RESUMO
Grain size and shape are critical agronomic traits that directly impact rice grain yield. Identifying genes that control these traits can provide new strategies for yield improvement. In this study, we characterized a rice mutant, reduced grain length (rgl), which exhibited decreased grain length due to reduced cell proliferation. Map-based cloning identified a base deletion in OsRGL2, a gene encoding a keratin-associated protein (KAP), as the cause of the mutant phenotype. CRISPR-Cas9-generated OsRGL2 knockout mutants also displayed reduced grain length, confirming its role. OsRGL2 transcripts were detected in various tissues, with relative higher gene expression in young panicles, and OsRGL2 was localized to the plasma membrane. Overexpression of OsRGL2 increased grain size by promoting cell proliferation in the spikelet hull and significantly enhanced grain yield per plant. Importantly, OsRGL2 was found to interact with RGB1, indicating that OsRGL2 positively regulates grain size and yield through its interaction with RGB1. Additionally, OsRGL2 regulated the expression of cell cycle-related genes, further elucidating its role in grain development. These findings demonstrate that OsRGL2 is a positive regulator of grain size in rice, and manipulating its expression may offer a novel strategy for enhancing rice grain yield.
Assuntos
Grão Comestível , Regulação da Expressão Gênica de Plantas , Mutação , Oryza , Proteínas de Plantas , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mutação/genética , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Queratinas/metabolismo , Queratinas/genética , Fenótipo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismoRESUMO
Existing clinical biomarkers do not reliably predict treatment response or disease progression in patients with advanced intrahepatic cholangiocarcinoma (ICC). Circulating neoplastic-immune hybrid cells (CHCs) have great promise as a blood-based biomarker for patients with advanced ICC. Peripheral blood specimens were longitudinally collected from patients with advanced ICC enrolled in the HELIX-1 phase II clinical trial (NCT04251715). CHCs were identified by co-expression of pan-cytokeratin (CK) and CD45, and levels were correlated to patient clinical disease course. Unsupervised machine learning was then performed to extract their morphological features to compare them across disease courses. Five patients were included in this study, with a median of nine specimens collected per patient. A median of 13.5 CHCs per 50,000 peripheral blood mononuclear cells were identified at baseline, and levels decreased to zero following the initiation of treatment in all patients. Counts remained undetectable in three patients who demonstrated end-of-trial clinical treatment response and conversely increased in two patients with evidence of therapeutic resistance. In the post-trial surveillance period, interval counts increased prior to or at the time of clinical progression in three patients and remain undetectable in one patient with continued long-term disease stability. Using our machine learning platform, treatment-resistant CHCs exhibited upregulation of CK and downregulation of CD45 relative to treatment-responsive CHCs. CHCs represent a promising blood-based biomarker to supplement traditional radiographic and biochemical measures.
Assuntos
Neoplasias dos Ductos Biliares , Biomarcadores Tumorais , Colangiocarcinoma , Células Neoplásicas Circulantes , Humanos , Colangiocarcinoma/sangue , Colangiocarcinoma/patologia , Biomarcadores Tumorais/sangue , Masculino , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/sangue , Neoplasias dos Ductos Biliares/diagnóstico , Neoplasias dos Ductos Biliares/metabolismo , Feminino , Pessoa de Meia-Idade , Prognóstico , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Idoso , Antígenos Comuns de Leucócito/metabolismo , Queratinas/metabolismoRESUMO
BACKGROUND: Chicken feathers contribute to large quantities of keratinaceous wastes that pose serious environmental problems and must be catered to properly. Chicken feathers are also a potential source of vital proteins, peptides, and amino acids, which could be used as low-cost animal feeds. Therefore, there has been increasing interest in keratinase-producing microbes for reprocessing and using keratinous biomaterials. METHODS: Among the five isolated keratinolytic microorganisms, one microbe, Bacillus XT 01, produced a significant amount of enzyme activity, which was partially characterized. The potential of this protease-producing microbe was investigated for converting feather keratin waste to valuable protein hydrolysate. RESULTS: Maximum keratinase production was observed after 5 days of incubating Bacillus XT 01 at an optimum temperature of 45 °C and pH 8.5. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and zymogram of ammonium sulfate precipitated culture supernatant showed the presence of several proteolytic enzymes with molecular weights between 30 and 60 kDa. The Bacillus strain caused almost complete feather degradation (98%) after 7 days of incubation at 45 °C in a shake culture medium. Antioxidant and reducing activities of the feather protein hydrolysate (FPH) elevated with increased cultivation time. Investigation of the effect of feather protein hydrolysate on plants indicated improved plant growth regarding the agronomic parameters, such as plant height, number of trifoliate leaves, number of pods, pod length, number of seeds per pod, and root length, which increased by 30.84%, 49.32%, 70.90%, 53.27%, 60.03%, and 54.71%, respectively. CONCLUSIONS: The prospective of Bacillus XT 01 for degrading feather waste keratin to highly valued hydrolyzed feather protein offers effectiveness in the poultry industry and ultimately decreases environmental pollution hazards.
Assuntos
Bacillus , Galinhas , Plumas , Queratinas , Peptídeo Hidrolases , Hidrolisados de Proteína , Plumas/química , Animais , Peptídeo Hidrolases/metabolismo , Bacillus/enzimologia , Hidrolisados de Proteína/metabolismo , Hidrolisados de Proteína/química , Queratinas/metabolismo , Concentração de Íons de HidrogênioRESUMO
Bacillus paralicheniformis T7, which exhibits high proteolytic and keratinolytic activities, was isolated from soil in Kazakhstan. Its secreted proteases were thermostable and alkaline, demonstrating maximum activity at 70 °C and pH 9.0. The proteases and keratinases of this strain were sensitive to Ni2+, Co2+, Mn2+, and Cd2+, with Cu2+, Co2+ and Cd2+ negatively affecting keratinolytic activity, and Fe3+ ions have a strong inhibitory effect on proteolytic and keratinolytic activity. Seven proteases were identified in the enzymatic extract of B. paralicheniformis T7: four from the serine peptidase family and three from the metallopeptidase family. The proteases hydrolyzed 1 mg of casein, hemoglobin, gelatin, ovalbumin, bovine serum albumin, or keratin within 15 s to 30 min. The high keratinolytic activity of this strain was confirmed through the degradation of chicken feathers, horns, hooves, wool, and cattle hide. Chicken feathers were hydrolyzed in 4 days, and the degrees of hydrolysis for cattle hide, wool, hoof, and horn after 7 days of cultivation were 97.2, 34.5, 29.6, and 3.6%, respectively. During submerged fermentation with feather medium in a laboratory bioreactor, the strain secreted enzymes with 249.20 ± 7.88 U/mL protease activity after 24 h. Thus, B. paralicheniformis T7 can be used to produce proteolytic and keratinolytic enzymes for application in processing proteinaceous raw materials and keratinous animal waste.
Assuntos
Bacillus , Peptídeo Hidrolases , Proteólise , Animais , Bacillus/enzimologia , Bacillus/metabolismo , Peptídeo Hidrolases/metabolismo , Bovinos , Queratinas/metabolismo , Plumas/metabolismo , Concentração de Íons de Hidrogênio , Galinhas , Hidrólise , Microbiologia do Solo , Fermentação , Proteínas de Bactérias/metabolismoRESUMO
The process of keratinization and cornification in the developing beak has been studied through immunofluorescence and immunogold electron microscopy in chick and zebrafinch embryos. After the curved beak anlagen appears at the tip of the maxillar bone, 5-8 layers of embryonic epidermis are generated from the basal layer of the epidermis. These cells are weakly immunoabeled for IFKs (Intermediate Filament Keratins) and more intensely for scaffoldin, a protein of the EDC (Epidermal Differentiation Complex) involved in the soft keratinization of the embryonic epidermis. Immunolabeling for CBPs (Corneous Beta Proteins) is visible in the transitional embryonic layers that are temporarily generated between the embryonic and definitive beak epidermis. The electron microscope reveals that intermediate layers contain immunolabeled periderm granules for scaffoldin mixed with bundles of corneous material immunolabeled for CBPs. Intense CBPs labeling occurs in the compacting corneous bundles of beta-keratinocytes in the definitive beak while scaffolding labeling disappears. The embryonic epidermis is sloughed before hatching. Sox (Sulfhydryl Oxidase) immunolabeling reveals that the enzyme is almost absent in embryonic layers but is present in transitional and definitive beta-keratinocytes. This indicates the formation of cross-linked disulfide bonds in the definitive corneous layer of the beak. Some calcium precipitation, suggested from von Kossa staining, occurs in the corneous layers only on the 18th day of development in the chick, in preparation for hatching.
Assuntos
Bico , Epiderme , Animais , Bico/embriologia , Epiderme/embriologia , Epiderme/ultraestrutura , Epiderme/metabolismo , Embrião de Galinha/embriologia , Imuno-Histoquímica , Queratinas/metabolismo , Microscopia de Fluorescência , Imunofluorescência , Microscopia ImunoeletrônicaRESUMO
Microbes within a consortium exhibit a synergistic interaction, enhancing their collective capacity to perform functions more effectively than a single species, especially in the degradation of keratin-rich substrates. To achieve a more stable and efficient breakdown of chicken feathers, a comprehensive screening of over 9,000 microbial strains was undertaken. This meticulous selection process identified strains with the capability to degrade keratin effectively. Subsequently, antagonistic tests were conducted to isolate strains of fungi and bacteria that were non-antagonistic, which were then used to form the artificial microbial consortia. The optimal fermentation conditions for the keratinophilic microbial consortia were determined through the optimization of response surface methodology. The results revealed that 11 microbial strains-comprising of 4 fungi and 7 bacteria-were particularly proficient in degrading chicken feathers. The artificially constructed microbial consortia (AMC) comprised two bacterial strains and one fungal strain. The optimal conditions for feathers degradation were identified as a 10 g/L concentration of chicken feathers, a 2.6% microbial inoculation volume and a fermentation fluid pH of 9. Under these conditions, the degradation rate for chicken feathers reached a significant 74.02%, representing an 11.45% increase over the pre-optimization rate. The AMC developed in this study demonstrates the potential for efficient and economical process of livestock and poultry feathers. It provides innovative insights and a theoretical foundation for tackling the challenging degradation of keratin-rich materials. Furthermore, this research lays the groundwork for the separation and purification of keratins, as well as the development of novel proteases, which could have profound implications for a range of applications.
Assuntos
Bactérias , Galinhas , Plumas , Fermentação , Fungos , Queratinas , Consórcios Microbianos , Plumas/microbiologia , Plumas/metabolismo , Animais , Galinhas/microbiologia , Queratinas/metabolismo , Fungos/metabolismo , Fungos/classificação , Fungos/isolamento & purificação , Fungos/genética , Bactérias/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Concentração de Íons de Hidrogênio , Biodegradação AmbientalRESUMO
Few studies have evaluated cytokeratin's (CK) staining patterns in atypical endometrial hyperplasia (AEH) coexisting with early-stage endometrial cancer (EC). We aimed to assess the staining patterns of selected CKs (CK7, CK19, CK20, CK AE1/AE3) in 74 patients with coexisting AEH and EC by independently analyzing both morphological variables. Specimens were collected from women with AEH and EC who underwent surgical interventions between 2012 and 2019 at the Department of Obstetrics and Gynecology of Vilnius University Hospital "Santaros Klinikos" in Vilnius, Lithuania. Immunostaining was also qualitatively classified as being heterogeneous or intense. The results revealed heterogeneous CK7 expression in all AEH cases and intense staining in 95.95% cases of AEH. The heterogeneous expression of CK7 was detected in all EC specimens. Intense CK7 expression was observed in 95.09% cases of EC G1 and in all G2 ECs. Heterogenous CK19 expression was present in all AEH specimens with intense staining in 92.42% of cases. Heterogeneous CK19 expression was observed in all EC samples with intense expression in 86.27% cases of EC G1 and 100% cases of EC G2. Interestingly, a significant relationship was found when comparing the heterogeneous expression of CK19 between AEH and well-differentiated EC. A significant difference was reported in the intense expression of CK AE1/AE3 (p = 0.031; p = 0.029) between AEH and G2 ECs and in the intense expression of CK AE1/AE3 between G1 and G2 ECs. CK20 staining was not a characteristic feature for AEH and early-stage EC. CK staining is present either in AEH or in early-stage endometrioid-subtype EC in different manners. Heterogeneous CK19 expression was significantly more common in AEH than in EC. CK20 expression was not associated with either AEH nor early-stage EC. An intense expression of CK AE1/AE3 was mainly present in moderately differentiated ECs, whereas the intense reactivity of AE1/AE3 showed a significant difference in well to moderately differentiated uterine tumors. The clinical implication of CK staining may aid in the more accurate diagnosis of AEH and early-stage EC as well as detect micrometastases leading to better oncological outcomes.
Assuntos
Hiperplasia Endometrial , Neoplasias do Endométrio , Humanos , Feminino , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Hiperplasia Endometrial/metabolismo , Hiperplasia Endometrial/patologia , Pessoa de Meia-Idade , Idoso , Queratinas/metabolismo , Adulto , Biomarcadores Tumorais/metabolismo , Queratina-20/metabolismo , Queratina-7/metabolismo , Imuno-HistoquímicaRESUMO
Chinese Tan sheep lambs are recognised for having tight 'spring-like' curly wool when young, but this phenotype disappears with age. This wool consists of shorter, fine wool fibres (which are usually unmedullated) and heterotypic hair fibres (which are frequently medullated), which are referred to as 'halo hair'. Both the wool and hair fibres consist of α-keratin proteins embedded in a keratin-associated protein (KAP) matrix. Of these KAPs, the KAP20-1 gene (designated KRTAP20-1) and its effect on four fibre traits (mean fibre curvature, mean fibre diameter, fibre diameter standard deviation, and coefficient of variation of fibre diameter) of Tan lambs was studied. Seven previously identified KRTAP20-1 variants (A, B, D, E, F, G, and H) of KRTAP20-1 were revealed, but the previously identified variant C was not present. Of the seven variants detected, only two (A and G) were common and present at frequencies greater than 5%, and the effect of these on the fibre traits of the finer wool fibres was assessed. It was found that variant G was associated with an increased mean fibre curvature in these wool fibres. This suggests that KRTAP20-1 might possibly be expressed differentially in the two fibre types, which may be of future value in breeding.
Assuntos
Fibra de Lã , Lã , Animais , Ovinos/genética , Lã/metabolismo , Fenótipo , Carneiro Doméstico/genética , Queratinas Específicas do Cabelo/genética , Queratinas/genética , Queratinas/metabolismo , Variação GenéticaRESUMO
Keratin is one of the major components of solid waste, and the degradation products have extensive applications in various commercial industries. Due to the complexity of the structure of keratin, especially the disulfide bonds between keratin polypeptides, keratinolytic activity is efficient with a mixture of proteins with proteases, peptidases, and oxidoreductase activity. The present work aimed to create an engineered chimeric protein with a disulfide reductase domain and a protease domain connected with a flexible linker. The structure, stability, and substrate interaction were analyzed using the protein modeling tools and codon-optimized synthetic gene cloned, expressed, and purified using Ni2+-NTA chromatography. The keratinolytic activity of the protein was at its maximum at 70 °C. The suitable pH for the enzyme activity was pH 8. While Ni2+, Mg2+, and Na+ inhibited the keratinolytic activity, Cu2+, Ca2+, and Mn2+ enhanced it significantly. Biochemical characterization of the protease domain indicated significant keratinolytic activity at 70 °C at pH 10.0 but was less efficient than the chimeric protein. Experiments using feathers as the substrate showed a clear degradation pattern in the SEM analysis. The samples collected from the degradation experiments indicated the release of proteins (2-fold) and amino acids (8.4-fold) in a time-dependent manner. Thus, the protease with an added disulfide reductase domain showed excellent keratin degradation activity and has the potential to be utilized in the commercial industries.
Assuntos
Peptídeo Hidrolases , Proteínas Recombinantes de Fusão , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética , Queratinas/química , Queratinas/metabolismo , Estabilidade Enzimática , Animais , Engenharia de Proteínas/métodos , Temperatura , Domínios Proteicos , Modelos Moleculares , Plumas/química , Especificidade por SubstratoRESUMO
The subareolar Sappey's plexus was studied using color lymphography and immunohistochemical methods with a panel of antibodies to podoplanin, smooth muscle actin, low molecular weight cytokeratin AE1/AE3, and GATA3 on archival material obtained during radical mastectomies and sectoral resections with lymph node dissection from 86 patients diagnosed with non-special type breast cancer. At the macro- and microscopic levels, the connection between the subareolar lymphatic plexus and the lymphatic system of the breast parenchyma has been demonstrated. In triple negative breast cancer with metastases to the axillary lymph nodes, the involvement of subareolar lymphatic plexus into lymphogenous metastasis to the lymph nodes of the axillary lymphatic collector was shown.
Assuntos
Axila , Neoplasias da Mama , Linfonodos , Metástase Linfática , Vasos Linfáticos , Humanos , Feminino , Metástase Linfática/patologia , Vasos Linfáticos/patologia , Linfonodos/patologia , Pessoa de Meia-Idade , Neoplasias da Mama/patologia , Actinas/metabolismo , Fator de Transcrição GATA3/metabolismo , Excisão de Linfonodo , Adulto , Linfografia/métodos , Neoplasias de Mama Triplo Negativas/patologia , Idoso , Queratinas/metabolismo , Glicoproteínas de MembranaRESUMO
BACKGROUND: Fungi clinically relevant to human skin comprise prevalent commensals and well-known pathogens. Only rarely human skin harbours fungi that evade identification. OBJECTIVE: To characterise an enigmatic specimen isolated from a skin lesion. METHODS: A comprehensive clinical and mycological workup including conventional methods for phenotypic characterisation and sequencing based on internal transcribed spacer (ITS) and large subunit (LSU) regions to infer a phylogenetic tree. RESULTS: Cultures on common solid media were macroscopically inconspicuous initially until mycelial tufts developed on the surface, notably on potato dextrose agar. Polymorphous chlamydospores were detected but no aleurospores and ascomata. At 26°C, the isolate grew on standard agars, plant materials and garden soil and utilised peptone, keratins, lipids, inulin, erythrocytes and cellulose. It also grew at 5°C and at 37°C. Nucleotide sequences of its ITS region showed 93% similarity to sequences of different Malbranchea species. The closest matches among LSU rRNA sequences were obtained with the genera Amauroascus, Arthroderma, Auxarthronopsis and Malbranchea (93%-95%). A combined phylogenetic analysis placed the fungus in a sister clade to Neogymnomycetaceae, classified as incertae sedis in Onygenales, on a large distance to either Diploospora rosea or 'Amauroascus' aureus. CONCLUSIONS: The genus Inopinatus gen. nov. (MB854685) with the species Inopinatus corneliae sp. nov. (MB854687) is introduced to accommodate our isolate (holotype: DSM 116806; isotypes: CBS 151104, IHEM 29063). Probably Inopinatus corneliae is a geophilic species that, although potentially harmful, was no relevant pathogen in our case. Its ecology, epidemiology and pathogenicity need to be further clarified.
Assuntos
DNA Fúngico , DNA Espaçador Ribossômico , Onygenales , Filogenia , Análise de Sequência de DNA , Pele , Humanos , Pele/microbiologia , Onygenales/genética , Onygenales/classificação , Onygenales/isolamento & purificação , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Dermatomicoses/microbiologia , Queratinas/metabolismo , DNA Ribossômico/genética , Masculino , Técnicas de Tipagem MicológicaRESUMO
The volume of difficult-to-process keratin waste is increasing as a result of rising global meat production. If not properly managed, this waste can contribute to environmental pollution and pose a threat to human and animal welfare. An interesting and more sustainable alternative is therefore the bioconversion of keratin using microorganisms and their enzymes. This work aimed to isolate bacteria from soil samples and zoonotic keratins and to evaluate their enzymatic capacity to degrade α- and ß-keratin wastes. A total of 113 bacterial strains were isolated from environmental samples and subjected to taxonomic identification using the MALDI-TOF MS technique and to a two-step screening for proteolytic and keratinolytic activity. The ability to degrade a ß-rich keratin substrate was observed in almost all of the strains isolated from soil and horsehairs. In contrast, when an α-rich keratin substrate was used, the highest levels of hydrolysis were observed only for Ker39, Ker66, Ker85, Ker100, and Ker101. Strains with the highest biodegradation potential were identified using molecular biology methods. Phylogenetic analysis of 16S rDNA gene sequences allowed the assignment of selected keratinolytic microorganisms to the genera Exiguobacterium, Priestia, Curtobacterium, Stenotrophomonas, Bacillus, Kocuria, or Pseudomonas. The results of this study are a promising precursor for the development of new, more sustainable methods of managing keratin waste to produce high-value hydrolysates.
Assuntos
Bactérias , Biodegradação Ambiental , Queratinas , Filogenia , Queratinas/metabolismo , Bactérias/metabolismo , Bactérias/genética , RNA Ribossômico 16S/genética , Microbiologia do Solo , Animais , Peptídeo Hidrolases/metabolismoRESUMO
The cytoskeleton of the cell is constantly exposed to physical forces that regulate cellular functions. Selected members of the LIM (Lin-11, Isl-1, and Mec-3) domain-containing protein family accumulate along force-bearing actin fibers, with evidence supporting that the LIM domain is solely responsible for this force-induced interaction. However, LIM domain's force-induced interactions are not limited to actin. LIMK1 and LMO1, both containing only two tandem LIM domains, are recruited to force-bearing keratin fibers in epithelial cells. This unique recruitment is mediated by their LIM domains and regulated by the sequences outside the LIM domains. Based on in vitro reconstitution of this interaction, LIMK1 and LMO1 directly interact with stretched keratin 8/18 fibers. These results show that LIM domain's mechano-sensing abilities extend to the keratin cytoskeleton, highlighting the diverse role of LIM proteins in force-regulated signaling.
Assuntos
Filamentos Intermediários , Queratinas , Proteínas com Domínio LIM , Quinases Lim , Proteínas com Domínio LIM/metabolismo , Humanos , Quinases Lim/metabolismo , Queratinas/metabolismo , Filamentos Intermediários/metabolismo , Ligação Proteica , Animais , Fatores de Transcrição/metabolismoRESUMO
Pityriasis rubra pilaris (PRP) is an inflammatory papulosquamous dermatosis, characterized by hyperkeratotic follicular papules and erythematous desquamative plaques. The precise pathogenic mechanism underlying PRP remains incompletely understood. Herein, we conduct a case-control study involving a cohort of 102 patients with sporadic PRP and 800 healthy controls of Han Chinese population and identify significant associations (P = 1.73 × 10-6) between PRP and heterozygous mutations in the Keratin 32 gene (KRT32). KRT32 is found to be predominantly localized in basal keratinocytes and exhibits an inhibitory effect on skin inflammation by antagonizing the NF-κB pathway. Mechanistically, KRT32 binds to NEMO, promoting excessive K48-linked polyubiquitination and NEMO degradation, which hinders IKK complex formation. Conversely, loss-of-function mutations in KRT32 among PRP patients result in NF-κB hyperactivation. Importantly, Krt32 knockout mice exhibit a PRP-like dermatitis phenotype, suggesting compromised anti-inflammatory function of keratinocytes in response to external pro-inflammatory stimuli. This study proposes a role for KRT32 in regulating inflammatory immune responses, with damaging variants in KRT32 being an important driver in PRP development. These findings offer insights into the regulation of skin immune homeostasis by keratin and open up the possibility of using KRT32 as a therapeutic target for PRP.
Assuntos
Queratinócitos , Pitiríase Rubra Pilar , Pele , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Adulto Jovem , Estudos de Casos e Controles , Homeostase , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinas/metabolismo , Queratinas/genética , Mutação com Perda de Função , Camundongos Knockout , NF-kappa B/metabolismo , Pitiríase Rubra Pilar/genética , Pitiríase Rubra Pilar/imunologia , Pitiríase Rubra Pilar/patologia , Pitiríase Rubra Pilar/metabolismo , Transdução de Sinais , Pele/patologia , Pele/imunologia , Pele/metabolismo , UbiquitinaçãoRESUMO
Water buffalo horn (WBH), a traditional Chinese medicine, is known for its antipyretic, anti-inflammatory and antioxidant properties. This study aims to investigate the therapeutic potential of WBH keratin (WBHK) and its derived thiol-rich peptide fractions (SHPF) for oxidative stress and inflammation. WBHK and SHPF were prepared and tested using various models including LPS-induced fever in rabbits, H2O2-induced oxidative damage in bEnd.3 cells, TNF-α-induced inflammation in bEnd.3 cells and LPS-induced inflammation in RAW 264.7 cells. Expression of key markers, such as Nrf2, Hmox-1 and NF-κB, were analyzed using qRT-PCR, ELISA and Western blotting. Label-free quantitative proteomic analysis was used to identify key differential proteins associated with the efficacy of SHPF. Our results demonstrated that treatment with WBHK significantly reduced body temperature after 0.5 h of administration in the fever rabbit model. SHPF could alleviate cellular inflammatory injury and oxidative damage by activating the key transcription factor Nrf2 and increasing the expression level of Hmox-1. SHPF could inhibit the NF-κB pathway by reducing IκB phosphorylation. It was also found that SHPF could reduce pro-inflammatory cytokine (IL-6, COX-2 and PGE2) and inhibit the expression of VCAM-1, ICAM-1, IL-6 and MCP-1. Proteomics analysis showed that SHPF could inhibit HMGB1 expression and release. The results indicated that SHPF could significantly reduce inflammation and oxidative stress by regulating the Nrf2/Hmox-1 and NF-κB pathways. These findings suggest the potential therapeutic applications of WBH components in the treatment of oxidative stress and inflammation-related diseases.
Assuntos
Heme Oxigenase-1 , Inflamação , Queratinas , Fator 2 Relacionado a NF-E2 , NF-kappa B , Estresse Oxidativo , Peptídeos , Transdução de Sinais , Animais , Estresse Oxidativo/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Coelhos , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Camundongos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Inflamação/induzido quimicamente , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/genética , Queratinas/metabolismo , Peptídeos/farmacologia , Búfalos , Células RAW 264.7 , Compostos de Sulfidrila/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Antioxidantes/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Cornos/química , Lipopolissacarídeos , Febre/tratamento farmacológico , Febre/induzido quimicamente , Febre/metabolismo , Peróxido de Hidrogênio/metabolismo , Masculino , Medicina Tradicional ChinesaRESUMO
BACKGROUND: Lower extremity wounds in patients with diabetes are difficult to heal due to an overabundance of pro-inflammatory M1 macrophages, reduced phagocytosis of necrosed cells, and circulatory issues. Keratin biomaterials have been shown to address some of these concerns by encouraging the proliferation of anti-inflammatory M2 macrophages, thereby creating more favorable conditions for wound healing resembling those of patients without diabetes. OBJECTIVE: To investigate the effect of a novel human keratin matrix (HKM) on wound healing. MATERIALS AND METHODS: Ten patients with diabetes with lower extremity wounds at risk for delayed healing underwent wound debridement and application of HKM. Patients received weekly follow-up care and reapplication of HKM until healing occurred; wound size at each visit was used to calculate healing rate. RESULTS: Increased healing rates were noted with HKM compared with standard of care (SOC), including debridement and collagen treatment in all 8 patients who had received SOC prior to HKM treatment. When HKM treatment was alternated with SOC in 2 patients due to other medical conditions, healing rates decreased with SOC and then increased after reintroduction of HKM applications. CONCLUSIONS: These results suggest that HKM may help regulate the pathological processes that contribute to wound chronicity to "kick-start" wound healing. This case series demonstrates that HKM is a promising technology to improve healing rates in nonhealing lower extremity wounds in patients with diabetes.
Assuntos
Desbridamento , Pé Diabético , Queratinas , Cicatrização , Humanos , Cicatrização/fisiologia , Cicatrização/efeitos dos fármacos , Masculino , Feminino , Pé Diabético/terapia , Pessoa de Meia-Idade , Idoso , Desbridamento/métodos , Queratinas/metabolismo , Resultado do Tratamento , Extremidade InferiorRESUMO
INTRODUCTION: The global poultry industry produces millions of tons of waste feathers every year, which can be bio-degraded to make feed, fertilizer, and daily chemicals. However, feather bio-degradation is a complex process that is not yet fully understood. This results in low degradation efficiency and difficulty in industrial applications. Omics-driven system biology research offers an effective solution to quickly and comprehensively understand the molecularmechanisms involved in a metabolic pathway. METHODS: In the early stage of this process, feathers are hydrolyzed into water-soluble keratin monomers. In this study, we used high-throughput RNA-seq technology to analyze the genes involved in the internalization and degradation of keratin monomers in Stenotrophomonas maltophilia DHHJ strain cells. Moreover, we used Co-IP with LC-MS/MS technology to search for proteins that interact with recombinant keratin monomers. RESULTS: We discovered TonB transports and molecular chaperones associating with the keratin monomer, which may play a crucial role in the transmembrane transport of keratin. Meanwhile, multiple proteases belonging to distinct families were identified as binding partners of keratin monomers, among which ATPases associated with diverse cellular activity (AAA+) family proteases are overrepresented. Four genes, including JJL50_15620, JJL50_17955 (TonB-dependent receptors), JJL50_03260 (ABC transporter ATP-binding protein), and JJL50_20035 (ABC transporter substrate-binding protein), were selected as representatives for determining their expressions under different culture conditions using qRT-PCR, and they were found to be upregulated in response to keratin degradation consistent with the data from RNA-seq and Co-IP. CONCLUSION: This study highlights the complexity of keratin biodegradation in S. maltophilia DHHJ, in which multiple pathways are involved such as protein folding, protein transport, and several protease systems. Our findings provide new insights into the mechanism of feather degradation.
Assuntos
Proteínas de Bactérias , Biodegradação Ambiental , Queratinas , Stenotrophomonas maltophilia , Stenotrophomonas maltophilia/metabolismo , Stenotrophomonas maltophilia/genética , Queratinas/metabolismo , Queratinas/genética , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Plumas/metabolismo , Plumas/microbiologia , Espectrometria de Massas em Tandem , Regulação Bacteriana da Expressão Gênica , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genéticaRESUMO
The purpose of this work is to produce keratin hydrolysate from sheep wool by alkaline hydrolysis and to assess its effectiveness in improving maize plant growth under greenhouse conditions. A hybrid response surface methodology with Box-Behnken design (RSM-BBD) was used to model and optimize the hydrolysis process. The synergistic effects between three critical independent variables including temperature, hydrolysis time, and concentration of KOH on the hydrolysis rate were statistically investigated and optimized. Under optimized conditions, a hydrolysis rate of 95.08% was achieved. The produced hydrolysate consists of water-soluble peptides, free amino acids and potassium ions, making it suitable to be used as a valuable agricultural input material for crop production. Amino acid analysis revealed high levels of proline and phenylalanine, which are responsible for water conditioning and the preservation of abiotic stress as readily available. The efficacy of the produced hydrolysate was assessed in the cultivation of maize as a crop model under greenhouse conditions. Results revealed that the application of the hydrolysate positively influenced the morphological traits of the maize crop such as plant height and leaf surface area. The magnitude of the response to the hydrolysate application depended on its concentration with the most positive effects observed at a dose 2 for the leaf's chlorophyll content, fresh shoot biomass and dry shoot biomass. The application of the hydrolysate improved fresh and dry shoot biomass by 32.5 and 34.4% compared to the control and contributed to the improvement of nitrogen use efficiency by the studied crop. The hydrolysate proved to be beneficial in improving overall plant growth and can be suitable and effective agricultural input for maize cultivation.
Assuntos
Queratinas , Lã , Zea mays , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Animais , Lã/crescimento & desenvolvimento , Queratinas/metabolismo , Ovinos , Hidrólise , Biomassa , Aminoácidos/metabolismoRESUMO
By analyzing the expression patterns of inner root sheath (IRS) specific genes during different developmental stages of hair follicle (HF) in Tan sheep embryos and at birth, this study aims to reveal the influence of the IRS on crimped wool. Skin tissues from the scapular region of male Tan sheep were collected at 85 days (E85) and 120 days (E120) of fetal development, and at 0 days (D0), 35 days (D35), and 60 days (D60) after birth, with four samples at each stage. Real-time quantitative polymerase chain reaction (RT-qPCR) was employed to determine the relative expression levels of IRS type I keratin genes (KRT25, KRT26, KRT27, KRT28), type II keratin genes (KRT71, KRT72, KRT73, KRT74), and the trichohyalin gene (TCHH) in the skin of Tan sheep at different stages. Results showed that the expression levels of all IRS-specific genes peaked at D0, with the expression of all genes significantly higher than at E85 (P < 0.01), except for KRT73 and TCHH. The expression levels of KRT25, KRT26, and KRT72 were also significantly higher than at E120 (P < 0.01). Furthermore, the expression levels of KRT27, KRT28, KRT71, and KRT74 were significantly higher than both at E120 and D35 (P < 0.01). The expression levels of other genes at different stages showed no significant difference (P > 0.05). Conclusion: The IRS-specific genes exhibit the highest expression levels in Tan sheep at the neonatal stage. The expression levels of KRT71, KRT72, and TCHH, which are consistent with the pattern of wool crimp, may influence the morphology of the IRS and thereby affect the crimp of Tan sheep wool.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Folículo Piloso , Animais , Folículo Piloso/metabolismo , Folículo Piloso/crescimento & desenvolvimento , Ovinos/genética , Ovinos/crescimento & desenvolvimento , Masculino , Lã/metabolismo , Lã/crescimento & desenvolvimento , Queratinas Tipo II/genética , Queratinas Tipo II/metabolismo , Queratinas/genética , Queratinas/metabolismo , Queratinas Tipo I/genética , Queratinas Tipo I/metabolismo , Proteínas de Filamentos IntermediáriosRESUMO
Keratinized tissue augmentation around implants guarantees long-term success and maintenance of implant rehabilitations. Free gingival grafting is often described as the gold standard, especially when dealing with limited residual keratinized tissue height. Traditionally, an epithelio-conjunctive graft is harvested, either on the palate or the tuberosity, to reconstruct the missing keratinized soft tissues. This article introduces an innovative approach to increase keratinized tissue around implants, benefiting from second-intention gingival healing. This original surgical approach is interesting because it does not involve autogenous grafting or biomaterials. Its main goals are to enhance predictability while reducing the numerous per and post-operative risks related to autogenous harvesting. The success of this technique depends on the observance of fundamental principles: protection against bacterial contamination (immunocompetence of the patient), phenotypic induction (preoperative presence of keratinized tissue), space maintenance, and stabilization of flaps.