N = 70) performed two isometric handgrip endurance trials separated by a Stroop task, which was either congruent low cognitive control (LCC) or incongruent (HCC). Motor coordination was assessed prior to the first endurance trial. Task self-efficacy for performing the second endurance trial was assessed following the Stroop task. Participants in the HCC condition reported lower task self-efficacy and showed a reduction in endurance exercise performance. Task self-efficacy mediated the cognitive control—performance relationship. Participants scoring lower on motor coordination showed the greatest declines in exercise performance following HCC, whereas motor coordination did not affect performance following LCC. The results of this study provide evidence that task self-efficacy and exercise performance are also negatively affected in children following HCC, and interestingly, these effects are exacerbated among those scoring lower in motor coordination. We recommend future research investigate motor coordination as a potential mechanism for the reductions in both cognitive and physical task performance following the prolonged exertion of HCC.Emerging research shows a strong connection between brain areas governing cognition and motor behavior. Indeed, research based on the Strength Model has shown that people perform worse on physical tasks following the exertion of high (compared to low) cognitive control which has been attributed to the dysregulation of neurophysiological processes within areas of the brain responsible for cognition. Yet, research investigating the negative aftereffects of high cognitive control (HCC) exertion on task performance has not considered the potential role of areas governing motor behavior. The present study investigated the effects of HCC exertion on task self-efficacy and exercise performance in children. A secondary purpose was to investigate whether motor coordination influences the change in exercise performance differently following low versus HCC exertion. Participants ( HoweverThe close connection between cognition and motor behavior is also a fundamental tenant in the theory of embodied cognition and critical during the developmental period from childhood to adolescence  would experience greater negative effects on exercise performance following HCC exertion compared to those with high motor coordination (HMC).n = 28 females; age range: 7–14; Mage = 10.14 ± 1.90) who were enrolled in a 2-week recreational summer sport day camp at a university and were tested in a lab at the same institution. The study utilized a single-blind, randomized experimental design with one independent variable: group, consisting of two levels—LCC exertion/HCC exertion; and two dependent measures: task self-efficacy and endurance exercise performance. The primary hypotheses related to expected changes in task self-efficacy and endurance exercise performance. For an overview of the experimental protocol as well as a timeline of manipulations and measures see Figure 1.Participants were 70 children  × 2 (LMC/HMC) univariate analysis of variance (ANOVA). Given the lack of previous research investigating the effects of motor coordination on performance following HCC, no effect size could be determined for a sample-size calculation. Therefore, the secondary analyses were considered exploratory.The effect sizes reported below, calculated using means and SDs, are based on The present study was approved by the Ethics Research Committee of McMaster University in accordance with the ethical standards of the Declaration of Helsinki 1, participants performed the congruent version of the Stroop task for 1 min and were asked to read aloud the words they saw printed. Based on recent recommendations for cognitive control manipulations  or a congruent Stroop color word task (LCC) for 5 min. However, prior to being randomized (stratified by sex) into their experimental condition using a random number generatorulations , we wantd = 0.40, Age Differences,” pp. 184–185). As such, pilot testing was conducted with the incongruent version of the Stroop task to investigate whether children in a sample equivalent to our sample  perceived the incongruent version of the Stroop task to be more challenging than the congruent version. Results of the pilot study revealed that children  rated the incongruent version of the Stroop task to be more challenging, with the 76% of children rating it as “a lot harder” and 24% rating it as “a little harder.”The incongruent version of the Stroop task  is desigFor the incongruent Stroop task , particiFor the congruent Stroop task (control), participants were presented with a list of words in which the print ink color and printed text were matched. Participants were asked to read aloud the word presented.The primary dependent measure was the change in the amount of time  participants maintained an isometric endurance handgrip contraction at 30% of their maximum voluntary contraction (MVC) using their dominant hand.Prior to the first endurance trial, participants performed 2, 4-s 100% MVCs (separated by 2 min) using a handgrip dynamometer  with graphic computer interface . The average force recording obtained from a 1-s window at the peak of each MVC was analyzed to determine peak force generation. The greatest peak force value was used to determine the 30% MVC target value for the endurance trials. The target force was shown as a static red horizontal line on a 17-inch computer monitor. The experimenter performed a 10-s demonstration of the endurance task, which was followed by a 10-s practice trial by the participant. This was done to ensure that the participants understood the task requirements and clarification (or another brief demonstration) was provided if necessary.In order to perform the endurance task, participants squeezed the handgrip dynamometer and were provided with visual feedback on the computer monitor in the form of a force tracing line . Participants were instructed to sustain a handgrip squeeze for as long as possible that kept their active force tracing line at, or slightly above, the static 30% criterion line. If the force tracing fell below the 30% criterion, participants were instructed to “squeeze harder so the line stays above the marker on the screen.” The trial ended when the active force tracing line fell below the criterion line for longer than 2 s despite corrective feedback or when participants voluntarily stopped. The experimenter followed a script and no verbal encouragement or motivational feedback was provided at any time. Participants had no knowledge of the magnitude of force generation or elapsed time during the endurance trials.I am confident that I can hold the handgrip for…”. The individual items represented gradations of performance that were relative to the participant’s performance on the previous trial. The scale began at (1) “Almost as long as last time” followed by (2) “As long as last time,” (3) “A little bit longer than last time,” and (4) “A lot longer than last time.” Participants rated their confidence for each item using an 11-point, 0  to 10 , scale. The task self-efficacy score was computed by averaging the ratings for each interval score to produce a scale value out of 10. Internal consistency of the scale was acceptable (Cronbach’s α = 0.75).Self-efficacy for task performance on the second endurance trial was assessed using a four-item scale adhering to recommendations by The Bruininks-Oseretsky Test of Motor Proficiency, 2nd Edition—Brief Form BOT-2,  was usedphysical exertion (RPE) using Borg’s CR-10 scale  to 12 (absolute maximum), with 10 (extremely strong) representing the highest physical exertion they had ever experienced.Following each endurance handgrip trial, participants rated their perceived 10 scale  in ordermental exertion (RPME) using an adapted version of nothing at all) to 12 (absolute maximum), with 10 (extremely strong) representing the highest mental exertion they had ever experienced. Numerous studies have used this version of Following each endurance trial and the Stroop task, participants rated their perceived Standing long jump, grip strength (MVC), and the 50-foot shuttle run (taken from the BOT-2 Complete form) served as indicators of muscular strength . Body coNever) to 3 (Often). An example item is: “Does not think before doing (is impulsive).” BRIEF2 scores were calculated for three indexes—the Behavior Regulation Index (BRI), the Emotion Regulation Index (ERI), and the Cognitive Regulation Index (CRI)—and an overall summary score, the Global Executive Composite (GEC). Scores were converted to standardized scores  to control for age and sex. The measure demonstrated excellent internal consistency (α = 0.95).The Behavior Rating Inventory of Executive Function, Second Edition (BRIEF2)—Parent Form  was usednot at all true) to 7 (very true). Each item was prefaced with the following stem “For the handgrip squeezing task I am about to do.” An example item is: “I am going to put a lot of effort into this.” Internal consistency estimates for the pre-task scales were good (α’s > 0.83).Motivation for performing the handgrip endurance task was assessed using two measures. Immediately prior to each endurance trial, participants completed the five-item effort and importance subscale from the Intrinsic Motivation Inventory IMI; . The effnot at all true) to 7 (very true). An example item is: “I enjoyed doing the task very much.” The questionnaire has four subscales: interest/enjoyment, perceived choice, perceived competence, and pressure/tension. The measure demonstrated good internal consistency (α = 0.81).Following the completion of the second endurance trial, participants completed the IMI Task Evaluation Questionnaire . The TasAn alternative account to the Strength Model  suggestsVery Bad) to +5 (Very Good). The Feeling Scale was originally developed to assess affect during exercise, but was adapted to assess affect immediately following physical  and cognitive  task performance. Participants were asked to verbally report a number corresponding to their current feeling state.Following each endurance trial and the Stroop task, participants rated their affective valence using an adapted version of at this momenthow tired or energized you feel ” by drawing a vertical line through a 100-mm line with the anchors ranging from “Extremely Tired” on the left hand side corresponding with 0, and “Extremely Energized” on the right hand side corresponding with 100. Scores were calculated from 0 to 100 by measuring the distance in millimeters the participants’ vertical line was placed from the left side of the scale.Following each endurance trial and the Stroop task, participants rated their perceptions of fatigue using a Visual Analogue Scale . ParticiFigure 1. Prior to taking part in the study, parents of the participants provided informed consent and completed the BRIEF2. Upon entering the lab, assent was obtained and the parameters of the study were explained. The BOT-2 was then administered, followed by two standing long jumps and then two 50-foot shuttle runs. Participants were then introduced to the handgrip task and completed two 100% MVC handgrip squeezes separated by 2 min of rest. Following the MVCs, the experimenter setup up the feedback monitor with a static horizontal red line showing the 30% MVC target value and provided a 10-s demonstration of the task. Participants then performed a 10-s practice trial to familiarize themselves with the task.The study protocol and timeline is presented in resist the temptation to quit. After finishing the first endurance trial there was a 2-min interval period before the Stroop task and participants were asked to provide ratings on the fatigue scale, RPE scale, RPME scale, and the Feeling Scale. They were then randomized (stratified by sex), using a random number generator, to either the HCC or LCC conditions.There was a 2-min rest period following the practice trial in which participants completed the pre-task effort and importance IMI subscale. Participants then completed the first endurance trial with the instructions to hold the contraction for as long as possible and Participants completed the congruent version of the Stroop task for 1 min and then provided a rating on the RPME scale. Participants then completed their respective experimental manipulation task for 5 min (incongruent Stroop task or congruent Stroop task) after which they provided ratings on the fatigue scale, RPME scale, and the Feeling Scale. There was then 5 min of rest in which all of the participants completed the task self-efficacy rating scale and the pre-task effort and importance IMI scale. Participants then completed the second endurance trial with the same instructions as the first. After finishing the trial, participants provided ratings on the fatigue scale, RPE scale, RPME scale, and the Feeling Scale. They then completed the IMI Task Evaluation Questionnaire prior to being debriefed and remunerated $15 (Indigo gift card). The total time to complete the study was approximately 53 min per participant.All statistical analyses were conducted using SPSS 25. Descriptive statistics were computed for all study variables. Separate one-way ANOVA models were computed to assess differences in means between conditions for age, potential covariates, manipulation checks (Stroop task performance and RPME scores), and ratings of perceived exertion (RPE and RPME) for exercise performance. The main hypotheses predicting between-group differences in self-efficacy and exercise performance (time to failure change) were evaluated using one-way ANOVAs.Residualized change scores for time to failure were also analyzed. Residualized change scores have been used in addition to raw change scores to determine endurance performance effects following HCC exertion e.g.,  and werePROCESS software macro (p < 0.05) indirect (mediation) effect. Effect size for mediation is reported as the completely standardized indirect effect (csie) as it can “generally be used in other situations where it is important to compare indirect effects”  effects were assessed using Model 4 in the re macro . As recoThe secondary hypothesis, investigating whether the change in endurance exercise performance  following cognitive control exertion is influenced by motor coordination, was evaluated using 2 (LCC/HCC) × 2 (LMC/HMC) univariate factorial ANOVAs. A median split was performed on motor coordination scores  to produce a dichotomized variable representing LMC and HMC. The median value was 48 which resulted in eight participants being removed to produce variables representing LMC and HMC at each level of cognitive control. Effect sizes for the 2 × 2 ANOVAs are reported as partial eta squared Table 1. Analyses revealed no significant differences (p > 0.05) between conditions. As previously mentioned, there has been considerable debate regarding the processes that are attributed to negative changes in performance following HCC exertion , completed fewer trials , and made more errors  on the incongruent version of the Stroop task compared to the LCC group.Descriptive statistics summarizing Stroop task RPME and performance scores are shown, by group, in Table 2. RPE and RPME scores not differ between the groups for either Trial 1 or Trial 2 , indicating participants in both groups reached equivalent levels of mental and physical exertion on both Trials.Descriptive statistics summarizing RPE and RPME scores for exercise performance on Trial 1 and Trial 2 are shown, by group, in Table 2. As seen in the table, there was a 9.78 s reduction in time to failure in the HCC group compared to an 8.40 s increase in the control (LCC) group. A one-way ANOVA revealed significant differences between conditions for both raw  and residualized change scores . Although Trial 1 times were significantly different and not statistically controlled for in the raw change analyses, the residualized change scores are adjusted scores that control for Trial 1 performance in a manner equivalent to ANCOVA  regardless of condition. However, it is important to note that, residualized change scores are arbitrary values and cannot be interpreted as absolute change in performance .Descriptive statistics summarizing the Trial 1 to Trial 2 raw and residualized endurance handgrip scores are presented in Table 2. As predicted, the HCC group reported lower self-efficacy for the second trial compared to controls . To evaluate the hypothesis that task self-efficacy mediates the effect of cognitive control exertion on exercise performance, indirect effects analyses were computed separately for raw time to failure change and residualized change scores. Preliminary examination of the correlation coefficients (Pearson’s r) between experimental conditions, time to failure change, and task self-efficacy showed significant (p < 0.01) bivariate relationships between all of the variables (see Table 3).Descriptive statistics summarizing the task self-efficacy scores are also presented in Figures 3), performance change score served as the dependent variable with experimental condition (LCC/HCC) specified as the independent variable and task self-efficacy as the mediator. Results indicated a significant indirect (mediation) effect of task self-efficacy for both raw time to failure change  as well as residualized change scores .In both mediation analyses  × 2 (LMC/HMC) univariate ANOVA for raw time to failure change scores showed an overall main effect for condition . As predicted, following HCC, levels of motor coordination had a large effect  on residualized change scores as LMC (n = 14) participants experienced greater reductions in residualized change scores when compared to HMC participants (n = 20). Alternatively, following LCC, levels of motor coordination had a small effect  on residualized change between LMC (n = 15) and HMC (n = 14) participants.The 2 × 2 univariate ANOVA for residualized change scores showed significant main effects for condition . However, self-efficacy scores across levels of motor coordination were nearly equal at each level of cognitive control . One interpretation of these findings is that participants higher in motor coordination may have drawn on their greater self-efficacy beliefs to persist longer at the task as it became more difficult to perform. Indeed, based on the change in exercise performance across levels of cognitive control and motor coordination  alongside self-efficacy scores , our findings suggest that self-efficacy beliefs among those high in motor coordination may buffer against the negative aftereffects of HCC.Our secondary (exploratory) analyses also provide insights into the unique relationships between motor coordination, task self-efficacy, and exercise performance following cognitive control exertion. Specifically, we found that self-efficacy mediated the cognitive control—performance relationship. We also found children scoring lower on motor coordination experienced greater declines in exercise performance following HCC exertion compared to those with higher motor coordination  perturbed the connection between brain regions governing cognition and motor behavior in a way that altered the efficiency of the signal. Dopamine is a neurotransmitter known to facilitate the communication between brain regions governing cognition and motor behavior  and attention deficit hyperactivity disorder (ADHD)) where comorbidity between impairments in motor coordination and cognitive control exist. For instance, children with DCD, a disorder that affects fine and gross motor skills, also show deficits in cognitive control . SimilarI believe in you,” “I know you can do this”) may increase task self-efficacy and, in turn, counter the negative aftereffects of HCC. Finally, although discussion pertaining to alterations in brain  may affect self-efficacy for exercise performance following HCC. Emerging research suggests children often rely on feedback from influential others  to gauge their self-efficacy when performing various sport and exercise tasks . Thus, irain see  and muscrain see ,b activaResearch should also examine specific sub regions within areas governing cognition  and motor behavior  using advanced neuroimaging techniques. For instance, research has shown that endurance handgrip squeezing elicits cortical activation within both the rostral prefrontal cortex and primary sensori-motor areas see . ImportaThe present study has provided the first evidence of the negative aftereffects of HCC exertion on self-efficacy and exercise performance in children. Interestingly, the negative aftereffects on exercise performance were the greatest among children scoring lower on motor coordination. These results highlight the unique relationship between brain areas governing cognitive control and motor behavior that have been largely studied separately. Thus, motor coordination provides an exciting new mechanism for the negative aftereffects of HCC that should be studied across a range of cognitive and physical behaviors.JG formulated the research question and study design, and was primarily responsible for data collection, analysis, and writing of the manuscript. YCL assisted with data collection and provided valuable comments during manuscript preparation. SB and JC contributed to study design, data analysis, interpretation of results, and writing of the manuscript. All the authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.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.


Human protein biomarker discovery relies heavily on pre-clinical models, in particular established cell lines and patient-derived xenografts, but confirmation studies in primary tissue are essential to demonstrate clinical relevance. We describe in this study the process that was followed to clinically translate a 5-protein response signature predictive for the activity of an anti-HER3 monoclonal antibody (lumretuzumab) originally measured in fresh frozen xenograft tissue. We detail the development, qualification, and validation of the multiplexed targeted mass spectrometry assay used to assess the signature performance in formalin-fixed, paraffin-embedded human clinical samples collected in a phase Ib trial designed to evaluate lumretuzumab in patients with metastatic breast cancer. We believe that the strategy delineated here provides a path forward to avoid the time- and cost-consuming step of having to develop immunological reagents against unproven targets. We expect that mass spectrometry-based platforms may become part of a rational process to rapidly test and qualify large number of candidate biomarkers to identify the few that stand a chance for further development and validation. In the last few years, pre-clinical research has gradually shifted from studying cell lines to patient-derived xenografts (PDX) as source of biomarkers, in particular in the oncology arena –3. In brAn attractive aspect of PDX models is that they may show comparable responses to the originating tumor, which make them suitable for screening against specific therapeutics  and for In a complementary approach, we recently identified a 5-protein signature predictive for lumretuzumab activity in cell line- and patients-derived xenograft tumors using a global proteomics strategy, sparking great interest whether the signature could be assessed and potentially confirmed in a breast cancer clinical trial. However, the signature needed to be translated from a fresh-frozen xenograft tissue (where it was discovered) to formalin-fixed tumor tissue  under very tight timelines, requiring a well devised strategy and careful considerations of methods and potential risks with respect to prevalence and modulation of the proteins of interest in the clinical samples.https://clinicaltrials.gov/ct2/show/NCT01918254; [In this study, we describe the development, qualification, and validation of a multiplexed targeted mass spectrometric (MS) assay to measure a response prediction protein signature in human clinical samples collected in a phase Ib trial . We firet al. [18 A.Q. reverse phase material (Dr. Maisch)) using 130 min gradients at a flow rate of 200 nL/min. The mass spectrometer was operated in a data-dependent acquisition mode to automatically switch between full scans in the Orbitrap mass analyzer  and the acquisition of CID fragmentation spectra  in the linear ion trap (LTQ). Protein identification and relative quantification to the internal SILAC-labelled proteins was performed essentially as described in [http://www.uniprot.org). Data were searched with a mass tolerance of 20 ppm for parent ions and 0.5 Th for fragment ions with methionines , lysines (labelled: +8.0142 Da) and arginines (labelled: +10.00827 Da) considered as differential modifications and cysteines considered as fully carbamidomethylated (+57.0199 Da). Peptides and proteins identifications were filtered at a False Discovery Rate (FDR) ≤1%.The discovery proteomics effort described here is performed similarly to the strategy described in Geiger et al.  except tribed in  using Mahttps://skyline.ms/project/home/software/Skyline/begin.view). SRM assays were qualified by generating reversed standard curves for the 17 peptides belonging to the 5 proteins of interest to define assay limit of detection (LOD) and lower limit of quantification (LLOQ). For each analyte, 1.5 amol—100 fmol (12 dilution points) of the synthetic heavy peptide were spiked into a pooled digest of the xenograft matrix containing 50 fmol of the light label peptide. LOD was determined by identifying the lowest concentration in the standard curve for which relative error and accuracy were lower than 25%. LLOQ was set as 3-fold LOD. Quantitation of the endogenous analytes was determined using the median of all measured transitions.Targeted quantitative analysis of the 5 putative protein candidates was performed using the same protein lysates as described above. A total of 0.5–2 μg protein digest was analyzed in triplicate on a HPLC system using a 75 μm inner diameter x 15 cm length chromatographic column packed with C18 resin  coupled to a triple quadrupole mass spectrometer . The mass spectrometer was operated in SRM mode using a total cycle time of 1 s and the two quadrupoles 1 and 3 were set at a width of 0.7 Th . For proThe SRM method development and qualification was performed in commercially acquired Breast Cancer and Non Small Cell Lung Cancer human tissue samples and the xenograft models  mentioned in the previous section.https://clinicaltrials.gov/ct2/show/NCT01918254; [The tumor biopsy samples investigated in this study were obtained from patients with HER3-positive, HER2-non-amplified metastatic breast cancer enrolled in the clinical trial BP27852, an open-label, multicenter, dose-escalation study to evaluate the safety, pharmacokinetics and activity of lumretuzumab 1918254; ). Local 18254; . 1918254; .Fit-for-purpose assay development and qualification for the 5 proteins constituting the response prediction signature was performed essentially as previously described . BrieflyA total of 1 μg protein digest was analyzed in triplicate on a nanoACQUITY liquid chromatography system  using a 100 μm inner diameter x 15 cm length chromatographic column packed with C18 resin  coupled to a triple quadrupole mass spectrometer . The mass spectrometer was operated in SRM mode using a total cycle time of 1 s and the two quadrupoles 1 and 3 were set at a width of 0.7 Th . For proP. furiosus protein matrix  containing 5 fmol of the heavy label peptide. LOD was determined by identifying the lowest concentration in the standard curve for which coefficient of variation (CV) and accuracy were lower than 20%, respectively better than 80%. LLOQ was determined by identifying the next highest concentration of the standard curve above the LOD. Quantitation of the endogenous analyte was determined using all available product ions.SRM assays were qualified by generating standard curves for the 14 peptides belonging to the 5 proteins of interest to define assay limit of detection (LOD) and lower limit of quantification (LLOQ). For each analyte, 25 amol—25 fmol (14 dilution points) of the synthetic light peptide were spiked into a SRM measurements for EGFR, HER2, and HER3 were performed concomitantly as described previously .Missing values for peptides that were measured below LLOQ (as determined by SRM) were imputed using the following procedure. Peptides measured with a concentration below LLOQ were assigned a random concentration drawn from a uniform distribution over a range from 0 to LLOQ amol/μg lysate. If more than one peptide value from the same protein was missing per measurement, an additional draw was then performed from a normal distribution, using the previously drawn random number as mean and a CV of 25%. This step was used to generate a variation assumed to be realistic for data at such low concentrations.http://www.jmp.com).All plots presented in this study have been generated using the JMP program version 10.0.1  or the Wilcoxon test (on median) was performed considering only protein groups with at least 75% of non-missing values per group. 12 proteins fulfilled a (predefined) absolute effect size criterion of >3-fold but without reaching significance (predefined as <0.05 FDR). In a second step, a response prediction signature was established using a support vector machine-based classifier. Feature selection was performed to select a sparse protein signature reaching predefined criteria of 95% sensitivity and 90% specificity. The resulting 5-feature signature  fulfilled the pre-set selection criteria, with median effect size greater than 1 (1.07–1.92) and a cross-validation accuracy of 99% . A prediThe 5-protein response prediction signature was then transferred onto a targeted MS platform to enable quantitative protein measurement from unfractionated lysates. A training set consisting of 9 responder and 17 non-responder xenograft models was used to re-train the classifier based on a multiplexed targeted SRM approach using the 3 best peptides of each of the target proteins as protein quantification reporters . Performper se not tissue- or peptide-dependent, prevalence of the proteins of interest in the target tumor tissue needed to be determined to ensure the feasibility of the approach. Also, the selection of the SRM reporter peptides required careful review as they might not perform equally well in fresh frozen and formalin-fixed tissue sources [The objective of this study consisted in translating a discovery, 5-plex SRM assay developed in fresh frozen xenograft tumor into a qualified assay for analyzing FFPE primary human tumors collected in a phase Ib clinical trial. While the mass spectrometric process is  sources . Finallyin silico digest of the 5 proteins and transition information was converted to a prototype SRM assay and confirmed by analyzing the recombinant protein digest by LC-MS. Each protein’s best 5 to 9 detected peptides were then assessed in 17 cell lines (various lineages) treated with formalin and in 10 Non Small Cell lung cancer and 10 breast cancer FFPE tissue samples obtained from commercial sources  were selected from an  sources . While s sources ; the selThe second step of the assay development focused on demonstrating that the response prediction signature was independent of the matrix and could be reproduced in the FFPE xenografts. For this purpose, 10 responder and 19 non-responder formalin-fixed xenograft samples  were selected to confirm the response prediction algorithm. Three sections of 10 μm thickness were cut from each block and the tumor area was isolated using laser-capture microdissection. The relative levels of the 5 proteins were measured against the spiked internal stable isotope labelled peptides using the prototype SRM assay. Similary to previous results in fresh frozen tumors, the 5 proteins were not consistently detected in all samples . HoweverP. furiosus lysate spanning a calibration range from 25 amol to 25 fmol peptide injected in 1 μg total lysate  of each protein were qualified with respect to linearity and lower quantification limits in a l lysate . For mosl lysate . The re-A total of 32 individual breast cancer tumor biopsies  were suThe suitability of the collected samples set for a MS-based approach was assessed by measuring the expression level of EGFR, HER2, and HER3 using established SRM assays as described previously . The resThe 5 proteins included in the response prediction signature were measured concomitantly in the SRM assay performed for each patient’s sample. Similar to what was observed in the assay development stages, DPYSL2, OAT and GM2A were measured and quantified in all samples while PADI and CLIC3 were most of the time below the assay’s sensitivity threshold. Consistency between peptides for each given protein was excellent (median %CV = 9.4), with a correlation between peptide concentrations that was always ≥ 0.9, indicating that the reported peptide levels were an appropriate surrogate for global protein concentration in a sample.The re-qualified response prediction signature in FFPE tissue was assessed in the donors’ samples and compared to their respective RECIST response status. Similar to what was implemented in the assay development phase, the missing PADI and CLIC3 quantification values were assumed to be informative missing (below LLOQ) and were imputed as described previously. The results for the 5-protein  and for the 3-protein  signatures are shown in In a heterogeneous disease such as breast cancer, histopathology-based factors such as tumor size, grade, nodal status, hormonal and HER2 receptor status are commonly used to guide treatment. However, patients with similar disease characteristics may still experience different outcomes, stressing the need for additional predictive, diagnostic and prognostic biomarkers (reviewed in ). In recIn this study, a targeted MS multiplexed approach was facilitated by the initial work conducted in the proteomics discovery step and the following SRM assay build performed in fresh frozen xenografts. Assay development and qualification in FFPE human tissue samples included re-qualification of the reporter peptides, prevalence analysis, qualification of the assay, and confirmation of the response prediction algorithm. We took advantage of the multiplexing capability of the assay including reporter peptides specific for EGFR, HER2 and HER3 to confirm overall suitability of the methodology by comparing the levels of these proteins with the IHC analysis in the same samples. The full assay transfer, development, and qualification was achieved within a 7 months period, a record time compared to other protein-centric technology platform, and suitable performance was demonstrated in the matrix of relevance confirming its classification as a Tier two “fit-for-purpose” assay . The conA discrepancy between pre-clinical models and the outcome in this study may be due to several factors, including biological (lineage) differences between xenografts, and primary human tumors. Also, the response signature was geared towards prediction of a monotherapy mode of action, which did not match the clinical situation where lumretuzumab was given in combination with paclitaxel and pertuzumab. Overall, it also can be argued that the confirmation of the response prediction signature  was hampered by the low number of responders observed in the trial, tumor heterogeneity, and by the challenge to translate findings obtained with single agent usage to a combination treatment setting in a clinical trial. However, most importantly, the low number of samples that could be effectively measured in the assay combined with a considerably larger biological heterogeneity than observed in the xenografts impaired an unbiased evaluation of the response signature. Residual material from pre-dose FFPE tumors was not available in sufficient amount due to high demand in tumor tissue for numerous testing or too heterogeneous to be dissected out using laser capture microdissection.In this study, we have demonstrated one possible avenue on how to enable a large number of biomarkers to be tested in clinical settings and to rapidly assess their intrinsic value. In particular, an innovative and rapid development of a targeted mass spectrometric-based assay to assess a response prediction signature was highly warranted as neither baseline HER2 or HER3 protein expression (as measured by IHC) nor Heregulin mRNA expression (as measured by PCR) were associated with clinical response to lumretuzumab in Breast Cancer . As poinS1 Fig(PPTX)Click here for additional data file.S2 Fig(PPTX)Click here for additional data file.S3 Fig(PPTX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.


Inhaled nitric oxide (iNO) is a selective pulmonary vasodilator with limited indications in adults. We present a patient with hypoxemia and right ventricular dysfunction due to submassive acute pulmonary emboli where iNO was used as a bridge to thrombolysis. A 29-year-old male was admitted to the intensive care unit (ICU) for alcohol intoxication complicated with aspiration pneumonia and acute respiratory failure requiring mechanical ventilation. His medical history included morbid obesity (BMI 43) and alcohol dependence syndrome. Nine days after admission, he developed severe acute hypoxia and tachycardia with arterial oxygen tension (PaO2) of 52 mmHg requiring a positive end-expiratory pressure (PEEP) of 14 cmH2O and fraction of inspired oxygen (FiO2) of 1. Chest computed tomography (CT) revealed a large embolus in the right main pulmonary artery and transthoracic echocardiogram (TTE) reported new right ventricular dilatation with decreased right ventricular function. Due to the severe hypoxemia, he was started on iNO via the breathing circuit of the ventilator at a concentration of 20 parts per million (ppm) with steady improvement in oxygenation after 1 hour with a PaO2 of 81 mmHg on the same ventilator setting. The patient was given thrombolysis with alteplase and the iNO was slowly tapered off during the subsequent four days with concomitant successful tapering of PEEP to 8 cmH2O and FiO2 of 0.45. Inhaled NO has been used to improve ventilation-perfusion matching and also to reduce pulmonary vascular resistance (PVR). Its effects on PVR may be useful in the setting of acute pulmonary emboli where vascular obstruction and vasoconstriction contribute to increased pulmonary arterial pressure and PVR which can present as acute right heart failure. We suggest that iNO, if available, could be considered in those patients with acute pulmonary emboli associated with significant hypoxemia as an adjunctive therapy or bridge to thrombolysis or thrombectomy. Nitric oxide (NO) is a naturally occurring gas, and at room temperature, it is both colorless and odorless, and when added to an inspired gas, it produces selective pulmonary vasodilation which improves pulmonary hypertension and decreases right ventricular (RV) afterload , 2. This2 and alcohol dependence syndrome.A 29-year-old male was admitted to the intensive care unit with acute respiratory failure requiring intubation in the field. The patient was found unresponsive on the floor of his apartment. His medical history included morbid obesity with a body mass index of 43 kg/mOn initial examination he was sedated on mechanical ventilation, afebrile (98 F), hypertensive with a blood pressure of 167/69 mmHg, tachycardic with heart rate of 111 beats per minute and a respiratory rate of 20 breaths per minute synchronous to the set rate on the ventilator. Peripheral capillary oxygen saturation (SPO2) was 98% with inspired oxygen saturation (FiO2) of 0.5. Lung examination revealed rales in the mid and lower zones of the right lung field. Cardiac examination revealed tachycardia with no murmurs. Abdominal, neurologic and skin exams were unremarkable.3 cells/ /μL), serum creatinine of 2.1 mg/dL (baseline 0.6 mg/dL), serum creatine kinase of 39, 552 units/L, serum lactic acid of 4.2 mmoles/L, and serum ethanol level of 316 mg/dL. Initial arterial blood gas analysis revealed a pH of 7.26, an arterial carbon dioxide (PCO2) level of 40.2 mmHg and oxygen level (PO2) of 108 mmHg on a FiO2 of 0.5, and positive end-expiratory pressure (PEEP) of 5. Chest X-ray (CXR) showed right upper, middle, and lower lobe infiltrates and computed tomography (CT) of the head demonstrated no acute infarcts or hemorrhage.Relevant laboratory on admission included the following: leukocytosis  on day 2 revealed an ejection fraction of 57% with normal right ventricular structure and function and normal pulmonary artery systolic pressure (PASP). The patient clinically and radiologically improved, he was awake and tolerating daily spontaneous breathing trials with FiO2 requirements of 0.4 to keep PO2 of 104.On day nine of admission, he was noted to be hypoxic with a PO2 of 52 mmHg with a SPO2 of 88%. He required a PEEP of 14 cmH20 and a FiO2 of 1 to achieve a PO2 of 66 mmHg. Due to body habitus prone position was not an option. Repeated CXR did not show any new abnormalities- resolving right-side infiltrates. In addition to the subcutaneous heparin started on admission for deep venous thrombosis (DVT) prophylaxis, a therapeutic dose of low molecular weight heparin (LMWH) at 1 mg/kg was administered with a presumptive diagnosis of acute pulmonary emboli as he was too unstable for transportation to radiology. A repeat TTE showed a PASP of 54 mmHg with new moderate dilatation of the RV and moderate tricuspid regurgitation  with a sChest CT angiogram revealed large embolus in the right main pulmonary artery . ThromboNitric oxide (NO) is a naturally occurring gas and at room temperature it is both colorless and odorless. It is produced endogenously from L-arginine by NO synthase and when added to an inspired gas, it produces selective pulmonary vasodilation. It activates guanylate cyclase which then activates cyclic guanosine monophosphate (cGMP). Cyclic GMP stimulates cGMP-dependent protein kinase that activates myosin light chain phosphatase, which dephosphorylates myosin light chains, leading to smooth muscle relaxation. Additionally, increased intracellular cGMP inhibits calcium entry into the cell, and decreases intracellular calcium concentrations further promoting relaxation of vascular smooth muscle . In the There are limited therapeutic indications for iNO in adults. Currently in the United States, it is licensed only for neonates with respiratory failure and persistent pulmonary hypertension. In adults, it has an established diagnostic role in vasodilator testing in patients with pulmonary arterial hypertension. iNO has been studied in patients with acute respiratory distress syndrome (ARDS) but with conflicting results. A meta-analysis by Sokol et al. reviewed five randomized control trials with a combined total of 535 patients with acute hypoxemic respiratory failure and found no significant effect in mortality with only transient improvement in oxygenation . The useThe estimated incidence of PE in the United States in one analysis looking at patients from 1998-2006 was 112 per 100, 000 adults with a 7.8% mortality rate . A suddeAssessing RV function is essential in the risk stratification of acute PE. In our case, we highlighted acute right ventricular dilatation with moderate tricuspid regurgitation. An emerging echocardiographic marker for patients with intermediate risk PE is the ratio of tricuspid regurgitation peak gradient (TRPG) to tricuspid annulus plane systolic excursion (TAPSE) or TRPG/TAPSE. A TAPSE of < 17 mmHg is suggestive of RV systolic dysfunction but when interpreted as a ratio to TRPG, a group of Polish researchers were able to demonstrate that a TRPG/TAPSE > 4.5 was associated with 21.1% risk of PE-related death or rescue thrombolysis . This ecThere are case reports of successful use of iNO as a therapeutic agent in patients with acute pulmonary embolism (PE). Inhaled nitric oxide may have a unique role as an adjunct to existing standard of care including anticoagulation, thrombolytics and surgery in the patient with acute PE and severe hypoxia, acute RV failure or hemodynamic instability. Its role as a selective pulmonary vasodilator may reduce RV afterload without causing systemic vasodilation which is beneficial in these critically ill patients. There are case reports of successful use of iNO as a therapeutic agent in patients with acute pulmonary embolism (PE) . A revieOur patient has similar characteristics to patients described in other reports with refractory hypoxemia and evidence of acute RV failure. The real-world challenge for the clinician practicing in a setting similar to ours, without on-site ECMO or advanced thoracic-vascular surgery, is to be able to stabilize the patient in order for safe transportation for diagnostic or therapeutic purposes. At our institution, iNO is available for use in neonates. We extrapolated data from pediatrics and from reported use in similar cases of intermediate risk PE with RV strain in our decision to use iNO and at what dose. There has been significant interest regarding the potential therapeutic use of iNO in PE with the first phase II double-blinded, randomized trial of iNO to treat acute PE recently completed by Kline et al. with the final analysis to be reported .Inhaled epoprostenol (iEPO) is another inhaled pulmonary vasodilator that like iNO has been used in patients with ARDS and refractory hypoxemia. It functions like iNO, causing selective pulmonary vasodilation when added to an inspired gas. Webb et al. described a case in 1996 where its use in a patient with massive PE resulted in a transient improvement in mean pulmonary arterial pressures but did not alter the clinical deterioration of the described patient . While tOur patient highlights the challenges in the evaluation and management of critically ill patients with acute pulmonary emboli and severe hypoxia and right-side failure.The use of iNO likely plays a role in the initial stabilization of these patients allowing time for more definitive therapy and may even decrease the need for more invasive therapeutic modalities. These potential benefits will need to be assessed in large prospective trials.


Multimorbidity is prevalent and adversely affects health outcomes. Foot pain is common and one of the primary reasons for utilisation of podiatry services. At present, little is known about the impact of multimorbidity on foot health and related outcomes following podiatric intervention. The aims of this study were to evaluate whether there is a difference in foot health outcomes following exposure to podiatric foot care for people with and without multimorbidity; and ii) to evaluate whether the presence or absence of multimorbidity affects patients’ perceptions of change in foot pain.The PROMFoot study is a prospective cohort study of adults with a new episode of foot pain attending the podiatry service within the NHS Greater Glasgow and Clyde health board. Baseline medical comorbidity status , longitudinal data on foot health measured using the Foot Health Status Questionnaire (FHSQ), and patient rating of change scores for foot pain were obtained from the PROMFoot study at baseline, and 3 and 6 months after podiatric intervention. Foot health scores  and perceptions of change for foot pain were compared between comorbidity groups.A total of 115 participants  with a mean age of 55 years were included. Multimorbidity was common, affecting 61 participants (53%); while 28 (24.3%) and 26 (22.6%) reported single or no medical comorbidities respectively. Significantly worse foot health scores for all FHSQ domains were observed for the multimorbidity group at baseline, 3 and 6 months. Change scores for foot pain were similar between groups and demonstrate modest improvements, however multimorbidity group membership was strongly associated with a perceptions of change in foot pain. Multimorbidity was independently associated with poorer foot function outcomes at 3 months, and poorer foot pain and foot function outcomes at 6 months.Multimorbidity was associated with poor foot health outcomes and lower rates of self-perceived improvement in foot pain over 6 months following podiatric intervention in a sample of patients attending podiatric biomechanics clinics for a new episode of foot pain.The online version of this article (10.1186/s13047-019-0346-x) contains supplementary material, which is available to authorized users. Foot pain is reported as common in the general population with prevalence estimates ranging from 17 to 30% , and thePatterns of diseases in multimorbidity can be highly variable as it can occur due to simple co-occurrence by chance, exposure to shared risk factors, and/or a pathogenic link between conditions . For exaAt present, little is known about the impact of multimorbidity on foot health and related outcomes following podiatric intervention. For MSK foot pain, the current literature is dominated by studies which focus on single diseases (such as rheumatoid arthritis) or non-disease-specific risk factors  and their associations with foot health outcomes such as foot pain and/or function , 13. WitParticipants in this study are from the Patient Reported Outcome Measures Foot (PROMfoot) Study. In short, PROMfoot is a longitudinal observational cohort study that was designed to evaluate measurement properties of four different foot-specific and generic patient reported outcome measures (PROMs) including the Foot Health Status Questionnaire (FHSQ), the Foot Function Index, the EuroQoL 5-dimensional questionnaire (EQ5D-5 L), and the Short-form 6-dimension questionnaire. The PROMfoot study was conducted within the National Health Service (NHS) Greater Glasgow and Clyde (GG&C) Health Board region. Patients with a new episode of foot pain who were referred to NHS GG&C Podiatric Biomechanics Clinics were the target population. Referrals to NHS GG&C Podiatric Biomechanics Clinics are largely comprised of patients with complex musculoskeletal foot pathology. Patients are allocated at the discretion of the podiatrist responsible following vetting of written referrals from general practice, orthopaedics, other health professions, or internal escalation following initial podiatry treatment. Patients were eligible to participate if they i) were scheduled to attend as a new patient for treatment at a podiatric biomechanics clinic within NHS GG&C; ii) self-reported current foot pain; iii) were aged 18 years or more; and iv) were willing and able to provide written informed consent. Patients were not eligible to participate if they failed to meet any of the above inclusion criteria. Ethical approval was obtained from the South East Scotland NHS Research Ethics Committee (16/SS/0193) on 2nd November 2016. Recruitment was undertaken between January and December 2017. For the purposes of addressing the aims of this study, longitudinal FHSQ data from PROMfoot are reported.Potentially eligible participants were identified by screening NHS GG&C Podiatric Biomechanics clinic lists using the TrakCare electronic patient management system by an NHS GG&C Podiatrist. Potentially eligible participants were sent an invitation letter and a study participant information sheet, and willing participants were invited to contact the researcher (LF). Telephone-based screening was undertaken to confirm study eligibility and patients’ willingness to participate. Patients who were willing to take part were invited to return signed consent forms to the researcher to confirm their participation in the study and complete baseline assessments via completion of a postal or web-based survey, according to their personal preference. Enrolment was confirmed upon receipt of written consent and completed baseline data.Study data were collected and managed primarily using the Research Electronic Data Capture (REDCap) secure electronic data capture tool hosted at Glasgow Caledonian University . Study dn = 0 conditions), 2) single condition (n = 1 condition), and 3) multimorbidity (> 1 condition). Foot pain severity experienced during the previous week was measured as a baseline descriptor using a numerical rating scale (NRS) from 0 to 10 . Regional foot pain location  was evaluated using a foot pain map developed previously and widely used for epidemiological research [Baseline demographic data collected included age (years), sex, employment status , and self-reported height and weight for calculation of body mass index (BMI). Participants’ post codes were collected in order to calculate indices of social deprivation –17. Thesresearch .The primary outcome of interest was foot health at 3 and 6 month follow-ups, which was evaluated using the Foot Health Status Questionnaire (FHSQ), a valid and reliable 13-item questionnaire with four domains including pain, function, footwear, and general foot health , 23. TheFollow-up study data were captured at 3 and 6 months after baseline measurements. Three and 6-month data included the 4 domains of the FHSQ and the 7-point PRCS. Podiatric foot care treatments received and self-care undertaken over the 6-month study period were recorded for each participant using a standard check-box list  and free text box for other treatments.Demographic and general health characteristics were summarized using descriptive statistics. Continuous data were screened for normality of distributions using the Kolmogorov-Smirnov test. Continuous variables were summarised using means and standard deviations (SD), and medians and inter-quartile ranges (IQR) where appropriate. For categorical and ordinal data, proportions were calculated and expressed as absolute frequencies (n) and percentages (%). Ordinal data from the PRCS were dichotomised and recoded as a categorical variable (improvement versus no change/deterioration). Differences in demographic and general health characteristics between groups were evaluated using Kruskal-Wallis and Mann-Whitney post hoc tests where appropriate.p values < 0.05 were considered to indicate statistical significance. All analyses were undertaken using IBM® SPSS® version 25.Differences between groups for discrete FHSQ domain scores at baseline, 3 and 6 months, and change scores between baseline, 3 and 6 months were evaluated using Kruskal-Wallis tests and Mann-Whitney post hoc tests where appropriate. To identify associations between multimorbidity status and participants’ self-perception of change in foot pain (dichotomised PRCS), Pearson’s chi-square tests were performed and presented as 3 × 2 contingency tables. Chi square test effect sizes were calculated using Cramer’s V. Multivariate linear regression analyses were undertaken to identify associations between multimorbidity status and FHSQ domain scores at 3 and 6 months with and without adjustment for covariates. All tests were two-tailed and Given the prospective nature of the study, some loss to follow-up was anticipated. Missing data were accounted for using 3 approaches. Secondary missing data analyses included 1) comparison of baseline demographic characteristics between the complete (referred to as closed cohort) and incomplete cohorts (open cohort); 2) graphical explorations of primary outcome data at discrete time points and change scores over time for closed and open cohorts; and 3) sensitivity analyses with repetition of primary and secondary outcome data analyses for the closed cohort . Of these, 154 (79.8%) were eligible, and of these 115 (74.7%) were enrolled  reported having no medical condition, while 28 (24.3%) and 61 (53%) reported having a single or multiple conditions respectively  of 6/10 (4) Table . Foot pap = 0.099 not significant (NS), BMI p = 0.041) and single condition group   and single conditions groups   Table .Baseline comparisons of demographic and clinical characteristics between the closed versus open cohort suggest cohorts were similar in terms of age, sex, BMI, HRQoL and employment status . At 6 months, FHSQ pain scores were significantly worse in the multimorbidity versus no conditions group (p < 0.01), and FHSQ footwear scores were significantly worse in the multimorbidity versus single conditions group (p < 0.01). Statistically significant differences were observed between groups for FHSQ general foot health at baseline, 3 months and 6 months, with the multimorbidity group having significantly worse scores than the no conditions (p < 0.01) and single conditions group (p < 0.05) at baseline, and the no conditions group at 3 and 6 months (both p < 0.05).Between group comparisons for foot health at discrete time points  are presented in Table p > 0.05). Modest improvements in FHSQ pain were observed for each group between 0 and 3 and 0–6 months. Modest improvements in FHSQ function were observed for no conditions and single condition groups only. Median scores of 0 suggest that FHSQ general foot health did not improve for any group. The FHSQ footwear domain scores improved between 0 and 6 months for the single condition group only.Between group changes scores are presented in Table p < 0.01), function (p < 0.01) and general health (p < 0.04) at 3 and 6 month follow ups (Table p = 0.01) at 3 months, and FHSQ pain (p = 0.02) and FHSQ function (p < 0.01) at 6 months  scores for FHSQ pain , 6.61, p = 0.037, medium effect size) and 0–6 months (X2 (2), 6.90, p = 0.032, medium-to-large effect size).Group comparisons for PRCS scores between 0 and 3 and 0–6 months are presented in Table p < 0.05), FHSQ pain at 6 months , and FHSQ general foot health at baseline . Analyses of change scores were consistent between closed and open cohorts with the exception of FHSQ footwear, which was significantly worse in the multimorbidity group versus no conditions group for the closed cohort. In spite of similarly high proportions of multimorbidity group reporting no improvement relative to no conditions and single condition groups within the closed cohort, associations between group membership and perceptions of change in foot pain were not statistically significant.Closed versus open cohort sensitivity analyses are presented in Additional files The results of this study demonstrate that people with multimorbidity had significantly poorer foot health for each domain of the FHSQ than people without, both before and after podiatric intervention. When adjusted for corresponding baseline domain FHSQ score, age and BMI, multimorbidity was independently associated with worse FHSQ outcomes for foot function at 3 months, and foot pain, foot function and footwear at 6 months. The multimorbidity group did not differ significantly from comparator groups for FHSQ domain change scores following intervention. Only modest improvements were observed for each group for the foot pain domain, with improvements in foot function observed in the no conditions and single conditions groups only. These improvements generally approached published minimal important difference (MID) values for the FHSQ . HoweverThere are several potential explanations for the persistent poor foot health observed in the multimorbidity group. Whilst we acknowledge that evaluation impact of specific patterns or clusters of multimorbidity on foot health were outwith the scope of the current study, we can tentatively deduce that multimorbidity may have involved the most prevalent and limiting conditions reported namely depression, osteoarthritis and back pain. These conditions have been identified previously as correlates of foot pain , 26, 27 Healthcare inequalities have been identified in the primary care management of patients with multimorbidity that include insufficient appointment times, higher stress levels amongst general practitioners (GP), and lower levels of patient enablement; defined as their ability to cope with and self-manage their conditions . To addrThe importance of self-management has been highlighted recently as a key strategy for meeting the needs of people living with multiple long-term conditions . The goaPodiatric treatments provided over the course of the study period most frequently involved provision of foot orthoses, footwear advice and exercises, which are largely consistent with a priori expectations of United Kingdom (UK) NHS MSK podiatry services. The results of this study raise questions concerning whether or not sufficient foot health benefits can be achieved in people who suffer from multimorbidity via interventions designed to address presenting foot pain complaints in isolation. The range of medical conditions observed in this study suggest that significant proportions of patients attending podiatric biomechanics services present with complex medical problems and poor general and foot health. Primary focus on foot and ankle biomechanics without consideration of other aspects of holistic management such as promotion of self-management techniques for broader health concerns may at least in part explain the poor outcomes observed. However cautious interpretation is warranted as details of treatments received were self-reported by participants as opposed to clinician-report or case note review which may have been more accurate. Nevertheless, recent primary care research has identified educational needs amongst GPs working with multimorbid patients including: - how to address low patient engagement with their health care and low health literacy . As suchThere are some limitations to this study that warrant further attention. Whilst the overall sample size was sufficient, the initial response rate to study invitations was lower than anticipated and as such cautious interpretation is warranted due to potential non-response bias. The implications of this are that those who responded (and those who were eligible and were subsequently enrolled) are not necessarily representative of the invited population who were eligible. It is acknowledged that not all of the 1329 patients invited would have been eligible to participate. Of those who were eligible, the recruitment rate was high at 74.7%. Whilst attrition rates were generally acceptable at less than 20% at each follow-up, some participant data sets were subject to sporadic missing data. However, loss to follow-up and missing data were accounted for collectively via 3 separate analysis techniques which suggested only modest differences between closed (full data) and open cohorts (incomplete data) and thus selection bias was concluded to be minimal. Another strength of this study was that participants were recruited from the routine NHS Podiatric Biomechanics setting by an NHS-employed podiatrist outwith the study team, and as such vulnerability to recruitment bias was minimised. Data were obtained from the NHS GG&C Health Board Region and we acknowledge that the socioeconomic composition of this region differs to that of the rest of Scotland and the UK, with higher levels of deprivation, poor health behaviours, and lower life expectancy –38. ThisMultimorbidity was associated with poor foot health and impacts negatively on foot health outcomes following podiatric intervention in a sample of patients attending podiatric biomechanics clinics for a new episode of foot pain. Multimorbidity was independently associated with poorer foot pain and foot function outcomes. Multimorbidity was also associated with lower rates of perceived improvement in foot pain following podiatric intervention. Current MSK foot care may be suboptimal and there are likely important training needs for podiatrists working with multimorbid patients. Further research is required to confirm associations between multimorbidity and poor foot health.Additional file 1: Table of demographic characteristics of the whole sample versus the closed cohort. Table comparing demographic characteristics between the whole sample which includes some missing data, and the closed cohort with complete data at each time point. (DOCX 12 kb)Additional file 2:Table of FHSQ domain scores for the closed cohort. Table displays FHSQ domain scores at each study time point and results of between group sensitivity analysis. (DOCX 12 kb)Additional file 3:Table of FHSQ domain change scores scores for the closed cohort. Table displays FHSQ domain change scores at each study follow-up and results of between group sensitivity analysis. (DOCX 12 kb)Additional file 4:Cross-tabulation contingency table of perceptions of foot pain change according to multimorbidity group. Table displays results of cross-tabulation frequencies and chi-square sensitivity analysis of associations between group membership and perceptions of foot pain change. (DOCX 12 kb)Additional file 5:Unadjusted and adjusted associations between multimorbidity and foot health outcomes. Table displays results of multivariate linear regression analyses for associations between multimorbidity and FHSQ outcomes at 3 and 6 months, which are unadjusted and adjusted for baseline FHSQ domain, age and BMI. (DOCX 14 kb)Additional file 6:Graph of FHSQ domain scores for closed versus open cohorts. Graphical representation of median FHSQ domain scores at each study time point for closed versus open cohorts. (TIF 252 kb)Additional file 7:Graph of FHSQ domain change scores for closed versus open cohorts. Graphical representation of median FHSQ domain change scores at each study follow-up for closed versus open cohorts. (TIF 132 kb)


Atoh1-null mice die at birth from respiratory failure, but the precise cause has remained elusive. Loss of Atoh1 from various components of the respiratory circuitry (e.g. the retrotrapezoid nucleus (RTN)) has so far produced at most 50% neonatal lethality. To identify other Atoh1-lineage neurons that contribute to postnatal survival, we examined parabrachial complex neurons derived from the rostral rhombic lip (rRL) and found that they are activated during respiratory chemochallenges. Atoh1-deletion from the rRL does not affect survival, but causes apneas and respiratory depression during hypoxia, likely due to loss of projections to the preBötzinger Complex and RTN. Atoh1 thus promotes the development of the neural circuits governing hypoxic (rRL) and hypercapnic (RTN) chemoresponses, and combined loss of Atoh1 from these regions causes fully penetrant neonatal lethality. This work underscores the importance of modulating respiratory rhythms in response to chemosensory information during early postnatal life. Breathing seems very simple: humans and other animals do it all the time without even thinking about it. Yet, many different cell types coordinate rhythmic breathing movements. Some cells set the breathing rhythm, motor neurons control the muscles, and other cells sense blood oxygen and carbon dioxide levels. Information about oxygen and carbon dioxide is necessary to trigger faster and deeper breaths when there is too little oxygen, for example, at high altitude. Or when there is too much carbon dioxide, for example, during exercise.At birth, most newborns can breathe as fast as needed because key genes oversee the development of all the cells involved in breathing. Learning more about these genes and what they do could lead to better understanding of why some newborns are at risk for sudden infant death or crib death. The Atoh1 gene, for example, helps carbon dioxide-sensing cells called retrotrapezoid neurons develop. Mice born without the Atoh1 gene are unable to breathe normally and die at birth. But when the gene is only deleted from these carbon dioxide-sensing cells in mice, just half of them die. This suggests that Atoh1 in other cells may also be important for breathing.Now, Van der Heijden and Zoghbi show that the Atoh1 gene also helps develop another set of cells that are essential for breathing called the parabrachial complex. These cells receive information from oxygen sensors and relay the information to cells that set breathing rhythms. Mice missing parabrachial complex cells do not breathe faster when oxygen levels in the air are low. Mice lacking Atoh1 from both the parabrachial complex cells and the retrotrapezoid cells have breathing problems and die at birth.Van der Heijden and Zoghbi show that the Atoh1 gene is essential for two cell types that make mice breathe faster when oxygen or carbon dioxide levels change. Together these two cell types are necessary for survival. The experiments also may provide insights into what goes wrong in babies who experience sudden infant death. Mutations in genes that are important to both cell types increase the risk of these infant deaths. Newborn babies with mutations in such key developmental genes will be at risk when in low oxygen or high carbon dioxide environments because their breathing systems are still maturing. This ability likely arises from a detailed genetic blueprint in the hindbrain respiratory circuit. Indeed, mapping the expression domains of key transcription factors in the developing hindbrain reveals a checkerboard pattern, with rostro-caudal and dorso-ventral stripes crisscrossing the entire region  die at birth of respiratory failure even though they generate some respiratory movements  and the retrotrapezoid nucleus (RTN)   (Huang eus (RTN) . The cheus (RTN) . Atoh1 lethality .Atoh1-lineage neurons contributing to neonatal survival and further delineate Atoh1’s function in respiratory development using intersectional genetics. We found that loss of Atoh1-lineage neurons developing from the rostral rhombic lip (rRL) impairs both respiratory rhythm and chemoresponsiveness. Some of these neurons are specifically activated during respiratory chemoresponses and project to the Atoh1-lineage paramotor nuclei (ITR and RTN) as well as the preBötC. Yet only the combined deletion of Atoh1 from the rRL and RTN recapitulated the fully penetrant lethality of Atoh1-null mice. This confirms that developmentally defined neural lineages have distinct roles in respiratory control and that, in neonatal mice, integration of chemosensory information is essential for survival.We therefore set out to find other Atoh1 is expressed along the entire rostro-caudal rhombic lip of the developing hindbrain , the rostral ventral respiratory group (rVRG) and the lateral reticular nucleus (LRt)  , red (Rous (LRt) . Two par preBötC , blue  and exposed these mice to either room air, hypoxia, or hypercapnia prior to staining for the neural activity marker cFos. We found tdTomato+, cFos+ double-positive cells in the medial and lateral parabrachial region as well as in the Kölliker Fuse after exposure to either hypoxia or hypercapnia, but not room air  and pituitary adenylate cyclase‐activating polypeptide (PACAP), because these peptides have been implicated in playing a role for hypercapnic and hypoxic responses respectively . We crossed Flox/FloxAtoh1 females  were born in Mendelian ratios , probably because the motor phenotypes impair their ability to get proper amounts of food and water. These phenotypes were likely the result of loss of Atoh1-lineage cerebellar neurons including glutamatergic deep cerebellar nuclei and cerebellar granule cells  at three weeks of age . These levels are similar to those observed in infants treated with caffeine , which are the sole output of the cerebellar cortex. PCs receive input from granule cells and directly project onto deep cerebellar nuclei. These Cre/+;Slc32a1Flox/FloxPcp2 mice have no defects in cell types other than PCs, and loss of PC signaling results in abnormal motor control including ataxia and poor performance on the rotarod  igh more . We alsotilation .Atoh1::En1-CKO mice with an initial increase in tidal volume and respiratory frequency, followed by respiratory depression  might indeed result from abnormal postnatal maturation, others  might be self-resolving with maturation. Yet at all ages tested rRL neurons are essential to prevent apneas and respiratory depression during hypoxia.Our results show that P7 Atoh1-lineage PBC neurons are necessary to prevent respiratory depression in response to hypoxia. Yet it is unknown whether they modulate respiratory rhythms through activation of downstream rhythmogenic or chemosensitive nuclei, or act directly as premotor neurons.Oxygen sensors in the carotid body communicate with the nucleus tractus solitarius (NTS) to integrate the sensory information into the central respiratory circuit, which is necessary for hypoxic chemoresponses . There iAtoh1-lineage rRL neurons, we made use of an intersectional reporter allele (Ai65) that expresses tdTomato only after removal of both an FRT-flanked and a loxP-flanked stop-cassette  and the chemosensitive retrotrapezoid nucleus (RTN)  . We alsous (RTN) , but detus (RTN) . We founircuitry . This shAtoh1-lineage intertrigeminal region (ITR)  , suggestelopment .Atoh1-lineage ITR neurons, we generated an ITR-specific conditional knockout mouse using the TGHoxA2::Cre mouse line that expresses Cre only in r2 neurons . Out of nearly four hundred offspring observed, no Atoh1-dCKO mice survived past P1 . Concomitant loss of Atoh1 from these two domains causes fully penetrant neonatal lethality. This study thus answers the decades-old question about the cause of respiratory failure in Atoh1-null mice: they die of an inability to modulate respiratory rhythms in response to hypoxic and hypercapnic conditions.Many of the transcription factors expressed in the developing hindbrain are necessary for respiratory circuit development and survival . In some neurons . The bropairment . Here weAtoh1::En1-CKO mice are primarily due to loss of Atoh1-lineage parabrachial neurons. First, even though multiple nuclei develop from the intersectional domain of Atoh1 and En1, the parabrachial neurons were the only Atoh1-lineage neurons that were activated specifically during hypercapnic and hypoxic chemochallenges  that only target a subset of PBC neurons. Furthermore, these genetic tools are less prone to potential off target effects caused by needle tracks and local injections. Therefore, we propose that Atoh1-lineage PBC neurons are an essential component in the chemosensory pathways that relays information from peripheral, and perhaps central, chemosensors to enhance the firing of central chemosensitive and rhythmogenic neurons within the respiratory network.Several pieces of evidence suggest that the abnormal chemoresponses we observed in the allenges . Second,allenges . Third, Atoh1-lineage, deep cerebellar nuclei contribute to the observed phenotypes. Atoh1-lineage parabrachial and deep cerebellar neurons are born around the same embryonic day (E9.5-E12.5)   and sincAtoh1 loss from the intertrigeminal region had little effect on respiratory chemoresponses. In agreement with earlier studies from our lab, we found that intertrigeminal neurons depend on Atoh1 expression for normal migration, which resulted in increased number of apneas and a shallower breaths. Previous studies suggested a role for intertrigeminal neurons in the attenuation of sleep and reflex apneas  results from abnormal RTN development.Likewise, x apneas , 2004. Wechanism . DespiteAtoh1-lineage nuclei that rely on Atoh1 expression for their function in respiratory chemoreflexes: the PBC and RTN. Removal of Atoh1 from both of these nuclei recapitulated the fully penetrant neonatal lethality of Atoh1-null mice, which we hypothesize is caused by combined loss of CO2-evoked glutamatergic signaling (RTN) and hypoxia-induced activation of PBC neurons. Previous studies in neonatal models have shown that arterial CO2 unloading removes respiratory drive and results in sustained apnea, in accordance with the notion that CO2 sensing is essential for neonatal breathing  are partially redundant, as would make sense for any circuit that controls a process as fundamental to survival as breathing. Indeed, en bloc recordings from Atoh1-null mice reveal that slow respiratory frequency can be fully restored by application of a glutamate reuptake inhibitor and partially restored by substance P application  cases  cases , we hypolsl-tdTomatoRosa (tm9(CAG-tdTomato)HzeGt(ROSA)26Sor, JAX:007914), CreAtoh1 (CreEn1 (tm2(cre)Wrst/JEn1, JAX:007916):, lsl-LacZRosa GlhGt(ROSA)26Sor, JAX:012429), LacZAtoh1, FloxAtoh1, CrePcp2 (B6.129-Tg(Pcp2-cre)2Mpin/J, JAX: 004146), FloxSlc32a1 , TGHoxA2::Cre (TGPhox2b::Cre (Tg(Phox2b-cre)3Jke, JAX:016223), FlpOAtoh1, Ai65 (tm65.1(CAG-tdTomato)HzeGt(ROSA)26Sor,JAX:021875), and TGSox2::Cre (Tg(Sox2-cre)1Amc, MGI:3801167). Ear tissue, collected from ear-marking, was used for PCR genotyping to minimalize stress on animals. For timed pregnancies, noon on the day of the vaginal plugging was set as embryonic day 0.5 (E0.5).All animals were housed in a Level 3, AALAS-certified facility on a 14 hr light cycle. Husbandry, housing, euthanasia, and experimental guidelines were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine. The following genetically engineered mouse lines were used: Atoh1Cre , En1Cre 2::CreTG , Phox2b:FlpO sequence from pQUAST-FLPo was ligated to a PGK-Neo cassette flanked by lox2722 sites in a pUC vector. This FlpO-PGK-Neo cassette was then cloned into a pre-existing pBlueScript II KS+ plasmid that contained the Atoh1 5’ and 3’ targeting arms without disrupting the Atoh1 transcriptional start site, identical to the approach previously described  and were validated previously (In situ hybridization (ISH) was performed on 25µm-thick sagittal sections cut from unfixed, fresh frozen E14.5 embryonic control and CKO mice covering the entire embryo. We generated digoxigenin (DIG)-labeled mRNA antisense probes against eviously . ISH waseviously .2, balance N2), or hypercapnia  for 1 hr. Animals were sacrificed within 30 min of exposure and tissue was processed as described in ‘Immunofluorescence (IF) Assays.’To analyze in vivo hypoxia or hypercapnia induced cFos expression, adult animals (6–8 weeks old) were habituated to the plethysmography chambers five hours the day prior to the experiment and one hour on the day of the experiment. Freely moving mice were placed in whole-body plethysmography chambers (Buxco) through which fresh air was pumped at a basal flow rate of 0.5 L/min. Next, animals were exposed to either room air, hypoxia  or hypercapnia . After this chemochallenge, we recorded breathing behavior for another fifteen-minute recovery period in room air. To determine changes in breathing behavior in response to the chemochallenge, we normalized all breathing parameters to baseline values using the following formula: normalized y(t)=y(x)/(average(y(xbaseline)), where y is the average value of a parameter during any given minute (x) and baseline is minutes one to twenty.Breathing analysis was performed using previously reported protocols with minor modifications . To testWe used Phonemah three software (DSI) to identify breath waveforms and used custom-written MATLAB (Mathworks) code to derive inspiratory time, expiratory time, and tidal volume . BreathsAtoh1-heterozygote animals and any of the Cre-lines, we grouped these animals together in the analysis.Since we did not observe any difference in breathing parameters between male and female mice, or between 2, balance N2) or hypercapnia ).Breathing analysis for neonatal mice was performed using a DSI pup-plethysmography set-up designed especially for these experiments. Specialized, small pup plethysmography chambers (DSI) were connected to a FinePointe Whole Body Plethysmography Unit with gas switch capability (DSI). The unit provided the pup plethysmograph chambers with a constant 1 L/min airflow and amplified breathing waveforms. P7 (day of birth is P0) animals were placed in the plethysmography chambers and could acclimate for 10 min, followed by 5 min of room air recording and a 5 min gas-challenge  was calculated as the percentage of the trace from which individual breaths were defined . For our final analysis, we included only those minutes for which the RINX was less than 40%. All animals from which we recorded fewer than three reliable minutes of respiratory rhythms in both room air and during chemochallenge (RINX <40%) were excluded from our analysis. The percentage of animals of each genotype included in the final analysis were similar (23 control mice out of 33 (70%); 12 Atoh1::En1-CKO mice out of 16 (75%); Chi-Square = 0.133, p=0.7149). RINX of included animals was also similar between genotypes . From the included minutes the following parameters were derived: inhalation time, exhalation time, breathing frequency, tidal volume, and minute ventilation. Apneas and sighs were determined by a trained observer who was blinded to the genotype of the mice.FinePointe software was used to define breaths and calculate basic breathing parameters. Breaths were defined as followed: (1) peak inspiratory flow (PIF) must be before peak expiratory flow (PEF); (2) expiration or inspiration is less than a couple samples; (3) Rpef, EF50, and Tr can be computed; (4) conditioning coefficient can be computed; (5) MinimumBoxFlowThreshold is larger than 0.005; (6) Start of expiration can be identified on the AC flow waveform. Breaths were rejected if TSource data from the pup plethysmography analysis can be found in the source data file: Caffeine was administered to pups through the drinking water of lactating dams (0.3 gram/L) from postnatal day three until weaning. Caffeine was dissolved in water provided by the animal facility and stored in special dark bottles to prevent photodegradation. Plasma levels of caffeine in pups were determined using a commercially available Caffeine/Pentoxifylline ELISA kit (Neogen). We obtained blood and isolated plasma from P19 pups raised in cages with normal water or caffeinated water.p-value was Bonferroni adjusted for multiple comparisons. Statistical significance was accepted at p<0.05 for all other tests.All quantification and statistical analyses were performed using MATLAB (Mathworks). Data in text and figures represent the mean ± standard error of the mean (SEM). Student’s t-test was used when comparing two independent groups. Chi-square test were performed to test Mendelian ratios of surviving offspring. We used two-way ANOVA to determine interaction effects between genotype  and treatment (no caffeine vs. caffeine), followed by a Tukey-Kramer post-hoc analysis in case significance was reached. To determine time-specific significant differences in respiratory chemoresponses, serial t-tests or ANOVA’s were performed and the  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Atoh1 from neurons regulating hypoxic and hypercapnic chemoresponses causes neonatal respiratory failure in mice" for consideration by eLife. Your article has been reviewed by two peer reviewers, including Jan-Marino Ramirez as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen Eve Marder as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Daniel K Mulkey (Reviewer #2).Thank you for submitting your article "Loss of The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Atoh1-lineage neurons in the PBC contribute to central and peripheral chemoreflexes and together with Atoh1 neurons in the retrotrapezoid nucleus are essential for survival. The reviewers were very impressed by this study: The experiments are well designed, controlled and the conclusions are generally well founded.This manuscript by Meike van der Heijden and Huda Zoghbi significantly contributes to our understanding of the role of the parabrachial complex. The parabrachial complex (PBC) is a sensory integration center at the interface between forebrain structures and the brainstem that has long been implicated in respiratory control including serving as a chemotransduction relay station. But, the problem is that this area is very heterogeneous, and most what we know about this area is based on somewhat outdated studies involving often crude lesioning and non-specific electrical or chemical stimulations. This study by van der Heijden and Zoghbi overcomes these limitations by showing Essential revisions:1) Apneas associated with sighs are mechanistically different from spontaneous apneic events. Indeed, the authors find in different mutants differential effects on the number of sighs versus apneas. Thus, we suggest that central apneas and apneas associated with sighs should be tabulated separately.2) The authors should justify why they define apnea as breaths lasting > 0.5s, specially since the apnea on Figure 2A is not that different from baseline. It seems like apneas should be defined for each genotype based on inter-breath interval. While clinicians often use a certain interval without a breath, traditionally, apneas in mice are defined by the prolonged expiratory time relative to baseline (e.g. 4 times the eupneic expiratory time). We don't ask the authors to reanalyze their data, but this should be clarified and discussed.3) Of interest are also the downward deflections in the plethysmograph recording : Were these expiratory efforts that are uncoupled with inspiration? This is just a comment and we don't ask the authors to analyze these downward deflections. But, we are curious to know whether expiration was abnormal in these mice.4) The authors note that some genotypes show more irregular breathing compared to control. However, it is not clear to me whether this is a bad thing. For example, high heart rate variability is considered more stable, whereas less variability is associated with ensuing heart problems. If the same is true for respiratory control then further explanation is needed.5) Baseline respiratory frequencies are relatively high compared to most mouse strains (PMID: 7977867). Also, the Materials and methods states that frequencies >500 are excluded; however, we would be concerned about anything >250 bpm, because the animals might have been stressed. Can the authors try some controls with extra-long acclimation periods, perhaps in an isolated room and cover the plethysmography chamber to prevent visual distractions? If frequencies are still high, then it would be more convincing that the frequencies that they observed were indeed baseline frequencies. The authors could also consider performing Poincaré analysis of respiratory variability as described (PMID: 26068853) to support the possibility that experiments were performed on quiescent mice.En1Atoh1CKO- mice but did is also improve life span? This would strengthen the link between irregular breathing and mortality.6) Caffeine treatment normalized apneas and irregular breathing in En1-CKOAtoh1 suggest differential roles of adenosine signaling. That would be very interesting particularly since adenosine is thought to contribute to respiratory roll-off during hypoxia. In any case, caffeine has been shown to increase baseline breathing and chemoreception in several animals including humans (PMID: 18703176) so we agree with the authors that it is very surprising caffeine decreased respiratory output in control mice: This unusual response could be due to the high baseline frequency. There are certainly also other explanations: caffeine also inhibits A2 receptors, which are excitatory so perhaps this is an A2 dependent effect? The authors should discuss these possibilities. Of course, the authors could also try selective A1 and A2 blockers to test this possibility, but we feel that this would exceed the scope of the present study.7) The differential effects of caffeine on control and 8) We would appreciate if the authors would also discuss whether they propose that PBC neurons are primary chemosensory neurons or a requisite component of the pathway.2/H+-sensitive PBC neurons are glutamatergic and express calcitonin gene-related peptide (CGRP) . While we don't ask for additional experiments, it would be helpful to know if these markers overlap with Atoh1.9) Previous evidence showed that CO2/H+-sensitive PBC neurons is maintenance of airway patency and there is evidence suggesting subsets of PBC neurons project to respiratory motor neurons (PMID: 25424719). However, results presented here  do not support this possibility. These differences should be discussed.10) A proposed role of COAtoh1 null mice is very interesting. Did the authors observe whether the mice took a breath? Did they turn blue? Clarifying this would be very important for follow up studies.11) The 100% neonatal death in  Essential revisions:1) Apneas associated with sighs are mechanistically different from spontaneous apneic events. Indeed, the authors find in different mutants differential effects on the number of sighs versus apneas. Thus, we suggest that central apneas and apneas associated with sighs should be tabulated separately.We thank the reviewers for this excellent suggestion. We have run our analysis again and this time we defined apneas are breaths lasting 0.5s and at least 2 times the length of the closest 6 breaths (3 previous breaths and 3 following breaths) . This definition of apnea was used in many previous published papers . We defined apneas associated with sighs as apneas where that breath also met the criteria of a sigh or that were preceded by a sigh or if the prior breath was a sigh. Spontaneous apneas were those not associated with a sigh. We updated Figure 2, Figure 3—figure supplement 1, and Figure 6 accordingly.2) The authors should justify why they define apnea as breaths lasting > 0.5s, specially since the apnea on Figure 2A is not that different from baseline. It seems like apneas should be defined for each genotype based on inter-breath interval. While clinicians often use a certain interval without a breath, traditionally, apneas in mice are defined by the prolonged expiratory time relative to baseline (e.g. 4 times the eupneic expiratory time). We don't ask the authors to reanalyze their data, but this should be clarified and discussed.See essential revision 1.3) Of interest are also the downward deflections in the plethysmograph recording : Were these expiratory efforts that are uncoupled with inspiration? This is just a comment and we don't ask the authors to analyze these downward deflections. But, we are curious to know whether expiration was abnormal in these mice.We did not find that these downward deflections were overrepresented in either genotype. We did not find any evidence that expiration was abnormal in these mice.4) The authors note that some genotypes show more irregular breathing compared to control. However, it is not clear to me whether this is a bad thing. For example, high heart rate variability is considered more stable, whereas less variability is associated with ensuing heart problems. If the same is true for respiratory control then further explanation is needed.2 and CO2 levels (PMID: 4571351). The irregularity score is thus another measure of in vivo respiratory control that can point to abnormalities in central respiratory control.Irregular breathing is often observed in neonates (PMID: 15050210), patients with Cheynes-Stokes breathing (PMID: 4571351), Rett syndrome patients (PMID: 9452925), and a mouse model for congenital central hypoventilation syndrome (PMID: 18198276). High number of apneas can cause a higher interbreath interval irregularity, because the apnea is often at least twice as long as the breath preceding and following the apnea. However, irregularity scores are more sensitive than any kind of automated apnea counter, because it also takes in account breaths that just fail to make a cut-off for the apnea definition but are still remarkably longer than the prior breath. For examples of such breaths, see outlier points in Figure 1—figure supplement 2, particularly in the plot for Trace #340. Abnormalities in chemosensation can cause irregular breathing because of a delayed respiratory response to fluctuating O5) Baseline respiratory frequencies are relatively high compared to most mouse strains (PMID: 7977867). Also, the Materials and methods states that frequencies >500 are excluded; however, we would be concerned about anything >250 bpm, because the animals might have been stressed. Can the authors try some controls with extra-long acclimation periods, perhaps in an isolated room and cover the plethysmography chamber to prevent visual distractions? If frequencies are still high, then it would be more convincing that the frequencies that they observed were indeed baseline frequencies. The authors could also consider performing Poincaré analysis of respiratory variability as described (PMID: 26068853) to support the possibility that experiments were performed on quiescent mice.We performed Poincaré analysis for five randomly picked mice from each group. We found only highly variable breathing in the conditional knockout mice, likely due to the high occurrence of apneas, most other points fell along the diagonal line . We alsoWe also performed experiments in mice with acclimation periods over 1.5 hours and did not observe a decline in respiratory frequencies. We have performed similar experiments in mature/adult mice and we found that these mice have a substantially smaller respiratory rate after the acclimation period used for this study (~200 breaths/minute). Furthermore, after acclimating mice for about 30 minutes they appeared quiescent and often asleep, suggesting that this relatively high baseline-breathing rate is a representation of age rather than stress.En1-CKO mice but did is also improve life span? This would strengthen the link between irregular breathing and mortality.6) Caffeine treatment normalized apneas and irregular breathing in Atoh1Caffeine treatment did not normalize lifespan. Because cerebellar neurons are lacking in these mice, we believe the lifespan is reduced because of impaired coordination and compromised access to food and water. This is stated more clearly now in the text as follows:En1-CKO“Atoh1mice developed severe ataxia, dystonia and tremor in the second to third week after birth and died shortly after weaning (P22-25), likely, because the motor phenotypes impairs food and water intake.”En1-CKO suggest differential roles of adenosine signaling. That would be very interesting particularly since adenosine is thought to contribute to respiratory roll-off during hypoxia. In any case, caffeine has been shown to increase baseline breathing and chemoreception in several animals including humans (PMID: 18703176) so we agree with the authors that it is very surprising caffeine decreased respiratory output in control mice: This unusual response could be due to the high baseline frequency. There are certainly also other explanations: caffeine also inhibits A2 receptors, which are excitatory so perhaps this is an A2 dependent effect? The authors should discuss these possibilities. Of course, the authors could also try selective A1 and A2 blockers to test this possibility, but we feel that this would exceed the scope of the present study.7) The differential effects of caffeine on control and Atoh1We agree with the reviewers that the observed effects of caffeine on respiratory output is unexpected and that the mechanism underlying this result is an interesting question for future studies. We now discuss the difference between A1 and A2 signaling as follows:“Caffeine treatment, along with glutamate and substance P, can stimulate endogenously slow respiratory rhythms in rats ; this is thought to be the mechanism by which caffeine reduces apneas of prematurity in newborn infants . […] Future studies with specific adenosine agonist and antagonist are needed to elucidate the specific effects of long-term caffeine treatment on respiratory control in neonatal mice.”8) We would appreciate if the authors would also discuss whether they propose that PBC neurons are primary chemosensory neurons or a requisite component of the pathway.We propose that PBC neurons are a requisite component of the pathway. We have clarified this in the Discussion as follows:Atoh1-lineage PBC neurons are an essential component in the chemosensory pathways that relays information from peripheral, and perhaps central, chemosensors to enhance the firing of central chemosensitive and rhythmogenic neurons within the respiratory network.”“Therefore, we propose that 2/H+-sensitive PBC neurons are glutamatergic and express calcitonin gene-related peptide (CGRP) . While we don't ask for additional experiments, it would be helpful to know if these markers overlap with Atoh1.9) Previous evidence showed that COAtoh1-lineage neurons with the substance P receptor NK1R and neuropeptides CGRP and PACAP, that have been implicated in the hypercapnic and hypoxic respiratory response, respectively. We have discussed this in the text as follows:We thank the reviewers for suggesting this experiment. We have now included a figure  that shows the overlap of Atoh1-lineage neurons might be important for respiratory responses through signaling with one or both of these neuro peptides.”“There are no previous reports that PBC neurons are intrinsically chemosensitive, so these neurons are likely activated by upstream neurons that are chemosensitive. […] Together, these results suggest that 2/H+-sensitive PBC neurons is maintenance of airway patency and there is evidence suggesting subsets of PBC neurons project to respiratory motor neurons (PMID: 25424719). However, results presented here  do not support this possibility. These differences should be discussed.10) A proposed role of COAtoh1-lineage vs. the whole PBC population) and because of the projection mapping technique . We addressed this in the Discussion as follows:We are aware of the findings that PBC neurons might project to respiratory motor neurons. We think that the difference between our findings and the findings of previous studies are caused by the difference in neurons targeted  and that the parabrachial nucleus projects to the ventrolateral medulla . […] Therefore, we propose that 11) The 100% neonatal death in Atoh1 null mice is very interesting. Did the authors observe whether the mice took a breath? Did they turn blue? Clarifying this would be very important for follow up studies.We added this clarification in the text as follows:dCKOAtoh1 mice were found dead in the cage on the day of birth. These animals had turned blue and there was no visible milk spot, suggesting that they died of respiratory failure before drinking any milk.”“We have not caught any of these animals alive. Several