Small-molecule agonist AdipoRon alleviates diabetic retinopathy through the AdipoR1/AMPK/EGR4 pathway.

BACKGROUND: Diabetes mellitus (DM) is a progressive disease that involves multiple organs due to increased blood glucose, and diabetic retinopathy (DR) is the main complication of DM in the eyes and causes irreversible vision loss. In the pathogenesis of diabetic vascular disease, oxidative stress caused by hyperglycemia plays an important role in Müller cell impairment. In recent years, AdipoRon, an adiponectin analog that demonstrated important physiological functions in obesity, diabetes, inflammation, and cardiovascular diseases, demonstrated cellular protection from apoptosis and reduced inflammatory damage through a receptor-dependent mechanism. Here, we investigated how AdipoRon reduced oxidative stress and apoptosis in Müller glia in a high glucose environment. RESULTS: By binding to adiponectin receptor 1 on Müller glia, AdipoRon activated 5' adenosine monophosphate-activated protein kinase (AMPK)/acetyl-CoA carboxylase phosphorylation downstream, thereby alleviating oxidative stress and eventual apoptosis of cells and tissues. Transcriptome sequencing revealed that AdipoRon promoted the synthesis and expression of early growth response factor 4 (EGR4) and inhibited the cellular protective effects of AdipoRon in a high-glucose environment by reducing the expression of EGR4. This indicated that AdipoRon played a protective role through the EGR4 and classical AMPK pathways. CONCLUSIONS: This provides a new target for the early treatment of DR.

Disruption of dNTP homeostasis by ribonucleotide reductase hyperactivation overcomes AML differentiation blockade.

Differentiation blockade is a hallmark of acute myeloid leukemia (AML). A strategy to overcome such a blockade is a promising approach against the disease. The lack of understanding of the underlying mechanisms hampers development of such strategies. Dysregulated ribonucleotide reductase (RNR) is considered a druggable target in proliferative cancers susceptible to deoxynucleoside triphosphate (dNTP) depletion. Herein, we report an unanticipated discovery that hyperactivating RNR enables differentiation and decreases leukemia cell growth. We integrate pharmacogenomics and metabolomics analyses to identify that pharmacologically (eg, nelarabine) or genetically upregulating RNR subunit M2 (RRM2) creates a dNTP pool imbalance and overcomes differentiation arrest. Moreover, R-loop-mediated DNA replication stress signaling is responsible for RRM2 activation by nelarabine treatment. Further aggravating dNTP imbalance by depleting the dNTP hydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) enhances ablation of leukemia stem cells by RRM2 hyperactivation. Mechanistically, excessive activation of extracellular signal-regulated kinase (ERK) signaling downstream of the imbalance contributes to cellular outcomes of RNR hyperactivation. A CRISPR screen identifies a synthetic lethal interaction between loss of DUSP6, an ERK-negative regulator, and nelarabine treatment. These data demonstrate that dNTP homeostasis governs leukemia maintenance, and a combination of DUSP inhibition and nelarabine represents a therapeutic strategy.

Effects of Lactobacillus acidophilus AC on the growth, intestinal flora and metabolism of zebrafish (Danio rerio).

The intensive aquaculture model has resulted in a heightened prevalence of diseases among farmed animals. It is imperative to identify healthy and efficacious alternatives to antibiotics for the sustainable progression of aquaculture. In this investigation, a strain of Lactobacillus acidophilus AC was introduced into the cultural water at varying concentrations (105 CFU/mL, 106 CFU/mL, 107 CFU/mL) to nourish zebrafish (Danio rerio). The findings revealed that L. acidophilus AC effectively increased the growth performance of zebrafish, improved the ion exchange capacity of gills, and enhanced hepatic antioxidant and immune-enzyme activities. Furthermore, L. acidophilus AC notably enhanced the intestinal morphology and augmented the activity of digestive enzymes within the intestinal tract. Analysis of intestinal flora revealed that L. acidophilus AC exerted a significant impact on the intestinal flora community, manifested by a reduction in the relative abundance of Burkholderiales, Candidatus_Saccharibacteria_bacterium, and Sutterellaceae, coupled with an increase in the relative abundance of Cetobacterium. Metabolomics analysis demonstrated that L. acidophilus AC significantly affected intestinal metabolism of zebrafish. PG (i-19:0/PGE2) and 12-Hydroxy-13-O-d-glucuronoside-octadec-9Z-enoate were the metabolites with the most significant up- and down-regulation folds, respectively. Finally, L. acidophilus AC increased the resistance of zebrafish to Aeromonas hydrophila. In conclusion, L. acidophilus AC was effective in enhancing the health and immunity of zebrafish. Thus, our findings suggested that L. acidophilus AC had potential applications and offered a reference for its use in aquaculture.

Integration of Vitreous Lipidomics and Metabolomics for Comprehensive Understanding of the Pathogenesis of Proliferative Diabetic Retinopathy.

As a vision-threatening complication of diabetes mellitus (DM), proliferative diabetic retinopathy (PDR) is associated with sustained metabolic disorders. Herein, we collected the vitreous cavity fluid of 49 patients with PDR and 23 control subjects without DM for metabolomics and lipidomics analyses. Multivariate statistical methods were performed to explore relationships between samples. For each group of metabolites, gene set variation analysis scores were generated, and we constructed a lipid network by using weighted gene co-expression network analysis. The association between lipid co-expression modules and metabolite set scores was investigated using the two-way orthogonal partial least squares (O2PLS) model. A total of 390 lipids and 314 metabolites were identified. Multivariate statistical analysis revealed significant vitreous metabolic and lipid differences between PDR and controls. Pathway analysis showed that 8 metabolic processes might be associated with the development of PDR, and 14 lipid species were found to be altered in PDR patients. Combining metabolomics and lipidomics, we identified fatty acid desaturase 2 (FADS2) as an important potential contributor to the pathogenesis of PDR. Collectively, this study integrates vitreous metabolomics and lipidomics to comprehensively unravel metabolic dysregulation and identifies genetic variants associated with altered lipid species in the mechanistic pathways for PDR.

Analysis of long noncoding RNAs in the aqueous humor of wet age-related macular degeneration.

Wet age-related macular degeneration (wAMD) is the main cause of irreversible blindness in the elderly, and its pathogenesis is still not fully understood. Long non-coding RNAs (lncRNAs) participated in the pathogenesis of a number of neovascular diseases, but their role in wAMD is less known. In order to reveal the potential role of lncRNAs in wAMD, we used high-throughput sequencing to assess lncRNAs and mRNAs expression profile in the aqueous humor of patients with wAMD and of patients with age-related cataract as control. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to identify the potential biological functions and signaling pathways of RNA. A coding-non-coding gene co-expression (CNC) network was constructed to identify the interaction of lncRNAs and mRNAs. Quantitative PCR was used to validate the expression of selected lncRNAs. We identified 1071 differentially expressed lncRNAs and 3658 differentially expressed mRNAs in patients with wAMD compared to controls. GO and KEGG analyses suggested that differentially expressed lncRNAs-coexpressed mRNAs were mainly enriched in Rab GTPase binding, GTPase activation, RAS signaling pathway and autophagy. The top 100 differentially expressed genes were selected to build the CNC network, which could be connected by 416 edges. LncRNAs are differentially expressed in the aqueous humor of patients with wAMD and they are involved in several pathogenetic pathways. These dysregulated lncRNAs and their target genes could represent promising therapeutic targets in wAMD.

Effects of Isaria cicadae on growth, gut microbiota, and metabolome of Larimichthys crocea.

The large yellow croaker (Larimichthys crocea) is the most productive mariculture fish in China, and its aquaculture scale is expanding along the southeastern coast of China, but that development is causing environmental damage by increasing the use of antibiotics and other chemicals. How to improve fish immunity through non-antibiotic substances is still a problem facing aquaculture industry. At present, the experiments have shown that Isaria cicadae spent substrate (IC) can improve the growth performance and immunity of Oreochromis niloticus. Therefore, I. cicadae may be a natural alternative to antibiotic for aquaculture. In order to study the effects of IC on growth performance, serum biochemical indices, intestinal microbiota, and intestinal metabolism of large yellow croakers, the fish were divided into three groups with three replicates in each group. Basal diet, basal diet with 2% and 6% IC supplementation (IC2 and IC6 groups), respectively. The results showed that weight gain rate (WG) and specific growth rate (SGR) of large yellow croaker significantly increased (P < 0.05) in IC6 group. The content of triglyceride (TG), low density lipoprotein cholesterol (LDL-C), total protein (TP) and albumin (ALB) increased significantly (P < 0.05), and total cholesterol (T-CHO) decreased significantly (P < 0.05) in IC2 group. Compared to IC0 group, the activity of malondialdehyde (MDA) , superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) increased significantly (P < 0.05) in IC2 group, the activity of total antioxidant capacity (T-AOC) and GSH-Px increased significantly (P < 0.05) in IC6 group, and the activity of lysozyme (LZM) increased significantly in IC2 and IC6 groups. The addition of IC in the diets significantly increased the diversity of the microbial community in the intestine of large yellow croaker (P < 0.05), significantly improved the relative abundance of Acidobacteriota (P < 0.05) at the phylum level, and reduced the relative abundance of Bacteroidota, Desulfobacterota, and Synergistota (P < 0.05). At the genus level, the relative abundance of Bacteroides, Cetobacterium and Mycoplasma, which are dominant bacteria in fish gut, significantly increased (P < 0.05). The relative abundance of Ruminofilibacter, Desulfomicrobium, DMER64, Syntrophomonas, Hydrogenophaga, and Aminobacterium reduced significantly (P < 0.05). Among them, Ruminofilibacter, DMER64, Syntrophomonas and Hydrogenophaga are bacteria that can participate in the hydrolysis and acidification of organic matter, while DMER64 is the hydrogen carrier. The intestinal metabolome analysis showed that IC could improve metabolic composition and function, which was related to host immunity and metabolism. In conclusion, I. cicadae can improve the growth performance, regulate the lipid metabolism and immune and antioxidant capacity of large yellow croakers by regulating intestinal microbiota and intestinal metabolism. This study provides a reference for the application of IC in aquaculture.

Dietary ingredient change induces a transient MyD88-dependent mucosal enteric glial cell response and promotes obesity.

OBJECTIVE: Consumption of a modern Western-type high-fat low-fiber diet increases the risk of obesity. However, how a host responds to such a diet, especially during the early period of dietary transition from a previous low-fat and fiber-rich diet, remains poorly explored. METHODS: Wild-type C57BL/6 mice were fed a normal chow diet or a high-fat diet. Enteric glial cell (EGC) activation was detected through quantitative real-time PCR (qRT-PCR), immunoblotting and immunohistology analysis. Fluorocitrate or genetic deletion of glial fibrillary acidic protein (GFAP)-positive glial-intrinsic myeloid differentiation factor 88 (Myd88) was used to inhibit EGC activation, and the effect of a high-fat diet on obesity was further investigated. The role of MYD88-dependent sensing of commensal products in adipocyte was observed to analyze the effect of obesity. RESULTS: A dietary shift from a normal chow diet to a high-fat diet in mice induced a transient early-phase emergence of a GFAP-positive EGC network in the lamina propria of the ileum, accompanied with an increase in glial-derived neurotrophic factor production. Inhibition of glial cell activity blocked this response. GFAP-positive glial Myd88 knockout mice gained less body weight after high-fat diet (HFD) feeding than littermate controls. In contrast, adipocyte deletion of Myd88 in mice had no effect on weight gain but instead exacerbated glucose intolerance. Furthermore, short-term fluorocitrate intervention during HFD feeding attenuated body weight gain. CONCLUSIONS: Our findings indicate that EGCs are early responders to intestinal ecosystem changes and the GFAP-positive glial Myd88 signaling participates in regulating obesity.

Serum Ang-1/Ang-2 ratio may be a promising biomarker for evaluating severity of diabetic retinopathy.

PURPOSE: To investigate the predictive role of serum angiopoietin-1 and angiopoietin-2 (Ang-1/Ang-2) in evaluating the severity of diabetic retinopathy (DR). METHODS: A total of 101 outpatients with type 2 diabetes mellitus (T2DM) were recruited and were further divided into the following five groups: T2DM without DR (non-DR), mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR and proliferative DR (PDR) in accordance with the International Clinical Diabetic Retinopathy Guidelines. Furthermore, 101 serum samples were included in the further analysis using enzyme-linked immunosorbent assays. A receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic value of each index. RESULTS: The expression of Ang-1 in the PDR group was significantly lower than that in the non-DR group, while Ang-2 showed an opposite upward trend (p < 0.05). The Ang-1/Ang-2 ratio of the non-DR group was significantly lower than that of the moderate NPDR, severe NPDR and PDR (p < 0.05, p < 0.01 and p < 0.01, respectively). Differences in the Ang-1/Ang-2 ratio were observed earlier than those in the individual Ang-1 and Ang-2 measurements. The maximal Youden index was 0.512 with a calculated area under the curve (AUC) value of 0.734 (p < 0.01). CONCLUSIONS: The Ang-1/Ang-2 ratio was helpful in assessing the severity of DR and may provide potential clinical benefits as a biomarker and early warning signs for DR diagnosis.

Hidden proteome of synaptic vesicles in the mammalian brain.

Current proteomic studies clarified canonical synaptic proteins that are common to many types of synapses. However, proteins of diversified functions in a subset of synapses are largely hidden because of their low abundance or structural similarities to abundant proteins. To overcome this limitation, we have developed an "ultra-definition" (UD) subcellular proteomic workflow. Using purified synaptic vesicle (SV) fraction from rat brain, we identified 1,466 proteins, three times more than reported previously. This refined proteome includes all canonical SV proteins, as well as numerous proteins of low abundance, many of which were hitherto undetected. Comparison of UD quantifications between SV and synaptosomal fractions has enabled us to distinguish SV-resident proteins from potential SV-visitor proteins. We found 134 SV residents, of which 86 are present in an average copy number per SV of less than one, including vesicular transporters of nonubiquitous neurotransmitters in the brain. We provide a fully annotated resource of all categorized SV-resident and potential SV-visitor proteins, which can be utilized to drive novel functional studies, as we characterized here Aak1 as a regulator of synaptic transmission. Moreover, proteins in the SV fraction are associated with more than 200 distinct brain diseases. Remarkably, a majority of these proteins was found in the low-abundance proteome range, highlighting its pathological significance. Our deep SV proteome will provide a fundamental resource for a variety of future investigations on the function of synapses in health and disease.

Effects of constant flow velocity on endurance swimming and fatigue metabolism in red drum and blackhead seabream.

Aquaculture has greater potential for seafood production than wild capture fisheries. To meet the growing demand for seafood, China's marine aquaculture industry has begun building deep-water cages in the open sea. However, under these conditions, fish encounter strong currents and waves, and ensuring their healthy growth is key to the farming process. To address these issues, it is necessary to study the sustained swimming abilities of cultured fish species. Blackhead seabream (Acanthopagrus schlegelii) and red drum (Sciaenops ocellatus) are traditional economic fish species in China; however, their sustained swimming ability under a constant current has been underexplored. Therefore, we examined the endurance swimming ability of three size classes of blackhead seabream and red drum at 20 °C. The fish were then subjected to swimming tests of 0, 30, 60, 90, 120, and 150 min at a constant swimming speed of 0.55 m/s (0.80 m/s), 0.65 m/s (0.90 m/s), and 0.70 m/s (0.98 m/s). The fish were then dissected to obtain muscle, blood, and liver samples; sample metabolite concentrations were measured at six time points, each of which guaranteed five sets of valid data. The results indicated that red drum has a significantly stronger swimming ability, and can be cultured in waters with a short-term flow rate not exceeding 0.75 m/s or 3.5 BL/s. Further, blackhead seabream can be cultured in waters with a flow velocity lower than 0.55 m/s or 2.5 BL/s. The species-related metabolic differences were mainly reflected in the hepatic glycogen and blood glucose concentrations, and those in swimming ability caused by body length were mainly reflected by the hepatic glycogen concentration. The hepatic glycogen concentration had the most significant effect on fish with body lengths >28 cm (P < 0.05). Overall, the experimental results indicated that the liver plays a major role in the physiological level of fish swimming fatigue, providing a direction for further research.

Does local government competition affect the dependence on polluting industries? Evidence from China's land market.

Many countries have formulated strict environmental regulation policies, but unfortunately, environmental pollution is still severe. We believe that this phenomenon may result from institutional factors. We study whether local government competition affects the dependence on polluting industries and test it with the transaction data of China's land market. We find that the decline in the economic ranking of cities increases the scale of land transferred to polluting industries, and the more the ranking decline, the larger the scale of land in polluting industries. Besides, we analyze the impact of the first decline in the urban economic ranking, finding that the land transfer scale of polluting industries exhibits a trend of slowing down after a rapid rise in the short term. Heterogeneity analysis shows that officials with lower education levels and before-retirement age will dependent more on polluting industries to a greater extent when they face local government competition, and cities with economic growth lower than the national average will increase their dependence on polluting industries to a greater extent. This paper provides new insights into the causes of the environmental governance dilemma, which also highlights the need to weaken economic indicators and strengthen environmental indicators in government performance assessment.

A Murine Commensal Protozoan Influences Host Glucose Homeostasis by Facilitating Free Choline Generation.

Recent progress indicates that the gut microbiota plays important role in regulating the host's glucose homeostasis. However, the mechanisms remain unclear. Here, we reported that one integral member of the murine gut microbiota, the protozoan Tritrichomonas musculis could drive the host's glucose metabolic imbalance. Using metabolomics analysis and in vivo assays, we found that mechanistically this protozoan influences the host glucose metabolism by facilitating the production of a significant amount of free choline. Free choline could be converted sequentially by choline-utilizing bacteria and then the host to a final product trimethylamine N-oxide, which promoted hepatic gluconeogenesis. Together, our data reveal a previously underappreciated gut eukaryotic microorganism by working together with other members of microbiota to influence the host's metabolism. Our study underscores the importance and prevalence of metabolic interactions between the gut microbiota and the host in modulating the host's metabolic health. IMPORTANCE Blood glucose levels are important for human health and can be influenced by gut microbes. However, its mechanism of action was previously unknown. In this study, researchers identify a unique member of the gut microbes in mice that can influence glucose metabolism by promoting the host's ability to synthesis glucose by using nonglucose materials. This is because of its ability to generate the essential nutrient choline, and choline, aided by other gut bacteria and the host, is converted to trimethylamine N-oxide, which promotes glucose production. These studies show how gut microbes promote metabolic dysfunction and suggest novel approaches for treating patients with blood glucose abnormality.

Protective effect of pentraxin 3 on pathological retinal angiogenesis in an in vitro model of diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is the most common retinal microvascular disease caused by diabetes. Previous studies indicated that Pentraxin 3 (PTX3), an acute phase reactant, was closely related to the development of DR. But the exact effect of PTX3 in diabetic retinopathy needs more investigations. METHODS: Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) analysis and western blot (WB) were used to detect the expression of PTX3 in vitro. The Ki67 immunofluorescent staining, scratch-wound migration assay, and tube formation experiments were performed to detect the effect of PTX3 knockdown and overexpression on the fibroblast growth factor (FGF)-induced proliferation, migration and tube-forming ability of human retinal microvascular endothelial cells (HRMECs). The phosphorylation levels of extracellular regulated protein kinases (ERK) and fibroblast growth factor receptor (FGFR) in HRMECs were detected by WB. RESULTS: In vitro, the mRNA and protein expressions of PTX3 in the high-concentration glucose condition group were upregulated compared with the normal group (p < 0.05). The proliferation, migration and tube-forming abilities of HRMECs exposed to high-concentration glucose were enhanced (p < 0.01, p < 0.01, p < 0.05 respectively), and the phosphorylation of FGFR and ERK1/2 were increased (p < 0.01, p < 0.05 respectively) compared with the normal condition group. Compared with the high glucose condition group, the proliferation, migration and tube-forming abilities of HRMECs in the high glucose + PTX3 siRNA condition group were further strengthened (p < 0.001, p < 0.0001, p < 0.05 respectively), and the phosphorylation of FGFR and ERK1/2 were increased (p < 0.001, p < 0.01 respectively). Compared with the high glucose condition group, the proliferation, migration and tube-forming abilities of HRMECs in the high glucose + PTX3 overexpression condition group were compromised (p < 0.001, p < 0.05, p < 0.01 respectively), and the phosphorylation of FGFR and ERK1/2 were inhibited (p < 0.001, p < 0.0001 respectively). Neither the scramble siRNA condition group nor the blank plasmid condition group showed significant difference on the proliferation, migration and tube-forming abilities of HRMECs compared with the high glucose condition group (p > 0.05). CONCLUSIONS: The upregulated expression of PTX3 may play a protective role on pathological angiogenesis in DR. PTX3 may serve as a new target for the treatment of DR.

Circ-LARP1B knockdown restrains the tumorigenicity and enhances radiosensitivity by regulating miR-578/IGF1R axis in hepatocellular carcinoma.

INTRODUCTION AND OBJECTIVES: Circular RNA La Ribonucleoprotein 1B (circ-LARP1B) was reported to serve as an oncogene in many types of cancers. Radiotherapy (RT) is an important element of the multimodal treatment concept in malignancies. Here, this work aimed to investigate the role of circ-LARP1B in the tumorigenesis and radiosensitivity of hepatocellular carcinoma (HCC). PATIENTS OR MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of genes and proteins. In vitro experiments were conducted using cell counting Kit-8 (CCK-8), colony formation, EDU, transwell, and tube formation assays, respectively. Dual-luciferase reporter assay was employed to identify the target relationship between miR-578 and circ-LARP1B or IGF1R (insulin-like growth factor 1 receptor). In vivo assay was performed using murine xenograft model. RESULTS: Circ-LARP1B was highly expressed in HCC tissues and cells, and high expression of circ-LARP1B was closely associated with poor prognosis. Functional experiments demonstrated that circ-LARP1B silencing impaired cell proliferation, invasion, angiogenesis and reduced radioresistance in vitro. Mechanistically, circ-LARP1B could competitively bind with miR-578 to relieve the repression of miR-578 on the expression of its target gene IGF1R. Further rescue assay confirmed that miR-578 inhibition reversed the inhibitory effects of circ-LARP1B knockdown on HCC cell malignant phenotypes and radioresistance. Moreover, miR-578 overexpression restrained tumorigenicity and enhanced radiosensitivity in HCC cells, which were attenuated by IGF1R up-regulation. Besides that, circ-LARP1B knockdown impeded tumor growth and enhanced irradiation sensitivity in HCC in vivo. CONCLUSIONS: Circ-LARP1B knockdown restrained HCC tumorigenicity and enhanced radiosensitivity by regulating miR-578/IGF1R axis, providing a new target for the treatment of HCC.

Deletion of YJL218W reduces salt tolerance of Saccharomyces cerevisiae.

The YJL218W open reading frame may be involved in peroxisomal biogenesis. However, whether it mediates salt tolerance is unclear. We found that after knockdown of YJL218W in Saccharomyces cerevisiae (S. cerevisiae), its salt tolerance was reduced and cell death was increased. Transcriptome sequencing and analysis further revealed that YJL218W knockdown mediated significant changes in the expression of 1432 messenger RNA (mRNAs), of which 603 were upregulated. KEGG enrichment analysis and polymerase chain reaction (PCR) assay indicated that YJL218W mediated the regulation of peroxisome-related genes. Therefore, YJL218W may regulate salt stress in S. cerevisiae by regulating peroxisome assembly.

Frequency-Dependent Block of Excitatory Neurotransmission by Isoflurane via Dual Presynaptic Mechanisms.

Volatile anesthetics are widely used for surgery, but neuronal mechanisms of anesthesia remain unidentified. At the calyx of Held in brainstem slices from rats of either sex, isoflurane at clinical doses attenuated EPSCs by decreasing the release probability and the number of readily releasable vesicles. In presynaptic recordings of Ca2+ currents and exocytic capacitance changes, isoflurane attenuated exocytosis by inhibiting Ca2+ currents evoked by a short presynaptic depolarization, whereas it inhibited exocytosis evoked by a prolonged depolarization via directly blocking exocytic machinery downstream of Ca2+ influx. Since the length of presynaptic depolarization can simulate the frequency of synaptic inputs, isoflurane anesthesia is likely mediated by distinct dual mechanisms, depending on input frequencies. In simultaneous presynaptic and postsynaptic action potential recordings, isoflurane impaired the fidelity of repetitive spike transmission, more strongly at higher frequencies. Furthermore, in the cerebrum of adult mice, isoflurane inhibited monosynaptic corticocortical spike transmission, preferentially at a higher frequency. We conclude that dual presynaptic mechanisms operate for the anesthetic action of isoflurane, of which direct inhibition of exocytic machinery plays a low-pass filtering role in spike transmission at central excitatory synapses.SIGNIFICANCE STATEMENT Synaptic mechanisms of general anesthesia remain unidentified. In rat brainstem slices, isoflurane inhibits excitatory transmitter release by blocking presynaptic Ca2+ channels and exocytic machinery, with the latter mechanism predominating in its inhibitory effect on high-frequency transmission. Both in slice and in vivo, isoflurane preferentially inhibits spike transmission induced by high-frequency presynaptic inputs. This low-pass filtering action of isoflurane likely plays a significant role in general anesthesia.

Microtubule and Actin Differentially Regulate Synaptic Vesicle Cycling to Maintain High-Frequency Neurotransmission.

Cytoskeletal filaments such as microtubules (MTs) and filamentous actin (F-actin) dynamically support cell structure and functions. In central presynaptic terminals, F-actin is expressed along the release edge and reportedly plays diverse functional roles, but whether axonal MTs extend deep into terminals and play any physiological role remains controversial. At the calyx of Held in rats of either sex, confocal and high-resolution microscopy revealed that MTs enter deep into presynaptic terminal swellings and partially colocalize with a subset of synaptic vesicles (SVs). Electrophysiological analysis demonstrated that depolymerization of MTs specifically prolonged the slow-recovery time component of EPSCs from short-term depression induced by a train of high-frequency stimulation, whereas depolymerization of F-actin specifically prolonged the fast-recovery component. In simultaneous presynaptic and postsynaptic action potential recordings, depolymerization of MTs or F-actin significantly impaired the fidelity of high-frequency neurotransmission. We conclude that MTs and F-actin differentially contribute to slow and fast SV replenishment, thereby maintaining high-frequency neurotransmission.SIGNIFICANCE STATEMENT The presence and functional role of MTs in the presynaptic terminal are controversial. Here, we demonstrate that MTs are present near SVs in calyceal presynaptic terminals and that MT depolymerization specifically prolongs the slow-recovery component of EPSCs from short-term depression. In contrast, F-actin depolymerization specifically prolongs fast-recovery component. Depolymerization of MT or F-actin has no direct effect on SV exocytosis/endocytosis or basal transmission, but significantly impairs the fidelity of high-frequency transmission, suggesting that presynaptic cytoskeletal filaments play essential roles in SV replenishment for the maintenance of high-frequency neurotransmission.

Targeting PRMT1-mediated FLT3 methylation disrupts maintenance of MLL-rearranged acute lymphoblastic leukemia.

Relapse remains the main cause of MLL-rearranged (MLL-r) acute lymphoblastic leukemia (ALL) treatment failure resulting from persistence of drug-resistant clones after conventional chemotherapy treatment or targeted therapy. Thus, defining mechanisms underlying MLL-r ALL maintenance is critical for developing effective therapy. PRMT1, which deposits an asymmetric dimethylarginine mark on histone/non-histone proteins, is reportedly overexpressed in various cancers. Here, we demonstrate elevated PRMT1 levels in MLL-r ALL cells and show that inhibition of PRMT1 significantly suppresses leukemic cell growth and survival. Mechanistically, we reveal that PRMT1 methylates Fms-like receptor tyrosine kinase 3 (FLT3) at arginine (R) residues 972 and 973 (R972/973), and its oncogenic function in MLL-r ALL cells is FLT3 methylation dependent. Both biochemistry and computational analysis demonstrate that R972/973 methylation could facilitate recruitment of adaptor proteins to FLT3 in a phospho-tyrosine (Y) residue 969 (Y969) dependent or independent manner. Cells expressing R972/973 methylation-deficient FLT3 exhibited more robust apoptosis and growth inhibition than did Y969 phosphorylation-deficient FLT3-transduced cells. We also show that the capacity of the type I PRMT inhibitor MS023 to inhibit leukemia cell viability parallels baseline FLT3 R972/973 methylation levels. Finally, combining FLT3 tyrosine kinase inhibitor PKC412 with MS023 treatment enhanced elimination of MLL-r ALL cells relative to PKC412 treatment alone in patient-derived mouse xenografts. These results indicate that abolishing FLT3 arginine methylation through PRMT1 inhibition represents a promising strategy to target MLL-r ALL cells.

PRMT1-mediated FLT3 arginine methylation promotes maintenance of FLT3-ITD+ acute myeloid leukemia.

The presence of FMS-like receptor tyrosine kinase-3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate primitive FLT3-ITD+ leukemia cells, which are potential sources of relapse. Thus, understanding the mechanisms underlying FLT3-ITD+ AML cell persistence is essential to devise future AML therapies. Here, we show that expression of protein arginine methyltransferase 1 (PRMT1), the primary type I arginine methyltransferase, is increased significantly in AML cells relative to normal hematopoietic cells. Genome-wide analysis, coimmunoprecipitation assay, and PRMT1-knockout mouse studies indicate that PRMT1 preferentially cooperates with FLT3-ITD, contributing to AML maintenance. Genetic or pharmacological inhibition of PRMT1 markedly blocked FLT3-ITD+ AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginine 972/973, and PRMT1 promoted leukemia cell growth in an FLT3 methylation-dependent manner. Moreover, the effects of FLT3-ITD methylation in AML cells were partially due to cross talk with FLT3-ITD phosphorylation at tyrosine 969. Importantly, FLT3 methylation persisted in FLT3-ITD+ AML cells following kinase inhibition, indicating that methylation occurs independently of kinase activity. Finally, in patient-derived xenograft and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD+ AML cells relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes AML maintenance and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to eliminate FLT3-ITD+ AML cells.

miR-483-5p promotes esophageal cancer progression by targeting KCNQ1.

OBJECTIVES: miR-483-5p has been reported to be an oncogene of various cancers, but its functional and regulatory mechanisms in esophageal cancer (EC) remain unclear. This study aimed to investigate the functional and molecular mechanisms of miR-483-5p in EC so as to provide a theoretical basis for exploring the therapeutic target for EC. METHODS: miRNA expression profiles were downloaded from the TCGA-ESCA dataset to screen the target miRNA. Real-time quantitative PCR was performed to detect the transcriptional levels of miR-483-5p and KCNQ1 in EC cells. Western blot was conducted to determine the protein expression of KCNQ1. Cell Counting Kit-8 assay was carried out to assess cell proliferation. Transwell assay was performed to evaluate cell migration and invasion. Dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-483-5p and KCNQ1. RESULTS: miR-483-5p was up-regulated in EC cells and could bind to the 3'-untranslational region of KCNQ1. Over-expressing miR-483-5p suppressed KCNQ1 expression. Besides, miR-483-5p over-expression facilitated EC cell proliferation, migration and invasion, while its down-regulation triggered opposite result. Over-expressing miR-483-5p and KCNQ1 simultaneously could weaken the promoting effect of miR-483-5p over-expression on EC cell proliferation, migration and invasion. CONCLUSION: miR-483-5p as an oncogene facilitated EC cell proliferation, migration and invasion by targeted silencing KCNQ1, which is likely to provide a basis for further exploring the molecular mechanism of EC progression.