Interferon-induced transmembrane protein 3 (IFITM3) limits lethality of SARS-CoV-2 in mice.

Interferon-induced transmembrane protein 3 (IFITM3) is an antiviral protein that alters cell membranes to block fusion of viruses. Conflicting reports identified opposing effects of IFITM3 on SARS-CoV-2 infection of cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with SARS-CoV-2 experience extreme weight loss and lethality compared to mild infection in wild-type (WT) mice. KO mice have higher lung viral titers and increases in inflammatory cytokine levels, immune cell infiltration, and histopathology. Mechanistically, we observe disseminated viral antigen staining throughout the lung and pulmonary vasculature in KO mice, as well as increased heart infection, indicating that IFITM3 constrains dissemination of SARS-CoV-2. Global transcriptomic analysis of infected lungs shows upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections in vivo.

Retention of the NLRP3 Inflammasome-Primed Neutrophils in the Bone Marrow Is Essential for Myocardial Infarction-Induced Granulopoiesis.

BACKGROUND: Acute myocardial infarction (MI) results in overzealous production and infiltration of neutrophils to the ischemic heart. This is mediated in part by granulopoiesis induced by the S100A8/A9-NLRP3-IL-1ß signaling axis in injury-exposed neutrophils. Despite the transcriptional upregulation of the NLRP3 (Nod Like Receptor Family Pyrin Domain-Containing 3) inflammasome and associated signaling components in neutrophils, the serum levels of IL-1ß (interleukin-1ß), the effector molecule in granulopoiesis, were not affected by MI, suggesting that IL-1ß is not released systemically. We hypothesize that IL-1ß is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. METHODS: Using a combination of time-dependent parabiosis and flow cytometry techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in parabiotic mice by permanent ligation of the left anterior descending artery and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in noninfarcted parabionts. Last, using multiple neutrophil adoptive and BM transplant studies, we studied the molecular mechanisms that govern reverse migration and retention of the primed neutrophils, IL-1ß secretion, and granulopoiesis. Cardiac function was assessed by echocardiography. RESULTS: MI promoted greater accumulation of the inflammasome-primed neutrophils in the BM. Introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited their ability to stimulate granulopoiesis in the noninfarcted parabionts. Previous priming of the NLRP3 inflammasome is not a prerequisite, but the presence of a functional CXCR4 (C-X-C-motif chemokine receptor 4) on the primed-neutrophils and elevated serum S100A8/A9 levels are necessary for homing and retention of the reverse-migrating neutrophils. In the BM, the primed-neutrophils secrete IL-1ß through formation of gasdermin D pores and promote granulopoiesis. Pharmacological and genetic strategies aimed at the inhibition of neutrophil homing or release of IL-1ß in the BM markedly suppressed MI-induced granulopoiesis and improved cardiac function. CONCLUSIONS: Our data reveal a new paradigm of how circulatory cells establish a direct communication between organs by delivering signaling molecules (eg, IL-1ß) directly at the sites of action rather through systemic release. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1ß release.

CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy.

Fibroblasts can be reprogrammed into cardiovascular progenitor cells (CPCs) using transgenic approaches, although the underlying mechanism remains unclear. We determined whether activation of endogenous genes such as Gata4, Nkx2.5, and Tbx5 can rapidly establish autoregulatory loops and initiate CPC generation in adult extracardiac fibroblasts using a CRISPR activation system. The induced fibroblasts (>80%) showed phenotypic changes as indicated by an Nkx2.5 cardiac enhancer reporter. The progenitor characteristics were confirmed by colony formation and expression of cardiovascular genes. Cardiac sphere induction segregated the early and late reprogrammed cells that can generate functional cardiomyocytes and vascular cells in vitro. Therefore, they were termed CRISPR-induced CPCs (ciCPCs). Transcriptomic analysis showed that cell cycle and heart development pathways were important to accelerate CPC formation during the early reprogramming stage. The CRISPR system opened the silenced chromatin locus, thereby allowing transcriptional factors to access their own promoters and eventually forming a positive feedback loop. The regenerative potential of ciCPCs was assessed after implantation in mouse myocardial infarction models. The engrafted ciCPCs differentiated into cardiovascular cells in vivo but also significantly improved contractile function and scar formation. In conclusion, multiplex gene activation was sufficient to drive CPC reprogramming, providing a new cell source for regenerative therapeutics.

Cardiac-Specific Expression of Cre Recombinase Leads to Age-Related Cardiac Dysfunction Associated with Tumor-like Growth of Atrial Cardiomyocyte and Ventricular Fibrosis and Ferroptosis.

Transgenic expression of Cre recombinase driven by a specific promoter is normally used to conditionally knockout a gene in a tissue- or cell-type-specific manner. In αMHC-Cre transgenic mouse model, expression of Cre recombinase is controlled by the myocardial-specific α-myosin heavy chain (αMHC) promoter, which is commonly used to edit myocardial-specific genes. Toxic effects of Cre expression have been reported, including intro-chromosome rearrangements, micronuclei formation and other forms of DNA damage, and cardiomyopathy was observed in cardiac-specific Cre transgenic mice. However, mechanisms associated with Cardiotoxicity of Cre remain poorly understood. In our study, our data unveiled that αMHC-Cre mice developed arrhythmias and died after six months progressively, and none of them survived more than one year. Histopathological examination showed that αMHC-Cre mice had aberrant proliferation of tumor-like tissue in the atrial chamber extended from and vacuolation of ventricular myocytes. Furthermore, the αMHC-Cre mice developed severe cardiac interstitial and perivascular fibrosis, accompanied by significant increase of expression levels of MMP-2 and MMP-9 in the cardiac atrium and ventricular. Moreover, cardiac-specific expression of Cre led to disintegration of the intercalated disc, along with altered proteins expression of the disc and calcium-handling abnormality. Comprehensively, we identified that the ferroptosis signaling pathway is involved in heart failure caused by cardiac-specific expression of Cre, on which oxidative stress results in cytoplasmic vacuole accumulation of lipid peroxidation on the myocardial cell membrane. Taken together, these results revealed that cardiac-specific expression of Cre recombinase can lead to atrial mesenchymal tumor-like growth in the mice, which causes cardiac dysfunction, including cardiac fibrosis, reduction of the intercalated disc and cardiomyocytes ferroptosis at the age older than six months in mice. Our study suggests that αMHC-Cre mouse models are effective in young mice, but not in old mice. Researchers need to be particularly careful when using αMHC-Cre mouse model to interpret those phenotypic impacts of gene responses. As the Cre-associated cardiac pathology matched mostly to that of the patients, the model could also be employed for investigating age-related cardiac dysfunction.

MG53 attenuates nitrogen mustard-induced acute lung injury.

Nitrogen mustard (NM) is an alkylating vesicant that causes severe pulmonary injury. Currently, there are no effective means to counteract vesicant-induced lung injury. MG53 is a vital component of cell membrane repair and lung protection. Here, we show that mice with ablation of MG53 are more susceptible to NM-induced lung injury than the wild-type mice. Treatment of wild-type mice with exogenous recombinant human MG53 (rhMG53) protein ameliorates NM-induced lung injury by restoring arterial blood oxygen level, by improving dynamic lung compliance and by reducing airway resistance. Exposure of lung epithelial and endothelial cells to NM leads to intracellular oxidative stress that compromises the intrinsic cell membrane repair function of MG53. Exogenous rhMG53 protein applied to the culture medium protects lung epithelial and endothelial cells from NM-induced membrane injury and oxidative stress, and enhances survival of the cells. Additionally, we show that loss of MG53 leads to increased vulnerability of macrophages to vesicant-induced cell death. Overall, these findings support the therapeutic potential of rhMG53 to counteract vesicant-induced lung injury.

The cell membrane repair protein MG53 modulates transcription factor NF-κB signaling to control kidney fibrosis.

Kidney fibrosis is associated with the progression of acute kidney injury to chronic kidney disease. MG53, a cell membrane repair protein, has been shown to protect against injury to kidney epithelial cells and acute kidney injury. Here, we evaluated the role of MG53 in modulation of kidney fibrosis in aging mice and in mice with unilateral ureteral obstruction (UUO) a known model of progressive kidney fibrosis. Mice with ablation of MG53 developed more interstitial fibrosis with age than MG53-intact mice of the same age. Similarly, in the absence of MG53, kidney fibrosis was exaggerated compared to mice with intact MG53 in the obstructed kidney compared to the contralateral unobstructed kidney or the kidneys of sham operated mice. The ureteral obstructed kidneys from MG53 deficient mice also showed significantly more inflammation than ureteral obstructed kidneys from MG53 intact mice. In vitro experiments demonstrated that MG53 could enter the nuclei of proximal tubular epithelial cells and directly interact with the p65 component of transcription factor NF-κB, providing a possible explanation of enhanced inflammation in the absence of MG53. To test this, enhanced MG53 expression through engineered cells or direct recombinant protein delivery was given to mice subject to UUO. This reduced NF-κB activation and inflammation and attenuated kidney fibrosis. Thus, MG53 may have a therapeutic role in treating chronic kidney inflammation and thereby provide protection against fibrosis that leads to the chronic kidney disease phenotype.

Membrane-delimited signaling and cytosolic action of MG53 preserve hepatocyte integrity during drug-induced liver injury.

BACKGROUND & AIMS: Drug-induced liver injury (DILI) remains challenging to treat and is still a leading cause of acute liver failure. MG53 is a muscle-derived tissue-repair protein that circulates in the bloodstream and whose physiological role in protection against DILI has not been examined. METHODS: Recombinant MG53 protein (rhMG53) was administered exogenously, using mice with deletion of Mg53 or Ripk3. Live-cell imaging, histological, biochemical, and molecular studies were used to investigate the mechanisms that underlie the extracellular and intracellular action of rhMG53 in hepatoprotection. RESULTS: Systemic administration of rhMG53 protein, in mice, can prophylactically and therapeutically treat DILI induced through exposure to acetaminophen, tetracycline, concanavalin A, carbon tetrachloride, or thioacetamide. Circulating MG53 protects hepatocytes from injury through direct interaction with MLKL at the plasma membrane. Extracellular MG53 can enter hepatocytes and act as an E3-ligase to mitigate RIPK3-mediated MLKL phosphorylation and membrane translocation. CONCLUSIONS: Our data show that the membrane-delimited signaling and cytosolic dual action of MG53 effectively preserves hepatocyte integrity during DILI. rhMG53 may be a potential treatment option for patients with DILI. LAY SUMMARY: Interventions to treat drug-induced liver injury and halt its progression into liver failure are of great value to society. The present study reveals that muscle-liver cross talk, with MG53 as a messenger, serves an important role in liver cell protection. Thus, MG53 is a potential treatment option for patients with drug-induced liver injury.

TRIC-A Channel Maintains Store Calcium Handling by Interacting With Type 2 Ryanodine Receptor in Cardiac Muscle.

RATIONALE: Trimeric intracellular cation (TRIC)-A and B are distributed to endoplasmic reticulum/sarcoplasmic reticulum intracellular Ca2+ stores. The crystal structure of TRIC has been determined, confirming the homotrimeric structure of a potassium channel. While the pore architectures of TRIC-A and TRIC-B are conserved, the carboxyl-terminal tail (CTT) domains of TRIC-A and TRIC-B are different from each other. Aside from its recognized role as a counterion channel that participates in excitation-contraction coupling of striated muscles, the physiological function of TRIC-A in heart physiology and disease has remained largely unexplored. OBJECTIVE: In cardiomyocytes, spontaneous Ca2+ waves, triggered by store overload-induced Ca2+ release mediated by the RyR2 (type 2 ryanodine receptor), develop extrasystolic contractions often associated with arrhythmic events. Here, we test the hypothesis that TRIC-A is a physiological component of RyR2-mediated Ca2+ release machinery that directly modulates store overload-induced Ca2+ release activity via CTT. METHODS AND RESULTS: We show that cardiomyocytes derived from the TRIC-A-/- (TRIC-A knockout) mice display dysregulated Ca2+ movement across sarcoplasmic reticulum. Biochemical studies demonstrate a direct interaction between CTT-A and RyR2. Modeling and docking studies reveal potential sites on RyR2 that show differential interactions with CTT-A and CTT-B. In HEK293 (human embryonic kidney) cells with stable expression of RyR2, transient expression of TRIC-A, but not TRIC-B, leads to apparent suppression of spontaneous Ca2+ oscillations. Ca2+ measurements using the cytosolic indicator Fura-2 and the endoplasmic reticulum luminal store indicator D1ER suggest that TRIC-A enhances Ca2+ leak across the endoplasmic reticulum by directly targeting RyR2 to modulate store overload-induced Ca2+ release. Moreover, synthetic CTT-A peptide facilitates RyR2 activity in lipid bilayer reconstitution system, enhances Ca2+ sparks in permeabilized TRIC-A-/- cardiomyocytes, and induces intracellular Ca2+ release after microinjection into isolated cardiomyocytes, whereas such effects were not observed with the CTT-B peptide. In response to isoproterenol stimulation, the TRIC-A-/- mice display irregular ECG and develop more fibrosis than the WT (wild type) littermates. CONCLUSIONS: In addition to the ion-conducting function, TRIC-A functions as an accessory protein of RyR2 to modulate sarcoplasmic reticulum Ca2+ handling in cardiac muscle.

The Prognostic Significance of FKBP1A and Its Related Immune Infiltration in Liver Hepatocellular Carcinoma.

Liver hepatocellular carcinoma (LIHC) remains a global health challenge with poor prognosis and high mortality. FKBP1A was first discovered as a receptor for the immunosuppressant drug FK506 in immune cells and is critical for various tumors and cancers. However, the relationships between FKBP1A expression, cellular distribution, tumor immunity, and prognosis in LIHC remain unclear. Here, we investigated the expression level of FKBP1A and its prognostic value in LIHC via multiple datasets including ONCOMINE, TIMER, GEPIA, UALCAN, HCCDB, Kaplan-Meier plotter, LinkedOmics, and STRING. Human liver tissue microarray was employed to analyze the characteristics of FKBP1A protein including the expression level and pathological alteration in cellular distribution. FKBP1A expression was significantly higher in LIHC and correlated with tumor stage, grade and metastasis. The expression level of the FKBP1A protein was also increased in LIHC patients along with its accumulation in endoplasmic reticulum (ER). High FKBP1A expression was correlated with a poor survival rate in LIHC patients. The analysis of gene co-expression and the regulatory pathway network suggested that FKBP1A is mainly involved in protein synthesis, metabolism and the immune-related pathway. FKBP1A expression had a significantly positive association with the infiltration of hematopoietic immune cells including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. Moreover, M2 macrophage infiltration was especially associated with a poor survival prognosis in LIHC. Furthermore, FKBP1A expression was significantly positively correlated with the expression of markers of M2 macrophages and immune checkpoint proteins such as PD-L1, CTLA-4, LAG3 and HAVCR2. Our study demonstrated that FKBP1A could be a potential prognostic target involved in tumor immune cell infiltration in LIHC.

Peptidylarginine Deiminases 2 Mediates Caspase-1-Associated Lethality in Pseudomonas aeruginosa Pneumonia-Induced Sepsis.

BACKGROUND: Pseudomonas aeruginosa (PA) is a pathogenic bacterium that causes severe pneumonia in critically ill and immunocompromised patients. Peptidylarginine deiminase (PAD) 2, PAD4, and caspase-1 are important enzymes in mediating host response to infection. The goal of this study was to determine the interplay between PAD2, PAD4, and caspase-1 in PA pneumonia-induced sepsis. METHODS: Pneumonia was produced in wild-type, Pad2-/-, and Pad4-/- mice by intranasal inoculation of PA (2.5 × 106 colony-forming units per mouse), and survival (n = 15/group) was monitored for 10 days. Bone marrow-derived macrophages (BMDMs) were isolated for in vitro studies. Samples were collected at specific timepoints for Western blot, bacterial load determination, and flow cytometry analysis. RESULTS: Caspase-1-dependent inflammation was diminished in PA-inoculated Pad2-/- mice, contributing to reduced macrophage death and enhanced bacterial clearance. In addition, Pad2-/- mice exhibited improved survival and attenuated acute lung injury after PA infection. In contrast, Pad4-/- mice did not display diminished caspase-1 activation, altered bacterial loads, or improved survival. CONCLUSIONS: Peptidylarginine deiminase 2 plays an essential role in the pathogenesis of pulmonary sepsis by mediating caspase-1 activation. This goes against previous findings of PAD4 in sepsis. Our study suggests that PAD2 is a potential therapeutic target of PA pneumonia-induced sepsis.

TRIC-A regulates intracellular Ca2+ homeostasis in cardiomyocytes.

Trimeric intracellular cation (TRIC) channels have been identified as monovalent cation channels that are located in the ER/SR membrane. Two isoforms discovered in mammals are TRIC-A (TMEM38a) and TRIC-B (TMEM38b). TRIC-B ubiquitously expresses in all tissues, and TRIC-B-/- mice is lethal at the neonatal stage. TRIC-A mainly expresses in excitable cells. TRIC-A-/- mice survive normally but show abnormal SR Ca2+ handling in both skeletal and cardiac muscle cells. Importantly, TRIC-A mutations have been identified in human patients with stress-induced arrhythmia. In the past decade, important discoveries have been made to understand the structure and function of TRIC channels, especially its role in regulating intracellular Ca2+ homeostasis. In this review article, we focus on the potential roles of TRIC-A in regulating cardiac function, particularly its effects on intracellular Ca2+ signaling of cardiomyocytes and discuss the current knowledge gaps.

MG53 suppresses tumor progression and stress granule formation by modulating G3BP2 activity in non-small cell lung cancer.

BACKGROUND: Cancer cells develop resistance to chemotherapeutic intervention by excessive formation of stress granules (SGs), which are modulated by an oncogenic protein G3BP2. Selective control of G3BP2/SG signaling is a potential means to treat non-small cell lung cancer (NSCLC). METHODS: Co-immunoprecipitation was conducted to identify the interaction of MG53 and G3BP2. Immunohistochemistry and live cell imaging were performed to visualize the subcellular expression or co-localization. We used shRNA to knock-down the expression MG53 or G3BP2 to test the cell migration and colony formation. The expression level of MG53 and G3BP2 in human NSCLC tissues was tested by western blot analysis. The ATO-induced oxidative stress model was used to examine the effect of rhMG53 on SG formation. Moue NSCLC allograft experiments were performed on wild type and transgenic mice with either knockout of MG53, or overexpression of MG53. Human NSCLC xenograft model in mice was used to evaluate the effect of MG53 overexpression on tumorigenesis. RESULTS: We show that MG53, a member of the TRIM protein family (TRIM72), modulates G3BP2 activity to control lung cancer progression. Loss of MG53 results in the progressive development of lung cancer in mg53-/- mice. Transgenic mice with sustained elevation of MG53 in the bloodstream demonstrate reduced tumor growth following allograft transplantation of mouse NSCLC cells. Biochemical assay reveals physical interaction between G3BP2 and MG53 through the TRIM domain of MG53. Knockdown of MG53 enhances proliferation and migration of NSCLC cells, whereas reduced tumorigenicity is seen in NSCLC cells with knockdown of G3BP2 expression. The recombinant human MG53 (rhMG53) protein can enter the NSCLC cells to induce nuclear translation of G3BP2 and block arsenic trioxide-induced SG formation. The anti-proliferative effect of rhMG53 on NSCLC cells was abolished with knockout of G3BP2. rhMG53 can enhance sensitivity of NSCLC cells to undergo cell death upon treatment with cisplatin. Tailored induction of MG53 expression in NSCLC cells suppresses lung cancer growth via reduced SG formation in a xenograft model. CONCLUSION: Overall, these findings support the notion that MG53 functions as a tumor suppressor by targeting G3BP2/SG activity in NSCLCs.

Butyrate Feeding Reverses CypD-Related Mitoflash Phenotypes in Mouse Myofibers.

Mitoflashes are spontaneous transients of the biosensor mt-cpYFP. In cardiomyocytes, mitoflashes are associated with the cyclophilin D (CypD) mediated opening of mitochondrial permeability transition pore (mPTP), while in skeletal muscle they are considered hallmarks of mitochondrial respiration burst under physiological conditions. Here, we evaluated the potential association between mitoflashes and the mPTP opening at different CypD levels and phosphorylation status by generating three CypD derived fusion constructs with a red shifted, pH stable Ca2+ sensor jRCaMP1b. We observed perinuclear mitochondrial Ca2+ efflux accompanying mitoflashes in CypD and CypDS42A (a phosphor-resistant mutation at Serine 42) overexpressed myofibers but not the control myofibers expressing the mitochondria-targeting sequence of CypD (CypDN30). Assisted by a newly developed analysis program, we identified shorter, more frequent mitoflash activities occurring over larger areas in CypD and CypDS42A overexpressed myofibers than the control CypDN30 myofibers. These observations provide an association between the elevated CypD expression and increased mitoflash activities in hindlimb muscles in an amyotrophic lateral sclerosis (ALS) mouse model previously observed. More importantly, feeding the mice with sodium butyrate reversed the CypD-associated mitoflash phenotypes and protected against ectopic upregulation of CypD, unveiling a novel molecular mechanism underlying butyrate mediated alleviation of ALS progression in the mouse model.

N-acetylcysteine prevents oxidized low-density lipoprotein-induced reduction of MG53 and enhances MG53 protective effect on bone marrow stem cells.

MG53 is an important membrane repair protein and partially protects bone marrow multipotent adult progenitor cells (MAPCs) against oxidized low-density lipoprotein (ox-LDL). The present study was to test the hypothesis that the limited protective effect of MG53 on MAPCs was due to ox-LDL-induced reduction of MG53. MAPCs were cultured with and without ox-LDL (0-20 µg/mL) for up to 48 hours with or without MG53 and antioxidant N-acetylcysteine (NAC). Serum MG53 level was measured in ox-LDL-treated mice with or without NAC treatment. Ox-LDL induced significant membrane damage and substantially impaired MAPC survival with selective inhibition of Akt phosphorylation. NAC treatment effectively prevented ox-LDL-induced reduction of Akt phosphorylation without protecting MAPCs against ox-LDL. While having no effect on Akt phosphorylation, MG53 significantly decreased ox-LDL-induced membrane damage and partially improved the survival, proliferation and apoptosis of MAPCs in vitro. Ox-LDL significantly decreased MG53 level in vitro and serum MG53 level in vivo without changing MG53 clearance. NAC treatment prevented ox-LDL-induced MG53 reduction both in vitro and in vivo. Combined NAC and MG53 treatment significantly improved MAPC survival against ox-LDL. These data suggested that NAC enhanced the protective effect of MG53 on MAPCs against ox-LDL through preventing ox-LDL-induced reduction of MG53.

MG53 protects against contrast-induced acute kidney injury by reducing cell membrane damage and apoptosis.

Mitsugumin 53 (MG53) is a tripartite motif family protein that has been reported to attenuate injury via membrane repair in different organs. Contrast-induced acute kidney injury (CI-AKI) is a common complication caused by the administration of iodinated contrast media (CM). While the cytotoxicity induced by CM leading to tubular cell death may be initiated by cell membrane damage, we wondered whether MG53 alleviates CI-AKI. This study was designed to investigate the effect of MG53 on CI-AKI and the underlying mechanism. A rat model of CI-AKI was established, and CI-AKI induced the translocation of MG53 from serum to injury sites on the renal proximal tubular (RPT) epithelia, as illustrated by immunoblot analysis and immunohistochemical staining. Moreover, pretreatment of rats with recombinant human MG53 protein (rhMG53, 2 mg/mL) alleviated iopromide-induced injury in the kidney, which was determined by measuring serum creatinine, blood urea nitrogen and renal histological changes. In vitro studies demonstrated that exposure of RPT cells to iopromide (20, 40, and 80 mg/mL) caused cell membrane injury and cell death, which were attenuated by rhMG53 (10 and 50 µg/mL). Mechanistically, MG53 translocated to the injury site on RPT cells and bound to phosphatidylserine to protect RPT cells from iopromide-induced injury. In conclusion, MG53 protects against CI-AKI through cell membrane repair and reducing cell apoptosis; therefore, rhMG53 might be a potential effective means to treat or prevent CI-AKI.

Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Zinc, an essential micronutrient, has a cancer preventive role. Zinc deficiency has been shown to contribute to the progression of esophageal cancer. Orai1, a store-operated Ca2+ entry (SOCE) channel, was previously reported to be highly expressed in tumor tissues removed from patients with esophageal squamous cell carcinoma (ESCC) with poor prognosis, and elevation of its expression contributes to both hyperactive intracellular Ca2+ oscillations and fast cell proliferation in human ESCC cells. However, the molecular basis of cancer preventive functions of zinc and its association with Orai1-mediated cell proliferation remains unknown. The present study shows that zinc supplementation significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, a specific Zn2+ chelator, whereas nontumorigenic esophageal epithelial cells are significantly less sensitive to zinc treatment. Fluorescence live cell imaging revealed that extracellular Zn2+ exerted rapid inhibitory effects on Orai1-mediated SOCE and on intracellular Ca2+ oscillations in the ESCC cells. Knockdown of Orai1 or expression of Orai1 mutants with compromised zinc binding significantly diminished sensitivity of the cancer cells to zinc treatment in both SOCE and cell proliferation analyses. These data suggest that zinc may inhibit cell proliferation of esophageal cancer cells through Orai1-mediated intracellular Ca2+ oscillations and reveal a possible molecular basis for zinc-induced cancer prevention and Orai1-SOCE signaling pathway in cancer cells.-Choi, S., Cui, C., Luo, Y., Kim, S.-H., Ko, J.-K., Huo, X., Ma, J., Fu, L.-W., Souza, R. F., Korichneva, I., Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice.

RATIONALE: Duchenne muscular dystrophy is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy patients, and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on Duchenne muscular dystrophy cardiac function has yet to be evaluated. Therefore, we assessed the therapeutic efficacy of CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing on dystrophin expression and cardiac function in mdx/Utr+/- mice after a single systemic delivery of recombinant adeno-associated virus. OBJECTIVE: To examine the efficiency and physiological impact of CRISPR-mediated genome editing on cardiac dystrophin expression and function in dystrophic mice. METHODS AND RESULTS: Here, we packaged SaCas9 (clustered regularly interspaced short palindromic repeat-associated 9 from Staphylococcus aureus) and guide RNA constructs into an adeno-associated virus vector and systemically delivered them to mdx/Utr+/- neonates. We showed that CRIPSR-mediated genome editing efficiently excised the mutant exon 23 in dystrophic mice, and immunofluorescence data supported the restoration of dystrophin protein expression in dystrophic cardiac muscles to a level approaching 40%. Moreover, there was a noted restoration in the architecture of cardiac muscle fibers and a reduction in the extent of fibrosis in dystrophin-deficient hearts. The contractility of cardiac papillary muscles was also restored in CRISPR-edited cardiac muscles compared with untreated controls. Furthermore, our targeted deep sequencing results confirmed that our adeno-associated virus-CRISPR/Cas9 strategy was very efficient in deleting the ≈23 kb of intervening genomic sequences. CONCLUSIONS: This study provides evidence for using CRISPR-based genome editing as a potential therapeutic approach for restoring dystrophic cardiomyopathy structurally and functionally.

Diabetes inhibits corneal epithelial cell migration and tight junction formation in mice and human via increasing ROS and impairing Akt signaling.

Corneal wounds usually heal quickly; but diabetic patients have more fragile corneas and experience delayed and painful healing. In the present study, we compared the healing capacity of corneal epithelial cells (CECs) between normal and diabetic conditions and the potential mechanisms. Primary murine CEC derived from wild-type and diabetic (db/db) mice, as well as primary human CEC were prepared. Human CEC were exposed to high glucose (30 mM) to mimic diabetic conditions. Cell migration and proliferation were assessed using Scratch test and MTT assays, respectively. Reactive oxygen species (ROS) production in the cells was measured using dichlorofluorescein reagent. Western blot was used to evaluate the expression levels of Akt. Transepithelial electrical resistance (TEER) and zonula occludens-1 (ZO-1) expression were used to determine tight junction integrity. We found that the diabetic CEC displayed significantly slower cell proliferation and migration compared with the normal CEC from both mice and humans. Furthermore, ROS production was markedly increased in CEC grown under diabetic conditions. Treatment with an antioxidant N-acetyl cysteine (NAC, 100 µM) significantly decreased ROS production and increased wound healing in diabetic CEC. Barrier function was significantly reduced in both diabetic mouse and human CEC, while NAC treatment mitigated these effects. We further showed that Akt signaling was impaired in diabetic CEC, which was partially improved by NAC treatment. These results show that diabetic conditions lead to delayed wound-healing capacity of CEC and impaired tight junction formation in both mice and human. Increased ROS production and inhibited Akt signaling may contribute to this outcome, implicating these as potential targets for treating corneal wounds in diabetic patients.

Comparative analysis of FKBP family protein: evaluation, structure, and function in mammals and Drosophila melanogaster.

BACKGROUND: FK506-binding proteins (FKBPs) have become the subject of considerable interest in several fields, leading to the identification of several cellular and molecular pathways in which FKBPs impact prenatal development and pathogenesis of many human diseases. MAIN BODY: This analysis revealed differences between how mammalian and Drosophila FKBPs mechanisms function in relation to the immunosuppressant drugs, FK506 and rapamycin. Differences that could be used to design insect-specific pesticides. (1) Molecular phylogenetic analysis of FKBP family proteins revealed that the eight known Drosophila FKBPs share homology with the human FKBP12. This indicates a close evolutionary relationship, and possible origination from a common ancestor. (2) The known FKBPs contain FK domains, that is, a prolyl cis/trans isomerase (PPIase) domain that mediates immune suppression through inhibition of calcineurin. The dFKBP59, CG4735/Shutdown, CG1847, and CG5482 have a Tetratricopeptide receptor domain at the C-terminus, which regulates transcription and protein transportation. (3) FKBP51 and FKBP52 (dFKBP59), along with Cyclophilin 40 and protein phosphatase 5, function as Hsp90 immunophilin co-chaperones within steroid receptor-Hsp90 heterocomplexes. These immunophilins are potential drug targets in pathways associated with normal physiology and may be used to treat a variety of steroid-based diseases by targeting exocytic/endocytic cycling and vesicular trafficking. (4) By associating with presinilin, a critical component of the Notch signaling pathway, FKBP14 is a downstream effector of Notch activation at the membrane. Meanwhile, Shutdown associates with transposons in the PIWI-interacting RNA pathway, playing a crucial role in both germ cells and ovarian somas. Mutations in or silencing of dFKBPs lead to early embryonic lethality in Drosophila. Therefore, further understanding the mechanisms of FK506 and rapamycin binding to immunophilin FKBPs in endocrine, cardiovascular, and neurological function in both mammals and Drosophila would provide prospects in generating unique, insect specific therapeutics targeting the above cellular signaling pathways. CONCLUSION: This review will evaluate the functional roles of FKBP family proteins, and systematically summarize the similarities and differences between FKBP proteins in Drosophila and Mammals. Specific therapeutics targeting cellular signaling pathways will also be discussed.

ROS-related mitochondrial dysfunction in skeletal muscle of an ALS mouse model during the disease progression.

In amyotrophic lateral sclerosis (ALS), mitochondrial dysfunction and oxidative stress form a vicious cycle that promotes neurodegeneration and muscle wasting. To quantify the disease-stage-dependent changes of mitochondrial function and their relationship to the generation of reactive oxygen species (ROS), we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1G93A and mt-cpYFP transgenes, in which mt-cpYFP detects dynamic changes of ROS-related mitoflash events at individual mitochondria level. Compared with wild type mice, mitoflash activity in the SOD1G93A (G93A) mouse muscle showed an increased flashing frequency prior to the onset of ALS symptom (at the age of 2 months), whereas the onset of ALS symptoms (at the age of 4 months) is associated with drastic changes in the kinetics property of mitoflash signal with prolonged full duration at half maximum (FDHM). Elevated levels of cytosolic ROS in skeletal muscle derived from the SOD1G93A mice were confirmed with fluorescent probes, MitoSOX™ Red and ROS Brite™570. Immunoblotting analysis of subcellular mitochondrial fractionation of G93A muscle revealed an increased expression level of cyclophilin D (CypD), a regulatory component of the mitochondrial permeability transition pore (mPTP), at the age of 4 months but not at the age of 2 months. Transient overexpressing of SOD1G93A in skeletal muscle of wild type mice directly promoted mitochondrial ROS production with an enhanced mitoflash activity in the absence of motor neuron axonal withdrawal. Remarkably, the SOD1G93A-induced mitoflash activity was attenuated by the application of cyclosporine A (CsA), an inhibitor of CypD. Similar to the observation with the SOD1G93A transgenic mice, an increased expression level of CypD was also detected in skeletal muscle following transient overexpression of SOD1G93A. Overall, this study reveals a disease-stage-dependent change in mitochondrial function that is associated with CypD-dependent mPTP opening; and the ALS mutation SOD1G93A directly contributes to mitochondrial dysfunction in the absence of motor neuron axonal withdrawal.