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One of the most unique coat color patterns in the domestic dog is merle (also known as dapple in the dachshund breed), characterized by patches of normal pigmentation surrounded by diluted eumelanin pigment. In dogs, this striking variegated pattern is caused by an insertion of a SINE element into the PMEL gene. Differences in the length of the SINE insertion [due to a variable-length poly(A)-tail] has been associated with variation in the merle coat color and patterning. We previously performed a systematic evaluation of merle in 175 Australian shepherds and related breeds and correlated the length of the merle insertion variants with four broad phenotypic clusters designated as "cryptic", "atypical", "classic", and "harlequin" merle. In this study, we evaluated the SINE insertions in 140 dachshunds and identified the same major merle phenotypic clusters with only slight variation between breeds. Specifically, we identified numerous cases of true "hidden" merle in dachshunds with light/red (pheomelanin) coats with little to no black/brown pigment (eumelanin) and thus minimal or no observable merle phenotype. In addition, we identified somatic and gonadal mosaicism, with one dog having a large insertion in the harlequin size range of M281 that had no merle phenotype and unintentionally produced a double merle puppy with anophthalmia. The frequent identification of cryptic, hidden, and mosaic merle variants, which can be undetectable by phenotypic inspection, should be of particular concern to breeders and illustrates the critical need for genetic testing for merle prior to breeding to avoid producing dogs with serious health problems.

CD8+ T cells are crucial to establish antitumor immunity, and their high infiltration associates with favorable prognoses. However, several CD8+ T cell subpopulations in the tumor microenvironment may play different roles in prognosis, progression, and immunotherapy. Here, we analyzed prior published single-cell RNA-sequencing (scRNA-seq) melanoma data to explore the heterogeneity of CD8+ T cell subpopulations and identified 7 major subpopulations. We found that high infiltration of exhausted CD8+ T cell subpopulation 2 would contribute to unfavorable prognoses. In contrast, a large proportion of naive/memory cells and cytotoxic CD8+ T cell subpopulation 3 would lead to favorable prognoses. Notably, the proportion of the cytotoxic CD8+ T cell subpopulation 3 would decrease in later-stage melanoma samples, while that of the exhausted CD8+ T cell subpopulation 2 would increase. We also found that high abnormal activities of metabolic pathways existed in exhausted CD8+ T cell subpopulation 1. Significantly, immunosuppressive checkpoints PD-1 and CTLA-4 signaling pathways were upregulated in exhausted CD8+ T cell subpopulations. In addition, a dynamic transcript landscape of immune checkpoints among different subpopulations was also depicted in this study. Moreover, we identified three overexpressed genes (PMEL, TYRP1, and EDNRB) that were significantly correlated to poor prognoses and only expressed in exhausted CD8+ T cell subpopulation 2. Importantly, they showed the highest expression in melanoma samples compared to other tumors. In general, we characterized the CD8+ T cell subpopulations in melanoma and identified that not only genes of immunosuppressive checkpoints but also PMEL, TYRP1, and EDNRB could serve as potential targets for melanoma therapy.

Genome editing provides opportunities to improve current cattle breeding strategies through targeted introduction of natural sequence variants, accelerating genetic gain. This can be achieved by harnessing homology-directed repair mechanisms following editor-induced cleavage of the genome in the presence of a repair template. Introducing the genome editors into zygotes and editing in embryos has the advantage of uncompromised development into live animals and alignment with contemporary embryo-based improvement practices. In our study, we investigated the potential to introduce sequence variants, known from the pre-melanosomal protein 17 (PMEL) and prolactin receptor (PRLR) genes, and produce non-mosaic, edited embryos, completely converted into the precision genotype. Injection of gRNA/Cas9 editors into bovine zygotes to introduce a 3 bp deletion variant into the PMEL gene produced up to 11% fully converted embryos. The conversion rate was increased to up to 48% with the use of TALEN but only when delivered by plasmid. Testing three gRNA/Cas9 editors in the context of several known PRLR sequence variants, different repair template designs and delivery as DNA, RNA or ribonucleoprotein achieved full conversion rates up to 8%. Furthermore, we developed a biopsy-based screening strategy for non-mosaic embryos which has the potential for exclusively producing non-mosaic animals with intended precision edits.

Background High-producing Holstein Friesian dairy cattle have a characteristic black and white coat, often with large proportions of black. Compared to a light coat color, black absorbs more solar radiation which is a contributing factor to heat stress in cattle. To better adapt dairy cattle to rapidly warming climates, we aimed to lighten their coat color by genome editing. Results Using gRNA/Cas9-mediated editing, we introduced a three bp deletion in the pre-melanosomal protein 17 gene (PMEL) proposed as causative variant for the semi-dominant color dilution phenotype observed in Galloway and Highland cattle. Calves generated from cells with homozygous edits revealed a strong color dilution effect. Instead of the characteristic black and white markings of control calves generated from unedited cells, the edited calves displayed a novel grey and white coat pattern. Conclusion This, for the first time, verified the causative nature of the PMEL mutation for diluting the black coat color in cattle. Although only one of the calves was healthy at birth and later succumbed to a naval infection, the study showed the feasibility of generating such edited animals with the possibility to dissect the effects of the introgressed edit and other interfering allelic variants that might exist in individual cattle and accurately determine the impact of only the three bp change.

Background The generation of functional human epidermal melanocytes (HEM) from stem cells provides an unprecedented source for cell-based therapy in vitiligo. Despite the important efforts exerted to obtain melanin-producing cells from stem cells, pre-clinical results still lack the safety and scalability characteristics essential for their translational application. Methods Here, we report a rapid and efficient protocol based on defined culture conditions capable of differentiating adult adipose-derived stem cells (ADSC) to scalable amounts of proliferative melanocyte precursors (PreMel) within 30 days. PreMel were characterized in vitro through qPCR, Western blot, flow cytometry, biochemical assays, and in vivo assays in immunocompromised mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, or NSG). Results After 30 days of differentiation, the stem cell-derived PreMel were defined as CD105neg CD73low according to immunophenotypic changes in comparison with parental stem cell markers. In addition, expression of microphthalmia-associated transcription factor (MITF), active tyrosinase (TYR), and the terminal differentiation-involved premelanosome protein (PMEL) were detected. Furthermore, PreMel had the potential to synthesize melanin and package it into melanosomes both in vitro and in vivo in NSG mice skin. Conclusions This study proposes a rapid and scalable protocol for the generation of proliferative melanocyte precursors (PreMel) from ADSC. These PreMel display the essential functional characteristics of bona fide HEM, opening a new path for an autologous cellular therapy for vitiligo patients.

Melanoma plasticity creates a plethora of opportunities for cancer cells to escape treatment. Thus, therapies must target all cancer cell subpopulations bearing the potential to contribute to disease. The role of the differentiation/pigmentation program in intrinsic and acquired drug resistance is largely uncharacterized. MITF level and expression of MITF-dependent pigmentation-related genes,MLANA,PMEL,TYR,andDCT, in drug-naïve and vemurafenib- or trametinib-treated patient-derived melanoma cell lines and their drug-resistant counterparts were analysed and referred to genomic alterations. Variability in execution of pigmentation/differentiation program was detected in patient-derived melanoma cell lines. Acute treatment with vemurafenib or trametinib enhanced expression of pigmentation-related genes in MITF-Mhighmelanoma cells, partially as the consequence of transcriptional reprograming. During development of resistance, changes in pigmentation program were not unidirectional, but also not universal as expression of different pigmentation-related genes was diversely affected. In selected resistant cell lines, differentiation/pigmentation was promoted and might be considered as one of drug-tolerant phenotypes. In other resistant lines, dedifferentiation was induced. Upon drug withdrawal (“drug holiday”), the dedifferentiation process in resistant cells either was enhanced but reversed by drug reexposure suggesting involvement of epigenetic mechanisms or was irreversible. The irreversible dedifferentiation might be connected with homozygous loss-of-function mutation inMC1R, as MC1RR151C +/+variant was found exclusively in drug-naïve MITF-Mlowdedifferentiated cells and drug-resistant cells derived from MITFhigh/MC1RWTcells undergoing irreversible dedifferentiation. MC1RR151C +/+variant might be further investigated as a parameter potentially impacting melanoma patient stratification and aiding in treatment decision.

The objective of this trial was to investigate the effects of using meloxicam as a pretransport or on arrival therapeutic on disease outcomes of bovine respiratory disease (BRD), biomarker outcomes associated with BRD, performance characteristics over the first 42 d on feed, and carcass traits at harvest in cross bred beef cattle. Multisourced, crossbred steer calves (n = 168) consisting of mainly British and British-Continental breeds were purchased from an auction market in central Missouri. Calves were processed prior to transportation and again upon feedlot arrival. Animals were randomized to 3 separate treatments: pretransport meloxicam (PMEL), arrival meloxicam (AMEL), and a control group receiving inactive excipient (CONT). Dosing at 1 mg/kg on weighted averaged administered per os. Animals were weighed and blood was collected pre- and post-transport. Haptoglobin (Hp)-matrix metaloproteinase (MMP)-9 complex, cortisol, and substance P were quantified. Weights were taken again at 42 d and at harvest. Clinical signs of BRD were monitored using indicators of depression, appetite, respiration, and temperature that qualified the animals for treatment. Harvest parameters were collected using a standardized United States Department of Agriculture grading system for quality grade and yield grade. Meloxicam did not have a significant effect on BRD morbidity over the course of the study and there was no significant effect on performance characteristics at 42 d (P > 0.10). Of the calves that did succumb to BRD, no significant differences were found in severity of disease (P > 0.10). Concentrations of substance P and Hp- MMP-9, were increased on arrival (P ≤ 0.05) however no significant treatment effect or interaction were found between AMEL, PMEL, CONT, or across different levels of biomarkers (P > 0.10). Meloxicam use prior to or on arrival does not mitigate disease or improve performance during the feeding period.

Tissue resident memory T cells (Trm) form in the skin in vitiligo and persist to maintain disease, as white spots often recur rapidly after discontinuing therapy. We and others have recently described melanocyte-specific autoreactive Trm in vitiligo lesions. Here, we characterize the functional relationship between Trm and recirculating memory T cells (Tcm) in our vitiligo mouse model. We found that both Trm and Tcm sensed autoantigen in the skin long after stabilization of disease, producing IFN-γ, CXCL9, and CXCL10. Blockade of Tcm recruitment to the skin with FTY720 or depletion of Tcm with low-dose Thy1.1 antibody reversed disease, indicating that Trm cooperate with Tcm to maintain disease. Taken together, our data provide characterization of skin memory T cells in vitiligo, demonstrate that Trm and Tcm work together during disease, and indicate that targeting their survival or function may provide novel, durable treatment options for patients.

Tanshinone IIA has been reported to exhibit anti-inflammatory effects, while it is not clear whether Tanshinone IIA has protective role in vitiligo. Premelanosome (PMEL) CD8+ T cells were adoptive transferred into Krt14- Kitl* mice with Kit ligand (KITL) over-expressed, to construct the vitiligo model. Pdk1fl/fl and Stat3fl/fl mice were crossed with Cd8cre mice to establish Pdk1TKO and Stat3TKO mice. Tanshinone IIA (200 μg) was intravenous injected to treat vitiligo in mice every 3 days. The accumulation of macrophages and CD8+ T cells in the ear skin was assayed by flow cytometry. Bone marrow-derived macrophages (BMDMs) were induced and stimulated with lipopolysaccharides (LPS) and IL-4. It was found that Tanshinone IIA alleviated the development of vitiligo, impaired PMEL CD8+ T cells accumulation in the ear skin, and inhibited LPS-induced TNF-α, IL-6, and IL-1β expression and secretion in BMDMs, which could also inhibit IL-4-induced Arg-1 and Mrc-1 expression in BMDMs. In addition, Tanshinone IIA could inhibit the proliferation and cytotoxic function of CD8+ T cells indicated by the expression of Perforin, Granzymeb, and IFN-γ. Furthermore, Tanshinone IIA treated Pdk1TKO mice, not Stat3TKO mice, showed impaired PMEL CD8+ T cells accumulation in the ear skin. In summary, Tanshinone IIA alleviates vitiligo development with impaired CD8+ T cells accumulation and activation of Pdk1-Akt pathway.

Vitiligo is an autoimmune skin disease, characterized by depigmentation and epidermal melanocytes loss. The specific mechanisms underlying vitiligo have not been fully understood. As a result, treating vitiligo is a dermatological challenge. Recently, much attention has been paid to the dysfunction and interaction of organelles under environmental stress. The impaired organelles could generate misfolded proteins, particularly accumulated toxic premelanosome protein (PMEL) amyloid oligomers, activating the autoimmune system and cause melanocyte damage. Unfolded protein response (UPR) dysfunction accelerates toxic PMEL accumulation. Herein, we presented a narrative review on UPR's role in vitiligo, the misfolded PMEL-induced attack of the autoimmune system under autophagy dysfunction caused by abnormal activation of transient receptor potential (TRP) channels and the background of UPR system defects in melanocytes. All of these mechanisms were integrated to form UPR/PMEL-TRP channels/autophagy axis, providing a new understanding of vitiligo pathogenesis.

GNAI2 (G protein subunit alpha i2) is a signaling modulator or transducer, involved in several transmembrane signaling systems, that plays a vital role in the melanogenesis signaling pathway. However, whether GNAI2 regulates cell proliferation and apoptosis in rabbit melanocytes is not known. We found that GNAI2 was differentially expressed in rabbits with different coat colors using qRT-PCR and Wes assays. Furthermore, it was observed that the rabbits with black skin had the highest GNAI2 levels, and those with white skin had the lowest expression. The coding sequence of GNAI2 was successfully cloned and inserted into pcDNA3.1 and pcDNA3.1-Myc vectors. It was observed that the GNAI2 protein was mainly localized in the cytoplasm using the indirect immunofluorescence staining assay. Overexpression of GNAI2 significantly increased melanin content, promoted melanocyte proliferation, and inhibited melanocyte apoptosis. On the contrary, the knockdown of GNAI2 using siRNA had the opposite effect. In addition, GNAI2 significantly increased the mRNA expression levels of the melanin-related genes TYR, GPNMB, PMEL, and DCT in rabbit melanocytes. The results suggested that GNAI2 regulated melanocyte development by promoting melanocyte proliferation and inhibiting apoptosis.

The ancient paralogs premelanosome protein (PMEL) and glycoprotein nonmetastatic melanoma protein B (GPNMB) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurodegenerative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolutionary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most ancient paralog of the family. By curating the functional domains in each paralog, we identified many commonalities dating back to the emergence of the gene family in basal metazoans. PMEL and GPNMB have gained functional domains since their divergence from TMEM130, including the core amyloid fragment (CAF) that is critical for the amyloid potential of PMEL. Additionally, the PMEL gene has acquired the enigmatic repeat domain (RPT), composed of a variable number of imperfect tandem repeats, this domain acts in an accessory role to control amyloid formation. Our analyses revealed the vast variability in sequence, length and repeat number in homologous RPT domains between craniates, even within the same taxonomic class. We hope that these analyses inspire further investigation into a gene family that is remarkable from the evolutionary, pathological and cell biology perspectives.

Chicken plumage color, the genetic basis of which is often affected by epistasis, has long interested scientists. In the current study, a population of complex epistasis was constructed by crossing dominant White Leghorn chickens with recessive white feather chickens. Through a genome-wide association study, we identified single nucleotide polymorphisms and genes significantly associated with white and colored plumage in hens at different developmental stages. Interestingly, white plumage in adulthood was associated with the recessive white feather gene (TYR), whereas white feathers at birth stage were associated with the dominant white feather gene (PMEL), indicating age-related roles for these genes. TYR was shown to exert an epistatic effect on PMEL in adult hens. Additionally, TYR had an epistatic effect on barred plumage, while barred plumage had an epistatic effect on black plumage. TYR had no epistatic effect on the yellow plumage. We confirmed that the barred plumage gene is CDKN2A, as reported in previous studies. Golgb1 and REEP3, which play important roles in the Golgi network and affect the formation of feather pigments, are important candidate genes for yellow plumage. The candidate genes for black plumage are CAMKK1 and IFT22. Further research is warranted to elucidate the molecular mechanisms underlying these traits.

Nuclear factor erythroid 2-related factor 2 (Nrf2), which is linked to autophagy regulation and melanogenesis regulation, is activated by marliolide. In this study, we investigated the effect of a marliolide derivative on melanosome degradation through the autophagy pathway. The effect of the marliolide derivative on melanosome degradation was investigated in α-melanocyte stimulating hormone (α-MSH)-treated melanocytes, melanosome-incorporated keratinocyte, and ultraviolet (UV)B-exposed HRM-2 mice (melanin-possessing hairless mice). The marliolide derivative, 5-methyl-3-tetradecylidene-dihydro-furan-2-one (DMF02), decreased melanin pigmentation by melanosome degradation in α-MSH-treated melanocytes and melanosome-incorporated keratinocytes, evidenced by premelanosome protein (PMEL) expression, but did not affect melanogenesis-associated proteins. The UVB-induced hyperpigmentation in HRM-2 mice was also reduced by a topical application of DMF02. DMF02 activated Nrf2 and induced autophagy in vivo, evidenced by decreased PMEL in microtubule-associated proteins 1A/1B light chain 3B (LC3)-II-expressed areas. DMF02 also induced melanosome degradation via autophagy in vitro, and DMF02-induced melanosome degradation was recovered by chloroquine (CQ), which is a lysosomal inhibitor. In addition, Nrf2 silencing by siRNA attenuated the DMF02-induced melanosome degradation via the suppression of p62. DMF02 induced melanosome degradation in melanocytes and keratinocytes by regulating autophagy via Nrf2-p62 activation. Therefore, Nrf2 activator could be a promising therapeutic agent for reducing hyperpigmentation.

Microphthalmia-associated transcription factor-M (MITF-M) is the key gene in the proliferation and differentiation of melanocytes, which undergoes an array of post-translation modifications. As shown in our previous study, deubiquitinase USP13 is directly involved in melanogenesis. However, it is still ambiguous that the effect of USP13-mediated MITF-M expression on melanocytes proliferation and apoptosis. Herein, we found that MITF-M overexpressing melanocytes showed high cell proliferation, reduced apoptosis, and increased melanin levels. Besides, melanin-related genes, TYR, DCT, GPNMB, and PMEL, were significantly up-regulated in MITF-M overexpressing melanocytes. Furthermore, Exogenous USP13 significantly upregulated the endogenous MITF-M protein level, downregulated USP13 significantly inhibited MITF-M protein levels, without altering MITF-M mRNA expression. In addition, USP13 upregulation mitigated the MITF-M degradation and significantly increased the half-life of MITF-M. Also, USP13 stabilized the exogenous MITF protein levels. In conclusion, the MITF-M level was regulated by USP13 deubiquitinase in melanocytes, affecting melanocytes proliferation and apoptosis. This study provides the theoretical basis for coat color transformation that could be useful in the development of the new breed in fur animals.

Background Glaucoma is a leading cause of visual disability and blindness. Release of iris pigment within the eye, pigment dispersion syndrome (PDS), can lead to one type of glaucoma known as pigmentary glaucoma. PDS has a genetic component, however, the genes involved with this condition are largely unknown. We sought to discover genes that cause PDS by testing cohorts of patients and controls for mutations using a tiered analysis of exome data. Results Our primary analysis evaluated melanosome-related genes that cause dispersion of iris pigment in mice (TYRP1, GPNMB, LYST, DCT, and MITF). We identified rare mutations, but they were not statistically enriched in PDS patients. Our secondary analyses examined PMEL (previously linked with PDS), MRAP, and 19 other genes. Four MRAP mutations were identified in PDS cases but not in controls (p = 0.016). Immunohistochemical analysis of human donor eyes revealed abundant MRAP protein in the iris, the source of pigment in PDS. However, analysis of MRAP in additional cohorts (415 cases and 1645 controls) did not support an association with PDS. We also did not confirm a link between PMEL and PDS in our cohorts due to lack of reported mutations and similar frequency of the variants in PDS patients as in control subjects. Conclusions We did not detect a statistical enrichment of mutations in melanosome-related genes in human PDS patients and we found conflicting data about the likely pathogenicity of MRAP mutations. PDS may have a complex genetic basis that is not easily unraveled with exome analyses.

The pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Excluded from the aggregate is the regulatory N-terminus, which nevertheless must be linked in cis to the CAF in order to drive amyloid formation. The domain is then likely cleaved away immediately before, during, or immediately after the incorporation of a new CAF subunit into the nascent amyloid. We had previously identified a 21 amino acid long region, which mediates the regulatory activity of the N-terminus towards the CAF. However, many mutations in the respective segment caused misfolding and/or blocked PMEL export from the endoplasmic reticulum, leaving their phenotype hard to interpret. Here, we employ a saturating mutagenesis approach targeting the motif at single amino acid resolution. Our results confirm the critical nature of the PMEL N-terminal region and identify several residues essential for PMEL amyloidogenesis.

Background Minxian black fur (MBF) sheep are found in the northwestern parts of China. These sheep have developed several special traits. Skin color is a phenotype subject to strong natural selection and diverse skin colors are likely a consequence of differences in gene regulation. Methods Skin structure, color differences, and gene expression (determined by RNA sequencing) were evaluated the Minxian black fur and Small-tail Han sheep (n = 3 each group), which are both native Chinese sheep breeds. Results Small-tail Han sheep have a thicker skin and dermis than the Minxian black fur sheep (P P OCA2 melanosomal transmembrane protein (OCA2), dopachrome tautomerase (DCT), tyrosinase (TYR) and tyrosinase related protein (TYRP1), melanocortin 1 receptor (MC1R), and premelanosome protein (PMEL). The results from our histological and transcriptome analyses will form a foundation for additional investigation into the genetic basis and regulation of pigmentation in these sheep breeds.

Skin color is an important economic trait in meat-type chickens. A uniform bright skin color can increase the sales value of chicken. Chickens with bright yellow skin are more popular in China, especially in the broiler market of South China. However, the skin color of chickens can vary because of differences in breeds, diet, health, and individual genetics. To obtain greater insight into the genetic factors associated with the process of skin pigmentation in chickens, we used a colorimeter and high-resolution skin photographs to measure and analyze the skin color of chickens. By analyzing 534 chickens of the same breed, age, and feed condition, we found that the yellowness values of the chickens varied within this population. A significant positive correlation was found between the cloacal skin yellowness values before and after slaughter, and the cloacal skin yellowness value of live chickens was positively correlated with the overall body skin yellowness value. Additionally, chicken skin yellowness exhibited low heritability, ranging from 0.07 to 0.27. Through RNA sequencing, 882 genes were found to be differentially expressed between the skin with the highest and lowest yellowness values. Some of these differentially expressed genes may play an important role in yellow pigment deposition in chicken skin, which included TLR2B, IYD, SMOC1, ALDH1A3, CYP11A1, FHL2, TECRL, ACACB, TYR, PMEL, and GPR143. In addition, we found that the expression and variations of the BCO2 gene, which is referred to as the yellow skin gene, cannot be used to estimate the skin yellowness value of chickens in this population. These data will help to further our understanding of chicken skin yellowness and might contribute to the selection of specific chicken strains with consistent skin coloration.

Selection by breeders modifies the morphology, behaviour and performance of domesticated species. Here, we examined signs of selection in Finnhorse, the only native horse breed in Finland. We first searched divergent genomic regions between Finnhorses and other breeds, as well as between different breeding sections of the Finnhorse with data from Illumina Equine SNP70 BeadChip, and then studied several of the detected regions in more detail. We found altogether 35 common outlier SNPs between Finnhorses and other breeds using two different selection tests. Many of the SNPs were located close to genes affecting coat colour, performance, size, sugar metabolism, immune response and olfaction. We selected genes affecting coat colour (KIT, MITF, PMEL), performance (MSTN) and locomotion (DMRT3) for a more detailed examination. In addition, we looked for, and found, associations with height at withers and SNPs located close to gene LCORL. Among the four breeding sections of Finnhorses (harness trotters, riding horses, draught horses and pony‐sized horses), a single SNP located close to the DMRT3 gene was significantly differentiated and only between harness trotters and pony‐sized horses.

Colors are important phenotypic traits for fitness under natural conditions in vertebrates. Previous studies have reported several functional genes and genetic variations of pigmentation, but the formation mechanisms of various skin coloration remained ambiguous in fish. Jinbian carp, a common carp variant, displays two colors (yellow and black) in the skin, thus, it is a good model for investigating the genetic basis of pigmentation. In the present study, using the Jinbian carp as model, isobaric tags for relative and absolute quantification (ITRAQ) proteomics analysis was performed for yellow and black skin, respectively. The results showed that 467 differentially expressed proteins (DEPs) were identified between the yellow skin and the black skin. Similar to mammals, the up-regulated DEPs in black skin included UV excision repair protein RAD23 homolog A (Rad23a), melanoregulin (mreg), 5,6-dihydroxyindole-2-carboxylic acid oxidase5 (tyrp1) and melanocyte protein PMEL (PMEL), which were mainly grouped into melanogenesis pathway. However, several up-regulated DEPs in yellow skin were mainly enriched in nucleotide metabolism, such as GTPase IMAP family member 5 (GIMAP5), AMP deaminase 1 (AMPD1), adenosylhomocysteinase b (ahcy-b), and pyruvate kinase (PKM). In summary, several candidate proteins and their enrichment pathways for color variation in Jinbian carp were identified, which may be responsible for the formation of different colorations.

Premelanosome protein (PMEL) is crucial for the formation of melanosomal fibrils through the transition from stage I to stage II melanosomes. It was used as a target antigen in some adoptive T-cell therapy of melanoma. The correlation of PMEL to prognosis and immune cell infiltration level are unknown in melanoma. The PMEL expression was evaluated via Tumor Immune Estimation Resource, Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA). We also evaluate the influence of PMEL on overall survival via GEPIA, PrognoScan, and immunohistochemistry in human tissue microarray. The correlation between PMEL expression level and immune cell or gene markers of immune infiltration level was explored on Tumor Immune Estimation Resource and GEPIA. PMEL expression was significantly higher in skin cutaneous melanoma (SKCM) and SKCM-metastasis in comparison with the other cancers. In SKCM, PMEL expression in high levels was associated with poor overall survival. In both SKCM and SKCM-metastasis patients, PMEL expression is negatively correlated with the infiltration cells of CD8+ T cells, macrophages, and neutrophils. Programmed cell-death protein 1 just showed response rates ranging from 20% to 40% in patients with melanoma, so it is critical to discover a new therapeutic target. PMEL is negatively associated with immune cell infiltration and can be as a negative prognosis marker or new immunotherapy target in SKCM and SKCM-metastasis.

High-producing Holstein Friesian dairy cattle have a characteristic black and white coat pattern where black frequently comprises a large proportion of the coat. Compared to a light coat color, black absorbs more solar radiation translating into radiative heat gain which is a contributing factor to heat stress in cattle, negatively impacting on their production levels, fertility and welfare. To better adapt dairy cattle to the rapidly changing climatic conditions with predictions for more frequent and prolonged hot temperature patterns, we aimed to lighten their coat color by genome editing. Using gRNA/Cas9-mediated editing, we introduced a three base pair (bp) deletion in the pre-melanosomal protein 17 gene (PMEL) proposed as the causative variant responsible for the semi-dominant color dilution phenotype seen in Galloway and Highland cattle. Calves generated from cells homozygous for the edited mutation revealed a strong color dilution effect. Instead of the characteristic black and white coat color patterning of control calves generated from unedited parental cells, the edited calves displayed a novel pattern of grey and white markings and absence of any black areas. This, for the first time, verified the causative nature of the PMEL mutation for diluting the black coat color in cattle. With these edited animals, it is now possible to dissect the effects of the introgressed edit and other interfering allelic variants that might exist in individual cattle and accurately determine the impact of only the three bp change on important health, welfare and production traits. In addition, our study proved targeted editing as a promising approach for the rapid adaptation of livestock to changing climatic conditions.

Melanoma progression is generally associated with increased transcriptional activity mediated by the Yes‐associated protein (YAP). Mechanical signals from the extracellular matrix are sensed by YAP, which then activates the expression of proliferative genes, promoting melanoma progression and drug resistance. Which extracellular signals induce mechanotransduction, and how this is mediated, is not completely understood. Here, using secretome analyses, we reveal the extracellular accumulation of amyloidogenic proteins, i.e. premelanosome protein (PMEL), in metastatic melanoma, together with proteins that assist amyloid maturation into fibrils. We also confirm the accumulation of amyloid‐like aggregates, similar to those detected in Alzheimer disease, in metastatic cell lines, as well as in human melanoma biopsies. Mechanistically, beta‐secretase 2 (BACE2) regulates the maturation of these aggregates, which in turn induce YAP activity. We also demonstrate that recombinant PMEL fibrils are sufficient to induce mechanotransduction, triggering YAP signaling. Finally, we demonstrate that BACE inhibition affects cell proliferation and increases drug sensitivity, highlighting the importance of amyloids for melanoma survival, and the use of beta‐secretase inhibitors as potential therapeutic approach for metastatic melanoma., The accumulation of PMEL fibrils in the metastatic melanoma microenvironment modulates YAP activity. Preventing aggregate formation reduces metastatic cell proliferation and chemosensitivity, suggesting beta‐secretase inhibition as treatment for metastatic melanoma.

Yellow plumage is common in chickens, especially in breeds such as the Huiyang Bearded chicken, which is indigenous to China. We evaluated plumage colour distribution in F1, F2, and F3 populations of an Huiyang Bearded chicken × White Leghorn chicken cross, the heredity of the yellow plumage trait was distinguished from that of the gold plumage and other known plumage colours. Microscopic analysis of the feather follicles indicated that pheomelanin particles were formed in yellow but not in white feathers. To screen genes related to formation of the pheomelanin particles, we generated transcriptome data from yellow and white feather follicles from 7- and 11-week-old F3 chickens using RNA-seq. We identified 27 differentially expressed genes (DEGs) when comparing the yellow and white feather follicles. These DEGs were enriched in the Gene Ontology classes ‘melanosome’ and ‘melanosome organization’ related to the pigmentation process. Down-regulation of TYRP1, DCT, PMEL, MLANA, and HPGDS, verified using quantitative reverse transcription PCR, may lead to reduced eumelanin and increased pheomelanin synthesis in yellow plumage. Owing to the presence of the Dominant white locus, both white and yellow plumage lack eumelanin, and white feathers showed no pigments. Our results provide an understanding of yellow plumage formation in chickens.