We analyzed DNA methylation patterns and transcriptomes of primary intestinal epithelial cells (IEC) of children newly diagnosed with inflammatory bowel diseases (IBD) to learn more about pathogenesis.We obtained mucosal biopsies (N = 236) collected from terminal ileum and ascending and sigmoid colons of children (median age 13 years) newly diagnosed with IBD , and 30 children without IBD (controls). Patients were recruited and managed at a hospital in the United Kingdom from 2013 through 2016. We also obtained biopsies collected at later stages from a subset of patients. IECs were purified and analyzed for genome-wide DNA methylation patterns and gene expression profiles. Adjacent microbiota were isolated from biopsies and analyzed by 16S gene sequencing. We generated intestinal organoid cultures from a subset of samples and genome-wide DNA methylation analysis was performed.We found gut segment-specific differences in DNA methylation and transcription profiles of IECs from children with IBD vs controls; some were independent of mucosal inflammation. Changes in gut microbiota between IBD and control groups were not as large and were difficult to assess because of large amounts of intra-individual variation. Only IECs from patients with CD had changes in DNA methylation and transcription patterns in terminal ileum epithelium, compared with controls. Colon epithelium from patients with CD and from patients with ulcerative colitis had distinct changes in DNA methylation and transcription patterns, compared with controls. In IECs from patients with IBD, changes in DNA methylation, compared with controls, were stable over time and were partially retained in ex-vivo organoid cultures. Statistical analyses of epithelial cell profiles allowed us to distinguish children with CD or UC from controls; profiles correlated with disease outcome parameters, such as the requirement for treatment with biologic agents.We identified specific changes in DNA methylation and transcriptome patterns in IECs from pediatric patients with IBD compared with controls. These data indicate that IECs undergo changes during IBD development and could be involved in pathogenesis. Further analyses of primary IECs from patients with IBD could improve our understanding of the large variations in disease progression and outcomes.  The intestinal epithelium is thought to play a critical role in the pathogenesis of inflammatory bowel Diseases (IBD), yet evidence derived from primary human tissue remain scarce.Purified intestinal epithelium from children newly diagnosed with IBD display distinct epigenetic and transcriptional alternations, which are partly retained in organoid cultures and correlate with disease outcome.Relatively small patient numbers require validation in additional cohorts.Stable epigenetic alterations in the intestinal epithelium of children with IBD may explain variations in disease outcome and have potential to be developed into disease prognostic biomarkers in the future.Inflammatory bowel diseases (IBD) cause chronic relapsing inflammation that can affect any segments of the digestive tract  or be restricted to the colon (ulcerative colitis [UC]).The etiology of IBD is multifactorial, although the interplay of factors is still poorly understood. Large-scale genome-wide association studies have helped to characterize the genetic risk, identifying over 200 disease-associated loci.Epigenetic mechanisms operate at the interface between genetic predisposition and our environment, capable of causing stable, potentially heritable changes of cellular function in response to environmental triggers.Genetic and functional studies predominantly using mouse models and cell linesTherefore, we simultaneously profiled genotype, epi-genotype , gene expression, and the adjacent gut microbiota of highly purified IEC, obtained from children newly diagnosed with IBD and a matched cohort of non-IBD controls. We analyzed genome-wide ‘omics’ layers for potential IBD-specific alterations and functional consequences, as well as cross-talk between layers. Additionally, we generated intestinal epithelial organoids from patient biopsy samples and investigated their epigenetic profiles. Lastly, we applied statistical models to genome-wide datasets to test their ability to distinguish between disease subtypes, as well as potential correlation with disease outcome measures.A cohort of 66 treatment-naïve children at diagnosis of their IBD, along with 30 age- and sex-matched non-inflammatory control children, were recruited by the Paediatric Gastroenterology team at Addenbrooke’s Hospital during 2013–2016. This study was conducted with informed patient and/or carer consent as appropriate, and with full ethical approval (REC-12/EE/0482). Sample and patient details are provided in °C to extract DNA from the adjacent microbiota. Further information is provided in the Biopsy samples were processed immediately and IECs purified using enzyme digestion and magnetic bead sorting for the epithelial cell adhesion molecule as described previously.Intestinal organoids were generated from mucosal biopsies by isolation of intestinal crypts and culturing as described previously and detailed in the DNA and RNA were extracted simultaneously from the same sample using the AllPrep DNA/RNA mini kit . DNA from the adjacent microbiota was extracted using QIAamp DNA Stool Mini Kit and from whole blood using the DNeasy Blood and Tissue Kit (both Qiagen). DNA was bisulfite-converted using Zymo DNA methylation Gold kit .Genome-wide DNA methylation was profiled using the Illumina Infinium HumanMethylation450 and EPIC BeadChip platforms . Sample numbers are provided in Locus-specific validation of DNA methylation profiles was performed on bisulfite-converted DNA after polymerase chain reaction amplification using the Pyromark Q24 (Qiagen) pyrosequencing system as described previously.Extensive details of the bioinformatics methods used in this publication are described and referenced in the A and Figure 1Bi and 1Bii). Moreover, samples derived from controls clustered closely both on an epigenetic (DNAm) and transcriptomic level for each gut segment. Interestingly, IBD-derived samples displayed more variation, with a subset of IBD samples distinctly separating from controls . However, analysis of the bacterial operational taxonomic unit by family abundance and alpha-diversity did reveal variation by gut segment  exceeds that of inflammation in the majority of datasets. This highlights the presence of disease-specific molecular alterations, which are partly independent of the current inflammatory activity. Full results of the variance decomposition analysis can be found in Figure 2B and C). Similarly, diagnosis explained 74% and 82% of the differentially expressed genes (DEGs) for CD and UC, respectively .Given the distinct clustering patterns we observed on a genome-wide scale, we next used a variance component model to assess the relative contribution of diagnosis and inflammation to the observed variance within each data layer, by gut segment. As shown in Figure 2Additionally, generating MDS plots by labelling samples according to gut segment, diagnosis, and inflammatory status did not show a clear separation between inflamed and non-inflamed samples . Lastly,In summary, these analyses demonstrate the presence of clear epigenetic, transcriptomic, and adjacent microbial alterations in the intestinal epithelium of children newly diagnosed with IBD, with a proportion being independent of intestinal inflammation.Next we performed differential DNAm and gene expression analyses by comparing control, CD-, and UC-derived datasets for each gut segment. When performing these analyses, inflammation was controlled for within the differential analysis thereby allowing us to focus on molecular alterations that occur in relative independence of mucosal inflammation. Additionally, in an attempt to connect epigenetic and transcriptomic signatures, we identified differentially methylated regions (DMRs) that were located within 10kb of the transcription start site of a DEG. Such regions were termed regulatory DMRs (rDMRs).Ai and Figure 3Aii and Figure 3B).Analysis of ileal IECs revealed CD-specific changes in both DNAm , a transcription regulator, where a decrease in DNAm matched the increase in gene expression levels in both CD and UC patients. Interestingly, a proportion of the CD-related changes identified in TI samples were also found to be present in SC samples  identified between the 2 diagnoses . Organoids derived from CD patients did not differ in their microscopic appearance or culturing behavior from those derived from healthy controls . However, assessing their genome-wide DNA methylation profiles revealed distinct alterations, suggesting that they retain a proportion of disease-associated epigenetic changes. Despite the relatively small sample number, CD-associated DMPs  of CD-associated DMPs compared with randomly selected CpGs . Using locus-specific pyrosequencing, we were able to validate a subset of CD-specific DMPs that were retained in organoid cultures .Next, we investigated the stability of IEC DNAm profiles in IBD patients over time. We obtained ileal and colonic biopsies (SC) from IBD patients both at diagnosis and at a later stage in their disease . Strikingly, CD- and UC-associated DMPs showed remarkable stability over time, demonstrated by the strong correlations of the methylation values at diagnosis and repeat endoscopy within each gut segment . The best model separating disease from control was based on DNAm data from the SC =40) with sensitivity of 75% and specificity of 100% . Importantly, the use of ileal DNAm datasets allowed separation between CD and UC with high precision (77%), sensitivity (57%), and specificity (93%)  . The accuracy of the TI DNAm signatures in distinguishing CD and UC was confirmed in a follow-up patient cohort, analyzed using a second DNAm array platform .Given the striking IBD-associated changes observed in intestinal epithelial DNAm and gene expression, we went on to test the ability of these signatures to predict diagnosis. Additionally, we hypothesized that variation observed within IBD-derived patient samples could be indicative of future disease behavior and outcome. To address these hypotheses, we applied a machine-learning model (random forest) to the individual omics data layers. The model identified those data points  that could predict disease status for each patient with high precision and accuracy see A. The beP < .05) with a number of disease outcome measures including the requirement for treatment with biologics and number of treatment escalations within the first 18 months following diagnosis (C). Interestingly, modules correlating with disease outcome measures did not show any correlation with gender, age, or disease phenotype at diagnosis . Clustering all samples according to expression levels of genes within the strongest modules separated CD patients in 2 groups (Di). Kaplan Meier curves for these groups demonstrated striking differences in both the requirement for biologics and time to third treatment escalation . In addition, we applied WGCNA to the DNAm data. Although the overall correlation of identified modules was less striking, separating samples according to DNAm profiles of the strongest module still demonstrated a significant difference in outcome measures between resulting patient groups . Similar results were obtained from UC patient-derived signatures in SC samples . Based on these preliminary results, both DNAm and RNAseq data contain signatures that accurately predict disease status and correlate with selected disease outcome parameters.To correlate genome-wide IEC profiles with clinical outcome measures  we used a Weighted Gene Co-expression Network Analysis (WGCNA) approach. WGCNA identifies patterns within a given dataset  and combines genes or CpGs that vary similarly across samples into modules. Each of these modules was then tested for a significant correlation with clinical outcomes. The application of WGCNA to RNAseq data derived from the TI of CD patients led to the identification of several gene modules  that correlate significantly , indicating a functional interconnection between the 2 data layers. Reassuringly, a number of identified genes had previously been reported, including APOA1The impact of the observed IEC-specific epigenetic alterations on IBD pathogenesis will depend at least in part on the stability of such molecular signatures. Investigating IEC DNAm profiles of the same patient at 2 time points  allowed us to demonstrate the strikingly high stability of disease-associated methylation signatures in small bowel and colonic IEC. This was despite changes in medication and mucosal inflammation over a period of up to 20 months. These findings suggest that stable epigenetic alterations may contribute to chronic relapsing inflammation by mediating altered IEC function. Interestingly, CD-derived epithelium appeared to retain a degree of disease-specific alterations even when cultured ex-vivo as organoids, further highlighting both their stability and relative independence of mucosal inflammation. Additionally, our findings add further support to recent reports on patient-derived intestinal organoids to be used as novel translational research tools.Despite the major success of genome-wide association studies in identifying disease-predisposing genetic loci, information on the functional consequences and cell specificity remain limited. Expression quantitative trait loci have been identified for a subset of the IBD risk loci from whole biopsiesAn additional major strength of our prospectively recruited pediatric patient cohort was the availability of detailed phenotype and disease outcome data, allowing us to test for potential correlation between molecular signatures and clinical phenotypes. Despite the relatively small sample numbers included in these analyses, both the potential diagnostic and prognostic value of our IEC signatures is evident. While current diagnostic approaches are sufficient for most patients, a minority of patients requires repeated and prolonged investigations to confirm a diagnosis. Additionally, it is frequently challenging to differentiate UC from CD in children, both at diagnosis and later in the disease course. Therefore, a diagnostic model to reliably differentiate CD from UC, such as the model built using DNAm data from the ileum with high sensitivity and specificity, could be of clinical value. Correlating genome-wide molecular signatures with clinical outcome measures continues to be a major challenge and a wide range of bioinformatics tools have been developed. We decided to utilize WGCNA, which has been successfully applied to both RNA-seq and DNAm datasets, allowing identification and correlation of individual gene modules with clinical parameters.As a limitation to our study, we acknowledge that the total number of patients included is relatively low and hence some of the analyses performed, particularly those that correlate signatures with clinical outcome, should be considered as preliminary. However, to the best of our knowledge this is the first and largest study applying a multi-omics profiling approach to a unique sample collection of highly purified intestinal epithelium. The fact that all patients were recruited at diagnosis (treatment-naïve) also represents an important strength. Last but not least, although our study was performed on a pediatric patient cohort, we consider our findings to be equally relevant to adult-onset IBD given the similarities in disease phenotype (particularly in teenage onset) and common concepts of disease pathogenesis.In summary, our study is the first to apply a multi-omics profiling approach to a large collection of purified intestinal epithelial samples. The findings clearly demonstrate disease-specific abnormalities in epithelial cell function in children with IBD. We also highlight how specific data signatures might be indicative of disease status and behavior and therefore have a potential to be of clinical relevance in the future.


Microcirculation disturbances are implicated in the prognosis of septicshock. Microvascular hyporesponsiveness can be assessed by anoximetry-derived perfusion index and reactive hyperemia. Using thisperfusion index, we investigated reactive hyperemia and its relationshipwith peripheral perfusion and clinical-hemodynamic parameters in septicshock.0-60) and metabolic mechanisms(ΔPI60-120). Correlation tests were performed betweenreactive hyperemia and clinical-hemodynamic data.Eighty-two patients were evaluated: 47 with septic shock and 35 controls.Tests were performed within 24 hours after admission. The perfusion indexwas evaluated before and after a 3-min blood flow occlusion using atime-response analysis for 5 min. The perfusion index was also evaluated inthe hyperemic phases and was mainly derived by mechanosensitive(ΔPI0-60 were lower in shock.However, ΔPI60-120 was similar between the groups . Thetime-to-peak of the perfusion index was correlated positively with the SOFAscores and negatively with C-reactive protein; the peak of the perfusionindex was positively correlated with vasopressor doses; and theΔPI60-120 values were positively correlated withC-reactive protein and vasopressor doses. No other significant correlationsoccurred.Reactive hyperemia measured by the perfusion index was significantly lower inpatients with septic shock, but this was only observed for the first 45seconds after cuff-deflation. In the remaining period, there were nostatistical differences between the groups. The peaks in the perfusion indexwere similar between groups, although the peak was reached more slowly inthe septic group. Values of ΔPIThis perfusion index-based study suggests that septic shock promotes initialperipheral vascular hyporesponsiveness and preserves posterior vascularreactivity to a considerable degree. These results demonstrate atime-dependent peripheral hyperemic response and a significant ischemicreserve in septic shock. Among the main disturbances are endothelialdysfunction and vascular hyporesponsiveness. Vascular hyporesponsiveness is caused bymultiple disruptions in cellular homeostasis and vasculardamages.(5) Clinical evidence strongly suggests that vascularhyporeactivity could contribute to peripheral hypoperfusion and the severity oforgan failure in septic shock when evaluated in nitric oxide (NO)-dependent arteriesand in skeletal muscle microcirculation.Septic shock, i.e., circulatory failure due to sepsis that leads to tissuehypoperfusion, is still a clinical syndrome with highmortality.(7) Another study showed that skin endothelialreactivity remains intact in septic shock.(8) Finally, septic neonates showed anincreased skin vascular reactivity compared to controls.(9) Recently, there is anincreasing interest in monitoring shock using non-vital vascular beds to assess skincirculation. Thus, a better understanding of peripheral vascularreactivity in septic shock is needed.Nevertheless, when performed in the skin tissue, some results seem contradictory. Ina study of critically ill patients (one-third with sepsis), the vascular reactivitywas not different between different degrees of organdysfunction. Multiple and organ-dependent mechanisms areinvolved, including NO bioavailability, sensory nerves, myogenic reflex,hyperpolarizing factors and cyclooxygenase metabolites.(12)Post-occlusive reactive hyperemia, a known vascular reactivity test, refers to anincrease in organ blood flow above the baseline levels following release from briefarterial occlusion. This flow increase estimates the vascular response to themaximal demand of tissue, making this test attractive for monitoring hypoperfusionstates as septic shock.(13) and indirectly measurethe perfusion variations.(14) Hypoperfusion measured with PI has been shown to bepredictive of mortality in sepsis,(15) and recent reports have shown that reactivehyperemia can be evaluated using PI.New oximeters can calculate the perfusion index (PI) from a pulsatilephotoplethysmography signal, mostly of skin microcirculation,Therefore, this preliminary study used PI to evaluate the global vascular reactivityin septic shock using a time-response analysis of the reactive hyperemia. It alsoaimed to verify the relationship between this parameter, peripheral perfusion,systemic macro-hemodynamics, vasopressors doses and organ failure scores.Universidade Federal doParaná (protocol number: 685.344/2014). Therefore, the studyprotocol was in accordance with the national and international ethical norms onresearch with human beings (conforming to the Declaration of Helsinki).All participants provided written informed consent, and the research was approved bythe Research Ethics Committee of the Hospital de Clínicas of the Universidade Federaldo Paraná from September 2014 to April 2016. The study selectedconsecutive adult patients admitted with the diagnosis of septic shock or within 24hours after septic shock onset in patients previously admitted for other causes.This transversal observational study was conducted in the intensive care unit (ICU)at  sepsis was defined based on clinical evidence ofinfection and 2 or more of the following: (1) fever axial temperature greater than38°C or hypothermia , (2) tachycardia (heart rate >90 beats per minute), (3) tachypnea (> 20 breaths per minute) or need formechanical ventilation, (4) leukocytosis  orleukopenia , or a ratio of greater than 10% bandcells to polymorphonuclear cells. Septic shock was defined as sepsis withhypotension and/or hypoperfusion represented by acute hyperlactatemia (irrespectiveof blood pressure), even after initial volume expansion, and requiringvasopressors.According to internationally accepted consensus definitions at the time of thebeginning of study,Exclusion criteria: Severe hepatopathy/coagulopathy, infective endocarditis, systemicsclerosis and severe obstructive arterial disease. These criteria were chosen toreduce the risks of possible hemorrhagic and ischemic complications of theprocedure.(17) the groups were approximatelymatched for these factors.Reactive hyperemia assessment using PI was also carried out in a conveniencesample of subjects matched by age, sex and without signs of clinical infectionor acute illness. Due to the importance of hypertension, diabetes mellitus andsmoking to microvascular reactivity,2) was > 70%. Initially, allpatients received 30mL/kg crystalloid fluid over 1 hour. Fluid administrationwas continued until there was no response to passive-leg raising or norespiratory variations of the inferior vena cava diameter . If MAP remained < 65mmHg after fluidadministration, a diagnosis of septic shock was made, and norepinephrine wastitrated to maintain MAP > 65mmHg. Intensivists were blinded to peripheralperfusion variables. The capillary refill time was also evaluated, and it wasconsidered prolonged if it was longer than 4.5 seconds.All patients received broad-spectrum antibiotic coverage. The local hemodynamicsupport was as follows: central venous pressure was 8 - 12mmHg, mean arterialpressure (MAP) was > 65mmHg, urine output was > 0.5mL/kg/hour, and centralvenous oxygen saturation  and Sequential Organ Failure Assessment (SOFA) score.Assessment of patients occurred within 24 hours after admission to the ICU witha diagnosis of shock or within 24 hours after the onset of shock in patients whowere previously admitted for other causes. All hemodynamic, metabolic and PIvariables were measured after fluid resuscitation.2) was calculatedas the difference between the partial pressures of central venous carbon dioxide(PcvCO2) and arterial carbon dioxide (PaCO2). We alsocollected other data, including primary site, type of infection and cause ofdeath.Simultaneous blood gases from arterial and central venous catheters wereobtained. Samples were taken in a 3mL heparinized syringe. Blood gas analysisand lactate concentration were determined using the GEM premier 3000 Gasometer. Central venous oxygen saturation was calculated from asample from the central venous catheter. The central venous-arterial bloodcarbon dioxide partial pressure difference The perfusion index was measured by attaching a pulse oximeter probe . The same researcher performed all tests. All studieswere performed after resuscitation and at least 1 h of hemodynamic stability (nochange in vasopressor dose or fluid boluses) in a controlled room (25°C). Thepulse oximeter was placed on the index finger. The PI was measured for a periodof 5 min  after signal stabilization. Subsequently, asphygmomanometer cuff was inflated around the homolateral arm, 30 - 50mmHg abovethe systolic pressure, to occlude the arterial flow for a period of 3min.Reactive hyperemia occurred on deflation of the cuff. The PI was determined every15 seconds for a period of 5 min to create a curve of PI variation (ΔPI)as a function of time. The variation in the PI (Δ) was calculated at eachassessed time point using the following formula:0-60) and 60 to 120 secondsafter cuff deflation (ΔPI60-120). These time intervals werespecially chosen to evaluate the phases of reactive hyperemia, which were mainlygenerated by mechanosensitive mechanisms and metabolic factors,respectively.(21) The ΔPI0-60 andΔPI60-120 were compared between groups.The peak of PI (ΔPI peak) and time to reach the peak (time to peak) werealso measured. Next, the mean of the delta of PI was determined between 0 and 60seconds after cuff deflation The Shapiro-Wilk test was used to test normalcy of the sample. Nonparametricvalues were expressed as medians/interquartile ranges and categorical variableswere expressed as percentages. The Mann-Whitney test and Chi-square test wereused to determine the significance of the differences in the nonparametric andcategorical variables, respectively. Spearman correlation analysis was conductedto determine the relationships between the reactive hyperemia and theclinical-hemodynamic parameters. The statistical program GraphPad Prism 3.02 wasused for analyses. The significance level sought was p < 0.05. Because thiswas the first study in the literature to use this design to evaluate septicshock , the sample size was not exactlycalculated. To minimize this issue, we observed the sample size selected byother similarly designed studies in the literature, which was between 15 and 42patients per septic group and between 15 and 38 patients in controlgroup.There were 47 septic shock patients included. In-hospital mortality of septic shockwas 46.8% (22/47). The clinical-demographic and hemodynamic data of the patients arelisted in 0-60) and at the second-minute interval(ΔPI60-120) after deflation of the sphygmomanometer cuff. Thepeaks of PI were similar between the groups, although the peak was reached moreslowly in the septic group. In addition, the reactive hyperemia was lower in theseptic group in the early/mechanosensitive phase, as represented byΔPI0-60. However, there were no statistically significantdifferences in the reactive hyperemia between the groups in the latter/metabolicphase, as represented by ΔPI60-120.0-60); and there were positivecorrelations between C-reactive protein, vasopressor doses, andΔPI60-120. There were no statistically significantcorrelations with regard to the other parameters.With regard to correlation analysis ,the orgThis is the first report to assess the time-response analysis of reactive hyperemiain septic shock evaluated with the PI. The main finding of this study is that septicshock, after fluid resuscitation, leads to peripheral hypoperfusion associated withearly hyporesponsiveness and considerably preserved vascular reactivity in theposterior phase. Thereby, it is possible to suggest the existence of atime-dependent peripheral hyperemic response in septic shock with a significantlymicrovascular ischemic reserve. One commonly found microcirculatory disturbance isimpaired vascular reactivity,(5) and it can be estimated with reactive hyperemia,which is the increase in organ blood flow following brief arterial occlusion. Itsmost common clinical use is the evaluation of endothelial function through the NObioavailability of conductance vessels.(22) Another potential clinical useconsists of analyzing the global tissue microvascular reactivity to represent theproportion of recruitable capillaries, arterioles and small arteries upon minimalflow delivery.(4)Multi-organ microcirculatory disturbances in sepsis, when not corrected, seem toexert a robust influence on outcomes.(6) Additionally, inskeletal muscle microcirculation, impaired reactive hyperemia was related to organfailure, suggesting a link between abnormal microvascular ischemic response andtissue hypoperfusion.(4) Inversely, our results demonstrated that reactivehyperemia, when measured in the fingertip with the PI, has a time-dependent dynamicin septic shock; it seems to be clearly reduced only in the first 45 seconds afterischemic stimulus and remains largely preserved after this period. In addition, thepeaks of reactive hyperemia were similar, although the peak was reached more slowlyin the septic shock group.Previous studies have shown that reactive hyperemia was clearly reduced in septicshock when evaluated in NO-dependent conduit arteries, demonstrating endothelialdamage as an early marker of unfavorable prognosis.(23) andhumans,(24) that the classic immune/inflammatory alterations ofsepsis also impact the skin microvasculature and adjacent tissues.On first view, these results could suggest that the peripheral microcirculation couldbe relatively spared from the severe vascular damages that occur in septic shock.However, previous reports consistently have shown, in animals vessel type and hyperemic phaseevaluated.(21) Therefore, the magnitude also depends on thedifferent metabolic pathways present in different tissues. While endothelial-derivedNO strongly mediates conduit vessel response, and cyclooxygenase influences muscle reactivehyperemia,(25) the reactive hyperemia of skin and adjacent tissuesis mediated by other mechanisms, including sensory nerves and hyperpolarizingfactors.(12) Curiously, many mediators that act ashyperpolarizing factors in microcirculation are increased in human sepsis andinclude neuro-mediators such as calcitonin gene-relatedpeptide(26) and oxidative stress-derived hydrogenperoxide. Elevated levels of hyperpolarizing factors couldexplain the relatively preserved reactivity of skin microcirculation despite thedamages of septic shock. An evaluation of these mediators and peripheral reactivehyperemia is needed to confirm this hypothesis.A possible explanation for these apparently contradictory results may be associatedwith the fact that reactive hyperemia magnitude depends on theorgan,(21) These facts could explain the previousdiverse results measured with near-infrared spectroscopy in sepsis (NIRS) thatevaluated only the initial seconds of the first hyperemicphase.(4) The evaluation of the initial hyperemic phase inseptic shock using PI (ΔPI0-60) corroborates the findings of theresults using the NIRS method and presumably adds new information about the latterhyperemic phases.The phase of hyperemic response is also important because early flow responses seemto be mainly derived by mechanosensitive mechanisms, while shear-stress andmetabolic factors affect late flow responses.(29) who demonstrated thatacute endotoxemia in humans, an acute phase sepsis model, selectively inhibits theNO-dependent vascular reactivity of human skin, while the post-ischemic reactivehyperemia remains preserved.Our results are also in line with those of Engelberger et al.,(30) our results showed a weak-to-moderate positivecorrelation at the metabolic phase of reactive hyperemia(ΔPI60-120), which also suggests an inverse response patternin these vascular territories.Interestingly, the systemic inflammation inferred by levels of C-reactive proteinalso seems to have a different influence on reactive hyperemia measured with the PI.While other reports showed a clear negative correlation with measurements in conduitvessels,60-120). In addition, as expected, weshowed reduced peripheral perfusion in septic shock patients compared to controls.It is well known that an intense redistribution of blood flow from non-vital organsto vital organs characteristically occurs in states of shock, causing peripheralhypoperfusion.(31) The peripheral hypoperfusion is generated by anincrease in sympathetic-neurohumoral activity and is associated with worse systemic perfusionand poor prognosis. Since previous reports showed that the PI is a verysensitive method for assessing adrenergic responses, the adrenergicstimulus (secondary to the sympathetic response to shock and to use of vasopressors)also becomes a direct hypothesis that could explain the relatively preservedperipheral vascular reactivity despite the peripheral hypoperfusion in the septicgroup: it implies the existence of a peripheral ischemic reserve in septicshock.With regard to adrenergic response, our results showed a positive correlation betweennoradrenaline doses, the peak of reactive hyperemia and the metabolic phase ofreactive hyperemia  Similar results occurred in conductancevessels(37) and muscle sympatheticnerves.(38) Even in the first minute after deflation, wherereactive hyperemia was reduced in the septic shock group(ΔPI0-60), there was no correlation with noradrenaline doses.Another interesting fact is that sepsis impairs adrenergic signaling and generatesreceptor hyporesponsiveness.(5) Thus, the requirement for increasing doses ofvasopressors could reflect adrenergic desensitization. Additionally, these resultsare not adequate to differentiate the sympathetic response to shock and the directadrenergic effects of vasopressors. Thus, the precise role of sympathetic stimulusin the hyperemic response of the PI needs to be elucidated further.Nevertheless, previous clinical reports consistently have shown that reactivehyperemia tends to be reduced, not increased, by direct α-adrenergic stimuluswhen measured in cutaneous circulation.2 and blood lactate. It isworth noting that this occurs despite the positive correlation of the early phase ofreactive hyperemia and arterial pressure. These results are compatible with thepreviously described uncoupling between macrocirculation and microcirculation inseptic shock.(39)Independent of the pathophysiologic cause, our results suggest that the presence ofthe early hyporesponsiveness or the non-utilization of the microvascular ischemicreserve does not seem to be associated with systemic anaerobic metabolism in septicshock after fluid resuscitation because there was not a correlation between reactivehyperemia , ScvO(16) we did not see any correlation between the SOFAscore and the peak of reactive hyperemia measured with PI. In addition, their reportfound higher values of reactive hyperemia in healthy subjects compared to those inthe septic group, probably due to methodological differences between the studies. Aspreviously described by our group, older patients present increased basal PI valuesand lower reactive hyperemia; further, cardiovascular risk factors such ashypertension, diabetes mellitus and smoking can influence the reactivitytest.(17) Thus, these factors were controlled in ourstudy.Finally, we found a weak but positive correlation between organ failure measured withSOFA score and time to peak of PI. Contrary to the report by He etal., Therefore, theseresults add important information to clinical monitoring because they show thedynamic response of peripheral reactive hyperemia in septic shock and suggest thatperipheral hypoperfusion is more functional and less structural than thoughtpreviously.This study had limitations. First, this was a monocentric preliminary study with ahigh mortality rate . We acknowledge that this sample wasrelatively limited and that some of our findings should be interpreted as generatinghypotheses. Thus, a bigger multicenter study, with a sample size calculation, isneeded to confirm these findings. Second, a single measure was obtained, thuslimiting our conclusions about intra-individual reproducibility. A temporalevolution study is needed to verify the potential of ΔPI as a dynamicparameter. Third, the number of patients did not allow to perform an outcomesanalysis in a prospective study. A study by our group is underway to verify theseaspects. Finally, one can argue that fingertip skin microcirculation is notrepresentative of vital organ microcirculation. However, monitoring non-vitalvascular beds has received increasing attention because these vascular beds areamong the first to deteriorate and the last to be restored after resuscitation incommon scenarios of shock and prediction outcome.In conclusion, in this preliminary study, we found that reactive hyperemia in septicshock patients, when evaluated with the perfusion index, appears to be impaired onlyin the early phase of the post-ischemic response and remains considerably preservedin the latter phase, despite the severe vascular damages of sepsis. The reactivehyperemia evaluation with the perfusion index does not seem to be correlated withsystemic macro-hemodynamics, and only the time-to-peak perfusion index is weaklycorrelated with organ failure, as assessed by the SOFA score. We hypothesize thatthe mechanisms responsible for these findings could be mediated by the sympatheticactivity or immunometabolic mediators. However, further investigations are necessaryto clarify these assumptions.


In the Philippines, non-therapeutic genital cutting is viewed as a culturally sanctioned rite of passage from boyhood to manhood. Strong social and peer pressure is exerted on boys aged between 8-16years to submit to destructive genital cutting, despite the fact that many men who have been subjected to genital cutting during infancy or childhood often describe their experiences in the language of violence, torture, mutilation, and sexual assault. Among a group of 505 Filipino boys subjected to ritual genital cutting (Tuli), 69% fulfilled the DSM-IV criteria for a diagnosis of PTSD, while among 1072 boys circumcised by medical operators or their assistants, 51% exhibited PTSD symptoms. Pursuant to ritual genital cutting, almost 3 out of every 4 boys exhibited PTSD-like symptoms. •1072 Filipino boys (11–16 years) from the Batangas had undergone medical circumcision and 505 ritual circumcision (Tuli).•69% of the Tuli group exhibited PTSD symptoms and 51% of boys circumcised by medical operators exhibited PTSD symptoms.•Boys subjected to Tuli exhibited PTSD comparable to Vietnam veteran inpatients at St. Cloud (Minnesota) VA Medical Center..•Both groups reported higher PTSD (p < .0001) than women subjected to “very distressing or terrifying” obstetric procedures.•A limitation of the study was its failure to examine ongoing levels of PTSD in both groups over increasing time intervals. PTSD caDespite ethical concerns about the child's right to bodily integrity , and theSabado de Gloria (Black Saturday) in a remote wooded area from which girls are excluded  deeply respect this fundamental tenet of international human rights law (UNESCO 2005) ,33.The results of this retrospective cohort study provide strong evidence of direct causal relationship between non-therapeutic genital cutting and the subsequent development of PTSD-like symptoms among Filipino boys. Destructive, non-therapeutic genital cutting of defenceless minors is a serious abuse of power enacted on children by attacking the root of their sexual identity. In males, circumcision irreversibly diminishes and damages their sexual organ with the guaranteed result of significantly reducing sexual sensation. Consequently, many young circumcised men suffer from premature ejaculation ,27, whilchildren's bodily integrity is a fundamental human right that must be respected. The societal pressure exerted on young healthy boys in the Philippines to endure destructive non-therapeutic genital cutting is clearly a flagrant abuse of power and violation of children's human rights [This large-scale retrospective cohort study empirically documents the clearcut psychosexual harm caused by both ritual and medical non-therapeutic genital cutting procedures. In this supposedly enlightened twenty-first century, destructive genital cutting of non-consenting minors, carried out as a rite of passage, is patently unethical and the time has come to recognize once and for all that n rights ,35.4.1The lack of a control group of genitally intact boys makes determining a baseline of PTSD in the male population aged between 11 and 16 years uncertain thereby making the observed results somewhat difficult to contextualise. As well, the different sample sizes of the two comparison groups (and the resultant heterogeneity of variance) may well have contributed to higher levels of statistical significance than otherwise might have been observed. Future research should analyse the severity of reported PTSD-like symptoms taking into account changing attitudes and social changes in the Filipino boys’ lives, how this affects their perceptions of genital cutting, and whether as adults they would also subject their own children to these destructive genital-cutting procedures. Replicating the present study among other populations and regions of the Philippines would be instructive in helping to provide external validation. Although both medically-performed and ritual genital cutting is a controversial topic, given widely held subjective views among Filipinos based on unchallenged cultural and religious beliefs, it is time to consider the human rights of Filipino boys and to cease this physically, sexually, and psychologically damaging practice.Not commissioned, externally peer reviewed.Bond University Human Research Ethics Committee.Ethical approval was granted by No sources of fundingNo conflicts of interest.The brief commentary was written entirely by Gregory J. Boyle.Samuel Ramos conducted the study under the supervision of Gregory J. Boyle.ACTRN12619000589189.Registration number: http://www.anzctr.org.au/TrialSearch.http://www.anzctr.org.au/TrialSearchGregory J. Boyle.


Evidence-based dietary recommendations during early complementary feeding are lacking. Despite extensive research of protein intake in infant formula on growth and risk of overweight, research focusing on the impact of protein intake during early complementary feeding is needed.Current literature, although limited, showed a potential effect of meat intake to promote infant linear growth (length gain), which in turn led to a decreased risk of overweight in some studies.Possible mechanisms regarding protein intake and infant growth are not clear and the early protein hypothesis cannot explain the differential effect of various sources of protein on infant growth during early complementary feeding. Emerging animal studies showed that the gut microbiome may mediate the dietary impact on infant growth.Z score, is strongly associated with obesity in young children and adolescents. Given the current obesity rates in U.S. children, identifying modifiable risk factors underpinning excessive weight and adiposity gain early in life are urgently needed. Although extensive research has been done on infant formula consumption and risk of overweight, a significant knowledge gap exists in the effects of complementary feeding on growth and risk of overweight during late infancy, especially regarding protein-rich foods. This review will present current literature on the impact of complementary foods of animal origin on growth trajectory and the risk of overweight in infants and discuss the potential mechanisms linking protein-rich complementary foods to infant growth and future research recommendations.Evidence-based consensus holds that the first year of life is critical in obesity programming and unfavorable infant growth patterns, namely, excessive weight gain in relation to length gain or increased weight-for-length It was first reported by Rolland et al.  represents the time when solid foods are progressively introduced to infants who no longer rely solely on breastmilk or formula . It is aCurrent national surveys indicate an overall low protein intake from animal origins in older infants. The Feeding Infants and Toddlers Study (FITS) is the largest dietary intake survey of infants and toddlers in the United States. FITS 2008 showed that less than 10% of older infants consumed meat which accounted for <5% of total energy (n = 50 per group), the small differences in meat intake between groups (12% vs. 8%) could have contributed to the nonsignificant findings. Some other studies  or dairy-based (n = 32) complementary diet from 5 to 12 mo of age. Total protein intake during the intervention was considered high at 15% of energy (~3 g/kg/d). The meat group consumed pureed beef, pork, and poultry (provided) and the dairy group consumed yogurt and cheese (provided). The same infant formula was provided for both treatments and fruits and vegetable consumptions were not restricted. Both groups consumed comparable amounts of total protein and minimum protein from the assigned  increased in the meat group and decreased in the dairy group over the course of the intervention, which resulted in half-inch (2.5% or 1.8 cm) longer length in the meat group at 12 mo  are scarce.The mechanisms linking protein intake and infant growth remain unclear. The Early Protein Hypothesis explained the observations that high-protein intakes accelerate weight gain in formula-fed infants. This hypothesis proposed that high protein intake may increase the secretion of insulin and insulin-like growth factor 1 (IGF-1) and result in rapid weight and fat gain in infants. The European Childhood Obesity Project tested this hypothesis in their multicountry randomized controlled trial using high- vs. low-protein infant formula . Weight-The role of the gut microbiome in human health and disease has been extensively examined, especially concerning body weight. A lower Bacteroidetes to Firmicutes ratio has been demonstrated as an “obesogenic” gut microbial profile and colonization of germ-free mice with “obesogenic” gut microbiota resulted in a significantly greater body fat increase than colonization with a “lean” microbiota. In preschool children, the dominating bacterial community tended to be less diverse in the obese/overweight children compared with those with normal BMI. Overall, the gut microbiota appears to have a significant impact on body weight regulation, as demonstrated in both human and animal studies.bifidobacteria can account for over 60% of the total fecal microbiota, while formula-fed infants tend to have more Bacteroides and Clostridium  is the best method for evaluating protein quality (higher DIAAS suggests greater digestibility). The DIAAS is 143 for dairy  111 for beef, and even lower for plants . Different DIAAS between dairy, beef, and plants suggest that at the same protein quantity, different types of protein may result in different amounts of substrates available for the gut microbiota. Amino acid compositions for various protein sources also differ. For example, meat, dairy, and plant proteins have varying amounts of branched-chain amino acids, with dairy being the highest. Branched-chain amino acid supplementation in mice increased the abundance of Ruminococcus gnavus, Clostridium symbiosum) could ameliorate the growth impairment in mice (Lactobacillus plantarum promoted juvenile growth in a strain-dependent manner. Interestingly, one animal study (Lactobacillus abundance in rats, which may partially contribute to some previous findings of meat promoting linear growth in infants. Overall, these studies suggest that the gut microbiota may have a direct impact on infant growth and could be a potential mediator linking the complementary feeding to infant growth trajectories.Besides having a significant impact on body weight and obesity in adults, emerging research suggests that the gut microbiota may also affect infant growth. A cohort study (Z score, but also body composition and neurodevelopment assessments. More importantly, mechanistic investigations of the impact of protein intake on infant growth are also lacking. Although imerging animal research showed a potential mediating effect of the gut microbiome in diet and growth, more human clinical studies that include longitudinal gut microbiome assessment are necessary.Early complementary feeding is a critical growth period that affects the etiology of obesity development. However, evidence-based dietary recommendations during early complementary feeding are lacking and more high-quality research is needed . SpecifiFunding was provided by NIH K01DK111665 to M.T.


Tropheus moorii across a wide sandy bay is linked to observed differences in mate preferences between the two lineages. This hypothesis predicts bi-directional nuclear gene flow and was rejected by congruent patterns of introgression in mtDNA, AFLP and microsatellite markers, with admixture confined to the populations west of the bay. This pattern can be explained on the basis of habitat changes in the course of lake level fluctuations, which first facilitated the development of a symmetric admixture zone including the area corresponding to the present sand bay and then shaped asymmetry by causing local extinctions and cessation of gene flow when this area became once more inhabitable. This conforms with previous assumptions that habitat dynamics are a primary determinant of population-level evolution in Tropheus. In this respect, Tropheus may be representative of species whose preferred habitat is subject to frequent re-structuring.Introgression patterns between divergent lineages are often characterized by asymmetry in the direction and among-marker variation in the extent of gene flow, and therefore inform on the mechanisms involved in differentiation and speciation. In the present study, we test the hypothesis that unidirectional introgression between two phenotypically and genetically distinct lineages of the littoral, rock-dwelling cichlid fish  Patterns of introgression between diverging lineages inform about mechanisms involved in differentiation and speciation. For instance, asymmetries in the direction of gene flow and among loci can elucidate various pre- and postzygotic processes that determine the evolutionary trajectories of the involved lineages. In animals, potential sources of asymmetric introgression include asymmetries in mate choice, driven, for example, by lineage-specific mating preferences and dominance relationships , geograpTropheus moorii, mitochondrial and phenotypic variation suggested unidirectional introgression between two genetically distinct color morphs across a major habitat barrier , which argues against cytonuclear incompatibilities and sex-specific  hybrid mortality that could otherwise explain the lack of introgressed mitochondrial haplotypes in the populations east of Mbete Bay. Lineage-specific dispersal behavior, i.e., a higher dispersal propensity of yellow-blotch than of bluish Tropheus, is rejected by similar and high levels of genetic differentiation among bluish-morph populations west of the bay  with a strict molecular clock . The various datasets required different run lengths, but all analyses were run until post-burn-in ESS for all parameters exceeded 200. The median and corresponding 95% highest posterior density (HPD) intervals were visualized with Tracer 1.6 (available from http://beast.bio.ed.ac.uk/tracer).Population size trajectories through time were inferred by means of Bayesian skyline plots BSPs;  in BEASTP values . Nuclear and mtDNA markers consistently reflected a cline in admixture between bluish and yellow-blotch Tropheus genomes in the populations west of Mbete Bay and an abrupt drop in admixture proportions in the populations east of the bay  was distinct and significant, except for weak or no differentiation across all three marker types between Chaitika and Nakaku as well as between Tanganyika Lodge and Kasakalawe Lodge; in both cases, sampling locations are connected by continuous rocky habitat. Additionally, mitochondrial differentiation estimated by ϕST was non-significant in three comparisons among adjacent populations  and narrowly missed significance based on FST estimates in three other population pairs .Both mitochondrial and nuclear markers displayed a high level of genetic diversity within and differentiation among populations  to a divergence time parameter of 0.489 (mean of four IMa2 runs). Higher divergence time parameter estimates (mean estimates of 0.905–1.016) were obtained between the “bluish lineage” haplotype groups from the different admixed locations west of Mbete Bay , as opposed to “pure bluish populations” (Nakaku and Chaitika) and “pure yellow-blotch populations” . Although Mbete Bay represents a divide between genetic and phenotypic clusters for several cichlid species  contains supplementary material, which is available to authorized users.Suppl material 3Suppl material 2Suppl material 1


Inflammation plays an essential role in the control of pathogens and in shaping the ensuing adaptive immune responses. Traditionally, innate immunity has been described as a rapid response triggered through generic and nonspecific means that by definition lacks the ability to remember. Recently, it has become clear that some innate immune cells are epigenetically reprogrammed or “imprinted” by past experiences. These “trained” innate immune cells display altered inflammatory responses upon subsequent pathogen encounter. Remembrance of past pathogen encounters has classically been attributed to cohorts of antigen-specific memory T and B cells following the resolution of infection. During recall responses, memory T and B cells quickly respond by proliferating, producing effector cytokines, and performing various effector functions. An often-overlooked effector function of memory CD4 and CD8 T cells is the promotion of an inflammatory milieu at the initial site of infection that mirrors the primary encounter. This memory-conditioned inflammatory response, in conjunction with other secondary effector T cell functions, results in better control and more rapid resolution of both infection and the associated tissue pathology. Recent advancements in our understanding of inflammatory triggers, imprinting of the innate immune responses, and the role of T cell memory in regulating inflammation are discussed. Advances on several research fronts have significantly broadened our understanding of the triggers and modulators of inflammation. Of importance to this review, we now appreciate that at sites of infection, adaptive immune memory cells regulate innate inflammatory responses that contribute to the control of pathogens. Herein, potential means to modulate inflammation for the optimal generation of protective immunity through vaccination are discussed.Mycobacterium tuberculosis [The ultimate goal of vaccination is to stimulate the generation of long-lived protective immunity without causing adverse clinical symptoms. Traditional vaccination strategies employing inactivated or attenuated pathogens or pathogen-derived protein antigens primarily target the generation of neutralizing antibody responses from B cells that act to prevent infection upon pathogen reencounter . These rrculosis , 6 have rculosis , and therculosis –11. In oWhen a pathogen breaches the initial barriers of the skin or a mucosal surface, both soluble and cellular innate defense mechanisms are encountered and an inflammatory response is rapidly initiated. Some of the most potent soluble antimicrobial factors encountered include complement, lysozymes, defensins, mucins, lectins, cathelicidins, and lipocalins –15. SeveSoluble inflammatory chemokines  and actiκb, AP-1, and interferon regulatory factors (IRFs) [In order for the inflammatory events discussed above to occur, pathogens must be detected in compromised tissues. Many different subsets of classic dendritic cells, plasmacytoid dendritic cells, and macrophages , 29 are s (IRFs) , 40, 41.s (IRFs) , 40, 42.s (IRFs) –11.Advancements in our knowledge of intracellular sensors of pathogens and host-derived stress products have revealed novel targets to modulate and improve vaccine efficacy , 44. A nIntracellular sensors interact with adaptor proteins such as apoptosis-associated speck-like protein containing a C-terminal caspase activation and recruitment domain (ASC)  to triggκb activation by the release of mitochondrial H2O2 in lung APC [Under normal circumstances, inhibitory “innate immune rheostats” act to prevent unnecessary inflammation at barrier surfaces , 58. Influng APC  and the lung APC –70 mitiglung APC  and exprlung APC  are primSafely overcoming these “rheostats” by targeted blockade of inhibitory molecules or by employing novel adjuvant formulations that facilitate the generation of protective local immunity through vaccination without causing damaging adverse effects is of paramount importance –75. IndeThe control of immune response gene expression by long noncoding (lnc) RNAs  is anothIn addition to the homeostatic mechanisms and negative feedback loops discussed above, which preserve vital functions of organs such as the lung and intestine, the timing of vaccination administration may also need to be taken into account. Patterns of expression of proteins such as IL-6, inflammatory monocyte chemokine ligand (CCL2), as well as Toll-like receptor (TLR) 9, which are regulated by circadian clock proteins , may expTo successfully generate protective immunity through vaccination, antigen-specific T cells must interact with activated APC displaying cognate antigen in the context of MHC. Such interactions result in the receipt of signal 1, the specific antigen, and signal 2, the costimulatory molecule-dependent signals, required for full T cell activation. Recognition of inflammatory cytokines by their corresponding cytokine receptors constitutes signal 3 that can amplify proliferation as well as effector functions in activated cells.Foreign antigens introduced by vaccination must reach the secondary lymphoid organs in order for T cell activation to occur. Antigen is delivered to draining lymph nodes via the lymph in particulate form or within migrating tissue-resident antigen-presenting cells that have egressed from the inflammatory site . ParticuExposure to and engulfment of pathogen-derived products at the site of vaccination or infection activates APCs and triggers their production of inflammatory cytokines. Cohorts of APC, once activated, will begin to migrate towards lymphoid organ chemokines CCL19 and CCL21 in a CCR7-dependent fashion , 98. Egrin vivo [The lifespan of activated tissue-migratory APCs within draining lymph nodes, especially the dendritic cell subset, is relatively short , and optin vivo , that emin vivo , or thatin vivo –10. Suchin vivo , 113.Following an acute infection or vaccination, the activation and expansion of naïve pathogen-specific T cells and the generation of effector cells generally occur within 7 days. Under normal circumstances, the majority of expanded effector cells that migrate to sites of infection or antigen administration undergo contraction following subsequent pathogen or antigen clearance. A small cohort of the expanded effectors will, however, survive to memory . These aSome antigen-specific memory T cells possess the ability to migrate throughout the body and are readily detected within tissues , 117. ThIn addition to rapidly producing cytokines upon recognition of cognate antigen, memory T cells perform many other effector functions to protect the host against infection . These fBoth memory CD4 , 133 andFor memory CD4 T cells, enhanced inflammatory responses are initiated in the lung following IAV infection independently of the classic PRR signaling molecules MYD88 and TRIF . Memory γ and does not require the receipt of CD80, CD86, and CD40 costimulatory molecule signals [in vivo are not affected by blockade of the CD28 costimulatory pathway [γ [in vivo [The ability of memory CD4 T cells to induce inflammatory responses upon pathogen detection is also independent of their production of the classic proinflammatory cytokines TNF and IFN- signals . That me pathway . The sen pathway , IFN-γ [thway [γ –144, GM-thway [γ , and pot[in vivo . Similar+ T effector cells that accumulate in the lung at the peak of the recall response [γ, is associated with the ability of memory T cell responses to protect against numerous viral, bacterial, and parasitic pathogens [Following secondary IAV infection, the earlier and more robust inflammatory response induced by memory CD4 T cells correlates with improved control of the virus in the lung . Our recresponse  (Figure athogens . Multicyathogens , 148. Inathogens , will fuAnother significant advance in our understanding of innate immunity is the knowledge that cells of the innate immune system are altered or “trained” by past experiences . For theIt has been long been appreciated that organs such as the lung remain in an altered state for an extended period of time following infection or insult , 155. Thin vitro [The protection afforded by “imprinted” innate immunity is associated with the presence of increased numbers of activated macrophages , 156, dein vitro .Pathogen-associated encounters may not be the only events capable of training innate immune cells. The engulfment of apoptotic host cells in the absence of infection has traditionally been considered an immunologically neutral event that fails to generate DAMP signals . Recent The altered inflammatory state that exists following the resolution of infection can also have alternative and undesired outcomes. For example, conditioning of innate immune cells by prior infection can result in increased susceptibility to secondary infection . IncreasWhile highly specific in nature, the adaptive immune response can also alter the outcome of infections with seemingly unrelated pathogens. This phenomenon, which has been termed heterologous immunity , is mediUnderstanding of the impact of prior pathogen encounter on both innate and adaptive immunity is imperative for the design of innovative vaccination regimes. Exciting developments in the field of macrophage and monocyte biology are changing the way memory is typically perceived in innate immune cells. The “training” of innate immunity must be further investigated in order to effectively implement these insights into improved vaccines that are better able to promote durable memory states. In addition, traditional paradigms of innate instruction of adaptive immunity must now appreciate that memory T cells regulate both the nature and shape of innate inflammatory responses through antigen-specific means. Furthermore, memory-regulated inflammatory responses can impact the development and functional potential of secondary effector T cells. Every infection, commensal interaction, and immunogenic vaccine thus has the potential to change the host tissue microenvironment as well as the adaptive immune T cell repertoire. Such changes can impart lasting immunological consequences that are able to influence subsequent responses to infection both positively and negatively.


We discuss the biophysical properties that determine the ability of microbial opsins to evoke reliable, precise stimulation or silencing of electrophysiological activity. We also review spectrally shifted variants offering possibilities for enhanced depth of tissue penetration, combinatorial stimulation for targeting different cell subpopulations, or all-optical read-in and read-out studies. Expression of the chosen optogenetic tool in the cardiac cell of interest then requires, at the single-cell level, introduction of opsin-encoding genes by viral transduction, or coupling “spark cells” to primary cardiomyocytes or a stem-cell derived counterpart. At the system-level, this requires construction of transgenic mice expressing ChR2 in their cardiomyocytes, or in vivo injection  of adenoviral expression systems. Light delivery, by laser or LED, with widespread or multipoint illumination, although relatively straightforward in vitro may be technically challenged by cardiac motion and light-scattering in biological tissue. Physiological read outs from cardiac optogenetic stimulation include single cell patch clamp recordings, multi-unit microarray recordings from cell monolayers or slices, and electrical recordings from isolated Langendorff perfused hearts. Optical readouts of specific cellular events, including ion transients, voltage changes or activity in biochemical signaling cascades, using small detecting molecules or genetically encoded sensors now offer powerful opportunities for all-optical control and monitoring of cellular activity. Use of optogenetics has expanded in cardiac physiology, mainly using optically controlled depolarizing ion channels to control heart rate and for optogenetic defibrillation. ChR2-expressing cardiomyocytes show normal baseline and active excitable membrane and Ca2+ signaling properties and are sensitive even to ~1 ms light pulses. They have been employed in studies of the intrinsic cardiac adrenergic system and of cardiac arrhythmic properties.Optogenetic techniques permit studies of excitable tissue through genetically expressed light-gated microbial channels or pumps permitting transmembrane ion movement. Light activation of these proteins modulates cellular excitability with millisecond precision. This review summarizes optogenetic approaches, using examples from neurobiological applications, and then explores their application in cardiac electrophysiology. We review the  Optogenetics offers techniques to modulate the activity of excitable cells using light, in a genetically specified manner. The method harnesses microbial proteins, known as opsins, which are light-activated proteins (channels or pumps) that permit transmembrane movement of ions. There are two types of opsins: Type I  and Type II . The opsins used most commonly in optogenetics fall under the category of Type I opsins  and are referred to as rhodopsins, meaning that they contain both an opsin protein (with seven transmembrane domains) as well as a light-sensitive chromophore. When activated by light, opsins cause depolarization or hyperpolarization of the cell membrane, with resulting cellular excitation or silencing on a millisecond time scale. Opsins were first expressed in mammalian neurons in the mid-2000s; since then the optogenetic toolkit has vastly expanded to address a number of different experimental demands. Aided by the recent elucidation of the crystal structure of opsin proteins, opsin engineering has led to a wide array of available tools with different properties, including ion conductances, kinetics, light sensitivity, and activation spectra. Although initial applications of optogenetics were primarily within the field of neuroscience, the technique soon became relevant to other excitable tissues. In this review, we summarize existing optogenetic tools and approaches and discuss their suitability for cardiac experimental questions. We then review existing cardiac applications and discuss some recent examples in cardiac optogenetics that have yielded new insights into cardiac physiology and cardiac disease.Any optogenetic experiment involves several practical considerations: first, the properties of the chosen opsin tool and whether it is suited to the cellular physiology of interest and the experimental application. This includes the direction of the ionic current, as well as the nature of the transported ion(s), in addition to the kinetics and temporal precision of the tool, the amplitude of the evoked current (which in turn depends on a number of factors described below), and its spectral properties. The next consideration is the ability to specifically target  the cell type of interest and deliver light into the experimental system of interest in a stable and spatially precise manner. Finally, the read-out of the system will depend on the experimental question being addressed; for example, this might be an electrophysiological recording, or a physiological imaging technique or an organ-level or behavioral output .via genetic or developmental targeting methods and spatial specificity through the use of patterned illumination. The combination of optogenetic tools with optical activity sensors  has also permitted realization of the highly desired “all-optical read-in and read-out” approach  were co-expressed in mammalian cultured hippocampal neurons . In addition, the channels must have a low rate of desensitization and/or recover rapidly from the desensitized state. It has been demonstrated that there is a strong correlation between light sensitivity and τoff, suggesting that the light sensitivity of a membrane-bound rhodopsin population is mainly accounted for by channel off-kinetics, although differences in single channel intrinsic light sensitivity and conductance may explain deviations from the curve . Others have noted that this suggests a theoretical trade-off between light sensitivity (volume of tissue activation) and channel kinetics (spiking precision)  , the single-channel conductance, and the degree of channel expression in the cell membrane of interest. Other contributing factors include the channel kinetics as well as the cell’s physiological response to the photocurrent. Rhodopsins with higher light sensitivity require a lower light intensity to reach maximal photocurrent amplitude, and thus may drive depolarization more readily across a range of light intensities or across a larger volume of tissue . Howeverecision) . Fast chto do so . In cont source) .τoff of the order of ~30 min can provide stable subthreshold excitation over long time periods, even in the absence of ongoing light stimulation . It is well expressed in cardiomyocytes and generates reliable action potentials in response to pulsed light , and the rise in intracellular Ca2+ through transmembrane influx and sarcoplasmic reticulum release generates the contractile force. A slow but sensitive opsin with large photocurrents and greater calcium ion permeability  , ArchT , eBR , and Mac (from Leptosphaeria maculans) also generate hyperpolarizing currents from outwardly directed proton pumping  . NpHR is pumping . More re pumping . Genetic pumping  has led  pumping . Therefo pumping . ThroughGuillardia theta (GtACR1 and GtACR2)  were isolated from chlorophyte algae,  GtACR2) . Their c GtACR2) . These c] was genetically modified to have a spectral activation peak of 460 nm (10 nm blue shifted relative to ChR2) as well as faster kinetics. This variant, known as CheRiff, was shown to have 9-fold higher light sensitivity than ChR2HR and had minimal optical cross-talk with red-shifted voltage indicators (Platymonas subcordiformis ChR (PsChR) (Tetraselmis striata ChR (TsChR)  through the modification of key residues of the rhodopsin molecules. This can be achieved by exchanging residues interacting with the retinal Schiff-base  or residues that influence the polarity along the retinal polyene chain . Wild-tydicators . Platymo (PsChR)  and Tetr (TsChR)  are the  (TsChR) .A major advantage of red-shifted variants is the possibility of combinatorial stimulation experiments using both blue light and yellow or red light to activate different cell populations within the same anatomical area, or for all-optical read-in and read-out methods, in which stimulation is combined with imaging techniques. One limitation of the early red-shifted options was that despite their red-shifted shoulder many retained some activity within the blue light range , making Another theoretical advantage of red-shifted spectra is volume and depth of tissue modulation. Since blue light is known to scatter more easily in opaque tissue such as the brain or cardiac tissue, red light has the potential to penetrate further with less scattering and thus may allow greater volume and depth of optogenetic modulation ,b, even in vitro or in vivo which we discuss below  to generate ChR2-expressing cardiomyocytes . The ESCThere have since been several additional reports of the use of embryonic stem cells to generate opsin-expressing cardiomyocytes. in vitro expression of ChR2 and NpHR in neonatal ventricular cardiomyocytes  formed from non-excitable  cells  transfected to express a light-sensitive ion channel (ChR2) coupled through gap junctions to excitable cardiomyocytes (CM) to form an optically controllable functional TCU . Dual whin vitro for longer periods of time than primary cardiomyocytes.Nussinovitch et al. demonstrated a similar TCU approach but this time using co-culture of ESC-derived cardiomyocytes with fibroblasts which were genetically engineered to simultaneously express both ChR2 and the red-shifted inhibitory opsin, ArchT. With this approach, they were able to generate bidirectional modulation, driving optogenetic pacing using blue light and optogenetic suppression of activity with red light . The advvia connexin 43 gap junctions in the AV node and exhibit synchronous membrane depolarization .The interactions between non-myocyte cardiac cells and cardiomyocytes have also been studied using this approach. For example, one study investigated the functional coupling between cardiac macrophages and cardiomyocytes in the AV node of mice, using a co-culture preparation. They demonstrated that the two cell types are electrically coupled rization . This apin vivo in a cell-type specific manner is through the generation of transgenic animals. This approach has been used to drive expression in both cardiomyocytes as well as cardiac non-myocytes. To date, optogenetic transgenic mice, zebrafish, and Drosophila have all been used in cardiac optogenetic experiments. One of the earliest cardiac optogenetic experiments was performed in transgenic zebrafish  were necessary for heart beat initiation.An effective approach to generate opsin expression ebrafish . The GalHR on exposure to Cre-recombinase (Ai27D mice). The resulting offspring expresses ChR2HR specifically in cardiac tissues  promoter are crossed with mice which conditionally express ChR2 tissues . This ap tissues . It has  tissues . Zaglia Several studies have also reported on transgenic methods for optogenetic targeting of cardiac non-myocytes, such as sympathetic cardiac neurons, parasympathetic neurons, cardiac neuroendocrine cells, and cardiac macrophages. Cardiac sympathetic neurons were targeted by cross-breeding mice expressing Cre-recombinase under the catecholaminergic-specific promoter tyrosine hydroxylase (TH) with mice that express ChR2 in a Cre-dependent manner, resulting in mice that express ChR2 only in TH-expressing catecholaminergic cells. This transgenic model was used to study the specific role of cardiac sympathetic nerve activation on cardiac contraction and arrhythmia . The rolPnmt+) cells by crossing Pnmt-Cre mice with mice that express ChR2 in a Cre-dependent manner . This has been used to study hyperpolarization as a mechanism for ventricular arrhythmia termination  were bred with mice expressing Cre-dependent ChR2, resulting in mice that express ChR2 specifically in AV node macrophages only after the administration of tamoxifen. This approach allowed the experimenters to causally assess the influence of AV node macrophage activation on atrial conduction in vivo and in a Langendorff-perfused heart preparation  at 30–50 beats per minute above the resting heart rate in approximately three quarters of the tested hearts. Lack of in vivo optogenetic responses was likely the result of low construct expression, as demonstrated by lower mCherry fluorescent signal in the ventricles. The team calculated that the minimal percentage of expressing cells for optogenetic pacing was 30–40% and demonstrated as proof of principle that this percentage could be achieved through a systemic viral injection. One disadvantage of this method is off-target expression in other organs, for example, they also found mCherry-labeled muscle fibers in the diaphragm and a small number of hepatocytes. It may be possible to circumvent this with the use of cell-type specific promoters.These studies represent the tip of the iceberg of genetically targeted cardiac cells. There are now a large number of cell specific promoters that may permit spatially constrained opsin expression in regions such as the sinus node, the atria, ventricles, conduction system, or progenitor cells. In addition, some genetic motifs may also permit subcellular targeting, for example, localizing the opsin to the plasma membrane, the sarcoplasmic reticulum or the mitochondria . In paraViral vector delivery has also been used in other animal models. For example, one group used AAV2/9 to transfect cardiac sympathetic neurons in the left stellate ganglion of dogs with the hyperpolarizing tool ArchT . The autin vitro cell culture or monolayer preparations, light delivery is relatively straight forward. For neurons, the light power density  required to elicit an action potential by wild type ChR2 has been estimated at ~1–5 mW/mm2 , digital micromirror devices (DMDs) have also been used to generate user-defined light patterns to focus the light on different anatomical targets  either singly or as an array have been proposed. To overcome the issue of blue light scattering and increase the depth of light penetration, these micro-LEDs could be used in combination with red-light shifted opsins which tend to be less vulnerable to light scattering and achieve a greater penetration depth  , they have yet to be successfully applied to cardiac tissue for optical stimulation. Recently, a multi-LED probe was used for the first time to drive intramural optical stimulation of ChR2 in isolated murine hearts, demonstrating optical pacing as proof of principle (For hrimson) . The devhrimson)  that couhrimson) . The devhrimson)  that canhrimson) . Novel bhrimson) . Howeverrinciple .The use of two-photon illumination for optogenetic experiments in neurons is reviewed in detail elsewhere . TherefoTwo-photon optogenetics also helps to circumvent challenges of spectral combinatorial approaches. For example, it permits the spatial segregation of optogenetic stimulation light from optical imaging light, such that one population of cells could be modulated optogenetically at the same time that the activity of a different but spatially intermingled population of cells is monitored with genetically encoded fluorescent activity sensors . This iIn cardiac experiments, two-photon stimulation thus presents an even greater challenge. It is unlikely that the small stimulation volumes generated by two photon techniques will be sufficient to pace the well-coupled syncytium of cardiomyocytes with low input resistance (from Kir2.1 potassium currents). So far, two photon approaches for optogenetic stimulation have not been shown to work in the heart, although they may be used for high resolution imaging purposes.An essential component of any optogenetic experiment is to monitor the response of the experimental set-up to the applied optogenetic modulation, also known as a “read out.” This can occur at many levels of the biological hierarchy – from single cell or tissue, to whole organ or organism responses .ex vivo Langendorff preparation with depth or surface electrodes, or in vivo in an anesthetized or awake animal using ECG recordings  and red-shifted genetically encoded calcium indicators (R-GECO) have been successfully used for optical stimulation and recording in primary ventricular cardiomyocytes  and the more recently developed genetically encoded calcium sensors (GECIs). All GECIs contain a calcium-binding domain fused to either a single or two fluorescent proteins (FRET-based fluorescence). The single fluorescent protein sensors, GCaMPs, are now widely used in neuroscience due to their high signal to noise ratio and fast kinetics; however, unlike the FRET-based sensors , they do not provide a ratiometric signal which can result in confounds during myocardial cell contraction. Despite this, GCaMPs have been used to record calcium transients in cardiomyocytes in vivo , even atin vivo ]. Initiain vivo , and neain vivo  have nowin vivo  and variin vivo . To datemyocytes .Another desirable readout is optical measurement of membrane voltage, as opposed to a surrogate cellular response such as calcium transients. To date, the most frequently used optical voltage sensors in cardiac optogenetics are red-shifted voltage sensitive dyes. These have been used to map action potential propagation as well as emergent properties such as excitation waves in cardiac tissue with high temporal resolution . The redL. maculans (MAC), and subsequently engineered non-pumping mutants, have an endogenous fluorescence that is modulated by changes in local membrane voltage thus permitting voltage sensing with optical resolution of single action potentials  have also seen great improvement over recent years. There are several different types of GEVI, including voltage-sensing fluorescent proteins (VSFP) (FRET-based or single fluorescent proteins) as well as opsin-based sensors. The proton-pumping microbial opsins such as archaerhodopsin (Arch), tentials . Much wotentials . In the  ex vivo  as well  ex vivo .Finally, there are a number of optical sensors designed to detect changes in activity in biochemical transduction cascades, for example, calmodulin-CAMKII/calcineurin, cAMP/PKA, and cGMP/PKG activity. These biological processes and thus their sensors act over a slower time course than ion and voltage sensors. A number of FRET-based and genetically encoded sensors are available and have been used in combination with optogenetic stimulation in neurons [e.g., ; reviewein vivo. Future endeavors, many of which are already in progress, fall under a wide range of areas.Emerging optogenetic techniques provide unprecedented opportunities to study cardiac physiology particularly with the capacity to deliver optogenetic control with spatiotemporal resolution and cell type specific precision. Over the last decade, there has been an explosion in the use of optogenetics in cardiac physiology, with applications ranging from investigating action potential propagation across cardiomyocyte monolayers to arrhythmia initiation and termination 2+ transients. Prolonged light stimulation induced action potentials followed by prolonged depolarizations. Such longer light stimulations resulted in prolonged elevations in intracellular levels of Ca2+ (Single ChR2-expressing ventricular cardiomyocytes can be isolated from adult transgenic mice (generated from ChR2-expressing embryonic stem cells) . These h of Ca2+ .in vitro cultures. The ChR2-expressing, ESC-derived cardiomyocytes were plated on multi-electrode arrays, where they formed two-dimensional functional syncytia of synchronously beating cells whose activity could be followed through local field potentials. Evoked electrical activity elicited by pulsed illumination spread to other regions and moving the illumination site correspondingly shifted the initial pacemaker site. Prolonged local illumination suppressed electrical activity in the illuminated area while sparing spontaneous activity in the non-illuminated areas consistent with this producing a sustained depolarization that would cause a local Na+ channel refractoriness.ChR2 has also been used for precise local stimulation in two-dimensional in vitro and in vivo, in investigations of physiological mechanisms involved in pacemaking or site-specific pro-arrhythmic effects of prolonged depolarizations and delayed afterdepolarizations. Thus, electrocardiographic studies of focal atrial ChR2 activation with pulsed blue illumination in anesthetized ChR2-EYFP expressing mice led to supraventricular pacing accompanied by prolonged (by ~50%) P-wave durations and PQ intervals (by ~20%) (via the conducting pathways of the heart. An illumination area of 0.05 mm2 (with a blue light intensity of 7.2 mW mm−2), corresponding to ~50 epicardial myocytes in addition to cardiomyocytes in deeper layers, sufficed to induce pacing, but higher light intensities were required with smaller areas of stimulation. The latencies of electrocardiographic responses in vivo were similar to the observed delays from onset of illumination to action potential peak in cardiomyocytes in vitro (~10 ms). Finally, prolonged illumination likely leading to long-term ChR2 activation and sustained depolarizations in ventricular regions disturbed the regular pattern of sinus rhythm and generated spontaneous ventricular extra beats.Illumination of ChR2 expressing cardiomyocytes permits introduction of novel stimulation patterns by ~20%) . Ventricin vivo approach in genetically wild-type rats utilized adeno-associated virus (AAV) intramyocardial transgene delivery to express the ChR2 transgene at one or more ventricular sites. Surface electrocardiographic measurements then revealed that focused blue light delivered via the optical fiber achieved efficient activation of the ventricle when applied at the apex (the site of ChR2-transgene delivery). It thus altered heart rates from a baseline sinus rhythm (~180 beats/min) to up to 300 beats/min during illumination. This did not occur when illumination was applied to remote myocardial areas in the same hearts or in animals injected with control AAV-CAG-GFP virus. Similar results were obtained in isolated perfused hearts. Other studies have demonstrated that ChR2 stimulation of cardiomyocytes is compatible with the use of long-wavelength voltage-sensitive dyes. These high resolution optical mapping studies confirmed that the ChR2-expressing cardiomyocytes were the source of pacemaker activity by demonstrating a shift in the site of the earliest ventricular activation – from the location of the conventional pacing electrode (at the side of the ventricles) to the site of ChR2-expression (at the apex)  .Applications of optogenetic techniques have been explored for their localizable, potentially translationally important, anti-arrhythmic effects . Classicin vitro cellular preparations, anti-arrhythmic effects of light were explored in neonatal rat atrial cardiomyocyte monolayers transduced with lentiviral vectors encoding light-activated Ca2+-translocating channelrhodopsin (CatCh-eYFP) or with eYFP controls. Light pulses  then triggered action potentials only in the CatCh-expressing cultures. Burst pacing induced spiral waves rotating around functional cores, modeling atrial fibrillation. Optical and multi-electrode array (MEA) mapping assessed effects of prolonged (500 ms) light pulses activating CatCh. These terminated re-entry in all CatCh-expressing cultures but none of the controls. This took place through a uniform depolarization depressing overall excitability (At the level of tability .2) in Langendorff perfused hearts. This termination was preceded by prolongation of the action potential (by ~14 ms) and pharmacological interventions that shortened the action potential duration were observed to prevent arrhythmia termination  produced its global cardiac, cardiomyocyte-restricted, and transgene expression. This permitted ReaChR-mediated depolarization and pacing. Ninety-seven percent of ventricular monomorphic and 57% of polymorphic ventricular tachycardias (VT) induced by burst pacing could then be terminated by single 470-nm light pulses  and the presence of the KATP channel activator pinacidil (100 μM). Thus, uniform light-induced depolarization proved effective for cardiac defibrillation. Explorations of light duration, intensity, and size of illuminated area indicated a requirement for illumination lasting for at least the duration of an entire VT cycle (~80 ms), over an area > 15 mm2, and of intensity ~0.4–1 mW/mm2, suggesting a requirement for a depolarization of a critical portion of myocardium. Such findings extended to experimental infarct-related ST-elevation associated arrhythmia, following left anterior descending coronary artery ligation. Sustained ventricular arrhythmia following forced electrical pacing was terminated by a single light pulse. Concordant findings emerged from WT hearts after adeno-associated virus induced ChR2 gene transfer  proved effective whether sustained ventricular arrhythmia was induced by electrical burst stimulation or S1-S2 protocols applied at the right ventricular base under conditions of reduced Ktransfer .single point and single line stimulation, triple-line pattern optical stimulation at 10mW/mm2 successfully achieved cardioversion, doing so while confining the irradiated epicardium with a reduction of total irradiation energy   .ex and in vivo hearts from transgenic and wild-type mice, respectively. Isolated ChR2 and pro-arrhythmic connexin 40, Cx-40-Ala96Ser expressing murine hearts perfused with low K+-Tyrode’s solution and an atrial KATP-channel activator showed a pro-arrhythmic shortened atrial refractoriness and slowed atrial conduction. Atrial arrhythmia was terminated using epicardially focused blue light  likely acting by light-induced block of electrical activity. This optical effect was reproduced in wild-type mice made to express ChR2 via adeno-associated virus (AAV) delivery  and VT through targeting specific cardiac regions if this was applied to human hearts. One study attempted to terminate re-entrant AT following rapid pacing by optogenetic stimulation of channelrhodopsin-2 (ChR2) expressed through gene delivery. This employed three patient-specific computational models reconstructed from late gadolinium-enhanced MRI scans . It comppagation .Optogenetics offers potential alternatives to drug or electrotherapy for treating arrhythmia. Drug therapy for atrial fibrillation can have serious side effects even including proarrhythmic effects. Patients at risk from potentially fatal ventricular arrhythmias currently receive implantable defibrillators delivering electrical shocks terminating arrhythmias on demand. However, such electrical shock therapy is associated with discomfort and tissue damage.in vivo (Optical pacing is compatible with energy requirements that compare favorably with electric pacing and optical fibers may be more biocompatible than electrodes. Optical pacing could potentially simultaneously pace multiple sites with advantages for cardiac resynchronization therapy over biventricular electrical pacemakers . Hypothein vivo .ex vivo Langendorff perfused hearts as well as in vivo hearts under anesthesia. Experiments have addressed fundamental questions about the physiology of the cardiac action potential and the spread of electrical activity across the heart as well as disease-focused questions such as mechanisms of arrhythmia initiation, propagation, and termination.In conclusion, the use of optogenetics in cardiology research has greatly accelerated over the last decade since the technique was first demonstrated as a proof of principle in zebrafish and mouse cardiomyocytes . The verOngoing work that will be shared with the neuroscience community will be the continued development and expansion of the optogenetic tool kit, and major goals include the development of red-shifted opsin that lacks a shoulder in the blue end of the spectrum and a single-component light activated potassium channel.in vivo light delivery with flexible illumination patterns and its combination with read out methods such as optical recordings for closed-loop stimulation paradigms. Improvements in optoelectronic device engineering with low power LED arrays for long-duration, multi-site stimulation, closed loop sensing and implantable or injectable flexible electronics that can mold to and adapt with the mobile myocardium will be necessary for cardiac optogenetics to become relevant for clinical applications. Finally, basic science and translational applications will continue to expand, for example, to include investigation of not only cellular but also subcellular mechanisms underlying myocardial excitability, contractility, rhythm generation and spread, in physiological as well as pathological states.Future cardiac directions will need to focus on further optimization of optogenetic tools specifically for myocardial cells such as modification of kinetics of depolarization/hyperpolarization to better suit the cardiac action potential and modulation of specific ion permeability to minimize off-target effects of non-physiologic changes in intracellular ion concentration. Other work will need to focus on improving genetic methods to specifically target different myocardial cell types and improving engineering approaches to permit chronic All three authors contributed to this review as a whole, with EF leading on the sections covering the principles and techniques bearing on optogenetic methods and XT and CH those aspects bearing on their cardiac application. Repeated revisions of the text were performed by EF and CH.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.