Evaluation of a resoruf in-based fluorescent probe for tyrosinase detection in skin pigmentation disorders

Purpose Skin pigmentation disorders,such as vitiligo and melasma,are difficult to diagnose in the early stages,but abnor-mal tyrosinase levels and tyrosinase activity are potential indicators.Some resorufin-based fluorescence probes(RBFPs)have been designed to detect tyrosinase in tumors,but they have not been used in skin pigmentation disorders.In this study,one of these RBFPs(synthesized by resorufin salt coupled with 3-(bromomethyl)phenol)was evaluated comprehensively.Methods The RBFP was tested in different kinds of mouse and human skin cells,as well as in in vivo models,including zebrafish,guinea pigs,and Sprague-Dawley rats.In addition,small interfering RNAs(siRNAs),kojic acid,and 1-phenyl-2-thiourea(PTU)were used to inhibit tyrosinase levels or tyrosinase activity.Results This probe successfully detected tyrosinase and emitted red fluorescence in melanoma cells and melanocytes.Fluorescence was also observable in zebrafish and on the skin of guinea pigs when using the RBFP.In mouse and human cells,the RBFP showed good selectivity to tyrosinase.Moreover,in the case of decreased tyrosinase levels or activity caused by siRNAs,kojic acid,or PTU,the probe was sensitive to these changes.Further,the RBFP showed no toxic effects at concentrations of<20μmol/L,both in vitro and in vivo.Conclusions Our findings indicate the value and limitations of the RBFP in tyrosinase detection,but suggest the need for further improvement of fluorescent probes in the diagnosis of skin pigmentation disorders.

Genetics and Characteristics of a Pigmentation Defective Laboratory Strain of the Lady Beetle, Coleomegilla maculata

Beetles in the family Coccinellidae, commonly known as ladybugs, lady beetles, or ladybirds, are easily identifiable and popular beneficial insects. Current research aims to support conservation efforts of beneficial insects in agroecosystems by exploring genetic processes related to nutrition. As a part of this research, colonies of Coleomegilla maculata have been maintained in culture and inbred over many generations since 2009. One result of this inbreeding has been the discovery of novel morphological phenotypes unique to laboratory strains or present in wild populations at such low levels that they have not yet been described. One such phenotype is described here. The strain described here, ye (yellow elytra and eyes) was characterized with classical Mendelian breeding and digital image analysis. This phenotype differs from wild populations by possessing yellow pigment in the elytra and pale grey to white eyes. In contrast, wild populations of C. maculata possess pink or red pigmented elytra with black spots, and black eyes. C. maculata is not known to exhibit polymorphism in the field. Inheritance is autosomal and recessive. This species was not previously known to exhibit the dramatic variation of color described here. The strain is stable in the homozygous recessive form, and retains laboratory rearing characteristics similar to the wild type laboratory strain.

Segmental Pigmentation Disorder with Congenital Heterochromia Iridis

We report the case of a 10-year-old girl with congenital complete heterochromia iridis and segmental pigmentation disorder in its hyperpigmented form. We have found no publication that mentions the combination of these 2 disorders.

Electron Probe Microanalysis of Exogenous Pigmentation of Oral Mucosa Originating from Dental Alloy: Two Case Reports

Pigmentation of the oral mucosa is relatively common and has a wide variety of etiologies. Although most pigmented lesions of the oral mucosa are associated with deposition of melanin and accidental displacement of a dental alloy, accurate differential diagnosis of a pigmented lesion is important, especially in the case of malignant melanoma. We report two cases of oral mucosal pigmentation associated with accidental displacement of a dental alloy in which malignant melanoma was suspected. Excisional biopsy was carried out in these cases with the incision line set at approximately 5 mm from the lesions. Histopathologically, brownish foreign substances were observed in the lamina propria. Metal quantitative analyses of the extracted specimens were carried out by electron probe microanalysis (EPMA). The metal components and the mass concentration revealed that the metals were derived from a dental casting silver alloy in Case 1 and from a gold-silver palladium alloy in Case 2. Although exogenous pigmentation originating from a dental alloy is not rare, differential diagnosis of oral pigmented lesions is sometimes very difficult. In such cases, histopathological examination may be necessary for the diagnosis to exclude melanocytic lesions and EPMA may be effective to identify the causative dental alloy.

Periorbital Post-Inflammatory Hyperpigmentation after Fractionated CO₂Laser Resurfacing in Asians

Background: Most data on laser resurfacing have come from studies of people with Fitzpatrick skin types 1 - 3;however, the world’s population is comprised mostly of Fitzpatrick skin types 4 - 6, which are more susceptible to post-inflammatory hyperpigmentation (PIH). Objective: For the purpose of expanding the expertise of plastic surgeons treating patients with darker skin types, this study examined the incidence of PIH in Asians who underwent laser resurfacing, including a histologic arm on fractional ablative resurfacing. Methods & Materials: The clinical study included six subjects of Vietnamese origin who underwent single-depth fractionated CO2 laser resurfacing. The histologic study involved a seventh subject. The MiXto SX&reg;laser with a new scanning handpiece was used, along with magnifying loupes to assess ablative depth after each of three laser passes performed. Photographs were taken at various postoperative intervals. Results: All six clinical subjects showed cosmetic improvement in skin texture and tone with no post-inflammatory hyperpigmentation. In the histologic study, H&E stained sections revealed uniform diathermy. Conclusion: It is possible to significantly reduce PIH in darker skinned subjects through use of a new scanning handpiece and a technique using loupes to assess the depth of ablative resurfacing. The histologic study confirms these findings.

Pigmentation Restored in Mutant Laboratory Strain of the Lady Beetle Coleomegilla maculata through Dietary Supplementation

A laboratory colony of Coleomegilla maculata (DeGeer), ye, selected for a pigmentation deficiency, was restored to near wild type cuticle coloration by adding crushed heads and wings of the red colored parental strain to the diet. While the wings and other colored portions of the cuticle re-gained the red color, the eyes of the pigmentation deficient insects were not changed from the pale mutant form. Plant derived carotenes lycopene and beta-carotene did not restore the mutant beetles to a visibly distinguishable red color. An additional pigmentation deficient mutant strain, gold, partially recovered red cuticle color when provided with diet containing pigmented insect particles. This work represents the first rescue of a color phenotype in a lady beetle.

Histological Study of the Liver Pigmentation of Chinese Fire-bellied Newt (Cynops orientalis) During Activity and Hibernation Periods

The pigmentation in the liver of Chinese fire-bellied newt(Cynops orientalis) was described during two periods of the annual cycle(summer activity and winter hibernation). A large number of melanin granules were gathered into clusters and distributed unevenly inside the pigment cells. Liver pigmentation(melanin content) was found unstable,varying during the annual cycle. During the hibernation period,pigmentation accumulation was shown to increase in the liver of the Chinese fire-bellied newt. Hepatocytes during the active period are approximately 14.64% larger than those in the hibernation period,while the nucleus is approximately 7.43% bigger during the active period when compared with that during the hibernation period. These findings indicate that variation in pigment distribution and hepatocyte morphology in Chinese fire-bellied newt liver may be an ecologically adaptive strategy to the adverse physiological conditions during hibernation.

Pigmentation Restored in Mutant Laboratory Strain of the Lady Beetle <i>Coleomegilla maculata</i>through Dietary Supplementation

A laboratory colony of Coleomegilla maculata (DeGeer), ye, selected for a pigmentation deficiency, was restored to near wild type cuticle coloration by adding crushed heads and wings of the red colored parental strain to the diet. While the wings and other colored portions of the cuticle re-gained the red color, the eyes of the pigmentation deficient insects were not changed from the pale mutant form. Plant derived carotenes lycopene and beta-carotene did not restore the mutant beetles to a visibly distinguishable red color. An additional pigmentation deficient mutant strain, gold, partially recovered red cuticle color when provided with diet containing pigmented insect particles. This work represents the first rescue of a color phenotype in a lady beetle.

The domestication-associated L1 gene encodes a eucomic acid synthase pleiotropically modulating pod pigmentation and shattering in soybean

Pod coloration is a domestication-related trait in soybean,with modern cultivars typically displaying brown or tan pods,while their wild relative,Glycine soja,possesses black pods.However,the factors regulating this color variation remain unknown.In this study,we cloned and characterized L1,the classical locus responsible for black pods in soybean.By using map-based cloning and genetic analyses,we identified the causal gene of L1 and revealed that it encodes a hydroxymethylglutaryl-coenzyme A(CoA)lyase-like(HMGL-like)domain protein.Biochemical assays showed that L1 functions as a eucomic acid synthase and facilitates the synthesis of eucomic acid and piscidic acid,both of which contribute to coloration of pods and seed coats in soybean.Interestingly,we found that L1 plants are more prone to pod shattering under light exposure than l1 null mutants because dark pigmentation increases photothermal efficiency.Hence,pleiotropic effects of L1 on pod color and shattering,as well as seed pigmentation,likely contributed to the preference forl1 alleles during soybean domestication and improvement.Collectively,our study provides new insights into the mechanism of pod coloration and identifies a new target for future de novo domestication oflegume crops.

In Vivo Modeling of Zebrafish Zinc Finger,MIZ-Type Containing 1 Expression and Its Effect on Pigmentation

Objective:The zinc finger,MIZ-type containing 1(ZMIZ1)gene has been identified as a possible susceptibility gene associated with vitiligo,therefore we conducted this study to investigate the role ofZMIZ1 in pigmentation.Methods:We generate a zebrafish loss-of-function model using morpholino oligonucleotides(MOs),and two orthologs of humanZMIZ1 have been annotated(ZMIZ1a andZMIZ1b).The expression profiles of ZMIZ1a and ZMIZ1b and their effects on the pigmentation in zebrafish were evaluated by using whole-mount in situ hybridization and melanin quantification.Statistical analysis was performed using the unpaired Studentt-test or one-way analysis.Results:Investigation of the temporal and spatial expressions of these two transcripts suggested that the expressions ofZMIZ1a andZMIZ1b in the brain start to emerge in a ubiquitous fashion from 2 days post-fertilization onwards.After the successful design and validation of MOs,we observed thatZMIZ1a andZMIZ1b MOs caused embryonic developmental delays and malformations in zebrafish.Further analysis of the melanin content in the morphants revealed thatZMIZ1a significantly(49.1%for 0.667 mmol/L inZMIZI1a group,P=0.03)reduced the melanin content in a dose-dependent manner,but only the highest concentration of injectedZMIZ1b MOs significantly(50%for 0.667 mmol/L inZMIZ1b group,P=0.02)reduced the melanin content.A tyrosinase inhibition assay indicated no significant difference between the morphants and wild-type zebrafish.Conclusion:This study successfully modeled a susceptibility gene identified by genome-wide association studies in a zebrafish loss-of-function model and provides insights into the biological mechanism of pigmentation.

Reduced pigmentation and thyroid hormone disruption in zebrafish embryos caused by industrial sludge near Bohai Bay,China

In recent years,pollution caused by the discharge of industrial wastewater into Bohai Bay has posed a potential threat to the health of surrounding residents.Sludge was collected from the outlet of a factory that discharges effluent into Bohai Bay,and alcohol extracts of sludge(SE)were prepared.We confirmed by UPLC-MS/MS analysis that the SE contained PAHs,including fluorene,pyrene,and phenanthrene.Zebrafish embryos as animal models were exposed to 0.1,0.3,0.5,1 and 5 mg/mL SE from 2 to 4 h post-fertilization(hpf)until 120 hpf.The results showed that SE caused a concentration-dependent increase in mortality and a decrease in hatchability.We found that SE significantly reduced eye pigmentation and decreased the movement of embryos and larvae.In addition,SE decreased triiodothyronine(T3)content and down-regulated the mRNA expression of some thyroid hormone-related genes including TPO and Thrβ,and caused the up-regulation of Dio2 and Dio3 at 120 hpf.Exposure to three individual PAHs found in SE,namely fluorene,pyrene,and phenanthrene,caused morphological and transcriptional changes that were similar to those caused by SE exposure.These findings indicate that PAHs in SE can reduce the pigmentation of zebrafish,which may be related to the genetic changes associated with thyroid hormones,and that zebrafish eye pigmentation can be used as an indicator of PAHs exposure.

The contribution of the melanin pathway to overall body pigmentation during ontogenesis of Periplaneta americana

The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ontogenesis is still lacking. To address this question we examined the roles of 2 key melanin genes （TH and DDC）, in embryonic and postembryonic development of the American cockroach, Periplaneta americana. Our results show that the melanin pathway does not contribute to the light brown coloration observed in the first nymphs. However, the dark brown coloration in mid nymphs and adults is produced solely from the melanin pathway. In addition, the DDC RNAi results reveal that it is dopamine melanin, not DOPA melanin, acts as the main contributor in this process. Overall, present study provides a new insight into insect pigmentation suggesting that genetic mechanisms of coloration can change during ontogenesis. Future studies of additional basal insect lineages will be required to assess in more details the generality of this phenomenon.

Effects of fish meal replacement with Chlorella meal on growth performance,pigmentation,and liver health of largemouth bass(Micropterus salmoides)

Chlorella meal is a potential protein source for aquafeeds.However,the physiological response of carnivorous fish fed Chlorella meal remains elusive.This study evaluated the effects of replacing dietary fish meal with Chlorella meal on growth performance,pigmentation,and liver health in largemouth bass.Five diets were formulated to replace dietary fish meal of 0%(C0,control),25%(C25),50%(C50),75%(C75),and 100%(C100)with Chlorella meal,respectively.Total 300 fish(17.6±0.03 g)were randomly assigned to 15 tanks(3 tanks/group).Fish were fed the experimental diet twice daily for 8 weeks.The increased dietary Chlorella meal quadratically influenced the final body weight(FBW),weight gain rate(WGR),specific growth rate(SGR),and feed intake(FI),which were significantly lower in the C100 group than in the other groups(P<0.05).The feed conversion ratio(FCR)increased linearly or quadratically with dietary Chlorella meal.Dietary Chlorella meal linearly or quadratically increased the lutein content of plasma,liver,and dorsal muscle of largemouth bass(P<0.05).Compared to the C0 group,all supplemented Chlorella meal groups significantly improved the yellowness(b*)of the dorsal body(1.5 to 2.0 fold),abdominal body(1.5 to 1.8 fold),and dorsal muscle(3.8 to 5.4 fold)of largemouth bass(P<0.05).In addition,compared to the C0 group,the liver vacuolation area of fish was significantly increased in the C75 and C100 groups(P<0.05).Transcriptional levels of apoptosis-related genes of b-cell lymphoma-2(bcl2),caspase-9-like(casp9),and caspase-3a(casp3)were markedly upregulated(0.9 to 1.6 fold)in the C100 group compared to the C0 group(P<0.05).Based on the quadratic regression analysis between FBW,WGR,or SGR and dietary Chlorella meal level,largemouth bass had the best growth when replacing 31.7%to 32.6%of fish meal with 15.03%to 15.43%dietary Chlorella meal.The present results indicated that dietary supplementation with Chlorella meal(11.85%to 47.45%)significantly enhanced the pigmentation;however,total replacement of fish meal(40%)with Chlorella meal(47.45%)caused growth retardation,apoptosis,and liver damage in largemouth bass.

CRISPR/Cas9-based mutation reveals Argonaute 1 is essential for pigmentation in Ostrinia fumacalis

Ostrinia fumacalis (Lepidoptera: Pyralidae) is one of the most destructive agricultural pests in Asia. Traditional pest-management methods include sex pheromone capture, transgenic crops that produce Bacillus thuringiensis toxin, and pesticides. Although these strategies control pest populations effectively, they also cause negative side effects, including dramatically increased pesticide resistance, severe pollution, and hazards for human health. Recently developed genome editing tools provide new prospects for pest management and have been successfully used in several species. However, few examples have been reported in the agricultural pest O. fumacalis due to a lack in genomic information. In this report, we identified only one transcript of O. fumacalis Argonaute 1 (OfAgo 1) gene from the genome and cloned the open reading frame. OfAgol presented the maximum expression at the embryo stage or in the fat body during the larval stages. To understand its function, an OfAgol mutant was constructed using the Clustered Regularly Interspaced Short Palindromic Repeat/RNA-guided Cas9 nuclease (CRISPR/Cas9). Mutagenesis of OfAgol disrupted cuticle pigmentation by dowregulating micro RNAs and pigmentation-related genes. This is the first report for the cloning and functional analysis of OfAgol, revealing a role of OfAgol in cuticle pigmentation. The current report also established a CRISPR/Cas9 system in O. fumacalisy providing a new insight for pest management.

Genome scan of pigmentation traits in Friesian-Jersey crossbred cattle

Pigmentation traits expressed in animals are visual characteristics that allow us to distinguish between breeds and between strains within breed. The objective of this study was to map quantitative trait loci （QTLs） affecting the pigmentation traits in approximately 800 F2 grand daughter dairy cattle from a Holstein-Friesian and Jersey cross breed cattle. Traits analyzed included pigmentation phenotypes on the body, teat and hoop. The phenoypes were collected from digital photos or visual inspection of live animals. QTL mapping was implemented using half-sib and line-of-descent inheritance models. Our analysis initially detected a number of significant QTLs on chromosomes： 2, 6, 13, 15, 18 and 22. The significant QTLs were divided into two groups： one group influencing the pigmentation color and the other group affecting the absence or level of pigmentation. The most significant QTL peaks were observed on Bovine taurus autosome 18 （BTA18） close to melanocortin 1 receptor （MC1R） for the color traits, on BTA6 close to the receptor tyrosine kinase （K/T） and BTA22 close to microphthalmia-associated transcription factor （M/TF） gene for the spotting traits. Association studies were conducted for candidate regions or genes known to affect pigmentation in dairy cattle.

Biomaterial engineering strategies for modeling the Bruch's membrane in age-related macular degeneration

Age-related macular degeneration,a multifactorial inflammatory degenerative retinal disease,ranks as the leading cause of blindness in the elderly.Strikingly,there is a scarcity of curative therapies,especially for the atrophic advanced form of age-related macular degeneration,likely due to the lack of models able to fully recapitulate the native structure of the outer blood retinal barrier,the prime to rget tissue of age-related macular degeneration.Standard in vitro systems rely on 2D monocultures unable to adequately reproduce the structure and function of the outer blood retinal barrier,integrated by the dynamic interaction of the retinal pigment epithelium,the Bruch's membrane,and the underlying choriocapillaris.The Bruch's membrane provides structu ral and mechanical support and regulates the molecular trafficking in the outer blood retinal barrier,and therefo re adequate Bruch's membrane-mimics are key for the development of physiologically relevant models of the outer blood retinal barrie r.In the last years,advances in the field of biomaterial engineering have provided novel approaches to mimic the Bruch's membrane from a variety of materials.This review provides a discussion of the integrated properties and function of outer blood retinal barrier components in healt hy and age-related macular degeneration status to understand the requirements to adequately fabricate Bruch's membrane biomimetic systems.Then,we discuss novel materials and techniques to fabricate Bruch's membrane-like scaffolds for age-related macular degeneration in vitro modeling,discussing their advantages and challenges with a special focus on the potential of Bruch's membrane-like mimics based on decellularized tissue.

Lutein-stevioside nanoparticle attenuates H_(2)O_(2)-induced oxidative damage in ARPE cells

In order to improve the bioavailability of lutein(LUT),a novel lutein-stevio side nanoparticle(LUT-STE)were prepared previously,but the information about LUT-STE on protecting of eye health was limited.This study investigated the effect of LUT-STE on antioxidant activity of H_(2)O_(2)-induced human retinal pigment epithelial(ARPE)cells.LUT and LUT-STE(final concentration of 5μg/mL)significantly enhanced cell viability from(74.84±5.10)%to(81.92±10.01)%(LUT)and(89.33±4.34)%(LUT-STE),and inhibited the cell apoptosis(P<0.05).After pretreatment with LUT-STE in ARPE cells,the levels of superoxide dismutase(SOD),catalase(CAT)and glutathion peroxidase(GSH-Px)in ARPE cells were significantly increased(P<0.05),the contents of reactive oxygen species(ROS)and malondialdehyde(MDA)were decreased.In addition,the vascular endothelial growth factor(VEGF)levels were inhibited by 13.61%and 17.39%,respectively,pretreatment with LUT and LUT-STE.Western blotting results showed that the pretreatment with LUT-STE inhibited the expression of caspase-9 and caspase-3 and up-regulated Bcl-2/Bax pathway to inhibit H_(2)O_(2)-induced apoptosis.In summary,the novel delivery LUT-STE had more pronounced inhibitory effect on H_(2)O_(2)-induced damage in human ARPE cells.

Regulatory factors of Nrf2 in age-related macular degeneration pathogenesis

Age-related macular degeneration(AMD)is a complicated disease that causes irreversible visual impairment.Increasing evidences pointed retinal pigment epithelia(RPE)cells as the decisive cell involved in the progress of AMD,and the function of anti-oxidant capacity of PRE plays a fundamental physiological role.Nuclear factor erythroid 2 related factor 2(Nrf2)is a significant transcription factor in the cellular anti-oxidant system as it regulates the expression of multiple anti-oxidative genes.Its functions of protecting RPE cells against oxidative stress(OS)and ensuing physiological changes,including inflammation,mitochondrial damage and autophagy dysregulation,have already been elucidated.Understanding the roles of upstream regulators of Nrf2 could provide further insight to the OS-mediated AMD pathogenesis.For the first time,this review summarized the reported upstream regulators of Nrf2 in AMD pathogenesis,including proteins and miRNAs,and their underlying molecular mechanisms,which may help to find potential targets via regulating the Nrf2 pathway in the future research and further discuss the existing Nrf2 regulators proved to be beneficial in preventing AMD.

An overview of pigment gland morphogenesis and its regulatory mechanism

Cotton has enormous economic potential,providing high-quality protein,oil,and fibre.But the comprehensive utilization of cottonseed is limited by the presence of pigment gland and its inclusion.Pigment gland is a common characteristic of Gossypium genus and its relatives,appearing as visible dark opaque dots in most tissues and organs of cotton plants.Secondary metabolites,such as gossypol,synthesized and stored in the cavities of pigment glands act as natural phytoalexins,but are toxic to humans and other monogastric animals.However,only a few cotton genes have been identified as being associated with pigment gland morphogenesis to date,and the developmental processes and regulatory mechanism involved in pigment gland formation remain largely unclear.Here,the research progress on the process of pigment gland morphogenesis and the genetic basis of cotton pigment glands is reviewed,for providing a theoretical basis for cultivating cotton with the ideal pigment gland trait.

Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

AIM:To explore the effect of epidermal growth factor receptor(EGFR)inhibition by erlotinib and EGFR siRNA on epidermal growth factor(EGF)-induced activation of retinal pigment epithelium(RPE)cells.METHODS:Human RPE cell line(ARPE-19 cells)was activated by 100 ng/mL EGF.Erlotinib and EGFR siRNA were used to intervene EGF treatment.Cellular viability,proliferation,and migration were detected by methyl thiazolyl tetrazolium(MTT)assay,bromodeoxyuridine(BrdU)staining assay and wound healing assay,respectively.EGFR/protein kinase B(AKT)pathway proteins and N-cadherin,α-smooth muscle actin(α-SMA),and vimentin were tested by Western blot assay.EGFR was also determined by immunofluorescence staining.RESULTS:EGF treatment for 24h induced a significant increase of ARPE-19 cells’viability,proliferation and migration,phosphorylation of EGFR/AKT proteins,and decreased total EGFR expression.Erlotinib suppressed ARPE-19 cells’viability,proliferation and migration through down regulating total EGFR and AKT protein expressions.Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin,α-SMA,and vimentin proteins.Similarly,EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation,viability,and migration,phosphorylation of EGFR/AKT proteins,and up-regulation of N-cadherin,α-SMA,and vimentin proteins.CONCLUSION:Erlotinib and EGFR-knockdown suppress EGF-induced cell viability,proliferation,and migration via EGFR/AKT pathway in RPE cells.EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy(PVR).