Nipple-sparing mastectomy (NSM) offers superior cosmetic outcomes and has been gaining wide acceptance. It has always been difficult to objectively quantify the risk of nipple-areola complex involvement (NACi). The goal was to develop a prediction model for clinical application.n = 578) and those who had NSM + immediate breast reconstruction (IBR) between January 2020 and January 2021 formed the validation cohort (n = 112). The prediction model was developed using univariate and multivariate logistic regression studies. Based on NACi risk variables identified in the development cohort, a nomogram was created and evaluated in the validation cohort. Meanwhile, stratified analysis was performed based on the model’s risk levels and was combined with intraoperative frozen pathology (IFP) to optimize the model.Patients who had a total mastectomy (TM) between January 2016 and January 2020 at a single institute formed the development cohort  > 4.0 cm, tumor-nipple distance (TND) ≤ 1.0 cm, clinical nodal status positive (cN +), and KI-67 ≥ 20% were revealed to be good predictive indicators for NACi. A nomogram based on these major clinicopathologic variables was employed to quantify preoperative NACi risk. The accuracy was verified internally and externally. The diagnostic accuracy of IFP was 92.9%, sensitivity was 64.3%, and specificity was 96.9% in the validation group. Stratified analysis was then performed based on model risk. The diagnostic accuracy rates of IFP and NACiPM in low-risk, intermediate-risk, and high-risk respectively were 96.0%, 93.3%, 83.9%, 61.3%, 66.7%, and 83.3%.We created a visual nomogram to predict NACi risk in breast cancer patients. The NACiPM can be used to distinguish the low, intermediate, and high risk of NAC before surgery. Combined with IFP, we can develop a decision-making system for the implementation of NSM. The surgical treatment of breast cancer has evolved from maximal radical cure to maximal preservation of shape and function. Breast surgeons and patients pay attention to the expected survival time while taking into account the improvement of postoperative quality of life . The surHowever, previous studies have found that the rate of occult (pre)-malignant invasion of a clinically normal nipple in mastectomy specimens ranged from 0 to 58% , 8, implNSM was provided to patients with a clinically normal NAC and no skin involvement. Although anomalous NAC clinical signs  are frequently thought to be significant evidence of NAC invasion, there is presently no consistent and efficient preoperative evaluation technique for clinically occult NAC invasion. Previous research has found that tumor size, tumor-to-nipple distance, lymph node status, and tumor location are all clinical characteristics that may be linked to nipple-areola complex involvement (NACi) , 13. PatNomograms based on clinical, imaging, and pathology data have been proven to provide more accurate predictions of tumor risk and treatment results for individual patients , 20. We Data were collected from 766 patients with breast cancer treated with total mastectomy (TM)/NSM + immediate breast reconstruction (IBR) between January 2016 and January 2021 at a single institute. The exclusion criteria: (1) unclear NAC pathological results, (2) abnormal clinical NAC  [Database research indicators include age, multifocal/single lesions, menopausal status, tumor location, nipple discharge, family history of cancer, clinical tumor size (CTS), TND, clinical nodal status (cN), mixed carcinoma in situ (MCIS), histological grade, pathological type, estrogen receptor (ER), progesterone receptor (PR), human-epidermal growth factor receptor 2 (HER2), and KI-67. Each variable can be accurately obtained preoperatively.To ensure study stability, we systematically identify and categorize included variables. When a cancer diagnosis has been made, multifocal tumors are characterized as two or more tumors in the same breast .The cylindrical breast tissue from under the areola to the chest wall under the NAC was defined as the central region . Nipple CTS was defined as the largest tumor diameter in unifocal instances and the maximum tumor diameter in multifocal situations. TND was defined as the lowest vertical distance from the tumor to the center of the ipsilateral nipple, with multifocal tumors having the shortest TND . The maxTM was approved in all patients in the development cohort, and NAC was given to a thorough pathological evaluation. NAC status was assessed in the validation cohort using intraoperative frozen pathology (IFP) and postoperative paraffin-fixed pathology. The subdermal glandular tissue was undermined in NAC, leaving 1 to 2 mm of the dermis intact. Then, after collecting a frozen section, a thin layer of glandular tissue was collected under the areola for evaluation [t test. Categorical variables were compared using the χ2 test or Fisher’s exact test. The clinical features data discrepancies between the development and validation cohorts were compared.The mean  of continuous variables is calculated and compared using an unpaired, two-tailed n = 1000).The development cohort was conducted to a univariate analysis to identify potential predictors of NAC positivity. Predefined variables were incorporated in a multivariate binary logistic regression analysis to determine corresponding regression coefficients with a 95% confidence interval (CI) for the development of the prediction model. These variables were used in a backward selection method to identify the independent risk factors for NAC involvement in a binary logistic regression analysis. Based on the backward stepwise regression method of R software, the effective variables were screened. A nomogram was developed to be a graphic representation of the model. For assessing the discriminative performance of the nomogram, the area under the curve (AUC) of the receiver operating characteristic (ROC) was measured. The calibration of the nomogram was performed internally in the development cohort and externally in the validation cohort, using a calibration plot with bootstrap sampling  or R version 4.1.2 software . All p > 0.05). The validation cohort, on the other hand, contained more postmenopausal, invasive cancer, and HER2-positive patients, and the difference was statistically significant (p < 0.05).Data from 578 patients in the development cohort and 112 individuals in the validation cohort were included in this study. Table p > 0.05). To forecast NACi risk, multivariable logistic regression was performed using the five factors presented in the nomogram.The development cohort’s potential predictors and the binary outcome, NAC involvement, were studied using univariable logistic regression analysis. Table p < 0.001). Central location tumor  and TND were highly correlated. According to the results, larger tumor size patients (> 4.0 cm) were more inclined to add the risk of NACi than small tumor size . Patients with cN + breast cancer were more likely to increase the risk of NACi than patients with cN- . In contrast, patients with the low KI-67 disease were more likely to reduce the risk of NACi than patients with high KI-67 .Based on the backward stepwise regression method of R software, the effective variables were screened. TND was the most effective factor for predicting NACi risk, .ROC analysis was performed to validate the nomogram internally in the development cohort Fig. A and extA 45-year-old patient was diagnosed with left-sided breast cancer. Imagine examination before surgery showed: CTS was 4.2*2.5 cm (MG), 3.50*1.05 cm (US), tumor central location, and TND was 1.21 cm (MG), 1.54 cm (US), cN ( −), Preoperative CNB biopsy: invasive ductal carcinoma, G2, ER +  + (85%), PR , HER-2 ( −), and KI67 (35%). NACi risk can be predicted by nomogram was 5.36%, and postoperative pathology confirmed NAC negative , and the accuracy of IFP in the intermediate-risk group was higher, the difference was statistically significant (p = 0.046)  positivity was defined as the NACi risk corresponding to the best cutoff value of the nomogram. We further combined the NACiPM with IFP and stratified analysis according to low-risk (NACi risk < 10%); intermediate-risk (10–50%); high risk (> 50%). The diagnostic accuracy rates of IFP and NACiPM in low risk, intermediate risk, and high risk respectively were 96.0% (72/75), 93.3% (70/75), 83.9% (26/31), 61.3% (19/31), 66.7% (4/6), and 83.3% (5/6). The diagnostic accuracy of IFP and NACiPM in the low-risk group and the high-risk group was comparable , NSM would be recommended. While patients with NACi risk > 50% and IFP ( +), SSM would be suggested. Patient-physician shared decision-making would be recommended in the rest of the situation Fig. .Fig. 7FoAt present, the commonly used clinical prediction tools for NACi include clinical physical examination, imaging, and pathological evaluation. Although the criteria used to select NSM for IBR have broadened over time, the consensus has not yet been reached regarding this issue , 17, 18.There is an urgent need for really simple and reliable prediction models that can be used in clinical practice. Hirohito Seki and colleagues have carried out a series of studies and achieved good prediction results , 18. CTSPublished studies show larger CTS and smaller TND are always more likely to lead to NAC positive, but different tumors in imaging, especially when combined with carcinoma in situ components, show great differences , 29. HowIn this study, according to the binary logistic regression model, it was concluded that CTS > 4.0 cm, TND ≤ 1.0 cm, cN( +), tumor central location, and ki-67 ≥ 20% were independent predictors of NACi risk. Although we analyzed more histopathological variables, we did not find significant associations between these factors and NAC involvement. The nomogram was produced drawn on different weight coefficients, and the model accuracy rate was 0.776 in the development group and 0.843 in the validation group. This may be the first visualization model for NACi risk prediction to our knowledge, the internal validation results of both groups were good. This model can visually analyze the data well, score each independent predictor separately, and calculate predicted probability. This simple and easy visualization process greatly facilitates preoperative doctor-patient dialogue and the prediction of risk assessment.The occult positive rate of NACi was 11.1% (64/578) in the development group and 12.5% (14/112) in the validation group, with no significant difference between the two groups. This indirectly demonstrates that IFP can accurately estimate the potential of NACi , 33. TheWe further conducted stratified analysis according to the model risk and set NACi risk < 10% as low-risk, 10–50% as intermediate-risk, and > 50% as high-risk. According to our research data, in the low-risk group, both IFP and the model had a certain false-negative rate, and IFP was higher, and the false-positive rate of both was very low. This may explain that the model can be complementary to IFP improvements in some patients. Because a higher false-positive rate means that more NAC may be resected incorrectly, the particularly pernicious false-positive rate is significantly greater than that of reoperation due to false negatives.In our study, the results suggested that the IFP diagnosis was negative in 5 patients, while the postoperative pathology was positive. One case of DCIS and 4 cases of lobular carcinoma in situ (LCIS) was diagnosed by postoperative pathology. For NAC diagnosed as LCIS, clinical management is relatively difficult, if LCIS is regarded as a precursor of invasive carcinoma ; thus, tThe model’s value was that it could appropriately screen the absolute low-risk and high-risk groups of NAC involvement preoperatively. For patients with NACi risk < 10% and IFP ( −) can implement NSM securely, while patients with NACi risk > 50% and IFP ( +) should be recommended SSM. For patients with intermediate-risk, or NACi risk < 10% and IFP ( +), or NACi risk > 50% and IFP ( −), these three scenarios should recommend patient-physician shared decision making. This prediction model is simple to calculate, which greatly facilitates the judgment of clinical surgeons in the assessment of the positive risk of NACi.In this study, we developed a NACiPM based on non-MRI data, an accurate and visual predictive model in NSM + IBR. The NACiPM can be used to distinguish the low, intermediate, and high risk of NAC before surgery. Combined with IFP, we can develop a decision-making system for the implementation of NSM.


Congenital heart disease (CHD) is the most common congenital anomaly at birth, affecting approximately 1% of live births. In recent decades great medical and surgical advances have significantly increased life expectancy, shifting healthcare professionals' and researchers’ interests in patients' Quality of Life (QoL). The main aims of our study were to evaluate generic and condition-specific QoL in a group of Italian children and adolescents with CHD and their parents and examine the level of agreement and directional disagreement between child/adolescent and parents reports on generic and condition-specific QoL.A cross-sectional study was designed with CHD children and adolescents and their parents referred to the Cardiology Department of “Bambino Gesù” Children’s Hospital. The PedsQL scale was used, including generic (PedsQL 4.0) and cardiac-specific modules (PedsQL 3.0) were administered to patients and caregivers. A Kruskal–Wallis test was used to compare generic and cardiac module scores between patients with different ages, CHD diagnoses, and between patients who underwent surgery interventions and/or are currently taking cardiac medications.498 families were enrolled in this study. On average, patients reported a good level of generic and condition-specific QoL, as well as their mothers and fathers. Children aged between 5–7 years old reported lower generic and cardiac-specific total QoL levels than children aged 8–12 years and adolescents (13–18 years). With regard to the agreement, patient-parent agreement on condition-specific QoL ranged from 25 to 75% while on generic QoL, it ranged from 19 to 76%. The highest percentage of disagreement between parents and children was found in patients aged 5–7 years old, both for condition-specific and generic QoL rates.Our study contributed to the growing body of knowledge on QoL in CHD, emphasizing the need for these families to receive support from multidisciplinary standardized care, including psychological consultations and support.The online version contains supplementary material available at 10.1186/s12872-022-02611-y. Congenital heart disease (CHD) is the most common congenital anomaly at birth, affecting approximately 1% of live births . In the As a result of their condition and of the special medical care that they require, CHD patients often have behavioural, emotional, and cognitive needs that may impact their own and their families’ QoL , 9–11. PConsidering that no previous studies have investigated the QoL of Italian children with CHD and adolescents, the first aim of the current study was to evaluate the generic and condition-specific QoL of pediatric CHD patients (reported both by patients and their parents) referred to our institution by examining potential differences in QoL levels between ages , between CHD diagnoses, and between patients who underwent surgical interventions and/or are taking cardiac medications at present, to inform clinicians on patients’ QoL.Even if patient self-reports should be preferred in clinical practice as the standard for evaluating their perceived QoL, there may be several situations in which parent proxy-reports are required. For example, when the children are too young, ill, fatigued, or cognitively impaired to complete the questionnaires. To date, while parental proxy-reports have been widely validated in the literature , 25, theTo the best of our knowledge, this is the first study describing the QoL of Italian CHD pediatric patients and directional disagreement between parents and children with CHD and adolescents, including both mothers’ and fathers’ perspectives.The current study represents the first step of a larger research project aiming at analysing the QoL and psychological adjustment of Italian CHD patients and their families referred to the Cardiology Department of “Bambino Gesù” Children’s Hospital, a tertiary pediatric hospital located in Rome, Italy. Specifically, in this cross-sectional study, children, adolescents, and their parents were recruited from January to December 2018 at our institution during the cardiological Day Hospital (DH). Cardiological DH is a follow-up appointment offered to all patients with CHD seen at our institution. It is placed in the setting of a multidisciplinary standardized follow-up care. During DH appointments, children and adolescents undergo a comprehensive evaluation by different physicians, including pediatric cardiologists, surgeons, and psychologists, at preset time frames. All patients enrolled in our study were followed up in the cardiology outpatient clinic. Clinical examination, 12 lead ECG and 2D echocardiography were performed at regular time intervals according to their cardiac conditions. In addition, psychological tests and consultations were performed on the same day of the cardiac evaluation. At the beginning of the psychological evaluation the clinician explained the study procedure to the patients and their parents and asked for their consent to participate. Considering that all the patients were minors at the time of recruitment (under the age of 18 years), parents signed the consent for themselves and their children, but only after obtaining verbal consent from the patient. After obtaining informed consent, the parents and patients completed the QoL questionnaires independently in two separate rooms. Due to their developing writing and reading skills, preschool-aged children were assisted in filling out the questionnaires by the clinician. Specifically, the clinician helped the children read the questions and signed the answers indicated by the children, as in the Italian validation study . Only afThe inclusion criteria were: 1) patients were aged between 2 and 18 years, and 2) parents and patients were able to understand the questionnaires. Non-Italian speakers and children diagnosed with severe neurodevelopmental disorders and/or with a diagnosis of intellectual disability were excluded from the study. These children had previously received their diagnosis, before the cardiological DH, and the psychologist was able to identify them through the priority path reserved to them for the DH appointments.Approval to conduct the study was given by the Ethics Committee of “Bambino Gesù” Children’s Hospital and all parents involved in the study signed written informed consent for them and their children to participate.PedsQL Generic Core Scales 4.0 scale [PedsQL 3.0 Cardiac Module [For QoL evaluation the .0 scale , 31-Italc Module , 33-Italc Module , 13.Kruskal–Wallis test was used to compare scores for generic and cardiac modules between patients of different ages , between CHD diagnoses, and between patients who underwent surgical interventions and/or are taking cardiac medications at present. For each Kruskal–Wallis test performed, follow-up analyses were conducted (pairwise comparisons in SPSS) to determine which comparisons were significant between groups [Model assumptions of normality were checked before statistical analyses exploring skewness (asymmetry) and kurtosis (kurtosis) of each variable . Descrin groups .To be consistent with the aim of obtaining a comprehensive understanding of patients’ QoL and to allow clinicians to understand the perception of both the patients and their parents, the comparisons were tested and reported for all patients, mothers and fathers.Aortopathies (Ao), which included all patients with aortic disease from the aortic valvular lesion to the aortic arch anomalies (i.e. aortic coartaction); Tetralogy of Fallot (ToF), independent from the surgical approach they had had (transannular or infundibular patch); Univentricular heart (UVH), which included all patients who have treated with Glenn or Fontan palliation according to the UVH physiology they had; Right ventricular-pulmonary artery conduit (RV-PA conduit), which included all patients requiring a connection from RV to PA by conduit instead of the natural RV outflow tract; Transposition to the Great Artery (TGA), which included all patients with atrioventricular concordance and ventricular artery discordance, treated with arterial switch operation; and Other Congenital Heart disease (oCHD), which included a miscellaneous of diseases not considered above.With regard to CHD diagnoses, patients were classified into six different categories, according to the congenital heart disease they had and the surgical treatment they had previously undergone. These 6 categories were: Finally, regarding the agreement and directional disagreement between child/adolescent and parent reports on generic and condition-specific, we calculated parent–child-disagreement as absolute and directional discrepancies , 36, 37.age = 46.15, SD = 5.30, range = 34–62), 323 fathers , 78 children aged 5–7 years old , 178 children aged 8–12 years old , and 195 adolescents aged 13–18 years old . Patients reported an average total generic QoL of 78.19 (SD = 12.63) and an average total condition-specific QoL of 78.82 (SD = 11.66). Mothers reported an average total patient-generic QoL of 79.22 (SD = 15.48) and an average total patient-condition-specific QoL of 73.17 (SD = 13.61). Fathers reported an average total patient-generic QoL of 82.17 (SD = 14.78) and an average total patient-condition-specific QoL of 76.51 (SD = 13.62). The sociodemographic and clinical characteristics of the sample are reported in Table A total of 578 families were admitted to the cardiologic DH when the psychologist responsible for this study was present. Twenty families were excluded because they were non-Italian speakers, and 50 families were excluded due to their children's diagnosis of severe neurodevelopmental disorders and/or intellectual disabilities. Ten families refused to participate. Finally, 498 families agreed to participate and were enrolled. No significant differences emerged between the families who refused to participate and the families enrolled. Specifically, our sample was composed of 429 mothers  = 32.003, p = 0.000) and cardiac-specific (H(3) = 7.873, p = 0.020) total QoL. In contrast, adolescents reported a worse perceived physical appearance than children in the other groups (H(3) = 25.146, p = 0.000). With regard to parent-reports, parents of children aged 5–7 years reported more problems with communication skills than parents of patients in the other groups (mothers: H(3) = 24.121, p = 0.000; fathers: H(3) = 12.805, p = 0.005), and parents of adolescents, reported a worse perceived physical appearance than parents of children aged 2–4 (mothers: H(3) = 45.649, p = 0.000; fathers: H(3) = 32.857, p = 0.000) and lower adherence to treatment than parents of children aged 2–4 and 5–7 (mothers: H(3) = 11.906, p = 0.008; fathers: H(3) = 17.287, p = 0.001). When CHD diagnoses were considered, significant differences emerged between UVH patients and other patients. Indeed, both UVH patients and their parents reported more cardiac symptoms (H(5) = 12.146, p = 0.033), worse perceived physical appearance (H(5) = 19.856, p = 0.001), and lower physical functioning (H(5) = 15.243, p = 0.009) than other patients. Finally, patients who underwent surgery and took medications at present and their parents reported more cardiac symptoms than patients who did not undergo surgery and did not take medications. Moreover, they perceived lower levels of physical appearance, a lower total score at the generic PedsQL and lower school functioning levels than children who take medications only.Results from the es Table , betweenes Table , and betes Table . These de.g., I get scared when I am waiting to see the doctor, I get scared when I have to go to the doctor). Moreover, children reported higher scores on cognitive problems  than both their mothers (disagreement rate 51%) and their fathers (disagreement rate 45%). Finally, an agreement of 23% was found between patient and father reports in communication skills.The agreement between mothers and fathers on generic QoL varied from 47 to 86%, while on condition-specific, it varied QoL between 52 and 100% .According to Varni et al.  and HuanNevertheless, the majority of these studies have mainly investigated the QoL of CHD patients through the PedsQL generic module, not including the cardiac-specific module. Moreover, they have focused only on specific age ranges and did not include both mothers and fathers in proxy-report evaluations. For these reasons, it is difficult to compare our results with this previous heterogeneous body of literature; moreover, no previous studies have been published on the QoL of Italian pediatric CHD patients. Our study has provided a comprehensive description of both generic and condition-specific QoL among Italian pediatric CHD patients at different ages (from 2 to 18 years), considering clinical factors such as CHD diagnosis and including both maternal and paternal reports. Our results can usefully inform clinical practice and guide clinicians’ efforts aimed at prevention and intervention.I feel I am not good looking, I don’t like other people to see my scars, I am embarrassed when others see my body) than children in the other groups. Adolescents with CHD not only have to face the challenges of living with a chronic condition but also need to accomplish the normative tasks of their developmental period, such as their body image development, which contributes in a fundamental way to the identity of adolescents. Due to their susceptibility to external influence and cultural values [With regard to the QoL levels of our patients in relation to their age and clinical factors, our results showed several differences between ages, CHD diagnoses, and between patients who underwent surgical interventions and/or are taking cardiac medications at present, highlighting the importance of considering sociodemographic and clinical factors when assessing the QoL of CHD patients. The main results showed that children aged 5–7 years reported the lowest generic- and cardiac-specific total QoL levels. During the transition from home to preschool or primary school, children should acquire skills that promote emotion recognition and regulation, empathy for others, problem-solving, and positive social interactions . Furtherl values , adolescl values . For thel values ; howeverl values . Howevere.g., I miss school because of not feeling well, I miss school to go to the doctor or hospital) than children who take medications only. These results should be interpreted with caution due to the small sample of the Medications-only group (n = 24). However, we may speculate that CHD patients who underwent surgery may have reported lower levels of school functioning because of the frequent absence from school related to medical examinations and/or hospitalizations.Our results also showed that significant differences emerged between UVH patients and other patients when clinical factors were considered. Previous studies have shown a correlation between QoL and the severity of CHD. For example, Marino and colleagues , using tIt is important to mention that the results from our study showed that beyond these differences based on the CHD diagnosis, interventions and medications, our patients and their parents reported high levels of QoL, above clinical cut-offs , 39. ThePediatric patient self-report should be considered the standard for measuring patients' QoL. However, there are situations in which parent proxy-reports are needed , 25, 59.Nevertheless, our results revealed a high disagreement rate between parents and patients aged between 5 and 7 years. Specifically, we observed that mothers underrated their children’s anxiety symptoms related to treatment for condition-specific QoL. Moreover, children reported higher scores on cognitive problems than both their mothers and their fathers, and a low agreement rate (23%) was found between patients and fathers in communicative skills. With regard to generic QoL both mothers and fathers showed a trend of underrating their 5–7-years-old children’s problems with generic physical, emotional, social and school functioning. According to previous studies, psychosocial domains related to thoughts and feelings  are often more difficult for parents to discern through observation, increasing the potential for discrepancies , 28, 60.For the first time, our study has explored agreement and directional disagreement between Italian CHD pediatric patients and their parents, both mothers and fathers, identifying possible conditions under which parent proxy-report instruments achieve better agreement with child self-report instruments that could facilitate clinicians’ interpretation of QoL outcomes. Including caregivers’ assessment can be an essential aspect in evaluating a child with CHD from the perspective of the use of healthcare facilities and the quality of communication between patients and their caregivers , 10. In The present study results could increase the use of patient-related outcomes in clinical practice and guide clinicians’ efforts aimed at prevention and intervention. Moreover, they could enable clinicians and researchers to broaden their understanding of CHD pediatric patients’ well-being  by proviOur study is not without limitations. First, it does not include a control group of healthy children. In future studies a multicenter approach should be taken into account in order to corroborate our findings. Second, it does not evaluate the role of potential risk and protective factors related to the QoL of CHD pediatric patients. Future studies should perform multivariate analyses, including as covariates patients’ age, type of CHD diagnosis, other clinical factors  and patients’ and parental psychological wellbeing. Another limitation is the cross-sectional research design. Follow-up measurements of patients’ QoL are needed to monitor patients’ QoL and familiar adjustment longitudinally. Finally, our study does not include an evaluation of QoL of CHD patients with neurodevelopmental disabilities and/or intellectual disabilities, and parental mental health.QoL levels in Italian children and adolescents with CHD were found to be high, above the clinical cut-offs , 39, accAdditional file 1. Supplementary Table 1. Differences between patients who underwent surgery interventions and/or are taking cardiac medications.Additional file 2. Supplementary Fig. 1. Agreement and directional disagreement between mothers and fathers of 2-4 y. patients on PedsQL Cardio. Supplementary Fig. 2. Agreement and directional disagreement between mothers and fathers of 2-4 y. patients on PedsQL General. Supplementary Fig. 3. Agreement and directional disagreement between mothers and fathers of 5-7 y. patients on PedsQL Cardio. Supplementary Fig. 4. Agreement and directional disagreement between mothers and fathers of 5-7 y. patients on PedsQL Generic. Supplementary Fig. 5. Agreement and directional disagreement between mothers and fathers of 8-12 y. patients on PedsQL Cardio. Supplementary Fig. 6. Agreement and directional disagreement between mothers and fathers of 8-12 y. patients on PedsQL Generic. Supplementary Fig. 7. Agreement and directional disagreement between mothers and fathers of 13-18 y. patients on PedsQL Cardio. Supplementary Fig. 8. Agreement and directional disagreement between mothers and fathers of 13-18 y. patients on PedsQL GenericAdditional file 3. Supplementary Fig. 9. Agreement and directional disagreement between 5-7 y. patients’ and parents’ reports on PedsQL Cardio. Supplementary Fig. 10. Agreement and directional disagreement between 5-7 y. patients’ and parents’ reports on PedsQL Generic. Supplementary Fig. 11. Agreement and directional disagreement between 8-12 y. patients’ and parents’ reports on PedsQL Cardio. Supplementary Fig. 12 Agreement and directional disagreement between 8-12 y. patients’ and parents’ reports on PedsQL Generic. Supplementary Fig. 13.Agreement and directional disagreement between 13-18 y. patients’ and parents’ reports on PedsQL Cardio. Supplementary Fig. 14. Agreement and directional disagreement between 13-18 y. patients’ and parents’ reports on PedsQL Generic.


An ‘on-demand’ contraceptive pill may suit women having infrequent sex. We assessed the feasibility of e-commerce pharmacy provision and acceptability of levonorgestrel (LNG) 1.5 mg for pericoital use in Kenya.A 12-month prospective, single-arm, open-label, interventional study conducted from August 2020 to August 2021. A total of 897 women aged 18–49 years at risk of pregnancy and having infrequent sex  were screened and enrolled. We undertook univariate and bivariate analysis on multiple data points: enrolment dataset, bimonthly surveys, extended survey around 6-month follow-up, and e-commerce pharmacy sales log.A total of 2291 pericoital LNG 1.5 mg pill doses were dispensed to 300 women over a 12-month period mainly via the USSD (Unstructured Supplementary Service Data) platform. Of the 266 women who obtained the pill and completed the survey, most (83%) were satisfied/very satisfied with e-commerce platform services, citing convenience and ease of use. The pill was also acceptable. Of the 266 women who used it at least once, 94% were satisfied/very satisfied, likely to continue using and would recommend it to others; 86% used it within 24 hours before/after sex as recommended and most of the time they had sex; and 147 (55%) experienced side effects, mainly nausea, headache, changes in menstrual pattern, cramps and dizziness that were mild and tolerable.E-commerce pharmacy provision of LNG 1.5 mg for pericoital use is feasible. In addition, a pericoital LNG 1.5 mg pill is acceptable among women having infrequent sex and could potentially address their unmet family planning needs. Levonorgestrel (LNG) 1.5 mg, a progestogen-only emergency contraceptive pill, is a potential method women can use on-demand or pericoitally .Evidence on feasibility of e-commerce pharmacy provision and acceptability of LNG 1.5 mg for pericoital use in low- and middle-income counties is scarce.E-commerce pharmacy provision of LNG 1.5 mg for pericoital use is feasible.LNG 1.5 mg for pericoital use is acceptable as a primary contraceptive method among women having infrequent sex.Adding the pericoital LNG 1.5 mg pill to the family planning method mix could expand method choice and address unmet need for family planning among women having infrequent sex including those who have never used, are dissatisfied or discontinued other modern methods.In 2019, an estimated 218 million women of reproductive age in low- and middle-income countries (LMICs) had an unmet need for family planning (FP) .Women having infrequent sex may be reluctant to take daily pills or use long-term methods due to intermittent risk of pregnancy or concerns about health risks such as interference with menstrual bleeding and infertility.Levonorgestrel (LNG), a progestogen-only emergency contraceptive pill (ECP), is a potential method women can use on-demand or pericoitally .This study assessed the feasibility of e-commerce provision and acceptability of LNG 1.5 mg for pericoital use as a primary contraceptive method based on an intervention study in two urban and peri-urban settings—Nairobi and Kiambu counties in Kenya. The findings can inform policy and programme discussions around the potential of LNG 1.5 mg for pericoital use as an additional contraceptive choice.Kasha—an e-commerce pharmacy platform distributing health, hygiene and self-care products in Kenya. Nairobi and Kiambu counties were selected due to relatively high modern CPR at 58% and 68%, respectively, and are currently served by Kasha distribution network that allows same day or next day delivery. Women were recruited from (a) 20 health facilities  by health providers working at FP service unit  and (b) in the community surrounding these facilities by trained community health volunteers (CHVs). CHVs are community members selected and trained to provide basic health information and services, including FP information, in the areas they live.The study was a 12-month prospective, single-arm, open-label, interventional study conducted in urban and peri-urban areas of Nairobi and Kiambu counties from August 2020 to August 2021. The intervention consisted of screening and recruitment of eligible women of reproductive age and distribution of pericoital LNG 1.5 mg contraceptive pill (study pill) through checklist to assess pregnancy at enrolment. In addition, women who reported having pre-existing conditions  were excluded.Women were eligible if they were aged 18–49 years, had infrequent sex , were not currently pregnant or using a modern method /HIV), and did not wish to get pregnant during the study period but would not consider it a serious issue. We used the World Health Organization (WHO)-endorsed pregnancy The study pill was only available via Kasha e-pharmacy. Women could order the study pill directly online through Kasha’s website, USSD (Unstructured Supplementary Service Data) or a call centre, using a unique enrolment ID obtained at enrolment. On ordering, women were counselled via telephone by trained Kasha pharmacists on how to use the LNG 1.5 mg pill, namely to take only one pill within 24 hours before or after sexual intercourse, and no more than six times per month, and use LNG 1.5 mg for pericoital use as their exclusive method. The study pill was delivered to the study participant at her choice of location, typically her home, for free using Kasha’s direct-to-consumer (D2C) distribution system.Enrolled participants were followed up every 2 weeks by telephone (bimonthly survey) until the end of the study (August 2021). Women were unenrolled from the study if they had become or wished to get pregnant, were no longer interested in participating, moved out of the study area permanently, or had switched/adopted a modern method (except condoms) during the study period.The study received ethical approval from the Population Council IRB (Protocol 890), the African Medical and Research Foundation Ethics and Scientific Review Committee (AMREF-ESRC P619/2019) and regulatory approval from the Pharmacy and Poisons Board (PPB//ECCT/20/02/03/2021). In addition, all study participants provided verbal informed consent at recruitment and written confirmation at delivery of the first order of the study pill.Women were interviewed via telephone at baseline  and twice a month  and once at the 6-month point (extended survey). The bimonthly surveys gathered information on the study pill use, satisfaction, and experience of side effects. The extended survey collected information on demographic background, contraceptives use, satisfaction with the study pill and services, and future contraceptive intentions. The e-commerce pharmacy sales registry dataset provided information on the quantity and doses dispensed.We calculated a sample size of 768 (384/site) women with unmet need for contraceptives and having infrequent sex based on prevalence formulae with 95% confidence level and 5% margin of error.Feasibility of e-commerce provision of LNG 1.5 mg for pericoital use was evaluated in terms of women’s ability to obtain the study pill from Kasha, and satisfaction with e-commerce pharmacy services. Acceptability of the pericoital LNG 1.5 mg pill was evaluated based on use of the study pill, self-reported adherence or compliance, satisfaction, the likelihood of future use and safety (self-reported side effects). We used descriptive and bivariate statistics with chi-square to test statistical significance differences between groups at p<0.05 and 95% confidence intervals. Data were analysed using Stata version 15.1.Of the 1164 women screened, 897 were eligible and enrolled in the study on consenting . Of thesMost of the participants wanted a child or another child, had ever used a modern method or did not perceive it as a problem if they became pregnant. Adopters were more likely than non-adopters to have more children and used a modern method , injectable, implant, daily pill). However, there were no significant variations between adopters and non-adopters by sociodemographic characteristics  .A total of 2291 doses of LNG 1.5 mg were dispensed to 300 women over a 12-month period . On average, women reported using the study pill 1.3 times per month. Most of the women reported using the study pill pericoitally, either within 24 hours before (average 4.9 hours) or after (average of 4.1 hours) coitus . Most woThe timing of use varied by background characteristics . For exaThere were 355 incidences of side effects reported by 147 women during the bimonthly surveys. The most reported side effect was nausea followed by headache, changes in bleeding pattern, cramps and dizziness. All side effects were reported as mild and tolerable .10.1136/bmjsrh-2022-201653.supp1Supplementary dataOver the past decade, there has been a call for developing and deploying an ‘on-demand’ pericoital pill for women having infrequent sex.Accessing LNG 1.5 mg for pericoital use through an e-commerce platform is feasible and offers women convenience, privacy and a home delivery option. The e-commerce pharmacy platform met pericoital LNG 1.5 mg pill adopters’ needs and further bolstered the rationale for providing the pill through pharmacies and digital platforms. Digital technologies and platforms can improve access to health information and services by removing or reducing barriers such as inconvenient service locations and hours of service or loss of privacy.Most women used the study pill within 24 hours before/after sexual exposure thus taking advantage of the pericoital nature of the LNG 1.5 mg. At least 40% of women exclusively used the study method within 24 hours before sex (the majority within 6 hours). Thus, women can distinguish between pericoital LNG 1.5 mg pill and postcoital EC pills. Our finding addresses the doubts among providers and FP stakeholders about the lack of distinctions between these two methods among users.Most adopters were satisfied or very satisfied with LNG 1.5 mg for pericoital use, were willing to continue using and would recommend it to others. Moreover, more than a third (37%) of adopters obtained the study pill more than once, suggesting potential demand and acceptability of a pericoital FP method. Our findings are consistent with a previous study that showed high acceptability of LNG 1.5 mg for pericoital use in multiple countries: Hungary, Thailand, Singapore and Brazil.LNG 1.5 mg for pericoital use could potentially address the unmet need for FP; 44% of adopters had never used a more effective modern method  while 11% had never used any method prior to enrolment in the study. Furthermore, women who had previously discontinued using modern methods were willing to use the LNG 1.5 mg pericoital pill as their primary contraceptive method. Our finding reflects the pericoital LNG 1.5 mg pill’s potential to fill the unmet FP need among women having infrequent sex or those dissatisfied with other modern methods. Therefore, the government should consider introducing the pericoital LNG 1.5 mg pill to expand FP method choice.The study has some limitations. Several other factors might have contributed to the high number of participants who despite enrolling initially, were no longer interested in the study, and hence discontinued or remained inactive . All participants were enrolled remotely, and data collection was telephone-based without face-to-face contact (due partly to the COVID-19 pandemic). For this reason, we posit that some women became reluctant to participate, lacked confidence, or were concerned about taking a new contraceptive pill to protect against a real risk . In addition, e-commerce provision, while beneficial in many ways, might have been imperfect (and new) for some participants. We interviewed women exiting the study; some participants became disinterested because they had infrequent sex and were not planning to engage in sexual activity during the remaining study period. Some participants, especially those without experience with the study pill, were demotivated by the frequent bimonthly telephone calls and therefore stopped picking up research assistants’ telephone calls or blocked the study lines. Similar challenges have been documented in other telephone-based surveys.E-commerce pharmacy provision of LNG 1.5 mg for pericoital use is feasible and a viable option for improving access to FP information and services. A pericoital LNG 1.5 mg pill is acceptable among women having infrequent sex, including those who have never used, are dissatisfied, or discontinued other modern methods. In addition, pericoital LNG 1.5 mg pill adopters experienced tolerable side effects, many of whom were satisfied with this method.


Cp gene knockout (Cp-/-) mice develop a neurodegenerative disease with aging and show iron accumulation in the brain. However, iron deficiency has also been observed in 3 M Cp-/- mice. The use of systemic Cp gene knockout is insufficient to reveal specific functions for CP in the central nervous system. Considering recent discoveries that astrocytes synthetize the majority of brain CP, we generated astrocyte conditional Cp knockout (CpGfapcKO) mice, and found that iron contents decreased in the cerebral cortex and hippocampus of young (6 M) and old (18 M) CpGfapcKO mice. Further experiments revealed that 6 M CpGfapcKO mice exhibited impaired learning and memory function, while 18 M CpGfapcKO mice exhibited improved learning and memory function. Our study demonstrates that astrocytic Cp deletion blocks brain iron influx through the blood-brain-barrier, with concomitantly increased iron levels in brain microvascular endothelial cells, resulting in brain iron deficiency and down-regulation of ferritin levels in neurons, astrocytes, microglia and oligodendrocytes. At the young age, the synapse density, synapse-related protein levels, 5-hydroxytryptamine and norepinephrine, hippocampal neurogenesis and myelin formation were all decreased in CpGfapcKO mice. These changes affected learning and memory impairment in young CpGfapcKO mice. In old CpGfapcKO mice, iron accumulation with aging was attenuated, and was accompanied by the alleviation of the ROS-MAPK-apoptosis pathway, Tau phosphorylation and β-amyloid aggregation, thus delaying age-related memory decline. Overall, our results demonstrate that astrocytic Cp deletion has divergent effects on learning and memory function via different regulatory mechanisms induced by decreased iron contents in the brain of mice, which may present strategies for the prevention and treatment of dementia.Ceruloplasmin (CP) plays an important role in maintaining iron homeostasis.  Ceruloplasmin (CP), first described by Holmberg and Laurell in 1948, is a ferroxidase . PreviouCP gene results in aceruloplasminemia, which is characterized by iron overload in the brain, leading to neurodegenerative diseases (NDs) with aging in humans and mice ) CpGfapcKO mice, learning and memory functions were impaired, whereas old (18 months old [18 M]) CpGfapcKO mice exhibited improved learning and memory, both of which are the result of iron depletion in the cerebral cortex or hippocampus, but affecting different regulatory pathways. Our results confirm that astrocyte-derived CP can facilitate iron uptake into brain parenchymal tissue from the circulation, and that specific knockout of Cp in astrocytes can inhibit iron uptake into the brain through the blood-brain-barrier (BBB). Decreased iron levels impaired learning and memory function via iron deficiency-induced synaptic formation, accompanied by a decline in myelination at a young age. However, decreased iron levels alleviated iron accumulation, reactive oxygen species (ROS) levels and age-related apoptosis in the CpGfapcKO mice.To examine the specific role of CP in the brain, we generated astrocyte-specific CpGfapcKO and Cpflox/flox mice, we first confirmed the conditional knockout of Cp gene with PCR  and CP exhibited significantly less colocalization in the cerebral cortex  mice. The CpGfapcKO mice spent significantly more time and covered a greater distance to find the hidden platform, during the five consecutive days of training  with young (6 M) ing Fig. . After tice Fig. . In the est Fig. , in contest Fig. . To examest Fig.  or in thest Fig. . These rTo study the mechanism of the above effect on learning and memory, we turned our attention to synaptic density and plasticity. Synaptophysin (SYN) is a presynaptic protein widely localized to synaptic vesicles . PostsynCpGfapcKO mice was lower than that of the control groups . The number of synapses in both the cerebral cortex and hippocampus of ups Fig. . Golgi sice Fig. .CpGfapcKO mice, both the NE and 5-HT levels were diminished in the cerebral cortex  and norepinephrine (NE), members of the monoamine neurotransmitter family, are associated with learning, memory function and anxiety-like behavior , 20. We tex Fig. , but in oup Fig. . These r+ immature neurons in the dentate gyrus (DG) of the hippocampus. We found a decreased number of DCX+ (immature neuron marker) and Ki67+  cells in the DG of CpGfapcKO mice  technology , and obsoup Fig S. We alsooup Fig S and induoup Fig S, and fouice Fig S.CpGfapcKO exhibited a significantly lower percentage of NeuN+ H-ferritin+ cells and a lower ratio of NeuN+ H-ferritin+ / NeuN+ both in the cerebral cortex and hippocampus  and L-ferritin in the cerebral cortex and hippocampus, and observed that the percentage of GFAP+L-ferritin+ cells and the ratio of GFAP+L-ferritin+ / GFAP+ were decreased in the hippocampus of CpGfapcKO mice compared to Cpflox/flox mice  participate in iron uptake from the peripheral blood into brain tissue , 6. We moup Fig. . H-ferrised Fig. . We alsoice Fig. , which i+HP+ cells and the ratio of NeuN+HP+ / NeuN+ were significantly increased in both the cerebral cortex and hippocampus in the CP-deficient astrocyte group  is another multi-copper ferroxidase involved in iron export from cells, and a homologue of CP . By the oup Fig. . To asseoup Fig. . To confice Fig. . Meanwhiice Fig S. These rCpflox/flox and CpGfapcKO mice. Interestingly, it took less time for the old 18 M CpGfapcKO mice to arrive at the hidden platform in the 2nd, 3rd, 4th and 5th day of training compared to the 18 M Cpflox/flox mice  staining. As expected, the amounts of apoptotic cells in these brain regions increased markedly with aging, while the number of apoptotic cells was significantly lower in the 18 M CpGfapcKO mice compared with the 18 M Cpflox/flox mice  to ferric iron (Fe3+) [3+ is then captured by transferrin in the interstitial fluid and distributed to glial cells and neurons in the CNS [Cp revealed that astrocytic CP plays critical roles in iron transport from the blood into the brain via the BBB, since the brain iron contents were decreased in CpGfapcKO mice with iron retention in BMVECs. Thus, a deletion of astrocyte Cp blocked the iron export via FPN1 in BMVECs into the hippocampus and cerebral cortex. Our further experimental data demonstrated that iron levels were decreased in neurons and glial cells. HP is predominantly expressed in neurons [Cp. Thus, the superposition decreased iron in the brain with enhanced of iron release from neurons via HP exacerbated the decrease of iron in neurons of CpGfapcKO mice.BBB is the gateway for iron entry into the brain from the blood. Astrocytes surrounding the BBB are the main cells responsible for CP synthesis, and astrocyte-secreted soluble CP and astrocyte GPI-CP are thought to facilitate the export activity of BMVEC FPN1 by catalyzing the oxidation of ferrous iron (Fen (Fe3+) . The Fe3 the CNS . However neurons , 36, whe neurons . Our resCpGfapcKO mice with iron deficiency in the cerebral cortex and hippocampus, the learning and memory ability was impaired, while, strikingly, the cognitive function improved in aged CpGfapcKO mice upon brain iron depletion in these regions.Iron is essential for cell metabolism and the synthesis of neurotransmitter and DNA, which are important for myelination, cell differentiation and neurogenesis. Long-term iron deficiency during development likely causes abnormal recognition memory processing and spatial learning . TherefoCp in oligodendrocytes induces iron overload, brain oxidative stress, myelin disruption and neurodegeneration in aged mice [CpGfapcKO mice, we found that the level of SYN, the number of spines of dendrites and the density of synapses were all decreased in the cerebral cortex. We also found that, in the hippocampus, the level of PSD95, the number of spines of dendrites and the density of synapsis were similarly decreased. These results suggest that synaptic plasticity declined after deletion of Cp in astrocytes, likely causing cognitive dysfunction. Considering that hippocampal neurogenesis dysfunction is correlated with learning and memory impairment [CpGfapcKO mice. In addition, iron deficiency in the brain has been shown to associate with the delay of myelin development and behavioral impairment [CpGfapcKO mice also exhibited decreased MBP expression and a thinner axonal myelin sheath compared to control mice. In CpGfapcKO mice, the iron was deficient in neurons and oligodendrocytes in the cerebral cortex and hippocampus, which may lead to impaired development of synapses and myelination. It is also reported that copper deficiency was correlated with myelin alterations [CpGfapcKO mice. Therefore, conditional knockout of Cp in astrocytes induced memory dysfunction was not related to copper alterations.The formation of learning and memory requires a complete synaptic framework and active circuit plasticity ; learninged mice . In our pairment , we assepairment –43; impapairment . The youerations , 45, ourCpGfapcKO mice, suggesting that astrocytic Cp deletion-induced iron deficiency may interfere with neurotransmitter production in the brain, which might be another important cause of cognitive function decline.Iron is a co-factor for many enzymes and participates in the synthesis of proteins in the CNS. Tyrosine hydroxylase is the rate-limiting enzyme in dopamine  and NE  synthesiCpflox/flox mice. Previous studies have shown that CP plays a key role in the prevention of neurodegenerative disease and free radical damage [Cp conditional knockout in astrocytes have a beneficial effect on the learning and memory abilities of old mice remains unclear.We were surprised to find that learning and memory were improved under astrocyte CP deficiency in 18 M mice compared to 18 M l damage , 12, 49.Cp deletion may increase brain iron and contribute to neurodegenerative diseases such as AD and PD [Cp-/- mice, leading to behavioral dysfunction, such as late cognitive impairment and cerebellar ataxia [CpGfapcKO mice compared to 18 M Cpflox/flox mice, with the expression of HP increased. Iron accumulation with aging and neurodegenerative damage have been tightly associated with ROS generation and subsequent cell death [CpGfapcKO mice, and found a significant decrease in apoptosis accompanied with decreased oxidative stress, increased Bcl-2/Bax ratio, decreased tau phosphorylation and diminished activity of the Erk/p38 MAPK pathway, all of which are linked to a delay in the pathogenesis of AD.It is known that the total iron content of the brain increases during aging , and it D and PD , 12, 51.r ataxia , 53. Howll death . Recent ll death , 54. Thell death . Iron hall death . Mitocholl death . Moreovell death . To inveCp is expected to limit the amount of iron in the brain, which is contrary to the results of iron deposition in the brain of global Cp knockout mice [CpGfapcKO mice, we examined the CP protein levels and iron contents in the liver and blood in the CpGfapcKO mice and controls  liver iron deposition causes the release of inflammatory cytokines [Cp-/- mice, the deficiency of neuronal CP may block neural iron export to induce brain iron overload.Although it is not possible to draw a definitive conclusion about the mechanisms underlying why global CP deficiency causes brain iron overload, while the opposite occurs in astrocyte-specific CP deficiency, we speculate that iron accumulation in the brain of global ytokines , and theytokines –61—the iytokines , 63; 3) ytokines , 65. NeuIn conclusion, deficiency of astrocyte CP led to a blockage of iron export via FPN1 from BMVECs into the cerebral cortex and hippocampus, with the iron remaining within in the endothelial cells, coupled with an increased expression of HP in neurons, ultimately causing cortical and hippocampal iron depletion. At a young age, the decreased iron contents may interfere with the synaptic formation, hippocampal neurogenesis, myelination and the synthesis of neurotransmitters, thus delaying learning and memory development. However, at old age, the decreased brain iron status prevents toxic iron accumulation and oxidative stress, which can alleviate MAPK-dependent apoptosis to improve spatial learning and memory Fig. . OverallCpflox/flox mice on C57BL/6 genetic background were obtained by a genetic targeting strategy by the Beijing Vitalstar Biotechnology Company; the Loxp sites were inserted around exon 2 by homologous recombination  and Cpflox/flox . The results and genotyping information can be found in Fig Sion Fig S; Gfap-crAfter neurobehavioral test as described as the following parts, mice were anaesthetized with 0.8% pentobarbital sodium, and then they received perfusion with pre-cooled saline to remove the blood until the fluid from the auricular sinus was clear. After that, sample preparation was performed according to the experimental requirements.st and 2nd day, while the platform was hidden under the water surface by 0.5–1.0 cm on the 3rd to 5th day; mice were allowed to find the platform located in the first quadrant. The escape latency time was recorded for up to 90 s. The test was conducted four times a day for five consecutive days, with a rest period of at least 30 min between two adjacent tests. The position of the escape platform was constant. On the sixth day, the platform was removed and the mice were allowed to swim freely. The swimming locus of each mouse was recorded for 90 s with a camera connected to a computer. The number of times a mouse passed the platform location and the percentage of target quadrant passing time were recorded by the computer and used to evaluate the learning and memory ability of each CpGfapcKO or Cpflox/flox mice.Spatial learning and memory function were assessed using the MWM, as described previously , 67. TheThis test was performed following as previously described, with modifications . BrieflyThe plastic chamber used in the open field test was also used in these tests. The mice were first accustomed to the apparatus for 5 min. Next, two objects were positioned 5 cm away from the two side walls. The mice were then placed in the apparatus and allowed to explore the two objects for 10 min. 24 h later, the mice were put in the same apparatus facing two objects, but one of the objects was replaced with a new object. The time spent exploring the novel object and familiar object was measured for 10 min.3 was used for suspending the dried tissues at room temperature overnight. The tissues were then heated at 90 °C for 20 min and hydrogen peroxide  was added for a further incubation at 70 °C for 15 min. The tissues were diluted in deionized water (≥18.2 MΩ) and detected by an ICP-MS spectrometer . The standards and digested samples were assessed at least in triplicate by ICP-MS.The total iron contents in the cerebral cortex, hippocampus and BMVECs were measured by ICP-MS as described previously . Tissuesg for 20 min and the supernatant was collected. Protein expression was assessed by western blot analysis as previously described [For western blot analysis, the mice were anaesthetized and perfused with cold normal saline to remove the blood until the fluid from the auricular sinus was clear. After perfusion, the cerebral cortex and hippocampus were removed, and tissue protein extraction was performed using RIPA buffer  containing protease inhibitor cocktail tablets . The lysates were centrifuged at 12,000 escribed , 70. Theβ-actin mRNA. The PCR primers used are as follows: Cp: Forward 5’- ACTGAAGCAGTCTGGGACTATGCT-3’, Reverse 5’- AGCCAGGCTGGTTTGTCTATAGTC -3’; β-actin: Forward 5’- AGGCCCAGAGCAAGAGAGGTA -3’, Reverse 5’- TCTCCATGTCGTCC CAGTTG -3’.Total RNA from cerebral cortex and hippocampus was extracted by TRIzol Reagent (Invitrogen) and complementary DNA (cDNA) was synthesized with a PrimeScript™ RT reagent Kit . Quantitative real-time PCR was performed using UltraSYBR Mixture  and a Bio-Rad CFX 96 detection system (Bio-Rad). The mRNA levels were normalized to 2 silicon drift detector  oriented at a 90° angle to the incident beam. A light microscope was attached to a computer for sample viewing. A monochromatic SR with photon energy of 15 keV was used to excite the samples. The XRF signals were collected for up to 20 s (live time) at each point. In order to correct the effect of the SR beam flux variation on the signal intensity, the Fe peak areas were normalized to the current intensity (I0) in an ionization chamber, which was placed in front of the samples. The results were analyzed and mapped using Origin 8.0 software .Mice were perfused with saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). The brain was removed, postfixed for 1.5 to 4 h and then soaked overnight in 30% sucrose until dropping to the bottom of the vessel. Serial coronal sections were cut at 50 μm intervals in a freezing microtome. Slices from the same coordinates were mounted onto Mylar film (polycarbonate). The brain sections were dried and put in a vacuum desiccator before analysis by μ-XRF. The experiment was performed on the 4W1B station at the Beijing Synchrotron Radiation Facility (BSRF). The continuous synchrotron X-rays were monochromatized by a Si (111) double crystal. The sample was placed at a 45° angle to the incident X-ray beam, and X-ray fluorescence was detected using a 50-mmThe brains were removed, postfixed in 4% PFA and cryoprotected in 30% sucrose. Coronal sections were cut at a 15.0 μm thickness in a freezing microtome and antigen retrieval was performed in 10.0 mM citrate buffer (pH 6.0) in a microwave oven at 96–98 °C for 10 min. The samples were blocked with normal goat serum, rabbit serum or donkey serum, according to the primary antibodies, and incubated overnight at 4 °C with a primary antibody: anti-GFAP monoclonal antibody , anti-CP antibody , anti-HP , anti-NeuN antibody , anti-Iba1 antibody , anti-CC1 antibody , anti-L-ferritin antibody , anti-H-ferritin antibody , anti-MBP antibody , anti-DCX antibody , anti-Ki67 antibody  and anti-4-HNE antibody . The following secondary antibodies were used and the incubations were at 37 °C for 50 min: Dylight 488 goat anti-mouse IgG  or Alexa Fluor 549 goat anti rabbit IgG . Sections were washed with 0.01 M PBS (pH 7.2–7.4) after each step. Finally, the sections were photographed with an Olympus FV3000 microscope.Golgi staining was examined by using the Frozen Section Neuron Golgi Staining Kit . According to the manufacturer’s instructions, mouse brains were immersed in a solution consisting of Solutions A and B (1:1) for 14 d in the dark. Next, the brains were transferred to Solution C for at least 48 h at 4°C in the dark. The brains were then sectioned in a microtome  to a thickness of 100 μm. Solutions D and E were used for staining the sections. Finally, the images of dendritic segments were scanned using an Axio Imager Z2 (Zeiss) attached to a tissue cytometer TissueFAXS .Male mice (6 M) were perfused with saline, followed by fixation with 2.5% glutaraldehyde and 2% PFA in 0.1 M PB (pH 7.4) overnight at 4 °C, post-fixed with 1% osmic acid in 0.1 M PB (pH 7.4) for 2 h, dehydrated in acetone and embedded in araldite. Ultrathin sections (50 nm) were then obtained with an ultramicrotome , stained with uranyl acetate and lead citrate, and images were acquired using a transmission electron microscope . The numbers of synapses in the cerebral cortex and hippocampus were counted and the G-ratio (inner diameter/outer diameter) of myelin were also calculated.Apoptosis in the cerebral cortex and hippocampus was determined using TUNEL method following the manufacturer’s protocol . Nuclei were stained with DAPI. The number of TUNEL-DAPI-positive neurons was quantified as described previously . The cou4•7H2O, 1.0 mM NaH2PO4•2H2O, 2.5 mM CaCl2, 10.0 mM glucose and 1.0 mM sodium pyruvate) with Dextran-T70  at a ratio of 1:3:4 (Brain tissues: Buffer: Dextran-T70). The homogenate was centrifuged at 7,450 g at 4 °C for 5 min. The white pellets were separated from the supernatant and homogenized again in 16% Dextran-T70 and centrifuged at 7,450 g at 4 °C for 5 min. The pellets were washed in the same buffer and centrifuged at 3, 500 g, at 4 °C for 5 min. The supernatants were removed and the BMVEC- containing pellet was retained for western blot analysis and ICP-MS.After anesthetization with 0.8% pentobarbital sodium, the mice were perfused with pre-cooled saline and the cerebral cortex and hippocampus were immediately removed. The cerebral cortex and hippocampus were weighed and homogenized in cold HBSS  from ThermoFisher. The mobile phase consisted of 0.05 M citric acid, 0.05 M sodium acetate and 0.5 mM sodium EDTA, 13% methanol, pH 3.1; the flow rate was 0.4 ml/min. The excitation wavelength used was 280 nm and the emission wavelength was 330 nm; the sample injection volume was 20 μL. The equipment included a Waters 717 Plus Autosampler System, a Waters 1525 Binary HPLC Pump and a Waters 2475 Multi λ Fluorescence Detector.The tissues were extracted and homogenized in ice-cold 0.1 M perchloric acid (v/w = 30:1) and immediately centrifuged at 15, 000 Astrocyte and neuron isolation was performed following the protocols of the Adult Brain Dissociation Kit . Briefly, adult mouse brains were dissociated by means of enzyme digestion and debris removal. Next, the astrocytes were further separated by using an Anti-ACSA-2 MicroBead Kit  and the neurons were further separated using a Neuron Isolation Kit . The number of cells was determined.TM RT reagent Kit with gDNA Eraser . The cDNA was applied in real-time quantitative PCR by Premix Ex Taq™ . The number of mRNA copies was calculated by the Cq value between samples and a standard substance. The following PCR primers were used Cp: , Heph .The total RNA was extracted from the cells via a TaKaRa MiniBEST Universal RNA Extraction Kit . As a template, total RNA was used to produce cDNA using a PrimeScriptThe levels of cortical or hippocampal MDA, ROS and total SOD (T-SOD) were measured as previously described ; the kitp value was less than 0.05.Statistical analyses were performed using GraphPad Prism 9.0 Software. All the data are presented as the mean ± SEM. The statistical analysis of each group was assessed using a two-tailed Student’s t-test. The difference between two groups was determined by Student’s t-test. The data were considered statistically significant when the Supplementary file Original Data FileThe reproducibility checklist


This study aimed to compare the short- and long-term outcomes of proximal repair vs. extensive arch surgery for acute DeBakey type I aortic dissection.From April 2014 to September 2020, 121 consecutive patients with acute type A dissection were surgically treated at our institute. Of these patients, 92 had dissections extending beyond the ascending aorta.Of the 92 patients, 58 underwent proximal repair, including aortic root and/or hemiarch replacement, and 34 underwent extended repair, including partial and total arch replacement. Perioperative variables and early and late postoperative results were statistically analyzed.p < 0.01). The overall operative mortality rate was 10.3% in the proximal repair group and 14.7% in the extended repair group (p = 0.379). The mean follow-up period was 31.1 ± 26.7 months in the proximal repair group and 35.3 ± 26.8 months in the extended repair group. During follow-up, the cumulative survival and freedom from reintervention rates at 5 years were 66.4% and 92.9% in the proximal repair group, and 76.1% and 72.6% in the extended repair group, respectively (p = 0.515 and p = 0.134).The duration of surgery, cardiopulmonary bypass, and circulatory arrest was significantly shorter in the proximal repair group (No significant differences were found in the rates of long-term cumulative survival and freedom from aortic reintervention between the two surgical strategies. These findings suggest limited aortic resection achieves acceptable patient outcomes. With improvements in the perioperative and postoperative management of acute type A aortic dissection (AADA), there has been a trend towards aggressive total arch replacement (TAR) rather than hemi-arch replacement to improve long-term outcomes, especially in DeBakey type I aortic dissection –5. HowevOur institute is in the northern Hokkaido and performs approximately 20 surgeries for AADA annually. At our institute, replacement of the ascending aorta with resection of the primary entry is the preferred surgical approach. TAR is performed only in cases of (1) an intimal tear localized along the greater curvature of the aortic arch, (2) malperfusion of the carotid arteries, (3) the critical stenosis of the true lumen, and/or (4) significant dilation of the aortic arch (exceeded diameter of 50 mm), and/or (5) patient age less than 60 years. If patients are not candidates for TAR, such as in cases of old age or preoperative patient status, hemi-arch replacement is performed to minimize the invasiveness of the surgery and maximize survival rates. Thus, the aim of the current study was to compare the short- and long-term outcomes of aortic arch surgery using our two surgical strategies in patients with DeBakey type I AADA.This study was approved by our institutional review board, which waived the requirement for informed patient consent, owing to the retrospective nature of the current study (IRB number: 19207).From April 2014 to September 2020, 121 adult patients with acute aortic dissection underwent urgent surgery at Asahikawa Medical University Hospital. Of these, 29 patients who underwent hemiarch replacement for DeBakey type II aortic dissection were excluded from this study. Of the 92 included patients, 58 underwent hemiarch replacement , whereas 34 underwent partial or total replacement .via the right superior pulmonary vein. The patient was cooled to the target rectal temperature of 26 °C. During systemic cooling, the ascending aorta was cross-clamped, and cardioplegic arrest was induced in the absence of a massive thrombus in the false lumen of the ascending aorta. Otherwise, cross-clamping was avoided and cardioplegia was administered after commencing hypothermic circulatory arrest. If the ascending aorta was cross-clamped, the proximal anastomosis was performed using a modified “turn up” technique. If cross-clamping of the ascending aorta was not performed, the distal anastomosis was completed first, and the proximal anastomosis was then completed with a separate graft using the modified “turn up” technique. Concomitant procedures, such as root replacement or coronary artery bypass grafting, were performed during cardiac arrest after the completion of the distal anastomosis.The operation was performed in the supine position. Median sternotomy was performed and cardiopulmonary bypass was established with arterial cannulation into the ascending aorta, femoral artery, or axillary artery determined by patient status, and bicaval venous drainage. A vent tube was inserted into the left ventricle Moderate hypothermic circulatory arrest with a rectal temperature of 26°C and antegrade selective cerebral perfusion are preferred for brain protection at our institute. In cases of hemi-arch replacement, a 1-branched prosthesis was used for the distal anastomosis. In cases of extended arch repair, a 4-branched prosthesis was used, and the arch vessels were reconstructed after commencing systemic perfusion during the re-warming phase.For TAR, a frozen elephant trunk  was used in most cases. In cases of a FET, the subclavian artery was reconstructed extra-anatomically using an 8 mm expanded polytetrafluoroethylene prosthesis  anastomosed to the left axillary artery through a separate incision. With this extra-anatomical procedure, all anastomoses can be performed in relatively safe position by avoiding deep distal anastomosis and avoiding in-situ reconstruction of the left subclavian artery, therefore we preferred it . If a FEFollow-up data on survival, the need for aortic reoperation, and the causes of death were determined from clinical records at our outpatient clinic or direct telephone interviews with patients or relatives. All required follow-up data were collected, and no patients were lost to follow-up. The mean duration of follow-up was 23.5 months (range 6.3–57.3) in the PR group and 32.5 months (range 27.8–60.5) in the ER group.n < 5). Survival and freedom from reintervention were analyzed using the Kaplan–Meier method and log-rank calculations. All statistical calculations were performed using IBM SPSS Statistics for Windows, version 22.0. . The significance level was set at p < 0.05.Continuous variables were presented as means and standard deviations or medians and first and third quartiles in cases of a skewed data distribution. Categorical variables were presented as frequencies and percentages. For comparison of continuous variables, the Student's t-test was applied for normal distributions, as verified using the Kolmogorov–Smirnov test. For non-normal distributions, the Mann–Whitney rank sum test was used. Categorical variables were compared using the chi-square test. Fisher's exact test was used for small sample sizes  an intimal tear localized along the greater curvature of the aortic arch, (2) malperfusion of the carotid arteries, (3) the critical stenosis of the true lumen, (4) significant dilation of the aortic arch (exceeded diameter of 50 mm), and/or (5) age under 60 years. The decision to perform extended arch repair was made intraoperatively for seven patients, despite not meeting inclusion criteria. Of the 58 patients in the PR group, 10 patients met the inclusion criteria, but two of these patients required preoperative cardiopulmonary resuscitation (CPR) and two patients experienced profound shock. These four patients therefore underwent hemi-arch replacement instead.p = 0.029). No significant differences in other preoperative factors were observed between the two groups.A comparison of the preoperative data between the 2 groups is shown in p = 0.440). The number of deaths after discharging from the hospital was 14. 11 patients in PR group were due to pulmonary (n = 4), intracranial bleed (n = 2), gastrointestinal (n = 2), cardiac (n = 1) and unknown cause (n = 2). 3 patients in ER group were due to mediastinitis (n = 1), gastrointestinal (n = 1) and stroke (n = 1). The estimated the 5-year survival rate based on Kaplan–Meier analysis was 66.4% ± 7.1% in the PR group vs. 76.1% ± 7.4% in the ER group, a difference that was not statistically significant . This result is in line with those of previous studies. Uchida et al. reported a higher survival rate in TAR at 5 years  (p = 0.54) (p = 0.01). The tendency for patients to have more favorable preoperative risk profiles in the ER group was also observed in the current study. The PR group was older and had a greater proportion of patients with preoperative shock, impaired consciousness, cardiac tamponade, or requiring CPR. At our institute, the surgical strategy was selected based not only on the location of the intimal tear, the range of dissection, the status of the true lumen, the diameter of aortic arch, and the age of the patient, but also on preoperative patient status. Therefore, with this unavoidable selection bias, preoperative patient status in the PR group was less favorable than that in the ER group and had more risk factors for mortality (such as need for CPR)  , and Omu = 0.54) . Other s = 0.54) , 13. How = 0.54) –13 lack for CPR) , 14, 15.p = 0.134), which has not been reported previously. Theoretically, incomplete resection of the dissected aortic arch may necessitate distal aortic reintervention (The PR group had a higher free from reintervention rate than the ER strategy group (92.9% ± 4.6% vs. 72.6% ± 10.8%, rvention , 16. TypConversely, ER was associated with a high risk of reintervention, which may be accounted for by the contents of late reintervention. As listed in 2 and populated by approximately 650,000 people. Because this area is large and has a low population density, long-distance transfers of patients with AADA to our institute are common. Although Andrew et al. (Our institute services a large area in northern Hokkaido, covering an area of 18,000 kmw et al. . Caleb ew et al. , which sw et al. , the medw et al. . The perOur study found that PR is less invasive than ER strategy and did not correlate with an increased risk of future surgical reintervention. In addition, reintervention in the PR group was performed electively. There was no perioperative mortality observed. Although the optimal extent of surgical resection and reconstruction of the arch in AADA is still under debate, and the surgical strategy should be selected taking into consideration multiple factors, our results suggest the adequacy of a limited approach for patients with a high risk profile, in preference for urgent life-saving therapy. In particular, in circumstances of relatively unfavorable preoperative patient status and a regional facility, limited aortic resection would be acceptable as not a palliative but a reasonable strategy.This study has several limitations. It was a retrospective analysis of our institutional database of prospectively collected observational data. Patient characteristics were not comparable between the study groups, and selection bias may exist regarding the choice of surgical strategy. Given the urgency of AADA, the potential for selection bias is unavoidable, and prospective randomization of surgical procedures would be impractical. Lower freedom from reintervention late in ER group was attributed partially to a dSINE following TAR with FET. In this respect, subsequent long-term follow-up are needed to confirm the result. Furthermore, our study was very limited by its small sample size. We believed it is worth revealing the “real-world” clinical outcomes in relatively small center, however, it is obvious that larger studies with longer follow-up periods are required to obtain more insights into this research area.No significant differences were found in the rates of long-term cumulative survival and freedom from aortic reintervention between the two surgical strategies. These findings support the theory that limited aortic resection achieves acceptable patient outcomes.


Medicago truncatula. A total of 219.49 Gb clean data were generated: 3,284 lncRNA genes were identified, of which 515 were differentially expressed, and 1,254 new genes were functionally annotated through database alignment. We further predicted and classified putative targets of these lncRNAs and found that they were enriched in biological processes and metabolic pathways such as plant hormone signal transduction, cell wall modification and the tricarboxylic acid (TCA) cycle. Finally, we characterized the functions of 2 Al-activated-malate-transporter-related lncRNAs in yeast. The recombinant plasmids of MSTRG.12506.5 and MSTRG.34338.20 were transformed into yeast, and these yeast exhibited better growth than those carrying empty vectors on medium supplemented with 10 μM AlCl3 and showed that they have biological functions affording Al stress tolerance. These findings suggest that lncRNAs are involved in regulating plant responses to Al stress. Our findings help to understand the role of lncRNAs in the response to Al stress in legumes and provide candidate lncRNAs for further studies.Numerous studies have shown that plant long noncoding RNAs (lncRNAs) play an important regulatory role in the plant response to environmental stress. However, there are no reports on lncRNAs regulating and enhancing aluminum (Al) stress tolerance in legumes. This study analyzed the role of lncRNAs in response to Al stress in the legume model plant  Micromotoxicity . Approxitoxicity . Previoul stress . Many pll stress . Among tl stress .Arabidopsis thaliana, ALUMINUM-ACTIVATED-MALATE-TRANSPORTER 1 (AtALMT1) was identified as a key gene for Al tolerance, and root malate secretion was shown to be regulated by the transcription factor SENSITIVE-TO-PROTON-RHIZOTOXICITY 1 (STOP1), which mediates Al-induced expression of AtALMT1 and is critical for Al resistance of A. thaliana (EXTENSIN-LIKE PROTEIN (ELP) and XYLOGLUCAN ENDOTRANSGLUCOSYLASE-HYDROLASE 17 (XTH17), which are responsible for cell wall modification, thereby balancing the Al distribution between the apoplast and symplast in the roots   and MtCBF, which play key roles in the cold stress response. However, lncRNA studies related to Al stress are rare.Noncoding RNAs (ncRNAs) longer than 200 nucleotides are called long noncoding RNAs (lncRNAs), have relatively low protein-coding capacity and compose the largest class of ncRNAs . LncRNAsinensis) . Yu et aM. truncatula, we performed high-throughput strand-specific RNA sequencing (RNA-seq) on root tip tissue under Al stress to study and characterize lncRNAs associated with the response to Al stress. Our findings provide new insights into the potential functions of lncRNAs during Al stress.To identify the lncRNAs corresponding to Al stress in M. truncatula A17 were surface-sterilized with 1% NaClO solution for 5 min, rinsed 5 times with distilled water, and germinated in the dark at 25°C for 3 days. Then, they were cultured in Hoagland nutrient solution (pH 5.8) at constant temperature  for 7 days, during which the nutrient solution was changed every 2 days (2 and 10 μM AlCl3 (pH 4.5) solution and one control (0 h) group, which was cultivated for 48 h in a 0.5 mM CaCl2 solution (pH 4.5). To reduce the circadian rhythm effects, the seedlings of control and 48 h groups were treated at the same time, and harvested after 48 h. For 4 h and 24 h treatment groups, their seedlings began to be treated 44 h and 24 h after the treat time of 48 h treatment group, respectively, and harvested at the same time as 48 h and control groups. Each 20 plants served as a replicate with three replicates per treatment. After the treatment, 1.5-cm-long root tips were taken, immediately frozen in liquid nitrogen, and stored at −80°C.The study was carried out in the laboratory of the College of Animal Science, Guizhou University. Seeds of y 2 days . Then, 2Root length was measured using a digital Vernier caliper, and root activity was measured using the naphthylamine microplate method . The malondialdehyde (MDA) content, peroxidase (POD) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, and soluble sugar (SS) content were measured using corresponding kits from Beijing Solarbio Science and Technology Co., Ltd. . Refer to manufacturer’s instructions for measurement method.RNA extraction, quantitative measurements and quality assessments were performed according to the methods described by M. truncatula reference genome MedtrA17_4.0 using Bowtie2 and TopHat2, after which they were assembled by StringTie   protein sequence, Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and SwissProt Protein databases ( < 10-5) . The Top < 10-5) . GO term–∆∆Ct method  and Power SYBR Green Master Mix (Applied Biosystems) in conjunction with an iQ 5 Multicolor Real-time PCR Detection System  were used for qRT–PCR analysis, and the primers used are designed by Premier 5 . The relt method .via seamless cloning, the results of which are shown in 3 and incubated at 28°C for 48 h, all of which was replicated 3 times.The full-length cDNA sequence of the genes were obtained 3, incubated at 28°C and 180 rpm for 24 h, and then analyzed spectrophotometrically to measure the OD600. The OD600 value of transgenic yeast grown in liquid media without AlCl3 was used as a control  was inoculated into SCu/2% (w/v) glucose liquid media supplemented with 10 μM AlCl control .M. truncatula and whether the redox system plays a role in the Al stress response, we measured the root length; root activity; POD, SOD and CAT activities; and SS and MDA contents. Root length and root activity had similar trends; both began to increase after 4 h of treatment, reached their highest values at 24 h, and then showed a decreasing trend (2O2) and hydroxyl radicals (-OH) accumulated in cells. The significant increase in SOD and CAT at 4 h after M. truncatula exposure to Al stress suggest that cell development and cellular antioxidant-reduction systems respond rapidly to stress.To investigate the Al stress tolerance of ng trend ,B. Compang trend . The SODng trend , and Theng trend . The SS ng trend . The MDAng trend . SOD andM. truncatula lncRNAs involved in the Al stress response, 12 cDNA libraries were used for RNA sequencing. A total of 54,448,590 to 71,603,639 bp of clean reads were generated from each sample, with a mean GC content and number of bases of 42.65% and 1,828.97 Mb, respectively, and a mean Q30 of 94.00%  and lncRNA (MSTRG.12506.5) was constructed using Cytoscape software , and two mRNAs  were measured by qRT–PCR. The expression trends of DElncRNAs were consistent with the results of RNA-seq, indicating the reliability of the expression analysis results .ALMT-related DElncRNAs (MSTRG.12506.5 and MSTRG.34338.20) were overexpressed in the Saccharomyces cerevisiae yeast strain INVSc1, and the growth characteristics of the transformants were observed. There was no difference in the growth rates of the yeast transformed with the empty and recombinant plasmids on plates of media without AlCl3 , salt stress  and cold stress   . These dstresses .M. truncatula, a total of 184 lncRNAs regulating plant hormone signal transduction were detected , gibberellin (GA) and cytokinin (CK). Another category is “stress hormones,” which include hormones such as abscisic acid (ABA), ethylene (ET) and jasmonic acid (JA) . IAAs ardetected . Among tdetected . We detedetected . Furtherdetected . In the detected , of whic3+ absorbed by plants is distributed in the apoplast of the cell, and pectin, the main cell wall polysaccharide of the apoplast, has many carboxyl groups and a strong affinity for Al3+  pathway; the other relies on the intracellular symplastic (cytosolic) pathway for detoxification and the mechanism of vacuolar sequestration . Most moechanism . In planom roots . In A. txudation . In this 3 mRNAs . Furtherand MATE . We deteand MATE  involvedALMT families can regulate malate production. Under conditions of 10 μM AlCl3, by targeting the lncRNAs MSTRG.12506.5 and MSTRG.34338.20 predicted to target the regulatory mRNAs MTR_1g077660 and MTR_1g077670, we performed transgenic yeast validation experiments. The results showed that growth of the transformants was significantly better than that of the empty vector-transformed controls, indicating that lncRNAs could adapt to Al stress by regulating malate synthesis-related genes and increasing malate secretion.Our validation experiments further demonstrated that DElncRNAs related to We obtained a total of 219.49 Gn of clean sequence data from 12 paired-end library sequences and identified 3,284 lncRNAs. Through GO and KEGG enrichment of lncRNA targets in response to Al stress, we found that these lncRNAs are involved in the regulation of plant hormone signal transduction and the TCA cycle. We further demonstrated that the lncRNAs MSTRG.12506.5 and MSTRG.34338.20 are involved in the regulation of the malate synthesis network. These results provide new insights into the function of lncRNAs in response to Al stress.https://www.ncbi.nlm.nih.gov/, PRJNA868891.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: RD and CC conceived the experiment. QG, ZY, FY, and SW carried it out. QG and ZY analyzed the data. RD and QG wrote the paper. All authors contributed to the article and approved the submitted version.This research was supported by the National Natural Science Foundation of China (32060392) and Support by Guizhou Province Science and Technology Projects (Qian Ke He Zhi Cheng [2020]1Y074).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.