ATM-AMPKα mediated LAG-3 expression suppresses T cell function in prostate cancer.

Immunotherapy for prostate cancer (PCa) faces serious challenges. Therefore, the co-inhibitory receptors that regulate T cell function of PCa must be elucidated. Here we identified that the inhibitory receptor LAG3 was significantly induced in T cells from PCa patients. Gene array analysis revealed that insufficient ataxia telangiectasia mutated (ATM) gene expression in PCa T cells was responsible for the elevated LAG3 expression. Mechanistically, insufficient ATM expression impaired its ability to activate AMPKα signaling and CD4+ T cell functions, which further enhances the binding of the transcription factors XBP1 and EGR2 to LAG3 promoter. Reconstitution of ATM and inhibition of XBP1 or EGR2 in PCa T cells suppressed LAG3 expression and restored the effector function of CD4+ T cells from PCa. Our study revealed the mechanism of LAG3 upregulation in CD4+ T lymphocytes of PCa patients and may provide insights for the development of immunotherapeutic strategies for PCa treatment.

Customized whole brain-covering 3D GRASE in multi-delay pseudo-continuous arterial spin labeling for duplex distinct hemodynamic mapping contrasts of brain tissues and circulation pathways.

Gradient and spin echo (GRASE) is widely employed in arterial spin labeling (ASL) as an efficient readout sequence. Hemodynamic parameter mappings of perfusion, such as cerebral blood flow (CBF) and arterial transit time (ATT), can be derived via multi-delay ASL acquisitions. Multi-delay ASL perfusion imaging inevitably suffers limited signal-to-noise ratio (SNR) since a motion-sensitized vessel suppressing module has to be employed to highlight perfusion signals. The present work reveals that in multi-delay ASL, manipulation of GRASE sequence on either planar imaging echo echo train for adjusted spatial resolutions or FSE echo train for modulated extent ofT2-blurring can significantly alter the mapping contrasts among tissues and among cerebral lobes under different pathways of blood circulation, and meanwhile regulates SNR. Four separate multi-delay ASL scans with different echo train designs in 3D whole brain covering GRASE were carried out for healthy subjects to evaluate the variations in regard to the parameter quantifications and SNR. Based on the quantification mappings, the GRASE acquisition with moderate spatial resolution (3.5 × 3.5 × 4 mm3) and segmentedkzscheme was recognized for the first time to be recommended for more unambiguous CBF and ATT contrasts between GM and WM in conjunction with more enhanced ATT contrast between anterior and posterior cerebral circulations, with reasonably good SNR. The technical proposal is of great value for the cutting-edge research of a variety of neurological diseases of global concerns.

CPT1A-Mediated Fatty Acid Oxidation Promotes Precursor Osteoclast Fusion in Rheumatoid Arthritis.

The overproduction of osteoclasts, leading to bone destruction in patients with rheumatoid arthritis (RA), is well established. However, little is known about the metabolic dysfunction of osteoclast precursors (OCPs) in RA. Herein, we show that increasing fatty acid oxidation (FAO) induces OCP fusion. Carnitine palmitoyltransferase IA (CPT1A), which is important for carnitine transportation and is involved in FAO in the mitochondria, is upregulated in RA patients. This metabolic change further increases the expression of clathrin heavy chain (CLTC) and clathrin light chain A (CLTA) by enhancing the binding of the transcription factor CCAAT/enhancer binding protein ß (C/EBPß) to the promoters of CLTA and CLTC. This drives clathrin-dependent endocytosis pathway, which attenuates fusion receptors in the cellular membrane and contributes to increased podosome structure formation. This study reveals a new mechanism through which FAO metabolism participates in joint destruction in RA and provides a novel therapeutic direction for the development of drugs against bone destruction in patients with RA.

Comparison of New Glucose-Lowering Drugs on the Risk of Pancreatitis in Type 2 Diabetes: A Network Meta-Analysis.

OBJECTIVE: To explore whether new glucose-lowering drugs increase the risk of pancreatitis in individuals with type 2 diabetes. This present network meta-analysis aimed to investigate the risk of pancreatitis associated with the use of glucagon-like peptide-1 (GLP-1) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors in the treatment of type 2 diabetes mellitus. METHODS: PubMed, Web of Science, Embase, and the Cochrane Library were searched. The literature was published from the date of their inception to July 21, 2021, including placebo-controlled or head-to-head trials of 2 new glucose-lowering drugs. The relative ratio (RR) and 95% confidence interval (CI) were used to assess the risk of GLP-1 agonists and DPP-4 inhibitors for pancreatitis or pancreatic cancer among patients with type 2 diabetes. RESULTS: Seventeen studies were identified, covered 102 257 participants. The pooled results showed a neutral relationship between GLP-1 agonists and pancreatitis (overall RR, 0.96; 95% CI, 0.31-3.00) or pancreatic cancer (overall RR, 1.10; 95% CI, 0.31-4.10) compared with placebo. Meanwhile, DPP-4 inhibitors were not associated with the increased risk of pancreatitis (overall RR, 1.60; 95% CI, 0.25-11.00) or pancreatic cancer (overall RR, 0.79; 95% CI, 0.26-2.40). Among them, lixisenatide and saxagliptin may be the safest drugs compared with other drugs according to the ranking of probability. Sensitivity and subgroup analysis confirmed the stability of the core results. CONCLUSION: The most obvious finding of this study is that GLP-1 agonists and DPP-4 inhibitors are safe with respect to the risk of pancreatitis and pancreatic cancer compared with placebo. This conclusion may provide useful evidence for correlated clinical researches.

ATPIF1 maintains normal mitochondrial structure which is impaired by CCM3 deficiency in endothelial cells.

BACKGROUND: Numerous signaling pathways have been demonstrated experimentally to affect the pathogenesis of cerebral cavernous malformations (CCM), a disease that can be caused by CCM3 deficiency. However, the understanding of the CCM progression is still limited. The objective of the present work was to elucidate the role of CCM3 by RNA-seq screening of CCM3 knockout mice. RESULTS: We found that ATPIF1 was decreased in siCCM3-treated Human Umbilical Vein Endothelial Cells (HUVECs), and the overexpression of ATPIF1 attenuated the changes in cell proliferation, adhesion and migration caused by siCCM3. The probable mechanism involved the conserved ATP concentration in mitochondria and the elongated morphology of the organelles. By using the CRISPR-cas9 system, we generated CCM3-KO Endothelial Progenitor Cells (EPCs) and found that the knockout of CCM3 destroyed the morphology of mitochondria, impaired the mitochondrial membrane potential and increased mitophagy. Overexpression of ATPIF1 contributed to the maintenance of normal structure of mitochondria, inhibiting activation of mitophagy and other signaling proteins (e.g., KLF4 and Tie2). The expression of KLF4 returned to normal in CCM3-KO EPCs after 2 days of re-overexpression of CCM3, but not other signaling proteins. CONCLUSION: ATPIF1 maintains the normal structure of mitochondria, inhibiting the activation of mitophagy and other signaling pathway in endothelial cells. Loss of CCM3 leads to the destruction of mitochondria and activation of signaling pathways, which can be regulated by KLF4.

METTL1-mediated m7G methylation maintains pluripotency in human stem cells and limits mesoderm differentiation and vascular development.

BACKGROUND: 7-Methylguanosine (m7G) is one of the most conserved modifications in nucleosides within tRNAs and rRNAs. It plays essential roles in the regulation of mRNA export, splicing, and translation. Recent studies highlighted the importance of METTL1-mediated m7G tRNA methylome in the self-renewal of mouse embryonic stem cells (mESCs) through its ability to regulate mRNA translation. However, the exact mechanisms by which METTL1 regulates pluripotency and differentiation in human induced pluripotent stem cells (hiPSCs) remain unknown. In this study, we evaluated the functions and underlying molecular mechanisms of METTL1 in regulating hiPSC self-renewal and differentiation in vivo and in vitro. METHODS: By establishing METTL1 knockdown (KD) hiPSCs, gene expression profiling was performed by RNA sequencing followed by pathway analyses. Anti-m7G northwestern assay was used to identify m7G modifications in tRNAs and mRNAs. Polysome profiling was used to assess the translation efficiency of the major pluripotent transcription factors. Moreover, the in vitro and in vivo differentiation capacities of METTL1-KD hiPSCs were assessed in embryoid body (EB) formation and teratoma formation assays. RESULTS: METTL1 silencing resulted in alterations in the global m7G profile in hiPSCs and reduced the translational efficiency of stem cell marker genes. METTL1-KD hiPSCs exhibited reduced pluripotency with slower cell cycling. Moreover, METTL1 silencing accelerates hiPSC differentiation into EBs and promotes the expression of mesoderm-related genes. Similarly, METTL1 knockdown enhances teratoma formation and mesoderm differentiation in vivo by promoting cell proliferation and angiogenesis in nude mice. CONCLUSION: Our findings provided novel insight into the critical role of METTL1-mediated m7G modification in the regulation of hiPSC pluripotency and differentiation, as well as its potential roles in vascular development and the treatment of vascular diseases.

METTL1 limits differentiation and functioning of EPCs derived from human-induced pluripotent stem cells through a MAPK/ERK pathway.

Transplantation of endothelial progenitor cells (EPCs) has high therapeutic potential for ischemia-related ailments like heart attacks and claudication. Due to limited EPC sources, direct reprogramming is a fast-developing way to convert human-induced pluripotent stem cells (hiPSCs) into EPCs fit for transplantation. However, the procedural efficacy was affected by multiple factors, including epigenetic modifications. Recent studies have shown that m7G methylation mediated by Methyltransferase like 1 (METTL1) is required for mouse embryonic stem cells (mESCs) to differentiate normally. Yet, its contributions to EPC differentiation still require elucidation. Here, using immunofluorescence microscopy and Fluorescence-activated Cell Sorting (FACS), we found that the typical EPC markers were significantly increased in METTL1 knockdown (METTL1-KD) hiPSCs-derived EPCs compared to those of control types. In addition, we found that METTL1 knockdown activates the MAPK/ERK signaling pathway during EPCs differentiation from hiPSCs. Furthermore, functional properties of METTL1-KD EPCs were significantly raised above those of control hiPSCs-derived EPCs. Moreover, we proved that METTL1-KD hiPSCs-derived EPCs significantly accelerate vascular smooth muscle cell proliferation and 'phenotype switching' through a co-culture system. To sum up, our results demonstrate that METTL1-KD significantly promotes the differentiation of EPCs along with their in vitro functions, and this effect may be achieved through activation of the MAPK/ERK signaling pathway. This enhances current knowledge of EPC generation from hiPSCs and presents a new therapeutic target of vascular diseases.

Adaptation in 5-HT2 receptors-CaMKII signaling in lateral habenula underlies increased nociceptive-sensitivity in ethanol-withdrawn rats.

Alcoholics often experience hyperalgesia, especially during abstinence, yet the underlying cellular and molecular bases are unclear. Recent evidence suggests that 5-HT type 2 receptors (5-HT2Rs) at glutamatergic synapses on lateral habenula (LHb) neurons may play a critical role. We, therefore, measured paw withdrawal responses to thermal and mechanical stimuli, and alcohol intake in a rat model of intermittent drinking paradigm, as well as spontaneous glutamatergic transmission (sEPSCs), and firing of LHb neurons in brain slices. Here, we report that nociceptive sensitivity was higher in rats at 24 h withdrawal from chronic alcohol consumption than that of alcohol-naive counterparts. The basal frequency of sEPSCs and firings was higher in slices of withdrawn rats than that of Naïve rats, and 5-HT2R antagonists attenuated the enhancement. Also, an acute ethanol-induced increase of sEPSCs and firings was smaller in withdrawal than in Naïve rats; it was attenuated by 5-HT2R antagonists but mimicked by 5-HT2R agonists. Importantly, intra-LHb infusion of 5-HT2R agonists increased nociceptive sensitivity in Naïve rats, while antagonists or 5-HT reuptake blocker decreased nociceptive sensitivity and alcohol intake in withdrawn rats. Additionally, KN-62, a CaMKII inhibitor, attenuated the enhancement of EPSCs and firing induced by acute alcohol and by 5-HT2R agonist. Furthermore, intra-LHb KN-62 reduced nociceptive sensitivity and alcohol intake. Quantitative real-time PCR assay detected mRNA of 5-HT2A and 2C in the LHb. Thus adaptation in 5-HT2R-CaMKII signaling pathway contributes to the hyper-glutamatergic state, the hyperactivity of LHb neurons as well as the higher nociceptive sensitivity in rats withdrawn from chronic alcohol consumption.

Impaired Tip60-mediated Foxp3 acetylation attenuates regulatory T cell development in rheumatoid arthritis.

In rheumatoid arthritis (RA), imbalanced T cells subsets play a critical role in sustaining chronic inflammatory responses in the synovium. Naïve T cells in RA patients undergo maldifferentiation, including an increase in the effector Th1/Th17 lineage and a reduction in regulatory T (Treg) cells. Upon stimulation, naïve CD4+CD45RO- T cells from RA patients exhibited insufficient expression of Foxp3, which induced a deficiency in Tregs production and an imbalance of Treg/Th17 differentiation. Further mechanistic study indicated that RA T cells failed to produce sufficient levels of the histone acetyltransferase Tip60, leading to reduced acetylation of Foxp3; this, in turn, decreased Foxp3 expression, impaired Treg commitment, and promoted Th17 production. Moreover, in human synovium chimeric mice, suppression of Tip60 activity in healthy T cells promoted tissue infiltration and arthritogenesis, while reconstitution of Tip60 in RA T cells suppressed synovitis and effector T cell infiltration. Our findings link T cell maldifferentiation and tissue infiltration with Tip60-mediated Foxp3 acetylation and identify Tip60 as a potential therapeutic target for suppression of tissue inflammation and autoimmunogenesis in RA.

Dynamic m6A mRNA methylation reveals the role of METTL3-m6A-CDCP1 signaling axis in chemical carcinogenesis.

N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNAs. Despite its functional importance in various physiological events, the role of m6A in chemical carcinogenesis remains largely unknown. Here we profiled the dynamic m6A mRNA modification during cellular transformation induced by chemical carcinogens and identified a subset of cell transformation-related, concordantly modulated m6A sites. Notably, the increased m6A in 3'-UTR mRNA of oncogene CDCP1 was found in malignant transformed cells. Mechanistically, the m6A methyltransferase METTL3 and demethylases ALKBH5 mediate the m6A modification in 3'-UTR of CDCP1 mRNA. METTL3 and m6A reader YTHDF1 preferentially recognize m6A residues on CPCP1 3'-UTR and promote CDCP1 translation. We further showed that METTL3 and CDCP1 are upregulated in the bladder cancer patient samples and the expression of METTL3 and CDCP1 is correlated with the progression status of the bladder cancers. Inhibition of the METTL3-m6A-CDCP1 axis resulted in decreased growth and progression of chemical-transformed cells and bladder cancer cells. Most importantly, METTL3-m6A-CDCP1 axis has synergistic effect with chemical carcinogens in promoting malignant transformation of uroepithelial cells and bladder cancer tumorigenesis in vitro and in vivo. Taken together, our results identify dynamic m6A modification in chemical-induced malignant transformation and provide insight into critical roles of the METTL3-m6A-CDCP1 axis in chemical carcinogenesis.

Mitochondria, Oxidative Stress and Innate Immunity.

Canonical functions of mitochondria include the regulation of cellular survival, orchestration of anabolic and metabolic pathways, as well as reactive oxygen species (ROS) signaling. Recent discoveries, nevertheless, have demonstrated that mitochondria are also critical elements to stimulate innate immune signaling cascade that is able to intensify the inflammation upon cytotoxic stimuli beyond microbial infection. Here we review the expanding research field of mitochondria and oxidative stress in innate immune system to highlight the new mechanistic insights and discuss the pathological relevance of mitochondrial dysregulation induced aberrant innate immune responses in a growing list of sterile inflammatory diseases.

Nonepimerizing Alkylation of H-P Species to Stereospecifically Generate P-Stereogenic Phosphine Oxides: A Shortcut to Bidentate Tertiary Phosphine Ligands.

The secondary RP-(-)-menthyl alkylphosphine oxide was confirmed as configurationally stable toward base and was used in base-promoted alkylation, stereospecifically affording P-retained bis or functional tertiary phosphine oxides in excellent yields. The alkylated products were deoxygenated using oxalyl chloride followed by ZnCl2-NaBH4 to form P-inversed bidentate phosphine boranes in high stereoselectivities.

Preparation of Optically Pure Tertiary Phosphine Oxides via the Addition of P-Stereogenic Secondary Phosphine Oxide to Activated Alkenes.

Functionalized P,C-stereogenic tertiary phosphine oxides were prepared by the addition of (RP)-menthyl phenylphosphine oxide to activated olefins, in high drP and drC, and were isolated in excellent yields. The reaction was readily catalyzed by Ca(OH)2 or occurred with gentle heating. A wide range of substrates, including vinyl ketones, esters, nitriles, and nitro alkenes, can be used in the reaction.

Double Asymmetric Induction During the Addition of (RP)-Menthyl Phenyl Phosphine Oxide to Chiral Aldimines.

P,C-Stereogenic α-amino phosphine oxides were prepared from the addition of (RP )-menthyl phenyl phosphine oxide to chiral aldimines under neat condition at 80 °C in up to 91:9 drC and 99% yields. The diastereoselectivity was mainly induced by chiral phosphorus that showed matched or mismatched induction with (S)- or (R)-aldimines, respectively.

One-pot process that efficiently generates single stereoisomers of 1,3-bisphosphinylpropanes having five chiral centers.

P,C-stereogenic 1,3-bisphosphinylpropanes 3 that have up to five stereogenic centers could be obtained stereoselectively in high yields by a one-step reaction of (RP)-menthylphenylphosphine oxide 1 with α,ß-unsaturated aldehydes 2 catalyzed by KOH at room temperature. A mechanism was proposed as to involve a stereoselective intermolecular 1,3'-phosphorus migration from the 1,2-adduct of 1 with 2 to another 2 generating a 1,4-adduct that subsequently reacts with 1 to produce 3.

The genome-wide transcriptional response to neonatal hyperoxia identifies Ahr as a key regulator.

Premature infants requiring supplemental oxygen are at increased risk for developing bronchopulmonary dysplasia (BPD). Rodent models involving neonatal exposure to excessive oxygen concentrations (hyperoxia) have helped to identify mechanisms of BPD-associated pathology. Genome-wide assessments of the effects of hyperoxia in neonatal mouse lungs could identify novel BPD-related genes and pathways. Newborn C57BL/6 mice were exposed to 100% oxygen for 10 days, and whole lung tissue RNA was used for high-throughput, sequencing-based transcriptomic analysis (RNA-Seq). Significance Analysis of Microarrays and Ingenuity Pathway Analysis were used to identify genes and pathways affected. Expression patterns for selected genes were validated by qPCR. Mechanistic relationships between genes were further tested in cultured mouse lung epithelial cells. We identified 300 genes significantly and substantially affected following acute neonatal hyperoxia. Canonical pathways dysregulated in hyperoxia lungs included nuclear factor (erythryoid-derived-2)-like 2-mediated oxidative stress signaling, p53 signaling, eNOS signaling, and aryl hydrocarbon receptor (Ahr) pathways. Cluster analysis identified Ccnd1, Cdkn1a, and Ahr as critical regulatory nodes in the response to hyperoxia, with Ahr serving as the major effector node. A mechanistic role for Ahr was assessed in lung epithelial cells, and we confirmed its ability to regulate the expression of multiple hyperoxia markers, including Cdkn1a, Pdgfrb, and A2m. We conclude that a global assessment of gene regulation in the acute neonatal hyperoxia model of BPD-like pathology has identified Ahr as one driver of gene dysregulation.