Hospitals in many countries are encouraged to develop audits to assess and improve the quality of care. Ward audit is a specific form of audit and feedback that is commonly used but little studied. The aim of this study is to describe the content and application of hospital ward audit in order to identify potential enhancements to such audits.n = 32), documentary analysis (n = 44) and 25 h of observations of healthcare workers involved in the design and implementation of ward audit. Data were analysed using framework analysis. Findings were presented iteratively to stakeholders who used them to develop a description of the content and delivery of ward audit.Multiple qualitative methods were used to study a diversity sample of four English National Health Service organisations over a 16-month period. We undertook semi-structured interviews  and discuss implications for theory. There is a need to reduce the use of punitive feedback.The online version contains supplementary material available at 10.1186/s12913-021-06239-0. Globally, hospitals have developed internal quality assurance and improvement structures, of which audit and feedback is a substantial part –4. AuditAudit and feedback can be an effective intervention to improve care, but its effectiveness varies  . There iThere have been calls to incorporate evidence and theory into the design of audit and feedback; to date much of this work has focused on national audits (e.g. ). The cuThis was a multi-site study using interviews, observations and documentary analysis, supported by two groups of stakeholders: a co-production group and an advisory group.We studied four English National Health Service (NHS) acute hospital provider organisations, with approximately 4750 beds in total and with each organisation including between one and four hospitals Table . Sites wThe research team, supported by stakeholder involvement, identified potential healthcare professional participants according to their role. Potential participants were given information about the study by the hospital research department. All interviewees gave informed consent. During the semi-structured interviews, the interviewer (MS) asked participants to describe what happens during ward audit, exploring how, when, where and why it is done and involving whom . The topWritten consent was sought for the observation of individuals. Where a group was observed, written, informed consent was sought from the senior person present, information was sent to those anticipated to be present (e.g. to meeting attendees) and posters were displayed giving information about the observations, the study and how to have data withdrawn from the study.During observations, the researcher asked occasional questions in order to better understand what was being observed and to develop rapport with participants . ObservaDocumentary analysis gathered data from secondary sources  . DocumenThe interviews and observations occurred during different site visits, over the same period as the documentary analysis (January 2018 to April 2019), thereby enabling each source of data to inform lines of enquiry.n = 3), clinical leads  (n = 3) and organisational clinical audit leads (n = 3). During the first four co-production group meetings (8 h), MS facilitated small group work and whole group discussion to develop a description of the group’s pre-study understanding of audit and feedback. MS facilitated the co-production group to: inform the sampling of documents, and interview and observation participants; review analysed research data; consider the differences and similarities between the research data and their pre-study views; identify challenges to the analysis and interpretation of data; propose further avenues to explore; and iteratively develop the description. An advisory group included patient, academic, professional body, audit provider and commissioner input; it provided consultation support to the co-production group via MS. The researcher (MS) is a nurse who had recently managed organisation-level quality assurance in the NHS and had training and experience in qualitative methods and group facilitation.Work to describe the audit was supported by two stakeholder groups . A co-prInterviews, observation field notes and reflective notes were transcribed, checked for accuracy and anonymised. Analysis involved familiarisation with the data followed by identifying initial themes within two interviews and two observations. MS compared and contrasted the themes across data sources and with the initial participant-described diagrams in order to create an initial framework. All transcripts were coded. Documents were pseudonymised, printed, coded and exemplar quotes extracted. Data were managed using Nvivo v12 . MS analysed the data using inductive framework analysis , with ovThe study forms part of a larger project to describe and enhance audit and feedback in dementia care in acute hospitals that included a focus on both ward audit and the national audit of dementia .MS interviewed 32 healthcare staff, undertook 19 observations (25 h) and analysed 44 documents  from acrFrom the analysis, we identified that ward audit involved clinical performance data being presented to ward managers on a monthly basis. This included performance on: medicine management (e.g. the percentage of medicine charts where the administered medicines have were correctly recorded); infection control ; nutrition ; bladder and bowel care (e.g. the percentage of patients with a urinary catheter who have catheter insertion date written in the records); and communication (e.g. the percentage of patients’ records with evidence that discharge has been discussed with carers or relatives).We identified two high-level themes: audit stages and punitive feedback. There were seven different stages to the audit Fig. : impetusimpetus was described as being both from within and outside the organisation. Internal drivers were to assure and improve care:The “Why have we got a [ward audit] tool? It’s for the ward sister who owns that environment, to demonstrate where she thinks she’s up to in that environment. And that’s the most important thing for me. The results, I won’t say, don’t matter, but the results are secondary to that. I don’t like to see a sea of green, I’m much happier if there is some amber and red on there” .Interviewer: “If you stopped doing [ward audit] tomorrow, what would be the impact?”Matron: “The impact on patients?”Interviewer: “On patients, on the staff, on the organisation.”Matron: “I honestly don’t think that there would be an impact, directly, on patients. I don’t think it would change the quality of the documentation or the quality of care that is given to the patients.”Interviewer: “Impact for staff?”Matron: “Possibly relief, they’re not having to do it. For the organisation, I suppose it’s the accountability for giving that assurance to external organisations. How they’re monitoring the quality, they would have very little to back that up” (Interview 15).The audit method was designed by directors of nursing or their deputies. Different organisations used different methods to gather data. One extracted audit data from the electronic record, the other three organisations used assessment of case notes by clinical staff. Two of the organisations also collected ward audit data through observations of practice, as well as staff and patient interviews.External impetus was to enable organisational managers to describe the monitoring of wards to the regulator:assessment of practice, participants described that it was important to involve those providing care as it gave them feedback on the care they deliver; however, participants reported that self-assessment may affect data quality. The accuracy of data collection is described below. Study observations at one site found that completing five patient interviews and five case note reviews took over five hours, excluding data entry or staff interview data collection. Immediate clinical issues were identified (e.g. asking for help applying cream) during the assessment of practice. However, there was evidence that some clinical issues  were not addressed.In the analysis involved colour coding the results . The targets used during the analysis may have been set higher than might be attained and were perceived as being aspirational . The accuracy of red- or amber-rated performance was explored with the team manager; the accuracy of green (high) performance was not explored. Some actions were described as being beyond the control of the ward team (e.g. replacing wash basins).The ward audit data feedback giving collated performance by multiple wards , whilst individual ward performance was sent to wards and matrons, directorate managers, directors of nursing and their deputies. The feedback did not include proposed solutions to address sub-optimal performance. At one organisation, ward-level comparison data were anonymised and presented bi-annually to ward managers.The organisational board and directorates received summary data “As the manager, the matron, we know how we’re doing. I don't know if I really need this to know how I'm doing.” Ward-level feedback was delivered verbally at a ward team meeting shortly after data were submitted and/or on receipt of analysed data. The verbal feedback was sometimes reiterated by email (Interview 21). Verbal feedback was repeated at daily huddle meetings of healthcare professionals, although this may depend upon the ward manager’s attendance at ward huddles . Verbal feedback to ward staff may be the only response to poor performance:“Sometimes all that’s needed is a butt kicking, if you know what I mean. A reminder that people need to get this done.” At two organisations, if a ward got consecutive red or amber results for the same standard, the matron  asked the ward manager to write an action plan. These may be escalated to positional leaders  or specialist teams. Actions may be documented in an action plan without anticipation of completion:There was lots of stuff on it [the action plan] that you can’t [get]… Estates work, splashbacks that are stained that there’s no way on God’s good green earth I’m going to get. This is an old building. We’ve actioned it.” .Organisation- and directorate-level groups reviewed ward audit data to look for trends across wards . Specialist groups  within the organisations may also review the data relevant to that group. In addition, the director or deputy director of nursing reviewed the data. They reported drawing upon other sources of information, including staff sickness, agency usage, patient acuity  and perceptions of team leadership  to assess whether and how to act. This triangulation was a reflective or discursive process .Feedback to ward managers may not be new information, and this may affect whether it leads to action:“I think there’s been a desire in the last five or ten years to develop a tool that provides objective measurement of the quality of nursing care. I think most people have developed something internally that approximates the sorts of things I’ve described” “(Ward audit) is talked about as part of the  account (for regulators and commissioners) … it’s on there. It doesn’t go into the detail, but it says if they want it, it’s available for them to talk about.” Each of these stages took place within a wider organisational assurance and improvement system, proposed and designed to meet the needs of senior nursing leaders and external stakeholders.Punitiveness was a cross-cutting theme that provided an explanatory link between stages. The data described the reported experience and anticipation of punitive feedback and negative consequences by those receiving the feedback. Punitiveness largely related to the nature of verbal feedback described using words such as a ‘kick’ , a ‘slap’ , or a ‘whipping’ . There were also non-verbal components, including additional scrutiny and senior management visits to the ward, which were described as intended, “to be persuasive or coercive. It depends on your viewpoint.” .Feedback from ward audit was reported to be punitive at three sites. There was evidence that this experience and anticipation of punitive feedback resulted in a range of unintended, potential consequences, Fig.  as descrWard manager: “They would be down on me like a tonne of bricks if I didn’t. If I hadn’t inputted the data, it would be, ‘Where’s your data? Why haven’t you done it?’ So, it’s just not worth not doing it.”Interviewer: “Practically, what would that 'tonne of bricks' involve?”Ward manager: “Oh, I would get…matron would be on my back. She’s really good, but somebody would be on her back. So, it would be like the house of cards that was toppled from the top and it would be all down. So, she would be under pressure and she would put that pressure on me to get that information where it should be, or some kind of explanation as to why it hadn’t been done. It’s just not worth it.” (Interview 10)“that ward would just be targeted as a failing ward. It’s got a lot of work to do. It shouldn’t be that.” The nature of the feedback may not be intentional. For example, senior nursing leaders described being aware of the feedback being experienced by recipients as punitive or inducing fear, and sought to balance this in their attempts to address complacency and produce performance data that they can, in turn, be described to the regulator:“There is a balance, isn’t there? Between, and I’m not suggesting we’re trying to make people fearful because we’re absolutely not. But I think, for me, if I’m responsible for something, I need to know what standards are expected of me. But I want to know, if I can’t reach that standard, that someone will support me. I don’t want to be allowed to do what I want. We don’t want a complacent environment; we want people who know they have to work to achieve something.” “We’re trying to reach standards right across the hospital and across the other hospitals that you’re providing the evidence to look at against, so there wouldn’t be anything [without ward audit]. And CQC (the regulator), when they come in, we’ve got something to show, ‘This is how we manage monitor, and manage- ‘, we’d have no evidence really.” Ward managers described both experiencing and anticipating punitive feedback from matrons and directors of nursing. The anticipation of this punitive feedback resulted in two main responses that sought to reduce such feedback. Firstly, ward managers provided their managers with information about actions being taken to improve sub-optimal care at the point of data entry . Secondly, the ward managers may seek to influence future performance by misrepresenting care.Ward manager: “I have been known to go, “Yes” (when it should be no) I’m not going to lie to you.”Interviewer: “How does that feel?”Ward manager: “It feels like it’s a waste of my time. I’m ticking a box.” (Interview 10)Misrepresentation may be related to fear of adverse comparative performance:“I think they (ward managers) would be honest if everybody was scoring low. When it’s one out of… If everybody scored low then it wouldn’t be a problem, because then everybody has got some improvements to do.” Hospital employees involved in the audit at different levels are aware that the results may not represent performance:Matron: “I don’t think that they’re honestly completed. I think they’re meaningless. “Interviewer: “And you know that because?”Matron: “Because you review notes, you look at notes. Just from ward to ward, notes that come from another ward – you know, patient transfers. There is no way that places could be getting 100%. Often, a lot of places are getting 100% in many of the domains – or above 98% all of the time.” (Interview 15)“ given the benefit of the doubt sometimes. I think it’s quite pedantic, the detail in the [ward audit] tool. So, if you’ve got 15 measures that tell you whether you get green on a chart, if one’s missing and you’re the ward sister, you might give green. Whereas if I’m an auditor, I probably won’t.” … “I think because it’s human nature. And if you ask someone to choose five sets of paperwork, which we do. Audit five sets of patient paperwork, that person might choose the worst five, they might choose the best five, and we have no control over that.” “There is sometimes a concern of course that people lie, and they submit data which paints them in a very positive light.” Some ward managers are aware of the temptation to misrepresent care and delegate responsibility to others:“sometimes it’s nice to put a junior member of staff doing it. They don’t realise what the consequences are.” Misrepresentation led to the audit report demonstrating high performance which was repeatedly described as a “sea of green” . There was little evidence that the report stimulated organisation-level improvement actions, as represented by a dotted line in Fig. Evidence from one site describes that punitive feedback was replicated down a communication chain. The links in the chain were from the director of nursing, to matrons, to ward managers, to ward staff attending ward huddle meetings. Punitive feedback was described as happening in response to: submitting data describing red or amber rated performance; not submitting data; and not improving performance:As described above, ward-level actions may be documented in an action plan, as a way to demonstrate a response to positional leaders, without anticipation of completion.“I sort of slap their wrists and tell them to wash their hands but really, I should mark them down then slap their wrist” Directors of nursing, who triangulate the data with other sources in order to determine the need for action, may not view the findings as credible:“You’ll see that the monthly return that I just showed you is effectively a sea of [perfect]. … I’m an inherent cynic so I tend to think that if it looks too perfect it probably isn’t.” External stakeholders (commissioners and regulators) were told about the presence of ward audit, but not given the results. That ward audit data may not represent actual care might not be noticed by the external stakeholders , as a result Fig. The ward manager may give ward staff feedback about actual performance but submit data to the corporate team that misrepresents performance:“I’m looking forward to a more mechanised tool which would reduce the audit burden, will release nurses to nurse, which is a good thing, but also might improve compliance and objectivity because it will be easier to do it properly and rather less easy to fake …. (to) enable them to be freed up to care for the patient directly.” Deputy director of nursing: “I think people just tick the box sometimes. They’re perhaps not doing the audits as thoroughly as they should.”Interviewer: “Why not?”Deputy director of nursing: “I don’t know really. Why would you not do it? Perhaps you want to be 100%, perhaps you’re frightened of the scrutiny it will bring you if you’re not 100%, perhaps it’s just the easiest thing to do.” (Interview 18)Other positional leaders  reported not being aware of how the feedback were perceived .We found participants reported different reasons for the data being inaccurate. They reported that people may submit inaccurate data to save time and/or due to fear of scrutiny:“People have been hesitant to recommend their ward for assessment despite the fact that when they do the audit and when they report the audit they’re indicating compliance but they’re nervous because they think just that one day when somebody else comes to look at it there could be that variance.” We found at least two organisational responses to address data quality. Firstly, there was evidence that organisations deliberately duplicated audits , such that the same topic was assessed by different people through different audits (e.g. Observation 29). More recently, organisations have adopted additional approaches to address data quality; for example, one organisation was purchasing hand-held computers. The reported rationale for the computers was that they would be more reliable as it would give ward managers “fewer reasons for them to wish to cheat” because it would be quicker and data entry could be monitored . One organisation had developed, and one was developing, an accreditation approach to ward performance monitoring. This approach involved wards seeking recognition for the quality of care on the ward. Whether the ward achieved the status of a high-performing ward was based upon an assessment of care undertaken by the corporate nursing team, rather than ward staff. This may have altered ward managers’ willingness to report high performance:We explored ward audit at four diverse NHS organisations through interviews, observations and documentary analysis. We used inductive framework analysis, iteratively presenting the findings to stakeholders for challenge, to direct further data collection and to integrate the findings into a description of how audit was undertaken. We found that reporting the presence of ward audit to external stakeholders, notably the regulator , was an important driver to positional leader support and organisational investment in the audit. The method of audit involved monthly data collection, typically by ward staff, in relation to topics including medicine management, infection control, nutrition, bladder and bowel care, and communication. We present the findings as a description of stages, before describing key findings regarding the nature of feedback and the accuracy of the data: Firstly, that feedback passing from more-senior to less-senior nurses was often perceived as punitive. Secondly, that ward collected data may not accurately represent the actual care provided. Furthermore, there was awareness at multiple levels that the results might not accurately represent clinical performance.In discussing our findings, we compare the current content of ward audit to evidence  and theoOur findings show that ward audit contains features consistent with audit and feedback evidence  and theogoal setting, assessment of practice involved data collection followed by analysis and feedback. Like CP-FIT, we describe unintended consequences where “health professionals may unethically manipulate data” ; We also describe motivations for doing so.Comparing our findings with CP-FIT  identifigoal setting, related not to “the clinical topic and its associated clinical behaviours or patient” , but to the rationale for undertaking the audit. We found that this included the motivation to meet the regulator’s requirements. We found non-acceptance by senior managers of high performance, which differs from the non-acceptance of low performance by recipients described by Brown et al. We found different levels of action , and describe differing responses by recipients at each of these levels. For example, triangulation at the organisational level demonstrated CP-FIT’s verification of all results, but only of ‘red’ results at the team-level.We found potential extensions to CP-FIT: we describe the impetus stage, which whilst apparently similar to verification perceptions, acceptance, intention and behaviour. In CP-FIT, this influence may represent an additional health professional variable acting through social influence, credibility and actionability upon the feedback cycle. Articulating the influence of the previous experience of feedback upon pre-conceptions and subsequent actions provides a valuable extension to CP-FIT.In contrast to Brown et al., we found that the target (green-rated care) was important, as it was a proxy for the avoidance of anticipated punitive feedback. We describe the role of previous feedback upon data collection and ward managers’ anticipation of future feedback. Brown et al. state, “health professionals often had profound emotional reactions to feedback, both positive and negative…, we found no reliable evidence that these directly influenced the feedback cycle.”(p15). We describe the influence of the ward managers’ anticipation of punitive feedback, a subsequent emotional response (fear) and a behavioural response (misrepresentation of performance). We found that these responses impacted upon organisation-level leaders’ In addition to not incorporating best practices, our findings suggest that ward audit has potential negative impacts upon care. We propose two mechanisms by which the audit may adversely affect patient care: opportunity cost and an erosion of standards of care. There is considerable opportunity cost and evidence that staff prioritise data collection over direct patient care. For example, observations at one site suggested that the gathering of case note data took over five hours per month per ward and was done by ward managers or their deputies, with further time spent interviewing staff and entering data. There was some evidence that clinical issues were identified and addressed during this time (e.g. a patient requiring help to apply a cream), but there was also evidence that staff prioritised data collection over patient care (e.g. not going to a patient who requested help). Whilst purposive sampling undermines the drawing of economic conclusions, we propose that the time cost may exceed the benefits seen from some limited changes occurring in care as a result of the audit.The requirement for nursing staff to spend time gathering ward audit data may have a more subtle effect: participants described ward audit as ‘a waste of my time’ and ‘meaningless’. We propose that the external prioritisation of perceived low value work may create feelings of discomfort in those being asked to prioritise time towards data collection for the audit. This discomfort may stem from being unable to meet personal goals relating to the delivery of high-quality care and externally set goals about the collection of data. It is possible that participants resolve this discomfort by downgrading their goals relating to standards of care . EvidencWe found evidence that feedback was experienced as punitive. This supports earlier findings describing punitive perceptions of audit . There iSecondly, we found that previous punitive feedback led to anticipated negative consequences which affected subsequent data quality. We found that the data was reported to be inaccurate and not credible. We propose that the lack of accuracy and credibility influenced commitment to change. Viewing the findings through the lens of clinical performance feedback intervention theory (CP-FIT), directors and deputy directors of nursing receive feedback through a table showing banded  performance of teams. Their goal is to have high performance that provides assurance about the quality of care. They pay attention to red and amber performance, reflectively and discursively triangulating it with other data sources including acuity (patient population) and their perceptions of ward staffing and leadership (team characteristics). However, the subsequent organisation-level action response was mediated by a lack of accuracy (most results were ‘green’ and as a result no action plan was developed) and may have been affected by the reported lack of credibility, resulting in reduced commitment to change. Punitive feedback has been described as being less actionable at the individual level . Our finFuture work should seek to implement enhancements to ward audit, including seeking to reduce the use of punitive feedback. Addressing punitive feedback may require consideration of the influences upon its use; for example, punitive feedback has been associated with leaders’ attributions that performance is related to individual, rather than system causes, or that performance is due to a lack of effort, rather than lack of ability or training . An inteReporting to the regulator was an important impetus to the ward audit. The regulator may also have a role in influencing the effectiveness of ward audit; for example, regulators could explore ward audit data quality through triangulation with other data, and/or by asking how ward audit stimulates organisation-level improvements based upon a review of influences upon performance.One organisation had developed, and one was developing, accreditation mechanisms, whereby corporate nursing teams assess performance. Such accreditation audits have since been nationally recommended . Our finWe used multiple methods and triangulated data to explore ward audit at diverse NHS organisations. The findings were presented to a stakeholder group for challenge, to identify where further data were needed and to synthesise the description. As part of this work to enhance the credibility of the findings, the research team and stakeholder group considered emergent findings against a framework for intervention description, and against previous evidence and theory.We do not provide identifiers as to which findings came from the which organisation in order to protect the anonymity of participants. The fourth site was sampled to test the description based upon data from earlier sites, and had the same CQC rating as an earlier site. We did not sample hospitals rated as ‘inadequate’; hospitals with this rating may have used different approaches to ward audit from the sites we studied. We describe reported perceptions of feedback. The nature of feedback beyond ward level was not observed and it may be that ward managers feel that the data profiles their personal performance, and that this makes feedback feel punitive . HoweverWe describe a communication chain at one site from director of nursing to ward staff. It is possible that there are other links in this chain. No assumption is made as to the intention or source of the nature of the feedback. In particular, it may be that the delivery, experience or anticipation of punitive feedback reflects organisational norms regarding attributions or patterns of communication; it may also reflect external influence .Organisations invest considerable resources in ward audit. We highlight evidence and theory-informed enhancements to ward audit. In particular, there is evidence that at some organisations the data are reported to be unreliable, that this is recognised by those involved and that data accuracy is associated with feedback being perceived as punitive. We recommend that recipients of ward audit data explore the reliability of the data and consider the potential role of punitive feedback as a factor undermining data quality and subsequent quality improvement. We propose national and organisation-level work to enhance ward audit, including through the implementation of non-punitive feedback.Additional file 1: Appendix A: The study sample (Note: Titles standardised in order to maintain anonymity).Additional file 2: Appendix B: Interview topic guide v3.Additional file 3: Appendix C: Example interview diagram.Additional file 4: Appendix D: A diagrammatic representation of study data sources.


Continent ileostomy (CI) was once a prevalent surgical technique for patients who required total proctocolectomy but then gave way to ileal pouch-anal anastomosis (IPAA) after 1980. Although IPAA has been the gold standard procedure preferred by most patients when total proctocolectomy is required, due to its imitation of physiological function of rectum and preserved function of anus, various complications have been observed with a relatively high rate of morbidity that could affect pouch longevity. Once serious complications such as pelvic abscesses and/or fistula occur, the pouch often needs to be removed. In addition, for some patients with a shortened small intestine or foreshortened mesentery, it is impossible for the ileal pouch to reach the pelvic floor, thus making the creation of an IPAA difficult. Previously, most of these patients would be referred for an end ileostomy, with an associated poor quality of life. In this circumstance, we propose that CI may deserve a reappraisal and serve as an alternative. In this article, we review the indications, contraindications, technique evolution, and outcomes of CI. The continent ileostomy (CI), first reported by Nils G. Kock in 1969, was an option for patients with ulcerative colitis (UC) or familial adenomatous polyposis (FAP) when they were referred to have a total proctocolectomy and permanent end ileostomy previously , 3. BecaCI can be performed either as a primary procedure for patients requiring restorative proctocolectomy or as a salvage procedure for those who have failed pelvic pouches.For patients who have not previously undergone abdominal surgery, the construction of a CI is mainly recommended to patients with difficult anatomy, such as a short small bowel mesentery, dysfunctional sphincter, or irradiated pelvis. A short small bowel mesentery, which makes the small bowel difficult to reach the pelvic floor, precludes an IPAA. Patients with dysfunctional anal sphincter are also not suitable for an IPAA since this is associated with poor postoperative functional outcomes. In addition, an irradiated pelvic floor, inducing inflammation or fibrosis, prevents access to the pelvis and hence the construction of an IPAA . SelecteOn the other hand, CI can be considered an option for patients when IPAA is contraindicated, or in IPAA failure when reduced small intestinal length impossible to reach pelvic floor prevents redoing the pelvic pouch surgery. IPAA is currently the gold standard for patients following a total proctocolectomy and is associated with fewer restrictions in sports and sexual activities than CI, offering a better QOL . HoweverWhen pouch failure occurs and a redo IPAA is not feasible or desirable, conversion of the original pouch to CI is preferred when expertise is available, since this allows conservation of intestinal length . While cPatients with problems of an end ileostomy and those seeking to avoid an external appliance and hence an improved body image can also be considered for CI.Indications for a CI was elucidated in Whether CD patients should be offered a CI remained controversial . PreviouPatients potential for short small bowel syndrome are not recommended for a CI since a relatively long segment of small bowel is required for constructing the pouch, which will increase the risk for short bowel syndrome if the operation were to fail.For acute severe colitis, CI is avoided in the acute setting given the additional time required in these often sick patients. Subtotal colectomy with an end ileostomy is the initial choice with a CI considered at the second stage of the surgery after the patients recover from their illness.It is important to note that the surgical procedure for CI is complex and the ability to handle intubation is acquired after surgery. Patients should be well informed prior to surgery, and the procedure should be contraindicated to those who are unable to understand such essential information. In addition, children and patients with learning difficulties or those with their extremities precluding intubation are unsuitable for CI.Contraindications for a CI was elucidated in The conventional continent ileostomy described by Kock is referred to as a Kock pouch in this review. In brief, after proctocolectomy, the penultimate 30-45 cm of the ileum is used to create a J- or S-shaped reservoir, and the terminal 15 cm used to create a nipple valve and exit conduit that traverses the abdominal wall. In obese patients, a greater length of small bowel should be reserved for the exit conduit. In the J-pouch design, the antimesenteric border of the two proximal 15 cm segments is apposed with suture after which, the lumen is opened. The posterior layer is sutured . After rTo deal with the high rate of valve slippage and fistula formation, Barnett created an isoperistaltic valve and collar  ApproximA total of 55 cm of ileum is needed for the T-pouch. The distal 15 cm segment of small bowel makes an efferent antireflux valve and ostomy, while the proximal 40 cm forms the pouch body, creating a “U” shape with each 20 cm limb. The blood supply to the mesentery of the distal 15 cm of small intestine is preserved with avascular mesenteric windows opened to increase the mobility of the bowel and to create space for sutures. The intestine is then used to make a valve segment between the two limbs of the U. The mesenteric border of the two limbs is approximated while the valve segment is fixed by a series of interrupted sutures passing through the avascular mesenteric windows . The twoSurgical complications occurred frequently in the initial reports of CI but later decreased due to refined techniques and increasing experience. Short-term complications include hemorrhage from the suture lines, anastomotic leakage, ischemia or necrosis of the intussuscepted valve, and intestinal obstruction of the small bowel with adherence to the pouch and fistula formation, while long-term complications include valve slippage and prolapse, fistula, volvulus, perforation hernia, valve stenosis, and pouchitis .Early complications can be severe and even lead to pouch excision. However, most patients are able to convalesce and keep their pouch. Parc et al.  reportedSimilar to our proposal for ileal pouch disorders, the long-term adverse sequelae of CI could also be classified as structural, inflammatory, functional, and neoplastic complications . Table .Valve malfunction includes slippage, prolapse, stenosis, and necrosis of the valve, which can cause difficulty in intubation, incontinent stoma, or even pouch failure. The weakest point of the valve is at the mesenteric aspect where intussusception produces a large bulk of fatty mesentery that prevents the two walls of the valve from firmly attaching to each other , 30.Nipple-valve slippage 29.7%) has been recognized as one of the most frequent causes for pouch revision or failure in patients with Kock pouch, followed by fistula formation (25.2%) 9.7% has , 31. It Valve prolapse occurs as complete slippage of the nipple valve when there is no adherence to the abdominal wall, often due to the fascial defect which is made too large at surgery. Difficulty with intubation often occurs, and surgical repair is usually necessary. Necrosis of the valve occurs from ischemia, often due to excessive sutures for valve fixation.With modifications in surgical technique, postoperative complication rates of the nipple valve have been reported to decrease from 41.1% to 4.8% as reported by Ecker . The ratFistula can develop anytime after surgery and occur in the valve, pouch, afferent limb, or in the collar in patients with BCIR , 32. WheApproximately 10% of patients with Kock pouch develop stoma stricture, compared to 25% of patients with a T-pouch . ParastoPouchitis, one of the major nonoperative complications, is the inflammation of pouch mucosa, usually accompanied by overgrowth of bacteria. The etiology of pouchitis has not been clearly elucidated. Several hypotheses for the development of pouchitis have been proposed, such as recurrence of UC, dysbiosis, deprivation of short-chain fatty acids, mucosal ischemia, host gene susceptibility, and immune dysregulation , 41. Thede novo after the construction of CI or in some cases, the pouch can intentionally be created in some CD patients without the involvement of small bowel or perianal disease. Clinically, CD of the pouch can be classified into inflammatory, fibrostenotic, or fistulizing phenotypes. As mentioned, CD was one of the main risk factors of pouch failure, and patients were 4.5 times more likely to develop pouch failure compared to those with FAP or UC in one study [CD of the pouch can occur ne study . The diane study .Since a part of the intestine has to be removed in patients with failed IPAA, and approximately 60 cm of intestine is needed for the construction of CI, patients being considered for CI may face with the risk of short bowel syndrome and its associated complications such as diarrhea, dehydration, weight loss, and nutrition deficiency . PatientIBD and FAP are associated with an increased risk of dysplasia and cancer . While pSeveral retrospective studies on CI with a large sample size reported that the rate of pouch revision was high, mainly due to valve dysfunction, mesh usage, fistula formation, and/or anastomotic leakage , 34. KocMost patients with CI can maintain a functional pouch for a prolonged period. Nessar et al.  reportedSeveral clinicopathological factors influencing pouch survival have been reported. A multivariate model to assess factors about pouch excision in patients with CI showed that the underlying disease, gender, fistula development, and body mass index (BMI) were potential risk factors . PatientPrevious studies showed that the majority of patients who responded to questionnaires declared satisfaction to the outcomes of surgeries, regardless of the types of CI  , 32, 35.For QOL, a study suggested that patients with Kock pouch were less likely to report dietary, social, work, and sexual restrictions than end ileostomy patients who needed more antidiarrheal medication and fiber intake and that hospitalization and pouch complications occurred less frequently in patients with Kock pouch . With BCCompared with end ileostomy, CI has noticeable advantages in QOL. The major merit of CI over end ileostomy is its ability to make patients free of external appliance and thus improve body image. For patients with a failed IPAA, when construction of a new IPAA is not feasible or desirable, CI may be an option . AlthougIn conclusion, CI is associated with acceptable outcomes in experienced hands and should be considered an alternative to end ileostomy and IPAA. CI is an option for patients with a failed IPAA when a new IPAA is not feasible or desirable.


Hepatocellular adenoma (HCA) is conventionally considered a rare benign liver tumor, but advanced studies have revealed that HCA is heterogeneous, and may include a type that is prone to malignant transformations. Differentiation between well-differentiated hepatocellular carcinoma and focal nodular hyperplasia is necessary to diagnose hepatocellular adenoma through imaging; however, the tumor marker of hepatocellular carcinoma, protein induced by vitamin K absence, or antagonist II (PIVKA-II), is rarely positive in hepatocellular adenoma.A 44-year-old woman presented to our hospital with complaints of loss of appetite and weight loss. Multidetector row computed tomography revealed a liver tumor  that was enhanced in the arterial phase. Her serum PIVKA-II level was very high (3327 mAU/mL). Based on the enlargement of the mass and the results of the diagnostic imaging, hepatocellular adenoma or hepatocellular carcinoma was suspected, and we considered the possibility of a malignant transformation due to the high level of serum PIVKA-II; thus, we performed hepatectomy. Histological examination showed brown pigment deposition in the hepatocytes, which was determined to be lipofuscin granules. Based on immunohistochemical findings, the diagnosis was unclassified hepatocellular adenoma. Immunohistochemical examinations revealed that the adenoma cells in the tumor were positive for PIVKA-II. Her serum PIVKA-II level returned to normal after the resection.We present a case of unclassified hepatocellular adenoma with brown pigment deposition and elevation of serum PIVKA-II level. For the differentiation of liver tumors with high levels of PIVKA-II and hypervascular mass, hepatocellular adenoma should be considered. Hepatocellular adenoma (HCA) is a rare benign epithelial liver tumor often observed in young women taking oral contraceptives. It is associated with oral contraceptive intake, glycogen storage disease types I and III, and a history of excess androgen exposure , 2. HCA 2. She had no history of liver disease, pregnancy , oral contraceptive, or anabolic steroid use. Her laboratory findings on admission showed mild liver and kidney dysfunction (Child-Pugh class A) (Table d-glucose was identified in the mass on positron emission tomography CT (not shown).A 44-year-old woman exhibiting weight loss and loss of appetite was referred to our hospital because of a lesion in the liver that was detected by ultrasonography. The patient’s body mass index (BMI) was 10.9 kg/mRadiologically, HCA and hepatocellular carcinoma (HCC) are considered different diseases, but accurate diagnosis based on the imaging findings alone is difficult. According to some studies –5, when The incidence of HCA in Western countries is 3–4 per 100,000 , 6–10. HCharacteristic with immunohistochemical staining, the positive expression of LFABP in normal hepatocytes becomes negative in HNF1α-inactivated HCA. HCC rarely accompanies this type of tumor. Furthermore, this type often occurs in multiple tumors; thus, it is called liver adenomatosis. In the inflammatory HCA, SAA and CRP are the inflammatory proteins that are overexpressed, and it is characterized histologically by inflammatory cell infiltration, bile ductular proliferation, and sinusoidal dilatation. This type usually occurs in overweight women and is often accompanied by bleeding. The β-catenin-activated types (b-HCA and b-IHCA) often show cellular and structural atypia due to the activation of the Wnt/β-catenin signaling pathway. Immunohistochemical staining indicated nuclear overexpression of β-catenin and GS, which is downstream from the β-catenin pathway. This type of mutation is caused by a deletion of CTNNB1, and most hotspot mutations in exon 3 lead to high levels of β-catenin activation and GS overexpression. However, exon 3 S45 and exon 7/8 mutations lead to moderate and weak activation of this pathway, respectively, resulting in diffuse heterogenous GS staining and weak patchy GS staining . Hence, 2) may be related to the lipofuscin granule deposition. However, cases of brown pigment deposition in adenoma cells have been reported as pigmented HCA in Eastern countries and Japan [A discriminative point for this case is the very high level of deposition of brown pigment in the adenoma cells. We used Fontana-Masson and iron stains, and the brown pigment was believed to be lipofuscin granules. Lipofuscin consists of lipid-containing residues from lysosomal digestion arising from aging and damage; hence, lipofuscin is called the “waste pigment” or “age pigment” –16. Furtnd Japan –20, and nd Japan , 18. Alt2, and there was comparative slimness. The mean PIVKA-II was 3254.3 mAU/mL . There have been two reported cases of malignant transformation of HCA [In this case, the serum level of PIVKA-II was very high, but it is usually negative in cases of HCA. A PubMed search of the keywords “hepatocellular adenoma” and “PIVKA-II or DCP”  yielded seven reports Table  19, 21––21–25. Tn of HCA , 21. Then of HCA . For tren of HCA . HoweverIn conclusion, we presented a case of unclassified HCA with brown pigment deposition and serum PIVKA-II level elevation. To differentiate liver tumors with high PIVKA-II levels and hypervascular masses by imaging, HCA should be considered.


Transcranial magnetic stimulation (TMS) is a non-invasive brain stimulation technique able to modulate cortical excitability. This modulation may influence areas and networks responsible for specific cognitive processes, and the repetition of the induced temporary changes can produce long-lasting effects. TMS effectiveness may be enhanced when used in conjunction with cognitive training focused on specific cognitive functions. Playing video games can be an optimal cognitive training since it involves different cognitive components and high levels of engagement and motivation. The goal of this study is to assess the synergistic effects of TMS and video game training to enhance cognition, specifically, working memory and executive functions. We conducted a randomized 2 × 3 repeated measures (stimulation × time) study, randomly assigning 27 healthy volunteers to an active intermittent theta-burst stimulation or a sham stimulation group. Participants were assessed using a comprehensive neuropsychological battery before, immediately after, and 15 days after finishing the video game+TMS training. The training consisted of 10 sessions where participants played a 3D platform video game for 1.5 h. After each gaming session, TMS was applied over the right dorsolateral prefrontal cortex (DLPFC). All participants improved their video gaming performance, but we did not find a synergistic effect of stimulation and video game training. Neither had we found cognitive improvements related to the stimulation. We explored possible confounding variables such as age, gender, and early video gaming experience through linear regression. The early video gaming experience was related to improvements in working memory and inhibitory control. This result, although exploratory, highlights the influence of individual variables and previous experiences on brain plasticity. Non-invasive brain stimulation techniques have become a step forward in cognitive neuroscience due to their ability to establish causal links between cognition and its neural substrate. Among these techniques, transcranial magnetic stimulation (TMS) allows modulation of cortical excitability in highly specific target regions, inducing changes in the associated cognitive functions and even enhancing them  or any of its sequels, regardless of the experience level, were also excluded.While 32 participants were recruited, five did not complete all the phases of the study . The final sample was therefore composed of 27 healthy adults (14 women and 13 men) aged 18–40 years (29.44 ± 6.28). Participants were excluded if they had neurological or psychiatric disorders, including depression, measured through Beck’s Depression Inventory  Ethics Committee. All participants gave written informed consent to participate in the study following the Declaration of Helsinki and received monetary compensation amounting to 80 euros.A randomized 2 × 3 repeated measures (stimulation × time) study was conducted. Participants were randomly allocated to one of two stimulation groups: Active or Sham and evaluated before, immediately after the training period, and 15 days after finishing the training. When comparing the outcome measures of the two experimental groups, differences after training were only found in one of the variables (see “Results” section). Therefore, we decided to further explore the data to identify possible confounding variables that might have influenced the results.ad hoc guided interview). The results of the logistic regression showed that early video gaming experience (in any kind of video game genre) predicted some of the performance changes observed in executive function tasks (see “Results” section). We, therefore, used this variable to classify the sample into four subgroups . Based on previous literature on video gaming and its effects on brain structure and function  after training ended (Post1), and 15 days after the end of the training . This assessment was performed using a scale created During training, participants played for 1.5 h at the rate of one session a day for 10 consecutive days excluding weekends  under the supervision of a researcher. Game sessions were also video recorded for later analysis. The only instructions given to participants were on how to use the controller and on what the main goals of the game were at the beginning of the training; otherwise, they played as they wished. The number of goals per session, the number of tries per goal, and the time needed to achieve each goal were measured. Overall performance was calculated by dividing the total number of goals achieved during the entire training period by the total number of attempts .motivation (their desire to play) before playing, and fun (enjoyment during gameplay) and frustration (dissatisfaction during gameplay) after playing and before TMS.The gaming experience was measured subjectively at each video game training session using a Likert scale of 1–5. Participants scored A total of 10 TMS sessions were conducted, one after each video game training session. In each TMS session, brief pre- and post-stimulation assessments were made, consisting of the Mini-Mental State Examination  coordinates Intermittent theta-burst stimulation  and one-way ANOVA (for comparisons of four subgroups). Gender differences between groups were calculated through Pearson’s chi-square test.We analyzed age differences between groups using Student’s Fitness to parametric assumptions was checked for all variables. To assess the normality of data distribution, the Shapiro–Wilk test was used. The homogeneity of variances was contrasted through the Levene’s test of equality of variances. Lastly, to test sphericity, Mauchly’s test was used when there were three levels of a repeated measure.When parametric assumptions were not satisfied, task scores were transformed, and/or alternative non-parametric tests were employed, as explained below.t-tests Bonferroni-corrected for multiple comparisons. In measurements with just a single assessment point, the differences between groups were analyzed using the Student’s t-test. To study differences between groups for variables that were not normally distributed, task scores were transformed calculating new scores reflecting pre- vs. post-training changes. Two difference scores between time points were calculated as follows: (1) pre-assessment vs. a first post-assessment difference (Pre-Post1); and (2) pre-assessment vs. follow-up assessment difference (Pre-Post2). ANOVAs were used to compare those difference scores between groups when parametric assumptions were satisfied, otherwise, we used the Mann–Whitney’s U. When the distribution was normal, but equality of variances was not satisfied, we used Welch’s ANOVA to compare scores between groups.To compare video gaming and cognitive performance between Active and Sham groups, and taking into account the assessment time points for the tasks (2 or 3 depending on the task), a repeated measures general linear model (rm GLM) was implemented for each variable. Significant effects and interactions were followed-up using paired t-test . Parametric assumptions were also assessed for each variable for the four groups and we implemented the same procedures as above when assumptions were not met. But when, after transforming the scores, parametric assumptions were still not met, we used the Kruskal–Wallis H test to compare the four four subgroups, and when significant differences were found we used Mann–Whitney’s U to compare the groups in pairs. Since performance for one of the variables was also predicted by gender (besides gaming experience), gender was included as a covariate in the corresponding analysis.Therefore, an rm GLM was used to compare the variables significantly influenced by early video game experience for the four subgroups resulting after combining stimulation type and early video gaming experience , public URL: t(25) = 0.35, p = 0.731) or between the four subgroups taking into account video gaming experience  = 0.89; p = 0.457). There were no significant gender differences between the two initial groups (Active vs. Sham: p = 0.842) or the four subgroups . Moreover, the number of participants with early video gaming experience was not significantly different between the Active and Sham groups (p = 0.863).Age was not significantly different between the Active and Sham groups (n = 14) and Sham (n = 13) groups, most differences were not statistically significant. See post hoc analyses.When comparing video game performance  and cognitive test performance  for the Active  = 101.95, p = 0.000, F = 6.16, p = 0.020, F = 4.85 p = 0.021, F = 5.88, p = 0.005, F = 3.82, p = 0.029, F = 3.35, p = 0.043, F = 4.3, p = 0.049, F = 6.33; p = 0.004, F = 4.45; p = 0.017, Post hoc analyses revealed that participants in the Active group had slower reaction times in the Post2 assessment compared to both the Pre  and the Post1 assessment . Interestingly enough, this effect was not observed for the more complex reaction time tasks  that were similar for all groups, probably due to training and practice effects. Since our main interest was to compare the effects of TMS combined with video game training, that main effect of time was not specifically explored but was observed as part of the GLM analysis results and thus is only reported for variables that were normally distributed. In particular, we observed improvement over time in video game skills  = 5.53, p = 0.027, F = 7.41, p = 0.012, ps < 0.043).We also found statistically significant differences between the Active and Sham groups in some tasks (main effect of the group), given the absence of interaction effect between group and time, this was probably due to differences in baseline performance. This effect was observed for the 3-back d’ score . The rest of the variables showing changes in the previous analyses obtained weak (BF 1–3) to moderate (BF 3–10) evidence , to find out whether these variables could predict the changes in cognitive performance—most especially in executive functions and working memory—after training .F = 3.36, p = 0.036, adjusted R2 = 0.21) and the d’ index  = 5.23, p = 0.007, adjusted R2 = 0.33); and the Stop-switching task SSRT  = 5.55, p = 0.005, adjusted R2 = 0.34). In particular, changes observed in the d’ index for the 3-back task were predicted only by early video gaming experience  = 3.51, p = 0.002), but not by age or gender (ps > 0.788). Similar results were obtained for the accuracy score for the 3-back task were only the p-value was close-to-significant for early video gaming experience  = 2.01, p = 0.056). SSRT changes were influenced by both early video gaming experience  = −3.78, p = 0.001) and gender  = 3.15, p = 0.004).The model was statistically significant for the 3-back task accuracy (n = 12) and non-experienced (n = 15) participants, finding that the difference was statistically significant (p = 0.013), with more male participants having early video gaming experience (69.23%) than women (21.43%).To further explore the influence of early video gaming experience and gender on cognitive performance, we first determined whether there was a gender imbalance between experienced . For the SSRT we also included gender as a covariate based on the results of the logistic regression.F = 5.6, p = 0.005, Post hoc analysis revealed differences between the Active+Exp subgroup and the two subgroups without early video gaming experience, namely, Sham+NoExp (p = 0.048) and Active+NoExp (p = 0.012). Furthermore, t-tests comparing Pre vs. Post1 3-back d’ scores within group, revealed significant differences for the two subgroups with early video gaming experience (Sham+Exp: t(5) = −3.28, p = 0.022 and Active+Exp: t(5) = −3.36, p = 0.020), but not for the two subgroups without early video gaming experience (Sham+NoExp: t(6) = −1.32, p = 0.236 and Active+NoExp: t(7) = 1.33, p = 0.225).For the 3-back task, results showed that, when comparing the change in d’ scores between pre and post1 assessment among groups, there was a statistically significant difference  = 7.71, p = 0.001, F = 5.37, p = 0.008, F = 2.2, p = 0.061, p = 0.033 and Active+NoExp, p = 0.001).For the SSRT, the GLM revealed a main effect of time (ps > 0.483) or between the four subgroups (ps > 0.522), not even for gender (p = 0.264).The levels of motivation before and fun and frustration after the training were not significantly different between the Active and Sham groups  specific cognitive abilities directly trained during the intervention ; and (2) other cognitive functions not directly trained . Results showed very limited changes that were mainly related to the near-transfer effect, i.e., variables related to video gaming skills.The near-transfer effect assessed using an alternative version of the video game that participants played before and after the intervention period, was present in both the Active and Sham stimulation groups. This result combined with the fact that there was no difference between those groups in video game achievements at the end of the training period, seems to indicate that the near-transfer effect was only due to the video game training and not mediated by TMS. To our knowledge, there is no literature in which TMS has shown improvements in video gaming performance, although some recent studies have documented positive near-transfer results using transcranial direct current stimulation , we observed an improvement in certain cognitive control tasks: digits forward, 3-back task, and accuracy and reaction times for switching trials in the stop-switching task. In contrast, we observed a deterioration in reaction times in go trials and the SSRT of this task. All these effects were present in both the Active and Sham groups, probably due to the video game training and the practice effects present in the neuropsychological tasks, observing, again, the lack of effect of the TMS stimulation. In any case, the near-transfer effects seemed more meaningful since the observed effect size  in this case was much bigger than that observed in the possible far-transfer effects , and the Bayesian analyses only showed weak to moderate evidence to support the alternative hypothesis of far-transfer effect.Since we did not find an effect of TMS on cognitive performance, we explored other variables that could influence the results. This analysis led us to observe that early video gaming experience may have mediated the improvement in certain cognitive functions. In particular, participants with early video gaming experience improved working memory performance (as evidenced by the increase in the d’ index of the 3-back task). This was a surprising result since far-transfer effects on visual working memory tasks as a result of video gaming play have not been widely documented in the literature. We have found one study in which working memory performance was enhanced after video game training in a first-person shooter action game . The 10-day training period would have made it easier for experienced players to improve their performance in specific executive function domains. In any case, given the small sample size of the subgroups included in the comparison of experienced vs. not experienced gamers, these results should be interpreted as exploratory.The observed lack of effect of TMS in any of the studied domains has several possible explanations. In the following paragraphs, we will explore different arguments and considerations previously documented in the literature to understand our results.First, we must consider whether the stimulation protocol (iTBS after training) and the targeted brain region (the DLPFC) were the most appropriate for the specific goals of this research. Although iTBS protocol is still not widely used in research focused on cognitive enhancement, it was chosen for its ability to induce longer-lasting effects over the cerebral cortex with shorter stimulation times compared to rTMS , public URL: The studies involving human participants were reviewed and approved by Universitat Oberta de Catalunya (UOC) Ethics Committee. The patients/participants provided their written informed consent to participate in this study.MP, DR-R, and EM conceptualized and designed the study. MP designed the experimental tasks. MP and RV-S acquired and analyzed the data, and all authors interpreted the results and drafted and reviewed the manuscript. All authors have approved the submitted version and have agreed to both to be personally accountable for the author’s contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Laodelphax striatellus  in a persistent propagative manner and causes severe losses in East Asian countries. To explore the expression and function of circRNAs in the regulation of virus infection, we determined the circRNA expression profile in RBSDV-free or RBSDV-infected L. striatellus midgut tissues by RNA-Seq. A total of 2,523 circRNAs were identified, of which thirteen circRNAs were differentially expressed after RBSDV infection. The functions of these differentially circRNAs were predicted by GO and KEGG pathway analyses. The expression changes of five differentially expressed circRNAs and eight parental genes were validated by RT-qPCR. The circRNAs-microRNAs (miRNAs) interaction networks were analyzed and two miRNAs, which were predicted to bind circRNAs, were differentially expressed after virus infection. CircRNA2030 was up-regulated after RBSDV infection in L. striatellus midgut. Knockdown of circRNA2030 by RNA interference inhibited the expression of its predicted parental gene phospholipid-transporting ATPase (PTA) and enhanced RBSDV infection in L. striatellus. However, none of the six miRNAs predicting to bind circRNA2030 was up-regulated after circRNA2030 knockdown. The results suggested that circRNA2030 might affect RBSDV infection via regulating PTA. Our results reveal the expression profile of circRNAs in L. striatellus midgut and provide new insight into the roles of circRNAs in virus–insect vector interaction.Circular RNAs (circRNAs) are endogenous RNAs that have critical regulatory roles in numerous biological processes. However, it remains largely unknown whether circRNAs are induced in response to plant virus infection in the insect vector of the virus as well as whether the circRNAs regulate virus infection. Rice black-streaked dwarf virus (RBSDV) is transmitted by  A high-proportion of genomes can be transcribed into RNA and the majority of these RNAs are non-coding RNAs (ncRNAs). The ncRNAs, can be divided into multiple types, including microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) . CircRNACDR1as can impact the development of zebrafish midbrain through sponging miR-7  infection  (S1 to S10) and encodes 13 proteins  . The genproteins . Most RBproteins . The P10iatellus .L. striatellus midgut during RBSDV infection by RNA-Seq. The expression of differentially expressed circRNAs and their parental genes was verified by quantitative real-time PCR (RT-qPCR). The functions of the differentially expressed circRNAs were predicted using GO and KEGG analyses. Also, the interactive networks between differentially expressed circRNAs and miRNAs were constructed. Furthermore, the function of circRNA2030 was investigated by RNA interference (RNAi). The results provide new insight into the functions of circRNAs in regulating plant virus infection in the insect vector.In its insect vector, RBSDV can move from midgut lumen to hemolymph or other tissues, then to salivary gland, and finally, infect the plant during insect feeding. The midgut is an important barrier for RBSDV infection . In thisL. striatellus used in this research were collected from Haian  and maintained in the incubator at 26 ± 1°C with 70%–80% humidity and 16 h light: 8 h dark photoperiod. The RBSDV-infected rice plants with typical dwarf symptom were collected for L. striatellus to acquire virus.The non-viruliferous populations of L. striatellus were reared on RBSDV-infected rice plants for 2 days and then transferred to healthy rice seedlings for another 2 days. Finally, the surviving nymphs of L. striatellus were collected as RBSDV-infected L. striatellus. The non-viruliferous 3rd-instar nymphs of L. striatellus were reared on healthy rice seedlings for 4 days, and the surviving nymphs of L. striatellus were collected as virus-free L. striatellus.The non-viruliferous 3rd-instar nymphs of L. striatellus nymphs for RNA-Seq analysis. For midgut dissection, the nymphs were rinsed with 75% ethanol and then washed three times using sterilized-deionized water. The midgut tissue was dissected in chilled 1× phosphate-buffered saline  with sterile forceps under a stereomicroscope. Each sample contained three independent biological repetitions.RBSDV-free (VF) and RBSDV-infected (RB) midguts were collected from 200 non-viruliferous or RBSDV-infected L. striatellus midgut was extracted using TRIzol reagent  according to the manufacturer’s instructions. The quantity and quality of total RNA was determined by spectrophotometry  and agarose gel electrophoresis, respectively.Total RNA of Ribosomal RNA in the total RNAs was eliminated by Epicentre Ribo-Zero Gold Kit . Thereafter, the cDNA libraries were constructed by the mRNA-Seq sample preparation kit using approximately 10 μg of RNA . Sequencing was carried out using the Illumina HiSeq 4000 platform, and the whole transcriptome was performed by Lianchuan Biotechnology .1. Bowtie2, Tophat2, and tophat-fusion were used to map sequences to the genome of L. striatellus (GenBank No.: GCA_003335185.2)  and Kyoto Encyclopedia of Genes and Genomes database (KEGG3) were performed to analyze the potential functions of circRNAs  . The map35185.2) . The unm35185.2) . The exp package . Gene OncircRNAs . The miRTM RT reagent kit with gDNA Eraser . SYBR PrimeScriptTM RT-PCR Kit  and IQTM5 multicolor real-time PCR detection system  were used and each experiment included three independent technical and biological replications. RT-qPCR was carried out for 40 cycles  after an initial denaturation step (94°C for 30 s). RPL5 (encoding ribosome protein L5) was used as an internal reference gene  was injected into the conjunction between the prothorax and mesothorax via capillary on FemtoJet  after being anesthetized with carbon dioxide. The dsRNA of enhanced green fluorescent protein (EGFP) was injected as a control. Each experiment was carried out with 30 nymphs and three independent biological repetitions.The T7 high yield transcription kit  was used to synthesize double-stranded RNA (dsRNA). The RBSDV-free 3rd-instar nymphs of L. striatellus were reared on RBSDV-infected rice plants for 2 days. Then, the nymphs were transferred to healthy rice seedlings for another 2 days, and the RBSDV accumulation was determined by detecting the gene and protein expression of P10 using RT-qPCR and immunofluorescence, respectively.The RNAi efficiency of circRNA2030 was determined by calculating its relative expression at 1, 3, and 5 days after injection of circRNA2030 dsRNA. Two days after microinjection, the nymphs of The midgut tissue was dissected in chilled 1× PBS under a stereomicroscope and fixed with 4% paraformaldehyde for 1 h. Then, the midguts were permeabilized with 2% Triton X-100 for 30 min and blocked with 3% BSA for 2 h. The primary antibody (anti-RBSDV P10 mouse mAb conjugated with FITC) was incubated with the midguts at 4°C overnight. Then, the midguts were incubated with TRITC phalloidin  for 10 min. An LSM 710  was used to view the confocal imaging.p-value < 0.05 and p-value < 0.01 were regarded as significant and very significant differences, respectively.SPSS 20.0  was used to perform the statistical analysis. One-way analysis of variance (ANOVA) with least significant difference (LSD) test was used in comparing the gene expression levels. The L. striatellus midguts and analyzed by RNA-Seq. The sequence was deposited in the National Center for Biotechnology Information (NCBI) with accession number GSE153102. A total of 2,523 circRNAs were identified (Six libraries were constructed from RBSDV-free (VF) and RBSDV-infected (RB) entified . In thisentified . Source entified . Due to L. striatellus midguts, including eight up-regulated and five down-regulated circRNAs  or its parental gene (PTA) were investigated. In general, if circRNA2030 could act as miRNAs sponges, the expression levels of the adsorbed miRNAs should be up-regulated when circRNA2030 was knocked down. However, none of the six related miRNAs was up-regulated after knockdown of circRNA2030 . In the Caenorhabditis elegans, and plants  can be found in the article/ JZ and QX conceived and designed the experiments. JZ, HW, WW, YD, and MW performed the experiments. JZ, HW, DY, and QX analyzed the data. JZ, HW, YZ, and QX wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Eucommia ulmoides Oliv. polysaccharides (EUPS), and induce an efficient humoral and cellular immune response against an antigen, we encapsulated the OVA and EUPS in long-circling nanoliposomes and conjugated it with anti-DEC-205 receptor antibody to obtain a DEC-205-targeted nanoliposomes (anti-DEC-205-EUPS-OVA-LPSM). The physicochemical properties and immune-modulating effects were investigated in vitro and in vivo by a series of the experiment to evaluate the targeting efficiency of anti-DEC-205-EUPS-OVA-LPSM. In vitro, anti-DEC-205-EUPS-OVA-LPSM (160 μg mL−1) could enhance DCs proliferation and increase their phagocytic efficiency. In vivo anti-DEC-205-EUPS-OVA-LPSM remarkably promoted the OVA-specific IgG and IgG isotypes levels, enhanced the splenocyte proliferation, and induced the NK cell and CTL cytotoxicity. Besides, the anti-DEC-205-EUPS-OVA-LPSM enhanced the maturation of DCs. These findings suggest that the DEC-205 receptor antibody-conjugated EUPS nanoliposome can act as an efficient antigen delivery system to enhance the cellular and humoral immune response by promoting DC maturation. This indicates that the anti-DEC-205-EUPS-OVA-LPSM has significant potential as an immune-enhancing agent and antigen delivery system.DEC-205 receptor-mediated dendritic cells (DC) targeting nanoliposomes is a promising delivery system in eliciting an immune response against pathogens. When this delivery system carries both antigen and immunomodulator, it can effectively regulate the DC function as well as the initial T cell response. To maximize the desired therapeutic effects of  Vaccination remains the most effective means to combat infectious diseases. Vaccination regimes are strictly followed in the farms; even then, in most of the cases, animals do not produce the desired immune response due to which most of the infections could not be completely controlled Monath, . ApplicaEucommia ulmoides Oliv. (EU) is Chinese traditional medicine with multiple pharmacological activities. In our previous study, we have found that the polysaccharides extracted from E. ulmoides Oliv. (EUPS) regulated DCs maturation and served as an immune-enhancer; besides, it activated the DCs, and promoted the antigen-specific humoral and cellular response  as an antigen-presenting cell (APC), can integrate innate and adaptive immune responses. The bone marrow hematopoietic progenitors derived immature DCs circulate in the blood and can be spotted in tissues as sentinel cells, and recognize antigen or take up the pathogen from the extracellular environment by employing the receptor-mediated endocytosis, pinocytosis, and micropinocytosis  were prepared by membrane dispersion method. The anti-DEC-205-EUPS-OVA-LPSM was prepared by conjugating the DEC-205 antibody to the EUPS-OVA-LPSM nanoliposome. The physicochemical property and the targeting effect of the anti-DEC-205-EUPS-OVA-LPSM nanoliposome were investigated by determining their effect on the humoral and cellular immune response in vivo. We found that the DEC-205 antibody conjugated nanoliposome could significantly enhance the cellular and humoral immune responses against OVA in the mice.We encapsulated the antigen and EUPS in nanoliposomes, and designed a DEC-205-targeted nanoliposomes to dendritic cells (DCs) to maximize the desired therapeutic effects of EUPS, and induce an efficient humoral and cellular immune response in response to antigen. In the present study, 2.2.1.The phospholipid, cholesterol, distearoyl phosphoethanolamine-PEG2000 (DSPE-PEG2000PDP), DSPC, SPDP crosslinker, FITC-dextran, concanavalin A (ConA), and lipopolysaccharide (LPS) were procured from Sigma Chemical Co. . Purified EUPS with a purity of ≥97% was procured from Tianqi Biotechnology Co., Ltd. . A standard high-performance liquid chromatography-refractive index detection method was utilized to purify the polysaccharide preparation (≥97% purity). Rat anti-mouse DEC-205 monoclonal antibody was purchased from Thermo Fisher Scientific Co., Ltd. . RPMI-1640, fetal bovine serum, were procured from Life Technologies Corporation . Cell Counting Kit-8 (CCK-8) was obtained from the Beyotime Institute of Biotechnology . Fluorescently-labeled anti-mouse monoclonal antibodies  were purchased from eBioscience .2.2.2.2.1.The mixture of DSPC, cholesterol, distearoyl phosphoethanolamine-PEG (2000) amine and DSPE-PEG (2000) PDP (molar ratio 45:30:2:0.4) was dissolved in chloroform was added to a small beaker. To fully dissolve the mixture, it was shaken for 15 min by utilizing the ultrasonic instrument propionate (SPDP) was employed to cross-link the DEC-205 antibody and liposomes , and the images were captured.2.2.4.Laser particle size analyzer  evaluated the particle size, zeta potential, and the colloidal dispersion system stability of the nanoliposomes.2.2.5.A ∞ (drug leakage amount at an infinite time). The fluorescence value of the samples at each time point was recorded as At (FITC dextran leakage of the samples at time t). The leakage rate of liposomes was calculated with the following equation: Leakage rate (%) = At/A ∞.The dialysis membrane method determined the leakage rate of liposome  of anti-DEC-205-EUPS-OVA-LPSM solution were added to the wells, and cultured for a further 24 h. Next, 10 μL of Cell Counting Kit-8 (CCK-8) reagent was added to each well and the OD was measured using a microplate reader at 450 nm after 4 h.The DCs single cells solution was prepared according to our previous report .2.3.3.The DCs were inoculated in a 6-well cell-culture plate with a round coverslip. After 24 h of culture, FITC-OVA, anti-DEC-205-EUPS-OVA-LPSM was added, and cultured at 37 °C for 12 h, then all supernatant was absorbed and discarded, PBS was washed three times, and 100 μL SDS cell lysate was added into each well to digest the cells. The fluorescence intensity  was measured by a fluorescent enzyme labeling instruments.2.3.4.6 cells/mL) were seeded to a 96-well plate, and anti-DEC-205-EUPS-OVA-LPSM (160 μg/mL) was added, and the plates were incubated for 72 h. the supernatant was harvested and the concentration of IL-12 was detected by ELISA kit  according to the manufacturer’s instructions.The single DCs suspension  were provided by Sichuan Laboratory Animal Center, (China). All animal procedures were performed as per internationally accepted principles, mentioned in the government of China issued Guidelines for Keeping Experimental Animals and approved by the IACUC, Southwest University. Eight groups containing ten female ICR mice in each group were formed by the random division. These eight groups were anti-DEC-205-EUPS-OVA nanoparticles group, naive group, EUPS group, OVA group, OVA plus EUPS group, blank LPSM group, OVA-EUPS-nanoparticles group, and OVA plus alum group. Mice were immunized on day 0 and 14 by administering the following vaccine formulations subcutaneously in the hind limb: 200 μL anti-DEC-205-EUPS-OVA nanoparticles , 200 μL saline, 0.1 mg EUPS alone, 200 μL OVA (50 μg), 200 μL OVA (50 μg) plus100 μg EUPS, 0.5 mg blank LPSM, 0.5 mg OVA-EUPS-nanoparticles , 200 μL OVA (OVA 50 μg) plus 200 μg alum.2.4.2.2SO4 supplementation. The optical density (OD) was measured at 450 nm by an ELISA reader .Serum samples were taken 2 weeks post-second booster dose to evaluate the OVA-specific IgG, IgG1, IgG2b, and IgG2a levels. Indirect ELISA was employed to measure the specific IgG titer and isotype levels, as described previously  levels were estimated in the serum utilizing the ELISA kits as per the manufacturer’s instructions.2.4.5.in vivo CTL assay was conducted as per our previous protocol . After 4 h, the mice spleen was removed, and the splenocytes were isolated and analyzed by flow cytometry . Specific lysis was determined by employing the following equation: Lysis ratio = Percentage CFSElow/Percentage CFSEhigh, Percentage specific lysis = [1 − (Unprimed ratio/Primed ratio) × 100].An 2.4.6.NK cell cytotoxicity was assessed as described previously  were stained with either anti-CD3-FITC, anti-CD8-APC, or anti-CD4-PE antibodies (10 μL) at 4 °C for 1 h. After washing twice, the cells were fixed by 1% PFA  and evaluated with a FACS Calibur (BD Biosciences). The proportion of CD4+, CD3+, and CD8+ T lymphocytes in the mice peripheral blood were examined by using the Cell Quest Pro software (BD Biosciences). The outcomes are demonstrated as the of CD4+ (%) and CD8+ (%).Peripheral blood samples or single-cell splenocyte suspensions were prepared as described previously . For multiple comparisons between groups, ANOVA with Bonferroni post-hoc test was used. Results are expressed as the mean ± standard deviation (SD) of the mean. The values with 3.3.1.3.1.1.The TEM findings indicated the morphological similarity of the anti-DEC-205-OVA-EUPS-LPSM, where both the liposomal entities appeared as the spherical or nearly spherical structure with the double membrane . The LPS3.1.2.p < .05, The dialysis membrane method was used to determine the leakage rate of liposomes . Therefore, a concentration of 160 μg mL−1 was selected for subsequent experiments.The 3.2.2.In order to explore the ability of DCs to take up anti-DEC-205-EUPS-OVA-LPSM, the phagocytic effect of DCs on antigens was observed using laser confocal scanning microscopy. Different fluorescence intensities indicate the uptake of different antigens by DCs. It can be seen in 3.2.3.p < .05). In the present study, the fluorescence intensity in the anti-DEC-205-EUPS-OVA-LPSM group was significantly higher than that of the EUPS-OVA-LPSM group (p < .05). Indicating that the phagocytic effect of anti-DEC-205-EUPS-OVA-LPSM group was significantly higher than that of EUPS-OVA-LPSM group. The anti-DEC-205 antibody conjugated nanoliposome can improve phagocytosis efficiency than those of nanoliposome. The anti-DEC-205 antibody conjugated nanoliposome can make the nanoliposome have active targeting and significantly improve the phagocytosis rate of the nanoliposome.As shown in 3.2.4.p < .05). The data suggested that anti-DEC-205-EUPS-OVA-LPSM can significantly stimulate DCs to secret the IL-12.As shown in 3.3.3.3.1.ELISA evaluated the effect of the anti-DEC-205-EUPS-OVA nanoparticles on the humoral immune response in mice. The blood sample of the ICR mice was collected 14 days post-second booster. The serum was separated and stored in a refrigerator at −20 °C to evaluate the antibody levels. The outcomes indicated that the antibody level of the anti-DEC-205-EUPS-OVA nanoparticles group was significantly higher than the naive, EUPS, OVA, OVA-EUPS, blank LPSM, OVA-EUPS-nanoparticles, and OVA-alum groups . It sugg3.3.2.p < .05). In the present study, the anti-DEC-205-EUPS-OVA-LPSM nanoparticles, as a novel delivery system, demonstrated a significant ability of splenocyte proliferation as compared to the control group.As depicted in 3.3.3.+ antigen serves as a T-lymphocytes biomarker. T lymphocytes can be categorized as T helper lymphocytes and inhibitor lymphocytes  in mice. We found that the killing percentage of NK cells in mice in the anti-DEC-205-EUPS-OVA-LPSM nanoparticles group and the OVA-EUPS-LPSM group was 57.8% and 52.8%, respectively. The killing percentages in the naive, EUPS, OVA, OVA-EUPS, LPSM, and OVA-alum groups were 7.21, 33.6, 25.5, 43.7, 7.55, and 27.6%, respectively. These findings showed that the killing activity of NK cells in the anti-DEC-205-EUPS-OVA-LPSM nanoparticles immunized mice was significantly enhanced as compared to the other groups. Also, the NK cells killing activity in the OVA-EUPS-LPSM group was substantially greater than the naive, OVA-EUPS, EUPS, OVA, LPSM, and OVA-alum groups. The Eucommia polysaccharide nanoparticles, coupled with the anti-DEC-205 antibody, showed significantly higher NK cells killing activity in mice immunized with activity .3.3.7.The MHC II, CD40+, CD86+, and CD80+ are the crucial bio-markers for the maturated DCs, and the up-regulation of these molecules indicates the maturity level of DCs. The MHC II, CD40+, CD86+, CD80+ expression levels were determined on day 3 after the first immunization to investigate the effect of anti-DEC-205-EUPS-OVA-LPSM nanoparticles on the DCs maturation in mice. The MHC II, CD40+, CD80+, CD86+ expression level in the ?? nanoparticles group was found to be substantially higher than the other groups . The exp4.p < .05), which was related to the increase of membrane fluidity and the decrease of the stability of liposome coalescence. The antibody on the surface of immunoliposome may also increase the interaction between liposomes and reduce the stability of the liposome membrane , in vitro. The outcomes demonstrated that the antibody conjugated DEC-205 enhanced the immature and mature MoDCcounts. Besides, CD1c+ BDC phagocytosing anti-hDEC-205 iLPSM and anti-hDEC-205 iLPSM were phagocytosed and made available for antigen processing by DC. It demonstrated that DEC-205 is an effective target receptor for delivering liposomes to human DCs  expression level in the anti-DEC-205-EUPS-OVA-LPSM nanoliposomes group was significantly higher than the OVA-EUPS-LPSM, OVA-EUPS, OVA-alum, and OVA groups. This suggested that the DEC-205 receptor-targeted nanoliposomes significantly facilitated the DC maturation. These findings revealed that the anti-DEC-205-EUPS-OVA-LPSM promoted the immune response by targeting the DEC-205 receptor, which in turn increased the polysaccharide and OVA delivery efficiency.DEC-205 receptor, also known as CD205, is a type I membrane protein and belongs to the C-type lectin superfamily. It has a relative molecular weight of 205 kD and 10 sugar recognition domains  can serve as an efficacious immune adjuvant to enhance immune responses. Their data showed that the liposomes containing OVA and LBP (LBPL-OVA) could effectively enhance the antigen specificity and further promote antibody production as well as CD4+ and CD8+ T cell proliferation in vivo. These results revealed that LBP (LBPL-OVA) elicited a significant humoral and cellular immune response , and its effect on the RGPL-induced immune response was evaluated. The outcome of this study demonstrated that the RGPL up-regulated the splenic lymphocyte proliferation level and OVA-specific IgG titers of ovalbumin-RGPL-vaccinated mice. It also increased the expression level of MHC II+ CD11c+ and CD86+ CD11c+ cells in the draining lymph nodes, central memory cells (TCM), and effector memory cells (TEM). RGPL could facilitate sufficient antigen exposure in lymph nodes. It suggests that RGPL could enhance dendritic cell maturation and functions , MHC II, and OVA-specific humoral and cellular immune responses.Certain natural polymers, which are used to design DC targeted nanoparticles, have strong immunoenhancement properties along with good safety and low toxicity . CTLs can identify and eliminate pathogen-infected cells. NK cells primarily participate in the innate immune response, and the NK cells mediated eradication of pathogens and tumor cells are nonspecific. They exercise cell-mediated cytotoxicity against tumor cells and infected cells and play a regulatory role by cytokines and chemokines secretion. The sinusoidal regions of the liver and the red pulp of the spleen under resting conditions are enriched with NK cells  are two primary lymphocytes (Schwarz et al., 5.In the current study, the anti-DEC-205-EUPS-OVA-LPSM nanoparticles significantly induced the cellular and humoral immune responses by facilitating the DC maturation. This study suggests that the application of the DEC-205 receptor-targeted nanoliposomes can increase the delivery efficiency of the conventional liposomes’ dendritic cell-targeted drugs and antigens. The targeted nanoparticles can further improve the efficiency of drug delivery and antigen presentation by passive targeting of conventional liposomes. This study has created a platform for future research studies on targeted drug and antigen delivery and the prolongation of their efficacy.