An in vitro nevus explant model for studying the effects of ultraviolet radiation.

Ultraviolet radiation (UVR) has been recognized as a potential trigger for the transformation of benign melanocytic nevi into melanoma. However, the mechanisms governing the formation and progression of melanocytic nevi remain poorly understood. This lack of understanding is partly due to the difficulty in isolating and culturing nevus tissues in vitro, resulting in a dearth of robust ex vivo models for nevi. Therefore, the establishment of a reliable melanocytic nevus model is imperative. Such a model is essential for elucidating nevus pathogenesis and facilitating the development of effective therapeutic interventions. Therefore, we have sought to establish an ex vivo nevus explant model to study UVR stimulation. And the structural integrity and tissue activity of the ex vivo nevi explant model was evaluated. We then observed melanogenesis and proliferation activity of the explants after UVR stimulation. There was less blister formation after Day 3 in nevi explants under our modified medium conditions. The nevi explant was able to maintain almost the same morphological structure and tissue activity as in vivo tissue within 24 h. Following UVR stimulation, we observed increased melanogenesis and proliferation activity in nevi explants. Nevi explants could serve as an ex vivo model for UVR-induced nevi stimulation research.

Clinical Misdiagnosis of Cutaneous Malignant Tumors as Melanocytic Nevi or Seborrheic Keratosis: A Retrospective Analysis of a Chinese Population.

Purpose: The rising incidence and mortality associated with cutaneous malignant tumours highlight the importance of early diagnosis of these tumors. In clinical practice, these tumors are often misdiagnosed as benign skin lesions such as melanocytic nevi (MN) and seborrheic keratosis (SK) because of their similar morphologic features. The incidence and clinicopathological subtypes of cutaneous malignancies in East Asia populations significantly differ from those in fair-skinned groups. However, studies on misdiagnoses in Eastern countries are lacking. Therefore, this study focused on the clinical and pathological features of cutaneous malignant tumors misdiagnosed as MN or SK in a Chinese population. Patients and Methods: A total of 4592 samples clinically diagnosed as MN (n = 3503) or SK (n = 1089) from July 2014 to June 2022 were collected and evaluated retrospectively. The clinical and pathological data were analyzed to identify associated factors. Results: Pathological assessments showed that 2.5% (86/3503) of the specimens clinically diagnosed as MN were malignancies, predominantly basal cell carcinoma (BCC, 84.9%, 73/86), followed by malignant melanoma (MM, 8.1%, 7/86) and squamous cell carcinoma (SCC, 7.0%, 6/86). Similarly, 5.7% (62/1089) of the specimens clinically diagnosed as SK were malignant tumors, of which BCC (50.0%, 31/62) was the most common, followed by SCC (41.9%, 26/62) and MM (8.1%, 5/62). In both types of specimens, advanced age and facial lesions were risk factors for malignancy misdiagnosis. The malignancy rate, mean age, and proportion of SCC in the specimens clinically diagnosed as SK were higher than those in the specimens clinically diagnosed as MN. Dermoscopy significantly reduced the rate of misdiagnosis of these tumors as MN or SK. Conclusion: In China, cutaneous malignant tumors misdiagnosed as MN or SK are not uncommon in clinical practice, and active introduction of noninvasive diagnostic techniques is essential to distinguish them.

Near-Infrared Light-Driving Organic Photothermal Agents with an 88.9% Photothermal Conversion Efficiency for Image-Guided Synergistic Phototherapy.

Photothermal agents (PTAs) with desirable near-infrared (NIR) absorption and excellent photothermal conversion efficiency (PCE) are ideal candidates for cancer treatment. However, numerous PTAs still require high-intensity and long-duration laser irradiation to completely ablate the tumor during the photothermal therapy (PTT) process, resulting in light damage to healthy skin and tissue as well as limiting their biomedical applications. Integrating intense NIR absorption and high PCE into a single small-molecule PTA is an important prerequisite for realizing efficient PTT, but is a serious challenge. Herein, a series of donor-acceptor type PTAs (CC1 to NC4) are designed through a molecular engineering strategy. Theoretical calculations and experimental results show that the NIR absorption and photothermal effect from CC1 to NC4 are significantly enhanced as expected. Notably, NC4 nanoparticles exhibit intense NIR absorption, superhigh PCE of up to 88.9% for PTT, photoacoustic imaging and photothermal imaging, and effective reactive oxygen species generation for photodynamic therapy (PDT). The superior PTT/PDT synergistic phototherapeutic efficacy is well demonstrated by the complete elimination of tumor in vivo upon one-time, low-intensity, and short-duration laser irradiation (808 nm, 330 mW cm-2, and 3 min). This work provides a valuable guideline for rational design of PTAs for cancer phototherapy.

Acceptor-donor-acceptor type organic photothermal agents with enhanced NIR absorption and photothermal conversion effect for cancer photothermal therapy.

Numerous photothermal agents (PTAs) require high-intensity and long-duration laser excitation for photothermal therapy (PTT), resulting in light damage to healthy skin and tissue as well as limiting their biomedical applications. Integrating desirable near-infrared (NIR) absorption and high photothermal conversion efficiency (PCE) into a single small-molecule PTA is an important prerequisite for realizing efficient PTT, but is a serious challenge. Herein, through molecular engineering strategy, an acceptor-donor-acceptor (A-D-A) type PTA (ADA3) was readily developed for 808 nm laser-driving photothermal imaging and PTT of tumor. Theoretical calculations and experiment results show molecular engineering strategy is significant in regulating the structure and energy gap of PTAs, so as to effectively induce a narrow band gap for NIR absorption and further optimize photothermal properties. ADA3 possesses molar extinction coefficient of 3.1 × 104 M-1 cm-1 at 808 nm, followed being assembled into nanoparticles, ADA3-NPs show high PCE of 80.3%. In vivo experiments indicate that ADA3-NPs have excellent antitumor capability under one-time, low-intensity and short-duration (808 nm, 330 mW/cm2, 3 min) laser irradiation. Therefore, this work definitely exemplifies the enormous potential of molecular engineering strategy and provides an effective method for developing small-molecule PTAs.

Expression Analysis of Retinal G Protein-coupled Receptor and its Correlation with Regulation of the Balance between Proliferation and Aberrant Differentiation in Cutaneous Squamous Cell Carcinoma.

Retinal G protein-coupled receptor (RGR), a photosensitive protein, functions as a retinal photoisomerase under light conditions in humans. Cutaneous squamous cell carcinoma (cSCC) is linked to chronic ultraviolet exposure, which suggests that the photoreceptor RGR may be associated with tumorigenesis and progression of squamous cell carcinoma (SCC). However, the expression and function of RGR remain uncharacterized in SCC. This study analysed RGR expression in normal skin and in lesions of actinic keratosis, Bowen's disease and invasive SCC of the skin with respect to SCC initiation and development. A total of 237 samples (normal skin (n = 28), actinic keratosis (n = 42), Bowen's (n = 35) and invasive SCC (n = 132) lesions) were examined using immunohistochemistry. Invasive SCC samples had higher expression of RGR protein than the other samples. A high immunohistochemical score for RGR was associated with increased tumour size, tumour depth, Clark level, factor classification, and degree of differentiation and a more aggressive histological subtype. In addition, RGR expression was inversely correlated with involucrin expression and positively correlated with proliferating cell nuclear antigen (PCNA) and Ki67 expression. Furthermore, RGR regulates SCC cell differentiation through the PI3K-Akt signalling pathway, as determined using molecular biology approaches in vitro, suggesting that high expression of RGR is associated with aberrant proliferation and differentiation in SCC.

In vitro differentiation of human amniotic epithelial stem cells into keratinocytes regulated by OPN3.

Human amniotic epithelial stem cells (hAESCs) are regarded as potential alternatives to keratinocytes (KCs) used for skin wound healing. Light is an alternative approach for inducing stem cell differentiation. Opsins (OPNs), a family of light-sensitive, G protein-coupled receptors, play a multitude of light-dependent and light-independent functions in extraocular tissues. However, it remains unclear whether the light sensitivity and function of OPNs are involved in light-induced differentiation of hAESCs to KCs. Herein, we determine the role of OPNs in differentiation of hAESCs into KCs through cell and molecular biology approaches in vitro. It is shown that mRNA expression of OPN3 in the amniotic membrane and hAESCs was higher than the other four primary OPNs by RT-qPCR analysis. Changes in OPN3 gene expression had a significant impact on cell proliferation, stemness and differentiation capability of hAESCs. Furthermore, we found a significant upregulation of OPN3, KRT5 and KRT14 with hAESCs treated at 3 × 33 J/cm2 irradiation from blue-light LED. Taken together, these results suggest that OPN3 acts as a positive regulator of differentiation of hAESCs into KCs. This study provides a novel insight into photosensitive OPNs associated with photobiomodulation(PBM)-induced differentiation in stem cells.

A pan-cancer analysis of RGR opsin expression and its downregulation associated with poor prognosis in glioma.

Retinal G protein-coupled receptor (RGR) serves a retinal photoisomerase function to mediate retinoid metabolism and visual chromophore regeneration in the human eyes. Retinoids display critical functions in cell proliferation, differentiation, and apoptosis. Abnormal retinoid metabolism may contribute to tumor development. However, in human tumor tissues, the expression of RGR remains uncharacterized. Herein, we performed the analysis of RGR expression in 620 samples from 24 types of tumors by immunohistochemistry (IHC) and 33 cancer types from the Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA), and Gene Expression Omnibus (GEO) databases by bioinformatic analyses. Furthermore, the biological role of RGR in glioma cells was investigated using molecular biology approaches in vitro. Notably, we found that brain lower grade glioma (LGG), in contrast to other tumor types, had the highest median score of IHC and RNA level of RGR expression. Survival analysis showed that low RGR expression was associated with worse overall survival in LGG (p<0.0001). RGR expression levels in glioma were also associated with pathological subtypes, grades, and isocitrate dehydrogenase (IDH) mutations. Moreover, its molecular function was closely associated with cadherin-related family member 1 (CDHR1), a tumor suppressive protein in glioma, suggesting that RGR might negatively regulate the tumorigenesis and progression of LGG through interacting with CDHR1. Our findings provide new insight into the role of RGR in human cancer, especially in glioma.

Opsin 3 expression in human Langerhans cell histiocytosis and its mediation of ELD-1 cellular function.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a type of -histiocytic disorder characterized by aberrant function, differentiation or proliferation of mononuclear phagocyte system cells, however, the pathogenesis is not fully understood. Opsin 3 (OPN3) plays an important role in regulating cell function. OBJECTIVES: We aimed to investigate OPN3 expression in LCH and Langerhans cells and evaluate its possible regulation of cellular function in a Langerhans cell-like cell line (ELD-1). MATERIALS & METHODS: Expression of OPN3 in LCH and paired adjacent healthy skin tissue was determined using microscopic tools (immunohistochemical and immunofluorescence staining) and RNA scope. OPN3 protein and mRNA levels in primary dendritic cells and ELD-1 were measured by real-time quantitative PCR and western blotting, respectively. The effects of reduced or over-expressed OPN3 mRNA level, via a lentiviral vector, were examined on ELD-1 proliferation, migration, cell cycle and apoptosis using the Cell Counting Kit 8, EdU-594 kit, Transwell assays and Cell Cycle Analysis Kit and Annexin V-PE apoptosis kit, respectively. Lastly, the signalling pathway mediating these functions was investigated via RNA sequencing and western blotting. RESULTS: OPN3 was highly expressed in human LCH tissue compared to healthy tissue, and was expressed in primary dendritic cells and ELD-1. Knockdown of OPN3 in ELD-1 inhibited cell proliferation, the cell cycle, and cell migration, while over-expression reversed these processes. These functions correlated with induction of the MAPK (p38/JNK/ERK) signalling pathway. CONCLUSION: Our results provide insight into the role of OPN3 in LCH which may become a molecular target for the clinical treatment of LCH.

'Seaweed appearance' in squamous cell carcinoma of the penis: A new dermoscopic finding.

Squamous cell carcinoma of the penis is an uncommon cancer. Vascular feature on dermoscopy is common in all forms of invasive squamous cell carcinoma, and the presence of the specific vascular features is often used to aid diagnosis. Here, we reported a new dermoscopic finding-seaweed-like vascular pattern in squamous cell carcinoma of the penis.

Affixing a Scalp Dressing With Hairpins.

Affixing a scalp dressing is challenging because of the presence of hair. Many methods have been recommended; their limitations include their effect on aesthetics and discomfort to the patient during the procedure. We describe a method for using hairpins to affix a dressing after scalp surgery or trauma that is sturdy, does not compromise aesthetics, and does not cause additional discomfort.

The necessity for popularizing varicella-zoster virus vaccine programs worldwide: An age-period-cohort analysis for the Global Burden of Disease study 2019.

BACKGROUND: Varicella-zoster virus (VZV) causes varicella and herpes zoster (VHZ), which is endemic worldwide. Although infection with VZV represents a considerable health threat, the global, regional and national burden of VZV infection, especially the probable relationship between VZV vaccines and the epidemiology of VZV infection, is poorly known. We sought to estimate the global spatial patterns and temporal trends of VHZ burden in 204 countries and territories from 1990 to 2019. METHODS: Numbers and age-standardized rates (ASR) of VHZ incidence, and disability-adjusted life years (DALYs) were estimated using data from the Global Burden of Diseases Study (GBD) 2019. Spatiotemporal trends in ASR were evaluated by estimated annual percentage change (EAPC). RESULTS: Worldwide, in 2019, there were approximately 84.0 million incidence and 0.9 million DALYs due to VHZ. The corresponding ASIR (age-standardized incidence rate) and ASDR (age-standardized DALY rate) drastically decreased in children (aged <20 years old), while the ASIR and ASDR of VHZ significantly increased in middle- and old-aged adults (aged >50 years old), with highest ASIR and ASDR in the High-income Asia Pacific and Western Sub-Saharan Africa, respectively. From 1990-2019, the corresponding EAPC in ASIR were 0.03 (95% uncertainty interval [UI]: 0.02-0.04). Whereas the global EAPC in ASDR decreased in all regions (-1.59, 95% UI: -1.64 to -1.55), expect for Australasia (0.46, [0.05, 0.88]). Notably, in 2019, age-specific rates of VHZ DALYs presented a rapid growth trend after 70 years old. CONCLUSION: The spatiotemporal trends of VHZ were heterogeneous across countries from 1990 to 2019. The spatiotemporal trend in ASIR is highest in the High sociodemographic index (SDI) region, however the EAPC in ASDR is lowest, in part probably due to VHZ vaccination. Therefore, reducing morbidity and burden strategies such as vaccines programs for the prevention of VHZ should be promoted in those regions with high growth incidence and/or burden, especially for the population after 70 years old.

Opsin 3 mediates UVA-induced keratinocyte supranuclear melanin cap formation.

Solar ultraviolet (UV) radiation-induced DNA damage is a major risk factor for skin cancer development. UV-induced redistribution of melanin near keratinocyte nuclei leads to the formation of a supranuclear cap, which acts as a natural sunscreen and protects DNA by absorbing and scattering UV radiation. However, the mechanism underlying the intracellular movement of melanin in nuclear capping is poorly understood. In this study, we found that OPN3 is an important photoreceptor in human epidermal keratinocytes and is critical for UVA-mediated supranuclear cap formation. OPN3 mediates supranuclear cap formation via the calcium-dependent G protein-coupled receptor signaling pathway and ultimately upregulates Dync1i1 and DCTN1 expression in human epidermal keratinocytes via activating calcium/CaMKII, CREB, and Akt signal transduction. Together, these results clarify the role of OPN3 in regulating melanin cap formation in human epidermal keratinocytes, greatly expanding our understanding of the phototransduction mechanisms involved in physiological function in skin keratinocytes.

High-Efficiency 3D-Printed Three-Chamber Electromagnetic Peristaltic Micropump.

This paper describes the design and characteristics of a three-chamber electromagnetic-driven peristaltic micropump based on 3D-printing technology. The micropump is composed of an NdFeB permanent magnet, a polydimethylsiloxane (PDMS) film, a 3D-printing pump body, bolts, electromagnets and a cantilever valve. Through simulation analysis and experiments using a single chamber and three chambers, valved and valveless, as well as different starting modes, the results were optimized. Finally, it is concluded that the performance of the three-chamber valved model is optimal under synchronous starting conditions. The measurement results show that the maximum output flow and back pressure of the 5 V, 0.3 A drive source are 2407.2 µL/min and 1127 Pa, respectively. The maximum specific flow and back pressure of the micropump system are 534.9 µL/min∙W and 250.4 Pa/W, respectively.

The effects of missense OPN3 mutations in melanocytic lesions on protein structure and light-sensitive function.

Opsin 3 (OPN3), a member of the light-sensitive, retinal-dependent opsin family, is widely expressed in a variety of human tissues and plays a multitude of light-dependent and light-independent roles. We recently identified five missense variants of OPN3, including p. I51T, p. V134A, p. V183I, p. M256I and p. C331Y, in human melanocytic tumours. However, it remains unclear how these OPN3 variants affect OPN3 protein structure and function. Herein, we conducted structural and functional studies of these variant proteins in OPN3 by molecular docking and molecular dynamics simulations. Moreover, we performed in vitro fluorescence calcium imaging to assess the functional properties of five single-nucleotide variant (SNV) proteins using a site-directed mutagenesis method. Notably, the p. I51T variant was not able to effectively dock with 11-cis-retinal. Additionally, in vitro, the p. I51T SNVs failed to induce any detectable changes in intracellular Ca2+ concentration at room temperature. Taken together, these results reveal that five SNVs in the OPN3 gene have deleterious effects on protein structure and function, suggesting that these mutations, especially the p. I51T variant, significantly disrupt the canonical function of the OPN3 protein. Our findings provide new insight into the role of OPN3 variants in the loss of protein function.

Identification and functional assays of single-nucleotide variants of opsins genes in melanocytic tumors.

Epidermal melanocytes sense solar light via the opsin-coupled signaling pathway which is involved in a range of biological functions, including regulating pigmentation, proliferation, apoptosis, and tumorigenesis. However, it remains unclear whether there are genetic variants within these opsins that affect opsin protein structure and function, and further melanocyte biological behaviors. Here, we examined single-nucleotide variants (SNVs) of five opsin (RGR, OPN1SW, OPN2, OPN4, and OPN5) genes in MM (malignant melanoma; n = 76) and MN (melanocytic nevi; n = 157), using next-generation sequencing. The effects of these pathogenic single-nucleotide variants (SNVs) on opsin structure and function were further investigated using molecular dynamics (MD) simulations, dynamic cross-correlation (DCC), and site-directed mutagenesis. In total, 107 SNV variants were identified. Of these variants, 14 nonsynonymous SNVs (nsSNVs) of opsin genes were detected, including three mutations in the RGR gene, three mutations in the OPN1SW gene, two mutations in the OPN2 gene, and six mutations in the OPN4 gene. The effect of these missense mutations on opsin function was then assessed using eight prediction tools to estimate the potential impact of an amino acid substitution. The impact of each nsSNV was investigated using MD simulations and DCC analysis. Furthermore, we performed in vitro fluorescence calcium imaging to assess the functional properties of nsSNV proteins using a site-directed mutagenesis method. Taken together, these results revealed that p.A103V (RGR), p.T167I (RGR), p.G141S (OPN1SW), p.R144C (OPN1SW), and p.S231F (OPN4) had more deleterious effects on protein structure and function among the 14 nsSNVs. Opsin gene alterations showed the low frequency of missense mutations in melanocytic tumors, and although rare, some mutations in these opsin genes disrupt the canonical function of opsin. Our findings provide new insight into the role of opsin variants in the loss of function.

OPN3 Regulates Melanogenesis in Human Congenital Melanocytic Nevus Cells through Functional Interaction with BRAFV600E.

OPN3, as a member of the opsin family, has various nonlight-dependent functions. Congenital melanocytic nevus (CMN) is a skin lesion with dark pigmentation that appears at birth and can be initiated by the BRAFV600E mutation in melanocytes. However, the role of OPN3 in BRAFV600E CMN cell melanogenesis has never been reported. In this study, we show that OPN3 acts as a negative regulator of melanin production by modulating BRAFV600E signaling in BRAFV600E CMN cells. Knocking down OPN3 expression can inhibit the BRAFV600E/extracellular signal‒regulated kinase signaling pathway and upregulate the expression of microcephaly-related transcription factors, tyrosinase, and TRP1 and TRP2, thus increasing melanin levels in BRAFV600E CMN cells. More remarkably, OPN3 and BRAFV600E were found to form a physical complex. Furthermore, a three-dimensional nevus model was used to further prove the negative regulatory effect of OPN3 on BRAFV600E CMN cell melanogenesis. Our study reveals a mechanism for OPN3-mediated melanogenesis in BRAFV600E CMN cells, and these results may lead to a more personalized and economically viable approach to treating BRAFV600E CMN.

Expression of Retinal G Protein-Coupled Receptor, a Member of the Opsin Family, in Human Skin Cells and Its Mediation of the Cellular Functions of Keratinocytes.

Background: Photoreceptive proteins play critical physiological roles in human skin cells. The retinal G protein-coupled receptor (RGR) is a photoisomerase in the human retina, but its expression and cellular functions in human skin cells have not been reported. Objectives: We aimed to detect RGR expression in various skin cells and evaluate its regulation of the cellular functions of keratinocytes. Methods: The expression, distribution, and subcellular location of the RGR in normal human epidermal keratinocytes and cells with pathological conditions including psoriasis, seborrheic keratosis, and squamous cell carcinoma were determined using microscopic tools (immunohistochemical staining, immunofluorescence staining, and immunoelectron microscopy) and Western blotting (WB). The protein levels of the RGR in primary human melanocytes, keratinocytes, and fibroblasts isolated from the neonatal foreskin were measured by WB. The expression and subcellular localization of the RGR in these cells were detected by immunofluorescence staining under a fluorescence microscope and laser scanning confocal microscope. Additionally, the levels of RGR expression in normal keratinocytes exposed to ultraviolet (UV)-A or total ultraviolet radiation (UVR) in the presence or absence of all-trans-retinal were measured by WB. Furthermore, the effects of the RGR on human keratinocyte functions including proliferation, migration, and apoptosis were evaluated using the Cell Counting Kit 8, wound healing, and Transwell assays after reducing the RGR mRNA level in keratinocytes using small interfering RNA technology. Results: The RGR was primarily located in the epidermal basal and spinous layers and skin appendages. Its expression increased in psoriatic lesions, seborrheic keratosis, and squamous cell carcinoma. Confocal microscopy showed that the RGR was located in the cell membrane and nucleus of keratinocytes, melanocytes, and fibroblasts. Keratinocytes had a higher expression of the RGR than melanocytes and fibroblasts, as well as nuclear expression, according to nuclear/cytoplasmic fractionation. Colloidal gold immunoelectron microscopy technology further confirmed that the RGR is mainly located in the nucleoplasm and mitochondria and is scattered in the cytoplasm and other organelles in the epidermal keratinocytes. Notably, RGR knockdown in keratinocytes led to the inhibition of cell proliferation and migration, augmenting cell apoptosis. Conclusions: This study is the first to demonstrate the presence of RGR in the human skin. Our findings indicate that the RGR may play a critical role in the physiological function of epidermal keratinocytes.

Tattoo-like dermatitis left after fire-needle therapy.

Traditional Chinese medical theory holds that fire acupuncture can relieve neuropathic pain, and in many Asian countries, acupuncture has been used as one of the methods to relieve herpes zoster nerve pain. Sometimes, the patterns left on the skin by the needles may be very confusing to the practitioner who sees the patient. Recently, we saw a patient with puzzling tattoo-like dermatitis on his skin, and upon further questioning, he had recently undergone a fire-needle treatment and was left with this pattern.