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2.
Sci Rep ; 14(1): 13066, 2024 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-38844764

RESUMO

The aim of this study was to assess the surface and tissue quality of keratinized mucosa grafts (KMG) obtained using the conventional scalpel and mucotome techniques. This was an experimental in vitro/ex vivo study involving six porcine hemi-mandibles. Specimens were harvested using both the mucotome and conventional scalpel techniques, with randomization determining the choice of technique for tissue removal. The specimens were prepared following predefined laboratory protocols and subsequently subjected to optical microscopy for evaluating epithelial and connective tissue and scanning electron microscopy for topographical and 3D profilometry analysis. Tissues harvested using the mucotome exhibited a linear base and uniform thickness, along with the presence of submucosa and fibrous connective tissue, all of which are ideal for graft success. Differences in the surface characteristics of specimens obtained through the two techniques were observed during a comparative analysis of images obtained through both microscopy types. KMG obtained using the mucotome technique displayed greater uniformity and reduced undesirable cell presence compared to the scalpel technique, thereby enhancing the likelihood of success in soft tissue graft surgical procedures. This study provides valuable insights to oral healthcare professionals and may contribute to future research aimed at achieving more successful surgeries, shorter postoperative recovery times, reduced discomfort, and an overall more positive patient experience.


Assuntos
Mandíbula , Mucosa Bucal , Animais , Suínos , Mucosa Bucal/transplante , Mucosa Bucal/citologia , Mandíbula/cirurgia , Queratinas/metabolismo , Microscopia Eletrônica de Varredura , Coleta de Tecidos e Órgãos/métodos
3.
Sci Rep ; 14(1): 12374, 2024 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-38811642

RESUMO

Circulating tumor cells (CTCs) have gathered attention as a biomarker for carcinomas. However, CTCs in sarcomas have received little attention. In this work, we investigated cell surface proteins and antibody combinations for immunofluorescence detection of sarcoma CTCs. A microfluidic device that combines filtration and immunoaffinity using gangliosides 2 and cell surface vimentin (CSV) antibodies was employed to capture CTCs. For CTC detection, antibodies against cytokeratins 7 and 8 (CK), pan-cytokeratin (panCK), or a combination of panCK and CSV were used. Thirty-nine blood samples were collected from 21 patients of various sarcoma subtypes. In the independent samples study, samples were subjected to one of three antibody combination choices. Significant difference in CTC enumeration was found between CK and panCK + CSV, and between panCK and panCK + CSV. Upon stratification of CK+ samples, those of metastatic disease had a higher CTC number than those of localized disease. In the paired samples study involving cytokeratin-positive sarcoma subtypes, using panCK antibody detected more CTCs than CK. Similarly, for osteosarcoma, using panCK + CSV combination resulted in a higher CTC count than panCK. This study emphasized deliberate selection of cell surface proteins for sarcoma CTC detection and subtype stratification for studying cancers as heterogeneous as sarcomas.


Assuntos
Biomarcadores Tumorais , Células Neoplásicas Circulantes , Sarcoma , Humanos , Células Neoplásicas Circulantes/patologia , Células Neoplásicas Circulantes/metabolismo , Sarcoma/patologia , Sarcoma/sangue , Sarcoma/diagnóstico , Sarcoma/metabolismo , Biomarcadores Tumorais/sangue , Feminino , Masculino , Proteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Queratinas/imunologia , Queratinas/metabolismo , Pessoa de Meia-Idade , Adulto , Vimentina/metabolismo , Vimentina/imunologia , Idoso , Anticorpos/imunologia , Linhagem Celular Tumoral
4.
Cells ; 13(10)2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38786031

RESUMO

The oral mucosa functions as a physico-chemical and immune barrier to external stimuli, and an adequate width of the keratinized mucosa around the teeth or implants is crucial to maintaining them in a healthy and stable condition. In this study, for the first time, bulk RNA-seq analysis was performed to explore the gene expression of laser microdissected epithelium and lamina propria from mice, aiming to investigate the differences between keratinized and non-keratinized oral mucosa. Based on the differentially expressed genes (DEGs) and Gene Ontology (GO) Enrichment Analysis, bone morphogenetic protein 2 (BMP-2) was identified to be a potential regulator of oral mucosal keratinization. Monoculture and epithelial-mesenchymal cell co-culture models in the air-liquid interface (ALI) indicated that BMP-2 has direct and positive effects on epithelial keratinization and proliferation. We further performed bulk RNA-seq of the ALI monoculture stimulated with BMP-2 in an attempt to identify the downstream factors promoting epithelial keratinization and proliferation. Analysis of the DEGs identified, among others, IGF2, ID1, LTBP1, LOX, SERPINE1, IL24, and MMP1 as key factors. In summary, these results revealed the involvement of a well-known growth factor responsible for bone development, BMP-2, in the mechanism of oral mucosal keratinization and proliferation, and pointed out the possible downstream genes involved in this mechanism.


Assuntos
Proteína Morfogenética Óssea 2 , Mucosa Bucal , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/genética , Mucosa Bucal/metabolismo , Animais , Camundongos , Queratinas/metabolismo , Queratinas/genética , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Ontologia Genética
5.
Mycopathologia ; 189(3): 37, 2024 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-38704808

RESUMO

Trichophyton rubrum is a human fungal pathogen that causes dermatophytosis, an infection that affects keratinized tissues. Integrated molecular signals coordinate mechanisms that control pathogenicity. Transcriptional regulation is a core regulation of relevant fungal processes. Previous RNA sequencing data revealed that the absence of the transcription factor StuA resulted in the differential expression of the MAPK-related high glycerol osmolarity gene (hog1) in T. rubrum. Here we validated the role of StuA in regulating the transcript levels of hog1. We showed through RT-qPCR that transcriptional regulation controls hog1 levels in response to glucose, keratin, and co-culture with human keratinocytes. In addition, we also detected hog1 pre-mRNA transcripts that underwent alternative splicing, presenting intron retention in a StuA-dependent mechanism. Our findings suggest that StuA and alternative splicing simultaneously, but not dependently, coordinate hog1 transcript levels in T. rubrum. As a means of preventing and treating dermatophytosis, our results contribute to the search for new potential drug therapies based on the molecular aspects of signaling pathways in T. rubrum.


Assuntos
Processamento Alternativo , Arthrodermataceae , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno , Tinha , Fatores de Transcrição , Humanos , Arthrodermataceae/genética , Arthrodermataceae/metabolismo , Glucose/metabolismo , Queratinócitos/microbiologia , Queratinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tinha/metabolismo , Tinha/microbiologia
6.
Nat Commun ; 15(1): 4174, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38755126

RESUMO

The transition from natal downs for heat conservation to juvenile feathers for simple flight is a remarkable environmental adaptation process in avian evolution. However, the underlying epigenetic mechanism for this primary feather transition is mostly unknown. Here we conducted time-ordered gene co-expression network construction, epigenetic analysis, and functional perturbations in developing feather follicles to elucidate four downy-juvenile feather transition events. We report that extracellular matrix reorganization leads to peripheral pulp formation, which mediates epithelial-mesenchymal interactions for branching morphogenesis. α-SMA (ACTA2) compartmentalizes dermal papilla stem cells for feather renewal cycling. LEF1 works as a key hub of Wnt signaling to build rachis and converts radial downy to bilateral symmetry. Novel usage of scale keratins strengthens feather sheath with SOX14 as the epigenetic regulator. We show that this primary feather transition is largely conserved in chicken (precocial) and zebra finch (altricial) and discuss the possibility that this evolutionary adaptation process started in feathered dinosaurs.


Assuntos
Galinhas , Plumas , Tentilhões , Animais , Plumas/crescimento & desenvolvimento , Plumas/metabolismo , Galinhas/genética , Tentilhões/genética , Regulação da Expressão Gênica no Desenvolvimento , Matriz Extracelular/metabolismo , Epigênese Genética , Redes Reguladoras de Genes , Via de Sinalização Wnt , Queratinas/metabolismo , Queratinas/genética , Evolução Biológica , Morfogênese/genética
7.
Int J Mol Sci ; 25(9)2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38732209

RESUMO

One of the primary complications in generating physiologically representative skin tissue is the inability to integrate vasculature into the system, which has been shown to promote the proliferation of basal keratinocytes and consequent keratinocyte differentiation, and is necessary for mimicking representative barrier function in the skin and physiological transport properties. We created a 3D vascularized human skin equivalent (VHSE) with a dermal and epidermal layer, and compared keratinocyte differentiation (immunomarker staining), epidermal thickness (H&E staining), and barrier function (transepithelial electrical resistance (TEER) and dextran permeability) to a static, organotypic avascular HSE (AHSE). The VHSE had a significantly thicker epidermal layer and increased resistance, both an indication of increased barrier function, compared to the AHSE. The inclusion of keratin in our collagen hydrogel extracellular matrix (ECM) increased keratinocyte differentiation and barrier function, indicated by greater resistance and decreased permeability. Surprisingly, however, endothelial cells grown in a collagen/keratin extracellular environment showed increased cell growth and decreased vascular permeability, indicating a more confluent and tighter vessel compared to those grown in a pure collagen environment. The development of a novel VHSE, which incorporated physiological vasculature and a unique collagen/keratin ECM, improved barrier function, vessel development, and skin structure compared to a static AHSE model.


Assuntos
Colágeno , Hidrogéis , Queratinócitos , Queratinas , Pele , Humanos , Hidrogéis/química , Colágeno/química , Colágeno/metabolismo , Queratinócitos/metabolismo , Queratinócitos/citologia , Pele/metabolismo , Pele/irrigação sanguínea , Queratinas/metabolismo , Diferenciação Celular , Proliferação de Células , Engenharia Tecidual/métodos , Matriz Extracelular/metabolismo , Células Cultivadas
8.
Curr Microbiol ; 81(7): 179, 2024 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-38761211

RESUMO

Enormous aggregates of keratinous wastes are produced annually by the poultry and leather industries which cause environmental degradation globally. To combat this issue, microbially synthesized extracellular proteases known as keratinase are used widely which is effective in degrading keratin found in hair and feathers. In the present work, keratinolytic bacteria were isolated from poultry farm soil and feather waste, and various cultural conditions were optimized to provide the highest enzyme production for efficient keratin waste degradation. Based on the primary and secondary screening methods, the potent keratinolytic strain (HFS_F2T) with the highest enzyme activity 32.65 ± 0.16 U/mL was genotypically characterized by 16S rRNA sequencing and was confirmed as Bacillus velezensis HFS_F2T ON556508. Through one-variable-at-a-time approach (OVAT), the keratinase production medium was optimized with sucrose (carbon source), beef extract (nitrogen source) pH-7, inoculum size (5%), and incubation at 37 °C). The degree of degradation (%DD) of keratin wastes was evaluated after 35 days of degradation in the optimized keratinase production medium devoid of feather meal under submerged fermentation conditions. Further, the deteriorated keratin wastes were visually examined and the hydrolysed bovine hair with 77.32 ± 0.32% degradation was morphologically analysed through Field Emission Scanning Electron Microscopy (FESEM) to confirm the structural disintegration of the cuticle. Therefore, the current study would be a convincing strategy for reducing the detrimental impact of pollutants from the poultry and leather industries by efficient keratin waste degradation through the production of microbial keratinase.


Assuntos
Bacillus , Biodegradação Ambiental , Meios de Cultura , Plumas , Queratinas , Peptídeo Hidrolases , Bacillus/metabolismo , Bacillus/genética , Bacillus/enzimologia , Queratinas/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética , Animais , Plumas/metabolismo , Meios de Cultura/química , Aves Domésticas , RNA Ribossômico 16S/genética , Bovinos , Microbiologia do Solo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Fermentação , Cabelo
9.
Biomed Mater ; 19(3)2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38626780

RESUMO

Wool derived keratin, due to its demonstrated ability to promote bone formation, has been suggested as a potential bioactive material for implant surfaces. The aim of this study was to assess the effects of keratin-coated titanium on osteoblast functionin vitroand bone healingin vivo. Keratin-coated titanium surfaces were fabricated via solvent casting and molecular grafting. The effect of these surfaces on the attachment, osteogenic gene, and osteogenic protein expression of MG-63 osteoblast-like cells were quantifiedin vitro. The effect of these keratin-modified surfaces on bone healing over three weeks using an intraosseous calvaria defect was assessed in rodents. Keratin coating did not affect MG-63 proliferation or viability, but enhanced osteopontin, osteocalcin and bone morphogenetic expressionin vitro. Histological analysis of recovered calvaria specimens showed osseous defects covered with keratin-coated titanium had a higher percentage of new bone area two weeks after implantation compared to that in defects covered with titanium alone. The keratin-coated surfaces were biocompatible and stimulated osteogenic expression in adherent MG-63 osteoblasts. Furthermore, a pilot preclinical study in rodents suggested keratin may stimulate earlier intraosseous calvaria bone healing.


Assuntos
Regeneração Óssea , Proliferação de Células , Materiais Revestidos Biocompatíveis , Queratinas , Osteoblastos , Osteogênese , Crânio , Titânio , Titânio/química , Osteoblastos/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/metabolismo , Regeneração Óssea/efeitos dos fármacos , Animais , Queratinas/química , Queratinas/metabolismo , Humanos , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Crânio/efeitos dos fármacos , Crânio/lesões , Osteogênese/efeitos dos fármacos , Ratos , Propriedades de Superfície , Masculino , Linhagem Celular , Adesão Celular/efeitos dos fármacos , Teste de Materiais , Sobrevivência Celular/efeitos dos fármacos , Ratos Sprague-Dawley
10.
J Colloid Interface Sci ; 666: 176-188, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38593652

RESUMO

AIM: Ultraviolet B (UVB) radiation can compromise the functionality of the skin barrier through various mechanisms. We hypothesize that UVB induce photochemical alterations in the components of the outermost layer of the skin, known as the stratum corneum (SC), and modulate its antioxidative defense mechanisms. Catalase is a well-known antioxidative enzyme found in the SC where it acts to scavenge reactive oxygen species. However, a detailed characterization of acute UVB exposure on the activity of native catalase in the SC is lacking. Moreover, the effects of UVB irradiation on the molecular dynamics and organization of the SC keratin and lipid components remain unclear. Thus, the aim of this work is to characterize consequences of UVB exposure on the structural and antioxidative properties of catalase, as well as on the molecular and global properties of the SC matrix surrounding the enzyme. EXPERIMENTS: The effect of UVB irradiation on the catalase function is investigated by chronoamperometry with a skin covered oxygen electrode, which probes the activity of native catalase in the SC matrix. Circular dichroism is used to explore changes of the catalase secondary structure, and gel electrophoresis is used to detect fragmentation of the enzyme following the UVB exposure. UVB induced alterations of the SC molecular dynamics and structural features of the SC barrier, as well as its water sorption behavior, are investigated by a complementary set of techniques, including natural abundance 13C polarization transfer solid-state NMR, wide-angle X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, and dynamic vapor sorption microbalance. FINDINGS: The findings show that UVB exposure impairs the antioxidative function of catalase by deactivating both native catalase in the SC matrix and lyophilized catalase. However, UVB radiation does not alter the secondary structure of the catalase nor induce any observable enzyme fragmentation, which otherwise could explain deactivation of its function. NMR measurements on SC samples show a subtle increase in the molecular mobility of the terminal segments of the SC lipids, accompanied by a decrease in the mobility of lipid chain trans-gauche conformers after high doses of UVB exposure. At the same time, the NMR data suggest increased rigidity of the polypeptide backbone of the keratin filaments, while the molecular mobility of amino acid residues in random coil domains of keratin remain unaffected by UVB irradiation. The FTIR data show a consistent decrease in absorbance associated with lipid bond vibrations, relative to the main protein bands. Collectively, the NMR and FTIR data suggest a small modification in the composition of fluid and solid phases of the SC lipid and protein components after UVB exposure, unrelated to the hydration capacity of the SC tissue. To conclude, UVB deactivation of catalase is anticipated to elevate oxidative stress of the SC, which, when coupled with subtle changes in the molecular characteristics of the SC, may compromise the overall skin health and elevate the likelihood of developing skin disorders.


Assuntos
Catalase , Raios Ultravioleta , Catalase/metabolismo , Catalase/química , Humanos , Epiderme/efeitos da radiação , Epiderme/metabolismo , Epiderme/enzimologia , Pele/efeitos da radiação , Pele/metabolismo , Pele/química , Queratinas/química , Queratinas/metabolismo
11.
Microb Cell Fact ; 23(1): 102, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38575972

RESUMO

BACKGROUND: Poultry feather waste has a potential for bioenergy production because of its high protein content. This research explored the use of chicken feather hydrolysate for methane and hydrogen production via anaerobic digestion and bioelectrochemical systems, respectively. Solid state fermentation of chicken waste was conducted using a recombinant strain of Bacillus subtilis DB100 (p5.2). RESULTS: In the anaerobic digestion, feather hydrolysate produced maximally 0.67 Nm3 CH4/kg feathers and 0.85 mmol H2/day.L concomitant to COD removal of 86% and 93%, respectively. The bioelectrochemical systems used were microbial fuel and electrolysis cells. In the first using a microbial fuel cell, feather hydrolysate produced electricity with a maximum cell potential of 375 mV and a current of 0.52 mA. In the microbial electrolysis cell, the hydrolysate enhanced the hydrogen production rate to 7.5 mmol/day.L, with a current density of 11.5 A/m2 and a power density of 9.26 W/m2. CONCLUSIONS: The data indicated that the sustainable utilization of keratin hydrolysate to produce electricity and biohydrogen via bioelectrical chemical systems is feasible. Keratin hydrolysate can produce electricity and biofuels through an integrated aerobic-anaerobic fermentation system.


Assuntos
Galinhas , Plumas , Animais , Anaerobiose , Galinhas/metabolismo , Hidrogênio/metabolismo , Queratinas/metabolismo , Metano/metabolismo , Biocombustíveis , Reatores Biológicos
12.
Int J Dev Biol ; 68(1): 39-45, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38591692

RESUMO

Keratin 17 (K17) is thought to be a candidate target gene for regulation by Lymphoid Enhancer Factor-1 (Lef-1). K17 is a marker that distinguishes junctional epithelium (JE) from epithelial rests of Malassez (ERM). However, the relationship of Lef-1 to K17 is not clear in this context. Moreover, the expression of other keratins such as K5, K6, K7 and K16 is not reported. Therefore, the aim of our study was to assay the expression of K5, K6, K7, K14, K16, K17 and Lef-1 in postnatal developing teeth, and clarify the corresponding immunophenotypes of the JE and ERM. Upper jaws of Wistar rats aged from postnatal (PN) day 3.5 to PN21 were used and processed for immunohistochemistry. K5 and K14 were intensely expressed in inner enamel epithelium (IEE), reduced enamel epithelium (REE), ERM and JE. There was no staining for K16 in the tissue, except for strong staining in the oral epithelium. Specifically, at PN3.5 and PN7, K17 was initially strongly expressed and then negative in the IEE. At PN16 and PN21, both REE and ERM were strongly stained for K17, whereas K17 was negative in the JE. In addition, K6, K7 and Lef-1 were not detected in any tissue investigated. REE and ERM have an identical keratin expression pattern before eruption, while JE differs from ERM in the expression of K17 after eruption. The expression of K17 does not coincide with that of Lef-1. These data indicate that JE has a unique phenotype different from ERM, which is of odontogenic origin.


Assuntos
Inserção Epitelial , Descanso , Ratos , Animais , Inserção Epitelial/metabolismo , Ratos Wistar , Epitélio/metabolismo , Imuno-Histoquímica , Queratinas/metabolismo
13.
BMC Biotechnol ; 24(1): 23, 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38671404

RESUMO

Volumetric loss is one of the challenging issues in muscle tissue structure that causes functio laesa. Tissue engineering of muscle tissue using suitable hydrogels is an alternative to restoring the physiological properties of the injured area. Here, myogenic properties of type I collagen (0.5%) and keratin (0.5%) were investigated in a mouse model of biceps femoris injury. Using FTIR, gelation time, and rheological analysis, the physicochemical properties of the collagen (Col)/Keratin scaffold were analyzed. Mouse C2C12 myoblast-laden Col/Keratin hydrogels were injected into the injury site and histological examination plus western blotting were performed to measure myogenic potential after 15 days. FTIR indicated an appropriate interaction between keratin and collagen. The blend of Col/Keratin delayed gelation time when compared to the collagen alone group. Rheological analysis revealed decreased stiffening in blended Col/Keratin hydrogel which is favorable for the extrudability of the hydrogel. Transplantation of C2C12 myoblast-laden Col/Keratin hydrogel to injured muscle tissues led to the formation of newly generated myofibers compared to cell-free hydrogel and collagen groups (p < 0.05). In the C2C12 myoblast-laden Col/Keratin group, a low number of CD31+ cells with minimum inflammatory cells was evident. Western blotting indicated the promotion of MyoD in mice that received cell-laden Col/Keratin hydrogel compared to the other groups (p < 0.05). Despite the increase of the myosin cell-laden Col/Keratin hydrogel group, no significant differences were obtained related to other groups (p > 0.05). The blend of Col/Keratin loaded with myoblasts provides a suitable myogenic platform for the alleviation of injured muscle tissue.


Assuntos
Queratinas , Desenvolvimento Muscular , Músculo Esquelético , Animais , Camundongos , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Queratinas/metabolismo , Linhagem Celular , Hidrogéis/química , Neovascularização Fisiológica/efeitos dos fármacos , Engenharia Tecidual/métodos , Modelos Animais de Doenças , Colágeno/metabolismo , Mioblastos/metabolismo , Mioblastos/citologia , Masculino , Alicerces Teciduais/química , Angiogênese
15.
Int J Biol Macromol ; 263(Pt 1): 130688, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38458294

RESUMO

This study reports the rational engineering of the S1' substrate-binding pocket of a thermally-stable keratinase from Pseudomonas aeruginosa 4-3 (4-3Ker) to improve substrate specificity to typical keratinase (K/C > 0.5) and catalytic activity without compromising thermal stability for efficient keratin degradation. Of 10 chosen mutation hotspots in the S1' substrate-binding pocket, the top three mutations M128R, A138V, and V142I showing the best catalytic activity and substrate specificity were identified. Their double and triple combinatorial mutants synergistically overcame limitations of single mutants, fabricating an excellent M128R/A138V/V142I triple mutant which displayed a 1.21-fold increase in keratin catalytic activity, 1.10-fold enhancement in keratin/casein activity ratio, and a 3.13 °C increase in half-inactivation temperature compared to 4-3Ker. Molecular dynamics simulations revealed enhanced flexibility of critical amino acid residues at the substrate access tunnel, improved global protein rigidity, and heightened hydrophobicity within the active site likely underpinned the increased catalytic activity and substrate specificity. Additionally, the triple mutant improved the feather degradation rate by 32.86 % over the wild-type, far exceeding commercial keratinase in substrate specificity and thermal stability. This study exemplified engineering a typical keratinase with enhanced substrate specificity, catalytic activity, and thermal stability from thermally-stable 4-3Ker, providing a more robust tool for feather degradation.


Assuntos
Queratinas , Peptídeo Hidrolases , Queratinas/metabolismo , Especificidade por Substrato , Peptídeo Hidrolases/metabolismo , Temperatura , Concentração de Íons de Hidrogênio
16.
Nat Commun ; 15(1): 2328, 2024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-38499530

RESUMO

Cornified skin appendages, such as hair and nails, are major evolutionary innovations of terrestrial vertebrates. Human hair and nails consist largely of special intermediate filament proteins, known as hair keratins, which are expressed under the control of the transcription factor Hoxc13. Here, we show that the cornified claws of Xenopus frogs contain homologs of hair keratins and the genes encoding these keratins are flanked by promoters in which binding sites of Hoxc13 are conserved. Furthermore, these keratins and Hoxc13 are co-expressed in the claw-forming epithelium of frog toe tips. Upon deletion of hoxc13, the expression of hair keratin homologs is abolished and the development of cornified claws is abrogated in X. tropicalis. These results indicate that Hoxc13-dependent expression of hair keratin homologs evolved already in stem tetrapods, presumably as a mechanism for protecting toe tips, and that this ancestral genetic program was coopted to the growth of hair in mammals.


Assuntos
Queratinas Específicas do Cabelo , Fatores de Transcrição , Animais , Humanos , Fatores de Transcrição/metabolismo , Pele/metabolismo , Cabelo/metabolismo , Queratinas/genética , Queratinas/metabolismo , Anfíbios , Mamíferos/metabolismo
17.
In Vitro Cell Dev Biol Anim ; 60(3): 236-248, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38502372

RESUMO

The epidermis is largely composed of keratinocytes (KCs), and the proliferation and differentiation of KCs from the stratum basale to the stratum corneum is the cellular hierarchy present in the epidermis. In this study, we explore the differentiation abilities of human hematopoietic stem cells (HSCs) into KCs. Cultured HSCs positive for CD34, CD45, and CD133 with prominent telomerase activity were induced with keratinocyte differentiation medium (KDM), which is composed of bovine pituitary extract (BPE), epidermal growth factor (EGF), insulin, hydrocortisone, epinephrine, transferrin, calcium chloride (CaCl2), bone morphogenetic protein 4 (BMP4), and retinoic acid (RA). Differentiation was monitored through the expression of cytokeratin markers K5 (keratin 5), K14 (keratin 14), K10 (keratin 10), K1 (keratin 1), transglutaminase 1 (TGM1), involucrin (IVL), and filaggrin (FLG) on day 0 (D0), day 6 (D6), day 11 (D11), day 18 (D18), day 24 (D24), and day 30 (D30) using immunocytochemistry, fluorescence microscopy, flow cytometry, qPCR, and Western blotting. The results revealed the expression of K5 and K14 genes in D6 cells (early keratinocytes), K10 and K1 genes in D11-D18 cells (mature keratinocytes) with active telomerase enzyme, and FLG, IVL, and TGM1 in D18-D24 cells (terminal keratinocytes), and by D30, the KCs were completely enucleated similar to cornified matrix. This method of differentiation of HSCs to KCs explains the cellular order exists in the normal epidermis and opens the possibility of exploring the use of human HSCs in the epidermal differentiation.


Assuntos
Telomerase , Animais , Humanos , Diferenciação Celular , Células Cultivadas , Células Epidérmicas/metabolismo , Epiderme/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Queratinócitos/metabolismo , Queratinas/metabolismo , Telomerase/genética , Telomerase/metabolismo
18.
Poult Sci ; 103(5): 103606, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38479096

RESUMO

The tons of keratin waste are produced by the poultry and meat industry which is an insoluble and protein-rich material found in hair, feathers, wool, and some epidermal wastes. These waste products could be degraded and recycled to recover protein, which can save our environment. One of the potential strategy to achieve this target is use of microbial biotreatment which is more convenient, cost-effective, and environment-friendly by formulating hydrolysate complexes that could be administered as protein supplements, bioactive peptides, or animal feed ingredients. Keratin degradation shows great promise for long-term protein and amino acid recycling. According to the MEROPS database, known keratinolytic enzymes currently belong to at least 14 different protease families, including S1, S8, S9, S10, S16, M3, M4, M14, M16, M28, M32, M36, M38, and M55. In addition to exogenous attack (proteases from families S9, S10, M14, M28, M38, and M55), the various keratinolytic enzymes also function via endo-attack (proteases from families S1, S8, S16, M4, M16, and M36). Biotechnological methods have shown great promise for enhancing keratinase expression in different strains of microbes and different protein engineering techniques in genetically modified microbes such as bacteria and some fungi to enhance keratinase production and activity. Some microbes produce specific keratinolytic enzymes that can effectively degrade keratin substrates. Keratinases have been successfully used in the leather, textile, and pharmaceutical industries. However, the production and efficiency of existing enzymes need to be optimized before they can be used more widely in other processes, such as the cost-effective pretreatment of chicken waste. These can be improved more effectively by using various biotechnological applications which could serve as the best and novel approach for recycling and degrading biomass. This paper provides practical insights about molecular strategies to enhance keratinase expression to effectively utilize various poultry wastes like feathers and feed ingredients like soybean pulp. Furthermore, it describes the future implications of engineered keratinases for environment friendly utilization of wastes and crop byproducts for their better use in the poultry feed industry.


Assuntos
Ração Animal , Peptídeo Hidrolases , Aves Domésticas , Animais , Ração Animal/análise , Galinhas/genética , Expressão Gênica , Queratinas/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética
20.
Eur J Surg Oncol ; 50(6): 108251, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38492258

RESUMO

OBJECTIVES: Sarcomatoid renal pelvis carcinoma (SRPC) is a rare variant of RPC. We aimed to summarize the clinicopathological features and prognostic factors of SRPC. METHODS: In this retrospective study, we collected data from 24 patients with SRPC who were treated at the Department of Urology, Affiliated Hospital of Qingdao University between 2008 and 2021. The clinicopathological features of the patients were obtained from their medical records to evaluate the diagnosis, prognostic factors, and response to systemic therapy. RESULTS: Immunohistochemical staining revealed that cytokeratin was expressed in 19 patients with SRPC, while vimentin was expressed in all patients. Computer tomography showed these tumors as low-density (n = 12) or mixed-density masses, with or without necrotic areas (n = 12). All patients showed different degrees of enhancement on computed tomography. Lymph node metastasis was present in 6 patients and distant metastasis in 5. The median survival of all patients was 28 months. Patients without metastasis had a median survival of 46 months compared with 18 months in those with metastasis (P < 0.05). Necrosis had no significant influence on prognosis (P > 0.05). The median survival of patients with and without hydronephrosis was 18 and 104 months (P < 0.05). Among patients without metastasis, those without hydronephrosis survived longer than those with hydronephrosis (104 vs 18 months, P < 0.05), and necrosis had no effect on prognosis. In patients with metastasis, necrosis and hydronephrosis had no effect on prognosis (P > 0.05). CONCLUSION: The prognosis of SRPC is poor, and the clinical stage, particularly the presence of distant metastasis, has a significant impact on prognosis.


Assuntos
Neoplasias Renais , Pelve Renal , Humanos , Feminino , Pessoa de Meia-Idade , Masculino , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Estudos Retrospectivos , Idoso , Pelve Renal/patologia , Adulto , Prognóstico , Metástase Linfática , Vimentina/metabolismo , Tomografia Computadorizada por Raios X , Taxa de Sobrevida , Queratinas/metabolismo , Hidronefrose/etiologia , Nefrectomia , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Necrose
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