Pembrolizumab cause immune‐related adverse events. We herein report a case of advanced bladder cancer, who treated with pembrolizumab and exhibited intriguing clinical course.A 63‐year‐old man with bladder carcinoma was treated by radical cystectomy, however, the bladder carcinoma recurred and invaded to the rectum. He was treated by combination therapy using gemcitabine and cisplatin, which were not effective for the tumor. He subsequently underwent treatment with pembrolizumab. In several 30 days, he suffered from the symptoms of myasthenia gravis. Serum levels of creatine kinase, its isozyme creatine kinase‐myocardial band, and troponin I were elevated. Electrocardiography showed a right bundle branch block. These findings suggested that he was myasthenia gravis with general myositis and presumable myocarditis. Oral prednisolone administration significantly attenuated these findings. The tumor drastically shrunk only by the single injection of pembrolizumab.Early intervention with corticosteroid was effective for neuromuscular complications due to pembrolizumab. AChR Abacetylcholine receptor antibodyCKcreatine kinaseCTcomputed tomographyECGelectrocardiographyirAEsimmune‐related adverse eventsMBmyocardial bandMGmyasthenia gravisMRImagnetic resonance imagingMuSK Abmuscle‐specific kinase antibodyPD‐1programmed cell death 1SIMC‐UroUro‐oncology Saitama Medical University International Medical CenterUCurothelial carcinomaImmunosuppressive treatment, for example, with corticosteroids, must be considered when we notice the sign of MG with myositis arises in a patient under treatment with a PD‐1 inhibitor. Immune‐mediated neuromuscular side effects vary in presentation, and differ from their idiopathic counterparts.Pembrolizumab, a humanized monoclonal antibody that inhibits PD‐1 receptors, is utilized as a second‐line therapy for advanced UC, which gives complete remission in approximately 7% of patients.A 63‐year‐old Japanese man  visited a nearby clinic due to intermittent macroscopic hematuria. He had a medical history of mild diabetes mellitus and surgery for colon carcinoma. Physical and laboratory findings were within normal limits except for the presence of microhematuria at the clinic. Cystoscopy revealed a broad‐base non‐papillary bladder tumor. CT #1,  and MRI 2, day 1, 8, and 15) and cisplatin , followed by radical cystectomy and extended pelvic lymph node dissection. Histopathological examination detected high‐grade UC in the previously resected portion even after chemotherapy, however, no cancer cells were detected in the surgical margin of the bladder specimen (pT1N0).He was referred to the SIMC‐Uro for the treatment of the invasive bladder tumor. Physical and laboratory findings were within normal limits . CT #2 at the SIMC detected no metastatic lesions. He underwent three cycles of monthly neoadjuvant chemotherapy using gemcitabine  which invaded to the rectum Fig. b. Transrhttps://www.eortc.be/services/doc/ctc/]) due to unknown reason. At the day of Grade 1‐diarrhea, laboratory data including serum CK level  ,From the course of the current case, we concluded that (i) regular screening using CK, CK‐MB, and troponin I is needed for early detection of neuromuscular complication, (ii) cardiac complications must be identified by repetitive ECG and echocardiography to avoid lethal cardiac damage by early intervention, and (iii) comprehensive management team play a pivotal role for these early detection and intervention. Further accumulation of case reports is needed to sophisticate cancer treatment using immune checkpoint inhibitors.The authors declare no conflict of interest.Table S1. Clinical course of the case.Click here for additional data file.


Evidence on the association between sleep duration and obesity among adults is inconsistent. Prospective studies investigating the association in Chinese adults have been limited. This study aims to prospectively evaluate sleep duration in relation to subsequent weight gain and general and central obesity risk among Chinese adults.A total of 21,958 participants aged 30 to 79 years reported their daily sleep duration. Obesity indicators were objectively measured; then significant weight gain (≥ 5 kg) and general and central obesity were modeled as the outcome. Logistic regression models were used to estimate odds ratios and 95% CIs.P = 0.04 for interaction). Short sleep duration was also associated with subsequent incident central obesity, with odds ratio of 1.13 (95% CI: 1.00‐1.28), but not with incident general obesity (P = 0.31).Average sleep duration was 7.5 hours at baseline. During 8.0 ± 0.8 years of follow‐up, participants who reported sleeping ≤ 6 hours had higher risk for significant weight gain than those who slept 7 hours . The association was stronger among those who were physically inactive at baseline (Compared with those who slept 7 hours per day, short sleepers had an increased risk of significant weight gain and central obesity. Previous studies, which are mostly cross‐sectional studies, have suggested that short sleep duration is associated with weight gain.Existing prospective observational studies have reported mixed results.Therefore, it remains unclear whether short sleep duration is associated with a higher risk for obesity and whether there exists a U‐shaped relationship in which long sleep duration is also associated with higher risk for obesity.In addition, large prospective studies that have examined the association of sleep duration with central obesity in middle‐aged and older adults are scarce.What is already known?Our results suggest that short sleep duration, one of products of fast‐paced life, is associated with gaining weight significantly, and the weight gain is likely to be accrued on the abdomen.To our knowledge, this is by far the largest prospective study reporting a negative association between sleep duration and central obesity in healthy adults who are free of common comorbidities.Our findings emphasize the important role of short sleep duration in weight control and central obesity management.What does this study add?Obesity has been a major public health issue worldwide and has nearly tripled in recent decades The potential link of sleep duration with obesity has been explored for years. However, large prospective studies conducted in the Chinese population have been lacking. Considering the Chinese population is currently in the process of fast socioeconomic growth and rapid nutritional transition, it is of importance to examine how short sleep duration, accompanied by the fast pace of life, might synergize with a potential increasing obesity trend. On the other hand, although several cross‐sectional studies have suggested a link between short sleep duration and the risk of obesity in adults n = 24,996) of randomly chosen surviving participants in the cohort.The present study was based on the China Kadoorie Biobank, which enrolled 500,000 adults aged 30 to 79 years in 10 survey areas . Detailed information about the study design has been reported elsewhere n = 171) and waist circumference (WC) (n = 3); those who had coronary heart diseases (n = 714), stroke (n = 223), cancer (n = 102), and chronic obstructive pulmonary disease ; and those who tried to reduce weight in the year prior to baseline (n = 255), leaving a total of 21,958 participants in the analysis of sleep duration with significant weight gain. As for the analysis of the association with incident general and central obesity, we further excluded participants with general obesity  or central obesity  at baseline, leaving 19,638 and 14,808 participants for each of the analyses, respectively.The present study included participants who attended both the baseline survey and the second resurvey. The follow‐up time started from the date they were enrolled at baseline (2004‐2008) until the date of the second resurvey (2013‐2014). We excluded those who had missing values on critical variables, such as BMI  and the Oxford Tropical Research Ethics Committee, University of Oxford (UK), approved the study.Physical examination at both surveys was performed by trained health workers following standardized procedures. Height was measured in centimeters using unified height meters and was reserved to 0.1 cm. Weight was measured with coat, shoes, and socks off, using Tanita TBF‐300GS constitution analyzer . The weight of clothes was subtracted according to different seasons. WC was measured using standard tape. Weight gain was defined as the increase of weight between baseline and second resurvey. A weight gain ≥ 5 kg was considered gaining weight significantly, as it has been used in previous studies At the baseline survey, participants were asked to report the number of hours they slept a day during the last 12 months. The question was phrased as “On average, how many hours of sleep do you get in a 24‐hour (h) period (including naps)?’’ Respondents could report in only 1‐hour increments. According to the American National Sleep Foundation’s sleep time recommendation, short sleep duration was defined as sleeping 6 hours or less, and long sleep duration was defined as sleeping 9 hours or more A laptop‐based questionnaire at baseline survey was administered by trained interviewers to collect information on sociodemographic factors, lifestyle, and health status. Sociodemographic factors included age, sex, study region, education level , and marital status ; lifestyle factors included smoking , alcohol drinking , tea drinking , diet frequency , and physical exercise ; and health status included snoring habit , depression , self‐rated health , and history of diabetes.We described baseline characteristics by sleep duration. Means or percentages were adjusted for age, sex, and study region using general linear models or logistic models. Incidences of significant weight gain, general obesity, and central obesity were compared across sleep duration groups adjusting for age, sex, and study region. Multiple logistic regression models were used to estimate odds ratios (OR) and 95% CIs to examine the association of sleep duration with obesity. Model 1 adjusted for age and sex. Further adjustment in model 2 included study region, education level, marital status, smoking, alcohol drinking, tea drinking, diet frequency, physical exercise, snoring, depression, and health rating. Model 3 additionally adjusted for history of diabetes, baseline weight and height, and BMI or WC.P < 0.05.Subgroup analyses according to baseline characteristics were conducted to explore the modification effects of these characteristics on the association between sleep duration and weight gain. Multiplicative interactions were tested by putting interaction terms in the fully adjusted models (model 3). To test the robustness of the association, a series of sensitivity analyses were performed by applying the following additional exclusion criteria: reported sleep difficulties  at baseline, diabetes at baseline, diagnosed depression at second resurvey, reported coffee drinking at second resurvey, and newly diagnosed stroke or transient ischemic attack cases during the follow‐up period. All analyses were conducted using SAS version 9.3 , and statistical significance was indicated by two‐tailed Among the 21,958 participants at baseline, average sleep duration was 7.5 hours; 20.8% of participants reported sleeping ≤ 6 hours, while 17.7% reported sleeping ≥ 9 hours. Participants who had shorter sleep durations were more likely to be older, living in urban areas, unmarried, and physically active but less likely to be snoring or drinking tea  participants had significant weight gain. Among 19,638 participants without general obesity at baseline, 5.5% developed incident general obesity; the corresponding incidence of central obesity was 24.6% among 14,808 participants without central obesity at baseline.P = 0.04 for interaction), with OR = 1.34 (95% CI: 1.07‐1.67).Compared with those who slept 7 hours, short sleepers had a significantly higher risk for significant weight gain in model 1 (OR = 1.15 [95% CI: 1.02‐1.29]), and the association did not change after further adjusting for potential confounders Table , except Although a higher incidence of general obesity among short sleepers was observed compared with 7‐hour sleepers (5.4% vs. 5.0%), the difference was not statistically significant after adjusting for potential confounders (OR = 1.11 [95% CI: 0.91‐1.35]). However, among participants without central obesity at baseline, short sleepers had a higher incidence of central obesity than 7‐hour sleepers (34.5% vs. 32.5%) and a 13% increase in the risk for developing central obesity (OR = 1.13 [95% CI: 1.00‐1.28])  To the best of our knowledge, the present study is the most extensive prospective design with objective measurements on obesity, which comprehensively controlled for potential confounders and applied strict exclusion criteria to avoid reverse causality. However, some limitations should be considered. First, sleep duration was self‐reported, which was moderately related to that measured by the objective method polysomnography In conclusion, the present study provided prospective evidence that short sleep duration (≤ 6 hours) was associated with weight gain ≥ 5 kg and a higher risk for developing central obesity, especially among participants who were physically inactive. Given the trend of sleeping less in the modern society and the high prevalence of central obesity in the Chinese population, the link of sleep with obesity needs to be recognized, and importance of sleep intervention in the context of weight management needs to be underscored.


To determine the relationship between maternal serum uric acid levels and fetal/neonatal complications in women with pre‐eclampsia/eclampsia, and to establish a predictive threshold value.ACOG criteria. Serum uric acid levels were measured by enzymatic colorimetric testing using a Cobas c 501 analyzer . Fetal complications included intrauterine growth restriction, preterm delivery, fetal death, and neonatal death.A diagnostic test and historical cohort study conducted by prospective cross‐sectional data collection on pregnant women with pre‐eclampsia/eclampsia at Hue University Hospital, Vietnam, between March 2015 and July 2017. Pre‐eclampsia was diagnosed based on AUC 0.752), with 64.4% sensitivity and 79.5% specificity. High uric acid level (≥393 μmol/L) resulted in increased risk of preterm birth , low Apgar scores , intrauterine growth restriction , and neonatal death . There was no relationship between uric acid level and fetal death .There were 205 women enrolled. Serum uric acid at a cutoff of 393 μmol/L is a good predictor of fetal/neonatal complications (Maternal serum uric acid concentration is a good predictor of fetal/neonatal outcomes in women with pre‐eclampsia/eclampsia. Maternal serum uric acid concentration is a good predictor of fetal/neonatal outcomes in pregnant women with pre‐eclampsia/eclampsia. Although most studies have shown that maternal uric acid levels play an important role in the prognosis of pre‐eclampsia,In Vietnam, although there have been many studies on pre‐eclampsia in terms of prevention, early diagnosis, and pregnancy outcomes, there are limited studies that have investigated uric acid concentration and its role in predicting severe fetal complications in pre‐eclamptic patients. The aim of the present study was to determine the relationship between maternal serum uric acid levels and fetal/neonatal complications in women with pre‐eclampsia/eclampsia, and to establish a predictive value for these complications.2A diagnostic test and historical cohort study conducted by prospective cross‐sectional data collection on singleton pregnant women with pre‐eclampsia/eclampsia admitted to the Department of Obstetrics and Gynecology, Hue University Hospital, Vietnam, between March 2015 and July 2017. The study excluded chronic hypertensive women, patients with metabolic disorders, and those with diseases that adversely affect serum uric acid levels in pregnancy, such as chronic kidney disease, acute and chronic nephritis, nephrotic syndrome, and kidney failure. Patients with cellular damage as a result of cancer and hemolytic diseases were also excluded. The study was approved by the Hue University of Medicine and Pharmacy Ethics Committee. Because uric acid sampling is indicated routinely for pregnant women with pre‐eclampsia in our clinical practice, informed consent was not required to obtain samples.Zα/22 × P × (1 − P)/Δ2, where α=0.05, Δ=0.05, and Zα/2=1.96. Based on the complication rate of intrauterine fetal death, neonatal death, small for gestational age or prematurity from the study by Livingston et al.P=11%). The minimum sample size required to identify the fetal/neonatal outcomes for women with pre‐eclampsia attending our hospital was 150 patients.To estimate the sample size required for this study, we used the equation n=Recruited patients were divided into two groups: pre‐eclampsia (group 1) or severe pre‐eclampsia and eclampsia (group 2), based on the criteria from the American College of Obstetricians and Gynecologists (ACOG).Fetal complications included IUGR, preterm delivery, fetal death, and neonatal death. Neonatal death was defined as death occurring during the first 4 weeks after birth, while fetal death was defined as intrauterine death before delivery (stillbirth).N‐ethyl‐N‐(2‐hydroxy‐3‐sulfopropyl)‐3‐methylaniline, and 4‐aminophenazone to form a quinonediimine dye. The intensity of the red color formed is proportional to the uric acid concentration and is determined photometrically; the methods used followed the manufacturer's instructions.Each patient was measured for height and weight. Body mass index (BMI) was calculated as body weight in kilograms divided by the square of height in meters. Serum uric acid levels were drawn intravenously at the hospital after each patient had rested for 30 minutes. The sterilization principle and compliance preservation regime were ensured during testing by use of the comparison color method (enzymatic colorimetric test) using a Coba c 501 analyzer  with UA2 ACN 700 (serum/plasma). The peroxide reacts in the presence of peroxidase, t test for independent samples. Values are given as mean and standard deviation (SD) or absolute number and percentage. Results are expressed as odds ratio (OR) with 95% confidence interval (CI) or two‐sided P value. P<0.05 was considered statistically significant.All analyses were performed using SPSS version 2.0 . Receiver operating characteristic (ROC) curves were constructed to examine the diagnostic test performance of uric acid for the prognosis of each fetal/neonatal outcome, and general complications defined as at least one of five events occurring, including: preterm birth, APGAR score less than 7 in the first minute, fetal death, IUGR, or neonatal death. Groups were compared using the 3During the study period, 205 pregnant women with pre‐eclampsia/eclampsia met the eligibility criteria and were recruited consecutively. The general characteristics of the study population and the fetal/neonatal complications are shown in Table P<0.001). IUGR was the most common fetal/neonatal complication, accounting for 25.9% (n=53), followed by preterm birth at 22.4% (n=46). Apgar score of less than 7 was established for 199 live neonates owing to 6 cases of fetal death.Uric acid concentration was significantly higher in group 2 compared with group 1 , followed by preterm birth , Apgar score <7 , and IUGR  ; low Apgar score ; IUGR ; and neonatal death . The only complication for which the difference was not significant was fetal death (P=0.773).The relationship between fetal outcomes and uric acid threshold of 393 μmol/L is shown in Table P<0.001).There was no association between uric acid level and maternal factors such as age, parity, or delivery method Table . The onlTable 4This cross‐sectional study of 205 pregnant women with pre‐eclampsia/eclampsia aimed to assess the role of maternal serum uric acid levels in predicting pregnancy outcomes. The study was also designed to establish a predictive threshold value of maternal uric acid levels and occurrence of fetal complications. The association of biomarkers, including uric acid, with adverse outcomes in pre‐eclamptic pregnant women is discussed in ACOG guidelinesThe occurrence of pregnancy outcomes in the present study were: preterm birth rate 22.4%), IUGR (25.9%), Apgar score less than 7 (9.0%), fetal death (2.9%), and neonatal death (4.9%). Other studies have reported similarly, with preterm birth ranging from 15% to 67%, IUGR ranging from 10% to 25%, and fetal mortality ranging from 1% to 2%.2.4%, IUGConcerning individual fetal/neonatal complications, the AUC of uric acid was highest for the prognosis of neonatal death, followed by preterm birth, Apgar score less than 7, and IUGR. ROC analysis showed that a serum uric acid threshold of 393 μmol/L was a good predictor of general fetal/neonatal complications (AUC 0.752), with 64.4% sensitivity and 79.5% specificity, in women with pre‐eclampsia/eclampsia.The results of our study are similar to those of Livingston et al.,Based on our threshold of 393 μmol/L, the risk of preterm birth was increased 6.367 times (95% CI 3.009–13.084), which was similar to Tejal's study ,In the present study, a threshold of 393 μmol/lL was not a predictive factor for fetal death . However, high uric acid concentration increased the risk of neonatal mortality by over sevenfold . This result is controversial owing to the wide confidence interval. In another study, maternal serum uric acid levels were increased in 86.4% of cases of perinatal.The uric acid threshold in the present study was a good predictor of severity of pre‐eclampsia/eclampsia . These findings were much higher than those of Hawkins et al.,In multivariable logistic regression analysis adjusted for maternal age, grade of pre‐eclampsia, and parity, serum uric acid was a good prognostic marker at the threshold of 393 μmol/L for general fetal/neonatal complications . For each individual outcome, serum uric acid was a good prognostic marker for IUGR, preterm birth, neonatal death, and low Apgar score.The present study had some limitations, such as missing maternal data for preconception and prenatal care, which made the assessment of maternal risks more difficult.In conclusion, maternal serum uric acid concentration was a good prognostic factor for monitoring and prognosis of fetal/neonatal outcomes in women with pre‐eclampsia/eclampsia. There was a relationship between high uric acid level and the risk of preterm birth, low Apgar index, IUGR, and neonatal death, but not fetal death.LMT contributed to study design, data analysis, manuscript drafting, and critical discussion. NHL, PLN, and NVQH contributed to data collection, analysis, manuscript drafting, and critical discussion. LDD contributed to data analysis, manuscript drafting, and critical discussion. TQV and CNT contributed to data interpretation, manuscript drafting, and critical discussion. All authors were involved in drafting the work or revising it critically.The authors have no conflicts of interest.


P = .251), internal validation , and external validation  cohorts in diagnosis of high malignancy potential of GISTs. We next focused on the NE‐RS. With 0.185 selected as the cutoff of NE‐RS for diagnosis of the malignancy potential of GISTs, accuracy, sensitivity, and specificity for diagnosis high‐malignancy potential GIST was 90.0%, 88.2%, and 92.3%, respectively, in the training cohort. For the internal validation set, the corresponding metrics are 89.1%, 94.9%, and 80.0%, respectively. The corresponding metrics for the external cohort are 84.6%, 76.1%, and 91.0%, respectively. Compared with only NE‐RS, the radiomics model increased the sensitivity in the diagnosis of GIST with high‐malignancy potential by 5.9% (P = .025), 2.5% (P = .317), 10.5% (P = .008) for the training set, internal validation set, and external validation set, respectively. The NE‐RS had comparable prediction efficiency in the diagnosis of high‐risk GISTs to CE‐RS. The NE‐RS and radiomics model both had excellent accuracy in predicting malignancy potential of GISTs.This work seeks the development and validation of radiomics signatures from nonenhanced computed tomography  to preoperatively predict the malignancy degree of gastrointestinal stromal tumors (GISTs) and the comparison of these signatures with those from contrast‐enhanced CT. A dataset for 370 GIST patients was collected from four centers. This dataset was divided into cohorts for training, as well as internal and external validation. The minimum‐redundancy maximum‐relevance algorithm and the least absolute shrinkage and selection operator (LASSO) algorithm were used to filter unstable features. (a) NE‐RS and radiomics signature from contrast‐enhanced CT (CE‐RS) were built and compared for the prediction of malignancy potential of GIST based on the area under the receiver operating characteristic curve (AUC). (b) The radiomics model was also developed with both the tumor size and NE‐RS. The AUC values were comparable between NE‐RS and CE‐RS in the training  algorithm, and the least absolute shrinkage and selection operator (LASSO) algorithm were used to filter unstable features. The area under the curve (AUC) values were comparable between radiomics signatures from non‐enhanced CT (NE‐RS) and radiomics signatures from enhanced CT (CE‐RS) in the three cohorts in diagnosis of high malignancy potential of GISTs. With 0.185 selected as the cut‐off of NE‐RS for diagnosis of the malignancy potential of GISTs, accuracy for diagnosis high‐malignancy potential GIST was 89%, 85% for the internal validation and external validation cohort. Compared with only NE‐RS, the radiomics model with combination of radiomic signature and tumor size, had increased accuracy of 91%, 89% for the internal validation and external validation cohort. The NE‐RS and radiomics model both had excellent accuracy in predicting malignancy potential of GISTs. Different treatment strategies have been applied to treat GIST, including close follow‐up, submucosal endoscopic dissection, endoscopic full‐thickness resection, and surgery.Radiomics represents an emerging tool for extracting numerous numerical features from medical images and has gained popularity in cancer diagnosis.We recruited 370 patients with GIST from four hospitals according to the following criteria: (a) patients who had surgeries or endoscopic resections; (b) both conventional CT and arterial phase CE‐CT examinations were done within a 15‐day period before treatment; (c) GIST diagnosis was carried out with histological and immunohistochemical tests; and (d) all reported clinical and pathological variables were available. Patients who received or were revived with imatinib preoperatively or those with multiple GIST detections were excluded. Ethical approvals were obtained for all four collaborating hospitals. The training cohort consisted of patients diagnosed consecutively between January 2011 and December 2016 in Renji Hospital. The internal validation cohort contained patients from January 2017 to June 2019 in Renji Hospital. The external validation cohort included patients from three hospitals diagnosed between January 2017 and June 2019. We collected clinical and pathological variables including patient gender, age, tumor location, size, and mitotic count. GISTs were classified according to the National Institutes of Health‐modified criteria into two groups, GISTs with low potential of malignancy and GISTs with high potential of malignancy, based on pathological tumor size, tumor location, and mitotic count.Figure P = .801) and external validation (P = .173), which indicated similar predictive efficacy of malignancy potential in GIST. To our knowledge, two previous studies have developed the radiomics signatures extracted from CE‐CT for predicting GIST with high malignancy potential.Then, we analyzed and compared the NE‐RS and CE‐RS performance in GIST malignancy prediction using the area under the receiver operating characteristic curve (AUC) on the internal and external validation cohorts. As shown in Figure Because nonenhanced CT is more commonly used for preoperative GIST diagnosis and is similar to CE‐CT in prediction efficacy, we focused on the radiomics signature from nonenhanced CT. The optimal threshold of the radiomics signature for GIST malignancy diagnosis was set at the point of the ROC curve with the highest Youden index.Similar to other studies,P = .317) and 10.5% (P = .008) for the cohorts of internal validation and external validation, respectively, in comparison to the case of GIST malignancy prediction with the NE‐RS thresholding. As shown in Table However, evaluation of malignancy only based on tumor size is not sufficient because tumors with mitotic count >5/50 HPF, regardless of tumor size, are considered aggressive with poor prognosis.With the advancement in resection methods, multiple resection methods could be performed for the treatment of GIST, including submucosal endoscopic dissection, full‐thickness endoscopic resection, and surgery. However, the choice of the resection method is based on preoperative evaluation of tumors, including evaluation of tumor size, growth pattern, and malignancy. It is recommended that endoscopic resection with choice of submucosal endoscopic dissection or endoscopic full‐thickness resection based on the clinical decision should be performed for GIST sized <5 cm without high‐malignant potential and that GIST sized >5 cm or with high‐malignant potential should be resected by surgery.In this study, radiomics features were extracted for a single two‐dimensional (2D) slice with the largest tumor area for each patient. No significant differences have been shown in the diagnostic efficacy between the texture analysis obtained from a single‐slice 2D slice versus a three‐dimensional volume.z‐score method was applied to standardize the training features by their respective means and standard deviations. The AUC of NE‐RS did not vary among different hospitals. This demonstrated that our normalization method was reliable. Third, the predictive model did not account for gene mutations. Nevertheless, these variables could not be acquired by preoperative clinical tests and thus were not considered. Further investigations are needed to the explore the relationship of gene mutation and radiomics features.The present work has some limitations. First of all, the dataset collection was done retrospectively. Thus, selective bias could not be eliminated. Nevertheless, patients were consecutively enrolled for bias reduction. Prospective studies are needed for the validation of our radiomics signature and model. Second, data heterogeneity bias resulted from CT parameter variations among the collaborating hospitals. Before the extraction of features, this bias was reduced through CT slice normalization and resampling. The In summary, we have developed and validated the NE‐RS with high performance in the diagnosis of GIST with high‐ and low‐malignancy potential and was comparable to the CE‐RS. Including tumour size and NE‐RS further increased the diagnostic accuracy in predicting GIST with high‐malignancy potential to make a preoperatively more precise clinical decision compared to only NE‐RS used.The study protocol was approved by the Ethics Committee of the Renji Hospital, Zhongshan Hospital, Sir Run Run Shaw Hospital, and First Affiliated Hospital, Wenzhou Medical University. Informed consent was obtained from each patient before performing CE‐CT examination.The datasets generated and/or analyzed during the current study are not publicly available due personal information involved but are available from the corresponding author on reasonable request.The authors declare no conflict of interest.Q‐WZ, JL, J‐RX, Y‐JG, and Z‐ZG conceptualized and designed the study. Q‐WZ, X‐XZ, R‐YZ, S‐LC, YZ, QL, and JW helped with generation, collection, assembly, analysis, and/or interpretation of the data. Q‐WZ, X‐XZ, R‐YZ, and S‐LC drafted and revised the manuscript. Q‐WZ, JL, J‐RX, Y‐JG, and Z‐ZG approved the final version of the manuscript.This work was supported by grants from the National Natural Science Foundation of China (No. 81670505 and 81772519), the Shanghai Municipal Education Commission: Gaofeng Clinical Medicine grant support (No. DLY201501), the 3‐year action plan for Shin Kang of Shanghai (No. 16CR3113B), Cross Medical Research Fund of Translational Medicine, Shanghai Jiao Tong University (No. ZH2018ZDA06), Shanghai Municipal Key Clinical Specialty (No. shslczdzk05902), and Shanghai Municipal Commission of Health and Family Planning (No. 2018YQ29).Supporting InformationClick here for additional data file.


Cervus nippon) is listed as a tonic in many ancient Chinese pharmaceutical. Many adulterants of sika deer products have been found in Chinese medicinal materials markets, which led to detrimental impacts in clinical treatment. However, it is lack of the rapid and effective identification method for sika deer. This study amplified 574 bp fragment of mtDNA COI region of 19 samples from seven Cervidae species, and the relevant five sequences from reindeer (Rangifer tarandus) were downloaded from GenBank. It was found that there were two SNP loci for sika deer. Phylogenetic analysis indicated that individuals from sika deer clustered together. Based on SNP locus, one pair specific primer for allele-specific PCR identification of sika deer was designed, which could be used to rapidly and accurately identify sika deer.Sika deer ( Cervus nippon) and red deer (Cervus elaphus Linnaeus) according to Chinese Pharmacopoeia , which no sequencing, is a simple, rapid, cheap, and reliable method for species identification. Since it was first used to identify black caviar   were downloaded from GenBank. Overall, we analyzed 24 partial sequences of COI region from eight Cervidae species. The phylogenetic analysis showed that individuals of C. nippon, C. elaphus, A. porcinus, D. dama, E. davidianus, E. cephalophus, R. unicolor, and R. tarandus were separately clustered within their corresponding clades, and C. nippon distinctly differs from other seven Cervidae species , 1 pmol of each primer, and approximately120 ng of genomic DNA. The conditions of running PCR were set as the following: initial denaturation at 94 °C for 3 min, 35 cycles of denaturation (94 °C for 30 s), annealing (60 °C for 30 s), extension (72 °C for 30 s), and a final extension at 72 °C for 10 min. For verifying the species specificity of the primers for the identification of sika deer, the same PCR reaction constitutes and conditions were used for the other six species of Cervidae in this study. The result of electrophoresis showed that only the genomic DNA of sika deer could be successfully amplified and produced a single band of about 500 bp , which iThis study designed one pair specific primer of sika deer based on SNP loci of COI sequences. It was proved that the primer had strong specificity and could be used to rapidly and effectively identify the products related to sika deer.


Breast milk is the optimal food for infant growth and development. The purpose of this study was to evaluate the association between breast milk macronutrient composition with dietary pattern among lactating women.A total of 220 lactating women from 2011 to 2012 in Changsha, a city of south-central China, was recruited using a multi-stage sampling method. Breast milk was collected, and the protein, fat, lactose, total dry matter, and energy contents of breast milk were measured. A 24 h recall method on three consecutive days was used to collect the dietary information of lactating women and an exploratory factor analysis was performed was to identify dietary patterns. The association between the concentration of a breast milk component and dietary pattern was assessed using a multivariable linear regression model.Three major dietary patterns were classified. Lactating women with dietary pattern 1 mainly ate fresh vegetables and fresh legumes. Those with dietary pattern 2 mainly ate red meat, cereals and eggs, and those with dietary pattern 3 mainly ate fungi and algae, dries legumes and soy milk. Pattern 2 was positively associated with the concentration of protein , total dry matter  and energy  in breast milk. Morever, lactation period was negatively associated with the protein and total dry matter concentrations and positively associated with lactose.The results show the lactation period was an important factor affecting milk composition and a dietary pattern with high intake of red meat, cereals, and eggs was associated with higher protein, total dry matter, and energy contents in breast milk. These findings show that the dietary patterns of lactating women can affect breast milk macronutrient composition and provide a foundation for improving child health. Breast milk is the optimal food for infant growth and development. According to the World Health Organization, mothers should breastfeed infants exclusively for at least 6 months . InfantsHuman milk contains all the energy, macronutrients, and micronutrients required for infant growth and development during the first 4 to 6 months of life. It also provides various immunological factors and bioactive components , 5. LactMost studies have analysed the association of individual nutrients or foods with the levels of breast milk components. However, dietary intake is a combination of multiple nutrients and foods. Considering the potential for interactions between total dietary intake and foods or nutrients, dietary pattern analysis is widely used to evaluate the impact of diet on health –14. TherBreast milk samples and dietary data were collected from 2011 to 2012 in Changsha, in Hunan Province, south-central China. This city has an integrated maternal and children’s health system that offers many free primary care services in local community health centres where the lactating women were recruited for our study using a multi-stage sampling method  was collected from 8 a.m. to 11 a.m. using an electric breast pump provided by our staff. The protein, fat, lactose, total dry matter, and energy contents of breast milk were measured within 4 h of sampling by Miris HMA, a breast milk analyser . The protein, fat, lactose, and total dry matter contents of breast milk were expressed as g/100 mL, and kcal/100 mL for energy.A 24 h recall method on three consecutive days was used to collect the dietary information of lactating women. Participants were asked to recall their detailed food intake and the estimated portion sizes over the previous three consecutive days using local weight units (1 Liang = 50 g). A total of 246 foods was identified in this study, and these were classified into 23 categories based on the similarity of ingredients and the Chinese Food Composition Table  , delivery mode , education level , occupation (housework and other), and family economic status. Lactation period was subdivided into six groups  according to the frequency distribution of all samples. Based on the per capita annual income of urban households in Hunan in 2013 , the totP <  0.1) of breast milk macronutrient composition in bivariate analyses were included as factors in the multivariate linear regression model. B with 95% CI was calculated to determine the strength of associations. P <  0.05 was considered indicative of statistical significance.The statistical analysis was performed using Statistical Product and Service Solutions 13.0. The data are expressed as numbers and percentages for categorical variables. The mean (SD) was used to express continuous variables with normally distributed data, and the median (IQR) to express skewed data. The concentration of breast milk components was compared according to the characteristics of the lactating women by ANOVA. Correlation analysis with Spearman’s correlation test was used to determine the correlations between lactation period and breast milk composition. Significant differences between the breast milk macronutrient composition concentrations and dietary patterns across four quartiles were assessed by ANOVA. The association between the concentration of a breast milk component and dietary pattern was assessed using a multivariate linear regression model. Significant predictors (n = 4) were excluded, as were those who were unable to provide breast milk samples (n = 10). After exclusion, 220 lactating women were included in the analysis. The median lactation period of the subjects was 46 (IQR 14–163) days, and their mean age was 27 ± 3 years. Table In total, 234 lactating women were invited to take part in this survey. Participants with incomplete dietary data  = 25.80, P <  0.001), fat  = 3.06, P = 0.01), lactose  = 10.81, P <  0.001), total dry matter  = 7.42, P <  0.001), and energy  = 3.49, P = 0.01) contents of breast milk during the lactation period. The highest contents were at seven and fewer days for protein, 8–15 days for fat, total dry matter, and energy, and over 181 days for lactose. Correlation analysis showed that the lactation period was negatively correlated with the contents of protein , total dry matter , and energy  in breast milk, but was positively correlated with the lactose content . Moreover, the lactating women with a college education or above  = 3.14, P = 0.04) and other  = 7.28, P = 0.01) as an occupation were more likely to have a higher protein concentration in breast milk. The lactating women from medium-income families had a higher total dry matter concentration  = 4.45, P = 0.01) and larger amount of energy  = 4.17, P = 0.02) in breast milk.Table Figure 0.05,  = 12.04, P < 0.001) and total dry matter  = 2.88, P = 0.04) in breast milk, while those in pattern 2 had higher concentrations of protein  = 6.09, P < 0.001) and total dry matter  = 3.98, P = 0.01). There were significant differences in the lactose  = 7.32, P < 0.001) level of breast milk among the quartiles of pattern 1; a higher dietary pattern score was associated with a higher lactose concentration. There were no significant differences in the fat and energy levels of breast milk across quartiles of the three patterns.Table Table We report here an association between maternal dietary pattern and breast milk macronutrient composition. The results showed that there are three major dietary patterns among lactating women in south-central China: dietary pattern 1 was mainly based on fresh vegetables and fresh legumes; dietary pattern 2 on red meat, cereals, and eggs; and dietary pattern 3 was based on fungi and algae, dried legumes, and soy milk. Moreover, the protein, total dry matter, and energy concentrations in breast milk were related to the dietary pattern.Macronutrients such as protein, fat, and carbohydrates are the main nutrients in breast milk. In this study, the concentrations in breast milk within 12 months of delivery were 1.37 ± 0.73 g/100 mL for protein, 3.20 ± 1.43 g/100 mL for fat, and 6.51 ± 0.40 g/100 mL for lactose. Yang et al. reported that the concentrations in breast milk within 2 to 4 months of delivery in eastern China were 0.8 ± 0.1 g/100 mL for protein, 3.1 ± 1.4 g/100 mL for fat, and 7.2 ± 0.3 g/100 mL for lactose  were 1.5In addition, the concentrations of protein, fat, and lactose differed significantly with the stage of lactation in bivariate analyses in our study. The lactation stage is the factor that affects milk composition most strongly; this has been shown in several studies , 20. In Many recent studies have reported associations between the maternal diet and milk macronutrient composition. Some studies reported a positive effect of maternal diet on breast milk composition, while others showed the opposite . In our The total dry matter content of breast milk is important for promoting infant growth and development. Wu et al. reported that the higher the total dry matter content of breast milk, the greater the height of the infant . HoweverFat is a major source of energy in human milk. Several studies found positive associations between dietary fatty acid intake and the fatty acid concentration in breast milk , 21, 26,Previous studies have shown that economic status is strongly associated with maternal diet, where family income influences the variety and quantity of food intake. Li et al. reported that family economic status was related to fruit intake in mothers , while WThis study had several highlights and limitations. First, to our knowledge, this is the first study of the macronutrient composition of breast milk of lactating women and its association with dietary pattern in south-central China, so the findings provide a valuable basis for improving maternal nutrition and infant feeding in this region. Second, we performed a principal component analysis to analyse dietary patterns, taking into account overall food intake and food interactions. Nevertheless, this study also had shortcomings. First, we used the 24 h recall method to collect dietary information, which may be subject to recall bias. Second, a small number of lactating women was found in the local communities and some were reluctant to enrol in the study. In total, only 220 lactating women were recruited. The sample size is too small to represent the general population, which may limit the generalizability of our findings. Third, we analysed only the association between macronutrients in breast milk and the dietary pattern. While micronutrients such as vitamins and minerals are also important components of breast milk, their contents may be affected by dietary intake –35, so tThree major dietary patterns were identified among lactating women in south-central China. Lactation period was an important factor affecting milk composition and a dietary pattern with high intake of red meat, cereals, and eggs was associated with higher protein, total dry matter, and energy contents in breast milk. These findings show that the dietary patterns of lactating women can affect breast milk macronutrient composition and provide a foundation for improving child health.Additional file 1: Table S1. Food grouping used in the dietary pattern analyses.Additional file 2. Lactating women dietary questionnaire.


Insight in the pathogenesis of intestinal fibrosis is an unmet medical need in inflammatory bowel diseases. Studies in murine models and human organ fibrosis point to a potential role of innate lymphoid cells (ILC) in chronic intestinal inflammation and fibrosis.−/− common γ chain deficient and anti‐CD90.2 monoclonal antibody treated RAG−/− mice. Inflammation was scored by macroscopic and histological examination and fibrosis was evaluated by hydroxyproline quantification and histology.Dextran sodium sulfate (DSS) in drinking water was used to induce chronic colitis and remodeling in C57Bl/6 wild type (WT), RAG‐deficient, RAG−/− mice , chronic intestinal inflammation and fibrosis developed similarly as in WT mice, with a relative increase in ILC2 during repeated DSS exposure. Chronic colitis could also be induced in the absence of ILC (RAG−/−γc−/− or anti‐CD90.2 treated RAG−/− mice) with no attenuation of fibrosis. Importantly, clinical recovery based on weight gain after stopping DSS exposure was impaired in ILC‐deficient or ILC‐depleted mice.In RAGThese data argue against a profibrotic effect of ILC in chronic colitis, but rather suggest that ILC have a protective and recovery‐enhancing effect after repeated intestinal injury. −/− mice we observed an enrichment of interleukin (IL)‐13 producing ILC2 with increasing numbers of dextran sodium sulfate (DSS) administration. However, upon antibody‐mediated depletion of all ILC, no attenuation of fibrosis could be observed. These results were validated in ILC deficient mice. However, in these mice we found that in absence of ILC, recovery after withdrawal of DSS is impaired and associated with a higher mortality.Identification of innate lymphoid cells (ILC) as mucosal, resident, cytokine‐producing cells, and players in liver and lung fibrosis, led us to the study of these cells in our murine model of chronic intestinal inflammation and fibrosis. In both wild type (WT) and RAG1 Throughout their disease course, one‐third of CD patients and 5% of UC patients will develop a fibrostenotic phenotype (strictures and bowel obstructions) due to excessive extracellular matrix deposition and muscularis propria hyperplasia in the bowel wall.Over the last few decades, most studies on IBD have focused on the identification of abnormal adaptive immune responses. More recently, the focus has shifted toward mucosal innate immune responses. Recent data suggested a causal link between defects in the resolution of intestinal inflammation and impaired bacterial clearance, excessive cytokine secretion, altered monocyte‐macrophage transition, and activation of tissue innate immune cells.Innate lymphoid cells (ILC) were discovered as an additional source of mucosal cytokines.ILC residing in the gut mucosa are important regulators of homeostasis and have the potential to control tissue remodeling.−/−) deficient mice , in anti‐CD90.2 injected RAG−/− mice and in RAG−/− common γ chain−/− (RAG−/−γc−/−) mice which both also lack the ILC population. This enabled us to separately analyse whether adaptive immunity and/or ILC are involved in colitis and fibrosis induction.Previously, we and others have shown that chronic dextran sodium sulfate (DSS) colitis induced by repeated cycles of DSS exposure is characterized by transmural inflammation and granuloma formation and followed by remodeling and fibrosis.22.1−/−tm/tm mice, lacking T and B cells, were obtained from Charles River (MA) (mice in figure 1 and 2) or were bred in our own animal facility (figures 3‐8).tm1Fwa Il2rgtm1Wjl (RAGγc−/−) mice were obtained from Taconic (New York). All animals were maintained in the Animal Care Facility of the Faculty of Medicine, University of Leuven (Belgium) and according to ARRIVE guideline. Colitis was induced with DSS as previously described.To induce DSS colitis, female 6‐week‐old C57BL/6OlaH WT mice were obtained from Envigo . RAG12.2Animals were euthanized with sodium pentobarbital  and Disease Activity Index (DAI) was determined based on body weight loss, stool consistency, and presence of gross blood in the stools. Next, inflammation in distal colon was evaluated as previously described.2.3YTS154 , to deplete CD90.2 expressing ILC, was produced at Bioceros and was used as previously described.2.42+/Mg2+ (Lonza) supplemented with 3 mg/mL dispase , 1 mg/mL collagenase D (Worthington), 0.2% DNase I , and 2% HEPES (Thermo Fisher Scientific). Lamina propria lymphocytes were filtered through a 70 µm cell strainer  and incubated for 30 minutes in fixable viability dye 780  at room temperature before staining with antibodies listed in Table S1 and Figure S1 to S3. ILC were gated as CD45+Lineage−  CD127+CD90.2+ cells. Within the ILC, ILC3 were defined as RORyt+, ILC2 were defined as Killer cell lectin‐like receptor subfamily G member (KLRG‐1)+RORyt−/dim and ILC1 were defined as KLRG‐1−RORyt−NK1.1+ cells. Myeloid cell types were defined based on the following markers: neutrophils (CD45+CD11b+Ly6G+), eosinophils (CD45+CD11b+ Siglec‐8+), mast cells (CD45+CD11b−CD117+Fcεr+), macrophages (CD45+CD11b+CD64+MHCII+), and monocytes (CD45+CD11b+Ly6C+). Intracellular staining of RORγt was performed according to manufacturing instructions of the Foxp3 staining kit (Invitrogen). For intracellular cytokine detection, cells were stimulated with phorbol myristate acetate 100 ng/mL and ionomycin 1 µg/mL (Sigma‐Aldrich) for 6 hours and monensin 0.7 µg/mL (Sigma‐Aldrich) for 4 hours followed by anti‐IL13 staining. After staining, cells were fixated in 1% paraformaldehyde.Distal colon sections were cut longitudinally and kept in complete medium  supplemented with 10% FBS  and 1% antibiotic‐antimyotic 100× (Thermo Fisher Scientific). To remove mucus and epithelial cells, samples were incubated twice under magnetic stirring for 20 minutes at 37°C in complete medium supplemented with 5 mM dithiothreitol  and 5 mM EDTA (Lonza). The epithelial fraction was discarded, and remaining tissue was digested for 40 minutes at 37°C under magnetic stirring in Hank's balanced salt solution with CaFlow cytometry was conducted on a BD LSR Fortessa instrument in accordance with standard methods. Calibration was performed before each acquisition by CS&T beads (BD). For fluorescence compensation settings, single color UltraComp eBeads compensation beads were used (Thermo Fisher Scientific). Fluorescence Minus One controls were included.2.5A 0.5 cm piece of affected distal colon was opened longitudinal and incubated for 24 hours at 37°C in complete medium before supernatant collection. Protein concentrations in the supernatants for interleukin 4 (IL‐4), IL‐5, IL‐6, IL‐12p70, IL‐22, IL‐23p19, TNF, and IFN‐γ were determined using multiplex meso scale discovery technologies. In short, samples were incubated overnight at 4°C on a U‐plex plate coated with biotinylated capture antibodies. After three wash steps in PBS 0.05% tween (Sigma‐Aldrich), detection antibodies were added for 1 hour at room temperature before activation of the plate electrodes resulting in a quantitative measurable emission of light.2.6P values for two group comparison were obtained using Mann‐Whitney U testing. The following methods were used: multiple comparison with Dunn's correction between multiple groups, Spearman for correlation testing, and Kaplan‐Meier curve for survival analysis. Differences were considered statistically significant at *P < .05, **P ≤ .01, and ***P ≤ .001.Statistical analysis were performed using GraphPad Prism 8 . Data are represented as medians (interquartile range) and the individual 2.7All studies were approved by and performed according to the local ethics committee for animal experimentation of the University of Leuven (P230‐2015).33.1−/− mice lacking B and T lymphocytes.−/− mice than in WT mice . Colon length decreased in both strains after 1 week of DSS exposure (WT: P = .006 and RAG‐1−/−: P = .001), but significantly more in RAG‐1−/− mice compared with WT mice  and monocytes (28.40% vs 8.16% of CD11b+ cells P = .004) were increased in WT mice after three cycles of DSS . In contrast, decreased proportions of eosinophils  and macrophages  were observed after three cycles of DSS in WT mice . Similarly, in RAG‐1−/− mice with chronic DSS colitis, neutrophils  and monocytes  were increased as compared with RAG‐1−/− mice without DSS exposure, while no significant effect was seen on eosinophils and macrophages .We also analysed the composition of inflammatory cells in the colonic mucosa. Gating of the different myeloid cell populations is illustrated in Figure S1. When compared with controls without DSS exposure, neutrophils  or after three cycles of DSS administration (chronic model), systemic and colonic inflammation were similar or slightly more severe in RAG‐13.2−/− mice as shown by MSB collagen quantification  and hydroxyproline measurements .To study whether ILC are involved in intestinal fibrosis in this model we first studied the distribution of mucosal ILC subsets during acute or repetitive DSS administration in WT mice. The gating strategy for ILC and the identification of subsets based on membrane markers and intracellular RORγt expression are shown in Figure S3. No changes were observed in the total ILC proportion in acute colitis or after two and three cycles of DSS exposure Figure . The per−/− mice, ILC represent 27.55% of CD45+ leukocytes in the colonic lamina propria. After two and three cycles of DSS, the increase in KLRG‐1+ ILC2 was confirmed in RAG‐1−/− mice as compared with RAG‐1−/− mice without DSS administration  suggesting a possible relation between ILC2 and fibrosis  depleting monoclonal antibody (mAB). To exclude effects of antibody treatment on CD90.2 expressing epithelial cells and fibroblasts, control RAG‐1−/− mice (not exposed to DSS) were injected at a similar dosing schedule.To further investigate the contribution of ILC to colitis and fibrosis, ILC were depleted in the chronic DSS model in RAG‐1+ cells, P = .002, P = .029, and P = .018, respectively; Figure +, P = .015, P = .006, and P = .010; Figure Efficacy of ILC depletion by anti‐CD90.2 (YTS) injection was confirmed by a reduction of ILC in the colon as compared with control mice and saline or isotype injected chronic DSS colitis mice  and eosinophils  after chronic DSS as compared with mice without DSS exposure  and IL‐22  and a tendency toward lower production of IFN‐γ , as compared with DSS exposed control groups , while a lower colon weight was observed in RAG−/−γc−/− mice compared with RAG−/− mice , no differences in colon weight and colon W/L ratio were observed  and monocytes  was more pronounced in RAG−/−γc−/− mice as compared with RAG‐1−/− mice , and as there was decreased production of IL‐5 and IL‐22 in the colonic mucosa of anti‐CD90 injected mice. As a relative shift from ILC3 predominance toward ILC2 predominance in the colon was observed both in WT and RAG−/− mice. RAG‐1−/− mice have a relative high proportion of ILC among CD45+ cells. To confirm the findings we also used a mouse strain lacking both ILC and adaptive immunity. Importantly, in both models no attenuation of fibrosis was observed neither after depletion of ILC nor in the absence of ILC, thus indicating that their role in fibrogenesis is negligible. However, in the absence of ILC a slower resolution of inflammation highlighted by a slower recovery of weight and higher mortality was observed rather pointing to a protective role of ILC.To know whether ILC contribute to tissue damage, fibrosis, and/or recovery in colitis, we then used a mAb to deplete ILC in RAG‐1Salmonella‐induced infection models of lung and colon fibrosis are prone to develop a type 2 immune response that can contribute to or be responsible for fibrosis, while DSS colitis is marked by a type I response.In contrast to murine models of lung and liver fibrosis, in our model depletion of ILC thus did not result in an attenuation of fibrosis.−/−γc−/− mice, suggesting a protective role for ILC. Protective effects of ILC via IL‐22 production have been shown in the recovery phase of acute intestinal injury.−/−γc−/− mice.Interestingly, slower recovery of weight after repeated inflammatory insults by DSS exposure was observed in RAGWe also evaluated expansion of neutrophils, eosinophils, and monocytes in chronic DSS colitis in the different strains. All three cell types were indeed expanded in each of the strains and also after YTS treatment. These inflammatory cells might contribute and/or be essential for the induction of chronic intestinal remodeling in an ILC‐independent innate immune process. Upon intestinal injury, neutrophils are the first cells recruited into the intestinal lining as a first line of response.−/− but not in RAG−/−γc−/− mice after three cycles of DSS can be explained by the absence of the common γc in the latter strain. Carlens et al−/−γc−/− mice, together with a reduction in CCL11 (eotaxin‐1). Eosinophils can also be drivers of inflammatory damage and potentially contribute to tissue repair and remodeling in IBD.The observed increase of eosinophils in RAG‐1−/− mice showed an exacerbation of acute DSS colitis due to decreased levels of KC and MIP‐2 and an impaired neutrophil resolution of damage, while exogenous IL‐33 administration attenuated chronic DSS inflammationAs administration of DSS leads to a disruption of the epithelial barrier and release of mature IL‐33, elevated levels of IL‐33 can be a potential driver of inflammation and remodeling in our model. However, IL‐33 plays a dual role as IL‐33In conclusion, our results show that DSS‐induced chronic murine colitis and fibrosis can develop independently of T cells, B cells, and ILC, pointing to an innate immune response mediated by neutrophils, macrophages, and/or eosinophils, as the drivers of inflammation and fibrosis. Our results do not exclude a contributing/modulatory role of ILC, especially of ILC2, but ILC2 are clearly not essential to the process of experimental intestinal fibrogenesis. On the other hand, ILC3 and their IL‐22 production might be protective against epithelial damage. We emphasize that these results were obtained in a particular murine model, which does not necessarily reflect the human situation. However, also in human IBD more data now support a primary role of myeloid cells.Marc Ferrante received financial support for research from Amgen, Biogen, Pfizer, Takeda, and Janssen; lecture fees: Abbvie, Amgen, Biogen, Boehringer‐Ingelheim, Falk, Ferring, Janssen, Lamepro, MSD, Mylan, Pfizer, and Takeda; consultancy fees: Abbvie, Boehringer‐Ingelheim, Janssen, MSD, Pfizer, Sandoz, and Takeda. Séverine Vermeire reports financial support for research: MSD, AbbVie, Takeda, Pfizer, and J&J; Lecture fees: MSD, AbbVie, Takeda, Ferring, Centocor, Hospira, Pfizer, J&J, and Genentech/Roche; Consultancy: MSD, AbbVie, Takeda, Ferring, Centocor, Hospira, Pfizer, J&J, Genentech/Roche, Celgene, Mundipharma, Celltrion, SecondGenome, Prometheus, Shire, Prodigest, Gilead, and Galapagos. Gert Van Assche reports financial support for research from Abbott and Ferring Pharmaceuticals; lecture fees from Janssen, MSD, and Abbott; consultancy fees from PDL BioPharma, UCB Pharma, Sanofi‐Aventis, Abbott, Abbvie, Ferring, Novartis, Biogen Idec, Janssen Biologics, NovoNordisk, Zealand Pharma A/S, Millennium/Takeda, Shire, Novartis, and Bristol Mayer Squibb. Christine Breynaert reports consultancy fees from Ablynx.All authors made substantial contributions to the submitted work. BC contributed to the study concept and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript, and statistical analysis. JC: acquisition data and interpretation of data. GDH: acquisition data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content. LB: material support. GvA: study concept and design, interpretation of data, and critical revision of the manuscript for important intellectual content. MF and SV: interpretation of data, and critical revision of the manuscript for important intellectual content. JLC: study concept and design, interpretation of data, and critical revision of the manuscript for important intellectual content. CB: study concept and design, analysis and interpretation of data, material support, drafting of the manuscript, and study supervision. All authors agreed with the final version of the manuscript. CB guarantor of the manuscript.Supporting informationClick here for additional data file.


Wolbachia and Spiroplasma are intracellular bacteria that are of great interest to entomologists, because of their ability to alter insect host biology in multiple ways. In the spider mite Tetranychus truncatus, co‐infection of Wolbachia and Spiroplasma can induce cytoplasmic incompatibility (CI) and fitness costs; however, little is known about the effect of co‐infection at the genetic level and the molecular mechanisms underlying CI. In this study, we explored the influence of the two symbionts on male mite host fitness and used RNA sequencing to generate the transcriptomes of T. truncatus with four different types of infection. In total, we found symbiont‐infected lines had a higher hatch proportion than the uninfected line, and the development time of the uninfected line was longer than that of the other lines. Co‐infection changed the expression of many genes related to digestion detoxification, reproduction, immunity and oxidation reduction. Our results indicate that co‐infection of Wolbachia and Spiroplasma confers multiple effects on their hosts, and helps illuminate the complex interactions between endosymbionts and arthropods. Wolbachia is an intracellular bacterium which infects a large number of terrestrial arthropods  on male mite host fitness and compared gene expression profiles among four types of males from different symbiont‐infected lines, including doubly infected (Iws), singly Wolbachia‐infected (Iw), singly Spiroplasma‐infected (Is) and uninfected (U) lines. We found that symbiont‐infected lines had a higher hatch proportion than the uninfected line, and the development time of the uninfected line was longer relative to the other lines. Co‐infection changed the expression of many genes, including genes related to digestion detoxification, reproduction, immunity and oxidation reduction. Our results indicate that co‐infection of Wolbachia and Spiroplasma confers multiple effects on their hosts and helps elucidate the complex interactions between endosymbionts and arthropods.Here, we explored the effects of the two symbionts  at 25 ±1 °C, 60% relative humidity and under 16 : 8 L : D conditions. Three lines, Iws, Iw and Is, were established as previously described .The effect of symbionts on male mite hatchability was assessed. For each line, a single 1‐day‐old adult virgin female was placed on a leaf disk and allowed to oviposit for 5 days. Each line used 36 leaf disks. To calculate hatch proportion, the eggs on the leaf disks were checked daily. Data were first tested for normality (Kolmogorov–Smirnov test) and homogeneity of group variances (Levene's test). When the data were normally distributed, they were analyzed with a one‐way analysis of variance with post‐hoc Tukey Honestly Significant Difference analysis for multiple comparisons. When the data did not follow a normal distribution, Kruskal–Wallis test with Bonferroni correction were used for multiple comparisons. All statistical analyses were done on SPSS 19.0 .To test whether symbiont infection influenced normal male development, we measured development time. For each line, approximately 150 3‐day‐old adult virgin females were placed on a leaf disk and allowed to oviposit for 3 h . Then, the females were removed and eggs were left. The eggs on the leaf disks were checked at 8:00, 16:00, and 22:00 hours every day to determine whether males underwent eclosion. Observed adult males were picked out and the number of adult males recorded. Development time was considered the time which from an egg was laid to adulthood. The differences of development time were analyzed using the Kruskal–Wallis test with Bonferroni correction. All statistical analyses were done on SPSS 19.0.One‐day‐old adult virgin males of the four lines were then collected. Samples were stored in liquid nitrogen for subsequent RNA isolation.Total RNA was extracted with the Trizol protocol according to manufacturer's instructions  for a total of 12 independent samples with three biological replicates per treatment. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system . Libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina®  following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. The complementary DNA (cDNA) library was then sequenced using Illumina Hiseq X Ten , and paired‐end reads were generated.Raw data (raw reads) in fastq format were first processed through in‐house perl scripts. In this step, clean data (clean reads) were obtained by removing reads that contained adapters, reads that contained poly‐N, and low‐quality reads from raw data. Simultaneously, Q20, Q30, GC‐content and sequence duplication levels of the clean data were calculated. All downstream analyses were conducted with high‐quality clean data.T. truncatus, a de novo transcriptome was built with Trinity v2.4.0 . KOBAS  of each primer, 7.2 μL ddH2O, 10 μL SYBR Green and 2 μL diluted cDNA. The concentration of cDNA was too high for qPCR, so we diluted cDNA 5‐fold by adding ddH2O in order to get a viable cycle threshold (Ct) value. The relative expression levels were calculated using the 2−ΔΔCt method  analysis, the expression levels of arbitrarily selected genes were measured by quantitative real‐time PCR (qPCR). The primer sequences are summarized in Table Wolbachia and Spiroplasma on host hatchability were measured. As shown in Figure P < 0.001), singly Wolbachia‐infected males (Iw: 0.9384 ± 0.0249) (P < 0.001) and singly Spiroplasma‐infected males (Is: 0.9227 ± 0.0246) (P < 0.05). There was no significant difference between hatch proportions of symbiont‐infected males.The effects of Wolbachia‐infected males (9.89 ± 0.043) (P < 0.05). Additionally, uninfected males (10.13 ± 0.046) developed significantly slower than males of other lines (Is: 9.80 ± 0.034) (P < 0.001).The development times of males from different lines are presented in Figure T. truncatus reference genome, all unigenes were annotated from the Nr (33827), Nt (11648), KO (21307), SwissProt (34170), PFAM (36542), GO (36868) and KOG (24304) databases  were constructed, and each contained 50–54 million reads. A total of 58 015 unigenes were generated from 193 528 396 bp, with a median sequence length of 2408 bp and an N50 of 5468 bp. Over 32 000 unigenes were larger than 2 kb in size  categories of amide biosynthetic process and ribosome biogenesis Fig. . In Iws In Iws versus U, KEGG pathway analysis showed that DEGs were involved in 277 different pathways, mainly including those related to ribosomes and glycolysis/gluconeogenesis Fig. . In Iws Wolbachia and Spiroplasma changed the expression of many more genes compared with other types of symbiont infection , glutathione S‐transferase, ABC transporter, carboxylesterase and multidrug resistance‐associated protein. Co‐infection of the two symbionts had different degrees of influence on the expression of these genes in a host. For CYP genes, six genes significantly changed in Iws, including four downregulated and two upregulated genes. Four genes that encode glutathione‐S‐transferase were downregulated . Additionally, for two genes that encode ABC transporters, one gene was upregulated and one gene was downregulated. Moreover, one carboxylesterase gene was downregulated. Many genes that encode multidrug resistance‐associated protein dramatically changed, including five downregulated and six upregulated genes. Several other genes were also downregulated, including those that encode phosphatase, canalicular multispecific organic anion transporter and P‐glycoprotein.The polyphagy of Cluster‐1760.0, Cluster‐6764.36412, Cluster‐11188.0, Cluster‐6764.8724, Cluster‐6764.43656); four of five of these genes were downregulated. We also observed downregulation of three genes related to vitellogenin. Two genes, Cluster‐6764.21118 and Cluster‐6764.22958, which participate in cuticle biosynthesis, were downregulated. Moreover, it is noteworthy that the genes that encode DNA topoisomerase 2‐alpha, which are essential for proper segregation of daughter chromosomes during mitosis and meiosis, were all substantially upregulated.Lipocalins are a family of proteins which transport small hydrophobic molecules. We found that the majority of genes which encode apolipoprotein D were downregulated and only one was upregulated. Several genes associated with larval molt were upregulated. There were several genes related to histone coding and histone modification . GAK is required for proper mitotic progression because GAK knockdown was shown to cause cell cycle arrest at metaphase (Shimizu et al., Wolbachia and Spiroplasma produced more eggs, and the development time of doubly infected males was relatively short compared with that of males infected with only Wolbachia, and only the Iws line induced CI. Further research is needed to determine the effect of co‐infection on the expressions of the above genes in T. truncatus.In males, development time was found to be is inversely correlated with the strength of CI (Yamada Some of the DEGs in the present study that are associated with the immune response were associated with co‐infection. These include the genes for the nuclear factor‐κB‐like transcription factor Relish, a Janus‐activated kinase/signal transducers and activators of transcription pathway ortholog of Dm stat92E, Toll‐like receptor Tollo and pelle. Moreover, a few genes related to autophagy and apoptosis also dramatically changed Table . We coulFurthermore, we observed numerous DEGs related to oxidoreductase activity due to co‐infection, such as the genes for dual oxidase, short‐chain dehydrogenase and peroxidase, which are important to maintain redox homeostasis.Wolbachia and Spiroplasma co‐infection affected transcripts that encode proteins involved in various biological processes in T. truncatus, such as digestion and detoxification, reproduction, the immune system and redox processes.In summary, we demonstrated that symbiont infection could increase male mite hatchability and accelerate the development from egg to adult. We found that Wolbachia and Spiroplasma co‐infection on arthropod hosts, and helps elucidate the complex interactions between symbionts and arthropods.Our study provides novel gene‐based research on the effects of The authors declare no conflict of interest.Table S1. Primers used for quantitative real‐time polymerase chain reaction of differentially expressed genes.Click here for additional data file.Table S2. Genes that were differentially expressed in response to symbiont infection in Tetranychus truncatus males.Click here for additional data file.


Auditory cortex is required for discriminative fear conditioning beyond the classical amygdala microcircuit, but its precise role is unknown. It has previously been suggested that Heschl's gyrus, which includes primary auditory cortex (A1), but also other auditory areas, encodes threat predictions during presentation of conditioned stimuli (CS) consisting of monophones, or frequency sweeps. The latter resemble natural prosody and contain discriminative spectro‐temporal information. Here, we use functional magnetic resonance imaging (fMRI) in humans to address CS encoding in A1 for stimuli that contain only spectral but no temporal discriminative information. Two musical chords (complex) or two monophone tones (simple) were presented in a signaled reinforcement context (reinforced CS+ and nonreinforced CS−), or in a different context without reinforcement , with an incidental sound detection task. CS/US association encoding was quantified by the increased discriminability of BOLD patterns evoked by CS+/CS−, compared to NS pairs with similar physical stimulus differences and task demands. A1 was defined on a single‐participant level and based on individual anatomy. We find that in A1, discriminability of CS+/CS− was higher than for NS1/NS2. This representation of unconditioned stimulus (US) prediction was of comparable magnitude for both types of sounds. We did not observe such encoding outside A1. Different from frequency sweeps investigated previously, musical chords did not share representations of US prediction with monophone sounds. To summarize, our findings suggest decodable representation of US predictions in A1, for various types of CS, including musical chords that contain no temporal discriminative information. As a result, the conditioned stimulus (CS) is predictive for the US and elicits a conditioned fear response. In nonhuman animals, there is a body of evidence that this type of learning requires synaptic plasticity in at least two amygdala nuclei , a wealth of evidence indicates plastic reconfiguration of ACX  during discriminative fear conditioning in humans to demonstrate encoding of US prediction, before US onset, in Heschl's gyrus  and a correlation between measurements in the two contexts of r = .25. Thus, to detect an effect of context on stimulus discriminability with 95% power and at an alpha level of .05 in a repeated‐measures ANOVA, a sample size of N = 20 was required.From the general and student population, we recruited 20 healthy, right‐handed participants  for the fMRI experiment. The fMRI experiment was preceded by a behavioral control experiment to ensure that the musical sounds were associable with an aversive US to the same extent as the monophone sounds. To this end, we recruited 22 different participants . The size of the fMRI sample was based on a power analysis using Gpower . The experiment and the form of taking written informed consent were in accordance with the Declaration of Helsinki and approved by the governmental ethics committee (KEK‐ZH 2013‐0258). Both experiments used a 3‐way factorial design with the within‐subjects factors stimulus , complexity  and context .2.2Sounds were 4 s long and either monophone sine sounds (simple) or the combination of three simultaneous monophone sine sounds . Participants in the fMRI experiments reported US habituation (excluding one subject due to loss of the electrode at the end of measurement), t18 = 4.83, p < .001. In the behavioral control experiment, five participants did not complete the re‐evaluation. The remaining 17 participants showed no habituation or sensitization to the US, t16 = 1.00, p = .33.US were applied for 500 ms and consisted of 3 square electric pulses to the forearm of 200 μs duration and 100 ms onset asynchrony . Stimulation current was determined in two phases: (1) staircase testing phase, to determine the maximum current tolerated by the participant; (2) random testing phase with 14 US intensities that were rated on a scale from 0 (not perceived) to 100 (clearly painful). Phase (2) was repeated immediately after the experiment. Final US current (mean ± SD: 7.40 ± 1.38 mA) was clearly uncomfortable but not painful  presented via headphones outside the MRI and using sounds similar to the ones used during fMRI. After a short training of five repetitions, participants were asked to press the correct button in at least 15 out of 20 repetitions.Next, in a training session without reinforcement, simple tones and their corresponding complex tones were introduced. Each sound was played twice and accompanied by a visual description, together with the instruction which button to press for each sound. The same sounds served as CS or NS during the subsequent acquisition phase.In the main task, CS+ was only reinforced in a reinforcement context, signaled by background color. In the nonreinforcement context, different sounds were presented. We instructed participants that US would never occur in the nonreinforcement context, while in reinforcement context, US probability depended on the type of CS but not on their behavior.The experiment was broken up into 8 blocks with average duration of 332 s, separated by short breaks. Each block of 24 trials took place in either reinforcement or nonreinforcement context, in alternating order Figure b. The fiF1, 3,795 = 5.79; p = .016). They also responded more accurately to simple (96.86% ± 4.63%) than to complex stimuli . Context had no effect on accuracy, and there were no interactions. In a linear mixed‐effects model with Satterthwaite approximation to degrees of freedom  and shorter for CS− and response‐matched NS than CS+ and response‐matched NS sounds . This was particularly pronounced in the reinforcement context . The fact that responses were also slower for CS+ response matched NS than CS− response matched NS may indicate some level of fear generalization via the associated motor response. Also, the CS+/CS− difference was more pronounced for simple sounds .Participants were instructed to indicate the identity of each sound with a key press, as trained beforehand. They received feedback (change in fixation cross color) at the end of the CS/NS if they pressed a wrong button or did not respond within 3 s of CS/NS onset. In the fMRI experiment, accuracy (hit rate) averaged per participant was between 87% and 99%. As a manipulation check, accuracy per condition was analyzed in a generalized linear mixed effects model with Satterthwaite approximation to degrees of freedom Luke, . This reom Luke, , reactio2.4We recorded skin conductance on left thenar/hypothenar  as described previously  whole‐body MRI scanner. We acquired two high‐resolution T1‐weighted scans . Functional images were acquired using a 1.5 mm isotropic T2*‐weighted echo‐planar (EPI) sequence  with partial brain coverage including the entire auditory cortex. Field of view was centered on ACX. We had no specific hypotheses about CS representation in the amygdala, and due to a technical oversight, slice tilt/phase‐encoding gradient polarity was not optimized to reduce susceptibility artifacts in this region. This is why our analysis focuses on A1, the primary region‐of‐interest.2.6We preprocessed MR images with the default procedures in SPM12 . Using a B0 scan for each participant, the SPM12 FieldMap toolbox, and the realign & unwarp procedure, we corrected all BOLD images for static distortions due to field inhomogeneities, for changes in these distortions due to head motion, and for head motion itself , we constructed a design matrix that contained one regressor per trial. To this end, a stick function for each event was convolved with the default canonical hemodynamic response function. This procedure appropriately estimates the trial‐by‐trial BOLD amplitude at the given inter‐trial‐interval  to estimate deformation parameters that map MNI space to each participant's native space.2.7http://surfer.nmr.mgh.harvard.edu/; Dale, Fischl, & Sereno, Automated parcellation of cortical structures on T1‐weighted images was performed in native space using the “recon‐all” pipeline in FreeSurfer Version 6.0  with a support vector machine  following a threefold cross‐validation scheme established in our previous work  per complexity condition and tested it on the remaining 12 CS , for each of the three folds. Because CS representations presumably change over time during learning, we did so by creating a running index over each individual stimulus and including every third stimulus into the test data set such that both training and test data were drawn from the entire duration of experiment. Because of the different numbers of CS− and unreinforced CS+ in the reinforced context, a binomial statistical test is not appropriate. We estimated the SVM performance under the null hypothesis of label exchangeability. To this end, we randomly permuted stimulus labels 1,000 times and repeated the classification procedure . For cross‐prediction, we used all simple CS or complex CS as training data set, and all CS from the other category as test data set. We did the same procedure for NS, where NS identities were matched between simple and complex by the required key press, to rule out that CS cross‐prediction is due to motor responses.Our main outcome measures are the difference in information content between two conditions, and cross‐prediction performance. The true value for both of these values can be above or below zero . Individual results were mapped to MNI spaces and analyzed with one‐sample t‐tests across the group.2.9http://pspm.sourceforge.net/; Bach et al., z‐scored across all trials  using linear mixed‐effects models . The random field theory‐based approach appropriately controls the false positive rate when used with these p values .Mass‐univariate results and searchlight‐results were performed in SPM. Group‐space images were smoothed with an 8 mm FWHM Gaussian kernel. For mass‐univariate analysis, we examined the planned contrasts (CS+ > CS−) > (NS1 > NS2) [analogous to the main effect of context in MVPA], (CS+ > CS−) > (NS1 > NS2) complex, (CS+ > CS−) > (NS1 > NS2) simple, (CS+ > CS−), (CS+ > CS−) complex, (CS+ > CS−) simple, (NS1 > NS2), (NS1 > NS2) complex, and (NS1 > NS2) simple. As a plausibility check, we furthermore report the contrast CS versus baseline and US/CS+ versus no US/CS+. We corrected for family‐wise error with a random field theory approach, a voxel‐inclusion threshold of 33.1t21 = 3.8, p < .001) and for complex sounds  in the behavioral sample, as well as in the fMRI sample . Furthermore, in both experiments, stimulus‐associated arousal was overall stronger in the reinforcement context than in the nonreinforcement context (main effect context) and for complex than for simple sounds (main effect complexity). In the fMRI experiment, arousal during complex sound presentation was higher than for simple sounds only in the reinforcement context (interaction context × complexity).Fear learning occurred in the reinforcement context (CS × context interaction) in both experiments Figure , Table 23.2p > .05 FWE‐corrected on cluster level and peak level across the whole brain, consistent with previous work  in A1 Figure , Table 3To compare this result with our previous work, we examined whether these results were specific to A1. We had previously investigated CS discriminability within a probabilistic definition of Heschl's gyrus . Thus, there was no evidence for stimulus‐invariant encoding with the specific types of sounds used here. As we had previously found such stimulus‐invariant encoding particularly in the left hemisphere, we repeated this analysis for the left hemisphere only, but did not find a significant cross‐classification . In the right hemisphere, there was no significant cross‐classification either .To examine whether CS/US association was represented in a similar way for both types of sounds, we used cross‐classification and investigated whether CS+/CS− patterns derived from simple sounds also predicted complex CS+/CS− identity, and vice versa. Because initiation of motor responses may influence this result, we compared these values against cross‐prediction of NS, matched for the motor response, as in our previous work. Different from our expectation derived from or previous experiment with frequency sweeps, we did not find that similar patterns distinguished CS+ and CS− for the two types of sounds: Simple CS+/CS− separation was not predicted from complex CS+/CS− separation or the other way around (main effect context: 3.4Finally, we investigated CS discriminability in the superior temporal gyrus excluding A1, using searchlight analysis. This revealed no clusters with a significant effect of context or context × complexity interaction. In a previous study, encoding of the CS/US association was found in a small cluster outside A1. Using this cluster as region of interest, there was no impact of context on classification performance, that is, no evidence for encoding of the CS/US association.4Auditory cortex is required for discriminative fear conditioning with sound CS, but its precise role is unclear until today. In our previous work (Staib & Bach, Three main findings emerge. First, encoding of a CS/US association for monophone sounds is demonstrated in a region of interest consistent with A1 on a single‐participant level, using a multivariate approach. This finding is replicated but much less pronounced when investigating a probabilistic definition of Heschl's gyrus, containing A1 but also other parts of ACX. This suggests that indeed CS/US association is encoded in, and possibly limited to, A1. A previous finding of CS encoding outside Heschl's gyrus was not replicated here. At the same time, univariate analysis revealed no CS+/CS− differences within our limited brain coverage, which notably excluded a number of areas previously reported in a meta‐analysis of CS+/CS− differences (Fullana et al., It has previously been shown for nondiscriminative fear conditioning with frequency sweeps that A1 relays US information during and shortly after the US occurrence to amygdala, and optogenetic inhibition of this information relay precludes fear conditioning (Letzkus et al., It is possible, however, that the current findings are not specific to fear conditioning. A role of early sensory areas in representing value properties of physical stimuli has become more apparent over the past years, including a representation of reward predictions and reward variance (Bach, Symmonds, Barnes, & Dolan, Two alternative interpretations for enhanced discrimination of CS‐evoked BOLD patterns appear unlikely to account for our results. First, ACX activity is known to be influenced by postconditioning receptive field mapping. However, this process is reported to start after about 20 min and to last for several hours (Froemke, Merzenich, & Schreiner, Taken together, we demonstrate auditory CS encoding in an anatomically defined A1 for monophone and musical sounds without temporal discriminative features. This extends a network perspective on fear conditioning (Herry & Johansen,