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BACKGROUND: Long QT syndrome (LQTS) is a hereditary disease that predisposes patients to life-threatening cardiac arrhythmias and sudden cardiac death. Our previous study of the human ether-à-go-go related gene (hERG)–encoded K(+) channel (K(v)11.1) supports an association between hERG and RING finger protein 207 (RNF207) variants in aggravating the onset and severity of LQTS, specifically T613M hERG (hERG(T613M)) and RNF207 frameshift (RNF207(G603fs)) mutations. However, the underlying mechanistic underpinning remains unknown. OBJECTIVE: The purpose of the present study was to test the role of RNF207 in the function of hERG-encoded K(+) channel subunits. METHODS: Whole-cell patch-clamp experiments were performed in human embryonic kidney (HEK 293) cells and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) together with immunofluorescent confocal and high resolution microscopy, auto-ubiquitinylation assays, and co-immunoprecipitation experiments to test the functional interactions between hERG and RNF207. RESULTS: Here, we demonstrated that RNF207 serves as an E3 ubiquitin ligase and targets misfolded hERG(T613M) proteins for degradation. RNF207(G603fs) exhibits decreased activity and hinders the normal degradation pathway; this increases the levels of hERG(T613M) subunits and their dominant-negative effect on the wild-type subunits, ultimately resulting in decreased current density. Similar findings are shown for hERG(A614V), a known dominant-negative mutant subunit. Finally, the presence of RNF207(G603fs) with hERG(T613M) results in significantly prolonged action potential durations and reduced hERG current in human-induced pluripotent stem cell–derived cardiomyocytes. CONCLUSION: Our study establishes RNF207 as an interacting protein serving as a ubiquitin ligase for hERG-encoded K(+) channel subunits. Normal function of RNF207 is critical for the quality control of hERG subunits and consequently cardiac repolarization. Moreover, our study provides evidence for protein quality control as a new paradigm in life-threatening cardiac arrhythmias in patients with LQTS.

Purpose Nonsteroidal anti-inflammatory drugs (NSAIDs) are among one of the most commonly prescribed medications for pain and inflammation. Diclofenac (DIC) is a commonly prescribed NSAID that is known to increase the risk of cardiovascular diseases. However, the mechanisms underlying its cardiotoxic effects remain largely unknown. In this study, we tested the hypothesis that chronic exposure to DIC increases oxidative stress, which ultimately impairs cardiovascular function. Methods and Results Mice were treated with DIC for 4 weeks and subsequently subjected to in vivo and in vitro functional assessments. Chronic DIC exposure resulted in not only systolic but also diastolic dysfunction. DIC treatment, however, did not alter blood pressure or electrocardiographic recordings. Importantly, treatment with DIC significantly increased inflammatory cytokines and chemokines as well as cardiac fibroblast activation and proliferation. There was increased reactive oxygen species (ROS) production in cardiomyocytes from DIC-treated mice, which may contribute to the more depolarized mitochondrial membrane potential and reduced energy production, leading to a significant decrease in sarcoplasmic reticulum (SR) Ca2+ load, Ca2+ transients, and sarcomere shortening. Using unbiased metabolomic analyses, we demonstrated significant alterations in oxylipin profiles towards inflammatory features in chronic DIC treatment. Conclusions Together, chronic treatment with DIC resulted in severe cardiotoxicity, which was mediated, in part, by an increase in mitochondrial oxidative stress.

Highlights • The usage of antihypertensive drugs is not associated with the risk and severity of COVID-19. • Hypertensive patients with COVID-19 benefit from prior usage of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers., Background The association of antihypertensive drugs with the risk and severity of COVID-19 remains unknown. Methods and Results We systematically searched PubMed, MEDLINE, The Cochrane Library, Cochrane Central Register of Controlled Trials (CENTRAL), ClinicalTrials.gov, and medRxiv for publications before July 13, 2020. Cohort studies and case-control studies that contain information on the association of antihypertensive agents including angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), calcium-channel blockers (CCBs), β-blockers, and diuretics with the risk and severity of COVID-19 were selected. The random-effects or fixed-effects models were used to pool the odds ratio (OR) with 95% confidence interval (CI) for the outcomes. The literature search yielded 53 studies that satisfied our inclusion criteria, which comprised 39 cohort studies and 14 case-control studies. These studies included a total of 2,100,587 participants. We observed no association between prior usage of antihypertensive medications including ACEIs/ARBs, CCBs, β-blockers, or diuretics and the risk and severity of COVID-19. Additionally, when only hypertensive patients were included, the severity and mortality were lower with prior usage of ACEIs/ARBs (overall OR of 0.81, 95% CI 0.66-0.99, p

Background Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) have recently emerged as potential antiviral and immunomodulatory options for the treatment of 2019 coronavirus disease (COVID-19). To examine the safety profiles of these medications, we systematically evaluated the adverse events (AEs) of these medications from published randomized controlled trials (RCTs). Methods We systematically searched MEDLINE, the Cochrane library, the Cochrane Central Register of Controlled Trials (CENTRAL), and the ClinicalTrials.gov for all the RCTs comparing CQ or HCQ with placebo or other active agents, published before June 20, 2020. The random-effects or fixed-effects models were used to pool the risk estimates relative ratio (RR) with 95% confidence interval (CI) for the outcomes. Results The literature search yielded 23 and 19 studies for CQ and HCQ, respectively, that satisfied our inclusion criteria. Of these studies, we performed meta-analysis on 6 studies for CQ and 18 studies for HCQ. We did not limit our analysis to published records involving viral treatment alone; data also included the usage of either CQ or HCQ for the treatment of other diseases. The trials for the CQ consisted of a total of 2,137 participants (n = 1,077 CQ, n = 1,060 placebo), while the trials for HCQ involved 2,675 participants (n = 1,345 HCQ and n = 1,330 control). The overall mild and total AEs were significantly higher in CQ-treated non-COVID-19 patients, HCQ-treated non-COVID-19 patients, and HCQ-treated COVID-19 patients. The AEs were further categorized into four groups and analyses revealed that neurologic, gastrointestinal (GI), dermatologic, and sensory AEs were higher in participants taking CQ compared to placebo, while GI, dermatologic, sensory, and cardiovascular AEs were higher in HCQ-treated COVID-19 patients compared to control patients. Moreover, subgroup analysis suggested higher AEs with respect to dosage and duration in HCQ group. Data were acquired from studies with perceived low risk of bias, so plausible bias is unlikely to seriously affect the main findings of the current study. Conclusions Taken together, we found that participants taking either CQ or HCQ exhibited more AEs than participants taking placebo or control. Precautionary measures should be taken when using these drugs to treat COVID-19. The meta-analysis was registered on OSF (https://osf.io/jm3d9). Registration The meta-analysis was registered on OSF (https://osf.io/jm3d9).

Malignant pleural mesothelioma (MPM) is an aggressive malignancy arising from mesothelial lining of pleura. It is associated with a poor prognosis, partly due to the lack of a precise understanding of the molecular mechanisms associated with its malignant behavior. In the present study, we expanded on our previous studies on cell cycle control of MPM cells by targeting CD26 molecule with humanized anti-CD26 monoclonal antibody (HuCD26mAb), focusing particularly on ubiquitin-specific protease 22 (USP22). We showed that USP22 protein expression is detected in clinical specimens of MPM and that USP22 knockdown, as well as CD26 knockdown, significantly inhibits the growth and proliferation of MPM cells in vitro and in vivo. Moreover, depletion of both USP22 and CD26 suppresses MPM cell proliferation even more profoundly. Furthermore, expression levels of USP22 correlate with those of CD26. HuCD26mAb treatment induces a decrease in USP22 level through its interaction with the CD26 molecule, leading to increased levels of ubiquitinated histone H2A and p21. By demonstrating a CD26-related linkage with USP22 in MPM cell inhibition induced by HuCD26mAb, our present study hence characterizes USP22 as a novel target molecule while concurrently suggesting a new therapeutic strategy for MPM.

Although the prognosis of acute lymphoblastic leukemia (ALL) has improved considerably in recent years, some of the cases still exhibit therapy-resistant. We have previously reported that CD9 was expressed heterogeneously in B-ALL cell lines and CD9(+) cells exhibited an asymmetric cell division with greater tumorigenic potential than CD9(-) cells. CD9(+) cells were also serially transplantable in immunodeficient mice, indicating that CD9(+) cell possess self-renewal capacity. In the current study, we performed more detailed analysis of CD9 function for the cancer stem cell (CSC) properties. In patient sample, CD9 was expressed in the most cases of B-ALL cells with significant correlation of CD34-expression. Gene expression analysis revealed that leukemogenic fusion proteins and Src family proteins were significantly regulated in the CD9(+) population. Moreover, CD9(+) cells exhibited drug-resistance, but proliferation of bulk cells was inhibited by anti-CD9 monoclonal antibody. Knockdown of CD9 remarkably reduced the leukemogenic potential. Furthermore, gene ablation of CD9 affected the expression and tyrosine-phosphorylation of Src family proteins and reduced the expression of histone-deubiquitinase USP22. Taken together, our results suggest that CD9 links to several signaling pathways and epigenetic modification for regulating the CSC properties of B-ALL.

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol naturally occurring in grapes and other plants, has cancer chemo-preventive effects and therapeutic potential. Although resveratrol modulates multiple pathways in tumor cells, how resveratrol or its affected pathways converge on chromatin to mediate its effects is not known. Using glioma cells as a model, we showed here that resveratrol inhibited cell proliferation and induced cellular hypertrophy by transforming spindle-shaped cells to enlarged, irregular and flatten-shaped ones. We further showed that resveratrol-induced hypertrophic cells expressed senescence-associated-β-galactosidase, suggesting that resveratrol-induced cellular senescence in glioma cells. Consistent with these observations, we demonstrated that resveratrol inhibited clonogenic efficiencies in vitro and tumor growth in a xenograft model. Furthermore, we found that acute treatment of resveratrol inhibited mono-ubiquitination of histone H2B at K120 (uH2B) in breast, prostate, pancreatic, lung, brain tumor cells as well as primary human cells. Chronic treatment with low doses of resveratrol also inhibited uH2B in the resveratrol-induced senescent glioma cells. Moreover, we showed that depletion of RNF20, a ubiquitin ligase of histone H2B, inhibited uH2B and induced cellular senescence in glioma cells in vitro, thereby recapitulated the effects of resveratrol. Taken together, our results suggest that uH2B is a novel direct or indirect chromatin target of resveratrol and RNF20 plays an important role in inhibiting cellular senescence programs that are intact in glioma cells.

Whole-genome sequencing projects have generated a wealth of gene sequences from a variety of organisms. A major challenge is to rapidly uncover gene regulatory circuits and their functional manifestations at the cellular level. Here we report the coupled fabrication of nanocraters ranging in size from 100 pL to 1.5 nL on permeable membranes for culturing cells. Using this approach, we developed bacterial and yeast cell microarrays that allowed phenotypic determinations of gene activities and drug targets on a large scale. Cell microarrays will therefore be a particularly useful tool for studying phenotypes of gene activities on a genome-wide scale.

Cellular therapy using embryonic stem cells has always been an area of great interest due to the pluripotent characteristics of stem cells. In 2006, Takahashi and Yamanaka (Cell 126, 663–676) demonstrated that somatic cells can be reprogrammed into a stem cell-like state, termed induced pluripotent stem (iPS) cells, by ectopic expression of Oct4, Sox2, Klf4 and c Myc. A later report (Nakagawa et al. Nat. Biotechnol.26:101–106, 2008) showed that iPS cells can be produced in the absence of the c Myc oncogene. We have used this latter strategy to successfully reprogram somatic cells derived from C57BL/6 mouse tail fibroblast to iPS cells. Retrovirus infected fibroblasts exhibited stem cell-like morphology by 14 days post infection. These iPS cells were then infected with a retrovirus that expressed HOXB4. Recombinant leukemia inhibitor factor (LIF) supplement was removed from media at this time and the cells allowed to differentiate into embryoid bodies. These cells were screened for specific differentiation stem cell markers, such as Oct4, Nanog, Sall4 and SSEA-1. iPS cells were converted into embryonic bodies and then infected with retroviruses expressing HOXB4. Embryoid bodies stably expressing HOXB4 were induced to hematopoietic differentiation by treatment of thrombopoietin (TPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), interferon gamma (IFNg) and fms-like tyrosine kinase (FLT3 ligand). Evaluation of iPS-derived hematopoietic cells on smears show strikingly similarity in morphology to the W4 mouse embryonic stem (ES) cells differentiated into hematopoietic cells as a control. Flow cytometry analysis of iPS-derived hematopoietic cells after 1 week exposure to cytokines revealed 7% B220+ cells (B cells), 11% Ter119+ cells (erythroid), and 13% Gr-1+ cells (granulocytes) similar to W4 ES cells. The iPS-derived hematopoietic cells were transplanted into irradiated immunodeficient mice via lateral tail vein injection. Transplantation of these iPS-derived hematopoietic progenitors tagged with GFP into irradiated SCID mice revealed that the hematopoietic progenitors were able to home to the bone marrow after 1 week of transplantation. Importantly, after 1 month, GFP+ engrafted cells remained in the bone marrow suggesting a long-term engraftment. This long term engraftment of the iPS-derived hematopoietic cells to the bone marrow constitutes an important step toward potential therapy of numerous patient-specific blood based diseases.