Althoug T2-weighted image (T2WI) can provide the area at risk in patients with acute myocardial infarction, the value of T2WI is still in dispute due to various limitations of T2WI. Quantitative T2 mapping can overcome the problems of T2WI and delineate myocardial edema with increased accuracy.Between Feb.2012, June.2012, 15 patients with acute MI underwent cardiac MRI after percutaneous primary coronary intervention (PPCI). Acute MI was diagnosed, based on EKG, coronary angiography and cardiac biomarker. In short axis view, every analyzed myocardium was subdivided into 30 sectors evenly using commercially available software . On CINE images, sector based myocardial contractility were calculated semi-automatically as a percentage of the systolic LV wall thickening /LV wall thicknessED ) x 100, ES ; end-systole, ED ; end-diastole). T2 value of myocardium was measured in each sector. Infarcted zone was defined as hyper-enhanced area on LGE . On T2 mapping, AAR was defined as sectors that had T2 value (ms) > 2SD from remote myocardium. On CINE imaging, dysfunctional myocardium was defined as sectors that had LV wall thickening less than 40% or wall thickening < 80% of remote myocardium. The size (% of involving sector) of infarcted zone, AAR, and dysfunctional myocardium were calculated and then compared.In all patients, AAR was delineated on T2 map with increased T2 values. The size of infarcted zone was smallest (24.6±11% ) and dysfunctional myocardium was largest (54.6±10.2%). The size of AAR on T2 map was between infarcted zone and dysfunctional myocardium (42.3±8%). The size differences  were statistically significant one another. (p<0.05).T2 map can delineate constantly myocardial edema, representing AAR in acute MI. The size of AAR quantified by T2 mapping was between infarcted zone and dysfunctional myocardium in re-perfused AMI.


Relationships between myocardial scintigraphic parameters and renal function have not been fully determined. We investigated correlations between estimated glomerular filtration rate (eGFR) and left ventricular (LV) diastolic function using stress electrocardiographic (ECG)-gated myocardial single photon emission computed tomography (SPECT).2. Patients were assigned to the following four groups .We enrolled 136 consecutive patients with suspected coronary artery disease (CAD) who were assessed using technetium-99m stress ECG-gated myocardial SPECT. We evaluated SPECT images using 17-segment defect scores graded on a 5-point scale, summed stress score, summed rest score and summed difference score (SDS). The parameters for assessing LV diastolic function were peak filling rate (PFR), 1/3 mean filling rate and time to peak filling. The CAD was defined as SDS ≥2. Chronic kidney disease (CKD) was defined as eGFR <60 mL/min/1.73 mp < 0.001 for both). Furthermore, PFR at rest positively correlated with eGFR  and inversely correlated with SDS . Multivariate stepwise regression analysis independently associated eGFR with PFR .The PFR was significantly impaired after stress in the CKD and CAD + CKD groups compared with controls (Our data suggest that impaired renal function is a significant determinant of LV diastolic dysfunction in patients with suspected CAD. Impaired renal function confers a higher risk of coronary artery disease (CAD) and/or chronic heart failure (CHF), independently of conventional cardiovascular risk factors in patients with chronic kidney disease (CKD) . Left veElectrocardiography (ECG)-gated myocardial single photon emission computed tomography (SPECT) with exercise or pharmacological stress can detect myocardial ischemia and abnormal global LV wall motion. Recent developments in quantitative gated SPECT (QGS) software allow quantitative measurement of LV diastolic function using 16-frame ECG-gated SPECT . AlthougThis prospective study included 387 consecutive patients with suspected CAD who had undergone stress ECG-gated myocardial SPECT at Akita University Hospital between July 2010 and March 2012. Patients older than 80 years of age were excluded. Moreover, we did not enroll patients with atrial fibrillation, major cardiovascular complications involving old myocardial infarction, idiopathic cardiomyopathy, significant valvular heart disease, depressed LV ejection fraction (EF) (<50 %) and hemodialysis. Patients with post-examination LV end-systolic volume (ESV) of <15 mL determined by QGS were also excluded to rule out patients with small hearts [99mTc) sestamibi or tetrofosmin proceeded using the 1-day protocol. A symptom-limited bicycle ergometer exercise test using the standard protocol with 12-lead ECG recordings being taken every minute was started in 65 % of patients. The ergometer exercise test was considered adequate if the patient reached >85 % of maximal predicted heart rate and/or developed chest pain. At near-maximal exercise, a 296–370 MBq dose of 99mTc sestamibi or tetrofosmin was injected, and exercise continued for 1 min thereafter. Images were acquired from 30 min after isotope administration. Whenever possible, beta-blockers, calcium channel blockers, and caffeine products were discontinued 24 h before testing.Stress-rest myocardial SPECT imaging using technetium-99m (99mTc sestamibi or tetrofosmin was injected 3 min after starting the adenosine infusion. The patients at rest were administered 3 h later with 740 MBq of 99mTc sestamibi or tetrofosmin.Forty-seven patients (35 % of the study population) that were deemed unable to tolerate the ergometer test or to achieve 85 % of the maximal predicted heart rate (HR) underwent a pharmacological stress test using the adenosine infusion protocol. Adenosine (0.120 mg/kg/min) was infused using an automatic pump over a period of 6 min and then 99mTc tracers during the imaging process and the maximal matrix size was 64 × 64. The R–R interval was divided by the R wave trigger into 16 equal portions while acquiring ECG-gated images.End-diastolic and end-systolic myocardial perfusion SPECT images were acquired using a Symbia T2 dual-headed gamma camera  with circular 180° acquisition for 36 projections at 20 s per projection and a low-energy, general purpose collimator. A 20 % window was centered on the 140-keV peak for We reconstructed the SPECT imaging data using a data processor combined with filtered back-projection. Summed stress (SSS), rest (SRS), and difference (SDS) scores were semi-quantified from the SPECT data according to a 17-segment, 5-point scoring system using automated Heart Score View software  , 12. SDSV/dt) curve, we obtained peak filling rate (PFR), mean filling rate during the first-third of diastole (1/3MFR) and time to peak filling (TPF) and used them to assess global LV diastolic function. We defined PFR as the greatest filling rate at early diastole and it corresponded to the first maximal dV/dt value normalized to EDV, and defined TPF as the interval from the time at end systole to that at PFR.Reconstructed short-axis ECG-gated stress and rest SPECT images were processed using the QGS software to automatically calculate LV end-diastolic volume (EDV), LVESV and LV ejection fraction. The LV time–volume curve was plotted from each of the 16 frames, and generated using Fourier transforms. From this time–volume curve and its first differentiated curve which was transformed into a LV time-filling rate  [2. Serum creatinine used to calculate eGFR was obtained within 32 ± 19 days from the time of myocardial SPECT.The glomerular filtration rate was estimated from the equation for Japanese patients as follows: eGFR = 194 × (serum creatinine) female) . Forty-fE) and late (A) transmitral flow velocities, the ratio of early to late peak velocities (E/A) and the deceleration time of E velocity [E′) was determined from spectral pulsed-wave tissue Doppler imaging (TDI) recordings and the mitral E/E′ ratio was calculated. The sample volume at the septal corner of the mitral annulus was used for the apical-four chamber view [Echocardiography proceeded using an iE33 cardiac ultrasound unit . Both LVEF and LV mass index (LVMI) were calculated from the standard M-mode echocardiograms. Mitral inflow velocity was traced and we derived peak early , CAD alone , CKD alone , and both CAD and CKD .Continuous variables are expressed as mean ± SD and categorical variables are expressed as numbers (%). Differences among groups were analyzed by one-way analysis of variance followed by a post hoc Tukey–Kramer test for multiple comparisons. Categorical variables were analyzed using the Table E/A values, whereas E/E′ was significantly higher in the CKD and CAD + CKD groups than the control group. E′ tended to decrease in the CKD and CAD + CKD groups, although the difference did not reach statistical significance. Only high-density lipoprotein cholesterol, serum creatinine and uric acid significantly differed among the biochemical parameters.Table Table Both LVEF and PFR significantly decreased after exercise stress or adenosine loading compared with global LV function at rest in the CAD and CAD + CKD groups, whereas TPF tended to be prolonged in these groups without reaching statistical significance Fig. .Fig. 2CoThe eGFR correlated significantly and positively with PFR at rest in all 136 patients Fig. .Fig. 3CoHere, we presented data regarding the relationship between renal dysfunction determined by eGFR and LV diastolic dysfunction determined by ECG-gated myocardial SPECT in patients with suspected CAD. Nearly 25 % of our patients referred for ECG-gated SPECT had results indicating CAD, and almost 30 % had CKD. These patients had a higher prevalence of LV diastolic dysfunction than those without CAD and CKD. On the other hand, LV systolic dysfunction was evident in patients who developed myocardial ischemia after stress. From the different specificities among parameters for determining LV diastolic function, we selected PFR for multivariate analysis, because it has been considered the most sensitive parameter which represents overall LV diastolic function . The resEchocardiographic investigations using conventional and pulsed-wave TDI have revealed abnormal LV diastolic function in patients with CKD . Since CSeveral pathophysiological conditions such as LV hypertrophy, CAD, microvascular abnormalities, interstitial fibrosis, altered fluid and electrolyte metabolism and neurohumoral alterations might contribute to the mechanisms of LV diastolic dysfunction in patients with CKD. Our clinical study cannot explain the different roles played by all of these factors. Left ventricular hypertrophy is one potential cause of LV diastolic dysfunction in patients with CKD, yet LVMI did not significantly differ among our study groups, making any significant contribution of its structural effect on LV function unlikely. One possible mechanism associated with CKD such as over-activation of the rennin–angiotensin–aldosterone system (RAAS) might help to explain our findings. In fact, the RAAS plays a key role among profibrotic factors , 25. Fur2. Therefore, some patients included in the control group might in fact have had CKD. The principal purpose of our study was to examine relationships between eGFR and scintigraphic findings in patients with various degrees of renal function. Further studies are needed to determine associations between albuminuria and scintigraphic parameters in patients with CKD. Previous studies of post-stress myocardial dysfunction focused on patients with exercise-induced ischemia, however, some reports have demonstrated post-ischemic stunning after vasodilator stress [This study has several important limitations. The small study cohort might have blunted between-group differences, especially those regarding possible additional influences of the duration of renal dysfunction, hypertension and diabetes on LV diastolic dysfunction. It is possible that LV diastolic functional parameters such as PFR and 1/3MFR might not show significant differences between the CAD and control groups because of an imbalanced inter-group population. Thus, our results and conclusions can only be regarded as preliminary. We have no information about albuminuria for the patients in whom eGFR was ≥60 mL/min/1.73 mr stress , 27–29. r stress . AlthougLeft ventricular diastolic dysfunction is more closely associated with eGFR than with myocardial perfusion abnormalities in patients with suspected CAD. Therefore, impaired renal function might predict LV diastolic dysfunction determined by ECG-gated myocardial SPECT more effectively than myocardial ischemia.


Myocardial function can be evaluated using color-coded tissue velocity imaging (TVI) to analyze the longitudinal myocardial velocity profile, and by expressing the motion of the atrioventricular plane during a cardiac cycle as coordinated events in the cardiac state diagram (CSD). The objective of this study was to establish gestational age specific reference values for fetal TVI measurements and to introduce the CSD as a potential aid in fetal myocardial evaluation.TVI recordings from 125 healthy fetuses, at 18 to 42 weeks of gestation, were performed with the transducer perpendicular to the apex to provide a four-chamber view. The myocardial velocity data was extracted from the basal segment of septum as well as the left and right ventricular free wall for subsequent offline analysis.During a cardiac cycle the longitudinal peak velocities of septum increased with gestational age, as did the peak velocities of the left and right ventricular free wall, except for the peak velocity of post ejection. The duration of rapid filling and atrial contraction increased during pregnancy while the duration of post ejection decreased. The duration of pre ejection and ventricular ejection did not change significantly with gestational age.Evaluating fetal systolic and diastolic performance using TVI together with CSD could contribute to increase the knowledge and understanding of fetal myocardial function and dysfunction. The pre and post ejection phases are the variables most likely to indicate fetuses with abnormal myocardial function. In the progression of fetal hemodynamic adaptation, the assessment of fetal myocardial function could be of crucial importance when evaluating fetuses at risk or suffering from intrauterine hypoxia ,2. AlthoThe cardiac state diagram (CSD) is a new visualization tool for the pumping and regulating function of the heart  that proThe study included 125 fetuses, at a gestational age of 18 to 42 weeks, all referred to the Centre of Fetal Medicine at the Karolinska University Hospital Huddinge during 2009–2012. Gestational age was determined according to ultrasound in the 16-18th week of gestation. Characteristics of the study population are presented in Table . The 2D and TVI sector widths were minimized to obtain a frame rate > 200 frames/s , which is necessary for adequate reconstruction of TVI data for the fetal myocardium [Tissue Doppler echocardiography data was obtained with a GE Vivid-i equipment , using a 3S-RS transducer. All recordings were performed with the transducer perpendicular to the apex, providing a view of the fetal myocardium equivalent to an apical four-chamber viewocardium . The recThe cardiac cycle was divided into six main phases: atrial contraction, pre ejection, ventricular ejection, post ejection, rapid filling and slow filling. Systole was defined as pre ejection and ventricular ejection, while diastole was defined as post ejection, rapid filling, atrial contraction and slow filling. The time intervals were measured according to the changes from static to dynamic work, based on the motion shifts of the AV-piston, in compliance with DAPP technology. This makes identification of the time intervals possible without a concurrent ECG signal. The measuring points are slightly different from prevalent measuring points, as the phases of pre and post ejection are defined according to heart mechanics unlike the isovolumetric contraction and relaxation, identified based on the zero axis crossing points of the velocity curve. This is usually carried out in combination with reference points in the ECG signal, and the occasional anatomical M-mode image, in order to acquire a precise electrophysiological timing. This is not possible when screening the fetus, which precludes accurate timing of the mechanical events. The phases are displayed in Figure Atrial contraction begins with an elevation of the AV-piston, during which tension forces are created in the ventricular walls. When these tension forces become greater than the contraction forces in the atria the AV-piston start to move down towards the apex and the atrial contraction phase ends. This ventricular reshaping together with the vortex formations created in the flow behind the atrioventricular valves initiate the closure of the atrioventricular valves and is the beginning of pre ejection - the transition phase (volume to tension) between atrial contraction and ventricular ejection. During pre ejection the atrioventricular valves close and the interventricular pressure rises. This phase ends with a powerful tension increase as the acceleration of blood starts to initiate the opening of the semilunar valves and ventricular ejection begins. The interventricular pressure exceeds the pressure in the aorta and the pulmonary artery, causing the semilunar valves to open and blood to be ejected into the systematic circulation. Ventricular ejection ends when there is no outflow from the semilunar valves and they are about to close just before a backflow into the ventricles is generated. This initiates post ejection – the transition phase (tension to volume) between ventricular ejection and rapid filling. During post ejection the ventricular myocardium starts to relax. The forces of the backflow overcome the tension in the muscles and the ventricular volumes increase. Rapid filling starts as the atrioventricular valves are about to open and a reverse volumetric reshaping process begins. At that time the interventricular pressure drops, muscle tension decrease and the AV-piston returns to its neutral position by the inflow controlled hydraulic return. This phase ends as soon as the dynamic and resilient forces outside the heart have ceased and slow filling begins. Static and dynamic forces generated by blood flow into the heart are now the only forces left to keep the heart in an expanded position. Once the slow filling phase ends a new cardiac cycle begins.The data set was analyzed using SPSS (PASW Statistics 18). Linear regression analyses were displayed in scatter plots as well as in tables presented as mean values with 95% confidence interval. For the variables of insignificant change, the results were presented as median values within a range.The myocardial time intervals and velocities measured during a cardiac cycle, as well as the Tei index and the e’/a’ ratio, extracted from septum as well as the left and right ventricular free wall, are presented in Table Fetal heart rate decreased from a mean 147 bpm range 133 to 164) at 18 weeks of gestation to a mean 137 bpm (range 117 to 142) at term (r = 0.401), equivalent to a prolonged duration of a fetal cardiac cycle with about 30 ms throughout gestation. This corresponded to an increase in diastolic duration, as the duration of systole did not vary significantly with gestational age, either during pre ejection or ventricular ejection. Figure 3 to 164 Multiple regression analysis showed that a combination of HR and gestational age enhanced the correlations for the duration of artial contraction  and rapid filling . However, no significant relationship was observed between HR and the post ejection phase or the systolic durations. Sex was not decisive for any of the measured variables even though HR was observed to be significantly higher for the female fetus up until 30 weeks of gestation.During a cardiac cycle the longitudinal peak velocities of septum showed a linear increase with gestational age, as did the peak velocities of the left and right ventricular free wall, except for the peak velocity of post ejection (post’). Figure The peak velocities of the right ventricular free wall were higher compared to the peak velocities of septum or the left ventricular free wall, and showed a higher rate of change with gestational age during ventricular ejection and rapid filling. This corresponded to a more prominent increase of the e’/a’ ratio observed in the right ventricular free wall compared to the left ventricular free wall and septum. The displacement of the AV-piston, i.e. the distance covered during ventricular ejection, also showed a linear increase more prominent in the right ventricular free wall as pregnancy advanced, as can be seen in Figure We could confirm the feasibility and potential use of the new technique, previously demonstrated in a study of non ST-elevation myocardial infarction in adults  and a stThe objective of this study was to establish gestational age specific reference data for TVI measurements of the fetal myocardium. These data also reveal some new information regarding prenatal cardiac function as this study is the first to assess myocardial mechanics during fetal development by evaluating the different time intervals during a cardiac cycle according to the motion shifts of the AV-piston. The finding that the post ejection phase, measured from septum and the left ventricular free wall, decreased with gestational age, might reflect lactate dependent myocardial performance. During this phase the ventricular myocardium starts to relax, which paradoxically is an energy consuming process, caused by the repolarization process of the heart muscle cells, and consequently this phase will be very sensitive to low oxygen levels. In accordance adults exhibit a prolongation of post ejection during exercise and controlled hypoxia resulting in lactecaemia . In a reIn concurrence with previous studies ,15-20 wePrevious studies that used spectral Doppler reported higher peak velocities compared to the present results ,19,20,23As long as the timing of myocardial events can be represented accurately, time intervals could be more robust measurements than myocardial velocities as these measurements are less angle dependant and less influenced by tethering effects. Furthermore, time intervals have been described as markers of cardiac performance, where the myocardial performance index or Tei index, reflecting the relationship between the transition phases (pre and post ejection) and ventricular ejection, has been evaluated as a marker of fetal cardiac dysfunction -34. ThesThe transition phases mirror the hearts mechanical function and have been shown to be lactate sensitive markers for asphyxia and metabolic acidosis ,36. LactThe timing of mechanical events has hitherto been difficult to ensure without a simultaneous ECG signal. To make the analysis of TVI measurements easier and more effective we introduced the CSD as a potential aid in fetal monitoring. Using this method we measure the time intervals according to the changes from static to dynamic work based on the DAPP-technology, and thus no longer require a simultaneous ECG signal. This means that in the future we can potentially provide a fully automated evaluation of the longitudinal myocardial velocity profile. Hypothetically this method could give early indications of myocardial dysfunction that should be beneficial in many patient groups where fetal myocardial function is affected and facilitate detection of risk pregnancies as well as aid in the assessment before and after intrauterine fetal therapy.The provided reference material demonstrated the feasibility of the technique and that the measurements are sensitive enough to yield insight into maturational changes in myocardial function, which provides a foundation that will enable further investigations and potentially allow identification of fetuses with myocardial dysfunction.The authors declare that they have no competing interests.NE, BL, LÅB and MW participated in the design of the study and the interpretation of data, as well as revised the maniscript. NE performed the offline measurements, statistical analysis and drafted the manuscript. JJ performed the software development, defined the timing of the mechanical events during a cardiac cycle, participated in the interpretation of data and revised the manuscript. KFW and LH performed the ultrasound image acquisition ande revised the manuscript. All authors read and approved the final manuscript.


Male humpback whales produce a long, complex, and stereotyped song on low-latitude breeding grounds; they also sing while migrating to and from these locations, and occasionally in high-latitude summer feeding areas. All males in a population sing the current version of the constantly evolving display and, within an ocean basin, populations sing similar songs; however, this sharing can be complex. In the western and central South Pacific region there is repeated cultural transmission of song types from eastern Australia to other populations eastward. Song sharing is hypothesized to occur through several possible mechanisms. Here, we present the first example of feeding ground song from the Southern Ocean Antarctic Area V and compare it to song from the two closest breeding populations. The early 2010 song contained at least four distinct themes; these matched four themes from the eastern Australian 2009 song, and the same four themes from the New Caledonian 2010 song recorded later in the year. This provides evidence for at least one of the hypothesized mechanisms of song transmission between these two populations, singing while on shared summer feeding grounds. In addition, the feeding grounds may provide a point of acoustic contact to allow the rapid horizontal cultural transmission of song within the western and central South Pacific region and the wider Southern Ocean. Male humpback whales produce a long, stereotyped and constantly evolving vocal breeding display, termed ‘song’ Within an ocean basin, populations in closer proximity to each other typically display a higher degree of song similarity Typically, humpback whales from different ocean basins sing distinctly different songs see . DespitePayne and Guinee Although high-latitude song is, compared to that on breeding grounds, relatively uncommon, such singing has been observed in spring, summer or autumn in the North Atlantic A number of humpback whale populations have been recognised around the world due to strong feeding and/or breeding ground site fidelity e.g.,e.g.,e.g.,Linkages between breeding and feeding grounds have been demonstrated in the South Pacific using ‘Discovery marks’, small metal cylinders shot into whales during whaling and subsequently recovered when whales were later killed Song sharing within the western and central South Pacific is very dynamic  Here we report the first recorded occurrence of humpback whale song in Antarctic Area V, south of the eastern Australian and New Caledonian breeding populations and their shared migratory corridor, New Zealand. Although song was available from only faint singers, we were able to identify the song as being humpback whale in its origin, and then to compare the song from both eastern Australia and New Caledonia in the previous and following breeding seasons, in an attempt to acoustically link breeding to feeding grounds.RV TangaroaIshmael software Ishmael software During the dedicated Australia-New Zealand Antarctic Whale Expedition on e.g., ‘ascending cry’, ‘moan’, ‘purr’; see Songs were viewed in Abode Audition (2.0 and CS5.5) using Blackman-Harris, 75% overlap, 2048 point fast Fourier transform (FFT) for the eastern Australian and New Caledonian recordings and 1024 point FFT for the Antarctic Area V recordings (to ensure a comparable frequency resolution), displaying approximately 30 seconds of song from 0–2.5 kHz. The nature of the Antarctic recording permitted only qualitative analysis of the song. First, to identify if the sounds were humpback whale song, all sound units were examined to see if there were any repeating patterns. Each sound type (‘unit’) was assigned a descriptive name based on the visual and aural qualities of the sound  were asked to match a number of themes. Ten themes in total (a reference set) were chosen from the song types presented in the current study. These were displayed  in Raven Pro 1.4  to allow the observers to both visually and aurally assess each theme. Each observer was given the test set containing 20 themes and was asked to assign each sample with a theme number or, in the case of one sample, no matching theme. The observers classified 89%, 90% and 95% of themes in a similar manner, resulting in an average 91% agreement in classification. Mismatches were between themes M1  and M2 , and through the evolution of the eastern Australian 2010 theme H to include ‘whoops’ in the squeak series. All matches pertaining to the 2009 eastern Australian, New Caledonian 2010 and Antarctic Area V themes resulted in 100% agreement in classification. Humpback whale song themes are known to be highly variable with a large number of sound types that can be combined in many different ways. For example, Oceania song over the period 1998 to 2008 has had at least 93 phrase types Due to the repetition of a stereotyped sequence of species-typical sounds, the recorded sounds from Antarctic Area V were identified as humpback whale song. At least four stereotyped phrases and thus themes (labelled H to M) were identified ; these qFour themes from the Antarctic Area V sample were seen repeatedly  matched between the eastern Australia 2009 and 2010 song  and 5. AHere we have presented the first example to our knowledge, of humpback whale song recorded on the Antarctic feeding grounds in the waters of eastern Antarctica. Themes identified from the Antarctic Area V recordings matched song themes from the two closest breeding populations which are likely to feed in this region, eastern Australia and New Caledonia. While feeding grounds along the coasts of North America (Pacific and Atlantic) are more accessible for song recording, the Southern Ocean (particularly away from the Western Antarctic Peninsula) is significantly more challenging as a study area, and this is why there is no previous record of humpback whale singing activity from this region. The opportunistic song recording documented here, and the high density of whales present near the Balleny Islands i.e., between-season movement, shared migration routes and summer feeding grounds); we merely present that song relevant to the surrounding populations is being produced on the summer feeding grounds, the pattern of song exchange indicates song was first present in eastern Australia before being recorded in Antarctica and then New Caledonia, and that this is the first time song has been recorded in the waters of eastern Antarctica.Payne and Guinee The sample sizes included in this study are relatively small. The sample from New Caledonia in 2009 contained three singers only , suggesti.e., the song type does not match within a season), it is likely that song was first learnt by the eastern Australian population and then transmitted at a later point in time to the New Caledonian population , at which point the two populations’ songs diverged. This could occur through the divergence of migratory streams, or the movement of a group of animals away from a shared feeding aggregation . Of particular note is the photo-identification recapture between New Caledonia (taken in 2007) and the Balleny Islands, taken on 1st March 2010 as part of the same expedition The 2009 New Caledonian song type was clearly different to the 2009 and 2010 eastern Australian, 2010 New Caledonian and 2010 Antarctic Area V song type. The New Caledonian 2010 song shared all six themes with the eastern Australian 2009 song. The Antarctic sample included theme I , and theme K , indicating it was more related to the 2009 eastern Australian/2010 New Caledonian song. Due to the time lag in song matching between eastern Australia and New Caledonia The present study suggests a plausible mechanism of song transmission that has major implications for our understanding of song similarity between seemingly geographically separated populations. The movement of individuals (facilitating song exchange), coupled with song transmission on the feeding grounds, may permit a rapid transfer of song across ocean basins and the circumpolar feeding grounds. Song has been introduced into the eastern Australian population from the western Australian population At the broader scale it is plausible that the movement of individuals, or song transmission on the circumpolar distribution of feeding grounds in the Southern Ocean, may allow the movement of different song types from one area to the next. The movement of different versions (song types) of this acoustic sexual display is a clear example of large-scale population-wide horizontal cultural transmission in a non-human animal Acoustic File S1Antarctic Area V 2010 song. A single representative phrase is provided for each theme and is the corresponding audio file for (WAV)Click here for additional data file.Acoustic File S2Eastern Australia 2009 song. A single representative phrase is provided for each theme and is the corresponding audio file for (WAV)Click here for additional data file.Acoustic File S3New Caledonia 2010 song. A single representative phrase is provided for each theme and is the corresponding audio file for (WAV)Click here for additional data file.Acoustic File S4Eastern Australia 2010 song. A single representative phrase is provided for each theme and is the corresponding audio file for (WAV)Click here for additional data file.Acoustic File S5New Caledonia 2009 song. A single representative phrase is provided for each theme and is the corresponding audio file for (WAV)Click here for additional data file.


Surgical ventricular restoration (SVR) after anterior myocardial infarction with post infarction aneurysm, significantly improves left ventricular (LV) performance. To describe mid-term postoperative changes of shape and performance of the newly created LV and mitral valve function through the classical (SI) and apical (ASI) left ventricular sphericity index, and determinants of mitral regurgitation.A total of 26 consecutive patients underwent SVR between November 2002 and January 2004 at our institution. Transthoracic echocardiogram (TTE) was performed before, 3.1 months).±immediately postoperatively and during follow up . EDV, ESV and EF improved , without significant change in E/A and DT  but with improvement in IMP±69,99 to 209,64±0,74 NS; 207,54±1,29 0.047).There was a statistically significant<0,15 p±0,21 to 0,501± and ASI  as in the decrease in degree of MR severity<0,13 p±0,10 to 0,67±0,76 0.018).SI<0,49 p±1,13 to 0,17±immediately postoperatively (0,76 and ASI enable simple and clinically useful definition of complex morphological and functional changes of the distal part of the left ventricle, as preoperatively, and postoperatively, after the SVR and formation of the new LV apex.


Methamphetamine (meth) is a stimulant used illegally around the world, including in Iran. Cardiomyopathy and cardiac failure may occur following chronic meth use and may cause the patients referred to the emergency department.A 28-year old man and two women, ages 29 and 31-year-old, with a history of meth use, were admitted to the emergency department with severe dyspnea at rest. Each had sinus tachycardia with tachypnea and an echocardiogram that showed severe systolic dysfunction consistent with heart failure. Additional evaluation in the hospital revealed cardiomyopathy with no other etiology other than the meth use.There are several reports that show an increase in frequency of meth use, suggesting that cardiomyopathy and acute heart failure may be a new medical concern. Substance misuse is a major health problem in all parts of the world. Methamphetamine (meth) is a synthetic amine stimulant that is a highly addictive stimulant, and is currently the most widespread illegal amine drug used in the United States . Its useChronic use results in a number of medical complications and fatalities . Meth diThree patients, a 28-year-old man, and a 29 and 31-year-old woman, were admitted separately in Loghman Hakim Hospital, a referral and tertiary care medical center in Tehran, Iran. All had a chief complain of severe dyspnea at rest. The 28-year-old man also complained of exertional chest pain which was typical ischemic chest pain. On arrival, all of them were conscious and followed commands. None of them had any underlying disease and the only pertinent positive in their medical history was meth use, which was supported by a urinary drug screen. The duration of meth use in the man was one year and was 2 and 3 years in the 29 and 31-year-old woman, respectively.Vital signs on arrival included: BP: 90/70 mmHg, 100/60 mmHg and 90/60 mmHg, PR: 120, 104 and 110 beats/min and RR: 26, 32 and 22 breaths/min, respectively. A chest radiograph showed pulmonary congestion and an electrocardiogram (ECG) revealed sinus tachycardia, poor R wave progression in the pericardial leads, and T wave inversions in precordial leads in all three patients. Also, the second patient showed right axis deviation (135°) and the third patient had QS wave in the inferior leads. Measurement of cardiac troponin, evaluation of renal and thyroid function tests, measurement of serum electrolytes, blood lipids and a complete blood cell count (CBC) are shown in Table The 28-year-old man underwent coronary angiography due to concomitant chest pain, which was normal. All three patients underwent two-dimensional echocardiography with Doppler during the initial evaluation, and revealed severe decrease in left ventricular ejection fraction (LVEF) in each.The patients were treated with angiotensin-converting enzyme inhibitors (ACEIs), beta-adrenergic antagonists, diuretics and digoxin and were discharged after 7, 10 and 20 days hospitalization. At the time of discharge, the 2 women were classified as having New York Heart Association class I (NYHA-I) and the man NYHA-III heart failure . OutpatiWritten informed consent was obtained from the patients for publication of this report and any accompanying images.Meth is relatively easy to manufacture making its production inexpensive and widely available. Its low cost and long duration of action have made it a very desirable drug for use . In addiMeth affects multiple organs, including the cardiovascular system . One repDiagnosing the etiology of dyspnea can be difficult, in part because several disorders may coexist. However, our patients were young and had no underlying medical problems except for meth use. The clinical evaluation found only left ventricular systolic dysfunction in all three patients. Although endomyocardial biopsies were not performed, the failure of clinical or echocardiographic improvement over time supports the diagnosis of meth-associated cardiomyopathy.The most probably mechanisms for meth cardiotoxicity relates to the potent central and peripheral sympathomimetic effects of meth . The incAs in our cases, patients with meth-associated cardiomyopathy have a significantly lower LVEF or more severe ventricular dilation when compared with patients with cardiomyopathy from other causes ,20. As tRecognition of cardiomyopathy and acute heart failure as a complication associated with the meth use may be a new medical concern.The authors declare that they have no competing interests.All of the authors had the same contribution in article. All authors read and approved the final manuscript.


Two weeks after QSYQ treatment, the following variables were assessed: myocardial blood flow (MBF) by Laser-Doppler Perfusion Imager, the ratio of heart weight to body weight (HW/BW), myocardial histology, myocardial content of ATP, AMP, free fatty acids (FFAs) and AMP/ATP by ELISA, and expression of PPARα, PGC-1α, and ATP 5D by Western blot. Statistical analysis was performed using one-way ANOVA followed by Turkey test for multiple comparisons. DOX challenge significantly increased left ventricular internal diameter and HW/BW and decreased the thickness of the left ventricular posterior wall, the left ventricle ejection fraction, and the left ventricle fractional shortening. DOX also increased AMP, FFA, and AMP/ATP, decreased ATP, and downregulated the protein content of ATP 5D, PPAR α, and PGC-1 α. All these DOX-induced cardiac insults were attenuated significantly by QSYQ treatment. These results show the potential of QSYQ to ameliorate DOX-induced disorders in cardiac structure and function; this effect may be related to the increase in myocardial ATP content via the upregulation of ATP 5D, PPAR α, and PGC-1 α and the oxidation of FFA.QiShenYiQi Pills (QSYQ) is a compound Chinese medicine used for treatment of cardiovascular diseases. The present study investigated the effects of QSYQ on the Doxorubicin- (DOX-) induced disorders in rat cardiac structure and function and the possible mechanism underlying. A total of 24 male Sprague-Dawley rats were administrated by intraperitoneal injections with DOX at a dose of 2.5 mg/kg, once every day for a total of 6 times. After the 6th injection, the rats were evaluated by echocardiographic analysis, and the animals with injured heart ( Cancer and cardiovascular diseases (CVDs) have become the main cause of death for the adults in China . More imα (PPAR α) in kidney [Doxorubicin (DOX), an anthracycline antibiotic , is one n kidney . These rα is known to mediate the expression of cardiac fatty acid oxidation (FAO) enzyme gene, while peroxisome proliferator-activated receptor-γ coactivator-1 α (PGC-1 α) is the essential regulator for cardiac metabolism and function. Coactivation of PGC-1 α and PPAR α can regulate FAO [δ (encoded by ATP 5D), as one of the subunits of ATP synthase, is critical for ATP synthesis [The activation of PPAR late FAO , which pynthesis . Howeverα, and PGC-1 α in rat heart.QSYQ is a compound Chinese medicine composing of Radix Astragali (RA), Salvia miltiorrhiza (SM), Panax notoginseng(PN) and rosewood. In 2003, QSYQ was approved for treating coronary heart disease and angina by the Chinese State Food and Drug Administration. Our laboratory has proved that QSYQ ameliorates pressure overload-induced cardiac hypertrophy, myocardial fibrosis, myocardial blood flow, and cardiac function . Zhang eMale Sprague-Dawley (SD) rats weighing 189 ± 12 g were purchased from the Animal Center of Peking University Health Science Center . The animals were housed in cages at 22°C ± 2°C and humidity of 40%  ± 5% under a 12-hour light/dark cycle, received standard diet and water ad libitum. The animals were fasted for 12 h before the experiment, while allowing free access to water. The experimental procedures were carried out in accordance with the European commission guidelines (2010/63/EU). All animals were handled according to the guidelines of the Peking University Animal Research Committee. The protocols were approved by the Committee on the Ethics of Animal Experiments of the Health Science Center of Peking University (LA2011-67).α and PGC-1 α were purchased from Abcam, Inc. . The antibody against ATP 5D was purchased from Santa Cruz Biotechnology . All other reagents were of reagent grade.QiShenYiQi Pills  was obtained from Tasly Pharmaceutical Co., Ltd. , 0.5 g per pouch. Doxorubicin Hydrochloride for Injection  was purchased from Pfizer Pharmaceuticals Ltd. , one bottle containing 10 mg of Doxorubicin Hydrochloride, 50 mg of lactose, and 1 mg of methylparaben. Isoflurane was purchased from Jiupai Pharmaceutical Co. Ltd. of HeBei . Tetramethylethylenediamine and ammonium persulfate were purchased from Sigma-Aldrich Co. . Supper ECL Plus was purchased from Pierce Biotechnology, Inc. . RIPA tissue lysate was purchased from Cell Signaling Technology, Inc. . Protease inhibitor was obtained from Merck Drugs & Biotechnology Co. . Protein marker was purchased from MBI Fermentas . Whole protein extracting kit was purchased from Bio-Rad Laboratories, Inc. . BCA protein assay kit was purchased from Bio-Rad Laboratories, Inc. . ELISA kit for ATP, AMP, and free fatty acid (FFA) of rat was purchased from R & D Systems, Inc. . The antibodies against PPAR n = 6), Control 4W (n = 6), DOX 2W (n = 14), DOX 4W + NS (n = 7), and DOX 4W + QSYQ group (n = 7). For the last three groups, twenty-four rats were administrated with DOX  by intraperitoneal injection once every other day for a total of 6 times, as described previously by others [Rats were randomly divided into 5 groups: Control 2W  with a 17.5 MHz linear array transducer (model 716). The following parameters were measured as indicators of cardiac function and remodeling: left ventricular internal diameter at end-diastole (LVIDd), left ventricular internal diameters at end-systole (LVIDs), left ventricular posterior wall at diastole (LVPWd), left ventricular posterior wall at systole (LVPWs), left ventricle ejection fraction (%EF), and left ventricle fractional shortening (%FS) .2 with the software LDPI win 3.1  [Images of MBF in the territory supplied by left anterior descending coronary artery were acquired by Laser-Doppler Perfusion Imager  equipped with a computer and evaluated on an area of 3 × 4 mm Sweden) .Rats were killed at the end of experiment, and the hearts were removed and washed with normal saline. Both body weight (BW) and heart weight (HW) were determined by an electronic balance , and the ratio of HW to BW (HW/BW) was calculated .μm) were rehydrated and stained with hematoxylin and eosin (HE). The images were captured by a digital camera connected to a stereo microscope  and an optical microscope  and processed with the software Image-Pro Plus 5.0 .Hearts were removed at the end of the experiment, fixed in 4% formaldehyde, and further prepared for paraffin sectioning. The paraffin sections  for 1 h at 37°C and washed with PBS. To label nucleus, the sections were incubated with Hoechst 33342  . Section3 was removed from the heart for the assessment of the concentrations of ATP, AMP, and FFA in myocardial tissues by ELISA kits according to the manufacturer's protocol. OD values were determined by enzyme microplate reader , with a detection wave length of 450 nm. The concentrations of ATP, AMP, FFA, and AMP/ATP were calculated based on the standard curves.At the end of the experiment, the rat was perfused with NS, and a tissue block about 2 mmn = 3). The whole protein was extracted. The concentration of whole protein was detected in duplicate with BCA protein assay kit according to instruction, and the mean values were computed. All the concentrations of whole proteins were adjusted to the lowest concentration detected and the samples were preserved at −80°C.A piece of about 200 mg of tissue was cut from the heart of each animal and preserved at −80°C (α (1 : 1000), PGC-1 α (1 : 500), and ATP5D (1 : 200). Following rinsing, the membranes were incubated for 1 h at room temperature with respective HRP-conjugated secondary antibody. The membranes were developed with ECL, exposed in dark box, and the protein signal was quantified by scanning densitometry in the X-film by bioimage analysis system . The result of each group was expressed as relative optical density compared with that from Control group. The whole protein was separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat dry milk or 3% BSA and, after washing, incubated overnight at 4°C with primary antibody against PPARAll parameters were expressed as mean ± S.E. Statistical analysis was performed using one-way ANOVA followed by Turkey test for multiple comparisons. A probability of less than 0.05 was considered to be statistically significant.The results of echocardiography analysis in various groups are displayed in Figures The ATP, AMP, FFA content, and the ratio of AMP/ATP were determined by ELISA at the end of the experiment in different groups . In compTo explore the cause of the observed change in ATP content, the expression of ATP 5D was detected by Western blots. As shown in α and PGC-1 α in myocardial tissues from different groups at the end of the experiment (α and PGC-1 α in DOX 4W + NS group were apparently decreased compared with Control 4W, while these decreases restored significantly in the DOX 4W + QSYQ group.Western Blotting was undertaken to assess the expression levels of PPAR periment . As notiα and PGC-1 α in the DOX-injured myocardium cells, facilitates the oxidation of fatty acid, degrades myocardial free fatty acid, and finally increases the content of ATP.The present study revealed that the intraperitoneal injection of DOX for 2 weeks in rats leads to an increase in LVID and HW/BW, a reduction of LVPW and LVPW, and a decrease in EF and FS. In addition, DOX caused reduction of ATP synthase subunit ATP 5D, the degradation of myocardial F-actin, and myocardium rupture. All the DOX-induced alterations can be evidently ameliorated by the administration of QSYQ for 2 weeks, suggesting the potential of QSYQ to relieve the DOX-induced cardiac insufficiency. In addition, we also found that QSYQ promotes the expression of PPAR Previous studies reported that intraperitoneal injection of DOX in rats provokes cardiomyopathy, exhibiting as a larger LVIDS and a thinner LVPW . In the α/PGC-1 α complex [α/PGC-1 α expression. These results suggest that QSYQ increases myocardial fatty acid beta-oxidation and ATP content and improves myocardial energy metabolism and cardiac function most likely through interference in PPAR α/PGC-1 α pathway.Fatty acid beta-oxidation takes place in mitochondrial matrix, a process that constitutes the major source of energy for myocardial cell activity . Numerou complex , with PG complex . In line complex . Importaδ which contributes to the synthesis of ATP [ATP 5D is the gene encoding ATP synthase subunit s of ATP . HoweverF-actins constitute myocardial thin filaments, which, alone with thick filament, are responsible for the actin-based myofilament motility . We demoHeart ejection function depends on cardiac energy supply and normal myocardial structure. We demonstrated that DOX caused cardiac energy depletion and myocardial structure damage, which contribute to the reduced heart ejection and perfusion function. QYSQ could restore the cardiac energy, myocardial structure and improve myocardial blood flow and cardiac function implying QYSQ as a promising remedy for reducing the adverse effects of DOX in clinic. α/PGC-1 α and fatty acid oxidation, reducing myocardial FFA and increasing ATP level. This result suggests QSYQ as a potential management to cope with the obstacle that DOX confronts in clinical use, and provides insight for better understanding the mechanism behind the QSYQ effect. Nevertheless, the detailed mechanisms thereby QSYQ protects heart from injury by DOX need further clarification, and mor studies, particularly using lager animals, are required to verify the feasibility for QSYQ application in clinic. In summary, QSYQ is able to ameliorate DOX-induced myocardial structure injury and cardiac dysfunction. This beneficial role of QSYQ is correlated with its potential to modulate energy metabolism, involving upregulating PPAR


Aortic stenosis (AS) affects 3 percent of persons older than 65 years and leads to greater morbidity and mortality than other cardiac valve diseases. Surgery with aortic valve replacement (AVR) for severe symptomatic AS is currently the only treatment option. Unfortunately, in patients with poor ventricular function, the mortality and long-term outcome is unsatisfied, and only a minority of these patients could bear surgery. Our previous studies demonstrated that vascular endothelial growth factor (VEGF) protects cardiac function in myocardial infarction model through classic VEGF-PI3k-Akt and unclear mitochondrial anti-apoptosis pathways; promoting cardiomyocyte (CM) proliferation as well. The present study was designed to test whether pre-operative treatment with VEGF improves AS-induced cardiac dysfunction, to be better suitable for AVR, and its potential mechanism.Adult male mice were subjected to AS or sham operation. Two weeks later, adenoviral VEGF (Ad-VEGF), enhanced green fluorescence protein  or saline was injected into left ventricle free wall. Two weeks after delivery, all mice were measured by echocardiography and harvested for further detection.AS for four weeks caused cardiac hypertrophy and left ventricular dysfunction. VEGF treatment increased capillary density, protected mitochondrial function, reduced CMs apoptosis, promoted CMs proliferation and eventually preserved cardiac function.Our findings indicate that VEGF could repair AS-induced transition from compensatory cardiac hypertrophy to heart failure. Aortic stenosis (AS) is the most common cardiac valve disease, affecting about 3 percent of persons older than 65 years. Although the survival rate in asymptomatic patients is comparable to that in age- and sex-matched control patients, the average overall survival rate in symptomatic patients is 2-3 years . For patAs the aortic valve area becomes smaller, the increased afterload on the left ventricle (LV) results in compensatory hypertrophy, which enables it to maintain systolic function. However, with time and sustained severe pressure overload, the LV dilates with impairment of contractile state and subsequent dysfunction. Although the molecular mechanisms involved in the transition from compensated hypertrophy to heart failure are poorly understood, the fundamental hypothesis is that, according to the nature of signaling stimulus, the cardiomyocytes (CMs) can either survive, leading to beneficial hypertrophy, or undergo apoptosis (programmed cell death), which promotes LV failure and dilation .Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which has been recognized to have both angiogenenic and nonangiogenic role for cardiovascular system. VEGF regulates multiple cellular stress responses, including survival, proliferation, migration, and differentiation. Our previous studies have shown that VEGF could facilitate CMs regeneration and protect it from apoptosis which was related with the activation of phosphatidylinositol-3 kinase (PI-3K) and the upregulation of Bcl-2 expression . RecentlTake advantage these features of VEGF, we hypothesized that pre-operative VEGF treatment could improves the AS patients' condition, especially those with severe cardiac hypertrophy, to avoid the worsening of LV function and better suitable for AVR. Thus, it is important to test this hypothesis in an animal model of AS, and the results may be useful in designing and justifying future clinical trials.ad libitum standard mouse food pellets and tap water, and housed in groups of four to five mice with 12:12 hour light-dark cycles.The experiment protocols were approved by Animal Care and Use Committee of Nanjing Medical University. Ten-week-old male C57BL/6 mice were obtained from the Experimental Animal Center of Nanjing University . Animals were fed 8 plaque-forming units of Ad-VEGF or Ad-EGFP into left ventricle free wall two weeks after aorta ligature.Recombinant human adenoviral vectors are the most efficient gene delivery vehicles currently used for gene transfer in preclinical gene therapy models and in clinical cardiovascular gene therapy protocols because of the ease of their production and the broad cell tropism, particularly within the cardiovascular system which makes them widely used in myocardial gene therapy. All major cardiac cell types can be efficiently transduced by adenoviral vectors, both in vitro and in vivo. With regard to CMs, efficient in vivo transduction has been demonstrated in gene therapy models from several mammalian species . In our Mice were randomly subjected to either aorta ligature-induced AS (n = 40) or sham operation (n = 10). Surgical mortality rates were 20% or 0%, respectively, for AS or sham operations. Two weeks after thoracic aortic constriction (TAC) (compensatory hypertrophy phase in this model), AS animals underwent midline sternotomy and further assigned to three groups: i) saline injected hypertrophied hearts (TAC group), ii) Ad-EGFP injected hypertrophied hearts (EGFP group), iii) Ad-VEGF injected hypertrophied hearts (VEGF group). Sham group mice also did midline sternotomy but no injection. Two weeks after viral delivery, all mice were examined by echocardiography and killed. The heart wet weight to body weight ratio and to tibia length ratio were calculated. Heart samples were frozen in liquid nitrogen and then stored at -80°C until analysis. Additional heart samples were used for electron microscopy and histological evaluation.Aortic stenosis was created by aorta ligature in accordance with method of transverse aortic constriction . In brieTwo weeks after aorta ligature and viral delivery, the mice were undergone cardiac function assessment by transthoracic echocardiography with 12-MHz phased-array transducer (Hewlett Packard). The heart was imaged in the cross-sectional mode in parasternal long- and short-axis views of the LV. Average interventricular septum diameter (IVSd), LV posterior wall thickness (LVPW), LV ejection fraction (LVEF) and LV fractional shortening (FS) were measured from three consecutive cardiac cycles. All measurements were done by two experienced echocardiographer who were blinded to treatment assignment.Tissue samples were taken from left ventricles, fixed with 4% formalin, embedded in paraffin, and cut into 3 μm thickness. Hematoxylin-eosin (HE) staining were performed using serial sections. CM cross-sectional area was measured by tracing the outlines of 100-200 CMs with a clear nucleus image per each heart using hematoxylin-eosin stained sections.For measurement of capillary density, sections taken perpendicular to the long axis of the LV were immunohistochemically stained with a specific primary antibody against von Willebrand Factor (vWF) . Capillary density was defined as the capillary to cardiomyocyte ratio.To detect whether VEGF promotes cardiomyocyte proliferation, immunohistochemical analysis was performed for Ki-67 . Only nuclei that were clearly located in cardiomyocytes were counted.Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay using a POD TUNEL kit . Apoptotic nuclei were identified manually to determine that only apoptotic cardiomyocyte nuclei were included. The number of TUNEL-positive cells was expressed as a percentage of total cells.3VO4), 1% (vol/vol) Triton X-100, 10% (vol/vol) glycerol, 1 mM dithiothreitol, 1 mM PMSF, and a protease inhibitor cocktail . The total protein homogenate (20-50 μg) was separated by SDS-PAGE and transferred onto PVDF membranes. The expression levels of important signaling molecules, VEGF, and apoptosis-related proteins were detected using antibodies against OPA1, Bax, Bcl-xL, Akt and p-Akt from Cell Signaling Technology.The LV tissue was homogenized with lysis buffer (pH 7.4) containing 25 mM Tris, 150 mM NaCl, 5 mM EDTA, 10 mM sodium pyrophosphate, 10 mM b-glycerophosphate, 1 mM sodium orthovanadate (NaStandard transmission electron microscopy (EM) was performed as previously described . DigitalData were analyzed using SPSS software package  and are reported as mean ± standard error of the mean. One-way ANOVA was used for comparison among and between groups, or Kruskal-Wallis test if normality was not passed, followed by Bonferroni or Dunn post-hoc analysis when appropriate. Values of P < 0.05 were considered statistically significant.P < 0.001) and similar LV fractional shortening (LVFS), LV ejection fraction (LVEF) (P = 0.92) compared with sham operated mice, indicating compensatory cardiac hypertrophy and normal systolic function . Treatment with Ad-VEGF prevented the reductions of LVEF and LVFS (P < 0.05), with no significant difference in LVPW, IVSd, heart weight/body weight ratio and heart weight/tibia length ratio, as compared to TAC animals , interventricular septal thickness (IVSd) P < 0.00 and simi) P = 0.9 compareds Figure .P < 0.05). VEGF treatment revealed an augmentation of neovascularization after induction of AS. In the VEGF group we observed a 45% increase in capillary density relative to TAC group (P < 0.01). The differences between TAC and EGFP group were not statistically significant.Figure P < 0.05, Figure P < 0.01). However, the numbers of TUNEL positive cells did not differ significantly between the TAC and EGFP groups.The number of TUNEL positive cells was significantly higher in mice with AS, compared to sham (P < 0.01). Additionally no significant difference was found between TAC and EGFP group.Immunostaining for the cell proliferation marker Ki-67 was used to confirm VEGF-induced CMs regeneration. Induction of AS resulted in a 2 fold increased in the expression of Ki-67 in the TAC group Figure . VEGF siEM was used to analyze mitochondrial fission and fusion changes in AS induced heart failure. The mitochondria in the TAC mice heart were disorganized and smaller Figure . The absP < 0.05). To investigate whether VEGF could activate PI3K-Akt signaling in the myocardium, we examined the level of phospho-Akt and Akt in the myocardium. As shown in Figure P < 0.05). Thus, Ad-VEGF injection increased expression of VEGF and phospho-Akt in myocardium.After 2 weeks injection of adenovirus containing VEGF in hypertrophied hearts, western blot analysis showed that levels of VEGF were increased in VEGF group compared with that in other groups (Fig AS-induced LV hypertrophy is an initially expected response in order for the CMs to generate additional force to overcome the increase in pressure load. The initial response may become decompensated when CMs degenerate as a result of inflammatory response, oxidative stress, apoptosis, fibrosis and progressive LV dilation with a progressive decline in cardiac pump function. In the present study, we found that two weeks AS mice presented cardiac hypertrophy and normal cardiac function. Whereas, at 4 weeks after aorta ligature, remarkable pulmonary congestion and LV dysfunction were exhibited in the TAC mice, underwent a transition from compensatory cardiac hypertrophy to heart failure. VEGF injection in compensatory hypertrophy heart was found to attenuate LV remodeling and to improve cardiac function through increased capillary density, preserved mitochondrial function, promoted CMs proliferation, as well as reduced CMs apoptosis.It is well known that myocardial apoptosis has been shown to be a critical determinant of unfavorable LV remodeling and play an important role in the progression of AS. However the underlying mechanisms by which the heart loses CMs in heart failure are not completely understood .One important component of the myocardial remodeling process is neoangiogenesis. After AS, neoangiogenesis is normally unable to compensate for the blood supply and to support the tissue growth required for contractile compensation and the greater demands of the hypertrophied myocardium; this may contribute to the death of myocardium, leading to progressive CMs apoptosis and secondary fibrosis replacement. Our study suggested an increase in angiogenesis in VEGF animals, and this may contribute importantly to the reduction in remodeling and apoptosis. Ferrata  demonstrMitochondria also have a critical role in regulating apoptosis. CMs function at a high metabolic state, requiring large amounts of high-energy phosphates, and as a consequence, mitochondria are abundant in these cells ,13. MitoPhosphoinositide 3-kinase and its downstream target serine/threonine kinase Akt are also recognized as another most critical pathways in regulating CMs activation, inflammatory responses and apoptosis ,19. ActiIn current, myocardial regeneration has become the hotspot and challenge of clinical treatment for heart failure. It may offer possibilities that could supplement apoptosis-conduced CMs shortage and maintain the absolute numbers of CMs, as well as improve cardiac function. Evidence is accumulating to suggest that VEGF exerts potent pleiotropic effects on the myocardium in the setting of acute myocardial infarction and chronic heart failure as well. In our previous study we demonstrated that overexpression VEGF could mobilize marrow stem cell and accelerate CMs regeneration in myocardial infarction model . FurtherIn summary, the results of the present study using the mouse model of AS bear out the cardioprotective, angiogenic, proliferative, and anti-apoptotic effects of VEGF and its possible molecular mechanism. It is known that reduced LV ejection fraction and increased LV cavity size before AVR are associated with poor postoperative recovery. Although the correlation to human or clinical data remains to be proved, it is our anticipation that the number of AS patients who are suitable for AVR will be increased, the mortality following AVR will be decreased, and postoperative recovery will be improved if VEGF pre-operative treatment can be demonstrated to be effective in clinical trials.AS: Aortic stenosis; AVR: Aortic valve replacement; VEGF: Vascular endothelial growth factor; CM: Cardiomyocyte; Ad-VEGF: Adenoviral VEGF; Ad-EGFP: Adenoviral enhanced green fluorescence protein; LV: Left ventricle; PI-3K: Phosphatidylinositol-3 kinase; IVSd: Interventricular septum diameter; LVPW: LV posterior wall thickness; LVEF: LV ejection fraction; FS: LV fractional shortening; HE: Hematoxylin-eosin; vWF: Willebrand Factor; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick-end labeling; EM: Electron microscopy.The authors declare that they have no competing interests.XHX, JX and YJC participated in the design of the study and coordination, LX and HLC participated in the data collect and modified the manuscript, XL performed the statistical analysis and helped to draft and modified the manuscript. All authors read and approved the final manuscript.


S)-PA-824 has potent antibacterial activity against Mycobacterium tuberculosisin vitro and in vivo and is currently in phase II clinical trials for tuberculosis (TB). In contrast to M. tuberculosis, where (R)-PA-824 is inactive, we report here that both enantiomers of PA-824 show potent parasiticidal activity against Leishmania donovani, the causative agent of visceral leishmaniasis (VL). In leishmania-infected macrophages, (R)-PA-824 is 6-fold more active than (S)-PA-824. Both des-nitro analogues are inactive, underlining the importance of the nitro group in the mechanism of action. Although the in vitro and in vivo pharmacological profiles of the two enantiomers are similar, (R)-PA-824 is more efficacious in the murine model of VL, with >99% suppression of parasite burden when administered orally at 100 mg kg of body weight−1, twice daily for 5 days. In M. tuberculosis, (S)-PA-824 is a prodrug that is activated by a deazaflavin-dependent nitroreductase (Ddn), an enzyme which is absent in Leishmania spp. Unlike the case with nifurtimox and fexinidazole, transgenic parasites overexpressing the leishmania nitroreductase are not hypersensitive to either (R)-PA-824 or (S)-PA-824, indicating that this enzyme is not the primary target of these compounds. Drug combination studies in vitro indicate that fexinidazole and (R)-PA-824 are additive whereas (S)-PA-824 and (R)-PA-824 show mild antagonistic behavior. Thus, (R)-PA-824 is a promising candidate for late lead optimization for VL and may have potential for future use in combination therapy with fexinidazole, currently in phase II clinical trials against VL.The novel nitroimidazopyran agent ( Leishmania donovani, is the second largest parasitic killer after malaria, with more than 200 million people currently at risk from infection. In 95% of cases, death can be prevented by timely and appropriate drug therapy , caused by the protozoan parasite  therapy ; however therapy , concert therapy . Problem therapy . Thus, t therapy .http://www.dndi.org).In the search for more-effective treatments for the neglected diseases, there has been renewed interest in the chemotherapeutic potential of nitroheterocyclic compounds. This interest largely stems from the success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of the Gambian form of human African trypanosomiasis (HAT). Treatment with NECT, consisting of oral nifurtimox, a nitrofuran drug also used against Chagas' disease, combined with eflornithine infusions, has resulted in cure rates of around 97%, leading to its inclusion on the WHO Essential Medicines List . The sucMycobacterium tuberculosis, the causative agent of tuberculosis (TB), and is currently being tested in phase II clinical trials -PA-824 does not involve the trypanosomatid type I nitroreductase (NTR) that activates fexinidazole and nifurtimox.As a result of our studies with fexinidazole, we searched the literature for other nitroimidazoles which may have potential for use in the treatment of visceral leishmaniasis. In this process, we noted the promising antitubercular drug PA-824 , 13. Thil trials –16. HereLeishmania donovani cell line LdBOB (derived from MHOM/SD/62/1S-CL2D) was grown as either promastigotes or axenic amastigotes in medium specific for each developmental stage  enzyme  enzyme  were culTrypanosoma brucei brucei S427 29-13 were grown in SDM-79 medium supplemented with 10% fetal calf serum (FCS) and hemin (100 mg ml−1). T. brucei bloodstream forms (S427 Single Marker) were cultured at 37°C in HMI9T medium (Trypanosoma cruzi Silvio-X10/7 clone A1 epimastigotes were grown in RTH-FCS at 28°C (T. cruzi amastigotes were maintained in Vero cells (African green monkey kidney cells) cultured in minimal Eagle's medium (MEM) supplemented with 1× GlutaMax (Life Technologies) and 10% FCS. HepG2 cells (hepatocyte carcinoma cells) were grown in MEM supplemented with 1× GlutaMax, 1× nonessential amino acids (Life Technologies), and 10% FCS. All mammalian cells were grown at 37°C, 5% CO2, in a humidified incubator.Procyclic trypomastigotes of T medium . Trypano at 28°C . T. cruzS)- and (R)-PA-824 were prepared using a four-step sequence from 2-bromo-4-nitroimidazole (scheme S1). In addition, (S)-PA-824 was prepared in a single step as previously described (scheme S2) (S)- and (R)-Des-nitro-PA-824 were prepared in 5 steps according to the method of Singh et al. , with all compounds found to be of ≥99% purity. Optical rotation measurements of (S)-PA-824 were in close agreement with published values, confirming a high level of optical purity (R)-PA-824 was equal and opposite to that of the S enantiomer, confirming its optical purity. Full experimental protocols and analytical data for all compounds are given in the supplemental material.(heme S2) . (S)- anh et al.   were grown in the presence of drug for 72 h, after which 50 μM resazurin was added to each well (0.5 mM in the case of T. cruzi epimastigotes) and fluorescence (excitation of 528 nm and emission of 590 nm) was measured after a further 4 h of incubation. Drug sensitivities of bloodstream-form T. brucei were determined as previously described  and fitted to a 2-parameter equation, where the data are corrected for background fluorescence, to obtain the effective concentration inhibiting growth by 50% (EC50):To examine the effects of test compounds on growth, triplicate cultures were seeded with 1 × 10escribed . T. brucI] represents inhibitor concentration and m is the slope factor. Experiments were repeated at least three times, and the data are presented as the weighted mean plus weighted standard deviation . Briefly, T. cruzi trypomastigotes (X10/7) clone A1 were used to infect Vero cells in 384-well plates (Greiner) and then exposed for 72 h to compounds followed by fixation, staining with Hoechst 33342, and evaluation of infection using an Operetta high-content imaging system (PerkinElmer).Details of the 3 per well) were plated in 384-well white clear-bottomed plates (Greiner) and cultured in minimal Eagle's medium with 10% FCS for 24 h at 37°C in 5% CO2. Compound dilution curves were then added to plates directly using a Labcyte Echo 550 acoustic dispenser (25 nl). After compound addition, plates were incubated for a further 69 h. Resazurin was then added to each well at a final concentration of 50 μM, and fluorescence was measured (excitation of 528 nm and emission of 590 nm) after 1.5 h of incubation.HepG2 cells (2.5 × 10R)-PA-824, (S)-PA-824, and fexinidazole sulfone were tested in fixed ratios based on their respective EC50s. The EC50s of each combination were determined after 72 h and plotted as an isobologram  harvested from the spleen of an infected hamster (−1 subcutaneously), with miltefosine , or with either (R)- or (S)-PA-824 . Miltefosine and sodium stibogluconate were administered once daily for 5 days, with (R)- and (S)-PA-824 administered twice daily over the same period. Drug dosing solutions were prepared fresh each day, and the vehicle for both enantiomers of PA-824 was 10% (vol/vol) dimethyl sulfoxide (DMSO), 40% polyethylene glycol 400, and 50% deionized water. On day 14 postinfection, all animals were humanely euthanized and parasite burdens were determined by counting the number of amastigotes/500 liver cells (Leishmania donovani units (LDU), mean number of amastigotes per liver cell × mg weight of liver  Act 1986 in accordance with the European Communities Council Directive (86/609/EEC). Groups of female BALB/c mice (5 per group) were inoculated intravenously with approximately 2 × 10 hamster . From daer cells . Parasitof liver .R and S enantiomers of PA-824 (50 mg kg−1) were orally administered to BALB/c mice. Blood samples (10 μl) were collected from the tail vein of each animal into sample storage tubes (Micronic BV) containing deionized water (20 μl) at defined intervals postdose and stored at −80°C until analysis. The level of each enantiomer in mouse blood was determined by UPLC-MS/MS (The ed water  μl at deR)- and (S)-PA-824. The plasma protein binding (PPB) of the enantiomers of PA-824 and its des-nitro analogues was determined by the equilibrium dialysis method (int), each enantiomer of PA-824 (0.5 μM) was incubated with female CD1 mouse liver microsomes  and the reaction was started by the addition of excess NADPH (8 mg ml−1). At time zero and then at 3, 6, 9, 15, and 30 min, an aliquot (50 μl) of the incubation mixture was mixed with acetonitrile (100 μl) to stop the reaction. A structural analogue was then added to all samples as an internal standard, the samples were centrifuged to sediment precipitated protein, and the plates were sealed prior to UPLC-MS/MS analysis using a Quattro Premier XE spectrometer . XLfit  was used to calculate the exponential decay and consequently the rate constant (k) from the ratio of peak area of test compound to internal standard at each time point. CLint was then calculated using the following equation: CLint (ml min−1 [g liver]−1) = k × V × microsomal protein yield, where V (ml mg protein−1) is the incubation volume per mg protein added and microsomal protein yield is taken as 52.5 mg protein per g liver. Verapamil (0.5 μM) was used as a positive control to confirm acceptable assay performance.The software package StarDrop by Optibrium was used to calculate physical parameters, including log P, molecular weight, and polar surface area (PSA) for (s method . The aquS)-PA-824, currently in phase II development by the TB Alliance, was synthesized and assessed for antileishmanial activity. The potency of (S)-PA-824 was determined in vitro against L. donovani (LdBOB) promastigotes and against intracellular amastigotes in peritoneal mouse macrophages. (S)-PA-824 showed antileishmanial activity against both developmental stages of the parasite, with EC50s of 0.9 ± 0.1 and 4.9 ± 0.3 μM against promastigotes and amastigotes, respectively (S)-PA-824 was largely inactive against the mammalian stages of T. brucei and T. cruzi, with EC50s of >30 μM in both cases. (S)-PA-824 was also found to be inactive  in a counterscreen against the mammalian cell line HepG2.The promise shown by fexinidazole as an oral treatment for visceral leishmaniasis prompted us to investigate the possibility that other nitroheterocyclic compounds may also have potential in treating this disease. With this in mind, the antitubercular clinical candidate  (R)-PA-824 was synthesized and its antileishmanial activity was assessed. In direct contrast to that seen in M. tuberculosis, (R)-PA-824 proved to be a ∼5-fold-more potent inhibitor of L. donovani growth in vitro than the S-enantiomer candidate, with EC50s of 0.16 ± 0.03 and 0.9 ± 0.1 μM against promastigotes and intracellular amastigotes, respectively (R)-PA-824 also showed slightly improved activity against the mammalian stages of T. brucei and T. cruzi while remaining inactive against the mammalian cell line HepG2 .The >100 μM) ; howeverectively . Indeed,S)-PA-824 is well tolerated in mice and can reduce M. tuberculosis infection levels after oral dosing (R)-PA-824 was similarly suitable for in vivo studies, the plasma protein binding (PPB), aqueous solubility, and CLint were measured and compared to those of the S enantiomer. Both enantiomers had good aqueous solubility (>250 μM) and similar low mouse PPB, with the fractions unbound for (S)- and (R)-PA-824 being 0.21 and 0.22, respectively. The fraction unbound for the (R)-des-nitro-PA-824 is 0.45. (R)-PA-824 shows higher mouse microsomal stability than does the S enantiomer (CLint = 0.65 versus 1.6 ml min−1 g−1). The calculated values for the partition coefficient, log P (2.7), and polar surface area (91 Å2) were identical for the two enantiomers.A number of studies have reported that - or (R)-PA-824 at 50 mg kg−1 showed a maximum concentration in blood after 4 h of 11,600 or 10,500 ng ml−1, respectively (t1/2), ∼5 h and 2 h for (S)- or (R)-PA-824, respectively], reaching undetectable levels at a time point between 8 and 24 h for (R)-PA-824. At 24 h, (S)-PA-824 still had a blood concentration of 900 ng ml−1.Blood levels of both enantiomers of PA-824 were determined after oral dosing to establish a suitable dosing regimen for VL efficacy studies. Mice dosed with (ectively . ThereafR)-PA-824 has an EC50 of 0.93 μM for L. donovani cultured in macrophages . After adjusting for plasma protein binding [(R)-PA-824 fraction unbound = 0.22], the estimated blood concentration required to exceed the EC90 becomes 5,600 ng ml−1. Comparison with the measured levels (R)-PA-824 blood levels exceed the EC90 1 h after dosing and remain above the EC90 for at least 7 h. Thus, 100 mg kg−1 twice daily was predicted to provide adequate exposure for (R)-PA-824. Although (S)- and (R)-PA-824 reach comparable blood levels, the lower potency of (S)-PA-824 (EC90 = 22 μM) results in a maximal free concentration below EC90  after oral dosing at 50 mg kg−1 ((rophages  with a Hd levels  shows th mg kg−1 .R)- and (S)-PA-824 was assessed in a mouse model of visceral leishmaniasis. Seven days following infection with L. donovani LV9 ex vivo amastigotes, groups of BALB/c mice were dosed orally with (R)- or (S)-PA-824 (30 or 100 mg kg−1) twice daily and for five consecutive days. Fourteen days following inoculation, parasite burdens in the livers of infected mice were determined. Current antileishmanial chemotherapies sodium stibogluconate (15 mg kg−1) and miltefosine (12 mg kg−1) were included in the study for comparison. Both enantiomers of PA-824 were extremely well tolerated by mice throughout the study, with no overt signs of toxicity. Mice dosed twice daily at 30 mg kg−1 with (R)- or (S)-PA-824 demonstrated similar efficacies, with both enantiomers suppressing infection in the murine model by approximately 35% compared to untreated controls (−1did not improve the efficacy of (S)-PA-824 in vivo, with L. donovani infection in this group of mice at levels comparable to those seen in drug-free animals. An earlier in vivo study, with mice given a range of doses down to 0.4 mg kg−1, demonstrated the apparent absence of a dose-dependent leishmanicidal effect with the S enantiomer of PA-824 . Interestingly, these findings mirror those seen in clinical trials of (S)-PA-824 in humans with tuberculosis (R)-PA-824 at 100 mg kg−1 effectively cured the murine model of infection, suppressing infection by 99.9%. Dosing with (R)-PA-824 at this level proved to be superior to treatment with current front-line drugs sodium stibogluconate (41.9% suppression) and miltefosine (68.7% suppression), underlining the therapeutic potential of this nitroimidazo-oxazine compound.The efficacy of both (controls . Adminisrculosis . In starS)-PA-284 is believed to function as a prodrug which requires bioreductive activation prior to exhibiting antitubercular activity -dependent nitroreductase (Ddn). In the absence of a Ddn homologue in the trypanosomatids, we hypothesized that reduction of (R)- and (S)-PA-824 in L. donovani may be catalyzed by the NADH-dependent nitroreductase, which has homology to bacterial type I nitroreductases and has already been shown to play a crucial role in the activation of fexinidazole and its metabolites (S)-PA-824  or (R)-PA-824    and C. TR)- and (S)-des-nitro-PA-824 were determined against L. donovani promastigotes and intracellular amastigotes. In both assays, both enantiomers of des-nitro-PA-824 were found to be inactive at the highest concentration tested (50 μM), suggesting that the nitro group either plays a crucial role in the mechanism of action of PA-824 or mediates the binding between PA-824 and its molecular target(s) in L. donovani.To determine whether the nitro group was important for the antileishmanial activity of PA-824, the potencies of (in vitro interactions of (R)-PA-824 in combination with fexinidazole sulfone and (S)-PA-824 against L. donovani promastigotes using the fixed-ratio isobologram method (R)-PA-824 and fexinidazole sulfone combinations against promastigotes revealed a near-linear relationship, suggesting that these two compounds act additively in combination (R)- and (S)-PA-824 demonstrated a more complex relationship. As the proportion of (R)-PA-824 in the mixture increases, the two enantiomers become progressively more antagonistic. A second experiment gave an identical pattern.Recent studies have illustrated the potential of drug combinations in the treatment of visceral leishmaniasis . Multidrm method . Isobolobination . HoweverS)- or (R)-PA-824 at concentrations equivalent to 10 times their respective EC50s (S)-PA-824, no intact parasites were visible at 36 h -PA-824. In comparison, the more rapid cytotoxic effect of (R)-PA-824 resulted in parasites losing viability by 24 h. The fact that both enantiomers of PA-824 are leishmanicidal rather than cytostatic is highly beneficial, since successful drug therapy with either compound is unlikely to depend on a fully functional patient immune response -PA-824 had been reported to possess an undefined level of activity against L. donovani axenic amastigotes 11), and stigotes . Both M.S)-PA-824 is active against L. donovani intracellular amastigotes at a potency comparable to that of fexinidazole sulfone , an active metabolite of the VL clinical candidate fexinidazole. In contrast to the structure-activity relationship for M. tuberculosis, where the S enantiomer of PA-824 is >100-fold more active than the R enantiomer (R)-PA-824 was 5-fold more potent than the S enantiomer in the L. donovani intramacrophage assay. Similarly, (R)-PA-824 was also more active against the disease-relevant stages of T. brucei and T. cruzi but showed no measurable activity in a mammalian cell counterscreen. Both enantiomers of PA-824 were also found to be leishmanicidal in vitro with good selectivity, good microsomal stability, good aqueous solubility, and low plasma protein binding.In the current study, we have shown that . Indeed, under this treatment regimen (R)-PA-824 almost completely cleared L. donovani parasites from the livers of infected mice. When either (R)- or (S)-PA-824 was administered at 30 mg kg−1, there was only a moderate reduction in the observed parasite burden (∼35%). Although we have not established linearity of exposure with dose for either compound, this modest efficacy is consistent with the anticipated sub-EC90 free inhibitor blood concentration. Surprisingly, (S)-PA-824 becomes less efficacious when administered at 100 mg kg−1. We hypothesize that this unexpected reduction in potency may be formulation related.Both enantiomers were orally bioavailable and were well tolerated in mice. The pharmacokinetic profiles of the two compounds were similar, but only the http://www.dndi.org/) for VL calls for an oral drug that can be used to treat immunosuppressed patients. Significantly, (R)-PA-824 is cytocidal rather than cytostatic, which is advantageous because treatment with this compound would not be dependent on the patient having a fully functional immune response. In addition, (R)-PA-824 is orally available. Collectively, our study indicates that (R)-PA-824 represents a strong lead compound with potential for development as a much-needed oral treatment for VL. However, it is important to note that the S, not the R, enantiomer of PA-824 has advanced into clinical trials for the treatment of TB. With this in mind, (R)-PA-824 should be considered a worthy candidate for lead optimization and not a classical drug-repurposing project.The target product profile - or (S)-PA-824 against L. donovani promastigotes. This demonstrates that (R)- and (S)-PA-824 are not activated by the same type I nitroreductase responsible for the bioactivation of the antileishmanial nitroimidazole fexinidazole. This is consistent with the observation that lab-generated strains of nifurtimox-resistant T. brucei (nifurtimox is activated by a type I NTR) were cross resistant to fexinidazole but showed no change in sensitivity to (S)-PA-824 (R)- and (S)-des-nitro-PA-824 were found to be inactive against L. donovani, suggesting that the nitro group does play a key role in the antileishmanial activity of this compound series. This result allows for the possibility that (R)-PA-824 relies on nitro group reduction to exert its antileishmanial activity. L. donovani does not possess a homologue of the M. tuberculosis deazaflavin nitroreductase (Ddn), which specifically reduces (S)-PA-824, but not the R enantiomer (Leishmania type I NTR cannot activate (R)-PA-824, this putative bioreduction would need to be mediated by an as-yet-unidentified nitroreductase. Alternatively, it is entirely possible that (R)-PA-824 exerts its activity by inhibiting the function of an essential protein. If that were the case, then the nitro substituent could mediate an important binding interaction with the target protein. It is known that nitro groups can play a role in small-molecule protein binding by forming hydrogen bonds, by polarizing the π system of an aromatic ring, or by reducing the basicity of an imidazole ring -PA-824 in Leishmania are under way.Overexpression of the )-PA-824 . Howeverantiomer , and sinole ring . We are R)-PA-824 and fexinidazole do not share a mechanism of action involving the Leishmania type I nitroreductase, despite both being nitroimidazoles. Therefore, a single mutation is less likely to confer resistance to both compounds. Combination studies demonstrated that fexinidazole and (R)-PA-824 have a pharmacological additive effect against L. donovani promastigotes, and so the two compounds could potentially be developed as an oral combination therapy for VL. Interestingly, a combination analysis between (R)- and (S)-PA-824 revealed that the two enantiomers were mildly antagonistic. Without knowing how these compounds exert their antileishmanial activity, it is difficult to draw conclusions from this result. However, it can be postulated that the less-active S enantiomer is competing with the R enantiomer for a common uptake mechanism, or for the postulated activating enzyme or other unknown target.The target product profile for VL also stipulates that new compound entities should be suitable for combination therapy. Our studies suggest that (R)-PA-824, or analogues thereof, are suitable oral development candidates for the treatment of VL, with the potential for use in combination with fexinidazole.In conclusion, we have identified bicyclic nitroimidazoles as potential therapeutics against VL with a mode of action that is distinct from that of fexinidazole. Our findings suggest that (