Loss of the m6A methyltransferase METTL3 in monocyte-derived macrophages ameliorates Alzheimer's disease pathology in mice.

Alzheimer's disease (AD) is a heterogeneous disease with complex clinicopathological characteristics. To date, the role of m6A RNA methylation in monocyte-derived macrophages involved in the progression of AD is unknown. In our study, we found that methyltransferase-like 3 (METTL3) deficiency in monocyte-derived macrophages improved cognitive function in an amyloid beta (Aß)-induced AD mouse model. The mechanistic study showed that that METTL3 ablation attenuated the m6A modification in DNA methyltransferase 3A (Dnmt3a) mRNAs and consequently impaired YTH N6-methyladenosine RNA binding protein 1 (YTHDF1)-mediated translation of DNMT3A. We identified that DNMT3A bound to the promoter region of alpha-tubulin acetyltransferase 1 (Atat1) and maintained its expression. METTL3 depletion resulted in the down-regulation of ATAT1, reduced acetylation of α-tubulin and subsequently enhanced migration of monocyte-derived macrophages and Aß clearance, which led to the alleviated symptoms of AD. Collectively, our findings demonstrate that m6A methylation could be a promising target for the treatment of AD in the future.

Risk factors of acute renal injury and in-hospital mortality in adult patients with postcardiotomy cardiogenic shock requiring veno-arterial extracorporeal membrane oxygenation: utility of MELD-XI score.

BACKGROUND: The suitability of model for end-stage liver disease excluding international normalized ratio (MELD-XI) score to predict the incidence of acute kidney injury (AKI) and in-hospital mortality in adult patients with postcardiotomy cardiogenic shock (PCS) requiring venoarterial extracorporeal membrane oxygenation (VA ECMO) remains uncertain. This study was performed to explore whether the MELD-XI score has the association with the incidence of AKI and in-hospital mortality in these patients. METHODS: Adult patients with PCS requiring VA ECMO from January 2012 to December 2017 were enrolled and first classified into AKI group (n = 151) versus no-AKI group (n = 132), then classified into survival group (n = 143) versus no-survival group (n = 140). Multivariate logistic regressions were performed to identify factors independently associated with AKI and mortality. Baseline data were defined as the first measurement available. RESULTS: Of 283 patients, the incidence of AKI was 53.36%. The in-hospital mortality rates were 63.58% and 33.33% in patients with and without AKI (p < 0.0001). Baseline MELD-XI score, baseline serum total bilirubin (T-Bil), baseline blood urea nitrogen (BUN), baseline left ventricular ejection fraction (LVEF), sequential organ failure assessment (SOFA) score, and lactate level at ECMO initiation were shown to be associated with the AKI. Vasoactive-inotropic score (VIS) and SOFA score at ECMO initiation as well as renal failure requiring renal replacement therapy (RRT) were shown to be associated with in-hospital mortality. CONCLUSIONS: The baseline MELD-XI score, baseline BUN, baseline T-Bil, baseline LVEF, SOFA score and lactate at the initiation of ECMO were associated with AKI. AKI, SOFA score, and VIS at the initiation of ECMO were associated with in-hospital mortality, whereas MELD-XI score was not found to be associated with in-hospital mortality. A specific MELD-XI score as a threshold, as well as its sensitivity and specificity, needs to be confirmed in further studies.

UPLC-MS/MS analysis of axitinib and pharmacokinetic application in beagle dogs.

Objective: To develop a method for determining the concentration of axitinib in beagle dog plasma and utilize this method to investigate the pharmacokinetics of orally administered axitinib in beagle dogs. Methods: Plasma samples were processed using acetonitrile precipitation and analyzed by UPLC-MS/MS with sunitinib as an internal standard (IS). Chromatographic separation was achieved on a Waters Acquisition UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 µm) with a gradient elution of acetonitrile and 0.1 % formic acid. Mass spectrometry uses an electrospray ion source for positive ion detection in a multiple reaction monitoring mode. The monitored ion transitions for axitinib and sunitinib were m/z 387 â†’ 355.96 and m/z 399.3 â†’ 282.96, respectively. Six beagle dogs were administered 0.33 mg/kg of axitinib orally, and venous blood samples were collected at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h post-dose for pharmacokinetic analysis. Results: The assay demonstrated a linear range of 0.5-100 ng/mL (r2 = 0.9992), and the lower limit of quantification was up to 0.5 ng/mL. Precision, as assessed by relative standard deviation (RSD), was within 8.64 % for both intraday and interday variability. The relative error (RE) for precision from -2.77 %-1.20 %. The recovery rate of the analytes exceeded 85.28 % and the matrix effect was approximately 100 %. Plasma samples maintained stability under various conditions, including room temperature storage for 12 h, processed on an automatic sampler at 4 °C for 6 h, three freeze-thaw cycles, and long-term storage at -80 °C for 60 days. Pharmacokinetic parameters were determined using DAS 2.0 software, revealing a half-life (T1/2) of 6.05 h and an area under the curve (AUC (0 → ∞)) of 97.13 ng h/mL for axitinib. Conclusions: The UPLC-MS/MS method developed in this study offers high specificity, rapid analysis, high recovery, excellent linearity, and minimal plasma volume requirements, making it well-suited for pharmacokinetic and drug interaction studies in beagles dogs.

Serum untargeted metabolomics analysis of the preventive mechanism of TAETEA Prebiotea on non-alcoholic fatty liver in rats.

Pu-erh tea belongs to the six tea categories of black tea, according to the processing technology and quality characteristics, is divided into two types of raw tea and ripe tea. Raw tea is made from fresh leaves of tea as raw materials, through the process of greening, kneading, sun drying, steam molding and other processes made of tightly pressed tea. Ripe tea is made from Yunnan large-leafed sun green tea, using a specific process, post-fermentation (rapid post-fermentation or slow post-fermentation) processing of loose tea and tightly pressed tea. TAETEA Prebiotea is Puerh Ripe Tea, TAETEA Prebiotea has the effect of increasing insulin level and improving hyperglycemia in mice, and it also has the effect of regulating blood lipids, which can reduce the level of serum total cholesterol (TC) and triglycerides (TG), increase the level of high-density lipoprotein cholesterol (HDL-C), and improve the metabolism of lipids. Therefore, further experiments were conducted by us, and TAETEA Prebiotea was formulated into a suitable dose for the intervention of non alcoholic fatty liver disease (NAFLD) model rats, and at the end of the experiments, the samples of each group of experiments were analyzed and detected by the method of UHPLC-Q-Exactive LC-MS liquid-mass spectrometry methodology, and the relevant metabolites as well as metabolic pathways were analyzed by the method of Non targeted metabolomics analysis. As a result, 71 differential metabolites could be screened, of which 35 differential metabolites were up-regulated after intervention and 36 differential metabolites were down-regulated after intervention. Based on the KEGG pathway enrichment and Pathway Impact bubble diagram analysis, glycine, serine, threonine metabolism, arginine and proline metabolism, protein digestion and absorption, and central carbon metabolism in cancer may be the main metabolic pathways in which TAETEA Prebiotea exerted preventive effects on NAFLD rats, C00148 (Proline), C00300 (Creatine) and C00719 (Betaine) are the differential metabolites that play important regulatory roles.

New insights into the endothelial origin of hematopoietic system inspired by "TIF" approaches.

Hematopoietic stem progenitor cells (HSPCs) are derived from a specialized subset of endothelial cells named hemogenic endothelial cells (HECs) via a process of endothelial-to-hematopoietic transition during embryogenesis. Recently, with the usage of multiple single-cell technologies and advanced genetic lineage tracing techniques, namely, "TIF" approaches that combining transcriptome, immunophenotype and function/fate analyses, massive new insights have been achieved regarding the cellular and molecular evolution underlying the emergence of HSPCs from embryonic vascular beds. In this review, we focus on the most recent advances in the enrichment markers, functional characteristics, developmental paths, molecular controls, and the embryonic site-relevance of the key intermediate cell populations bridging embryonic vascular and hematopoietic systems, namely HECs and pre-hematopoietic stem cells, the immediate progenies of some HECs, in mouse and human embryos. Specifically, using expression analyses at both transcriptional and protein levels and especially efficient functional assays, we propose that the onset of Kit expression is at the HEC stage, which has previously been controversial.

Human yolk sac-derived innate lymphoid-biased multipotent progenitors emerge prior to hematopoietic stem cell formation.

Hematopoietic stem cell (HSC)-independent lymphopoiesis has been elucidated in murine embryos. However, our understanding regarding human embryonic counterparts remains limited. Here, we demonstrated the presence of human yolk sac-derived lymphoid-biased progenitors (YSLPs) expressing CD34, IL7R, LTB, and IRF8 at Carnegie stage 10, much earlier than the first HSC emergence. The number and lymphopoietic potential of these progenitors were both significantly higher in the yolk sac than the embryo proper at this early stage. Importantly, single-cell/bulk culture and CITE-seq have elucidated the tendency of YSLP to differentiate into innate lymphoid cells and dendritic cells. Notably, lymphoid progenitors in fetal liver before and after HSC seeding displayed distinct transcriptional features, with the former closely resembling those of YSLPs. Overall, our data identified the origin, potential, and migratory dynamics of innate lymphoid-biased multipotent progenitors in human yolk sac before HSC emergence, providing insights for understanding the stepwise establishment of innate immune system in humans.

Identification and validation of a novel prognostic circadian rhythm-related gene signature for stomach adenocarcinoma.

Circadian rhythm genes were reported to be strongly associated with the development and prognosis of circadian rhythm disorders related to stomach adenocarcinoma (STAD), which is one of the most prevalent cancers. This study aimed to identify a circadian rhythm-related gene signature that could help predict STAD outcome. Using bioinformatics analysis approaches, 105 genes were examined in 350 patients with STAD. Overall, six hub-type circadian rhythm-associated genes (GNA11, PER1, SOX14, EZH2, MAGED1, and NR1D1) were identified using univariate and multivariate Cox regression analyses. These genes were then used to build a genetic predictive model, which was further validated using a publicly available dataset (GSE26899). Overall, genes associated with the circadian rhythm were found to be substantially correlated with the characteristics of the STAD patients (grade, sex, and M stage). In addition, the circadian rhythm-related gene signature was significantly associated with the MAPK and Notch signaling pathways, which are known risk factors for poorer STAD outcome. Taken together, these findings suggest that the herein proposed prognostic model based on six circadian rhythm-associated genes may have predictive value and potential application for clinical decision-making and for personalized treatment of STAD.

Comparative analysis of pumpkin rootstocks mediated impact on melon sensory fruit quality through integration of non-targeted metabolomics and sensory evaluation.

Melon fruits are popular because of sweet taste and pleasant aroma. Grafting has been extensively used for melons to alleviate abiotic stresses and control soil borne diseases. However, use of grafting for vegetable fruit quality improvement is less studies. In modern age fruit quality particularly sensory quality characteristics have key importance from consumer eye lens. We performed liquid chromatography-mass spectrometry and metabonomic analysis to examine sensory fruit quality of melon grafted onto ten different pumpkin rootstocks. Bases on the result of our study, 478 metabolites were detected and 184 metabolites consisting of lipids, amino acids and organic oxygen compounds were differentially expressed in grafted melon fruits. The results from metabolomic, physiochemical and sensory analysis explain the differences in melon fruit flavor from two contrasting rootstocks. In conclusion the fruits from Tianzhen No. 1 rootstock exhibited better organoleptic characteristics and higher soluble sugars content [glucose (19.87 mg/g), fructose (19.68 mg/g) and sucrose (169.45 mg/g)] compared with other rootstocks used in this study. Moreover, the contents of bitterness causing amino acids such as L-arginine, L-asparagine, Histidinyl-histidine and Acetyl-DL-valine were found lower in Tianzhen No. 1-grafted melon fruits compared with Sizhuang No. 12-grafted melon fruits. These fruit quality characteristics made Tianzhen No. 1 rootstock suitable for commercial cultivation of Yuniang melon.

Heterogeneity in endothelial cells and widespread venous arterialization during early vascular development in mammals.

Arteriogenesis rather than unspecialized capillary expansion is critical for restoring effective circulation to compromised tissues in patients. Deciphering the origin and specification of arterial endothelial cells during embryonic development will shed light on the understanding of adult arteriogenesis. However, during early embryonic angiogenesis, the process of endothelial diversification and molecular events underlying arteriovenous fate settling remain largely unresolved in mammals. Here, we constructed the single-cell transcriptomic landscape of vascular endothelial cells (VECs) during the time window for the occurrence of key vasculogenic and angiogenic events in both mouse and human embryos. We uncovered two distinct arterial VEC types, the major artery VECs and arterial plexus VECs, and unexpectedly divergent arteriovenous characteristics among VECs that are located in morphologically undistinguishable vascular plexus intra-embryonically. Using computational prediction and further lineage tracing of venous-featured VECs with a newly developed Nr2f2CrexER mouse model and a dual recombinase-mediated intersectional genetic approach, we revealed early and widespread arterialization from the capillaries with considerable venous characteristics. Altogether, our findings provide unprecedented and comprehensive details of endothelial heterogeneity and lineage relationships at early angiogenesis stages, and establish a new model regarding the arteriogenesis behaviors of early intra-embryonic vasculatures.

RNA m6A methylation orchestrates cancer growth and metastasis via macrophage reprogramming.

N6-methyladenosine (m6A) is a reversible mRNA modification that has been shown to play important roles in various biological processes. However, the roles of m6A modification in macrophages are still unknown. Here, we discover that ablation of Mettl3 in myeloid cells promotes tumour growth and metastasis in vivo. In contrast to wild-type mice, Mettl3-deficient mice show increased M1/M2-like tumour-associated macrophage and regulatory T cell infiltration into tumours. m6A sequencing reveals that loss of METTL3 impairs the YTHDF1-mediated translation of SPRED2, which enhances the activation of NF-kB and STAT3 through the ERK pathway, leading to increased tumour growth and metastasis. Furthermore, the therapeutic efficacy of PD-1 checkpoint blockade is attenuated in Mettl3-deficient mice, identifying METTL3 as a potential therapeutic target for tumour immunotherapy.

Embryonic endothelial evolution towards first hematopoietic stem cells revealed by single-cell transcriptomic and functional analyses.

Hematopoietic stem cells (HSCs) in adults are believed to be born from hemogenic endothelial cells (HECs) in mid-gestational embryos. Due to the rare and transient nature, the HSC-competent HECs have never been stringently identified and accurately captured, let alone their genuine vascular precursors. Here, we first used high-precision single-cell transcriptomics to unbiasedly examine the relevant EC populations at continuous developmental stages with intervals of 0.5 days from embryonic day (E) 9.5 to E11.0. As a consequence, we transcriptomically identified two molecularly different arterial EC populations and putative HSC-primed HECs, whose number peaked at E10.0 and sharply decreased thereafter, in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region. Combining computational prediction and in vivo functional validation, we precisely captured HSC-competent HECs by the newly constructed Neurl3-EGFP reporter mouse model, and realized the enrichment further by a combination of surface markers (Procr+Kit+CD44+, PK44). Surprisingly, the endothelial-hematopoietic dual potential was rarely but reliably witnessed in the cultures of single HECs. Noteworthy, primitive vascular ECs from E8.0 experienced two-step fate choices to become HSC-primed HECs, namely an initial arterial fate choice followed by a hemogenic fate conversion. This finding resolves several previously observed contradictions. Taken together, comprehensive understanding of endothelial evolutions and molecular programs underlying HSC-primed HEC specification in vivo will facilitate future investigations directing HSC production in vitro.

Small molecule inhibitor of TGF-ß signaling enables robust osteogenesis of autologous GMSCs to successfully repair minipig severe maxillofacial bone defects.

BACKGROUND: Clinically, for stem cell-based therapy (SCBT), autologous stem cells are considered better than allogenic stem cells because of little immune rejection and no risk of communicable disease infection. However, severe maxillofacial bone defects restoration needs sufficient autologous stem cells, and this remains a challenge worldwide. Human gingival mesenchymal stem cells (hGMSCs) derived from clinically discarded, easily obtainable, and self-healing autologous gingival tissues, have higher proliferation rate compared with autologous bone marrow mesenchymal stem cells (hBMSCs). But for clinical bone regeneration purpose, GMSCs have inferior osteogenic differentiation capability. In this study, a TGF-ß signaling inhibitor SB431542 was used to enhance GMSCs osteogenesis in vitro and to repair minipig severe maxillofacial bone defects. METHODS: hGMSCs were isolated and cultured from clinically discarded gingival tissues. The effects of SB431542 on proliferation, apoptosis, and osteogenic differentiation of hGMSCs were analyzed in vitro, and then, SB431542-treated hGMSCs composited with Bio-Oss® were transplanted into immunocompromised mice subcutaneously to explore osteogenic differentiation in vivo. After that, SB431542-treated autologous pig GMSCs (pGMSCs) composited with Bio-Oss® were transplanted into circular confined defects (5 mm × 12 mm) in minipigs maxillary to investigate severe bone defect regeneration. Minipigs were sacrificed at 2 months and nude mice at 8 weeks to retrieve specimens for histological or micro-CT or CBCT analysis. Effects of SB431542 on TGF-ß and BMP signaling in hGMSCs were investigated by Western Blot or qRT-PCR. RESULTS: One micromolar of SB431542 treatment induced a robust osteogenesis of hGMSCs in vitro, without adverse effect on apoptosis and growth. In vivo, 1 µM SB431542 treatment also enabled striking osteogenesis of hGMSCs subcutaneously in nude mice and advanced new bone formation of pGMSCs in minipig maxillary bone defect model. In addition, SB431542-treated hGMSCs markedly increased bone-related proteins expression, and BMP2 and BMP4 gene expression. Conversely, SMAD3 protein-dependent TGF-ß signal pathway phosphorylation was decreased. CONCLUSIONS: Our study show that osteogenic differentiation of GMSCs treated with TGF-ß signaling inhibitor SB431542 was increased, and SB431542-treated autologous pig GMSCs could successfully repair minipig severe maxillofacial bone defects. This preclinical study brings about a promising large bone regeneration therapeutic potential of autologous GMSCs induced by SB431542 in clinic settings.