Propensity score matching showed that increased weight gain was associated with a longer hospital length of stay (LOS) , an increased number of patients who received pRBCs (L 311 (36%) vs. M 429 (50%), p < 0.0001), and a higher incidence of postoperative acute kidney injury (AKI) (L 84 (9.8%) vs. M 165 (19.2%), p < 0.0001). Weight gain can easily represent fluid overload. Fluid overload after cardiac surgery is common and is associated with prolonged hospital LOS and increases the incidence of AKI.Optimal fluid therapy during perioperative care as part of enhanced recovery after cardiac surgery (ERACS) should improve the outcome. Our objective was finding out the effects of fluid overload on outcome and mortality within a well-established ERACS program. All consecutive patients undergoing cardiac surgery between January 2020 and December 2021 were enrolled. According to ROC curve analysis, a cut-off of ≥7 kg  and <7 kg  was defined. A moderate correlation was shown between weight gain and fluid balance r = 0.4, and a simple linear regression was significant  Fluid overload after cardiac surgery (CABG) is common and increases postoperative complications . To dateGoal directed fluid therapy (GDT) has been recommended during ERACS  within tFluid overload could be minimized using a restrictive fluid therapy protocol. However, studies comparing liberal versus restrictive fluid therapy in cardiac  and non-Postoperative fluid overload can be quantified either by calculating the fluid balance or by measuring postoperative weight gain. The fluid balance calculation seems to be more precise but time consuming and less reliable due to charting errors and gaps . ConversThe aim of this retrospective study was to assess the impact of fluid overload on outcome and mortality in a well-established ERACS program.The primary endpoints are the incidence of weight gain after cardiac surgery and the correlation between postoperative weight gain and fluid balance. The secondary endpoints are the effects of fluid overload on hospital length of stay (LOS), fast track failure (FTF), acute and chronic kidney injury (AKI), postoperative pulmonary complications, packed red blood cells (pRBCs) transfusion, and mortality.This retrospective observational study was performed at a university affiliated Heart Center and began after approval by the local ethics committee (registration number 552/20-ek).We selected all consecutive patients who followed the Leipzig ERACS protocol from January 2020 to December 2021 undergoing elective or emergency cardiac surgery. Exclusion criteria were: patients ineligible for ERACS, younger than 18 years, non-cardiac surgery patients , and incomplete data.Patients without any complications were transferred to the postanesthetic care unit (PACU) directly from the operating room (OR). Once in the PACU, they were stabilized and extubated, hereafter transferred to the intermediate care unit (IMC) on the same day (POD 0). Patients were then transferred to the ward at the earliest on POD 1. The definition of a fast-track failure (FTF) is the unplanned transfer from the ward to the ICU or IMC at any time during the same hospital stay .Patients were allowed to drink clear fluids, e.g., water, clear juices, and tea up to 2 h before surgery.2) were collected every 30 min. A transesophageal echocardiography (TOE) probe was inserted in all patients, if not contraindicated . Swan-Ganz catheters were spared to patients with a low ejection fraction (EF) of ≤35% or impaired right ventricle (RV) function or tricuspid valve (TV) surgery. More details regarding anesthesia and postoperative analgesia were published previously [After anesthesia induction, a three-lumen central venous catheter and an 8.5 F introducer sheath were placed in the internal jugular vein under ultrasound guidance, and an arterial catheter was inserted for blood pressure monitoring. Body temperature was measured through a nasopharyngeal temperature probe and core temperature through a urinary catheter. Arterial and central venous blood gas analysis including hemoglobin (Hb), hematocrit (Hct), serum lactate, and central venous oxygen saturation  was used in all patients.The blood transfusion threshold was a hematocrit less than 20% during CBP and less than 25% after weaning from CBP and during off-pump surgery. Cell saver CATSmartThe following data were routinely collected intraoperatively: the amount of crystalloid/colloid infusion, packed red blood cells (pRBCs), fresh frozen plasma (FFP), platelets, cell saver, and fluid volume of the heart-lung machine during surgery . The outputs included urine output, remaining volume of the heart-lung machine, and ultrafiltration.2), TOE, transthoracic echocardiography (TTE), lung ultrasound (LUS), and hematocrit level (Hct). Swan-Ganz parameters were added for patients with reduced EF. The passive leg raising (PLR) maneuver, Trendelenburg position, or fluid challenge test using 200 mL of electrolyte crystalloids solution, e.g., Jonosteril® , are used if the TTE, TOE, and LUS results are not conclusive , no severe bleeding, core temperature ≥ 36 °C, and clinical judgement and communication between anesthesiologist and surgeon ,13.The PACU, managed by the department of anesthesia, includes 8 beds and is staffed with a physician to patient ratio of 1:4 and a nurse-to-patient ratio of 1:3. The PACU opening hours were from 10:00 a.m. to 10:30 p.m. only during weekdays.Intraoperative fluid therapy monitoring continued in the PACU. Arterial and venous blood gas analysis and UOP were documented hourly. Combined transthoracic echocardiography (TTE) and lung ultrasound (LUS) imaging were performed for all patients at least twice in the PACU (before extubation and before transfer to the IMC) and if otherwise indicated .2 > 90 mmHg and PaCO2 < 46 mmHg, SpO2 > 96% on O2 flow 2–6 L/min), urinary output ≥ 0.5 mL/kg/h, no significant bleeding (<50 mL/h), normal serum lactate, normal SvO2, cardiac enzymes, and chest X-ray [Patients were extubated after fulfilling the predefined extubation criteria , and thest X-ray .In the IMC, patients are fully monitored including invasive BP. Blood gas analysis was assessed every 3 h and POCUS (point of care ultrasound) if indicated.Postoperative fluid intake  and output , feces) were collected until discharge from the IMC to the ward, where the first postoperative weight was measured, which mostly represented the maximum recorded postoperative weight.® system  for all off-pump CABG for continuous stroke volume variation and cardiac index monitoring. Since 2018, intraoperative TOE, with certain indications for a Swan-Ganz catheter (see above), replaced Vigileo®.We started with the fast-track protocol in November 2005 with 3 beds in the PACU completely separated from the ICU. The early experimental results were published in 2008 . Many asThe patients’ body weight was measured preoperatively as the baseline value and after transfer from the IMC to the ward.Weight gain is defined as the difference between the preoperative weight and maximum postoperative weight.Complex surgery is defined as any surgery including more than one procedure, e.g., CABG + Valve/s or 2 or more valves.Postoperative respiratory complications included any cause of pneumonia, ARDS, and severe airway edema. The categorization of acute kidney injury follows the KDIGO stadium classification . AccordiCalculated fluid balance is defined as the mathematical subtraction of every single fluid output from every single fluid input. Measured fluid balance is the sum of all fluid balances documented in the nursing documentation sheet.®  and the machine-readable patient’s chart Medlinq® . Anesthesia and PACU observation sheets were corrected manually by the anesthesiologist directly after scanning. Data of cardiopulmonary bypass were documented by a special machine operator in the Connect Manager . SPSS  and StatsDirect  were used for statistics.Data were retrieved from the clinical information system iMedOne2 test or Fisher’s exact test where appropriate. Means of maximum weight gain for each postoperative day (POD) were compared with a one-way ANOVA with a Tukey–Kramer post-hoc test to show the differences between each pair. A p-value < 0.05 was considered statistically significant. A Pearson correlation coefficient (r) and simple linear regression was used for the relationship between weight gain and fluid balance.Due to a number of studies with a difference in the time of collecting postoperative weight ,22,23,24p = 0.05.According to our sample size analysis, 1538 patients had to be included if the hospital length of stay would have been shorter by one day (15 days vs. 14 days) ± 7 with a power of 80% and Propensity score matching was performed to minimize the bias that might result from differences in demographic, preoperative, and intraoperative data, especially for the complexity of the surgeries. The variables used for the logistic regression model were: age, gender, day of maximum recorded weight, EuroScore II, surgery complexity, x-clamp time, CPB time, and length of surgery. We used one to one matching, paring each subject to the closest propensity score subject from the other group. Based on the pre-matching range of baseline variable differences, the maximum caliper width for pair-matching was defined as 0.2 of the pooled logit score standard deviation.A total of 6437 patients were operated between January 2020 to December 2021. In total, 3754 were admitted to the PACU. We included 2213 patients with complete data and who completed the ideal fast-track pathway from the PACU to the IMC and thereafter to the ward .According to ROC curve analysis see , a cut-oDemographic data, baseline characteristics, and type of surgery before and after PSM are summarized in p < 0.001, R2 = 0.16). A total of 1675 patients was included in the fluid balance analysis (538 patients were excluded because of incomplete data). Inconsistency between the measured and calculated fluid balance was found in 26% of the patients.The mean increase in weight was +7.6 ± 4.1 kg, while the mean intra- and postoperative fluid balance was +8.6 ± 3.7 L and the mean of the difference between fluid balance and weight gain was 1.0 ± 4.3 kg. Group L had a weight gain of 4.1 ± 1.8 kg, whereas group M had an average weight gain of 10.6 ± 3.1 kg. Only a moderate correlation was shown between weight gain and fluid balance (r = 0.4). A simple linear regression for weight change and fluid balance was significant (p < 0.05 when compared with each day from POD1 to POD4).The mean of maximum weight gain is comparable for patients weighed from POD1 to POD4 (7.6–7.8 kg)  with a dp < 0.001) in the day of maximum recorded weight. After PSM, no significant difference (p = 0.3) was found between groups (n groups .p < 0.0001). Postoperative acute kidney injury at stages I and II, but not stage III, were significantly higher in group M. AKI was almost twice as high in group M compared to group L.Postoperative outcome and mortality are summarized in In our study, 54% of our patients experienced postoperative weight gain ≥7 kg, which was associated with a longer hospital length of stay, increased number of received pRBCs, and higher incidence of postoperative acute kidney injury in enhanced recovery after cardiac surgery. We could find only a moderate correlation between the postoperative change of weight and perioperative fluid balance.Engelman et al.  publisheUnfortunately, there are few studies describing the effects of these elements on perioperative fluid management. GDT in cardiac surgery was described by Osawa et al.  and showp < 0.0001) that received less fluid boluses ; however, no impact was found on hospital LOS and postoperative complications. The hospital mortality rate was higher in the intervention group. Again, Parke et al. [Liberal versus restrictive fluid therapy after surgery was also not well studied in cardiac surgery. Parke et al.  conductee et al.  limited e et al.  and Messe et al.  studied AKI stage I and II, but not III, were higher in our patients with more volume overload. Although it may be a causal bias , we do not think so because volume overload would be more obvious in AKI III, which is insignificantly different between both groups. Furthermore, preoperative serum creatinine was comparable between both groups. This is supported by Chen et al.  and ShenNo difference in the postoperative respiratory complications is also consistent with several studies ,7,27. ThThe difference in type of surgery between two groups is striking. There is significantly more aortic valve replacement (AVR) in group M. Patients with AV disease (mostly aortic stenosis) usually presented with LV hypertrophy and a degree of diastolic dysfunction. These patients, with good EF, tend to receive more fluids postoperatively as the sole treatment for any hemodynamic instability. POCUS examination of these patients mostly reveals LV hypertrophy and kissing papillary muscles that mandate fluid therapy. The presence of more on-pump CABG in group L is unexpected because the priming fluid, inflammation, and fluid shift effects of cardiopulmonary bypass may lead to fluid overload. Nevertheless, on-pump CABG is the most common surgical procedure in cardiac surgery, and the physicians may feel more confident in managing these patients, aiming for earlier hospital discharge compared to other types of procedures. Minimally invasive procedures including the thoracotomy approach and partial sternotomy were comparable in both groups. As in most minimally invasive cardiac surgery studies ,31, thesp = 0.19, vs. r = 0.4, p < 0.001 in our study). This could be explained by the complex calculation of fluid balance, which is more reliable for human error and is demonstrated in our study by the difference between the calculated and measured fluid balance of 26%. Furthermore, the insensible fluid loss is either ignored or imprecisely calculated. Therefore, we believe that weight gain is more representative of fluid balance after surgery. Köster et al. [The discrepancy between the calculated and measured fluid balance and the correlation between fluid balance and body weight gain in our study is in concordance with the literature; Eastwood et al.  concluder et al.  showed tA higher number of patients receiving transfusion can be noticed in our group M in contrast to group L. As per departmental SOPs, we have a restrictive threshold for transfusion. However, 36% of the group L still received at least one pRBC, which is still in the range of pRBCs transfused in major randomized studies comparing restrictive vs. liberal transfusion after cardiac surgery (52.3% and 25%) ,33.Physician compliance and adherence to fluid management protocols could not be assessed because of the nature of retrospective studies. Implementation of fluid management protocols  are reinforced with regular audits, availability of diagnostic tools (ultrasound machines), and documentation in the local manual for cardiac anesthesiologists. Nevertheless, “lack of compliance” with failure to adhere to the standardized protocol could not be excluded as reason for fluid overload. In the anesthesia setting, implementation of quality indicators was confronted with poor employee compliance .The main limitation of our study is the retrospective design through which we cannot fully exclude the risk of unknown biases. This study did not take into account intraoperative insensible fluid loss and intraoperative bleeding not processed through the cell saver system. We could not precisely assess adherence to the fluid management protocol. It is difficult to compare hospital LOS in this study with studies from other countries. In Germany, the hospital LOS depends not only on the patients’ general condition, but also on the German payment system. Minimum hospital LOS between 4–12 days, according to the cardiac procedure, is required for full payment from health insurance companies. In 2018, the average length of stay for cardiac surgery patients in Germany was 11 days ,36.Volume overload after cardiac surgery is also quite common within a well-established ERACS program, and it is associated with a prolonged hospital length of stay, higher incidence of postoperative AKI, and blood transfusion. Weight gain is likely to estimate the fluid overload in a better way compared to complex fluid balance calculations. Individual ERACS elements must be prospectively studied to examine its effect on postoperative fluid overload.


To explore the effect of liraglutide treatment on serum adropin and its relationship to the liver fat content in newly diagnosed patients with type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated fatty liver disease (MAFLD).Serum adropin level and liver fat content were assessed in patients with T2DM and MAFLD (n = 22), along with healthy controls (n = 22). Afterward, the patients received liraglutide treatment for 12 weeks. Serum adropin levels were examined by a competitive enzyme-linked immunosorbent assay. Liver fat content was quantified via magnetic resonance imaging-estimated proton density fat fraction (MRI-PDFF).P < 0.05] and higher liver fat content , compared to healthy controls.We found that patients with newly diagnosed T2DM and MAFLD had lower serum adropin levels [2.79 ± 0.47 vs. 3.27 ± 0.79 ng/mL, P < 0.001), and liver fat content decreased from 18.04 to 7.74 % (P < 0.001) in patients with T2DM and MAFLD. Furthermore, increases in serum adropin were strongly associated with decreases in liver fat content , liver enzyme and glucolipid metabolism parameters.Following 12-week liraglutide treatment, serum adropin levels increased from 2.83 to 3.65 ng/mL (The increase in serum adropin level following liraglutide treatment was strongly correlated with the reduction in liver fat content and glucolipid metabolism. Hence, adropin might be a potential marker for the beneficial effects of liraglutide on treating T2DM and MAFLD. Metabolic dysfunction-associated fatty liver disease (MAFLD), formerly known as non-alcoholic fatty liver disease (NAFLD), has become a leading cause of chronic liver disease in the world –3. MAFLDAdropin, encoded by the energy homeostasis-associated (Enho) gene, is highly expressed in the liver and brain . AccumulThis study was designed to explore the effect of liraglutide treatment on serum adropin and its relationship to the liver fat content in newly diagnosed patients with T2DM and MAFLD. Given the invasiveness, high cost and complications of liver biopsy, we conducted magnetic resonance imaging-estimated proton density fat fraction (MRI-PDFF), an emerging surrogate to diagnose and monitor the treatment response of NAFLD, to qualify liver fat content of all participants.2 ; fasting blood glucose (FBG); fasting insulin (FINS); HbA1c; liver function test ; and free fatty acid (FFA). Homoeostasis model assessment of insulin resistance (HOMA-IR) = FPG (mmol/L) × FINS (mU/L)/22.5; Homoeostasis model assessment of β-cell function (HOMA-β) = 20 × FINS (mU/L)/FPG (mmol/L)—3.5  and higher liver fat content  than the control group , whereas correlated positively with HDL-C , as described in Table P < 0.01), negatively associated with serum adropin level.In T2DM group, serum adropin levels correlated inversely with BMI, LDL-C, TG, HbA1c, TBA, FFA and liver fat content .Table P < 0.001, Fig. P < 0.001, Figs. In addition, serum adropin levels elevated from 2.83 to 3.65 (P = 0.013), HbA1c , FBG , AST , FFA . In model 2, adjusted for sex and age, the results of BMI, HbA1c, FBG, and AST remained similar to model 1 except for FFA  and ALT .In T2DM group, longitudinal changes by treatment were assessed by linear mixed effects models. Two-class linear mixed models were fitted, and the data were summarized in Table P < 0.001).Moreover, we observed that increases in serum adropin were strongly associated with decreases in liver fat content during 12-week liraglutide treatment even after adjusting for sex and age  exacerbated in the adropin deficient mice fed with either western diet or methionine-choline deficient diet . In factse model , 33. Adrse model . In our Our study has some limitations. First, liver fat content was quantified via MRI-PDFF rather than the gold standard liver biopsy. Given the exploratory nature of our study, performing liver biopsies even in a research setting of T2DM and MAFLD comorbidity would hardly accepted by patients who are newly diagnosed. Second, owing to the small sample size and non-randomization design, bias may have been presented; hence, larger randomized trials are warranted to validate our findings. Furthermore, further animal and cell-based experiments are required to explore the underlying molecular mechanism of liraglutide and other GLP-1 receptor agonists  on adropin and liver fat content. Nonetheless, our findings reflected the real-world clinical settings and exhibited adequate reliability with sophisticated statistical methods.In conclusion, our study indicated that liraglutide significantly elevated serum adropin levels in patients with T2DM and MAFLD. The increase in serum adropin level following liraglutide treatment was strongly correlated with the improvement of liver fat content and glucolipid metabolism. Hence, adropin might be a potential marker involved in the beneficial effects of liraglutide on treating T2DM and MAFLD.


Development of automated segmentation models enabling standardized volumetric quantification of fibroglandular tissue (FGT) from native volumes and background parenchymal enhancement (BPE) from subtraction volumes of dynamic contrast-enhanced breast MRI. Subsequent assessment of the developed models in the context of FGT and BPE Breast Imaging Reporting and Data System (BI-RADS)-compliant classification.For the training and validation of attention U-Net models, data coming from a single 3.0-T scanner was used. For testing, additional data from 1.5-T scanner and data acquired in a different institution with a 3.0-T scanner was utilized. The developed models were used to quantify the amount of FGT and BPE in 80 DCE-MRI examinations, and a correlation between these volumetric measures and the classes assigned by radiologists was performed.p < 0.0001), whereas BPE amounted to 0.37 (p = 0.0006).To assess the model performance using application-relevant metrics, the correlation between the volumes of breast, FGT, and BPE calculated from ground truth masks and predicted masks was checked. Pearson correlation coefficients ranging from 0.963 ± 0.004 to 0.999 ± 0.001 were achieved. The Spearman correlation coefficient for the quantitative and qualitative assessment, i.e., classification by radiologist, of FGT amounted to 0.70 (Generalizable algorithms for FGT and BPE segmentation were developed and tested. Our results suggest that when assessing FGT, it is sufficient to use volumetric measures alone. However, for the evaluation of BPE, additional models considering voxels’ intensity distribution and morphology are required.A standardized assessment of FGT density can rely on volumetric measures, whereas in the case of BPE, the volumetric measures constitute, along with voxels’ intensity distribution and morphology, an important factor.• Our work contributes to the standardization of FGT and BPE assessment.• Attention U-Net can reliably segment intricately shaped FGT and BPE structures.• The developed models were robust to domain shift.The online version contains supplementary material available at 10.1186/s13244-023-01531-5. The BPE, after visual assessment, should be classified into (a) minimal, (b) mild, (c) moderate, and (d) marked categories [Breast cancer is the most common cancer in the female population worldwide . Screenitegories . As showtegories . This istegories . Currenttegories . To achiThe main challenge is that breast MRI acquisition is not standardized: different institutions and doctors use different equipment, magnetic field strength, protocols (including the timing and the amount of the post-contrast acquisitions), different contrast agents, and patient positioning, to acquire and process the data. As a result, MRI data exhibit different resolution, contrast, noise level, and artifacts leading to high variability in the quality and reliability of the diagnosis.Over the past few years, the field of CNN-based segmentation models for medical imaging data has witnessed remarkable advancements. Among these, the U-Net architecture  and its It has been shown that the CNN-based segmentation architectures can reliably segment the FGT tissue from native MRI sequences, with the DSC values ranging from 0.81 ± 0.11 to 0.87 ± 0.08 –25. RegaThe primary objective of the first part of this study was to develop two automated and generalizable segmentation models: one segmenting the FGT from native volumes, the other segmenting the BPE from subtraction volumes. In the second part of the study, the resulting models were applied to 80 DCE-MRI examinations of 75 patients from our institution, with the aim to assess the correlation between the volumes of FGT and BPE and classes assigned visually by radiologists.This retrospective study has been approved by the local ethics committee. The main parameters of the datasets utilized in this study are provided in Table To curate datasets 1–3, we conducted a search in the Picture Archiving and Communication System (PACS) of our institution. The search aimed to identify DCE-MRI examinations fulfilling following inclusion criteria: (a) age above 18 years, (b) absence of implants, (c) availability of assessments for FGT and BPE classes in the corresponding radiological report, and (d) BI-RADS Assessment Category indicating a likelihood of malignancy of 1 or 2, corresponding to a 0% likelihood [Dataset 1 was curated by searching through examinations acquired between September 2013 and October 2015 using a 3.0-T scanner. This dataset was used for model training, validation, and testing. For volumetric analysis, the search through examinations acquired between September 2020 and October 2022 continued until a total of 80 eligible examinations resulting in possibly well-equalized distributions of FGT and BPE classes were identified. In this way, dataset 2, containing examinations acquired with 1.5-T scanner, and dataset 3, containing examinations acquired with 3.0-T scanner, were obtained. Examinations from dataset 2 were additionally used for testing models’ performance.almost entirely fat FGT and minimal BPE. Another advantage of using two separate models is that potential errors in FGT segmentation do not affect BPE segmentation. For instance, artifacts or extreme superior and inferior regions with higher intensity on native volume may be mistakenly included in the FGT segmentation.The design of AI model development differed from previous works by utilizing two separate models: one for FGT segmentation from the native volume, the other one for BPE segmentation from the subtraction volumes. This approach allows for an investigation of all subtraction images, without the need for precise registration. This is important, as even a small misalignment between sequences , we chose to train the models slice by slice Fig. . Our appThe ground truth masks were created in 3D Slicer  by S.N..Dataset 1 was split into a patient-stratified train-validation-test sets, and the FGT model was trained using 2112 slices from 20 patients for training, 416 slices from 4 patients for validation, and 520 slices from 5 patients for testing. The model was additionally evaluated on dataset 2 and dataset 4, which comprised 1004 slices from 6 patients. Importantly, the subtraction volumes exhibit lower contrast and signal-to-noise ratio compared to native volumes. To account for this, a larger amount of data was utilized for the BPE model. The total training set for the BPE model comprised 11,829 slices from 54 patients, with a validation set of 2469 slices from 12 patients, and a testing set of 3095 slices from 16 patients. Subsequently, the BPE segmentation model was tested on dataset 2 and dataset 4, which collectively contained 2672 slices from 6 patients.α parameter of the loss to 0.99 and the β parameter to 0.01. Additionally, during training, brightness augmentation in the 0.2–1.8 range delivered best performance on the test set. All final hyperparameters together with average inference runtimes are reported in Table SAll the data were rescaled to 0–1 range prior to training. A subset of the dataset was used for hyperparameter tuning using fivefold cross validation. The best hyperparameters obtained in this way were then fine-tuned during training using the entire dataset. Five rounds of training of native and subtraction models were then performed using best fine-tuned hyperparameters. Noteworthy, the best performance was achieved with focal Tversky loss  functionbreastvol, FGT(%) (1)/BPE(%) (2), derived from ground truth and predicted masks.The model obtained in each training run was evaluated on the test data coming from three datasets: dataset 1, 2, and 4 (cf. Table r). Secondly, the volumetric DSC was computed for the breast and the FGT/BPE masks. Additionally, a weighted DSC was calculated, with the weights proportional to FGT(%)/BPE(%). This adjustment was made to account for the higher penalization of small shifts in case of lower FGT(%)/BPE(%). The models were additionally evaluated with Bland–Altman plots. Lastly, the overlays of the ground truth and the predicted masks were assessed visually.Their correlation was plotted, followed by linear fit and calculation of Pearson correlation coefficient (FGT(%) according to Eq. (BPE/FGT (%) according to Eq. , taking into account errors in the calculation of FGT(%) and BPE/FGT(%). These were calculated by propagation of uncertainty from mean absolute errors of the native and subtraction model using the test set.Next, the correlation between those quantitative measures and qualitative assessment by radiologists was assessed by using Spearman correlation coefficient . These models were characterized by DSC of 0.950 ± 0.002 for the breast segmentation and by DSC and weighted DSC of 0.820 ± 0.005 and 0.864 ± 0.004 for the FGT segmentation, respectively. The r for the subtraction models amounted to 0.992 ± 0.001 for the breast and 0.963 ± 0.004 for the BPE(%). These models featured DSC of 0.927 ± 0.001 for the breast segmentation and DSC and weighted DSC of 0.628 ± 0.018 and 0.715 ± 0.015 for the BPE segmentation, respectively.The performance metrics of the models trained with the best fine-tuned hyperparameters is reported in Table r and DSC values as well as the visual assessment. Analysis of the volumetric correlations between ground truth and predicted masks for the test set for the chosen models is displayed in Fig. FGT% with the limits of agreements (LoA) of − 7.3% and 6.5%. The bias in the estimation of BPE% amounted to − 0.98% with LoA of − 7.2% and 5.2%. Figure For further analysis, one native model and one subtraction model was chosen. The choice was made based on FGT(%) and BPE/FGT(%), calculated from the predicted masks with the qualitative classification into four classes by the radiologists. In the case of FGT(%) distribution, the mean values increase with the increasing FGT class, which is not the case for the mean BPE/FGT(%) values and the increasing BPE class. The FGT(%) distribution of almost entirely fat and scattered FGT classes fully overlap. This is also the case for BPE/FGT(%) in case of minimal, mild, and moderate BPE classes. The coefficient ρ for FGT(%) amounted to 0.70 (p < 0.0001), whereas for BPE/FGT(%) amounted to 0.37 (p = 0.0006).Figure The attention U-Net models developed in this study accurately segmented FGT and BPE structures from breast DCE-MRI data. Table ρ = 0.70 (p < 0.0001), which is statistically strong. The box plots summarizing the FGT(%) distribution for each class  ranges for each class could be defined, thus allowing reproducible and standardized breast density assessment. This could be further explored in the investigation of breast cancer risk factors enabling triaging of the patients.The comparison of quantitative measures and qualitative assessment of breast tissue density revealed a correlation characterized by ρ = 0.37, p = 0.0006, cf. Figure It is crucial to note that the statistically low correlation between quantitative and qualitative assessment of contrast uptake , which might be particularly significant in case of entirely fat FGT and minimal BPE. Table S1. Overview of the Data, Hardware, Best Hyperparameters and Inference Time for the models reported in the main text.


IPD remains a major cause of morbidity and mortality globally despite the implementation of PCVs in childhood immunization programs, due to the emergence of non-vaccine serotypes (NVTs) and persisting vaccine types. Newly approved vaccines such as PCV15 and PCV20 may address some of these issues. This study aimed to describe the epidemiology of IPD in Canada from 2000-2019 after PCV7 and PCV13 approval in 2001 and 2010, respectively, and evaluate the potential future impact of PCV15 and PCV20.Data were collected from IPD cases in children and adults captured by the Pneumococcal Reference Laboratory in Alberta and the National Microbiology Lab during 2000-2019; this includes the majority of isolates from IPD across Canada, excluding Quebec. We calculated annual age-, and serotype-specific incidence rates and serotype distribution using population estimates from Statistics Canada.A total of 37,921 isolates from IPD cases were included in the analyses. After PCV7 introduction, IPD due to PCV7 serotypes decreased by 64%  from 2001 to 2010, while PCV13/non-PCV7 serotypes increased between 2004 and 2010 from 1.0 to 3.8 per 100,000 per year. After PCV13 introduction, IPD due to PCV13/non-PCV7 serotypes decreased by 53%  between 2010 and 2019. The overall rate initially decreased from 8.7 to 7.6 per 100,000 between 2010-2014 but increased to 9.6 per 100,000 in 2019. From 2010 to 2019, IPD incidence increased by 63%  for PCV15/non-PCV13 serotypes and by 33%  for PCV20/non-PCV15 serotypes. The proportion of serotypes in 2019 covered by PCV15 and PCV20 was 32% and 45% in infants aged < 2 years and 43% and 56% in adults aged ≥65 years, respectively. There was an increased IPD burden observed for some PCV13 serotypes: serotype 3 increased from 0.7 to 1.1 per 100,000 per year between 2010 and 2019 and serotype 4 increased from 0.2 to 0.8 per 100,000 per year between 2014 and 2019.IPD rates in Canada continue to increase despite the widespread use of PCV13. While PCV15 and PCV20 hold promise in addressing prevalent recently circulating serotypes, there remains a need to develop vaccines with broader coverage considering the constant emergence of NVTs.Nirma Khatri Vadlamudi, PhD, MPH, Pfizer Canada: Grant/Research Support Greg Tyrrell, PhD, Merck Canada: Advisor/Consultant|Merck Canada: Grant/Research Support Manish Sadarangani, BM BCh, FRCPC, DPhil, GlaxoSmithKline: Grant/Research Support|Merck: Grant/Research Support|Moderna: Grant/Research Support|Pfizer: Grant/Research Support|Sanofi Pasteur: Grant/Research Support|Seqirus: Grant/Research Support|Symvivo: Grant/Research Support|VBI Vaccines: Grant/Research Support


Parainfluenza virus (PIV) is an important cause of acute respiratory illness in children; however, scarce data have been reported on the clinical significance of PIV co-detection with other viruses.Between 12/01/2016 and 03/31/2020, we conducted active surveillance for children who presented to the emergency department or were hospitalized with fever or respiratory symptoms at seven U.S. medical centers within NVSN. Demographic and clinical data were collected through parent/guardian interviews and chart abstraction. Nasal and/or throat swabs were tested for PIV types 1–4; respiratory syncytial virus (RSV); rhinovirus or enterovirus (RV/EV); adenovirus (AdV); common cold coronaviruses (ccCoV) 229E, HKU1, NL63, and OC43; SARS-CoV-2; influenza (Flu) A, B, and C; and human metapneumovirus (hMPV). We used a generalized linear mixed-effects model with a logit link to compare the odds of hospitalization between children < 2 years old with PIV-only detection and those with PIV co-detection. Our analysis included age, underlying conditions, and preterm birth as fixed effects and study site as a random effect.Table 1.Comparison of the demographic and clinical characteristics of children with PIV-only detection and those with respiratory virus co-detection, NVSN, 2016–2020.Figure 1.Relative frequencies of respiratory viruses co-detected with PIV in U.S. children <2 years old, stratified by highest* level of care received, NVSN, 2016–2020. Abbreviations: PIV, parainfluenza virus; RV/EV, rhinovirus or enterovirus; RSV, respiratory syncytial virus; AdV, adenovirus; ccCoV, common cold coronaviruses; hMPV, human metapneumovirus; Flu, influenza.Table 1. Notably, children with PIV co-detection were more likely to attend daycare, preschool, or school. Compared with PIV-only detection, the odds of hospitalization were higher for PIV/RSV  and PIV/Flu , lower for PIV/AdV, and comparable for the remaining pairs including PIV/RV/EV, the most common co-detection.Of 17,850 children tested for PIV, 1,641 (9.2%) were positive: 1,116 (68.0%) with PIV-only detection and 525 (32.0%) with PIV co-detected with at least one other virus. The demographic and clinical characteristics of children with single PIV detection and those with co-detection are compared in Table 2.Multivariable logistic regression model of hospitalization in U.S. children with PIV-only detection and those with PIV co-detection with one other respiratory virus, NVSN, 2016–2020.The association between PIV co-detection and odds of hospitalization was virus-specific for children < 2 years old. Further investigation is warranted to determine if these findings are driven by RSV and influenza's impact on illness severity or if they are indicative of a potential virus–virus interaction that exacerbates PIV disease.Janet A. Englund, MD, Ark Biopharma: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant John V. Williams, MD, Merck: Grant/Research Support|Quidel: Board Member Marian G. Michaels, MD, MPH, Merck: Grant/Research Support|Viracor: Grant/Research Support Elizabeth P. Schlaudecker, MD, MPH, Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant Mary A. Staat, MD, MPH, CDC: Grant/Research Support|Cepheid: Grant/Research Support|Merck: Grant/Research Support|NIH: Grant/Research Support|Pfizer: Grant/Research Support|Up-To-Date: Honoraria Pedro A. Piedra, MD, Ark Bioscience: Advisor/Consultant|Ark Bioscience: Grant/Research Support|GSK: Grant/Research Support|Icosavax: Advisor/Consultant|Icosavax: Grant/Research Support|Mapp Biologics: Grant/Research Support|Meissa Vaccines: Grant/Research Support|Moderna: Advisor/Consultant|Novavax: Advisor/Consultant|Novavax: Grant/Research Support|Sanofi-Pasteur: Grant/Research Support|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Takeda: Advisor/Consultant Rangaraj Selvarangan, BVSc, PhD, D(ABMM), FIDSA, FAAM, Abbott: Honoraria|Altona Diagnostics: Grant/Research Support|Baebies Inc: Advisor/Consultant|BioMerieux: Advisor/Consultant|BioMerieux: Grant/Research Support|Bio-Rad: Grant/Research Support|Cepheid: Grant/Research Support|GSK: Advisor/Consultant|Hologic: Grant/Research Support|Lab Simply: Advisor/Consultant|Luminex: Grant/Research Support Natasha B. Halasa, MD, MPH, Merck: Grant/Research Support|Quidell: Grant/Research Support|Quidell: donation of kits|Sanofi: Grant/Research Support|Sanofi: vaccine support Geoffrey A. Weinberg, MD, Merck & Co: Honoraria


P-T-X) properties of the Na2CO3-H2O system up to 700°C and 11.0 kbar using a hydrothermal diamond anvil cell and a Raman spectrometer. Our results show that Na2CO3 becomes increasingly soluble under high P-T conditions, leading to the disappearance of melt-fluid immiscibility and the continuous transition from Na2CO3 melts to hydrothermal brines under deep crustal conditions. Given the abundance of Na2CO3 in highly evolved carbonatitic systems, we suggest that the continuous melt-fluid transition in deep-seated carbonatites results in REEs being sufficiently concentrated in the brine-melts to form economic ore bodies, whereas in shallow systems, REEs preferentially partition into carbonatitic magmas over synmagmatic brines and disperse in carbonatite rocks that underwent limited fractionation.Carbonatites are the most important primary sources for the rare earth elements (REEs). While fractional crystallization of carbonate minerals results in the enrichment of volatiles, alkalis, and REEs in the remaining melts, the transition from carbonatitic magmas to hydrothermal brines remains unclear. Here, we investigated the pressure-temperature-composition ( Distinct melt-fluid transitions in carbonatites: Continuous evolution in the deep, fluid exsolution in the shallow. Carbonatites are igneous rocks containing carbonate minerals that crystallized from melts with carbonate predominating over all other anions combined  at 0°C to 30.85 mol/kg (81.0 wt %) at 450°C under vapor pressure and can be extrapolated to the K2CO3 melting point at 891°C  at 100°C to almost 0 around the critical point of H2O  under higher P-T conditions  for melt-fluid transition in deep-seated carbonatites  under vapor pressure , the phase changes observed in all loads include dehydration from thermonatrite (Na2CO3·H2O) into anhydrous natrite (Na2CO3) around 110°C  at 100°C to around 1.0 mol/kg (9.6 wt %) at 300°C  with fluid pressure below 1.0 kbar at 400°C, hydrous melting of natrite occurred between 500° and 530°C under 1.6 kbar  were considerably inaccurate, due to the very weak CO32− Raman signal in the aqueous fluids or the weak and wide O-H band in the Na2CO3 melts . Nevertheless, by comparing the Na2CO3 concentration in the homogeneous melt in load #2 (13.52 mol/kg or 58.9 wt %) and the aqueous fluids that coexisted with a melt in loads #1 to 3 , the fluid-melt distribution coefficient of Na  or calcite  to the Na2CO3-H2O system did not preclude the occurrence of melt-fluid immiscibility under shallow crustal conditions. However, while Na2CO3 was loaded with K2CO3 solution (1.0 mol/kg) in load #4, the CO32− concentration between 200° and 400°C was higher than that in the Na2CO3-H2O system  by 0.42 to 1.08 mol/kg; meanwhile, the natrite melting temperature also decreased by about 50°C. The addition of calcite in load #5 did not cause detectable variations in fluid composition at T ≤ 350°C, whereas under higher P-T conditions, the formation of nyerereite [Na2Ca(CO3)2] from reaction between Na2CO3 and calcite (fig. S1) resulted in the CO32− concentration decreasing by 0.26 to 0.79 mol/kg between 400° and 500°C. Regardless of the uncertainties in Raman quantification results, the fluid compositions in loads #1 to 5 were consistent at T > 500°C .The addition of K > 500°C , indicat2CO3-H2O loads (#6 to 12) with fluid pressure greater than 1.0 kbar at 400°C, the natrite crystals were observed to dissolve progressively into the liquids at temperatures exceeding liquid-vapor homogenization (P-T conditions ranging from 381° to 555°C and between 2.8 and 6.6 kbar (32− Raman peak intensity increased accordingly (2CO3 solubility from retrograde to intensely prograde with increasing pressure. The CO32− concentration in the homogeneous liquids ranged between 8.75 and 20.19 mol/kg (48.1 to 68.2 wt %), comparable to the brine-melt obtained in load #2 (13.52 mol/kg or 58.9 wt %), and are much greater than the supercritical liquid at the upper critical end point  into the high-pressure Na2CO3-H2O system did not cause obvious variation in CO32− concentration (T > 300°C (fig. S2) suppressed the increase in CO32− concentration with increasing P-T condition. In contrast to the CO32− concentration in load #6, which increased from 2.86 mol/kg (23.3 wt %) at 300°C and 0.7 kbar to 6.59 mol/kg (41.1 wt %) at 400°C and 2.5 kbar, that in load #14 was lower by 0.53 mol/kg at 350°C and 2.0 kbar, and by 0.98 mol/kg at 384°C and 2.6 kbar (table S1). With the further increase in P-T condition, nyerereite dissolved under 550°C and 5.6 kbar, which resulted in a slight increase in CO32− concentration by another 0.33 mol/kg  and those from previous studies  or with a vapor-like supercritical fluid , which implies the release of H2O as steam from carbonatitic subvolcanoes. The 1.5-kbar section . Meanwhile, the melt-fluid immiscibility curve deviates from the solubility curve and lies beneath it, leading to occurrence of the Na2CO3 brine-melt either as a single phase , or equilibrated with a natrite solid  or an aqueous fluid , or absence of the Na2CO3 brine-melt but an aqueous fluid with limited Na2CO3 concentration  instead. Under higher pressures  or as a homogeneous liquid, which can be called a brine-melt  or an aqueous fluid , with no boundary between these two phases.To better illustrate the phase changes in the Na section  is uniqure range . The BM 2CO3-H2O system under deep and shallow crustal conditions reveal that hydrothermal brines can be generated from continuous fractionation of carbonatitic magmas or through fluid exsolution, depending on whether the P-T condition is greater or not than the supercritical curve of the Na2CO3-H2O system. Estimated emplacement depths of carbonatites span a wide range from extrusive volcanoes to deep-seated intrusions  and very high salinities [up to 80 wt % NaCl-equivalent (NaCleq)] in the melt-fluid inclusions  located well in the melt-fluid immiscibility zone  brine-melts enriched in alkali carbonates, sulfates, and REEs to hydrothermal brines with moderate temperatures (300° to 400°C) and salinities (10 to 30 wt % NaCleq) after the precipitation of REE minerals  were prepared by loading analytical grade anhydrous Na2CO3 pellets (Na2CO3 ≥ 99.9 wt %) with deionized water into HDAC under room P-T conditions, together with air bubbles to control the P-T paths of the fluids  or with H2O and Iceland spar fragments (#5 and 14), to investigate the effects from K2CO3 or CaCO3 on the phase transitions and the fluid or melt compositions under different P-T conditions. During the experiment, each sample was heated at ~5°C/min, with Raman spectrum of the solution being measured after a 5-min suspension at each temperature point with intervals of 50°C. Because of the minerals used for pressure calibration in HDAC experiments   and Na2CO3-K2CO3-H2O (#4 and 13) loads was estimated from the liquid-vapor homogenization temperature using isochoric P-T curves defined for the NaCl-H2O system  with equivalent mass concentrations (2CO3 + CaCO3 + H2O loads (#5 and 14), error between the fluid pressures as calibrated using the microthermometric method and from the frequency shift in the ν1, 1086 Raman band of calcite  laser (100 mW) and collected using a Renishaw inVia Raman spectrometer installed at the Institute of Mineral Resources, Chinese Academy of Geological Sciences. The spectrometer is equipped with a Leica ×10 long-working distance objective , a 2400 gr/mm grating and a 1024 × 256 pixel charge-coupled device detector, providing a spectral pixel resolution of 1.2 cm−1. Each spectrum was acquired with five accumulations of 10 s each in the 100 to 4200 cm−1 spectral range, with the focus position of the objective being fixed ~20 μm below the interface between fluid and upper diamond anvil (32− (ν1) and HCO3− (ν5) were fitted using Gaussian + Lorentzian profiles, after baseline corrections using quadratic curves to remove the interference from the ν1 band of diamond; the O-H stretching bands of water were fitted using three Gaussian subbands, after baseline corrections using a linear function (fig. S4) (Unpolarized Raman spectra of Na32− concentration in high P-T aqueous fluids was carried out using the integrated peak intensity of the O-H band of water (sum of three subbands) as an internal standard (32−-to-H2O Raman peak intensity ratio (Rintensity) with temperature and CO32− concentration was calibrated in advance, by using the same instrument and 1.03 to 18.80 mol/kg (12.45 to 72.18 wt %) K2CO3 solutions in the 23° to 700°C temperature range . The CO32−-to-HCO3− Raman scattering factor ratio has been shown to vary less than 10% from 1.46 at P-T conditions up to 600°C and 1 GPa , error in Raman quantification results for CO32− concentration in a high P-T alkali carbonate  solution was generally below ±0.3 mol/kg.Raman quantification for CO


Tumour hypoxia is a negative predictive and prognostic biomarker in colorectal cancer typically assessed by invasive sampling methods, which suffer from many shortcomings. This retrospective proof-of-principle study explores the potential of MRI-derived imaging markers in predicting tumour hypoxia non-invasively in patients with colorectal liver metastases (CLM).A single-centre cohort of 146 CLMs from 112 patients were segmented on preoperative T2-weighted (T2W) images and diffusion-weighted imaging (DWI). HIF-1 alpha immunohistochemical staining index (high/low) was used as a reference standard. Radiomic features were extracted, and machine learning approaches were implemented to predict the degree of histopathological tumour hypoxia.p = 0.002) and ADC  were significantly predictive of tumour hypoxia. Morphological T2W TE75  and functional DWI b0  and b800  images also provided predictive information. T2W TE300  and b = 10  images were not predictive of tumour hypoxia.Radiomic signatures from DWI b200 .The online version contains supplementary material available at 10.1186/s13244-023-01474-x. Tumour hypoxia is a valuable prognostic/predictive biomarker in colorectal cancer.This proof-of-principle study demonstrates non-invasive associations between MR-radiomics and tumour hypoxia.Radiomics from DWI b200, ADC, and T2W TE75 predicted tumour hypoxia.The online version contains supplementary material available at 10.1186/s13244-023-01474-x. Tumour hypoxia is a valuable prognostic and predictive biomarker in colorectal cancer (CRC) –4. GenomPolarographic electrodes inserted directly into the tumour are considered the gold standard for measuring tumour hypoxia. However, in the routine clinical workflow, histopathological analysis is more commonly performed, with tissue hypoxia markers, such as hypoxia-inducible factor-1 (HIF-1) alpha, being the most relevant . Both apAs medical image analysis research has gained recognition in the clinical world, increasingly relevant applications of radiomics coupled with machine learning have emerged. Radiomic features and signatures have been associated with long-term prognosis and response to local and systemic therapy –28. PromPast literature has attempted to perform histological/radiological correlation using radiomics. More specifically, CT-derived radiomic features have been associated with PET imaging of hypoxia . Other sIn this proof-of-principle project, we aimed to use MRI-derived radiomic features and novel machine learning approaches to non-invasively predict the degree of histopathological tumour hypoxia in patients with colorectal liver metastases. We hypothesise that by leveraging medical image analysis techniques and ubiquitous imaging modalities, non-invasive insight into CRC hypoxia can be obtained.n = 143), missing or old MRIs , non-cancerous liver lesions (n = 15) and mucinous subtype (n = 14). Patients that had received prior systemic therapy were excluded due to the expected impact the different treatments would have on the microenvironment, introducing noise and variability. Cases where accurate matching between imaging and pathology was not possible were immediately excluded (n = 9). Reasons for failed matching included the presence of multiple lesions within the same liver segment or a discrepancy in liver segments reported between the pathology and MRI report. Figure We retrospectively collected clinical patient data from all colorectal cancer patients with known liver metastases who had undergone liver resection at our institution  between April 2015 and January 2020. Our initial cohort comprised 370 patients, from which 248 subjects were excluded for prior systemic therapy (The Picture Archive and Communication System (PACS) was retrospectively queried to retrieve the preoperative MRI scans for the patients in our cohort. For each liver resection, we selected the MRI scan closest to the date before the procedure. Apart from the echo time, two T2-weighted sequences were used  with a field-of-view of 400 × 400 × 233 (AP × RL × FH) mm, a reconstructed in-plane voxel of 1 × 1 (AP × RL) mm, slice thickness of 5 mm FH, echo times of 75 and 300 ms respectively and a repetition time of 2725 ms, without fat suppression. Five routinely available sequences were collected from the diffusion-weighted scan  with a field-of-view of 450 × 400 × 251 (AP × RL × FH) mm, reconstructed in-plane voxel size of 1.75 × 1.75 (AP × RL) mm, echo time of 86 ms, repetition time of 1447 ms, and a SPAIR fat suppression pulse was used—leading to a maximum of seven sequences available per liver resection. The selected echo times and b-values were based on the standard MRI protocols at our centre for the evaluation of colorectal liver metastases, established through a combination of clinical practice guidelines and the need to balance image quality and acquisition time, in accordance with the preferences of our institution's radiology department. All four b-value sequences plus ADC were collected to explore all possible associations between diffusion-weighted imaging and hypoxia. The scans were acquired on a 3 T Philips Achieva and a 3 T Philips Ingenia , with an anterior and built-in posterior coil as receiver coils. Gadolinium-containing contrast agents used in our liver examination were administered after the included T2W and DWI sequences were acquired. 3DSlicer (v.4.10.2) was used to view the radiological images and perform the manual segmentation . As a single radiologist performed all the segmentations, we were able to mitigate the risk of inter-reader variability. Each sequence was segmented independently. No assumptions were made regarding the possibility of generalising segmentations between sequences. Additional file Similar to how the two largest lesions (when present) were resected and subsequently selected for staining, an expert radiologist applied the same criteria to CLM delineation on MRI images. Liver metastases were matched between MRI and pathology based on lesion location. Segmentations of the two largest liver metastases were performed to account for inter-tumoural heterogeneity. Pairwise analysis was performed to assess the degree of correlation between the segmentations of different sequences . The extracted features included first-order statistics, two-dimensional and three-dimensional shape features, grey level dependence matrix (GLDM) features, grey level run length matrix (GLRLM) features, grey level co-occurrence matrix (GLCM) features, grey level size zone matrix (GLSZM) features, and neighboring grey tone difference (NGTDM) matrix features, each with a set of filters applied to them . The dataset was normalised by centering the data at the mean with standard deviation to account for potential scanner level differences. Each radiomic feature vector represented the morphological phenotype of the segmented region of interest. A complete description of the radiomic features generated by PyRadiomics can be found in van Griethuysen et al. .The resulting feature space suffered from the curse of dimensionality, where the number of features was greater than the samples. This situation typically results in increased noise in the data and makes the machine learning model more prone to overfitting the training data and less likely to generalise to unseen information. We performed ensemble feature selection to identify the most representative/relevant features within the sizable radiomic feature space. In this method, seven supervised feature selection approaches  would independently select the top 100 most relevant features for the specific outcome (e.g. hypoxia staining index high vs. low). Subsequently, only the features chosen by the majority of methods (> = 4/7 approaches) would be included in the analysis. Ensemble feature selection approaches help ensure that only the features with the maximum relevance would be used in the study by leveraging the strengths of specific selection methods to overcome weaknesses in others , 38. TheOur institutional biobank was queried to obtain archival resection specimens for the patients included in the final cohort. As a critical regulator of cellular response to hypoxia, HIF-1 alpha is a reliable and permanent histopathological marker of tumour hypoxia in colorectal cancer, which can be detected accurately after tissue excision and storage . Tumour The analysis was performed on data from 116 resections consisting of a reference standard on pathology  paired with a pre-resection MRI scan (with the available sequences). This data was divided into a training set (75%) to train and optimise the machine learning algorithm and an independent test set (25%) to evaluate the model’s ability to generalise to unseen data. To discover the best machine learning pipeline  to model the data, a tree-based evolutionary algorithm was used  . We suppp values above the conventional threshold (0.05) were considered to be statistically significant.To assess predictive performance, we measured the area under the receiver operating curve (AUC). Additionally, we computed precision-recall AUC (PR AUC), accuracy, sensitivity, specificity, the F1 score, the negative predictive value (NPV), and the positive predictive value (PPV). Bootstrapping with 1000 samples was performed to compute 95% confidence intervals for all the evaluation metrics. Mann–Whitney U test was used to compare the predictions between the classes and test for significance . p valueShapley values were calculated for the features used by the machine learning models to gain insight into the AI predictions on the unseen test set. The features with the most impact on the prediction were plotted for each MRI sequence. Figure n = 68, 59%). All patients in our dataset underwent liver resection. In cases where only a single lesion was resected , that lesion would be directly included in the study. In patients where > 1 lesion was resected , the largest two lesions > 1 cm were included. Median time gap between the pre-resection MRI acquisition and liver resection was 25.5 days (IQR = 16.5–35 days). HIF-1 alpha staining was performed on the archival specimens from the NKI Core Facility—Molecular Pathology and Biobank  or “hypoxia high”  , T2W TE300 , DWI b0 , DWI b10 , DWI b200 , DWI b800 , and ADC , when available. Detailed characterisation of our patient cohort can be found in Table Pre-resection MRI scans were then delineated by a radiologist focusing on T2W TE75  and ADC  were significantly predictive of tumour hypoxia. Anatomical imaging in the form of T2W TE75  and functional b0  and b800  images showed trends toward predictive ability. Although the p values did not reach the conventional threshold for statistical significance, they are close to this threshold, suggesting that there may be some predictive power associated with these imaging sequences, particularly in the context of our modest dataset.Radiomic signatures from DWI b200  and b10  images were poorly predictive of tumour hypoxia  scans in the clinical workflow, T2-weighted imaging and DWI have the most profound biological connection to hypoxia –45. PrecIn our cohort of baseline colorectal liver metastases, radiomic signatures derived from the ADC map and DWI b200 image were significantly associated with histopathological tumour hypoxia. Imaging markers derived from T2W TE75, DWI b0, and DWI b800 were also predictive of the HIF-1 alpha staining index, albeit narrowly short of statistical significance. In general, lower spin echo times and higher b-value images were able to encode more information regarding the hypoxia status. These findings are in line with our current understanding of hypoxia under MRI.acute hypoxia and the latter being associated with chronic hypoxia [Tumour hypoxia is generally influenced by perfusion and diffusion, with the former being related to  hypoxia , 49. As  hypoxia . The ebbAlongside diffusion-weighted functional sequences, T2W imaging can also be linked to hypoxia. During acquisition, the balance between oxygen-related paramagnetic molecules influences the T2 time constant of nearby tissue. The presence of electrons defines such molecules. Prominent examples include molecular oxygen, deoxyhaemoglobin, methaemoglobin and oxygen-related diamagnetic molecules such as oxyhaemoglobin. The higher the concentration of paramagnetic molecules, the shorter the T2 time constant , 52. T2 We observed that the radiomic features selected for each sequence collectively encoded morphological heterogeneity . Exploration of association with survival was also not possible due to the heterogeneity of this modestly-sized retrospective cohort. Patients whose baseline tissue and MR scans were included in this study would later receive different treatments making the comparison challenging, especially since the hypoxia status is derived from a lesion while the survival can be confounded by other patient-level differences . Prospective studies with detailed patient inclusion and matching could explore the potential of hypoxia AI models to predict survival.Notwithstanding these limitations, we could identify promising radiomic signatures for tumour hypoxia on T2W and DWI, especially on b200 and ADC. In clinical practice, the results of this study could open the gates for further research aimed at improving patient selection for various liver-directed therapies and prompt treatment modification by more aggressive tumour targeting or by the use of hypoxia-modifying drugs.Morphological phenotypes, as quantified by radiomic signatures, were found to predict tumour hypoxia. These findings suggest that anatomical T2W and functional DWI MRI sequences hold information that can be used for the non-invasive prediction of tumour hypoxia. Radiogenomic prediction of microenvironmental characteristics such as hypoxia could add significant insight into tumour biology, especially in a longitudinal setting. However, as this study was proof of concept, large-scale prospective multicentre studies would be needed to develop and validate robust hypoxia predictive algorithms.Additional file 1. Supplementary material containing the correlation between sequences, radiomic signatures, staining protocol, and selected model/hyperparameters.


Liquid biopsy is a minimally-invasive method of sampling bodily fluids, capable of revealing evidence of cancer. The distribution of cell-free DNA (cfDNA) fragment lengths has been shown to differ between healthy subjects and cancer patients, whereby the distributional shift correlates with the sample’s tumour content. These fragmentomic data have not yet been utilised to directly quantify the proportion of tumour-derived cfDNA in a liquid biopsy.We used statistical learning to predict tumour content from Fourier and wavelet transforms of cfDNA length distributions in samples from 118 cancer patients. The model was validated on an independent dilution series of patient plasma.This proof of concept suggests that our fragmentomic methodology could be useful for predicting tumour content in liquid biopsies. For the past decade, the primary goal in the analysis of plasma cfDNA has been the detection of tumour material. To this end, extremely sensitive assays have been developed to classify samples as healthy or with cancer –6, with Monitoring strategies require tumour-specific features that reflect tumour burden quantitatively. The main genomic approaches that have been used until now have focussed on mutations or copy-number alterations (CNAs). Mutation-based monitoring requires sufficiently high sequencing depth to accurately determine mutations and their allele frequency . To propFollowing observations that the distribution of cfDNA lengths differs between fragments of tumour and non-tumour origin , we reasWe analysed data published by Tsui et al.  consistiMethods section for details and a schematic). For 41 patients, designated cohort A, there was at least one point mutation in copy-number neutral regions, enabling direct estimation of tumour content from the variant allele frequencies  had a particularly high loading  only in copy-number neutral regions . Assuming each point mutation to be heterozygous, the proportion of the tumour harbouring each mutation is twice the mutation allele frequency. Mutations found in all clones cluster at the largest allele frequency, and the tumour content of the sample can be obtained by doubling this number. We performed this analysis for each patient, clustering mutations by their allele frequency with a Chinese restaurant process from the R package cloe . The maxlm and fft functions in R [wavelets [We used the discrete Fourier transform to extract features from the oscillatory pattern at 81–141 bp after regressing out a second-degree polynomial fit to the fragment length distribution on a log scale. That is, for each sample we considered the proportion ons in R . Motivatwavelets .Fig. 3MoichorCNA [betaboost [stabs [We used beta regression  to modelichorCNA . As the etaboost . The pact [stabs  was usedFinally, we applied the trained model to an independent test set, consisting of a dilution series. The predicted tumour content was compared with the dilution, and the concordance was assessed using linear regression with or without an intercept term.Additional file 1. Supplementary text and figures.Additional file 2. A knitted R Markdown document for reproducing the main results.


The data provide important information for understanding the characteristics of UHP fluids, especially supercritical fluids. Supercritical fluids with high dissolved contents of carbon (2 wt.%) and sulfur (3 wt.%) released during deep subduction could cause efficient migration of carbon and sulfur from slabs. This improves their recycling efficiency in the subduction zone, playing a vital role in the deep cycling of these elements. The contribution of supercritical fluids to the deep carbon and sulfur cycle may have been significantly underestimated previously.Our research presents a method that more accurately determines the quantitative composition of ultrahigh-pressure (UHP) fluid [high H Due to their low viscosity, high mobility, and high element contents, supercritical fluids are important agents in the cycling of elements. However, the chemical composition of supercritical fluids in natural rocks is poorly understood. Here, we investigate well-preserved primary multiphase fluid inclusions (MFIs) from an ultrahigh-pressure (UHP) metamorphic vein of the Bixiling eclogite in Dabieshan, China, thus providing direct evidence for the components of supercritical fluid occurring in a natural system. Via the 3D modeling of MFIs by Raman scanning, we quantitatively determined the major composition of the fluid trapped in the MFIs. Combined with the peak-metamorphic pressure–temperature conditions and the cooccurrence of coesite, rutile, and garnet, we suggest that the trapped fluids in the MFIs represent supercritical fluids in a deep subduction zone. The strong mobility of the supercritical fluids with respect to carbon and sulfur suggests that such fluids have profound effects on global carbon and sulfur cycling. Fluids released from slabs at different subduction depths may show large differences in chemical composition and speciation, and they can be subdivided into hydrous melts, aqueous fluids, and supercritical fluids, with the latter being released at high-temperature and high-pressure (HP) conditions . Their h2O systems  inclusions. For continental subduction zones, multiphase inclusions that may be related to supercritical fluids have been documented in multiple metamorphic belts worldwide. The typical multiphase solid–fluid inclusions in Dabie-Sulu . Omphacite and rutile are mainly distributed along the vein wall in the form of large euhedral grains (1 to 3 cm)  . Coesiteto 3 cm) C, with r. The vein-forming fluid should be in equilibrium with the host eclogites and not present reactivity. Therefore, the studied metamorphic vein represents a relatively primitive UHP fluid released during deep subduction.The vein has a sharp and distinct boundary with the surrounding eclogites, without an evident reaction halo. A previous study also supports this observation and showed that the silicate minerals and newly grown zircons in the vein show similar O isotope and Hf isotope compositions to those in the eclogites . These rSI Appendix, Fig. S6). Moreover, MFIs in garnet are far fewer and more irregular than those in omphacite. These healed cracks filled with H2O and signs of fluid leakage   indicate that the primary inclusions were cracked during postentrapment. We believe a possible mechanism is that unlike omphacite, which only forms under metamorphic conditions above eclogite-facies, garnet grows over a wide range of temperatures (as low to 400 °C) and pressure (amphibolite-facies). This means that garnet in metamorphic veins could have experienced complex multiple growth stages. Therefore, the UHP MFIs trapped in early-stage garnet were most likely to be affected by the later growth (extrusion deformation) of the mineral, which resulted in the very common occurrence of postentrapment modifications or fluid leakage of UHP MFIs in garnet.A large number of MFIs are found in omphacite, and they show a preferred directional distribution that is parallel to mineral elongation D. Most iSI Appendix, Fig. S2) demonstrate that the daughter mineral assemblages are similar for all suitable fluid inclusions (~40), including quartz, calcite, sulfates , silicates , and H2O  in omphacite and anhydrite, calcite, silicates , hematite, and H2O in garnet. The newly developed 3D Raman scanning and modeling allowed us to clarify the chemical characteristics of supercritical fluid and further examine data obtained from laboratory experiments. Nine inclusions with good morphology and large size were selected for 3D imaging and modeling analysis. 3D imaging (2O), occasional small cavities could be observed within the inclusions, and they were likely caused by volume contraction during cooling.The Raman analysis results  indicate constant bulk chemical compositions of the inclusions in omphacite  and higher contents of MgO (5.3 to 18.7 wt.%), Al2O3 (5.3 to 11.8 wt.%), and TFe2O3 (9.3 to 11.0 wt.%). The MFIs in the omphacite and garnet had the same general H2O content.The results of 3D modeling and calculations  are least affected by postentrapment modifications  mostications  and do nications ; and iv)ractions D–F. TherTheoretically, the mineral assemblages of MFIs in omphacite and garnet should be exactly the same; however, nonnegligible differences are observed between them. This significant difference cannot be the result of low-degree reaction. According to kinetics theory , crystal2, 13.2 wt.% CaO, 7.0 wt.% SO3, 5.5 wt.% CO2, 3.4 wt.% Al2O3, 4.5 wt.% MgO, 1.3 wt.% TFe2O3, 0. 6 wt.% BaO, and 41.6 wt.% H2O, with trace amounts of Li, Na, and K. As shown in 342 content of the captured fluid in the MFIs clearly conforms to this range. For the critical H2O content parameter, our results are compatible with the supercritical fluid reported by Mibe et al. in their experiments at 3.6 GPa (H2O: 27 to 35 wt%) (2O solubility (2 and H2O) calculated by MFIs has values of 4 GPa and 900 °C to 5 GPa and 900 °C, thus indicating a subsolidus fluid composition, which is closer to that at 5 GPa and 900 °C. This means that the present study may have documented more of subsolidus aqueous fluids close to the SCEP.According to 3D modeling and calculations, the calculated average composition of the captured fluids can be estimated as 22.5 wt.% SiO 35 wt%) . Comparelubility , the flu2O system for producing supercritical fluid is at pressure >5.5 GPa and temperature >1,050 °C (2O–CO2) and our natural samples . Therefore, there may be some discrepancies between the actual SCEP of the studied sample and the experimental results. Combined with Elazar’s experimental data  in UHP rocks can be an intuitive indicator for the preliminary identification of supercritical fluid activity. Other discoveries of multiphase inclusions with similar characteristics in UHP rocks from subduction zones worldwide  imply thCompared with the dilute slab-derived fluid (<15 wt.%) at forearc depths of <80 km , the supIn addition to the silicate components, large amounts of calcite (~10 wt.%) and anhydrite (15 to 20 wt.%) have been identified in the MFIs, indicating that the calculated supercritical fluid shows extreme high solubility for volatile elements such as carbon and sulfur. The solubility of carbon and sulfur in slab-derived fluids is pivotal to control and regulate the carbon and sulfur cycle in subduction zones. The solubility of calcite and anhydrite in the fluids is mainly controlled by pressureand differs by several orders of magnitude between the conditions under the surface and at UHP , 43. TheThe present study provides critical information for revealing the quantitative principal composition of supercritical fluid in subduction zones. Our data demonstrate the quantitative composition of supercritical fluids and solubility of carbon and sulfur. Moreover, the findings confirm the large-scale mobilization of carbon and sulfur by supercritical fluid released from slabs under UHP conditions. The extremely strong mobility of carbon and sulfur indicates that supercritical fluids play a vital role in the carbon and sulfur cycle. Moreover, the contribution of supercritical fluids to the deep carbon and sulfur cycle may have been significantly underestimated in the past.The major element mapping analysis of MFIs and host minerals  was undertaken at the CAS Key Laboratory of Crust-Mantle Materials and Environments at the University of Science and Technology of China, Hefei, using a JXA-8530F Plus Hyper Probe. The operating conditions for the mapping analyses were an accelerating voltage of 15 kV and a probe current of approximately 200 nA. For the element mapping, the spatial resolution and dwell time of single point acquisitions were 0.2 μm and 10 ms, respectively.−1. For 3D scanning, the spatial resolution of single-point acquisitions along the x and y axis was 0.5 μm, while that along the z axis was 1 μm. The time of single-point acquisitions was 0.5 to 2 s, and the time of single-inclusion scanning was approximately 20 h.Laser Raman analysis of the MFIs in the omphacite and garnet was performed at the CAS Key Laboratory of Crust-Mantle Materials and Environments at the University of Science and Technology of China, Hefei, using a Nicolet FT-Raman 960 spectrometer with a 532-nm Ar laser excitation. 3D imaging of MFIs was performed using a WITec alpha300R confocal Raman microscope (WITec GmbH) with 532 nm cobalt laser excitation at the State Key Laboratory of Lithospheric Evolution, Institute of Geology and Geophysics, Chinese Academy of Sciences. The energy of the laser source was 25 mW, and the spectral collection range was 100 to 4,000 cmSI Appendix, Fig. S5) can obtain the absolute volume of each daughter mineral phase in MFIs. Combined with the density of daughter minerals, the mass fraction of each mineral phase can be calculated. According to the ideal chemistry of daughter minerals, we can quantitatively calculate the composition characteristics of the fluid trapped during the crystallization of host minerals . The ideal chemistry of daughter minerals is as follows: calcite: CaCO3; quartz: SiO2; anhydrite: CaSO4; ferro-holmquistite: Li2(Fe2+3Al2)Si8O22(OH)2; barite: BaSO4; muscovite: KAl2(Si3Al)O10(OH)2; clinochlore: Mg5Al(AlSi3O10)(OH)8; pargasite: NaCa2(Mg4Al)(Si6Al2)O22(OH)2; diopside: CaMgSi2O6; actinite: Ca2(MgFe2+)5[Si4O11]2(OH)2; grossular: Ca3Al2(SiO4)3; and hematite: Fe2O3. The data source is https//rruff.info. We assumed that the fluid in the inclusions was pure water when calculating the composition of MFIs. Although it has been reported that the fluid inclusions of Bixiling contain high NaCl contents .The Raman data analysis was performed with WITec Project plus software. 3D imaging and modeling analyses of the MFIs were performed using Image J software. 3D modeling Click here for additional data file.


Carcinus maenas and compared to native Canadian crustaceans. l-leucine transport and accumulation throughout the body of the green crab is also evaluated in various gill and organ tissues in fed and fasted states.Branchial amino acid uptake is evaluated in the invasive  Carcinus maenas) is an arthropod native to European waters; however, it is now known to be a globally invasive species. Recently, it was discovered that the C. maenas could transport nutrients in the form of amino acids across their gill from the surrounding environment, a feat previously thought to be impossible in arthropods. We compared the ability for branchial amino acid transport of crustacean's native to Canadian Pacific waters to that of the invasive C. maenas, determining if this was a novel pathway in an extremely successful invasive species, or a shared trait among crustaceans. Active transport of l-leucine was exhibited in C. maenas, Metacarcinus gracilis, Metacarcinus magister, and Cancer productus across their gill epithelia. Carcinus maenas exhibited the highest maximum rate of branchial l-leucine transport at 53.7 ± 6.24 nmolg−1 h−1, over twice the rate of two native Canadian crustaceans. We also examined the influence of feeding, gill specificity, and organ accumulation of l-leucine. Feeding events displayed a heavy influence on the branchial transport rate of amino acids, increasing l-leucine transport rates by up to 10-fold in C. maenas. l-leucine displayed a significantly higher accumulation rate in the gills of C. maenas compared to the rest of the body at 4.15 ± 0.78 nmolg−1 h−1, with the stomach, hepatopancreas, eyestalks, muscle tissue, carapace and heart muscle exhibiting accumulation under 0.15 nmolg−1 h−1. For the first time, the novel transport of amino acids in Canadian native arthropods is described, suggesting that branchial amino acid transport is a shared trait among arthropods, contrary to existing literature. Further investigation is required to determine the influence of environmental temperature and salinity on transport in each species to outline any competitive advantages of the invasive C. maenas in a fluctuating estuarine environment.Many aquatic species are well known as extremely successful invaders. The green crab ( Carcinus maenas) is now found in coastal waters worldwide, including the Pacific and Atlantic coasts of Canada. Primary observations of the C. maenas' arrival on Pacific North American coasts were in 1989 in San Francisco Bay, CA, spreading 300 km north to Coos Bay, OR, by 1997, and quickly spreading to Vancouver Island, BC, by 1998 (C. maenas outcompete juvenile Dungeness crab (Metacarcinus magister) for habitat and food resulting in population decline and over $6 million in losses per year to the British Columbia decapod fishery (C. maenas displaces the American lobster (Homarus americanus), resulting in a commercial loss of $44 to 114 million per year  meadows that many estuarine organisms rely on for food and shelter. In Atlantic Canada, eelgrass meadows have experienced biomass declines between 50% and 100% when C. maenas invades their ecosystem  from the aqueous environment similarly to other marine invertebrates , Cancer productus (red rock crab), and Metacarcinus magister (Dungeness crab)), of the Pacific can transport amino acids, such as l-leucine across the gill epithelium in comparison to C. maenas. We hypothesized that arthropod species native to the Pacific coast of Canada would be able to transport l-leucine across the gill epithelium alongside C. maenas due to their shared gill structures, but with a lower capacity to do so, given C. maenas' increased ability to regulate internal osmolytes  were collected in accordance with Fisheries and Oceans Canada license XR 1352020, off the western side of Vancouver Island in Barkley Sound (N49802.274–W125820.710 and N49801.749–W125821.515). Captured animals were transported to the Bamfield Marine Science Centre . Animals were held in various mesh-covered outdoor 2000 L tanks, on a flow through system of filtered seawater pumped from the ocean . Animals were subjected to natural summer lighting  and fed salmon to satiety every 3 days. A minimum of 7 days prior to experimentation, animals were transported into indoor carbon filtered tanks, fasted, and held at 31.7 ± 0.2 ppt at 17.7 ± 0.8°C. All collection and holding was performed in accordance with University of Alberta, Bamfield Marine Sciences Centre, and Canadian Council on Animal Care guidelines.Invasive adult male 2, 12 MgCl2, 11 KCl, 9 NaHCO3, 0.1 NH4Cl, 0.3 glucose, 0.1 glutathione, 0.5 glutamine, mOsm = 954 ± 2.6) at a rate of approximately 127 ± 1.7 μL/min using a 12-channel peristaltic pump . Gills were then placed in 50 mL of bathing solution comprised of seawater  spiked with 14C radiolabelled l-leucine , at concentrations of 0, 1, 10, 100, and 1000 μM and perfused for 1 h, based on previous work by Gill perfusion experiments were performed in accordance with the methods described by l-leucine, C. maenas, M. gracilis and M. magister, were fed salmon tissue to satiety in the hour leading up to euthanization. Posterior gills were then dissected and perfused in 2 μM radiolabelled l-leucine following the procedure above. The influence of feeding on the branchial uptake of l-leucine was not evaluated in C. productus as a result of limited animal availability. Each treatment was replicated between four and six times, depending on individual species gill availability.In order to compare the influence of feeding on the branchial uptake of Calculations and Data Analysis). All “zeros” were considered true zeros as gills were perfused without radioisotope and individual beta emissions were recorded at background levels.A 1-mL subsample of perfusate was collected at the end of each treatment for radioisotope analysis and mixed with 4 mL of Optiphase  liquid scintillant. Individual beta emissions were measured via counts per minute (CPM) of each sample using a TriCarb 29 000 TR liquid Scintillation Analyzer. Gills were weighed and amino acid transport rates were then calculated for each gill (see l-leucine was made from a concentrated l-leucine stock of 10 mM (Sigma Aldrich), and added to the seawater 24 h before experimentation to equilibrate. Thirty minutes before the start of the experiment, 1 μCi−1 of 14C radiolabelled l-leucine  was added to each exposure chamber for a final concentration in the chambers of 300 μM l-leucine. Water samples of 1 mL were taken at t = 0 and t = 24 to monitor radioisotope concentration. Carcinus maenas were starved for 4 days prior to exposure to whole animal experiments. Five individual crabs were then randomly placed into the containers and allowed to rest for 24 h in the aerated mixture. Upon termination of the exposure, crabs were washed (1 min) in a highly concentrated nonradiolabelled l-leucine wash (10 mM) for displacement of any radiolabelled leucine that was loosely bound to the outside of the animal, they were then washed in 100% seawater for 1 min, and immediately following that, crabs were placed on ice for 15 mins and euthanized as stated above. Once crabs were euthanized, they were dissected and the following organs were weighed and digested as described below for radioisotope analysis: gill 5 (G5), gill 8 (G8), stomach (ST), hepatopancreas (HP), eyestalk (ES), muscle (MU), carapace (CA) and haemolymph (HL).l-leucine accumulation in the gills of C. maenas, the procedure above was also conducted using C. maenas that had been fed to satiety 1 h prior to experimental exposure. Five individual fasted crabs and five individual fed crabs were then randomly placed into the containers and allowed to rest for 24 h in the aerated mixture. Once crabs were euthanized, they were dissected and gills 2 to 9 (G2–G9) were weighed and digested as described below for radioisotope analysis.In order to compare the impacts of feeding on 3  at the three to five times (exact volume recorded) the weight of the tissue. All vials were sealed and placed in an incubator at 65°C for 48 h, with vortexing after 24 h. Scintillation fluor was then added to the scintillation vials  in a 5:1 ratio of volume to tissue and assayed for radioactive beta-emission counts on a TriCarb 29000 TR liquid Scintillation Analyzer. Samples were standardized to a common counting efficiency (quench corrected), using a quench curve constructed from tissue digests. Water samples (1 mL) were treated as above, where Optiphase  liquid fluor was added at a volume of 2:1 ratio of volume to water and run for emission counts on the Liquid Scintillation Analyzer above.Tissues were weighed and collected in 20 mL scintillation vials and digested with 2N HNOSpecific activity is the ratio of measured radioactivity of the stock solution to the total concentration (μM) of amino acid present in the experimental exposure:−1 based on individual gill weights. Finally, amino acid uptake (nmol g−1 h−1) or tissue accumulation (nmol g−1 h−1) was determined by accounting for total perfusion time in hours (t):To calculate amino acid uptake, measured perfusate or tissue radioactivity (CPM) was divided by the specific activity (SA) of the exposure solution. To account for the variable gill sizes among species, amino acid concentration was converted into nmol gl-leucine and concentration-dependent uptake was evaluated and plotted using SigmaPlot Version 11.0 . Uptake at each given amino acid concentration (μM) versus branchial transport rate (nmol g−1 h−1) for the four arthropod species at high and low environment concentrations were plotted on raw data using Sigmaplot Version 11.0.Outliers were identified and removed using Grubbs' test, and l-leucine accumulation in various C. maenas organs , exhibiting a significantly higher  branchial transport rate when compared to M. gracilis (20.3 ± 4.53 nmolg−1 h−1) and M. magister (15.9 ± 5.22 nmol−1−1). While C. maenas showed no significant difference to C. productus (43.8 ± 10.6 nmol−1−1) at 1000 μM. Notably, no significant differences (p = 0.076) were seen between any crustacean’s branchial transport at 1 μM exposure. Contrarily, at 10 μM exposure, M. magister (7.08 ± 2.27 nmol−1−1) was noted to have significantly higher branchial transport of l-leucine  compared to both C. maenas (0.83 ± 0.20 nmol−1−1) C. productus (1.36 ± 0.68 nmol−1−1). Finally, at 100 μM exposure C. maenas (44.8 ± 14.6 nmol−1−1) exhibited significantly higher branchial transport , when compared to both M. gracilis (5.31 ± 1.20 nmol−1−1) and M. magister (2.69 ± 1.73 nmolg−1 h−1).-leucine  was noteMetacarcinus gracilis  and M. magister  exhibited significantly higher branchial uptake of l-leucine immediately after feeding, increasing from a fasted value of 1.19 ± 0.278 nmol−1−1 to 3.40 ± 0.853 nmol−1−1, and 0.48 ± 0.23 nmol−1−1 to 2.00 ± 0.28 nmol−1−1, respectively (C. maenas did not exhibit a significant difference (p = 0.054) between fed and fasted gills, a general trend can be seen increasing an order of magnitude from fasted gills with a transport rate of 0.132 ± 0.025 nmol−1−1 to fed gills with a transport rate of 1.37 ± 0.85 nmol−1−1.ectively . While CC. maenas whole-body exposures gill 5 and gill 8 showed significantly higher  uptake of l-leucine when compared with all other organs, with total accumulation rate at 4.15 ± 0.78 nmol−1−1 and 2.90 ± 0.47 nmol−1−1, respectively (−3 ± 2.86−3 nmolg−1 h−1), hepatopancreas (1.34 × 10−2 ± 2.86 × 10−3 nmol−1−1), eyestalks (1.25 × 10−1 ± 1.81 × 10−2 nmol−1−1), muscle (2.27 × 10−2 ± 3.40 × 10−3 nmol−1−1), carapace (1.54 × 10−1 ± 1.04 × 10−2 nmol−1−1), haemolymph (1.22 ± 0.38 nmol−1−1) and heart (8.42 × 10−3 ± 2.39 × 10−3 nmol−1−1).Notably, throughout ectively . No othel-leucine accumulation in C. maenas, no significant interaction was found between the fed state and gill number (p = 0.4). Likewise, no significant differences were seen between gill number and l-leucine accumulation in either fed or fasted gills (p > 0.05).When evaluating how feeding state and gill number influence branchial C. maenas haemolymph, M. gracilis haemolymph, C. productus haemolymph and M. magister haemolymph, or saltwater (32 ppt) osmolarity . It was hypothesized that, given C. maenas' ability to acquire l-leucine from the environment, native Pacific arthropods would also share this ability and transport l-leucine across their gill epithelium but at a lower rate of transport than C. maenas. Our hypothesis was partially supported in that l-leucine was transported across both the anterior and posterior gill membranes of all four species. The noted uptake suggests that M. gracilis, C. productus, M. magister and C. maenas have the ability to actively transport l-leucine from the environment through their gills. However, C. maenas was only noted to have an increased capacity for transport over M. magister at high environmental concentrations of 100 and 1000 μM, showing a lower transport rate than M. magister at 10 μM and was otherwise comparable to the Pacific native species. Notably, after being transported from the environment, amino acids were shown to remain largely in the gill, with lower concentrations noted in the haemolymph and throughout the organs. Branchial amino acid transport rates were also shown to increase in C. maenas, M. gracilis and M. magister immediately following a feeding event.Marine invertebrates utilize waterborne amino acids in various physiological purposes, such as osmoregulation and nutrient uptake, increasing their survival and optimal habitat range . Of the l-leucine in the invasive green crab at environmentally relevant amino acid concentrations. Notably, our results show that branchial amino acid uptake is not only displayed in a single arthropod species, but likely a shared trait among all marine crustaceans. M. gracilis, C. productus, M. magister, as well as C. maenas all display an active transport pathway for the uptake of free l-leucine environmental concentrations ranging from 0 to 1000 μM , C. maenas exhibited higher rates of transport compared to two of the three native species (i.e. M. gracilis and M. magister)  pathway could offer greater physiological significance to arthropods based on normal environmental amino acid concentrations than the higher 100 to 1000 μM treatments , C. maenas may have a competitive advantage over M. gracilis and M. magister.It was hypothesized that agister) . Intereseatments . Given tnditions . This fuM. gracilis and M. magister both exhibited significant increases in branchial l-leucine uptake immediately after a feeding event , indicating little specificity between l-leucine accumulation and unique organ tissues similar to the results of this study. After the gills, the highest rate of accumulation throughout the body can be seen in the haemolymph. This elevated level of free amino acids within the circulatory system of C. maenas after a 24-h exposure period suggests that the primary utilization of l-leucine within the green crab, may not be protein synthesis. In C. maenas, protein synthesis is known to return to fasted levels within 16 h of a single meal, peaking at a rate nearly four times that of fasted crustaceans  and many marine bivalves , using free l-alanine as an osmolyte in muscle tissues to regulate osmolarity in an estuarine environment (Procambarus clarkia) exhibits a 5.4-fold increase in amino acid accumulation in response to increasing salinity in both muscle tissue and the hepatopancreas (Crassostrea gigas) and the blue mussel M. galloprovincialis exhibit increased amino acid transporter expression in the gill epithelia, adductor muscle and kidney in response to both hypoosmotic and hyperosmotic stress (It is therefore possible that these amino acids are being utilized as an osmolyte throughout the body, not unlike the osmoregulatory strategy of the Japanese mitten crab (pancreas . LikewisC. maenas. The invasive green crab is a known euryhaline species with survivable salinity ranging from 4 to 54 ppt (Metacarcinus gracilis, Metacarcinus magister, and Cancer productus, which all possess a lower ability to regulate internal ions. M. magister in particular displays a moderate ability for osmoregulation, surviving prolonged periods in salinity ranging from 12 to 35 ppt (M. gracilis and C. productus have weak osmoregulatory abilities, unable to withstand extended periods in salinities lower than approximately 18 to 20 ppt and 13 to 16 ppt, respectively (C. maenas leaves no surprise surrounding its ability to thrive in vastly different coastal waters around the world (l-leucine when compared with Canadian native species at high amino acid concentrations, it showed no advantage at environmental levels. However, in the presence of a feeding cloud, or localized area of high amino acid concentrations, the competitive ability of C. maenas could be greatly increased. l-leucine was determined to accumulate throughout all tested tissues within the body of C. maenas, suggesting widespread utilization. While the exact utilization of these amino acids remains unknown, a deeper understanding of branchial transport could improve clarity regarding success of crustacean species in variable habitats and offer insight into the future spread of C. maenas.In order to conserve the diversity and abundance of native species, it is crucial to understand any and all physiological factors that influence species interactions and resultant survival. Throughout this study, we sought to expand our understanding of marine crustacean ion transport processes, and more importantly, gain insight into a potential key factor in the invasive success of the globally dominant This work was supported by the Natural Sciences and Engineering Research Council Discovery RGPIN-2020-04153 grant awarded to Dr Blewett.Supplementary data file contains raw isotope counts, calculations for branchial gill perfusions, and whole body accumulation studies presented in this article.We have no competing interests.https://doi.org/10.5061/dryad.4mw6m90f8The data underlying this article are available in the Dryad Digital Repository, at All procedures were approved by the Bamfield Animal Research Ethics Board and the University of Alberta, and were in accordance with the Guidelines for the Canadian Council on Animal Care.T.A.B conceived the design of the study. R.A.G, A.B, A.W, and T.A.B, carried out all experimental procedures. R.A.G carried out all statistical analyses and drafted the manuscript. T.A.B coordinated the study and helped draft the manuscript. R.A.G, A.B, A.W, and T.A.B edited the M.S. All the authors have given their approval for this publication.Web_Material_coad015Click here for additional data file.