Chronic pressure overload is a key risk factor for mortality due to its subsequent development of heart failure, in which the underlying molecular mechanisms remain vastly undetermined. In this review, we updated the latest advancements for investigating the role and relevant mechanisms of oxidative stress involved in the pathogenesis of pressure-overload-induced cardiomyopathy and cardiac dysfunction, focusing on significant biological sources of reactive oxygen species  production, antioxidant defenses, and their association with the cardiac metabolic remodeling in the stressed heart. We also summarize the newly developed preclinical therapeutic approaches in animal models for pressure-overload-induced myocardial damage. This review aims to enhance the current understanding of the mechanisms of chronic hypertensive heart failure and potentially improve the development of better therapeutic strategies for the associated diseases. Oxidative stress, defined as the disturbed homeostasis between the production and detoxification of reactive oxidative species (ROS) in biological systems, has been considered a major pathological factor in many diseases. ROS or free radicals collectively refer to any chemical species containing unpaired electrons that highly increase the chemical reactivity of an atom or molecule, such as the hydroxyl radical, superoxide anion , uncoupled nitric oxide synthases, xanthine oxidase (XO), monoamine oxidases, and cytochrome P450 oxidase, which are most commonly characterized in controlling cardiac redox signaling ,4. Reciprocally, the heart has an integrated complex antioxidant defense system comprised of enzymatic and non-enzymatic antioxidants to counteract ROS accumulation and neutralize ROS into nontoxic molecules. Enzymatic antioxidants form the first line of defense against ROS, such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidases (GPx), paraoxonase (PON), and thioredoxin (Trx). In contrast, non-enzymatic antioxidants constitute the second line of defense by interrupting free radical chain reactions . Pressure overload in the heart, for instance, caused by hypertension or aortic stenosis, induces structural and functional remodeling in response to the increased workload. As initially compensated concentric hypertrophy gradually progresses to decompensated eccentric hypertrophy under chronic pressure overload, the heart undergoes a series of pathological remodeling processes in terms of contractile dysfunction, electrophysiological abnormalities, and metabolic inflexibility ,7. Pressure overload is considered to stimulate cardiomyocyte growth, known as hypertrophy, which leads to enlargement and thickening of ventricular walls accompanied by increased synthesis and deposition of collagen and extracellular matrix proteins, termed fibrosis . PressurIn contrast, chronic pressure-overload-induced oxidative stress leads to excessive mitochondrial DNA damage, defective mitochondrial ATP generation, overproduction of free radicals, and impaired contractile dysfunction due to ROS-mediated nonenzymatic modifications of proteins central to excitation-contraction coupling ,14. MoreThese primary pathological responses, free radicals, and their sources implicate secondary injury responses under chronic pressure overload, which progressively aggravates myocardial function and eventually leads to heart failure. Myocardial antioxidants are believed to act concertedly to reduce ROS-induced cell damage through diverse avenues, such as inhibiting metal-catalyzed ROS formation, promoting the antioxidant generation, upregulating antiapoptotic protein expression, and scavenging ROS precursors and free radicals . Under pressure overload or ischemic insults, the endogenous antioxidant mechanisms in the heart tend to be overwhelmed by ROS overflow, resulting in diminished antioxidant capacity . TherefoThis review summarizes the recent experimental evidence on the molecular mechanisms regulating oxidative stress in pressure-overloaded hearts. We also discuss emerging therapeutic approaches that can effectively improve pressure-overload-induced adverse cardiac remodeling by reducing excessive oxidative stress, thus, providing a greater perspective on novel treatment strategies for pressure-overload-induced heart conditions. Oxidative stress has been extensively characterized for its detrimental effects on the functional and structural integrity of the heart in various diseases. This review mainly highlighted the new progress during the past five years in the regulatory mechanisms of ROS production and antioxidative defense under the condition of pressure overload. In addition, studies have shown that pressure overload induces a significant metabolic remodeling in the myocardium, which eventually results in a deficient energy supply in the heart and subsequent cardiac dysfunction ,22. EmerSince mitochondria are the major primary source of ROS production in the heart under cardiac stress, numerous studies have been conducted to explore the molecular mechanisms regulating ROS production in the heart’s mitochondria. Here, we included recent discoveries of mitochondrial proteins or mitochondrial redox signaling-associated proteins exerting protective or detrimental effects on the heart under pressure overload. Recent studies have identified several mitochondrial proteins regulating pressure-overload-induced oxidative stress. For example, mitochondrial GTPases 1 (MTG1) protein expression has been found to be significantly increased in failing human hearts and pressure-overloaded mouse hearts after aortic banding (AB) . MTG1 ovIn contrast, MTG1 deficiency exacerbated pressure-overload-induced mitochondrial dysfunction and cardiomyopathy in MTG1 knockout (KO) mice, which was significantly attenuated with the treatment of N-acetyl-L-cysteine, a powerful antioxidant . These rFurthermore, TSPO decreased mitochondrial membrane potential (ΔΨm) and lowered excessive superoxide production in the heart of TSPO KO mice subjected to transverse aortic constriction (TAC) . These fIn addition, some proteins were found to be involved in the cardiac protection against pressure-overload-induced cardiac hypertrophy and dysfunction through regulating mitochondrial redox signaling. For instance, cardiac-specific overexpression of valosin-containing protein (VCP), an ATPase associated with various activities, has been found to ameliorate persistent pressure-overload-induced maladaptive hypertrophic remodeling and contractile dysfunction in transgenic (TG) mice subjected to TAC . The further genomic analysis identified a marked downregulation of cardiac mitochondrial genes involved in oxidative stress, especially several supernumerary subunits of complex I, III, and IV in VCP TG mice under pressure overload than the sham controls, accompanied by remarkably lowered oxidative markers in stressed hearts. These results suggest that VCP could protect the heart against pressure-overload-induced oxidative injury . Unlike the mitochondria that generate ROS as a secondary effect, Nox enzymes produce ROS as a primary function . The phaMultiple studies have demonstrated that Nox’s play a negative role in the heart subjected to pressure overload insult. The expression and functional significance of Nox5 in cardiac hypertrophy were recently studied using human cardiac tissues, transgenic mice, and neonatal rat cardiomyocytes . In fail2− production in the hearts of Nox5-Tg mice than their WT littermates which was consistent with observations made in vitro. Nox5-derived ROS stress was associated with altered MAPK signaling in the pressure-overloaded heart. Furthermore, Nox5 overexpressing transgenic (Nox5-Tg) mice subjected to either TAC or Angiotensin II (AngII) infusion exhibited worsened cardiac hypertrophy and myocardial contractile dysfunction compared with TAC or AngII-treated wild-type (WT) mice. TAC also caused a significant increase in NADPH-induced OAside from increased Nox5 expression in failing human hearts, Nox2 expression was also upregulated in human cardiac tissues from patients with dilated cardiomyopathy . GeneticIn addition, multiple studies demonstrate genes or proteins regulating pathological cardiac remodeling under pressure-overload through Nox-mediated ROS production. For example, cardiac expression of ubiquitin-specific protease 2 (USP2) was significantly diminished in pressure-overloaded mouse hearts after TAC. Overexpression of USP2 via tail injection of recombinant adeno-associated virus serotype 9 (AAV9) in TAC-treated male mice has been reported to attenuate pressure-overload-induced ventricular dysfunction, cardiac fibrosis, inflammation, and overall oxidative stress compared with AAV9 control-treated counterparts . Specifiβ3-adrenergic receptor (AR) stimulation was found to alleviate pressure-overload-induced cardiac hypertrophy, fibrosis, and cardiomyocyte apoptosis while improving cardiac dysfunction . In cont−/− mice [Furthermore, genetic deletion of low-density lipoprotein receptor (LDLr) by using adeno-associated virus vector serotype 8 (AAV8) has been found to counteract pressure-overload-induced cardiac hypertrophy, cardiomyocyte apoptosis, diastolic dysfunction, and interstitial and perivascular myocardial fibrosis in TAC-treated female LDLr−/− mice . −/− mice with mild hypercholesterolemia. LDLr deletion dramatically attenuated pressure-overload-induced elevation in plasma lipid peroxidation malondialdehyde (MDA) level, plasma XO activity, nitro-oxidative stress markers, and Nox2 and Nox4 protein expression levels in the myocardium . LDLr deThe heart has a complex and multilevel network of antioxidant defense to neutralize ROS and detoxify ROS accumulation to counteract ROS overproduction from various resources and biological activities under extreme stress conditions. Major endogenous enzymatic defenses, such as SOD, CAT, GSH, GPx, PON, and Trx, form the front line of protection against ROS-mediated injury in all living organisms. Here, we updated recently identified new targets that were involved in cardiac antioxidant responses in experimental models of pressure overload and their relevant regulatory mechanisms.Nrf2, also known as nuclear factor erythroid-derived 2-like 2, is a basic leucine zipper protein that functions as a critical transcription factor regulating the expression of antioxidant proteins that protect against oxidative damage. Emerging evidence has revealed a vital role of Nrf2 activation in protecting against maladaptive cardiac remodeling by regulating the transcriptional activities of a myriad of antioxidant response element (ARE)-dependent genes. These ARE-dependent genes encode detoxification enzymes and cytoprotective proteins. As such, Nrf2/ARE pathway activation mediates the induction of NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) to elicit powerful detoxication and elimination abilities to remove electrophiles and reactive oxidants, thus, conferring cellular resistance to oxidative stress ,35. Kelc−/− mice compared to pressure overload WT hearts after aortic constriction [−/− mice. Previously, Nrf2 deletion has been found to aggravate pressure-overload-induced myocardial hypertrophy, source substantial mitochondrial damage, promote cardiomyocyte apoptosis, and contractile dysfunction in Nrf2triction . More in−/− mice with worsened contractile dysfunction and hypertrophy compared with Nox4TG/Nrf2+/+ mice that exhibited considerably less adverse cardiac remodeling. These findings revealed a Nox4-dependent Nrf2 upregulation in the heart against chronic pressure overload, and induction of this cytoprotective pathway may represent a new therapeutic approach.Apart from this, cardiac-specific Nox4 overexpression-induced protective qualities against chronic pressure overload were significantly attenuated in Nox4TG/Nrf2Recent studies have identified manifold molecules in the heart that modulate cardiac antioxidative responses through the Nrf2-mediated signaling against pressure-overload-induced oxidative stress. One example is peroxiredoxin-1 (Prdx1), an efficient antioxidant enzyme previously upregulated in failing mouse hearts, which was studied in pressure-overloaded hearts of TAC-treated male mice . Myocardial Prdx1 overexpression via AAV9-Prdx1 injection significantly inhibited pressure-overload-induced pathological cardiac remodeling post-TAC, including myocardial hypertrophy, dysfunction, fibrosis, inflammation, and oxidative stress compared with control groups. Mechanistically, Prdx1 overexpression significantly diminished TAC-induced cardiac ROS production, and activity of both MDA and Nox, with improved SOD and CAT activity. This also reversed pressure overload-induced decrease in Nrf2 and HO-1 levels and increased nuclear translocation of Nrf2 from the cytosol . FurtherAnother example involves Sestrin2 (Sesn2), a highly conserved stress-inducible metabolic protein and known free radical repressor, whose expression was significantly elevated in the hypertrophic hearts of AB-treated male mice compared to sham-operated control . The obsCorrelatively, tripartite motif containing-21 (TRIM21), an interferon-inducible E3 ligase, may function as a negative regulator of Nrf2 signaling in pressure-overloaded hearts. Genetic ablation of TRIM21 fully preserved cardiac contractility under severe TAC in TRIM21 knockout mice compared with TAC-treated control littermates . DeletioAnoctamin-1 (ANO1), a voltage-gated calcium-activated anion channel protein highly expressed in epithelial cells, appears to be significantly reduced in myocardial tissues following TAC-induced pressure overload . InverseMLK3−) into TAC-treated mice (TAC + AAVMLK3−) was found to significantly alleviate pressure-overload-induced cardiac dysfunction, fibrosis, pyroptosis, and ferroptosis more than control mice (TAC + AAVNC) [In contrast to a strongly positive role of ANO1 against pressure overload in the heart, mixed lineage kinase 3 (MLK3), a member of the MAP3K family, has been previously characterized as a negative regulator in protecting against cardiomyocyte injury ,42,43. R+ AAVNC) . The expMLK3− exhibited significantly improved SOD and GSH levels but substantially decreased MDA levels in the heart compared with TAC + AAVNC. Cyclooxygenase 2, JNK, and p53, whose expressions mediate oxidative stress-induced ferroptosis, were significantly reduced in MLK3 KO TAC mice, revealing additional cardioprotective effects involving MLK3 inhibition against pressure overload.Beyond this, TAC + AAV2O2), has been reported to ameliorate TAC-induced increase in myocardial NADPH level, superoxide production, and lipid peroxidation in p66Shc KO mice compared with WT littermates. Contrarily, p66Shc overexpressed mice exhibited an aggravated phenotype [Additionally, the deletion of P66Shc, a mitochondrial redox signaling protein that produces hydrogen peroxide , and a series of ventricular oxidative stress markers, such as 4-HNE, were attenuated in p66Shc KO mice and augmented in p66Shc overexpressed mice. Treatment with SOD mimetic M40401 or PDE5 inhibitor sildenafil could significantly decrease ventricular oxidative stress markers. This study demonstrated a regulatory role of p66Shc in pressure-overload-induced oxidative stress through downstream of SOD and PDE5, thus, bringing potential therapeutic value for pressure-overload-induced heart failure. In summary, these studies from animal models provided new evidence further supporting the contribution of oxidative stress in the pathogenesis of cardiomyopathy and dysfunction caused by pressure overload and the development of heart failure. These new discoveries also highlighted the significance of potential therapeutic approaches to maintain the homeostasis between ROS production and detoxification by targeting both mechanisms. The newly identified potential targets are summarized in Perturbations in myocardial energy metabolism have been considered causative for cardiac pathogenesis, culminating in detectable tissue injury and malfunction. Unlike other tissues in the body, the heart prioritizes long-chain fatty acids as the primary cardiac energy source to generate ATP through mitochondrial beta-oxidation. Alternatively, glucose is less favored by the myocardium. Under the stress of pressure overload, the energy-deficient heart switches from fatty acid oxidation (FAO) to the more oxygen-efficient glucose oxidation . Such metabolic shift is adaptive in the early stage of hypoxic cardiac tissue since higher oxygen efficiency could ameliorate ischemia but not in the chronic pressure-overloaded heart. Many studies indicated that metabolic remodeling induced by chronic pressure overload is associated with mitochondrial damage and impaired oxidative capacity that inevitably results in excessive ROS formation ,46,47,482O2-induced oxidative stress in WT cardiac myocytes whose expression of HO-1 was uniquely upregulated. Fatty acid metabolism: Recently, free fatty acid receptor 4 (Ffar4) deficiency was discovered to aggravate pressure-overload-induced cardiac dysfunction following exacerbated development of concentric hypertrophy in Ffar4KO male mice subjected to chronic TAC . Treatme18-HEPE was barely detected in Ffar4KO high-density lipoprotein, suggesting an indispensable role of Ffar4 in its basal production and a novel cardioprotective role via inducing the 18-HEPE expression and mitigating oxidative stress . In addiIn contrast, its knockdown significantly elevated levels of MDA and ROS in the hearts of Plin5-null mice subjected to TAC. The activity of the antioxidant enzyme SOD, on the contrary, was significantly decreased, suggesting a higher oxidative burden due to excessive FAO in the TAC-treated Plin5-deficient heart. This study provided insights into how cardiac lipid metabolic dysfunction contributed to the pathogenesis of cardiac pressure overload, in which enhanced FAO and subsequent oxidative stress played a central role. Glucose metabolism: Sodium-glucose cotransporter 1 (SGLT1), a major glucose carrier in the heart, has been lately linked to increased myocardial nitro-oxidative stress in rats subjected to TAC-induced chronic pressure overload compared with sham control . The expAnother freshly identified protein is TP53-induced glycolysis and apoptosis regulator (TIGAR), which possesses an intrinsic fructose bis-phosphatase activity that lowers glycolysis. One study showed that, compared to sham control, TAC induced a progressive upregulation of TIGAR expression in WT mice. On the other hand, TIGAR deficiency in TIGAR-KO TAC myocardium eliminated significant ROS damage by promoting its secondary antioxidant property by upregulating the pentose phosphate pathway to produce NAPDH, which suggests a novel therapeutic approach to counteract pressure-overload-induced oxidative injury . ciKO) with augmented protein carbonylation, an irreversible oxidative protein modification [Additionally, cardiac-specific deletion of sirtuin 1 (Sirt1), a critical metabolic sensor of glucose and lipid metabolism, has been found to induce mild left ventricular (LV) systolic dysfunction and increased propensity to produce ROS in cardiac mitochondria of Sirt1 KO mice  expression and expectedly increased ketone body utilization, both of which were observed in Bdh1 overexpressed TG hearts with and without being subjected to TAC . 8-HydroIn vitro, adenovirus-mediated Bdh1 overexpression was found to markedly reduce intracellular ROS production and cardiomyocyte apoptosis in β-hydroxybutyrate-treated H9c2 cells while enhancing the mRNA expression of crucial antioxidants and antioxidative enzymes, such as metallothionein 2 (MT2), CAT, SOD, and forkhead box O3A. As baseline mRNA expression of MT2 and CAT did not differ significantly between Bdh1 TG mice and WT mice, these results indicate that Bdh1 overexpression-mediated cardioprotection through the enhanced antioxidative defenses may emerge in the condition of sufficient ketone bodies in response to significant energy deprivation, such as after TAC.−/−Cbs+/− mice treated with TAC [Amino acid metabolism: An increased plasma concentration of homocysteine has long been considered to independently predict major adverse cardiac events, such as the development of chronic heart failure, especially in females . Therefowith TAC . −/−Cbs+/− mice fed with a methionine-rich, folate-deficient diet to establish mild hyperhomocysteinemia presented with worsened cardiac hypertrophy, diastolic dysfunction, interstitial fibrosis, and apoptosis than control TAC mice fed with a standard chow and diet AdCBS TAC mice. Furthermore, significantly reduced plasma concentrations of GSH and cysteine while GPx and SOD activities detected in diet TAC mice were markedly elevated in control TAC mice and diet AdCBS TAC mice. TAC-induced elevated plasma lipid peroxidation and reactive nitrogen species formation were significantly lower in control TAC mice and diet AdCBS TAC mice than in diet TAC mice. Collectively, CBS ablation could counteract TAC-induced oxidative stress and augment antioxidant defenses, which confer beneficial effects on pressure-overload-induced cardiomyopathy.TAC LdlrIn recent years, there has also been rapid progress in branched-chain amino acids (BCAA) metabolism involved in cardiac metabolic remodeling. Germ-line knockout of Krüppel-like factor 15 controlled BCAA catabolic gene protein phosphatase 2C in mitochondria (PP2Cm) has been associated with promoting cardiac dysfunction and accelerating cardiac hypertrophy in TAC-treated PP2Cm KO mice when compared with WT TAC-treated control . BrancheIn recent years, there has been vast research investigating potential pharmacological targets of oxidative stress that can effectively provide cardioprotection. Many studies have identified additive cardioprotective effects against pressure-overload-induced oxidative stress from previously approved medications prescribed for treating other diseases, e.g., the repurposed pharmacological agents. In addition, many natural organic compounds extracted from herbs and species have demonstrated powerful antioxidant effects to counteract pressure-overload-induced oxidative stress. Aside from pharmacological approaches, nonpharmacological interventions, such as calorie restriction, play an important therapeutic role in pressure-overload-induced cardiac dysfunction. This updated information is presented in Several approved drugs have been identified to improve pressure-overload-induced cardiac injury in animal models outside of their initial medical indication. These repurposed pharmacological agents offer significant advantages to developing an entirely new drug for treating pressure overload-associated cardiac diseases.Raloxifene, a previously approved drug for the prevention and treatment of osteoporosis in postmenopausal women, has been found to considerably reduce pressure-overload-induced myocardial hypertrophy, fibrosis, inflammation, and attenuate cardiac dysfunction in TAC mouse hearts through intragastric administration compared with control-treated counterparts . RaloxifFasudil is a potent Rho-kinase inhibitor and vasodilator that has been used to treat cerebral vasospasm since it was discovered. Rats subjected to TAC exhibited significantly decreased cardiac antioxidant activities of SOD, CAT, and GPx with increased MDA content. Furthermore, pretreatment with fasudil via subcutaneous injection could dramatically boost antioxidant activities and reduce MDA levels in the hearts of TAC rats compared with vehicle-treated counterparts . FasudilCelecoxib, a nonsteroidal anti-inflammatory drug, exhibited cardioprotection from pressure-overload-induced hypertrophic remodeling, contractile dysfunction, cardiomyocyte apoptosis, and interstitial fibrosis in rats subjected to abdominal aortic constriction (AAC) . CelecoxSacubitril/Valsartan (LCZ696) is the first agent approved as an angiotensin receptor neprilysin inhibitor (ARNI) to treat patients who have chronic heart failure with reduced ejection fraction . AdminisMoreover, TAC caused downregulation in the expression levels of antioxidants manganese superoxide dismutase (MnSOD) and sirtuin3 (Sirt3) were markedly reversed by LCZ696 treatment. In contrast, Sirt3 knockdown aggravated phenylephrine (PE)-induced oxidative stress in cultured cardiomyocytes and abolished LCZ696 conferred cardioprotection. This study demonstrated that LCZ696 could mediate an antihypertrophic effect in TAC-treated mice by upregulating the MnSOD/Sirt3 pathway against myocardial oxidative stress . Oral adMoreover, LCZ696 was found to reduce mitochondrial superoxide levels greater than valsartan or sacubitril alone in ventricular myocytes isolated from pressure-overloaded rat hearts and AngII-treated primary adult ventricular myocytes, revealing a powerful antioxidative effect against cardiac oxidative stress. In addition, treatment with MitoTEMPO, a potent mitochondria-targeted antioxidant, nearly abolished AngII-stimulated increase in total cellular superoxide production in these myocytes, confirming the mitochondrial origin of ROS production in response to pressure overload .A great deal of research has explored potential treatments for pressure-overload-related heart diseases using natural, organic components derived from plants. Here, we summarized the latest findings in this field.Oridonin, an active diterpenoid extracted from the Chinese herbal medicine Rabdosia was currently found to inhibit pressure-overload-induced myocardial hypertrophy and fibrosis while attenuating cardiac oxidative stress in mice subjected to AB . Oridoni2− and MDA levels [Apocynin, a natural organic compound structurally related to vanillin, has been isolated from various plant sources and serves as the most employed Nox inhibitor. Previously, apocynin has been able to ameliorate pressure-overload-induced myocardial hypertrophy in rats subjected to AAC . Recent A levels . This stCarnosic acid (CA), a natural phenolic terpenoid isolated from Rosmarinus officinalis previously characterized as an effective lipid peroxidation inhibitor, was recently investigated for its potential effects on cardiac remodeling in mice subjected to aortic banding-induced pressure overload . Orally Stachydrine treatment, a constituent of the Chinese herb Leonurus heterophyllus sweet, via oral gavage alleviated pressure-overload-induced cardiac hypertrophy, contractile dysfunction, and excessive autophagic activity in TAC-treated rats . In vitrNobiletin (NOB), a polymethoxy flavonoid isolated from citrus peels, has been discovered to attenuate pressure-overload-induced cardiac hypertrophy, fibrosis, endoplasmic reticulum stress, and cardiomyocyte apoptosis while improving cardiac dysfunction in AB-treated mice via oral gavage . NOB als2O2 content in myocardial mitochondria and abolished the augmented cardiomyocyte apoptosis in AS-IV-treated TAC mice. Such protective effects of AS-IV were associated with the regulation of sentrin-specific protease 1 expression.Through intraperitoneal injection, treatment with astragaloside IV (AS-IV), an active substance isolated from Astragalus membranaceus, has been shown to improve pressure-overload-induced cardiac dysfunction, reverse cardiomyocyte hypertrophy, and myocardial mitochondrial damage in TAC-treated male mice . AS-IV a2O2, and Nox4 considerably decreased in SOD and GSH content along with an increased MDA level indicative of direct oxidative damage. These pathological changes were remarkably reversed after Cam treatment. This study is the first to provide evidence on the potential cardioprotective effect of Cam in heart failure due to chronic pressure overload [Once more delivered by intraperitoneal injection, treatment with cardamonin (Cam), a chalconoid extracted from several plants, such as Alpinia katsumadai, has been demonstrated to significantly alleviate pressure-overload-induced cardiac hypertrophy, fibrosis, cardiomyocyte apoptosis, and myocardial dysfunction in TAC-treated mice . TAC-indoverload .Aucubin (AUB), an iridoid glycoside in plants, established itself to suppress PE-induced cardiomyocyte hypertrophy in vitro and significantly attenuate chronic pressure-overload-induced cardiac hypertrophy, fibrosis, inflammation, and cardiac dysfunction in AB-treated male mice via intraperitoneal injection . MoreoveHispidulin, a naturally occurring flavonoid in different plants, was shown to attenuate pressure-overload-induced myocardial hypertrophy and improve cardiac dysfunction in male mice subjected to descending AB through intraperitoneal injection in comparison to their vehicle-treated AB mice counterparts . In addiLycium barbarum L., now a popular food supplement, have been recently evaluated for their antioxidative effects in the hearts of Wistar rats subjected to abdominal aorta banding (AAB) [In addition to natural organic ingredients derived from plants, many other natural organic compounds or food supplements have been investigated for their potential value in protecting the heart from pressure overload. For example, barbarum polysaccharides (LBPs) isolated from a traditional Chinese herb ng (AAB) . Commercng (AAB) .Lycopene, a type of organic pigment especially abundant in tomatoes and a powerful natural antioxidant, has been displayed to suppress pressure-overload-induced myocardial hypertrophic responses and cardiac dysfunction in AB-treated mice . LycopenFurthermore, lycopene pretreatment could improve the PE-inhibited expression of HO-1, SOD1, and CAT in cardiomyocytes at the mRNA level, indicating that both ARE activity and ARE-mediated antioxidant enzyme expression were upregulated by lycopene. Lycopene-mediated antioxidant actions were associated with downregulated phosphorylation of ERK1/2, p38, and JNK, as well as Akt (Thr308) and GSK3β of the pro-hypertrophic signaling pathway .Intraperitoneal injection of fisetin, a plant flavonol and dietary antioxidant, has been reported to inhibit pressure-overload-induced myocardial hypertrophy while mitigating cardiac dysfunction in male mice subjected to descending AB . In vitrVitamin D (VD) administered via oral gavage in TAC-treated male mice has been reported to significantly inhibit pressure-overload-induced cardiac hypertrophy, fibrosis, and inflammation while improving consequent cardiac dysfunction compared with the vehicle-treated control . LikewisIntravenous injection of irisin, an isolated hormone from mouse skeletal muscle, in TAC-treated male rats significantly lowered TAC-induced increase in mRNA expression of Nox2 and XO while increasing SOD1 and plasma GPx levels compared with vehicle-treated TAC rats . In addiInhibition of cathelicidin-related antimicrobial peptide (CRAMP) by transfecting AB-treated mice with recombinant AAV9-shCRAMP via intraperitoneal injection has been reported to ameliorate pressure-overload-induced cardiac hypertrophy, inflammation, and oxidative stress . SpecifiOral administration of Qindan capsule (QC), a compound used in traditional Chinese medicine to treat hypertension, has been found to significantly attenuate pressure-overload-induced cardiac hypertrophy and fibrosis while improving the impaired cardiac function and myocardial ultrastructure in TAC-treated male mice compared to vehicle-treated control . MoreoveIntragastric administration of taurine, an organic compound widely prevalent in animal tissues, has exhibited abilities to prevent TAC-induced deterioration of cardiac function in TAC-treated male mice and significantly attenuate cardiomyocyte hypertrophy and fibrosis compared with vehicle-treated control . FurtherMany chemical compounds that potentially interact with ROS signaling have been identified as promising therapeutic targets to treat pressure-overload-induced heart diseases in the future.Exogenous alpha-calcitonin gene-related peptide (a-CGRP) administration was shown to enhance cardiac function while attenuating myocardial hypertrophy and fibrosis in TAC-treated mice . Treatme2O2− treated rat neonatal cardiac myocytes, suggesting a cardioprotective effect in cardiomyocytes against oxidative stress.Treatment by the hippo pathway inhibitor, XMU-MP-1, which targets the expression of mammalian Ste-20-like kinase 1 and mammalian Ste-20-like kinase 2, was found to attenuate myocardial hypertrophic responses and reduce cardiac apoptosis and fibrosis. XMU-MP-1 improved cardiac function in TAC-treated mice . XMU-MP-A small Wnt inhibitor molecule called Wnt-C59 constrained porcupine required for Wnt palmitoylation and was discovered to significantly attenuate pressure-overload-induced hypertrophic responses while improving cardiac dysfunction and enhancing the survival of adult male mice subjected to TAC . Wnt-C59Treatment with mitoquinone (MitoQ), a robust mitochondria-targeted antioxidant, via oral gavage in AAC-treated male mice has improved pressure-overload-induced LV contractile dysfunction and mitochondrial depolarization. MitoQ preserved disrupted mitochondrial networks . MitoQ t+ T-cell receptor activation in pressure-overloaded hearts [Isolevuglandins (isoLGs) adducts have recently been discovered to modify cardiac proteins into neoantigens that subsequently engage in CD4d hearts . Orally d hearts .On top of these medications, studies have shown that non-pharmacological interventions play a beneficial role in response to pressure overload. For example, calorie restriction (CR) has been studied in mouse hearts subjected to pressure overload following ascending aortic constriction (AAC) . Mice feExcessive Nox-dependent and mitochondrial superoxide production detected in the AL + AAC group were significantly reduced in the CR + AAC group. On the contrary, cardiac GPx and SOD activities were substantially increased in the CR + AAC group but not in the AL + AAC group. It is important to note that the initial two weeks of CR before AAC did not bring any significant differences in cardiac morphological, functional, or redox changes between AL and CR intake groups, suggesting that short-term CR could be applied as a valuable strategy to manage pressure-overload-induced cardiac injury .Recently, preconditioning with two weeks of dietary restriction (DRPC) has also been reported to alleviate pressure-overload-induced maladaptive cardiac remodeling in the left ventricle of DRPC-treated male mice subjected to 2 weeks of AB with the AL + AAC group . AAC-indCardiac pressure overload is a common pathological condition in clinical settings, and this condition results in the concentric hypertrophy of cardiac muscles with contractile dysfunction that eventually progresses to heart failure, thus, contributing to high morbidity and mortality rates among elderly patients. Recent experimental studies in animal models, using either genetic modification or pharmacologic intervention on target genes, demonstrated that pressure-overload-induced oxidative stress plays a vital role in adverse cardiac remodeling and the subsequent progression to heart failure.Cardiac oxidative stress is tightly associated with a maladaptive metabolic remodeling in response to pressure overload, further complicating the pathogenesis and consequences of pressure overload in associated heart diseases. Thus, numerous studies have explored potential therapeutic strategies to inhibit oxidative stress by enhancing antioxidant responses while suppressing ROS production.Emerging therapeutic strategies involve repurposed pharmacological agents, naturally occurring organic compounds and ingredients, synthetic chemical compounds, and calorie restriction. These new advances offer different perspectives for understanding the pathogenesis of pressure overload-associated heart diseases and provide novel evidence for developing strategies to combat chronic heart failure. The therapeutic targets summarized above potentially unlocked more treatment options that could develop in the future.For example, Nox isoforms have been extensively reported to be upregulated in the stressed heart, fueling ROS production. Thus, there has begun a quest for developing selective Nox inhibitors, such as Nox-specific peptidic inhibitors, Nox2ds-tat, and small-molecule inhibitors . In addiDespite recent progress in elucidating the mechanisms of pressure-overload-induced oxidative stress in the heart, many aspects remain undetermined and require further investigation. First, although various methods for assessing ROS production are available for in vitro studies, direct detection of ROS in vivo has presented challenges due to its very short half-life and high reactivity. Further endeavors are required to develop methods for in situ, real-time detection of ROS in vivo.Second, although animal studies using transgenic or knockout mouse models have provided strong evidence for the regulatory mechanisms of pressure-overload-induced cardiac injury, much work remains to be done to translate these experimental observations into clinical use. Third, current animal models of experimental pressure overload are generated by surgically restricting aortic blood flow to increase heart afterload, which only somewhat mimics the complex structural and functional changes that develop in response to pressure overload in human hearts.Therefore, there is a constant need to develop more reliable and informative animal models that can better mimic pressure-overload-induced heart failure’s multifactorial and combinatorial pathogenicity. Fourth, the therapeutic effects of newly developed drugs or compounds in animal experiments are rather unspecific or indirectly related to antioxidative efforts against pressure-overload-induced cardiac oxidative stress.Although targeting antioxidant mechanisms has been considered a promising cardioprotective strategy to combat adverse effects of excessive ROS, a plethora of clinical trials have produced mixed results regarding their efficacy . For exaNrf2, a previously characterized master regulator of antioxidative responses, has recently been incriminated in inducing myocardial damage and dysfunction, revealing a dark side in the heart . These iAlthough this review focuses on oxidative stress and antioxidant defenses involved in pressure-overload-induced cardiac injury, oxidative stress with shared mechanisms of action is substantially implicated in a range of cardiovascular conditions. For instance, multiple Nox isoforms previously identified to be variably elevated in hypertensive animal models were comprehensively reviewed. Nox5 has been most recently highlighted as a crucial mediator of vascular oxidative stress, and Nox4 plays an especially important role in salt-sensitive hypertension .Additionally, mitochondrial-targeted antioxidants, such as the aforementioned MitoQ, have been shown to reduce hypertension and hypertension-associated organ damage ,96. The


Soft tissue sarcoma is a rare entity that accounts for 1% of adult cancers but represents 20% of paediatric solid tumours. Overall prognosis in advanced disease remains poor. MicroRNAs (miRNAs) are short non-coding RNAs that target mRNAs and control gene expression and may exert both oncogenic and tumour suppressor functions in cancers. The deregulation of miRNAs in soft tissue sarcomas may be exploited in the development of miRNA-based strategies for the prognostication of disease outcomes, identification of treatment resistance and new-generation therapeutics.Soft tissue sarcomas are highly aggressive malignant neoplasms of mesenchymal origin, accounting for less than 1% of adult cancers, but comprising over 20% of paediatric solid tumours. In locally advanced, unresectable, or metastatic disease, outcomes from even the first line of systemic treatment are invariably poor. MicroRNAs (miRNAs), which are short non-coding RNA molecules, target and modulate multiple dysregulated target genes and/or signalling pathways within cancer cells. Accordingly, miRNAs demonstrate great promise for their utility in diagnosing, prognosticating and improving treatment for soft tissue sarcomas. This review aims to provide an updated discussion on the known roles of specific miRNAs in the pathogenesis of sarcomas, and their potential use in prognosticating outcomes and prediction of therapeutic resistance. Sarcomas are malignant neoplasms of mesenchymal origin with over 70 histologic subtypes and may be broadly divided into two categories: soft tissue sarcomas  and bone sarcomas  1]. The. The1]. 0 resections. Radiotherapy and chemotherapy are typically reserved for advanced disease; radiotherapy is usually provided in high-risk tumours that are large, deep and/or high grade [Traditionally, soft tissue sarcomas are managed by wide excisional surgery for localized disease. Surgery may also be used as a palliative procedure in metastatic disease . With thgh grade , while agh grade . While agh grade , a largegh grade . Increasgh grade . The medgh grade ,12,13. Egh grade ,14, thusMicroRNAs (miRNAs) are short non-coding RNAs of 19–25 nucleotides that regulate post-transcriptional gene expression. Mature miRNAs bind to complementary sites on target mRNAs, usually at the 3′ UTR, thereby suppressing mRNA translation or causing degradation of the mRNA transcript . BecauseThe role of miRNAs in the diagnosis of soft tissue sarcomas has been proposed and discussed elsewhere ,22; howemiRNAs mediate soft tissue sarcoma progression by influencing various pathogenic processes, thereby acting as oncogenes or tumour suppressors. As the clinical behaviours underlying pathogenesis and management of soft tissue sarcomas differ between subtypes, the following discussion will review the roles of relevant miRNAs within each soft tissue sarcoma subtype. Gastrointestinal stromal tumour (GIST) is the most common mesenchymal tumour specific to the gastrointestinal tract and is commonly characterized by activating mutations in the KIT or PDGFRA receptor tyrosine kinases . KIT is Downregulation of miR-494 has been observed in GIST cell lines, and miR-494 overexpression in GIST cells triggered apoptosis and inhibited cell growth ,24. miR-Other miRNAs that are downregulated in GIST include miR-17, miR-20a, miR-4510, miR-133b, miR-152, miR-518-5p and miR-137 ,32,33,34On the contrary, oncogenic miRNAs promote the development of GIST. miR-374b was highly expressed in GIST tissues, and its expression increased the mRNA and protein levels of various molecules in the PI3K/AKT cell survival pathway in GIST cells . miR-374Liposarcoma is one of the most common soft tissue sarcomas, and may be classified into four subtypes based on its pathological and molecular genetic characteristics: pleomorphic (PLPS), myxoid/round cell (MLPS/RLPS), dedifferentiated (DDLPS) and well-differentiated (WDLPS). The round cell component in MLPS/RLPS is thought to be associated with metastasis and poorer prognosis . The regmiR-155 is known to act as an oncogene in multiple malignant tumours . It has Other miRNAs which are overexpressed in LPS and play a role in invasion and metastasis include miR-135b, miR-25-3p and miR-92a-3p ,52,53. mOn the contrary, certain miRNAs have been found to inhibit the progression of LPS through suppression of proliferation and induction of apoptosis. miR-143 is downregulated in both WDLPS and DDLPS tumours and cell lines ,39. RestSome miRNAs may also regulate LPS progression by interfering with tumour cell metabolism and introducing oxidative stress. miR-133a, significantly underexpressed in DDLPS tissues, has been found to modulate DDLPS cell metabolism, with enforced expression of miR-133a resulting in decreased glycolysis and increased oxidative phosphorylation. This was coupled with impaired cell proliferation and cell cycle progression . miR-193Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in paediatric patients and young adults. RMS can be classified into two major histological subtypes: embryonal rhabdomyosarcoma (ERMS) and alveolar rhabdomyosarcoma (ARMS). RMS may be further classified based on clinical outcome into fusion-positive RMS or fusion-negative RMS based on the presence or absence of either PAX3-FOXO1 or PAX7-FOXO1 gene fusions. As these gene fusions are absent in ERMS, ERMS patients are all fusion-negative, while majority of ARMS patients are fusion positive ,118,119.In recent studies, miRNAs which have been identified to play a role in skeletal muscle proliferation and differentiation such as miR-1, miR-133, miR-206 and miR-29 ,124, havAnother notable miRNA in RMS regulation is miR-1 ,55,63. BSeveral miRNAs share similar targets in the regulation of RMS. miR-29 is a key miRNA that is epigenetically silenced in RMS tissues and cell lines ,64,65. IOther miRNAs which are downregulated in RMS cell lines and tissue samples have also demonstrated anti-tumour effects in RMS by inducing apoptosis and myogenic differentiation, as well as impairing cell proliferation, invasion and metastasis. In miR-7 transfected RMS cells, miR-7 acts via its target mitochondrial proteins solute carrier family 25 member 37 (SLC25A37) and translocase of inner mitochondrial membrane 50 (TIMM50) to promote apoptosis and necroptosis , and alsIn contrast, miR-27a and miR-486-5p were discovered to be upregulated in the more aggressive fusion-positive RMS samples and cell lines ,79. miR-Malignant peripheral nerve sheath tumour (MPNST) is an aggressive soft tissue sarcoma arising from peripheral nerves or deep neurofibromas. It has a poor prognosis due to its propensity for metastasis and local recurrence. MPNSTs may occur sporadically, but around half of MPNST cases arise in patients with the autosomal dominant genetic disorder neurofibromatosis type 1 (NF1) ,131. MosmiR-204 was found to be downregulated in both NF1 and non-NF1 MPNST tissues and cell lines . RestoraLikewise, certain miRNAs in MPNSTs are upregulated. miR-21 expression levels in MPNST clinical samples were significantly higher compared to NF samples , congrueLeiomyosarcomas (LMS) are highly aggressive malignancies of smooth muscle tissues which account for approximately 10% of all STS . UterineThere are statistically significant differences in the expression of multiple miRNAs between LMS and smooth muscle samples , endometSynovial sarcoma is a high-grade mesenchymal neoplasm that accounts for 10% to 20% of all soft tissue sarcomas in adolescents and young adults ,142. Downregulation of miR-494-3p and miR-126 was discovered in synovial sarcoma tumours ,93. Re-eLikewise, oncogenic miRNAs involved in the potentiation of synovial sarcoma were also reported. Overexpression of let-7e microRNA and miR-99b in synovial sarcoma were found, and the downregulation of the two miRNAs using miRNA inhibitors resulted in the suppression of cell proliferation, accompanied by an increased expression of their putative targets, high mobility group (HMGA2) and SMARCA5 , both ofFibrosarcomas are defined as a malignant neoplasm composed of fibroblasts with variable collagen production . miR-197Angiosarcomas are vascular sarcomas of endothelial cell origin . Three mRecent studies have also reported the correlations between miRNA expression and metastatic risk, tumour grade, overall survival and recurrence-free survival, indicating the possible utility of miRNA-guided prognostication of soft tissue sarcomas. A summary of the miRNAs involved in the prognostication of soft tissue sarcomas is found in In GIST, smaller tumour size and a lower mitotic rate correspond with lower metastatic risk . NegativKaplan–Meier survival analysis revealed that overexpression of miR-26a-2 was significantly correlated with poor patient survival in WDLPS, DDLPS and MLPS . The expIn RMS, certain miRNAs are differentially expressed between fusion-negative RMS and fusion-positive RMS (RMS with either PAX3-FOXO1 or PAX7-FOXO1 fusion oncogenes) and therefore point toward varied clinical outcomes. Low miR-206 expression was correlated with poor overall survival and was an independent predictor of shorter survival in metastatic ERMS and fusion-negative ARMS. Low miR-206 expression also significantly correlated with high SIOP stage and the presence of metastases at diagnosis . Lower lIn LMS, miR-181b-5p was associated with recurrence-free survival, and high miR-181b levels were found to be an independent predictor of recurrence-free survival regardless of LMS subtype and tumour size . FurtherIn metastatic tumour samples of synovial sarcoma, downregulation of miR-494-3p and increased expression of its potential target CXCR4 were more pronounced than in non-metastatic tumours and healthy tissues . High exmiRNAs have also been shown to play a role in influencing the resistance of soft tissue sarcomas to various forms of treatment . ReversaImatinib, a tyrosine kinase inhibitor, works by inhibiting KIT activation, thereby blocking the activation of the downstream MAP kinase and PI3K-AKT cell survival pathways. Due to the prevalence of GISTs harbouring activating mutations in KIT, clinical management of metastatic or recurrent GIST usually involves the use of a tyrosine kinase inhibitor such as imatinib mesylate . HowevermiR-218 expression was found to be decreased in imatinib-resistant cell lines, but subsequent ectopic overexpression of miR-218 in imatinib-resistant cells under the effect of imatinib mesylate resulted in significantly decreased cell viability and increased apoptosis. It was further suggested that the PI3K/AKT signalling pathways could play a role in this mechanism . SimilarIn contrast, overexpression of miR-125a-5p and miR-107 was associated with imatinib resistance in GIST specimens. It was further shown that expression of the miR-125a-5p target PTPN18 was suppressed in imatinib-resistant GIST samples, and that the silencing of PTPN18 expression increased cell viability in GIST882 cells with a homozygous KIT mutation subsequent to imatinib treatment. However, miR-125a-5p expression did not modulate imatinib sensitivity in GIST48 cells with double KIT mutations . It was The use of miRNAs as potential biomarkers of imatinib resistance in GIST was studied by Kou et al. who found that serum miR-518e-5p could discriminate imatinib-resistant GIST patients from healthy controls and imatinib-sensitive GIST patients . This coIn ARMS, the PAX3-FOXO1 fusion oncogene regulates chemotherapy and radiotherapy tolerance . RepressHigher levels of MELK were associated with doxorubicin chemoresistance in ULMS cells. MELK overexpression in ULMS could induce M2 macrophage polarization via the miR-34a/JAK2/STAT3 pathway, contributing to doxorubicin chemoresistance in the tumour microenvironment . In synoA summary of the miRNAs implicated in treatment resistance in soft tissue sarcomas may be found in The exciting field of miRNA research has seen miRNA-based technology entering pre-clinical and clinical settings as diagnostic and therapeutic tools for various diseases in recent years. At present, several miRNA-targeted therapeutics for cancer have reached clinical development, including the use of an miR-34 mimic (MRX34) encapsulated in lipid nanoparticles for the treatment of multiple solid tumours and an miR-16 mimic for the treatment of lung cancer . HoweverIn this review, three key areas in which miRNAs may be utilised in the management of soft tissue sarcomas have been discussed: (i) prognostication, (ii) prediction of treatment resistance, and (iii) therapeutics. The use of miRNAs in the prognostication of soft tissue sarcomas could guide clinical management by identifying patients who have higher metastatic risk and thus require closer surveillance or a lower threshold for adjuvant treatment. Studying miRNA expression in patient serum could also serve as a biomarker for the earlier detection of disease relapse. Distinguishing which cancers are more amenable to treatment options based on their miRNA signature could also increase the overall efficacy of soft tissue sarcoma therapy. Furthermore, the prediction of treatment resistance to specific agents can potentially guide systemic treatment choices in advanced soft tissue sarcomas. Finally, miRNA-based therapies offer an appealing approach to cancer treatment because a single miRNA can regulate multiple target genes and/or signalling pathways within cancer cells. While few miRNA-based strategies have reached clinical routine, continuous advancements in this field offer great promise for their utility in diagnosing, prognosticating and improving treatment outcomes for soft tissue sarcomas.


Despite arduous and time-consuming experimental efforts, protein–protein interactions (PPIs) for many pathogenic microbes with their human host are still unknown, limiting our understanding of the intricate interactions during infection and the identification of therapeutic targets. Since computational tools offer a promising alternative, we developed an R/Bioconductor package, HPiP (Host–Pathogen Interaction Prediction) software with a series of amino acid sequence property descriptors and an ensemble machine learning classifiers to predict the yet unmapped interactions between pathogen and host proteins.Mycobacterium tuberculosis and human proteins. Collectively, our fully documented HPiP software will hasten the exploration of PPIs for a systems-level understanding of many understudied pathogens and uncover molecular targets for repurposing existing drugs.Using severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) or the novel SARS-CoV-2 coronavirus-human PPI training sets as a case study, we show that HPiP achieves a good performance with PPI predictions between SARS-CoV-2 and human proteins, which we confirmed experimentally in human monocyte THP-1 cells, and with several quality control metrics. HPiP also exhibited strong performance in accurately predicting the previously reported PPIs when tested against the sequences of pathogenic bacteria, https://github.com/BabuLab-UofR/HPiP) as well as on Bioconductor (http://bioconductor.org/packages/devel/bioc/html/HPiP.html).HPiP is released as an open-source code under the MIT license that is freely available on GitHub (Bioinformatics Advances online. Changes in the patterns of microbial infections, as well as the emergence of multidrug-resistant strains (superbugs) among pathogens, have deteriorated the efficacy of existing therapeutics and limited current treatment options . This nede novo PPIs based on the primary sequences of proteins. Second, continuously running iterations by sequence-based computational methods can become cumbersome due to sizeable run time required to train a scoring model, and when handling large number of sequences. Third, while HP-PPI predictions using sequence-based methods are available through a few web server platforms, such as PredHPI, they lack programming interfaces and are not designed to analyze a sizeable number of sequences in a single batch. Also, in such web applications, it is often unclear the type of reference interactions used to evaluate HP-PPI predictions, especially when PPIs are unavailable for many host–pathogen systems.While querying protein pairs using 3D structures is limited by low proteome coverage, computational approaches using protein sequence information are more amenable to investigating PPI networks due to the advancements in next-generation sequencing data of pathogens in recent years. However, there are a few shortcomings. First, only a small selection of physicochemical descriptors such as amino acid composition (i.e. di- or tri-peptide), as well as k-Spaced amino acid pairs and Conjoint Triad have been considered, but combining these with other descriptors can detect Conversely, progress in machine learning (ML)-based methods using supervised learning algorithms such as Support Vector Machine (SVM), Random Forest (RF) and Logistic Regression (LR) is emerging as an alternate approach to predict PPIs based on physicochemical properties of amino acid sequences, and discern interacting from non-interacting protein pairs using known interactors as a training set. Nevertheless, most studies reported only the results of cross-validation (CV), with no reference to the evaluation of test prediction results on a new set of data using rigorous experimental or external validations. While these issues may challenge the interpretability of prediction performance in deciphering genuine interactions, with an ensemble ML methods and experimental evaluation, prediction accuracy and coverage of PPIs can be enhanced. However, current ML approaches that predict HP-PPIs are not automated, and therefore end-user must obtain suitable training or test datasets, and needs in-depth ML knowledge.Mycobacterium tuberculosis and human protein sequences. Source code, standalone executable programs, usage instructions, illustrative dataset and output for testing are provided at https://github.com/BabuLab-UofR/HPiP and in In this study, we address the aforesaid gaps by introducing an R/Bioconductor software package, HPiP (Host–Pathogen Interaction Prediction), built on an ensemble approach with three  different ML classifiers, and a pipeline to either automatically or manually upload the training dataset, and predict PPIs based on more than a dozen amino acid sequence property descriptors of pathogen and host proteins. As a case study, using SARS-CoV-1 or the SARS-CoV-2-human PPI training set, HPiP achieved a good performance with PPI prediction between SARS-CoV-2 and human proteins using ensemble ML method. Besides verifying PPIs with 10-fold CV, published datasets and quality control metrics, we experimentally confirmed the predicted PPIs in human THP-1 monocytes by affinity purification-mass spectrometry (AP/MS). Similar high performance on the PPI prediction by HPiP was achieved when tested against the bacterial pathogen, k-fold CV, but other ML classifiers can be substituted with parameters supported by the caret package (https://github.com/topepo/caret/). The best parameters are defined based on the model prediction, while a model’s predictive performance was evaluated using precision versus recall, or sensitivity versus 1-specificity plots. In the final Step 5, either individual or ensemble (i.e. average of predictions) of tuned ML classifiers is subjected to the test set for scoring putative HP-PPIs. The resulting interactions are split into complex membership using fast greedy, walk-trap, label propagation, multilevel community and Markov clustering algorithms that are available in HPiP package, though users can define complexes using other clustering methods such as ClusterONE via an open-source Cytoscape platform. HPiP then uses scored PPIs or predicted complexes to perform functional enrichment using Gene Ontology  or KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway terms to define bioprocesses involving host-dependency factor for pathogens.The HPiP software is composed of a five-step automated computational workflow  to prediTo illustrate our approach, we optimized HPiP performance to predict SARS-CoV-2-human host PPIs in two ways. In the first method (referred as ‘prediction 1’), 1562 interactions were gathered for training from the closely related SARS-CoV-1 coronavirus with human host proteins .Evaluation of sequence descriptors by 10-fold CV in training showed a robust performance of 0.76 area under the receiver operating characteristic curve (auROC) can be achieved when all the descriptors were combined compared to 0.75 average training accuracy for each descriptor . AssessmP-value ≤ 10−3) by random sampling. These filtered associations tend to decrease gradually as ensemble classifier score was elevated  84elevated . Confirmelevated  such as elevated , implyinOne-fifth  of the remaining interacting proteins colocalized to the same cellular compartment , while 1 or SARS-CoV-2 (prediction 2)vbac038_Supplementary_DataClick here for additional data file.


Aging is associated with an increased incidence of co-morbidities, including detrusor underactivity (DU). DU is defined as the failure to create sufficient and durable expulsive force to adequately empty the urinary bladder during a normal voiding timespan. DU is prevalent in older adults, as evidenced by its prevalence in nearly two-thirds of nursing home residents. Current treatments are mostly palliative or come with many side effects. β-adrenoceptor-mediated relaxation is the primary mechanism of detrusor relaxation, and Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels have previously been identified by us and others as a very important mediator of this relaxation, however the role of HCN in detrusor relaxation has not been elucidated. Hence, we seek to characterize its role in adrenergic relaxation mechanisms and spontaneous myocyte activity. Male and female 10–12-month-old C57Bl/6 mice were used for this study. Pharmacomyography studies were performed to assess the effect of different drugs that act at various steps along the adrenergic relaxation pathway, +/- CsCl, an HCN blockade at [5mM]. As expected, we saw that increasing HCN opening probability by isoproterenol or forskolin (adenylyl cyclase/cAMP-agonist) or lamotrigine (HCN-activator) resulted in decreases in tonic tension, but were diminished in the presence of CsCl. Mechanisms modulated by H89 (PKA-inhibitor) and NS1619 (BK-channel-agonist) show no change in tonic tension, however spontaneous phasic activity significantly increases. These data support increased cAMP, not hyperpolarization, as the key inductor of HCN in adrenergic relaxation.


This case highlights a 77-year-old woman with polymyalgia rheumatica (PMR) following coronavirus disease 2019 (COVID-19) vaccination without shoulder pain and its characteristic positron emission tomography-computed tomography (PET-CT) findings. Some cases of PMR following COVID-19 vaccination have been reported. However, patients without shoulder pain or PET-CT findings without uptake at the shoulders are not described. Our case suggests that PMR should be considered in the differential diagnosis of patients with neck pain following COVID-19 vaccination, even if they lack shoulder pain. Polymyalgia rheumatica (PMR) is a common inflammatory rheumatic disorder that affects older adults. It is characterized by pain and stiffness of the shoulders and pelvic girdle . AlthougA 77-year-old Japanese woman with no past medical history presented with a four-week history of neck pain. Two days after receiving the second COVID-19 mRNA vaccine [/BioNTech ], she developed myalgia at the posterior and lateral neck, which worsened in the morning. Her symptoms persisted for four weeks; low-grade fever and tiredness in the thighs accompanied them; however, she did not have shoulder pain. Physical examination revealed tenderness at the cervical spinous processes, the sternocleidomastoid muscles, and the greater trochanters, not at the shoulders or hips. The range of motion of the shoulders was not limited. The painful arc test was negative. Muscle strengths were normal. Laboratory tests showed a C-reactive protein (CRP) level of 4.32 mg/dL, an erythrocyte sedimentation rate (ESR) of 100 mm/h, and a normal level of creatine kinase. The test results for rheumatoid factor, anti-cyclic citrullinated peptide antibodies, and the other autoantibodies related to myopathy were negative. The blood culture did not grow any bacteria. Ultrasonography of the shoulders detected no abnormal findings. Positron emission tomography-computed tomography (PET-CT) revealed increased 18F-fluorodeoxyglucose (FDG) uptake at the cervical and lumbar interspinous bursae, ischial tuberosities, trochanteric areas, and hips, but not at the shoulders , which recognizes viral single-strand RNA, and potential saturation of TLR7 signaling have been observed in the peripheral B cells, T cells, and monocytes of patients with PMR . A recenThis case demonstrates that PMR lacking shoulder pain developed following COVID-19 vaccination. PMR should be considered in the differential diagnosis of patients with neck pain and raised markers of inflammation following COVID-19 vaccination, even if they lack shoulder pain. As this is a rare adverse event of the COVID-19 vaccine and COVID-19 poses a much greater risk than PMR, the vaccine’s benefits should not be dismissed.


In all known geneticpolymers, molecular recognitionvia hydrogenbonding between complementary subunits underpins their ability toencode and transmit information, to form sequence-defined duplexes,and to fold into catalytically active forms. Reversible covalent interactionsbetween complementary subunits provide a different way to encode information,and potentially function, in sequence-defined oligomers. Here, weexamine six oligoarylacetylene trimers composed of aniline and benzaldehydesubunits. Four of these trimers self-pair to form two-rung duplexstructures, and two form macrocyclic 1,3-folded structures. The equilibriumproportions of these structures can be driven to favor each of theobserved structures almost entirely depending upon the concentrationof trimers and an acid catalyst. Quenching the acidic trimer solutionswith an organic base kinetically traps all species such that theycan be isolated and characterized. Mixtures of complementary trimersform exclusively sequence-specific 3-rung duplexes. Our results suggestthat reversible covalent bonds could in principle guide the formationof more complex folded conformations of longer oligomers. Nucleic acid polymers play a central rolein the biochemistry ofall known life. Their structures underlie the mechanism by which evolutionsculpts macromolecular catalysts within a vast sequence space. Arguably,the essential feature of these polymers is the molecular recognitionbetween complementary nucleobase subunits that enables base-pairing.It is base-pairing that allows for the transfer of information throughtemplated polymerization and the folding of nucleic acid polymersinto catalytically active structures.1 In support of this, both information transfer2 and the assembly of catalytically active ribozymes havebeen demonstrated in nonenzymatic RNA systems.3 However, many alternative RNA-like polymers, collectively referredto as XNAs, are also capable of both information transfer and function.4 Thus, at least some structural variation canbe tolerated while maintaining these core properties. We are interestedin whether synthetic sequence-defined polymers with fundamentallydifferent molecular designs might also exhibit the properties of informationtransfer and function.The RNA-world hypothesissuggests that RNA innately possesses themeans to initiate the transition from chemistry to biology withoutthe complex biomachinery of extant biology.5 informationstoring sequence-defined polymers can now be made to considerablelengths,6 polymers that mimic the foldingbehavior of enzymes and ribozymes are built with ever-increasing sophistication,7 and many synthetic sequence-dependent duplexforming structures have been reported.8To fully mirror the evolutionary behaviorof nucleotide polymers,synthetic polymers must be able to store and propagate information,and either directly through folding  or indirectlythrough translation , the information they store mustencode structures with varying fitness. For a synthetic polymer, thisrequires overcoming challenges in building monodisperse sequence-definedpolymers that self-assemble into chemically active configurations and that can undergo template-directed polymerization.Compounding these challenges, the presence of the complementary recognitionsubunits required for templated polymerization means that geneticpolymers will contain mutually reactive subunits with the potentialto self-associate and fold in a multitude of ways. Despite these challenges,significant strides have been made in recent decades in the synthesisof polymers that mimic biomolecules:9 and the dynamictemplated self-assembly of helices by Nitschke and co-workers.10 In contrast to the chemistry developed herein,these systems do not utilize molecules that store sequence information.As such, they do not possess an obvious mechanism for the transferof information like the nonenzymatic-templated polymerization observedfor nucleobase polymers.Reversible covalent bonds have been remarkably useful in producinga range of life-like complex behaviors in synthetic systems. Two notableexamples are the self-replication of disulfide-bound macrocycles reportedby Otto and co-workers11 To our knowledge, this is one of the two examplesof sequence information transfer using reversible covalent bonds.12 To expand our system to longer oligomers, wethen developed a solid-phase synthesis of sequence-defined anilinebenzaldehyde oligoarylacetylenes (ABOs).13 During that work, it became apparent that controlling the interactionsof the reactive aniline A and benzaldehyde B subunits was essential to increasing the complexity of these systems.Recently, we reported that reversiblecovalent imine bonds cantemplate sequence information transfer in macrocyclic dimers.AAB (ss-AAB) formed a two-rungduplex with unpaired sticky ends (ds-AAB) and folded into a macrocycle with an unpaired internal monomer(fold-AAB).To that end, we have been exploring sequence-defined ABO trimers1. ABOs a8j but, to the best of our knowledge, these interactions have not beendescribed in reversible covalent chemistry systems with sequence-definedoligomers. Aniline benzaldehyde oligomers with a peptoid backbonehave been shown to self-sort into sequence-encoded polyimine duplexesin peptoid pentamers by the Scott laboratory.8i While self-association and folding behavior were not the focus oftheir report, the authors mention that they suspect some amount offolding likely occurred under the conditions they studied.Self-association and folding behavior have been exploredextensivelyin synthetic sequence-defined oligomers with noncovalent H-bondingrecognition subunits,14Asimine bonds are only conditionally reversible, they offer someadvantages over H-bonding systems; their covalent base-paring interactionsdo not rely on association constants but can be driven to completionby the removal of water; furthermore, aromatic imine bonds do nottypically equilibrate if they are protected from catalysts, water,and harsh conditions.AAB is the same as BAA).Notably,these molecules differ from biopolymers and many syntheticinformational polymers in that they lack a directional polarity. Thisgreatly simplifies their dynamic behavior as duplexes do not haveparallel or antiparallel geometries. Likewise, the lack of directionalityrestricts the size of the sequence space due to symmetry considerations as an imine bond-forming acid catalyst. The results of theseexperiments are presented in the next section.As they are homopolymers, ABA and BAB, we foundthat formation of the partially double-stranded duplex structures ds-ABA and ds-BAB .For theisolated ABO trimers d ds-BAB 2 was str1H NMRspectrum of ss-ABA (3.5 mM in CDCl3) is shownin ds-ABA appeared, ds-ABA is favored over ss-ABA by 10 > 1. Reactions run at higherconcentrationsof ABA (7.5 and 15 mM) gave a similar ratio of ds- to ss-ABA. At 15 mM, weobserved a small amount of an uncharacterized species  which may represent a higher ordermultiplex structure.The aromatic region of the Figure S4). We suspected that protonation of the basic imine and aniline siteson ds-ABA promotes the aggregation ofthese molecules and is responsible for the peak broadening and chemicalshift changes.16 Accordingly, an NMR spectrumof ds-ABA with sharp peaks was recoveredby quenching the TFA with a 3-fold excess of triethylamine (TEA).Basified solutions were kinetically stable and could be washed withwater, concentrated to dryness, or purified on silica gel withoutany apparent imine hydrolysis or the formation of higher order multiplexes.Conversely, if solutions of ds-ABA inthe presence of TFA were not quenched with TEA, adding water led toimmediate hydrolysis to ss-ABA  in dry CDCl3 wasfirst treated with TFA (2 mM), concentrated to dryness to remove water,and then redissolved in dry CDCl3 to the same concentration(3.5 mM) gave complete conversion to ds-ABA. Upon quenching with TEA, this procedure gave pure kinetically stable ds-ABA, which could be isolated and characterized and a spin system witha singlet, doublet, doublet, triplet pattern (consistent with A). In the spectrum, there is another upfield shifted spinsystem consistent with A, which does not have ROSEY correlationsto the imine protons but instead correlates to an upfield resonanceconsistent with NH2 .For with NH2 3. Taken AAB and ABB, proved the most complicated andinteresting.Like BAB and ABA, these trimers can self-associateto form two-rung duplexes with unpaired sicky ends. In addition, sincethe terminal subunits of the single-stranded trimers are complementary,they can fold into macrocyclic structures with the internal monomerunpaired  andnearly identical to the strain incurred in forming the double-strandedstructures . In contrast, minimizationof the 1,2-folded AAB structure shows significant strainon folding , and 1,2-folding wasnot observed for any sequence in this study.While these folded structureswere initially a surprise, simpleMM2 energy minimizations show that the steric stain incurred by this1,3-folding is relatively low . The ratioof folded to double-stranded structures was determined from the relativeintegral of the imine protons of each species. As expected, lowertrimer concentrations favor the folded structures and higher concentrationsfavor the double-stranded structures . Thedifferent equilibria may reflect this difference in acid sensitivity.We expectedthat the equilibrium ratio of ructures 4. At theABA and BAB trimers,quenching solutions of ABB and AAB withan organic base gave kinetically stable solutions, such that the ds- and fold-trimers could be isolatedwithout re-equilibrating. Using Figure S18) and 2D NMR experiments  provided strong evidencefor the structural assignments of all four structures ds-AAB, fold-AAB, ds-ABB, and fold-ABB. As described above for ds-ABA, theanalysis uses 1H–1H ROSEY NMR to identifymonomers that are paired through imine bonds. The splitting patternsof those resonances and 1H–1H COSY NMRwere then used to assign those monomers to internal or terminal positions.For all ds- and fold-ABOs, we observedstrong and uniform 1H–1H ROSEY correlationsbetween the imine proton and all four protons ortho to the imine bond. This suggests that rotation around the iminebond is relatively unhindered in these structures; if a single iminerotamer was strongly favored, only two of the four protons would belikely to be in a range to produce a strong ROE.As was the case with the 1H NMR resonances of fold-ABB as theconcentration was lowered. We suspected that this might be the resultof increasing acidity at lower trimer concentrations as there is lesstrimer to buffer the acidity.While investigatingthe concentration dependence described above,we observed a surprisingly dramatic shift in the downfield 1H NMR spectrum as TFA was titrated intosolutions of ABB. With increasing acidity, we observeddramatic changes in the chemical shift of all resonances and a shiftin the ds to fold equilibrium toentirely fold-ABB. 1H NMRspectra of a solution of ABB (0.55 mM in CDCl3) as TFA was added to the solution ([TFA] 2 to 20 mM)). The largedownfield shift in the imine resonance of fold-ABB , followed by quenching with a 3-foldexcess TEA .Quenching acidic solutionsof the acid 5, bottom18 we investigated the potentialrole of the trifluoroacetate counterion in the formation and aggregationof fold-ABB and ds-ABB. Titration experiments with tetraethylammonium trifluoroacetateshowed no change in the 1H NMR spectra of fold-ABB  or ds-ABB . This strongly suggests that trifluoroacetate does not bind tothe unprotonated molecules in chloroform. Similarly, a titration withtetraethylammonium trifluoroacetate preformed in the presence of TFAshowed that additional trifluoroacetate merely acts as a general base,and the results mirror the TFA quenching observed with triethylamine. Notably, the pKa of TFA in acetonitrile is reported to be 12.65,19 so the ability of trifluoroacetate to functionas a competent base in organic solvent is not surprising. Taken together,these titration experiments suggest that protonation is the drivingforce for the observed behaviors and that trifluoroacetate is a passivebystander.Becausemany macrocycles have been shown to tightly bind negativelycharged ions,ds- to fold-AAB is similarly sensitiveto the TFA concentration,we repeated the titration experiment with AAB. Surprisingly,we found the opposite result as with ABB. At higher concentrationsof TFA, ds-AAB is favored almost completelyover fold-AAB .In contrast to ABB, dramatic chemical shift changes werenot observed in these spectra, and only a peak broadening and lossof the ds-ABB signal were observed inthe NMR spectrum as the TFA concentration was increased. Sharp peakscould be recovered by quenching with TEA. We suggest that acid-mediatedaggregation is also the driving force for this change in equilibriumwith the ds-AAB aggregate being favoredover fold-AAB.To see if the equilibrium of ss-AAA and ss-BBB to form ds-AAA·BBB would be the moststraightforward as these sequences do not have competing self-associationor folding behavior, and duplexes of similar polymers have been reportedby Moore and co-workers.20 However, experimentswhere an equimolar mixture of ss-AAA (480 uM) and ss-BBB (480 uM) in CDCl3 was treated with TFA (1 mM) overnight gave NMR spectra withbroad uninterpretable resonances, and quenching the TFA with an excessof TEA only had a small effect on sharpening the peaks . We found that both dilution andheat caused a downfield shift and a sharpening of the NMR resonances,consistent with concentration-dependent aggregation through π-stacking.17a The NMR spectra indicate a nearly completeconversion to ds-AAA·BBB; no aldehyde peaks are present nor any upfield peaks from unpairedaniline subunits, and two imine peaks are present in a 1:2 ratio consistentwith the three imine protons expected for ds-AAA·BBB. MALDI-TOF HRMS also supports theexclusive formation of the 3-rung duplexes .Suspecting that thepeak broadening might bethe result of aggregatesformed through π-stacking interactions of the 3-rung duplexes,we acquired spectra over a dilution series at rt 7 and 50 ss-ABA with ss-BAB and ss-AAB with ss-ABB . Remarkably, conversionto the 3-rung duplexes did not require the starting materials to bein their single-stranded states. A mixture of ds-ABA, ss-ABA, ds-BAB, and ss-BAB gavecomplete conversion to ds-ABA·BAB under the imine formation conditions, and a mixture of ds-AAB, ss-AAB, fold-AAB, ds-ABB, ss-ABB, and fold-ABB gave complete conversion to ds-AAB·BBA. This strongly suggests thatimine exchange reactions occur quickly in the presence of TFA andthe 3-rung products are thermodynamically favored to the exclusionof the 2-rung partial duplexes and folded structures. The 1H NMR spectrum for ds-ABA·BAB shows a small amount (approx. 10% by integration) of broadpeaks of unknown species. It is possible these are the result of higherorder polymers; however, the MADLI-TOF MS data only shows peaks consistentwith the 3-rung duplexes.For the remaining two mixtures of complementarysequences, h ss-ABB 2 and 3. K model of indefiniteassociation.211H resonances of ds-AAB·ABB acquired in CDCl3 at rt. Curves for all three duplexesacquired at rt and 50 °C can be found in the Supporting Information. From the similarities in Ke . Interestingly, under higher [TFA](20 mM), the NMR shows a minor product consistent with barbell-AAB·BBB; both fold-AAB and fold-ABB havea downfield shifted aromatic proton at 8.3 ppm which is not presentin the ladder or partial ladder spectra. It is likely that these downfieldshifted resonances would be present in barbell-AAB·BBA as we observe under high acid conditions(20 mM TFA). Unfortunately, our efforts to further characterize thisminor product have been unsuccessful, potentially due to the easeof hydrolysis of the linking imine bond.Given the propensity for -AAB·BBA 5, whose Understandingand controlling the interactionsof complementaryrecognition units within and between defined sequences are an essentialstep toward building synthetic polymers that will mimic the propertiesof genetic polymers. Our work shows that for ABO trimers, not onlyare both double-stranded and folded structures accessible, but theirequilibrium can also be modulated by controlling their sequence andenvironment, and that mixtures of sequence complementary trimers formsequence paired duplexes.ds and fold structures that we observed in the limitedABO trimer sequence space,four could be generated nearly quantitatively without the need forpurification, while the remaining two could be formed in good yieldby controlling the concentration of TFA and ABO and then purifiedto give pure compounds (Of the six higher order ompounds 2.fold-AAB and fold-ABB). This behavior has important implications for longer oligomers.While covalent base pairs are likely to make longer oligomers moresusceptible to inescapable kinetic traps,22 they may also confer the ability to exhibit useful and diverse foldingbehavior in much shorter systems.The aciddependence of imine equilibrium reactionsis a usefulfeature of this system, allowing simple quenching with TEA to kineticallytrap all species. No good analogy to this exists for short oligonucleotidesor synthetic oligomers that use H-bonding recognition subunits; solution-phaseequilibria are always likely to reassert themselves until oligomersare sufficiently long to become kinetically metastable. In contrast,for reversible covalent imine bonds, a single base pair is sufficientto form a kinetically stable and isolable structure (e.g., AAB and BBA trimers, the formationof partial two-rung duplexes and 1,3-folded structures did not impedethe formation of 3-rung duplexes in the presence of the fully complementarystrand. This sequence-specific self-sorting matches the behavior ofshort polyimine peptoids;8i however, itdiffers from nucleotide polymers of a similar length as a three-base-pairRNA duplex is highly unstable.For the Ultimately, the utility of longeroligomers will depend on howeasily their many competing interactions can be controlled. The methodsdescribed herein offer a necessary level of control and synthetictractability in mixed sequence oligomers that use reversible covalentbonds as recognition subunits. Further elucidating the behaviors ofoligomers in this family of molecules is likely to offer insightsinto the chemical space of genetic polymers beyond the nucleic acidsand provide access to novel synthetic polymers that mimic the behaviorsof biopolymers.


Background. Pericardial tamponade is a known life-threatening condition rarely reported in COVID-19 but has not been reported following asymptomatic SARS-CoV-2 infection. Its pathomechanism is still elusive. Case Summary. We report the case of a 66-year-old man with progressive shortness of breath and leg swelling due to new-onset heart failure and pericardial tamponade following asymptomatic SARS-CoV-2 infection. Ultrasound-guided placement of a pericardial drainage led to significant improvement of symptoms and revealed an exudative effusion. Throughout the diagnostic process, we were confronted with a systemic inflammatory syndrome suspicion of an induced autoimmune condition. After steroid pulse therapy and oral anticoagulation for subclavian vein thrombosis, the patient was discharged and followed in our outpatient clinic. Discussion. Patients with asymptomatic SARS-CoV-2 infection are at risk for developing life-threatening complications. Induced autoimmune conditions could be a potential explanation for late-onset pericardial tamponade in this population. A multimodal imaging approach is crucial in the diagnosis and characterization of cardiac inflammation. An interdisciplinary approach is essential. Awareness of uncommon cardiac complications following a SARS-CoV-2 infection is crucial for the initial assessment and the appropriate treatment of these patients. Pericardial tamponade is a known life-threatening condition rarely reported in COVID-19 but has not been reported following asymptomatic SARS-CoV-2 infection. We report the case of a 66-year-old man with myopericarditis, pericardial tamponade, and polyserositis within five weeks after asymptomatic infection with SARS-CoV-2. Throughout the diagnostic process, we were confronted with a systemic inflammatory syndrome suspicious of an induced autoimmune condition. This report highlights a dedicated diagnostic workup by multimodal cross-sectional imaging studies. Induced autoimmune conditions could be a potential explanation for late-onset pericardial tamponade in this population.A 66-year-old man without any known comorbidities or allergies presented to our emergency department with ten-day progressive shortness of breath without chest pain, abdominal distension, decreased effort tolerance, and 12 kg weight gain within the last ten days. The first assessment showed an afebrile patient (36.5°C) with sinus tachycardia (108 bpm), hypertension (153/110 mmHg), respiratory rate 15/min, SpO2 87% on room air, signs of low voltage in ECG Figures , anasarc39 days prior, the unvaccinated patient was tested positive for SARS-CoV-2 (by RT-PCR) as part of a health care workers' screening. Prior to the infection, the patient was healthy and fit, doing cycling regularly. During the infection, the patient remained asymptomatic. On admission, the patient was tested negative for influenza, respiratory syncytial virus, and SARS-CoV-2 by RT-PCR. SARS-CoV-2-IgG was positive. Laboratory assessment  showed lA CT-thorax scan showed bilateral lower lobe collapse consolidations, large bilateral pleural effusion, pericardial effusion, and ground-glass opacification in the left upper lobe. Dedicated echocardiography  remained negative as well as serology for cardiotropic pathogens. Interferon-y (QuantiFERON) testing was positive. After pericardiocentesis, symptoms improved. Cytologic evaluation of the pericardial fluid was suspicious for high malignant lymphoma. Further flow cytometry and immunophenotyping excluded a monoclonal cell population as well as PET-CT showed no pathologic extracardiac FDG uptake, which could have indicated a highly proliferative tumor mass or active tuberculosis. Pleurocentesis was performed for diagnostic purpose and symptomatic treatment. The exudative fluid showed no evidence of malignancy or infection but indicating polyserositis. After removal of at least 1850 ml in total over 9 days, echocardiography showed minimal residual effusion and the drainage was removed. Left ventricular function recovered, but the right ventricular global function was still impaired. Echocardiography revealed hypertrophy of the free right ventricular wall. Cross-sectional studies  confirmeA duplex and compression sonography of the upper extremities was performed due to new-onset pain in both arms. It confirmed thrombosis of the left subclavian vein and symmetrical thrombophlebitis of the cephalic vein. An extensive vasculitis and collagenosis screening revealed elevated ANA titer (1 : 100) with high positivity for Ro52, borderline positivity for nRNP/Sm, negative anti-ds-DNA, anti-Sm-antibodies, and normal complement C3/C4. Direct Coombs test (anti-Fy(b)) was positive without evidence of hemolysis. An antiphospholipid syndrome evaluation revealed a slight elevated anti-cardiolipin-IgM titer. Serology for hepatitis A, B, C, and E and HIV was negative. Urine analysis and sediment showed no evidence of concomitant nephropathy. Past medical history was negative for joint pain, skin rash or mucosal lesions, alopecia, and neuropsychiatric abnormalities. Although ACR/EULAR criteria for SLE were negative, 100 mg oral prednisolone was administered for seven days in the set of acute myopericarditis and suspected autoimmune etiology. Within the next three weeks, the patient recovered slowly with loss of about 15 kg along with diuretics and guideline-directed medical heart failure therapy. Left and right ventricular function improved. The elevated liver enzymes had normalized along the improvement of cardiac function suggesting congestive hepatopathy. The patient was discharged after 23 days and followed in our outpatient clinic.Two weeks later, the patient reported an improvement of his physical performance without any complaints. The laboratory assessment revealed a rise in the ANA titer up to 1 : 320 with persisting high positivity for Ro52 and a positive RT-PCR for SARS-CoV-2 in a nasopharyngeal swab with a cycle threshold of 35.5 indicating low viral shedding. Echocardiography showed a persisting moderate reduced right ventricular function and a pericardial effusion of 8 mm around the right ventricle. A rheumatologic examination was intended but is still pending due to patient's concerns. At a six-month telephone follow-up, the patient was asymptomatic and again at his prior exercise capacity but refused any further medical consultation.We report the case of a healthy 66-year-old male patient with myopericarditis, pericardial tamponade, and polyserositis within five weeks after asymptomatic infection with SARS-CoV-2.A negative SARS-CoV-2 PCR and a positive IgG titer at admission indicated a postinfection condition on the background of a positive RT-PCR 39 days prior. At follow-up two weeks after discharge, SARS-CoV-2 PCR was positive with a cycle threshold of 35.5. Repositive tests by RT-PCR are very common in recovered COVID-19 patients. The reason is still unclear, but false RT-PCR results, intermittent virus shedding, viral reactivation, or reinfection with another SARS-CoV-2 strain are currently being discussed [Hypertension is not a characteristic feature of tamponade but is not uncommon in subacute pericardial tamponade , 3. SympWe used a step-by-step approach for excluding the most common etiologies for pericardial effusion, which are cancer (10-25%), pericarditis and infectious causes (15-30%), and connective tissue diseases (5-15%), while tuberculosis is the dominant etiology in developing countries (>60%). Nevertheless, up to 50% still remain idiopathic .Initial cytology findings of the pericardial fluid were suspicious for aggressive lymphoma. Atrial and right heart involvement in our cross-sectional studies may be suggestive for cardiac lymphoma as malignant pericardial effusions can result in tamponade, but primary lymphoma of the heart is rare. Common imaging features in these cases are ill-defined infiltrative masses, tumor extension along the epicardial surfaces, and pericardial thickening . Saab etAn active extrapulmonary tuberculosis was discussed, but neither microscopy nor culture or PET-CT could confirm this hypothesis. On the other hand, the professional background (volunteer in a nursing home) and the positive QuantiFERON test are still suggestive and should prompt further diagnostic workup if disease progress is suspected, but the uneventful time course argued against an active disease process.There was no evidence for any common infectious myocarditis, but SARS-CoV-2 infection remained a potential cause.Cardiac involvement is a salient feature in COVID-19 and is associated with a worse outcome. Pericardial tamponade is a known but rarely reported complication of myopericarditis in COVID-19. Its underlying pathomechanism is still elusive . Farina To our knowledge, there are only two cases in literature reporting asymptomatic SARS-CoV-2 infection with pericardial tamponade , 11. OneHowever, in contrast to both cases, our patient revealed noticeable increased autoimmune laboratory abnormalities. Berger and Volc proposed a model for viral-induced autoimmunity by type I interferon production and reported an increased frequency of lupus anticoagulant, ANA, anti-erythrocyte antibodies, SSA/Ro, and anti-cardiolipin antibodies in patients with COVID-19 . Thus, tThere are two reported cases of COVID-19 associated tamponade with elevated ANA titer; one was diagnosed with SLE with possible antiphospholipid syndrome , and theA multicentric lupus myocarditis case series reported myocarditis as the first manifestation of SLE in 58.6% of patients and endomyocardial biopsy (EMB) was low yield in the diagnostic process . In our We are aware that despite the extensive workup, the origin of myopericarditis in our case remains unclear particularly due to the lacking histologic evidence. Regardless of this, a direct or indirect association with SARS-CoV-2 still remained a possible candidate in our diagnostic journey as other differential diagnoses were ruled out and disease progression occurred in the context of a recent asymptomatic infection. Further studies are needed to show whether asymptomatic SARS-CoV-2-infected patients are at risk for potential life-threatening sequelae.The key feature of this case was the diagnostic process in a subacute to late-onset myopericarditis following an asymptomatic SARS-CoV-2 infection and the differentiation between directly linked infectious organ damage, malignancy, and a triggered autoimmune condition. These patients need close follow-up, and an interdisciplinary approach is essential. Awareness of cardiac complications following a SARS-CoV-2 infection is crucial in the diagnostic and therapeutic approach. Due to millions of patients infected in the COVID-19 pandemic, this diagnostic dilemma will be challenging in the future.