Jak2/STAT6/c-Myc pathway is vital to the pathogenicity of Philadelphia-positive acute lymphoblastic leukemia caused by P190BCR-ABL.

BACKGROUND: The Philadelphia chromosome encodes the BCR-ABL fusion protein, which has two primary subtypes, P210 and P190. P210 and P190 cause Philadelphia-positive chronic myeloid leukemia (Ph+ CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL), respectively. The Ph+ ALL is more malignant than Ph+ CML in disease phenotype and progression. This implies the key pathogenic molecules and regulatory mechanisms caused by BCR-ABL in two types of leukemia are different. It is reported that STAT6 was significantly activated only in P190 transformed cells. However, the potential role and the mechanism of STAT6 activation in Ph+ ALL and its activation mechanism by P190 are still unknown. METHODS: The protein and mRNA levels of STAT6, c-Myc, and other molecules were measured by western blot and quantitative real-time PCR. The STAT6 inhibitor AS1517499 was used to specifically inhibit p-STAT6. The effect of p-STAT6 inhibition on Ph+ CML and Ph+ ALL cells was identified by CCK-8 and FCM assay. Dual luciferase reporter and ChIP assay were performed to confirm the direct binding between STAT6 and c-Myc. The impact of STAT6 inhibition on tumor progression was detected in Ph+ CML and Ph+ ALL mouse models. RESULTS: Our results demonstrated that P210 induced CML-like disease, and P190 caused the more malignant ALL-like disease in mouse models. STAT6 was activated in P190 cell lines but not in P210 cell lines. Inhibition of STAT6 suppressed the malignancy of Ph+ ALL in vitro and in vivo, whereas it had little effect on Ph+ CML. We confirmed that p-STAT6 regulated the transcription of c-Myc, and STAT6 was phosphorylated by p-Jak2 in P190 cell lines, which accounted for the discrepant expression of p-STAT6 in P190 and P210 cell lines. STAT6 inhibition synergized with imatinib in Ph+ ALL cells. CONCLUSIONS: Our study suggests that STAT6 activation plays an essential role in the development of Ph+ ALL and may be a potential therapeutic target in Ph+ ALL. Video abstract.

DNMT3A controls miR-200b in cardiac fibroblast autophagy and cardiac fibrosis.

AIM AND OBJECTIVE: Regulation of microRNA gene expression by DNA methylation may represent a key mechanism to drive cardiac fibrosis progression. Cardiac fibroblast autophagy is the primary source of cardiac fibrosis, but the mechanisms underlying this process are incompletely understood. Here we found that DNMT3A suppression of the microRNA-200b (miR-200b) through pathway leads to cardiac fibroblast autophagy in cardiac fibrosis. METHODS: To understand the impact of DNMT3A on miR-200b at cardiac fibrosis, the rat cardiac fibrosis model was established via the abdominal aortic coarctation. Cardiac fibroblasts (CFs) were harvested from SD neonate rats and cultured. The expression of DNMT3A, miR-200b, collagen I was measured by western blotting, immunohistochemistry and qRT-PCR. Gain- or loss-of-function approaches were used to manipulate DNMT3A and miR-200b. RESULTS: DNMT3A level was upregulated and negatively correlated with miR-200b expression in fibrosis tissues and cardiac fibroblast. We found that autophagy was activated by miR-200b inhibitor and inactivated by miR-200b mimic in the rat cardiac fibroblast. Knockdown of DNMT3A notably increased the expression of miR-200b. CONCLUSIONS: Taken together, these findings indicate that DNMT3A regulation of miR-200b controls cardiac fibroblast autophagy during cardiac fibrosis and provide a basis for the development of therapies for cardiac fibrosis.

Symptomatic intracerebral hemorrhage after intravenous thrombolysis in Chinese patients: comparison of prediction models.

BACKGROUND: To assess the performance of risk scores in predicting symptomatic intracranial hemorrhage (SICH) after intravenous thrombolysis (IVT). METHODS: A multicenter prospective study was performed in 811 patients who underwent IVT with standard-dose recombinant tissue plasminogen activator within 4.5 hours of acute ischemic stroke (AIS) onset in 67 stroke centers involved in the Thrombolysis Implementation and Monitor of acute ischemic Stroke in China program from May 2007 to April 2012. SEDAN (blood sugar, early infarct signs, [hyper]dense cerebral artery sign, age) score, Safe Implementation of Thrombolysis in Stroke (SITS)-SICH score, Glucose Race Age Sex Pressure Stroke Severity (GRASPS) score, Multicenter Stroke Survey (MSS) score, and Stroke Prognostication using Age and National Institutes of Health Stroke Scale (SPAN)-100 index were calculated in selected patients, and their predictive performance for SICH was compared according to the National Institute of Neurological Disorders and Stroke (NINDS), Safe Implementation of Thrombolysis in Stroke-Monitoring Study (SITS-MOST), and European Cooperative Acute Stroke Study (ECASS)-II criteria. RESULTS: For predicting the risk of SICH (NINDS definition) after IVT, the area under the receiver operating characteristic (ROC) curve of MSS score was the highest (.71, P < .0001). For predicting the risk of SICH (SITS-MOST definition) after IVT, the area under the ROC curve of GRASPS score was the highest (.73, P = .005). For predicting SICH (ECASS-II definition) after IVT, the area under the ROC curve of MSS score was the highest (.73, P < .0001). CONCLUSIONS: SITS-SICH, GRASPS, and MSS scores predicted the risk of SICH after IVT in patients with AIS, but only the latter 2 were better in the Chinese population. MSS score had the best predictive performance for SICH using NINDS and ECASS-II definitions, whereas GRASPS score was the best for SICH using the SITS-MOST definition.

mTOR inhibition by AZD2014 alleviates BCR::ABL1 independent imatinib resistance through enhancing autophagy in CML resistant cells.

Chronic myeloid leukemia (CML) is a common hematopoietic malignancy in adults. Great progress has been made in CML therapy with imatinib. However, resistance to imatinib may occur during treatment. BCR::ABL1 dependent imatinib resistance has been well resolved with more potent tyrosine kinase inhibitors, but BCR::ABL1 independent resistance still remains to be resolved. This study is devoted to find novel targets for BCR::ABL1 independent imatinib-resistant patients. It is reported BCR::ABL1 independent resistance is mainly related to the activation of alternative survival pathway, and mTOR is an important regulator for cell growth especially in tumor cells. Hence, we explored the role of mTOR in BCR::ABL1 independent resistance, the possibility of mTOR to be a therapeutic target for imatinib resistant patients and the related mechanism. We found mTOR was upregulated in imatinib-resistant cells. mTOR inhibition by AZD2014 led to growth inhibition and synergized with imatinib in apoptosis induction in K562/G01. AZD2014 exerted its anti-leukemia effect through enhancing autophagy. mTOR signal pathway is poorly inhibited by imatinib and AZD2014 shows little effect on BCR::ABL1 signal pathway, which indicates that mTOR is involved in imatinib resistance via a BCR::ABL1 independent manner. Taken together, mTOR represents a potential target to overcome BCR::ABL1 independent imatinib resistance.

The HSP90 inhibitor KW-2478 depletes the malignancy of BCR/ABL and overcomes the imatinib-resistance caused by BCR/ABL amplification.

BACKGROUND: With the widespread clinical application of tyrosine kinase inhibitors (TKIs), an increasing number of chronic myeloid leukaemia (CML) patients have developed resistance or intolerance to TKIs. BCR/ABL is the oncoprotein of CML. HSP90 is an essential chaperone of BCR/ABL and plays an important role in protein folding and the function of BCR/ABL. Therefore, inhibiting the chaperone function of HSP90 may be an effective strategy for CML treatment and to overcome TKI resistance. METHODS: The effect of KW-2478 on CML cell viability, apoptosis and cell cycle progression was detected by CCK-8 assay or flow cytometry. The levels of BCR/ABL, HSP90 and other signalling proteins were detected by western blots. The mitochondrial membrane potential was detected by flow cytometry combined with JC-1 staining. The interaction between BCR/ABL and HSP90α was detected by coimmunoprecipitation. The effect of KW-2478 on BCR/ABL carcinogenesis in vivo was investigated in CML-like mouse models. RESULTS: KW-2478 inhibited growth and induced apoptosis of CML cells. KW-2478 inhibited the chaperone function of HSP90α and then weakened the BCR/ABL and MAPK signalling pathways. This treatment also caused an increase in p27 and p21 expression and a decrease in cyclin B1 expression, which led to G2/M phase arrest. The mitochondrial pathway was primarily responsible for KW-2478-induced apoptosis. KW-2478 had a synergistic effect with imatinib in growth inhibition. Notably, KW-2478 had a stronger effect on growth inhibition, apoptosis induction and cell cycle arrest of K562/G01 cells than K562 cells. KW-2478 could effectively prolong the mouse lifespan and alleviate disease symptoms in CML-like mouse models. CONCLUSIONS: This finding demonstrated that KW-2478 had anticancer properties in imatinib-sensitive and imatinib-resistant CML cells and illustrated the possible mechanisms. This study provides an alternative choice for CML treatment, especially for TKI-resistant patients with BCR/ABL amplification and TKI-intolerant patients.

Discrimination of isomeric monosaccharide derivatives using collision-induced fingerprinting coupled to ion mobility mass spectrometry.

Because of the isomeric heterogeneity that is ubiquitous in analytical science, a formidable analytical challenge is to fully discriminate multiple isomers, especially those candidate isomers with various biological functions. Ion mobility mass spectrometry (IM-MS) has gained impressive advances for gaining molecular conformations, whereas coexisting structurally similar isomers often make unambiguous discrimination impossible due to the limited IM resolution of commercially available instruments. Herein, we demonstrate an energy-resolved collision-induced fingerprint (CIF) method to fully discriminate isomeric monosaccharide derivatives that differ in terms of composition, connectivity and configuration without complex instrument modifications. By simply increasing the collisional energy in the trap cell, the full width at half maximum (FWHM) of IM peaks can be markedly narrowed by at least 2-fold. Given the excellent reproducibility of CIF measurements, the full discrimination of isomers can benefit from their unique feature values and root-mean square deviation (RMSD) in CIF spectra. Moreover, rapid discrimination of each monosaccharide derivate isomer from binary mixtures is demonstrated. This strategy will expand the horizons of IM-MS platform in the rapid differentiation of a wider range of isomers more than monosaccharide derivatives in complex systems, which facilitates the identification and evaluation of innovative isomer candidates with unexplored functions.

Insights into the degradation and toxicity difference mechanism of neonicotinoid pesticides in honeybees by mass spectrometry imaging.

Honeybees are essential for the pollination of a wide variety of crops and flowering plants, whereas they are confronting decline around the world due to the overuse of pesticides, especially neonicotinoids. The mechanism behind the negative impacts of neonicotinoids on honeybees has attracted considerable interest, yet it remains unknown due to the limited insights into the spatiotemporal distribution of pesticides in honeybees. Herein, we demonstrated the use of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) for the spatiotemporal visualization of neonicotinoids, such as N-nitroguanidine (dinotefuran) and N-cyanoamidine (acetamiprid) compounds, administered by oral application or direct contact, in the whole-body section of honeybees. The MSI results revealed that both dinotefuran and acetamiprid can quickly penetrate various biological barriers and distribute within the whole-body section of honeybees, but acetamiprid can be degraded much faster than dinotefuran. The degradation rate of acetamiprid is significantly decreased when piperonyl butoxide (PBO) is applied, whereas that of dinotefuran remains almost unchanged. These two factors might contribute to the fact that dinotefuran affords a higher toxicity to honeybees than acetamiprid. Moreover, the toxicity and degradation rate of acetamiprid can be affected by co-application with tebuconazole. Taken together, the results presented here indicate that the discrepant toxicity between dinotefuran and acetamiprid does not lie in the difference in their penetration of various biological barriers of honeybees, but in the degradation rate of neonicotinoid pesticides within honeybee tissues. Moreover, new perspectives are given to better understand the causes of the current decline in honeybee populations posed by insecticides, providing guidelines for the precise use of conventional agrochemicals and the rational design of novel pesticide candidates.

Spatiotemporal Visualization of Insecticides and Fungicides within Fruits and Vegetables Using Gold Nanoparticle-Immersed Paper Imprinting Mass Spectrometry Imaging.

Food safety issues caused by pesticide residue have exerted far-reaching impacts on human daily life, yet the available detection methods normally focus on surface residue rather than pesticide penetration to the internal area of foods. Herein, we demonstrated gold nanoparticle (AuNP)-immersed paper imprinting mass spectrometry imaging (MSI) for monitoring pesticide migration behaviors in various fruits and vegetables (i.e., apple, cucumber, pepper, plum, carrot, and strawberry). By manually stamping food tissues onto AuNP-immersed paper, this method affords the spatiotemporal visualization of insecticides and fungicides within fruits and vegetables, avoiding tedious and time-consuming sample preparation. Using the established MSI platform, we can track the migration of insecticides and fungicides into the inner region of foods. The results revealed that both the octanol-water partition coefficient of pesticides and water content of garden stuffs could influence the discrepancy in the migration speed of pesticides into food kernels. Taken together, this nanopaper imprinting MSI is poised to be a powerful tool because of its simplicity, rapidity, and easy operation, offering the potential to facilitate further applications in food analysis. Moreover, new perspectives are given to provide guidelines for the rational design of novel pesticide candidates, reducing the risk of food safety issues caused by pesticide residue.

microRNA-29a inhibits cardiac fibrosis in Sprague-Dawley rats by downregulating the expression of DNMT3A.

OBJECTIVE: This study aims to investigate the effect of miR-29a targeting the regulation of DNMT3A on the development of cardiac fibrosis in Sprague-Dawley (SD) rats. METHODS: In vivo experiment: SD rats were randomly divided into model and control groups. The cardiac and left ventricular indices in each group were calculated. The pathological changes of the myocardium were observed. The expression levels of miR-29a, CollA1, α-SMA, and DNMT3A in the myocardium of each group were detected. In vitro experiment: The cardiac fibroblasts (CFs) of SD rats were isolated from the myocardial tissue of SD rats and cultured. The miR-29a mimics, inhibitors, DNMT3A-siRNA, and control-siRNA were transfected into CFs. The expression levels of miR-29a, DNMT3A, CollA1, and α-SMA were detected, and the proliferation of CFs after transfection was observed. RESULTS: The heart weight index of the rats in the model group increased significantly compared with that in the control group. Obvious collagen deposition was observed in the myocardial tissue of the model group. The expression levels of CollA1, α-SMA, and DNMT3A in the model group were significantly higher than those in the control group (p<0.05). CONCLUSION: miR-29a reduced the activation and proliferation of CFs to improve cardiac fibrosis probably by the downregulation of DNMT3A.

LncRNA GAS5 controls cardiac fibroblast activation and fibrosis by targeting miR-21 via PTEN/MMP-2 signaling pathway.

Long noncoding RNAs (LncRNAs) are aberrantly expressed in many diseases including cardiac fibrosis. LncRNA growth arrest-specific 5 (GAS5) is reported as a significant mediator in the control of cell proliferation and growth; however, the role and function in cardiac fibrosis remain unknown. In this study, we confirmed that GAS5 was lowly expressed in cardiac fibrosis tissues as well as activated cardiac fibroblast. Overexpression of GAS5 inhibited the proliferation of cardiac fibroblast. Moreover, microRNA-21 (miR-21) has been reported to be overexpressed in cardiac fibrosis tissues as well as activated cardiac fibroblast, which is responsible for the progression of cardiac fibrosis. We found that up-regulated GAS5 decreased the expression of miR-21 significantly. Furthermore, GAS5 that upregulated or downregulated the expression of PTEN through miR-21 in cardiac fibroblasts. Taken together, GAS5 plays a suppressive role in cardiac fibrosis via negative regulation of miR-21. These results indicated that GAS5 may be a novel therapeutic target for further research of cardiac fibrosis.

Hyperphosphorylation of tau protein in the ipsilateral thalamus after focal cortical infarction in rats.

Hyperphosphorylation of tau has been considered as an important risk factor for neurodegenerative diseases. It has been found also in the cortex after focal cerebral ischemia. The present study is aimed at investigating changes of tau protein expression in the ipsilateral thalamus remote from the primary ischemic lesion site after distal middle cerebral artery occlusion (MCAO). The number of neurons in the ventroposterior thalamic nucleus (VPN) was evaluated using Nissl staining and neuronal nuclei (NeuN) immunostaining. Total tau and phosphorylated tau at threonine 231 (p-T231-tau) and serine 199 (p-S199-tau) levels, respectively, in the thalamus were measured using immunostaining and immunoblotting. Moreover, apoptosis was detected with terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL) assay. It was found that the numbers of intact neurons and NeuN(+) cells within the ipsilateral VPN were reduced significantly compared with the sham-operated group, but the levels of p-T231-tau and p-S199-tau in the ipsilateral thalamus were increased significantly in rats subjected to ischemia for 3 days, 7 days and 28 days. Furthermore, the number of TUNEL-positive cells was increased in the ipsilateral VPN at 7 days and 28 days after MCAO. Thus, hyperphosphorylated tau protein is observed in ipsilateral thalamus after focal cerebral infarction in this study. Our findings suggest that the expression of hyperphosphorylated tau protein induced by ischemia may be associated with the secondary thalamic damage after focal cortical infarction via an apoptotic pathway.