Like other post-translational modifications (PTMs) of proteins, S-nitrosylation has been considered a key regulatory mechanism of multiple cellular functions in many physiological and disease conditions. Emerging evidence has demonstrated that S-nitrosylation plays a crucial role in regulating redox homeostasis in the stressed heart, leading to discoveries in the mechanisms underlying the pathogenesis of heart diseases and cardiac protection. In this review, we summarize recent studies in understanding the molecular and biological basis of S-nitrosylation, including the formation, spatiotemporal specificity, homeostatic regulation, and association with cellular redox status. We also outline the currently available methods that have been applied to detect S-nitrosylation. Additionally, we synopsize the up-to-date studies of S-nitrosylation in various cardiac diseases in humans and animal models, and we discuss its therapeutic potential in cardiac protection. These pieces of information would bring new insights into understanding the role of S-nitrosylation in cardiac pathogenesis and provide novel avenues for developing novel therapeutic strategies for heart diseases. Physiological nitric oxide (NO) is synthesized by L-arginine through three types of nitric oxide synthases, namely neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) . NO is aThe term S-nitrosylation was first coined by Jonathan S. Stamler in 1992 to describe the reversible formation of the S-nitrosothiols (SNOs) of reduced thiols from protein sulfhydryl groups that were exposed to NO . Most prLike other PTMs of proteins, such as phosphorylation, S-nitrosylation has emerged as a key regulatory mechanism in various cellular functions and has been studied in many physiological and disease conditions. It has been reported that S-nitrosylation plays an important role in cellular functions in aspects of gene regulation, immune modulation, vascular homeostasis, and respiratory and neuronal signaling. Conversely, its impairment has been implicated in many diseases such as neurodegenerative diseases, airway diseases, diabetes, and endotoxic/septic shock. In recent years, the significance of protein S-nitrosylation has been revealed in the heart, leading to a great number of discoveries in the mechanisms underlying the pathogenesis of heart diseases. Emerging evidence indicates that S-nitrosylation is a potential target of cardiac protection that would provide new avenues for developing new therapeutic strategies.In this review, we summarize the recent studies in understanding the molecular and biological basis of S-nitrosylation, including its formation, spatiotemporal specificity, homeostatic regulation by transnitrosylation and denitrosylation, and its association with cellular redox status. We also outline the currently available methods that have been applied to detect SNO. We additionally sum up the up-to-date studies in the implications of S-nitrosylation in cardiovascular diseases and discuss its therapeutic potential in cardiac protection. This information could bring new insights into understanding the role of SNOs in cardiac function and protection against heart diseases.S-nitrosylation, by definition, is the covalent attachment of endogenous NO to the thiol (or “sulfhydryl”) side chain of cysteine to form an SNO. Endogenous SNO moieties are much more stable than NO itself, serving as NO donors and participating in NO metabolism . S-nitroCysteine is unique among the coded amino acids of a protein. It is the only one that carries a thiol group, an organic compound containing a sulfhydryl (-SH) functional group with a sulfur atom bonded to a hydrogen atom . This hiIn addition to the SNO in proteins, SNO also occurs in low-molecular-weight (LMW) thiols that produce SNO-LMW, such as S-nitrosocysteine (CysNO), S-nitrosoglutathione (GSNO), and S-nitroso-CoA (SNO-CoA) . Glutath2), nitrogen trioxide (N2O3), or peroxynitrite (ONOO−) [2+/3+, Zn2+, and Cu2+. Transition metals incorporated within metalloenzymes have different oxidation states and can rapidly transfer electrons to and from the metal, making them efficient catalysts for redox reactions. More specifically, the transition metals can catalyze the one-electron oxidation of NO to nitrosonium (NO+), which can nitrosate a cysteine thiol located close to the catalytic center to form a nitrosothiol [2-independent transnitrosylation [Several mechanisms have been reported to be involved in SNO formation. First, NO is a poor oxidant that rarely reacts spontaneously with thiol residues in the physiological milieu. Therefore, SNO mostly occurs after oxidative reactions that render NO to higher oxides of nitrogen with strong nitrosylating effects, such as nitrogen dioxide (NO (ONOO−) . Secondl (ONOO−) . Third, osothiol . SNOs gesylation . In 2011sylation .Like other signaling pathways mediated by NO, SNO has a typical spatiotemporal specificity, and its pKa-dependent thiol reactivity has been reported to be one of the major determinants to interpret this specificity . Another2 exhibit higher solubility in hydrophobic environments in which they tend to accumulate and concentrate.The discovery of the two main consensus motifs surrounding cysteine residues, the acid-base motif and the ‘hydrophobic compartment’ motif, introduced another level of target specificity of S-nitrosylation. The acid-base motif model was first proposed in 1997 by Stamler et al. . They prAll the determinants of SNO specificity mentioned above have been considered to help explain SNO specificity. In 2010, Marino et al. conducted a comprehensive analysis of 70 NO-Cys sites in 55 proteins regarding the general features of SNO that were reported in previous literature . The proThe cellular homeostasis of SNO is regulated by two main mechanisms: transnitrosylation and denitrosylation. Transnitrosylation is the reversible transfer of an NO group from one cysteine residue to another, thereby allowing for the effective propagation of SNO-based signals . A recenIn contrast, denitrosylation is the reverse reaction of S-nitrosylation via the removal of an SNO moiety from either SNO-proteins or SNO-LMW. From another perspective, transnitrosylation can be considered a denitrosylation process of the donor protein.S-nitrosylation is generally a nonenzymatic reaction. However, there is accumulating evidence pointing to enzymatic regulations of S-nitrosylation via both S-nitrosylases and denitrosylases, which cooperatively control steady-state levels of SNO .Ⅲ) initially binds weakly to GSH (CytFeⅢ-GSH). Then ferric cytochrome c is synergically reduced by NO and GSH to cytochrome c (CytFeⅡ), resulting in GSNO formation [S-nitrosylases: S-nitrosylases can enzymatically mediate the transfer of an NO moiety from either Metal-to-Cys or Cys-to-Cys. Nitrosylase metalloproteins, such as hemoglobin and cytochrome c, can transfer NO groups from redox-active transition metals to cysteine thiol  via the metal-catalyzed reaction. For example, in mammalian hemoglobin, oxidized hemes (Fe(III)) catalyzes auto-S-nitrosylation of Cys93 (CysβSNO) in a hemoglobin (Hb) β-chain via the intramolecular transfer of NO from iron-nitrosyl hemoglobin (HbFeNO) to Cys93. SNO-Hb also exhibits Cys-to-Cys transnitrosylase activity, which transfers NO to an N-terminal erythrocytic membrane protein, AE1 (anion exchange protein 1) . Cytochrormation .Cys-to-Cys nitrosylases are SNO-proteins that transfer their NO groups to acceptor cysteines on the target protein (Cys-to-Cys transfer) . AlongsiDenitrosylases: Reciprocally, denitrosylation can be either an enzymatic or nonenzymatic reaction. Nonenzymatic denitrosylation can spontaneously occur under the nucleophilic attack of cytosolic reductants (such as ascorbate), which cleaves the S–N bond of the S-nitrosyl group . EnzymatTrxs are a group of ubiquitous small redox proteins with a general disulfide reductase activity. There are two distinct classes of mammalian thioredoxins, Trx1 and Trx2. Trx1 is mainly localized in the cytoplasm, while Trx2 is restricted to mitochondria. Notably, Trx1 can also translocate to the nuclear compartment or be exported out of cells under certain conditions . Trxs haGSNO denitrosylation is catalyzed by the main cellular denitrosylase S-nitrosoglutathione reductase (GSNOR), which is usually coupled with NADPH consumption. GSNO can be irreversibly reduced to glutathione disulfide (GSSG) by GSNOR in the presence of nicotinamide adenine dinucleotide (NADH) and GSH. GSSG can be further catalyzed by NADPH-dependent glutathione reductase (GR) to regenerate GSH ,45. TherAdditionally, under oxidizing conditions, the resulting GSSG from GSNOR denitrosylation can go through protein S-glutathionylation by reversibly forming a mixed disulfide (PSSG) between protein thiols (P-SH) and GSH . The degIn summary, as shown in The detection of SNO in biological samples tends to be difficult due to its particularly liable nature and low abundance. However, the development of a great number of reliable laboratory techniques has made it possible for the characterization, identification, and quantification of both SNO-proteins and SNO-sites. The applied methods are summarized below and also in Biophysical techniques such as X-ray crystallography and NMR spectroscopy provide powerful methods for the structural characterization of single, isolated SNO-proteins by high-resolution crystal structure analysis . HoweverThe development of SNO-specific antibodies, such as SNO-cysteine- bovine serum albumin (BSA), provides a direct method to detect SNO-proteins from the immunohistochemical approach . HoweverMS-based technologies have been used to directly detect SNO-proteins, especially after protease digestion and purification, such as electrospray ionization MS (ESI-MS), a ‘soft ionization’ technique with a very gentle ionization process resulting in little or no cleavage of SNO-bonds . The pro2−) and NO-based assays that measure NO or NO2− released upon S-NO cleavage [2+) or homolytically by UV light. They are then subjected to various NO detection methods such as a Griess-Saville assay, ozone-based chemiluminescence, and fluorescence assays using 4,5-diaminofluorescein (DAF-2) or its analogous derivatives [Indirect detection methods for the absolute quantification of protein-bound SNOs include a series of nitrite  is a sulfur-based assay that has been most commonly used for the highly specific identification of individual SNOs. BST was initially developed by Jaffrey et al. in 2001 . The iniRecently emerging direct methods use alternatives to ascorbate-based enrichment strategies, such as gold nanoparticles (AuNPs) capture and organomercury resin capture (MRC). Such methods can achieve the identification of SNO-sites via the formation of stable SNO-conjugates that can be subjected to MS analysis . MoreoveThe bioorthogonal cleavable-linker and switch technique (Cys-BOOST) combined with LC-MS/MS has recently been reported for the quantitative analysis of SNO sites and the detection of consensus motifs . In Cys-12C) or heavy (13C) isotopes. The isotopes can differentially label nascent free thiols to determine the relative abundance of proteins in paired samples. A cICAT contains an additional acid-cleavable linker that can remove the biotin tag before MS analysis to improve MS spectra.The application of iodoacetamide analogs such as isotope-coded affinity tag (ICAT) and second-generation cleavable ICAT (cICAT) have been intensively used for both the qualitative and quantitative proteomic analysis of SNO-peptides with LC-MS/MS . The strOther than identifying specific SNO-proteins and SNO-SID, cysteine-reactive tandem mass tag (CysTMT) labeling combined with large-scale MS analysis has been used to determine the SNO-occupancy of each modifiable Cys in the myocardium for measurement of thiol-reactivity to SNO-modification . SNO occ2+ homeostasis. Aberrant or dysregulated SNO-dependent NO signaling has been linked to heart diseases and related conditions such as myocardial ischemia, heart failure, and atrial fibrillation [The heart is one of the most affected organs by SNOs, and a large number of SNOs have been reported in cardiomyocytes ,71. SNOsillation . HoweverThe studies related to SNO protein in the hearts are concentrated in the ischemic myocardial injury. Several SNO proteins have been found to play a protective role against ischemia-reperfusion (IR) injury. On the contrary, some increased SNO proteins have been found to be associated with cardiac hypertrophy.Increased SNO formation has been linked to cardioprotection against ischemic myocardial injury. Nadtochiy et al. investigated the protective efficacy of the mitochondrial S-nitrosating agent S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) in vivo using murine models of myocardial ischemia (MI) with permanent left anterior descending artery (LAD) ligation . Their rStudies have also been conducted to explore the mechanism of SNO involved in cardiac protection. It has been demonstrated that aside from entering the cell nucleus as a nuclear transnitrosylase in the form of SNO-GADPH, GADPH can also be imported into the mitochondria and modulate mitophagy and apoptosis after myocardial IR injury . Kohr et−/− mouse hearts subjected to IPC treatment exhibited a loss of cardioprotection and the rise of SNO induced by IPC. Thus, SSM could be considered a favored target of the caveolae/eNOS/NO/SNO signaling in IPC-induced cardioprotection. Notably, the gap junction (GJ) protein connexin 43 (Cx43) expressed in myocardial mitochondria exists exclusively at the inner membrane of SSM and serves as a marker for SSM [ATP channels (mKATP) [2) in the Langendorff-perfused mouse heart model [+ influxes, and ROS formation, thus participating in the cardioprotective signal transduction cascade.Sun et al. reported that methyl-β-cyclodextrin (MβCD) treatment could disrupt myocardial caveolae structure at the sarcolemma . The dis for SSM . Mitocho (mKATP) . Soetkamrt model . Their rG-Nitroarginine methyl ester (L-NAME), a constitutive NO synthase inhibitor. Furthermore, PostC-induced cardioprotection could be comparably mimicked by an NO donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP). In the meantime, PostC-induced cardioprotection could not be reversed by inhibitors of the sGC/cGMP-dependent NO signaling pathway. These results suggest the involvement of NO-mediated SNO signaling instead of sGC/cGMP/PKG signaling in PostC-induced cardioprotection. Sun et al. explored the cross-talk between S-sulfhydration (SSH) mediated by hydrogen sulfide (H2S) and S-nitrosylation in PostC hearts using the Langendorff-perfused mouse hearts and I/R protocol [Sun et al. also set foot in clarifying the participation of the two major NO-dependent signaling pathways, soluble guanylyl cyclase/cyclic guanosine monophosphate/protein kinase G (sGC/cGMP/PKG) and protein S-nitrosylation, in NO-mediated acute IPC-induced cardioprotection in Langendorff-perfused mouse hearts . Their rprotocol . Their rTang et al. explored the role of the S-nitrosylation of muscle LIM protein (SNO-MLP) involved in the pathogenesis of myocardial hypertrophy. They examined the SNO-MLP level in myocardial samples acquired from patients diagnosed with myocardial hypertrophy, transverse aortic constriction (TAC) mice, angiotensin II/phenylephrine-treated neonatal rat cardiomyocytes, and TLR3 (Toll-like receptor 3) knockout mice. A significant increase of SNO-MLP on Cysteine 79 (Cys79) was observed in hypertrophic myocardium. Increased SNO-MLP promoted the binding of Cys79 with downstream TLR3, followed by TLR3-mediated RIPK3 (receptor-interacting protein kinase 3) and NLRP3 (NOD-like receptor pyrin domain containing 3) inflammasome activation . Therefo2+ handling protein RyR2, suggesting a direct association between NOS1-dependent protein S-nitrosylation and contractility. Moreover, ROS inhibition with Tempol, a free radical scavenger, reduced not only protein S-nitrosylation and cardiac contractility but also inotropic responses to ISO. Tempol strongly mimicked the effects of NOS-1 inhibitors, indicating that ROS production could link between NOS1-mediated S-nitrosylation and βA stimulation.Vielma et al. explored the relationship between NOS isoforms and isoproterenol (ISO)-induced S-nitrosylation by using selective pharmacological inhibitors of NOS-1 and NOS-3 . Their rLike the SNO proteins, SNO-LMWs have also been heavily studied in the stressed heart, particularly the GSNO. Since GSNOR plays a crucial role in GSNO denitrosylation and in maintaining the equilibrium between SNO-proteins and GSNO, various studies have been conducted in the hearts with GSNOR knockout (KO) and transgenic (TG) mouse models.2O2-induced mPTP opening in WT mouse embryonic fibroblasts (MEFs). The demonstrated inhibition of mPTP opening with GSNO treatment confirmed the cytoprotective role of S-nitrosylated CypD. Additionally, CypD−/− MEFs transfected with either WT CypD or C203S-CypD generated by the site-directed mutation of Cys 203 of CypD to a serine showed a similar level of inhibition on mPTP opening, suggesting that the inhibitory effects on the mPTP opening by SNO of Cys-203 was similar to that of C203S-CypD or CypD deletion. Amanakis et al. recently identified Cys-202 of CypD as a redox-sensitive site for multiple PTMs, including S-nitrosylation [Sun et al. showed that the effects of IPC on protein S-nitrosylation and cardioprotection against myocardial IR injury were mimicked by GSNO . They resylation .2+ leak in GSNOR−/− cardiomyocytes and, subsequently, compromised cardiac contractility. Hatzistergos et al. found that, aside from a generally improved cardiac performance, post-MI adult heart from GSNOR−/− knockout mice also exhibited increased regenerative and proliferative activity of adult cardiac progenitors, mesenchymal stem cells, and cardiomyocytes compared with WT controls [Since GSNOR is key to maintain the equilibrium between SNO-proteins and GSNO, it has been extensively studied and is considered an essential element in cardiovascular health and diseases. Lima et al. found that normoxic myocardium of GSNOR KO mice after an LCA (left coronary artery) ligation exhibited a significantly improved cardiac performance compared to a control group . Their rcontrols . This di2+, suppressing the Ca2+-calcineurin signaling and leading to the induction of left ventricular hypertrophy (LV) and dysfunction under chronic ISO challenge. Additionally, GSNOR overexpression or pharmacologic NOS inhibition was found to block the increase of the S-nitrosylation of phospholamban (PLN), NCX (Na+/Ca2+ exchanger), and cTnC (cardiac troponin C) in the heart upon ISO stimulation. In particular, their results showed that the formation of a functionally inactive PLN pentamer under ISO stimulation required not only the phosphorylation of inhibitory monomeric PLN at Ser16 and Thr17 but also the S-nitrosylation of PLN at Cys36 or Cys41, while the S-nitrosylation of cTnC at Cys84 led to decreased βAR-induced responsiveness.Irie et al. explored the role of β-adrenergic receptor (β-AR) signaling in the pathophysiology of cardiomyocyte hypertrophy and heart failure in mice . Their r−/−, and GSNOR-CMTg mice. GSNOR−/− mice, on the other hand, showed markedly worsened LV hypertrophy than WT controls, indicating that increased denitrosylation by GSNO, although not able to prevent LV dysfunction, could reduce the degree of cardiac hypertrophy during chronic pressure overload. GSNOR-Tg mice were also reported to be cardioprotective against sepsis-induced myocardial depression under a lipopolysaccharide (LPS) challenge [Mori et al. reported that the specific overexpression of GSNOR in cardiomyocytes (GSNOR-CMTg) exhibited mitigated LV dysfunction and hypertrophy compared to WT controls during chronic pressure overload . Pressurhallenge .It is generally believed that women tend to develop heart diseases ten years later than men and have significantly better overall cardiac function and survival against heart diseases. The selective activation of estrogen receptor-β (ER-β) has been closely linked to cardioprotection observed in females following MI . Lin et Shao et al. investigated the cardioprotective effects of pharmacologic preconditioning in male and female hearts using an adenosine A1 receptor agonist N6-cyclohexyl adenosine (CHA) in a murine model of I/R injury . They idTo clarify the molecular signature of failing hearts, Menazza et al. employed oxidation RAC (Ox-RAC) and SNO-RAC in the detection of protein oxidation and S-nitrosylation levels in left ventricular samples acquired from biopsies in dilated cardiomyopathy (DCM) and heart failure (HF) as well as from healthy male and female donors . The sex−/−) in male hearts exhibited significantly reduced infarct size and enhanced post-ischemic functional recovery. In contrast, female hearts yielded the opposite results, with a worsened infarct size and functional recovery post-MI. Moreover, the cardioprotective effects induced by a GSNOR blockade in male hearts can be attributed to the increased pre-ischemic SNO levels of ND3 Cys39 and a substantially decreased post-ischemic H2O2 production. Additionally, they found that GSNOR inhibition could impair GSNOR metabolism as a formaldehyde dehydrogenase in the heart, especially in female hearts where post-ischemic free formaldehyde levels were significantly elevated after N6022 treatment. Excessive mitochondrial formaldehyde was found to exert a detrimental impact on female hearts, which could be rescued by an aldehyde dehydrogenase (ALDH) agonist Alda-1.Based on their previous findings that female hearts exhibit higher protein-SNO levels and significantly increased GSNOR activity at baseline compared to male hearts, Casin et al. investigated the sex-dependent cardioprotection of GSNOR against I/R injury in males and female mice with both ex vivo and in vivo models of myocardial I/R injury . Their r2) before and during a 25 min period of LAD occlusion (NaNO2-PO group) and 10 min prior to reperfusion (NaNO2-PR group). Their results showed that both NaNO2-treated groups exhibited a significantly attenuated severity of ischemia following coronary occlusion compared to sham controls, with the NaNO2-PR group gaining the highest survival rate of 92%. Meanwhile, sodium nitrite also markedly reduced the total number of ventricular premature beats (VPBs) and the incidence and number of ventricular tachycardia (VT) episodes. SNO proteins and peptides were remarkably increased in the NaNO2-PR group but not in the control and NaNO2-PO groups, although the results were not statistically significant. Their research provided the first evidence that the inorganic sodium nitrite could protect against the I/R-induced severe ventricular arrhythmia. This cardioprotective effect was associated with increased protein SNO levels only when sodium nitrite was administered prior to reperfusion. More preclinical research will be expected based on the increasing studies in this field.Inorganic nitrites and nitrates, such as sodium nitrite that was used as pharmacological preconditioning in the above experiment, have long been reported to have cardioprotective effects against myocardial I/R injury . AdditioA large number of current discoveries regarding S-nitrosylation have emphasized the heart itself. Meanwhile, the role of S-nitrosylation in other diseases that may cause cardiomyopathies, such as hypertension, atherosclerosis, stroke, and diabetes, have been also studied. However, the underlying molecular mechanisms remain largely undetermined.2 into two-kidney, one-clip (2K1C) renovascular hypertensive rats could restore cardiac performance along with increased protein S-nitrosylation in LV myocardium [Choi et al. explored the role of S-nitrosylation in hypertension by using the murine angiotensin II (AngII) infusion model . They reocardium .−/− apolipoprotein E (ApoE)−/− double knockout mice subjected to HHcy challenge exhibited much less T-cell activation and a significantly mitigated amount of total lesions of HHcy-induced atherosclerosis compared with GSNOR+/+ApoE−/− mice. Lin et al. reported that the administration of an H2S donor, NaHS, could generate a significant anti-atherosclerotic effect against atherosclerotic aortic lesions and the accumulation of lipid-laden macrophages in apoE−/− mice subjected to an atherogenic diet [2S-producing enzyme, resulted in the opposite effects to NaHS. The anti-atherosclerotic effect was attributed to increased plasma NO and protein S-nitrosylation in aorta vascular smooth muscle cells (VSMCs). Wang et al. further examined the role of H2S in ApoE−/− CSE−/− knockout mice [2S against atherogenesis through upregulating protein S-nitrosylation. Their study provided insights into the therapeutic potential of H2S-releasing drugs that could bypass the defects of NaHS.In addition, Li et al. found that hyperhomocysteinemia (HHcy) could aggravate atherosclerosis by upregulating GSNOR activity in T cells and switching the S-nitrosylation of protein kinase B/Akt Cys 224 to the phosphorylation of Akt Ser473 . The phonic diet . Furtherout mice . They coFurthermore, Shi et al. reported that, during acute ischemic stroke, the excessive activation of neuronal N-methyl-D-aspartate receptors (NMDARs) could result in NO overproduction and the subsequent S-nitrosylation of tyrosine phosphatase-2 (SHP-2) . S-nitroBesides these cardiac-associated vascular diseases, abnormal S-nitrosylation has also been related to vascular lesions and insulin resistance associated with type 2 diabetes and obesity. Wadham et al. reported that the exposure of endothelial cells to high glucose could significantly reduce the S-nitrosylation of proteins involved in regulating vascular functions, such as eNOS . MoreoveIncreasing evidence has demonstrated that S-nitrosylation is an essential PTM in maintaining normal cardiac cellular function and plays a key role in regulating the pathogenesis of cardiac diseases or cardiac stresses. S-nitrosylation is becoming a potential target of cardiac protection that could provide new avenues for developing new therapeutic strategies.The field of protein S-nitrosylation has proved to be rapidly evolving over the last several years. Despite the importance of this PTM in various cellular functions, SNO regulation in the heart and the mechanisms underlying the cardiac pathogenesis and protection remain largely unknown. Several key aspects need to be considered in the future directions. First, despite the considerable progress made in identifying SNO-proteins and motifs in vitro, the real-time detection of cellular SNO in the heart is still a big challenge. More reliable and in situ detective strategies need to be developed, especially in vivo measurements. The development of these advanced techniques would significantly contribute to our current understanding of this emerging paradigm redox signaling, especially in the setting of heart diseases. Second, extensive research efforts are needed to elucidate the complex molecular mechanisms underlying the bioactivity of S-nitrosylation, including the regulations on its formation, spatiotemporal specificity, homeostasis, and the association with cellular redox status and various stresses, as well as the cross-talk with other PTMs in proteins. Third, the identification of specific SNO proteins and their biological function in the heart will help develop new knowledge for the treatment and prevention of myocardial diseases and apply these findings to clinical and translational medicine. Further studies in these fields would bring new insights into preventing and treating heart diseases.


Chimeric antigen receptors (CAR) are fusion proteins engineered from antigen recognition, signaling, and costimulatory domains that can be used to reprogram T cells to specifically target tumor cells expressing specific antigens. Current CAR-T cell technology utilizes the patient’s own T cells to stably express CARs and has achieved exciting clinical success in the past few years. However, current CAR-T cell therapy still faces several challenges, including suboptimal persistence and potency, impaired trafficking to solid tumors, local immunosuppression within the tumor microenvironment and intrinsic toxicity associated with CAR-T cells. This review focuses on recent strategies to improve the clinical efficacy of CAR-T cell therapy and other exciting CAR approaches currently under investigation, including CAR natural killer (NK) and NKT cell therapies. Chimeric antigen receptor T (CAR-T) cells-based treatments have made promising advances in cancer immunotherapy. Endowed with the specificity for any cell-surface antigen, these cytotoxic T cells offer potentially curative therapeutic options to patients with previously incurable malignancies. Conceptually, CAR-T therapy requires taking a patient’s native T cells and coupling them with more robust cytotoxic functions. The production of CAR-T cells then requires six steps—apheresis collection from the patient, T cell enrichment, gene modification, activation and ex vivo expansion, quality assessment, and reinfusion into patients. Structurally, a CAR-T cell is a T cell that has been genetically engineered to express an antigen-specific, non-major histocompatibility (MHC)-restricted receptor, which is composed of the single-chain variable fragment (scFv) of an antibody fused to a transmembrane domain and a signaling domain. Initial design of CAR-T cells contained only the CD3-ζ domain, the initiator of T cell receptor signaling intracellularly . These fAs of December 2019, there are over 600 active clinical trials along with more than 200 completed trials investigating CAR-T cells around the globe . To dateAnother recent trial used anti-CD19 CAR-T cells in 21 patients with R/R FL and found that CD19 CAR-T immunotherapy was highly effective in adults with clinically aggressive FL with durable remission . In addiSome recent successes have also been shown in solid tumors though no results from large-scale trials have been reported. In a phase-I dose-escalation trial, Her2-specific CAR-T cells were infused in patients with progressive glioblastoma and found to be safe and clinically efficacious, though no patient achieved complete response . IntracrDespite exciting progress reported in the last few years for CAR-T therapy, there are a number of challenging obstacles researchers and patients face. For one, toxicity related to therapy is still of concern to clinicians. Additionally, efficacy is limited by non-optimal persistence and potency combined with impaired trafficking to the tumor site in solid tumors. Lastly, tumor heterogeneity and immunosuppression from the tumor microenvironment can further dampen the effects of CAR-T cells.Major toxicities observed in clinical trials of CARs for the treatment of ALL and B cell lymphoma have been cytokine-release syndrome (CRS), a condition characterized by high levels of inflammatory cytokines that result in a sepsis-like state in patients and CAR-T cell-related encephalopathy syndrome (CRES), a type of neurotoxicity . CRS usuMany factors are thought to be contributive to the development of CRS or CRES in patients, some of which are related to treatment regimen and patient characteristics while others are more intrinsic to the therapy itself. Higher cell doses, expansion, and conditioning chemotherapy with fludarabine have been associated with the development of severe CRS and/or CRES ,36,37. Itumor-specific cell-surface antigens and using CARs targeting tumor-associated cell-surface antigens could inadvertently damage healthy cells expressing the same antigen. In patients with renal cell carcinoma (RCC) treated with a first-generation CAR-T against carbonic anhydrase IX (CAIX), some patients experienced liver enzyme disturbances that necessitated treatment cessation, a toxicity event that could be eliminated by pretreatment with an anti-CAIX monoclonal antibody [CAR-T-related adverse effects in solid tumor patients are often due to “on-target, off-tumor” effects. In contrast with hematologic malignancies, there is few antibody . Thereforeg), which have been shown to negatively impact adoptive T cell transfer [Currently, the degrees of T cell persistent and expansion in vivo are still not optimized, limiting their clinical efficacy, especially in solid tumors ,41,42,43transfer . DisappoIn addition to persistence issues, potency of CAR-T cells is limited by T cell exhaustion. This can be induced by excessive stimulation due to high disease burdens and antigen-independent signaling triggered by aggregation of CAR receptors ,45,46. COne major obstacle of using CAR-T cells in solid tumors is inefficient localization and infiltration into the tumor stroma. Tissue homing and infiltration require proper expression and precise pairing of adhesion molecules on both the T cells and the vasculature to facilitate leukocyte extravasation towards a chemokine gradient established by tumor cells. However, perfect matching between chemokine receptors on CAR-T cells and the chemokines secreted by tumor cells rarely happen. In addition, recent studies reported reduced chemokine productions as a result of local tumor microenvironment (TME) suppression ,50. ThisUnlike leukemias and lymphomas, solid tumors often lack specific cell surface markers. Instead, solid tumors are distinguished by anatomic locations, histologic features, molecular mutations, and markers that can be expressed on the surface or intracellularly. Therefore, discovering tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs) that allow for a high-degree of tumor-targeting effects while sparing healthy tissues is one of the most challenging aspects in developing CAR-T cells for solid tumors. Furthermore, finding an ideal antigen that is primarily expressed on the cell surface rather than expressed intracellularly makes the process even more daunting. Though several surface TSAs have been discovered, it was found that there is a great degree of tumor heterogeneity, even among patients suffering from the same type of cancer. Ideally, due to the antigen heterogeneity, it is prerequisite to identify a TSA for each patient and then proceed to generate specific CAR T cells. However, this can be a very complicated engineering process associated with unsustainable high costs for patients and manufacturers. Targeting TAAs, on the other hand can potentially lead to “on-target, off-tumor” effects . Regardlreg have all been shown to secrete anti-inflammatory and pro-angiogenic soluble factors to further impede T cell functions [Once at the tumor site, CAR-T cells must also overcome immunosuppressive molecules and cells that could further impede its engagement with a target antigen and/or suppress its cytotoxic functions. In addition, the TME is characterized by harsh conditions, including oxidative stress, nutrient deprivation, acidic pH, and tissue hypoxia, all of which can reduce CAR-T survival and proliferation. Furthermore, tumor cells can upregulate the expression of programmed death ligand 1 (PD-L1) and galectin-9 that are known to suppress T cell functions through checkpoint inhibitory receptors. Lastly, the vast immunosuppressive stromal and immune cell types within the TME, such as cancer-associated fibroblasts (CAFs), tumor endothelial cells (TECs), tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), and Tunctions . As a reunctions . This coTo better combat these challenges, additional genetic engineering of CARs combined with synthetic biology and the use of combinational therapy hold the key to provide cellular immunotherapy with novel attributes necessary to overcome its hypo-functionality, trafficking issues, and the immunosuppressive forces in the TME. In addition, efforts to enhance the safety profile of CARs with better spatial and temporal control of their activity and persistence after deployment are also under investigation. The following section highlights some of the recent developments and notable clinical trials are summarized in Switchable CARs” that do not target any specific surface target antigen and only become operational in the presence of a bispecific adaptor molecule that mediates connections between components of the CAR-T construct , that produced significantly lower levels of cytokines than the prototype CD19-BBz while retained potent catalytic activity. As a result, a phase I study with 25 patients of B cell lymphoma revealed no significant CRS or CRES events while achieving a CR rate of 54.5% . Others onstruct B [59,60,Armored CARs” that constitutively secrete cytokines such as IL-12. IL-15 and IL-18 also provided further hope that more sophisticated engineering of CAR-T cells can overcome the persistence issue all together  locus, resulting in enhanced potency and antitumor activity [TET2) locus, leading to impressive potency of the particular clone in a CLL patient [Decreased potency of CAR-T cells can also be prevented by site-directed integration of CAR-T construct that simultaneously eliminates specific genes that are known to suppress T cell functions. Using the CRISPR/Cas9 technology, Eyquem et al. were able to direct a CD19-targeting CAR to the T cell receptor α constant  resulted in increased expression of CXCR3 in CAR-Ts, which was associated with improved targeting to the tumor site and tumor control [+ non-Hodgkin’s lymphoma (NHL) but more clinical studies are needed to further investigate the therapeutic advantages of chemokine-receptor-expressing CAR-T cells [Based on insights from mechanistic studies that showed chemokine receptors CXCR3, CCR2, and CCR5 are critical for T cell trafficking to the tumor site , several control . A curre-T cells . Another-T cells . Early c-T cells . Regiona-T cells . Two rec-T cells ,84. Curr-T cells .Tandem CARs”, which are bispecific CAR-T cells that have two scFvs against two different antigens linked by a single molecule in the same CAR-T construct or bicistronic CARs in which two monospecific CARs are expressed from the same vector  inhibitors, including antibodies against PD-1 and CTLA-4. Co-administration of PD-1/PD-L1 blocking antibodies with CAR-T cells was shown to restore CD28 CAR-T cells’ persistence and cytotoxicity in the TME in a mouse model of mesothelioma. Clinically, using CD19-targeting CAR-T cells combined with anti-PD-L1 antibodies in patients with R/R DLBCL showed promising results with a 90% ORR and six patients in CR . Additio studies ,95,96,97toxicity . In othetoxicity ,100. Curtoxicity . CurrentLastly, dominant negative genes have been successfully introduced into CAR-T cells to overcome immunosuppression in the TME. Kloss et al. engineered prostate membrane antigen (PSMA)-targeting CAR-T cells to co-express a dominant-negative transforming growth factor-beta (TGFβRII) which negates the suppressive effects of TGFβ signaling in T cells. When tested in a mouse model of prostate cancer, investigators found that these TGFβRII-expressing CAR-T cells demonstrated enhanced persistence and potency and are more resistant to exhaustion. A clinical trial investigating its effects in patients with metastatic prostate cancer is currently underway . RecentlThe role of NK and NKT cells has been studied in cancer. The ability of NK cells to exert cytolytic functions on cancerous cells through the release of perforin and granzyme B (GzmB) as well as through FasL–Fas interactions, combined with its non-MHC-restricted pattern of activation make it an attractive option for cellular immunotherapy . On the Each cell type is an appealing alternative for CAR engineering for different reasons. First, allogeneic NK cells do not cause graft-versus-host disease (GVHD) as shown in clinical studies using haploidentical and cord blood-derived NK cells in patients with hematologic malignancy or solid tumors ,106. SecEngineering of CAR-NK cells include the same manufacturing scheme as CAR-T cells and a recent novel approach that substitutes NK-specific intracellular activation domain for CD3-ζ A. This mCAR-NKTs also showed promising results in several recent studies. Heczey et al. engineered CAR-expression NKT cells targeting GD2 in neuroblastoma and CD19 in lymphoma and observed great success in preclinical studies. These CAR-NKT cells not only had high selectivity for target tumor antigens but can also proliferate and produce high levels of cytokines efficiently within the TME . CurrentOverall, recent advances made in the field of CAR-based therapy are exciting and impressive. Many patients with hematologic malignancies have greatly benefitted from CAR-T cell treatments. Though there is less success in solid tumors, better engineering techniques, delivery, and combinational therapies may hold the key for improved outcomes in these patients. Lastly, the emergence of other CAR-based cellular therapies, including CAR-NK and CAR-NKT cells shed additional insights and provided hope that advances in bioengineering may enable broader and more effective usage of these immune cells in cancer.


International guidelines recommend the initiation of empirical antibiotherapy for possible associated bacterial pneumonia in COVID-19 critically ill yet further suggesting a rapid reassessment upon source documentation [plus test  was performed. The FA-PNEU is an automated multiplex PCR test allowing direct detection of 15 bacteria with a semi-quantitative value, 3 atypical bacteria, 9 viruses, and 7 antimicrobial resistance genes within 1 h and 15 min [This preliminary analysis was conducted over a 1-month period at the intensive care unit (ICU) of the Cliniques universitaires Saint-Luc and included all COVID-19 adult patients from whom a lower respiratory tract sample could be obtained. Specimens were conveyed to the microbiology laboratory where a FilmArray Pneumonia Panel d 15 min . FA-PNEUStaphylococcus aureus, Haemophilus influenza, and Moraxella catarrhalis were the principal bacteria identified with significant genome copies. None of the 32 FA-PNEU tests identified atypical bacteria neither other respiratory viruses. Direct communication of FA-PNEU results led to speeded-up antibiotic modifications in 15/32 (46.9%) patients.Forty-one COVID-19 patients were admitted to ICU, and 32 could be included upon respiratory sample availability. The study population was comparable to previously described COVID-19 critically ill in terms of age, sex ratio, severity scores, comorbidities, and symptoms [It is a known difficulty to adjudicate on the presence of a co-infection in COVID-19 patients particularly in critically ill. Clinical presentation, inflammatory markers, and bilateral radiological infiltrates lead to misperception and cannot be used in the diagnosis of a bacterial superinfection. As a consequence, empirical antibiotherapy is quasi-systematically initiated until microbiological documentation of co-infecting pathogens. Yet, current data on co-infections is limited. With the focus on intensive care settings, a case series in February 2020 analyzing 21 COVID-19 ICU patients reported no bacterial respiratory co-infections but 3 influenza infections . A similAs rapid molecular testing was performed within the shortest possible time following ICU admission, a majority of our patients did not receive empirical antibiotherapy while awaiting FA-PNEU result. Ultimately one third of the patients remained antibiotic-free over the entire process, and 5 patients had their antibiotics stopped following a negative FA-PNEU result. These antibiotic savings are crucial for COVID-19 critically ill known to have a long ICU stay with reported nosocomial infection rates as high as 31% .To conclude, bacterial documentation is essential to assess co-infection in COVID-19 critically ill. The use of molecular diagnostic tools and the initiation of narrow-spectrum antibiotics are key elements of COVID-19 antimicrobial stewardship guidelines in critically ill. Studies on larger populations and in different geographical areas should be performed to outline analogous antibiotic saving strategies.


Positive psychiatry offers a unique approach to promote brain health and well-being in aging populations. Health interventions are increasingly becoming available using self-guided apps, however, little is known about the effectiveness of app technology or the difference between in-person versus self-guided app methodology for behavioural activation. The objective of this study was to investigate the difference in users and outcomes between two formats of a positive psychiatry intervention to promote brain health and well-being in later-life: (1) clinician-assisted, and (2) independent app use for self-management. As part of a larger national knowledge translation intervention two methods of a behavioural activation intervention (Clinician-assisted vs. Independent app use) were retrospectively compared. Main outcomes were patient characteristics , psychological outcomes , and behavioural outcomes . Clinician-assisted patients (n=254) were more likely to be male, older, and had lower health and resilience scores at baseline than Independent app users (n=333). Clinician-assisted patients had notably higher completion rates (99.2% vs. 10.8%). Psychological outcomes were similar regardless of intervention method for those who completed the intervention. Clinician-assisted patients had higher rates of goal attainment and goal SMART-ness. A preliminary goal setting methodology for effective behavioural activation, to promote brain health and wellness, is given. Clinician-patient relationships were found to be an important factor for intervention completion, caution is given for app use referral. Results indicate a need for further exploration to determine best practices for health app use in clinical practice.


Human metapneumovirus is a common virus associated with acute lower respiratory infections (ALRIs) in children. No global burden estimates are available for ALRIs associated with human metapneumovirus in children, and no licensed vaccines or drugs exist for human metapneumovirus infections. We aimed to estimate the age-stratified human metapneumovirus-associated ALRI global incidence, hospital admissions, and mortality burden in children younger than 5 years.We estimated the global burden of human metapneumovirus-associated ALRIs in children younger than 5 years from a systematic review of 119 studies published between Jan 1, 2001, and Dec 31, 2019, and a further 40 high quality unpublished studies. We assessed risk of bias using a modified Newcastle-Ottawa Scale. We estimated incidence, hospital admission rates, and in-hospital case-fatality ratios (hCFRs) of human metapneumovirus-associated ALRI using a generalised linear mixed model. We applied incidence and hospital admission rates of human metapneumovirus–associated ALRI to population estimates to yield the morbidity burden estimates by age bands and World Bank income levels. We also estimated human metapneumovirus-associated ALRI in-hospital deaths and overall human metapneumovirus-associated ALRI deaths . Additionally, we estimated human metapneumovirus-attributable ALRI cases, hospital admissions, and deaths by combining human metapneumovirus-associated burden estimates and attributable fractions of human metapneumovirus in laboratory-confirmed human metapneumovirus cases and deaths.In 2018, among children younger than 5 years globally, there were an estimated 14·2 million human metapneumovirus-associated ALRI cases (uncertainty range [UR] 10·2 million to 20·1 million), 643 000 human metapneumovirus-associated hospital admissions (UR 425 000 to 977 000), 7700 human metapneumovirus-associated in-hospital deaths (2600 to 48 800), and 16 100 overall  human metapneumovirus-associated ALRI deaths (5700 to 88 000). An estimated 11·1 million ALRI cases (UR 8·0 million to 15·7 million), 502 000 ALRI hospital admissions (UR 332 000 to 762 000), and 11 300 ALRI deaths (4000 to 61 600) could be causally attributed to human metapneumovirus in 2018. Around 58% of the hospital admissions were in infants under 12 months, and 64% of in-hospital deaths occurred in infants younger than 6 months, of which 79% occurred in low-income and lower-middle-income countries.Infants younger than 1 year have disproportionately high risks of severe human metapneumovirus infections across all World Bank income regions and all child mortality settings, similar to respiratory syncytial virus and influenza virus. Infants younger than 6 months in low-income and lower-middle-income countries are at greater risk of death from human metapneumovirus-associated ALRI than older children and those in upper-middle-income and high-income countries. Our mortality estimates demonstrate the importance of intervention strategies for infants across all settings, and warrant continued efforts to improve the outcome of human metapneumovirus-associated ALRI among young infants in low-income and lower-middle-income countries.Bill & Melinda Gates Foundation. Acute lower respiratory infections (ALRIs) are one of the leading causes of morbidity and mortality in children globally, accounting for 10% of deaths in children younger than 5 years in 2017.Evidence before this studyHuman metapneumovirus is a common virus in children with acute lower respiratory infection (ALRI). Two pooled analyses have shown that human metapneumoviruses are identified in 6·1–6·4% of ALRIs in patients younger than 20 years globally. However, no global estimates of human metapneumovirus-associated ALRI burden are available for children younger than 5 years. The estimation of global human metapneumovirus burden is challenging due to the paucity of population-based studies on the incidence of human metapneumovirus infections.Added value of this studyWe did a meta-analysis based on published and unpublished data on laboratory-confirmed human metapneumovirus-associated ALRI incidence, hospital admissions, and mortality burden. We included 159 studies with data on human metapneumovirus-associated ALRI community incidence, hospital admission rates, proportion of human metapneumovirus-positive cases among hospitalised ALRI cases, and in-hospital case-fatality ratios, which included 40 high quality unpublished studies that provided data stratified by narrow age groups in children younger than 5 years. 27% of the studies were from low-income and lower-middle-income countries. Our estimates of human metapneumovirus-associated ALRI burden were stratified by the level of child mortality of the setting and by age groups, from which we identified the population subgroups most susceptible to human metapneumovirus-associated ALRI morbidity and mortality, identifying target populations and settings for future intervention studies and informing intervention strategies. Since the presence of human metapneumovirus in the upper respiratory tract in children with ALRIs does not indicate causation, we reported the global burden of ALRIs that are causally attributable to human metapneumovirus (human metapneumovirus-attributable ALRI burden), which could aid understanding with regard to the involvement of this virus in causing childhood ALRI. We estimated that human metapneumovirus could be identified in 11% of ALRI cases, 4–13% of hospital admissions due to ALRIs, and 2% of ALRI deaths among children under 5 years globally, using our estimates in conjunction with previously published all-cause ALRI burden estimates. After accounting for the causal attribution of human metapneumovirus, we estimated that 8% of ALRI cases, 3–10% of hospital admissions due to ALRIs, and 1% of ALRI-associated mortality could be attributed to human metapneumovirus. About 58% of these hospital admissions occurred in the first year of life. About 64% of the in-hospital deaths occurred in infants younger than 6 months, including 79% occurring in low-income and lower-middle-income countries.Implications of all the available evidenceTo our knowledge, our systematic review provides the first estimates of global human metapneumovirus-associated ALRIs and human metapneumovirus-attributable ALRI morbidity and mortality in children under 5 years overall and stratified by narrow age bands . Our results indicate that infants younger than 1 year have an increased risk of severe human metapneumovirus-associated ALRIs compared with older children. Infants younger than 6 months, especially those in low-income and lower-middle-income countries, are at greater risk of death from human metapneumovirus-associated ALRIs than older infants and those from upper middle-income countries and high-income countries. These findings warrant continued efforts to develop targeted intervention strategies to protect infants from human metapneumovirus infections, and to improve the outcome of infants with human metapneumovirus-associated ALRIs, especially in low-income and lower-middle income countries. Such strategies could include specific antiviral therapeutics, monoclonal antibodies, or vaccination for children and their mothers during pregnancy.A live-attenuated recombinant human metapneumovirus vaccine has been assessed in a phase 1 clinical trial; however, study findings showed that the vaccine had insufficient immunogenicity in children aged 6–59 months.16We aimed to estimate the global and regional number of human metapneumovirus-associated ALRI cases, hospital admissions, and mortality by age strata in children under 5 years for 2018, using published and unpublished data on laboratory-confirmed  human metapneumovirus morbidity and mortality. Since the presence of human metapneumovirus in the upper respiratory tract in children does not indicate that ALRI symptoms were directly caused by these viruses, we also estimated the global burden of ALRI that is attributable to human metapneumovirus by accounting for the causal attribution of human metapneumovirus. The estimates would provide evidence for the further development of targeted interventions and treatment.We did a systematic review of the human metapneumovirus-associated ALRI disease burden in children under 5 years . We searWe included studies that reported any of the following data in children younger than 5 years: community incidence rates of ALRIs . We included hCFR data for any length of study period. We included data on the proportion of human metapneumovirus-positive cases among hospitalised ALRI cases if reported for at least one full calendar year.We excluded studies: without a clear denominator population at risk ; those in which human metapneumovirus was not the primary outcome; those reporting modelled burden estimates; those in which human metapneumovirus infections were diagnosed on the basis of serology alone; and those only including population subgroups with high-risk conditions.We supplemented data from published studies with high quality unpublished data  using agreed standardised approaches and definitions within the collaborative Respiratory Virus Global Epidemiology Network.We used a modified Newcastle-Ottawa Scale to assess the risk of bias in seven domains, including study design, adjustment for health utilisation, patient groups excluded, definition, sampling strategy, diagnostic testing, and hypoxaemia ascertainment .18, 2020Our burden estimation approaches, including main analyses and sensitivity analyses, are summarised in the After meta-analyses, we estimated number of cases and number of hospital admissions using Monte Carlo simulation, which enabled the combination of meta-estimates and population estimates (UN 2018 population estimates).We estimated human metapneumovirus-associated ALRI in-hospital deaths by combining the estimates of hospital admissions and hCFRs of human metapneumovirus-associated ALRIs.Many child deaths from ALRIs occur outside of hospitals, especially in resource-limited settings due to low care-seeking or restricted access to care. Thus, we estimated an inflation factor ratio of overall ALRI deaths to in-hospital ALRI deaths at eight sites in six countries with high child mortality .The presence of human metapneumovirus in the upper respiratory tract of a child with ALRI does not indicate that human metapneumovirus caused the ALRI symptoms in every instance. Thus, we estimated the ALRI burden that is causally attributable to human metapneumovirus  by combining the human metapneumovirus-associated ALRI burden estimates and the proportion of laboratory-confirmed human metapneumovirus ALRI cases or deaths that are attributable to human metapneumovirus (the attributable fraction of human metapneumovirus in human metapneumovirus-associated ALRI cases or deaths). We used a median value of 78% as the input for the attributable fraction of human metapneumovirus-associated ALRI cases based on three multicountry studies.We did sensitivity analyses for human metapneumovirus-associated ALRI morbidity and in-hospital deaths , and estChild Health and Mortality Prevention Surveillance . Ten studies provided data for the 0–59-month age group after imputation, including six studies from settings with high child mortality; five studies were from lower-middle-income countries, two studies from upper-middle-income countries, and three studies from high-income countries . Four stWe identified 39 studies reporting human metapneumovirus-associated ALRI hospital admission rates, including 29 studies reporting data by three narrow age groups  and 502 000 ALRI hospital admissions (UR 332 000–762 000) could be attributed to human metapneumovirus in children under 5 years . We estiWe did several sensitivity analyses to estimate hospital admissions, in-hospital deaths, and overall human metapneumovirus-associated ALRI deaths, and of human metapneumovirus-attributable ALRI deaths (by country development strata and World Bank income level) using different approaches . For gloTo our knowledge, this is the first study to report global human metapneumovirus-associated ALRI burden estimates both among children younger than 5 years overall and stratified by narrow age groups . When compared with our previously published all-cause ALRI burden estimates, human metapneumovirus is identified in 11% of ALRI cases, 4–13% of ALRI hospital admissions, and 2% of ALRI deaths among children under 5 years globally.Similar to respiratory syncytial virus and influenza, we found that the human metapneumovirus-associated ALRI hospital admission rate was much higher in infants than older children, with about 58% of hospital admissions and 71% of in-hospital deaths due to human metapneumovirus-associated ALRIs in children under 5 years occurring in the first year of life.Our hCFR estimates show that young infants aged 0–5 months are at an increased risk of human metapneumovirus–associated ALRI mortality compared with infants older than 5 months. hCFR estimates for infants aged 0–5 months varied substantially across settings, possibly reflecting the differences in disease severity at admission and in the quality of hospital care. Increased disease severity at presentation could be associated with the high prevalence of certain underlying conditions and delays in care-seeking.For settings with high child mortality, the inflation factor was based on scarce data.Our study had several other limitations. Little data were available for Europe and Latin America, which could have affected the generalisation of the estimates. Moreover, heterogeneity in the methodology  existed across studies for each outcome and is likely to have biased our estimates . We adjuOur results show that infants younger than 1 year have disproportionately high risks of severe human metapneumovirus infections across settings. Infants aged 0–5 months in low-income and lower-middle-income countries have an increased risk of mortality from human metapneumovirus-associated ALRIs compared with those in upper-middle-income countries and high-income countries, accounting for nearly eight of every ten global deaths occurring in this age group. These estimates highlight the importance of interventions for infants. In low-income and lower-middle-income countries, continued efforts are needed to reduce human metapneumovirus-associated ALRI mortality among young children, especially infants, in hospitals and communities through improving access to care and case management in hospitals. Considering the paucity of data at the regional level, these estimates should be viewed as preliminary estimates. In the future, additional high quality data on childhood human metapneumovirus-associated ALRI cases, hospital admissions, and mortality, especially age-stratified data, would help refine the estimates and track changes in human metapneumovirus-associated ALRI morbidity and mortality over time.


In superficial temporal artery-to-middle cerebral artery (STA-MCA) bypass surgery, indocyanine green video angiography (ICG-VA) is usually used to verify bypass patency. Less-commonly reported is the ability to use this technique to evaluate candidate recipient vessels based on either collateral flow or identification of the distal branch of interest.An 82-year-old man presented with progressive cerebral infarction due to infarction of the M2 inferior trunk of the right middle cerebral artery. He underwent superficial temporal artery-middle cerebral artery bypass to prevent further ischemia 1 week after the initial stroke. In the surgery, M4 cortical arteries fed by the inferior trunk could not be identified as recipient arteries. Intraoperative ICG-VA showed delayed luminescence of some M4 arteries. Because the M4 arteries fed by the inferior trunk showed delayed retrograde flows from the anterior cerebral artery on preoperative digital subtraction angiography, the M4 arteries with delayed luminescence on ICG-VA were considered to be M4 arteries fed by the inferior trunk and selected as the recipient arteries.ICG-VA shows differences in flow speed as delayed luminescence. This finding may be useful for detecting target vessels. Microscope-integrated near-infrared indocyanine green video angiography (ICG-VA) is a simple, reliable, fast, and noninvasive technique introduced into neurosurgery for intraoperative observation and documentation of blood flow –5. In suA case of progressive cerebral infarction due to arteriosclerotic occlusion of the M2 inferior trunk of the right MCA, which required STA-MCA bypass to prevent further ischemia, is reported. Although it was difficult to identify the M4 cortical recipient arteries fed by the inferior trunk on gross examination, delayed luminescence on ICG-VA helped identify the correct recipient arteries.An 82-year-old man presented to our hospital with weakness of the right side of his body. He had a past history of hypertension and gastric cancer. On admission, neurological examination showed mild right hemiparesis  4). The NIH Stroke Scale (NIHSS) score was 3. Magnetic resonance imaging (MRI) showed a small acute cerebral infarction of the deep white matter in the right parietal lobe Fig. . MR angiWe obtained consent from the patient for publication of this case report. The case report was approved by the institutional review board of the Eisyokai Yosida Hospital.In selective-targeted STA-MCA bypass, an essential step is to identify the correct recipient artery, because revascularization into a wrong territory is ineffective, with a risk of severe ischemic complications . To targOn the other hand, it is controversial to perform urgent STA-MCA bypass like our case. However, efficacy of urgent STA-MCA bypass has been reporte –8. We haIn conclusion, ICG-VA shows differences in flow speed as delayed luminescence. This finding may be useful for detecting target vessels.


We present the case of a critically ill infant with presumed COVID-19, presenting with a clinical picture of bronchiolitis. This case highlights that, while exercising precautions for COVID-19 in critically ill children, it is essential to consider other more likely diagnoses.On March 11, 2020, the World Health Organization declared the coronavirus disease 2019 (COVID-19) a global pandemic, with over 10,000,000 cases and 500,000 deaths worldwide by July. Under 5% of COVID-19 infections are among children, most of whom remain asymptomatic or experience mild disease.2 50.4; HCO3 22.3). The white blood cell count was normal. An initial nasopharyngeal polymerase chain reaction (PCR) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported as positive . On the following day, a multiplex viral PCR was positive for respiratory syncytial virus (RSV), with two subsequent negative SARS-CoV-2 PCRs at 12-hour intervals.On March 20, a 4-month-old girl was brought to a community emergency department with 5 days of cough, poor feeding, and fever. There was an afebrile 2-year-old sibling with nasal congestion but no contact with known COVID-19-infected individuals. The patient presented clinical features of bronchiolitis and was hemodynamically stable but in moderate respiratory distress, requiring 2-litres/minute of oxygen. Upon transfer to our tertiary/quaternary pediatric hospital, she was placed in an airborne infection isolation room (AIIR) and cared for using airborne droplet and contact precautions. Nebulized hypertonic saline was avoided, and deep nasal suctioning was minimized. She was maintained on low-flow oxygen via nasal prongs for all transfers and placed only on non-invasive ventilation with continuous positive airway pressure (CPAP) through a full-face mask upon arrival in an intensive care AIIR. The chest X-ray  demonstrThe patient required continuous sedation to tolerate CPAP (minimizing risk of aerosolization and improving CPAP efficacy) and was gradually weaned from respiratory support, then discharged home directly from intensive care after 72 hours (minimizing transmission risk on an inpatient ward). SARS-CoV-2 testing of both parents was negative, but the sibling was not tested. The parents remained with their child, and caution was taken to restrict out-of-room time to a dedicated COVID-19 suspected/confirmed restroom wearing a face mask with strict hand hygiene.1,2 COVID-19 most frequently produces only mild symptoms in children.1 Given the relative rarity of critical illness among pediatric patients with COVID-19, few guidelines exist for this patient population.3 Importantly, infants presenting with severe respiratory distress remain far more likely to have common pediatric respiratory syndromes  than COVID-19.Our report of a critically ill infant with presumed COVID-19 illustrates important pediatric-specific considerations for diagnosis, management, and infection control in the pandemic context. This infant was the first critically ill infant in Canada with a positive SARS-CoV-2 PCR result. This previously healthy infant had no risk factors for critical illness from COVID-19, nor fit the clinical picture of pediatric COVID-19 from epidemiological reports in which nearly none have required intensive care or respiratory support.4 Multiplex PCR was positive for RSV (Ct of 26), and two subsequent SARS-CoV-2 PCRs were negative. It is possible that this child presented with RSV bronchiolitis, and a low SARS-CoV-2 viral load. Alternatively, the detection of SARS-CoV-2 may have been low-level viral shedding as seen with other ubiquitous respiratory viruses , rather than any real clinical contribution by COVID-19. Also, despite high sensitivity and specificity of PCR-based assays in general, a false-positive test result could be possible, further introducing clinical uncertainty.This case highlights the diagnostic uncertainty when assessing critically ill children during the COVID-19 pandemic. Despite a presentation consistent with bronchiolitis, the initial positive SARS-CoV-2 PCR shifted the presumptive diagnosis to RSV/SARS-CoV-2 co-infection. Clinicians should avoid premature closure (a form of cognitive bias), searching for other viruses even when faced with a positive SARS-CoV-2 test in the context of severe respiratory disease. In this case, initial nasopharyngeal PCR was positive for SARS-CoV-2 at a Ct value of 35. Higher viral loads are inversely related to Ct values and vary by technique, with values among COVID-19 patients typically between 22–28 and 30–32 for nasopharyngeal and oropharyngeal swabs, respectively.5 However, computed tomography should be avoided for this indication in children, as this would lead to significant ionizing radiation exposure for a majority of children with seasonal viral illness.Adult studies suggest that chest computed tomography is more sensitive than PCR for a diagnosis of COVID-19, and chest imaging abnormalities have been described on computed tomography in children.Emergency management for bronchiolitis was modified due to presumptive COVID-19. The transmission of SARS-CoV-2 occurs via respiratory droplets and contact with contaminated fomites; airborne spread can occur with aerosol-generating procedures (AGPs). Procedures such as nasopharyngeal suctioning, nebulization-delivered therapies, high-flow nasal oxygen, CPAP, and tracheal intubation are considered AGPs. The advantages of these therapies for bronchiolitis must be balanced against the risk of SARS-CoV-2 aerosolization, particularly when stores of personal protective equipment (PPE) are limited. For therapies deemed necessary, clinicians should don appropriate PPE and proceed. However, nebulized hypertonic saline, for example, may modestly benefit infants with bronchiolitis, but its routine use would be difficult to justify in the pandemic context. The risk of viral dispersion from CPAP depends ultimately on several factors, including flow, duration, and patient cooperation; therefore, the risk of airborne transmission must be weighed against the benefit of avoiding intubation. Ultimately, AGPs should be used very judiciously and, when undertaken, should be performed in AIIR under airborne, droplet, and contact precautions. Emergency management of critically ill children may require departures from pediatric standards of care, such as the separation of child-and-parent to limit nosocomial transmission or limiting blood gas measurements due to additional risks of lab contamination, exposure for personnel, and PPE consumption.During the COVID-19 pandemic, physicians caring for critically ill children will have to remain vigilant with infection control precautions, while addressing more likely underlying etiologies.J Clin Epidemiol 2014;67(1):46-51).Informed consent was obtained from the family. Manuscript was prepared in accordance with CARE guidelines for case reports (Gagnier JJ, Kienle G, Altman DG, et al.; and CARE Group. The CARE guidelines: consensus-based clinical case report guideline development.


Introduction: Treatment of methicillin-resistant (MR) staphylococcal prosthetic joint infections (PJIs) remains a matter of discussion, with vancomycin-rifampin combination therapy being the preferred treatment for DAIR and one-stage exchange arthroplasty strategies. This study analyzes the outcomes of patients with chronic methicillin-resistant coagulase-negative staphylococcal PJIs treated with vancomycin-minocycline combination therapy.Methods: This prospective, single center cohort study included all chronic MR coagulase-negative staphylococcal PJIs (01/2004-12/2014) treated with exchange arthroplasty and at least 4 weeks of minocycline-vancomycin. The following endpoints were considered: reinfection including relapse (same microorganism) and a new infection (different microorganism) and PJI-related deaths. Their outcomes were compared with PJIs treated with rifampin-vancomycin during the same period.Results: Thirty-four patients  with 22 hip and 12 knee arthroplasty infections were included. Sixteen (47%) had previously been managed in another center. Median vancomycin MIC of strains was 3 mg/L. Nineteen underwent one-stage, 15 two-stage exchange arthroplasty. After a median [IQR] follow-up of 43 [26-68] months, 2 patients relapsed and 6 developed a new PJI. Compared to 36 rifampin-vancomycin treated PJIs, relapse- or reinfection-free survival rates didn't differ, but more new infections developed in the minocycline group .Conclusions: Minocycline-vancomycin combination therapy for chronic MR coagulase-negative staphylococcal PJIs seems to be an interesting therapeutic alternative. Vancomycin monotherapy failed to treat experimental staphylococcal bone infection Medical and surgical treatments of chronic prosthetic joint infection (PJI) are still a matter of debate. They combine complete removal of the infected device and effective antibiotic therapy. Staphylococci are the most frequent microorganisms isolated, with 15-50% of the isolates being methicillin-resistant In vitro, minocycline remains effective against tetracycline-resistant isolates Minocycline, a tetracycline antibiotic, is naturally effective against methicillin-resistant staphylococci, including multiresistant isolates. The aim of this study was to analyze the outcomes of patients treated with combination antibiotic regimen including minocycline for chronic methicillin-resistant staphylococcal PJI.This prospective cohort study was conducted in a French National Referral Center for Bone-and-Joint Infections from January 2004 to December 2014. All patients with PJIs admitted to the Center are registered in our prospective PJI cohort . Data were extracted from that database. Written informed consent was obtained from all patients and the cohort was approved by the Île-de-France Ethics Committee.All patients treated for chronic, i.e., lasting >1 month, methicillin-resistant staphylococcal PJIs who underwent 1- or 2-stage exchange arthroplasty were eligible. To be included in the minocycline group, patients had to be ≥18 years, and receive at least 4 weeks of first-line antibiotics combining oral minocycline and continuous intravenous (IV) vancomycin. PJI was defined Patients with polymicrobial PJIs, including microorganism(s) other than staphylococci, were not included.S. aureus were non-included, because they were very few (n=3).Finally, patients with methicillin-resistant . Drug-resistance patterns were determined with the disk-diffusion method for all specimens and for each colony with a different morphology. Vancomycin MICs were determined with E-test . When a given patient had several different isolates identified, we retained the most resistant staphylococcal strain for treatment and description.Preoperative joint aspirates and intraoperative samples were handled as previously described 3Staphylococcus.All minocycline group patients received initial combination antibiotic therapy with high-dose continuous IV vancomycin and oral minocycline for 4-6 weeks, followed by 6-8 weeks of an oral regimen. Vancomycin treatment modalities and drug monitoring have previously been described Reasons using minocycline instead of rifampin-combination therapy were rifampin resistance, drug interaction, previous intolerance, reported allergy to rifampin, or previous treatment failure with rifampin.Minocycline was taken orally: 100 mg thrice daily for patients weighing <90 kg and 200 mg twice a day for those >90 kg. To avoid its malabsorption, all concomitant medications associated with decreased drug absorption  were stopped. Treatment compliance and gastrointestinal tolerance were monitored throughout the entire treatment duration.Adverse drug-reaction severity was assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) One-stage exchange arthroplasty was performed, provided that no bone reconstruction was necessary and that the microorganism had been isolated from pre-operative joint aspirates Patients were discharged at the end of IV antibiotic therapy and then seen as outpatients, at 3, 6, 12 and 24 months, and then every 2 years. For patients not seen for >2 years, they or their primary physicians were contacted by telephone. Outcome analysis required a minimum of 2 years post-surgery to the last follow-up visit. The following endpoints were considered: reinfection including relapse with the same microorganism as the initial PJI or a new infection with a microorganism different from the initial PJI, and PJI-related death (infection- or treatment-related).The primary outcome of interest was the cumulative probability of reinfection, i.e., relapse or new infection.2 tests. Quantitative variables are expressed as median , first assessed for normality and then compared with either Student or Mann-Whitney U-test. The relapse- and reinfection-free survival rate was analyzed using Kaplan-Meier method and expressed as the percentage ± standard deviation. The Mantel-Cox log-rank test was used to test the between-group survival-distribution difference. A P value < 0.05 was considered significant. All statistical tests were performed with SPSS.20 software.Qualitative variables are expressed as number (%) and compared using χTo assess minocycline-combination-therapy efficacy and tolerance, we compared outcomes of these patients to those receiving initially 4-6 weeks of continuous IV vancomycin and rifampin-combination therapy, followed by 6-8 weeks of an oral regimen and managed during the same period. These patients were defined as the rifampin group. We chose rifampin-combination therapy as comparator according to IDSA guidelines, as it is the preferred therapy for methicillin-resistant staphylococcal PJIs treated with DAIR and one-stage implant exchange During the study period, among 832 PJIs managed in our Center, 90 10.8%) were chronic methicillin-resistant staphylococcal PJIs, who underwent one or two-stage exchange arthroplasty and received minocycline-combination therapy , vancomycin-fusidic acid (n=2), vancomycin-fosfomycine (n=2), vancomycin-gentamicin (n=2), or vancomycin (n=1) or linezolid monotherapy (n=1).S. aureus PJI were included in this group.To avoid confusion biases, 6 minocycline patients who had received rifampin during the oral therapy, were excluded from the analysis. Three methicillin-resistant Finally, 34 (5%) patients were included in the minocycline group. Baseline characteristics of the patients are reported in Table Reasons not to use initial rifampin combination therapy were rifampin resistance in 26, potential drug interaction in 1, previous treatment failure with rifampin in 2, drug intolerance in 2, allergy in 1 and not determined in 2 patients.S. epidermidis (91%). Vancomycin MIC was >2 mg/L for more than two-thirds (n=24) of the isolates. Resistance patterns to other antibiotics are reported in Table The staphylococcal species isolated in the minocycline group are shown in Table Durations of IV and total antibiotics, and surgical interventions in the minocycline group are detailed in Table The initial IV antibiotic therapy was followed by 6-8 weeks of an oral regimen with minocycline monotherapy, except 7 who received linezolid (n=2) or combination therapy with minocycline and clindamycin (n=5).All the patients received minocycline 100 mg, thrice daily, except an 88-year old woman given 100 mg twice daily and 5 others taking 200 mg twice daily.Median [IQR] follow-up was 43 Staphylococcus epidermidis knee-arthroplasty infections relapsed. Both had undergone 2 or 3 prior surgeries for their PJIs. Their staphylococci were only susceptible to cyclines, linezolid and glycopeptides (vancomycin MIC 2 mg/L). One underwent one-stage, the other two-stage exchange arthroplasty. Both received vancomycin-minocycline for 6 weeks, followed by minocycline for further 6 weeks and relapsed 1 month after stopping antibiotics. No minocycline resistance was observed. One patient's vancomycin MIC increased from 2 to 4 mg/L.Only 2 patients with methicillin-resistant Pseudomonas aeruginosa, Enterobacter cloacae, Cutibacterium acnes or polymicrobial flora, respectively. Acute hematogenous PJIs occurred in 2 other patients one year after their first PJIs and were due to susceptible S. aureus and Proteus mirabilis.Six patients with prosthetic hip infections developed new infections: 4 were late chronic infections that developed 2-4 years after the first PJI and were caused by Three patients experienced minocycline adverse events, necessitating drug withdrawal for one with thrombopenia and another with cholestatic hepatitis. Three patients developed grade 2 renal insufficiency attributed to vancomycin.Five patients initially treated with vancomycin-minocycline were not included in the study because they took minocycline less than 4 weeks , but fewer hip infections , their isolated strains had higher vancomycin MICs , and with a trend towards more patients having undergone 3 or more surgeries for their PJIs . Two-stage exchange arthroplasty was done more frequently in the minocycline group, without reaching significance . Although more new infections occurred in the minocycline group , neither relapse- nor reinfection-free survival rates differed significantly between the 2 groups  relapsed and 80% of the patients were infection-free at 2 years of follow-up, even though 47% of them had already undergone at least 1 surgery for their PJIs and multiresistant staphylococci prevented the use of "gold standard" antibiotics, e.g. fluoroquinolones or rifampin To enhance the message of these outcomes, we compared the minocycline treated group to patients treated during the same time period with vancomycin-rifampin, the preferred treatment for methicillin-resistant staphylococcal PJI S. aureus (MRSA) skin-and-soft-tissue infections in vitro activity against MRSA embedded in biofilm. Our Referral Center's long experience using minocycline started in 1984, following Biddle's presentation at the 24th ICAAC, to treat methicillin-resistant staphylococcal bone-and-joint infections, although further and larger data in humans are lacking. Pertinently, minocycline was always used in combination with vancomycin during the first weeks of treatment.Minocycline has been used for many years to treat multiresistant staphylococcal infections in vitro susceptibility rates than tetracycline and doxycycline Staphylococcus strain susceptible to minocycline; none of them relapsed. That finding highlights minocycline efficacy even against tetracycline- or doxycycline-resistant strains tet(K) and tet(L) genes coding for drug-efflux pumps that confer resistance to tetracycline and doxycycline, but not minocycline or tigecycline Among the tetracycline-family members, minocycline has higher Other important advantages of minocycline are its low cost, its oral administration and good bone diffusion. To treat these complex bone and joint infections we used higher minocycline dosage than recommended in order to reach effective minocycline bone concentrations. Among our patients, we observed high minocycline bone penetration in 6 (unpublished data), with bone minocycline concentrations well above those of serum . No study on minocycline bone diffusion has been published yet and data on diffusion of other tetracyclines are scarce S. aureus infections. Data on other combination therapies are lacking.All our patients received vancomycin-combination therapy, even those treated with two-stage exchange. IDSA guidelines recommend combination therapy with rifampin only in case of implant retention (DAIR) or one-stage exchange arthroplasty. But experiences in animals showed that vancomycin monotherapy failed to sterilize staphylococcal bone infections Tigecycline, a glycylcycline antibiotic derived from minocycline, is effective against a broad-spectrum of bacteria, including methicillin-resistant staphylococci. Success rates of 76-85% after tigecycline treatment of bone-and-joint infections in 2 case series have been reported Drug tolerance in our study was acceptable, despite the use of higher dosages. Adverse event and withdrawal rates in the minocycline treated group are lower than those reported for other anti-staphylococcal antibiotics S. aureus infections, because they were only very few in the cohort and S. aureus is a notably different organism to treat, nor polymicrobial infections with non-staphylococcal species to avoid confusing results. Fourth, the study was not randomized and the risk of bias exists. Indeed, some patients' characteristics differ between the groups, as already discussed. Finally, to analyze minocycline efficacy, we excluded patients who had received it for less than 4 weeks, including those who stopped minocycline because of adverse events, and those who received rifampin during oral treatment. But to examine tolerance more thoroughly, we separately analyzed the patients with less than 4 weeks of minocycline and addressed those observations in the discussion of tolerance.Our single center, observational, comparative study, has several limitations. First, all the patients received combination therapy with vancomycin, limiting the evaluation of minocycline efficacy alone. However, previous studies, especially animal models, showed that vancomycin monotherapy cannot cure chronic staphylococcal osteomyelitis In conclusion, minocycline-vancomycin combination therapy to treat complex chronic methicillin-resistant coagulase-negative staphylococcal PJIs managed with exchange arthroplasty achieved 80% favorable outcomes at 2 years. Minocycline appears to be an effective therapeutic alternative for these difficult-to-treat infections, especially when rifampin cannot be used. More data from large prospective randomized trials are necessary to confirm our preliminary observations.


To assess the effect of prone positioning therapy on intubation rate in awake patients with COVID-19 and acute respiratory failure.This is a two-center parallel group, superiority, randomized  controlled trial.All patients admitted to the Hospital Civil de Guadalajara and Hospital General de Occidente in Mexico for COVID-19 associated acute respiratory failure and in need of supplementary oxygen through high-flow nasal cannula are screened for eligibility.Inclusion criteria: all adult patients admitted to the COVID-19 unit who test positive for COVID-19 by PCR-test and in need for oxygen are eligible for inclusion. Randomization starts upon identification of requirement of a fraction of inspired oxygen ≥30% for an oxygen capillary saturation of ≥90%Exclusion criteria: less than 18 years-old, pregnancy, patients with immediate need of invasive mechanical ventilation , vasopressor requirement to maintain median arterial pressure >65 mmHg, contraindications for prone positioning therapy  guidelines (Additional file The online version contains supplementary material available at 10.1186/s13063-020-04882-2. Additional file 1Additional file 2


Science by Fiachra Humphries et al.1 reveals an important role for the metabolic intermediate fumarate in reducing gasdermin D-mediated pyroptosis. Their work adds a new inhibitor to the growing toolkit with the potential for treating pyroptosis-driven inflammatory diseases.A recent study in Pyroptosis is an inflammatory programmed cell death pathway initiated by direct sensing of pathogen-derived ligands or indirect sensing of changes to the normal cell state through sensors that can activate the inflammasome. Caspase-1 is recruited to the multimeric inflammasome to become activated and direct downstream pyroptosis and maturation of the proinflammatory cytokines IL-1β and IL-18. Similarly, caspase-11 directly senses cytosolic lipopolysaccharide (LPS) to drive pyroptosis. The lytic cell death associated with pyroptosis mediates clearance of pathogens and release of inflammatory cytokines. However, dysregulation of inflammasome activation is associated with many autoinflammatory diseases and cancer progression.3 Cleavage of the linker domain of GSDMD releases the autoinhibitory C-terminal domain and allows the N-terminal domain to oligomerize and form pores on the plasma membrane of cells. The pore-forming activity of GSDMD during pyroptosis was found to be critical for cell death and facilitating the release of IL-1β and IL-18. The gasdermin (GSDM) family in humans includes GSDMA, GSDMB, GSDMC, GSDMD, GSDME (or DFNA5), and PJVK (or DFNB59), while mice lack GSDMB but possess GSDMD, GSDME, and GSDMA (GSDMA1-3), and GSDMC (GSDMC1-4) homologs. The N-terminal domains of GSDMA, GSDMB, GSDMC, and GSDME can also induce cell death similar in morphology to GSDMD-mediated pyroptosis, but the conditions under which these other GSDM family members are activated are still poorly understood. Recently, caspase-3-mediated cleavage of GSDME was found to promote a lytic form of cell death similar to GSDMD-mediated pyroptosis. Gsdmd−/− mice are viable and developmentally normal, suggesting GSDMD is largely involved in regulating inflammatory cell death. Whether GSDM family members have a role in non-immune cells requires further study.In 2015, researchers identified gasdermin D (GSDMD) as a key substrate of caspase-1 and caspase-11 (mouse) or the human homolog caspase-4/5.4 Treating mice with NSA protected them from LPS shock.4 The FDA-approved drug disulfiram was also recently found to specifically inhibit GSDMD pore formation but not caspase-1 or GSDMD cleavage by covalent addition of dithiodiethylcarbamoyl (DTC) at Cys191/192.5 Interestingly, this study found that NSA treatment inhibited the cleavage of caspase-1, GSDMD, and IL-1β, while disulfiram treatment only inhibited GSDMD pore formation. Disulfiram, similar to NSA, also reduced mortality in a mouse model of LPS shock. Together, these studies also established a critical role for the Cys191/192 residue in GSDMD inhibitor function.Recent studies have identified inhibitors of GSDMD function. In 2018, necrosulfonamide (NSA) was found to inhibit GSDMD function, likely through alkylating a key cysteine residue (Cys191 in human or Cys192 in mouse), which disrupts the ability of the N-terminal domain of GSDMD to oligomerize and form membrane pores.1 in Science, the citric acid cycle metabolic intermediate fumarate was also shown to reduce pyroptosis -cysteine. This modification broadly prevented GSDMD-mediated cell death and cytokine release. DMF treatment also modified other GSDMD cysteine residues, suggesting succination may have other off-target effects. Treatment with exogenous DMF reduced both the cleavage of GSDMD and the interaction between GSDMD and caspase-1 while also leaving caspase-1 activation intact. Further experiments showed that succination broadly alters GSDMD protein-protein interaction and function. In addition to limiting GSDMD activity, DMF also reduced the cleavage of GSDME in GSDMD-deficient cells treated with the pyroptosis-inducing trigger LPS plus nigericin, potentially via succination of GSDME reactive cysteines. Treatment of mice with fumarate reduced mortality following LPS shock and limited disease in mouse models of familial Mediterranean fever (FMF) and experimental autoimmune encephalitis (EAE).In a recent publication by Fiachra Humphries et al.The expanding repertoire of molecules that inhibit pyroptosis through different mechanisms and at different steps has important implications for treating inflammatory diseases. Multiple inhibitors have been identified that target upstream inflammasome activation. Notably, NLRP3 inhibitors  have been proposed to treat autoinflammatory diseases including multiple sclerosis and arthritis. Additionally, monoclonal antibodies and recombinant proteins that inhibit IL-1 signaling  have been used extensively to treat autoinflammatory diseases and may have a role in limiting cancer. Because GSDM family members (including GSDMD and GSDME) mediate both the membrane lysis of dying cells as well as the release of inflammatory cytokines and DAMPs, there is also continued interest in identifying inhibitors that regulate GSDM pore-dependent release of inflammatory mediators (including HMGB1 and inflammasome-dependent cytokines) or alter pore-mediated changes in ion homeostasis. While current GSDMD inhibitors all functionally modify Cys191/192 to achieve their inhibitory activity, making synergy between them unlikely, differences in bioavailability/pharmacokinetics in tissues may constrain their use. The development of novel inhibitors that target the GSDM family or combining inhibitors that target discrete steps in pyroptosis signaling may prove beneficial.1 which further indicates off-target effects may need to be overcome. This work also found that inhibiting the citric acid cycle enzyme fumarase could inhibit GSDMD-dependent cell death,1 suggesting alternative strategies for inhibiting pyroptosis. Further study into the molecular mechanisms of action for the Cys-reactive inhibitors that have been identified will clarify how they function differently from one another. While major hurdles remain in understanding the specific role each of these different inhibitors may have in treating autoinflammatory disease, research into this area is likely to have major therapeutic implications.One of the major limitations of inflammasome inhibitors has been off-target effects on other programmed cell death pathways and inflammatory signaling pathways. Indeed, current GSDMD inhibitors each modify reactive cysteines, which is likely to also affect caspases, which are cysteine-dependent proteases. Necrosulfonamide has also been shown to inhibit necroptosis by targeting a reactive cysteine residue of another pore-forming executioner protein, MLKL. Additionally, DMF inhibited both GSDMD and GSDME cleavage and modified multiple cysteine residues on each,


Relapse remains the main cause of treatment failure in acute myeloid leukemia (AML) undergoing allogeneic hematopoietic stem cell transplantation . Emerging evidence has demonstrated that AML patients might benefit from maintenance therapy post-transplantation, especially for high-risk AML patients. In this mini-review, we will summarize targeted drugs, such as hypomethylating agents, FLT3 inhibitors and isocitrate dehydrogenase inhibitors, as maintenance therapy post-transplantation in AML patients undergoing allo-HSCT. Acute myeloid leukemia (AML) is a heterogeneous group of clonal diseases. Conventional chemotherapy can result in the complete remission (CR) rate of approximately 70–80% in AML patients , 2. LeukThe broad definition of maintenance therapy includes preemptive therapy based on measurable residual disease (MRD) and prophylactic therapy not based on MRD. The narrow definition of maintenance therapy refers only to prophylactic therapy. It remains under discussion whether preemptive or prophylactic therapy is superior in reducing relapse and improving survival for AML patients , 11. HerHypomethylating agents such as azacitidine and decitabine have shown favorable efficacy and tolerability as induction therapy in AML patients, especially elderly patients unable to tolerate intensive chemotherapy , 13. SomFLT3 internal tandem duplication (FLT3-ITD) mutations occur in approximately 25% of adults with AML. In contrast to AML with FLT3 wild-type, AML patients with FLT3-ITD mutations have shorter remissions and higher relapse rates , 24. AllSorafenib, a multi-kinase inhibitor, has shown promising efficacy in the treatment of FLT3-ITD AML, including maintenance post-transplantation –33. ReceSome retrospective and single-arm exploratory studies revealed that post-transplantation maintenance of other FLT3 inhibitors could also reduce relapse and improve survival for patients with FLT3-ITD AML , 41. TheApproximately 20% of AML genomes harbor mutations in one of two isoforms of IDH (IDH1 or IDH2). IDH mutation reduces α-ketoglutarate to 2-hydroxyglutarate, leading to histone hypermethylation and a block in myeloid differentiation . AML patVenetoclax, a selective small-molecular inhibitor of B-cell lymphoma-2 (Bcl-2), has been demonstrated that single-agent or combined with other agents are effective and tolerable to AML patients including relapsed or refractory AML patients, especially in elderly patients unfit for intensive chemotherapy, with overall response rate of 19–72% –50. A phHistone deacetylase inhibitors (HDACi) are epigenetic modifiers that are shown to induce cell-cycle arrest, cell differentiation and apoptosis of AML cells. Panobinostat is an oral HDACi that has been reported to have moderate antileukemia activity in advanced AML and MDS patients . A phaseThe Hedgehog signaling pathway plays a critical role in embryonic development and aberrant Hedgehog signaling are deemed to contribute to the survival and expansion of leukemia stem cells . GlasdegTo date, the duration of maintenance of targeted drugs post-transplantation is not firmly established. In some retrospective and prospective studies, targeted drugs are usually maintained for 1–2 years after allo-HSCT , 8, 23. Traditional immunotherapies such as donor lymphocyte infusion, interleukin-2 and interferon-α might play a role in preventing relapse for AML patients after allo-HSCT , 58. HowOver the past decade, some novel targeted drugs have been explored for post-transplant maintenance for AML patients, and have shown promising results. Two back-to-back RCTs both reveal that sorafenib maintenance after alloHSCT prevents relapse in patients with FLT3ITD AML, resulting in an OS benefit. However, most of the studies about maintenance therapy in AML patients after allo-HSCT are retrospective and non-randomized. Currently, there is no consensus on the optimal duration of targeted drugs to prevent relapse, and it remains unclear whether long-term exposure to targeted drugs induces drug resistance. And which specific subpopulations of AML patients will benefit post-transplant maintenance most remains to be further investigation. Therefore, well-designed, fully powered, prospective trials are needed to address the unsettled questions to further improve the outcomes post-transplantation. Except for targeted drugs, some immunotherapies including CAR T-cell therapy, antibody-directed therapy, immune checkpoint inhibitors and vaccines have also attracted board attention.