Novel strategy for applying hierarchical density-based spatial clustering of applications with noise towards spectroscopic analysis and detection of melanocytic lesions.

Advancements in dermoscopy techniques have elucidated identifiable characteristics of melanoma which revolve around the asymmetrical constitution of melanocytic lesions consequent of unfettered proliferative growth as a malignant lesion. This study explores the applications of hierarchical density-based spatial clustering of applications with noise (HDBSCAN) in terms of the direct diagnostic implications of applying agglomerative clustering in the spectroscopic analysis of malignant melanocytic lesions and benign dermatologic spots. 100 images of benign (n = 50) and malignant moles (n = 50) were sampled from the International Skin Imaging Collaboration Archive and processed through two separate Python algorithms. The first of which deconvolutes the three-digit tupled integer identifiers of pixel color in image composition into three separate matrices corresponding to the red, green and blue color channel. Statistical characterization of integer variance was utilized to determine the optimal channel for comparative analysis between malignant and benign image groups. The second applies HDBSCAN to the matrices, identifying agglomerative clustering in the dataset. The results indicate the potential diagnostic applications of HDBSCAN analysis in fast-processing dermoscopy, as optimization of clustering parameters according to a binary search strategy produced an accuracy of 85% in the classification of malignant and benign melanocytic lesions.

Melanocyte progenitor cells reside in human subcutaneous adipose tissue.

Based on the assumption that some progenitor cells in an organ might reside in neighboring adipose tissue, we investigated whether melanocyte progenitor cells reside in human subcutaneous adipose tissue. First, we examined the expression of human melanoma black 45 (HMB45) and microphthalmia-associated transcription factor (MITF) in undifferentiated adipose-derived stem cells (ADSCs) by immunostaining, RT-PCR, and western blotting. These two markers were detected in undifferentiated ADSCs, and their expression levels were increased in differentiated ADSCs in melanocyte-specific culture medium. Other melanocytic markers (Melan A, MATP, Mel2, Mel EM, tyrosinase, KIT, and PAX3) were also detected at variable levels in undifferentiated ADSCs, and the expression of some markers was increased during differentiation into the melanocyte lineage. We further showed that ADSCs differentiated in melanocyte-specific culture medium localized in the basal layer and expressed tyrosinase and HMB45 in a 3D epidermal culture system. Melanin deposits were also induced by ultraviolet-light-B (UVB) irradiation. These results demonstrate that melanocyte progenitor cells reside in human subcutaneous adipose tissue and that these cells might have the potential to differentiate into mature melanocytes. Melanocyte and keratinocyte progenitors residing in human subcutaneous tissue can be used for the treatment of skin diseases and skin rejuvenation in the future.

Dermal Fibroblasts Internalize Phosphatidylserine-Exposed Secretory Melanosome Clusters and Apoptotic Melanocytes.

Pigmentation in the dermis is known to be caused by melanophages, defined as melanosome-laden macrophages. In this study, we show that dermal fibroblasts also have an ability to uptake melanosomes and apoptotic melanocytes. We have previously demonstrated that normal human melanocytes constantly secrete melanosome clusters from various sites of their dendrites. After adding secreted melanosome clusters collected from the culture medium of melanocytes, time-lapse imaging showed that fibroblasts actively attached to the secreted melanosome clusters and incorporated them. Annexin V staining revealed that phosphatidylserine (PtdSer), which is known as an 'eat-me' signal that triggers the internalization of apoptotic cells by macrophages, is exposed on the surface of secreted melanosome clusters. Dermal fibroblasts were able to uptake secreted melanosome clusters as did macrophages, and those fibroblasts express TIM4, a receptor for PtdSer-mediated endocytosis. Further, co-cultures of fibroblasts and melanocytes demonstrated that dermal fibroblasts internalize PtdSer-exposed apoptotic melanocytes. These results suggest that not only macrophages, but also dermal fibroblasts contribute to the collection of potentially toxic substances in the dermis, such as secreted melanosome clusters and apoptotic melanocytes, that have been occasionally observed to drop down into the dermis from the epidermis.

Cell-substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime.

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.

H2 O2 -induced oxidative stress disrupts mitochondrial functions and impairs migratory potential of human epidermal melanocytes.

Reactive oxygen species (ROS) have already been demonstrated to impede the migratory ability in non-melanocytic cell lines by depleting mitochondrial ATP production. Therefore, understanding the mitochondrial metabolic response to migration in the presence of ROS should be a key to understanding repigmentation in vitiligo. This study aimed to investigate the energy mechanism associated with the ROS-mediated attenuation of melanocyte migration. After melanocytes were pretreated with H2 O2 , their ATP production, migratory ability, ultrastructural changes and Mitochondrial Permeability Potential were analysed. The results showed that, in parallel with the decreased ATP production, the migratory ability of melanocytes was significantly inhibited by oxidative stress. Supplementation with exogenous ATP reversed the suppressed ATP-dependent migration of melanocytes. Melanocytes were then stressed with H2 O2 and Agilent Whole Human Genome microarray analysis identified 763 up-regulated mRNAs and 1117 down-regulated mRNAs. Among them, 11 of the encoded proteins were involved in mitochondrial ATP production and their expression levels were verified. The decreased expression of NADH dehydrogenase 2(ND2) , cytochrome c oxidase 1(COX1) and cytochrome c oxidase 3(COX3) was shown to be involved in the depletion of mitochondrial ATP production, which was coupled with the impaired migratory potential. These results indicate that the migration of melanocytes relies heavily on an inexhaustible supply of ATP from mitochondria.

Aberrant ETS-1 signalling impedes the expression of cell adhesion molecules and matrix metalloproteinases in non-segmental vitiligo.

Cell adhesion is a complex process that involves multiple molecules on the cell surface (ie cell adhesion molecules [CAMs]), surrounding cells and extracellular matrix (ECM). Repigmentation in vitiligo is dependent on the ECM remodelling and cellular migration, primarily attributed to the transcriptional activation of matrix metalloproteinases (MMPs). In this study, we aimed to demonstrate the role of ETS-1 signalling in the regulation of MMPs and CAMs. Therefore, we studied the expression of ETS-1, MMPs (MMP-2, MMP-9) and CAMs including E-cadherin, ITGA-1 and ICAM-1 in vitiligo (both active and stable) ex vivo. Further, we compared melanocyte morphology and their adhesion towards collagen IV and laminin between control and vitiligo groups in vitro. Also, we silenced ETS-1 in melanocytes cultured from control skin and observed post-silencing effect on above-mentioned MMPs and CAMs. We perceived absent ETS-1 and significantly reduced CAMs and MMPs in vitiligo compared with normal skin. Scanning electron microscopy (SEM) revealed a translucent material surrounding individual melanocytes in stable vitiligo and controls, whereas active vitiligo melanocytes demonstrated loss of this extracellular substance. Adhesion assays revealed significantly decreased binding of cultured melanocytes to collagen IV and laminin V plates in both stable and active vitiligo. Importantly, ETS-1 silencing resulted in significantly reduced expression of CAMs and MMPs. In conclusion, absent ETS-1 expression in both stable and active non-segmental vitiligo seems to impede the expression of CAMs, apart from MMPs, probably leading to progressive depigmentation in active disease and absence of spontaneous repigmentation in stable disease.

BAP1-inactivated melanocytic tumor with preserved BAP1 expression? Morphology to the rescue!

BAP1-inactivated melanocytic tumors typically present with distinctive histopathological changes and loss of nuclear BAP1 protein expression. Rare cases exhibit the typical morphology but with preserved expression of BAP1. In the current issue of Journal of Cutaneous Pathology, Linos et al. describe such a case and provide a comprehensive molecular-genetic exploration to explain such a phenomenon.

Characterization of Induced Pluripotent Stem Cells from Human Epidermal Melanocytes by Transduction with Two Combinations of Transcription Factors.

OBJECTIVE: In order to generate induced Pluripotent Stem Cells (iPSCs) more efficiently, it is crucial to identify somatic cells that are easily accessible and possibly require fewer factors for conversion into iPSCs. METHODS: Human epidermal melanocytes were transduced with lentiviral vectors carrying 3 transcription factors (OCT-4, KLF-4 and c-MYC, 3F) or 4 transcription factors (OCT-4, KLF-4, c-MYC and SOX-2, 4F). Once the clones had formed, assays related to stem cell pluripotency, including alkaline phosphatase staining, DNA methylation levels, expression of stem cell markers and ultrastructure analysis were carried out. The iPSCs obtained were then induced to differentiate into the cells representing the three embryonic layers in vitro. RESULTS: Seven days after the transduction of epidermal melanocytes with 3F or 4F, clones were formed that were positive for alkaline phosphatase staining. Fluorescent staining with antibodies against OCT-4 and SOX-2 was strongly positive, and the cells showed a high nucleus-cytoplasm ratio and active karyokinesis. No melanosomes were found in the cytoplasm by ultrastructural analysis. There were obvious differences in DNA methylation levels between the cloned cells and their parental cells. However, there was not a significant difference between 3F or 4F transfected clonal cells. Meanwhile, the iPSCs successfully differentiated into the three germ layer cells in vitro. CONCLUSION: Human epidermal melanocytes do not require ectopic SOX-2 expression for conversion into iPSCs, and may serve as an alternative source for deriving patient-specific iPSCs with fewer genetic elements.

Extracellular vesicles released by melanocytes after UVA irradiation promote intercellular signaling via miR21.

Skin pigmentation is controlled by complex crosstalk between melanocytes and keratinocytes and is primarily induced by exposure to ultraviolet (UV) irradiation. Several aspects of UVA-induced signaling remain to be explored. In skin cells, UVA induces plasma membrane damage, which is repaired by lysosomal exocytosis followed by instant shedding of extracellular vesicles (EVs) from the plasma membrane. The released EVs are taken up by neighboring cells. To elucidate the intercellular crosstalk induced by UVA irradiation, EVs were purified from UVA-exposed melanocytes and added to keratinocytes. Transcriptome analysis of the keratinocytes revealed the activation of TGF-ß and IL-6/STAT3 signaling pathways and subsequent upregulation of microRNA (miR)21. EVs induced phosphorylation of ERK and JNK, reduced protein levels of PDCD4 and PTEN, and augment antiapoptotic signaling. Consequently, keratinocyte proliferation and migration were stimulated and UV-induced apoptosis was significantly reduced. Interestingly, melanoma cells and melanoma spheroids also generate increased amounts of EVs with capacity to stimulate proliferation and migration upon UVA. In conclusion, we present a novel intercellular crosstalk mediated by UVA-induced lysosome-derived EVs leading to the activation of proliferation and antiapoptotic signaling via miR21.

Autophagy induction can regulate skin pigmentation by causing melanosome degradation in keratinocytes and melanocytes.

Autophagy regulates cellular turnover by disassembling unnecessary or dysfunctional constituents. Recent studies demonstrated that autophagy and its regulators play a wide variety of roles in melanocyte biology. Activation of autophagy is known to induce melanogenesis and regulate melanosome biogenesis in melanocytes. Also, autophagy induction was reported to regulate physiologic skin color via melanosome degradation, although the downstream effectors are not yet clarified. To determine the role of autophagy as a melanosome degradation machinery, we administered several autophagy inducers in human keratinocytes and melanocytes. Our results showed that the synthetic autophagy inducer PTPD-12 stimulated autophagic flux in human melanocytes and in keratinocytes containing transferred melanosomes. Increased autophagic flux led to melanosome degradation without affecting the expression of MITF. Furthermore, the color of cell pellets of both melanocytes and keratinocytes was visibly lightened. Inhibition of autophagic flux by chloroquine resulted in marked attenuation of PTPD-12-induced melanosome degradation, whereas the expression of melanogenesis pathway genes and proteins remained unaffected. Taken together, our results suggest that the modulation of autophagy can contribute to the regulation of melanocyte biology and skin pigmentation.

Melanocyte spheroids are formed by repetitive long-term trypsinization.

BACKGROUND: Autologous melanocyte transplantation plays an important role in the treatment of vitiligo. OBJECTIVE: Previous studies have indicated that, compared with melanocytes growing in monolayers, melanocyte spheroids have a better survival in growth factor- and serum-deprived conditions. METHODS: Melanocyte spheroids were obtained from human epidermis by repetitive long-term trypsinization and maintained an aggregated morphology for a short period in certain conditions. RESULTS: Melanocyte spheroids were capable of growing into normal dendritic melanocytes in monolayer when they were harvested and reinoculated in 24-well plates. Immunohistochemical analysis of the melanocyte spheroids revealed that they were positive for HMB45, a melanosome-specific marker. No melanomas occurred when melanocyte spheroids were transplanted into mice. CONCLUSION: Our study provides a promising approach for melanocyte transplantation to treat vitiligo.

The distribution of melanocytes and the degradation of melanosomes in fetal hair follicles.

There have been many studies about the formation, storage, transport and degradation of melanosomes in epidermal melanocytes but studies of melanocytes and melanosomes in fetal hair follicles (HFs) have been limited and ambiguous. The goal of this study was to investigate the distribution of melanocytes and the degradation of melanosomes in fetal HFs. After obtaining approval and informed consent for the study, a scalp specimen from a 5 month gestational age fetus was obtained and was divided into two parts. One part was subjected to immunohistochemical staining with the melanocyte-specific marker HMB-45 and was then observed by light microscopy to detect the distribution of melanocytes in HFs. The other part underwent conventional processing for transmission electron microcopy (TEM). Subsequently, the morphology of melanosomes in HF melanocytes and their degradation in cortical keratinocytes were observed. Immunohistochemically, scattered round melanocytes lacking dendrites were mainly observed along the outer root sheath of the lower part of the HF. A few fusiform or tri-dendritic melanocytes were located at the bottom of the hair bulbs. Significantly melanized melanocytes with multiple dendrites were concentrated in the pigmented area in the center of the hair bulbs, only above the dermal papilla. Analysis by TEM revealed melanocytes containing melanosomes at all stages of development. Autophagosomes containing stage mature IV melanosomes were observed in some melanocytes. Many phagolysosomes containing numerous melanosomes were observed in the cortical keratinocytes. Some phagolysosomes were concentrically surrounded by 3-5 layers of endoplasmic reticulum. Melanosomes that had been degraded or were being degraded in phagolysosomes in keratinocytes had lost their integrity and had become an ill-defined melanosomal dust that were arranged irregularly. Partial melanin particles were released into the cytosol. Melanocytes in different regions of fetal HFs had different morphologies and were at various stages of differentiation. Fetal HF melanocytes contained not only melanosomes at different developmental stages, but autophagosomes were seen occasionally. Melanosomes were degraded into irregular pigment particles in the phagolysosomes of cortical keratinocytes. These results provide important clues to elucidate the mechanism of melanosome biodegradation.

Resolution of conjunctival melanoma with topical interferon alpha 2b in a patient with mitomycin C intolerance. / Resolución de un melanoma conjuntival con interferón tópico alfa 2b en un paciente con intolerancia a la mitomicina C.

OBJECTIVE: To describe the clinical and histological resolution of a case of an inexcisable conjunctival melanoma using topical interferon alpha 2b (INFα2b) in a patient with mitomycin C (MMC) intolerance. CASE REPORT: Conjunctival melanoma is a rare, but potentially sight- and life-threatening, tumour. In cases of multiple lesions, or when surgical excision is not possible, topical combination chemotherapy with MMC and INFα2b has been described as first line therapy. The case is presented of a 77 year-old woman with a multifocal conjunctival in situ melanoma, who was intolerant to initial treatment with MMC and was switched to long-term INFα2b therapy, with a good outcome. CONCLUSIONS: When topical MMC is given as chemotherapy treatment for primary acquired melanosis with atypia or in situ melanoma is not well tolerated, switching to INFα2b seems to be a good option. This approach could replace surgical management of pigmented tumours, especially the larger ones, with potential benefits that include less dependence on surgical margins. This report prompts a need for prospective studies designed to examine the role of INFα2b as primary treatment for heavily pigmented conjunctival tumours avoiding the ocular surface toxicity caused by MMC.

Degraded melanocores are incompetent to protect epidermal keratinocytes against UV damage.

Melanosomes are membrane-bound intracellular organelles that are uniquely generated by melanocytes (MCs) in the basal layer of human epidermis. Highly pigmented mature melanosomes are transferred from MCs to keratinocytes (KCs), and then positioned in the supra-nuclear region to ensure protection against ultraviolet radiation (UVR). However, the molecular mechanism underlying melanosome (or melanin pigment) transfer remains enigmatic. Emerging evidence shows that exo-/endo-cytosis of the melanosome core (termed melanocore) has been considered as the main transfer manner between MCs and KCs. As KCs in the skin migrate up from the basal layer and undergo terminal differentiation, the melanocores they have taken up from MCs are subjected to degradation. In this study, we isolated individual melanocores from human MCs in culture and then induced their destruction/disruption using a physical approach. The results demonstrate that the ultrastructural integrity of melanocores is essential for their antioxidant and photoprotective properties. In addition, we also show that cathepsin V (CTSV), a lysosomal acid protease, is involved in melanocore degradation in calcium-induced differentiated KCs and is also suppressed in KCs following exposure to UVA or UVB radiation. Thus, our study demonstrates that change in the proportion of melanocores in the intact/undegraded state by CTSV-related degradation in KCs affects photoprotection of the skin.

Unveiling skin macrophage dynamics explains both tattoo persistence and strenuous removal.

Here we describe a new mouse model that exploits the pattern of expression of the high-affinity IgG receptor (CD64) and allows diphtheria toxin (DT)-mediated ablation of tissue-resident macrophages and monocyte-derived cells. We found that the myeloid cells of the ear skin dermis are dominated by DT-sensitive, melanin-laden cells that have been missed in previous studies and correspond to macrophages that have ingested melanosomes from neighboring melanocytes. Those cells have been referred to as melanophages in humans. We also identified melanophages in melanocytic melanoma. Benefiting of our knowledge on melanophage dynamics, we determined the identity, origin, and dynamics of the skin myeloid cells that capture and retain tattoo pigment particles. We showed that they are exclusively made of dermal macrophages. Using the possibility to delete them, we further demonstrated that tattoo pigment particles can undergo successive cycles of capture-release-recapture without any tattoo vanishing. Therefore, congruent with dermal macrophage dynamics, long-term tattoo persistence likely relies on macrophage renewal rather than on macrophage longevity.

Dysregulation of autophagy in melanocytes contributes to hypopigmented macules in tuberous sclerosis complex.

BACKGROUND: Tuberous sclerosis complex (TSC) gene mutations lead to constitutive activation of the mammalian target of rapamycin (mTOR) pathway, resulting in a broad range of symptoms. Hypopigmented macules are the earliest sign. Although we have already confirmed that topical rapamycin treatment (an mTOR inhibitor) protects patients with TSC against macular hypopigmentation, the pathogenesis of such lesions remains poorly understood. OBJECTIVE: Recently emerging evidence supports a role for autophagy in skin pigmentation. Herein, we investigated the impact of autophagic dysregulation on TSC-associated hypopigmentation. METHODS: Skin samples from 10 patients with TSC, each bearing characteristic hypopigmented macules, and 6 healthy donors were subjected to immunohistochemical and electron microscopic analyses. In addition, TSC2-knockdown (KD) was investigated in human epidermal melanocytes by melanin content examination, real-time PCR, western blotting analyses, and intracellular immunofluorescence staining. RESULTS: Activation of the mTOR signaling pathway decreased melanocytic pigmentation in hypopigmented macules of patients with TSC and in TSC2-KD melanocytes. In addition, LC3 expression (a marker of autophagy) and autophagosome counts increased, whereas, intracellular accumulation of autophagic degradative substrates (p62 and ubiquitinated proteins) was evident in TSC2-KD melanocytes. Furthermore, depigmentation in TSC2-KD melanocytes was accelerated by inhibiting autophagy (ATG7-KD or bafilomycin A1-pretreatment) and was completely reversed by induction of autophagy via mTOR-dependent (rapamycin) or mTOR-independent (SMER28) exposure. Finally, dysregulation of autophagy, marked by increased LC3 expression and accumulation of ubiquitinated proteins, was also observed in melanocytes of TSC-related hypopigmented macules. CONCLUSION: Our data demonstrate that melanocytes of patients with TSC display autophagic dysregulation, which thereby reduced pigmentation, serving as the basis for the hypomelanotic macules characteristic of TSC.

Effect of urea-extracted sericin on melanogenesis: potential applications in post-inflammatory hyperpigmentation

BACKGROUND: Hyperpigmentation disorders such as post-inflammatory hyperpigmentation are major concerns not only in light-skinned people but also in Asian populations with darker skin. The anti-tyrosinase and immunomodulatory effects of sericin have been known for decades. However, the therapeutic effects of sericin on hyperpigmentation disorders have not been well documented. METHODS: In this study, we used an in vitro model to study the anti-tyrosinase, tolerogenic, and anti-melanogenic effects of sericin on Staphylococcus aureus peptidoglycan (PEG)-stimulated melanocytes, dendritic cells (DCs), and artificial skin (MelanoDerm™). Enzyme-linked immunosorbent assay, conventional and immunolabeled electron microscopy, and histopathological studies were performed. RESULTS: The results revealed that urea-extracted sericin has strong anti-tyrosinase properties as shown by a reduction of tyrosinase activity in melanin pigments both 48 h and 10 days after allergic induction with PEG. Anti-inflammatory cytokines including interleukin (IL)-4, IL-10, and transforming growth factor-p were upregulated upon sericin treatment (10, 20, and 50 µg/mL), whereas production of allergic chemokines, CCL8 and CCL18, by DCs was diminished 48 h after allergic induction with PEG. Moreover, sericin lowered the expression of micropthalmia-associated transcription factor (MITF), a marker of melanogenesis regulation, in melanocytes and keratinocytes, which contributed to the reduction of melanin size and the magnitude of melanin deposition. However, sericin had no effect on melanin transport between melanocytes and keratinocytes, as demonstrated by a high retention of cytoskeletal components. CONCLUSION: In summary, sericin suppresses melanogenesis by inhibition of tyrosinase activity, reduction of inflammation and allergy, and modulation of MITF function.

Classical autophagy proteins LC3B and ATG4B facilitate melanosome movement on cytoskeletal tracks.

Macroautophagy/autophagy is a dynamic and inducible catabolic process that responds to a variety of hormonal and environmental cues. Recent studies highlight the interplay of this central pathway in a variety of pathophysiological diseases. Although defective autophagy is implicated in melanocyte proliferation and pigmentary disorders, the mechanistic relationship between the 2 pathways has not been elucidated. In this study, we show that autophagic proteins LC3B and ATG4B mediate melanosome trafficking on cytoskeletal tracks. While studying melanogenesis, we observed spatial segregation of LC3B-labeled melanosomes with preferential absence at the dendritic ends of melanocytes. This LC3B labeling of melanosomes did not impact the steady-state levels of these organelles but instead facilitated their intracellular positioning. Melanosomes primarily traverse on microtubule and actin cytoskeletal tracks and our studies reveal that LC3B enables the assembly of microtubule translocon complex. At the microtubule-actin crossover junction, ATG4B detaches LC3B from melanosomal membranes by enzymatic delipidation. Further, by live-imaging we show that melanosomes transferred to keratinocytes lack melanocyte-specific LC3B. Our study thus elucidates a new role for autophagy proteins in directing melanosome movement and reveal the unconventional use of these proteins in cellular trafficking pathways. Such crosstalk between the central cellular function and housekeeping pathway may be a crucial mechanism to balance melanocyte bioenergetics and homeostasis.

Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection.

The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. IMPORTANCE: The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic neuralgia (PHN) is a common complication of shingles that manifests as prolonged excruciating pain, which has proven difficult to treat. The formation of fused multinucleated cells in ganglia might be associated with this condition. An effective vaccine against VZV is available but not recommended for immunocompromised individuals, highlighting the need for new therapies. This study investigated the viral and cellular responses to hyperfusion, a condition where the usual constraints of cell membranes are overcome and cells form multinucleated cells. This process hinders VZV and is regulated by a viral glycoprotein, gB. A combination of live-cell imaging and next-generation genomics revealed an alteration in viral and cellular responses during hyperfusion that was caused by the loss of gB regulation. These studies reveal mechanisms central to VZV pathogenesis, potentially leading to improved therapies.

[Toll-like receptor 4 activation inhibits melanogenesis in melanocytes].

Objective To explore the effect of Toll-like receptor 4 (TLR4) activation on melanogenesis in melanocytes. Methods The primary melanocytes were isolated, cultured and then stimulated by TLR4 agonist lipopolysaccharide (LPS). Firstly, the melanin content changes were detected in melanocytes, and then quantitative real-time PCR and Western blotting were employed to determine the expression levels of pre-melanosomal protein (Pmel17), tyrosinase (TYR) and microphthalmia-associated transcription factor (MITF). Finally, the melanosomes were observed by electron microscopy. Results After stimulated by LPS, melanin formation in melanocytes was significantly reduced. The expressions of Pmel17 and TYR were markedly suppressed, which depended on the regulation of transcription factor MITF. The electron microscopy showed that melanosome synthesis was inhibited. Conclusion TLR4 activation reduces melanin synthesis in melanocytes, which indicates that the bacterial products may affect melanin synthesis through innate immunity.