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.

Changes to insulin sensitivity in glucose clearance systems and redox following dietary supplementation with a novel cysteine-rich protein: A pilot randomized controlled trial in humans with type-2 diabetes.

We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m2/min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation AktSer437 and AS160Thr642 were inconclusive, but there was good evidence for increased IRSSer312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1Thr423/2Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake.

Chryseobacterium aquifrigidense keratinase liberated essential and nonessential amino acids from chicken feather degradation.

Keratinous biomass valorization for value-added products presents a high prospect in ecological management and the advancement of the bio-economy. Consequently, soil samples from the poultry dumpsite were collected. The bacteria isolated on the basal salt medium were screened for keratinolytic activity. The potent chicken feathers degrading bacteria were identified through 16S rRNA gene sequencing and phylogenetic analysis. Fermentation process conditions were optimized, and the amino acid compositions of the feather hydrolysate were likewise quantified. Ten (10) proteolytic bacteria evaluated on skimmed milk agar showed intact chicken feather degradation ranging from 33% (WDS-03) to 88% (FPS-09). The extracellular keratinase activity ranged from 224.52 ± 42.46 U/mL (WDS-03) to 834.55 ± 66.86 U/mL (FPS-07). Based on 16S rRNA gene sequencing and phylogenetic analysis, the most potent keratinolytic isolates coded as FPS-07, FPS-09, FPS-01, and WDS-06 were identified as Chryseobacterium aquifrigidense FANN1, Chryseobacterium aquifrigidense FANN2, Stenotrophomonas maltophilia ANNb, and Bacillus sp. ANNa, respectively. C aquifrigidense FANN2 maximally produced keratinase (1460.90 ± 26.99 U/mL) at 72 h of incubation under optimal process conditions of pH (6), inoculum side (5%; v/v), temperature (30°C), and chicken feather (25 g/L). The feather hydrolysate showed a protein value of 67.54%, with a relative abundance of arginine (2.84%), serine (3.14%), aspartic acid (3.33%), glutamic acid (3.73%), and glycine (2.81%). C. aquifrigidense FANN2 yielded high keratinase titre and dismembered chicken feathers into amino acids-rich hydrolysate, highlighting its significance in the beneficiation of recalcitrant keratinous wastes into dietary proteins as potential livestock feed supplements.

Sargassum horneri extract containing polyphenol alleviates DNCB-induced atopic dermatitis in NC/Nga mice through restoring skin barrier function.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by skin barrier dysfunction. Sargassum horneri (S. horneri) is a brown alga that has been widely used in traditional medicine of eastern Asian countries. Recent studies proved that a brown alga S. horneri has anti-inflammatory activity. In this study, we investigated the effect of S. horneri ethanol extract (SHE) against AD in 2,4-dinitrobenzene (DNCB) induced AD in NC/Nga mice. We observed that SHE treatment decreased the epidermal thickness and epidermal hyperplasia that had been worsened through DNCB application. Moreover, SHE significantly inhibited the proliferation of mast cells and decreased the expression of IL-13 on CD4⁺ cells prompted by elevated thymic stromal lymphopoietin (TSLP) expression in DNCB-induced AD in mice. We also demonstrated that SHE directly inhibited the expression of keratinocyte-produced TSLP known to exacerbate skin barrier impairment. Especially, the decrease of filaggrin, an integral component of proper skin barrier function through a function in aggregating keratin filaments, observed in DNCB-induced AD mice was significantly improved when treated with SHE. More importantly, we proved that SHE was able to decrease the serum levels of IgG1 and IgG2ₐ, two crucial factors of AD, indicating the protective effect of SHE. Taken together, our findings suggest that SHE may protect NC/Nga mice against DNCB-induced AD via promoting skin barrier function.

Vitamin D3 supplementation alleviates chemically-induced cirrhosis-associated hepatocarcinogenesis.

Vitamin D3 (VD3) deficiency has been associated with increased risk for cirrhosis and hepatocellular carcinoma, a highly incident malignant neoplasia worldwide. On the other hand, VD3 supplementation has shown some beneficial effects in clinical studies and rodent models of chronic liver disease. However, preventive effects of dietary VD3 supplementation in cirrhosis-associated hepatocarcinogenesis is still unknow. To investigate this purpose, male Wistar rats submitted to a combined diethylnitrosamine- and thioacetamide-induced model were concomitantly supplemented with VD3 (5,000 and 10,000 IU/kg diet) for 25 weeks. Liver samples were collected for histological, biochemical and molecular analysis. Serum samples were used to measure 25-hydroxyvitamin D [25(OH)D] and alanine aminotransferase levels. Both VD3 interventions decreased hepatic collagen deposition and pro-inflammatory p65 protein levels, while increased hepatic antioxidant catalase and glutathione peroxidase activities and serum 25(OH)D, without a clear dose-response effect. Nonetheless, only the highest concentration of VD3 increased hepatic protein levels of VD receptor, while decreased the number of large preneoplastic glutathione-S-transferase- (>0.5 mm²) and keratin 8/18-positive lesions, as well the multiplicity of hepatocellular adenomas. Moreover, this intervention increased hepatic antioxidant Nrf2 protein levels and glutathione-S-transferase activity. In summary, dietary VD3 supplementation - in special the highest intervention - showed antifibrotic and antineoplastic properties in chemically-induced cirrhosis-associated hepatocarcinogenesis. The positive modulation of Nrf2 antioxidant axis may be mechanistically involved with these beneficial effects, and may guide future clinical studies.

Curcuma's extraction attenuates propranolol-induced psoriasis like in mice by inhibition of keratin, proliferating cell nuclear antigen and toll-like receptor expression.

Curcuma was the dried rhizomes of Curcuma kwangsiensis S.G. Lee et C.F. Liang (Chinese name: e zhu), have been used in China for thousands of years. There are some reports have shown that curcumin, the major component of curcuma, has a good curative effect on psoriasis, but the mechanism is still unknown, so the present study was designed to investigate the effect of curcuma's extraction on psoriasis-like mouse, and to explore the mechanisms of therapy. First, we observed that curcuma's extractions effect on mitosis of mouse vaginal epithelial cells; then making psoriasis like model and measuring the score of skin damage on days 7 and 14; finally, we observed the expression of immune factors (CK14, CK16, CK17, PCNA, TLR-2, TLR-4, TLR-9) in propranolol induced psoriasis like rats. Curcuma's extraction prohibited the mitosis of mouse vaginal epithelial cells; curcuma's extractions have a significantly efficacy and dose dependent inhibition on imiquimod induced psoriasis like rats; and the expression of immune factors (CK14, CK16, CK17, PCNA, TLR-2, TLR-4, TLR-9) was decreasing in the curcuma's extraction treated groups compared with normal groups. Our research proved that curcuma's extractions have a significantly efficacy on psoriasis like rats, thus, curcuma's extractions can be a potential novel treatment for psoriasis. Furthermore, the expression of immune factors was decreasing after treatment with curcuma's extraction suggest us cytokines has strong relation with the mechanism of therapy for psoriasis. Our results contribute towards validation of curcuma in the treatment of psoriasis and other joint disorders.

Sustainable production, biochemical and molecular characterization of thermo-and-solvent stable alkaline serine keratinase from novel Bacillus pumilus AR57 for promising poultry solid waste management.

The increasing amount of recalcitrant keratinous wastes generated from the poultry industry poses a serious threat to the environment. Keratinase have gained much attention to convert these wastes into valuable products. Ever since primitive feathers first appeared on dinosaurs, microorganisms have evolved to degrade this most recalcitrant keratin. In this study, we identified a promising keratinolytic bacterial strain for bioconversion of poultry solid wastes. A true keratinolytic bacterium was isolated from the slaughterhouse soil and was identified and designated as Bacillus pumilus AR57 by 16S rRNA sequencing. For enhanced keratinase production and rapid keratin degradation, the media components and substrate concentration were optimized through shake flask culture. White chicken feather (1% w/v) was found to be the good substrate concentration for high keratinase production when supplemented with simple medium ingredients. The biochemical characterization reveals astounding results which makes the B. pumilus AR57 keratinase as a novel and unique protease. Optimum activity of the crude enzyme was exhibited at pH 9 and 45 °C. The crude extracellular keratinase was characterized as thermo-and-solvent (DMSO) stable serine keratinase. Bacillus pumilus AR57 showed complete degradation (100%) of white chicken feather (1% w/v) within 18 h when incubated in modified minimal medium supplemented with DMSO (1% v/v) at 150 rpm at 37 °C. Keratinase from modified minimal medium supplemented with DMSO exhibits a half-life of 4 days. Whereas, keratinase from the modified minimal medium fortified with white chicken feather (1% w/v) was stable for 3 h only. Feather meal produced by B. pumilus AR57 was found to be rich in essential amino acids. Hence, we proposed B. pumilus AR57 as a potential candidate for the future application in eco-friendly bioconversion of poultry waste and the keratinase could play a pivotal role in the detergent industry. While feather meal may serve as an alternative to produce animal feed and biofertilizers.

Anti-psoriatic properties of paeoniflorin: suppression of the NF-kappaB pathway and Keratin 17.

BACKGROUND: Psoriasis is a common inflammatory skin disease characterized by hyperproliferation of epidermal keratinocytes, however, there is still no curative therapy for psoriasis. Paeoniflorin (PF), a Chinese herbal medicine, has shown anti-inflammatory effects. OBJECTIVES: We aimed to illustrate the effect and associated mechanism of PF on keratinocyte proliferation using the IMQ-induced psoriasis mouse model. MATERIALS AND METHODS: The anti-psoriatic effect of PF in vivo and in vitro was assessed by western blot, RT-PCR, immunofluorescence, cell counting kit-8 and haematoxylin/eosin staining. RESULTS: In vivo, epidermal thickness, dermal infiltrated lymphocytes and the level of several antimicrobial peptides, pro-inflammatory cytokines, and K17 were significantly reduced in mice with topical application of PF. In vitro, PF inhibited the proliferation of HaCaT cells in a dose-dependent manner, down-regulated K17 expression, and suppressed NF-kappaB activation. CONCLUSION: Our findings indicate that PF may inhibit the proliferation of keratinocytes by targeting K17, suggesting that PF might be a potential target in the treatment of psoriasis.

Strategical isolation of efficient chicken feather-degrading bacterial strains from tea plantation soil sample.

Chicken feather waste is generally insufficiently utilized despite its high content of protein, constituting an environmental issue. Biodegradation of the waste with enabling microbes provides an advantageous option among the available solutions. In this study, an efficient whole feather-degrading strain was strategically isolated from a soil sample taken from a local tea plantation that has little or nothing to do with feathers. The strain was identified as Bacillus thuringiensis (designated as FDB-10) according to the cloned complete 16S rRNA sequence. The FDB-10 could efficiently degrade briefly heat-treated whole feather (102 °C, 5 min; up to 90% of a maximum concentration of 30 g/L) in a salt medium supplemented with 0.1 g/L yeast extract within 24 h (37 °C, 150 rpm). Addition of carbon sources (glycerol, glucose, starch, Tween 20, Tween 80, 1.25 g/L as glycerol) to the fermentation medium could improve the degradation. However, significant inhibition could be observed when the added carbon source reached the amount usually adopted in the investigation of carbon source preference (1%). Nitrogen source (NH4Cl, (NH4)2SO4, peptone) adversely influenced the performance of the strain. When the molar concentrations of NH4+ were equal for the two salt, the inhibitory effect on degradation of whole feathers was similar. Entirely different from other reported feather-degrading strains showing a preference to melanin-free feather substrates, the strain isolated in this study could degrade melanin-containing feather equally efficiently, and higher protease activity could be detected in the digest mix. As a plus, the strain could degrade feathers in rice wash produced in daily cooking, indicating its potential use in the simultaneous treatment of rice cooker wastewater produced by a rice processing plant. All these results imply that the FDB-10 is a strain with great potential in the biodegradation of feather waste.

Intense Pulsed Light: Friend or Foe? Molecular Evidence to Clarify Doubts.

BACKGROUND/AIM: Intense pulsed light (IPL) has been extensively applied in the field of dermatology and aesthetics; however, the long-term consequences of its use are poorly unknown, and to the best of our knowledge there is no study on the effect of IPL in neoplastic lesions. In order to better understand the molecular mechanisms underlying IPL application in the skin, we used an animal model of carcinogenesis obtained by chemical induction with 12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). MATERIALS AND METHODS: Institute of Cancer Research (ICR) mice were administered DMBA and/or TPA and treated with IPL. Skin was evaluated by histopathology and 2DE-blot-MS/MS analysis. RESULTS: Our data evidenced an inflammatory response and a metabolic remodeling of skin towards a glycolytic phenotype after chronic exposure to IPL, which was accomplished by increased oxidative stress and susceptibility to apoptosis. These alterations induced by IPL were more notorious in the DMBA sensitized skin. Keratins and metabolic proteins seem to be the more susceptible to oxidative modifications that might result in loss of function, contributing for the histological changes observed in treated skin. CONCLUSION: Data highlight the deleterious impact of IPL on skin phenotype, which justifies the need for more experimental studies in order to increase our understanding of the IPL long-term safety.

In Vitro Human Onychopharmacokinetic and Pharmacodynamic Analyses of ME1111, a New Topical Agent for Onychomycosis.

ME1111 is a novel antifungal agent currently under clinical development as a topical onychomycosis treatment. A major challenge in the application of topical onychomycotics is penetration and dissemination of antifungal agent into the infected nail plate and bed. In this study, pharmacokinetic/pharmacodynamic parameters of ME1111 that potentially correlate with clinical efficacy were compared with those of marketed topical onychomycosis antifungal agents: efinaconazole, tavaborole, ciclopirox, and amorolfine. An ME1111 solution and other launched topical formulations were applied to an in vitro dose model for 14 days based on their clinical dose and administration. Drug concentrations in the deep layer of the nail and within the cotton pads beneath the nails were measured using liquid chromatography-tandem mass spectrometry. Concentrations of ME1111 in the nail and cotton pads were much higher than those of efinaconazole, ciclopirox, and amorolfine. Free drug concentrations of ME1111 in deep nail layers and cotton pads were orders of magnitude higher than the MIC90 value against Trichophyton rubrum (n = 30). Unlike other drugs, the in vitro antifungal activity of ME1111 was not affected by 5% human keratin and under a mild acidic condition (pH 5.0). The in vitro antidermatophytic efficacy coefficients (ratio of free drug concentration to MIC90s against T. rubrum) of ME1111, as measured in deep nail layers, were significantly higher than those of efinaconazole, tavaborole, ciclopirox, and amorolfine (P < 0.05). This suggests that ME1111 has excellent permeation of human nails and, consequently, the potential to be an effective topical onychomycosis treatment.

Low-level red LED light inhibits hyperkeratinization and inflammation induced by unsaturated fatty acid in an in vitro model mimicking acne.

BACKGROUND AND OBJECTIVE: Acne vulgaris is a chronic inflammatory disease of the pilosebaceous units (PSU), associated with increased sebum production, abnormal follicular keratinization (hyperkeratinization), follicular overgrowth of Propionibacterium acnes (P. acnes), and increased inflammatory mediator release. Light therapy has attracted medical interests as a safe alternative treatment for acne. Both blue and red light therapies at high doses >10 J/cm2 have demonstrated marked effects on inflammatory acne lesions. However, few studies have investigated the effects of lower doses of light. The aim of this study is to investigate the biological effects of lower doses of red light at 0.2-1.2 J/cm2 for acne using an in vitro model previously developed to mimic the inflammation and hyperkeratinization observed clinically in acne. MATERIALS AND METHODS: Human epidermal equivalents were topically exposed to an unsaturated fatty acid, oleic acid (OA), followed by red light-emitting diode (LED) light treatments (light-plus-OA treatments). Endpoints evaluated included the proinflammatory cytokine IL-1α, epidermal barrier integrity, as measured by transepithelial electrical resistance (TEER), and stratum corneum (SC) thickness to monitor hyperkeratinization. RESULTS: OA-induced IL-1α release was significantly (P < 0.05) reduced following red LED light at 0.2, 0.5, and 1.2 J/cm2 , from 266 ± 11 pg/ml of no-light-plus-OA-treated (OA treatment without light) controls to 216 ± 9, 231 ± 8, and 212 ± 7 pg/ml, respectively. Histological examination showed that SC thickening following OA treatment was reduced from 43% of total epidermis for no-light-plus-OA treatment to 37% and 38% of total epidermis following 0.5 and 1.1 J/cm2 red light plus OA treatment, respectively (P < 0.05). Moreover, 1.1 J/cm2 red-light-plus-OA treatment improved OA-induced TEER changes from 29% of baseline for no-light-plus-OA treatment, to 36% of baseline. CONCLUSION: Low level red LED light therapy could provide beneficial effects of anti-inflammation, normalizing pilosebaceous hyperkeratinization, and improving barrier impairment in Acne vulgaris. Lasers Surg. Med. 50:158-165, 2018. © 2017 Wiley Periodicals, Inc.

Production and Characterization of Keratinolytic Proteases by a Chicken Feather-Degrading Thermophilic Strain, Thermoactinomyces sp. YT06.

Thermoactinomyces sp. strain YT06 was isolated from poultry compost and observed to degrade integral chicken feathers completely at 60°C, resulting in the formation of 3.24 mg/ml of free amino acids from 50 ml of culture containing 10 g/l chicken feathers. Strain YT06 could grow and secrete keratinase using feather as the only carbon and nitrogen sources without other supplement, but complementation of 10 g/l sucrose and 4 g/l NaNO3 increased the production of the keratinolytic enzyme. The maximum protease activity obtained was 110 U/ml and for keratinase was 42 U/ml. The keratinase maintained active status over a broad pH (pH 8-11) and temperature (60-75°C). It was inhibited by serine protease inhibitors and most metal ions; however, it could be stimulated by Mn²âº and the surfactant Tween-20. A reductive agent (ß-mercaptoethanol) was observed to cleave the disulfide bond of keratin and improve the access of the enzyme to the keratinaceous substrate. Zymogram analysis showed that strain YT06 primarily secreted keratinase with a molecular mass of approximately 35 kDa. The active band was assessed by MALDI-TOF mass spectrometry and was observed to be completely identical to an alkaline serine protease from Thermoactinomyces sp. Gus2-1. Thermoactinomyces sp. strain YT06 shows great potential as a novel candidate in enzymatic processing of hard-to-degrade proteins into high-value products, such as keratinous wastes.

Vitis vinifera peel and seed gold nanoparticles exhibit chemopreventive potential, antioxidant activity and induce apoptosis through mutant p53, Bcl-2 and pan cytokeratin down-regulation in experimental animals.

Several studies suggest surface modifications of gold nanoparticles (AuNPs) by capping agents or surface coatings could play an important role in biological systems, and site directed delivery. The present study was carried out to assess the antioxidant and apoptotic activities of the Vitis vinifera peel and seed gold nanoparticles in experimentally induced cancer in Swiss albino mice. 12-dimethylbenz [a] anthracene (DMBA) (single application) and 12-O-tetradecanoylphorbol 13-acetate (TPA) (thrice a week) were applied on the dorsal area of the skin to induce skin papillomagenesis in Swiss albino mice for 16 weeks. Gold nanoparticles were synthesized using Vitis vinifera peel and seed aqueous extracts and characterized by Transmission electron microscopic (TEM) analyses. On topical application, peel and seed gold nanoparticles demonstrated chemopreventive potential by significantly (p<0.05) reducing the cumulative number of tumors while increasing the antioxidant enzyme activities in the gold nanoparticles treated mice. The down-regulated expression of mutant p53, Bcl-2 and the levels of pan-cytokeratins might have facilitated the process of apoptosis in the chemical carcinogenesis process. The results were supported by the histopathological evaluation which exhibited mild dysplasia and acanthosis in the skin tissues of Vitis vinifera peel and seed AuNPs treated mice. Based on the present study, the chemopreventive action of Vitis vinifera peel and seed AuNPs is probably due to its ability to stimulate the antioxidant enzymes within the cells and suppressed abnormal skin cell proliferation that occurred during DMBA-induced skin papillomagenesis.

Randomized, split-body, single-blinded clinical trial of topical broccoli sprout extract: Assessing the feasibility of its use in keratin-based disorders.

BACKGROUND: Epidermolysis bullosa simplex is a skin-blistering disorder caused by mutations in keratin (K)14 or K5. Treatment with nuclear factor (erythroid-derived 2)-like 2 inducer sulforaphane ameliorated skin blistering in Krt14-null mice, correlating with induction of K17. To be therapeutically useful for epidermolysis bullosa simplex, topical broccoli sprout extract (BSE), enriched for sulforaphane, would ideally induce the expression of homologous keratins (eg, K6, K17, K16) in the basal layer of human epidermis without impacting expression of defective keratins (K5/K14). OBJECTIVE: The purpose of this 1-week, randomized, split-body, single-blinded, placebo-controlled trial was to assess the impact of BSE on keratin expression. METHODS: Five subjects (34-71 years old) applied BSE (500 nmol of sulforaphane/mL) or vehicle alone to the inner aspect of the arm daily. Expression of keratin, nuclear factor (erythroid-derived 2)-like 2, and other markers was assessed using reverse transcription-polymerase chain reaction and indirect immunofluorescence. RESULTS: One subject (age 71 years) was excluded a posteriori because of poor tissue quality. Topical BSE activated nuclear factor (erythroid-derived 2)-like 2 and up-regulated K17 in the epidermis of all subjects, had variable effects on K16 and K6 expression, and did not alter expression of K14 or K5. LIMITATIONS: Small sample size is a limitation. CONCLUSION: BSE represents an attractive therapeutic candidate for K14-associated epidermolysis bullosa simplex.

Cysteic Acid in Dietary Keratin is Metabolized to Glutathione and Liver Taurine in a Rat Model of Human Digestion.

Poultry feathers, consisting largely of keratin, are a low-value product of the poultry industry. The safety and digestibility of a dietary protein produced from keratin (KER) was compared to a cysteine-supplemented casein-based diet in a growing rat model for four weeks. KER proved to be an effective substitute for casein at 50% of the total dietary protein, with no changes in the rats' food intake, weight gain, organ weight, bone mineral density, white blood cell counts, liver glutathione, or blood glutathione. Inclusion of KER in the diet reduced total protein digestibility from 94% to 86% but significantly increased total dietary cysteine uptake and subsequent liver taurine levels. The KER diet also significantly increased caecum weight and significantly decreased fat digestibility, resulting in a lower proportion of body fat, and induced a significant increase in blood haemoglobin. KER is therefore a safe and suitable protein substitute for casein, and the cysteic acid in keratin is metabolised to maintain normal liver and blood glutathione levels.

Characterization of Antifungal Activity and Nail Penetration of ME1111, a New Antifungal Agent for Topical Treatment of Onychomycosis.

Fungal nail infection (onychomycosis) is a prevalent disease in many areas of the world, with a high incidence approaching 23%. Available antifungals to treat the disease suffer from a number of disadvantages, necessitating the discovery of new efficacious and safe antifungals. Here, we evaluate the in vitro antifungal activity and nail penetration ability of ME1111, a novel antifungal agent, along with comparator drugs, including ciclopirox, amorolfine, terbinafine, and itraconazole. ME1111 showed potent antifungal activity against Trichophyton rubrum and Trichophyton mentagrophytes (the major etiologic agents of onychomycosis) strains isolated in Japan and reference fungal strains with an MIC range of 0.12 to 0.5 mg/liter and an MIC50 and MIC90 of 0.5 mg/liter for both. Importantly, none of the tested isolates showed an elevated ME1111 MIC. Moreover, the antifungal activity of ME1111 was minimally affected by 5% wool keratin powder in comparison to the other antifungals tested. The ME1111 solution was able to penetrate human nails and inhibit fungal growth in a dose-dependent manner according to the TurChub assay. In contrast, 8% ciclopirox and 5% amorolfine nail lacquers showed no activity under the same conditions. ME1111 demonstrated approximately 60-fold-greater selectivity in inhibition of Trichophyton spp. than of human cell lines. Our findings demonstrate that ME1111 possesses potent antidermatophyte activity, maintains this activity in the presence of keratin, and possesses excellent human nail permeability. These results suggest that ME1111 is a promising topical medication for the treatment of onychomycosis and therefore warrants further clinical evaluation.

Sodium selenite/selenium nanoparticles (SeNPs) protect cardiomyoblasts and zebrafish embryos against ethanol induced oxidative stress.

Alcoholic cardiomyopathy is the damage caused to the heart muscles due to high level of alcohol consumption resulting in enlargement and inflammation of the heart. Selenium is an important trace element that is beneficial to human health. Selenium protects the cells by preventing the formation of free radicals in the body. In the present study, protein mediated synthesis of SeNPs was investigated. Two different sizes of SeNPs were synthesized using BSA and keratin. The synthesized SeNPs were characterized by scanning electron microscopy (SEM) with elemental composition analysis Energy Dispersive X-ray spectroscopy(EDX) and X-ray diffraction (XRD). This study demonstrates the in vitro and in vivo antioxidative effects of sodium selenite and SeNPs. Further selenium and SeNPs were evaluated for their ability to protect against 1% ethanol induced oxidative stress in H9C2 cell line. The selenium and SeNPs were found to reduce the 1% ethanol-induced oxidative damage through scavenging intracellular reactive oxygen species. The selenium and SeNPs could also prevent pericardial edema induced ethanol treatment and reduced apoptosis and cell death in zebrafish embryos. The results indicate that selenium and SeNPs could potentially be used as an additive in alcoholic beverage industry to control the cardiomyopathy.

Fusarium spp. is able to grow and invade healthy human nails as a single source of nutrients.

Onychomycosis caused by Fusarium spp. is emerging, but some factors associated with its development remain unclear, such as whether this genus is keratinolytic. The main aim of the present study was to evaluate the ability of Fusarium to use the human nail as a single source of nutrients. We also performed an epidemiological study and antifungal susceptibility testing of Fusarium spp. that were isolated from patients with onychomycosis. The epidemiological study showed that Fusarium species accounted for 12.4 % of onychomycosis cases, and it was the most common among nondermatophyte molds. The most frequent species identified were F. oxysporum (36.5 %), F. solani (31.8 %), and F. subglutinans (8.3 %). Fluconazole was not active against Fusarium spp., and the response to terbinafine varied according to species. Fusarium was able to grow in vitro without the addition of nutrients and invade healthy nails. Thus, we found that Fusarium uses keratin as a single source of nutrients, and the model proposed herein may be useful for future studies on the pathogenesis of onychomycosis.

Transcriptomic Analysis Reveals Wound Healing of Morus alba Root Extract by Up-Regulating Keratin Filament and CXCL12/CXCR4 Signaling.

Facilitation of the wound healing process is important because a prolonged wound site increases pain and the risk of infection. In oriental medicine, an extract of Morus alba root (MA) has usually been prescribed as traditional treatment for accelerating wound healing, and it has been proven to be safe for centuries. To study the molecular mechanism of MA-mediated skin wound healing, we performed a primary cell culture and a skin explant culture and observed significant difference between the groups with and without MA extract. In the cellular system, a real-time cell analysis and real-time quantitative PCR were performed. It was found that MA extract enhanced proliferation in a dose-dependent manner on Kera-308 cell line, and up-regulated keratin expression including wound-induced Krt6a. In skin explant culture, the mRNA level derived from cell outgrowth displayed a tendency toward more up-regulated mRNA associated keratin filaments and toward a more up-regulated mRNA level of C-X-C motif chemokine 12 (CXCL12) and a chemokine receptor 4 (CXCR4) axis signaling pathway downstream. In this process, we concluded that MA extract had a scientific possibility of wound repair by increasing intracellular and extracellular supports and by inducing a CXCL12/CXCR4 signaling pathway.