Monocytes Release Pro-Cathepsin D to Drive Blood-to-Brain Transcytosis in Diabetes.

BACKGROUND: Microvascular complications are the major outcome of type 2 diabetes progression, and the underlying mechanism remains to be determined. METHODS: High-throughput RNA sequencing was performed using human monocyte samples from controls and diabetes. The transgenic mice expressing human CTSD (cathepsin D) in the monocytes was constructed using CD68 promoter. In vivo 2-photon imaging, behavioral tests, immunofluorescence, transmission electron microscopy, Western blot analysis, vascular leakage assay, and single-cell RNA sequencing were performed to clarify the phenotype and elucidate the molecular mechanism. RESULTS: Monocytes expressed high-level CTSD in patients with type 2 diabetes. The transgenic mice expressing human CTSD in the monocytes showed increased brain microvascular permeability resembling the diabetic microvascular phenotype, accompanied by cognitive deficit. Mechanistically, the monocytes release nonenzymatic pro-CTSD to upregulate caveolin expression in brain endothelium triggering caveolae-mediated transcytosis, without affecting the paracellular route of brain microvasculature. The circulating pro-CTSD activated the caveolae-mediated transcytosis in brain endothelial cells via its binding with low-density LRP1 (lipoprotein receptor-related protein 1). Importantly, genetic ablation of CTSD in the monocytes exhibited a protective effect against the diabetes-enhanced brain microvascular transcytosis and the diabetes-induced cognitive impairment. CONCLUSIONS: These findings uncover the novel role of circulatory pro-CTSD from monocytes in the pathogenesis of cerebral microvascular lesions in diabetes. The circulatory pro-CTSD is a potential target for the intervention of microvascular complications in diabetes.

Structural basis for catalysis and substrate specificity of human ACAT1.

As members of the membrane-bound O-acyltransferase (MBOAT) enzyme family, acyl-coenzyme A:cholesterol acyltransferases (ACATs) catalyse the transfer of an acyl group from acyl-coenzyme A to cholesterol to generate cholesteryl ester, the primary form in which cholesterol is stored in cells and transported in plasma1. ACATs have gained attention as potential drug targets for the treatment of diseases such as atherosclerosis, Alzheimer's disease and cancer2-7. Here we present the cryo-electron microscopy structure of human ACAT1 as a dimer of dimers. Each protomer consists of nine transmembrane segments, which enclose a cytosolic tunnel and a transmembrane tunnel that converge at the predicted catalytic site. Evidence from structure-guided mutational analyses suggests that acyl-coenzyme A enters the active site through the cytosolic tunnel, whereas cholesterol may enter from the side through the transmembrane tunnel. This structural and biochemical characterization helps to rationalize the preference of ACAT1 for unsaturated acyl chains, and provides insight into the catalytic mechanism of enzymes within the MBOAT family8.

TRPML3/BK complex promotes autophagy and bacterial clearance by providing a positive feedback regulation of mTOR via PI3P.

TRPML3 is a Ca2+/Na+ release channel residing in both phagophores and endolysosomal membranes. It is activated by PI3P and PI3,5P2. Its activity can be enhanced by high luminal pH and by replacing luminal Na+ with K+. Here, we report that big-conductance Ca2+-activated potassium (BK) channels form a positive feedback loop with TRPML3. Ca2+ release via TRPML3 activates BK, which in turn facilitates TRPML3-mediated Ca2+ release, potentially through removing luminal Na+ inhibition. We further show that TRPML3/BK and mammalian target of rapamycin (mTOR) form another positive feedback loop to facilitate autophagy induction in response to nutrient starvation, i.e., mTOR inhibition upon nutrient starvation activates TRPML3/BK, and this further reduces mTOR activity, thereby increasing autophagy induction. Mechanistically, the feedback regulation between TRPML3/BK and mTOR is mediated by PI3P, an endogenous TRPML3 activator that is enriched in phagophores and is up-regulated by mTOR reduction. Importantly, bacterial infection activates TRPML3 in a BK-dependent manner, and both TRPML3 and BK are required for mTOR suppression and autophagy induction responding to bacterial infection. Suppressing either TRPML3 or BK helps bacteria survival whereas increasing either TRPML3 or BK favors bacterial clearance. Considering that TRPML3/BK is inhibited by low luminal pH but activated by high luminal pH and PI3P in phagophores, we suggest that TRPML3/BK and mTOR form a positive feedback loop via PI3P to ensure efficient autophagy induction in response to nutrient deprivation and bacterial infection. Our study reveals a role of TRPML3-BK coupling in controlling cellular homeostasis and intracellular bacterial clearance via regulating mTOR signaling.

IRE1α Mediates the Hypertrophic Growth of Cardiomyocytes Through Facilitating the Formation of Initiation Complex to Promote the Translation of TOP-Motif Transcripts.

BACKGROUND: Cardiomyocyte growth is coupled with active protein synthesis, which is one of the basic biological processes in living cells. However, it is unclear whether the unfolded protein response transducers and effectors directly take part in the control of protein synthesis. The connection between critical functions of the unfolded protein response in cellular physiology and requirements of multiple processes for cell growth prompted us to investigate the role of the unfolded protein response in cell growth and underlying molecular mechanisms. METHODS: Cardiomyocyte-specific inositol-requiring enzyme 1α (IRE1α) knockout and overexpression mouse models were generated to explore its function in vivo. Neonatal rat ventricular myocytes were isolated and cultured to evaluate the role of IRE1α in cardiomyocyte growth in vitro. Mass spectrometry was conducted to identify novel interacting proteins of IRE1α. Ribosome sequencing and polysome profiling were performed to determine the molecular basis for the function of IRE1α in translational control. RESULTS: We show that IRE1α is required for cell growth in neonatal rat ventricular myocytes under prohypertrophy treatment and in HEK293 cells in response to serum stimulation. At the molecular level, IRE1α directly interacts with eIF4G and eIF3, 2 critical components of the translation initiation complex. We demonstrate that IRE1α facilitates the formation of the translation initiation complex around the endoplasmic reticulum and preferentially initiates the translation of transcripts with 5' terminal oligopyrimidine motifs. We then reveal that IRE1α plays an important role in determining the selectivity and translation of these transcripts. We next show that IRE1α stimulates the translation of epidermal growth factor receptor through an unannotated terminal oligopyrimidine motif in its 5' untranslated region. We further demonstrate a physiological role of IRE1α-governed protein translation by showing that IRE1α is essential for cardiomyocyte growth and cardiac functional maintenance under hemodynamic stress in vivo. CONCLUSIONS: These studies suggest a noncanonical, essential role of IRE1α in orchestrating protein synthesis, which may have important implications in cardiac hypertrophy in response to pressure overload and general cell growth under other physiological and pathological conditions.

Early onset of pathological polyploidization and cellular senescence in hepatocytes lacking RAD51 creates a pro-fibrotic and pro-tumorigenic inflammatory microenvironment.

BACKGROUND AND AIMS: RAD51 recombinase (RAD51) is a highly conserved DNA repair protein and is indispensable for embryonic viability. As a result, the role of RAD51 in liver development and function is unknown. Our aim was to characterize the function of RAD51 in postnatal liver development. APPROACH AND RESULTS: RAD51 is highly expressed during liver development and during regeneration following hepatectomy and hepatic injury, and is also elevated in chronic liver diseases. We generated a hepatocyte-specific Rad51 deletion mouse model using Alb -Cre ( Rad51 -conditional knockout (CKO)) and Adeno-associated virus 8-thyroxine-binding globulin-cyclization recombination enzyme to evaluate the function of RAD51 in liver development and regeneration. The phenotype in Rad51 -CKO mice is dependent on CRE dosage, with Rad51fl/fl ; Alb -Cre +/+ manifesting a more severe phenotype than the Rad51fl/fl ; Alb -Cre +/- mice. RAD51 deletion in postnatal hepatocytes results in aborted mitosis and early onset of pathological polyploidization that is associated with oxidative stress and cellular senescence. Remarkable liver fibrosis occurs spontaneously as early as in 3-month-old Rad51fl/fl ; Alb -Cre +/+ mice. While liver regeneration is compromised in Rad51 -CKO mice, they are more tolerant of carbon tetrachloride-induced hepatic injury and resistant to diethylnitrosamine/carbon tetrachloride-induced HCC. A chronic inflammatory microenvironment created by the senescent hepatocytes appears to activate ductular reaction the transdifferentiation of cholangiocytes to hepatocytes. The newly derived RAD51 functional immature hepatocytes proliferate vigorously, acquire increased malignancy, and eventually give rise to HCC. CONCLUSIONS: Our results demonstrate a novel function of RAD51 in liver development, homeostasis, and tumorigenesis. The Rad51 -CKO mice represent a unique genetic model for premature liver senescence, fibrosis, and hepatocellular carcinogenesis.

A novel RIPK1 inhibitor reduces GVHD in mice via a nonimmunosuppressive mechanism that restores intestinal homeostasis.

Intestinal epithelial cells (IECs) are implicated in the propagation of T-cell-mediated inflammatory diseases, including graft-versus-host disease (GVHD), but the underlying mechanism remains poorly defined. Here, we report that IECs require receptor-interacting protein kinase-3 (RIPK3) to drive both gastrointestinal (GI) tract and systemic GVHD after allogeneic hematopoietic stem cell transplantation. Selectively inhibiting RIPK3 in IECs markedly reduces GVHD in murine intestine and liver. IEC RIPK3 cooperates with RIPK1 to trigger mixed lineage kinase domain-like protein-independent production of T-cell-recruiting chemokines and major histocompatibility complex (MHC) class II molecules, which amplify and sustain alloreactive T-cell responses. Alloreactive T-cell-produced interferon gamma enhances this RIPK1/RIPK3 action in IECs through a JAK/STAT1-dependent mechanism, creating a feed-forward inflammatory cascade. RIPK1/RIPK3 forms a complex with JAK1 to promote STAT1 activation in IECs. The RIPK1/RIPK3-mediated inflammatory cascade of alloreactive T-cell responses results in intestinal tissue damage, converting the local inflammation into a systemic syndrome. Human patients with severe GVHD showed highly activated RIPK1 in the colon epithelium. Finally, we discover a selective and potent RIPK1 inhibitor (Zharp1-211) that significantly reduces JAK/STAT1-mediated expression of chemokines and MHC class II molecules in IECs, restores intestinal homeostasis, and arrests GVHD without compromising the graft-versus-leukemia (GVL) effect. Thus, targeting RIPK1/RIPK3 in IECs represents an effective nonimmunosuppressive strategy for GVHD treatment and potentially for other diseases involving GI tract inflammation.

BRG1 mediates epigenetic regulation of TNFα-induced CCL2 expression in oral tongue squamous cell carcinoma cells.

Strong evidence has indicated that upregulation of chemokine (CC motif) ligand-2 (CCL2) expression and the presence of an inflammatory tumor microenvironment significantly contribute to the migratory and invasive properties of oral squamous cell carcinoma, specifically oral tongue squamous cell carcinoma (OTSCC). However, the precise epigenetic mechanism responsible for enhanced CCL2 expression in response to the inflammatory mediator tumor necrosis factor alpha (TNF-α) in OTSCC remains inadequately elucidated. We have demonstrated that the production of CCL2 can be induced by TNF-α, and this induction is mediated by the chromatin remodel protein BRG1. Through the use of a chromatin immunoprecipitation (ChIP) assay, we have found that BRG1 was involved in the recruitment of acetylated histones H3 and H4 at the CCL2 promoter, thereby activating TNF-α-induced CCL2 transcription. Furthermore, we have observed that recruitment of NF-κB p65 to the CCL2 promoter was increased following BRG1 overexpression and decreased after BRG1 knockdown in OTSCC cells. Our Re-ChIP assay has shown that BRG1 knockdown completely inhibits the recruitment of both acetylated histone H3 or H4 and NF-κB to the CCL2 promoter. In summary, the findings of our study demonstrate that BRG1 plays a significant role in mediating the production of CCL2 in OTSCC cells in response to TNF-α stimulation. This process involves the cooperative action of acetylated histone and NF-κB recruitment to the CCL2 promoter site. Our data suggest that BRG1 serves as a critical epigenetic mediator in the regulation of TNF-α-induced CCL2 transcription in OTSCC cells.

Protein Tyrosine Amination: Detection, Imaging, and Chemoproteomic Profiling with Synthetic Probes.

Protein tyrosine nitration (PTN) by oxidative and nitrative stress is a well-known post-translational modification that plays a role in the initiation and progression of various diseases. Despite being recognized as a stable modification for decades, recent studies have suggested the existence of a reduction in PTN, leading to the formation of 3-aminotyrosine (3AT) and potential denitration processes. However, the vital functions of 3AT-containing proteins are still unclear due to the lack of selective probes that directly target the protein tyrosine amination. Here, we report a novel approach to label and enrich 3AT-containing proteins with synthetic salicylaldehyde (SAL)-based probes: SALc-FL with a fluorophore and SALc-Yn with an alkyne tag. These probes exhibit high selectivity and efficiency in labeling and can be used in cell lysates and live cells. More importantly, SALc-Yn offers versatility when integrated into multiple platforms by enabling proteome-wide quantitative profiling of cell nitration dynamics. Using SALc-Yn, 355 proteins were labeled, enriched, and identified to carry the 3AT modification in oxidatively stressed RAW264.7 cells. These findings provide compelling evidence supporting the involvement of 3AT as a critical intermediate in nitrated protein turnover. Moreover, our probes serve as powerful tools to investigate protein nitration and denitration processes, and the identification of 3AT-containing proteins contributes to our understanding of PTN dynamics and its implications in cellular redox biology.

Fabrication of Carboxylate-Functionalized 2D MOF Nanosheet with Caged Cavity for Efficient and Selective Extraction of Uranium from Aqueous Solution.

The efficient removal of radioactive uranium from aqueous solution is of great significance for the safe and sustainable development of nuclear power. An ultrathin 2D metal-organic framework (MOF) nanosheet with cavity structures was elaborately fabricated based on a calix[4]arene ligand. Incorporating the permanent cavity structures on MOF nanosheet can fully utilize its structural characteristics of largely exposed surface area and accessible adsorption sites in pollutant removal, achieving ultrafast adsorption kinetics, and the functionalized cavity structure would endow the MOF nanosheets with the ability to achieve preconcentration and extraction of uranium from aqueous solution, affording ultrahigh removal efficiency even in ultra-low concentrations. Thus, more than 97% uranium can be removed from the concentration range of 50-500 µg L-1 within 5 min. Moreover, the 2D nano-material exhibits ultra-high anti-interference ability, which can efficiently remove uranium from groundwater and seawater. The adsorption mechanism was investigated by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR) analysis, and density functional theory (DFT) calculations, which revealed that the cavity structure plays an important role in uranium capture. This study not only realizes highly efficient uranium removal from aqueous solution but also opens the door to achieving ultrathin MOF nanosheets with cavity structures, which will greatly expand the applications of MOF nanosheets.

A distributed data processing scheme based on Hadoop for synchrotron radiation experiments.

With the development of synchrotron radiation sources and high-frame-rate detectors, the amount of experimental data collected at synchrotron radiation beamlines has increased exponentially. As a result, data processing for synchrotron radiation experiments has entered the era of big data. It is becoming increasingly important for beamlines to have the capability to process large-scale data in parallel to keep up with the rapid growth of data. Currently, there is no set of data processing solutions based on the big data technology framework for beamlines. Apache Hadoop is a widely used distributed system architecture for solving the problem of massive data storage and computation. This paper presents a set of distributed data processing schemes for beamlines with experimental data using Hadoop. The Hadoop Distributed File System is utilized as the distributed file storage system, and Hadoop YARN serves as the resource scheduler for the distributed computing cluster. A distributed data processing pipeline that can carry out massively parallel computation is designed and developed using Hadoop Spark. The entire data processing platform adopts a distributed microservice architecture, which makes the system easy to expand, reduces module coupling and improves reliability.

Asymptomatic norovirus infection during outbreaks in China: A systematic review and meta-analysis.

Acute gastroenteritis outbreaks may be caused by the excretion of norovirus (NoV) from asymptomatic individuals. Despite numerous studies involving asymptomatic NoV infection during outbreaks in China, a comprehensive assessment of its role has not been conducted, which is critical for emergency management. Our objective was to assess the prevalence of asymptomatic NoV infection during outbreaks in China. We conducted a comprehensive search of multiple databases, including PubMed, Web of Science, Cochrane Library, China National Knowledge Infrastructure, China Wanfang, and China Weipu, between January 1, 1997 and June 19, 2023. The retrieved articles and their references underwent screening, which utilized polymerase chain reaction-based assays for the detection of NoV in asymptomatic individuals during outbreaks that occurred in China. The primary summary data were the prevalence of asymptomatic NoV infection in outbreaks. We generated pooled estimates of asymptomatic prevalence in the population as a whole and in subgroups by using random-effect models. Of the 97 articles included, the pooled asymptomatic prevalence of NoV among 5117 individuals in outbreaks was 17.6% (95% confidence interval [CI]: 14.1-21.3). The asymptomatic prevalence of NoV GII (17.1%, 95% CI: 12.9-21.5) was similar to that of NoV GI (22.0%, 95% CI: 12.8-32.4). However, the proportion of asymptomatic individuals involved in NoV GII (57.44%) was significantly higher than that of NoV GI (5.12%), and NoV GII (75.26%) was reported much more frequently than NoV GI (14.43%) in the included articles. Meta-regression analysis of 11 possible influencing factors (geographic region, setting, season, sample type, genotype, transmission route, occupation, age, per capita income, study quality, and cases definition) showed that the source of heterogeneity might be related to the outbreak settings, per capita income, and study quality (p = 0.037, 0.058, and 0.026, respectively). Of particular note was the asymptomatic prevalence peaked in preschoolers (27.8%), afterward, it fell into trough in elementary and junior school children (10.5%), before the second peak located in adults (17.8%), and the elderly (25.2%). Prevalent genotypes reported include GII.4, followed by GII.17, GII.2, GII.3, GII.6, and so forth. The estimated asymptomatic prevalence of NoV during outbreaks in China was as high as 17.6%, with NoV GII dominating. In addition, genetic subtypes of NoV in outbreaks should be detected whenever possible. The role of asymptomatic individuals in NoV outbreaks cannot be ignored. This knowledge will help governments develop public health policies and emergency response strategies for outbreaks, assess the burden, and develop vaccines.

Characteristics of Molecular Genetic Mutations and Their Correlation with Prognosis in Adolescent and Adult Patients with Acute Lymphoblastic Leukemia.

INTRODUCTION: The prognosis of acute lymphoblastic leukemia (ALL) in adolescents and adults is poor, and recurrence is an important cause of their death. Changes of genetic information play a vital role in the pathogenesis and recurrence of ALL; however, the impact of molecular genetic mutations on disease diagnosis and prognosis remains unexplored. This study aimed to explore the frequency spectrum of gene mutations and their prognostic significance, along with the minimal residual disease (MRD) level and hematopoietic stem cell transplantation (HSCT), in adolescent and adult patients aged ≥15 years with ALL. METHODS: The basic characteristics, cytogenetics, molecular genetics, MRD level, treatment regimen, and survival outcome of patients with untreated ALL (≥15 years) were collected, and the correlation and survival analysis were performed using the SPSS 25.0 and R software. RESULTS: This study included 404 patients, of which 147 were selected for next-generation sequencing (NGS). NGS results revealed that 91.2% of the patients had at least one mutation, and 67.35% had multiple (≥2) mutations. NOTCH1, PHF6, RUNX1, PTEN, JAK3, TET2, and JAK1 were the most common mutations in T-ALL, whereas FAT1, TET2, NARS, KMT2D, FLT3, and RELN were the most common mutations in B-ALL. Correlation analysis revealed the mutation patterns, which were significantly different between T-ALL and B-ALL. In the prognostic analysis of 107 patients with B-ALL, multivariate analysis showed that the number of mutations ≥5 was an independent risk factor for overall survival and the RELN mutation was an independent poor prognostic factor for event-free survival. DISCUSSION: The distribution of gene mutations and the co-occurrence and repulsion of mutant genes in patients with ALL were closely related to the immunophenotype of the patients. The number of mutations ≥5 and the RELN mutation were significantly associated with poor prognosis in adolescent and adult patients with ALL.

Role of Additional MRI-Based Morphologic Measurements on the Performance of VI-RADS for Muscle-Invasive Bladder Cancer.

BACKGROUND: Vesical Imaging-Reporting and Data System (VI-RADS) is a pathway for the standardized imaging and reporting of bladder cancer staging using multiparametric (mp) MRI. PURPOSE: To investigate additional role of morphological (MOR) measurements to VI-RADS for the detection of muscle-invasive bladder cancer (MIBC) with mpMRI. STUDY TYPE: Retrospective. POPULATION: A total of 198 patients (72 MIBC and 126 NMIBC) underwent bladder mpMRI was included. FIELD STRENGTH/SEQUENCE: 3.0 T/T2-weighted imaging with fast-spin-echo sequence, spin-echo-planar diffusion-weighted imaging and dynamic contrast-enhanced imaging with fast 3D gradient-echo sequence. ASSESSMENT: VI-RADS score and MOR measurement including tumor location, number, stalk, cauliflower-like surface, type of tumor growth, tumor-muscle contact margin (TCM), tumor-longitudinal length (TLL), and tumor cellularity index (TCI) were analyzed by three uroradiologists (3-year, 8-year, and 15-year experience of bladder MRI, respectively) who were blinded to histopathology. STATISTICAL TESTS: Significant MOR measurements associated with MIBC were tested by univariable and multivariable logistic regression (LR) analysis with odds ratio (OR). Area under receiver operating characteristic curve (AUC) with DeLong's test and decision curve analysis (DCA) were used to compared the performance of unadjusted vs. adjusted VI-RADS. A P-value <0.05 was considered statistically significant. RESULTS: TCM (OR 9.98; 95% confidence interval [CI] 4.77-20.8), TCI (OR 5.72; 95% CI 2.37-13.8), and TLL (OR 3.35; 95% CI 1.40-8.03) were independently associated with MIBC at multivariable LR analysis. VI-RADS adjusted by three MORs achieved significantly higher AUC (reader 1 0.908 vs. 0.798; reader 2 0.906 vs. 0.855; reader 3 0.907 vs. 0.831) and better clinical benefits than unadjusted VI-RADS at DCA. Specially in VI-RADS-defined equivocal lesions, MOR-based adjustment resulted in 55.5% (25/45), 70.4% (38/54), and 46.4% (26/56) improvement in accuracy for discriminating MIBC in three readers, respectively. DATA CONCLUSION: MOR measurements improved the performance of VI-RADS in detecting MIBC with mpMRI, especially for equivocal lesions. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY: Stage 2.

Empagliflozin ameliorates vascular calcification in diabetic mice through inhibiting Bhlhe40-dependent NLRP3 inflammasome activation.

Type 2 diabetes mellitus (T2DM) patients exhibit greater susceptibility to vascular calcification (VC), which has a higher risk of death and disability. However, there is no specific drug for VC therapy. NLRP3 inflammasome activation as a hallmark event of medial calcification leads to arterial stiffness, causing vasoconstrictive dysfunction in T2DM. Empagliflozin (EMPA), a sodium-glucose co-transporter 2 inhibitor (SGLT2i), restrains hyperglycemia with definite cardiovascular benefits. Given the anti-inflammatory activity of EMPA, herein we investigated whether EMPA protected against VC in the aorta of T2DM mice by inhibiting NLRP3 inflammasome activation. Since db/db mice receiving a normal diet developed VC at the age of about 20 weeks, we administered EMPA (5, 10, 20 mg·kg-1·d-1, i.g) to 8 week-old db/db mice for 12 weeks. We showed that EMPA intervention dose-dependently ameliorated the calcium deposition, accompanied by reduced expression of RUNX2 and BMP2 proteins in the aortas. We found that EMPA (10 mg·kg-1·d-1 for 6 weeks) also protected against VC in vitamin D3-overloaded mice, suggesting the protective effects independent of metabolism. We showed that EMPA (10 mg·kg-1·d-1) inhibited the abnormal activation of NLRP3 inflammasome in aortic smooth muscle layer of db/db mice. Knockout (KO) of NLRP3 significantly alleviated VC in STZ-induced diabetic mice. The protective effects of EMPA were verified in high glucose (HG)-treated mouse aortic smooth muscle cells (MOVASs). In HG-treated NLRP3 KO MOVASs, EMPA (1 µM) did not cause further improvement. Bioinformatics and Western blot analysis revealed that EMPA significantly increased the expression levels of basic helix-loop-helix family transcription factor e40 (Bhlhe40) in HG-treated MOVASs, which served as a negative transcription factor directly binding to the promotor of Nlrp3. We conclude that EMPA ameliorates VC by inhibiting Bhlhe40-dpendent NLRP3 inflammasome activation. These results might provide potential significance for EMPA in VC therapy of T2DM patients.

Indoxyl sulphate-TNFα axis mediates uremic encephalopathy in rodent acute kidney injury.

Acute kidney injury (AKI) is often accompanied by uremic encephalopathy resulting from accumulation of uremic toxins in brain possibly due to impaired blood-brain barrier (BBB) function. Anionic uremic toxins are substrates or inhibitors of organic anionic transporters (OATs). In this study we investigated the CNS behaviors and expression/function of BBB OAT3 in AKI rats and mice, which received intraperitoneal injection of cisplatin 8 and 20 mg/kg, respectively. We showed that cisplatin treatment significantly inhibited the expressions of OAT3, synaptophysin and microtubule-associated protein 2 (MAP2), impaired locomotor and exploration activities, and increased accumulation of uremic toxins in the brain of AKI rats and mice. In vitro studies showed that uremic toxins neither alter OAT3 expression in human cerebral microvascular endothelial cells, nor synaptophysin and MAP2 expressions in human neuroblastoma (SH-SY5Y) cells. In contrast, tumour necrosis factor alpha (TNFα) and the conditioned medium (CM) from RAW264.7 cells treated with indoxyl sulfate (IS) significantly impaired OAT3 expression. TNFα and CM from IS-treated BV-2 cells also inhibited synaptophysin and MAP2 expressions in SH-SY5Y cells. The alterations caused by TNFα and CMs in vitro, and by AKI and TNFα in vivo were abolished by infliximab, a monoclonal antibody designed to intercept and neutralize TNFα, suggesting that AKI impaired the expressions of OAT3, synaptophysin and MAP2 in the brain via IS-induced TNFα release from macrophages or microglia (termed as IS-TNFα axis). Treatment of mice with TNFα (0.5 mg·kg-1·d-1, i.p. for 3 days) significantly increased p-p65 expression and reduced the expressions of Nrf2 and HO-1. Inhibiting NF-κB pathway, silencing p65, or activating Nrf2 and HO-1 obviously attenuated TNFα-induced downregulation of OAT3, synaptophysin and MAP2 expressions. Significantly increased p-p65 and decreased Nrf2 and HO-1 protein levels were also detected in brain of AKI mice and rats. We conclude that AKI inhibits the expressions of OAT3, synaptophysin and MAP2 due to IS-induced TNFα release from macrophages or microglia. TNFα impairs the expressions of OAT3, synaptophysin and MAP2 partly via activating NF-κB pathway and inhibiting Nrf2-HO-1 pathway.

Dysregulation of AMPK-mTOR signaling leads to comorbid anxiety in Dip2a KO mice.

Autism is often comorbid with other psychiatric disorders. We have previously shown that Dip2a knockout (KO) induces autism-like behaviors in mice. However, the role of Dip2a in other psychiatric disorders remains unclear. In this paper, we revealed that Dip2a KO mice had comorbid anxiety. Dip2a KO led to a reduction in the dendritic length of cortical and hippocampal excitatory neurons. Molecular mechanism studies suggested that AMPK was overactivated and suppressed the mTOR cascade, contributing to defects in dendritic morphology. Deletion of Dip2a in adult-born hippocampal neurons (Dip2a conditional knockout (cKO)) increased susceptibility to anxiety upon acute stress exposure. Application of (2R,6R)-hydroxynorketamine (HNK), an inhibitor of mTOR, rescued anxiety-like behaviors in Dip2a KO and Dip2a cKO mice. In addition, 6 weeks of high-fat diet intake alleviated AMPK-mTOR signaling and attenuated the severity of anxiety in both Dip2a KO mice and Dip2a cKO mice. Taken together, these results reveal an unrecognized function of DIP2A in anxiety pathophysiology via regulation of AMPK-mTOR signaling.

Design, synthesis and biological evaluation of MNK-PROTACs.

Mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) can regulate cellular mRNA translation by controlling the phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E), which plays an important role in tumor initiation, development, and metastasis. Although small-molecule MNK inhibitors have made significant breakthroughs in the treatment of various malignancies, their clinical application can be limited by drug resistance, target selectivity and other factors. The strategy of MNK-PROTACs which selectively degrades MNK kinases provides a new approach for developing small-molecule drugs for related diseases. In this study, DS33059, a small-molecule compound modified based on the ongoing clinical trials drug ETC-206, was chosen as the target protein ligand. A series of novel MNK-PROTACs were designed, synthesized and evaluated biological activity. Several compounds showed good inhibitory activities against MNK1/2. Besides, compounds exhibited moderate to excellent anti-proliferative activity in A549 and TMD-8 cells in vitro. In particular, compound II-5 significantly inhibited A549 (IC50 = 1.79 µM) and TMD-8 (IC50 = 1.07 µM) cells. The protein degradation assay showed that compound II-5 had good capability to degrade MNK1. The MNK-PROTACs strategy represents a new direction in treating tumors and deserves further exploration.

Association between obstructive sleep apnea severity and depression risk: a systematic review and dose-response meta-analysis.

Obstructive sleep apnea (OSA) has received considerable attention as a potential risk factor for depressive symptoms. The systematic review was conducted to confirm the dose‒response connection between OSA severity and depression risk. A systematic literature search of English and Chinese articles published in PubMed, EMBASE, Scopus, Web of Science, Cochrane Library, China National Knowledge Infrastructure (CNKI), and SinoMed databases from their inception to 28 August 2023 was conducted. An evaluation using the Newcastle‒Ottawa Scale was performed. A meta-analysis was used to evaluate the impact of OSA severity. A random-effects dose‒response model was conducted to evaluate the linear and nonlinear dose‒response connections. We evaluated publication bias by funnel plots, and symmetry by Egger's test. We identified 18 cross-sectional researches. 3143 participants which were involved in the dose‒response meta-analysis. Contrasted with mild OSA, individuals with severe OSA had a higher adjusted risk of depression (rate ratio: 1.34, 95% confidence interval = 1.05-1.70), with substantial heterogeneity (I2 = 70.9%, Pheterogeneity<0.001). There is a significant linear connection between OSA severity and depression risk. The depression risk increased by 0.4% for every 1 event per hour increase in the apnea-hypopnea index (AHI). The protocol for this unfunded research was drafted and registered at PROSPERO (ID CRD42023474097).

Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation.

Inducible regulatory T (iTreg) cells play a crucial role in immune suppression and are important for the maintenance of immune homeostasis. Mounting evidence has demonstrated connections between iTreg differentiation and metabolic reprogramming, especially rewiring in fatty acid oxidation (FAO). Previous work showed that butyrate, a specific type of short-chain fatty acid (SCFA) readily produced from fiber-rich diets through microbial fermentation, was critical for the maintenance of intestinal homeostasis and capable of promoting iTreg generation by up-regulating histone acetylation for gene expression as an HDAC inhibitor. Here, we revealed that butyrate could also accelerate FAO to facilitate iTreg differentiation. Moreover, butyrate was converted, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote FAO and thereby iTreg differentiation. Mutation of CPT1A at Arg243, a reported amino acid required for MCoA association, impaired both MCoA and BCoA binding, indicating that Arg243 is probably the responsible site for MCoA and BCoA association. Furthermore, blocking BCoA formation by ACSS2 inhibitor compromised butyrate-mediated iTreg generation and mitigation of mouse colitis. Together, we unveil a previously unappreciated role for butyrate in iTreg differentiation and illustrate butyrate-BCoA-CPT1A axis for the regulation of immune homeostasis.

Delineating the heterogeneity of matrix-directed differentiation toward soft and stiff tissue lineages via single-cell profiling.

Mesenchymal stromal/stem cells (MSCs) form a heterogeneous population of multipotent progenitors that contribute to tissue regeneration and homeostasis. MSCs assess extracellular elasticity by probing resistance to applied forces via adhesion, cytoskeletal, and nuclear mechanotransducers that direct differentiation toward soft or stiff tissue lineages. Even under controlled culture conditions, MSC differentiation exhibits substantial cell-to-cell variation that remains poorly characterized. By single-cell transcriptional profiling of nonconditioned, matrix-conditioned, and early differentiating cells, we identified distinct MSC subpopulations with distinct mechanosensitivities, differentiation capacities, and cell cycling. We show that soft matrices support adipogenesis of multipotent cells and early endochondral ossification of nonadipogenic cells, whereas intramembranous ossification and preosteoblast proliferation are directed by stiff matrices. Using diffusion pseudotime mapping, we outline hierarchical matrix-directed differentiation and perform whole-genome screening of mechanoresponsive genes. Specifically, top-ranked tropomyosin-1 is highly sensitive to stiffness cues both at RNA and protein levels, and changes in TPM1 expression determine the differentiation toward soft versus stiff tissue lineage. Consistent with actin stress fiber stabilization, tropomyosin-1 overexpression maintains YAP1 nuclear localization, activates YAP1 target genes, and directs osteogenic differentiation. Knockdown of tropomyosin-1 reversed YAP1 nuclear localization consistent with relaxation of cellular contractility, suppressed osteogenesis, activated early endochondral ossification genes after 3 d of culture in induction medium, and facilitated adipogenic differentiation after 1 wk. Our results delineate cell-to-cell variation of matrix-directed MSC differentiation and highlight tropomyosin-mediated matrix sensing.