Pigment Cell & Melanoma Research

I.Cutaneous skin melanomas with wild-type BRAF alleles ("BRAF-WT melanomas") remain relatively difficult to treat, even though they typically possess driver mutations in a RAS gene or NF1. For example, these tumors respond relatively poorly to combinations of MEK and BRAF inhibitors, and their response to ICIs is muted compared to the response of BRAF-mutant melanomas. ERBB2 and ERBB4, which encode receptor tyrosine kinase genes, are necessary and sufficient for the proliferation of multiple BRAF-WT melanoma cell lines. Consequently, we have postulated that ERBB4-ERBB2 heterodimerization drives BRAF-WT melanomas. This mechanism is consistent with the observation that elevated ERBB4 transcription or ERBB4 mutations are found in a significant fraction of BRAF-WT melanoma tumor samples. Moreover, a subset of ERBB4 mutations found in BRAF-WT melanoma samples increases proliferation in a BRAF-WT melanoma cell line. Because the elevated ERBB4 transcription observed in BRAF- WT melanomas is typically insufficient to cause ligand-independent ERBB4 signaling, we have postulated that ligands for ERBB family receptors drive the elevated ERBB4-ERBB2 heterodimerization responsible for the proliferation of BRAF-WT melanoma cell lines. We have explored this hypothesis by analyzing data found in the Broad Institutes Cancer Cell Line Encyclopedia. These data suggest that some EGF family hormones are required for the proliferation of BRAF-WT melanoma cell lines. Likewise, the G11/Gq pathway, which can stimulate cleavage and maturation of EGF family hormones, is also required for the proliferation of BRAF-WT melanoma cell lines. Thus, these data suggest additional therapeutic targets in BRAF-WT melanomas. Moreover, because many uveal (ocular) melanomas possess elevated G11/Gq signaling, these data suggest that ligand stimulation of ERBB receptor signaling may contribute to uveal melanomagenesis or progression.

Deleterious CDKN2A germline variants account for ~40% of familial melanoma cases, while rare variants in CDK4, BAP1, and telomere-maintenance genes collectively attribute ~10% of familial risk. We sought to identify new high-penetrance susceptibility variants by sequencing 305 melanoma cases from 89 multi-case families negative for known predisposition gene variants. In one family, cutaneous melanoma co-segregated with a rare variant in DMRTA1 (p.Glu383Gln), located less than 480 kb upstream of CDKN2A on chromosome 9. Whole-genome sequencing then revealed an intergenic 234kb deletion that co-segregated with melanoma in 18 out of 21 cases across four generations. Further investigations revealed a further 10 families carrying this deletion, co-segregating with melanoma. The deleted region was predicted to encompass regulatory sequences and to interact with the CDKN2A promoter region. Tiled CRISPR inhibition of the predicted enhancer region confirmed interactions between the distant upstream deletion with CDKN2A resulting in decreased p16 transcript mRNA expression. Deletion carriers exhibited nearcomplete loss of p16 mRNA expression from the affected chromosome. This distant noncoding deletion is one of the most common founder variants predisposing to melanoma and reveals a new mechanism controlling p16 expression. Routine screening for this deletion in individuals with perceived high risk of melanoma is warranted.

Importance: Non-medullary thyroid cancer (NMTC) and melanoma are associated with inherited long telomeres due to germline pathogenic/likely pathogenic variants (PV/LPV) in POT1, TINF2, and ACD resulting in long-telomere syndrome (LTS) and they commonly have somatic TERT promoter mutations. The genetic relationship between these variants and their clinical associations are defined incompletely and may inform clinical practice. Objective: To test the hypothesis that germline LTS-associated PV/LPV are exclusive from functional somatic TERT variants and assess clinical/genetic associations. Design: Retrospective observational cohort study with/without germline LTS variants, that have somatic sequencing and pathology data. Setting: Participants were enrolled through 18 cancer centers participating in the Oncology Research Information Exchange Network (ORIEN). Participants: 995 adults with NMTC and 993 with melanoma between 2013 and 2025. All adult patients at an ORIEN center were offered enrollment Exposures: All patients with NMTC or melanoma are included. There are no required exposures. Main Outcomes and Measures: The presence/absence of a germline or somatic long-telomere variant; secondary outcomes are associations with tumor stage, telomerase expression, and oncogenes. Results: Germline and somatic variants in POT1/TINF2/ACD, somatic TERT promoter variants, TERT fusions, oncogenes, and telomerase mRNA expression were evaluated in 995 NMTC and 993 melanoma patients. In NMTC, 13 (1.5%) had a germline LTS variant while 0/12 with tumor sequencing had somatic TERT promoter variants/fusions. In melanoma, 7 (0.7%) had a LTS variant; 0/2 with tumor sequencing had a TERT promoter variant/ fusion. Meta-analysis including NMTC and melanoma in the current study, a recent thyroid cancer study, and thyroid TCGA, germline LTS-associated PV/LPV and somatic TERT variants/fusions were mutually exclusive (p=0.036). High telomerase mRNA levels were associated with TERT promoter variants/fusions (p<4e-11) and larger NMTC/distant metastases (p=0.016), but not germline LTS variants. NMTCs with somatic TERT promoter variants/fusions had higher tumor mutation burden (p<0.02) versus tumors from patients with a germline LTS variant. TERT promoter mutant variant allele frequency was lower in smaller and non-metastatic vs larger/metastatic NMTC. Conclusion and Relevance: Germline LTS-associated variants appear to be exclusive from somatic TERT promoter variants/fusions but are not associated with aggressive NMTC, suggesting common roles in tumorigenesis but different biological impacts.

Background: Human papillomaviruses (HPVs) pose a severe threat to global public health by driving nonmelanoma skin cancer (NMSC) and cervical cancer, with NMSC being one of the most common cancers worldwide. Epidermodysplasia verruciformis (EV) is an inborn error of immunity characterized by an increased susceptibility to persistent infection of cutaneous HPV and a high risk of NMSC. The genetic basis remains unknown in many patients with EV. Methods: We collected four unrelated pedigrees with EV. Genetic analysis identified five variants in JAK1 encoding the Janus kinase 1. Ex vivo models and patient-derived tissue were employed to evaluate the functional effects of JAK1 variants and delineate the pathogenic mechanisms. Results: We identified different variants in JAK1 in four pedigrees with dominant EV. Genetic analysis revealed five novel variants in JAK1, three of which resulted in nonsense-mediated mRNA decay (NMD). Functional assays identified a decreased phosphorylation of the signal transducers and activators of transcription (STATs), impaired interferon responses, and defective T cell activation. Immune dysregulation in patients, characterized by a reduced CD4/CD8 T cell ratio, decreased CD8 naive T cell proportion, and accumulated memory T cells, implies impaired antiviral immunity against HPV. Conclusions: Our findings confirm that JAK1 loss-of-function (LOF) variants underlie susceptibility to cutaneous HPV infection. [Funded by the National Natural Science Foundation of China (81788101, 81230015, 82394420, and 82394423), the National Key Research and Development Program of China (2022YFC2703900), the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-018), and the Regione Lombardia, Italy (Innovative Research Project 1137-2010)].

Mutations in MAP3K7 are responsible for two distinct syndromes Cardiospondylocarpofacial (CSCF) and Frontometaphyseal dysplasia 2 (FMD2). Both are characterized by skeletal malformations, facial dysmorphisms, hearing loss, and mild intellectual disability. While cardiac defects are predominant in CSCF, keloid scar is a distinct feature in FMD2. Problem with gonadal development and disorders of sexual development (DSD) have not been previously chracterized. Here we report three syndromic cases of 46,XY DSD with CSCF or FMD2, each carrying a novel heterozygous missense variants in MAP3K7 (NM_145331.3:c.250G>A; p.V84M, NM_145331.3:c.195A>G; p.I65M, and NM_145331.3: c.574A>G; p.S192G). The DSD phenotypes include cryptorchidism, micropenis, small testis, and hypospadias. In silico tools predict all three variants are deleterious. All three MAP3K7 variants occur in the kinase domain at highly conservative positions among mammals. MAP3K7 is highly expressed in human fetal Sertoli cells. MAP3K7 knock-out in HEK293T cells led to downregulation of GATA4 and FOG2 expression by RNA-Seq. Like MAP3K1, MAP3K7 phosphorylated p38 while all three MAP3K7 variants did not alter phosphorylated p38 compared to wildtype in HEK293TMAP3K7-/- cells. Two MAP3K7 missense mutants (p.V84M and p.I65M) ectopically activate ovarian beta catenin/ Wnt signalling in TOPFLASH assays. Our data suggest that MAP3K7 contributes to male sex differentiation by increasing expression of pro-testis genes GATA4 and FOG2 in HEK293TMAP3K7-/- cells and antagonizing pro-ovarian beta-catenin signalling, and that one or more of these activities were likely affected in 3 cases of 46,XY DSD with CSCF/FMD2 during sex development.

BackgroundHeterozygous c.283+1G>A and c.283G>A variants in the THRB gene, encoding for thyroid hormone receptor (TR){beta}1 and {beta}2, lead to autosomal dominant macular dystrophy (ADMD). We report the detailed clinical characterization of two first-degree relatives with ADMD, heterozygous for THRB c.283+1G>A, and an unrelated ADMD patient with a novel variant, c.283G>C. The genomic and molecular consequences of both variants were studied. MethodsgDNA and mRNA were obtained from leukocytes. Clinical characterization included biochemistry, bone density and body composition, ECG, echocardiography, ultrasound, audiometry and color-vision. In vitro assays investigated TR function and DNA binding. ResultsThe patients manifested no resistance to thyroid hormone beta (RTH{beta}) and had normal FT4 and TSH. Detailed studies in two patients showed no goiter, tachycardia, hypercholesterinemia or hepatic steatosis. Hearing was not impaired. Both had impaired color vision and reduced bone density. RT-PCR from all three patients revealed skipping of exon 4 exclusive to TR{beta}1, producing a deletion of 87 amino acids in the N-terminal domain (TR{beta}1{Delta}NTD). In vitro, DNA-binding affinity of TR{beta}1{Delta}NTD to DR4-TRE with or without RXR was comparable to TR{beta}1WT. Surprisingly, TR{beta}1{Delta}NTD was transcriptionally twice more active than TR{beta}1WT with a similar EC50 for T3, demonstrating gain-of-function of TR{beta}1{Delta}NTD. THRA expression in leukocytes was increased by 3-fold compared to unrelated controls and different from RTH{beta} patients. ConclusionThese THRB splice site variants produce TR{beta}1 exon 4 skipping, resulting in a gain-of-function mutant, TR{beta}1{Delta}NTD. This explains the dominant ADMD phenotype devoid of RTH{beta} and suggests a TR{beta}1 gain-of-function syndrome.

BackgroundThe diagnostic yield for 46,XY disorders of sex development (DSD) remains limited. Whole-genome sequencing (WGS) improves detection of both coding and non-coding variants that may be missed by routine testing. Cytochrome b5, encoded by CYB5A, is an essential co-factor for CYP17A1-mediated 17,20-lyase activity. We report on WGS on a Vietnamese family with 46,XY DSD with two siblings presenting with female external genitalia. MethodsClinical assessment and hormone profiling were conducted. WGS was conducted on peripheral blood DNA, in two affected siblings followed by variant annotation and ACMG-based classification. A minigene RNA splicing assay in HEK293 cells was used to evaluate the functional impact of the CYB5A intronic variant. ResultsThe patients hormone profile showed low testosterone and estradiol. WGS identified compound-heterozygous CYB5A variants: a paternally inherited missense variant (p.Val34Glu, likely pathogenic) and a maternally inherited deep intronic deletion (c.129+862_129+863del) for which SpliceAI predicted aberrant splicing. Minigene assays confirmed that the intronic deletion creates cryptic splice sites, resulting in pseudoexon inclusion and a premature stop codon, consistent with nonsense-mediated decay. The intronic variant meets ACMG criteria for pathogenicity. ConclusionThis family expands the spectrum of CYB5A-related DSD and demonstrates that compound-heterozygous variants, including deep intronic defects, can lead to a disruption in 17,20-lyase activity. These findings highlight the importance of WGS and functional assays for identifying clinically relevant non-coding variants in DSD.

Purpose Patients carrying Germline Pathogenic Variants (GPVs) in multiple cancer susceptibility genes (CSGs) can be described within the context of Multi-locus Inherited Neoplasia Allele Syndrome (MINAS). The role of each GPV is typically interpreted based on clinical phenotypes. Here, we used tumor sequencing, particularly mutational signatures, to investigate the contribution of GPVs in MUTYH and PALB2 to colorectal polyposis and breast cancer in a single patient at a molecular level. Methods We analyzed tumor sequencing data, including mutational signatures and genomic scars, of a breast tumor and a colorectal polyp from a patient with biallelic GPVs in MUTYH and a heterozygous GPV in PALB2. Results The colorectal polyp showed a dominant contribution of MUTYH-associated Base Excision Repair deficiency (BERd) mutational signatures, with no evidence of Homologous Recombination Repair Deficiency (HRD). In contrast, the breast tumor showed both MUTYH-driven BERd and HRD-associated signatures, including SBS3, ID6 and an elevated HRD score, despite the absence of a detectable second hit in PALB2. These findings suggest a differential contribution from the CSGs, with MUTYH contributing to both lesions and PALB2 contributing specifically to the breast tumor. The observed pattern does not align with the additive or synergistic models described in MINAS. Conclusions Our study provides evidence that mutational signatures can elucidate the contribution of multiple CSGs to tumorigenesis within a single patient. These findings extend current interpretations of MINAS beyond additive or synergistic phenotypes, which may help to better understand tumor etiology, with potential clinical implications, including eligibility for targeted therapies.

Melanoma is a highly plastic cancer characterized by distinct cellular phenotypes associated with broadly unique gene expression profiles. TRIM9 is a brain-enriched E3 ubiquitin ligase detected in melanoma, but how TRIM9 expression regulates melanoma phenotype is unknown. Using two metastatic human melanoma cell lines and a mouse melanoma model, we found that TRIM9 promoted melanoma proliferation and altered cell morphology. In cell lines, TRIM9 promoted cellular blebbing and negatively regulated adhesion, secretion, and mesenchymal motility. TRIM9 interacted with VASP in melanoma cells, altering VASP modification, localization, and dynamics. In the absence of TRIM9, cells had an altered actin organization and more focal adhesions, where VASP accumulated and exhibited rapid turnover. We find the alterations in actin architecture and adhesion associated with TRIM9 deletion were coincident with increased motile and contractile mesenchymal behavior in vitro. In vivo loss of TRIM9 in melanoma slowed tumor growth and altered metastasis frequency, size, and destination. Our findings indicate TRIM9 alters the proliferative and morphological phenotypes of metastatic melanoma cells to influence disease progression.

Vitiligo is an acquired pigmentary disorder of the skin and mucus membranes. Previous study has demonstrated that autologous cultured epithelial grafts (ACEG) is an effective treatment for stable vitiligo. However, extraction of full-thickness skin might result in scar formation at donor site, which have hindered the wider application of this technology, especially for patients requiring large-area transplantation. Hair follicle as a source of keratinocyte and melanocyte, could be potential source of cells for preparation of autologous cultured sheet. Through culture system optimization, we have demonstrated maintenance of undifferentiated hair follicle-derived cells in feeder-independent culture system. After expansion, the hair follicle cells were directed to differentiate into a multi-layered, epidermis-like sheet. Cell identity, viability, purity, genomic stability, and antiseptic testing for hair follicle-derived epithelial sheet (HFES) were evaluated to ensure its safety. Immunofluorescence staining showed that basal keratinocytes were the main cell type of the autologous HFES. Optimization of culture conditions leads to increased melanocyte proliferation and functionality. Transcriptomic analysis confirmed upregulation of melanosome maturation genes. The proportions of cells are also similar to composition of cells under physiological conditions. Transplantation of HFES to depigmented areas in patients with stable vitiligo results in skin repigmentation. This technology provides a novel therapeutic option for vitiligo management.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary neuromuscular disorder. The Russian FSHD Patient Registry was established in 2019 following the development of a PCR-based method for genetic confirmation of the diagnosis. Results: The registry included 470 participants (51% male). Genetic confirmation was obtained for 76% (n=356), the remainder were included based on clinical and anamnestic data. Clinical assessment forms and patient-reported questionnaires were analyzed for 310 and 142 patients, respectively. D4Z4 repeat unit (RU) distribution showed patterns consistent with European cohorts, with a predominance of patients with 3 RUs. A moderate inverse correlation was found between RUs number and clinical severity scales. Periscapular weakness was the most common onset manifestation (46.8%), followed by facial weakness (31.6%) which was often unnoticed by patients. The mean age in the Russian cohort was 37.8 years (range 0-97), indicating a younger cohort compared to international data. A delta-adjusted cluster analysis (n=215) identified three distinct trajectories: a classic phenotype with onset before age 14 and early involvement of various muscle groups (n=177), and two clusters characterized by either facial or periscapular onset with slow progression. Conclusion: The Russian FSHD registry provides a comprehensive characterization of a large national cohort, revealing a predominance of patients with 3 D4Z4 repeats and a younger demographic profile compared to international data. Cluster analysis identified three heterogeneous disease trajectories, offering a framework for improved patient stratification.

Celiac disease (CD) is strongly associated with specific HLA DQ heterodimers, formed by HLA DQA1 and HLA DQB1 proteins. In particular DQ2.5 (DQB1*02 associated to DQA1*05) and DQ8 (DQB1*03:02 with DQA1*03) are present in virtually all celiac patients. HLA DQB1*02 is considered the main single genetic susceptibility marker and has been reported in 90 to 95% of CD patients. However, the distribution of these alleles may vary across populations, potentially impacting the performance of genetic screening strategies. In this study, we evaluated the prevalence of HLA DQ2.5 and DQ8 genotypes in celiac patients (n = 41) and an unbiased general population cohort (n = 60) from San Luis, Argentina, using a PCR-based genotyping approach. In addition, we assessed the feasibility of a simplified saliva direct PCR protocol for large scale testing. Overall, 95.1% of CD patients carried DQ2.5 and/or DQ8. Notably, 41.5% of patients were DQ8(+)/DQ2.5(-), and 36.6% lacked the DQB1*02 allele, indicating that DQB1*02 based screening alone would have reduced sensitivity in this population. In the general population, 53.3% of individuals carried CD associated genotypes, with a markedly higher prevalence of DQ8 compared to European cohorts. Genotype distributions deviated from Hardy Weinberg equilibrium in CD patients but not in the general population. We show that DQB1*03:02 is a reliable proxy for DQ8, allowing simplification of genotyping strategies, whereas DQA1*05 typing remains essential to discriminate DQ2.5 from other lower risk DQB1*02 carrying heterodimers. We also describe a saliva direct PCR approach showing a performance comparable to purified DNA based assays. These findings highlight the importance of population specific genetic data for optimizing CD screening strategies and foster the development of simplified, cost effective genotyping approaches for large scale applications.

Wolfram syndrome is a rare genetic disorder characterized by antibody-negative early-onset atypical diabetes mellitus, optic nerve atrophy, sensorineural hearing loss, diabetes insipidus (arginine vasopressin deficiency), and progressive neurodegeneration, with significant variability in disease severity. We assessed the accuracy of a genotype-based severity scoring system to predict the onset of cardinal symptoms in Wolfram syndrome. This system is based on the type of WFS1 variants (in-frame or out-of-frame) and their location relative to transmembrane domains. Severity scores were assigned to 324 patients with documented onset ages for diabetes mellitus, optic atrophy, hearing loss, and diabetes insipidus. Our analysis revealed a clear correlation between severity scores and earlier onset of diabetes mellitus and optic atrophy. Patients with in-frame variants outside transmembrane domains exhibited milder symptoms, especially WFS1 c.1672C>T (p.Arg558Cys) variant, whereas those with out-of-frame variants showed the earliest onset. Severity scores 3 and 4 did not follow the expected progression, suggesting that transmembrane domain involvement in both alleles may result in greater severity. These findings suggest that this scoring system provides valuable insights into the progression of Wolfram syndrome and may guide clinical care. Further refinement may improve its utility for predicting the onset of non-diabetic symptoms.

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer associated with substantial morbidity and mortality in advanced stages. Despite its well-described stepwise progression from actinic keratosis to invasive disease, robust molecular markers for stage discrimination and clinical decision-making remain limited. We sought to define the transcriptional continuum underlying cSCC progression, identify stage-associated biomarkers, and assess the broader relevance of these programs across human malignancies. Bulk RNA sequencing (HTG EdgeSeq) and spatial transcriptomics (GeoMx) were performed on biopsies from eight patients, each presenting multiple disease stages (healthy skin, premalignant lesion, tumor core, and invasive front) within the same lesion field, enabling within-patient analysis of progression. Spatial transcriptomic analyses identified more than 2,000 differentially expressed genes whose expression varied across disease stages. These genes were organized into 18 coordinated expression programs reflecting progressive biological rewiring during tumor evolution. Proliferation, extracellular matrix remodeling, inflammation, and stress-response pathways were progressively upregulated, whereas epithelial differentiation and metabolic processes, including lipid and amino acid metabolism, were downregulated. Macrophages exhibited distinct metabolic reprogramming, with increased purine metabolism, glycolysis, and pyruvate metabolism across progression. To evaluate the broader clinical relevance of these progression-associated programs, we developed a reproducible Snakemake pipeline to systematically screen 32 solid and hematologic malignancies from The Cancer Genome Atlas (TCGA). A combined cSCC-progression signature was significantly associated with poor overall survival (P < 0.05) in 10 additional cancer types. Finally, we identified 12 stage-informative biomarkers, whose spatially restricted expression patterns were validated using Visium HD. This study provides a spatially resolved and stage-aware transcriptomic map of cSCC progression, identifies coordinated gene programs underlying disease evolution, and defines progression-associated signatures with prognostic relevance across multiple cancers, highlighting their potential translational value.

Adult females of the two-spotted spider mite, Tetranychus urticae Koch, enter a photoperiodically induced diapause to overwinter. Diapause in T. urticae is accompanied by reproductive arrest and the orange body coloration that arises from the accumulation of astaxanthin esters. How these two traits are coordinated at the molecular level remains poorly understood. Here, we compared the proteomes of adult females reared under diapause-inducing (long-night) and non-diapause-inducing (short-night) photoperiods using liquid chromatography-tandem mass spectrometry, followed by RNA interference (RNAi) of candidate genes. The carotenoid biosynthesis enzymes phytoene desaturase (TuPDS) and lycopene cyclase/phytoene synthase (TuLCPS), both encoded by genes horizontally transferred from fungi, were more abundant in diapausing females than in non-diapausing females. RNAi of the genes encoding TuPDS and TuLCPS markedly reduced orange pigmentation as well as {beta}-carotene and astaxanthin contents, demonstrating that these enzymes are required for diapause-associated pigmentation. Our proteomic analysis further identified a single PLAT (Polycystin-1, Lipoxygenase, Alpha-toxin) domain protein, TuPLAT10, as one of the most strongly upregulated proteins in diapausing females. The PLAT domain is a lipid-binding module, suggesting a role for TuPLAT10 in lipid metabolism. In addition to the suppression of orange pigmentation, RNAi of the TuPLAT10 gene restored reproduction even under diapause-inducing conditions and selectively reduced TuPDS and TuLCPS protein levels, despite the absence of sequence similarity to their genes. We propose that TuPLAT10 acts as a lipid-allocation switch that, in response to photoperiodic information, partitions fatty acids between astaxanthin esterification and yolk lipid supply, thereby coupling reproductive arrest and carotenoid pigmentation during diapause in T. urticae.

Background: Although thin, T1 melanomas have an excellent cure rate with surgery alone, >25% of melanoma deaths originate from thin melanomas (TMs). There is, therefore, an urgent need to improve the identification and management of patients with TMs at high risk of recurrence. Methods: Patients with T1 melanoma and recurrence [&le;] 2 years of diagnosis (T1 rapid group) were compared to patients with T1 melanoma and recurrence [&ge;]10 years after diagnosis (T1 late group). Results: 442 patients from 14 sites were included: 310 and 132 patients in the T1 rapid and late groups, respectively. Median age at primary melanoma diagnosis was 51 years [15-85], 272 (62%) male, 254 (58%) superficial spreading and 101 (23%) head/neck primary. The majority (73%) of recurrences in the T1 rapid group were locoregional. Using univariable logistic regression analysis, age >65 years (p<0.0001), lentigo maligna (LM) melanoma subtype (p=0.025), head/neck primary site (p=0.0065), mitoses [&ge;]1/mm2 (p=0.0181) and ulceration (p=0.0087) were significantly associated with T1 rapid recurrence compared to T1 late recurrence. Using multivariable analysis, age >65 years (p=0.0010), mitoses [&ge;]1/mm2 (p=0.049) and ulceration (p=0.037) remained significant. Conclusions: Rapid recurrence of TM is associated with age >65 years, LM subtype, head/neck primary site, mitoses [&ge;]1/mm2 and ulceration.

Mesenchymal stem cells (MSCs) have garnered significant attention over the past three decades due to their robust regenerative potential, primarily mediated by their paracrine activity by releasing soluble bioactive factors and extracellular vesicles (EVs). The MSC secretome plays a pivotal role in wound healing by influencing cellular migration, inflammation, angiogenesis, extracellular matrix (ECM) remodeling, and re-epithelialization. SPARC (Secreted Protein Acidic and Rich in Cysteine), a multifunctional ECM glycoprotein involved in tissue repair and remodeling, regulates key processes such as cell migration, proliferation, angiogenesis, and survival. Despite its known role in ECM dynamics, the impact of SPARC expression on the regenerative properties of MSCs remains underexplored. In this study, we hypothesized that SPARC overexpression in MSCs enhances their secretomes regenerative capacity. Using lentiviral systems, we generated SPARC-overexpressing (+SPARC) and SPARC-knockdown (KD-SPARC) MSCs to investigate SPARCs role in wound healing. Conditioned media (CM) derived from these MSCs were analyzed in vitro for their effects on human skin keratinocytes and fibroblasts. Our results revealed that SPARC expression significantly influences cell-specific migration and cell cycle. Furthermore, in an in vivo wound healing model, CM from +SPARC MSCs accelerated regeneration, while SPARC absence in MSCs CM delayed the healing process. These findings underscore the critical role of SPARC in modulating MSC secretome composition and enhancing its regenerative efficacy. This study highlights SPARC as a promising therapeutic target for the development of advanced regenerative therapies aimed at improving cutaneous wound healing outcomes.

BackgroundDuring 2021-2022, high pathogenicity avian influenza (HPAI) caused mass mortality in wild birds across Europe, with Northern Gannets (Morus bassanus) among the most affected. Following the outbreak, unusual alterations in the species characteristic pale iris were observed in some individuals. MethodsOpportunistically captured gannets on Bass Rock (n=52), selected to represent a range of iris pigmentation, were examined. Slit-lamp biomicroscopy, indirect ophthalmoscopy, rebound tonometry and photography were performed. Iris pigmentation was classified as normal, mottled or black. Eleven birds underwent avian influenza virus (AIV) serology. Histopathology was performed on two eyes. ResultsAbnormal iris pigmentation was found in 74% of adult and immature gannets, with 61% affected bilaterally. Additional signs consistent with uveitis were present in 77% of affected birds. Iris pigmentation abnormalities were positively associated with AIV H5 seropositivity (Fishers exact test, P=0.018). Histopathology from affected eyes showed increased melanin deposition and disorganisation, including loss of a distinct anterior layer of melanocytic cells and hypertrophy of melanocytes within the iris stroma. LimitationsField conditions limited uniform lighting and concurrent serology. ConclusionsIris pigmentation changes were associated with prior HPAI exposure and frequently accompanied by signs of uveitis, suggesting iris alterations may indicate past infection and potential chronic sequelae.

An expectation of a hypothesis that proposes cell-to-cell signalling pathways are redundant due to the redundancy of pathway terminal transcription factors (TFs) was tested by screening 35 signalling ligands (SLs) for rescue of a decapentaplegic (dpp) hypomorphic wing growth phenotype. The screen identified three examples of partial rescue: Hedgehog (HH), Semphorin 1a (SEMA1A) and Wnt ortholog 2 (WNT2). HH overexpression with dppGAL4 may increase the expression of DPP activity from the hypomorphic dpp alleles. However, SEMA1A and WNT2 did not phenocopy ectopic expression of HH or DPP and neither SEMA1A nor WNT2 were required for wing growth suggesting substitution of DPP for partial restoration of wing growth. The WNT2 rescue was dependent on the Frizzled 4 (FZ4) WNT receptor excluding the possibility that WNT2 weakly binds the DPP receptor. Although examples of phenotypic nonspecificity of SL function were identified, this is an expectation, and not direct proof, of the hypothesis of TF redundancy. Screen Report SummaryAn expectation of a hypothesis proposing that cell-to-cell signalling pathways are redundant due to the redundancy of the pathway terminal transcription factors was tested by screening for replacement of one signalling ligand (DPP; SLa) with another SLb for wing growth. Three non-DPP SLs were identified in the screen of 35SLs: HH, SEMA1A and WNT2. Genetic analysis of Sema1a and Wnt2 suggests functional complementation of dpp for wing growth suggesting that SEMA1A and WNT2 partially replace DPP for wing growth. Therefore, an expectation of the hypothesis is met.

Optic fissure (OF) is a transient structure in the ventral optic cup, which acts as a conduit for periocular mesenchyme cells to enter the eye, forming hyaloid vasculature and retinal ganglion axons to exit. Optic fissure closes to form a continuous layer of retinal pigment epithelium and neural retina. Failure of OF closure results in coloboma, which is mostly genetic in nature. The severity of blindness depends on the tissue it effects and accounts for 10% of childhood blindness. In the current study, we describe coloboma pathogenesis caused by hippo effectors yap1 and wwtr1. Both the paralogs are expressed in the OF edges, possibly in the pioneer cells. wwtr1 homozygotes do not have coloboma, while yap1 homozygotes have coloboma and pigment defects which are exacerbated by absence of one copy of wwtr1 (yap1-/-; wwtr1+/-). The coloboma observed in these mutants is not due to defective optic cup morphogenesis nor an overgrown optic nerve. The pigment defects are more pronounced at the OF with complete absence of RPE specific transcription factors mitfA, tfec, and pigmentation gene dct. On the other hand, NR specific genes are upregulated and the unpigmented region at the OF have transdifferentiated retinal ganglion cells, amacrine, and photoreceptor cells. Our observations indicate that in the absence of yap1 and wwtr1, the cells at the OF cannot attain a conducive state to fuse nor they maintain the RPE specific fate and instead they transdifferentiate into unpigmented retina, causing a steric block for fusion, resulting in coloboma.