Acinetobacter baumannii (MDRAB) strains R1 and R2. The bacterial growth of biofilm-dispersed MDRAB was inhibited by CST or TGC. However, the inhibitory effects were attenuated by a combination of CST and low concentrations of TGC. The bactericidal effects of CST, but not TGC, were observed on biofilm-dispersed MDRAB. Notably, the bactericidal effects increased with a combination of CST and high concentrations of TGC, whereas they were attenuated with the combination of CST and low concentrations of TGC. Although biofilm formation by MDRAB decreased with increasing concentrations of CST or TGC, there was no complete disruption of the biofilms. Additionally, the biofilms increased with a combination of 1–2 μg/mL CST and TGC at 2 μg/mL and 2–4 μg/mL for strains R1 and R2, respectively. Biofilm-embedded MDRAB was eradicated with CST, but not TGC. Notably, the eradication effects increased with a combination of CST and high concentrations of TGC, whereas attenuation happened with the combination of CST and low concentrations of TGC. These results provide information on the combined effects of CST and TGC in the treatment of biofilm-associated MDRAB infection.We investigated the antimicrobial effects of colistin (CST) and tigecycline (TGC), either alone or in combination, on biofilm-dispersed and biofilm-embedded multidrug-resistant  Acinetobacter baumannii is an important opportunistic pathogen associated with nosocomial infections, such as bacteremia, pneumonia, meningitis, urinary tract infections, and wound infections2. Additionally, A. baumannii is included among the six nosocomial pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) that acquire multidrug resistance and virulence4. Therefore, A. baumannii, especially multidrug-resistant A. baumannii (MDRAB), has gained importance as a human pathogen in hospital environments.A. baumannii is regarded as a low-virulence pathogen5, recent studies have shown that A. baumannii exhibits several forms of pathogenicity, such as biofilm formation, adherence, invasion of lung epithelial cells, host cell death, and iron acquisition6. Recently, Colquhoun et al. hypothesized that biofilms are, at least in part, responsible for the high prevalence of A. baumannii nosocomial and recurrent infections because they frequently contaminate hospital surfaces and patient indwelling devices7. A. baumannii is capable of forming biofilms6; biofilm persister cells are protected from antimicrobial agents and immune responses and are therefore difficult to eliminate8, suggesting bacterial colonization in hosts and promoting persistent infections. Therefore, understanding A. baumannii biofilm formation, maturation, and dispersal is important for developing effective strategies to inhibit its growth and proliferation.Although 9, although there have been reports of antibiotic resistance against these antibiotics worldwide11. Previous studies have suggested that a combination of TGC with CST, levofloxacin (LVX), amikacin (AMK), and imipenem (IPM) may be an effective therapy to synergistically prevent the emergence of resistance during treatment of MDRAB infections13. Moreover, Wang et al. reported the synergistic effects of the combinations of meropenem (MEPM), IPM, sulbactam (SBT), CST, and TGC on biofilm-embedded carbapenem-resistant A. baumannii and carbapenem-susceptible A. baumannii strains14. Meanwhile, other studies reported that no synergistic effects of CST and TGC were observed in A. baumannii clinical isolates16. Therefore, the combined effects of antibiotics on A. baumannii may depend on bacterial strains. Additionally, we have reported that clinical isolates of MDRAB showed different degrees of biofilm formation in the presence of sub-minimum inhibitory concentrations (sub-MICs) of CST and TGC17, suggesting that A. baumannii is emerging as a highly pathogenic bacterium and that the characteristics of A. baumannii vary under different environmental stress conditions, such as in the presence of multiple antimicrobial agents. Therefore, in this study, we focused on the combined effects of CST and TGC on MDRAB biofilms, that is, the inhibitory and bactericidal effects of these antibiotics on biofilm-dispersed and biofilm-embedded MDRAB. Furthermore, we analyzed the combined effects of CST and TGC on biofilms formed by MDRAB.Colistin (CST) and tigecycline (TGC) are the last resort antibiotics used against a number of multidrug resistant bacteria17. The MICs of CST on MDRAB strains R1 and R2 were at 2 μg/mL, and those of TGC were at 0.5 μg/mL, respectively 20. Moreover, catheter-related urinary or bloodstream infections are caused by biofilm-forming strains21. Therefore, biofilm formation is thought to be an important pathogenic feature, especially in relation to intravascular line infections and VAP, so treatment strategies for inhibition of biofilm formation may lead to a reduced risk of severe A. baumannii infection.9. CST is an old antibiotic member of the polymyxin family, which are membrane-active peptides with bactericidal capabilities that can disrupt the outer bacterial cell membrane22. It is currently used as a last-resort antibiotic for the treatment of multidrug-resistant gram-negative bacterial infections23. Meanwhile, TGC is a member of the glycylcycline class of semisynthetic antimicrobial agents developed for the treatment of polymicrobial infections caused by multidrug-resistant gram-positive and gram-negative bacteria and acts as a bacteriostatic agent against these bacteria24. In fact, the bactericidal effects of CST were observed on biofilm-dispersed and biofilm-embedded MDRAB, whereas TGC had no bactericidal effect on these cells. These results reflect the reasonable action of these antibiotics. Furthermore, we demonstrated that the combination of CST and TGC exhibited synergistic bactericidal effects on biofilm-dispersed and biofilm-embedded MDRAB. However, although the synergistic effects of CST on these cells increased when used in combination with high concentrations of TGC, they were attenuated when used in combination with low concentrations of TGC. It remains unclear why the antibiotic activity of CST on MDRAB is attenuated when used in combination with low concentrations of TGC. Efflux pumps play an important role in antimicrobial resistance and virulence for A. baumannii25. Lin et al. reported that EmrB-deleted mutant of A. baumannii was more susceptible to CST, suggesting that EmrAB pump systems in A. baumannii contribute to the resistance to CST26. Additionally, Cheng et al. reported that the expression of efflux pumps including EmrAB in A. baumannii upregulated under the pressure of TGC at a low concentration27. With considering these results, we speculate that bactericidal effects of CST were attenuated when used in combination with low concentrations of TGC because of EmrAB efflux pumps upregulated by TGC at low concentrations. Additionally, these results suggest that the antibiotic resistance characteristics of A. baumannii vary under different environmental stress conditions . We previously reported that clinical isolates of MDRAB showed different degrees of biofilm formation in the presence of sub-MICs of CST and TGC17. Moreover, we demonstrated that biofilm formation by MDRAB increased even more upon treatment with CST in combination with TGC at concentrations less than the MBC, suggesting that the characteristics of A. baumannii vary in the presence of antibiotics at low concentrations. We have reported that the number of biofilm cells was positively and significantly correlated with the mRNA levels of genes encoding efflux pumps (adeB and adeG) and autoinducer synthase (abaI), which are biofilm-related genes in MDRAB strain R2 in the presence of CST17. Additionally, several reports demonstrated that mRNA levels of genes encoding efflux pumps, porins, virulence factors, and biofilm-related genes in A. baumannii were altered in the presence of various antibiotics at sub-MICs29. Therefore, we speculate that the expression of biofilm-related genes in biofilm-embedded MDRAB is altered in the presence of CST and TGC because these cells are exposed to low concentrations of antibiotics that slightly infiltrate into biofilms. Therefore, exposure to a lethal dose of bactericidal concentrations of CST and TGC in patients is necessary for the treatment of A. baumannii infection.CST and TGC remain the only effective antibiotics for the treatment of MDRABA. baumannii in the presence of antibiotics.In summary, we demonstrated the combined effects of CST and TGC on biofilm-dispersed MDRAB, biofilm-embedded MDRAB, and biofilms formed by MDRAB. Since MDRAB clinical isolates showed different characteristics upon treatment with antibiotics, further studies are required to understand the mechanism of the biofilm-related characteristics of All methods were carried out in accordance with relevant guidelines and regulations.A. baumannii were isolated from the Teikyo University Hospital during an outbreak that occurred around 2010. The bacteria were isolated on CHROMagar™ Acinetobacter and incubated for 24 h at 37 °C. The R1 strain was isolated from the sputum of a patient with interstitial pneumonia. The R2 strain was isolated from a urine sample of a patient with malignant lymphoma and pneumonia. The isolates were streaked onto blood agar plates and cultivated for 24 h to obtain monoclonal colonies and were identified as A. baumannii through DNA sequencing of the partial RNA polymerase β-subunit (rpoB) gene30. Additionally, the isolates were confirmed to be non-clonal via pulsed-field gel electrophoresis (data not shown). After identification, the isolates were stored in glycerol stocks at − 80 °C at the Department of Microbiology & Immunology, Teikyo University School of Medicine. Antimicrobial susceptibility testing was performed using two strains of A. baumannii based on the MICs of IPM, AMK, and ciprofloxacin (CPFX). Against these two strains, the MICs of IPM, AMK, and CPFX were > 8, > 32, and > 2 mg/L, respectively. Thus, these strains were identified as MDRAB strains. The MIC of CST against MDRAB in strains R1 and R2 was determined to be 2 mg/L17. The MIC of TGC against these strains was found to be 0.5 mg/L17. These bacteria were cultured on Luria–Bertani (LB) agar plates  for 16 h at 37 °C. Thereafter, the bacteria were suspended in LB broth at a concentration of OD595 = 0.1, then the concentration was adjusted via optical density (OD) measurements at 595 nm. The bacterial suspensions obtained were used for biofilm formation.R1 and R2 strains of 31. Then, the plate was covered with a 96-pin microtiter plate lid and incubated for 24 h at 37 °C. After incubation, the 96-pin lid with established biofilms was washed three times with sterile PBS and then placed onto a fresh 96-well microtiter plate with each well containing LB with various concentrations of antibiotics. After incubation for 24 h at 37 °C, to analyze the inhibitory effects of antibiotics on biofilm-dispersed MDRAB, the 96-pin lid was removed and the culture broth was measured at A595. To analyze the bactericidal effects of antibiotics on biofilm-dispersed MDRAB, 5 μL of culture broth was plated onto LB agar, and the presence of viable cells was confirmed through the growth from the bacterial suspension after 24 h of incubation at 37 °C. To analyze the number of biofilm cells, the 96-pin lid was washed three times with sterile PBS and then placed onto a fresh 96-well microtiter plate with each well containing 0.1% crystal violet solution. After staining for 30 min, the 96-pin lid with stained biofilms was washed three times with sterile PBS to remove unreacted crystal violet. Then, the 96-pin lid was placed onto a fresh 96-well microtiter plate with each well containing 99.5% ethanol and extracted for 15 min. The amount of crystal violet extracted was measured using A595. The viability of biofilm-embedded MDRAB was determined using the Biofilm Viability Assay Kit (DOJINDO) according to the manufacturer’s protocol. After the incubation of biofilms in the presence of antibiotics, the 96-pin lid was washed three times with sterile PBS to remove planktonic and nonattached cells and then placed onto a fresh 96-well microtiter plate with each well containing LB broth with WST solution. After incubation for 24 h, the 96-pin lid was removed, and the LB broth was measured at A450.To analyze the combined effects of CST and TGC on biofilm-dispersed MDRAB, biofilm-embedded MDRAB, and biofilms formed by MDRAB, bacterial strains were added to 96-well microtiter plates containing the Biofilm Formation Assay Kit or Biofilm Viability Assay Kit


Pseudomonas aeruginosa. We characterized the correlates of protection with MEDI3902, a bispecific human IgG1 monoclonal antibody that targets the P. aeruginosa type 3 secretion system PcrV protein and the Psl exopolysaccharide, in a rabbit model of ventilator-associated pneumonia using lung-protective, low-tidal-volume mechanical ventilation. Rabbits infused with MEDI3902 prophylactically were protected, whereas those pretreated with irrelevant isotype-matched control IgG (c-IgG) succumbed between 12 and 44 h postinfection . Lungs from rabbits pretreated with c-IgG, but not those pretreated with MEDI3902, had bilateral, multifocal areas of marked necrosis, hemorrhage, neutrophilic inflammatory infiltrate, and diffuse fibrinous edema in alveolar spaces. All rabbits pretreated with c-IgG developed worsening bacteremia that peaked at the time of death, whereas only 38% of rabbits pretreated with MEDI3902 (3/8 rabbits) developed such high-grade bacteremia . Biomarkers associated with acute respiratory distress syndrome were evaluated longitudinally in blood samples collected every 2 to 4 h to assess systemic pathophysiological changes in rabbits pretreated with MEDI3902 or c-IgG. Biomarkers were sharply increased or decreased in rabbits pretreated with c-IgG but not those pretreated with MEDI3902, including the ratio of arterial oxygen partial pressure to the fraction of inspired oxygen of <300, hypercapnia or hypocapnia, severe lactic acidosis, leukopenia, and neutropenia. Cytokines and chemokines associated with acute respiratory distress syndrome were significantly downregulated in lungs from rabbits pretreated with MEDI3902, compared with c-IgG. These results suggest that MEDI3902 prophylaxis could have potential clinical utility for decreasing the severity of P. aeruginosa ventilator-associated pneumonia.Ventilator-associated pneumonia is an important clinical manifestation of the nosocomial pathogen  Pseudomonas aeruginosa, is recognized as the most common agent in nosocomial infections  pathogens, poses a significant challenge to modern medicine . While that proof-of-concept study did not achieve its primary endpoint, evidence of a responder population was identified, suggesting that this molecule has a path forward for continued clinical development  targeting Psl exopolysaccharide and PcrV , and it elopment . Both Psn system , 11. Comn system , 12.P. aeruginosa ventilator-associated pneumonia (–2O (The objective of this study is to compare the protective efficacy of MEDI3902 and an irrelevant isotype-matched control IgG (c-IgG) in a recently described rabbit model of ventilator-associated pneumonia . Althoug14–neumonia –19. To beumonia –, our rabnia  of 0.35, which achieved an end-tidal CO2 goal of 35 to 45 mm Hg at baseline before infection  (study 1) or 60 hpi (study 2). All 8 rabbits that were pretreated with c-IgG died between 12 and 44 hpi, whereas all 8 of those pretreated with MEDI3902 survived to 36 h or 60 hpi, when they were euthanized    and E.Of the rabbits that were pretreated with c-IgG, 50% (4/8 rabbits) had lung weight (LW) to body weight (BW) ratios exceeding 10 g/kg  and F, aMacroscopically, gross images of lungs from rabbits pretreated with c-IgG, but not those pretreated with MEDI3902, showed extensive areas of lung hemorrhage and necrosis . Microscopically, lungs from rabbits pretreated with c-IgG demonstrated multifocal areas of marked necrosis, hemorrhage, neutrophilic inflammatory infiltrate, and diffuse fibrinous edema in alveolar spaces  and C; bP = 0.026) (All rabbits that were pretreated with c-IgG (8/8 rabbits) developed progressively worsening bacteremia, which peaked at the time of death, whereas only 38% of rabbits pretreated with MEDI3902 (3/8 rabbits), denoted MEDI3902(4), MEDI3902(5), and MEDI3902(6), developed high-grade bacteremia   and G. B= 0.026)  and H.2)/FiO2 ratio from preinfection baseline throughout the 36-h or 60-h monitoring period, whereas 75% of those pretreated with c-IgG (6/8 rabbits) developed moderate to severe acute respiratory distress syndrome (ARDS), with PaO2/FiO2 ratios of <200 at the time preceding death  showed little change in the arterial oxygen partial pressure (PaOng death  and E. Dng death  and F anng death  and G.2) showed little change from preinfection baseline for all rabbits pretreated with MEDI3902 (8/8 rabbits), 63% of rabbits pretreated with c-IgG (5/8 rabbits) developed hypercapnia and 25% (2/8 rabbits) developed hypocapnia .Rabbits pretreated with c-IgG developed severe leukopenia, neutropenia, and monocytopenia, as well as modest decreases in platelet counts, whereas white blood cell (WBC) counts remained little changed from preinfection baseline for those pretreated with MEDI3902  for analysis of differential expression of 84 inflammatory cytokines and receptors . Notably24–P. aeruginosa strain 6077. Inasmuch as ExoU is critical for P. aeruginosa to cause lethal pneumonia in a nonventilated mouse model and a ventilated rabbit model  pretreated with c-IgG , but none of those pretreated with MEDI3902, developed septic shock-like syndrome, including severe hypotension, hypocapnia, and multiple organ dysfunction . Septic MEDI3902 also protected against acute lung inflammation , therebyP. aeruginosa strain 6077 in both rabbit models, MEDI3902 prophylaxis resulted in greater protection against acute lung injury in the rabbit model of ventilator-associated pneumonia, as evidenced by a LW/BW ratio of 4.91 g/kg for ventilated lungs versus 8.88 g/kg for nonventilated lungs from rabbits pretreated with MEDI3902  and a humanized dosing regimen of standard-of-care antibiotic, will allow preclinical testing of MEDI3902 in a manner that mimics how this novel molecule could be used clinically to treat ventilator-associated pneumonia . Second,neumonia , we did neumonia , which wP. aeruginosa-induced ventilator-associated pneumonia.Taken together, our results showed here that MEDI3902 prophylaxis conferred significant protection in a clinically relevant rabbit model of ventilator-associated pneumonia, supporting the continued development of this molecule for reducing the severity of P. aeruginosa strain 6077, a cytotoxic strain that expresses the type III secreted toxins ExoU, ExoT, and ExoY, was used in the rabbit model of ventilator-associated pneumonia. Strain 6077 was a gift from Joanna Goldberg (Emory University). Strain 6077 was prepared as described previously for infection of rabbits (600) of approximately 1.7. Bacteria were collected by centrifugation at 16°C, washed once, and then resuspended in lactated Ringer’s solution (LRS). The washed cells were then diluted in LRS to a concentration of 9 × 107 CFU/1.8 mL for the rabbit study. The number of bacteria in the inoculum was confirmed by serial dilution on 5% sheep blood agar plates. MEDI3902 was expressed and purified as described previously  were used in all animal studies. Rabbits were housed in single stainless-steel cages in a climate-controlled housing room with a daily 12-h light/12-h dark cycle. They were provided rabbit food pellets and water We used 8 rabbits for study 1, in which the animals were monitored for 36 hpi, and 8 rabbits for study 2, in which the animals were monitored for 60 hpi. For each of the two studies, 4 of the rabbits were randomized for prophylactic intravenous administration of 15 mg/kg MEDI3902 at 24 h before infection, whereas the other 4 rabbits were treated with 15 mg/kg R347, an irrelevant isotype c-IgG.2O, PEEP of 6 cm H2O, flow rate of 4 L/min, respiratory rate of 30 to 35 breaths/min, and FiO2 of 0.35, with 2.00% isoflurane to maintain general anesthesia . The pressure-controlled ventilator was set to deliver a peak inspiratory pressure (PIP) of 15 cm Hesthesia . A neonaAseptic surgical techniques were used to cannulate the right carotid artery with an 18-gauge arterial line catheter, which was connected to a pressure monitoring kit with the Safedraw blood sampling system (Argon) for continuous invasive blood pressure monitoring and arterial blood sampling. The patency of these lines was maintained by continuous flushing with 0.9% saline containing 1 U/mL heparin in a 1,000-mL bag pressurized to 300 mm Hg.For fluid replacement, the marginal ear vein was cannulated with a 22-gauge intravenous catheter for continuous infusion of Normosol-R with 5% dextrose at 2 mL/kg/h using an intravenous infusion pump (Hospira).After approximately 2 to 3 h of mechanical ventilation, the rabbit was injected with a mixture of 12 mg/kg ketamine and 1.7 mg/mL xylazine, and then a 1.8-mL bacterial inoculum was instilled directly into the lungs through the endotracheal tube. The rabbit was then placed in the right lateral decubitus position for the duration of the study. Rabbits were euthanized with an overdose of sodium pentobarbital while still under general anesthesia after the predetermined monitoring period of either 36 hpi (study 1) or 60 hpi (study 2) or after attainment of humane endpoints, which were defined as arterial oxygen saturation of <60% and arterial blood gas analysis showing lactate levels of >15.0 mmol/L and base excess less than −10.0 mmol/L.2, partial pressure of O2, Na+, K+, Cl−, ionized Ca2+, creatinine, glucose, lactate, pH, and base excess using the Element POC rapid blood analyzer (Heska); and (iii) alanine aminotransferase, amylase, and creatinine levels using the Comprehensive Diagnostic Profile rotor with the VetScan VS2 system (Abaxis).Arterial blood samples were collected from the carotid artery at baseline (30 to 60 min before infection), every 2 h for the first 24 hpi, and then every 4 h for 28 to 60 hpi. Each blood sample was characterized for (i) 5-part WBC differential and red blood cell and platelet parameters using the Element HT5 veterinary hematology analyzer (Heska); (ii) partial pressure of COLungs, spleen, and kidneys were removed aseptically from euthanized animals, weighed, and immediately processed. The right lung, spleen, and kidneys were cut into <0.5-cm pieces, and 0.2- to 0.3-g samples were homogenized in 0.9% saline, followed by quantification of CFU by serial dilutions on 5% sheep blood agar (Remel). Arterial blood collected every 2 to 4 h for biomarker analysis (see above) was diluted (100 μl of blood in 900 μl of sterile water) and plated for quantitative culture on 5% sheep blood agar (Remel).2) Profiler kit  as described previously (TC) values were used for each tissue, using a threshold of 0.051. The mean TC values for four rabbit housekeeping genes, ACTB, GAPDH, LDHA, and loc100346936, were used for normalization. For each gene, the fold regulation was computed as the fold change in the lungs of rabbits pretreated with MEDI3902, relative to those pretreated with c-IgG, using normalized TC values.Right lung pieces were preserved in RNAlater (Thermo Fisher Scientific) within 5 to 7 min after euthanasia. RNA was extracted, and differential expression of 84 genes encoding cytokines and receptors was then evaluated using the rabbit-specific inflammatory cytokines and receptors real-time reverse transcriptase , thus allowing evaluation of fold gene regulation of MEDI3902-treated animals relative to the pooled c-IgG animals. The 95% confidence interval (CI) for fold regulation and the false-discovery rate (FDR)-adjusted P values are reported  model with heterogeneous within-group variance showed no difference between the c-IgG groups used in the two rabbit studies (reported , 41.


Therefore, there is an urgent need to develop new cardioprotective therapies for IHD and HF. Compelling evidence from animal studies and pilot clinical trials in HF patients suggest that urocortin (Ucn) isoforms, which are peptides associated with stress and belonging to the corticotropin releasing factor family, have promising potential to improve cardiovascular functions by targeting many signaling pathways at different molecular levels. This review highlights the current knowledge on the role of urocortin isoforms in cardioprotection, focusing on its acute and long-term effects.Despite the considerable progress in strategies of myocardial protection, ischemic heart diseases (IHD) and consequent heart failure (HF) remain the main cause of mortality worldwide. Several procedures are used routinely to guarantee the prompt and successful reestablishment of blood flow to preserve the myocardial viability of infarcted hearts from ischemia injuries. However, ischemic heart reperfusion/revascularization triggers additional damages that occur when oxygen-rich blood re-enters the vulnerable myocardial tissue, which is a phenomenon known as ischemia and reperfusion (I/R) syndrome. Complications of I/R injuries provoke the adverse cardiac remodeling, involving inflammation, mishandling of Ca Actually, one of the critical factors involved in I/R syndrome is the cytoplasmic Ca2+ overload produced by the abnormal Ca2+ homeostasis. Isolated cardiac myocytes after I/R presents an increase in diastolic [Ca2+]i, a decrease in the systolic [Ca2+]i transients, and a reduction of SR Ca2+ load, which correlate with a decrease in the activity of Na+/Ca2+ exchanger (NCX) i) . The pater (NCX) ,10. All er (NCX)  as illuser (NCX) ,12. Sequelae of acute myocardial infarction (AMI) are known to trigger the adverse cardiac remodeling, which is the basic mechanism underlying the progression to HF considered the end stage of various types of cardiovascular disease, including ischemic heart disease (IHD) . TherefoIn the last two decades, Urocortin (Ucn) isoforms, peptides related to stress, arose as potential therapeutic drugs to improve performances of heart in I/R and under HF ,17. ThisUcn is a mammalian peptide member of the corticotropin-releasing factor (CRF) family. Three isoforms of Ucn have been described so far, Ucn1, Ucn2, and Ucn3 ,19,20,21Ucn2 is formed by 38 aa showing homologies with rat and human CRF 34%) and Ucn1 (43%). Meanwhile, Ucn3 (38–41 aa) has 37–40% homology with Ucn2 ,20,21. T% and UcnUcn isoforms have high affinity to CRF receptors CRFR1 and CRFR2, which are G-protein coupled receptors that trigger various downstream signal transduction pathways. CRFR1 is mainly expressed in the brain, while CRFR2 is highly expressed in cardiac cells and peripheral tissue . CRFR2 hStructurally, CRFR1 and CRFR2 are approximately 70% identical at the aa level, but they exhibit considerable divergence at the N-terminal extracellular domain. Hence, they display ligand selectivity between Ucn members . Indeed,Altogether, signaling pathways activated downstream CRFR2 play a critical role in different aspects of myocardial function, which explain the many effects that Ucn isoforms have on the cardiovascular system.2+ overload and recovered completely the amplitude of [Ca2+]i transients; thus, it improved [Ca2+]i handling and improved heart contractility i homeostasis, inflammation, and changes in the expression of different genes related to cell death, hypertrophy, and extracellular matrix components, particularly the generation of collagen, increasing fibrosis in the heart. These events evoke heart dysfunction, leading to HF [The early adverse cardiac remodeling after an ischemic event is characterized by the alteration in [Cang to HF .In vivo studies have been performed to investigate the effects of Ucn administration in the adverse cardiac remodeling and cardiac function . Most st2+]i transient, and modulated the expression of several proteins related to [Ca2+]i homeostasis, such as TRPC5 and Orai1 channels [In a rat model of I/R, the i.v. infusion of Ucn2 (150 µg/Kg) right before heart reperfusion recovered significantly cardiac contractility and prevented fibrosis. Ucn2 administration recovered left ventricle ejection fraction and shortening, improved the amplitude of [Cachannels . Moreovechannels . 2+ handling and left ventricle function, as compared with HF mice. In addition, mRNA expression of hypertrophy markers and stress as BNP, ANF, α-skeletal actin, and β-MHC was lower in mice transfected with Ucn3 gene than in HF mice, preventing adverse cardiac remodeling and apoptosis [Interestingly, a recent study demonstrated that the Ucn3 gene transfected using adeno-associated viruse 8 (AAV8) in mice after 3 weeks of AMI improved Capoptosis .Preclinical studies proposed a positive therapeutic potential of Ucn’s family in cardiovascular diseases since they increased cardiac output, left ventricular ejection fraction (LVEF) ,63, and In the case of Ucn2, increased blood levels were observed in patients with systolic dysfunction but not in those with coronary artery disease without myocardial infarction . A recenOn the other hand, independent reports analyzed the effect of the i.v. infusion of Ucn on hemodynamic parameters in healthy and HF patients. For instance, that i.v. administration of a bolus of 50 µg of Ucn1 to healthy volunteers, compared to placebo administration, resulted in increased plasma levels of corticotropin (ACTH), cortisol, ANP, and decreased in ghrelin level . By contOther studies evaluated the use of i.v. administration of Ucn2 to avoid Ucn1 effects on the central nervous system. For example, the i.v. administration of Ucn2 (25 and 100 µg) in healthy volunteers and in patients with congestive HF increased cardiac output, heart rate, and LVEF in a dose-dependent manner; meanwhile, it decreased systemic vascular resistances . SimilarTo summarize, even if the effect of Ucn infusion in patients suffering AMI has not been addressed yet, data from clinical trials in HF patients provided evidence that Ucn isoforms efficiently decrease peripheral vascular resistances and the mean arterial pressure. The lowering of peripheral vascular resistances will be beneficial for AMI patients since it may promote a decrease in the afterload. At the same time, cardiac output increase by Ucn might be beneficial to compensate for cardiac myocyte loss during AMI, although this effect may also increase the oxygen consumption of the myocardium. Moreover, Ucn’s ability to regulate the post-transcriptional and translational processes involved in cardiac remodeling is worth being deeply investigated. Actually, understanding the mechanisms and downstream targets of Ucn in preclinical studies holds promise to allow fine-tuning of the signaling pathway activated under I/R, which can be hopefully translated soon to clinical trials. Future detailed clinical trials using a large number of patients are eagerly needed to determine whether Ucn could be useful as a sensitive biomarker for the adverse cardiac remodeling post-AMI or as therapeutic drugs that will mitigate AMI injuries to avoid the evolution of adverse cardiac remodeling toward HF.


Brain ischemia can be simulated in vitro by oxygen/glucose deprivation, which can be reversible by the re-establishment of physiological conditions. Accordingly, we examined the effects of glucose deprivation on the PI3K/Akt survival signaling pathway and its crosstalk with HIF-1α and Ca2+ homeostasis in SH-SY5Y human neuroblastoma cells. It was found that glucose withdrawal decreased HIF-1α protein levels even in the presence of the ischemia-mimicking CoCl2. On the contrary, and despite neuronal death, we identified a strong activation of the master pro-survival kinase Akt, a finding that was also confirmed by the increased phosphorylation of GSK3, a direct target of p-Akt. Remarkably, the elevated Ca2+ influx recorded was found to promptly trigger the activation of Akt, while a re-addition of glucose resulted in rapid restoration of both Ca2+ entry and p-Akt levels, highlighting the plasticity of neurons to respond to ischemic challenges and the important role of glucose homeostasis for multiple neurological disorders.The two crucial cellular insults that take place during cerebral ischemia are the loss of oxygen and loss of glucose, which can both activate a cascade of events leading to neuronal death. In addition, the toxic overactivation of neuronal excitatory receptors, leading to Ca Cerebral ischemia is a state of extreme metabolic stress occurring when the supply of oxygen and glucose is interrupted in a part of the brain, mainly due to the blockade of a cerebral artery . NeuronaThe heterodimeric hypoxia-inducible factor (HIF) is a transcription factor that is predominantly connected to the hypoxic status of cells and tissues mainly through its subunit HIF-1α , which iOne of the most prominent pro-survival signaling pathways is PI3K/Akt. Akt, a serine/threonine-specific protein kinase associated with the plasma membrane, is induced by the activation of phosphatidylinositol 3-kinase (PI3K), following stimulation of growth factor receptors with tyrosine kinase activity or G-protein-coupled receptors ,14. Once2+ homeostasis i, reduced ER Ca2+ content, and increased SOCE , moves close to the plasma membrane and facilitates Ca2+ entry through the ORAI1 component of the store-operated channel. We noticed unchanged levels of STIM1 after glucose deprivation and Tg treatment for 24 h  to simulate ER Carivation . Cells wrivation B. Of notrivation . Upon ERfor 24 h D, demons2+ entry, immediate or subsequent to ER Ca2+ depletion, could account for the increase in p-Akt levels observed during glucose deprivation. For this, we used cells re-suspended in KRH solution containing 3 mM CaCl2 and then incubated either with ionomycin (IONO) or miltefosine (HePC) for 0, 3, 6, or 10 min , and particularly its subunit HIF-1α, is the master regulator of cellular adaptation in a low oxygen environment. HIF-1α is required for cellular protection and metabolic alterations and, as such, is considered one of the many downstream targets of Akt. We and several other groups have established that under conditions that promote cell survival, there is a dose-dependent increase in both the protein levels of HIF-1α and p-Akt ,40,41,422+ signaling, as highlighted by a number of studies i) was measured in SH-SY5Y cells using the Ca2+-sensitive dye, Fura-2. Briefly, cells were detached, centrifuged, and resuspended in complete Krebs–Ringer–Henseleit (KRH) buffer  containing 50 μΜ DTPA. Afterwards, cells (10 × 106 cells/mL) were loaded with 5 μM Fura-2 AM  in KRH containing 200 μΜ sulfinpyrazone and incubated at 37 °C for 30 min. After washing, cells were centrifuged and resuspended in KRH buffer (1 × 106 cells/sample) or in KRH without glucose in case of simulating ischemic conditions. Cell aliquots were transferred to a thermostatted cuvette in a Perkin Elmer LS-50 fluorometer  and maintained at 37 °C, under continuous stirring. Fura-2 fluorescence was monitored with excitation and emission wavelengths of 340 nm and 510 nm respectively. Ca2+ entry was measured in cell samples resuspended in a Ca2+-free KRH containing 10 μM EGTA. Thapsigargin, the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase blocker, was added to induce depletion of intracellular Ca2+ stores. The recording was continued for up to 10 min until the end of the first peak and Ca2+, at a final concentration of 3mM, was then added. Traces were recorded and analysed as previously described [2+]i = Kd × (F − Fmin)/(Fmax − F), where Kd is the dissociation constant of the Fura-2–Ca2+ complex that was assumed to be 225 nM. The minimum values (Fmin) were determined by an intrinsic calibration procedure after the addition of 3 mM EGTA whereas, to estimate the maximal values (Fmax), CaCl2 (13 mM) was added after cell lysis with 0.1% Triton X-100. Changes in [Ca2+]i were determined by measuring peak or plateau values and expressing them as the change from basal levels.Intracellular Caescribed  using th4 cells/well) were seeded into 96-well plates and cultured in RPMI 1640 medium for 24 h at 37 °C. Afterwards, cells were treated with increasing concentrations of CoCl2 (6.25–400 μM), glucose (0–2 mg/mL) in RPMI containing 10% FBS for 24 h. Cell viability was determined using the MTT colorimetric method. Briefly, at the end of the incubation period 2.5 mg/mL MTT  solution was added to the culture medium and cells were incubated at 37 °C for 4 h. Afterwards, the cultured medium was removed and 100 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance of each sample was measured at 570 nm in an ELISA plate reader . Cell viability was expressed as the percentage of the absorbance value compared to control cells.SH-SY5Y cells  were expressed as fold changes of control samples (set at 1).p) values < 0.05 were considered significant. Statistical analysis was performed using the GraphPad Prism software .All data shown are representative of at least three independent experiments and are presented as mean ± standard error of the mean (S.E.M.). Statistical significance of differences was evaluated using one-way ANOVA, followed by Dunnett’s multiple comparison test. Probability (


Nearly perfectly unidirectional second harmonic generation of light is realized in a magnetoelectric material. 2O4 exhibits SHG transmission changes by almost 100% upon reversal of a magnetic field of just ±10 mT. The observed nonreciprocity results from an interference between magnetic-dipole and electric-dipole SHG. Although the former is inherently weaker than the latter, a resonantly enhanced magnetic-dipole transition has a comparable amplitude as a nonresonant electric-dipole transition, thus maximizing the nonreciprocity. Multiferroics and magnetoelectrics are an obvious materials platform to exhibit nonreciprocal nonlinear optical functionalities.Mirror symmetries are of particular importance because they are connected to fundamental properties and conservation laws. Spatial inversion and time reversal are typically associated to charge and spin phenomena, respectively. When both are broken, magnetoelectric cross-coupling can arise. In the optical regime, a difference between forward and backward propagation of light may result. Usually, this nonreciprocal response is small. We show that a giant nonreciprocal optical response can occur when transferring from linear to nonlinear optics, specifically second harmonic generation (SHG). CuB Unidirectional manipulation of photons is a key issue in modern information technology as exemplified by optical isolators used for lasers and in optical networks. While conventional optical isolators are the composite of a magneto-optical medium, magnets, and polarizers, recent studies show that such a one-way flow of photons can be achieved in single bulk magnetoelectric materials, where time-reversal and space-inversion symmetries are simultaneously broken. Such nonreciprocal propagation occurs because the optical properties change with the reversal of the propagation direction of light d-d transition of Cu2+ holes in CuB2O4 (Second harmonic generation (SHG), which denotes the frequency-doubling of a light wave in a material, is one of the simplest nonlinear optical processes. SHG can be classified into two types depending on its origin. One is electric-dipole (ED)–SHG, in which the frequency-doubled electric polarization  M→i(2ω) . Here, wI(2ω) is enhanced by constructive interference, but can be extinguished just by the reversal of i-type tensor and MD-SHG is a c-type tensor ; the latter changes sign under the reversal of the magnetic field. However, experimental realization of such a huge modulation of the SHG yield  remains a challenge. ED-SHG usually dominates MD-SHG because in the expansion of the electromagnetic field potential, with respect to The SHG intensity on the detector will be ch other . Obvious2O4, where the SHG intensity changes by 97% upon the reversal of an external magnetic field. The effect is most pronounced for light around 1.4 eV, which is resonant with the lowest intra-atomic d-d transition of the Cu2+ ions. We found that the MD-SHG process is resonantly enhanced at this d-d transition, in contrast to the ED-SHG process. The resultant resonant MD-SHG and nonresonant ED-SHG contributions are of the same order of magnitude, yielding a large nonreciprocal signal via their interference.Here, we report on the observation of nonreciprocal SHG in noncentrosymmetric CuB2O4 crystalizes in a noncentrosymmetric tetragonal structure with the space group symmetry Id  ions occupy two inequivalent sites denoted as A and B  in an external magnetic field in the ab plane, where θ denotes the angle between the crystal’s [010] axis and the direction of the magnetic field , the electric polarization is absent for H parallel to the [010] axis . This results in the absence of ED-MD interference and, hence, of a change in SHG intensity upon the reversal of the magnetic field . The remaining dependence on the direction of the magnetic field is caused by the small shift of the resonance energy experiences with temperature  at photon energies below (above) the MD resonance, which explains the sign change of the nonreciprocal effect across the resonance. Notably, the amplitudes of the MD- and ED-SHG contributions become the same at the positions indicated by dotted circles in Next, we discuss the observed spectral shape. 2O4. The SHG intensity changes by almost 100% upon the reversal of a small magnetic field of 10 mT. The observed nonreciprocity originates from the interference between ED-SHG and MD-SHG. The MD-SHG process is resonantly enhanced at the d-d transition of Cu2+ holes and becomes comparable in amplitude to the nonresonant broadband ED-SHG process. This work establishes that nonreciprocal wave-propagation effects are not limited to linear optics but are also present as nonlinear optical effects and exhibit a magnitude up to the optical-diode-like unidirectional propagation of light.To conclude, we experimentally demonstrate the nearly perfectly unidirectional propagation of SHG light waves in CuB2O4 were grown by a flux method. Powders of CuO (7.250 g), B2O3 (15.228 g), and LiCO3 (4.041 g) were mixed. They were heated in air at 1020°C, subsequently cooled down to 800°C at a rate of 1.1°C/hour, and then cooled to room temperature at a rate of 16°C/hour. A single crystal was oriented by using a Laue x-ray diffractometer and cut into thin plates of 50 μm thickness with the widest (100) faces. The sample surfaces were specularly polished by alumina lapping films.Single crystals of CuBNote added in proof: We have recently become aware of the related work by J. Mund et al. (arXiv: 2005.05393 2020).


Our study suggests that almost 80% of AML patients can undergo salvage therapy following relapse after front-line HDCT/autologous HCT. Allogeneic HCT can provide cure in one third of patients relapsing after front-line HDCT/autologous HCT.Autologous hematopoietic cell transplantation (HCT) is suitable for consolidation of favorable-/intermediate-risk AML patients in CR1. However, ~50% of AML patients relapse after autologous HCT, and efficacy of subsequent salvage strategies including allogeneic HCT remains unclear. We studied 123 consecutive patients with newly diagnosed AML undergoing high-dose chemotherapy (HDCT)/autologous HCT in CR1. In relapsing patients afterwards, we analyzed salvage treatments and outcomes focusing particularly on salvage allogeneic HCT. Of 123 patients, 64 (52%) relapsed after autologous HCT. Subsequently, 13 (21%) received palliative therapy, whereas 51 (79%) proceeded to salvage therapy with a curative intent. Of the 47 patients with a curative intent and who did not proceed directly to allogeneic HCT, 23 (49%) achieved CR2 or had ongoing hematologic CR1 despite molecular relapse. Finally, 30 patients (47%) received allogeneic HCT with estimated 3-year leukemia-free and overall survival rates of 33% and 43%. Hematologic remission at allogeneic HCT and lack of acute GvHD had a positive impact on OS and LFS ( NPM1-mutated AML [Although up to 80% of AML patients less than 60 years of age achieve complete hematological remission CR1) following intensive induction chemotherapy  followin. Consoliated AML . Allogenated AML , 11 cannNevertheless, relapse remains the major problem for patients with AML. This is also true for patients after HDCT/autologous HCT, with relapse rates up to 50% . MRD tesReluctant use of autologous HCT for consolidation of CR1 in AML is driven by concerns of the lacking GvL effect, the possibility of autologous graft contamination by leukemic stem cells, and the toxicity of the HDCT. Moreover, the preferred re-induction strategies in the relapse situation after autologous HCT are a matter of continuous debate, especially regarding the feasibility of subsequent salvage allogeneic HCT and considering the toxicity of the previous HDCT/autologous HCT –21. To fde novo, secondary (s-AML) following myelodysplastic syndrome, therapy-associated (t-AML), and extramedullary manifestation (chloroma), as well as biphenotypic leukemia. (B) Patients must have received two cycles of anthracycline/cytarabine-based induction therapy followed by consolidation by HDCT/autologous HCT. (C) The rationale for HDCT/autologous HCT was the achievement of MRD-negative CR1 by flow cytometry and molecular genetics (if appropriate markers were available) in AML patients with favorable of intermediate-risk profile with adequate performance status. Only occasionally, patients with adverse risk who either lacked a suitable donor or refused allogeneic HCT in CR1 were also included. (D) CD34+ stem cell mobilization with granulocyte colony-stimulating factor and stem cell harvest were performed after the second induction cycle [This retrospective single-center study included 123 consecutive adult patients (≥18 years) diagnosed with AML and undergoing HDCT/autologous HCT in CR1 between 2000 and 2018 at the Department of Medical Oncology, University Hospital of Bern, Switzerland. Inclusion criteria were: (A) all types of AML including on cycle , 23. HDCon cycle , 25.Patients with evidence of relapse  occurring after consolidation by HDCT/autologous HCT in CR1 were subsequently analyzed. All allogeneic HCTs were performed at the Department of Hematology, University Hospital of Basel, Switzerland. European LeukemiaNet  criteria were used for genetic risk stratification of patients . The stuThis retrospective analysis aimed to study relapse after autologous HCT consolidation in CR1 with a special focus on feasibility and outcome of allogeneic HCT as a salvage procedure. The following endpoints were considered: (A) cumulative incidence of relapse in patients with consolidation by HDCT/autologous HCT in CR1; (B) frequency of relapsing patients effectively receiving allogeneic HCT, interval from first relapse to allogeneic HCT, and NRM following allogeneic HCT; and (C) overall survival (OS) and leukemia-free survival (LFS) in patients with salvage allogeneic HCT after autologous HCT in CR1.Staging and morphologic response criteria in AML were based on the 2017 ELN AML recommendations . MoleculFor allogeneic HCT, myeloablative conditioning (MAC) was defined as a regimen containing either a total dose of greater than 6.4 mg/kg busulfan i.v. or two alkylating agents. Regimens containing lower conditioning intensities were defined as reduced-intensity conditioning (RIC) . Acute aCategorical variables were summarized as frequencies and percentages, and continuous variables were summarized as median and range. Probabilities of OS and LFS were calculated using the Kaplan–Meier method. All analyses were performed using Statistical Software for Social Sciences version 26.0  and R Software for Statistical Computing and Graphics (R version 3.6.2).De novo AML was the most common type  followed by s-AML , t-AML , and extramedullary manifestation/chloroma . Six (5%) patients had biphenotypic acute leukemia. A total of 102 (83%) patients had either favorable or intermediate-risk AML , whereas 21 patients (17%) were considered as adverse risk.This study included 123 consecutive AML patients undergoing intensive induction therapy with subsequent consolidation by HDCT/autologous HCT in CR1. We summarized patient and disease characteristics at diagnosis in Table Remission status following consolidation by HDCT/autologous HCT in CR1 and eventual relapse treatment are presented in Supplementary Table In total, 23 of 47 patients (49%) receiving salvage treatment with a curative intent at relapse  achieved CR2 or had ongoing hematologic CR1 under bridging therapy despite molecular relapse.n = 6/30) or undergoing preceding intensive relapse treatment (n = 24/30)  Fig. . The medOut of 30 patients, 22 (74%) received grafts from HLA-identical donors, equally distributed between related and unrelated donors (11 each). Eight patients (26%) had either mismatched (4/8), haploidentical (2/8) donors, or umbilical cord blood (2/8) as hematopoietic stem cell source. The remission status at the time of allogeneic HCT was as follows: 83% (25/30) were in CR2 or in CR1 with molecular relapse, 3% (1/30) had PR, and 14% (4/30) had refractory disease.The median interval since allogeneic HCT to last follow-up (FU) was 28 months. At last FU, 11 patients were alive  and in remission. Of these 11 patients with remission post-allogeneic HCT, 5 patients underwent conditioning treatment directly (4/5) or had bridging treatment with enasidenib (1/5) due to molecular relapse. Eleven patients (37%) suffered from post-transplant relapse or progression with a median time of 6 months following allogeneic HCT. Mortality was due either to non-relapse reasons  or relapsed/refractory disease  Fig. . Mortalip = 0.032) and presence of severe acute GvHD (grade II–IV) negatively influenced both OS (p = 0.006) and LFS (p = 0.025). In contrast, complete hematologic remission at allogeneic HCT contributed to better LFS (p = 0.036) and OS (p = 0.011). None of the other risk factors  had a significant impact on OS and LFS.Applying univariate risk factor analysis  following relapse treatment after consolidation by HDCT/autologous HCT in CR1 are depicted in Supplementary Table In patients without relapse after consolidation by HDCT/autologous HCT in CR1, 50 out of 59 (85%) were alive in CR of AML with a median FU of 7 years. Of the remaining 9 patients (15%), 4 patients (7%) died from transplant-related mortality due to infection in aplasia in the early post-autologous HCT phase, 3 patients (5%) died due to co-morbidities , and 2 (3%) due to a secondary malignancy occurring after autologous HCT.HDCT followed by autologous HCT represents a suitable therapeutic option for consolidation of CR1 in good and intermediate-risk AML patients , 31–33. First, the relapse rate after consolidation with HDCT/autologous HCT in CR1 (52%) within our patient cohort was in accordance with previous data on autologous HCT-based consolidation in AML , 31, 35.Forty-nine percent of patients relapsing after first-line HDCT/autologous HCT and undergoing salvage re-induction were achieving CR2 in our cohort. This was similar to the CR2 rates reported by others for AML patients relapsing after conventional chemotherapy and receiving salvage chemotherapy treatment , 38. In Importantly, the 3-year OS rate in the salvage-allo-cohort was 43%, which is comparable to reports in the literature in patients undergoing salvage allogeneic HCT with preceding chemotherapy consolidation of CR1 . In all,In comparison to front-line allogeneic HCT, salvage allogeneic HCT is known to be associated with higher NRM up to 46% in poor risk AML patients in the post-transplant FU –43. AccoImprovement and integration of NGS technologies in the diagnostic work-up have enabled to depict a unique genetic make-up for each individual AML case. In some situations, a molecular relapse may allow timely planning of allogeneic HCT, even before it becomes apparent at the morphological level –46. ThisIn conclusion, our study demonstrates that consolidation by HDCT/autologous HCT in CR1 is not negatively affecting feasibility and efficacy of subsequent allogeneic HCT for those patients relapsing after autologous HCT, and more than one third of patients relapsing after consolidation by HDCT/autologous HCT in CR1 can be rescued by salvage allogeneic HCT. As there is no curative alternative to allogeneic HCT in relapsing AML patients, the indication to salvage allogeneic HCT must be considered by treating physicians for patients relapsing after consolidation of CR1 with HDCT/autologous HCT in patients who are fit for this approach.Supplementary Tables 1–3


The etiology of ionic imbalances resulting from stroke-induced ischemia and acidosis includes the dysregulation of multiple plasma membrane transport proteins, such as increased activity of sodium-potassium-chloride cotransporter-1 (NKCC-1). Experiments using NKCC1 antagonists, bumetanide (BMN) and ethacrynic acid (EA), were carried out to determine if inhibition of this cotransporter affects Na+ and Ca2+ overload observed following in vitro ischemia-acidosis. Fluorometric Ca2+ and Na+ measurements were performed using cultured cortical neurons, and measurements of whole-cell membrane currents were used to determine target(s) of BMN and EA, other than the electroneutral NKCC-1. Both BMN and EA depressed ischemia-acidosis induced [Ca2+]i overload without appreciably reducing [Na+]i increases. Voltage-gated Ca2+ channels were inhibited by both BMN and EA with half-maximal inhibitory concentration (IC50) values of 4 and 36 μM, respectively. Similarly, voltage-gated Na+ channels were blocked by BMN and EA with IC50 values of 13 and 30 μM, respectively. However, neither BMN nor EA affected currents mediated by acid-sensing ion channels or ionotropic glutamatergic receptors, both of which are known to produce [Ca2+]i overload following ischemia. Data suggest that loop diuretics effectively inhibit voltage-gated Ca2+ and Na+ channels at clinically relevant concentrations, and block of these channels by these compounds likely contributes to their clinical effects. Importantly, inhibition of these channels, and not NKCC1, by loop diuretics reduces [Ca2+]i overload in neurons during ischemia-acidosis, and thus BMN and EA could potentially be used therapeutically to lessen injury following ischemic stroke.One consequence of ischemic stroke is disruption of intracellular ionic homeostasis. Intracellular overload of both Na For example, the Na+-K+-Cl- cotransporter 1 (NKCC1)  X.  promotesll death . In contrivation .2+ dysregulation evoked by ischemia in cortical neurons, enhancing neuronal death  rats were isolated and cultured as previously described . Briefly2+ concentrations, [Ca2+]i, were measured in isolated cortical neurons using fluorescent imaging techniques and the Ca2+ sensitive dye, fura-2. Cells were loaded using the membrane permeable ester form of fura-2, fura-2 acetoxymethyl ester (fura-2 AM), as previously described . Cells plated on coverslips were incubated for 2 h at room temperature in Neurobasal media with 10 μM SBFI AM, 0.4% DMSO, 0.5% bovine serum albumin (BSA) and 0.1% pluronic. The coverslips were then washed in SBFI AM free physiological saline solution (PSS) prior to experiments being performed. The same microscope, objective, filter set, light source and camera as used for Ca2+ imaging were used. Images were acquired at 0.3 Hz.Changes in intracellular Nas) were monitored throughout experiments for stable values ≤30 MΩ and were compensated at 40% . When using the perforated-patch whole-cell configuration, electrical access was achieved with a pipette solution containing amphotericin B , 5 mM TEA, 5 mM MgCl2 and 1 mM ethylene glycol tetraacetic acid (EGTA), requiring the use of conventional whole-cell patches to allow diffusion into the clamped cells. To activate voltage-gated sodium currents, cells were held at −70 mV and stepped from −50 to +5 mV for 50 msec in 5 mV increments. All membrane currents were amplified and filtered at 5 kHz with an Axon 200 amplifier, digitized at 10 kHz with a Digidata 1322A, and acquired using Clampex 10 (Axon) software. Currents were leak subtracted using the Clampex P/4 protocol.Membrane currents were recorded using the conventional and perforated, whole-cell patch clamp configurations as previously described . The conericin B . An amph2, 1.0 MgCl2, 20 glucose, and 25 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH to 7.4 with NaOH). Acidosis was produced using this solution adjusted to pH 6.0. Ischemia-acidosis was produced using glucose-free PSS with 4 mM sodium azide added and adjusting the solution to pH 6.0. The control pipette solution consisted of (in mM): 75 K2SO4, 55 KCl, 5 MgSO4, and 10 HEPES (titrated to pH 7.2 with N-methyl-d-glucamine). External solutions were applied using a rapid application system as previously described : 140 NaCl, 5.4 KCl, 1.3 CaClescribed . To accoescribed . BumetanAll chemicals used in this investigation were of analytical grade. The following reagents were used: TEA ; TTX ; Antibiotic/Antimycotic, BSA, DMEM, DMSO, FBS ; B-27, Fura-2 AM, Neurobasal ; Ethacrynic Acid ; and Bumetanide, EGTA, HEPES, L-Glutamine, Poly-L-Lysine .340/R380) and converted to [Ca2+]i using the Grynkewicz equation with constant parameters Rmin, Rmax and β determined using calibration solutions containing fura-2 salt. Differences in the values of these parameters from those obtained using an in situ protocol are controlled for by normalizing responses in the same cell. However, since [Ca2+]i is not a linear function of R340/R380, this calculation is necessary to preserve the dynamic changes produced in the cell. For sodium imaging experiments, because the emission intensity of SBFI excited by 340 nm light is highly pH sensitive , divided by the emission intensity of the cell excited by 380 nm light as a function of time, I380(t) (RSBFI = I340(0)/I380(t)). Analyses of [Ca2+]i, RSBFI and electrophysiological recordings were performed using Clampfit 10.5 (Axon Instruments). Statistical analysis was conducted using SigmaPlot 11 and SigmaStat 3 software . Statistical differences were determined using paired and unpaired t-tests for within group and between group experiments, respectively, and were considered significant if p < 0.05. For multiple group comparisons 1- and 2-way ANOVAs, with or without repeat measures, were used, as appropriate. When significant differences were determined with an ANOVA, post-hoc analyses were conducted using a Dunn Test to determine differences between individual groups.Imaging data files were collected with SlideBook 4.02 . Fluorescence emission intensities of individual fluorescent cells, measured using circular regions of interest (ROIs) placed over cell bodies, were collected as functions of time. For calcium imaging experiments, emission intensities were exported to SigmaPlot11, ratioed  induced a rapid, transient increase in [Ca2+]i in cortical neurons followed by a slow steady rise in the continued presence of the Isc. pH 6 solution. Upon washout of the solution, the [Ca2+]i rebounded to a second, slower decaying peak, before returning to near baseline levels  relative to Control , is dependent on the transient increases and as well as the degree of recovery during the recording period. For cells incubated in bumetanide, the decreased elevations in [Ca2+]i occurring during ischemia-acidosis were offset by the reduced recovery during washout. Thus, bumetanide failed to reduce net [Ca2+]i increases relative to Control. In contrast, ethacrynic acid, which produced a similar degree of inhibition of recovery as bumetanide, but a greater block of initial peak, sustained and rebound peak [Ca2+]i than bumetanide, reduced the net [Ca2+]i increase produced by ischemia-acidosis   . Since ts (ASIC) . Bumetans (ASIC) . In conts (ASIC) . These rvely see , which s2+]i . Previous studies in our laboratory have shown VGCC are activated downstream of ASIC following ischemia-acidosis (50), Hill coefficients and non-reducible current (asymptotic minimum) values of 8 μM, 0.41 and 0.37, respectively for BMN (blue circles and line) and 36 μM, 0.75 and 0.0 for EA (red circles and line)  . Thus, e2+]i triggered by acidosis can also be reduced via the inhibition of voltage-gate Na+ channels (VGSC). Furthermore, blocking of VGSC has been shown to lessen neuronal [Ca2+]i increases observed after oxygen-glucose deprivation  values of 19 μM, 0.46 and 0.21, respectively for BMN (blue circles and line) and 36 μM, 0.72 and 0.22, respectively for EA (red circles and line). These results indicate nearly 20% of the VGSC current is BMN and EA-insensitive.Our laboratory has shown that elevations of i overload. Neither loop diuretic significantly blocked acidosis- or glutamate-activated whole cell inward currents, suggesting that the inhibition of ischemia-acidosis induced [Ca2+]i overload was not due to block of ASIC or glutamatergic ion channels. In addition, neither BMN nor EA significantly altered ischemia-acidosis induced increases in [Na+]i. Thus, the increase in [Na+]i induced by ischemia-acidosis is not mediated by Na+ influx through either VGSCs or NKCC. These observations further suggest that loop diuretic suppression of [Ca2+]i overload during ischemia-acidosis is not a downstream effect of mechanisms activated to preserve [Na+]i homeostasis.The primary finding of this study is the loop diuretics bumetanide and ethacrynic acid inhibit voltage-gated Ca2+]i overload  blocked nearly all of the activity of both NKCC1 and NKCC2  similar to its IC50 for NKCC1 and NKCC2. In contrast to observations of VGCC block by bumetanide in the current study, a population of cortical neurons expressed VGSC that were resistant to bumetanide block, even at the highest concentrations (1 mM). In both bumetanide-sensitive and bumetanide-insensitive cells, VGSC currents were blocked by >70% by TTX (100 nM). Bumetanide application did not have an additive effect with TTX, suggesting bumetanide specifically inhibits one of the TTX-sensitive channel subtypes expressed in cortical neurons. Cortical neurons express a variety of TTX-sensitive VGSC, including NaV1.1, NaV1.2, NaV1.3, NaV1.6 and NaV1.7 and the TTX-insensitive NaV1.9  was more potent than its block of NKCC1 but comparable to its block of NKCC2. Like bumetanide, EA failed to completely block VGSC currents, with approximately 20% of the current remaining at 1 mM EA.The ethacrynic acid block of VGSC . Both ischemia and acidosis are known to evoke increases in [Na+]i resulting in reverse-mode activity of the Na+/Ca2+ exchanger (+ by NCX produces Ca2+ influx and [Ca2+]i elevations. Given [Na+]i is not affected by the loop diuretics, it does not appear a reduction in [Na+]i produced by inhibition of VGSC and concomitant lessening of Ca2+ influx via NCX can explain the ability of these loop diuretics to mitigate ischemia-acidosis evoked [Ca2+]i overload. NKCC activity has been implicated in the [Na+]i accumulation that precedes NCX activity and [Ca2+]i overload in astrocytes (+]i elevation in neurons suggesting NKCC is not a major conduit for ischemia-acidosis induced Na+ influx which leads to [Ca2+]i overload. The lack of NKCC contribution to [Na+]i increases may be due to reduced activity of the cotransporter during ischemia-acidosis. It was previously reported the activity of NKCC1 is reduced by ∼80% when extracellular pH approaches 6.0 (Inhibition of voltage-gated sodium channels by BMN and EA did not alter [Naxchanger . Extrusitrocytes . Neitherches 6.0 .+]i in neurons and astrocytes appeared during re-oxygenation rather than during the ischemic event (H. +]i accumulation during ischemia-acidosis. While reduced edema associated with NKCC1 inhibition by bumetanide may lessen stroke injury (2+]i overload in response to ischemia-acidosis by BMN would also improve outcomes in stroke. Similarly, inhibition of voltage-gated channels may explain how bumetanide reduces glutamate-mediated excitotoxicity (Stroke-induced gray and white matter injury in mice was shown to be reduced in NKCC knockout animals H. . Similarevent H. , consiste injury , the blutoxicity .2+ channels is known to contribute to the actions of antiepileptic drugs, such as levetiracetam (Bumetanide has been shown to decrease seizure activity in humans . Resultsiracetam . Similariracetam . Finallyiracetam . Therefo+, K+, and Cl− from the tubular fluid. Loop diuretics have additional effects, such as the lowering of blood pressure, which is often observed even prior to diuresis (2+]i overload reported here.Clinically, bumetanide and ethacrynic acid, are used in edematous states, such as heart failure, to promote diuresis and natriuresis . This efdiuresis . The effdiuresis , and thu2+]i overload in neurons at concentrations near their respective IC50 values for NKCC inhibition. These effects appear to be in part mediated via the inhibition of voltage-gated Ca2+ and Na+ channels and are not due to any direct effects on ionotropic glutamatergic receptors or acid-sensing ion channels. Furthermore, the inability of the loop diuretics to reduce ischemia-acidosis evoked [Na+]i elevations suggest NKCC cotransporters are not involved in neuronal ionic imbalances during ischemia-acidosis; and inhibition of these transporters does not account for the beneficial effects of bumetanide and ethacrynic acid under these conditions. However, the ability of these loop diuretics to lessen ischemia-acidosis induced [Ca2+]i overload suggests that they may be useful for reducing stroke injury and that their effect on Ca2+ and Na+ channels may in part explain observations made in previous studies.In conclusion, the loop diuretics, bumetanide and ethacrynic acid effectively suppress ischemia-acidosis induced [Ca


TMCO1 is an ER Ca2+ leak channel which facilitates Ca2+ leak upon ER “overload” through the novel Ca2+ load activated Ca2+ mechanism. This mini-review brings together the clinical and scientific advances made since the discovery of TMCO1 deficiency in disease, including broadened phenotype, understanding of pathophysiology, and implications to patient management of TMCO1 defect syndrome.A decade has passed since transmembrane coiled-coil domains 1 (TMCO1) defect syndrome was identified in 11 undiagnosed patients within the Old Order Amish of Northeastern Ohio—a disorder characterized by a distinctive craniofacial dysmorphism, skeletal anomalies and global developmental delay. Twenty seven patients, from diverse ethnic groups, have been reported with pathogenic TMCO1 variants now recognized to cause cerebrofaciothoracic dysplasia (CFTD). The implication of previously uncharacterized TMCO1 within disease has instigated a 10-year journey to understand the function of TMCO1 protein in Ca Beginning in the 1960s, inherited disorders among Old Order Amish communities were the subject of exceptional observational studies  and manyThe Amish communities of North America represent unique populations due to their closed communities and small founding populations. They descend from Swiss Anabaptist immigrants who came to the New World in the early eighteenth century. There is increased incidence of rare recessive disorders in Amish populations, caused by founder effects. The majority of affected individuals are homozygous for their respective pathogenic variant, which resides within a relatively large homozygous haplotype block. Biological and social aspects of the Amish communities make them ideal participants for studies in population genetics and genomic medicine.High density single-nucleotide polymorphism (SNP) microarray genotyping has been successfully used in linkage analysis and genome-wide homozygosity mapping for undiagnosed recessive conditions within the Amish communities. Using SNP array genotyping data, a disease locus can be mapped to one specific chromosome region with a few affected patients. This historic breakthrough was particularly significant in identifying genetic causes of rare conditions and gene discovery before the era of next-generation sequencing .In 2010, we identified a novel autosomal recessive condition in a group of 11 undiagnosed patients within the Old Order Amish of Northeastern Ohio with similar clinical phenotypes . All werGenome-wide SNP array genotyping and autozygosity mapping identified a single shared homozygosity region of 3.3 Mb in all patients at chromosome 1q23.3-q24.1 containing 23 candidate genes . DNA seq2+-selective cation channel. The negative charge of pore-lining residue D140 is required for Ca2+ permeation, with a charge-neutralizing mutation abrogating Ca2+ extrusion from the ER. No currents are detectable through the TMCO1 (p.Ser47Ter) mutant protein  pumps, and release through Ca2+ leak channels. ER Ca2+ leak is currently not well defined.The ER is a critical intracellular Ca2+ leak channel  mouse showed severe ER Ca2+ mishandling within osteoblasts and granulosa cells, although no overload was observed in germinal vesicles (TMCO1 has been established as an ER Ca channel . Knockdontration . TMCO1 Avesicles . This su2+-load activated Ca2+ (CLAC) mechanism. Increases of ER Ca2+ concentrations to “overload” levels result in endogenous TMCO1 monomers and dimers undergoing oligomerization to form homotetramers. Tetrameric TMCO1 channel facilitates active Ca2+ leak from the ER to reduce Ca2+ load to resting ER concentrations as illustrated in 2+-sensing mechanism of TMCO1 has not yet been investigated. Further studies are required to determine if TMCO1 can self-detect ER Ca2+ load or whether a secondary Ca2+-sensing protein is required.TMCO1 functions in a novel Ca2+ homeostasis is important in maintaining ER protein synthesis and membrane lipid biogenesis. The activity of many ER chaperones and foldases utilize Ca2+as a cofactor, binding Ca2+ with low affinity  and Ca2+-induced Ca2+ release (CICR) signaling. SOCE is an important signaling mechanism in non-excitable cells: facilitated by the Ca2+-release activated Ca2+ (CRAC) channel. CICR is thought to play an important role in Ca2+ signaling in cardiac smooth muscle.ER Ca2+ homeostasis on downstream functions is critical to understand TMCO1 deficiency pathophysiology. TMCO1-KO mouse displays a disease phenotype consistent with TMCO1 defect syndrome, including delayed osteogenesis, cognitive disability, and unstable gait  and ER stress-mediated apoptosis through the IRE1 pathway  . They prTwenty-seven patients, from diverse ethnic backgrounds, are reported in literature as having TMCO1 defect syndrome. Five further pathogenic variants have been identified. All identified variants result in a premature termination codon (PTC), including a splicing variant which results in exon five skipping and early termination in exon six. Nonsense- mediated mRNA decay has been proposed for 4/5 variants, with 1/5 variants predicted to produce a non-functional protein product . It is tAn additional five variants have been reported as pathogenic on ClinVar but not reported in literature . 4/5 varThe increase in patient cohort size has improved understanding of both disease genotype and phenotype. Full reported clinical manifestation information is available within Key features initially reported by Xin et al have been reinforced by these additional cases. Most notable craniofacial features include low hairline, low-set ears, highly arched bushy eyebrows with synophrys, orbital hypertelorism, and brachycephaly with a flat face. Skeletal anomalies affecting the spine, ribs and scapula are common, including rib fusions and spine fusions. Severe intellectual difficulties have been reported in all cases. Developmental milestones have not been consistently addressed in clinical reports; however, 50% of individuals have been reported as non-verbal, with verbal individuals reported to have delayed first words, speech defects, and a small limited vocabulary. Developmental motor milestones are also delayed, with some individuals unable to walk and significant delay in age of independent sitting reported. Behavioral problems, such as anxiety and self-mutilation, have been described .TMCO1 defect syndrome phenotype has been well described; however, disease development over time has been scarcely reported in literature. Further case reports of individuals may provide insight into disease progression and the effectiveness of treatments and interventions. With that in mind, this mini-review recommends clinical management based on current evidence.Assessment of skeletal and neurological abnormalities should be completed. Speech and language therapy and physical therapy may provide benefit to individuals. Genital urinary tract abnormalities have been identified in 37% individuals, including kidney anomalies in four individuals. Assessment by a renal team is therefore advisable.Cardiac anomalies have been reported in three individuals, including a narrow patent ductus arteriosus (PDA) present at age 4, hypertrophic left ventricle at 9 months, a systolic heart murmur at birth . A smallA history of spontaneous abortions has been reported in families affected by TMCO1 defect syndrome . TMCO1 dTMCO1 has been linked with other conditions. Primary open-angle glaucoma (POAG) is a heritable common cause of blindness, with a single modifiable risk factor of intraocular pressure (IOP), which is also highly heritable . GWAS st2+ homeostasis, although how the deficiency results in the clinical presentation of CFTD is not fully understood. Improving our understanding of the pathophysiology of CFTD will be significant in developing potential treatments to alleviate symptoms.It is now understood that TMCO1 is a CLAC channel with a role in maintaining ER Ca2+ leak, including TMCO1 Ca2+ sensing mechanism and the impact of store overload on store dependent Ca2+ signaling pathways. Ca2+ signaling has been shown to be altered due to TMCO1 deficiency but has not been investigated in depth (2+ signaling is associated with disease. Deficiencies of CRAC channel components, Orai and STIM, are well known in their link to innate and acquired immunity. They have also been associated with congenital myopathies, anhydrosis, dental defects, pancreatitis, and kidney issues (Further research is required into the regulation of TMCO1-dependent Cain depth . Aberrany issues . Some phTMCO1 is reported to be ubiquitously expressed in both embryonic and adult tissues . TMCO1 d2+ homeostasis has been shown to increase ER stress, resulting in UPR, ERAD, and IRE1-dependent apoptosis and is clearly linked to the pathophysiology of TMCO1 defect syndrome (2+ signaling may contribute to TMCO1 defect syndrome phenotype, it seems likely that perturbations in other ER processes through ER stress mechanisms play a more principle role. Further translational research with the emphasis on the normalization of CLAC channel and ER Ca2+ homeostasis as potential therapeutic approaches is certainly our ultimate hope for the affected patients and future research direction.Loss of ER Casyndrome . This is2+ homeostasis, has significantly improved our understanding of the pathophysiology of CFTD. This is an example of the impact of rapidly evolving genomic technologies on gene discovery as well as the consequent improvement of patient management. It also demonstrates how rare disease studies may help us understand essential functional pathways in cell biology.A decade-long journey has defined TMCO1 defect syndrome as a form of CFTD, and has instigated research in to the function of TMCO1 protein. The characterization of TMCO1 function as a novel CLAC channel, with a role in maintaining ER CaHW initiated the concept and outline. HB-R drafted the manuscript. All authors contributed ideas, edited, and approved the final 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.


This suggests that astrocytic Ca2+ signals also contribute to pathological processes in the CNS. In this context, the molecular mechanisms by which aberrant astrocytic Ca2+ signals trigger dopaminergic neuron loss during Parkinson’s disease (PD) are only beginning to emerge. Here, we provide an evidence-based perspective on potential mechanisms by which aberrant astrocytic Ca2+ signals can trigger dysfunction in three distinct compartments of the brain, viz., neurons, microglia, and the blood brain barrier, thereby leading to PD. We envision that the coming decades will unravel novel mechanisms by which aberrant astrocytic Ca2+ signals contribute to PD and other neurodegenerative processes in the CNS.Astrocytes display a plethora of spontaneous Ca These cuffering . The cri2+ signals in situ and in vivo. Astrocytic Ca2+ signals respond to a variety of pharmacological and behavioral stimuli  and reversal of the Na+/Ca2+ exchanger, NCX. The presence of distinct compartments, mechanisms and sources for astrocytic Ca2+ signals strongly suggests that these signals modulate a diverse array of signaling pathways not only in the astrocytes themselves, but also in the neural circuits within which they reside.Unlike neurons, astrocytes are not electrically excitable, which has necessitated inquiry into the molecular machinery utilized by astrocytes to exert their functional effects on neurons and neural circuits. In this regard, studies utilizing genetically encoded calcium indicators (GECIs) such as GCaMPs have shown that astrocytes possess a plethora of spontaneous Ca stimuli , and are stimuli . In addiulum ER;  and mitoulum ER; , as well sources . At a surocesses . For exa2+ signals in CNS function are bolstered by studies showing that these signals regulate the probability of neurotransmitter release  (i) The targeted knockdown of EAAT2 in astrocytes causes degeneration of SNc DA neurons in a mouse model of PD (ii) Exposure of rodents to the PD toxin 6-hydroxydopamine (6-OHDA) causes a downregulation of EAAT2  . Therefo (EAAT2) . With reel of PD  and (ii)of EAAT2 .2+-dependent manner   activate mitochondrial dehydrogenases in order to generate the co-factors required for oxidative phosphorylation and ATP generation  , suggesters MCU; , suggesters MCU; , and sponeration . Disrupt2+ signals in mitochondria within astrocytic processes as it relates to α-syn pathology. Recent studies have shown that astrocytes readily take up extracellular α-syn aggregates, and that α-syn can damage astrocytic mitochondria  in the pathogenesis of PD, it is pertinent to discuss aberrant Cachondria , as welleostasis . Togetheunctions . Therefo2+ signals in astrocytes could play a role in initiating microglial activation during PD. In the sections below, we present two potential scenarios in which aberrant changes in spontaneous astrocytic Ca2+ signals could lead to abnormal crosstalk between astrocytes and microglia, thus accelerating neuronal loss in PD.Microglia are classically viewed as the immune surveillance cells of the brain, with functions that include phagocytosis and synaptic pruning . In the i) Humans with Lewy body pathology in the neocortex demonstrate a negative correlation between AQP4 expressing astrocytes and α-synuclein aggregates, such that astrocytes with AQP4 expression do not appear in areas with abnormal α-synuclein expression (ii) Exposure of AQP4 knockout (KO) mice to the PD toxin, MPTP causes an increase in the susceptibility of SNc DA neurons to degeneration (iii) AQP4 KO mice show diminished differences between ventral tegmental area (VTA) and SNc DA neurons in their susceptibility to MPTP-induced neurodegeneration (iv) AQP4 knockout mice display significant increases in microglial reactivity following exposure to MPTP when compared to wildtype littermates. In this case, the study also shows that the increase in microglial reactivity occurs due to secretion of neuroinflammatory molecules such as interleukin 1β (IL1β) and tumor necrosis factor α  is a tetrameric water channel, abundantly expressed in astrocytes . Emerginpression , (ii) Exneration , (iii) Aneration , and  gene, ApoE4 significantly increases the risk for Alzheimer’s disease AD; . In the 2+-dependent manner  on Ca2+ (2+ signals in altering TJP biology in PD, the use of new imaging modalities such as multiphoton microscopes in combination with astrocyte-specific transgenic mice (2+ sensors in astrocytes should enable an understanding of the role of aberrant astrocytic endfoot Ca2+ signals in TJP and BBB function during PD.Together, the findings described above lead to the important question of whether or not disruptions in Ca on Ca2+ , therebynic mice  and gene2+ signals cause either neuronal dysfunction, microglial activation, or a loss of BBB integrity (2+ signals in astrocytes during PD in the first place, molecules such as ApoE4 and α-synuclein likely initiate abnormal Ca2+ signaling in astrocytes via multiple and distinct mechanisms. It is therefore reasonable to hypothesize that once they are initiated, abnormal astrocytic Ca2+ signals cause further abnormalities in ApoE4 or α-synuclein, thereby setting up a vicious feedback loop between aberrant astrocytic Ca2+ signaling and ApoE4 or α-synuclein pathology in PD.In this perspective review, we discuss potential pathological mechanisms during PD in which aberrant astrocytic Cantegrity . Althoug2+ signals modulate multi-tiered interactions between these seemingly distinct CNS compartments, eventually leading to neurodegeneration.An additional point to note is that neurons, microglia and the BBB are also capable of directly interacting with each other, which would result in a complex network of multi-tiered pathological interactions. Based on this view, we predict that the coming decades will unravel specific mechanisms by which aberrant astrocytic CaRS conceived and wrote the manuscript. RS and EB compiled and edited the manuscript. EB created the figures with input from RS. Both authors contributed to the article 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.