Mapping the immune cell landscape of severe atopic dermatitis by single-cell RNA-seq.

BACKGROUND: Efforts to profile atopic dermatitis (AD) tissues have intensified, yet comprehensive analysis of systemic immune landscapes in severe AD remains crucial. METHODS: Employing single-cell RNA sequencing, we analyzed over 300,000 peripheral blood mononuclear cells from 12 severe AD patients (Eczema area and severity index (EASI) > 21) and six healthy controls. RESULTS: Results revealed significant immune cell shifts in AD patients, including increased Th2 cell abundance, reduced NK cell clusters with compromised cytotoxicity, and correlated Type 2 innate lymphoid cell proportions with disease severity. Moreover, unique monocyte clusters reflecting activated innate immunity emerged in very severe AD (EASI > 30). While overall dendritic cells (DCs) counts decreased, a distinct Th2-priming subset termed "Th2_DC" correlated strongly with disease severity, validated across skin tissue data, and flow cytometry with additional independent severe AD samples. Beyond the recognized role of Th2 adaptive immunity, our findings highlight significant innate immune cell alterations in severe AD, implicating their roles in disease pathogenesis and therapeutic potentials. CONCLUSION: Apart from the widely recognized role of Th2 adaptive immunity in AD pathogenesis, alterations in innate immune cells and impaired cytotoxic cells have also been observed in severe AD. The impact of these alterations on disease pathogenesis and the effectiveness of potential therapeutic targets requires further investigation.

The N-terminal cysteine is a dual sensor of oxygen and oxidative stress.

Cellular homeostasis requires the sensing of and adaptation to intracellular oxygen (O2) and reactive oxygen species (ROS). The Arg/N-degron pathway targets proteins that bear destabilizing N-terminal residues for degradation by the proteasome or via autophagy. Under normoxic conditions, the N-terminal Cys (Nt-Cys) residues of specific substrates can be oxidized by dioxygenases such as plant cysteine oxidases and cysteamine (2-aminoethanethiol) dioxygenases and arginylated by ATE1 R-transferases to generate Arg-CysO2(H) (R-CO2). Proteins bearing the R-CO2 N-degron are targeted via Lys48 (K48)-linked ubiquitylation by UBR1/UBR2 N-recognins for proteasomal degradation. During acute hypoxia, such proteins are partially stabilized, owing to decreased Nt-Cys oxidation. Here, we show that if hypoxia is prolonged, the Nt-Cys of regulatory proteins can be chemically oxidized by ROS to generate Arg-CysO3(H) (R-CO3), a lysosomal N-degron. The resulting R-CO3 is bound by KCMF1, a N-recognin that induces K63-linked ubiquitylation, followed by K27-linked ubiquitylation by the noncanonical N-recognin UBR4. Autophagic targeting of Cys/N-degron substrates is mediated by the autophagic N-recognin p62/SQTSM-1/Sequestosome-1 through recognition of K27/K63-linked ubiquitin (Ub) chains. This Cys/N-degron-dependent reprogramming in the proteolytic flux is important for cellular homeostasis under both chronic hypoxia and oxidative stress. A small-compound ligand of p62 is cytoprotective under oxidative stress through its ability to accelerate proteolytic flux of K27/K63-ubiquitylated Cys/N-degron substrates. Our results suggest that the Nt-Cys of conditional Cys/N-degron substrates acts as an acceptor of O2 to maintain both O2 and ROS homeostasis and modulates half-lives of substrates through either the proteasome or lysosome by reprogramming of their Ub codes.

Impact of ultraviolet radiation on cardiovascular and metabolic disorders: The role of nitric oxide and vitamin D.

BACKGROUND/PURPOSE: Ultraviolet (UV) radiation has both harmful and beneficial effects on human skin and health. It causes skin damage, aging, and cancer; however, it is also a primary source of vitamin D. Additionally, UV radiation can impact energy metabolism and has protective effects on several cardiovascular and metabolic disorders in mice and humans. However, the mechanisms of UV protection against these diseases have not been clearly identified. METHODS: This review summarizes the systemic effects of UV radiation on hypertension and several metabolic diseases such as obesity, diabetes, and nonalcoholic fatty liver disease (NAFLD) in mice, and we also consider the mechanisms of action of the related regulators nitric oxide (NO) and vitamin D. RESULTS: UV exposure can lower blood pressure and prevent the development of cardiovascular diseases and metabolic disorders, such as metabolic syndrome, obesity, and type 2 diabetes, primarily through mechanisms that depend on UV-induced NO. UV radiation may also effectively delay the onset of type 1 diabetes through mechanisms that rely on UV-induced vitamin D. UV-induced NO and vitamin D play roles in preventing and slowing the progression of NAFLD. CONCLUSION: UV exposure is a promising nonpharmacological intervention for cardiovascular and metabolic disorders. NO and vitamin D may play a crucial role in mediating these effects. However, further investigations are required to elucidate the exact mechanisms and determine the optimal dosage and exposure duration of UV radiation.

8-Shogaol inhibits rheumatoid arthritis through targeting TAK1.

Rheumatoid arthritis (RA) is a chronic immune-mediated disorder, mainly characterized by synovial inflammation and joint damage. If insufficiently treated, RA can lead to irreversible joint destruction and decreased life expectancy. While better understanding of the pathologies and the development of new antirheumatic drugs have improved the outcome of individuals with RA, many patients still cannot achieve remission and experience progressive disability. Fibroblast-like synoviocytes (FLS) have gained attention due to its pivotal role in RA pathogenesis and thus targeting FLS has been suggested as an attractive therapeutic strategy. To identify candidate molecules with strong inhibitory activity against FLS inflammation, we tested the effect of 315 natural extracts against IL-17-mediated IL-6 production. Zingiber officinale was found as the top hit and further analysis on the active compound responsible led to the discovery of 8-shogaol as a potent molecule against synovitis. 8-Shogaol displayed significant inhibitory effects against TNF-α-, IL-1ß-, and IL-17-mediated inflammation and migration in RA patient-derived FLS (RA-FLS) and 3D synovial culture system. 8-Shogaol selectively and directly inhibited TAK1 activity and subsequently suppressed IKK, Akt, and MAPK signaling pathways. Moreover, treatment with 8-shogaol reduced paw thickness and improved walking performance in the adjuvant-induced arthritic (AIA) rat model. 8-Shogaol also reversed pathologies of joint structure in AIA rats and decreased inflammatory biomarkers in the joints. Collectively, we report a novel natural compound that inhibits RA through reversing pathologies of the inflamed synovium via targeting TAK1.

Clinical features and natural course of pediatric longitudinal melanonychia: A retrospective cohort study in Korea.

BACKGROUND: Large studies on the clinical features and natural course of pediatric longitudinal melanonychia (LM) are lacking. OBJECTIVE: To investigate the clinical features and natural course of pediatric LM. METHODS: Retrospective cohort analysis of pediatric patients (age ≤ 18 years) with LM. RESULTS: We examined 703 LM lesions in 381 children. Single, narrow, and homogeneously pigmented fingernail lesions were most frequently observed. Our results suggested that within 3, 4.5, and 9.5 years after onset, approximately 3%, 5%, and 10% of LM lesions, respectively, will completely regress and that single, left-sided, and homogeneously pigmented lesions are more likely to disappear completely. The age of onset, sex, finger/toe position, Hutchinson's sign, and nail dystrophy were not associated with complete regression. During follow-up, most cases demonstrated no change in color or width between the first and last visit, and early darkening/widening before stabilization or lightening/narrowing was common. The lightning of pigmentation was associated with complete regression, whereas change in width was not. LIMITATIONS: Retrospective study at a tertiary center. CONCLUSION: Our results suggest that clinicians ought to follow pediatric patients with LM without intervention for several years even if lesions grow darker or wider. Single, left-sided, and homogeneously colored lesions are more likely to regress.

Targeted PMP22 TATA-box editing by CRISPR/Cas9 reduces demyelinating neuropathy of Charcot-Marie-Tooth disease type 1A in mice.

Charcot-Marie-Tooth 1A (CMT1A) is the most common inherited neuropathy without a known therapy, which is caused by a 1.4 Mb duplication on human chromosome 17, which includes the gene encoding the peripheral myelin protein of 22 kDa (PMP22). Overexpressed PMP22 protein from its gene duplication is thought to cause demyelination and subsequently axonal degeneration in the peripheral nervous system (PNS). Here, we targeted TATA-box of human PMP22 promoter to normalize overexpressed PMP22 level in C22 mice, a mouse model of CMT1A harboring multiple copies of human PMP22. Direct local intraneural delivery of CRISPR/Cas9 designed to target TATA-box of PMP22 before the onset of disease, downregulates gene expression of PMP22 and preserves both myelin and axons. Notably, the same approach was effective in partial rescue of demyelination even after the onset of disease. Collectively, our data present a proof-of-concept that CRISPR/Cas9-mediated targeting of TATA-box can be utilized to treat CMT1A.

Analysis of multi-omics data on the relationship between epigenetic changes and nervous system disorders caused by exposure to environmentally harmful substances.

Environmentally hazardous substances and exposure to these can cause various diseases. Volatile organic compounds can easily evaporate into the atmosphere, thereby exerting toxic effects through either the skin or respiratory tract exposures. Toluene, a neurotoxin, has been widely used in various industries. However, it has a detrimental effect on the nervous system (such as hallucinations or memory impairment), while data on the mechanism underlaying its harmful effects remain limited. Therefore, this study investigates the effect of toluene on the nervous system via epigenetic and genetic changes of toluene-exposed individuals. We identified significant epigenetic changes and confirmed that the affected abnormally expressed genes negatively influenced the nervous system. In particular, we confirmed that the miR-15 family, upregulated by toluene, downregulated ABL2, which could affect the R as signaling pathway resulting in neuronal structural abnormalities. Our study suggests that miR-15a-5p, miR-15b-5p, miR-16-5p, miR-301a-3p, and lncRNA NEAT1 may represent effective epigenomic markers associated with neurodegenerative diseases caused by toluene.

Efficacy and Safety of a New Botulinum Toxin (HU-014) Versus Existing Onabotulinumtoxin A in Subjects With Moderate to Severe Glabellar Lines.

BACKGROUND: Recently, the safety of a new botulinum toxin (HU-014) was confirmed through animal experiments. The evaluation of the efficacy and safety of this newly introduced botulinum toxin is required considering the risk of adverse events (AEs) and need for standardization before its universal use. OBJECTIVE: The aim of this multicenter, double-blind, randomized, parallel, active-controlled phase III clinical trial was to investigate the noninferiority of HU-014 versus existing onabotulinumtoxin A for the treatment of moderate to severe glabellar lines. METHODS: In total, 267 subjects were randomized to either the test (HU-014) or control (onabotulinumtoxin A) group. At the baseline and at weeks 4, 8, 12, and 16, investigator's live assessment, independent photographic assessment, subjects' improvement assessment, subjects' satisfaction assessment, and safety assessment were performed. RESULTS: At week 4, the response rate was 90.15% and 92.31% in the test and control groups, respectively, as per investigator's live assessment while frowning, without a significant difference. Both groups also showed no significant differences in response rates in the other assessments. In addition, no serious AEs were reported. CONCLUSION: HU-014 was noninferior to existing onabotulinumtoxin A in the treatment of glabellar lines at a 1:1 dose ratio, and both products were well tolerated.

Phase I/III Clinical Trial to Evaluate the Safety and Efficacy of a New Botulinum Toxin (HU-014) Versus OnabotulinumtoxinA in Subjects With Moderate-to-Severe Crow's Feet Lines.

BACKGROUND: HU-014, a newly introduced botulinum toxin type A, has not been investigated for its efficacy and safety in crow's feet line (CFL) treatment. OBJECTIVE: Here, we compared the efficacy and safety of HU-014 and onabotulinumtoxinA in CFL treatment. METHODS: This was a randomized, double-blind, active drug-controlled, multicenter, 16-week, Phase I/III study designed to determine the noninferiority of HU-014 compared with onabotulinumtoxinA in moderate-to-severe CFL treatment. In the Phase III study, 290 subjects were randomized at a 1:1 ratio to receive a single treatment of HU-014 or onabotulinumtoxinA. The primary endpoint was the proportion of subjects achieving Grade 0 or 1 in the facial wrinkle scale on maximum smile at Week 4. RESULTS: The primary endpoint was achieved by 72% of the subjects with HU-014 and onabotulinumtoxinA treatments, supporting the noninferiority of HU-014 compared with onabotulinumtoxinA. All secondary efficacy outcomes were achieved by the subjects. The 2 groups showed no significant differences in the safety analysis. CONCLUSION: HU-014 has noninferior efficacy and safety compared with onabotulinumtoxinA in the treatment of CFL.

Integrated analysis of multi-omics data on epigenetic changes caused by combined exposure to environmental hazards.

Humans are easily exposed to environmentally hazardous factors in industrial sites or daily life. In addition, exposure to various substances and not just one harmful substance is common. However, research on the effects of combined exposure on humans is limited. Therefore, this study examined the effects of combined exposure to volatile organic compounds (VOCs) on the human body. We separated 193 participants into four groups according to their work-related exposure (nonexposure, toluene exposure, toluene and xylene exposure, and toluene, ethylbenzene, and xylene exposure). We then identified the methylation level and long noncoding RNA (lncRNA) levels by omics analyses, and performed an integrated analysis to examine the change of gene expression. Thereafter, the effects of combined exposure to environmental hazards on the human body were investigated and analyzed. Exposure to VOCs was found to negatively affect the development and maintenance of the nervous system. In particular, the MALAT1 lncRNA was found to be significantly reduced in the complex exposure group, and eight genes were significantly downregulated by DNA hypermethylation. The downregulation of these genes could cause a possible decrease in the density of synapses as well as the number and density of dendrites and spines. In summary, we found that increased combined exposure to environmental hazards could lead to additional epigenetic changes, and consequently abnormal dendrites, spines, and synapses, which could damage motor learning or spatial memory. Thus, lncRNA MALAT1 or FMR1 could be novel biomarkers of neurotoxicity to identify the negative health effects of VOC complex exposure.

Differences in foot skin microbiomes between patients with type 2 diabetes and healthy individuals.

Impaired immunity and changes in the microenvironment in patients with diabetes might influence the composition of the cutaneous microbiome. However, data on the cutaneous microbiome of these patients are scarce. This study compared the fungal and bacterial components of the skin microbiome between patients with type 2 diabetes mellitus (DM) and healthy individuals. We obtained skin swab samples from the plantar forefoot of 17 patients with DM and 18 healthy individuals to conduct a cross-sectional study. The samples were profiled with culture-independent sequencing of the V3 to V4 regions of the bacterial 16S rRNA gene and the fungal ITS2 region, followed by direct DNA extraction and molecular polymerase chain reaction (PCR). We observed a differential cutaneous microbiome, especially for fungi, in patients with type 2 diabetes compared to that in healthy controls. Trichophyton rubrum was more abundant in DM samples. The Shannon diversity index for fungi was lower in the DM patients. Principal coordinate analysis plots and permutational multivariate analysis of variance (PERMANOVA) tests based on Bray-Curtis distances between samples supported the association of the fungal microbiome with DM at the species level. The results suggest that clinicians should pay attention to both fungi and bacteria and provide appropriate prevention and therapeutic strategies for diabetic cutaneous complications including diabetic foot ulcers. These data also contribute to future research associated with diabetes and cutaneous microbiomes.

UVB-induced depletion of donor-derived dendritic cells prevents allograft rejection of immune-privileged hair follicles in humanized mice.

Dendritic cells (DCs) are key targets for immunity and tolerance induction; they present donor antigens to recipient T cells by donor- and recipient-derived pathways. Donor-derived DCs, which are critical during the acute posttransplant period, can be depleted in graft tissue by forced migration via ultraviolet B light (UVB) irradiation. Here, we investigated the tolerogenic potential of donor-derived DC depletion through in vivo and ex vivo UVB preirradiation (UV) combined with the injection of anti-CD154 antibody (Ab) into recipients in an MHC-mismatched hair follicle (HF) allograft model in humanized mice. Surprisingly, human HF allografts achieved long-term survival with newly growing pigmented hair shafts in both Ab-treated groups (Ab-only and UV plus Ab) and in the UV-only group, whereas the control mice rejected all HF allografts with no hair regrowth. Perifollicular human CD3+ T cell and MHC class II+ cell infiltration was significantly diminished in the presence of UV and/or Ab treatment. HF allografts in the UV-only group showed stable maintenance of the immune privilege in the HF epithelium without evidence of antigen-specific T cell tolerance, which is likely promoted by normal HFs in vivo. This immunomodulatory strategy targeting the donor tissue exhibited novel biological relevance for clinical allogeneic transplantation without generalized immunosuppression.

Comparison of the Treatment Outcome of Oral Tofacitinib with Other Conventional Therapies in Refractory Alopecia Totalis and Universalis: A Retrospective Study.

Treatment of alopecia totalis and alopecia universalis is often challenging and unsatisfactory. Recently, Janus kinase inhibitor has shown promising results. The aim of this study is to compare the efficacy and tolerability of oral tofacitinib and conventional modalities for treating refractory alopecia totalis/universalis. A total of 74 patients (18 treated with tofacitinib, 26 treated with conventional oral treatment (steroid ± cyclosporine), and 30 treated with diphenylcyclopropenone) were included in the study. The patients' medical records were reviewed retrospectively. After 6 months, 44.4% of patients in the tofacitinib group, 37.5% in the conventional oral treatment group, and 11.1% in the diphenylcyclopropenone group achieved 50% improvements in the Severity of Alopecia Tool score. During treatment, 10% of patients in the tofacitinib group, 73.1% in the conventional oral treatment group, and 10% in the diphenylcyclopropenone group experienced adverse drug reactions. In conclusion, oral tofacitinib was more effective than diphenylcyclopropenone immunotherapy and more tolerable than conventional oral treatment after 6 months of treatment.

Quercetin Directly Targets JAK2 and PKCδ and Prevents UV-Induced Photoaging in Human Skin.

Quercetin is a naturally occurring polyphenol present in various fruits and vegetables. The bioactive properties of quercetin include anti-oxidative, anti-cancer, anti-inflammatory, and anti-diabetic effects. However, the effect of quercetin on skin aging and the direct molecular targets responsible have remained largely unknown. Herein, we investigated the protective effect of quercetin against UV-mediated skin aging and the molecular mechanisms responsible. Treatment with quercetin suppressed UV-induced matrix metalloproteinase-1 (MMP-1) and cyclooxygenase-2 (COX-2) expression and prevented UV-mediated collagen degradation in human skin tissues. Quercetin exerted potent inhibitory effects towards UV-induced activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) activity. Further examination of the upstream signaling pathways revealed that quercetin can attenuate UV-mediated phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N terminal kinases (JNK), protein kinase B (Akt), and signal transducer and activator of transcription 3 (STAT3). Kinase assays using purified protein demonstrated that quercetin can directly inhibit protein kinase C delta (PKCδ) and Janus kinase 2 (JAK2) kinase activity. Quercetin was observed to bind to PKCδ and JAK2 in pull-down assays. These findings suggest that quercetin can directly target PKCδ and JAK2 in the skin to elicit protective effects against UV-mediated skin aging and inflammation. Our results highlight the potential use of quercetin as a natural agent for anti-skin aging applications.

Pmp22 mutant allele-specific siRNA alleviates demyelinating neuropathic phenotype in vivo.

Charcot-Marie-Tooth disease (CMT) is a genetic disorder that can be caused by aberrations in >80 genes. CMT has heterogeneous modes of inheritance, including autosomal dominant, autosomal recessive, X-linked dominant, and X-linked recessive. Over 95% of cases are dominantly inherited. In this study, we investigated whether regulation of a mutant allele by an allele-specific small interfering RNA (siRNA) can alleviate the demyelinating neuropathic phenotype of CMT. We designed 19 different allele-specific siRNAs for Trembler J (Tr-J) mice harboring a naturally occurring mutation (Leu16Pro) in Pmp22. Using a luciferase assay, we identified an siRNA that specifically and selectively reduced the expression level of the mutant allele and reversed the low viability of Schwann cells caused by mutant Pmp22 over-expression in vitro. The in vivo efficacy of the allele-specific siRNA was assessed by its intraperitoneal injection to postnatal day 6 of Tr-J mice. Administration of the allele-specific siRNA to Tr-J mice significantly enhanced motor function and muscle volume, as assessed by the rotarod test and magnetic resonance imaging analysis, respectively. Increases in motor nerve conduction velocity and compound muscle action potentials were also observed in the treated mice. In addition, myelination, as evidenced by toluidine blue staining and electron microscopy, was augmented in the sciatic nerves of the mice after allele-specific siRNA treatment. After validating suppression of the Pmp22 mutant allele at the mRNA level in the Schwann cells of Tr-J mice, we observed increased expression levels of myelinating proteins such as myelin basic protein and myelin protein zero. These data indicate that selective suppression of the Pmp22 mutant allele by non-viral delivery of siRNA alleviates the demyelinating neuropathic phenotypes of CMT in vivo, implicating allele-specific siRNA treatment as a potent therapeutic strategy for dominantly inherited peripheral neuropathies.

ADSSL1 mutation relevant to autosomal recessive adolescent onset distal myopathy.

OBJECTIVE: Distal myopathy is a heterogeneous group of muscle diseases characterized by predominant distal muscle weakness. A study was done to identify the underlying cause of autosomal recessive adolescent onset distal myopathy. METHODS: Four patients from 2 unrelated Korean families were evaluated. To isolate the genetic cause, exome sequencing was performed. In vitro and in vivo assays using myoblast cells and zebrafish models were performed to examine the ADSSL1 mutation causing myopathy pathogenesis. RESULTS: Patients had an adolescent onset distal myopathy phenotype that included distal dominant weakness, facial muscle weakness, rimmed vacuoles, and mild elevation of serum creatine kinase. Exome sequencing identified completely cosegregating compound heterozygous mutations (p.D304N and p.I350fs) in ADSSL1, which encodes a muscle-specific adenylosuccinate synthase in both families. None of the controls had both mutations, and the mutation sites were located in well-conserved regions. Both the D304N and I350fs mutations in ADSSL1 led to decreased enzymatic activity. The knockdown of the Adssl1 gene significantly inhibited the proliferation of mouse myoblast cells, and the addition of human wild-type ADSSL1 reversed the reduced viability. In an adssl1 knockdown zebrafish model, muscle fibers were severely disrupted, which was evaluated by myosin expression and birefringence. In these conditions, supplementing wild-type ADSSL1 protein reversed the muscle defect. INTERPRETATION: We suggest that mutations in ADSSL1 are the novel genetic cause of the autosomal recessive adolescent onset distal myopathy. This study broadens the genetic and clinical spectrum of distal myopathy and will be useful for exact molecular diagnostics.

Clinical characterization and genetic analysis of Korean patients with X-linked Charcot-Marie-Tooth disease type 1.

Mutations in the gap junction protein beta 1 gene (GJB1) cause X-linked Charcot-Marie-Tooth disease type 1 (CMTX1). CMTX1 is representative of the intermediate type of CMT, having both demyelinating and axonal neuropathic features. We analyzed the clinical and genetic characterization of 128 patients with CMTX1 from 63 unrelated families. Genetic analysis revealed a total of 43 mutations including 6 novel mutations. Ten mutations were found from two or more unrelated families. p.V95M was most frequently observed. The frequency of CMTX1 was 9.6% of total Korean CMT family and was 14.8% when calculated within genetically identified cases. Among 67 male and 61 female patients, 22 females were asymptomatic. A high-arched foot, ataxia, and tremor were observed in 87%, 41%, and 35% of the patients, respectively. In the male patients, functional disability scale, CMT neuropathy score, and compound muscle action potential of the median/ulnar nerves were more severely affected than in the female patients. This study provides a comprehensive summary of the clinical features and spectrum of GJB1 gene mutations in Korean CMTX1 patients.