Epilepsy is a neurological disorder that affects millions of people worldwide. Anti-epileptic drugs (AEDs) are critical for their management. However, the therapeutic window is narrow, and traditional laboratory-based therapeutic drug monitoring (TDM) methods can be time consuming and unsuitable for point-of-care testing. To address this issue, we developed a disposable sensor chip based on molecularly imprinted polymer-modified carbon paste electrodes (MIP-CPs) for the TDM of AEDs such as phenobarbital (PB), carbamazepine (CBZ), and levetiracetam (LEV). In this work, functional monomers (methacrylic acid) and crosslinking monomers (methylene bisacrylamide and ethylene glycol dimethacrylate) were copolymerized in the presence of the AED template and grafted on the graphite particles by simple radical photopolymerization. The grafted particles were mixed with silicon oil, dissolving ferrocene as a redox marker to make the MIP-carbon paste (CP). Disposable sensor chips were fabricated by packing the MIP-CP into the base made of poly  (PET) film. The sensor’s sensitivity was determined using differential pulse voltammetry (DPV), carried out on a single sensor chip for each operation. Linearity was obtained from 0–60 μg/mL in PB and LEV and 0–12 μg/mL in CBZ, covering their respective therapeutic range. The time taken for each measurement was around 2 min. The experiment using whole bovine blood and bovine plasma indicated that the existence of species that interfered had a negligible effect on the test’s sensitivity. This disposable MIP sensor provides a promising approach for point-of-care testing and facilitating the management of epilepsy. Compared with existing tests, this sensor offers a faster and more accurate way to monitor AEDs, which is crucial for optimizing therapy and improving patient outcomes. Overall, the proposed disposable sensor chip based on MIP-CPs represents a significant advancement in AED monitoring, with the potential for rapid, accurate, and convenient point-of-care testing. Anti-epileptic drugs (AEDs) are the primary method of therapy for epilepsy, a persistent neurological brain illness affecting about 50 million people worldwide ,2,3. TheElectrochemical sensors have recently gained attention as a promising alternative for analyzing the AEDs ,11,12. ECarbon paste electrodes are widely used in electrochemical analysis because of their high electrical conductivity, low cost, and ease of preparation ,16,17. COne of the several techniques of carbon surface modification is the molecular imprinting technique. It is a process in which a specific molecule is used as a template to create a molecular imprint in a polymer matrix. The resulting molecularly imprinted polymer (MIP) has the unique ability to selectively recognize and capture the target molecule in a complex mixture, providing a highly selective and sensitive method for its analysis. Because of this, MIPs have demonstrated immense potential in a wide range of applications, including chemical and biological sensing such as nanosensing ,24, sepaIn recent years, there have been several studies on using MIPs for AED detection, with some researchers reporting detection limits in the nanomolar range and being capable of detection in biological fluids. However, despite these promising results, there is still a need for the development of MIP sensors that can provide reliable and accurate detection of AEDs in a wide range of conditions, including those that may affect sensor performance, such as pH, temperature, and interference from other substances. One of the significant limitations of present MIP-based sensors is the lack of robustness and reproducibility of MIP synthesis protocols, which can lead to batch-to-batch variation in sensor performance. Additionally, many MIP-based sensors need more selectivity and sensitivity, mainly when applied to complex matrices such as biological fluids. There is also a need for further validation of MIP-based sensors against established analytical methods and clinical samples to assess their accuracy and reliability in real-world settings. Finally, there need to be more studies that explore the scalability and commercial viability of MIP-based sensors, which is a critical aspect of their widespread adoption in clinical practice.In this study, we aimed to develop a disposable chip sensor for therapeutic drug monitoring (TDM) of AEDs by utilizing a molecularly imprinted polymer (MIP)-modified carbon paste electrode. The MIP-CP-based sensing material on a disposable chip sensor provides a simple and cost-effective fabrication process for the sensor, making it more accessible for scale-up and mass production. Furthermore, this sensor offers several advantages, such as portability for field-based applications, reduced sample contamination, reagentless sensing, and selective analyte detection in a wide range of biological matrices. Although this work focused on the phenobarbital sensor, we also presented preliminary work on levetiracetam and carbamazepine sensors. N,N’-methylene bisacrylamide (MBAA), acrylamide (AAm), ethylene glycol dimethacrylate (EDMA), phenobarbital sodium salt (PB), and carbamazepine (CBZ) were purchased from Wako Pure Chemical Industry . Levetiracetam (LEV) and ferrocene were purchased from Tokyo Chemical Industry . N,N-dimethylformamide (DMF) was purchased from Kanto Chemical Co., Ltd. . Bovine blood for testing was bought from the Tokyo Shibaura Zoki Corporation . The graphite particles  were generously donated by Ito Graphite Co., Ltd. . Silicone oil (300 cs viscosity) was purchased from Shin-Etsu Chemical Co., Ltd. Heat-adhesive polyethylene terephthalate (PET) sheets  were purchased from Amazon online shop. Conductive carbon ink (RAFS 090) was obtained from Toyo Ink Co., Ltd., Tokyo, Japan. Silver/Silver Chloride was purchased from ALS Co., Ltd. . Distilled water was prepared automatically from WG-204 .Methacrylic acid (MAA), epronics Co., Ltd., Tokyo, Japan) printer was used to print conductive ink  on the specified PET sheets . Fellowes-Jupiter Plus laminator was used for laminating the sheets. A fabool-CO2 laser cutter  was used for cutting the PET sheets. For all the electrochemical analyses, Compactstat. h. potentiostat  installed with IviumSoft version 4.1084 [JSM-6010LV (JEOL) was used for scanning electron microscopy images. Hamamatsu L9588 LightningCure spot-light source Model LC8 was used for the UV source. JET-Circuit (To create the initiator graphite (IG), a diethyldithiocarbamate methylene group was added to the surface of the graphite particle via chloromethylation through the procedure described previously . A gener2 bubbling, and saturated with the solvent. After two hours, the template was extracted using a vacuum filtration method. The suspension was cleaned in sequence using (a) 20 mL DMF, (b) 100 mL 1 M NaCl aq. at 60 °C, (c) 100 mL distilled water, and (d) 100 mL methanol. The cleaned MIP was dried using vacuum drying. Non-imprinted polymer (NIP) for PB was created using the same method as MIP, except no template was added.The functional monomer MAA  and cross-linking monomers MBAA  and EDMA  were dissolved in 1 mL distilled water and 10 mL dichloroethane. To this, 0.1 g of sodium phenobarbital (PB) was added and mixed thoroughly. Finally, 0.25 g IG was added to this solution, kept in a quartz crystal test tube , and bubbled with nitrogen for 30 min deoxygenation. The quartz tube was placed from the tip of the light guide 2 cm before the UV source for two hours, under continuous stirring and N2 bubbling, the quartz tube was positioned 5 cm in front of the UV source. The template was removed using a vacuum filtering technique after two hours. The suspension was cleaned one at a time using 30 mL DMF, 200 mL 1.5 M NaCl at 70 °C, 200 mL distilled water, and 10 mL ethanol. The cleaned MIP was dried using vacuum drying.In 10 mL of DMF, the cross-linking monomer MBAA , the functional monomer MAA , and the functional monomer EDMA  were dissolved. Levetiracetam (LEV) was thoroughly dissolved with 0.1025 g (0.60 mmol). Lastly, 0.24 g of IG was added to this solution, which was then placed in a quartz tube and bubbled with nitrogen for 20 min. Throughout the two hours of continuous stirring and N2. A vacuum filtering technique was used to retrieve the template after two hours. The suspension was cleaned in sequence using the following solutions: (a) 30 mL DMF, (b) 200 mL 1.5 M NaCl at 70 °C, (c) 300 mL distilled water, and (d) 10 mL ethanol. Vacuum drying was used to dry the cleaned MIP.The cross-linking monomers MBAA , EDMA , and the functional monomer MAA  were dissolved in 10 mL DMF. This was carefully combined with 0.1184 g (0.501 mmol) of carbamazepine (CBZ). Lastly, 0.25 g of IG was added to this solution and bubbled with nitrogen for 20 min in a quartz tube. The quartz tube was held 5 cm before the UV source for two hours while stirring and bubbling N2 laser cutter and heat-sealed again over the electrode holes as in The entire process for fabricating the chip sensor is shown schematically in The original picture of the disposable PET chip can be seen in Because of its excellent sensitivity, differential pulse voltammetry (DPV) was chosen as the electrochemical method for measuring drug responses in the saline buffer and the blood. Iterative optimization of the electrochemical setup was performed to achieve the best possible outcomes. All the DPVs were performed against silver–silver chloride (Ag/AgCl) reference electrodes, varying the potential from 0.0 V to 0.9 V with a step potential of 10 mV and a scan rate of 20 mV/s. The connection of the chip to the potentiostat is seen in Unless otherwise stated, phosphate-buffered saline (pH 7.4) containing 0.1 M NaCl and a 0.05 M mixture of potassium dihydrogen phosphate and disodium hydrogen phosphate were used for the entire experiment to create various concentrations of the analyte under test. The target analyte was first dissolved in 100 μL of physiological saline to prepare the stock solutions for spiked bovine blood samples.R2) value of 0.99. As the functional monomer, MAA allowed the polymer to interact specifically with the carbonyl group and nitrogen in the phenobarbital molecule. They formed hydrogen bonds with the carboxyl group of MAA, forming a stable complex. This interaction created a specific binding cavity to phenobarbital with high affinity.Further, non-imprinted polymers were created as a control experiment to confirm the effect of imprinting, and the results are plotted in Selectivity is an important parameter when deciding on an ideal sensor. Therefore, we performed a selectivity test of the sensor in the presence of phenytoin (PHY), a structurally similar drug, as shown in It is essential to extensively understand the sensor’s selectivity when targeting the use of the sensor in whole blood, as the various components of blood and other contaminants of the blood may interact with the sensor’s sensitivity. We tested the sensor in whole bovine blood and bovine plasma to verify whether our sensor works in such a complex matrix. The result of this is shown in −1 and 0.8461, respectively. The binding between the analyte molecule and the MIP cavities results from various chemical interactions, such as hydrogen bonding, electrostatic interactions, and van der Waals forces. When rebinding, it is possible that the specific functional groups in the LEV molecule, such as amine and carbonyl, form hydrogen bonds with the complementary functional groups in the MIP cavities.The prepared levetiracetam MIP was used as the electrode for the sensor chip. The voltammogram and the calibration curve obtained in buffer saline are shown in The LEV-MIP was tested for selectivity against phenobarbital, and it was found that the response of the sensor in PB was unstable, accounting for the excellent selectivity of the designed MIP. Thus, the MIP sensor for LEV can be implemented for further analysis. Additionally, we observed that the same DPV parameters may only work for some types of MIP sensors . Therefo−1 and 0.9578, respectively. The selectivity test of carbamazepine and phenobarbital showed that the MIP was highly selective towards CBZ. Both the sensitivity and selectivity plots can be seen in MIP-carbon paste sensors for carbamazepine were tested for sensitivity. We tried the sensor only once and found it highly sensitive to CBZ. The current was measured, and the intensity at 0.8 V was plotted on the calibration curve. The calculated sensitivity and correlation coefficient of the CBZ sensor were 0.1346 A·mL·gInterestingly, the sensitivity trends of LEV and CBZ at higher concentrations were similar and tended to be lower than levetiracetam MIP obtained at low concentrations. One possible explanation for the high sensitivity and selectivity of the CBZ MIP sensor is the specific interaction between the functional monomer and the CBZ molecule. The functional monomer used in the synthesis of the MIP was methacrylic acid (MAA), which has a carboxylic acid group and is thought to be hydrogen bonded to the nitrogen of the seven-membered ring of CBZ , leading to the formation of selective recognition sites (cavities) within the polymer matrix. Further, the lack of sensitivity of CBZ and LEV MIPs to phenobarbital could be due to the differences in the chemical structure and binding modes of PB compared with the target molecules, or the suboptimal cavity size and shape of the MIPs for PB binding.It should be noted that for sample preparation, we used 1 mL DMF to dissolve 10 mg of CBZ and then dissolved it in 200 mL buffer saline to dilute it and make several smaller concentration samples. Although the amount of DMF is so little and diluted, there may be a possibility that the polymer matrix of the MIP is affected by the presence of DMF in the measurement solution, which may thus affect the sensitivity. The best method to dissolve CBZ needs to be established. Further, optimizing the MIP composition and measurement parameters is essential for a sensitive MIP. We plan to improve the MIP and the measurement parameters to obtain a stable and sensitive CBZ sensor. The outcomes of the tests performed on our MIP-based AED sensors have proven entirely satisfying. The sensor quickly determines the concentration without reagents, thus reducing the complexities of adding a biological agent for detection. We used ferrocene in silicone oil as the redox marker in our experiment; the details can be found in our previous work . When feThe PB sensor has a high selectivity in addition to having a high sensitivity. In most instances, the degree of sensitivity exhibited by MIPs is determined by the monomers chosen for polymerization. However, it can be attributed to several factors, including the imprinted cavities’ specificity, the imprinted polymer’s large surface area, and its high binding affinity for the target molecule. The specificity of the imprinted cavities is achieved through the precise control of the monomer composition and polymerization conditions, which dictate the size and shape of the imprinted cavities. The large surface area of the imprinted polymer provides many recognition sites for the target molecule, increasing the likelihood of binding and leading to a higher signal. Additionally, the high binding affinity of the imprinted cavities is due to the formation of hydrogen bonds and van der Waals forces between the target molecule and the imprinted cavities.Nonetheless, when measured in an aqueous media, the sensitivity is hindered by the high polarity of water because water tends to weaken the hydrogen bonds between the MIP and the template. This could be one of the reasons for observing saturated or lower responses of levetiracetam and carbamazepine at higher concentrations. Optimizing the monomers (ratio and choice of the monomers), polymerization time, porogen selection, etc., can improve the MIP-CP. An expanded study on selectivity, NIP-CP, and results in whole bovine blood or rat plasma will be communicated soon.


Complete resection of the tumor and the ipsilateral thyroid lobe at the primary surgery is the “gold standard” for the treatment of parathyroid carcinoma (PC). However, differences in the overall survival (OS) of patients with PC who underwent partial and total surgical resection remain to be determined.https://medicine.yale.edu/lab/rimm/research/software/) was used to define the optimal cut-off values for continuous variables. The inverse probability of treatment weighting (IPTW) method was used to reduce the selection bias. IPTW-adjusted Kaplan–Meier curves and Cox proportional hazards models were used to compare the OS of patients with PC in the partial and total surgical resection groups.Data on patients with PC who underwent partial and total surgical resection were extracted from the Surveillance, Epidemiology and End Results (SEER) database (2000–2018). The X-tile software . The optimal cut-off values for age at diagnosis were 53 and 73 years, respectively, while that for tumor size was 34 mm. In both the Kaplan–Meier analysis and univariable Cox proportional hazards regression analysis before IPTW, the difference in OS between the partial and total surgical resection groups was not statistically significant (p>0.05). These findings were confirmed in the IPTW-adjusted Kaplan–Meier analysis and multivariate Cox proportional hazards regression analysis (p>0.05). Subgroup analysis revealed that total surgical resection was beneficial for OS only in the subgroup with unknown tumor size.There was no significant difference in the prognosis of patients who underwent partial and total surgical resection. This finding may provide a useful reference for the treatment of PC. Parathyroid carcinoma (PC) is one of the rarest malignancies of the endocrine system. It accounts for 0.005% of all malignancies in this system, and only approximately 5% of all cases of primary hyperparathyroidism. Since 2001, the incidence of PC has remained relatively stable  . The cauSurgery is an important predictor of PC prognosis and the only curative treatment for this disease . PreviouOwing to the rarity of PC and the lack of prospective studies, investigations based on small samples produce conflicting results. The prognostic impact of the extent of surgery on patients with early-stage PC is unclear. Therefore, our study was designed to assess the difference in the long-term prognosis of patients with early-stage PC who underwent partial and total surgical resection. For this purpose, we used data from the Surveillance, Epidemiology, and End Results (SEER) database. Inverse probability of treatment weighting (IPTW) was used to reduce the selection bias.https://seer.cancer.gov/seerstat/). SEER is an open-access public database, and private patient information has been officially withheld from this database. Hence, approval from an institutional review board was not required.This study cohort was retrieved from the SEER program; this database records patient data from 18 population-based cancer registries, covering approximately 27.8% of the population of the United States of America . The SEEThe criteria for data extraction were as follows (1): date from 2000 to 2018; (2) primary tumor site was C75.0 - parathyroid gland; and (3) diagnosis of parathyroid cancer by experienced pathologists. We extracted patient demographic variables , tumor characteristics , treatment status , months of survival, and survival status. The exclusion criteria were: (1) unknown duration of survival; (2) surgical procedures other than partial and total resection; (3) presence of lymph node metastases; (4) lymph nodes and distant metastases at the time of diagnosis; and (5) incomplete information Figure 1https://medicine.yale.edu/lab/rimm/research/software/). Marital status was categorized as “married at diagnosis” and “other”, the number of malignancies was categorized as “single” and “multiple”, radiotherapy was categorized as “yes” and “no/unknown”, and regional nodes examined were categorized as “yes” or “no”; the above variables were binary processed. According to the SEER Program Surgery Codes Manual, surgical procedure codes 30 and 40 denote partial and total surgical resection. In addition, the patient profiles were screened for lymph nodes and distant metastases based on variables, such as the extent of disease for regional nodes. According to the explanation of variables in the SEER database, overall survival (OS) was the endpoint of interest in this study. OS was defined as the period from the date of PC diagnosis to that of death from any cause.Variable reassignment was not performed for sex and race in the extracted data. Age at diagnosis and tumor size were converted to categorical variables after determining the cut-off values using the X-tile software  in the RStudio environment . All statistical analyses involved two-sided testing, and p-values <0.05 denoted statistically significant differences.Categorical variables are shown as frequencies and their proportions. The baseline characteristics of patients before and after matching were assessed using standard mean difference (SMD); a value >0.1 indicated that the information was unbalanced between the groups . AdjustmA total of 334 PC patients met the inclusion criteria with a median follow-up time of 205 months. Of those, 183 and 151 patients underwent partial and total surgical resection, respectively. After processing, 53 and 73 years were identified as the best cut-off values for age at diagnosis, while 34 mm was the best cut-off value for tumor size. Of the patients, 53.6% were male, 36% were aged <53 years, 51% were aged 53–73 years, and 62% were married at the time of diagnosis. Moreover, 50% of patients had a tumor size <34 mm, and 74% had a single primary malignancy. Radiotherapy and regional lymph node testing were performed in 10% and 34% of the patients, respectively. In the unweighted data, the SMDs of age at diagnosis, sex, race, tumor size, and radiotherapy were >0.1, thereby denoting bias in the original data. After IPTW adjustment, the SMDs of all variables were <0.1, indicating that the partial and total surgical resection groups were comparable Table 1.Kaplan–Meier analysis did not show a statistical difference in the OS of patients with PC between the partial and total surgical resection groups before IPTW (p=1.000) Table 2.The forest plot did not show a difference in prognosis between the partial and total surgical resection groups, except for the subgroup of unknown tumor size. In the group with unknown tumor size, total surgical resection was associated with better OS than partial surgery . In the subgroup with tumor size >34 mm, the p-value was 0.053, almost reaching the threshold of statistical significance. In the subgroup analysis of other variables, there was no difference in the prognosis for the partial and total surgical resection groups Figure 3In this study, we sought to identify differences in the prognostic impact of partial and total surgical resection on the OS of patients with early-stage PC by analyzing data from the representative SEER database. Owing to the wide coverage of the SEER database and the relatively complete data collection, it is possible to investigate rare types of cancer, such as PC. To the best of our knowledge, this is the first study using the IPTW method to achieve the aforementioned objective.Due to the rarity of PC, the available literature on this disease mostly comprises reviews and case reports. Nevertheless, several studies have investigated the prognostic factors of OS and cancer-specific survival in patients with PC, providing data to support the treatment and management , 22–25. Surgery is an important factor in the prognosis of patients with PC. Compared with partial surgical resection, primary surgery involving total surgical resection provides the best chance of cure and is associated with a significantly better prognosis , 13. HowAlthough we applied the IPTW method to correct the baseline features and obtained a relatively reliable conclusion. The limitations of this population-based database investigation should be acknowledged. Firstly, this study involved a retrospective analysis, which may have been biased by the removal of cases with incomplete information. Secondly, several important indicators that have been associated with PC  are not included in the SEER database. Therefore, we were unable to examine the effects of such indicators on the prognosis of patients with PC in relation to the surgical approach. Thirdly, the SEER database does not provide PC gene information, especially the CDC73 mutation status. Again, due to the rarity of PC cases, even national databases do not include many cases , which may lead to biased study results. Finally, clinical and oncology variables require precise coding for analysis. For example, the SEER definition of surgical modality is imprecise, and the “Surgical Codes Other Sites 2023” manual, which is available on the official website of the database, does not specifically address the coding of parathyroid surgery.In patients with early-stage PC, there was no significant difference in OS following partial and total surgical resection. Our findings may provide insights and would assist in the selection of the appropriate surgical approach for patients with PC.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants or the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.SJ and CZ were responsible for the conception and design of the study. SJ also collected data and performed the statistical analysis. KX, WC and JY wrote and edited the manuscript. WC also made contributions to manuscript review and revision. CZ provided financial support. All authors contributed to the article and approved the submitted version.


The study investigated the emergence of toddlers’ regulatory strategies in aversive contexts. Forty-two toddlers were observed at 24 and 30 months of age using two paradigms designed to elicit fear and anger. We examined toddlers’ use of regulatory strategies at these two stages of life regarding the frequency of self-versus other-oriented strategies and of reactive versus more controlled behaviors. Results showed that the type and level of control of strategies used in toddlerhood in managing negative emotions depend on emotion  and age. Toddlers used self-oriented strategies to regulate fear and other-oriented strategies to regulate anger. To manage fear, when toddlers got older, they increased the use of reactive strategies  and decreased the use of more purposeful strategies . In contrast, to regulate anger, toddlers utilized an intermediate level of control  and increased the use of this strategy with age. In addition, toddlers were able to select appropriate strategies for different stressors, and they increased with age the ability to adapt the strategies to the environmental conditions. Theoretical and practical implications are discussed. Emotion regulation (ER), defined as behaviors and strategies that serve to modulate the expression of emotion and affect, develops over the first years of life and is thought to be foundational for social adjustment in the preschool and school years . AccordiAdaptive emotion regulation is a critical developmental goal underlying numerous psychological outcomes, including optimal cognitive performance in executive tasks, effective social strategies with peers, and management of stressful experiences in domestic and educational settings . While tThe first one is the ability to rely on their own rather than others’ resources for regulation. Following Eisenberg and Morris , childreThe second ability involved in ER development during toddler years is the ability to move from reactive to more purposeful responses. The advancements of motor and linguistic skills contribute to widening the set of regulatory strategies that are available for managing emotion in different situations ,5,11, esEmotion regulation strategies have been typically assessed in aversive contexts, as these situations require the greatest mobilization of regulatory resources to manage negative affect. According to the functionalist perspective , despiteThe literature highlighted that toddlers use more primitive strategies to manage fear while using more complex strategies to manage anger, comparing the aversive contexts eliciting fear and anger . Diener In line with the literature ,25, the n = 25) were male; 63% (n = 24) had one or more siblings, and 11% (n = 4) attended a daycare center. Children were considered for inclusion if they were healthy, with no developmental delays, according to their family pediatrician. Average maternal age was 35.17 years old , and mothers had an average of 13.95 years of education . All fathers and 76% of the mothers (n = 29) were employed with a stable job. Parents were informed that the aim of the study was to examine how children develop the ability to cope with emotional situations with or without their support, and their written consent was obtained. The families were not compensated for their participation, but they received a DVD with all videos collected at the lab. The sample was treated in accordance with the ethical standards outlined by the American Psychological Association and the Italian Association of Academic Psychologists, and the study was approved by the Department Ethics Review Board of G. D’Annunzio University of Chieti and Pescara. At 24 and 30 months of children’s age (+/−1 week of age), mothers were invited to the lab to participate in the Emotion Regulation Paradigm , or instead, she/he freezes or retreats from the aversive stimulus . Self 2: Self-soothing behaviors. The child actively engages in repetitive motor behaviors, such as sucking on fingers, twisting the hands or hair, scratching the head, and pulling the ear. Self 3: Dealing with the aversive situation. The child tries to engage with the stimulus, i.e., touching, handling, labeling, or speaking with the toy.Other-Oriented Regulatory Strategies. Other 1: Fussing at the mother. The child demonstrates motor and/or emotional agitation while looking at the mother. Other 2: Drawing mother’s attention to themself. The child actively tries to engage the mother by looking at her and/or calling her. Other 3: Object/situational management by the mother. The child tries to engage the mother’s attention with the aim of solving the problem, e.g., pointing to the stimulus and looking at her alternatively, telling her something related to the actual event, and asking the mother to get the stimulus or for information about it.The above regulatory strategies were coded dichotomously (present vs. absent) every 15 s in the anger and fear contexts from videotaped observations. All regulatory behaviors which occurred in the context were coded for each interval. The codes are exhaustive; that is, toddlers engaged in at least one of these behaviors during each 15 s interval. In addition, the codes are not mutually exclusive; that is, multiple codes could occur in each interval but not simultaneously. The scores for each of the regulatory behaviors were summed and divided by the total number of 15 s intervals in the context. The resulting proportion variables had a possible range of 0–1. Two separate trained coders coded all videos for fear and anger contexts, respectively. Interrater reliability was calculated based on ten cases at 24 months (25%) and nine cases at 30 months (23%) for each emotion regulation strategy in each context. The average Cohen’s Kappa value among strategies at 24 months was 0.94 (range: 0.92–0.96) and 0.95 (range: 0.89–0.96) in the fear context and anger context, respectively; at 30 months, it was 0.93 (range: 0.85–0.96) in the fear context and 0.92 (range: 0.82–96) in the anger context.Preliminary analyses examined differences between males and females using the different emotion regulation strategies at each age and in each context using independent-sample t-tests. In addition, Pearson correlations were conducted to explore the associations between strategies at each age and across ages and contexts. The main analyses were run with the linear mixed model procedure of SPSS with REML as the method for estimation . To reprThe t-tests did not show any significant difference between males and females regarding the regulation strategy used in fear and anger contexts at both ages (24 and 30 months). This variable was not included in the other analysis. Correlations among the frequencies of self-oriented strategies are reported in Correlations among the frequencies of other-oriented strategies are reported in p < 0.01, with a higher use of this strategy in the Fear than in the Anger context at 30 months, F = 18.18, p < 0.01. The main effects of age and context were significant in the Self 2 strategy, showing that the frequency of this strategy decreased over time in both contexts, and it was higher in the anger context than in the Fear context at both ages. Finally, the two-way interaction between context and age was significant in the Self 3 strategy. Children use this strategy more during Fear, F = 33.03, p < 0.01, than Anger, F = 9.23, p < 0.01, context with a decrease over age in both contexts, but stronger in the fear context . The first set of mixed models examined the effects of context, age, and their interaction on self-oriented emotion regulation strategies. The two-way interaction between context and age was significant in the Self 1 strategy cf. . Childrep = 0.03, with higher use in the Fear than in the Anger context at 30 months, F = 6.47, p = 0.02.The second set of analyses explored the effects of context, age, and their interaction on other-oriented emotion regulation strategies cf. . The twop = 0.03; 30 months: F = 20.68, p < 0.01) with an increase of use in the Anger context over age, F = 7.98, p =< 0.01  at 30 months and more controlled strategies (Self 2 and Self 3 strategies) at earlier ages  in managing feelings in a fear context. While this may seem counter-intuitive, with older toddlers using more primitive strategies than younger toddlers, our findings align with previous research. Diener and Mangelsdorf  examinedThe study suggested that toddlers utilized self-oriented strategies to manage fear and utilized other-oriented strategies to manage anger, thus selecting appropriate strategies for different stressors. Moreover, we found that toddlers increased with age the strategies that were more effective in dealing with emotional demands and environmental conditions, although less advanced in the ability to monitor emotion internally. It suggests that the development of emotion regulation is signaled not simply by the cognitive level expressed by the regulatory strategies used at a given age but rather by their capacity to meet the goals of a given emotional situation. Both findings are in line with Buss , suggestSome limitations of the study need to be considered in the interpretation of the results and as directions for future research. First, due to the small sample size and the lack of a longitudinal assessment of ER strategies, including a third time point, such as 36 months of child’s age, we cannot generalize the results. Further studies should replicate our findings in a larger sample and a longer period of time. Second, we examined children’s strategies based on literature showing their role in emotion regulation. Therefore, due to our interest in the developmental change in these strategies across two different contexts, we did not assess how these strategies were effective in reducing the negative effect elicited by our paradigm. Future research should improve the investigation by testing the impact of these putative regulatory strategies on toddlers’ emotion regulation. Finally, despite a growing body of research indicating the importance of contextual effects on behavioral strategies , we measured the direct association between age and the strategy without examining potential mediating or moderator factors . The lac


To investigate the causal association between Osteoarthritis (OA) and five comorbidities: depression, tiredness, multisite chronic pain, irritable bowel syndrome (IBS) and gout.This study used two-sample Mendelian Randomisation (MR). To select the OA genetic instruments, we used data from the largest recent genome-wide association study (GWAS) of OA (GO Consortium), with a focus on OA of the knee , hip  and hand . Genetic associations for comorbidities were selected from GWAS for depression , tiredness , multisite chronic pain , IBS  and gout . We performed a bidirectional MR analysis using the inverse variance weighted method, for both joint specific and overall OA.Hip OA had a causal effect on multisite chronic pain . Multisite chronic pain had a causal effect on knee (odd ratio (OR) 2.74, 95% CI 2.20 to 3.41), hip , hand  and overall OA . In addition, depression and tiredness had causal effects on knee and hand, but not hip, OA.Apart from Hip OA to multisite chronic pain, other joint OA did not have causal effects on these comorbidities. In contrast, multisite chronic pain had a causal effect on any painful OA. In our are valid instrumental variables for the exposure being measured [The reason for the frequent co-occurrence of OA with comorbidities remains largely unknown. It may be because of shared risk factors such as ageing, or because of OA itself, the comorbidities themselves or the medication used. However, using observational studies to investigate such concurrence always runs the risk of bias due to unmeasured confounding. An alternative method for estimating causal effects is Mendelian Randomisation (MR), where genetic variants are used as instrumental variables for a given exposure, which are randomly assigned and are not influenced by later environmental confounders. However, this rests on the assumption that the genetic variants used measured . OA is rEven though we previously explored the bidirectional association between OA and different comorbidities using an epidemiological observational study design, the causal relationship is difficult to explain . We ther2This study used a Two-Sample MR framework, where genetic variants proxying the exposure are identified in one population sample, and their effects on an outcome are tested in a separate sample, as well as making use of available summary statistics from GWAS. MR hinges on three core assumptions, specifically: (i) the genetic variants are robustly associated with the exposure being measured; (ii) the genetic variants are not associated with known confounding variables; and (iii) the genetic variants only affect the outcome through the exposure  . This st2.12.1.12.1.1.1This analysis used the most recent and largest GWAS of OA to identify the genetic instruments used, which to date is the Genetics of OA (GO) Consortium meta-analysis . GO Cons2.1.1.2We reported findings of five comorbidities, namely fibromyalgia , depresTo test the effect of comorbidities on OA, we selected genetic instruments from the largest available GWAS that had published its results in a peer reviewed journal, or was made available for external analysis, up until July 2022.A summary of the different data sources used for this study can be found in 2.22.2.1−8) SNPs extracted from the data set, we used LD clumping to identify the lead SNPs, excluding all non-independent SNPs at a threshold of r2 ≤ 0.001 within an LD window of 1000 kb. For datasets accessible on the MRC IEU database, this was achieved in R (version 4.1.2) using the TwoSampleMR R package [We extracted the genetic instruments from the genome-wide data set for the exposure being measured. Of the genome wide significant ), to be equivalent to the change per 2-fold increase in the binary exposure [Once the instrumental variables were selected, we independently extracted individual SNP-outcome associations and individual SNP-exposure associations from the data sets. These associations were measured either in the natural-log odds ratio (logOR), or per unit change. We then calculated the ratio of the SNP-outcome association over the SNP-exposure association  and pooled the results by joint site . In addition, we also pooled the Wald ratio results by each comorbidity  to get the Average Causal Effect of/on OA. We used the TwoSampleMR package  to estimexposure . Where t2.2.3with UK Biobank (which produced more genome-wide significant hits) as the main result. Nonetheless, as a sensitivity analysis, we independently used GWAS data sets from the GO Consortium meta-analysis GWAS without UK Biobank  [In Two Sample MR, there is a risk that if the two samples have an overlapping population, the MR result will be biased towards the confounded association when using weak instruments. This is an issue when the GO Consortium GWAS includes UK Biobank, and the selected comorbidity GWAS datasets are 45–100% UK Biobank participants. However, if the genetic instruments are strong (F-statistic >10) then this bias is less likely to be an issue , therefoontrols)  for bothIn addition, we undertook two other methods to verify the results, specifically: i) MR-Egger, which uses the SNP-exposure and SNP-outcome associations as equivalent to points for linear regression, with the intercept value being used to determine the presence of horizontal pleiotropy ; and ii)2.3Three PPI representatives with OA were involved in this study through group meetings. They provided their inputs at each step of the study, including discussions about the struggles of living with multiple conditions, the lack of research in causal relationship and management of multimorbidity, and the importance for this study. The results of the study were shared with the members in lay-person language and their inputs were considered in writing the manuscript.3We performed bi-directional MR, estimating the causal effects both of OA on comorbidities  and como3.1There was inconsistent evidence of a causal effect of Knee OA on depression, multisite chronic pain, and gout . There w3.2There was consistent evidence of a causal effect of depression on Knee OA, Hand OA, and Pooled OA, but inconsistent evidence of a causal effect on Hip OA , Fig. 6.3.3For the MR-Egger analysis, there was no evidence of horizontal pleiotropy for most of the consistent causal effects in the main analysis, apart from the effect of multisite chronic pain on Hand OA, where pleiotropy was detected, and the direction of effect changed. There was also evidence of a protective effect of Hand OA on depression and multisite chronic pain when adjusting for pleiotropy, and a protective effect of IBS on all OA subtypes.For the weighted median analysis, all the consistent causal effects from the main analysis were replicated, apart from the effect of depression and tiredness on Hand OA . Evidence was also found for an effect of Knee OA on multisite chronic pain, IBS and gout, Hand OA on tiredness and gout on Hip OA.4UK Biobank data, as well as supported by the other two MR analyses, apart from the horizontal pleiotropy bias observed for Hand OA. Some consistent evidence was also observed for an Average Causal Effect on OA for depression, tiredness and multisite chronic pain, but that needs to be taken with caution due to the heterogeneity between, and double counting SNPs for the different phenotypes of OA.We used two sample MR methods to investigate bidirectional causal relationships between subtypes of symptomatic OA and five common comorbidities. The key findings are: (i) there was a bidirectional causal association between Hip OA and multisite chronic pain, (ii) there was consistent evidence of causal effect of multisite chronic pain on all types of OA, and (iii) there was a causal effect of depression and tiredness on Knee OA and Hand OA. The findings were supported by the sensitivity analysis with and without the The general finding of the effects of comorbidities on OA being stronger than the effects of OA on comorbidities is consistent with a previous study by Barowsky et al., 2021  for deprLarge estimates detected for depression, tiredness and multisite chronic pain on OA is consistent with an important role of central pain sensitisation in symptomatic OA and the association with depression and anxiety and other pain traits . Pain seFor the effect of Hip OA on multisite chronic pain, it is possible that pain and biomechanical changes associated with Hip OA cause secondary biomechanical insults to other lower limb joints and to the lumbar spine. This may lead to additional pain at these sites and has been suggested as a likely explanation for the association between Hip OA and lower back pain .4.1This study used the largest available GWAS of OA, including the largest non-UK Biobank GWAS of OA for the sensitivity analysis . All ourGiven the evidence found in this study for a causal effect of comorbidities linked to central pain sensitisation on all types of OA, it is possible that psychological interventions to help patients deal with pain could be effective at reducing the pain due to OA . HoweverIn conclusion, we have found consistent evidence of bidirectional causal effect of Hip OA and multisite chronic pain, consistent evidence of a causal effect of multisite chronic pain on all types of OA, and depression and tiredness on Knee and Hand OA. However, this is just the first step to investigate the causality between OA and five common comorbidities. Further study is needed to confirm the results and to understand the mediating pathways of these causal relationships and potential targets for further treatment.CC, AK, SZ and SS. SBZ and JR supervised AK in her work. WDT wrote the first draft of the paper with support from WZ, MD, CC, SZ and SS; all authors read and made critical revisions to the paper. WDT, WZ, MD, CC, SZ and SS are guarantors of the papers integrity.WZ, MD and WDT designed this study, with further development coming from CFK, SS and SZ. WDT, WZ, SS and AK wrote the statistical analysis plan, based on a previous analysis plan written by AK. WDT undertook most of the analyses with support from WZ, MD, This study used publicly available summary data and did not involve contact with participants; thus, no extra ethical approval or informed consent was required. The research complies with the Declaration of Helsinki.This study uses two-sample MR, using summary statistics from predominately publicly available GWAS datasets.mskkp.org) in March 2022 from https://msk.hugeamp.org/dinspector.html?dataset=Boer2021_OA_Mixed_Main.The summary statistics for the main GO Consortium GWAS of osteoarthritis were downloaded from the Musculoskeletal Knowledge Portal . WZ has also received funding from 10.13039/501100012041Versus Arthritis (grant no. 21595 and 20777), the Football Association regarding Foot and ankle Osteoarthritis and Cognitive impairment in retired UK Soccer players (FOCUS), the 10.13039/501100000265Medical Research Council regarding the Alleviate Pain Data Hub (MR/W014335/1), and the 10.13039/501100000272National Institute for Health Research for the Allopurinol Treat to Target clinical trial and the Research for Patient Benefit fund (19023). SSZ is supported by a 10.13039/501100000272National Institute for Health Research Clinical Lectureship and works in centres supported by 10.13039/501100012041Versus Arthritis .As part of this study, MD has received consultation fees from AstraZeneca, Grunenthal, and Mallinckrodt. WZ has received consultation fees from AstraZeneca, Grunenthal, Eli Lilly and Regeneron advisory boards, and speaker fees from Xiangya Hospital and Shenzhen Rheumatological Meeting. The other authors declare no competing interests.


Purpose: At present, there is a lack of accurate early diagnostic markers for ischemic stroke.Methods: By using dimensionality reduction cluster analysis, differential expression analysis, weighted co-expression network analysis, protein-protein interaction network analysis, cell heterogeneity and key pathogenic genes were identified in ischemic stroke. Immunomicroenvironment analysis was used to explore the immune landscape and immune associations of key genes in ischemic stroke. The analysis platform we use is R software (version 4.0.5). PCR experiments were used to verify the expression of key genes.Results: Single cell sequencing data in ischemic stroke can be annotated as fibroblast cells, pre-B cell CD34, neutrophils cells, bone marrow (BM), keratinocytes, macrophage, neurons and mesenchymal stem cells (MSC). By the intersection of differential expression analysis and WGCNA analysis, 385 genes were obtained. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these genes were highly correlated with multiple functions and pathways. Protein-protein interaction network analysis revealed that MRPS11 and MRPS12 were key genes, both of which were down-regulated in ischemic stroke. The Pseudo-time series analysis found that the expression of MRPS12 decreased gradually with the differentiation of pre-B cell CD34 cells in ischemic stroke, suggesting that the downregulation of MRPS12 expression may play an important role in ischemic stroke. At last, PCR showed that MRPS11 and MRPS12 were significantly down-regulated in peripheral blood of patients with ischemic stroke.Conclusions: Our study provides a reference for the study of pathogenesis and key targets of ischemic stroke. Ischemic stroke is a medical condition that occurs when the blood flow to a part of the brain is blocked, typically by a blood clot . This laOverall, research into ischemic stroke is ongoing and has made significant progress in recent years. While there is still much to study the pathogenesis and early robust markers of ischemic stroke for improved outcomes for those affected by stroke . TranscrThe development of high-throughput sequencing technology, also known as next-generation sequencing, has revolutionized the field of genomics by allowing rapid and cost-effective sequencing of entire genomes and transcriptomes . TranscrIn this study, we analyzed single-cell sequencing data and high-throughput sequencing data for ischemic stroke, combined with a variety of methods, including dimensionality reduction clustering and weighted co-expression network analysis (WCGNA), and we found that MRPS11 and MRPS12 are robust markers of ischemic stroke, providing reference for its prevention and treatment.For a vast variety of disorders, the GEO database contains single-cell sequencing and transcriptome sequencing data. The blood single cell sequencing dataset GSE174574 on ischemic stroke, which included three mouse model of middle cerebral artery occlusion (MACO) with ischemic stroke and three normal control samples with sham operation, was downloaded for this investigation . The dathttp://117.50.127.228/CellMarker/index.html).We first standardize the data using the “LogNormalize” method of the “Seurat” package. The number of high-variable genes was then set to 3000, and the setting technique was changed to “vst” to conduct additional research on high-variable genes. The “Seurat” package’s SCTransform function was used to recombine data from several samples and remove cell cycle and mitochondrial interference. The data dimensions were reduced by using the UMAP technique with dims set to 30. The cells were clustered using the “KNN” algorithm, with the following settings: k.Paam = 30, dims = 20, resolution = 1.0, random.seed = 2023. Then, the annotations on cells were made using “SingleR” and the CellMarker website  is a database produced by the Gene Ontology Consortium, seeking to establish a database relevant to various species, which can define and describe the activities of genes and proteins and is applicable to various species. Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) are the three categories. The GO enrichment analysis was performed using the enrichGO function from the ClusterProfile package. We extract the top 10 signaling pathways with the smallest p value for display. p < 0.05 and the Log |FC| > 0 are thought to be statistically significant.The KEGG (Kyoto Encyclopedia of Genes and Genomes) database included metabolic pathways database, hierarchical categorization database, gene database, genome database, etc. It was a database that systematically analyzed gene functions, genomic information, and functional information. By comparing the examined genes with the pathway gene set in the database, the enriched pathways were produced. The KEGG enrichment analysis employed the ClusterProfile R package's enrichKEGG function. We extract the top 10 signaling pathways with the smallest https://cn.string-db.org/) was used for PPI analysis. The top 20 genes’ interactions were shown using the cytoHubba plugin of the Cytoscape program, which was used to visualize the results.Proteins that interact with one another and take part in numerous parts of life processes, such as biological signal transduction, gene expression regulation, energy and substance metabolism, and cell cycle regulation, make up protein-protein interaction networks (PPI). Understanding the working principle of proteins in biological systems, the reaction mechanism of biological signals, the metabolism of energy and materials in particular physiological states like diseases, as well as the functional relationships between proteins, all depend on systematic analysis of the interaction between a large number of proteins in biological systems. In this study, the STRING database , extracted RNA, and synthesized cDNA using reverse transcription reagent (Vazyme Biotech). PCR analysis was then performed using SYBR Green Premix reagent (Vazyme Biotech) and PCR system . Relative gene expression was calculated by 2p < 0.05 was defined as statistically significant.In single-cell sequencing data, we searched for differentially expressed genes associated with stroke by using the FindMarkers function of the “Seurat” package. In transcriptome data, WGCNA analysis was used to search for gene modules associated with stroke. The difference in gene expression between stroke and normal samples was analyzed using the rank sum test. All analyses were performed using R software, version 4.0.5. Unless otherwise noted, Our entire workflow was summarized in After quality control of ischemic stroke and control samples, we obtained a total of 18,676 genes and 58,469 cells. As shown in p < 0.05, with a correlation of 0.56 and 0.54, respectively. Therefore, the genes of the black and red modules were included in the follow-up analysis. There were 4909 genes in total, among which 1219 were in black module and 3690 were red genes. In the analysis of GSE58294 dataset, we found that the optimal soft threshold was also 7. As shown in p < 0.05, with a correlation of 0.77 and 0.72, respectively. Therefore, the genes of blue and greenyellow modules were included in the follow-up analysis, and there was a total of 4260 genes, including 3926 genes of black and 334 genes of red. In order to find genes relatively associated with ischemic stroke, the intersection of genes obtained from single-cell analysis and genes obtained from transcriptome WGCNA analysis was performed, as shown in To further explore differences between ischemic stroke and normal control samples in transcriptome sequencing, WGCNA analysis was then performed. In the WGCNA of GSE16561, we found that the optimal soft threshold value was 7, and the data matched the power law distribution, which was suitable for subsequent analysis. As shown in To further clarify the mechanism of action mediated by these 385 key genes in ischemic stroke, enrichment analysis was performed. As is shown in p < 0.001). And we found that, as shown in As shown in pre-B cell CD34 plays an important role in ischemic stroke, and the above analysis showed that both MRPS11 and MRPS12 were mainly expressed in pre-B cell CD34. Therefore, we extracted pre-B cell CD34 cells from ischemic stroke samples and used the “monocle2” package for pseudo-time series analysis. As shown in Through the above analysis, it is found that the down-regulation of MRPS12 may play an important role in the occurrence and development of ischemic stroke, and may be the target of its diagnosis and treatment. Subsequently, in transcriptome sequencing data, we further explored the association between MRPS12 and immune cells in GSE58294. **P < 0.01, ***P < 0.001).Finally, in order to verify the expression of MRPS11 and MRPS12 in peripheral blood of patients with ischemic stroke, clinical samples were collected and PCR experiments were conducted. The results showed that compared with healthy controls, MRPS11 and MRPS12 were significantly down-regulated in peripheral blood of patients with ischemic stroke  family, which is involved in the assembly of mitochondrial ribosomes and protein synthesis within the mitochondria –25. MitoResearch on the MRPS family has focused on their role in mitochondrial function and their potential as therapeutic targets for various diseases . ChangesIn particular, MRPS11 and MRPS12 have been implicated in the pathogenesis of cancer. A study found that the interaction between LncRNA ZFHX4-AS1 and MRPS11 may be related to the immune microenvironment in ovarian cancer and promote ovarian cancer progression . AnotherMultiomics analysis, a comprehensive approach that integrates multiple omics technologies such as genomics, transcriptomics, proteomics, and metabolomics, holds significant promise in the field of ischemic stroke research. By examining various molecular levels simultaneously, multiomics analysis enables a deeper understanding of the complex pathophysiological mechanisms underlying ischemic stroke. It allows the identification of key genetic variants, gene expression patterns, protein alterations, and metabolite profiles associated with stroke, providing valuable insights into the molecular pathways involved in the disease. This integrative approach can aid in the discovery of novel biomarkers for early diagnosis, prognosis, and personalized treatment strategies, ultimately leading to improved patient outcomes and the development of targeted therapeutic interventions for ischemic stroke.Overall, the MRPS family, including MRPS11 and MRPS12, plays a critical role in mitochondrial function and has been linked to various diseases. The identification of MRPS11 and MRPS12 as potential markers in transcriptomic analysis of ischemic stroke highlights the importance of these genes in neurological disorders and their potential as therapeutic targets.Supplementary Figure 1Supplementary Table 1


This study aims to investigate the relationship and mechanisms of action among academic stress, academic anxiety, academic self-efficacy, and academic burnout among adolescents.A study of 929 Chinese adolescents  was conducted using the Study Stress Questionnaire, Academic Anxiety Subscale, Junior Middle School Students’ Learning Weariness Scale, and the Academic Self-efficacy Questionnaire.①Academic stress was significantly and positively correlated with academic anxiety and academic burnout, and significantly and negatively correlated with academic self-efficacy. ②Academic anxiety partially mediated the relationship between academic stress and academic burnout. ③Academic self-efficacy significantly moderated the direct effect of academic stress on academic burnout, and higher academic self-efficacy could buffer the negative effect of academic stress. ④Academic self-efficacy significantly moderated the second half of the mediated model’s path (academic anxiety → academic burnout), that is, low academic self-efficacy amplified the risk effect of academic anxiety on academic burnout.Academic anxiety partially mediates the relationship between academic stress and academic burnout, and this mediating role is moderated by academic self-efficacy. Results of a survey conducted by the China Care for the Next Generation Working Committee in 2020 showed that more than 30% of elementary school students were bored with school and more than 70% of adolescent students have academic burnout, and many students experienced severe burnout or even refused to go to school due to the home quarantine during the epidemic that interrupted the normal rhythm of school attendance for adolescents . RegardiPrevious studies have shown that students’ own poor learning foundation, low self-evaluation, and lack of interest and initiative in learning can lead to academic tiresome . AcademiAcademic anxiety is a negative emotional state that students experience more frequently in academic situations , encompaAcademic self-efficacy (ASE) is the judgment and confidence in an individual’s ability to believe that he or she can successfully complete a specific academic task at a specific stage of learning  and is tFurthermore, academic self-efficacy is considered to be a predictor and protective factor for adolescents’ internalizing and externalizing problems . TherefoWe applied a survey design to achieve our research objectives. We used a whole-group sampling method and approached a junior high school in Shandong Province to collect data from their students, a sample of adolescents from this junior high school that was well represented. Participants were invited to provide their voluntary consent and then complete these measures. Participants did not provide any personal information that would allow researchers or organizations to identify them. Participants were led into the school’s microcomputer room by a schoolteacher and completed an electronic questionnaire on a computer after a trained master examiner read the instructions. All participation was voluntary, and each participant received a ballpoint pen as payment at the end of the test.Using the whole-group sampling method, all students in grades 6 and 7 of a junior high school in Shandong Province, a total of 982 adolescents, were selected to complete the questionnaire survey, and a total of 929 valid questionnaires were returned, with a valid return rate of 94.60%. Among them, 499 (53.71%) were male and 430 (46.29%) were female; 488 were in the first grade and 441 were in the second grade, aged 11 to 15 (11.94 ± 0.77). These adolescents came from urban, rural and rural areas, and it can be confirmed that the sample drawn is highly representative of the adolescent population.p < 0.001, RMSEA = 0.075, SRMR = 0.042, GFI = 0.882, AGFI = 0.851, CFI = 0.922, TLI = 0.911, indicating that the results of CFA had good fitting indicators.This study used the study stress questionnaire for middle school students  to measuIn this study, the academic anxiety subscale of the Mental Health Diagnostic Test (MHT), adapted by Zhou Bucheng , was usep < 0.001, RMSEA = 0.081, SRMR = 0.046, GFI = 0.906, AGFI = 0.874, CFI = 0.938, TLI = 0.927, indicating that the results of CFA had good fitting indicators.This study used Junior Middle School Students’ Learning Weariness Scale  to measup < 0.001, RMSEA = 0.082, SRMR = 0.064, GFI = 0.865, AGFI = 0.834, CFI = 0.922, TLI = 0.912, indicating that the results of CFA had good fitting indicators.The academic self-efficacy questionnaire, developed by First, the common method deviation test was performed using SPSS 22.0, and descriptive statistics and correlation analysis were performed for the main variables. The common method bias test calculated according to Harman’s one-way test showed that there were 10 factors with eigenvalues greater than 1. The first principal component explained 29.89% of the total variance, which is below the critical value of 40%, so it can be concluded that there is no significant common method bias problem in this study. After that, Model 15 in SPSS macro program process v3.3 prepared by As shown in β = 0.38, t = 13.40, p < 0.001), and the predictive effect of academic stress on academic burnout remained significant when the mediating variable academic anxiety was introduced , academic stress had a significant predictive effect on academic anxiety , and academic anxiety had a significant positive predictive effect on academic burnout . The upper and lower limits of Bootstrap 95% confidence intervals for the direct effect of academic stress on academic burnout and the mediating effect of academic anxiety did not contain 0  = 323.24, p < 0.001) and learning stress was a significant positive predictor of learning anxiety . Equation 2 was significant overall  = 169.84, p < 0.001), with learning stress significantly and positively predicting academic burnout  and learning anxiety significantly and positively predicting academic burnout , thus learning anxiety mediated the relationship between learning stress and academic burnout. The interaction term between academic stress and academic self-efficacy was a significant negative predictor of academic burnout , and the interaction term between academic anxiety and academic self-efficacy was a significant positive predictor of academic burnout , indicating a significant moderating effect of academic self-efficacy. The results are shown in Model 15 in the process macro program prepared by Hayes was used to test for a moderating mediating effect. The results showed that equation 1 was significant overall (Z ≦ -1SD) and high (Z ≧ 1SD) subgroups according to the standardized scores controlling for academic self-efficacy for simple slope analysis. First, we explored how academic self-efficacy moderated the direct effect of academic stress on academic burnout. The results found  and insignificant for adolescents when the level of academic self-efficacy was high . This indicates that the lower the academic self-efficacy of middle school students, the greater the effect of academic stress on academic burnout, and conversely, at high levels of academic self-efficacy, the effect of academic stress on academic burnout was not significant.After standardizing the study variables, the study divided the subjects into low ; for adolescents with high academic self-efficacy, academic anxiety was a significant positive predictor of academic burnout . This suggests that the higher the academic self-efficacy of middle school students, the greater the effect of learning anxiety on academic burnout may be, and on the contrary, at low levels of academic self-efficacy, the effect of learning anxiety on academic burnout may not have a significant effect.The study further went on to analyze the moderating effect of academic self-efficacy on the relationship between academic anxiety and academic burnout in the second half of the mediated model path see . It was β = 0.011, SE = 0.027, 95% CI = −0.040 to 0.065; medium level of academic self-efficacy: β = 0.038, SE = 0.015, 95% CI = 0.010 to 0.067; high level of academic self-efficacy: β = 0.064, SE = 0.017, 95% CI = 0.032 to 0.100). The results support the hypotheses developed in this research.The bias-corrected bootstrap confirmed that the indirect relationship between academic stress and academic burnout through anxiety was moderated by academic self-efficacy. To be specific, with the improvement of self-efficacy, the indirect effect was stronger  suggests that the protective effect of protective factors is undermined when risk factors are too high , and leaIn this study, a moderated mediation model was constructed to examine the mediating process of “academic stress → academic anxiety → academic burnout” and the moderating role of academic self-efficacy. The results showed that the mediating effect of academic anxiety between academic stress and academic burnout was significant, and the moderating effect of academic self-efficacy in the direct path and the second half of the mediating path was significant, which helps to understand the relationship between learning stress and academic burnout and its internal mechanisms. There are several limitations of this study that could be improved in further research. First, this study used a cross-sectional design and was unable to test the stability of this mechanism of action across time; future studies could be administered to these participants again after a certain period of time to explore the stability of this mechanism of action across time; second, all of the variables explored in this study were related to academics, and according to ecosystem theory , home enNevertheless, this study has strong practical implications. Because of the current high levels of academic burnout among some adolescents, this study can provide targeted suggestions and insights for preventing and intervening in adolescents’ academic burnout. The results of the study show that, first, we can reduce the level of academic burnout by reducing the academic stress of adolescents, such as reducing extracurricular assignments. Second, we can equip adolescents with ways to regulate their emotions and reduce academic boredom by reducing academic anxiety. In addition, according to the results of the study, academic self-efficacy is effective in boosting confidence and coping with external stress effectively, but if students have high levels of academic anxiety, instantly students with high academic self-efficacy will increase the expression of academic burnout as their anxiety level increases. Therefore, for adolescents with high academic self-efficacy, although they can cope with external pressure effectively, they need to pay attention to the regulation of their academic anxiety, so it is more important to provide them with emotional psychological guidance to alleviate their academic anxiety levels; for adolescents with low academic self-efficacy, they need to stimulate their academic self-efficacy as well as pay attention to their own internal emotional regulation. In conclusion, the intervention and prevention of academic burnout in junior high school students should not focus on one aspect only, but should be carried out in an integrated and systematic way from three aspects: academic stress, academic anxiety, and academic self-efficacy.The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the ethics committee at Shandong Normal University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.XG: conceptualization, methodology, supervision, validation, resources, data curation, formal analysis, investigation, validation, and writing—original draft.This study was financially supported by National College Students’ Innovation and Entrepreneurship Training Program of China [grant number: 202210445011]. Shandong Normal University Undergraduate Research Fund Project in 2023 [grant number: BKJJ2022083].The author declares 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.


Objectives: The purpose of this paper is to describe blood services in the Aba Tibetan and Qiang Regions, (hereinafter referred to as Aba Prefecture), a region of China’s Qinghai–Tibetan Plateau, the third largest area of Tibet and the main inhabited area of the Qiang people. Design: We present a comprehensive investigation into blood donations, donors, screening and supply in the 13 counties of Aba Prefecture based on data from 2013 to 2018. Geography and population were also used to analyze the differences in blood services among different regions. Participants: The number of blood donors totaled 19,047. Results: Over the past 6 years, blood donations have increased by 29 and clinical blood usage by 45%. The blood donation rate was 3.4‰ and per capita blood use was 1.04 mL, both of which were significantly lower than the national average, and blood donation decreased with altitude. It should be noted that the donation rate of the Tibetan and Qiang peoples is much lower than that of the Han population. Moreover, the rejection rate of blood in laboratory testing was found to be higher than the national average, especially in counties located at higher altitudes. Conclusions: Blood donations and usage increased every year in Aba Prefecture, but blood shortage is still an important issue. In addition, the prevalence of transfusion–transmitted diseases is relatively high, which may be linked to lower-education and unfavorable geographical and medical conditions. The Aba Tibetan and Qiang regions, commonly known as Aba Prefecture, is a region in Sichuan province, on the southeastern edge of the Qinghai–Tibetan Plateau. Aba Prefecture is the third-largest Tibetan area and the main inhabited area for China’s Qiang people. It covers 13 counties covering an area of 84,200 square kilometers with a population of 940,000. The region is predominantly characterized by plateaus and mountain valleys. The average altitude is 3500–4100 m, and the altitude of the county seats (the major population centers) varies from 1326 to 3406 m [p-value of less than 0.05 was considered statistically significant.The data of blood donation, donor testing and supply in the 13 counties were collected by the Aba Prefecture blood center. Geographical and population data for each county in 2018 were obtained from the people’s government of Aba Prefecture. All data were entered and encoded using Microsoft Excel and analyzed using IBM SPSS 25.0 software . Descriptive statistics were displayed, and the sociodemographic characteristics of blood donors,  were analyzed. Additionally, multi-group comparisons for blood donation and blood use among the different counties were performed using one-way ANOVA, followed by Tamhane’s T2 comparison test as the heterogeneity of variance . A PearsThere was no direct patient or public involvement in this study, and all data were collected online. Whole blood donations between 2013 and 2018 were extracted from the Aba Prefecture blood management information system.It should be noted that all donations (whole blood with no apheresis components) were collected by the Aba Prefecture central blood center. Furthermore, the majority of donations were collected on mobile donation vehicles on the street. Data on geography, population, blood donation and blood use of the 13 counties are presented in p < 0.05). In six counties––Barkam, Wenchuan, Li, Mao, Songpan and Jiuzhaigou––the donation rate per 1000 was higher than 2.9, while the rate was lower than 2.5 in the other seven. The comparison between counties showed that the average donation rate in Barkam and Wenchuan was significantly higher. Moreover, the donation rate in Hongyuan, Rangtang and Aba, with county-seat altitude exceeding 3000 m, was significantly lower. Barkam serves as the capital of Aba Prefecture, while the only higher-education institute is situated in Wenchuan. These factors may account for the higher number of donors and greater amount of donated blood in these two countries. To estimate the relative importance of county-seat altitude on donation rate, a Pearson correlation analysis was conducted using donation data from counties other than Barkam or Wenchuan. The results indicated a significant negative correlation between the donation rates and the altitude of their respective county seats , which implied that as the altitude of the county seats increased, the donation rates decreased.The number of blood donations varied across different counties. Before donating, blood donors underwent pre-screening consisting of a medical history questionnaire, physical examination and rapid tests that measured hemoglobin concentration (Hb), hepatitis B surface antigen (HBsAg), alanine aminotransferase (ALT) and ABO blood group. A pre-screening kit for HBsAg was conducted using a rapid detection kit that produced a small but expected rate of false-negatives. After donation, laboratory tests for ABO group, HBsAg, hepatitis C virus (HCV) antibodies, ALT, Human immunodeficiency virus (HIV)-1 and HIV-2 antibodies and treponemal (TP) antibodies were conducted. Tests for transfusion-transmitted disease (TTD) used two rounds of enzyme-linked immunosorbent assays (ELISA) with two test kits from different manufacturers. If one of the tests yielded a positive result, the corresponding blood products were disqualified from transfusions. Subsequently, the samples were sent to the Chengdu Blood Center for nucleic acid testing (NAT) of hepatitis B virus (HBV), HCV and HIV. As shown in There were almost no whole blood or platelet components used in Aba Prefecture during 2013–2018. Red blood cells and plasma were the main components, and in this study, the quantity of red blood cells represented the total quantity of supplied blood. A total of 5805.6 L of blood was supplied for clinical use by the blood center from 2013 to 2018. The 1138.8 L supplied in 2018 represented an increase of 45.07% over the 785.0 L in 2013 . On averMaintaining a safe and adequate blood service is an important responsibility for every country . China h2) [Most people in Aba Prefecture live at an altitude of 2000–4000 m in remote rural mountainous areas that have poor natural and traffic conditions. The average population density in this area is 11 people per square kilometer, far below that of Sichuan Province (171 people/km2) . With th2) ,13,14. M2) . TherefoIn 2018, the per capita blood use per year of Aba Prefecture was 1.03 mL, which is far lower than for Sichuan Province (2.8 mL) or China (3.2 mL), but higher than for Tibet (0.3 mL) ,6,7. ThiThe risk of transfusion-transmitted disease (TTD) still challenges blood services in China , but tesBlood supply is an essential part of public health-care services, but outside help is only temporary; therefore, substantial improvement in autogenous capacity to guarantee a safe blood supply is still needed in Aba. Under the support of local government, blood collection and supply institutions hold publicity and advocacy events to enhance and encourage the public’s awareness and participation in voluntary non-remunerated blood donation. Every year, through mainstream media platforms––radio, television, newspapers, magazines––and modern communications services like QQ and WeChat, a diversity of events is launched on World Blood Donor Day, World Red Cross Day and other publicity days, as well as at festivals such as the Spring Festival. We hope to establish a safe and adequate blood service system in Aba Prefecture through the close collaboration and efforts of all relevant parties, including researchers, blood bank staff and government.We conducted a systematic and comprehensive study on the status of blood service in the Aba Tibetan and Qiang Regions (Aba Prefecture) from 2013 to 2018 that included geography, population, voluntary non-remunerated blood donation, donors, screening and supply.The special characteristics of Aba Prefecture make the blood donation service there different from that in other part of China.The results may only reflect blood donation in Aba Tibetan and Qiang Regions, not be generalizable over other minority groups at high-altitude regions.Our survey was a preliminary investigation into blood services ranging from blood donation, donors, screening and supply in Aba Prefecture, a region of China’s Qinghai-Tibetan Plateau. Further research is needed, and measures should be taken to control the transmission of epidemics and to ensure adequate and sustainable availability of blood.