Crucial role of SWL1 in chloroplast biogenesis and development in Arabidopsis thaliana.

KEY MESSAGE: The disruption of the SWL1 gene leads to a significant down regulation of chloroplast and secondary metabolites gene expression in Arabidopsis thaliana. And finally results in a dysfunction of chloroplast and plant growth. Although the development of the chloroplast has been a consistent focus of research, the corresponding regulatory mechanisms remain unidentified. In this study, the CRISPR/Cas9 system was used to mutate the SWL1 gene, resulting in albino cotyledons and variegated true leaf phenotype. Confocal microscopy and western blot of chloroplast protein fractions revealed that SWL1 localized in the chloroplast stroma. Electron microscopy indicated chloroplasts in the cotyledons of swl1 lack well-defined grana and internal membrane structures, and similar structures have been detected in the albino region of variegated true leaves. Transcriptome analysis revealed that down regulation of chloroplast and nuclear gene expression related to chloroplast, including light harvesting complexes, porphyrin, chlorophyll metabolism and carbon metabolism in the swl1 compared to wild-type plant. In addition, proteomic analysis combined with western blot analysis, showed that a significant decrease in chloroplast proteins of swl1. Furthermore, the expression of genes associated with secondary metabolites and growth hormones was also reduced, which may be attributed to SWL1 associated with absorption and fixation of inorganic carbon during chloroplast development. Together, the above findings provide valuable information to elucidate the exact function of SWL1 in chloroplast biogenesis and development.

Performance of machine learning-based coronary computed tomography angiography for selecting revascularization candidates.

BACKGROUND: Limited studies have investigated the accuracy of therapeutic decision-making using machine learning-based coronary computed tomography angiography (ML-CCTA) compared with CCTA. PURPOSE: To investigate the performance of ML-CCTA for therapeutic decision compared with CCTA. MATERIAL AND METHODS: The study population consisted of 322 consecutive patients with stable coronary artery disease. The SYNTAX score was calculated with an online calculator based on ML-CCTA results. Therapeutic decision-making was determined by ML-CCTA results and the ML-CCTA-based SYNTAX score. The therapeutic strategy and the appropriate revascularization procedure were selected using ML-CCTA, CCTA, and invasive coronary angiography (ICA) independently. RESULTS: The sensitivity, specificity, positive predictive value, negative predictive value, accuracy of ML-CCTA and CCTA for selecting revascularization candidates were 87.01%, 96.43%, 95.71%, 89.01%, 91.93%, and 85.71%, 87.50%, 86.27%, 86.98%, 86.65%, respectively, using ICA as the standard reference. The area under the receiver operating characteristic curve (AUC) of ML-CCTA for selecting revascularization candidates was significantly higher than CCTA (0.917 vs. 0.866, P = 0.016). Subgroup analysis showed the AUC of ML-CCTA for selecting percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) candidates was significantly higher than CCTA (0.883 vs. 0.777, P < 0.001, 0.912 vs. 0.826, P = 0.003, respectively). CONCLUSION: ML-CCTA could distinguish between patients who need revascularization and those who do not. In addition, ML-CCTA showed a slightly superior to CCTA in making an appropriate decision for patients and selecting a suitable revascularization strategy.

Origin of Deformation Twinning in bcc Tungsten and Molybdenum.

Twinning is profuse in bcc transition metals (TMs) except bulk W and Mo. However, W and Mo nanocrystals surprisingly exhibit twinning during room temperature compression, which is completely unexpected as established nucleation mechanisms are not viable in them. Here, we reveal the physical origin of deformation twinning in W and Mo. We employ density functional theory (DFT) and a reduced-constraint slip method to compute the stress-dependent generalized stacking fault enthalpy (GSFH), the thermodynamic quantity to be minimized under constant loading. The simple slipped structures and GSFH lines show that compressive stresses stabilize a two-layer twin embryo, which can grow rapidly via twinning disconnections with negligible energy barriers. Direct atomistic simulations unveil the explicit twinning path in agreement with the DFT GSFH lines. Twinning is thus the preferred deformation mechanism in W and Mo when shear stresses are coupled with high compressive stresses. Furthermore, twinnability can be related to the elastic constants of a stacking fault phase (SFP). The hcp phase may serve as a candidate SFP for the {112}⟨1[over ¯]1[over ¯]1⟩ twinning system in bcc TMs and alloys, which is coincident with the {111}⟨112[over ¯]⟩ twinning in fcc structures.

Pyrenoid: Organelle with efficient CO2-Concentrating mechanism in algae.

The carbon dioxide emitted by human accounts for only a small fraction of global photosynthesis consumption, half of which is due to microalgae. The high efficiency of algae photosynthesis is attributed to the pyrenoid-based CO2-concentrating mechanism (CCM). The formation of pyrenoid which has a variety of Rubisco-binding proteins mainly depends on liquid-liquid phase separation (LLPS) of Rubisco, a CO2 fixing enzyme. At present, our understanding of pyrenoid at the molecular level mainly stems from studies of the model algae Chlamydomonas reinhardtii. In this article, we summarize the current research on the structure, assembly and application of Chlamydomonas reinhardtii pyrenoids, providing new ideas for improving crop photosynthetic performance and yield.

Comprehensive analysis of lncRNA-mRNA regulatory network in Populus associated with xylem development.

Long noncoding RNAs (lncRNAs) play essential roles in numerous biological processes in plants, such as regulating the gene expression. However, only a few studies have looked into their potential functions in xylem development. High-throughput sequencing of P. euramericana 'Zhonglin46' developing and mature xylem was performed in this study. Through sequencing analysis, 14,028 putative lncRNA transcripts were identified, including 4525 differentially expressed lncRNAs (DELs). Additional research revealed that in mature xylem, a total of 2320 DELs were upregulated and 2205 were downregulated compared to developing xylem. Meanwhile, there were a total of 8122 differentially expressed mRNAs (DEMs) that were upregulated and 16,424 that were downregulated in mature xylem compared with developing xylem. The cis- and trans-target genes of DELs were analyzed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, which indicated that these DELs participate in controlling the phenylpropanoid and lignin biosynthesis pathway as well as the starch and sucrose metabolism pathway. Among the cis-regulated DELs, LNC_006291, LNC_006292, and LNC_006532 all participate in regulating multiple HCT gene family membranes. As targets, POPTR_001G045900v3 (CCR2) and POPTR_018G063500v3 (SUS) both have only one cis-regulatory lncRNA, referred to as LNC_000057 and LNC_006212, respectively. Moreover, LNC_004484 and two DELs named LNC_008014 and LNC_010781 were revealed to be important nodes in the co-expression network of trans-lncRNAs and mRNAs associated to the lignin biosynthesis pathway and cellulose and xylan biosynthetic pathways, respectively. Finally, quantitative real-time PCR (qRT-PCR) was used to confirme 34 pairs of lncRNA-mRNA. Taken together, these findings may help to clarify the regulatory role that lncRNAs play in xylem development and wood formation.

Prognostic value of coronary CTA-based classifications for predicting major events without obstructive coronary artery disease.

We aim to explore the classifications based on coronary computed tomography angiography (CTA) for predicting the risk of major adverse cardiovascular events (MACE) in patients with suspected non-obstructive coronary artery disease (CAD) and compare with traditional non-obstructive CAD (NOCAD) classification, Duke prognostic NOCAD index, Non-obstructive coronary artery disease reporting and data system (NOCAD-RADS). 4378 consecutive non-obstructive CAD patients were assessed by coronary CTA for traditional NOCAD classification, Duke prognostic NOCAD index, NOCAD-RADS and a new classification (stenosis proximal involvement, SPI) from two medical centrals. We defined proximal involvement as any plaque was present in the main or proximal segments of coronary artery (left main, left anterior descending artery, left circumflex artery, or right coronary artery). The main outcome was MACE. During a median follow-up of 3.7 years, a total of 310 patients experienced MACE event. Kaplan-Meier survival curves showed the cumulative events increased significantly associated with traditional NOCAD, Duke NOCAD index, NOCAD-RADS and SPI classifications (all P < 0.001). In multivariate Cox regressions, the risk for the events increased from HR 1.20 (95% CI 0.78-1.83, P = 0.408) for SPI 1 to 1.35 (95% CI 1.05-1.73, P = 0.019) for SPI 2, using SPI 0 as the reference group. Coronary CTA based SPI classification provided important prognostic information for all cause-mortality risk and MACE prediction in patients with non-obstructive CAD, which was non-inferior than traditional NOCAD, Duke NOCAD Index and NOCAD-RADS classifications. The plaque location information by coronary CTA may provide additional risk prediction in patients with non-obstructive CAD.

Non-obstructive CAD and Risk of All-cause Mortality in Middle-aged and Older Patients: a Nine-Year Follow-up and Multicentre Study.

AIM: We aimed to examine all-cause mortality risk in relation to the extent of non-obstructive coronary artery disease (CAD) by coronary computed tomography angiography (CTA) in Chinese middle-aged and older patients in a multicenter study with nine-year follow-up. METHODS: This was a retrospective, observational, multicentre study. The study population consisted of 3,240 consecutive middle-aged and older patients (age ≥ 40 years) with suspected CAD who underwent coronary CTA between June 2011 and December 2013 at three hospitals in Wuhan, China. Patients were grouped according to CAD extent for the final analysis: no CAD, 1-vessel non-obstructive CAD, 2-vessels non-obstructive CAD, and 3-vessels non-obstructive CAD. The primary endpoint was all-cause mortality. Kaplan-Meier method and Cox proportional hazards regression models were used for analysis. RESULTS: A total of 2,522 patients were included in the present analysis. Of these, 188 (7.5%) deaths occurred during the median 9.0 years (interquartile range 8.6-9.4) of study follow-up. The annualized all-cause mortality rate was 0.54 (95% CI: 0.44-0.68), 0.91 (95% CI: 0.68-1.21), 1.44 (95% CI: 1.01-1.93), and 2.00 (95% CI: 1.46-2.69) for the no CAD, 1-vessel non-obstructive CAD, 2-vessels non-obstructive CAD, and 3-vessels non-obstructive CAD group, respectively. Kaplan-Meier survival curves showed a significant increase in the cumulative events associated with the extent of non-obstructive CAD (P < 0.001). In multivariate Cox regression, after adjustment for age and sex, the presence of 3-vessels non-obstructive CAD was a significant predictor of all-cause mortality (HR 1.60, 95% CI: 1.04-2.45, P = 0.032). CONCLUSION: In this cohort of Chinese middle-aged and older patients undergoing coronary CTA, the presence and extent of non-obstructive CAD, compared to no CAD, were associated with a significantly greater nine-year risk of all-cause mortality. The present findings suggest the clinical importance of the stage of non-obstructive CAD and warrant investigation of the optimal risk stratification to improve outcomes among these patients.

ALKBH5-YTHDF2 m6A modification axis inhibits rheumatoid arthritis progression by suppressing NLRP3.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. Recently, NLRP3 has been demonstrated to be closely related to RA. The objective of our research was to analyze the specific mechanism of NLRP3 in RA. The m6A levels of NLRP3 was detected with methylated RNA immunoprecipitation (MeRIP) kit. The mRNA and protein levels of related genes were tested with RT-qPCR and Western blot. The inflammatory factors levels were detected with ELISA kits. The cell proliferative ability was measured with CCK-8 and EdU staining assays. NLRP3 levels was prominently in synovial tissues and fibroblast-like synoviocytes (FLS) from RA patients. NLRP3 silencing suppressed FLS proliferation and inflammatory factor levels. Additionally, ALKBH5 was found to bind with NLRP3, and ALKBH5 silencing suppressed FLS proliferation and inflammatory factor levels while NLRP3 overexpressing neutralized the role of ALKBH5 in FLS. Furthermore, m6A modified induced by ALKBH5 suppressed NLRP3 mRNA level through YTHDC2 in RA, and NLRP3 is a hinge factor in RA progression.

[The expression of long non-coding RNA human leukocyte antigen complex P5(lncRNA HCP5) in synovial tissue of patients with rheumatoid arthritis is up-regulated and correlated with immune cell infiltration].

Objective To identify the potential long non-coding RNA (lncRNA) expressed in rheumatoid arthritis (RA) synovium key to RA onset and investigate its association with immune cell infiltration. Methods RA synovium data were downloaded from the GEO database and normalized. The lncRNAs key to RA onset were identified using multiple machine learning methods. Infiltration of 22 immune cell populations in RA synovium was measured by cell-type identification by estimating relative subsets of RNA transcripts (CIBER-SORT). The relationship between the key lncRNA and infiltrating immune cells was analyzed. Finally, real-time quantitative PCR was applied to validate the expression of the key lncRNA in RA synovial cells. Results lncRNA human leukocyte antigen complex P5(HCP5) was identified as the key lncRNA associated with RA onset. Infiltration analysis revealed increased abundance of CD8+ T cells, γδ T cells, and M1 macrophages while decreased abundance of M2 macrophages in RA synovial tissue. Correlation analysis demonstrated that the lncRNA HCP5 expression was positively associated with the infiltration abundance of CD8+ T cells, γδ T cells, and M1 macrophages in RA synovial tissue. Furthermore,the expression of lncRNA HCP5 in RA synovial cells was up-regulated. Conclusion lncRNA HCP5 expression is up-regulated in RA synovial tissue and potentially associated with immune cells infiltration.

Comparison of prognostic value between CAD-RADS 1.0 and CAD-RADS 2.0 evaluated by convolutional neural networks based CCTA.

Objectives: The aim of the present study was to investigate the prognostic value of the novel coronary artery disease reporting and data system (CAD-RADS) 2.0 compared with CAD-RADS 1.0 in patients with suspectedcoronary artery disease (CAD) evaluated by convolutional neural networks (CNN) based coronary computed tomography angiography (CCTA). Methods: A total of 1796 consecutive inpatients with suspected CAD were evaluated by CCTA for CAD-RADS 1.0 and CAD-RADS 2.0 classifications. Kaplan-Meier and multivariate Cox models were used to estimate major adverse cardiovascular events (MACE) inclusive of all-cause mortality or myocardial infarction (MI). The C-statistic was used to assess the discriminatory ability of the two classifications. Results: In total, 94 (5.2%) MACE occurred over the median follow-up of 45.25 months (interquartile range 43.53-46.63 months). The annualized MACE rate was 0.014 (95% CI: 0.011-0.017). Kaplan-Meier survival curves indicated that the CAD-RADS classification, segment involvement score (SIS) grade, and Computed Tomography Fractional Flow Reserve (CT-FFR) classification were all significantly associated with the increase in the cumulative MACE (all P < 0.001). CAD-RADS classification, SIS grade, and CT-FFR classification were significantly associated with endpoint in univariate and multivariate Cox analysis. CAD-RADS 2.0 showed a further incremental increase in the prognostic value in predicting MACE (c-statistic 0.702, 95% CI: 0.641-0.763, P = 0.047), compared with CAD-RADS 1.0. Conclusions: The novel CAD-RADS 2.0 evaluated by CNN-based CCTA showed higher prognostic value of MACE than CAD-RADS 1.0 in patients with suspected CAD.

Graft Patency Assessment with CCTA Using a Comprehensive Singlebranch Bridging Score.

AIMS: This study aims to assess the prognostic value of graft patency with coronary computed tomography angiography (CCTA) using a comprehensive single-branch targeted atherosclerotic risk score (CSBS) in patients before coronary artery bypass grafting (CABG). METHODS: This retrospective study contains a total of 88 patients who underwent clinical CCTA before off-pump CABG surgery between 2015 and 2018. Graft failure was defined as patients with missing multi-slice CCTA or coronary angiography. The predictive value of CSBS (ranging from 0-70 and divided into 2 groups: < 20 and ≥20) was analyzed using Kaplan-Meier analysis and Cox regression models. RESULTS: Patients' mean age was 61.2 ± 10.5 years, with a mean follow-up of 20.4 ± 15.2 months. A total of 203 grafts (21.5% arterial grafts) were analyzed and 30 of the vessels were occluded (14.8%). There was no significant difference in graft occlusion among the three targeted vessel groups. The Cox proportional hazard analysis showed that CSBS < 20 was a significant predictor of graft failure. CONCLUSION: Lower comprehensive single-branch targeted atherosclerotic risk score evaluated by CCTA is an independent prognostic factor for graft failure in patients before CABG surgery.

Genome-wide identification of PLATZ genes related to cadmium tolerance in Populus trichocarpa and characterization of the role of PtPLATZ3 in phytoremediation of cadmium.

Plant AT-rich sequence and zinc-binding (PLATZ) proteins are a class of plant-specific zinc finger transcription factors that perform critical functions in plant development and resistance. However, the function of PLATZs in heavy metal tolerance has not yet been investigated. Moreover, only a few PLATZ proteins have been functionally characterized in tree species. In this study, we identified 18 PtPLATZ genes in Populus trichocarpa, an important woody model plant, and classified them into five groups. PtPLATZ genes attributed to the same clade usually possess similar exon-intron structures containing two or three introns, as well as a similar motif composition. Furthermore, chromosomal location analysis indicated an uneven distribution of PtPLATZ genes on 13 of the 19 Populus chromosomes. Promoter cis-acting element prediction and gene expression analysis showed that PtPLATZ genes were highly responsive to heavy metal stress. Heterologous yeast expression revealed that PtPLATZ1, PtPLATZ2, PtPLATZ3, PtPLATZ4, PtPLATZ8 and PtPLATZ9 are significantly involved in Cd tolerance. In addition, transgenic expression of PtPLATZ3 significantly enhanced Cd tolerance and accumulation, slowed the decline in chlorophyll content, maintained membrane integrity in Populus, and increased the expression of genes related to Cd tolerance and accumulation. In conclusion, our results suggest the potential of PtPLATZ3 to improve Cd tolerance and accumulation in Populus, which is of great significance for phytoremediation.

Clinical Evaluation of the Automatic Coronary Artery Disease Reporting and Data System (CAD-RADS) in Coronary Computed Tomography Angiography Using Convolutional Neural Networks.

RATIONALE AND OBJECTIVES: The coronary artery disease reporting and data system (CAD-RADS™) was recently introduced to standardise reporting. We aimed to evaluate the utility of an automatic postprocessing and reporting system based on CAD-RADS™ in suspected coronary artery disease (CAD) patients. MATERIALS AND METHODS: Clinical evaluation was performed in 346 patients who underwent coronary computed tomography angiography (CCTA). We compared deep learning (DL)-based CCTA with human readers for evaluation of CAD-RADS™ with commercially-available automated segmentation and manual postprocessing in a retrospective validation cohort. RESULTS: Compared with invasive coronary angiography, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the DL model for diagnosis of CAD were 79.02%, 86.52%, 89.50%, 73.94%, and 82.08%, respectively. There was no significant difference between the DL-based and the reader-based CAD-RADS™ grading of CCTA results. Consistency testing showed that the Kappa value between the model and the readers was 0.775 (95% confidence interval [CI]: 0.728-0.823, p < 0.001), 0.802 (95% CI: 0.756-0.847, p < 0.001), and 0.796 (95% CI: 0.750-0.843, p < 0.001), respectively. This system reduces the time taken from 14.97 ± 1.80 min to 5.02 ± 0.8 min (p < 0.001). CONCLUSION: The standardised reporting of DL-based CAD-RADS™ in CCTA can accurately and rapidly evaluate suspected CAD patients, and has good consistency with grading by radiologists.

Single-Molecule and Vesicle Trafficking Analysis of Ubiquitination Involved in the Activity of Ammonium Transporter AMT1;3 in Arbidopsis under High Ammonium Stress.

Plants absorb nitrogen from the soil using ammonium transporters (AMTs). Plants can precisely regulate AMT1;3 levels using sophisticated regulatory systems, ensuring adequate nitrogen uptake without hazardous ammonium production. Here, we demonstrated that ubiquitylation can contribute to AMT1;3 degradation under high ammonium stress. Using the ubiquitin site mutant AMT1;3K75R,K233R-EGFP, we demonstrated that the loss of ubiquitination affects the dynamic characteristics of AMT1;3 proteins on the plasma membrane and markedly inhibits the endocytosis of AMT1;3 proteins under high ammonium stress. AMT1;3K75R,K233R-EGFP plants also showed inhibition of protein degradation that targets the vesicular pathway after being exposed to high levels of ammonium. Our findings showed that the dynamic properties, endocytosis, and vesicle trafficking pathways of AMT1;3 proteins are altered in AMT1;3K75R,K233R-EGFP under high ammonium conditions.

Development of chloroplast transformation and gene expression regulation technology in land plants.

Chloroplasts in land plants have their own small circular DNA that is presumed to have originated from cyanobacteria-related endosymbionts, and the chloroplast genome is an attractive target to improve photosynthetic ability and crop yield. However, to date, most transgenic or genetic engineering technologies for plants are restricted to manipulations of the nuclear genome. In this review, we provide a comprehensive overview of chloroplast genetic engineering and regulation of gene expression from the perspective of history and biology, focusing on current and latest methods. In addition, we suggest techniques that may regulate the chloroplast gene expression at the transcriptional or post-transcriptional level.

Atomic-scale observation of dynamic grain boundary structural transformation during shear-mediated migration.

Grain boundary (GB) structural change is commonly observed during and after stress-driven GB migration in nanocrystalline materials, but its exact atomic scale transformation has not been explored experimentally. Here, using in situ high-resolution transmission electron microscopy combined with molecular dynamics simulations, we observed the dynamic GB structural transformation stemming from reversible facet transformation and GB dissociation during the shear-mediated migration of faceted GBs in gold nanocrystals. A reversible transformation was found to occur between (002)/(111) and Σ11(113) GB facets, accomplished by the coalescence and detachment of [Formula: see text]-type GB steps or disconnections that mediated the GB migration. In comparison, the dissociation of (002)/(111) GB into Σ11(113) and Σ3(111) GBs occurred via the reaction of [Formula: see text]-type steps that involved the emission of partial dislocations. Furthermore, these transformations were loading dependent and could be accommodated by GB junctions. This work provides atomistic insights into the dynamic structural transformation during GB migration.

Ginsenoside Rk2 Protects against Ulcerative Colitis via Inactivating ERK/MEK Pathway by SIRT1.

BACKGROUND: Chinese traditional medicine is widely used in the treatment of ulcerative colitis (UC). Ginsenoside Rk2 is a newly discovered dammarane triterpenoid saponin isolated from ginseng. Our study aimed to investigate the effects of Ginsenoside Rk2 on UC. METHODS: Human clones of colorectal adenocarcinoma Caco-2 cells and human intestinal epithelial THP-1 cells were co-cultured to establish a UC model in vitro. Cell viability and apoptosis were analyzed by cell counting kit 8 (CCK-8) and flow cytometry assay, respectively. Inflammatory cytokines' mRNA levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Western blot was applied to examine the protein expression of apoptosis-associated proteins and the activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MEK) pathway. Furthermore, fisetin, an ERK kinase activator, was used to carry out rescue experiment. SRT1720, an activator of SIRT1, was applied to increase the SIRT1 protein levels while SIRT1 inhibitor nicotinamide (NAM) exerted the opposite effect. RESULTS: Ginsenoside Rk2 promoted cell viability, suppressed cell apoptosis, and reduced the release of pro-inflammatory cytokines including interleukin (IL)-1ß, IL-6, IL-10, and tumor necrosis factor-α (TNF-α) of HT-29 cells in UC model in a concentration-dependent manner. Meanwhile, the inhibitory effects of Ginsenoside Rk2 on the ERK/MEK pathway strengthened with the increase of concentration, and was verified by fisetin application. Furthermore, the upregulation of SIRT1 induced by Ginsenoside Rk2 prompted dephosphorylation of ERK and MEK to attenuate ERK/MEK pathway activation and reduced inflammatory progress, which was confirmed by SRT1720 as well as NAM. CONCLUSIONS: Ginsenoside Rk2 inactivated ERK/MEK pathway by regulating SIRT1 to restore the cellular function of human intestinal epithelial THP-1 cells. Therefore, Ginsenoside Rk2 may be effective in the treatment of UC.

Curcumin relieved the rheumatoid arthritis progression via modulating the linc00052/miR-126-5p/PIAS2 axis.

Curcumin, with its antioxidant, anti-inflammatory, and antitumor properties, is widely used in the treatment of bone disorders, including rheumatoid arthritis (RA). We investigated the effects of curcumin on fibroblast-like synoviocytes in RA and its underlying mechanism. mRNA and microRNA (miRNA) expression levels were determined using reverse transcription-quantitative polymerase chain reaction. Cellular functions were detected using cell counting kit-8, 5-ethynyl-2'-deoxyuridine, Transwell, and flow cytometric assays. Enzyme-linked immunosorbent assay was performed to measure the cytokine release. Western blotting was used to determine the protein expression levels. An in vivo assay was performed to verify the role of linc00052 in RA. Curcumin promoted apoptosis and inhibited the growth, migration, and invasion of RA fibroblast-like synovial (RAFLS) cells. Curcumin treatment suppressed the inflammatory response of RAFLS cells. Moreover, curcumin increased linc00052 levels, and linc00052 knockdown reversed the effects of curcumin. Additionally, linc00052 functioned as a competing endogenous RNA to upregulate the expression of the protein inhibitor of activated STAT 2 (PIAS2) by sponging miR-126-5p. Curcumin inhibited the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. In vivo assays showed that curcumin decreased the arthritis score and improved inflammatory infiltration and synovial cell proliferation. These results reveal that curcumin protects against RA by regulating the inc00052/miR-126-5p/PIAS2 axis through JAK2/STAT3 signaling pathway.

Identification of Synovial Fibroblast-Associated Neuropeptide Genes and m6A Factors in Rheumatoid Arthritis Using Single-Cell Analysis and Machine Learning.

OBJECTIVES: Synovial fibroblasts (SFs) play an important role in the development and progression of rheumatoid arthritis (RA). However, the pathogenic mechanism of SFs remains unclear. The objective of this study was to investigate how neuropeptides and N6-methyladenosine (m6A) played an important role in the underlying pathogenic processes of SFs that contribute to the development of RA. METHODS: Single-cell RNA sequencing data were examined using single-cell analysis and machine learning. SF subgroups were identified based on the clustering and annotation results of the single-cell analysis. Moreover, cell-cell communication was used to analyse neuropeptide-related receptor and ligand pairs on the surface of SF cell membranes. Machine learning was used to explore the m6A factors acting on these neuropeptide genes. RESULTS: NPR3, GHR, BDKRB2, and CALCRL, four neuropeptide genes, were shown to be differently expressed among SF subgroups. Further investigation of receptor-ligand interactions found that NPR3 (in conjunction with NPPC, OSTN, NPPB, and NPPA) and GHR (in conjunction with GH1 and GH2) may have a role in SF interactions. As predicted by machine learning, IGFBP2 and METTL3 were identified as key factors regulating m6A of NPR3 and GHR. The expression levels and enrichment pathways of METTL3 and IGFBP2 were different among SF subgroups. CONCLUSIONS: Single-cell analysis and machine learning efficiently identified neuropeptide genes and m6A factors that perform important regulatory functions in RA. Our strategy may provide a basis for future studies to identify pathogenic cell subpopulations and molecular mechanisms in RA and other diseases.