Efficacy and safety of probiotics in irritable bowel syndrome: A systematic review and meta-analysis.

BACKGROUND: Irritable bowel syndrome (IBS) is a common gastrointestinal disease characterized by abdominal pain, distension, and altered bowel habits. Probiotics may alleviate IBS symptoms, but clinical trials remain conflicting. AIMS: To conduct a systematic review and meta-analysis of clinical trials to evaluate the efficacy and safety of probiotics for IBS patients. METHODS: We searched relevant trials in PubMed, Web of Science, Embase, Cochrane Library, and Google Scholar from 2000 to June 2023. Standardized mean difference (SMD) and 95% confidence interval (CI) were calculated for continuous outcomes. A risk ratio (RR) and a 95% CI were calculated for dichotomous outcomes. RESULTS: A total of 20 studies involving 3011 patients were obtained. The results demonstrated that probiotics are more effective than placebo in reducing global IBS symptoms improvement rate (RR = 1.401, 95% CI 1.182-1.662, P < 0.001) and quality of life scores (SMD = 0.286, 95% CI = 0.154-0.418, P < 0.001). Subgroup analyses showed that a shorter treatment time (less than eight weeks) could reduce distension scores (SMD = 0.197, 95% CI = 0.038-0.356, P = 0.015). High doses (daily dose of probiotics ≥ 10ˆ10) or multiple strains of probiotics exhibit beneficial effects on abdominal pain (SMD = 0.412, 95% CI = 0.112-0.711, P = 0.007; SMD = 0.590, 95% CI = 0.050-1.129, P = 0.032; respectively). However, there was no significant benefit on global symptom scores (SMD = 0.387, 95% CI 0.122 to 0.653, P = 0.004) with statistically high inter-study heterogeneity (I2 = 91.9%, P < 0.001). Furthermore, there was no significant inter-group difference in terms of adverse events frequency (RR = 0.997, 95% CI 0.845-1.177, P = 0.973). CONCLUSION: Probiotics are effective and safe for IBS patients. High doses or multiple probiotic strains seem preferable, but definite conclusions are challenging due to the high heterogeneity. Large-scale, well-designed, and rigorous trials are needed to confirm their effectiveness.

Timing of MeCP2 Expression Determines Midbrain Dopamine Neuron Phenotype Specification.

Midbrain dopamine (DA) neurons are associated with locomotor and psychiatric disorders. DA phenotype is specified in ancestral neural precursor cells (NPCs) and maintained throughout neuronal differentiation. Here we show that endogenous expression of MeCP2 coincides with DA phenotype specification in mouse mesencephalon, and premature expression of MeCP2 prevents in vitro cultured NPCs from acquiring DA phenotype through interfering NURR1 transactivation of DA phenotype genes. By contrast, ectopic MeCP2 expression does not disturb DA phenotype in the DA neurons. By analyzing the dynamic change of DNA methylation along DA neuronal differentiation at the promoter of DA phenotype gene tyrosine hydroxylase (Th), we show that Th expression is determined by TET1-mediated de-methylation of NURR1 binding sites within Th promoter. Chromatin immunoprecipitation assays demonstrate that premature MeCP2 dominates the DNA binding of the corresponding sites thereby blocking TET1 function in DA NPCs, whereas TET1-mediated de-methylation prevents excessive MeCP2 binding in DA neurons. The significance of temporal DNA methylation status is further confirmed by targeted methylation/demethylation experiments showing that targeted de-methylation in DA NPCs protects DA phenotype specification from ectopic MeCP2 expression, whereas targeted methylation disturbs phenotype maintenance in MeCP2-overexpressed DA neurons. These findings suggest the appropriate timing of MeCP2 expression as a novel determining factor for guiding NPCs into DA lineage.

Esketamine alleviates postoperative depression-like behavior through anti-inflammatory actions in mouse prefrontal cortex.

The rising incidence of postoperative depression (POD) in recent years has placed a heavy burden on patients' physical and mental health. At this point in time, however, POD pathogenesis remains poorly understood and novel therapeutic strategies are being sought. The present study aimed to clarify esketamine's protective effects and possible mechanisms of action in POD. To this avail, we used an animal model of postoperative depression to analyze behavioral, parameters, plus the inflammatory response in serum and in the medial prefrontal cortex (mPFC). Using immunofluorescence staining, we detected the number of microglia and parvalbumin (PV) in mPFC, and determined changes in neuronal dendritic spine density via Golgi staining. Expression of Iba1, PSD95 and NF-κB was examined by Western blot analysis. Our results show that esketamine can significantly improve depression-like symptoms caused by anesthesia and surgery. In addition, esketamine administration reversed the decrease in the density of PV neurons and restored synaptogenesis in mPFC which had been perturbed by inflammation. The evidence obtained suggests esketamine's anti-inflammatory effects may be mediated by the BDNF/TrkB signaling pathway and possibly by attenuation of the nuclear factor κB (NF-κB) pathway. These data warrant further investigations into the interplay of esketamine, and microglia in the modulation of POD symptomatology.

Differentiation Between Primary Central Nervous System Lymphoma and Atypical Glioblastoma Based on MRI Morphological Feature and Signal Intensity Ratio: A Retrospective Multicenter Study.

OBJECTIVES: To investigate the value of morphological feature and signal intensity ratio (SIR) derived from conventional magnetic resonance imaging (MRI) in distinguishing primary central nervous system lymphoma (PCNSL) from atypical glioblastoma (aGBM). METHODS: Pathology-confirmed PCNSLs (n = 93) or aGBMs (n = 48) from three institutions were retrospectively enrolled and divided into training cohort (n = 98) and test cohort (n = 43). Morphological features and SIRs were compared between PCNSL and aGBM. Using linear discriminant analysis, multiple models were constructed with SIRs and morphological features alone or jointly, and the diagnostic performances were evaluated via receiver operating characteristic (ROC) analysis. Areas under the curves (AUCs) and accuracies (ACCs) of the models were compared with the radiologists' assessment. RESULTS: Incision sign, T2 pseudonecrosis sign, reef sign and peritumoral leukomalacia sign were associated with PCNSL (training and overall cohorts, P < 0.05). Increased T1 ratio, decreased T2 ratio and T2/T1 ratio were predictive of PCNSL (all P < 0.05). ROC analysis showed that combination of morphological features and SIRs achieved the best diagnostic performance for differentiation of PCNSL and aGBM with AUC/ACC of 0.899/0.929 for the training cohort, AUC/ACC of 0.794/0.837 for the test cohort and AUC/ACC of 0.869/0.901 for the overall cohort, respectively. Based on the overall cohort, two radiologists could distinguish PCNSL from aGBM with AUC/ACC of 0.732/0.724 for radiologist A and AUC/ACC of 0.811/0.829 for radiologist B. CONCLUSION: MRI morphological features can help differentiate PCNSL from aGBM. When combined with SIRs, the diagnostic performance was better than that of radiologists' assessment.

A novel histone deacetylase inhibitor-based approach to eliminate microglia and retain astrocyte properties in glial cell culture.

The close association between astrocytes and microglia causes great difficulties to distinguish their individual roles in innate immune responses in central nervous system. Current chemical-based methods to eliminate microglia in glial cell culture introduce various molecular and functional alterations to astrocytes. Here, we describe a novel two-step approach to achieve a complete elimination of microglia without affecting the biological properties of co-cultured astrocytes by temporal treatment of histone deacetylase inhibitor trichostatin A (TSA). We verify TSA as a potent inducer for microglial-specific apoptotic cell death, which also causes comprehensive gene expression changes in astrocytes. However, withdrawal of TSA not only ensures no microglia repopulation, but also restores all the gene expression changes in terms of astrocyte functions, including neurotrophic factors, glutamate and potassium transporters, and reactive astrocyte subtypes. By contrast, withdrawal of PLX5622, the commonly used colony-stimulating factor 1 receptor inhibitor neither prevents microglia repopulation nor restores the gene expression changes mentioned above. Using this method, we are able to discriminate differential roles of microglia and astrocytes in the induced expression of antiviral and pro-inflammatory cytokines upon various pathological stimuli including the spike protein of SARS-CoV-2. This simple and efficient method can be customized for the understanding of microglia-astrocyte interaction and the development of epigenetic therapies that target over-activated microglia in neuroinflammation-related diseases.

A novel histone deacetylase inhibitor-based approach to eliminate microglia and retain astrocyte properties in glial cell culture.

The close association between astrocytes and microglia causes great difficulties to distinguish their individual roles in innate immune responses in central nervous system. Current chemical-based methods to eliminate microglia in glial cell culture introduce various molecular and functional alterations to astrocytes. Here, we describe a novel two-step approach to achieve a complete elimination of microglia without affecting the biological properties of co-cultured astrocytes by temporal treatment of histone deacetylase inhibitor trichostatin A (TSA). We verify TSA as a potent inducer for microglial-specific cell death, which also causes comprehensive gene expression changes in astrocytes. However, withdrawal of TSA not only ensures no microglia repopulation, but also restores all the gene expression changes in terms of astrocyte functions, including neurotrophic factors, glutamate and potassium transporters, and reactive astrocyte subtypes. By contrast, withdrawal of PLX5622, the commonly used colony-stimulating factor 1 receptor inhibitor neither prevents microglia repopulation nor restores the gene expression changes mentioned above. Using this method, we are able to discriminate differential roles of microglia and astrocytes in the induced expression of antiviral and pro-inflammatory cytokines upon various pathological stimuli including the spike protein of SARS-CoV-2. This simple and efficient method can be customized for the understanding of microglia-astrocyte interaction and the development of epigenetic therapies that target over-activated microglia in neuroinflammation-related diseases.

Crosstalk between cardiomyocytes and noncardiomyocytes is essential to prevent cardiomyocyte apoptosis induced by proteasome inhibition.

Heart is a multi-cellular organ made up of various cell types interacting with each other. Cardiomyocytes may benefit or suffer from crosstalk with noncardiomyocytes in response to diverse kinds of cardiac stresses. Proteasome dysfunction is a common cardiac stress which causes cardiac proteotoxicity and contributes to cardiac diseases such as heart failure and myocardial infarction. The role of crosstalk between cardiomyocytes and noncardiomyocytes in defense of cardiac proteotoxicity remains unknown. Here, we report a cardiomyocyte-specific survival upon proteasome inhibition in a heterogeneous culture consisting of cardiomyocytes and other three major cardiac cell types. Conversely, cardiomyocyte apoptosis is remarkably induced by proteasome inhibition in a homogeneous culture consisting of a majority of cardiomyocytes, demonstrating an indispensable role of noncardiomyocytes in the prevention of cardiomyocyte apoptosis resulting from proteasome inhibition. We further show that cardiomyocytes express brain natriuretic peptide (BNP) as an extracellular molecule in response to proteasome inhibition. Blockade of BNP receptor on noncardiomyocytes significantly exacerbated the cardiomyocyte apoptosis, indicating a paracrine function of cardiomyocyte-released extracellular BNP in activation of a protective feedback from noncardiomyocytes. Finally, we demonstrate that proteasome inhibition-activated transcriptional up-regulation of BNP in cardiomyocytes was associated with the dissociation of repressor element 1 silencing transcription factor (REST)/ histone deacetylase 1 (HDAC1) repressor complex from BNP gene promoter. Consistently, the induction of BNP could be further augmented by the treatment of HDAC inhibitors. We conclude that the crosstalk between cardiomyocytes and noncardiomyocytes plays a crucial role in the protection of cardiomyocytes from proteotoxicity stress, and identify cardiomyocyte-released BNP as a novel paracrine signaling molecule mediating this crosstalk. These findings provide new insights into the key regulators and cardioprotective mechanism in proteasome dysfunction-related cardiac diseases.

Luteoloside Exerts Analgesic Effect in a Complete Freund's Adjuvant-Induced Inflammatory Model via Inhibiting Interleukin-1ß Expression and Macrophage/Microglia Activation.

BACKGROUND: Flavonoid monomers are proved to have an anti-inflammatory effect and may also be promising for chronic pain treatment. In the present study, the analgesic effect and the relevant mechanisms of luteoloside, one of the flavonoid monomers, were investigated. METHODS: The analgesic effect of luteoloside was first evaluated in complete Freud's adjuvant induced inflammatory model by von Frey test and Hargreaves test in both male and female mice. The interleukin-1ß levels in plantar tissue, serum, dorsal root ganglion, and the dorsal horn of the spinal cord were determined by enzyme-linked immunosorbent assay or immunofluorescence. The activation of macrophage/microglia was tested by Iba-1 staining. RESULTS: Our data showed that luteoloside exhibited both acute and chronic analgesic phenotypes. Every single dose of luteoloside solution reached the peak transient analgesic effect 2 h after administration and lasted less than 6 h. About 14 consecutive days administration (one dose per day) later, luteoloside showed a sustained analgesic effect which lasted more than 24 h. Celecoxib 20 mg/kg combined with luteoloside 40 mg/kg achieved a similar analgesic effect as celecoxib 40 mg/kg alone. Luteoloside inhibited interleukin-1ß expression in plantar tissue, dorsal root ganglion, the dorsal horn of spinal cord, and serum, after 14 days of continuous administration. Furthermore, our results also showed that the activation of macrophage/microglia in dorsal root ganglions were significantly inhibited 2 h after each single dose in daily luteoloside administration and recovered to a higher level 6 h later. These findings might be involved in the mechanisms of the acute analgesic effect of luteoloside. CONCLUSION: Luteoloside presents an analgesic effect via anti-inflammatory and other mechanisms such as inhibiting the activation of macrophage/microglia.

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

LESS with Suture Suspension for Early-Stage Adnexa Cancer Staging.

BACKGROUND AND OBJECTIVE: Laparoendoscopic single-site surgery has been used in treating gynecologic diseases including early-stage cervical and endometrial cancer, but less so in early-stage adnexal cancer. We aimed to demonstrate the use of laparoendoscopic single-site surgery with suture suspension for staging of early-stage ovarian/fallopian-tube cancer and describe the study results. METHODS: Seven patients with early-stage adnexal cancer underwent staging surgery via laparoendoscopic single-site surgery at West China Second University Hospital of Sichuan University from November 2017 to September 2018. RESULTS: All cases were successfully staged via this technique. Two patients underwent the high-level para-aortic lymphadenectomy up to the infrarenal vein, and four patients underwent para-aortic lymphadenectomy at the level of the inframesenteric artery; one patient underwent the para-aortic lymph node sampling. The operation time was 305-365 minutes. The estimated intraoperative blood loss was 50-200 mL. No intra-operative complications occurred; one patient developed pneumonia 48 hours postoperation. The number of pelvic and para-aortic nodes was 15-39 and 1-18, respectively. Pain scores 12 and 24 hours postsurgery were 2-3 and 1-2 with the use of butorphanol tartrate, respectively. On 4-14 months followup, the umbilical incision had good cosmesis; no umbilical hernia or vaginal dehiscence and no neoplasm recurrence were noted. CONCLUSION: Laparoendoscopic single-site surgery may be a feasible and safe technique for staging early-stage ovarian/tubal cancer. This approach has some advantages included providing easier access to the upper abdominal regions when performing high-level infrarenal para-aortic lymphadenectomy; the 2-cm elastic incision favors fast specimen extraction and colpotomy are avoided. However, the long-term oncologic outcomes need to be further investigated.

STAT3 activation regulated circ-STAT3.46 promote expression of IGF1R by sponging of miR-139-5p in human colon cancer.

BACKGROUND: Recently the roles of circRNAs were extensively studied within human malignancies. Now we explored a potential regulatory axis consisted of circ-STAT3.46-miR-139-5p-IGF1R in human colon cancer. METHODS: The expression of circ-STAT3.46-miR-139-5p-IGF1R were determined by using real-time PCR in human colon cancer (n=56) and adjacent normal tissues. The relationship between clinical characters and tissue or serum exosome circ-STAT3.46 were studied. The detailed regulation within circ-STAT3.46-miR-139-5p-IGF1R was verified by in vitro studies. RESULTS: Aberrant expression of circ-STAT3.46, miR-139-5p, and IGF1R were spotted between colon cancer tissues and control. A significantly negative correlation between circ-STAT3.46 and miR-139-5p were verified within human colon cancer tissues. Expression of circ-STAT3.46 in colon cancer tissue and serum exosome were associated with TMN stage and bad prognosis of post-surgery colon cancer patients. IGF1R was positively correlated to circ-STAT3.46 in human colon cancer tissues. Moreover, the transcription of circ-STAT3.46 was regulated by IGF1/IGF1R/STAT3 signaling. Overexpression of circ-STAT3.46 can decrease miR-139-5p in colon cancer cells, meanwhile, increased miR-139-5p were found in circ-STAT3.46 knockdown cells. RNA pull-down assay revealed that circ-STAT3.46 could sponge miR-139-5p, and luciferase reporter assay indicated that miR-139-5p could further downregulate IGF1R transcription by binding to its 3'UTR in human colon cancer cells. CONCLUSIONS: circ-STAT3.46 was regulated by IGF/IGF1R/STAT3 activation, and overexpression of circ-STAT3.46 can up-regulate IGF1R by sponging of miR-139-5p within human colon cancer.

Vitamin C Promotes Astrocyte Differentiation Through DNA Hydroxymethylation.

Previous studies have reported that vitamin C (VC) promotes neural stem/precursor cell (NSC) differentiation toward dopamine (DA) neurons via DNA hydroxymethylation-induced transcriptional activation of DA neuron-specific genes. To further understand the VC effects on NSC differentiation, we profiled the transcriptome and DNA methylome/hydroxymethylome using high-throughput sequencing. Interestingly, RNA sequencing analyses have shown that, in addition to DA neuronal genes, astrocytic genes Gfap, Slc1a3, and S100a16 were also upregulated in NSC cultures differentiated with VC treatment. Consistently, enhanced GFAP+ astrocytic yields were manifested in the differentiated cultures with VC treatment, collectively indicating that VC promotes astrocytic differentiation. In genome-wide hydroxymethylome analyses, VC treatment induces enrichment of DNA hydroxymethylation (5-hydroxymethyl cytosine; 5hmC) near the consensus binding motifs of nuclear factor I (NFI). Furthermore, we showed that VC significantly enhanced recruitment of NFI and STAT3, key transcription factors for astrogenesis, in the 5hmC-enriched regions of the astrocyte-specific genes. These findings suggest that VC play important roles in astrocytogenesis during brain development. Stem Cells 2018;36:1578-1588.

In vitro immunotherapy potency assays using real-time cell analysis.

A growing understanding of the molecular interactions between immune effector cells and target tumor cells, coupled with refined gene therapy approaches, are giving rise to novel cancer immunotherapeutics with remarkable efficacy in the clinic against both solid and liquid tumors. While immunotherapy holds tremendous promise for treatment of certain cancers, significant challenges remain in the clinical translation to many other types of cancers and also in minimizing adverse effects. Therefore, there is an urgent need for functional potency assays, in vitro and in vivo, that could model the complex interaction of immune cells with tumor cells and can be used to rapidly test the efficacy of different immunotherapy approaches, whether it is small molecule, biologics, cell therapies or combinations thereof. Herein we report the development of an xCELLigence real-time cytolytic in vitro potency assay that uses cellular impedance to continuously monitor the viability of target tumor cells while they are being subjected to different types of treatments. Specialized microtiter plates containing integrated gold microelectrodes enable the number, size, and surface attachment strength of adherent target tumor cells to be selectively monitored within a heterogeneous mixture that includes effector cells, antibodies, small molecules, etc. Through surface-tethering approach, the killing of liquid cancers can also be monitored. Using NK92 effector cells as example, results from RTCA potency assay are very well correlated with end point data from image-based assays as well as flow cytometry. Several effector cells, i.e., PBMC, NK, CAR-T were tested and validated as well as biological molecules such as Bi-specific T cell Engagers (BiTEs) targeting the EpCAM protein expressed on tumor cells and blocking antibodies against the immune checkpoint inhibitor PD-1. Using the specifically designed xCELLigence immunotherapy software, quantitative parameters such as KT50 (the amount of time it takes to kill 50% of the target tumor cells) and % cytolysis are calculated and used for comparing the relative efficacy of different reagents. In summary, our results demonstrate the xCELLigence platform to be well suited for potency assays, providing quantitative assessment with high reproducibility and a greatly simplified work flow.

Remote limb ischemic postconditioning promotes motor function recovery in a rat model of ischemic stroke via the up-regulation of endogenous tissue kallikrein.

AIMS: Remote ischemic conditionings, such as pre- and per-conditioning, are known to provide cardioprotection in animal models of ischemia. However, little is known about the neuroprotection effect of postconditioning after cerebral ischemia. In this study, we aim to evaluate the motor function rescuing effect of remote limb ischemic postconditioning (RIPostC) in a rat model of acute cerebral stroke. METHODS: Left middle cerebral artery occlusion (MCAO) was performed to generate the rat model of ischemic stroke, followed by daily RIPostC treatment for maximum 21 days. The motor function after RIPostC was assessed with foot fault test and balance beam test. Local infarct volume was measured through MRI scanning. Neuronal status was evaluated with Nissl's, HE, and MAP2 immunostaining. Lectin immunostaining was performed to evaluate the microvessel density and area. RESULTS: Daily RIPostC for more than 21 days promoted motor function recovery and provided long-lasting neuroprotection after MCAO. Reduced infarct volume, rescued neuronal loss, and enhanced microvessel density and size in the injured areas were observed. In addition, the RIPostC effect was associated with the up-regulation of endogenous tissue kallikrein (TK) level in circulating blood and local ischemic brain regions. A TK receptor antagonist HOE-140 partially reversed RIPostC-induced improvements, indicating the specificity of endogenous TK mediating the neuroprotection effect of RIPostC. CONCLUSION: Our study demonstrates RIPostC treatment as an effective rehabilitation therapy to provide motor function recovery and alleviate brain impairment in a rat model of acute cerebral ischemia. We also for the first time provide evidence showing that the up-regulation of endogenous TK from remote conditioning regions underlies the observed effects of RIPostC.

SNP rs3202538 in 3'UTR region of ErbB3 regulated by miR-204 and miR-211 promote gastric cancer development in Chinese population.

BACKGROUND/AIMS: ErbB3 is an oncogene which has proliferation and metastasis promotion effects by several signaling pathways. However, the individual expression difference regulated by miRNA was almost still unknown. We focused on the miRNAs associated SNPs in the 3'-UTR of ErbB3 to investigate the further relationship of the SNPs with miRNAs among Chinese gastric cancer (GC) patients. METHODS: We performed case-control study including 851 GC patients and 799 cancer-free controls. Genotyping, real-time PCR assay, cell transfection, the dual luciferase reporter assay, western-blot, cell proliferation and trans-well based cell invasion assay were used to investigate the effects of the SNP on ErbB3 expression. Moreover, a 5-years-overall survival and relapse free survival were investigated between different genotypes. RESULTS: We found that patients suffering from Helicobacter pylori (Hp.) infection indicated to be the susceptible population by comparing with controls. Besides, SNP rs3202538 (G/T) in ErbB3 3'-UTR was involved in the occurrence of GC by acting as tumor risk factors. SNP rs3202538 (G/T) could be regulated by both miR-204 and miR-211 which caused an upregulation of ErbB3 in patients. Furthermore, the carriers of T genotype was related to the significantly high expression of ErbB3, and to big tumor size, poor differentiation as well as the high probability of metastasis. Both miR-211 and miR-204 can significantly decrease cell proliferation, metastasis as well as downstream AKT activation through G but not T allele of ErbB3 3'UTR. Moreover, the SNP of G/T was associated with shorter survival of post-surgery GC patients with 5 years of follow up study. CONCLUSION: In conclusion, our findings have shown that the SNP rs3202538 (G/T) in ErbB3 3'-UTR acted as promotion factors in the GC development through disrupting the regulatory role of miR-204 and miR-211 in ErbB3 expression.

A Central Role for Phosphorylated p38α in Linking Proteasome Inhibition-Induced Apoptosis and Autophagy.

Autophagy and the ubiquitin proteasome system (UPS), as two major protein degradation pathways, coordinate with each other in regulating programmed cell death. Autophagy can compensate for the UPS impairment-induced cell dysfunction and apoptosis. However, it is not clear how cells maintain the delicate balance between UPS-related apoptosis and autophagy. Here, we showed that proteasome inhibition-mediated UPS impairment can activate the phosphorylated p38α (p-p38α)-dependent apoptotic pathway and autophagy pathway in both neuroblastoma cell line N2a and primary cortical neuronal cells. Multiple indices were utilized for the autophagy detection including LC3II transition, acidic vesicle formation, lysosomal accumulation, and p62 reduction. Blockade of autophagy flux with autophagy inhibitor 3-methyladenine or bafilomycin A1 resulted in further phosphorylation of p38α, polyubiquitinated protein aggregation, and greater apoptotic cell death. On the contrary, enhancement of autophagy by rapamycin attenuated the cell loss by lowering p-p38α level and degrading protein aggregates, indicating a protective role of autophagy in cell stress and apoptosis. Moreover, de-activation of p38α with pharmaceutical p38α inhibitor BIRB796 greatly increased autophagy activation, reduced protein aggregates, and attenuated cell loss, suggesting a bidirectional regulation between p-p38α and autophagy. In addition, manipulation of p-p38α by BIRB796 or p38α knockdown decreased the phosphorylation of key components of the mammalian target of rapamycin (mTOR)-dependent pathway, indicating that the mTOR pathway mediates the p-p38α regulation on autophagy. Overall, our data emphasize p-p38α as a key mediator in the antagonistic interaction between apoptosis and autophagy in response to UPS impairment. Centering p-p38α as a potential regulatory target may provide a dual advantage of proteostasis maintenance and cell survival for simultaneous inhibition of apoptosis and activation of autophagy.

AC0010, an Irreversible EGFR Inhibitor Selectively Targeting Mutated EGFR and Overcoming T790M-Induced Resistance in Animal Models and Lung Cancer Patients.

AC0010 is a pyrrolopyrimidine-based irreversible EGFR inhibitor, structurally distinct from previously reported pyrimidine-based irreversible EGFR inhibitors, such as osimertinib and rociletinib. AC0010 selectively inhibits EGFR-active and T790M mutations with up to 298-fold increase in potency compared with wild-type EGFR. In a xenograft model, oral administration of AC0010 at a daily dose of 500 mg/kg resulted in complete remission of tumors with EGFR-active and T790M mutations for over 143 days with no weight loss. Three major metabolites of AC0010 were tested and showed no wild-type EGFR inhibition or off-target effects, such as inhibition of IGF-1R. AC0010 is safe in non-small cell lung cancer (NSCLC) patients at a dose range between 50 and 550 mg once per day, and no hyperglycemia or other severe adverse effects were detected, such as grade 3 QT prolongation. The objective responses were observed in NSCLC patients with EGFR T790M mutation. Mol Cancer Ther; 15(11); 2586-97. ©2016 AACR.

Multi-parametric assessment of cardiomyocyte excitation-contraction coupling using impedance and field potential recording: A tool for cardiac safety assessment.

INTRODUCTION: The ICH S7B guidelines recommend that all new chemical entities should be subjected to hERG repolarization screening due to its association with life-threatening "Torsades de Pointes" (TdP) arrhythmia. However, it has become evident that not all hERG channel inhibitors result in TdP and not all compounds that induce QT prolongation and TdP necessarily inhibit hERG. In order to address the limitations of the S7B/E14 guidelines, the FDA through a public/private partnership initiated the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative to examine the possible modification and refinement of the ICH E14/S7B guidelines. One of the main components of the CiPA initiative is to utilize a predictive assay system together with human cardiomyocytes for risk assessment of arrhythmia. METHOD: In this manuscript we utilize the xCELLigence® CardioECR system which simultaneously measures excitation-contraction coupling together with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to assess the effect of 8 reference compounds across 3 different independent sites. These 8 compounds were part of Phase I CiPA validation study. RESULTS: Our data demonstrate that hERG channel blockers, such as E4031 and moxifloxacin, prolonged field potential duration (FPD) at low concentration and induced arrhythmic beating activity as measured by field potential (FP) recording and impedance (IMP) recordings at higher concentrations. On the contrary, nifedipine, an inhibitor of calcium channel, didn't disrupt the periodicity of cell beating and weakened cell contractile activity and shortened FPD. Multichannel inhibitors, such as flecainide, quinidine and mexiletine, not only increased FPD and induced arrhythmia but also significantly reduced the amplitude of FP spike. JNJ303, an IKs inhibitor, only affected FPD. Comparison of the compound effect on FPD across the 3 different sites is consistent in terms of trend of the effect with observed 3-10 fold differences in minimal effective concentration at which a minimum of 10% response is detected. In addition, pentamidine, a hERG trafficking inhibitor which induced irregular beating activity over a more prolonged duration of time was readily flagged in this assay system. Taken together, this multi-parameter assay using hiPSC-CMs in conjunction with simultaneous measurement of ion channel activity and contractility can be a reliable approach for risk assessment of proarrhythmic compounds.