Endothelin-1 is a potent endogenous vasoconstrictor that contributes to renal microcirculatory impairment during endotoxemia and sepsis. Here we investigated if the renal circulatory and metabolic effects of endothelin during endotoxemia are mediated through activation of endothelin-A receptors.−1, n = 8) or served as endotoxin-treated controls (n = 8). Renal artery blood flow, diuresis and creatinine clearance decreased in response to endotoxemia. Perfusion in the cortex, as measured by laser doppler flowmetry, was reduced in both groups, but TBC 3711 attenuated the decrease in the medulla (p = 0.002). Compared to control, TBC 3711 reduced renal oxygen extraction as well as cortical and medullary lactate/pyruvate ratios (p<0.05) measured by microdialysis. Furthermore, TBC 3711 attenuated the decline in renal cortical interstitial glucose levels (p = 0.02) and increased medullary pyruvate levels (p = 0.03). Decreased creatinine clearance and oliguria were present in both groups without any significant difference.A randomized experimental study was performed with anesthetized and mechanically ventilated pigs subjected to Escherichia coli endotoxin infusion for five hours. After two hours the animals were treated with the selective endothelin receptor type A antagonist TBC 3711 (2 mg⋅kgThese results suggest that endothelin released during endotoxemia acts via endothelin A receptors to impair renal medullary blood flow causing ischemia. Reduced renal oxygen extraction and cortical levels of lactate by TBC 3711, without effects on cortical blood flow, further suggest additional metabolic effects of endothelin type A receptor activation in this model of endotoxin induced acute kidney injury. Sepsis is a common cause of acute kidney injury (AKI). The pathophysiology of septic AKI remains elusive but renal impairment during sepsis has been postulated to be mediated by kidney hypoperfusion owing to excessive renal vasoconstriction We have previously shown that treatment with tezosentan, a dual ETA/ETB antagonist, improves renal artery blood flow and renal cortical microcirculation in endotoxemia, but has no effect on urine production The experiments were conducted in accordance with “The European Convention for Protection of Vertebrate Animals used for Experimental and other Scientific Purposes” . The study protocol was approved by the Stockholm South Regional Ethics Committee for Experiments in Animals, Huddinge District Court, Stockholm, Sweden . Sixteen crossbred (Landrace/Yorkshire/Hampshire) female pigs weighing 33.3±0.7 kg were used. Animals were fasted for solid food but had free access to water for 24 h before surgery.−1⋅h−1) and midazolam (0.15 mg⋅kg−1⋅h−1) was infused throughout the experiment as a complement to sevoflurane and to reduce surgically induced pain. Pancuronium bromide (0.5 mg⋅kg−1⋅h−1) was given for muscle paralysis. After oral intubation, ventilation with oxygen in air (FiO2 0.44) and a PEEP of 4 cm H2O was initiated. Ventilation was adjusted to reach a PCO2 of 4.7–5.3 kPa at baseline and the ventilator settings were then kept constant throughout the experiment. During the surgical preparation hydroxyethyl starch  and saline with glucose (25 mg⋅ml−1) was infused continuously at 10 ml⋅kg−1⋅h−1 and 20 ml⋅kg−1⋅h−1, respectively.Anesthesia was initiated and maintained as previously described in detail −1 with a perfusion solution . A stabilization period of 60 minutes was allowed before baseline measurements. Samples were collected for 10 minutes at baseline, 120 and 300 minutes after start of endotoxemia. Adjustment for time delay of samples reaching the vials was done. All samples were analyzed immediately on a bench top analyzer . After surgical preparation, the abdomen was carefully closed and the animals were allowed 60 minutes of recovery before the initiation of the experimental protocol. A continuous infusion of Ringeŕs Acetate (15 ml⋅kg−1⋅h−1) and saline with glucose 25 mg⋅ml−1 (5 ml⋅kg−1⋅h−1) was started directly after the surgery and kept constant throughout the experiment.Animals were placed in a supine position and the left carotid artery was cannulated with a single lumen catheter for measurement of arterial pressure and blood sampling. The jugular vein on the left side was also catheterized and used for fluid administration. A balloon-tipped pulmonary artery catheter  was inserted into the pulmonary artery via an introducer in the right jugular vein. The position in the pulmonary artery was determined by pressure guidance on a monitor. The different blood pressures were measured via pressure transducers . The pressure transducers were calibrated to atmospheric pressure at the level of the heart and to 100 mmHg or 25 mmHg using a saline column. After a midline laparotomy, an ultrasonic flow probe , was placed around the left renal artery and connected to a recorder . The left renal vein was visualized and an 18 Gauge catheter was inserted into the vein and carefully fixated. The urinary bladder was catheterized with a Foley catheter for hourly urine collection. The left kidney was then exposed and a laser Doppler probe  was sutured to the surface for cortical measurements. A needle laser Doppler probe  was then inserted 10–12 mm into the kidney for medullary measurements. Position was confirmed visually by opening the kidney post mortem. The two probes were connected to a Periflux 5001 base unit . Calibration was performed according to the manufacturer's instructions. Two microdialysis catheters  was inserted in the left kidney. First a small opening of the capsule was made with a needle, thereafter one probe was inserted in the cortical region of the kidney. The second microdialysis catheter was inserted in a right angle in a separate opening of the capsule and placed in the renal medulla. Position was confirmed visually by opening the kidney post mortem. The two probes were continuously perfused  at a speed of 1 µl⋅minHemodynamic measurements were acquired online  with acquisition software  and stored on a computer. Microcirculatory measurements were recorded online with Perisoft for Windows (Perimed AB) data acquisition software. Cardiac output was indexed to body surface area Blood samples were collected in pre-chilled EDTA tubes and immediately centrifuged at 3000 r.p.m. (200 g) for 15 min at 4°C to obtain plasma. The plasma was stored at 80°C until assayed. Plasma creatinine concentration was analyzed by the Jaffe method . The carotid blood samples were used for immediate arterial blood gas analyses . In addition, lactate  were analyzed using arterial blood. Renal vein samples were analyzed for blood gases and lactate at baseline, 120 and 300 minutes after the infusion of endotoxin was initiated. ET-1 levels in arterial blood and renal vein was measured by radioimmunoassay as described earlier by Hemsen −1 endotoxin, Sigma-Aldrich Sweden AB, Stockholm, Sweden). Endotoxin infusion was started at 0.3125 µg ⋅kg−1⋅h−1 and was increased stepwise until reaching 2.5 µg⋅kg−1⋅h−1 after 30 min. It was then kept constant throughout the experiment. After 120 minutes of endotoxemia animals were randomized to receive treatment with the ETA antagonist TBC 3711  2 mg⋅kg−1 (n = 8) or no treatment . The dose of TBC 3711, not having any ETB effect, was based on the results of an earlier study by our group Baseline data were collected immediately prior to endotoxemia. After baseline measurements all animals received a continuous infusion of endotoxin  and the graphs were created with Sigma Plot 11.0 . Data are expressed as means ± standard error of the mean (SEM). Changes in parameters over time were analysed according to a two-way repeated measures ANOVA. Main effects (time and treatment) were analyzed with time as a repeating variable before intervention, which included the time-points baseline, 60 and 120 minutes, and after intervention, which included the time-points 120, 180, 240 and 300 minutes, as within effects and treatment (control/TBC 3711) as between effects. A significant interaction between time and treatment was interpreted as a difference in the response to E.coli LPS over time between the groups. The significance level was set at sys and ERO2sys showed no significant change but systemic oxygen uptake, VO2sys increased and base excess decreased (data not shown).Infusion of endotoxin reduced cardiac output, pH and increased heart rate, body temperature and arterial lactate . Furthermore, we used a slightly longer observation period and as evident by The current results suggest that endothelin reduces renal medullary perfusion in normotensive endotoxemic pigs due to activation of ETA-receptors. This is in contrast to previous findings in rats, where no change in medullary circulation was observed as an effect of treatment with the ETA antagonist BQ-123 in endotoxemia The maintained medullary perfusion in the treatment group was not perfusion pressure dependent, since arterial pressure was significantly reduced by TBC 3711. Moreover, no difference in total renal blood flow was detected between groups indicating that the effect of TBC 3711 was on a microcirculatory level. Under normal physiological conditions intravenous infusion of ET-1 reduces medullary blood flow predominantly via ETA-activation total renal oxygen delivery and consumption, we cannot conclude if this reduction was a local medullary effect or if the renal cortex was affected as well. The most obvious explanation for a difference in renal oxygen consumption is a discrepancy in reabsorption of sodium, as this is the most oxygen demanding process in the kidney. However, there were no differences in sodium handling between the groups that could explain the difference in renal VO2. The natriuretic effect of ET-1 is also assumed to be mediated largely through activation of ETB 2. However, the control animals developed a more significant acidosis compared to TBC 3711-treated pigs, which may influence renal oxygen handling In the early phase of endotoxemia, oxygen delivery to the kidney was increased, indicating that oxygen supply was not impaired at the onset of renal damage. ETA antagonism was associated with a reduced total renal oxygen extraction. As we measured Though no significant change in renal vein lactate was found in these experiments, local interstitial lactate-differences in cortex and medulla was observed with the use of microdialysis catheters. Analysis of interstitial fluid with microdialysis makes it possible to gain information about local metabolic changes and have been used previously in endotoxemia The hypothesis that selective ETA antagonism would improve diuresis by preserving ETB function was not supported by the experimental results. This may be due to an inadequate observation period, but may also suggest that the relative importance of using a specific ETA-antagonist, and thus not inhibiting ETB, for maintaining diuresis during endotoxemia is limited.per se has the potential to impair renal function in endotoxemia per se had an effect on some of the measured variables herein. This is a potential limitation as it is impossible to entirely rule out that ETA-antagonism attenuated a reduction in renal microcirculation caused by surgery. However, we find this unlikely as a study using a 22-hour porcine model of endotoxemia with similar instrumentation as the present investigation could not demonstrate an effect of surgery It is relevant to point out some limitations of this study. First, this study was performed in anaesthetized animals and anaesthesia We conclude that TBC 3711, a selective ETA antagonist, attenuates endotoxemia induced microcirculatory impairment and ischemia in the renal medulla. Reduced total renal oxygen extraction and cortical L/P-ratio by TBC 3711, without effects on cortical blood flow, further suggest additional metabolic effects of ETA activation. However, in the short-term perspective, selective ETA-antagonism does not improve renal function in this model of endotoxin induced acute kidney injury.


The efficacy of antiretroviral therapy depends on patient adherence to a daily medication regimen, yet many patients fail to adhere at high enough rates to maintain health and reduce the risk of transmitting HIV. Given the explosive global growth of cellular-mobile phone use, text-messaging interventions to promote adherence are especially appropriate. This meta-analysis synthesized available text messaging interventions to promote antiretroviral therapy adherence in people living with HIV.We performed Boolean searches of electronic databases, hand searches of recent year conference abstracts and reverse searches. Included studies (1) targeted antiretroviral therapy adherence in a sample of people living with HIV, (2) used a randomized-controlled trial design to examine a text messaging intervention, and (3) reported at least one adherence measurement or clinical outcome.CI = 1.18, 1.64). Sensitivity analyses of intervention characteristics suggested that studies had larger effects when interventions (1) were sent less frequently than daily, (2) supported bidirectional communication, (3) included personalized message content, and (4) were matched to participants’ antiretroviral therapy dosing schedule. Interventions were also associated with improved viral load and/or CD4+ count .Eight studies, including 9 interventions, met inclusion criteria. Text-messaging interventions yielded significantly higher adherence than control conditions (OR = 1.39; 95% Text-messaging can support antiretroviral therapy adherence. Researchers should consider the adoption of less frequent messaging interventions with content and timing that is individually tailored and designed to evoke a reply from the recipient. Future research is needed in order to determine how best to optimize efficacy. The efficacy of antiretroviral therapy (ART) depends on patient adherence to a daily medication regimen, yet many patients fail to adhere at high enough rates to maintain health and reduce the risk of transmitting HIV The current standard of care in promoting adherence is patients’ contact with their health care providers. Although research on adherence behavior shows the importance of adherence counseling on biological outcomes  implementation costs prove increasingly onerous as healthcare providers continue to be pressured to do “more with less” both in developed countries One promising approach is the use of electronic text-messaging to deliver behavioral interventions Intervention trials reported in English were included if they (1) targeted ART adherence in a sample of people living with HIV (PLWH), (2) used a randomized-controlled trial to examine an electronic text messaging intervention, and (3) reported at least one adherence measurement  or biological outcome . Studies were excluded when reported data were insufficient to calculate effect sizes and contacted authors were unable to provide the necessary additional data. Listings of excluded studies with justification for exclusion are included as supplemental tables in Relevant studies were located through multiple strategies. We conducted serial searches of PsycINFO, PubMed, CINAHL, and ProQuest Dissertations and Theses for the years 1990 through October 2013, using Boolean strategy with the following terms: (cellphone OR “cellular phone” OR “mobile phone” OR “text message” OR “simple message service” OR SMS OR pager OR “two-way electronic messaging system”) AND (HIV OR HIV+ OR HIV-positive OR “people living with HIV/AIDS” OR “human immunodeficiency virus positive” OR “human immunodeficiency virus-positive” OR PLWHA) AND  AND (adherence OR “medication adherence” OR MNA OR “medication non-adherence”). Unpublished literature was sought both through the ProQuest electronic database described above and through reviewing abstracts from conferences with a focus on HIV and adherence over the past five years and contacting authors to request inclusion of their work in this synthesis. Finally, the reference sections of all relevant studies were searched for any additional relevant literature. The numbers of results returned by each electronic database are included in Two reviewers independently abstracted data from the studies using a standardized coding form, including general information , participant characteristics , design parameters , messaging intervention features , and outcomes . Across coded variables there was a 94% reviewer agreement rate; coding discrepancies were reconciled through discussion.kappa = 0.633; 95%CI = 0.515, 0.751; p<0.001). We report each of the instrument’s five sub-scales separately in order to provide a clearer appraisal of individual methodological elements than may be gained by a summary score Description of included studies’ methodological quality is an important facet of the systematic review. We assessed for risk of bias within individual studies using Downs and Black’s methodological quality scale k = 3), some in purely dichotomous terms (k = 4) and one reported a combination of continuous and dichotomous outcomes. For analyses, ESs were estimated with the standardized mean difference (d) dCox transformation Trials reported different numbers and types of outcomes; most reported multiple adherence outcomes . To uphold the assumption of independence, effect sizes (ESs) were averaged across adherence measures. When trials reported multiple follow up assessments, the last follow up was used in order to assess the greatest persistence and sustainability of the intervention effect. There was variation in the operationalization of adherence in this sample. Some studies reported outcomes entirely as continuous measures  of included studies were conducted in North America, 3 took place in Africa, and 1 in South America. Most data (70%) were collected during or after the year 2007 (range = 1999–2010). In total, 1,785 individuals consented and 1,463 (82%) of these were retained to at least one follow-up time point. The sample was 49% female and African participants made up 65% of the sample. Mean participant age was 40.00 years (range = 36.21–42.95). In those trials that reported it, mean baseline CD4+ was 291.83 cells/µL  and viral load was 4.34 log10 copies/mL .Eight studies M = 177.49 days; range = 14–365). The mean number of outcomes reported was 2.33 (range = 1–4). The majority of interventions (k = 5) used multiple adherence measurements while three studies relied on a single form of adherence measurement: self-report k = 6), followed by biological measures . A minority of studies employed in-person pill counts (k = 2) or pharmacy refill data (k = 1) to operationalize adherence. Among the 6 (67%) interventions that dichotomized adherence, threshold values ranged from 80 to 100% (median = 95%).The average length of intervention was about six months . Messages were sent daily in 5 (56%) interventions. Message timing was matched to dosing schedule in 4 (44%) interventions. Three (33%) interventions personalized message content to the individual participant. Five (56%) interventions supported bidirectional communication in the design .Seven (78%) interventions used cellular-mobile phones k = 9; OR = 1.39; 95% CI = 1.18, 1.64). k = 3; OR = 1.56; 95% CI = 1.11, 2.20); individual outcome measures did not always achieve statistical significance, but it should be noted that the numbers of available studies limited statistical power. A forest plot depicting the distribution of individual study ESs, averaged across available adherence measures, appears in M = 9.25; SD = 1.58). There was also low risk of measurement bias  and sampling bias  among individual studies. However, external validity was low in most studies  and a substantial proportion of individual studies  were underpowered.In Given the small sample size, determining moderators of ES magnitude would typically seem ill-advised; yet, an important question to be answered through this meta-analysis involves determining the active ingredients of the interventions. In addition, several design variations were fairly evenly distributed across studies in this review. We therefore stratified analyses across those intervention design parameters that were reasonably well-distributed in the sample. The results of these sensitivity analyses appear in The main result of this meta-analysis is that text-messaging interventions improve HIV treatment outcomes. In this sample, compared to control groups, those receiving text messages’ support were more likely to maintain adherence thresholds at follow up and meet the clinical goals of lower viral load and higher CD4+ count see . In addiAnother design element in need of further exploration is that of directionality. Designs that allowed, encouraged, or required message recipients’ response exhibited better outcomes than “one-way” reminder messages, which may have occurred because of enhanced engagement. Engagement is the “tuning in” of the recipient to the communication and has been identified as a necessary prerequisite for health behavior change A third element of messaging design investigated here was that of individual tailoring of message content. Interventions that used participant input to generate personalized message content saw larger effects on outcomes than those that sent uniform messages to all participants. While providers are certainly the experts of disease, patients are the experts of themselves. Interventionists who sought the participants’ own words and preferences may have produced intervention content that was less intrusive and more relatable for participants.The final facet of intervention design considered in this review is messaging that is matched to dosage timing. A greater effect was observed in studies where messaging corresponded to the time of an ART dose, an effect that seems akin to that of receiving a timed reminder There are several limitations of the current study. Although inclusion criteria were kept as broad as possible while maintaining focus on the research question, only 8 studies representing 9 interventions met inclusion criteria. Although 5 additional reports see  met critThe sampling frame is another important contextualizing factor through which to view these findings. The majority of intervention trials sampled an ART-naïve HIV+ population and only 2 interventions targeted individuals with demonstrated non-adherence. It is unclear what effect a predominantly ART naïve sample may have had on the overall effect size estimates. If baseline adherence behavior approaches a normal distribution in this population, a “signal-noise” effect may occur with inherently adherent individuals in the intervention group attenuating the perceived effect, which has been observed elsewhere Although the effect size associated with text-messaging is of fairly modest size, one should recognize that many of the control groups in the sampled trials received standard of care interventions, which logically would enhance adherence and thereby decrease the effect size in the treatment vs. control comparisons that the current meta-analysis examined Variation in the operationalization and measurement of adherence has been described before in the adherence literature The implementation of adherence thresholds also varied in our sample. The task of operationalizing “sufficient” adherence is necessarily fraught with multiple contributing factors. Providers historically have set a goal of perfect (100%) adherence k = 3) trials Finally, this review combined results from first-wave large scale trials with early pilot studies, resulting in a wide range of intervention and follow up durations . We atteen masse using technology that even in the developing world is increasingly mainstream makes it exceedingly cost-effective to implement; this is in addition to the savings in medical cost offset secondary to increased ART adherence. All of this bodes well for the eventual translation of this intervention strategy from research into practice. Future research is needed to model the impact of this type of intervention on health care systems. A formal cost-effectiveness analysis can compare the costs to implement electronic messaging with the benefits of enhanced ART adherence upon (1) drug resistance and the attendant escalation of pharmacotherapy; (2) the number of HIV-related hospitalizations/care; and (3) HIV transmission rates.Electronic messaging interventions can help PLWH achieve enhanced adherence to ART and improved clinical outcomes. The simple fact that such interventions can be disseminated In conclusion, there is good reason for optimism about text-messaging interventions to promote ART adherence. Researchers should consider the adoption of a less than daily frequency of messaging that is individually timed and tailored and designed to evoke a reply from the recipient. Future research is needed to formally compare these design and intervention characteristics described and analyzed here in order to titrate each of these parameters to an optimal effect.Appendix S1Literature Search Results by Database.(DOCX)Click here for additional data file.Appendix S2Reports excluded after full-text review with justification.(DOCX)Click here for additional data file.Appendix S3Reports excluded based on title and abstract with justification.(DOCX)Click here for additional data file.Checklist S1PRISMA reporting checklist.(DOC)Click here for additional data file.


The hypothesis examined in this study was the α1G TTCCs' influence in myocyte maturation and their rapid withdrawal from the cell cycle after birth.T-type Ca2+ currents were measured.Cardiac myocytes were isolated from neonatal and adult wild type (WT), α1G−/− and α1G over expressing (α1GDT) mice. Bromodeoxyuridine (BrdU) uptake, myocyte nucleation, cell cycle analysis, and T-type CaAll myocytes were mono-nucleated at birth and 35% of WT myocytes expressed functional TTCCs. Very few neonatal myocytes had functional TTCCs in α1G−/− hearts. By the end of the first week after birth no WT or α1G−/− had functional TTCCs. During the first week after birth about 25% of WT myocytes were BrdU+ and became bi-nucleated. Significantly fewer α1G−/− myocytes became bi-nucleated and fewer of these myocytes were BrdU+. Neonatal α1G−/− myocytes were also smaller than WT. Adult WT and α1G−/− hearts were similar in size, but α1G−/− myocytes were smaller and a greater % were mono-nucleated. α1G over expressing hearts were smaller than WT but their myocytes were larger.The studies performed show that loss of functional TTCCs is associated with bi-nucleation and myocyte withdrawal from the cell cycle. Loss of α1G TTCCs slowed the transition from mono- to bi-nucleation and resulted in an adult heart with a greater number of small cardiac myocytes. These results suggest that TTCCs are involved in the regulation of myocyte size and the exit of myocytes from the cell cycle during the first week after birth. After 6–7 min of perfusion the heart softened and the ventricles were minced and filtered through macroporous filter (mesh opening: 100 μm). Isolated ventricular myocytes were equilibrated in Tyrode solution  and 0.5% bovine serum albumin. Myocytes were seeded (4 × 104) in polystyrene, nonpyrogenic 12-well culture plates precoated with 40 μg/ml laminin.Mice were anesthetized with sodium pentobarbital . Hearts were perfused via the aorta with Tyrode solution . Cells were fixed with 70% ethanol, pH 2, permeabilized with 0.05% Triton X-100 and incubated with a mouse anti-actin antibody. Cardiomyocytes were identified by primary antibody (anti-actin) and secondary antibody (APC). Age matched WT and α1G−/− mice were studied on Day 1   or on Day 7 after birth .Flow cytometry was performed on myocytes from neonatal mouse hearts: neonatal mouse hearts were isolated under a dissecting microscope, gently minced and then dissociated to single cell solution with DNase (0.2 mg/ml) and trypsin (3 mg/ml) buffer solution  [2 2 mM, CsCl 5.4 mM, 4-aminopyridine 2 mM, MgCl2 1.2 mM, glucose 10 mM, HEPES 5 mM, NMDG 150 mM and pH 7.4 with CsOH. An Axopatch 2B voltage-clamp amplifier was used to control the membrane potential and membrane currents were analyzed with pClamp 8 software. Total cell membrane T-type currents and L-type currents were measured from a holding potential of −90 mV using square wave pulses from −70 mV to +50 mV, in 10 mV increments. ICaL was measured from a holding potential of −50 mV using identical square wave pulses. ICaT was calculated by subtracting the ICaL measured from a holding potential of −50 mV from the total Ca2+ current measured from a holing potential of −90 mV.L-  had a measureable ICaT. No ICaT was found at day 7 in the 27 α1G−/− myocytes studied. These results show that there is a significant loss of Ca2+ influx through TTCC in WT myocytes and α1G−/− myocytes have little or no Ca2+ entry via TTCCs during the first week of life. The ICaT found in the single α1G−/− myocyte at day 1 likely represents current through α1H TTCC, the other channel that has been found in neonatal hearts [TTCC and LTCC currents were measured in neonatal myocytes 1 and 7 days after birth in WT and α1G−/− mice . All myoer birth . ICaT waat day 7 . Signifil hearts ,24. ThesNeonatal mice were injected with BrdU for 7 days and then BrdU+ myocyte nuclei were measured in the intact heart, with immunostaining , or in iDefining myocyte specific nuclei in tissue sections can be challenging , so to cBrdU incorporation into myocyte DNA during the first week of life could represent myocyte proliferation or the doubling in myocyte DNA content that takes place with binucleation, which has been associated with exit from the cell cycle ,26. To eMyocytes were isolated from WT and α1G−/− hearts 1 and 7 days after birth to unambiguously determine the percentage of mono and binucleated myocytes . These sWe also determined if there were differences in myocyte size by the end the first week of life  in WT anWe next explored if any of the differences in nucleation and myocyte size observed in neonatal myocytes were still present in the adult animal. These studies were performed at 2 months of age at a time when we have shown that these hearts have normal (WT and α1G−/−) or enhanced (α1GDT) . We adde2+ currents were measured in adult WT and α1GDT myocytes  adult myocytes. Large ICaT was observed in α1GDT myocytes, as we have reported previously [Camyocytes . ICaT waWe have previously reported that heart weight, normalized to body weight or tibial length, is not different in 2 month old α1G−/−, α1GDT and WT mice . In the Myocyte nucleation (mono- versus bi-nucleation) was also measured in myocytes isolated from WT, α1G−/− and α1GDT animals. More than 90% of all myocytes were binucleated in WT and α1GDT hearts. The % of mononucleated myocytes was significantly greater in α1G−/− myocytes versus that of the other two groups , consistIn summary, these experiments show that neonatal myocytes without α1G TTCC are smaller than normal and appear to have a slower than a normal exit from the cell cycle. The adult heart has a normal size but has myocytes that are smaller than normal and there is a greater percentage of mononucleated myocytes.2+ current entry they are not involved in the excitation–contraction coupling [2+ influx through TTCCs is linked to cell proliferation and to specific phases of the cell cycle [T-type Ca channels are expressed in cardiac myocytes in the fetal and neonatal hearts , but arecoupling . There ill cycle ,31,12,322+ influx through α1G TTCCs regulates the transition from a proliferative fetal myocyte to a terminally differentiated binucleated adult ventricular myocyte during the first week of neonatal life.The major findings of this study are that 1) on Day 1 after birth about 35% of ventricular myocytes had functional TTCCs, and this % decreases during the first week of life. 2a) by two months of age normal ventricular myocytes do not have functional TTCCs; 2b) α1G TTCCs appear to be the major TTCC expressed during the first week of life. 3) BrdU is incorporated into ventricular myocytes during the first week of life and this is associated with a 2N to 4N transition and binucleation, representing myocyte withdrawal from the cell cycle. 4) Loss of α1G TTCCs was associated with a slower transition from mononucleation to binucleation and reduced BrdU incorporation in the first week after birth; and 5) loss of α1G TTCC resulted in an adult heart with more myocytes than are found in the WT heart and these myocytes are smaller and a greater % are mononucleated. Collectively these data suggest that Ca2+ influx through TTCCs regulates myocyte growth, the 2N to 4N (binucleation) transition and the associated exit of cardiac myocytes from the cell cycle. If true this would suggest that α1G−/− hearts have a longer than a normal time period during which their ventricular myocytes can proliferate.Our studies show that 1 day after birth most of the myocytes in the neonatal heart are mononucleated and a fraction (about 35%) have functional TTCCs and almost all myocytes are mononucleated. By Day 7 after birth very few myocytes express TTCCs and many of the myocytes have become binucleated, which we interpret as a sign that they have withdrawn from the cell cycle ,33. WhenHeart size is not significantly altered by either the gain or loss of α1G TTCCs, in spite of the fact that the α1GDT mice have a modest hypercontractile phenotype . The fac2+ influx through TTCCs regulates myocyte growth as well as the transition from mono- to bi-nucleation and the associated exit of myocytes from the cell cycle soon after birth. α1G−/− myocytes appear to have a reduced rate of cell cycle withdrawal, resulting in an adult heart with a normal size but with an increased number of small, mononucleated myocytes. α1GDT myocytes are larger than normal, yet their hearts are also normal in size. This is a complex phenotype that could result from a premature exit of myocytes from the cell cycle, resulting in a reduced number of myocytes per unit mass. The α1GDT phenotype is more complicated because these myocytes (and hearts) are hypercontractile [The major findings of this study are all consistent with the idea that Catractile  and thisThere are a number of limitations in our study that largely result from the fact that it is challenging to study cell cycle activity in the heart and to determine the source of new cardiac myocytes. Our results are consistent with the idea that there may be a greater number of smaller, mononucleated myocytes in the α1G−/− adult heart. It is unclear if the basis for this is enhanced myocyte proliferation and if this occurs during fetal life or during the maturation of the adult heart. How α1G TTCCs are linked to the alterations in myocyte size, nucleation and the capacity for proliferative activity is, in our view, an issue deserving study.2+ influx is involved in the regulation of myocyte growth and the exit of neonatal myocytes from the cell cycle. Understanding the molecular bases of these processes could lead to novel strategies to coax myocytes into or out of the cell cycle to generate new cardiac myocytes in times of stress.In summary, these experiments suggest that TTCCs are expressed transiently in cardiac myocytes and the resultant Ca1


Pantala flavescens) appear in the Maldives every October–December. Since they cannot breed on these largely waterless islands, it has recently been suggested that they are “falling out” during a trans-oceanic flight from India to East Africa. In addition, it has been suggested that this trans-oceanic crossing is just one leg of a multi-generational migratory circuit covering about 14,000–18,000 km. The dragonflies are presumed to accomplish this remarkable feat by riding high-altitude winds associated with the Inter-tropical Convergence Zone (ITCZ). While there is considerable evidence for this migratory circuit, much of that evidence is circumstantial. Recent developments in the application of stable isotope analyses to track migratory dragonflies include the establishment of direct associations between dragonfly wing chitin δ2H values with those derived from long-term δ2H precipitation isoscapes. We applied this approach by measuring wing chitin δ2H values in 49 individual Pantala flavescens from the November–December migration through the Maldives. Using a previously established spatial calibration algorithm for dragonflies, the mean wing δ2H value of −117±16 ‰ corresponded to a predicted mean natal ambient water source of −81 ‰, which resulted in a probabilistic origin of northern India, and possibly further north and east. This strongly suggests that the migratory circuit of this species in this region is longer than previously suspected, and could possibly involve a remarkable trans-Himalayan high-altitude traverse.Large numbers of the Globe Skimmer dragonfly ( Danaus plexippus) which completes a multi-generational migratory circuit between natal sites in Canada and the United States and wintering sites in Mexico Pantala flavescens) is widely distributed throughout the tropics and many temperate areas, and is thought to undertake extensive migrations following the Inter-tropical Convergence Zone (ITCZ). This meteorological phenomenon is marked by a band of clouds around the globe near the equator, which moves north and south with the seasons. For a dragonfly, Pantala flavescens has a remarkably short larval life  values in wing chitin of two samples of migrating P. flavescens that were collected in the Maldives  to infer their natal origins, based on well-known spatial patterns of δ2H in precipitation and in major surface waters across the Indian subcontinent. Pantala flavescens arrives in Maldives across the ocean from southern India We used stable-hydrogen isotope  is neither endangered nor protected. It is globally distributed and has been assessed by IUCN as Least Concern.No specific permits were required for the described field studies. No specific permissions were required to collect dragonflies in the Maldives . We confirm that neither collection site is protected. At one site , collection was made on public land. At the other site (Kunfunadhoo), collection was made in the grounds of a private resort hotel, with the approval and assistance of the resort environmental officer (Anke Hofmeister). The species collected (2 derived from high-temperature (1350°C) flash pyrolysis of 350 µg wing subsamples using continuous-flow isotope-ratio mass spectrometry. All results for non-exchangeable δ2H are expressed in the typical delta notation, in units of per mil (‰), and normalized on the Vienna Standard Mean Ocean Water – Standard Light Antarctic Precipitation (VSMOW-SLAP) standard scale. Two keratin laboratory reference materials  were used to normalize the results, using the previously assigned δ2H values of –197.0 and −54.1 ‰, respectively. A δ2H calibration equation directly correlating measured dragonfly wing chitin (δ2Hwing) to precipitation-driven ambient natal water (δ2Hp) was derived by δ2Hwing = 0.91(δ2Hp) –42.54. We used this relationship to convert our measured δ2Hwing values of P. flavescens to ambient natal water equivalent.Dragonflies were collected by RCA in the Maldives on Malé (4°11′N 73°31′E) between 7–9 November 2010 (n = 22) and on Kunfunadhoo Island in Baa Atoll (5°07′N 73°05′E) on 10 December 2009 (n = 27) during the annual migration event P. flavescens prior to adult arrival on the Maldives in November-December . We did not attempt an elevation-corrected model since the topography of India involves relatively low relief over much of the continent south of the Himalayas, with the only relatively negative precipitation δ2H occurring in the mountains  to assess the likelihood that any given cell within the δ2H wing isoscape could represent the potential origin of each dragonfly individual using the normal probability density function. We used an expected mean wing δ2H based on that predicted by the calibrated isoscape and a standard deviation of 19.1 ‰ estimated from the standard deviation of the residuals for the regression linking δ2Hwing and δ2Hpδ2H isoscape to obtain the likely natal origin of the population where each cell then represented the number of dragonflies assigned to that location given our odds ratio.A likelihood-based assignment method that incorporated estimates of uncertainty was used to link dragonfly wing ontinent . We usedδ2H patterns, with peninsular India generally having considerably higher δ2H values compared to the low δ2H values in the mountainous regions farther to the north. Our November sample of P. flavescens  did not differ from the December sample . The overall mean of −117±16 ‰ corresponded to predicted natal waters of −81 ‰ . This raises the intriguing possibility that at least some P. flavescens might complete a trans-Himalayan migration. This species is known to fly higher than any other odonate Secondly, while the most parsimonious natal area for our samples of P. flavescens arriving in Maldives in November-December from the far north of the subcontinent, and not central or southern India? One possible answer follows from Anderson’s P. flavescens might only be transported in large numbers from East Africa to northern Indian subcontinent, and not to central or southern India.Thirdly, our results raise an interesting question: if our two populations are representative, then why are P. flavescens arriving in Maldives during November–December, and has extended the likely distances over which they migrate while recognizing that individuals may well be coming from natal origins even further north and east. We have further provided indirect support for the proposed migration of P. flavescens across the Arabian Sea from East Africa to northern Indian subcontinent during June–July. The use of stable isotope analyses to further unravel the behavior of these migratory dragonflies has been amply justified, and further studies of P. flavescens involving additional areas, seasons and isotopes are planned.In conclusion, this study supports the hypothesis of Indian subcontinent origins of Table S1Summary of available surface water δ2H data for India.(DOC)Click here for additional data file.Table S2Summary of available amount-weighted precipitation δ2H for July-September for India and Pakistan.(DOC)Click here for additional data file.Text S1Precipitation and surface water δ2H summary(DOC)Click here for additional data file.


Artemisinin and artemisinin semi-synthetic derivatives (collectively known as endoperoxides) are first-line antimalarials for the treatment of uncomplicated and severe malaria. Endoperoxides display very fast killing rates and are generally recalcitrant to parasite resistance development. These key pharmacodynamic features are a result of a complex mechanism of action, the details of which lack consensus. Here, we report on the primary physiological events leading to parasite death.m) and plasma membrane (ΔΨp) electrochemical potentials were measured using real-time single-cell imaging following exposure to pharmacologically relevant concentrations of endoperoxides . In addition, mitochondrial electron transport chain components NADH:quinone oxidoreductase , bc1 (complex III) and cytochrome oxidase (complex IV) were investigated to determine their functional sensitivity to the various endoperoxides.Parasite mitochondrial (ΔΨm and ΔΨp. The rate of depolarization was decreased in the presence of a reactive oxygen species (ROS) scavenger and Fe3+ chelators. Depolarization of ΔΨm by endoperoxides is not believed to be through the inhibition of mitochondrial electron transport chain components, owing to the lack of significant inhibition when assayed directly.Parasite exposure to endoperoxides resulted in rapid depolarization of parasite ΔΨm and ΔΨp is shown to be mediated via the generation of ROS that are initiated by iron bioactivation of endoperoxides and/or catalysed by iron-dependent oxidative stress. These data are discussed in the context of current hypotheses concerning the mode of action of endoperoxides.The depolarization of ΔΨ Both radicals subsequently rearrange to primary or secondary carbon-centred radicals via either β-scission or a -H shift. In support of this hypothesis, evidence of the formation of these carbon-centred radical intermediates has been provided using electron paramagnetic resonance spin-trapping techniques.11 It has been proposed that these C-centred radicals are capable of haem and/or protein alkylation; whilst Meunier and coworkers12 have provided evidence for haem alkylation, there are only a few reports of model studies on protein alkylation involving reactions with ferrous salts in the presence of cysteine (iron–sulphur chelates).13The mechanism(s) of activation and subsequent biological target(s) of endoperoxides continue to be debated.JAC OnlineFigure S1, available as Supplementary data at ).15 The ring opening involves heterolytic cleavage of the endoperoxide bridge followed by interaction with water generating an open, unsaturated hydroperoxide, capable of direct oxidation of protein residues. Fenton degradation of the oxygen-centred radical intermediate can provide hydroxyl radicals (HO·) highly reactive against amino acids, lipids or nucleic acids.In the second model, it is hypothesized that iron acts as a Lewis acid to facilitate ionic activation of antimalarial trioxanes generating downstream reactive oxygen species .18 The cofactor model of artemisinin activation, however, is not restricted to mitochondrial flavoenzymes but rather implicates cytosolic flavoenzymes and also rejects the direct requirement for either Fe2+ and/or non-haem iron activation.An alternative pathway of artemisinin bioactivation has been suggested via electron transport chain (ETC) components causing downstream ROS production and membrane depolarization in isolated 20For models involving iron-based activation, the origin of the iron available for bioactivation is also a point of debate. There is experimental evidence for the involvement of both haem and non-haem iron in the bioactivation.P. falciparum cultures.6 By using [14C]artemisinin, Maeno et al.21 showed its accumulation in the digestive food vacuole where haemoglobin degradation leads to the release of soluble haem. More recently, inhibition of haemoglobin digestion by the genetic deletion of cysteine protease falcipain-2 was shown to significantly attenuate in vitro endoperoxide-mediated parasite kill.22 Haem has been observed to enhance the oxidizing effects of endoperoxide drugs.23 As noted above, the reaction between haem and artemisinin has been confirmed in vitro and in vivo.25 In addition, it has been proposed that haem is the primary activator of artemisinin, reacting with it more efficiently than other iron-containing molecules .26 It has also been reported that antimalarial trioxanes accumulated into the food vacuole are activated by neutral lipid-associated haem and induce oxidative membrane damage.28The involvement of haem in the activation of endoperoxide compounds was first proposed following isolation of haem–artemisinin adduct in 30 Additionally, Haynes et al.32 have proposed that artemisinins do not inhibit haemozoin formation and cannot react with haem according to conventional chemistry models. Several studies have shown that iron chelation, selective for non-haem iron, antagonizes the accumulation and antimalarial activity of endoperoxide antimalarials in vitro and can prevent their toxic effects in mice.33However, using radio- or fluorescent-labelled artemisinins and microscopy, two studies contest the accumulation of endoperoxide drugs in the food vacuole and their possible interaction with haem.23 the translationally controlled tumour protein (PfTCTP),34 the sarco/endoplasmic reticulum membrane calcium PfATPase630 and the parasite mitochondrion. Specific targeting of parasite mitochondria by endoperoxides was reported initially based on morphological changes to mitochondria following exposure to artemether.36 Zhao et al.37 reported a specific inhibitory effect by artemether against Plasmodium cytochrome c oxidase (complex IV) that was later also reported by Krungkrai et al.38 based on measurements of O2 consumption. Li et al.16 hypothesized a role for parasite mitochondria, specifically the type II NADH:quinone oxidoreductase (PfNDH2),40 by acting both as a target and as an activator of endoperoxides via electron donation by the ETC. Conflicting data have since emerged, with Crespo et al.41 only observing mitochondrial dysfunction (as determined by rhodamine 123 fluorescence) upon exposure to artemisinin after 8 h but not after 4 h, interpreting this as a downstream effect rather than an initiator of killing. Conversely, Wang et al.17 reported depolarization of the membrane potential of isolated parasite mitochondria by artemisinin following 2 h of incubation.Once activated, endoperoxide antimalarials have been reported to disrupt a number of parasite functions and enzymes, including the haem detoxification pathway,ρ0 cells, which are devoid of a functioning ETC, it was demonstrated that the ETC does not have any role in the reductive activation of the endoperoxide to cytotoxic carbon-centred radicals.42Studies of artemisinin toxicity in human cells indicate that respiring mitochondria play an essential role in endoperoxide-induced cytotoxicity via the generation of ROS; however, using HeLa m) and plasma membrane potential (ΔΨp)44 and reported malaria parasite ETC activities including PfNDH2.45 Using these approaches, we have re-examined the effect of endoperoxides on parasite bioenergetic functions and discuss our data in the context of the current hypotheses.We have previously described the use of a real-time single-cell imaging method for monitoring malaria parasite mitochondrial  cultures consisted of a 2% (v/v) suspension of O+ erythrocytes in RPMI 1640 medium  supplemented with 10% pooled human AB+ serum, 25 mM HEPES (pH 7.4) and 20 μM gentamicin sulphate.46 Cultures were grown under a gaseous headspace of (in v/v) 4% O2 and 3% CO2 in N2 at 37°C. Parasite growth was synchronized by treatment with sorbitol.47 Drug susceptibilities were determined with an inoculum size of 0.5% parasitaemia (ring stage) and 1% haematocrit and were assessed by the measurement of fluorescence after the addition of SYBR Green I as described previously.48 Drug 50% inhibitory concentration (IC50) values were calculated from the log of the dose–response relationship, as fitted with Grafit software . Drug stock solutions (1–10 mM) were prepared in DMSO. The results are given as the means of at least three separate experiments.9 cells/mL) by adding 5 volumes of 0.15% (w/v) saponin in phosphate-buffered saline  for 5 min, followed by three washes by centrifugation and resuspension in HEPES (25 mM)-buffered RPMI containing a protease inhibitor cocktail . Cell extract was prepared by repeated freeze–thawing in liquid N2, followed by disruption with a sonicating probe.Free parasites were prepared from aliquots of infected erythrocytes , an analogue of ubiquinone , was dissolved (10 mg) in 400 μL of nitrogen-saturated hexane. An equal volume of aqueous 1.15 M sodium dithionite was added and the mixture shaken vigorously until colourless. The upper, organic phase was collected and the decylubiquinol recovered by evaporating off the hexane under N2. The decylubiquinol was dissolved in 100 μL of 96% ethanol (acidified with 10 mM HCl) and stored in aliquots at −80°C. Decylubiquinol concentration was determined spectrophotometrically from absolute spectra, using ɛ288–320 = 4.14 mM−1 cm−1.The artificial quinol electron donor was prepared based on a previously described method.c oxidoreductase (bc1 protein) and cytochrome c oxidase (complex IV) activity were assayed in a Cary 4000 spectrophotometer . The bc1 reaction buffer consisted of 50 mM potassium phosphate (pH 7.5), 2 mM EDTA, 10 mM KCN  and 30 μM horse heart cytochrome c (oxidized) (Sigma).49 The complex IV mixture is composed of 50 mM potassium phosphate (pH 7.5), 2 mM EDTA and 5 μM antimycin. Inhibitors were added prior to the addition of substrate. The reaction volume was 700 μL and assays were performed at room temperature. P. falciparum bc1 and complex IV were assayed from cell-free P. falciparum extract at a total protein concentration of 30–60 μg/mL. Cytochrome c reductase (bc1) and oxidase (complex IV) activities were initiated, respectively, by the addition of 50 μM decylubiquinol (dQH2) and 30 μM equine cytochrome c (reduced). The horse heart cytochrome c was reduced by sodium dithionite and then passed through a PD-10 desalting column . Activities were measured by monitoring the cytochrome c (reduced) concentration at 550–542 nm (ɛ550–542 = 18.1 mM−1 cm−1).Decylubiquinol:cytochrome PfNDH2 enzyme activity was determined based on a modification of the NADH:quinone oxidoreductase assay previously described.50 Enzyme activity was measured in a buffered solution  containing 50 mM potassium phosphate (pH 7.5), 2 mM EDTA, 10 mM KCN and 50 μM coenzyme Q1 at room temperature. Recombinant PfNDH2 enzyme was added as an Escherichia coli crude membrane preparation at a total protein concentration of between 10 and 20 μg/mL.40 Inhibitors were added before initiation of the reaction by addition of 200 μM NADH. PfNDH2 activity was measured spectrophotometrically by monitoring the decrease of NADH concentration at 340 nm (ɛ340 = 6.22 mM−1 cm−1) and Q1 concentration at 283 nm (ɛ283 = 8.1 mM−1 cm−1).The rhodamine derivative tetramethyl rhodamine ethyl ester (TMRE) was used to monitor the membrane potential of the cytoplasm and mitochondria from malaria-infected red blood cells. TMRE is cationic and reversibly accumulates inside energized membranes according to the Nernst equation. For the experiment, suspensions (1%) of infected erythrocytes in HEPES-buffered RPMI medium (no serum) were loaded with 250 nM TMRE  for 10 min at 37°C. For imaging, malaria parasite-infected erythrocytes were immobilized using polylysine-coated coverslips in a Bioptechs FCS2 perfusion chamber  and maintained at 37°C in growth medium (no serum). Inhibitors were added to the perfusate and the membrane potential-dependent fluorescence responses were monitored in real time. During all manipulations, the concentration of TMRE in the perfusate was kept at 250 nM. The fluorescence signals from malaria-infected erythrocytes were collected on a Zeiss Pascal confocal laser scanning microscope through a Plan-Apochromat 63× 1.2 numerical aperture water objective. Excitation of TMRE was performed using the HeNe laser line (543 nm). Emitted light was collected through a 560 nm long-pass filter from a 543 nm dichroic mirror. Photobleaching (the irreversible damage of TMRE producing a less fluorescent species) was assessed by continuous exposure (5 min) of loaded cells to laser illumination. For each experiment, the laser illumination and microscope settings that gave minimal reduction in signal were used. Data capture and extraction were carried out with Zeiss Pascal software and plot design was performed with Kaleidagraph .PfNDH2, bc1 complex and cytochrome c oxidase, were measured directly in the presence of a number of endoperoxides. As described in the Materials and methods section, the bc1 complex (complex III) and cytochrome c oxidase (complex IV) were measured directly from parasite cell-free extracts, whilst PfNDH2 activity was measured from membrane preparations of a heterologous expression system described previously.40 No or relatively weak (∼20%) inhibition of the individual respiratory components was observed for all the endoperoxides tested at comparatively high fixed doses , corresponding to the addition of plasma and mitochondrial membrane potential. For all assays, the fluorescence dynamic range was set up so that untreated TMRE-loaded cells were regarded as having complete fluorescence (100%), whereas the baseline (0%) was set by the addition of 10 μM H+ ionophore carbonyl cyanide p-(trifluoromethyl)phenylhydrazone (FCCP).To determine the effect of endoperoxides on membrane potential, a real-time single-cell imaging approach was used. The measurement is based on the accumulation of the cationic fluorescence probe TMRE according to the Nernst equation. Due to dynamic fluorescence measurements, the probe is subject to photobleaching. To minimize this, several parameters of the confocal laser scanning microscope  were optimized before recording each experiment. Upon addition of TMRE to bc1 complex inhibitor, decreased the total membrane potential-dependent fluorescence by 30%  and mitochondrial membrane (ΔΨm) potentials to TMRE accumulation, further experiments were performed to measure ΔΨp and ΔΨm independently.Although these experiments demonstrate for the first time the rapid depolarization of membrane potential upon exposure to endoperoxides, since there is a contribution from both plasma , the fluorescence intensity from the cytosol decreases ∼70%–80%,43 leaving a local and strong signal originating from the parasite mitochondrion, as demonstrated in Figure m-dependent fluorescence, concanamycin A-treated parasites were normalized to 100% and the baseline (0%) was set by FCCP addition (10 μM). Atovaquone addition rapidly (≈3 min) reduced concanamycin-independent TMRE fluorescence by 70%  was set by FCCP addition (10 μM). The vacuolar H+-ATPase is involved in transforming the energy of ATP hydrolysis to generate the electrochemical potential at the surface of the malaria parasite through the transport of H+ across the plasma membrane.51 As expected, the addition of concanamycin A, a well-known V-type ATPase inhibitor, rapidly decreased ΔΨp-dependent fluorescence by 60%  membrane potential depolarization.DFO and DFP, two chelating agents selective for non-haem Fe50) using both iron chelators revealed moderate antimalarial activity against 3D7 P. falciparum of 17.3 ± 2 μM (DFO) and 111.8 ± 2 μM (DFP). However, during the short time period of the single-cell assays (<6 min), the addition of 100 μM DFO or 100 μM DFP to the perfusate did not result in a decrease of parasite ΔΨm and ΔΨp-dependent fluorescence signal. However, addition of DFO and DFP (100 μM) to the perfusate was observed to significantly minimize the depolarization caused by either artemisinin .Tiron, a cell membrane-permeable superoxide scavenger, was added (100 μM) to the perfusate and endoperoxide-induced ΔΨe Figure . In contPlasmodium cytochrome c oxidase (complex IV)38 and type II NADH:quinone oxidoreductase (PfNDH2).16 In agreement with previous studies, we also noted inhibition of complex IV but only to a maximum of 20% at relatively high concentrations (1 μM) of a range of endoperoxides  of ∼−95 mV.54 It is generated by V-type H+-ATPases, which transfer protons out of the parasite cytosol.56 Localized in the plasma membrane, the V-type H+-ATPase also plays a role in regulating a neutral cytosolic pH (near 7.3), crucial for enzyme function, incorporation of vitamin B5 and generation of a pH gradient across membranes of internal organelles.57 The maintenance of a ΔΨp is also important in mediating the influx of K+ in the parasite cytosol and the uptake of nutrients such as choline.59 The direct inhibition of the V-type H+-ATPase by specific inhibitors such as bafilomycin A1 or concanamycin A has been demonstrated to depolarize the plasma membrane potential and disrupt the physiology of the parasite, leading to its death.57 The parasite mitochondrial membrane potential (ΔΨm) is generated via the ETC, through the activity of the bc1 complex and cytochrome c oxidase.60 An essential function of the mitochondrion for parasite survival during the intraerythrocytic stages is the provision of orotate for pyrimidine biosynthesis through the activity of dihydroorotate dehydrogenase.62In unicellular organisms, life can be defined as the maintenance of an electrochemical potential across the organism's outer membrane (negative entropy).P. falciparum-infected erythrocytes to pharmacologically relevant concentrations of endoperoxides resulted in a rapid loss of membrane potential-dependent accumulation of TMRE , it was not possible from these experiments to distinguish whether the depolarization was on account of an effect on the ΔΨp alone and/or the ΔΨm. Inhibition of the parasite V-type H+-ATPase by addition of concanamycin A to the perfusate ‘unmasked’ the parasite to reveal the ΔΨm-dependent accumulation of TMRE , where R, T, F, Din and Dout represent the universal gas constant, the absolute temperature, the Faraday constant and the intra- and extracellular probe concentrations, respectively. The washing step would therefore affect the distribution of the probe in a time-dependent manner and may account for the discord between the data of Crespo et al.41 and those presented here and by Wang et al.17Exposure of E Figure . On accoE Figure . With thd Figure . These dp alone. Exposure to endoperoxides resulted in the rapid depolarization of parasite ΔΨp  are sufficient for parasite kill, albeit with stage-dependent differences.67 It is also noteworthy that disruption of the parasite transmembrane pH gradient via inhibition of the V-type ATPase has been reported to drop the cytosolic pH by 0.4 pH units in <3 min and 0.5–0.6 units within 20 min,57 leading to the inhibition of parasite growth within 30 min to 4 h (depending on inhibitor concentration).57Pretreatment of parasites with atovaquone in the perfusate further allowed the measurement of ΔΨp Figure . The rapp or ΔΨm  either directly, by activation of the endoperoxides by Fe2+5 and/or haem,70 or indirectly through the ability of Fe3+ to oxidize cytosolic (reduced) cofactors.18 In this study, we used DFO and DFP, two known chelators of Fe3+,71 to determine the effect on endoperoxide-induced ΔΨp depolarization. Addition of the chelators to the perfusate was clearly observed to confer a protective effect to the parasite upon exposure to the endoperoxides  and/or flavin enzymes. In previous experiments performed in situ using single-cell imaging, we observed that endoperoxide–acridine adducts could be completely washed out in the presence of DFO, but remained irreversibly bound in the absence of DFO, suggesting that the drugs were being activated by iron to form stable covalent adducts.52 It is therefore possible that the protective effect of the iron chelators observed in our studies here is a result of a decrease in the bioactivation of the endoperoxides. An alternative or additional explanation is that the iron chelators conferred a protective effect by decreasing the generation of ROS through Fe2+-mediated oxidative stress such as the Fenton reaction, generating the highly membrane damaging hydroxyl radical (·OH). It is also noteworthy that DFO has been reported to be able to directly scavenge ROS.72 It should also be noted that in the experiments presented here, intracellular haem concentrations cannot be selectively and rapidly manipulated; therefore, the potential role of haem in the depolarization of ΔΨp by endoperoxides cannot be elucidated.The parasite's unique ability to digest haemoglobin is generally accepted to confer selective toxicity to endoperoxides  scavenger, showing a decreased rate of membrane depolarization following exposure to endoperoxides .This work was supported by grants from the None to declare.JAC Online .Figure S1 is available as Supplementary data at


Numerous studies have reported widespread increases in iWUE coinciding with rising atmospheric CO2 over the past century. While this could represent a coherent global response, the fact that increases of similar magnitude were observed across biomes with no apparent effect on tree growth raises the question of whether iWUE calculations reflect actual physiological responses to elevated CO2 levels.The analysis of tree-ring carbon isotope composition  are used in place of actual tree-ring δ13C measurements. From simulated data sets we calculated non-physiological changes in iWUE from 1900 to present and across a 4000 m altitudinal range. This generated results strikingly similar to those reported in recent studies encompassing 22 species from tropical, subtropical, temperate, boreal and mediterranean ecosystems. Only 6 of 49 surveyed case studies showed increases in iWUE significantly higher than predicted from random values.Here we use Monte Carlo simulations to test if an artifact of calculation could explain observed increases in iWUE. We show that highly significant positive relationships between iWUE and CO13C occur independently of changes in 13C discrimination that characterize physiological responses to elevated CO2. Due to a correlation with CO2 concentration, which is used as an independent factor in the iWUE calculation, any tree-ring δ13C data set would inevitably generate increasing iWUE over time. Therefore, although consistent, previously reported trends in iWUE do not necessarily reflect a coherent global response to rising atmospheric CO2. We discuss the significance of these findings and suggest ways to distinguish real from artificial responses in future studies.Our results reveal that increases in iWUE estimated from tree-ring δ Nearly identical increases in iWUE have been reported across biomes Anthropogenic activities have substantially altered atmospheric composition and climate with important implications for terrestrial biomes. Forest ecosystems are expected to be the most responsive, as tree species show higher increases in productivity and greater reductions in transpiration than any other functional type measured in large-scale (e.g. FACE sites) elevated CO2 stimulation, may lead to increased iWUE while limiting tree growth 2 based on the classic calculation of iWUE from tree-ring δ13C. For example, it has been suggested that due to a correlation with CO2, increases in iWUE would occur regardless of source to product (i.e. atmosphere to plant biomass) changes in isotopic fractionation that characterize physiological responses to environmental change 13C do not reflect actual shifts in either carbon uptake or water loss through transpiration. More importantly, it would imply that responses to rising CO2 have been globally overestimated, possibly explaining the lack of a clear effect on tree growth.These findings have generally been interpreted as evidence of large scale (warming-induced) drought stress, which despite CO13C data and classic equations to determine how iWUE values relate to CO2 levels when there is no physiological change in source to product 13C fractionation. Based on simulated tree-ring δ13C data and actual atmospheric δ13C and CO2 measurements, we calculate changes in iWUE over the past century and across a wide altitudinal range. We then compare responses generated from simulated data with results from actual tree-ring δ13C obtained from the recent literature. We discuss our results and their implications for future research, focusing on the significance of previously observed responses and suggesting ways to validate changes in iWUE, testing the effect of atmospheric CO2 on natural forest ecosystems.In this paper, we examine whether artifacts of calculation could explain increasing trends in iWUE reported in the recent literature. We use simulated (random) δ13C) in tree-rings 13C/12C ratio in trees, and other C3 plants, is controlled at the leaf level by the ratio of intercellular (Ci) to ambient (Ca) CO2 concentrations. If Ci is high relative to Ca, strong discrimination against 13C yields isotopically light (12C enriched) biomass. Conversely, if Ci is low there is less discrimination against 13C resulting in higher δ13C values. Regardless of growth rates or net changes in productivity, any change in carboxylation and/or stomatal conductance that alters Ci/Ca is recorded as a change in δ13C 13C also varies over time, tree-rings must be analyzed in relation to atmospheric 13C abundance at the moment of its assimilation. For example, anthropogenic CO2 emissions have decreased the δ13C composition of the atmosphere 13C) result in a reduction of 13CO2 relative to 12CO2. Therefore, physiological changes that occur in coincidence with anthropogenic emissions can only be assessed after changes in atmosphere to plant biomass discrimination are accounted for, which is done as follows 13C is discrimination against 13C, δ13Cair is the carbon isotope ratio of air (the source) and δ13Cplant is the carbon isotope ratio of the product (plant biomass). To be translated into physiologically relevant information Δ can be expressed as:a is the discrimination against 13CO2 during diffusion through the stomata (−4.4‰) and b is the net discrimination due to carboxylation (−27‰). Real increases in carboxylation rates or reduction in conductance, expected in responses to rising CO2 levels, would result in a distinct shift in Δ13C gCO2(Ca−Ci)), Δ13C values can be converted into plant's intrinsic water use efficiency (iWUE) at the moment of biomass production g is the leaf stomatal conductance and 0.625 is the relation between conductance for CO2 molecules and water vapor.The most widely used method to estimate changes in iWUE in natural ecosystems is the analysis of stable carbon isotope ratios  to observed tree-ring data. This method allowed us to build a probability distribution and study what features of the distribution are essential for describing previously reported (observed) patterns. The underlying assumption is that the simulated distribution represents the observed data well enough so that variation in randomized and measured data is the same. Real tree-ring δ13C ranges from about −20 to −30‰, which typically corresponds to water-stressed and unstressed conditions respectively. To represent different portions of this spectrum, we used simulations of δ13C ranging from −20 to −21‰, −25 to −26‰ and −29 to −30‰. At each range we calculated 13C discrimination (Δ) in relation to a constant atmospheric δ13C composition (−8‰), determining changes in iWUE in response to atmospheric CO2 as the only varying factor.To test whether correlations involved in the calculation of iWUE could explain systematic  increases in response to rising CO13Cair and Ca13C data sets. To describe changes in CO2 partial pressure and 13C content with altitude, we relied on well-established relationships between δ13Cplant and Ca partial pressure. Globally, δ13Cplant increases ∼0.8‰ for every 1000 m of altitudinal gain, a pattern that holds independently of plant species 13C data coupled with 13C enrichment expected with altitude (δ13CAlt = 0.0008*Alt+δ13Cplant). We then constructed a simple three-dimensional model of changes in iWUE over the past century and across altitudinal gradients as follows:−1) are a function of both CO2 (ppm) and altitude (m). We then regressed annual percent increases in iWUE calculated from simulated data sets (predicted) against iWUE measured from tree-ring δ13C (observed) in 49 recent studies encompassing 22 species from tropical, subtropical, temperate, boreal and mediterranean ecosystems   , which i2 levels . Randomit change  and 2 an has been generally attributed to decreased carboxylation velocity and investment in Rubisco 2 allows plants to reduce stomatal conductance (g) less than plants growing under ambient CO2g acclimate as suggested by experimental results, maintenance rather than continuous increases in iWUE should be observed as a result of decadal to centennial CO2 stimulation. Our comparisons between predicted and observed iWUE data suggest that this occurred in most case studies surveyed here. The few cases that showed increases in iWUE significantly higher than predicted by simulated data include deciduous and coniferous species, growing in temperate and tropical regions ranging from 100 to about 1600 m asl  Indicates studies where annual percent change in iWUE was reported in the original text; (**) indicates studies where only δ13C series were presented. In all case studies iWUE was determined based on tree-ring δ13C following classic calculations Click here for additional data file.


Mus musculus musculus) in which females were experimentally mated either with their preferred or non-preferred male, and their offspring were infected with a mouse pathogen, Salmonella enterica (serovar Typhimurium).It is often suggested that mate choice enhances offspring immune resistance to infectious diseases. To test this hypothesis, we conducted a study with wild-derived house mice (We found that offspring sired by preferred males were significantly more likely to survive the experimental infection compared to those sired by non-preferred males. We found no significant differences in the pathogen clearance or infection dynamics between the infected mice, suggesting that offspring from preferred males were better able to cope with infection and had improved tolerance rather than immune resistance.Our results provide the first direct experimental evidence within a single study that partner preferences enhance offspring resistance to infectious diseases. The resulting offspring (F2 generation) were used for experimental infection (see below). All F2 mice were weaned at the age of 21 ± 1 days and were kept individually in standard mouse cages (26.5 × 20.7 × 14 cm) with wooden bedding (Abedd: aspen wood chips), enrichment material consisting of nesting material (Abedd: aspen wood shavings), and nest boxes. Food (Altromin rodent diet 1324) and water were provided ad libitum. A standard 12:12 h light cycle was maintained and temperature ranged from 22 to 25°C. After experimental infection animals were housed individually in ventilated cages . All other housing conditions remained identical to conditions prior to infection.The mice used in this experiment were F2 descendants of wild-trapped house mice  and two unrelated males (N = 60). Prior to the social preference test, all females successfully mated and raised one litter to avoid any confounds from females’ sexual experience. Males were sexually mature , however they had no mating experience. On the day of the experiment, all mice were weighed. Mice were brought to the testing room at 12:00 to allow the animals to acclimate for six hours (five triplets were tested per day). During acclimation, male mice were stimulated with 5 μl of pooled female’s urine on filter paper . The experiments started in the dark phase (under red light conditions) and were conducted between 18:00 and 22:00. All trials were recorded with a D-link camera (DCS-3710 Day & Night WDR network camera) sensitive to red light. To avoid any effects from the presence of an observer on mouse behavior all experiments were simultaneously observed outside the experimental room on a monitor connected to the video camera system. Later, recorded videos were analyzed using the program Noldus Observer XD 9.0 to verify initial partner preference of females based on direct monitor observations.The experimental apparatus consisted of three cages Figure : one cenSalmonella enterica serovar Typhimurium . The bacteria (stored as slants at 4°C and originated from frozen stocks at -80°C) were cultured in 7.5 ml of heart-brain infusion at 37°C for 13 h (overnight) while shaking at 170 rpm. The overnight solution was diluted with sterile phosphate buffered saline (PBS) until the desired dilution of 103. To verify the concentration, serial plating of the dilution (50 μl per plate and three plates per solution) was performed.To assess pathogen clearance of F2 offspring from P versus NP sires, a total of 72 offspring resulting from 23 families were experimentally infected on two consecutive days. The number of individuals was balanced for partner preference (P/NP) and sex. All mice were adults (15-21 weeks old) and received an intraperitoneal (IP) infection of 200 μl 2. Additionally, we also monitored the survival of additional P (N = 64) and NP (N = 44) offspring in the colony (same age class), which were not experimentally infected. To determine pathogen loads of experimentally infected animals, spleens of euthanized animals were immediately removed and homogenized  in 1 ml PBS under sterile conditions. Afterwards, 50 μl of each homogenate was plated on selective agar plates , and incubated overnight for 18 hours at 36°C. Pathogen loads per spleen were determined by calculating bacterial concentration (cfu/spleen) of spleen homogenates [All animals (N = 72) were divided into three groups to obtain a sample size of 24 individuals per week during the experimental period of three weeks. Groups were balanced for partner preference and sex. Individuals were euthanized and dissected over three weeks: group 1 after seven days (N = 24), group 2 after 14 days (N = 24), and group 3 21 days post infection (N = 24). The health condition of mice was examined daily until the end of the experiment. All animals were weighed weekly and euthanized humanely using an overdose of COogenates ,37 usingTo assess differences in female reproductive success when paired with P versus NP we compared the number of litters produced in the different treatments using a χ2 test. Differences in litter sizes were analysed using a general linear model (GLM) with pairing  as a fixed factor and female body mass as a covariate. Mean pup weight  was calculated as the total weight of litters divides by litter size at weaning. We compared mean pup weight of litters sired from P versus NP males in using a GLM with pairing  as a fixed factor and female body mass as a covariate. Sex ratio was analysed using a generalized linear model (GZLM) with a binomial distribution and a logit link function. The number of males within a litter was included as the dependant variable and litter size as binomial denominator. Pairing  was integrated as a fixed factor and female body mass as a covariate. These statistical analyses were performed using IBM SPSS® version 19  software.For subsequent pathogen and survival models we tested whether initial body mass was randomly distributed across experimental groups by using a linear mixed-effects model (LMM) with offspring sex and partner preference  as fixed effects and week  as a covariate. We also entered the day of experimental infection as a random effect to control for statistical non-independence of trials started on one or the other date. Additionally, to control for sex difference in body mass, we calculated residuals of body mass on sex and used these residuals as sex-independent measure of body mass. We transformed individual body mass and count data (pathogen load) using log-transformation to enhance normality and homogeneity of variances.To assess individual pathogen load, we applied a LMM, entering partner preference (P/NP), and sex as fixed effects, week as a covariate and starting date of the experiments and family (N = 23) as random effects. We analysed survival using a generalized linear mixed-effects model (GZLMM) with binomial error distribution, entering partner preference treatment, and sex as fixed effects, week and body mass as covariates and starting date of the experiments and family ID as random effects. Statistical analyses were performed using ‘R’ (version 2.14.1). We implemented LMMs using the ‘lme’ function of the ‘nlme’ package, and GZLMM using the ‘glmmPQL’ function of the ‘MASS’ package.We included all two-way interactions into initial models and applied a backward stepwise removal procedure  to avoidThe authors declare that they have no competing interests.SR and DJP designed the study, SR, SS, KET, MT and FW collected the data, SR and AH performed the analysis. All authors contributed towards writing the manuscript and approved its final version.


M−H]− is one of the most dominant anions formed in dissociative electron attachment to various small biomolecules like nucleobases and single amino acids. In the present study, we investigate the [M−H]− channel for the dipeptide dialanine by utilizing an electron monochromator and a two-sector-field mass spectrometer. At electron energies below 2 eV, the measured high-resolution ion-efficiency curve has a different shape to that for the single amino acid alanine, which is explained by the altered threshold energies for formation of [M−H]− determined in quantum chemical calculations. Moreover, the structure of the formed [M−H]− anion is further studied by investigating the unimolecular and collision-induced decay of this anion. Trajectory calculations have been carried out to aid the interpretation of the experimentally observed fragmentation patterns.The dehydrogenated parent anion [ M−H]− − ion yield as a function of the electron energy for the dipeptide dialanine  to alanine shows at electron energies below 3 eV an asymmetric peak structure with the steep onset starting at about 0.8 eV, a maximum at about 1.25 eV, and at least one shoulder at about 1.52 eV on the high-energy side. Other aliphatic amino acids like valine,[M−H]− at low energies. Lowering the electron-energy-beam resolution to about 50 meV led to the same principal shape[ν=4. For molecules with a carboxyl group  resonance is solely involved in the dissociative electron-attachment process leading to [M−H]−.−.,42 The lo [M−H]−. Concernio [M−H]−. for glyco [M−H]−. it was so [M−H]−. However,M−H]−. These thresholds have been computed by using G4(MP2).[M−H]− ion yield may a priori be assigned to a vibrational structure of the amide ν(NH) stretch or the ν(OH) stretch, which have very similar frequencies. We ruled out the latter stretching mode because although the calculated thresholds tend to be in general higher than the experimental ones, the calculated threshold for the loss of hydrogen from the carboxyl group (Hcbx) does not match the first peak at 0.81. However, based on the results discussed below, we favor an alternative explanation for the peak structure in that a site-selective bond-cleavage process leading to different [M−H]− isomers at the two resonances takes place. Hence, we assign the first feature at 0.81 eV to the isomer with loss of hydrogen from the amide group (Hamide) and the 1.17 eV peak to the isomer with loss of Hcbx.This leads to the question as to whether the features observed here for dialanine at 0.81 and 1.17 eV can be assigned like in the case of amino acids to a vibrational structure? We calculated the thermodynamic thresholds for dialanine for the various isomers of [ G4(MP2). This quaamide. To solve this question we utilized the two-sector-field mass spectrometer and studied the fragmentation pattern of the anion [M−H]− formed at low electron energies. Such studies utilizing the mass-analyzed ion kinetic energy (MIKE) scan technique  provide insight into the composition of [M−H]−. Unfortunately, the two-sector-field mass spectrometer has an energy resolution of about 1 eV, that is, both resonances (0.81 and 1.17 eV) will contribute unresolved to the ion yield. It turned out that at the electron energy close to 1 eV, at which [M−H]− is formed most efficiently  of the dehydrogenated anion formed at this energy. Thus one can assume that a relaxed [M−H]− anion exits the ion source and does not decay spontaneously on the way to the detector.From the high-resolution measurements one cannot reach a definite conclusion as to whether the peak structure is formed by two different isomers or from the isomer with loss of HM−H]− (m/z 159) between the magnetic and electric sectors may give insight about such structural isomerism. Therefore we carried out investigations on CID and the corresponding spectrum for the 1 eV resonance of [M−H]− is shown in Figure m/z 42, 44, 71, 72, 87, 88, 98, 99, 115, 142, and 144 with the highest ion signal for m/z 88 and m/z 115. Dissociation after collision-induced activation can be understood as a statistical reaction, in which after initial electronic excitation the excess energy is randomly distributed over the vibrational degrees of freedom. An indication of such a process can also be found in the Gaussian shape of the decay peaks in the MIKE spectra, that is, the kinetic-energy release follows a Maxwell–Boltzmann distribution.− without Hcbx, Hamide, hydrogen from an amino group (Hamin), and hydrogen from two carbon sites , respectively. The technical details are described in the section below. General observations were that the lowest-energy set of trajectories showed no dissociations but only several hydrogen transfers. Trajectories leading to fragmentation are listed in Table 3)COOH]−. The simulations frequently showed such proton-transfer reactions, which in many cases led to stabilizations of the [M−H]− anions. No trajectory without initial removal of Hamin led to dissociation. The most abundant fragment anion in CID is observed at m/z 115. The simulations elucidate two possible reaction pathways leading to this anion: 1) initial hydrogen loss from the amide site, subsequent proton transfer from the carboxyl group (two trajectories), and CO2 removal or 2) loss of CO2 after hydrogen loss from the carboxyl group (one trajectory). The former trajectory is shown as an example in Figure We note that several peaks are observed in the CID spectrum, in which an instantaneous assignment of a peak to a certain fragment anion is not straightforward. Hence we carried out trajectory calculations for [M−H]− isomer without Hamide and Hcbx are also observed in CID , which may obscure slow, sequential decay reactions. However, the present findings show that at the electron energy close to 1 eV a new fragmentation channel is available that is not present for amino acids. Such a remarkable site-selective fragmentation process in biomolecules within a relatively narrow energy range was previously observed in nucleobases.−. This leads to the formation of a number of smaller fragment anions formed promptly in the ion source  of 88 and 115 can be observed. These metastable decay channels have also been recently reported in reference −. In previous electron-attachment studies with dialanine the anion at m/z 88 was also observed abundantly at electron energies between 5 and 6 eV,[3)COOH]− in reference [M−H−CO2]−[20] at m/z 115 and [M−H−H2O]− at m/z 141, respectively. We also measured the electron energy dependence of the metastable decays of [M−H]− into m/z 88 and m/z 115, respectively (not shown). In agreement with reference − formed at the electron energy of approximately 5.5 eV. The resulting spectrum is shown in Figure m/z 88, 115, and 141. At this point the question arises as to why we do not observe in the CID at 5.5 eV a rich fragmentation pattern like at low electron energies. In general, one possible explanation may be that different isomers of [M−H]− are formed at the two different electron energies. In this case the trajectory calculation would again shed light on the isomers involved. For example, the presence of the isomer with loss of Hcarboxyl and Hamide could be excluded, since otherwise the CID at approximately 5.5 eV would show the fragments listed in Table m/z 141 and m/z 88  simulation for the decay of [M−H]− for the single amino acid valine showed only one weaker fragmentation channel (COOH−) but not the main fragmentation channel.[Thus another explanation based on the consideration of the excess energy of the [ channel. NevertheM−H]− isomers without the hydrogen from the carboxyl group and the amide group are formed in the low-energy region. We confirmed this hypothesis by experimental collision-induced decay spectra and MD simulations. This formation of two different isomers is in marked contrast to single amino acids, in which exclusive hydrogen loss from the carboxyl group is possible at low electron energies due to energetic reasons. In contrast to the [M−H]− anion at approximately 1 eV, the [M−H]− anion formed at an electron energy of about 5.5 eV is highly unstable upon fragmentation and represents an intermediate product of sequential decays.We have investigated the formation and decay of the dehydrogenated parent anion formed through electron attachment to the dipeptide dialanine. High-resolution measurements combined with quantum chemical calculations of the threshold energies of different isomers indicate that the [− from CCl4. This resonance at about 0 eV was also used for the calibration of the energy scale.The high-resolution electron-attachment experiment was performed with a crossed-beam setup in combination with a quadrupole mass spectrometer (for more details see reference ). A commM−H]− were studied with a two-sector-field instrument[M−H]− in the field-free region between the magnetic and electric sector were monitored by MIKE scans.[m0 (in the present study [M−H]−) and then scanning the electric-sector field voltage U. Whereas a non-decaying anion with mass m0 passes the electric sector at U0, a fragment anion with mass m1 formed by a decay of the precursor anion with m0 only passes the electric sector at the reduced sector field voltage :M−H]− in the time window between about 10 and 30 μs were studied. Collision-induced decays of [M−H]− were also investigated using the MIKE scan method. However, in this case a gas cell between the magnetic and electric sector was filled with stagnant nitrogen gas inducing decays through collisions.Metastable (unimolecular) decomposition pathways of [Trajectory calculations: We tried to obtain qualitative insight into the fragmentation process of the [M−H]− anions by performing a limited set of trajectory calculations. In one set of calculations [M−H] was described quantum chemically by the B3LYP[the B3LYP density the B3LYP basis sethe B3LYP density the B3LYP,55 The pthe B3LYP was used