Multimodal analgesia consists of the combination of analgesic drugs at low doses to act in different places along the path of pain. Studies with continuous infusion of analgesic drugs in cats are not common. This study aimed to evaluate the analgesic effect of maropitant, lidocaine and ketamine alone or in combination (intravenous bolus + subsequent continuous intravenous infusion) in the management of acute postoperative pain in cats undergoing ovariohysterectomy. Seventy healthy cats undergoing an ovariohysterectomy received a standard anesthetic protocol consisting of acepromazine and morphine, propofol (anesthesia induction), and isoflurane (anesthesia maintenance). The animals were stratified into seven groups (n = 10 in each group): control (CG), maropitant (MG), lidocaine (LG), ketamine (KG), maropitant + lidocaine (LMG), maropitant + ketamine (KMG), and maropitant + lidocaine + ketamine (LKMG). All drugs were injected first as an intravenous bolus and then by continuous intravenous infusion. During surgery, esophageal temperature, respiratory rate, heart rate, oxygen saturation, expired isoflurane concentration, and partial pressure of carbon dioxide at the end of expiration were evaluated at 7 time points. Postoperative pain was evaluated for 6 h after extubation using the visual analogue scale and the UNESP-Botucatu multidimensional composite pain scale for assessing postoperative pain in cats.Adverse effects related to maropitant, lidocaine and ketamine infusion were not observed. Pain scores were lower in the MG, KG and LG groups when compared to the CG group using both scales. Although pain scores were also lower in all combination groups than CG, more animals in these groups required rescue analgesia compared to MG. This indicates that the postoperative analgesic effect of all drugs, either alone or in combination, confers analgesia, although the combinations did not promote greater analgesia.Continuous intravenous infusion of maropitant, lidocaine, and ketamine alone induces postoperative analgesic effect in cats undergoing ovariohysterectomy, but combinations of these drugs did not increase the analgesic effect. No adverse effect was observed with any drug or their combination. Surgical procedures elicit harmful stimuli, which, if not prevented, can alter the neuroplasticity of the spinal cord and generate central sensitization. This sensitization process involves several receptors and excitatory neurotransmitters in the dorsal horn of the spinal cord. Thus, novel strategies are needed to prevent central sensitization and improve the management of postoperative acute pain .Multimodal analgesia involves the use of multiple analgesic drugs, usually at low doses, with different mechanisms for managing pain –5. CombiLidocaine blocks sodium channels, preventing the propagation of action potential, and consequently generates sensory and motor blockade for local and regional anesthesia. It can induce local anesthesia and can be used as an analgesic through continuous intravenous (IV) infusion in dogs and humans both intra- and post-operatively , 9. In cN-methyl-d-aspartate (NMDA) receptors  of Ringer’s solution with lactate.Maropitant group (MG): IV bolus (1 mg/kg) + maropitant  IV continuous infusion (1.67 µg/kg/min).Lidocaine group (LG): IV bolus (1.5 mg/kg) + continuous IV lidocaine  infusion (50 µg/kg/min)Lidocaine and maropitant group (LMG): IV bolus (1.5 mg/kg lidocaine + 1 mg/kg maropitant) + continuous IV infusion of lidocaine and maropitant Ketamine group (KG): IV bolus (1 mg/kg) + continuous IV ketamine  infusion (10 µg/kg/min)Ketamine and maropitant group (KMG): IV bolus (1 mg/kg ketamine + 1 mg/kg maropitant) + continuous IV infusion of ketamine and maropitant Lidocaine, ketamine, and maropitant group (LKMG): IV bolus (1.5 mg/kg lidocaine + 1 mg/kg ketamine + 1 mg/kg maropitant) + continuous IV infusion of lidocaine, ketamine, and maropitant The cats were divided into seven groups (10 in each group) by block randomization. Cats received the same final volume as the control group, according to the weight of each animal:The bolus dose  was diluted in Ringer’s solution with lactate  and administered intravenously  over one minute. The injections were administered sequentially for cases in which more than one drug was involved . After IV bolus administration of drugs, the animals were anesthetized and continuous IV infusion of one or more drugs was immediately initiated. Notably, maropitant was always infused alone, whereas lidocaine and ketamine were infused together in the same syringe. All drugs used for continuous IV infusions were diluted (Ringer’s solution with lactate) to a final volume of 20 mL. Continuous IV infusions were maintained until the end of surgery with two syringe-infusion pumps .All cats were premedicated with intramuscular injections of acepromazine  and morphine . A 24-gauge catheter  was inserted into the cephalic vein 20 min after the pre-anesthetic medications and used for drug administration and fluid therapy using Ringer’s solution with lactate (Fresenius Kabi Brazil Ltda) at a rate of 5 mL/kg/h. Immediately after catheterization, anesthetic induction was performed with propofol administered to effect , and the cat’s trachea was intubated . Isoflurane (1.5 vol.%)  with 100% oxygen (300 mL/kg/min) was administered via a non-rebreathing system  for maintenance anesthesia. Cephalothin (30 mg/kg)  was administered intravenously as the prophylactic antibiotic therapy before the surgery. The animals were placed in the dorsal recumbency position and antisepsis of the surgical site was performed. Ovariohysterectomy was performed via midline incision caudal to the umbilicus by an experienced surgeon in all animals.2), expired isoflurane concentration (Etiso) , and partial pressure of carbon dioxide at the end of expiration (EtCO2). All values were obtained with a multi-parameter monitor (Mindray BeneView T8). Systolic blood pressure (SBP) was measured using a vascular ultrasonic Doppler . A blood pressure cuff was placed on the proximal third of the radioulnar region and a Doppler crystal placed over the median artery. If the animals presented physiological changes such as hypotension and hypercapnia, physiological support such as medications  and assisted ventilation could be provided.The following variables were evaluated: esophageal temperature (T), respiratory rate (RR), heart rate (HR), oxygen saturation  to the surgical stimulus observed by considering an increase of 20% relative to basal values. Not necessarily all three variables  increased together. The baseline values were collected from the anesthetized animals before the start of the surgical procedure.Pain was evaluated postoperatively by only one evaluator, who was blinded to the treatment performed. Pain assessment was done 1 h after extubation, and then every hour for 6 h. Two pain assessment scales were used to assess the postoperative pain in cats: the visual analogue scale (VAS) and the UNESP-Botucatu multidimensional composite pain scale for assessing postoperative pain in cats .Whenever the value obtained on the multidimensional scale for postoperative pain assessment was ≥ 10  and/or V2, EtCO2, and T) were subjected to two-way analysis of variance, and their means were compared using Bonferroni test. Non-parametric data  of the groups were subjected to the Kruskal–Wallis test for between-group comparisons. When these groups were compared with the control group, the Mann Whitney test was used; Fisher’s exact test was used to compare the number of rescue treatments. For all tests, the significance level was set at P < 0.05.All data collected were analyzed using Prism for Windows . The data were tested for normal distribution using the Shapiro–Wilk test. Normally distributed data , surgery (25.3 ± 0.9 min), and extubation (8.8 ± 1.1 min). The body weight of the animals did not differ significantly between the groups (mean 2.9 ± 0.2 kg).HR of animals in the CG group was significantly higher at the time of ovarian pedicles ligation when compared with those in the LMG, KMG, and LKMG groups. Animals in the MG group had a significantly higher HR than those in the LMG and KMG groups. Animals in the LG group showed no significant difference in HR when compared with the other groups Table . The RR 2, EtCO2, Etiso, and T showed no significant difference between the groups. These variables presented the following minimum and maximum values: SBP, 55–133 mmHg; SpO2, 97–100%; EtCO2, 35–45 mmHg; Etiso, 1–2 V%, and T, 35–38.8 °C.SBP, SpOThe pain scores were lower in the MG group than in the CG group at all assessment time points for both scales used. The pain score of MG group did not differ from the other groups that received lidocaine or ketamine alone and their combinations, at any time point. Pain scores in the other groups differed statistically from that of the CG group P = 0.01)  were obtained from the literature . The bolCats belonging to MG, LG, and KG had lower HR and RR than those in the CG group, especially at the time of ovarian pedicle traction. Mesovary traction and ligation generate noxious stimuli, which can increase the HR and RR due to the autonomic nervous system response , 26. In Cats that received lidocaine alone or in combination with other drugs were stable in HR, SBP, and RR. The plasma concentration of lidocaine was probably < 5 µg/mL, and therefore at a level not expected to influence HR or SBP . PypendoIn the present study, HR and SBP remained unaltered despite the use of ketamine as also observed by Gutierrez-Blanco et al. , but dif2, T, and ETCO2. These results are in congruence with findings reported previously [Continuous IV infusions of maropitant, ketamine, lidocaine, and their combinations did not significantly change SpOeviously , 21 for eviously , 28–30.All treatment groups showed an antinociceptive effect when compared with CG. It has been shown that isolated or combined administration of drugs reduce pain, indicated by the low numbers of analgesic rescues and lower scores in the pain assessment scales. Lidocaine and ketamine are indicated for the management of acute pain in dogs resulting from surgical procedures . IV admiWhen analgesic drugs are not used, nociceptive signaling generated during surgical procedures induce central sensitization as well as neuroplasticity in the dorsal horn of the spinal cord , 33. CenContinuous IV infusion of maropitant, lidocaine, or ketamine alone promoted an antinociceptive effect. In dogs, continuous infusions of ketamine and lidocaine alone or in combination or with other drugs induce antinociceptive effects and decrease analgesic requirements , 37. TheOur study shows that ketamine produced an isolated antinociceptive effect, probably because the IV bolus administration, followed by continuous IV infusion throughout the surgical procedure, maintained adequate plasma concentrations for sustained analgesia. A study of ketamine (0.5 mg/kg) as the pre-anesthetic medication in cats showed that ketamine alone does not produce satisfactory analgesia postoperatively . We beliAlthough Kinobe and Miyake  reportedMaropitant produced a postoperative antinociceptive effect in cats undergoing OHE. This effect was also observed with the administration of lidocaine and ketamine alone, which is consistent with the findings of Corrêa et al. .Continuous IV infusion of maropitant, lidocaine and ketamine alone in domestic cats generated an analgesic effect in the postoperative period after OHE. Combination of these drugs, however, did not significantly increase the analgesic effects. The study provides insight into the use of these drugs for multimodal analgesia in cats, mainly by demonstrating that maropitant, lidocaine and ketamine did not generate adverse effects or other types of alterations indicative of intoxication.


To identify differences in quality of life (QoL) of patients with inflammatory bowel disease (IBD) between diagnosis (Crohn’s Disease and Ulcerative Colitis), gender , treatment condition (with and without surgery), and attachment styles ; to examine associations between QoL, sociodemographic, clinical, and positive psychological variables; to determine whether sociodemographic, clinical, and positive psychological variables predict QoL.Mage = 43.37 years, SD = 12.81), of whom 71.4% were females and 67.1% had Crohn’s Disease. Positive psychological variables , sociodemographic  and clinical variables  were assessed as independent variables. QoL was the dependent variable, analyzed through four domains .The sample included 70 participants diagnosed with IBD  than participants with a preoccupied attachment style. Strong positive correlations were found between positive psychological variables and QoL. Body appreciation was a significant predictor of three QoL domains . Meaning in life made a unique contribution to the social QoL regression model, and it was also a significant predictor of psychological QoL. Body acceptance by others was a significant predictor of physical QoL, whereas disease duration and education predicted environment QoL. Attachment styles did not predict any QoL domain.The most significant predictors of QoL in patients with IBD were body appreciation and meaning in life. Body acceptance by others and body appreciation were the main predictors of physical QoL. Psychological interventions for patients who suffer from IBD should address body appreciation and meaning in life. Inflammatory Bowel Disease (IBD) represents a group of gastrointestinal disorders that cause chronic and recurrent inflammation of the gastrointestinal tract . Patients were included in the study if they were 18 years of age or older, were diagnosed with IBD, and did not suffer from any other chronic condition. Three participants did not meet the inclusion criteria, as they presented other comorbidities, such as obesity, obsessive-compulsive disorder, and psoriasis. The final sample was thus composed of 70 participants.SD = 12.81), ranged from 18 to 73 years of age. Most participants were married . The mean years of schooling was 15.16 (SD = 3.51), with an education range of 7 (basic education) to 25 years (higher education).SD = 128.15; range 2–500 months). Thus, the sample included participants who were recently diagnosed, but also individuals who have lived with the diagnosis for 41 years. Most participants have not been submitted to surgery .The mean of months elapsed between receiving the diagnosis and the data collection (disease duration) was approximately 164 .Meaning in Life Scale . The Body Acceptance by Others Scale .Two scales were used to measure positive body image. The Body Appreciation Scale-2 (BAS-2)  . Data was collected in collaboration with the Portuguese Association of Inflammatory Bowel Disease (APDI) between January and April 2020. Invitations to complete the survey were sent by APDI. The members received an email containing a brief description of the study and a link to a secure online survey. Participants were informed, online, about the ethical principles of the study . After providing digital informed consent, participants were asked to complete the instruments described above.Statistics 26).Data were analyzed using the Statistical Package for the Social Sciences, 26.0 version for Windows (IBM SPSS η2) was interpreted according to the commonly used Cohen’s  and the following criteria: absolute skewness (Sk) and kurtosis (K) values lower than 3.0 and 8.0, respectively Kline, . No miss Cohen’s  guidelins Cohen, . Multipls Cohen, . A signiDescriptive statistics  for positive psychological variables are displayed in n = 43), followed by preoccupied (n = 13), disconnected (n = 11) and fearful (n = 3). Thus, subjects who presented a fearful attachment style were excluded from this analysis due to the small subsample size. Individuals with a secure attachment style reported a significantly higher physical (F (2.64) = 6.03, p = .004, η2 = .16), psychological (F (2.64) = 4.82, p = .011, η2 = .13), social QoL (F (2.54) = 6.57, p = .003, η2 = .17) than participants with a preoccupied attachment style, but there were no differences for the environment QoL  = 1.35, p = .226, η2 = .41). Data not demonstrated.The most frequent attachment style was secure  and environment QoL domains  were positively correlated with education. The environment domain was also found to be positively correlated with the disease duration .All positive psychological variables were significantly correlated with QoL domains, with exception of psychological QoL that was not significantly correlated with body acceptance and of environment QoL domain that was not significantly correlated with anxiety attachment factor. Regarding sociodemographic and clinical variables, psychological  = 3.08, p < .01) and body acceptance  = 2,41, p = .02) made a statistically significant contribution to the physical QoL regression model. The model was significant and explained 43.1% of the variance in physical QoL  = 14.042, p <.001). Variance Inflation Factor (VIF) values were analyzed, and it was concluded that the model did not exhibit multicollinearity.β = .36, t (65) = 3,48, p < .01) and ML  = 4,41, p < .01) made a statistically significant contribution to the psychological QoL regression model. The model was significant and explained 57.8% of the variance in psychological QoL  = 24.622, p < .001). VIF values were analyzed, and it was concluded that the model did not exhibit multicollinearity.Body appreciation  = 2.31, p = .02). The model was significant and explained 23.2% of the variance in social QoL  = 6.198, p < .001). VIF values were analyzed, and it was concluded that the model did not exhibit multicollinearity.The four variables that showed the highest correlation with social QoL  were included in this model. ML made a statistically significant contribution to the social QoL regression model (p < .001), but body appreciation was the only variable that made a unique contribution to the model  = 3.12, p < .01). Therefore, we decided to improve the environment QoL regression model, by including the most relevant sociodemographic and clinical variables for this domain according to the literature. The second model explained a higher variance (43.7%) in environment QoL  = 18.304, p < .001). Body appreciation  = 6,06, p < .01), education  = 2.58, p < .01), and disease duration  = 2.02, p = .05) made a statistically significant contribution to the environment QoL regression model. The model VIF values were analyzed, and it was concluded that the model exhibited no multicollinearity.Body appreciation, body acceptance by others, Trust in others, and ML were initially included into the environment QoL regression model, as they were the variables that showed the highest correlation with environment QoL. The results showed the model was significant and explained 36.8% of the variance in environment QoL  = 11.055, The present study aimed to identify differences in QoL according to patients’ diagnosis, gender, treatment condition, and attachment styles. No significant differences were found between CD and UC on QoL. This result is in line with the studies conducted by Amorim and Guerra  and by RRegarding gender, some studies have reported a lower QoL in females diagnosed with IBD than in males  may limit the generalization of the results to the Portuguese population diagnosed with IBD. Thus, it would be important to collect a larger sample with more heterogeneity regarding gender in order to achieve more reliable conclusions. It would also be important to collect data about the stage of the disease (active or remission) to better understand if this variable contributes to the improvement of QoL (Tylka & Wood-Barcalow, This study added a positive perspective about the factors that influence QoL of individuals diagnosed with IBD. It also enhances the need to promote, in clinical practice, a positive body image of patients, to help them seek for ML, and build positive attachment styles, in order to improve QoL. The findings revealed that all independent variables included in this study were significant predictors of at least one of the four domains of QoL, which reinforces the protective factors complexity in patients with a chronic disease. This study can contribute to the advancement of scientific research in the field of positive psychology and to the development of clinical practices that help improving QoL in patients with IBD.


To investigate the population pharmacokinetics of posaconazole gastroresistant tablets in children with cystic fibrosis (CF) and perform simulations to recommend optimal doses.Children from a paediatric CF centre who had received posaconazole tablets and underwent therapeutic drug monitoring were identified from pharmacy records. Relevant clinical data were collated from case notes and electronic patient records and used to develop an allometrically scaled population pharmacokinetic model. A stepwise covariate model-building exercise evaluated the influence of interacting medicines and liver function.2 = 0.98). Simulations showed that trough concentrations of >0.75 mg/L would exceed an AUC of 30 mg·h/L in 90% of patients.One hundred posaconazole serum concentrations were collected from 37 children with a median age of 14 years (range 7–17). Posaconazole pharmacokinetics were adequately described by a one-compartment model with inter-individual variability on clearance. Dose simulations demonstrated a 77%–83% probability of attaining a trough target of 1 mg/L with a dose of 300 mg every 12 h for two doses then 300 mg once daily (OD) in children aged 6–11 years; and 86%–88% with a dose of 400 mg every 12 h for two doses then 400 mg OD in adolescents aged 12–17 years. This dose scheme also yielded a 90% probability of achieving an AUC of 30 mg·h/L. AUC and trough concentration were highly correlated (rA starting dose of 300 mg OD in those aged 6–11 years and 400 mg OD in those aged 12–17 years (following loading doses) yields a 90% probability of attaining an AUC of 30 mg·h/L. Aspergillus fumigatus is one of the most commonly isolated fungi in sputum from CF patients.,Aspergillus grown in 9.4% of children (aged 0–15 years) in the 12 months prior to the 2019 annual review. This is proportionally higher in older age groups, with positive sputum samples found in 10.1% of children aged 8–11 years and 18.9% in those aged 12–15 years.Scedosporium species and Exophiala dermatitidis.Cystic fibrosis (CF) is a common autosomal recessive genetic disorder, resulting from the defective CF transmembrane conductance regulator (CFTR) gene, which causes abnormal mucous secretions in multiple organs, notably the lungs, liver and pancreas. Lung disease is characterized by chronic pulmonary infection by opportunistic bacteria and fungi, leading to inflammation and a progressive decline in lung function. ,Aspergillus diseases and British Society of Medical Mycology antifungal TDM guidelines suggest a posaconazole treatment target trough concentration of >1 mg/L, with no upper limit yet shown to be associated with toxicity.,Triazole antifungal medicines, such as posaconazole, are used for the treatment of lung disease caused by these fungi in children with CF. Although there are no national or international guidelines specific to CF, this is in line with international guidelines for the wider population.Cmin for the tablet formulation was found to be 48% lower in patients with CF.A. fumigatus isolates from CF patients were found to be resistant to azole antifungals.People with CF are considered to be a unique population, displaying altered pharmacokinetics for some drugs, most likely as a result of comorbidities affecting drug absorption and disposition.A recently published population pharmacokinetic model provided dose recommendations for a liquid formulation of posaconazole in immunocompromised children.2-receptor antagonists, the indication for posaconazole, details of the dosing regimen (including formulation taken) and sampling time/date. Samples were excluded from the analysis if sample timing was not known or if preceding doses were identified as not administered.Retrospective patient data collected during the routine care of children aged between 5 and 17 years (both inpatients and outpatients) within the paediatric CF clinic at a tertiary centre, who had received posaconazole gastroresistant tablets for treatment of CF-related fungal lung disease between 2014 and 2020, were collated. The study and use of anonymized clinical data was approved by the Trust’s Research and Development Office and Health Research Authority, reference number 20/HRA/3760. As the study involved the retrospective use of anonymized data, formal participation/parental consent was not required. Patients were identified by matching pharmacy and clinical biochemistry records by pharmacy staff who routinely care for children with CF. Patients who had at least one posaconazole TDM sample taken were included. The time and date of the posaconazole TDM sample, along with the reported concentration, were extracted from electronic patient records. Samples that were below the limit of quantification in the analysis were recorded in the dataset as LOQ/2. For inpatients, dosing history was taken from the electronic nursing administration record, capturing doses given in the preceding 48 h before sampling. For outpatients, the time of the last dose, if not recorded, was presumed to be at 8 am on the day of the sample and assumed to be at steady state based on the patient’s drug history. The model was run with and without these samples to check if this assumption led to biased parameter estimates. Additional data collected from electronic patient records were basic demographic information, liver function at the time of sampling, concomitant interacting medications, use of concomitant proton pump inhibitors and histamine HPosaconazole analysis was carried out using 2D TurboFlow™ HPLC tandem MS (2D HPLC-MS/MS). This was on a Thermo Fisher Scientific Transcend™ prior to 2017 and a Prelude SPLC™ HPLC system coupled with a TSQ Endura™ triple quadruple mass spectrometer  thereafter.,Analysis was carried out by Trust laboratories using in-house methodology, developed in line with FDA and EMA guidelines.2O) and 10 nM ammonium acetate in methanol and posaconazole was detected using atmospheric chemical ionization in positive ion mode.Samples were stored at −20°C prior to analysis. Posaconazole was extracted from serum via protein precipitation with acetonitrile. Posaconazole-d4 was used as the corresponding internal standard. Chromatography mobile phases were 10 nM ammonium acetate in ultrapure water ,a prioriAllometric size scaling of clearance and volume terms were added P < 0.01. A stepwise covariate model (SCM)-building exercise with Perl Speaks NONMEM (PsN)P value of ≤0.05 and backwards elimination with a P value of ≤0.01. The following covariates were tested on clearance: liver function abnormality ; and potential interacting medicines that were identified in patients included in the dataset: Orkambi® (lumacaftor/ivacaftor), rifampicin, rifabutin, clarithromycin, histamine H2-receptor antagonists and proton pump inhibitors.For nested models, significant model improvement through additional parameters was evaluated with the likelihood ratio test, the difference in −2 log-likelihood  of the models being asymptotically ,,Dosing simulations were performed in R.Final model evaluation consisted of plotting predictions versus observations, and conditional weighted residuals versus time and predictions. A visual predictive check with 1000 samples was performed to check predictive performance, and a non-parametric bootstrap (1000 samples) was conducted to evaluate parameter robustness.The initial dataset included 109 posaconazole serum concentrations from 41 children, obtained during 65 treatment courses. From this, nine serum concentrations were excluded . The final dataset therefore included 100 levels from 37 children, with a median age of 14 years (range 7–17). There were 29 samples taken from children aged 7–11 years, with median weight of 31.5 kg (range 25–58). The median posaconazole serum concentration for this age group was 3.08 mg/L (range 0.2–8.91) from a median dose of 300 mg (range 100–300). Of 71 samples taken from children aged 12–17 years, with median weight of 50 kg (range 34.7–82.8), the median posaconazole serum concentration was 2.07 mg/L (range 0.2–4.96) from a median dose of 300 mg (range 200–600). There were 47 samples from outpatients where the time of the last dose was presumed to be at 8 am on the day of the sample. Study population characteristics are detailed in Table a) was 0.16 h−1, apparent clearance (CL/F) was 8.43 L/h and apparent volume (V/F) was 186 L, standardized to a 70 kg individual. The corresponding estimates for the reduced dataset (including only those samples with known dose times) were: 0.13 h−1, 7.93 L/h and 108 L for Ka, CL/F and V/F, respectively, and hence there was no apparent influence of assuming dose time so all samples were included.The pharmacokinetics of posaconazole in children with CF were adequately described by a one-compartment model with IIV on clearance and additive and proportional residual error. Goodness-of-fit plots, and a visual predictive check indicating an acceptable model fit, are given in Figure 2 = 0.98)  followed by 300 mg once daily (OD) was 77% in children aged 9–11 years and 83% in children aged 6–8 years; using a starting dose of 400 mg every 12 h for two doses followed by 400 mg OD, the probability of attaining a trough target of 1 mg/L was 88% in adolescents aged 12–14 years and 86% in adolescents aged 15–17 years. However, simulations using an alternative AUC target showed that with these dosing regimens there was a greater probability (more than 90% in both age groups) of attaining an AUC of 30 mg·h/L. Plots of simulated target attainment versus dose are shown in Figure Here we report the first population pharmacokinetic analysis of posaconazole gastroresistant tablets in 37 children with CF, 14 of whom were less than 12 years old, who received posaconazole to treat CF-related fungal lung disease. A one-compartment model adequately described the data  are within the stated 95% CIs for CL/F derived by Boonsathorn (6.3–34.1), the lower value in our model may be explained by the difference in formulations used by the patients in each of the models, with lower bioavailability of the suspension used in the model in children without CF leading to a higher estimate of CL/F.et al.−1).a to 0.588 h−1 had limited impact on model fit and other parameter estimates (ΔOFV +2.64 points when fixing Ka), indicating inferences on AUC or trough concentration would not be affected.The population pharmacokinetics of posaconazole in children with CF from our model are broadly in line with the parameters ascertained by Boonsathorn Staphylococcus aureus and non-tuberculous mycobacteria including Mycobacterium avium complex. Recommended treatment for these includes rifampicin and rifabutin (inducers of these pathways) and clarithromycin (an inhibitor), which respectively have the potential to either reduce or increase serum concentrations of posaconazole if given concomitantly. Many patients with CF are now treated with CFTR modulators, which target the basic CFTR channel defect. One of these, Orkambi®, a combination of ivacaftor and lumacaftor, can reduce posaconazole serum concentrations by induction of UGT via the lumacaftor component, and concomitant use with posaconazole is not recommended.®, though due to the induction effect of the lumacaftor component of Orkambi® on CYP3A enzymes at steady state the overall exposure of ivacaftor with concomitant posaconazole does not exceed that achieved in patients receiving ivacaftor monotherapy.® if initiating posaconazole in patients already commenced and stabilized on this medicine, as was the case in our cohort. We tested these interacting drugs as covariates in the analysis as it was hypothesized that these may be associated with changes in posaconazole CL/F. However, none were statistically significant. It is likely that the small percentage of samples included within the analysis was insufficient to detect any effects. We also tested the effect of concurrent use of proton pump inhibitors and histamine H2-receptor antagonists as a covariate, and no effect on the CL/F of the tablet formulation was seen, confirming the findings already seen in healthy subjects.et al.Posaconazole is metabolized via UDP glucuronidation  and is a substrate for p-glycoprotein (P-gp) efflux.,Aspergillus, which states this value as it is associated with a 75% response rate in patients receiving posaconazole as salvage therapy for invasive aspergillosis and is equivalent to a Cavg of 1.25 mg/L.,et al.A. fumigatus invasive pulmonary aspergillosis, an AUC greater than 30 mg·h/L was associated with adequate resolution of the galactomannan index—a surrogate marker of antifungal response against Aspergillus species. For azole antifungals, the pharmacodynamics and thus response to treatment are ultimately related to the MIC of an organism, and specifically the AUC0–24/MIC ratio, which is the pharmacodynamic index for posaconazole consistently shown to be most closely correlated with efficacy. Gastine et al.A. fumigatus strain with an MIC of 0.25 mg/L in line with the EUCAST epidemiological cut-off value for this organism, resulting in an AUC0–24/MIC ratio of 120. Recommendations for the AUC0–24/MIC target range between 100 and 200 for treatment of A.fumigatus.,,Dosing simulations were carried out against a target trough concentration of 1 mg/L, as per established guidelines,Aspergillus-related lung disease, and to evaluate response for less susceptible strains of A. fumigatus where we would expect to increase the AUC attained to maintain an AUC0–24/MIC ratio of >100. Our study also included children with lung disease associated with Scedosporium species (Lomentospora prolificans and Scedosporium apiospermum) and E. dermatitidis. Unlike Aspergillus, pharmacodynamic targets have not been elucidated for these organisms and there is a need to identify these and produce recommendations on the optimal use of posaconazole in children with CF with these organisms.Our simulations showed that an AUC of 30 mg·h/L was easier to attain with ‘standard’ dosing, that is doses in a similar range to those licensed in adults, than a trough concentration of 1 mg/L. With the number of samples required to determine AUC accurately being impractical in clinical use, our simulations showed that with AUC and trough concentrations highly correlated, trough concentrations of >0.75 mg/L  would exceed an AUC of 30 mg·h/L in 90% of patients. Future work is required to evaluate whether a trough concentration of 0.75 mg/L, as a surrogate for AUC, is sufficient to optimize outcome in this cohort for the treatment of This study is limited by its retrospective nature and ideally a prospective study with optimal sample timing should be carried out. However, given the difficulty in recruiting to studies requiring multiple blood samples in children, it is likely that our study included a larger cohort than might otherwise have been possible. Our simulations used recommended, though unvalidated, targets in our cohort of patients. There is a need to refine and validate these according to the organism, site of action and disease state, and most importantly assess the clinical outcome in children with CF.Aspergillus infections.,Our study confirms that the population pharmacokinetics of posaconazole in children with CF are in line with data published in other paediatric populations. Our dose simulations suggest that a starting dose of 300 mg every 12 h for two doses (loading) then 300 mg OD in children aged 6–11 years and 400 mg every 12 h for two doses then 400 mg OD in adolescents aged 12–17 years will ensure a 90% probability of attaining an AUC of 30 mg·h/L, a target which is known in other patient populations to be associated with a favourable outcome when treating A. fumigatus, including whether a trough concentration of 0.75 mg/L is sufficient to optimize outcome for the treatment of Aspergillus-related lung disease.There is a need to establish pharmacodynamic targets for other fungi commonly found in children with CF and to evaluate clinical outcomes using both these targets, and those already recommended for This study was supported by the National Institute of Health Research (NIHR) through a Health Education England/NIHR Pre-doctoral Clinical Academic Fellowship , a Senior Investigator Award  and the Imperial Biomedical Research Centre. J.F.S. and S.G. were supported at an institution level by the NIHR Great Ormond Street Biomedical Research Centre and J.F.S. received a UK Medical Research Council fellowship (MR/M008665/1).None to declare.JAC Online.The NONMEM model code is available as dkab312_Supplementary_DataClick here for additional data file.


T‐score of > −2.5 over 3 years at the total hip (TH) or lumbar spine (LS) in women with osteoporosis, ≥55 years of age, after the following treatment sequences: 1 year romosozumab followed by 2 years denosumab , 1 year romosozumab followed by 2 years alendronate, or alendronate‐only for 3 years . Probabilities of attaining the BMD target within 1 year of treatment were also determined. At both skeletal sites, in women with a baseline Tscore ≥ −2.7, there was >50% probability of achieving the BMD target with any 3‐year regimen. The probability of achieving the target BMD in those with a baseline TH Tscore equal to −3.0 was 61% with romosozumab/denosumab, 38% with romosozumab/alendronate, and 9% with alendronate. In those with a baseline LS Tscore equal to −3.0, the probability of achieving a T‐score > −2.5 was 93% with romosozumab/denosumab, 81% with romosozumab/alendronate, and 55% with alendronate. With 1 year of treatment, in patients with a baseline TH T‐score equal to −2.7, the probability of reaching the target Tscore with romosozumab was 71% to 78% and 38% with alendronate. For patients with an initial LS T‐score equal to −3.0, the probability of achieving the target T‐score over 1 year was 85% to 86% with romosozumab and 25% for alendronate. Our findings suggest baseline BMD and the probability of achieving BMD T‐score goals are factors to consider when selecting initial treatment for patients with osteoporosis. As baseline T‐score falls below −2.7 (TH) and −3.0 (LS), alendronate has <50% likelihood of achieving a BMD goal above osteoporosis range, whereas these probabilities remain relatively high for regimens beginning with romosozumab. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Increases in bone mineral density (BMD) with osteoporosis treatment are associated with reduced fracture risk. Increasing BMD is therefore a goal of osteoporosis therapy. Here, we compare the probability of achieving a  Traditionally, first‐line treatment has consisted of an antiresorptive , but recent studies have highlighted the need to begin with anabolic therapy to obtain optimal treatment outcomes in individual patients. Fracture risk has emerged as a key variable to help select the initial therapy. In patients at very high risk of fragility fracture, including those with a recent fracture, available data and emerging guidelines are increasingly supportive of initial treatment with an anabolic agent. Currently available anabolic treatment options include abaloparatide or teriparatide, administered for up to 2 years, and romosozumab, administered for 1 year. These treatments are usually followed by an antiresorptive treatment using bisphosphonates or denosumab.Current guidelines for the treatment of postmenopausal women with osteoporosis in the United Kingdom, United States, and Europe are not fully aligned.2, 3 Although low BMD is certainly not the only factor contributing to fracture risk, studies have confirmed the importance of the relationship between BMD level attained after or during therapeutic intervention and fracture risk reduction. Overall, the findings suggest that total hip BMD has the strongest association with subsequent fracture risk and may be a useful clinical target for a goaldirected treatment approach for patients with osteoporosis.A recent meta‐regression analysis including 38 randomized controlled trials (RCTs) linked the magnitude of treatment‐related changes in bone mineral density (BMD) with anti‐fracture efficacy, particularly for vertebral and hip fractures.14 AltT‐score above −2.5 has been suggested as a minimum treatment goal, but higher total hip BMD targets may be more appropriate for women with additional independent fracture risk factors such as advanced age, underlying disease, or frequent falls. As such, it is critical that treatment goals are tailored to the patient, with treatment decisions individualized accordingly. Goal‐directed therapy may be a useful concept to communicate the need for aggressive, early therapy for some patients with very low BMD or very high imminent fracture risk, and for critically evaluating initial treatment decisions. Finally, sharing a specific therapeutic target with the patient could be useful for optimizing adherence to therapy.A total hip nt falls.21 As  Because of its dual effect, romosozumab produces substantial gains in BMD over short periods of time, making it favorably indicated in patients at high imminent risk of fracture. The most recent guidelines from the American Association of Clinical Endocrinologists (AACE) now include romosozumab treatment within their recommended first‐line treatment options for postmenopausal women at very high risk of fracture .The bone‐forming agent romosozumab, approved for treating postmenopausal osteoporosis by FDA, EMA, PMDA, and other health authorities, is a humanized monoclonal antibody to the osteocyte protein sclerostin. By inhibiting sclerostin activity, romosozumab exerts a dual effect to stimulate modeling‐based bone formation and inhibit bone resorption.23 BecT‐score > −2.5 at the total hip or lumbar spine over 3 years with alendronate treatment alone versus treatment sequences of 12 months of romosozumab followed by 2 years of alendronate, or 12 months of romosozumab followed by 2 years of denosumab. An additional objective was to compare the probabilities of achieving non‐osteoporotic BMD T‐scores over 1 year of treatment with romosozumab compared with alendronate.Since BMD on therapy serves as an indicator of treatment success, one of the factors that should be considered to determine choice of treatment is the probability of achieving specific BMD treatment targets. The primary objective of these post hoc analyses was to compare the probabilities of achieving a  {FIG1}Briefly, women in the FRAME trial received either romosozumab or placebo for 1 year, each followed by open‐label denosumab for 2 years (1 year in FRAME and a second year in the study extension). In ARCH, women received either romosozumab or alendronate for 1 year, followed by 2 years of open‐label alendronate in both groups; further study details have been previously described.These post hoc analyses were based on the FRAME NCT01575834) and ARCH (NCT01631214) phase 3, multicenter, international, randomized, double‐blind trials in postmenopausal women with osteoporosis  at baseline and every 12 months thereafter using Hologic  or GE/Lunar  densitometers. Scans were processed and analyzed blinded to treatment at a central imaging vendor  as previously described22; asAll patients from the ARCH trial and women randomized to romosozumab from the FRAME trial, with total hip BMD values at baseline and at least one evaluable total hip BMD value at month 12 or 36, or with lumbar spine BMD values at baseline and at least one evaluable lumbar spine BMD value at month 12 or 36, were included in these post hoc analyses.T‐score > −2.5 with the three treatment sequences at month 36 was estimated based on a logistic regression model, adjusted for baseline total hip or lumbar spine T‐score within each treatment group, respectively. Similar analyses were performed for probability estimates over 1 year with either romosozumab treatment (from either ARCH or FRAME) or alendronate treatment (from ARCH). In addition, the probability of achieving a total hip or lumbar spine T‐score > −2 with the three regimens at 1 and 3 years was analyzed because patients with high risk of fracture or other risk factors may warrant a higher BMD target to achieve an acceptably reduced risk of fracture. To deal with missing data, only patients with all the required variables available were included in each analysis (pairwise deletion). The baseline threshold T‐scores equal to −2.7, −3.0, and −3.5 were not prespecified. At a certain point for both total hip and lumbar spine, the probability of achieving a Tscore > −2.5 was below 50% when treated with alendronate alone. As such, these example baseline T‐score thresholds were selected logically to demonstrate a range of probabilities of achieving a T‐score > −2.5.The probability of achieving a total hip or lumbar spine T‐scores for both total hip and lumbar spine among those enrolled in FRAME  compared with ARCH    and 38% with alendronate. For patients with initial total hip Tscore equal to −3.0 or below, the probability of achieving a total hip T‐score above −2.5 after 1 year of either treatment was low  and 64% for alendronate. For patients with an initial lumbar spine T‐score equal to −3.0, the probabilities of achieving a lumbar spine Tscore > −2.5 were 86% and 85% for those treated with romosozumab in FRAME and ARCH, respectively, but only 25% for alendronate‐treated patients. In patients who began with a lumbar spine T‐score equal to −3.5, the probabilities of achieving a lumbar spine T‐score > −2.5 at 1 year were 32% and 40% for romosozumab‐treated patients  and 2% for alendronate‐treated patients.In patients with a baseline lumbar spine Tscore equal to −2.7 treated initially with romosozumab, there was a 28% and 12% probability of achieving a T‐score ≥ −2.0  compared with 2% for alendronate‐only treatment. Patients with baseline total hip T‐scores below −2.7 had a low probability of achieving a total hip T‐score ≥ −2.0, regardless of treatment  and 15% with alendronate alone .For patients with initial lumbar spine T‐score as low as −2.7 in the total hip or −3.0 in the lumbar spine have more than a 50% chance of achieving a Tscore above −2.5 with all three treatment regimens over 3 years. However, as baseline T‐scores fall below −2.7 to −3.0 in the total hip and below −3.0 to −3.5 in the lumbar spine, the probability of achieving a non‐osteoporotic BMD T‐score within 3 years of alendronate alone falls to about 10% at both sites; in contrast, the probabilities of achieving T‐scores above osteoporotic range remain high if the initial treatment is romosozumab.In these analyses, we show that women with a baseline T‐scores of −2.7 or below in the total hip and −3.0 or below in the lumbar spine, the probability of attaining a nonosteoporotic BMD Tscore was low with alendronate alone. In contrast, with 1 year of romosozumab treatment, the probabilities of attaining non‐osteoporotic Tscores were more than 70% for the total hip and about 85% for the lumbar spine. Whenever possible, women who have a baseline T‐score below −2.7 at the total hip or below −3.0 at the lumbar spine should be considered for romosozumab therapy first in order to provide a high likelihood of attaining BMD levels above the osteoporotic range within 1 to 3 years.With just 1 year of treatment in patients with baseline T‐score > −2.5 because achieving this is a logical first step for patients who present with a BMD in the osteoporotic range. However, many fractures occur in women with T‐scores above −2.5, and for these and other individuals, attaining a T‐score of −2.0 or higher might be appropriate; we therefore also investigated the probability of achieving this BMD target. Although there was a lower probability of achieving this more stringent goal across all treatments, those treated initially with romosozumab had a much higher probability at both skeletal sites than alendronate alone. The goal of therapy should be amenable to personalization based on the presence of other independent risk factors for fracture. Having a goal can help close the recognized gap in long‐term adherence to treatment, supporting lifelong management of osteoporosis.We investigated the probability of achieving a target ove −2.5,3 and  Similarly, reduced fracture incidence was found in women originally randomized to romosozumab versus alendronate in the ARCH study during years 2 and 3, while all women were on alendronate. These findings suggest that the rebuilding of bone with romosozumab can lead to a persistent benefit after transition to an antiresorptive therapy. One year of treatment with romosozumab resulted in higher BMD compared with alendronate in the ARCH study, and higher BMD, particularly in the total hip, was associated with lower subsequent risk of both nonvertebral and vertebral fracture. In women who begin with a total hip T‐score of −2.7, 1 year of treatment with alendronate is unlikely to achieve the target BMD level, but the probability with 1 year of romosozumab exceeds 70%.In patients who have a very high risk of fracture in the next 1 or 2 years, it is important to consider selection of a pharmacologic treatment that improves BMD and thereby reduces fracture risk as quickly as possible. In the FRAME trial, fewer fractures were observed in the second year, while all women were on denosumab, among patients who initially received romosozumab.5 SimiTscore to begin with, particularly at the total hip (T‐score below −3.5), it may be difficult to achieve non‐osteoporotic BMD goals within 3 years, regardless of treatment. Still, a treatment strategy utilizing romosozumab initially followed by denosumab has the greatest probability of improving BMD to treatment targets within 3 years. Future research could illuminate whether multiple courses of romosozumab or other sequential, or even combination, treatments may provide further improvements.The results presented here also demonstrate that if a patient has a very low  A further limitation of this study is that the analyses limit the achievement of improved BMD T‐scores to > −2.5 or ≥ −2.0. Achieving these BMD targets may not be adequate for patients who may have high fracture risk primarily from non‐BMD factors. Finally, it should be noted that here we assessed which treatment sequence was associated with the greatest chance of achieving a specific T‐score. Of course, the choice of therapeutic agent for each individual patient should take into consideration factors other than just BMD.The large number of patients included in the FRAME and ARCH studies allowed a large sample size to be used in these post hoc analyses, increasing the robustness of the findings. Limitations included the different study designs of FRAME and ARCH. As the study design and eligibility criteria are different across the FRAME and ARCH studies, post hoc comparisons made across trials are indirect and descriptive. Additionally, in the present study, we did not analyze the relationship between the BMD achieved and the decrease in the risk of subsequent fracture, although this has been evaluated previously in ARCH and other studies.28 A fT‐score above osteoporosis range at the total hip and lumbar spine within 3 years, with the romosozumab/denosumab sequence yielding the greatest probability of achieving this goal in patients with the lowest T‐scores. Over 1 year of therapy, the probabilities of achieving BMD levels above the osteoporosis range at total hip and lumbar spine were much higher with romosozumab than alendronate. When selecting initial treatment for patients with osteoporosis, especially in those with a high risk of imminent or long‐term fragility fracture, the BMD at baseline and probability of achieving their BMD goal should be considered.In patients with osteoporosis, treatment with romosozumab followed by an antiresorptive agent provided the greatest probability of achieving a target CD, ZW, ZY: Employees of Amgen. CL: Employee and stockholder of UCB Pharma. EH: Consulting/speaker fees from Amgen, UCB Pharma, Lilly, Ipsen, Theramex, AgNovos, and Alexion. EL: Consulting/speaker fees from Amgen, Lilly, Theramex, and UCB Pharma. FC: Received institutional grants and research support from Amgen; has served as a consultant for Amgen and Radius Health; and has served on the speakers' bureaus for Amgen and Radius Health. FB: Consulting/speaker fees from Amgen, UCB Pharma, Lilly, Abiogen, Astellas, Janssen, and Chiesi Farmaceutici. J‐EBJ: Advisory boards: Amgen, Eli Lilly, UCB Pharma, and Gedeon Richter; lectures: Amgen, Eli Lilly, UCB Pharma, Gilead, and Otsuka. PP: Consulting/speaker fees from Amgen, UCB Pharma, Lilly, Kyowa and Kirin. SF: Received research and/or consulting grants from Amgen, UCB Pharma, Radius, Agnovos, Alexion, and Gedeon Richter. SRC: Consultant and honoraria for speaking from Amgen.www.Vivli.org and a signed data sharing agreement will need to be executed. All documents are available in English only, for a prespecified time, typically 12 months, on a password‐protected portal.Underlying data from this manuscript may be requested by qualified researchers 6 months after product approval in the US and/or Europe, or global development is discontinued, and 18 months after trial completion. Investigators may request access to anonymized IPD and redacted study documents, which may include raw data sets, analysis‐ready data sets, study protocol, blank case report form, annotated case report form, statistical analysis plan, data set specifications, and clinical study report. Before use of the data, proposals need to be approved by an independent review panel at https://publons.com/publon/10.1002/jbm4.10546.The peer review history for this article is available at Appendix S1. Supplementary InformationClick here for additional data file.


Autophagy‐related long non‐coding RNAs (lncRNAs) disorders are related to the occurrence and development of breast cancer. The purpose of this study is to explore whether autophagy‐related lncRNA can predict the prognosis of breast cancer patients. The autophagy‐related lncRNAs prognostic signature was constructed by Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression. We identified five autophagy‐related lncRNAs  associated with prognostic value, and they were used to construct an autophagy‐related lncRNA prognostic signature (ALPS) model. ALPS model offered an independent prognostic value , where this risk score of the model was significantly related to the TNM stage, ER, PR and HER2 status in breast cancer patients. Nomogram could be utilized to predict survival for patients with breast cancer. Principal component analysis and Sankey Diagram results indicated that the distribution of five lncRNAs from the ALPS model tends to be low‐risk. Gene set enrichment analysis showed that the high‐risk group was enriched in autophagy and cancer‐related pathways, and the low‐risk group was enriched in regulatory immune‐related pathways. These results indicated that the ALPS model composed of five autophagy‐related lncRNAs could predict the prognosis of breast cancer patients. Autophagy is a dynamic equilibrium process that degrades cellular material under cellular pressure.In this study, we hypothesized that a variable model composed of multiple autophagy‐related lncRNAs could be used to predict the prognosis of breast cancer patients. The lncRNA, mRNA expression dataset and clinical pathological features of breast cancer from The Cancer Genome Atlas (TCGA), were used to assess prognostic value of autophagy‐related lncRNAs. Finally, we employed an autophagy‐related lncRNA prognostic signature (ALPS) model to effectively predict the prognosis of breast cancer patients.22.1https://cancergenome.nih.gov/). Ensembl human genome browser, GRH38.p13 (http://asia.ensembl.org/index.html), was used to annotate and classify 14,142 lncRNAs and 19,658 protein‐coding genes. Male subjects or patients with less than 30 days overall survival (OS) were excluded, 1,027 breast cancer patients were used in the present study. The patients were randomly divided into a training and testing group. After excluding patients with incomplete clinical pathological data, this study enrolled 569 patients for subsequent analysis.RNA‐seq expression and clinical information of 1,108 breast cancer patients were obtained from The Cancer Genome Atlas (TCGA) data portal (2.2http://www.autophagy.lu/index.html). Moussay et al detailed descriptions of human autophagy‐related genes.P < 0.001.232 autophagy‐related genes come from the Human Autophagy Database . A Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression analysis of prognostic‐related autophagy‐associated lncRNAs using the ‘glmnet’ package was performed in R software. To evaluate its independent prognostic effect on survival, multivariate Cox regression analysis was used to analyse autophagy‐related lncRNAs candidates. Therefore, an ALPS model composed of five autophagy‐related lncRNAs was constructed. This ALPS model selects the best lncRNA prognostic markers based on the lowest Akaike information criterion (AIC) value. The risk score of each patient was calculated according to the following formula: Risk Score = ∑k=0ncoefk∗xk, where coef(k) and x(k) represent regression coefficient and the expressive value of each autophagy‐related lncRNA, respectively.The univariate Cox regression model was used to analyse the relationship between the expression level of autophagy‐related lncRNA and the OS in breast cancer patients  and 95% confidence intervals (CI), and potential covariates involved age, TNM stage, tumour size (T), lymph node metastasis (N), distant metastasis (M), risk score, ER status, PR status and HER2 status. The accuracy of each clinicopathological feature and risk score in predicting survival time was evaluated by the receiver operating characteristic (ROC) curve.2.5Nomogram was utilized to predict the probable 1‐year, 3‐year and 5‐year survival of breast cancer patients. A nomogram was constructed by integrating with clinical pathological variables such as age, stage, T stage, N stage, M stage, ER status, PR status, HER2 status and the risk score derived from the prognostic signature.2.6P‐value < 0.05 and false discovery rate  <0.25 were considered significantly enriched.PCA was used to investigate the distribution of patients with different risk score. GSEA version 4.0.3  was used to analyse the genes that were differentially expressed between the high‐ and low‐risk group patients. 1000 permutations were selected, and Affymetrix was used as the chip platform for the calculation of the normalized enrichment score (NES). Normal 2.7http://www.cytoscape.org/) and ggalluvial R package were used to visualize the co‐expression network and Sankey Diagram.The correlation between autophagy‐related lncRNA and its co‐expressed mRNA was analysed by co‐expression network and Sankey Diagram. Cytoscape software . P < 0.05 was regarded as statistically significant.The statistical analysis of all data was performed using R software . ROC curve analysis showed that the AUC value of the risk score based on ALPS model was greater than 0.7, which was greater than most other clinical prognostic indicators, such as stage and T stage, N stage, M stage, ER status, PR status and HER2 status , and risk score of T3‐4 is significantly higher thanT‐2’s (P = 0.01), etc Figure P < 0.05). Figure To further explore whether the ALPS model was associated with the characteristics of breast cancer, we evaluated the relationship between the risk score of ALPS model and clinical characteristics. The risk score of ALPS model was significantly correlated with Stage, ER status, PR status and HER2 status Table : risk sc3.4P < 0.001) and the risk score of ALPS model  were independently associated with OS  model could well predict prognosis of breast cancer patients. Notably, the predictive function of ALPS model was independent of other clinical pathological features.Currently, the most valuable prognostic factors of breast cancer were included Nottingham Prognostic Index (NPI), and pathological features of stage, ER, PR, HER2, while the treatment strategy of patients depends on their pathological characteristics.In our present study, we identified an ALPS model composed of five autophagy‐related lncRNAs from the TCGA dataset. Our results suggested that the ALPS model is a clinical valuable prognostic biomarker in breast cancer. Specifically, the risk score of ALPS model could divide the population of breast cancer into two prognostically distinct groups. To accomplish this, the assay integrates five lncRNAs and presented a single prognostic score as a continuous variable and proposes specific cut‐offs (risk score = 1.3528). Importantly, a nomogram that was integrated with multiple variables (including the risk score) can predict the survival of breast cancer patients. Finally, an intriguing finding was that the distribution of five lncRNAs from the ALPS model tend to be low‐risk, which was consistent with other studies. GSEA analysis also showed that based on the ALPS model, the high‐risk group was enriched in tumour‐related pathways, while the low‐risk group was positively correlated with immune function pathways.Of the five autophagy‐related lncRNAs in the ALPS model, only MAPT‐AS1, LINC0187 and AL122010.1 have been studied in breast cancer or other cancers. MAPT‐AS1 is reported as an independent prognostic marker of clear cell renal cell carcinoma (ccRCC), inhibiting the proliferation and invasion of ccRCC.The advantage of our study is that a small amount of lncRNA (only five lncRNA) can well predict the survival of breast cancer patients, so they can be better applied in clinical practice. Taken together, relevant research reports of these five lncRNAs further conform that our research direction is promising. This study also has several limitations. Firstly, that only TCGA dataset was used to internally validate the predictive accuracy of ALPS model but lacked cross‐validation externallyNeither METABRIC dataset nor GEO dataset obtained these five lncRNAs spontaneously. Secondly, the predict benefits of endocrine therapy and chemotherapy were not explored in ALPS model. Consequently, in the future research, it may be necessary to recruit cohorts of breast cancer patients (or drug‐resistant patients) to validate the predictive accuracy of the ALPS model, especially for predicting benefits of endocrine therapy and chemotherapy.In conclusion, development of ALPS model integrating five lncRNAs , derived from machine learning based screening, could well predict the survival of breast cancer patients. Future prospective clinical trials are needed to further consolidate the effectiveness of the ALPS model.The authors declare that they have no competing interests.Qianxue Wu: Conceptualization (lead); Data curation (lead); Formal analysis (lead); Methodology (lead); Writing‐original draft (lead). Qing Li: Data curation ; Formal analysis (supporting). Xiang Zhang: Supervision ; Writing‐review & editing . Wenming Zhu: Data curation ; Formal analysis (supporting). Hong‐Yuan Li: Supervision (lead); Writing‐review & editing (supporting).Fig S1Click here for additional data file.Fig S2Click here for additional data file.Table S1Click here for additional data file.


Epicardial adipose tissue (EAT) has been implicated in the pathophysiology of heart failure (HF) with left ventricular ejection fraction (LVEF) >40%, but whether this is due to a regional or global effect of EAT remains unclear.Regional EAT is associated with alterations in local cardiac structure and function.Patients with HF and LVEF >40% were studied. Cardiac Magnetic Resonance imaging was used to localize EAT surrounding the right ventricle (RV) and LV separately, using anterior‐ and posterior interventricular grooves as boundaries. Atrial‐ and ventricular EAT were differentiated using the mitral‐valve position. All EAT depots were related to the adjacent myocardial structure.p < .001). RV‐EAT showed a strong association with increased RV mass  and remained associated with RV mass after adjusting for total EAT, sex, N‐terminal prohormone of brain natriuretic peptide (NT‐proBNP), renal function and blood glucose. LV‐EAT showed a similar association with LV mass in univariable analysis, albeit less pronounced . Atrial EAT was increased in patients with atrial fibrillation compared to those without atrial fibrillation , whereas ventricular EAT was similar .102 consecutive HF patients were enrolled. The majority of EAT was present around the RV (42% of total EAT, Regional EAT is strongly associated with local cardiac structure and function in HF patients with LVEF >40%. These data support the hypothesis that regional EAT is involved in the pathophysiology of HF with LVEF >40%. HF with mid‐range EF [HFmrEF] and preserved EF [HFpEF]) is an emerging heart disease with poor prognosis for which currently no evidence based therapies exists.EAT is a layer of visceral fat that is in direct contact with the heart that has the potential to influence myocardial structure and function. For instance, experimental and clinical studies have also shown that local EAT can affect the electrophysiological properties of the atrial myocardium, resulting in slowed conduction and fractionated electrograms.The total EAT volume is markedly increased in HFmrEF and HFpEF patients compared to healthy controls, and is associated with disease severity.22.1We studied symptomatic HF patients with an LVEF>40% with an N‐terminal prohormone of brain natriuretic peptide (NT‐proBNP) of >125 pg/ml and echocardiographic evidence for LV diastolic dysfunction, left atrial dilatation and/or LV hypertrophy, according to current European Society of Cardiology criteria.The Institutional Review Board of the University Medical Center Groningen approved the retrospective study and the need for individual informed consent was waived. The present study was in concordance with the principles outlined in the Declaration of Helsinki. The present analysis was based on a database previously used by our group, and was extended with 38 more cases.2.22.2.1CMR was performed using a standard protocol for the acquisition of cardiac volumes and functional parameters, as previously published.2.2.2Cine loop images were analyzed off‐line by two observers (G.W. and T.M.G.) using dedicated software . Papillary muscles were included when measuring mass, and excluded when measuring volumes.EAT is the adipose tissue situated between the outer wall of the myocardium and the visceral layer of the pericardium.2.3t‐test, the Wilcoxon rank test, the Chi‐squared test or the Fisher's exact test where appropriate. Associations between EAT and cardiac structure and function were analyzed using univariable and multivariable linear regression models. In the multivariable regression model, the association between local EAT with myocardial structure was adjusted for the four clinical characteristics that were most strongly associated with total EAT; with forced entry of total EAT. Prior to regression analysis, covariates that were non‐normally distributed were log transformed. As a sensitivity analysis, we also assessed the relationship between EAT and local cardiac structural and functional abnormalities in the normal body mass index (BMI) range (BMI < 25 kg/m2). Intraobserver and interobserver variability for EAT and RV mass measurements were assessed using the intraclass correlation coefficient. Statistical analyses were performed using SPSS . Statistical significance was considered achieved at a p‐value <.05.Data are presented as numbers (percentage), mean ± SD or median with interquartile ranges. Differences between groups were analyzed using the independent samples 33.1p < .05. The EAT volume around the RV was significantly higher than the EAT volume around the LV and the atria  .We studied 102 consecutive HF patients with LVEF >40%. Mean age was 70 ± 10 years, 49% were female and the median NT‐proBNP was 1331 [inter quartile range 683–2531] pg/ml. Other patient characteristics are shown in Table 3.2The associations between total EAT and clinical characteristics are shown in Table 3.3β = 0.60, p < .001), and remained significantly associated with RV mass after adjusting for total EAT, sex, NTproBNP, eGFR, and HbA1c . The association between RV‐EAT and RV mass was comparable between patients with a BMI above and below 25 kg/m2 . RV‐EAT was not independently associated with other indices of RV‐structure and function. RV‐EAT was not associated with LV or atrial structure and function in multivariable analysis , but this relation was lost in multivariable analysis . Instead, a more robust association was detected between LV‐EAT and increased LV volumes  and LV end‐systolic volume β = 0.22, p = .002 in multivariable analysis. Upon echocardiographic analysis, EAT around the LV was inversely related to lateral e′ velocity  but not with septal e′ velocity  (data not shown). LV‐EAT was not independently associated with right‐ventricular or atrial structure and function , despite having comparable BMI . LV and RV EAT volumes were not significantly different between patients with atrial fibrillation and those without atrial fibrillation  volumes in heart failure patients with or without atrial fibrillation. (*p < .05). Volumes are indexed for body surface area.Click here for additional data file.Table S1 Associations between total EAT and clinical characteristics.Click here for additional data file.Table S2 Associations between EAT location and opposite myocardial structure on CMR.Click here for additional data file.


GBA1 gene encoding glucocerebrosidase cause Gaucher's disease, whereas heterozygous carriers are at risk for Parkinson's disease (PD). Glucosylsphingosine is a clinically meaningful biomarker of Gaucher's disease but could not be assayed previously in heterozygous GBA1 carriers.Biallelic mutations in the GBA1 N370S carriers with and without PD.The aim of this study was to assess plasma glucosylsphingosine levels in Glucosylsphingosine, glucosylceramide, and four other lipids were quantified in plasma from N370S heterozygotes with (n = 20) or without (n = 20) PD, healthy controls (n = 20), idiopathic PD (n = 20), and four N370S homozygotes  using quantitative ultra‐performance liquid chromatography tandem mass spectrometry.Plasma glucosylsphingosine was significantly higher in N370S heterozygotes compared with noncarriers, independent of disease status. As expected, Gaucher's/PD cases showed increases in both glucocerebrosidase substrates, glucosylsphingosine and glucosylceramide.GBA1‐PD. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.Plasma glucosylsphingosine accumulation in N370S heterozygotes shown in this study opens up its future assessment as a clinically meaningful biomarker of  GBA1 gene encodes the lysosomal enzyme, acid β‐glucocerebrosidase . Biallelic mutations in GBA1 cause the lysosomal lipid storage disorder, Gaucher's disease (GD).GBA1, N370S, is pathogenic for GD and represents one of the most common genetic risk factors for Parkinson's disease (PD) in the heterozygous carriers,The GBA1, including N370S, decrease GCase activity. GCase metabolizes glycosphingolipids, a class of lipids essential for membrane structure and a range of cellular functions. The prevailing hypothesis for PD associated with GBA1 mutations  is that a decrease in GCase activity alters the lipid composition of membranes and results in increased α‐synuclein aggregation and accumulation in the lysosomes.GBA‐PD. However, accumulation of GluCer or GluSph in GBA‐PD brain or cerebrospinal fluid (CSF) has not been unequivocally demonstrated,GBA‐PD.Pathogenic variants in GBA‐PD.GluCer is by far more abundant than GluSph and is shown to accumulate in macrophages and plasma of people with GD. However, it is not a clinically useful biomarker for GD.GBA1 N370S mutation carriers and noncarriers with and without PD. We aimed to test whether GluSph levels were elevated in GBA1 N370S mutation carriers and whether levels were associated with PD status. We hypothesized, based on the GD literature, that plasma GluSph would be a superior marker of reduced GCase activity in comparison with plasma GluCer levels.In this study, we measured GCase substrates, GluSph and GluCer, as well as the product, ceramide, and additional lipids  as negative controls in a cohort of GBA1 pathogenic variants, including 135 carriers with PD and 83 carriers without PD.GBA1 carriers with and without PD were recruited among Columbia University Irving Medical Center patients with PD and spouse controls, and GBA1 carriers without PD were recruited among family members of patients with GD  at the Lysosomal Storage Diseases Program at ISMMS. Demographics, as well as the Unified Parkinson's Disease Rating Scale (UPDRS) score in the on state and the Montreal Cognitive Assessment score, were collected from all participants. To reduce heterogeneity, we included only GBA1 N370S heterozygotes in this study (and not other GBA1 pathogenic variants). We further excluded LRRK2 G2019S mutation carriers. In addition, we included four samples of N370S homozygotes with PD, who also by definition have GD . Two of the four GD/PD participants were treated with enzyme replacement therapy at the time of recruitment. A total of 10 mL of whole blood per subject was collected in Ethylenediaminetetraacetic acid (EDTA) tubes, which were centrifuged, and from which 1 mL plasma aliquots per subject were extracted within 60 minutes of collection. Samples were stored in a −80°C freezer until processing. All clinical study procedures were approved by the Columbia University Institutional Review Board , and all participants signed informed consents. All samples were shipped to Nextcea  on dry ice in one batch.Participants in this study were participants of the Spot study, which was previously described.The bioanalytics were conducted by Nextcea, where scientists remained blinded to participants' PD status and genotype before analysis.A multiplexed quantitative ultraperformance liquid chromatography tandem mass spectrometry method was used to simultaneously quantitate plasma ceramides , GluCers , galactosylceramides , GluSph (GluSph 18:1), galactosylsphingosine , and glucosylcholesterol. The GluSph (GluSph 18:1) was well separated from galactosylsphingosine . Standard curves were prepared from related standards using a class‐based approach. Internal standards were used for each analyte reported. A SCIEX Triple Quad 7500 mass spectrometer was used in positive electrospray ionization mode for detection . Injections were made using a Shimadzu Nexera XR UPLC (ultraperformance liquid chromatography) system . The instruments were controlled by SCIEX OS 2.0 software.The parameters for assay validation included 3‐day intra‐assay and interassay accuracy and precision, sensitivity (lower limit of quantitation [LLOQ]), specificity, carryover, recovery, matrix effect, dilution integrity, and stability in human plasma. The LLOQ for all lipids measured was 5 pg/mL. Interassay and intra‐assay coefficients of variation for all dilutions (including LLOQ) were ≤20%. The specificity of the GluSph versus GluCer assay was evaluated at the LLOQ in six different lots of human plasma with stable isotope‐labeled GluCer and GluSph, where we observed no interference.x2) weighting. The concentrations of all lipids were quantitated in nanograms per milliliter (ng/mL) of plasma.The intensities of the analytes and internal standards were determined by integration of extracted ion peak areas using SCIEX OS 2.0 software. Calibration curves were prepared by plotting the peak area ratios for each analyte to internal standard versus concentration. The model for the calibration curves was linear with . Groups were a priori matched by sex and age (PD− versus PD+). We then compared the lipid species concentrations across the four groups. We computed total concentrations of ceramides, GluCer and GalCer, by summing all fatty acid analogs for each species. Although primary analyses compared the four groups, we included the four GD/PD subjects (positive controls) in Figure 1 for illustrative purposes. We further constructed regression models to predict GBA1 mutation status (outcome), including sex, age, PD status, and lipid concentrations as predictors. Analyses were computed in SPSS. Figure 1  was computed using GraphPad Prism version 9.1.1 for macOS, GraphPad Software .Demographics and disease characteristics were compared across the four groups based on PD status and genotype  are presented in Table GBA1 N370S heterozygotes and noncarriers with and without PD and in four patients with GD/PD. The plasma lipid concentrations across the four groups are presented in Table GBA1 status and plasma lipid concentrations, including age, sex, PD status, and the six lipid groups tested. GluSph was the only variable (and the only lipid) associated with GBA mutation status (P < 0.001).Demographic and disease characteristics of the four groups , but not the mild GBA mutation, N370SGBA‐PD. However, the exact mechanism by which GBA mutations cause PD remains to be determined. For example, it is possible that reduced activity of GCase leads to lower intralysosomal levels of ceramide, which negatively affect the maturation and activation of cathepsin‐D.It is important to note that GluSph, rather than GluCer, is emerging as a more disease‐relevant biomarker. A recent study reported higher levels of GluCer in the CSF in conjunction with a decrease in blood GCase activity in GBA mutation carriers and noncarriers will be needed to investigate whether GluSph levels are associated PD status. Furthermore, future studies should include a wide range of GBA1 pathogenic variants  to test potential correlation between variant severity and GluSph levels. Studies that will examine GluSph levels in the brain and CSF in addition to blood plasma may firmly test its relevance as a disease marker, as is the case for GD.We have not identified an association between plasma GluSph levels and PD status in the current samples of N370S carriers or with idiopathic PD. However, because GluSph levels were numerically higher in carriers with PD compared with carriers without PD (0.82 ± 0.24 versus 0.73 ± 0.20), and in noncarriers with PD compared with controls (0.50 ± 0.14 versus 0.48 ± 0.14), we posit that a larger cohort of In summary, these results indicate that plasma GluSph levels may be a useful target engagement/modulation biomarker for interventions aiming to increase GCase activity. Studies on larger cohorts would be required to test whether elevated GluSph levels are associated with PD risk or progression.Study concept and design: R.N.A., F.H., K.S.M., K.M.M., S.P.Subject recruitment, assessment, genotyping, and blood collection: M.S., M.B., Z.G.‐O.Biomarker assays: F.H., T.H., L.S.Statistical analysis of data: R.N.A.Initial drafting of manuscript: R.N.A., K.M.M., M.S., A.H.Editing and approval of submitted manuscript: all authors.Disclosures unrelated to the study: Manisha Balwani: research support , honoraria . Cheryl Waters: research support ; consulting fees ; speaker's honoraria . Ziv Gan‐Or: research support , consulting fees . Kalpana M. Merchant: research support (The Michael J. Fox Foundation), advisor/paid consultant . Roy N. Alcalay: research support , consultation fees .Table S1 Concentrations of each lipid analyzed in carriers and non‐carriers with and without PDa P value represents overall effect by ANOVA.Values are presented as mean (standard deviation). Data were analyzed by one‐way ANOVA. Participants were grouped by the presence or absence of either Parkinson's disease (PD− or PD+) or the GBA1 N370S mutation (GBA− or GBA+). Click here for additional data file.


Prenatal and postnatal conditions are crucial for the development of calves. Primiparous cows are still maturing during pregnancy, thus competing with the nutritional needs of their offspring. Therefore, mature cows might provide a superior intrauterine condition. Furthermore, weaning calves at an older age might affect them positively as well by reducing stress and offering time for various organs and their functions to develop. We aimed to evaluate effects of mothers’ parity and calves’ weaning age on gastrointestinal development and corresponding acid–base balance. Fifty-nine female German Holstein calves (about 8 days old) were investigated in a 2 × 2 factorial experiment with factors weaning age (7 vs. 17 weeks) and parity of mother (primiparous vs. multiparous). Calves were randomly assigned to one of these four groups. Animal behavior that was observed included resting, chewing and active behavior.Behavioral patterns were interactively affected by time and weaning age. Rumen sounds per 2 min increased in early-weaned calves during their weaning period. In late-weaned calves a consistently increase in rumen sounds was already recorded before their weaning period. Urinary N-containing compounds  were interactively affected by time and weaning age. Concentrations of all measured compounds except urea increased during early weaning. All except hippuric acid concentration decreased in early-weaned calves after weaning. In late-weaned calves allantoin and uric acid increased before weaning and did not change during weaning.These results suggest that late-weaned calves developed adequate rumen functions and acid–base balance, whereas early-weaned calves might have suffered from ruminal acidosis and catabolism. Weaning calves at 7 weeks of age might be too early for an adequate rumen development. The prenatal and early postnatal periods are critical times of development. Any intervention may cause life-long changes in metabolism, which is termed metabolic imprinting, and therefore have a great impact on health and performance in later life . The parAll data according to feed intake was previously published in Schwarzkopf et al. . In summp < 0.001). Late-weaned calves increased their time spent with chewing at d 105 of the trial compared to d 1 (p = 0.002). Time spent with chewing exceeded 20% after d 21 (earlyMC and earlyPC), 28 (lateMC) or 49 (latePC). Time spent with active behavior increased over time , whereas late-weaned calves did not decrease the percentage of time spent resting until d 112 (p = 0.005).All observed behavior patterns changed over time and were interactively influenced by time and weaning age Table . Percent p = 0.00. Time sp p = 0.00. Time spp < 0.001), whereas in late-weaned calves it increased already before weaning  and therefore weaning caused no further increase . Rumen fill increased from d 1 to d 14 in both weaning groups . It was also influenced by parity  and then from d 42 to d 140 (p = 0.011). Interaction between time and weaning age was significant for pH of feces (Table p < 0.001). The highest pH values were observed at d 28 of the trial with no significant differences between the groups . Afterwards, it decreased over time. The steepest pH decrease was observed during weaning in both weaning groups . Hence, it was different between weaning groups on experimental d 98 (p = 0.043). However, late-weaned calves had a significant decrease in feces pH before weaning .The pH value in saliva increased over time and was not affected by weaning age or mother’s parity Table . It incrp < 0.001).Whole blood pH increased over time Fig.  C and wap = 0.010) and then increased within the experimental period from d 42 to d 70 (p < 0.001). Afterwards, it remained stable for the rest of the experimental time. For late-weaned calves urinary pH stayed low until d 98  and then increased during weaning  without dropping down before.The pH value in urine was interactively affected by time and weaning age Table . During p < 0.001; data not shown). It increased in early-weaned calves between d 28 and 42 (p < 0.001). Therefore, it was higher in early-weaned calves than in late-weaned calves on experimental d 42 (p = 0.005). In late-weaned calves, it increased until d 70 (p < 0.001) and then remained stable on this level. Furthermore, specific density did not change through weaning in late-weaned calves (p = 0.1).Specific density of urine was interactively influenced by time and weaning age (p < 0.001). Urinary allantoin . Urinary allantoin concentration was lower at the end of the experimental period (d 140) compared to the d after weaning . Urinary hippuric acid concentration increased in early-weaned calves until d 70 (d 28 vs. d 70 p < 0.001) and did not change significantly thereafter. It also increased in late-weaned calves form d 28 to d 70 (p = 0.020), while the steepest increase occurred during weaning . The urinary allantoin concentration increased before weaning (d 28 vs. d 70 p < 0.001). Urea concentration did not change significantly during weaning in both weaning groups and was not different between the groups on any experimental d.Nitrogen-containing compounds in urine  were interactively affected by time and weaning age Table . Concentp < 0.001; Fig. p = 0.005; Fig. p < 0.001). Therefore, they were higher in these than in late-weaned calves on d 70 . NABE values increased in late-weaned calves before weaning .All variables of urine were standardized to the corresponding specific density (data not shown), but no differences to unstandardized data regarding statistical data evaluation were observed. The NABE . To provoke an increase in concentrate intake at this early age, MR consumption has to be reduced. As a result, concentrate intake increased to 1 kg/d. In calves at 15 wk of age, the concentrate intake was twice as high compared to the early-weaned calves. Thus, at the beginning of weaning, the concentrate intake was limited to 1 kg/d in the late-weaned group. After weaning, all calves received hay and a TMR. The effects of two different rearing regimens and mother’s parity are discussed in the following.Rumen maturation at early age was assessed by behavioral observations, rumen sounds and scoring of rumen fill. Closely related to rumen maturation, acid base homeostasis and N metabolism have to adapt to ruminant status. Thus, the pH in blood, urine, feces and saliva was measured to assess changes in systemic acid–base-metabolism going along with rumen maturation. Furthermore, the concentration of several N-containing compounds  in urine was determined to estimate the onset of microbial activity in the gastrointestinal tract, especially in the rumen, and to assess the development of early N metabolism in growing calves.p = 0.018). This indicated that rearing conditions have a greater impact on rumen development, maturation of acid–base homeostasis and N metabolism than mother’s parity. Rumen fill score was greater in PCs, which might be caused by their smaller body seize paired with the same feed intake as MCs.Mother’s parity had no effect on any parameter except rumen fill score  and then again from d 42 to d 140 (p = 0.01) in all groups. Therefore, there might be an ontogenetic window for development of ruminant salivary buffer composition during these time points. The salivary gland responsible for secreting large amounts of buffer in ruminants is the Glandula parotis. The main chemical components of Parotis saliva are Ca2+, Na+, K+, Urea, HCO3−, HPO42−, Cl− and water. In general, the concentration of these components did not vary much between healthy adult cows and were unaffected by diet. Only urea concentration was affected by diet in adult cows [3− and HPO42− [Parotis, to produce adequate volumes of saliva to buffer VFA production in the rumen was not assessed in this study.Maturation of the gastrointestinal tract might be associated with maturation of systemic acid–base homeostasis. To assess its development, the pH values in saliva, blood, urine, and feces were measured during early life in all calves. As pH in saliva increased over time, being unaffected by weaning age or mothers’ parity, it seemed to be determined solely by the evolutionary blueprint. The first significant increase in pH compared to experimental d 1 was observed at d 42 , which indicated that both groups reached a concordant status of rumen development. Therefore, rumen and its functions appeared to be equally developed at the age of 5 months, which corresponds to experimental d 140. Late-weaned calves showed no sign of impaired rumen development at that age despite of the higher weaning age. Ad libitum intake of MR in the first 5 wk of life did not retard rumen development . High MR40, whichWeaning calves at 7 wk of age appears to be too early for an adequate rumen development according to the evolutionary blueprint. This became apparent in an inefficient rumen function and a not fully developed metabolic adaption in early-weaned calves before experimental d 70, although they were fully weaned with 42 d, corresponded to an age of 7 wk. This indicated that calves needed more time to mature. They reached functional ruminant status at about 11 wk of age, which might be a more appropriate age for weaning. None of the response variables of behavior, rumen development, and urinary excretion of N-containing compounds were significantly different at the end of the trial. This indicated an equal functional status of the rumen despite the prolonged MR feeding in late-weaned calves. However, long-lasting effects of a metabolic imprinting for later life in terms of health and performance are possible for these future dairy cows and need to be studied.In accordance with the German Animal Welfare Act and approved by the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), Oldenburg, Germany, the present trial was carried out at the experimental station of the Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Brunswick, Germany .n = 59) were studied from the age of (mean ± standard deviation) 8 ± 2 days (d) until d 149 ± 2 of life. All calves originated from one established herd of German Holstein cows and were born within a seasonal calving period of 3 months (October - December). After the feeding trial, the animals remained in the institute´s own herd. They received 3 L of colostrum through a nipple bucket within 2 h after birth. Consistent quality of colostrum was evaluated using a colostrum densimeter  and had to be defined as good at a value greater than 1035 g/L. In the pre-experimental feeding period, milk replacer  was mixed to the pooled herd milk, starting at the age of 3 d, with gradually increasing amounts from 0.30 kg MR powder/d (d 3 after birth) to 0.90 kg MR/d (d 5 after birth), when the maximum of 6 L liquid feed with a concentration of 150 g/L MR powder was available. Calves entered the study at an average live weight of 44.5 ± 5.2 kg at experimental d 1 and were moved into straw-bedded stables with provision of MR and concentrate through self-feeding systems . They were randomly allocated to either early weaning at 7 wk of age  and primiparous cows ) or late weaning at 17 wk of age  and primiparous cows ) group assuring an equal allocation of calves from primiparous cows  and calves from multiparous cows . Early weaning was conducted at the age of 7 weeks, as this is a common management decision take on dairy farms, while late weaning was executed at the age of 17 weeks, because the reticulorumen volume approximately reaches adult proportions [Detailed information about the study design were described elsewhere . In shorCalf behavior was recorded using instantaneous scan sampling . Scoring and measuring of rumen sounds were done on experimental d 1, 7, 14, 28, 42, 56, 70, 84, 98, 112, 126 and 140.Rumen motility was assessed by counting the number of rumen sounds within 2 min with a stethoscope in the rdhuizen  . Urine was stored at -20 °C before analysis of net acid base excretion (NABE), urea, allantoin, creatinine, hippuric acid and uric acid were done. NABE and the base–acid ratio (BAR) were determined by the titrimetric method as described by Kutas [All samples were collected between 8 AM and 1 PM. Feces and urine were collected after spontaneous release or manual stimulation and filled in clean and sealable tubes. The pH value in feces and urine was measured 1 to 4 h after collection with a pH meter . For collection of saliva, two sterile cotton wool swabs were offered each animal on a clamp for chewing. Afterwards they were put into a commercially available salivette  and cooled on ice. Salivettes were centrifuged at 2000 × by Kutas . Followiby Kutas . Urinaryby Kutas . The equby Kutas . Ruminalby Kutas .The pH value in feces and saliva was measured in two samples per animal and point in time, and the mean value was used for further analyses. NABE and BAR of the urine were calculated as:NABE = base excess – (acid excess + ammonium ions) BAR = sum of bases/sum of acids.p < 0.05 was the level of significance. Visualization was done using GraphPad Prism 6.0 , whereby measurements on serial time points were interpolated linearly.All variables, except for variables in ruminal fluid, were evaluated as repeated measures using PROC MIXED procedure in SAS . The model included fixed factors of time, weaning age, parity of the mother and their interactions. Results were presented as least squares means (LSMeans) with standard errors (SE). All pair-wise differences of LSMeans were tested with the Tukey–Kramer procedure. Variables in ruminal fluid were evaluated by two-way ANOVA with weaning age, mother’s parity and their interaction as independent variables using SAS . For all statistical tests,