Regulatory gene network for coffee-like color morph of TYRP1 mutant of oujiang color common carp.

BACKGROUND: Neither a TYRP1-mediated highly conserved genetic network underlying skin color towards optimum defense nor the pathological tendency of its mutation is well understood. The Oujiang Color Common Carp (Cyprinus carpio var. color) as a model organism, offering valuable insights into genetics, coloration, aquaculture practices, and environmental health. Here, we performed a comparative skin transcriptome analysis on TYRP1 mutant and wild fishes by applying a conservative categorical approach considering different color phenotypes. RESULTS: Our results reveal that an unusual color phenotype may be sensitized with TYRP1 mutation as a result of upregulating several genes related to an anti-inflammatory autoimmune system in response to the COMT-mediated catecholamine neurotransmitters in the skin. Particularly, catecholamines-derived red/brown, red with blue colored membrane attack complex, and brown/grey colored reduced eumelanin are expected to be aggregated in the regenerated cells. CONCLUSIONS: It is, thus, concluded that the regenerated cells with catecholamines, membrane attack complex, and eumelanin altogether may contribute to the formation of the unusual (coffee-like) color phenotype in TYRP1 mutant.

Screening of Key Components for Melanogenesis Inhibition of Polygonum cuspidatum Extract Based on the Spectrum-Effect Relationship and Molecular Docking.

Polygonum cuspidatum (PC) extract has been listed in the "Catalog of Used Cosmetic Ingredients (2021 Edition)", which can inhibit melanogenesis, thus exerting a whitening effect, and has been widely used in cosmetics. However, there are currently no quality standards for PC extract used in cosmetics, and the bioactive components associated with anti-melanogenesis remain unclear. In view of this, the present study was the first to investigate the spectrum-effect relationship between fingerprints of PC extract and melanogenesis inhibition. Ten batches of PC extract fingerprints were established by HPLC. Pearson's correlation analysis, gray correlation analysis (GRA) and orthogonal partial least squares regression analysis (OPLSR) were used to screen out resveratrol, emodin and physcion as the main whitening active ingredients using the inhibition of tyrosinase in B16F10 cells as the pharmacological index. Then, the melanogenesis inhibitory effects of the above three components were verified by tyrosinase inhibition and a melanin content assay in B16F10 cells. The interaction between small molecules and proteins was investigated by the molecular docking method, and it was confirmed by quantitative real-time PCR (qRT-PCR) that resveratrol, emodin and physcion significantly down-regulated the transcript levels of melanogenesis-related factors. In conclusion, this study established a general model combining HPLC fingerprinting and melanogenesis inhibition and also analyzed the spectrum-effect relationship of PC extract, which provided theoretical support for the quality control of PC extract in whitening cosmetics.

Ssc-mir-221-3p regulates melanin production in Xiang pigs melanocytes by targeting the TYRP1 gene.

BACKGROUND: MicroRNAs (miRNAs) are small endogenous non-coding RNAs that regulate gene expression by down-regulating it. Several studies have suggested that miRNAs plays a crucial role in mammalian skin color production. The TYRP1 gene, a member of the tyrosine family, is an important candidate gene that affects melanogenesis. This study aimed to identify genes and miRNAs that affect melanin production in Xiang pigs by transcriptome sequencing, and to validate their targeted regulatory relationships. RESULTS: 17 miRNAs and 1,230 genes were significantly differentially expressed (P < 0.05) in the black and white skin tissues of Jianbai Xiang pigs. miRNA-221-3p was identified as a candidate miRNA for melanin formation and its target gene, TYRP1, was selected. The TYRP1 gene is a member of the TYR gene family, which evolved from the TYR gene through chromosome segmental duplication. The function of the gene was highly conserved throughout the evolutionary process. overexpression of TYRP1 gene significantly increased the expression of TYR, TYRP1, and DCT genes P < 0.01, which led to an increase in the relative content of melanin. Silencing of TYRP1 through the use of TYRP1-siRNA significantly reduced the expression of TYR, TYRP1, and DCT genes in Jianbai Xiang pig melanocytes P < 0.01, which in turn decreased the relative melanin content. The targeted binding relationship between ssc-miR-221-3p and TYRP1 gene was validated. After transfection of porcine melanocytes with ssc-miR-221-3p mimic, the expression of ssc-miR-221-3p was significantly up-regulated (P < 0.01). Furthermore, the mRNA and protein levels of TYR, TYRP1, and DCT genes were significantly down-regulated (P < 0.01), and melanin content in cells was significantly reduced (P < 0.01). CONCLUSION: The TYRP1 gene affects melanogenesis in melanocytes of Jianbai Xiang pigs, and ssc-miR-221-3p targets the TYRP1 gene to regulate melanogenesis in melanocytes of Jianbai Xiang pigs.

Integration of GWAS and eGWAS to screen candidate genes underlying green head traits in male ducks.

Sexually dimorphic plumage coloration is widespread in birds. The male possesses more brightly colored feathers than the female. Dark green head feathers comprise one of the most typical appearance characteristics of the male Ma duck compared with the female. However, there are noticeable individual differences observed in these characteristics. Herein, genome-wide association studies (GWAS) were employed to investigate the genetic basis of individual differences in male duck green head-related traits. Our results showed that 165 significant SNPs were associated with green head traits. Meanwhile, 71 candidate genes were detected near the significant SNPs, including four genes (CACNA1I, WDR59, GNAO1 and CACNA2D4) related to the individual differences in the green head traits of male ducks. Additionally, the eGWAS identified three SNPs located within two candidate genes (LOC101800026 and SYNPO2) associated with TYRP1 gene expression, and might be important regulators affecting the expression level of TYRP1 in the head skin of male ducks. Our data also suggested that transcription factor MXI1 might regulate the expression of TYRP1, thereby causing differences in the green head traits among male ducks. This study provided primary data for further analysis of the genetic regulation of duck feather color.

Rab32/38-Dependent and -Independent Transport of Tyrosinase to Melanosomes in B16-F1 Melanoma Cells.

B16-F1 melanoma cells have often been used as a model to investigate melanogenesis, but the evidence that melanosome biogenesis and transport occur by the same mechanisms in normal melanocytes and B16-F1 cells is insufficient. In this study, we established knockout B16-F1 cells for each of several key factors in melanogenesis, i.e., tyrosinase (Tyr), Hps4, Rab27A, and Rab32·Rab38 (Rab32/38), and then compared their phenotypes with the phenotypes of corresponding mutant mouse melanocyte cell lines, i.e., melan-c, melan-le, melan-ash, and Rab32-deficient melan-cht cells, respectively. The results showed that Tyr and Rab27A are also indispensable for melanin synthesis and peripheral melanosome distribution, respectively, in B16-F1 cells, but that Hps4 or its downstream targets Rab32/38 are not essential for Tyr transport in B16-F1 cells, suggesting the existence of a Rab32/38-independent Tyr transport mechanism in B16-F1 cells. We then performed comprehensive knockdown screening of Rab small GTPases and identified Rab10 and Rab24, previously uncharacterized Rabs in melanocytes, as being involved in Tyr transport under Rab32/38-null conditions. Our findings indicate a difference between the Tyr transport mechanism in melanocytes and B16-F1 cells in terms of Rab32/38-dependency and a limitation in regard to using melanoma cells as a model for melanocytes, especially when investigating the mechanism of endosomal Tyr transport.

Variation in Genetic Mechanisms for Plumage Polymorphism in Skuas (Stercorarius).

Coloration is evolutionarily labile and so provides an excellent trait for examining the repeatability of evolution. Here, we investigate the repeatability of the evolution of polymorphic variation in ventral plumage coloration in skuas (Stercorarius: Stercorariidae). In 2 species, arctic (S. parasiticus) and pomarine skuas (S. pomarinus), plumage polymorphism was previously shown to be associated with coding changes at the melanocortin-1 receptor (MC1R) locus. Here, we show that polymorphism in a third species, the south polar skua (S. maccormicki), is not associated with coding variation at MC1R or with variation at a Z-linked second candidate locus, tyrosinase-related protein 1 (TYRP1). Hence, convergent evolution of plumage polymorphisms in skuas is only partly repeatable at the level of the genetic locus involved. Interestingly, the pattern of repeatability in skuas is aligned not with phylogeny but with the nature of the phenotypic variation. In particular, south polar skuas show a strong sex bias to coloration that is absent in the other species, and it may be that this has a unique genetic architecture.

Tyrp1 Mutant Variants Associated with OCA3: Computational Characterization of Protein Stability and Ligand Binding.

Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions.

GIF-2209, an Oxindole Derivative, Accelerates Melanogenesis and Melanosome Secretion via the Modification of Lysosomes in B16F10 Mouse Melanoma Cells.

Melanogenesis and melanosome secretion are regulated by several mechanisms. In this study, we found that the oxindole derivative GIF-2209 accelerated melanogenesis associated with the discrimination in the expression and intracellular distributions of two melanogenic enzymes, tyrosinase (TYR) and tyrosinase-related protein-1 (TYRP-1). GIF-2209 upregulated the expression of TYR via a microphthalmia transcription factor (MITF)-independent mechanism, leading to high expression of protein. In contrast, GIF-2209 did not alter the mRNA levels of TYRP-1 and suppressed its protein levels. GIF-2209 induced the dissociation of TYR from TYRP-1 but did not alter the association between TYR and CD63, a melanosome and lysosome marker. The protein levels of CD63 were also upregulated by GIF-2209. GIF-2209 induced lysosome expansion and redistribution in all areas of the cytosol, accompanied by autophagy acceleration (upregulation of LC3BII protein levels and downregulation of p62 protein levels). In addition, GIF-2209 stimulated the secretion of melanosomes containing high levels of TYR, TYRP-1, and CD63 proteins. The GIF-2209 mediated melanosome secretion was sensitive to the lysosome inhibitor chloroquine. These results suggest that GIF-2209 may activate lysosomal functions with TYR gene expression, while it accelerates melanosome secretion, which finally leads to the depletion of intracellular melanogenic enzyme, especially TYRP-1 protein.

In Vitro TyRP-1 Knockdown Based on siRNA Carried by Liquid Crystalline Nanodispersions: an Alternative Approach for Topical Treatment of Vitiligo.

PURPOSE: Vitiligo is a skin disease characterized by depigmentation and the presence of white patches that are associated with the loss of melanocytes. The most common explanation for the cause of this condition is that it is an autoimmune condition. TyRP-1 is involved in melanin pigment synthesis but can also function as a melanocyte differentiation antigen. This protein plays a role in the autoimmune destruction of melanocytes, which results in the depigmentation, characteristic of this disease. In this study, we evaluated liquid crystalline nanodispersions as non-viral vectors to deliver siRNA-TyRP-1 as an alternative for topical treatment of vitiligo. METHODS: Liquid crystalline nanodispersions were obtained and characterized with respect to their physical-chemical parameters including size, PdI and zeta potential, as well as Small Angle X-ray Scattering and complexing to siRNA. The effects of the liquid crystalline nanodispersions on the cellular viability, cell uptake and levels of the knockdown target TyRP-1 were evaluated in melan-A cells after 24 h of treatment. RESULTS: The liquid crystalline nanodispersions demonstrated adequate physical-chemical parameters including nanometer size and a PdI below 0.38. These systems promoted a high rate of cell uptake and an impressive TyRP-1 target knockdown (> 80%) associated with suitable loading of TyRp-1 siRNA. CONCLUSIONS: We demonstrated that the liquid crystalline nanodispersions showed promising alternative for the topical treatment of vitiligo due to their physical parameters and ability in knockdown the target protein involved with autoimmune destruction of melanocytes.

Molecular cloning and expression analysis of tyr and tyrp1 genes in normal and albino yellow catfish Tachysurus fulvidraco.

The full-length complementary DNA of two genes related to vertebrate albinism, the tyrosinase gene tyr and tyrosinase-related protein 1 gene tyrp1, were cloned and analysed from normal and albino yellow catfish Tachysurus fulvidraco. The open reading frames (ORF) of tyr and tyrp1 encode putative peptides of 533 and 526 amino acids (amino-acid), both of which possess two conserved copper binding sites. The homologous identities of deduced amino-acid sequences showed that both Tyr and Tyrp1 of T. fulvidraco share considerable similarity with that of channel catfish Ictalurus punctatus. Both tyr and tyrp1 were expressed in a wide range of adult tissues. Tyr gene had the highest expression level in the brain of both normal and albino T. fulvidraco. Tyrp1 had the highest expression level in the skin of normal groups, and the fin of albino groups. The messenger (m)RNA expressions of tyr and tyrp1 were detectable at different early developmental stages and varied with embryonic and larval growth. Tyr and tyrp1 mRNA have obvious tissue specificity both in normal and albino T. fulvidraco and higher expression levels were detected in the normal group revealing that tyr and tyrp1 may have an important role in pigmentation. These results will provide useful data for understanding the molecular mechanism of melanin formation and the occurrence of albinism in T. fulvidraco.

STX13 regulates cargo delivery from recycling endosomes during melanosome biogenesis.

Melanosomes are a class of lysosome-related organelles produced by melanocytes. Biogenesis of melanosomes requires the transport of melanin-synthesizing enzymes from tubular recycling endosomes to maturing melanosomes. The SNARE proteins involved in these transport or fusion steps have been poorly studied. We found that depletion of syntaxin 13 (STX13, also known as STX12), a recycling endosomal Qa-SNARE, inhibits pigment granule maturation in melanocytes by rerouting the melanosomal proteins such as TYR and TYRP1 to lysosomes. Furthermore, live-cell imaging and electron microscopy studies showed that STX13 co-distributed with melanosomal cargo in the tubular-vesicular endosomes that are closely associated with the maturing melanosomes. STX family proteins contain an N-terminal regulatory domain, and deletion of this domain in STX13 increases both the SNARE activity in vivo and melanosome cargo transport and pigmentation, suggesting that STX13 acts as a fusion SNARE in melanosomal trafficking pathways. In addition, STX13-dependent cargo transport requires the melanosomal R-SNARE VAMP7, and its silencing blocks the melanosome maturation, reflecting a defect in endosome-melanosome fusion. Moreover, we show mutual dependency between STX13 and VAMP7 in regulating their localization for efficient cargo delivery to melanosomes.

The Recent Crystal Structure of Human Tyrosinase Related Protein 1 (HsTYRP1) Solves an Old Problem and Poses a New One.

Show your metal: l-Tyrosine is converted into the protective antioxidative polymer melanin in a sequence of reactions. In humans, the catalytic pathway starts with the tyrosinase HsTYR and two tyrosinase-related proteins HsTYRP1 and HsTYRP2. All three enzymes have the same active site but the latter two contain two zinc ions instead of copper ions.

Candidate Gene Analysis Suggests Untapped Genetic Complexity in Melanin-Based Pigmentation in Birds.

Studies on melanin-based color variation in a context of natural selection have provided a wealth of information on the link between phenotypic and genetic variation. Here, we evaluated associations between melanic plumage patterns and genetic polymorphism in the Réunion grey white-eye (Zosterops borbonicus), a species in which mutations on MC1R do not seem to play any role in explaining melanic variation. This species exhibits 5 plumage color variants that can be grouped into 3 color forms which occupy discrete geographic regions in the lowlands of Réunion, and a fourth high-elevation form which comprises 2 color morphs (grey and brown) and represents a true color polymorphism. We conducted a comprehensive survey of sequence variation in 96 individuals at a series of 7 candidate genes other than MC1R that have been previously shown to influence melanin-based color patterns in vertebrates, including genes that have rarely been studied in a wild bird species before: POMC, Agouti, TYR, TYRP1, DCT, Corin, and SLC24A5 Of these 7 genes, 2 (Corin and TYRP1) displayed an interesting shift in allele frequencies between lowland and highland forms and a departure from mutation-drift equilibrium consistent with balancing selection in the polymorphic highland form only. Sequence variation at Agouti, a gene frequently involved in melanin-based pigmentation patterning, was not associated with color forms or morphs. Thus, we suggest that functionally important changes in loci other than those classically studied are involved in the color polymorphism exhibited by the Réunion grey white-eye and possibly many other nonmodel species.

Pleiotropic effects of coat colour-associated mutations in humans, mice and other mammals.

The characterisation of the pleiotropic effects of coat colour-associated mutations in mammals illustrates that sensory organs and nerves are particularly affected by disorders because of the shared origin of melanocytes and neurocytes in the neural crest; e.g. the eye-colour is a valuable indicator of disorders in pigment production and eye dysfunctions. Disorders related to coat colour-associated alleles also occur in the skin (melanoma), reproductive tract and immune system. Additionally, the coat colour phenotype of an individual influences its general behaviour and fitness. Mutations in the same genes often produce similar coat colours and pleiotropic effects in different species (e.g., KIT [reproductive disorders, lethality], EDNRB [megacolon] and LYST [CHS]). Whereas similar disorders and similar-looking coat colour phenotypes sometimes have a different genetic background (e.g., deafness [EDN3/EDNRB, MITF, PAX and SNAI2] and visual diseases [OCA2, RAB38, SLC24A5, SLC45A2, TRPM1 and TYR]). The human predilection for fancy phenotypes that ignore disorders and genetic defects is a major driving force for the increase of pleiotropic effects in domestic species and laboratory subjects since domestication has commenced approximately 18,000 years ago.

BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells.

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.

Class III ß-tubulin, a novel biomarker in the human melanocyte lineage.

It is generally thought that class III ß-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III ß-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III ß-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.

Visual input regulates melanophore differentiation.

Introduction: Developmental processes continue in organisms in which sensory systems have reached functional maturity, however, little research has focused on the influence of sensory input on cell and tissue development. Here, we explored the influence of visual system activity on the development of skin melanophores in Xenopus laevis. Methods: Melanophore number was measured in X. laevis larvae after the manipulation of visual input through eye removal (enucleation) and/or incubation on a white or black substrate at the time when the visual system becomes functional (stage 40). To determine the developmental process impacted by visual input, migration, proliferation and differentiation of melanophores was assessed. Finally, the role of melatonin in driving melanophore differentiation was explored. Results: Enucleating, or maintaining stage 40 larvae on a black background, results in a pronounced increase in melanophore number in the perioptic region within 24 h. Time lapse analysis revealed that in enucleated larvae new melanophores appear through gradual increase in pigmentation, suggesting unpigmented cells in the perioptic region differentiate into mature melanophores upon reduced visual input. In support, we observed increased expression of melanization genes tyr, tyrp1, and pmel in the perioptic region of enucleated or black background-reared larvae. Conversely, maintaining larvae in full light suppresses melanophore differentiation. Interestingly, an extra-pineal melatonin signal was found to be sufficient and necessary to promote the transition to differentiated melanophores. Discussion: In this study, we found that at the time when the visual system becomes functional, X. laevis larvae possess a population of undifferentiated melanophores that can respond rapidly to changes in the external light environment by undergoing differentiation. Thus, we propose a novel mechanism of environmental influence where external sensory signals influence cell differentiation in a manner that would favor survival.

Gut Microbiome-Driven metabolites influence skin pigmentation in TYRP1 mutant Oujiang Color Common Carp.

The gut microbiome plays a key role in regulating the gut-skin axis, and host genetics partially influence this regulation. The study investigated the role of gut microbiota and host genetics in the gut-skin axis, focusing on the unusual "coffee-like" color phenotype observed in TYRP1 mutant Oujiang Color Common Carp. We employed comparative high-throughput omics data from wild-type and mutant fish to quantify the influence of both genetics and gut microbes on skin transcriptomic expression and blood metabolites. We found 525 differential metabolites (DMs) and 45 distinct gut microbial genera in TYRP1 mutant fish compared to wild type. Interaction and causal mediation analyses revealed a complex interplay. The TYRP1 mutation likely triggers an inflammatory pathway involving Acinetobacter bacteria, Leukotrience-C4 and Spermine. This inflammatory response appears to be counterbalanced by an anti-inflammatory cardiovascular genetic network. The net effect is the upregulation of COMT, PLG, C2, C3, F10, TDO2, MHC1, and SERPINF2, leading to unusual coffee-like coloration. This study highlights the intricate interplay between gut microbiota, host genetics, and metabolic pathways in shaping complex phenotypes.

Phase 1, first-in-human study of TYRP1-TCB (RO7293583), a novel TYRP1-targeting CD3 T-cell engager, in metastatic melanoma: active drug monitoring to assess the impact of immune response on drug exposure.

Introduction: Although checkpoint inhibitors (CPIs) have improved outcomes for patients with metastatic melanoma, those progressing on CPIs have limited therapeutic options. To address this unmet need and overcome CPI resistance mechanisms, novel immunotherapies, such as T-cell engaging agents, are being developed. The use of these agents has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs), which is challenging to predict preclinically and can lead to neutralization of the drug and loss of efficacy. Methods: TYRP1-TCB (RO7293583; RG6232) is a T-cell engaging bispecific (TCB) antibody that targets tyrosinase-related protein 1 (TYRP1), which is expressed in many melanomas, thereby directing T cells to kill TYRP1-expressing tumor cells. Preclinical studies show TYRP1-TCB to have potent anti-tumor activity. This first-in-human (FIH) phase 1 dose-escalation study characterized the safety, tolerability, maximum tolerated dose/optimal biological dose, and pharmacokinetics (PK) of TYRP1-TCB in patients with metastatic melanoma (NCT04551352). Results: Twenty participants with cutaneous, uveal, or mucosal TYRP1-positive melanoma received TYRP1-TCB in escalating doses (0.045 to 0.4 mg). All participants experienced ≥1 treatment-related adverse event (TRAE); two participants experienced grade 3 TRAEs. The most common toxicities were grade 1-2 cytokine release syndrome (CRS) and rash. Fractionated dosing mitigated CRS and was associated with lower levels of interleukin-6 and tumor necrosis factor-alpha. Measurement of active drug (dual TYPR1- and CD3-binding) PK rapidly identified loss of active drug exposure in all participants treated with 0.4 mg in a flat dosing schedule for ≥3 cycles. Loss of exposure was associated with development of ADAs towards both the TYRP1 and CD3 domains. A total drug PK assay, measuring free and ADA-bound forms, demonstrated that TYRP1-TCB-ADA immune complexes were present in participant samples, but showed no drug activity in vitro. Discussion: This study provides important insights into how the use of active drug PK assays, coupled with mechanistic follow-up, can inform and enable ongoing benefit/risk assessment for individuals participating in FIH dose-escalation trials. Translational studies that lead to a better understanding of the underlying biology of cognate T- and B-cell interactions, ultimately resulting in ADA development to novel biotherapeutics, are needed.

QTL and association analysis for skin and fibre pigmentation in sheep provides evidence of a major causative mutation and epistatic effects.

The pursuits of white features and white fleeces free of pigmented fibre have been important selection objectives for many sheep breeds. The cause and inheritance of non-white colour patterns in sheep has been studied since the early 19th century. Discovery of genetic causes, especially those which predispose pigmentation in white sheep, may lead to more accurate selection tools for improved apparel wool. This article describes an extended QTL study for 13 skin and fibre pigmentation traits in sheep. A total of 19 highly significant, 10 significant and seven suggestive QTL were identified in a QTL mapping experiment using an Awassi × Merino × Merino backcross sheep population. All QTL on chromosome 2 exceeded a LOD score of greater than 4 (range 4.4-30.1), giving very strong support for a major gene for pigmentation on this chromosome. Evidence of epistatic interactions was found for QTL for four traits on chromosomes 2 and 19. The ovine TYRP1 gene on OAR 2 was sequenced as a strong positional candidate gene. A highly significant association (P < 0.01) of grandparental haplotypes across nine segregating SNP/microsatellite markers including one non-synonymous SNP with pigmentation traits could be shown. Up to 47% of the observed variation in pigmentation was accounted for by models using TYRP1 haplotypes and 83% for models with interactions between two QTL probabilities, offering scope for marker-assisted selection for these traits.