NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

Homeostatic and early-recruited CD101- eosinophils suppress endotoxin-induced acute lung injury.

INTRODUCTION: Acute lung injury (ALI) is a fatal but undertreated condition with severe neutrophilic inflammation, although little is known about the functions of eosinophils in the pathogenesis of ALI. Our objectives were to investigate the roles and molecular mechanisms of eosinophils in ALI. METHODS: Pulmonary eosinophils were identified by flow cytometry. Mice with abundant or deficient eosinophils were used. Cellularity of eosinophils and neutrophils in bronchoalveolar lavage fluid, inflammatory assessment, and survival rate were determined. Human samples were also used for validating experimental results. RESULTS: Blood eosinophils were increased in surviving patients with acute respiratory distress syndrome (ARDS) independent of corticosteroid usage. There existed homeostatic eosinophils in lung parenchyma in mice and these homeostatic eosinophils, originating from the bone marrow, were predominantly CD101-. More CD101- eosinophils could be recruited earlier than lipopolysaccharide (LPS)-initiated neutrophilic inflammation. Loss of eosinophils augmented LPS-induced pulmonary injury. Homeostatic CD101- eosinophils ameliorated, while allergic CD101+ eosinophils exacerbated, the neutrophilic inflammation induced by LPS. Likewise, CD101 expression in eosinophils from ARDS patients did not differ from healthy subjects. Mechanistically, CD101- eosinophils exhibited higher levels of Alox15 and Protectin D1. Administration of Protectin D1 isomer attenuated the neutrophilic inflammation. CONCLUSIONS: Collectively, our findings identify an uncovered function of native CD101- eosinophils in suppressing neutrophilic lung inflammation and suggest a potential therapeutic target for ALI.

Uncovering fungal community composition in natural habitat of Ophiocordyceps sinensis using high-throughput sequencing and culture-dependent approaches.

BACKGROUND: The fungal communities inhabiting natural Ophiocordyceps sinensis play critical ecological roles in alpine meadow ecosystem, contribute to infect host insect, influence the occurrence of O. sinensis, and are repertoire of potential novel metabolites discovery. However, a comprehensive understanding of fungal communities of O. sinensis remain elusive. Therefore, the present study aimed to unravel fungal communities of natural O. sinensis using combination of high-throughput sequencing and culture-dependent approaches. RESULTS: A total of 280,519 high-quality sequences, belonging to 5 fungal phyla, 15 classes, 41 orders, 79 families, 112 genera, and 352 putative operational taxonomic units (OTUs) were obtained from natural O. sinensis using high-throughput sequencing. Among of which, 43 genera were identified in external mycelial cortices, Ophiocordyceps, Sebacinia and Archaeorhizomyces were predominant genera with the abundance of 95.86, 1.14, 0.85%, respectively. A total of 66 genera were identified from soil microhabitat, Inocybe, Archaeorhizomyces, unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, unclassified Ascomycota and unclassified fungi were predominant genera with an average abundance of 53.32, 8.69, 8.12, 8.12, 7.21, 4.6, 3.08 and 3.05%, respectively. The fungal communities in external mycelial cortices were significantly distinct from soil microhabitat. Meanwhile, seven types of culture media were used to isolate culturable fungi at 16 °C, resulted in 77 fungal strains identified by rDNA ITS sequence analysis, belonging to 33 genera, including Ophiocordyceps, Trichoderma, Cytospora, Truncatella, Dactylonectria, Isaria, Cephalosporium, Fusarium, Cosmospora and Paecilomyces, etc.. Among all culturable fungi, Mortierella and Trichoderma were predominant genera. CONCLUSIONS: The significantly differences and overlap in fungal community structure between two approaches highlight that the integration of high-throughput sequencing and culture-dependent approaches would generate more information. Our result reveal a comprehensive understanding of fungal community structure of natural O. sinensis, provide new insight into O. sinensis associated fungi, and support that microbiota of natural O. sinensis is an untapped source for novel bioactive metabolites discovery.

A predictive model for diagnosis of lower extremity cellulitis: A cross-sectional study.

BACKGROUND: Cellulitis has many clinical mimickers (pseudocellulitis), which leads to frequent misdiagnosis. OBJECTIVE: To create a model for predicting the likelihood of lower extremity cellulitis. METHODS: A cross-sectional review was performed of all patients admitted with a diagnosis of lower extremity cellulitis through the emergency department at a large hospital between 2010 and 2012. Patients discharged with diagnosis of cellulitis were categorized as having cellulitis, while those given an alternative diagnosis were considered to have pseudocellulitis. Bivariate associations between predictor variables and final diagnosis were assessed to develop a 4-variable model. RESULTS: In total, 79 (30.5%) of 259 patients were misdiagnosed with lower extremity cellulitis. Of the variables associated with true cellulitis, the 4 in the final model were asymmetry (unilateral involvement), leukocytosis (white blood cell count ≥10,000/uL), tachycardia (heart rate ≥90 bpm), and age ≥70 years. We converted these variables into a points system to create the ALT-70 cellulitis score as follows: Asymmetry (3 points), Leukocytosis (1 point), Tachycardia (1 point), and age ≥70 (2 points). With this score, 0-2 points indicate ≥83.3% likelihood of pseudocellulitis, and ≥5 points indicate ≥82.2% likelihood of true cellulitis. LIMITATIONS: Prospective validation of this model is needed before widespread clinical use. CONCLUSION: Asymmetry, leukocytosis, tachycardia, and age ≥70 are predictive of lower extremity cellulitis. This model might facilitate more accurate diagnosis and improve patient care.

Effect of CYP2D6 variants on venlafaxine metabolism in vitro.

1. CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme superfamily, we recently identified 22 CYP2D6 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of venlafaxine in vitro. 2. The wild-type and 24 CYP2D6 variants were expressed in insect cells, and each variant was characterized using venlafaxine as the substrate. Reactions were performed at 37 °C with 5-500 µM substrate (three variants was adjusted to 1000 µM) for 50 min. By using high-performance liquid chromatography to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of O-desmethylvenlafaxine were determined. 3. Among the 22 CYP2D6 variants, the intrinsic clearance (Vmax/Km) values of all variants were significantly decreased (from 0.2% to 84.5%) compared with wild-type CYP2D6*1. In addition, the kinetic parameters of two CYP2D6 variants could not be detected because they have no detectable enzyme activity. 4. The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards venlafaxine in vivo.

[Damage and changes in expression of autophagy related factors LC3 and p62 of condylar cartilage in fluorosis mouse].

PURPOSE: To study the damage and the expression of LC3 and p62 of condylar cartilage in fluorosis mouse. METHODS: Thirty 4-week-old male C57BL/6 mice were randomly divided into control group and the experimental group with 15 animals in each group. The control group received regular drinking water and the experimental group received a fluoride concentration of 75 mg/L drinking water for 8 weeks. The structure of condylar cartilage was observed through modified safranine O-fast green FCF cartilage stain kit. Immunohistochemistry was used to detect the expression of MMP-13, type Ⅱ collagen and LC3 and p62. Two-way analysis of variance test was conducted for analysis of semi-quantitative results of immunohistochemistry using SPSS 22.0 software package. RESULTS: Compared with the control group, the fibrocartilage layer of the experimental group became thinner, the condrocytes were smaller, and the staining became deeper.Immunohistochemistry results showed that the expression of MMP-13 and LC3 increased; the expression of type Ⅱ collagen and p62 decreased in the experimental group. CONCLUSIONS: There was degeneration of the condylar cartilage and autophagy in mice with drinking water containing 75 mg/L fluoride.

Partial Alleviation of Homologous Superinfection Exclusion of SeMNPV Latently Infected Cells by G1 Phase Infection and G2/M Phase Arrest.

Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p < 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation.

Erratum: Author correction to "The FAPα-activated prodrug Z-GP-DAVLBH inhibits the growth and pulmonary metastasis of osteosarcoma cells by suppressing the AXL pathway" [Acta Pharm Sin B 12 (2022) 1288-1304].

[This corrects the article DOI: 10.1016/j.apsb.2021.08.015.].

Rapamycin Exacerbates Staphylococcus aureus Pneumonia by Inhibiting mTOR-RPS6 in Macrophages.

Purpose: This study aimed to explore the effect of Rapamycin (Rapa) in Staphylococcus aureus (S. aureus) pneumonia and clarify its possible mechanism. Methods: We investigated the effects of Rapa on S. aureus pneumonia in mouse models and in macrophages cultured in vitro. Two possible mechanisms were investigated: the mTOR-RPS6 pathway phosphorylation and phagocytosis. Furthermore, for the mechanism verification in vivo, mice with specific Mtor knockout in myeloid cells were constructed for pneumonia models. Results: Rapa exacerbated S. aureus pneumonia in mouse models, promoting chemokines secretion and inflammatory cells infiltration in lung. In vitro, Rapa upregulated the secretion of chemokines and cytokines in macrophages induced by S. aureus. Mechanistically, the mTOR-ribosomal protein S6 (RPS6) pathway in macrophages was phosphorylated in response to S. aureus infection, and the inhibition of RPS6 phosphorylation upregulated the inflammation level. However, Rapa did not increase the phagocytic activity. Accordingly, mice with specific Mtor knockout in myeloid cells experienced more severe S. aureus pneumonia. Conclusion: Rapa exacerbates S. aureus pneumonia by increasing the inflammatory levels of macrophages. Inhibition of mTOR-RPS6 pathway upregulates the expression of cytokines and chemokines in macrophages, thus increases inflammatory cells infiltration and exacerbates tissue damage.

Delayed diagnosis of arytenoid cartilage dislocation after tracheal intubation in the intensive care unit: A case report.

BACKGROUND: Arytenoid cartilage dislocation is a rare and often overlooked complication of tracheal intubation or blunt laryngeal trauma. The most common symptom is persistent hoarseness. Although cases of arytenoid dislocation due to tracheal intubation are reported more frequently in otolaryngology, reports on its occurrence in the intensive care unit (ICU) are lacking. We report a case of delayed diagnosis of arytenoid cartilage dislocation after tracheal intubation in the ICU. CASE SUMMARY: A 20-year-old woman was referred to the ICU following a fall from a height. Her voice was normal; laryngeal computed tomography showed unremarkable findings on admission. However, due to deterioration of the patient's condition, tracheal intubation, and emergency exploratory laparotomy followed by laparoscopic surgery two d later under general anesthesia were performed. After extubation, the patient was sedated and could not communicate effectively. On the 10th day after extubation, the patient complained of hoarseness and coughing with liquids, which was attributed to laryngeal edema and is common after tracheal intubation. Therefore, specific treatment was not administered. However, the patient's symptoms did not improve. Five d later, an electronic laryngoscope examination revealed dislocation of the left arytenoid cartilage. The patient underwent arytenoid closed reduction under general anesthesia by an experienced otolaryngologist. Reported symptoms improved subsequently. The six-month follow up revealed that the hoarseness had resolved within four weeks of the reduction procedure. CONCLUSION: Symptoms of arytenoid cartilage dislocation are difficult to identify in the ICU leading to missed or delayed diagnosis among patients.

A study on UHPLC-MS/MS analyses of DNA and RNA oxidative damage metabolites in patients with cervical carcinoma: 8-oxoG in urine as a potential biomarker of cervical carcinoma.

Objective: The purpose of this study was to compare the levels of 8-oxoG and 8-oxodG in urine of patients with cervical carcinoma and healthy women to evaluate their influences on cervical carcinoma. Methods: In this study, urine samples were collected from 70 patients with cervical carcinoma, 24 patients with one-year follow-up, and 100 healthy women. The contents of 8-oxodG and 8-oxoG in urine were assayed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Results: The levels of 8-oxoG and 8-oxodG were higher in patients with cervical carcinoma (P < 0.000), while AUC of 8-oxoG and 8-oxodG was higher than 0.7. Specifically, the high-risk HPV (HR-HPV) positive group had higher 8-oxoG levels (P < 0.000), but there was no difference in 8-oxodG levels. Yet, 8-oxoG level was associated with lymphatic metastasis, lymph-vascular space infiltration (LVSI) and stromal infiltration, while 8-oxodG level was affected by the differentiation degree and stromal infiltration. According to statistics, the distinct cut-off index of lymphatic metastasis was 7.282 nmol/mmol creatinine. After operation, the concentrations of 8-oxoG and 8-oxodG dropped significantly (8-oxoG P < 0.000, 8-oxodG P = 0.004). Except for chemotherapy group, the urinary 8-oxoG dose of all treatment groups and 8-oxodG dose of chemo-radiotherapy group declined obviously. Conclusions: 8-oxoG may be a potential biomarker for cervical carcinoma.

Conformal PEDOT Coating Enables Ultra-High-Voltage and High-Temperature Operation for Single-Crystal Ni-Rich Cathodes.

Single-crystal Ni-rich Li[NixMnyCo1-x-y]O2 (SC-NMC) cathodes represent a promising approach to mitigate the cracking issue of conventional polycrystalline cathodes. However, many reported SC-NMC cathodes still suffer from unsatisfactory cycling stability, particularly under high charge cutoff voltage and/or elevated temperature. Herein, we report an ultraconformal and durable poly(3,4-ethylenedioxythiophene) (PEDOT) coating for SC-NMC cathodes using an oxidative chemical vapor deposition (oCVD) technique, which significantly improves their high-voltage (4.6 V) and high-temperature operation resiliency. The PEDOT coated SC LiNi0.83Mn0.1Co0.07O2 (SC-NMC83) delivers an impressive capacity retention rate of 96.7% and 89.5% after 100 and 200 cycles, respectively. Significantly, even after calendar aging at 45 °C and 4.6 V, the coated cathode can still retain 85.3% (in comparison with 59.6% for the bare one) of the initial capacity after 100 cycles at a 0.5 C rate. Synchrotron X-ray experiments and interface characterization collectively reveal that the conformal PEDOT coating not only effectively stabilizes the crystallographic structure and maintains the integrity of the particles but also significantly suppresses the electrolyte's corrosion, resulting in improved electrochemical/thermal stability. Our findings highlight the promise of an oCVD PEDOT coating for single-crystal Ni-rich cathodes to meet the grand challenge of high-energy batteries under extreme conditions.

Association between circadian rhythm disruption and polycystic ovary syndrome.

OBJECTIVE: To explore the association of circadian rhythm disruption with polycystic ovary syndrome (PCOS) and the potential underlying mechanism in ovarian granulosa cells (GCs). DESIGN: Multicenter questionnaire-based survey, in vivo and ex vivo studies. SETTING: Twelve hospitals in China, animal research center, and research laboratory of a women's hospital. PATIENTS/ANIMALS: A total of 436 PCOS case subjects and 715 control subjects were recruited for the survey. In vivo and ex vivo studies were conducted in PCOS-model rats and on ovarian GCs collected from women with PCOS and control subjects. INTERVENTION(S): The PCOS rat model was established with the use of testosterone propionate. MAIN OUTCOME MEASURE(S): Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), RNA sequencing, rhythmicity analysis, functional enrichment analysis. RESULT(S): There was a significant correlation between night shift work and PCOS. PCOS-model rats presented distinct differences in the circadian variation of corticotropin-releasing hormone, adrenocorticotropic hormone, prolactin, and a 4-h phase delay in thyrotropic hormone levels. The motif enrichment analysis of ATAC-seq revealed the absence of clock-related transcription factors in specific peaks of PCOS group, and RNA sequencing ex vivo at various time points over 24 hours demonstrated the differential rhythmic expression patterns of women with PCOS. Kyoto Encyclopedia of Genes and Genomes analysis further highlighted metabolic dysfunction, including both carbohydrate and amino acid metabolism and the tricarboxylic acid cycle. CONCLUSION(S): There is a significant association of night shift work with PCOS, and genome-wide chronodisruption exists in ovarian GCs.

Epitope spreading toward wild-type melanocyte-lineage antigens rescues suboptimal immune checkpoint blockade responses.

Although immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1), can deliver durable antitumor effects, most patients with cancer fail to respond. Recent studies suggest that ICI efficacy correlates with a higher load of tumor-specific neoantigens and development of vitiligo in patients with melanoma. Here, we report that patients with low melanoma neoantigen burdens who responded to ICI had tumors with higher expression of pigmentation-related genes. Moreover, expansion of peripheral blood CD8+ T cell populations specific for melanocyte antigens was observed only in patients who responded to anti-PD-1 therapy, suggesting that ICI can promote breakdown of tolerance toward tumor-lineage self-antigens. In a mouse model of poorly immunogenic melanomas, spreading of epitope recognition toward wild-type melanocyte antigens was associated with markedly improved anti-PD-1 efficacy in two independent approaches: introduction of neoantigens by ultraviolet (UV) B radiation mutagenesis or the therapeutic combination of ablative fractional photothermolysis plus imiquimod. Complete responses against UV mutation-bearing tumors after anti-PD-1 resulted in protection from subsequent engraftment of melanomas lacking any shared neoantigens, as well as pancreatic adenocarcinomas forcibly overexpressing melanocyte-lineage antigens. Our data demonstrate that somatic mutations are sufficient to provoke strong antitumor responses after checkpoint blockade, but long-term responses are not restricted to these putative neoantigens. Epitope spreading toward T cell recognition of wild-type tumor-lineage self-antigens represents a common pathway for successful response to ICI, which can be evoked in neoantigen-deficient tumors by combination therapy with ablative fractional photothermolysis and imiquimod.

Topical treatment strategies to manipulate human skin pigmentation.

Skin pigmentation is a result of melanin produced by melanocytes in the epidermis. Melanocyte activity, along with the type and distribution of melanins, is the main driver for diversity of skin pigmentation. Dark melanin acts to protect against the deleterious effects of ultraviolet (UV) radiation, including photo-aging and skin cancer formation. In turn, UV radiation activates skin melanocytes to induce further pigmentation (i.e., "tanning pathway"). The well-characterized MSH/MC1R-cAMP-MITF pathway regulates UV-induced melanization. Pharmacologic activation of this pathway ("sunless tanning") represents a potential strategy for skin cancer prevention, particularly in those with light skin or the "red hair" phenotype who tan poorly after UV exposure due to MC1R inactivating polymorphisms. Skin hyperpigmentation can also occur as a result of inflammatory processes and dermatological disorders such as melasma. While primarily of cosmetic concern, these conditions can dramatically impact quality of life of affected patients. Several topical agents are utilized to treat skin pigmentation disorders. Here, we review melanogenesis induced by UV exposure and the agents that target this pathway.

AT-rich interactive domain1A determines sensitivity to oxaliplatin in gastric cancer cells.

BACKGROUND: Gastric cancer is a highly heterogeneous disease and its traditional histopathological classification is difficult to meet clinical needs. Oxaliplatin is an antitumor drug with high efficiency and low toxicity. Therefore, the insensitivity or secondary drug resistance of oxaliplatin to gastric cancer is vital for tumor progression. The aim of this study was to investigate the sensitivity of gastric cancer cells to oxaliplatin after ARID1A (AT-rich interactive domain1A gene) gene silencing. METHODS: MGC-803 and AGS cells were selected as gastric cancer cells for study. ARID1A protein and mRNA expression was detected by Western blot and quantitative reverse-transcription PCR (qRT-PCR). The short hairpin RNA (shRNA) fragment of ARID1A gene silencing was constructed and introduced into gastric cancer cells. The cell proliferation activity was calculated using CCK8 and the IC50 was calculated. The flow cytometry was used to detect the cell cycle and apoptosis rate. The ability of cell invasion was detected by transwell method. Cells were treated with different concentrations of oxaliplatin. RESULTS: The proliferation of gastric cancer cells was promoted by ARID1A gene silencing (P<0.01), the quantity of cells in S phase increased (P<0.05), and the invasive ability increased (P<0.05). After treatment with oxaliplatin at different concentrations, ARID1A gene silencing reduced the inhibition rate of oxaliplatin on gastric cancer cells and apoptosis rate (P<0.05), and increased IC 50 (P<0.01). CONCLUSIONS: ARID1A gene silencing, a factor promoting proliferation of gastric cancer cells, would reduce the sensitivity of gastric cancer cells to oxaliplatin, which can provide a basis for the exploration of targeted drugs for individualized treatment of gastric cancer.

Hdac3 is an epigenetic inhibitor of the cytotoxicity program in CD8 T cells.

Cytotoxic T cells play a key role in adaptive immunity by killing infected or cancerous cells. While the transcriptional control of CD8 T cell differentiation and effector function following T cell activation has been extensively studied, little is known about epigenetic regulation of these processes. Here we show that the histone deacetylase HDAC3 inhibits CD8 T cell cytotoxicity early during activation and is required for persistence of activated CD8 T cells following resolution of an acute infection. Mechanistically, HDAC3 inhibits gene programs associated with cytotoxicity and effector differentiation of CD8 T cells including genes encoding essential cytotoxicity proteins and key transcription factors. These data identify HDAC3 as an epigenetic regulator of the CD8 T cell cytotoxicity program.

Gain-of-Function Genetic Alterations of G9a Drive Oncogenesis.

Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/ß-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations. SIGNIFICANCE: Oncogenic G9a abnormalities drive tumorigenesis and the "cold" immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore "hot" tumor immune microenvironments.This article is highlighted in the In This Issue feature, p. 890.

The Salvia miltiorrhiza NAC transcription factor SmNAC1 enhances zinc content in transgenic Arabidopsis.

NAC transcription factors play important roles in plant biological processes, including plant development, environmental stress responses and element enrichment. A novel NAC transcription factor gene, designated SmNAC1, was isolated from Salvia miltiorrhiza. SmNAC1 was localized in the nucleus in onion protoplasts and exhibited transcriptional activation activities in yeast. In addition, the SmNAC1 protein could specifically bind to the cis-elements of the NAC proteins. SmNAC1 was expressed at a higher level in the leaves of S. miltiorrhiza, indicating that SmNAC1 might be involved in the transportation of zinc. To examine the function of SmNAC1, transgenic Arabidopsis plants overexpressing SmNAC1 were generated. Zinc content assays in the transgenic plants demonstrated that overexpressed SmNAC1 plants had enhanced tolerance to high zinc concentrations, and zinc was enriched in the shoot tissues. Our results demonstrate that SmNAC1 plays important roles in the response to zinc stress. Zinc was mainly enriched in the leaves of S. miltiorrhiza and the shoot tissues of transgenic Arabidopsis plants. SmNAC1 might participate in zinc transportation from the roots to the shoots, that constitutes a useful gene for improving zinc content in plants.