Myocardial infarction (MI) with non-obstructive coronary arteries (MINOCA) is an important clinical problem especially in the era of extensive utilization of coronary angiography in MI patients. Its pathophysiology is poorly understood which makes diagnostics and treatment of MINOCA challenging in everyday clinical practice. The aim of the study was to assess characteristics of MINOCA patients in Poland based on data from the Polish National ORPKI Registry. In 2016, 49,893 patients with non-ST-segment elevation (NSTEMI) or ST-segment elevation (STEMI) myocardial infarction entered the ORPKI registry. MINOCA was defined as a non-obstructive coronary artery disease (CAD) and a lack of previous coronary revascularization. MINOCA was identified in 3924 (7.8%) patients and clinical presentation was more often NSTEMI than STEMI . MINOCA patients were younger and more often females with significantly lower rates of diabetes, smoking, arterial hypertension, kidney disease, previous MI and previous stroke comparing to patients with obstructive CAD. Myocardial bridge was visualized in angiography more often in the MINOCA group . Additional coronary assessment inducing fractional flow reserve, intravascular ultrasound, optical coherence tomography was marginally (< 1%) used in both groups. Periprocedural mortality was lower in MINOCA group . MINOCA patients represent a significant proportion of MI patients in Poland. Due to multiple potential causes, MINOCA should be considered rather as a working diagnosis after coronary angiography and further efforts should be taken to define the cause of MI in each individual patient. Myocardial infarction with non-obstructive coronary arteries (MINOCA) is an important clinical problem in the era of extensive usage of coronary angiography.Many of MINOCA causes are treatable, so additional diagnostics beyond coronary angiography is important for final diagnosis and treatment selection.In large-scale national registry patients with MINOCA represent a significant proportion of MI patients in Poland .Due to multiple potential causes, MINOCA should be considered rather as a working diagnosis after coronary angiography and further efforts should be taken to define the cause of MI in each individual patient.Myocardial infarction (MI) with non-obstructive coronary arteries (MINOCA) is an important and common clinical problem as according to current knowledge, almost every patient with the diagnosis of MI should be referred to coronary angiography. An invasive strategy is also a frequent approach in unclear cases, in which after ruling out the obstructive coronary artery disease (CAD) further diagnostics should be applied. The pathophysiology of MINOCA is multifactorial and poorly understood with several proposed mechanisms. This makes diagnostics and treatment of MINOCA challenging in daily clinical practice. That is why the problem of MINOCA diagnostics was highlighted in the recent European Society of Cardiology Clinical Practice Guidelines on acute myocardial infarction in patients presenting with ST-segment elevation . HoweverData on patients with the diagnosis of ST-segment elevation (STEMI) or non-ST-segment elevation myocardial infarction (NSTEMI) included into the ORPKI between January and December 2016 (12 months) were analyzed. The ORPKI is a national electronic database operated by the Jagiellonian University Medical College in Krakow collecting data on all percutaneous procedures in interventional cardiology performed in Poland . All medIn 2016, 49,893 patients with STEMI or NSTEMI entered the ORPKI registry. MINOCA was identified in 3924 (7.8%) patients  as well as a meta-analysis of clinical studies about 6%) , 6.% , 6.6].Importantly, MINOCA is just an initial and general diagnosis which does not describe underlying pathophysiology. The potential pathophysiological mechanisms of clinical scenario when MI may be diagnosed according to the definition but there is a lack of the obstructive coronary artery disease are quite complex. These include both coronary and non-coronary pathologies. The coronary causes comprise of several different mechanisms. Thromboembolism may be an underlying pathological factor by itself or may be caused by plaque rupture or coronary spasm. This includes also thrombotic disorders (hereditary or acquire) . Plaque In line with previous reports, we found MINOCA patients to be younger, rather with an initial diagnosis of NSTEMI than STEMI and to be more often female comparing to patients with obstructive CAD. Our analysis also showed that MINOCA patients had a lower risk profile concerning other CAD risk factors. However, in meta-analysis of MINOCA studies there was no significant difference in arterial hypertension, diabetes mellitus, smoking, and family history of CAD comparing to obstructive CAD patients .Patients with MINOCA have lower mortality comparing to obstructive CAD patients with MI in 12-month follow-up. However, in-hospital mortality of about 1% and 3.5% at 12-month is still high especially as compared to stable non-MI patients with normal coronaries in angiography . This unPresented analysis has several limitations. Angiograms were assessed locally by operators, and not by an independent image analysis core laboratory. The ORPKI for the moment does not collect data beyond the cathlab. So, it was impossible to assess further diagnostics done during hospitalization and after discharge and to analyze data according to final diagnosis as well as provide event rates at follow-up. In addition, there is a potential bias from unmeasured confounding factors not included in this analysis. Despite all these limitations our study reflects the outcome of a “real-world”. Thus, data could be extrapolated to the general population.Patients with MINOCA represent a significant proportion of MI patients in Poland. Due to multiple potential causes, MINOCA should be considered as a working diagnosis after coronary angiography and further efforts should be taken to define the cause of MI in each patient.


Many Service members and Veterans with lower-limb amputations have the potential for high function and the desire to resume physically demanding occupations that require them to carry heavy loads . However, it is currently unclear which prosthetic feet best accommodate heavy load carriage while also providing good overall function and mobility during unweighted activities. The main objective of this study was to investigate the ability of currently available prosthetic ankle-foot systems to accommodate weighted walking by examining the mechanical characteristics  and dynamic function  of prosthetic feet designed for high activity users. Load versus deflection curves were obtained for nine prosthetic ankle-foot systems using a servohydraulic test frame and load cell. Effective roll-over shape characteristics and late-stance energy return measures were then obtained using quantitative gait analysis for three users with unilateral, transtibial amputation. Results from mechanical and dynamic testing showed that although forefoot stiffness varied across the nine feet investigated in this study, changes measured in roll-over shape radius and effective foot length ratio were relatively small in response to weighted walking. At the same time, prosthetic feet with more compliant forefoot keel structures appeared to provide more late-stance energy return compared to feet with stiffer forefoot keel structures. These results suggest that prosthetic ankle-foot systems with compliant forefoot keel structures may better accommodate weighted walking by reducing the metabolic cost of physically demanding activities. However, to more fully understand the biomechanical and functional implications of these results, other factors, such as the residual-limb strength of the user and the overall stiffness profile of the prosthetic foot, should also be considered. The ability to wear or carry loads beyond body weight is important for functional independence and is required in many occupational contexts . With regard to the military, the mass of protective gear and weapons/ammunition alone can be upwards of 21 kg 46 lb) and light infantry troops often carry loads of 45 kg (100 lb) or more during dismounted operations 6 lb and ,8. The lClinicians treating Service members and Veterans with a lower-limb amputation have a wide variety of prosthetic components to choose from; yet there remains a general lack of objective criteria for evaluating and prescribing prosthetic ankle-foot components ,10, and Accordingly, the main objective of this study was to investigate the ability of currently available prosthetic ankle-foot systems to accommodate weighted walking by examining the mechanical characteristics  and dynamic function  of prosthetic feet designed for the highest activity users. In order to evaluate forefoot stiffness, load versus deflection curves were obtained for nine different prosthetic ankle-foot systems using a servohydraulic test frame and load cell. Following mechanical testing, three research participants were recruited to walk with each prosthetic ankle-foot system and quantitative gait analysis was used to obtain effective roll-over shape and energy return data. We hypothesized that for prosthetic ankle-foot systems with compliant forefoot keel structures, added loads would be associated with larger deformations of the prosthetic forefoot, thereby reducing roll-over shape radii compared to unweighted walking , red. InNine prosthetic ankle-foot systems, all marketed for users in the highest Medicare Functional Classification Level (MFCL K4), were investigated in this study: 1) Renegade AT , 2) Thrive , 3) Variflex XC , 4) Soleus Tactical , 5) Triton Heavy Duty , 6) All Pro , 7) Rush Foot , 8) Trekk , and 9) Panthera CFII . Prosthesis selection was based on several factors important for Service members returning to active duty: 1) no moving parts, which generally require less maintenance and are less prone to failure over time, 2) feet with long, spring-like keel structures, which should experience less strain for a given deflection and therefore be more robust, and 3) feet with thick keel structures, which are recommended for high impact activities required in active duty. Despite the potential for all nine feet to be appropriate for MFCL K4 users, distinguishing features in their mechanical design  suggested that some feet may exhibit more consistent roll-over shapes when walking with added loads than others. Three units were purchased for each ankle-foot type based on the body weight and foot length of three users identified for human subject testing. Accordingly, twenty-seven ankle-foot prostheses were characterized during mechanical and human subject testing.Prior to human subject testing, load versus deflection profiles for each foot were obtained using a servohydraulic universal test frame  with axial/torsional capabilities, a computer-based data acquisition system , and a load cell . The load frame applied a uniaxial load to the foot as shown in To approximate conditions expected during active duty military activities, each foot was tested in a “use state” . A sheet of adhesive-backed polytetrafluoroethylene (PTFE) was also placed on the tread of the boot to provide a low friction interface with the loading platform. All feet were mounted in a fixture such that the plantar surface of the boot was set at a 20-degree angle from horizontal, simulating forefoot loading as defined by the ISO 10328 standard . DiffereEach foot was loaded to body weight + vest weight (22 kg), approximating loading conditions associated with the second peak of the vertical ground reaction force of transtibial prosthesis users walking at speeds between 1.2–1.6 m/s . Test loABW). Stiffness values were normalized by body weight in order to calculate means across subjects.To compare the mechanical properties of each foot, load versus deflection profiles from the ninth loading cycle were plotted from 50 N of applied load to the maximum load. The ninth loading cycle was used for analysis to allow the foot to settle into a consistent position on the load frame. The best-fit linear slope of the load versus deflection curve was then calculated between body weight and 22 kg above body weight for each respective subject, corresponding to forefoot stiffness at loads above body weight  mounted flush within a surrounding 3.4 m walkway while an 8-camera Oqus 100 motion analysis system  tracked the reflective markers on their socket. Subjects walked at their normal speed until at least five clean force platform hits had been collected. During this first condition, the subject’s walking speed was recorded; during all subsequent trials, walking speed was monitored to ensure that the subject maintained a comparable speed. Subjects were then fitted with a weighted (22 kg) vest  for further analysis. To calculate the roll-over shape of each prosthetic foot, the center of pressure of the ground reaction force was transformed into a socket-based coordinate system with its origin at the knee center [Raw marker data were processed using Qualisys Track Manager (QTM 2.11), then exported into MATLABe center . Given te center . To quanFrom roll-over shape data, the effective foot length ratio of each foot was also calculated according to methods described previously . This meFinally, a unified deformable (UD) segment analysis  was usedData were collected from three male subjects with unilateral, transtibial amputation. Amputation etiology included trauma (2 subjects) and bone lesion (1 subject). The mean age, mass, and height of the subject population was 39 ± 6 years, 85 ± 14 kg, and 1.76 ± 0.05 m, respectively . All sub2) of the best-fit linear slope of the load versus displacement curve above body weight was 0.996 ± 0.006 (range: 0.969–1.000) across all feet.Mean calculated forefoot stiffness is quantified in Despite differences observed in forefoot stiffness during mechanical testing, roll-over shape profiles measured during human subject testing appeared relatively similar between the weighted and unweighted walking conditions for all 27 ankle-foot prostheses tested in this study. Finally, While many Service members and Veterans with lower-limb amputation have the potential for high function, objective criteria for evaluating and prescribing appropriate ankle-foot prostheses is currently lacking. Specifically, for Service members with a lower-limb amputation, it is unclear which prosthetic ankle-foot systems best accommodate load carriage while also providing good overall function and mobility for unweighted activities. Compared to healthy, intact ankle-foot systems that adapt to added load by maintaining similar ankle motion and effective rocker shapes during walking , most prABW was relative to the users’ body weight, potentially resulting in load-dependent variability within each foot type. Larger differences in mechanical designs between foot type resulted in larger variations in forefoot stiffness across feet. According to the results shown in As expected, mechanical testing revealed that forefoot stiffness varied across all ankle-foot systems, both within foot type and between foot type. Variations within foot type are evident from the different order (left to right) of load versus deflection curves shown for each subject in ABW  and were similar in magnitude to the approximate change (0.015) in roll-over shape radius observed in the ankle-foot system of an able-bodied population walking with a comparable 23-kg weighted vest [Contrary to our hypothesis, this result was not readily apparent in the subsequent analysis of ankle-foot roll-over shape data. Instead, while appreciable changes in forefoot stiffness were observed across all feet during mechanical testing, roll-over shape profiles appeared largely insensitive to the effects of load carriage. ted vest . AccordiThe clinical implications of varying rocker radius and foot length on the energetic cost of walking have been investigated previously by Adamczyk and Kuo , who fouBeyond these studies, others have shown that reductions in effective foot length may also contribute to a drop-off effect that could lead to a shorter step length on the contralateral foot and a more forceful loading of the sound side limb during weighted walking activities ,16,21. FThe apparent insensitivity of roll-over shape parameters to weighted walking in the present study suggests that a more important consideration in prescribing prosthetic feet for high activity users may instead be the effect of forefoot stiffness on late-stance energy return. Indeed, late-stance energy return appeared highly sensitive to forefoot stiffness, with the least stiff feet  providing the most late-stance energy return and the stiffest feet  providing the least late-stance energy return. These results are also in agreement with those of a previous study by Fey et al. , which fCollectively, these study results highlight several important paths for future investigation. In the current study, three users with different body weights and activity levels each walked with nine commercially available prosthetic feet, applying functionally relevant loading profiles to a total sample size of 27 different ankle-foot systems. This study design allowed for a thorough investigation of prosthetic feet designed for MFCL 4 users, guiding future clinical testing of these systems. However, to understand the statistical and clinical significance of changes observed in the rocker shape and late-stance energy return of these feet, future studies should include activities beyond that of level walking and involve a larger, more diverse study population. Using the roll-over shape radius data collected in this study, we ran a power analysis for a one-tailed paired t-test using G*Power 3.1. Effect size (d) was calculated using the mean and standard deviation difference in rocker radius between the unweighted and weighted conditions (across subjects) for one foot with the consistently lowest (Soleus) and one foot with the consistently highest (Thrive) forefoot stiffness according to Future studies should also consider the effect of weight distribution about the torso, walking speed, and prosthetic alignment on the dynamic characterization of prosthetic ankle-foot systems designed for high activity users. Indeed, several previous studies of weighted walking have used different weight distribution methods  to simulate common scenarios of load carriage e.g., ,6,7). Wh,7. Wh6,7With regard to mechanical testing, it is important to note that only one angle was used to test and analyze forefoot deflection, and that more comprehensive testing configurations, such as those outlined by ISO 22675, may provide additional insight into the overall dynamic response of prosthetic feet designed for high activity users. Furthermore, future analyses should consider the contribution of overall stiffness and vertical compliance on the assessment of prosthetic feet designed to accommodate heavy load carriage and to what extent heel stiffness affects weighted walking.According to the mechanical and human subject testing performed in this study, prosthetic feet with a range of forefoot stiffness profiles exhibited minimal changes in roll-over shape radii and effective foot length ratio measured during weighted walking compared to unweighted walking. At the same time, prosthetic feet with more compliant forefoot keel structures appeared to provide more late-stance energy return compared to feet with stiffer keels, both during the weighted and unweighted walking conditions. The results of this study may be useful in providing a guide for the prescription of prosthetic feet for high activity users. For examples, prosthetic feet that feel too soft or too stiff can be replaced with other foot types that are stiffer or more compliant according to the data presented in S1 File(XLSX)Click here for additional data file.


Preoperative hyponatremia is associated with an increased risk of mortality on the liver transplantation (LT) waiting list. We sought to investigate the impact of pre- and intraoperative serum sodium levels on the one-year mortality after LT. We identified 1,164 patients for whom preoperative and intraoperative serum sodium levels were available. Cox regression analysis with multivariable adjustment was performed for one-year mortality. A propensity score matching analysis was performed for preoperative and intraoperative serum sodium groups to compare one-year survival. The cutoff of sodium level with minimal p-value was 130 mEq/L for both preoperative and intraoperative sodium. Intraoperative hyponatremia was an independent predictor of one-year mortality in the multivariable Cox regression analysis, while preoperative hyponatremia was not. Kaplan-Meier curve showed that there was a significant difference in the one-year mortality between preoperative and intraoperative serum sodium groups. However, after propensity score matching, there was no difference in the one-year mortality among the preoperative sodium groups, while there was a significant difference among the intraoperative sodium groups. Intraoperative hyponatremia defined by mean sodium <130 mEq/L was independently associated with a significantly high one-year mortality. Mean intraoperative serum sodium levels may be a better prognostic predictor than preoperative serum sodium levels. Almost half of the patients with cirrhosis have serum sodium concentrations below the lower limit of the normal range (<135 mEq/L)3. Hyponatremia in patients with ESLD is associated with increased mortality independently of other indicators of liver disease severity such as the Model for End-Stage Liver Disease (MELD) score10. Also, preoperative hyponatremia of <130 mEq/L was associated with decreased post-liver transplantation (LT) survival12. Meanwhile, several studies also reported no significant association between preoperative hyponatremia and postoperative mortality15. Furthermore, there was no difference in the 6-month survival rate between those for whom MELD-Na score was used for liver allocation and those for whom the standard MELD score was used16. Therefore, the association of preoperative hyponatremia with postoperative mortality continue to be debated.Hyponatremia is a common yet ominous sign in patients with end-stage liver disease (ESLD)17. However, in this study, the impact of intraoperative hyponatremia during transplantation surgery was not evaluated. Intraoperative hyponatremia may be more closely related to the posttransplant outcomes than preoperative hyponatremia because the intraoperative serum sodium fluctuates more due to administration of a large amount of fluid, sodium bicarbonate, and diuretics. During surgery, frequent preload change occurs by clamping of inferior vena cava and bleeding and metabolic acidosis develops during anhepatic and reperfusion of liver graft. Therefore, these significant change in sodium homeostasis during surgery may be more strongly associated with postoperative mortality compared to preoperative serum sodium levels.Additionally, the impact of intraoperative hyponatremia on post-LT mortality has not been evaluated. A previous retrospective study reported that a larger increase in serum sodium concentration after surgery compared to preoperative baseline concentrations was associated with worse recipient short-term outcomes18, although the lower limit of normal serum sodium is 135 mEq/L. Accordingly, many studies use <130 mEq/L to define hyponatremia14, while other studies still use <135 mEq/L as a cutoff of hyponatremia19. However, there have been no studies that attempted to find optimal cutoff of serum sodium that is associated with increased risk of post-LT mortality.Additionally, among the studies reporting the impact of hyponatremia on mortality in patients with ESLD, the level of serum sodium used to define hyponatremia does not seem to be standardized. Hyponatremia in patients with decompensated cirrhosis was defined as a serum sodium level <130 mEq/L in previous studiesTherefore, in this study, we attempted to conduct a retrospective observational study (1) to address whether preoperative and intraoperative hyponatremia during LT are associated with diminished post-LT survival and (2) to find the optimal cutoff of preoperative and intraoperative serum sodium concentration that is associated with poor posttransplant survival.A flow diagram showing the inclusion of participants in the study and the results of propensity score matching are shown in Supplemental Fig. To evaluate the cutoff of pre- and intraoperative sodium levels that is associated with one-year mortality, the minimal p-value approach was used. The results of serial multivariable Cox regression analyses according to the different cutoffs of pre- and intraoperative serum sodium are shown in Table Using the cutoff of 130 mEq/L of pre- and intraoperative hyponatremia, both preoperative and intraoperative hyponatremia was entered into the multivariable Cox regression analysis for one-year mortality and logistic regression analysis for in-hospital mortality. The result of Cox regression analysis is shown in Table The median follow-up after LT was 365 days . 112 (9.6%) of the 1164 patients (9.6%) died in-hospital, and 132 patients (11.3%) died within one year after LT. Figures Table Supplemental Figure We investigated the association between preoperative and intraoperative serum sodium levels and one-year all-cause mortality after liver transplantation. The main finding of our analysis was that intraoperative serum hyponatremia defined as <130 mEq/L was an independent predictor of one-year mortality after adjustment of MELD score and other perioperative covariates of the severity of illness. This cutoff was determined according to the minimal p-value approach. Although preoperative hyponatremia was also associated with poor one-year survival, the association was not significant after propensity score matching and was not an independent predictor of one-year mortality in the multivariable Cox regression analysis. Therefore, our data indicated that intraoperative hyponatremia determined by a mean value < 130 mEq/L may be a stronger prognostic factor that is associated with one-year mortality after liver transplantation.12, other studies reported no impact of preoperative hyponatremia on survival14. A recent large retrospective study of 47,254 patients who underwent LT in the United States reported no association of preoperative hyponatremia and 90-day mortality after LT15.It has gained consensus that preoperative hyponatremia adversely affects the survival of end-stage liver patients, including those on the LT wait list. However, the impact of preoperative hyponatremia on post-LT survival has been debated. While several studies reported a significant association between preoperative hyponatremia and decreased short-term post-LT survivalWe hypothesized that the mean intraoperative serum sodium levels may be significantly and independently associated with one-year posttransplant mortality. As shown in our results, the intraoperative serum sodium levels tended to be lower than the preoperative value. Despite frequent metabolic acidosis during surgery and sodium bicarbonate administration, a large amount of crystalloid and colloid administration by hemodynamic derangement during surgery may lead to dilutional hyponatremia. Also, the amount of fluid administration tend to be excessive before declamping of inferior vena cava, and diuretics used to treat fluid overload may also contribute to intraoperative hyponatremia. This frequent and severe hyponatremia during surgery may be strongly associated with postoperative mortality than preoperative hyponatremia.19. Preoperative sodium levels obtained in our study was also from a single measurement in most of the cases. Meanwhile, intraoperative serum sodium levels were obtained during standardized pre-specified time points during surgery in our study. Mean sodium levels calculated from eight intraoperative values contain information of sodium homeostasis during the surgery including the important periods of graft anastomosis and reperfusion. Mean sodium levels may reflect the overall status of sodium homeostasis during LT with high validity. Second, although preoperative serum sodium concentration may vary depending on the factors including intravascular volume status and the amount of administered diuretics at the time of measurements, previous studies did not extensively adjust the potential covariates through propensity score matching17. Adjustment of many confounding factors may have revealed the more strong association between intraoperative sodium levels and mortality.There could be another explanation for the strong association between intraoperative hyponatremia and postoperative mortality. First, the preoperative serum sodium levels investigated in previous studies were obtained at a single time point with varied interval from the time of transplantation22. Our analysis of the cause of death during one year after transplantation showed that the incidence of hepatic failure and renal failure were significantly higher in the patients with intraoperative hyponatremia, which was the same in the propensity score-matched cohort. This means that intraoperative hyponatremia may be associated with the prognosis of liver graft and risk of postoperative renal failure. Furthermore, our sensitivity analysis of first intraoperative sodium groups showed that there was a significant difference in survival between groups. This difference was also observed after propensity score matching. This result supports the hypothesis that the intraoperative sodium levels may be more important to predict one-year mortality than the preoperative serum sodium level.It is not clear whether the association between serum sodium and mortality is attributable to hyponatremia per se or if it simply reflects the fact that patients with poorly maintained serum sodium concentration during the transplantation surgery tend to have a baseline and operation-related characteristics that are related to poor patient survival. Hyponatremia is associated with hepatorenal syndrome, ascites, and mortality from liver disease23. More patients in the intraoperative hyponatremia group developed osmotic demyelination syndrome than normonatremia group. The comparison of baseline characteristics of our study population according to mean intraoperative sodium levels showed significant differences in the baseline medical status of patients. The patients with mean hyponatremia had higher MELD scores, received large crystalloid infusions and had more frequent intraoperative transfusions. Although these covariates were adjusted to evaluate the association and were mated using propensity score, our results may have been biased by other unknown or unmeasured baseline and intraoperative characteristics.Specifically, intraoperative hyponatremia is associated with postoperative central pontine myelinolysis that could influence the overall mortality17. They compared 30-day and one-year mortality according to the delta sodium, a difference between pre- and postoperative serum sodium levels. A larger increase in serum sodium was associated with prolonged intubation time, and ICU stay, but not mortality.To our knowledge, there is no previous study that reported the prognostic value of mean intraoperative serum sodium. A previous small retrospective study of 164 patients evaluated the influence of a change in serum sodium after surgery on the clinical outcomes after LT14. However, little is known about how the correction of intraoperative hyponatremia affects long-term outcomes after LT, except the risk of rapid correction of hyponatremia.The present study has several important limitations. First, this was a single-center retrospective study of a relatively small sample size. Our data showed that intraoperative serum sodium levels tend to become lower than the preoperative value during transplantation surgery. This may be because the patients become sicker at the time of transplant and/or fluid administration during anesthesia induction may cause hemodilution and a decrease in serum sodium concentration. With the same sample size, the more frequent intraoperative hyponatremia may be strongly associated with mortality compared to preoperative hyponatremia. Second, our data came from the institutional database of more than ten years. The policy of administrating colloid, sodium bicarbonate or diuretics, and the choice of the type of main crystalloid solution may have changed over this extended period. Third, the clinical implication of our finding of an association between intraoperative hyponatremia and mortality is uncertain. A recent study reported that patients with hyponatremia corrected before LT had no survival benefit compared with the patients with normal sodium homeostasisIn conclusion, the present study demonstrated that intraoperative hyponatremia independently predicts one-year mortality after LT, while preoperative hyponatremia did not. The cutoff of hyponatremia during LT was determined to be 130 mEq/L according to the minimal p-value approach. Intraoperative mean serum sodium levels may have prognostic value independent of other predictors of patient survival.This retrospective observational study was approved by the institutional review board of Seoul National University Hospital (1803-096-930). All methods were carried out in accordance with the approved guidelines. The need for informed consent was waived given the study’s retrospective design. We retrospectively reviewed the prospectively collected liver transplantation data base of our institution of 1211 consecutive adult patients (≥18 years old) who underwent liver transplantation for the first time at our institution between January 2005 and December 2015. Patients who did not have preoperative or intraoperative serum sodium concentrations reported (n = 31) were excluded. The remaining 1180 patients were included.During the study period, organs for non-living related transplant were allocated according to the policy of Korea Network for Organ Sharing (KONOS). According to the rules of KONOS, organs are allocated according to the grading of emergency. The grade of emergency ranges from 1 to 5. The emergency grade 1 is determined when fulminant hepatic failure develops in a patient admitted in the intensive care unit with more than one of the following conditions including ventilator therapy, hemodialysis and prolonged prothrombin time: international normalized ratio (>2.0). The grade 1 is determined regardless of the MELD score. The emergency grades 2 to 5 is determined according to the MELD score: grade 2 (MELD 38 to 40), grade 3 (MELD 31 to 37), grade 4 (MELD 21 to 30), grade 5 (MELD < 20).2 of 0.5. Radial and femoral arterial pressures were continuously monitored. A Swan-Ganz catheter was inserted through a 9 Fr Advanced Venous Access catheter  that was placed into the right internal jugular vein. Continuous cardiac index and right ventricle-associated variables were monitored using the Vigilance II monitor . Ephedrine and continuous infusion of dopamine and/or norepinephrine and/or epinephrine were used to maintain hemodynamics according to the monitored cardiac index, mixed venous oxygen saturation, and systemic vascular resistance. Donor grafts were prepared with histidine–tryptophan–ketoglutarate solution. The piggyback technique was used to anastomose the graft and donor vessels. End-to-end anastomosis of the hepatic artery and duct-to-duct anastomosis of the bile duct were carried out in succession. During the surgery, immunosuppression was induced with 1000 mg methylprednisolone  and 20 mg basiliximab I.V. . Intraoperative diuretics of furosemide 10 to 20 mg were used in patients with persistent positive fluid balance and when volume overload was suspected with pulmonary artery occlusion pressure being greater than 18 mmHg. Sodium bicarbonate of 40 ml was administered when base excess was less than −10 mEq/L. Vasopressin was not used intraoperatively during the study period.The anesthesia protocol of our institution during the study period was as follows. Anesthesia was induced with propofol and rocuronium, and was maintained with sevoflurane and remifentanil. Volume controlled ventilation was maintained with a tidal volume of 6–8 ml/kg and a FiO24. Hyperglycemia-corrected serum sodium levels were used to classify and match the different sodium groups.Demographic, clinical, and laboratory data at the time of LT were extracted from our liver transplantation database including MELD score and underlying liver disease diagnoses. Preoperative serum sodium levels measured within one month before transplantation were collected. If there were more than one measurements of serum sodium during the month before surgery, the average value was used for preoperative serum sodium. Mean intraoperative serum sodium levels were calculated as an average of the following eight values routinely measured according to our anesthesia protocol of our institution: after anesthesia induction, 1 hour after anesthesia induction, 10 min after the beginning of the anhepatic phase, 5 min before and after graft reperfusion, 20 min after reperfusion, 5 min after the start of biliary reconstruction, and at the end of surgery. All perioperative and intraoperative serum sodium levels were corrected for hyperglycemiaThe primary outcome variable was one-year all-cause mortality after liver transplantation. Secondary outcomes included in-hospital mortality, the incidence of osmotic demyelination syndrome, length of ICU and hospital stay, and the incidence of postoperative renal replacement therapy. Patient survival was calculated as the interval between LT and the last known follow-up or death.P < 0.05 was considered statistically significant. The Shapiro-Wilk test was used to determine the normality of the data.SPSS software version 23.0  and STATA/MP version 15.1  were used to analyze the data. For all analyses, First, we determined the appropriate serum sodium cutoff level that is associated with posttransplant all-cause 1-yr mortality. A minimal p-value approach was used to determine the cutoff of sodium levels with a range of 120 to 140 mEq/L. We did not evaluate cutoff >140 because we intended to find the cutoff of hyponatremia. Multivariable Cox proportional hazard regression analyses were performed using different cutoffs for both preoperative and intraoperative serum sodium, separately. Cox regression analyses included the following covariates that may be associated with postoperative mortality: living versus deceased donor, recipient age, sex, body-mass index, history of hypertension, diabetes mellitus, preoperative diuretics administration, hepatorenal syndrome, cold-ischemic time, warm ischemic time, MELD score, graft-recipient body weight ratio, ABO-incompatibility transplantation, preoperative left ventricular ejection fraction, operation time, red blood cell transfusion during surgery, intraoperative mean blood glucose, preoperative hemoglobin and preoperative albumin.Second, we attempted to evaluate the association between preoperative and mean intraoperative sodium levels and postoperative in-hospital and one-year mortality. Multivariable logistic regression analysis was performed for in-hospital mortality, while multivariable Cox regression analysis was performed for one-year mortality. Both analyses included the same covariates listed above and also included preoperative and intraoperative hyponatremia using the cutoff determined by the above analysis.Third, a propensity score matching analysis was performed between hyponatremia and normonatremia groups for both preoperative and intraoperative serum sodium levels using the cutoffs determined by the minimal p-value approach. This analysis was performed to reduce any confounding factors that contribute to the postoperative morality. After propensity score matching, the survival between the different sodium groups and the secondary outcomes variables were compared between the matched groups using the Kaplan-Meier curve. Multivariable logistic regression analysis was used to determine the probabilities of hyponatremia and normonatremia group assignment, which was used for matching. The following covariates were considered for the propensity score matching: living versus deceased donor transplantation, recipient age, sex, body-mass index, history of hypertension, diabetes mellitus, preoperative diuretics administration, hepatorenal syndrome, cold-ischemic time, warm ischemic time, MELD score, graft-recipient body weight ratio, ABO-incompatibility transplantation, preoperative left ventricular ejection fraction, operation time, red blood cell transfusion during surgery, intraoperative mean blood glucose, preoperative hemoglobin and preoperative albumin.2 of regression model were 0.31 and 0.30 for preoperative and intraoperative matching, respectively. There was no imbalance between the matched groups .Patients were matched 1:1 using a greedy nearest neighbor algorithm with a caliper of 0.1 standard deviations of the logit-transformed propensity score. A total of 428 patients were matched in the preoperative serum sodium groups while 658 patients were matched between the two intraoperative serum sodium groups (Supplemental Figs Fourth, a Kaplan-Meier curve analysis of one-year mortality was performed for both the preoperative and intraoperative serum sodium groups. The log-rank test was used to determine statistical significance between the different serum sodium groups.Fifth, as a sensitivity analysis, we evaluated the association between first intraoperative sodium levels and one-year mortality. We performed a propensity score matching between first intraoperative sodium groups. A Kaplan-Meier curve analysis of one-year mortality was performed between the sodium groups before and after matching.The missingness for age, sex, body-mass index, MELD components, serum sodium, length of hospital stay, and mortality was 0%. Missing values of other covariates were less than 8%. We imputed the missing values according to the incidence of missing. If the incidence of missing was <3%, the missing was substituted by the mean for continuous variable and by the mode for incidence variable. Missing values of variables with a ratio of missing was >3% and <8% were replaced by hot-deck imputation.Supplementary materials.


The optimal approach to airway management during in-hospital cardiac arrest is unknown.To describe hospital-level variation in endotracheal intubation during cardiopulmonary resuscitation (CPR) for in-hospital cardiac arrest and the association between hospital use of endotracheal intubation and arrest survival.Retrospective cohort study of adult patients suffering in-hospital cardiac arrest at Get With The Guidelines-Resuscitation participating hospitals between January, 2000, and December, 2016. Hospitals were categorized into quartiles based on the proportion of in-hospital cardiac arrest patients managed with endotracheal intubation during CPR. Risk-adjusted mixed models with random intercepts were created to assess the association between hospital quartile of in-hospital arrests managed with endotracheal intubation during CPR and survival to hospital discharge.Hospital rate of endotracheal intubation during CPR for in-hospital arrestSurvival to hospital dischargep value < .001). This association was modified by the presence of respiratory failure prior to arrest (p for interaction < .001), and stratified analyses demonstrated lower patient survival at hospitals with higher rates of endotracheal intubation was limited to patients without respiratory failure prior to cardiac arrest.Among 155,252 patients suffering in-hospital cardiac arrest at 656 hospitals, 69.7% of patients received endotracheal intubation during CPR and overall survival to discharge was 24.8%. At the hospital level, the median rate of endotracheal intubation use was 71.2% . We found a strong inverse association between hospital rate of endotracheal intubation and survival to discharge , 0.74 to 0.90; In a national sample of patients suffering IHCA, the use of endotracheal intubation during CPR varied across hospitals. We found a strong inverse association between hospital use of endotracheal intubation during CPR and survival to discharge, but this association was confined to patients without respiratory failure prior to arrest. Identifying the optimal approach to airway management for in-hospital cardiac arrest may have a significant impact on patient survival. More than 200,000 patients suffer in-hospital cardiac arrest (IHCA) annually in the USA with an in-hospital mortality that approaches 80% , 2. AirwAlthough prior patient-level studies of IHCA suggest a negative association between endotracheal intubation and patient survival , less isAccordingly, we analyzed data from the Get With The Guidelines-Resuscitation (GWTG-R) registry to describe hospital rates of endotracheal intubation during IHCA and evaluated the association between hospital rates of intubation and survival outcomes. We hypothesized that hospitals with lower rates of intubation during IHCA would be associated with greater survival. Prior studies suggest the association between intubation and arrest outcomes may be modified by the presence of respiratory failure and arrest rhythm, with greater potential harm of intubation with ventricular tachycardia or ventricular fibrillation (VT/VF) arrest or non-respiratory arrest. Accordingly, we also assessed for modification of the association between airway management and patient outcomes by initial arrest rhythm and the presence or absence of respiratory failure. We hypothesized that the inverse association between hospital rates of intubation and patient outcomes would be stronger for VT/VF arrests and non-respiratory arrests.8 registry, an American Heart Association sponsored prospective, multi-site registry of in-hospital cardiac arrest events. The GWTG-R has been described previously in detail [We analyzed data from the GWGT-Rn detail . Brieflyn detail .We included patients aged 18 years or older who experienced IHCA at a GWTG-R participating hospital from January 1, 2000, to December 31, 2016. If a patient had multiple IHCAs, we excluded data from subsequent episodes to focus on the index event. Patients with pre-existing invasive ventilation prior to the arrest were excluded from the analysis. We also excluded patients with missing data on airway management, first pulseless rhythm, or survival outcomes. As our analyses were conducted at the hospital level, we excluded hospitals with fewer than 10 arrests to avoid inflation of variation due to small sample sizes.We identified patients who underwent placement of an endotracheal tube during CPR. Use of a bag valve mask, nasal mask, mouth-to-mouth ventilation, or a laryngeal mask airway during CPR was considered non-tracheal intubation approaches to airway management and not included in the exposure group.The primary outcome measure was survival to hospital discharge. The secondary outcomes included return of spontaneous circulation and survival to 24 h.We determined the proportion of patients treated with endotracheal intubation during CPR at GWTG-R participating hospitals. We then categorized hospitals into quartiles based on the proportion of patients who received tracheal intubation during CPR. We compared patient and hospital-level characteristics across hospital quartile of intubation during CPR using Cochrane-Armitage test for categorical variables and simple linear regression for continuous variables.We next constructed a two-level hierarchical multivariable model to evaluate the risk-adjusted association between the hospital quartile of intubation during CPR and our primary and secondary outcomes. In these models, hospital site was added as a random effect and we adjusted for patient-level variables and hospital quartile of endotracheal intubation as fixed effects. Patient-level covariates for risk adjustment were chosen from previously validated survival models  using thWe evaluated the association between quartiles of hospital intubation rates and study outcomes using hierarchical logistic regression models with hospital-specific random intercepts. We also examined for effect modification (interaction) of the association between hospital intubation rates and patient outcomes by initial arrest rhythm and the presence or absence of respiratory failure. Further stratified analyses were planned if the interaction term was significantly associated with survival to discharge, stratified by presence or absence of ventricular tachycardia or ventricular fibrillation as the initial presenting rhythm and presence or absence of respiratory failure prior to arrest. For all analyses, the null hypothesis was evaluated at a two-sided significance level of 0.05. SAS Software Version 9.4 .We identified 155,252 index IHCAs at 656 hospitals . Similar trends were observed for our secondary outcomes of ROSC and survival to 24 h  of patients survived to hospital discharge. The unadjusted discharge survival decreased from 27.2% in the hospital quartile with the lowest intubation rate to 22.4% in the hospital quartile with the highest intubation rate (Table 4.8% of pp < .0001) and presenting arrest rhythm . The association between hospital endotracheal intubation use and outcomes was modified by the presence of respiratory failure  but not presenting rhythm (P = 0.30). Stratified on the absence or presence of respiratory failure, patients without respiratory failure were more likely to survive IHCA at hospitals with low rates of tracheal intubation use, but no association was observed between hospital rates of tracheal intubation use and survival of IHCA among patients with respiratory failure  such that subgroup analysis on different types of airways was not feasible. Fourth, we cannot exclude the potential for resuscitation time bias in which an exposure is more likely to occur the longer the cardiac arrest continues . HoweverAmong more than 150,000 patients suffering in-hospital cardiac arrest at more than 650 hospitals, the rate of tracheal intubation use during resuscitation efforts varied from 27 to 100%. Hospitals with less frequent use of tracheal intubations during CPR were associated with better patient survival. This above association was modified by the presence or absence of respiratory failure with the apparent benefit of lower hospital rates of tracheal intubation use being limited to patients without respiratory failure. A better understanding of optimal airway management in the care of patients suffering in-hospital cardiac arrest is needed.


While imaging of peripheral tissues has provided an abundance of data with regards to immune cell dynamics, central nervous system (CNS) imaging is comparatively complicated by tissue accessibility and manipulation. Despite these challenges, the results of dynamic intravital neuro-immune imaging thus far have provided foundational insights into basic CNS biology. Utilizing a combination of intravital and ex vivo 2P-LSM, we have observed novel pathways allowing immune cells, stromal cells, cancer cells and proteins to communicate between the CNS parenchyma and peripheral vasculature. Similar to what has been reported in the intestinal tract, we have visualized myeloid cells extend dendritic processes across the blood brain barrier (BBB) into pial blood vessels. Furthermore, transient vessel leaks seen during systemic inflammation provide opportunities for cellular protein to be exchanged between the periphery and CNS. These insights provide new, visual information regarding immune surveillance and antigen presentation within the CNS. Furthermore, when combining intravital 2P-LSM and microfluidic devices complexed with mathematical modeling, we are gaining new insights into the intravascular behavior of circulating immune cells. This new knowledge into the basic mechanisms by which cells migrate to and interact with the CNS provide important considerations for the design of neuro-electronic biomaterials that have the potential to connect the peripheral-neural microenvironments into a unique, artificial interface.Evolving technologies and increasing understanding of human physiology over the past century have afforded our ability to intervene on human diseases using implantable bio-materials. These bio-electronic devices present a unique challenge through the creation of an interface between the native tissue and implantable bio-materials: the generation of host immune response surrounding such devices. While recent developments in cancer immunology seek to stimulate the immune system against cancer, successful long-term application of implantable bio-material devices need to durably minimize reactive immune processes at involved anatomical sites. Peripheral immune system response has been studied extensively for implanted bio-materials at various body sites. Examples include tooth composites , inguina The use of implantation devices dates back to ancient Egypt with archeological discovery of a toe prosthesis . More reMeanwhile, challenges remain in the field of implantable neural devices, whether in applying fine sensor arrays for recording neural activity, or in reconstituting neural activities into motor commands to circumvent deficits resulting from spinal cord injury or stroke. Numerous factors contribute to this ongoing challenge, but two are classically identified as being of major importance: tissue stiffness mismatch and breakdown of blood-brain barrier (BBB). Critically, both of these issues interrupt normal neural physiology, implying that simple strategies to prevent tissue fibrosis, such as scavenging of cytokines like TGF-β, will not be sufficient. Such an approach may reduce impedance from tissue fibrosis, but would result in faithfully reporting deranged neural activity.First and foremost, these devices do not match tissue stiffness of the brain, an organ with the lowest stiffness second only to blood. The importance of tissue stiffness is underscored by the broad success of bony prostheses dating back before recorded history, whereas for blood, the most pliable tissue, even state-of-the art intravascular devices such as extracorporeal membrane oxygenation remain plagued by tissue reaction. This issue is well established in neural technology and extensively reviewed elsewhere .Second, and unique to brain tissue, device implantation invariably disrupts the BBB. This disruption leads to dramatic alterations in the function of all intra-parenchymal cells, whose physiology is predicated on residing within an environment separated from the intravascular milieu. Microglia, astrocytes, neurons, pericytes, and brain endothelial cells are all known to trigger inflammatory programs in response to serum proteins present within the brain parenchyma. In turn, this leads to further inflammation as intra-parenchymal cytokine release promotes further increase in BBB permeability, resulting in accumulation of peripheral blood immune cells such as monocytes, a cell type that is typically excluded from the immune-privileged brain parenchyma. This inflammation occurs via the coordinated synergistic efforts of multiple mechanisms, most of which follow patterns well established in other tissues.+ cells were responsible for MEA device failure that was associated with the destruction of neuronal circuits in close proximity to the implanted bio-materials  and circulating monocytes serve as the major source of antigen presenting cells (APCs), which recognize damage-associated molecular patterns (DAMPs) or pathogen-associate molecular patterns (PAMPs), while presenting processed antigenic peptides in the draining lymph nodes . However per day . Microgl per day . Microgl per day . Because per day , implyinSince microglia play an integral role in the CNS immune response, both biological interference and mechanical device design play a central role in manipulating the neuro-electronic interface. For example, anti-Parkinson’s disease monoamine oxidase B inhibitors have been shown to dampen the inflammatory response of microglia by regulating voltage-gated sodium channels, reducing the neuro-inflammatory reaction to MPTP . Another3CR1+/Ly6C+/CD45hi/CCR2+ blood derived macrophages that can be found in the perivascular and parameningeal spaces and can sample cerebrospinal fluid (CSF) in the arachnoid and Virchow-Robin spaces (3CR1loCCR2hiGR1+Ly6Chi) that are short-lived and home to inflamed tissue, or a patrolling subset (CX3CR1hiCCR2loGR1-Ly6Clo) that are long-lived and are recruited to non-inflamed tissue , we found that the number of extensions doubled in the cortex by day 9 of induction and doubled in the spine by day 12, even though clinical symptoms usually only appear by day 12. In the cortex, projections could be seen in vessels of both small and large caliber, while in the spinal cord, they were only seen in association with small caliber vessels. In contrast to this EAE model, studies of traumatic spinal cord injury showed a 40% reduction in projections that slowly recovered to 85% of baseline by day 8 after injury. Projections in spinal cord injury were associated with both large and small caliber vessels. Of interest, in a third model of primary CNS malignancy in the gray matter of the cortex, we observed dendritic extensions into the tumor neo-vasculature with a frequency of less than half of baseline (73/mm2 vs. 175/mm2). These intriguing observations required further investigations in order to understand the molecular and cellular signaling between microglia, surrounding CNS parenchyma, and endothelial tissues that regulate this behavior under physiological and different disease states.Recently, we described a unique mechanism whereby microglia extend their dendritic processes through the basement membrane and endothelium into the surrounding pial vessels, thereby providing a new potential avenue for immune cell interactions . While e3CR1+ cells extravasated from blood vessels into the lesion and increased their abundance by six-fold in 5 days. Parenchymal microglia, on the other hand, did not accumulate at the injury site. While imaging animals on days 2, 5, and 8 post-injury, we observed axon-macrophage contacts that were associated with morphologic changes of the axon, ultimately resulting in either thinning of the axon or destruction within an hour.Differential contribution of resident microglia and blood-derived monocytes/macrophages to neuron loss during CNS trauma was also demonstrated by direct intravital observation by 2P-LSM. Using a traumatic spinal crush injury model, we investigated the role of inflammatory monocytes in the process of post-traumatic axonal dieback . Immedia3CR1 microglia phagocytose blood vessel contents, which lead to their activation in the first 3 days following induction of EAE. The phagocytic CX3CR1+ microglia were then associated with an increase in CD11c+ DCs by day 6 post induction, which was then followed by infiltration and accumulation of antigen-specific T cells by day 9. Both antigen-specific T-cells and DCs congregated around areas of previous vessel leak, forming immune clusters within the CNS parenchyma. Following these observations of immune clusters, clinical EAE symptoms then started around day 10–12. Of interest, treatment with hydroxyzine ultimately decreased microglia activation and phagocytic capacity by day 3 and resulted in decreased accumulation of peripheral immune cells by day 6 , psychosis, and systemic lupus erythematosus . Indeed,While our understanding of the role of different immune subsets involved in implantable bio-material device failure will continue to evolve, one constant parameter is the absolute requirement for circulating cells to successfully navigate the vast vasculature as they traverse and home to sites of insult; either in the periphery or in the CNS. When combining 2P-LSM with resonance scanning, we were able to visualize cellular flow within post-capillary venules in the CNS in real time . Our iniin vivo (Several recent studies have implicated a potential new role for the deep cervical lymph nodes in regulating CNS immunity . Until rin vivo .The above insights from 2P-LSM intravital imaging provide several key characteristics to consider for proper design and insertion of implantable bio-materials that could preserve functional integrity without interference from intrinsic host tissue-derived immune response. First, the possibility that resident microglia can participate in direct antigen presentation by extending their processes into intact blood vessels provides a potential mechanism for recruiting and mediating additional peripheral immune cell-CNS parenchyma contact. This argues that while coating of electrodes with non-immunogenic materials and rationale device design may decrease local inflammation, the addition of systemic therapy aimed at reducing inflammatory co-factors in the peripheral circulation may provide added benefit in prolonging device longevity. Additionally, considering that microglia are more concentrated in white matter , surgicaAs tissue reactions and immune responses are highly sensitive to tissue trauma within the CNS, care must be taken in conducting experiments where disruption of CNS tissues is involved  in oAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.This work was supported in part by a Sponsored Research Agreement from Biogen Idec.


Children born preterm, less than 37 weeks’ gestation, are at increased risk of viral respiratory infections and associated complications both during their initial birth hospitalisation and in their first years following discharge. This increased burden of viral respiratory infections is likely to have long term implications for lung health and function in individuals born preterm, particularly those with bronchopulmonary dysplasia. Several hypotheses have been put forward to explain the association between early life viral respiratory infection and development of suboptimal lung health and function later in life following preterm birth. Although preterm infants with diminished lung function, particularly small airways, might be particularly susceptible to asthma and wheezing disorders following viral infection, there is evidence that respiratory viruses can activate number of inflammatory and airway re-modelling pathways. Therefore, the aim of this review is to highlight the perinatal and early life risk factors that may contribute to increased susceptibility to viral respiratory infections among preterm infants during early life and to understand how respiratory viral infection may influence the development of abnormal lung health and function later in life. Every year, more than 11% of children are born preterm; equating to an estimated 15 million of the global population. Complications of prematurity underlie more than half of all neonatal deaths worldwide, such that over one million infants die each year due to complications of preterm birth –4. The rChronic lung disease of prematurity, or BPD, was first described by Northway over 50 years ago as a severe pulmonary disease primarily caused by aggressive mechanical ventilation and prolonged exposure to high oxygen concentration in an era before the widespread use of antenatal steroid and postnatal surfactant . The sigAlongside the structural immaturity associated with preterm birth and subsequent development of BPD, many preterm infants have insufficient production and composition of pulmonary surfactant . SurfactIn addition to pulmonary structural and functional immaturity, preterm birth is also associated with developmental immaturity of innate and adaptive immune systems as well as a significant lack in the functional interaction between the two systems as outlined in Other perinatal and neonatal factors associated with preterm birth have also been associated with altered immune programming during early life, which may result in lifelong adverse immune consequences among survivor of preterm birth . ExposurThe disruption to lung development and diminished immune responses place survivors of preterm birth, at increased risk of severe respiratory infection during early life. Early-life exposure to such pathogens is likely to have lifelong respiratory consequences. Indeed, even in healthy term-born infants, hospitalisation with acute respiratory viruses in early life is associated with increased rates of childhood asthma, wheezing disorders and suboptimal lung function –31. WhilThe role of immature respiratory and immune systems has a critical impact on susceptibility to nosocomial infections and infection-related complications during the birth hospitalisation . AlthougFollowing the neonatal hospitalisation, the overall risk of re-hospitalisation with an acute respiratory infection remains at least three times higher during the first year of life compared to infants born at term ,3,38,39.Reduced gestational age is another well-known risk factor for respiratory infection-related re-hospitalisations during early life , with coP < 0.001) [Preterm infants with certain coexisting morbidities, including BPD, congenital heart disease or chronic oxygen dependency are particularly at risk for severe infections and infection-related hospitalisation during early life . Preterm< 0.001) . Similar< 0.001) .Several other factors have been attributed to the substantial vulnerability to respiratory infection and infection-related hospitalisation in preterm infants. Previous genetic studies have demonstrated a significant association between the inherited polymorphisms in several immunological and surfactant protein genes and the incidence of severe viral infection and level of infection-related morbidity among preterm infants ,51. AlsoP < 0.001) as well as more frequent (41.4 vs. 12.6%) and longer stays in the NICU for invasive respiratory support , compared to term-born infants [Nearly, all children become infected with RSV during the first 2 years of life, yet only 1% requires hospitalisation . However infants .P < 0.001), when compared with non RSV-related hospitalisation [P = .001), compared to non RSV-related hospitalisation [Increased clinical care for preterm infants hospitalised with RSV translates to increased morbidity, healthcare utilisation and costs of care for respiratory illnesses later in life ,66. Earllisation . In addilisation .Early-life exposure to severe RSV infection has also been identified as a main contributor in the development of suboptimal lung health and function later in life, with several studies reporting increased risks of late-onset or persistent wheezing phenotypes, asthma diagnosis, and impaired lung function in adolescents and young adults –31. EvenAlthough it is possible that the immature lungs in preterm infants might be particularly susceptible to long-term damage following severe RSV-related respiratory illness during early life ,73, RSV Several mechanistic pathways have been hypothesised to describe the potential role of severe RSV-related respiratory illnesses in the pathogenesis of asthma and wheezing disorders later in life, including persistent activation of atypical immune responses and alterations to the structure and function of the developing airway ,72,74. MProtection against RSV using palivizumab, a humanised monoclonal antibody for RSV prevention, can result in significant reductions in both acute and chronic morbidity following RSV-related illnesses in preterm infants, with previous studies reporting up to a 50% reduction in the frequency of hospitalisation and a 60 to 80% reduction in the relative risk of recurrent wheezing disorders during the first years of life –84. HoweStudies of the burden and impact of non-RSV respiratory viral illnesses on the subsequent development of chronic respiratory morbidity in preterm infants have been limited. Rhinovirus (RV) is a major cause of acute respiratory illnesses and wheezing episodes in infants and young children and has been associated with an increased health burden, particularly among preterm infants –94. Althp < 0.05); even though the majority of these illnesses did not require hospitalisation [Similar to RSV, RV-related respiratory illness during early life has also been associated with a high risk of suboptimal lung health and function later in life –102. Inslisation . Anotherlisation . Similarlisation  as well lisation .The long-lasting impact of early-life RV-related respiratory illnesses among preterm infants remains to be elucidated. Furthermore, despite strong epidemiological evidence supporting the role of RV in the pathogenesis of asthma, airway reactivity and airflow limitation, the underlying mechanisms are unclear. Although damage to the developing airways and altered immune responses are thought to be the main mechanisms through which RV could induce asthma and airway hyperreactivity later in life ,108, othThe synergistic interaction between viral infections, most commonly RV, and sensitization to aeroallergens during early life has also been linked to the risk of developing asthma later in life in term-born children .104,112.Influenza-related respiratory infection has long been considered as a leading cause of morbidity, hospitalisations and mortality among the elderly. However, equivalent levels of morbidity, complications, hospitalisations and costs of care have been attributed to influenza-related respiratory illnesses in young children –121. TheThe significance of several previously under-recognised and newly emerging viruses is yet to be investigated. Human metapneumovirus (hMPV) was first isolated in 2001 from young children with a spectrum of clinical disease similar to that of RSV  and has Human bocavirus (hBoV) was first identified in 2005 from the nasopharyngeal aspirate of patients with unresolved respiratory infection . HBoV haIn summary, knowledge on the long-term impact of viral infections during early life, particularly among preterm infants remains limited. The vast improvements in molecular detection techniques during the past few decades have shed light on several previously under-recognised circulating respiratory viruses as well as improved the characterisation of newly emerging viruses. However, the disease burden and long-term clinical and functional impact of a variety of early-life viral infections among preterm infants, particularly those with BPD, has not been comprehensively assessed.Despite mounting clinical and experimental evidence, the potential association of viral-related acute respiratory illnesses during early life and the subsequent development of suboptimal lung health and function is still debated. It remains unclear whether early-life infection with respiratory viruses may induce long-term deficits in the structure and function of the respiratory system through damaging the airways, and altering immune responses or whether these illnesses simply unmask a pre-existing tendency for chronic respiratory morbidity in children at risk, including preterm infants. Although it is likely that the significant immaturity of the structure and function of the respiratory system in preterm infants contributes to chronic respiratory morbidity later in life, viral-induced acute respiratory illnesses during a critical period of lung growth and development may also be an important stimulus for persistent airway remodelling. Additional studies are needed to characterise and assess the consequences of early-life infection with respiratory viruses among preterm infants – both symptomatic and asymptomatic. Identifying the mechanistic role of respiratory viruses in the development of chronic respiratory morbidity is equally important and may aid the design of more targeted therapeutic strategies to reduce virus-induced pulmonary morbidity, and potentially halt the progression of, or even prevent the development of irreversible airway disease.


Precise coordination of cellular processes requires prompt specification of protein function in response to various stimuli. This specification includes regulating protein abundance, localization, catalysis, and binding. Post-translational modifications (PTMs) provide cells the plasticity for dynamic and reversible control of protein function. Viral infections provide an exciting lens through which to study PTMs, since PTMs contribute to both cellular responses to infection and viral hijacking of the host. PTMs enhance the already multifunctional nature of viral proteins and offer another level of functional diversity within limited genetic space. Influenza virus protein functions are fine-tuned by diverse types of PTMs, including phosphorylation, ubiquitination, SUMOylation, neddylation, ISGylation, glycosylation, ADP-ribosylation, palmitoylation, and acetylation. All of the major viral proteins are subject to at least one type of PTM. Additionally, as influenza viruses encode no known protein-modifying enzymes, all of these PTMs are mediated by host machinery. Here, we use influenza virus and its proteins as exemplars for how PTMs impact virus replication .Influenza virus transcribes and replicates its negative single-stranded RNA genome via a virally encoded RNA-dependent RNA polymerase (RdRp). This is performed by the viral ribonucleoprotein complex (RNP), containing genomic RNA encapsidated by the viral nucleoprotein (NP) and bound at both termini by a single heterotrimeric RdRp. While all components of the RNP are post-translationally modified, mechanistic consequences of NP PTMs have been particularly well described. During infection, newly made NP traffics from the cytoplasm into the nucleus and then back to the cytoplasm as either free NP or assembled RNPs. NP trafficking is regulated by both phosphorylation and SUMOylation ,2. PhospNuclear NP oligomerizes along the length of newly synthesized genomic RNA. This process is negatively regulated for both influenza A and B viruses by NP phosphorylation at conserved sites on apposing sides of the homotypic interface ,4. PreveAll RNP components are ubiquitinated, and perturbing global ubiquitination impairs RNP function . In certFunctions of additional RNP PTMs are less well defined. Phosphorylation of the three polymerase subunits—PB1, PB2, and PA—has unknown consequences . PB1, PAIn addition to regulating RNPs, PTMs impact genome trafficking and evasion of antiviral responses. RNPs assemble in the nucleus and are exported to the cytoplasm, where they traffic to sites of assembly and budding. Export requires the viral matrix protein (M1) and the nuclear export protein (NEP). Current data support a daisy-chain model in which the RNP interacts with M1, M1 interacts with NEP, and NEP interacts with the cellular export machinery. Phosphorylation of M1 enhances import . M1 phosPTMs are important for the two main viral proteins that engage the host immune response: the nonstructural protein 1 (NS1) and hemagglutinin (HA). NS1 is the canonical antagonist of innate immune responses. NS1 sequesters dsRNA to avoid detection by host sensors and also antagonizes and directly binds these sensors, including RIG-I and TRIM25. Phosphorylation and ISGylation of NS1 disrupt protein–RNA interactions, while phosphorylation also disrupts protein–protein interactions –27. In tHost cell reprograming during infection often focuses on wholesale changes in gene transcription, such as the induction of interferon-stimulated genes. Remodeling of the host cell might be more broadly considered to also include changes in protein degradation, subcellular localization, and differential activation of cell signaling cascades. Cellular proteins and their PTMs regulate reprogramming events, a feature exploited by influenza virus. Influenza infection results in global changes in PTMs of the host proteome, including triggering kinase cascades , reprogrInfluenza viruses indirectly utilize PTMs to co-opt cellular machinery. The ubiquitin machinery plays key roles at multiple steps during entry . InfectiStudies of PTMs have shed light on our understanding of the influenza replication cycle while also raising exciting new questions. Whereas PTMs have been mapped to all viral proteins—with most being modified at multiple sites by diverse PTMs—the host effectors and functional outcomes of most PTMs remain unknown. While some of the PTMs have clearly defined activities (see above), perhaps the largest question is whether all of the modifications discovered so far have functional impacts during infection. Even if all of the PTMs are functionally important, it is possible that some modifications are required only under discrete circumstances but dispensable at other times, making this a more complicated question to address. We also have little appreciation for how these modifications change temporally during infection. It has been proposed that dynamic PTMs could dictate progression of the replication cycle. Indeed, it is the reversible nature of these PTMs that make them attractive mechanisms for dynamic regulation. Yet, how specific PTMs are orchestrated during influenza virus infection and whether this serves to temporally order viral processes has only begun to be explored.Viral proteins are notoriously multifunctional, and understanding how the varied tasks are separated remains elusive. An exciting possibility is that PTMs parse these different functions by establishing distinct populations of the same viral protein. For example, the separation of viral transcription from genome replication during influenza virus infection is incompletely understood. Viral proteins, small viral RNAs, and various host proteins have all been implicated in biasing polymerase output. The proteins of the viral RNP are modified by multiple PTMs, and PTMs could provide additional mechanisms to establish discrete populations of transcribing versus replicating RNPs.The roles of PTMs have generally been characterized in isolation, raising the question of how these modifications work in concert. Moreover, since certain amino acid residues can be subject to many different PTMs, a single residue may be competitively or differentially modified over time. Several examples of PTM cross-talk on influenza virus proteins have already been identified ,12,16, s


Mycobacterium bovis BCG with these mechanistically different classes of cell wall inhibitors at MIC caused a 4 to 5-fold increase in intrabacterial ATP concentration. This effect on ATP homeostasis was specific to inhibitors of cell wall synthesis and not observed for other anti-tuberculosis drugs. Treating M. bovis BCG with sub-MIC concentrations of the ATP synthase inhibitor bedaquiline or the uncoupler carbonyl cyanide 3-chlorophenylhydrazone suppressed drug induced ATP surge, suggesting that the increase in ATP concentration was due to increased oxidative phosphorylation. Pharmacological suppression of the ATP burst attenuated bactericidal activity of the cell wall-targeting drugs up to 100-fold, suggesting that increased ATP levels are associated with the lethal effect of these antibiotics. Interestingly, inhibition of the ATP burst also suppressed induction of the promoter of the cell envelope stress response operon iniBAC by cell wall inhibitors suggesting a link between ATP surge and iniBAC expression. In conclusion, we show that treatment of M. bovis BCG with inhibitors of cell wall synthesis causes a burst of intrabacterial ATP by increasing oxidative phosphorylation. This ATP surge appears to be required for induction of the iniBAC cell envelope stress response operon and to contribute to drug induced cell death. Hence, this work revealed links between inhibition of cell wall synthesis, oxidative phosphorylation, iniBAC induction and cell death. The identification of the molecular mechanisms linking these processes may reveal novel targets for the discovery of bactericidal antibiotics.Mycobacterial cell wall inhibitors interfere with targets involved in synthesis of mycolic acids, arabinogalactan and peptidoglycan. These antibiotics corrupt structural integrity of the cell envelope and this is believed to be the cause of drug mediated cell death. Here, we show that treatment of  Mycobacterium tuberculosis resulting in more than a million deaths per year  was obtained from the American Type Culture Collection. M. bovis BCG Pasteur (ATCC #35734) piniBAC-RFP harbors the coding sequence of the red fluorescent protein mCherry under control of the iniBAC promoter and was constructed as previously described  and incubated for 7 days at 37°C and 80 rpm prior to turbidity determination . Percentage growth was determined compared to untreated control and plotted as a function of drug concentration (Graph Pad Prism 5 software). The concentrations that inhibit 90% of growth were recorded as MIC.Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method . ExponenM. bovis BCG were treated for 24 h with stated concentrations of drugs. Treated cultures were then diluted and plated on agar. Colonies were counted after 2–3 weeks of incubation at 37°C.Cidal effects of drugs were determined by CFU enumeration on agar plates after exposure to stated concentrations of test compounds as described previously . BrieflyTM Microbial Cell Viability Assay kit from Promega according to the manufacturer’s instructions and as previously described  to minimize effects of growth (or drug induced cell death). OD600 of cultures was measured at the beginning and end of the experiments and the observed OD600 increase was < / = 2-fold as expected. 25 μl of samples  were mixed with an equal volume of freshly prepared BacTiter-GloTM reagent in white flat bottomed 96 well plates (Corning) and lysis was carried out for 10 min at room temperature with shaking at an amplitude of 3 mm inside the M200Pro plate reader (Tecan). Emitted luminescence was displayed as relative light units (RLU) as described previously  and displayed as relative fluorescence unit (RFU) as described previously . After 24 h, ATP content of the cultures was determined with the BacTiter-GloTM assay with RLU as readout. Surprisingly, we not only found that some drug treatments resulted in a 4 to 5-fold increase of intrabacterial ATP levels, but that all cell wall synthesis inhibitors, independent of the nature of their primary target, had this effect . In contrast, all tested non-cell wall synthesis inhibitors had either no effect on ATP levels or, in the case of inhibitors of oxidative phosphorylation, reduced intrabacterial ATP concentration as expected . These results show that treatment of M. bovis BCG with different classes of cell wall synthesis inhibitors cause an increase in intrabacterial ATP concentration, not observed upon exposure to non-cell wall inhibitors.To determine whether any of the anti-mycobacterial drugs cause an increase in intrabacterial ATP concentration, we treated exponentially growing M. bovis BCG cultures with the MIC of the mycolic acid synthesis inhibitor INH, causing a surge of ATP, and increasing – but sub-MIC – concentrations of the ATP synthase inhibitor BDQ for 24 h. Co-treatment of cultures with INH at MIC with sub-MICs of BDQ indeed reduced the ATP surge in a dose dependent manner . At 0.016 μM BDQ, a concentration which corresponds to 1/16th its MIC (0.26 μM), the ATP surge was completely suppressed .Increase in intrabacterial ATP concentration could be due to reduced ATP utilization or due to increased ATP synthesis. If the ATP surge is due to increased ATP synthesis via oxidative phosphorylation, inhibiting F-ATP synthase should prevent the surge. To determine whether inhibition of ATP synthase prevents cell wall synthesis inhibitor-induced ATP increase we co-treated Figure 2B).To provide BDQ and ATP synthase independent evidence that inhibition of oxidative phosphorylation suppresses the INH-induced ATP burst, we carried out the same experiments with a compound targeting a different component of the oxidative phosphorylation process. Co-treatment of cultures with INH and sub-MICs of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a protonophore which reduces the proton concentration gradient across the bacterial membrane and thus affects ATP synthesis, resulted in suppression of the ATP surge .To determine whether these findings can be extended to mechanistically different cell wall synthesis inhibitors, we tested the effects of oxidative phosphorylation inhibitors on the ATP surge caused by the arabinogalactan synthesis inhibitor EMB and found the same effects: BDQ or CCCP at sub-MIC prevented EMB induced ATP increase  or CCCP  reduced the bactericidal effect of INH in a dose dependent manner up to 100-fold. The dose dependent increase of bacterial survival by BDQ or CCCP co-treatment was associated with a dose dependent suppression of the ATP burst . To determine whether this finding extends to other cell wall synthesis inhibitors, we tested the effects of BDQ and CCCP on the bactericidal activity of EMB and found the same effects: co-treatment with EMB and inhibitors of oxidative phosphorylation at sub-MICs increased survival and reduced the ATP surge .Tomioka and colleagues  showed tThese results show that sub-inhibitory concentrations of oxidative phosphorylation inhibitors attenuate the bactericidal activity of cell wall synthesis inhibitors. Suppression of cell death is associated with suppression of drug induced ATP burst, suggesting that cell death is mediated in part by the ATP surge.iniBAC, which is transcriptionally induced by cell wall synthesis inhibitors, independent of their specific targets . Thus, there was a strict correlation between drugs that trigger an increase of ATP concentration and piniBAC promoter activity . To determine whether increase in piniBAC activity depends on the increase of ATP levels, we co-treated M. bovis BCG piniBAC-RFP cultures with INH and increasing sub-MIC concentration of BDQ or CCCP for 24 h and measured fluorescence of the cultures. Sub-inhibitory concentrations of BDQ or CCCP that abrogated the ATP surge also abrogated piniBAC induction . The same results were obtained when INH was substituted with a different cell wall synthesis inhibitor, EMB .To measure transcriptional activation of BAC-RFP, ). Inductcultures . To confiniBAC cell envelope stress promoter follows the drug treatment dependent ATP concentration patterns: Drug induced ATP surge was associated with increased iniBAC promoter activity and pharmacological suppression of ATP increase suppressed iniBAC promoter activity. This suggests a link between ATP level and iniBAC expression. These results are consistent with a model in which ATP represents a signaling molecule for iniBAC induction. Whether the iniBAC regulator IniR is indeed the mechanistic link and senses the ATP burst via its AAA ATPase domain remains to be determined.Together, these results show that activity of the Antibacterials inhibit usually well-defined primary targets preventing the synthesis of some cell components. Studies over the past decade have shown that inhibition of an individual enzyme often appears to be insufficient in itself to cause rapid bacterial cell death. Rather, death is achieved by complex intracellular “corruption” events, which follow on from the initial target modulation by the drug, resulting, for instance, in accumulation of broadly toxic intermediates or suicidal derailing of signaling systems that ultimately cause collapse of central homeostatic systems within the bacteria .A key metabolite in bacterial physiology is ATP. Like all cells, bacteria need to maintain their intracellular ATP concentration within a certain range to maintain viability. In mycobacteria, reduction of intrabacterial ATP below a lower threshold is exploited by the anti-tuberculosis drug BDQ. BDQ is an inhibitor of F-ATP synthase and thus affects ATP levels directly by blocking regeneration of ATP via oxidative phosphorylation . Interesall cell wall synthesis inhibitors, but not other antibiotics tested, triggered a 4 to 5-fold increase in intrabacterial ATP concentration. This drug induced ATP burst could be suppressed by inhibitors of oxidative phosphorylation, suggesting that cell wall synthesis inhibitors trigger a common cell envelope stress response involving increased ATP production by oxidative phosphorylation. Importantly, pharmacological suppression of the ATP surge attenuated the kill effect of cell wall antibiotics, indicating that ATP increase contributes to the lethal effect of the drugs. Thus, “death by (excess) ATP” appears to be a strategy utilized by inhibitors of cell wall synthesis to kill mycobacteria.Here, we asked whether increase in ATP to toxic levels may be part of the intracellular follow-on events triggered by some anti-mycobacterials. Surprisingly, we found that Mycobacterium smegmatis (Mycobacterium, suggesting that the ATP burst triggered by cell wall synthesis inhibition may be conserved across different mycobacterial species.Our finding is consistent with recent observations made in single cells analyses of megmatis . In thisiniBAC (iniBAC (iniBAC promoter induction on the intrabacterial ATP level.Interestingly, pharmacological suppression of drug-induced ATP surge also prevented induction of the cell envelope stress response operon iniBAC . The funiniBAC . The faciniBAC . Recentl (iniBAC . This reiniBAC appears to require drug induced increase of ATP. Hence, this work reveals links between four processes, namely inhibition of cell wall synthesis, oxidative phosphorylation, cell death and iniBAC induction. The identification of the molecular mechanisms linking these processes, and how ATP burst contributes to cell death may reveal novel drug induced death pathways and targets that can be exploited for the discovery of bactericidal antibiotics. This work may also have clinical implications. Our in vitro data suggest antagonism between Bedaquiline and cell wall synthesis inhibitors. Hence, combining these two drug classes should be carefully considered. Co-treatment of patients with Bedaquiline and cell wall synthesis inhibitors may be counterproductive.In conclusion, we show that “death by ATP” appears to be an extended mechanism of action employed by inhibitors of mycobacterial cell wall synthesis to exert their bactericidal activity. Interestingly, induction of the cell envelope stress operon AS carried out the experiments. AS and TD designed the work and wrote the manuscript.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.