The acrosome reaction (AR) is one of the important factors in assessing sperm infertility. However, the accuracy of these assessments may be influenced by the subjective judgments of experts. Addressing the issue of subjectivity in the assessment of the AR, we developed the Acrosome Reaction Classification System (ARCS). This system enables automatic calculation of the AR ratio using deep learning, which not only detects AR sperm by identifying micro-changes in the plasma membrane (PM), but also offers improved speed and performance compared to experts. Moreover, we established the need for independent ARCS with appropriate magnifications to detect AR sperm across various magnifications. The ARCS also offers consistent analysis for AR sperm detection and reduces misrecognition due to human error. In conclusion, our proposed methodology has the potential to contribute to the development of deep learning-based diagnostic models for sperm characteristics in pigs and other species, while the ARCS can be utilized in artificial intelligence-based infertility diagnoses within reproductive medicine.The analysis of AR is widely used to detect loss of acrosome in sperm, but the subjective decisions of experts affect the accuracy of the examination. Therefore, we develop an ARCS for objectivity and consistency of analysis using convolutional neural networks (CNNs) trained with various magnification images. Our models were trained on 215 microscopic images at 400× and 438 images at 1000× magnification using the ResNet 50 and Inception–ResNet v2 architectures. These models distinctly recognized micro-changes in the PM of AR sperms. Moreover, the Inception–ResNet v2-based ARCS achieved a mean average precision of over 97%. Our system’s calculation of the AR ratio on the test dataset produced results similar to the work of the three experts and could do so more quickly. Our model streamlines sperm detection and AR status determination using a CNN-based approach, replacing laborious tasks and expert assessments. The ARCS offers consistent AR sperm detection, reduced human error, and decreased working time. In conclusion, our study suggests the feasibility and benefits of using a sperm diagnosis artificial intelligence assistance system in routine practice scenarios. Sperm infertility diagnosis is accompanied by laborious manual work that requires accurate assessment of thousands from billions of sperm . The accThe sperm head is covered with a plasma membrane (PM) and contains an acrosome and a nucleus . In partFrom a reproductive biology perspective, when sperm is exposed to high levels of bicarbonate and reactive oxygen species, the PM becomes loose and fragmented . After tVisual assessment of sperm AR is typically performed manually by experts, heavily relying on their subjective judgment. Additionally, the manual process has a low throughput as it involves inspecting only tens of cells from a specimen containing tens of millions. To address these issues, research on automatic sperm detection and classification in computer vision has been conducted for decades. Various studies have focused on threshold-based segmentation, edge detection ,7, regioObject detection in computer vision has gained significant attention in recent years, mainly due to the remarkable progress in convolutional neural networks (CNNs)  and theiFrom a deep learning perspective, the accuracy and precision of a CNN architecture are typically improved by increasing the number of the hidden layers . HoweverThe number of studies utilizing deep learning for sperm classification and object detection in microscopic images has been steadily increasing. In particular, deep learning-based studies focusing on DNA status classification  and headWe developed an Acrosome Reaction Classification System (ARCS) that comprised three main steps. The first step involved collecting datasets at two magnifications: 400× (400-mag) and 1000× (1000-mag). Images containing both AR and non-AR boar sperms were collected using a microscopic imaging system A. In the7 sperm/mL. To prepare the samples for detecting various AR sperm patterns, the diluted semen samples were centrifuged at 410 g for 5 min. The supernatant was removed, and the pellets were resuspended in 0.1 M phosphate buffer solution (PBS) to obtain a concentration of 1.5 × 107 sperm/mL. Following a previous study [7 sperm/mL. For sample preparation, the samples were smeared onto slide glasses (Sigma) and dried. They were then washed three times with distilled water. After air-drying, the samples were stained with a 0.25% Coomassie Brilliant Blue  solution for 5 min and dried at room temperature. Following that, the samples were washed three times with distilled water and dried again. Digital images of the samples were captured using a digital camera  mounted on an optical microscope . In terms of sperm classification, we considered intact PM and acrosomes as non-AR sperm, while swollen PM and released acrosomes were classified as AR sperm . Semen samples were collected from the pigs  using the glove-hand method. The samples were then diluted with semen extender  to achieve a concentration of 1.5 × 10us study , the samAR sperm .We obtained a total of 653 images from the sperm samples of the 10 pigs, each image having dimensions of 1200 × 800 pixels . Out of For the 400-mag images, a random split was performed, with 215 images allocated to the training dataset (172 images) and the test dataset (43 images). Similarly, for the 1000-mag images, a random split was conducted, resulting in 438 images assigned to the training dataset (351 images) and the test dataset (87 images) . To achiThe procedure was implemented using TensorFlow 1.14.0 in Python 3.7.4. Two different CNN architectures, namely, ResNet 50  and InceThe accuracy, precision, recall, F1 score, and mean average precision (mAP) at an intersection over union (IoU) of 0.5 were selected as measures to evaluate the performance of trained models. The IoU is defined as follows (1):With an IoU threshold of 0.5, the accuracy (2), precision (3), and recall (4) were calculated, and the corresponding F1 score (5) could be defined asThe precision–recall curve is computed from a method’s ranked output, and recall is defined as the proportion of all positive examples ranked above a given rank . The AP eeding r :(7)PinteLastly, the mAP (8) was calculated as an average of APs for all object classes:The performance results of the 5-fold cross-validation are presented as the mean ± standard deviation. The training and validation processes were treated as two separate procedures, and detection boxes were drawn using the trained models on the validation dataset with a score threshold of ≥0.8. The mean average precision (mAP) was evaluated on the validation dataset at every 500 iterations to fine-tune the hyperparameters. After several attempts, the following hyperparameters were determined: a maximum number of iterations of 30,000; an initial learning rate of 0.0003, which was then reduced to 0.00003 after 10,000 iterations. Subsequently, an experiment was conducted to determine the optimal CNN architecture for ARCS. The performances of the ResNet 50 and Inception–ResNet v2 architectures were measured and compared.After the completion of training and validation, the models were evaluated using the test dataset, which consisted of 43 (400-mag) and 87 (1000-mag) images. To assess the performance of the trained models, three expert embryologists  were involved in the evaluation. These experts did not see the image data prior to annotating the test dataset and possessed approximately 3 to 6 years of experience. The human annotation process followed the same rules as described in the previous section. To ensure accuracy, the experimenters thoroughly reviewed the annotations, correcting any potential mistakes before final submission. The annotations provided by the embryologists were then compared with the ground truth of the test dataset in order to calculate accuracy, precision, recall, and F1 scores. Furthermore, a comparison was made between the number of boxes assigned to the AR/non-AR classes and the AR sperm ratio, considering both the detected boxes by the trained models and the annotations provided by the three embryologists. This analysis was performed across all ranges of score thresholds and IoU values greater than 0.5.N represents the number of images in the test dataset, and i-th image in the test dataset, respectively.The detected boxes of the AR/non-AR were drawn with the minimum score threshold Statistical analyses were performed using SAS v. 9.4 . The ResNet-400-mag, ResNet-1000-mag, and ResNet-400 + 1000-mag datasets were evaluated using one-way analysis of variance (ANOVA). To compare the trained models based on ResNet 50 and Inception–ResNet v2, a t-test was employed. The results are presented graphically using a scatter dot plot generated with Graphpad Software Inc., San Diego, CA, USA, and the data are displayed as the mean ± standard error of the mean.ResNet-400-mag and ResNet-1000-mag classified the AR sperm when the validation datasets of the 400-mag and 1000-mag were evaluated . SpecifiThe models were trained on the training datasets of the 400-mag, 1000-mag, and 400 + 1000-mag using the ResNet 50 architecture, referred to as ResNet-400-mag, ResNet-1000-mag, and ResNet-400 + 1000-mag, respectively. Losses for both the training and the validation datasets stabilized after 10,000 iterations for all tested models. Notably, the ResNet-1000-mag exhibited the most stable learning curve, with the initial drop in loss falling below 0.5 A–C. The p < 0.05) compared to the other models (p < 0.05) than in the other models  in the 400-mag validation dataset compared to the 1000-mag validation dataset (p < 0.01) in the 400-mag validation dataset compared to the 1000-mag validation dataset  higher than that of ResNet-400-mag  exhibited higher accuracy, precision, recall, F1, and mAP compared to ResNet-400-mag . In part-400-mag A. Additi-400-mag . Moreovep < 0.01) reduced by 1.75 times in Incep-Res-400-mag compared to ResNet-400-mag and by 1.69 times in Incep-Res-1000-mag compared to ResNet-1000-mag (Next, we report the median mAP values of the models in the 5-fold cross-validation process. The accuracy, precision, recall, F1, and mAP were higher in Incep-Res-400-mag and Incep-Res-1000-mag than in ResNet-400-mag and ResNet-1000-mag in the test datasets . However1000-mag . DespiteThe detection performances of Incep-Res-400-mag (Lastly, we compared the automatically calculated AR ratios of Incep-Res-400-mag and Incep-Res-1000-mag with three experts. Additionally, to facilitate the analysis of AR through visual assessment, we printed information about the detected boxes on the images. As a result, the test images successfully display information about the classes, the number of detected sperm, and the AR ratio on the upper right side F and 0.5The accuracy of cytological sperm diagnosis has advanced in conjunction with the development of microscopic resolving powers. However, despite these advancements, detecting micro-changes in organelles such as the PM, nucleus, and mitochondria still requires professional knowledge in the field of sperm analysis. Furthermore, to ensure the reliability of sperm analysis in experimental and clinical settings, hundreds or even thousands of specimens need to be evaluated. The requirement for specialized domain knowledge and the demanding nature of these tasks can lead to misidentification and subjective judgments by experts, resulting in inaccurate sperm analysis results. Our system consistently recognizes micro-changes in the PM of microscopic images on the basis of pixel information and correctly classifies them as AR sperm. Moreover, our system can effectively distinguish initial AR sperm even at 400-mag. This implies that our approach can objectively identify micro-modifications in the PMs of sperm, which are challenging for experts to diagnose accurately across numerous specimens at 400× magnification. Therefore, our system has the potential to replace manual labor in sperm diagnosis. Additionally, we intentionally did not utilize 100× and 200× magnification images for model training due to their low resolving power, making it difficult to discern micro-changes in the PM.The seminal plasma comprises various inorganic substances, including white blood cells, microorganisms, and tissue and cell debris . These sSperm analysis based on visual assessment is broadly classified into two main methods: motility analysis and morphological analysis . MotilitTherefore, we developed the ARCS, which automatically calculates the AR ratio on the basis of deep learning. It not only detects AR sperm with micro-changes in the PM but also exhibits competitive speed and performance compared to experts. Additionally, we emphasize the need for independent ARCS tailored to appropriate magnifications for detecting AR sperm in various scenarios. Thus, our model can replace the tedious tasks of detecting sperm in numerous specimens and the subjective assessment of experts in determining the AR status of micro-changed PMs in sperm heads using CNN-based object detection and classification. Moreover, the ARCS contributes to consistent AR sperm detection and reducing human errors in misrecognition. In conclusion, the proposed methodology contributes to the development of a deep learning-based diagnostic model for detecting sperm characteristics in pigs and other species. The ARCS can be utilized in artificial intelligence-based infertility diagnosis in reproductive medicine.


Previous research in this field has not examined the significance of each of the basic psychological needs (BPNs) on changes in the physical activity level, adherence to the Mediterranean diet (AMD), kinanthropometric and derived variables, and the physical fitness of adolescents. Therefore, the purpose of this study was (a) to examine the variances in physical activity, AMD, and kinanthropometric and derived variables, as well as fitness levels, among adolescents with varying degrees of satisfaction regarding each of the BPNs and (b) to assess the differences in the study variables among adolescents based on whether the BPNs are satisfied individually or jointly. The sample consisted of 791 adolescents . The findings indicated that adolescents in the highest percentiles (75–100) of competence, autonomy, or relatedness showed higher scores in physical activity and AMD and better kinanthropometric and physical fitness variables than adolescents in the lowest percentiles (0–25). Adolescents who showed joint satisfaction of all BPNs showed the best results on all variables analyzed. In addition, it should be noted that competence played the most relevant role. Self-determination theory (SDT) defines autonomy, competence, and relatedness as the three basic psychological needs (BPNs) . SatisfaIn addition, it should be noted that in previous cross-sectional research, the authors showed the relationship between LBW and certain healthy lifestyle habits, such as the practice of physical activity  or adherFurthermore, the preliminary findings from pilot studies conducted by the researchers have identified additional personal factors that play a crucial role in maintaining good health and the prevention of chronic diseases in adolescents, such as kinanthropometric and derived variables  and the In the psychological field, the importance of joint or individual satisfaction with BPNs has been analyzed . HoweverBased on the objectives of the present research, as well as previous scientific literature, the following research hypotheses (H) are proposed: (H1) adolescents who experience higher levels of satisfaction with BPNs are expected to exhibit increased levels of physical activity, greater AMD, better physical performance, and better kinanthropometric and derived variables; and (H2) in the absence of previous research that would allow us to establish an accurate hypothesis as to which BPN is more relevant to the study variables, we hypothesize that adolescents who have all BPNs satisfied collectively will demonstrate elevated levels of physical activity, AMD, and physical performance and improved kinanthropometric and derived variables.The current study employed a cross-sectional design and utilized non-probability convenience sampling. Four compulsory secondary schools in various regions of the Region of Murcia, Spain, were involved in the study. The research adhered to the STROBE statement , guidingThe final sample consisted of 791 adolescents  aged 12–16 years old (mean age: 14.39 ± 1.26 years old). These adolescents willingly participated in the research after providing informed consent forms signed by both themselves and their parents. The sample size was determined using the methodology employed in previous studies  and the The participants included in the study satisfied the following inclusion criteria: (a) completion of all questionnaires, kinanthropometric measurements, and physical tests in their entirety; (b) not presenting any disease or surgical operation that prevented participation in any of the tests; (c) age range between twelve and sixteen years old; and (d) attending compulsory secondary education.To assess competence, autonomy, and relatedness (BPNs), the Spanish version of the Basic Psychological Needs Scale (BPNS)  was emplBased on previous research , the anaIn addition, BPNs were considered as satisfied when the score was above the 50th percentile, resulting in the following classification: none (no BPN satisfied), competence (competence satisfied only), autonomy (autonomy satisfied only), relatedness (relatedness satisfied only), competence and autonomy satisfied, competence and relatedness satisfied, autonomy and relatedness satisfied, and all BPNs satisfied.To evaluate the participants’ physical activity level, the Spanish version of the “Physical Activity Questionnaire for Adolescents” (PAQ-A) was employed ,15. ThisThe assessment of the participants’ adherence to the Mediterranean diet was conducted using the “Mediterranean Diet Quality Index for children and adolescents” (KIDMED) questionnaire . This quIn accordance with the protocol established by the International Society for the Advancement of Kinanthropometry (ISAK) , three aThe instruments utilized for the measurements were as follows: a TANITA BC 418-MA Segmental  (body mass); a SECA stadiometer 213  (height and sitting height); a Harpenden skinfold caliper  (skinfolds); and an inextensible tape, Lufkin W606PM  (girths).The derived variables calculated with the final values of the kinanthropometric measurement were: BMI, waist-to-hip ratio (waist girth/hip girth) , correctIn accordance with the methodology employed in prior studies , the folData collection for this research took place in each of the high schools, specifically during the physical education classes. The tests were conducted in a covered sports pavilion to minimize the potential interference of confounding variables. It is important to note that all tests were performed on the same day to maintain consistency in data collection.The protocol, based on previous research , consist2), with values classified as small (ES ≥ 0.10), moderate (ES ≥ 0.30), large (ES ≥ 1.2), or very large (ES ≥ 2.0). The significance level was set at p < 0.05 [Initially, the normality of the data was evaluated using the Kolmogorov–Smirnov test, as well as an analysis of skewness and kurtosis. As the data demonstrated a normal distribution, parametric tests were employed for subsequent analyses. Descriptive statistics, including the mean (M) and standard deviation (SD), were utilized to summarize the analyzed variables. The internal consistencies of self-reported measures were evaluated using Cronbach’s alpha. Subsequently, four one-way ANOVA tests were conducted. The first ANOVA aimed to identify differences in the study variables among different percentiles in the competence variable. The second ANOVA focused on the autonomy variable, while the third ANOVA examined the relatedness variable. Lastly, the fourth ANOVA was conducted to determine differences in the study variables based on the BPNs satisfied by the adolescents. For statistically significant variables, Bonferroni’s pairwise comparison was employed. The effect size (ES) was calculated using partial eta squared , the PAQ-A reported 0.840, and the AMD reported 0.756. A scale has an acceptable internal consistency when the Cronbach’s alpha value is greater than 0.70 , which iThe post hoc analysis of the differences between the satisfaction percentiles of the competence variable is shown in The differences in the studied variables, according to the percentile of satisfaction of the autonomy variable, are found in The subsequent post hoc analysis  showed tThe differences in the studied variables according to the BPNs satisfied in the adolescents are shown in It is worth noting that the level of physical activity was significantly lower in adolescents with no basic psychological needs satisfied, as compared to those with satisfied competence, competence and autonomy, competence, and relatedness, or all BPNs. In addition, adolescents with satisfied competence showed higher scores than those with satisfied relatedness, while those with satisfied competence and relatedness showed higher scores than those with satisfied autonomy and relatedness. On the other hand, adolescents who had all BPNs satisfied showed higher scores than those who only had autonomy, relatedness, or autonomy and relatedness satisfied. Regarding the AMD, adolescents with no BPNs satisfied showed lower scores than those with competence and relatedness satisfied or those with all BPNs satisfied .The results from the kinanthropometric and derived variables showed that adolescents who did not have any BPNs satisfied had lower scores than adolescents who had competence and autonomy satisfied in height, corrected arm girth, and muscle mass and lower scores than adolescents who had competence and relatedness satisfied in corrected thigh girth and muscle mass but higher scores in the sum of three skinfolds and fat mass, as well as lower scores than adolescents who had all BPNs satisfied in height, corrected thigh girth, and muscle mass .As for the physical condition variables, performance was significantly lower in VO2 max, handgrip, CMJ, and the 20 m sprint in adolescents who had no BPNs satisfied, as compared to those who had competence together with autonomy or relatedness, as well as compared to those who had all BPNs satisfied. In addition, it is worth noting that in VO2 max, adolescents who had satisfied competence showed higher scores than those who did not have any BPNs satisfied; in addition, adolescents who had satisfied autonomy and relatedness showed lower performance than those who had competence and relatedness or all BPNs satisfied .The objectives established for the present research were (a) to determine the differences in the level of physical activity, AMD, kinanthropometric and derived variables, and fitness among adolescents with different degree levels of satisfaction of each of the BPNs and (b) to establish the differences in the level of physical activity, AMD, kinanthropometric and derived variables, and physical fitness of adolescents according to whether the BPNs are satisfied individually or jointly. The results indicate that adolescents who practice more physical activity have a greater satisfaction of the BPNs, which is similar to the results of previous research . HoweverIn addition, another novelty found in the present investigation is the determinant role played by the satisfaction of the need for competence in the practice of physical activity during adolescence. Thus, adolescents who had all BPNs satisfied jointly or those who had satisfied competence individually or together with autonomy or relatedness practiced more physical activity as compared to those who did not have any satisfied BPNs or satisfied autonomy or relatedness individually. This could be due to the fact that a greater perception of competence would lead adolescents to seek optimal challenges to maintain or improve their skills , with spRegarding AMD, the lowest percentile 0–25) of satisfaction of the competence, autonomy, or relatedness variables showed a significantly lower adherence to these nutritional guidelines compared to the other percentiles. However, no significant differences were found in the level of AMD in any of the BPNs when comparing the other BPN satisfaction percentiles . In previous research, the authors showed a significant and positive relationship between BPN satisfaction and AMD  of satis. The preSimilar to the practice of physical activity, differences were also found in AMD according to individual or joint satisfaction of BPNs. Adolescents who did not have any satisfied BPNs scored lower in AMD as compared to adolescents who had satisfied all BPNs and those who had competence and relatedness jointly satisfied. A possible explanation for these results would be that the acquisition of a certain healthy lifestyle habit favors the acquisition of other healthy habits , with thAccording to the kinanthropometric and derived variables, adolescents in the lower percentiles of satisfaction (0–25) of the BPNs showed a lower height and sitting height, greater body fat accumulation, and less muscle development than adolescents in the higher percentiles (50–75 and 75–100). Authors of previous research have shown that autonomy is related to body mass and body size discrepancies , but theIn fact, adolescents who had satisfied all the BPNs, or competence individually or together with autonomy or relatedness, were those who showed better kinanthropometric and derived variables, as compared to adolescents who did not have any satisfied BPNs. This could be due to the fact that a good physical self-concept improves the perception of competence and is related to the measurement of subjective psychological well-being in the adolescent population . AnotherRegarding physical fitness, adolescents in the lowest percentile (0–25) of BPN satisfaction showed worse performances in VO2 max, handgrip strength, CMJ, and 20 m sprint tests, as compared to the highest satisfaction percentiles (50–75 and 75–100). In addition, competence seemed to be the most important BPN for physical fitness, as adolescents who had all BPNs satisfied, or those with satisfied competence independently or together with autonomy or relatedness, showed better performances. Only one previous research study is known in which the authors analyzed the relationship between BPNs and the physical fitness of adolescents, and although it showed that environments that satisfy BPNs may favor increased fitness, it did not include differences in specific physical fitness tests, as it only calculated a total fitness score . In prevThe findings from this investigation provide support for the first research hypothesis (H1), as they indicate that adolescents who experience higher levels of satisfaction with BPNs demonstrate a higher level of physical activity, greater AMD, better physical performance, and better kinanthropometric and derived variables, and it was the adolescents in the highest percentiles of each of the BPNs who showed higher scores in each of the study variables. The results of this study confirm the acceptance of the second research hypothesis (H2). The findings demonstrate that adolescents who have all BPNs collectively satisfied exhibit a higher level of physical activity, AMD, physical performance, and improved kinanthropometric and derived variables. In addition, the results obtained allow us to give greater relevance to the need for competence, since when not all BPNs were satisfied, the satisfaction of competence alone or together with relatedness or autonomy showed significant differences.This research is similar to those previously carried out in the adolescent population ,27,54 buRegarding the limitations of the present study, it should be noted that, unlike other psychological scales, the BPNS  does not have a classification scale to determine the degree of satisfaction of each of the BPNs, so percentiles had to be established according to the scores of the adolescents in the sample. The single use of questionnaires is an aspect to be considered in future research, since more information on the satisfaction of the BPNs would be obtained through the combined use of the questionnaire and interviews or focus groups on the adolescent population. Since this was a cross-sectional study, causality cannot be established in the results found, so longitudinal studies are required to establish the indicated relationships between the BPNs and other psychological variables, as well as between BPNs and changes in the level of physical activity, AMD, kinanthropometric and derived variables, and physical fitness.To conclude, the present study shows that adolescents in the highest percentiles (50–75 and 75–100) of BPN satisfaction had better levels of physical activity, AMD, kinanthropometric and derived variables, and physical fitness. Furthermore, it is noteworthy that adolescents who had all BPNs satisfied showed higher levels of physical activity, AMD, better kinanthropometric and derived variables, and better physical fitness, with competence being the most relevant; when not all BPNs were satisfied, the satisfaction of competence alone or together with autonomy or relatedness reported the greatest benefits.The relevance of the present research lies in the fact that it establishes the importance that BPNs can have in the healthy development of the adolescent population beyond the sports field in which this relationship had been widely studied. Thus, the results show the relevance of the satisfaction of the BPNs in the adoption of healthy lifestyle habits, which is fundamental in a society in which the adolescent population has considerably reduced its practice of physical activity and its adherence to adequate nutritional patterns. Therefore, this research may be of relevance to all professionals working with adolescents .


Some impediments in language learning may have a detrimental impact on learners’ actual performance on the test and lead to anxiety and demotivation. Language achievement is influenced by self-assessment (SA), academic resilience (AR), academic motivation (AM), and test-taking skills (T-TS) among other factors. Considering the relevance of these factors in language achievement, the current investigation aims to delve into the probable interactions of SA, AR, AM, T-TS, and test anxiety (TA) management among English as a foreign language (EFL) learners. A model was devised and evaluated using confirmatory factor analysis (CFA) and structural equation modeling (SEM) to achieve this objective. This research collected 512 by distributing online questionnaires to fifteen approved private institutions which applied Telegram-based language learning. The study findings reflected that SA, AR, and AM could predict EFL learners’ T-TS. It was also confirmed that SA, AR, and AM modulated EFL learners’ TA. The implications of the study are presented and accompanied by some future research proposals as well as instructional consequences. Several factors impact the decisions that educators and test developers must make to establish and administer an effective education and assessment program. Both emotive and cognitive characteristics of the learners are critical in successful language testing. Practically speaking, learning-oriented evaluation in the classroom operates reasonably well to evaluate language learners’ language progress and observe their mental and emotional well-being  in terms of their T-TS and TA, this research sought to explore the contribution of SA, AM, AR to T-TS, and TA management. Holding these standpoints into consideration led to the formulation of the following research questions:H01: EFL learners’ SA does not provide insight into their T-TS in Telegram-assisted-language learning.H02: EFL learners’ SA does not provide insight into their TA in Telegram-assisted-language learningH03: EFL learners’ AR does not provide insight into their T-TS in Telegram-assisted-language learning.H04: EFL learners’ AR does not provide insight into their TA in Telegram-assisted-language learningH05: EFL learners’ AM does not provide insight into their T-TS in Telegram-assisted-language learningH06: EFL learners’ AM does not provide insight into their TA in Telegram-assisted-language learningIn this respect, the following null hypothesis are proposed:As a response to these inherent gaps, a model was developed to illustrate the probable linkages between SA, AR, AM, T-TS, and TA. This model was developed based on pertinent theories and contemporary literature see Fig. . Using CThe term “assessment” encompasses an array of methods that are used to analyze and draw conclusions about the advancement that pupils have made in their studies Bachman, . Many alResilience has been conceptualized as the ability to sustain normal development and make positive adaptations while facing significant adversity , which was first proposed by Deci and Ryan , is now TA may be experienced by learners before, during, and after the test. Horwitz et al.  introducThe COVID-19 pandemic has interrupted education in many countries; thus, as an emergent reaction, several governments have prioritized the implementation of virtual instructional strategies. During the COVID-19 outbreak, online courses and social media facilitated two-way communication between pupils and their instructors Peng, . In receTelegram is compatible with the operating systems used by Android, iOS, and Windows Phone, as well as Mac and Windows devices. Furthermore, the Telegram app may be used concurrently from a number of different devices . Among the subcomponents of AR, Perceived Happiness  received the highest mean score. Regarding AM, the mean score was . Furthermore, During-Test was the highest  among T-TS. In the final variable, TA, Incapacity was different . Then, the normality of the data was assessed and reported in Table As Table Based on what Table β = 0.84, t = 29.54) and TA  was positive. The effect of AR on T-TS  and TA  was significant. The same was true about the impact of AM on T-TS  and TA .The causal relationships among the variables are portrayed in Figs. The data in Table β = 0.89, t = 29.67), Time Management , During-Test , After-Test , Incapacity , and Worry and Dread . The association between AR, TS, and TA subfactors is as follows: AR and Before-Test , Time Management , During-Test , After-Test , Incapacity , and Worry and Dread . Additionally, AM is linked with TS, and TA subfactors are as follows: Before-Test , Time Management , During-Test , After-Test , Incapacity , and Worry and Dread .In model 2 which is illustrated via Figs. As the final step, a Pearson product-moment correlation was run to address the correlation between SA, AR, AM, T-TS, and TA subfactors.Table This research intended to investigate the impact of the SA, AR, and AM on T-TS and TA management in academic settings. To accomplish this goal, an empirical study was carried out with EFL students enrolled in English language institutions learning English via Telegram. The analysis of the data revealed that enhancement in SA, AR, and AM enables learners to pass their tests skillfully and manage their possible test anxiety. These findings underlined the crucial part that social media, specifically the Telegram app, plays in improving students’ mental and emotional health, and the suggested model Fig.  was confTaking into consideration the first and the second research questions, which were “RQ1:Can EFL learners’ SA provide insight into their T-TS in online classes? and RQ2: Can EFL learners’ SA provide insight on their TA in online classes?”, the findings of this exploration show that SA can play a moderator function in T-TS and TA management (model 1 & model 2). It is possible to infer that how students approach self-evaluation may help them build or restore a positive self-image and a strong sense of self-worth, which in turn helps to reinforce the accomplishment of language learning objectives. Furthermore, SA helps learners cultivate an internalized confidence in their abilities and a perception of effectiveness in their skills to complete assignments effectively . Based on the opportunities provided by the Telegram app, learners can easily communicate, and the anxiety of being judged by their peers would decrease. This outcome is in accordance with Esmailzade Ashini et al. , who conThe results of the fifth and sixth research questions revealed that AM played a significant role in increasing EFL learners’ T-TK and decreasing TA. That is, AM inspires students to act with vigor and build a good self-image and gives them assistance and engagement (model 1 & model 2). Both Dörnyei et al.  and Al-HIn a nutshell, the purpose of this inquiry was to uncover the connection between SA, AR, AM, T-TS, and TA management in the EFL context. In this respect, a model was hypothesized and then verified using CFA and SEM in the proper manner. Furthermore, this study was to provide the spotlight on the advantages that Telegram-based training may bestow to language assessment when applied in an EFL environment. The obtained data provide support for the proposed model and reflect the predictive capacity of SA, AR, and AM, respectively.In light of the results of the research, there are a number of pedagogical recommendations that may be made. Implementation of MALL into the curriculum has the potential to boost and extend teachers’ and student access to course data and foster LOA whenever and wherever, even outside of the classroom, as the outcome of the swift development of emerging technologies and the need for hosting online and virtual courses. This enhancement is of great importance in the realm of language learning and assessment. As it was displayed in this the current research, language learners felt more secure and less anxious. As such, educators need to learn the digital literacy necessary to use MALL platforms, notably the Telegram app, for the purpose of language instruction and assessment. It is necessary for both instructors and language learners to acquire an awareness of the self-aid conceptions and the attributions such constructs have on their well-being and LOA enhancement. It is important for the academic courses to either directly or indirectly include training in the relevant methods.It is recommended that those responsible for developing academic curricula and materials make changes to such materials and take these results into consideration when working on other academic fields. It is advised that policymakers, curriculum designers, material producers, test developers, and language teachers give some thought to language instruction based on MALL and teaching self-aid practices. This will ensure the academic success of the learners, LOA, and, more significantly, the well-being of society. In addition, practical tactics that may be used to cultivate and put into practice SA, AR, AM, and T-TS should be introduced into higher education. Language teachers may get the necessary training via pre-service and in-service training courses, respectively.Although this research provides interesting insights into the topic in question, it faces some limitations, which need to be addressed. To get things started, all of the learners who participated in our investigation were aged between 17 and 23 with similar English proficiency. In forthcoming studies, it may be possible to evaluate the correlations between SA, AR, AM, T-TS, and TA management in a variety of educational environments and among students majoring in a variety of different fields of study. In addition, due to the fact that all of the information gleaned from this study was obtained via the self-reporting of the learners on questionnaires, the generalizability of the findings is under question. In future research, incorporating qualitative and quantitative methods may provide more thorough findings. This study was limited to telegram-assisted language learning. As a future research avenue, it is suggested to investigate the effects of applying other apps in language learning enhancement and psychological health of the learners. Moreover, an additional survey is suggested to triangulate the study findings and to undertake a comprehensive analysis of the degree to which demographic factors of the students may have an impact on the interrelationship among SA, AR, AM, T-TS, and TA management.


Lasso transmission is a method for realizing long-distance flexible transmission and lightweight robots. However, there are transmission characteristic losses of velocity, force, and displacement during the motion of lasso transmission. Therefore, the analysis of transmission characteristic losses of lasso transmission has become the focus of research. For this study, at first, we developed a new flexible hand rehabilitation robot with a lasso transmission method. Second, the theoretical analysis and simulation analysis of the dynamics of the lasso transmission in the flexible hand rehabilitation robot were carried out to calculate the force, velocity, and displacement losses of the lasso transmission. Finally, the mechanism and transmission models were established for experimental studies to measure the effects of different curvatures and speeds on the lasso transmission torque. The experimental data and image analysis results show torque loss in the process of lasso transmission and an increase in torque loss with the increase in the lasso curvature radius and transmission speed. The study of the lasso transmission characteristics is important for the design and control of hand functional rehabilitation robots, providing an important reference for the design of flexible rehabilitation robots and also guiding the research on the lasso regarding the compensation method for transmission losses. Stroke is a medical condition that can lead to the disability of middle-aged and elderly people. The seventh census found that China’s population is rapidly aging. The population aged 60 and above is 264 million, accounting for 18.70% of the total population, and the population aged 65 and above is 191 million, or 13.5% of the total population. Compared with the last decade, the increase rates for these two age groups are 2.51% and 2.72%, respectively. With the increase in the elderly population, stroke has become a common condition, and it often leads to motor dysfunction or even lifelong disability [Stroke has the characteristics of a high incidence rate, disability rate, mortality rate, and recurrence rate. Most survivors of stroke experience the sequelae of hand dysfunction, and hand movement accounts for 80% of human upper limb movement, making it indispensable in daily life. Moreover, in recent years, the age of the incidence of stroke has decreased, and the rehabilitation training for the hand function of stroke survivors has become a major social problem to be solved ,4,5. A lThe transmission structure of an exoskeleton hand rehabilitation machine is generally rigid, and the linkage mechanism is usually used as the transmission structure to realize the underdrive design of fingers and to ensure the compactness of the structure and the number of motors in order to meet the requirements of lightweight design. It is easier to control than soft equipment. However, its limitation is that it is difficult to align with people’s finger joints and is uncomfortable and heavy ,14,15,16Flexible exoskeleton hand function robots and flexible gloves mostly use rope transmission to provide power, and flexible gloves also have a way of transmitting force through compressed air ,20,21. FFrom the above analysis, it can be seen that structural design is important for the development of the hand-functional robot. The connecting rod structure is relatively thick and hard; therefore, it is difficult to realize multi-degree-of-freedom movement and maintain a large working space. Pneumatic and hydraulic transmission cannot be limited to plane motion, and its material is elastic and can be twisted, compressed, and wrinkled. This kind of motion can be regarded as providing an infinite number of degrees of freedom, which makes the control of the robot very challenging and requires new modeling, control, dynamics, and advanced planning methods. A lasso-driven rehabilitation hand is the most similar to the muscle drive of the human hand, which can realize movements similar to the human hand, easily produce higher fingertip force, and easily realize lightweight design ,33. ThisEach finger has multiple degrees of freedom, and each finger can be regarded as having three degrees of freedom after simplification. The fingers of a normal person’s hand are composed of four bones except the thumb, and the thumb is composed of three bones, which is one less bone than the other four fingers, but the metacarpals of the other four fingers are all fixed on the palm of the hand, and the degree of freedom is not increased because of the extra phalanges. Therefore, in the study of human hand movement, the thumb, like other fingers, can be considered as a joint structure with three degrees of freedom. As shown in Once the finger is disabled due to stroke or nerve injury, and if the patient has not used it for a long time, the patient’s hand behavior will be suppressed, the finger movement ability will be covered up, and inertia behavior will occur, which will lead to learned waste. In order to avoid this symptom and help the affected limb carry out rehabilitation training, continuous passive rehabilitation therapy was put forward. By assisting the patient’s fingers to carry out continuous passive exercise training, it can help the patient recover from the hand dysfunction as soon as possible. However, the number of traditional doctors is limited, and it is difficult for them to continue long-term and high-intensity rehabilitation training. The hand functional rehabilitation robot can solve this problem well. As long as the doctors set the rehabilitation mode, they can drive the patient’s fingers to carry out rehabilitation training.The overall structure of the hand rehabilitation robot is shown in As shown in As shown in The structure and parameters of the lasso affect its performance, but there is still a lack of relevant research. In this study, through the process of structural modeling, simulation, and mathematical modeling of the lasso, the influence of the parameters of the lasso on its performance is studied.The initial pre-tightening force of the rope is zero, and given any positive displacement of the rope, the end of the rope responds to the output. The rope elements are so small that each segment is a plane curve, as shown in Here, we have established an ideal transmission model. Assuming that there are no factors, such as backlash, collisions, and deformation, we can build a simplified model only considering the influence of friction factors in the transmission process. As shown in When From the analysis of the rope micro-element in the moving state, the relationship between the rope force and displacement output at point When the whole rope moves, all the micro-elements move in the same direction. When the elongation of the rope increases  that the spatially arbitrary curvilinear lasso transmission system can be regarded as a series connection of two springs with elastic coefficients We used the LS-DYNA module in ANSYS Workbench to simulate the dynamic process of lasso transmission, and the results are shown in As shown in To determine the relationship between the input and output characteristics of lasso transmission, we set up a test platform for actual measurement and then observed and recorded the effects of different speeds and total curvatures on the rope transmission effect. The test bed was mainly composed of a speed-regulating motor (rotating speed range 7–14 r/min), a reducer, coupling, a dynamic torque sensor, a lasso transmission system, and load weight. The motor provided different speeds for the transmission experiment, weights were loaded on the load wheel to ensure a constant load, and the monitor recorded the real-time values of the dynamic torque and speed of the lasso input and output. The three-dimensional model diagram and the physical diagram of the lasso transmission characteristic analysis test bed are shown in From the results of the dynamic simulation analysis, the data of the resultant force of steel wire input and output with time were extracted, and Y-axis displacement and speed of steel wire input and output with time were extracted, and Y-axis displacement of steel at the input end is 49.7 mm, and the Y-axis displacement of steel at the output end is 20.6 mm. Steel loses about 39.1 mm in displacement after lasso transmission. The measurement of the element divided into grids shows that each element stretches about 0.025 mm, indicating that the loss of speed and displacement of steel from the input end to the output end can be attributed to the elastic deformation of the elastomer, and the rest reflects the deformation of the lasso.From the simulation results, the data of In this paper, the lasso system test platform we built was used to measure the input and output characteristic data of the lasso. First, under the conditions of a curvature radius of R = 20 mm, a load of 1 kg, and a speed of 6 rpm, and the data of 17 sets of lasso transmission input and output torques were recorded, as shown in From the above experimental data and image analysis, it can be said that there is torque loss in the process of lasso transmission, and with the increase in the lasso curvature radius and transmission speed, the torque loss increases correspondingly. This provides a theoretical reference for us to design a lasso drive robot.The actuator of the hand functional rehabilitation robot is a glove, and the whole flexible actuator looks like a glove. However, flexible electronics products, sensors, and ropes are embedded in the glove to help the hand functional rehabilitation robot control the bending angle of fingers through the ropes. Therefore, the workspace of the rehabilitation robot is consistent with the human hand.For patients with hand dysfunction, grasping ability is one of the abilities they need most, because grasping exercise is the most commonly used action in daily life. Patients’ illness will lead to muscle weakness or stiffness in their hands, which makes it difficult for them to complete this action, resulting in anxiety-related psychological problems, which are unfavorable for the recovery of finger function. The primary function of the hand function robot we designed is to help the patient complete the grasping exercise training. As shown in In addition to grasping experiment, we have also experimented with finger movement training, for which we set up three finger-pointing training modes. More diverse training methods are helpful to the recovery of patients. As shown in When the hand functional rehabilitation robot drives the fingers to train, we collect the pulling force data provided by the hand functional rehabilitation robot for the fingers. As shown in Based on the above contents, we can draw a conclusion. From the experiment of rehabilitation training mode of hand functional rehabilitation robot, we can see that the robot can drive fingers to carry out rehabilitation training and provide patients with the help needed to meet the hand movement requirements in their daily life.In this paper, based on the structural design of hand functional rehabilitation robot driven by a lasso, the influence of the movement speed and total curvature of the lasso on the transmission characteristics of the lasso was studied. By analyzing the transmission characteristics of the lasso, the research provided a compensation basis and guidance for the movement control of the lasso system, reduced the kinematic loss of the lasso in the transmission process, made the lasso transmission system more time-effective, and improved the accuracy of the movement speed and transmission torque. There has been a lack of research on lasso transmission characteristics with different loads, material properties, and friction coefficients in the experiment. Because of the great difference in loads under different working conditions, it is necessary to analyze the specific conditions. Therefore, future research will focus on the influence of different material properties and transmission system friction on transmission characteristics to obtain a more reliable lasso transmission system.This paper focused on the sequelae of hand dysfunction in stroke survivors and designed the structure of a hand rehabilitation robot. The transmission mode is flexible lasso transmission, and the static micro-element analysis of the lasso transmission model was carried out. The results indicate that the input and output characteristics of an arbitrary curve lasso transmission system in space are related to the elastic modulus of the rope, cross-sectional area, and friction coefficient between the lasso contact surfaces and the total curvature.The lasso transmission model was established, and the dynamic simulation analysis and data acquisition for the test were carried out. The dynamic solution results and experimental data indicate that the lasso system can meet the speed and force required by the hand rehabilitation robot as a transmission mechanism, but there were response losses of force, speed, and displacement in the lasso transmission of steel, and the reasons for the loss can be analyzed, which provided a basis for the optimization of the lasso transmission system of the hand functional rehabilitation robot.After the design and manufacture, the hand functional rehabilitation robot was tested by patients, and the rehabilitation experiments of finger grasping training and finger training mode were carried out respectively. The experimental results show that the hand functional rehabilitation robot based on lasso transmission can help fingers to carry out continuous and passive hand functional rehabilitation training.


Immunoblotting and fluorescence image analyses indicated that NPR1-GFP, with and without cTP, had similar molecular weights, suggesting that the chloroplast-targeted NPR1-GFP is likely translocated from the chloroplasts to the nucleus after processing in the stroma. Translation in the chloroplast is essential for nuclear NPR1 accumulation and stress-related expression of nuclear genes. An overexpression of chloroplast-targeted NPR1 enhanced stress tolerance and photosynthetic capacity. In addition, compared to the wild-type lines, several genes encoding retrograde signaling-related proteins were severely impaired in the Arabidopsis npr1-1 mutant, but were enhanced in NPR1 overexpression (NPR1-Ox) transgenic tobacco line. Taken together, chloroplast NPR1 acts as a retrograding signal that enhances the adaptability of plants to adverse environments.Chloroplasts play crucial roles in biotic and abiotic stress responses, regulated by nuclear gene expression through changes in the cellular redox state. Despite lacking the N-terminal chloroplast transit peptide (cTP), nonexpressor of pathogenesis-related genes 1 (NPR1), a redox-sensitive transcriptional coactivator was consistently found in the tobacco chloroplasts. Under salt stress and after exogenous application of H A fundamental question in plant physiology and cell biology is the communication between organelles and the nucleus, which coordinates genetic programs and cellular functions . In all The communication between chloroplasts and the nucleus occurs through retrograde signaling, which involves an ROS-mediated redox cascade. This cascade activates protective mechanisms and modulates hormonal biosynthesis ,4. ProteArabidopsis thaliana [Arabidopsis, our previous work has shown that NPR1, which accumulates in chloroplasts under salt stress, exhibits antioxidant and chaperone activities [The nonexpressor of pathogenesis-related genes 1 (NPR1) acts as a transcriptional coactivator and is a key component of plant immunity through salicylic acid (SA)-mediated defense responses and systemic-acquired resistance (SAR) in thaliana . Pathogethaliana . The degtivities . In thisNicotiana tabacum Wisconsin-38 was used as the wild-type (WT) plant to create the transgenic lines. Arabidopsis thaliana Col-0 WT and npr1-1 mutant  were used in this study. Tobacco and Arabidopsis seeds were cultured on half-strength Murashige and Skoog medium under photoperiod  at 25 °C. The seedlings were transferred to soil as required, and grown for 3–5 weeks under 16L/8D light cycles. Fully mature WT and transgenic plants were subjected to various chemical treatments, including salt stress. The tobacco plants were treated with 200 mM NaCl, while the Arabidopsis plants were treated with 100 mM NaCl.p35S::NPR1-GFP and p35S::NPR1-Ox transgenic plants were previously described [NPR1 was amplified by polymerase chain reaction (PCR) and cloned into a pMBP vector containing the 35S promoter-driven green fluorescent protein (GFP) gene and NOS terminator. The native NPR1 promoter was amplified via PCR from genomic DNA of N. tabacum. To generate pNPR1::NPR1-GFP transgenic plants, a 0.8 kbp fragment of the NPR1 promoter was amplified via PCR and cloned into a promoter-less NPR1-GFP construct, which was obtained by deleting the 35S promoter region from p35S::NPR1-GFP. To generate p35S::cTP-NPR1-GFP transgenic plants, a 237 bp PCR product of the chloroplast transit peptide  from the small subunit of RuBisCo (GenBank AY220079) was cloned between the end of the 35S promoter and the 5′-end of NPR1 in the p35S::NPR1-GFP construct. To generate the p35S::NPR1(∆nls)-GFP construct, a 54 bp nuclear localization sequence (NLS) from nucleotide position 1612 to 1665 in the NPR1 gene was deleted from p35S::NPR1-GFP using the GeneArt Site-directed Mutagenesis PLUS kit . The NPR1-GFP constructs were cloned into pBI121 and utilized for the generation of transgenic plants. The transgenic plants were advanced to the T3 generation. Despite being T3 homozygous, they were used as an experimental plant after confirming kanamycin resistance. escribed . The opeTM cDNA Synthesis Kit  in a 20 μL reaction volume at 42 °C for 30 min, following the instructions of the manufacturer. Gene-specific PCR primers for qPCR were designed based on sequence information from the GenBank database, using stringent criteria , using a TP950 thermal cycler . Real Time System III Software version 6.0  was used, and data was exported to Microsoft Excel. The transcription levels were expressed relative to the reference gene (β-actin) level. Expression analysis was performed using three independent triplicate experiments to determine the mean relative transcript expression levels for each gene in the WT, NPR1 overexpression (NPR1-Ox) transgenic plants, and npr1-1 mutants. The expression ratio of each gene was calculated by comparing the WT versus NPR1-Ox or Col-0 WT versus npr1-1 mutants. The transcription profiles of the genes involved in retrograde communication for chloroplast development and operational signaling in response to salt stress were determined.Total RNA was isolated using TRIzol reagent . For the analysis of relative transcript levels using real-time RT-PCR,3 μg, the total RNA from leaves was reverse-transcribed using the high-fidelity PrimeScriptcriteria : a prediw/v) trypan blue for 1 min. After cooling to room temperature, the plants were submerged in 70% (w/v) chloral hydrate. The stained plants were decolorized overnight and then photographed using a digital camera.To assess plant cell death, tobacco plants treated with salt stress were immersed in a boiling solution containing 10 mL lactic acid, 10 mL glycerol, 10 g phenol, and 0.4% (−2 s−1). A gas stream  was continuously supplied to the photosynthesis unit using a mass flow control system. The temperature inside the growth chamber was maintained at 25 °C, and the humidity was kept at 70 ± 1%.To measure steady-state net photosynthesis, eight-week-old plants were subjected to a gas exchange measuring station  equipped with a built-in light source  and a fluorescence microscope  equipped with a high-resolution CCD camera  were used to visualize the GFP and CFP fluorescence in the cells. Excitation and emission were set at 488 and 520 nm, respectively, for GFP, and at 450 and 470 nm, respectively, for CFP. Red chlorophyll fluorescence was visualized by exciting at 458 nm and a detecting emission at 647–720 nm. The intensity of fluorescence was quantified using the Image J software . Thirty images were acquired per independent experiment, and each experiment was repeated more than three times .The expression of NPR1 tagged with either GFP (NPR1-GFP) or cyan fluorescent protein (CFP) (NPR1-CFP) was investigated in both intact leaves and protoplasts of stable 2− and H2O2 levels were determined using 0.2% nitroblue tetrazolium solution in 50 mM sodium phosphate buffer (pH 7.5) and 1 mg mL−1 diaminobenzidine staining solution in distilled water, respectively.To determine the total amount of ROS, epidermal strips were peeled from tobacco leaves after exposure to salt stress for different time intervals, and immersed in a 50 μM 2′,7′-dichlorofluorescin diacetate (DCFH-DA) solution . ROS levels were observed using a fluorescence microscope  equipped with a CCD camera . The O2, 1 mM NaF, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 80 μM MG115, 80 μM MG132, and a protease inhibitor cocktail tablet . After centrifugation of the lysate at 15,000× g for 15 min, the supernatant was collected as a chloroplast stroma protein fraction.For chloroplast isolation, intact leaves were used and a chloroplast isolation kit  was employed. Intact chloroplasts were collected using a 40/80% Percoll gradient. The chloroplasts were suspended in a chloroplast lysis buffer containing 0.5 mM HEPES–KOH (pH 7.5), 2 mM MgClg for 10 min, using 2.3 M sucrose. Nuclear proteins were extracted from the pellet using the Working Extraction Buffer, and then centrifuged to obtain a pure supernatant as the soluble nuclear protein. Nuclear proteins were extracted using a plant nuclei isolation/extraction kit , following the manufacturer’s instructions. First, intact leaves were incubated with the nuclei isolation buffer, and then the resulting cell lysate was filtered using a mesh. A pure preparation of the nuclei was obtained via centrifugation at 12,000× g for 15 min, and the mitochondrial fraction was removed at 20,000× g for 15 min. The final supernatant was obtained as the crude cytosolic fraction. To extract cytosolic proteins from tobacco plants, frozen leaves were homogenized with a homogenization buffer containing 50 mM HEPES–KOH (pH 7.5), 250 mM sorbitol, 50 mM potassium acetate, 2 mM magnesium acetate, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 80 μM MG115, 80 μM MG132, and a protease inhibitor cocktail tablet. The crude nuclear and chloroplast fractions were removed via centrifugation at 1000× TM FEMTO Chemiluminescent Sustrate  on X-ray film. All experiments were repeated at least three times, and the representative data are presented. The intensity of the protein bands was quantified using the Image J software .The proteins were separated using 4–12% or 3–8% Bis-Tris Plus gels, and transferred onto iBlot 2 NC Regular Stacks . Ponceau S staining was performed to ensure equal loading. The blots were blocked using iBind Cards (Novex), according to the manufacturer’s instructions. NPR1-GFP proteins were detected by incubating the blots with a mouse monoclonal anti-GFP antibody . Anti-Toc75  antibodies were used as the marker for the chloroplast outer membrane, while anti-RbcL (Agrisera) or anti-histon3 (Agrisera) primary antibodies were used as markers for the chloroplast stroma and the nucleus, respectively. The bands were visualized using West Glow2, 20 mM MES, 0.1% BSA, 1% cellulase R-10, and 0.25% macerozyme R-10. After 30 min stabilization on ice, 106 mL−1 protoplasts were used for further transient transformation. Protoplasts (300 µL) in W5 buffer were gently pipetted into a pre-chilled 0.4 cm electroporation cuvette, and 50 µg of the plasmid construct in 10 µL TE buffer was added. Electroporation was performed using the Gene Pulser Xcell System  at 160 V/960 μF (voltage/capacitance), according to the manufacturer’s instructions. The protoplasts were then transferred to a conical tube containing 500 μL K3 medium ) using a glass Pasteur pipette, and observed using confocal microscopes .Mesophyll protoplasts were isolated from both WT and transgenic tobacco leaves using enzyme solution containing 0.5 M mannitol, 1 mM CaClp-values and statistical significance. At each time point, statistically significant differences between wild-type and transgenic plant lines or each treatment group, and the corresponding control group are indicated by * p < 0.05 or ** p < 0.01. The experiments were repeated at least three times with three replicates each time. The data from a representative experiment are presented after confirming that similar results were obtained. Statistical analyses were performed using a paired two-sample Student’s t-test, and the data are presented as the mean ± standard deviation (SD). The figure legends provide detailed statistical parameters, including NPR1 and GFP driven by the NPR1 promoter (pNPR1::NPR1-GFP) [pNPR1::NPR1-GFP transgenic tobacco plants after salt treatment  . AlthougPR1-GFP) , it was reatment a. The amreatment b.cTP sequence (79 amino acid residues) of tobacco RbcS (GenBank accession number AY220079) to the 5′ end of NPR1-GFP to generate p35S::cTP-NPR1-GFP transgenic tobacco lines  (We attached the 9.8 kDa co lines c. We useco lines d. Notabl2.3-kDa) d. The in2O2 and 1-amino-1-cyclopropane carboxylic acid (ACC), an ethylene precursor, using confocal laser scanning microscopy . Fluorescence of GFP alone was consistently detected in the cytoplasm and nucleus of 35S::GFP transgenic plants, and it did not merge with red autofluorescence from chloroplasts, indicating that GFP rarely entered the chloroplasts in tobacco leaves  in the chloroplast protein fractions from eatments . The amoeatments g.2O2-treated leaves. The fluorescent NPR1 signals in chloroplasts were significantly enhanced after both treatments  to salt stress or exogenous ACC or H2O2 application, the GFP fluorescence levels of nuclear NPR1 from chloroplast-targeted NPR1-GFP appeared distinctly in mesophyll cells  genes, including NtACS4 in the early phase at 1–3 h and NtACS1 in the late phase at 48–72 h after pathogen infection [NtACS4- and NtACS1-silenced transgenic plants  via RNA interference-mediated repression [NtACS4i and NtACS1i transgenic plants , an NADPH oxidase inhibitor , was adm nucleus b, indicapNPR1::NPR1-GFP) (pNPR1:: NPR1(∆5′35aa)-GFP), exhibited strong fluorescence in the chloroplasts  . Additioroplasts . HoweverpNPR1::NPR1(Δnls)-GFP, was transiently co-expressed with p35S::NPR1-CFP in mesophyll protoplasts isolated from WT tobacco (pNPR1::NPR1(Δnls)–GFP transient expression were much higher than those in the p35S::NPR1-CFP transient protoplasts. NPR1(Δnls)-GFP fluorescence intensity in the chloroplasts continuously increased until 36 h. These results suggest that stress-induced NPR1 export from chloroplasts requires an NLS.NPR1 is controlled by the NLS at the C-terminus, and functions as a transcriptional coactivator in the nucleus . We dele tobacco . NPR1-CFpNPR1::NPR1(Δnls)-GFP and p35S::NPR1-CFP, were transiently co-expressed in mesophyll protoplasts isolated from WT tobacco leaves  fraction throughout the experiment, NPR1 levels in the sediment fraction without cytosol increased 3–6 h after exposure to stress. Notably, the levels of the NPR1 dimer and low-molecular-weight proteins increased in this fraction d. These The study further investigated the movement of vesicles containing NPR1-GFP molecules around intact chloroplasts and in the perinuclear region of the protoplasts in salt-stressed mesophyll cells e, indicapNPR1::NPR1-GFP transformants using fluorescence microscopy . Surprisingly, the NPR1 vesicles approaching the nucleus were observed to fuse with the nucleus, leading to a rapid disappearance of NPR1-GFP fluorescence, indicating that the NPR1 protein was imported into the nucleus  through the mechanism of liquid–liquid phase separation  and posipNPR1::NPR1-GFP and p35S::cTP-NPR1-GFP transformants under salt stress -dependent pathways ,24. Sevesis NPR1 .http://rarge-v2.psc.riken.jp/chloroplast/, accessed on 2 February 2023), which provides information on Arabidopsis mutants of nuclear-encoded chloroplast proteins [npr3 mutants exhibit a pale green color phenotype. Phylogenetic analysis revealed that NPR3 and NPR4 are NPR1 paralogs that act as SA receptors [Although NtNPR1 lacks cTP and signal peptides, NPR1-GFP fluorescence, but not that of GFP alone, was observed in the chloroplasts, as evidenced by the yellow signal in the merged image with chloroplast autofluorescence . NPR3  under salt stress, as evidenced by chlorophyll fluorescence measured using the PAM 2000 photosynthesis yield analyzer   a. Constihout cTP . Howeverhaperone .2+ and ROS in guard cell and their potential co-amplification effects [Guard cells initiate a rapid physical reaction used by land plants to alter their interactions with the environment. Several studies have demonstrated the interplay between Ca effects . At the 2·− and H2O2, respectively. Leaves with increased NPR1 expression showed decreased ROS accumulation in the chloroplast compared to controls under salt stress  under salt stress. Real-time quantitative RT-PCR (qRT-PCR) was performed on genes encoding RuBisCo, core complex, and PS I and II antenna proteins. At 3 and 12 h after salt stress, transcript levels of almost all genes were higher in transgenic plants than those in WT plants. In particular, compared to WT, the transcript levels of all tested nuclear-encoded genes in p35S::cTP-NPR1-GFP were significantly higher 12 h after salt stress.In ynthesis e. To eluPR) gene [PR gene transcription under salt stress, GFP transcript levels in p35S::cTP-NPR1-GFP and p35S::NPR1-GFP transgenic plants with or without Lin treatment were examined using qRT-PCR. GFP transcription in both transformants, with or without Lin treatment, did not change, implying that 35S promoter was not affected by NPR1 localization  gene . To invelization a. NPR1 transcript levels increased in both transformants under salt stress only in the absence of Lin, which may be due to the stress-induced NPR1 transcript itself , EXECUTER1 (EX1), EX2, GENOMES UNCOUPLED1 (GUN1), and the single-stranded DNA-binding protein, WHIRLY 1, have been implicated in chloroplast retrograde signaling ,40,41. Mn at 1 h . In partlymerase .NPR1-Ox plants than those in WT plants , CIA2-like (CIL), WHIRLY, and plant homeodomain (PHD), exhibit dual nuclear–plastid localization, suggesting their possible involvement in retrograde signaling . In ArabNonetheless, the exact regulatory mechanism underlying the mediation of plastid-to-nucleus retrograde signaling remains unknown. NPR1 and WHIRLY1 exhibit similarities in that both proteins display dual functions and localization in the chloroplasts and nucleus. The possibility that NPR1 and WHIRL1 exist as two isoforms cannot be completely ruled out, given their compartment-specific molecular functions .Our study aimed to investigate the functional significance of the chloroplast NPR1 and its role in retrograde signaling, which plays a critical role in regulating proper allocation under stress conditions. We explored the intracellular translocation of NPR1 as a potential physiological strategy for functional switching between dual localizations. Retrograde NPR1 signaling, which is triggered by redox status or stress-induced components in chloroplasts, is a crucial environmental stress regulatory mechanism in plants. Chloroplast-to-nucleus retrograde signaling is essential for adaptable plant growth and development. Our findings suggest that the chloroplast NPR1 may serve as a retrograde communicator for the protective machinery from the chloroplasts to the nucleus in a redox-mediated manner, particularly in photosynthetically active leaf cells under adverse environmental conditions.


Assisted reproduction technology (ART) has advanced significantly, raising concerns regarding its impact on the secondary sex ratio (SSR), which is the sex ratio at birth in offspring. This study aimed to explore factors affecting SSR in singletons, singletons from twin gestation, and twins from twin gestation within the context of ART.A retrospective analysis was conducted on data from 8335 births involving 6,223 couples undergoing ART. Binary logistic regression assessed relationships between parental and embryonic factors and SSR in singletons and singletons from twin gestation. Multinomial logistic regression models were utilized to identify factors influencing SSR in twins from twin gestation.Secondary infertility , advanced paternal age , and blastocyst embryo transfer  were associated with an increased SSR, while frozen embryo transfer (FET) showed a negative association with SSR  in singletons. A longer duration of gonadotropin (Gn) usage reduced SSR in singletons  and singletons from twin gestation . In singletons from twin gestation, male-induced infertility  and higher Gn dosage  were significantly associated with an increased SSR. Women aged > 35 years and intracytoplasmic sperm injection (ICSI) were associated with lower SSR . In twins from twin gestation, paternal age exceeded maternal age  and higher Gn dosage  were associated with a higher proportion of male twins. Cleavage stage transfer  resulted in a higher percentage of boy-girl twins compared to blastocyst transfer.This study demonstrates the complex interplay of various factors in determining the SSR in ART, highlighting the importance of considering infertility type, paternal age, fertilization method, embryo transfer stage, and Gn use duration when assessing SSR. Nevertheless, further research with a large sample size is necessary to confirm and expand upon the findings of this study.The online version contains supplementary material available at 10.1186/s12978-023-01723-8. Assisted reproductive technology (ART) has evolved from a marvel innovation to a conventional medical treatment for infertility. Since the first successful in vitro fertilization (IVF) treatment in 1978, it was estimated that at least 5 million infants worldwide had been born by ART . IntracyThe human sex ratio, critical for gender balance, consists of primary sex ratio (PSR) and secondary sex ratio (SSR). The PSR can deviate from 1:1 in theory, sometimes reaching as high as 170 males to 100 female . HoweverEven though numerous studies have explored potential factors of SSR in the ART population and have observed skewed gender distribution in the ART population compared to naturally conceived population, conclusive findings about the influence of ART on SSR remain elusive. Additionally, few studies have investigated SSR in the context of singletons and multiple births based on the premise that all mothers received double embryo transfer. Furthermore, there has been no investigation into the SSR of singletons born from twin pregnancies. Thus, this retrospective study, including 6223 cycles and 8335 babies, was conducted to investigate potential factors influencing the SSR of singletons, singletons from twin gestation, and twins from twin gestation. Singletons denote infants born from a single pregnancy where the mother carried only one child. Singletons from twin gestation refer to cases in which mothers with twin pregnancies successfully delivered only one child, while twins from twin gestation pertain to situations in which mothers with twin pregnancies successfully delivered two children.A total of 27,202 couples who underwent assisted reproductive technology (ART) at the Centre for Reproductive Medicine of the Third Affiliated Hospital of Guangzhou Medical University, China, between January 2010 and January 2015, were recruited in the study. The inclusion criteria were as follows: (1) couples who received ART treatment at the Third Affiliated Hospital of Guangzhou Medical University; (2) ART cycles from double embryo transfer; (3) singleton pregnancies and twin pregnancies; (4) live births of singleton or twins; (5) subjects who completed the study follow-up. Moreover, for singleton pregnancies, only singleton births were included. The exclusion criteria are detailed in Fig. In this research, various variables were examined, including the type of infertility, infertility factor, maternal age group, paternal age group, age disparities between partners, fertilization method, maternal body mass index (BMI), embryo transfer stage, dosage of gonadotropin (Gn), and days of Gn use. These variables were extracted from the hospital electronic medical records system of the Third Affiliated Hospital of Guangzhou Medical University. Besides, data regarding the gender of newborns was collected from the hospital’s electronic medical records system and telephonic follow-up.The study received ethical approval from the ethics committee of the third Affiliated Hospital of Guangzhou Medical University. Informed consent was provided by all study participants.2, 18.5 < BMI < 23.9 kg/m2, and BMI ≥ 24 kg/m2, according to the Chinese criteria[The secondary sex ratio (SSR) was defined as the ratio of male to female births in this study. Maternal age was classified into either < 35 or ≥ 35 years . Paterna criteria. Age dif2 test or Fisher’s exact test would be used to compare differences between groups in categorical variables. Logistic regression models, adjusted for selected characteristics, were used to estimate the associations between parental and embryonic factors with SSR. Odds ratios (ORs) with a 95% confidence interval (CI) were calculated. All statistical analyses were conducted using SPSS software . A two-tailed P value < 0.05 was considered statistically significant.Shapiro–Wilk test was used to assess the normal distribution of continuous variables. For normally distributed continuous variables, t-tests were used, and the results were expressed as means with standard deviations. In cases where continuous variables did not meet a normal distribution, the Mann–Whitney U test was used, and continuous variables were presented as medians . Categorical variables were presented as numbers and proportions. Based on the expectation value, the Pearson χIn this study, a total of 8335 babies were included from 6223 cycles. The secondary sex ratio (SSR) of singletons, singletons from twin gestation, and twins from twin gestation were 124.1: 100, 134.2: 100 and 109.7: 100, respectively. For singletons, a significantly higher SSR was observed in births where parents had experienced secondary infertility compared to those with primary infertility . Additionally, paternal age ≥ 37 years was associated with a higher likelihood of live male births compared to other paternal age groups . The median gonadotropin (Gn) usage was 2100 IU/L (25th-75th percentile: 1500–2775), and the median days of Gn use were 12 days (25th-75th percentile: 11–13). In singletons from twin gestation, the median Gn was 2025 IU/L (25th-75th percentile: 1462.4–2850), and the median days of Gn use were 12 days (25th-75th percentile: 11–13). For twins from twin gestation, there was a significantly higher proportion of males with blastocyst stage embryo transfer compared to cleavage stage transfers . In addition, the median Gn usage for this group was 2062.5 IU/L (25th-75th percentile: 1575–2700), with a median duration of Gn use of 12 days (25th-75th percentile: 11–13)  is an important implication for population health and fertility. In this hospital-based retrospective study, we investigated the factors related to the sex ratio of newborns born to mothers who underwent assisted reproductive technology (ART) and achieved singleton or twin deliveries. Our findings shed light on various factors that influence SSR in singletons and twins, underscoring the impact of a specific ART protocol on sex ration of newborns. These factors included infertility type, paternal age group, fertilization method, embryo transfer stage, course of gonadotropin (Gn), infertility factor, maternal age group, and age difference.In terms of the infertility type, our findings suggest that, in singleton pregnancies, secondary infertility is more prone to having male offspring through ART compared to primary infertility. Primary infertility typically affects young patients who have never experienced a pregnancy. The causes of infertility in this group are often complex and diverse, making them more susceptible to sperm-ovum fertilization failure and low fertilization rates during their first in vitro fertilization (IVF)-assisted pregnancy . Our resIn addition, the characteristics of the ART treatment can alter the proportion of male infants in our research. Previous studies show that the use of BT is associated with an increase in SSR, while intracytoplasmic sperm injection (ICSI) is related to a reduction in SSR . HoweverOur study revealed that prolonged gonadotropin use was linked to a higher proportion of female infants in both singletons and singletons from twin gestation. The underlying reasons for the discrepancy remain unclear. However, it is unlikely that prolonged gonadotropin use would cause sex-selective abortions by adversely affecting male-fertilized eggs . Thus, fConsistent with existing literature, blastocyst embryos were found to be correlated with a higher proportion of male offspring in contrast with cleavage stage embryos. Male embryos tend to develop faster than female embryos do in IVF or ICSI, and as a result, operators are more inclined to select embryos with fast embryonic development and favorable morphology for transfer , 30. MorFor singletons from twin gestation, except for the same impact of gonadotropin days on the sex ratio of the newborn, there are additional factors on the sex ratio of the newborn. Our research aligns with several studies , 34 thatRegarding infertility factors, our study identified a higher percentage of male babies born to ART patients solely attributed to male factors compared to those with other infertility factors. However, Dean J H et al.  claimed For twins from twin gestation, our results suggest that older fathers tend to have a higher chance of having twin boys. Nevertheless, further data is needed to support our findings. Several factors may be associated with SSR in this context. Firstly, older fathers are more likely to have boys. A cross-sectional study conducted in China supported this observation, revealing that the SSR in the oldest group of fathers was higher than that of the youngest age group, which was consistent with our results . HoweverFurthermore, this study is the first time to suggest that cleavage stage transfer may have a positive impact on the birth of boy-girl twins, an interesting finding as this phenomenon has not been previously reported in the literature. However, this finding might be due to chance, given the relatively small sample size of the study. A larger sample size and more rigorous multi-center collaboration are needed to further investigate the reasons for this result.At present, many parents undergoing ART treatments hold preferences for gender of their offspring. However, Chinese law prohibits gender selection through ART. Nonetheless, clinicians can provide patients with an expected probability based on this study. Moreover, it is essential to recognize that the global fertility rate is continuously decreasing, and an increasing number of infertile individuals are opting for ART treatment. Consequently, the impact of the SSR on population gender balance will become increasingly pronounced. Clinicians can play a key role in mitigating any gender imbalances by considering to the potential influencing factors discussed in this study.Three models were constructed to explore the potential association between SSR and various factors related to both parents and embryos. Several factors were found that had a significant effect on SSR. In singletons, secondary infertility, paternal age ≥ 37 years and blastocyst transfer were linked with a higher likelihood of male births compared to primary infertility, paternal age < 37 years, and cleavage stage transfer, respectively, Conversely, FET and increased duration of Gn use were linked to a decreased SSR. For singletons from twin gestation, male-induced infertility, and a higher dosage of Gn were positively correlated with SSR, while maternal age > 35 years, ICSI, and the duration of Gn use were associated with a decrease of SSR compared to maternal age ≤ 35 years and IVF, respectively. In twins from twin gestation, groups where paternal age exceeded maternal age were more likely to have male twins than to have boy-girl twins compared to groups with no age difference. Cleavage stage transfer had a higher probability of resulting in boy-girl twins compared to blastocyst transfer. While the study acknowledges the need for more extensive sample size for future research, the impacts of infertility factors, age difference and duration of Gn use on SSR remain important considerations. Understanding which factors within ART process can affect SSR holds significance for maintaining gender balance in the population.This study has several limitations. Firstly, our samples were derived from a single reproductive center, and our sample size is relatively small. Thus, to validate present findings, it’s necessary to expand the sample size in future analyses. Secondly, due to the retrospective nature of this analysis, there are inherent limitations concerning the integrity and homogeneity of the data. Lastly, the data was extracted based on the hospital's system, without the use of a detailed questionnaire survey. This method may lead to the missing of certain important information. In the future, we plan to perform multicenter research to satisfy sample diversity and meet sample capacity requirements, enhancing the richness and credibility of the research evidence. Besides, we intend to conduct laboratory research to figure out whether infertility factors, age difference and duration of Gn use indeed impact SSR and to explore the relevant mechanisms. With the improvement of our research, we will perform a more comprehensive analysis of the factors influencing SSR.Additional file 1: Table S1. Univariate logistic regression analyses of different variables with SSR in Singletons.Additional file 2: Table S2. Univariate logistic regression analyses of different variables with SSR in Singletons from twin gestation.Additional file 3: Table S3. Univariate logistic regression analyses of different variables with SSR in twins from twin gestation.


Subsequently, key transcription factors were identified by a combined analysis using transcriptome and in silico datasets. Gene ontology: bioprocess (GO: BP) analysis using differentially expressed genes (DEGs) revealed that immune response-related genes were significantly enriched in AR compared with HC. Among them, IL1RL1, CD274, and CD44 were significantly higher in AR patients. We also identified key transcription factors between HC and AR using the in silico dataset and found that AR samples frequently express KLF transcription factor 4 (KLF4), which regulates immune response-related genes including IL1RL1, CD274, and CD44 in human nasal epithelial cells. Our integrative analysis of transcriptomic regulation provides new insights into AR, which may help in developing precision management for patients with AR.This study is aimed at investigating the potential molecular features of allergic rhinitis (AR) and identifying gene signatures and related transcription factors using transcriptome analysis and in silico datasets. Transcriptome profiles were obtained using three independent cohorts  comprising healthy controls (HC) and patients with AR. The pooled dataset ( Rhinitis is a highly prevalent multifactorial disease with heterogeneous conditions, consisting of allergic rhinitis (AR), nonallergic rhinitis, and infectious rhinitis. AR is characterized by inflammation of the nasal mucosa ; howeverVEGFB, 322A>C) and/or Integrin Subunit Alpha 2  mainly in AR patients [POSTN) gene was enhanced in AR compared to non-AR [To date, next-generation sequencing and bioinformatics analysis have been used to identify genetic and transcriptomic alterations involved in the development and progression of the disease and to classify patients for therapeutic prediction , 7. Prevpatients . In addio non-AR . HoweverKFL4) as a potential candidate associated with immune- and cytokine-related AR subtypes. KLF4 can act as a transcription factor regulating the expression of various target genes and is involved in the development of several epithelial tissues, such as skin and lungs [KLF4 is a critical regulator of monocyte differentiation [β , a proinflammatory cytokine induced by monocyte and dendritic cells, can enhance KLF4 expression, resulting in upregulation of immune-related genes such as IL1RL1, CD274, and CD44. Our findings provide new insights into potential biomarkers for AR diagnosis and potential targeted therapy.In this study, to determine the AR-related molecular signatures and genes, we performed transcriptome analysis of a pooled dataset consisting of 43 healthy controls (HC) and 39 AR samples. Our analysis exhibited an altered expression pattern of immune response genes in AR. In addition, using consensus clustering, we identified two AR subtypes (AR1 and AR2) and identified KLF transcription factor 4 . Raw data were preprocessed by log2 transformation and quantile normalization, and the batch effects were corrected using empirical Bayes methods implemented in a sva R package “Combat” [Public datasets GSE101720, GSE19190, and GSE46171 were obtained from the NCBI Gene Expression Omnibus database (“Combat” . All dathttp://geneontology.org/) and REAC (http://www.reactome.org/) databases. Coordinated gene regulation was identified using GSEA  [Gene set analyses were performed using the gProfileR (0.7.0) package in R software and data from gene ontology: biological processes  . A genetion 3.0) .https://maayanlab.cloud/chea3/). ChEA3 was performed as previously described [http://gene-regulation.com/pub/programs/alibaba2/index.html) [Transcription factors were predicted using ChIP-X Enrichment Analysis version 3  .2. The cells were treated with inflammatory cytokines such as IL-1β for 24 h.The human nasal epithelial cell line (HNEpC) purchased from Promocell  was mainly grown in DetachKit (Promocell) at 37°C in a humidified atmosphere with 5% COKLF4 coding gene sequences into pcDNA3.1 (-) , the KLF4 gene was amplified using total RNA extracted from the HEK293 cell line. PCR was performed using specific primers containing a 5′-extension and XbaI  and NotI (NEB) restriction sites with CloneAmp HiFi PCR Premix . Subsequently, the amplicons were digested with XbaI and NotI and cloned into the pcDNA3.1 (-) vector using an In-Fusion® cloning system according to the manufacturer's recommendations.For the cloning of tagged KLF4 coding sequence using Lipofectamine® 3000  according to the manufacturer's recommendations. Overexpression effects were validated by quantitative real-time PCR analysis using the iQ™ SYBR Supermix . The primers used in this study are summarized in Supplementary Table HNEpC cells were transfected with pcDNA3.1 (-) containing the tagged t-test, permutation t-test, or one-way ANOVA test.Statistical analysis was performed using R software . Statistical analyses were performed using Student's n = 82). Next, we established the pooled transcriptome data using GEO datasets (n = 237) or “down” (n = 197)  > 0.3; P < 0.01, maximum P value with a minimum set size > 10). Gene set enrichment analysis revealed the enriched expression of immune- and cytokine-related genes in AR. We found that the tissue development-related signatures were frequently contained in the “down” genes , including 43 cases of HC and 39 cases of AR only composed of the nasal mucosa and nasal airway epithelium using GEO , CD274 (FC = 0.90), and CD44 (FC = 0.40) were the most significantly altered genes between the HC and AR groups . We analyzed the proportion of ambiguous clustering (PAC) score using the CDF curve and found that the optimal K was 2 with the lowest PAC value  and AR2 (n = 18). We found that most of the immune- and cytokine-related signatures were significantly altered between AR1 and AR2  from healthy controls to AR1 and then AR2  using ChEA3  plays potential driver roles in immune response, we investigated the underlying TFs and the putative regulatory elements of immune-related genes. We identified 1,632 TFs predicted to bind to the promoter region of immune response-related genes   , top. KLrt site) , bottom.KLF4 expression can affect the immune response-related genes, we established a KLF4 overexpression system in a human nasal epithelial cell line (HNEpC) that has lower expression levels of KLF4  which are associated with the KLF4 transcription factor using transcriptome analysis based on an in silico database.In this study, we established a pooled transcriptome data using three independent cohorts  are associated with AR risk [CD274 and CD44 have emerged as critical immune regulators associated with T cell receptor blockade and the development of airway inflammation [IL1RL1, CD274, and CD44 compared to AR1 and HC. This finding implies that AR is divided by immune traits, although further elucidation is required.As the epithelium of the AR is a region where the population of allergen-presenting cells is concentrated, the AR immune system has been associated with the expression of cytokines . AR indier genes , 28. A rto IL-33 . Moreove AR risk , 30, 31.ammation , 32. In KLF4 transcription factor was significantly associated with immune response genes, and it was highly expressed in AR2. As KLF4 is known to be an immune marker for dendritic cell differentiation and essential for inflammatory monocyte differentiation [IL1RL1, CD274, and CD44 are strongly related to KLF4. However, it should be noted that we did not evaluate the validity of our results using an independent rhinitis cohort, which requires further extended studies in the near future. In addition, the various multifaced functions of the KLF4 have been known previously, including cell proliferation, differentiation, development, and transcription [Via in silico analysis, we found that the ntiation , 34, ourcription , which sKLF4 is an independent predictor of AR. KLF4 expression is associated with IL-1β, a proinflammatory cytokine induced by monocyte and dendritic cells, and may contribute to the expression of immune response-related gene transcription. Thus, targeting KLF4 could be a promising therapeutic strategy for AR patients.In conclusion, we suggest that the expression of