Common hyper-entropy patterns identified in nicotine smoking, marijuana use, and alcohol use based on uni-drug dependence cohorts.

Substance use disorders present similar behaviors and psychopathologies related to impaired decision making/inhibition control and information processing, suggesting common alterations in frontal and limbic brain areas. To test this hypothesis, we identified three uni-substance use cohorts with dependence to only one type of substance from the Human Connectome Project: marijuana dependence, nicotine dependence, and alcohol dependence. Fifty-nine marijuana uses, 34 nicotine smokers, 35 alcohol drinkers, and their age and sex-matched non-substance use controls were identified. We used brain entropy mapping to probe brain alterations in substance use disorders. Compared to non-substance use individuals, all three substance use disorder cohorts had increased brain entropy. Marijuana dependence and nicotine dependence showed overlapped hyper-brain entropy in bilateral dorso-lateral prefrontal cortex, anterior cingulate cortex, and right insula. Hyper-brain entropy in marijuana dependence and alcohol dependence overlap in left insula, left doso-lateral prefrontal cortex, and posterior cingulate. Hyper-brain entropy in nicotine dependence and alcohol dependence overlap only in left dorso-lateral prefrontal cortex. Hyper-brain entropy in those areas was correlated with increased impulsivity or reduced inhibition control in substance use disorder but not in controls. Drug dependence is associated with hyper-brain entropy in the prefrontal cortex and the meso-limbic system, independent of a specific addictive drug. Brain entropy in this circuit provides a sensitive marker to detect brain and behavioral alterations in substance user disorders.

YTHDF2 Regulates Macrophage Polarization through NF-κB and MAPK Signaling Pathway Inhibition or p53 Degradation.

Macrophages are heterogeneous cells that can be polarized into M1 or M2 phenotype. m6A "reader" YTH domain family protein 2 (YTHDF2) has been the m6A binding protein with the highest activity, which can recognize and disturb m6A-containing mRNA in processing bodies to reduce mRNA stability. YTHDF2 is recently identified as an effective RNA binding protein that modulates inflammatory gene levels within inflammatory responses. However, the role of YTHDF2 in M1/M2 macrophage polarization has not been reported. We established a M1/M2 macrophage polarization model using bone-marrow-derived macrophages and found that the expression levels of YTHDF2 in M1/M2 macrophages were both elevated. YTHDF2-knockdown macrophage polarization model was then established, and through qPCR, ELISA, and FACS, we discovered that suppressing YTHDF2 encouraged M1 polarization but restrained M2 polarization. In M1 macrophages, YTHDF2 silencing had no significant effect on p53 expression; however, in YTHDF2 knockdown, M2 macrophage p53 expression was remarkably upregulated. p53 inhibitor PFT-α was then applied and revealed that suppressing p53 simultaneously promoted YTHDF2-silenced M1 polarization and facilitated M2 macrophage polarization. Actinomycin D assays were further utilized to examine the mRNA degradation level of different cytokines, and p53 mRNA degradation in YTHDF2-depleted M2 cells was discovered impeded. Western Blot analysis also implied that a deficit in YTHDF2 expression may activate MAPK and NF-κB pathways. In this study, YTHDF2 induces M2 macrophage polarization by promoting the degradation of p53 mRNA. YTHDF2 suppresses M1 macrophage polarization by inhibiting NF-κB, p38, and JNK signaling pathways, yet p53 remains unaffected in YTHDF2-silenced M1 macrophages.

METTL3 Modulates Osteoclast Differentiation and Function by Controlling RNA Stability and Nuclear Export.

Osteoclast differentiation and function are crucial for maintaining bone homeostasis and preserving skeletal integrity. N6-methyladenosine (m6A) is an abundant mRNA modification that has recently been shown to be important in regulating cell lineage differentiation. Nevertheless, the effect of m6A on osteoclast differentiation remains unknown. In the present study, we observed that the m6A level and methyltransferase METTL3 expression increased during osteoclast differentiation. Mettl3 knockdown resulted in an increased size but a decreased bone-resorbing ability of osteoclasts. The expression of osteoclast-specific genes (Nfatc1, c-Fos, Ctsk, Acp5 and Dcstamp) was inhibited by Mettl3 depletion, while the expression of the cellular fusion-specific gene Atp6v0d2 was upregulated. Mechanistically, Mettl3 knockdown elevated the mRNA stability of Atp6v0d2 and the same result was obtained when the m6A-binding protein YTHDF2 was silenced. Moreover, the phosphorylation levels of key molecules in the MAPK, NF-κB and PI3K-AKT signaling pathways were reduced upon Mettl3 deficiency. Depletion of Mettl3 maintained the retention of Traf6 mRNA in the nucleus and reduced the protein levels of TRAF6. Taken together, our data suggest that METTL3 regulates osteoclast differentiation and function through different mechanisms involving Atp6v0d2 mRNA degradation mediated by YTHDF2 and Traf6 mRNA nuclear export. These findings elucidate the molecular basis of RNA epigenetic regulation in osteoclast development.

Cerebral functional activity and connectivity changes in anti-N-methyl-D-aspartate receptor encephalitis: A resting-state fMRI study.

BACKGROUND: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis showing severe neuropsychiatric symptoms is the most common type of autoimmune encephalitis. However, the corresponding standard clinical magnetic resonance imaging (MRI) presents normal or atypical in the majority of patients with anti-NMDAR encephalitis. Here, this study aimed to investigate the alterations in brain functional activity in patients with anti-NMDAR encephalitis and whether these alterations contributed to cognition and mood disorders. METHODS: Seventeen patients with anti-NMDAR encephalitis and eighteen gender, age and education-matched healthy controls were recruited. All participants underwent neuropsychological tests (including Montreal Cognitive Assessment (MoCA), Hamilton Anxiety Scale (HAMA), and Hamilton Depression Scale (HAMD24)) and resting-state functional MRI. MRI data was firstly analyzed by amplitude of low-frequency fluctuation (ALFF), and brain regions with altered ALFF between groups were selected as regions of interest for the further functional connectivity (FC) analysis. Correlation analyses were performed to investigate the associations between brain dysfunction and neuropsychological performance. RESULTS: Relative to the healthy controls, patients with anti-NMDAR encephalitis performed inferiorly in the MoCA score, and showed anxiety and depression disorders with higher HAMA and HAMD24 scores (all p < 0.05). In the brain functional activity analysis, the patients showed decreased ALFF values in the bilateral posterior cingulate gyrus, left precuneus, and bilateral cerebellum (false- discovery- rate corrected, p < 0.05). Furthermore, relative to the control group, the patients showed significantly increased FC between the left posterior cingulate cortex (PCC) and the bilateral lingual gyrus, right calcarine, right cuneus, also between the right PCC and the right fusiform gyrus, bilateral lingual gyrus, left calcarine, left cuneus, and right posterior central gyrus (false- discovery- rate corrected, p < 0.05). FC strength between the left posterior cingulate gyrus and right cuneus, and between the right posterior cingulate gyrus and left cuneus were both positively correlated with MoCA memory scores (r = 0.485, p = 0.048; r = 0.550, p = 0.022). CONCLUSION: The present study highlight that decreased spontaneous neural activities and abnormal FC exhibited in the patients with anti-NMDAR encephalitis, which may participate in the process of cognition and emotion deficits. These results may help to elucidate the clinical radiological contradictions in anti-NMDAR encephalitis and contribute to deeper understanding of the pathophysiological mechanism of the disease.

N6-methyladenosine demethylase FTO promotes M1 and M2 macrophage activation.

Macrophage polarization is the driving force of various inflammatory diseases, especially those involved in M1/M2 imbalance. N6-methyladenosine (m6A) is the most prevalent internal mRNA modification in eukaryotes that affects multiple biological processes, including those involved developmental arrest and immune response. However, the role of m6A in macrophage polarization remains unclear. This study found that FTO silencing significantly suppressed both M1 and M2 polarization. FTO depletion decreased the phosphorylation levels of IKKα/ß, IκBα and p65 in the NF-κB signaling pathway. The expression of STAT1 was downregulated in M1-polarized macrophages while the expression of STAT6 and PPAR-γ decreased in M2 polarization after FTO knockdown. The actinomycin D experiments showed that FTO knockdown accelerated mRNA decay of STAT1 and PPAR-γ. Furthermore, the stability and expression of STAT1 and PPAR-γ mRNAs increased when the m6A reader YTHDF2 was silenced. In conclusion, our results suggest that FTO knockdown inhibits the NF-κB signaling pathway and reduces the mRNA stability of STAT1 and PPAR-γ via YTHDF2 involvement, thereby impeding macrophage activation. These findings indicated a previously unrecognized link between FTO and macrophage polarization and might open new avenues for research into the molecular mechanisms of macrophage polarization-related diseases.

DNA methyltransferase DNMT1 inhibits lipopolysaccharide­induced inflammatory response in human dental pulp cells involving the methylation changes of IL­6 and TRAF6.

Dental pulp inflammation is a pathological process characterized by local lesions in dental pulp and the accumulation of inflammatory mediators. DNA methylation of cytosine residues is a key epigenetic modification that is essential for gene transcription, and plays pivotal roles in inflammatory reactions and immune responses. However, the function of cytosine DNA methylation in the innate immune defense against the inflammation of dental pulp is poorly understood. To investigate the effect of DNA methylation in inflamed dental pulp upon innate immune responses, expression levels of the DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) in human dental pulp cells (hDPCs) after lipopolysaccharide (LPS) stimulation were evaluated by western blotting and reverse transcription­quantitative (RT­q) PCR. Only DNMT1 expression was decreased, while the transcription of inflammatory cytokines was increased. In the immune responses of LPS­induced hDPCs, the results of RT­qPCR and ELISA showed that DNMT1 knockdown promoted the production of the pro­inflammatory cytokines, interleukin (IL)­6 and IL­8. Western blotting demonstrated that DNMT1 knockdown increased the phosphorylation levels of IKKα/ß and p38 in the NF­κB and MAPK signaling pathways, respectively. Furthermore, MeDIP and RT­qPCR analysis demonstrated that the 5­methylcytosine levels of the IL­6 and TNF receptor­associated factor 6 (TRAF6) promoters were significantly decreased in DNMT1­deficient hDPCs. Taken together, these results indicated that the expression of DNMT1 was decreased after LPS stimulation in hDPCs. DNMT1 depletion increased LPS­induced cytokine secretion, and activated NF­κB and MAPK signaling; these mechanisms may involve the decreased methylation levels of the IL­6 and TRAF6 gene promoters. This study emphasized the role of DNMT1­dependent DNA methylation on the inflammation of LPS­infected dental pulp and provides a new rationale for the investigation of the molecular mechanisms of inflamed dental pulps.

Voxel-based analysis and multivariate pattern analysis of diffusion tensor imaging study in anti-NMDA receptor encephalitis.

PURPOSE: This study aimed to investigate brain white matter (WM) changes and their relationship to cognition in patients with anti-N-methyl-D-aspartate (anti-NMDA) receptor encephalitis. Multivariate pattern analysis (MVPA) was used to explore brain regions that play an important role in classification. METHODS: Fifteen patients and fifteen controls underwent Montreal Cognitive Assessment (MoCA) and diffusion tensor imaging. Based on fractional anisotropy (FA) and mean diffusivity (MD) for MVPA classification, the weights of each brain region were calculated. RESULTS: Compared with the controls, the patients showed an FA reduction in right middle temporal gyrus, left middle cerebellar peduncle, right praecuneus, and an MD increase in left medial temporal gyrus and left frontal lobe. The MoCA score for patients was lower than controls, especially in executive function, fluency, delayed recall and visual perception items. The FA value of right praecuneus was positively correlated with total MoCA score and fluency score. The MD of left frontal lobe was negatively correlated with total MoCA score, and MD of the left medial temporal gyrus was positively correlated with delayed recall. The accuracy, sensitivity and specificity of classification based on FA were 70%, 60% and 80%, respectively. Based on MD, they were each 80%. The brain regions with large weights from FA and MD overlap in temporal lobe, cerebellum and hippocampus. CONCLUSIONS: These results suggest that WM changes are associated with cognitive deficits. MVPA based on FA and MD has good classification ability. Our study may provide new insights into the pathophysiological mechanisms of residual cognitive deficits.

The effect of DNA methylation on the miRNA expression pattern in lipopolysaccharide-induced inflammatory responses in human dental pulp cells.

Endodontic infection is a widespread oral problem. DNA methylation is a key epigenetic modification that plays important roles in various inflammatory responses, but its role in dental pulp inflammation is poorly understood. In this study, we assessed the expression of DNA methyltransferases (DNMTs) in human dental pulp cells (hDPCs) during lipopolysaccharide (LPS)-induced inflammation and found that DNMT3B mRNA expression was reduced and DNMT1 mRNA and protein levels decreased significantly. Pretreatment with the DNMT inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) significantly enhanced the expression of the inflammatory cytokines IL-6 and IL-8 in LPS-stimulated hDPCs, indicating that DNA methylation may play a role in hDPC inflammation. Studies have reported that some microRNAs (miRNAs) are involved in dental pulp infection. DNA methylation can modulate the inflammatory response by regulating miRNA expression, but this phenomenon has not yet been reported in pulp inflammation. The present study used next-generation sequencing to examine the effect of 5-Aza-CdR on the miRNA expression profile of LPS-treated hDPCs, and the results showed that 5-Aza-CdR pretreatment changed the miRNA expression pattern in hDPCs during inflammation. Among the changed miRNAs, miR-146a-5p, which is a pulp inflammation-related miRNA, demonstrated the most noticeably altered expression. miR-146a-5p could be induced by LPS in hDPCs, and 5-Aza-CdR preincubation or DNMT1 knockdown markedly increased its expression level. However, no significant difference was found in the methylation pattern of the MIR146A promoter with 5-Aza-CdR pretreatment or DNMT1 knockdown in LPS-stimulated hDPCs. These results indicate that DNA methylation may regulate the LPS-induced inflammatory response by changing the miRNA expression in hDPCs.

m6A Reader YTHDF2 Regulates LPS-Induced Inflammatory Response.

N6-methyladenosine (m6A) is an abundant mRNA modification that affects multiple biological processes, including those involved in the cell stress response and viral infection. YTH domain family 2 (YTHDF2) is an m6A-binding protein that affects the localization and stability of targeted mRNA. RNA-binding proteins (RBPs) can regulate the stability of inflammatory gene mRNA transcripts, thus participating in the regulation of inflammatory processes. As an RBP, the role of YTHDF2 in the LPS-induced inflammatory reaction has not been reported. To elucidate the function of YTHDF2 in the inflammatory response of macrophages, we first detected the expression level of YTHDF2 in RAW 264.7 cells, and found that it was upregulated after LPS stimulation. YTHDF2 knockdown significantly increased the LPS-induced IL-6, TNF-α, IL-1ß, and IL-12 expression and the phosphorylation of p65, p38, and ERK1/2 in NF-κB and MAPK signaling. Moreover, the upregulated expression of TNF-α and IL-6 in cells with silenced YTHDF2 expression was downregulated by the NF-κB, p38, and ERK inhibitors. YTHDF2 depletion increased the expression and stability of MAP2K4 and MAP4K4 mRNAs. All of these results suggest that YTHDF2 knockdown increases mRNA expression levels of MAP2K4 and MAP4K4 via stabilizing the mRNA transcripts, which activate MAPK and NF-κB signaling pathways, which promote the expression of proinflammatory cytokines and aggravate the inflammatory response in LPS-stimulated RAW 264.7 cells.

METTL3 Regulates Osteoblast Differentiation and Inflammatory Response via Smad Signaling and MAPK Signaling.

Osteoblasts are crucial bone-building cells that maintain bone homeostasis, whereas inflammatory stimuli can inhibit osteogenesis and activate inflammatory response. N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes and plays important roles in multiple biological processes. However, whether m6A modification affects osteoblast differentiation and inflammatory response remains unknown. To address this issue, we investigated the expression of the N6-adenosine methyltransferase METTL3 and found that it was upregulated during osteoblast differentiation and downregulated after LPS stimulation. We then knocked down METTL3 and observed decreased levels of osteogenic markers, ALP activity, and mineralized nodules, as well as Smad1/5/9 phosphorylation, in LPS-induced inflammation. METTL3 knockdown promoted the mRNA expression and stability of negative regulators of Smad signaling, Smad7 and Smurf1, the same regulatory pattern identified when the m6A-binding protein YTHDF2 was silenced. Moreover, METTL3 depletion enhanced proinflammatory cytokine expression and increased the phosphorylation of ERK, p38, JNK, and p65 in MAPK and NF-κB signaling pathways. The increase in cytokine expression was inhibited after MAPK signaling inhibitor treatment. All data suggest that METTL3 knockdown inhibits osteoblast differentiation and Smad-dependent signaling by stabilizing Smad7 and Smurf1 mRNA transcripts via YTHDF2 involvement and activates the inflammatory response by regulating MAPK signaling in LPS-induced inflammation.

CIA2 deficiency results in impaired oxidative stress response and enhanced intracellular basal UPR activity in Saccharomyces cerevisiae.

Yeast Cia2p is a component of the cytosolic Fe/S protein assembly (CIA) machinery. Initial studies of the CIA machinery were performed in yeast, but the precise role of Cia2p in this eukaryote is still unknown. We report that CIA2 deficiency results in impaired oxidative stress response, as evidenced by increased sensitivity to the oxidant cumene hydroperoxide (CHP), impaired activities of superoxide dismutases and aconitase and decreased replicative lifespan in the mutants. Moreover, intracellular reactive oxygen species levels were significantly increased in CIA2-deficient cells after treatment with CHP. We also show that CIA2-deficient cells display an increased resistance to tunicamycin-induced endoplasmic reticulum (ER) stress, as evidenced by the upregulated splicing of the mRNA of HAC1, which encodes a functional transcription factor that regulates the transcription of unfolded protein response (UPR) target genes, suggesting enhanced intracellular UPR activity. Furthermore, the transcription of several canonical UPR target genes is strongly induced in CIA2-deficient cells as compared with wild-type controls. Taken together, these results suggest the involvement of Cia2p in oxidative and ER stress responses in yeast.