The fundamental role of the inward Na+/Ca2+ exchanger (NCX) is firmly established. However, little is known about the reverse mode exchange. A simulation study attributed important role to reverse NCX activity, however experimental evidence is still missing. Whole-cell and perforated patch-clamp experiments were performed on rabbit SN cells supplemented with fluorescent Ca2+-tracking. We established 2 and 8 mM pipette NaCl groups to suppress and enable reverse NCX. NCX was assessed by specific block with 1 μM ORM-10962. Mechanistic simulations were performed by Maltsev–Lakatta minimal computational SN model. Active reverse NCX resulted in larger Ca2+-transient amplitude with larger SR Ca2+-content. Spontaneous action potential (AP) frequency increased with 8 mM NaCl. When reverse NCX was facilitated by 1 μM strophantin the Ca2+i and spontaneous rate increased. ORM-10962 applied prior to strophantin prevented Ca2+i and AP cycle change. Computational simulations indicated gradually increasing reverse NCX current, Ca2+i and heart rate with increasing Na+i. Our results provide further evidence for the role of reverse NCX in SN pacemaking. The reverse NCX activity may provide additional Ca2+-influx that could increase SR Ca2+-content, which consequently leads to enhanced pacemaking activity.Sinus node (SN) pacemaking is based on a coupling between surface membrane ion-channels and intracellular Ca X1 or later called IKr)1 and the cAMP-dependent hyperpolarization activated funny-current2 has a major role forming the DD. This mechanism of pacemaking, driven by transmembrane ion channels with Hodgkin-Huxley kinetics was termed later as “membrane clock” (M clock). Further studies which identified rhythmic, spontaneous subsarcolemmal Ca2+ releases (LCR) generated by the sarcoplasmic reticulum (SR) via ryanodine receptors during the DD (“Ca2+ clock”) have challenged the dominant role of membrane clock in the spontaneous automaticity of the SN5. After intense debate regarding the underlying mechanism of spontaneous SN pacemaking, a large body of evidence proved that the M clock and Ca2+ clock are functionally tightly coupled, so the two mechanisms form the latest concept of SN automaticity which is called the coupled clock system. This coupling is based on numerous time-, voltage- and Ca2+-dependent mechanisms7 including a major role of the L-type Ca2+ current (ICaL) that “resets” and ”refuels” the Ca2+ clock6.The SN AP is characterized by its slow diastolic depolarization (DD) phase starting from the maximal diastolic potential (MDP) and ending at the AP threshold of about − 40 mV. It was suggested that the decaying delayed rectifier potassium current  NCX in the SN pacemaking as part of the CaCaL. ORM-10962 is a novel and fully selective NCX inhibitor without any effect on the other currents at a concentration of 1 μM where ORM-10962 causes about 80% inhibition of both modes (forward and reverse) of NCX17.However, the direct experimental validation of these interesting in silico results were hampered so far because of the lack of selective NCX inhibitors, i.e. the absence of a NCX blocker without major effects on ITherefore, the aim of this study was (i) to experimentally investigate the possible existence of reverse NCX in SN cells and (ii) to explore its probable functional role in SN pacemaking mechanism.Guide for the Care and Use of Laboratory Animals  and conformed to Directive 2010/63/EU of the European Parliament. The protocols were approved by the Review Board of the Department of Animal Health and Food Control of the Ministry of Agriculture and Rural Development, Hungary (XIII./1211/2012). Animal studies were carried out in compliance of ARRIVE guidelines.All experiments were conducted in compliance with the The measurements were performed on spontaneously beating SN cells obtained from sinus node tissue of young New Zealand white rabbits (Innovo Ltd.) from both genders weighing 2.0–2.5 kg.+ level of the cells, while in the other group, the pipette solution was reduced to 2 mM NaCl aiming to suppress the reverse mode of the NCX without completely abolishing the exchanger function.In this study, our aim was to establish two experimental groups throughout the study: one with active reverse mode and the other with blunted reverse exchange. In order to achieve this, the pipette solutions contained 8 mM NaCl and 2 mM NaCl, respectively. 8 mM NaCl were used to approximate the physiological Na18. Rabbits were sacrificed by concussion after intravenous administration of 400 IU/kg heparin. The chest was opened and after the quick removal of the heart, it was placed into an isolation solution containing in mM: 135 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 10 HEPES, 4.4 NaHCO3, 10 glucose, 1.8 CaCl2 (titrated to pH 7.2 with NaOH). The heart was mounted on a 60 cm high modified Langendorff column and perfused with oxygenated isolation solution at 37 °C. First, the blood was washed out (3–5 min), then the heart was perfused with nominally Ca2+-free solution until the heart stopped contracting (4–5 min). The enzymatic digestion was performed by perfusion with the same isolation solution supplemented with 1.8 mg/ml (260 U/ml) collagenase  and 33 µM CaCl2. After 13–14 min, the heart was taken off the cannula. The right atrium of the heart was cut and the SN region was excised and cut into small pieces. The strips were placed into the enzyme free isolation solution containing 1 mM CaCl2 and equilibrated at 37 °C for 15 min. The cells were separated by filtering through a mesh and were stored at room temperature.Isolated single SN cells were obtained by enzymatic dissociation as described previouslypip and to investigate the effectiveness of the selective NCX inhibitor ORM-10962. From a holding potential of − 40 mV, the membrane was depolarized to 30 mV with a slope of 0.7 V/s, then hyperpolarized to − 70 mV. The reverse mode was calculated at + 25 mV while the forward operation was calculated at − 60 mV, both during the downhill phase of the current. The pipette solution contained (in mM): 125 CsCl, 20 TEACl, 5 MgATP, 10 HEPES, 8 or 2 NaCl, titrated to pH 7.2 with CsOH. The free intracellular Ca2+ was buffered to ~ 100 nM  to approximate a normal diastolic Ca2+ value. The composition of the external solution was: 135 mM NaCl, 10 mM CsCl, 0.33 mM NaH2PO4, 10 mM TEACl, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, 1 mM CaCl2, 20 µM ouabain, 50 µM lidocain, 1 µM nisoldipin, titrated to pH 7.4.As a standard pharmacological approach, a voltage-ramp protocol was used to measure the NCX current and to verify the differences in the two experimental groups containing 2 mM or 8 mM Nacycle length: 410 ms, maximal diastolic potential: − 57 mV, overshoot: 24 mV, action potential duration: 180 ms, diastolic depolarization slope: 0.124 mV/ms.During the voltage clamp experiments from Figs. 2 sensitive current under the predefined AP waveform was measured. The extracellular solution contained: 135 mM NaCl, 10 mM CsCl, 0.33 mM NaH2PO4, 10 mM TEACl, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, 1.8 mM CaCl2, 0.2 mM BaCl2, 20 µM ouabain, 50 µM lidocain, 1 µM nisoldipine, 1 µM mibefradil, titrated to pH 7.4. The intracellular solution contained (in mM): 125 CsCl, 20 TEACl, 5 MgATP, 10 HEPES and 10 EGTA titrated to pH 7.2 with CsOH, and 2 or 8 mM NaCl was added respectively.In the case of experiments demonstrated in Fig. 2PO4, 10 mM CsCl, 10 mM TEACl, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, 1 mM CaCl2, 20 µM ouabain, titrated to pH 7.4. The intracellular solution contained (in mM): 125 CsCl, 5 MgATP, 20 TEACl, 10 HEPES titrated to pH 7.2 with CsOH.The compositions of the applied solution in experiments depicted in Figs. 2+ was unbuffered allowing Ca2+ transients (CaT) that were monitored by Fluo-4 AM (5 µM) fluorescent dye. The isolated SN cells were loaded with the dye for 20 min at room temperature in darkness. Fluorescence measurements were performed as it was described in our previous study18. Experiments were carried out on the stage of an Olympus IX 71 inverted fluorescence microscope. The dye was excited at 480 nm and the emitted fluorescence was detected at 535 nm. Optical signals were sampled at 1 kHz and recorded by a photon counting photomultiplier . Amplitudes of the Ca2+ transients were calculated as differences between systolic and diastolic values. To measure Ca2+ changes, the cells were damaged by a patch pipette at the end of the experiment to obtain maximal fluorescence (Fmax). Ca2+ was calibrated using the following formula: Kd(F − Fmin)/(Fmax − F). The Kd of the Fluo-4 AM was set to 335 nM.During these measurements, the intracellular Ca19. The membrane potential was recorded in current clamp configuration using a gap-free acquisition protocol. Normal Tyrode’s solution was used, containing (in mM): 144 NaCl, 0.4 NaH2PO4, 4 KCl, 0.53 MgSO4, 1.8 CaCl2, 5.5 glucose and 5 HEPES, titrated to pH 7.4. The patch pipette solution contained (in mM): 120 K-gluconate, 2.5 NaCl, 2.5 MgATP, 2.5 Na2ATP, 5 HEPES, 20 KCl, titrated to pH 7.2 with KOH. 35 µM β-escin was added to the pipette solution to achieve the membrane patch perforation. The cells were loaded with Fluo-4 AM to measure intracellular Ca2+ changes following the method that was described here previously : 120 K-gluconate, 2.5 MgATP, 2.5 KMembrane voltage or ionic currents were obtained by using an Axoclamp 1-D amplifier  connected to a Digidata 1440A  analogue–digital converter. The membrane voltage was recorded by Clampex 10.0 .Maximum diastolic potential (MDP) was calculated as the most negative potential reached before the AP depolarization.Take off potential (TOP) defined as the voltage measured at the time when the voltage derivative exceeded 0.5 mV/ms.The slope of depolarization was calculated as the mean voltage derivative of the AP between MDP and take off potential.Action potential duration (APD) defined as the time interval between TOP and the next MDP.Cycle length was measured between the peaks of two consecutive APs.All experiments in this study were performed at 37 °C.The parameters of the APs were calculated as follows:p < 0.05) was assessed using Student’s t-test, or repeated measures ANOVA. Data are presented as mean ± S.E.M.Normal distribution of the data was verified by using Shapiro–Wilk test. In this study, we used hierarchical analysis: the technical replicates obtained from the same heart were averaged providing n = 1. Therefore, the experimental number ‘n’ refers the number of hearts used. Thus, all experiments can be considered independent. For the experiments, 3–5 sinus node cells were used from each rabbit. Statistical significance . All model codes are available in the Supplement.To mechanistically underpin our experimental findings, we conducted numerical simulations using the Maltsev et al. minimal modelpip conditions, respectively. We also compared the effect of 1 µM ORM-10962 on the NCX current in the presence of 2 and 8 mM [Na]pip to exclude any Na-dependent action of ORM.In the first set of experiments conventional NCX-ramp protocol was used to study the absence and presence of a reverse current in 2 and 8 mM [Na]2  the current lacks the outward component. The total NCX current was calculated as a difference of the control and the Ni2+-insensitive current . A marked difference between the reversal potentials of the experimental groups was also observed: in the 8 mM [Na]pip group the reversal potential well approximates the calculated reversal potential, indicating the existence of a thermodynamical possibility for the reverse mode operation .After registration of the control current  in the presence of 8 mM [Na]pip did not differ from the current measured in the presence of 2 mM [Na]pip indicating that the operation of the forward modes was identical between groups.Indeed, the reversal potential in the 2 mM [Na]+-dependent effect of the ORM prior to these experiments. The effect of ORM-10962 was calculated as a ratio between the total current (control vs 10 mM NiCl2) and the ORM-inhibited fraction (control vs 1 µM ORM-10962). As Fig. pip groups . Since we did not observe reverse NCX in the presence of 2 mM NaCl, we could not quantify ORM effect in this case. The effect of ORM on the reverse mode in the presence of 8 mM [Na]pip was: 72.5 ± 5%, n = 7.Since we planned to use ORM to inhibit NCX (in the later part of the study), we considered important to exclude any possible Napip, both ORM-10962 and NiCl2 dissected a notable outward current from the control, which may reflect the potential existence of reverse NCX that may be Na-dependent and increases as the membrane potential becomes more positive.In the presence of 8 mM [Na]pip group as an experimental condition where the reverse mode is active, and the 2 mM [Na]pip group as having suppressed or no reverse exchange activity.Therefore, based on these results, we consider the 8 mM [Na]2+ was buffered by using 10 mM EGTA. The K+-currents, ICaL, ICaT, INa/K were inhibited during the experiments. After recording the control current, the NCX current was estimated by application of 10 mM NiCl2 . In the presence of 8 mM [Na]pip, this net outward charge transport was significantly larger . This value is slightly smaller than the one predicted by the Maltsev–Lakatta model (2.45 pC).In the next set of experiments, we aimed to investigate whether the reverse NCX current develops under a SN action potential. In these experiments, a previously recorded canonical SN AP waveform was used Fig. a as comm2+ release was blocked due to Ca2+-channel inhibition and intracellular Ca2+ buffering. It was necessary, since 10 mM NiCl2 (concentration needed to complete inhibition of NCX) also suppresses the ICaL, which would seriously contaminate the results. Therefore, this method allows estimating the total carried charges through the reverse mode, however, lacks the Ca2+ release, which is a crucial component of the NCX driving force. Thus, 1 µM ORM-10962 was used to explore the reverse NCX in the presence of Ca2+ release . The diastolic Ca2+ level did not change . The duration of the caffeine-induced Ca2+ transient at 50% of the amplitude did not change between the groups  and 2 mM [Na]pip .The larger Ca2+ refilling after a caffeine application. Thermodynamical considerations dictate, when SR is empty, the initially small Ca2+ releases cause a negative shift of the NCX reversal potential. This may provide a large thermodynamical driving force for the reverse operation during the early phase of refilling. Therefore, an additional Ca2+ influx through the reverse NCX may improve the intracellular Ca2+ gain compared to cells where reverse exchange is blunted. This question was addressed by reapplication of the consecutive AP commands immediately after 10 mM caffeine application. During voltage command the Ca2+ transients were monitored, and the change of diastolic Ca2+ level and Ca2+ transient amplitude were analysed. Since the absolute values of both the diastolic Ca2+ and Ca2+ transient amplitudes were generally decreased after the application of caffeine, we normalized both variables for the values obtained during the first pulses.It was hypothesized that the functional role of the reverse NCX could be further demonstrated during SR Ca2+ level was analysed after the establishment of the steady-state (at pulse No14) while the amplitude was analysed in all pulses. In agreement with Ca2+ transient experiments presented in Fig. 2+ did not show difference under steady state , but the Ca2+ transient amplitude  exerted faster increase during refilling .In order to address the functional importance of reverse mode in spontaneous pacemaking we compared the AP and CaT characteristics between 2 and 8 mM [Na]In order to further validate AP data, perforated patch experiments were also performed with 2 and 8 mM NaCl in the pipette. The cycle length, APD and diastolic slope changed similarly between groups as was observed under experiments with the whole cell configuration . As a consequence, it shortened the cycle length , INa/K, Ib(Ca). A specific feature of this model is the prominent reverse NCX activity during the initial phase of the action potential.Maltsev and Lakatta established a “minimal set” of sarcolemmal ion currents and intracellular Cai changes on AP cycle length, intracellular Ca2+ levels, as well as NCX and ICaL , however the carried charge, in contrast, increased , suggesting that Ca2+ influx through reverse mode of the exchanger is essential for maintaining a stable SR Ca2+ level, or in other words, the Ca2+ influx provided by the ICaL per se is insufficient to maintain pacemaking in this model.The role of reverse NCX in SN pacemaking was further elucidated by disabling reverse NCX in the model  a voltage-, and Na+i dependent outward current was found during the initial part of the SN action potential which was sensitive to ORM-10962 and NiCl2. (2) Higher SR Ca2+ content and (3) faster heart rate was observed in the presence of active reverse NCX.In this study we characterized the reverse NCX and its functional role during the SN action potential. In accordance with previous numerical simulations+/Ca2+ exchanger of the cardiac sarcolemma transporting 3 Na+ for 1 Ca2+ represents the main Ca2+ extrusion mechanism of the cell. The NCX exerts a thermodynamically defined reversal potential (ENCX) based on the transport stoichiometry: ENCX = 3ENa − 2ECa. This means when the actual membrane potential  is more positive than ENCX, Ca2+ entry and consequential outward current happens via the reverse mode of NCX. When membrane potential falls below ENCX, the direction of the transport changes to Ca2+ efflux and inward current is carried by forward mode.The Na+/Ca2+ exchange in a given cell type is defined by the actual driving force of the NCX. Based on previous model simulations14, reverse operation of the NCX is expected when the membrane potential is more positive than 0 mV. When our experimental conditions were used for calculations to approximate the NCX equilibrium, the reverse mode was favoured in the first 55–65 ms from the AP upstroke  but in the presence of 2 mM Na+i the outward current was suppressed  the Ni2+- and ORM-sensitive outward current component was only detectable in the presence of 8 mM pipette Na+ and disappeared when low Na+ was applied. This indicates that the outward component of the Ni2+- or ORM-sensitive current is Na+i dependent. (2) As it was thermodynamically predicted, this outward component of the Ni2+- or ORM-sensitive current appeared only at positive membrane potentials, during the very first section of the AP.Taking together, these results support previous modelling simulations21 and models22,23 remains to be studied.How these findings in rabbit SN cells and models relate to human SN cells2+ content, it may imply an important functional role of reverse NCX in cardiac SN pacemaking mechanism.Considering that SN pacemaking is critically based on the actual SR Ca2+ content in the presence of active reverse NCX . Based on thermodynamical considerations, the lower Ca2+i levels during the couple of first stimuli shifts the NCX equilibrium toward more negative values favouring an initially large, than gradually decreasing outward NCX parallel with the increase of Ca2+i. It is supposed that this initially large reverse component may boost up the recovery of CaT after caffeine application. As Fig. 2+ level and CaT amplitude was found. Taken together these results, an important functional role of reverse NCX could be considered since: (1) the Ca2+ transient amplitude was higher in the presence of active reverse mode and (2) in line with this, the SR Ca2+ content increased. (3) The cells with active reverse exchange exerted faster refilling of Ca2+ in the initial steps after caffeine application, indicating synergistic effect between reverse NCX and ICa during SR Ca2+ loading.In line with these numerical calculations, in our experiments a larger CaT amplitude was found  and in the absence of reverse NCX  facilitates forward NCX leading to net Ca2+ loss while higher Na+-level inhibits the forward mode causing net Ca2+ gain. Therefore, in the case of 8 mM Napip both the Na+-mediated reverse mode activation and forward mode suppression could be able to increase the Ca2+i. However, in SN the forward mode activity has considerable contribution to the diastolic depolarization. Therefore, if Ca2+ increase was caused by forward mode inhibition the diastolic slope would decrease as an indicator of inward current reduction.One can speculate that the observed results could be attributable to the NCX forward mode activity. Theoretically, the reduced Na+i increases. As Fig. CaL are increased and could account for the accelerated pacemaking. However, as Fig. 2+ content and abrupt termination of spontaneous pacemaking. Obviously, the charge carried by ICaL also reduced in this case, but it may imply that Ca2+ influx by the reverse activity contributes in setting the SR Ca2+ content, suggesting important indirect role in setting the actual heart rate. This is underpinned by the fact that SR Ca2+ loss and failure of automaticity could be rescued by improved SERCA activity , the ICaL/reverse NCX Ca2+ influx ratio is 17.2 compared to 3.3 found in this study for SN cells (at Na+i of 8 mM) under in silico condition, and 2.81 under in vitro condition.The CaCaL and a maintained Ca2+ influx. In SN cells, the absence of the plateau phase considerably restricts the ICaL opening time limiting ICaL Ca2+ influx. This may explain that under normal condition, in the ventricular myocytes, the ICaL is markedly dominating over the reverse NCX but it may represent an important source of Ca2+ influx in SN cells since the Ca2+ entry via ICaL is restricted due to the characteristic SN AP waveform.This large discrepancy between ventricular myocytes and SN cells could be the consequence of the long plateau phase in the ventricular cells providing long-lasting opening of the I38). The action potentials recorded from the central region markedly differ from the action potentials obtained from the transitional or peripheral zone39. In context of the reverse mode, two important differences should be mentioned: from the centre region to the distal areas the (1) action potential upstroke becomes significantly larger and (2) the cycle length shortens. These differences could be explained by the influence of the atrial muscle40, the higher role of If in the periphery42, and the different role of the ICaL in pacemaking from the centre to the periphery43.There is a large body of evidence that the SN exerts considerable electrical heterogeneity (reviewed by:Considering the results of this study, it is feasible that the small upstroke in the centre region considerably restricts the reverse function, while the distal areas may enable increasing amount of reverse mode. This increasing reverse NCX gradient from the centre to the periphery may contribute in the observed AP cycle length shortening in the transitional zone and the periphery, therefore the reverse NCX could contribute to the electrophysiological heterogeneity of the SN.+- and voltage-dependent, ORM-10962 and Ni2+-sensitive outward current component appearing during the early SN action potential that could be considered as reverse NCX.In this study, we identified a Na2+ influx through the reverse NCX may contribute to the setting of the SR Ca2+ content and might therefore represent an additional mechanism of the SN coupled-clock pacemaking system that contributes to the control of the heart rate in SN cells.Our results suggest that CaCa. Considering these limitations, the aim of the ORM-sensitive current was to demonstrate the existence of the reverse NCX current during the initial phase of the AP.It is important to note that ORM-sensitive currents (in Supplementary Fig. In the experiments, we used only one canonical action potential waveform. Since the SN is a heterogeneous system, our results can be interpreted as representative for SN cells that have a relatively large action potential overshoot and should not be generalized to the entire SN.Na may change the intracellular Na+-level of the cells that could influence the function of the NCX.A possible contribution of the I+ level could also influence the INa, Na/K pump, Na/H pump and If.In the experiments, we used 2 and 8 mM NaCl in the patch pipette. This change in the intracellular NaSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.


To use the logistic regression model to evaluate the value of ultrasound characteristics in the Ovarian-Adnexal Reporting and Data System ultrasound lexicon in determining ovarian solid component-containing mass benignancy/malignancy.  We retrospectively analyzed the data of 172 patients with adnexal masses discovered by ultrasound, and diagnosis was confirmed by postoperative pathological tests from January 2019 to December 2021. Thirteen ovarian tumor-related parameters in the benign and malignant ovarian tumor groups were selected for univariate analyses. Statistically significant parameters were included in multivariate logistic regression analyses to construct a logistic regression diagnosis model, and the diagnostic performance of the model in predicting ovarian malignancies was calculated. p < 0.05). Logistic regression analyses showed that maximum solid component diameter, whether acoustic shadows were present in the solid component, number of papillae, and presence/absence of ascites were included in the logistic regression model, and the area under the receiver operating characteristic curve for this regression model in predicting ovarian malignancy was 0.962 . Logit (p) ≥ −0.02 was used as the cutoff value, and the prediction accuracy, sensitivity, specificity, positive predictive value, and negative predictive values were 93.6%, 86.8%, 98.1%, 96.7%, and 91.9%, respectively.  Of the 172 adnexal tumors, 104 were benign, and 68 were malignant. There were differences in cancer antigen 125, maximum mass diameter, maximum solid component diameter, multilocular cyst with solid component, external contour, whether acoustic shadows were present in the solid component, number of papillae, vascularity, presence/absence of ascites, and presence/absence of peritoneal thickening or nodules between the benign ovarian tumor and malignancy groups ( The logistic regression model containing the maximum solid component diameter, whether acoustic shadows were present in the solid component, number of papillae, and presence/absence of ascites can help in determining the benignancy/malignancy of solid component-containing masses. The mortality rate of ovarian cancer is the highest among gynecological cancers. The latest study reported that there were 313,959 new cases of ovarian cancer globally in 2020 and 207,252 deaths. Hence, ovarian cancer is an important disease that threatens women's health [We retrospectively analyzed the data of patients who underwent surgery due to the discovery of adnexal masses by ultrasound from January 2019 to December 2021. Patients underwent preoperative CA125 test, and ultrasound examination was performed 1 week before surgery. The ultrasound images were clear, and diagnosis report descriptions were complete. There were clear postoperative pathological results. After excluding patients with typical benign lesions, unilocular cysts, and multilocular cysts defined in the O-RADS ultrasound lexicon, a total of 172 patients with adnexal masses containing solid components were enrolled. The ages of patients ranged from 15 to 85 years, and the mean age was 40.05 ± 13.11 years. This study was approved by the ethics committee of the hospital.The ultrasound equipment used were Samsung UGEO WS80A, Canon Aplio300, and Mindray Resona8 Elite color Doppler ultrasound diagnostic devices equipped with an abdominal probe and intraluminal probe. The intraluminal probe frequency was 3–10 MHz, and the abdominal probe frequency was 1–5 MHz. Patient images were viewed on the Picture Archiving and Communication System imaging system. The Alinity i system fully automated analyzer  was used to measure serum CA125 levels.Two experienced ultrasound physicians , who were blinded to the pathological results, conducted classification and interpretation of image features. When the imaging results were different, the physicians discussed until a consensus was reached before recording the interpretation. Thirteen parameters, namely, age, hysterectomy or not, menopause status, CA125, mass characteristics, maximum mass diameter, maximum solid component diameter, external contour, whether acoustic shadows were present in the solid component, number of papillae, vascularity signal grade, presence/absence of ascites, and presence/absence of peritoneal thickening or nodules of all patients, were recorded. Menopause was defined as the absence of menstruation for more than 12 months.The mass characteristics were as follows. (1) Solid unilocular mass: the unilocular cyst contains a measurable solid component or at least one papillary projection, and the maximum diameter of the solid component or papillary projection is ≥3 mm; (2) solid multilocular mass: multilocular cysts with measurable solid component or at least one papillary projection and the maximum diameter of the solid component or papillary projection are ≥3 mm; and (3) solid mass: two-dimensional grayscale ultrasound showed that the solid component in the mass accounted for 80% of the tumor and could include a papillary projection or small cysts protruding into the mass. External contours were smooth (regular outer margin) or irregular (nonuniform outer margin), and a lobulated outer margin was considered irregular. Acoustic shadows are artifacts caused by acoustic attenuation after tissues absorb sound waves. Papillae are solid components with cystic wall or high septa ≥ 3 mm that protrude into the cyst and should be counted as the number of papillae. Ascites refers to fluid extending above the uterine fundus beyond the pouch of the Douglas or cul-de-sac when anteverted/anteflexed and anterior/superior to uterus when retroverted/retroflexed. Peritoneal thickening or nodules are nodularity or diffuse thickening of the peritoneal lining(s) or along the bowel serosal surface or peritoneum associated with peritoneal carcinomatosis. Vascularity is as follows: color scores 1–4, overall assessment of color Doppler flow within the entire lesion: 1-no flow, 2-minimal flow, 3-moderate flow, and 4-very strong flow.t-test was used for intergroup comparison. The mean maximum diameter is expressed as the median (M), and Mann–Whitney U test was used for intergroup comparisons. Qualitative data are expressed as the number of patients, and the χ2 test was used for univariate analyses. Pathological results were used as a dependent variable to construct the logistic regression diagnosis model. The corresponding regression coefficient (β), standard error (SE), and OC  were obtained. The receiver operating characteristic (ROC) curve of the logistic regression model in ovarian malignancy diagnosis was plotted, and its diagnostic performance was analyzed. p < 0.05 was considered statistically significant.SPSS20.0 statistical software was used for the statistical analyses. Age was quantitative data, expressed as Of the 172 adnexal tumors, postoperative histopathology tests showed that 104 were benign and 68 were malignant. Of the benign tumors, 29 were fibrothecomas, 28 were benign teratomas, 14 were mucinous and serous cystadenomas, 12 were serous and mucinous adenofibromas, 7 were fallopian tube effusion or empyema, 6 were ovarian endometriomas, 4 were simple cysts, 2 were fallopian tube tuberculosis, and 2 were Brenner tumors. Of the malignant tumors, 30 were serous, mucinous, and endometrioid borderline tumors, 9 were serous and mucinous cystoadenocarcinomas, 8 were endometrioid adenocarcinomas, 6 were clear cell carcinomas, 5 were adult granulosa cell tumors, 3 were metastases, 3 were mixed germ cell tumors, and 1 patient each had stromal carcinoid of ovary, yolk sac tumor, immature teratoma, and moderately differentiated Sertoli-Leydig cell tumor.p < 0.05, 1Comparison of the benign and malignant groups showed that there were differences in CA125, maximum diameter, maximum solid component diameter, multilocular cyst with solid component, external contour, whether acoustic shadows were present in the solid component, number of papillae, vascularity, presence/absence of ascites, and presence/absence of peritoneal thickening or nodules between the benign ovarian tumor and malignancy groups, and these differences were statistically significant  = −2.261 + 0.037∗X3 − 5.318∗X6 + 4.280∗X7 + 3.678∗X9. The area under the ROC curve for this regression model in predicting ovarian malignancy was 0.962 . Logit (p) ≥ −0.02 was used as the cutoff value and the prediction accuracy, sensitivity, specificity, positive predictive value, and negative predictive value were 93.6%, 86.8%, 98.1%, 96.7%, and 91.9%, respectively , logit (p) ≥ −0.02 was used as the cutoff value, and the prediction accuracy, sensitivity, specificity, positive predictive value, and negative predictive value were 93.6%, 86.8%, 98.1%, 96.7%, and 91.9%, respectively, demonstrating good diagnostic performance. Thus, the four included factors may help in O-RADS4 category subclassification.In this study, logistic regression analyses were used to identify the following four independent risk factors predicting ovarian malignancy by screening: maximum solid component diameter, whether acoustic shadows were present in the solid component, number of papillae, and presence/absence of ascites. The results showed that of the 63 masses with acoustic shadows, only 2 were confirmed to be malignant ovarian tumor after surgery. Hence, acoustic shadows may be a critical characteristic for ruling out malignancy, consistent with the results of Cao et al. . HoweverOne limitation of this study is that it was a retrospective analysis, which may have incomplete image information and possibly some bias due to the low sample size. A large prospective study should be carried out in the future to examine the correlation between O-RADS 4 category subclassification and menopause age and malignant ovarian tumor.In summary, the logistic regression model containing the maximum solid component diameter, whether acoustic shadows were present in the solid component, number of papillae, and presence/absence of ascites can help in determining the benignancy/malignancy of solid component-containing masses, and acoustic shadow is a critical characteristic for excluding malignant tumors.


Na) and late sodium current (INa-late) in the isolated single male and female right ventricular outflow tract (RVOT) cardiomyocytes before and after LiCl. Male rabbits (n = 9) were more prone to developing lithium-induced Brugada-pattern ECG changes  with fatal arrhythmia  than in female (n = 7) rabbits at 10 mmol/kg (but not 1 or 3 mmol/kg). Compared to those in the female RVOT cardiomyocytes, LiCl (100 μM) reduced INa to a greater extent and increased INa-late in the male RVOT cardiomyocytes. Moreover, in the presence of ranolazine , LiCl (10 mmol/kg) did not induce Brugada-pattern ECG changes (p < 0.005). The male gender is much predisposed to lithium-induced Brugada-pattern ECG changes with a greater impact on INa and INa-late in RVOT cardiomyocytes. Targeting INa-late may be a potential therapeutic strategy for Brugada syndrome-related ventricular tachyarrhythmia. Lithium intoxication induces Brugada-pattern ECG, ventricular arrhythmia, and sudden death with the predominant preference for the male over the female gender. This study investigated the mechanisms of gender difference in lithium-induced arrhythmogenesis. The ECG parameters were recorded in male and female rabbits before and after the intravenous administration of lithium chloride (LiCl) . Patch clamps were used to study the sodium current (I However, cardiac adverse effects, including both benign electrocardiographic (ECG) changes and near-fatal arrhythmias, have been reported . Lithiumtability . Some litability ,4.+ channel inhibition, and the electrical changes imposed by lithium are determined by the duration of lithium treatment and serum lithium concentration [Lithium has been proven to unmask type 1 Brugada-pattern ECG changes, which was suggested to be caused by its effect on Nantration . Lithiumntration ,6. In thntration . As in tntration . 2+) content [The lithium-related Brugada-pattern ECG changes are notable for a coved ST segment, J point elevation, and T-wave inversion in the right precordial leads V1 and/or V2 ,10. The  content . The str content . There a content . In pati content . 2+ handlings in the cardiomyocytes between the sexes [+ current (INa-late) have been reported in mice ventricular cells, which was translated into effects on cardiac AP durations [There are different ionic profiles and Cahe sexes . Male prhe sexes . Gender-urations . Our preurations . HoweverAll the experiments involving animals absolutely complied with the Guide for the Care and Use of Laboratory Animals and were approved by a local ethics review committee , National Defense Medical Center, Taiwan. Na-late inhibition on LiCl-induced Brugada-pattern ECG changes and arrhythmogenesis, LiCl (10 mmol/kg) was administered in male rabbits 5 min after being treated with ranolazine  [Rabbits of both sexes, which weighed between 2.0 and 2.5 kg, were anesthetized by the inhalation of 2.0–2.5% isoflurane. At least 30 min were allowed for the rabbits’ hemodynamics to stabilize under continuous ECG monitoring. The limb leads were attached as described in the literature , and then later) . The Brun later) .2 1.8, MgCl2 0.5, and glucose 11. All traces of blood being washed out, the hearts were perfused for 8–12 min with oxygenated Ca2+-free Tyrode’s solution containing collagenase   and protease  , for enzymatic dispersion to take place [2+-free oxygenated Tyrode’s solution until the isolation of single cardiomyocytes. Next, the solution was gradually switched to oxygenated normal Tyrode’s solution.Rabbits that weighed 2.0–2.5 kg of both sexes were anesthetized with overdosing isoflurane (5% in oxygen) delivered from a precision vaporizer. We confirmed the adequacy of the anesthesia by the lack of corneal reflex and by monitoring the response to painful stimuli elicited by a scalpel tip. Single RVOT cardiomyocytes were enzymatically dissociated from the rabbits as previously described . Brieflyke place . Then, t90, APD50, and APD20, respectively.As described previously, the whole-cell patch clamp experiment was performed in the isolated RVOT cardiomyocytes by using an Axopatch 200B amplifier  at 35 ± 1 °C. Borosilicate glass electrodes  with a tip resistance of 3–5 MΩ were used. Before the formation of the membrane-pipette seal, the tip potentials were zeroed in Tyrode’s solution. The junction potentials between the bath and pipette solution (9 mV) were corrected for the AP recordings. The resting membrane potential (RMP) was measured during the period from the last repolarization to the next AP. The amplitude of AP (APA) was defined from the RMP to the peak of depolarization. The AP duration (APD) obtained at 90%, 50%, and 20% repolarization of APA was defined as APDNa) was recorded by using 40-msec. pulses from a holding potential of −120 mV to the test potentials, which varied between −80 and 0 mV in 10 mV increments at a frequency of 3 Hz at room temperature (25 ± 1 °C). The external solution contained (in mM): CsCl 133, NaCl 5, HEPES 5, glucose 5, MgCl2 2, CaCl2 1.8, and nifedipine 0.002 (pH 7.3). Micropipettes were filled with a solution containing (in mM) CsCl 133, TEACl 20, EGTA 10, NaCl 5, MgATP 5, and HEPES 5 (pH 7.3 with CsOH). The INa-late was recorded with an external solution containing : NaCl 140, CsCl 5, HEPES 5, MgCl2 2, CaCl2 1.8, nicardipine 0.002, and glucose 5. The amplitude of the INa-late was measured at a voltage of −20 mV as the mean value between 200 and 250 ms after the membrane depolarization by a 2000-ms. pulse from −140 mV stepped to −20 mV. The sodium current   or an unpaired t-test was used for comparing differences between the RVOT cardiomyocytes of different groups. The effects of LiCl on ionic currents were compared using the Mann–Whitney rank-sum test or unpaired At the baseline, there were no significant differences in the heart rate, QT intervals, QTc intervals, P wave amplitudes, or T wave amplitudes between the sexes. LiCl of 1 and 3 mmol/kg did not significantly change the ECG parameters in male and female rabbits. The QRS duration at 10 mmol/kg of LiCl was significantly longer in male rabbits than in female rabbits A. No sig90, APD50, and APD20 between genders both at the baseline and after LiCl (100 μM) administration .As shown in In this study, for the first time, we successfully provoked Brugada-pattern ECG changes in male rabbits via the administration of high-dose LiCl. This finding is consistent with the known risk of lithium intoxication in humans. The simplicity of lithium administration to produce Brugada-pattern ECG changes and ventricular tachyarrhythmia in rabbits may provide a platform for mechanistic insights and pharmacological intervention in ventricular arrhythmogenesis during lithium intoxication. + channel causing discrete interstitial changes in RV with associated reentry and ventricular fibrillation originating from RVOT subendocardium has been documented [+– Ca2+ exchangers and Na+/K+ pump could induce the changing physiology of the cellular membrane. Lithium can decrease the intracellular K+ concentration, replace intracellular Ca2+ and cause hypercalcemia, and induce various ECG changes [Na at subtherapeutic concentrations [Na and INa-late in male and female RVOT cardiomyocytes. These findings suggest that lithium exhibits sex differences in modulating the INa and INa-late of RVOT cardiomyocytes, which may explain the lithium-provoked Brugada-pattern ECG changes and ventricular tachyarrhythmias in the male rabbits in our study. The greater INa inhibition caused by 100 μM of lithium in male RVOT cardiomyocytes might have led to greater ECG changes and a greater incidence of Brugada-pattern ECG changes and tachyarrhythmia in male rabbits. The experiments in canine epicardial and endocardial layers show that male cardiomyocytes had a larger INa amplitude than female cardiomyocytes [Na may potentially play a role in the higher risk of the Brugada syndrome in male individuals. Accordingly, we hypothesized that the male sex would increase the arrhythmogenic potential of Brugada syndrome via its greater impact on channelopathy in cardiomyocytes. The INa-late, though a relatively small current relative to the peak INa, was sufficiently large during the AP plateau to affect the AP duration. An enhanced INa-late in cardiomyocytes can lower the threshold of the AP, prolong the AP duration, and increase the intracellular Ca2+ concentration, which may initiate diastolic depolarization and increase excitability, leading to increased arrhythmogenesis [Na-late has not been specifically established [Na-late in only male RVOT cardiomyocytes. This finding indicates the impact of gender on the incidence of lithium-induced Brugada-pattern ECGs or ventricular tachyarrhythmia, suggesting that the INa-late may play a crucial role in the ventricular arrhythmogenesis of lithium intoxication. Gender-specific differences in INa-late have been reported in mice ventricular cells [Na-late by lithium is greater in males resulting in triggered activity caused by intracellular Ca2+ overload with the activation of the Na+/Ca2+ exchange [2+-dependent receptor inactivation, which further accumulated intracellular Ca2+ by the inhibition of a Ca2+ extrusion via the Na+/Ca2+ exchange [2+ overload is usually related to a prolonged QTc, multiple case series have shown no significant changes in the QTc intervals of patients before and after initiating lithium therapy [Kr) and INa-late. The inhibition of IKr by ranolazine prolongs AP duration, while its effect to inhibit INa-late abbreviates the AP duration. The clinical net effect of the inhibition of these ion channel currents sums up to a modest increase in the mean QTc interval over the therapeutic range of ranolazine [Na-late inhibition may have a therapeutic implication for the management of lithium-induced ventricular arrhythmogenesis.Syncope or cardiac arrest was mostly found in male patients with lithium-induced type 1 Brugada-pattern ECG changes ,6,27. Incumented . Lithium changes . Unmaskimyocytes . These fogenesis . Howeverablished . We founar cells . The augexchange . Moreoveexchange . Althoug therapy ,37. A pr therapy . Within + current activity of RVOT cardiomyocytes. Theoretically, the histological presentation and the expression of proteins or mRNA levels would not be changed in a short period of time [This study has some potential limitations. First, since the lithium dosage used in this study was relatively large, it is unclear whether our study may well correlate to lithium intoxication in clinical scenarios. Second, the anti-arrhythmogenic potential of ranolazine in lithium-induced Brugada-pattern ECG changes in rabbits might not be translated to the human Brugada syndrome, which is majorly caused by channelopathy. Moreover, our study just investigated the acute response of lithium on the Na of time ,40. It iNa and INa-late in male RVOT cardiomyocytes than in females (Na-late may be a potential therapeutic strategy for Brugada syndrome-related ventricular tachyarrhythmia.Male sex is predisposed to lithium-induced Brugada-pattern ECG changes and ventricular conduction delay, with lithium having a greater impact on the I females . Targeti


Combining with mutation profiles, the results show that top recurrent AML mutations were unbiasedly allocated into M2 and M5 territories, indicating the t-SNE instructed transcriptomic stratification profoundly outperforms mutation profiling in the FAB system. Further functional data mining prioritizes several myeloid-specific genes as potential regulators of AML progression and treatment by Venetoclax, a BCL2 inhibitor. Among them two encode membrane proteins, LILRB4 and LRRC25, which could be utilized as cell surface biomarkers for monocytic AML or for innovative immuno-therapy candidates in future. In summary, our deep functional data-mining analysis warrants several unappreciated immune signaling-encoding genes as novel diagnostic biomarkers and potential therapeutic targets.Acute myeloid leukemia (AML) is a blood cancer with high heterogeneity and stratified as M0–M7 subtypes in the French-American-British (FAB) diagnosis system. Improved diagnosis with leverage of key molecular inputs will assist precisive medicine. Through deep-analyzing the transcriptomic data and mutations of AML, we report that a modern clustering algorithm, The online version contains supplementary material available at 10.1007/s12672-022-00516-y. t-SNE instructed transcriptomic stratification discriminates FAB subgroups of AML.Potential association of the prioritized genes with Venetoclax resistance at single-cell resolution.The online version contains supplementary material available at 10.1007/s12672-022-00516-y. Acute leukemia is a group of clonal hematologic malignancies originated from aberrant hematopoietic stem and progenitor cells (HSPCs) with significant heterogeneity. Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Based on hematopoietic cellular morphology, immunophenotype, cytogenetic and genetic alterations, AML has been stratified into several subtypes. Although the AML diagnosis has been just recently updated by WHO and ELN, the FAB diagnosis system is still the most popular guideline in clinic since 1970s and classifies AML into 8 subtypes, M0–M7 .AML with certain monocytic differentiation (M-AML) accounts for about 30% of all AML cases. It is diagnosed primarily by morphological and immunophenotypic characteristics, such as the degree of bone marrow differentiation and leukemia blast cell count. M-AML mainly includes acute myelomonocytic leukemia FAB M4) and acute monoblastic leukemia FAB M5) [ and acut [ and acAt present FAB M3 subtype patients  could be treated by arsenic trioxide combined with all-trans retinoic acid dual induction therapy to achieve clinical cure. However, treatment of other FAB subtype patients remains challenging –3. In adTo overcome the heterogeneity of AML in diagnosis and improve the therapeutic efficiency of Venetoclax, identifying novel molecular biomarkers of FAB_M5 therefore appears urgent and important. The present study integrates clinical information, mutation profiles, AML patient transcriptome of pooled samples or single cell samples, datasets of Venetoclax drug tailoring and knowledge from animal AML models. We finally prioritized several novel biomarkers for AML patients, especially for FAB M5 subtypes. Their prognosis values were also verified at the pooled-cell or single-cell resolution.All of the datasets in the study had been published and are available for download online. See Supplemental Table 1 for the DOI address of the discovery datasets. If not stated elsewhere, gene expression level was recorded and normalized as Reads Per Kilobase of exon model per Million mapped reads (RPKM) in RNA-seq datasets for comparing analysis. Initial discoveries were focused on the full datasets of The Cancer Genome Atlas Leukemia (TCGA_LAML) and Beat_AML , 11, inct-distributed Stochastic Neighbor Embedding, t-SNE; Uniform Manifold Approximation and Projection, UMAP. Although PCA visualization in 2D failed to stratify M2, M3 and M5, both t-SNE and UMAP have good performance in dimension reduction and visualization. For convenience, only t-SNE displays in 2D were reported in the study for visualizing transcriptome at the single patient level. Once the 2D matrix of t-SNE plotting is determined, other information (i.e. mutation profiles or gene expression levels) were concatenated to the matrix and further projected to the same t-SNE plot by R package ggplot2.All of the visualization results were generated in R (version 4.0) using proper packages. See Supplemental Table 2 for the software information of R packages for clustering and following analysis. Since that Microarray or RNA-seq based transcriptomic data are high-dimension matrix, three popular dimension reduction algorithms were implemented to visualize the data in the 2-dimentional (2D) setting, one is the classical Principal Component Analysis (PCA) and the other two are developed and applied wildly in singe-cell RNA-seq (scRNA-seq) data analysis: NPM1_Group1 vs. NPM1_Group2 or in the comparison between FAB_M5 vs. FAB_M2 were calculated by the R package limma. Top 50 variable genes, volcano plots, Venn diagram and correlation analysis of DEGs were performed in R using proper packages. See Supplemental Table 2 for the software information of R packages. To calculate DEGs’ HR value, clinical information including patient survival status and following-up days was downloaded from TCGA. Overall survival (OS) was used as a prognostic endpoint. In survival analysis using the TCGA dataset, patients with following-up days less than 30 days were excluded. The R packages survival and survminer were used for univariate Cox hazard regression analysis to evaluate OS-related risk genes. Gene expressional level-based Kaplan–Meier survival plots and the p values were also calculated and visualized accordingly.DEGs in the comparison between The R package clusterProfiler and the software GSEA 4.1.0 were used for enrichment analysis of altered biological pathways. Supplemental Table 2 for the software information and original publications. Alterations with normalized enrichment score (NES) > 1.5 or < −1.5 and p < 0.05 and were considered statistically significant.Expression of the prioritized genes in monocytic cells or progenitors were validated using public datasets of both healthy and leukemic samples, including in the platforms of single cell or pooled RNA-seq and ATAC-seq (Assay for Transposase-Accessible Chromatin with high throughput sequencing). See the Supplemental Table 1 for the DOI address of these datasets. Correlation of the genes in AML risk stratification was also validated using a longitudinal scRNA-seq dataset of a patient with two stages: Diagnosis (Dx) and Relapse (Rl). The risk value of each cell in the patient was inferred by R package Scissor. After normalization of the Dx/Rl dataset by R package harmony, identities of each cluster were annotated by R package celldex. Plots of gene and protein expression level at single cell level were generated by R package Seurat4.Human normal 293 T cells and AML cell lines THP‑AML (THP‑1) were purchased from The Type Culture Collection of the Chinese Academy of Medical Sciences . AML cells were maintained in RPMI‑1640 medium (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum (Thermo Fisher Scientific). 293 T cells were maintained in DMEM supplemented with 10% FBS. We maintained cell lines at 37˚C in a 5% CO2 cell culture incubator and tested all cell lines routinely for mycoplasma contamination.LILRB4 were cloned into the pSin-Flag (Addgene) vector (GenScript). Positive clones were screened using ampicillin as a selection pressure (Sigma‑Aldrich) and identified by Sanger sequencing on recombinant plasmid. To produce lentiviral particles, 4 × 106 293 T cells in a 10-cm dish were co-transfected with LILRB4-pSin-Flag, psPAX2 and pVSV-G (Addgene) plasmids. The supernatant containing viral particles was harvested twice at 48 h and 72 h after transfection, then filtered through Millex-GP Filter Unit . Viral particles were concentrated in 4 °C high speed centrifuge at 12,000 rpm for 6 h, resuspended in DMEM and stored at −80 °C until use.Plasmids expressing human 5 cells/well were plated in a final volume of 1 ml culture media. For infection, THP-1 cells were plated onto 6-well plates at 2 × 105 cells/well and infected with lentiviral stocks at a multiplicity of infection of 100 in the presence of polybrene . Puromycin at a final concentration of 1 µg/ml (Sigma‑Aldrich) was added to the media 72 h after transfection to select for stably transfected cells.A total of 2.5 × 10® (GenStar). Reverse transcription was performed at 37 °C for 15 min and then 85 °C for 15 min using a StarScript III RT Kit.Total RNA was extracted from AML cell lines using TRIzolLILRB4 expression levels were calculated and normalized using the comparative quantification cycle 2−ΔΔCt method relative to an endogenous control ACTB. SYBR qPCR Mix (GenStar) was used to detect and quantify the expression of the target gene. The following primers were used: ACTB, 5′-CTGGAACGGTGAAGGTGACA-3′ (forward) and5′-CGGCCACATTGTGAACTTTG-3′ (reverse); LILRB4, 5′- CATCCATGACAGAGGACTATGC-3′ (forward) and 5′- GGGCTGAAAGGGTGGGTTTA-3′ (reverse).(GenStar). The relative 4/ml. The cell suspension was seeded into a 96-well plate (100 µl/well). Subsequent to adding 10 µl Cell Counting Kit‑8  solution to each well, the plates were incubated for 1 h in the incubator and the absorbance at 450 nm was measured with a microplate reader after0, 24, 48, 72 and 96 h. Cell proliferation and survival curves were plotted based on the absorbance values.The cells were counted, and the cell suspension was diluted to a final concentration of 2 × 10THP-1 cells were incubated for appropriate time in RPMI 1640- medium supplemented with 10% FBS and titrated concentrations of Venetoclax . Venetoclax is 10 mmol/L in DMSO. To calculate the IC-50 of the drug, drug treatments with different concentrations were designed. The treatments in Fig. All statistical calculations in the study were performed with proper R packages. Sizes of samples and p values were labeled accordingly. If not stated elsewhere, difference with p < 0.05 was recognized as statistically significant.All scripts for computational analysis and visualization were written in R and available upon reasonable request. The study did not generate new software or algorithms.TET2- and PTPN11-related murine models of myeloid leukemia [TET2- and PTPN11-related animals manifest features of FAB_M4/M5 rather than FAB_M0/M1/M2 since they are also hot mutations in chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia (CMML and JMML). To extract key features of FAB_M4/M5, we performed the present study in silico as outlined in Fig. Datasets of AML generated in TCGA and other centers such as BeatAML and Leucegene provides invaluable resources with easy access for further comprehensive data-mining. In previous studies, we and other laboratories have reported numerous leukemia –15. Howet-SNE and UMAP, which are now widely implemented in single cell RNA-seq analysis (scRNA-seq) [t-SNE display . These results suggest that transcriptomic inputs-based t-SNE clustering successfully demarcates M3, M2 and M5 subtypes in AML stratification.As an initial discovery, transcriptomic datasets in the form of RNA-seq  of AML patients were downloaded from TCGA . InstrucRNA-seq) . Surprislay Fig. A. Due tot-SNE clusters or FAB subtypes, we projected the mutation information to the same t-SNE plot. Top recurrent AML mutations in genes which involves in epigenetic regulations, cell signaling, transcriptional programming were selected for such analysis: NPM1, DNMT3A, FLT3, KIT, KRAS, NRAS, PTPN11, CEBPA and RUNX1. When combination of mutation was not considered, patients with mutations in KIT, NRAS, PTPN11 and CEBPA appear to be allocated in the region above the dashed line (territory of M2) . For convenience in following studies, the AML patients with NPM1 mutations were divided into two subgroups in the TCGA dataset, namely NPM1_Group1 whose territory bias to FAB_M5, and NPM1_Group2 whose territory bias to FAB_M2 .To test if any AML recurrent mutations bias to certain NPM1_Group1 and FAB-M5 in biological aspects, we compared the molecular differences between NPM1_Group1 vs. Group2 and also between FAB_M5 vs. M2 at the level of genes and biological pathways. Using NPM1_Group2 AML patients as the control group, differentially expressed genes (DEGs) were identified and as visualized in the heatmap (for top 50 variable genes) and volcano plot   Fig. A and B. TA2 Fig. A and B. NPM1_Group1 vs. Group2 and FAB_M5 vs. M2 was then analyzed at the entire transcriptome level . As shown in Fig. NPM1_Group1 vs. Group2 turns to be among the upregulated DEGs in FAB_M5 vs. M2. More than 400 upregulated DEGs and more than 80 upregulated biological pathways in NPM1_Group1 (and FAB_M5) were extracted (fold change or enrichment score > 2 or < -2 and p < 0.05), including TNF signaling pathway, inflammatory pathway, interferon gamma pathway and IL6/STAT pathway. In contrast, pathways involved in ribosome-related transcriptional regulation and RNA surveillance were enriched in NPM1_Group2 (and FAB_M2) . In leukemic datasets, these fours were also enriched in myeloid leukemia . Interestingly, LILRB4 is also enriched in bulk RNA-seq sample of acute lymphoid leukemia .We then validated the correlation between the 6 genes and FAB_M5 or general AML through different datasets. First, we analyzed the expression pattern of the 6 genes in various hematopoietic developmental stages using both pooled sample and single cell in the form of RNA-seq and ATAC-seq , 22. As analysis –26. The LRRC25 was positively correlated with the count of neutrophils and monocytes in the peripheral blood of AML patients . These results validate those three out of four genes have important and significant prognosis value in the TCGA dataset.Furthermore, based on the Beat_AML clinical datasets, the expression level of LILRB4, LRRC25, NCF2 and RAB31 all are positively correlated with Venetoclax insensitivity.The drug Venetoclax, an inhibitor of BCL2, has shown profound therapeutic efficacy in refractory AML treatment and been approved by FDA recently . HoweverBased on a scRNA-seq dataset with two disease stages: diagnosis and relapse after Venetoclax, we then explore the potential correlation of the four genes with Venetoclax drug resistance during treatment in a published dataset generated from a monocytic AML patient . As showLILRB4 for in vitro validation since it was reported that LILRB4 is a therapeutic target of FAB M5 [LILRB4 were increased in the LILRB4-transfected THP-1 cells compared with the control (empty vector)  and then cultured the THP-1 cells in a medium with this concentration of Venetoclax. As shown in Fig. LILRB4 maintain increased survival rate compared to the control.To verify the function of the four risk genes in AML and their resistance to Venetoclax, we chose f FAB M5 . Expressor) Fig. A. In a rrol Fig. B. To ass μM Fig. C and theTet2-KO; Tet3-KO, Tet2-KO;Flt3-ITD, Tet2-KO; Ins2Akita/+); and (c) datasets from healthy and other leukemic donors, including both pooled samples and single cell samples. The cross-center and cross-species usage of various datasets at the levels of both single patient or single cell warrant the outcome of our data-mining in a form with great conservation and accuracy. Since it has been recently reported that LILRB4 is an immune therapeutic target for M5 AML [LRRC25, NCF2 and RAB31), i.e. to access if they also function as therapeutic targets for monocytic AML as LILRB4.Overall, the present study is a comprehensive data-driven in silico research for prioritizing mammal-conserved risk genes (biomarkers), which may play essential role in diagnosis, progression and treatment management of AML, mainly in the form of FAB M5 subtypes. As outlined in Fig. r M5 AML , 29, theNPM1_Group1 vs. NPM1_Group2. Due to the challenging batch effect of transcriptomic data, we did not include GTEx datasets of healthy donors to the TCGA_LAML (data not shown). Concurrent collection of both leukemic samples and healthy samples are required in a future study design and it will further assist the prioritization of M2 or M5 features when compared with healthy donors.Of note, there are no healthy donors in the TCGA_LAML cohort and our characterization had to mainly focus on comparing FAB_M5 vs. FAB_M2 or t-SNE and UMAP clustering outperform the traditional PCA since they can discriminate M2, M3 and M5 in the 2D space  diagnosis system.Since s of AML , 31. Gens of AML  is with Furthermore, as shown in Fig. LILRB4, LRRC25, NCF2, RAB31, LCP1 and HCK. Interestingly five of the gene are specifically expressed in myeloid cells or progenitors, except that LCP1 is expressed in both myeloid lineage and lymphoid lineage (Fig. LILRB4 is a novel immune therapy target for FAB_M5 AML [LCP1 and HCK in leukemia or lymphoma has been sporadically reported [LRRC25, NCF2 and RAB31 is largely unknown and the experimental validation are demanded in future study to facilitate these proteins used in clinic [After several rounds of filtering and prioritization, six genes were finally presented with conservation and positive HR value: B_M5 AML . The rolreported –26. Howen clinic –38. As aIn summary, the present in silico study integrated several aspects of AML datasets, including transcriptome, mutation, clinical information, single cell data, risk stratification with overall survival. We finally prioritize and validate four immune signaling-related genes as novel markers for FAB_M5 AML. The working flow and outcome will also guide future research and stratification analysis of other diseases such as autoimmune disorders and other types of blood cancers.Additional file 1.Additional file 2.Additional file 3.Additional file 4.


Vibrio cholerae is the etiologic agent of the severe human diarrheal disease cholera. To colonize mammalian hosts, this pathogen must defend against host-derived toxic compounds, such as nitric oxide (NO) and NO-derived reactive nitrogen species (RNS). RNS can covalently add an NO group to a reactive cysteine thiol on target proteins, a process called protein S-nitrosylation, which may affect bacterial stress responses. To better understand how V. cholerae regulates nitrosative stress responses, we profiled V. cholerae protein S-nitrosylation during RNS exposure. We identified an S-nitrosylation of cysteine 235 of AphB, a LysR-family transcription regulator that activates the expression of tcpP, which activates downstream virulence genes. Previous studies show that AphB C235 is sensitive to O2 and reactive oxygen species (ROS). Under microaerobic conditions, AphB formed dimer and directly repressed transcription of hmpA, encoding a flavohemoglobin that is important for NO resistance of V. cholerae. We found that tight regulation of hmpA by AphB under low nitrosative stress was important for V. cholerae optimal growth. In the presence of NO, S-nitrosylation of AphB abolished AphB activity, therefore relieved hmpA expression. Indeed, non-modifiable aphBC235S mutants were sensitive to RNS in vitro and drastically reduced colonization of the RNS-rich mouse small intestine. Finally, AphB S-nitrosylation also decreased virulence gene expression via debilitation of tcpP activation, and this regulation was also important for V. cholerae RNS resistance in vitro and in the gut. These results suggest that the modulation of the activity of virulence gene activator AphB via NO-dependent protein S-nitrosylation is critical for V. cholerae RNS resistance and colonization. Vibrio cholerae have developed anti-RNS systems. The flavohemoglobin HmpA is an NO detoxification protein, playing key roles in V. cholerae RNS resistance. HmpA is transcriptionally activated by the NO sensor NorR. Here we discovered that the V. cholerae virulence regulator AphB is a repressor of hmpA, and a protein S-nitrosylation of AphB serves as an off switch to derepress hmpA expression and protects V. cholerae from nitrosative stress. AphB also promotes V. cholerae virulence gene expression, and an S-nitrosylation of AphB abolished the production of virulence factors. Using a mouse model, we further explored the importance of the defensive and offensive flexibility afforded by AphB, showing that NO-dependent protein S-nitrosylation is critical for V. cholerae’s war strategy.Beset by constantly evolving armies of potential pathogenic invaders, human hosts deploy a variety of countermeasures, including the production of host-derived toxic compounds nitric oxide (NO) and NO-derived reactive nitrogen species (RNS). In response, pathogens such as  Vibrio cholerae is the causative agent of cholera, a severe watery diarrhea disease. There are millions of cases of cholera and thousands of deaths caused by V. cholerae each year [V. cholerae uses sophisticated signal transduction pathways to activate a set of virulence factors [toxT requires ToxR and TcpP, and TcpP is regulated by AphA and AphB. Under anaerobic conditions, tcpP expression increases, which is directly regulated by AphB [tcpP.ach year . To colo factors ,3. The m by AphB ,5, a LysV. cholerae must also coordinate the sequential expression of a series of factors required for defense against host-derived toxic compounds. One of these compounds elevated during infection is nitric oxide (NO), a toxic radical that disrupts the function of proteins containing cysteine residues, enzymes catalyzing iron-dependent reactions, and members of the electron transport chain [V. cholerae encounters NO during human infections.In addition to activating virulence genes, rt chain . Furtherrt chain . It has rt chain –10, suggV. cholerae, HmpA, a member of the flavohemoglobin family of enzymes, plays a key role in resistance of NO by catalyzing the conversion of NO to nitrous oxide or nitrate [hmpA expression is activated by the NO sensor NorR, a predicted σ54-dependent transcriptional regulator [nnrS, which encodes a novel protein that is important for RNS resistance [V. cholerae protein S-nitrosylation, we performed a proteomic study of V. cholerae exposed to NO using a modified iodoacetyl isobaric tandem mass tags (iodoTMT) approach [hmpA expression and that nitrosative stress inactivated AphB via protein S-nitrosylation. We also showed that AphB nitrosylation was important for V. cholerae RNS resistance and pathogenesis in vitro and in vivo.To cope with NO produced during infection, many pathogenic bacteria have evolved mechanisms to convert NO into other, less toxic, nitrogen oxides . In V. c nitrate . In the egulator . NorR alsistance . NnrS dosistance . NO alsosistance . To inveapproach . We founV. cholerae protein S-nitrosylation, we applied a modified iodoTMT approach [V. cholerae protein S-nitrosylation in the presence of NO (V. cholerae under a virulence-inducing condition (AKI medium) [To investigate how NO affects approach  to identce of NO . First,  medium)  and trea medium) . We also medium) ,5. AphB C76S/C94S mutants, but not in AphBC235S mutants , which encodes a key NO detoxifying enzyme [hmpA promoter, interaction was assessed by electrophoretic mobility shift assays (EMSA) using purified AphB and biotinylated hmpA promoter DNA fragments which contains the putative AphB binding site , the AphB binding of hmpA was abolished ) and western blot analyses were then performed. In the presence of NO, wildtype AphB dimer formation was significantly reduced, whereas similar numbers of dimers of AphBC235S mutant proteins were formed without or with NO  inhibited tcpP expression when wildtype aphB was present  to bypass the requirement of AphB activation. These data suggest that inactivation of AphB by S-nitrosylation leads to a reduction of virulence gene expression.We then measured S-nitrosylation of AphB on  present , circles mutants , squarestac-tcpPH strains. We found that the mutants displayed a slight growth defect in the absence of NO, but the viability of Ptac-tcpPH strains was significantly reduced , but not in that of RNS-mitigated mice (+AG)  Figs  and S3C.V. cholerae cells were exposed to nitric oxide, the key virulence regulator AphB was nitrosylated at Cys235, which inactivated AphB and allowed expression of the AphB-repressed hmpA. HmpA contains an iron-heme moiety that directly catalyzes the NO-decomposition reaction as well as a flavoreductase domain that mediates the transfer of the electrons to and from NO [V. cholerae RNS resistance in vitro and in vivo  type III secretion system, inhibits the DNA-binding capacity of SsrB, and exhibits increased fitness when exposed to RNS in vivo [S. aureus, S-nitrosylation of the quorum sensing (QS) regulator AgrA leads to decreased transcription of the agr operon and quorum sensing-activated toxin genes. In addition, in E. coli, nitrosylated OxyR activates genes that encode enzymes controlling S-nitrosylation and protecting against nitrosative stress under anaerobic conditions [In this study, we found that when  from NO . HmpA plenterica –28. HmpAid hosts . Previou in vivo . In S. anditions . Interesnditions ,33.V. cholerae deploys AphB to repress hmpA, which is only activated by NorR in the presence of NO [Salmonella, HmpA production needs to be tightly regulated [hmpA increases bacterial susceptibility to ROS. We showed here that constitutive expression of hmpA in V. cholerae was detrimental to bacterial survival against ROS attacks or when bacteria were grown to stationary phase  regulating hmpA [Ralstonia, Burkholderia, Bordetella; γ-proteobacteria such as all members in Enterobacteria, Shewanella, all other Vibrionales except V. cholerae. The only exception is that in Pseudomonas, hmpA is predicted to be activated by NorR, as in the case of V. cholerae. NsrR belongs to the Rrf2 family of transcriptional regulators. It acts as a strong repressor of hmpA transcription and uses the iron-sulfur cluster to sense NO and relieve the repression of hmpA [V. cholerae recruits AphB to serve as a repressor to perform the function of NsrR. It would be interesting to test if a similar repressor is present in other bacteria lacking NsrR, such as Pseudomonas, as well.It is intriguing that ce of NO . In otheegulated ,24,34 bery phase , suggesting hmpA . These i of hmpA . In gene of hmpA , leadingV. cholerae resides in both aquatic environments and in human small intestines. AphB plays a pivotal role in the life cycle of V. cholerae. During the transition from oxygen-rich environments to the oxygen-poor intestine, AphB Cys235 is reduced and can activate downstream virulence genes. The rate of reduction of AphB is low and another redox-sensing regulator, OhrR, is needed to jump-start virulence gene expression [ohrA, encoding an organic hydroperoxide resistant protein [aphBC235S mutant colonization was not fully restored in the +AG mice  that was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania.V. cholerae El Tor C6706 [V. cholerae and E. coli were propagated in LB (Luria-Bertani) Miller medium with appropriate antibiotics at 37°C, unless otherwise noted. AKI medium was used to induce virulence gene expression [or C6706  was usedpression . CultureaphB (VC1049), PBAD-aphB wildtype and cysteine to serine mutant derivatives, and plasmids to make recombinant AphB proteins (AphB-His6) were constructed as described previously [hmpA- (VCA0183), nnrS- (VC2330), and norR- (VCA0182) LacZ reporters, and hmpA in-frame deletion were constructed as described in [tac-hmpA plasmid was constructed by cloning hmpA coding sequences into pMal-c2x (New England Biolab) and then subcloned to pSRK-Gm [aphB variants used for mouse experiments were constructed by the Tn7-based genomic integration system [aphB wildtype or C235S mutant with the native promoter (341 bp upstream of ATG) were PCR amplified and cloned into the pGP704-mTn7 vector via NotI restriction cloning. The insertion of the aphB wildtype or C235S mutant into the V. cholerae genome was done by triparental mating among V. cholerae ΔaphB, E. coli S17-1 λpir carrying the helper plasmid pUX-BF13, and E. coli SM10 λpir carrying aphB variants on pGP704-mTn7. Exconjugants were selected on thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates containing gentamicin (20 μg/ml). The insertion of mini Tn7 cassette at the intergenic region of VC0487 and VC0488 was confirmed by PCR and sequencing using flanking primers of insertion site. The chromosomal lacZ::Ptac-tcpPH strain was constructed by the multiplex genome editing by natural transformation (MuGENT) method [tac promoter and tcpPH sequence were PCR amplified from pMal-c2x (New England Biolab) and V. cholerae C6706 genome respectively, stitched with the lacZ UP and Down 3kb arms, cotransformed into V. cholerae C6706 with VC1807::spec DNA, and selected on LB agar plates containing spectinomycin (100 μg/ml). The insertion of Ptac-tcpPH was confirmed by PCR and sequencing. The primer sequences used for the study and further details about the constructions are available upon request.In-frame deletion of eviously . Transcrribed in . Ptac-hm pSRK-Gm . Chromosn system . Briefly) method . BrieflyV. cholerae were grown in AKI medium without shaking for 4 hrs and then challenged with 100 μM DEA NONOate for 1 hr. The cell lysates were first incubated with 20 mM methyl methanethiosulfonate (MMTS) (Pierce) and then reduced with 20 mM sodium ascorbate. Proteins containing reduced thiols were then labeled with iodoTMTzero and digested with trypsin (8 μg trypsin/100 μg proteins). IodoTMTzero-labeled peptides were enriched by using the Anti-TMT Resin and eluted by TMT elution buffer (Thermo Scientific). The eluted peptides were submitted to Poochon Scientific . Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed using a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) coupled with an UltiMate 3000 nano UPLC system (Thermo Scientific). Peptide sequences were identified using the Proteome Discoverer 2.4 software  based on the SEQUEST and percolator algorithms. MS/MS spectra were searched against a UniProt V. cholerae protein database and a common contaminants database using full tryptic specificity with up to two missed cleavages. Variable modifications included in the search were an addition mass of TMTzero (324.2 Da) on cysteine. Consensus identification lists were generated with false discovery rates set at 1% for protein, peptide, and site identifications.Iodoacetyl isobaric tandem mass tags (iodoTMT) labeling of S-nitrosylated thiols was performed by modifying the method described previously ,48. BrieΔaphB with PBAD-aphBWT, PBAD-aphBC76S/C94S, and PBAD-aphBC235S were inoculated into the AKI medium and incubated microaerobically at 37°C for 4 hrs. 0.1% arabinose and, when indicated, 100 μM DEA-NONOate were added and further incubated for 1 hr. Bacterial cells were collected and subjected to Cayman Chemical’s S-nitrosylated protein detection assay kit (biotin switch) [Overnight cultures of  switch)  with modWT-His6 and AphBC235S-His6 proteins were expressed and purified on nickel columns according to the manufacturer’s instructions (Qiagen). Double-stranded biotin-labeled hmpA (CAATAAATTAGGTTTTGGCATGCAATCTGCTCATTAAGAAGGCTTGTGTAGAGTGGCTCA) and tcpP (GCAATTAAGTTCTCATTATCAACTGCAGAATTAGATTGCAAATAATTATATTAAAAAAAA) probes were prepared by annealing the single-stranded 5’biotin-labeled sense and antisense 50mer oligos after denaturation. Binding reactions contained various amount of AphB proteins and 20 nM of DNA in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 50 mM KCl, 100 μg/ml BSA, 10 μg of poly dI-dC/ml, and when indicated, 100 mM dithiothreitol (DTT). After 20 minutes of incubation at 25°C, samples were size-fractionated using 5% polyacrylamide gels in 0.5X Tris-acetate-EDTA (TAE) buffer. The biotin-labeled free DNA and protein-DNA complexes were electrophoretic transferred via binding reactions to nylon membrane and visualized using horseradish peroxidase (HRP) conjugated streptavidin (Pierce). To S-nitrosylate purified AphB proteins, DTT was first removed from AphBWT and AphBC235S by dialysis. 1 μM proteins were then incubated with 1 mM S-Nitroso-L-glutathione (GSNO)  for 15 mins at 25°C.AphBΔaphB with vector pBAD24, PBAD-aphBWT, or PBAD-aphBC235S harboring different lacZ reporters were inoculated 1:100 into the minimal medium described in [hmpA and tcpP transcripts, the above cultures were treated with DEA-NONOate for one hour before bacterial cells were harvested and total RNA was purified by using RNeasy Kit (Qiagen). RNA reverse transcription was performed by using the SuperScript II Kit (Invitrogen). Quantitative realtime qPCR was performed on a CFX96 Real-Time system (BioRad) using primers specific for hmpA and tcpP. The 16S rRNAs were used as internal controls in all reactions.Overnight cultures of ribed in  containiWhole-cell lysates were prepared from bacterial cultures and samples were normalized to the amount of total protein as assayed by the Biorad protein assay (Biorad). The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% polyacrylamide gel and transferred to nitrocellulose membrane for western blot analysis using polyclonal rabbit anti-TcpA  or AphB ΔaphB containing PBAD-aphBWT or PBAD-aphBC235S plasmids were grown in the AKI medium for 4 hrs. 0.1% arabinose and when indicated, 50 μM DEA NONOate were then added and all the cultures were incubated for an additional 2 hrs. Cells were resuspended in PBS and treated with 0.8 mM crosslinking reagent DSP (dithiobis(succinimidyl propionate))(Thermo Fisher) on ice for 2 hrs. The cross-linking reactions were terminated by adding 50 mM Tris-HCl (pH 7.5). Cell pellets were resuspended in SDS loading buffer without reducing agents and AphB multimers were detected by western blots using AphB antibody.V. cholerae containing a vector control or Ptac-hmpA plasmid were grown in LB with 0.5 mM IPTG at 37°C for 8 hrs with aeriation. Cell pellets were washed with PBS and resuspended in PBS with 20 μM of the redox-sensitive, cell-permeable dye 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) (Abcam). Cells were then stained for 1 hr at 37°C, and DCFDA-stained cells were subjected to flow cytometry  for ROS detection using the 488 nm laser for excitation and detected at 535 nm.V. cholerae RNS resistance in vivo. Briefly, six-week-old CD-1 mice were used. 0.5% (wt/vol) streptomycin and 0.5% aspartame were added to the drinking water throughout the experiment. After 3 days of streptomycin treatment, 1 mg/ml aminoguanidine  was added to the drinking water of +AG group of mice and throughout remainder of the experiment. Additionally, the equivalent of 1 mg of AG was orally gavaged into each mouse in the +AG group of mice daily throughout the remainder of the experiment. One day after starting the AG treatment, approximately 108 CFU of each of the two differentially labeled strains (wildtype lacZ+ and mutant lacZ-) were mixed at a 1:1 ratio and intragastrically administered to each mouse. Fecal pellets were collected from each mouse at the indicated time points, resuspended in LB, serially diluted, and then plated on plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside  and appropriate antibiotics. At day 7, small intestine tissues were harvested and CFU of small intestine-colonized V. cholerae were enumerated. The competitive index was calculated as the ratio of mutant to wildtype colonies normalized to the input ratio.The streptomycin-treated adult mouse model was used to examine S1 FigA. Specificity of AphB binding of hmpA. AphB-His6 proteins were mixed with 20 nM 50mer biotin-labeled double-stranded hmpAp DNA in the absence or in the presence of 1 μg unlabeled hmpA promoter DNA. B. AphB on tcpP. AphB-His6 proteins added (from left to right) at concentrations of 0, 0.125, 0.25, 0.5, 1, and 2 μM, respectively. 20 nM 50mer dsDNA containing the tcpP promoter region were used in each lane.(PDF)Click here for additional data file.S2 Fig6 proteins added (from left to right) at concentrations of 0, 0.125, 0.25, 0.5, 1, and 2 μM, respectively. 20 nM 50mer dsDNA containing hmpA (A) or tcpP (B) promoter region were used in each lane.AphB-His(PDF)Click here for additional data file.S3 Fig8 cells of wildtype and ΔhmpA mutants (A) or wildtype and aphBC235S mutants (B) or wildtype and Ptac-tcpPH mutants (C) were mixed 1:1 and intragastrically administered to mice without aminoguanidine (AG) treatment (-AG) and mice with AG (+AG). V. cholerae CFU from fecal samples was determined daily. The competitive index (CI) was calculated as the ratio of mutant to wildtype colonies normalized to the input ratio. Horizontal line: mean CI of 5 mice. *: p <0.05; **: p <0.005; ***: p <0.001 (Mann-Whitney U test).10(PDF)Click here for additional data file.S4 FiglacZ::Ptac-tcpPH mutants or wildtype and lacZ::Ptac-tcpPH/ΔtcpA mutants were cocultured in the AKI medium microaerobically for 8 hrs in the absence and in the presence of 100 μM DETA NONOate. CFU was then determined. Data are the means ± SD from 6 independent experiments. ***: p <0.005 (Ordinary one-way ANOVA).Wildtype and (PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Arabidopsis thaliana seedlings expressing the Ca2+-sensitive photoprotein aequorin in the cytosol were challenged with water activated by low-power non-thermal plasma generated by a dielectric barrier discharge (DBD) source. PAW sensing by plants resulted in the occurrence of cytosolic Ca2+ signals, whose kinetic parameters were found to strictly depend on the operational conditions of the plasma device and thus on the corresponding mixture of chemical species contained in the PAW. In particular, we highlighted the effect on the intracellular Ca2+ signals of low doses of DBD-PAW chemicals and also presented the effects of consecutive plant treatments. The results were discussed in terms of the possibility of using PAW-triggered Ca2+ signatures as benchmarks to accurately modulate the chemical composition of PAW in order to induce environmental stress resilience in plants, thus paving the way for further applications in agriculture.Non-thermal plasma technology is increasingly being applied in the plant biology field. Despite the variety of beneficial effects of plasma-activated water (PAW) on plants, information about the mechanisms of PAW sensing by plants is still limited. In this study, in order to link PAW perception to the positive downstream responses of plants, transgenic  Calcium signalling constitutes a universal and versatile transduction mechanism across the tree of life, ranging from procaryotic to eukaryotic organisms ,3,4,5,6.2+ elevations in the model plant Arabidopsis thaliana (Arabidopsis) in response to plasma-activated water (PAW) generated by plasma torches, providing evidence for the involvement of Ca2+ signalling in the perception of PAW by plants cyt), 1-week-old seedlings of Arabidopsis thaliana stably expressing the bioluminescent Ca2+ reporter aequorin in the cytosol cyt  were responsible for the induction of [Ca2+]cyt transients that quickly dissipated within 10 min. The Ca2+ transients observed with the 10 min DBD-PAW showed an intermediate Ca2+ dynamics at both frequencies, thereby representing a transition state between the short and long treatments.In all cases, the DBD-PAWs were found to be able to induce cytosolic Ca signals , whereasCa2+]cyt . The dynreatment a–d, with2+]cyt elevations  was found to correlate with the plasma discharge frequency settings of the DBD device, indicating the occurrence of significantly more intense Ca2+ signals in the samples treated with DBD-PAW at 20 kHz, in comparison with those treated with DBD-PAWs at 12 kHz, at least for the medium and long time intervals   .2O , the measured pH values indicated a more pronounced acidity for PAW samples generated at 20 kHz in comparison with those obtained at 12 kHz .3− enrichment in DBD-PAW  instead seemed to increase in the first 10 or 15 min  of the cold plasma treatment and then decrease  when the plasma activation was protracted up to 30 min  and nitrites (358 nm) ( DBD-PAW c. NO2− co 30 min d. The presence of dissolved ozone in DBD-treated water cannot be excluded because of the well-known characteristic odour after the treatment and the masked shoulder at about 260 nm [3−]) and nitrites (measured as the sum of [HNO2] and [NO2−]), showing a clear correspondence between PAW exposure time and nitrate abundance for DBD-PAWs obtained at plasma discharge frequencies of 12 kHz  and 20 kHz , medium (10 and 15 min) and long (30 min) time intervals)  a,b. Conctervals) c; howevetervals) d.3− concentrations at each fixed time always differed between the two discharge frequency settings , at different time intervals  after the first treatment. In response to several elicitor treatments, plants can respond by inducing a temporary refractory state, i.e., the failure to react to a second dose of the same stimulus ,28,29,302O to cold plasma for a short time interval (3 min) was selected  in order to avoid triggering sustained [Ca2+]cyt dynamics that could mask the effect of the second PAW administration.For the present analysis, PAW obtained by exposing deionized H2+]cyt variations revealed that the second treatment resulted in the induction of Ca2+ signals characterized by an increased magnitude, as well as a slower onset  between the two DBD-PAW applications, which can also be inferred from the progressively increased slope of the [Ca2+] increase trace  or a plasma torch  for the same time interval (3 min). Striking differences were found both in terms of the dynamics and amplitude of the evoked [Ca2+]cyt signals. In particular, in aequorin-expressing Arabidopsis plants, PT-PAW triggered a much more pronounced and sustained cytosolic Ca2+ elevation cyt. In particular, a DBD was developed to perform operations at relatively low power, allowing investigations into the potential Ca2+-mediated perception of PAW generated by low-energy water activation. Indeed, the comparison between the cytosolic Ca2+ signals induced by PAW obtained by either the DBD or a plasma torch (PT) indicated that the Ca2+-inducing activity exerted by the PT was comparable with that of the DBD for longer activation times (and thus the transferred energy) and tended to become saturated, whereas for shorter activation times (and thus lower transferred energy) the behaviour was quite sensitive, even to small changes in this parameter.To elucidate the biochemical and molecular basis of the so-called plant “plasma vaccination” , we focuevations . In thisIn general, the DBD system is a versatile plasma source which, in principle, allows the power to be finely adjusted, so that a wide range of chemical mixtures at relatively low doses can be obtained and finely tuned. Moreover, it offers the advantage of being easily scalable, as the electrode surface can be tailored to the specific scope. The DBD was caged in a support structure for easy handling, so that it could operate close to the biological set up and/or the chemistry facilities, to minimize the time between the generation, plant treatment and chemical analysis steps. 2+ indicator aequorin. Aequorin-based Ca2+ measurements provide a sensitive and reliable assay to quantitatively monitor and analyse PAW-induced intracellular Ca2+ changes. The relative insensitivity of aequorin to pH cyt elevations that quickly dissipated, whereas longer time intervals of water exposure to DBD plasma  induced higher and more prolonged Ca2+ increases. These results seem to indicate that the two different Ca2+ signature trends observed may underlie the different outcomes in the plant, suggesting a dose–response relationship to stressors according to the hormesis theory cyt measurement assays, reconstituted seedlings were gently rinsed with deionized H2O to eliminate any residual excess coelenterazine, then placed individually in the wells of a CulturePlate-24  and allowed to rest floating in 150 μL of deionized H2O for an additional 10 min. PAW samples (or deionized H2O as a negative control) were applied to each seedling through injection with an EnVision 2105 XCite multimode plate reader (Perkin Elmer), which was also used to collect and integrate (over 500 ms) the emitted luminescence. The experiments were terminated by administering 150 μL of a discharge solution  ethanol) to each well in order to fully consume the aequorin pool and properly calibrate the recorded light signals into [Ca2+]cyt values, according to an algorithm based on the calibration curve of aequorin cyt traces obtained in this way with those measured with a custom-built luminometer  [Ten minutes prior to the [Caaequorin . A high-dge, UK) .The simplest method of estimating the energy transferred to the water is to consider the equation of heat transfer, taking the heating and loss terms into account. In particular, during the heating phase, the loss terms responsible for deviations from a linear increase were estimated from the time-resolved measurements of the water temperature. In this way, it is possible to use a unique parameter to compare the results obtained using the different plasma sources and the different experimental conditions, including the different power and activation times. Therefore, the following heat transport equation was applied:This equation has the following solution:If we compare the results for the different sources at different operational parameters and measure the temperature increase during the heating phase and the temperature decrease during the following decay, the effective power transferred to the water can be derived and, therefore, by multiplying by the activation time , the corresponding energy transferred to the water was obtained. 2+-mediated perception of PAW generated by a DBD in Arabidopsis seedlings. The quantitative analysis of the evoked Ca2+ dynamics highlighted significant correlations between the elicited Ca2+ responses and the DBD’s discharge frequency, activation time, as well as temporal delay between subsequent plant treatments. A comparison with the Ca2+ changes recorded after plant treatment with a different plasma-generating source, i.e., a plasma torch, was also presented and discussed. Our results, by providing fundamental insights into the sensing mechanisms of PAW by plants, may lay the ground for a better application of PAW in agriculture.The data obtained in this work demonstrate the Ca