Torsade de pointes arrhythmias (TdP) in the chronic atrioventricular block (CAVB) dog model result from proarrhythmic factors, which trigger TdP and/or reinforce the arrhythmic substrate. This study investigated electrophysiological and arrhythmogenic consequences of severe bradycardia for TdP.Dofetilide (25 μg/kg per 5 min) was administered to eight anesthetized, idioventricular rhythm (IVR) remodeled CAVB dogs in two serial experiments: once under 60 beats per minute (bpm), right ventricular apex paced (RVA60) conditions, once under more bradycardic IVR conditions. Recordings included surface electrocardiogram and short-term variability (STV) of repolarization from endocardial unipolar electrograms. TdP inducibility (three or more episodes within 10 min after start of dofetilide) and arrhythmic activity scores (AS) were established. Mapping experiments in 10 additional dogs determined the effect of lowering rate on STV and spatial dispersion of repolarization (SDR) in baseline.p < 0.05). Mapping: Pacing from high (128 ± 10 bpm) to middle (88 ± 10 bpm) to experimental rate (61 ± 3 bpm) increased all electrophysiological parameters, including interventricular dispersion, due to steeper left ventricular restitution curves, and intraventricular SDR: maximal cubic dispersion from 60 ± 14 (high) to 69 ± 17 (middle) to 84 ± 22 ms (p < 0.05 vs. high and middle rate).IVR-tested animals had longer baseline RR-interval  and repolarization intervals than RVA60 animals. Dofetilide increased STV similarly under both rhythm strategies. Nevertheless, TdP inducibility and AS were higher under IVR conditions (6/8 and 37 ± 27 vs. 1/8 and 8 ± 12 in RVA60, respectively, both In CAVB dogs, severe bradycardia increases the probability and severity of arrhythmic events by heterogeneously causing electrophysiological instability, which is mainly reflected in an increased spatial, and to a lesser extent temporal, dispersion of repolarization. Ventricular arrhythmias comprise a multitude of life-threatening conditions that often result in sudden cardiac death (SCD) . Much reThe employment of the chronic atrioventricular block (CAVB) dog model has resulted in great advances in the research field of ventricular arrhythmias. The reproducibility of torsade de pointes arrhythmias (TdP) in this model results from complex ventricular adaptation processes, initiated by ablation of the proximal His bundle. The subsequent chronic drop in heart rate and thus cardiac output, combined with an altered ventricular activation pattern, initiate contractile, structural, and electrical remodeling processes. Cumulatively, these adaptations reestablish an adequate cardiac output but simultaneously, and adversely, also increase susceptibility for TdP .IKr and IKs, causes this reserve to become chronically impaired. As such, electrical instability induced by additional debilitating factors, such as infusion of a proarrhythmic drug, is inadequately compensated for, resulting in the appearance of triggered activity and TdP  conditions, anesthesia, and dofetilide inducing further slowing of IVR. In the other experiment, all animals were continuously paced at 60 beats per minute (bpm) from the right ventricular apex (RVA60). Additionally, detailed in vivo mapping experiments were conducted under different pacing frequencies to elucidate the effect of pacing frequency on SDR.This study therefore specifically aimed to All experiments were approved by the Committee for Experiments on Animals of Utrecht University, Netherlands. Animal handling and care wEre in accordance with the European Directive for the Protection of Vertebrate Animals Used for Experimental and Scientific Purposes 2010/63/EU of the European Parliament and Council of September 22, 2010. This study included a total of 18 adult purpose-bred mongrel dogs .ad libitum access to water and were provided with dog food pellets twice a day. Daily checks on health and comfort were performed; weight was measured once a week.Animals were housed in conventional dog kennels enriched with wooden bedding and playing tools. If possible, dogs were housed in pairs and let out of the kennel at least once a day to go outside and play. The animals had 2 and N2O (1:2).Premedication consisted of 0.5 mg/kg methadone, 0.5 mg/kg acepromazine, and 0.02 mg/kg atropine [intramuscular (i.m.)]. In addition, prophylactic antibiotic ampicillin  was given before and after the experiment, and perioperative analgesics consisted of meloxicam (Metacam) (0.2 mg/kg subcutaneously preoperatively) and buprenorphine . General anesthesia was induced using pentobarbital sodium [25 mg/kg intravenously (i.v.)] and maintained with mechanical ventilation of isoflurane (1.5%) in a mixture of OA pacemaker screw-in lead was introduced through the jugular vein and positioned in the RVA after which it was connected to an internal pacemaker . Subsequent radiofrequency ablation of the proximal His-bundle induced complete AV block. All animals were allowed a minimum remodeling period of 3 weeks. All animals remodeled under continuous IVR conditions except for three dogs used in the mapping experiments, which remodeled under continuously RVA paced conditions at the lowest captured rate. Experiments included in the serial study were performed 5.6 ± 1.8 weeks after creation of AV block, whereas the mapping experiments were performed 14.1 ± 4.6 weeks after creation of AV block.Electrophysiological recordings were continuously made during all experiments and consisted of a standard six-lead electrocardiogram (ECG) with four additional precordial leads, right ventricular (RV) monophasic action potential (MAP) catheters , and either a left ventricular (LV) MAP catheter or a duo-decapolar catheter  recording unipolar electrograms (EGMs) from 10 distinct endocardial regions in the LV. The measurements derived from a MAP or an EGM catheter are interchangeable .Eight animals were subjected to a dofetilide challenge twice; in one experiment, animals were in IVR, anesthesia, and dofetilide slowing heart rate, whereas the other experiment was performed under RVA60 conditions.IKr blocker, to assess inducibility for TdP. Electrophysiological recordings were continued for a minimum of 10 min following start of infusion. In experiments where TdP occurred within 5 min of dofetilide infusion, administration was immediately discontinued. In addition, persistent arrhythmias (>10 s) were manually terminated by electrical cardioversion, applied via thoracic patches.The dofetilide challenge was performed as previously described by In vivo mapping experiments were performed in 10 other animals to assess the effects of acute bradycardia on SDR under baseline conditions. In these experiments, 56 needles with each four recording electrodes (interelectrode distance 0.4 mm) were evenly inserted into the LV and RV walls  were analyzed using EPTracer software . All intervals were determined from lead II of the surface ECG and measured manually from five consecutive beats during baseline and either prior to the first ectopic beat or at 5 min following start of dofetilide infusion. QT interval was corrected for heart rate (QTc) using the Van der Water formula . AdditioActivation time (AT) was manually determined as the time difference between the start of the QRS complex (IVR) or pacing spike (RVA60) and the steepest upstroke of the MAP or the steepest downstroke of the QRS in the EGM . The MAPSTV of repolarization was calculated from 30 consecutive beats using the formula:D being LV ARI or LV MAPD (LV ARI/MAPD) or RV MAPD  . For allAnimals were defined to be inducible when three or more episodes of TdP occurred during the 10-min period after the onset of dofetilide administration. TdP were defined as a polymorphic ventricular tachycardia of five beats or more, twisting around the isoelectric line.n + 1 rule, wherein n reflects the number of ectopic beats. Persistent TdP were scored according to the number of defibrillation shocks needed to terminate the TdP; one defibrillation was awarded 50 points, two cardioversions were given 75 points, and three or more shocks were given 100 points. AS was then calculated by averaging the scores of the three most severe arrhythmic episodes within the 10-min interval  was calculated to quantify the severity of the arrhythmic activity in the experiments. Ectopic events were scored according to the interval .t-tests. Inducibility was analyzed using the McNemar test. AS was analyzed with the Wilcoxon signed rank test, and the Mann–Whitney U test was used for the number of TdP. P < 0.05 was considered statistically significant.All obtained data are expressed as mean ± standard deviation (SD). Statistical analyses and comparison of serial electrophysiological data were performed with (un)paired Student p = 0.07), but this was not accompanied by changes in QRS morphology. In comparison to the IVR experiments, pacing from the RVA  significantly delayed LV activation  and shortened LV MAPD/ARI and RV MAPD from 313 ± 45 and 251 ± 29 ms to 254 ± 28 and 228 ± 25 ms, respectively (both p < 0.05 vs. IVR experiments). The differences in pacing rate did not affect temporal dispersion of repolarization, as baseline LV and RV STV did not differ between both rhythm strategies . Moreover, dofetilide caused a substantial prolongation of LV MAPD/ARI, RV MAPD, and ΔMAPD under both rhythm strategies   in IVR experiments was significantly higher than the 13% (1/8) observed in the RVA60 experiments ( < 0.05) . Moreoven = 10) established the effects of stepwise lowering heart rate on spatial heterogeneity in repolarization. Lowering pacing rate from high to middle rate pacing prolonged repolarization as demonstrated by QTc and LV ARI from 344 ± 12 and 202 ± 15 to 365 ± 15 and 229 ± 17, respectively, both p < 0.05 (p < 0.05). Subsequent lowering to experimental rate caused a further prolongation of repolarization; LV ARI to 260 ± 19 ms (p < 0.05 vs. high and middle rate) and RT to 325 ± 20 ms (LV) and 279 ± 15 ms (RV) (both p < 0.05 vs. high and middle rate). Moreover, although insignificant, a modest trend toward increasing temporal dispersion seemed to appear as rate was decreased from high to middle rate .Detailed mapping experiments (p < 0.05 . This efARI restitution curves were dissimilar for the LV and the RV. The steeper slope of the LV curve reflected the greater prolongation of LV ARI in response to increasing cycle lengths . A compap < 0.05 vs. high and middle rate).Intraventricularly, SDR in the LV increased as pacing rate was stepwise decreased, starting from a rate similar to canine sinus rhythm. This increase was most prominent under the most bradycardic conditions, as average and maximal SDRs were significantly increased in all orientations compared to both high- and middle-rate pacing . This efin vivo that this can be explained by (3) bradycardia-induced increases in SDR in three dimensions, and (4) to a much lesser extent temporal dispersion of repolarization.This study established the contribution of severe bradycardia in arrhythmogenesis and correlated its proarrhythmic effects with changes in electrophysiological parameters. The results have confirmed that severe bradycardia increases (1) the likelihood of arrhythmia development and (2) the severity of arrhythmic events and show for the first time The two main characteristics of the CAVB dog—the compensated heart failure combined with its increased susceptibility for TdP—which both result from ventricular remodeling, make this animal model unique in the research field of ventricular arrhythmias. Also in humans, cardiac remodeling that results in compensated heart failure is associated with an increased risk of SCD as the result of ventricular arrhythmias.This disposition for ventricular arrhythmias arises from multiple electrical alterations that cumulatively augment the likelihood that ectopic stimuli are generated and enhance spatial differences that facilitate the spread and perpetuation of such stimuli. Hence, it creates a substrate, which facilitates the development of ectopic beats and TdP. However, an additional trigger is needed to provoke such events. These triggers initiate ectopic stimuli by destabilizing cellular electrophysiology to such extent that early or late after-depolarization is provoked; this increasing instability is reflected by the temporal dispersion of repolarization, quantified as the STV . In mostIn the serial comparison, dofetilide induced ectopic events in all animals, regardless of rhythm strategy. Nevertheless, in RVA60-paced animals, these events were limited to single and multiple ectopic beats, whereas TdP were abundantly present in the IVR tested animals . This diHowever, the increased incidence and severity of arrhythmic events in IVR experiments were not reflected in the dofetilide-induced increases in repolarization duration and/or STV . This diCorrespondingly, the additional mapping experiments that studied SDR under baseline conditions demonstrated that heterogeneities in interventricular and intraventricular repolarization durations increased as pacing frequency was gradually lowered. Interventricular dispersion of repolarization is known to be of importance in TdP arrhythmogenesis and to be a bradycardia-dependent phenomenon . As ΔAPDin vivo in a guinea pig model of long-QT syndrome three. They demonstrated that bradycardia augmented the baseline heterogeneities in APD, which promoted ventricular arrhythmogenesis. However, in contrast to the current study, these studies were limited in their assessment of repolarization to either a small number of RV sites or the epicardium, precluding the evaluation of global ventricular changes. Hence, while prior studies have underlined the importance of bradycardia as a modulator of arrhythmogenesis and demonstrated its effects on SDR, the current study is unique in its high-resolution, global, three-dimensional evaluation of these effects in an in vivo large animal model.These results correspond to prior studies reporting an increased SDR under bradycardic conditions. For example, Nevertheless, even though the aforementioned studies and the current study demonstrate a clear correlation between heart rate and changes in dispersion of repolarization, instability of other electrical properties, for example, myocardial activation, might have also played into the observed episodes of arrhythmogenesis.Interestingly, dofetilide tended to prolong the PP intervals more in the RVA experiments than in the IVR experiments. Hence, a heightened sympathetic tone in the more bradycardic IVR experiments could have promoted the more arrhythmogenic outcome of these experiments.For the experimental setting, these results emphasize the importance of carefully considering rhythm control in studies in the field of arrhythmogenesis and antiarrhythmic interventions. For example, in studies testing drugs, it is important that one is able to discriminate between arrhythmogenic effects related to the drug tested and experimental intrinsic effects, such as the administration of anesthetics . The ideIn the clinical setting, it is important to understand how heart rate directly affects cardiac electrophysiology and how it can both contribute to and impede ventricular arrhythmogenesis. Especially the latter observation is of great clinical interest. The observation that arrhythmogenesis can be impeded by increasing pacing rate highlights the potential of pacemakers to treat chronic cardiac conditions wherein there is an increased risk of ventricular arrhythmogenesis.Inconsistent ventricular activation is known to cause electrophysiological instability, possibly evoking an electrical storm. In IVR experiments, the uncontrollability of ventricular activation focus might have been an additional proarrhythmic factor. However, no changes in QRS morphologies were observed in the serial comparisons, implying that instability of ventricular activation is unlikely to have caused the increased arrhythmic activity observed in the IVR experiments.In addition, as dofetilide infusion is stopped prematurely when TdP occur within the 5-min window of infusion, less dofetilide was administered in IVR experiments. Therefore, the obtained result might still be an underestimation of the proarrhythmic effects of severe bradycardia.Moreover, pacing the animals in the serial experiments could have been a confounding factor as eliciting an activation front may have interfered with arrhythmogenesis. Nevertheless, as we see a similar increase in STV and observe a comparable initiation of ectopic events, we believe that the increased TdP incidence in IVR experiments is a result of the greater increase in SDR. As we show that the extent of SDR closely correlates to heart rate, we do not believe that cardiac pacing was responsible for the difference in arrhythmogenesis.Lastly, the frequency–dependency of SDR was only studied under baseline conditions and not additionally tested following the administration of dofetilide. Hence, these mapping experiments demonstrate that bradycardia induces proarrhythmic increases in SDR but do not directly demonstrate its role in arrhythmogenesis.Severe bradycardia in the CAVB dog increases the probability of arrhythmia development and the severity of arrhythmic events by heterogeneously causing cellular electrophysiological instability, which is mainly reflected in an increased spatial, and to a lesser extent temporal, dispersion of repolarization.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by The Committee for Experiments on Animals of Utrecht University, Netherlands.VW, AD, AB, RV, VM, and JBe carried out the experiments. VW wrote the manuscript with support from MH and MV. JBa and RC helped supervise the project and revise the manuscript. MV supervised the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The participants performed an exercise test with a gradually increasing intensity until the individual maximum exercise capacity was reached, before and after 8-d fast. Blood samples were collected before, immediately after, and 3 h after exercise. Gene expression was analyzed by reverse-transcription quantitative polymerase chain reaction and protein levels were analyzed by immunobloting. Eight days of total starvation diet affected the body composition and decreased exercise capacity. Further, fasting decreased the expression of genes associated with iron storage and export, and increased the expression of genes involved in iron acquisition. Conversely, only PCBP2 protein increased after fasting; however, an upward trend was apparent for all proteins. In conclusion, the body adapts to starvation by adjusting iron economy.The popularity of fasting and restricted food intake is increasing. While the body’s adaptability to dietary insufficiency is crucial for health, molecular mechanisms of adaptive changes are not well understood. Here, we compared the effects of fasting and exercise on the expression of leukocyte genes and proteins involved in the storage, export, and acquisition of iron, an essential element with physiological roles. Healthy men participated in the study (age, 30–70 years; body weight, 60–100 kg; body mass index, 20–29.9 kg/m Periods of starvation were natural for human ancestors. Indeed, calorie restriction is associated with improved health, weight reduction, increased insulin sensitivity, diabetes control, or cancer prevention . The humIn conditions of iron deficiency synthesis of proteins responsible for iron incorporation into the cell is activated. These proteins include the transferrin receptor (TFRC) and divalent metal transporter (DMT1). Cellular iron is then used up and its excess stored in ferritin. Ferritins (FTH and FTL) play a crucial role in iron storage and play important antioxidative role by regulating the labile iron pool (LIP). In addition, chaperone proteins, human poly(rC)-binding proteins (PCBPs 1–4) bind iron; however, the physiological role of these proteins is not fully understood. In mammalian cells, only PCBP1 and PCBP2 are expressed at high levels . AccordiDespite notable interest in the changes in the body elicited by fasting, data on the changes in iron metabolism are not conclusive, and mostly pertain to alterations in serum or plasma iron levels. For example, Afolabi et al. (2007) showed that 5 d of total starvation exerts only a small effect on iron serum levels and plasma transferrin levels. Similarly, a 2-d fast does not significantly affect iron levels in human. By contrast, in women who starved for 2 d over an 8-d period in a 48-d study, a significant decrease of serum and hair iron levels was observed, as well as a decrease of ferritin and hemoglobin (Hb) levels, hematocrit (Hct), red blood cells, and total iron binding capacity . The aboFasting leads to a body energy deficit and should reduce the exercise capacity. Maintaining the appropriate level of iron is important because of its role in oxygen transport via Hb and myoglobin, and in energy production, which highlights its crucial role in physical activity . MoreovePrevious study on animal model showed that prolonged fasting could activate hypoxia inducible factor  . This trIndependent of fasting, acute physical activity affects iron and iron-regulatory protein status by increasing oxidative stress and inflammation ,26,27. IFerritins and APP mRNA levels decreased significantly after starvation period  and 3 h after exercise (p = 0.006) while FTL mRNA levels significantly decreased 3 h after exercise (p = 0.048). Minor changes in APP and TFRC mRNA levels were noted at the same time.Before starvation, exercise caused a decrease in A levels A,B. In dFTL mRNA level 3 h from the end of the effort (p = 0.05) (p = 0.03) immediately after the effort. No effects of exercise were observed for other tested genes.After starvation, exercise caused a significant decrease in  = 0.05) B and a sp = 0.02) and a significant decrease 3 h after the exercise . No such changes were observed for exercise performed after 8 d of total starvation diet. Exercise before fasting did not affect FTH levels. Interestingly, despite the lack of statistical significance, an upward trend was noted for both ferritin levels in response to fasting  (p = 0.03) (p = 0.047).Changes in the intracellular levels of tested proteins were largely not statistically significant. However, exercise before fasting resulted in a significant increase in FTL levels immediately after the exercise  . We also = 0.03) . FurtherIn the current study for the first time we demonstrate that 8-d fasting significantly modulate gene and protein expression involved in iron metabolism in leucocytes. In addition, the effects of exercise before and after starvation has been explored. The studied genes and proteins can be divided into those involved in import of iron into a cell and its storage  and those responsible for its excretion like APP, FPN, PCBP3, PCBP4.ferritins, APP, PCBP1 and PCBP2 mRNAs rapidly decreased after fasting, while protein levels remained unchanged. Our results presenting FTH, PCBP1 and PCBP2 mRNA levels in response to starvation were similar to those described by Grzybkowska et al. (2019) where authors investigated changes in expression of the same genes in leukocytes of healthy individuals in response to a 100 km marathon [FTH and FTL genes in leukocytes after 8 d of total starvation. One of the reasons of this changes could be related to reduction of labile iron pool (LIP) levels within cells [FTH and FTL translation and protects TRFC against degradation Furthermore, observed increase in TFRC mRNA after starvation support this conclusion as it has been reported that activation of IRP leads to upregulation of the receptor as IRP protects TFRC mRNA from degradation [APP translation [APP mRNA after starvation indirectly points that it is possible that decrease LIP in this cells also decreased. Furthermore, IRE regions have been discovered in several transcripts of genes not associated with iron metabolism, such as: mitochondrial aconitase or hypoxia inducible factor 2 alpha (HIF2α) [HIF-1 alpha mRNA, which may indirectly indicate hypoxia conditions within cells. It has been shown that hypoxia significantly reduced transcription all ferritins in cultured lens epithelial cells, however this effect was stronger for FTH [FTH and FTL mRNA after starvation decreased comparably, and this difference could be associated with specificity of various types of cells. Moreover, similarly to our results, Goralska et al. (2014) observed the hypoxia-induced reduction in ferritin gene transcripts not corresponding to expression of ferritin proteins [FTH, but increased its translation through decreased IRP1-IRE interaction [FTRC similar to Goralska et al. (2014), which also indicate that during fasting there was hypoxia conditions within WBC.The data referring to changes in the expression of genes related to iron metabolism and intracellular proteins in leukocytes are limited . To the marathon . The autmarathon . On fissmarathon . In our in cells  however in cells . Under iradation . Consistnslation . Observe (HIF2α) . For exa (HIF2α) . After 8 for FTH . Our resproteins . Accordieraction . FurthemHIF-2a mRNA contains IRE which indicates its dependence on iron level [Because iron deficiency influences energy metabolism, mechanisms described above can lead to a significant inhibition of energy-consuming processes such as translation in cells and this can explain the discrepancy between transcript and protein levels. Significant increase in HIF-1a influence on energy production within cells. On a cellular level, reaction in which ATP is utilized are suppressed . Literaton level , howeveron level . Increason level ,44. Durion level . Moreoveon level . It is pon level . It is dPCBP1 and PCBP2 which encode iron chaperone proteins but have different roles. PCBP1 is mainly involved in the transfer of iron to ferritin, while PCBP2 is involved in iron export from a cell [PCBP2 and PCBP1, and increase protein level of PCBP2 after fasting. PCBP2 binds to ferroportin and participates in the export of iron from the cell; it also binds to DTM1 and plays a role in the intracellular transport of iron or iron delivery to metalloenzymes [PCBP1 and PCBP2 mRNA levels. This decrease could be associated with the use of transcripts for protein biosynthesis (a significant increase in PCBP2 levels was observed). Moreover, it is likely that gene transcription was inhibited during energy deficiency. Collectively, observations of the current study indicate that during fasting, mRNAs of genes associated with iron homeostasis are degraded in response to iron starvation. Physiological meaning of these changes remains to be studied. PCBP4 can protect from iron toxicity as mention above most of the data indicates that LIP decrease as a result of fasting. However, fasting can also induce autophagy of iron containing proteins thus iron trafficking what may enhance iron toxicity [Other genes of our interest were m a cell . Besidesm a cell . Interesm a cell . Here weoenzymes ,7. Howevtoxicity . Converstoxicity .All of these data indicate that during starvation leucocytes adopt to scarcity of iron by lowering FTH, FTL and APP expression and increase expression of TFRC. The changes are probably trigger by decrease in LIP.In the current study, fasting significantly reduced exercise performance in all study subjects. Increase in FTL levels 1 h after the exercise was the only detected difference in the levels of proteins involved in iron metabolism. FTL is mainly responsible for iron storage and its synthesis is controlled on both, transcriptional and translational levels. Previously, we have demonstrated that during stress, when stress activated protein kinases are functioning, ferritin undergoes partial degradation and releases iron . IncreasThe study was approved by the Committee for Ethics in Scientific Research of Jan Dlugosz University in Czestochowa . The research project was performed respecting and applying the principles formulated in the World Medical Association Declaration of Helsinki—Ethical Principles for Medical Research Involving Human Subjects. All study participants have been informed about the purpose of the study and the associated risks of research methods used. Study participants had practiced fasting many times before. Their next voluntary fasting session was combined with the presented research. Qualified medical personnel informed the surveyed men about possible negative health effects of such practice. The subjects remained under medical care throughout the entire period of fasting. The participants gave their written, voluntary, and informed consent to participate in the study.2; (5) no chronic diseases; (6) systolic blood pressure in the range of 100–140 mmHg and diastolic blood pressure in the range of 60–90 mmHg. The Exclusion criteria were: (1) the presence of chronic diseases; (2) smoking cigarettes; (3) the use of medications, strong stimulants or psychoactive substances; (4) failure to complete the test procedure.Thirteen volunteers, healthy men who had previously practiced starvation diet (for 3–42 d) participated in the study. The number of fasts previously held by the individual respondents ranged from 3 to 12. Each participant avoided starvation in the 6 months before the study. All participants underwent a medical examination and no contraindications to performing exhausting exercise were found. The subjects stated that they had practiced regular physical activity in the form of moderate-intensity yoga; their level of physical fitness was not controlled by the authors of the study. The inclusion criteria were: (1) previous experience in undertaking CWF of more than three days; (2) age range of 30–70; (3) body weight between 60–100 kg; (4) body mass index—BMI—20–29.9 kg/mThe basic somatic parameters of the participants are summarized in The participants, in good physical and mental health, arrived in the laboratory in the morning, after a 12-h fast and a night’s rest, having abstained from alcohol, medication, and exercise for the previous 2 d. At the beginning of the study, the subjects’ age and basic somatic data were recorded , were determined via bioelectric impedance analysis using the Tanita TBF 300 A body composition analyzer .The participants performed an exercise test of gradually increasing intensity until the individual maximum exercise capacity was reached (until exhaustion) using Excalibur Sport cyclo ergometer . The initial load was 60 W, gradually increasing by 30 W every 3 min. The exercise was stopped when the subject was unable to maintain the set pedaling rhythm; when the oxygen uptake began to stabilize at its maximum (or decreased); or when the heart rate did not increase or stabilized at the maximum level (or began to decrease). After 8 d of complete fasting, during which the subjects only consumed ad libitum mineral water with an average ionic content, the procedures described above were repeated.For biochemical and gene expression analyses, 10 mL of blood was collected from the venous vein. For Hb, Hct, β-hydroxybutyrate ), and uric acid level  determinations, the blood was collected twice: at baseline (before the first exercise) and after 8 d of starvation (before the second exercise).g, 10 min), and the obtained leukocyte pellet was divided in two , and processed as described in To assess leukocyte gene expression and intracellular protein levels, the blood was collected at three time points: before each exercise (before and after starvation), and 3 min and 3 h after the test to refuse of work. Immediately after collection, 4 mL of the whole blood were mixed with 20 mL of the red blood cell lysis buffer , and incubated on ice for up to 20 min. The samples were then centrifuged  as instructed by the manufacturer.g, 30 min) and the aqueous phase was collected. RNA was precipitated by the addition of 500 µL of isopropanol. After 15-min incubation at room temperature (22 °C), the samples were centrifuged again . The obtained pellet was rinsed in 1 mL of 75% ethanol and centrifuged . After pouring out the ethanol and drying, the pellet was suspended in 25 µL of water. The RNA quantity and quality were checked using a photometer . For all samples, the A230/A260 and A230/A320 read ratios were over 1.7. Applied procedure was previously described [RNA was isolated as described by Chomczyński and Sacchi (1987). Briefly, the leukocytes were lysed in 800 µL of Fenozol . After 10-min incubation at 50 °C to separate the aqueous phase containing RNA, 200 µL of chloroform were added. Next, the samples were centrifuged  with oligo-dT primers. Gene expression was analyzed by RT-qPCR . Each sample was analyzed in triplicate. Polymerase  was used on Eppendorf Mastercycler Gradient 5331.TUBB) was used as a housekeeping gene. The following specific primers were used (Primer 3 Web tool)  :g for 20 min. Protein concentration in the lysates was determined using a Bradford method [w/v) solution of dried skim milk in TBS buffer containing 0.1% (v/v) Tween 20, the membrane was incubated with specific primary antibodies, overnight at 4 °C. The following primary, anti-rabbit antibodies (1:1000 dilution) were used: anti-SLCA40A1 ; anti-hnRNP E1 ; anti-hnRNP E2 ; anti-FTH1 ; and anti-Ferritin Light Chain . All antibodies have been validated in previous cell line studies. The membrane was then incubated with anti-rabbit secondary antibody (1:20 000 solution) . The immunoreactive bands were visualized using enhanced chemiluminescence and Chemi Doc MP imaging system. Changes in protein levels were normalized to total protein load in each lane using Image Lab Software (Bio-Rad).The leukocytes were prepared as described in d method  and sampt-test was used. Additionally, two-way ANOVA was performed to determine the effects of fasting and exercise. To calculate the effect size, Cohen’s d was used. Standardized effects were classified as small (>0.2), moderate (>0.5), and large (>0.8). All calculations were performed in GraphPad Prism 6.0 .Basic statistical analyses of all parameters were performed, and the mean values and SEM were calculated. To check the normality of the distribution, Shapiro–Wilk test was used. To compare the values at the same time points before and after starvation or before and after exercise, paired p < 0.05.Gene expression was calculated as a comparative Ct method described by Schmitten and Livak (2008). Housekeeping gene TUBB was selected experimentally. In order to calculate relative expression, we measured Ct values of target and housekeeping genes. The difference between the threshold cycles was displayed as binary logarithm (log2). The relative expression was calculated in Microsoft Excel 2010. After data transformation to linear values, and statistical significance was evaluated using the Shapiro–Wilk test to assess for normal distribution and the Wilcoxon test for comparison of results before and after fasting. All calculations and graphics were performed using GraphPad Prism 6.0. Differences were considered statistically significant at a level of To determine changes in blood and plasma volume, Dill and Costill  formula In conclusion, our data revel that fasting induce significant changes in genes expression and only small changes in proteins involved in iron metabolism in human leukocytes. Observed decrease in genes involved in iron storage and export and increase in those responsible for iron import into a cell resemble those data when only iron deficiency was applied. Conversely, we did not observe significant changes in proteins like ferritin H, L and ferroportin after 8 d of fasting what has been shown in iron deficiency state induced e.g., by iron chelator . CertainOur study has some limitations: We investigated changes in mRNA and proteins only in WBC. Our results concern only healthy men adapted to starvation.


CNR1, GRIN2A, PENK, and PDYN genes and analyzed two peripheral pain sensation-related traits and seven analgesia-related traits in postoperative pain management. A total of 192 volunteers who underwent the peripheral pain sensation tests and 139 and 252 patients who underwent open abdominal and orthognathic cosmetic surgeries, respectively, were included in the study. None of the four STRs or VNTRs were associated with peripheral pain sensation. Short tandem repeats in the CNR1, GRIN2A, and PENK genes were associated with the frequency of fentanyl use, fentanyl dose, and visual analog scale pain scores 3 h after orthognathic cosmetic surgery , analgesic dose, including epidural analgesics after open abdominal surgery , and visual analog scale pain scores 24 h after orthognathic cosmetic surgery , respectively. The associations between STRs in the CNR1 gene and the frequency of fentanyl use and fentanyl dose after orthognathic cosmetic surgery were confirmed by Holm’s multiple-testing correction. These findings indicate that STRs in the CNR1 gene influence analgesia in the orofacial region.Short tandem repeats (STRs) and variable number of tandem repeats (VNTRs) that have been identified at approximately 0.7 and 0.5 million loci in the human genome, respectively, are highly multi-allelic variations rather than single-nucleotide polymorphisms. The number of repeats of more than a few thousand STRs was associated with the expression of nearby genes, indicating that STRs are influential genetic variations in human traits. Analgesics act on the central nervous system via their intrinsic receptors to produce analgesic effects. In the present study, we focused on STRs and VNTRs in the  Pain sensation is an inherent mechanism to avoid tissue damage from injuries and burns. It is an essential system for the living body, but excessive pain is a harmful sensation that evokes unpleasant and aversive reactions. Therefore, there is a clinical need to adequately control severe pain. Opioids, including morphine and fentanyl, have been used as analgesics to alleviate severe pain in many medical situations, such as cancer pain treatment and postoperative pain management. Opioid analgesic consumption varies widely among countries. Palliative care has been reported to be practiced adequately or moderately in the United States, Canada, Europe, and Australia . HoweverOPRM1) gene is the most studied genetic variation that is associated with individual differences in opioid analgesia (ABCB1), adrenoceptor β1 (ADRB1), catechol-O-methyltransferase (COMT), cytochrome P450 family 2 subfamily D member 6 (CYP2D6), cytochrome P450 family 3 subfamily A member 4 (CYP3A4), solute carrier family 22 member 1 (SLC22A1), and UDP glucuronosyltransferase family 2 member B7 (UGT2B7) genes were also shown to be associated with analgesic effects of opioids , short tandem repeats (STRs), variable number of tandem repeats (VNTRs), copy number variations (CNVs), and retrotransposons, exist in the human genome. Short tandem repeats and VNTRs are typically classified as repetitive sequence motifs of less than six base pair (bp) nucleotides and more than six bp, respectively. Short tandem repeats and VNTRs were identified in the human genome at approximately 0.7 and 0.5 million loci, respectively, which are less than the 85 million loci for SNPs . Howeverneration . The genCNR1), glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A), prodynorphin (PDYN), and proenkephalin (PENK) genes were reported to be associated with the vulnerability to schizophrenia and substance dependence , which is a subunit of NMDA receptor channels. GRIN2A gene expression is abundant in the central nervous system and increases during development in humans and mice  score after surgery in postoperative pain management.In the present study, STRs and VNTR in the The present study was conducted with clinical data from 192 participants to assess peripheral pain sensation, 139 patients who underwent major abdominal surgery, and 252 patients who underwent orthognathic cosmetic surgery. Volunteers who lived in the Kanto area of Japan were enrolled in the study to assess peripheral pain sensation. The patients who underwent major open abdominal surgery with combined general and epidural anesthesia were enrolled at the Research Hospital, Institute of Medical Science, The University of Tokyo, or Toho University Sakura Hospital. Patients who were scheduled to undergo orthognathic cosmetic surgery  for mandibular prognathism were enrolled at Tokyo Dental College Suidoubashi Hospital. All of the subjects provided written informed consent before participating in the study.The study protocol was approved by each Institutional Review Board at the Institute of Medical Science, The University of Tokyo , Toho University Sakura Hospital , Tokyo Dental College , and the Tokyo Metropolitan Institute of Medical Science .In the study of peripheral pain sensation, latencies to cold and mechanical pain perception were measured in the cold pressor- and mechanical stimulation-induced pain tests, respectively. The cold pressor-induced pain test was slightly modified from original methods . BrieflyPostoperative pain management after open abdominal surgery was previously described . BrieflyThe clinical data on postoperative pain management after orthognathic cosmetic surgery were previously reported . BrieflyEither whole blood or oral mucosa samples were collected from all subjects for the genetic analysis. DNA was extracted from whole blood or oral mucosa samples using the conventional phenol-chloroform method or QIAamp DNA Mini Kit  according to the manufacturer’s instructions.CNR1, GRIN2A, and PENK genes was performed by Aoba Genetics, Inc., . Approximately 15 ng of genomic DNA from each participant was used as the template for genotyping using polymerase chain reaction (PCR) with fluorescent dye-labeled primers and an ABI PRISM 3130xl Genetic Analyzer . Briefly, PCRs were performed with a 5′-VIC-labeled specific forward primer for each STR and Invitrogen AccuPrime Taq DNA polymerase, High Fidelity , under the following conditions: 94°C for 2 min, 35 cycles of 94°C for 30 s, 57°C for 30 s, and 68°C for 30 s, followed by 68°C for 7 min. The specific primers for STRs in the CNR1, GRIN2A, and PENK genes were the following: CNR1 STR , GRIN2A STR , PENK STR forward, 5′-TAATAAAGGAGCCAGCTATG-3′; reverse, 5′-ACATCAGATGTAAATGCAAGT-3′; Genotyping STRs in the PDYN gene was previously described . All of the clinical data, including the participants’ demographic data and analgesic requirements of opioids after open abdominal surgery and orthognathic cosmetic surgery, were first analyzed for normal probability distributions and homoscedastic variance using the Kolmogorov-Smirnov test and p < 0.001, Mann-Whitney U test). The distribution of the participants’ ages in the study of peripheral pain sensation was significantly associated with latencies to cold and mechanical pain perception . The latency to mechanical pain perception were significantly different between sexes . The latency to cold pain perception was associated with mechanical pain perception . The distribution of ages of patients who underwent open abdominal surgery was significantly associated with the frequency of analgesic use during the 24 h postoperative period . The body weight and BMI distributions for patients who underwent orthognathic cosmetic surgery were significantly associated with 24 h postoperative fentanyl dose .The present study included three association analyses with clinical data on peripheral pain sensation and analgesic requirements of opioids after open abdominal surgery and orthognathic cosmetic surgery . The disCNR1, GRIN2A, and PENK genes and VNTRs in the PDYN gene and examined whether STRs and VNTRs in these genes were associated with peripheral pain sensation and analgesic requirements of opioids. Short tandem repeats in the CNR1, GRIN2A, and PENK genes were AAT trinucleotide, GT dinucleotide, and CA dinucleotide repeat polymorphisms, respectively. The VNTR in the PDYN gene was 68 bp repeats (We focused on STRs in the  repeats . These rCNR1 gene showed a large repeat number and wide variation as 9–18 repeats (6-7 variations) in the both S and L alleles , the distributions of repeat numbers were different between the S and L alleles . Variations of the STR in the PENK gene and VNTR in the PDYN gene were as low as five variations (11–15 repeats) and four variations (1–4 repeats), respectively. Associations between STRs or VNTRs and clinical data on peripheral pain sensation, including latencies to cold and mechanical pain perception, were examined using the Spearman’s rank correlation test after the correction for age and sex. The repeat number of the L allele of the CNR1 STR was slightly associated with the latency to mechanical pain perception in male volunteers , but this association was not confirmed after multiple-testing correction using the Holm method for each STR.The distribution of the STR in the  alleles . In contGRIN2A STR was significantly associated with analgesic dose, including epidural analgesics , but this association was not confirmed after multiple-testing correction using the Holm method for each STR.Repeat numbers of STRs and VNTRs in patients who underwent open abdominal surgery showed a similar distribution as participants whose peripheral pain sensation was assessed . The surPENK STR was significantly associated with VAS pain score at 24 h , but no significant association was found after Holm’s multiple-testing correction. The repeat number of the S allele of the CNR1 STR was significantly associated with the frequency of fentanyl use, fentanyl dose, and VAS pain score at 3 h . Associations with the frequency of fentanyl use and fentanyl dose were also statistically significant after Holm’s multiple-testing correction.Repeat numbers of the STRs and VNTRs in patients who underwent orthognathic cosmetic surgery showed a similar distribution as participants whose peripheral pain sensation was assessed . The indρ = 0.324, p < 0.001, and ρ = 0.185, p < 0.05, respectively). Thus, pain sensitivity was considered to decrease with age. The age of the patients who underwent open abdominal surgery was also negatively associated with the frequency of analgesic use . The lower frequency of analgesic use would be caused by lower pain sensitivity in older patients. The latency to mechanical pain perception in females was lower than in males , but no difference in cold pain perception was found between sexes, indicating that females are more sensitive to mechanical pain but not too cold pain compared with males (data not shown). The latency to cold pain perception was measured by the cold-pressor test immediately before surgery in patients who underwent orthognathic cosmetic surgery. In patients who underwent open abdominal surgery, the latencies to cold and mechanical pain perception were not examined. The cold-pressor test was slightly different from the peripheral pain perception test that was performed in volunteers , but these associations were not confirmed after multiple-testing correction using the Holm method to analyze the latency to cold pain perception with four clinical parameters, including the frequency of fentanyl use, fentanyl dose, VAS pain score at 3 h, and VAS pain score at 24 h. The distribution of the latency to cold pain perception in patients who underwent orthognathic cosmetic surgery was not associated with the patients’ ages . The low standard deviation of the patients’ ages , 18–35 repeats (18 variations), and 11–15 repeats (five variations), respectively. On the contrary, the repeat number and variation of the VNTR in the PDYN gene was 1-4 repeats and four variations. The repeat numbers of STRs in the CNR1, GRIN2A, and PENK genes were large, rather than the VNTR in the PDYN gene. Among these STRs, high variations of repeat numbers of STRs were found in the CNR1 and GRIN2A genes rather than the PENK gene, indicating that not all STR variations are high. Replication slippage events outnumber point mutations by one or two orders of magnitude in STRs  at over 20 nucleotides of the total length of STRs, although the repeat length and differences in STR variations  could be influenced by other factors, such as chromosomal background near the STRs. The STR in the CNR1 gene showed multiple peaks in the distribution of repeat numbers at 9–12 and 15 repeats. This unusual allelic distribution of the STR in the CNR1 gene was described in previous studies, which suggests our data on allelic distribution in the CNR1 gene was not a genotyping error  was significantly lower than in patients with short AAT repeats (homozygous or heterozygous for alleles with ≤11 repeats; CNR1 gene would be recessive for phenotypes that are related to the CNR1 gene. AAT STRs are the most representative trinucleotide repeats in the human genome, but they are less frequent in exons (CNR1 STR may be caused by the lower stability of CNR1 mRNA.Short tandem repeats have been reported to be associated with gene expression and human traits . Approxiin exons . AAT STRCNR1 gene were reported to be associated with the vulnerability to irritable bowel syndrome (CNR1 STR (CNR1 STR. Additionally, CNR1 colocalized with μ-opioid receptors in the same presynaptic nerve terminals in the nucleus accumbens, caudate putamen, and dorsal horn in rats (via CNR1 in modulating the analgesic effects of opioids for orofacial surgery.Short tandem repeats in the syndrome , multiplsyndrome , 2013, ssyndrome , and subsyndrome . The T2-CNR1 STR . The pre in rats . A posit in rats . These rCNR1 gene that contributes to analgesic effects of opioids, replication studies will be required in different races/ethnicities with sufficient sample sizes for each effect size. Additionally, elucidating the molecular mechanisms that underlie the association between repeat polymorphisms, including STRs and VNTRs, and analgesic effects of opioids may lead to a precise understanding of individual differences in analgesic effects of opioids and improve treatment strategies for pain.Genetic contributions to analgesic effects of opioids are influenced by psychological and environmental factors, including race, ethnicity, culture, and pain sensation status. To further elucidate genetic variability of the STR in the


Pontine infarction is the major subtype of posterior circulation stroke, and diabetes is more common in pontine infarction patients than in anterior circulation stroke patients. Whether the prevalence of diabetes remains homogenous within the posterior circulation stroke population is unclear. The present study is aimed at investigating the prevalence of diabetes in pontine infarction and comparing it to other subtypes of posterior circulation stroke.  We conducted a multicenter case-control study. Patients with posterior circulation stroke were screened. The subjects were divided into pontine infarction and nonpontine infarction groups. P < 0.05). After adjusting for confounding factors, diabetes was an independent risk factor for pontine infarction . For small vessel occlusion, diabetes was also more common in the pontine infarction group . Multivariate analysis also showed that diabetes was an independent risk factor for pontine infarction .  From November 1, 2018, to February 28, 2021, a total of 6145 stroke patients were screened and 2627 patients had posterior circulation strokes. After excluding cardioembolic stroke, as well as its other determined and undetermined causes, 1549 patients with 754 pontine infarctions were included in the analysis. The prevalence of diabetes in the pontine infarction group was higher than that in the nonpontine infarction group (42.7% vs. 31.4%,  In comparison with the nonpontine infarction subtype of posterior circulation stroke, patients with pontine infarction had a higher prevalence of diabetes, and diabetes was an independent risk factor for pontine infarction. As the population ages, the prevalence of diabetes has increased dramatically in recent decades. It has increased from less than 1% to 11.6% in China, which has the largest diabetic population . Many stWithin the posterior circulation, the anatomy and physiology of the artery were heterogeneous among the different segments. Thus, the prevalence of diabetes may vary among the different subtypes of posterior circulation stroke. Pontine infarction is the most common subtype and accounts for more than 40% of posterior circulation strokes . The preIn the present study, we aimed to investigate the prevalence of diabetes in pontine infarction and to compare it with that in nonpontine subtypes of posterior circulation stroke.This was a multicenter case-control study, which was conducted from November 1, 2018, to February 28, 2021. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Guangzhou Medical University and Longhua Hospital. Informed consent was signed by the patient or the patient's authorized person.Patients at the Second Affiliated Hospital of Guangzhou Medical University and Longhua Hospital were consecutively screened and enrolled if they met the following inclusion criteria: (1) older than 18 years old; (2) symptoms of acute stroke; (3) new infarction identified by diffusion-weighted imaging (DWI) within 7 days of stroke onset; (4) lesion located in the territory of posterior circulation; (5) intracranial and extracranial cerebral vessels evaluated by ultrasound, CTA, MRA, or DSA; and (6) infarction caused by atherosclerosis and arteriosclerosis. Patients were excluded if they met any of the following criteria: (1) missing clinical or imaging information; (2) nonatherosclerotic stroke; (3) brain tumor; (4) intracranial metastatic tumor; (5) intracerebral hemorrhage; or (6) traumatic brain injury.A current smoker is defined as someone who smokes on some days or every day. An ex-smoker is defined as someone who has smoked ≥100 cigarettes in their lifetime but has not smoked in the past 28 days .A former drinker is defined as someone who once drank but who did not drink during the past year; a current light drinker is defined as someone who in the past year has had 1-12 drinks but has 3 drinks or fewer per week on average; and a current heavy drinker is defined as someone who has had at least 3 drinks per week on average over the past year .Hypertension is diagnosed when someone's systolic blood pressure in the office or clinic is ≥140 mmHg and/or their diastolic blood pressure is ≥90 mmHg following repeated examinations .Diabetes is diagnosed when someone has the classical symptoms of hyperglycemia or hyperglycemic crisis with HbA1c ≥ 6.5% (the test should be performed in a laboratory using a method that is NGSP certified and standardized to the DCCT assay), fasting plasma glucose ≥ 7.0 mmol/L , 2 h plasma glucose ≥ 111 mmol/L during a 75 g oral glucose tolerance test (OGTT), or a random plasma glucose ≥ 11.1 mmol/L. Poorly controlled diabetes was defined as HbA1c ≥ 7%. Someone with an HbA1c level of less than 7% is considered well controlled .The classification of stroke subtypes using Trials of Org 10172 in Acute Stroke Treatment (TOAST) is as follows: (1) large-artery atherosclerosis, (2) cardioembolism, (3) small-vessel occlusion (SVO), (4) stroke of other determined etiology, and (5) stroke of undetermined etiology . Stroke Information about medication usage was collected before stroke onset.Pontine infarction was defined as an acute infarction located in the pons regardless of whether there were infarctions in other areas. Nonpontine infarction was defined as an acute infarction located in the extrapontine area.t-test was used when the normality assumption was met; otherwise, the equivalent nonparametric test was used. Differences in the categorical variables were compared using Pearson's chi-square test with post hoc analysis. Missing data points and risk factor variables recorded as unknown did not exceed 3% for any single variable. A univariate binary logistic regression analysis was performed to determine the effects of the independent variables. Individual variables with a P value < 0.1 in the univariate analysis were used in the multivariable regression analysis, and the results were expressed as odds ratios (ORs) and 95% confidence intervals (CIs).Differences in the continuous variables were compared using the test of homogeneity of variance. Student's As shown in P < 0.05). Multivariate analysis showed that diabetes was an independent risk factor for pontine infarction  had diabetes, which was higher than the percentage of diabetic patients with nonpontine infarction . Most of the patients with diabetes were diagnosed previously. The baseline characteristics are shown in P < 0.05, P < 0.05, A total of 580 patients had pontine infarction caused by SVO, and there was a higher prevalence of diabetes in these patients than in patients with nonpontine infarction  to detect branch artery disease . Some ofIn conclusion, the prevalence of diabetes was higher in pontine infarction than in the other subtypes of posterior circulation stroke, and diabetes was an independent risk factor for pontine infarction.


Under the 2 °C target, carbon mitigation costs could be more than offset by health co-benefits in 2050, bringing a net benefit of $393–$3,017 billion . This study not only provides insight into potential health benefits of an early peak in China, but also suggests that similar benefits may result from more ambitious climate targets in other countries.Recent evidence shows that carbon emissions in China are likely to peak ahead of 2030. However, the social and economic impacts of such an early carbon peak have rarely been assessed. Here we focus on the economic costs and health benefits of different carbon mitigation pathways, considering both possible socio-economic futures and varying ambitions of climate policies. We find that an early peak before 2030 in line with the 1.5 °C target could avoid ~118,000 and ~614,000 PM 2 emissions peak before 2030.Understanding benefits of carbon mitigation is an important impetus for governments to adopt more ambitious climate targets. Here, the authors show positive air quality and health co-benefits are possible if China’s CO Failure to act on climate change would result in huge socio-economic losses, potentially far outweighing the mitigation costs2. It is thus urgent to implement climate policies to curb the continuous rise in emissions of greenhouse gases (GHGs). As the largest carbon emitter in the world, China recently vowed to peak its carbon dioxide emissions before 2030 and achieve carbon neutrality before 2060, strengthening the earlier commitments of its nationally determined contribution (NDC) under the Paris Agreement to peak carbon “around” 2030. The carbon neutrality pledge also signaled that China is scaling up the ambition of its NDC as agreed in Paris.According to the Intergovernmental Panel on Climate Change (IPCC) 1.5 °C Special Report, the rise in global average surface temperatures could reach 1.5 °C between 2030 and 2052, posing great threats to human and natural systems2 reductions could bring health co-benefits from abated air pollution4. At a global level, the costs of reducing GHGs emissions could be more than offset by the health co-benefits by 20505. Yet the cost-benefit ratios of achieving NDC targets differ between developed and developing countries. Developing countries like India and China would gain considerable health benefits via air pollution abatement, up to 3–9 times the implementation costs in 20506, whereas the comparable ratios in the European Union and USA would be relatively modest, at 7–84% and 10–41%, respectively5.Given many shared anthropogenic sources of GHGs and air pollutants, mitigation policies aimed at CO7, and other recent studies have likewise shown realistic paths to surpass the NDC target9. With the peak year moving forward significantly, existing literature based on China’s NDC commitment likely overestimates its projected cumulative CO2 emissions and underestimates related co-benefits in air quality and public health10. Investigation into the social and economic impacts of such an early carbon peak in China, moreover, is still scarce. Besides, consideration of the diversity of climate change paths remains insufficient in the existing studies on the synergistic impact of climate policy on air quality11.Notably, China’s carbon emissions peak is now estimated to occur ahead of 2030, the year of its NDC target, owing to structural economic changes and enhanced emission reduction actions. As projected in our previous research, emissions for China may peak before 2025, at least 5 years ahead of China’s Paris target of 20302.5 concentrations and the mortality from long-term PM2.5 exposure. We find that implementation of climate policies in China would reduce PM2.5-related mortality and yield huge health co-benefits, especially in the long run. The more stringent the climate policy is, the greater the health benefits it would bring. The health co-benefits could fully offset the carbon mitigation costs in the long-term, to 2050. However, the co-benefits of climate policies alone will not be enough to realize China’s air quality goals and prevent the growth of the PM2.5-related health burden before 2050, especially given the increasing population aging.This study, a comprehensive assessment of air quality and health co-benefits of climate policies in China, applies an integrated assessment framework  is imposed as a quantitative constraint on the SSPs , the cumulative carbon emissions by 2100 are fixed to meet that RF level for various scenarios, labeled accordingly as SSP1_RCP2.6, SSP2_RCP2.6, and SSP5_RCP2.6 in Table Nine scenarios combining the characters of socio-economic developments and the ambitions of climate policies are considered in this study, represented respectively by the Shared Socioeconomic Pathways (SSPs) and Representative Concentration Pathways (RCPs). When a specific long-term radiation forcing (RF) target , the fossil fuel share of total primary energy would decline from 91.6% under SSP1_REF to 83.8% under SSP1_RCP1.9 in 2030, and from 85.2% to 42.9% in 2060  and biomass fuels (~92%) used in electricity generation and industry would be captured and stored by 2050 , meeting China’s newer peak year commitment of “before 2030.” Note also that China would be projected to achieve another NDC commitment of cutting CO2 emissions per unit gross domestic product (GDP) to 0.47 t-CO2/1000 USD, a reduction of 60% from 2005 levels, by 2030 in all scenarios , decrease with reductions of CO2 emissions as the climate policy becomes more stringent. But the level of mitigation differs by pollutant species, mainly reflecting their specific sectoral sources , nitrogen oxides (NOX) and CO2 emissions12, CO2 mitigation measures such as reduced coal consumption bring great co-reductions in SO2 and NOX emissions. As the major sources of volatile organic compounds (VOCs), black carbon (BC) and organic carbon (OC) emissions do not overlap with major sources of CO2  produces almost all (>99%) of NH3 emissions, which are therefore barely affected by policies to mitigate CO2 in the scenarios. In fact, separate from the growing food demand that accompanies economic development, large amounts of biomass fuels are required in all climate policy scenarios (both RCP2.6 and RCP1.9)  was 53.0 μg/m3 in China. The implementation of different climate policies will result in different levels of PM2.5 mitigation relative to the REF scenario  than that of SSP1_REF in 2030 , recognizing that in reality the mortality impacts of chronic exposure to air pollution may be distributed over years or even decades after exposure19. In 2030, they grow to 3.6–3.9 million across all scenarios and increase to 6.4–7.5 million in 2050, with the elderly (>65 yr) particularly at risk  and 8.7%  less than those in SSP1_REF, in 2030 and 2050, respectively  discussed in “Methods”, our analysis shows that the health co-benefits brought by CO2 emissions is almost certain to occur at some point during 2020–2030. China could realize its NDC commitment by peaking CO2 emissions around 2030 under the 2 °C scenarios, and earlier, before 2030, under the 1.5 °C scenarios. The stricter the climate policy and the cleaner the socio-economic pathway, the earlier the peak of CO2 emissions will occur and the larger the air quality and public health co-benefits achieved in the long run. Such health co-benefits could substantially if not fully offset the costs of more stringent climate policies and bring significant net economic benefits in 2050.The peak of China’s CO2.5-related mortality in China, and it is also necessary to adopt other measures, including further tightened conventional pollution controls, greening infrastructure with alternative building materials, and designing cities to encourage low-emission lifestyles.China’s Ministry of Ecology and Environment has set up a Department of Climate Change to promote synergies between climate change mitigation and air pollution control policies. Our results imply that the path of sustainable, low-carbon social and economic development (SSP1) will bring great environmental and health benefits for China at an affordable cost, especially in the long run. Implementing climate policies as soon as possible, especially decarbonizing the energy structure and applying CCS in industry and power sectors, can not only ensure achievement of the emission reduction target set in China’s NDC, but also bring more net co-benefits in air quality, public health and the economy. However, relying on the air pollution co-benefits of such climate policies alone is insufficient to prevent the growth of PM3 emissions, potentially exacerbating PM2.5 pollution and related health burdens in some regions. Measures should be taken to effectively control NH3 emissions. These could include enhancing the nitrogen use efficiency of fertilizer application and optimizing the scale of agricultural operations25, which may also reduce emissions of N2O, another greenhouse gas26. Informed by improved understanding of complex air pollution formation mechanisms , future policy analyses should also attach greater importance to both synergies and trade-offs between climate change mitigation and air pollution control. Only through policies that coordinate control of air pollution and carbon emissions can policymakers maximize their total benefits.A climate policy that increases the share of biomass fuels will likely combine with growing food demand to increase use of chemical fertilizer and, therefore, NH2.5 exposures to estimate the total health burden caused by air pollution. While ignoring health effects of exposure to other air pollutants  may lead to underestimation, it is likely to be small because the health burden of PM2.5 is estimated to be around 6.2-fold greater than that of O3 on average27 and controversy exists about whether health effects of NOX double-count those of nitrate PM2.528. Also, the common approach of using mass concentrations of PM2.5 was used, disregarding composition, sources and particle size distribution. All of these characteristics may have different impacts on health29, but are commonly ignored in such studies as the epidemiological evidence is currently limited and inconsistent31.We used effects of PM32. As we attempted to isolate the impact of climate policy on air pollution, we also did not consider special policies of future air quality control in our scenario analysis. For example, air quality could be further improved if stronger agricultural nitrogen management is implemented in the future, for example increasing the efficiency of nitrogen use during fertilizer application and agricultural waste management33. Nevertheless, the GCAM results for different scenarios here are reasonable, showing consistent trends in emissions of CO2 and air pollutants with the results of the other five integrated assessment models selected by the IPCC23.In addition, uncertainties may be introduced by various factors in the integrated assessment model, GCAM, including methods of model application, selection of representative technologies and parameterization, and choice of the base yearFinally, since we mainly focus on the air pollution and health co-benefits of carbon reduction, the meteorological fields (from 2015) were assumed to remain the same for all scenarios and years, to eliminate possible confounding effects of climate change. Similarly, due to the lack of projected baseline mortality data for 2030 and 2050, we assume that the baseline mortality rates will remain the same as 2015 in the future.2 and air pollutant emissions under SSP-RCP scenarios were obtained from GCAM. Then, the ambient PM2.5 concentrations and related mortalities under each scenario were quantified by combining the Weather Research and Forecasting Model Coupled with Chemistry (WRF-Chem) and the Global Exposure Mortality Model (GEMM). Finally, the health benefits were monetized using VSL, and then compared to the costs of implementing the climate policies.In this study, an integrated assessment framework was applied to investigate the economic costs and health benefits under different emission mitigation pathways in China Fig.. Estimat34. The SSPs are defined along two different axes: challenges to climate mitigation and challenges to adaptation  are developed with a set of qualitative assumptions given by the SSPs in the absence of climate polices. As the long-term radiation forcing target (from an RCP) is imposed on the SSP baselines as a quantitative constraint, the cumulative carbon emissions by 2100 are fixed  curves to change emission factors over time , but not the full macroeconomic costs in terms of GDP or consumption losses23. The model provides the mitigation costs of different energy and climate policies for each specific region.In general, carbon emission controls are applied to each activity and are strengthened as GDP rises, representing the historical trend that more stringent pollution control measures will be adopted as income levels increase40. The model has been demonstrated to reproduce pollution in China well and is widely used in previous studies, including our own42.According to the ambition of the landmark 2015 Paris Agreement to limit the increase in global average surface temperatures well below 2 °C, we compared the scenarios with or without climate policies seeking a maximum 2 °C temperature rise, i.e., RCP2.6 and REF, under the least and greatest socio-economic challenges to mitigation, i.e., SSP1 and SSP5, respectively. In addition, the SSP1_RCP1.9 scenario was added, in line with China’s raised recent ambitions and the stricter goal of 1.5 °C in the Paris Agreement. Thus, 11 parallel experiments were designed, including the base run in 2015 and 10 runs for 5 selected scenarios , in 2030 and 2050, respectively. Coupled dynamical and chemical simulations were conducted using WRF-Chem, version 3.6.1, and incorporated additional pathways of secondary pollution enhancement in China based on our previous studies43. The domain covers the Greater China region, including mainland China and part of East Asia, at a grid resolution of 20 km × 20 km. MIX, the mosaic Asian anthropogenic emission inventory developed by Tsinghua University44, was applied for the 2015 base run, while emissions in future scenarios were developed from GCAM model results37. The estimated biogenic inventory provided by MEGAN (the Model of Emissions of Gases and Aerosols from Nature) was also used45. The model configurations are given in detail in Supplementary Note Consistent with previous studies, we selected the meteorological fields of a given year (2015) and conducted simulations of four representative months  for all scenarios2.5 concentration against ground-based observations from almost 1500 stations  and lower respiratory infections (LRIs) in China under the various scenarios50. The relative risk (RR) functions for GEMM were constructed on the basis of 41 cohort studies of outdoor air pollution exposures in 16 countries, including a recent cohort study in China51. The attributable-fraction type relationship presented in Eq. .3 is the threshold concentration; j provided by Burnett et al. (2018).Exposure to ambient air pollution, especially PM53. Age structure in 2015 and future scenarios are from GBD2017 and SSP database35, respectively.Here, we apply the disease incidence in 2015 derived from the Global Burden of Disease Results Tool 2017 version (GBD2017). For population size and spatial distribution, the LandScan Global Database (v2018) is adopted for 2015, and the projected population scenarios in SSPs are from NCAR’s IAM group and the City University of New York Institute for Demographic ResearchThe health effects of the climate policies are represented by differences  function are one the most important source of uncertainties in health impacts analysis2.5 pollution and other sources . As suggested by a census-based epidemiological study, the GEMM results are better aligned with outcomes than the IER results in China56. The differences between effect estimates given by different relative risk functions  are analyzed in Supplementary Note In contrast with GEMM’s exclusive reliance on outdoor air pollution, the integrated exposure–response (IER) model adopted by the GBD incorporates risk information from both ambient PM58. Given the huge disparities between VSL values derived from different studies, even by one order of magnitude55, we calculated the VSL results in target years based several local studies in China for comparison. Moreover, since diverse VSL calculation methods may also lead to inconsistent results, we also calculated results on the basis of US EPA methodology59 in Supplementary Note 60. See details in Supplementary Note To quantify the benefit of avoided mortality risks, we applied the Value of Statistical Life (VSL) to monetize the estimated health impacts. The VSL values in each scenario and year are calculated via Eq. ,6\documeFurther information on research design is available in the Supporting informatonReporting Summary


Additionally, a special emphasis has been put on elemental composition characterization in cracked grain boundaries in order to quantify possible Si or Zr enrichment. It has been also studied the differences of solidification ranges and grain structures between both samples as a consequence of different minor alloying elements concentration in order to analyze their effect on crack susceptibility. In this sense, Scheil-Gulliver simulation results have shown that samples with higher Si and Zr contents presented higher solidification range temperature. This fact, as well as an increase of the presence of high angle grain boundaries (HAGB), leaded to an increment in the crack formation during solidification. Therefore, in this research work, an understanding of the factors affecting crack phenomenon in the LPBF manufactured IN738LC was accomplished.Inconel 738LC (IN738LC) is a nickel-based superalloy specially used in the hot section components of turbine engines. One of its main drawbacks relies on the cracking susceptibility when it is manufactured by laser powder bed fusion (LPBF). This paper analyzes the influence of minor alloying element concentration on cracking tendency of IN738LC superalloy manufactured by LPBF. For that objective, samples were manufactured using two powders, which presented different minor alloying elements concentration . It was shown that the samples crack tendency was very different depending on the powder used for their manufacturing. In fact, the measured crack density value was 2.73 mm/mm Among A process .(1)Ed=P10Mg [® [6Al4V [One of the main drawbacks of LPBF technology is the limited materials that can be manufactured without defect formation. In this context, the most commonly manufactured materials by LPBF are AlSi10Mg  and Scal10Mg [®  aluminum10Mg [® , Hastell10Mg [®  and Inco10Mg [®  nickel a® [6Al4V  titanium3 [Inconel 738LC (IN738LC) is a nickel based superalloy with high creep properties and hot corrosion resistance up to 980 °C temperature . Based o3 . Further3 . However3 . In fact3 . This ki3 . Due to 3 . Neverth3 ,19. Addi3 ,21. It iAccording to the literature, there are several factors that could influence solidification crack formation in the IN738LC superalloy. Among these factors, it seems that the most important ones are the superalloy composition and the superalloy final grain structure. In this sense, M. Cloots et al.  investigAs previously defined, the other factor that affects crack formation in LPBF manufactured samples is the grain structure, which is defined by the grains size, morphology and misorientation. In the LPBF samples, the formation of large columnar grains in the building direction is favored due to the layer-by-layer manufacturing process . It is wIn this research work, two IN738LC powders with different Si, Zr and B minor alloying elements content were manufactured by LPBF technology. Therefore, the effect of these minor elements on concentration and grain structure on crack susceptibility of IN738LC superalloy has been studied. A special emphasis has been put on elemental composition analysis of grain boundaries close to cracks. Furthermore, the crack susceptibility coefficient was calculated for samples with differences in minor alloying elements concentration and the importance of grain boundaries misorientation on crack formation was highlighted.Two IN738LC powders produced by gas atomization were used as feedstock material for LPBF manufacturing. One batch was supplied by Aubert and Duval (A&D) from Eramet Group, whereas the other was supplied by LPW Technology Ltd. from Carpenter Technology Innovation . Powder particles morphology was analyzed by scanning electron microscopy as shown in Samples with the dimensions described in the image of 3COOH + 10 mL HNO3 + 15 mL HCl.Samples were cut in the building direction (x–z plane) for defect quantification and microstructural analysis. Subsequently, they were grinded up to 2500 µm SiC paper and polished with 6, 3 and 1 µm diamond paste. Grain structure was revealed by chemical etching for few seconds with this specific reagent: 10 mL CH2 was studied in each sample and then the area porosity percentage was calculated. In order to dismiss the presence of cracks in this step, a circularity threshold value of 0.7 was established. Cracks quantification was carried out following the methodology described by Carter et al. [Porosity and cracking quantification were performed in non-etched samples to evaluate possible differences due to minor alloying elements addition. Porosity was analyzed by image analysis using the optical microscope (GX51 Olympus) and the AnalySIS Docu software. In fact, an area of 10 mmr et al.  and the 2 using 1 µm step size and noise reduction was performed up to six neighbors. Additionally, samples were characterized using the Zeiss Ultra Plus Scanning Electron Microscope (FEG-SEM) in secondary electron (SE) mode at 15 kV to analyze the morphology and particle size. Furthermore, inverse pole figure (IPF) maps were measured by electron backscattered diffraction (EBSD). Both analyses were performed with Aztec, Oxford Instruments, NanoAnalysis software and for EBSD map, the scanned area was 300 × 300 µmTransmission electron microscopy (TEM) was used to investigate more accurately the elemental composition at grain boundary. A lamella of ~50 nm thickness was prepared in front of a crack tip, as illustrated in Samples were manufactured using both IN738LC compositions and the process parameters summarized in 2 for LPW and A&D samples. As shown in With respect to crack tendency, crack density was measured also in both samples and the obtained values were 2.73 and 0.25 mm/mm2 and an aspect ratio of 3 ± 1, whereas A&D grains mean area is 687 ± 70 and aspect ratio 4 ± 1. Thus, compared to LPW grains, A&D grains are more elongated and present a higher area. Comparing both IPF maps, it is obvious that the A&D sample exhibits more texture in <001> direction than LPW sample. Additionally, in the LPW IPF map, a crack is observed at a grain boundary with 40° misorientation between adjacent grains, which evidences that crack formation occurs intergranularly [As it has been explained in the introduction, the typical grain structure of LPBF samples consists of large columnar grains . Actuallanularly . This efAs explained in the experimental section, several lamellas were obtained by FIB technique in order to analyze the LPW and A&D samples grain boundary elemental composition by TEM. Some representative images of the grain structure of both samples are shown in In order to study segregation phenomena at cell and grain boundaries, the elemental composition maps were obtained by BF-STEM EDS in all cases. Apart from these types of segregations, other authors have also observed the presence of titanium carbides in GB of IN738LC samples . Our samWith respect to A&D sample, the same analysis as for LPW sample was performed; however, in this case, grain and cell boundaries were localized in the same picture as shown in With the aim of performing a more localized composition analysis, TEM EDS line-scans were performed in LPW sample cell boundary and in A&D sample grain boundary. In fact, the selected zones are presented in In this aspect, as shown in Two different IN738LC compositions were employed to manufacture samples by LPBF technology. After chemical composition analysis, it was observed that the IN738LC powder supplied by LPW presented higher concentration of Si, Zr and B elements, which are supposed to increase the cracking tendency of this superalloy. Actually, it was experimentally demonstrated that LPW sample presented much more cracks than A&D sample, which indicates that minor additions of Zr, Si and B play an important role on crack tendency of IN738LC superalloy. In the literature, some authors have analyzed the effect of composition on cracking tendency by atom probe tomography (APT) ,19,27. T1/2 (fs refers to solid fraction) curve in the range between 0.94–0.97 solid fraction (fs). In order to verify if the studied cases satisfy this criterion, Scheil-Gulliver simulations were performed with ThermoCalc software and TCNI10 database . On the other hand, when the liquid volume is between 0.1–0.01, they established that material is in the vulnerable region and the time spent in this range is defined as tV. Thus, the relation between tV and tR results in the CSC, implying that a higher CSC results in higher crack tendency.Finally, in order to compare the crack susceptibility of both samples, crack susceptibility coefficient (CSC) was calculated based on Clyne and Davies research . These r1 (primary dendrite arm space) must be measured. Secondly, the solidification time with respect to liquid fraction (fL) can be calculated  were calculated following the method described by Guraya et al. , where λlculated  using thApart from minor alloying elements segregation, in the literature it is suggested that grain boundary (GB) misorientation plays an important role in the cracking susceptibility of a material . Based ogb to twice the solid-liquid interface energy γsl. Therefore, when γgb < 2 γsl, the liquid film will be unstable and coalescence between dendrites will occur, which is the case of attractive GB. On the other hand, in the repulsive GBs, γgb > 2 γsl, the liquid film remains stable at lower temperatures; thus, grain boundary remains wetted until lower temperatures. This effect could explain the increase of crack formation in the LPW sample compared to A&D sample, based on the fact that LPW sample has GBs with higher misorientation than A&D.Rappaz et al.  discusseFurthermore, melt pools from the last layer of LPW a and A&DThus, a higher content of minor alloying elements implies an expansion of the alloy solidification range, which favors the formation of repulsive grain boundaries. Indeed, these repulsive grain boundaries lead to the formation of adjacent grains with higher misorientation and also facilitate the stray grains formation. As a result, a less textured grain structure with smaller and more misoriented grains is formed. Thus, these factors acting together involve higher crack susceptibility.In this research work, it was presented that using the same LPBF process parameters, LPW sample, which contains higher Si and Zr content, exhibited much more solidification cracks than A&D sample. This fact implies that minor alloying elements addition has huge influence on crack formation. In all the cases, solidification cracks were developed at grain boundaries; therefore, composition map measurements were performed in these zones using TEM EDS. The results showed that there were no significant segregation differences between LPW and A&D samples at grain and cell boundaries. Nevertheless, Scheil-Gulliver simulations indicated that LPW sample presented higher solidification range than A&D sample, which is known to be an important factor affecting crack formation. Furthermore, crack susceptibility coefficient was calculated using the results from the simulation. In this sense, the CSC value obtained for LPW sample was higher than the one obtained for A&D sample. Thus, LPW sample will be more prone to cracking than A&D sample because it spends more time in the vulnerable region. Additionally, the effect of samples grain structure was investigated with respect to cracking tendency. It was observed that LPW sample presents GBs with higher misorientation than A&D sample. This is an indicative that samples with more misoriented grains are more prone to cracking probably due to the existence of liquid films at lower temperatures. This fact provokes that GBs remain wetted during more time, which increases the possibility of crack formation. Therefore, it was concluded that controlling minor alloying elements composition of IN738LC is critical to avoid crack formation during LPBF manufacturing. Furthermore, it was observed that minor alloying elements concentration affects both, sample grain structure and solidification range, which are factors that increase crack formation tendency.


Elephas maximus) anaesthetised with azaperone-etorphine, medetomidine and an etorphine constant rate infusion in lateral recumbency for a dental procedure. The hypoxaemia was corrected utilising positive end-expiratory pressure (PEEP) of 5 cm – 10 cm H2O, a technique that has not previously been reported in the management of anaesthetised elephants. PEEP decreases atelectasis, shunt fraction, and increases lung compliance. Positive end-expiratory pressure was achieved by partial occlusion of the tailpiece of a manually triggered demand valve ventilator during expiration. This is a simple effective method of generating PEEP and correcting hypoxaemia without the need for any additional specialised equipment. However, PEEP decreased arterial blood pressure and should be implemented with caution if arterial blood pressure is not monitored.Hypoxaemia is a common complication in anaesthetised or immobilised elephants. It is presumably because of hypoventilation and ventilation-perfusion mismatch. To prevent hypoxaemia, orotracheal intubation and positive pressure ventilation are recommended. This case report describes a hypoxaemic period despite positive pressure ventilation in a 46-year-old female Asian elephant ( Elephas maximus).Hypoxaemia is a frequently reported complication in captive and free-ranging immobilised elephants  was anaesthetised for extraction of the right mandibular molar 3. Food had been withheld for 17 h and water withheld for 15 h prior to anaesthesia. The elephant was immobilised with 3 μg/kg etorphine IM  and 9 μg/kg azaperone IM  administered by hand injection. Fifteen minutes after initial immobilisation agents were delivered, profound sedation was achieved and the animal was guided into left lateral recumbency onto a deep sand bed using a rope casting technique. The left auricular vein was catheterised and 1.2 μg/kg medetomidine intravenous (IV)  was administered. Anaesthesia was maintained using an etorphine constant rate infusion (0.6 μg/kg/h – 1.5 μg/kg/h IV). The etorphine was diluted in 1 L of lactated ringers solution and run through a fluid pump to ensure it was delivered at a constant rate. Heart rate (HR), pulse oximetry (SpO2), respiratory rate (RR), end-tidal CO2, inspired oxygen concentration (FiO2), electrocardiography and invasive blood pressure (BP) (18 g catheter in the auricular artery) were monitored using a calibrated multi-parameter anaesthetic monitor . Arterial blood gas tests were performed using a handheld blood gas monitor iStat, Abbott Laboratories, United States (US).A 46-year-old female μg/kg atipamezole was administered IV . Shortly after reversal, a 30-mm internal diameter (ID) cuffed orotracheal tube was placed and connected to a manually triggered Mega-Vertebrate demand ventilator . With a peak inspiratory pressure (PIP) of 15 cm H2O, RR of 7 and an inspiratory: expiratory ratio (I:E) of 1:1, it allowed the maintenance of normocapnia (EtCO2 between 35 millimetre of mercury [mmHg] and 45 mmHg), while providing a FiO2 of about 33%.As a result of difficulties with intubation and a period of apnoea that developed after the medetomidine administration, 6 2 value decreased from 100% to 92%. A leak around the orotracheal tube was detected and another 30-mm ID tube with a slightly larger external diameter was placed. Although the PIP was increased to 20 cm H2O, the SpO2 value declined between 86% and 89%. An arterial blood gas analysis confirmed a diagnosis of hypoxaemia with a PaO2 of 49 mmHg and a PaCO2 of 55 mmHg , and shortly after, the SpO2 reached 97%; however, further blood gas analysis was not available.To treat the hypoxemia, it was decided to add PEEP. At the end of the inspiration phase, the anaesthetist let the air pressure decrease until it reached 5 cm – 10 cm HWhile oxygenation improved with PEEP, BP declined from a mean arterial pressure of 132 mmHg immediately before PEEP to as low as 70 mmHg during PEEP. In preparation for recovery, the arterial catheter was removed and PEEP was discontinued because of the risk of unmonitored hypotension.E. maximus, lateral recumbency for 15 min is also associated with a significant decrease in PaO2 when compared with standing. This reduction in PaO2 can be as significant as 30% but is usually not accompanied by any change in PaCO2 (Honeyman et al. The potent opioid etorphine is often used to anesthetise elephants either alone or in combination with other drugs (Honeyman et al. As a result of the use of potent respiratory depressants and the rapid development of V/Q mismatch, oxygen supplementation is recommended for all immobilised or anaesthetised elephants (Horne & Loomis 2O, which did not allow for a proper recruitment manoeuvre and was followed by a further decrease in SpO2.In this case, a combination of drug-induced hypoventilation and the development of V/Q mismatch are the likely causes of the observed hypoxaemia. Correction of hypoventilation by providing IPPV and increasing PIP was unsuccessful in correcting hypoxaemia. In anaesthetised horses, atelectasis forms rapidly, and the early implementation of IPPV is recommended to maintain and improve oxygenation (Moens et al. 2 was 33%, although a FiO2 of 42% is reported by the manufacturer (In Case of Anaesthesia, 2 is associated with increased shunt fraction in other species, the lower FiO2 provided by this ventilator may have been beneficial in preventing further atelectasis (De Monte et al. This case utilised a commercially available assisted demand ventilator. The design of this specific ventilator is such that rapid and high flow oxygen delivery into the ventilator generates negative pressure at the tailpiece of the ventilator and draws in atmospheric air through entrainment. The increase of gas flow permits a quicker delivery of the required tidal volume. The resulting FiOPositive end-expiratory pressure, which is defined as the residual alveolar pressure above atmospheric pressure at the end of expiration, improved oxygenation (Ambrosio et al. 2O prevents atelectasis, although a higher PEEP is required to reopen closed alveoli (Osbertg et al. 2O results in a significant reduction of atelectasis and an increase in lung volume by up to 60%, while in horses, titration of the PEEP improves oxygenation, reduces shunt fraction and increases lung compliance (De Monte et al. Positive end-expiratory pressure improves pulmonary gas exchange in multiple species under anaesthesia (Ambrosio et al. On a modern mechanical ventilator, PEEP is a basic setting that applies pressure at the end of expiration. Otherwise, commercial PEEP valves or handmade water column can be adapted onto the expiratory limb of the circuit or the scavenge tubing of the anaesthesia machine (Allison et al. 2 to 40% was shown to improve and maintain oxygenation and respiratory compliance in dogs (De Monte et al. 2 provided by the ventilator may have aided the improved oxygenation in this elephant.The use of PEEP in elephants may be of particular use in recruiting collapsed alveoli and improving oxygenation because of their unique respiratory system. Elephants lack a pleural space, instead, they possess a distensible collagen fibre network that adheres to the pleura of the chest wall. In addition, the parenchyma itself is supported by an elastic septum that limits alveoli collapse (Browne et al. As a result of the increased intrathoracic pressures generated by PEEP, cardiac output and consequently, BP often declines (Luecke & Pelosi Although the application of PEEP may impair cardiac output, the period of hypotension in this animal may have been exacerbated by other factors. The withholding of water prior to anaesthesia may have produced a mild hypovolaemia resulting in hypotension. In addition, the use of azaperone and atipamezole may have caused vasodilation, thus further reducing BP. The aetiology of the hypotension was likely multifactorial and PEEP impaired the ability of the animal to compensate adequately for its impaired cardiovascular status.E. maximus undergoing general anaesthesia.In conclusion, PEEP could easily be generated on the Mega-Vertebrate demand ventilator and was effective at improving oxygenation in laterally recumbent