Deciphering DED assembly mechanisms in FADD-procaspase-8-cFLIP complexes regulating apoptosis.

Fas-associated protein with death domain (FADD), procaspase-8, and cellular FLICE-inhibitory proteins (cFLIP) assemble through death-effector domains (DEDs), directing death receptor signaling towards cell survival or apoptosis. Understanding their three-dimensional regulatory mechanism has been limited by the absence of atomic coordinates for their ternary DED complex. By employing X-ray crystallography and cryogenic electron microscopy (cryo-EM), we present the atomic coordinates of human FADD-procaspase-8-cFLIP complexes, revealing structural insights into these critical interactions. These structures illustrate how FADD and cFLIP orchestrate the assembly of caspase-8-containing complexes and offer mechanistic explanations for their role in promoting or inhibiting apoptotic and necroptotic signaling. A helical procaspase-8-cFLIP hetero-double layer in the complex appears to promote limited caspase-8 activation for cell survival. Our structure-guided mutagenesis supports the role of the triple-FADD complex in caspase-8 activation and in regulating receptor-interacting protein kinase 1 (RIPK1). These results propose a unified mechanism for DED assembly and procaspase-8 activation in the regulation of apoptotic and necroptotic signaling across various cellular pathways involved in development, innate immunity, and disease.

Study of individual domains contributing to MALT1 dimerization in BCL10-independent and dependent assembly.

The CARMA-BCL10-MALT1 (CBM) signalosome functions as a pivotal supramolecular module, integrating diverse receptor-induced signaling pathways to regulate BCL10-dependent NF-kB activation in innate and adaptive immunity. Conversely, the API2-MALT1 fusion protein in t(11; 18)(q21; q21) MALT lymphoma constitutively induces BCL10-independent NF-kB activation. MALT1 dimer formation is indispensable for the requisite proteolytic activity and is critical for NF-kB activation regulation in both scenarios. However, the molecular assembly of MALT1 individual domains in CBM activation remains elusive. Here we report the crystal structure of the MALT1 death domain (DD) at a resolution of 2.1 Å, incorporating reconstructed residues in previously disordered loops 1 and 2. Additionally, we observe a conformational regulation element (CRE) regulating stem-helix formation in NLRPs pyrin (PYD) within the MALT1 DD structure. The structure reveals a stem-helix-mediated dimer further corroborated in solution. To elucidate how the BCL10 filament facilitates MALT1 dimerization, we reconstitute a BCL10-CARD-MALT1-DD-IG1-IG2 complex model. We propose a N+7 rule for BCL10-dependent MALT1 dimerization via the IG1-IG2 domain and for MALT1-dependent cleavage in trans. Biochemical data further indicates concentration-dependent dimerization of the MALT1 IG1-IG2 domain, facilitating MALT1 dimerization in BCL10-independent manner. Our findings provide a structural and biochemical foundation for understanding MALT1 dimeric mechanisms, shedding light on potential BCL10-independent MALT1 dimer formation and high-order BCL10-MALT1 assembly.

Associations between cardiometabolic indices and the risk of diabetic kidney disease in patients with type 2 diabetes.

BACKGROUND: This study was designed to assess the associations between emerging cardiometabolic indices-the atherogenic index of plasma (AIP), the stress hyperglycemia ratio (SHR), the triglyceride-glucose (TyG) index, and the homeostasis model assessment of insulin resistance (HOMA-IR)-and the incidence of diabetic kidney disease (DKD) in type 2 diabetes (T2D) patients. METHODS: We consecutively enrolled 4351 T2D patients. The AIP, SHR, TyG index, and HOMA-IR were calculated from baseline parameters. DKD was defined as a urine albumin/creatinine ratio > 30 mg/g or an eGFR < 60 mL/min per 1.73 m. All participants were categorized into tertiles based on the cardiometabolic indices. Multivariate logistic regression models, restricted cubic splines, and receiver operating characteristic (ROC) curves were used for analysis. RESULTS: A total of 1371 (31.5%) patients were diagnosed with DKD. A restricted cubic spline showed a J-shaped association of the AIP and TyG index with DKD, a log-shaped association between HOMA-IR and DKD, and a U-shaped association between the SHR and DKD incidence. Multivariate logistic regression revealed that individuals in the highest tertile of the four cardiometabolic indices had a significantly greater risk of DKD than did those in the lowest tertile (AIP: OR = 1.08, 95% CI = 1.02-1.14, P = 0.005; SHR: OR = 1.42, 95% CI = 1.12-1.81, P = 0.004; TyG index: OR = 1.86, 95% CI = 1.42-2.45, P < 0.001; HOMA-IR: OR = 2.24, 95% CI = 1.52-3.30, P < 0.001). The receiver operating characteristic curves showed that the HOMA-IR score was better than other indices at predicting the risk of DKD, with an optimal cutoff of 3.532. CONCLUSIONS: Elevated AIP, SHR, TyG index and HOMA-IR are associated with a greater risk of DKD in patients with T2D. Among these indices, the HOMA-IR score demonstrated the strongest association with and predictive value for DKD incidence.

Synergistic anti-tumor effect of dual drug co-assembled nanoparticles based on ursolic acid and sorafenib.

Both ursolic acid (UA) and sorafenib (Sora) have been generally utilized in cancer treatment, and the combination of the two has also shown a good anti-tumor effect. However, single-agent therapy for Hepatocellular carcinoma (HCC) has the disadvantages of multi-drug resistance, poor water solubility and low bioavailability, and the application of traditional nanocarrier materials is limited due to their low drug loading and low carrier-related toxicity. Therefore, we prepared US NPs with different proportions of UA and Sora by solvent exchange method for achieving synergistic HCC therapy. US NPs had suitable particle size, good dispersibility and storage stability, which synergistically inhibited the proliferation of HepG2 cells, SMMC7721 cells and H22 cells. In addition, we also proved that US NPs were able to suppress the migration of HepG2 cells and SMMC7721 cells and reduce the adhesion ability and colony formation ability of these cells. According to the results, US NPs could degrade the membrane potential of mitochondrial, participate in cell apoptosis, and synergistically induce autophagy. Collectively, the carrier-free US NPs provide new strategies for HCC treatment and new ideas for the development of novel nano-drug delivery systems containing UA and Sora.

Dual-drug controllable co-assembly nanosystem for targeted and synergistic treatment of hepatocellular carcinoma.

The effectiveness of chemotherapeutic agents for hepatocellular carcinoma (HCC) is unsatisfactory because of tumor heterogeneity, multidrug resistance, and poor target accumulation. Therefore, multimodality-treatment with accurate drug delivery has become increasingly popular. Herein, a cell penetrating peptide-aptamer dual modified-nanocomposite (USILA NPs) was successfully constructed by coating a cell penetrating peptide and aptamer onto the surface of sorafenib (Sora), ursolic acid (UA) and indocyanine green (ICG) condensed nanodrug (USI NPs) via one-pot assembly for targeted and synergistic HCC treatment. USILA NPs showed higher cellular uptake and cytotoxicity in HepG2 and H22 cells, with a high expression of epithelial cell adhesion molecule (EpCAM). Furthermore, these NPs caused more significant mitochondrial membrane potential reduction and cell apoptosis. These NPs could selectively accumulate at the tumor site of H22 tumor-bearing mice and were detected with the help of ICG fluorescence; moreover, they retarded tumor growth better than monotherapy. Thus, USILA NPs can realize the targeted delivery of dual drugs and the integration of diagnosis and treatment. Moreover, the effects were more significant after co-administration of iRGD peptide, a tumor-penetrating peptide with better penetration promoting ability or programmed cell death ligand 1 (PD-L1) antibody for the reversal of the immunosuppressive state in the tumor microenvironment. The tumor inhibition rates of USILA NPs + iRGD peptide or USILA NPs + PD-L1 antibody with good therapeutic safety were 72.38 % and 67.91 % compared with control, respectively. Overall, this composite nanosystem could act as a promising targeted tool and provide an effective intervention strategy for enhanced HCC synergistic treatment.

A erythrocyte-platelet hybrid membrane coated biomimetic nanosystem based on ginsenosides and PFH combined with ultrasound for targeted delivery in thrombus therapy.

Thrombus is one of the culprits for global health problems. However, most current antithrombotic drugs are limited by restricted targeting ability and a high risk of systemic bleeding. A hybrid cell membrane-coated biomimetic nanosystem (PM/RM@PLGA@P/R) was constructed in this paper to fulfil the targeted delivery of ginsenoside (Rg1) and perfluorohexane (PFH). Poly lactic-co-glycolic acid (PLGA) is used as carriers to coat Rg1 and PFH. Thanks to the camouflage of erythrocyte membrane (RM) and platelet membrane (PM), the nanosystem in question possesses remarkable features including immune escape and self-targeting. Therefore, a compact nano-core with PLGA@P/R was formed, with a hybrid membrane covering the surface of the core, forming a "core-shell" structure. With its "core-shell" structure, this nanoparticle fancifully combines the advantages of both PFH (the low-intensity focused ultrasound (LIFU)-responsive phase-change thrombolysis) and Rg1(the antioxidant, anti-inflammatory and anticoagulant abilities). Meanwhile, PM/RM@PLGA@P/R nanoparticles exhibits superior in-vitro performance in terms of ROS scavenging, anticoagulant activity and immune escape compared with those without cell membranes (PLGA@P/R). Furthermore, in the animal experiment in which the tail vein thrombosis model was established by injecting k-carrageenan, the combined treatment of LIFU and PM/RM@PLGA@P/R showed a satisfactory antithrombotic efficiency (88.20 %) and a relatively higher biological safety level. This strategy provides new insights into the development of more effective and safer targeted biomimetic nanomedicines for antithrombotic treatments, possessing potential application in synergistic therapy field.

Lysine Deprivation Suppresses Adipogenesis in 3T3-L1 Cells: A Transcriptome Analysis.

Growing evidence proves that amino acid restriction can reverse obesity by reducing adipose tissue mass. Amino acids are not only the building blocks of proteins but also serve as signaling molecules in multiple biological pathways. The study of adipocytes' response to amino acid level changes is crucial. It has been reported that a low concentration of lysine suppresses lipid accumulation and transcription of several adipogenic genes in 3T3-L1 preadipocytes. However, the detailed lysine-deprivation-induced cellular transcriptomic changes and the altered pathways have yet to be fully studied. Here, using 3T3-L1 cells, we performed RNA sequencing on undifferentiated and differentiated cells, and differentiated cells under a lysine-free environment, and the data were subjected to KEGG enrichment. We found that the differentiation process of 3T3-L1 cells to adipocytes required the large-scale upregulation of metabolic pathways, mainly on the mitochondrial TCA cycle, oxidative phosphorylation, and downregulation of the lysosomal pathway. Single amino acid lysine depletion suppressed differentiation dose dependently. It disrupted the metabolism of cellular amino acids, which could be partially reflected in the changes in amino acid levels in the culture medium. It inhibited the mitochondria respiratory chain and upregulated the lysosomal pathway, which are essential for adipocyte differentiation. We also noticed that cellular interleukin 6 (IL6) expression and medium IL6 level were dramatically increased, which was one of the targets for suppressing adipogenesis induced by lysine depletion. Moreover, we showed that the depletion of some essential amino acids such as methionine and cystine could induce similar phenomena. This suggests that individual amino acid deprivation may share some common pathways. This descriptive study dissects the pathways for adipogenesis and how the cellular transcriptome was altered under lysine depletion.

Loss of TRIM24 promotes IL-10 expression via CBP/p300-dependent IFNß1 transcription during macrophage activation.

BACKGROUND: As an anti-inflammatory cytokine, interleukin 10 (IL-10) plays a vital role in preventing inflammatory and autoimmune pathologies while also maintaining immune homeostasis. IL-10 production in macrophages is tightly regulated by multiple pathways. TRIM24, a member of the Transcriptional Intermediary Factor 1 (TIF1) family, contributes to antiviral immunity and macrophage M2 polarization. However, the role of TRIM24 in regulating IL-10 expression and its involvement in endotoxic shock remains unclear. METHODS: In vitro, bone marrow derived macrophages cultured with GM-CSF or M-CSF were stimulated with LPS (100ng/ml). Murine models of endotoxic shock were established by challenging the mice with different dose of LPS (i.p). RTPCR, RNA sequencing, ELISA and hematoxylin and eosin staining were performed to elucidate the role and mechanisms of TRIM24 in endotoxic shock. RESULTS: The expression of TRIM24 is downregulated in LPS-stimulated bone marrow-derived macrophages (BMDMs). Loss of TRIM24 boosted IL-10 expression during the late stage of LPS-stimulation in macrophages. RNA-seq analysis revealed the upregulation of IFNß1, an upstream regulator of IL-10, in TRIM24 knockout macrophages. Treatment with C646, a CBP/p300 inhibitor, diminished the difference in both IFNß1 and IL-10 expression between TRIM24 knockout and control macrophages. Loss of TRIM24 provided protection against LPS-induced endotoxic shock in mice. CONCLUSION: Our results demonstrated that inhibiting TRIM24 promoted the expression of IFNß1 and IL-10 during macrophage activation, therefore protecting mice from endotoxic shock. This study offers novel insights into the regulatory role of TRIM24 in IL-10 expression, making it a potentially attractive therapeutic target for inflammatory diseases.

Imperatorin inhibits LPS-induced bone marrow-derived macrophages activation by decreased NF-κB p65 phosphorylation.

BACKGROUND: Imperatorin (IMP) is a secondary metabolite of plants and is the most abundant in Angelica dahurica. Previous studies showed that IMP exhibited anti-inflammatory activity in RAW264.7 cell line. Here, we aim to investigate the roles and mechanisms of IMP in bone marrow-derived macrophages (BMDMs), in view of the difference between primary macrophages and cell lines. METHODS: BMDMs were stimulated with LPS for the inflammation model. Flow cytometry was performed with BMDMs treated with different doses of IMP (0-20mg/L) within staining Annexin V-APC for 5 min. The cytokines and inflammatory mediators were detected by RT-PCR or ELISA. RNA-seq was performed in IMP-treated BMDMs or control, stimulated with LPS for 6h. Western blotting is carried out to determine the phosphorylation of p65, ERK1/2, JNK1, p38, and Akt. RESULTS: Our results showed that IMP inhibited IL-12p40, IL-6, TNF-α and IL-1ß in LPS-stimulated BMDMs. RNA-seq analysis suggested that IMP inhibits Toll-like receptor signaling pathway (KEGG), TNF signaling pathway (KEGG), NF-κB signaling pathway (KEGG), Inflammatory Response (GO). In addition, IMP inhibited myd88, tpl2, cxcl1, ptgs2(COX-2) expression in mRNA level. Finally, we found decreased phosphorylation of NF-κB p65 in IMP-treated BMDMs, after stimulated with LPS. CONCLUSION: IMP inhibits IL-12p40, IL-6, TNF-α, and IL-1ß expression in LPS-stimulated BMDMs. IMP inhibits macrophage activation, which maybe resulted in decreased phosphorylation of NF-κB p65. Furthermore, IMP may protect against the progress of inflammatory-related diseases.

Isocorydine Ameliorates IL-6 Expression in Bone Marrow-Derived Macrophages and Acute Lung Injury Induced by Lipopolysaccharide.

Isocorydine (ICD) is a type of isoquinoline alkaloid originating from Corydalis edulis, which has been used to relieve spasm, dilate blood vessels, and treat malaria as well as hypoxia in clinic. However, its effect on inflammation and underlying mechanisms remains unclear. The aim of our study was to determine the potential effects and mechanisms of ICD on pro-inflammatory interleukin-6 (IL-6) expression in bone marrow-derived macrophages (BMDMs) and acute lung injury mouse model. A mouse model of acute lung injury was established by intraperitoneal injection of LPS and treated with different doses of ICD. The body weight and food intake of mice were monitored to determine the toxicity of ICD. The tissue samples of lung, spleen and blood were taken to assess the pathological symptoms of acute lung injury and the expression levels of IL-6. Further, BMDMs isolated from C57BL/6 mice were cultured in vitro and treated with granulocyte-macrophage colony-stimulating factor (GM-CSF), LPS and different doses of ICD. CCK-8 assay and flow cytometry were performed to assess the viability of BMDMs. The expression of IL-6 was detected by RT-PCR and ELISA. RNA-seq was carried out to detect the differential expression genes of ICD-treated BMDMs. Western blotting was used to detect the change in MAPK and NF-κB signaling pathways. Our findings show that ICD ameliorates IL-6 expression and attenuates phosphorylation of p65 and JNK in BMDMs, and can protect mice from acute lung injury.

Experimental and computational models to investigate intestinal drug permeability and metabolism.

Oral administration is the preferred route for drug administration that leads to better therapy compliance. The intestine plays a key role in the absorption and metabolism of oral drugs, therefore, new intestinal models are being continuously proposed, which contribute to the study of intestinal physiology, drug screening, drug side effects, and drug-drug interactions.Advances in pharmaceutical processes have produced more drug formulations, causing challenges for intestinal models. To adapt to the rapid evolution of pharmaceuticals, more intestinal models have been created. However, because of the complexity of the intestine, few models can take all aspects of the intestine into account, and some functions must be sacrificed to investigate other areas. Therefore, investigators need to choose appropriate models according to the experimental stage and other requirements to obtain the desired results.To help researchers achieve this goal, this review summarised the advantages and disadvantages of current commonly used intestinal models and discusses possible future directions, providing a better understanding of intestinal models.

Hepatoenteric recycling is a new disposition mechanism for orally administered phenolic drugs and phytochemicals in rats.

Many orally administered phenolic drugs undergo enterohepatic recycling (EHR), presumably mediated by the hepatic phase II enzymes. However, the disposition of extrahepatically generated phase II metabolites is unclear. This paper aims to determine the new roles of liver and intestine in the disposition of oral phenolics. Sixteen representative phenolics were tested using direct portal vein infusion and/or intestinal perfusion. The results showed that certain glucuronides were efficiently recycled by liver. OATP1B1/1B3/2B1 were the responsible uptake transporters. Hepatic uptake is the rate-limiting step in hepatic recycling. Our findings showed that the disposition of many oral phenolics is mediated by intestinal glucuronidation and hepatic recycling. A new disposition mechanism 'Hepatoenteric Recycling (HER)", where intestine is the metabolic organ and liver is the recycling organ, was revealed. Further investigations focusing on HER should help interpret how intestinal aliments or co-administered drugs that alter gut enzymes (e.g. UGTs) expression/activities will impact the disposition of phenolics.

Ibrutinib and venetoclax target distinct subpopulations of CLL cells: implication for residual disease eradication.

Ibrutinib inhibits Bruton tyrosine kinase while venetoclax is a specific inhibitor of the anti-apoptotic protein BCL2. Both drugs are highly effective as monotherapy against chronic lymphocytic leukemia (CLL), and clinical trials using the combination therapy have produced remarkable results in terms of rate of complete remission and frequency of undetectable minimal residual disease. However, the laboratory rationale behind the success of the drug combination is still lacking. A better understanding of how these two drugs synergize would eventually help develop other rational combination strategies. Using an ex vivo model that promotes CLL proliferation, we show that modeled ibrutinib proliferative responses, but not viability responses, correlate well with patients' actual clinical responses. Importantly, we demonstrate for the first time that ibrutinib and venetoclax act on distinct CLL subpopulations that have different proliferative capacities. While the dividing subpopulation of CLL responds to ibrutinib, the resting subpopulation preferentially responds to venetoclax. The combination of these targeted therapies effectively reduced both the resting and dividing subpopulations in most cases. Our laboratory findings help explain several clinical observations and contribute to the understanding of tumor dynamics. Additionally, our proliferation model may be used to identify novel drug combinations with the potential of eradicating residual disease.

Rapid intestinal glucuronidation and hepatic glucuronide recycling contributes significantly to the enterohepatic circulation of icaritin and its glucuronides in vivo.

Icaritin (ICT), a prenylflavonoid derivative extracted from the Epimedium genus, has exhibited antitumor effects in hepatocellular carcinoma (HCC) cells and safety and tolerance in clinical settings. However, ICT exhibits low blood concentration and the in vivo dominant plasma species of ICT is glucuronides [icaritin-3-glucuronide (G1), icaritin-7-glucuronide (G2) and icaritin-3, 7-diglucuronide (DIG)]. Therefore, how ICT reaches the liver and exerts its effect with low toxicity remains unknown. Therefore, pharmacokinetic experiments (p.o. 5 mg/kg with/out 50 mg/kg inhibitor combo), intestinal perfusion (2 µM ICT), portal vein infusion (1.6 µM ICT, 7.1 µM G1, 6.8 µM G2 and 4.4 µM DIG), and in vitro studies (the concentration range of substrates: 0.3-10 µM) were conducted in the present study. Ultimately, ICT was shown to undergo glucuronidation by the intestine and subsequent uptake by hepatocytes via organic anion transporting peptides (OATPs) as conjugates, followed by biliary excretion mainly as diglucuronide. In conclusion, we found for the first time that the intestine is considered as the major metabolic organ, liver as the main recycling organ for the enterohepatic recycling (EHR) of ICT. Moreover, DIG is the main species in the systemic circulation following oral administration of ICT which explains the low toxicity of ICT in clinical settings.

Development and validation of an LC-MS/MS method for the quantification of flavonoid glucuronides (wogonoside, baicalin, and apigenin-glucuronide) in the bile and blood samples: Application to a portal vein infusion study.

Glucuronidation is one of the major metabolic pathways for flavonoids. However, quantification of flavonoid glucuronides in biological samples, especially in the bile, is sometimes challenging due to signal suppression by bile acids. The purpose of this study is to establish a robust LC-MS/MS method for directly measuring flavonoid glucuronides in bile and blood. Wogonoside (wogonin-7-O-glucuronide), baicalin (baicalein-7-O-glucuronide) and apigenin-7-O-glucuronide were used as the model compounds and taurocholic acid (T-CA) were used as the model bile acid to establish the method. Bile samples were processed using solid phase extraction (SPE) and blood samples were prepared using protein precipitation method. The analytes were separated on a Resteck HPLC (50 mm × 2.1 mm ID, 1.7 µm) column using acetonitrile and 0.1% formic acid in water as the mobile phases. The mass analysis was performed in an AB Sciex 5500 Qtrap mass spectrometer via multiple reaction monitoring (MRM) in the positive mode. The results showed that the linear range of the above three analytes were 10 nM-5000 nM in the bile and 1.56 nM-4000 nM in the blood, respectively. The recoveries of three glucuronides were >85% and the matrix effects were <20% at low, medium and high concentrations in the bile and the blood. The results also showed that >90% of these bile acids were removed by the selected SPE procedure to facilitate glucuronide analysis. The validated method was successfully applied to a portal vein infusion study using rats to quantify baicalin, wogonoside, and apigenin-glucuronide in bile and blood samples.

Development and Validation of a Radiomic-Based Model for Prediction of Intrahepatic Cholangiocarcinoma in Patients With Intrahepatic Lithiasis Complicated by Imagologically Diagnosed Mass.

BACKGROUND: This study was conducted with the intent to develop and validate a radiomic model capable of predicting intrahepatic cholangiocarcinoma (ICC) in patients with intrahepatic lithiasis (IHL) complicated by imagologically diagnosed mass (IM). METHODS: A radiomic model was developed in a training cohort of 96 patients with IHL-IM from January 2005 to July 2019. Radiomic characteristics were obtained from arterial-phase computed tomography (CT) scans. The radiomic score (rad-score), based on radiomic features, was built by logistic regression after using the least absolute shrinkage and selection operator (LASSO) method. The rad-score and other independent predictors were incorporated into a novel comprehensive model. The performance of the Model was determined by its discrimination, calibration, and clinical usefulness. This model was externally validated in 35 consecutive patients. RESULTS: The rad-score was able to discriminate ICC from IHL in both the training group (AUC 0.829, sensitivity 0.868, specificity 0.635, and accuracy 0.723) and the validation group (AUC 0.879, sensitivity 0.824, specificity 0.778, and accuracy 0.800). Furthermore, the comprehensive model that combined rad-score and clinical features was great in predicting IHL-ICC (AUC 0.902, sensitivity 0.771, specificity 0.923, and accuracy 0.862). CONCLUSIONS: The radiomic-based model holds promise as a novel and accurate tool for predicting IHL-ICC, which can identify lesions in IHL timely for hepatectomy or avoid unnecessary surgical resection.

Roux-en-Y gastric bypass enhances insulin secretion in type 2 diabetes via FXR-mediated TRPA1 expression.

OBJECTIVE: Roux-en-Y gastric bypass surgery (RYGB) improves the first phase of glucose-stimulated insulin secretion (GSIS) in patients with type 2 diabetes. How it does so remains unclear. Farnesoid X receptor (FXR), the nuclear receptor of bile acids (BAs), is implicated in bariatric surgery. Moreover, the transient receptor potential ankyrin 1 (TRPA1) channel is expressed in pancreatic ß-cells and involved in insulin secretion. We aimed to explore the role of BAs/FXR and TRPA1 in improved GSIS in diabetic rats after RYGB. METHODS: RYGB or sham surgery was conducted in spontaneous diabetic Goto-Kakizaki (GK) rats, or FXR or TRPA1 transgenic mice. Gene and protein expression of islets were assessed by qPCR and western blotting. Electrophysiological properties of single ß-cells were studied using patch-clamp technique. Binding of FXR and histone acetyltransferase steroid receptor coactivator-1 (SRC1) to the TRPA1 promoter, acetylated histone H3 (ACH3) levels at the TRPA1 promoter were determined using ChIP assays. GSIS was measured using enzyme-linked immunosorbent assays or intravenous glucose tolerance test (IVGTT). RESULTS: RYGB increases GSIS, particularly the first-phase of GSIS in both intact islets and GK rats in vivo, and ameliorates hyperglycemia of GK rats. Importantly, the effects of RYGB were attenuated in TRPA1-deficient mice. Moreover, GK ß-cells displayed significantly decreased TRPA1 expression and current. Patch-clamp recording revealed that TRPA1-/- ß-cells displayed a marked hyperpolarization and decreased glucose-evoked action potential firing, which was associated with impaired GSIS. RYGB restored TRPA1 expression and current in GK ß-cells. This was accompanied by improved glucose-evoked electrical activity and insulin secretion. Additionally, RYGB-induced TRPA1 expression involved BAs/FXR-mediated recruitment of SRC1, promoting ACH3 at the promoter of TRPA1. CONCLUSIONS: The BAs/FXR/SRC1 axis-mediated restoration of TRPA1 expression plays a critical role in the enhanced GSIS and remission of diabetes in GK rats after RYGB.

Myeloid translocation gene CBFA2T3 directs a relapse gene program and determines patient-specific outcomes in AML.

CBFA2T3 is a master transcriptional coregulator in hematopoiesis. In this study, we report novel functions of CBFA2T3 in acute myeloid leukemia (AML) relapse. CBFA2T3 regulates cell-fate genes to establish gene expression signatures associated with leukemia stem cell (LSC) transformation and relapse. Gene set enrichment analysis showed that CBFA2T3 expression marks LSC signatures in primary AML samples. Analysis of paired primary and relapsed samples showed that acquisition of LSC gene signatures involves cell type-specific activation of CBFA2T3 transcription via the NM_005187 promoter by GCN5. Short hairpin RNA-mediated downregulation of CBFA2T3 arrests G1/S cell cycle progression, diminishes LSC gene signatures, and attenuates in vitro and in vivo proliferation of AML cells. We also found that the RUNX1-RUNX1T1 fusion protein transcriptionally represses NM_005187 to confer t(8;21) AML patients a natural resistance to relapse, whereas lacking a similar repression mechanism renders non-core-binding factor AML patients highly susceptible to relapse. These studies show that 2 related primary AML-associated factors, the expression level of CBFA2T3 and the ability of leukemia cells to repress cell type-specific CBFA2T3 gene transcription, play important roles in patient prognosis, providing a paradigm that differential abilities to repress hematopoietic coregulator gene transcription are correlated with patient-specific outcomes in AML.

Oolong Tea Extract Induces DNA Damage and Cleavage and Inhibits Breast Cancer Cell Growth and Tumorigenesis.

BACKGROUND/AIM: Breast cancer is the most prevalent and devastating malignant disease among women worldwide. Green tea has been extensively studied for its anti-cancer effects, however, existing literature on the correlation of other types of tea with breast cancer is very limited. MATERIALS AND METHODS: We used six different breast cancer cell lines (ER+, PR+ or HER2+ and triple-negative), treated under different concentrations of green, oolong, black and dark tea extracts, and determined their biological effects. RESULTS: We determined cell viability, observed the changes of cell morphology, measured DNA damage and cleavage, and analyzed the effect on soft agar colony formation and growth. CONCLUSION: Oolong tea, same as green tea, can induce DNA damage and cleavage, play an inhibitory role in breast cancer cell growth, proliferation and tumorigenesis, and was a great potential as a chemo-preventive agent against breast cancer.