Lactate modulates RNA splicing to promote CTLA-4 expression in tumor-infiltrating regulatory T cells.

RNA splicing is involved in cancer initiation and progression, but how it influences host antitumor immunity in the metabolically abnormal tumor microenvironment (TME) remains unclear. Here, we demonstrate that lactate modulates Foxp3-dependent RNA splicing to maintain the phenotypic and functional status of tumor-infiltrating regulatory T (Treg) cells via CTLA-4. RNA splicing in Treg cells was correlated with the Treg cell signatures in the TME. Ubiquitin-specific peptidase 39 (USP39), a component of the RNA splicing machinery, maintained RNA-splicing-mediated CTLA-4 expression to control Treg cell function. Mechanistically, lactate promoted USP39-mediated RNA splicing to facilitate CTLA-4 expression in a Foxp3-dependent manner. Moreover, the efficiency of CTLA-4 RNA splicing was increased in tumor-infiltrating Treg cells from patients with colorectal cancer. These findings highlight the immunological relevance of RNA splicing in Treg cells and provide important insights into the environmental mechanism governing CTLA-4 expression in Treg cells.

USP47 inhibits m6A-dependent c-Myc translation to maintain regulatory T cell metabolic and functional homeostasis.

The functional integrity of Tregs is interwoven with cellular metabolism; however, the mechanisms governing Treg metabolic programs remain elusive. Here, we identified that the deubiquitinase USP47 inhibited c-Myc translation mediated by the RNA N6-methyladenosine (m6A) reader YTHDF1 to maintain Treg metabolic and functional homeostasis. USP47 positively correlated with the tumor-infiltrating Treg signature in samples from patients with colorectal cancer and gastric cancer. USP47 ablation compromised Treg homeostasis and function in vivo, resulting in the development of inflammatory disorders, and boosted antitumor immune responses. USP47 deficiency in Tregs triggered the accumulation of the c-Myc protein and in turn exacerbated hyperglycolysis. Mechanistically, USP47 prevented YTHDF1 ubiquitination to attenuate the association of YTHDF1 with translation initiation machinery, thereby decreasing m6A-based c-Myc translation efficiency. Our findings reveal that USP47 directs m6A-dependent metabolic programs to orchestrate Treg homeostasis and suggest novel approaches for selective immune modulation in cancer and autoimmune diseases by targeting of USP47.

TRAF3/STAT6 axis regulates macrophage polarization and tumor progression.

Converting tumor-associated macrophages (TAMs) from the M2 to the M1 phenotype is considered an effective strategy for cancer therapy. TRAF3 is known to regulate NF-κB signaling. However, the role of TRAF3 in TAM polarization has not yet been completely elucidated. Here, we found that ablation of TRAF3 increased M1 markers, iNOS, FGR and SLC4A7, while down-regulated M2 markers, CD206, CD36 and ABCC3, expression levels in macrophages. Moreover, TRAF3 deficiency enhanced LPS-induced M1 and abolished IL-4-induced macrophage polarization. Next, quantitative ubiquitomics assays demonstrated that among the quantitative 7618 ubiquitination modification sites on 2598 proteins, ubiquitination modification of IL-4 responding proteins was the most prominently reduced according to enrichment analysis. STAT6, a key factor of IL-4 responding protein, K450 and K129 residue ubiquitination levels were dramatically decreased in TRAF3-deficient macrophages. Ubiquitination assay and luciferase assay demonstrated that TRAF3 promotes STAT6 ubiquitination and transcriptional activity. Site mutation analysis revealed STAT6 K450 site ubiquitination played a vital role in TRAF3-mediated STAT6 activation. Finally, B16 melanoma mouse model demonstrated that myeloid TRAF3 deficiency suppressed tumor growth and lung metastasis in vivo. Taken together, TRAF3 plays a vital role in M2 polarization via regulating STAT6 K450 ubiquitination in macrophages.

TRK-fused gene (TFG) regulates ULK1 stability via TRAF3-mediated ubiquitination and protects macrophages from LPS-induced pyroptosis.

TRK-fused gene (TFG) is known to be involved in protein secretion and plays essential roles in an antiviral innate immune response. However, its function in LPS-induced inflammation and pyroptotic cell death is still unknown. Here, we reported that TFG promotes the stabilization of Unc-51 like autophagy activating kinase (ULK1) and participates in LPS plus nigericin (Ng) induced pyroptotic cell death. Our results showed that TFG-deficient THP-1 macrophages exhibit higher mitochondrial ROS production. LPS/Ng stimulation triggers a much higher level of ROS and induces pyroptotic cell death. ULK1 undergoes a rapid turnover in TFG-deficient THP-1 cells. TFG forms complex with an E3 ligase, tumor necrosis factor receptor-associated factor 3 (TRAF3), and stabilizes ULK1 via disturbing ULK1-TRAF3 interaction. Knockdown of TFG facilitates the interaction of ULK1 with TRAF3 and subsequent K48-linked ULK1 ubiquitination and proteasome degradation. Rescue of ULK1 expression blocks LPS/Ng-induced cell death in TFG-deficient THP-1 macrophages. Taken together, TFG plays an essential role in LPS/Ng-induced pyroptotic cell death via regulating K48-linked ULK1 ubiquitination in macrophages.

Millifolide A, a dimeric ether of degraded sesquiterpene lactones, inhibited the proliferation of human lung cancer cell line A549.

The inhibitory effect of three degraded sesquiterpene lactones, iso-seco-tanapartholide, arteludooicinolide A and millifolide A isolated from Achillea millefolium L., on anti-human lung cancer cells was examined using MTT and reporter gene assays. Millifolide A has significant inhibitory effects on the proliferation of human lung cancer cells probably through inducing cell apoptosis.

Gene mutations of esophageal squamous cell carcinoma based on next-generation sequencing.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers without effective therapy. To explore potential molecular targets in ESCC, we quantified the mutation spectrum and explored the relationship between gene mutation and clinicopathological characteristics and programmed death-ligand 1 (PD-L1) expression. METHODS: Between 2015 and 2019, 29 surgically resected ESCC tissues and adjacent normal tissues from the Fourth Hospital of Hebei Medical University were subjected to targeted next-generation sequencing. The expression levels of PD-L1 were detected by immunohistochemistry. Mutational signatures were extracted from the mutation count matrix by using non-negative matrix factorization. The relationship between detected genomic alterations and clinicopathological characteristics and PD-L1 expression was estimated by Spearman rank correlation analysis. RESULTS: The most frequently mutated gene was TP53 (96.6%, 28/29), followed by NOTCH1 (27.6%, 8/29), EP300 (17.2%, 5/29), and KMT2C (17.2%, 5/29). The most frequently copy number amplified and deleted genes were CCND1/FGF3/FGF4/FGF19 (41.4%, 12/29) and CDKN2A/2B (10.3%, 3/29). By quantifying the contribution of the mutational signatures to the mutation spectrum, we found that the contribution of signature 1, signature 2, signature 10, signature 12, signature 13, and signature 17 was relatively high. Further analysis revealed genetic variants associated with cell cycle, chromatin modification, Notch, and Janus kinase-signal transducer and activator of transcription signaling pathways, which may be key pathways in the development and progression of ESCC. Evaluation of PD-L1 expression in samples showed that 13.8% (4/29) of samples had tumor proportion score ≥1%. 17.2% (5/29) of patients had tumor mutation burden (TMB) above 10 mut/Mb. All samples exhibited microsatellite stability. TMB was significantly associated with lymph node metastasis (r = 0.468, P = 0.010), but not significantly associated with PD-L1 expression (r = 0.246, P = 0.198). There was no significant correlation between PD-L1 expression and detected gene mutations (all P > 0.05). CONCLUSION: Our research initially constructed gene mutation profile related to surgically resected ESCC in high-incidence areas to explore the mechanism underlying ESCC development and potential therapeutic targets.

Clinical and imaging features of pediatric COVID-19.

BACKGROUND: Pediatric COVID-19 is relatively mild and may vary from that in adults. This study was to investigate the epidemic, clinical, and imaging features of pediatric COVID-19 pneumonia for early diagnosis and treatment. METHODS: Forty-one children infected with COVID-19 were analyzed in the epidemic, clinical and imaging data. RESULTS: Among 30 children with mild COVID-19, seven had no symptoms, fifteen had low or mediate fever, and eight presented with cough, nasal congestion, diarrhea, headache, or fatigue. Among eleven children with moderate COVID-19, nine presented with low or mediate fever, accompanied with cough and runny nose, and two had no symptoms. Significantly (P < 0.05) more children had a greater rate of cough in moderate than in mild COVID-19. Thirty children with mild COVID-19 were negative in pulmonary CT imaging, whereas eleven children with moderate COVID-19 had pulmonary lesions, including ground glass opacity in ten (90.9%), patches of high density in six (54.5%), consolidation in three (27.3%), and enlarged bronchovascular bundles in seven (63.6%). The lesions were distributed along the bronchus in five patients (45.5%). The lymph nodes were enlarged in the pulmonary hilum in two patients (18.2%). The lesions were presented in the right upper lobe in two patients (18.1%), right middle lobe in one (9.1%), right lower lobe in six (54.5%), left upper lobe in five (45.5%), and left lower lobe in eight (72.7%). CONCLUSIONS: Children with COVID-19 have mild or moderate clinical and imaging presentations. A better understanding of the clinical and CT imaging helps ascertaining those with negative nucleic acid and reducing misdiagnosis rate for those with atypical and concealed symptoms.

Xylomexicanins I and J: Limonoids with Unusual B/C Rings from Xylocarpus granatum.

Two tetranortriterpenoids with new skeletons, xylomexicanins I and J (1 and 2), were isolated during the investigation of chemical constituents from seeds of the Chinese mangrove, Xylocarpus granatum. Xylomexicanin I (1) is an unprecedented limonoid with bridged B- and C-rings. A biosynthesis pathway for 1 from xylomexicanin F is proposed.

Xylomexicanins C and D, new mexicanolide-type limonoids from Xylocarpus granatum.

Two new limonoids, named xylomexicanins C and D, were isolated from a dichloromethane extract of the seeds of Xylocarpus granatum cultivated in Hainan, China, and their structures were elucidated on the basis of one- and two-dimensional NMR (including 1H, 13C-NMR, DEPT, 1H-1H COSY, HSQC, HMBC, and NOESY) and confirmed by high-resolution mass spectrometry. Xylomexicanin C exhibited antiproliferative activity against human breast carcinoma cells (KT).

Antitumour activities of sesquiterpene lactones from Inula helenium and Inula japonica.

Eight sesquiterpene lactones were isolated from the roots of Inula helenium and flowers of I. japonica. Among them, isoalantolactone (3) and santamarine (6) exhibited significant growth inhibitory activities against gynecologic cancer cell lines, while others weakly inhibited the growth of the cell lines (IC50 < or = 100 microM). In addition, 3 significantly inhibited the tumour growth of S180 tumour-bearing mice. Compounds 3 and 6 were not toxic to human embryonic lung fibroblast cells in vitro. These results demonstrated that the antitumour activities are closely related to the structures of the compounds, that is, an alpha-exomethylene-gamma-lactone ring is necessary for these activities.

Cholecystokinin receptor-1 mediates the inhibitory effects of exogenous cholecystokinin octapeptide on cellular morphine dependence.

BACKGROUND: Cholecystokinin octapeptide (CCK-8), the most potent endogenous anti-opioid peptide, has been shown to regulate the processes of morphine dependence. In our previous study, we found that exogenous CCK-8 attenuated naloxone induced withdrawal symptoms. To investigate the precise effect of exogenous CCK-8 and the role of cholecystokinin (CCK) 1 and/or 2 receptors in morphine dependence, a SH-SY5Y cell model was employed, in which the µ-opioid receptor, CCK1/2 receptors, and endogenous CCK are co-expressed. RESULTS: Forty-eight hours after treating SH-SY5Y cells with morphine (10 µM), naloxone (10 µM) induced a cAMP overshoot, indicating that cellular morphine dependence had been induced. The CCK receptor and endogenous CCK were up-regulated after chronic morphine exposure. The CCK2 receptor antagonist (LY-288,513) at 1-10 µM inhibited the naloxone-precipitated cAMP overshoot, but the CCK1 receptor antagonist (L-364,718) did not. Interestingly, CCK-8 (0.1-1 µM), a strong CCK receptor agonist, dose-dependently inhibited the naloxone-precipitated cAMP overshoot in SH-SY5Y cells when co-pretreated with morphine. The L-364,718 significantly blocked the inhibitory effect of exogenous CCK-8 on the cAMP overshoot at 1-10 µM, while the LY-288,513 did not. Therefore, the CCK2 receptor appears to be necessary for low concentrations of endogenous CCK to potentiate morphine dependence in SH-SY5Y cells. An additional inhibitory effect of CCK-8 at higher concentrations appears to involve the CCK1 receptor. CONCLUSIONS: This study reveals the difference between exogenous CCK-8 and endogenous CCK effects on the development of morphine dependence, and provides the first evidence for the participation of the CCK1 receptor in the inhibitory effects of exogenous CCK-8 on morphine dependence.

Novel taxa-4(20),12-diene and 2(3→20)abeotaxane from needles of Taxus canadensis.

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7ß,9α,10ß-triacetoxytaxa-4(20),12-diene-2α,5α,11ß-triol (1) and 2α,7ß,10ß-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.

A novel taxane 13-glucoside and other taxanes from the leaves of Taxus cuspidata.

Three new taxanes, 2 α,9 α,10 ß-triacetoxy-13 α-( ß-D-glucopyranosyloxy)taxa-4(20),11-dien-5 α-ol, 5 α,10 ß,13 α-triacetoxytax-11-ene-2 α,7 ß,9 α,20-tetraol, and 5 α,10 ß,13 ß-triacetoxy-2 α,7 ß-dihydroxy-2(3→20) abeotaxa-4(20),11-dien-9-one, were isolated from the leaves of the Japanese yew, Taxus cuspidata. Compound 1 is the first example of a taxane with 13-glycosidic linkage.

Secondary metabolites of plants from the genus Saussurea: chemistry and biological activity.

The Asteraceae family comprises ca. 1000 genera, mainly distributed in Asia and Europe. Saussurea DC., as the largest subgenus of this family, comprises ca. 400 species worldwide, of which ca. 300 species occur in China. Most plants in China grow wild in the alpine zone of the Qingzang Plateau and adjacent regions at elevations of 4000 m. Plants of the genus Saussurea (Asteraceae) are used in both traditional Chinese folk medicine and Tibet folklore medicine, since they are efficacious in relieving internal heat or fever, harmonizing menstruation, invigorating blood circulation, stopping bleeding, alleviating pain, increasing energy, and curing rheumatic arthritis. A large number of biologically active compounds have been isolated from this genus. This review shows the chemotaxonomy of these compounds (215 compounds) such as sesquiterpenoids (101 compounds), flavonoids (19 compounds), phytosterols (15 compounds), triterpenoids (25 compounds), lignans (32 compounds), phenolics (23 compounds), and chlorophylls (11 compounds). Biological activities (anti-inflammatory, anticancer, antitumor, hepatoprotective, anti-ulcer, cholagogic, immunosuppressive, spasmolytic, antimicrobial, antiparasitic, antifeedant, CNS depressant, antioxidant, etc.) of these compounds, including structure-activity relationships, are also discussed.

Taxusecone, a novel taxane with an unprecedented 11,12-secotaxane skeleton, from Taxus cuspidata needles.

Taxusecone, 2alpha,7beta,9alpha,10beta-tetraacetoxy-5alpha,12-dihydroxy-11,12-secotax-4(20)-ene-11,13-dione (1), a novel taxane with an unprecedented skeleton, was isolated from the needles of Taxus cuspidata.

[Methylation status of the IL-10 gene promoter in the peripheral blood mononuclear cells of rheumatoid arthritis patients].

Interleukin 10(IL-10), as an immunoregulatory cytokine, plays an important role in rheumatoid arthritis (RA). IL-10 gene silencing is associated with the chromatin remodeling in differentiated Th1 and Th2 cells. To explore the relationship between IL-10 promoter methylation and gene silencing in the pathogenesis of RA, IL-10 mRNA, protein expression and promoter methylation status were analyzed in the peripheral blood mononuclear cells (PBMC) of 34 RA patients and 30 healthy controls by reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and methylation specific polymerase chain reaction (MSP), respectively. The results showed that IL-10 mRNA and protein expression in RA patients seemed to be lower than that in healthy controls, but there was no statistically significant difference (P>0.05). IL-10 promoter was methylated at a frequency of 85.29% in RA cases, which was significantly higher than the percentage in healthy controls (43.33%) (c 2 =12.439, P=0.000). IL-10 promoter methylation and mRNA expression showed a strong negative correlation (r=-0.579, P=0.001). IL-10 promoter methylation, but not mRNA expression, also correlated statistically with the number of arthritic joints. However, there were no statistical correlations between IL-10 promoter methylation (or mRNA expression) and clinical indices of RA, such as the levels of erythrocyte sedimentation rate (ESR), C reactive protein (CRP) and rheumatic factor (RF) or age (P>0.05). These findings suggest that promoter methylation may be a crucial mechanism of IL-10 gene inactivation in RA and IL-10 promoter CpG island hypermethylation might be involved in the occurrence and development of RA.

[Cholecystokinin octapeptide augments expression of anti-inflammatory cytokines in mice challenged by lipopolysaccharide].

OBJECTIVE: To observe the effects of cholecystokinin octapeptide (CCK-8) on the expression of anti-inflammatory cytokines interleukin-4 (IL-4) and IL-10 in lipopolysaccharide (LPS) challenged mice. METHODS: Kunming mice were randomly assigned into four groups (each n=7): (1) Intraperitoneal injection of LPS (10 mg/kg), and the times of peak level of IL-4 and IL-6 expression in serum and lung tissue were noted at 0, 2, 4, 6 and 12 hours after challenge. (2) Control group ( intraperitoneal injection of normal saline 0.2 ml). (3) LPS+CCK-8 group ( intraperitoneal injection of CCK-8 60 microg/kg 30 minutes before giving LPS). (4) CCK-8 group (intraperitoneal injection injection CCK-8 60 microg/kg) at different time points. The expressions of IL-4 and IL-10 in the serum and lung tissues were assayed by enzyme linked immunoadsorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Two hours after LPS challenge, IL-4 and IL-6 were increased significantly in serum and lung tissue. At 4 hours and 6 hours, serum IL-4 and IL-6 reached their peak levels, while in lung tissue they reached their peak levels at 6 hours. Pre-treatment of CCK-8 augmented IL-4 and IL-10 expressions in LPS challenged mice (both P<0.01). But single CCK-8 injection showed no significant effect on IL-4 and IL-10 in serum and lung tissue. CONCLUSION: CCK-8 is involved in the anti-inflammatory response by increasing the expressions of IL-4, IL-10 in LPS challenged mice, and in turn it alleviates the inflammatory response in lung tissue.