However, the viability of using μEMA to capture behaviors and states in a large-scale longitudinal study has not yet been demonstrated.Ecological momentary assessment (EMA) uses mobile technology to enable in situ self-report data collection on behaviors and states. In a typical EMA study, participants are prompted several times a day to answer sets of multiple-choice questions. Although the repeated nature of EMA reduces recall bias, it may induce participation burden. There is a need to explore complementary approaches to collecting in situ self-report data that are less burdensome yet provide comprehensive information on an individual’s behaviors and states. A new approach, microinteraction EMA (μEMA), restricts EMA items to This paper describes the μEMA protocol currently used in the Temporal Influences on Movement & Exercise (TIME) Study conducted with young adults, the interface of the μEMA app used to gather self-report responses on a smartwatch, qualitative feedback from participants after a pilot study of the μEMA app, changes made to the main TIME Study μEMA protocol and app based on the pilot feedback, and preliminary μEMA results from a subset of active participants in the TIME Study.The TIME Study involves data collection on behaviors and states from 246 individuals; measurements include passive sensing from a smartwatch and smartphone and intensive smartphone-based hourly EMA, with 4-day EMA bursts every 2 weeks. Every day, participants also answer a nightly EMA survey. On non–EMA burst days, participants answer μEMA questions on the smartwatch, assessing momentary states such as physical activity, sedentary behavior, and affect. At the end of the study, participants describe their experience with EMA and μEMA in a semistructured interview. A pilot study was used to test and refine the μEMA protocol before the main study.single-question selection method and smartwatch vibrotactile prompting. We also added sensor-triggered questions for physical activity and sedentary behavior. As of June 2021, a total of 81 participants had completed at least 6 months of data collection in the main study. For 662,397 μEMA questions delivered, the compliance rate was 67.6% (SD 24.4%) and the completion rate was 79% (SD 22.2%).Changes made to the μEMA study protocol based on pilot feedback included adjusting the temporally dense intensive longitudinal self-report data in a sustainable manner. Data suggest that μEMA may be valuable for understanding behaviors and states at the individual level, thus possibly supporting future longitudinal interventions that require within-day, temporally dense self-report data as people go about their lives.The TIME Study provides opportunities to explore a novel approach for collecting  Mobile technologies create new opportunities to design personalized health interventions in 2 broad ways. First, mobile technologies such as smartphones and smartwatches can be used to improve the assessment of behavioral, psychological, and contextual states, reducing reliance on known-to-be-problematic retrospective recall surveys that may poorly capture within-day variations -3. Seconwe currently need self-reportinput to measure many subjective experiences  that play an important role in predicting health behavior changes  there is a survey, I answer the question and I am done...The watch questions are actually less burdensome than the phone questions. Watch questions are pretty convenient because you can just touch [tap] on the watch and you’re done basically.F, aged 24 yearsHowever, several participants felt that the μEMA question did not stay available for long enough. A participant stated as follows:I like how it was really simple, it wasn’t like a series of questions. They were just “yes” or “no.” What made it difficult was the timing...it seems like it only lasts for five seconds and it disappears. Sometimes I may be doing something like resting and then survey comes up. Then I have to stick my arm out of the blanket to answer but then it goes away before I could.F, aged 22 years.” Although the smartwatch vibration creates a quiet buzzing sound when on the arm, if the smartwatch is sitting on a surface , the resulting surface vibration can make a surprisingly loud sound that is difficult to ignore.Initially, the prompt only remained active for 15 seconds, which we later updated during the pilot to 20 seconds. Participants generally noticed the smartwatch vibration on their wrists with a delay, despite a relatively aggressive vibration pattern. In fact, pilot participants described the μEMA vibration pattern  as “too loudParticipants reported difficulty answering μEMA questions when both hands were occupied, especially while driving. However, they were instructed to only answer μEMA questions when it was safe to do so, and they were given the specific example of driving as a situation in which they should ignore the prompts. Participants also reported difficulty answering μEMA questions while doing other cognitively demanding activities such as writing. In fact, a participant mentioned missing prompts while being in an examination room, stating as follows:Sometimes if I was taking a quiz or test in class, I didn’t want to be caught using my watch, so I didn’t touch  it.M, aged 19 yearsParticipants found the ability to undo their responses useful, especially when there were accidental taps on the smartwatch. A participant stated as follows:Yes [Undo] was useful because sometimes on the watch...maybe because of the clothes I’m wearing...but [if] an option would be chosen so I’d be able to go “undo” and choose the right answer.F, aged 24 yearsSort of answer option helpful in instances when they were not sure of their experience at the moment and would have had difficulty entering a more limited Yes or No response. A participant stated as follows:Participants also found the Sort of” made me think about my answer a little more rather than an outright yes or no, which is how I kind of determine how I’m going to answer yes or no...if you’re feeling such and such, [then] I do like the “sort of” option because you’re not feeling fully something. And you are not “not” feeling it completely either. So [“Sort of”] is a pretty easy option for people who are feeling like in the middle. “middle answer such as sort of.In response to such observations with the goal of achieving cognitive simplicity to support microinteraction, we designed all the questions to have a In the pilot study, each question presented was randomly sampled from the pool of questions. This resulted in questions either being overrepresented or repeated too consecutively for some individuals, adding monotony, which some participants noted. A participant stated as follows:I think there were questions based on time or place. Questions like how was your day?...It would ask multiple times a day. I don’t know if it was intentional, but it feels like I’m answering the same question over and over again. Same thing for places. It would ask me multiple times “is this your home?” or something like this.M, aged 18 yearsIn the pilot study, each μEMA prompt selected a question at random. As a result, the app had less control over preventing overrepresentation of questions within the day. On the basis of the interviews, we learned that participants found this repetitiveness of the questions monotonous. Thus, for the main study we developed a new question-selection algorithm that (1) guarantees that a given question is never asked consecutively and (2) ensures that a question is not answered more than a predetermined number of times.only when it was contextually relevant.In the pilot study, we randomly sampled questions on PA, SB, and the participant’s current location. However, these questions also felt repetitive to the pilot participants. Thus, in the main study protocol, these questions were asked when the passive sensors (using accelerometer and GPS) detected a relevant event, gathering information validation questions to check whether the participants are paying attention to the μEMA prompts. Second, we added questions related to the slowly changing characteristics of the participants; these questions add variety to the question pool while also gathering useful information. Finally, on the μEMA undo screen, we added more variety in the thank-you messages that participants receive after answering a μEMA prompt. As the main study would last for 12 months per participant, we deemed reducing burden and increasing engagement to be important considerations.We added 2 additional types of questions for μEMA. First, we included We increased the time duration that a μEMA question stays visible on the smartwatch face from 15 seconds to 20 seconds. To address participant concerns about the loud vibration, we changed the pattern from an 11-second-long vibration to a 6-second pattern of 2-second vibrations separated by 1-second pauses.The protocol described here is based on changes made to the μEMA app after the pilot study feedback.The TIME Study uses a nested design with multiple measurement bursts of smartphone-based EMA across a 12-month study period . Each meHere we describe the self-report measures, question-selection algorithms, and simulation results to validate the filtering algorithm to select questions for μEMA.Core-construct questions measure different behaviors, momentary states, and contexts that cannot be objectively captured using sensors and are expected to change throughout the day. The questions are selected to capture reflective processes, reactive processes, and internal and external processes that can predict or explain behavior adoption and maintenance . Examplesurprising participants with approximately 4 novel questions a day. These questions follow the wording requirements of other μEMA questions . The system uses a question bank of >280 questions so that they will not repeat for approximately 3½ months (assuming 60 μEMA prompts per day).In addition to the core-construct questions, 8% (5/62) of the scheduled prompts during waking hours are used to present questions about person-level characteristics. These are questions that gather data on attributes such as whether a participant owns a car, cares for a pet, self-describes as a morning person, or has other specific personality characteristics. The questions serve two purposes: gathering information that changes relatively slowly and breaking monotony  by 1 + 3 = 5?—Yes or No), trivial trivia questions (Sun rises in the east?—Yes or No), intentionally silly questions (Do pigs fly?—Yes or No), and simple attention-assessment questions . To break the question monotony and enhance engagement, validation questions rarely repeat. Our item bank has >300 validation questions for an estimated 7½ months of question prompting without repetition.The remaining 2% (1/62) of the waking-day prompts are used to present validation questions designed to assess whether participants are paying attention to the μEMA questions and answering them carefully . The validation questions are like other μEMA questions—cognitively simple single questions with 2-3 answers. However, these questions always have an unambiguously correct answer to verify response accuracy. Validation questions include simple math problems . In real time, we compute the area under the curve (AUC) of the high-passed raw accelerometer signal for 10-second epochs . The AUConly when a specific sensor event of interest occurs that suggests contextual relevance. The questions are Physically active [Δ] min ago? and Sedentary [Δ] min ago?, with answer options Yes, Sort of, and No, where Δ is the time difference from the current time to the end of the activity plus 25% of the activity bout length . This additional 25% of bout length is used to ensure that the question is asked at approximately the moment in time when the behavior was likely taking place but not at the behavioral transition.Sensor-triggered questions are prompted At each scheduled μEMA prompt time, if there is an eligible sensor-triggered question, the system will trigger the question only 50% of the time. This is done to avoid continuous and monotonous sensor-triggered prompting for prolonged sedentary and physical activities. When the sensor-triggered questions are eligible to be prompted, they take priority over the core-construct, validation, and engagement questions. For instance, if the next scheduled prompt has a core-construct question queued but the app has identified a PA event, then 50% of the time, that core-construct question is replaced by the PA confirmation question. If both PA and SB are identified, the question about the most recent event is presented.A way to gather self-report data on a comprehensive set of behaviors using μEMA is to randomly select a question to ask the user—as was done in our pilot study. However, it may be useful to measure internal factors  more frequently within a day rather than external factors . The app uses a strategic sampling algorithm to maximize the value of data collected about some constructs by limiting presentation of some questions, ensure that questions differ within an hour, and guarantee that each day includes questions from each of four broad construct categories .Each μEMA day, from our bank of 30 μEMA questions of core constructs, the app selects 4 (13%) questions related to internal factors, 2 (7%) to external factors, 2 (7%) to reflective processes, and 2 (7%) to reactive processes. More internal factors than other categories are selected because they are expected to vary throughout the day . Rather Feeling stressed?) than questions that assess more stable constructs . If the number of answers received for a question reaches the question’s limit, that question is no longer presented that day.Each μEMA question is assigned a maximum number of presentations per day . This enFeeling stressed? is answered, it is not asked again for at least one hour. However, if the question is not answered, it could be presented at the next prompt, within the hour.Once a particular μEMA question has been answered, it will not be presented again until at least 60 minutes have elapsed. For instance, if the question μEMA_prompting_day, line 1), the app first generates a day-level subset  of 10 questions from the pool of 30 μEMA questions . Next, at each μEMA prompt, day-level questions that have reached their limit  or have been asked recently  are filtered. From the remaining list, a question is selected at random . If the question prompted is answered, the question’s prompt count is updated , as is the last-answered time . If the question is not answered, then it is available for selection in the next scheduled μEMA prompt.The app uses the following algorithm to select μEMA questions . For eacAlgorithm used to select μEMA questionsFor each μEMA_prompting_day:day_level_subset=subset For each μEMA prompt:filtered_questions=remove_maxed_out_questions (day_level_subset)filtered_questions=remove_questions_answered_within_one_hour (filtered_questions)question_to_ask=select_question_at_random (filtered_questions)if question_to_ask is answered:update_prompt_count (question_to_ask)update_answer_time (question_to_ask)To verify our filtering algorithm , we simuWe simulated question selection for 365 days for 300 users, where 261 days were reserved for μEMA prompting, resulting in 78,300 total μEMA days. We assumed 8 hours of sleep and 16 hours of wake time per day, resulting in 15½ hours available for μEMA prompting. Targeting 4 prompts an hour results in 62 μEMA prompting opportunities. Of these, 90% (55/62) were available for core constructs, resulting in 55 slots for core-construct questions each day. We assumed that 75% (46/62) of the scheduled prompts would be answered .Compared with random sampling , the filParticipants were eligible for the TIME Study main trial if they (1) owned an Android smartphone running Android version ≥6.0 as their only personal mobile phone with no intention to switch to a non-Android smartphone, (2) did not wear a smartwatch already, (3) were aged 18-29 years and living in the United States, (4) were currently engaged in recommended levels of PA (or intended to within the next 12 months) , (5) spoBecause of COVID-19 restrictions on in-person recruitment for human subjects research, all participant recruitment and onboarding was conducted remotely. After screening, research staff individually met with each participant remotely (through Zoom) to obtain informed consent. Staff then guided the participants through the TIME app installation on their personal smartphones. Researchers then observed EMA burst compliance for the first 4-day EMA burst  during the next 2 weeks. If the compliance for the run-in period was <8 surveys per day on all days, the participants were withdrawn from the study. Otherwise, they could continue in the study and were mailed a smartwatch (Fossil Gen 4 or Gen 5 model). Once the participants received the smartwatch, a staff member scheduled a second remote orientation session with the participant to guide them through the TIME smartwatch setup. Expectations for smartwatch wear (including wearing it during sleep), charging it, and answering μEMA questions were explained. Participants were instructed that to remain compliant they should wear the smartwatch for 23 hours a day and charge it every day. They were also instructed to wear the smartwatch while sleeping. For μEMA prompting, participants were instructed as follows:“You will be prompted on the smartwatch with single questions of the type Are you walking right now? Yes | Sort of | No. These questions will take only 2-4 seconds to respond to—just like checking time on the smartwatch. There may be up to six such question prompts in an hour, on average, and the frequency of question prompts may vary at different times of your waking day. All the smartwatch questions will be outside of the 4-day smartphone survey burst periods. Each watch prompt will wait for up to 20 seconds for you to respond; if the question is not answered within 20 seconds, the question may disappear from the watch. Unlike phone surveys, watch questions are not re-prompted.”After the smartwatch orientation, participants could use the smartwatch as they deemed fit , while answering μEMA questions when they appeared. Participants receive US $20 per month for wearing the smartwatch for 23 hours on at least 24 days of the month. Participants could earn up to an additional US $80 per month for EMA burst compliance . In addition, if at the end of the study, participants have >50% compliance with μEMA, they were able to keep the smartwatch as their personal device. However, no monthly or regular compensation was provided for μEMA compliance. Participants do not receive feedback on their μEMA compliance, and their withdrawal from the study does not depend on μEMA compliance.We measure compliance rate, completion rate, undo rate, and validation rate to characterize participant response behavior when answering μEMA questions.Compliance rate is measured as the percentage of μEMA questions answered out of all the scheduled questions, including those not prompted because of the device being turned off or in DND mode.Completion rate is measured as the percentage of μEMA questions answered out of all the delivered questions, excluding those not prompted because of the device being turned off or in DND mode.Undo? option was selected after providing an initial answer.Undo rate is measured as the total number of μEMA questions answered when the Validation rate is measured as the percentage of μEMA validation questions answered correctly out of all the validation questions answered.Overall, the compliance rate (n=81 at ≥6 months) for μEMA was 67.7% (SD 13.7%) and the completion rate was 80.23% (SD 13.3%). The μEMA validation rate for the participants (n=81) was 92.9% (SD 23.7%)—indicating that they were paying attention to the μEMA question content and not carelessly tapping to dismiss them.Table 4 presents a summary of the response behavior. We also verified the filter-based question-selection algorithm’s performance against the real-world data from 2% (2/81) of the study participants who completed 6 months in the study . We founOur work with μEMA demonstrates that the technique may enable temporally dense ILD collection with manageable burden to support longitudinal studies or interventions, although some limitations of this work will require further investigation.Traditional mobile phone–based EMA can lead to interruption and response burden. To prevent this burden, researchers often compromise on the temporal density of prompts  and reduce the number of questions or constructs being measured. Complementary forms of EMA are needed where both the researchers’ needs for comprehensive understanding of health behaviors and users’ concern for burden are taken into account. μEMA provides such an opportunity, where the quick microinteractions not only make it less burdensome for the users to answer questions on the smartwatch, but also enable data gathering at a higher temporal density.The TIME μEMA substudy combines smartphone-based EMA with low-burden μEMA on a smartwatch to gather real-time self-report data from naturalistic settings using the personal smartphones of participants. Participants are encouraged to use their smartphone and the loaned smartwatch normally with only a few limitations . Participants are incentivized to wear the smartwatch, but they can pause smartwatch survey prompting as needed. Participants are not directly compensated for high μEMA compliance, although they are told that they can keep the smartwatch if they have >50% μEMA compliance at the end of 1 year. EMA compliance, by contrast, is reinforced with an explicit and compliance-contingent reward provided at regular monthly intervals.Our preliminary results from the pilot and ongoing TIME μEMA substudy show that despite a high interruption rate, μEMA could be sustainable for gathering self-report data longitudinally. Controlled filtering of μEMA questions enables measurement of a comprehensive set of behaviors repeatedly during waking hours. The validation questions make it possible to identify careless responding on both μEMA and smartphone-based EMA.The TIME μEMA substudy can be used to assess the utility and sustainability of μEMA. Comparing response rates with the qualitative data from the exit interviews, for example, suggests that μEMA can be used to gather temporally dense self-report information. In addition, passively collected data on wrist motion (using accelerometers) and location (using GPS) may help explore contextual factors that affect μEMA compliance. The filtering-based μEMA question-selection algorithm may be used to capture diurnal patterns of different constructs during waking hours. These diurnal patterns could be used to explore (1) different clusters of individuals who have similar diurnal patterns for variables of interest  and (2) Overall, the purpose of μEMA is not to replace traditional EMA but to complement it. The TIME Study’s μEMA substudy protocol provides an opportunity to explore how small amounts of information gathered at high frequency can help us learn about health behavior change and maintenance, even as constructs related to behavior change deviate by the hour. Beyond observation studies, TIME Study data could enable exploration of the viability of μEMA for longitudinal interventions , where self-report on many different behaviors may be required. For instance, when optimizing mobile health interventions as part of microrandomized trials , μEMA maThere will be several limitations of the TIME μEMA substudy that will provide opportunities for future research. Findings about the μEMA methodology may not generalize to other population age groups because the main protocol is designed to study health behavior adoption and maintenance in young adults (age 18-29 years). There has been an increased interest in exploring the acceptance of wearable technologies in older adults -62, youtWe had to restrict our data collection to Android users because our research app used advanced sensor capabilities not programmatically available on iOS devices; thus, the TIME μEMA substudy excludes iOS users. Nevertheless, we observed more demographic diversity in the Android users who participated in our screening surveys  versus the iOS users . We have no reason to believe that the platform itself would affect μEMA response patterns.Yes, Sort of, and No) that may be less sensitive to construct variance than mobile phone–based EMAs with more response options. Yet, with a median of ≥4 measurements per construct within a day .The TIME μEMA substudy provides an opportunity to explore a new method of collecting temporally dense self-report data. Although EMA provides information on temporal dynamics of behavior, the burden of accessing the mobile phone, unlocking it, and then answering multiple complex questions compromises engagement. In this formative research, we explored the feasibility of using μEMA to measure multiple constructs in an ILD study. When deploying μEMA, each prompt is a cognitively simple single question that can be answered with a quick glanceable microinteraction with an always-accessible smartwatch. Because of this simplicity, μEMA permits a higher interruption rate than EMA without a corresponding increase in perceived burden. The TIME Study is the first ILD study to deploy μEMA for an entire year; thus, it could provide insights on methodological properties of μEMA—especially the ability to sustain participant engagement. Data from the study could be used to study what influences μEMA compliance, what can be learned about an individual’s behavior using μEMA, and how μEMA might be used along with EMA and passive sensors to richly characterize human behavior, especially how it may change throughout the day in response to rapidly changing contexts.


Staphylococcus aureus (MSSA) is the most frequent organism, and the majority are endogenous. Decolonisation reduces PJIs but there is a paucity of evidence comparing treatments. Aims; compare 3 nasal decolonisation treatments at (1) achieving MSSA decolonisation, (2) preventing PJI.Periprosthetic joint infection (PJI) causes significant morbidity. Methicillin sensitive Our hospital prospectively collected data on our MSSA decolonisation programme since 2013, including; all MSSA carriers, treatment received, MSSA status at time of surgery and all PJIs. Prior to 2017 MSSA carriers received nasal mupirocin or neomycin, from August 2017 until August 2019 nasal octenidine was used.P < 0.0001). There was no difference in PJI rates (P = 0.452).During the study period 15,958 primary hip and knee replacements were performed. 3200 (20.1%) were MSSA positive at preoperative screening and received decolonisation treatment, 698 mupirocin, 1210 neomycin and 1221 octenidine. Mupirocin (89.1%) and neomycin (90.9%) were more effective at decolonisation than octenidine  and PJI rates. Further research is needed to compare alternative MSSA decolonisation treatments. Hip and knee arthroplasty offers excellent outcomes for end-stage osteoarthritis and other painful conditions . PeriproStaphylococcus aureus (MSSA) is the most common organism isolated in early postoperative PJI [Methicillin sensitive tive PJI , accounttive PJI . A link tive PJI . DNA anative PJI , 10, 11.tive PJI , 11, 12.S. aureus decolonisation is effective at reducing PJIs [There is strong evidence that nasal ing PJIs , 14. Howing PJIs . There iing PJIs .Octenidine HCl is a topical antiseptic with activity against both gram-positive and gram-negative bacteria. As an antiseptic nasal gel octenidine can be supplied by the surgical pre-assessment team without prescription, streamlining the process, and reducing cost. Although it has less potential for inducing resistance than mupirocin , there iNeomycin is an aminoglycoside antibiotic active against both gram-positive and gram-negative bacteria. There is limited research into the efficacy of neomycin ointment for nasal MSSA decolonisation. Leigh et al. showed neomycin achieved nasal decolonisation in 61% of cases compared to 95% with mupirocin at 8 days after treatment . ResistaThis study aims to compare the efficacy of nasal mupirocin, neomycin or octenidine, as part of an MSSA eradication bundle with octenidine skin wash, at both achieving MSSA decolonisation and reducing rates of MSSA PJI in primary hip or knee replacement.S. aureus media  without pre-enrichment. Plates were read at 18 and 24 h, further identification was performed using the VITEK MS  and sensitivity testing using the VITEK 2. Clinical and Laboratory Standards Institute (CLSI) breakpoint standards were used to define methicillin sensitivity. Screening was performed around 14 days prior to surgery, if surgery was delayed beyond 28 days then screening was repeated. Patients testing positive for MRSA were excluded from this study.In 2010 our organisation introduced an MSSA screening and decolonisation programme for patients undergoing elective hip and knee joint arthroplasty. Patients underwent seperate groin and nose swabs for detection of MSSA during their preoperative assessment. Liquid swabs  were originally used until a change to flocked swabs  in June 2018. Swabs were directly streaked onto Colorex Patients testing positive for MSSA carriage were given nasal decolonisation treatment to use 5 days prior to surgery and 5 days after . Group one, from 2010 until August 2017, used nasal mupirocin 2% . Group two, from August 2017, used an octenidine HCl 0.1% nasal decolonisation agent . Group three used neomycin 0.5% and Chlorhexidine 0.1% nasal cream , this was used as an alternative to mupirocin due to periods of national supply shortage. All patients received an octenidine HCl body wash  for daily use 5 days prior to surgery.Nasal cavity cleared if needed.Cream introduced into the anterior nasal cavity with a finger or cotton bud for 2 min.Cream spread further by pressing the sides of the nose together.Any excess cream removed.Patients received verbal and written instructions on how to apply both the nasal treatment and the body wash. Octenisan body wash was applied, once per day, to wet hair and body using a wash cloth, after 1 min skin contact this was washed off. Hair was only included on alternate days (days two and four). Mupirocin and octenidine were both applied twice daily, neomycin was applied four times per day. The steps for nasal treatment are:Primary outcome was decolonisation efficacy, measured by MSSA positive culture on the day of surgery. The secondary outcome was MSSA PJI.Data was collected prospectively by the SSI surveillance team locally on all MSSA carriers from September 2013. This data has been retrospectively analysed for this study. Four groups of interest were identified from this database. The first three groups were positive at initial screening and treated as per protocol at the time, receiving nasal mupirocin, octenidine or neomycin. The fourth were negative at initial screening but positive when swabbed on the day of surgery  criteria . Cases oStatistical analysis was performed using Statistical Package for the Social Sciences (SPSS Version 26). All results quoted to two significant figures. Categorical data was analysed using chi squared test.Data for cases from September 2013 to August 2019 was analysed. Groups one and two covers a period of 47 months, group three covers 24 months . Significantly more patients remained colonised with MSSA after treatment with octenidine (50.0%) than with mupirocin  or with neomycin . There was no statistical difference between mupirocin and neomycin (P = 0.393).Microbiology records were reviewed, in total 1776 patients (56.8%) were swabbed for MSSA on day of surgery , MSSA was isolated from deep tissue samples in ten cases (0.23% MSSA PJI rate). There was no statistical difference between the four groups . This is likely to represent both false negatives at screening and intermittent carriers.Staphylococcus aureus is recognised as the most common causative organism in early postoperative PJI [tive PJI . Since ttive PJI , 21, 22,tive PJI , 24). Sitive PJI . Decolontive PJI .The topical antibiotic mupirocin is the only treatment that currently has good study data. The majority of existing research focuses on decolonisation as a method to reduce SSI and nosocomial infections i.e. an active treatment is compared to a non-active control. There are very few studies comparing different treatment options. In the UK, National Institute for Clinical Excellence (NICE) guidance states mupirocin should be considered whenever MSSA is a likely cause of SSI .Mupirocin is a topical antibiotic working through inhibition of bacterial isoleucyl-tRNA synthetase . WidesprS. aureus infections, nasal decolonisation rates were 95% for mupirocin and 61% for neomycin 8 days after treatment [In our cohort of 3129 patients, mupirocin and neomycin were significantly more effective at MSSA decolonisation (89% and 91% of patients decolonised respectively) than octenidine (50% decolonised). On the day of surgery patients were 5.1 times more likely to be positive on swab culture when treated with octenidine. In the largest study of nasal decolonisation Perl et al. compared mupirocin to placebo in over 4000 patients . Mupirocreatment . The autreatment , 33. OurDespite the significant difference in decolonisation efficacy, in our study, this did not translate into a difference in MSSA PJI rate. There was no statistical difference in MSSA PJI rate between the three treatment groups or with group 4. The authors acknowledge that the sample size is too small to draw firm conclusions on PJI.Interestingly, all three infected cases in group 3 (octenidine) were caused by MSSA. There were no PJI caused by other organisms. Octenidine has a broad spectrum of activity , it may A meta-analysis in 2020 showed that mupirocin nasal decolonisation reduced PJI compared to control groups (placebo or no treatment) . Using aS. aureus SSI. Povidone-iodine is of particular interest as it is applied as a single treatment on the day of surgery potentially reducing issues of compliance. MSSA decolonisation is dependent on the total number of doses received [Phillips et al. compared mupirocin to a povidone-iodine solution for nasal decolonisation in a cohort undergoing arthroplasty or spinal fusion . Similarreceived . Our insThe European Medicines Agency (EMA) has recommended that, based on a presumed impact on clinical end points, microbiological outcomes can used when evaluating nasal decolonisation for orthopaedic or cardiac surgery . Our resth group also had no statistical difference in deep infection rates. Bacterial density clearly plays a significant role that is not fully understood but could explain why intermittent carriers and incomplete eradication may not increase the risk of PJI. Around one third of patients with MSSA carriage appear to have intermittent carriage which can be missed on screening. This supports the concept of universal decolonisation for all patients undergoing high risk surgery without screening, or the use of more sensitive detection techniques such as PCR. Previous cost analysis published from our instituition showed screening costs of £8 per patient compared to £7.73 for decolonisation treatment. Universal decolonisation without screening is cheaper (£8 saving for carriers and £0.27 for non-carriers) and avoids missed carriers. The potential downside is the development for increased resistance with increased use of mupirocin. This further highlights the need for research to compare alternative agents.MSSA carriers represented 20% of our cohort, with a further 9.1% newly positive on day of surgery, possibly representing intermittent carriers. Despite commencing treatment peri-operatively, this 4P = 0.108). This reduction could have impacted on the MSSA PJI rates, concealing a higher PJI rate with octenidine. The changes referred to would not be expected to impact on the efficacy of the MSSA decolonisation regime used at the time. As a result of this analysis our organisation returned to using mupirocin as the primary agent. This change was again audited to ensure its effectiveness, 6/72 (8.3%) were positive on admission for surgery. This is consistent with the original decolonisation efficacy suggesting there are no confounding factors that changed over the time period of the study. Over the course of the study the swabs used to acquire samples were changed. Regular internal validation was performed to ensure there was no change in the sensitivity of the techniques and equipment used.The main limitation of this study is the lack of randomisation. The data covers 6 years, during which multiple changes were made to standard practice and surgical techniques to improve patient care including reducing infections. The PJI rate did decrease markedly over this time, this is evident in the difference in overall PJI rate between the two treatment groups in Table Over time the proportion of patients re-swabbed on admission increased, as such there is a statistically significant difference in the proportion between the groups. This is a potential source of bias. The research team discussed this with the nursing staff responsible for admitting patients, it appears the decision was down to the member of staff’s interpretation of the protocol and not any patient factors.Due to the low incidence, this study was likely to be underpowered to detect a difference in the secondary outcome, MSSA PJI. A Post-hoc power analysis was performed for our study, for the group 2 versus group 3 MSSA PJI analysis we calculated the power to be 0.27. A sample size of 14,094 would be needed to achieve a power of 0.8.There is now good evidence to support MSSA decolonisation therapy prior to major joint arthroplasty and other high risk surgeries. Mupirocin is currently the only agent with strong clinical evidence but mupirocin, neomycin and octenidine are in common use. There is a significant lack of evidence to compare different decolonisation agents. In our non-randomised cohort of 3129 patients we have studied the efficacy of mupirocin, neomycin and octenidine, and found no statistical difference in efficacy between mupirocin and neomycin. However, they were both significantly more effective at MSSA decolonisation than octenidine, with 5.1 times more patients remaining MSSA positive after treatment with octenidine. Any possible differences in the agents and decolonisation did not translate to a difference in deep infection rate.With the threat of drug resistance further randomised research is needed to establish effective alternatives to mupirocin. The importance of bacterial load must be better understood to develop improved screening and treatment protocols.


Graphene, a novel form of the hexagonal honeycomb two-dimensional carbon-based structural material with a zero-band gap and ultra-high specific surface area, has unique optoelectronic capabilities, promising a suitable basis for its application in the field of optical fiber sensing. Graphene optical fiber sensing has also been a hotspot in cross-research in biology, materials, medicine, and micro-nano devices in recent years, owing to prospective benefits, such as high sensitivity, small size, and strong anti-electromagnetic interference capability and so on. Here, the progress of optical fiber biochemical sensors based on graphene is reviewed. The fabrication of graphene materials and the sensing mechanism of the graphene-based optical fiber sensor are described. The typical research works of graphene-based optical fiber biochemical sensor, such as long-period fiber grating, Bragg fiber grating, no-core fiber and photonic crystal fiber are introduced, respectively. Finally, prospects for graphene-based optical fiber biochemical sensing technology will also be covered, which will provide an important reference for the development of graphene-based optical fiber biochemical sensors.  In 2004, graphene was separated by the 2010 Nobel Prize winners Andre Geim and Konstantin Novoselov [Graphene is a two-dimensional single-crystal functional material with a hexagonal honeycomb lattice composed of carbon atoms with spovoselov . Moreoveovoselov . Becauseovoselov ,14,15,16Optical biochemical sensors have broad development prospects because of their advantages of non-destructive detection, high sensitivity and fast detection speed, etc. . The opt2/Vs) at room temperature, a high specific surface area and a unique Dirac cone structure leading to zero-band gaps. All these contribute to absorption on the surface of biomolecules, leading to extremely sensitive biomolecule sensing. In recent years, graphene biochemical sensors have been explored by numerous scholars due to their obvious advantages, high sensitivity, and label-free, real-time detection of bio-logical molecules  [As a new functional 2D material, graphene can be easily integrated into optical fiber because of its high electrical conductivity, flexibility, strength, and lack of dangling bonds. Then, graphene has no mass and has an ultra-fast migration speed  ,28,29. FA, etc.) . In 2017A, etc.) . In 2021A, etc.) . In 2019A, etc.) . In 2017A, etc.) . MoreoveA, etc.) . ThroughIn this paper, development of optical fiber biochemical sensors based on graphene is reviewed. Firstly, four basic methods of preparing graphene, including mechanical the exfoliation method, chemical vapor deposition method, epitaxial growth method, and oxidation-reduction method are introduced, and the advantages and disadvantages of each method are stated. The next section describes the graphene-based optical fiber-sensing mechanism, including sensing properties of graphene and the graphene optical fiber-sensing mechanism. In the following sections, we review recent progresses in different various typical types of optical fiber biochemical sensors, covering the long-period fiber-grating sensor, Bragg fiber-grating sensor, no-core fiber sensor, and photonic crystal fiber sensor. Finally, the issues of the graphene optical fiber biochemical sensor are proposed and the progress to be made in the future is described, which not only clearly clarifies the current development stage, but also provides an efficient feasible direction and guidance for the future exploration of graphene-based op-tical fiber biochemical sensors.Graphene, as one of the 2D-layered nanomaterials, can be synthesized in two techniques: top-down exfoliation and bottom-up growth. The top-down method, including the mechanical exfoliation method, liquid phase exfoliation technique and oxidation-reduction method (redox), is used to prepare single or few-layer 2D nanomaterials by destroying the van der Waals forces between the layers of 2D materials. The bottom-up method is the synthesis of 2D nanomaterials at the molecular level by chemical means, including chemical vapor deposition, epitaxial growth method, hydrothermal method, and pulsed magnetron sputtering, pulsed laser deposition, etc. ,38,39,40The mechanical exfoliation method, also known as the micromechanical cleavage technique, is an effective way to exfoliate graphene into single- or few-layer nanomaterials by applying mechanical force on layered bulk crystals. 2SO4, NaNO3, and KMnO4. Then, reduction can obtain graphene by using hydrazine hydrate and ammonia water as reducing agents and mixing the reducing agent with the GO solution by ultrasonic dispersion. Redox has the advantages of low environmental pollution, high efficiency, high feasibility, low cost, and large-scale production in the industry, etc. However, it is easy to produce the pollution of liquid waste during the synthesis process and to destroy the electronic structure of the graphene crystal due to strong oxidizers, which affects the performance of graphene and limits its development in the field of microelectronics.At present, the redox method is a relatively commonly used method for preparing graphene . Figure CVD synthesizes graphene by the chemical decomposition material of carbon source under a certain temperature or in an external field and deposition on the substrate surface ,48,49; iIn 2006, Bergeretal C. et al. used the epitaxial growth method to prepare monolayer and multilayer graphene sheets . The thiGraphene has been considered as a multifunctional material because of its 2D planar structure. It has excellent potential applications in wide range of fields, such as photonics, optoelectronics, sensors, flexible electronics and nanocomposites, etc. . In part2/(Vs). Because of its excellent carrier mobility and mechanical properties, graphene is widely favored by researchers in condensed matter physics and materials science. Graphene has been widely used in the field of biochemical sensing based on its good photoelectric characteristics and excellent high-energy transfer efficiency, large surface area, and biocompatibility [The structure of graphene is shown in tibility . This seGraphene has excellent optical absorption characteristics. The absorption rate of monolayer graphene to visible light is only 2.3% , and thePhotoluminescence (PL) is an optical phenomenon that semiconductors launch light emissions by absorbing incident light, in which semiconductors emit light by absorbing incident light with energy higher than the energy band gap of the semiconductor. In the mechanism of PL, the excited electrons generated by optical excitation will return to the ground state with the emission of photons . Graphen5 cm2/(V·s), which is about 100 times that of the electron in silicon and the resistivity is about 10−6 Ω·cm, which is lower than that of metallic copper or silver. Therefore, it is the material with the lowest resistance at room temperature, and its conductive density is one million times that of copper. As the number of graphene layers increases, its conductivity gradually decreases, and then tends to remain unchanged at eight monolayers [Graphene’s unique structure enables it to have special properties, such as the perfect quantum tunneling effect and quantum Hall effect, etc. The optical conductivity of graphene depends on electron density, layer number and width at low temperatures. When electrons are transmitted in the graphene layer, the interference is very small and it is not easy to be scattered. The mobility can reach 2 × 10nolayers . The optnolayers ; the optGraphene is an excellent plasmon material , which iThe energy band of graphene has a linear dispersion relation, and its Dirac cone structure provides a unique zero band gap, large specific surface area, ultra-broadband optical spectrum, and strong nonlinear optical properties, which enable it to have a certain optical transmission ability. In this section, we will mainly introduce the energy band theory and sensing mechanism of graphene.2 hybrid orbital. Kx and Ky are the components of the wave vector k. As k and k’ are symmetric, and the conduction and valence bands at two points in the Brillouin zone are degenerate, the linear dispersion relationship of the energy bands of graphene is obtained. Hence, electrons can be considered to be massless relativistic particles, namely Dirac fermions, and the dispersion relation of the two-dimensional electron energy is isotropic, and is called Dirac cones. [It can be observed from c cones. ,82. Figuc cones. ,84,85,86c cones. ,88.(1)σ0 = πe2/2/π, which can be controlled by chemical doping or applied voltage. There is no intra-band contribution in the µ = 0 of pristine graphene. It can be observed from the theoretical expression and experimental results of optical absorption that the intra-band photoconductivity σ in the terahertz and far-infrared bands dominates due to the appearance of the zero-band gap, while the total conductivity variation in the near-infrared and visible light bands mainly depends on the transition process between the bands. Particularly, the propagation of surface plasmons in graphene has relations to the intra-band contribution.From the above formular, it can be observed that the intra-band and inter-band optical conductivities of graphene are concerned with μ and ω. The μ of doped graphene is controlled by the carrier concentration nThe optical fiber evanescent wave biochemical sensor works based on the principle of the evanescent wave . The evaIt is well known that biochemical sensors have been widely used in medical diagnosis, environmental monitoring, food safety, genetic engineering and other fields, and the accuracy and requirements of detection are becoming higher and higher with the development of artificial intelligence technology. Graphene and its derivatives (graphene oxide-GO), as new sensitive materials, are rapidly becoming a popular material for fiber-optic biochemical sensors. Based on the two-dimensional monolayer honeycomb lattice structure, GO is formed by enriching oxygen-containing functional groups (hydroxyl and carboxyl groups) on the surface of graphene through oxidation. Compared with graphene, the functional groups of GO are highly dispersed, hydrophilic and modifiable. Its unique two-dimensional structure and abundant oxy-gen-containing functional groups on the surface have presented unique advantages in biochemical-sensing applications. As shown in In recent years, researchers have discovered that the change in the effective refractive index of the optical fiber evanescent field caused by the combination of the substance to be tested; and the molecular film can be used to achieve detection, which involves using ultraviolet light to modify the refractive index of the fiber core on a regular basis in order to create a spatial phase grating . Fiber gLong-period fiber gratings are also called transmission fiber gratings, and their period is relatively long, which is generally hundreds of microns. In 2017, Liu Chen’s research team studied biosensors based on graphene oxide nanosheets that functionalized dual-peak long-period grating (dLPG), and proposed a label-free biosensor based on GO-coated dLPG for the real-time detection of the biological affinity between the antibody and the antigen; this was through a new GO deposition technology that combines chemical bonding and physical adsorption, enabling GO to bind to dLPG firmlyand uniformly with good stability and high repeatability. −1 to the change of methane gas concentration, which is 2.96 times and 1.31 times higher than the traditional long-period fiber-grating sensor and the silver-plated long-period fiber-grating SPR sensor, respectively. The performance of the LPFG SPR sensor can be improved by covering the graphene. It has good response characteristics and repeatability, which provides a broad prospect for high-sensitivity graphene gas sensors [In 2017, Wei Wei’s research team proposed a SPR sensor based on graphene LPG for high-sensitivity gas-sensing detection. A layer of single-molecule graphene is coated on the surface of the Ag film of the long-period optical fiber SPR sensor to enhance the evanescent electric field intensity on the surface of the optical fiber, thereby enhancing the interaction between the SPR wave and the molecule.  sensors .In 2020, Wang’s research team developed a graphene oxide micro-tapered long-period fiber-grating (MTLPG) biosensor for the detection of hemoglobin molecules . The resIn 2021, Esposito’s research team developed a long-period fiber-grating biosensor based on the graphene oxide double-clad structure for the detection of C-reactive protein (CRP) in human serum . The minBragg fiber gratings are also called reflective or short-period fiber gratings, and the grating period is only a few hundred nanometers. In 2014, Sridevi et al. developed a graphene oxide-etched Bragg fiber-grating biosensor for the detection of the protein concanavalin A (Con A) . In 20152 gas in room temperature sensing. 2 gas-sensing mechanism on RGO coated eFBG. 2 gas molecules on RGO changes the refractive index by increasing and decreasing the local carrier concentration. The RGO-coated eFBG sensor can detect sub-ppm level NO2 gas. By using multiple gratings in the same fiber, the performance of the sensor can be improved to sub-ppb level, and the response time can be shortened, providing more effective biochemical-sensing reference data [In 2016, the Sridevi research team studied an etched fiber Bragg grating (eFBG) chemical sensor based on the redox graphene (RGO) coating for NOnce data .The classic approach of improving the sensitivity of optical fiber sensors involve corroding the structure of fiber cladding . HoweverYang HongYan’s research team designed a single-mode no-core single-mode (SNS) fiber step-index distribution sucrose sensor for glucose detection. Photonic crystal fiber (PCF) has been proven to be a good prism. Single-mode transmission, excellent birefringence, and tunable dispersion characteristics are all advantages of its unique structure and flexible design . The com−1 and a resolution of 3.97 × 10−5 RIU [−1 in the refractive index range of 1.39–1.42 and the maximum sensitivity reaches 10,000 nmRIU−1; the resolution is 1 × 10−6 RIU in the range of 1.43–1.46 [In 2014, Dash et al. proposed a birefringent PCF-SPR biochemical sensor based on a graphene-silver layer ; graphen10−5 RIU . In the .43–1.46 .−1, resolution is 7.1 × 10−5 RIU, wavelength sensitivity is 2000 nmRIU−1, and resolution is 5 × 10−5 RIU. Lou et al. proposed an eccentric PCF-SPR biochemical sensor based on graphene-gold coated on the outer surface of PCF in 2019 [−1; the maximum sensitivity is 8600 nmRIU−1.Rifat et al. coated graphene copper on the outer surface of PCF in 2016, and constructed a PCF-SPR biochemical sensor based on the graphenecopper layer . Within  in 2019 . After gIn 2018, Wang qi’s research team proposed and developed a sensitivity-enhanced SPR biochemical sensor based on the GO complex and staphylococcal protein A (SPA) co-modified PCF for detecting human IgG. . The schCompared with circular-shaped PCF, D-shaped PCF has an asymmetric cross-sectional structure and a wide and flat side polishing surface, which is not only conducive to the transfer and deposition of graphene, but also make it easy to reprocess the graphene on the side-polishing surface . Chemica−1, and a resolution of 2 × 10−5 RIU. The sensor can be applied to real-time detection of biomolecules and tiny drug molecules due to the long-distance transmission, high sensitivity and resolution.In 2018, Tong Kai’s research team proposed a D-shaped PCF biosensor based on SPR graphene coating . Graphen−1 and a maximum amplitude sensitivity of 192 RIU−1 in the refractive index range of 1.27–1.37 in the internal filling detection scheme; the maximum sensitivity is 22,400 nmRIU−1 and the maximum quality factor (FOM) is 127 in the external-filling detection scheme.Yashar et al. proposed a D-shaped PCF biochemical sensor based on SPR in 2021 . They st−1 and a maximum resolution of 8.33 × 10−6 RIU in the refractive index range of 1.21–1.32. This type of chemical sensor offers good sensing performance and a lot of economic promise in biochemical sensing and environmental monitoring and assessment. However, it cannot obtain excellent sensing performance in the entire effective sensing range which should be the direction of our future work.Instead of using metal layers as plasma materials, some researchers employed two-dimensional materials and put a layer of graphene on the film for study. In 2021, Tian et al. proposed a graphene-coated D-shaped PCF chemical sensor based on SPR, which uses indium tin oxide (ITO) instead of metal as the plasma material . The senFor the past few years, graphene-based fiber-optic biochemical sensors have demonstrated excellent performance in genetic engineering, medical diagnosis, environmental monitoring, and food safety, etc. However, the widespread application of graphene-based commercial fiber biochemical sensors still faces challenges, and the following key issues need to be solved: Firstly, the current research on graphene is still limited to theories and laboratories-the existing preparation methods of graphene are not perfect enough. How to prepare high-quality graphene with controllable size and thickness, no defect, no pollution, and uniform distribution at a low cost is one of the urgent problems to be solved. Secondly, when graphene is transferred to the surface of the fiber, it is easy to be contaminated and damaged. How to realize the lossless and high-quality coupling between graphene and fiber is the second key problem to be solved. Thirdly, graphene’s flexible photoelectric characteristics make it difficult to find a perfect solution to the cross-sensitive detection. For example, temperature can interfere with the gas sensor when measuring gas concentrations. Finally, the repeatability and stability of graphene sensors are not optimistic either. When the external environment causes the electronic structure of graphene to change, it will result in graphene taking a certain time to recover. It is difficult to automatically restore it to the original state, which leads to the poisoning of the sensor when graphene adsorbs chemical molecules.As the world is suffering from COVID-19, the public health security system of mankind is facing unprecedented challenges. If the research of the graphene fiber biochemical sensor can break through the limitation of theory and experiment and put it in to large-scale industrial production, it will rapidly improve the pace of human intelligent life. Here, based on the above reasons, by choosing three graphene-based fiber biochemical sensors with special fiber structures  as breakthrough points, we reviewed the new research development and existing problems of the graphene-based fiber biochemical sensor in detail, which provides reliable data and novel ideas for the practical application of the graphene-based fiber biochemical sensor in order to achieve a major breakthrough in the fiber biochemical sensor. 2) and black phosphorus (BP), etc., which is the multifunctional integration with optical fiber to realize biochemical sensing. Moreover, graphene, as a modifier combined with semiconductor materials, in order to realize the application of graphene photoelectric sensors and graphene-based flexible implantable biochemical sensors, is mushrooming development. In the future, graphene will present extraordinary talents in the field of artificial intelligence and wearable sensing as a new material for fiber sensors.Although much work still need to be conducted for the application of the graphene-based fiber biochemical sensor, the future for graphene-sensing devices appears very exciting. In order to improve the performance of graphene-based fiber-optic biosensors, there have been many improved progresses in the preparation of graphene, the structural form of graphene and the design of graphene-based fiber-optic biosensors. For example, functionalized graphene is modified by other molecules or atoms, such as doping molecules and adding substrates, etc., in order to improve the performance of graphene. So far, in addition to graphene and its derivatives, there have been many reports on novel two-dimensional materials, such as molybdenum disulfide (MoS


Vascular smooth muscle cells (VSMCs) are stromal cells of the vascular wall and are continually exposed to mechanical signals. The loss of VSMCs is closely related to the occurrence of many vascular diseases, such as aortic aneurysms and aortic dissection. The proliferation and apoptosis of VSMCs are mechanically stimulated. Yes-associated protein (YAP), one of the core components of the Hippo pathway, plays a key role in the response of VSMCs to mechanical signals. In this study, we tested the impact of different intensities of mechanical stretch on the proliferation and apoptosis of VSMCs, as well as YAP. We tested VSMCs’ proliferation and apoptosis and YAP reaction via immunocytochemistry, western blotting, CCK-8 and flow cytometric analysis. We found that 10% elongation could increase the phosphorylation of YAP and prevent it from entering the nucleus, as well as inhibit cell proliferation and promote apoptosis. However, 15% elongation reduced YAP phosphorylation and promoted its nuclear entry, thereby promoting cell proliferation and inhibiting apoptosis. Accordingly, YAP knockdown suppressed the phenotype of VMSCs induced by 15% elongation. Taken together, YAP regulates proliferation and apoptosis of VSMCs differently under different intensity of mechanical stretch. Mechanical stretch with appropriate intensity can promote the proliferation and inhibit apoptosis of VSMCs by activating YAP. Aortic aneurysms are associated with aortic dissection and rupture. There is currently no effective treatment to prevent or cure aortic aneurysms. At present, both the pathogenesis and pathophysiology of ascending aortic aneurysms are not entirely clear. Vascular smooth muscle cells (VSMCs) have been recognized as the most important factor in the development of ascending aortic aneurysms. Aortic aneurysms can occur due to loss of VSMCs in the media layer of the aortic wall, leading to progressive aortic dilation –3. We obAortic walls are subjected to various mechanical stimuli from the bloodstream, such as shear and mechanical stretch. Stretch sensing is generally known as an integrin-mediated pathway, which is coupled to cell contractile activity, and thus shares many mechano-transduction pathways with the rigidity sensing process in translating mechanical stimuli into intracellular biochemical signals –6. The rDrosophila by genetic mosaic screening following identification of a loss-of-function mutation of Hippo that led to a strong overgrowth phenotype , anti-Lats , anti-p-Lats , anti-Rho , anti-ROCK , and anti-β-actin ). The membranes were incubated with IRDye 800CW goat anti-rabbit IgG (H+L)  at room temperature for 1 h. Bands were detected using the chemiluminescent imaging system.P values < 0.05 were considered to indicate statistical significance.One-way analysis of variance was performed in SPSS software  to evaluate group differences. Data are expressed as means ± standard deviation, and All data generated or analyzed during this study are included in this published article.To determine the changes in proliferation and apoptosis of VSMCs under different intensities of mechanical stretch, we applied 0, 10, 15, and 20% elongation forces to VSMCs . We usedTo test whether YAP was involved in the effects of mechanical stress on VSMCs, we first performed immunofluorescence staining to determine the intracellular localization of YAP. YAP was both in the nucleus and the cytoplasm under 0% elongation –2C. YAP To determine the effects of the Hippo pathway in VSMCs, we knocked down YAP by siRNA. We tested the efficiency of different YAP-targeting siRNAs using western blotting to generate optimal knockdown of YAP. Our results showed that YAP siRNA1 (ACAGCAGGAGTTATTTCGG) resulted in the greatest knockdown of YAP . To testTo better understand the role of the Rho-ROCK-Hippo pathway in the effects of mechanical stretch on YAP, we inhibited the Rho-ROCK pathway using Y27632. We performed immunocytochemistry staining to determine the intracellular localization of YAP. The results showed that more YAP stayed in the cytoplasm under 0% elongation following inhibition of the Rho-ROCK pathway (group 2) compared with 0% elongation (group 1) , 4B. Y27One emerging concept is that changes in the VSMCs phenotype in response to mechanical forces are important in vascular diseases, such as hypertension, atherosclerosis, aortic aneurysms, and age-dependent arterial stiffening , 26, 27,Studies have found that mechanical stimulation can inhibit the Hippo pathway, increase nuclear levels of YAP, promote cell proliferation, and inhibit apoptosis. , 28–31 YIn addition, we tested the activity of the Rho-ROCK pathway and found that 10% elongation stimulated the expression of related proteins to decrease, while 15% elongation stimulated the expression of related proteins to increase. After blocking the Rho-ROCK pathway, the effects of different intensities of stretch on YAP were weakened and phosphorylated YAP was decreased in each group. Overall, inhibition of the Rho-ROCK pathway combined with 10% mechanical stress led to activation of the Hippo pathway, which in turn reduced nuclear localization of YAP. YAP failed to bind to TEAD; this led to a decrease in ccnd1 expression, inhibition of VSMCs proliferation, and promotion of apoptosis. When the mechanical stress was increased to 15%, we observed the opposite effects.Through our experiments, we found that the mechanism underlying the effects of mechanical stress on VSMCs is as follows: after being stimulated by physiological 10% elongation stretch, VSMCs are inhibited in various ways. Inhibition of Rho-ROCK leads to a decrease in LATS1/2 levels, which in turn leads to an increase in the phosphorylation of YAP. Phosphorylated YAP cannot enter the nucleus nor bind to TEAD. On the other hand, our ongoing experiment shows that 10% elongation stretch may downregulates the expression of miR130a, which increases the amount of VGLL4. In the nucleus, VGLL4 may competes with YAP for the opportunity to bind TEAD. As a result of these two mechanisms, 10% elongation reduced ccnd1, resulting in decreased VSMCs proliferation and increased apoptosis, while a stimulus that is lower or higher than physiological elongation resulted in opposing effects, eventually promoting the proliferation of VSMCs and reducing apoptosis.On conclusion, our study demonstrates the possibility of preventing or treating diseases such as aortic aneurysms by controlling blood pressure. The Rho-ROCK pathway can be activated by mildly adjusting the blood pressure. Activation of the Rho-ROCK pathway, in turn, inhibits the Hippo pathway and ultimately promotes VSMCs proliferation and inhibits apoptosis. Our findings may provide insight into the pathogenesis prognosis of these diseases, as well as into therapeutic interventions.


In situ intercalative polymerization has been investigated as a strategic way to obtain poly (PPF) and poly (PHF) nanocomposites with different graphene types and amounts. Graphene (G) has been dispersed in surfactant stabilized water suspensions. The loading range in composites was 0.25–0.75 wt %. For the highest composition, a different type of graphene (XT500) dispersed in 1,3 propanediol, containing a 6% of oxidized graphene and without surfactant has been also tested. The results showed that the amorphous PPF is able to crystallize during heating scan in DSC and graphene seems to affect such capability: G hinders the polymer chains in reaching an ordered state, showing even more depressed cold crystallization and melting. On the contrary, such hindering effect is absent with XT500, which rather induces the opposite. Concerning the thermal stability, no improvement has been induced by graphene, even if the onset degradation temperatures remain high for all the materials. A moderate enhancement in mechanical properties is observed in PPF composite with XT500, and especially in PHF composite, where a significative increase of 10–20% in storage modulus E’ is maintained in almost all the temperature range. Such an increase is also reflected in a slightly higher heat distortion temperature. These preliminary results can be useful in order to further address the field of application of furan-based polyesters; in particular, they could be promising as packaging materials. Nowadays, there is a growing interest in the preparation of new chemicals and materials based on renewable resources, as biomass-derived fuel and chemicals are a promising alternative to fossil-based materials. Chemicals from vegetable feedstocks like sugars, vegetable oils, organic acids, glycerol, and others have been proposed as monomers for polymers production ,2,3,4. 2Concerning composites, the state of the art reports furan-based resin composites with clays, basalt, glass, or flax fibers ,16,17,182 carbons organized in a honeycomb structure. It has been considered the building block of all other graphitic carbon allotropes such as graphite, fullerenes, carbon nanotubes (CNT), and nanoribbons, even if, however, these carbon allotropes are not synthesized from graphene (except for nanoribbons). Graphite indeed is a naturally occurring material, single-walled CNT was first synthesized in 1991 following the discovery of fullerenes in 1985. With Young’s modulus of 1 TPa and ultimate strength of 130 GPa, single-layer graphene is the strongest known material. Moreover, single-layer graphene has very high electrical conductivity, up to 6000 S/cm, and a thermal conductivity of 5000 W/mK. These excellent properties, in addition to extremely high surface area (2600 m2/g) and gas impermeability indicate graphene as the perfect candidate for improving mechanical, electrical, thermal, and gas barrier properties of polymers [Among fillers commonly used in composites, graphene, with its combination of extraordinary physical properties and ability to be dispersed in various polymer matrices, is a promising candidate for a new class of furan-based polymer nanocomposites. Graphene is an atomically thick, two-dimensional sheet composed of space area 00 m2/g aace area 00 m2/g apolymers ,28,29,30ace area 00 m2/g apolymers , nylon 6polymers , poly, diffractometry (XRD), thermogravimetry (TGA), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMTA), and scanning electron microscopy (SEM).In particular, in the present work, poly (PPF) and poly (PHF) have been taken into consideration. PPF is a promising sustainable alternative of poly (PET), while PHF, in comparison with PEF, PPF, and PBF, shows minor tensile modulus and strength. Jiang et al.  indeed, 2O3, purity 99%) was supplied by Carlo Erba. The reactants have been used as received. Graphene (G) dispersions in water, exfoliated using a proprietary surfactant, were kindly provided by Graphene-XT . The industrial grade graphene G from Graphene-XT had an average lateral width of about 0.2 μm and 2–10 nm thickness. The concentration of the undisclosed suspending agent was below 1% w/w; for further information, refer to the technical data sheet [1,3-propanediol  and 1,6-hexanediol  were purchased from Sigma-Aldrich; 2,5-furandicarboxylic acid  was provided by Aldrich; antimony trioxide (Sbta sheet . Graphen−3 mol) of Sb2O3 corresponding to 1 wt % respect to the theoretical final polymer, were charged into a glass reactor (250 mL capacity). The ratio PD/FDCA was 1.8. The reactor was closed with a three-necked flat flange lid equipped with a heating band (T = 80 °C), a mechanical stirrer and a torque-meter (which gave an indication of the viscosity of the reaction melt). The system, under nitrogen flux, was then connected to a water-cooled condenser and immersed in a thermostatic salt-bath at 190 °C, while the stirrer was switched on at 200 rpm. The temperatures of band and bath were gradually increased up to 100 and 220 °C, respectively. All the water distilled off after less than 2 h. The reactor was then connected to a liquid nitrogen-cooled condenser. Dynamic vacuum was applied in 2 h down to 0.14 mbar, while the temperature was gradually increased up to 250 °C for the salt-bath and 120 °C for the band. When the torque of the melt was around 4 mN, a viscous, brown and transparent melt was discharged from the reactor. The molecular structure of PPF was confirmed by 1H-NMR.The polycondensation procedure was described in previous papers ,44,45. 21 H NMR , 600 MHz): δ 2.28 , 4.55 , 7.31 . The carbon labels are referred to the chemical structure reported in The composites have been similarly prepared by adding the dispersion containing graphene into the reactor. The compositions have been calculated in wt % respect to the theoretical final polymer. The samples are denoted PPF-XYw(d), where X is the wt % of graphene, Y indicate the graphene type G or XT500, and w or d indicates a water or diol dispersion.−3 mol) of Sb2O3, were charged into a glass reactor. The ratio HD/FDCA was 1.2. The reactor was closed with a three-necked flat flange lid equipped with a heating band (T = 80 °C), a mechanical stirrer, a torque-meter (which gave an indication of the viscosity of the reaction melt). The system was kept under nitrogen flow, then connected to a water-cooled condenser and immersed in a thermostatic salt-bath at 180 °C, while the stirrer was switched on at 200 rpm. The temperature was gradually increased up to 220 °C, as well as the heating band (100 °C), until almost all the water distilled off, after less than 2 h. The reactor was then connected to a liquid nitrogen-cooled condenser. Dynamic vacuum was applied in 2 h down to 0.23 mbar, while the temperature was increased up to 250 °C and the lid was set at 120 °C. When the torque of the melt was around 8 mN, a viscous, dark brown and transparent melt was discharged from the reactor. The molecular structure of PHF was confirmed by 1H-NMR.28.3 g (0.181 mol) of FDCA, 25.7 g (0.218 mol) of HD and 407 mg , 600 MHz): δ 1.49 , 1.82 , 4.41 , 7.30 . The carbon labels are referred to the chemical structure reported in The composite was similarly prepared by adding the water dispersion containing G into the reactor. The sample is named PHF-0.75Gw and corresponds to 0.75 wt % of graphene respect to the theoretical final polymer.1 H NMR spectra were recorded on Varian Inova 600 spectrometer ; the solvent used was CDCl3.3 and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) 95/5 as eluent and to dissolve polymer samples. A UV detector was used and a calibration plot was constructed with polystyrene standards.Gel permeation chromatography (GPC) measurements were performed on a HP 1100 Series using a PL gel 5 µm Minimixed-C column with a solution of CHCl−1 heating rate from 50 to 900 °C. The onset degradation temperatures (Tonset) were taken from the intersections of the tangents of the initial points and the inflection points. The 10% mass loss temperatures (T10D) and the temperature at maximum process rate (Tmax) were also measured.Thermogravimetric analysis (TGA) was performed in nitrogen atmosphere, using a Perkin Elmer TGA7 apparatus (gas flow 40 mL/min) at 10 °C min−1 from 20 to 220 °C, kept at high temperature for 2 min and then cooled to 0 °C at 10 °C min−1. After this thermal treatment, the samples were analyzed by heating from 0 °C to 220 °C at 10 °C min−1 (second scan). The PHF samples were heated at 20 °C min−1 from 0 to 180 °C, kept at high temperature for 2 min, then cooled to −20 °C at 10 °C min−1, and heated from −20 °C to 180 °C at 10 °C min−1 (second scan). During the cooling scan the crystallization temperature (Tc) and the enthalpy of crystallization (∆Hc) were measured. During the second scan, the glass transition temperature (Tg), the melting temperature (Tm) and the enthalpy of fusion (∆Hm) were measured. Tg was taken as the midpoint of the heat capacity increment associated with the glass-to-rubber transition.The calorimetric analysis (DSC) was carried out by means of a Perkin-Elmer DSC6. Measurements were performed under nitrogen flow. In order to delete their previous thermal history, the PPF samples (ca. 10 mg) were first heated at 20 °C minThe X-ray diffraction (XRD) analysis was carried out at room temperature by means of a PANalytical X’Pert PRO diffractometer equipped with an X’Celerator detector. Data were acquired by exposing the samples to Cu-Kα X-ray radiation, which has a characteristic wavelength (λ) of 1.5418 Å. X-rays were generated from a Cu anode supplied with 40 kV and a current of 40 mA. The 2θ plots of X-ray spectra have been obtained over 2θ range of 2.1–35°. For each step of 0.067°, the signal has been counted for 41 s, to increase its intensity. PPF film samples, pressed at 200 °C were analyzed. PHF samples, were pressed at 100 °C, obtaining samples with almost 1 mm of thickness and a smooth and regular surface to analyze.−1 in the temperature range starting from −120 °C to 110 °C.Physical and mechanical properties were determined using a Rheometric Scientific DMTA IV Dynamic Mechanic Thermo analysis instrument with a dual cantilever testing geometry. Typical test samples were bars obtained by injection molding at 190 °C using a Minimix Molder with PHF. PPF sample bar have been obtained by compression molding at 200 °C. The analysis was carried out at a frequency of 3 Hz, at 3 °C minFracture surfaces of samples were obtained under cryogenic fracture by liquid nitro-gen to avoid plastic deformations. Sections were analyzed by scanning electron microscopy  to investigate their morphology.In a previous work by the same Authors , it was 1 H NMR. PPF and PHF homopolymers spectra are showed in The molecular structure was checked by The samples have high molecular weight within the range 50,000–70,000 g/mol  with a pg) of 53 °C. During the cooling scan from the melt at 10 °C/min, PPF is unable to crystallize; it only presents, during the second heating scan (at 10 °C/min) a cold crystallization process at 138 °C and a melting peak at 172 °C. All the PPF composites with G are substantially amorphous too, but some observations can be done. The samples PPF-Gw during the second heat scan present low enthalpy values (ΔHcc and ΔHm) with respect to the homopolymer. Moreover, the shape of the peaks become broader, showing cold crystallization and melting processes more and more depressed. The polymer chains in such samples have even more difficulties in reaching an ordered state; therefore, Gw hinders the crystallization of the matrix occurring during heating. This behavior can be due to the presence of surfactant used to stabilize the G suspension. On the other hand, in PPF-0.75XT500d, cold crystallization and melting processes are more intense than those of PPF and of the corresponding PPF-0.75Gw. The graphenic sample XT500 seems to favor the rearrangement of the polymer chains during heating, probably due to the absence of surfactant and/or to the presence of 6 wt % of graphene oxide, and/or to the larger lateral width respect to G. In contrast, PHF is semicrystalline (c) and melts at 145 °C (Tm). The glass transition temperature is 13 °C. No significant differences are present in the composite: graphene does not seem to affect the thermal behavior of PHF. The DSC data are consistent with literature [The thermal behavior of the investigated samples was assessed. Calorimetric data are summarized in stalline : it crysterature ,47. Moreg, no substantial changes respect to both PPF and PHF were highlighted. Graphene does not seem to affect the mobility of the chains neither as plasticizing neither as nucleating agent.Concerning the T2O3 around 2θ = 28°. The reflection attributed to (002) diffraction peak of graphene, at 26.6° [2O3 is similarly visible around 2θ = 28°, as well as the (002) diffraction peak of graphene in PHF-0.75Gw [at 26.6° , is visiat 26.6° . The catF-0.75Gw .onset (369 °C) and Tmax (391 °C). Tonset, Tmax, and T10D of all the composites remain almost similar to the respective homopolymers, no significant differences are present. Thus, graphene does not seem to improve the thermal stability of the polyester matrix: indeed, decomposition curves shift toward slightly lower temperature region. On the contrary, slight retardation in decomposition were reported by Paszkiewicz et al. for PPF composites with few layer graphene [The thermal stability of homopolymers and their nanocomposites with graphene was investigated by TGA under inert nitrogen atmosphere. The thermogravimetric curves are shown in graphene , while mgraphene . The nongraphene ,42 and agraphene .Temperature and frequency dependent mechanical relaxation data for amorphous PPF and semicrystalline PHF were recorded via storage modulus (E′) and tan δ, which is the ratio of the loss modulus to storage modulus. Such data can provide indications about the mechanical reinforcement, which is correlated to a good interface interaction filler/matrix due to the presence of van der Waals forces or π–π interactions . In FiguSimilarly, a slight reinforcing effect of graphene in E’ was reported with PMMA . The autIn the zoomed image of For all samples, the glass transition temperature is recognized by the large drop in storage modulus and by the corresponding peak maximum in tan δ c. The vag, all samples are substantially similar to the homopolymer.Above TCrystallization is observed by the increase in storage modulus around 100 °C for all samples. As already expressed, PPF is able to crystallize under specific conditions, as for example during DMTA scan at 3 °C/min, after the glass-rubber transition. Such phenomenon is more pronounced for PPF-0.75XT500d, probably due to a considerably higher crystallization rate induced under heating by graphene XT500, as already evidenced by DSC analysis.In case of PHF samples , the comMoreover, heat distortion temperature (HDT), corresponding to log E’ = 8.9 (1.82 MPa) according to Scobbo  was calcPPF and PHF composites containing graphene were prepared by in situ polymerization. Graphene (G) was dispersed in surfactant stabilized water suspensions. For one PPF composition, a different type of graphene was also tested, XT500, dispersed in 1,3-propanediol without any surfactant. In this case, it is notably to observe that 1,3-propanediol is also a monomer for the PPF synthesis. PPF samples result to be amorphous, while PHF samples are semicrystalline materials. Graphene seems to affect the ability of PPF to crystallize during heating: the nanocomposites containing G show a depressed cold crystallization and melting processes. On the contrary, XT500 seems to help the rearrangement of the polymer chains in an ordered state.-affects the ability of PPF to crystallize during heating, while it does not affect the thermal transitions of PHF;-does not affect the thermal stability neither of PPF neither of PHF;-determines a mechanical reinforcement especially in PHF.Concerning the thermal stability, no improvement has been induced by graphene; however, the onset degradation temperatures still remain high for all the materials, around 370 °C. A moderate enhancement in mechanical properties is observed in PPF-0.75XT500d (around 6–12% of E’ increase), and especially in PHF composite, where an adequate increase of 10–20% in E’ is maintained in almost all the temperature range. Such increase is also reflected in a slightly higher HDT (38 vs. 34 °C). In terms of mechanical performances, for PPF composites 1,3-propanediol results to be a better medium to suspend graphene respect to surfactant stabilized water dispersions. PD in fact, is a monomer in PPF polycondensation and this helps in preventing the restacking of the sheets. In summary, graphene:Such preliminary results can be useful in order to further address the field of application of furan-based polyesters. Indeed, the above materials could be potentially used for packaging application, where high barrier properties—potentially further improved by graphene—are required. Moreover, it is noteworthy that the in situ polymerization procedure avoids the use of powders, employed in the melt compounding techniques, and the use of a large amount of solvent, employed for solvent casting methods, representing an advantage in term of safety and sustainability.


Long interspersed element 1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome that can serve as an endogenous upstream activator of cytoplasmic nucleic acid sensing pathways to elicit an antiviral immune response. In this study, we investigated the influence of enteroviral infection on L1 mobility. The results showed that infection with different enteroviruses, both EV-D68 and EV-A71, blocked L1 transposition. We screened diverse viral accessory proteins for L1 activity and identified EV-D68 2A, 3A, 3C, and EV-A71 ORF2p proteins as viral L1 inhibitors. EV-D68 2A suppressed L1 mobility by expression suppression of L1 proteins. Viral proteins 3A and 3C restricted ORF2p-mediated L1 reverse transcription in isolated L1 ribonucleoproteins. The newly identified enteroviral protein ORF2p inhibited the expression of L1 ORF1p. Altogether, our findings shed light on the strict modulation of L1 retrotransposons during enterovirus replication. Transposable elements have been recognized as major contributors to mammalian genomes since the discovery of mobile DNA . They arL1 retrotransposition is largely restricted in normal tissues but can be activated in response to stress, aging, or disease . RecentlEnteroviruses (EVs) of the Picornaviridae family, small, non-enveloped, icosahedral viruses which comprises non-enveloped RNA viruses, provoke a diverse array of clinical symptoms . The genHEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 100 μg/mL penicillin-streptomycin. Human LINE-1 constructs 99 PUR L1RP EGFP, 99 PUR JM111 EGFP (JM111), the pc-L1-1FH plasmid, and pEGFP-N1-ORF1-EGFP plasmids were kindly provided by Dr. Haig H. Kazazian, Jr., and John L. Goodier . The JM1EV-D68 prototype Fermon (2014) isolated US/MO/14-18947 (MO) and US/KY/14-18953 (KY) were purchased from ATCC. Professor Cheng Tong kindly provided the EV-A71 prototype Anhui2007, and prototype CC063 was isolated in our laboratory. Viruses propagated in RD cells. The supernatants of enterovirus-infected cells were harvested, filtered through a 0.22 mm filter, and centrifuged through a 20% sucrose cushion in an SW28 rotor at 28,000 rpm for 1.5 h. Purified pellets were stored at −80°C.HEK293T cells were seeded in 24 well plates and transfected with 1 μg of L1RP EGFP or JM111 constructs. Cells were then puromycin-selected (3 μg/mL) 48 h post-transfection. The percentage of GFP(+) cells was measured using a BD FACSCalibur Flow Cytometer after 48 h of puromycin selection. The plasmid JM111 construct was used as a negative control. A total of 10,000 single-cell events were gated using flow cytometry. The percentage of GFP(+) cells was analyzed using the CellQuest Pro (v.5.2).®  from a single sample.HEK293T cells seeded in 24-well plates were transfected with the pYX014 or pYX017 plasmids. The cells were then puromycin selected 24 h post-transfection. After 3 days of puromycin selection, dual-luciferase assays were performed according to the manufacturer’s instructions (Promega). Firefly and Renilla luciferase were measured using Promega GloMaxHEK 293T cells were pre-infected or pre-transfected. The cells were seeded in 96-well plates 1 day post infection/transfection. Then the cells were cultured for 3 days before assessing thier viability. Absorbance was detected at a wavelength of 490 nm using a BioTek ELISA reader  on adding 3--5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt (MTS)  to each well.Cell samples were harvested and lysed in RIPA buffer . The cell lysate was separated on 12% SDS-PAGE gels and then transferred to nitrocellulose membranes using a semidry apparatus (Bio-Rad). Anti-HA was purchased from Thermo Fisher Scientific . The anti-GAPDH antibody and anti-α-Tubulin antibody were purchased from GenScript Biotech Corp . Anti-EV-D68 VP1 antibody and anti-EV-A71 VP1 antibody were purchased from GeneTex .HEK 293T cells were transfected with pEGFP-N1-ORF1-EGFP plasmids in the absence or presence of enterovirus protein-expressing plasmids (HA-tagged). After 2 days, cells were fixed with 4% paraformaldehyde in PBS for 30 min, permeabilized in 0.1% Triton X-100 in PBS for 10 min, and blocked in 5% BSA solution for 1 h. Then, the cells were incubated with anti-HA antibody overnight at 4°C. Cells were incubated with Alexa Fluro 594 conjugated goat anti-rabbit IgG antibody  for 1 h at 4°C. Nuclei were counterstained with 4’6-diamidino-2-phenylindole (DAPI). Fluorescence imaging was performed using a fluorescence microscope (OLYMPUS) with a maximum magnification of 40 X.2, 500 mM Tris–HCl (pH 7.5), 1M DTT, RNasin (40U/μL), 0.05%(v/v) Tween 20, and dNTP] using the LEAP primer: 5′-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTT TTTTTVN-3′. L1 mRNA was reversed-transcribed to L1 cDNA by L1 ORF2p in the RNPs at 37°C for 1 h. To detect the level of the L1 mRNA, L1 RNA was extracted from the L1 RNP and reverse-transcribed with the same primer using MuLV RT . The synthesized cDNA from both LEAP assay and MuLV RT was then analyzed by quantitative real-time PCR (qRT-PCR) using the following primers: linker PCR primer, 5′-GCGAGCACAGAATTAATACGACT-3′; L1 3′-end primer, 5′-GGGTTCGAAATCGATAAGCTTGGATCCAGAC-3′, with a standard three-step method  as previously described  and reverse (5′-CTTCCTGTGTCCATGTGATCTCA-3′); L1 ORF2p, forward (5′-AGGAAATACAGAGAACGCCACAA-3′) and reverse (5′-GCTGATATGAAATTCTGGGTTGA-3′); GAPDH, forward (5′-GCAAATTCCATGGCACCGT-3′) and reverse (5′-TCGCCCCACTTGATTTTGG-3′); EV-D68, forward (5′-TGTTCCCACGGTTGAAAACAA-3′) and reverse (5′-TGTCTAGCGTCTCATGGTTTTCAC-3′). Endogenous mRNA levels of GAPDH were used as the loading control and are not shown unless otherwise indicated.Total RNA from the samples of interest was first isolated using TRIzol (Life Technologies) and then subjected to reverse transcription using MonScript RTIII All-in-One Mix with dsDNase according to the manufacturer’s instructions. The qRT-PCR was performed using MonAmpt-test was performed on the data between the two groups. P < 0.01 was considered significant in groups.This study’s statistical data were analyzed using GraphPad Prism software . Data are described as the M ± SD from three replicates of each experiment. Unpaired Student’s We used a well-established EGFP reporter system in HEK293T cells to evaluate the effect of enterovirus infection on L1 retrotransposition . The L1RSubsequently, we used individual EV-D68 non-structural protein-expressing constructs to determine which of the following EV-D68 protein suppresses L1 activity: 2A, 2B, 2C, 3A, 3C, or 3D. These EV-D68 protein-expressing plasmids were co-transfected with pL1-EGFP into HEK293T cells, and GFP-positive cells were determined 96 h after transfection. The results showed that three of these EV-D68 proteins, 2A, 3A, and 3C, significantly reduced L1 mobility . We idenin vitro LEAP reverse transcriptase (RT) assay to assess the function of ORF2p in LINE-1 ribonucleoprotein (RNP) complexes  for L1 retrotransposition  to confiomplexes . We founomplexes , which iomplexes . CollectWe confirmed L1 inhibition by 3A in L1-EGFP and L1-Luciferase assays . We thenNext, all the L1-EGFP and L1-Luciferase assays demonstrated that 3C robustly impaired LINE-1 mobility . The resEnterovirus protein ORF2p, also called UP, a newly identified viral protein encoded by certain enteroviruses, such as EV-A71 but not EV-D68, is essential for the release of viral particles in human gut cells . Thus faLong interspersed element 1s is an active human DNA parasite that shapes the human genome’s structure, function, and evolution . AberranA series of reports have shown that many host antiviral factors, such as APOBEC3s, ZAP, SAMHD1, MX2/MxB, and TREX1, potently suppress the transposition activity of L1-and L1-dependent non-autonomous retrotransposons . When inEnterovirus 2A is a cysteine proteinase that is important for viral replication and pathogenesis. Besides viral proteinase 2A, which contributes to shearing viral poly-proteins, it can also cleave host proteins to facilitate viral infection . 2A-depeEnterovirus 3A protein, a membrane-bound protein, can recruit acyl-coenzyme A-binding domain-containing 3 and phosphatidylinositol-4-kinase IIIβ to facilitate the viral assembly replication organelles . These oEnterovirus 3C is another cysteine protease encoded by all the enteroviruses. Similar to 2A, 3C plays a determinant role in processing viral precursor polyproteins. Both 3A and 3C inhibit the RT activity in L1 RNP in a ORF1p-independent manner. As we could not obtain an effective antibody against L1 ORF2p, it is still necessary to investigate L1 ORF2 expression and the potential interaction between viral proteins and L1 ORF2p in future studies. Interestingly, we noted that the expression of 3A and 3C could increase the intracellular accumulation of L1 ORF1p proteins , which mRecently, we and others identified a novel enterovirus protein, named ORF2p or UP, which is encoded by a second open reading frame in the genome of most enteroviruses . Viral pHence, we suggest that enteroviruses encoding diverse viral proteins can negatively modulate L1 mobility by targeting different processes of L1 transposition. The use of different viral proteins acting at different steps of L1 transposition together to control LINE-1 transposition reveals unignorable selection pressure from mobile DNA on virus replication.flowrepository.org/id/RvFrXk8O1mykIRfUw1uiFiENBO6PdSQkCbXsCGOalFx8QcNOi7rFutb1zJJ3JNr7.Data from flow cytometry experiments was submitted to the FlowRepository. More information may be accessed via the following URL: WW conceived and designed the experiments. YL, SS, and HG participated in multiple experiments. YL, SS, HG, ZZ, LZ, QY, YG, JN, and WW analyzed the data. YL, HG, and WW wrote the manuscript with help from all authors. All authors contributed to the article and approved the submitted version.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.