The present study identified the boundary condition that determines when disfluent texts benefit learning. We used eye-tracking to examine the joint influence of text legibility (fluent vs. disfluent) and signaling  on multimedia learning. The results revealed that both disfluent text and signaling led to better transfer test performance, and there was also an interaction between them. Specifically, the disfluent text led to better learning outcomes with or without signaling; however, in the fluent text condition, only signaling facilitated learning. Eye movement analyses indicated that signaling guided learners to pay more attention to important content in the learning materials. The current results suggest that signaling can enhance individuals’ perceived fluency or familiarity to the material and guide the attention during multimedia learning, and the positive impact of disfluency on multimedia learning seems to be more stable and ubiquitous. We discuss these under the framework of disfluency effect and attention-guiding effect.Do students learn better with texts that are  Multimedia learning refers to the presentation of words and pictures that are intended to foster learning . The worPerceptual fluency is generally defined as the subjective experience of ease or speed in processing perceptual information. It is usually regarded as a metacognitive experience . StudiesCognitive load theory argues that the extra cognitive burden stimulated by perceptual disfluency hinders learning. According to the traditional conception of CLT , three sContrary to the theory of impediments to disfluency, it is believed that the extra cognitive burden stimulated by disfluency promotes learning. Disfluency theory is based on dual-system processing theory. Dual-system processing theory argues that humans have two independent processing systems: System 1, which leads to quick, effortless, more associative and intuitive processing, and System 2, which leads to slow and effortful, more analytic and deliberate processing . Alter eDesirable difficulty theory argues that memory in the learning process includes both storage and retrieval strength. Storage strength refers to the depth of memory of learning content, and retrieval strength references difficulties in extracting content from memory . AccordiMetacognition is the process of monitoring cognitive activities. The judgment of learning (JOL) is an important form of metacognition monitoring and refers to judgments of the recall possibilities for memory tests after the study phase . JOL sigSignaling is an instructional design method that uses non-content information  in multimedia learning to attract learners’ attention, guiding them to focus on key information and promoting learning effects , 2010. TIn multimedia learning, fluency coding can cause learners to overestimate their mastery of learning materials, resulting in metacognitive bias. Disfluent manipulation can reduce learners’ metacognitive bias, stimulate System 2 to initiate analytical thinking, and encourage learners to invest more mental effort in processing learning materials, thus improving the learning effect. However, there are drawbacks to the facilitation and hindrance theories of disfluency. For example, some scholars have found that there is no difference between fluent and disfluent materials . HoweverSD=1.88), and there were 47 females and 17 males. Sixteen participants were randomly assigned to each of the four groups derived from a 2×2 between-subjects design with signaling  and fluency (disfluent vs. fluent) as factors. G*Power 3.1 was used to calculate the numbers of participants with a large effect size of f=0.40 with power set at 0.80 and alpha set at 0.05 . The mean age was 20.61years  to 7 (disfluent). Mental effort was assessed by “how much mental effort did you invest?” , cognitiIt comprises one question : “PleaseIt consists of the following four questions: “How can the intensity of lightning be reduced?”; “Suppose you see clouds in the sky and there is no lightning. Why”; “What does air temperature have to do with lightning?”; and “What causes lightning?” According to The material concerned how lightning develops was adapted from Eye movements were recorded with an EyeLink 1000 Plus Desktop Eye Tracker  with a 1,000Hz sample rate. The experimental materials were presented on a 21-inch monitor with a resolution of 1,280×1,024. The distance between each participant’s eyes and the screen was approximately 65cm. A chin rest was used to minimize head movement.At first, participants were asked to complete a test about knowledge of meteorology. Then, the participants were told that they will learn an article about how lightning is formed, which consists of five pages. Each page will last 90s and will automatically turn to the next page. They cannot return to read it when time is over. Therefore, they should focus on reading and try their best to understand every step of lightning formation. After the learning phase, students were asked to complete the subjective rating questionnaire to measure their perceptual fluency, mental effort, cognitive load, and the JOL they experienced during learning. Thereafter, they have 15min to complete tests of retention (6min) and transfer (9min).We also used eye-tracking system to record eye movement data during the reading process. So, after the prior knowledge test, participants were told that they will attend an eye movement calibration test. During calibration, they will be asked to stare at the red dot on the screen until it disappears and to keep their head as still as they can throughout the experiment.F=161.378, p<0.001, ηp2=0.729], and the clarity score of the fluent group participants  was significantly higher than that of the disfluent group . Because the subjective rating scale for disfluency used a seven-point scoring mechanism (1=fluency and 7=disfluency), manipulation of the disfluent materials met the definition of disfluent operability and is consistent with the classification of fluency.Because previous studies have demonstrated that the disfluency effect and signaling effect were stronger for low-experience learners than for high-experience learners, we included only low-experience students in our study. On the pre-test knowledge questionnaire, the scale we used to measure participants’ prior knowledge is a five-point Likert type (from 1 to 5). We set the screening criteria as mean rating across seven items lower than 3, indicating that they have a low-experience in meteorology . ParticiF=0.167, p>0.05], nor of fluency . But a significant interaction was observed . A simple effect analysis revealed that under the condition of signaling, the fluency group’s mental effort was significantly higher than that of the disfluency group ; in the case of non-signaling, there was no significant difference between the fluency group and the disfluency group . The average mental effort of the four groups was greater than seven points, indicating that the mental effort of the four groups was high. In terms of the materials’ difficulty, there was neither a main effect of signaling , nor a main effect for fluency , nor an interaction . Concerning JOL, the main effect of signaling  and fluency , and the interactions of signaling and fluency  were all non-significant. The average value of the task difficulty for the four groups was between 5 and 6, indicating that the learning content was moderately difficult.Two-factorial ANOVAs with signaling and fluency as independent variables and scores for mental effort, materials’ difficulty, JOL, retention, and transfer performance as the dependent variable were conducted see . On the F=2.360, p=0.130], nor a main effect for fluency , nor an interaction . Concerning transfer, a 2×2 ANOVA revealed a main effect of signaling , with learners studying with signaling outperforming those with non-signaling. There was a main effect for fluency , with learners studying with disfluency outperforming those with fluency. There also revealed an interaction between signaling and fluency , and a simple effect analysis revealed that for the fluent condition, the transfer score of the signaling group was significantly higher than that of the non-signaling group ; for the disfluent condition, there was no significant difference in transfer performance . The interaction between signaling and fluency is shown in Concerning retention, there was neither a main effect for signaling , but there is a significant difference on transfer score , and the bias index of the fluent condition  was significantly higher than that of the disfluent condition .F=1.040, p>0.05], nor a main effect for fluency , nor an interaction . With regard to the proportion of the count of fixations on the picture area, a 2×2 ANOVA showed the main effect of signaling  and fluency , and the interaction of signaling and fluency  were all non-significant. With regard to the number of transitions between the text and picture area of interest, a 2×2 ANOVA showed neither a main effect for signaling , nor a main effect for fluency , nor an interaction .To explore whether an improvement in learners’ academic performance could be caused by processing more pictures, the interest areas of particular pictures were analyzed, and the results are shown in F=5.674, p=0.020, ηp2=0.086], with learners studying the signaling gaze longer than did the non-signaling group, but there was no main effect for fluency , and no interaction . Concerning count of fixations, results revealed a significant main effect of signaling , with learners studying the signaling gaze longer than did the non-signaling group. Moreover, there was no main effect of fluency , and no interaction . Concerning the number of transitions between signaling and non-signaling areas of interest, there was a significant main effect of signaling , with learners studying under the signaling condition having more transitions between text and picture than did the non-signaling group. But the main effect of fluency  and the interaction of signaling and fluency  were all non-significant.To explore the influence mechanism of signaling on learning results, this study considered the area of the signaling in the material as the area of interest and compared the differences in eye movement among different experimental groups, the results are shown in This research investigated the impact of perceptual fluency and signaling on the processing of multimedia learning graphics and explored the role of color signaling in multimedia learning. The results show that the transfer scores for the disfluency group were significantly higher than for the fluency group, and the scores for the signaling group were significantly higher than those for the non-signaling group. Under fluent conditions, the transfer performance of the signaling group was significantly higher than that of the non-signaling group, while under disfluent conditions, there was no difference.In multimedia learning, retention tests mainly determine learners’ memorization abilities as reflected by the number of items learned or how much each learner memorizes. The retention test only requested participants recall what they had learned, which may have been affected by each learner’s working memory capacity. In multimedia learning, the transfer score reflects the learner’s ability to understand the learning materials and use them in new situations and indicates the quality of the learning. This study found that the transfer score of the disfluency group was significantly higher than that of the fluency group, which is consistent with previous research  and indiThere was no significant difference in retention performance between the signaling and non-signaling groups, but there was a significant difference in the transfer performance. The transfer performance of the signaling group was significantly higher than that of the non-signaling group, indicating that color signaling had an impact on deeper processing, and reasonable inclusion of signaling in learning materials can enhance the perceptual salience of key information. In other words, color signaling has an attention-guiding effect, and it can maintain learner’s attention on that information, causing them to invest more attention resources on it and promoting learning effects; this is consistent with previous studies . In termThis research also determined that the interaction between signaling and fluency was significant in terms of transfer scores, and color signaling being added to a disfluent text will not significantly increase or decrease test scores. This study also found that adding color signaling to a fluent text would improve test scores. Disfluency theory argues that disfluent texts manipulate the surface complexity of the material. When the learning material becomes blurred, the learner’s “perceived difficulty” increases, which reduces the cognitive bias of learning caused by fluency and thereby alleviates overconfidence. At the same time, disfluency manipulation activates analytical thinking of System 2 and makes learners invest more mental effort on processing information and improving their understanding of learning materials. Therefore, regardless of whether there is a signaling condition, disfluent manipulation makes learners experience “perceptual difficulty” at a metacognitive level, stimulates System 2, and activates analytical thinking. That is, regardless of the conditions, disfluency manipulation promotes improvement in test scores. This reflects the stability and universality of the disfluency effect in the multimedia learning framework. Under fluency conditions, learners do not perceive the difficulty caused by characteristics external to the material at the metacognitive level, so System 2 will not be stimulated to activate analytical thinking. Thus, under the condition of non-signaling, performance is poor. Under the signaling condition, although fluent material fails to stimulate System 2 and initiate analytical thinking, signaling has an attention-guiding effect in multimedia learning that can guide learners to pay attention to related information and simplify their visual search process. From another angle to the interaction, we found that signaling can improve learning effect whether the text is fluent or not, it also reflected the stability of the signaling effect, and combining with the eye movement indicators, we conclude that signaling can guide the learners’ attention and enhance individual’ perceived fluency and familiarity to the material. Thus, it can help learners find important information, release working memory to integrate information and construct mental representations.Results about JOLs showed that, learners’ JOL and test scores were significantly positively correlated under disfluent conditions, which suggests that disfluent operation may improve the accuracy of participants’ JOL. According to the result of the bias index, we found there is a significant difference in transfer score, and the bias index of the fluent condition was significantly higher than that of the disfluent condition, indicating that perceptually fluent learning materials are prone to producing metacognitive illusions (over-confidence) during the learning process. Although the data showed that disfluent manipulation alleviated the learner’s over-confidence, we only observed a significant difference in transfer (not retention) performance test. We speculated that this might be because the retention test mainly examined the verbatim remembering ability which is relatively less susceptible to metacognition; however, the transfer test reflects the learner’s ability to understand the material and apply what they learned to solve problems which may be greatly influenced by over-confidence illusion  in the teaching practice, we can get some inspiration based on this research. For example, set up questions with desirable difficulty for students to make them involve in mental effort during learning.Under the conditions of this research, the following conclusions can be drawn. Firstly, in multimedia learning, signaling can effectively guide learners’ attention and promote depth of learning and materials processing; this is supported by eye movement research, and signaling also can enhance individuals’ perceived fluency or familiarity to the learning materials. Therefore, in the teaching process, educators can use color signaling to highlight important knowledge points when presenting teaching content with complex elements. Secondly, metacognition caused by experiencing disfluency can reduce learners’ cognitive bias and may become a cue for learners to process more deeply. This work also illustrates the stability and universality of the positive impact of disfluency on multimedia learning. Although it seems unreasonable to make the text Some limitations of this study should be acknowledged. First, this study verified that mental effort benefits transfer learning; however, based on the current data, we barely know the underlying mechanism. The eye movement only revealed learners’ attention pattern between the signaled text and unsignaled text, and most of the discussions were inferred from behavioral data. Second, the transfer tasks employed in this study were more of a near transfer, extend them to far transfers will promote educational and practical implications of the research.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Fujian Normal University. The patients/participants provided their written informed consent to participate in this study.TL and JZ developed the study concept and contributed to the study design. Testing and data collection were performed by TL. TL performed the data analysis and interpretation under the supervision of JZ, and TL drafted the manuscript, and JZ provided critical revisions. All authors contributed to the article and approved the submitted version.This work was supported by the Key Research Institute of Humanities and Social Sciences, Ministry of Education, China (Grants 16JJD190004).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Drosophila melanogaster, the maternally inherited endosymbiont Wolbachia pipientis interacts with germline stem cell genes during oogenesis. One such gene, bag of marbles (bam) is the key switch for differentiation and also shows signals of adaptive evolution for protein diversification. These observations have led us to hypothesize that W. pipientis could be driving the adaptive evolution of bam for control of oogenesis. To test this hypothesis, we must understand the specificity of the genetic interaction between bam and W. pipientis. Previously, we documented that the W. pipientis variant, wMel, rescued the fertility of the BWbam hypomorphic mutant as a transheterozygote over a bam null. However, BWbam was generated more than 20 years ago in an uncontrolled genetic background and maintained over a balancer chromosome. Consequently, the chromosome carrying BWbam accumulated mutations that have prevented controlled experiments to further assess the interaction. Here, we used CRISPR/Cas9 to engineer the same single amino acid bam hypomorphic mutation (L255Fbam) and a new bam null disruption mutation into the 1118w isogenic background. We assess the fertility of wildtype bam, L255F/bamnullbam hypomorphic, and L255F/bamL255Fbam mutant females, each infected individually with 10 W. pipientis wMel variants representing three phylogenetic clades. Overall, we find that all of the W. pipientis variants tested here rescue bam hypomorphic fertility defects with wMelCS-like variants exhibiting the strongest rescue effects. In addition, these variants did not increase wildtype bam female fertility. Therefore, both bam and W. pipientis interact in genotype-specific ways to modulate female fertility, a critical fitness phenotype.In  Wolbachia pipientis is a maternally inherited endosymbiotic bacteria that infects over 65% of insect species and manipulates reproduction in a myriad of ways in order to ensure its transmission through the female germline (W. pipientis induces in its hosts include cytoplasmic incompatibility (CI), in which embryos of matings between infected males and uninfected females die; male killing, in which male embryos die; feminization of embryos; and manipulation of germline stem cell (GSC) differentiation in order to increase female fertility  and then protect the insect from viruses that can also cause human illness such as Dengue, Zika, and Chikungunya  that cause CI have been identified and functionally validated in D. melanogaster . Infection with the wMel variant rescues the fertility defect of females transheterozygous for the classic bam hypomorphic allele (BWbam) and a classic bam null allele (Δ59bam)  and wMelCS-like variants  that are estimated to have diverged 80,000 fly generations before present  been replaced by wMel-like variants world-wide in the late 20th century  .We confirmed that the amino acid replacement results in the wMel W. pipientis variants tested here do not increase fertility in the wildtype bam background, but all wMel W. pipientis variants rescue the fertility of bam mutant females, with wMelCS-like W. pipientis showing the highest rescue effects. Therefore, the fertility rescue of the bam hypomorph by W. pipientis is not due to an overall increase in fertility modulated by W. pipientis, but a specific interaction between bam and W. pipientis genotypes.We find that all bam alleles, Δ59bam (null) and BWbam (hypomorph) are described on Flybase  that contained the single amino acid mutation of the original BWbam hypomorphic allele (CTT->TTT) as well as a single synonymous change to the closest preferred codon in order to kill the gRNA site upon homology directed repair. We used 80 bp of homology on each side of our targeted change , and an siRNA for Lig4 . For the L255Fbam allele, the edited nucleotide change disrupts an AflII restriction site, allowing us to screen F1’s by PCR (Promega GoTaq mastermix) followed by restriction digests with AflII (NEB). We prepared genomic DNA using the Qiagen PureGene kit.For the 1118w isogenic males for three generations, and then crossed the mutants to the 1118w; TM2/TM6 line to maintain the bam mutants over the TM6 balancer. All CRISPR/Cas9 edits in the lines were confirmed by Sanger sequencing (Cornell BRC Genomics Facility).We backcrossed females of all CRISPR/Cas9 mutants to W. pipientis, we used the Zymo quick DNA miniprep kit to prepare DNA from three replicate samples each with three female flies. For each sample, we used three different primers for endpoint or qPCR , washed with PBST , and then mounted in ProLong Glass with NucBlue (ThermoFisher). We imaged ovaries on a Zeiss i880 confocal microscope with a 405 nm laser line at 10X  and 40X (described above). We analyzed and edited images using Fiji (ImageJ). Developing ovaries consist of cysts containing differentiated nurse cells which are polyploid and feature large, easily identifiable nuclei. In contrast, GSC daughter differentiation is blocked in bam loss-of-function ovaries resulting in cysts filled with small GSC-like cells with small nuclei. To quantify the rescue of bam's differentiation function, we counted the number of nurse cell positive egg chambers (cysts) per ovary for uninfected females and females infected with each W. pipientis genotype .We used the same assay we previously described to assess the rescue of the original 1118w isogenic bam genotypes for the fertility assays: 1118w; bam+/bam+, 1118w; L255Fbam/null-In2-3xP3-DsRedbam, and 1118w; L255Fbam/L255Fbam. In addition, we performed a small control fertility assay using combinations of the alleles described above and the classic BWbam hypomorph and Δ59bam alleles.We used the following 1118w; bam+/bam+ and 1118w; L255Fbam/null-In2-3xP3-DsRedbam in three batches to reduce technical error from too large of an experiment. We included separate uninfected controls in each batch of W. pipientis variants and used these to make statistical comparisons of mean progeny per female.We performed the fertility assays for BWbam and Δ59bam analysis) as follows , and allow us to understand the magnitude of the effect of W. pipientis variation on the bam phenotype. We display the data with a swarm plot that shows all of the data points and either a Cumming estimation plot (for more than two sample comparisons) or a Gardner-Altman plot (two sample comparisons) that shows the effect size for each sample compared to the control and 95% bootstrap confidence interval. In text, we report significance as a mean difference (effect size) outside the 95% bootstrap confidence interval.For the nurse cell assay, egg laying assay, and fertility assays we used estimation statistics to assess the mean difference (effect size) of nurse cells, eggs, and adult progeny between infected and uninfected lines. All counts for nurse cells, eggs, and progeny from these assays are reported in bam hypomorphic mutation as the original BWbam allele  as well as assess the phenotype of the homozygous bam hypomorphic genotype which has never been assessed before.We used CRISPR/Cas9 to recreate the same ckground . Therefo1118w; L255Fbam allele recapitulated the BW/bamΔ59bam mutant phenotypes by assessing the phenotype of a transheterozygous 1118w; L255Fbam hypomorph over a bam null allele. Since we wanted all of our alleles in the same genetic background and there were no existing bam null alleles in the 1118w isogenic background, we used CRISPR/Cas9 to knock in a 3xP3-DsRed cassette into the second intron of bam, which resulted in a bam null allele in the same 1118w background marked with a trackable eye marker . Therefore, two copies of the L255Fbam allele are sufficient for GSC daughter differentiation and fertility.As the  females . These ose cells . Previouifferent . While wbam genotype on fertility between lines with different genetic backgrounds, we did perform a small control experiment to ask if our new L255Fbam hypomorphic allele over the classic BWbam hypomorphic allele also show similar fertility to wildtype females as we observed for L255Fbam/L255Fbam females. We observed that L255Fbam/BWbam females and homozygous L255Fbam females did not show significantly different fertility from 1118 bam+/bam+w females (L255Fbam/null-In2-3xP3-DsRedbam genotype showed a similar fertility defect to L255Fbam/Δ59bam (classic null) and found that both showed severely reduced fertility compared to wildtype . Over 17 days, we counted the progeny produced by each test female infected individually for each of the 10 W. pipientis variants and assessed the mean difference of adult progeny from uninfected control females. We found that none of the W. pipientis variants increased female fertility in the wildtype bam background, and some lines showed a significant decrease in total progeny per female across all days measured   . Of the n nature . The var1118w; +bam/bam+ fertility assay indicated that none of the W. pipientis variants tested have an overall increase in fertility in females   there were outlier females who had large rescue of fertility, some in the range of what we observe for wildtype bam fertility  . This feertility . In all ertility . Howeverbam hypomorphic fertility phenotype by W. pipientis because we can also compare this to the effect of W. pipientis on wildtype bam fertility, we also wanted to know if genetic variation in W. pipientis affected the rescue of the bam hypomorphic cytological phenotype. Because fertility is affected by environmental and genetic differences that act in later developmental stages outside of bam’s function early in gametogenesis, we also measured the effect of W. pipientis variation on the rescue of the cytological bam mutant phenotype. Since the partial loss of function of bam results in over-proliferation of GSCs and reduced differentiation, we can quantify bam function by assaying for differentiated germ cells. The cytological bam hypomorphic phenotype in females consists of tumorous ovarioles filled with GSC-like cells, with few properly developing egg chambers  showed the highest rescue  rescue and a low fertility (adult progeny) rescue? Measuring the impact of W. pipientis on multiple stages of oogenesis and reproduction therefore gives us insight into any further complexities in the interaction between GSC genes and W. pipientis.We previously showed that a single t rescue  and 6. TwMelCS-like variants had the highest rescue effect on bam function, another example of a W. pipientis induced D. melanogaster fitness phenotype of which wMelCS-like variants exhibit the highest effects. The wMelCS-like variants tested here also confer the highest levels of viral resistance to D. melanogaster males  replaced by wMel-like variants in natural populations of D. melanogaster .In addition, we could utilize existing W. pipientis loci contributing to the different magnitude of bam rescue. However, as has been previously pointed out by wMel clades and wMelCS clades there are eight indels and 108 SNPs, including differences in the coding sequence of 58 genes. So further work would have to be done to narrow down which variants contribute to the degree of rescue. A next step could be moving out to more divergent W. pipientis wMel variants  to ask Fly lines and plasmids used in this study are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.G3 online.jkab312_Supplementary_DataClick here for additional data file.


Xiaoxuming decoction (XXMD) has been traditionally used to manage stroke though debates on its clinical efficacy were present in the history. Till nowadays, it is still one of the most commonly used herbal recipes for stroke. One of the reasons is that a decent proportion of ischemic stroke patients still have residue symptoms even after thrombolysis with rt-PA or endovascular thrombectomy. Numerous clinical studies have shown that XXMD is an effective alternative therapy not only at the acute stage, but also at the chronic sequelae stage of ischemic stroke. Modern techniques have isolated groups of compounds from XXMD which have shown therapeutic effects, such as dilating blood vessels, inhibiting thrombosis, suppressing oxidative stress, attenuating nitric oxide induced damage, protecting the blood brain barrier and the neurovascular unit. However, which of the active compounds is responsible for its therapeutic effects is still unknown. Emerging studies have screened and tested these active compounds aiming to find individual compounds that can be used as drugs to treat stroke. The present study summarized both clinical evidence of XXMD in managing stroke and experimental evidence on its molecular mechanisms that have been reported recently using advanced techniques. A new perspective has also been discussed with an aim to provide new targets that can be used for screening active compounds from XXMD. Stroke is the second leading cause of death worldwide, most of which is ischemic strokes . Stroke Currently, the diagnosis of stroke has been standardized with the use of CT, MR and other imaging methods. The course of this disease can be divided into the hyperacute (24 h), acute (1–7 days), subacute (7 days–6 months), and the sequelae stages (6 months later) , among wFor patients who are ineligible for thrombolysis within the time window or arriving at the hospital 24 h after stroke onset, current antiplatelet therapies have shown benefit in reducing mortality and recurrence of stroke. Although fibrin lowering agents and anticoagulants have been proven to be effective, their actual clinical use is limited due to their bleeding risk . Other mGlycyrrhiza glabra L. [Fabaceae]). The composition of XXMD seems strange and difficult for later generations of doctors to understand. In combination with the debate on the etiology of stroke , many doctors were concerned to use XXMD. Fortunately, this continuous debate has furthered insights into its composition and indications. Recently, clinical application of XXMD has been expanded. The addition and subtraction of certain medicines from XXMD based on the Six Meridians are more consistent with the material basis of TCM therapy for stroke and similar diseases. In this review, clinical studies on XXMD, active compounds of XXMD, and the underlying mechanisms of XXMD will be elaborated, which might point to a new direction for drug development to target stroke patients with residual symptoms and a new perspective was raised with an aim to accelerate the development of new drugs. To find publications on XXMD, 4 English databases , 4 Chinese databases , and 2 online clinical trial registration websites  were searched up to June 30, 2021. Related trials and systematic reviews were obtained by searching the references of the included studies. Different searching strategies were used, including: “Xiaoxuming Decoction” or “Xiaoxuming Tang” or “Xuming Tang” and “acute cerebral infarction” or “Stroke” or “ischemic attack”. No language restriction was used.Ischemic stroke belongs to the category of Wind induced diseases in Traditional Chinese Medicine. XXMD is an effective prescription for the treatment of stroke, which is a breakthrough in the management of stroke in the history of Chinese medicine. It was recorded in “Beiji Qianjin Yaofang Zhufeng” of Qianjin Yaofang (Tang dynasty- Simiao Sun), and later included in “Medical Prescriptions” and “Tangtou Gejue”. Due to its remarkable effect on stroke, it was regarded as “the most important recipe among effective therapies” by many doctors at that time and was prescribed as the first-line wind-dispelling agent, reflecting its importance in the treatment of stroke before Tang and Song Dynasties . After TIn the past few decades, modern TCM clinical practice has revealed new indications of XXMD. For this reason, XXMD has been widely used to treat not only stroke but also other diseases. Based on current understanding of the course of ischemic stroke in modern medicine, XXMD has been used to treat ischemic stroke patients at both the acute-subacute and the sequelae stages. Over 320 articles have been published in Chinese, but the majority of them were more like case reports. Therefore, only a few articles that are more or less meeting the criteria of randomized controlled clinical trials were selected to demonstrate the efficacy of XXMD in managing ischemic stroke.A number of studies showed that both the National Institutes of Health Stroke Scale (NIHSS) and the TCM syndrome score of XXMD treated patients were better than those who were treated with western medicine alone. It is proven that XXMD can effectively restore patients’ neurological functions, alleviate clinical symptoms, and accelerate the process of physical recovery.Pueraria montana var. lobata (Willd.) Maesen and S.M.Almeida ex Sanjappa and Predeep] Decoction  . It was found that alteplase plus XXMD significantly improved tissue perfusion in the infarct center and the surrounding ischemic area. Meanwhile, it can significantly improve cerebral artery blood supply and hemodynamics, promoting the recovery of neurological functions . In anotecoction , or withs Bunge) , or withs Bunge)  to treatFor patients at the sequelae stage of ischemic stroke (>6 months), it was also found that the addition of XXMD to conservative treatments significantly decreased the NIHSS score. Zeng treated 35 patients with stroke sequelae with XXMD and found that it was more superior to conventional western medicines alone in improving neurological functions . Hu 201.Ischemic stroke is divided into wind syndrome, heat syndrome, phlegm syndrome, blood stasis syndrome, Qi deficiency syndrome, Yin deficiency and Yang hyperactivity syndrome. XXMD is often used to treat patients with phlegm syndrome, Qi deficiency and blood stasis syndrome. It is especially  effectivXXMD has been proven an effective therapy for ischemic stroke. Development of modern medicine has deepened our understanding of the traditional medicine in many ways. Many researchers have extensively explored pharmacological mechanisms underlying the therapeutic effect of XXMD using emerging chemical and pharmacological techniques. This study aimed to summarize the composition of XXMD, active compounds, pharmacological actions, and pharmacokinetics of these compounds, which provided a comprehensive review of XXMD, an efficacious alternative therapy for ischemic stroke.Aconitum carmichaeli Debeaux, Neolitsea cassia (L.) Kosterm, Glycyrrhiza glabra L, Conioselinum anthriscoides “Chuanxiong,” Paeonia lactiflora Pall, Scutellaria baicalensis Georgi  Maesen and S.M.Almeida ex Sanjappa and Predeep, Gentiana macrophylla Pall.1) Wind-dispelling herbs are commonly used, such as Angelica biserrata (R.H.Shan and C.Q.Yuan) C.Q.Yuan and R.H.Shan, Hansenia weberbaueriana (Fedde ex H. Wolff) Pimenov and Kljuykov, Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk, Stephania tetrandra S. Moore, 2) Warming and cooling herbs are simultaneously used. For example, Zingiber officinale Roscoe, Aconitum carmichaeli Debeaux, and Scutellaria baicalensis Georgi are prescribed together.3) Tonifying and purging herbs are prescribed together. Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk and Stephania tetrandra S. Moore [Menispermaceae] dispel the wind, whereas Panax ginseng C.A.Mey, Zingiber officinale Roscoe, Aconitum carmichaeli Debeaux tonify Qi, increase Yang, and replenish the blood.4) Simultaneous targeting Qi and Blood. Panax ginseng C.A.Mey, Zingiber officinale Roscoe, Aconitum carmichaeli Debeaux can tonify Qi and increase hemopoiesis.The characteristics of the formula can be summarized as follows:Development of modern TCM clinical practice as well as chemical and pharmacological analysis has advanced our understanding of XXMD, which prompts new perspectives to understand XXMD and stroke treatment. Based on the composition of XXMD, functional characteristics of Chinese medicines as well as purified compounds, scientists have systematically studied the active ingredients of XXMD using emerging medical and chemical techniques.Li et al.  studied In order to accurately understand the material basis of the pharmacological actions of TCM prescriptions, The main pathogenic mechanism of ischemic stroke is acute interruption of cerebral blood flow caused by thrombosis, leading to ischemia, hypoxia, degeneration, and necrosis of the local brain tissue. As a result, neurological impairment is presented. The altered state of blood lipid metabolism and hypercoagulability are closely related to the prognosis of patients. Western medicine mainly focuses on symptomatic relief by prescribing anticoagulants, antiplatelets, statins, nutritional support, and early rehabilitation programs, which play an important role in patients’ recovery. However, adverse reactions also occur in some patients, and certain patients do not achieve the expected benefit from these therapies. Studies have shown that XXMD can significantly dilate blood vessels, inhibit thrombosis, improve blood coagulation in blood vessels and regulate lipid metabolism .Vasodilation: insufficient blood supply caused by cerebral vasoconstriction is one of the pathophysiological mechanisms of ischemic stroke. Vasodilating drugs can restore blood supply and subsequent energy supply to ischemic regions, which provides material basis for subsequent anti-ischemia-reperfusion injury therapies and recovery of neurological functions. Studies have shown that XXMD can significantly improve cerebrovascular blood supply in animals with cerebral ischemia and cerebral hemorrhage, reduce the permeability of capillaries in brain tissues, and alleviate the pathological changes of the blood brain barrier . Lu et aIn vitro administration of XXMD significantly suppressed the maximum platelet aggregation rate induced by ADP and prolonged the maximum platelet aggregation time by 50%. Interestingly, intravenous administration of XXMD significantly increased the blood flow of the internal carotid artery in dogs, reduced the cerebrovascular resistance, slightly accelerated the heart rate, without apparent influence on the blood pressure. In addition, studies found that the NIHSS, levels of fibrinogen and D-dimers were significantly lower in the group managed with conventional treatments plus XXMD than in the control group managed with conventional treatments alone, indicating that XXMD has a significant therapeutic effect on acute cerebral infarction. It can effectively suppress coagulation and improve neurological functions  significantly decreased levels of the total cholesterol and triglycerides observed in the high-fat group . It is kin vivo and in vitro. The therapeutic effect of XXMD was evaluated by assessing neural response to ischemic injury, the activity of caspase-3 in the hippocampal CA1 region, and the expression of Bcl-2. Cognitive performance was assessed using the Morris water maze test 7 days after the ischemic injury. It was found that XXMD increased the density of viable neurons in the hippocampal CA1 region, attenuated spatial cognitive deficits, inhibited the activation of caspase-3, and up-regulated the expression of Bcl-2. These indicate that anti-apoptosis is one of the mechanisms underlying the therapeutic effect of XXMD /Bax-apoptosis related genes in the brain and inhibiting the downstream cascade of caspase induced apoptosis . Zhu et  of XXMD .Anti-oxidative stress: it is well known that blood flow is interrupted after ischemic stroke onset, and reperfusion to the brain tissue triggers a series of mechanisms, such as accumulation of large amounts of calcium ions, massive production of oxygen free radicals, and acute cell edema, which leads to necrosis of neural cells and damage of the neurovascular unit. Thrombolytic therapies or intravascular recanalization rapidly increased the blood flow to the brain tissue, which potentially aggravated the injury of the ischemic tissue and brain edema. Consequently, brain tissue damage became more severe, which is an important factor contributing to the deterioration of ischemic stroke patients. However, for the majority of ischemic stroke patients, recanalization therapies generally improve the neurological functions of these patients. Timing of starting these therapies must be an important determinant of the outcome of these patients. The balance between increased blood supply to the microcirculation along with the subsequent improvement of neuronal metabolism and ischemia-reperfusion induced injury remains to be investigated.Animal experiments showed that the effective ingredients of XXMD at both low and medium doses could inhibit lipid peroxidation, increase the activity of glutathione-peroxidase (GSH-Px), leading to suppression of oxidative stress response. Cheng et al. found that the addition of XXMD to conventional treatments significantly restored neurological functions as shown by the decreased NIHSS and the increased BI score. This was mainly due to suppression of levels of superoxide dismutase and chemokine MCP-1, increased levels of BDNF, and increased blood supply to the brain as well as improved hemodynamics . In addiAnti-nitric oxide (NO) induced damage: nitric oxide is a highly active and diffusible molecule with a very short half-life. NOS is present in endothelial cells, neural cells, nerve fibers, and glial cells, and it has a variety of physiological functions in organisms, such as dilating blood vessels, lowering the blood pressure, suppressing platelet aggregation and adhesion. When cerebral ischemia occurs, NO has a dual effect. For example, experimental data have shown that NO exerts a protective effect at the early stage of ischemia and a neurotoxic effect at the late stage. Improving microcirculation in the ischemic penumbra salvages the dying neurons, whereas necrosis in the infarct core aggravates tissue damage. Wang and Tang found that the level of plasma NO and the activity of NOS in stroke model rats were significantly decreased after intragastric administration of XXMD, suggesting that the reduction of plasma NO content and NOS activity in rats with acute ischemic stroke might be one of the mechanisms of XXMD to ameliorate cerebral ischemia injury . It has in vitro and in vivo experiments (Anti-neuroinflammation: it is clear that cerebral infarct activates microglia and astrocytes to engulf the necrotic tissue. This consequently results in the development of fibrotic scars. This neuroinflammatory response is further aggravated by cytokines released by microglia and astrocytes . Therefoeriments . A netwoeriments . In hemoeriments . This waeriments . These sProtection of the neurovascular unit: in an experimental study, Wang et al. found that the effective compounds of XXMD alleviated the reduction of cerebrovascular reserve and the reduction of the number of neurons as well as the altered morphology due to chronic ischemia in rats. It also effectively decreased the activation of astrocytes . The othProtection of the blood-brain barrier: intragastric infusion of different doses of XXMD to the ischemia-reperfusion stroke model rats resulted in not only reduced size of cerebral infarcts, improved behavioral performance, but also decreased cell ultrastructural damage, and decreased blood-cerebrospinal fluid barrier permeability . It mighIn conclusion, XXMD is an effective alternative therapy for ischemic stroke through regulating the blood flow of patients and protecting the brain tissue. It can dilate blood vessels, reduce thrombosis, regulate blood coagulation and blood lipids to improve the blood flow status of patients, exerting the protective effect. On the other hand, XXMD can not only inhibit the oxidative reaction, calcium overload, inflammatory reactions secondary to neuronal injury, protect the neurovascular unit, but also reduce brain edema and protect the blood-brain barrier.XXMD has a long history and modern clinical evidence in the treatment of ischemic stroke, but the results of multi-center randomized clinical trials are still lacking. Modern biochemical methods have deepened the understanding of the composition and effective compound groups of this traditional recipe, but the individual effective ingredients have not been confirmed.Although the mechanism of XXMD in managing ischemic stroke has been widely explored, there are still many challenges. Studies in recent years have shown that microvascular pericytes will contract after ischemia , and eryAs mentioned above, XXMD is not only effective for ischemic stroke, but also effective for hemorrhagic stroke . This ra


In meat processing, changes in the myofibrillar protein (MP) structure can affect the quality of meat products. High hydrostatic pressure (HHP) has been widely utilized to change the conformational structure  of MP so as to improve the quality of meat products. However, a systematic summary of the relationship between the conformational structure (secondary and tertiary structure) changes in MP, gel properties and product quality under HHP is lacking. Hence, this review provides a comprehensive summary of the changes in the conformational structure and gel properties of MP under HHP and discusses the mechanism based on previous studies and recent progress. The relationship between the spatial structure of MP and meat texture under HHP is also explored. Finally, we discuss considerations regarding ways to make HHP an effective strategy in future meat manufacturing. Protein is one of the most important elements of nutrition for humans, providing energy and essential amino acids. Meat is the most valuable livestock product and, therefore, the preferred protein source of many people . In addiHHP treatment is a non-thermal processing method that has been widely used in the fields of seafood, fruit, vegetable and meat products . HHP is Treatment with HHP is not limited by the shape or size of the material, and the original shape of the solid food can be maintained . Meat isMuscle protein mainly consists of myofibril protein, comprising two-thirds of the total proteins in lean muscle. In meat product processing, changes in MP will alter the quality of meat, including its appearance, hardness, springiness, chewiness, juiciness, taste and other texture characteristics ,15,16. TMyofibrillar protein, a salt-soluble protein, accounts for approximately 55% of muscle protein content and mainly includes tropomyosin, myosin, myogenic protein, and actin . Myosin HHP can dissociate the non-covalent, ionic, hydrophobic, and hydrogen bonds to change the secondary, tertiary, and quaternary structure of MP . ChangesBy using available computational methods, detailed pressure-induced changes in protein secondary structure from characteristic shifts in the band frequencies that are recorded via various techniques, such as Fourier-transform infrared spectroscopy (FTIR), Raman spectra and circular dichroism (CD) . The secMost studies have shown that 100 MPa pressure can change the secondary structure of MP in meat products, with specific details according to various levels of pressure outlined in In a previous report, the α-helix structure of MP was significantly reduced by HHP, with corresponding increases in the contents of β-turn and random coil . Some st0) and the contents of the free sulfhydryl group (−SH) reflect changes in the tertiary structure of proteins and are closely related to their functional properties [Changes in surface hydrophobicity  causes MP aggregation due to the intermolecular interactions that dominate [re above 0 MPa caudominate ,48. OverThe gel properties of MP determine the texture, adhesion and water retention of meat products. The formation of protein gels usually involves denaturation and aggregation, that is, the formation of a network structure, through physical and chemical reactions, that is capable of retaining water, fat, or other components . In orde2 can change the physical properties of meat products, mainly because, at pressures under 200 MPa, a large amount of myosin heavy chains and actin are dissolved, protein aggregation ability is reduced, and the tyrosine and tryptophan residues are exposed due to the unfolding of the tertiary structure [Oreochromis niloticus) at 0° [As discussed in tructure . Hsu et s) at 0° . At 500 s) at 0° . Chen ets) at 0° . The ress) at 0° . The enhs) at 0° . Zhang fs) at 0° . Some sts) at 0° ,61. Howes) at 0° . This res) at 0° . It is gs) at 0° ,68,69.HHP treatment can lead to protein unfolding . Figure HHP treatment of meat has no significant effect on its flavor compounds, amino acids, vitamins or other small molecular substances when compared with traditional thermal processing ,71. As aThe use of HHP as a non-thermal sterilization technique, with minimal impact on sensory quality and nutritional value, has received wide approval, and a variety of meat products have benefited from the application of HHP to ensure food safety and the extension of shelf life ,73. The Color is considered to be the most important factor influencing consumers to judge the freshness of food products. There are three main parameters for evaluating color, namely L* value (brightness), A* value (redness) and B* value (yellowness). Under the condition of 4 °C and 100–200 MPa for 15 min, the muscle brightness of flounder increased continuously, and the brightness of cod also increased continuously under the conditions of 220–300 MPa for 5 min . The treProcambarus clarkia) initially increased sharply, then decreased slightly, but all were higher than those of the control [Texture is one of the important indexes of meat products, among which hardness and elasticity are the most studied . Hardnes control . In our  control ,36. As f control ,36. The  control ,36, furtProtein oxidation not only leads to the deterioration of food color and texture, but also to the loss of nutrients  and a reduction in protein digestibility ,84. In aHHP has been proven to be effective in improving the color and texture of meat products . GeneralAlthough HHP is considered to be an effective technology for improving the quality of meat as well as for the production of muscle-gelled foods, a certain threshold of treatment intensity is required for meat processing. However, the change in MP structure is revealed to be the main factor underlying the improvement in the quality of meat products. The spatial conformation of MP changes under HHP treatment, leading to changes in the product color and texture, and the mechanisms demonstrate the interdependence of the processes. However, meat is a complex system, containing not only MP but also other macromolecular proteins, polysaccharides and small molecules. Thus, the interactions between the different components under HHP treatment, together with their effects on the quality of meat products, require further in-depth research, particularly with regard to the molecular mechanism of the process.


P < 0.05). A backward, stepwise, multiple regression analysis was applied to define a model with high relation between in vivo  and in vitro  fertility data. The analysis produced two models that accounted for more than 78% of the variation of ERCR , identifying a suitable combination of parameters useful to predict bull fertility. The predictive equation on CG samples included eight variables: four kinetic parameters and four DNA integrity indicators. For the HQS fraction, the predictive equation included five variables: three kinetic parameters and two DNA integrity indicators. A significant relationship was observed between real and predicted fertility in CG (R2 = 0.88) and HQS fraction (R2 = 0.82). We identified 15 differentially expressed miRNAs between high- and low-fertility bulls, nine of which are known  while the remaining are novel. The multidimensional preference analysis model partially separates bulls according to their fertility, clustering three semen quality variable groups relative to motility, DNA integrity, and viability. A positive association between field fertility, semen quality parameters, and specific miRNAs was revealed. The integrated approach could provide a model for bull selection in AI centers, increasing the reproductive efficiency of livestock.Predicting bull fertility is one of the main challenges for the dairy breeding industry and artificial insemination (AI) centers. Semen evaluation performed in the AI center is not fully reliable to determine the level of bull fertility. Spermatozoa are rich in active miRNA. Specific sperm-borne miRNAs can be linked to fertility. The aim of our study is to propose a combined flow cytometric analysis and miRNA profiling of semen bulls with different fertility to identify markers that can be potentially used for the prediction of field fertility. Sperm functions were analyzed in frozen-thawed semen doses (CG: control group) and high-quality sperm (HQS) fraction collected from bulls with different field fertility levels (estimated relative conception rate or ERCR) by using advanced techniques, such as the computer-assisted semen analysis system, flow cytometry, and small RNA-sequencing. Fertility groups differ for total and progressive motility and in the abnormality degree of the chromatin structure ( Prediction of bull fertility is one of the crucial factors dictating optimum efficiency in the livestock production system. Considering that, in dairy cattle, the selection and reproductive management are based on the use of artificial insemination (AI), the assessment of the level of bull fertility nowadays is one of the main challenges for AI centers and cattle breeders.At present, quality parameters routinely applied in semen evaluation tests performed in AI centers are not fully reliable to determine a bull's fertility level. Field fertility, in terms of the ability of bulls to make cows pregnant through AI, is affected by a wide range of factors, such as health status, genetic traits, full functionality of reproductive organs, herd management, semen quality, and cryopreservation protocols. Reproductive efficiency of AI bulls is predicted by direct measure of fertility. The most utilized method of measuring male fertility is related to the non-return rate (NRR) although low-to-moderate correlation has been observed between the semen quality criteria currently used and the NRR . The estin vitro semen quality with field data, with the aim to exclude low-potential-fertility bulls from breeding programs, thus saving considerable time and economic resources. As genomic selection for male fertility is not widely implemented, semen evaluation for the prediction of male fertility has a greater impact on breeding outcomes  range.Several advanced technologies can be used to examine the quality of spermatozoa—such as computer-assisted semen analysis (CASA) and flow cytometry (FCM)—which can provide accurate and objective evaluation of sperm function. With the existing methods, it is easier to identify low-fertility (LF) bulls, but ranking those with moderate-to-high fertility is more complicated . Some reMoreover, the integration of several tests, from the evaluation of semen quality with advanced technologies to sperm molecular investigation, such as small RNA profiling, is a promising approach to achieve a better understanding of sperm functions as well as to evaluate semen quality and predict bull fertility .Mature spermatozoa contain coding and non-coding RNAs, some of which are involved in the regulation of many physiological pathways related to spermatogenesis, sperm maturation, and early embryonic development with the potential to modulate sperm morpho-functional features as well as male reproductive ability . Among sIn our study, we hypothesize that bulls with moderate-to-high fertility can be identified by a difference in semen quality and by the analysis of miRNA expression.An integrated approach between advanced semen quality analysis and miRNA profiling of cryopreserved semen in HF and LF bulls is proposed to identify markers and build a model that can be potentially used for the prediction of male field fertility.6 spermatozoa), frozen with a commercial egg yolk–based extender, and stored in liquid nitrogen. For each bull, the frozen-thawed semen doses were evaluated before (control group: CG) and immediately after Percoll density gradient centrifugation .Frozen semen samples from 10 mature progeny-tested Italian Holstein–Friesian bulls of known reproductive ability were purchased from INSEME S.p.A bull AI center . The bulls were classified according to field fertility data expressed as the ERCR, provided by the Italian Holstein Breeders Association (ANAFI). To calculate the ERCR, the 56-day NRR to service was adjusted for several factors, including farm, year and month of insemination, parity, days open of the cow at first insemination, bull, bull progeny, and cow. The ERCR represents the effect of the bull on the NRR (expressed as a percentage) of cows inseminated in the herd and is determined by the difference between that NRR value for a particular bull and the average NRR values obtained with the semen of other bulls. As its calculation is based on a large number of services from many different herds, the ERCR is considered a highly accurate measurement to identify HF and LF bulls . On aver6 cells per dose) were simultaneously thawed in a water bath at 37°C for 20 s and pooled. The pool (5 mL) of each bull was split into two aliquots of 2.5 mL that were overlaid on a dual-layer (90–45%) discontinuous Percoll gradient  in two 15-ml conical tubes and centrifuged at 700 × g for 30 min at 20°C. The Percoll layers were prepared by diluting Percoll solution in as indicated by Parrish et al.  and after Percoll separation in the HQS fraction obtained.® v1, Proiser, R + D S.L., Paterna, Spain) combined with a phase contrast microscope (Nikon Optiphot) equipped with a negative phase contrast 10 × objective and integrated warmer stage and connected to a video camera .Total motility and sperm kinetics parameters were assessed by the CASA system , progressive motility , curvilinear velocity , straight-line velocity , average path velocity , linearity coefficient , amplitude of lateral head displacement , straightness coefficient , wobble coefficient , and beat cross frequency .Semen from CG was evaluated as raw without dilution, and 5 μl of semen pellet obtained after Percoll density gradient centrifugation (HQS fraction) were diluted in 5 μl TALP sperm medium  prewarme® EasyCyte™ 5HT microcapillary flow cytometer  equipped with fluorescent probes excited by a 20-mW argon ion laser (488 nm). Forward-scatter (FSC) vs. side-scatter (SSC) plots were used to separate sperm cells from debris. Non-sperm events were excluded from further analysis. Fluorescence detection was set with three photomultiplier tubes: detector FL-1 (green: 525/30 nm), detector FL-2 (yellow/orange: 586/26 nm), and detector FL-3 (red: 690/50 nm). Calibration was carried out using standard beads . A total of 5,000 sperm events per sample for each bull were analyzed at a flow rate of 200 cells/s. Compensation for spectra overlap between fluorochromes was set according to the procedures outlined by Roederer , respectively.® Sperm Viability Kit  was used for the analysis of plasma membrane integrity as described by Gliozzi et al. . Both dyes can be used to label DNA. After staining, live sperm cells with intact cell membranes fluoresced bright green, whereas cells with damaged cell membranes fluoresced red. Aliquots of semen extended with Easy Buffer (2.0 × 105 spermatozoa/ml) were supplemented with SYBR®14 0.1 μM and PI 12 μM  according to manufacturer instructions. After gentle mixing and 10 min incubation at 37°C in the dark, three replicates per sample were performed. Debris particles were gated out according to the intensity of green and red fluorescence. Three sperm populations were detected on the FL-1/FL-3 dot plot: viable (green), dead (red), and moribund (double-stained) spermatozoa.The LIVE-DEADi et al. . The kit® EasyCyte™ 5HT microcapillary flow cytometer, and a total of 5,000 events were acquired. Percentages of sperm with either intact or damaged plasma membrane as well as intact or damaged acrosome were computed. Spermatozoa with disrupted acrosomes were labeled with a green probe. Dead spermatozoa with damaged plasma membrane were labeled with a red fluorochrome.Acrosomal membrane integrity was assessed using Easykit 5 . This kit is used to measure the level of disrupted acrosome within viable or dead sperm populations. The ready-to-use 96-well plate was filled with 200 mL of embryonic holding solution , and 40,000 sperm were added during 45 min at 37°C protected from light in basal condition. The signal was read with the Guava5 cells were diluted in 200 μl of TNE buffer  and added to 400 μl of an acidic solution . After 30 s, cells were stained with 1.2 ml of acridine orange solution . Samples were protected from light and incubated at room temperature for 2.5 min; after that, two replicates per sample were acquired and analyzed using, respectively, CytoSoft and EasyCompDNA software  and expressed as Alpha-T, which is indicative of the shift from green to red fluorescence, is expressed as the ratio of red to total fluorescence intensity [red/(red + green)], and quantifies the degree of abnormal chromatin structure with an increased susceptibility to acid-induced denaturation; standard deviation of Alpha-T (ATSD), which shows the extent of abnormality in the chromatin structure within a population; DNA fragmentation index (%DFI), which indicates the percentage of sperm with fragmented DNA; and percentage of sperm with high green fluorescence (%HG), which is representative of the percentage of immature cells with reduced nuclear condensation (incomplete histone-protamine exchange). The FCM instrument was AO-saturated prior to analysis by running the AO equilibration solution.The Sperm Chromatin Structure Assay was assessed as previously described in other studies of ours , 15 by un = 10) collected in two seasons (summer and winter) were used for RNA isolation. RNA was extracted as previously described . RNA concentration and quality were determined by the Agilent 2100 Bioanalyzer   from each bull sperm  . The iso®AMPure® XP  (1 Vol. sample: 1.8 Vol. beads), thus obtaining two pools of libraries representing the two technical replicates. The two pools were purified on a Pippin Prep system  to recover the 125–167 nt fraction containing mature miRNAs. The quality and yield after sample preparation were measured with an Agilent 2200 Tape Station, High Sensitivity D1000. Libraries were sequenced on two lanes of Illumina Hiseq 2000 .Libraries were generated using the Illumina Truseq Small RNA Preparation kit according to the manufacturer's instructions and the protocol described by Capra et al. . LibrariBos taurus miRNAs available at miRBase (http://www.mirbase.org/) were used to accomplish known miRNA detection on the trimmed sequences. Known miRNAs from related species  available at miRBase were also input into Mirdeep2 to support the individuation of novel miRNAs. The Mirdeep2 quantifier module was used to quantify expression and retrieve counts for the detected known and novel miRNAs. Differential expression analyses between the HF and LF bulls and in different seasons were run with the Bioconductor edgeR package  were analyzed using the SAS™ package v 9.4  with different statistical models to evaluate the following:Data obtained from Isolation of sperm fractions and evaluation of sperm separation.The general linear model procedure (PROC GLM) was used to (i) evaluate the effect of technical replicates on semen quality parameters in the HQS fraction—the model included as fixed effects the bull and the replicate nested in the sperm fraction—and (ii) verify the efficiency of the sperm separation from the CG to HQS fractions. The model included the fixed effect of the sperm fraction and of the bull.in vitro semen quality and in vivo fertility.Relations between in vitro sperm quality in function of the ERCR (HF and LF bulls). The model included the fixed effects of fertility rank, categorized as LF (ERCR ≤ 1.0) and HF (ERCR ≥ 1) and season of semen collection .PROC GLM was used to assess in vitro sperm parameters and repeatedly eliminated non-significant variables, using a significance level of P < 0.05 to retain variables. The real values of the significant variables obtained from each bull as determined by backward elimination were included in an equation  to calculate the predicted fertility values. Correlation between real and predicted fertility values was then assessed.A backward stepwise multiple regression analysis (PROC REG) was used to obtain a model with high relation between sperm quality parameters and ERCR. The redundant parameters were excluded from analysis. The procedure started with a model that contained all in vitro semen quality traits and miRNA expression that correlated with the level of bull fertility. MDPREF is a PCA of data performed using the PROC PRINQUAL procedure of SAS  was then conducted to explore the e of SAS . This prResults are given as adjusted least squares means ± standard error means (LSM ± SEM).P < 0.05) improvement of the sperm quality occurred in the HQS fraction with respect to the CG.No significant differences were observed among technical replicates concerning semen quality parameters in HQS fractions . Sperm cin vitro semen quality were observed according to the semen collection season. In vitro sperm quality parameters according to the fertility level, assessed on both the CG and HQS fraction, are presented in P < 0.05) improvement of MOT TOT and PROG was found in CG/HF bulls with respect to the CG/LF group. Significant differences were also observed for Alpha-T, showing a major abnormality in the degree of chromatin structure in LF bulls. In the CG/HF bulls, higher values were found with respect to LF bulls for VIAB and VIA variables as well as a lower percentage of sperm with fragmented DNA (%DFI) and percentage of sperm with immature cells with reduced nuclear condensation (%HG) although not with significant values. In the HQS fraction, an increase (P < 0.05) of the abnormality degree of chromatin structure (Alpha-T) was also noticed in LF bulls with respect to the HF bulls.No significant effects on in vitro  and in vivo (ERCR) fertility on both CG and HQS fraction. The initial model applied on both CG and HQS fraction was ERCR= MOT TOT + PROG + VSL + VCL + VAP + LIN + STR + WOB + ALH + BCF + VIAB + VIA + VDA + ALPHAT + ATSD + %DFI + %HG.A backward elimination analysis was performed to obtain a fertility prediction model between R2 = 0.88; HQS fraction: R2 = 0.78). The predictive equation of the final model on CG included eight variables (ERCR = ALPHAT + %DFI + ATSD + %HG + STR + VAP + VCL + WOB): four DNA integrity indicators , and four kinetic parameters .The analysis produced two final models that accounted for more than 78% of the variation of ERCR , and three kinetic parameters .On HQS fraction, the predictive equation of the final included five variables (ERCR= ALPHAT + %DFI + MOT TOT + PROG + VSL): two DNA integrity indicators  between real and predicted fertility found in CG and HQS fraction, respectively.Bos taurus miRNAs, 98 were homologous of known miRNAs from other species, and 246 were new candidate miRNAs , the second one relative to DNA integrity indicators , and the last one relative to sperm viability indicators . The DNA integrity indicators (considered as damage) showed a positive relation with the group of bulls with LF, indicating also a positive relation with bta-miR-378c, bta-miR-423-3p, and bta-miR-486. Sperm viability indicators showed a positive relation with the group of HF bulls and miR-191. In MDPREF was used to study the relationship between ertility . In the in vitro sperm characteristics, assessed with advanced techniques such as CASA and FCM as well as miRNA expression by small RNA-sequencing with field fertility (ERCR).The current study was conducted with the aim to identify semen quality and miRNA biomarkers with the final goal to predict the fertility status in bulls. To categorize the most suitable parameters and provide a reliable prediction of bull fertility (among HF and LF bulls), we combined several In our study, the HQS fraction was successfully fractioned after Percoll gradient, showing good reproducibility between technical replicates and attenuating sample variabilities , 21. Thein vivo fertilization, and early embryo development. Considering the HQS fraction, we observed that the Alpha-T variable can significantly discriminate among bull fertility levels, showing a lower DNA damage in bulls with superior fertility. In this group also total motility, viability, and %DFI have shown better values although not with statistically significant values.As reproductive success depends on the ability of the sperm cell to transfer information to the developing embryo, we analyzed many variables strictly related to sperm functions. According to our results, in the CG group, descriptors such as total and progressive motility were significantly higher in bulls with superior fertility. These results are in agreement with Gliozzi et al.  and in cR2 = 0.83–0.84. Our model developed in an earlier study , miR-2285n, miR-378 and miR-486 have already been proven to change their expression levels between low and high motile sperm populations in our previous study  previously reported to be differentially expressed between the low and high motile sperm fractions .in vitro semen quality and in vivo fertility. Through an advanced semen quality assessment, which combines kinetic semen parameters originating from the CASA system and DNA analysis based on FCM, supported by statistical models, it was possible to establish that semen from LF bulls differs from that of HF individuals mainly in terms of sperm DNA integrity. Also viability and acrosome integrity concur to discriminate bulls according their fertility level.In this study, we provide a reliable model to predict bull fertility, easily implementable in bull AI centers, which routinely evaluate sperm motility and may instruct external laboratories to assess DNA integrity, confirming the relationship between bull in vitro quality parameters to propose a solution to predict fertility that potentially could be used for further studies in the field in a wider population. As a novelty, the application of the MDPREF analysis demonstrated that a positive correlation was present among specific in vitro quality parameters (as motility viability and DNA integrity indicators), specific miRNAs, and bull fertility status.We investigated differentially expressed miRNAs in the high-quality sperm fraction of bulls as candidate markers for evaluating fertility status. Fifteen miRNAs were differentially expressed in bulls with contrasting fertility. With our study, we applied a first statistical model integrating the relative expression of known DE-miRNAs and semen Further studies are needed to evaluate these specific miRNAs as qualitative biomarkers for the prediction of assessment of sperm quality and the prediction of bull fertility. The integrated approach between advanced semen quality analysis and miRNA profiling could provide a model for prediction of fertility that might be beneficial to AI centers in the selection of bulls and facilitate the elimination of subfertile subjects or ejaculates with low fertilizing capacity.PRJNA727990.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: the Sequence Read Archive (SRA) database, on NCBI server, and accession number: Ethical review and approval was not required for the animal study because the commercial frozen semen doses used in this study were acquired from an approved commercial artificial insemination bull centers.EC, FP, FT, and AS conceived the study. FT isolated the spermatozoa fractions through Percoll gradient and evaluated sperm characteristics and carried out the statistical analysis. EC and PC performed the RNA extraction, libraries preparation and sequencing. BL carried out the bioinformatic analysis. EC carried out pathway analysis. FP acquired the funding and supervised the project. EC and FT wrote the manuscript and generated the figures. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Viral and cellular gene expression are regulated by epigenetic alterations, including DNA methylation, histone modifications, nucleosome positioning, and chromatin looping. Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic retrovirus associated with inflammatory disorders and T-cell lymphoproliferative malignancy. The transforming activity of HTLV-1 is driven by the viral oncoprotein Tax, which acts as a transcriptional activator of the cAMP response element-binding protein (CREB) and nuclear factor kappa B (NFκB) pathways. The epigenetic effects of Tax and the induction of lymphoproliferative malignancy include alterations in DNA methylation and histone modifications. In addition, alterations in nucleosome positioning and DNA looping also occur in HTLV-1-induced malignant cells. A mechanistic definition of these effects will pave the way to new therapies for HTLV-1-associated disorders. The Greek prefix “epi” means “over, outside of, around” . Thus, eAll major classes of cancer-causing agents are associated with epigenetic re-programming. This includes cancers caused by chemical carcinogens, hormones, heavy metals, radiation, viruses, and bacteria . There hHuman T-cell leukemia virus type 1 (HTLV-1) is the first discovered human retrovirus, and it has infected 15–20 million individuals worldwide . HTLV-1 hbz gene from the minus strand of the proviral genome that encodes HBZ, the helix basic zipper protein . Li. Li12].  protein . Tax has several activities that contribute to transformation . Tax is hbz RNA and HBZ protein are critical for proliferation and tumor cell maintenance [However, Tax is also a key target of CD8+ CTL responses . Tax is ntenance ,28.DNA methylation results from the covalent transfer of a methyl group from S-adenosyl-L-methionine to cytosine in CpG dinucleotides . Promotegag, pol, and env genes of the provirus, and then it extends in the 5′ and 3′ directions in vivo, and when the 5′ LTR becomes methylated, viral transcription is silenced [Most HTLV-1-infected cells in vivo harbor a provirus that is transcriptionally silent . There asilenced . Provirusilenced . Alternasilenced .ATLL genomes are characterized by a prominent promoter-associated CpG island and DNA hypermethylation; the level of hypermethylation is correlated with poor prognosis . MutatioWatanabe et al. identified differentially methylated positions (DMPs) specific to HTLV-infected cells, and identified those that correlated with ATLL development and progression, including CpG islands in genes encoding thymocyte-expressed molecule (THEMIS), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and ring-type E3 ubiquitin transferase 130 (RNF130), which negatively regulate T-cell receptor (TCR) signaling . Other gPost-translational modifications of histones play an important role in the epigenetic regulation of chromatin . The mosTranscriptional initiation, elongation, and mRNA processing are co-regulated through the posttranslational modification of the RNA Pol II (RNAPII) C-terminal domain (CTD). CTD is phosphorylated by transcription initiation factor IIH (TFIIH) and positive transcription elongation factor b (pTEFb), and bound by negative elongation factor A (NELF-A) and positive elongation factor Spt5 . NELF-A Tax interacts with multiple histone-modifying enzymes. This includes histone acetylases (HATs) and deacetylases (HDACs). Tax binds histone acetylases, CREB-binding protein (CBP), and p300 in order to transactivate the viral promoter . This waTax binds the histone methyltransferases SUV39H1 and SET and MYND domain-containing protein (SMYD3) . Tax binThe suppression of incoming DNA by transcriptional silencing is an important mechanism, whereby invading viruses block the initiation of the cellular innate immune response. This has been shown for the murine leukemia virus  and manyH3K27me3 is a mark of chromatin condensation and gene silencing, and it is regulated by enhancer of zeste homolog 1 (EZH1) and EZH2, which are core components of polycomb repressive complex PRC2 with embryonic ectoderm development protein (EED) and suppressor of zeste 12 homolog (SUZ12). Several drugs have been identified as EZH1 or EZH2 inhibitors, including the FDA-approved EZH2 inhibitor tazemetostat . H3K27meH2AK119ub1, a signature of PRC1 resistance, is enriched on the latent HTLV-1 provirus . It is iIn summary, the inhibition of histone-modifying enzymes is an active area of investigation in ATLL and other T-cell lymphomas.Nucleosomes are composed of an octomer of histone proteins, with two copies each of the histones H2A, H2B, H3, and H4 . Linker Tax induces the clearance of nucleosomes from the HTLV-1 promoter . NucleosStudies of ATLL DNA and chromatin modifications demonstrated a sharp boundary in the provirus near nucleotide 6718 (NC_001436.1), with the 5′ portion, but not the 3′ portion, of the genome being silenced in most patient samples . This suAn insulator is a genetic boundary element that blocks interactions between enhancers and promoters . The besCTCF binds 55,000 DNA sites in 19 diverse cell types  to multiCTCF regulates the expression of many DNA viruses. EBV has 19 CTCF-BS, with most of them at key regulatory regions  . With thHerpes simplex virus type 1 has seven CTCF-BS, including three sites within control locus 2 (CTRL2) downstream of the latency-associated transcript (LAT) enhancer . MutatioThe boundary between endogenous retroviruses and adjacent genes is enriched in CTCF-BS . CTCF sicis at non-contiguous loci up to >300 kb from the integration site [A single conserved CTCF binding site (vCTCF-BS) has been identified in the integrated HTLV-1 provirus, flanking methylated proviral DNA and closed chromatin in the 5′ provirus and unmethylated DNA and open chromatin in the 3′ provirus . Miura eion site . These fThe study of the epigenetic regulation of HTLV-1 is a burgeoning field of investigation. Many questions remain to be answered. What are the dynamics of the epigenetic modulation of the HTLV-1 genome before and after integration? Are there differences in epigenetic alterations in malignant compared to non-malignant infected cells? What is the importance of epigenetic alterations reported in cell culture studies to those present in vivo in animal model studies and patients? Does epigenetic regulation differ in asymptomatic infected individuals compared to the patients with HAM, ATLL, or other inflammatory diseases? How does epigenetic regulation of the provirus and the cellular genome differ in ATLL cases wherein the Tax gene is intermittently expressed and those cases in which it is deleted or permanently silenced? Are there key cellular genes that determine pathogenesis that are regulated by epigenetic alterations in HTLV-associated disease? Are there specific therapeutic approaches that disrupt epigenetic regulation in HTLV-1-infected cells? Can such maneuvers have a role in the treatment or prevention of diseases? Can one reduce the proviral load in infected individuals? Do approaches for disrupting or reinforcing latency that are being tested in HIV-1-infected individuals have applications in HTLV-1 disease? How does CTCF regulate the integrated provirus and the cellular genome in cultured cells and in vivo? Does it work primarily as a transcriptional repressor, or do its activities in promoting chromatin loops or interactions with the nuclear laminin contribute to its activity? Do interactions with cohesin components contribute to the role of CTCF? Are there chromatin insulators, other than CTCF, contributing to the regulation of HTLV-infected cells? Answers to these and other questions should provide novel insights into our understanding of this complex retrovirus.HTLV-1 is a complex retrovirus associated with ATLL through the actions of Tax and HBZ oncoproteins as well as through the integration of site-specific effects on the host cell. Alterations in DNA methylation, histone modifications, and DNA looping play key roles in modifying viral and host cell gene expression. Future studies will uncover the molecular underpinnings of these observations and their pathogenic effects.