In both cohorts, habituation responses at 5 months significantly predicted deferred imitation responses at 12 months of age in both cohorts. Furthermore, ERP habituation responses explained a unique proportion of variance in deferred imitation scores which could not be accounted for by a neurobehavioural measure  conducted at 5 months of age. Our findings highlight the potential for ERP markers of habituation and novelty detection measured before 6 months of age to provide insight into later imitation abilities and memory development across diverse settings.Across cultures, imitation provides a crucial route to learning during infancy. However, neural predictors which would enable early identification of infants at risk of suboptimal developmental outcomes are still rare. In this paper, we examine associations between ERP markers of habituation and novelty detection measured at 1 and 5 months of infant age in the UK ( A significant proportion of human learning is facilitated through imitation and observational learning . InsteadSince Piaget’s first observations of infant imitation, proposing a sudden onset of imitation around 18–24 months of age  of the contributing processes, such as infants’ ability to encode and retain stimuli can provide insight into infant memory development. Individual differences in these neural markers have been shown to be associated with concurrent imitation behaviours. Bauer et al.  found thOur previous work examining neural markers of habituation and novelty detection across two cohorts in the UK and The Gambia has shown a protracted developmental trajectory of robust novelty and habituation responses in the Gambian compared to the UK cohort ,39. In aBuilding on this background, this paper is the first to describe longitudinal associations between the ERP markers of habituation and novelty detection collected at 1 and 5 months of age, and the deferred imitation scores at 8 and 12 months of age. We hereby aim to assess this link in both the UK and The Gambia, to answer a common set of questions: through an analysis of such mechanistic associations in the UK cohort, we aim to expand the current literature from other high-income study sites cohorts by examining whether reported brain-behaviour associations ,37 hold https://www.globalfnirs.org/the-bright-project/, accessed on 30 June 2022) which longitudinally assessed two parallel infant cohorts in the UK (n = 61) and The Gambia (n = 214), with longitudinal visits at 7–14 days, 1, 5, 8, 12, 18 and 24 months of infant age. The protocol incorporated neuroimaging  and eye tracking alongside behavioural measures with the MSEL performed at 5, 8, 12, 18 and 24 months of infant age. The EEG assessments were run at 1, 5 and 18 months; here, data from the 1- and 5-month age point are considered to enable prediction of deferred imitation scores gathered at 8 and 12 months of age. In the current analysis, we examine associations between the 5-month age point of the MSEL, the EEG responses at 1 and 5 months, and deferred imitation scores at 8 and 12 months of age. These associations were examined separately for the UK and the Gambian cohort to find out whether some mechanistic associations generalise across these two diverse settings, indicating a degree of universality. We hypothesise that:The current study was conducted as part of the Brain Imaging for Global Health project . At both sites, participants were eligible if (1) mothers were above 18 years of age, (2) living primarily in the study’s catchment area, (3) infants were born at term  and (4) infants were not identified with neurological deficits during neonatal checks. In the UK, only infants with a birthweight of >2.5 kg were enrolled. Furthermore, in The Gambia only predominantly Mandinka speaking families were recruited, who represent the majority ethnic group in the region data collection took place, to avoid confounds of translating and adapting language-based measures into multiple languages. No restrictions were made on infants’ birthweight in this cohort, because multiple environmental factors  prevalent in this region frequently lead to growth faltering with regard to both infants’ length and weight over the first months of life . FurtherFrequent sounds , Infrequent sounds  and Trial Unique sounds (presented at 10% probability). Trial Unique stimuli consisted of a range of different sounds, such as clicks, tones, digitized vocalizations and syllables  and werData were recorded via a Neuroelectrics Enobio8 system, Neurolectrics, Barcelona, Spain) with eight electrodes placed at Fz, FC1/2, C1/z/2 and CP1/2. Data were recorded in reference to infants’ left mastoid and this reference was retained throughout data analysis. At the 1-month age point, infants were asleep during data acquisition, whereas at 5 months infants were assessed while awake and quietly interacting with one of the researchers. As discussed in Katus et al. , this diData were pre-processed via customised Matlab routines . A bandpass filter of 0.5–30 Hz was applied , data were then offset corrected for a 32 ms timing delay and segmented from 200 ms pre- to 800 ms post-stimulus onset. Artifacts were removed via a simple voltage threshold of ±100 µV. Flatlining epochs with a change of less than 0.1 µV in an epoch were also rejected. The condition with fewest valid trials was identified and a random sample of the same size was chosen from both other conditions. Datasets with <15 trials per condition were discarded. To enable the extraction of habituation markers, data sets with <45 Frequent stimuli were discarded.Scoring and outcome variables. As a measure of novelty detection, we calculated the difference between P3 mean amplitudes to Trial Unique and Frequent sounds, which was normalised for individual variance in overall ERP amplitudes by dividing responses by amplitudes to Frequent sounds . As a measure of habituation, we examined the difference in responses to trials 1–15 and 30–45, normalised by responses to the first 15 trials .Infants completed the deferred imitation task at their 8- and 12-month visits. Stimuli for this study were designed to be (1) motorically manageable for young infants, (2) novel to both infants in the UK and The Gambia and (3) independent of whole-body movements, as fNIRS data were recorded during this task in the UK cohort. Here, we examine only the behavioural data. Items and target actions are illustrated in baseline phase). After 30 s, the experimenter demonstrated the target action three times (demonstration phase). This procedure was repeated for all six items. For half of the items, the demonstration phase was followed by an immediate imitation phase, during which the experimenter presented the infant with the item directly following the demonstration for this item. Across participants, it was counterbalanced which half of items were presented during the immediate imitation phase. After a 20 min delay, infants were in turn presented with each item and their performance of the target action was recorded. The procedure is illustrated in Infants sat on their parents’ lap at a table facing the experimenter. First, infants were presented with an item and were given time to freely explore it . Training on this task took the form of a detailed protocol description, one in-person training and several regular check-up meetings to discuss questions and provide feedback on specific items. As part of these discussions, scoring criteria were simplified and made more objective, leading to high inter-rater reliability rates (post hoc video-based scoring yielded ICC’s > 0.8).Our scoring procedures followed common principles applied in deferred studies . The majority of actions had only one step and were scored as 0 (not completed) or 1 (completed). For some items, two steps were scored . Responses during the baseline and imitation phases were all scored according to the number of steps completed. Infants’ scores during the baseline phase were then subtracted from those in the immediate and the deferred phase, obtaining an imitation score that disregarded the number of target actions completed prior to demonstration.The MSEL was performed at the 5-, 8-, 12-, 18-, and 24-month visits of the BRIGHT project. Here, data from the 5-month age point are presented. The administration of the MSEL as well as the adaptations made for use in the Gambian context are described in Milosavljevic et al. . The MSEBased on prior work using both a verbal and non-verbal outcome measure on basis of the MSEL , we obtaFor the deferred imitation task, baseline corrected scores were modelled in one repeated measures ANOVA per site by Condition (immediate/deferred) and Age (8 months/12 months), followed by appropriate post hoc comparisons. For the ERP task, a detailed component analysis is reported in Katus et al. . Here, wSample characteristics for infants included and excluded in the analyses can be found in Data rejection/retention rates for both cohorts can be found in Raw scores for the baseline, immediate and deferred condition of the deferred imitation task are visualised in F1,15 = 23.388, p < 0.001, pη2 = 0.609) but not Age , and no interaction was observed . The condition effect was driven by higher scores in the deferred compared to the immediate condition at both the 8-month  and 12-month  age point. In The Gambian cohort, we found main effects for Condition  and Age , but no interaction effect . Post hoc tests showed that main effects were driven by higher scores in the deferred compared to the immediate condition at the 8 month  and the 12-month  age point, and that scores were higher at 12 months compared to 8 months in both the immediate  and the deferred imitation condition .Deferred imitation scores were modelled in a repeated measures ANOVA with within factors Condition (immediate/deferred) and Age (8 month/12 month). In the UK cohort, a main effect was found for Condition  or the Gambian cohort . To assess whether these non-significant results presented evidence for a true null effect, we calculated the Bayes Factor testing the likelihood of the current data being obtained under the null compared to the alternative hypothesis (BF01) and found it to be high for both the UK (BF01 = 4.179) and the Gambian (BF01 = 3.616) cohort. A BF > 3, indicates that the observed data is at least three times more likely under the null- than under the alternative, and is generally accepted as indicating reliable evidence for the null hypothesis to be true [t46 = −0.302, p = 0.764, BF01 = 3.766) or 12 months .To help contextualise these findings, exploratory analyses were conducted: while items were designed to be appropriate to the motor abilities to infants at each age point, we examined whether older infants found it easier to handle the presented objects and perform target actions. To assess this, we compared baseline scores across the age points. We found no evidence for differences in baseline scores between the 8- and 12-month age point in either the UK cohort . In the UK cohort, we found a positive correlation between the ERP habituation index at 5 months and deferred imitation scores at 12 months . No such associations were found for the habituation scores at 1 month, or novelty detection scores at either age point. In the Gambian cohort, we found associations between the ERP habituation  and novelty detection  indices at 5 months, but not at 1 month, with deferred imitation scores at 12 months. Associations between deferred imitation at 12 months and ERP responses at 5 months are illustrated in To assess whether our two measures of infant memory development (ERP markers and deferred imitation scores) showed the anticipated associations at both sites, we examined correlations between the ERP habituation and novelty detection indices at 1 and 5 months, with deferred imitation scores at 8 and 12 months. Visual inspection revealed one outlying value in the UK cohort for the habituation response of the EEG . In both cohorts, we found that MSEL scores were not significant as predictors of deferred imitation scores at 12 months of age . In both cohorts, ERP habituation markers at 5 months, but not at 1 month, were significant predictors . While the ERP novelty detection marker correlated significantly with deferred imitation scores at 12 months in the Gambian cohort, it was not found to be a significant predictor in the hierarchical regression model .Since no correlations were found between the ERP markers and the deferred imitation scores at 8 months, we only proceeded to model the 12-month deferred imitation scores as an outcome for all further analyses. Hereby, we were interested to find out whether the ERP indices explained variance over and above what could be accounted for by a broader index of neurodevelopmental status, since ERP markers and deferred imitation scores were hypothesised to tap the same underlying domain of memory development. In hierarchical regression models (one per site), with 12-month deferred imitation scores as an outcome, we entered MSEL scores at 5 months to the baseline model, and then, in a second step entered ERP indices at 1 and 5 months of age. The variance accounted for increased with addition of these markers . This association was not found for the 12-month immediate imitation, and for neither age point in the UK cohort. In a hierarchical regression model, ERP habituation at 5 months was not a significant predictor of immediate imitation at 8 months, the association did not hold in this more stringent analysis.We conducted an additional exploratory analysis, to assess whether the fact that scores in the immediate imitation condition being reduced relative to the deferred imitation condition could index infants’ habituation to the presented objects over the baseline, demonstration and immediate imitation phase. We found that ERP habituation correlated significantly with Immediate imitation at 8 months in the Gambian cohort only . Moreover, counterintuitively, we observed more imitation behaviours in the deferred relative to the immediate condition in both the UK and the Gambian cohort at both 8 and 12 months of age. While this finding seems to be at odds with the differential memory demands placed on infants in these conditions, observations from assessments suggest that it can be attributed to infants’ decreasing interest in the presented objects over the baseline, demonstration, and immediate imitation phase. After the 20 min delay (in which infants switched to a screen-based passive task), they generally showed an increased engagement with the imitation task compared to the immediate imitation phase. This is in part corroborated by our exploratory analysis, showing a correlation between infants ERP habituation responses and immediate imitation scores at eight months of age in the Gambian cohort.Overall, we only found evidence for our hypothesis that ERP markers would predict deferred imitation; while we did not find associations between the ERP markers and deferred imitation at 8 months at either site, we did find links with the ERP at 5 months and deferred imitation at 12 months. Several potential reasons could explain this. First, our outcome measures were quite stringently scored, by correcting deferred imitation scores for spontaneous performance of target actions during the baseline phase per infant (as is common practice in these kinds of tasks) . AdditioIn a similar way, we found ERP markers at one month were not associated with deferred imitation at either eight or twelve months. In our previous work, we found that in both the UK cohort and The Gambian cohort, infants at one month did not yet show either novelty or habituation responses based on their ERP. This is in line with prior research suggesting that these robustly emerge by two months of age at the earliest ,48. It mWhile we hypothesised to see similar associations of ERP markers and deferred imitation scores across the domains of habituation and novelty detection due to the complementary nature of these processes, we found the most robust associations with infants’ habituation responses. In the UK, only ERP habituation responses were associated with deferred imitation at 12 months and provided a significant predictor in the regression models. In The Gambia, both habituation and novelty detection at 5 months were correlated with deferred imitation at 12 months, but only habituation responses significantly predicted variance over and above MSEL scores in the regression models. In this context, it needs to be noted that in our prior work using fNIRS we found habituation responses early on in development, whereas novelty detection only emerged at the 18-month age point . It may Even though a strong argument can be made for deferred imitation to tap the same cognitive resources as recall memory and verbal recall  ,49 we diFirstly, sample characteristics need to be considered when interpreting these results. We have achieved the longitudinal collection of neurophysiological and behavioural data across two geographically and socio-demographically diverse cohorts. However, since longitudinal associations required contribution of data across several age points and assessment modalities, there was a marked reduction in sample size for the overall smaller UK cohort. Cohorts were designed to be different in size as the main goal of the BRIGHT project was to tease apart longitudinal developmental trajectories in The Gambia. Especially analyses examining changes across age points may therefore be underpowered, which might also have contributed to non-significant findings . While this may limit the robustness of the findings from the UK cohort, it is important to note that several prior studies ,37 suffeSecondly, while items of this task were developed in parallel across sites, the overall reduced familiarity with toy play in the Gambian cohort and resulting situational novelty needs to be considered. While this paper was primarily interested in understanding mechanistic links between early ERP markers and deferred imitation without a direct site comparison, individual differences in the Gambian cohort may to a greater part reflect which infants were better able to cope with a novel situation than in the UK. However, our additional exploratory analyses allowed us to contextualise our findings in a way that highlights several commonalities across age points and study sites: first, we did not find site differences with regard to infants’ engagement with the presented objects. This is not to say that more subtle differences  do not differ across contexts, though such an analysis is beyond the aims of this specific study. Thirdly, while confounds with motor development may come into play, our analyses comparing baseline performance did not show systematic differences, and the lack of an association between the MSEL PDQ, which includes fine motor scores further corroborates this.Lastly, performance of target action during the baseline performance was high. This was due to the need to design items easy enough to be motorically manageable for infants of this young age group, which for the most part resulted in one-step actions which infants some of the time would perform during baseline. Other studies  of infaOur findings highlight the potential for ERP markers of habituation and novelty detection measured before six months of age to provide insight into later imitation abilities and memory development across settings. Such markers may thus be a useful addition to behavioural markers in identifying infants developing sub-optimally to provide early intervention.


Previously reported results have shown that, compared with neoadjuvant chemoradiotherapy alone, total neoadjuvant therapy improves tumor response in locally advanced rectal cancer. This article reports results of a retrospective case series and provides updated information on tumor response and survival. Total neoadjuvant therapy (TNT) improves tumor response in locally advanced rectal cancer (LARC) patients compared to neoadjuvant chemoradiotherapy alone. The effect of TNT on patient survival has not been fully investigated.χ2 test. Disease-free survival (DFS), local recurrence-free survival, distant metastasis-free survival, and overall survival were assessed by Kaplan-Meier analysis and log-rank test. Cox regression models were used to further evaluate DFS.This was a retrospective case series of patients with LARC at a comprehensive cancer center. Three hundred and eleven patients received chemoradiotherapy (chemoRT) as the sole neoadjuvant treatment and planned adjuvant chemotherapy, and 313 received TNT . These patients then underwent total mesorectal excision or were entered in a watch-and-wait protocol. The proportion of patients with complete response (CR) after neoadjuvant therapy  was compared by the P=.05). DFS, local recurrence-free survival, metastasis-free survival, and overall survival were no different. Disease-free survival was not associated with the type of neoadjuvant treatment .The rate of CR was 20% for chemoRT and 27% for TNT (Although TNT does not prolong survival than neoadjuvant chemoradiotherapy plus intended postoperative chemotherapy, the higher response rate associated with TNT may create opportunities to preserve the rectum in more patients with LARC. This study shows that, despite the higher treatment compliance and early delivery of systemic chemotherapy, patients living with LARC treated with TNT (induction chemotherapy and chemoRT) do not have longer survival than those treated with chemoRT and intended adjuvant chemotherapy. Although the effect on survival may be negligible, TNT improves the likelihood of achieving CR and thus should be strongly considered in patients that are more likely to benefit from organ preservation.2 Unfortunately, over a quarter of patients treated with chemoradiotherapy and TME develop distant metastasis, which remains the leading cause of death in these patients.3 On the basis of the benefit observed in patients with colon cancer, adjuvant systemic chemotherapy is recommended for patients living with LARC treated with chemoradiotherapy and curative-intent TME.4 However, the benefit of adjuvant chemotherapy in these patients has not been conclusively determined.6Neoadjuvant chemoradiotherapy followed by total mesorectal excision (TME) is highly effective in providing local tumor control of locally advanced rectal cancer (LARC).7 However, these results were criticized as the study included trials with poor-quality TME as well as patients treated with postoperative chemoradiotherapy.8 A more recent meta-analysis of individual patient data from 4 prospective randomized trials suggested that adjuvant fluorouracil-based chemotherapy did not improve survival in patients with mid or low rectal cancer treated with neoadjuvant chemoradiotherapy followed by good-quality TME,9 but the compliance with adjuvant chemotherapy in the trials was low.9-11 The inconclusive evidence on the benefit of adjuvant chemotherapy in patients with LARC has resulted in different treatment guidelines.13A systematic review of 21 randomized trials found longer disease-free survival (DFS) and longer overall survival after curative surgery in patients who received adjuvant chemotherapy compared with patients who did not.14-18 Because of the growing interest in preserving the rectum in patients with a clinical complete response (cCR) to neoadjuvant therapy, tumor response is an important clinical outcome.19–22 While the evidence on the effect of TNT on tumor response has been accumulating over the years,14-18 whether TNT improves survival compared to chemoradiotherapy and intended postoperative chemotherapy is still controversial.23 We had previously reported the results of the adoption of TNT for LARC and found that TNT was associated with a higher tumor response rate compared to chemoRT.16 In this current study, we provide updated information on the tumor response and evaluate DFS, local recurrence-free survival, metastasis-free survival, and overall survival. We also study the relationship between the tumor response and DFS in the overall patient cohort and by the neoadjuvant treatment group.The delivery of systemic chemotherapy before chemoradiotherapy and surgery—known as TNT—aims to enhance primary tumor response, improve compliance with chemotherapy, and treat potential micrometastases early.16 Locally advanced rectal cancer was defined as stage II  or III  invasive rectal adenocarcinoma within 15 cm from the anal verge in accordance with the American Joint Committee on Cancer guidelines. The locoregional staging was based on endorectal ultrasound (ERUS) or magnetic resonance imaging (MRI). Patients with a history of pelvic radiation, polyposis syndromes, inflammatory bowel disease, recurrent rectal cancer, metastatic disease, or other primary tumors within the previous 5 years were excluded. Three hundred and eleven patients received neoadjuvant chemoradiotherapy with an intention to receive adjuvant chemotherapy (chemoRT) and 313 received TNT . Nine out of the 313 patients in the TNT group also received postoperative chemotherapy. The study was approved by the Institutional Review Board of Memorial Sloan Kettering Cancer Center.This study population consisted of patients diagnosed with LARC at Memorial Sloan Kettering Cancer Center between June 1, 2009, to March 1, 2015, as in our previous study.2 daily or oral capecitabine at 825 mg/m2 twice daily. Patients generally received a radiation dose of 45 Gy with a sequential or integrated boost of 5-11 Gy to the tumor. Patients treated with chemoRT were recommended to receive additional chemotherapy as adjuvant treatment for a total of 3 to 4 months in accordance with the guidelines of the National Comprehensive Cancer Network.12 In the TNT group, TNT was planned as 4 months of induction chemotherapy in the form of mFOLFOX6  or CAPOX . Chemoradiotherapy was given 2 to 3 weeks after completing the induction chemotherapy.16Chemoradiotherapy consisted of 25 to 28 fractions of 1.8 Gy with concurrent infusional fluorouracil at 225 mg/m19 Patients with cCR who chose surgery, patients without a cCR at restaging, and patients in whom the tumor regrew during WW underwent TME. Some of the patients did not undergo TME: 4 patients (1%) in the TNT group underwent local excision, 1 patient (0.3%) in the chemoRT group, and 2 patients (0.6%) in the TNT group were deemed unresectable, and 2 patients (0.8%) in the chemoRT group and 9 patients (3%) in the TNT group declined resection.In both groups, patients with cCR at the completion of neoadjuvant therapy were given the option to enter a watch-and-wait (WW) protocol to preserve the rectum.25) or cCR sustained for 2 years (based on previously described criteria27). Clinical complete response was determined based on endoscopic findings such as a flat, white scar plus a normal digital rectal exam as well as radiographic findings on pelvic MRI that were not concerning for lymphadenopathy or residual tumor. Survival was measured from the first day of neoadjuvant treatment. Local recurrence-free survival included local recurrence after TME, non-salvageable regrowth in WW patients, or death as events. Metastasis-free survival included distant metastasis and death as events. Disease-free-survival included local recurrence after TME, non-salvageable regrowth in WW patients, distant metastasis, or death as events. Overall survival included death as the event.Complete response was defined as pathological CR  results of the univariable analysis, (2) known prognostic factors, and (3) variables found to be different by the group. When fitting these multivariable models, collinearity, sparse cells, and nonproportional hazards were evaluated. Additionally, variables with many missing values were excluded to maintain a robust sample size in the multivariable models. In an exploratory analysis, the interaction between neoadjuvant treatment group and tumor response was evaluated in a multivariable Cox model. For all analyses, P-values less than 0.05 were deemed statistically significant. All analyses were conducted with SAS, version 9.4, and R, version 3.1.1, software.Patient and treatment characteristics were compared by treatment group using the χn = 311) and the TNT group (n = 313) are listed in P < .001). Most patients in the 2 groups were men . The proportion of patients with cT4 and patients with node-positive disease were higher in the TNT group than in the chemoRT group. A greater proportion of patients in the TNT group compared with the chemoRT group was staged by MRI . The mean tumor distance from the anal verge did not differ significantly between the 2 groups.The clinicopathologic characteristics and treatment details for the chemoRT group (P < .001). The mean total duration of chemotherapy (months of neoadjuvant chemotherapy plus months of adjuvant chemotherapy) was longer in the TNT group .The mean dose of radiation received was similar in both groups. All patients in the TNT group started chemotherapy, whereas 26% of patients in the chemoRT group did not receive any postoperative chemotherapy (P = .05).The number of patients with a sustained cCR in the chemoRT group dropped from 19 (6%) at 1 year after completion of neoadjuvant therapy to 14 (5%) at 2 years. In the TNT group, the number of patients with a sustained cCR dropped from 70 (22%) at 1 year after completion of neoadjuvant therapy to 39 (13%) at 2 years. The overall rate of CR  at 2 years was still higher in the TNT group compared with the chemoRT group . No clinically meaningful difference in the rates of local recurrence-free survival and metastasis-free survival was observed between the groups  and B. TP<.001) and CR  (P = .066). We then performed a multivariable analysis by selecting baseline clinical variables that were imbalanced between the groups or showed associations with DFS in the univariable analysis (P = .012) and cT4 tumors  had significant associations with DFS. We also incorporated tumor response into the multivariable model (P < .001) as well as male sex and cT4 tumors remained statistically significant. No associations were observed between DFS and the type of neoadjuvant treatment, tumor distance from the anal verge, cN status, locoregional staging method, or total duration of chemotherapy.In univariable analysis, DFS was associated with cT4 classification  . Male seanalysis . Male sele model  and founP < .0001)  (P = .021) even after adjusting for clinical and demographic covariates, indicating that the separation of DFS curves in complete versus incomplete responders was more pronounced in patients who received TNT compared to chemoRT.To further interrogate the relationship between tumor response and DFS, we analyzed survival by tumor response (CR vs. incomplete response) in the entire cohort and in each neoadjuvant treatment group separately . We foun< .0001) , Fig. 2Actively)  and C. Vctively)  was foun28 However, the neoadjuvant treatment modality did not appear to have an impact on survival. This is in line with previous reports showing similar long-term outcomes of TNT versus chemoRT.29Our study shows that despite the higher treatment compliance and early delivery of systemic chemotherapy, patients living with LARC treated with TNT (induction chemotherapy and chemoRT) do not have longer survival than patients treated with chemoRT and intended adjuvant chemotherapy. While some patients in WW developed tumor regrowth with a longer follow-up, CR was still higher for the TNT group compared with the chemoRT group. In our cohort, cT4 tumors and CR were independent factors associated with DFS similar to previous literature reports.30-35 A randomized prospective trial failed to show improvements in response in patients with LARC treated with 2 cycles of induction mFOLFOX6 plus chemoRT compared with chemoRT alone, and thus closed before completing accrual.36 The GCR-3 phase II trial that randomized patients with LARC to TNT (4 cycles of CAPOX followed by chemoRT) or the conventional arm (chemoRT followed by 4 cycles of CAPOX) reported similar response and survival rates despite higher compliance with chemotherapy in the TNT group.29 However, this study was not powered to detect differences in survival. Consistent with our results, a retrospective review of patients with LARC from the National Cancer Database has shown equivalent survival outcomes for patients treated with systemic chemotherapy before chemoRT and TME compared to a propensity score-matched cohort of patients treated with chemoRT and TME.23 Also similar to our study, patients treated with systemic chemotherapy before chemoRT had a greater response rate but the difference did not reach statistical significance.23Single-arm case series have shown that induction chemotherapy followed by chemoRT was well tolerated, effective for early symptomatic relief, and provided excellent tumor response in patients with LARC, but did not provide data on survival compared with patients treated with chemoRT.37 The PRODIGE-23 phase III trial randomized patients with LARC to the control arm  or the experimental arm . This study reported a higher response rate and improved 3-year DFS rate  and 3-year metastasis-free survival rate  in the experimental arm compared with the control arm.38 While this study is the first to report an improvement in DFS in patients treated with induction chemotherapy, it did not test a true TNT strategy and incorporated a different chemotherapy agent only in the experimental arm. Therefore, it is possible that the differences in metastasis-free survival and DFS may be due to the addition of irinotecan to the experimental arm rather than the treatment sequence. Despite the treatment intensification, the study did not find a difference in overall survival.The recently published RAPIDO trial found an improved disease-related treatment failure in patients with LARC treated with short-course radiation followed by 4 months of FOLFOX or CAPOX compared with patients treated with chemoRT, TME, and optional adjuvant chemotherapy. Despite the greater dose of chemotherapy given in the experimental arm, the study failed to show a difference in overall survival.28 While one may assume that increasing the number of complete responders would result in an improved survival for the entire group, our data do not support this assumption. The higher rate of response in the TNT group did not translate into better survival compared with the chemoRT group. These results are consistent with several prospective randomized trials that have shown equivalent overall survival for treatment arms associated with different CR rates.39-41 Our analysis of survival by response according to the treatment group provides a plausible explanation for the apparent discrepancy between tumor response and patient survival. The greater separation in the DFS Kaplan-Meier curves between the complete responders and incomplete responders in the TNT group compared to the chemoRT group suggests that TNT increases the proportion of complete responders from a pool of biologically favorable tumors and concentrates the patients with worse survival in the incomplete responder group. These findings have important clinical implications as complete tumor response has been considered a surrogate of patient survival in rectal cancer patients and is even incorporated as an endpoint in clinical trials.Our study confirms that patients with a CR to neoadjuvant therapy demonstrate correlation with significantly better survival compared with patients with an incomplete response.42 As patients with excellent response to chemoRT derive no benefit from postoperative adjuvant chemotherapy,9 the widespread use of TNT will inadvertently overtreat some patients living with LARC. On the other hand, the increase in the response rate in the TNT group could increase the proportion of patients living with LARC who may benefit from organ preservation. The preliminary results of the OPRA trial suggest that at least 40% of patients living with LARC treated with induction chemotherapy and chemoRT can preserve the rectum, provided that they are given enough time for the tumor to respond.43 Therefore, although the effect on survival may be negligible, TNT should be given strong consideration in patients that are more likely to benefit from organ preservation such as those with low rectal cancer that may otherwise require a coloanal anastomosis or a permanent stoma. In addition, starting TNT with induction chemotherapy opens the possibility of skipping chemoradiation—and avoiding radiation-related toxicity—in patients with higher tumors who can safely undergo sphincter preserving TME.45Although our study did not collect treatment toxicity information, a higher total dose of chemotherapy is likely to be associated with greater toxicity.43 it is possible that the greater proportion of WW patients in the TNT group could have influenced survival outcomes. While providing chemoradiation followed by chemotherapy in the neoadjuvant setting has been associated with higher response rates,43 the impact of the sequence of TNT on survival was not evaluated in this study. Although we attempted to adjust for possible confounding factors in a multivariate analysis, we cannot exclude the possibility of patient selection bias or other unaccounted factors contributing to survival.Our study has several limitations due to its retrospective design. The neoadjuvant therapy for rectal cancer at our institution has evolved during the study period. Total neoadjuvant therapy was initially introduced to treat younger patients with more advanced tumors. This may explain some of the differences in patient age and clinical stage between the groups. In addition, the tools used to stage rectal cancer also changed during the study period. Endorectal ultrasound, which was commonly used in the initial years of the study, was later replaced by MRI. The possibility that the broader view of the mesorectum and the mesorectal fascia provided by MRI compared with ERUS may account for some of the differences in tumor stage between the groups. Furthermore, the recent increase in the number of young patients with rectal cancer may also account for the age differences seen between the groups. Another limitation of our study is the increased adoption of WW in recent years reflected in the higher proportion of WW patients in the TNT group versus the chemoRT group. While WW appears to be safe,Our analyses suggest that TNT is associated with an improvement in the likelihood of a CR, which may allow increased rates of organ preservation with WW, but is not associated with an improvement in survival compared with conventional chemoRT followed by adjuvant chemotherapy.


USP6 gene on chromosome 17 remains to be the most commonly accepted reasoning as ABC’s etiology remains unknown. As the radiographic appearance of ABC is quite similar to other kinds of bone cysts, a histological diagnosis is often required to attain a definitive diagnosis. Curettage remains the gold standard treatment with high chances of local recurrence. Evidence has shown the beneficial applications of administering a sclerosing agent. Further trials would improve the level of evidence available for physicians to make a better management plan. We have demonstrated the treatment of an aneurysm bone cyst of the proximal humerus on a 5-year-old female in this report, which might be utilized as a reference for future procedures.An aneurysmal bone cyst (ABC) is a non-malignant, skeletal tumor that is extremely rare and most commonly presents within the first two decades of life. Genetic mutation of the  USP6) gene on chromosome 17. Although ABCs are the primary cause of presentation in 70% of cases and secondary associations of other tumors in 30% of cases, the primary presentation has a low incidence rate of 0.14 to 0.32 per 100,000 individuals [An aneurysmal bone cyst (ABC) is a non-malignant, blood-filled, rare skeletal tumor first described by Jaffe and Lichtenstein in 1942 due to its cyst-like appearance inside the bone. They are most commonly presented as an expanding mass and are often painful. Children below the age of 20 have an increased predisposition to develop ABC due to their rapid growth rate. Since the cause of ABC is known, the most widely accepted reasoning is the genetic mutation of the ubiquitin-specific peptidase 6  x 5.1 (tr) x 5.9 (ap) cm , and functional score. A study done with 72 patients has shown a cure rate of 97%. Studies involving pediatric cases for sclerotherapy have also shown similar results ,7. One oAn ABC is a highly destructive benign bone tumor with an unfavorable prognosis if not treated early, despite surgical treatment. Proper follow-up should be scheduled at least every three months for the first two years of treatment. In our patient, we surgically treated the tumor first by sclerotherapy after which we curetted and filled it with autogenous bone graft followed by chemical cauterization in order to restrict the aggressive tumor from causing a pathological fracture. The patient responded well to the treatment and is under follow-up.


An audiogram is not sufficient to indicate cochlear dead regions.To investigate cochlear dead regions in sensorineural hearing loss subjects using the TEN test. Site: CEDALVI/ HRAC-USP-Bauru/Sao Paulo/Brazil, August 2003 to February 2004.A contemporary cross-sectional cohort study. Material and Methods: The TEN test was applied in three groups: G1; G2; G3.In the G1 group the TEN value required to eliminate the test tone was, on average, close to the absolute threshold for all frequencies. No dead regions were found in the ears tested in group G2. 76 ears were tested in group G3, and six showed no evidence of dead regions in the cochlea.The TEN test was an effective test to indicate a dead region in the cochlea of subjects with sloping sensorineural hearing loss. There is evidence that pure-tone detection is different for subjects with high frequency sensorineural hearing loss and flat hearing loss; we observed a significant difference between the masked threshold and the absolute threshold only in sloping hearing loss and not for flat hearing loss. The cochlear region where internal ciliated cells (ICC) are injured, inactive or absent, and the neurons that innervate this region are inactive or even degenerate, has been named dead region of the cochlea, or dead region.3Although the dead region concept was elaborated many years ago by Gravendeel and Plomp,6Audiogram configurations have also been considered as evidence or dead regions, such as sloping audiograms, where thresholds worsen abruptly with increasing pitch (over 50 dB/octave) at high frequencies. Hearing losses of 40-50 dB at low frequencies and hearing close to normal in medium and high frequencies may suggest dead regions at low frequencies. This may also be the case when there is hearing loss over 50 dB at low frequencies with improved thresholds at higher frequencies. A U-shaped audiogram, with better hearing at low and high frequencies and a dead region at middle frequencies, is rare and generally does not interfere significantly with speech recognition. Residual hearing allows good recognition, meaning that some of these cases do not require hearing aids .Huss et al. investigated pure tone perception in subjects with and without dead regions diagnosed by psychophysical tuning curves and the threshold-equalizing-noise (TEN) test, in which participants were asked to score the sharpness of a pure tone on a scale from sharp (1) to noisy (7). The authors concluded that the pure tone subjective impression of sharpness is not a consistent indicator of dead regions in the cochlea, as higher scores were seen not only in ears with dead regions but also in normal ears at similar frequencies and at higher thresholds.It is clear that patient reports or the audiogram by themselves are not sufficient to establish or exclude the presence and extension of dead regions. Thus, masking has been used in some studies to investigate this condition in greater detail.The investigation of dead regions uses ipsilateral masking, in which the test signal and the masking noise are presented to the same ear. The idea is to raise the threshold of non-tested frequencies so that they do not respond to excitation diffusion of the signal that is being tested at a specific frequency.Moore et al. developed the Threshold-Equalizing-Noise test to investigate the presence of dead regions. Although masking is defined as a procedure used in audiological evaluation, and not a test in itself, TEN is considered a test by its authors. It is based on the detection of pure tones presented simultaneously with a wide band noise (TEN) which produces practically the same level of masking (measured in dBNPS) throughout audiogram frequencies  in normal hearing or hearing-impaired individuals with no dead regions.5In Brazil Eguti investigated the efficiency of the masking technique using white noise to identify dead regions in the cochlea of 32 adult individuals that presented acquired sensorineural or mixed hearing loss and a sloping audiometric configuration. Moore'sThe diagnosis of dead regions in the cochlea is important in medical practice, as studies have shown that its presence limits the use of hearing aids, since these regions respond minimally or not at all to sound amplification.9Our aim was to use the TEN test to investigate the presence of dead regions in the cochlea in subjects with sensorineural hearing loss seen at the Center for Audition, Vision and Language Disorders (CEDALVI) of the Sao Paulo University Craniofacial Anomalies Rehabilitation Hospital/Bauru Campus (HRAC-USP-Bauru).This study was undertaken at the CEDALVI/HRAC/USP-Bauru between August 2003 and February 2004. According to the routine at CEDALVI, patients are monitored by a multidisciplinary audiology team. Participants were assessed by an ENT specialist who did otoscopy and decided whether conditions were appropriate for the TEN test, namely intact tympanic membranes and no external and middle ear compromise.Cases for this study are shown on a)G1: pure tone air conduction thresholds within normal limits (≤15dBNA);b)G2: mild flat configuration sensorineural hearing loss (Davis and Silverman);c)G3: moderate to severe sloping sensorineural hearing loss.The selection of subjects was based on the following inclusion criteria:Participants signed a free informed consent form approved by the Research Ethics Committee of the HRAC-USP, number 263/2004-UEP-CEP, agreeing to take part in the study and to allow publication of collected data.a)TEN biological calibrationWe used the Veniarb)TEN testThe TEN test was applied using a two-channel audiometer, PAC 2000 model (Acustica Orlandi) with a TDH-39 P supra-aural earphone and a Sony Discman digital MEGA BASS model, in a soundproofed cabin. Moore et al. (2000) recorded the test on a CD with noise (TEN) on one channel and a digitally generated test signal (pure tone) recorded on another CD channel. This was done to simplify and increase the clinical availability of the TEN test.(1)the CD player output plug was connected to the audiometer right and left input socket . The first CD track is a calibrating tone, which was played to adjust the audiometer right and left channel VU meters at -5 dB, or 5 dB below zero; this was done to calibrate the signal-to-noise ratio, or the intensity difference between the pure tone and noise. For the remaining tracks the noise intensity level in the right channel was read as 10 dB better than the indicated level on the dial, and the left channel pure tone was read as 10 dB worse. We could then adjust the audiometer controls for the desired intensities for pure tones and noise. Thus, if the pure tone and noise intensities were 50 and 40dBNPS, the dial was adjusted to 40 and 50 dB, respectively.(2)Air conduction absolute thresholds (ATs) and masked thresholds (MTs) were obtained by manual audiometry, according to Carhart and Jerger,(3)MTs with noise (TEN) were assessed at the same frequencies, according to the audiometric configuration, with both channels operating. The TEN intensity depended on the AT by frequency in each patient, and was 10 dB better than the AT, that is, if the AT was 50 dB, the TEN was adjusted to 40 dB on the dial.(4)If the MT was only 5 dB worse than the AT, the test was repeated with higher masking noise intensity.The following test procedures were undertaken: a)Biological calibration of the TENThe minimal effective masking level considered was the amount of noise (TEN) that was needed to eliminate the test tone (30 dBNPS) for each frequency.b)TEN testAccording to Moore et al.'sc)Statistical methodThe following statistical tests were used: t test , and Spearman's rank correlation coefficient. The null hypothesis rejection value (Ho) was 5%.As seen on According to Moore et al.'sA frequency of occurrence analysis was made in group G3 , as show Statistical analysis using the t test (Ho= A=B and H1= B>A) was applied to group G3, in which the tests suggested dead regions in the cochlea according to Moore et al.'sThe t test (Ho= A=B e H1= B>A) was applied to group G3 to check whether there was any significant difference between the MT and the AT after applying the TEN test. No significant difference was seen between the MT and the AT at all frequencies.Spearman's rank correlation coefficient (rs) (Ho= rs = 0 and H1= rs ≠ 0) was calculated to check whether dead regions could be correlated with different frequencies, in cases of sloping sensorineural hearing loss. Differences between the MT and the AT were reported by frequency in group G3.The correlation is significant only for differences between the MT and the AT between frequencies of 1 kHz and 2 kHz, 6 kHz and 8 kHz, and 3 kHz and 4 kHz for group G3. The correlation is not significant between frequencies of 1 kHz and 8 kHz, and 1 kHz and 6 kHz.A 5dB variation over the absolute auditory threshold following presentation of TEN was considered as indicating the absence of dead regions in the cochlea. This variation was observed in the biological calibration procedure for ten normal ears (group G1; According to Moore et al.'sStatistical analysis showed that the difference between the MT and the AT was higher only for 2 kHz when compared to 1 kHz. Moore et al.Equality relations between 8 kHz and 6 kHz, and 4 kHz and 3 kHz could be associated with the closeness of theses frequencies; a larger difference has been observed from 2 kHz upwards, which suggests constancy in the variation of masked thresholds for high frequencies.Inconclusive results were seen more frequently at frequencies of 6 kHz and 8 kHz , mostly Spearman's rank correlation coefficient was calculated for group G3 to check whether dead regions could be associated with different frequencies in sloping sensorineural hearing loss. There was no significant correlation between distant frequencies, such as between 1 kHz and 8 kHz, and 1 kHz and 6 kHz. For close frequencies, such as 1 kHz and 2 kHz, and 3 kHz and 4 kHz for G3, and 6 kHz and 8 kHz, there was a significant correlation, which could be explained by the excitation diffusion phenomenon in the case of dead regions within the nervous fiber response area. According to Henderson et al.,White noise tests for dead regions, however, require further validation, such as comparisons with PTC results. PTC is a proven and internationally accepted method to study cochlear tuning curves (HalpinSummers et al.The TEN test described by Moore et al.During the TEN test dial readings can by somewhat confusing; according to Moore el al.'sTo address these difficulties in the TEN test, MooreAlthough we saw a significant difference between the MT and the AT at all frequencies, the number of inconclusive results  and the As Moore-Dead regions in the cochlea were present in 92% of cases of sloping sensorineural hearing loss and absent in flat hearing loss.-The TEN test is effective to suggest the presence of dead regions in the cochlea in individuals with sloping sensorineural hearing loss.A critical analysis of our results in this study allows us to conclude that:


While fibroblast growth factor/extracellular signal-regulated kinase (FGF/ERK) signalling is a key regulator of ESC differentiation and PrE specification, we find it is not just responsible for ESCs heterogeneity, but also the inheritance of similar cell cycles between sisters and cousins. Taken together, our results indicate a tight relationship between transcriptional heterogeneity and cell cycle regulation in lineage specification, with primed cell populations providing a pool of flexible cell types that can be expanded in a lineage-specific fashion while allowing plasticity during early determination.During embryonic development cells acquire identity as they proliferate, implying that an intrinsic facet of cell fate choice requires coupling lineage decisions to cell division. How is the cell cycle regulated to promote or suppress heterogeneity and differentiation? We explore this question combining time lapse imaging with single-cell RNA-seq in the contexts of self-renewal, priming, and differentiation of mouse embryonic stem cells (ESCs) towards the Primitive Endoderm (PrE) lineage. Since ESCs are derived from the inner cell mass (ICM) of the mammalian blastocyst, ESCs in standard culture conditions are transcriptionally heterogeneous containing dynamically interconverting subfractions primed for either of the two ICM lineages, Epiblast and PrE. Here, we find that differential regulation of cell cycle can tip the balance between these primed populations, such that naïve ESC culture promotes Epiblast-like expansion and PrE differentiation stimulates the selective survival and proliferation of PrE-primed cells. In endoderm differentiation, this change is accompanied by a counter-intuitive increase in G1 length, also observed  Naïve mouse embryonic stem cells (mESCs) are karyotypically normal, immortal cell lines derived from the inner cell mass (ICM) of pre-implantation embryos . ESCs arUnder specific conditions, mESCs can recapitulate several aspects of the ICM specification. When cultured in serum and the cytokine LIF, they constitute a dynamically heterogeneous cell culture model that contains populations that are primed, but not committed, for both primitive endoderm (PrE) and Epiblast. These populations exhibit biases in differentiation but will readily interconvert when left in self-renewing culture . While tin vivo. Although they have explored the role of pluripotency factors in lineage priming , in addition to LIF (2i/LIF) . Taken t priming , they haIn vivo, the segregation of PrE from Epiblast is regulated by the FGF/ERK pathway  is an important component of the defined ESC culture system known as 2i/LIF  was found to support the more rapid proliferation of the Epiblast-primed population, and PrE differentiation (RACL) promotes the more rapid proliferation of cells primed for endoderm differentiation. We found that FGF signalling regulates cell states and proportions through both coordinating inheritance of cell cycle lengths as well as rates of endoderm priming. This functional regulation of cell cycle in heterogeneous cell cultures indicates that cell cycle synchronization supports self-renewal as well as preparing cells for further differentiation. Furthermore, we found that G1 length is adjusted independently from the cell cycle, pointing to a model where cells receive signals from cytokines during G1, while still actively proliferating. Taken together, our work suggests that cell cycle length is not only tightly regulated by the culture context, but that this coordination has a functional role in both heterogeneity and lineage choice.in vitro to those occurring in vivo, we performed single-cell RNA sequencing by MARS-seq  PrE-like cells and follow their differentiation using time lapse microscopy. We exploited a mESC line that couples a sensitive PrE reporter, Hhex-mCherry, with a second that enables lineage tracing, H2B-Venus  . These ck-means clustering and used this clustering to assign identity in differentiation  (As possible contributors to the growing PrE population across n day 3) , Table 2n day 3)  and suggHhex expression during the first 72 hr, thereby transitioning into the PrE cluster. However, after the 72-hr time point, only 1.9 ± 1.2% of the cells were able to transit between clusters. This suggested that competence to enter the endoderm lineage was lost after the first 3 days of the differentiation, indicating that proliferation was enhanced following lineage choice. Hence, even though only a small population of cells entered differentiation, the selective expansion of these cells allowed them to take over the final culture.As these cultures started from a homogeneous population of 2i/LIF cells, we assumed some conversion to endoderm fate preceded the acceleration of proliferation and assessed the probability of cell state transition before and after day 3 (72 hr). We found that 12.6 ± 0.6% of the total population upregulated t in s in d in To assess whether the combination of this small amount of lineage conversion followed by enhanced proliferation was sufficient to account for the behaviour of differentiating cultures, we sought to generate a simple model introducing the transition probability observed in time lapse t in . This miHhex at higher levels (0.18) compared to cells that are non-endodermal progenitors  . Thus, we took the dataset from day 2 of differentiation and asked whether low level reporter expression correlated with later differentiation by asking which cells would prospectively give rise to PrE (PrE Fate) and which would never become endoderm by day 3 (NEDiff Fate). We found that there was a tendency for PrE progenitor cells (PrE Fate) to express e, 0.15) , Table 3ulations .in vitro PrE differentiation was similar to the spontaneous dynamic priming that occurs in standard ESC culture, but primed PrE remained a minority of the culture, we hypothesized that ESC culture might favour the proliferation of the undifferentiated or Epiblast-like population. As ESC culture can be supported by the same cytokines but with a different base media , the Hhex-expressing primed population had a short cell cycle, but in NACL, where ESC self-renewal predominates, we found the opposite. Here, the Low Hhex cells exhibited significantly shorter division times than the High Hhex cells  have a higher ratio of G1 to cell division than slow cells (0.15 ± 0.06 vs. 0.14 ± 0.04), suggesting Low Hhex cells have a longer G1. Taken together, our results suggest that PD03 promotes the faster proliferating Low Hhex population, but that the decreased cell cycle time in this population is not a result of stimulating G1/S transition, consistent with the role of this drug in inhibiting it  such that endodermal identity accrues with each cell cycle. Primed cells that fail to enhance endodermal gene expression tend to die. By day 3 in differentiation, all the future founder cells in the culture are set, and further commitment does not occur. At this point, a combination of selective cell survival, increased rates of proliferation and enhanced endodermal transcription leads to the final differentiated cell populations. By the later time points in differentiation, the combination of single-cell sequencing, time lapse imaging, and mathematical modelling, suggests that cells that fail to enter the endoderm program selectively die, but at early time cell division dominates. Moreover, while differentiation promotes more rapid cell cycling, it also produces apparently contradictory increases in G1 length.What are the factors that govern a cell’s choice to differentiate? An increase in G1 would give cycling cells more time to respond to inductive signals, ultimately leading to increased responsiveness to differentiation cues. This would suggest that cells progress into early differentiation, lengthening their G1 phase, thereby improving accessibility to cues promoting commitment, and enabling signalling dependent transcription factors to accumulate ahead of replication. While the link between G1 phase and commitment of pluripotent stem cells into differentiation was first described in 1987 in embryonal carcinoma cells , the recin vivo, where there is a progressive increase in G1 despite the robust activation of ERK signalling. Cell cycle progression is tightly regulated by the activity of cyclin-dependent kinases (CDKs), and inhibition of cyclin E-CDK2 produces G1 lengthening and spontaneous differentiation in pluripotent stem cells  by ERK , this doem cells . In mESCem cells , we propMammalian cells share the striking quality of being synchronized in cell cycle length between members of the same generation (sisters and cousins), but not with the previous generation (mother–daughter). This was originally demonstrated in both a lymphoblast cell line and ESCs . MoreoveIn vivo, where PrE differentiation occurs in a salt and pepper pattern in three dimensions, synchronization could enable cells to retain the memory of a signal regardless of their position and despite cell mixing. Moreover, if G1 represents the window of opportunity for cells to act upon signalling cues, we envision cell synchronization could represent inheritance of signalling opportunity, promoting similar durations of active transduction in clones of differentiating cells. If ERK is regulating both differentiation and cell cycle synchronization via G1, it is also possible that cell cycle synchronization is a consequence of ERK-mediated alterations to the temporal window over which a cell responds to cytokine signalling.Given that cell cycle synchronization can be observed on both fast and slow proliferating cells, its relevance to differentiation is difficult to discern. If changes in proliferation are a response to differentiation cues, then synchronous inheritance could ensure that daughter cells retain an equivalent enhanced cell cycle state from their parents regardless of their position relative to the source of the signal. in vitro occurs over several days, while in embryonic development the segregation of Epiblast from PrE occurs over a much shorter time scale, approximately 24 hr. While this difference limits the extent to which our observations in vitro can be extrapolated to embryonic development, the similarities between both transcriptome and G1 regulation are striking. Therefore, we speculate that cell cycle synchronization could facilitate a coordinated change in cell division time and G1 length in the context of embryonic lineage specification, enabling coordinated signalling responses.Differentiation in vivo and in vitro differentiation is different, it appears that the use of increasing G1 to drive increasing levels of commitment is a fundamental facet of cell fate choice.Altogether, our data suggest that ESC cultures and their differentiation are complex models that involve an interplay of both priming-based selection and proliferation. We find that cell state changes are major drivers of culture-specific changes in proliferation that, in turn, contribute to the final state of a culture. Despite the increasing rates of proliferation, cells exploit G1 to integrate the signals coming from their environment and evaluate their choices. Although the time scale of the Mouse ESC lines were cultured in standard conditions in NACL as previously described in Mouse ESCs lines used in this study were generated in E14JU ESCs. This line was derived from a 129/Ola background.Hhex transcription can be visualized by translational amplification of Hhex, which drives the expression of the monomeric fluorescent protein mCherry (The cell line HFHCV was previously reported in  mCherry .CpoI (RsrII) and KlfI enzymes and inserted upstream of the Hygromycin resistance gene cassette of the ES-FUCCI plasmid (Addgene #62451). FCXCT2 ESCs (BglI-linearized FUCCI-H2B-miRFP670 DNA (25 μg), and stable transfectants were selected for with hygromycin (125 μg/ml). Cell lines were validated by flow cytometry, fluorescence microscopy, and karyotyping.The FUCCI-H2B-miRF670 reporter construct was constructed as follows. The H2B and miRFP670 sequences were fused by overlap extension PCR. The miRFP670 sequence was cloned from pY42-pmiRFP670AAA-NLS-Myc, a kind gift from YH Kim. The resulting fragment H2B-miRFP670 was inserted into the PCR-Blunt II-TOPO backbone by TOPO cloning. The H2B-miRFP670 fragment was digested with the CT2 ESCs  were eleAll cell lines have been routinely tested for mycoplasma.2 and 20% O2 at 37°C under a Deltavision Widefield Screening microscope. ESCs were seeded at 300 cells/cm2 48 hr before the beginning of the time lapse for NACL and PD03 experiments, or at 30,000 cells/cm2 24 hr before for PrE differentiation. For PD03 experiments, cells were cultured in NACL for at least two passages and PD03 was added just before starting the time lapse. For PrE experiments, cells were cultured in 2i/LIF for 2 passages and then changed to RPMI minimal medium just before starting the experiment. Activin A, Chiron, and LIF (RACL) were added 24 hr after the time lapse started. In all experiments, the media was changed every day of the time lapse. To test that the laminin coating was not affecting the behaviour of the HFHCV cell line, we sorted Hhex-high and low populations seeded in both gelatine and laminin and analysed them 24 and 48 hr later to test that their re-equilibration rates are the same.Mouse ESC lines were cultured in NACL or 2i/LIF medium on Laminin 511 (BioLamina)-coated 8-well slides (Ibidi) and imaged at 20 min intervals for 6 days in mCherry and Venus fluorescent light channels, in 5% COCells were collected by trypsinization and stained against a marker of undifferentiated mESCs, Pecam-1 , or a marker for PrE differentiated cells, PDGFRA , and DAPI  to exclude dead cells. mESCs were stained for 15 min at 4°C before being washed and resuspended in FACS buffer  in phosphate-buffered saline) with DAPI. Flow cytometry analysis was carried out using a BD LSR Fortessa, and flow cytometry sorting was carried out in a BD FACS Aria III. Data analysis was carried out using FCS Express 6 Flow software (De Novo Software) by gating on forward and side scatter to identify a cell population and eliminate debris, then gating DAPI negative, viable cells before assessing the levels of GFP, mCherry, or APC.Mouse ESCs were cultured in 8-well slides (Ibidi). ESC immunostaining was carried out as previously described in Hhex in each time point. Only cells with completed cell cycle information were used for cell cycle and compartment analysis.We performed manual cell tracking using Imaris v9.5 (Bitplane). Nuclei were segmented using the H2B marker, and we measured the Hhex-mCherry fluorescence intensity of a circular area of 50 μm diameter inside the segmented nuclei. For each area measured, we took the median fluorescence intensity as the measure for that given data point. Intensity measurements were linked to its time point and lineage, allowing us to infer the division time for each cell that was tracked, as well as the expression level of Data mining was performed with Matlab. A script was generated to take the separated output data frames from Imaris and convert into a single file containing all the dataset information, by cell. This file also contains the lineage tree information, mean fluorescence intensities, and division times. Alive and dead cells, as well as cells without complete cell cycle information, are located into groups for further analysis. Another file organized by time frame is generated in order to perform time dynamics analysis.Hhex compartments were determined by dividing the Hhex-mCherry intensity distribution into quartiles. All cells above the first quartile (0.1648) are considered High, and all cells below the third quartile (0.1376) are Low. The same quartiles are used for the PD03 dataset  and x is the number of days, starting with 1. The number of NEDiff cells can then be described as follows, where 1s is their survival rate:The model describes the growth of the NEDiff population and PrE populations as exponential growth functions. Starting from t and consequently divide with division time d2 and survive with survival rate s2:PrE cells are described as the sum of cells that transition from NEDiff to PrE at each generation with rate The sum was solved using Wolfram Alpha Mathematica.CellCycleScoring function.Single-cell libraries were sequenced using Illumina NextSeq 500. Pre-processing steps and quality control were done as described previously in p value = 0.05) we define these clusters transcriptionally similar.To estimate similarity between clusters we used CAT . First, https://github.com/SilasBoyeNissen/TranscriptionalHeterogeneityAndCellCycleRegulationAsCentralDeterminantsOfPrimitiveEndodermPriming, , different culture conditions favor acceleration/stimulation of cell cycle of different cell populations, and that only a small population of cells from the original culture enters a differentiation process which is followed by selected expansion and/or survival of their progeny. They also observed that during endodermal specification (towards PrE), the cell cycle was shortened with a proportional relative increase of G1 phase length and that FGF activity is responsible for cell cycle synchronization and the inheritance of similar cell cycles between sisters and cousins. Together these finding indicate a close relationship between transcriptional heterogeneity and cell cycle regulation during lineage priming. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Transcriptional Heterogeneity and Cell Cycle Regulation as Central Determinants of Primitive Endoderm Priming." for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:eLife once the below concerns have been addressed.This paper probes the link between lineage priming, lineage specification, and cell cycle in the ESCs. The authors report a number of interesting findings, including that: differential regulation of the cell cycle can tip the balance between populations of cells primed to different cell fate choices ( PrE vs Epi), different culture conditions favor acceleration/stimulation of cell cycle of different cell populations, and that only a small population of cells from the original culture enters a differentiation process which is followed by selected expansion and/or survival of their progeny. They also observed that during endodermal specification (towards PrE), the cell cycle was shortened with a proportional relative increase of G1 phase length and that FGF activity is responsible for cell cycle synchronization and the inheritance of similar cell cycles between sisters and cousins. The reviewers agree that this work is interesting and well done and would be appropriate for publication in 1) There was concern about the massive amount of cell death in the time-lapse data underlying Figures 2 and 3. The media-specific effects on cell survival need to be explained and discussed in a revised manuscript.2) There was also concern about the extent to which parallels can be drawn between findings in ESCs and embryonic development. For example, ESCs have a normal growth phase and so all these cells in culture have the same size but early embryo cleavages occur without a growth phase. Similarly, the timeline of PrE vs Epi specification in vivo and in vitro are completely different. In embryos, PrE is specified within 24h, whereas in vitro it takes 6 days. Thus it is difficult to see how these two timelines together with the different cell cycle lengths can be reliably compared. These limitations should be discussed in a revised manuscript.3) At least for the PrE differentiation conditions, the authors need to better emphasize the strong survival effects of the media and discuss the relative contributions of selective cell death and cell cycle changes for generating a homogeneously differentiated PrE culture.4) The authors should address whether RACL is purely selective through changes to cell survival and cell cycle, or does it in addition trigger an increase in priming towards the Hhex-positive population before the acceleration of the cell cycle between days 2 and 3? It would be informative to compare transition rates towards the Hhex-high state between NACL and RACL to address this question5) The authors should clarify their reasoning as to why cell cycle synchronization is relevant for the differentiation of a culture, or lineage priming . How does the absence or presence of cell cycle synchronization between daughter cells impact the composition of cell populations at a steady state or upon differentiation, as both long and short cell cycles appear to be equally well correlated?6) The use of the term "differentiation" is somewhat confusing . As this is an initially heterogeneous cell population transiting to a more homogeneous, different state there may be a better way of describing this without terming it differentiation.7) The data underlying the table in Figure S5C seem important as they address how state transitions shape the population structure in addition to the cell cycle properties of the different compartments. This should be discussed more extensively in a revised manuscript and perhaps moved to the main Results section. It would also be interesting to quantify residence times and rates of state transitions in units of time-1. These analyses could then be quantitatively compared to corresponding transitioning rates in the presence of PD03 8) In Line 24 of the abstract the authors state that there is a lengthening of G1 in PrE-favouring conditions, but in the main body of the text it is the proportion between G1 and the rest of the cycle that is changing, "). Similar claims about the lengthening of the G1 are made in the Discussion. The authors should ensure that the result is described consistently throughout the manuscript9) In Line 123 of the Results section, the authors state that: "The identity and distribution of PrE and NEDiff states were confirmed with GATA6 and NANOG staining ." but the data presented by the authors are not sufficient to support this claim. Pairwise correlation between individual marker intensity needs to be calculated to prove this point with appropriate controls (for example with the use of the same primary antibody with two different secondary antibodies)10) In Line 274 of the Results section, the authors state: "As we cannot combine the G1 reporter with Hhex lineage, we cannot assess whether it is the High Hhex or Low Hhex cells which present a longer G1." – I wonder why the authors cannot use here the same combination of the time-lapse plus immunostaining images as in the earlier experiments.11) The figure legend for figure S1 needs more detail so that it can be followed without cross-referencing with the main text and figure 1. Essential revisions:1) There was concern about the massive amount of cell death in the time-lapse data underlying Figures 2 and 3. The media-specific effects on cell survival need to be explained and discussed in a revised manuscript.We acknowledge that there is a large amount of cell death in the beginning of differentiation and believe this could be a response to changes in media and cytokines. This is also observed in other differentiation and reprogramming protocols .While our original analyses were focused on cell cycle, we did not mean to imply that survival is irrelevant to selecting the correct populations at the end of differentiation. Although there is clearly an increase in NEDiff cell death, these rates fluctuate during differentiation (Table 2) and it is difficult to make any hard conclusions about the timing of selection. In addition, we find no signature for apoptosis in all scRNA-seq clusters associated to early NEDiff. However, we thank the reviewers for raising this issue and we address the issue of survival more extensively in the revised manuscript .To further explore when relative survival is most important for effective differentiation, we have reformulated our model. Our new modelling results show that changes in the survival rates have stronger impact shifting the cell proportions at later stages of differentiation. Phase diagrams in Figure 3- Supplement 1 show that at day 5 reaching an appropriate proportion of PrE requires less change in the survival rates than in doubling times, suggesting that survival can have greater impact on the ratio than cell division. However, at day 3, the modelling suggests the opposite, consistent with a first wave of a cell cycle regulation and PrE fastest division time . We have discussed this in the Results section, lines 162-168.We have added survival alongside proliferation to the abstract.2) There was also concern about the extent to which parallels can be drawn between findings in ESCs and embryonic development. For example, ESCs have a normal growth phase and so all these cells in culture have the same size but early embryo cleavages occur without a growth phase. Similarly, the timeline of PrE vs Epi specification in vivo and in vitro are completely different. In embryos, PrE is specified within 24h, whereas in vitro it takes 6 days. Thus it is difficult to see how these two timelines together with the different cell cycle lengths can be reliably compared. These limitations should be discussed in a revised manuscript.We were aware of the difficulties inherent in this comparison and apologize if our statements were not sufficiently conditional. We have now added caveats to the Results section (line 328) and a revised discussion that explicitly discusses the difference in time lines (lines 404-409).On line 111, we added a clarification that our in vitro model is a tool for deconstructing “transcriptional signatures” as this is precisely what we measure both in vivo and in vitro.On line 322 we clarified that even though the time frames are different, we found a similar G1 signature trend in our in vitro dataset than the one found in the in vivo published dataset.On line 337 we amended the sentence to clarify that some aspects of cell cycle regulation found in vitro could be also exploited in vivo.On line 414 we have added a caveat line explaining that the use of G1 to drive increasing levels of commitment is a facet of cell fate choice both in vivo and in vitro, even though the time scales are different.3) At least for the PrE differentiation conditions, the authors need to better emphasize the strong survival effects of the media and discuss the relative contributions of selective cell death and cell cycle changes for generating a homogeneously differentiated PrE culture.As discussed above, we have now added discussion of the contribution of cell survival  and adapted our model to evaluate contributions of differences in survival and doubling times to differentiation (see Point 1 above).4) The authors should address whether RACL is purely selective through changes to cell survival and cell cycle, or does it in addition trigger an increase in priming towards the Hhex-positive population before the acceleration of the cell cycle between days 2 and 3? It would be informative to compare transition rates towards the Hhex-high state between NACL and RACL to address this questionTo address this point we compared transition rates towards the High Hhex state in the different media conditions, and found that there is an increase in priming towards the High Hhex population in days 2 and 3 of PrE differentiation (compared to NACL). We thank the reviewer for this comment and we now discuss this point (lines 223-225) and have added this to Figure 4 – Supplement 1D.5) The authors should clarify their reasoning as to why cell cycle synchronization is relevant for the differentiation of a culture, or lineage priming . How does the absence or presence of cell cycle synchronization between daughter cells impact the composition of cell populations at a steady state or upon differentiation, as both long and short cell cycles appear to be equally well correlated?This is a very good question and we can only speculate as to how cell synchronization impacts on population composition. We have added a discussion of this point (lines 393-403) and quote it here for convenience.“If changes in proliferation are a response to differentiation cues, then synchronous inheritance could ensure that daughter cells retain an equivalent enhanced cell cycle state from their parents regardless of their position relative to the source of the signal. in vivo, where PrE differentiation occurs in a salt and pepper pattern in three dimensions, synchronization could enable cells to retain the memory of a signal regardless of their position and despite cell mixing. Moreover, if G1 represents the window of opportunity for cells to act upon signalling cues, we envision cell synchronization could represent inheritance of signalling opportunity, promoting similar durations of active transduction in clones of differentiating cells. If ERK is regulating both differentiation and cell cycle synchronization via G1, it is also possible that cell cycle synchronization is a consequence of ERK mediated alterations to the temporal window over which a cell responds to cytokine signalling”6) The use of the term "differentiation" is somewhat confusing . As this is an initially heterogeneous cell population transiting to a more homogeneous, different state there may be a better way of describing this without terming it differentiation.We have changed the phrasing on line 136, now corresponding to line 139. Current phrasing is as follows:“This is the point in time when PrE clones became distinguishable from NEDiff cells in the time lapse video (Video 1) and suggested that culture conditions promoted endoderm specification by providing an environment that favoured the proliferation of a PrE-primed population.”7) The data underlying the table in Figure S5C seem important as they address how state transitions shape the population structure in addition to the cell cycle properties of the different compartments. This should be discussed more extensively in a revised manuscript and perhaps moved to the main Results section. It would also be interesting to quantify residence times and rates of state transitions in units of time-1. These analyses could then be quantitatively compared to corresponding transitioning rates in the presence of PD03 We have moved Figure S5C to Figure 4E and discussed it further in the text (lines 213-216). We have also calculated transitions times using the division times from the dataset, and we added this to Figure 4 – Supplement 1C (and lines 217-220) for NACL and Figure 5 – Supplement 2A for PD03 (line 247). We observed the same trend for transition time than what we described for transition probability.We have also calculated residence times for each state in NACL and PD03 (Table 4) and discussed it further in the text (Lines 221-223 for NACL and 250 for PD03). We calculated residence times based on generations and in hours.Based on this information, it appears that the major impact of PD03 is to reduce the residence time in the High Hhex state while increasing the residence time in the Low Hhex state.8) In Line 24 of the abstract the authors state that there is a lengthening of G1 in PrE-favouring conditions, but in the main body of the text it is the proportion between G1 and the rest of the cycle that is changing, "). Similar claims about the lengthening of the G1 are made in the Discussion. The authors should ensure that the result is described consistently throughout the manuscriptWhile the claim in line 24 refers to PrE differentiation conditions, where we see a lengthening of G1 phase , the line referring to G1 being marginally increased refers to Figure 6A, where G1 and division times are measured in steady state conditions . We apologize if our claims were confusing and we have added a clarification in lines 297 and 318.9) In Line 123 of the Results section, the authors state that: "The identity and distribution of PrE and NEDiff states were confirmed with GATA6 and NANOG staining ." but the data presented by the authors are not sufficient to support this claim. Pairwise correlation between individual marker intensity needs to be calculated to prove this point with appropriate controls (for example with the use of the same primary antibody with two different secondary antibodies)We confirmed the progressive acquisition of GATA6 cells and the loss of NANOG cells at different time points of PrE differentiation by quantifying multiple images from the immunostaining to Figure 2 —figure supplement 2B, together with a negative control corresponding to a sample without a primary antibody. The plots show individual datapoints corresponding to single cells, segmented based on DAPI staining.To address this point we have also revised the text (line 127) to read “The identity and time evolution of PrE and NEDiff states were confirmed with GATA6 and NANOG staining ." We did this as we realized the word “distribution” may be misleading.10) In Line 274 of the Results section, the authors state: "As we cannot combine the G1 reporter with Hhex lineage, we cannot assess whether it is the High Hhex or Low Hhex cells which present a longer G1." – I wonder why the authors cannot use here the same combination of the time-lapse plus immunostaining images as in the earlier experiments.High and Low Hhex states are too dynamic to infer cell states based on antibody staining of ESC culture. If we stain with Hhex at the end of a FUCCI time lapse, there is no way to infer whether that High Hhex cell was also High 3 days prior to the staining. In the case of GATA6, we were labelling cells at the end of differentiation, and then inferring the nature of progenitors of these differentiated cells.Our means of assigning dynamic identity in ESCs therefore required a different approach. Here, we know that fast dividing cells correspond to the Low Hhex, and slow dividing cells represent the High Hhex fraction. We therefore used division time to infer cell identity and found that fast dividing cells have longer G1 Ratio than slow cells (0.15 ± 0.055 vs 0.141 ± 0.043), suggesting Low Hhex cells have a longer G1, as indicated in the Results section, line 303-305.11) The figure legend for figure S1 needs more detail so that it can be followed without cross-referencing with the main text and figure 1.We have supplied detail.References:Nature 576 (7787): 487–91. https://doi.org/10.1038/s41586-019-1825-8.Argelaguet, Ricard, Stephen J. Clark, Hisham Mohammed, L. Carine Stapel, Christel Krueger, Chantriolnt-Andreas Kapourani, Ivan Imaz-Rosshandler, et al. 2019. ‘Multi-Omics Profiling of Mouse Gastrulation at Single-Cell Resolution’. 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