Identification of behavioral mechanisms underlying psychopathology is essential for the development of novel targeted therapeutics. However, this work relies on rigorous, time-intensive, clinic-based laboratory research, making it difficult to translate research paradigms into tools that can be used by clinicians in the community. The broad adoption of smartphone technology provides a promising opportunity to bridge the gap between the mechanisms identified in the laboratory and the clinical interventions targeting them in the community. The goal of the current study is to develop a developmentally appropriate, engaging, novel mobile application called CALM-IT that probes a narrow biologically informed process, inhibitory control. We aim to leverage the rigorous and robust methods traditionally used in laboratory settings to validate this novel mechanism-driven but easily disseminatable tool that can be used by clinicians to probe inhibitory control in the community. The development of CALM-IT has significant implications for the ability to screen for inhibitory control deficits in the community by both clinicians and researchers. By facilitating assessment of inhibitory control outside of the laboratory setting, researchers could have access to larger and more diverse samples. Additionally, in the clinical setting, CALM-IT represents a novel clinical screening measure that could be used to determine personalized courses of treatment based on the presence of inhibitory control deficits. Precise identification of behavioral mechanisms underlying psychopathology is essential to the assessment and development of novel, targeted therapeutics –4. DespiInhibitory control a well-defined and brain-related construct capturing an individual’s ability to resist or modulate impulses and suppress prepotent or automatic behavioral responses . InhibitInhibitory control also has broad clinical relevance. Research indicates that youth with psychopathology, including mood and attentional difficulties, have exhibit difficulty inhibiting responses, and exhibit aberrant neural activity when attempting to inhibit a motor response –10. EmotThere is a profound need for mental health treatment research efforts to probe underlying mechanisms to inform diagnosis and treatment decisions –4. CliniThe goal of the present work is to leverage the strong psychometric properties of a canonical inhibitory control task in the creation of CALM-IT. By targeting a precise pathophysiologically-informed mechanism, and using a platform that is highly engaging and easily disseminated in pediatric samples, the mobile application CALM-IT was designed to bridge the gap between precise mechanisms-driven basic science research and community-based assessment of childhood psychopathology. CALM-IT is designed to leverage the strong methodological design of well-established laboratory based inhibitory control tasks while increasing participant engagement through gamification of the tasks. By using a mobile platform, participant interaction with CALM-IT mirrors those with other mobile-based games with the goal of increasing participant engagement via dynamic stimuli and in-game incentives. The development of CALM-IT would allow clinicians and researchers to screen for and track inhibitory control deficits in the community. This could extend the investigation of inhibitory control beyond the confines of the laboratory setting, therefore facilitating access to a larger and more diverse sample of patients. Ultimately, CALM-IT could lead to novel clinical screening measures for inhibitory control deficits that could be used to inform a personalized course of treatment.Our first aim is to assess the feasibility of CALM-IT as a mobile-paradigm. We will assess the ease of dissemination, mobile application engagement, and ability to extract mobile application-based behavioral measures of inhibitory control within a large transdiagnostic pediatric sample.Next, we will assess the reliability of mobile application-based behavioral measures of inhibitory control across two sessions of play, spaced one week apart.Our second aim is to validate mobile application-based measures of inhibitory control against measures of inhibitory control extracted from four canonical laboratory-based tasks: the Antisaccade Task , the AX Finally, our third aim is to identify clinical correlates of mobile application-based measures of inhibitory control. Specifically, we will examine associations between mobile application-based behavioral measures of inhibitory control with shared vs. unique variance of three symptoms present across a wide range of pediatric psychopathologies: ADHD, anxiety, and irritability symptoms.Youth aged 8–18 years old will be recruited from the community to participate in research at the National Institute of Mental Health (NIMH). Participants will be recruited as part of a larger protocol that aims to specifically recruit youth with a primary diagnosis of an Anxiety Disorder, Disruptive Mood Dysregulation Disorder (DMDD), ADHD, and youth with no psychiatric diagnosis. Past work within this sample has found that recruiting samples characterized by these clinical diagnoses has resulted in the full range of irritability, anxiety, and ADHD symptoms ,59–62. APrior to participation, parents will provide written informed consent and youth will provide written assent. Participants will complete up to three different testing sessions. Administration of CALM-IT will occur outside of the laboratory setting. Families will be sent instructions on how to download CALM-IT onto their mobile device. As such, CALM-IT testing sessions will be completed within a community setting (e.g. at home) at whatever time is most convenient for the family. Parents will be instructed that CALM-IT should be played only by the child with no assistance. Adherence to this procedure will be assessed in a follow up interview. Families will also have the option of coming in for in-laboratory testing where children will complete four canonical inhibitory control tasks (detailed below). Families will receive monetary compensation for completion of CALM-IT. All study procedures have been approved by the NIMH Institutional Review Board.Aim 1a), we will recruit 200 youth. For investigation of the stability of CALM-IT (Aim 1b), we will invite 75 youth out of the overall sample of 200 to play CALM-IT twice. For validation of mobile application-based behaviors of inhibitory control (Aim 2), we will invite 100 youth out of the overall sample of 200 to complete a battery of in-laboratory inhibitory control paradigms. We evaluated each of these proposed sample sizes using the pwr package in R to determine the size of the effect that the sample would allow us to detect using a significance level of 0.05 and a power level of 0.80 Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.Reviewer #1: Partly**********2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions  to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.Reviewer #1: Partly**********3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?Reviewer #1: Yes**********4. Have the authors described where all data underlying the findings will be made available when the study is complete?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: No**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.Reviewer #1: This manuscript is a protocol for a validation study of a mobile application for inhibitory control evaluation in young people. It presents an interesting idea of measuring inhibitory control via a mobile application which presumably is a more feasible way in comparison to other experimental approaches when used in the community settings. The protocol summarises the main details of the proposed validation study. Several important details in the methods are missing which should be clarified. I recommend this protocol for publication after major revision.Abstract:The abstract could be more specific and include the importance of this research and the possibility of future application of the CALM-IT in clinical practice.Introduction:Page 4: “However, these tasks tend to be time-intensive, repetitive, and expensive, making them infeasible in a community setting”. Can the authors add an example of the tasks which are not feasible to conduct in a community setting and why? In what sense is the new application CALM-IT less repetitive? My understanding is that it comes from well-established paradigms  which are repetitive but their use in the community is more about the use of appropriate device rather than creating a whole new paradigm or measuring instrument.Page 5: “While literature examining treatment of youth psychopathology using interventions specifically targeting inhibitory control is limited, previous work shows a reduction of mood symptoms following simulant medication treatment for co-occurring attentional .”. What is exactly meant by youth psychopathology? Diagnoses of mental illness or behavioural problems ? Could you please provide examples of specific psychopathology conditions in youth treated in inhibitory control and how are these findings connected with the present study?Methods: This part is described into considerable details, however, to make the methodology replicable several details are missing.Participants: The protocol does not clearly state the age range of participants the authors plan to recruit for their study and the process of their recruitment and selection . How will the authors assure that the participants will represent a whole range of symptoms? Moreover, if the participants will be under-aged, I would recommend clarifying the role of parents or legal guardians in the process. Whether they will be present or not during the use of the app and additional evaluations and how the authors plan for potential interference from the parents in the app testing . It is unclear if the app will be tested/used at home or in a controlled environment .What is the rationale behind selecting the particular diagnoses of anxiety (but not depression), ADHD (including ADD?), DMDD? The topic of inhibitory control problems in children with these selected diagnoses could be more elaborated also in the introduction. To summarise the existing evidence of this problem may give a better understanding of the importance of this study and the CALM-IT application.Stimuli: The application seems to present a limited number of trials and stimuli types. Did the authors consider using different colours and shapes of stimuli for each application testing session to avoid habituation for specific stimuli? For example, the no-go stimuli are always yellow stars, changing the colour or type of the no-go stimulus for each level may reflect more on the flexibility of each participant to inhibit inappropriate response in a variety of different scenarios.Effect size calculation: What is the rationale behind selecting the effects of r between .20 and .32. Could the authors give examples of similar studies or otherwise justify the selected effects and/or the effect size calculation?Symptom measures: I would recommend to the authors to consider measuring the impulsivity of participants also by using standardised questionnaires to investigate the different facets of impulsive behaviour. Some established measures as UPPS-P can be used in children.Assessing the engagement: This seems to be assessed only based on the performance metrics from the app. I would recommend to the authors considering running a focus group or using a questionnaire of user experience to gain more information on the engagement and the usability of the app. This can be an important source of information for adjusting apps in development based on real user-experience feedback. If the app engagement is assessed only based on the performance metrics, how will the authors distinguish between poor performance in the app based on boredom or lack of engagement versus a poor performance caused by very poor inhibitory control abilities?**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  16 Apr 2021Editor’s Comments:https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf1.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at We have reviewed the PLOS ONE style templates and ensured that our manuscript meets the PLOS ONE’s style requirements.http://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-software) for manuscripts whose main purpose is the description of a new software or software package. In this case, new software must conform to the Open Source Definition (https://opensource.org/docs/osd) and be deposited in an open software archive. Please see http://journals.plos.org/plosone/s/materials-and-software-sharing#loc-depositing-software for more information on depositing your software.2.Please note that PLOS ONE has specific guidelines on software sharing , and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.4. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: We apologize for any overlapping text in the submitted manuscript with our prior work. After reviewing the two manuscripts closely it appears that the overlapping text is in the description of the antisaccade and flanker in-laboratory tasks. We have revised the language to no longer overlap while maintaining accuracy and clarity:(p. 12): “Each trial begins with a preparatory period during which participants are presented with either a green or red instructional fixation cross indicating that the next trial will either be a prosaccade or antisaccade trail, respectively. During the prosaccade and antisaccade trials, a yellow visual target is presented for 1 second in a pseudorandomized location 630 pixels or 315 pixels to the left or right of the center of the screen for 1 second. The number of trials with the visual target in each location is equal across prossacade and antisaccade trials. The testing session consists first of a practice block. After completing the practice, participants will complete 3 experimental blocks each with 16 antisaccade and 16 prosaccade trials. The EyeLink 1000 Plus eye tracking system will be used to collect and process eye gaze data .”(p. 13): “Participants will be instructed to press the left or right arrow button to indicate the direction of the central arrow in series of five side-by-side arrows centered on the screen. The trial will terminate upon response and participants will be instructed to respond as quickly as they can. Trials are categorized as either congruent or incongruent trials based on the direction of the flanking arrows. Congruent trials correspond to trials where the flanking arrows all point in the same direction as the central arrow. The congruency of the visual stimuli therefore facilitates the correct motor response. In contrast, incongruent trials correspond to trials where the flanking arrows all point in the opposite direction of the central arrow. The incongruency of the visual stimuli therefore interferes with the execution of the correct motor response.”Reviewer #1 :This manuscript is a protocol for a validation study of a mobile application for inhibitory control evaluation in young people. It presents an interesting idea of measuring inhibitory control via a mobile application which presumably is a more feasible way in comparison to other experimental approaches when used in the community settings. The protocol summarises the main details of the proposed validation study. Several important details in the methods are missing which should be clarified. I recommend this protocol for publication after major revision.1. The abstract could be more specific and include the importance of this research and the possibility of future application of the CALM-IT in clinical practice.We have expanded the abstract to now include more specific language and a description of the importance of this research to future application in clinical practice (p. 2):“The development of CALM-IT has significant implications for the ability to screen for inhibitory control deficits in the community by both clinicians and researchers. By facilitating assessment of inhibitory control outside of the laboratory setting, researchers could have access to larger and more diverse samples. Additionally, in the clinical setting, CALM-IT represents a novel clinical screening measure that could be used to determine personalized courses of treatment based on the presence of inhibitory control deficits.”2. Page 4: “However, these tasks tend to be time-intensive, repetitive, and expensive, making them infeasible in a community setting”. Can the authors add an example of the tasks which are not feasible to conduct in a community setting and why? In what sense is the new application CALM-IT less repetitive? My understanding is that it comes from well-established paradigms  which are repetitive but their use in the community is more about the use of appropriate device rather than creating a whole new paradigm or measuring instrument.We appreciate the opportunity to provide more clarity to this statement. We have revised the above referenced sentence (p. 4):“However, these tasks tend to be time intensive, repetitive, and expensive, making them difficult to effectively deploy within a community setting.”Additionally, we believe that there are two critical aspects that need to be more effectively communicated in our introduction. First, we now provide an example of the types of demands that traditional in-laboratory tasks have that make it difficult to translate to the community setting (p. 4):“For example, some tasks rely on eye-tracking technology  that requires equipment and specific environmental controls  while others involve large numbers of repetitive trials presenting single simplistic stimuli one at a time  that require long periods of sustained attention .”Second, we now more explicitly discuss the aspects of CALM-IT that allow it to be both more easily accessible and engaging while still leveraging the methodological designs from well-established paradigms (p. 6-7):“CALM-IT is designed to leverage the strong methodological design of well-established laboratory based inhibitory control tasks while increasing participant engagement through gamification of the tasks. By using a mobile platform, participant interaction with CALM-IT mirrors those with other mobile-based games with the goal of increasing participant engagement via dynamic stimuli and in-game incentives.”3. “While literature examining treatment of youth psychopathology using interventions specifically targeting inhibitory control is limited, previous work shows a reduction of mood symptoms following simulant medication treatment for co-occurring attentional .”. What is exactly meant by youth psychopathology? Diagnoses of mental illness or behavioural problems ? Could you please provide examples of specific psychopathology conditions in youth treated in inhibitory control and how are these findings connected with the present study?We apologize for the omission. We now specifically state the youth psychopathology referenced in the cited work (p. 5):“While literature examining treatment of youth psychopathology, such as attention deficit hyperactivity disorder (ADHD) and irritability, using interventions specifically targeting inhibitory control is limited, previous work shows a reduction of mood symptoms, including anxiety, following simulant medication treatment for co-occurring attentional . These findings provide some evidence suggesting that inhibitory control may reflect a key behavioral mechanism underlying the emergence of mood symptoms and treatment response.”4. The protocol does not clearly state the age range of participants the authors plan to recruit for their study and the process of their recruitment and selection . How will the authors assure that the participants will represent a whole range of symptoms? Moreover, if the participants will be under-aged, I would recommend clarifying the role of parents or legal guardians in the process. Whether they will be present or not during the use of the app and additional evaluations and how the authors plan for potential interference from the parents in the app testing . It is unclear if the app will be tested/used at home or in a controlled environment .The participants section has been updated to provide more information regarding recruitment procedures and the targeted age range and clinical psychopathology (p. 8):“Youth aged 8 – 18 years old will be recruited from the community to participate in research at the National Institute of Mental Health (NIMH). Participants will be recruited as part of a larger protocol that aims to specifically recruit youth with a primary diagnosis of an Anxiety Disorder, Disruptive Mood Dysregulation Disorder (DMDD), ADHD, and youth with no psychiatric diagnosis. Past work within this sample has found that recruiting samples characterized by these clinical diagnoses has resulted in the full range of irritability, anxiety, and ADHD symptoms .”We also now include a discussion of the role of parents or legal guardians and the testing environment in the discussion of our procedures (p. 9):“Prior to participation, parents will provide written informed consent and youth will provide written assent. Participants will complete up to three different testing sessions. Administration of CALM-IT will occur outside of the laboratory setting. Families will be sent instructions on how to download CALM-IT onto their mobile device. As such, CALM-IT testing sessions will be completed within a community setting (e.g. at home) at whatever time is most convenient for the family. Parents will be instructed that CALM-IT should be played only by the child with no assistance. Adherence to this procedure will be assessed in a follow up interview. Families will also have the option of coming in for in-laboratory testing where children will complete four canonical inhibitory control tasks (detailed below). Families will receive monetary compensation for completion of CALM-IT. All study procedures have been approved by the NIMH Institutional Review Board.”5. What is the rationale behind selecting the particular diagnoses of anxiety (but not depression), ADHD (including ADD?), DMDD? The topic of inhibitory control problems in children with these selected diagnoses could be more elaborated also in the introduction. To summarise the existing evidence of this problem may give a better understanding of the importance of this study and the CALM-IT application.The reviewer raises an important point about the relevance of depression. Epidemiological studies demonstrate that youth with anxiety and DMDD often develop depression . As such, depression may be a relevant construct within the proposed sample. We now include a secondary analysis of depression using the Mood and Feelings Questionnaire:(p. 15): “For secondary analyses, depression symptoms will be measured using the parent-report Mood and Feelings Questionnaire . Total scores on the MFQ will be calculated for each participant as a measure of depressive symptoms.”(p. 19): “Finally, secondary analyses will be run examining bivariate correlations between measures of hyperactive-impulsivity, inattentiveness, and depression.”We now also include additional discussion of the prevalence and comorbidity of anxiety, ADHD, and DMDD in childhood and adolescence and associations with inhibitory control in the introduction (p. 4-5):“The proposed work could be of particular importance in relation to anxiety, irritability, and ADHD symptoms as these symptoms are common, co-occurring symptoms that are present across a wide range of childhood psychopathology , and are implicated in the later development of mood disorders including depression . Furthermore, aberrant cognitive control more broadly has been implicated as potential mechanism associated with the presentation of anxiety , irritability , and ADHD  symptoms.”6. Stimuli: The application seems to present a limited number of trials and stimuli types. Did the authors consider using different colours and shapes of stimuli for each application testing session to avoid habituation for specific stimuli? For example, the no-go stimuli are always yellow stars, changing the colour or type of the no-go stimulus for each level may reflect more on the flexibility of each participant to inhibit inappropriate response in a variety of different scenarios.The reviewer brings up a very interesting recommendation regarding the study of the flexibility of inhibitory control in the context of changing task demands/stimuli characteristics. We believe that the first critical step is to first establish validity of CALM-IT as a measure of baseline inhibitory control and thus have limited versions of the game to two, one modeled after go-no-go and one modeled after stop signal delay paradigms. This also allows us to keep the game play to a reasonable length of time while maximizing the amount of data collected. However, the reviewer’s suggestion would be a very important avenue of future work as well as potentially other manipulations of the stimuli that could be leveraged to train inhibitory control. While this is out of the scope of the current proposed project, we now include additional discussion of potential future versions of CALM-IT should the proposed study confirm valid measurement of inhibitory control using the current proposed version of CALM-IT (p. 19):“The present protocol would lay the groundwork for an important line of future work that could provide researchers and clinicians a multifaceted tool to measure multiple aspects of inhibitory control. For example, future versions of the app could include manipulation of the stimuli presented such that participants are required to update the which stimuli represents the stop-stimuli, thus targeting one’s ability to flexibly deploy inhibitory control. Furthermore, an adaptive version of CALM-IT that increases difficulty based on participants’ individual performance could function as an intervention aimed at improving inhibitory control.”7. Effect size calculation: What is the rationale behind selecting the effects of r between .20 and .32. Could the authors give examples of similar studies or otherwise justify the selected effects and/or the effect size calculation?We realize that the language used in the description of the power analyses suggests that we began with a priori effect sizes of r between .20 and .32 however, those effects were reported as a function of the sample size to provide a reader with context or the size of effects that could be detected with a power of .80 at a significance level of alpha = .05. We have revised the language in this section to provide more clarity (p. 9-10):“To investigate the feasibility of CALM-IT (Aim 1a), we will recruit 200 youth. For investigation of the stability of CALM-IT (Aim 1b), we will invite 75 youth out of the overall sample of 200 to play CALM-IT twice. For validation of mobile application-based behaviors of inhibitory control (Aim 2), we will invite 100 youth out of the overall sample of 200 to complete a battery of in-laboratory inhibitory control paradigms. We evaluated each of these proposed sample sizes using the pwr package in R to determine the size of the effect that the sample would allow us to detect using a significance level of 0.05 and a power level of 0.80 . For the feasibility of CALM-IT (Aim 1a), our proposed sample size is sufficient to detect effects of r≥0.20. For the stability of CALM-IT (Aim 1b), our proposed sample size is sufficient to detect effects of r≥0.32. Finally, for the validation of application-based behaviors of inhibitory control (Aim 2), the proposed sample size is sufficient to detect effects of r≥0.28. Thus all proposed samples would allow us sufficient power to detect small to medium effect sizes that are similar to those found within comparable samples .”8. Symptom measures: I would recommend to the authors to consider measuring the impulsivity of participants also by using standardised questionnaires to investigate the different facets of impulsive behaviour. Some established measures as UPPS-P can be used in children.Unfortunately, due to demands placed on our pediatric patients with severe clinical impairment within the current protocol, we are hesitant to add additional measures. However, we appreciate the important point made by the reviewer regarding impulsivity specifically and have added secondary analyses in which we will specifically examine associations with the two components of ADHD (impulsive hyperactivity and inattentiveness) as measured by the Conners Comprehensive Behavior Ratings Scale. Moreover, we will look specifically at the clinician endorsed items for on the ADHD module of the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime version.(p. 15): “Secondary analyses will be conducted examining associations with the two components of ADHD: hyperactive-impulsivity and inattentiveness. For these analyses, the CBRS DSM-IV Hyperactive-Impulsive and CBRS DSM-IV Inattentive subscales will be used.”(p. 19): “Finally, secondary analyses will be run examining bivariate correlations between measures of hyperactive-impulsivity, inattentiveness, and depression.”9. Assessing the engagement: This seems to be assessed only based on the performance metrics from the app. I would recommend to the authors considering running a focus group or using a questionnaire of user experience to gain more information on the engagement and the usability of the app. This can be an important source of information for adjusting apps in development based on real user-experience feedback. If the app engagement is assessed only based on the performance metrics, how will the authors distinguish between poor performance in the app based on boredom or lack of engagement versus a poor performance caused by very poor inhibitory control abilities?The degree to which participants engage in experimental tasks is a persistent issue across measurement and construct. Unfortunately, this issue is present even for the most canonical in-laboratory measures of inhibitory control. One of central aims of CALM-IT is to create a more engaging measurement of inhibitory control that could limit confounding issues of loss of attention or limited motivation. Thus, we agree with the reviewer that it is critical that we evaluate the degree to which CALM-IT was in fact engaging. To improve our evaluation of engagement we now additional propose to examine our measure of app engagement across levels (p. 16):“Average percent targets hit and percent stars hit will also be examined across each level to assess maintenance of attention and engagement across all levels of the task.”Additionally, we will now include a follow up interview as part of our procedures to provide feedback from participants regarding their experience playing the game and specifically the degree to which they report being engaged (p. 16):“Finally, a follow-up interview will be conducted to collect a qualitative assessment of CALM-IT engagement from each.”AttachmentResponseToReviews_CALMIT.docxSubmitted filename: Click here for additional data file. 12 May 2021Rationale and validation of a novel mobile application probing motor inhibition: Proof of concept of CALM-ITPONE-D-20-39885R1Dear Dr. Cardinale,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Veena KumariAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 25 May 2021PONE-D-20-39885R1 Rationale and validation of a novel mobile application probing motor inhibition: Proof of concept of CALM-IT Dear Dr. Cardinale:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Veena Kumari Academic EditorPLOS ONE


A. baumannii multidrug-resistant (MDR) isolate. The MIC of halicin was found to be 16 μg/mL for S. aureus (ATCC BAA-977), 32 μg/mL for E. coli (ATCC 25922), 128 μg/mL for A. baumannii (ATCC BAA-747), and 256 μg/mL for MDR A. baumannii. Upon storage, the MICs were increased, suggesting instability of the drug after approximately a week of storage at 4 °C. MICs and zones of inhibition were found to be high (R = 0.90 to 0.98), suggesting that halicin has a promising antimicrobial activity and may be used as a wide-spectrum antibacterial drug. However, the drug’s pharmacokinetics have not been investigated, and further elucidation is needed.Artificial intelligence (AI) is a new technology that has been employed to screen and discover new drugs. Using AI, an anti-diabetic treatment  was nominated and proven to have a unique antibacterial activity against several harmful bacterial strains, including multidrug-resistant bacteria. This study aims to explore the antibacterial effect of halicin and microbial susceptibility using the zone of inhibition and the minimum inhibition concentration (MIC) values while assessing the stability of stored halicin over a period of time with cost-effective and straightforward methods. Linear regression graphs were constructed, and the correlation coefficient was calculated. The new antibacterial agent was able to inhibit all tested gram-positive and gram-negative bacterial strains, but in different concentrations—including the  The rise of antibiotic-resistant bacteria is considered a crucial health threat globally . The WorA team of scientists at MIT has recently reported using artificial intelligence to repurpose a new compound with antibacterial activities against broad spectrum bacteria, including MDR ,6. The tAs a result of a recent discovery, a couple of studies have evaluated the antibacterial activity of the drug. Unfortunately, the standard guidelines reference lacks the antibacterial activity of halicin against pathogenic bacteria, making it difficult to validate its antibacterial efficacy. Here, halicin MIC was validated and determined. The results of the antibacterial activity were correlated with the disc diffusion assay against gram-positive and gram-negative bacteria, including multidrug-resistant bacterium, to construct linear regression graphs of the halicin antibacterial drug.Halicin was purchased from Carbosynth , while Mueller-Hinton broth was bought from Scharlab . Distilled water was generated through Milli Q  and had been used throughout this study. The measurement was made with CytationTM 3 Cell Imaging Multi-Mode Reader .Staphylococcus aureus , Escherichia coli , and Acinetobacter baumannii . Bacterial suspensions were adjusted to a turbidity equivalent to that of a 0.5 McFarland Standard and incubated overnight under aerobic conditions at 37 °C. The media were prepared according to the manufacturer Scharlab’s recommendations [The American Type Culture Collection (ATCC) bacteria were used in this study and MDR clinical isolate. Muller-Hinton broth was used to revive the bacterial strains, 6 colony-forming-unit/mL (CFU/mL) was used, and incubated overnight at 37 °C with a continuous shaking speed of 120 RPM [The MIC of halicin was determined by preparing a serial twofold dilution of the drug, ranging from 256 to 0.125 μg/mL, in Mueller–Hinton broth, and filtered by a 0.22 μm filter and added into 96-well plates containing bacterial suspensions. The total culture volume was 200 μL. A final bacterial inoculum of 1 × 10 120 RPM . In addiS. aureus (ATCC BAA-977) and E. coli (ATCC 25922), while the concentration range was 256 to 16 μg/mL against A. baumannii (ATCC BAA-747 and 3086 MDR isolate), according to the MIC results. A final concentration of 1 × 106 CFU/mL inoculum was equally distributed on the Mueller-Hinton agar surface. The diameters of the clear areas of no growth were recorded in millimeters (mm). This test was performed in independent triplicates.The zone of inhibition assay was performed with discs impregnated with halicin solutions, at a twofold concentration range of 128 to 16 μg/mL against 2) were calculated using Microsoft Excel 2016 software . A t-test was used to compare the results’ variance, in which p < 0.05 was considered as statistically significant.The mean, standard deviation (SD), regression equation, correlation coefficient, and (R) coefficient of determination  helped clarify the MIC with a sensitive inhibition zone diameter. Furthermore, these assays are characterized by their simplicity; they are fast and cost-effective compared to other microbiological tests. All of these factors are essential for the reproducibility of the data, mainly when testing different phenotypic bacteria.S. aureus (ATCC BAA-977), 32 μg/mL for E. coli (ATCC 25922), 128 μg/mL for A. baumannii (ATCC BAA-747), and 256 μg/mL for MDR A. baumannii (MDR 3086), as shown in S. aureus, significantly increased to be 64 μg/mL for E. coli, insignificantly changed from 128 μg/mL for A. baumannii, and significantly increased to be ≥256 μg/mL for MDR A. baumannii  reference platforms. On the other hand, the MIC test is relatively expensive since a higher amount of a drug is required to be measured. There is no reference evidence for halicin effectiveness against bacteria; hence, standard strains were used in this study to estimate the MIC and determine the region of inhibition on agar plates .S. aureus BAA-977, 20 mm for E. coli ATCC 25922, and 20 mm for A. baumannii ATCC BAA-747. However, the MDR A. baumannii was only sensitive at >128 μg/mL, with a diameter of 22 mm for a halicin concentration of 256 μg/mL. The inhibition zones demonstrated that halicin could inhibit all tested gram-positive and gram-negative bacterial strains, but with variable efficacy. The diameter of the zone of inhibition at a halicin concentration of 128 μg/mL was recorded as 29 mm for strains of 2) between halicin MIC and its bacterial inhibitory effect was also shown in 2 ≥ 0.95) against E. coli ATCC 25922 and A. baumannii ATCC BAA-747, and fairly strong (R2 ≥ 0.8 and <0.9) against S. aureus ATCC BAA-977 and MDR A. baumannii MDR 3086.The statistical coefficient of determination , while E. coli was sensitive at a diameter of ≥14 mm at MIC 32 μg/mL. The A. baumannii ATCC strain was also sensitive at an inhibition diameter of ≥20 mm at MIC 128 μg/mL. In contrast, the MDR strain was resistant at the same concentration but sensitive at a higher concentration (265 μg/mL), which gave an inhibition diameter of >20 mm. These variable data suggest that the pharmacokinetics of the halicin drug may behave differently with sensitive and drug-resistant bacteria.Many factors might affect the reproducibility, susceptibility, and accuracy of the agar diffusion assay. Therefore, the interpretations consider all the variable factors . The corS. aureus’s sensitivity was ≥16 mm, while both A. baumannii strains demonstrated a sensitivity of ≥20 mm. The E. coli’s sensitivity was ≥14 mm. Therefore, this study could be a reference for halicin antibacterial activity that will offer a guide when evaluating this drug’s pharmacological and toxicological windows. Nevertheless, the absence of a substantial analytical study and stability study is unexpected. Therefore, there is a need for a thorough investigation before the clinical use of halicin against MDR infections.Halicin is effective against the tested gram-positive and gram-negative bacteria;


Avian infectious bronchitis virus (IBV) is an important gammacoronavirus. The virus is highly contagious, can infect chickens of all ages, and causes considerable economic losses in the poultry industry worldwide. In the last few decades, numerous studies have been published regarding pathogenicity, vaccination, and host immunity-virus interaction. In particular, innate immunity serves as the first line of defense against invasive pathogens and plays an important role in the pathogenetic process of IBV infection. This review focuses on fundamental aspects of host innate immune responses after IBV infection, including identification of conserved viral structures and different components of host with antiviral activity, which could provide useful information for novel vaccine development, vaccination strategies, and intervention programs. Gammacoronavirus. This virus infects the upper respiratory tract, reproductive system, and kidneys of chickens [The avian infectious bronchitis virus (IBV) belongs to the genus chickens . Dependichickens , intestichickens , enterocchickens , and glachickens . Chickenchickens . After Ichickens ,8. Therechickens .The IBV viral genome is an unsegmented, single-stranded positive-sense RNA of 27.6 kb in length . The 3′-As for control of IBV infection, strict biosecurity on poultry farms is required. Because no effective drugs against IBV infection have been reported so far, vaccination is the most efficient approach to prevent production losses . HoweverDue to the requirement for reducing and limiting antibiotics in the poultry industry, IBV control is of great importance in terms of the increasing demand for antibiotic-free chickens. Because unsuccessful vaccination will lead to failure in IBV control, in the last few decades, numerous studies have presented the function of host innate immunity in resistance towards IBV. The innate immune system, containing various cells and molecules, non-specifically targets invading pathogens and serves as the first line of defense. Therefore, besides the efforts in novel vaccine development and prophylactic measures, the enhancement of innate immune responses in resistance to IBV have gained increasing attention in the design of novel prevention approaches.Although IBV was the first coronavirus discovered, its functional host cell receptor is still in debate. Previous research suggests that Neu5Acα2-3Galβ1-3GlcNAc (Neu5Ac)  could be a potential receptor, as it bound specifically to the S1 protein of the M41 IBV strain . AminopeIBV primarily targets epithelial cells, causing mucosal pathological lesions, including epithelial hyperplasia, ciliary loss and necrosis, and inflammatory cell infiltration . After vHeterophils, counterpart to mammalian neutrophils, are main granulocytes in poultry blood and form the polymorphonuclear cell population of chicken ,22. In SMacrophages are involved in recognition, phagocytosis, and degradation of invading pathogens. The number of macrophages in respiratory lavage fluid, embryonic tissues, trachea, and lung was increased after IBV inoculation ,26. In r+CD3− NK cells in the lungs and blood from 1 dpi [Other innate immune cells, such as natural killer (NK) cells, are also activated at an early IBV infection stage. For instance, 5-week-old chickens infected with M41 IBV strain via nasal–tracheal route induced a rapid activation of CD107om 1 dpi . These com 1 dpi . TherefoTo summarize, innate immune cells including heterophils, macrophages, and NK cells are recruited rapidly (24 hpi) at the sites of IBV infection. Based on the antiviral function of these cells, including pathogen recognition, phagocytosis, cytokine production, apoptosis regulation, etc., rapid activation of these cells is important for virus clearance and resistance to IBV.Though less studied, chicken innate immunity functions are similar to mammalian innate immunity with certain differences. Pattern recognition receptors (PRRs) are important components in host innate immunity, which are expressed on cell membranes, endosome membranes, and lysosome membranes, as well as in cytoplast. Based on the mammalian literature, toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and NOD-like receptors (NLRs) are the most common host PRRs responding to viruses ,38. All identified TLRs are type I transmembrane proteins, enriched on the cell surface or membrane of endosomes . In chicIn mammals, TLR3 and TLR7 are well known for recognition of RNA viruses . chTLR3 The mammalian RLR family has three members: RIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) . HoweverNLRs could also be additional candidate receptors in chicken because NLR elements  are identified in the chicken genome . ExpressLack of available cell lines of IBV strains has limited investigation of avian PRRs sensing the virus, and cell-adapted Beaudette IBV strains are extensively used in the research. In vivo studies using CEK cells and tracheal organ culture (TOC) presented elevated levels of chTLR3 and chMDA5 expression at an early stage of infection (9 hpi) by different respiratory or nephropathogenic IBV strains ,56, whilIn general, in vivo experiments presented activated PRR (mostly on chTLRs) expression after infection. For instance, in tracheas of 3-week-old and 6-day-old SPF chickens, up–regulation of chTLR3 and chTLR7 was observed at 1 dpi, when inoculated with respiratory attenuated Mass and Conn IBV strains, respectively , while dThough these studies presented spatial and temporal differences in PRR regulation after IBV infection, due to the importance of PRRs in pathogen recognition, activation of PRRs is still important in IBV control. Therefore, to rapidly activate PRRs, TLR ligands have drawn attention in novel vaccine design. For instance, administration of TLR7 agonist R848 (resiquimod) with live or inactivated Mass IBV strain significantly enhanced the sIgA responses through upregulation of TGF-β4 . In addiIn general, PRR activation mediates IFN pathways via TRIF or MyD88. Subsequently, these signaling cascades promote the NF-κB and IFN regulatory factor (IRF) family, resulting in expression of type I IFNs and then transcription of interferon-stimulated genes (ISGs) with antiviral activity . ISGs haIn chickens, eight members of the IRF family were identified, whereas IRF3 and IRF9 are absent . chIRF7 IFNs, including type I, type II, and type III IFNs , are mulThe type I IFN functions as a powerful antiviral mechanism, which has been shown to be involved in host response after IBV infection. It was shown that IFN-α could inhibit respiratory Beaudette or Gray IBV strains both in vitro and in vivo . In vitrIn vivo studies showed a more complex result, in that the expression level of IFN-α was significantly upregulated at 1 dpi in spleens after virulent respiratory IBV infection , while iSimilarly to type I IFNs, at an early stage of infection (12 hpi), after inoculation with the respiratory Conn IBV strain, IFN-γ was significantly downregulated in tracheas and lungs of the infected chicken . At 2–3 To summarize, though the responses of IFNs after IBV infection vary in a strain-dependent and time-dependent manner, in general, activation of IFNs is restrained at a very early stage of IBV infection to allow viral replication. Upregulation of IFNs is often observed with activated ISGs after infection is established, when the innate immunity responds for viral clearance. Therefore, early intervention and activation of IFNs are critical in control of the disease. Other cytokines and chemokines are also crucial regulators of innate immune responses against viral infection. For instance, correlated with recruited macrophages, production of IL-1β was involved in reducing IBV viral loads in the respiratory tract . In addiDepending on the IBV strain, it was reported that proinflammatory cytokine expression was induced differently in different tissues. In tracheas, at an early infection stage (1–3 dpi), expression of IL-1β, IL-10R2, IL-6, and LITAF was elicited after inoculation with either respiratory or nephropathogenic IBV strains . The expIn kidneys, regulation of these cytokines was not significantly affected following infection with respiratory IS/885/00-like (885), M41, and nephropathogenic QX-like IBV strains . ChickenChemokines orchestrate migration of cells during immune surveillance. Mass IBV strain stimulated gene expression of CXCR4, CCR6, chemokine-like receptor 1/CHEMR23, and Matrix metalloproteinase (MMPs) from an early phase of viral infection (1 dpi) in tracheas . These cBased on these observations, after IBV infection occurs, the innate immunity is activated, resulting in recruiting of innate cells at the infection sites and upregulation of different PPRs, cytokines, and chemokines, etc. However, downregulation of PPRs (TLR7), IFNs , and other cytokines (IL-1β) was still observed at a very early stage of infection (12 hpi) by respiratory IBV strains, suggesting inhibition of the innate immunity is important to establish successful IBV infections, which may reflect common strategies the coronaviruses might take to avoid detection by the host innate immunity. Because most studies were carried out using respiratory IBV strains, it would be important to gain more information about the antiviral host responses using the nephropathogenic IBV strains, which may help to develop vaccination strategies and other intervention programs. A general description of the innate immune responses against IBV infection is shown in Apoptosis is one of the primary mechanisms that animals use to combat viral infections. It can also facilitate virus spread at a later stage of infection . There hIt is suggested that the IBV ORF1b region is responsible for triggering apoptosis . In mammE. roratus) showed 100% homology with the IBV GI-13 lineage [Since first being documented in the United States in 1931, IBV has become endemic throughout the poultry industry . It has  lineage . WhetherAlongside research on vaccination and prevention measures, in recent years more attention has been focused on understanding the early immune responses after IBV infection, as this would expand our knowledge of the pathology of the virus, which in turn could benefit the development of prevention and control strategies. Innate immunity contributes to a network by utilizing PPRs to detect conserved PAMPs, where different components such as IFNs and proinflammatory cytokines play essential roles in antiviral activity. Several reviews regarding chicken immune responses to IBV infection are recommended for a comprehensive understanding of the virus-host immunity interaction .Given the vast diversity of IBV strains, innate immune responses evoked by IBV infection vary in a strain-dependent and time-dependent manner. Still, early intervention and activation of the innate immunity is essential for control of the disease. To evoke an early innate immune response, agonists of PRRs and IFNs have drawn more attention in novel vaccine design. In addition, population diversity of the virus also contributes to the enhancement of host immunity, as a more diverse viral population in the vaccine induced stronger innate immune responses . TherefoThough the information on IBV-host innate immunity interaction is still limited due to lack of experimental measures in chicken, it is well-recognized that the innate immunity contributes not only to prevention strategy development, but also to the pathogenicity of the virus. For effective control of the virus, early enhancement of the host innate immunity is critical. Furthermore, because the chicken innate immunity acts in a strain-dependent and time-dependent manner after IBV infection, early diagnosis of the IBV strain is also important for better control of the virus. Further investigation is required to explore differences in immune response triggered by different IBV strains with differing genotypes and pathogenicity.


Glacier ice archives information, including microbiology, that helps reveal paleoclimate histories and predict future climate change. Though glacier-ice microbes are studied using culture or amplicon approaches, more challenging metagenomic approaches, which provide access to functional, genome-resolved information and viruses, are under-utilized, partly due to low biomass and potential contamination.Janthinobacterium, Polaromonas, Herminiimonas, Flavobacterium, Sphingomonas, and Methylobacterium as the dominant genera, while microbial communities were significantly different between two ice cores, associating with different climate conditions during deposition. Separately, ~355- and ~14,400-year-old ice were subject to viral enrichment and low-input quantitative sequencing, yielding genomic sequences for 33 vOTUs. These were virtually all unique to this study, representing 28 novel genera and not a single species shared with 225 environmentally diverse viromes. Further, 42.4% of the vOTUs were identifiable temperate, which is significantly higher than that in gut, soil, and marine viromes, and indicates that temperate phages are possibly favored in glacier-ice environments before being frozen. In silico host predictions linked 18 vOTUs to co-occurring abundant bacteria , indicating that these phages infected ice-abundant bacterial groups before being archived. Functional genome annotation revealed four virus-encoded auxiliary metabolic genes, particularly two motility genes suggest viruses potentially facilitate nutrient acquisition for their hosts. Finally, given their possible importance to methane cycling in ice, we focused on Methylobacterium viruses by contextualizing our ice-observed viruses against 123 viromes and prophages extracted from 131 Methylobacterium genomes, revealing that the archived viruses might originate from soil or plants.We expand existing clean sampling procedures using controlled artificial ice-core experiments and adapted previously established low-biomass metagenomic approaches to study glacier-ice viruses. Controlled sampling experiments drastically reduced mock contaminants including bacteria, viruses, and free DNA to background levels. Amplicon sequencing from eight depths of two Tibetan Plateau ice cores revealed common glacier-ice lineages including Together, these efforts further microbial and viral sampling procedures for glacier ice and provide a first window into viral communities and functions in ancient glacier environments. Such methods and datasets can potentially enable researchers to contextualize new discoveries and begin to incorporate glacier-ice microbes and their viruses relative to past and present climate change in geographically diverse regions globally.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-021-01106-w. Howetabolism . In the tabolism  through Glaciers potentially archive environmental conditions and microbes over tens to hundreds of thousands of years. Unfortunately, glaciers around the world, including those from Tibetan Plateau and Himalaya, are rapidly shrinking, primarily due to the anthropogenic-enhanced warming of Earth’s ocean-atmosphere system . Such meFuture work will benefit from emerging technologies to detect microbial growth , betterAn artificial ice core was constructed from sterile water, which was pre-filtered through a Millipore system  outfitted with a 0.22-μm mesh final filter and autoclaved at 121°C for 30 min, then frozen at −34°C for 12–24 h in a 2-L sterile plastic cylinder . The cylinder was transferred from −34 to −5°C and kept at that temperature overnight to reduce the possibility of fracturing (which is caused by sudden temperature changes) before placing it at room temperature for about 30 min to melt the surface ice and expose the underlying ice core.Cellulophaga baltica strain #18  was cultured in MLB medium , 0.5 g bacto peptone, 0.5 g yeast extract, 0.5 g casamino acids, 3 ml glycerol, and 1000 ml water) stationary overnight at room temperature. The cell concentration was measured by epifluorescence microscopy after the cells were captured on a 0.22-μm-pore-sized filter  and stained by SYBR Green  as described previously [2 cover slip. Cells on the filter were counted using epifluorescence microscopy (Zeiss Axio Imager.D2) with >350 cells or >20 fields counted, which was a reliable threshold to estimate the total bacterial abundance [eviously  with sombundance .Pseudoalteromonas phages strain PSA-HP1 (NCBI: txid134839) were harvested from 95% lysed plaque assays (agar overlay technique). The concentration of PSA-HP1 was counted by a wet-mount method using SYBR Gold  staining and glass beads as described previously [9 copies/μl; genome size 4.8 kb) was purchased from Life Technologies (Cat. No. P7589). Above components  were combined in 1 ml ddH2O, which contained 1.00×106 cells, 4.48×107 viruses, and 1.88×108 copies of lambda DNA to make the mock contaminants. The concentration of contaminant cells is approximate to the cell numbers in glacier ice (~102–104 cells/ml [2–105 cells/ml [eviously . The lamcells/ml ). The 1 The decontamination procedure consisted of three steps Fig.  followinTwo artificial ice core sections (sections 1 and 2) were processed using the decontamination procedure described above Fig. . The iceThe plateau shallow core  and the summit core 3  were drilled on the Guliya ice cap in 1992 and 2015, respectively Fig. . Both co3 of air were collected over 4 days of continuous sampling in the room using an air sampler (SKC Inc.) as described previously [Four controls were used to trace possible sources of background contamination during ice sample processing as described previously . First, eviously , during 2 in area) room that is reserved for microbial experiments with low-biomass samples. The hood was exposed with UV light for more than 1 h before experiments.A total of 300 ml of artificial ice, 300 ml of the blank control, and 100–300 ml each of the glacier ice samples were filtered through sterilized polycarbonate 0.22-μm-pore-sized filters  to collect microbes including all bacterial/archaeal cells larger than 0.22 μm. The filters were preserved at −20°C until DNA extraction (within 24 h). Viruses in the filtrate of two samples (D25 and D49) were concentrated to 0.8 ml using 100 kDa Amicon Ultra Concentrators  and preserved at 4°C until DNA extraction (within 24 h). To check for possible cross contamination among samples and potential viral contaminants introduced to the samples during processing, 1 ml of 0.22-μm-pore-size filtrate from the water of the Olentangy River  was co-processed in parallel with samples D25 and D49 throughout the entire analyses. All the biological work in this study after the ice sampling in the cold room laboratory was performed in a hood within a small  according to the manufacturer’s instructions, with an additional step of beating with beads to disrupt bacterial spores and Gram-positive cells before cell lysis by homogenizing at 3400 RPM for 1 min with 100 mg of autoclaved (121°C for 30 min) 0.1-mm-diameter glass beads  in a MiniBeadBeater-16 .2O. The amplification included a 5-min denaturation step at 95°C, followed by 40 cycles of 30 s at 95°C, 30 s at 56°C, and 50 s at 72°C, with a final extension of 5 min at 72°C. For the nested PCR, the reaction mixture was identical to the first PCR, except that 5.0 μl of the first PCR product and 1.25 μl of each nested primer  were included. The amplification conditions were also identical to the first PCR except for the extension time of 20 s at 72°C for 40 cycles of amplifications.Nested PCR experiments  were per4, 103, 102, 101, 100 and 10−1 copies, respectively) as templates to serve as a reference. A negative control was conducted using 5 μl of ddH2O as template.For each of artificial ice section samples , 0.5 μl of each primer (10 μM), 3 μl of template DNA, and 6 μl of RNase-free water. All reactions were performed in triplicate, using an Illumina Eco cycler (Cat No. 1010180).Total bacterial and archaeal biomasses of the glacier ice samples and the “background” controls were estimated using qPCR after isolating DNA. The primer set 1406f (5′-GYACWCACCGCCCGT-3′) and 1525r (5′-AAGGAGGTGWTCCARCC-3′) was used to amplify bacterial and archaeal 16S rRNA genes . ThermocTotal numbers of CBA 18 in each of the artificial ice samples  using weighted UniFrac metrics was performed to distinguish general distribution patterns of microbial profiles among all samples. The Mantel tests were conducted to evaluate the linkage between the microbial community structure and environmental parameters. The significance of the difference in microbial community between grouped samples (PS versus S3 core samples) was evaluated by analysis of similarity statistics , which was performed using functions in the Vegan package version 2.4-4 in R version 3.4.2 .The viral genomic DNA from three samples  was subjected to low-input library preparation pipeline using the Nextera® XT Library Prep Kit  in the clean room, according to our methods described previously , 47, 63.Synechococcus phages, Cellulophaga phages, and Pseudoalteromonas phages) using Blastn. Then, they were clustered into vOTUs if viral contigs shared ≥95% nucleotide identity across 80% of their lengths as described previously [All metagenomic analyses were supported by the Ohio Supercomputer Center. Viromic sequence data was processed using iVirus pipeline with default parameters described previously , 137. Breviously , 49. TheA total of 33 and 107 vOTUs (length ≥10 kb) were obtained for two glacier ice samples D25 and D49) and the river water control (RiverV) viromes, respectively. Mapping the quality-controlled reads of the 3 viromes to the 140 vOTUs (33+107) showed that the viral communities in the glacier ice samples were completely different from those in the river water control . Only two genes from two contigs (one gene per contig) had significant hits to eukaryotic viruses (bit score 128 and 164). In addition, two other efforts were made to detect eukaryotic viruses (chloroviruses) in the glacier ice samples: (a) the four known chlorovirus hosts, including or virus  and (b) or virus  and CHL or virus . However−3, and average nucleotide identity of ≥70% across ≥2,000 bp with the host genomes [Taxonomy assignments were performed using vConTACT v2.0 . Briefly genomes . Finally genomes ;) and coThe putative AMGs were identified and evaluated according to our previously established methods . SpecialTo explore the geographic distribution of glacier viruses, the genome fragments of 33 vOTUs were used as baits to recruit reads from 225 previously published viromes from a wide range of environments including global oceans (145 viromes of GOV 2.0) , Arctic Methylobacterium. In addition, Methylobacterium viruses (prophages) were also extracted from 131 bacterial genomes within Methylobacterium species, which were obtained from the RefSeq database (release v99). These efforts identified 484 Methylobacterium phages, which were used for genome-based network analyses to evaluate their relationship with five glacier-ice viruses infecting Methylobacterium, using vConTACT version 2 [Methylobacterium viruses of interest were evaluated and illustrated by Easyfig version 2.2.5 [Methylobacterium viruses was performed as described above for AMGs.The 123 previously published viromes  were reersion 2 , 86. Theon 2.2.5 . The phyAdditional file1: Table S1 Relative abundance and taxonomy of 33 most abundant OTUs in Inner, Wash and Cut parts of two glacier-ice sections. Table S2 Microbial community composition of eight glacier-ice samples at genus level. Table S3 Physical and chemical characteristics of ice samples from the Guliya ice cap. Table S4 Significance of each parameter's influence on the distribution of microbial communities by Mantel tests. Table S5 General statistics for two viromes. Table S6 Taxonomic assignments and viral cluster information of 33 glacier-ice vOTUs and 2,304 RefSeq genomes. Table S7 Adjusted coverage of 33 vOTUs per gigabase of MetaG for two glacier-ice viromes. Table S8 Predicted hosts taxonomy by sequence similarity method (Blastn). Table S9 Predicted hosts taxonomy by sequence composition method (VirHostMatcher). Table S10 Predicted hosts taxonomy by CRISPR similarity method. Table S11 Temperate viruses predicted by VIBRANT from the 33 vOTUs. Table S12 Methylobacterium viruses identified from glacier ice, bacterial genomes in RefSeq database and environmental metagenomes. Table S13 Annotations of all genes from 33 vOTUs. Table S14 Four AMGs identified from glacier-ice viruses. Table S15 Annotations of 70 viral contigs’ genes from the Global Ocean Viromes 2.0 dataset.Additional file2: Figure S1 Ice core sampling and preparation in the laboratory. (a) The cold work room (−5°C) with band saw, BioGard laminar flow hood and wash systems. (b) the outer layer of the ice section being removed by the band saw. (c) The ice section being washed with 95% ethanol and (d) with water. (e) The “clean” inner ice is preserved in the autoclaved beakers or bottles. Figure S2 Microbial communities at genus level (a) and overlapped OTUs (b) of removed and inner ice samples collected during decontamination procedures. The most abundant genera (n = 30) and OTUs (n = 33) are illustrated. Cut, Wash and Inner represent ice samples collected from band saw scrapping, water washing and the inner ice, respectively. Figure S3 Rarefaction curves of two glacier-ice viromes by vOTU numbers. Rarefaction curves were constructed by the change of vOTUs (≥10 kb) number along sequencing depth  obtained by subsampling quality-controlled reads. Figure S4 The unrooted neighbor-joining phylogenetic tree of Mu N genes from eight Methylobacterium viruses. The tree was constructed using the predicted amino acid sequences of the N genes from two glacier ice viruses  and six prophages identified from bacterial genomes. Each viral contig contains two copies of N genes. Viruses belonged to the same VC  are indicated in the same color. Bootstrap values  are shown at the branch points. The scale bar indicates a distance of 0.2. Figure S5 Characterization of virus-encoded auxiliary metabolic genes (AMGs). (a) Genome map of glacier-ice virus D25_22_20338 encoding AMGs (motility genes motA and motB). CheckV was used to assess host-virus boundaries and remove potential host fractions on the viral contig (See ntig See . Genes wntig See . The gen


The recreational runners were found to have a smaller dorsiflexion angle, initial dorsiflexion contact angle, ankle inversion, knee adduction, range motion in the frontal knee plane and hip frontal plane. The running kinematics data were influenced by running experience. The findings from the study provide a better understanding of the kinematics variables for competitive and recreational runners. Thus, these findings might have implications for reducing running injury and improving running performance.Kinematics data are primary biomechanical parameters. A principal component analysis (PCA) of waveforms is a statistical approach used to explore patterns of variability in biomechanical curve datasets. Differences in experienced and recreational runners’ kinematic variables are still unclear. The purpose of the present study was to compare any differences in kinematics parameters for competitive runners and recreational runners using principal component analysis in the sagittal plane, frontal plane and transverse plane. Forty male runners were divided into two groups: twenty competitive runners and twenty recreational runners. A Vicon Motion System  captured three-dimensional kinematics data during running at 3.3 m/s. The principal component analysis was used to determine the dominating variation in this model. Then, the principal component scores retained the first three principal components and were analyzed using independent  Long-distance running is a convenient sport that is popular with numerous runners globally ,2. With A number of studies have investigated biomechanical running experience. It was noted that running experience might influence the risk of running injuries. It has been proposed that novice runners were more prone to lower limb injuries than recreational runners per 1000 h running . FollowiSome researchers have also proposed that running experience was not able to influence running biomechanics variables. The previous study demonstrated that running kinematics were influenced by age, which might cause knee flexion reduction and increase contact time during running. With increased years of running experience, the kinematics variables of spatiotemporal variables, kinematics variables and lower limb joint ankle were unchanged . In addiIn recent years, new analysis techniques, such as machine learning, have been used to analyze running biomechanics. A support vector machine and principal component scores have been applied to the kinematics parameters to determine the differences between competitive and recreational runners. The results favored the support vector machine (SVM) to achieve cross-fold classification accuracies of between 80.00–100% in knee flexion angle in the sagittal plane, ankle angle in the frontal plane and pelvis in the sagittal plane . Eneida Principal component analysis is a method used to identify data patterns and express data to highlight the similarities and differences between data. It has been widely used in many research fields involving data analysis and preprocessing . PrincipThe PCA algorithm has been proposed for biomechanics applications. Many previous studies have shown that principal component analysis could distinguish different gait dynamics more sensitively than traditional parameter analysis techniques ,24,25,26Few studies have found kinematics variable differences between different level runners in previous research. However, in some investigations, PCA can extract the key features for the joint angle, joint moment and joint force and distinguish the groups differently. A principal component analysis is a reduction technique that might reduce the time-wave dimensionality. To compare the variables of running lower limb kinematics, the purpose of the present study was to utilize PCA to identify the ankle, knee and hip joint differences during the running phase between competitive runners and recreational runners. We used the PCA waveform analysis to extract primary features that can quantify differences for competitive runners and recreational runners. We hypothesized that lower limb joint angle differences would exist between experienced runners and reactional runners during the running stance phase.Twenty healthy male competitive runners  and twenty male recreational runners  were recruited from the running club and the university running association to participate in this study. Competitive runners had to satisfy the following conditions: rearfoot strike pattern, run at least 15 km per week and have running experience of more than five years . They diPrior to running data collection, all participants were required to wear uniform running shoes  and tight pants and t-shirts, and were fully familiarized with testing procedures. Thirty-six retroreflective markers were fixed to anatomical landmarks. Then, the spherical reflective (14 mm) markers were put on the bilateral lower limbs. The marker position defined by skeletal landmarks included: right/left anterior superior iliac spine, right/left posterior superior iliac spine, right/left femur lateral epicondyle, right/left femur medial epicondyle, right/left first and fifth metatarsal heads, right/left distal interphalangeal joint of the second toe, right/left medial and lateral malleoli, right/left medial and lateral epicondyle of the femur. Clusters of 4 markers were placed laterally on the right and left thigh and shank segments .The participants were required to stand on the force plate following the reflective marker placement for static model data collection. In addition, before the experiment, all the participants were asked to run and jog for warm-up activities on the ground. Participants were then asked to right foot run through the force plate at a speed of 3.3 m/s. Kinematics data were captured using an eight-camera motion system  at a sampling rate of 200 Hz. Each participant completed five successful running trials over a 15 m runway at 3.3 m/s in a standard biomechanics laboratory. A speed measuring instrument  was placed on both sides of force plate to control running speed. A successful run required the participants to run with the entire right foot placed on the force plate at the running speed of 3.3 m/s.The 6DOF lower limb model was used to calculate three-dimensional kinematic variables . The oriPCA can use data compression and dimensionality reduction methods to present the original data as a set of orthogonal variables called principal components (PCs) in a new hyperplane and then classify the prominent waveform characteristics . TherefoPCA was used to identify dominant modes of variation for the three angles  in three-dimensional planes , calculating waveforms in nine analyses . The reason for using this approach is that this method was more sensitive in terms of separating the difference in the lower limb angles change in three-dimensional planes. At first, the captured motion kinematics data for the sagittal, frontal and transverse planes were normalized to 101 time points of running stance .X) X).p represents 200 trials , and where n represents 101 data points over running stance phase.For each PCA analysis, data were created into a X to remove the influence of features with larger overall values on the proportion of variance in the calculation. The mean of each joint angle was calculated First, data preprocessing was performed on the original data Then, X was preprocessed, it was expressed as:After the original data matrix X was calculated, and the obtained X′:The covariance between each dimension of ‘The eigenvalues and eigenvectors of the covariance matrix were then calculated. First, the eigenvalue of and corresponding eigenvectors To reduce the n-dimensional raw data to d-dimensions, the first d columns of the principal component matrix calculated in Equation (6) were extracted to obtain the dimensionality reduction matrix:X was multiplied with the reduced matrix Z:The original data matrix k is the number of selected principal components, n is the total number of all principal components.The eigenvalues of the covariance matrix and its corresponding eigenvectors reflect a linear transformation process of the original data. The eigenvectors represent the main transformation direction, while the eigenvalues represent the degree of transformation of the corresponding eigenvectors. It can also be described that the eigenvectors represent the angles of the matrix space features, and the corresponding eigenvalues represent the variance of each angle. However, there are several common criteria for selecting principal components: cumulative contribution margin criterion and Bartlett test criterion Kaiser method. In this paper, the number of principal components was selected using the cumulative contribution rate. The number of principal components can be selected according to the actual needs, for example, if the cumulative contribution of the first k principal components reaches 85%. Then, the number of principal components is determined ask Cumulative Contribution Ratio (CCR) is obtained from the following equation. In the present study, the first three PCs were conducted because they represent the primary variables of kinematics data. We used the PCA approach by MATLAB  .(9)Σi=1Principal component scores of waveforms were calculated using the mean and standard deviation for sagittal ankle plane, frontal ankle plane, ankle transverse, knee sagittal, knee frontal plane, knee transverse, hip sagittal, hip frontal and hip transverse. PC1 represented the overall amplitude and shape in three planes, and PC2 was the range of motion for the ankle, knee and hip joint. PC3 represented the initial contact angle during the running stance.t-tests using SPSS23.0 software. We compared principal component scores for three planes waveforms. Statistically significant p-values were set at 0.005.To compare the differences between the competitive runners and recreational runners, we used independent t-tests; the p-values and data are described in In the nine kinematics waveforms, principal components models included the nine joint angles waveforms in All the joint ankle means and original waveforms for the sagittal plane, frontal plane and transverse plane are depicted in p = 0.001). Therefore, the recreational runners exhibited a smaller dorsiflexion angle than the competitive runners during the running stance. In addition, high PC3 indicated a more significant change in the dorsiflexion during the initial contact (0–80%) to plantarflexion during the swing phase in p = 0.001). The recreational runners exhibited greater dorsiflexion during the initial contact than the competitive runners.Differences were found between the competitive and recreational runners for the ankle sagittal plane . For thep = 0.001). Therefore, the recreational runners exhibited a smaller inversion angle than the competitive runners during the running stance. In addition, high PC2 indicated range of motion changes during the running stance phase. The competitive runners showed negative PC2 in p = 0.001). The competitive runners exhibited a smaller ROM than the recreational runners during the running stance in the ankle frontal plane.Differences were found between the competitive and recreational runners for the ankle frontal plane . For thep = 0.001). The competitive runners exhibited a smaller ROM than the recreational runners during the running stance in the transverse ankle plane.Differences were found between the transverse ankle plane’s competitive and recreational runners . For thep = 0.001). Therefore, the recreational runners exhibited a larger knee flexion angle than the competitive runners during the running stance.One difference was found between the competitive and recreational runners for the knee sagittal plane . For thep = 0.001). Therefore, the recreational runners exhibited less knee adduction angle than the competitive runners during the running stance.One difference was found between the competitive and recreational runners for the sagittal knee plane . For thep = 0.001). Therefore, the recreational runners exhibited a greater internal rotation angle than the competitive runners during the running stance. In addition, high PC2 indicated range of motion changes during the running stance phase. The competitive runners showed negative PC2 in p = 0.001). The competitive runners exhibited greater ROM than the recreational runners during the running stance in the knee transverse plane.Differences were found between the competitive and recreational runners for the ankle frontal plane (p = 0.001). Therefore, the recreational runners exhibited a larger hip flexion angle than the competitive runners during the running stance.One difference was found between the competitive and recreational runners for the hip sagittal plane . For thep = 0.001). Therefore, the recreational runners exhibited a greater adduction angle than the competitive runners during the running stance. In addition, high PC2 indicated the range of motion changes during the running stance phase. The recreational runners showed negative PC2 in p = 0.001). Thus, the competitive runners exhibited greater ROM than the recreational runners during the running stance in the hip frontal plane.Differences were found between the competitive and recreational runners for the hip frontal plane . For thep = 0.001). Therefore, the recreational runners exhibited less hip internal rotation angle than the competitive runners during the running stance.One difference was found between the competitive and recreational runners for the hip transverse plane . For PC1The present study aimed to compare the variables of running lower limb kinematics using the principal component analysis approach between competitive runners and recreational runners. Our hypothesis was that lower limb joint angle differences would exist between competitive runners and recreational runners during the running stance phase. This study has observed that lower limb kinematics data derived from PCA might be more sensitive than traditional technology. This resulted in differences in lower limb joint kinematics variables during the running between the competitive and recreational runners being revealed.The basic idea of principal component analysis is to reduce the dimensionality of the original data into a small number of unrelated composite indicators to replace the original indicators. Through the compression and dimensionality reduction of the original data, the time required for data analysis can be reduced, and the original complex problems can be simplified. The dimensionality reduction can make the data retain as much information of the original data as possible to reflect the real situation to the maximum. Each principal component has its corresponding eigenvalue, and the size of the eigenvalue reflects the variance contribution of the principal component. The larger the eigenvalue, the stronger the degree of analysis of the original data, that is, the more information the principal component contains about the original data. This method allows the analysis of the motion patterns over time and is well suited for biomechanical analysis . In addiPrevious studies have found greater plantarflexion and dorsiflexion of good runners during the running swing stance phase. This study was consistent with previous research that compares the elite runners and recreational runners’ sagittal kinematics variables, which were reflected in ankle sagittal plane PC2 . HoweverThe hip joint performs a crucial role in lower limb motion, and instability of the hip joint is considered an essential mechanism of lower limb injury . IncreasWhile acknowledging the results of this study, some limitations should be considered. Firstly, kinetic parameters were not collected, and competitive runners and recreational runners may have a different ground reaction force, although all the runners were rearfoot strikers. Secondly, we used the same running speed to capture the kinematics data without considering the influence of different running speeds on running kinematics in lower limb variables. Lastly, in this study, all the runners were male. In future research, focus and attention should consider different genders to compare the kinematics and kinetics variables of the lower limb joints.The present study aimed to utilize PCA to identify the kinematics parameters differences during the running stance phase between competitive runners and recreational runners. Using the principal component scores, the critical features for the joint angle were obtained, which demonstrated differences in the lower limb kinematics for the competitive and recreational runners. The recreational runners’ essential differences included a smaller dorsiflexion angle, initial dorsiflexion contact angle, ankle inversion, knee adduction, range motion in the frontal knee plane and hip frontal plane during the running stance phase. The findings from the study provide a better understanding of the kinematics variables for competitive and recreational runners. Thus, these findings might have implications for reducing running injury and improving running performance.