles and gain new insight into epithelial biology. Re-analysis of single-cell RNAseq data collected from human and mouse skin has confirmed long-held views regarding the quantitative importance and pairwise regulation of specific keratin genes in keratinocytes of surface epithelia. Furthermore, such analyses confirm and extend the notion that changes in keratin gene expression occur gradually as progenitor keratinocytes commit to and undergo differentiation, and challenge the prevailing assumption that specific keratin combinations reflect a mitotic versus a post-mitotic differentiating state. Our findings provide a blueprint for similar analyses in other tissues, and warrant a more nuanced approach in the use of keratin genes as biomarkers in epithelia.


Assuntos
Queratinócitos , Queratinas , Camundongos , Animais , Humanos , Queratinas/genética , Queratinas/metabolismo , Epitélio/metabolismo , Queratinócitos/metabolismo , Células Epiteliais/metabolismo , Diferenciação Celular/genética
13.
Int J Biol Macromol ; 222(Pt A): 938-949, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36183757

RESUMO

The complex chemical structure of polypeptide and the imperfection of processing technology cause the mechanical properties of regenerated keratin to be hard and brittle. This defect seriously affects the application prospects of keratin materials. To solve the above problems, α-lipoic acid modified keratin (KER) was blended with Polyamide 6 (PA6) and prepared into composite fibers via the wet-spinning method in this work. The spinnability and spinning conditions of the KER/PA6 blend solution were analyzed by rheological theory. The results illustrated that keratin solution will easily form a gel state under certain temperatures and concentrations, which was not conducive to the preparation of regenerated fiber. When the temperature was 45 °C and the mass fraction was 10 %, the viscosity and rheology of the solution were appropriate. The rheological properties of the blend solution showed that too much keratin would make the solution easy to gel, which was not conducive to the preparation of regenerated fibers and may affect the fiber properties. On this basis, the prepared composite fibers were characterized to explore the macromolecular aggregation state of keratin and PA6 in fibers. FT-IR and XRD results proved that there was no chemical reaction between keratin and PA6 in the composite fibers, which belonged to physical blending. At the same time, the two polymers had good compatibility and can be blended at the molecular level. SEM, DSC, and tensile strength test results indicated that when the proportion of keratin was too high, the structure and properties of the composite fibers will have obvious defects, which was consistent with the rheological analysis. Therefore, the blend ratio of keratin/PA6 was determined to be 3:7. Under this condition, the fibers exhibited a homogeneous structure and good thermal properties, especially its mechanical properties were close to wool fibers. The KER/PA6 composite fibers show important research value and can also provide technical reference for the development of regenerated biomass materials.


Assuntos
Queratinas , Nylons , Animais , Queratinas/química , Espectroscopia de Infravermelho com Transformada de Fourier , Queratina-6 , Polímeros/química , Reologia
14.
J Environ Manage ; 324: 116380, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36208515

RESUMO

Keratinase production by Bacillus cereus IIPK35 was investigated under solid-state fermentation (SSF) and the maximum titer of 648.28 U/gds was revealed. Feather hydrolysates obtained from SSF exhibited paramount antioxidant properties in ABTS [2,2'-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid], FRAP [Ferric ion reducing antioxidant power], and DPPH [2,2,-Diphenyl-1-picrylhydrazyl] assay. The keratinase was purified up to homogeneity have a molecular weight of 42 kDa, and showed its stability between pH 6.5-10.0 and temperature 35-60 °C with optimum enzyme activity at pH 9.0 and 55 °C. The catalytic indices viz. Km of 9.8 mg/ml and Vmax of 307.7 µmol/min for keratin were determined. Besides keratin, the enzyme displayed broad and proteolytic activity towards other proteinaceous substrates such as casein, skim milk, gelatin, and bovine serum albumin. Pure keratinase activity was stimulated in presence of Ca2+ and Mg2+ ions, while it was strongly inhibited by both iodoacetamide and EDTA, indicating it to be a metallo-serine protease in nature. Circular dichroism study endorses the structural stability of the secondary structure at the said range of pH and temperature. The IIPK35 keratinase is non-cytotoxic in nature, shows remarkable storage stability and is stable in presence of Tween 80, Triton X 100, and sodium sulfite. Furthermore, it showed excellent milk clotting potential (107.6 Soxhlet Unit), suggesting its usefulness as an alternative milk clotting agent in the dairy industry. This study unlocks a new gateway for keratinase investigation in SSF using chicken feathers as substrate and biochemical and biophysical characterization of keratinase for better understanding and implication in industrial applications.


Assuntos
Plumas , Queratinas , Animais , Bacillus cereus , Antioxidantes , Leite , Serina , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases , Temperatura , Galinhas
15.
J Immunother Cancer ; 10(10)2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36252995

RESUMO

BACKGROUND: Immunotherapy is currently part of the standard of care for patients with advanced-stage non-small cell lung cancer (NSCLC). However, many patients do not respond to this treatment, therefore combination strategies are being explored to increase clinical benefit. The PEMBRO-RT trial combined the therapeutic programmed cell death 1 (PD-1) antibody pembrolizumab with stereotactic body radiation therapy (SBRT) to increase the overall response rate and study the effects on the tumor microenvironment (TME). METHODS: Here, immune infiltrates in the TME of patients included in the PEMBRO-RT trial were investigated. Tumor biopsies of patients treated with pembrolizumab alone or combined with SBRT (a biopsy of the non-irradiated site) at baseline and during treatment were stained with multiplex immunofluorescence for CD3, CD8, CD20, CD103 and FoxP3 for lymphocytes, pan-cytokeratin for tumors, and HLA-ABC expression was determined. RESULTS: The total number of lymphocytes increased significantly after 6 weeks of treatment in the anti-PD-1 group (fold change: 1.87, 95% CI: 1.06 to 3.29) and the anti-PD-1+SBRT group (fold change: 2.29, 95% CI: 1.46 to 3.60). The combination of SBRT and anti-PD-1 induced a 4.87-fold increase (95% CI: 2.45 to 9.68) in CD103+ cytotoxic T-cells 6 weeks on treatment and a 2.56-fold increase (95% CI: 1.03 to 6.36) after anti-PD-1 therapy alone. Responders had a significantly higher number of lymphocytes at baseline than non-responders (fold difference 1.85, 95% CI: 1.04 to 3.29 for anti-PD-1 and fold change 1.93, 95% CI: 1.08 to 3.44 for anti-PD-1+SBRT). CONCLUSION: This explorative study shows that that lymphocyte infiltration in general, instead of the infiltration of a specific lymphocyte subset, is associated with response to therapy in patients with NSCLC.Furthermore, anti-PD-1+SBRT combination therapy induces an immunological abscopal effect in the TME represented by a superior infiltration of cytotoxic T cells as compared with anti-PD-1 monotherapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Anticorpos Monoclonais Humanizados , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Fatores de Transcrição Forkhead , Humanos , Queratinas , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Microambiente Tumoral
16.
Kyobu Geka ; 75(12): 1059-1061, 2022 Nov.
Artigo em Japonês | MEDLINE | ID: mdl-36299163

RESUMO

A 67-year-old woman was referred to our hospital for cough and fever. Chest computed tomography (CT) showed some masses showing slightly enhanced effect in the pericardium. FDG-PET showed the accumulation of FDG in the masses. Thoracoscopic surgical biopsy was performed to establish the diagnosis. The histological study showed proliferation of short spindle-shaped cells surrounded by lymphocyte, and the spindle cells were immunohistochemically positive for cytokeratin AE1/AE3, WT-1, D2-40, CAM5.2, intelectin-1 and negative for CEA, TTF-1, napsin A, claudin-4, calretinin, MUC4, PAX8, CD30. These findings were compatible with epithelial pericardial malignant mesothelioma.


Assuntos
Neoplasias Cardíacas , Neoplasias Pulmonares , Neoplasias do Mediastino , Mesotelioma Maligno , Mesotelioma , Neoplasias do Timo , Feminino , Humanos , Idoso , Mesotelioma/diagnóstico por imagem , Mesotelioma/cirurgia , Calbindina 2 , Fluordesoxiglucose F18 , Neoplasias Pulmonares/patologia , Claudina-4 , Queratinas
17.
PLoS One ; 17(10): e0275367, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36251712

RESUMO

A 3-months old Chinese shar-pei puppy with ichthyosis was investigated. The dog showed generalized scaling, alopecia and footpad lesions. Histopathological examinations demonstrated a non-epidermolytic hyperkeratosis. The parents of the affected puppy did not show any skin lesions. A trio whole genome sequencing analysis identified a heterozygous de novo 3 bp deletion in the KRT1 gene in the affected dog. This variant, NM_001003392.1:c.567_569del, is predicted to delete a single asparagine from the conserved coil 1A motif within the rod domain of KRT1, NP_001003392.1:p.(Asn190del). Immunohistochemistry demonstrated normal levels of KRT1 expression in the epidermis and follicular epithelia. This might indicate that the variant possibly interferes with keratin dimerization or another function of KRT1. Missense variants affecting the homologous asparagine residue of the human KRT1 cause epidermolytic hyperkeratosis. Histologically, the investigated Chinese shar-pei showed a non-epidermolytic ichthyosis. The finding of a de novo variant in an excellent functional candidate gene strongly suggests that KRT1:p.Asn190del caused the ichthyosis phenotype in the affected Chinese shar-pei. To the best of our knowledge, this is the first description of a KRT1-related non-epidermolytic ichthyosis in domestic animals.


Assuntos
Hiperceratose Epidermolítica , Ictiose , Queratina-1 , Animais , Cães , Humanos , Lactente , Asparagina/genética , China , Hiperceratose Epidermolítica/genética , Hiperceratose Epidermolítica/patologia , Hiperceratose Epidermolítica/veterinária , Ictiose/genética , Ictiose/veterinária , Queratina-1/genética , Queratina-10/genética , Queratinas/genética , Mutação
18.
PLoS One ; 17(10): e0273807, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36240204

RESUMO

Keratoconus (KC) is a multifactorial progressive ectatic disorder characterized by local thinning of the cornea, leading to decreased visual acuity due to irregular astigmatism and opacities. Despite the evolution of advanced imaging methods, the exact etiology of KC remains unknown. Our aim was to investigate the involvement of corneal epithelium in the pathophysiology of the disease. Corneal epithelial samples were collected from 23 controls and from 2 cohorts of patients with KC: 22 undergoing corneal crosslinking (early KC) and 6 patients before penetrating keratoplasty (advanced KC). The expression of genes involved in the epidermal terminal differentiation program and of the oxidative stress pathway was assessed by real time PCR analysis. Presence of some of the differentially expressed transcripts was confirmed at protein level using immunofluorescence on controls and advanced KC additional corneal samples. We found statistically significant under-expression in early KC samples of some genes known to be involved in the mechanical resistance of the epidermis (KRT16, KRT14, SPRR1A, SPRR2A, SPRR3, TGM1 and TGM5) and in oxidative stress pathways (NRF2, HMOX1 and HMOX2), as compared to controls. In advanced KC samples, expression of SPRR2A and HMOX1 was reduced. Decreased expression of keratin (KRT)16 and KRT14 proteins was observed. Moreover, differential localization was noted for involucrin, another protein involved in the epidermis mechanical properties. Finally, we observed an immunofluorescence staining for the active form of NRF2 in control epithelia that was reduced in KC epithelia. These results suggest a defect in the mechanical resistance and the oxidative stress defense possibly mediated via the NRF2 pathway in the corneal keratoconic epithelium.


Assuntos
Epitélio Corneano , Ceratocone , Córnea/metabolismo , Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Epitélio Corneano/metabolismo , Humanos , Queratinas/metabolismo , Ceratocone/genética , Ceratocone/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/genética
19.
BMC Cancer ; 22(1): 1082, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36271354

RESUMO

BACKGROUND: The tumour-stroma ratio (TSR) is identified as a promising prognostic parameter for breast cancer, but the cutoff TSR value is mostly assessed by visual assessment, which lacks objective measurement. The aims of this study were to optimize the cutoff TSR value, and evaluate its prognosis value in patients with breast cancer both as continuous and categorical variables. METHODS: Major clinicopathological and follow-up data were collected for a series of patients with breast cancer. Tissue microarray images stained with cytokeratin immunohistochemistry were evaluated by automated quantitative image analysis algorithms to assess TSR. The potential cutoff point for TSR was optimized using maximally selected rank statistics. The association between TSR and 5-year disease-free survival (5-DFS) was assessed by Cox regression analysis. Kaplan-Meier analysis and log-rank test were used to assess the significance in survival analysis. RESULTS: The optimal cut-off TSR value was 33.5%. Using this cut-off point, categorical variable analysis found that low TSR (i.e., high stroma, TSR ≤ 33.5%) predicts poor outcomes for 5-DFS (hazard ratio [HR] = 2.82, 95% confidence interval [CI] = 1.81-4.40, P = 0.000). When TSR was considered as a continuous parameter, results showed that increased stroma content was associated with worse 5-DFS (HR = 1.71, 95% CI = 1.34-2.18, P = 0.000). Similar results were also obtained in three molecular subtypes in continuous and categorical variable analyses. Moreover, in the Kaplan-Meier analysis, log-rank test showed that low TSR displayed a worse 5-DFS than high TSR (P = 0.000). Similar results were also obtained in patients with triple-negative breast cancer, human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and luminal-HER2-negative breast cancer. CONCLUSION: TSR is an independent predictor for 5-DFS in breast cancer with worse survival outcomes in low TSR. The prognostic value of TSR was also observed in other three molecular subtypes.


Assuntos
Células Estromais , Neoplasias de Mama Triplo Negativas , Humanos , Intervalo Livre de Doença , Células Estromais/patologia , Prognóstico , Neoplasias de Mama Triplo Negativas/patologia , Queratinas
20.
Front Immunol ; 13: 951817, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36263036

RESUMO

Primary pulmonary lymphoepithelioma-like carcinoma (PLELC) is an Epstein-Barr virus (EBV)-related, rare subtype of non-small-cell lung cancer (NSCLC). Immune checkpoint inhibitors (ICI) show durable responses in advanced NSCLC. However, their effects and predictive biomarkers in PLELC remain poorly understood. We retrospectively analyzed the data of 48 metastatic PLELC patients treated with ICI. Pretreated paraffin-embedded specimens (n = 19) were stained for PD-1, PD-L1, LAG3, TIM3, CD3, CD4, CD8, CD68, FOXP3, and cytokeratin (CK) by multiple immunohistochemistry (mIHC). Next-generation sequencing was performed for 33 PLELC samples. Among patients treated with ICI monotherapy (n = 30), the objective response rate (ORR), disease control rate (DCR), median progression-free survival (mPFS), and overall survival (mOS) were 13.3%, 80.0%, 7.7 months, and 24.9 months, respectively. Patients with PD-L1 ≥1% showed a longer PFS (8.4 vs. 2.1 months, p = 0.015) relative to those with PD-L1 <1%. Among patients treated with ICI combination therapy (n = 18), ORR, DCR, mPFS, and mOS were 27.8%, 100.0%, 10.1 months, and 19.7 months, respectively. Patients with PD-L1 ≥1% showed a significantly superior OS than those with PD-L1 <1% (NA versus 11.7 months, p = 0.001). Among the 19 mIHC patients, those with high PD-1/PD-L1 and LAG3 expression showed a longer PFS (19.0 vs. 3.9 months, p = 0.003). ICI also showed promising efficacy for treating metastatic PLELC. PD-L1 may be both predictive of ICI treatment efficacy and prognostic for survival in PLELC. PD-1/PD-L1 combined with LAG3 may serve as a predictor of ICI treatment effectiveness in PLELC. Larger and prospective trials are warranted to validate both ICI activity and predictive biomarkers in PLELC. This study was partly presented as a poster at the IASLC 20th World Conference on Lung Cancer 2019, 7-10 September 2019, Barcelona, Spain.


Assuntos
Antineoplásicos Imunológicos , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Infecções por Vírus Epstein-Barr , Neoplasias Pulmonares , Humanos , Antígeno B7-H1 , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Receptor de Morte Celular Programada 1 , Inibidores de Checkpoint Imunológico/uso terapêutico , Receptor Celular 2 do Vírus da Hepatite A , Antineoplásicos Imunológicos/uso terapêutico , Estudos Retrospectivos , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Estudos Prospectivos , Biomarcadores Tumorais , Herpesvirus Humano 4 , Carcinoma de Células Escamosas/tratamento farmacológico , Queratinas , Fatores de Transcrição Forkhead
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