Emergency medical service (EMS) providers and other health care professionals.In 2019 alone, 656,000 children in the United States were victims of child abuse and neglect.By the end of the escape room, the learner should be able to: 1) understand the national and local prevalence of child maltreatment; 2) understand the different types of child maltreatment and common associated presentations; 3) know the local EMS agency reporting requirements; 4) understand when to make base hospital contact with respect to concern for maltreatment; 5) collaborate effectively as a team.Child maltreatment can be a sensitive and challenging topic. In this class, we presented learners with a short, 15-minute lecture (see Pre-Escape Room Lecture PowerPoint) followed by an escape room activity. The Pre-Escape Room Lecture PowerPoint includes suggestions on the type of image and/or statistics to include on each slide, which can be taken from your site’s available de-identified photos and information. The lecture included material describing national and local statistics on child maltreatment, definitions of abuse, and techniques to help identify concern for maltreatment. Learners were free to ask questions following lecture. They were then divided into their assigned crews/teams for the escape room activity. The puzzles in the escape room served to reinforce concepts and details presented in lecture. We held a debrief after the escape room activity to discuss puzzle answers and address any follow-up questions.Learners completed a program evaluation after the activity. These questions assessed the learners’ perception of the importance and applicability of the content presented, the escape room format, and what they felt was the most significant and helpful to their practice.Learners reported enjoying the activity and felt the escape room-based approach allowed for deeper engagement with the topic since the serious nature of child maltreatment can sometimes make this difficult.Pediatric abuse and neglect is a serious and often heavy topic to present to healthcare providers. While we took into consideration that presenting a sensitive topic such as child abuse in an escape room format may be perceived as insensitive or display a lack of insight or respect for the topic, we also understood that the way we built out the clues and puzzles would be important in how the game was perceived by the participants. By building the puzzles to be factual and not overly excessive, we allowed the learners to interact with the information and practice identifying possible cases of abuse and how and when to report suspicions in a manner that did not trivialize the seriousness of the topic or take away from the fact that they were competing in a game. We used a PowerPoint lecture to present the foundation of the content and then lightened the learning session with the use of the escape room activity. The level of competition and comradery lightened the overall mood, and the learners left the class on a high note.Child abuse recognition, escape room activity, small-group activity, prehospital, neglect, physical abuse, emotional abuse, sexual abuse, mandated reporter. Learner Audience:EMT and ParamedicTime Required for Implementation:Lecture preparation: Adapting the Pre-Escape Room Lecture PowerPoint will depend on your access to images and case studies from your site. We suggest reaching out to your child abuse specialists, utilizing journal articles, and an internet search for such images recommended in the PowerPoint. Supply preparation: Initially our team borrowed boxes and locks from our educational department. We then purchased our own boxes, locks, UV lights, blank puzzle kit, and invisible ink pens from Amazon which shipped within the week. We utilized the emergency department laminator to laminate some of our clues. The initial build of the escape room puzzles took approximately one day of preparation, and we created two identical escape rooms to run simultaneously. The two escape rooms took between 30–45 minutes to set up on the day of the session.The lecture prior to the escape room was 15 minutes in duration. The run time of the escape room is approximately 20 minutes. The debrief takes approximately 10 minutes.Recommended Number of Learners per Instructor:One instructor per room. The instructor should have basic knowledge of prehospital county-specific policy with regards to online medical control (consultation between EMS and a physician to guide care) access and child abuse policies. The maximum number of learners in the escape room should not be more than six. A typical fire-based EMS crew is four members with potentially two ambulance providers.Topics:Child abuse recognition, escape room activity, small-group activity, prehospital, neglect, physical abuse, emotional abuse, sexual abuse, mandated reporter.Objectives:By the end of the escape room the learner should be able to:Understand the national and local prevalence of child maltreatment.Understand the different types of child maltreatment and common associated presentations.Know the local EMS agency reporting requirements.Understand when to make base hospital contact with respect to concern for maltreatment.Collaborate effectively as a team.Puzzle number 1: Kid Doe’s Suspected Child Abuse Report. This puzzle exposes the learner to the documentation submitted when reporting suspected abuse to Child Protective Services (CPS). This puzzle meets objectives 3 and 5. Puzzle number 2: Child Abuse Stats and the Hidden Answers. This puzzle helps the learner understand the scope of the problem by reviewing statistics on child abuse. This puzzle meets objectives 1 and 5  and 2. PThe local EMS Agency policy on managing pediatric abuse cases and when online medical control is required. Our recommendation for instructors is also to be familiar with all content included in the supplemental PowerPoint presentations prior to giving the presentation and conducting the escape room activity.The learners are mandated child abuse reporters and should have a basic understanding of abuse recognition and reporting.The escape room puzzles are placed on a desk in the room. The child abuse statistics are individually printed and posted on the walls around the room. We created two duplicate escape rooms to run simultaneously.If budget is a factor, and purchasing locks and boxes are not an option, the box and lock items on this list can be exchanged for envelopes that the escape room facilitator can provide to the learners as they solve the clues. We also recommend laminating the paper clues the learners will interact with to improve the longevity of the game pieces.https://www.amazon.com/Plano-131252-Storage-Emergency-Marine/dp/B009YSFT7S/ref=pd_lpo_1?pd_rd_i=B08PL8248S&psc=1Dry storage locking box. We used the Plano 131252 Dry Storage Emergency Marine Box. At: https://www.amazon.com/Puroma-Combination-Padlock-Toolbox-Storage/dp/B075DFPR2W/ref=sr_1_1?crid=1PRQXNL0I54LA&dchild=1&keywords=puroma%2B2%2Bpack%2Bcombination%2Block%2B4%2Bdigit%2Bpadlock&qid=1635194958&sprefix=puroma%2B2%2Bpack%2B%2Caps%2C114&sr=8-1&th=14-digit lock. We used Puroma 2 pack combination lock. At: https://www.amazon.com/Juvale-Wooden-Boxes-Hinged-Lid-Unfinished/dp/B07C2K4G4P/ref=sr_1_5?crid=1C5YZ9AE0OEEH&dchild=1&keywords=juvale+unfinished+boxes&qid=1635195100&sprefix=juvale+unfinished+boxes%2Caps%2C118&sr=8-5Unfinished wooden boxes. We used Juvale boxes. At: https://www.amazon.com/Ceiling-Pieces-Screw-Hanging-Decorations/dp/B081T1Z8C3/ref=sr_1_3?crid=133BVKAFL9E1B&dchild=1&keywords=mini%2Bceiling%2Bscrew%2Bhooks&qid=1635195281&sprefix=mini%2Bceiling%2Bscew%2Bhooks%2Caps%2C122&sr=8-3&th=1Pack of mini screw hooks – we used these to be able to attach locks to the wooden boxes. At: https://www.amazon.com/Eilin-Combination-School%E3%80%81Home%E3%80%81Office%E3%80%81Storage-Lockers%E3%80%81Gym-Lockers%E3%80%81Drawers%E3%80%81Cabinets%E3%80%81Toolboxes%E3%80%81Luggage/dp/B07L6XGWC4/ref=sr_1_16?crid=OIBQHZT25BGR&dchild=1&keywords=3-digit+combination+lock+pack&qid=1635195417&sprefix=3-digit+combination+lock+packs%2Caps%2C135&sr=8-163-digit lock. Any lock brand will work for the escape room. At: https://www.amazon.com/Vaultz-Locking-Supply-Inches-VZ03708/dp/B015MPUBEM/ref=sr_1_26?crid=1OBX0MAUWMH12&dchild=1&keywords=kids%2Block%2Bbox%2Bwith%2Bkey&qid=1635195603&sprefix=kids%2Block%2Bbox%2Caps%2C150&sr=8-26&th=1Lock box with key. Any brand will work for the escape room. At: https://www.amazon.com/dp/B00I7DUEQ2/ref=redir_mobile_desktop?_encoding=UTF8&aaxitk=eab7ecdcfd63f528fc919a1674258132&hsa_cr_id=9121159900501&pd_rd_plhdr=t&pd_rd_r=2ba60569-ac99-4dc3-90f7-82b0902fb590&pd_rd_w=SxAO7&pd_rd_wg=gY6Lx&ref_=sbx_be_s_sparkle_mcd_asin_1_img&th=1Blank puzzle pieces. We used Hygloss 12-piece puzzles. At: https://www.amazon.com/STENDA-Invisible-Blacklight-Christmas-Thanksgiving/dp/B08XW8L2NX/ref=sr_1_4?crid=2UHCL09IJ1TV6&dchild=1&keywords=invisible+ink+pens+with+uv+light+for+kids&qid=1635195889&sprefix=ivisible+ink+pens%2Caps%2C124&sr=8-4Invisible ink pens with UV light. Any brand will work for the escape room. At: https://www.amazon.com/Play-Doh-Party-Dough-assortedcolors/dp/B01J7WB866/ref=sr_1_23?crid=1XVVRP43V7BLP&dchild=1&keywords=small%2Bplaydough%2Btubs&qid=1635196035&sprefix=small%2Bplay%2Bough%2Btubs%2Caps%2C123&sr=8-23&th=1Play-doh assorted colors. We used the party pack containing 12 different colors. At: https://frugalfun4boys.com/code-activity-kids-make-spy-decoder/Decoder wheel template. At: https://www.amazon.com/Stainless-Cutting-Carving-Scrapbooking-Creation/dp/B0899VKWMB/ref=asc_df_B0899VKWMB/?tag=hyprod-20&linkCode=df0&hvadid=475811287390&hvpos=&hvnetw=g&hvrand=17695763987983061778&hvpone=&hvptwo=&hvqmt=&hvdev=c&hvdvcmdl=&hvlocint=&hvlocphy=9031600&hvtargid=pla-1027999032707&th=1Hobby knife. Any brand will work to create the cutouts for the first puzzle. At: Letter-sized card stock colored paper, three sheets.Two letter-sized envelopes.Printer to print clues. Scotch tape. Laminator with pouches.This escape room was administered to 130 EMTs and paramedics in 12 sessions. Each learner was given an evaluation form to complete at the end of the class. Learners were asked to rate three questions using the following scale:1 = should improve2 = satisfactory3 = average4= excellentQuestion 1: Information presented was important and pertinent to my practice: 95% of learners rated this as excellent (4). Less than 1% of learners rated this question as average (3) and less than 1% rated this question as satisfactory (2). Question 2: Forum provided good information and communication: 95% of learners rated this as excellent (4) and less than 1% rated this question as satisfactory (2). Question 3: Program met stated objectives: 95% of learners rated this as excellent (4) and less than 1% rated this question as satisfactory (2). Learners were able to write in comments and 48 of them felt that the information presented on recognizing child abuse was the most significant item in the class. Thirty-one participants commented “great class” and that the escape room was fun. One of the learners appreciated the team-building aspect of the escape room.Reference tool used to summarize important information covered in the escape room activity.


This interactive and entirely virtual escape room based on high yield gastrointestinal/abdominal board review material is a didactic activity for emergency medicine residents of all postgraduate years and third- or fourth-year medical students. This escape room can be completed in small teams or as individuals in a remote, in-person, or mixed location setting.2020 is a year that will forever change medical education. The novel coronavirus 2019 pandemic caused many national, state, local, or hospital specific recommendations for transition of meetings to be completely virtual or to only allow for limited in-person meetings. In order to abide by these recommendations, the majority of medical education transitioned to online platforms. Now more than ever, creative and engaging methods for expanding clinical knowledge and teaching teamwork as well as unique integrations of technology for medical education delivery are needed.By the end of this didactic activity, learners will be able to:Identify causes of upper gastrointestinal bleeding.Recall test-taking buzzwords for infectious causes of diarrhea.Acknowledge the correct hepatitis B titers that correspond with various clinical scenarios.Describe the management for alkali caustic ingestions.Determine the components of Maddrey Discriminant Function Score, Charcot’s triad, Ranson’s Criteria for Pancreatitis, and Glasgow-Blatchford Score.Diagnose specific gastrointestinal diseases from a clinical description.Choose the correct gastrointestinal diagnosis based on clinical image findings.Demonstrate teamwork in solving problems.“Gamification” is the use of game elements in a nongame context.The interactive virtual escape room provided is a no cost, unique alternative educational activity that can be done either entirely or partly remotely. In order to replicate this team-based didactic exercise, the instructor needs to simply divide residents and students into small groups and then share with each group the link provided.A five-point Likert scale survey was distributed to participating learners after completing this activity to assess relevance and satisfaction of the high yield gastrointestinal/abdominal board review escape room. Thirty-two of forty-two participating residents/medical students completed this seven-question survey.All participating resident/medical students who completed the survey enjoyed this activity and that this was a unique learning experience, and the majority of participating residents/medical students thought this interactive virtual escape room was easy to use, learned something, thought this was a helpful way to review for emergency medicine boards or in-training exams, and preferred a game like this to a standard lecture. Additionally, over half of survey respondents said they are going to try to come up with a game for their assigned board review presentations.Escape room gamification of high yield gastrointestinal/abdominal board review material was an engaging, fun, and effective distance learning activity for residents and medical students. This didactic activity not only promoted interactive learning but also encouraged virtual teamwork among small groups.Gastroenterology, GI, abdominal, board review, in-training exam review, escape room, virtual didactics, distance learning, high yield, upper GI bleeding, esophageal varices, diarrhea, alkali ingestion, volvulus, foreign body, intussusception. Learner Audience:Medical Students, Interns, Junior Residents, Senior ResidentsTime Required for Implementation:50 Minutes total: 10 minutes to explain this virtual interactive escape room and divide into small teams, 40 minutes for learners to complete the escape room.Recommended Number of Learners per Instructor:It is recommended that learners are pre-sorted into groups of 4–6 residents of various post-graduate years and 1–2 medical students per team so that teams are small enough that everyone can participate. One instructor could provide requested support to all groups simultaneously, or there can be one facilitator assigned to each small group in case the team gets stuck on a particular clue.Topics:Gastroenterology, GI; abdominal, board review, in-training exam review, escape room, virtual didactics, distance learning, high yield, upper GI bleeding, esophageal varices, diarrhea, alkali ingestion, volvulus, foreign body, intussusception.Objectives:By the end of this didactic activity, learners will be able to:Identify causes of upper gastrointestinal bleeding.Recall test-taking buzzwords for infectious causes of diarrhea.Acknowledge the correct hepatitis B titers that correspond with various clinical scenarios.Describe the management for alkali caustic ingestions.Determine the components of Maddrey Discriminant Function Score, Charcot’s triad, Ranson’s Criteria for Pancreatitis, and Glasgow-Blatchford Score.Diagnose specific gastrointestinal diseases from a clinical description.Choose the correct gastrointestinal diagnosis based on clinical image findings.Gamification and technology-enhanced active learning for medical education are the two main conceptual frameworks utilized in creating this interactive and virtual escape room based on high yield gastrointestinal/abdominal board review material.7The Clue Master Document, supplemental file, details how the escape room works and where clues are located in the escape room and the answers to these clues. It is also recommended that the facilitator trial the escape room link themselves (available at bit.ly/giescaperoom) to familiarize themselves with where the patient information, six clues, and final clues are linked within the virtual escape room , 2. Plea5https://bit.ly/giescaperoomThis interactive virtual escape room was trialed on individual residents and faculty members prior to using this didactic exercise on forty-two total residents and medical students during a weekly didactic conference. There were thirty-five residents and seven medical students that participated in this virtual escape room, and thirty-two of these forty-two total residents/medical students completed the post escape room survey. This post escape room survey consisted of a five-point Likert scale to answer seven questions to assess relevance and satisfaction of the high yield gastrointestinal/abdominal board review escape room. The Likert scale options included “very much,” “a lot,” “no opinion,” “not really,” and “this was lame” as options and in this order represented strongly agree to strongly disagree .Of the thirty-two survey respondents, 100% said they “very much” enjoyed this activity and that this was a unique learning experience. Twenty-eight of thirty-two, or 87.5%, said they “very much” thought this interactive virtual escape room was easy to use. Twenty-four of thirty-two, or 75%, “very much” learned something, thought this was a helpful way to review for emergency medicine boards or in-training exams, and preferred a game like this to a standard lecture. Nineteen of thirty-two, 59.4% of survey respondents said they are “very much” going to try to come up with a game for their assigned board review presentations.In terms of tips for successful utilization of this interactive virtual escape room, participating residents/medical students should have access to a laptop or desktop. Please note that although this escape room could be used on a tablet or phone, it is optimal and easier to use this virtual escape room on a laptop or desktop because each clue that is clicked will open a new tab and you will need to click back to the escape room tab after completing clues. It took all of the individual or group participants 30–35 minutes to complete so it is recommended to allow for 40 minutes to ensure completion by all participants. It is also recommended to allow for 10 minutes before starting the escape room to explain this didactic activity. It should be explained that participants will be divided into small teams via Zoom break out rooms , a link will be provided which will be a photo of a resuscitation bay that has links embedded in certain aspects of the photo that the teams will have to click on to find patient information, six clues, and a final clue that uses information provided from solving the previous six clues to successfully “escape the room.” The easiest way for the small group break outs to work together is to designate one person of the group to share their screen and be the clicker/typer as the team works their way through the escape room clues –7. Of noThe facilitator should in advance divide participants into groups of 4–6 residents of various post-graduate years and 1–2 medical students per team so that teams are small enough that everyone can participate, and the teams are roughly even in terms of medical knowledge. This didactic activity can be performed completely virtually or in-person or as a mix of in-person and virtual settings. Participants will need access to a laptop/computer regardless of setting to be able to utilize the escape room and video conference to work as part of a team.Upon completion of the escape room, participants who escape successfully by solving all clues, including the final clue, are rewarded with a high yield gastrointestinal study outline compiled from Dr. Carol Rivers' Written Board Review resources via Ohio ACEP app, “The Ultimate Emergency Medicine Guide: The only EM book you need to succeed,” by Dr. Sajid Khan, Rosh Review Question Bank, and Hippo EM Board Review Videos. This study guide should be emailed along with the link for the escape room  to all participants so they can review the study guide outline and go through the escape room individually if desired. Participants were also encouraged to take group photographs/screenshots after successfully “escaping” as you do for a real Escape Room.Learning points of this didactic activity are to review a high yield gastrointestinal/abdominal information. All participants should receive a link to a shared Google Document that is a comprehensive GI study outline compiled from Dr. Carol Rivers' Written Board Review resources via Ohio ACEP app, “The Ultimate Emergency Medicine Guide: The only EM book you need to succeed,” by Dr. Sajid Khan, Rosh Review Question Bank, and Hippo EM Board Review Videos.


Scientific literature is published at a rate that makes manual data extraction a highly time-consuming task. The Comprehensive Antibiotic Resistance Database (CARD) utilizes literature to curate information on antimicrobial resistance genes and to enable time-efficient triage of publications we have developed a classification algorithm for identifying publications describing first reports of new resistance genes. Trained on publications contained in the CARD, CARD*Shark downloads, processes and identifies publications recently added to PubMed that should be reviewed by biocurators. With CARD*Shark, we can minimize the monthly scope of articles a biocurator reviews from hundreds of articles to a few dozen, drastically improving the speed of curation while ensuring no relevant publications are overlooked.Database URL http://card.mcmaster.ca Antimicrobial resistance (AMR) is a health-care crisis estimated to cause the death of 10 million individuals and cost $100 trillion worldwide by 2050 , 4. WithUnderstanding the mechanisms underlying AMR is of great importance for the targeted treatment of bacterial infections and for community risk assessment. With an understanding of the suite of AMR genes (ARGs) found in a pathogen together with the antibiotics they confer resistance toward, we come closer to predicting the phenotypic antibiogram of a pathogen . As suchCARD is an ontological-driven database of ARGs and individual mutations conferring resistance in a broad range of bacterial species, including those found in clinical, agricultural and environmental settings . PoweredThe CARD team initially attempted the use of word-association scoring matrices to prioritize papers in PubMed. After two rounds of development, the Python-based CARD*Shark 2 algorithm guided curators to relevant papers using an algorithm that scored papers based on their abstract similarity to papers previously curated in CARD for ARGs. For example, for macrolide antibiotics, using the relative frequencies of each word in the abstracts of all papers associated with macrolides in CARD, CARD*Shark 2 calculates a score for each macrolide-associated paper by taking the sum of relative frequencies of all abstract words. Based on the score, it assigned new papers in PubMed to a low- or high-level group that dictates the papers’ relevance to curators if the score falls below or above a cut-off, respectively. For our analysis, papers assigned to the high-level group are considered positive predictions, and low-level papers are considered negative predictions. Although the algorithm provided curators with a reasonable set of papers to triage, there were a few key limitations. First, CARD*Shark 2 pulled papers from PubMed on a monthly basis using only drug class terms found in CARD’s ARO as query terms, ignoring words for individual antibiotics or ARGs and thus potentially missing important papers when an abstract does not discuss a drug class directly. In addition, papers in PubMed can include multiple drug class terms and are thus scored differently by each drug class–specific scoring matrix. Such papers could be reported under multiple drug class search results, creating redundancy and inefficiency for curators, but also inconsistency as papers could appear low level in one drug class group but high level in another. Finally, every paper CARD*Shark 2 examined was considered a positive result even if the initial PubMed query result included non-AMR papers, yet the scoring was often not discriminatory and forced a manual review of all papers by curators. With these flaws in mind, we were interested in implementing a machine learning version of CARD*Shark that reviewed all recently added PubMed articles monthly and identified all relevant publications with high recall, while not overburdening the curator with too many false positives, i.e. low precision.The type of problem CARD*Shark 2 aims to solve is one of binary classification: given an input of publications newly added to PubMed, we wish to assign a pass/fail on whether they contain valuable new information for curation into CARD. Many machine learning algorithms can solve binary classification problems and we examined these as possible improvements upon CARD*Shark 2. Machine learning classification algorithms fall into two broad categories: supervised learning and unsupervised learning . UnsuperFor the classification of the scientific literature, we examined supervised classification algorithms including logistic regression, naive Bayes, random forest, extreme gradient boosting and support vector machines to determine the best-performing approach to guide the curation of papers into CARD. To create prediction models for classification tasks, we used PubMed’s Entrez application programming interface (API) to retrieve abstracts and metadata from PubMed’s database  as a steOnce preprocessed, the procedure of converting data into usable vectors for machine learning is called feature extraction and aims to reduce the dimensionality of data for algorithmic use. The two major types of dimensionality reduction that can be applied to text include bag-of-words (BOW) and term frequency–inverse document frequency (TF–IDF) , 20. BOWF1 statistic . For comparison, we also scored the 9710 papers using CARD*Shark 2 with 5-fold stratified cross-validation.To determine the best classifier and using scikit-learn , we traiPapers for September and November 2019 were obtained from PubMed using the MeSH ‘[Date—Create]’ query via the Entrez API (i.e. ‘start date’ [Date - Create]: ‘end date’ [Date - Create]). We processed these papers, as outlined earlier, and classified them using the models generated from cross-validation. For independent human validation of the classifiers, 100 papers were randomly selected from the September logistic regression predictions, with an even 50/50 split between classification results (i.e. 50% predicted positive and 50% predicted negative), while for the November set, a total of 330 papers were selected for validation . Subsequently, a group of 11 experienced CARD curators reviewed each paper by answering a series of three questions to generate binary labels for each paper, with two individuals reviewing each paper. The subset of algorithms examined during human validation was based on the best-performing approaches during initial investigations, instead of all possible algorithms, to reduce the overall effort required by our human curators. The three questions asked of the human curators were the following: ‘Is there an AMR gene described in the abstract’, ‘Is there an elevated MIC anywhere in the paper’ and ‘Would you curate the gene into CARD based solely on the abstract?’ If conflicting labels arose, the paper was not included in the final validation results; otherwise, the human curator results were compared to the supervised model predictions as an independent assessment of performance.F1 results of CARD*Shark 2 underperformed in comparison to most supervised learning models  area under the curves of >0.94, while CARD*Shark 2 obtained an area under the curve of 0.88 . Similarg models , althougF1 values, we find logistic regression, naive Bayes, random forest and extreme gradient boosting each have one model that performs with the highest F1 score, with random forest on TF–IDF on bi/trigrams having the best overall performance . During this time, CARD*Shark 2 flagged 22 196 unique papers, 66 of which were added to CARD by the curators . The benOverall, we have found that supervised learning applications to rapidly triage thousands of publications can viably reduce the burden associated with curating data. However, due to the limited scope associated with CARD’s curation goal (i.e. identifying new ARGs only), models perform with poor precision but high recall. To compensate for this precision, a combination of CARD*Shark 2 and the supervised learning models will be incorporated into CARD by ranking publications based on model agreement to maintain high recall while prioritizing high-value publications (i.e. publications with the highest model agreement are reviewed first). As such, a computer-guided curation paradigm that centers ultimately on expert, human biocuration allows CARD to provide comprehensive, high-value, trustworthy data for genomic surveillance of AMR.


Pelvic radiotherapy is used to treat a range of cancers. Radiotherapy can damage surrounding, non-cancerous tissue and organs, causing long-term problems, including bowel symptoms such as bleeding, pain, and incontinence. The provision of support and treatment for those affected as well as shared decision making regarding treatment should be informed by a solid understanding of the prevalence, nature, and severity of symptoms. We conducted a systematic review of population-based studies presenting patient-reported bowel symptoms to synthesize evidence on symptom prevalence and severity following pelvic radiotherapy. Multiple different bowel symptoms have been reported, and prevalence varies from 1% (bleeding) to 59% . We found substantial variation in the reported methods and few data pertaining to cancers other than prostate. Our review supports the view that bowel symptoms are a significant problem following pelvic radiotherapy and highlights limitations of the evidence base that should be addressed in future research. n = 39; gynecological, n = 6) reporting 19 datasets were included. Studies were methodologically heterogeneous. Most frequently assessed was bowel function . Also assessed was urgency, diarrhea, bleeding, incontinence, abdominal pain, painful hemorrhoids, rectal wetness, constipation, mucous discharge, frequency, and gas. Prevalence ranged from 1% (bleeding) to 59% . In total, 10 papers compared radiotherapy with non-cancer comparators and 24 with non-radiotherapy cancer patient groups. Symptom prevalence/severity was greater/worse in radiotherapy groups and symptoms more common/worse post-radiotherapy than pre-diagnosis/treatment. Symptom prevalence varied between studies and symptoms. This review confirms that many people experience chronic bowel symptoms following pelvic radiotherapy. Greater methodological consistency, and investigation of less-well-studied survivor populations, could better inform the provision of services and support.Pelvic radiotherapy can damage surrounding tissue and organs, causing chronic conditions including bowel symptoms. We systematically identified quantitative, population-based studies of patient-reported bowel symptoms following pelvic radiotherapy to synthesize evidence of symptom type, prevalence, and severity. Medline, CINAHL, EMBASE, and PsychINFO were searched from inception to September 2022. Following independent screening of titles, abstracts, and full-texts, population and study characteristics and symptom findings were extracted, and narrative synthesis was conducted. In total, 45 papers (prostate,  The term pelvic radiation disease has been applied to a group of chronic symptoms that may arise following pelvic radiotherapy ,2. TheseAcute bowel symptoms often arise during treatment and, for many, resolve within three months of the cessation of radiotherapy . ChronicTreatment options for post-radiotherapy bowel symptoms remain limited and are not without risk ,8. NovelThis systematic review aimed to identify data on chronic bowel symptoms reported by survivors following pelvic radiotherapy. Specific objectives were to synthesize data on symptom type, prevalence, and severity; temporal patterns in symptoms post-treatment; and survivor factors associated with increased prevalence and/or severity of symptoms. We focused on data from survivors recruited from population-based sampling frames to reduce the influence of selection biases on eligible studies.This review was registered (CRD42021274083) with the Prospective Register for Systematic Reviews (PROSPERO) and conducted and reported in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines .Systematic searches were carried out in Medline, the Excerpta Medica Database (EMBASE), American Psychological Association (APA) PsychINFO, and the Cumulative Index of Nursing and Allied Health Literature (CINAHL). The search, developed with input from a medical librarian, covered three concepts: radiotherapy, pelvic cancer, and bowel symptoms . No dateAfter de-duplication, titles and abstracts were screened independently by two reviewers . Full texts of papers considered potentially eligible by reviewers one or two were obtained and assessed, again by two reviewers, against the inclusion criteria. Discrepancies were resolved by discussion, with the third reviewer (LS) consulted in the event of disagreement.A paper was eligible if it was a primary, peer-reviewed research article, available in English which reported the results of a population-based quantitative study that included at least 100 survivors exposed to pelvic radiotherapy, this was to ensure focus on studies large enough to provide some confidence in precision of findings. Papers had to report the prevalence and/or severity of chronic symptoms  and theA paper was excluded if (1) the study population was participating in a clinical trial, as survivors may have experienced more intensive follow-up or intervention than constitutes routine care; (2) bowel symptoms were based on data abstracted from clinical records or clinician’s assessment; (3) outcomes were not collected in a standardized way across all participants; (4) it reported qualitative results only or a case study; (5) the study included survivors treated with different modalities, and it was not possible to disaggregate the results for irradiated survivors; (6) it was not possible to disaggregate chronic from acute symptoms; (7) the study was of colorectal cancer survivors due to the problem of distinguishing effects of radiotherapy from the effects of the cancer and/or associated surgery on the bowel; or (8) data for small sub-groups of irradiated survivors could not be readily combined to achieve a sample size ≥ 100. Criteria 1–6 were specified a priori, and criteria 7 and 8 were added following initial scoping searches. Data extraction from eligible papers was conducted by one reviewer onto a structured form and checked by another (AB and IB) with discrepancies resolved by discussion. We extracted data describing study characteristics , population characteristics , and findings  or univariate comparisons with groups not exposed to pelvic radiotherapy ; and any comparisons of prevalence/severity between subgroups defined by survivor characteristics).Quality appraisal used an adapted 10-item version of the Methodological Index for Non-Randomized Studies (MINORS) tool . Each itPapers reporting results from survivors drawn from the same data source(s) and treated/diagnosed during the same period were regarded as a single study reporting on the same dataset. Papers using the same data source but covering different periods of diagnosis/treatment were considered separate studies.Heterogeneity with respect to cancer sites, symptoms assessed, data collection tools, and assessment time points precluded meta-analysis. Instead, narrative synthesis was undertaken . We presFollowing de-duplication, 9031 citations underwent title and abstract screening ; 79 weren = 5 datasets; n = 20 papers) [n = 4 datasets of which n = 1 reported with New Zealand; n = 5 papers) [n = 2 datasets; n = 2 papers) [n = 2 datasets; n = 6 papers) [n = 2 datasets; n = 6 papers) [n = 1 dataset; n = 1 paper) [n = 1 dataset reported together; n = 2 papers) [n = 1 dataset; n = 1 paper) [n = 1 dataset; n = 2 papers) [n = 1 [n = 1 [n = 4 [Papers reported on datasets from nine countries: USA ( papers) ,56,57,59 papers) ,44,51,54 papers) ,58, Swed papers) ,24,25,55 papers) ,45,46,471 paper) , Ireland papers) ,27, Finl1 paper) , and Nor papers) ,34. A to1 [n = 1 , mixed g1 [n = 4 ,24,25,55n = 12 datasets) reported cross-sectional analyses [n = 9 datasets; all prostate) reported longitudinal analyses [n = 23 papers) [n = 4) [n = 2) [n = 2) [n = 2) [n = 1) [n = 1) [In total, 22 papers  ,38,43,54 [n = 2) ,40, Euro [n = 2) , EORTC Q [n = 2) , EORTC Q [n = 1) . Thirtee [n = 1) ,52,53,55n = 26 papers) [n = 19) [n = 16) [n = 10) [n = 8) [n = 6) [n = 5) [n = 3) [n = 3) [n = 2) [n = 2) [n = 1) [n = 1) [The most frequently reported bowel outcome was a bowel function score n =  papers [ papers) ,57,58,59[n = 19) ,52,53,54[n = 16) ,50,52,55[n = 10) ,48,50,55 [n = 8) ,53,55, d [n = 6) ,49,50,58 [n = 5) ,49,50, p [n = 3) ,49,50, r [n = 3) ,49,50, c [n = 2) ,23, muco [n = 1) , and gasn = 8 with a comparison group) were scored as high-quality papers [Nine papers  to 15 years. The prevalence of symptoms six months post-treatment/diagnosis ranged from 1% for bleeding  to 46% fAll ten papers ,53,54,59Overall, papers suggested a peak in the prevalence of most symptoms at six months (compared with baseline) and some decrease thereafter . Exceptin = 2 prostate; n = 3 gynecological) reported comparisons of irradiated populations with non-cancer comparators [n = 2 gynecological) with a non-radiotherapy group (surgery or no treatment) diagnosed with the same cancer as the irradiated group [Five papers  ,47,48,59A summary of findings by symptom is presented in Bowel function was reported as a score or a prevalence (sometimes at a stated degree of bother or problem) of experiencing bowel dysfunction.n = 20 papers) [Bowel function score: Twenty-six papers  reported on bowel function score ,57,58,59 papers) ,56,57,59 papers) ) to 90.3 papers) ).Two papers  ,28 preseFossa et al.  comparedVan de Poll-Franse et al.   ,52,53,54Seven papers from three prostate datasets compared pre- and post-treatment ,35,48,53Fossa et al.  reportedBergmark  reportedSixteen papers  from six datasets  reported on urgency ,50,52,55Three papers  ,26,55 coTen papers  ,34,53,55All four papers  which compared incontinence in irradiated survivors with non-cancer comparators reported higher prevalence in the cancer group. This was statistically significant in the three gynecological papers. Two prostate papers from different datasets, and one gynecological paper, reported higher prevalence of fecal incontinence in irradiated compared to non-irradiated patients. This was statistically significant in the prostate papers.Two papers  ,53 reporSeven papers  ,50,53,58One prostate paper compared diarrhea prevalence pre- and post-radiotherapy and reported a statistically significant increase of 7.6 percentage points .Three papers  compared radiotherapy with other treatments ,50,53. DTwo gynecological papers from two datasets reported worse/higher prevalence of diarrhea in radiotherapy and non-radiotherapy groups ,58; statFive papers  reported on abdominal pain or painful bowel movements ,28,49,50One prostate paper reported a higher prevalence of symptoms at six months post-treatment compared with the baseline  .Two papers reported data from the same prostate dataset ,50. The One prostate paper comparing a radiotherapy and a non-cancer group reported higher prevalence in the radiotherapy group  .Two high-quality prostate papers from one dataset reported no statistical difference in the prevalence of pain in a radiotherapy group versus a surgery group ,50. BergThree papers from one prostate dataset  ,49,50 reTwo of these papers  ,50 also Three papers ,49,50  compared radiotherapy and surgery groups ,50 reporDrummond et al.  found stTwo papers  ,55 from One paper  reportedOne gynecological paper  identifiThis systematic review aimed to identify and synthesize population-based data on the type, prevalence, and severity of bowel symptoms following pelvic radiotherapy, temporal trends in prevalence/severity, and associations with survivor characteristics. In total, 45 eligible papers reported on 12 distinct symptoms, though 7 were reported in fewer than 5 papers. The most commonly reported symptom was bowel function (often measured using EPIC-26), but even for this single symptom several different measures of function were reported .Prevalence of problems varied by symptom, ranging from 1% for bleeding  to 59% (n = 7) and marked by low prevalence. Previous studies have reported higher prevalence of these particular bowel symptoms [Bleeding and fecal incontinence are particularly problematic or distressing to cancer survivors ,61 and psymptoms . These dsymptoms . Furthersymptoms , it may In the papers we reviewed, there was little systematic documentation of temporal trends in symptom prevalence post-treatment, and studies varied greatly in the time-points at which they assessed outcomes. Some papers suggested a peak in symptom prevalence around six months ,33, and Previous, small, non-population-based studies have reported on survivor characteristics that may be risk or protective factors for bowel problems post-radiotherapy . A largeWe selected studies for review that used population-based sampling frames and patient-reported outcome measures. This was intended to increase the homogeneity and comparability of included studies. However, we found marked heterogeneity across multiple dimensions, including symptoms assessed, data collection tools used, comparator groups included, assessment time points, and cancer site treated . Additionally, radiotherapy regimens varied over time and institution. This heterogeneity precluded a statistical combination of study findings in a meta-analysis. However, our review highlights some specific gaps that might be addressed through future data collection and research to build a more consolidated evidence base.Most of the 45 eligible papers from 19 datasets pertained to prostate cancer . There were fewer papers and only two datasets pertaining to gynecological cancer survivors . There is a need for more population-based studies of gynecological cancers, for which the extent of damage to surrounding organs might be greater than for prostate cancer due to treatment usually involving irradiation of a larger area. There is also a need for more studies of less-researched cancers such as bladder cancer, the treatment of which, with radiotherapy, is also associated with bowel symptoms , but forThe potential for learning from future studies would be improved through greater consistency in data collection methods across studies, including in the timing of data collection relative to treatment, the choice of outcomes, and data collection instrument. Regarding the latter, EPIC-26, though widely used, has possible shortcomings, including poor construct validity . An alteOur review did not consider the lived experiences of survivors with post-radiotherapy bowel problems. Evidence  suggests chronic bowel problems may have a substantial impact on peoples’ quality of life, but symptoms are under reported, under-recognized, and under-treated by health professionals ,72,73. TThis review confirms that, following pelvic radiotherapy, cancer survivors may live, sometimes long-term, with a variety of chronic bowel symptoms. Worldwide, there are more than eight million survivors of prostate, endometrial, and cervical cancer , many of


In vivo co-immunoprecipitation data further shows Dbp5 is recruited to tRNA independent of Los1, Msn5 (another tRNA export factor), or Mex67 (mRNA export adaptor), which contrasts with Dbp5 recruitment to mRNA that is abolished upon loss of Mex67 function. However, as with mRNA export, overexpression of Dbp5 dominant-negative mutants indicates a functional ATPase cycle and that binding of Dbp5 to Gle1 is required by Dbp5 to direct tRNA export. Biochemical characterization of the Dbp5 catalytic cycle demonstrates the direct interaction of Dbp5 with tRNA (or double stranded RNA) does not activate Dbp5 ATPase activity, rather tRNA acts synergistically with Gle1 to fully activate Dbp5. These data suggest a model where Dbp5 directly binds tRNA to mediate export, which is spatially regulated via Dbp5 ATPase activation at nuclear pore complexes by Gle1.Cells must maintain a pool of processed and charged transfer RNAs (tRNA) to sustain translation capacity and efficiency. Numerous parallel pathways support the processing and directional movement of tRNA in and out of the nucleus to meet this cellular demand. Recently, several proteins known to control messenger RNA (mRNA) transport were implicated in tRNA export. The DEAD-box Protein 5, Dbp5, is one such example. In this study, genetic and molecular evidence demonstrates that Dbp5 functions parallel to the canonical tRNA export factor Los1.  Saccharomyces cerevisiae, intron-containing tRNAs are spliced in the cytoplasm on the mitochondrial surface by the tRNA Splicing Endonuclease (SEN) complex following nuclear export (Key to the production of proteins is the delivery of amino acids to ribosomes by transfer RNAs (tRNAs). Precursor tRNAs (pre-tRNAs) are first transcribed by RNA Polymerase III with a 5´ leader and a 3´ trailer sequence . To funcr export . Furtherr export . As tRNAr export . The proS. cerevisiae, to identify additional factors responsible for regulating nucleocytoplasmic dynamics of tRNA localization and processing  export (6) at the cytoplasmic face of the nuclear pore complex (NPC) to promote the directional export of mRNA  may support tRNA export. However, a mechanistic understanding of how Dbp5 supports these diverse functions and whether they are regulated by similar co-factors and enzymatic activity is largely not understood.While non-coding RNA (ncRNA) and mRNA export pathways have long been hypothesized to be regulated by independent processes, recent studies indicate the participation of numerous mRNA export factors in regulation of diverse ncRNAs . In addi) export . Similar) export . Dbp5 is of mRNA . In addi of mRNA . Notably of mRNA , suggestdbp5L12A) that disrupt a nuclear export sequence (NES) recognized by Crm1 to promote Dbp5 transport out of the nucleus. In contrast, another mutant, dbp5R423A, was found to be impaired in nuclear import from the cytoplasm. Importantly, neither of these mutations disrupt the essential mRNA export functions of Dbp5; however, limited nuclear access of dbp5R423A induced tRNA export defects suggesting a potentially novel role for nuclear Dbp5 in tRNA export. In support of this hypothesis, a physical interaction between Dbp5 and tRNA was also characterized, which was elevated upon nuclear accumulation of Dbp5 in a dbp5L12A mutant . In contrast to single stranded RNA substrates , this study further demonstrates an interaction of Dbp5 with tRNA and double stranded RNA (dsRNA) that leads to robust ATPase activation only in the presence of Gle1/ InsPresearch , suggestlos1Δ, the NPC localization of GFP-Dbp5 was not altered . Double mutants of los1Δ with dbp5L12A (increased in the nucleus) or dbp5R423 (depleted from the nucleus) exhibited no growth defects  in single and double mutants. The los1Δ dbp5R423A double mutant exhibited significantly stronger nuclear accumulation of pre-tRNA as compared to single mutants or control after a 4-hour incubation at 37°C  that are end-processed in the nucleus to generate intron-containing intermediates (I) that can be detected by Northern blotting . When nuIleUAU. The abundance of the pre-tRNAIleUAU target in each IP was normalized to the abundance of the target in the corresponding input sample to control for changes in gene expression. Relative enrichment of the target RNA was then compared to the background signal obtained from RNA IPs in a common untagged control. In a los1Δ strain, a ~2-fold reduction in the relative amount of pre-tRNA co-immunoprecipitated with Dbp5 (~9.5-fold enrichment in WT to ~4.5-fold in los1Δ) was observed, but importantly the deletion failed to abolish the Dbp5 pre-tRNA in vivo interaction , ATPase activity  and have a dominant-negative effects on mRNA export status when overexpressed   . Unlike epleted) .DBP5, dbp5R426Q, dbp5R369G, or dbp5E240Q were integrated at the URA3 locus under regulation of the inducible pGAL promoter. Protein expression was induced for 6 hours by shifting cells from raffinose to galactose containing media. Induction was followed by 1 hour of growth in glucose to halt expression and relieve potential changes in tRNA export induced by a change in the primary carbon source. Using this approach, all proteins were expressed as indicated by western blotting   /D. Thesedbp5R426Q and dbp5R369G overexpression on tRNA export in vivo, the Dbp5 ATPase cycle and role of Gle1 in the context of tRNA was further investigated in vitro. Previous studies have characterized Dbp5 binding to single stranded RNA (ssRNA) substrates and demonstrated Dbp5 binding to ssRNA is linked to the nucleotide state of the enzyme, with the highest affinity for ssRNA occurring when Dbp5 is ATP bound .in vitro, electrophoretic mobility shift assays (EMSA) were performed in the presence of different nucleotide states and tRNA substrates. Consistent with published observations of complex formation between Dbp5 and ssRNA . From quantification of EMSAs, an estimated Kd of ~150nM (Phe tRNA) and ~130nM (mixed tRNA) was obtained , were fained (3 . These dInteraction with ssRNA substrates has been well documented to stimulate the Dbp5 ATPase cycle . As such6) has been shown to synergistically activate Dbp5 ATPase activity at low RNA concentrations  to a level like that of ssRNA (1.11 +/− 0.07) (6 and tRNA is mirrored by poly(I:C) and observed with both mixed yeast tRNAs as well as yeast Phenylalanine tRNA  with RNase T1 for 2 hours at 37°C. RNase T1 degrades ssRNA, not dsRNA, allowing determination of whether RNA activation observed in the presence of Gle1/InsP6 is specific to properly folded tRNA and poly(I:C). As expected, treatment of the ssRNA (pA) with RNase T1 resulted in the loss of RNA-stimulated Dbp5 ATPase activity (0.68 +/− 0.06 before RNase T1 treatment to 0.17 +/− 0.01 after). Furthermore, RNase T1 treatment reduced pA/Gle1/InsP6 activation (0.98 +/− 0.17 before treatment to 0.69 +/−0.07 after) to that of Dbp5 and Gle1/InsP6 alone (0.62 +/− 0.03) (6 (1.19+0.11 without vs 1.18+/−0.19 ATP/s with RNase T1 treatment). This was recapitulated with poly(I:C) (1.26 +/− 0.11 ATP/s without and 1.46 +/− 0.135 ATP/s with RNase T1), confirming that the synergistic activation of Dbp5 observed in the presence of Gle1/InsP6 is dependent on the structured tRNA or dsRNA. These findings provide for the possibility that Dbp5 engages tRNA in the cell without ATPase activation and could remain bound until the Dbp5-tRNA complex reaches Gle1 at the cytoplasmic side of a nuclear pore complex. In support of this model, a strong additive defect in pre- tRNAIleUAU processing was observed by Northern blotting when los1Δ was combined gle1–4  . The synine tRNA . To conf/− 0.03) . HoweverS. cerevisiae, Los1 and Msn5, are non-essential and the los1Δ/msn5Δ double mutant is viable despite the essential role of tRNA export (in vivo and in vitro data provide evidence that (1) Dbp5 and Gle1 have functions independent of Los1 in tRNA export; (2) Dbp5 can bind tRNA directly in vitro and does so in a manner that does not require Los1, Msn5, and Mex67 in vivo; and (3) the Dbp5 ATPase cycle is uniquely modulated by Gle1/InsP6 in the presence of structured RNA (e.g. tRNA or dsRNA) in vitro and the ATPase cycle supports tRNA export in vivo.The best characterized tRNA export factors in A export . These dA export . However6 and mRNA binding  in the presence of Gle1/InsP6 suggests the interaction is sequence independent and is not driven by elements unique to tRNAs. Collectively, the in vitro characterization of Dbp5 and tRNA described here suggest two non-mutually exclusive possibilities for why Dbp5 is only activated by tRNA/dsRNA in the presence of Gle1/InsP6. First, it may be that Dbp5 binds tRNA to form an inhibited intermediate that is relieved by Gle1/InsP6. This is an attractive hypothesis that is unique from how Dbp5 engages ssRNA  and potentially supports previously proposed export models that suggest Dbp5 may bind and travel with a tRNA from nucleus to cytoplasm  broadly interact with RNA via the phosphate backbone . The obs binding . While fytoplasm . A secong states . Future g states , as such6 is absent, Dbp5 is able to bind tRNA in vitro and in vivo. The ability of a tRNA to bind but not stimulate the ATPase cycle of Dbp5 has important implications for mechanisms by which Dbp5 may function in tRNA export. For example, in mRNA export it is thought that Dbp5 functions are localized at NPCs  and the DEAD-box protein at its core, eIF4AIII, that acts as a platform for the binding of other proteins to an mRNA . For example, it will be important for future studies to address the composition of pathway specific export-competent tRNA-protein complexes and how Dbp5 contributes to changes in the architecture of such a complex as they move from nucleus to cytoplasm and back again.Overall, this study and other recent publications support a general function for both Dbp5 and Mex67 in RNA export . While tdbp5R426Q, dbp5R369G, or dbp5E240Q were integrated at URA3 locus under control of pGAL promoter in a wild-type strain with Dbp5 expressed from its endogenous locus and promoter. pGAL expression was induced by first derepressing the promoter with growth in YP media with 2% raffinose overnight followed by a shift to 2% galactose containing media for 6 hours. Overexpression was halted by addition of 2% glucose and further incubation for 1 hour.A list of all yeast strains used in this study is provided in Yeast were pre-cultured to saturation overnight, diluted, and 1:10 serial dilutions were made with the most concentrated wells representing a concentration of 0.25 OD/ml. 3 μL of each dilution was spotted on YPD and grown at indicated temperatures for 2 days.Imaging experiments were carried out using the confocal configuration of an Andor Dragonfly microscope equipped with an EMCCD camera driven by Fusion software (Andor) using a 60x oil immersion objective  1.4). Images were acquired from cells grown to mid log phase in SC media at 25°C and shifted to 37°C for indicated time or treated with DMSO or 500uM Auxin and 10 uM InsP6 for relevant experiments. Cells were immobilized in 384-well glass bottomed plates (VWR) that were pre-treated with Concanavalin A.+ membrane (Amersham). RNA was crosslinked to membranes at 2400J/m2 using UV Crosslinker (VWR) and tRNA were detected using digoxigenin-labeled (DIG) probes. Mean integrated intensity values were measured for I and P bands using FIJI  and incubated with rotation at 25°C for 3 hours. Cells were then washed twice with Buffer B , 0.5mM MgCl2). For spheroplasting cell pellets were resuspended in a 1ml Buffer B solution containing 0.05% beta-mercaptoethanol. 15ul of 20mg/mL zymolyase T20 was then added to each sample and were subsequently incubated at 37°C for 45min. After washing once with Buffer B, cells were adhered to eight well slides (Fisher Scientific) pre-coated with Poly-L-Lysine. Slides were placed sequentially in 70%, 90%, and 100% ethanol for 5min and then allowed to dry. Samples were next incubated at 37°C for 2 hours in pre-hybridization buffer ). Pre-hybridization solution was then aspirated and replaced with pre-warmed pre-hybridization solution that contained either 0.25uM Cy3 tRNA probe or 0.025uM FITC dT probe and further incubated at 37°C overnight. Wells were then sequentially washed for 5min once with 2x SSC, three times with 1xSSC, and once with 0.5x SSC. Slides were dipped in 100% ethanol, air-dried, and mounting media with 4’, 6-diamidino-2-phenylidole (DAPI) was applied to each well prior to sealing of coverslip onto slide. Imaging was performed using Andor Dragonfly equipped with Andor iXon Ultra 888 EMCCD camera driven by Fusion software (Andor) using a 60× 1.4 N.A. oil objective. Images were processed in FIJI  (FISH experiments were carried out as described in previous publications with minor modifications  using did SRIM03  with Cy3ections) . Quantifections) . NuclearRIPs were conducted as previously described with minor modifications . Pull doIleUAU Unspliced Forward: GCTCGTGTAGCTCAGTGGTTAGtRNAIleUAU Unspliced Reverse: CTTTTAAAGGCCTGTTTGAAAGtRNAFBA1 Forward: CGAAAACGCTGACAAGGAAGFBA1 Reverse: TCTCAAAGCGATGTCACCAG~5 OD pellets were lysed in laemmli buffer and loaded onto 10% SDS PAGE. Protein transfer was performed onto nitrocellulose membrane using cold wet tank transfer protocol. Western blotting was performed with 1:5000 dilution of mouse monoclonal anti-DBP5 and 1:10000 dilution of mouse monoclonal anti-GAPDH (Thermo Fisher) primary antibody overnight at 4°C and 1:10000 anti-mouse DyeLight 650 secondary to detect protein.in vitro ATPase assays and fluorescence polarization using full-length Dbp5 performed as described previously . All ATPase assays with RNA titrations were fit to Michaelis Menten or allosteric sigmoidal models (when Gle1/InsP6 was added) in GraphPad Prism.Protein purification, eviously . Commerc2, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 0.5% NP-40) in the presence of indicated nucleotide and loaded on to 6% native polyacrylamide el (0.5 x TBE). Gels were stained with Apex gel stain to visualize RNA. Mean integrated intensity of bands were quantified in FIJI (Recombinant full length Dbp5 was purified as above and described previously. RNA bin in FIJI  and normSupplement 1Supplement 2


Heart failure with preserved ejection fraction (HFpEF) is a leading cause of death and disability, with its prevalence surpassing that of heart failure with reduced ejection fraction. Obesity and hypertension are often associated with HFpEF. HFpEF can be modeled through simultaneous metabolic and hypertensive stresses in male C57BL/6N mice provoked by a combination treatment of a high-fat diet (HFD) and constitutive nitric oxide synthase inhibition by Nω-nitro-L-arginine methyl-ester (L-NAME). Ubiquitin-proteasome system (UPS) dysfunction was detected in many forms of cardiomyopathy, but whether it occurs in HFpEF remains unknown. We report successful modeling of HFpEF in male FVB/N mice and, by taking advantage of a transgenic UPS reporter mouse, we have detected myocardial UPS functioning impairment during HFpEF, suggesting a pathogenic role for impaired protein degradation in the development and progression of HFpEF. Heart failure with preserved ejection fraction (HFpEF) is a lethal clinical syndrome associated with poor quality of life and substantial healthcare resource utilization comprising over half of all heart failure but with very limited pharmaceutical interventions . From clThe pathophysiology of HFpEF is complex and is not well understood. However, some hallmark characteristics remain. End-diastolic pressure elevation occurs due to an intricate interplay between diastolic malfunction, elusive systolic malfunction, and reduced atrial, LV, and arterial compliance . Most HFThe molecular pathophysiology of HFpEF is aberrantly complex and fluid, citing endothelial dysfunction in the setting of metabolic inflammation, abnormalities in nitric oxide (NO) synthesis and bioavailability , impededR120G-based cardiac proteinopathy  is cardioprotective against numerous challenges without altering normal protein turnover or cardiac function . Potentiinopathy ; and inhthy mice .in vivo UPS performance is the transgenic mouse model with ubiquitous expression of a green fluorescence protein (GFP) that has been modified with carboxyl fusion of degron CL-1  and the Institutional Animal Care and Use Committee of the University of South Dakota. The creation and validation of the GFPdgn transgenic mice for monitoring the dynamic changes in UPS performance in vivo was previously described  was conducted after 6-h fasting. Blood from the tail was collected and measured immediately before (0 min) and 15, 30, 60, 90, and 120 min after the injection using Bayer ContourMice ran on an Exer-3/6 treadmill with Stimulus Detection  on a 10-degree incline. The day before the test, animals were subjected to an acclimation at 5 m/min for 4 min, then increased to 10 m/min for 10 min. For the final test, mice were randomly grouped in a lane and perform at 5 m/min for 4 min then at 14 m/min for 2 min. Speed was increased by 2 m/min every 2 min until exhaustion. Exhaustion is defined as the animal getting shocked continuously for 5 s. The Treadmill Software  measured running distance, time ran, and exhaustion.The tail-cuff method was used to measure systolic blood pressure. Animals were trained in the instrument for at least 2 months before the final test. The mice were acclimated in the temperature-controlled chamber for 15 min, and results were measured, then another 15 min were allowed for resting data. Blood pressure was then recorded for 4 days, and readings averaged.Echocardiography was performed as previously reported in a double-blinded manner . In brieVentricular myocardial tissues were homogenized in 1x loading buffer . The homogenates were boiled for 5 min, then centrifuged for 20 min at 4°C at 14,000 rpm, and the supernatant was collected. Protein concentration of the samples were measured using Pierce BCA Protein Assay Reagent . Equal amounts of proteins were loaded to each lane of a 10% SDS-PAGE gel and fractionated by electrophoresis, then electrically transferred to a PVDF membrane overnight in 4°C. The PVDF membrane was washed and incubated in blocking buffer for 60 min before being probed with the primary antibody overnight followed by incubation with HRP-conjugated secondary antibodies. The bound secondary antibodies on the PVDF were then detected with the SuperSignal West Pico PLUS Chemiluminescent Substrates (ThermoScientific) and imaged with a ChemiDoc MP imaging system (Bio-Rad). Densitometry quantification was performed using the ImageLab software (Bio-Rad).® . Thermo Scientific™ NanoDrop 2000 UV Spectrophotometer was used to determine the concentration and purity of the RNA. Reverse transcription utilized the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems™) and a total of 900 ng of extracted RNA as template. qPCR reactions used 2 μL of 1:10 nuclease-free water diluted cDNA solution and PowerUp SYBR Green master mix (Applied Biosystems) for the following gene products: Atrial Natriuretic Factor (Nppa), Brain Natriuretic Peptide (Nppb), GFPdgn, Phospholamban (Pln), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All reactions for target genes  were performed in duplicate. The 2−ΔΔCT relative quantification method using GAPDH as the normalization gene was used to compute the relative expression of the target genes. The primer sequences for the qPCR reactions are shown in Total RNA was extracted from left ventricular myocardium using TRI Reagentp < 0.05). Two-tailed unpaired Student’s t-test was used unless indicated otherwise. In addition, the Welches correction for unequal variance was used when analyzing gene expression results. Researchers were blinded to the individual genotypes during the data collection and data analysis. A p-value or adjusted p-value of <0.05 was considered statistically significant.GraphPad Prism software 9.2.0  was used for all statistical analysis. Results are presented as the mean ± SEM. Datum was deemed an outlier according to the Grubbs test (p > 0.9999) but became significant from week 1 to week 17 , between week 0 and week 2 (p = 0.0891 and 0.0001), or between week 0 and week 18 (p = 0.4778 and 0.0033) are also summarized in p = 0.0171, HFD can induce obesity and the latter inevitably increases body weight (BW). Thus, we measured mouse BW immediately before (week 0) and weekly after initiation of the HFD+L-NAME treatment. No significant changes in BW were observed in the Chow group throughout the 18 weeks of study . The dif week 17 . Self-cop = 0.0474, Obesity can cause glucose intolerance; hence, we performed glucose tolerance tests on these animals at weeks 6 and 15. A bolus intraperitoneal injection of glucose induced increases in blood glucose levels in both groups, but the peak increase was significantly greater (week 6) and the increases lasted longer (weeks 6 and 15) in the HFD+L-NAME group compared with the Chow group , indicatSerial echocardiography was performed to evaluate the dynamic changes in cardiac morphometry and function in live mice. Consistent with an increase in systolic blood pressure observed in the HFD+L-NAME group, left ventricular (LV) concentric hypertrophy was detected at both week 7 and week 15. This is evidenced by significantly greater LV end-diastolic posterior wall thickness LVPW; d,  and a teNppb (p = 0.1092) and Nppa (p = 0.1327) tended to be higher and Pln mRNA levels (p = 0.1154) lower in the HFD+L-NAME group than in the Chow group  and fractional shortening (FS) were not decreased throughout the study compared with those of the Chow control group , indicatp = 0.241) but significantly elevated GFPdgn protein levels (p = 0.0497) and greater GFPdgn protein to mRNA ratios (p = 0.0379) in the HFD+L-NAME treatment group compared with the CHOW group  models, and aging models . The novThe UPS is the primary pathway responsible for the targeted degradation of abnormal, misfolded, damaged, or oxidized proteins, in addition to for the degradation of normal but no longer needed proteins . In respEvidence of impaired UPS functioning in HFpEF beckons for further investigation to provide a deeper understanding of the pathogenesis and devise new treatment strategies for HFpEF. For example, it will be important to elucidate the main factors that impair UPS performance during HFpEF. To this end, both diabetes and hypertension have been shown to impair myocardial UPS performance and proteasome functional insufficiency , but morNppa, Nppb, Pln, and GFPdgn may have been more suited for normalization with 36B4 rather than GAPDH as GAPDH has been shown to change during obesity and metabolic syndrome (This study has potential limitations. While the power of the study was sufficient for the primary readouts, the number of mice per group is not enough to decipher whether the GFPdgn transgenic expression has an impact on the occurrence of HFpEF. No female mice were included, leaving it untested whether the “double hit” method could cause HFpEF in female FVB/N mice or not. Additionally, mice were treated with HFD+L-NAME for up to 18 weeks. Despite both Schiattarella et al. and our results providing convincing evidence of HFpEF within this time frame, the pathogenesis of HFpEF is known to develop over the course of decades in humans . It begssyndrome . DespiteIn summary, the present study demonstrates for the first time that impaired UPS performance can occur in an animal model of HFpEF; thus, further investigation into a potential cause-effect relationship between UPS impairment and HFpEF is warranted.


Whole pancreas transplantation provides durable glycemic control and can improve survival rate; however, it can carry an increased risk of surgical complications. One devastating complication is a duodenal leak at the site of enteroenteric anastomosis. The gastroduodenal artery (GDA) supplies blood to the donor duodenum and pancreas but is commonly ligated during procurement. Since we have not had expressive changes in pancreatic back table surgical techniques in the recent decades, we hypothesized whether back table GDA reconstruction, improving perfusion of the donor duodenum and head of the pancreas, could lead to fewer surgical complications in simultaneous pancreas-kidney (SPK) transplants.2 tissue saturation, and patient morbidity through the Comprehensive Complication Index (CCI®).Between 2017 and 2021, we evaluated demographic information, postoperative complications, intraoperative donor duodenum, recipient bowel Op = 0.03913). Intraoperative tissue oxygen saturation was higher in the GDA-R group than in the GDA-L . We observed an increase in transfusion rate in GDA-R (p < 0.05), which did not result in a higher rate of exploration (p = 0.38). CCI® patient morbidity was also significantly lower in the GDA-R group (s < 0.05).A total of 26 patients were included: 13 underwent GDA reconstruction (GDA-R), and 13 had GDA ligation (GDA-L). There were no pancreatic leaks in the GR group compared to 38% (5/13) in the GDA-L group (This study identified improved intraoperative duodenal tissue oxygen saturation in the GDA-R group with an associated reduction in pancreatic leaks and CCI® morbidity risk. A larger prospective multicenter study comparing the two methods is warranted. The treatment of diabetes mellitus brings many challenges for both healthcare professionals and patients. From the system perspective, this silent illness demands a large part of the healthcare budget, including treatment, chronic/long-term care, rehabilitation, and hospitalizations of patients affected by this disease. Type 1 diabetes and its sequelae also significantly impact the quantity and quality of life of affected individuals [The worldwide incidence of diabetes has increased in recent decades, with an estimated 463 million peoplebeing affected by this disease in 2019 [Whole-pancreas transplantation continues to play an essential role in treating patients with type 1 diabetes. Patients with type 1 diabetes mellitus and end-stage renal disease (ESRD) may be eligible for simultaneous pancreas and kidney (SPK) transplantation. Whole-pancreas transplant cures patients with type 1 diabetes mellitus by returning them to normoglycemia . This prThe gastroduodenal artery (GDA), which constitutes the main blood supply to the duodenum in 7–30% of patients , 9, is tAs established in the literature, a good blood supply is crucial for adequate tissue oxygen saturation, critical for wound healing and the prevention of anastomotic leaks . GDA recDespite the theoretical improvement in enteric perfusion by GDA reconstruction, its clinical benefits remain uncommon due to the limited contemporary evidence available on its efficacy in reducing postoperative complications . It was,This study received ethical approval from the institutional review board (REB-103025). We reviewed all adult patients who underwent pancreatic transplantation at our center from February 2017 to August 2021. All recipients were thoroughly assessed and independently cleared to undergo simultaneous pancreas and kidney transplantation by nephrology and transplant surgery teams. Grafts were procured from neurological determination of death (NDD) donors and donors after circulatory death (DCD).The pancreas was retrieved in a similar fashion by a multi-visceral surgical team during the study period. The transplant surgeon performed back-table graft evaluation and preparation after randomly designating the patient to either GDA reconstruction (GDA-R) or non-reconstruction (GDA-L). Neither patient characteristics nor the surgeon’s preference determined the preference for reconstruction or ligation. Recipient and donor demographics, intraoperative characteristics and postoperative complications were recorded and analyzed.One team completed a standard extraperitoneal kidney transplant to the left external iliac artery and vein prior to the pancreas implantation, whereas a second team prepared the pancreas, which was always implanted intraperitoneally in the right iliac vessels. Our center commonly employs a two-surgical team approach to pancreas and kidney transplants. The decision to assign a reconstruction cohort was randomly made before the pancreas back table and not influenced by donor or recipient factors. A Y-graft from the donor iliac artery  is used for vascular reconstruction of all pancreas grafts. In the GDA-L group, the external/internal iliac artery branch of the Y graft is anastomosed end-to-end with the donor superior mesenteric artery (SMA) and end-to-end with the splenic artery. The common iliac artery branch of the Y-graft is then anastomosed to the recipient’s right common or external iliac artery Fig. . The porIn the GDA-R group, the Y-graft is positioned and carefully evaluated the calibre of the GDA, SMA, and splenic artery before the anastomosis Fig. . One endAfter reperfusion, a device using near-infrared time-resolved spectroscopy (tNIRS) was used intraoperatively to determine tissue oxygen saturation . The tNIWe routinely left surgical drains next to the pancreas and the kidney grafts. We measured amylase or lipase in the pancreatic drain on days 3, 7, 10, 15 and or until the amylase or lipase level reached the serum level, which was used as a reference for drain removal and to define biochemical leak or pancreatic fistula grade B with a persistent drain left in place or percutaneously repositioned after three weeks or grade C if a surgical approach was deemed . The patBlood sugar levels were measured every 15 min for the first two hours after reperfusion and then every four h for 24 h. Doppler ultrasound was performed routinely on the first postoperative or if sugar did not drop as expected or if it rose after the immediate postoperative period. A doppler ultrasound, as well as CT pelvis, demonstrating the patent gastroduodenal artery and its communications in the transplant are provided in Fig. Our standard immunosuppression induction protocol for SPK transplants included methylprednisone (250 mg IV) and rabbit antithymocyte globulin (rATG) at a dose of 1.5 mg/kg/day for a cumulative target dose of 5–6 mg/kg. We did routinely anticoagulate SPK recipients using a modified low-dose heparin protocol, as previously published by our group . FollowiAn infectious complication was routinely investigated with a CT-scan to determine the presence of intrabdominal collection. A procedure intervention was defined as opening the wound, need for an interventional radiologist to insert a percutaneous abdominal drain, or returning to the operating room. A pancreatic or enteric leak was documented respectively if a fluid collection contained a concentration of amylase and/or lipase in the drain fluid that was three times higher compared with serum and persisted over three weeks or if any enteric content, requiring open or percutaneous repair or drainage . Bowel cStatistical analysis was conducted using the R software . Patient demographic data, as well as continuous postoperative data, were summarized as mean ± SD. Unpaired t-tests were conducted for continuous variables, and Fisher’s exact tests were conducted for categorical variables.The cumulative graft and recipient survival rates were assessed using Kaplan–Meier analysis, and the log-rank test was used to compare the two groups statistically.The severity of surgical complications was based on the modified Clavien-Dindo adjusted for SPK transplantation over a period of 90 days –21. MoreP < 0.05.Statistical significance was set at Between February 2017 and August 2021, we performed 26 pancreas transplants, including one pancreas after kidney transplantation, one pancreas alone, one repeat simultaneous kidney and pancreas transplantation, and 23 de novo simultaneous kidney and pancreas transplants. The groups were balanced, with 13 patients in each group. All patients experienced normoglycemia within the first 48 h after pancreatic reperfusion.The mean follow-up period post-transplant was 923 days (56–1711 days). Early graft failure (first 90 days) occurred in 15.4% (2/13) of patients in the GDA-L group, and no early graft loss occurred in the GDA-R group. Late graft failure (> 90 days) occurred in one patient in the GDA-R group (acute rejection after one year) and one patient in the GDA-L group (patient had severe acute rejection five years after transplantation). The all-cause mortality rate in this study was 3.8% (1/26).p = 0.30). The cumulative graft survival rate was 76.9% in the GDA-L group and 92.3% in the GDA-R group .Kaplan–Meier analysis divided by the groups showed a cumulative recipient survival rate of 92.3% in GDA-L and 100% in GDA-R Fig. A . The Comprehensive Complication Index was obtained for each patient, and the mean CCI® was significantly lower in the GDA-R group compared to GDA-L group (p = 0.0492)  in GDA-R was clinically significant hematomas. The two cases that required treatment with minimally invasive intervention  turned out to be one infected small hematoma and a small collection, which was neither confirmed to be a pancreatic leak nor an infected collection. The most frequent surgical complication was pancreatic leaks in GDA-L, depicted in Table p < 0.05), which did not result in a higher re-exploration rate. The rate of post-transplant exploratory laparotomy was 15.4% and 38.5% in the GDA-R and GDA-L groups, respectively (p = 0.38).   compared with 76.8% in the GDA-L group (standard deviation: ± 17.0) p = 0.004 (Table Humar et al. showed that 6.5% of pancreatic transplant patients develop a pancreatic leak, a life-threatening complication . Other aOur study suggests that the adoption of back-table arterial reconstruction of the GDA to improve perfusion of the donor duodenum and pancreatic head could lead to a significant positive impact on pancreatic transplant outcomes. Previous work demonstrates similar benefits for GDA reconstruction, such as reducing pancreatitis, leakage, thrombosis, or bleeding , 12, 15.Diverse factors, including ischemia–reperfusion injury, donor factors, and technical features, can cause posttransplant duodenal and pancreatic leaks. We adopted the recommendations of the last ISGPS update to address issues associated with pancreatic complications. Singh et al.  reportedFor the first time, we used a clinical tissue oximeter using tNIR time-resolved spectroscopy to document the superior tissue saturation levels in the donor duodenum in the GDA-R group compared to the GDA-L group. In fact, the donor duodenum's tissue oxygenation was not different compared to the oxygenation noted in the recipient jejunum in the majority of patients in the GDA-R group. Although we did not assess blood flow, we confirmed that GDA-R improves perfusion and re-establishes normal oxygenation levels into the tissue, preventing duodenal leak or pancreatic leak, such a devastating complication. Existing literature has shown that a colonic oxygen saturation level below 90%, measured through tissue oximetry, is predictive of an anastomotic leak . A systeThis study had some significant limitations. The small sample size tempers our findings from a single center in our study. In addition, although data on tissue saturation were prospectively collected, complications and graft survival data were retrospectively collected. Despite these limitations, our results support future prospective, multicenter, and randomized studies.Reconstruction of the GDA resulted in better perfusion of the donor duodenum, as indicated by tissue perfusion spectroscopy. This may, in turn, reduce the incidence of pancreatic or duodenal leaks, preventing the need for further imaging studies, the need for procedural intervention, or allograft pancreatectomy secondary to an untreatable fistula. The rate of patient morbidity, calculated using the CCI®, was also significantly lower in the GDA reconstruction group. GDA reconstruction was also linked to fewer pRBCs transfused units, reducing the risk of transfusion-associated compilations. A randomized, prospective, multicenter study is warranted to address this question definitively.


Caenorhabditis elegans strain. Mechanistically, RTN3 regulated these events through its interactions with glucose‐regulated protein 78 (GRP78), which further inhibited the adenosine 5 monophosphate‐activated protein kinase (AMPK)–isocitrate dehydrogenase 2 (IDH2) pathway. In the end, knockout of RTN3 relieved fatty liver and mitochondrial dysfunction. Our study indicated that RTN3 was important in NAFLD and lipid catabolism and that an increase in RTN3 in the liver might be a risk factor for nonalcoholic steatohepatitis and NAFLD.Reticulon 3 (RTN3), an endoplasmic reticulum protein, is crucial in neurodegenerative and kidney diseases. However, the role of RTN3 in liver tissues has not been described. Here, we employed public datasets, patients, and several animal models to explore the role of RTN3 in nonalcoholic fatty liver disease (NAFLD). The underlying mechanisms were studied in primary hepatocytes and L02 cells in vitro. We found an increased expression of RTN3 in NAFLD patients, high‐fat diet mice, and oxidized low‐density lipoprotein‐treated L02 cells. The RTN3 transgenic mice exhibited the phenotypes of fatty liver and lipid accumulation. Single‐cell RNA sequencing analysis indicated that increased RTN3 might induce mitochondrial dysfunction. We further showed this in primary hepatocytes, the L02 cell line, and the  This study suggested that RTN3 can interact with GRP78 on the endoplasmic reticulum. The increased expression of RTN3 can facilitate interactions between RTN3 and GRP78, thereby decreasing the activity of GRP78 in regulating AMPK phosphorylation, which further reduces the expression IDH2, finally resulting in mitochondrial dysfunction, increased reactive oxygen species, and NAFLD. A mechanistic study revealed that the overexpression of RTN3 inhibited the adenosine 5 monophosphate‐activated protein kinase (AMPK)–isocitrate dehydrogenase 2 (IDH2) pathway by altering the interaction between RTN3 and glucose‐regulated protein 78 (GRP78), inducing mitochondrial dysfunction, which ultimately promotes the genesis and development of NAFLD. Finally, we found that reducing the expression of RTN3 can rescue the NAFLD and mitochondrial dysfunction caused by a high‐fat diet (HFD) by activating the AMPK–IDH2 pathway. Hence, our study indicated that overexpression of RTN3 in the liver might be a risk factor for NAFLD, and RTN3 may be a potential therapeutic target for NAFLD.This study showed a strong correlation between increased RTN3 levels and NAFLD. Both transgenic mouse models overexpressing the wild‐type (WT) human RTN3 gene (Tg‐RTN3) and 22.1n = 6) than in the ad libitum diet (ALD‐WT) group (n = 6) . The results showed that the RNA levels of RTN3 were increased dramatically in the NAFLD patient group, HFD mouse group, and steatosis‐steatohepatitis diet (SSD) mouse group compared to healthy controls Figure . We then) Figure . Accordi) Figure . Finally) Figure . IHC ana) Figure . These f2.2n = 6) presented with overt fat accumulation at 4 months of age with standard chow (n = 6)  Figure . The liv) Figure . Simulta) Figure . In addi) Figure . These o2.3Next, to further identify RTN3‐expressing hepatocyte subpopulations, a supervised analysis was performed on a previously published single‐cell RNA sequencing (scRNA‐seq) dataset isolated from the live tissues of chow diet, 15‐ and 30‐week high‐fat high‐fructose diet (HFHFD) mice, in which hepatocyte subpopulations unique to NASH were reported.2.4Because scRNA‐seq indicated that increased RTN3 was related to mitochondrial respiration in NASH mouse models, we detected the morphology and function of mitochondria in the hepatocytes of Tg‐RTN3 mice. Transmission electron microscopy (TEM) exhibited that the size and density of hepatocyte mitochondria were significantly decreased in Tg‐RTN3 mice compared to controls Figure . MoreoveC. elegans. In our generated Tg‐Ret1 C. elegans, Oil red O staining indicated that the number of large lipid drops in Tg‐Ret1 was much greater than that in the WT  was used to detect the candidate RTN3‐interacting molecules in WT liver tissues. Interestingly, GRP78 was found to be a possible RTN3‐interacting protein (data not shown). Coimmunoprecipitation (Co‐IP) analysis further validated the interaction between RTN3 and GRP78 in primary hepatocytes Figure . GRP78, Collectively, our data suggested that RTN3 can interact with GRP78 on the ER. The overexpression of RTN3 can promote the interactions between RTN3 and GRP78, thereby reducing the ability of GRP78 in regulating AMPK phosphorylation, which further reduces the expression IDH2, finally resulting in mitochondrial dysfunction, increased ROS, and NAFLD Figure .2.6n = 6) exhibited less fat accumulation in liver tissues than WT mice (n = 6) after 3 months of HFD feeding  mice were also generated to further confirm the relationship between RTN3 and NAFLD Figure . We foung Figure . The livg Figure . Liver fg Figure . In addig Figure . All theg Figure . SimilarR Figure . FinallyR Figure .C. elegans, studies have indicated that reducing the expression of RTN3 can rescue lipid accumulation, liver function, and mitochondrial dysfunction caused by HFD by activating the AMPK–IDH2 pathway. Reducing the expression of RTN3 in the liver may be a potential therapeutic strategy for treating NAFLD.In RTN3 KO mice and related 3C. elegans, cell lines, and NAFLD patient samples, which help us understand how RTN3 and its interacting proteins contribute to mitochondrial dysfunction and NAFLD. Drug development for related targets such as RTN3 may be a potential therapeutic strategy for treating NAFLD.As an ER membrane protein, RTN3 plays a significant role in the nervous system.In our study, we revealed that increased RTN3 expression may lead to mitochondrial dysfunction in hepatocytes. Previous studies reported that structural and functional alterations of mitochondria in hepatocytes contribute to the pathogenesis of NAFLD.By employing biochemical approaches, we identified overtly decreased expression of IDH2 in the liver tissues of Tg‐RTN3 mice. Previous studies have demonstrated that a reduction in IDH2, an enzyme located in mitochondria, may lead to mitochondrial dysfunction in hepatocytes and cardiomyocytes.C. elegans models.Recently, some studies have focused on the role of RTN3 in physiology and pathology. Mutations in RTN3 have been detected in early‐onset Alzheimer's disease.4In summary, our study suggests that high expression of RTN3 is a possible lesion for lipid deposition in the liver by impairing mitochondria and increasing ROS levels, partly through competitive binding with GRP78 to attenuate the GRP78‐mediated AMPK–IDH2 pathway. Reducing the expression of RTN3 may be a potential therapeutic method for NAFLD. Our findings also imply that RTN3 is a pivotal upstream regulator of the AMPK–IDH2 pathway that functions in mitochondrial biological homeostasis.55.1Human liver sections were collected from Xiangya Hospital and the Second Xiangya Hospital. This study was approved by the Institutional Review Board committee of Central South University in China .5.2Tg‐RTN3 and RTN3 KO mice were described previously.C. elegans (N2 strain), Tg‐Ret1 C. elegans strain, and Daf‐22 C. elegans strain were purchased or generated as previously described.The WT 2‐controlled atmosphere (Thermo Fisher Scientific).Primary hepatocytes were isolated from mouse liver tissues as follows: fresh liver tissues were separated from newborn mice and cut up by scissors. The fragmented tissues were washed with phosphate‐buffered saline with 100 U/mL penicillin and 100 μg/mL streptomycin and digested with collagenase/hyaluronidase. After filtration and centrifugation, the separated cells were cultured with complete medium. The L02 cell line was purchased from the GuangZhou Jennio Biotech Co., Ltd.  and cultured with complete medium. Both primary hepatocytes and the L02 cell line were maintained at 37°C in a humidified, 5% COThe RTN3 antibody was generated in the Yan laboratory.5.3Tissues were fixed with formalin and embedded in paraffin. Six‐micrometer sections were prepared for baking and dewaxing. After antigen retrieval and blocking, the RTN3 antibody was incubated for 8 h. Then, a broad spectrum IHC kit was used for the next steps including secondary antibody incubation, 3,3′‐diaminobenzidine staining, and hematoxylin staining. Finally, the slides were examined by routine light microscopy .5.4g for 120 min at 4°C to separate the supernatants. The protein concentrations were detected by bicinchoninic acid protein assays and analysis kits. A total of 30 μg protein lysates were separated by 4%–12% NuPAGE Bis‐Tris gel electrophoresis by standard methods with the primary and secondary antibodies mentioned above. Finally, the bands were detected in iBright Gel imaging system (Thermo Fisher Scientific).Tissues or cells were dissociated on ice in radio‐immunoprecipitation assay lysis buffer with protease inhibitor cocktail for 1 h. The homogenates were centrifuged at 15,000 ×5.5For HE staining, paraformaldehyde‐fixed liver tissue was embedded in paraffin and sliced into 6‐μm sections. The slides were baked at 60°C and dewaxed by dimethylbenzene. After alcohol treatment with an inverse concentration gradient, the slides were stained by HE with HE Stain Kit according to established protocols. Finally, the slides were examined by routine light microscopy .For Oil red O staining, paraformaldehyde‐fixed tissues were embedded in optimal cutting temperature compound and sliced into 10‐μm sections. The cells were cultured in a Nunc Lab‐Tek Chamber Slide System (Thermo Fisher Scientific). The slides were treated with isopropanol for 5 min. Oil Red O staining, hematoxylin staining, and slide sealing were performed with an Oil Red O Stain Kit according to established protocols. Finally, the slides were examined by routine light microscopy .5.6g for 5 min at 4°C, and the supernatants were separated and treated with ALT or AST assay kits according to established protocols. Finally, the levels of ALT or AST were detected with a colorimeter  at a wavelength of 505 nm.For ALT and AST assays, mouse serum was extracted at 1000 ×g for 120 min at 4°C, and the supernatants were collected and treated with a Triglyceride Detection Kit according to established protocols. Levels of TAG were detected with a colorimeter (Cary 60 UV‒Vis) at a wavelength of 505 nm.For TAG detection, the liver tissues were dissociated on ice in isopropanol. The homogenates were centrifuged at 15,000 ×5.72 transformation. The R (version 4.0.4) and RStudio (version 1.2.5033) were used to address all the data in this study. GSE185051 contained 52 NAFLD samples and five healthy liver samples, GSE200482 contained four normal chow diet C57BL6J mouse liver samples and five SSD C57BL6J mouse samples, and GSE200409 contained 12 mouse liver samples from mice fed a normal or HFD for different times.The RNA‐seq datasets of GSE185051 and GSE200482, as well as microarray dataset GSE200409, were obtained from the Gene Expression Omnibus (GEO) database with logscRNA‐seq data from the GSE166504 dataset were obtained from the GEO database.p‐value was set at 0.05.The “clusterProfiler” (version 3.18.1) R package was used for GO enrichment analyses, which included biological process, molecular function, and cellular component. The 5.8The samples were treated 4% glutaraldehyde as we described previously.5.9Liver tissues or cells were homogenized on ice with extraction solution. After centrifugation, the supernatants were collected for detection. The ATP levels were measured by phosphomolybdic acid colorimetry with an ATP array kit at a wavelength of 340 nm with a colorimeter (Cary 60 UV‒Vis). The ROS levels were measured by fluorometric analysis using 2,7‐dichlorofluorescein diacetate with an ROS array kit. The ROS levels were detected by colorimeter (Cary 60 UV‒Vis) with 488 nm excitation wavelength.5.10C. elegans.The HT115 bacteria which included the plasmids that can express the dsRNAs to target the ret‐1 and silence the expression of ret‐1 were prepared for adult The WT RTN3 oding sequence (CDS) with a C‐terminal Flag‐tag in pcDNA3.1+ was designed and structured. The L02 cells were transiently transfected with pcDNA3.1‐blank and pcDNA3.1‐RTN3 using Lipofectamine™ 3000 CD Transfection Reagent following the manufacturer's instructions.5.11(−△△Ct) methods were used to compare the RNA levels of each group.Total RNA was isolated from tissues or cells with an RNA isolation kit. The RNA‐seq and bioinformatics analyses were performed by the BerryGenomics Biotech company . cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit with 1 μg RNA and prepared with Maxima SYBR Green/ROX qPCR Master Mix (2×). Finally, the Fast 7500 RT‐PCR Systems (Applied Biosystems) and 25.12The protein supernatants of WT mouse liver tissues were extracted as described in the WB methods. A total of 500 μg lysates in 1 mL were used for MS and Co‐IP with primary antibody (IgG or anti‐RTN3) and Protein A + G beads overnight. After denaturation by heating and centrifugation, the supernatants were separated by 4%–12% NuPAGE Bis‐Tris gel electrophoresis. For MS analysis, the gel was stained with Coomassie blue staining solution R250 , and the potential bands were cut and sent to Novogene Bioinformatics Institute  for further MS analysis. For Co‐IP, the supernatants were detected by standard WB methods with antibodies against RTN3 and GRP78. The bands were detected in iBright Gel imaging system (Thermo Fisher Scientific).5.13t‐test. p < 0.05 was considered significant.The data were subjected to statistical analysis with GraphPad Prism 6 (GraphPad Software). Five independent repetitions were conducted for each group in this study. Image J was used to perform the WB grayscale analysis. All data are presented as means ± standard deviation. Data were analyzed using paired Student's H.H. and S.G. wrote the draft of the manuscript and performed the bioinformatic analysis and cell and molecular experiments. Y.‐Q.C. enrolled the patient's samples. Y.‐X.L. and J.‐Y.J. performed hematoxylin–eosin and immunohistochemistry. Y.L. performed animal feeding. L.‐L.F. and R.X. revised the manuscript and designed and supported the project. All authors approved the final manuscript.The authors declare no conflicts of interest.All procedures followed were in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 2000. This study was approved by the Institutional Review Board committee of the Central South University in China . All patients provided written informed consent.Supporting InformationClick here for additional data file.