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Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia disorder associated with lethal arrhythmias. Most CPVT cases are caused by inherited variants in the gene encoding ryanodine receptor type 2 (RYR2)., Objective: The goal of this study was to investigate the structure-activity relationship of tetracaine derivatives and to test a lead compound in a mouse model of CPVT., Methods: We synthesized >200 tetracaine derivatives and characterized 11 of those. The effects of these compounds on Ca 2+ handling in cardiomyocytes from R176Q/+ mice was tested with confocal microscopy. The effects of lead compound MSV1302 on arrhythmia inducibility and cardiac contractility were tested by programmed electrical stimulation and echocardiography, respectively. Plasma and microsomal stability and cytotoxicity assays were also performed., Results: Ca 2+ imaging revealed that 3 of 11 compounds suppressed sarcoplasmic reticulum Ca 2+ leak through mutant RyR2. Two compounds selected for further testing exhibited a half-maximal effective concentration of 146 nM (MSV1302) and 49 nM (MSV1406). Whereas neither compound altered baseline electrocardiogram intervals, only MSV1302 suppressed stress- and pacing-induced ventricular tachycardia in vivo in R176Q/+ mice. Echocardiography revealed that the lead compound MSV1302 did not negatively affect cardiac inotropy and chronotropy. Finally, compound MSV1302 did not block I Na , I Ca,L , or I Kr ; it exhibited excellent stability in plasma and microsomes, and it was not cytotoxic., Conclusion: Structure-activity relationship studies of second-generation tetracaine derivatives identified lead compound MSV1302 with a favorable pharmacokinetic profile. MSV1302 normalized aberrant RyR2 activity in vitro and in vivo, without altering cardiac inotropy, chronotropy, or off-target effects on other ion channels. This compound may be a strong candidate for future clinical studies to determine its efficacy in CPVT patients., Competing Interests: Disclosures R.M.S. and X.H.T.W. are founding partners and M.S.-V., R.M.S., and X.H.T.W. were shareholders of Elex Biotech Inc, a start-up company that developed drug molecules that target ryanodine receptors to treat cardiac arrhythmia disorders. The other authors do not have relevant disclosures., (Copyright © 2024 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Coronary heart disease (CHD) is a prevalent cardiac disease that causes over 370,000 deaths annually in the USA. In CHD, occlusion of a coronary artery causes ischemia of the cardiac muscle, which results in myocardial infarction (MI). Junctophilin-2 (JPH2) is a membrane protein that ensures efficient calcium handling and proper excitation-contraction coupling. Studies have identified loss of JPH2 due to calpain-mediated proteolysis as a key pathogenic event in ischemia-induced heart failure (HF). Our findings show that calpain-2-mediated JPH2 cleavage yields increased levels of a C-terminal cleaved peptide (JPH2-CTP) in patients with ischemic cardiomyopathy and mice with experimental MI. We created a novel knock-in mouse model by removing residues 479-SPAGTPPQ-486 to prevent calpain-2-mediated cleavage at this site. Functional and molecular assessment of cardiac function post-MI in cleavage site deletion (CSD) mice showed preserved cardiac contractility and reduced dilation, reduced JPH2-CTP levels, attenuated adverse remodeling, improved T-tubular structure, and normalized SR Ca 2+ -handling. Adenovirus mediated calpain-2 knockdown in mice exhibited similar findings. Pulldown of CTP followed by proteomic analysis revealed valosin-containing protein (VCP) and BAG family molecular chaperone regulator 3 (BAG3) as novel binding partners of JPH2. Together, our findings suggest that blocking calpain-2-mediated JPH2 cleavage may be a promising new strategy for delaying the development of HF following MI., Competing Interests: Declaration of competing interest XHTW is a consultant for Pfizer and Rocket Pharmaceuticals, and a founding partner and board member of Elex Biotech Inc., a start-up company that developed drug molecules that target ryanodine receptors to treat cardiac arrhythmia disorders. LZ is the co-founder and consultant for Pelagos Pharma Inc., a start-up company that develop drug molecules that target nuclear receptors to treat heart failure and neurodegenerative disorders. Other authors have no potential conflicts to disclose. AI-assisted technology is not used in the preparation of this work (except checking grammar and spelling)., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

During synthetic textile washing, rubbing between fibres or against the washing machine, exacerbated by the elevated temperature, initiates the release of millions of microplastic fibres into the environment. A general tribological strategy is reported that practically eliminates the release of microplastic fibres from laundered apparel. The two-layer fabric finishes combine low-friction, liquidlike polymer brushes with "molecular primers", that is, molecules that durably bond the low-friction layers to the surface of the polyester or nylon fabrics. It is shown that when the coefficient of friction is below a threshold of 0.25, microplastic fibre release is substantially reduced, by up to 96%. The fabric finishes can be water-wicking or water-repellent, and their comfort properties are retained after coating, indicating a tunable and practical strategy toward a sustainable textile industry and plastic-free oceans and marine foodstuffs., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

Background: Accessory pathways are a common cause of supraventricular tachycardia (SVT) and can lead to sudden cardiac death in otherwise healthy children and adults when associated with Wolff-Parkinson-White syndrome. The goal of this study was to identify genetic variants within a large family with structurally normal hearts affected by SVT and Wolff-Parkinson-White syndrome and determine causality of the gene deficit in a corresponding mouse model., Methods: Whole exome sequencing performed on 2 distant members of a 3-generation family in which multiple members were affected by SVT or Wolff-Parkinson-White pattern (preexcitation) on ECG identified MRC2 as a candidate gene. Serial electrocardiograms, intracardiac electrophysiology studies, echocardiography, optical mapping studies, and histology were performed on both Mrc2 mutant and WT (wild-type) mice., Results: A rare HET (heterozygous) missense variant c.2969A>G;p.Glu990Gly (E990G) in MRC2 was identified as the leading candidate gene variant segregating with the cardiac phenotype following an autosomal-dominant Mendelian trait segregation pattern with variable expressivity. In vivo electrophysiology studies revealed reentrant SVT in E990G mice. Optical mapping studies in E990G mice demonstrated abnormal retrograde conduction, suggesting the presence of an accessory pathway. Histological analysis of E990G mouse hearts showed a disordered ECM (extracellular matrix) in the annulus fibrosus. Finally, Mrc2 knockdown in human cardiac fibroblasts enhanced accelerated cell migration., Conclusions: This study identified a rare nonsynonymous variant in the MRC2 gene in individuals with familial reentrant SVT, Wolff-Parkinson-White ECG pattern, and structurally normal hearts. Furthermore, Mrc2 knock-in mice revealed an increased incidence of reentrant SVT and bypass tract formation in the setting of preserved cardiac structure and function., Competing Interests: Dr Lupski has stock ownership in 23andMe (Sunnyvale, CA), is a paid consultant for the Regeneron Genetics Center (Tarrytown, NY), and is a coinventor on multiple US and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, and bacterial genomic fingerprinting. The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing conducted at Baylor Genetics (BG) Laboratories (Houston, TX). Dr Lupski serves on the Scientific Advisory Board of BG. Dr Wehrens is a cofounder and board member of Elex Biotech LLC, a start-up company developing RyR2 (ryanodine receptor type-2) modifying drugs for heart disease. Dr Wehrens is also a consultant at Pfizer and Rocket Pharmaceuticals. The other authors report no conflicts.

High concentrations of ammonium, phosphate, and phenol are recognized as water pollutants that contribute to the degradation of soil acidity. In contrast, small quantities of these nutrients are essential for soil nutrient cycling and plant growth. Here, we reported composite materials comprising biochar, chitosan, ZrO, and Fe 3 O 4 , which were employed to mitigate ammonium, phosphate, and phenol contamination in water and to lessen soil acidity. Batch adsorption experiments were conducted to assess the efficacy of the adsorbents. Initially, comparative studies on the simultaneous removal of NH 4 , PO 4 , and phenol using CB (biochar), CBC (biochar + chitosan), CBCZrO (biochar + chitosan + ZrO), and CBCZrOFe 3 O 4 (biochar + chitosan + ZrO + Fe 3 O 4 ) were conducted. The results discovered that CBCZrOFe 3 O 4 exhibited the highest removal percentage among the adsorbents ( P  < 0.05). Adsorption data for CBCZrOFe 3 O 4 were well fitted to the second-order kinetic and Freundlich isotherm models, with maximum adsorption capacities of 112.65 mg/g for NH 4 , 94.68 mg/g for PO 4 and 112.63 mg/g for phenol. Subsequently, the effect of CBCZrOFe 3 O 4 -loaded NH 4 , PO 4 , and phenol (CBCZrOFe 3 O 4 -APP) on soil acidity was studied over a 60-day incubation period. The findings showed no significant changes ( P  < 0.05) in soil exchangeable acidity, H + , Mg, K, and Na. However, there was a substantial increase in the soil pH, EC, available P, CEC, N-NH 4 , and N-NO 3 . A significant reduction was also observed in the available soil exchangeable Al and Fe ( P  < 0.05). This technique demonstrated multi-functionality in remediating water pollutants and enhancing soil acidity.

Background: Overexpression of the CREM (cAMP response element-binding modulator) isoform CREM-IbΔC-X in transgenic mice (CREM-Tg) causes the age-dependent development of spontaneous AF., Purpose: To identify key proteome signatures and biological processes accompanying the development of persistent AF through integrated proteomics and bioinformatics analysis., Methods: Atrial tissue samples from three CREM-Tg mice and three wild-type littermates were subjected to unbiased mass spectrometry-based quantitative proteomics, differential expression and pathway enrichment analysis, and protein-protein interaction (PPI) network analysis., Results: A total of 98 differentially expressed proteins were identified. Gene ontology analysis revealed enrichment for biological processes regulating actin cytoskeleton organization and extracellular matrix (ECM) dynamics. Changes in ITGAV, FBLN5, and LCP1 were identified as being relevant to atrial fibrosis and structural based on expression changes, co-expression patterns, and PPI network analysis. Comparative analysis with previously published datasets revealed a shift in protein expression patterns from ion-channel and metabolic regulators in young CREM-Tg mice to profibrotic remodeling factors in older CREM-Tg mice. Furthermore, older CREM-Tg mice exhibited protein expression patterns reminiscent of those seen in humans with persistent AF., Conclusions: This study uncovered distinct temporal changes in atrial protein expression patterns with age in CREM-Tg mice consistent with the progressive evolution of AF. Future studies into the role of the key differentially abundant proteins identified in this study in AF progression may open new therapeutic avenues to control atrial fibrosis and substrate development in AF., Competing Interests: Declaration of competing interest XHTW is a founding partner and shareholder of Elex Biotech Inc., a start-up company that developed drug molecules that target ryanodine receptors to treat cardiac arrhythmia disorders. Other authors have no conflict of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: In heart failure, signaling downstream the β2-adrenergic receptor is critical. Sympathetic stimulation of β2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind β2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear., Methods: cAMP accumulation in real time downstream of the β2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12 OX ) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure., Results: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12 OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12 OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12., Conclusions: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis., Competing Interests: Disclosures H. Qasim was employed by IonOptix LLC following the initial article submission. H. Qasim, M. Rajaei, Y. Xu, and B.K. McConnell have a Provisional Patent Application (no. 63/621 310; Modulation of AKAP12 Signalosome; UH Technology Disclosure ID no. 2024-023). The other authors report no conflicts.

Myocardial lipid metabolism is critical to normal heart function, whereas altered lipid regulation has been linked to cardiac diseases including cardiomyopathies. Genetic variants in the JPH2 gene can cause hypertrophic cardiomyopathy (HCM) and, in some cases, dilated cardiomyopathy (DCM). In this study, we tested the hypothesis that JPH2 variants identified in patients with HCM and DCM, respectively, cause distinct alterations in myocardial lipid profiles. Echocardiography revealed clinically significant cardiac dysfunction in both knock-in mouse models of cardiomyopathy. Unbiased myocardial lipidomic analysis demonstrated significantly reduced levels of total unsaturated fatty acids, ceramides, and various phospholipids in both mice with HCM and DCM, suggesting a common metabolic alteration in both models. On the contrary, significantly increased di- and triglycerides, and decreased co-enzyme were only found in mice with HCM. Moreover, mice with DCM uniquely exhibited elevated levels of cholesterol ester. Further in-depth analysis revealed significantly altered metabolites from all the lipid classes with either similar or opposing trends in JPH2 mutant mice with HCM or DCM. Together, these studies revealed, for the first time, unique alterations in the cardiac lipid composition-including distinct increases in neutral lipids and decreases in polar membrane lipids-in mice with HCM and DCM were caused by distinct JPH2 variants. These studies may aid the development of novel biomarkers or therapeutics for these inherited disorders., (© 2024 Lahiri et al.)

Introduction: Heterozygous autosomal-dominant single nucleotide variants in RYR2 account for 60% of cases of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia disorder associated with high mortality rates. CRISPR/Cas9-mediated genome editing is a promising therapeutic approach that can permanently cure the disease by removing the mutant RYR2 allele. However, the safety and long-term efficacy of this strategy have not been established in a relevant disease model., Aim: The purpose of this study was to assess whether adeno-associated virus type-9 (AAV9)-mediated somatic genome editing could prevent ventricular arrhythmias by removal of the mutant allele in mice that are heterozygous for Ryr2 variant p.Arg176Gln (R176Q/+)., Methods and Results: Guide RNA and SaCas9 were delivered using AAV9 vectors injected subcutaneously in 10-day-old mice. At 6 weeks after injection, R176Q/+ mice had a 100% reduction in ventricular arrhythmias compared to controls. When aged to 12 months, injected R176Q/+ mice maintained a 100% reduction in arrhythmia induction. Deep RNA sequencing revealed the formation of insertions/deletions at the target site with minimal off-target editing on the wild-type allele. Consequently, CRISPR/SaCas9 editing resulted in a 45% reduction of total Ryr2 mRNA and a 38% reduction in RyR2 protein. Genome editing was well tolerated based on serial echocardiography, revealing unaltered cardiac function and structure up to 12 months after AAV9 injection., Conclusion: Taken together, AAV9-mediated CRISPR/Cas9 genome editing could efficiently disrupt the mutant Ryr2 allele, preventing lethal arrhythmias while preserving normal cardiac function in the R176Q/+ mouse model of CPVT., Competing Interests: DECLARATIONS Conflicts of interest Wehrens XHT is a consultant for Pfizer and Rocket Pharmaceuticals, and a founding partner and board member of Elex Biotech Inc., a start-up company that developed drug molecules that target ryanodine receptors to treat cardiac arrhythmia disorders. The other authors do not have relevant disclosures.

Chronic kidney disease (CKD) is associated with a higher risk of atrial fibrillation (AF). The mechanistic link between CKD and AF remains elusive. IL-1β, a main effector of NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, is a key modulator of conditions associated with inflammation, such as AF and CKD. Circulating IL-1β levels were elevated in patients with CKD who had AF (versus patients with CKD in sinus rhythm). Moreover, NLRP3 activity was enhanced in atria of patients with CKD. To elucidate the role of NLRP3/IL-1β signaling in the pathogenesis of CKD-induced AF, Nlrp3-/- and WT mice were subjected to a 2-stage subtotal nephrectomy protocol to induce CKD. Four weeks after surgery, IL-1β levels in serum and atrial tissue were increased in WT CKD (WT-CKD) mice versus sham-operated WT (WT-sham) mice. The increased susceptibility to pacing-induced AF and the longer AF duration in WT-CKD mice were associated with an abbreviated atrial effective refractory period, enlarged atria, and atrial fibrosis. Genetic inhibition of NLRP3 in Nlrp3-/- mice or neutralizing anti-IL-1β antibodies effectively reduced IL-1β levels, normalized left atrial dimensions, and reduced fibrosis and the incidence of AF. These data suggest that CKD creates a substrate for AF development by activating the NLRP3 inflammasome in atria, which is associated with structural and electrical remodeling. Neutralizing IL-1β antibodies may be beneficial in preventing CKD-induced AF.

Textiles with a wicking finish transport moisture away from the skin, such that it is exposed to the environment for fast evaporation, aiding in thermophysiological comfort. Once saturated, such as in highly humid environments or if the wearer dons multiple layers, the efficacy of such a finish is substantially reduced. Here, we develop a new type of fluid transport textile design by combining physical and chemical wettability patterns to transport and remove liquids like sweat. First, a non-toxic, superhydrophobic fabric finish is developed that retains the air permeability of the fabric. Next, two superhydrophobic fabric layers are threaded together, containing wettability channels patterned at the inner/interior side of the fabrics. This design allows for liquid transport through the stitches to the interior channels and keeps both external faces dry. The developed strategy enables directional fluid transport under highly humid conditions, resulting in a ∼20 times faster transport rate than evaporation-based methods. The design principles described here can be used to provide thermophysiological comfort for users in extreme conditions, such as firefighters, law enforcement personnel, and health workers wearing personal protective ensembles.

Plastics are widely employed in modern civilization because of their durability, mold ability, and light weight. In the recent decade, micro/nanoplastics research has steadily increased, highlighting its relevance. However, contaminating micro/nanoplastics in marine environments, terrestrial ecosystems, and biological organisms is considered a severe threat to the environmental system. Geographical distribution, migration patterns, etymologies of formation, and ecological ramifications of absorption are just a few topics covered in the scientific literature on environmental issues. Degradable solutions from material science and chemistry are needed to address the micro/nanoplastics problem, primarily to reduce the production of these pollutants and their potential effects. Removing micro/nanoplastics from their discharge points has been a central and effective way to mitigate the adverse pollution effects. In this review, we begin by discussing the hazardous effect on living beings and the identification-characterization of micro/nanoplastics. Then, we provide a summary of the existing degradation strategies, which include bio-degradation and advanced oxidation processes (AOPs), and a detailed discussion of their degradation mechanisms is also represented. Finally, a persuasive summary of the evaluated work and projections for the future of this topic is provided., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction: Heart failure (HF) is the leading cause of death worldwide. Most large and small animal disease models of HF are based on surgical procedures. A common surgical technique to induce HF is transverse aortic constriction (TAC), which induces pressure overload. The conventional TAC (cTAC) procedure is a highly invasive surgery that is associated with severe inflammation and excessive perioperative deaths., Aim: To establish an improved, minimally invasive TAC (mTAC) procedure that does not require thoracotomy., Methods and Results: Following anesthesia, mice were intubated, and a small incision was made at the neck and chest. After cutting the sternum about 4 mm, the aortic arch was approached without opening the pleural cavity. A suture was placed between the brachiocephalic artery and the left common carotid artery. This model was associated with low perioperative mortality and a highly reproducible constriction evidenced by an increased right-to-left carotid blood flow velocity ratio in mTAC mice (5.9 ± 0.2) vs . sham controls (1.2 ± 0.1; P < 0.001). mTAC mice exhibited progressive cardiac remodeling during the 8 weeks post-TAC, resulting in reduced left ventricular (LV) contractility, increased LV end-systolic diameter, left atrial enlargement and diastolic dysfunction, and an increased heart weight to tibia length ratio (mTAC: 15.0 ± 0.8 vs . sham: 10.1 ± 0.6; P < 0.01)., Conclusion: Our data show that the mTAC procedure yields a highly reproducible phenotype consisting of LV contractile dysfunction and enlargement, combined with left atrial enlargement and diastolic dysfunction., Potential Impact of the Findings: This model may be used to test the molecular mechanisms underlying atrial remodeling associated with HF development or to evaluate therapeutic strategies to treat these conditions., Competing Interests: Conflicts of interest Xander H.T. Wehrens is a founding partner and director of Elex Biotech, a start-up company that developed drug molecules that target ryanodine receptors to treat cardiac arrhythmia disorders. Reddy AK is a consultant to Indus Instruments, the company that currently makes and sells the Doppler system and the rodent monitoring system.

Coke-oven wastewater (CW), containing an array of toxic pollutants above permissible limits even after conventional primary and secondary treatment, needs a tertiary (polishing) step to meet the statutory limit. In the present study, a suitable bacterial-microalgal consortium (Culture C) was constructed using bacterial (Culture B: Bacillus sp. NITD 19) and microalgal (Culture A: a consortium of Chlorella sp. and Synechococcus sp.) cultures at different ratios (v/v) and the potential of these cultures for tertiary treatment of CW was assessed. Culture C4 (Culture B:Culture A = 1:4) with inoculum size: 10% (v/v) was selected for the treatment of wastewater since the maximum growth (3.08 ± 0.57 g/L) and maximum chlorophyll content (4.05 ± 0.66 mg/L) were achieved for such culture in PLE-enriched BG-11 medium. During treatment of real secondary treated coke-oven effluent using Culture C4 in a closed photobioreactor, the removal of phenol (80.32 ± 2.76%), ammonium ions (47.85 ± 1.83%), fluoride (65.0 ± 4.12%), and nitrate (39.45 ± 3.42%) was observed after 24 h. In a packed bed bioreactor containing immobilized C4 culture, the maximum removal was obtained at the lowest flow rate (20 mL/h) and highest column bed height (20 cm). Artificial intelligence-based techniques were used for modeling and optimization of the process.

Aims: Abnormal intracellular calcium (Ca2+) handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signalling pathway contributes to atrial remodelling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models., Methods and Results: Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF (cAF). NFATc1 and NFATc2 mRNA levels were elevated in cAF patients compared with those in normal sinus rhythm (NSR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM:NFATc2-/- mice. Programmed electrical stimulation revealed that CREM:NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with wild-type controls, which was reversed in CREM:NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM:NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on ryanodine receptor type 2 (RyR2) in CREM:RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras and EF-hand domain-containing protein (Rasef) as a direct target of NFATc2-mediated transcription., Conclusion: Our findings reveal activation of the NFAT signalling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model., Competing Interests: Conflict of interest: X.H.T.W. is a co-founder and Scientific Advisory Board member of Elex Biotech, a drug development company focused on novel compounds for the cardiac arrhythmia disorders and heart failure. All other authors declared no conflict of interest., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Introduction: Abnormal calcium signaling between organelles such as the sarcoplasmic reticulum (SR), mitochondria and lysosomes is a key feature of heart diseases. Calcium serves as a secondary messenger mediating inter-organellar crosstalk, essential for maintaining the cardiomyocyte function., Areas Covered: This article examines the available literature related to calcium channels and transporters involved in inter-organellar calcium signaling. The SR calcium-release channels ryanodine receptor type-2 (RyR2) and inositol 1,4,5-trisphosphate receptor (IP 3 R), and calcium-transporter SR/ER-ATPase 2a (SERCA2a) are illuminated. The roles of mitochondrial voltage-dependent anion channels (VDAC), the mitochondria Ca 2+ uniporter complex (MCUC), and the lysosomal H + /Ca 2+ exchanger, two pore channels (TPC), and transient receptor potential mucolipin (TRPML) are discussed. Furthermore, recent studies showing calcium-mediated crosstalk between the SR, mitochondria, and lysosomes as well as how this crosstalk is dysregulated in cardiac diseases are placed under the spotlight., Expert Opinion: Enhanced SR calcium release via RyR2 and reduced SR reuptake via SERCA2a, increased VDAC and MCUC-mediated calcium uptake into mitochondria, and enhanced lysosomal calcium-release via lysosomal TPC and TRPML may all contribute to aberrant calcium homeostasis causing heart disease. While mechanisms of this crosstalk need to be studied further, interventions targeting these calcium channels or combinations thereof might represent a promising therapeutic strategy.

The present work emphasizes the development of a generic methodology that addresses the core issue of any running chemical plant, i.e., how to maintain a delicate balance between profit and environmental impact. Here, ethylene oxide (EO) production plant has been taken as a case study. The production of EO takes place in a multiphase catalytic reactor, the reliable first principle-based model of which is still not available in the literature. Artificial neural network (ANN) was therefore applied to develop a data-driven model of the complex reactor with the help of actual industrial data. The model successfully built up a correlation between the catalyst selectivity and other operational parameters. This model was used to establish two objective functions, profit and environmental impact. In this paper, the negative environmental impact has been designated by Eco-indicator 99, which considers all the negative health and environmental impacts of a certain product. A recently developed metaheuristic optimization technique, namely multi-objective firefly (MOF) algorithm, was used to develop Pareto diagram of profit vs. Eco-99. The Pareto diagram will help the plant engineers to make strategy on what operating conditions to be maintained to make a delicate balance between profit and environmental impact. It was also found that by applying this modeling and optimization technique, for a 130 kTA EO plant, approximately 7048 t/year of carbon dioxide can be saved from emission into the atmosphere., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Frying affects the nutritional quality of fish detrimentally. In this study, using Catla catla and mustard oil, experiments were carried out in varying temperatures (140-240 °C), times (5-20 min), and oil amounts (25-100 ml/kg of fish) which established drastic reduction of 44.97% and 99.40% for polyunsaturated fatty acid (PUFA)/saturated fatty acids (SFA) and index of atherogenicity (IA) profile, respectively. Artificial neural network (ANN) was implemented successfully to provide an association between the independent inputs with dependent outputs (values of R 2 were 0.99 and 0.98; RMSE were 0.038 and 0.046; and performance were 0.038 and 0.067 for PUFA/SFA and IA, respectively) by exhaustive search of various algorithms and activation functions available in literature. ANN model-based meta-heuristic, stochastic optimization formalisms, genetic algorithm (GA) and particle swarm optimization (PSO), were applied to optimize the combination of cooking parameters for improving the nutritional quality of food which improved the nutritional value by maximizing the PUFA/SFA profile up to 63.05% and minimizing the IA profile to 99.64%. Multi-objective genetic algorithm (MOGA) was also employed to tune the inputs by maintaining a balance between the contrasting outputs and enhance the overall food value simultaneously with multi-objective (beneficial for health, economic, and environment-friendly) proposal. MOGA was able to improve the PUFA/SFA profile up to 44.76% and reduce the IA profile to 92.94% concurrently with the reduction of wastage of culinary media and energy consumption, following the optimized cooking condition (118.92 °C, 6.06 min, 40 ml oil/kg of fish)., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Post-therapeutic relapse remains the biggest challenge to breast cancer management. The re-initiation of proliferation of dormant tumor cells in either metastatic or primary tumor location marks the final rate-limiting step of malignancy and mortality. The underlying molecular mechanisms remain poorly understood. We have recently demonstrated that KLF8 promotes breast cancer metastasis via CXCR4 upregulation. Here we report a role and mechanisms for KLF8 in driving the recurrence-like tumor outgrowth in both secondary and primary sites in a CXCR4-dependent manner. Treatment of an MDA-MB-231 breast cancer cell variant with the CXCR4 ligand, CXCL12, induces formation of filopodia in monolayer culture and filopodium-like protrusions (FLPs) in 3D culture. The FLP + cells proliferate significantly faster than FLP- cells in the 3D culture supplemented with CXCL12. Both the FLP formation and enhanced proliferation in the 3D culture can be prevented by silencing KLF8 expression in the cells. From this prevention, the cells can be rescued by overexpressing wild-type CXCR4 but not its inactive mutant form in the cells. Overexpression of KLF8 or CXCR4 in the cells dramatically enhances their invasive outgrowth and metastasis after being implanted into immunocompromised mice. Mechanistically, we found that the activated FAK was recruited to the nascent FLPs and that proliferation of the cells was completely prevented with a FAK-specific inhibitor. Taken together, these results shed new light on the role of KLF8 in promoting breast cancer recurrence, the fatal episode of the disease, by inducing CXCR4-dependent FLP formation., Competing Interests: None., (AJTR Copyright © 2022.)

Loss of atrioventricular conduction system (AVCS) cells due to either inherited or acquired deficits leads to conduction diseases, which can deteriorate into fatal cardiac arrhythmias and sudden death. In this issue of the JCI, Wang et al. constructed a mouse model of atrioventricular block (AVB) by inducing AVCS cell-specific injury using the Cx30.2 enhancer to drive expression of diphtheria toxin fragment A. AVCS cell ablation in adult mice led to irreversible AVB. jkjkIn contrast, AVCS cell injury in neonatal mice was followed by spontaneous recovery in a subset of mice, revealing a limited postnatal time window during which the regeneration of AVCS cells can occur as a result of cellular plasticity. This exciting study paves the way for future research into biological or cellular treatment approaches for cardiac conduction diseases by exploiting the regenerative potential of AVCS cells.

A novel copper-based catalyst supported by a long-chain hydrocarbon stearic acid (Cu x O@C 18 H 36 O 2 ) was synthesized by a hydrothermal method and double replacement reactions. The as-prepared catalyst is shown as self-assembled hierarchical nanoflakes with an average size of ∼22 nm and a specific surface area of 51.4 m 2 g -1 . The catalyst has a good performance on adsorption as well as Fenton-like catalytic degradation of Rhodamine B (RhB). The catalyst (10 mg/L) showed an excellent adsorption efficiency toward RhB (20 mg/L) for pH ranging from 5 to 13, with the highest adsorption rate (99%) exhibited at pH 13. The Fenton-like catalytic degradation reaction of RhB (20 mg/L) by Cu x O@C 18 H 36 O 2 nanoflakes was effective over a wide range of pH of 3-11, and • OH radicals were generated via Cu 2 O/H 2 O 2 interactions in acidic conditions and CuO/H 2 O 2 reactions in a neutral solution. The highest efficiency catalytic degradation of RhB (20 mg/L) was 99.2% under acidic conditions (pH = 3, H 2 O 2 = 0.05 M), with an excellent reusability of 96% at the 6th cycle. The results demonstrated that the as-prepared Cu x O@C 18 H 36 O 2 nanoflakes are an efficient candidate for wastewater treatment, with excellent adsorption capacity and superior Fenton-like catalytic efficiency and stability for RhB., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Hexavalent chromium has high toxic effect on the ecological system. The aim of the present study is to isolate and characterize the bacteria that can reduce the toxicity of hexavalent chromium from liquid effluent. The bacterial isolate was identified as Bacillus sp. ltds1 after 16 S rRNA gene sequencing, and annotation has been submitted in National Center for Biotechnology Information (NCBI) GenBank. The bacterial strain was found able to grow in Luria Broth medium at 100 mg/L Cr 6+ concentration. A maximum Cr 6+ bioremediation (95.24 ± 2.08 %) could be achieved using the said isolate at 40 mg/L, pH 7, and inoculum concentration 4 % at 24 h. The residual chromium was found in the form of less toxic trivalent chromium (Cr 3+ ), which confirms that the bacterial isolate can transform toxic Cr 6+ to non-toxic Cr 3+ . Fourier Transform Infra-Red (FTIR) study was performed to analyze the functional groups and overall nature of chemical bonds involved in the remediation process, whereas, Energy-Dispersive Spectroscopy (EDS) studies of native and treated cells showed the changes in elemental composition in response to metal stress. Artificial Neural Network (ANN) based prediction model is developed based on experimental points. The developed model was found to predict the bioremediation of Cr 6+ at various operating conditions. Particle Swarm Optimization (PSO) is used to optimize the variables like the initial concentration of metal, pH, temperature, and inoculum concentration for the said bacterial strain. The results showed that the isolate could be applied as a potential bioremediation agent for Cr 6+ removal.

This study reports a novel cross-linking approach to fabricate the hydrothermally neutralized silica hydrogel of SiO 2 -H 3 BO 3 -hexadecyltrimethoxysilane by grafting alkylsilane groups onto the nanoporous silica. The synthesized silica hydrogel possessed a large specific surface area of 51.3 m 2 g -1 and showed excellent dye adsorption capability of cationic dyes in neutral (pH 7) and alkaline (pH 9) medium from wastewater. The colloidal electrokinetic potential analysis revealed that the outstanding adsorption efficiency of cationic dyes over anionic dyes strongly relies on the surface charge of the hydrogels. Moreover, the hydrophobic interactions between the dye molecules and the hydrogels were studied, and it was found that the dye adsorption performance can be tuned by altering the concentration of hydrophobic reagents of the hydrogel. The dye adsorption mechanism was established, and the kinetic study suggested that the adsorption is a pseudo-second-order reaction. Adsorption isotherms at various equilibrium conditions fitted well with the Langmuir isotherm. Therefore, this strongly supports the promising and practical application of the prepared silica hydrogel. The recyclability of the hydrogel was studied, and it showed 90% adsorption efficiency by the regenerated gel up to 6 cycles, which has a high potential in wastewater treatment.

Cardiomyocyte contraction relies on precisely regulated intracellular Ca 2+ signaling through various Ca 2+ channels and transporters. In this article, we will review the physiological regulation of Ca 2+ handling and its role in maintaining normal cardiac rhythm and contractility. We discuss how inherited variants or acquired defects in Ca 2+ channel subunits contribute to the development or progression of diseases of the heart. Moreover, we highlight recent insights into the role of protein phosphatase subunits and striated muscle preferentially expressed protein kinase (SPEG) in atrial fibrillation, heart failure, and cardiomyopathies. Finally, this review summarizes current drug therapies and new advances in genome editing as therapeutic strategies for the cardiac diseases caused by aberrant intracellular Ca 2+ signaling.

Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans 1 . Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies 2 . Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism 3 , is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.

Background: Enhanced diastolic calcium (Ca 2+ ) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca 2+ leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca 2+ /calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis., Methods: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca 2+ leak in atrial cardiomyocytes was assessed using confocal Ca 2+ imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9., Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca 2+ spark frequency in atrial cardiomyocytes with Ca 2+ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca 2+ leak, as well. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF., Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated sarcoplasmic reticulum Ca 2+ release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. Patients with HF exhibit a loss of junctophilin-2 (JPH2), a structural protein critical in forming junctional membrane complexes in which excitation-contraction takes place. Several mechanisms have been proposed to mediate the loss of JPH2, one being cleavage by the calcium-dependent protease calpain. The downstream mechanisms underlying HF progression after JPH2 cleavage are presently poorly understood. In this study, we used Labcas to bioinformatically predict putative calpain cleavage sites on JPH2. We identified a cleavage site that produces a novel C-terminal JPH2 peptide (JPH2-CTP) using several domain-specific antibodies. Western blotting revealed elevated JPH2-CTP levels in hearts of patients and mice with HF, corresponding to increased levels of calpain-2. Moreover, immunocytochemistry demonstrated nuclear localization of JPH2-CTP within ventricular myocytes isolated from a murine model of pressure overload-induced HF as well as rat ventricular myocytes treated with isoproterenol. Nuclear localization of JPH2-CTP and cellular remodeling were abrogated by a genetic mutation of the nuclear localization sequence within JPH2-CTP. Taken together, our studies identified a novel C-terminal fragment of JPH2 (JPH2-CTP) generated by calpain-2 mediated cleavage which localizes within the cardiomyocyte nucleus during HF. Blocking nuclear localization of JPH2-CTP protects cardiomyocytes from isoproterenol-induced hypertrophy in vitro. Future in vivo studies of the nuclear role of JPH2-CTP may reveal a causal association with adverse remodeling during HF and establish CTP as a therapeutic target.

Background: Abnormal calcium (Ca 2+ ) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca 2+ cycling, the type-2 ryanodine receptor (RyR2) and phospholamban (PLN), enhances the susceptibility to AF, but the underlying mechanisms remain unclear. Protein phosphatase 1 (PP1) limits steady-state phosphorylation of both RyR2 and PLN. Proteomic analysis uncovered a novel PP1-regulatory subunit (PPP1R3A [PP1 regulatory subunit type 3A]) in the RyR2 macromolecular channel complex that has been previously shown to mediate PP1 targeting to PLN. We tested the hypothesis that reduced PPP1R3A levels contribute to AF pathogenesis by reducing PP1 binding to both RyR2 and PLN., Methods: Immunoprecipitation, mass spectrometry, and complexome profiling were performed from the atrial tissue of patients with AF and from cardiac lysates of wild-type and Pln-knockout mice. Ppp1r3a-knockout mice were generated by CRISPR-mediated deletion of exons 2 to 3. Ppp1r3a-knockout mice and wild-type littermates were subjected to in vivo programmed electrical stimulation to determine AF susceptibility. Isolated atrial cardiomyocytes were used for Stimulated Emission Depletion superresolution microscopy and confocal Ca 2+ imaging., Results: Proteomics identified the PP1-regulatory subunit PPP1R3A as a novel RyR2-binding partner, and coimmunoprecipitation confirmed PPP1R3A binding to RyR2 and PLN. Complexome profiling and Stimulated Emission Depletion imaging revealed that PLN is present in the PPP1R3A-RyR2 interaction, suggesting the existence of a previously unknown SR nanodomain composed of both RyR2 and PLN/sarco/endoplasmic reticulum calcium ATPase-2a macromolecular complexes. This novel RyR2/PLN/sarco/endoplasmic reticulum calcium ATPase-2a complex was also identified in human atria. Genetic ablation of Ppp1r3a in mice impaired binding of PP1 to both RyR2 and PLN. Reduced PP1 targeting was associated with increased phosphorylation of RyR2 and PLN, aberrant SR-Ca 2+ release in atrial cardiomyocytes, and enhanced susceptibility to pacing-induced AF. Finally, PPP1R3A was progressively downregulated in the atria of patients with paroxysmal and persistent (chronic) AF., Conclusions: PPP1R3A is a novel PP1-regulatory subunit within the RyR2 channel complex. Reduced PPP1R3A levels impair PP1 targeting and increase phosphorylation of both RyR2 and PLN. PPP1R3A deficiency promotes abnormal SR-Ca 2+ release and increases AF susceptibility in mice. Given that PPP1R3A is downregulated in patients with AF, this regulatory subunit may represent a new target for AF therapeutic strategies.

Krüppel-like factor 8 (KLF8) plays many important roles in various diseases, especially cancer. Previous studies have shown that KLF8 is regulated by ubiquitylation. The molecular mechanism underlying this posttranslational modification of KLF8, however, has not been investigated. Reported here is our identification of the neural precursor cell expressed, developmentally down-regulated 4 (NEDD4) as the E3 ubiquitin ligase for this modification. By co-immunoprecipitation and ubiquitylation assays, we determined that KLF8 interacts with NEDD4 and is ubiquitylated by NEDD4. By site-directed mutagenesis and pharmacological inhibition of MEK, we found that the ubiquitylation of KLF8 by NEDD4 depends upon the phosphorylation of KLF8 at serine 48 by ERK. Cycloheximide chase analysis, target gene promoter reporter assay and fluorescent staining indicated that NEDD4 plays a critical role in promoting the stability and transcriptional activity of KLF8 in the nucleus. Taken together, this work identified NEDD4 as a novel E3 ubiquitin ligase for KLF8 that provides insights into targeting the KLF8-NEDD4 axis to treat various types of cancer associated with overexpression of both proteins., Competing Interests: None.

Limited robustness is a serious drawback for superhydrophobic coatings and degrades the performance of superhydrophobic surfaces in practical applications. Although fluororeagents have excellent durability for superhydrophobicity, their use has been restricted due to various health and environmental concerns. In this work, we describe a facile and efficient fabrication strategy for creating robust fluorine-free superhydrophobic composite coatings that are prepared by a simple dip-dry method, in which the H 3 BO 3 -incorporated SiO 2 -alkyl-silane coatings are deposited on woven cotton fabric surfaces followed by polydimethylsiloxane modification. The coated surface shows a large water contact angle of 157.95 ± 2° and a small sliding hysteresis angle (SHA) of 3.8 ± 0.6°, demonstrating excellent superhydrophobicity. The coated fabric surface also exhibited robustness and durability, withstanding a tape-peeling test (under 48.05 kPa) for around 80 repetitions and sandpaper rubbing (loaded 100 g) for 40 cycles. Furthermore, the coated fabric surface displayed self-healing and oil-water separation capacities. The developed superhydrophobic coatings in this study are robust, environmentally benign, and easy to fabricate, showing promising applications in textile industries.

Background: Heart failure (HF) is a complex disease with a rising prevalence despite advances in treatment. Protein phosphatase 1 (PP1) has long been implicated in HF pathogenesis, but its exact role is both unclear and controversial. Most previous studies measured only the PP1 catalytic subunit (PP1c) without investigating its diverse set of interactors, which confer localization and substrate specificity to the holoenzyme. In this study, we define the PP1 interactome in cardiac tissue and test the hypothesis that this interactome becomes rearranged during HF progression at the level of specific PP1c interactors., Methods: Mice were subjected to transverse aortic constriction and grouped on the basis of ejection fraction into sham, hypertrophy, moderate HF (ejection fraction, 30%-40%), and severe HF (ejection fraction <30%). Cardiac lysates were subjected to affinity purification with anti-PP1c antibodies followed by high-resolution mass spectrometry. PP1 regulatory subunit 7 (Ppp1r7) was knocked down in mouse cardiomyocytes and HeLa cells with adeno-associated virus serotype 9 and siRNA, respectively. Calcium imaging was performed on isolated ventricular myocytes., Results: Seventy-one and 98 PP1c interactors were quantified from mouse cardiac and HeLa lysates, respectively, including many novel interactors and protein complexes. This represents the largest reproducible PP1 interactome data set ever captured from any tissue, including both primary and secondary/tertiary interactors. Nine PP1c interactors with changes in their binding to PP1c were strongly associated with HF progression, including 2 known (Ppp1r7 and Ppp1r18) and 7 novel interactors. Within the entire cardiac PP1 interactome, Ppp1r7 had the highest binding to PP1c. Cardiac-specific knockdown in mice led to cardiac dysfunction and disruption of calcium release from the sarcoplasmic reticulum., Conclusions: PP1 is best studied at the level of its interactome, which undergoes significant rearrangement during HF progression. The 9 key interactors that are associated with HF progression may represent potential targets in HF therapy. In particular, Ppp1r7 may play a central role in regulating the PP1 interactome by acting as a competitive molecular "sponge" of PP1c.

Rationale: Autosomal-dominant mutations in ryanodine receptor type 2 ( RYR2) are responsible for ≈60% of all catecholaminergic polymorphic ventricular tachycardia. Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue catecholaminergic polymorphic ventricular tachycardia., Objective: To determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct catecholaminergic polymorphic ventricular tachycardia arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+)., Methods and Results: Guide RNAs were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the SaCas9 ( Staphylococcus aureus Cas9) genome editing system. AAV serotype 9 was used to deliver Cas9 and guide RNA to neonatal mice by single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV serotype 9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wild-type littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wild-type Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca 2+ spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca 2+ transients., Conclusions: AAV serotype 9-based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia.

Side effects of radiation therapy (RT) remain the most challenging issue for pancreatic cancer treatment. Cerium oxide nanoparticles (CONPs) are currently being tested in pre-clinical trials as an adjuvant to sensitize pancreatic cancer cells to RT and protect normal tissues from the harmful side effects. CONPs were not able to significantly affect RT-induced DNA damage in cancer cells, thereby ruling out sensitization through increased mitotic catastrophe. However, activation of c-Jun terminal kinase (JNK), a key driver of RT-induced apoptosis, was significantly enhanced by co-treatment with CONPs and RT in pancreatic cancer cells in vitro and human pancreatic tumors in nude mice in vivo compared to CONPs or RT treatment alone. Further, CONP-driven increase in RT-induced JNK activity was associated with a marked increase in Caspase 3/7 activation, indicative of apoptosis. We have previously shown that CONPs increase reactive oxygen species (ROS) production in cancer cells. ROS has been shown to drive the oxidation of thioredoxin 1 (TRX1) which results in the activation of apoptosis signaling kinase 1 (ASK1). The increase in ASK1 activation following the co-treatment with CONPs followed by RT suggests that the increased JNK activation is the result of increased TRX1 oxidation. The ability of CONPs to sensitize pancreatic cancer cells to RT was mitigated when the TRX1 oxidation was prevented by mutagenesis of a cysteine residue or when the JNK activation was blocked by an inhibitor. Taken together, these data demonstrate an important mechanism for CONPs in specifically killing cancer cells and provide novel insights into the utilization of CONPs as a radiosensitizer and therapeutic agent for pancreatic cancer.

Background The medicinal properties of Inula cappa, a perennial shrub, are ascribed to its roots. In this article, we study the anti-inflammatory and immunomodulatory activities of I. cappa root extracts based on its utilization in traditional medicine-treatment of rheumatoid arthritis, menoxenia, fever, jaundice and many others. Methods Anti-inflammatory and immunomodulatory activities of I. cappa extracts were studied. The anti-inflammatory activity was determined by carrageenan-induced rat paw edema and cotton pellet-induced granulation method while the immunomodulatory activity was estimated by phagocytic assay method, hemagglutinating antibody (HA) titer assay, delayed-type hypersensitivity assay method, plaque forming cell assay and determination of immunoprophylatic activity. Results The methanolic extract showed maximum reduction in the rat paw edema and showed significant inhibition of the cotton pellet-induced granulomas in rats. The methanolic extract also showed potential immunomodulatory activity in all the assays performed. Two sesquiterpenes, isoalantolactone and germacranolide were also isolated from the methanolic extract. Conclusions The present study supports the evidence that the roots of I. cappa can be used as a potent anti-inflammatory and immunomodulatory agent.

Recently, there is a growing concern about stress during undergraduate medical training. The objectives of our study were to assess perceived stress among undergraduate medical students and to find out academic factors as determinants. A cross-sectional, questionnaire-based survey was carried out among undergraduate medical students of R. G. Kar Medical College, India, during July 2011-June 2012. Perceived stress was assessed using the Perceived Stress Scale-14. A 10-item questionnaire was used to assess academic sources of stress and their severity. The overall mean perceived stress score was 29.58 (standard deviation [SD] = 6.60), and 46.3% of the participants were in the group of more stressed (perceived stress score ≥28). The academic stressor factors predicted 78% of variability of perceived stress. A higher level of perceived stress was reported by the students. The students should be taught different stress management techniques to improve their ability to cope with a demanding professional course.

Krüppel-like factor 8 (KLF8) plays important roles in cancer and is strictly regulated by various post-translational modifications such as sumoylation, acetylation, ubiquitylation and PARylation. Here we report a novel phosphorylation of KLF8 by ERK2 responsible and critical for the stability of KLF8 protein. The full-length KLF8 protein displays a doublet in SDS-PAGE gel. The upper band of the doublet, however, disappeared when the N-terminal 50 amino acids were deleted. In its full-length the upper band disappeared upon phosphatase treatment or mutation of the serine 48 (S48) to alanine whereas the lower band was lost when the S48 was mutated to aspartic acid that mimics phosphorylated S48. These results suggest that S48 phosphorylation is responsible for the motility up-shift of KLF8 protein. Pharmacological and genetic manipulations of various potential kinases identified ERK2 as the likely one that phosphorylates KLF8 at S48. Functional studies indicated that this phosphorylation is crucial for protecting KLF8 protein from degradation in the nucleus and promoting cell migration. Taken together, this study identifies a novel mechanism of phosphorylation critical for KLF8 protein stabilization and function.

Krüppel-like factor 8 (KLF8) has been strongly implicated in breast cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we report a novel signaling from KLF8 to C-X-C cytokine receptor type 4 (CXCR4) in breast cancer. Overexpression of KLF8 in MCF-10A cells induced CXCR4 expression at both mRNA and protein levels, as determined by quantitative real-time PCR and immunoblotting. This induction was well correlated with increased Boyden chamber migration, matrigel invasion and transendothelial migration (TEM) of the cells towards the ligand CXCL12. On the other hand, knockdown of KLF8 in MDA-MB-231 cells reduced CXCR4 expression associated with decreased cell migration, invasion and TEM towards CXCL12. Histological and database mining analyses of independent cohorts of patient tissue microarrays revealed a correlation of aberrant co-elevation of KLF8 and CXCR4 with metastatic potential. Promoter analysis indicated that KLF8 directly binds and activates the human CXCR4 gene promoter. Interestingly, a CXCR4-dependent activation of focal adhesion kinase (FAK), a known upregulator of KLF8, was highly induced by CXCL12 treatment in KLF8-overexpressing, but not KLF8 deficient cells. This activation of FAK in turn induced a further increase in KLF8 expression. Xenograft studies showed that overexpression of CXCR4, but not a dominant-negative mutant of it, in the MDA-MB-231 cells prevented the invasive growth of primary tumor and lung metastasis from inhibition by knockdown of KLF8. These results collectively suggest a critical role for a previously unidentified feed-forward signaling wheel made of KLF8, CXCR4 and FAK in promoting breast cancer metastasis and shed new light on potentially more effective anti-cancer strategies., Competing Interests: The authors declare no conflicts of interest.

Krüppel-like factor 8 (KLF8) is a dual transcriptional factor critical for breast cancer progression. Epidermal growth factor receptor (EGFR) is frequently overexpressed in aggressive such as triple-negative breast cancer and associated with poor clinical outcomes. Here we report a novel KLF8-EGFR signaling axis in breast cancer. We identified a highly correlated co-overexpression between KLF8 and EGFR in invasive breast cancer cells and patient tumor samples. Overexpression of KLF8 in the non-tumorigenic MCF-10A cells induced the expression of EGFR, whereas knockdown of KLF8 from the MDA-MB-231 cells decreased it. Promoter activation and binding assays indicated that KLF8 promotes the EGFR expression by directly binding its gene promoter. We also revealed that KLF8 directly represses the promoter of miR141 and miR141 targets the 3'-untranslational region of EGFR transcript to inhibit EGFR translation. Treatment with the EGFR inhibitor AG1478 or overexpression of miR141 blocked the activity of ERK downstream of EGFR and inhibited KLF8-depndent cell invasiveness, proliferation and viability in cell culture and invasive growth and lung metastasis in nude mice. Conversely, overexpression of an inhibitory sponge of miR141 led to the opposite phenotypes. Taken together, these findings demonstrate a novel KLF8 to miR141/EGFR signaling pathway potentially crucial for breast cancer malignancy.

Krüppel-like factor 8 (KLF8) is a transcriptional factor critical for metastatic progression of breast cancer. Epithelial stromal interaction 1 (EPSTI1), a recently identified stromal fibroblast-induced gene in non-invasive breast cancer cells is highly overexpressed in invasive breast carcinomas. The function and regulation of EPSTI1, however, remain largely unknown. In this paper, we report a novel KLF8 to EPSTI1 signaling pathway in breast cancer. Using various expression analyses, we revealed a high co-overexpression of KLF8 and EPSTI1 in invasive human breast cancer cells and patient tumors. Ectopic overexpression of KLF8 in the non-invasive MCF-10A cells induced the EPSTI1 expression, whereas KLF8 knockdown from the invasive, MDA-MB-231 cells decreased the EPSTI1 expression. Promoter activation and binding analyses indicated that KLF8 promoted the EPSTI1 expression by directly acting on the EPSTI1 gene promoter. EPSTI1 knockdown dramatically reduced the KLF8-promoted MCF-10A cell invasion, and ectopic expression of EPSTI1 in the non-invasive MCF-7 cells is sufficient to induce the cell invasion. Experiments using nude mice demonstrated that the ectopic EPSTI1 granted the MCF-7 cells capability of both invasive growth in the breasts and metastasis to the lungs. Using co-immunoprecipitation coupled with mass spectrometry, we discovered that EPSTI1 interacts with the valosin-containing protein (VCP), resulting in the degradation of IκBα and subsequent activation of NF-κB in the nucleus. These findings suggest a novel KLF8 to EPSTI1 to VCP to NF-κB signaling mechanism potentially critical for breast cancer invasion and metastasis.

Krüppel-like factor 8 (KLF8) regulates critical gene transcription associated with cancer. The underlying mechanisms, however, remain largely unidentified. We have recently demonstrated that KLF8 expression enhances the activity but not expression of matrix metalloproteinase-2 (MMP2), the target substrate of MMP14. Here, we report a novel KLF8 to MMP14 signaling that promotes human breast cancer invasion and metastasis. Using cell lines for inducible expression and knockdown of KLF8, we demonstrate that KLF8 promotes MMP14 expression at the transcriptional level. Knocking down KLF8 expression inhibited the breast cancer cell invasion both in vitro and in vivo as well as the lung metastasis in mice, which could be rescued by ectopic expression of MMP14. Promoter reporter assays and oligonucleotide and chromatin immunoprecipitations determined that KLF8 activates the human MMP14 gene promoter by both directly acting on the promoter and indirectly via promoting the nuclear translocation of β-catenin, the expression of T-cell factor-1 (TCF1) and subsequent activation of the promoter by the β-catenin/TCF1 complex. Inhibition of focal adhesion kinase (FAK) using pharmacological inhibitor, RNA interference or knockout showed that the cell surface presentation of active MMP14 downstream of KLF8 depends on FAK expression and activity. Taken together, this work identified novel signaling mechanisms by which KLF8 and FAK work together to promote the extracellular activity of MMP14 critical for breast cancer metastasis.