Circular RNA-encoded oncogenic PIAS1 variant blocks immunogenic ferroptosis by modulating the balance between SUMOylation and phosphorylation of STAT1.

BACKGROUND: The clinical response rate to immune checkpoint blockade (ICB) therapy in melanoma remains low, despite its widespread use. Circular non-coding RNAs (circRNAs) are known to play a crucial role in cancer progression and may be a key factor limiting the effectiveness of ICB treatment. METHODS: The circRNAs that were downregulated after coadministration compared with single administration of PD-1 inhibitor administration were identified through RNA-seq and Ribo-seq, and thus the circPIAS1 (mmu_circ_0015773 in mouse, has_circ_0008378 in human) with high protein coding potential was revealed. Fluorescence in situ hybridization (FISH) assays were conducted to determine the localization of circPIAS1 in human and mouse melanoma cells, as well as its presence in tumor and adjacent tissues of patients. Validation through dual-luciferase reporter assay and LC-MS/MS confirmed the ability of circPIAS1 to encode a novel 108 amino acid polypeptide (circPIAS1-108aa). Specific antisense oligonucleotides (ASOs) targeting the junction site of circPIAS1 were developed to reduce its intracellular levels. Proliferation changes in melanoma cells were assessed using CCK8, EdU, and colony formation assays. The impact of circPIAS1-108aa on the ferroptosis process of melanoma cells was studied through GSH, MDA, and C11-BODIPY staining assays. Western Blot, Immunoprecipitation (IP), and Immunoprecipitation-Mass Spectrometry (IP-MS) techniques were employed to investigate the impact of circPIAS1-108aa on the P-STAT1/SLC7A11/GPX4 signaling pathway, as well as its influence on the balance between STAT1 SUMOylation and phosphorylation. Additionally, a melanoma subcutaneous transplanted tumor mouse model was utilized to examine the combined effect of reducing circPIAS1 levels alongside PD-1 inhibitor. RESULTS: Compared with the group treated with PD-1 inhibitor alone, circPIAS1 was significantly down-regulated in the coadministration group and demonstrated higher protein coding potential. CircPIAS1, primarily localized in the nucleus, was notably upregulated in tumor tissues compared to adjacent tissues, where it plays a crucial role in promoting cancer cell proliferation. This circRNA can encode a unique polypeptide consisting of 108 amino acids, through which it exerts its cancer-promoting function and impedes the effectiveness of ICB therapy. Mechanistically, circPIAS1-108aa hinders STAT1 phosphorylation by recruiting SUMO E3 ligase Ranbp2 to enhance STAT1 SUMOylation, thereby reactivating the transduction of the SLC7A11/GPX4 signaling pathway and restricting the immunogenic ferroptosis induced by IFNγ. Furthermore, the combination of ASO-circPIAS1 with PD-1 inhibitor effectively inhibits melanoma growth and significantly enhances the efficacy of immune drugs in vivo. CONCLUSIONS: Our study uncovers a novel mechanism regarding immune evasion in melanoma driven by a unique 108aa peptide encoded by circPIAS1 in melanoma that dramatically hinders immunogenic ferroptosis triggered by ICB therapy via modulating the balance between SUMOylation and phosphorylation of STAT1. This work reveals circPIAS1-108aa as a critical factor limiting the immunotherapeutic effects in melanoma and propose a promising strategy for improving ICB treatment outcomes.

[Simultaneous determination of seven artemisinin-related compounds in Artemisia annua by UPLC-QQQ-MS/MS].

A variety of compounds in Artemisia annua were simultaneously determined to evaluate the quality of A. annua from multiple perspectives. A method based on ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-QQQ-MS/MS) was established for the simultaneous determination of seven compounds: amorpha-4,11-diene, artemisinic aldehyde, dihydroartemisinic acid, artemisinic acid, artemisinin B, artemisitene, and artemisinin, in A. annua. The content of the seven compounds in different tissues(roots, stems, leaves, and lateral branches) of A. annua were compared. The roots, stems, leaves, and lateral branches of four-month-old A. annua were collected and the content of seven artemisinin-related compounds in different tissues was determined. A multi-reaction monitoring(MRM) acquisition mode of UPLC-QQQ-MS/MS was used, with a positive ion mode of atmospheric pressure chemical ion source(APCI). Chromatographic separation was achieved on an Eclipse Plus RRHD C_(18) column(2.1 mm×50 mm, 1.8 µm). The gradient elution was performed with the mobile phase consisted of formic acid(0.1%)-ammonium formate(5 mmol·L~(-1))(A) and the methanol(B) gradient program of 0-8 min, 55%-100% B, 8-11 min, 100% B, and equilibrium for 3 min, the flow rate of 0.6 mL·min~(-1), the column temperature of 40 ℃, the injection volume of 5 µL, and the detection time of 8 min. Through methodological investigation, a method based on UPLC-QQQ-MS/MS was established for the simultaneous quantitative determination of seven representative compounds involved in the biosynthesis of artemisinin. The content of artemisinin in A. annua was higher than that of artemisinin B, and the content of artemisinin and dihydroartemisinic acid were high in all the tissues of A. annua. The content of the seven compounds varied considerably in different tissues, with the highest levels in the leaves and neither artemisinene nor artemisinic aldehyde was detected in the roots. In this study, a quantitative method based on UPLC-QQQ-MS/MS for the simultaneous determination of seven representative compounds involved in the biosynthesis of artemisinin was established, which was accurate, sensitive, and highly efficient, and can be used for determining the content of artemisinin-related compounds in A. annua, breeding new varieties, and controlling the quality of Chinese medicinal materials.

Regioselective Dehydrogenative Reverse Prenylation of Indoles with 2-Methyl-2-butene.

Bulk chemical 2-methyl-2-butene, one of the main C5 distillates of the petrochemical industry, has scarcely been utilized directly in synthesizing high-value-added fine chemicals. Herein, we use 2-methyl-2-butene as the starting material to develop a palladium-catalyzed highly site- and regio-selective C-3 dehydrogenation reverse prenylation of indoles. This synthetic method features mild reaction conditions, a broad substrate scope, atom- and step-economies.

Synthesis of 5,6-Dihydrophenanthridines via Palladium-Catalyzed Intramolecular Dehydrogenative Coupling of Two Aryl C-H Bonds.

5,6-Dihydrophenanthridines are common aza heterocycle frameworks of natural products and pharmaceuticals. Herein, we reported the first palladium-catalyzed intramolecular C-H/C-H dehydrogenative coupling reaction of two simple arenes to generate 5,6-dihydrophenanthridines. The approach features a broad substrate scope and good tolerance of functional groups, offering an efficient alternative synthesis route for important 5,6-dihydrophenanthridine compounds.

[Genome-wide identification of bZIP family genes and screening of candidate AarbZIPs involved in terpenoid biosynthesis in Artemisia argyi].

Artemisia argyi is an important medicinal and economic plant in China, with the effects of warming channels, dispersing cold, and relieving pain, inflammation, and allergy. The essential oil of this plant is rich in volatile terpenoids and widely used in moxi-bustion and healthcare products, with huge market potential. The bZIP transcription factors compose a large family in plants and are involved in the regulation of plant growth and development, stress response, and biosynthesis of secondary metabolites such as terpenoids. However, little is known about the bZIPs and their roles in A. argyi. In this study, the bZIP transcription factors in the genome of A. argyi were systematically identified, and their physicochemical properties, phylogenetic relationship, conserved motifs, and promoter-binding elements were analyzed. Candidate AarbZIP genes involved in terpenoid biosynthesis were screened out. The results showed that a total of 156 AarbZIP transcription factors were identified at the genomic level, with the lengths of 99-618 aa, the molecular weights of 11.7-67.8 kDa, and the theoretical isoelectric points of 4.56-10.16. According to the classification of bZIPs in Arabidopsis thaliana, the 156 AarbZIPs were classified into 12 subfamilies, and the members in the same subfamily had similar conserved motifs. The cis-acting elements of promoters showed that AarbZIP genes were possibly involved in light and hormonal pathways. Five AarbZIP genes that may be involved in the regulation of terpenoid biosynthesis were screened out by homologous alignment and phylogenetic analysis. The qRT-PCR results showed that the expression levels of the five AarbZIP genes varied significantly in different tissues of A. argyi. Specifically, AarbZIP29 and AarbZIP55 were highly expressed in the leaves and AarbZIP81, AarbZIP130, and AarbZIP150 in the flower buds. This study lays a foundation for the functional study of bZIP genes and their regulatory roles in the terpenoid biosynthesis in A. argyi.

Chronic microglial inflammation promotes neuronal lactate supply but impairs its utilization in primary rat astrocyte-neuron co-cultures.

Neuronal activity is closely associated with energy metabolism. In addition to glucose, astrocyte-derived lactate serves as an energy source for neurons. Chronic inflammation is a common pathological event that is associated with aging and neurodegenerative diseases. However, the mechanisms underlying inflammation-induced neuronal injury are not fully understood. Both microglia and astrocytes participate in the regulation of neuronal functions; therefore, we used astrocyte-neuron co-cultures to investigate the effects of chronic microglial activation on neuronal lactate metabolism. Chronic low-grade inflammation was induced by repeated stimulation of primary rat microglia with low-dose lipopolysaccharide (LPS, 10 ng/mL). The medium from the LPS-activated microglia was collected and used to mimic the inflammatory environment in primary cultures. In monocultures exposed to an inflammatory environment, intracellular lactate decreased in neurons but increased in astrocytes. However, astrocyte-neuron co-cultures exhibited increased lactate levels in neurons and decreased lactate levels in astrocytes when exposed to an inflammatory environment. Inhibition of lactate transporters expressed on neurons or astrocytes reduced the intracellular lactate in co-cultured neurons exposed to inflammation, but not in those exposed to physiological conditions. Adenosine triphosphate (ATP) production was reduced in both mono-cultured and co-cultured neurons. These results indicate that a chronic inflammatory environment increases neuronal lactate supply by promoting the astrocyte-neuron lactate shuttle, but it impairs lactate oxidation in neurons. Additionally, chronic inflammation disrupts the neuronal cytoskeleton. This study highlights the importance of glial cells in regulating neuroenergetics and neuronal function and provides a comprehensive explanation for the neurotoxic effects of neuroinflammation.

Se-(Fluoromethyl) Benzenesulfonoselenoates: Shelf-Stable, Easily Available Reagents for Monofluoromethylselenolation.

A new class of electrophilic monofluoromethylselenolation reagents, Se-(fluoromethyl) benzenesulfonoselenoates, has been developed. They can be readily prepared from sodium benzenesulfinates, Se powder and ClCFH2 in one step under mild reaction conditions. Se-(fluoromethyl) benzenesulfonoselenoates are efficient electrophilic monofluoromethylselenolation reagents for a wide range of nucleophiles including indole, 6-azaindole, pyrrole, thiophene, electron-rich arene, aryl boronic acid and alkyne. The monofluoromethylselenolation approach features mild and environmentally friendly reaction conditions, good tolerance of various functional groups, and broad substrate scope.

Synthesis of Acyl Hydrazides via a Radical Chemistry of Azocarboxylic tert-Butyl Esters.

A new chemistry of azo compounds, that is, addition of free radicals generated in situ to access various acyl hydrazides, has been developed. The protocol provides a novel strategy for the synthesis of valuable acyl hydrazides. The transformation features mild reaction conditions, good tolerance of functional groups, and a broad substrate scope. In view of the importance of acyl hydrazides in functional materials and medicinal chemistry, this approach would find broad applications.

Switchable, Reagent-Controlled C(sp3)-H Selective Iodination and Acetoxylation of 8-Methylquinolines.

An efficient Pd-catalyzed C(sp3)-H selective iodination of 8-methylquinolines is reported herein for the first time. Because of the versatility of organic iodides, the method offers a facile access to various C8-substituted quinolines. By slightly switching the reaction conditions, an efficient C(sp3)-H acetoxylation of 8-methylquinolines has also been enabled. Both approaches feature mild reaction conditions, good tolerance of functional groups, and a broad substrate scope.

[Chemical constituents from Urtica dioica fruits].

The chemical constituents in Urtica dioica fruits were investigated by silica gel chromatography, preparative HPLC, NMR, and HR-MS for the first time. As a result, 21 compounds were isolated from the fruits of U. dioica and identified 7R,8S,8'R-olivil(1), oleic acid(2), α-linoleic acid(3), palmic acid(4), methyl palmitate(5), α-linolenic acid(6), α-linolenic acid methyl ester(7), 5-O-caffeoyl-shikimic acid(8), vanillic acid(9), p-coumaric acid(10), 5-O-p-coumaroylshikimic acid(11), cinnamic acid(12), quinic acid(13), shikimic acid(14), ethyl caffeate(15), coniferyl ferulate(16), ferulic acid(17), caffeic acid(18), chlorogenic acid(19), pinoresinol(20), and quercetin(21). Compound 1 was a new compound and compounds 2-16 were isolated from U. dioica for the first time.

[Establishment and verification of quality evaluation method of Jizhi Syrup based on quantitative analysis of multi-components by single marker].

A quantitative analysis of multi-components by single marker(QAMS) method was established for the simultaneous determination of ephedrine hydrochloride, protocatechuic acid, 5-caffeoylquinic acid, 4-hydroxybenzoic acid, naringin, neohesperidin, glycyrrhizic acid, and praeruptorin A in Jizhi Syrup by high performance liquid chromatography(HPLC) with ultraviolet multi-wavelength detection system, and its feasibility in quality evaluation of Jizhi syrup was verified. With naringin as the internal reference substance, the relative correction factors and chromatographic peak localization methods of other seven components were respectively established at 210, 254, 280, and 320 nm. The method reproducibility was validated, and the result of QAMS were compared with those obtained by the external standard method(ESM) to verify the accuracy and feasibility of the method. The relative correction factors of ephedrine hydrochloride, protocatechuic acid, 5-caffeoylquinic acid, 4-hydroxybenzoic acid, neohesperidin, glycyrrhizic acid, and praeruptorin A with naringin as reference were 0.846, 0.582, 0.608, 0.293, 0.913, 2.207, and 0.940, respectively, which presented excellent reproducibility under different experimental conditions. Furthermore, QAMS and ESM showed no significant difference in the results for 15 batches of samples. Except protocatechuic acid and 5-caffeoylquinic acid, other six compounds were the exclusive components of single medicinal materials. In addition, glycyrrhizic acid and praeruptorin A were identified in the Jizhi Syrup for the first time, filling up the blank of no component detected in Glycyrrhizae Radix et Rhizoma and Peucedani Radix. The method established in this study is convenient, efficient, specific, accurate, and reliable, which can comprehensively and effectively evaluate the quality of Jizhi Syrup to ensure the safety and efficacy of this drug in clinical application.

[Active constituents of Urtica fissa in inhibition of benign prostatic hyperplasia].

The present study investigated the material basis of Urtica fissa for the inhibition of benign prostatic hyperplasia(BPH). The active fractions were screened, and the extracts of dichloromethane and ethyl acetate exhibited significantly inhibitory activities against 5α-reductase in vitro and BPH in model rats. The chemical constituents in the active fractions were systematically investigated, and 28 compounds were obtained, which were identified as lobechine methyl ester(1), dibutyl-O-phthalate(2), 1-monolinolein(3), epipinoresinol(4), 5-hydroxy-3,4-dimethyl-5-pentanyl-2(5H)-furanone(5), E-7,9-diene-11-methenyl palmitic acid(6), evofolin B(7), ficusal(8), threo-2,3-bis-(4-hydroxy-3-methoxyphenyl)-3-ethoxypropan-1-ol(9), α-viniferin(10),(9R,7E)-9-hydroxy-5,7-mengatigmadien-4-one-9-O-ß-D-glucopyranoside(11), indole-3-carboxaldehyde(12), p-hydroxy ethyl cinnamate(13), benzyl alcohol-O-ß-D-glucoside(14), L-methionine(15), 4-methoxyaniline(16), 6-aminopurine(17), 8'-acetyl oilvil(18), 4-methoxyl-8'-acetyl oilvil(19), vanillic acid(20), ß-hydroxypropiovanillone(21), 7-hydroxy-6-methoxycoumarin(22), p-hydroxybenzaldehyde(23), pinoresinol(24), erythro-1,2-bis-(4-hydroxy-3-methoxyphenyl)-1,3-propanediol(25), urticol(26), urticol-7-O-ß-D-glucopyranoside(27), and lobechine(28). Compounds 1-17 were isolated from U. fissa for the first time. Meanwhile, compound 1 was a new natural product. Compounds 10, 11, 19, 21, and 27 exhibited significant inhibitory effects on 5α-reductase.

[Construction strategies and prospect of the Global Medicinal Plant Stem Cell Bank].

Medicinal plant stem cells are separated from the meristem and vascular cambium of medicinal plants, which can produce active components for preventing and treating diseases and improving body physical functions under certain conditions. Medicinal plant stem cells come from a broad category of medicinal plants, including ethnic medicinal plants, folk medicinal plants, original plants of health products, vegetables, fruits, and other potential medicinal plants. At present, the techniques for the isolation, identification, preservation and culture of medicinal plant stem cells have become increasingly mature, and the mechanism of stem cell differentiation, growth and regulation of secondary metabolites has been studied in depth. Medicinal plant stem cells have a broad application prospect in medicine, health food, food and medical beauty products. As a strategic resource, the construction of the "Global Medicinal Plant Stem Cell Bank" was first proposed to preserve various kinds of medicinal plant resources in the world, and it will go global relying on the internationalization strategy of traditional Chinese medicine. The bank should follow safety, environmental protection, advanced and practical design principles. The main construction contents include the original plant bank, stem cell bank, component resource bank, gene bank, database and resource sharing system, with genetic and data resources incorporated into the scope of protection and utilization. The bank will establish a new strategy for medicinal plant resources protection and regeneration, and provide a new resource for natural products drug discovery and a technology sharing platform for various medicinal plant stem cells. As a resource treasury, a source of innovative technologies and a center of cooperation, it will become the core driving force of the global medicinal plant stem cell industry.

[Screening of reference genes for quantitative real-time PCR in Artemisia argyi].

Artemisia Argyi Folium, a traditional Chinese medicine of important medicinal and economic value, sees increasing demand in medicinal and moxibustion product market. Screening stable and reliable reference genes for quantitative real-time PCR(qRT-PCR) is a prerequisite for the analysis of gene expression in Artemisia argyi. In this study, eight commonly used reference genes, Actin, 18s, EF-1α, GAPDH, SAND, PAL, TUA, and TUB, from the transcriptome of A. argyi, were selected as candidate genes. The expression of each gene in different tissues(roots, stems, and leaves) of A. argyi and in leaves of A. argyi after treatment with methyl jasmonate(MeJA) for different time(0, 4, 8, 12 h) was detected by qRT-PCR. Then, geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder were employed to evaluate their expression stability. The results demonstrated that Actin was the most stable reference gene in different tissues and in leaves treated with MeJA, and coming in the second was SAND. Furthermore, the expression of DXS and MCT which are involved in terpenoid backbone biosynthesis was detected in different tissues and after MeJA treatment. The results showed that the expression patterns of DXS and MCT in different tissues and under MeJA treatment calculated with Actin and SAND as internal reference genes were consistent, which validated the screening results. In conclusion, Actin is the most suitable reference gene for the analysis of gene expression in different tissues of A. argyi and after MeJA treatment. This study provides valuable information for gene expression analysis in A. argyi and lays a foundation for further research on molecular mechanism of quality formation of Artemisia Argyi Folium.

[Lignans with inhibitory effect on 5α-reductase from Urtica cannabina].

The lignans in Urtica cannabina were isolated by preparative HPLC, silica, and ODS column chromatographies, and identified by NMR and HR-MS. The inhibitory activities on 5α-reductase were evaluated in vitro. As a result, ten secolignans,(2R,4S)-2,4-bis(3-methoxyl-4-hydroxyphenyl)-3-butoxypropanol(1), 3,4-trans-3-hydroxymethyl-4-[bis(3,4-dimethoxyphenyl)methyl] butyrolactone(2), 3,4-trans-3-hydroxymethyl-4-[(3,4-dimethoxyphenyl)(3-methoxyl-4-hydroxyphenyl)methyl] butyrolactone(3), 3,4-trans-3-hydroxymethyl-4-[bis(3-methoxyl-4-hydroxyphenyl)methyl] butyrolactone(trans urticol, 4), 3,4-trans-3-hydroxymethyl-4-[bis(3,4-dimethoxyphenyl)methyl] butyrolactone-3-O-ß-D-glucopyranoside(5), 3,4-trans-3-hydroxymethyl-4-[(3,4-dimethoxyphenyl)(3-methoxyl-4-hydroxyphenyl)methyl]butyrolactone-3-O-ß-D-glucopyranoside(6), 3,4-trans-3-hydroxymethyl-4-[bis(3-methoxyl-4-hydroxyphenyl)methyl]butyrolactone-3-O-ß-D-glucopyranoside(trans-urticol-7-O-ß-D-glucopyranoside, 7), cycloolivil-4-O-ß-D-glucopyranoside(8), isolariciresinol-4'-O-ß-D-glucopyranoside(9), and olivil-4'-O-ß-D-glucopyranoside(10), together with a polyphenol [α-viniferin(11)], were isolated from U. cannabina for the first time. Compound 1 was a new lignan. Compound 7 was potent in inhibiting 5α-reductase.

Synthesis of trifluoromethylthioesters from aldehydes via a visible light-promoted radical process.

We report herein an efficient, economical, and scalable trifluoromethylthiolation of aldehydes to generate trifluoromethylthioesters via a visible light-promoted radical process. The transformation features cheap reagents, simple operation, a broad substrate scope, and especially no metal involved in the reaction. Trifluoromethylthiolations of several complex aldehyde-containing bioactive compounds have been realized; thus the approach has the potential to be an important tool for the late-stage functionalization of advanced synthetic intermediates and bioactive molecules, and should have many applications in medicinal chemistry.

New lignans from Urticae Fissae Herba.

Two new lignans named neourticol A (1) and neourticol B (2), together with seven known compounds (3-9), were isolated from Urticae Fissae Herba, a folk medicine for rheumatism arthritis in China. The active evaluation results showed that 1 and 2 possessed the potent anti-complement and anti-inflammatory activities.

The chemical constituents with cytotoxicity from Urtica fissa seed.

Phytochemical investigation of Urtica fissa seed led to the isolation of a new secolignan (1) and a new glycoalkaloid (2), together with 16 known compounds (3-18). The subsequent active evaluation indicated that two lignans (1, 12), two ceramides (3, 4), and the glycoalkaloid (2) possessed the significant cytotoxicity. They could obviously inhibit the proliferation of tumor cells HeLa and CCRF-CEM cells, with IC50 values as low as 1.5 µM.

The bioassay-guided isolation of antifungal saponins from Hosta plantaginea leaves.

Four new steroidal saponins hostaside Ⅰ (1), hostaside Ⅱ (2), hostaside Ⅲ (3), and hostaside Ⅳ (4), together with five known steroidal saponins (5-9), were isolated by the bioassay-guided fractionation from the leaves of Hosta plantaginea (Lam.) Aschers, a worldwide well-known ornamental plant. Hostasides Ⅰ and Ⅱ showed significant antifungal activities, and they could inhibit the growth of Candida albicans and Fusarium oxysporium with MIC values as low as 4 µg/ml.

The chemical constituents from Urtica fissa leaves.

A new ceramide urticamide (1), two new secolignans urticalactones I (2) and Ⅱ (3), and a new flavonoid glycoside urticaside (4), together with 15 known compounds (4-19), were isolated from the leaves of Urtica fissa, a folk medicine for rheumatism arthritis in China. The active evaluation results showed that 1, 2, 3, 8, and 13 possessed the potent anti-inflammatory. They could inhibit the release of NO and TNF-α in lipopolysaccharide (LPS) stimulated RAW 264.7 cells, with IC50 values less than 4.0 µM.