Little is known on how diet, physical activity (PA) and sedentary behavior (SB) changes during pregnancy and after childbirth in primiparous couples. Moreover, it is unclear how potential behavioral changes are associated with changes in BMI. This study examined changes in diet, PA and SB, and their association with changes in BMI in couples transitioning to parenthood.Dietary intake (FFQ), PA, SB (both Actigraph GT3X accelerometers) and BMI of women and men were assessed at 12 weeks of gestation, 6 weeks and 6 months postpartum. Data were analyzed using dyadic longitudinal data analyses techniques.In women, a decrease in fruit intake, an increase in alcohol intake, an increase of light-intensity PA, and a decrease in SB were observed from the beginning of pregnancy up to 6 months postpartum. Decreases in fruit intake between 6 weeks and 6 months postpartum was associated with increases in BMI. Men did not show significant dietary changes, while an increase in light-intensity PA and a decrease in moderate-to-vigorous PA (MVPA) was observed at 6 months postpartum when compared to 12 weeks of gestation. Paternal increases in “avoidance food group” intake were associated with increases in BMI between baseline and 6 weeks postpartum. No associations of changes in BMI and changes in PA and SB were found.Not only mothers but also fathers experienced unfavorable changes in lifestyle during the transition to parenthood, with impact on BMI changes. This highlights the need to monitor unhealthy changes in lifestyle and body weight in both parents when expecting a child and after childbirth.Clinicaltrials.gov, NCT03454958. In thIndeed, changes in EBRB during the transition to parenthood have been described in previous research , 8. HoweFinally, most research focused on changes in BMI and EBRB without looking into associations between changes in BMI and changes in EBRB . This kn2.2.1.Clinicaltrials.gov, NCT03454958) with a focus on changes in body weight, body composition and EBRB during the transition to parenthood was set up. For more details on the study protocol and methodology, the interested reader is referred to the earlier published study protocol , anthropometric measurements were performed and participants received an accelerometer which they had to wear for seven consecutive days, starting the day after the study visit. An online questionnaire was sent to the participants 1 week following the study visit (day 8 after each study visit), and a reminder to complete the questionnaire was sent three times with intervals of 1 week.2.2.2.Physical activity (PA) and sedentary behavior (SB) were measured using accelerometers , worn on the right hip for seven consecutive days for at least 12 h/day. Couples were instructed to wear the accelerometers in the week following the anthropometric measurements and to keep a daily log to provide information on activities during which the accelerometer was removed . Daily logs were used for manual data-cleaning and to assess if registered non-wear time (≥60 min of consecutive zeros) was indeed non-wear time or sedentary time, making use of Actilife Software. Days during which accelerometers were worn for <10 h were removed, and participants with less than 5 days of valid accelerometer data were excluded from the analysis . Time spOne week after the study visit, an online questionnaire to assess dietary intake, socio-demographic characteristics and child feeding practices (the latter only for PP6WK – PP6M) was sent to the participants. Dietary intake was assessed over the last 30 days using a validated 22-items Food Frequency Questionnaire (FFQ), of which questioned food groups were based on the national Food Based Dietary Guidelines . For eacSocio-demographics  were assessed at baseline (PG12) and weeks of exclusive breastfeeding were questioned at follow-up (PP6WK – PP6M). Sex and gestational age of the new-born and data of the last recorded weight of the mother before delivery were retrieved from the medical records. The latter was needed to calculate gestational weight gain (GWG) (kg), which was done by subtracting the last recorded body weight before delivery with body weight measured at PG12.2.3.p-values < 0.05 were considered statistically significant. For data-cleaning and exploration the R packages dplyr, tidyverse, and ggplot2 were used  had to be excluded for using the multilevel models. For this purpose, data patterns were tested using R package mice and margin plots were created using the R package VIM , 25. MisFor the first and primary aim, investigating changes in EBRB, the data were analyzed using a multilevel model for each EBRB variable  for longitudinal dyadic data that treats the three levels in the data (repeated measures nested within persons nested within couples) as two levels of random variation . This bep > 0.10 where removed, and EBRB predictors with both two- and three-way interactions with p > 0.10 were removed. In a third step, level two variables  were added. Finally, two-way covariate interactions with p > 0.10 were removed from the models.For the second aim, investigating how changes in BMI are associated by changes in EBRB over time, difference scores for the outcome  and predictor variables  variables were calculated by subtracting variables measured at PP6WK with those at PG12 and variables measured at PP6M with those at PP6WK, after which continuous predictor variables were grand mean centered. Covariates which were considered  were also grand mean centered. Data were imputed based on grand mean centered values. As difference scores could not be calculated for participants of which only the baseline measurement was available, only a total of 133 out of 144 couples were included in the analysis. Two models were built, one to investigate the association between changes in BMI and changes in dietary intake (which we will refer to as the diet model), and one to investigate the association between changes in BMI and changes in PA and SB (which we will refer to as the PA&SB model). Similar models were used as described above, i.e., two-level two-intercept unstructured models estimated with REML. The models were built using a stepwise approach, during each step, two-way interaction terms between sex  and time , two-way interaction terms between sex and the EBRB predictors or covariates , as well as three-way interaction terms between sex, time and EBRB predictors  were added. In a first step, level one variables  were added. Secondly, two-way covariate interactions with 3.3.1.n = 2 couples), with IVF pregnancy, as this might already have affected women’s body weight irrespective of the pregnancy and postpartum period itself (n = 3 couples) or with a history of bariatric surgery in the mother (n = 3 couples) were excluded together with their partner from the analyses. A total of 15 women and 17 men were lost-to follow-up because they could not be contacted again or did not want to participate in the follow-up measurements . Sample characteristics for women and men at baseline are shown in Of the initial 152 couples measured at baseline, 144 were eligible to be included in the analyses. Participants who underwent a miscarriage : −60.9 g/day (14.0), p < 0.001), and significantly increased their alcohol intake from baseline to postpartum (PP6WK – PG12 (SE): +12.3 g/day (3.8), p = 0.001; PP6M – PG12 (SE): +21.0 g/day (3.7), p < 0.001). A trend towards a significant increase for total energy intake was found at 6 weeks postpartum compared to baseline (PP6WK – PG12 (SE): +69.9 kcal/day (40.2), p = 0.083). No significant changes were found for women’s changes in vegetables group intake and “avoidance food group” intake, nor for fruit, vegetables, “avoidance food group” and total energy intake for men during the transition to parenthood . A trend towards a significant decrease was found for alcohol intake at 6 months postpartum compared to baseline (PP6M – PG12 (SE): −19.2 g/day (10.2), p = 0.062).Result for this first and primary aim are shown in p = 0.030; PP6M – PG12 (SE): +51.8 min/day (7.3), p < 0.001). Maternal MVPA levels at 6 weeks postpartum significantly decreased (PP6WK – PG12 (SE): −4.5 min/day (2.0), p = 0.021) compared to baseline, but this effect disappeared at 6 months postpartum (PP6M – PG12 (SE): −2.4 (2.0), p = 0.226). Also women’s SB decreased (PP6WK – PG12 (SE): −2.5%/day (0.9), p = 0.004; PP6M – PG12 (SE): −5.4%/day (0.9), p < 0.001). For men, a significant increase in LIPA was found at 6 months postpartum compared to baseline (PP6M – PG12 (SE): +23.1 min/day (9.1), p = 0.011), while a significant decrease was found for MVPA (PP6M – PG12 (SE): −5.4 min/day (2.7), p = 0.047). No significant changes in men’s SB were found. Results from the different models on changes in dietary intake are shown in In terms of PA and SB, both women and men experienced significant changes at 6 weeks and 6 months postpartum compared with the beginning of pregnancy. Women significantly increased LIPA-levels (PP6WK – PG12 (SE): +15.6 min/day (7.2), r = 0.18).The ICC for all dietary intake, PA and SB variables are small but positive. For example, fruit intake of men and women within couples is positively correlated  and predictor EBRB variables can be found in 2 (p = 0.031) in that period. Moreover, changes in BMI (PG12 – PP6WK/PP6M) could be explained by associations with some of the investigated covariates . Each €1000/month higher household family income corresponded with an average decrease in BMI of 0.18 kg/m2 (p = 0.029), and for each additional kg of GWG there was an average increase in BMI of 0.04 kg/m2 (p = 0.009).Results of the diet model can be found in womanPG12PP6WK), and between 6 weeks postpartum and 6 months postpartum (BMIwomanPP6WKPP6M) can be predicted as follows:After excluding from the model all non-significant predictor variables and interactions, an exact prediction of changes in BMI can be made. Women’s changes in BMI between 12 weeks of gestation and 6 weeks postpartum  + 0.04*GWG (kg).BMIBMIwomanPP6WKPP6M= −0.36–0.19*household family income  + 0.05*GWG (kg) – 0.25*fruit group intake (100 g/day).2 (0.04), p = 0.083). A significant positive association was found between changes in BMI and changes in “avoidance food group” intake between 12 weeks of pregnancy and 6 weeks postpartum; an increase of average daily “avoidance food group” with 100 g in that period corresponded to an increase in BMI of 0.08 kg/m2 (p = 0.029).For men, a negative trend toward significance was found for the paternal association between changes in BMI and changes in fruit group intake (−0.15 kg/mmanPG12PP6WK) can be predicted as follows:Men’s changes in BMI between 12 weeks of gestation and six weeks postpartum (BMImanPG12PP6WK = 0.37 + 0.08* “avoidance food group” intake (100 g/day).BMI4.This study aimed to investigate how EBRB changes in both members of a couple during the transition to parenthood, and how changes in BMI were associated with changes in EBRB. Our findings showed that mothers decreased their fruit intake from the beginning of pregnancy up to 6 months postpartum while increasing their alcohol intake. No changes in vegetables, “avoidance food group” and total energy intake were found. Moreover, mothers increased their LIPA over the study period. MVPA decreased at 6 weeks postpartum compared to the beginning of pregnancy, but restored to the initial level at 6 months postpartum. Finally, also maternal SB levels decreased across the transition to parenthood. For fathers, no average changes in dietary intake were observed. Paternal LIPA increased at 6 months postpartum compared to the beginning of pregnancy, while MVPA decreased. Paternal SB did not change significantly over the period studied. Furthermore, changes in maternal BMI were positively associated with GWG and negatively associated with household family income and changes in daily fruit intake. Paternal changes in BMI were positively associated with daily changes in “avoidance food group” intake. No associations between changes in BMI and changes in PA and SB were found for mothers, nor for fathers.Only limited studies are available studying changes in EBRB during the transition to parenthood, while most studies investigated changes during pregnancy, or studied differences between mothers and non-mothers , 33–35. 2 from 12 weeks of gestation to 6 weeks postpartum) might clarifying why a positive association between changes in “avoidance food group” intake and BMI was found during this period. Attention is thus needed for unhealthy changes on an individual basis in paternal diet during the pregnancy period, as this might set the basis for unhealthy habits and body weight on the longer term. Follow-up of fathers’ body weight and lifestyle is not part of standard care yet. Based on these and previous results indicating unhealthy changes in paternal body weight during the transition to parenthood, it is highly recommended to include fathers in pre- and postnatal care trajectories.For fathers, we did not find any changes in dietary intake during the transition to parenthood. This is in line with studies showing no differences in paternal dietary intake  from pregnancy to 1 year after birth , but conr = 0.28 and r = 0.27 respectively). Even though these correlations are rather low, they are substantial and should not be statistically neglected when analyzing EBRB in couples. Moreover, concordance of dietary behaviors within couples and the association between partner support and PA behavior has been described  , which wescribed , 53. ThiAs expected, maternal and paternal LIPA increased and maternal SB decreased during the transition to parenthood. This confirms the results from an earlier focus group study during which parents explained that they became more active and less sedentary because of the baby  . At 6 weInadequate diet, lack of engagement in PA or excessive SB during pregnancy are associated with negative health and pregnancy outcomes for both mother and child , 58, 59.4.1.p-values are biased. A dyadic research approach takes statistically into account that two couple members come from the same dyad and allows to investigate women’s and men’s data simultaneously. This is a unique aspect of this research, as existing studies in parents expecting a child have to the best of our knowledge not yet investigated data on a dyadic level. Second, BMI, PA and SB were measured objectively. Objective measurements exclude social desirability bias, and provide more reliable insights into actual levels of PA in a population of pregnant women  data was manually imputed. As this was only for a limited number of activities (between 1.1 and 2.0% depending of sex and measurement moment), we would expect limited bias from this imputation. Moreover, no distinction could be made between different domains of PA. For example, it might be that we would have observed changes in leisure-time PA and/or household-related PA. Secondly, as there was no data collection phase at the end of the pregnancy phase or in the first days postpartum, it cannot be derived whether described changes in EBRB at 6 weeks postpartum when compared to 12 weeks of gestation reflect changes during pregnancy or changes within the first weeks following birth. We would expect that changes are overestimated  , and LIP5.Women decreased their fruit intake during the transition to parenthood, which was associated with increased BMI values between 6 weeks and 6 months postpartum. Men did not show significant changes in dietary intake during the transition to parenthood. However increases in paternal BMI between the beginning of pregnancy and 6 weeks postpartum were found to be associated with increases in “avoidance food group” intake and changes in BMI. For both parents, increases in LIPA from the beginning of pregnancy up to 6 months postpartum were observed, and mothers also decreased their SB during this period. Fathers decreased their MVPA levels from the beginning of pregnancy up to 6 months postpartum. No associations between changes in BMI and changes in PA and SB were found. Not only mothers but also fathers are vulnerable for unhealthy EBRB changes associated with changes in BMI. This has implications on the design and delivery of lifestyle interventions during the pregnancy and postpartum period. Researchers, healthcare providers, or policy makers involved in the development of lifestyle interventions during this critical life phase can use these results to adequately support both parents in order to obtain or maintain a healthy body weight and lifestyle, instead of a focus solely on the mother. This can be done by using a multilevel couple- or family-based approach. Moreover, HCPs could play a crucial role in providing additional support to fathers(-to-be). Recommendations for HCPs are thus needed to facilitate couple-focused interventions.The raw data supporting the conclusions of this article will be made available by the authors, subject to data protection requirements. The anonymized dataset and a data dictionary defining each variable of the dataset can be made available with publication upon reasonable request to the corresponding author. Data will only be shared after approval of the ethical committee that handled the review of the project for which the data would be needed.The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the University Hospital of Vrije Universiteit Brussel , approved on May 16th 2018 (B.U.N. 143201835875). The patients/participants provided their written informed consent to participate in this study.VV, DA, TD, LG, RD, PC, and AB contributed to the conception and design of the study. VV recruited and measured the participants and was responsible for data extraction and wrote the original manuscript. VV and LS performed the statistical analyses. VV, LS, DA, TD, CM, LG, RD, PC, and AB were responsible for reflection and interpretation of the data. LS, DA, TD, CM, LG, RD, PC, and AB revised the manuscript critically. All authors contributed to the article and approved the submitted version.This research was supported by a research grant from The Research Foundation—Flanders (“Fonds voor Wetenschappelijk Onderzoek” (FWO)) with project number G033418 N. RD is holder of an FWO Fundamental Clinical Investigatorship with number 1803311 N.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Thyroid dysfunction might have a negative impact on the prognosis of patients with heart failure (HF) and affect the lipid metabolism. The aim of our study was to investigate the prognostic role of thyroid dysfunction and its relationship with lipid profile in hospitalized HF patients.Thyroid dysfunction strongly correlates with prognosis of HF patients and combination with lipid profile improves the prognostic value.We performed a single‐center retrospective cohort study including hospitalized HF patients between March 2009 and June 2018.p < .001), elevated TSH , LT3S , overt hyperthyroidism , subclinical hypothyroidism  and overt hypothyroidism  independently increased the risk of composite endpoint defined as the combination of all‐cause mortality, heart transplantation, or left ventricular assist device requirement. Higher total cholesterol  was still a protective factor in HF patients. When divided into four groups by fT3 and median lipid profiles, comparison of Kaplan–Meier survival curves for various groups showed good risk stratification (p < .001).Among enrolled 3733 patients, low fT3  patients. Among enrolled 3733 patients, low fT3 and elevated TSH independently increased the risk of composite endpoint defined as the combination of all‐cause mortality, heart transplantation, or left ventricular assist device requirement. Higher total cholesterol was still a protective factor in HF patients and showed good risk stratification combined with fT3 in Kaplan–Meier survival curves. Therefore, the occurrence of thyroid dysfunction can favor the onset and progression of several cardiovascular diseases, such as atrial or ventricular arrhythmia, dyslipidemia, atherosclerosis, hypertension, and heart failure (HF), determining a worse prognosis in terms of hospitalization, heart transplantation, and even mortality.Multiple cohort studies have examined the relationship between thyroid function, and incident atrial fibrillation, HF, and coronary heart disease. In particular, subclinical hypothyroidism and hyperthyroidism were known to be associated with worsening left ventricular systolic and diastolic function, heart rate, and systemic vascular resistance, especially when thyroid‐stimulating hormone (TSH) > 10 mLU/L or <0.1 mLU/L.THs have multiple effects on lipid metabolism, including synthesis, mobilization, and degradation.With complete information on thyroid function in a large cohort of HF patients, we aimed to investigate the prevalence and prognostic effect of thyroid hormone abnormality in hospitalized patients and its relationship with lipid metabolism.22.1We enrolled 5124 patients hospitalized in Heart Failure Care Unit (HFCU), Fuwai Hospital, for symptomatic HF from March 2009 to June 2018. The diagnosis of HF was made by two independent cardiologists based on guidelines.2.2The thyroid function measurement was evaluated at admission to HFCU. Twelve‐hour fasting blood samples were drawn, and serum‐free T3 (fT3), fT4, total T3 (TT3), total T4 (TT4), and TSH were measured by using radioimmunoassay  in the Nuclear Medicine Department of Fuwai Hospital. The reference ranges for fT3, fT4, and TSH were 2.3–4.2 pg/mL, 0.89–1.76 ng/dL, and 0.55–4.78 mLU/L, respectively. Patients were divided according to thyroid function tests as follows2.3Follow‐up data were obtained by reviewing the patients’ hospital records, interviewing the patients via telephone, and examining the outpatient record. The composite endpoint was defined as the combination of all‐cause mortality, heart transplantation, or left ventricular assist device requirement. The data was primarily obtained from death certificates, post‐mortem reports, and medical records. Death caused by accidents was excluded. The median follow‐up duration was 2.79  years.2.4t‐test, skewed variables were tested with the Mann–Whitney U test, and categorical variables were tested with chi‐squared tests as appropriate. Bonferroni's correction was performed in multiple comparisons between thyroid dysfunction and euthyroidism groups, and a p < .01 was considered statistically significant. LDL‐C levels of the whole population were grouped into quartiles. We also grouped LDL‐C, HDL‐C, TC, and TG levels of the whole population into two groups based on median value. Cox proportional hazards models were performed to assess the association between thyroid dysfunction or lipid profile and risk of death. Model 1 indicated unadjusted; Model 2 was adjusted for age, sex, and baseline BMI; Model 3 was further adjusted for systolic blood pressure (SBP), heart rate (HR), serum sodium, total bilirubin (TBiL), alanine transaminase (ALT), TC, creatinine, log transformed N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP), left ventricular ejection fraction (LVEF), New York Heart Association (NYHA) functional class, ischemic causes, comorbidities of hypertension, diabetes or atrial fibrillation (AF), medication of β‐blocker, angiotensin‐converting enzyme inhibitor (ACEI), angiotensin receptor antagonist (ARB), and aldosterone receptor antagonist (MRA). In addition, we conducted a subgroup analysis to assess the potential heterogeneity of association between low T3 and composite endpoint. Interactions between low T3 and relevant variables were estimated by means of Cox proportional hazard analysis. Kaplan–Meier analysis was used to assess the cumulative survival of fT3, TSH, and lipid profiles. Furthermore, the association between the level of thyroid hormones and the endpoint was evaluated on a continuous scale with a restricted cubic spline curve based on Cox proportional hazards models adjusted by Model 2. The association between thyroid function and lipid profile was determined using Spearman's correlation analysis and Kaplan–Meier analysis. Two‐tailed p values < .05 were assumed as statistical significance. All analyses were performed using R 3.6.2 .Descriptive statistics were used to examine the baseline characteristics of the study population at baseline. Data were described as the mean ± standard deviation for normally distributed continuous parameters, as the median (interquartile range) for skewed distributed variables, and frequencies (percentages) for categorical variables. Continuous normally distributed variables were tested with the Student's 33.1The included 3733 patients were divided into the following subgroups based on thyroid status: euthyroidism, LT3S, subclinical hypothyroidism, overt hypothyroidism, subclinical hyperthyroidism, overt hyperthyroidism, and undetermined. The most frequent thyroid dysfunction was LT3S 16.3%), followed by subclinical hypothyroidism (6.3%), subclinical hyperthyroidism (6.0%), overt hypothyroidism (4.4%), and overt hyperthyroidism (1.1%). The baseline characteristics of patients in each group are shown in Table 6.3%, fol3.2p < .001), as well as abnormal TSH . In multivariate analysis, low fT3  and elevated TSH  were still independent predictors of worse outcome even after adjustment for the maximum number of variables of Model 3. Kaplan–Meier survival curves about thyroid hormones were shown in Figure p < .001). These results were corroborated with regression splines that demonstrated a linear trend for risk of composite outcome across fT3 and a U‐shape trend for death risk across TSH , overt hyperthyroidism , subclinical hypothyroidism  and overt hypothyroidism  were all independent risks of composite endpoint adjusted for Model 3. In the subgroup analysis, lower fT3 was independently associated with composite endpoint across all patient subgroups, including lipid subgroups  was a protective factor in HF patients even after adjustment for the maximum number of variables, see Model 3. We observed a notable association between LDL‐C and composite outcome; patients with LDL‐C values in Q2 (1.89 mmol/L ≤ LDL‐C < 2.41 mmol/L), and Q4 (LDL‐C ≥ 3.03 mmol/L) had 0.86  and 0.67  lower risk compared with patients in Q1(LDL‐C < 1.89 mmol/L), respectively. Higher TG seemed to be a protective factor in HF patients both in univariate  and multivariate Cox regression analysis  adjusted for Model 2, however not significant in Model 3 (p = .284). Figure p < .001).In the analysis of the lipid profile shown in Table 3.4ρ = .195, p < .001), TG , HDL‐C  and LDL‐C . Significant positive relationships were still detected between TT3, TT4, and the lipid profile mentioned above (p < .001). As shown in Figure p < .001). Similar results were observed in fT3 combined with TG, LDL‐C, and HDL‐C (p < .001).Analysis regarding the association between thyroid hormones and the parameters of lipid profile was shown in Table 4As reported in the present study, LT3S, overt hyperthyroidism, subclinical hypothyroidism, and overt hypothyroidism were independently associated with composite endpoints including all‐cause mortality, heart transplantation, or left ventricular assist device requirement in hospitalized HF patients. Lower fT3 and elevated TSH were independent prognosticators of worse outcomes in HF. Moreover, fT3 was positively related to lipid profiles, and fT3 combined with lipid profile improved risk stratification of the composite endpoint.Thyroxine (T4) and triiodothyronine (T3), produced peripherally by T4 5′‐monodeiodination, have multiple effects on cardiovascular function, mediated by different signaling pathways that have been clustered into genomic and non‐genomic action. Thyroid hormones bind to thyroid hormone receptor‐α and β in cardiac myocytes to regulate gene expression.Both overt and subclinical hypothyroidism are associated with several cardiovascular risk factors such as dyslipidemia, hypertension, accelerated atherosclerosis, and coronary artery disease,An increased TSH, indicating a deficiency of circulating thyroid hormones, was independently associated with a higher incidence of composite outcomes, including death, urgent heart transplantation, or hospitalization because of worsening HF.Thyroid hormones, even within the normal ranges, may contribute to the alternation in lipid metabolism. Hypothyroidism and subclinical hypothyroidism have significantly elevated levels of TC, TG, and LDL‐CThough we explored the prognostic value of thyroid dysfunction in patients with pre‐existing HF requiring intense care and evaluated the association between the lipid profile and thyroid hormones in HF patients for the first time. Some limitations could not be neglected. First, it is a single‐center retrospective study, the possibility of residual confounding cannot be excluded and need to be validated in larger cohort studies. Secondly, thyroid function was assessed only once at baseline. We were unable to assess the prognostic value of transient changes in thyroid function in HF patients. Thirdly, there was heterogeneity in the cause of HF, and the medication after discharge was unavailable.5The presence of LT3S, overt hyperthyroidism, and overt and subclinical hypothyroidism on admission for HF requiring intense care were independent poor prognostic indicators. Low fT3 and elevated TSH could serve as promising markers to predict adverse outcomes in HF patients. Low fT3 seemed to be associated with a lower level of lipid profiles in HF, and the combination of fT3 and parameters of lipid profile improves the power of risk stratification.Yuhui Zhang and Jian Zhang designed and supervised the study. Ping Zhou, Liyan Huang, Mei Zhai, Yan Huang, Xiaofeng Zhuang, and Huihui Liu performed sample and data acquisition. Ping Zhou and Liyan Huang performed data analysis and interpretation. Ping Zhou wrote the manuscript. Yuhui Zhang and Jian Zhang approved manuscript submission. All authors read and contributed to the manuscript.The authors have no potential conflict of interest to declare.Figure S1. Kaplan–Meier analysis for composite endpoint on the basis of lipid profile in heart failure.Click here for additional data file.Supplementary information.Click here for additional data file.


Validation of the method with subject‐specific computational fluid dynamics and application to synthetic velocity waveforms in the common carotid, brachial, and femoral arteries reveals that the Poiseuille solution underestimates peak WSS 38.5%–55.1% during the acceleration and deceleration phases of systole and underestimates or neglects retrograde WSS. Following evidence that oscillatory shear significantly augments vasodilator production, it is plausible that mischaracterization of the shear stimulus by assuming parabolic flow leads to systematic underestimates of important biological effects of time‐varying blood velocity in conduit arteries.Wall shear stress (WSS)—a key regulator of endothelial function—is commonly estimated in vivo using simplified mathematical models based on Poiseuille's flow, assuming a quasi‐steady parabolic velocity distribution, despite evidence that more rapidly time‐varying, pulsatile blood flow during each cardiac cycle modulates flow‐mediated dilation (FMD) in large arteries of healthy subjects. More exact and accurate models based on the well‐established Womersley solution for rapidly changing blood flow have not been adopted clinically, potentially because the Womersley solution relies on the local pressure gradient, which is difficult to measure non‐invasively. We have developed an open‐source method for automatic reconstruction of unsteady, Womersley‐derived velocity profiles, and WSS in conduit arteries. The proposed method (available online at  We developed an open‐source method for automatic reconstruction of pulsatile velocity profiles and wall shear stress from one‐dimensional velocity waveforms in conduit arteries obtained from clinically available time‐averaged diameter and time‐varying one‐dimensional velocity data, acquired in vivo with non‐invasive medical imaging. This new method of data analysis can provide substantially more accurate estimates of oscillatory shear stress affecting the arterial endothelium.1Endothelial dysfunction precedes asymptomatic vascular remodeling and is involved in atherogenesis, leading to profound clinical manifestations of cardiovascular disease  centerline or mean velocity waveforms. Such waveforms can be acquired in vivo with non‐invasive medical imaging. The method was validated with subject‐specific computational fluid dynamics (CFD) simulations driven by in vivo pulsed‐wave Doppler (PWD) velocity recordings.As representative test cases, we compared the time‐varying Womersley method to the quasi‐steady Poiseuille reconstruction using computer‐generated velocity waveforms for the right common carotid (RCC), brachial (RBRC), and femoral (RF) arteries at rest and during cardiovascular stress . To check for accuracy, we compared the computer‐generated velocity profiles to experimental data from the brachial artery in a single test subject. Results show that estimating WSS based on the steady flow assumptions of Poiseuille's flow fails to capture physiologic levels of oscillatory shear stress.2The authors declare that all supporting data are available within the article.2.1ρ) of 1060 kg/m3 and viscosity (μ) of 4.5 mPa·s. The flow in all segments is assumed to be laminar.To compare WSS estimation methods for a variety of major arteries in the human body under varying conditions, we employed a previously developed reduced‐order model of the major systemic vessels within the trunk, limbs, and head at rest, during a fight‐or‐flight response , and during moderate aerobic exercise ), as shown in Figure https://doi.org/10.5281/zenodo.4630326.Input 1D velocity waveforms (2.2U1D(t) is acquired. Assuming a parabolic radial velocity profile, the steady component of blood flow is expressed by Poiseuille's flow following Equation (U¯ is time‐averaged mean velocity of U1D(t) and r is the radial location ranging from 0 to the vessel radius R . Steady flow rate (Q¯) is obtained by integrating the velocity field on the cross‐section:Poiseuille's flow accounts for the steady, fully developed motion of flowing blood . The following equations indicate a separate problem for each axial location where τ¯w) is:U1D(t) rather than U¯), thereby producing terms with time dependency: U¯, Q¯(t), and τ¯w(t). This “quasi‐steady” approximation assumes frequency content is negligible and refers to reconstruction of transient flow profiles with respect to Poiseuille's flow ), flow rate (Q(t)), and WSS (τw(t)) at any arterial segment based on previous work ) as depicted in Figure https://doi.org/10.5281/zenodo.7576408) has been simplified to require only time‐averaged vessel diameter and time‐varying 1D velocity data from the user. The Fourier and Bessel functions representing the velocity profile below are automatically computed from input data, allowing users to interact minimally with the mathematical details of the method. In particular, the Womersley velocity profile is expressed by Equations 1/2, defines the shape of the radial velocity profile through relation of vessel radius, pulsatile flow frequency, and viscous forces. Here ω is the angular frequency of the cardiac pulse, t is time, n is the natural numbers, and i is the imaginary number. Bn is Fourier coefficients that decompose the time‐varying U1D(t) wave. J0 and J1 denote Bessel functions of the first kind of order 0 and 1, respectively. The first term in Equation  components as defined in Equations (Equations  and 7)  7) are squations  and 3)  n = 0th 2.4T is the period of the cardiac cycle. The OSI ranges from 0, indicating non‐reversing WSS, to a maximum value of 0.5.The oscillatory shear index (OSI) characterizes how the WSS vector deflects from the predominant direction of flowing blood across the cardiac cycle  and ultrasound imaging studies used to validate the WSS predictions of the velocity‐driven Womersley solution with solutions of three‐dimensional CFD WSS.2.5.1Time‐of‐flight MRA in the right arm of a healthy male subject (27 y.o.) was obtained on a 3T MAGNETOM Prisma scanner (Siemens) at the Purdue MRI Facility, Purdue University. MRA scan parameters were repetition time (TR), 27 ms; echo time (TE), 3.58 ms; flip angle, 18°; pixel bandwidth, 185; and spatial resolution, 0.38 × 0.38 × 0.44 mm. High‐resolution color and PWD velocity recordings were obtained in the same subject with a Vevo 3100 imaging platform and a 15–30 MHz frequency linear array transducer . Collected PWD waveforms were extracted with a custom detection script   of 1060 kg/m3 and viscosity (μ) of 4.5 mPa·s. The 1D time‐varying velocity waveform extracted from PWD . These discrepancies can be attributed to the pressure gradient obtained from the solution of one‐dimensional equations of an elastic tube, indicating nontrivial dependence on axial position and diameter as in  during fear  and aerobic exercise . These values indicate that the velocity fields in these larger arteries are defined by the unsteadiness of blood flow rather than viscous forces. When the Womersley number is close to 1, the velocity profile is closely approximated by Poiseuille's flow. However, for larger Womersley numbers, pulse frequency and phase lag between flow and pressure waveforms dictate the shape of the velocity profile and subsequent WSS.To expand the pool of test data to a larger set of arteries, we applied the Poiseuille and Womersley reconstructions to time‐averaged mean velocity waveforms from our previously published reduced‐order model of the normal human arterial tree . The Womersley profiles for the RF artery are similar for rest and fear states; however, during moderate aerobic exercise, significant increase in forward blood flow to the skeletal muscle prevented retrograde shear. In contrast, the flow reversal for the Poiseuille velocity model is possible only when mean velocity is reversed  stimulated a 14‐fold increase in NO synthesis  neglect the unsteady, pulsatile component of flow and potentially underestimate maximum WSS and OSI in large arteries  associated with Womersley's solution. The analytical solutions for reconstructing the instantaneous radial velocity profile, flow rate, and WSS at any arterial segment detailed here are driven by 1D velocity waveforms as acquired in vivo with Doppler ultrasound  requires only time‐averaged diameter of the vessel and time‐varying 1D velocity data from the user—readily acquired in vivo with non‐invasive medical imaging such as Doppler ultrasound. The method was validated with subject‐specific CFD simulations driven by in vivo PWD velocity recordings. In addition, we applied the method to synthetic 1D velocity waveforms in conduit arteries at rest and during two states of cardiovascular stress . Relative to Womersley approximations, Poiseuille's flow underestimated systolic WSS, overestimated diastolic WSS, and failed to capture negative values of WSS during the systolic deceleration phase. These results are consistent across all representative cases . In comparison to the Womersley solution, Poiseuille‐based calculations underestimated range of the WSS stimulus at the onset and offset of flow  by as much as 63.1% and underestimated or failed to capture near‐wall reversal of WSS. Since fluctuations in shear patterns modulate vasodilation in vivo, these findings suggest that intra‐ and inter‐subject variability associated with FMD may be better related to transient WSS and oscillatory shear stress calculated with Womersley theory, rather than similar values calculated with the parabolic flow assumption of Poiseuille's flow.Underlying assumptions influence estimates of WSS in large conduit arteries. The assumption of a quasi‐steady, parabolic velocity profile leads to systematic bias and an underestimation of pulsatile WSS. To support clinical adoption of more accurate pulsatile flow solutions, we developed an open‐source method for automatic reconstruction of velocity profiles in time and space, based on the well‐established Womersley solution for pulsatile blood flow. The publicly available code (J. C. Muskat, C. F. Babbs, and V. L. Rayz conceived and designed research; J. C. Muskat and C. F. Babbs performed experiments; J. C. Muskat analyzed data; J. C. Muskat, C. F. Babbs, C. J. Goergen, and V. L. Rayz interpreted results of experiments; J. C. Muskat, C. F. Babbs, C. J. Goergen, and V. L. Rayz prepared figures; J. C. Muskat drafted manuscript; J. C. Muskat, C. F. Babbs, C. J. Goergen, and V. L. Rayz edited and revised manuscript; J. C. Muskat, C. F. Babbs, C. J. Goergen, and V. L. Rayz approved final version of manuscript.No conflicts of interest, financial or otherwise, are declared by the authors.Data S1Click here for additional data file.


PLA2G6) encodes an enzyme that catalyzes the hydrolytic release of fatty acids from phospholipids. Four neurological disorders with infantile, juvenile, or early adult‐onset are associated with PLA2G6 genetic alterations, namely infantile neuroaxonal dystrophy (INAD), atypical neuroaxonal dystrophy (ANAD), dystonia‐parkinsonism (DP), and autosomal recessive early‐onset parkinsonism (AREP). Few studies in Africa reported PLA2G6‐associated disorders and none with parkinsonism of late adult onset.The phospholipase A2 group VI gene . Brain MRI without contrast was performed. Genetic testing was done using a custom‐made Twist panel, screening 34 known genes, 27 risk factors, and 8 candidate genes associated with parkinsonism. Filtered variants were PCR‐amplified and validated using Sanger sequencing and also tested in additional family members to study their segregation.PLA2G6, an in‐frame deletion NM_003560:c.2070_2072del  and a missense variant NM_003560:c.956C>T (p.Thr319Met). Both variants were classified as pathogenic.Two siblings born to consanguineous parents developed parkinsonism at the age of 58 and 60 years, respectively. MRI showed an enlarged right hippocampus in patient 2, but no overt abnormalities indicative of INAD or iron deposits. We found two heterozygous variants in PLA2G6 is associated with late‐onset parkinsonism. Functional analysis is needed to confirm the dual effect of both variants on the structure and function of iPLA2β.This is the first case in which  PLA2G6) encodes an enzyme that catalyzes the hydrolytic release of fatty acids from phospholipids, producing lysophospholipids, which are used in deacetylation/reacetylation reactions. It possesses a calcium‐dependent phospholipase activity, which has a role in phospholipid remodeling, an attribute consistent with cellular and mitochondrial membrane integrity and signal transduction.The Phospholipase A2 group VI gene (PLA2G6‐associated neurodegeneration (PLAN) can be categorized into 4 phenotypes: infantile neuroaxonal dystrophy (INAD), atypical neuroaxonal dystrophy (ANAD), adult‐onset dystonia‐parkinsonism (DP), and autosomal recessive early‐onset parkinsonism (AREP). Symptoms of INAD and ANAD appear in infancy to early childhood and manifest mainly as progressive psychomotor deterioration, ataxia, spasticity, and axial dystonia.According to the age of onset and clinical features, DP usually occurs between 20 and 40 years of age and manifests with parkinsonism, dystonia, and sometimes epilepsy. Earlier presentations in the second decade of life are also documented. In addition, impaired cognitive functions are common phenomena, and iron deposition incidence is scant.PLA2G6‐associated AREP patients exhibit bradykinesia, lower limb tremor, hypomimia, and gait disturbance. With the progression of the symptoms, additional features are observed in some cases, such as dystonia, dysarthria, dysphagia, cognitive impairment, and extrapyramidal signs and symptoms. Non‐parkinsonian features may develop at later stages and may encompass cerebellar ataxia and autonomic dysfunction. Similar to DP, iron accumulation in the brain is rare. Instead, frontal and generalized white matter atrophy may be observed.In the early stages of their disease, PLA2G6‐associated parkinsonism have been reported in Africa. Here, we introduce the first case of genetically confirmed autosomal recessive parkinsonism with a late‐onset disease in the sixth and seventh decades of life, identified in two siblings from a Sudanese family.To the best of our knowledge, no cases of adult‐onset A clinical diagnosis of PD was made in 2 patients using the United Kingdom Parkinson's Disease Society Brain Bank criteriaThe Research Ethics committee for Medical and Health studies at the Faculty of Medicine, University of Khartoum, Sudan, approved the study. Written informed consent was obtained from all participants.® Discover. DNA purification was carried out following prepIT® L2P manual protocol. DNA quality and quantity were performed using agarose gel electrophoresis and spectrophotometry .Five individuals  donated saliva for DNA extraction. One to two milliliters of saliva were collected using OrageneWe utilized Twist custom panel, which allows screening of 34 genes, 27 risk variants, and 8 candidate genes, which are mostly associated with Parkinsonism Filtered variants were PCR‐amplified and validated (Sanger sequencing) to confirm the genotypes in affected individuals. The variants were Sanger‐sequenced in three unaffected relatives  to study the segregation. Forward and reverse primers were designed using Primer3 online tool.Two siblings born to consanguineous parents  developed the first symptom at the age of 60 years in the form of resting tremor at her left arm. Her condition worsened within five years as the tremor markedly interfered with most of the daily activities, in addition to developing intermittent freezing of gait. Symptoms initially improved with levodopa; however, at the time of examination, the patient suffered from marked end‐of‐dose motor fluctuations and Levodopa‐induced dyskinesia. This patient also reported frequent hallucinations, sleeping problems (mainly insomnia), and inability to control urination. Upon assessment, she had hypomimia , dysarthria and showed symptoms of dopamine dysregulation syndrome.Upon assessment “on” levodopa, mild bradykinesia was observed in patient 1 while more progressive bradykinesia was observed in patient 2; neither of the two patients showed resting tremor while moderate rigidity in both patients was found in all extremities and neck. We were unable to perform a formal detailed cognitive assessment.We performed fundoscopy, lipid profile, high‐resolution lipoprotein electrophoresis, peripheral blood smear, serum iron, serum ferritin, and transferrin saturation studies for both patients. Prompted by a history of hearing impairment in patient 2, we performed Ear, Nose, and Throat examination as well as audiometric testing. All these investigations were unremarkable for both patients. We found moderate hippocampal expansion on T2‐weighted MRI brain scans of patient 1  and NM_003560.4: c.956C>T (p.Thr319Met)) were found in both siblings (Patient 1 and Patient 2) showing late onset parkinsonism. List of variants found and their pathogenecity scores are found in Table Two heterozygous variants in PLA2G6 were validated using Sanger sequencing. Segregation analysis , this segregation pattern is highly suggestive of an autosomal recessive inheritance in a compound heterozygous state. The variants were classified as pathogenic in these patients based on the ACMG criteria given in Supplementary Both variants found in sis Fig.  showed tPLA2G6 in a compound heterozygous state in two patients with late onset parkinsonism. Pathogenic variants in this gene have been associated with autosomal recessive juvenile onset or early‐onset parkinsonism . To our knowledge, this is the first case in which a PLA2G6 variant is associated with late‐onset autosomal recessive presentation. Yet, it is still consistent with the complex nature of PLA2G6‐associated phenotypes and its tendency to feature multiple neurological disorders despite the rarity of its mutations.In a consanguineous Sudanese family, we identified two variants in PLA2G6‐associated INAD was also reported in another Sudanese family, where a homozygous pathogenic variant affecting a canonical splice site (NM_003560.2:c.1427+2T>C) was found in the two affected siblings.The first variant we identified is a known pathogenic in‐frame deletion  which, unlike our study, was reported in a homozygous state in all previous studies, and all reported individuals were cases of INAD.The second variant is also a known missense variant  located in exon 7, which was associated with several cases presenting with the same phenotype.PLA2G6‐related parkinsonism in general. PLA2G6‐associated hippocampal changes have been previously described in number of previous studies.PLA2G6 mutation in a 35‐year‐old patient with adult‐onset autosomal recessive parkinsonism and early stages of dementia.Apart from the unusually late age of onset, the presentation of the disease was consistent with the phenotypes of previous patients carrying p.Val691del or p.Thr319Met, and also with what is known about adult‐onset This study had limitations. Although one of our patients had a history of psychiatric manifestations, it was not feasible to perform a formal, detailed cognitive and psychiatric assessment. Also, iron deposition in the brain cannot be reliably ruled out as T2* and SWI MRI sequences were not obtained. Last, using a panel limits the chance of detecting any other possible molecular findings  which are classically detectable with WES or WGS.PLA2G6 variants in two patients from a consanguineous Sudanese family, consistent with a genetic diagnosis of autosomal recessive parkinsonism. Although both variants were previously reported, these patients had an unusually late onset of disease in the sixth decade of life, expanding the age range of the PLA2G6‐associated parkinsonism. Functional analysis of the effects of these variants on the structure and function of iPLA2β may unravel the mechanisms of this variability.We identified two Conception and design: UW, YB, CT, J‐C C, SL, OS, LEOE; Primary clinical evaluation: OS and YB. Secondary clinical assessment , participant recruitment and sampling: YB, MI, KE, and IE. DNA preparation and PCR: YB: Target NGS analysis: CT. Data analysis and interpretation: YB, CT, J‐C C, SL and UW. Writing the first draft: YB. Writing with critical revision: YB, UW, J‐C C, and LEOE. All authors revised and approved the final version of the manuscript.The authors declare no conflict of interest.Program “Investissements d'Avenir” ANR‐10‐IAIHU‐06, and from a grant from France Parkinson: to J‐C C, SL, and CT. UKB: PSP‐Element A‐397.0003 mit der Kostenstelle 993000: for UW and YB. Princess Nourah bint Abdulrahman University Researchers supporting project number PNURSP2022R172, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia to LEOE.Appendix S1.Click here for additional data file.Appendix S2.Click here for additional data file.Appendix S3.Click here for additional data file.Figure S1.Click here for additional data file.


A DDC founder mutation c.714 + 4A > T (IVS6 + 4A > T) is prevalent in the Chinese population. This study investigated the epidemiology of AADC deficiency in Taiwan by analyzing data from National Taiwan University Hospital (NTUH), a central institution for diagnosing and treating the disease. From January 2000 to March 2023, 77 patients with AADC deficiency visited NTUH. Among them, eight were international patients seeking a second opinion, and another two had one or both non‐Chinese parents; all others were ethnically Chinese. The c.714 + 4A > T mutation accounted for 85% of all mutated alleles, and 94% of patients exhibited a severe phenotype. Of the 77 patients, 31 received gene therapy at a mean age of 3.76 years (1.62–8.49) through clinical trials, and their current ages were significantly older than those of the remaining patients. Although the combined incidence of AADC deficiency in this study (1:66491 for 2004 and later) was lower than that reported in newborn screening (1:31997 to 1:42662), case surges coincided with the launch of clinical trials and the implementation of newborn screening. Currently, many young patients are awaiting for treatment.Aromatic  AADC deficiency was first identified in 1990 by Hyland and Clayton.Aromatic DDC) gene, which encodes the AADC enzyme.DDC founder mutation c.714 + 4A > T (IVS6 + 4A > T) is prevalent in the Chinese population.DDC mRNA but may still allow for a small amount of normally spliced mRNA product.AADC deficiency arises from biallelic pathological mutations in the dopa decarboxylase .o‐methyldopa (3‐OMD) can serve as a convenient biomarker for AADC deficiency,National Taiwan University Hospital (NTUH) plays a significant role in the medical care of AADC deficiency patients in Taiwan. As medical treatment with dopamine agonists and other adjunctive drugs is ineffective for the severe form of AADC deficiency prevalent in Taiwan,22.1DDC variations (NM_000790.4), and phenotype (mild or severe). If survival status or date of death was unavailable, the date of the last follow‐up was used for statistical purposes. We describe all patient outcomes as known on March 1, 2023. DDC variant data were also retrieved from our laboratory's internal records for verification. This study was part of the long‐term follow‐up for patients with AADC deficiency approved by the Institutional Review Board (NTUH 201303100RIND), and informed consent was obtained from all patients included in the study.We reviewed internal records of patients with AADC deficiency who visited NTUH from January 2000 to March 2023. Patients' demographic data Table  were ver2.2https://pop-proj.ndc.gov.tw/). The Statistical Report of Rare Disease Confirmed Cases in Taiwan was downloaded from the Health Promotion Administration, Ministry of Health and Welfare (https://www.hpa.gov.tw/). The Rare Disease and Orphan Drug Act was passed in 2000, requiring doctors to register Taiwanese patients with rare diseases with the government. However, the registration system was not immediately available following the Act's passage.Yearly birth numbers from 1996 to 2022 were obtained from the National Development Council , and the 33.1Between January 2000 and March 2023, 77 patients with AADC deficiency visited NTUH. The gender distribution was balanced, with 41 females (53%) and 36 males (47%). Among them, eight were international patients seeking a second opinion, and another two had one or both non‐Chinese parents; all others were ethnically Chinese. Gene therapy clinical trials for AADC deficiency began in 2010. Of the 77 patients, 31  received gene therapy. Thirty‐eight patients never received gene therapy; 18 are alive, and 20 are deceased or lost to follow‐up. The survival status of the eight oversea patients who visited NTUH only once was unknown. The most recent (March 2023) Statistical Report of Rare Disease Confirmed Cases from the Health Promotion Administration recorded 63 patients with AADC efficiency, of whom 20 have died. Although not all patients with AADC deficiency in Taiwan visited NTUH, our case number still exceeded that of the Statistical Report of Rare Disease Confirmed Cases, likely because the latter only included Taiwanese nationals and may have missed early cases. Unfortunately, we could not obtain individual case information from the Health Promotion Administration for a detailed comparison.3.2n = 24) was 10.0 years (range: 3.0–17.7), while the mean age of deceased patients who received gene therapy (n = 7) was 11.7 years (range: 3.7–17.7); there was no statistical difference between the two groups (p = 0.39). Among those who did not receive gene therapy, the mean age of living patients (n = 18) was 4.9 years (range: 0.4–17.2), the mean age of death or the last visit of deceased patients (n = 15) was 3.9 years (range: 1.2–7.5), and mean age of the last visit of patients with unknown survival status (n = 13) was 5.5 years (range: 1.9–14.7). Consequently, patients who have received gene therapy survived longer than those who did not, regardless of whether they are alive, deceased, or have an unknown survival status .The earliest patient in this cohort was born in 1996. The mean current age of living patients who received gene therapy  to 1:210383 (0.48 per 100 000) . The c.714 + 4A > T mutation accounted for 85% of all alleles , while all other 72 patients (94%) exhibited a severe phenotype. Sequencing data were available for 59 ethnically Chinese patients, encompassing 118 alleles , and informed consent was obtained from all patients included in the study.


Acquired resistance represents a bottleneck to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment in lung cancer. Our study aimed to explore the efficacy of antiangiogenic‐based therapy in osimertinib‐resistant NSCLC patients and assess the efficacy of anlotinib in vitro study.Our multicenter study retrospectively collected 268 osimertinib‐resistant NSCLC patients with EGFR T790M mutation and explored the efficacy of anlotinib in patients and in vitro.p = 0.050) and the chemotherapy group . Both the ORR and DCR of the antiangiogenic‐based group were higher than the immunotherapy group and the chemotherapy group. Subgroup analysis showed a trend of more benefits from the anlotinib‐based therapy than the bevacizumab‐based therapy in terms of PFS  and OS . In vitro assays verified that anlotinib alone or combined with osimertinib exerted potent cytotoxicity to T790M‐mutant H1975 cell line with acquired osimertinib resistance.PFS was significantly longer in the antiangiogenic‐based therapy group than in the immunotherapy group (HR: 0.71, Our study suggested that antiangiogenic‐based therapy might improve PFS and OS in EGFR‐mutant NSCLC patients with acquired resistance to osimertinib. Moreover, anlotinib‐based therapy could be a promising effective treatment for this group of patients. Our study suggested that antiangiogenic‐based therapy might improve PFS and OS in EGFR‐mutant NSCLC patients with acquired resistance to osimertinib. Moreover, we also conducted preliminary in vitro assays to evaluate the efficacy of Anlotinib and in combination with osimertinib in EGFR T790M‐mutant osimertinib‐resistant NSCLC cell line H1975OR. At our center, most patients resistant to Osimertinib regularly receive antiangiogenic‐based therapy, immunotherapy, chemotherapy, or immunotherapy combined with chemotherapy under the guidance of physician evaluation. For antiangiogenic‐based therapy, anlotinib and bevacizumab were two common choices. However, recent results from multiple clinical trials suggested that the combination of osimertinib and bevacizumab failed to improve the PFS of patients with EGFR‐mutant non‐squamous NSCLC as first‐line treatment as well as of patients previously treated with EGFR‐TKIs.Therefore, the standard care for a broader range of patients with acquired resistance to Osimertinib needs to be discovered. In this study, we compared the efficacy of regular regimens patients received at our center and investigated the potential antitumor effect of anlotinib or in combination with osimertinib in EGFR‐T790M mutant osimertinib‐resistant NSCLC cell line.22.1Two hundred sixty‐eight patients form the First Affiliated Hospital of USTC and Shanghai Chest Hospital met the following inclusion criteria in this multicenter study: (1) advanced NSCLC with secondary EGFR exon 20 T790M mutation, (2) received at least one line of chemotherapy and osimertinib therapy, (3) received antiangiogenic therapy, immunotherapy, or chemotherapy after osimertinib resistance, (4) Eastern Cooperative Oncology Group performance status (ECOG PS) 0–1. The therapeutic schedule was decided by the physician under the principle that patients at high risk of bleeding should not be treated with antiangiogenic drugs, and patients with severe adverse effects to previous chemotherapy should not choose chemotherapy as a priority. This study was approved by the Ethics Committee of the Shanghai Chest Hospital (NO.KP17‐20) and The First Affiliated Hospital of USTC (202304062238000354629). The need for informed consent was waived due to the retrospective nature of the study.2.2EGFR detection was performed by next‐generation sequencing (NGS) or amplification refractory mutation system (ARMS). The disease stage was decided on the 8th edition of the American Joint Committee on Cancer International Association for the Study of Lung Cancer (AJCC) (IASLC) tumor‐node‐metastasis (TNM) classification. Enhanced chest computed tomography (CT) and abdominal ultrasound scans were performed every 4–6 weeks or when symptoms progressed significantly. Enhanced brain magnetic resonance imaging (MRI) was examined every 4–6 months if there was no lesion at baseline and no symptoms thereafter.2.32.Human lung adenocarcinoma osimertinib‐resistant cell line H1975OR was generously provided by the department of pharmacology, school of medicine, Shanghai Jiaotong University. Cells were cultured in RPMI‐1640 culture medium  added with 10% fetal bovine serum (Gemini Bio) at 37 °C with 5% of COOsimertinib was purchased from Selleck Chemical LLC. Anlotinib was generously offered by Chia Tai Tianqing Pharmaceutical Group Co., Ltd . Osimertinib and anlotinib were dissolved with DMSO (Sigma) and stored at −20°C until use. CCK‐8 assay (Proteintech) was utilized to determine cell viability.2.4Briefly, cells were seeded 4000 per well in 96‐well plates and treated with indicated agents. For cell growth assay, plates were placed in Incucyte® ZOOM Live‐Cell Analysis System (Essen BioScience) for 3 days, and phase object confluence was calculated by analysis software under the guidance of the manual. For cell viability assay, after being cultured with indicated agents for 3 days, cells were treated with CCK‐8 reagents, and the optical density at 450 nm was measured with a plate reader according to the manufacturer's instructions.2.5Cells were seeded 4000 per well in six‐well plates and treated with indicated agents. After being cultured for 7 days, cells were fixed with 4% paraformaldehyde fix solution (Biosharp) for 30 min and stained with crystal violet staining solution (Beyotime) overnight. The colony area was determined using Fiji ImageJ software.2.6The categorical variables were compared by chi‐square test. The endpoints were PFS ; OS  and ORR . Hazard ratios (HR) and 95% confidence intervals were performed by Cox proportional hazards regression model, and the survival difference was visualized by Kaplan–Meier curve and log‐rank test. To avoid the influence of confounding factors, factors with p values less than 0.1 in univariate analysis were included in multivariate analysis. To reduce the selection bias as much as possible, propensity score matching (PSM) method was applied to anlotinib‐based and bevacizumab‐based group patients at a 1:1 ratio via nearest‐neighbor method. Propensity scores for included patients were calculated by multiple logistic regression with adjustment for clinical characteristics, including age, sex, smoking history, surgery history, number of metastatic lesions, brain metastasis, and EGFR mutation subtype. A total of 42 pairs were successfully matched.Quantitative data from in vitro assays were shown as mean ± SD. The statistical difference between groups was determined using the Mann–Whitney test, chi‐square test, or Kruskal–Wallis test. All statistical analyses were performed using SPSS version 22.0 (IBM Corporation) and GraphPad Prism 9.33.1L858R/T790M (57.1%). Most patients (N = 145) received antiangiogenic‐based therapy, including 103 anlotinib‐treated patients and 42 bevacizumab‐treated patients. 47 received anti‐PD‐1 therapy or anti‐PD‐1 therapy combined with chemotherapy. 76 patients received chemotherapy alone.Two hundred sixty‐eight patients who met the eligibility criteria were included in this study. The clinicopathologic characteristics of eligible patients are shown in Table 3.2p = 0.072) and treatment  were associated with PFS (Table During the median follow‐up of 9.63 months (range: 0.63–20.17) for all the 268 patients, 190 (70.1%) patients developed PD. The overall median PFS was 4.50 months (95% CI: 3.93–5.08) Figure . The unip (antiangiogenic‐based therapy vs. chemotherapy) <0.001, p (immunotherapy vs. chemotherapy) <0.001)], indicating that treatment was an independent prognostic factor of OS.Death occurred in 128 (47.8%) patients in the overall population. The median OS was 9.78 (95% CI: 8.38–11.17) Figure . Univari3.3p = 0.050) and the chemotherapy group . Meanwhile, the median OS was 12.18 (95% CI: 11.36–13.00) months in the antiangiogenic‐based therapy group, 11.41 (95% CI: 6.14–16.68) months in the immunotherapy group, and 5.5 (95% CI: 4.85–6.15) months in the chemotherapy group , while there was no significant difference between the antiangiogenic‐based therapy group and the immunotherapy group . We also compared the overall response rate (ORR) and disease control rate (DCR) of the three groups. Results showed that both the ORR and DCR of the antiangiogenic‐based group were longer than the immunotherapy group and the chemotherapy group . The median OS was 12.81 (95% CI: 10.31–15.32) months in the anlotinib‐based group and 10.46 (95% CI: 9.75–11.16) months in the bevacizumab‐based group , but the survival curve showed a trend of more benefits from the anlotinib‐based group. After PS matching, the median PFS was 5.93 (95% CI: 4.56–7.31) months in the anlotinib‐based group and 5.75 (95% CI: 0.88–10.62) months in the bevacizumab‐based group , and the survival curve also showed a trend of more benefits from the anlotinib‐based group. Results above suggested that NSCLC patients resistant to osimertinib might benefit more from anlotinib as a later‐line treatment.Next, we analyzed the difference between two antiangiogenic regimens—anlotinib and bevacizumab. To minimize the selection bias, we performed PS matching to the two subgroups. Clinicopathologic characteristics before and after PS matching are shown in Table p Figure . There wp Figure . The medp Figure . There w3.550 = 6.113 μM, Figure 50 = 1.513 μM; H1975 IC50 = 2.300 μM, Figure To investigate the potential antitumor effect of anlotinib and in combination with osimertinib in osimertinib‐resistant EGFR‐T790M mutant NSCLC, we performed cell growth and viability assay in H1975 and osimertinib‐resistant H1975 cell line (H1975OR) treated with anlotinib and/or osimertinib. Our results confirmed that H1975OR cell line was resistant to osimertinib compared to parental cell line .p = 0.83, HR: 0.96 [95% CI 0.68–1.37]). And the WJOG8715 trial also showed that compared with osimertinib alone, mPFS was not longer with osimertinib plus Bevacizumab .As mentioned above, the WJOG9717L trial firstly investigated whether osimertinib plus bevacizumab was better than osimertinib alone for untreated EGFR‐mutant NSCLC patients. Results showed no significant difference between the two groups in terms of PFS .On the other hand, immunotherapy is also under investigation in the setting of post‐osimertinib treatment. Previously study suggested that the efficacy of immunotherapy as monotherapy decreased for patients harboring EGFR mutations.Based on the findings from our retrospective study, we also conducted preliminary in vitro assays to evaluate the efficacy of anlotinib and in combination with osimertinib in EGFR T790M‐mutant osimertinib‐resistant NSCLC cell line H1975OR. And our results indicated that anlotinib alone or combined with osimertinib showed potent cytotoxicity against H1975OR, providing a basis for further preclinical and clinical investigations.However, this study had several limitations. First, this was a retrospective, non‐randomized study that selection bias existed inevitable due to unavoidable missing data. However, the baseline clinical characteristics of patients in the different groups were balanced well or were processed by PS matching, indicating that no large selection bias existed. Secondly, antiangiogenic‐based combination therapy in our study achieved a better efficacy while adverse events increased unavoidable. Due to the missing data in our retrospective study, adverse events and their impact on the patients' quality of life were not assessed. Clinical trials are needed to further explore the adverse effects and the efficacy. Thirdly, our study lacked in vivo experiments to further verify the efficacy of anlotinib alone or combined with osimertinib. Finally, more and more EGFR‐mutated, advanced NSCLC patients receive osimertinib as first‐line treatment, whether the antiangiogenic‐based therapy is also applicable to first‐line osimertinib resistant patients remains unclear, which needs to be further explored.Collectively, our study showed that anlotinib alone or combined with osimertinib might be an effective treatment for patients with acquired osimertinib‐resistant EGFR T790M mutant NSCLC.Ya Chen: Conceptualization ; data curation ; writing – original draft . Hongyu Liu: Conceptualization ; writing – review and editing . Nana Hu: Methodology ; validation . Yanan Wang: Formal analysis ; investigation . Zhengyu Yang: Supervision ; validation . Junqiang Zhang: Writing – review and editing . Baohui Han: Writing – review and editing .This research received funding from Science and Technology Commission of Shanghai Municipality (20YF1428100) and National Multi‐disciplinary Treatment Project for Major Diseases (2020NMDTP).The authors declares no conflict of interest.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study. If the research was not conducted in accordance with the Helsinki Declaration, the authors must explain the rationale for their approach, and demonstrate that the institutional review body explicitly approved the doubtful aspects of the study.


Klebsiella variicola in Enterobacteriaceae is isolated exclusively in CD‐MAT and can induce a pro‐inflammatory response in vitro and exacerbates colitis both in dextran sulfate sodium (DSS)‐induced colitis mice model and IL‐10−/− spontaneous colitis mice model. Mechanistically, active type VI secretion system (T6SS) is identified in the genome of K. variicola, which can impair the intestinal barrier by inhibiting the zonula occludens (ZO‐1) expression. Dysfunction of T6SS by CRISPR interference system alleviates the inhibitory effect of K. variicola on ZO‐1 expression and attenuated colitis in mice. Overall, these findings demonstrate that a novel colitis‐promoting bacteria exist in the mesenteric adipose tissue of CD, opening a new therapeutic avenue for colitis management.Mesenteric adipose tissue (MAT) in Crohn's disease (CD) is associated with transmural inflammation. Extended mesenteric excision can reduce surgical recurrence and improve long‐term outcomes, indicating that MAT plays an important role in the pathogenesis of CD. Bacterial translocation has been reported to occur in the MAT of patients with CD (CD‐MAT), but the mechanisms by which translocated bacteria lead to intestinal colitis remain unclear. Here it is shown that members of Enterobacteriaceae are highly enriched in CD‐MAT compared with non‐CD controls. Viable  Klebsiella variicola isolated from CD‐MAT promotes host intestinal inflammation through type VI secretion system (T6SS), which impairs intestinal barrier by inhibiting the expression of zonula occludens‐1 (ZO‐1). Dysfunction of T6SS alleviates the inhibitory effect of K. variicola on ZO‐1 expression and attenuates colitis in mice.Enterobacteriaceae is highly enriched in the mesenteric adipose tissue from patients with Crohn's disease (CD‐MAT).  Therefore, MAT of patients with CD (CD‐MAT) has been regarded as a pathological feature of CD. Due to that extended mesenteric excision has been proven to reduce surgical recurrence and improve long‐term outcomes, CD‐MAT has recently emerged as an independent risk factor for the pathogenesis of CD. However, how does CD‐MAT influence the pathogenesis of CD and the underlying mechanisms remain to be demonstrated.Mesenteric adipose tissue (MAT) around the involved intestine in Crohn's disease (CD) form a special extra‐intestinal manifestation called “creeping fat.”1 It i Pro‐inflammatory cytokines, such as C‐reactive protein, resistin, leptin, and adiponectin, produced locally in MAT have been suggested to induce a systemic inflammatory response and intestinal colitis. Considering the initial of inflammatory response in CD‐MAT, bacteria existed in MAT are of great concern. Currently, there is growing evidence that bacteria translocated in MAT occurs in host intestinal inflammation. Ha et al. (2020) reported that specific immune responses elicited by the translocated bacteria (Clostridium innocuum) could induce the formation of “creeping fat.” Additionally, using multidimensional data, we previously reported that the existence of the MAT microbiome contributed to alterations in the metabolic and transcriptional characteristics of CD‐MAT, and certain isolated pathogenic bacteria in MAT could exacerbate colitis in mice. However, the mechanism by which the translocated bacteria promote intestinal colitis remain unclear.An “outside‐in effect” has been proposed to be involved in MAT‐induced intestinal inflammation.6 Pro‐ Expansion of Enterobacteriaceae has been regarded as a dominant dysbiosis characteristic in inflammatory bowel disease (IBD) patients. Studies have revealed the existence of elevated numbers of mucosa‐associated Escherichia coli (E. coli) in IBD patients, and an enrichment of adherent invasive E. coli (AIEC) strains in the ileac mucosa of patients with CD. Bacteria in MAT has been proposed to be originally translocated from the intestinal lumen. Hence, we proposed that MAT may act as another hidden place for CD‐related pathobionts. The epithelial barrier, maintained by the regulation of tight junctions (TJs) and adherens junctions (AJs), plays a critical role in protecting the body from exogenous elements including pathogenic bacteria, and breakdown of this barrier is a hallmark of IBD. Intestinal pathobionts  have been shown to destabilize host intestinal mucosal barrier by secreting enzymes or toxins that cleave the mucus or directly disrupt tight junctions in the underlying epithelial cells. Secretion system is widespread in intestinal associated pathobionts, which dedicated to delivery toxin into prokaryotic and eukaryotic cells. Recent studies have shown that the type VI secretion system (T6SS) is involved in suppressing host immune response and reorganizing the actin cytoskeleton within host cells. Actin cytoskeleton regulates and disrupts TJs and AJs that maintain epithelial barrier. However, it is unknown whether T6SS is involved in the pathogenesis of CD.Transmural inflammation commonly occurs during CD development.11 ExpHere, we analyzed the bacterial community structure in MAT and identified the CD‐MAT specific bacteria by 16S rRNA gene sequencing and culture‐omics. The role of our identified bacteria in inducing intestinal barrier dysfunction and promoting inflammation was examined in vitro and in vivo. We reported that the pro‐inflammatory and barrier disruption roles of our candidate bacteria were mediated by T6SS. Additionally, we also demonstrated that T6SS was highly expressed in the fecal microbiome of patients with CD.22.1FigureStreptococcus, Burkholderia, Pseudomonas, Acinetobacter, Geobaccillus, and Thermus was associated with Enterobacteriaceae accumulation in CD‐MAT  analysis (LDA ≥ 4) to identify the differentially abundant taxa in the MAT of CD at multiple levels. It was observed that Enterobacteriales, Enterobacteriaceae , and uncultured_f_Enterobacteriaceae were consistently significantly enriched in the mesenteric tissue of patients with CD Figure1A, indiT Figure , which mT Figure . In addiT Figure . Overall2.2FigureEscherichia fergusonii, Klebsiella variicola, Shigella flexneri, and Klebsiella pneumoniae)  using Klebsiella‐specific probes under sterile conditions  under anaerobic and aerobic conditions and picked 229 colonies (174 from CD and 55 from control) with different colony appearances on different culture plates. 16S rRNA gene sequencing revealed that these colonies were classified into 17 families Figure2A, with) Figure . As showD Figure . Observas Figure .Klebsiella pneumoniae and Klebsiella oxytoca in the initiation and perpetuation of colitis have been revealed, however, the pro‐inflammatory role of K. variicola was rarely mentioned. To determine the pro‐inflammatory effect of our 4 isolated strains of K. variicola , a bacteria‐cell  co‐culture experiment was carried out. The result showed that, despite the extent of their pro‐inflammatory ability differed with each other, 4 strains of K. variicola could respectively evoke a strong pro‐inflammatory response toward IEC‐6 cell, in comparison to a commensal bacterial strain of E. fergusonii . Whole‐genome sequencing of K. variicola was performed, phylogenetic identity of K. variicola was confirmed again based on a set of conserved functional genes   was also confirmed  quantitative PCR. K. variicola was positively detected in 16 out of 47 (34.04%) CD‐MAT samples, in comparison to 4 out of 18 (22.22%) in the non‐CD‐MAT. The distribution of K. variicola in CD‐MAT was in the range of 14–66 537 CFU mg−1, with a median abundance being 65.5 CFU mg−1 (Table K. variicola. The results showed that K. variicola was positively detected in 7 out of 16 (43.75%) CD‐intestinal tissues, in comparison to 1 out of 8 (12.5%) in the non‐CD‐intestinal tissues. The distribution of K. variicola in terminal ileum mucosa from CD patients was in the range of 9–13 718 CFU mg−1, with a median abundance being 112 CFU mg−1  mice spontaneously developed chronic enterocolitis similar with chronic IBD in humans, when exposed to normal commensal bacteria. Thus, to better understand the inflammation‐promoting role of K. variicola, an IL‐10‐deficient mouse model was used in this study , resulting in an increased DAI in K. variicola‐treated group  daily into SPF mice treated with a 5‐day antibiotic cocktail for seven consecutive days. Mice were given drinking water instead of 3% DSS to exclude damage from medicinal chemicals , Occludin, Claudin1, and Synaptopodin (SYNPO) expression levels. As shown in Figure ZO‐1 was significantly suppressed in K. variicola‐treated mice compared to those treated with E. fergusonii or PBS, but not for the expression of Occludin and Claudin1 and SYNPO. Immunofluorescence staining confirmed the downregulation of ZO‐1 in mouse colonic tissues  system to knockdown ClpV, a core gene in T6SS   Figure  and K. vs Figure . Moreoves Figure . This isn Figure . In the 3K. variicola was exclusively isolated from CD‐MAT, which exacerbated colitis in mice through the disruption of the intestinal barrier by T6SS. Moreover, a higher abundance K. variicola was also observed in the gut microbiome of CD patients compared to healthy controls, suggesting that K. variicola may play an active role in CD pathogenesis.In this study, we demonstrated the existence of colitogenic pathobionts in the MAT of patients with CD and explained their detrimental role in promoting intestinal inflammation. We showed that members of Enterobacteriaceae were significantly enriched in CD‐MAT. A newly defined intestinal pathogen in Enterobacteriaceae— obesity and type 2 diabetes, and IBD patients. Of them, Ha et al. (2020) first conducted the functional analysis of the translocated bacteria, which showed that members of Clostridium in CD‐MAT could promote the restructuring of MAT and the formation of “creeping fat.” Considering that extended mesenteric excision is associated with reduced surgical recurrence, we speculated that pathogenic bacteria might be involved in the pathogenesis of CD in CD‐MAT. Our previous study highlighted that Proteobacterium plylum is correlated with CD recurrence and discovered that Achromobacter pulmonis isolated from CD‐MAT could exacerbate colitis in mice. Nevertheless, as a newly defined microbial niche, there might still be numerous undiscovered microorganisms with specific functional roles in the mesenteric tissue. An expansion of Enterobacteriaceae has been regarded as a dominant dybiosis characteristic in IBD patients, indicating its significant role in colitis promotion. Additionally, the migration of Enterobacteriaceae from lumen into different niches of gut  has been reported frequently. Here, we observed that Enterobacteriaceae were significantly enriched in MAT of CD patients, consistent with results from a previous study in which the mesenteric bacteria were translocated from the intestinal lumen. Additionally, we observed heterogeneity in the distribution of Enterobacteriaceae in CD‐MAT. MAT in the high‐E group had much lower bacterial diversity, indicating an intensive translocation of a specific bacterial group. Besides, pathogenic or opportunistic bacteria  accumulated in the bacterial community from the high‐E group, suggesting that Enterobacteriaceae may play a detrimental role in the pathogenesis of CD.The existence of bacterial translocation in MATs has been previously identified, including in DSS‐treated colitis in mice,37 obeK. variicola belonging to Enterobacteriaceae existed exclusively in CD‐MAT. K. variicola is a member of the Klebsiella genus, which was initially regarded as a plant‐associated pathogen and subsequently recovered from human clinical specimens. It has been suggested that approximately 20% of human isolates previously thought to be K. pneumoniae are K. variicola/KpIII or K. quasipneumoniae/KpI. In the current study, through whole‐genome sequencing and genotyping‐based phylogenic analysis, we were able to differentiate K. variicola from the other three species. Genes associated with adhesion, invasion, and motility were observed in the K. variicola genome, indicating its translocation ability. Additionally, the genomic data of K. variicola displayed a wide metabolic versatility (including lipid metabolism) and anti‐oxidative damage ability, which may help enable K. variicola to successfully survive in the MAT niche. Additionally, although not clearly explained by Ha et al. (2020), Klebsiella spp. was also exclusively isolated from the MAT of patients with IBD, confirming that Klebsiella spp. commonly co‐existed with mesentery tissue.Based on the culture‐omics data, we identified that pecimens.40 It K. pneumoniae and K. oxytoca in Klebsiella spp. have been reported to have colitogenic roles in the initiation and perpetuation of intestinal inflammation. However, whether K. variicola has a colitogenic role remains unknown. Here, we isolated 4 strains of K. variicola from 3 different CD‐MAT samples and observed that they all could induce a significant pro‐inflammatory phenotype upon cell‐bacterial co‐culture in vitro. Furthermore, we confirmed that K. variicola exacerbates colitis in mice. To our knowledge, this is the first evidence that colitogenic K. variicola exists in the mesenteric tissue. Previous studies have shown that mice deficient in Muc2 have increased translocation of both commensal and pathogenic bacteria, supporting that epithelial barrier dysfunction can accelerate passive migration. Considering K. variicola is a colitogenic pathogen translocated from the intestinal lumen, we propose that K. variicola may promote intestinal barrier dysfunction. As expected, we observed that K. variicola promoted intestinal barrier disruption in a mouse model without DSS treatment. Bacterial toxins or effectors produced by the bacterial secretion system contribute to the intestinal barrier disruption. For example, the zonula occludens toxin, a toxin produced by Vibrio cholerae, interacts with the extracellular domains of tight junction proteins  to disrupt epithelial integrity. Actin cytoskeleton regulate and stabilize TJs and AJs. C. rodentium uses type III secretion systems to reorganize the cytoskeleton and consequently alter the architecture and integrity of tight junction proteins. T6SS in V. cholerae can induce actin cross‐linking, which is associated with intestinal inflammation. In this study, using whole‐genome sequencing, we identified the presence of gene clusters encoding core T6SS components in K. variicola. Meanwhile, the existence of representative genes of T6SS were also identified in the other 3 stains of K. variicola. ClpV ATPase is crucial for T6SS functioning, and the inhibition of ClpV significantly reduced the K. variicola induced inflammatory effect in vitro and in vivo and alleviated its inhibitory effect on ZO‐1 expression. Importantly, we validated that K. variicola and ClpV were enriched in the gut microbiome of patients with CD, suggesting that pathogenic bacteria with barrier disruption role as well as T6SS are closely related to the pathogenesis of CD.Previously, ammation.27 HowK. variicola is translocated from the intestinal lumen, but the fecal samples paired with MAT specimens are not available. The existence of higher level of K. variicola was thus validated by using published data (PRJEB15371). Additionally, we identified that T6SS plays an active role in promoting inflammation; however, the function of the specific core protein in T6SS remains to be demonstrated.However, there were limitations to this study. We proposed that In conclusion, this study provides a framework to show that colitogenic pathobionts exist in the CD mesentery, which can induce a breakdown of the epithelial barrier and the release of pro‐inflammatory cytokines . The diagnosis of CD was based on an integrated observation of endoscopy, clinical, and histopathological data. Clinical information of the recruited cohort is listed in Table Klebsiella variicola was identified as a CD‐specific species for functional and mechanistic analysis in the current study. In total, 4 strains of K. variicola were isolated from 3 different CD‐MAT samples, which were named as K. variicola ZSLY01, K. variicola ZSLY02, K. variicola ZSLY03, and K. variicola ZSLY04. All of them showed pro‐inflammatory ability toward the intestinal cell line, IEC‐6 (detected by cell‐bacteria co‐culture experiment). K. variicola ZSLY01 was chosen as representative strain for the subsequent in vivo and in vitro experiments. In the following context, all K. variicola mentioned refers to K. variicola ZSLY01 unless otherwise specified. Additionally, the following reference strains were used in this study: reference adherent/invasive Escherichia coli strain LF82, E. coli DH5α and a commensal strain of Escherichia fergusonii isolated from SPF C57BL/6 mice. Escherichia coli strain LF82 had been reported associated with ileac Crohn's disease, which is a lactose fermenter that grows in pink colonies on MacConkey's agar and was used as a prey cell in the inter‐bacterial killing assay; while the latter two were used as a non‐pathogenic bacterial control in the authors’ cell and mouse experiments. These bacteria were grown in brain‐heart infusion (BHI) medium under aerobic conditions at 37 °C for 14–16 h.The strains isolated from MATs are listed in Table  Mixtures with denatured amplicons and 20% PhiX Control v.3 were then sequenced on Hiseq 2500 . Quantitative Insights into Microbial Ecology 2 (QIIME II) using reference parameters was used for demultiplexing and quality filter.DNA was extracted from MAT samples using the bead‐beating method with a DNeasy Blood and Tissue Kit . The hypervariable V4 region of the 16S rRNA gene was amplified by PCR using dual barcode primers as previously described.46 MixKlebsiella spp.  were diluted to a final concentration of 5 ng µL−1 in the hybridization buffer . After hybridization, the sections were rinsed with wash buffer  for three times (5 min/time), with Tris‐buffer for three times (3 min/time) at 48 °C and 1 min in ddH2O. Dry the slides in an oven at 46 °C for 10 min, and then mounted the slides by Prolong anti‐fade mounting media with DAPI (Life Technologies). Microscopic observations were performed by Leica Laser Scanning confocal microscope .Paraffin‐embedded MAT histopathological sections were de‐waxed and hydrated first, followed by the overnight hybridization step performed at 46 °C. EUB338 probe  and specific probe designed for K. variicola ZSLY01, K. variicola ZSLY02, K. variicola ZSLY03, K. variicola ZSLY04, E. fergusonii, K. variicola‐vector, or K. variicola‐dcas‐ClpV) at a multiplicity of infection (MOI) of 20 at 37 °C, 5% CO2 for 4 h. Then, they were washed with warm PBS containing antibiotics . Cells were then cultured for another 1 h in DMEM (Gibco) containing the antibiotics mentioned above. RNA from cells was extracted using TRIzol reagent (Thermo Fisher Scientific) and the gene expression was measured by qPCR.Murine preadipocyte 3T3L1 or rat intestinal epithelial cell line IEC‐6 were co‐cultured with bacteria . To assemble the filtered data, the Canu v1.5 package was used. Racon v3.4.3 and Circlator v 1.5.5 were employed to check the quality of the assembly. Gene prediction was carried out using the Prodigal (v2.63) software program. RepeatMasker (v4.0.5) was used to predict repetitive sequence.[50] PhiSpy v2.3 and CRT v1.2 were used for prophage prediction and CRISPR identification, respectively. Functional characterization of the identified genes was completed by diamond with the E‐value threshold of 1E‐5 against the databases of the NCBI Non‐Redundant protein database (NR), Swiss‐Prot, Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO).The whole genome sequencing of was used.47 Rac−/− mice (6–8 weeks old) were purchased from Gem Pharmatech Co. Ltd. (Guangzhou) and Beijing HFK Bioscience Co. Ltd. (Beijing) and were housed under a 12‐h light‐dark cycle in specific pathogen‐free (SPF) facilities. All animal study was performed in accordance with guidelines approved by the ethics committee of the South China Agricultural University (No. 2019c026). To explore the pro‐inflammatory role of K. variicola in the pathogenesis of colonic inflammation, the dextran sulfate sodium (DSS)‐induced colitis model and spontaneously developing colitis model (IL‐10−/− mice) were used. For DSS‐induced colitis model, male C57BL/6 mice were daily gavaged with bacteria (1×109 CFU/mouse) for 14 days after being treated with the antibiotic cocktail  in the drinking water for 5 days. 3% DSS was administered on day 0, which lasted for seven consecutive days, and then changed by regular water for two days. The mice were monitored for weight loss, stool consistency, and hemoccult during the experiments, and the disease activity index (DAI) was calculated from the observations above. For spontaneously developing colitis model, IL‐10−/− mice were treated with the above antibiotic cocktail in the drinking water for 5 days, followed by daily gavage of bacteria (1×109 CFU/mouse) for 21 consecutive days. During the experiment, mice were monitored for weight loss, stool consistency, and hemoccult, and the DAI was calculated from the observations above. In addition, to explore the impact of K. variicola on intestinal barrier function, male SPF C57BL/6 mice were gavaged with K. variicola for 7 consecutive days after a 5‐day‐antibiotic cocktail treatment.C57BL/6 and IL‐10ere used.40 For Briefly, E. coli LF82 and K. variicola , which were defined as prey and predator cells, respectively, were harvested after overnight growth, washed, and concentrated to (OD600) in PBS. Predator cells were well mixed with E. coli at a ratio of 1:1, which were then spotted onto pre‐warmed LB agar plates. The plates were incubated at 37 °C for 4 h and then each spot was cut out in the LB plate and suspended in sterile PBS. The number of prey cells recovered on the MAC agar plate was counted to determine the colony‐forming units per mL.The contact‐dependent neighbor‐killing assay was performed according to a previously established protocol, with minor modifications.53 Brihttp://www.rgenome.net/cas‐designer/) targeting the gene ClpV was cloned into plasmid #62226 with a single SpeI restriction site. Construction of the complete N20 containing plasmid was conducted by single digestion with SpeI, template excision with DpnI, and fragment ligation with T4 DNA ligase (Thermo Scientific). This plasmid was subsequently transformed into E. coli DH5α and subjected to Sanger sequencing to confirm successful insertion (RuiBiotech). To create K. variicola‐dcas‐ClpV, competent K. variicola cells were first transformed with dcas9 with kanamycin resistance and then prepared for competent cells for the second transformation with N20‐pTargetF with spectinomycin resistance. The original pTargetF vector without N20 was transformed into K. variicola‐dcas competent cells to obtain the K. variicola‐vector as a negative control. The inhibition efficiency of ClpV was evaluated by qPCR.The CRISPR interference (CRISPR i) system was used to inhibit ClpV expression in T6SS. This system worked from two plasmids: one plasmid encoding constitutive dCas9 , and another encoding constitutively expressed sgRNA targets . The newly designed sgRNA on the website (https://www.omicstudio.cn/tool). Significance between two groups was determined by an unpaired two‐tailed Student's t‐test. Significance between data that did not pass the normal distribution was determined with the unpaired two‐tailed Mann–Whitney test. For comparisons between multiple groups, an ordinary one‐way or two‐way analysis of variance (ANOVA) was utilized, followed by Tukey's and Dunnett's tests. For comparison between multiple groups with two factors, two‐way ANOVA was used, followed by Tukey's multiple comparison test. The specific statistical details were shown in the figure captions and the source data. Differences were regarded as significant when p < 0.05. Unless otherwise indicated, data for all experiments are presented as the mean ± standard error of the mean (SEM).GraphPad Prism 8 was used for statistical analyses, except for ROC, which was analyzed using an online bioinformatics analysis platform . J.Y. and J.K. helped with sample collection. Y.X. helped with clinical data collection. J.W. and Y.C. helped with data analysis. All authors read, revised, and approved the final manuscript.Supporting InformationClick here for additional data file.