To explore the risk probability and main influencing factors of stroke in followed-up hypertension patients through the analysis of long-term followed-up cohort data.The method of followed-up observation cohort was used to collect the information of 168,417 followed-up hypertension patients from 2002 to 2020 in Jiading District in Shanghai. Kaplan–Meier method was used to analyze the risk probability of stroke complications in long-term followed-up HTN patients, and the influencing factors were analyzed by Cox proportional risk model.p < 0.001), body mass index , family history of stroke  and blood pressure grade .Among 168,417 followed-up hypertension patients, 11,143 cases had suffered stroke, and the cumulative incidence rate of stroke was 6.62% . With the extension of the hypertension years, the cumulative risk probability of stroke in HTN patients would continue to increase and the interval was not equidistant. The total cumulative risk probability of stroke in HTN patients was 78.9% . During the period of hypertension, the risk occurring probability of stroke was not fixed, but fluctuating. There were 4 onset peaks, which were in 8 years (probability was 4.2%), 15 years (probability was 14.0%), 22 years (probability was 6.0%) and 26 years (probability was 13.9%). The highest risk probability of male patients was in 26 years (probability was 23.1%), and the second peak was in 15 years (probability was 15.6%). The highest risk probability of female patients was in 15 years (probability was 12.9%), and the second peak was in 26 years (probability was 8.7%). The risk probability of different gender, BP grade and BMI was different, the male were at higher risk than the female, stage 3 HTN was higher than stage 2 and stage 1 HTN, obese people and underweight people were at higher risk than those who have normal weight. The main factors closely related to the occurrence of stroke complications were age , and would continue to increase disproportionately during period of hypertension (4 different onset peaks). With the persistence of hypertension, the risk probability of stroke would increase continuously. Multivariate Cox regression analysis showed that male patients, patients with HBP, abnormal BMI and positive family history were main factors closely related to the occurrence of stroke complications. Stroke is an acute cerebrovascular disease caused by many factors, and it is the first cause of death in China and the second largest cause of death in the world –3. HyperThe main outcome of hypertension is stroke. Untreated hypertension increases the risk of stroke seven times . HyperteAfter understanding the relationship between hypertension and stroke and the association with different types of hypertension and stroke, it is necessary to further explore the risk probability of stroke in hypertension, is it 100% or low probability? This study mainly explored from the method the risk probability of stroke in primary hypertension patients and the specific value of the risk probability and main influencing factors of stroke in followed-up primary hypertension patients. In order to better observe and explore the risk probability and risk factors of stroke in hypertension patients, the data of a long-term follow-up cohort of hypertensive patients in Jiading district in Shanghai was used in this study, and the risk probability and influencing factors of stroke in followed-up hypertension patients was analyzed and explored. We hope that these results can provide some help for the prevention and control of stroke in community in the future.All patients (Note: hypertension patient in this article is primary hypertension) were from the Hypertension Follow-up Management System in Jiading district in Shanghai China. The Follow-up Management System of hypertension have been formally established and implemented since from 2002. The Hypertension Followed-up Management System Database records the medical information of all registered and followed-up hypertension patients. According to the guidelines and policies of hypertension prevention and control, the hypertension patients in communities were registered and followed up, family doctors and public health personnel in the community health service center are responsible for the registration, follow-up and management of hypertension patients in the community, the center for disease control and prevention is responsible for the formulation of technical scheme, the training of personnel and the quality control of follow-up data. All patients had primary hypertension. All medical followed-up information of patients must be truthfully recorded in the system database by followed-up doctors. A total of 168 417 hypertension patients were entered into the database of the observation as the deadline of September 30, 2020. All about stroke occurred in followed-up patients must be recorded in detail. Stroke must be diagnosed and confirmed by a senior hospital. This study was approved by Science and Technology Commission, Health Commission and CDC in Jiading district in Shanghai (No JDCDC-2020-0036). All methods were carried out in accordance with relevant guidelines and regulations.The registered information and followed-up records of hypertension patients were recorded in the system, mainly including the date of birth, gender, ID number, residential address, occupation, education level, family history, date of establishment, termination date and reasons for termination, blood pressure and blood pressure at each follow-up, history of stroke , lifestyle  and so on. Considering the integrity and continuity of the data, some incomplete data and later supplementary variables were not included in this study. The date of occurrence of stroke in hypertension patients must be later than the date of registration. If the date of onset of stroke was earlier than or close to the date of registration, this patient data would not be included.The diagnosis of stroke was based on the clinical diagnostic criteria  –13. The The measurement and classification of blood pressure and the diagnosis of hypertension were carried out according to the standard of Chinese guidelines for the prevention and treatment of hypertension –15. BMI The queue observed was a dynamic queue, and the time when the observed patients entered and exit the queue was inconsistent. Therefore, the observation starting point defined in this paper refers to the time point when patients were diagnosed with hypertension, diagnostic time. Observation end point refers to the time when hypertension patients have expected outcome events (stroke) or withdraw from the observation queue due to loss of follow-up. Follow up time or observation duration refers to the time difference between the end point and the starting point (observation end point minus observation starting point).2) test was used to compare the data between different groups. The risk probability was analyzed by Kaplan–Meier methods, the related influencing factors were analyzed by Cox regression. The end event was whether the patient had stroke (Yes 1/ No 0), the difference between the end-point time of observation minus the time of diagnosis of hypertension was used as the observation time or survival time (unit: year), no stroke occurred or lost follow-up or withdrawal during the observation period were regarded as censoring data. Select the observation time to enter the "Time", stroke event (Yes 1/ No 0) to enter the "Status", categorical variable to enter the "Covariates" and take the minimum value as the reference group for multi categorical variables, “Methods” select Forward LR. Bilateral test, p < 0.05 for the difference was statistically significant.According to the deadline, all the recorded data in the hypertension follow-up management system before the deadline were exported to the Microsoft Excel database, and then the corresponding logic check, data screening and conversion were carried out. Finally, the sorted database was imported into the SPSS statistics software package (IBM SPSS statistics version 21) for statistical analysis. The mean and standard deviation of age, blood pressure and other quantitative data were calculated. The number and frequency (%) were calculated according to gender, number of patients, etc. The chi-square , through the grouping data comparison of age, BMI, blood pressure, smoking habit and other variables. See TableAmong selected variables of registration information, nationality groups cannot be grouped because they all are Han. In the early occupation registration, because the registration was not standardized, it was difficult to classify and did not enter the analysis. Considering the elderly patients basically in low education level group, the educational level was not included in the analysis. The results showed that the difference of stroke in these variable groups was still obvious  was selected as the horizontal axis of time, and the occurrence of stroke  was defined as the event state, and Kaplan–Meier method was used to analyze the risk occurred probability of stroke. The calculated risk probability of each time point was made as a longitudinal axis and a dynamic curve was drawn. See Fig. 2 = 113.570, p < 0.001). By calculating the cumulative risk probability of stroke in hypertensive patients of different genders, the results showed that the total cumulative risk probability of stroke in hypertension patients was 0.789 (78.9%), and male was 0.910 (91.0%), female was 0.707 (70.7%).The result of Fig. 2 = 189.139, p < 0.001). The risk probability of stage 2 BP patients was basically similar to that of stage 1 BP patients at the beginning, but the risk of stroke will suddenly increase after 22 years. It is unclear why. In patients, it may be superimposed or caused by some other factors, which needs further observation and research.The result of Fig. 2 = 86.431, p < 0.001).Figure 2 = 2.432, p = 0.119).The result of Fig. 2 = 14.257, p < 0.001).The result of Fig. p < 0.05), but the direction and degree of these factors were different. At the same time, considering that age, body mass index and blood pressure have a great impact on the occurrence of stroke, in order to better show the impact of different levels of age, body mass index, blood pressure and HP control level, we further made a more detailed analysis of the impact of different levels of them. See Table In this study, some long term followed factors including sex, age, smoking habit, drinking habit, physical activity, body mass index, blood pressure grad and family history (including family history of hypertension and stroke) were selected. Cox regression analysis was used to explore the influence of these factors on the occurrence of stroke complications. From the results of Cox regression analysis, these factors have different degrees of influence. Sex, age, smoking habit, drinking habit, physical activity, body mass index, blood pressure grad, family history of stroke, family history of hypertension and BP control level were the influencing factors of stroke in hypertension patients , diabetes mellitus 34.94%), heart damage (13.93%), kidney diseases (1.04%), fundus damage (0.30%) and peripheral vascular diseases (0.04%). Cardiovascular and cerebrovascular diseases were the main complications in patients with hypertension in community . In this%, diabet4.94%, heAt present, there were many reports on the risk probability analysis or prediction of stroke with hypertension, and the methods used include Logistic Regression analysis, Framinghan evaluation method and ESRs score method –22. The The main type of stroke was cerebral infarction in hypertension in Jiading Shanghai, the proportion of ischemic cerebrovascular, hemorrhagic cerebrovascular and unclassified stroke was respectively 71.18%, 5.95% and 22.87% in hypertensive stroke [p < 0.05). According to the results of the long-term observation of registered hypertension patient, the major influencing factors of stroke complications were age (RR = 2.917), body mass index (RR = 1.654), family history of stroke (RR = 1.386), blood pressure grad , hypertension duration (RR = 1.071) and family history of hypertension (RR = 1.051). By further classifying levels of age, blood pressure and body mass index, the results showed that different blood pressure grades, age groups and body mass index had different effects on the occurrence of stroke. The higher blood pressure, the older age and the higher body mass index was, the higher risk of stroke was in hypertension patients. These results were consistent with the above risk occurred probability of stroke in hypertension patients and literature reports. The influence of sex, smoking habit, drinking habit and physical activity on stroke complications was statistically significant, the lower limit value of RR of were less than 1. This also means that the direction of action of these factors was protective factors or opposite effect. The reason might be affected by the assignment of data classification, for example, male was assigned as 1 and female was assigned as 2 in sex classification, but the risk of male was actually high than that of female. It might also be caused by other factors. This needs further study, but it is undeniable that sex, smoking habit, drinking habit and physical activity are the influencing factors of stroke, which must be paid attention to in community prevention and control.Stroke is a group of cerebrovascular circulation disorders caused by various reasons, manifested as focal neurological deficit, and even accompanied by disturbance of consciousness. Because of its sudden onset, it is also called cerebrovascular accident. There were many risk factors for stroke, such as age, heredity, hypertension, heart disease, arrhythmia, diabetes, hyperlipidemia, smoking, drinking, obesity, high salt, high animal oil diet, excessive physical activity, etc. , 23–30. Atrial fibrillation (AF) is the most common arrhythmia and has significant morbidity. Morphological voltage P wave duration (MVP) ECG score is of great significance in predicting ischemic stroke hospitalization and long-term atrial fibrillation . A scoreIn short, hypertension patients were prone to stroke, and the total cumulative occurred probability of stroke in followed-up HTN patients was 0.789 (78.9%), and male was 0.910 (91.0%), female was 0.707 (70.7%). The risk probability of stroke among hypertension patients was high and would continue to increasing disproportionately during period of hypertension, outcome of stroke in HTN patients would have four different onset peaks. Male, blood pressure level (stage 3 BP), abnormal weight (underweight and overweight) and blood pressure control level could increase the risk probability of stroke. The results of multivariate Cox regression analysis showed that male patients, patients with high blood pressure, abnormal body mass index and positive family history were high-risk objects of stroke. Paying attention to blood pressure, weight and male are important and effective measure to prevent stroke in community.


Most conventional post-stroke rehabilitation treatments do not involve imposed constraints of the unaffected limb. In contrast, Constraint-Induced Movement Therapy (CIMT) is comprised of massed task practice with the affected limb and constraint of the unaffected limb. CIMT is a promising rehabilitation technique used for motor recovery of affected limbs after stroke, but its effectiveness and mechanism are not fully understood. We compared the effects of the two exercise modes on limb function post-stroke in animal models and human subjects, and investigated whether oxidative stress response was involved in regulating the effects. We first conducted a randomized controlled trial (RCT), in which 84 subjects with cerebral infarction were assigned to dose-matched constraint-induced movement therapy (CIMT), or unconstraint exercise (UE), or conventional rehabilitation treatment. Motor functions of the limb are primary outcomes of the RCT measured using Brief Fugl–Meyer upper extremity score (FMA-UE), Ashworth score, and Barthel scale. Psychological influence of CIMT and UE was also examined using Self-Rating Depression Scale (SDS). Next, we investigated the effects of CIMT and UE in rats undergoing middle cerebral artery occlusion and reperfusion (MCAO/R). Motor function, infarct volume, and pathohistological changes were investigated by mNSS, MRI, and histological studies. The role of Keap1-Nrf2-ARE was investigated using qRT-PCR, Western blot, immunochemistry, immunofluorescence, and ELISA experiments. In RCT, patients taking CIMT had a higher score in FMA-UE, Barthel index, and SDS, and a lower score in modified Ashworth, compared to those taking UE. In rats receiving CIMT, motor function was increased, and infarct volume was decreased compared to those receiving UE. The expression of Keap1 protein and mRNA in the peri-infarct tissue was decreased, and Nrf2 and ARE protein and mRNA were increased in rats receiving CIMT compared with UE. Nrf2 agonist t-BHQ increased the benefits of CIMT. In conclusion, CIMT is more effective than UE in improving upper limb motor function, reducing muscle spasm in patients with cerebral infarction compared to UE, but patients receiving CIMT may feel depressed. Moreover, both CIMT and UE are beneficial to limb function recovery and limit the infarct expansion in MCAO/R rats, but CIMT was more effective than UE. Oxidative stress reaction has an essential role in regulating the CIMT induced benefits. Stroke is the second leading cause of death and disability worldwide . The higConstraint-induced movement therapy (CIMT) has been developed as a rehabilitation exercise to improve the function of the upper limb in survivors of stroke ,12. The Treadmill training is an unconstraint exercise (UE) mode commonly practiced to improve limb function, which has a higher exercise intensity and lower frequency compared to CIMT ,16. The 2O2), superoxide anion (O2−), and hydroxyl radicals (-OH), further destroys cell functions. Increasing studies have confirmed that oxidative stress is one of the important pathological mechanisms of neuronal cell damage and death after stroke [Oxidative stress reaction is the imbalance of synthesis and clearance of reactive oxygen species (ROS), causing peroxidation of biomacromolecules such as nucleic acids, lipids, and proteins. It can lead to the production of toxic substances, thereby causing cell dysfunction, damage, or apoptosis . ROS pror stroke . Ischemir stroke ,20. Therr stroke ,22. GiveHere, we conducted an RCT and animal study to evaluate the effects of two exercise modes: CIMT and UE, in limb dysfunction of stroke survivors, and to investigate the relationship between CIMT/UE and oxidative stress reaction. Our study attempts to provide evidence for the clinical practice of stroke rehabilitation.The protocol of the RCT was approved by the ethical committee of our institution , and the study was registered on Chinese clinical registry (ChiCTR2100042339).Patients with cerebral infarction were recruited between December 2020 and June 2021, from the Department of Rehabilitation Medicine of West China Hospital, Sichuan University, and the Department of Rehabilitation Medicine of Affiliated Hospital of Chengdu University. Patients who were eligible should age between 30 and 80, and were diagnosed with cerebral infarction in accordance with the Chinese guideline for the diagnosis and treatment of ischemic stroke , with a Patients were excluded if they (1) were in unstable condition, or in the progressive state of stroke, or have cerebral hemorrhage, or (2) had a previous history of stroke, or limb motor dysfunction prior to the onset of cerebral infarction; or (3) had any serious concomitant systemic disease, condition, or disorder that, in the opinion of the investigator, was not appropriate for this study; or (4) had bilateral hemispheric injury.If participants were eligible after being screened based on the inclusion and exclusion criteria, they were asked to sign the informed consent and were randomized into the CIMT group, NE group, or control group. Randomized assignments were generated using a random number table and placed in sealed envelopes, which were then opened by an independent study coordinator in consecutive order.CIMT group received constraint induced movement therapy plus conventional rehabilitation training; UE group received upper limb task-oriented training plus conventional rehabilitation training; and Control group received conventional rehabilitation training only.All three groups went through conventional rehabilitation training program. The program comprised joint range of motion training, bed activities and transfer training, locomotor training, and physical factor therapy  at physical therapists’ discretion. The participants underwent conventional rehabilitation therapy for 30 min, once a day, five times a week, for 3 weeks.Participants in the UE group received upper limp task-oriented training . The traParticipants in the CIMT group underwent constraint-induced movement therapy. The patients wore mittens for 1 h each time to limit the use of their unaffected hand; thus, they were forced to use affected hands to perform specific tasks. Three to five tasks  were selected for each patient based on the functional assessment by the therapist. Before the training, the patients were given instructions on how to use the mitten to improve safety and adherence. The training lasts for 1 h each time, once a day, five times a week, for 3 consecutive weeks ,28.The middle cerebral artery occlusion and reperfusion (MCAO/R) model was used in the animal study. After anesthesia, a middle incision was made in the neck of the rats, and the right common carotid and external carotid arteries were carefully exposed. A suture with a rounded tip coated with poly-L-lysine was inserted approximately 20 mm through the internal carotid arteries. The middle cerebral artery was distally occluded. After 90 min, the suture was withdrawn. Sham operation was conducted by exposing the carotid arteries without occlusion of the middle cerebral artery.According to our design, we used Nrf2 antagonist ML385, and Nrf2 agonist t-BHQ to evaluate the regulatory role of Nrf2 in mediating CIMT or NE-induced neurological functional recovery. ML385 was dissolved in dimethyl sulfoxide (DMSO) and diluted with saline to 5% DMSO concentration for use. ML385 was injected intraperitoneally with a dose of 30 mg/kg every day, from days 1 to 14 after MCAO/R.Another group of rats were injected intraperitoneally with t-BHQ. The t-BHQ was dissolved in DMSO and diluted with PBS to 1% DMSO concentration. The single dose was 16.7 mg/kg, and the injections were given every 8 h for 3 times after MCAO.After successful model establishment, rats were randomly assigned to model group, or CIMT group, or UE group, or normal control (NC) group, or sham group. The interventions began 24 h after MCAO. Rats in the CIMT group were forced to use the affected limbs during the training, by fixation of the unaffected limbs in natural flexion position with a plaster cast. A cotton lining was used to prevent damage to the skin. The animals wore the plaster cast throughout the 14-day intervention phase. To increase the intensity of the CIMT, the rats underwent a 30-min treadmill training every day, and the treadmill speed was set as 8 m/min for days 1–3, 12 m/min for days 4–7, and 15 m/min for days 7–14 . For ratThe mNSS test was performed to evaluate neurological functional outcome on days 1, 4, and 14. The mNSS included motor, sensory, reflex, and balance tests. The score of the mNSS test ranges from 0 to 18, and a higher score indicates a worse neurological function.max × W × T/2, where Lmax and W is the maximum and minimum diameter, respectively, of the slice with the largest infarction area, and T is the product of the number of slices and slice thickness.MRI was used to assess the infarct volume of the brain. Fifteen slices of T2-weighted images were acquired with 0.8 mm thickness, repetition time 2500 msec, and echo time 33 msec. The infarct volume was quantified with the following formula: infarct volume = LThe rats were sacrificed and brain tissues were collected for further histological analysis. The brains were perfused with saline and 4% paraformaldehyde, and then were fixed in 10% neutral buffered formalin for 24 h. Then, the brain tissues were dehydrated, embedded in paraffin, and cut into 4 μm slices.HE staining was performed according to standard protocols. Briefly, after dewaxing with xylene, the slides were stained with hematoxylin for 2 min and thereafter with eosin. After dehydration, neutral gum was used to seal the slides for microscopic analysis.Nissl staining was performed to assess neurological survival. The slides were deparaffinized and stained with cresyl violet for 1 min. Then, the slides were rinsed with distilled water, dehydrated with alcohol, and mounted with neutral gum for later analysis.For immunofluorescence (IF), the slides were deparaffinized and rehydrated following standard protocol. Then, the slides were blocked with 10% goat serum at room temperature, and incubated with a primary antibody at 4° overnight, then with a fluorescent secondary antibody for 1 h at room temperature. The nuclei were stained with DAPI. Then, the slides were mounted with anti-quenching medium and were observed under a fluorescent microscope.For immunohistochemistry (IHC), after dewaxing and rehydration, the slides were immersed in citric acid and heated in micro-oven for antigen retrieval. Endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide for 30 min. The primary antibody was incubated overnight and the secondary antibody for 1 h. The DAB staining was then performed using a commercial DAB kit. Nuclei were colored with hematoxylin. The slides were sealed and dry for 12 h for microscopic analysis.The border zone tissue was immersed in liquid nitrogen and grounded to powder. Total RNA was extracted using the TRIzol method. The cDNAs were obtained with a reverse transcription kit (Takara) following the manufacturer’s instructions. QRT-PCR was performed with TB GreenTM Premix Ex TaqTM II (Takara) on the CFX96 Real-time PCR Detection System (Bio-Rad). The relative expression of the mRNAs was analyzed by the ∆∆Cq method. The primer sequences are presented in We extracted total proteins from the border zones in the right hemisphere of the brains, and then loaded the protein samples onto an SDS-PAGE gel. After separation by electrophoresis, the protein samples were transferred to polyvinylidene fluoride (PVDF) membranes. Nonspecific binding was prevented using 5% nonfat milk to block the membranes at room temperature for 1 h, followed by incubation with the primary antibodies and the secondary antibody. The immunoblots were detected using an enhancement detection system, and the proteins were quantified with the ImageJ software.Commercial ELISA kit was used to measure levels of MDA in the rat peri-infarct zone of the brain samples. All steps were performed following manufacturer’s instructions. The levels of MAD were expressed as mmol/L.For the RCT, we calculated the sample size with the Fugl–Meyer score as the primary outcome. Eighty-four participants would be required considering a 1:1:1 allocation ratio, a test power of 80%, a significant level of 5%, a standardized deviation (SD) of 3.4, and a dropout rate of 20%.t-test were used for intra-group statistical analysis as appropriate; ANOVA or rank-sum test was selected for inter-group comparison as appropriate, with a test level of 0.05. For categorical variables, a Fisher exact probability test was used.Continuous data are presented as the mean ± SD. A rank-sum test or In the RCT, a total of 84 patients were eligible at enrollment. Each group recruited 28 patients, and 3 patients drop out in the control group, 4 in the CIMT group and 2 in the UE group. Nine patients dropped out . Finally, 75 patients completed the intervention and were analyzed.p > 0.05). The demographic characteristics are depicted in p < 0.001), while inter-groups analysis demonstrated that FMA-UE was the highest in the CIMT group compared to the other two groups , and CIMT was more effective than UE  staining revealed swollen, stained cytoplasm, nuclear pyknosis, nuclear fragmentation, vacuole structure, and disturbed structure in the peri-infarct zone after the induction of MCAO/R, which were alleviated was in the CIMT group compared to control or UE group. Nissl staining suggested an increase in the number of neurons in CIMT group compared with UE or control group (p = 0.0004), while the nucleic Nrf2 mRNA level was increased in CIMT compared to the UE group (p = 0.0094). Western-blot analysis demonstrated similar changes: that Keap1 protein level was decreased (p = 0.0317) and the nucleic Nrf2 protein level was increased (p = 0.0174) in the CIMT group. HO-1 was increased in the CIMT group compared with the UE group (p = 0.0285). IF and IHC revealed that the protein levels of Keap1 and HO-1 were increased, and Nrf2 was decreased in the CIMT group. The production of MDA was suppressed in the CIMT group, compared with UE or control (p = 0.0377, p = 0.0194, Next, we investigated the role of oxidative stress signaling in the modulation of CIMT or UE induced effects. RT-qPCR suggested that Keap1 mRNA expression was lower in CIMT group than in the UE group (We used Nrf2 antagonist ML385 and agonist t-BHQ to investigate the role of Nrf2. After administration with t-BHQ, the motor function was significantly recovered as suggested by a lower score of mNSS in the CIMT+t-BHQ group than in the model, CIMT, and CIMT + ML385 groups. The CIMT induced motor function recovery was counteracted by ML385. Moreover, the administration of t-BHQ in rats undergoing CIMT resulted in a smaller infarct volume. Rats undergoing CIMT had a smaller infarct volume compared to those receiving CIMT plus ML385.Using qRT-PCR, Western-blot, and IF, we found that the administration of t-BHQ in rats undergoing CIMT resulted in an increased expression of Nrf2 in protein and mRNA levels. Furthermore, the expression of HO-1 was increased in the CIMT+t-BHQ group compared with CIMT and CIMT+ML385 groups. The injection of t-BHQ in rats undergoing CIMT also resulted in a lower production of MDA .Our study investigated the clinical efficacy and possible mechanism of constraint and unconstraint exercise mode on limb dysfunction after stroke, in the hope of providing a theoretical basis for the selection of exercise mode in clinical practice. We first conducted an RCT to compare effects of CIMT and UE directly. It was preliminarily found that, compared to UE, CIMT has more significant effects in improving upper limb function and reducing spasm. However, CIMT may cause patients to feel depressed, which may be because some patients did not adapt to the constraint device. Next, in the animal experiment, we studied the improvement of motor function of MCAO/R rats by constraint and unconstraint exercise therapy. Motor function was improved more in CIMT than in UE group as assessed by an mNSS test. Furthermore, the Keap1-Nrf2-ARE signaling pathway was found to play a regulatory role revealed by RT qPCR, Western blot, immunofluorescence, and immunohistochemistry, which was further confirmed in gain of function and loss of function experiments. MDA, the downstream product of oxidative stress, was suppressed in the CIMT and UE group, and CIMT group had a lower level of MDA compared to the UE group. Neural regeneration and apoptosis were also studied, and a higher level of neural regeneration and lower level of apoptosis were observed in the CIMT group than in UE group. Overall, CIMT could significantly improve the motor function of MCAO/R rats, which has a close link to oxidative stress, regeneration, and apoptosis.Exercise therapy is recognized as an important component of treatment for post-stroke rehabilitation and is crucial in determining patient activity of daily living and quality of life . It is dExercise therapy is closely related to oxidative stress. Previous studies have shown that exercise can increase the production of ROS and lead to oxidative stress in many tissues, including blood and skeletal muscle. The main mechanism is that muscle contraction stimulates the production of ROS in active muscle fibers ,37. FurtCIMT is characterized with forced use of impaired limbs to avoid learned non-use phenomenon. Most previous RCTs did not compare CIMT directly with UE, but with conventional treatment protocols instead, and they did not investigate psychological change after wearing the constraint device ,12,42. MOur study has some limitations. There are great differences in the exercise therapy applied in the RCT and the animal study, regarding the exercise type. In the clinical trial, we only follow up with the patients for 3 weeks, and the long-term effects of CIMT and UE remain to be studied. Moreover, we did not analyze whether CIMT had different effects on different types of cerebral infarction. Previous study indicated that the spontaneous, functional prognosis varies among different types of strokes . The RCTIn conclusion, CIMT is more effective than UE in improving upper limb motor function and reducing muscle spasm in patients with cerebral infarction than UE, but CIMT may cause the patients to feel depressed. Moreover, both CIMT and UE are beneficial to limb function recovery and limit the infarct expansion in MCAO/R rats, but CIMT was more effective than UE. Oxidative stress reaction has an essential role in regulating the CIMT induced benefits.


Sorghum bicolor) is an economically important cereal crop that can be used as human food, animal feed, and for industrial use such as bioenergy. In sorghum breeding programs, development of cultivars with desirable seed quality characteristics is important and development of rapid low-cost screening methods for seed nutritional traits are desired, since most standard methods are destructive, slow, and less environmentally friendly. This study investigates the feasibility of single kernel NIR spectroscopy (SKNIRS) for rapid determination of individual sorghum seed components. We developed successful multivariate prediction models based on partial least squares (PLS) regression for protein, oil, and weight in sorghum. The results showed that for sorghum protein content ranging from 8.92% to 18.7%, the model coefficients of determination obtained were Sorghum ( Sorghum bicolor) ranks fifth in world cereal production after corn, wheat, rice, and barley, with over 66 million tons produced  to assess the feasibility of using SKNIRS for small-seeded crops; and 2) to evaluate calibration models to predict protein, oil, and weight in single intact sorghum seeds that could be used to indicate seed trait variability in samples as well as potentially sorting seeds by traits for breeders.Sorghum bicolor) were selected and obtained from the U.S. Department of Agriculture (USDA) National Germplasm Center . The samples were selected to maximize diversity based on size and country of origin, A collection of 108 widely diverse sorghum accessions  was measured from single whole seeds using a Leco FP-628 nitrogen combustion analyzer  according to AACC Method 46-30.01 as described elsewhere . IndividOil content  of the intact seeds was measured by a Bruker MiniSpec mq20 NMR Analyzer  as described previously . The oilSpectral data were analyzed using Unscrambler X software . Standard normal variate (SNV) was used as a pre-processing method for oil and protein. Partial least squares (PLS) regression was performed and evaluated using leave-one-out cross validation to select the optimal models. The samples were further subdivided into training  and validation  sets based on creating equal variability, as much as possible, in the biochemical seed parameter reference values. Model evaluation and selection were based on model factor levels as suggested by the Unscrambler software and evaluation of independent spectral data in the validation sets. For protein models, reference protein was the average protein from the three kernels from each accession. These values were indexed to the nine averaged spectra from the same kernels. For oil models, the nine averaged spectra were indexed to the oil content measured by NMR as previously described. Weight models indexed the three spectra average of a single seed to its true seed weight. p< 0.01; Seed parameters of 108 diverse sorghum accessions represented by their means, standard deviations (SD), and ranges are summarized in vs. reference plots of external validation sets for protein, oil, and weight models are shown in 2 = 0.87, RMSEP= 0.69, 2 = 0.71, RMSEP= 0.41, 2 = 0.63, RMSEP= 0.007, The predicted 2 = 0.23, RMSEP= 1.96, The protein model from single sorghum seeds showed poor prediction . The peaks for oil were observed for calibration models around 3rd overtone C-H stretching (992-1025 nm and 1210 nm) and 3rd overtone O-H stretching (960 nm), To the best of our knowledge, this is the first study which has applied SKNIRS to predict sorghum seed protein, oil, and weight. Our findings in this paper are applicable to a broad range of sorting implementations in the field, industry, as well as sorghum breeding programs. The SKNIRS technique provides numerous advantages including: i) as wet chemistry methods of sorghum quality assessments are slow and costly, the SKNIRS method is a suitable, non-chemical alternative for classification or screening for protein, oil, and weight, nondestructively; ii) SKNIRS may provide a powerful, fast, high-throughput seed nutrient quality technique for sorghum breeding as well as uniformity screening activities; iii) SKNIRS can simultaneously quantify sorghum seed constituents; and iv) SKNIRS does not require sample preparation and therefore minimizes potential errors in the sample preparation process.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.GH developed the research hypothesis, designed the research methodology, performed the research, analyzed the data, and wrote the original draft with the contributions from PA, PM, and BS. PA developed the methodology and draft. PA and PM interpreted data analysis, helped with methodology and software. All authors contributed to the article and approved the submitted version.This study was supported by the United States Department of Agriculture (USDA-ARS) 1890 Research Sabbatical program to ARS Project 3020-43440-010-002-S and a subsequent Non-Assistance Cooperative Agreement with GH (FAMU).We thank USDA National Plant Germplasm Center  for providing the seeds. We thank Kevin Fay  for his excellent technical help with nitrogen combustion. The mention of a proprietary product or trade name does not constitute a recommendation or endorsement by the US Department of Agriculture. The USDA is an equal opportunity provider and employer.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.


Fungal microbiota's involvement in the pathogenesis of Crohn's disease (CD) is incompletely understood. The terminal ileum is a predilection site both for primary involvement and recurrences of CD. We, therefore, assessed the mucosa-associated mycobiota in the inflamed and non-inflamed ileum in patients with CD.The mucosa-associated mycobiota was assessed by ITS2 sequencing in a total of 168 biopsies sampled 5 and 15 cm proximal of the ileocecal valve or ileocolic anastomosis in 44 CD patients and 40 healthy controls (HC). CD patients with terminal ileitis, with endoscopic inflammation at 5 cm and normal mucosa at 15 cm and no history of upper CD involvement, were analyzed separately. The need for additional CD treatment the year following biopsy collection was recorded.Malassezia and a depletion of Saccharomyces, along with increased abundances of Candida albicans and Malassezia restricta. Malassezia was associated with the need for treatment escalation during follow-up. Current anti-TNF treatment was associated with lower abundances of Basidiomycota. The alpha diversity of the inflamed and proximal non-inflamed mucosa within the same patients was similar. However, the inflamed mucosa had a more dysbiotic composition with increased abundances of Candida sake and reduced abundances of Exophiala equina and Debaryomyces hansenii.CD patients had reduced mycobiota evenness, increased Basidiomycota/Ascomycota ratio, and reduced abundance of Chytridiomycota compared to HC. The mycobiota of CD patients were characterized by an expansion of Malassezia was associated with an unfavorable disease course.The ileal mucosa-associated mycobiota in CD patients is altered compared to HC. The mycobiota in the inflamed and proximal non-inflamed ileum within the same patients harbor structural differences which may play a role in the CD pathogenesis. Increased abundance of  Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) characterized by transmural and segmental inflammation of the gastrointestinal tract. Currently, CD is thought to develop in genetically susceptible individuals exposed to environmental factors and gut microbiota, causing an aberrant immune response that leads to inflammation and subsequent tissue damage . The preSaccharomyces cerevisiae antibodies (ASCA) were early proposed as a diagnostic biomarker of CD (CARD9 single-nucleotide polymorphism (rs4077515 creating substitution p.S12N) to be associated with CD  or a single-balloon enteroscope (Olympus SIF-Q180). A total of six ileal pinch biopsies were collected from each study participant, three biopsies from approximately 5 and 15 cm proximal of the ileocecal valve or ileocolic anastomosis, respectively. In CD patients with terminal ileitis, the 5-cm samples were taken from an endoscopically inflamed area and 15-cm samples from normal-appearing mucosa. In CD patients categorized as having active disease, both biopsy locations (5 and 15 cm) were endoscopically inflamed. For CD patients in remission and the HC group, both biopsy locations (5 and 15 cm) appeared endoscopically normal. Endoscopic inflammation was evaluated using Rutgeerts score , whetheracterial  and fungFormalin-fixed biopsies were stained with hematoxylin and eosin (H&E). Histological examination was performed blinded for phenotype by an experienced pathologist and scored according to the Global Histologic Disease Activity Score (GHAS) and Robarts score , 28, 29.The fungal cell wall is particularly robust and is known to be hard to lyse , 32. A DITS2 metagenomic sequencing libraries were prepared according to the “Illumina Metagenomics Sequencing Demonstrated Protocol”  with minhttp://frogs.toulouse.inra.fr/). Five biopsy samples were removed from the study due to a low number of sequences. This included two 5-cm samples from HC and three 5-cm samples from CD patients. Phyloseq Package for R analysis was used for alpha and beta diversity analyses as well as illustration. Deseq2 package for R analysis was used for differential analysis of OTUs with respect to the different phenotypes  was used for statistical analysis apart from analyses of sequencing data. Demographic and clinical characteristics are presented as % (n) for categorical variables, median [interquartile range (IQR)] for skewed distributed variables, and mean value [standard deviation (SD)] for normally distributed variables. Accordingly, the chi-squared test, Mann–Whitney The study was approved by the Regional Committee for Medical and Health Research Ethics, Central Norway . All study participants provided written informed consent.p = 0.017). The groups were otherwise similar. The bacterial microbiota characteristics in this cohort have been described previously , whereas the observed numbers of operational taxonomic units (OTUs) were similar (p = 0.21). This implies that the fungal species richness was similar, but that the evenness of fungi was reduced within the CD group compared to HC  according to the Jaccard index  and Jaccard index (p =0.051) did not differ significantly between CD patients with and without the need for treatment escalation  of anti-TNF treatment, n = 21 , inflammation (both endoscopic and histologic) was not associated with an altered mycobiota diversity or composition, based on observed OTUs, Simpson index, and Bray–Curtis dissimilarity  was not associated with an altered fungal microbiota diversity or composition in the CD cohort (n = 44) (Within the whole CD cohort (milarity . This ar(n = 44) . These f(n = 44) .Malassezia and loss of Saccharomyces. At species level, Malassezia was identified as Malassezia restricta and Malassezia sympodialis. The expansion of Malassezia restricta in CD has been reported previously  in UC patients has been associated with therapeutic response, and effects of FMT may be mediated by a reduction in Candida abundance . The patients/participants provided their written informed consent to participate in this study.MO, TM, and RF were responsible for the study design. MO and RF were responsible for data acquisition. MO, MR, OR, VB, AG, and ER were responsible for analyses of biological material and data analysis. MO, MR, and RF interpreted the results and drafted the manuscript. MO, MR, VB, AG, ER, TM, AS, HS, and RF contributed to the critical revision of the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by the Liaison Committee between Central Norway Regional Health Authority (RHA), the Norwegian University of Science and Technology (NTNU) (2016/29014), and by St. Olav's Hospital, Trondheim University Hospital. The GCF is funded by the Faculty of Medicine and Health Sciences at NTNU and Central Norway Regional Health Authority.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.


Introduction: Migraine is one of the most common neurological diseases. The pathophysiology of migraine has not yet been fully elucidated. There is increasing evidence supporting the relationship between oxidative stress and migraine.Method: This is a retrospective, cross-sectional and observational study. The patients were divided into two groups, episodic migraine and chronic migraine. Episodic migraine patients were divided into two subgroups, migraine with aura and migraine without aura. Serum Albumin, total bilirubin, uric acid levels, and migraine clinical findings were obtained from medical records.Results: A total of 181 participants, 88 patients and 93 controls, were included in the study. Serum albumin levels were lower in the patient group than in the control group, they did not reach statistical significance (p=0.082). There was no significant difference between the patient and control groups for total bilirubin levels (p=0.785). Serum uric acid levels in the patient group were found to be significantly lower than in the control group (p<0.001). Measured levels were similar in chronic and episodic migraine groups, and migraine with aura and migraine without aura subgroups.Conclusion: We thought this oxidative stress marker may be associated with the presence of migraine, but this is not significant for migraine subtypes and migraine progression. Migraine is a chronic neurological disease characterized by throbbing headache attacks, usually unilateral, aggravated by physical activity, accompanied by photophobia, phonophobia, nausea, and vomiting . It has The pathophysiology of migraine has not yet been fully elucidated. Different mechanisms have been proposed to date including vascular dysfunction, neurogenic inflammation, and activation of the trigeminovascular pathway . There iSerum albumin (ALB), total bilirubin (TBIL), and uric acid (UA) are major non-enzymatic antioxidants in human plasma and may play important roles in scavenging endogenous reactive oxygen species (ROS) . UA is tStudies measuring serum levels of antioxidants in migraine are limited. In this study, it was aimed to investigate the effects of major non-enzymatic antioxidants such as ALB, TBIL, and UA in the serum on the pathogenesis and chronicity of the disease by measuring the levels in migraine patients.In this retrospective, cross-sectional and observational study, a total of 88 patients and 93 healthy control groups included, aged between 18-60 years, admitted to the neurology headache outpatient clinic of a tertiary university hospital between 01 January 2018 and 01 January 2022 and were diagnosed with migraine according to the International Classification of Headache Disorders (ICHD)-3 beta criteria .Those with chronic systemic disease , malignancy, acute or chronic infection, endocrine-metabolic disease, rheumatic-immunological disease, and hematological disease , those who use cigarette alcohol, those who use topiramate and diuretic drugs, patients with gastrointestinal insufficiency and malnutrition, and patients whose medical records could not obtain serum ALB, TBIL and UA values and migraine clinical findings were not included in the study. In addition to the selection criteria used for the patient group, the control group consisted of healthy individuals who did not have a history of any type of headache and gave blood samples for routine health screening. Blood samples were taken from the antecubital vein from the patient in the attack-free period and control groups after 8-10 hours of fasting and sent to the hospital laboratory.The study was conducted in accordance with the Declaration of Helsinki guidelines and approval was obtained from the ethics committee of Gaziantep University Faculty of Medicine .Statistical analysisStatistical analyses were performed with the help of SPSS Statistics, version 22.0 . Mean and standard deviation values were used for descriptive analyses. The conformity of the quantitative variables to the normal distribution was analyzed by kurtosis and skewness tests. Categorical variables were compared with the Chi-square Test. The independent T-test was used to compare the quantitative variables with normal distribution, and the Mann Whitney U Test was used to compare the quantitative variables that did not fit the normal distribution. Comparisons between categorical variables with more than two groups were made with one-way ANOVA and Kruskal Wallis tests. The inter-correlation of quantitative data was analyzed with Spearman’s and Pearson’s correlation tests. A p-value below 0.05 was considered statistically significant.A total of 181 participants, 88 patients and 93 controls, were included in the study. While 55 migraineurs were in the EM group, 33 were in the CM group. Twelve of the EM patients were in the MwA subgroup and 43 were in the MwoA subgroup. There were 72 (81.8%) women in the patient group and 76 (81.7%) in the control group. The mean age of the patient group was 35.28, and the mean age of the control group was 35.15. The patient and control groups are similar in terms of age and gender distribution. Serum ALB, TBIL, and UA values were 4.16 g/dl, 0.50 mg/dl, 3.91 mg/dl in the patient group, and 4.26 g/dl, 0.53 mg/dl, and 4.90 mg/dl in the control group, respectively. Although serum ALB values were lower in the patient group than in the control group, they did not reach statistical significance (p=0.082). There was no significant difference between the patient and control groups for TBIL values (p=0.785). Serum UA values in the patient group were found to be significantly lower than in the control group (p<0.001). Demographic characteristics and laboratory data of the patient and control groups are shown in Table There was no significant difference between the EM and CM groups for serum ALB, TBIL, and UA values . Migraine clinical findings and laboratory data of EM and CM patients are shown in Table In the EM subgroup, no significant difference was found between MwA and MwoA for serum ALB, TBIL, and UA values . Laboratory data of MwA and MwoA patients are shown in Table Oxidative stress occurs as a result of insufficient antioxidants in the elimination of ROS and causes various diseases if not controlled . NeuronsThe precise mechanisms by which oxidative stress may play a role in the pathophysiology of headaches have not been clearly defined. It is hypothesized that the increased production of ROS may lead to peroxidation of cell membrane phospholipids, destruction of intracellular molecules , and consequently deterioration in the normal function of cells . In partIt has been thought that oxidative stress and insufficient antioxidant defense may play a role in the chronicity of migraine . PreviouDetermination of oxidative changes in patients with migraine may contribute to the determination of treatment planning and treatment success in addition to its diagnostic contributions. Approaches that increase antioxidant capacity such as agents with antioxidant content and lifestyle changes such as dietary supplements, regular physical exercise and elimination of psychosocial stressors may be promising in migraine prophylaxis. To date, possible antioxidant treatment options such as dietary supplements containing riboflavin, coenzyme Q10, melatonin, magnesium, vitamin D, and polyunsaturated fatty acids have been recommended in migraine ,29.Our study has some limitations. First of all, our retrospective, cross-sectional and observational study does not provide conclusive evidence that oxidative stress plays a role in the pathogenesis of migraine. The results of our study, which included a small patient population from a single center, cannot be generalized. Differences such as diet, physical activity level, and psychosocial factors may have affected the results of the study. In addition, valuable parameters such as total antioxidant levels, total oxidant levels, and oxidative stress index were not measured.In our study, we found that serum UA levels were low in migraine patients. We thought that this oxidative stress marker may be associated with the presence of migraine, but this is not significant for migraine subtypes and migraine chronicity. Studies focusing on oxidative balance may be valuable in understanding the underlying mechanism of migraine. In addition, investigating the effects of the agents used in the treatment of migraine on oxidative stress biomarkers may further elucidate the pathogenesis of the disease. We think that prospective cohort studies with a large population will be useful.


Journal for Immunotherapy of Cancer. Adam E Snook was the most prolific writer, while Dung T. Le was the most commonly co-cited author. “T cell”, “MMI” and “PD-1blocked” were the most widely studied aspects of CRC immunotherapy. “Immune checkpoint inhibitor”, “combination therapy”, “drug therapy” and “liver metastases” were current research hotspots. “Tumor microenvironment”, “neutrophils”, “tumor-associated macrophages”, and “suppressor cell” have emerged as research hotspots in recent years. “Gut microbiota”, “nanoparticle” and “tumor mutational burden” as recently emerged frontiers of research that should be closely monitored.An increasing number of studies have shown that immunotherapy serves a significant role in treating colorectal cancer (CRC) and has become a hotspot. However, few studies used the bibliometric method to analyze this field comprehensively. This study collected 1,899 records of CRC immunotherapy from 2012 to October 31, 2021, and used CiteSpace to analyze regions, institutions, journals, authors, and keywords to predict the latest trends in CRC immunotherapy research. The United States and China, contributing more than 60% of publications, were the main drivers in this field. Sun Yat-sen University was the most active institution, while the National Cancer Institute had the highest frequency of citations. Most publications were published in the  CRC is one of the most common cancers in the world. According to statistics, there have 1.9 million new cases of CRC in 2020, and it is expected to reach 2.5 million in 2035 .Ioannides and Whiteside first introduced the tumor microenvironment (TME), which refers to the local biological environment during tumor development, providing a scaffold and barrier for tumor cell growth and generating immune-exempt areas to provide a ‘breeding ground’ for tumors. TME helps tumor cells evade immune surveillance by providing an environment for tumor growth and suppressing local immune responses , 6. A baOn May 23, 2017, the FDA approved Pembrolizumab for use in adult or pediatric patients with unresectable or metastatic, MSI-H/dMMR solid tumors, including colorectal cancer. On July 31 of the same year, the FDA approved Nivolumab to treat patients aged 12 years or older with MSI-H/dMMR colorectal cancer , 9. IpilImmune checkpoint inhibitors (ICIs), modulating the interaction of T cells, antigens-presenting cells (APCs) and tumor cells to help unleash suppressed immune responses, emerged as a very effective therapy for patients with mCRC that is mismatch-repair-deficient (dMMR) or microsatellite instability-high (MSI-H) (termed dMMR/MSI-H mCRC). Due to the efficacious, stable and durable responses, pembrolizumab and nivolumab (with or without Ipilimumab) were approved by US Food and Drug Administration (FDA) to treat these patients. However, mCRC is characterized by insufficient mutated tumor antigen , thus thBibliometric analysis adopts the number of citations as a proxy measure of research quality, which is a useful tool for assessing the trend of research efforts statistically and qualitatively. It provides an objective assessment of the contributions of academic groups and individual researchers through a comprehensive analysis of the authors, countries, journals, citations and publication dates of selected articles, thus providing a method for understanding trends in specific fields and ranking academic groups and individuals . In addiThere was currently no similar analysis in CRC. This paper utilized bibliometric analysis to describe the literature related to CRC immunotherapy over the last decade to understand its features and forecast future research trends and hotspots. Therefore, to better capture the current status and trends in CRC immunotherapy research, the purposes of this study were to reveal present study trends, explore into possible research hotspots and guide researchers in their future work by using bibliometric methods to visualize references.The Web of Science (WOS) core database from Clarivate Analytics was deemed the best for bibliometric analysis , 17, so The annual publication was analyzed and plotted using GraphPad prism v8.0.2. Furthermore, bibliometric methods were used to gather the research foundation, frontier knowledge, and trends. In this study, the acquired bases were imported into the CiteSpace (5.6.R3) software , a literg(v) represents the centrality value of the vertex v. stσ(v) represents the number of shortest paths passing through v between vertex s and vertex t. stσ represents the number of all shortest paths from vertex s to vertex t. Burst detection is used to detect significant changes in the number of references at a given time. It is used to discover the decline or rise of a certain subject term or keyword. The algorithm invented by Kleinberg, J was used in cite space to predict the change of keywords at a certain stage and thus to predict the field’s future development , and all options in the term source were selected, node types were selected one at a time, selection criteria . Each node in the figure indicated an observation including country, institution, author, co-cited literature and keywords. If the publication is issued in cooperation with several countries, institutions and authors, then each country, institution and author is taken into account, which is the basis for the network of institutional, national and author cooperation.The node size represented the frequency of occurrence; the larger the node, the higher the frequency of occurrence. Connections between nodes represented collaborative, co-occurring or co-referential relationships. The different colors of nodes represented different years; the different colors of circles from inside to outside indicated the years from 2012 to 2021. The outermost purple ring indicated that the node has very high centeredness and was often regarded as a critical point in a specific domain . The impA total of 1899 publications on CRC immunotherapy were published on the web of science from 2012 to October 31, 2021, including 1454 (76.57%) ARTICLES and 445 REVIEWS (23.43%). The literature covered 66 countries or regions and 367 institutions.The annual number of publications for immunotherapy on CRC from 2012 to 2021 as shown in The annual trend publications associated with immunotherapy for CRC by country from 2012 to 2021 were presented in Among the top 10 countries/regions regarding publications, the U.S. had 19,051 citations, far more than all other countries, while it had a high citation/publication ratio (31.65). Although China had many citations (9285), its citation/publication rate (15.68) was lower than other countries. It is to be noted that France had the highest citation/publication ratio (52.32) among the ten countries, despite the relatively low number of publications, which also indicated the high quality of its published papers.The analysis of the international collaboration network  published the most documents in this field, followed by Oncoimmunology , Cancer , Frontiers in Immunology (57 articles. 3.00%) and Cancer Immunology Immunotherapy . Among the top ten most prolific journals, the Journal for Immunotherapy of Cancer had the highest IF of 13.751 and Cancer Research had the highest citation/publication rate (52.33). Of the top 10 journals, 80% were classified as Q1 (top 25% of IF distribution), remaining two journals were Q2 (between 25th and 50th quartiles) and Q4 (between 50th and 100th quartiles). The most frequently co-cited journal was the Journal of Clinical Oncology (4270 citations), followed most frequently by Clinical Cancer Research (3841 citations) and Cancer Research (3807 citations). Among the top 10 co-cited journals, New England Journal of Medicine was cited 3640 times with the highest IF of 91.253. Except for Journal of Immunology, the rest of the journals were in Q1.The top ten most prolific and co-cited journals were listed in The subject distribution of academic journals was depicted by a dual-map overlay of the journals   was the most cited article by Dung T. Le. In addition, 2 of the top 5 co-cited papers were written by Dung T. Le and published in different journals.The co-cited literature network diagram was composed of 850 nodes and 4135 links, with a time slice set to 1 year and a period set to 2012-2021 7, PD-1 Moreover, we performed a temporal co-citation analysis and mapped the timeline view of co-cited references Figure 6Based on the number of citations and centrality analysis of keywords through CiteSpace Table 8,CiteSpace designed the keyword time zone view to show high-frequency keywords’ evolution clearly. The keywords were situated in the years in which they first appeared together, and colors of the links represented the years when the two keywords first appeared together. High-frequency keywords (T > 80) were displayed in We use CiteSpace to detect emergent keywords to identify the frontiers of research in this field. Among the 103 keywords with high citation outbreaks, we focused on those with bursts starting in 2020 Figure 9In this study, we obtained 1899 publications on colorectal cancer immunotherapy from 2012 to 2021 by searching the web of science core database and manual screening. In terms of annual publications, publications increased rapidly after 2015. In terms of national and regional distribution, the United States has been the driving force behind the highest academic contribution to colorectal cancer immunotherapy in the last decade, as reflected by the number of publications, centrality, citation frequency and citation publication ratio. China’s annual publications proliferated after 2016, and since 2020 China has surpassed the United States. Although the average citation was relatively low, the higher total number of citations showed the high contribution of China to the development of the field. France, Spain, Italy and South Korea have lower publication numbers, but their publications have multiplied in recent years, related to the cooperation with the United States.90% of the 10 most productive institutions were from China and the United States, indicating that China and the United States played significant roles in the academic development of the field. Interestingly, Sun Yat-sen University, the most productive institution globally, had the lowest average citation among the top ten institutions. The National Cancer Institute (NCI) had the highest total citations (1556) and average citations (53.7) among the top 10 institutions, indicating that NCI published higher quality articles in the field and played a crucial role in promoting the development of the field.Journal for Immunotherapy of Cancer (66 articles), Oncoimmunology (63 articles), Cancer (57 articles), Frontiers in Immunology (57 articles) and Cancer Immunology Immunotherapy (53 articles) published more than 50 papers. The Journal of Clinical Oncology received the highest citations. Notably, both Clinical Cancer Research (66.11) and Cancer Research (52.33) had more than 50 citations per article and ranked 2nd and 3rd, respectively, among co-cited journals. thus, they are recognized as research resources for CRC immunotherapy.Among the ten most active journals, Most of the top 10 productive journals were related to immunology and clinical, while most of the 10 highly co-cited journals were related to biology, which was consistent with the dual-map overlay analysis. The dual-map overlay represented the subject distribution of academic journals. New England Journal of Medicine (IF=91.253) and Science (IF=47.728), respectively. Researchers first evaluated the clinical activity of PD-1 inhibitors in patients for progressive CRC with/without mismatch repair (MMR) and found that microsatellite instable (MSI) CRC (MSI-CRC) were more sensitive to PD-1 blockade responses compared to microsatellite stable (MSS) tumors  tumors . TherefoWe found that most 5 co-cited articles with the highest frequency and centrality were related to immune checkpoint inhibitors (ICIs) and MSI, which indicated the importance of ICIs and MSI in the field. Among the top five co-cited references, the article titled “Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries” was focused on a global review of cancer prevalence and treatment measures . Based oThe timeline view of the co-cited literature showed that most of the studies were published after 2007. The prognosis (Cluster0) has the darkest color and the most published literature and has been a consistent focus of CRC immunotherapy. Microsatellite instability (Cluster1), programmed cell death protein ligand1 (Cluster2) have appeared in recent years and have larger nodes and warmer colors, indicating that the problems under these clusters were hot research topics in recent research years. Microsatellite instability (Cluster1), programmed cell death protein ligand1 (Cluster2) have emerged in recent years and have larger nodes and warmer colors, indicating that the problems under these clusters were hot spots of research in recent years, which was consistent with the above highly co-cited literature focusing on PD-1/PD-L1 and mismatch repair.In addition, in recent years, cytokine-induced killer cells (Cluster3) and t cell exhaustion (Cluster9) may be new research hotspots in CRC immunotherapy. It has been shown that gut microbes can promote colorectal carcinogenesis by stimulating specific immune responses . Gut micIn bibliometric analysis, keyword bursts can reflect research hotspots in academic fields , and timAccording to the citation count and keyword centrality analysis, which mainly included keywords related to immunotherapy mechanism of CRC included “Microsatellite instability”, “T cell”, “PD-1blockade”. It was similar to Nicolas J. Llosa et al. , who repSeveral studies have confirmed the significant anti-tumor effects of PD-1blockade in dMMR/MSI-H CRC immunotherapy , 34, furin vitro effects. In a study by Aparna Raj Parikh et al.  can be detected either by the lack of immunohistochemical staining of the MMR proteins MLH1, MSH2, MSH6 or PMS2 or by PCR-identified alterations in the lengths of microsatellites between a patient’s tumour and a sample of normal tissue or blood. The use of immunotherapy for dMMR/MSI-H CRC may lead to better outcomes, and the combination of multiple immune checkpoint inhibitors may achieve satisfactory results compared to single agents. Patients should be observed for immune-related side effects during treatment, such as immune-related nephritis, immune-related endocrine disorders and toxicity. However, there are still some challenges in the clinical treatment of this population, such as the keyword “resistance” that appeared in We should explain the limitations of the study. On the one hand, these data are collected from a single source, the web of science core, only, leading to the omission of articles from other sources. In addition, the manual removal of papers unrelated to the study by the investigator may lead to selection bias. Last, only English articles were entered in this study, which may lead to source bias.To our knowledge, our study was the first comprehensive metrological and statistical analysis of CRC immunotherapy over the past 10 years. In this study, we found that the United States has contributed significantly to the advancement of the field by publishing the most significant number of articles in the field and keeping the quality of articles high. China’s publications in this field snowballed after 2018, surpassing the U.S. since 2020, and maintaining a rapid growth trend that may be related to government financial support. The rapid growth of publications also indicated that CRC immunotherapy was becoming of increasing interest worldwide.Our research showed a trend for cooperation between various countries and institutions. However, there was a still lack of intensive cooperation between different institutions/countries. Notably, the National Cancer Institute (NCI) and Sun Yat-sen University have made significant contributions to the progress of the field. Therefore, institutions can strengthen their cooperation with them to promote the future development of the field better.Journal of Clinical Oncology and Journal for Immunotherapy of Cancer were the most cited and most published journals. In addition, the most prolific and co-cited authors were Adam E Snook and Dung T. Le, respectively. Thus, researchers can better grasp the research progress in this field through articles published in these journals or authors who will be potential collaborators in the field.Our research indicated that nanomaterials might be the most advanced and popular technology in recent research. In addition, CRC immunotherapy is associated with microsatellite status. Research on immunotherapy for MSI-CRC has focused on PD-1blockade, T cell and combination with other ICIs. Research on immunotherapy for microsatellite stabilized CRC has focused on immune checkpoint inhibitors, combination therapy, and drug therapy. These aspects will remain significant research hotspots in the future. Colorectal cancer liver metastasis has received increasing attention from researchers due to its characteristics. In addition, research on the immune microenvironment has drawn attention to its vital role in immunotherapy, and improving the effectiveness of immunotherapy by modulating the immune microcircuit has become increasingly important in the treatment of CRC. We can predict that the research on tumor microenvironment will become more enthusiastic in the future. The role of gut microbes in CRC immunotherapy may soon become a research hotspot and should be closely monitored.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 in the article/supplementary material.LM: Writing- Original draft preparation, Investigation, table and figure preparation. JM: Investigation and table preparation. MT : Supervision. YL: Conceptualization, Methodology, Supervision. All authors contributed to the article and approved the submitted version.This work was supported by National Natural Science Foundation of China (31770537) and Major Science and Technology Special Project of Gansu Province (20ZD7FA003).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.


The research indicates that academic skills and family function may influence academic achievement. The Covid 19 epidemic has impacted regular academic and family function. However, there is a dearth of studies evaluating the relevance of academic skills and family function on college students' academic achievement. This research was done to determine the levels and relationships between academic achievement and study skills and family functions. Two hundred seventy-nine college students were assessed with Sociodemographic and academic proforma, Study Skills Assessment Questionnaire (SSAQ), and The McMaster Family Functioning Scale (MFFS).Results revealed that the mean score of SSAQ and MFFS were 179.92 and 17.88, respectively. Multiple regression analysis showed that the score of reading skills and the MFFS score statistically significantly predicted the score of the exam.On the basis of this study's results, it is possible to conclude that reading skills and family functioning may influence academic success. Academic performance may be impacted by poor sleep quality, somatization, depression, anxiety, post-traumatic stress disorder, and psychosis after infection [Academic performance is usually the most important element in deciding whether or not to offer a university degree. One of the most important markers of a student's academic performance is their Grade Point Average (GPA). A grade point average (GPA) is a numerically weighted average of a student's grades for the course of his studies that can be used to assess a student's academic performance and position. Students' academic performance may be influenced by internal factors such as study skills and their immediate environment, such as family, as the Covid-19 epidemic impacted a significant proportion of the Arabic population [nfection , 3. Siminfection . Infectinfection . During nfection , 6. Thusnfection . Thus, tStudy skills are an array of skills that requires training and practice with specific techniques that help a learner acquire, organize, retain, and use information . Study sAfter obtaining approval from the institutional ethics committee, this cross-sectional study was conducted at Shaqra University in Saudi Arabia. An online Google form was created to collect data, and a link to a WhatsApp group of students from various colleges at—University was provided. The form did not include any items that could be used to identify the respondent, and there was an option to opt-out of the study.Sociodemographic and academic proforma: It consisted of age, gender, college of affiliation, level, GPA score, experiencing any difficulty in the study, difficulty in understanding teaching, perceived course suitability, and perceived favorable family environment. Prior semester exam score was GPA that ranged from 1 to 5 and was obtained by dividing the course credit hours by course point.Study Skills Assessment Questionnaire (SSAQ): The tool assesses eight domains: time management and procrastination, concentration and memory, study aids and note-taking, test strategies and test anxiety, organizing and processing information, motivation, and attitude, reading and selecting the main idea, and writing . Eight iThe McMaster Family Functioning Scale (MFFS): This scale assesses the degree to which a family has constructive and supportive relationships . This 12The research began on April 12, 2021, and ended on August 31, 2021. The study included participants of any gender who were members of Shaqra University's colleges and had completed a 6-month course. Individuals with a broken family or who live separately were excluded. By the deadline, 279 responses had been received from individuals who met the study's inclusion criteria. The assessment tools were:IBM SPSS version 25.0 was used to analyze the data. The demographic, study skills and family function variables were analyzed using descriptive statistics. Independent Student’s T-tests were used to determine the relationships between study skills and family function with demographic and academic variables. Multiple regression analysis was used to determine whether the value of the SSAQ and MFFS scores can predict the value of the exam score. The significance of statistical analysis was 0.05.Sociodemographic features characterized by the majority being female, single, from health science, did not report major difficulty in the study, satisfied with the course they were perusing, the family was supportive for their academic course and good study skills Table .Table 1SThe mean GPA, MFFS, and SSAQ scores were 4.24, 17.88, and 179.92, respectively. The student has a better score on SSAQ subscale Concentration/Memory (23.58), Readiness to take an exam (23.65), Attitude/Motivation (23.42); while lesser in Time management (22.08), Aid/Notetaking (22.03), Organizing information (22.57), Reading skill (21.89); and lowest in Writing skill (20.67) , Note taking , Readiness to take an exam , Organizing information , Attitude Motivation , main idea/self testing , and Writing skill   and Attitude/Motivation by supportive family environment  , difficulty in Study , Perceived course suitability  and Supportive family environment   was done using the SSAQ subscale score and MFFS score as the predictor variable and exam score as the dependent variable. Value on SSAQ subscale Reading skill score statistically significantly negatively predicted the value on the Score of exam score (p = 0.018). Similarly, the value of the MFFS Score statistically significantly negatively predicted the value of the Score of exam score (p = 0.003) (Table Multiple linear regression analysis (Adjusted The study examined the relationship between academic performance, study skills, and family functioning during the Covid 19 pandemic. There is a paucity of studies reporting these variables in Arabic countries to compare the findings. The academic skills score obtained in this study was comparable to another study . They obA significant finding of this study was the presence of a significant relationship between difficulty in comprehending the teaching and the majority of the study skills subscale score. A meta-analysis reveals that motivation and social support aid in academic performance and learning . AccordiAnother significant finding of this study was the occurrence of a significant relation between exam scores with reading skills and family function. Similar to this study's findings, an earlier report from South Africa indicates that reading skills and habits are associated with academic performance among undergraduate students , 27. EarAs a result of the findings, it may be concluded that family function might influences the academic performance. The findings imply that family function should be examined when college students' academic performance is impaired, and that family members should be educated about the family's role in academic performance. This conclusion must be confirmed through additional research.The study's findings should be interpreted in the background of the study's limitations, including a cross-sectional online study that relied on snowball sampling and could not determine the response rate, and there was no control group. Future studies should address these issues along with the other determinant of family function and academic skills and performance.


Sarcopenia and cognitive impairment are the most common causes of disability in the aging population. The potential role of sarcopenia in the development of cognitive impairment remains poorly understood. A cross-sectional analysis was performed using nationally representative data to evaluate associations between sarcopenia and cognition in China.We included 2,391 participants  who were at least 60 years of age in 2015 from the China Health and Retirement Longitudinal Study (CHARLS). Muscle strength, appendicular skeletal mass (ASM), and physical performance measurements, were measured to diagnose sarcopenia according to the Asian Working Group for Sarcopenia 2019 (AWGS2019). Cognitive function was assessed by 10 items in the Telephone Interview for Cognitive Status (TICS-10), delayed word recall, and graph drawing. Based on cognitive score tertiles, data were divided into three groups. Multiple linear and logistic regression models were used to assess the relationship between sarcopenia and cognition.p < 0.001) and negatively associated with components of sarcopenia in male group. The results remained consistent in male after further adjusting for creatinine, uric acid, blood sugar, etc. Low cognitive function in female was only associated with low muscle strength . In addition, participants with possible sarcopenia had greater risk of cognitive decline than those without sarcopenia . However, the same association was not significant in female group.The prevalence of possible sarcopenia was 27.16% for men and 27.46% for women. Cognitive decline was significantly associated with sarcopenia status (β = −0.88, We suggest that sarcopenia might be associated with cognition function, with possible sarcopenia being significantly associated with higher cognition risk in China population, which providing a further rationale for timely recognition and management of sarcopenia. Sarcopenia is a skeletal muscle disease rooted in the lifelong accumulation of adverse muscle changes due to increased protein catabolism or increased anabolic resistance, both common conditions in older adults, and it can increase the incidence of poor clinical outcomes, such as falls, fractures, physical disability, and even mortality . StudiesAt present, accumulating evidence suggests that sarcopenia may be associated with an increased risk of cognitive impairment in older adults, although the findings are inconsistent , 9. A meThe CHARLS is a longitudinal study of people over the age of 45 in China. This study was first established in 2011 and is an ongoing nationally representative longitudinal survey that aims to explore the socioeconomic determinants and consequences of aging . Brieflyn = 4,689), lack of information on blood tests and health status , lack of data on sarcopenia status , lack of information on age or age <60 (n = 339), missing physical examination and blood test data (n = 53), and missing cognitive data . Thus, a total of 2,391 participants were included the cross-sectional analysis. The specific screening process is shown in In this study, CHARLS data from 2015 were used. The inclusion criteria for this study were as follows: (a) individuals who were at least 60 years of age at the time of the 2015 CHARLS study and (b) individuals with available data on their sarcopenia status. The exclusion criteria were as follows: individuals with (a) missing sarcopenia status data and cognitive data; (b) missing age data; (c) age <60 years; (d) no physical examination data or blood data. In total, 21,095 participants were interviewed by the CHARLS in 2015. From the CHARLS data booklet we can also derive a blood response rate of only 64%, which may be due to the fact that the collection of blood samples was invasive. In addition, the purpose of the CHARLS data study was to provide high quality data on households and individuals aged 45 years and older in China, so we found 7,222 individuals younger than 60 years of age, and we had to exclude data from these groups as well. During our screening stages, some of the participants were excluded due to a lack of physical examination data  as hard as they could . A cut-oIn our article, we used an anthropometric equation to estimate the muscle mass, which has previously been validated in Chinese individuals , 20. Thewhere ASM is in kg, height is in cm, weight is in kg, age is in years, and sex is represented by 1  or 2 .2 = ASM/height (m)2. The cut-off point for low muscle mass was based on the lowest 20% percentile of ASM/Ht2 in the study population. Since our data are derived from the 2015 CHARLS data, we refer to the criteria of Wu et al.  was used to measure the participant's usual walking speed (m/s) over a 2.5-m distance. Participants walked the 2.5-m distance at normal speed, once back and forth , timed by a stopwatch; the average of the two recorded values was used . RepeateThis study measured four dimensions of cognitive function, including orientation, attention, episodic memory, and visuospatial abilities. Orientation and attention were evaluated by the TICS-10, on a scale of 0 to 10 . AttentiSociodemographic and health-related factors were included as covariates. Sociodemographic variables included age, sex and educational attainment . Health-related factors consist of body mass index (BMI), smoking , and blood measurements include blood glucose, total cholesterol (TC), low-density lipoprotein (LDL), creatinine, uric acid, C-reactive protein (CRP) and glycated hemoglobin (GHB).t-tests, and analyses of variance (ANOVAs). Second, the subjects were divided into two groups according to the diagnostic criteria: those without sarcopenia and those with possible sarcopenia. Additionally, the subjects were divided according to sex and analyzed separately to determine their baseline characteristics and differences in cognitive scores between the two sarcopenia groups. Multiple linear regression models were performed to analyze the relationship between sarcopenia (and its defining components) with the cognitive scores. And logistic regression analysis was used to calculate odds ratios (ORs) with 95% confidence intervals (CIs). The covariables models adjusted were as follows: Model 1: adjusted for smoking, education and age; Model 2: as model 1 and additionally adjusted for BMI; Model 3: as Model 2 and additionally adjusted for creatinine, uric acid, blood sugar, LDL, TC, CRP, GHB.Categorical data are presented as proportions, and continuous data are reported as means ± standard deviations or medians and tertile range for continuous variables and as percentages for categorical variables. First, based on cognitive score tertiles, data were divided into three groups. The baseline characteristics of the cross-sectional samples were summarized and compared between these groups using chi-square tests, Student's n = 1,539), 418 (27.16%) had possible sarcopenia, and among the female subjects (n = 852), 234 (27.46%) had possible sarcopenia. Regardless of sex, there were significant differences between those with and without possible sarcopenia in terms of age, height, weight, muscle mass, muscle strength, and physical activity (p < 0.05). Furthermore, man in the no possible sarcopenia group had higher level of education (p < 0.001). The cognitive scores of males with possible sarcopenia (12.00) were significantly lower (p < 0.001) than those of males without possible sarcopenia (13.00) in males; the same pattern was observed in females.The baseline characteristics of the study subjects according to sex and likelihood of sarcopenia are shown in p < 0.01). Among male subjects, the percentage of those with the lowest cognitive scores with possible sarcopenia (41.9%) was higher than that of those without possible sarcopenia (32.6%). Comparable results were also observed in females (p < 0.05).p < 0.05). After adjusted for age, education, smoking, BMI, significant negative correlations (correlation coefficient > 0.5) were observed between cognitive function and sarcopenia, cognitive function and low muscle mass, cognitive function and low GS (p = 0.02) in the fully adjusted model (Model 3).d low GS . This efp = 0.001). In females, the risk of low cognitive scores in those with possible sarcopenia was 1.69 times  (p = 0.005) greater than that in those without sarcopenia. But after adjusting for blood-related variables in females (Model 3), no significant associations were observed.Across all study subjects, those with possible sarcopenia were 1.40  times more likely to have cognitive scores decline than subjects without sarcopenia . SubjectWe found that the presence or absence of sarcopenia in an older population may have a differential impact on cognitive performance. According to this cross-sectional study, among the older population in China, those with possible sarcopenia are at high likelihood of having low cognitive function than those without sarcopenia. The present study showed that the prevalence of possible sarcopenia in female subjects was higher than that in male subjects (27.46 vs. 27.16%); this difference may be due to the larger sample size of males than females in this study. The prevalence of sarcopenia was recently found to be 21.7 and 33.3% in females and males, respectively, but the population in that study was aged 80–99 years . HoweverThis article also examined the relationship between various components of sarcopenia and cognitive scores, finding that low muscle mass, low muscle strength, and slow pace  were all negatively correlated with cognitive scores. These results are consistent with those of other studies. For example, studies have shown that in older Chinese men, lower muscle mass was associated with higher depression scores . SeveralIn addition, the present study, which adjusted for sex, found that possible sarcopenia was significantly associated with low cognitive function in men and that participants with possible sarcopenia were at greater risk of having low cognitive function than those without sarcopenia. However, sarcopenia was not associated with low cognitive function in female in fully adjusted model. One possible reason for this difference may be the small sample size in females. In addition, the Partial Androgen Deficiency in Older Men (PADAM) study showed that older men experience a partial, gradual and variable decline in testosterone, manifested by depression, lack of motivation and energy, and lower mental vitality, with age . Sex horThere are some limitations in this study. First, a significant amount of data in CHARLS is missing or incomplete, which could lead to biases. We also did a comparison of basic information between the excluded and included samples, there were still differences in their gender and age. However, we also found the average age of the included participants was 67.04 ± 5.57 years, which was similar to a previous study (68.13 ± 6.46 years) using the 2015 CHARLS data . Second,The present study found that sarcopenia and low cognitive function were correlated in older individuals and that sarcopenia is a risk factor for low cognitive function in older individuals. Thus, sarcopenia prevention and treatment can be used as a therapeutic measure to prevent or delay low cognitive function in older individuals, to control and manage sarcopenia or reduce the risk of sarcopenia in high-risk groups, and to enable early detection and treatment. Sarcopenia prevention and treatment can reduce low cognitive function in older individuals; prevent cognitive impairment; reduce the burden on individuals, families and society; improve the living standards of older individuals; and provide new methods and ideas for improving the health of older individuals.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 in the article/supplementary material.The studies involving human participants were reviewed and approved by Ethics approval for the CHARLS study was obtained from the Institutional Review Board (IRB) at Peking University. The IRB approval number for the main household survey was IRB00001052-11015 and for the biomarker collection was IRB00001052-11014. The patients/participants provided their written informed consent to participate in this study.HD designed the study and wrote the paper. MY performed the statistical analyses. HX revised the manuscript. XL collected and interpreted data. YC contributed to the interpretation of study results. ZL contributed to acquisition of funding, study design, and provided administrative support. All authors have read and agreed to the published version of the manuscript.