Low expression of miR-182 caused by DNA hypermethylation accelerates acute lymphocyte leukemia development by targeting PBX3 and BCL2: miR-182 promoter methylation is a predictive marker for hypomethylation agents + BCL2 inhibitor venetoclax.

BACKGROUND: miR-182 promoter hypermethylation frequently occurs in various tumors, including acute myeloid leukemia, and leads to low expression of miR-182. However, whether adult acute lymphocyte leukemia (ALL) cells have high miR-182 promoter methylation has not been determined. METHODS: To assess the methylation status of the miR-182 promoter, methylation and unmethylation-specific PCR analysis, bisulfite-sequencing analysis, and MethylTarget™ assays were performed to measure the frequency of methylation at the miR-182 promoter. Bone marrow cells were isolated from miR-182 knockout (182KO) and 182 wild type (182WT) mice to construct BCR-ABL (P190) and Notch-induced murine B-ALL and T-ALL models, respectively. Primary ALL samples were performed to investigate synergistic effects of the hypomethylation agents (HMAs) and the BCL2 inhibitor venetoclax (Ven) in vitro. RESULTS: miR-182 (miR-182-5P) expression was substantially lower in ALL blasts than in normal controls (NCs) because of DNA hypermethylation at the miR-182 promoter in ALL blasts but not in normal controls (NCs). Knockout of miR-182 (182KO) markedly accelerated ALL development, facilitated the infiltration, and shortened the OS in a BCR-ABL (P190)-induced murine B-ALL model. Furthermore, the 182KO ALL cell population was enriched with more leukemia-initiating cells (CD43+B220+ cells, LICs) and presented higher leukemogenic activity than the 182WT ALL population. Furthermore, depletion of miR-182 reduced the OS in a Notch-induced murine T-ALL model, suggesting that miR-182 knockout accelerates ALL development. Mechanistically, overexpression of miR-182 inhibited proliferation and induced apoptosis by directly targeting PBX3 and BCL2, two well-known oncogenes, that are key targets of miR-182. Most importantly, DAC in combination with Ven had synergistic effects on ALL cells with miR-182 promoter hypermethylation, but not on ALL cells with miR-182 promoter hypomethylation. CONCLUSIONS: Collectively, we identified miR-182 as a tumor suppressor gene in ALL cells and low expression of miR-182 because of hypermethylation facilitates the malignant phenotype of ALL cells. DAC + Ven cotreatment might has been applied in the clinical try for ALL patients with miR-182 promoter hypermethylation. Furthermore, the methylation frequency at the miR-182 promoter should be a potential biomarker for DAC + Ven treatment in ALL patients.

Low-dose hypomethylating agents cooperate with ferroptosis inducers to enhance ferroptosis by regulating the DNA methylation-mediated MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway in acute myeloid leukemia.

BACKGROUND: Ferroptosis is a new form of nonapoptotic and iron-dependent type of cell death. Glutathione peroxidase-4 (GPX4) plays an essential role in anti-ferroptosis by reducing lipid peroxidation. Although acute myeloid leukemia (AML) cells, especially relapsed and refractory (R/R)-AML, present high GPX4 levels and enzyme activities, pharmacological inhibition of GPX4 alone has limited application in AML. Thus, whether inhibition of GPX4 combined with other therapeutic reagents has effective application in AML is largely unknown. METHODS: Lipid reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) assays were used to assess ferroptosis in AML cells treated with the hypomethylating agent (HMA) decitabine (DAC), ferroptosis-inducer (FIN) RAS-selective lethal 3 (RSL3), or their combination. Combination index (CI) analysis was used to assess the synergistic activity of DAC + RSL3 against AML cells. Finally, we evaluated the synergistic activity of DAC + RSL3 in murine AML and a human R/R-AML-xenografted NSG model in vivo. RESULTS: We first assessed GPX4 expression and found that GPX4 levels were higher in AML cells, especially those with MLL rearrangements, than in NCs. Knockdown of GPX4 by shRNA and indirect inhibition of GPX4 enzyme activity by RSL3 robustly induced ferroptosis in AML cells. To reduce the dose of RSL3 and avoid side effects, low doses of DAC (0.5 µM) and RSL3 (0.05 µM) synergistically facilitate ferroptosis by inhibiting the AMP-activated protein kinase (AMPK)-SLC7A11-GPX4 axis. Knockdown of AMPK by shRNA enhanced ferroptosis, and overexpression of SLC7A11 and GPX4 rescued DAC + RSL3-induced anti-leukemogenesis. Mechanistically, DAC increased the expression of MAGEA6 by reducing MAGEA6 promoter hypermethylation. Overexpression of MAGEA6 induced the degradation of AMPK, suggesting that DAC inhibits the AMPK-SLC7A11-GPX4 axis by increasing MAGEA6 expression. In addition, DAC + RSL3 synergistically reduced leukemic burden and extended overall survival compared with either DAC or RSL3 treatment in the MLL-AF9-transformed murine model. Finally, DAC + RSL3 synergistically reduced viability in untreated and R/R-AML cells and extended overall survival in two R/R-AML-xenografted NSG mouse models. CONCLUSIONS: Our study first identify vulnerability to ferroptosis by regulating MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway. Combined treatment with HMAs and FINs provides a potential therapeutic choice for AML patients, especially for R/R-AML.

Prognostic value analysis of cholesterol and cholesterol homeostasis related genes in breast cancer by Mendelian randomization and multi-omics machine learning.

Introduction: The high incidence of breast cancer (BC) prompted us to explore more factors that might affect its occurrence, development, treatment, and also recurrence. Dysregulation of cholesterol metabolism has been widely observed in BC; however, the detailed role of how cholesterol metabolism affects chemo-sensitivity, and immune response, as well as the clinical outcome of BC is unknown. Methods: With Mendelian randomization (MR) analysis, the potential causal relationship between genetic variants of cholesterol and BC risk was assessed first. Then we analyzed 73 cholesterol homeostasis-related genes (CHGs) in BC samples and their expression patterns in the TCGA cohort with consensus clustering analysis, aiming to figure out the relationship between cholesterol homeostasis and BC prognosis. Based on the CHG analysis, we established a CAG_score used for predicting therapeutic response and overall survival (OS) of BC patients. Furthermore, a machine learning method was adopted to accurately predict the prognosis of BC patients by comparing multi-omics differences of different risk groups. Results: We observed that the alterations in plasma cholesterol appear to be correlative with the venture of BC (MR Egger, OR: 0.54, 95% CI: 0.35-0.84, p<0.006). The expression patterns of CHGs were classified into two distinct groups(C1 and C2). Notably, the C1 group exhibited a favorable prognosis characterized by a suppressed immune response and enhanced cholesterol metabolism in comparison to the C2 group. In addition, high CHG score were accompanied by high performance of tumor angiogenesis genes. Interestingly, the expression of vascular genes (CDH5, CLDN5, TIE1, JAM2, TEK) is lower in patients with high expression of CHGs, which means that these patients have poorer vascular stability. The CAG_score exhibits robust predictive capability for the immune microenvironment characteristics and prognosis of patients(AUC=0.79). It can also optimize the administration of various first-line drugs, including AKT inhibitors VIII Imatinib, Crizotinib, Saracatinib, Erlotinib, Dasatinib, Rapamycin, Roscovitine and Shikonin in BC patients. Finally, we employed machine learning techniques to construct a multi-omics prediction model(Risklight),with an area under the feature curve (AUC) of up to 0.89. Conclusion: With the help of CAG_score and Risklight, we reveal the signature of cholesterol homeostasis-related genes for angiogenesis, immune responses, and the therapeutic response in breast cancer, which contributes to precision medicine and improved prognosis of BC.

DNA hypermethylation-induced miR-182 silence targets BCL2 and HOXA9 to facilitate the self-renewal of leukemia stem cell, accelerate acute myeloid leukemia progression, and determine the sensitivity of BCL2 inhibitor venetoclax.

Rationale: microRNAs (miRNAs) are frequently deregulated and play important roles in the pathogenesis and progression of acute myeloid leukemia (AML). miR-182 functions as an onco-miRNA or tumor suppressor miRNA in the context of different cancers. However, whether miR-182 affects the self-renewal of leukemia stem cells (LSCs) and normal hematopoietic stem progenitor cells (HSPCs) is unknown. Methods: Bisulfite sequencing was used to analyze the methylation status at pri-miR-182 promoter. Lineage-negative HSPCs were isolated from miR-182 knockout (182KO) and wild-type (182WT) mice to construct MLL-AF9-transformed AML model. The effects of miR-182 depletion on the overall survival and function of LSC were analyzed in this mouse model in vivo. Results: miR-182-5p (miR-182) expression was lower in AML blasts than normal controls (NCs) with hypermethylation observed at putative pri-miR-182 promoter in AML blasts but unmethylation in NCs. Overexpression of miR-182 inhibited proliferation, reduced colony formation, and induced apoptosis in leukemic cells. In addition, depletion of miR-182 accelerated the development and shortened the overall survival (OS) in MLL-AF9-transformed murine AML through increasing LSC frequency and self-renewal ability. Consistently, overexpression of miR-182 attenuated AML development and extended the OS in the murine AML model. Most importantly, miR-182 was likely dispensable for normal hematopoiesis. Mechanistically, we identified BCL2 and HOXA9 as two key targets of miR-182 in this context. Most importantly, AML patients with miR-182 unmethylation had high expression of miR-182 followed by low protein expression of BCL2 and resistance to BCL2 inhibitor venetoclax (Ven) in vitro. Conclusions: Our results suggest that miR-182 is a potential therapeutic target for AML patients through attenuating the self-renewal of LSC but not HSPC. miR-182 promoter methylation could determine the sensitivity of Ven treatment and provide a potential biomarker for it.

Clinical significance of bladder training in preoperative localization of high-intensity focused ultrasound ablation of uterine fibroids.

OBJECTIVES: High-intensity focused ultrasound (HIFU) is widely used to treat uterine fibroids. HIFU preoperative localization of uterine fibroids can be used to determine whether the patient is a suitable candidate for HIFU treatment. This study investigated the clinical significance of bladder training in improving the success rate of HIFU preoperative localization uterine fibroids. MATERIAL AND METHODS: Our sample consists of patients who planned to undergo HIFU treatment in our hospital but who were failed in previous HIFU preoperative localization. They were recruited between July 2021 and April 2022, and randomly divided into experimental and control groups. A total of 150 patients were enrolled. Each group consisted of 75 patients. The patients in the experimental group adopted the procedure of drinking water multiple times and retaining urine. The training program lasted three days. The patients in the control group were required to keep regular drinking and urination habits without any special instructions or requirements. RESULTS: There were no statistically significant differences between the two groups in maximum bladder capacity, residual urine volume of bladder, bladder filling levels, and bladder shape change. After bladder training, the maximum bladder capacity and the degree bladder shape change of the patients in the experimental group were improved significantly. The success rate of HIFU preoperative localization in the patients in the experimental group was significantly higher than that of the control group. CONCLUSIONS: Bladder training can effectively improve the success rate of HIFU preoperative localization of uterine fibroids.

Specific detection of fusion transcripts based on a duplex-specific nuclease and isothermal exponential amplification reaction.

Fusion genes are mostly found in tumor tissues, but are in low expression levels in healthy tissues, making them good candidate biomarkers for tumor diagnosis and therapy. Here, we propose a duplex-specific nuclease-isothermal exponential amplification reaction (DSN-IEXPAR) method for the detection of fusion transcripts. A DNA probe is specifically designed for fusion transcript recognition and hybridization, and DSN cleavages the DNA probe in the DNA/RNA duplex. Through controlling the recognition and cleavage temperature, DSN can only cut the DNA probe fully matched with the target fusion transcript rather than other transcripts containing partial the same sequence, endowing the proposed method with high specificity to the fusion transcript in the presence of homologous sequences. The truncated DNA probe after cutting can subsequently trigger IEXPAR as a probe, so as low as 100 fM fusion transcript can be detected with the proposed DSN-IEXPAR. The evaluation of the analytical performance of DSN-IEXPAR demonstrates that it can provide an effective platform for fusion transcript detection in the ordinary laboratory and clinical diagnosis.

Sensitive detection of fusion transcripts with padlock probe-based continuous cascade amplification (P-CCA).

Gene fusion, resulting from chromosomal rearrangements, is the juxtaposition of two or more original genes into the same set to form a functional gene. The significant specificity of fusion genes for tumor cells makes them promising candidates for diagnostic biomarkers and therapeutic targets. The sensitive detection of fusion transcripts is of great significance in biological research and disease diagnosis. Here, we propose a method for the sensitive detection of PML-RARα gene fusion transcripts by the direct ligation of the padlock probe at the junction site, and the cyclized DNA then triggers a continuous cascade amplification of two subsequent amplification reactions: rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP). Due to the ability of the ligation reaction to differentiate mismatched sequences and the high amplification efficiency of continuous cascade amplification reactions, the proposed method can detect as low as 1 fM targets with high specificity, and has been successfully applied to real samples. Through a facile design of the triggering sequence in padlock probes, the cascade RCA and LAMP can be integrated into one-tube isothermal reactions with a simple one-step operation. Therefore, this work provides a convenient padlock probe-based continuous cascade amplification (P-CCA) method for the detection of fusion transcripts, and offers a fast and reliable platform for the early clinical diagnosis of gene fusion-related cancers.

Characteristics of systemic inflammation and brain iron deposition in Parkinson's disease patients.

OBJECTIVE: This study aimed at determining the characteristics of systemic inflammation and brain iron deposition in Parkinson's disease (PD) patients. METHODS: Thirty two PD patients and 30 gender- as well as age-matched controls were enrolled. Serum interleukin (IL)-1ß, IL-33, tumor necrosis factor (TNF)-α, IL-6, IL-10, ferritin, iron, and total iron binding capacity (TIBC) levels were assayed. Quantitative susceptibility mapping (QSM) was used to quantitatively analyze brain iron accumulation in the regions of interest (ROIs). Correlations between concentrations of inflammatory cytokines and biomarkers for peripheral iron metabolism, brain iron deposition were evaluated in the PD group. RESULTS: Serum concentrations of IL-1ß and IL-33 were found to be significantly elevated in the PD group compared to the control group, and in early-stage PD group compared to advanced-stage PD group. Total QSM value for bilateral ROIs was significantly elevated in the PD group compared to the control group, and in advanced-stage PD group compared to early-stage PD group. There was a significant inverse correlation between serum IL-1ß concentration and total QSM value for bilateral ROIs, between serum ferritin, iron, TIBC concentrations, and total QSM value for bilateral ROIs in PD patients. However, there was no significant correlation between serum IL-1ß concentrations and serum ferritin, iron, TIBC concentrations in PD patients. INTERPRETATION: The inflammatory state and chronic brain iron deposition progression in PD patients might be asynchronous. Alterations in systemic inflammation were not correlated with peripheral iron metabolism and might not contribute to the aggravation of brain iron deposition in PD patients.

CT-based radiomics to predict development of macrovascular invasion in hepatocellular carcinoma: A multicenter study.

BACKGROUND: Macrovascular invasion (MaVI) occurs in nearly half of hepatocellular carcinoma (HCC) patients at diagnosis or during follow-up, which causes severe disease deterioration, and limits the possibility of surgical approaches. This study aimed to investigate whether computed tomography (CT)-based radiomics analysis could help predict development of MaVI in HCC. METHODS: A cohort of 226 patients diagnosed with HCC was enrolled from 5 hospitals with complete MaVI and prognosis follow-ups. CT-based radiomics signature was built via multi-strategy machine learning methods. Afterwards, MaVI-related clinical factors and radiomics signature were integrated to construct the final prediction model (CRIM, clinical-radiomics integrated model) via random forest modeling. Cox-regression analysis was used to select independent risk factors to predict the time of MaVI development. Kaplan-Meier analysis was conducted to stratify patients according to the time of MaVI development, progression-free survival (PFS), and overall survival (OS) based on the selected risk factors. RESULTS: The radiomics signature showed significant improvement for MaVI prediction compared with conventional clinical/radiological predictors (P < 0.001). CRIM could predict MaVI with satisfactory areas under the curve (AUC) of 0.986 and 0.979 in the training (n = 154) and external validation (n = 72) datasets, respectively. CRIM presented with excellent generalization with AUC of 0.956, 1.000, and 1.000 in each external cohort that accepted disparate CT scanning protocol/manufactory. Peel9_fos_InterquartileRange [hazard ratio (HR) = 1.98; P < 0.001] was selected as the independent risk factor. The cox-regression model successfully stratified patients into the high-risk and low-risk groups regarding the time of MaVI development (P < 0.001), PFS (P < 0.001) and OS (P = 0.002). CONCLUSIONS: The CT-based quantitative radiomics analysis could enable high accuracy prediction of subsequent MaVI development in HCC with prognostic implications.

A LRRK2 GTP Binding Inhibitor, 68, Reduces LPS-Induced Signaling Events and TNF-α Release in Human Lymphoblasts.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. Common genetic variation in LRRK2 modifies susceptibility to immunological disorders including Crohn's disease and leprosy. Previous studies have reported that LRRK2 is expressed in B lymphocytes and macrophages, suggesting a role for LRRK2 in immunological functions. In this study, we characterized the LRRK2 protein expression and phosphorylation using human lymphoblasts. Lipopolysaccharide (LPS), a proinflammatory agent, induced the increase of LRRK2 expression and kinase activities in human lymphoblasts in a time-dependent manner. Moreover, LPS activated the Toll-like receptor (TLR) signaling pathway, increased TRAF6/LRRK2 interaction, and elevated the phosphorylation levels of MAPK (JNK1/2, p38, and ERK1/2) and IkBα. Treatment with LRRK2 inhibitor 68 reduced LPS-induced TRAF6/LRRK2 interaction and MAPK and IkBα phosphorylation, thereby reducing TNF-α secretion. These results indicate that LRRK2 is actively involved in proinflammatory responses in human lymphoblasts, and inhibition of GTP binding by 68 results in an anti-inflammation effect against proinflammatory stimuli. These findings not only provide novel insights into the mechanisms of LRRK2-linked immune and inflammatory responses in B-cell-like lymphoblasts, but also suggest that 68 may also have potential therapeutic value for LRRK2-linked immunological disorders.

Characterization of the microstructure and hardness of case-carburized gear steel.

The microstructure and hardness of case-hardened steel were investigated after carburizing and austenitizing at 820-900 °C, and oil quenching and tempering at 180 °C. The carburized case had a multiphase microstructure consisting of martensite, carbides, and retained austenite, and the maximum content of the retained austenite was 30%; the particle size range was 2-3 µm. The nano-hardness decreased from about 12 GPa near the surface to about 7 GPa in the core, and the microhardness decreased from 800 HV0.2 to 450 HV0.2. The in-depth distribution of the microhardness and nano-hardness showed a similar trend, and the ratio of nano-hardness to microhardness was about 15. The results were attributed to the fine particle size of the retained austenite and its even distribution in the martensite matrix and it could not lower the nano-hardness. The nano-hardness was relatively low in areas of the retained austenite (about 5.5 GPa), and pop-in effects were observed, indicating the phase transformation of the retained austenite during nanoindentation loading.

Overexpression of Slit2 decreases neuronal excitotoxicity, accelerates glymphatic clearance, and improves cognition in a multiple microinfarcts model.

BACKGROUND: Cerebral microinfarcts (MIs) lead to progressive cognitive impairments in the elderly, and there is currently no effective preventative strategy due to uncertainty about the underlying pathogenic mechanisms. One possibility is the dysfunction of GABAergic transmission and ensuing excitotoxicity. Dysfunction of GABAergic transmission induces excitotoxicity, which contributes to stroke pathology, but the mechanism has kept unknown. The secreted leucine-rich repeat (LRR) family protein slit homologue 2 (Slit2) upregulates GABAergic activity and protects against global cerebral ischemia, but the neuroprotective efficacy of Slit2 against MIs has not been examined. METHODS: Middle-aged Wild type (WT) and Slit2-Tg mice were divided into sham and MI treatment groups. MIs were induced in parietal cortex by laser-evoked arteriole occlusion. Spatial memory was then compared between sham and MI groups using the Morris water maze (MWM) task. In addition, neuronal activity, blood brain barrier (BBB) permeability, and glymphatic clearance in peri-infarct areas were compared using two-photon imaging, while GABAergic transmission, microglial activation, neuronal loss, and altered cortical connectivity were compared by immunofluorescent staining or western blotting. RESULTS: Microinfarcts increased the amplitude and frequency of spontaneous intracellular Ca2+ signals, reduced neuronal survival and connectivity within parietal cortex, decreased the number of GABAergic interneurons and expression of vesicular GABA transporter (VGAT), induced neuroinflammation, and impaired both glymphatic clearance and spatial memory. Alternatively, Slit2 overexpression attenuated dysfunctional neuronal Ca2+ signaling, protected against neuronal death in the peri-infarct area as well as loss of parietal cortex connectivity, increased GABAergic interneuron number and VGAT expression, attenuated neuroinflammation, and improved both glymphatic clearance and spatial memory. CONCLUSION: Our results strongly suggest that overexpression of Slit2 protected against the dysfunction in MIs, which is a potential therapeutic target for cognition impairment in the elderly.

SWE combined with ACR TI-RADS categories for malignancy risk stratification of thyroid nodules with indeterminate FNA cytology.

OBJECTIVES: To compare the diagnostic efficacy of shear wave elastography (SWE) comnined with ACR TI-RADS categories for malignancy risk stratification of thyroid nodules with interminate FNA cytology. METHODS: The clinical data, sonographic features, ACR TI-RADS grading and shear wave elastography images of 193 patients of surgical pathologically proven thyroid nodules with interminate FNA cytology were retrospectively analyzed. The diagnostic efficacy of ACR TI-RADS categories, the maximum Young's modulus (Emax) of SWE and the combination of the two were calculated respectively. RESULTS: The ROC curves were drawn using surgical pathology results as the gold standard. The ROC curves indicated that the cut-off value of ACR TI-RADS and Emax of SWE was TR5 and 41.2 kPa respectively, and the area under the ROC curve (AUC) was 0.864 (95% CI: 0.879-0.934) and 0.858 (95% CI: 0.796-0.920) respectively. The diagnostic sensitivity, specificity and accuracy of ACR TI-RADS was 81.4% (127/156), 84.8% (31/37), and 81.9% (158/193), respectively. That of SWE Emax was 80.8% (126/156), 78.4% (29/37), and 80.3% (155/193), respectively. After SWE combined with ACR TI-RADS, the sensitivity, specificity and accuracy was 94.2% (147/156), 75.7% (28/37), and 90.7% (175/193), respectively. CONCLUSIONS: ACR TI-RADS classification system and shear wave elastography had high diagnostic efficacy for thyroid nodules with interminate FNA cytology. The combination of the two could improve diagnostic sensitivity and accuracy, and could help to differentiate benign and malignant thyroid nodules with interminate FNA cytology.

Whole body deletion of Gpr68 does not change hematopoietic stem cell function.

G protein-coupled receptor 68 (GPR68) responds to extracellular protons, thus called the proton-sensing G protein-coupled receptor (GPCR), leading to activation of the phospholipase C-ß (PLCß)/calcium (Ca2+) pathway or the adenylyl cyclase (AC)/cyclic AMP (cAMP) pathway. We recently found that whole body deletion of Gpr68 (Gpr68-/- mice) reduced the number of B lymphocytes with age and during hematopoietic regeneration, such as in response to fluorouracil (5-FU) administration. This prompted us to characterize the hematopoietic stem cell (HSC) phenotype in Gpr68-/- mice. Despite high level of Gpr68 protein expression on HSC in bone marrow (BM), the pool size of HSC was unaltered in Gpr68-/- mice either under steady state or upon stress, including aging and 5-FU treatment. HSC from Gpr68-/- mice exhibited comparable cellular features, such as cell cycle quiescence and cell survival. HSC from Gpr68-/- mice also exhibited comparable competitiveness after serial transplantation. Surprisingly, cytosolic Ca2+ accumulation was increased in HSC from Gpr68-/- mice. In contrast, cAMP levels were reduced in hematopoietic stem and progenitor cells (HSPC) from Gpr68-/- mice. Intriguingly, we found high level of Gpr68 protein expression on non-hematopoietic cells in BM, especially endothelial cells that function as HSC niche. In addition, expression of other proton-sensing GPCR was upregulated in HSPC from Gpr68-/- mice. Our studies suggest that Gpr68-/- mice display insignificant phenotype on HSC biology, possibly due to the function of Gpr68 in non-hematopoietic cells and/or the compensatory effects from other proton-sensing GPCR.

Cyclosporine enhances the sensitivity to lenalidomide in MDS/AML in vitro.

Our previous study revealed that expression of G protein-coupled receptor 68 (GPR68) was upregulated in MDSL cells, a cell line representing myelodysplastic syndromes (MDS), in response to lenalidomide (LEN), and mediated a calcium/calpain proapoptotic pathway. Isx, a GPR68 agonist, enhanced the sensitivity to LEN in MDSL cells. The fact that Isx is not a U.S. Food and Drug Administration-approved drug prompts us to look for alternative candidates that could enhance the sensitivity of LEN in MDS as well as other hematologic malignancies, such as acute myeloid leukemia (AML). In the study described here, we found that regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of calcineurin (CaN), was upregulated in MDSL cells in response to LEN, possibly through degradation of IKZF1. Consistently, cyclosporin (Cys), a pharmacological inhibitor of CaN, inhibited the activity of CaN and induced apoptosis in MDSL cells, indicating that CaN provided a prosurvival signal in MDSL cells. In addition, Cys enhanced the cytotoxic effect of LEN in MDS/AML cell lines as well as primary bone marrow cells from MDS patients and AML patient-derived xenograft models. Intriguingly, pretreatment with LEN reversed the suppressive effect of Cys on T-cell activation. Our study suggests a novel mechanism of action of LEN in mediating cytotoxicity in MDS/AML via upregulation of RCAN1, thus inhibiting the CaN prosurvival pathway. Our study also suggests that Cys enhances the sensitivity to LEN in MDS/AML cells without compromising T-cell activation.

G protein-coupled receptor 68 increases the number of B lymphocytes.

G protein-coupled receptor 68 (GPR68) is a proton sensor that is activated upon binding to extracellular protons. We have previously found that GPR68 induces a proapoptotic pathway in bone marrow (BM) cells from the patients with myelodysplastic syndromes (MDS) after treated with lenalidomide. However, the function of GPR68 in normal hematopoietic cells remains unclear. With genetic loss of function approach, we found reduced frequency and number of B lymphocytes in the peripheral blood (PB) of whole body Gpr68-/- mice compared to control littermates upon aging. During hematopoietic regeneration, such as in response to fluorouracil (5-FU), we also found reduced frequency and number of B lymphocytes in Gpr68-/- mice compared to wild type mice. Mechanism studies revealed that Gpr68 expression was upregulated in B lymphocytes of BM during aging and in hematopoietic progenitor cells after treatment with 5-FU. In addition, activation of Gpr68 by its activators increased the frequency and number of B lymphocytes. Our studies indicate that Gpr68 expression is upregulated in hematopoietic cells upon aging and during hematopoietic regeneration that ends up with increased number of B lymphocytes.

Extracellular ASC exacerbated the recurrent ischemic stroke in an NLRP3-dependent manner.

Using a photothrombotic mouse model of single stroke, we show that a single stroke onset increases the nuclear factor-κB (NF-κB), NLR family CARD domain containing protein 4 (NLRC4), and absent in melanoma 2 (AIM2) inflammasomes, as well as the mRNA levels of NLRP3. Next, using a photothrombotic mouse model of recurrent stroke, we found that recurrent strokes increased the activation of NLRP3, exacerbated the brain damage and the pro-inflammatory response in wild type (WT) mice, but not in NLRP3 knockout (NLRP3 KO) mice. Additionally, we found that apoptosis-associated speck-like protein containing a CARD (ASC) protein level surrounding the infarct area was comparatively increased, but that ASC specks outside of microglia in both the ipsilateral and contralateral of stroke site were decreased in NLRP3 KO mice relative to wild-type (WT) controls, and the number of ASC specks surrounding the second infarct area was positively correlated to the damage scores. Mechanistically, we found that recombinant ASC (RecASC) activated NLRP3 and induced pro-inflammatory responses, exacerbating the outcome of ischemic stroke, in WT mice, but not in NLRP3 KO mice. We therefore conclude that the NLRP3 inflammasome is activated by two attacks of stroke, which act together with ASC to exacerbate recurrent strokes.

Blockade of IL-17A/IL-17R Pathway Protected Mice from Sepsis-Associated Encephalopathy by Inhibition of Microglia Activation.

Sepsis-associated encephalopathy (SAE) is a poorly understood condition that leads to long-term cognitive impairment and increased mortality in survivors. Recent research revealed that IL-17A/IL-17R might serve as a checkpoint in microglia-mediated neuroinflammation. The present study was designed to determine the specific role of IL-17A-mediated microglia activation in the development of SAE. A mouse model of SAE was induced by cecal ligation and puncture (CLP), and behavior performance was evaluated by the inhibitory avoidance test and the open field test. Cytokine expression and microglia activation in brain tissue were determined at 6 h, 12 h, 24 h, 48 h, and day 7 post surgery. Further, septic mice were intracerebral ventricle- (i.c.v.-) injected with recombinant IL-17A, anti-IL-17A ab, anti-IL-17R ab, or isotype controls to evaluate the potential effects of IL-17A/IL-17R blockade in the prevention of SAE. Septic peritonitis induced significant impairment of learning memory and exploratory activity, which was associated with a higher expression of IL-17A, IL-1ß, and TNF-α in the brain homogenate. Fluorescence intensity of Iba-1 and IL-17R in the hippocampus was significantly increased following CLP. Treatment with recombinant IL-17A enhanced the neuroinflammation and microglia activation in CLP mice. On the contrary, neutralizing anti-IL-17A or anti-IL-17R antibodies mitigated the CNS inflammation and microglia activation, thus alleviating the cognitive dysfunction. Furthermore, as compared to the sham control, microglia cultured from CLP mice produced significantly higher levels of cytokines and expressed with higher fluorescence intensity of Iba-1 in response to IL-17A or LPS. Pretreatment with anti-IL-17R ab suppressed the Iba-1 expression and cytokine production in microglia stimulated by IL-17A. In conclusion, blockade of the IL-17A/IL-17R pathway inhibited microglia activation and neuroinflammation, thereby partially reversing sepsis-induced cognitive impairment. The present study suggested that the IL-17A/IL-17R signaling pathway had an important, nonredundant role in the development of SAE.

Xyloketal derivative C53N protects against mild traumatic brain injury in mice.

PURPOSE: Mild traumatic brain injury (mTBI), the most common type of TBI, can result in prolonged cognitive impairment, mood disorders, and behavioral problems. Reducing oxidative stress and inflammation can rescue the neurons from mTBI-induced cell death. Xyloketal B, a natural product from mangrove fungus, has shown good antioxidative and neuroprotective effects in several disease models. Here, we investigated the potential protection afforded by a xyloketal derivative, C53N, in a closed-skull mTBI model. MATERIALS AND METHODS: Skulls of mice were thinned to 20-30 µm thickness, following which they were subjected to a slight compression injury to induce mTBI. One hour after TBI, mice were intraperitoneally injected with C53N, which was solubilized in 0.5% dimethyl sulfoxide in saline. In vivo two-photon laser scanning microscopy was used to image cell death in injured parenchyma in each mouse over a 12-hour period (at 1, 3, 6, and 12 hours). Water content and oxidation index, together with pathological analysis of glial reactivity, were assessed at 24 hours to determine the effect of C53N on mTBI. RESULTS: Cell death, oxidative stress, and glial reactivity increased in mTBI mice compared with sham-injured mice. Treatment with 40 or 100 mg/kg C53N 1 hour after mTBI significantly attenuated oxidative stress and glial reactivity and reduced parenchymal cell death at the acute phase after mTBI. CONCLUSION: The present study highlights the therapeutic potential of the xyloketal derivative C53N for pharmacological intervention in mTBI.

Chronic colitis induces meninges traffic of gut-derived T cells, unbalances M1 and M2 microglia/macrophage and increases ischemic brain injury in mice.

Ischemic stroke is one of the most common diseases leading to death and is the primary cause of physical handicap. Recent studies have reported that chronic colitis increases the risk of ischemic stroke, but it is unknown whether chronic colitis participates in ischemic brain injury directly. A combined mouse model of chronic colitis induced by dextran sodium sulfate (DSS) and ischemic stroke induced by photochemical infarction was used in this study. We demonstrated that chronic colitis significantly increased the infarction volume, activated microglia/macrophage numbers, proliferation of M1 microglia/macrophage, non-gut-derived CD4+ T lymphocyte penetration and decreased neuron numbers in the peri-infarction at 7 d after stroke. Furthermore, gut-derived CD4+ T cell accumulation on the meninges was observed at 7 d after stroke. In addition, selective depletion of meningeal macrophages resulted in a reduction of infarction volume and the non-gut-derived CD4+ T lymphocyte penetration. We concluded that chronic colitis exacerbated ischemic stroke by promoting CD4+ T cell migration from the gut to the meninges and disequilibrium of M1 and M2 microglia/macrophages. We speculated that the gut-derived CD4+ T cells may interact with meningeal macrophages and result in non-gut-derived CD4+ T lymphocyte infiltration that aggravated brain injury in ischemic stroke.