People in green professions are exposed to a variety of risk factors, which could possibly enhance the development of depression. Amongst possible prevention approaches, internet- and mobile-based interventions (IMIs) have been shown to be effective and scalable. However, little is known about the effectiveness in green professions. The aim of the present study is to examine the (cost-)effectiveness of a tailored IMI program for reducing depressive symptoms and preventing the onset of clinical depression compared to enhanced treatment as usual (TAU+).N = 360) with sufficient insurance status and at least subthreshold depression (PHQ-9 ≥ 5) are eligible for inclusion. Primary outcome is depressive symptom severity (QIDS-SR16). Secondary outcomes include incidence of depression (QIDS-SR16), quality of life (AQoL-8D) and negative treatment effects (INEP). A health-economic evaluation will be conducted from a societal perspective. The IMI program is provided by psychologists of an external service company and consists of six guided IMIs  targeting different symptoms . Intervention choice depends on a screening of participants’ symptoms and individual preferences. The intervention phase is followed by a 12-months consolidating phase with monthly contact to the e-coach.A pragmatic randomized controlled trial (RCT) will be conducted to evaluate a tailored and therapeutically guided preventive IMI program in comparison to TAU+ with follow-ups at post-treatment (9 weeks), 6-, 12-, 24-, and 36-months. Entrepreneurs in green professions, collaborating spouses, family members and pensioners (This is the first pragmatic RCT investigating long-term effectiveness of a tailored guided IMI program for depression prevention in green professions. The present trial builds on a large-scale strategy for depression prevention in green professions. The intended implementation of the IMI program with a nationwide rollout has the potential to reduce overall depression burden and associated health care costs in case of given effectiveness.DRKS00014000. Registered on 09 April 2018.German Clinical Trial Registration:  Major depressive disorder (MDD) is a highly prevalent disorder in the general population: a 12-month prevalence of MDD amounts to 6.7% , 4 and hAn estimation of prevalence rates in the specific target group of green professions in Germany has yet to be conducted. However, people in green professions are exposed to a variety of risk factors, which might increase vulnerability for developing MDD. Risk factors include financial pressure, high administrative workload, long working hours, high stress levels, part-time jobs off the farm, health problems and preceding work accidents , 8, as wPsychological interventions for subthreshold depression as indicated prevention have been shown to significantly reduce depressive symptom severity . AdditioAlthough face-to-face psychotherapy represents an effective treatment option, impact of such on-site offers is restricted. MDD is still an underdiagnosed and undertreated condition with only approximately 50% of persons with 12-month MDD receiving some kind of treatment, and only approximately 20% of affected persons receiving an appropriate treatment concordant with treatment guidelines . In GermInternet- and mobile-based interventions (IMIs) have already shown to be effective in reducing subthreshold depression –25 and tIMIs constitute a promising approach for providing an effective and easily accessible prevention offer in routine health care , 28. As http://www.svlfg.de/). The SVLFG currently establishes a comprehensive prevention model project  for their insurees with the offered services of guided IMIs, personalized tele-based coaching as well as on-site prevention group workshops, provided by different service providers in the health care sector. Parallel to a nationwide rollout, the single internet- or telephone-based prevention offers will be evaluated in randomized controlled clinical trials (RCTs) in order to establish evidence on the clinical and cost-effectiveness. Eligible for this trial are policyholders of the SVLFG who are entrepreneurs, family members, spouses or have retired in one of the three sectors of agriculture, forestry or horticulture (referred to as “green professions”).To improve the delivery situation in rural areas, and specifically for the target group of green professions, this study will be conducted in the framework of a national depression prevention program for people in green professions, implemented by the Social Insurance for Agriculture, Forestry and Horticulture . Even though these IMIs differ regarding their focus on mental health problems, they will be evaluated as part of a depression prevention program. The underlying idea follows the rational of a transdiagnostic approach: Transdiagnostic research has already yielded promising evidence that treatment protocols based on transdiagnostic maintaining factors and treatment concepts can lead to symptom reduction comparable to disorder-specific treatments , 31. ThiEspecially in prevention context, it is desirable to reach and provide preventive interventions for a large population sample. Low adherence and high dropout rates , 33 restIs the IMI program effective in reducing depressive symptom severity compared to TAU+?Is the IMI program effective in preventing the onset of clinical depression as compared to TAU+?Is the IMI program effective in reducing various mental health outcomes  compared to TAU+?Is there a differential or rather an undifferentiated, general effect of specific IMIs on different mental health outcomes?Is the IMI program superior in terms of cost-effectiveness and QALY health gains compared to TAU+?Which variables moderate and mediate the effects of the IMI program?What is the level of intervention satisfaction, adherence and acceptance to the IMI program?Are there negative effects of the IMI program?The aim of the present study is to investigate whether a tailored IMI program targeting a variety of specific complaints associated with clinical depression is useful in reducing depressive symptoms and preventing onset of clinical depression as compared to enhanced treatment as usual (TAU+) within a 36-month follow-up period. We propose, that depressive symptomology will be reduced to a greater extent in the intervention group (e.g. IMI program) than in the control condition (e.g. TAU+) post-treatment (T1). The following research questions will be investigated in this pragmatic randomized controlled trial over a follow-up period of 36-months:A two-armed pragmatic randomized controlled trial (RCT) will be conducted comparing the clinical and cost-effectiveness of a tailored, guided IMI program to a control group (CG) receiving enhanced treatment-as-usual (TAU+). Primary and secondary outcomes will be assessed over a period of 36 months. Assessments will take place at baseline (T0), post-treatment , as well as at 6-month (T2), 12-month (T3), 24-month (T4) and 36-month (T5) follow-up.This clinical trial has been approved by the ethics committee of University of Ulm No. 454/17) and will be reported in accordance with the Consolidated Standards of Reporting Trials (CONSORT) Statement 2010 and the extension for reporting pragmatic trials –44 as we4/17 and We will include a) agriculturists, foresters and horticulturists with sufficient insurance status of a social insurance company (SVLFG) in Germany. The study is accessible for entrepreneurs, collaborating spouses and family members as well as pensioners who do no longer contribute to the production process with sufficient insurance status. Participants are required to b) be age 18 or above, c) show an indication for at least subthreshold depression (PHQ-9 ≥ 5), d) have internet access, and e) be willing to give informed consent. Participants will be excluded a) if they are currently receiving psychotherapy (self-report), b) if they are not able to distance themselves from suicidal ideation (not able to sign a non-suicide contract), and c) if they have comorbid chronic pain symptomology (Chronic Pain Grade Questionnaire (CPG) ≥ Grade II + chronic pain longer than six months) and prefer to participate in the parallel clinical trial PACT-A on improving pain related distress in people with chronic pain : study protocol of a 36-month follow up pragmatic randomized controlled trial, in preparation).This clinical trial is embedded in a large-scale mental health initiative of the SVLFG with the aim to improve the prevention of mental health problems in agriculturists, foresters and horticulturists. Participants are recruited via several articles in the members-journal (quarterly circulation 1.3 million), postal mailings as well as information on associated websites. Since interested persons are provided with various ways of contact  and an alternatively direct access to the screening assessment (via QR-Code and URL), a low-threshold access into the study is provided. Recruitment started in January 2018 and was concluded in April 2019  and chronic pain symptomology (CPG ≥ Grade II + chronic pain longer than six months), as well as possible suicidal ideation. Online questionnaires include self-report of age, insurance status, and current psychotherapy. If the eligibility criteria are fulfilled, applicants will receive an online information letter including detailed information about study procedure and informed consent. They will be informed that they can withdraw from the intervention and/or study at any time without any negative consequences. After giving informed consent, participants will enter the study and complete baseline assessment (T0).www.sealedenvelope.com) is used to perform permuted block randomization with randomly arranged block sizes  and an allocation ratio of 1:1.Subsequently, participants will be randomly allocated to intervention or control condition and will be informed about group membership via e-mail. Randomization will take place at an individual level. Randomization will be conducted by a person who is blind regarding all processes within the intervention. Whereas blinding of participants is not possible, data collectors are blinded regarding group membership. Group membership is only known by the persons administering allocated treatments to participants. The automated web-based programme sealed envelope  participatory selection of a suitable (guided) IMI and (3) access to IMI. The IMI program will be carried out by the external service company GET.ON without involvement from the study team. Thus, the GET.ON institute is responsible for providing an adequate care of the participants against the background of the psycho-diagnostic assessment and the information they receive in the course of patient contact. This includes for example the handling of adverse events in the course of e-coaching. The study team evaluates in this setting the standard procedure of the GET.ON institute for the allocation of the different trainings and the treatment of participants in the course of the IMI program.psycho-diagnostic assessment is based on a selection of self-report questionnaires measured as part of baseline assessment (T0). Relevant baseline assessment values are transmitted electronically to GET.ON for each participant to allow for psycho-diagnostic assessment based on allocation rules applied in supply routine of GET.ON. Each participant is assigned to a personal e-coach after signing in to the intervention platform. E-coaches are trained psychologists, who are in training for a further qualification in psychotherapy and supervised by an experienced psychological therapist.The participatory selection of a suitable (guided) IMI.Participants are contacted via telephone or private message function on the intervention platform by their personal e-coach for GET.ON Mood Enhancer) or in the context of a comorbid diabetes mellitus (GET.ON Mood Enhancer Diabetes), by improving sleep quality (GET.ON Recovery), or by reducing subjective stress load (GET.ON Stress), panic symptoms (GET.ON Panic), or harmful alcohol use (GET.ON Be smart - Drink less). These interventions have already been shown to effectively reduce depressive symptom severity as primary [The IMI program entails six different IMIs with the aim to reduce subthreshold depression by focusing on depressed mood in general the participants will receive access to the selected IMI. The IMIs each consist of 6–8 lessons with a duration of 30–60 min. Participants are advised to do one lesson per week. Thus, completion of the intervention is expected to take 6–8 weeks. Depending on the individual pace of the participant, the completion of the intervention can differ. After each lesson, participants get an individualized feedback by their personal e-coach. Guidance is employed to improve adherence of participants and decrease dropout [Directly afterwards Participants in the intervention group (IG) will be transferred to the service provider GET.ON to get access to a tailored and guided IMI program with the option of conducting one of six available IMIs. The IMI program consists of a 3-step intervention entailing (1) GET.ON Mood Enhancer, GET.ON Mood Enhancer Diabetes, GET.ON Recovery, GET.ON Panic, GET.ON Be smart - drink less) and on the transactional model of stress and coping [GET.ON Stress).GET.ON Mood Enhancer aims to reduce depressed mood by promoting the uptake of positive activities and the learning of problem-solving strategies [rategies .GET.ON Mood Enhancer Diabetes is an adaptation of GET.ON Mood Enhancer and aims to reduce depressed mood in the context of diabetes mellitus [mellitus .GET.ON Recovery is directed towards persons with clinical insomnia complaints and aims to prevent depression by improving participants’ sleep quality, especially through modifying coping behaviour regarding rumination and worrying. This is achieved by assignments such as keeping a diary about ruminating thoughts [thoughts .GET.ON Stress aims to reduce depressive symptomology by addressing chronic stress and reducing stress-related complaints. The training instructs to apply problem- and emotion-oriented stress management strategies based on the distinction between solvable and unsolvable problems [problems .GET.ON Panic aims to reduce depressive symptoms by addressing panic symptoms and agoraphobia. Participants are instructed to perform in-vitro and in-vivo exposition. Further, the training comprises information and exercises for coping with negative automatic thoughts and for establishing positive thought processes [rocesses . If thisGET.ON Be smart - drink less was developed to prevent depression by reducing harmful alcohol use in participants, who do not yet meet clinical criteria for alcohol dependence. This training aims to identify emotions and situations associated with alcohol consumption by keeping an alcohol-diary. Further, alternative exercises for actions for coping with negative emotions and problems are included [included . This trThe IMIs are based on principles of cognitive behavioural therapy  or mobile device . The IMIs contain psycho-educative text material, exercises, and testimonials of exemplary personas, who accompany the participants throughout the intervention. Additionally, interactive elements in form of auditory material and videos clips are included. Audio clips for example are used to convey relaxation techniques, while video clips often serve to give a comprehensible overview about theoretical concepts by experts. Since a focal point is the transfer of content and strategies learned into every-day life, many application examples, instructions and exercises, as well as homework assignments for practising between lessons are included. The content of the online-trainings has been specifically adapted to the target population by inclusion of fitting picture and video material, as well as exemplary testimonials and fictional personas living and working in the agricultural setting. These personas describe the struggle with everyday problems and challenges in green professions, e.g. generational conflicts or handing over of the company to the younger generation.Subsequent to the completion of the IMI, participants are followed up for 12 months by their individual e-coach to consolidate the treatment effect. During this phase, participants are contacted once a month through telephone or the internal messaging system by their personal e-coach to review their current state, report problems, and give account regarding the transfer of strategies learned into the daily routine.Both participants of the IG and the CG will have unrestricted access to treatment-as-usual (TAU). Participants of the CG will receive an online information letter with psycho-educative information about stress, depression and chronic pain. Additionally, a link to an online freely available audio CD with information about stress reduction is provided, as well as a link to a database with different health services and information about further treatment options in standard care  for the CG).TAU will not follow a standardized protocol, however, health care consumption will be measured with the TiC-P   , who repol group , we assun the CG . Based on the CG , 48–55, n the CG . To compAll assessments will be conducted online. For an overview of instruments at screening, baseline (T0), post-treatment T1), and follow-up assessments (T2-T5), see Table , and folThe preliminary screening assesses sociodemographic variables  and information about various recruitment channels.The German Version of the Patient Health Questionnaire (PHQ-9)  is adminThe Chronic Pain Grade Scale (CPG)  is appliThe primary outcome is depressive symptom severity at post-treatment (T1), assessed with the German Version of the Quick Inventory Depressive Symptomology (QIDS-SR16) . The 16-Depressive symptom severity will be measured over a period of 36 months at 6-month (T2), 12-month (T3), 24-month (T4) and 36-month (T5) follow-up with QIDS-SR16.Clinical significance of depression response will be determined with QIDS-SR16 scores according to Jacobson and Truax .Additionally, QIDS-SR16 will be used to classify participants regarding presence of clinical depression at all measurement points (T0-T5). A comparison of QIDS-SR16 scores to current and lifetime diagnosis based on Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID) as measure criterion showed that QIDS-SR16 is a reliable screening instrument for diagnosis of clinical depression . Cut-offFurther, Items adapted from the Composite International Diagnosis Interview version 3.0 (CIDI 3.0) and Screening Scales (CIDI-SC) , and theThe 10-item version of the perceived stress scale (PSS-10) , 82 willInsomnia Severity will be measured using the Insomnia Severity Index (ISI) . The ISIThe GAD-7  will be The severity of panic and agoraphobic symptoms will be assessed with the self-report version of the panic and agoraphobia scale (PAS) , 92. TheAssessment instruments for measurement of pain intensity and pain associated disability were selected under consideration of the recommendations of the Initiative on Methods, Measurement and Pain Assessment in Clinical Trials (IMMPACT) , 94.The Multidimensional Pain Interference Scale (MPI) , 96 is ahttp://auditscreen.org/cmsb/uploads/audit-german-m-nster.pdf).The Alcohol Use Disorder Identification Test (AUDIT)  will be Emotional exhaustion as the basic dimension of the burnout construct will be measured with a subscale of the Maslach Burnout Inventory (MBI-GS) . In the Health-related quality of life will be assessed with the self-report questionnaireassessment of Quality of Life (AQoL-8D). This questionnaire consists of 35 items, covering the three physical dimensions “independent living”, “pain”, and “senses”, as well as the five psycho-social dimensions “mental health”, “happiness”, “coping”, “relationships”, and “self-worth” . The AQoWork capacity will be measured with the German version of the Subjective Prognostic Employment Scale (SPE) . The SPEThe provider (GET.ON institute) will provide information about the intervention costs, including costs pertaining initial phone calls by psychologists, documentation and coaching  provided by qualified psychologists, technical support, server for hosting the interventions and overhead. Cost evaluation will be based on the German version of the Dutch cost questionnaire “Trimbos Institute and Institute of Medical Technology Questionnaire for Costs Associated with Psychiatric Illness” (TiC-P) . In thishttp://wai.profhorvath.com/) at T1 and T3. This will allow us to compare how the therapeutic relationship is experienced by client and by e-coach, to gain a differentiated and comprehensive picture of the experienced working alliance. The WAI-SR and the WAI-SRT were adapted in wording for the current study investigating therapeutic alliance in guided IMIs. The items were changed to refer to e-coaches instead of therapists and to online-trainings instead of therapy. Additionally, the Technological Alliance Inventory - Online Therapy (TAI-OT) will be administered to assess the technological alliance between client and the online-intervention. The TAI-OT is a new self-report questionnaire developed by Labpsitec (http://www.labpsitec.uji.es/esp/index.php) consisting of 12 items and measures the degree to which the online-program is perceived as helpful in achieving therapeutic goals.The short version of the Working Alliance Inventory (WAI-SR)  will be Intervention satisfaction will be assessed using a German Version of the Client Satisfaction Questionnaire . The INEP records whether any negative changes, which are experienced during or after the treatment in the social and/or work environment, are attributed on the psychotherapeutic intervention . In thisAdditionally, an open question will be included for qualitative assessments of negative side effects of IMIs. Participants will describe experienced negative events and side effects, their time of beginning, their frequency, and their duration. Two further questions rate the negative impact of these events in the past and at present time.Participants will be requested to visit a general practitioner and return a standardized formula for clarification of medical condition. This procedure is applied to ensure participants suffering from severe medical conditions receive appropriate medical care. Since the aim of the present study is to evaluate effectiveness (not efficacy) of online-trainings in standard care, the GP visit as well as the return of the formula is left to the responsibility of the participant. Further, the presence of diabetes mellitus will be assessed using a self-report item as part of the psycho-diagnostic assessment for the IMI program.As potential moderating variables, demographic information , information about the agricultural farm , and about the situation of the entrepreneurial family  will be recorded at baseline. Further, a variety of predictors  will be included to assess relevant factors for development of depressive symptomology.Suicidal ideation will be screened using PHQ-9 in screening assessment and QIDS-SR16 for further assessment points (T0-T5). A score ≥ 1 on suicide item of PHQ-9 (“Thoughts that you would be better off dead or of hurting yourself in some way?”) or QIDS-SR16 , leads to BDI-II suicide item . A scoreParticipants receive an online information letter with detailed information on available health services and the advice to seek professional help if symptoms increase. The wording of the online information letter is adapted in emphasis, depending on the severity of the indicated suicidality . This procedure will be administered on all assessment points and independent of study inclusion.Eligible participants will be asked to return a signed non-suicide contract as part of screening and baseline (T0), if they are able to distance themselves from suicidal ideation. Participants are informed that the IMI program is not provided as intervention for problems accompanied by persisting suicidal ideation.For BDI > 1, a detailed clarification of self-endangerment will be performed for all screened participants independent of study inclusion status and assessment point. A telephone contact will be announced via e-mail in case of not returning a given text module or sending an informal answer in response to the online information letter within 48 h of undergoing the assessment point. If self-endangerment remains unclear, psychotherapists (at least) in training will contact the participant via telephone and initiate further actions. Further actions will be determined depending on perceived endangerment. If participant remains not contactable for further 48 h, the police department will be informed. All steps are closely monitored and supervised by a licenced psychological psychotherapist.All statistical analyses will be performed based on intention-to-treat (ITT) principle. Patterns of missing data will be examined and analyses will be corrected for missing data by applying multivariate imputation according to Van Buuren and Groothuis-Oudshoorn . For thoIG– CostsCG)/(EffectsIG– EffectsCG), where Costs are the annual per-participant costs and Effects are the unit of effect in IG and CG. In the CUA, the ICER will be expressed as incremental costs per quality adjusted life year (QALY) gained as based on the AQoL-8D. Sampling uncertainty in the ICER will be handled using nonparametric bootstrapping by resampling on patient-level. In addition, confidence intervals for ICERs will be obtained by bootstrapped quantiles. The bootstrapped ICERs will be plotted in a cost-effectiveness plane where the horizontal axis reflects differences in effects and the vertical axis differences in costs. The bootstrapped ICERs will also be shown in a cost-effective acceptability curve disclosing the probability that the intervention is cost-effective for a range of willingness-to-pay ceilings. To test the robustness of the base-case findings, multi-way sensitivity analyses will be done. Several assumptions made in the base-case scenario (e.g. about cost prices and volumes) will be changed to assess their impact on the ICERs.The health-economic evaluation will involve a combination of a cost-effectiveness analysis (CEA) and a cost-utility analysis (CUA). The economic evaluation will be performed from a societal perspective  and a public health care perspective  with a time horizon of 36 months. In the cost-effectiveness analysis, the incremental cost-effectiveness ratio (ICER) will be based on the incremental costs per unit of effect gained (e.g. reliably improved case based on RCI). The corresponding equation is ICER = effectiveness of the proposed IMI program. Especially participants of the CG are expected to be at risk for study dropout, since they do not get access to the IMI program during the follow-up period of 36 months. To improve study adherence, incentives for completion of online-assessments and standardized reminding procedures are applied. To reduce intervention dropout, IMIs have been adapted in content for green professions and therapeutic guidance is deployed. Dropout can be reduced by providing therapeutic guidance . AdditioInternet-based preventive interventions have the potential to improve the general care situation substantially, specifically in rural areas, provided that the proposed health care offer encounters reasonable acceptance and utilization in green professions. The proposed tailored prevention approach is thought to improve dissemination of preventive interventions aiming to overcome depressive symptoms or risk factors for development of depression. Such prevention approaches can contribute to reduction of chronification and overall depression burden in green professions. This in turn can result in decrease of sick days, incapacity to work and early retirement, and thus a substantial cost reduction in the health care sector of green professions. If proven effective, implementation of the proposed IMI program into routine health care of green professions will have a large public health impact.The study is currently ongoing. Recruitment started in January 2018 and was concluded in April 2019.


Alternative polyadenylation is commonly examined using cDNA sequencing, which is known to be affected by template-switching artifacts. However, the effects of such template-switching artifacts on alternative polyadenylation are generally disregarded, while alternative polyadenylation artifacts are attributed to internal priming.Here, we analyzed both long-read cDNA sequencing and direct RNA sequencing data of two organisms, generated by different sequencing platforms. We developed a filtering algorithm which takes into consideration that template-switching can be a source of artifactual polyadenylation when filtering out spurious polyadenylation sites. The algorithm outperformed the conventional internal priming filters based on comparison to direct RNA sequencing data. We also showed that the polyadenylation artifacts arise in cDNA sequencing at consecutive stretches of as few as three adenines. There was no substantial difference between the lengths of poly(A) tails at the artifactual and the true transcriptional end sites even though it is expected that internal priming artifacts have shorter poly(A) tails than genuine polyadenylated reads.Our findings suggest that template switching plays an important role in the generation of spurious polyadenylation and support the need for more rigorous filtering of artifactual polyadenylation sites in cDNA data, or that alternative polyadenylation should be annotated using native RNA sequencing. The majority of human genes utilize alternative polyadenylation (APA) sites , 2, whicTS refers to the ability of DNA polymerase to discontinue elongation while still binding the newly synthesized strand and to reinitiate synthesis at a homologous locus of another nucleic acid strand . Fig. 11b. Both We hypothesized that artifactual polyadenylation events at shorter stretches of adenines are caused by TS. To characterize artifactual and genuine transcriptional end sites, we analyzed publicly available datasets in which both direct (d) RNA and cDNA sequencing data were available for the same cell lines. Potential pA sites were determined based on the cDNA sequencing data and then compared with direct (d) RNA sequencing data to identify artifacts in the cDNA sequencing results. In total, 87,980 and 403 potential pA sites were identified in the human cDNA dataset  and huma. Nevertheless, many putative artifacts were detected in regions with as few as 3–5 As, and the likelihood of these artifactual pA sites to be detected by dRNA sequencing did not decrease when these sites contained more As in a 20-nt window . The algorithm considered the number of As in the region immediately upstream of a pA site, the number of reads in the proximity of the pA site that fell outside of A-rich regions, and the ratio of polyadenylated reads to the coverage of the region The putative TESs identified by the algorithm differed greatly from the putative TS artifacts Fig. . The nuc+ cDNA sequencing data of two species (human and HCMV), stemming from three different long-read sequencing platforms , generated by three different library preparation methods -selection, as well as only poly(A)-selection), and then compared them to dRNA sequencing data obtained by the MinION platform. Our analyses confirmed that artifacts arising in A-rich regions complicate the study of alternative polyadenylation. This phenomenon is generally accredited to internal priming [We analyzed poly(A) priming . Given oEven though it was not part of the filtering criteria, sites that the algorithm classified as TESs were likely to contain consensus polyadenylation motifs. This result indicates that machine learning algorithms can distinguish even more specifically between TESs and artifacts using more sequence information. However, a large training dataset would be required for machine learning to be efficient, and such datasets are not available at the time.We have shown that the TS is prevalent in both the viral and the human dataset. Nevertheless, large differences were observed in the proportion of detected artefactual and genuine poly(A) sites. These could potentially be attributed to the fact that the human genome has a lower GC-content (40.9%) than the HCMV (57.2%), therefore there are more A-rich regions in the human genome. However, another explanation is that the number of genuine polyadenylation sites is finite. Once all the genuine polyadenylation sites have been detected, any increase in coverage can only increase the number of false positives. In the small viral genome, it is more feasible to capture all the genuine sites than in the large human genome. The large overall coverage of cDNA reads, especially the high cDNA to dRNA ratio in the HCMV dataset are likely to have contributed to the lower positive and higher negative predictive values in the HMCV samples.Our findings were obtained using long-read sequencing datasets. While it may seem sensible to extend our conclusions to short-read sequencing data and other results obtained by cDNA sequencing, it must be noted that some aspects of long-read sequencing promote the production of template-switching artifacts. Firstly, long-read sequencing usually necessitates reverse transcription of the whole transcript, not only its most 3′ fragment, which is an option for short-read sequencing. Reverse-transcribing more genomic regions provides more potential templates for TS. Secondly, SMART technology, which is widely used in long-read sequencing studies to produce full-length transcripts, requires ideal conditions for TS , 30. WheTS is known to produce cDNA artifacts, however its effects on the analysis alternative polyadenylation have never been discussed until now. Considering that the poly(A) tail is likely the most frequent template in most transcriptomic libraries, polyadenylation artifacts may be even more prevalent than the more reviewed splicing artifacts. The effects of TS on short-read sequencing can be mitigated by higher reverse-transcription temperatures  or by emhttps://github.com/nanopore-wgs-consortium/NA12878/blob/master/RNA.md) were generated by extracting RNA from the GM12878 human cell line (Ceph/Utah pedigree) and sequenced on MinION flow cells (FLO-MIN106) using R9.4 chemistry (SQK-RNA001 and SQK-LSK108 kits) [https://www.ebi.ac.uk/ena/data/view/PRJEB22072) and PRJEB25680 (https://www.ebi.ac.uk/ena/data/view/PRJEB25680). RNA was isolated from HCMV-infected  human embryonic fibroblast cells  and sequenced on the RSII and Sequel platforms of Pacific Biosciences using the Iso-Seq library preparation protocol and on the MinION platform using the SQK-RNA001 and SQK-LSK108 kits and another cDNA library was prepared combining the SQK-LSK108 and the TeloPrime Full-Length cDNA Amplification Kit (Lexogen) to select for capped RNA molecules. In this latter experiment, the TeloPrime kits own enzymes were used for poly(A)-selection. This dataset containing results from five sequencing libraries  is referred to in the text as the HCMV dataset.Two long-read cDNA and dRNA sequencing datasets were downloaded and analyzed during the study. (1) The human cDNA and dRNA sequencing FASTQ reads of the Nanopore WGS Consortium (08 kits) . This da08 kits) , 33 dataThe computational pipeline of the study is summarized in Additional file https://github.com/zsolt-balazs/LoRTIA) was used to identify potential poly(A) sites in the cDNA sequencing data. A genomic locus was considered a potential poly(A) site when at least two reads and at least 0.1% of the overlapping reads ended at a given nucleotide. In a 21-nt window, the genomic position with the highest number of poly(A) + reads was selected as the potential poly(A) site. The separate experiments of the HCMV dataset were analyzed separately and the results were joined to create the list of potential HCMV poly(A) sites. The LoRTIA toolkit was also used to mark reads which ended in A-rich genomic regions . When characterizing high confidence calls only the sites where more than ten reads ended in a 21-nt window around the locus were analyzed.The LoRTIA toolkit (We have deployed a slightly different definition of A-rich regions than it is commonly used in the literature. A common approach is to count the number of consecutive As in a region surrounding the poly(A) site. Another method is to count the number As in a given, often 20-nt-long window (because 20 nt is the primer length). Instead, we iterated the 20 nt upstream of a potential poly(A) site and incremented a counter each time an A was iterated, all the other nucleotides were counted as − 1. If the counter reached − 1, the iteration was halted and the highest count was regarded as the A-count of the region  sites which did not meet these requirements were classified as TS artefacts. The genomic loci which contained at least 100 times more 5′-ends from the opposite strand than 3′-ends were also classified as artefactual poly(A) sites. The sites which were classified as TS artefacts, but were no further than 50 nt away from sites that were classified as TES, were excluded from comparisons and sequence analyses in order to prevent the characteristics of the TES to interfere with the characteristics of TS artefacts .The potential poly(A) sites which were not at A-rich loci, were accepted by our script as transcriptional end sites. The potential poly(A) sites at A-rich loci were accepted as TES if the number of reads in a 21-nt window around that loci contained either more reads which ended in a non-A-rich region than reads which ended in an A-rich region or a proportion of overlapping reads greater than The 12 most commonly utilized human PAS  were seaThe length of the sequenced poly(A) tail of the cDNA reads was measured for each read that contained a poly(A) tail recognized by the LoRTIA toolkit. The sequence containing the 3′-terminal 30 nt of the mapped part of the read and the first 150 nt of the 3′-terminal soft clipped (unmapped) part of the read was aligned to a stretch of 180 As using the Smith-Waterman algorithm . The number of As in the best local alignment was counted as the length of the poly(A) tail. Measuring the poly(A) tail length this way may overestimate poly(A) tail length in A-rich regions, however it allows for a more accurate measurement in error prone reads.http://exon.umdnj.edu/polya_db/v3/misc/download.php). Potential poly(A) sites were considered confirmed by the PolyA_DB dataset if the database contained poly(A) sites in the 21-nt window of the potential poly(A) sites.The PolyA_DB data   sites. In addition to Fig. n = 403) and the human  datasets. (b) The performance of the different filtering methods. The left side shows the true positive predictive value of the internal-priming (red) and template-switching (blue) filters based on the dRNA sequencing results . The human potential poly(A) sites were filtered using SQANTI (yellow) and also based on whether or not they were found in the PolyA_DB (green). The right side of the panel shows the proportion of potential poly(A) sites filtered out by the different filtering options, which were not supported by dRNA sequencing . (c) Barplot of the number of potential poly(A) sites in regions with different adenine-content in the HCMV (left) and (right) human dataset. The features that the filtering algorithm characterized as TES are marked in blue, the putative artefacts are marked in red. (d) The positive predictive value of the different filtering methods is shown as a function of the adenine content. The HCMV results are not detailed because the low number of TESs contained in that dataset would not provide for a meaningful analysis.Additional file 2:Figure S2. Overview of the analysis. (a) A summary of the pipeline. Specific steps are explained in detail in boxes. (b) Identifying potential poly(A) sites. The criteria used by the LoRTIA toolkit to detect poly(A) sites are listed. The number of polyadenylated reads ending at a given nucleotide had to be at least two and at least 0.1% of the overlapping reads for the site to be considered a potential poly(A) site. If multiple sites fulfilled these criteria in a 21-nt window, the site with the highest number of supporting reads was accepted. (c) The algorithm for determining the adenine content of a region. The counting started from the poly(A) site, and each A increased, while every different letter decreased the counter by one. The counting continued until maximum 20 nucleotides or until the counter reached − 1. The highest value of the counter was regarded as the adenine content. (d) Criteria for characterizing a potential poly(A) site as a TES. One criterion was that the number of reads ending in not A-rich regions (less than three As) in a 21-nt window, had to be higher than the number of reads ending in A-rich regions (three or more As). The second criterion was that the number of supporting reads had to be higher than the percentage of overlapping reads calculated by a logistic function that takes into consideration the adenine content of the region. A potential poly(A) site was classified as a TES if any of the two criteria was fulfilled.Additional file 3:Table S1. Negative predictive value in A-rich regions in the human dataset. The percentage of putative TS artifacts not supported by the dRNA sequencing experiment  is presented in A-rich regions of different adenine content calculated by two algorithms. If artifacts were mainly caused by internal priming, it would be expected that true negative calls accumulated in regions which have a high number of adenines despite having few consecutive adenines close to the artifactual pA site. The cells are colored according to their negative predictive value.


Tongue twister error data described here were collected from 44 unimpaired speakers producing six repetitions each of 40 tongue-twisters that manipulated the position of error-prone segments within two prosodic domains: intonational phrases and utterances. Data are counts of perseveration and anticipation errors on the initial segments of phrase-initial words or phrase-final words. The location of the phrase-level prominence and other factors associated with speech errors were carefully controlled. For more details about the design, materials and methods, and for interpretation and discussion, see Beirne and Croot (2018)  Specifications TableValue of the data•Tongue twister errors elicited in generally meaningful connected speech are available for comparison with similar data elicited in lists.•The tongue twisters in which these data were elicited control the location of the phrase-level prominence such that it does not fall on the tongue twister words, allowing comparison with errors elicited in tongue twisters with other prosodic structures.•The tongue twister errors were collected using impressionistic broad phonetic transcription, allowing comparison with errors observed using other measurement techniques.1Data are counts of segmental speech errors presented in four worksheets within an excel workbook according to whether they were produced in the phrase-initial or phrase-final condition, and whether the rows show participants or items.2The tongue twister error data reported here were collected from 44 undergraduate psychology students with English  as their first language .The experimental stimuli were 40 tongue-twister items, each composed of two syntactically well-formed 5-word clauses, designed to be elicited with the prosodic structure of two intonational phrases within an utterance. Items were generally meaningful, with some semantic anomalies. Each intended intonational phrase  contained a set of four monosyllabic, singleton onset “tongue-twister words,” and a number or function word. Tongue-twister words in each item began with a “target segment” (henceforth ‘B’), or one of two “confusable segments” (‘A’ and ‘C’) that were equally likely to participate in an error with B The function word or number included in each phrase had initial segments unlikely to participate in errors with the A, B or C segments Participants read each tongue twister item aloud six times in succession at an approximately constant speech rate guided by a metronome of 160 beats per minute (BPM). Responses were recorded and errors were transcribed from the recordings using broad phonetic transcription.Errors were substitutions of a target segment by one of the confusable segments, additions of a confusable segment including segments that were doubly-articulated with the target, or part of a larger error unit. They were self-corrected, or corrected-to-error, and occurred in completed and incomplete words. If an error on the target segment B had its source on the initial segment of preceding confusable word, it was classed as a perseveration error. If the source was the initial segment of the following word, it was classed as an anticipation error. Tongue twister words within items were matched on median frequency and mean neighbourhood density Note that there is an inconsistency in the data in the number of errors contained in the Phrase-initial error spreadsheet arranged by participants compared with the Phrase-initial error spreadsheet arranged by items. The inconsistency lies in the cell measuring anticipation errors in Phrase 1 on Repetition 6, where the by-participants spreadsheet shows 4 errors and the by-items spreadsheet shows 5 errors. This inconsistency is present back through our earliest versions of the data and we are unable to further determine which count is correct and which incorrect. There were 466 or 465 errors in total: 79 or 80 in the phrase-initial condition, 386 in the phrase-final condition.For more details about the design, materials and methods, see


Dengue virus (DENV) is the etiological agent of dengue fever. Severe dengue could be fatal and there is currently no effective antiviral agent or vaccine. The only licensed vaccine, Dengvaxia, has low efficacy against serotypes 1 and 2. Cellular miRNAs are post-transcriptional regulators that could play a role in direct regulation of viral genes. Host miRNA expressions could either promote or repress viral replications. Induction of some cellular miRNAs could help the virus to evade the host immune response by suppressing the IFN-α/β signaling pathway while others could upregulate IFN-α/β production and inhibit the viral infection. Understanding miRNA expressions and functions during dengue infections would provide insights into the development of miRNA-based therapeutics which could be strategized to act either as miRNA antagonists or miRNA mimics. The known mechanisms of how miRNAs impact DENV replication are diverse. They could suppress DENV multiplication by directly binding to the viral genome, resulting in translational repression. Other miRNA actions include modulation of host factors. In addition, miRNAs that could modulate immunopathogenesis are discussed. Major hurdles lie in the development of chemical modifications and delivery systems for in vivo delivery. Nevertheless, advancement in miRNA formulations and delivery systems hold great promise for the therapeutic potential of miRNA-based therapy, as supported by Miravirsen for treatment of Hepatitis C infection which has successfully completed phase II clinical trial. MicroRNAs (miRNAs) are a class of small non-coding RNAs (18–25 nucleotides) which function as key post-transcriptional regulators of gene expression. By binding to the complementary 3′-untranslated regions (3’UTR) of specific messenger RNAs (mRNAs), miRNAs prevent translational events, either through mRNA degradation or translational repression .Viruses are obligate intracellular parasites that rely highly on host metabolic processes to create specific microenvironments required for viral replication and infectivity. In the event of a viral infection, miRNAs are actively involved in the interplay between viruses and hosts, from viral attachment to disease progression. The presence of microRNA Response Elements (MREs) which are generally located in the 5’UTR and 3’UTR of the viral genome, has supported the underlying mechanism of miRNA-mediated antiviral activities . One of Similar to other viruses, miRNA-related research of Dengue virus (DENV) has been growing in recent years. DENV is a mosquito-transmitted, positive sense virus which belongs to the Flavivirus genus of the Flaviviridae family. There are four distinct, but closely related serotypes of the dengue virus, namely DENV-1, DENV-2, DENV-3 and DENV-4. Over the last few decades, dengue has become one of the major public health challenges in the world, especially in tropical and subtropical countries. The World Health Organization  reported that the incidence of dengue has increased 30-fold in the recent 50 years . Bhatt aAedes aegypti or Aedes albopictus. Upon DENV inoculation into the skin by a mosquito bite, the virus is taken up by the tissue-resident dendritic cells or the macrophages via clathrin-dependent receptor-mediated endocytosis. DENV attaches to dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) on dendritic cells [DENV has a 10.7-kb capped RNA with 5′ and 3′ UTRs flanking a single open reading frame that encodes three structural proteins  and seven non-structural proteins  . Dengue ic cells . The manic cells .Upon DENV entry into cells, the viral RNA is uncoated and following replication, the dsRNA can be recognized by the host pattern recognition receptors (PRRs). The PRRs include the Toll-like receptors  and the host RNA sensors comprising the retinoic acid-inducible gene I protein (RIG-1)-like receptors and melanoma differentiation-associated protein 5 (MDA5) . The cytToll-like receptor 3 (TLR3) was shown to have a pivotal role in the recognition and restriction of DENV replication in several cellular lineages , 18, 19.To date, a number of DENV proteins have been identified as the inhibitors of IFN-α/β signaling pathway by interfering with different targets along the pathway. DENV NS2A, NS4A and NS4B have been shown to work in concert to block the phosphorylation of the signal transducer and activator of transcription 1 (STAT1) and nuclear translocation . AnotherDengue fever is a non-fatal disease with symptoms like high fever, headache, vomiting, pain behind the eyes, muscle and joint pain as well as a characteristic skin rash. Unfortunately, a small but significant proportion of dengue patients could develop the Dengue Haemorrhagic Fever (DHF) and the Dengue Shock Syndrome (DSS) which are life-threatening. The pathogenesis of severe dengue has been attributed to immunopathogenesis which elicited an abrupt “cytokine storm”, leading to vascular leakage, hypotension and shock . The comThe biogenesis of miRNAs in humans involves 4 key enzymes- Drosha, Exportin 5, Dicer and Argonaute (Ago) 2 , 36. TheIn general, there are two miRNA-based therapeutic modalities: miRNA antagonists and miRNA mimics. The former approach could be applied to inhibit miRNAs with undesired functions such as miRNAs which are proviral. A miRNA antagonist is usually introduced as a chemically modified miRNA which is known as anti-miR or antagomiR that could complementarily bind to the mature miRNA strand. To date, several chemical modifications have been introduced to antimiR oligonucleotides with the aim of increasing their binding affinity, stability and in vivo delivery. The most commonly used chemical modifications include locked nucleic acid (LNA), a bicyclic RNA analogue in which the ribose is locked in a C3’-endo conformation, 2’ribose modifications such as 2-O-methyl (2-MO), 2-fluoro (2-F), 2-O-methoxyethyl (2-MOE) and the modification of phosphorothioate (PS) backbone . Among tmiRNA mimics are synthetic RNA duplexes which mimic endogenous miRNAs. They are introduced to replenish or to further enhance the levels of miRNAs which are crucial to control disease progression. These miRNA mimics provided a promising proof-of-concept for miRNA replacement therapy. The tumor suppressor miR-34 mimic (MRX34) is by far the most clinically advanced therapeutic mimic. Although the MRX34 phase I clinical trial was halted in September 2016 due to five cases of immune-related events , the queSeveral studies have implied the important roles of miRNAs in viral infections. Mutations in the main catalytic components of the RNA interference (RNAi) pathway led to an increase in replication of DENV , vesiculInterestingly, it is apparent that an effective disruption of the host RNAi machinery is one of the pathogenic strategies of viruses to mitigate the host response. Several viruses have been reported to produce viral suppressors of RNAi proteins that could suppress miRNA-mediated silencing of the host RNAi machinery by interfering with RISC loading or by inhibiting the slicing activity of the Ago protein, a component of RISC –66. For MicroRNA-like small RNAs have been reported to be produced by RNA viruses such as DENV, West Nile and H5N1 influenza viruses –70. HussmiRNAs generally induce translational repression by binding to MRE sites of the targeted mRNAs. Consistent with this mode of action, it is not surprising that most of the miRNAs identified to date have been reported to affect DENV replication by targeting the viral genome sequences. Evidence gathered independently pointed to the fact that miRNAs complementarily bind to the 5’UTR or 3’UTR of DENV genome and thereby inhibiting DENV replication. The first evidence that a miRNA could suppress DENV multiplication by directly binding to the viral genome was represented by miR-548 g-3p . In the Castrillon-Betancur and Urcuqui-Inchima  revealedIn another study, miR-252 was found to be highly induced in a DENV-2 (strain New Guinea C) infection model of the mosquito C6/36 cell line and was able to suppress DENV replication by targeting the DENV-2 E gene . The ideAs viruses are dependent on the host machinery for replication and infection, it is of no surprise that a number of miRNAs have been demonstrated to indirectly regulate DENV replication through the modulation of the host factors or immune response. These indirect effects included modulation of expression of a cellular transcript encoding a host factor required for one or a few steps in the viral cycle. Modulation of receptor expression could regulate the entry of the virus, thus affecting tropism and cofactors vital for replication complexes or translation that could impair or increase viral replication as well as viral protein production, respectively. In addition, miRNAs are also known to enhance or restrict cell responses to the infection like immune response or defense mechanisms .For example, the highly expressed let-7c has been speculated to protect the infected Huh-7 cells from oxidative stress and inflammation response following DENV infection . Let-7c Zhu et al. (2014) demonstrated that the highly expressed miR-30e* during dengue infection suppressed DENV replication by enhancing the host antiviral immune response . When unApart from modulating the host immune response, a study by Wu et al. (2014) provided evidence to link the antiviral role of miRNA to perturbations of other host factors. For example, miR-223 was reported to be capable of inhibiting DENV-2 (strain TR1751) replication and its antiviral effect was likely associated with dampened expression of the microtubule-destabilizing protein, stathmin 1 (STMN1), a key microtubule regulatory protein that controls microtubule dynamics . The exaDiosa-Toro (2017) identified a miRNA with moderate but significant antiviral property against DENV. In the study, miRNA-3614-5p was found to be upregulated in DENV-negative cells and its overexpression reduced DENV infectivity. This miRNA-3614-5p was found not only to reduce DENV but also West Nile virus infectivity. Adenosine deaminase acting on RNA 1 (ADAR1) was analyzed as one of the targets of miR-3614-5p and was demonstrated to stimulate DENV infectivity at early time points post-infection. Taken together, the study extended the knowledge of the contribution of human miRNAs in building the network of interactions between DENV and its human host cells .In contrast, miRNAs could also modulate the host factors or immune response in a way that favour viral replication. Several miRNAs have been reported to positively modulate viral replications, such as miR-1 in Hepatitis B and miR-122 in HCV infections. Nevertheless, miR-146a was the only miRNA identified to date that had a proviral effect in response to dengue infection . InteresmiRNAs identified to date are mostly associated with DENV replication, either via direct targeting of the viral genome or through modulation of the host mediators which are essential for DENV multiplication. Although some studies have shown that the DENV viral load had a positive correlation with dengue severity –100, othIn light of this, a few studies were devoted to understand the effects of miRNAs on cytokine dysregulation during dengue infection. Chen et al. (2014)  demonstrEfforts were also directed to examine the association between differentially expressed miRNAs and highly elevated cytokines following DENV-2 (strain New Guinea C) infection . Qi et aOverall, the host miRNAs can act as either proviral or antiviral via several mechanisms Table . DependiPresent understanding of the role of miRNAs in the pathogenesis of dengue is restricted by insufficient characterization of the miRNA-associated molecular pathways in dengue infection, replication and host immunity . The detIn addition, miRNA might also activate an innate immune response which is nonspecific and is associated with the structure, length, chemical modification, cellular localization and concentration instead of the base sequence. Immune activation triggered by miRNAs could have a negative effect on the health of the recipient. Thus, a few novel strategies have been developed to reduce the miRNA induced immune responses such as through the use of modified nucleotides (LNAs). Although it has not progressed to clinical trials, in vivo animal experimentation on the use of LNA-based miR-146a inhibitor against EV-A71 showed that it could prevent death in mice by restarting the production of type I IFN. At the early stage of viral infection, prophylactic administration of LNA antagomiR-146a was effective. This demonstrates the potential in vivo application of LNA for treatment against EV-A71 infection. Since miR-146a is also present in DENV, in vivo experimentation in the murine model is much needed to evaluate the therapeutic potential of miR-146a.Molecular understanding of host-virus interaction is essential in the development of more efficient antiviral strategies. Considering the intrinsic regulatory role of miRNAs, the hypothesis that miRNAs are playing an important role in regulating viral pathogenesis has been the basis of miRNA-related studies on viral infections. Indeed, this hypothesis has been well supported in dengue infection and the roles of miRNAs are discussed in this review. Understanding and elucidating the roles of miRNAs in fundamental processes associated with DENV infection are necessary to fully characterize their potentials in disease diagnosis and prognosis as well as disease treatment. The recent study of miRNAs might provide the tools for identifying common key genes and pathways that could be activated, repressed or enhanced in members of Flaviviruses. This would facilitate the identification of common targets for therapeutic interventions. Despite the continued efforts of the research community to seek antiviral agents against dengue infections, there is currently no effective antiviral treatment against all four DENV serotypes. As such, miRNAs that have been shown to affect DENV replication and pathogenesis do present opportunities as the targets for miRNA-based antiviral therapeutics for DENV infection. Nevertheless, the existing miRNA research on DENV remains at the target identification stage. In light of the advanced stage of development of Miravirsen, an oligonucleotide which has been shown to inhibit the function of miR-122a in HCV, this miRNA-based therapeutic has been proven to be a promising strategy to delve into, especially at a stage where an effective anti-DENV therapy is still lacking. miR-146a could be taken up as low-hanging fruit for research in the near future as it is the only human miRNA induced by different flaviviruses such as DENV and Japanese encephalitis virus (JEV), as well as in several other viral infection models including enterovirus, VSV and hepatitis B virus –110. MamTo date, there is no in vivo studies in animals or clinical trials being initiated for any of the miRNAs that have been observed to have antiviral activity against dengue infection. Further studies are needed to verify whether the miRNAs known to have antiviral effects against DENV serotypes in vitro will exhibit anti-DENV effects in vivo and whether they are able to protect mice from lethal challenges with DENV serotypes 1–4. Without known targets in vivo in an animal model, the development of miRNAs as promising targets of intervention against dengue is still exploratory. Moreover, research should focus on special delivery systems that target specific tissues to reduce therapeutic doses and unwanted side effects. The selection of the lead miRNA to develop into an antiviral therapeutic will have to depend on both in vitro and in vivo performance in animal models. Thus, efficient in vivo delivery is critical for their successful use in an animal model. The use of nanocarriers and new non-invasive imaging techniques should be developed to monitor the in vivo distributions of the therapeutic nanocarriers that might help to optimize the treatment efficacy. The miRNAs have to maintain high stability in tissues by having good pharmacological properties and displaying no toxicity at the concentrations being administered. Besides these desirable attributes, the miRNA should not display off-target effects. Ongoing development of modified miRNAs and suitable nanocarriers are required to overcome these problems. The development of non-viral systems for the delivery of miRNA has undergone extensive expansion in recent years, providing solutions for the challenges hampering miRNA therapeutic success. The most advanced approach for systemic delivery was via lipid nanoparticles as a carrier system which is currently ongoing in Phase I-III human clinical trials. Nevertheless, the delivery of miRNA to organs other than the liver has been challenging and the toxicity of the nanocarriers is of concern. Thus, preclinical evaluations of miRNA administration using animal models is necessary to determine the efficiency of miRNA delivery to other organs besides the liver, as well as examining the potential safety.


The successful polymerization of the as-prepared monomer was accomplished without acid additives. Its post-polymerization modification with strong Lewis acid boron tribromide furnished unique poly (aniline quinone/hydroquinone) with desired properties for all-solid-state reference electrode (RE) applications. The electrochemical responses from the conjugated polyaniline backbone in this unique polymer have been “suppressed” by the quinone pendant. The resulting poly (aniline quinone) showed a quasi-reversible redox process from the redox behavior of the pendant quinone. The stable electrode potential of this poly (aniline quinone/hydroquinone) suggested that it was a single phase in which the amounts of totally reduced and totally oxidized species could be maintained at a constant in various solvents and electrolytes. Its electrochemical stability was excellent with 95% peak current retention after continuous cyclic voltammetric testing. The aniline and quinone moieties in poly (aniline quinone/hydroquinone) render it to have both hydrophilic and hydrophobic compatibility. It showed excellent behavior as a reference electrode in aqueous and non-aqueous media and can be used in both non-zero current and zero-current conditions, providing a stable potential with a maximum potential drift of ~4.7 mV over ten consecutive days.This work reports the design, synthesis, and characterization of a novel redox-active conjugated polyaniline containing quinone moiety as a solid state reference electrode. The union of electro-active quinone with π-conjugated polyaniline was created by the first chemical synthesis of  Electrochemistry is a discipline that studies chemical reactions that involve the exchange of electric charges between two substances. Electrochemistry has significant applications, ranging from batteries, sensors, corrosion control to electrolysis . A referOne of the major research areas in electrochemistry is to develop miniaturized reference electrodes that can provide highly stable electrode potential with ease of fabrication at low cost for electrochemical micro-sensor development. Electrochemical micro-sensors are one of the most promising sensor platforms due to their high portability, low cost, simple structure, and fast response time. They are being developed for a broad range of applications, including ambient air-quality monitoring, occupational health and safety, biomedical diagnostics, industrial process control, and anti-terrorism ,8. A ver2Cl2, Ag/AgCl or Fe(CN)63−/4−anions) in conductive polymers to enhance their stability and adjust the ion transports. The second was to use bilayer or multi-layer conductive polymers as part of the RE system. The third was to directly use the conductive polymers as REs [TFSI]). The results showed better performance than the conventional metal wires, and it was stable for seven days. But it is still a quasi-RE, not a real RE  ionic liquid. The FcMeOH’s reversible pair was observed in different media and the peak potential of FcMeOH has been summarized in 3CN/PBS (1:1) and CH3CN were different from these polar organic solvents as they were either aqueous or miscible with water. This finding suggests that poly (aniline quinone/hydroquinone) shows an ideal Nernst behavior that is solvent independent under certain conditions. This is in great contrast to the traditional redox or conjugated polymer in which non-ideal behavior, the solvent dependence of all the equilibrium constants prevents meaningful comparison of electrochemical potential data obtained with a polymer film in different solvents. P1 was also tested in water (PBS aqueous buffer) and water/organic solution (PBS/acetonitrile mixture) and was found to provide the stable and reproducible peak potentials in these systems as well  P1 and its innovative polymerization methods described in this work will enable a new class of all-solid-state reference electrode that can be implemented in various electrochemical micro-devices.The combination of redox-active quinone and polyaniline was accomplished to give a novel redox conjugated polyaniline. The post-polymerization modification with boron tribromide furnished poly (aniline quinone/hydroquinone)


Anxiety affects a quarter of strokes. It can be disabling even after mild stroke and transient ischaemic attack (TIA). It is not feasible to deliver conventional psychological therapies to the large population of anxious stroke and TIA patients. We are testing the feasibility of a web-enabled randomised controlled trial (RCT) to compare an individualised telemedicine cognitive behavioural therapy (CBT)-based intervention with a self-guided web-based relaxation programme. This study aims to evaluate the feasibility of novel trial procedures and the delivery of the TASK interventions in stroke and TIA patients.We aim to recruit 40 community-based stroke and TIA patients experiencing anxiety at least 1 month post-discharge in Lothian, Scotland. We will assess the (1) recruitment number per month; (2) percentage completion of electronic consent; (3) time taken for remote eligibility confirmation; (4) percentage completion of follow-up surveys: modified Rankin scale, EuroQol-5D5L, 7-item generalised anxiety disorder, Patient Health Questionnaire-2 and modified fear questionnaire; (5) data capture of intervention fidelity and (6) use of actigraph smartwatches to obtain continuous data of rest/activity.The current study will provide feasibility data on streamlined web-enabled trial procedures and the use of smartwatches to obtain objective measures in stroke and TIA patients, offering potential for large efficient RCTs to be conducted centrally and remotely with far fewer resources in the future. This study will inform further refinements of the TASK interventions before evaluation in a definitive RCT.NCT03439813. Retrospectively registered on 20/2/2018.Clinicaltrials.gov The online version of this article (10.1186/s40814-018-0329-x) contains supplementary material, which is available to authorized users. There are more than 100,000 strokes per year and 1.2 million stroke survivors in the UK .Anxiety is a universal emotion that helps people adapt to changing situations. However, it can become maladaptive when anxiety becomes pervasive or out-of-proportion to the danger posed by a situation. When maladaptive anxiety starts to interfere with a person’s occupational or social functioning, it is considered an anxiety disorder. Anxiety can be broadly divided into two clinical subtypes—phobic and generalised. Phobic anxiety is characterised by a disproportionate fear of well-defined situations or stimuli and marked avoidance of those situations . By contPatients with anxiety disorder reported more avoidance in agoraphobia-related situations, e.g. going out alone, going to crowded places and travelling on public transport; social situations and specific situations, e.g. physical exertion, having sex, being alone at home and activities related to fear of having a headache, another stroke or a fall . In our Younger people and those with a previous history of anxiety or depression are more likely to develop anxiety after stroke , 5. LongInadequate service provision in psychological care post stroke across the UK is evident from the reports of the Sentinel Stroke National Audit Programme in England, Wales and Northern Ireland  and the Other potential barriers to accessing psychological therapy include the inability to attend face-to-face appointments due to physical immobility and social stigma attached to seeking psychological treatment. Reluctance to travel in agoraphobia and the fear of being judged in social phobia are diagnosis-specific barriers to attending face-to-face sessions. People with social phobia symptoms were three times more likely to report a fear of what others might think or say about them if they sought psychological treatment compared to people with other anxiety disorders . To dateTelemedicine could offer a way to overcome the barriers in accessing psychological therapy by treating patients remotely while maintaining an individualised therapist-patient alliance that is integral to CBT. Meta-analyses demonstrated guided internet-based CBT to be superior to waitlist control and as efficacious as face-to-face CBT in treating anxiety disorders or depression in general adults, with face-to-face CBT spending seven times more therapist time than guided internet-based CBT , 14. It Delivering CBT remotely via telemedicine would enable centralisation of resources for staff, training, quality monitoring and cross-covering of different geographical areas, reducing travel time for therapists as well as patients. Digital content and treatment approaches in a telemedicine intervention could be updated and refined easily, thus, making use of the latest best evidence. The provision, maintenance and updating of digital content could be commissioned or delivered via charitable organisations. Organisation delivering the intervention can continue to invest in research and development to utilise the latest technology to deliver the treatment content at lower costs. At present, it is not yet clear whether telemedicine-delivered CBT is cost effective in stroke and TIA patients.We developed the treating anxiety after stroke (TASK) CBT intervention following the UK Medical Research Council’s framework for complex intervention development , elaboraA definitive RCT needs to recruit and retain large number of patients to provide sufficient power to detect a clinically relevant treatment effect. Web-enabled and automated trial procedures can centralise processes and resources, offering the potential for scaling up an RCT with far fewer resources and costs than conventional procedures. For instance, one of the major costs of traditional multicentre trials is the setting up of multiple centres which includes establishing legal contract between the sponsor and local sites, identification and training of local staff, site initiation visits, monitoring, local closeout and archiving. Adopting a novel centralised system could avoid the need for these and streamline a large trial. Feasibility of remotely performed trial procedures need to be tested in stroke and TIA patients: online self-screening and recruitment, remote eligibility confirmation, electronic informed consent, remote intervention delivery, automated self-reported outcome measures and data capture of intervention fidelity.Anxious patients after stroke showed higher levels of avoidant behaviour across a range of situations, e.g. going out alone, physical exertion and social situations compared with those who were not anxious . DisturbThis study aims to evaluate the feasibility of (i) novel web-enabled trial procedures, (ii) the TASK-CBT intervention and (iii) actigraph smartwatches to collect continuous data throughout the entire trial in stroke/TIA patients.We report the protocol of the TASK feasibility RCT in accordance with the SPIRIT checklist  AdditionThe TASK feasibility trial is a parallel two-armed RCT comparing TASK-CBT with TASK-Relax. Figure www.task4stroke.org) [We designed the TASK trial procedures using Research Electronic Data Capture (REDCap), a secure web-based database management application . We creaoke.org) , one foroke.org) , a devicoke.org) . ContinuWe aim to recruit 40 community-based residents within NHS Lothian (United Kingdom postcodes EH and FK1) who are aged 18 or above, with a diagnosis of stroke or TIA , at least 1 month after being discharged to the community from clinic or hospital ward. Participants need to have capacity to give informed consent, be able to communicate in English on the telephone, have internet access and report at least one positive response on our 6-item anxiety screening questions , followed by randomisation.We designed the TASK-CBT intervention to be delivered via telemedicine—telephone, website, email and mobile text. This enables remote delivery of the intervention while preserving the individualised therapeutic alliance between the therapist and the patient that is integral to CBT. The TASK-CBT intervention represents a low-intensity, guided self-help psychological intervention in a stepped care model . A healtWe summarise the key ‘active ingredients’ of the TASK-CBT intervention in Fig. TASK-Relax is a web-based self-guided relaxation programme. Relaxation therapy is a commonly used comparator in RCTs of CBT in psychiatry research . The TASParticipants of both arms receive weekly mobile text reminder and a participant record card to record progress and completion of follow-up surveys. Data collection occurs at T1(week 6) and T2  via emailed links to self-completed electronic surveys. Once the follow-up survey at T2 is complete, we offer all participants access to the website given to the other group for a further 10 weeks.Concomitant standard clinical care and interventions  for anxiety or mood disorders, e.g. antidepressants, benzodiazepines are permitted and recorded in our follow-up surveys.A member of the research staff not involved in conducting the TASK trial generates a permuted block randomisation sequence with random block sizes using STATA14 . The seqThe TASK researcher (HYC) enrolling participants and delivering the allocated intervention is not blinded to the treatment group assignment. The TASK researcher informs participants that they will be randomly allocated to one of two anxiety interventions by the computer and receive the login details to their allocated website via email. Participants are masked to the contents and type of anxiety intervention allocated to the other group. This method attempts to mask participants to our hypothesis that one treatment is superior to the other.In a sub study embedded within the TASK trial, all TASK participants are invited to wear the actigraph smartwatch. Once consented, the smartwatch is posted to the participant with simple care instructions. At 2 months, the battery of the smartwatch will run out. With the participant’s agreement and on the safe return of the first smartwatch, a second smartwatch will be sent to the participant, so he/she can wear it for the rest of the trial. All smartwatches are returned using prepaid special delivery envelopes.The current trial assesses the feasibility of the TASK-CBT intervention and the technology-enabled trial procedures. Feasibility data collected: recruitment (number per month), percentage of completed consent (online or by post), time taken to complete remote eligibility confirmation via electronic health records (date of randomisation–date of ‘Sign Up and Consent Form’ received), percentage drop out after fewer than three telephone TASK-CBT sessions and self-completion of electronic follow-up surveys at T1 and T2 (percentage of completed surveys).We define a lack of feasibility of TASK-CBT and the current trial design as (1) recruitment number of < 2 per month, (2) > 50% of TASK-CBT patients dropping out after fewer than three telephone sessions, (3) > 10% non-completion of follow up surveys at T2 and (4) participants reporting harm from the intervention.Treatment relevant outcomes Table  include We will assess the percentage of TASK participants who also consent to wearing the smartwatch, the duration of the smartwatch being worn by each participant using the data recorded on the smartwatch, percentage of participants of this sub study who agree to wear the smartwatch again after 2 months, percentage of participants who did not return smartwatch (attrition).The therapist for this feasibility trial is a stroke physician (HYC) who has received training on delivering CBT from and has ongoing supervision by a consultant neuropsychiatrist (AJC) at weekly meetings during the TASK trial. For intervention fidelity, we will assess (i) percentage agreement between the therapist’s record and transcripts of audio-recording of telephone sessions, assessed by a clinician independent of the study; (ii) automated data capture of online task completion; (iii) summary web usage data from Google Analytics.We post a participant record card to every participant and send weekly text reminders to their mobile phone throughout their participation. All participants will receive text and email reminders to complete the follow-up surveys at T1 and T2. Non-responders will receive further text and email reminders and a final phone call from a researcher blinded to the treatment allocation to complete unfilled online surveys over the telephone.Participants are free to withdraw from the study at any point, or a participant can be withdrawn by one of the TASK trial investigators if he/she loses capacity during the study period.A trained medical doctor delivers the TASK-CBT intervention in the current feasibility trial. All participants have contact details of the TASK research team from the start of the trial. They are informed that if severe cases of anxiety or depression are identified during the trial, the TASK research team will liaise with their general practitioner to arrange appropriate care. Trial investigators will assess these cases and report as adverse events to the sponsor according to good clinical practice.All study data are collected and managed using REDCap electronic data capture tools hosted at University of Edinburgh. REDCap (Research Electronic Data Capture) is a secure, web-based application designed to support data capture for research studies . IdentifThere is no data monitoring committee planned for this study.As this is a feasibility study, we did not perform power calculation. Feasibility outcomes will be summarised descriptively. All electronic items on the follow-up surveys must be scored to permit submission, preventing any missing values. Only the trial investigators have access to the final trial dataset. Actigraphy data from the smartwatch device will be sent anonymously for time-series analysis at the Centre for Medical Informatics, Usher Institute, University of Edinburgh.Using innovative information technology, we designed a streamlined web-enabled trial to be tested for the first time in stroke and TIA patients: online self-screening and recruitment, remote eligibility confirmation, electronic informed consent, remote intervention delivery, automated self-reported outcome measures and data capture of intervention fidelity. Our trial design offers a number of potential advantages. For instance, a centralised model could enable recruitment of eligible patients anywhere in the country, or anywhere in the world without the need to have locally-based principal investigators or research staff. Larger sample sizes could be reached more easily without significantly raising extra costs. Potential participants with proof of identity and eligibility could sign up themselves regardless of their locations. This model of recruitment could empower patients to choose to participate in a clinical trial without being screened or ‘selected’ by local healthcare or research staff. Generalisability could be increased as a result. While this method of recruitment remains aspirational for clinical trialists at present, owing to the regulatory and administrative challenges in conducting multicentre RCTs, it is of vital importance that we continue to evaluate the feasibility of novel procedures that would increase the ease of recruiting participants to large-scale RCTs, regardless of where they live. A centralised system of intervention delivery could be easily scaled up but also quality assured—a particular challenge in clinical trials where many therapists are employed to deliver the intervention in different centres.There has been limited use of wearable devices to assess therapeutic outcomes in RCTs in stroke patients and RCTs of psychological interventions . We receAnxiety is one of the commonest neuropsychiatric complications post stroke or TIA. Psychological care post stroke/TIA is inadequate, and the traditional paradigm of delivering face-to-face CBT at clinic is not feasible for the large population of stroke and TIA patients owing to limited resources and other barriers related to anxiety after stroke, e.g. agoraphobia and social phobia. Innovative ways of delivering psychological intervention remotely to the patient’s home, e.g. individualised CBT via telemedicine could be a feasible model to treat the large population of stroke and TIA patients experiencing anxiety. If feasible, the TASK-CBT intervention could provide an exemplar model for delivering psychological intervention for a range of post-stroke psychological complications. Web-enabled features embedded in our TASK-CBT and TASK-Relax websites, e.g. data capture of completion of weekly online tasks and web usage analytics, provide novel ways of measuring intervention fidelity objectively—an important aspect of internal validity that can be challenging to assess and is infrequently reported in trials of complex intervention .The TASK trial design has the potential to be applied in RCTs of any self-management or guided self-management interventions for a range of patient groups. Our design has the obvious limitation of excluding people who have no access to the internet, or those who do not have a minimum level of skills in information technology. This is likely to be overcome by the trend towards wider adoption of home broadband and user-friendly devices amongst seniors , and theThe current trial represents feasibility testing of the TASK-CBT intervention and the technology-enabled trial procedures. This will lead to further refinements of intervention design and trial procedures. Our current trial does not inform the efficacy of TASK-CBT. A number of important aspects of the TASK trial design will need to be considered in the next phase, the TASK II trial, before proceeding to the definitive trial, TASK III.In TASK II, we will determine an appropriate primary outcome measure to be used in the definitive trial. We will consider a range of candidate outcome measures of anxiety, functional independence, quality of life and participation. We will select the primary outcome based on the measure’s sensitivity to change/difference between groups, its practicality for use in large-scale RCT (to minimise attrition) and its clinical meaningfulness. Our observational data suggested that stroke/TIA patients with anxiety disorder reported a statistically significantly higher level of functional dependence on the modified Rankin Scale, a poorer quality of life on EurolQol-5D-5L and worse participation on a the Work and Social Adjustment Scale, compared to those without anxiety .Patients after stroke/TIA can present, to varying degree, predominantly phobic, predominantly generalised, or mixed anxiety types. This poses an additional challenge as different anxiety measures are required to assess the two anxiety subtypes. It may be necessary to have different primary outcomes defined for different anxiety subtypes.Other considerations include defining the responder’s status: for example, whether to use the minimal relevant change from baseline on an anxiety measure, a dichotomised outcome, or an absolute anxiety level post-intervention to assess between-group difference.Our choice of using TASK-Relax, a web-guided relaxation programme as an active control was to avoid exaggerating the effect size—a known effect of using ‘waitlist’ or ‘treatment as usual’ as control condition in RCTs of psychological interventions . We acceWe will refine the TASK-CBT intervention based on participants’ feedback from this current trial. The TASK therapist training materials will be developed based on our experience and the use of anonymised audio-recording transcripts. We will consider using audio recording for within-trial training, maintenance and enhancement of intervention fidelity in the next phase of TASK, where therapists will be employed to deliver the TASK-CBT intervention.As it is not yet clear whether TASK-CBT is cost-effective in stroke and TIA patients, health economics analysis of the TASK-CBT intervention using EuroQoL-5D-5L data will be conducted in the definitive RCT.Testing the feasibility of self-recruitment and remote eligibility confirmation in our local centre represents only the first step in finding novel efficient ways of recruiting patients to large-scale nationwide RCTs. We believe all patients should be empowered to self-recruit to an RCT that they are eligible to regardless of where they live. Verifying eligibility remotely using electronic health records across different health boards in RCTs could be a viable option in the future but at present remains aspirational due to administrative barriers.Protocol version: AC17087 version 3, 1/12/2017.Recruitment commenced on 17/1/2018. Estimated completion date 1/5/2018.Additional file 1:TASK-CBT Intervention development using the 6SQuID approach. (PDF 725 kb)Additional file 2:Report of patient involvement in TASK. (DOCX 463 kb)Additional file 3:SPIRIT checklist items. (DOC 120 kb)Additional file 4:TiDier template items. (DOCX 28 kb)Additional file 5:Screening questions in ‘Sign up and consent form’. (PDF 173 kb)Additional file 6:Research ethics approval letter. (PDF 134 kb)


No-reflow is a serious and frequent event during primary percutaneous coronary intervention (PPCI) for acute ST segment elevation myocardial infarction (STEMI). The aim of this study was to identify possible predictors for no-reflow.We investigated 218 patients with acute anterior STEMI who underwent PPCI from December 2016 to December 2018. No-reflow was defined as a coronary TIMI flow grade of ≤ 2. TIMI flow grade 3 was defined as normal reflow.In our study, the no-reflow phenomenon was observed in 39 patients (18%) during angiography. The patients of no-reflow group were found to be more older, diabetics, longer pain-to-balloon time, lower blood pressure, higher platelet counts and higher levels of D-Dimer and Cystatin C (Cys-C). In multivariate logistic regression analysis, only diabetes , longer pain-to-balloon time  and higher Cys-C level  were predictors for no-reflow.Cys-C might be a useful predictor for the no-reflow phenomenon after PPCI in STEMI patients. It might help to screen STEMI patients with high risk of no-reflow on admission. The best treatment of ST-segment elevation myocardial infarction (STEMI) is reperfusion of ischemic myocardium as soon as possible. Primary percutaneous coronary intervention (PPCI) has become the preferred strategy for reperfusion and the current standard care for STEMI. NeverthCystatin C (Cys-C) is the most important inhibitor of endogenous cysteine proteases and serves as a marker of renal function. EpidemiThe protocol of the present study was approved by the Ethical Committees of the Second Hospital of Shandong University  and the study conformed to the Declaration of Helsinki Principles. Informed written consent was obtained from all participants. The survey was completely anonymous and did not ask for identifying information.The study population was composed of 218 patients with acute anterior STEMI who underwent PPCI in the Second Hospital of Shandong University from December 2016 to December 2018. Venous blood samples were obtained at admission. All patients met standard diagnostic criteria and received PPCI of the left anterior descending artery (LAD) within 12 h from symptoms onset. The key exclusion criteria were as follows: age >75 years; PPCI was performed after 12 h from symptoms onset or no stent was implanted during the PCI; thrombolysis failure and rescue PCI; cardiogenic shock; acute pulmonary edema; ventricular septal rupture; cardiac tamponade; severe respiratory, renal, or hepatic dysfunction or failure; history of thromboembolic disease and imflammatory process.All patients received 300 mg acetylsalicylic acid (ASA), 600 mg clopidogrel and 40mg atorvastatin as a loading dose on admission and intravenous standard heparin (70 U/kg of body weight) before CAG/PCI. The procedure was performed with standard technique and the radial artery approach was the first choice. The glycoprotein IIb/IIIa receptor inhibitor tirofiban was administered during the PPCI according to the operator’s preference. Balloon predilatation or postdilatation, the type of stents, and thrombus aspiration were used according to the operator’s discretion. The blood flow in the infarct-related artery (IRA) was measured following stenting during the angiography according to the Thrombolysis in Myocardial Infarction (TIMI) grading system. No-reflBlood sample was drawn from the antecubital vein in the emergency department. A routine laboratory blood work-up was conducted for all of the patients.t test when the variables were normally distributed, and Mann–Whitney U test was used for abnormal distribution. Multivariable logistic regression analysis was applied to identify independent predictors of no-reflow. Variables that could be a predictor of no-reflow with a significant p value were entered into multivariate analysis. The results of univariate and multivariate regression analyses were presented as odds ratio with 95% CI. A two-tailed p value of < 0.05 was considered statistically significant. The above statistical analyses were performed using SPSS 19.0 .All the descriptive variables are expressed as the mean ± standard deviation (SD). Categorical data are expressed as frequencies and percentages and the Chisquare test or Fisher’s exact test was used for the analysis. Comparisons between 2 groups were conducted using the Student’s PPCI was performed in 218 STEMI patients during 2016–2018. The 218 patients were divided into two groups according to the final TIMI flow after the PPCI. No-reflow was seen in 39 (18%) patients during angiography. The clinical characteristics of the subjects in two groups were shown in As presented in The effects of different variables on no-reflow were analyzed by using univariate and multivariate logistic regression analyses as shown in To further evaluate the value of using Cys-C as a predictive marker for the no-reflow phenomenon after PCI, we performed the ROC analysis. As shown in We have demonstrated in the present study that Cys-C is associated with no-reflow phenomenon in patients with PPCI. Our results suggest that serum Cys-C level may be a new predictor of no-reflow in STEMI patients treated by PPCI.Early revascularization of infarct-related artery by PPCI has become the most effective strategy in STEMI patients. But no-reflow phenomenon significantly limits the benefits of PPCI therapy. Detecting clinical predictors for insufficient myocardial reperfusion may help STEMI patients with no-reflow. In our study, the rate of no-reflow after PPCI is about 18%, and it is similar to the rates of no-reflow reported in previous studies varied from 2.3 to 39.9% –18. PatiRecent evidence suggests that high circulating Cys-C is associated with cardiovascular diseases, independent of creatinine-based renal function estimates. In addition, heritability analyses indicate that Cys-C and cardiovascular diseases share genetic influences. Cys-C iBased on our results, we conclude that Cys-C might be a useful predictor for the no-reflow phenomenon after PPCI in STEMI patients. It might help to screen STEMI patients with high risk of no-reflow on admission and help to choose the best treatment.Several limitations should be mentioned for the present study. First is its modest sample size, further studies in more patients are needed to confirm our data. Second, for all the enrolled patients, LAD was the culprit vessel. Our results might be confirmed with other coronary arteries. Third, the association and changes in Cys-C over time were not studied. The prognostic impact of Cys-C in no-reflow of STEMI patients remains to be examined in future studies.S1 Data Set(SAV)Click here for additional data file.