Nutrition support is a necessary therapy for critically ill cardiac surgery patients. However, conclusive evidence for this population, consisting of well-conducted clinical trials is lacking. To clarify optimal strategies to improve outcomes, an international multidisciplinary group of 25 experts from different clinical specialties from Germany, Canada, Greece, USA and Russia discussed potential approaches to identify patients who may benefit from nutrition support, when best to initiate nutrition support, and the potential use of pharmaco-nutrition to modulate the inflammatory response to cardiopulmonary bypass. Despite conspicuous knowledge and evidence gaps, a rational nutritional support therapy is presented to benefit patients undergoing cardiac surgery. Patients undergoing cardiac surgery are routinely exposed to significant systemic inflammation, causing organ injury and dysfunction. Cardiopulmonary bypass (CPB) triggers systemic inflammatory response syndrome (SIRS) with release of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and mainly pro-inflammatory cytokines –3. This Underfeeding is a major issue in cardiac surgery patients , 6. TablPreoperative fasting sets the stage for catabolic stress , insulinConsidering the postoperative course, in a retrospective analysis of about 5400 mechanically ventilated patients, cardiac surgery was most associated with iatrogenic malnutrition  evaluateAs society ages, older patients are presenting for cardiac surgery with an increased prevalence of comorbidities. In addition, the number of patients with advanced heart failure has increased and the implantation of pulsatile-flow ventricular assist devices (VAD) has become an established therapeutic strategy to improve survival rates and quality of life . MalnutrFigure Selection of patients who will benefit most from nutrition support in the postoperative period is critical, but has not been established or standardized. When developed, this selection process would be based on a combination of clinical and biochemical parameters related to validated risk scores for nutrition, cardiac surgery, critical illness, and emerging markers of systemic inflammation, particularly those related to cardiopulmonary bypass and postoperative ICU pharmacology and technology.have already reached an undernourished general state. To foresee an aggravation in the nutritional status, an assessment of nutritional risk must also identify patients at a pre-critical level of malnutrition, who will benefit (and not be harmed) by nutrition support. The Malnutrition Universal Screening Tool (MUST), the Mini Nutritional Assessment (MNA), the Short Nutritional Assessment Questionnaire (SNAQ), the Malnutrition Screening Tool (MST), and the Subjective Global Assessment (SGA) [Several scores or assessment tools have been introduced to enable the quantification of nutrition risk. These tools were neither developed for nor validated in critically ill patients . Therefont (SGA)  are wellLomivorotov and colleagues demonstrated that in patients undergoing cardiac surgery, detection of malnutrition is associated with prolonged ICU stay (>2 days) and both MUST and MNA have independent predictive accuracy with regard to postoperative complications . In a suAs the aforementioned tools consider all critically ill patients at high risk for malnutrition, the Nutrition Risk in the Critically ill (NUTRIC) score was developed to define nutrition risk in the critically ill ICU patients –19. The Considering the critically ill, the Nutritional Risk Score (NRS)-2002 must be interpreted cautiously as an Acute Physiology and Chronic Health Evaluation (APACHE) score >10 in cardiac surgery patients indicates that a patient is already at high risk for malnutrition. Patients with an NRS-2002 score >3 are defined as at “risk for malnutrition”, and those with an NRS-2002 score ≥5 as “high risk for malnutrition”, which may be too broad and nonspecific for directing aggressive nutrition support. In contrast, the NUTRIC score was demonstrated in five prospective, but non-randomized studies, as a sensitive measure to identify the nutritional risk and to discriminate between patients with high risk for malnutrition and those likely to benefit from aggressive nutrition therapy . AdditioUltrasound of the quadriceps muscle is an easy-to-use and readily available tool to measure muscle mass and determine changes in muscle and fat tissue –26. CompPreoperative: at least 2–7 days before surgeryEarly preoperative: ≤24 hours before surgeryEarly postoperative: ≤24 hours after ICU admissionPostoperative: >24 hours after ICU admissionDetermining the ideal time to start feeding in relation to cardiac surgery represents a crucial factor for nutritional support to be effective. Until now, only a few studies have addressed this question. In regards to timing, the following time windows may be of particular relevance:current practice currently only allows practitioners to initiate the nutrition therapy on an individualized patient-tailored consideration.One challenging aspect facing perioperative nutritional support is the fact that over half the patients having cardiac surgery are admitted as outpatients on the day of surgery, creating a significant challenge to preoperative nutritional risk assessment and timely intervention. If a beneficial role for a preoperative approach is determined, clinicians will need to overcome this challenge and consider an outpatient approach to optimize the nutritional status prior to admission. In the meantime, the best assessment and treatment window for now is immediately after surgery or soon after arrival at the ICU. Due to the limited evidence on the preoperative or early postoperative identification of these patients, Considering international guidelines, there is a strong consensus and most experts will report that EN is always preferred to PN. Following cardiac surgery, critically ill patients are frequently on vasopressor treatment because of an inflammatory response syndrome, vasoplegia, and/or postoperative low output syndrome due to myocardial stunning. The need for vasopressor support further results in marked changes in energy expenditure and frequent intolerance to oral feeding, leading to significant energy/protein deficits and increased risk of malnutrition. Although proven safe , EN is oper se is not a contraindication to EN. In hemodynamically unstable critically ill or cardiac surgery patients, there is at least some evidence that early EN is absorbed and metabolized without any harmful effect on systemic measurements of oxygenation and perfusion and supportive evidence from a large-scale observational study that this translates into an advantage in terms of mortality [Given the current evidence, vasopressor use ortality . TherefoCardiac surgery with myocardial ischemia/reperfusion and use of CPB is known to be associated with deleterious consequences, resulting from the inflammatory response during cardiac surgery. The duration of CPB time correlates with the extent of the inflammatory response. Furthermore, surgical trauma, ischemia/reperfusion, and contact activation with the CPB circuit result in the release of mainly pro-inflammatory markers, reactive oxygen species, and reactive nitrogen species that contribute to the development of organ dysfunction . In thisIn a recent clinical trial, perioperative nutritional therapy was administered in the cardiac surgery ICU in order to increase myocardial and plasma arginine/asymmetric dimethylarginine ratio and other amino acids . The invSo far, preliminary results from small phase II trials on fish oil (FO)-containing emulsions have demonstrated that preoperative FO application is a promising strategy to modulate the biological and clinical response to cardiac surgery . Berger Pharmaco-nutrition offers a promising approach to enhance the body’s defense mechanisms and to attenuate the deleterious effects of SIRS and improve outcomes. This may be of particular relevance for high-risk patients undergoing complex procedures with prolonged CPB duration and an overwhelming release of pro-inflammatory mediators.Whenever possible, preoperative optimization of the nutritional state should be targeted in the malnourished patient undergoing cardiac surgery. The increasing number of patients with advanced heart failure and planned VAD implant represent a subpopulation that may as well benefit from optimization of the nutritional state. Thus, determination of nutritional risk, preferably using a structured scoring tool, should be part of the patient’s preoperative assessment.To reach maximum benefit, preoperative nutritional therapy should be initiated in malnourished patients after cardiac surgery at least 2–7 days before surgery  .Monitoring of nutrition intake should be routinely assessed daily in patients after cardiac surgery during the ICU stay. In particular, on day 3 all patients should be carefully evaluated as to their nutrition risk and effort should be made to achieve at least 80% of their prescribed protein/energy requirements, either by enteral or parental feeding, as soon as possible.Postoperative nutrition support should be initiated early (0–24 hours after surgery) in patients at high nutritional risk with an expected prolonged ICU stay.Attention to refeeding syndrome may be of importance for patients in whom nutrition support is started after a prolonged period of starvation or in patients with preexisting malnutrition, respectively. In those patients, advancement of feeding should be slower, taking 3–4 days to reach goal, and targeting to adapt to both macronutient and micronutrient special needs .If initiated early postoperatively within <24 hours after ICU admission, an additional immune-modulating component  to nutrition may be considered for patients with complex and prolonged surgical procedures, to counteract the overwhelming inflammatory response.The few randomized trials of nutrition support in patients undergoing cardiac surgery are limited to small numbers of patients and demonstrate heterogeneous results, so the experts felt unable to give strong recommendations for clinical practice. Nevertheless, six key messages have been identified by the experts, which are thought to be of clinical relevance in the treatment of these patients:In extension to the need of reliable data, international standardized procedures such as the ESPEN and IASMEN endorsed strategy for Enhanced Recovery After Surgery (ERAS) are warranted to optimize nutrition support in cardiac surgery patients. In view of the heterogeneous standards of perioperative care in these patients and lack of evidence provided by large-scale RCTs, the multi-modal ERAS program for optimal perioperative care may help to reduce surgical stress, maintain physiological functional capacity, and facilitate postoperative recovery by providing the best available evidence .Targeting preoperative optimization of the nutritional state may result in improved postoperative outcome. Structured scoring tools should be validated and implemented as part of preoperative assessment and to monitor the efficacy of nutrition therapy.In identified patients, the feasibility and clinical significance of early-initiated postoperative nutrition support needs to be evaluated.Dose-finding studies for both macronutrients and micronutrients are needed to answer the questions of “how to supplement patients after cardiac surgery” and “with which combination of nutrients”.To counteract the frequently occurring inflammatory response, the clinical significance of an immune-modulating component  should be evaluated in patients with complex and prolonged surgical procedures.Validated and reliable assessment of energy requirement in patients after cardiac surgery need to be developed.The role trophic EN might play in the hemodynamically stable patient after initial stabilization needs further evaluation.Furthermore the multidisciplinary group identified six important topics for future research:Valid and reliable data are urgently needed to improve the so far non-standardized clinical practice of nutrition screening, assessment, and support in patients after cardiac surgery. Although both inflammatory response and postoperative complications are predictable, clinical practice has several restrictions, limiting optimal nutrition therapy. The accurate identification of patients who benefit most from nutritional therapy presents a clinical imperative requiring validation by adequately powered clinical studies.


Our understanding of the relative importance of these two populations in the distinct processes of pathogenesis and resolution of inflammation during neurodegenerative disorders was limited by a lack of tools to specifically manipulate each cell type. During recent years, the development of experimental cell-specific depletion models has enabled this issue to be addressed. Herein we compare and contrast the different depletion approaches that have been used, focusing on the respective functionalities of microglia and monocyte-derived macrophages in a range of neurodegenerative disease states, and discuss their prospects for immunotherapy.While bone marrow-derived Ly6C Monocytes are constantly turned over and generally have limited life spans in the circulation.“Myeloid cell” is a collective term that encompasses many innate immune cells including macrophages and monocytes. Resident macrophages are specialized subsets defined by their organ location  microglia, liver Kupffer cells), having been differentiated in these specialized microenvironments during development from (in the majority of cases) embryonic precursors. They primarily conduct homeostatic functions in the tissues, but their numbers and properties can change during a local inflammatory process, and they can proliferate within tissues in order to maintain homeostasis. In contrast, circulating monocytes are derived from hematopoietic stem cells in the bone marrow and are generally divided into two subsets in the blood of mice based on their expression of Ly6C. Ly6Chi monocytes can be accomplished by antibody-mediated depletion  under the CD11b-promoter , acute neurodegeneration  or chronic neurodegeneration , Alzheimer’s disease, Prion disease). We do not review the role of microglia in development or homeostasis and refer the reader to separate reviews addressing these areas of research . Mice accumulate mutant SOD1 aggregates in the spinal cord, leading to motor neuron death and recapitulation of the central aspects of ALS pathology including an age-dependent accumulation of CD11b−/− mice with wildtype bone marrow extends survival compared to mice transplanted with mSOD1 bone marrow  of mSOD1 mice, reaching as much as 30% of the CD11b+ compartment in late stage animals, and that their antibody-mediated depletion attenuated motor neuron death and delayed disease onset and mortality. Such monocyte-specific contribution to disease progression is very interesting, since most previous studies have used targeting systems that do not discriminate between microglia and monocytes. However, this study would benefit from confirmation in other laboratories since the presence of spinal cord monocyte-derived macrophages is contradicted by parabiosis experiments  is a microtubule-stabilizing protein that during the course of AD becomes hyperphosphorylated, a process leading to dissociation from microtubules and aggregation into paired helical filaments and formation of neurofibrillary tangles  exacerbated prion disease and reduced survival, suggesting a neuroprotective role for microglia. Since the two studies have used different scrapie strains, different strategies to limit the microglial response and different timing of the microglial targeting, it is difficult to consolidate the data sets. However, it could indicate strain-specific microglial responses or suggest that microglia possess neuroprotective and neurotoxic reactions to prions at different time points during the disease course.Prion disease is a transmissible neurodegenerative disorder affecting both animals and humans. The contribution of myeloid subsets to disease progression has been characterized using mouse models in which prion disease is produced by intracerebral inoculation of scrapie protein strains. While microglial proliferation and activation occurs irrespective of the inoculated prion strain  and its mouse model EAE is initiated by autoreactive, myelin-specific CD4It has been demonstrated that EAE severity correlates with numbers of monocytes infiltrating the spinal cord and that prevention of this infiltration protects against EAE development  phases, or their lack of achieving this role during chronic disease states, warrants further investigation.Stroke is caused by severe occlusion or stenosis of a cerebral artery and is the second leading cause of death and disability worldwide. Brain resident microglia become activated by various cytokines and damage-associated molecular patterns which are released by necrotic or apoptotic tissues  rather than solely their previously perceived role in amyloid phagocytosis  programmes that we are currently unaware of but which have significant bearing on the interpretation of the studies.full microglial functionality. Exactly how the CNS regulates the repopulation process and senses when the niche is replenished are important unanswered questions.The enormous propensity of the CNS to repopulate with myeloid cells, be they surviving microglia colonies that hyperproliferate, or CNS-adapted infiltrating monocytes that fill up the available niche, indicates that a myeloid-deficient CNS is a highly non-physiological condition. While the infiltrating monocytes co-occupy the CNS and begin to express microglia-specific proteins, it still remains to be proven if they develop What is clear from our review is that there is some disparity between the results of different microglia depletion systems in the same disease model. Discerning the underlying molecular mechanisms that lead to these different outcomes will serve to further our knowledge of the pathogenesis that we wish to target therapeutically. In particular, the relative timing of microglial depletion in relation to the insult might explain some of the disparity, and deserves further consideration.While the homeostatic functions of microglia are well understood, these cells always having activity in surveying the CNS, the molecular basis for these functions are less well characterized. TGFβ appears to be a key cytokine in preventing microglia and other infiltrating myeloid cells from pro-inflammatory (pathogenic) activation  or replacement with CNS-adapting macrophages are intriguing prospects for immunotherapy. We have previously reported that immunosuppressive macrophages can down-modulate pro-inflammatory macrophage and T cell activities in settings of experimental MS (Zhang et al., All authors have had similar roles in defining the literature to be reviewed, preparing individual sections of the text and working up the final version of 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.


The incidence of delayed graft function in patients undergoing kidney transplantation remains significant. Optimal fluid therapy has been shown to decrease delayed graft function after renal transplantation. Traditionally, the perioperative volume infusion regimen in this patient population has been guided by central venous pressure as an estimation of the patient’s volume status and mean arterial pressure, but this is based on sparse evidence from mostly retrospective observational studies. Excessive volume infusion to the point of no further fluid responsiveness can damage the endothelial glycocalyx and is no longer considered to be the best approach. However, achievement of adequate flow to maintain sufficient tissue perfusion without maximization of cardiac filling remains a challenge. Novel minimally invasive technologies seem to reliably assess volume responsiveness, heart function and perfusion adequacy. Prospective comparative clinical studies are required to better understand the use of dynamic analyses of flow parameters for adequate fluid management in kidney transplant recipients. We review perioperative fluid assessment techniques and discuss conventional and novel monitoring strategies in the kidney transplant recipient. Despite improvements in outcomes for patients undergoing renal transplantation, delayed graft function (DGF) remains a significant complication and is a predictor of the subsequent clinical course . DelayedThe kidneys receive approximately 25% of the body’s total cardiac output (CO) and are essential for maintaining tonicity of body fluids and for adjusting extracellular volume. Patients with renal failure often have electrolyte imbalances and tend to oscillate between hypovolemia and hypervolemia . This reCentral venous pressure (CVP)-guided volume infusion is the traditional approach in renal transplantation , 8 and iPrevious studies on the assessment of intravascular volume and optimization of CO and renal blood flow have indicated that conventional monitoring provides insufficient data for adequate fluid management –16. HoweIn this article, we review perioperative fluid assessment techniques and discuss conventional and novel monitoring strategies in this challenging patient population.The definition of DGF is not consistent in the literature. At least 18 different heterogeneous criteria were identified in a systematic review . DelayedThe reported incidence of DGF varies from 2 to 70% in deceased kidney transplants , 23, notThe underlying pathophysiology of DGF can be donor, recipient or surgeon related and can be due to hemodynamic (ischemic–reperfusion) or immunological (particularly T-lymphocyte) processes. A key factor is occult imbalance between oxygen delivery and consumption in the graft .Acute tubular necrosis (ATN) is the most common cause of DGF. Acute tubular necrosis can be already present at the time of organ procurement if the deceased donor’s kidney is injured. Prolonged warm and cold ischemia time, as well as the manner of preservation, also can lead to ATN . Acute tSeveral epidemiological studies in different patient populations have confirmed an association between stages of AKI and short-term and long-term outcomes –33 as weIn summary, DGF is predominantly caused by ATN. Adequate perfusion of the transplanted kidney is required, but to date no specific therapeutic intervention has reduced the incidence of ATN and recovery . The rolEuvolemia: a state in which the vascular volume is adequate for filling of the heart and maintenance of an appropriate CO to generate the appropriate oxygen supply for tissue needs.Fluid responsiveness: a state in which an increase in vascular volume increases the stroke volume (SV) and CO. The notion of fluid responsiveness does not necessarily mean that the patient will benefit from or need fluid therapy. Parameters of perfusion should guide the decision to assess the fluid response rather than responsiveness in a compromised patient.Fluid overload: a state in which excessive accumulation of fluid in the vascular system is caused by excessive parenteral infusion or deficiencies in cardiovascular or renal fluid volume regulation.Hypovolemia: a state in which effective circulatory volume is decreased and there is insufficient oxygen delivery to tissues, possibly resulting in organ dysfunction.The assessment of a patient’s intravascular volume for achievement of appropriate fluid management is a major challenge for the clinician and has been shown to improve postoperative outcomes . UnderstDespite a clear definition, there is no gold standard for the diagnosis and treatment of hypovolemia. Both acute and chronic conditions interact with fluid homeostasis, and medical history must always be considered. Progressive loss of renal function results in various adaptive and compensatory changes to maintain homeostasis with glomerular filtration rates below 10 ml/min, and these abnormalities typically have clinical consequences. A patient with end-stage renal disease has a disturbed acid–base and electrolyte balance and compromised hemodynamic autoregulation. Inflammatory mediators can alter renal hemodynamics and cause glomerular, tubular and interstitial damage, if uncontrolled .In addition to the uncertainty associated with the patient’s volume state, appropriate targets remain unclear. To determine which monitoring method provides the most accurate information in the context of kidney transplantation, and which target to rely on, we will review previously implemented methods, their advantages and disadvantages, and their relative utility in predicting fluid responsiveness and tissue perfusion. Methods range from conventional techniques such as clinical history and physical examination, measurement of blood pressure (BP), heart rate (HR), urine output (UOP), CVP and pulmonary artery pressure (PAP), to novel technologies such as echocardiography, cardiorespiratory interactions and dynamic analyses of flow parameters.Clinical experience and use of traditional parameters such as BP, HR, UOP, CVP  and PAP Hypovolemia in the perioperative setting often can be misdiagnosed clinically  due to aIn the case of hypovolemia and hypoperfusion, physiological compensatory mechanisms can maintain effective vascular volume and perfusion by increasing the vascular retention of an infused volume and decreasing vascular capacitance. Plasma levels of renin and aldosterone increase and atrial natriuretic factor decreases, followed by arterioconstriction which decreases precapillary hydrostatic pressure and filtration , 42. A 3Blood pressure is the most commonly used vital parameter for organ perfusion, and functions as an indirect measurement of renal perfusion in clinical practice. Although classic physiological studies suggest that autoregulation of renal blood flow is maintained at lower levels of mean arterial pressure (MAP 50–60 mmHg) , 45, retGiven the expected large oscillations in hemodynamic parameters during kidney transplantation, invasive arterial BP monitoring is commonly used. In a recent study of the biochemical outcomes of renal transplant recipients, no difference was observed in the creatinine levels of patients with a MAP between 95 and 131 mmHg, but MAP was continuously maintained at > 95 mmHg via the judicious use of fluids and dopamine . ConsistEarlier studies have indicated that the magnitude of UO is one of the most clinically used predictors for graft function immediately after reperfusion  and propA decrease in CVP in the post-transplant period is observed in a majority of kidney recipients, independent of their intravascular fluid status , but thiUse of pulmonary artery catheters has fallen out of favor due to concerns about complications such as pulmonary embolism among high-risk patients , pulmonaStatic cardiac filling pressures such as CVP and PAP correlate poorly with the intravascular volume and are notoriously unreliable in predicting accurate fluid responsiveness compared to dynamic parameters , 60. In Thus, the CVP should not be used alone to guide fluid responsiveness  and shouThe primary goal in the perioperative setting is to avoid tissue hypoxia, the major cause of organ dysfunction. Traditional indicators can be normal in the presence of tissue hypoxia and cannot be used to predict an imbalance between oxygen demand and consumption, particularly if they are not interpreted in the context of perfusion markers such as CO, lactates and central venous saturation –15, 61.Sufficient oxygenation and tissue perfusion are crucial for all metabolic needs of cells and are based on hemoglobin concentration, arterial oxygen content and CO. Therefore, the percent change in any one of these three variables equally alters oxygen delivery, with CO usually having the largest changes . As CO iAccording to Frank–Starling’s law of the heart, administering fluids can increase the preload and thereby increase SV and CO. Under physiological conditions, both ventricles work on the ascending portion of the Frank–Starling curve, and afterload, contractility and HR are assumed to remain constant during the volume infusion . This meAmong the many devices and monitors currently available for measuring cardiac function and output, we will review the intraoperative use of transesophageal echocardiography (TEE) and emphasize the utility of noninvasive devices that calculate reliable trends in CO and dynamic indices based on simple physiology.Transesophageal echocardiography can be a useful diagnostic tool for evaluating right and left heart function, potential outflow tract obstruction and the presence of a pericardial effusion. It also can be used to obtain Doppler measurements of CO. However, TEE is operator dependent, and errors in the measurement of the diameter of the left ventricular outflow tract skew results. Presence of an irregular HR also affects the final calculation of CO.Use of intraoperative TEE on renal allograft outcomes has only been evaluated in one study, in which living donor renal transplants were examined. Guiding fluid therapy in 110 patients by corrected flow time obtained with continuous transesophageal Doppler (TED) monitoring did not improve immediate graft function compared to fluid administration guided by CVP. However, significantly less fluid was used in the TED-monitored group, and the incidence of postoperative complications related to fluid overload was reduced .Transesophageal eechocardiography does not permit continuous monitoring for instantaneous evaluation of fluid responsiveness. Transesophageal echocardiography is also time consuming and expensive, and requires intensive operator training. Careful attention must be given to the limitations of the assessment of CO by TEE, which may be beyond the level of expertise of a physician with only moderate experience. New studies with more robust data are required to support the use of TEE for fluid management in this patient population.Cardiac output alone can be a poor predictor of fluid responsiveness, although its response to a preload-modifying maneuver such as mechanical ventilation can provide useful information about fluid responsiveness without the administration of fluid .2 rebreathing [Currently there are several noninvasive CO monitoring technologies including pulse contour analysis (PCA), pulse wave transit time, thoracic electrical bioimpedance/bioreactance and COreathing . We willreathing .Dynamic variation of arterial waveform-derived parameters , pulse pressure variation (PPV) and stroke volume variation (SVV)) in mechanically ventilated patients provides a precise indication of fluid responsiveness, particularly when compared to static indices . HoweverThe fundamentals underlying these parameters are based on simple physiology and are well represented in the Frank–Starling curve Fig. . The cycDynamic analyses of flow parameters need to be restricted to patients under controlled mechanical ventilation with tidal volumes between 8 and 10 ml/kg ideal body weight because heart–lung interactions are more complicated with spontaneous breathing efforts with or without a ventilator . When riThe use of PPV may be preferable to SPV and SVV because it is measured directly from the arterial pressure tracing and uses more advanced digital software . DespiteIn a recent study from Japan, SVV was compared with CVP and pulmonary artery diastolic pressure as estimates of the right and left ventricular preload in patients undergoing renal transplantation. As expected, SVV better predicted volume responsiveness . A retroAn overview of studies monitoring and targeting fluid therapy in kidney transplantation and their main outcomes is presented in Table Accumulating evidence supports the concept that fluid therapy should be individualized and based on dynamic indices of the intravascular volume. Conventional monitoring does not provide sufficient information for this approach, but novel technologies may allow titration of therapeutic interventions to ensure that oxygen delivery matches tissue need. One approach using dynamic indices has been goal-directed therapy (GDT). Goal-directed therapy decreased postoperative complications, hospital length of stay, mortality and hospital costs in high-risk surgical patients –19. HoweIn contrast to GDT, which is guided by “goals”, flow-directed therapy dictates fluid administration based on “triggers” . The triBy optimizing perioperative hemodynamic management in kidney transplantation, we hopefully can improve patient and kidney outcomes. The most recent studies of fluid therapy in the perioperative setting provide more questions than answers and challenge faith in traditional concepts. Effective perioperative fluid administration in kidney transplantation remains a black box, reinforced by the fact that fluid requirements are highly variable among patients and surgical procedures and the assessment of an individual patient’s intravascular volume remains challenging. It is unlikely that there will be a standardized all-inclusive algorithm, and a more highly individualized evidence-based approach adapted to each patient’s physiological needs and responses to volume and pharmacological therapy might be more helpful.Prospective comparative clinical studies are warranted to better understand the use of dynamic analyses of flow parameters for optimal fluid management in specific patient populations such as kidney transplant patients, who often have impaired cardiovascular physiology and reduced hemodynamic autoregulation due to end-stage kidney disease.


In this Perspective on the research article by William Rudgard and colleagues, Priya Shete and coauthor discuss the challenges of measuring the impact of social protection programs such as cash transfers. Tuberculosis (TB) disproportionately affects the poorest and most vulnerable individuals—people who face tremendous economic barriers to accessing TB diagnosis and treatment . Even thPLOS Medicine, William Rudgard and colleagues evaluate the association between cash transfer programs  and catastrophic costs, defined as total costs in excess of 20% of annual household income [If we are to demonstrate that social protection programs improve TB outcomes, we must first define an appropriate outcome measure that balances health status  and economic vulnerability . In the present issue of d income . This oud income ) incorpod income .Rudgard and colleagues suggest that a TB-specific cash transfer approach, in which cash transfers are provided specifically to TB patients, has the potential to prevent patients with TB from suffering catastrophic costs more than TB-sensitive cash transfers, in which TB patients are eligible for cash transfers based on other measures of vulnerability but not specifically targeted by virtue of their TB status . This imUnfortunately, demonstrating that social protection improves a specific downstream health outcome is a very challenging task. Ecological studies have shown association between increases in national spending on social protection and decreases in TB burden . HoweverAs we seek to build a stronger case for such social protection interventions improving TB outcomes, a number of elements must be addressed. First, we need better evidence to support consensus measured outcomes. In the case of catastrophic costs, for example, there are ongoing efforts to better measure TB-related costs across a variety of country contexts and to define the most appropriate threshold in terms of association with TB outcomes . Not onlUltimately, if social protection is to help eliminate TB or any other disease of poverty, concerted effort is required between epidemiologists, clinicians, policy makers, economists, and health systems experts to create the evidence base necessary to demonstrate a causal link between the two. Progress in this regard is not only possible; it is necessary. The TB and global health communities must continue to integrate disease-specific health priorities within the larger health systems goals of UHC and global priorities outlined in the Sustainable Development Goals (SDG) agenda. Attempting to achieve these goals in the setting of limited data should reaffirm the importance of supporting research that is both methodologically rigorous and politically relevant. Only through such a coordinated approach can we hope to eliminate specific diseases such as TB while also achieving sustainable multisector development targeting those populations most affected by diseases of poverty and the poverty of disease.


Dear Sir:Acute liver failure secondary to dengue virus infection is a rare but catastrophic event, which is poorly understood. In the December issue of the Journal, Kye Mon and others describe liver-related complications among 1,926 patients with serologically confirmed dengue infection in Thailand.We explored other studies that reported acute liver failure with dengue infection, as summarized by Kye Mon and others; only one of these studies mentioned assessment of alcohol intake. This study indicated that “Although we attempted to gather information on prior alcohol consumption, the response rate was poor and the data collected were believed to be unreliable, precluding analysis.”2Evaluating the presence of alcohol intake, either regularly or in binge, is critical whenever a provider addresses liver-related disease. In the case of acute hepatitis or liver failure from dengue infection, this seems particularly important. First, as the authors mention, hepatocytes and Kupffer cells are important targets for the virus.We applaud the efforts of Kye Mon and others to shed light on a devastating aspect of dengue virus infection. Nonetheless, we believe that studies should better evaluate alcohol intake when addressing infection-related liver failure or hepatitis in tropical settings. This approach will likely clarify several aspects of this deleterious complication of viral infection and might emphasize a potential point of prevention for liver disease.


It has been suggested that the evolution of protein complexes is significantly influenced by stochastic, non-adaptive processes. Using ligand binding as a proxy of function, we show that the structure of ligand-binding sites significantly influences the evolution of protein complexes. We show that homomers with multi-chain binding sites (MBSs) evolve new functions slower than monomers or other homomers, and those binding cofactors and metals have more conserved quaternary structure than other homomers. Moreover, the ligands and ligand-binding pockets of homologous MBS homomers are more similar than monomers and other homomers. Our results suggest strong evolutionary selection for quaternary structure in cofactor-binding MBS homomers, whereas neutral processes are more important in complexes with single-chain binding sites. They also have pharmacological implications, suggesting that complexes with single-chain binding sites are better targets for selective drugs, whereas MBS homomers are good candidates for broad-spectrum antibiotic and multitarget drug design.  •Ligand binding site structure significantly influences protein function evolution•MBS homomers have more similar ligand binding pockets than monomers and other homomers•Cofactor and metal-binding MBS homomers have more conserved QS than other homomers•MBS homomers are promising targets for developing antibiotics and multitarget drugs Homomers with ligand binding sites involving multiple protein chains (MBS homomers) evolve new functions slower than other homomers and monomers, and the ones binding cofactors/metals also have more conserved quaternary structure (QS). These complexes are likely to be promising targets for antibiotics and multitarget drugs. The majority of genomic traits show dramatic changes with organismal complexity and genome size . Due to There is, however, a notable exception to this pattern: protein complexes. The majority of proteins do not function in isolation but form complexes, which can be either homomeric, i.e., are formed by several units of the same protein, or heteromeric, which are formed by several different proteins . Whereas protein-ligand interactions have been studied for decades and are of fundamental importance in drug discovery  ontology database . ChEBI pZ score > 2) of homologs with sequence similarity between 30% and 49%. Because RMSD depends on the size of the superposed structures, we used the size of the matching pocket as a covariate. We expected that hits to binding sites in proteins with more different ligands and binding sites will have higher RMSD. As predicted, we found a significant difference between MBS and SBS homomers  of the significant hits  dehydrogenase activity, transaminase activity, thiamine pyrophosphate binding, cofactor binding, transmembrane transport, ion binding, transporter activity, and other functions frequently involving cofactor binding . Next, wThe patterns described above could also potentially emerge if MBS homomers are significantly younger and faster evolving than SBS monomers or homomers, which could result in the observed lower chemical variability, as younger protein families are expected to have less diverse functions . To testArel). Essentially, Arel is the ratio of the solvent accessible surface area observed for a polypeptide chain (ignoring intermolecular interactions) to the value expected for a folded protein of the same molecular weight . The highest frequencies of pathogenic mutations are in the binding sites of heteromers , using the sequence pairs with 50%–30% identity. We used the size of the match as a covariate because RMSD is not a size-independent measure; it was measured as the number of aligned ProBiS vertices. We performed similar searches between the ligand binding (holo) structures and ligand-free (apo) structures of the same proteins when they were both available in the PDB. In these searches, no significance cutoff was used. Variability in the structure of the binding sites was measured as the average RMSD of the best hits, using the size of the match as covariate.Structural variability of the binding sites of homologs was performed using the RMSD of the significant hits  and role (“has_role”) ontologies. The enrichment of terms was also calculated with GeneMerge, which is a generic tool for term-enrichment analysis of ranked lists. Enriched terms (after correction for multiple testing with FDR) were visualized with a method conceptually similar to the graphical output of GOrilla , with diThe evolutionary age of human proteins was based on the ProteinHistorian database , using tThe list of disease-causing mutations was downloaded from Ensembl (variation dataset) and was mapped to the PDB structures of human proteins or their homologs if the sequence similarity between the human protein and its homolog was higher than 90% and the mutated amino acid was identical. The set of putative neutral mutations is based on ExAC variants  that mapAll analyses and statistical tests (except GO and ChEBI enrichment) were performed with in-house Perl scripts and R. Protein structures were visualized with UCSF Chimera .


Humanitarian aid workers caring for Syrian refugees face major stressors as they attend to refugees’ needs on the field. Without adequate psychosocial support, evidence has shown that fieldworkers experience high burnout and turnover as well as long-term poor mental health. Unfortunately, scarce training in this regard leaves them ill-equipped to care for themselves and practice resilience while handling trauma in the field. This paper highlights our reflection on working with mindfulness programs during humanitarian crises, specifically how our program, Stress Management and Relaxation Response Training (SMART), has helped over time fieldworkers and the community they cared for. We propose that programs targeting the wellbeing of fieldworkers should be prioritized as part of efforts to improve the international aid response although they may require impeccable coordination and generous resources. We encourage donors to fund those projects viewed as special social protection programs building resilience and strengthening within system support. We argue that this will increase the efficacy of the crisis intervention and work towards sustainable peace building. In this paper, we will examine field interventions throughout the Syrian refugees crisis in Lebanon starting in 2014. As such, we will analyze two principal subjects: 1) the limited attention to stress management training for fieldworkers despite their work in a space of daily trauma, and 2) evidence that the Stress Management and Resilience Training Relaxation Response and Resilience Program (SMART-3RP) and other mind-body programs could fill in this gap as a public health intervention. By providing context in these areas, we seek to develop potential policy suggestions and next steps to improve the effectiveness of crisis interventions with renewed attention to mental health in the field [Aid workers in humanitarian crises face tremendous challenges in facilitating relief and resettlement , 3. EvenRelief workers in international conflicts are susceptible to both primary and secondary trauma through their work, and are increasingly becoming targets of violent attacks, abductions, and other tactics meant to intimidate local populations and foreign intervention , 5. SincAlthough there is still a dearth of research into trauma and mental health issues among staff in today’s humanitarian crises, available literature reveals a disproportionate risk of depression, anxiety, and especially posttraumatic stress disorder (PTSD) . This meThese gravely poor health outcomes could endanger not just those entering the aid workforce, but also disrupt the efficacy of larger coordinated humanitarian operations.Despite the overwhelming prevalence of mental health problems, there still exists a stigma in discussing vulnerability or trauma. In a survey of aid workers conducted by The Guardian, interviewees reported a “culture of silence,” with workers feeling self-conscious when speaking up about their mental issues for fear of not seeming like a “true humanitarian” . Even inStudies have shown the significant value of psychological support for staff at the level of their organization. In one study, Sri Lankan humanitarian workers who reported high levels of support from their organizations were less at risk for symptoms of depression and PTSD . This suThe complexity of the Syrian conflict puts humanitarian staff at a uniquely acute risk for trauma-related mental health problems. There were reported to be nearly 5 million registered refugees in Turkey, Lebanon, Jordan, Iraq, and Egypt by the end of 2016, with over 200 NGOs and agencies working tirelessly to provide medical care, food, water, and protection . CommuniEarlier this year, our group published our work in developing a psychosocial training intervention to improve mental health outcomes of Lebanese fieldworkers during the Syrian refugee crisis. We translated and adapted the SMART-3RP program, developed by the Benson-Henry Institute for Mind Body Medicine at Massachusetts General Hospital . SMART- The SMART-3RP blends techniques in stress management, cognitive behavioral therapy, and positive psychology. It centers on coping and increasing resiliency capabilities by teaching a relaxation response (RR) . The RR Though it is not a treatment for depression, the mind-body program has been shown to significantly decrease depressive symptoms . ImproveIn 2014, our group translated the SMART training program into Arabic and adapted it to suit the challenges of aid workers, consulting The Syrian Project Desk at the Lebanese Ministry of Social Affairs (MOSA) for cultural appropriateness and wording. Social workers were recruited from regions across Lebanon through flyers prepared and submitted by the MOSA. The recruitment was open to all social workers interested in the program with priority given to social and fieldworkers involved with Syrian refugees. One hundred twenty participants enlisted and all underwent the training. Of the 120 fieldworkers who underwent SMART training, 100 opted to participate in the survey portion of our research with 20 abstaining from enrolling in the study and their reservations were noted. Our results noted that the SMART program was helpful in encouraging mindfulness and self-reflection. When applied to their stressful environments, staff reported that these practices allowed them to be more aware of their negative emotions and think more positively. They also voiced improved problem-solving capabilities, with greater patience in overcoming challenging situations [The survey responses from the SMART-3RP trained humanitarian workers in Lebanon also highlighted interpersonal skills that developed in parallel with personal stress management—a valuable capacity for working with such a large and vulnerable refugee population. Participants reflected that thinking about their own emotions helped them better understand other refugees, and also that they intended to use their SMART strategies to show empathy . LiteratAs previously discussed, there is a significant lack of psychosocial support mechanisms for aid workers in the field. Few organizations report hands-on training of their personnel in stress management, or screen their staff for potential risk factors in stress responses . DespiteAs the Syrian conflict enters its seventh year, it is essential that the international response puts new support mechanisms in place for fieldworkers, and fundraises for self-care and resilience programs. Stress management training represents an alternative way to improve wellbeing and enable better communication and understanding in host community-refugee interactions. There is no doubt that humanitarian workers continue to be frequently targeted during conflicts . They arOur reflections from the field in Lebanon following the Syrian refugee crisis highlight the tremendous psychosocial burden put on humanitarian workers, and invite the crisis intervention community to reinvest both in fieldworkers’ well-being and the resilience of host and refugee communities. Delivering trainings in mindfulness and self-care such as SMART-3RP would encourage and protect the quality of life for all in the field. As humanitarian missions scale up, this new resource would be a valuable addition to known manuals and workshops in the humanitarian field. We invite the field to open the debate and facilitate research to study those outcomes in depth.


Page kidney refers to a clinical picture characterized by acute onset of hypertension due to external compression of the kidneys from hematoma, tumor, lymphocele, or urinoma. Hypertension is believed to result from renin-angiotensin-aldosterone activation triggered by renal hypoperfusion and microvascular ischemia. Renal failure, in addition to hypertension, may occur in the setting of a single functional kidney or a diseased contralateral kidney. We report a case of a patient who had a transplant kidney biopsy complicated by a subcapsular perinephric hematoma. The patient presented with an acute increase in blood pressure and a rapid rise in serum creatinine following a transplant kidney routine biopsy. He underwent emergent evacuation of the perinephric hematoma, with consequent decrease of his blood pressure and return of serum creatinine back to his baseline level. Early recognition and rapid intervention are needed in order to correct hypertension and reverse acute renal failure in Page kidney occurring in renal transplant recipients.


Microorganisms play a crucial role in the sustainability of the various ecosystems. The characterization of various interactions between microorganisms and other biotic factors is a necessary footstep to understand the association and functions of microbial communities. Among the different microbial interactions in an ecosystem, plant–microbe interaction plays an important role to balance the ecosystem. The present review explores plant–microbe interactions using gene editing and system biology tools toward the comprehension in improvement of plant traits. Further, system biology tools like FBA , OptKnock, and constraint-based modeling helps in understanding such interactions as a whole. In addition, various gene editing tools have been summarized and a strategy has been hypothesized for the development of disease free plants. Furthermore, we have tried to summarize the predictions through data retrieved from various types of sources such as high throughput sequencing data  and metabolic models have been reconstructed from such sequences for species communities. It is well known fact that systems biology approaches and modeling of biological networks will enable us to learn the insight of such network and will also help further in understanding these interactions. Arabidopsis. Furthermore, several different bacterial species have been isolated from a single plant. Conventionally, to investigate the various plant–microbial interactions use of a number of laborious laboratory experiments such as growth assays and pot house experiments are required  or interiorly (endophytes). Microbial communities can affect the plant physiology either positively or negatively in direct or indirect ways by various interactions mutualism, commensalism, amensalism, and pathogenic consequences. Endophytic bacteria is an example of plant–microbe interaction wherein bacteria live in a non-competitive environment of host plant tissue without any major damage to the host cell . Endophyrequired . Howeverrequired . These prequired ,b, 2016.hed data . This reFigure 1 showing both phyllospheric and rhizospheric microorganisms. The nutritional enriched environment around roots creates a favorable environment for the growth of soil microorganisms, which includes rhizosphere and the rhizoplane soil microbial communities. A number of microorganisms interact with different plant tissues or cells with various level of dependence. These interactions may be beneficial, harmful, or neutral for one or both the organisms on the basis of this attribute plant–microbe interactions are known as amensalism , antagonism (negative–positive), commensalism , competition (negative–negative), mutualism (positive–positive), and neutralism . The commensalism or mutualism are more frequent interactions found in plants, in which either one or both species gain benefit from the relationship respectively  of active proteins, crucial metabolites within the interacting cell are more inconsistent than the topology of the metabolic model. This indicates a clear overview about that existence and these factors define the network topology. Furthermore, the amount of each active element in such system has varied significantly so such attributes are accessed by metabolomics, transcriptomics, and proteomics and these can be taken as significant markers to explicit the host–microbial interactions. There are examples in which microbes dominated the over the molecular control of the host and resulted in exceptional results including production of “zombie ants” and mimicry of flowers by the fungi Ophiocordyceps unilateralis , transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR-Cas) that introduce double strand break (DSB) in the target gene, which are repaired by the error-prone non-homologous end joining (NHEJ) pathway or homology-directed repair HDR; .viz. zinc finger domains can be artificially engineered to target specific sequences in the host. It consists of two DNA binding domains, the domain one is comprised of eukaryotic transcription factors and contain a zinc finger. The second domain includes the catalytic component, the nuclease FokI restriction enzyme that catalyzes the specific DNA sequences. ZFNs have successfully performed well in defining the functions of various genes from diverse organism, including proven highly valuable in defining the roles of numerous genes in cells from a variety of organisms, including fruit flies, humans, mice, and higher plants  in ∼33–35 amino acid conserved target sequence. The progress in gene editing tools and development of various methods for easy synthesis and assembly of TALENs, allows the efficient editing at multiple sites. There have been various examples of the success of TALENs like knockout of the CCR5 gene for HIV resistance in human cells ; destrucGene editing has been highly appreciated for their ability to change the desired DNA fragment using engineered nucleases often called as molecular scissors. Since it edits the product according to fitment of the process it has various applications in a diversity of areas. The CRISPR-Cas system has been evidenced as most efficient, easy and simple . CRISPR-Figure 5) The guide RNAs (gRNAs) are derived from CRISPRs. CRISPRs consists of tandem arrays of a 30–40 bp short, direct repeat sequence which are separated by spacer sequences that matches the foreign sequence. Further transcription and processing of CRISPR produces mature CRISPR (cr)RNAs, the sequence flanked by signature CRISPR repeat tag at 5′ and 3′ end. The CRISPR (cr)RNAs form complex with Cas proteins to form a ribonucleoprotein (crRNP) that introduce cleavage in the DNA/RNA of the invader (Cas9 is a DNA endonuclease guided by RNA to target foreign DNA for inhibition ( invader . One of  invader . The gen invader . Suitabl invader . The imp invader .Table 2). Additionally, multiple editing in the same cell is possible using multiple gRNA that show various applications, like mutation in genes which are functionally related to control complex traits . The bacterial CRISPR-Cas could be used to inhibit the viral genetic material with the action of Cas9 as a nuclease thereby curtailing the establishment of viral infection in the plant , CBM, and OptKnock has been described to understand the metabolic pathways involved in plant–microbe interactions. Furthermore, gene editing tools such as TALENs and CRISPER-Cas have been described to control the pathogen interactions with plants to obtain customized plants. A snapshot of gene editing tools has been described to obtain disease free customized plants. There should be a better understanding of signaling pathways and metabolic networks to have an understanding of plant–microbial interactions. A combinatorial approach of computational biology and genomic tools has proven supportive to understand the communication pathway and metabolic pathway and provides an alternative to regulate these pathways to get a beneficial effect on plants with ecological sustainability.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Large cancer genome sequencing initiatives have led to the identification of cancer driver genes based on signals of positive selection in somatic mutation data. Additionally, the identification of purifying (negative) selection has the potential to identify essential genes that may be of therapeutic interest. The most widely used way of quantifying selection pressures in protein-coding genes is the dN/dS metric, which compares non-synonymous to synonymous substitution rates. In this study, we examine whether and how this metric is influenced by the mutational processes that have been active during tumor evolution. We use exome sequencing data from six different cancer types from The Cancer Genome Atlas (TCGA) and demonstrate that dN/dS in its basic form, where uniform base substitution probabilities are assumed, is in fact strongly biased by these mutational processes. This is particularly true in malignant melanoma, where the mutational signature is characterized by a high amount of UV-induced cytosine to thymine mutations at dipyrimidine dinucleotides. This increases the likelihood of random synonymous mutations occurring in hydrophobic amino acid codons, leading to reduced dN/dS ratios in genes encoding membrane proteins and falsely suggesting purifying selection in these genes. When this effect is corrected for by taking mutational signature-derived substitution probabilities into account, purifying selection was found to be limited and similar in all cancer types studied. Our results demonstrate that it is crucial to take mutational signatures into account when applying the dN/dS metric to cancer somatic mutation data. Carcinogenesis is an evolutionary process resulting from the accumulation of somatic mutations in cancer genes  that the overall mutational patterns observed in tumors are also strongly influenced by heterogeneous mutational processes underlying their development, and that cancer types are characterized by different mutational signatures , stomach and esophageal adenocarcinoma (STES), and lung adenocarcinoma and squamous cell carcinoma (LUNG) were concatenated. Mutation data that were annotated in hg18 were converted to hg19 using UCSC's liftOver  mutation annotation format (maf) files were downloaded from Broad Institute [Broad Institute TCGA Genome Data Analysis Center 2016): Firehose stddata__2016_01_28 run. Broad Institute of MIT and Harvard. doi: 6: FirehoAdditional somatic mutation data, called from high-coverage whole genome sequencing (WGS) data from 38 TCGA malignant melanoma samples as reported earlier  was calculated for each gene that contained at least 10 point mutations across samples within the cancer under analysis , s as the number of observed synonymous mutations, N as the number of non-synonymous positions and S as the number of synonymous positions.Where To determine the number of (non-)synonymous positions, the three possible point mutations for each genomic position in a specific gene were simulated . The number of non-synonymous and synonymous positions was determined after annotating the simulated mutations with ANNOVAR synonymous mutations in a gene, given a prior mutational probability determined by the specific mutational process that has been operative in a specific cancer type. Ni and Si are the number of (non-)synonymous class i substitutions per site for a given gene. Pi is the probability of substitution class i.Where A one-tailed binomial test was used to check whether (corrected) dN/dS ratios were significantly lower than 1. False discovery rate corrections were done using the Benjamini–Hochberg method  and lung cancer .To unveil which cellular processes might be underlying this apparent purifying selection, we performed a gene ontology (GO) gene set enrichment analysis (GSEA) and found strong enrichments for membrane-related activities like membrane transporters and ion channels Table . FurtherTo compare the different cancer types, we repeated the GSEA for the 100 most significant genes in each cancer type. This analysis showed that the membrane enrichment was not present for any other cancer type and hence specific for malignant melanoma Table .The previous results indicate that the high number of genes with low dN/dS values that were identified in malignant melanoma are not correlated to gene essentiality and might hence not be due to purifying selection. We next checked whether differences in underlying mutational processes and hence mutational signatures might be responsible for the observed differences in dN/dS between the analyzed cancers.We first compared the proportions of the six main substitutions  between the different cancer types. While in most cancers the main substitution found was C>T, as expected, this was most pronounced in malignant melanoma with 87.6% of all somatic mutations being C>T substitutions Figure . In lungMS/SMS in Figure The basic calculation of dN/dS boils down to normalizing the ratio of observed non-synonymous (n) to synonymous (s) mutations to the ratio of expected non-synonymous (N) to synonymous sites (S) in a gene Figure . This und nucleotide positions, this decrease in dN/dS is most likely caused by the higher occurrence of synonymous mutations for C>T and T>C substitutions as compared to the other substitution classes  of all genes having simulated dN/dS values below 1 Figure . While tCCR7, one of the genes with the lowest melanoma 96-class simulated dN/dS values  in this part of the protein, as exemplified in Figure As it is obvious from the previous results that differences in mutational signatures have a strong effect on the observed dN/dS ratios when using a uniform model, this parameter should be used with care when examining selection processes in cancer somatic mutation data. This is illustrated by the striking resemblance between the simulation results from this study and the earlier reported findings of purifying selection in genes coding for membrane proteins in melanoma based on the (uniform) dN/dS metric synonymous sites but the expected number of (non-)synonymous mutations at these sites, given the probabilities derived from the specific mutational signature. These corrected dN/dS values were found to be higher (median 0.97 vs. 0.54) than the uncorrected dN/dS values in malignant melanoma Figure , and no These results suggest that purifying selection is overall rather limited in cancer and not different between cancer types. A potential issue is the intrinsic assumption that selection processes do not have a major influence on the observed mutational signature in a cancer type itself. While this assumption seems solid for positive selection, where the majority of mutations have been shown to be passenger events (Vogelstein et al., Because a minimal amount of somatic mutations is required for dN/dS to be reliably calculable within a gene, our analysis was restricted to genes containing a minimal number of somatic mutations across samples. This implies certain genes under purifying selection might be excluded from analysis, possibly leading to an overestimation of dN/dS when comparing cancers in Figure In this study, we have shown that differences in mutational processes that have been active during tumor evolution can have a large impact on the expected number of synonymous and non-synonymous mutations in a gene. While this is a global analysis, where subclonality and intratumoral heterogeneity have not been taken into account, it is clear that not considering the resulting differences in mutational signatures might lead to false conclusions regarding selection pressures as quantified using the dN/dS metric. In conclusion, it is critical that mutational signatures are taken into account when calculating dN/dS values based on somatic mutation data from tumors.JV and EL designed the study. JV was responsible for data analysis and drafted the manuscript. Both authors discussed the results and finalized 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.


Drug-induced liver injury (DILI) is a major cause of drug attrition. Testing drugs on human liver models is essential to mitigate the risk of clinical DILI since animal studies do not always suffice due to species-specific differences in liver pathways. While primary human hepatocytes (PHHs) can be cultured on extracellular matrix proteins, a rapid decline in functions leads to low sensitivity (<50%) in DILI prediction. Semiconductor-driven engineering tools now allow precise control over the hepatocyte microenvironment to enhance and stabilize phenotypic functions. The latest platforms coculture PHHs with stromal cells to achieve hepatic stability and enable crosstalk between the various liver cell types towards capturing complex cellular mechanisms in DILI. The recent introduction of induced pluripotent stem cell-derived human hepatocyte-like cells can potentially allow a better understanding of interindividual differences in idiosyncratic DILI. Liver models are also being coupled to other tissue models via microfluidic perfusion to study the intertissue crosstalk upon drug exposure as in a live organism. Here, we review the major advances being made in the engineering of liver models and readouts as they pertain to DILI investigations. We anticipate that engineered human liver models will reduce drug attrition, animal usage, and cases of DILI in humans. Drug-induced liver injury (DILI) is a leading cause of pharmaceutical attrition and acute liver failures in the US . In partIn the case of the liver, several different model systems have been developed to provide human-specific data on drug behavior . These i in vitro. Here, we will describe the most recent advances in engineered human liver models that have utility for early DILI prediction and to obtain a better understanding of the diverse mechanisms underlying different forms of DILI. We begin with a brief description of conventional/traditional culture models and then discuss engineered liver models starting with static micropatterned cocultures (2D), followed by static spheroids and bioprinted liver models (3D). We then discuss perfused culture platforms that are more technologically complex than static plate-based systems but can be adapted to both 2D and 3D cultures. Precision cut liver slices and humanized liver rodent models are discussed as the most complex and in vivo-like liver models currently available. High content readouts and in silico predictions are briefly discussed as they pertain to DILI detection. Finally, A spectrum of human liver model systems is being developed to address the above mentioned challenges using engineering tools  that enable greater control over the cellular microenvironment to influence cell functionsCulture of hepatic cell lines and primary hepatocytes on adsorbed or gelled extracellular matrix (ECM) has been carried out for several decades and other reviews cover the genesis and development of this field . Here, w in vitro . Ev. Ev in vHepatocyte spheroids have shown improved functions over conventional 2D pure monolayers for drug screening applications, likely due to the establishment of homotypic cell-cell interactions and the presence of ECM within and around the spheroids . Cell liEngineered scaffolds and channels can aid in the assembly of spheroids that are more consistent in size than is possible with random configurations. For instance, Kostadinova et al. first deposited a mixture of liver stromal cells onto a porous nylon scaffold, followed by seeding of PHHs . Secretiμm). These spheroids are then transferred to another multiwell plate for drug testing [A specialized plate has been developed for creating hanging drops of mixed liver cells such as PHHs, endothelial cells, and Kupffer macrophages that can lead to the formation of spheroids of controlled diameters . Ma et al. utilized rapid, digital 3D bioprinting to print iPSC-HHs, endothelial cells, and adipose-derived stem cells in a microscale hexagonal architecture embedded in hydrogel that mimics the liver lobule architecture [Bioprinting can also be utilized to create spheroidal structures by positioning liver stromal cells  relative to hepatocytes, which can lead to a compartmentalized architecture and microvascular networks . These sitecture . WhetherNaturally derived  and synthetic biomaterials  can be utilized for embedding aggregated hepatocytes . Use of Even though hepatocytes in the liver are protected from flow-induced shear stress by the endothelial fenestrae, flow can cause gradients of oxygen, nutrients, and hormones, which have been shown to lead to zonation or differential functions in hepatocytes across the length of the sinusoid . DILI caIn addition to inducing zonal hepatic functions, several investigators have postulated that flow can allow better nutrient exchange and removal of waste products, which can lead to higher hepatic functions than static cultures. Novik et al. observed production of drug metabolites at greater rates in PHH/endothelial cell cocultures subjected to flow as compared to static cocultures . Sivaram in vivo rat studies.In addition to perfusion of culture medium, microfluidic devices can also be utilized to control the spatial arrangement of cells to yield the type of architecture  that has been shown to improve liver functions . For insMicrofluidic devices are inherently low-throughput for testing a large panel of drugs and are more difficult to set up and handle relative to industry-standard multiwell plates. Therefore, incorporation of real-time monitoring of toxicity biomarkers in microfluidic devices can not only aid in ease of usability, but also provide more rapid assessment of drug effects than is possible with conventional assays. For instance, Bavli et al. assessed mitochondrial function and glucose metabolism in real time on a liver-on-a-chip device where 3D aggregates of HepG2/C3A cells were exposed to rotenone and troglitazone for 24 hours . Oxygen Microfluidic perfusion is also useful to create organs-on-a-chip platforms in which the liver compartment is linked to compartments containing cells of other tissue types towards measuring how drug metabolism by liver cells affects other cell types . Viravai in vitro human liver model for testing specific hypotheses in a 24–72-hour timeframe.In contrast to many cell-based models, precision cut liver slices (PCLS) retain tissue architecture and contain all cell types of the liver . However in vivo context [ in vivo-like levels [ in vitro human hepatocyte data to assess the mode of action by which sodium phenobarbital produces tumors in rodents and the relevance of this data for human risk assessment [In certain types of rodent models, the liver can be repopulated with PHHs such that human-specific drug metabolites and DILI can be measured in an context , 74. In e levels , 76. Sevsessment . Xu et asessment . In partsessment . Serum AHumanized rodent models in which only PHHs are used cannot recapitulate the types of human DILI where the adaptive immune system plays an important role. For example, although Kakuni et al. were able to recapitulate troglitazone toxicity in a chimeric mouse with a humanized liver, immune-mediated reactions associated with troglitazone toxicity could not be studied due to the use of an immunodeficient SCID mouse . Therefo in vivo [Some groups have implanted PHHs in ectopic sites instead of directly transplanting them into a compromised rodent liver. For instance, Chen et al. first cultured PHHs and supportive stromal cells  in PEG hydrogels before implanting these constructs in the subcutaneous or intraperitoneal regions of immune-competent mice . The hum in vivo .High content screening (HCS) of multiplexed fluorescent readouts can be used to obtain an understanding of mechanisms underlying DILI at the organelle level. HCS systems  couple automated and multispectral epifluorescent microscopy with software for real-time analysis of fluorescent intensities within individual cells. Here, we will focus on HCS studies in which toxicity tests were performed on human-relevant cells and will refer the reader to several other reviews that cover HCS technologies in greater detail –88.Cmax of a candidate compound in humans during the early stages of drug development.HCS for DILI detection was initially implemented by O'Brien et al. using HepG2 cells  and late in vivo studies in rats if the drug also produced significant elevation of ALT or TBL  in the animal [Another type of high content readout involves toxicogenomics (TGx), which combines genomics  and bioinformatics analyses to characterize genes and pathways underlying drugs' effects on cells . Changese animal .Human liver cultures can potentially complement TGx studies when no liver enzyme elevation is observed in animals. Rodrigues et al. exposed PHHs, HepaRG, HepG2, and human skin-derived precursor hepatic progenitor cells to acetaminophen . TranscrGene expression can also be complemented with proteomics and metabolomics to allow for the study of DILI pathogenesis at multiple levels. We refer the readers to other reviews on the development and application of these tools for DILI detection , 101. He in silico models based on chemical descriptors and in vitro toxicity endpoints and found that utilizing both the descriptors and the endpoints resulted in better toxicity prediction as compared to using the chemical descriptors alone [ in vivo hepatotoxicity data and used a machine learning approach to create models that are useful for the in silico safety assessment of new molecular entities during the early stages of drug development [ in vitro assays that provide information on diverse molecular pathways that are modulated upon dosing with industrial chemicals and reference pharmaceuticals [Quantitative structure-activity relationships (QSAR) can be used to determine whether any property of the chemical  is an indicator of its potential to cause drug toxicity. There are multiple computational systems currently in use, some of which assess liver-specific toxicity . For insrs alone . Mullineelopment . Anotherelopment . In thiseuticals . in vitro data, can model some species-specific aspects of mitochondrial effects, bile acid toxicity, and innate immune responses [ in vitro assays, provide mechanistic insights into how a drug impacts known DILI pathways [Cmax values of drugs that have gone through human clinical trials, in a prospective drug screening campaign, physiologically based pharmacokinetic (PBPK) modeling can be useful to extrapolate between in vitro and in vivo exposure conditions [Some groups are creating computational models that quantitatively integrate mechanistic pathways implicated in DILI. For instance, DILIsym software simulates pathways and progression of endpoints pertinent in DILI . This soesponses . The “Vipathways . Finallynditions , 111. Su in vitro using microfluidic perfusion and in vivo with humanized rodent livers.Human DILI is a major global health burden and it has become clear over many drug failures that animal studies are not sufficient to fully predict and understand human-relevant outcomes . Further in vitro can be useful for modeling certain types of DILI where heterotypic cell-cell communication between two or more liver cell types is important. For instance, activation of Kupffer macrophages into a more inflamed state can downregulate certain CYP450s in PHHs, which can modulate the toxicity of drugs that are metabolized by those enzymes [Over many years of research, the field of engineered liver models has come to realize some important considerations in the design of such models. First, PHHs can be functionally stabilized for many weeks even when the culture model does not always mimic the exact architecture or composition of the liver  . Second, enzymes . Additio enzymes . It is nCmax information for a compound, it is important to determine a safety margin using in vitro toxicity data and binding affinity of the drug to the molecular target of interest. PBPK modeling can also aid in extrapolating critical information on pharmacokinetic parameters that could be potentially important in vivo [The aforementioned technological developments have already and will continue to improve the sensitivity of human DILI detection and provide insights into the mechanisms of different types of DILI. However, with several models now available in the marketplace, selection criteria need to be applied to select appropriate models for specific phases of the drug development pipeline. In our view, the choice of the culture model is dependent on the hypotheses being tested and the confidence that the chosen culture model has the levels of sensitivity and specificity that are acceptable for the type of throughput desired. For example, renewable iPSC-HHs or even hepatic cell lines  when cultured in engineered platforms to improve their differentiated functions can be used to identify highly toxic compounds very early in the drug development pipeline with good specificity  . These c in vivo , 111. In in vivo . As a le in vivo . There i in vivo , 115. in vivo-like phenotype and which pathways are affected more than others. With the increased use of commercially available cryopreserved cells, fresh tissues/cells are not always available. In that case, we believe that, at the very least, the gene expression of cultured cells over time should be compared against cells immediately after thawing. Finally, whether 3D architecture in the form of spheroids and bioprinted constructs will yield greater advances for DILI prediction than engineered 2D models is not yet clear. We anticipate that consortia led by pharmaceutical companies, which are already evaluating different engineered systems against their drugs in-house, would be highly beneficial towards addressing this question. Ultimately, in vitro liver culture, whether 2D or 3D, is likely not going to mimic the in vivo liver phenotype perfectly but the degree to which it does will determine its utility for testing specific hypotheses in drug development and mechanistic inquiries into DILI.Even with considerable progress in the development of increasingly complex human liver platforms, some key questions/issues will need to be addressed moving forward. First, it will be important to rely on similar endpoints and data normalization schemes  when showing functionality and stability of a given culture system so that the data can be compared across different laboratories using the same system and across different types of engineered systems. Some markers, such as albumin and CYP3A4 activity, are commonly employed for appraising PHH functions, but the community will need to agree upon which PHH markers are appropriate for specific applications and how best to demonstrate the phenotype of other cell types in the liver. Second, it is important to compare gene expression and functions of liver cells in culture over time to fresh tissue and freshly isolated cell counterparts (prior to any plating) from the same donor(s) in order to determine the extent to which cultured cells deviate from the in vitro, we anticipate that consensus will also have to be reached on nonhepatic endpoints that are important for the prediction of those types of DILI in which heterotypic cell-cell communication plays an integral role.It would be highly beneficial to reach a consensus as to which biomarkers to utilize to validate the utility of a platform for predicting different forms of DILI. Typically, endpoints such as ATP, albumin, urea, ALT, and lactate dehydrogenase (LDH) can be used to appraise the level of hepatic injury nondestructively in the same culture over time. Some combination of these endpoints used with stable PHH cultures can provide upwards of ~70% sensitivity for identification of drugs from several different classes as “toxic” . HCS pro in vitro approaches which specific individuals will go on to adapt to cell stress and which individuals will experience severe DILI. Creation of hundreds and even thousands of iPSC lines from different individuals, some with greater susceptibility to liver toxicity due to certain drugs, may ultimately be necessary to fully understand interindividual variations in DILI outcomes due to genetic makeup. Dosing iPSC-HH cultures with drugs under different diseased backgrounds  could also provide clues as to patient-specific DILI. However, iPSC-HH functions need to be further improved to be similar to PHHs before their potential for investigating DILI can be fully realized. Engineering tools have shown great promise in improving iPSC-HH functions, especially for DILI detection [Several studies utilizing cell lines, PHHs and iPSC-HHs, have shown that even some so-called “idiosyncratic” toxins  can be detected using cellular stress markers , 89, potetection ; howeverIn conclusion, different engineered human liver models can now be utilized in specific phases of drug development based on the posed hypotheses, throughput requirements, and budgetary constraints. Continued development and validation of such models will provide higher sensitivity in the prediction of different types of DILI and provide a better understanding of factors that can cause idiosyncratic DILI in certain patients. Ultimately, engineered human liver models will reduce the usage of animals in preclinical drug development and mitigate the risk of DILI to human patients in clinical trials and in the marketplace.


Effective and efficient transitions in care between emergency medical services (EMS) practitioners and emergency department (ED) nurses is vital as poor clinical transitions in care may place patients at increased risk for adverse events such as delay in treatment for time sensitive conditions  or worsening of status . Such transitions in care are complex and prone to communication errors primarily caused by misunderstanding related to divergent professional perspectives leading to misunderstandings that are further susceptible to contextual factors and divergent professional lenses. In this systematic review, we aim to examine (1) factors that mitigate or improve transitions in care specifically from EMS practitioners to ED nurses, and (2) effectiveness of interventional strategies that lead to improvements in communication and fewer adverse events.We will search electronic databases ; gray literature ; and reference lists and conduct forward citation searches of included studies. All English-language primary studies will be eligible for inclusion if the study includes (1) EMS practitioners or ED nurses involved in transitions for arriving EMS patients; and (2) an intervention to improve transitions in care or description of factors that influence transitions in care . Two reviewers will independently screen titles/abstracts and full texts for inclusion and methodological quality. We will use narrative and thematic synthesis to integrate and explore relationships within the data. Should the data permit, a meta-analysis will be conducted.This systematic review will help identify factors that influence communication between EMS and ED nurses during transitions in care, and identify interventional strategies that lead to improved communication and decrease in adverse events. The findings can be used to develop an evidence-informed transitions in care tool that ensures efficient transfer of accurate patient information, continuity of care, enhances patient safety, and avoids duplication of services. This review will also identify gaps in the existing literature to inform future research efforts.CRD42017068844PROSPERO  Transitions in care between healthcare providers are complex processes that can result in vital information being lost or altered –3. ThreeEMS practitioners and ED nurses have different professional perspectives. EMS practitioners in the pre-hospital setting focus on information that enables them to determine underlying pathology/injury, required treatments, and destination –12. TheyLoss of information during transitions in care may lead to incorrect triage decisions in terms of acuity rating or prioritization of order to be examined by a physician, delayed time to treatment, or the patient being triaged to an inappropriate area of the ED , 17. UltPreliminary findings from two studies conducted by GR—one of decision-making by paramedics (in progress) and one of decision-making by ED triage nurses—indicate that EMS-triage nurse communication is problematic . ParamedA related UK-based literature review  identifiThe objectives of this systematic review are to assess factors that mitigate or improve transitions in care from EMS practitioners to ED nurses and to identify effective interventional strategies that lead to improvements in communication and a reduction in adverse events.what works to understand the perceptions, attitudes, and beliefs of those directly involved in transitions in care. Based on a preliminary scoping search, we expect diverse observational and qualitative studies of transitions in care. As such, we will conduct a mixed systematic review  , which can be used to assess multiple study designs and has evidence of validity and reliability.  Each of esearch.  This coh, which cTo assess confidence in the evidence in the syntheses, we will use the GRADE  . We expect considerable heterogeneity between studies; thus, meta-analysis may not be appropriate. In this case, we will aggregate the quantitative findings with the qualitative findings using data-based convergent synthesis , wherebylow quality  will be established a priori. This comparison can provide insight into whether the low quality studies contribute unique information and how they may impact the generalizability of the findings. [Providing that there is sufficient information, we will consider comparisons of several subgroups: (1) study types, (2) paramedics and nurses, (3) different countries, (4) transitions in care at triage and bedside, and (5) barriers and facilitators. We will also conduct a sensitivity analysis to examine the influence of studies with a low quality rating on the robustness of review findings. , 36 To dindings. We are using an integrated knowledge translation  approachThe findings from this systematic review will result in an in-depth understanding of the factors and interventional strategies that lead to improved communication and decrease in adverse events. To date, although EMS and ED nurses generally exhibit good working relationships, there is also a level of mistrust and sense of not being heard , 2. Our The potential heterogeneity of the literature including varying study designs, study quality, and outcomes reported may constrain our ability to draw consistent and clear conclusions on the outcomes. Regardless of study heterogeneity, this systematic review will provide a meaningful analysis regarding the existing evidence on transitions in care between EMS and ED nurses and identify areas for future research.