Genetic distribution, characterization, and function of Escherichia coli type III secretion system 2 (ETT2).

Many Gram-negative bacteria use type Ⅲ secretion system (T3SS) to inject effector proteins and subvert host signaling pathways, facilitating the growth, survival, and virulence. Notably, some bacteria harbor multiple distinct T3SSs with different functions. An extraordinary T3SS, the Escherichia coli Type III Secretion System 2 (ETT2), is widespread among Escherichia coli (E. coli) strains. Since many ETT2 carry genetic mutations or deletions, it is thought to be nonfunctional. However, increasing studies highlight ETT2 contributes to E. coli pathogenesis. Here, we present a comprehensive overview of genetic distribution and characterization of ETT2. Subsequently, we outline its functional potential, contending that an intact ETT2 may retain the capacity to translocate effector proteins and manipulate the host's innate immune response. Given the potential zoonotic implications associated with ETT2-carrying bacteria, further investigations into the structure, function and regulation of ETT2 are imperative for comprehensive understanding of E. coli pathogenicity and the development of effective control strategies.

Crotonoids A-H, Labdane-Type Diterpenoids with Anti-Inflammatory Activity from Croton sublyratus.

Croton sublyratus (Euphorbiaceae) is a traditional medicinal plant used by the Thai populace to treat helminthic infections and dermatologic conditions. In present study, eight new labdane-type diterpenoids, crotonoids A-H (1-8) and one known analogue (9) were isolated from the aerial parts of C. sublyratus. Compounds 6 and 7 belong to the rare class of 14,15-dinor-labdane diterpenoids. Compound 8 exhibited a rare 14,15,17-trinor-labdane skeleton. The structures of all these diterpenoids were elucidated by spectroscopic data analysis, electronic circular dichroism calculations, and single-crystal X-ray diffraction analysis. Compound 9 exhibited moderate anti-inflammatory activity via the inhibition of NO production in lipopolysaccharide-induced RAW 264.7 cells.

Value of quantitative microsurface structure analysis for evaluating the invasion depth of type 0-II early gastric cancer.

Background and Aim: The microsurface structure reflects the degree of damage to the glands, which is related to the invasion depth of early gastric cancer. To evaluate the diagnostic value of quantitative microsurface structure analysis for estimating the invasion depth of early gastric cancer. Methods: White-light imaging and narrow-band imaging (NBI) endoscopy were used to visualize the lesions of the included patients. The area ratio and depth-predicting score (DPS) of each patient were calculated; meanwhile, each lesion was examined by endoscopic ultrasonography (EUS). Results: Ninety-three patients were included between 2016 and 2019. Microsurface structure is related to the histological differentiation and progression of early gastric cancer. The receiver operating characteristic curve showed that when an area ratio of 80.3% was used as a cut-off value for distinguishing mucosal (M) and submucosal (SM) type 0-II gastric cancers, the sensitivity, specificity, and accuracy were 82.9%, 80.2%, and 91.6%, respectively. The accuracies for distinguishing M/SM differentiated and undifferentiated early gastric cancers were 87.4% and 84.8%, respectively. The accuracy of EUS for distinguishing M/SM early gastric cancer was 74.9%. DPS can only distinguish M-SM1 (SM infiltration <500 µm)/SM (SM infiltration ≥500 µm) with an accuracy of 83.8%. The accuracy of using area ratio for distinguishing 0-II early gastric cancers was better than those of using DPS and EUS (P < 0.05). Conclusion: Quantitative analysis of microsurface structure can be performed to assess M/SM type 0-II gastric cancer and is expected to be effective for judging the invasion depth of gastric cancer.

Curcumin suppresses the malignant phenotype of laryngeal squamous cell carcinoma through downregulating E2F1 to inhibit FLNA.

Curcumin is a kind of polyphenol substance extracted from the rhizome of Curcuma longa. Because of its good biological activity and pharmacological effects, it has been used in anti-tumor research. The aim of this study was to investigate the anti-cancer mechanism of curcumin on laryngeal squamous cell carcinoma (LSCC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to check the expression level of transcription factor E2F1 (E2F1) and filamin A (FLNA) mRNA. E2F1 and FLNA protein and proliferation-associated protein were detected through western blot. Cell viability was showed by MTT assay, and flow cytometry was used to exhibit cell cycle distribution and cell apoptosis. Tube formation assay was used to detect the angiogenesis ability of cells. Transwell was used as a method to observe cell migration and invasion. The online website JASPAR predicted the binding site of E2F1 and FLNA promoter, and chromatin immunoprecipitation (ChIP) and dual-luciferase report experiment verified the combination. Curcumin treatment made LSCC cells viability reduce, cell cycle retardant, angiogenesis decrease, metastasis inhibition and apoptosis increase. And curcumin treatment could downregulate the expression of E2F1, and E2F1 overexpression would reverse the influence of curcumin treatment in LSCC cells. Moreover, E2F1 could bind to FLAN promoter and promote FLNA expression. The expression level of FLNA was higher in LSCC tissue and cells compared with normal tissue and cells. E2F1 knockdown inhibited malignant phenotype of LSCC cells, which would be reversed by FLNA addition. In addition, FLNA had high level in LSCC tissue and cells. Curcumin regulated FLNA expression via inhibiting E2F1. Finally, in vivo assay showed that curcumin inhibition restrained LSCC tumor formation. Curcumin downregulated FLNA expression through inhibiting E2F1, thereby suppressing the malignant phenotype and angiogenesis of LSCC cells, which was a new regulatory pathway in LSCC.

Genome-wide characterization and comparison of endogenous retroviruses among 3 duck reference genomes.

Endogenous retroviruses (ERV) are viral genomes integrated into the host genome and can be stably inherited. Although ERV sequences have been reported in some avian species' genome, the duck endogenous retroviruses (DERV) genome has yet to be quantified. This study aimed to identify ERV sequences and characterize genes near ERVs in the duck genome by utilizing LTRhavest and LTRdigest tools to forecast the duck genome and analyze the distribution of ERV copies. The results revealed 1,607, 2,031, and 1,908 full-length ERV copies in the Pekin duck (ZJU1.0), Mallard (CAU_wild_1.0), and Shaoxing duck (CAU_laying_1.0) genomes, respectively, with average lengths of 7,046, 7,027, and 6,945 bp. ERVs are mainly distributed on the 1, 2, and sex chromosomes. Phylogenetic analysis demonstrated the presence of Betaretrovirus in 3 duck genomes, whereas Alpharetrovirus was exclusively identified in the Shaoxing duck genome. Through screening, 596, 315, and 343 genes adjacent to ERV were identified in 3 duck genomes, respectively, and their functions of ERV neighboring genes were predicted. Functional enrichment analysis of ERV-adjacent genes revealed enrichment for Focal adhesion, Calcium signaling pathway, and Adherens junction in 3 duck genomes. The overlapped genes were highly expressed in 8 tissues (brain, fat, heart, kidney, liver, lung, skin, and spleen) of 8-wk-old Mallard, revealing their important expression in different tissues. Our study provides a new perspective for understanding the quantity and function of DERVs, and may also provide important clues for regulating nearby genes and affecting the traits of organisms.

Genome-wide association study for bone quality of ducks during the laying period.

The cage-rearing model of the modern poultry industry makes the bones of birds, especially egg-laying birds, more vulnerable to fracture, which poses serious damage to the health of birds. Research confirms that genetic material plays an important role in regulating bone growth, development, and remodeling. However, the genetic architecture underlying bone traits is not well understood. The objectives of this study are to identify valuable genes and genetic markers through a genome-wide association study (GWAS) for breeding to improve the duck bone quality. First, we quantified the tibia and femur quality traits of 260 laying ducks. Based on GWAS, a total of 75 SNP loci significantly associated with bone quality traits were identified, and 67 potential candidate genes were annotated. According to gene function analysis, genes P4HA2, WNT3A, and BST1 et al may influence bone quality by regulating bone cell activity, calcium and phosphate metabolism, or bone collagen maturation and cross-linking. Meanwhile, combined with the transcriptome results, we found that HOXB cluster genes are also important in bone growth and development. Therefore, our findings were helpful in further understanding the genetic architecture of the duck bone quality and provided a worthy theoretical basis and technological support to improve duck bone quality by breeding.

The aroA and luxS Double-Gene Mutant Strain Has Potential to Be a Live Attenuated Vaccine against Salmonella Typhimurium.

Salmonella Typhimurium (S. Typhimurium) is a zoonotic pathogen posing a threat to animal husbandry and public health. Due to the emergence of antibiotic-resistant strains, alternative prevention and control strategies are needed. Live attenuated vaccines are an ideal option that provide protection against an S. Typhimurium pandemic. To develop a safe and effective vaccine, double-gene mutations are recommended to attenuate virulence. In this study, we chose aroA and luxS genes, whose deletion significantly attenuates S. Typhimurium's virulence and enhances immunogenicity, to construct the double-gene mutant vaccine strain SAT52ΔaroAΔluxS. The results show that the mutant strain's growth rate, adherence and invasion of susceptible cells are comparable to a wild-type strain, but the intracellular survival, virulence and host persistence are significantly attenuated. Immunization assay showed that 106 colony-forming units (CFUs) of SAT52ΔaroAΔluxS conferred 100% protection against wild-type challenges; the bacteria persistence in liver and spleen were significantly reduced, and no obvious pathological lesions were observed. Therefore, the double-gene mutant strain SAT52ΔaroAΔluxS exhibits potential as a live attenuated vaccine candidate against S. Typhimurium infection.

Deterministic reprogramming of neutrophils within tumors.

Neutrophils are increasingly recognized as key players in the tumor immune response and are associated with poor clinical outcomes. Despite recent advances characterizing the diversity of neutrophil states in cancer, common trajectories and mechanisms governing the ontogeny and relationship between these neutrophil states remain undefined. Here, we demonstrate that immature and mature neutrophils that enter tumors undergo irreversible epigenetic, transcriptional, and proteomic modifications to converge into a distinct, terminally differentiated dcTRAIL-R1+ state. Reprogrammed dcTRAIL-R1+ neutrophils predominantly localize to a glycolytic and hypoxic niche at the tumor core and exert pro-angiogenic function that favors tumor growth. We found similar trajectories in neutrophils across multiple tumor types and in humans, suggesting that targeting this program may provide a means of enhancing certain cancer immunotherapies.

Features of acute COVID-19 associated with post-acute sequelae of SARS-CoV-2 phenotypes: results from the IMPACC study.

Post-acute sequelae of SARS-CoV-2 (PASC) is a significant public health concern. We describe Patient Reported Outcomes (PROs) on 590 participants prospectively assessed from hospital admission for COVID-19 through one year after discharge. Modeling identified 4 PRO clusters based on reported deficits (minimal, physical, mental/cognitive, and multidomain), supporting heterogenous clinical presentations in PASC, with sub-phenotypes associated with female sex and distinctive comorbidities. During the acute phase of disease, a higher respiratory SARS-CoV-2 viral burden and lower Receptor Binding Domain and Spike antibody titers were associated with both the physical predominant and the multidomain deficit clusters. A lower frequency of circulating B lymphocytes by mass cytometry (CyTOF) was observed in the multidomain deficit cluster. Circulating fibroblast growth factor 21 (FGF21) was significantly elevated in the mental/cognitive predominant and the multidomain clusters. Future efforts to link PASC to acute anti-viral host responses may help to better target treatment and prevention of PASC.

HIPK3 maintains sensitivity to platinum drugs and prevents disease progression in gastric cancer.

In the realm of cancer therapeutics and resistance, kinases play a crucial role, particularly in gastric cancer (GC). Our study focused on platinum-based chemotherapy resistance in GC, revealing a significant reduction in homeodomain-interacting protein kinase 3 (HIPK3) expression in platinum-resistant tumors through meticulous analysis of transcriptome datasets. In vitro and in vivo experiments demonstrated that HIPK3 knockdown enhanced tumor proliferation and metastasis, while upregulation had the opposite effect. We identified the myocyte enhancer factor 2C (MEF2C) as a transcriptional regulator of HIPK3 and uncovered HIPK3's role in downregulating the morphogenesis regulator microtubule-associated protein (MAP7) through ubiquitination. Phosphoproteome profiling revealed HIPK3's inhibitory effects on mTOR and Wnt pathways crucial in cell proliferation and movement. A combined treatment strategy involving oxaliplatin, rapamycin, and IWR1-1-endo effectively overcame platinum resistance induced by reduced HIPK3 expression. Monitoring HIPK3 levels could serve as a GC malignancy and platinum resistance indicator, with our proposed treatment strategy offering novel avenues for reversing resistance in gastric cancer.

Transcriptome-based comparison reveals key genes regulating allometry growth of forelimb and hindlimb bone in duck embryos.

Allometric growth of the forelimb and hindlimb is a widespread phenomenon observed in vertebrates. As a typical precocial bird, ducks exhibit more advanced development of their hindlimbs compared to their forelimbs, enabling them to walk shortly after hatching. This phenomenon is closely associated with the development of long bones in the embryonic stage. However, the molecular mechanism governing the allometric growth of duck forelimb and hindlimb bones is remains elusive. In this study, we employed phenotypic, histological, and gene expression analyses to investigate developmental differences between the humerus (forelimb bone) and tibia/femur (hindlimb bones) in duck embryos. Our results revealed a gradual increase in weight and length disparity between the tibia and humerus from E12 to E28 (embryo age). At E12, endochondral ossification was observed solely in the tibia but not in the humerus. The number of differentially expressed genes (DEGs) gradually increased at H12 vs. T12, H20 vs. T20, and H28 vs. T28 stages consistent with phenotypic variations. A total of 38 DEGs were found across all 3 stages. Protein-protein interaction network analysis demonstrated strong interactions among members of HOXD gene family (HOXD3/8/9/10/11/12), HOXB gene family (HOXB8/9), TBX gene family (TBX4/5/20), HOXA11, SHOX2, and MEIS2. Gene expression profiling indicated higher expression levels for all HOXD genes in the humerus compared to tibia while opposite trends were observed for HOXA/HOXB genes with low or no expression detected in the humerus. These findings suggest distinct roles played by different clusters within HOX gene family during skeletal development regulation of duck embryo's forelimbs versus hind limbs. Notably, TBX4 exhibited high expression levels specifically in tibia whereas TBX5 showed similar patterns exclusively within humerus as seen previously across other species' studies. In summary, this study identified key regulatory genes involved in allometric growth of duck forelimb and hindlimb bones during embryonic development. Skeletal development is a complex physiological process, and further research is needed to elucidate the regulatory role of candidate genes in endochondral ossification.

The functions of EF-hand proteins from host and zoonotic pathogens.

EF-hand proteins not only regulate biological processes, but also influence immunity and infection. In this review, we summarize EF-hand proteins' functions in host and zoonotic pathogens, with details in structures, Ca2+ affinity, downstream targets and functional mechanisms. Studies entitled as EF-hand-related but with less solid features were also discussed. We believe it could raise cautions and facilitate proper research strategy for researchers.

LncRNA MIR31HG fosters stemness malignant features of non-small cell lung cancer via H3K4me1- and H3K27Ace-mediated GLI2 expression.

Non-coding RNAs are responsible for oncogenesis and the development of stemness features, including multidrug resistance and metastasis, in various cancers. Expression of lncRNA MIR31HG in lung cancer tissues and peripheral sera of lung cancer patients were remarkably higher than that of healthy individuals and indicated a poor prognosis. Functional analysis showed that MIR31HG fosters stemness-associated malignant features of non-small cell lung cancer cells. Further mechanistic investigation revealed that MIR31HG modulated GLI2 expression via WDR5/MLL3/P300 complex-mediated H3K4me and H3K27Ace modification. In vivo MIR31HG repression with an antisense oligonucleotide attenuated tumor growth and distal organ metastasis, whereas MIR31HG promotion remarkably encouraged cellular invasion in lung and liver tissues. Our data suggested that MIR31HG is a potential diagnostic indicator and druggable therapeutic target to facilitate multiple strategic treatments for lung cancer patients.

Therapeutic effect and transcriptome-methylome characteristics of METTL3 inhibition in liver hepatocellular carcinoma.

Methyltransferase-like 3 (METTL3) is the key subunit of methyltransferase complex responsible for catalyzing N6-methyladenosine (m6A) modification on mRNA, which is the most prevalent post-transcriptional modification in eukaryotes. In this study, we utilized online databases to analyze the association between METTL3 expression and various aspects of tumorigenesis, including gene methylation, immunity, and prognosis. Our investigation revealed that METTL3 serves as a prognostic marker and therapeutic target for liver hepatocellular carcinoma (LIHC). Through experimental studies, we observed frequent upregulation of METTL3 in LIHC tumor tissue and cells. Subsequent inhibition of METTL3 using a novel small molecule inhibitor, STM2457, significantly impeded tumor growth in LIHC cell lines, spheroids, and xenograft tumor model. Further, transcriptome and m6A sequencing of xenograft bodies unveiled that inhibition of METTL3-m6A altered genes enriched in SMAD and MAPK signaling pathways that are critical for tumorigenesis. These findings suggest that targeting METTL3 represents a promising therapeutic strategy for LIHC.

A lytic phage to control multidrug-resistant avian pathogenic Escherichia coli (APEC) infection.

The inappropriate use of antibiotics has led to the emergence of multidrug-resistant strains. Bacteriophages (phages) have gained renewed attention as promising alternatives or supplements to antibiotics. In this study, a lytic avian pathogenic Escherichia coli (APEC) phage designated as PEC9 was isolated and purified from chicken farm feces samples. The morphology, genomic information, optimal multiplicity of infection (MOI), one-step growth curve, thermal stability, pH stability, in vitro antibacterial ability and biofilm formation inhibition ability of the phage were determined. Subsequently, the therapeutic effects of the phages were investigated in the mice model. The results showed that PEC9 was a member of the siphovirus-like by electron microscopy observation. Biological characterization revealed that it could lyse two serotypes of E. coli, including O1 (9/20) and O2 (6/20). The optimal multiplicity of infection (MOI) of phage PEC9 was 0.1. Phage PEC9 had a latent period of 20 min and a burst period of 40 min, with an average burst size of 68 plaque-forming units (PFUs)/cell. It maintained good lytic activity at pH 3-11 and 4-50°C and could efficiently inhibit the bacterial planktonic cell growth and biofilm formation, and reduce bacterial counts within the biofilm, when the MOI was 0.01, 0.1, and 1, respectively. Whole-genome sequencing showed that PEC9 was a dsDNA virus with a genome of 44379 bp and GC content of 54.39%. The genome contains 56 putative ORFs and no toxin, virulence, or resistance-related genes were detected. Phylogenetic tree analysis showed that PEC9 is closely related to E. coli phages vB_EcoS_Zar3M, vB_EcoS_PTXU06, SECphi18, ZCEC10, and ZCEC11, but most of these phages exhibit different gene arrangement. The phage PEC9 could successfully protect mice against APEC infection, including improved survival rate, reduced bacterial loads, and organ lesions. To conclude, our results suggest that phage PEC9 may be a promising candidate that can be used as an alternative to antibiotics in the control of APEC infection.

Comprehensive transcriptomic and metabolomic analysis of the effect of feed restriction on duck sternal development.

Skeletal characteristics are important to the growth and development of poultry. In feeding management, constant free feeding (FF) of poultry may lead to imbalance between bone development and weight gain. Feed restriction (FR), to a certain extent, is one way to solve this problem. However, the effect of feed restriction on poultry bone development needs further elucidation at the molecular level. Therefore, in the present study, we investigated the effects of different levels of feed restriction (60% FR, 70% FR, 80% FR, and FF) on the sternum development of ducks at 7 and 8 wk old. In the seventh wk, with increasing feed restriction, the values of traits including body weight, breast muscle weight, sternal weight, keel length, and calcified keel length decreased. However, in the eighth wk, the sternum weight and keel length of ducks treated with 60% FR were unexpectedly higher than those of FF individuals, indicative of catch-up growth. Then, we conducted RNA-seq and metabolomic analysis on sterna from 7- and 8-wk-old FF and 60% FR ducks. The results identified multiple differentially expressed genes (DEGs) associated with sternum development that were influenced by feed restriction. Among them, we found that the mRNA expression levels of the chondroitin sulfate synthase 3 (CHSY3) and annexin A2 (ANXA2) which are involved in glycosaminoglycan biosynthesis and bone mineralization, had smaller changes over time under FR treatment than under FF treatment, implying that the FR treatment to a certain extent prevented the premature calcification and prolonged the development time of duck sternum. In addition, the metabolomic and integrative analyses revealed that several antiaging-related metabolites and genes were associated with sternal catch-up growth. Pyrimidine metabolism was identified as the most significant pathway in which most differential metabolites (DMs) between FF and 60% FR were enriched. The results from integrative analysis revealed that the content and expression of 4-aminobutyric acid (GABA) and its related genes showed relatively higher activity in the 60% FR group than in the FF group. The present study identifies multiple biomarkers associated with duck sternum development that are influenced by feed restriction and suggests the potential mechanism of feed restriction-associated duck sternal catch-up growth.

Antitumor immunity as the basis for durable disease-free treatment-free survival in patients with metastatic urothelial cancer.

Cisplatin-based chemotherapy has been associated with durable disease control in a small subset of patients with metastatic urothelial cancer. However, the mechanistic basis for this phenomenon has remained elusive. Antitumor immunity may underlie these exceptional responders. In a phase II trial evaluating a phased schedule of gemcitabine and cisplatin followed by gemcitabine and cisplatin with ipilimumab for metastatic urothelial cancer, 4 of 36 patients achieved durable disease-free treatment-free survival (DDFTFS) and remain in remission over 5 years after enrolment on the study. We sought to identify the genomic and immunological mechanisms associated with functional cures of such patients. Whole exome sequencing was performed on pretreatment archival tumor tissue. Neoantigen prediction and ranking were performed using a novel pipeline. For a subset of patients with available biospecimens, selected peptides were tested for neoantigen-specific T cell reactivity in peripheral blood CD4+ and CD8+ T cells cultured with autologous antigen-presenting cells at baseline, postchemotherapy, and postchemotherapy and ipilimumab timepoints. Multiplex assays of serum protein analytes were also assessed at each time point. Serum proteomic analysis revealed that pretreatment, patients achieving DDFTFS demonstrated an immune activated phenotype with elevations in TH1 adaptive immunity, costimulatory molecules, and immune checkpoint markers. After combination cisplatin-based chemotherapy and ipilimumab treatment, DDFTFS patients again displayed enrichment for markers of adaptive immunity, as well as T cell cytotoxicity. CD27 was uniquely enriched in DDFTFS patients at all timepoints. Neoantigen reactivity was not detected in any patient at baseline or post two cycles of chemotherapy. Both CD4+ and CD8+ neoantigen-specific T cell reactivity was detected in two of two DDFTFS patients in comparison to zero of five non-DDFTFS patients after combination cisplatin-based chemotherapy and ipilimumab treatment. Antitumor immunity may underlie functional cures achieved in patients with metastatic urothelial cancer treated with cisplatin-based chemotherapy and immune checkpoint blockade. Probing the mechanistic basis for DDFTFS may facilitate the identification of biomarkers, therapeutic components, and optimal treatment sequences necessary to extend this ultimate goal to a larger subset of patients.

Multi-omic longitudinal study reveals immune correlates of clinical course among hospitalized COVID-19 patients.

The IMPACC cohort, composed of >1,000 hospitalized COVID-19 participants, contains five illness trajectory groups (TGs) during acute infection (first 28 days), ranging from milder (TG1-3) to more severe disease course (TG4) and death (TG5). Here, we report deep immunophenotyping, profiling of >15,000 longitudinal blood and nasal samples from 540 participants of the IMPACC cohort, using 14 distinct assays. These unbiased analyses identify cellular and molecular signatures present within 72 h of hospital admission that distinguish moderate from severe and fatal COVID-19 disease. Importantly, cellular and molecular states also distinguish participants with more severe disease that recover or stabilize within 28 days from those that progress to fatal outcomes (TG4 vs. TG5). Furthermore, our longitudinal design reveals that these biologic states display distinct temporal patterns associated with clinical outcomes. Characterizing host immune responses in relation to heterogeneity in disease course may inform clinical prognosis and opportunities for intervention.

IL-27 enhances peripheral B cell glycolysis of rheumatoid arthritis patients via activating mTOR signaling.

Our previous study found that increased serum IL-27 could promote rheumatoid arthritis (RA) B cell dysfunction via activating mTOR signaling pathway. This study aimed to explore the effects of IL-27 on B cell metabolism and clarify the mechanisms via which IL-27 enhancing glycolysis to induce B cells hyperactivation. Peripheral CD19+ B cells were purified from healthy controls (HC) and RA patients and then cultured with or without anti-CD40/CpG and glycolysis inhibitor 2-deoxy-D-glucose (2-DG) or mTOR inhibitor rapamycin. Furthermore, the isolated CD19+ B cells were treated by HC serum or RA serum in the presence and absence of recombinant human IL-27 or anti-IL-27 neutralizing antibodies or 2-DG or rapamycin. The B cell glycolysis level, proliferation, differentiation and inflammatory actions were detected by qPCR, flow cytometry or ELISA. We found that the glycolysis in RA B cells was increased significantly compared with HC B cells. Glycolysis inhibition downregulated the proliferation, differentiation, and inflammatory actions of RA B cells. RA serum and IL-27 promoted B cell glycolysis, which could be obviously rescued by anti-IL-27 antibodies or mTOR inhibitor rapamycin. Our results suggest that the enhanced cellular glycolysis of RA B cells induced by IL-27 may contribute to B cells hyperactivation through activating the mTOR signaling pathway.

N6-methyladenine-mediated aberrant activation of the lncRNA SOX2OT-GLI1 loop promotes non-small-cell lung cancer stemness.

Despite the advent of precision medicine and immunotherapy, mortality due to lung cancer remains high. The sonic hedgehog (SHH) cascade and its key terminal factor, glioma-associated oncogene homolog 1 (GLI1), play a pivotal role in the stemness and drug resistance of lung cancer. Here, we investigated the molecular mechanism of non-canonical aberrant GLI1 upregulation. The SHH cascade was upregulated in stem spheres and chemo-resistant lung cancer cells and was accountable for drug resistance against multiple chemotherapy regimens. GLI1 and the long non-coding RNA SOX2OT were positively regulated, and the GLI1-SOX2OT loop mediated the proliferation of parental and stem-like lung cancer cells. Further mechanistic investigation revealed that SOX2OT facilitated METTL3/14/IGF2BP2-mediated m6A modification and stabilization of the GLI1 mRNA. Additionally, SOX2OT upregulated METTL3/14/IGF2BP2 by sponging miR-186-5p. Functional analysis corroborated that GLI1 acted as a downstream target of METTL3/14/IGF2BP2, and GLI1 silencing could block the oncogenicity of lung cancer stem-like cells. Pharmacological inhibition of the loop remarkably inhibited the oncogenesis of lung cancer cells in vivo. Compared with paired adjacent normal tissues, lung cancer specimens exhibited consistently upregulated GLI1/SOX2OT/METTL3/14/IGF2BP2. The m6A-modified GLI1-SOX2OT loop may serve as a potential therapeutic target and prognostic predictor for lung cancer therapy and diagnosis in the clinic.