To which extent trauma- induced disturbances in ionized calcium (iCa2+) levels have a linear relationship with adverse outcomes remains controversial. The goal of this study was to determine the association between the distribution and accompanying characteristics of transfusion-independent iCa2+ levels versus outcome in a large cohort of major trauma patients upon arrival at the emergency department.® (2015–2019) was performed. Adult major trauma patients with direct admission to a European trauma centre were selected as the study cohort. Mortality at 6 h and 24 h, in-hospital mortality, coagulopathy, and need for transfusion were considered as relevant outcome parameters. The distribution of iCa2+ levels upon arrival at the emergency department was calculated in relation to these outcome parameters. Multivariable logistic regression analysis was performed to determine independent associations.A retrospective observational analysis of the TraumaRegister DGU® 30 183 adult major trauma patients were found eligible for inclusion. iCa2+ disturbances affected 16.4% of patients, with hypocalcemia (< 1.10 mmol/l) being more frequent (13.2%) compared to hypercalcemia .In the TraumaRegister DGUP < .001) to have severe injury, shock, acidosis, coagulopathy, transfusion requirement, and haemorrhage as cause of death. Moreover, both groups had significant lower survival rates. All these findings were most distinct in hypercalcemic patients. When adjusting for potential confounders, mortality at 6 h was independently associated with iCa2+ < 0.90 mmol/L , iCa2+ 1.30–1.39 mmol/L , and iCa2+  ≥ 1.40 mmol/L . Moreover, an independent relationship was determined for iCa2+ 1.00–1.09 mmol/L with mortality at 24 h , and with in-hospital mortality . Both hypocalcemia < 1.10 mmol/L and hypercalcemia ≥ 1.30 mmol/L had an independent association with coagulopathy and transfusion.Patients with hypo- and hypercalcemia were both more likely (Transfusion-independent iCa2+ levels in major trauma patients upon arrival at the emergency department have a parabolic relationship with coagulopathy, need for transfusion, and mortality. Further research is needed to confirm whether iCa2+ levels change dynamically and are more a reflection of severity of injury and accompanying physiological derangements, rather than an individual parameter that needs to be corrected as such.The online version contains supplementary material available at 10.1186/s13054-023-04541-3. Disturbances in ionized calcium iCa2+) levels are a frequent finding in critically ill and severely injured patients , 2. It i+ levels In contrast to the protein-bound fraction of calcium, the unbound ionized free calcium in the extracellular space is biologically active in a variety of critical physiological pathways including blood coagulation, muscular and cardiac contraction, neuromuscular transmission, and hormone secretion . In majoThe subsequent hypocalcemia can cause disturbances in muscular contractility with decreased cardiac function, vasodilation, and respiratory failure –10. FurtPrevious research has indicated that transfusion-related hypocalcemia during trauma resuscitation is associated with increased mortality, coagulopathy, and blood transfusion requirements , 13–17. However, most of these findings are reported in small cohort studies, with only moderate evidence . The queThe aim of this study was to determine the association between the distribution and accompanying characteristics of transfusion-independent ionized calcium levels versus outcome in a large European cohort of major trauma patients upon arrival at the emergency department (ED).®.For this retrospective observational analysis, data was retrieved from the TraumaRegister DGU® of the German Trauma Society  is a multicentre database of pseudonymized and standardized documentation of severely injured patients. The inclusion criteria are ED admission to hospital, with life signs on arrival, and subsequent intensive care unit (ICU) management or reaching hospital with vital signs but dying before ICU admission. The participating hospitals are primarily located in Germany (90%), but an increasing number of hospitals from other countries contribute data as well. The ‘full dataset’ is obligatory for all supra-regional trauma centres, only hospitals certified as a regional or local trauma centre within the TraumaNetzwerk DGU® are allowed to complete a ‘basic dataset’.The TraumaRegister DGULaboratory values on admission are defined as the first documented values at time of first blood collection upon arrival at the ED.A detailed description of the dataset is provided in Additional file ® and is registered under the TR-DGU project ID 2020-030.The present study is in line with the publication guidelines of the TraumaRegister DGU® ‘version 2015’ dataset (accident years 2015 until 2019). The selection of the study sample is shown in Fig. The study cohort was selected from the TraumaRegister DGUTo check for potential bias, additional analysis of demographic and outcome data was performed for patients who were excluded because of missing/invalid iCa2+ levels.The distribution of documented ionized calcium levels was analysed by measuring the proportion of each level in the study cohort. Ionized calcium levels were categorized as (mmol/l) < 0.9 (< 0.90), 0.9 (0.90–0.99), 1.0 (1.00–1.09), 1.1 (1.10–1.19), 1.2 (1.20–1.29), 1.3 (1.30–1.39), and ≥ 1.4 (≥ 1.40).For each of these levels, mortality at 6 h, 24 h, and in-hospital mortality was determined. Furthermore, the incidence of coagulopathy upon arrival and transfusion requirement were determined. Coagulopathy was defined as a partial thromboplastin time (PTT) ≥ 40 s or an international normalized ratio (INR) ≥ 1.4, according to the Berlin definition of polytrauma . TransfuNext, three subgroups were defined, based on the incidence of adverse outcome and in line with previous reports on this topic: normocalcemia (1.10–1.29 mmol/l), hypocalcemia (< 1.10 mmol/l), and hypercalcemia (≥ 1.30 mmol/l) .P value ≤ 0.05 was considered as statistically significant. However, in large samples small differences could become statistically significant, even without any clinical importance. Therefore, P values are not shown in the presentation of the demographics and clinical characteristics in Table Relevant patient demographics, clinical characteristics and outcome parameters were retrieved from the registry for further analysis. Continuous variables were recorded as means with standard deviation (SD) if approximal normally distributed and median with interquartile range (IQR) otherwise. Categorical variables were presented as numbers with percentages. Differences were assessed by Student’s t test/Mann–Whitney U-test for continuous and Chi-squared test for categorical variables. A Kaplan–Meier survival curves were used to compare differences in time to death, and differences were evaluated with a log rank test.Multivariable models have been calculated for mortality , coagulopathy, and blood transfusion as dependent endpoints. The categorical variable iCa2+ 1.1 mmol/L was considered as the reference group.From the about 150 variables in the registry, potential predictors were selected a priori to the analysis. Because of the large sample size, statistical significance did not always correlate with clinical significance. Therefore, these variables were chosen based on previous literature studying prediction models for mortality, coagulopathy, and blood transfusion in major trauma patients. Moreover, prior expert discussion and the availability of the data within the registry were considered in the selection of these predictors.For prediction of blood transfusion and coagulopathy, the following independent predictors were considered: age, male sex, anticoagulation therapy before the accident, blunt trauma, relevant injuries (AIS ≥ 3) in different body regions, Injury Severity Score (ISS), prehospital Glasgow Coma Scale (GCS) ≤ 8, prehospital systolic blood pressure ≤ 90 mmHg, prehospital volume administration of more than 1000 ml. Age was considered in 4 subgroups , identical to previous studies, due to the known non-linear effect on mortality. Sample size above 59 years was calculated to ensure it was still large enough to yield statistically stable results . All the® since 2003 and was updated in 2014 to a second version (RISC II). This model predicts hospital mortality after trauma and was repeatedly validated in the years after its introduction. The current model consists of the following predictors: worst and second-worst injury (AIS severity level), head injury, age, sex, pupil reactivity and size, pre-injury health status, blood pressure, acidosis (base excess), coagulation (INR), haemoglobin, and cardiopulmonary resuscitation  ≥ 1), GCS ≤ 8, and hypothermia (temperature < 35 °C). Laboratory tests revealed lower haemoglobin levels and a higher incidence of coagulopathy and acidosis (base excess < − 6). During pre-hospital resuscitation, both groups were more likely to get intubated and to receive catecholamines. Prior to ICU admission, there was a higher incidence of transfusion, massive transfusion (packed red blood cells ≥ 10units), and Ca2+ supplementation. All these findings were statistically significant (P < 0.001), but not for the hypocalcemia group (P = 0.088).Within the abnormal ionized calcium level groups, the hypercalcemic group had more distinct findings for all the above-described parameters when compared to the hypocalcemia group, except for prehospital GCS ≤ 8. This discrepancy was mainly prominent for coagulopathy (31.9% vs. 21.9%) and acidosis (40.1% vs. 25.3%). Compared to normocalcemia, the incidence of prehospital volume administration > 1000 ml was significantly higher for the hypercalcemia group (P < 0.001).Haemorrhage as cause of death was more likely to occur in the hypercalcemia (13.1%) and hypocalcemia (10.7%) groups compared to the normocalcemia (4.9%) group , coagulopathy, and transfusion are presented in Table P < 0.001), iCa2+ 1.30–1.39 mmol/L , and iCa2+  ≥ 1.40 mmol/L . Moreover, an independent relationship was determined for iCa2+ 1.00–1.09 mmol/L with mortality at 24 h , and with in-hospital mortality .When adjusting for potential confounders, mortality at 6 h was independently associated with iCa2+  < 0.90 mmol/L  than hypercalcemia (3.2%). In this large European cohort, the reported incidence of hypocalcemia was remarkably lower compared to previous studies, with hypocalcemia being as frequent as 23% up to 74% [Reports on hypercalcemia are sparse because most studies seem to include hypercalcemia in the normocalcemia group. However, MacKay et al.  identifiThe differences in reported hypo- and hypercalcemia levels compared to those in previous studies are likely related to the different selection criteria used , 19, 31.It is well known that calcium levels may decrease rapidly during massive transfusion of large volumes of citrate containing blood products . HoweverIn contrast to a recent retrospective cohort study by Chanthima, in which the first measured iCa2+ nor the administered calcium dose for citrate load correction were significantly associated with in-hospital mortality, different previous studies have demonstrated an association between hypocalcemia and mortality , 4, 13. Based on these findings for hypocalcemic patients one could argue for early empirical calcium administration in bleeding trauma patients to avoid hypocalcemia and promote the priming effect in platelets . HoweverIncreasing iCa2+ levels were associated with an increasing incidence of coagulopathy, transfusion requirement, and mortality . The cumulative survival at 30d was even lower compared to hypocalcemia, with survival differences again being mostly related to differences in early mortality within the first day. Mortality within the first day was more than double in hypercalcemic compared to normocalcemic patients (13.2% vs. 6.0%). Likewise, MacKay et al.  also ideHowever, the independent association of hypercalcemia with coagulopathy and transfusion conflicts with the theory of calcium-induced platelet priming and thus requires different explanations. It is known that in critical ill patients the fraction of free ionized calcium changes due to alterations in serum protein concentrations and acid–base balance disturbances . In the Our findings do not support routine early empiric calcium supplementation. There is no simplistic linear relation between iCa2+ levels and outcome. Both hypo- and hypercalcemia have an independent association with coagulopathy and transfusion requirement. Despite a similar parabolic relation with unadjusted mortality, this parabolic relationship could not be demonstrated for 24 h or in- hospital mortality in a multivariable logistic regression model. This finding demonstrates that there is also no evidence to assume that early strict management of iCa2+ levels, with aggressive correction of both hypo- and hypercalcemia, has a beneficial effect on survival.The injury itself, the accompanying shock, and subsequent resuscitation will all affect the iCa2+ levels after trauma. Therefore, iCa2+ levels might be a much more dynamic concept and the importance during trauma resuscitation might be much more complex than is believed to date.A retrospective observational study design comes with its inherent limitations. Associations can be determined but causality cannot be proven, therefore result should be interpreted with caution and in their clinical context.Additionally, some specific limitations for this study need to be addressed.First, measurement errors cannot be excluded. Moreover, the exact timing of the measurement after arrival may slightly differ between samples. However, obtaining the first blood samples before extensive resuscitation is considered common practice and the large sample size used in this analysis will likely compensate for possible errors.Second, the exclusion of patients with missing iCa2+ levels could induce some bias in generalizing the results to the entire trauma population. No information was available whether this missing value was because of not measuring a blood gas sample or if this was a registration error. This excluded group had a slightly lower injury severity. Likely, the threshold to perform a blood gas analysis on arrival will be higher in less severely injured patients. Nevertheless, we believe that the sample we present in this study is representative for the overall major trauma population.Third, data about prehospital transfusion was not available in the dataset. However, in a recent survey by the European Society of Anaesthesiology only 6% of respondents from German Helicopter Emergency Medical Services (HEMS) had access to pre-hospital blood products. Moreover, in the overall European cohort, the majority of services with blood products available only used them very rarely, also including non-trauma cases , 36. The2 levels were available, the contribution of respiratory acidosis or alkalosis could not be assessed.Fourth, base excess was used as a marker of metabolic acidosis. As no pH or pCOIonized calcium levels in major trauma patients upon arrival at the ED have a parabolic relationship with outcome. In this multicentre analysis of 30 183 patients with major trauma, both hypo- and hypercalcemia were associated with an increased incidence of coagulopathy, need for transfusion, and mortality . In a multivariable logistic regression model, this parabolic relationship with coagulopathy, transfusion requirement, and 6 h mortality was confirmed. However, no parabolic or linear relationship with 24 h and in-hospital mortality could be demonstrated.This study shows no convincing evidence to support routine empirical administration of calcium. Further research is needed to confirm whether altered ionized calcium levels are more a reflection of severity of injury and accompanying physiological derangements, rather than an individual parameter that needs to be corrected as such. To better understand the dynamic alterations in iCa2+ levels, prospective trials should monitor iCa2+ levels at different time points after injury, in relation to acid–base status and resuscitation strategies.Additional file 1. Description of the TraumaRegister DGU®.Additional file 2. Distribution and accompanying outcome parameters of ionized calcium levels in adult major trauma patients.Additional file 3. Demographics and clinical characteristics for different ranges of ionized calcium levels.


Here, mass shifts of cluster ions, commonly used as calibrants, and other analytes are qualitatively evaluated on the Bruker amaZon spherical ion trap (QIT) and the Finnigan LXQ linear ion trap (LIT). To extend the mass range from previous experiments 3, and NaCF3COO cluster ions, CF3COO−, Na+, and Cs+ adduct ions, protonated commercial calibration solutions and peptides, and deprotonated peptides were investigated on the Bruker amaZon speed QIT and some of these were also investigated on the Finnigan LXQ LIT.Chemical mass shifts of CsI, Y(HCOO)m/z became apparent as m/z approached 1000. To some extent, the issues were more severe at slower scans. Peak distortions included loss of resolution, tailing, or fronting and were different between the amaZon QIT and the LXQ LIT. The noncluster and nonadduct ions analyzed showed no obvious mass shifts or peak distortions under the same analysis conditions.On both instruments, peak distortions and mass shifts toward lower m/z up to 4000 on both instruments. Peak distortions were different between the amaZon QIT and the LXQ LIT, and were not always visible despite mass shifts. Both mass shifts and peak distortions make cluster ions and some adduct ions unsuitable for ion trap calibration.As expected, the ion traps investigated here showed mass shift and peak distortion issues, and such issues persisted at  These manifest as mass shifts toward lower m/z (forward scan), but analysis can also be performed scanning from higher to lower m/z (reverse scan). It appears that reverse scans require lower auxiliary field amplitudes compared to forward scans for optimal ejection, thus reverse scanning may in some cases be used for ejection with less dissociation and less mass shift of fragile ions. However, some loss of resolution has been observed for the reverse scans.m/z in the reverse scan.Analyses in ion traps are commonly done by scanning from lower to higher In other cases not discussed here, (minor) chemical mass shifts have been attributed to nondissociating collisions with buffer gas (damping) and depend on collisional cross‐section. Such shifts show opposite signs for forward and reverse scans, respectively, so it has been proposed that accuracy can be increased by combining forward and reverse scans.m/z ranges (m/z < 3000), but extended mass range modes in commercial traps may allow analysis to m/z 6000, but little experimental investigation has been performed on mass shifts at high m/z. For calibration of high m/z ranges in elecrospray ionization mass spectrometry (ESI‐MS), cluster ions are the most common calibrants.m/z calibration of the amaZon speed QIT.m/z signals and experienced memory effects, so that was not tested here. Instead, high mass dendrimers found to yield high m/z signals were used for comparison to cluster ions at m/z 2000–4000. Despite the potentially severe memory effects of poly(propyleneglycol) and poly(ethyleneglycol),m/z calibration of ion traps than the dimers suggested by Bruker.Ion traps commonly operate in the lower 22.1Peptides, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), ESI tuning mix (perfluorinated phosphazenes), and CsI were from Sigma Aldrich. MeOH and NaHCOO were from Merck, MeCN was from Fisher, and HCOOH was from Fluka. Pierce™ LTQ ESI Positive Ion Calibration Solution  was from Thermo Scientific. Water was purified in a Synergy 185 system (Millipore) to a resistivity of 18.2 MΩ.cm. Dendrimers were provided by Polymer Factory Sweden AB.2.2The QIT used was the Bruker amaZon speed RF 0212G003 2013 spherical ion trap. Its ring electrode main radio frequency (RF) is 781 kHz, with the endcap auxiliary RF 1/3 of this. It was operated using TrapControl 8.0.25 .The LIT was the Finnigan LXQ 10149 linear ion trap. In this trap, the X‐rods (with ejection slits) are distanced 4.76 mm from the trap center, while the Y‐rods are 4 mm from the center. Its main RF is 1.2 MHz. It was operated using Tune Plus 2.2 (Thermo Electron).Both instruments used He as buffer gas. The pressure readings are for the high vacuum chamber, not the trap itself, where the pressure is higher.2.33) was prepared by suspending 200 mg/ml Y2O3 in HCOOH, then diluting 100× into 9:1 MeOH:H2O.8 ESI tuning mix stock solution was diluted 20× in 19:1 MeCN:H2O (5× recommended concentration). Pierce was used as delivered or, to obtain CsI‐clusters simultaneously as the calibrant signals, mixed with 5 mM CsI in MeCN 9:1 (amaZon) or 4:1 (LXQ). The peptide mixture consisted of Leucine‐enkephalin, Angiotensin II, Angiotensin I, Bombesin, and adrenocorticotropic hormone (ACTH) amino acids18–39 at 0.8 μM each, in 3:2 MeCN:H2O, with 0.1 vol% HCOOH. Low mass Na+‐adducted dendrimers  were prepared according to the literature.128H240O18 (“D2089”), C52H34O13I12 (“D2412”), C100H82O30I10 (“D3055”), and C124H106O37I12 (“D3732”), 100 μg/ml each, with 5 μg/ml NaHCOO, dissolved in THF:DMSO 1:1 and injected as such. Cs+ adducts of the dendrimers were obtained by adding 2 vol% 5 mM CsI. On the LXQ, both Na+, and Cs+ adducts were obtained simultaneously by infusion of 5 mM CsI in MeCN without rinsing before infusion of the dendrimer mixture.CsI was dissolved in MeCN to 5 mM . Positive mode spectra on the amaZon were calibrated against ESI tuning mix  and negative mode spectra against a peptide mixture . The high m/z ranges of both the LXQ and the amaZon were calibrated against the four Na+‐adducted dendrimers . Since it is not certain that the Na+‐adducted dendrimers show no shift themselves, it must be emphasized that all shifts shown are relative. Reported m/z values are averages of three spectra  and error bars show one standard deviation. All peak shapes shown are from averaging several spectra.All spectra were re‐calibrated against a seemingly relatively stable ion series after acquisition  and shifts shown are thus those relative to the calibrants. Calibration results such as curve fit and residual error can be seen in m/z 100–3000, and for m/z 2000–4000 Extended mass range (27 000 u/s) scan mode (the only available mode for the high mass range) was used. The default values used for low mass cutoffs were m/z 100, 70, 50, 50, and 200 for Xtreme, Ultra, Enhanced resolution, Maximum resolution, and Extended mass range, respectively. He pressure difference to setpoint was 1.80 × 10−9 bar for all modes except Maximum resolution, were it was 0.90 × 10−9 bar.On the amaZon, ion charge control (ICC) was set to 100 000 (200 000 default) and 20 scans/spectrum, and on the LXQ automatic gain control (AGC) was set to 15 000 and 10 scans/spectrum. On the amaZon, Xtreme (52 000 u/s), Ultra (32 500 u/s), Enhanced resolution (8100 u/s), and Maximum resolution (5200 u/s) scan modes were used for m/z 150–2000, and for m/z 2000–4000 Turbo, Normal, and Zoom scan modes were used (Enhanced scan mode is not available for the high mass range). The default values used for low mass cutoffs were m/z 50 for all scan speeds in the normal mass range and 100 for all scan speeds in extended mass range. The high vacuum was 9.7 × 10−9 bar.On the LXQ, Turbo (128 000 u/s), Normal (16 600 u/s), Enhanced (5000 u/s), and Zoom (1100 u/s) scan modes were used for 3, NaCF3COO‐clusters, protonated and CF3COO−‐adducted ESI tuning mix, Na+‐adducted low mass dendrimers of SpheriCal®‐ESI, and protonated and deprotonated peptides were analyzed in all four scan speeds on the amaZon. CsI‐clusters and the CsI + Pierce mixture were analyzed in all four scan speeds of both the amaZon and the LXQ, and protonated Pierce on the LXQ.For the low mass range, Y(HCOO)+ and Cs+ adducts, and CsI clusters were analyzed. In high mass range on the LXQ, the four dendrimers as both Na+ and Cs+ adducts in the same spectra, and CsI clusters were analyzed in all three scan speeds. The experiments are summarized in Table In the Extended mass range mode on the amaZon, the four dendrimers as Nam/z values were calculated from recently published masses and weights.Theoretical Calculated average mass was used in Turbo scan and Extended mass range scan, and for multiply charged peptides when isotope clusters were not resolved. In all other cases, the calculated monoisotopic mass was used. amaZon spectra were processed in DataAnalysis 4.2  and LXQ spectra were processed in Qual Browser 2.0 (Thermo Electron). Data analysis was performed in LibreOffice Calc 6 and 7.3m/z show similar absolute m/z errors. In addition to chemical mass shift toward lower m/z and peak distortion for fragile ions, some mass shift toward higher m/z, loss of resolution, and tailing can be expected for high‐intensity signals.m/z ions experience less space charge as the lower m/z ions are ejected before them.Although some signals were of low intensity and some spectra were noisy, we refrained from measuring these again to avoid bias; it could be argued that a mix of high intensity and low intensity signals is closer to real applications. However, all data from 1 day was discarded as instrument performance in terms of resolution and sensitivity was unsatisfactory, probably due to unstable He pressure after start‐up. Importantly, all mass shifts discussed are relative the calibration ions. In the absence of mass shifts, it is expected that analytes at similar For the amaZon, the mass shifts and peak distortions of the CsI clusters in positive mode were representative of the other cluster ions and some of the adducts  in the higher m/z ranges in at least some scan modes. All cluster ions analyzed, and some of the adduct ions, showed mass shifts in the higher m/z ranges. Mass shifts at lower m/z ranges (m/z < 1000), if present, were less clear. Adduct ions that showed shifts included CF3COO−‐adducted ESI tuning mix signals 6Cs+ has previously been shown to be more stable in CID compared to other clusters in the range,m/z shows less shift compared to the nearby analytes. Interestingly, there were large differences between the mass shifts of (CsI)nCs+ and (CsI)nCs22+ on both instruments, with the latter showing larger shift toward lower m/z In the low mass range on the LXQ, CsI cluster ions showed fronting, increasing with decreasing scan speed, again except for the Zoom scan Figure , right, +‐ and Cs+‐adducted dendrimers , showed tailing in Turbo scan, but fronting in Normal scan nCs22+ cluster signals showed narrow peak shapes, but the largest mass shifts, while (CsI)nCs+ cluster signals showed severe tailing and broadening, yet smaller mass shift. Additionally, while peak distortion was more severe on the amaZon QIT, mass shifts were not always worse. Thus, it appears that it is not always possible to predict mass shift severity from the peak shape.Some peak distortion often accompanied mass shifts, but was different between the amaZon QIT and the LXQ LIT. Importantly, mass shifts did not always correlate well with peak distortion. For example, on the LXQ the Csm/z analysis and high‐resolution measurements. The latter may be of additional importance since high resolution is often associated with high accuracy, while in some instances it may instead result in worse accuracy for fragile ions. Notable, however, is the relatively good performance of the Maximum resolution and the Zoom scans compared to the Enhanced scans.This suggests that ion trap users should look for peak distortions and mass shifts especially during high All analyzed heavy cluster ions and some adduct ions showed at least some mass shifts. Cluster ions seem unsuitable for calibration of ion traps and adducts should only be used with caution. Similarly, analysis of such ions may yield inaccurate results. For suitable calibrants, we refer to a recent review .The authors declare no conflicts of interest.Three documents with supporting information are included: Supplementary calibration fit contains more detailed results and evaluation of the calibrations. Supplementary mass shifts contains plots of mass shifts not shown in the main article. Supplementary peak shapes shows more peak shapes in addition to those shown in the main article.https://publons.com/publon/10.1002/rcm.9436.The peer review history for this article is available at DATA S1 Supplementary calibration fitClick here for additional data file.DATA S2 Supplementary mass shiftsClick here for additional data file.DATA S3 Supplementary peak shapesClick here for additional data file.


MHA) to primiparous, spring-calving beef females on dam and progeny performance. Angus heifers (n = 60) were blocked by expected parturition date, stratified by body weight (BW) and body condition score (BCS), and randomized to 1 of 15 pens. Pens were randomly assigned to 1 of 3 dietary treatments: a basal diet supplemented with 0 (M0), 15 (M15), or 30 (M30) g/animal/d of MHA . Diets were fed from 45 ± 13 (SD) d pre-calving through 81 ± 13 d postpartum (DPP), after which all cow–calf pairs were managed as a single group on pasture until weaning (199 ± 13 DPP). Dam BW, BCS, and blood samples were taken at 6 predetermined timepoints. Progeny data were collected at birth, 2 intermediate timepoints, and at weaning. Milk samples were collected for composition analysis at 7 ± 2 DPP and at 55 ± 5 DPP. Serial progesterone samples were analyzed to establish resumption of cyclicity, and ultrasonography was performed at 55 ± 5 DPP to evaluate ovarian function. Cows were bred via artificial insemination at 82 ± 13 DPP and subsequently exposed to bulls for a 55-d breeding season. Pen was the experimental unit, and preplanned orthogonal contrasts were tested (linear effect and M0 vs. M15 + M30). Dam BW and BCS were not affected by treatment (P ≥ 0.29) throughout the study. Week 1 milk fat concentration increased linearly (P = 0.05) and total solids tended to increase linearly (P = 0.07) as MHA increased; however, no other milk components were affected (P ≥ 0.16). Treatment did not affect (P ≥ 0.16) dam reproductive parameters or progeny growth from birth until weaning. Post-calving, circulating methionine equivalents tended to linearly increase (P = 0.10) with increasing MHA supplementation. At breeding, plasma urea N linearly decreased (P = 0.03) with increased supplementation of MHA, and plasma non-esterified fatty acids were less (P = 0.04) in MHA-supplemented dams compared with dams receiving no MHA. Maternal circulating glucose, glutathione peroxidase, and thiobarbituric acid-reactive substances were not affected (P ≥ 0.15) by treatment at any point. These data indicate that peripartum supplementation of MHA may increase milk fat composition shortly after calving, but MHA supplementation did not improve progeny growth or dam reproductive performance in the current study.The objective was to evaluate the effects of peripartum supplementation of a methionine hydroxy analogue ( Proper peripartum nutrition is necessary for maximizing beef cattle performance, not only for fetal development, milk production, and progeny growth but also for repairing the reproductive tract and preparing to establish the subsequent pregnancy. Nutrient requirements increase exponentially during late gestation concomitant with rapid fetal growth and then remain high or even increase during early lactation. Primiparous females face the greatest challenge during this time, not only needing to meet their requirements for maintenance, pregnancy, and lactation but also their own growth requirements. Methionine is often considered the first limiting or co-limiting amino acid in cattle for growth , pregnanMHA). In a meta-analysis of lactational performance in dairy cows, ADG) and reproductive tract scores.Providing free amino acid sources becomes wasteful due to a substantial proportion being degraded in the rumen and not reaching the small intestine intact. Ruminally protected forms, such as amino acids encapsulated with lipids or pH-sensitive materials and synthesized amino acid analogues, have been devised for this reason . The mosThe objectives of this study were to evaluate dam and progeny performance of primiparous females fed to meet their nutrient requirements and supplemented with MHA during the last month of gestation and continuing until subsequent breeding. These measures included maternal performance, milk composition, ovarian function, reproductive success, metabolic status, and oxidative stress in the dam and size at birth, pre-weaning growth, and metabolic status in the progeny. The hypothesis was that maternal supplementation of MHA would result in improved calf growth through increased nutrient concentrations in milk without hindering reproductive success.n = 60; body weight [BW] = 452 ± 28 [SD] kg; body condition score [BCS] = 6.0 ± 0.5) and managed as a single group from weaning until treatment initiation were used in a randomized complete block design to elucidate the effects of MHA during late gestation and early lactation. Heifers were blocked by expected parturition date using ultrasonographic fetal age estimates , stratified by BW and BCS, and randomized to 1 of 15 pens . Pens were randomly assigned to 1 of 3 dietary treatments (5 pens per treatment) consisting of a basal diet supplemented with 0 (M0), 15 (M15), or 30 (M30) g/animal/d of MHA . The initiation of treatments began 45 ± 13 d prior to the average parturition date and continued until breeding at 81 ± 13 d postpartum. The basal diet  at the McNay Memorial Research and Demonstration Farm in Chariton, Iowa from February to October 2017. Angus heifers of similar genetic background . During this grazing period, no supplementation was offered other than free choice mineral and vitamin supplementation.Initial BW was determined as the average preprandial BW measured on the day prior to and the day of treatment initiation , with identical methodology at the initiation of synchronization of ovulation (73 and 74 ± 13 d postpartum) to determine overall treatment effects without differences in gut fill associated with estrus. A single pre-calving BW was taken at 18 ± 13 d prior to average parturition date, and a single post-calving BW collection occurred at 7 ± 2 d postpartum. A BCS assessment was conducted according to where CW = conceptus weight, kg; CBW = calf body weight, kg; and DP = days pregnant. For females bred by natural service, days pregnant were estimated based on ultrasonographic fetal age estimates.While on pasture, dam BW and BCS were collected again at the first pregnancy detection (117 ± 13 d postpartum) and at weaning (199 ± 13 d postpartum).Ten females were removed due to lack of complete study data ; however, there was no evidence to suggest those problems resulted from treatment . When cattle were removed from their pen, the feed and supplement delivery to that pen were adjusted accordingly.Calf sex and BW were recorded within 24 h of birth. At 7 ± 2 d of age, progeny body measurements were taken using a tape measure, which included biparietal width , front cannon length , and body length . Calf BW was measured again at 55 ± 5 d of age (week 8 milk sampling), 117 ± 12 d of age (first pregnancy detection), and 199 ± 12 d of age (weaning).DM) per calf was added to the daily feed delivery of each pen, in identical proportions to the cow diet, to account for the feed consumption of calves and to prevent hindrance of targeted dam performance.As progeny became older, they were observed consuming the dams’ ration because creep feed was not available. As a result, from 61 ± 13 d of age until 81 ± 13 d of age, 0.91 kg of additional dry matter  and 55 ± 5 d postpartum (week 8). Milk samples were taken preprandial, with the week 1 sample collected on the Thursday falling between 3 and 9 d postpartum to ensure sufficient time for all colostrum to be removed from the mammary gland. The week 8 sample was taken on the Thursday that was closest to 60 d postpartum. The rear right quarter of each cow’s udder was cleaned and then milked completely by hand. If there was not enough sample volume obtained, the same procedure was administered to the rear left quarter, and the collections were mixed. Milk samples were transferred to plastic tubes  containing potassium dichromate preservative and immediately frozen. Frozen milk samples were transported to Dairy Lab Services and milk composition was analyzed via infrared instrumentation. The 2 samples collected from each cow were analyzed separately.AFC), ovarian length, and ovarian height. Ultrasound examinations were performed chute-side by the same trained investigator. All follicles visible to the naked eye (>3 mm) were counted on each ovary in real time. The AFC of each ovary was recorded separately. Additionally, a clear freeze frame of the whole ovary was used along with the caliper function of the ultrasound machine to determine the ovarian height and length. Both ovaries were measured separately; however, statistical analysis was performed only on the average height and length of the 2 ovaries combined. Cows were not synchronized prior to ovarian ultrasonography, resulting in cows on random and unquantified days of their estrous cycle.At the weekly data collection timepoint closest to 60 d postpartum (55 ± 5 d postpartum), cows were subjected to transrectal ultrasonography  for determination of antral follicle count (CIDR) protocol. At CIDR removal, an Estrotect heat detection aid  was placed on the tailhead of each cow. Seventy-two hours after CIDR removal, all cows were bred via fixed-time AI . At FTAI, heat detection patches were assessed as an indicator of riding incidences associated with expression of estrus before FTAI . Seven days after FTAI, cows were exposed to a group of 3 Angus bulls for a 55-d breeding season. Conception to FTAI and overall pregnancy rates were determined at 117 ± 13 and 199 ± 13 d postpartum, respectively. Cows that remained in the final analysis of the current study and calved the following year were included in calculating the calving interval between the 2017 and 2018 calving season.At 74 ± 13 d postpartum, all cows were synchronized for ovulation using the 5-d CO-Synch + controlled internal drug release  and immediately placed on ice. Blood samples for serum analysis were collected via jugular venipuncture into 10-mL serum blood collection tubes (BD Vacutainer), and blood was allowed to clot at room temperature prior to being placed on ice for transportation. All blood samples were centrifuged at 2,500 × Concentrations of 2-hydroxy-4-(methylthio)butanoic acid, or MHA, and methionine in serum were obtained using a liquid chromatography mass spectrometry method developed internally by Novus International Inc. The MHA calibration was conducted in MHA-free plasma spiked with MHA over a range of 25 to 2000 µg/kg. The relative standard deviation was 1.2% and the percent recovery of the internal standard averaged 104.3%. Methionine calibration was conducted in diluent over a range of 500 to 5000 µg/kg. The relative standard deviation was 1.3% and the percent recovery of the internal standard averaged 107.0%. Data are reported as concentrations of methionine equivalents in serum which represents the sum of MHA and methionine.CV) was 3.8% and the interassay CV was 9.0%. For progeny plasma urea N, the average intraassay CV was 4.1% and the interassay CV was 5.2%. Plasma non-esterified fatty acid (NEFA) concentration was determined using a commercially available kit (HR Series NEFA-HR (2), Wako Pure Chemical Industries, Ltd., Mountain View, CA). The average intraassay CV was 2.5% and the interassay CV was 1.5%. Plasma samples were analyzed for glucose with a commercially available kit . The average intraassay CV was 3.7% and the interassay CV was 3.6%For all plasma analyses, samples were read in duplicate in 96-well plates on a microplate reader. Plasma samples were analyzed for urea N with a commercially available kit . For dam plasma urea N, the average intraassay coefficient of variation (GPX) and thiobarbituric acid-reactive substance (TBARS) concentrations by Novus International Inc. Serum GPX and TBARS were only quantified at initiation of the study, at first pregnancy detection, and at weaning. The concentration of GPX was determined using a bovine glutathione peroxidase ELISA kit  with an intraassay CV of 7.5%. The concentration of TBARS was determined using a commercially available kit  with an intraassay CV of 5.0%.To determine the effects of MHA supplementation on maternal oxidative stress status, serum samples were evaluated for glutathione peroxidase  to determine the duration of the anestrous period. Blood samples were collected using the methodology described above for plasma recovery. Plasma samples were evaluated for progesterone concentration via radioimmunoassay using methodology previously described by P > 0.10 for a specific variable. For all progeny-related variables, the statistical model also included the fixed effect of sex. For progeny BW, day of age was always included as a covariate.Effect of treatment for binomial data  was analyzed using the GLIMMIX procedure of SAS 9.4 . The remaining reproductive parameters, milk composition, maternal performance, and progeny performance were analyzed using the MIXED procedure of SAS. For all dependent variables of interest, pen served as the experimental unit, the main fixed effect was treatment, and the random effect was pen. For all maternal variables, days postpartum was initially run as a covariate; however, it was removed from the model when Maternal and progeny metabolite concentrations were analyzed with the MIXED procedure of SAS as repeated measures using the compound symmetry covariance structure (based on having the lowest Bayesian Information Criterion for most variables). The model included the fixed effects of treatment and day, as well as the treatment × day interaction.P ≤ 0.05 and tendencies were declared when 0.05 < P ≤ 0.10.Two preplanned orthogonal contrasts were decided a priori: 1) testing a linear effect, and 2) testing the difference between the control treatment (M0) and the 2 treatments containing MHA (M15 and M30). Significance was declared when P ≥ 0.53; P ≥ 0.29) by MHA supplementation at any timepoint during either the dietary treatments or until weaning.As designed, dam BW and BCS were not different  in the biparietal width of offspring at birth with increasing MHA supplementation to dams. Conversely, there was a tendency for increased (P = 0.06) front cannon length in progeny born to MHA-supplemented dams compared with those not fed MHA. Offspring body length was not affected (P ≥ 0.39) by treatment. Progeny BW was not affected (P ≥ 0.31) by maternal MHA supplementation at any of the interim measurements or at weaning. Progeny ADG  was not affected (P ≥ 0.32) by treatment.Progeny birth weight was not affected  in cows supplemented with MHA compared with dams receiving no MHA. Additionally, there was a tendency for a linear increase (P = 0.07) in percent milk total solids as dams received greater concentration of MHA. Milk protein, lactose, other solids, and urea N were not affected (P ≥ 0.25) due to dietary treatment at the week 1 measurement. At the week 8 (55 ± 5 d postpartum) milk collection, milk composition was not affected (P ≥ 0.16) by treatment.At the week 1 (7 ± 2 d postpartum) milk sampling, milk fat linearly increased  by treatment.The percentage of primiparous females that had resumed cyclicity by the beginning of the synchronization of ovulation protocol was not affected  by treatment.When ovarian ultrasonography was performed at 55 ± 5 d postpartum, AFC for each ovary and total AFC were not affected  by dietary treatment. Additionally, the calving interval was not affected  for all dam metabolite concentrations but will not be discussed for brevity. There tended to be a treatment × day interaction  with increasing supplementation of MHA. Concentration of serum methionine equivalents was not affected (P ≥ 0.28) by MHA supplementation at treatment initiation or after dietary treatments ended. Concentration of plasma urea N was not affected by the treatment × day interaction . Plasma urea N linearly decreased (P = 0.03) at breeding (the conclusion of dietary treatments) with increased supplementation of MHA, and MHA-supplemented dams tended to have lower (P = 0.07) plasma urea N compared with control dams. There was a treatment × day interaction  in cattle supplemented with MHA compared with dams receiving no MHA, and plasma NEFA tended to linearly decrease (P = 0.06) with increased MHA supplementation. Plasma NEFA were not affected due to treatment at any other timepoint (P ≥ 0.22). Plasma glucose concentration was not affected by the treatment × day interaction . Serum glutathione peroxidase activity and TBARS concentrations were not affected by the treatment × day interaction .The effect of day was significant (P < 0.001) for all progeny metabolite concentrations but will not be discussed for brevity. There was a treatment × day interaction for progeny plasma glucose concentration  at any timepoint. Progeny plasma urea N concentration was not affected by the treatment × day interaction (P = 0.82) or by treatment (P = 0.57).The effect of day was significant  products likewise observed no effect on BW and BCS due to treatment during the peripartum period , it had a weak positive relationship to progeny birth weight , while iPre-weaning progeny performance was not affected by MHA supplementation, which concurs with previous results . In MoisMilk fat concentration increased with increasing MHA supplementation at the week 1 sampling in the current study and is likely what accounted for the tendency for total milk solids to also increase. In a meta-analysis, Multiple hypotheses exist for the mechanism in which milk fat increases due to RPMet supplementation. These include alleviating the effects of ruminal biohydrogenation-induced milk fat depression , methionLH) concentration in the anterior pituitary must increase in the few weeks after parturition, otherwise inadequate LH pulsatility and concentration will result in the failure of dominant follicles to undergo terminal maturation prior to successful ovulation  tended to linearly increase as MHA supplementation increased at the post-calving timepoint; however, methionine equivalents were not affected before or after the dietary treatments in the current study. Interestingly, It is widely accepted that substantial increases in dietary crude protein, specifically rumen degradable protein, can elevate plasma urea N concentration. The MFP product is formulated to contain 84% MHA, which is estimated to have a 40% rumen bypass rate . In the B. indicus-influenced beef cows did not alter NEFA , when NEFA were lower in MHA-supplemented cattle. Peripartum MHA supplementation in ter NEFA . Socha eter NEFA . In contter NEFA . By suppter NEFA . Cows coter NEFA . This liB. indicus-influenced beef heifers (Glucose concentration was not affected by dietary treatment, agreeing with previous MHA supplementation in  heifers  and earl heifers . CollinsIn the current study, GPX activity was not affected at any point throughout the study. In the high-producing dairy cow, there is a drastic increase in metabolic activity after parturition and as lactation begins, leading to increased production of reactive oxygen species and concurrent depletion of antioxidants . ConsideAnalysis of TBARS was conducted to look at lipid peroxidation and to serve as a secondary indicator for oxidative stress status. Thiobarbituric acid-reactive substance concentration was not affected by treatment at initiation of the study or at either the first pregnancy detection or weaning. To our knowledge, no prior work has been completed studying TBARS in periparturient cattle receiving MHA supplementation.No similar data pertaining to progeny pre-weaning plasma urea N concentration after maternal peripartum supplementation with MHA products have been identified. Increased intake of nitrogenous compounds is usually associated with increased blood urea N; however, in the current study as milk urea N concentration numerically increased at 7 ± 2 d postpartum with MHA supplementation, progeny plasma urea N values collected on the same day decreased with increasing maternal supplementation with MHA.Progeny glucose concentration was not affected by treatment at any point, which agrees with Based on data from the present study, primiparous females fed a basal diet to meet or exceed nutrient requirements and supplemented with MHA at 15 or 30 g daily during late gestation and early lactation may increase milk fat concentration shortly after parturition and alter maternal circulating metabolites around the time of breeding. Plasma methionine equivalents tended to be greater post-calving due to MHA supplementation; however maternal BW, BCS, and reproductive performance were not affected. Additionally, the increase in milk fat did not translate to improved progeny pre-weaning performance. Methionine hydroxy analog supplementation of females already fed to meet peripartum nutrient requirements saw minimal improvement; however, research on MHA supplementation of females fed poor quality forage or experiencing nutrient restriction, especially primiparous dams, is warranted.


People often try to improve others’ emotions. However, it is unclear which interpersonal emotion regulation strategies are most effective and why. In 121 candid dyadic conversations between undergraduate students via video conferencing, target participants recounted a stressful event to regulator participants. Three strategies used by regulators during these conversations to change targets’ emotions were obtained from the regulator after the conversation: extrinsic reappraisal, extrinsic suppression, and extrinsic acceptance. Perceived regulator responsiveness was obtained from targets to examine the social consequences of extrinsic emotion regulation and its mediating role in successful extrinsic emotion regulation. We found that regulators’ extrinsic reappraisal use was associated with improved target emotions measured across two distinct classes of outcomes: targets’ emotions during the conversation and targets’ perception that the regulator improved their emotions. Regulators’ extrinsic suppression and acceptance, in contrast, were not related with improved target emotions or perceptions of improvement. Instead, all extrinsic regulatory strategies were associated with improved targets’ emotions when mediated by targets’ perceptions of regulator responsiveness. Finally, observer-ratings of regulators’ extrinsic reappraisal and suppression use were found to be consistent with regulators’ self-ratings and follow the same pattern of results on the outcome measures. These findings provide insight into why the social regulation of emotions can succeed or fail and hold implications for interventions aimed at guiding people toward more successfully improving others’ emotions.The online version contains supplementary material available at 10.1007/s42761-023-00183-4. Overwhelmed with his new job, Tyler divulges his fears and doubts to his coworker Rebecca. Tyler fears failing the people who gave him this job opportunity, and he doubts that he can keep up with the accumulating workload. When Rebecca hears about his distress, she considers using three different strategies to help him. She could remind him that many people struggle at first in a new job, so it is okay to feel this way , she could scold him for being so negative at work because it looks bad for him , or she could suggest that he reframe his situation as a learning experience in which it is okay to make mistakes . Ultimately, she chooses the latter strategy, which proves effective in easing Tyler’s negative emotions.This example highlights the importance of interpersonal emotion regulation, a process in which one person’s emotions are regulated by another person  as in the case of the social sharing of emotions Rimé,  or may bSimilar to intrapersonal cognitive reappraisal which works through the reinterpretation of an emotion-eliciting situation Gross, , 2015, eWhen using extrinsic suppression, a regulator signals for a target person to hide their emotion expression through indirect means  or direct means . By suppressing a target’s expressions of negative emotions, a regulator may improve a target’s emotions by preventing detrimental social behaviors such as co-rumination Boren,  or ventiIn the case of extrinsic acceptance, we conceptualize this strategy as one in which a regulator embraces the emotions of the target without attempts to control the target’s emotions which may or may not result in the target also accepting their own emotions. Extrinsic acceptance may result in greater emotion awareness and expression ; these instructions/signals can influence the target’s cognitions and regulatory actions, potentially improving the target’s emotions. For example, a regulator may try to help a target downregulate their negative emotions due to a recent breakup by telling them that they “will regret” feeling like this later . For instance, if extrinsic reappraisal is associated with worse perceived regulator responsiveness, we would also expect it to be associated with worse target affect. To determine target emotion improvement, we measured two types of outcomes from the targets: their emotions during the conversation and their perception of the regulators’ success at improving how they feltf = 0.3 with 80% power. We thus aimed to collect data from at least 120 dyads and more if possible, terminating data collection at the conclusion of the academic quarter. Twenty-six participants were excluded due to internet connectivity issues or other technical issues during the study. Four participants were excluded due to knowing the other participant in the dyad. All other participants were paired with stranger participants. The final sample consisted of 242 participants .Two hundred seventy-two undergraduate students at the University of California, San Diego participated in this social interaction study in same-gender dyads. Participants received course credit. A power analysis, using G*Power, indicated that a sample size of 120 dyads (40 dyads per cell) would be sufficient to detect a medium effect size of f2 = 0.08 with 80% power.Within each dyad, one participant was randomly assigned a regulator role, and the other participant was assigned a target role. To examine questions about extrinsic emotion regulation, we used multivariate linear regression models to determine the extent that independent variables from the regulator participants influenced the dependent variables from the target participants. A sensitivity analysis using G*Power indicated that for a two-predictor linear regression model, the present sample size of 121 dyads would be sufficient to detect a small effect size of We originally intended a between-subject design with three extrinsic prohedonic goal conditions for the regulator participants . However, the manipulation of regulator goals was deemed to have failed because it did not influence regulators’ self-reported goals or their use of extrinsic emotion regulation strategies . Given this, and because of the rich nature of the dataset, which involved real, ecologically valid extrinsic emotion regulation actions that participants self-generated to improve targets’ emotions alongside measurements from both dyadic members, we decided it was most appropriate to analyze the data based on participants’ natural variability in interpersonal emotion regulation actions collapsing across the originally intended manipulation.https://osf.io/kazep/.Data, code, and online supplemental materials are available on OSF at To get comfortable with the experimental environment and with each other, each pair of participants first interacted together in a 5-min fast friends task  nor did they significantly predict any of the outcome measures . After these instructions were given, the target was moved to a private Zoom breakout room for 5 min to brainstorm what they wanted to discuss during the stress sharing conversation.For the stress sharing conversation, participants were randomly assigned to be the regulator or the target. In the instructions given to the participants, the regulator was referred to as the friend, and the target was referred to as the sharer to minimize any demand effects. Both participants were told that the role of the target is to speak about a stressor they are experiencing in their life right now, to discuss the details of why that experience happened, and to share how they feel. The stressor could include anything they perceive to be stressful at work, school, or home. Then, both participants were told that the role of the regulator is to talk with the target about that stressor. All regulators were given the goal to have a natural conversation with the target participants, and they were encouraged to share their thoughts and impressions, give advice, and ask questions. Participants who were not in the control condition were also told to either increase the positive emotions or decrease the negative emotions of the target participants. Compared to the control condition, these instructions did not successfully change regulators’ extrinsic prohedonic goals (Regulators rated the following three extrinsic emotion regulation strategies that they used to manage the emotions of targets during the stress sharing conversation on 1 (strongly disagree) to 7 (strongly agree) scales: extrinsic reappraisal  to 5 (strongly agree) scales (“The other participant succeeded in making me feel more positive emotions” and “The other participant succeeded in making me feel less negative emotions”). In figures and tables, these two items are referred to as extrinsic positive emotion upregulation and extrinsic negative emotion downregulation, respectively.Targets rated the responsiveness of the regulators using adapted items from the Perceived Partner Responsiveness Scale  to 5 (strongly agree) scales.Both regulator and target rated their closeness and their motivation to affiliate at the end of the conversation. Closeness was measured using the Inclusion of Other in Self scale  to 5 (strongly disagree) scale.Regulators rated to what extent they believed they successfully improved the targets’ positive and negative emotions on 1 (strongly disagree) to 5 (strongly agree) scales (“I succeeded in making the other participant feel more positive” and “I succeeded in making the other participant feel less negative”).In order to account for targets’ emotions potentially influencing the regulators’ extrinsic strategy choices, at the beginning of the study, targets rated how much they felt positive and negative affect in general (in their daily life) on 1  to 5 (very much) scales. The five items for the positive emotion composite (α = .74) were “proud / good about yourself,” “compassionate / sympathetic / touched,” “grateful / appreciative / thankful,” “inspired / uplifted / elevated,” and “happy / pleased / joyful.” The six items for the negative emotion composite (α = .85) were “angry / irritable / frustrated,” “anxious / nervous,” “distressed / upset,” “guilty / embarrassed / ashamed,” “sad / down,” and “resentful / bitter / annoyed.”We obtained observer ratings of regulators’ strategy use from three trained behavioral coders. Behavioral coders watched videos of the entire conversation and rated both extrinsic reappraisal and extrinsic suppression on 1  to 5 (behavior is very frequently present) scales . We assessed inter-rater reliability on 13% of the data for extrinsic reappraisal (ICC = .75) and extrinsic suppression (ICC = .78). Six dyads were excluded due to improper recording or uploading of the video data.Additionally, we attempted to obtain observer ratings of two forms of regulator extrinsic acceptance: regulators attempt at accepting the targets’ emotions (measured through self-report) and regulators attempt to lead the targets to accept their emotions (not measured through self-report). Due to low inter-rater reliability, we did not proceed with coding these variables.b = 0.32, t(119) = 3.72, 95% CI , p < 0.001, and at downregulating their negative emotions, b = 0.21, t(119) = 2.32, 95% CI [0.03 0.39], p = .022.First, we tested the relationship between regulator extrinsic reappraisal use and extrinsic emotion improvement using univariate linear regression models without control variables  = 2.69, 95% CI , p = .008, and less negative emotion, b =  − 0.12, t(119) =  − 2.47, 95% CI , p = .015, during the stress sharing conversation.Then, we tested the relationship between regulator extrinsic reappraisal use and target emotions using univariate linear regression models without controls. When regulators used more extrinsic reappraisal, targets experienced significantly more positive emotion, b =  − 0.32, t(117) =  − 2.04, 95% CI , p = .044. Target trait negative affect was not significantly associated with regulator extrinsic reappraisal use, b =  − 0.08, t(117) =  − 0.62, 95% CI , p = .54. This suggests that regulators used more extrinsic reappraisal on those who entered the study in a worse affective state and, on average, successfully uplifted them during the conversation. Finally, we controlled for target trait affect in our main analyses. When we did, we still found that when regulators used more extrinsic reappraisal, targets experienced significantly more positive emotion, b = 0.27, t(117) = 3.45, 95% CI , p < .001, and less negative emotion, b = 0.12, t(117) =  − 2.49, 95% CI , p = .014, indicating that targets’ affect at the start of the study could not explain the main results.Additionally, we examined the alternative explanation for these results in the other causal direction—that regulators used more extrinsic reappraisal on targets with better affect . We did not find any support for this alternative explanation. To examine this, we tested how target trait affects predicted regulator extrinsic reappraisal use. We found that targets with less positive affect at beginning of the study received significantly greater extrinsic reappraisal from regulators, Neither extrinsic suppression nor extrinsic acceptance was significantly associated with targets’ emotions or extrinsic emotion improvement  = 9.08, 95% CI , p < .001, and lower negative emotion, b =  − 0.20, t(119) =  − 2.51, 95% CI , p = .013. When targets perceived greater regulator responsiveness, they also reported that regulators were significantly more successful at upregulating their positive emotions, b = 0.63, t(119) = 4.85, 95% CI , p < .001, and at downregulating their negative emotions, b = 0.71, t(119) = 5.64, 95% CI , p < .001.When targets perceived greater regulator responsiveness, they experienced significantly greater positive emotion, b = 0.13, t(119) = 2.24, 95% CI , p = .027, and more extrinsic acceptance, b = 0.20, t(119) = 3.47, 95% CI , p < .001, whereas targets perceived less regulator responsiveness when regulators used more extrinsic suppression, b =  − 0.14, t(119) =  − 2.44, 95% CI , p = .01 , sense of connection , and regulator empathy  and regulator extrinsic suppression use  positively correlated with regulators’ self-ratings of these strategies, respectively (r(113) = .40, p < .001 and r(113) = 0.38, p < .001). Furthermore, we obtained similar results when using observer ratings to predict emotion outcomes and perceived partner responsiveness  interpersonal goals. Thus, attempts at changing targets’ emotions may have been conversational-directed depending on the targets’ disclosure instead of goal-directed. This raises the question—what trigger regulators to have these goals? In future research, it would be useful to examine whether regulators set these goals because of their own motivations or because of the type of disclosure by the target. For example, a target person may seek emotional support but receive problem-oriented support Below is the link to the electronic supplementary material.


Jejunal diverticula are often asymptomatic and rare, so they can go unnoticed until serious complications like obstruction, bleeding, perforation, volvulus, or diverticulitis occur. Elderly people over 60 are more likely to have this condition. Jejunal diverticula are often asymptomatic and rare, so they can go unnoticed until serious complications like obstruction, bleeding, perforation, volvulus, or diverticulitis occur. Elderly people over 60 are more likely to have this condition. Two diverticula were discovered 1 and 2 feet away from the duodenojejunal (DJ) junction. Due to excessive ileal edema, ileal resection, and a double barrel ileostomy was performed.Small bowel diverticula are a rare disease, with a reported incidence of 0.3%–1.3% at autopsies and 2.0%–2.3% radiographically.2A 75‐year‐old male with no known comorbidities arrived at the emergency department complaining of constipation, nausea, and vomiting for the past 3–4 days. The patient was in his usual state of health when he experienced constipation. He has not passed flatulence for the past 3–4 days. The patient also complained of localized pain in the lower abdomen, which was gradual in onset, dragging in character, and non‐radiating. The patient had 3–4 episodes of vomiting, which was non‐projectile and did not contain any blood or mucous. He had a prior history of on and off constipation.On general physical examination, the patient was anemic and dehydrated. His blood pressure was 149/76 mm of Hg and his heart rate of 94 beats/min. On examination, the abdomen was firm and swollen (distended), with discomfort in the lower abdomen on palpation. The bowel sounds were audible. Laboratory results showed a total WBC count of 1.9 B/L, hemoglobin 13.7 g/dL, RBC count of 4.12 B/L, platelet count of 122 B/L, absolute neutrophil count of 1520, sodium 138 mEq/L, potassium 3.5 mEq/L, bicarbonate 21 mEq/L, chloride 106 mEq/L, total bilirubin 2.6 mg/dL, direct bilirubin 1.5 mg/dL, and alkaline phosphatase 150 IU/L. X‐RAY imaging of the abdomen in supine posture indicated a few dilated bowel loops and a few air‐fluid levels, suggesting most likely sub‐acute blockage  was discovered 1 foot (1 cm) from the ileocecal junction. One diverticulum was 1 foot (1 cm) away from the duodenojejunal (DJ) junction, while the other was 2 feet (2 cm) away from the DJ junction. There was an edematous ileal wall at the site of the perforation. Significant ileal edema necessitated ileal resection and a double barrel ileostomy Figure . The pat3Most often, diverticula are found in the jejunum, 15% are found in the ileum, and 5% are distributed in both. Jejunal Diverticulosis is mostly a silent disease, and only 29% of patients are symptomatic, while only 10% of patients proceed to develop complications including, obstruction, fistula formation, peritonitis, lower gastrointestinal bleeding, and perforation.Krishnamurthy et al.Some patients respond to the brief cessation of enteral nourishment, alleviation of gastrointestinal symptoms provided by a nasogastric tube, and the use of empirical, broad‐spectrum antibiotics, although 8%–30% of patients experience problems necessitating surgical intervention. In the event of a perforation, exploratory laparotomy with resection of the affected intestinal segments and primary anastomosis is necessary. The extent of the intestinal resection depends on the length of the bowel affected, with a more conservative resection of only perforated intestinal segments preferred in cases of involvement of long sections of the bowel, which was the situation in our case.Acquired jejunoileal diverticulosis has a wide range of clinical manifestations. As a result, it may be challenging to diagnose the illness. Up to 90% of patients have been shown to present with symptoms similar to those of irritable bowel syndrome, including intermittent abdominal pain, constipation, and diarrhea. Imaging tests primarily have an atypical appearance without essential diagnostic features and may not correspond with the clinical symptoms.4To conclude, a high index of suspicion for jejunal diverticulitis should be maintained in patients presenting with acute abdominal symptoms to avoid misdiagnosis and prevent adverse outcomes. Careful clinical assessment coupled with timely radiological evaluation can aid in early detection and appropriate management of this condition, potentially saving lives. Therefore, it is crucial for healthcare providers to remain vigilant and consider jejunal diverticulitis as a possible differential diagnosis in all cases of acute abdomen.Aleeza Jawed: Writing – original draft; writing – review and editing. Areesha Jawed: Writing – original draft; writing – review and editing. Sandhya Kumari: Writing – original draft; writing – review and editing. Omer Ahmed Shaikh: Writing – original draft; writing – review and editing. Abdulqadir J. Nashwan: Writing – original draft; writing – review and editing.None.All authors declared no conflict of interest.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


Genetic and pharmacological inhibition of IDH1 was then combined with extracellular flux analyses and gas chromatography–mass spectrometry for metabolomic analyses and cancer cell proliferation in vitro and in vivo. In PCa cells, more than 90% of the total IDH activity is mediated through IDH1 rather than its mitochondrial counterparts. This profile seems to originate from the specialized prostate metabolic program, as observed using mouse prostate and PDOs. Pharmacological and genetic inhibition of IDH1 impaired mitochondrial respiration, suggesting that this cytoplasmic enzyme contributes to the mitochondrial tricarboxylic acid cycle (TCA) in PCa. Mass spectrometry‐based metabolomics confirmed this hypothesis, showing that inhibition of IDH1 impairs carbon flux into the TCA cycle. Consequently, inhibition of IDH1 decreased PCa cell proliferation in vitro and in vivo. These results demonstrate that PCa cells have a hybrid cytoplasmic–mitochondrial TCA cycle that depends on IDH1. This metabolic enzyme represents a metabolic vulnerability of PCa cells and a potential new therapeutic target.The androgen receptor (AR) is an established orchestrator of cell metabolism in prostate cancer (PCa), notably by inducing an oxidative mitochondrial program. Intriguingly, AR regulates cytoplasmic isocitrate dehydrogenase 1 (IDH1), but not its mitochondrial counterparts IDH2 and IDH3. Here, we aimed to understand the functional role of IDH1 in PCa. Mouse models,  The prostate exhibits a unique truncated mitochondrial tricarboxylic acid (TCA) cycle to produce and secrete citrate. Here, we show that during carcinogenesis, prostate cancer cells develop a hybrid cytoplasmic–mitochondrial TCA cycle to reactivate the TCA cycle. This cycle notably depends on the cytoplasmic enzyme isocitrate dehydrogenase 1, which represents a metabolic vulnerability that could be targeted for therapeutic purposes. Indeed, luminal epithelial cells of the prostate can produce and secrete massive amounts of citrate through a truncated tricarboxylic acid cycle (TCA) , 3, 4, 5per se does not demonstrate restoration of a canonical TCA cycle. In accordance, detailed respiratory assays of mitochondria isolated from PCa tumours have shown a metabolic shift towards higher succinate oxidation . No. NoMPC2 IDH1 in LNCaP cells and was significantly decreased in VCaP cells after the knockdown of IDH1  causing oncogenesis [IDH1 knockdown . In LNCaP cells, AR activation, which stimulates respiration Fig. , also in13C‐labelled glucose to directly study TCA cycle activity in LNCaP cells. Carbon flux from glucose in all the detected intermediates from the TCA cycle significantly increased following AR activation . As expected, AR activation induced a significant increase in proliferation in these cell lines . Several KO clones with deletions altering the open reading frame of IDH1 were obtained  of ned Fig. , indicatned Fig. . Indeed,ity Fig. , consistity Fig. , possiblity Fig. . Moreovells Fig. . ConsequIDH1 KO clones. Surprisingly, we observed no effect of IDH1 KO on the proliferation of these clones , for example, is a transcriptional cofactor that promotes mitochondrial biogenesis and activity. Its overexpression in a breast cancer model in vitro decreased proliferation despite significantly increasing mitochondrial content and respiratory capacity of cancer cells [in vivo context, the same cancer cells exhibited a reverse phenotype, leading to an increased growth advantage compared to control cells in this more restrictive environment [in vivo in a xenograft assay. In this in vivo context, the two IDH1 KO clones showed a significant delay in tumour growth and a longer time required to reach the xenograft study's endpoint compared to control cells  are required to form the IDH3 mitochondrial complex. At the mRNA level, they were all significantly higher in the IDH1‐KO1 tumours compared to control tumours, a phenomenon not observed in IDH1‐KO2 tumours  upstream of IDH1 as well as specific mitochondrial transporters to allow shuttling of metabolites between subcellular compartments. The identification of these components remains to be fully determined. We believe that the establishment of this unusual cancer cell metabolic program originates from the unique prostate‐specific truncated TCA cycle, as recently studied by Frégeau‐Proulx et al. .IDH1 is rarely mutated in PCa cells, we repurposed mutant IDH1 inhibitors in our models to induce a pharmacological blockade of IDH1. Concentrations used herein (1–5 μm) to inhibit PCa cells with wild‐type IDH1 correspond to the concentrations used to specifically kill acute myelogenous leukaemia (AML) cells with mutant IDH1 with no impact on AML cells with wild‐type IDH1 [Mutant IDH1 has been extensively studied over the past decade and is an established clinical target in various brain and blood cancers. Indeed, studies report that when mutated, IDH1 will produce 2‐HG in sufficient amounts to competitively counteract αKG, a necessary cofactor for the activation of dioxygenase enzymes involved in genomic stability. Accumulation of 2‐HG thus disrupts the integrity of the genome and the epigenome, eventually amounting to oncogenesis , 43. As ype IDH1 . Thus, Pype IDH1 , and bec5In conclusion, the current study demonstrates that PCa cells exhibit a hybrid cytoplasmic–mitochondrial TCA cycle that depends on IDH1, an actionable target representing an important PCa metabolic vulnerability.The authors declare no conflict of interest.KG performed experiments, developed methods, analysed results, and wrote the manuscript. CW, LB, CJ, and MH performed experiments, developed methods, analysed results, helped write the manuscript and provided intellectual input. AL and CL performed experiments and provided intellectual input. BN and MS helped with method development and provided intellectual input. JL and AB provided intellectual input. YF, LL, JR, EL, CA, and FP performed surgery and tissue harvesting, helped with method development, and provided intellectual input. J‐PL and MP provided equipment and provided intellectual input. EA‐W conceived and supervised the project, performed experiments, analysed data, secured funding, and wrote the manuscript. All authors contributed to the manuscript and approved it for submission.https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/1878‐0261.13441.The peer review history for this article is available at Fig. S1. IDH activity in PCa models in support of Fig. 1.Fig. S2. IDH expression in mouse and human prostate models in support of Fig. 2.Fig. S3. The TCA cycle of PCa cells functions through an IDH1‐mediated hybrid cytoplasmic‐mitochondrial pathway .Fig. S4. Establishment of a cell model genetically invalid for IDH1 in support of Fig. 5.Table S1. Human qRT‐PCR primers.Table S2. Human shRNA sequences.Click here for additional data file.


Assessing the consistency of quantitative MRI measurements is critical for inclusion in longitudinal studies and clinical trials. Intraclass coefficient correlation and coefficient of variation were used to evaluate the different consistency aspects of diffusion‐ and myelin‐based MRI measures. Multi‐shell diffusion and inhomogeneous magnetization transfer data sets were collected from 20 healthy adults at a high‐frequency of five MRI sessions. The consistency was evaluated across whole bundles and the track‐profile along the bundles. The impact of the fiber populations on the consistency was also evaluated using the number of fiber orientations map. For whole and profile bundles, moderate to high reliability of diffusion and myelin measures were observed. We report higher reliability of measures for multiple fiber populations than single. The overall portrait of the most consistent measurements and bundles drawn from a wide range of MRI techniques presented here will be particularly useful for identifying reliable biomarkers capable of detecting, monitoring and predicting white matter changes in clinical applications and has the potential to inform patient‐specific treatment strategies. Using intraclass correlation coefficient (reliability) and coefficient of variation (variability) we evaluate the different consistency aspects of diffusion‐ and myelin‐based MRI measures data sets (multi‐shell diffusion and inhomogeneous magnetization transfer) collected from 20 healthy adults at a high frequency (five MRI sessions over 6 months). The consistency was evaluated firstly, across whole bundles and track‐profiles along bundles, and secondly, we also investigated the impact of the fiber populations. We report moderate to high reliability and low variability of diffusion and myelin measures for whole and profile bundles. Higher reliability of measures for multiple fiber populations than single was observed. In this context, longitudinal studies of WM have become increasingly important. Currently, various magnetic resonance imaging (MRI) techniques are used to assess different properties of WM tissues such as axonal density, fiber organization, or myelin  although the recent studies include a reasonably large sample size On the other hand, none of these studies evaluated the impact of local WM complexity on consistency, particularly the number of fiber populations. Indeed, these studies consider each voxel as a single entity with a homogeneous fiber population. However, it has been described that voxels contain multiple fiber populations, i.e., between 66% and 90% of white matter voxels cannot be assumed to contain a single coherently oriented axon bundle  MRI data sets in “high‐frequency”—i.e., a high number of MRI acquisitions over a short period of time (6 months) for 20 healthy subjects. All subjects were scanned five times with an average interval of 4 weeks for a total of 100 MRIs. This high‐frequency data set thus generates enough data per subject to allow a relevant assessment of the consistency of different brain MRI measurements. The reliability and variability were evaluated using the intraclass coefficient correlation (ICC) value and within‐ and between‐subject coefficient of variation . Then, the consistency of MRI measurements was evaluated across the bundles as a tracts‐of‐interest analysis approach—i.e., averaging voxels within each WM bundle. To go further, the consistency of each WM measure was also evaluated as a profile along the bundle using 2https://high-frequency-mri-database-supplementary.rtfd.io.Method and results are documented and available at 2.1Twenty healthy adults  were recruited from the environment of the University of Sherbrooke and the Centre Hospitalier Universitaire of Sherbrooke (CHUS). The study was designed with this proportion of male and female subjects to match a future MS group. Participants were screened for eligibility to undergo MRI, no history of brain disease or injury, left‐handedness and received financial compensation for their participation. The study was approved by the ethics committee of the CHUS (Comité d'éthique de la recherche du CIUSSS de l'Estrie) in Sherbrooke, Canada and all participants gave prior informed written consent.2.2Whole‐brain MRI data were acquired using a clinical 3 T MRI scanner  using a 32‐channel head coil. Each MRI session was repeated five times over 6 months and a 4‐week interval +/− 1 week). For each participant, images were acquired at approximately the same time of day to avoid potential diurnal effects . All MRI data acquisitions were aligned on the anterior commissure‐posterior commissure plan (AC‐PC) and included (a) anatomical 3D T1‐weighted, (b) multi‐shell diffusion‐weighted images (DWI), (c) inhomogeneous magnetization transfer (ihMT) for a total time of acquisition of 20 min  3D T1‐weighted MPRAGE image was acquired axially at 1.0 mm isotropic resolution, repetition time (TR)/echo time (TE)/inversion time (TI) = 7.9/3.5/950 ms, field‐of‐view (FOV) = 224 × 224 mm2, 66 slices for an acquisition time of 9 min 19 s. The data included 100 unique directions uniformly distributed over each shell and across the three shells at b = 300 mm2/s (n = 8 directions), b = 1000 mm2/s (n = 32 directions), b = 2000 mm2/s (n = 60 directions), with non‐diffusion‐weighted images b = 0 mm2/s (n = 7), for a total of 107 total diffusion volumes  Multi‐shell DWI images were acquired with a single‐shot EPI spin‐echo sequence at 2.0 mm isotropic resolution, TR/TE = 4800/92 ms, SENSE factor = 1.9, Multiband‐SENSE factor = 2, flip angle of 90°, FOV = 224 × 224 mm(c) Inhomogeneous MT images were acquired using a 3D segmented‐EPI gradient‐echo sequence with different MT preparation pulses with first TE/TR = 3.6/112 ms, 2 × 2 mm resolution, flip angle of 15°, FOV = 224 × 224 mm, 65 slices of 2 mm of thickness and three echoes with echo spacing 6.0 ms for an acquisition time of 6 min 04 s , was used to process DWI and T1w images. This pipeline generates both diffusion measures and tractography of WM, from raw DWI, T1w, bvec/bval files and the reversed phase‐encoded b = 0 and has been proved to be highly reproducible in time and immediate test–retest , which used Accelerated Microstructure Imaging via Convex Optimization . For each echo, raw ihMT images were firstly co‐registered using ANTs linear registration  a multi‐atlas and multi‐parameter version of RecoBundles . The processed b0 images resampled at 1 mm isotropic from Tractoflow were used as an input with four iterations with decreasing degrees of downsampling and smoothing. All subject‐specific measures maps and bundles were aligned in the common diffusion space using the resulting nonlinear registration. Finally, averaged maps were computed for each measure using all subjects and sessions and shown in Figure https://high-frequency-mri-database-supplementary.readthedocs.io/en/latest/results/average_maps.html.To perform consistency voxel‐based analysis, all images were registered in a common space. Symmetric diffeomorphic normalization (SyN) of ANTs is used to build a template in diffusion space based on our population. For the registration, we used iterative rigid, affine, and SyN (neighborhood cross‐correlation) transformations with optimal similarity measures for the linear   the bundle‐averaged that is, one measure for the whole bundle and (2) along the bundle as a profile, also called tract‐profile or connectometry .Many studies have reported that voxels containing multiple fiber populations  was used to evaluate the covariance of the averaged diffusion measures for all bundles. For this, we used each bundle's averaged measure, extracted from each voxel and averaged along bundles. Pearson correlations were computed for each session and then averaged across sessions to generate an average correlation map for all sessions. The average correlation interactive map and correlation interactive maps corresponding to the sessions are available at 2.112.11.1The reliability of computed measures was investigated using the Image Intra‐Class Correlation coefficient . To avoid overloading, confidence intervals and p‐values are only reported for selected bundles and the whole bundle analysis . SLF 1 was not excluded from the analysis, but its results should be taken with caution  and a lower association with FA  and AD . MD and RD also show strong associations with ECvf and ICvf (r > .8). On the other hand, AD (except for RD and MD), OD and the two measures of HARDI seem uncorrelated, either between or within a model . MTI measurements show a strong association between ihMTdR1sat and MTsat (r = .88), ihMTR (r = .81); and much weaker correlation between ihMTdR1sat and MTR (r = .45). MTsat appears to be the only MTI measure that correlates, albeit moderately, with diffusion measures (r ranging from .3 to .6). This weak correlation between MTI measures and diffusion measures shows that these are sensitive to different microstructural features.Pearson's correlations of all measurements across WM bundles are shown in Figure https://high-frequency-mri-database-supplementary.readthedocs.io/en/latest/results/measure.html#whole-bundle-measures.Based on these results, subsequent analyses will present the following measurements: RD, AFD total, NuFO, ISOvf, MTR and ihMTdR1sat were extracted from the 4 selected bundles: Arcuate Fasciculus (AF), Section 3 of the Corpus Callosum (CC), and Cortico‐Spinal Tract (CST). These measures were selected either because they show strong correlations with other measures and may share overlapping information which can cause redundancies, or because, in the opposite case, they show a pattern of decorrelation with other measures and may therefore provide different information  followed by MTR (ICC ~ 0.67 across bundles). Both MT measures showed the lowest variability with CVs <5% for all bundles. Noted that MTsat showed higher variability (CVw ~ 2.8% and CVb ~ 4%) compared to MTR (CVw ~ 1.9% and CVb ~ 2.1%) .The distribution of measures along the bundle is provided here s Figure . The difMore precisely, similarly to whole bundle results, consistency of DTI measures quantified along the bundles most often displays very stable profiles with higher ICCs (mean across section = 0.8 [0.64–0.95]) and lower CVs values  with respect to other measures  wRegarding the MTI measures, the consistency exhibits globally stable profiles for most bundles, with more stable CV profiles than ICC profiles. As for the whole bundle results, the highest ICCs and lowest CVs values are found for MTsat and MTR measures, while ihMT measures showed lower reliability (ICC ~ 0.51 [0.3–0.82]) and higher variability (CVw ~ 7.6% [3.1%–15.6%] and CVb ~ 6.5% [1.9%–15%]) . Compared with ICC computed from average bundle, the compartmentalization revealed higher ICCs  and, equal to lower CVs across bundles . More precisely, ICCs measures from multi‐compartment were higher, while the ICCs value observed for single compartment is lower and inversely for CVs (see https://high‐frequency‐mri‐database‐supplementary.readthedocs.io/en/latest/results/fiber_population_consistency.html#whole‐bundle‐consistency). Differences between single and multi‐compartments can be moderate, especially for DTI measurements, or more important such as MTI measurements. Some measures exhibit a different pattern than others. This is the case for the OD measurement of the NODDI model, whose reliability pattern is inverted. As expected, since OD is a measure of the dispersion of fiber orientation in the voxel, a higher or equal ICCs to the average for single compartment compared with multiple compartments for most bundles is not surprising. In addition, the impact of compartmentalization has a more moderate effect on the consistency of SLF bundles measures. Indeed, the values of ICCs and CVs are generally equal (or present a slight difference) between the two compartments. This moderate effect is also found for DTI measures, for most, but not all, bundles . Globally, there is a decrease (or increase depending on the measure) in the mean values of the measures with increasing fiber populations. The distribution profile of measures is available https://high‐frequency‐mri‐database‐supplementary.readthedocs.io/en/latest/results/fiber_population_consistency.html#profile‐bundle‐consistency). The single‐compartment profile is generally close to or equal to the whole bundle profile, while the multi‐compartment is more distant from the average profile. Some bundles present a different pattern for the inferior (sections 1–3) and superior  sections compared to middle sections. For example, CST exhibits a higher ICC in the single compartment for inferior sections (1–2), whereas the superior sections (8–10) show weak differences between the compartments compared to the middle part of the bundle  across the bundles and especially excellent for most DTI measures as well as AFD total and OD index from the HARDI and NODDI models, respectively . In addition, the profile consistencies of these measures are comparable to the whole bundles, with voxel values well above ICC >0.7 and CVw <4% along the bundle. In contrast, MTI showed good reliability and variability for MT measurements  and moderate for ihMT measurements . We also showed that the number of fiber populations affects the consistency of the measurements, with a moderate effect on the DTI and HARDI measurements, and a more important effect on the NODDI and MTI measurements. Finally, SLF 2–3 and CC 4 to CC 6 bundles showed the most consistent MRI measurements followed by AF, CST, CC 7, OR, CC3, IFOF and CG, while CC 2a, CC 2b, UF and ILF have more moderate consistency measurements Figure .In this study, some choices have been made for acquisition schemes, MRI techniques or processing tools to do all the consistency analyses. We know that many other options are available for similar tasks and ways to assess reliability and variability. Our protocol is a compromise between the literature and the constraints imposed by the scanner. Indeed, the choice of b‐values at 300, 1000 and 2000 with a specific non‐equal distribution of the 100 directions is optimized for the balance between signal‐to‐noise ratio (SNR) and angular contrast ratio, while maintaining a reasonably short acquisition time. Low b‐values of 300 and 1000 with 8 and 32 directions (respectively) are used to obtain a robust and unbiased estimate of the DTI and the mono‐exponential part of the signal , the higher the ICC and lower the within‐ and between‐subject CV (and inversely). Despite it seems like counter‐intuitive results, a smaller range of values for the compartment with multiple fiber populations for most measures compared to the single compartment explains the greater consistency (CST FA range in multi: 0.34–0.42 vs. single 0.48–0.61). Thus, the intrinsic variability of the measurements in the multi‐compartment is already more restricted than in the single compartment. Although the method and the variability values are not directly comparable, Volz et al. also report greater variability of the compartment with a single fiber population compared to compartments with multiple fiber populations  and high between‐variability . This variability pattern is a necessary criterion for the high validity of a biomarker, which can be used to diagnose, monitor and predict neurological consequences by clarifying the effects of a disease or treatment. However, another important point in clinical practice is the distinction between healthy and pathological states, for which high between‐variability is not required.As expected, the measures corresponding to this criterion are the “simplest” that is, the DTI‐derived measures, particularly FA, RD, AD and MD. However, more advanced measures could also be promising candidates as biomarkers. Indeed, axonal density and neuroinflammation indices can be interesting in pathologies where the axons are altered and disrupted, such as Alzheimer's disease . Again, this indicates that MTsat would be a more favorable biomarker than MTR. Moreover, recent studies have shown that MTsat is more sensitive than MTR in MS  , the Université de Sherbrooke Institutional Chair in Neuroinformatics from Pr Descoteaux (www.usherbrooke.ca) and Mitacs Accelerate program (www.mitacs.ca). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Part of this research was supported by the NSERC Discovery grant . Federico Spagnolo is an employee of F. Hoffmann‐La Roche Ltd. Muhamed Barakovic is an employee of Hays plc and a consultant for F. Hoffmann‐La Roche Ltd. Stefano Magon is an employee and shareholder of F. Hoffmann‐La Roche Ltd. Guillaume Gilbert is an employee of Philips Healthcare. Guillaume Theaud, Matthieu Dumont, Jean‐Christophe Houde and Loika Maltais are employees at Imeka Solutions Inc.Maxime Descoteaux is co‐owner and chief scientific officer at Imeka Solutions Inc. (available online: https://github.com/scilus. Operations like generating and eroding masks were done using Scilpy scripts available at https://github.com/scilus/scilpy. Consistency analysis code is available at https://github.com/AlexVCaron/longitudinal_image_statistics.All Nextflow and code used to process the T1 and DWI images, ihMT images, RecoBundlesX and Tractometry are available at Data S1: Supporting InformationClick here for additional data file.