Development of a Static Avascular and Dynamic Vascular Human Skin Equivalent Employing Collagen/Keratin Hydrogels.

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.

Design, fabrication, and evaluation of keratin and pectin incorporated supramolecular structured zero-oxidation state selenium nanogel blended 3D printed transdermal patch.

This study investigates the synthesis of selenium nanoparticles (SeNPs), owing to the low cost and abundance of selenium. However, the toxicity of SeNP prompts the development of a selenium nanocomposite (SeNC) containing pectin, keratin, and ferulic acid to improve the bioactivity of Se[0]. Further, incorporating the SeNC in a suitable formulation for drug delivery as a transdermal patch was worth studying. Accordingly, various analytical techniques were used to characterize the SeNPs and the SeNC, confirming successful synthesis and encapsulation. The SeNC exhibited notable particle size of 448.2 ± 50.2 nm, high encapsulation efficiency (98.90 % ± 2.4 %), 28.1 ± 0.45 drug loading, and sustained drug release at pH 5.5. Zeta potential and XPS confirmed the zero-oxidation state. The supramolecular structure was evident from spectral analysis endorsing the semi-crystalline nature of the SeNC and SEM images showcasing flower-shaped structures. Further, the SeNC demonstrated sustained drug release (approx. 22 % at 48 h) and wound-healing potential in L929 fibroblast cells. Subsequently, the SeNC loaded into a gelling agent exhibited shear thinning properties and improved drug release by nearly 58 %. A 3D printed reservoir-type transdermal patch was developed utilizing the SeNC-loaded gel, surpassing commercially available patches in characteristics such as % moisture uptake, tensile strength, and hydrophobicity. The patch, evaluated through permeation studies and CAM assay, exhibited controlled drug release and angiogenic properties for enhanced wound healing. The study concludes that this patch can serve as a smart dressing with tailored functionality for different wound stages, offering a promising novel drug delivery system for wound healing.

The StuA Transcription Factor and Alternative Splicing Mechanisms Drive the Levels of MAPK Hog1 Transcripts in the Dermatophyte Trichophyton rubrum.

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.

Biodegradation of Keratin Waste by Bacillus velezensis HFS_F2 through Optimized Keratinase Production Medium.

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.

Conserved regulatory switches for the transition from natal down to juvenile feather in birds.

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.

Developmental relationship between junctional epithelium and epithelial rests of Malassez.

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.

Unraveling UVB effects: Catalase activity and molecular alterations in the stratum corneum.

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.

Keratin Formed Bioadhesive Ophthalmic Gel for the Bacterial Conjunctivitis Treatment.

Keratin has the potential to function as the gel matrix in an ophthalmic formulation for the encapsulation of the macrolide antibiotic azithromycin. The quality of this formulation was thoroughly evaluated through various analyses, such as in vitro release assessment, rheological examination, intraocular retention studies in rabbits, assessment of bacteriostatic efficacy, and safety evaluations. It is worth mentioning that the gel demonstrated shear thinning properties and exhibited characteristics of an elastic solid, thereby confirming its structural stability. The gel demonstrated a notable affinity for mucosal surfaces in comparison to traditional azithromycin aqueous solutions. In vitro release testing revealed that drug release transpired via diffusion mechanisms, following a first-order kinetic release pattern. Additionally, the formulated gel exhibited remarkable antibacterial efficacy against Staphylococcus aureus and Pseudomonas aeruginosa in bacteriostatic evaluations. Lastly, safety assessments confirmed that the gel eye drops induced minimal irritation and displayed no apparent cytotoxicity, indicating their good safety and biocompatibility for ocular application. Thus, these findings indicated that the prepared azithromycin gel eye drops complied with the requisite standards for ophthalmic preparations.

The effects of keratin-coated titanium on osteoblast function and bone regeneration.

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.

Bioenergy production from chicken feather waste by anaerobic digestion and bioelectrochemical systems.

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.

Multiprotein collagen/keratin hydrogel promoted myogenesis and angiogenesis of injured skeletal muscles in a mouse model.

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.

Fabrication of silver nanoparticles decorated on sodium alginate microbeads enriched with keratin and investigation of its catalytic and antioxidant activity.

In this study, an environmentally friendly, effective, easily synthesizable and recoverable nano-sized catalyst system (Ag@NaAlg-keratin) was designed by decorating Ag nanoparticles on microbeads containing sodium alginate (NaAlg) and keratin obtained from goose feathers. The structure, morphology and crystallinity of the Ag@NaAlg-keratin nanocatalyst were evaluated by XRD, FT-IR, FE-SEM, EDS/EDS mapping and TEM analyses. Catalytic ability of designed Ag@NaAlg-keratin nanocatalyst was then investigated against 4-nitrophenol (4-NP) and methyl orange (MO) reductions. Ag@NaAlg-keratin nanocatalyst effectively reduced 4-NP in 6 min and MO in 5 min, with rate constants of 0.17 min-1 and 0.16 min-1, respectively. Additionally, activation energies (Ea) were found as 39.8 kJ/mol for 4-NP and 37.9 kJ/mol for MO. Performed recyclability tests showed that the Ag@NaAlg-keratin nanocatalyst was easily recovered due to its microbead form and successfully reused five times, maintaining both its activity and structure. Furthermore, antioxidant activity of Ag@NaAlg-keratin nanocatalyst was the highest (73.16 %).

Peptide recovery from chicken feather keratin and their anti-biofilm properties against methicillin-resistant Staphylococcus aureus (MRSA).

Bacteria have the potential to adhere to abiotic surfaces, which has an undesirable effect in the food industry because they can survive for sustained periods through biofilm formation. In this study, an antibacterial peptide (ABP), with a molecular mass of 3861 Da, was purified from hydrolyzed chicken feathers using a locally isolated keratinolytic bacterium, namely Rhodococcus erythropolis, and its antibacterial and antibiofilm potential were investigated against planktonic and biofilm cells of Methicillin-Resistant Staphylococcus Aureus (MRSA). The results demonstrated that purified ABP showed the growth inhibition of MRSA cells with the minimum inhibitory concentration (MIC) of 45 µg/ml and disrupted MRSA biofilm formation at a concentration of 200 ug/ml, which results were confirmed by scanning electron micrograph (SEM). Moreover, the secondary structures of the peptide were assessed as part of the FTIR analysis to evaluate its mode of action. ExPASy tools were used to predict the ABP sequence, EPCVQUQDSRVVIQPSPVVVVTLPGPILSSFPQNTA, from a chicken feather keratin sequence database following in silico digestion by trypsin. Also, ABP had 54.29% hydrophobic amino acids, potentially contributing to its antimicrobial activity. The findings of toxicity prediction of the peptide by the ToxinPred tool revealed that ABP had non-toxic effects. Thus, these results support the potential of this peptide to be used as an antimicrobial agent for the treatment or prevention of MRSA biofilm formation in feed, food, or pharmaceutical applications.

Wound Dressing Based on Silver Nanoparticle Embedded Wool Keratin Electrospun Nanofibers Deposited on Cotton Fabric: Preparation, Characterization, Antimicrobial Activity, and Cytocompatibility.

Wool keratin (WK) protein is attractive for wound dressing and biomedical applications due to its excellent biodegradability, cytocompatibility, and wound-healing properties. In this work, WK-based wound dressings were prepared by depositing WK/poly(vinyl alcohol) (PVA) and silver nanoparticle (Ag NP)-embedded WK/PVA composite nanofibrous membranes on cotton fabrics by electrospinning. Ag NPs were biosynthesized by reduction and stabilization with sodium alginate. The formed Ag NPs were characterized by ultraviolet-visible and Fourier transform infrared (FTIR) spectroscopy, and their size was determined by transmission electron microscopy and image analysis. The formed Ag NPs were spherical and had an average diameter of 9.95 nm. The produced Ag NP-embedded WK/PVA composite nanofiber-deposited cotton fabric surface was characterized by FTIR and dynamic contact angle measurements, and the nanofiber morphologies were characterized by scanning electron microscopy. The average diameter of the nanofibers formed by 0.1% Ag NP-embedded WK/PVA solution was 146.7 nm. The antibacterial activity of the surface of cotton fabrics coated with electrospun composite nanofibers was evaluated against the two most common wound-causing pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. The cotton fabric coated with 0.1% Ag NP-embedded WK/PVA nanofibers showed very good antibacterial activity against both pathogens, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results showed good cytocompatibility against L-929 mouse fibroblast cells. However, the increase in Ag NP content in the nanofibers to 0.2% negatively affected the cell viability due to the high release rate of Ag ions. The results achieved show that the developed wound dressing has good potential for wound healing applications.

Keratin-based bioplastics extracted from chicken feathers: Effect of chitosan concentration on the structural, chemical bonding, and mechanical properties of bioplastics.

Keratin was synthesized by alkaline hydrolysis from chicken feathers and then continue by casting method for producing bioplastics with additional various amounts of chitosan as a filler, polyvinyl alcohol (PVA) and glycerol as a plasticizer. The main purpose is analysis the effect of chitosan on the structural properties using quantitative analysis of X-ray diffraction (XRD) spectra, chemical bonding by Fourier transforms infrared (FTIR) spectra, and mechanical properties by texture analyser to the keratin-based bioplastics. Biodegradation of bioplastics was analysed from the loss of weight by burying in the soil. It's found that, the additional of chitosan (0 %, 2 %, 5 %, and 8 %) increased the crystallinity of bioplastics by 11.83 %, 11.12 %, 18.99 %, and 17.03 %, respectively, but decreasing tensile strength and elasticity of bioplastics. Degradation of bioplastic keratin-based shows that the addition of chitosan can reduce the degradation time which is directly proportional to the loss of CO bonds. The highest degradation rate is 89.29 % in 49 days for keratin-based bioplastics with 8 % chitosan, indicated that high potential for future production.

Molecular strategies to enhance the keratinase gene expression and its potential implications in poultry feed industry.

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.

In vitro generation of epidermal keratinocytes from human CD34-positive hematopoietic stem cells.

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.

EBS in Children with De Novo Pathogenic Variants Disturbing Krt14.

Epidermolysis bullosa simplex (EBS) is a dermatological condition marked by skin fragility and blister formation resulting from separation within the basal layer of the epidermis, which can be attributed to various genetic etiologies. This study presents three pathogenic de novo variants in young children, with clinical manifestations appearing as early as the neonatal period. The variants contribute to the EBS phenotype through two distinct mechanisms: direct keratin abnormalities due to pathogenic variants in the Krt14 gene, and indirect effects via pathogenic mutation in the KLHL24 gene, which interfere with the natural proteasome-mediated degradation pathway of KRT14. We report one severe case of EBS with mottled pigmentation arising from the Met119Thr pathogenic variant in KRT14, another case involving a pathogenic KLHL24 Met1Val variant, and a third case featuring the hot spot mutation Arg125His in KRT14, all manifesting within the first few weeks of life. This research underscores the complexity of genetic influences in EBS and highlights the importance of early genetic screening for accurate diagnosis and management.

Efficacy of in-office lysis of clitoral adhesions with excision of keratin pearls on clitoral pain and sexual function: a pre-post interventional study.

BACKGROUND: Keratin pearls are foci of central keratinization within concentric layers of squamous cells that can form under the clitoral prepuce and cause pain (clitorodynia); in-office removal of keratin pearls may reduce clitoral pain and improve sexual function. AIM: This study aims to investigate clitoral pain and sexual function in women with partial clitoral phimosis and keratin pearls before and after in-office lysis of clitoral adhesions with keratin pearl excision (LCA-KPE). METHODS: A pre-post interventional study evaluated patients who underwent LCA-KPE between January 2017 and February 2023 in 2 metropolitan gynecology clinics specializing in vulvar pain. Patients presenting with keratin pearls and partial clitoral phimosis identified through retrospective chart review were asked to complete postprocedure questionnaires and provide subjective responses on clitoral discomfort, sexual function, sexual distress, and their experience with in-office LCA-KPE. Bivariate analyses with paired t tests were conducted to determine the effect of LCA-KPE. Qualitative data were analyzed with thematic coding. OUTCOMES: An 11-point pain visual analog scale was utilized to determine pre- and postprocedure clitoral discomfort and difficulty with orgasm. Female sexual dysfunction was measured with the Female Sexual Function Index (FSFI) and Female Sexual Distress Scale-Revised. RESULTS: A total of 32 of 74 patients who met inclusion criteria completed postprocedure surveys (43% response rate). Mean clitoral pain for respondents was 6.91 at baseline and 2.50 after LCA-KPE (P < .001). Mean difficulty with orgasm was significantly decreased from 5.45 at baseline to 3.13 after LCA-KPE (P < .001). Participants had a mean FSFI total score of 17.68 after treatment compared with a mean total baseline FSFI of 12.12 (P = .017). The mean FSFI score for pain was 2.43 at follow-up compared with 1.37 at baseline (P = .049). There was no significant difference in the mean Female Sexual Distress Scale-Revised score before vs after the procedure (P = .27). Qualitative themes described the procedure as painful but worthwhile, with 77% of participants reporting the overall experience as positive. Recurrence rate overall was 28%, with a median of 2 repeat procedures. CLINICAL IMPLICATIONS: Recognizing keratin pearls as a structural cause of clitoral pain and offering in-office treatment is an important tool in addressing clitorodynia and improving sexual function. STRENGTHS AND LIMITATIONS: This is the largest study to date documenting the occurrence, identifying associated pain conditions, and evaluating procedural outcomes for clitoral keratin pearls. This study was limited by a relatively small sample size. CONCLUSION: In-office LCA-KPE significantly reduced clitoral discomfort and difficulty with orgasm.