Higher-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) of the elderly exhibit several commonalities, including first line treatment with hypomethylating agents (HMA) like azacitidine (AZA) or decitabine (DAC). Until today, response to treatment occurs in less than 50 percent of patients, and is often short-lived. Moreover, patients failing HMA have a dismal prognosis. Current developments include combinations of HMA with novel drugs targeting epigenetic or immunomodulatory pathways. Other efforts focus on the prevention of resistance to HMA using checkpoint inhibitors to enhance immune attack. This review focuses on recent advances in the field of HMA-based front-line therapies in elderly patients with myeloid diseases. SRSF2, U2AF1, SF3B1) across WHO-defined blast thresholds. Studies have demonstrated that SF3B1-mutant HR-MDS and AML patients are clinically, cytologically, and molecularly highly similar; this overrides the artificial separation between both diseases [de novo AML type [SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR and STAG2, have been specifically linked to secondary AML as opposed to de novo AML patients, suggesting that they may primarily drive the specific biology of the disease [At diagnosis, median age of patients with acute myeloid leukemia (AML) is 67 years, which means that more than half the cases are elderly adults ,2. The sdiseases . AdditioAML type . Moreove disease .n = 463) [Over the last years, treatment outcomes have improved in younger patients, but remain poor in older adults with HR-MDS or AML ,11. A ren = 463) , althougTreatment algorithms of older patients had remained essentially uniform for decades, but changed after the introduction of hypomethylating agents (HMAs) . HMAs suThus, in this review, we will discuss upcoming novel treatment strategies beyond single agent HMAs for older patients unsuitable for standard ICT.2 AZA for 7 days every 28 days, over 6 cycles involving 43 patients with HR-MDS had been published. In the trial, 49% of patients responded, and results have been very closely reproduced in subsequent phase II and III trials [AZA and DAC are both cytosine analogues developed in the 1960s ; howeverI trials . In 2009I trials . Median I trials .2 IV over 4 h three times a day for 3 days, in 6-week cycles, was compared to BSC only in 233 HR-MDS patients older than 60 and not eligible for ICT, in a randomized phase III trial. There was no significant prolongation of median OS comparing DAC to BSC , but progression-free survival (PFS) was significantly longer in patients treated with DAC compared to BSC  [In 2011, low-dose DAC 15 mg/mctively) . Responsctively) . Also bactively) .de novo versus secondary AML, baseline BM blasts above 30%, intermediate- versus poor-risk cytogenetics and ECOG PS of 2 versus 0 to 1 [de novo or secondary AML, who were ineligible for treatment by standard ICT. In 2012, DAC was evaluated in a multicenter randomized phase III trial in 485 older (≥65 years), newly diagnosed AML patients with poor or intermediate- risk cytogenetics compared to CCR like BSC or LDAC. A post-hoc analysis revealed a significant increased OS rate in favor of DAC , including a better CR rate  . In an es 0 to 1 . In factIn 2015, the AZA-AML-001 phase III trial, copying the trial design of the respective MDS study , investiHMAs are given continuously until progression and median response duration is about one year. However, a subset of patients has long lasting remission, in rare cases for more than 3–4 years ,18. NeveThe European ALMA score (E-ALMA) categorized three risk groups with different survival and response rates to AZA , based on ECOG, white blood cell counts (WBC) before AZA onset, and cytogenetics . AZA seems to be a reasonable treatment option, especially for patients with a favorable or intermediate E-ALMA scores . Bores eMoreover, recent developments in the molecular characterization of MDS and AML have revealed the presence of the mutations involved in epigenetic processing . The impact of different mutations on response to AZA in MDS or AML has been evaluated in several studies. One of the first studies sequenced the TET2 gene in 86 MDS and AML patients with up to 20–30% blasts treated with AZA. While 15% of these patients carried TET2 mutations, ORR including HI was 82% in TET2 mutated versus 45% in wild-type patients. Thus, mutated TET2 and favorable cytogenetic risk independently predicted a higher response rate to AZA treatment. However, response duration and OS were not different . Bejar eWelch et al. investigated molecular determinants of clinical responses after a 10-day DAC regimen in 116 patients with AML, including 26 patients with MDS. The clearance of leukemia-specific mutations correlated closely with morphologic and cytogenetic responses. Especially mutations in TP53 and SF3B1 genes showed consistent, rapid reductions in variant allele frequency, to levels of less than 5% after DAC treatment. Surprisingly, all patients with TP53 mutations had a response after DAC treatment with BM blast clearance, as compared to 47% of patients without TP53 mutations . In otheCurrently, there are no prospective data reporting on a direct comparison between AZA and DAC. However, retrospective data do not indicate a clear superiority of either compound .Guadecitabine, a dinucleotide of DAC and deoxyguanosine, is a new-generation DNA hypomethylating drug that shows similar potency, but improved pharmacokinetic properties. The agent was designed to prolong the exposure of tumor cells to its active metabolite DAC . Guadeci2 or 90 mg/m2 guadecitabine on days 1–5 (5-day regimen). The second cohort was treated with a 10-day regimen: 60 mg/m2 guadecitabine on days 1–5 and days 8–12, for up to 4 cycles, followed by 60 mg/m2 for 5 days in subsequent cycles. It was shown that 23% of all patients had CRc to guadecitabine, and the median survival was 6.6 months; 19% of patients were alive after 2 years [2 per day subcutaneously for 5 days in 28-day cycles. There were no significant differences between these two doses in overall CR rates of all treated patients [2 per day subcutaneously versus the physician’s choice of treatment , in patients with previously untreated AML who are ineligible for ongoing treatment with ICT  in 800 treatment naïve AML patients, who are ineligible for treatment with ICT. Guadecitabine is also under investigation in a phase III trial , including 404 AML patients who failed or relapsed following prior ICT.In solid tumors, there are discussions about whether guadecitabine might sensitize tumor cells to immunotherapeutics, and resensitize resistant cancer cells to chemotherapeutic drugs ,34. Another exciting, “novel” HMA is an oral formulation of 5-azacitidine (CC-486), which may provide a more convenient means of administration than the conventional AZA SC . FurtherBased on these results, the randomized, placebo-controlled phase III QUAZAR LR-MDS trial  with a target enrollment of 386 LR-MDS patients with red blood cell transfusion depended anemia and thrombocytopenia was initiated, comparing 300 mg CC-486 for 21 days of repeated 28-day cycles plus BSC versus placebo plus BSC. Low-dose CC-486 (200 or 300 mg for the first 7 or 14 days of each 28-day cycle) is also currently being investigated in a phase I/II trial  examining its use as a maintenance therapy following HSCT in ~30 patients with MDS or AML (QUAZAR Post-Transplant Study). Additionally, a phase III study is ongoing, where oral AZA (300 mg for 14 days of 28-day cycles) is evaluated as a maintenance therapy after ICT without transplant in AML patients . 2 IV) daily, on days 1−5, or AZA  daily, on days 1−7 of each 28-day cycle, in combination with once-daily oral VEN (400 mg or 800 mg). Preliminary data shows 41% CR in patients with intermediate- and 30% CR in patients with poor-risk cytogenetics. Median OS in all patients was 17.5 months. A subgroup analysis comparing AZA to DAC, and VEN 400 mg versus 800 mg is currently ongoing. Using VEN, the most common treatment emergent adverse events were nausea and febrile neutropenia [BCL-2 is a pro-survival protein preventing apoptotic cell death and facilitating MYC-induced transformation . The protropenia . Another phase Ib/II open-label study (NCT02287233) evaluated VEN combined with LDAC in patients ≥65 years and previously untreated AML, who were ineligible for treatment with ICT. Among patients given VEN 600 mg and LDAC, 62% achieved CR/Cri , with a median duration of CR/CRi of 14.9 months . In the Lenalidomide (LEN) belongs to a class of compounds called immunomodulatory derivatives (IMiDs). LEN has been shown to possess anti-angiogenic activity through the inhibition of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and TNF-alpha induced endothelial cell migration . In addiA phase III randomized study evaluated LEN in 205 IPSS low-/intermediate-1-risk del5q MDS patients with RBC transfusion-dependency. Patients received a placebo or LEN 10 mg/day on days 1–21, or 5 mg/day on days 1–28, of a 28-day cycle. More patients in both LEN arms achieved RBC-transfusion independence (TI) compared to patients receiving a placebo (56.1% and 42.6% versus 5.9%). After LEN 10 mg/day, cytogenetic response rates were 50.0% versus 25.0% after LEN 5 mg/day . As a reIn other clinical trials, LEN monotherapy has been evaluated extensively in previously untreated, older (>60 years) AML patients, with or without del(5q). In one of these studies, AML patients with del(5q) were treated with LEN 50 mg daily for 28 days as induction therapy, and 10 mg daily for 21 days of a 28-day cycle, as maintenance therapy. Among the 37 evaluable patients, 14% achieved a PR or CR, with relapse-free survival of about 5 months. Median OS was 2 months for the entire population, showing a modest activity of LEN monotherapy in older del(5q) AML patients [Another study evaluated 33 newly diagnosed AML patients ≥60 years, without isolated 5q abnormalities. Again, patients received LEN at 50 mg daily for up to two 28-day cycles, and 10 mg LEN daily as maintenance therapy. After LEN induction therapy, the overall CR/CRi rates were 30% and 53% respectively, albeit with a median OS from study enrollment of about 4 months only .The combination of AZA and LEN is supported by reasonable single-agent activities across all stages of MDS and AML, irrespective of their different and not overlapping modes of action. Cumulative hematological toxicity seemed to be the only clinical limitation of this combinatorial approach. While LEN is known for its inhibition of cell-cycle progression, and promotion of apoptosis in cells that are not actively dividing, AZA’s effects depend on actively dividing cells . The comTP53 mutations in patients achieving complete cytogenetic response and hematological remission after AZA and LEN, suggesting the potential benefit of combining both agents. This effect was not reported with LEN alone; therefore, whether the addition of AZA might improve treatment outcome remains to be determined in future studies [We evaluated the combination of AZA and LEN in 20 del(5q) HR-MDS or AML patients (median age 69 years) within a phase I trial, including 35% of patients with AML . In the  studies ,52. However, the largest randomized study so far failed to show a marked benefit from this combination in HR-MDS patients. Sekeres et al. evaluated AZA alone versus a combination with LEN or vorinostat in HR-MDS and CMML, in a phase II study including 277 patients. Differences in ORR were not statistically significant, with 38% for patients receiving AZA, 49% for AZA plus LEN, and 27% for AZA plus vorinostat . FurtherA phase II randomized Australian study (ALLG MDS4) also evaluated the regimen AZA ± LEN in 160 HR-MDS and AML patients with low blast count. AZA monotherapy showed an ORR of 56% versus 69% in the combination arm. Nevertheless, PFS demonstrated no benefit through the addition of LEN to AZA in this population .Ades et al. evaluated escalating doses of LEN combined with ICT in a phase II study including 82 patients with HR-MDS or AML and 5q deletion. In the study, 46% of patients achieved CR, and the ORR was 58.5%. The study included 62 patients with complex karyotype, in which 44% achieved CR, median OS was 8.2 months . 2 iv five days of a 4-week cycle) and sapacitabine  [Sapacitabine is a novel oral nucleoside analog with a unique ability to induce single-strand DNA breaks after incorporation into DNA , and prok cycle) . Of the k cycle) .Transcriptional silencing of tumor-suppressor genes and other genes involved in differentiation and apoptosis is a result of DNA promotor hypermethylation and post-translational modification, like deacetylation of histone tails. Thus, inhibition of histone deacetylation and DNA hypermethylation can induce re-expression of silenced genes in leukemia in a synergistic fashion. In HR-MDS and AML, monotherapy with histonedeacetylaseinhibitors (HDACi) such as valproic acid, entinostat, vorinostat, panobinostat, or pracinostat has demonstrated only limited efficacy in phase I and II clinical trials ,60. TherIn another phase II study, 184 patients with HR-MDS or CMML were randomly assigned to AZA ± vorinostat. With a median follow-up of 23 months, the ORR was 38% for patients receiving AZA monotherapy, versus 27% for AZA plus vorinostat . AdditioIn contrast, first data of the phase II study evaluating the combination regimen of pracinostat plus AZA in elderly patients with AML who were not eligible for induction chemotherapy are much more promising. For 50 patients who received the combination, CR, CRi and a morphologic leukemia free state was achieved in 42%, 4% and 6% of patients, respectively. The median duration of CR was 13.2 months, OS was 19.1 months after approximately 21 months of follow-up, and the estimated one year survival was 62% . Thus, tde novo AML, and around 7.5% in de novo MDS patients [IDH1- and IDH2-mutations are gain of function mutations, leading to the conversation of α-ketoglutarate to β-hydroxyglutarate (2-HG) . 2-HG inpatients . Furtherpatients .Enasidenib (AG-221) emerged as the first IDH mutation–specific inhibitor, and is a covalent inhibitor of IDH2 mutation . In a phIvosidenib (AG-120) is one of several IDH1 inhibitors  currently under development. In a phase I/II study exclusively enrolling IDH1-mutated patients with advanced hematologic malignancies, the ORR was 38.5%, with 17.9% achieving a CR. Moreover, one third of responding patients experienced elimination of IDH1 mutation . In bothBecause IDH inhibitors target mechanisms in epigenetic regulation, potential synergistic effects with HMAs are assumed, and are currently under investigation .+ and CD8+ T cell infiltrates in the BM prior therapy, and responders showed a progressive increase in BM CD8+ and CD4+ T cell infiltrates [Under normal physiological conditions, immune checkpoints like CTLA-4, PD-1, and PD-L1 are crucial for the maintenance of self-tolerance to prevent autoimmunity, and to protect tissues from damage. In many tumors, the expression of checkpoint receptors is upregulated to evade immune attack. During an active anti-tumor immune response, checkpoint receptor expression can be increased in response to IFNγ, as a mechanism of adaptive immune resistance . ICI tariltrates . At a meiltrates . A studyiltrates . In summiltrates .Compared to younger patients, older individuals with HR-MDS and AML have poor outcomes when conventional treatment strategies are used. Thus, there is a crucial need to improve frontline approaches for these patients. Presently, HMA leads to responses in less than half of patients, and all of them will eventually relapse. Due to dismal outcomes among older patients, new effective and tolerable treatment regimens improving survival and quality of life are needed. One of the most promising strategies currently under investigation is the combination of AZA or DAC with new investigational therapies, aiming to achieve long term synergistic activity and better patient outcomes. Approaches intended to prevent HMA resistance, e.g., by using checkpoint inhibitors, will determine the future of clinical research. Recent data also suggest a potential renaissance of intensive treatment strategies like CPX-351, a novel liposomal formulation of cytarabine and daunorubicin . Partici


A phase I study with a selective LRP1 agonist has been completed showing no toxicity. These findings may open the way to early phase clinical studies with pharmacologic LRP1 activation in patients with acute myocardial infarction (AMI).Cardioprotection refers to a strategy aimed at enhancing survival pathways in the injured yet salvageable myocardium following ischemia-reperfusion. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor that can be targeted following reperfusion, to induce a cardioprotective signaling through the activation of the reperfusion injury salvage kinase (RISK) pathway. The data from preclinical studies with non-selective and selective LRP1 agonists are promising, showing a large therapeutic window for intervention to reduce infarct size after ischemia-reperfusion. A pilot clinical trial with plasma derived  Acute myocardial infarction (AMI) is a leading cause of morbidity and mortality world-wide . The priProgrammed cell death plays a central role in determining the fate of the injured-yet-salvageable myocardium ,10,11. CWe herein describe the potential role of low-density lipoprotein receptor-related protein 1 (LRP1) signaling, and its modulation in the regulation of cell survival pathways and reduction of IRI.Ischemic necrosis is preventable and/or minimized by reperfusion. However, reperfusion itself paradoxically accelerates a series of events that result in cellular injury and death ,4,5. Sev2+ phosphate levels, and adenosine nucleotide depletion create the optimal conditions for a prolonged mitochondrial permeability transition pore (mPTP) opening through the matrix protein cyclophilin D (CypD), leading to an energetic collapse and irreversible cell damage and death [Reperfusion may exacerbate the damage induced by ischemia through further enhancement of necrotic cell death . Necrosind death ,15.Apoptosis is a finely regulated form of programmed, non-inflammatory, cell death, triggered by reperfusion and due to the rapid restoration of the mitochondrial activity, pH imbalance, and adenine nucleotides synthesis and release, following a period of prolonged ischemia . The opePyroptosis is a form of inflammatory programmed cell death, morphologically and functionally different from apoptosis, and mediated by caspase-1 . CaspaseNumerous studies have been performed to determine the nature of the key death and survival pathways following myocardial ischemia and reperfusion, and several attempts to improve the outcome of reperfusion strategies have followed ,19. IdenThe RISK pathway refers to a complex signaling cascade that combines two main pro-survival pathways, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and PI3K-protein kinase B (Akt), together with the mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase 1/2 (ERK1/2) ,20,21.The evidence supporting the key role for the RISK pathway derives from the cardioprotective effects observed with pharmacological agents such as urocortin, which are abolished by the co-administration of PI3K-Akt and MEK-ERK1/2 inhibitors . SeveralRecent studies have explored the molecular mechanisms downstream of PI3K-Akt and the ERK1/2, and their interplay to mediate cardioprotection at reperfusion . In brieThe PI3K-Akt signal transduction cascade is mediated by class IA PI3K. PI3K p110α is coupled to the p101 adaptor protein that is conventionally activated by growth factors through G-protein-coupled receptors (GPCR), participating in the regulation of cell survival, growth, and metabolism [Similarly, the MEK-ERK1/2 pathway  is conventionally activated by tyrosine kinase receptors, G-protein-coupled receptors, and environmental stimuli, and controls cell proliferation, differentiation, and survival ,35,36. TBoth Akt and ERK promote mTORC1 signaling, a crucial pathway in cell growth, proliferation, and metabolism, through phosphorylation on the distinct sites of a repressor of mTORC1 activity, a GTPase activator protein, named tuberous sclerosis complex 2 (TSC2) . These eThe endogenous activation of the RISK pathway during the early reperfusion phase following ischemia limits reperfusion injury. Therefore, the pharmacological stimulation of the RISK pathway represents a therapeutic strategy to reduce reperfusion-mediated cardiomyocyte death. The activation of the PI3K and ERK1/2 kinase cascades appears to constitute a universal pro-survival kinase cascade that mediates cardioprotection at reperfusion ,22.Necrosis, apoptosis, and pyroptosis are distinct forms of regulated cell death that, however, are not completely independent from each other . Outer mNowadays, several approaches have been described to increase the activity of the component of the RISK pathway and to reduce the cell death. For the purpose of this review, we will focus on a potential novel modulator of the IRI, LRP1 .α2-macroglobulin receptor (A2MR) or cluster of differentiation 91 (CD91), is an ubiquitous multifunctional membrane receptor of the LDL receptor family, mostly known for its role in lipoprotein scavenging [LRP1, also known as avenging ,43,44,45avenging ,47,48,49avenging ,51.Three important properties of LRP1 dictate its diverse role in biology, namely: (1) as a scavenger receptor, LRP1 binds and internalizes a variety of ligands for degradation in lysosomes and recycling through its extracellular domain, such as apolipoprotein E and A2MG; (2) as a signaling receptor, it interacts with numerous adaptor proteins through its cytoplasmic domain in a phosphorylation-specific manner and transduces multiple intracellular signals involved in numerous biological processes, as well as regulates nuclear signaling through the small fragment-LRP1 C-terminal intracellular domain (LRP1-ICD) generated by γ-secretases, and then translocated from the cytoplasm to nucleus; and (3) as a scaffold receptor, it modulates the activity of other membrane proteins such as integrins and receptor tyrosine kinases ,47,52,53LRP1 is a type 1 transmembrane protein encoded by the Lrp1 gene located on chromosome 12q13-14 . LRP1 deIII). Certain ligands  function as LRP1 agonists and stimulate LRP1 direct signaling or transactivate signal pathways through LRP1 co-receptors [The LRP1 receptor interacts with over 40 distinct molecules, including several plasma proteases . Also kneceptors .Several in vitro and in vivo studies have shown that LRP1 signaling is crucial in inhibiting death pathways and promoting cell survival ,74,75,76LRP1 expression is necessary for the platelet-derived growth factor (PDGF)-mediated activation of the ERK1/2 protein kinase. LRP1 associates with the PDGF receptor-β (PDGFR-β) in the endosomal compartments to modulate its signaling activity on the mitogen-activated protein kinases of the (MAPK)/ERK and PI3K-Akt pathways . The LRPIn peripheral nerve injury, LRP1 interacted with various ligands, such as tPA, A2MG, and MMP9, to promote Schwann cells (SCs) survival through Akt and ERK1/2 signal pathways ,69,70. TIn macrophages, LRP1 deficiency increased apoptotic cell death and inflammation by impairing Akt activation . LRP1 meThus, LRP1 appears to serve as a common receptor of multiple ligands to mediate their cytoprotective effects by activating different pro-survival signaling cascades. However, the specific contribution of LRP1 in the regulation of other cell death processes, such as necrosis and pyroptosis, has not been studied, and, therefore, whether LRP1 signaling also affects these phenomena is currently unknown.LRP1 signaling also modulates the inflammatory response. LRP1 ligands increase in injured tissues and accelerate the resolution of the inflammatory response . In macrLRP1 regulates the signaling of various membrane proteins, including the cell surface calreticulin (CRT) and the transforming growth factor-β (TGF-β) receptor. LRP1 acts as a signaling co-receptor for CRT and mediates the effects of the matricellular soluble protein thrombospondin 1 (TSP1), which regulates cell adhesion and migration . LRP1 siAltogether, these experimental evidences allow one to speculate that LRP1 represents a key mechanism inhibiting cell death, and promoting the resolution of the inflammation and the formation of a scar following tissue injury . These fLRP1 expression is markedly enhanced during hypoxemia and/or ischemia ,89,90 unLRP1 was also strongly up-regulated in the myocardial tissue in the animal models of ischemia-reperfusion ,90,91, aMultiple gain- and loss-of-function studies indicated that LRP1 protects against cardiomyocyte death and dysfunction through its ability to promote Akt- and ERK1/2-dependent survival pathways ,77,78 T, consistLRP1 initiates the PI3K-Akt and MEK1/2-ERK1/2 signaling cascades shortly after the incubation of rat ventricular cardiomyocytes with A2MG, and induces protein synthesis and cell growth . TreatmeOhashi et al. have previously shown that adiponectin through LRP1 and CRT promotes the neovascularization of ischemic muscle through a cyclooxygenase 2(COX-2)-dependent mechanism in endothelial cells . It was 2+− dependent non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2), which in turn overloads the nucleus with the NF-kB and HIF-1α transcription factors, repressing sarcoplasmic reticulum Ca2+ ATPase (SERCA2) transcription and activity in HL-1 cardiomyocytes [LRP1 has also been reported to be involved in cardiomyocyte lipid metabolism and calcium handling under ischemic conditions ,91,93 T. Hypoxiamyocytes . This maFurthermore, various studies have highlighted the role of LRP1 in cardiac remodeling after AMI ,94. EnhaIII, or with recombinant human AAT, significantly limited IRI [III reduced the infarct size in mice, independent of its anticoagulant activity [The binding of SERPINs to LRP1 was shown to induce a pro-survival signal in several experimental settings, though its effects in a myocardial ischemia-reperfusion model remained unexplained until recently. LRP1 non-selective stimulation by SERPINs, such as with plasma-derived AAT, A2MG, and ATited IRI ,97,98,99ited IRI , and theited IRI . The useited IRI . ATIII ractivity .The short peptide LRP1 agonist, SP16, administered in mice intraperitoneally after reperfusion, significantly reduced the infarct size and preserved the left ventricular systolic function in a dose-dependent fashion . The carFollowing IRI, the inflammatory response is necessary to promote infarct healing, but at the same time, the activation of key pro-inflammatory pathways induces a further loss of viable myocardium, leading to adverse ventricular remodeling and heart failure after AMI. Besides its pro-survival activity, LRP1 agonism has also proven to quench inflammatory signals. In vitro, the LRP1 agonist SP16 inhibited NLRP3 inflammasome activation and down-regulated NF-κB signaling following lipopolysaccharide (LPS) and Gp96 stimulation in a leukocyte assay, and treatment with the LRP1 blocking antibody dissipated these anti-inflammatory effects . This is12 receptor antagonists, which have been shown to independently provide, at least in part, some cardioprotective effects [Promising pre-clinical studies with cyclosporine and other mitochondrial membrane permeability inhibitors failed to translate from bench to bedside, possibly as a result of inconsistent efficacy, dose- and time-dependent limitations, and concomitant standard-of-care treatment with P2Y effects ,102,103.clinicaltrials.gov, NCT01936896) [Clinical data with plasma derived AAT, which functions as a non-selective LRP1 agonist, in patients with ST segment elevation acute myocardial infarction (STEMI), are limited to a pilot clinical feasibility trial  . A singl1936896)  and redu1936896) . There w1936896) .clinicaltrials.gov, NCT03651089) [Selective LRP1 agonists are under clinical development. A phase I clinical trial testing with SP16 in healthy volunteers has been completed showing no toxicity(3651089) .12 receptor antagonists as adjuvant antiplatelet therapy for all patients with AMI, has been proposed by some authors as a major factor for this lack of success in translating cardioprotective strategies into clinical settings [12 receptor antagonists have potent anti-platelet effects and proposed cardioprotective effects, not attributable to the inhibition of platelet aggregation, through the modulation of signaling involving the RISK pathway [12 antagonists have already maximally triggered the RISK pathway, and other therapeutic interventions targeting the same pathway would give little additional benefit [The research field of cardioprotection has for decades been searching for a suitable strategy that could minimize, or prevent, lethal IRI after AMI. Despite many encouraging pre-clinical studies, which identified interventions that dramatically lower the infarct size, these studies have not translated into a clinically applicable drug or intervention in patients with AMI ,5,7. Thisettings ,103. P2Y pathway ,103. Hen benefit .12 receptor antagonist in order to determine whether the new experimental approach would produce a synergic, more powerful, cardioprotective effect. It is worth noting, however, that in the pilot clinical trial with the LRP1 agonist, AAT, all of the patients had been pre-treated with a P2Y12 receptor antagonist, yet a benefit for the LRP1 agonist appeared to be maintained [Future pre-clinical investigations would therefore need to further explore the cardioprotective molecular mechanisms of LRP1 modulation independently of the RISK pathway, including inducible cardiomyocyte-, endothelial-, leukocyte-, and fibroblast-restricted LRP1 deletion, in order to evaluate the cell-specific contribution of LRP1 stimulation during AMI, as well as testing in combination with a P2Yintained ,105.LRP1 is a multifunctional receptor that can be targeted following reperfusion to induce a cardioprotective signaling through the RISK pathway. The data from preclinical studies with non-selective and selective LRP1 agonists are promising, showing a large therapeutic window for intervention to reduce infarct size after ischemia reperfusion. A pilot clinical trial with plasma derived AAT, a naturally occurring LRP1 agonist, supports the potential translational value of LRP1 as a novel therapeutic target for cardioprotection. A phase I study with a selective LRP1 agonist has been completed showing no toxicity. These findings may open the way to early phase clinical studies with pharmacologic LRP1 activation in patients with AMI.


Ischemic stroke is a clinically common cerebrovascular disease whose main risks include necrosis, apoptosis and cerebral infarction, all caused by cerebral ischemia and reperfusion (I/R) injury. This process has particular significance for the treatment of stroke patients. Notoginseng leaf triterpenes (PNGL), as a valuable medicine, have been discovered to have neuroprotective effects. However, it was not confirmed that whether PNGL may possess neuroprotective effects against cerebral I/R injury. To explore the neuroprotective effects of PNGL and their underlying mechanisms, a middle cerebral artery occlusion/reperfusion (MCAO/R) model was established. In vivo results suggested that in MCAO/R model rats, PNGL pretreatment  remarkably decreased infarct volume, reduced brain water content, and improved neurological functions; moreover, PNGL  significantly alleviated blood-brain barrier (BBB) disruption and inhibited neuronal apoptosis and neuronal loss caused by cerebral I/R injury, while PNGL with a different concertation  significantly reduced the concentrations of IL-6, TNF-α, IL-1 β, and HMGB1 in serums in a dose-dependent way, which indicated that inflammation inhibition could be involved in the neuroprotective effects of PNGL. The immunofluorescence and western blot analysis showed PNGL decreased HMGB1 expression, suppressed the HMGB1-triggered inflammation, and inhibited microglia activation (IBA1) in hippocampus and cortex, thus dose-dependently downregulating inflammatory cytokines including VCAM-1, MMP-9, MMP-2, and ICAM-1 concentrations in ischemic brains. Interestingly, PNGL administration (146 mg/kg) significantly downregulated the levels of p-P44/42, p-JNK1/2 and p-P38 MAPK, and also inhibited expressions of the total NF-κB and phosphorylated NF-κB in ischemic brains, which was the downstream pathway triggered by HMGB1. All of these results indicated that the protective effects of PNGL against cerebral I/R injury could be associated with inhibiting HMGB1-triggered inflammation, suppressing the activation of MAPKs and NF-κB, and thus improved cerebral I/R-induced neuropathological changes. This study may offer insight into discovering new active compounds for the treatment of ischemic stroke. Nearly 6.2 million people die from stroke each year, and it is estimated that the lifetime risk for stroke is 8% to 10%. Ischemic stroke accounts for 85% of all strokes [poptosis ,4. Althopoptosis ,8.The high mobility group box-1 protein (HMGB1), as a nuclear DNA-binding protein and an important damage associated molecular pattern (DAMP), is released from necrotic and dying neural cells in the ischemic brain, leading to the activation of microglia and the expression of inflammatory factors in ischemic brain, and it may activate TLR 2/4 and RAGEs signaling following rapid translocation to the cytoplasm or release from dying cells after cerebral ischemia ,10, whicPanax notoginseng (Burk) F. H. Chen is a commonly used Chinese medicinal herb and plant, the roots and stems of which have been used for the treatment of cardiovascular disease in many Asian countries [panax notoginseng, and their extracts, panax notoginseng saponins (PNS) possess many beneficial effects, such as neuroprotective, antiinflammatory, antiapoptotic [2+ influx into neurons [Traditional Chinese medicines, used in China for thousands of years with high efficiency and low toxicity, have attracted great interest in recent years ,23,24,25ountries . Currentpoptotic , and antpoptotic ,28,29. A neurons ,31.panax notoginseng (Burk) F. H. Chen. However, the panax notoginseng (Burk) F. H. Chen stems and leaves were often ignored. Annual production of Panax notoginseng stems and leaves is over 25 million kilograms in China, but its effective utilizing rate is under 5%, so many panax notoginseng resources are wasted. In contrast, panax notoginseng stems and leaves contain high protein, crude fiber, vitamin C and carotenoids, low fat and rich mineral element, among which contents of zinc, iron and manganese are remarkably high. The contents of protein, carotenoids and vitamin C are higher than those of ordinary vegetables, thus, panax notoginseng stems and leaves have a higher nutritional value. Its major active ingredients in panax notoginseng stems and leaves are saponins, the chemical structure is mainly protopanaxadiol type that has functions of sedative-hypnotic action, analgesia, blood lipid regulating, anti-inflammatory/retarding the aging process. And panax notoginseng stems and leaves has been confirmed as the raw materials of production of advanced cosmetics, functional foods and common foods [It was known that PNS were mainly obtained and purified from the roots of on foods ,33,34,35on foods ,32,33. ABase on the reported neuroprotective effects of PNS against cerebral I/R injuries, we hypothesized that PNGL may have neuroprotective effects of relieving cerebral I/R injury with inhibiting HMGB1-triggered inflammation and apoptosis by regulating the MAPK and NF-κB signaling pathways. Therefore, middle cerebral artery occlusion/reperfusion (MCAO/R)-operated rats were used to explore the effects of the PNGL against cerebral ischemia stroke. In additions, our chemical researchers have found that the content of total saponins of panax notoginseng stems and leaves is 4% to 6%. Among them, monomeric saponins contained are mainly 20(s)-protopanaxadiol saponins, and contains almost no ginseng triol saponins which is the biggest difference between PNS and PNGL. As shown in Male Sprague Dawley (SD) rats  were used in this study, and they were purchased from Beijing Vital Lihua Experimental Animals Co., Ltd. Rats were housed at an ambient temperature of 20 ± 1 °C and a relative humidity of 55 ± 15% with artificial light for 12 h each day and free access to a standard laboratory chow diet and sterilized drinking water throughout the experiments. All efforts were made to minimize the number of animals used and their suffering. The study was conducted in accordance with the Declaration of Helsinki. The protocol was approved by the Laboratory Animal Ethics Committee of the Institute of Medicinal Plant Development, Peking Union Medical College, and conformed to the Guide for the Care and Use of Laboratory Animals (Permit Number: SYXK 2017-0020).Rats were divided into seven groups (10 for each group), namely, a sham-operated group, a MCAO/R model group, a 73 mg/kg dose of PNGL group, a 146 mg/kg dose of PNGL group, a 192 mg/kg dose of PNGL group, and a positive drug butylphthalide  group and an aspirin group , according to a random number table. The PNGL, NBP, and ASP samples were all dissolved in normal saline. Then, rats were intragastrically administered daily in a constant volume. Moreover the animals in the sham and MCAO/R groups were given an equal volume of saline water. Drug administration groups were exposed to continuous gastric administration (once per day) for 7 days before MCAO/R operations.As previously described ,36, MCAOAs shown in Rats were monitored by video camera after surgery, and the time of death was recorded. Neurological deficits at 22 h after MCAO were assessed using Zea Longa Scores ,38,39. B-1) via intramuscular injection . All brains were sliced into 2 mm sections, incubated for 20 min in a 2% solution of TTC (37 °C) temperature, and then fixed in 4% paraformaldehyde. The infarct areas on each slice were quantified by using the ImageJ 1.44p software .As previously described ,10,36, 2As previously described ,10,36, aBBB integrity and permeability was quantitatively assessed by measuring the extravasation of Evans blue dye, which serves as a marker of albumin leakage. As previously described . Evans b−1 for 20 min, the blood serums were obtained and stored at −80 °C for further measurement. Furthermore, the hippocampus and cortex tissues were rapidly removed and carefully dissected on an ice plate, frozen in liquid nitrogen and then stored at −80 °C until the assays were performed. Then, the hippocampus and cortex samples were partly weighed and homogenized by sonication in a specified amount of normal saline (100 microliters/ 10 milligrams). The homogenate was kept at 4 °C for 30 min and then centrifuged at 12,000 rpm for 3 min at 4 °C. The supernatant was collected, obtained and stored at −20 °C and then used to measure the inflammatory cytokines by using assay kits.The blood samples from each groups were collected ,40,41, aThe protein concentration of the collected supernatants was determined via the BCA protein assay kit . The inflammatory cytokines from the ischemic brains and the blood serums were analyzed using ELISA kits . All experimental steps were performed according to the kit operation specifications. OD values were measured by a microplate reader.Giemsa staining was commonly used to revealed morphological features of injured neurons in the cerebral cortex. As previously described ,10,36, t2O2 for 5 min and incubated by equilibration buffer and terminal deoxynucleotidyl transferase to inactivate endogenous peroxidases. Then, anti-digoxigenin-peroxidase conjugates were employed to incubate the sections. Finally, the utilization of diaminobenzidine demonstrated peroxidase activity in all tissue sections, and the slices were counterstained with hematoxylin. TUNEL-positive cells were visualized by using a Leica microscope , and images were randomly selected for image analysis via ImageJ software.To measure the extent of neuronal apoptosis in cerebral I/R injury, an in situ apoptosis detection kit  was employed to discover neuronal apoptosis caused by ischemia. A TUNEL analysis was carried out with minor modification according to the manufacturer’s instructions. Firstly, the sections were installed on the slides and permeabilized by incubating them with 100 µL of 20 µg/mL proteinase K solution for 15 min. Next, the sections were incubated with 100 µL of 0.3% HTo explore the effects of PNGL on HMGB expression and IBA1-marked microglia activation in CUMS-induced rats ,43, immuWestern blotting was performed as previously reported ,30,36,46F, 1:2000). Then, the membrane was incubated at room temperature for 2 h with horseradish peroxidase conjugated antibodies at a 1:2000 dilution. Protein expression was detected by an enhanced chemiluminescence method and imaged by using the ChemiDoc XRS instrument . To eliminate variations in protein expression, three independent experiments were performed.p < 0.05 was considered statistically significant.Data are presented as the mean values ± standard deviation (SD) or standard error of the mean (SEM). All analyses were performed by using GraphPad Prism 8.0 statistical software . Two-way analysis of variance (ANOVA) was used with drug (PNGL versus Vehicle) and treatment (MCAO/R vsrsus Control) as independent factors. Group differences after significant ANOVAs were measured by post hoc Bonferroni test, and p < 0.05; 146, ** p < 0.01; 292, ** p < 0.01, p < 0.05; 292, ** p < 0.01, To assess the neuroprotective effects of PNGL on focal brain ischemic injury, SD rats were subjected to 2 h of MCAO and 24 h of reperfusion. Extensive infarction was detected by TTC staining in the cerebral cortical and subcortical areas over a series of sections of the ipsilateral hemisphere in rats subjected to MCAO A, and thp < 0.01, p < 0.05; 146, ** p < 0.01; 292, ** p < 0.01, Moreover, the neurological deficit scores were evaluated using the scoring criteria of Zea-Longa. Longa scores suggested that MCAO/R significantly increased neurological deficit scores in rats . In contrast, PNGL  pretreatment exhibited strong staining and possessed neurons arranged regularly in the hippocampus and cortex (p < 0.01). In addition, PNGL (146 mg/kg) showed similar improvements of nervous density in the hippocampus regions with significant differences (p < 0.01), and ASP (10 mg/kg) were equivalent to those of PNGL in improving neuron density, indicating that PNGL decreases neuronal apoptosis and loss caused by CIRI.The CA1 and CA3 areas of the hippocampus are commonly considered to be sensitive to ischemic injury ,48,49,50d cortex A. PNGL . Moreover, ASP (10 mg/kg) evidently inhibited the neuronal apoptosis in hippocampus CA1, CA3, and cortex regions, which nearly corresponded to PNGL (146 mg/kg).Contrary to Giemsa staining, TUNEL Staining B,D showeResearches exhibited that the inflammatory factors were induced not only in microglia of the CA1 region in the ischaemic hippocampus but also of the CA3 and DG regions ,43, so tTo further explore neuropathological alterations of neurogenic inflammation in ischemic brains from all groups, inflammatory cytokines and microglia activation in ischemic brains were also examined after 24 h reperfusion following 2 h of MCAO. Immunofluorescent results showed that the MCAO/R model rats exhibited more IBA1-positive neurons in the ischemic hippocampus CA1, dentate gyrus (DG), and cortex regions than the sham-operated group A. Howevep < 0.05, p < 0.05, p < 0.05, p < 0.01, Similarly, the MCAO/R group significantly increased the inflammatory cytokines in ischemic brains than did the sham group , phosphorylated P44/42 (p-P44/42), and phosphorylated P38 (p-P38) significantly increased in the MCAO/R model (It was proven that HMGB-1 triggers the NF-κB, MAPKs, STAT3, and AP-1 signaling via activating TLR4 signaling, which in turn increases innate and acquired immunity, and further heightens the post-ischemic neuroinflammation response. Accordingly, intercepting the HMGB-1/TLR4 signaling pathway is a promising therapeutic strategy for alleviating cerebral stroke injury and limiting neuroinflammation.p < 0.05, p < 0.01, p < 0.05, p < 0.01, p < 0.05, p < 0.01, As shown in Ischemic stroke remains one of the leading causes of death worldwide, which mainly caused by cerebral ischemia and reperfusion injury ,3,4. NumPNGL, as the total saponins of Panax notoginseng stem and leaf, has already been shown to exert potent neuroprotective and anti-apoptotic properties. Since the first beneficial effect of PNGL was detected on nervous diseases, several reports have not only revealed its diverse pharmacological properties but have also raised some speculative proposals concerning its mechanism of action. Moreover, currently there had been no clear evidence on whether PNGL has neuroprotective effects against cerebral I/R injury. Meanwhile, our research found that PNGL pretreatment improved the neurological functions, attenuated the brain swelling, and reduced the cortex infarct volume in MCAO/R rats . Additio2+ overload promotes the activation of complements, platelets and endothelial cells, activates inflammatory transcription factors and the release inflammatory signals [Immunity and inflammation are key elements that contribute to the pathobiology of stroke, and its caused-CI/RI and the secondary damage to brain tissues are closely associated with immunity and inflammation responses ,6,7,8. P signals , and gen signals ,56,57. IHMGB1 is a nuclear DNA-binding protein and an important DAMP, which activates TLR2/4 and RAGEs signaling following rapid translocation to the cytoplasm or release from dying cells after cerebral ischemia ,10. AddiFurther research results suggested PNGL pretreatment dose-dependently inhibited the HMGB-1/TLR4 signaling-triggered downstream proteins including MAPKs, NF-κB, and cleaved caspase 3/8 ,7,8, sugThese findings, together with our results, supported the neuroprotective effects of PNGL against cerebral I/R injury and neuronal apoptosis via decreasing the HMGB levels, inhibiting HMGB1-triggered inflammation, reduced the production of pro-inflammatory mediators, and suppressing the activation of MAPKs and NF-κB signaling pathways. However, neuroprotective effects of PNGL has not been convincingly confirmed by in vitro models of ischemic brain, and the mechanisms was not evidently sufficient to explain on how PNGL may regulate the subsequent immune and neuroinflammatory responses caused by the HMGB1/TLR2/4 pathway. Hence, further investigations are needed to elucidate the mechanisms more deeply and to investigate the clinical applications of PNGL.Nevertheless, our results suggest that PNGL represents a potential treatment value for cerebral ischemic infarctions, and works through a mechanism of HMGB1-triggered inflammation inhibition through the MAPK and NF-κB pathwayIn summary, this study supported the neuroprotective effects of PNGL on cerebral I/R injury, and the potential mechanisms may be largely associated with the inhibition of HMGB1-triggered inflammation, reduction of pro-inflammatory mediators including IL-6, TNF-α, IL-1 β, MMP-9, MMP-2, and ICAM-1, and attenuation of neuronal apoptosis and loss caused by ischemia via suppressing the activation of MAPKs and NF-κB signaling pathways. Although further studies are needed to elucidate the roles of MAPK signaling pathways in the cross talk between pro-inflammatory mediators and apoptosis, our findings may represent a novel mechanism of PNGL in focal cerebral I/R injury in rats, and provide new insights into therapeutic targets for ischemia stroke patients.


CHCHD2 and CHCHD10 proteins, which are homologous proteins with 54% identity in amino acid sequence, belong to the mitochondrial coiled-coil-helix-coiled-coil-helix (CHCH) domain protein family. A series of studies reveals that these twin proteins form a multimodal complex, producing a variety of pathophysiology by the disease-causing variants of these proteins. In this review, we summarize the present knowledge about the physiological and pathological roles of twin proteins, CHCHD2 and CHCHD10, in neurodegenerative diseases.Mutations of  A mitochondrion is an essential organelle that converts nutrients into chemical energy by a oxidative phosphorylation (OXPHOS) reaction to provide ATP as energy in eukaryotic cells. Mitochondrion also plays multifaceted roles including cellular stress/immune response, metal ion metabolism, calcium signaling/buffering, apoptosis, and cytosolic protein degradation ,2. MitocMitochondrial dysfunction is a hallmark of aging and a variety of disorders including ischemia/reperfusion injury in stroke and myocardial infarction, metabolic syndromes, and neurodegeneration ,5. Mitoc9C motifs ((CX9C)2) are imported to the mitochondrial intermembrane space (IMS) and are folded by the disulfide relay-dependent Mia40 machinery ..CG5010 iCHCHD2 and/or CHCHD10 knockout cultured cells revealed that CHCHD2 promotes CHCH10 oligomerization [Drosophila, CG5010 also responds to mitochondrial DNA damage and mitochondrial unfolded protein stress, and is stabilized on the PD-associated PINK1, Parkin, or DJ-1-defefient genetic backgrounds [CHCHD2 and CHCHD10 form both homodimer and heterodimer, although the physiological meanings remain unclear ,51,56,58rization . Endogenrization ,70,71. Trization ,72. In Dp32 causes developmental defects in mouse embryos with severe dysfunction of the mitochondrial respiratory chain due to impaired mitochondrial protein synthesis, suggesting that p32 is involved in mitochondrial translation mediated by mitoribosomes [Drosophila, suggesting that p32 regulates the Ca2+-buffering ability of mitochondria [Drosophila failed to detect significant genetic interaction between p32 and CG5010, in spite of the conserved physical association (Both CHCHD2 and CHCHD10 also associate with a mitochondrial matrix protein p32/HABP1/C1qBP/gC1qR, although the binding mode of IMS-resident CHCHD2 and CHCHD10 with p32 remains unclear ,56,73. Tibosomes . The rolchondria . Althougociation .CHCHD2 and CHCHD10 appear to reside in a high molecular weight complex of approximately 220 kDa . In a glPINK1 and Parkin, are well recognized to regulate mitochondrial quality control, which includes the arrest of mitochondrial transport and removal of damaged mitochondria through mitophagy [TBK1, Optineurin, VCP/p97, and SQSTM1/p62, which are causative genes of ALS–FTD, have been shown to be involved in PINK1-Parkin-mediated mitophagy [DJ-1, demonstrates resistance to oxidative stress [Mitochondrial defects have long been implicated in the etiology of PD as genetic evidence. Early-onset PD-causative genes, itophagy . Interesitophagy ,78,79. Ae stress ,81.CG5010 is upregulated in PINK1-, Parkin-, and DJ-1-deficient flies, probably due to chronic reduction of ∆ψm, there was no evidence that CHCHD2/CHCHD10 and PINK1-Parkin signaling or DJ-1 directly interact [CG5010 mitochondrial phenotypes, suggesting that PINK1-Parkin signaling tried to remove all of the defected mitochondria by CG5010 loss. A similar conclusion was reported by another study using human cultured cells [Although ed cells .TDP-43 is a heterogeneous ribonucleoprotein that plays a major role in regulating RNA splicing, stability, and transport. In ALS and FTD patients, the accumulation of TDP-43 in cell bodies of neurons in affected regions is often observed, indicating that a unifying pathology underlies the ALS–FTD spectrum. Mitochondrial mislocalization of TDP-43 and its involvement in neurotoxicity of ALS-FTD have also been reported . Loss ofCHCHD2- and CHCHD10-linked neurodegenerative diseases are dominantly inherited, the findings of their mutations leading to premature protein truncation suggest mitochondrial phenotypes arise from haploinsufficiency. Supporting this idea, the expression of PD-associated human CHCHD2 mutants on a wild-type genetic background, but not CG5010 null background, does not produce obvious mitochondrial phenotypes in Drosophila [Drosophila study showed that the overexpression of human CHCHD2 mutants on a wild-type genetic background leads to mitochondrial degeneration and dopaminergic neuron loss. This may be attributed to higher levels of CHCHD2 ectopic expression and Myc-tag addition to CHCHD2, as even wild-type CHCHD2 expression caused a reduced electrophysiological response and shorter lifespan compared with a no transgene control. However, the finding that CHCHD2 T61I, which can form dimers with CHCHD2 [Although h CHCHD2 ,83, tendh CHCHD2 . Similarh CHCHD2 . Thus, tCHCHD2 and CHCHD10 is relatively low, the fact that many pathogenic and risk variants of the two genes have been isolated from patients with a variety of neurodegenerative diseases strongly suggests that the dysfunctions of these twin proteins underlie the pathogenesis of major neurodegenerative diseases. Considering the observations that CHCHD2 and CHCHD10 form different patterns of mutant complex, there could be multiple modes of pathogenesis in these diseases. In addition, not a few mutations of both genes are found in sporadic neurodegenerative diseases, raising the possibility that the two genes are key factors in polygenic hereditary diseases. Thus, it would be important to examine both gene mutations simultaneously. Finally, what mechanism by the dysfunctions of the twin proteins determines the affected nervous system? The issue is the “twin paradox” in current neurology.Although the mutation frequency of


Meanwhile, ANO1 has been examined in more detail, and the role of ANO1 in cell proliferation and the development of different types of malignomas is now well established. While ANO5, ANO7, and ANO9 may also be relevant for growth of cancers, evidence has been provided for a role of ANO6 (TMEM16F) in regulated cell death. The cellular mechanisms by which anoctamins control cell proliferation and cell death, respectively, are just emerging; however, the pronounced effects of anoctamins on intracellular Ca2+ levels are likely to play a significant role. Recent results suggest that some anoctamins control membrane exocytosis by setting Ca2+i levels near the plasma membrane, and/or by controlling the intracellular Cl− concentration. Exocytosis and increased membrane trafficking induced by ANO1 and ANO6 may enhance membrane expression of other chloride channels, such as CFTR and volume activated chloride channels (VRAC). Notably, ANO6-induced phospholipid scrambling with exposure of phosphatidylserine is pivotal for the sheddase function of disintegrin and metalloproteinase (ADAM). This may support cell death and tumorigenic activity of IL-6 by inducing IL-6 trans-signaling. The reported anticancer effects of the anthelminthic drug niclosamide are probably related to the potent inhibitory effect on ANO1, apart from inducing cell cycle arrest through the Let-7d/CDC34 axis. On the contrary, pronounced activation of ANO6 due to a large increase in intracellular calcium, activation of phospholipase A2 or lipid peroxidation, can lead to ferroptotic death of cancer cells. It therefore appears reasonable to search for both inhibitors and potent activators of TMEM16 in order to interfere with cancer growth and metastasis.Before anoctamins (TMEM16 proteins) were identified as a family of Ca The human homologue of Drosophila tweety and the bestrophin family of channels were shown to operate as Ca2+ activated Cl− channels  and head and neck cancers i), and therefore proposed this as a major mechanism by which ANO1, and possibly other anoctamins, affect cell proliferation and a number of other cellular properties i, i.e., basal [Ca2+]i as well as receptor mediated Ca2+ signals depend on expression of anoctamins i, which is an essential regulator of exocytosis. Thus compartmentalized [Ca2+]i close to the plasma membrane is required for docking of exocytic vesicles and granules, respectively. This process requires the so-called Munc13 proteins and the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor machinery [We observed that animals lacking expression of ANO1 in epithelial cells of airways and intestine accumulate mucus in club (Clara) and goblet cells . We founachinery ,183.7-agonist ATP [Anoctamins were shown to have additional impact on cancer related events that involve plasma membrane function. Both ANO1 and ANO6 support cell migration and metastasis ,73,184. nist ATP . ANO6 alnist ATP ,108. Impnist ATP ,186,187.nist ATP ,188,189.nist ATP  and ANO1nist ATP  for the nist ATP ,193,194.nist ATP . Interesnist ATP  and necrnist ATP  a role onist ATP ,198, whinist ATP ,199. Finnist ATP ,176,200 The current data suggest that upregulated ANO1 in rapidly growing cells and tumors, supports proliferation, while expression in differentiated non-proliferating cells is generally much lower and may enable cells to perform specific tasks such as signaling, contraction, or secretion of electrolytes and mucus. Proliferation and inflammation/hypoxia are intimately connected through multiple signaling pathways including JACK/STAT ,202,203.All living cells are able to maintain a constant cell volume. According to a general concept, regulatory volume decrease (RVD) prevents cell swelling and necrotic cell death, while regulatory volume increase (RVI) prevents cell shrinkage and apoptotic cell death ,210. The− channels such CFTR, bestrophin, and anoctamins, as well as electroneutral KCl co-transporter [2+ activated ANO1 Cl− currents: With increased activation of CaCC, VRAC decreases and vice versa [2+ activated Cl− currents. Vice versa, overexpression of LRRC8A enhanced Ca2+ activated Cl− currents. Because LRRC8A and ANO1 could be co-immunoprecipitated, a co-localization of both anion channels is proposed, with membrane insertion of LRRC8A being supported by ANO1 [Although rather abundant, LRRC8/VRAC may not be essential for RVD and thus cells may be able to control their cell volume in the absence of VRAC ,226,227.nsporter ,226,227.ce versa ,233. We ce versa , while Z by ANO1 . Apart f by ANO1 . Because by ANO1 ,234,235.2+ from the ER-store [Taken together, a functional relationship exists between VRAC and ANO1, possibly because activation of both channels involves release of CaER-store ,236. As ER-store ,234,236.2+ can lead to senescence or cell death [7-mediated increase of intracellular Ca2+ leads to cell death of macrophages and lymphocytes expressing endogenous ANO6, and of HEK293 cells overexpressing ANO6 [− channel ORCC or ICOR, and has a role in cell shrinkage and programmed cell death [Sustained large increase in intracellular Call death ,238. We ing ANO6 ,239. ANOll death ,241. Expll death . Moreovell death .− currents produced by both proteins [Although many studies demonstrated the pro-proliferative role of ANO1, a pro-apoptotic function of ANO1 has also been reported ,242,243.proteins . Meanwhile, a number of independent regulated cell death pathways have been identified . Initial− currents have been activated in cells overexpressing ANO1, ANO6, ANO9 and ANO10 [2) [2-dependent activation of metalloproteinase is essential for this effect [Pro-apoptotic Clnd ANO10 ,123,227.NO10 [2) ,200. Nots effect ,250,251.c−. This leads to depletion of glutathione (GSH) and inactivation of the phospholipid peroxidase glutathione peroxidase 4 (GPX4). In addition, GPX4 can be directly inhibited by RSL3 [Anoctamins are also activated through reactive oxygen species and by lipid peroxidation. This may lead to inflammation and proliferation, ion secretion and ferroptosis, depending on the cell type, the anoctamin paralogue being activated, and the strength of peroxidation ,252,253. by RSL3 . 2 or through lipid peroxidation [Cell death can be induced in cancer cells by activation of ANO6 through melittin-induced PLAxidation . This maxidation ,247. Lipxidation . Howeverxidation . ROS levxidation . A numbexidation ,261,262.xidation ,247. − and cation-permeable channels. They demonstrate impressive effects on basic cell properties, and support both cell proliferation and regulated cell death. Clearly more work is required to be able to define the cellular functions of anoctamins, and their role in proliferation and cancer development. Despite the plethora of underlying cell specific signaling pathways, it will be interesting to learn whether common mechanisms exist for the cellular effects induced by anoctamins, such as enhanced intracellular Ca2+ signaling. Blocking ANO1 appears feasible to interfere with cancer growth. Proteins of the anoctamin/TMEM16 family scramble membrane phospholipids and operate as Cl2+, phospholipid scrambling, membrane blebbing and membrane shedding. It may all contribute to ANO6-induced cell death. Thus, direct activation of ANO6 may be a promising new strategy to induce cell death in cancer cells.In contrast to the pro-proliferative effect of ANO1, ANO6 seem to contribute to different types of regulated cell death . Activat


Dear Editor,Mammarenavirus genus, Arenaviridae family. Arenaviruses are classified into two main groups—Old World (OW) and New World (NW)—based on virus genetics, serology, antigenic properties and geographical relationships. The OW LASV and Lujo virus (LUJV), as well as NW Junín virus (JUNV), Machupo virus (MACV), Guanarito virus (GTOV), Sabiá virus (SABV) and Chapare virus (CHAPV), are known to cause severe hemorrhagic fever and are listed as biosafety level 4 (BSL-4) agents. The arenavirus glycoprotein complex (GPC) contains three subunits—the retained stable-signal peptide (SSP), the receptor-binding subunit GP1, and the membrane fusion subunit GP2  belongs to the 50 values < 1 μmol/L are presented in Table S1. Hit compounds 21, 29 and 57 exhibiting robust inhibition of the LASV pseudotype virus  entry with IC50 values lower than 0.2 nmol/L  was used as a control. As a result, two non-synonymous substitutions—L428S and F446L—were obtained in the compound 21 and the compound 29, 57 and 72 treatment groups, respectively  in the presence of 1 μmol/L of any of the compounds 21, 29, and 72, or 10 nmol/L of compound 57, respectively, which approximately corresponded to the ICt-Bu) moiety in compound 57 might broaden the antiviral spectrum of the backbone . Moreover, compound 57 blocked NW GPC-mediated membrane fusion. As shown in Figure Since the parent compound, ST-161, possessed specific antiviral activity against LASV, we investigated whether the four hit compounds extended their antiviral activities to other pathogenic arenaviruses. As shown in Figure S2, compounds 21, 29 and 72 largely maintained LASV specificity. In contrast, compound 57 showed promising inhibitory effects on the entry of NW pathogenic viruses, with a sharp blockage on the entry of GTOVpv and MACVpv in a picomolar range, as well as CHAPVpv, JUNVpv and SABVpv in a nanomolar range, suggesting that the tert-butyl , the National Natural Sciences Foundation of China (Grant No. 31670165), the Open Research Fund Program of CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, and the Open Research Fund Program of Wuhan National Bio-Safety Level 4 Lab of CAS (NBL2017008), the Open Research Fund Program of the State Key Laboratory of Virology of China (2018IOV001).Guangshun Zhang, Junyuan Cao, Yan Cai, Yang Liu, Yanli Li, Peilin Wang, Jiao Guo, Xiaoying Jia, Mengmeng Zhang, Gengfu Xiao, Yu Guo and Wei Wang declare that they have no conflict of interest.Supplementary material 1 (PDF 819 kb)Below is the link to the electronic supplementary material.


In this study, transcriptome analysis of PPRV infected PBMC subsets—T helper cells, T cytotoxic cells, monocytes, and B lymphocytes was done to delineate their role in host response. PPRV was found to infect lymphocytes and not monocytes. The established receptor for PPRV—SLAM was found downregulated in lymphocytes and non-differentially expressed in monocytes. A profound deviation in the global gene expression profile with a large number of unique upregulated genes (851) and downregulated genes (605) was observed in monocytes in comparison to lymphocytes. ISGs—ISG15, Mx1, Mx2, RSAD2, IFIT3, and IFIT5 that play a role in antiviral response and the genes for viral sensors—MDA5, LGP2, and RIG1, were found to be upregulated in lymphocytes and downregulated in monocytes. The transcription factors—IRF-7 and STAT-1 that regulate expression of most of the ISGs were found activated in lymphocytes and not in monocytes. Interferon signaling pathway and RIG1 like receptor signaling pathway were found activated in lymphocytes and not in monocytes. This contrast in gene expression profiles and signaling pathways indicated the predominant role of lymphocytes in generating the antiviral response against PPRV in goats, thus, giving us new insights into host response to PPRV. Peste des petits ruminants (PPR) is an acute, highly contagious viral disease with high mortality (90%) and morbidity (100%) in sheep and goats and is classified as an OIE listed disease due to its economic relevance and severity  is a lymphotropic and epitheliotropic cell-associated virus, which uses peripheral blood mononuclear cell (PBMCs) as a vehicle for its dissemination within the host , 4. SLAMted goat –15.The host immune response is often complicated due to differences in how different cell types receive a signal from different classes of receptors and produce distinct effector molecules . The indKR140086.1) that is being maintained at National Morbillivirus Referral Laboratory, Indian Veterinary Research Institute, Muktehswar by animal-to-animal passages (n = 4) that were negative for PPRV antibody by competitive-ELISA  in heparin-coated vacutainer vials. PBMCs were isolated by using Ficol histopaque gradient method. PBMCs were strained through cell strainer of 0.40 micron. The PBMCs subsets were enriched by positive selection using indirect MACS technology (Milteny Biotech). Initially, the cell-specific surface marker FITC-conjugated primary antibodies, anti CD4+ , anti CD8+ , anti CD14+ , and anti CD21+ , were used. Subsequently, the cells were magnetically labeled with anti—FITC MicroBeads. Then the cell suspension was loaded on a miniMACS® column which was placed in the magnetic field of a MACS Separator. The magnetically labeled cells were retained in the column while the unlabeled cells run through. After removal of the column from the magnetic field, the magnetically retained cells were eluted as positively selected cell fraction. Cell sorting was done as per the manufacturer's protocol. Primary FITC-conjugated antibodies were titrated to determine the optimal dilution. Cells were kept on the ice and cold buffers were employed to minimize alterations in gene expression during labeling and sorting. The purity of the cells was further checked by flow cytometer. The cells were stored in RNA later for further use at −80°C.Blood was collected from goats  DEGs were functionally annotated  is a repository of protein-protein and genetic interactions for many species. Based on the interactions available in the BioGRID database, PPI network among the selected genes is retrieved . Initial species , ortholo species . The int species .AJ849636.2)  were mapped to PPRV reference genome (GenBank: 49636.2)  through QIAGEN's IPA  was used to analyze the data of T helper cells, T cytotoxic cells, B lymphocytes, and monocytes. IPA has got its own database, Ingenuity Pathways Knowledge Base (IKB) that along with the list of DEGs were used to identify the canonical pathways and the most significant biological processes. Core analysis for each dataset was performed to know activated (Z score > 2) or inactivated (Z score < −2) canonical pathways. Also, upstream regulators  were identified.GAPDH was taken as the internal control as it was found to be the best suitable endogenous control in earlier studies in PPRV  to test the significance of difference. Differences between groups were considered significant at P ≤ 0.05.To validate the expression of some selected genes, qRT-PCR was performed on Applied Biosystems 7500 Fast system.  in PPRV . The prolibrator . StudentTo uncover the transcriptional variation underlying the host response to PPRV at PBMCs subtype level, enriched T helper cells, T cytotoxic cells, B lymphocytes, and monocytes were obtained by using MACS technology. The purity was found to be >90% through flow cytometry (data not shown). Then, we performed the global transcriptome profiling of T helper cells, T cytotoxic cells, and B lymphocytes and monocytes at 0 day (control) and 9 dpi.Following alignment with Bowtie 2, several reads in the infected T helper cells, T cytotoxic cells, and B lymphocytes mapped to PPRV reference genome but no reads from infected monocyte mapped to PPRV genome . PPRV N The number of DEGs in T helper cells were 2,214 , T cytotoxic cells−1,948 , monocytes−2,451 , and B lymphocytes−2,463 . The number of upregulated genes was larger than the numbers of downregulated genes in all the cells. Furthermore, Venn diagrams were generated to examine the overlapping mRNA profiles between cells. This revealed 467 genes being common all subsets and 555, 225, 964, and 527 genes being unique in T helper cells, T cytotoxic cells, monocytes, and B lymphocytes, respectively . Upon coISG15, ISG20, IRF7, IFIT1, IFIT3, IFIT5, MX1, MX2, RELA, RSAD2, DDIT4, EIF2AK2, IFI6, OAS1X, PML, and RTP4. Monocytes showed a contrasting ISGs profile with most of the genes downregulated , T cytotoxic cells (Log2 fold change −1.185), and B lymphocytes (Log2 fold change −2.204). On the contrary, it was not found to be differentially expressed in monocytes. Further, granzyme B (GZB) was found to be non-differentially expressed in lymphocytes and monocytes.ISGs highly expressed in lymphocytes were, egulated . Among tonocytes . More nuectively . Among Vnfection . IFN α aphocytes . SLAM waISG15, ISG20, IFIT3, MX2, IFIT1, IFIT2, IFIT5,OAS1X, MX1, DHX58, DDX58, HSPB1, DDIT4, RSAD2, EIF2AK2, NPC2, C19ORF66, ADAR, and DDX3X were found to be upregulated in lymphocytes and downregulated in monocytes. Whereas, as CXCL10, HMOX1, TMPRSS2, TNF, ICAM1, C1QBP, LEF1, EIF3D, and SNW1 were found to be upregulated in lymphocytes and not differentially expressed in monocytes  were downregulated in monocytes and upregulated in lymphocytes.DEGs regulating the immune processes were selected through functional analysis. The number of immune-related genes with cut off fold change ≥±1.5 and degree ≥5 were 269, 250, 70, 356 in T helper cells, T cytotoxic cells, monocytes, and B lymphocytes, respectively, and were designated as differentially expressed highly connected (DEHC) genes. As the number of interactions was higher in monocytes, the cut off was increased to degree ≥10 reducing the number of DEHC genes to 194. The PPI networks were constructed between immune DEHC genes; and between the genes selected through knowledge-based approach (106 genes) and DEGs, which revealed common hubs . In the Core analysis for each dataset was performed to know activated (Z score > 2) or inactivated (Z score < −2) canonical pathways. Moreover, the focus was on the apoptosis, cellular immune response, humoral immune response, cytokine signaling, and pathogen influenced signaling ingenuity canonical pathways.Canonical pathways associated with T helper cells in infected goats at 9 dpi are represented in Canonical pathways associated with T cytotoxic cells in infected goats at 9 dpi are represented in Canonical pathways associated with monocytes in infected goats at 9 dpi are represented in Canonical pathways associated with B lymphocytes in infected goats at 9 dpi are represented in Comparison of canonical pathways in T helper cells, T cytotoxic cells, monocytes, and B lymphocytes at 9 dpi in goats is represented in GIP2 (ISG15), IFIT1, IFIT3, MX1, IFI6, IFITM1, IFI35, STAT1, and STAT2 were significantly downregulated in monocytes. However, all these genes were upregulated in T helper cells, T cytotoxic cells, and B lymphocytes with varying degree of expression  in comparison to T helper cells (Z score 0.77), T cytotoxic cells (Z score 1.88), and B lymphocytes (Z score 3.44). Genes in the interferon signaling network pression . Oncostaphocytes . Role ofphocytes . Complemonocytes .KLF4, IRF7, XBP1, and MYC; in T cytotoxic cells were KLF4, ETS2, NFKB1A, and IRF7; in monocytes were EGR1, PPP1R13L, and STAT3 and in B Lymphocytes were MXD1, KLF4, NFKBIA, and BCL6 (The number of TFs (activated/inactivated) differentially expressed and governing the DEGs in T helper cells, T cytotoxic cells, monocytes, and B lymphocytes were 21, 24, 10, and 30, respectively. The top four upregulated TFs in T helper cells were and BCL6 .IRF7—transcription factor and STAT1—a transcription regulator, were found to be activated in T helper cells, T cytotoxic cells, and B lymphocytes and inactivated in monocytes. The genes governed by the IRF7 and STAT1 were mostly ISGs including IFIT3, MX1, and ISG15  and not in monocytes which is in concordance with the alignment of reads with PPRV genome. Moreover, maximum viral transcripts were found in T helper cells in goats. These results indicate that the PPRV virulent virus probably replicates in lymphocytes and not in monocytes. The closely related viruses, Canine distemper virus (CDV) and Measles virus (MV) are reported to infect and replicate in T cells and not in monocytes –39. SLAMGlobal transcriptome of monocytes indicated profound deviation from the lymphocytes as evident from a large number of unique upregulated genes (851) and downregulated genes (605) under PPRV infection. Differential expression analysis of immune cell types in trivalent inactivated influenza vaccine-induced immune response also showed unique transcriptomic expression profiles as well as changing biological networks . Among t(IFIH1), and R1G-1 (DDX58) activate NFKB and IRF3/7  were not differentially expressed in lymphocytes or monocytes. The upstream regulator analysis in lymphocytes revealed activation of transcription factors, IRF-7, and STAT-1 that regulate most of the ISGs under PPRV infection. Thus, it expected that ISGs are transcriptionally induced more by IRF7 and STAT1 than by stimulation of interferons in PPRV infected lymphocytes. The presence of STAT1, which plays a vital role in interferon type I (IFN-α/β) and type II (IFN-γ) signaling  . Any kinCXCL10 that was upregulated in the T helper cells, T cytotoxic cells, and B lymphocytes and not differentially expressed in monocytes have been reported to have chemoattractant  stimulates the hemagglutination and neutralization activity .The study was carried out after obtaining permission from Indian Veterinary Research Institute Animal Ethics Committee (IVRI—IAEC) under the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The protocols were approved vide letter no 387/CPSCEA.RS, BPM, and RG conceived and designed the research. KR and DM performed the vaccine testing experiment. YS maintained the server for analysis. SW, ARS, DC and SS conducted the wet lab work. SW, RK, ARS, AP, VS, SK, and RG analyzed the data. SW, RG, RK, WM, APS, NH, AT, BS, and BM helped in manuscript drafting and editing. RS, BPM, and RG proofread the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer NS and handling editor declared their shared affiliation.


Ictalurus punctatus), a species native to North America, is one of the most important commercial freshwater fish in the world. Outside of the United States, China has become the major producer and consumer of channel catfish after experiencing rapid development in the past three decades. In this study, based on restriction site associated DNA sequencing (RAD-seq), a high-density genetic linkage map of channel catfish was constructed by using single nucleotide polymorphisms (SNPs) in a F1 family composed of 156 offspring and their two parental individuals. A total of 4,768 SNPs were assigned to 29 linkage groups (LGs), and the length of the linkage map reached 2,480.25 centiMorgans (cM) with an average distance of 0.55 cM between loci. Based on this genetic linkage map, 223 genomic scaffolds were anchored to the 29 LGs of channel catfish, and a total length of 704.66 Mb was assembled. Quantitative trait locus (QTL) mapping and genome-wide association analysis identified 10 QTLs of sex-related and six QTLs of growth-related traits at LG17 and LG28, respectively. Candidate genes associated with sex dimorphism, including spata2, spata5, sf3, zbtb38, and fox, were identified within QTL intervals on the LG17. A sex-linked marker with simple sequence repeats (SSR) in zbtb38 gene of the LG17 was validated for practical verification of sex in the channel catfish. Thus, the LG17 was considered as a sex-related LG. Potential growth-related genes were also identified, including important regulators such as megf9, npffr1, and gas1. In a word, we constructed the high-density genetic linkage map and developed the sex-linked marker in channel catfish, which are important genetic resources for future marker-assisted selection (MAS) of this economically important teleost.A high-density genetic linkage map is of particular importance in the fine mapping for important economic traits and whole genome assembly in aquaculture species. The channel catfish ( Genetic-map construction is a critically important tool for further genomic studies, as well as for genetic breeding of economically important aquatic species. It has been employed for genome assembly , comparaChlamys farreri) (Siniperca chuatsi) (Oreochromis niloticus L.)  (Eriocheir sinensis) (With the rapid development of next-generation sequencing (NGS), an increasing number of methodologies have been applied for cost-effective development and genotyping of thousands of single nucleotide polymorphisms (SNPs) in non-model animals, such as genome resequencing , transcrfarreri) , mandarichuatsi) , tilapiaicus L.) , Asian scarifer) , and Chiinensis) .Pelteobagrus fulvidraco)  (Cynoglossus semilaevis) (Hippoglossus hippoglossus)  (Cyprinus carpio)  and breeding in many economically important aquaculture species. Sex is one of the most basic characteristics of organisms. Many species of teleost fish have sexually dimorphic growth patterns  with sigvidraco) , Japanesivaceus) , half-smilaevis) , and Atlglossus) . Growth glossus)  with extglossus) , salmon o salar) , and com carpio) . In addi carpio) .Traditional strategies for genetic improvement of growth-related traits have mainly relied on phenotypic data, which increased time and cost for breeding. However, MAS using marker-linked QTLs has accelerated genetic improvement with high accuracy of selection. Since genetic linkage map and QTLs allow to identify molecular markers or candidate genes associated with traits , they haIctalurus punctatus) has been popular in the worldwide. Especially in the native United States, it accounts for more than 60% of the US annual aquaculture production  in separate tanks. After approximately 1 week, a random collection of approximately 5,000 larvae was stocked in separate larvae-culture tanks (3.0 m × 1.0 m × 0.5 m). Zooplankton was fed at the first 10 days, and then with formula feed. After 20 days, a random collection of approximately 1,000 larvae was transferred to an outdoor pond (about 667 square meters) for further culturing. Until December 2016, these offspring individuals at the age of 18 months were measured for growth-related traits including body height (BH), body length (BL), body weight (BW), and body width (WD), and the genders of these F1 individuals were also identified at the same time.A FAfter investigating the relationships among the growth-related traits with R3.3.1 software  to calcuEcoRI  , was incubated at 65°C for 10 min. Barcode adapters with a sample-specific nucleotide code were designed, following the standard Illumina adapters design flow. Unique barcode adapter (10 μmol) for each DNA sample was added to individual reaction system. Twelve DNA samples were pooled per tube. In order to obtain more sequencing data for two parents, they were pooled in triplicates. Therefore, there were 162 DNA samples used for pooling. Fourteen pools were collected and the fragments at the size range of 300–500 bp were chosen. Fourteen libraries were independently sequenced using the 150-bp pair-end sequencing method on an Illumina HiSeqX-ten platform .DNAs of the total 158 individuals (156 offspring and two parents) were used to construct RAD libraries, which was prepared via a previously published protocol . In brieAfter removal of sequencing adapters, low-quality reads  were filtered using the SOAPnuke software . The remGenome-wide SNPs were identified using a stringent SNP discovery filtering method within the software SOAPsnp . In ordeP-value = 0.01) was used to discard highly segregated markers . Subsequently, the separate LGomosomes module was used to assign markers into LGs and then execute with logarithm of odds (LOD) scores for recombination fraction. A range of LOD scores from 5 to 15 incrementing by 1 were tested for linkage grouping. The final LOD score (15) was selected based on whether the number of LGs matched the number of chromosomes of channel catfish and the assembled chromosomes showed the 1:1 synteny relationship with the Linkage groups (LGs) were assigned using JoinMap4.1 software  under thp-value of 0.02. Significant LOD thresholds were calculated by permutation test of α < 0.05 and n = 1,000 for significant linkages. The software also calculated phenotypic variation that resulted from growth- and sex-related QTLs. These markers were mapped on the channel catfish genome (We employed MapQTL6.0  to perfoh genome  and upstDanio rerio) and channel catfish , 0.1 mol/L KCl, 3.0 × 10-3 mol/L MgCl2; Vazyme, Nanjing, China], and sufficient ddH2O. Touchdown PCR was initiated at 94°C for 30 s. The annealing temperature of these reactions decreased from 60°C to a touchdown 50°C at the cooling rate of 1°C in every cycle; followed 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 20 cycles; and final extension steps at 72°C for 10 min. For fragment length analysis, PCR products were genotyped on a ABI PRISM 3730XL DNA Sequencer  with GS500 marker as an internal size standard. The allelic sizes were determined using GeneMarker version 1.5 .According to a previous report , we obtar = 0.8292-0.9312, P-value < 0.001 for all). The highest correlation value 0.9312 was observed between BW and BL A total of 1,367,192 SNPs in all individuals were identified using SOAPSnp, in which 10,661 SNPs passed through the filtering criteria . These SNPs were classified into three categories: paternal heterozygous , maternal heterozygous , and heterozygous in both . Among them, 4,768 SNPs were consistent with Mendelian segregation pattern, and then they were used for subsequent linkage map construction using a pseudo-testcross strategy .These SNPs were finally grouped into 29 LGs , which iIn this study, four growth-related traits including BW, BL, BH, and WD were measured. Six QTLs associated with growth-related traits were identified on the LG28, which were detected between the narrow span of 32.53–45.29 cM . Among tP < 0.02, LOD > 3.3). Among these QTLs, the highest LOD value of 31.01  of channel catfish with a total length of 704.66 Mb were assembled, which comprised 83.39% of the assembled scaffold sequences  and 18,1There were 16,197 synteny blocks between the assembled genomes of channel catfish and zebrafish, and 15 of the 29 LGs of channel catfish had relatively conserved collinear blocks on zebrafish chromosomes . The totspata2), spata5, splicing factor 3 (sf3), and forkhead box (fox). Five sex-related genes including Wilms tumor protein 1-interacting protein (wt1), spata2, probable ATP-dependent DNA helicase DDX11 (ddx11), zinc finger homeobox protein 3-like isoform X1 (zfhox3), and forkhead box protein F1 (foxf1) were located near four sex-related QTLs   .megf9), neuropeptide FF receptor 1 (npffr1), and growth arrest-specific protein 1 (gas1), were found in two (qGW_1 and qGW_3) of the six growth-related QTL regionso (npffr1 is receptor of neuropeptide FF and RFamide-related peptide (rfrp), which are involved in control of feeding behavior both in certain invertebrates and in vertebrates , which is approximately 25 kb in length and consists of six exons and five introns       (S. chuatsi)  (C.c. haematopterus)  (Trachinotus blochii)   , genetic47 SNPs) , blunt s48 SNPs) , mandari83 SNPs) , common 95 SSRs) , and pom58 SNPs) .In the present study for channel catfish, we employed the RAD sequencing technology to construct a high-density genetic linkage map with 4,768 SNPs, reaching the total length to 2,480.25 cM with an average SNP distance of 0.55 cM. Using the markers to target specific scaffolds from previous study , we anchP-value < 0.05, P. fulvidraco) (O. niloticus)   and Nileloticus) . The higS. chuatsi) one significant QTL for sex determination was identified on LG23; genotypes of all the female fish on r1_33008 marker were heterozygous, and all males were homozygous, thus this sex-specific marker can be used to identify male and female individuals of mandarin fish; meanwhile, 11 significant QTLs for growth traits were also detected on four LGs (H. hippoglossus) (Sparus aurata)   and gilt aurata) .M. amblycephala), three QTLs related to gonad development were detected on LG13, LG12, and LG1  and intraspecific hybridization of channel catfish close to 1:1, and the offspring in gynogenetic families were all females  (Actinidia chinensis Planchon)   and kiwilanchon) , and theThe good resolution and high density of our genetic linkage map provide an effective support for QTL mapping of economically traits, as well as for genome assembly. QTL fine mapping and positional cloning of candidate genes have been an efficient approach for breeding programs in aquaculture animals, especially for investigation of quantitative traits . Interesspata, wt1, and foxl1. Six QTLs for growth were detected on the LG28, on which three growth-related genes were identified within the QTL intervals. A previously reported sex-linked marker was confirmed on the LG17, which can effectively identify male and female individuals of channel catfish from difference genetic resources. In summary, we provide a valuable genetic resource for future molecular breeding of this economically important fish species.In the present study, we employed RAD sequencing to construct a high-density genetic linkage map with 4,768 SNPs for the channel catfish. Ten sex-related QTLs were detected on the LG17, on which 23 genes related to sex development were annotated, such as https://db.cngb.org/cnsa/), CNP0000229.The datasets generated for this study can be found in CNGB Nucleotide Sequence Archive (XY, SZ, QS, WB, and XC conceived the ideas and designed the investigations. XZ, TX, and SZ analyzed the data. SZ, MW, QQ, LZ, and HJ collected and processed the samples. HL, JS, and ZZ performed the experiments. XZ, SZ, and XY wrote the manuscript. XY and QS revised the manuscript. All authors read and approved the final manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors XY.


PTPN11, SOS1, RIT1, A2ML1, BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, RAF1, SHOC2, and SPRED1. The median age at diagnosis was 4.5 years (range: 1 month−51 years). Individuals of mixed-race ancestry were most represented (53.8%), followed by black Africans (30.8%). Our cohort revealed a lower frequency of pulmonary valve stenosis (34.6%) and a less severe developmental milestones phenotype. Molecular analysis found variants predicted to be pathogenic in 5 / 16 cases (31.2%). Among these mutations, two were previously reported: MAP2K1-c.389A>G (p.Tyr130Cys) and PTPN11 - c.1510A>G ; three are novel: CBL-c.2520T>G (p.Cys840Trp), PTPN11- c.1496C>T (p.Ser499Phe), and MAP2K1- c.200A>C (p.Asp67Ala). Molecular dynamic simulations indicated that novel variants identified impact the stability and flexibility of their corresponding proteins. Genotype-phenotype correlations showed that clinical features of NS were more typical in patients with variants in MAP2K1. This first application of targeted NGS for the molecular diagnosis of NS in South Africans suggests that, while there is no major phenotypic difference compared to other populations, the distribution of genetic variants in NS in South Africans may be different.Noonan Syndrome (NS) is a common autosomal dominant multisystem disorder, caused by mutations in more than 10 genes in the Ras/MAPK signaling pathway. Differential mutation frequencies are observed across populations. Clinical expressions of NS are highly variable and include short stature, distinctive craniofacial dysmorphism, cardiovascular abnormalities, and developmental delay. Little is known about phenotypic specificities and molecular characteristics of NS in Africa. The present study has investigated patients with NS in Cape Town (South Africa). Clinical features were carefully documented in a total of 26 patients. Targeted Next-Generation Sequencing (NGS) was performed on 16 unrelated probands, using a multigene panel comprising 14 genes:  KRAS, NRAS, RIT1, and RRAS), or modulators of Ras function  and downstream signal transducers   is a common autosomal dominant condition, with an estimated global incidence of 1:1,000 to 1:2,500 live births . Written informed consent was obtained from the parents and/or the patient prior to their involvement into the study, including permission to publish photographs.This study was conducted in Cape Town, South Africa, and included 26 participants (20 children and six adults); among them, 20 were unrelated. Patients were recruited through the University of Cape Town (UCT) affiliated Hospitals, namely Red Cross War Memorial Children's Hospital (RCWMCH) and Groote Schuur Hospital (GSH). Patients were selected retrospectively and prospectively using the Van der Burgt scoring system for clinical diagnosis of NS -Molecular Genetics Laboratory, GSH, following the manufacturer's instructions, the standard Maxwell 16 protocol .A2ML1, BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, RIT1, SHOC2, SOS1, and SPRED1. This multigene panel is predicted to cover 100% of the targeted regions, in 268 amplicons . Pre-designed primers for Ion AmpliSeq Noonan Research Panel were used . The primers amplify exons and intron/exon boundaries of 14 genes known to be associated with NS and related conditions, including All variants identified and predicted to be pathogenic were subsequently confirmed with Sanger capillary direct cycle sequencing and capillary electrophoresis using standard protocols. Depending on availability of family members, segregation studies were performed: the proband's parents were screened to ascertain the origin of the variant. In addition, and where possible, other available family members clinically affected or not, were screened for the identified variant, using Sanger direct cycle sequencing.PTPN11 protein SHP-2 (PDB id: 2SHP)  simulations were conducted to assess the effect of novel variants on proteins function. The tertiary structure of the n = 20; 77%). The majority were children  with a median age at clinical diagnosis of 4.5 years (range: 1 month−51 years). Our cohort had preponderance of individuals of mixed-ancestry background (53.8%), followed by black Africans (30.8%). There was a slight predominance of males .A total of 26 patients were included in this study, mostly unrelated (n = 16/26) of cases. Six  patients were unable to walk after 24 months. Speech delay was reported in 50% (13/26) of cases. Craniofacial features were widely variable, but more characteristic in infants , with widely spaced eyes, epicanthic folds and ptosis found in 75% of cases , PTPN11 c.1496C>T (p.Ser499Phe), and MAP2K1 c.200A>C (p.Asp67Ala). Analysis confirmed segregation of three novel variants with the disease in the respective families. Segregation analysis revealed a possible case of germline mosaicism in a family where the unaffected father does not carry the variant  found in his two affected children. These children were half-brothers, born from two separate mothers.Following targeted NGS of 16 DNA samples from unrelated patients, ic 7/16; . The novMAP2K1 c.389A>G (p.Tyr130Cys) and PTPN11 c.1510A>G . Two identified CBL variants were predicted to be pathogenic by prediction tools, but were reported as benign: c.1858C>T (p. p.Leu620Phe) and as variant of uncertain significance: c.2345C>T (p.Pro782Leu) in ClinVar. Correlatively, CBL c.2345C>T failed to segregate with the disease in a large dominant family (The two variants previously reported in ClinVar as pathogenic are t family .Molecular dynamic (MD) simulations  showed tFor MAP2K1, substitution of the negatively charged and hydrophilic amino acid Asp67 with the non-polar and hydrophobic amino acid Ala67 could impact binding interactions; for SHP-2, substitution of the polar and hydrophilic amino acid Ser499 with the non-polar and highly hydrophobic amino acid Phe499 in the active site of the protein could impact binding interactions and the stability of structure.MAP2K1 were diagnosed earlier (mean age at diagnosis: 1 year) and presented with more typical clinical features of NS, followed by patients with variants in PTPN11  known to be frequently associated with Cardiofaciocutaneous syndrome , compared to the expected >70% when using whole exome sequencing (WES) or a comprehensive multigene panel testing . Written informed consent was obtained from the parents and/or the patient prior to their involvement into the study, including permission to publish photographs.CT-N, AE, and AW contributed to conception and design of the study. CT-N and AW collected data; CT-N, GA, and CB performed molecular analysis and interpretation of data. CT-N wrote the first draft of the manuscript and CB wrote a section of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.