Ligand Modulates Cross-Coupling between Riboswitch Folding and Transcriptional Pausing.

Numerous classes of riboswitches have been found to regulate bacterial gene expression in response to physiological cues, offering new paths to antibacterial drugs. As common studies of isolated riboswitches lack the functional context of the transcription machinery, we here combine single-molecule, biochemical, and simulation approaches to investigate the coupling between co-transcriptional folding of the pseudoknot-structured preQ1 riboswitch and RNA polymerase (RNAP) pausing. We show that pausing at a site immediately downstream of the riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced sequence resembling the pause consensus, and electrostatic and steric interactions with the RNAP exit channel. While interactions with RNAP stabilize the native fold of the riboswitch, binding of the ligand signals RNAP release from the pause. Our results demonstrate that the nascent riboswitch and its ligand actively modulate the function of RNAP and vice versa, a paradigm likely to apply to other cellular RNA transcripts.

When Perfect Is the Enemy of Good: Results of a RAND Appropriateness Panel on Treat to Target in Asymptomatic Inflammatory Bowel Disease.

BACKGROUND: A treat-to-target strategy for inflammatory bowel disease (IBD) recommends iterative treatment adjustments to achieve clinical and endoscopic remission. In asymptomatic patients with ongoing endoscopic activity, the risk/benefit balance of this approach is unclear, particularly with prior exposure to advanced therapies. METHODS: Using the RAND/University of California Los Angeles Appropriateness Method, 9 IBD specialists rated appropriateness of changing therapy in 126 scenarios of asymptomatic patients with ulcerative colitis and Crohn's disease and active endoscopic disease. Disease extent and behavior, prior treatment, prior complications, and recent disease progression were considered, as were factors that might influence decision-making, including age and pregnancy. Ratings were collected through anonymous survey, discussed at an in-person meeting, and finalized in a second anonymous survey. RESULTS: Panelists rated change in therapy as appropriate (i.e., expected benefit sufficiently outweighs potential harms from continuing therapy) in 96/126 scenarios, generally in patients with progressive, complicated, and/or extensive disease, while changing therapy was rated uncertain in 27 scenarios of mild and/or stable disease. Changing therapy was rated inappropriate in ulcerative colitis patients with mild and stable disease previously exposed to ≥3 therapies or with improved endoscopic activity, and in Crohn's disease patients with only scattered aphthous ulcers. The validated threshold for disagreement was not crossed for any scenario. Patient age older than 65 years and a plan for pregnancy in the next year might influence decision-making in some settings. DISCUSSION: Appropriateness ratings can help guide clinical decision-making about changing therapy to achieve endoscopic remission in asymptomatic patients with IBD until data from ongoing randomized studies are available.

Endoscopic Phenotype of the J Pouch in Patients With Inflammatory Bowel Disease: A New Classification for Pouch Outcomes.

BACKGROUND & AIMS: Pouchitis is a common complication of ileal pouch-anal anastomosis (IPAA) in patients with ulcerative colitis who have undergone colectomy. Pouchitis has been considered a single entity despite a broad array of clinical and endoscopic patterns. We developed a novel classification system based on the pattern of inflammation observed in pouches and evaluated the contributing factors and prognosis of each phenotype. METHODS: We identified 426 patients (384 with ulcerative colitis) treated with proctocolectomy and IPAA who subsequently underwent pouchoscopies at the University of Chicago between June 1997 and December 2019. We retrospectively reviewed 1359 pouchoscopies and classified them into 7 main pouch phenotypes: (1) normal, (2) afferent limb involvement, (3) inlet involvement, (4) diffuse, (5) focal inflammation of the pouch body, (6) cuffitis, and (7) pouch with fistulas noted 6 months after ileostomy takedown. Logistic regression analysis was used to assess factors contributing to each phenotype. Pouch survival was estimated by the log-rank test and the Cox proportional hazards model. RESULTS: Significant contributing factors for afferent limb involvement were a body mass index of 25 or higher and hand-sewn anastomosis, for inlet involvement the significant contributing factor was male sex; for diffuse inflammation the significant contributing factors were extensive colitis and preoperative use of anti-tumor necrosis factor drugs, for cuffitis the significant contributing factors were stapled anastomosis and preoperative Clostridioides difficile infection. Inlet stenosis, diffuse inflammation, and cuffitis significantly increased the risk of pouch excision. Diffuse inflammation was associated independently with pouch excision (hazard ratio, 2.69; 95% CI, 1.34-5.41; P = .005). CONCLUSIONS: We describe 7 unique IPAA phenotypes with different contributing factors and outcomes, and propose a new classification system for pouch management and future interventional studies.

Recommendations on the Appropriate Management of Steroids and Discharge Planning During and After Hospital Admission for Moderate-Severe Ulcerative Colitis: Results of a RAND Appropriateness Panel.

INTRODUCTION: Limited guidance exists for the postdischarge care of patients with ulcerative colitis hospitalized for moderate-severe flares. METHODS: RAND methodology was used to establish appropriateness of inpatient and postdischarge steroid dosing, discharge criteria, follow-up, and postdischarge biologic or small molecule initiation. A literature review informed on the panel's voting, which occurred anonymously during 2 rounds before and after a moderated virtual session. RESULTS: Methylprednisolone 40-60 mg intravenous every 24 hours or hydrocortisone 100 mg intravenous 3 times daily is appropriate for inpatient management, with methylprednisolone 40 mg being appropriate if intolerant of higher doses. It is appropriate to discharge patients once rectal bleeding has resolved (Mayo subscore 0-1) and/or stool frequency has returned to baseline frequency and form (Mayo subscore 0-1). It is appropriate to discharge patients on 40 mg of prednisone after observing patients for 24 hours in hospital to ensure stability before discharge. For patients being discharged on steroids without in-hospital biologic or small molecule therapy initiation, it is appropriate to start antitumor necrosis factor (TNF) therapy after discharge for anti-TNF-naive patients. For anti-TNF-exposed patients, it is appropriate to start vedolizumab or ustekinumab for all patients and tofacitinib for those with a low risk of adverse events. It is appropriate to follow up patients clinically within 2 weeks and with lower endoscopy within 4-6 months after discharge. DISCUSSION: We provide recommendations on the inpatient and postdischarge management of patients with ulcerative colitis hospitalized for moderate-severe flares.

Using Wearable Biosensors to Predict Length of Stay for Patients with IBD After Bowel Surgery.

BACKGROUND AND AIMS: It remains unknown whether ambulation or sleep predicts postoperative length of stay for patients with IBD. We aim to identify the utility of wearable biosensors in predicting postoperative length of stay for patients with IBD. METHODS: Associations of postoperative length of stay with step count/sleep duration/sleep efficiency measured by wearable biosensors were examined. The best-fitting multivariable linear regression model predicting length of stay was constructed using stepwise model selection. RESULTS: Final sample included 37 patients. Shorter sleep duration on postoperative day 4 (r = 0.51, p = 0.043) or 5 (r = 0.81, p = 0.0045) or higher sleep efficiency on postoperative day 5 (r = - 0.77, p = 0.0098) was associated with a shorter length of stay. Additionally, a more positive change in sleep efficiency from postoperative day 4-5 was associated with a shorter length of stay (r = - 0.77, p = 0.024). The best-fitting multivariable linear regression model revealed Clavien-Dindo grade 1 (p = 0.045) and interaction between Clavien-Dindo grade 2/3a and mean daily steps (p = 0.00038) are significant predictors of length of stay. The following variables were not significantly associated with length of stay: mean daily steps/sleep duration/sleep efficiency, average rate of change in these three variables, and changes in step count between successive postoperative days 1-5, sleep duration between successive postoperative days 2-5, and sleep efficiency between successive postoperative days 2-4. CONCLUSION: We demonstrated the utility of activity and sleep data from wearable biosensors in predicting length of stay. Patients with more severe complications may benefit more (i.e., reduced postoperative length of stay) from increased ambulation. However, overall, sleep duration/efficiency did not predict length of stay.

Histopathology of Colectomy Specimens Predicts Endoscopic Pouch Phenotype in Patients with Ulcerative Colitis.

BACKGROUND: The endoscopic appearance in patients with "pouchitis" after ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) can be quite heterogenous. Patients with an endoscopic phenotype resembling Crohn's disease (CD) are at high risk of pouch loss. AIMS: We aimed to assess how the histopathology of colectomy specimens predicts endoscopic pouch phenotypes in UC. METHODS: We retrospectively assessed pouchoscopies from patients with UC who underwent IPAA and classified pouch findings into 7 main phenotypes: (1) normal, (2) afferent limb involvement, (3) inlet involvement, (4) diffuse, (5) focal inflammation of the pouch body, (6) cuffitis, and (7) pouch with fistulas noted ≥ 6 months from ileostomy takedown. We assessed the clinical and pathological data including deep, focal inflammation, granulomas, and terminal ileal involvement in the colectomy specimens. Logistic regression analysis was performed to identify contributing factors to each phenotype. RESULTS: This study included 1,203 pouchoscopies from 382 patients with UC. On multivariable analysis, deep inflammation was significantly associated with pouch fistulas (Odds ratio 3.27; 95% confidence interval 1.65-6.47; P = 0.0007). Of the 75 patients with deep inflammation, only two patients (2.7%) were diagnosed with CD based on pathology review. Terminal ileal involvement significantly increased the risk of afferent limb involvement (Odds ratio 2.96; 95% confidence interval 1.04-8.47; P = 0.04). There were no significant associations between other microscopic features and phenotypes. CONCLUSIONS: We identify histologic features of colectomy specimens in UC that predict subsequent pouch phenotypes. Particularly, deep inflammation in the resected colon was significantly associated with pouch fistulas, a pouch phenotype with poor prognosis.

Effectiveness of Ustekinumab Dose Escalation in Patients With Crohn's Disease.

BACKGROUND & AIMS: A subset of patients with Crohn's disease (CD) do not respond to ustekinumab at the standard dose of 90 mg every 8 weeks. Little is known about the efficacy of shortening the interval between doses. METHODS: We performed a retrospective study to determine the effectiveness of ustekinumab dose interval shortening, collecting data from 506 patients with CD who received subcutaneous ustekinumab 90 mg every 8 weeks at a single center. We obtained data from 110 patients who initially received subcutaneous ustekinumab 90 mg every 8 weeks and then had their interval shortened to every 4 weeks. Harvey Bradshaw Index (HBI) scores before and after the dose interval shortening was available for 78 patients in the cohort (71%), levels of C-reactive protein (CRP) for 60 patients (55%), and levels of fecal calprotectin for 8 patients (7%). RESULTS: Following dose interval shortening, the patients' median HBI decreased from 4.5 to 3 (P = .002), the median level of CRP decreased from 8 mg/L to 3 mg/L (P = .031), and median level of fecal calprotectin decreased from 378 µg/g to 157 µg/g (P = .57). Among patients who had an HBI >4, a level of CRP ≥5mg/dL, a level of fecal calprotectin >250ug/g, or endoscopic evidence for disease activity before dose interval shortening, after the dose interval was shortened, 28% achieved clinical remission (an HBI score ≤4), 22% had a normal level of CRP (<5 mg/dL), 50% had reduced levels of fecal calprotectin, and 36% achieved endoscopic remission. CONCLUSIONS: Shortening the ustekinumab 90 mg dose interval to 4 weeks for patients with CD who did not respond to doses every 8 weeks improved clinical and biological indices of disease activity. Patients who lose response to the standard dose of ustekinumab might benefit from dose interval shortening, which was effective and safe.

Versatile transcription control based on reversible dCas9 binding.

The ability to control transcription in a time-dependent manner in vitro promises numerous applications in molecular biology and nanotechnology. Here we demonstrate an approach that enables precise, independent control over the production of multiple RNA transcripts in vitro using single guide RNA (sgRNA)-directed transcription blockades by catalytically dead Streptococcus pyogenes CRISPR-Cas9 enzyme (dCas9). We show that when bound to a DNA template, the dCas9:sgRNA complex forms a robust blockade to transcription by RNA polymerases (RNAPs) from bacteriophages SP6, T3, and T7 (>99.5% efficiency), and a partial blockade to transcription by Escherichia coli RNAP (∼70% efficiency). We find that all three bacteriophage RNAPs dissociate from the DNA template upon encountering the dCas9 blockade, while E. coli RNAP stays bound for at least the 90-min duration of our experiments. The blockade maintains >95% efficiency when four mismatches are introduced into the 5' end of the sgRNA target sequence. Notably, when using such a mismatched blockade, production of specific RNA species can be activated on demand by addition of a double-stranded competitor DNA perfectly matching the sgRNA. This strategy enables the independent production of multiple RNA species in a temporally controlled fashion from the same DNA template, demonstrating a new approach for transcription control.

Black Race and Public Insurance Are Predictive of Inappropriate Evaluation of Iron Deficiency Anemia and Diarrhea.

BACKGROUND AND AIMS: Socioeconomic status, race, and insurance can impact healthcare delivery and utilization in several chronic disease states. The primary aim of our study was to determine whether race and insurance status are predictors of having an appropriate workup for celiac disease and inflammatory bowel disease (IBD) when presenting with iron deficiency anemia (IDA) and chronic diarrhea. METHODS: Medical records of patients seen at the University of Chicago Medical Center between January 1, 2006, and September 20, 2017, were reviewed. Patients with two separate encounters within 6 months associated with the diagnosis codes for both IDA and chronic diarrhea were identified. Patients without a diagnosis code for IBD and celiac disease were further grouped as those that had an "appropriate" workup and those that did not. Factors associated with the appropriate evaluation were analyzed by univariate and multivariate logistic regression. RESULTS: In total, 899,701 records were searched. A total of 83 patients fit inclusion into the study (8 IBD, 3 CD, 72 neither IBD or CD). Black race was associated with a 91% decreased odds of having the appropriate workup on univariate (OR 0.090, 95%CI 0.017-0.475, p = 0.005) and age-adjusted multivariate analysis (OR 0.095, 95% CI 0.017-0.527, p = 0.007). Public insurance status was significantly associated with a 90% decreased odds of appropriate workup on univariate (OR 0.102, 95% CI 0.024-0.438, p = 0.002) and age-adjusted multivariate analysis (OR 0.104, 95% CI 0.021-0.513, p = 0.005). CONCLUSIONS: Black race and public insurance were significantly associated with not having an appropriate workup for IBD and celiac disease when presenting with iron deficiency and chronic diarrhea.

Follow-Up of Patients With Ulcerative Colitis and Histological Normalization.

The natural history of ulcerative colitis (UC) follows a relapsing and remitting course of inflammation and is accompanied by associated mucosal injury and historically, microscopic features of chronicity that were the sine qua non for the diagnosis.1 As goals for the management of UC have evolved to include objectively measured endoscopic improvement of the mucosa, there also has been a move to include histological endpoints in assessment of disease activity.2,3 However, there remain a number of unanswered questions about histology in UC and this is not yet a specific treatment goal.4.

Meeting report: SMART timing--principles of single molecule techniques course at the University of Michigan 2014.

Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines.

Recommendations for Broadening Eligibility Criteria in Inflammatory Bowel Disease Clinical Trials.

BACKGROUND: Clinical trial recruitment for patients with inflammatory bowel disease (IBD) has become more challenging over time. We aimed to develop recommendations for broadening IBD clinical trial eligibility to improve the inclusion of a more representative patient population in a more efficient timeline. METHODS: We applied the RAND/UCLA Appropriateness Method focused on broadening IBD clinical trial eligibility. A literature review was performed for 7 domains, each representing a different area related to trial recruitment. Based on these domains, 32 statements were developed. A questionnaire was sent to IBD specialists to anonymously vote on each statement with regards to its appropriateness and feasibility. After the first round of voting, participants met for a moderated discussion to review all statements. At the end of the discussion a second round of anonymous voting led to the final recommendations. RESULTS: The final round of voting resulted in 26 statements. All were rated as feasible and 25 of 26 rated as appropriate. Recommendations generally are to be more inclusive of complicated disease phenotypes, more liberal around safety criteria, to recognize the importance of non-invasive imaging and biomarkers, to minimize the washout period and to not enforce a minimum or maximum number of prior medications, to allow a recently recorded colonoscopy to count as a baseline study, and to be less restrictive of age. CONCLUSION: Recommendations to broaden clinical trial eligibility were found to be both appropriate and feasible with a high degree of agreement amongst an international group of IBD specialists.

A Comparative Analysis of Clinical Symptoms and Modified Pouchitis Disease Activity Index Among Endoscopic Phenotypes of the J Pouch in Patients With Inflammatory Bowel Disease.

Background: The modified pouchitis disease activity index (mPDAI) based on clinical symptoms and endoscopic findings is used to diagnose pouchitis, but validated instruments to monitor pouchitis are still lacking. We recently established an endoscopic classification that described 7 endoscopic phenotypes with different outcomes. We assessed symptoms and compared mPDAIs among phenotypes in inflammatory bowel disease (IBD). Methods: We retrospectively reviewed pouchoscopies and classified them into 7 main phenotypes: normal (n = 25), afferent limb (AL) involvement (n = 4), inlet involvement (n = 14), diffuse (n = 7), focal inflammation of the pouch body (n = 25), cuffitis (n = 18), and pouch-related fistulas (n = 10) with a single phenotype were included. Complete-case analysis was conducted. Results: One hundred and three IBD patients were included. The median mPDAI was 0 (IQR 0-1.0) in patients with a normal pouch. Among inflammatory phenotypes, the highest median mPDAI was 4.0 (IQR 2.25-4.75) in cuffitis, followed by 3.0 (IQR 2.5-4.0) in diffuse inflammation, 2.5 (IQR 1.25-4.0) in inlet involvement, 2.5 (IQR 2.0-3.5) in AL involvement, 2.0 (IQR 1.0-3.0) in focal inflammation, and 1.0 (IQR 0.25-2.0) in the fistula phenotype. Perianal symptoms were frequently observed in pouch-related fistulas (8/10, 80%) and cuffitis (13/15, 87%). Among patients with cuffitis, all had incomplete emptying (6/6, 100%). Conclusions: We correlated the mPDAI with the endoscopic phenotypes and described the limited utility of symptoms in distinguishing between inflammatory phenotypes. Further studies are warranted to understand which symptoms should be monitored for each phenotype and whether mPDAI can be minimized after pouch normalization.

Endoscopic Normalization and Transition of J-Pouch Phenotypes Over Time in Patients With Inflammatory Bowel Disease.

BACKGROUND: Patients with inflammatory bowel disease (IBD) who undergo proctocolectomy with ileal pouch-anal anastomosis may develop pouchitis. We previously proposed a novel endoscopic classification of pouchitis describing 7 phenotypes with differing outcomes. This study assessed phenotype transitions over time. METHODS: We classified pouch findings into 7 main phenotypes: (1) normal, (2) afferent limb (AL) involvement, (3) inlet (IL) involvement, (4) diffuse, (5) focal inflammation of the pouch body, (6) cuffitis, and (7) pouch-related fistulas noted more than 6 months after ileostomy takedown. Among 2 endoscopic phenotypes, the phenotype that was first identified was defined as the primary phenotype, and the phenotype observed later was defined as the subsequent phenotype. RESULTS: We retrospectively reviewed 1359 pouchoscopies from 426 patients (90% preoperative diagnosis of ulcerative colitis). The frequency of primary phenotype was 31% for AL involvement, 42% for IL involvement, 28% for diffuse inflammation, 72% for focal inflammation, 45% for cuffitis, 18% for pouch-related fistulas, and 28% for normal pouch. The most common subsequent phenotype was focal inflammation (64.8%), followed by IL involvement (38.6%), cuffitis (37.8%), AL involvement (25.6%), diffuse inflammation (23.8%), normal pouch (22.8%), and pouch-related fistulas (11.9%). Subsequent diffuse inflammation, pouch-related fistulas, and AL or IL stenoses significantly increased the pouch excision risk. Patients who achieved subsequent normal pouch were less likely to have pouch excision than those who did not (8.1% vs 15.7%; P = .15). CONCLUSIONS: Pouch phenotype and the risk of pouch loss can change over time. In patients with pouch inflammation, subsequent pouch normalization is feasible and associated with favorable outcome.

Endoscopic pouch phenotypes can change over time and subsequent development of diffuse inflammation, pouch-related fistulas, and afferent limb/inlet stenoses significantly worsen pouch outcomes. In patients with pouch inflammation, subsequent pouch normalization is feasible and associated with favorable outcomes.

The C-terminal tail of polycystin-1 suppresses cystic disease in a mitochondrial enzyme-dependent fashion.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent potentially lethal monogenic disorder. Mutations in the PKD1 gene, which encodes polycystin-1 (PC1), account for approximately 78% of cases. PC1 is a large 462-kDa protein that undergoes cleavage in its N and C-terminal domains. C-terminal cleavage produces fragments that translocate to mitochondria. We show that transgenic expression of a protein corresponding to the final 200 amino acid (aa) residues of PC1 in two Pkd1-KO orthologous murine models of ADPKD suppresses cystic phenotype and preserves renal function. This suppression depends upon an interaction between the C-terminal tail of PC1 and the mitochondrial enzyme Nicotinamide Nucleotide Transhydrogenase (NNT). This interaction modulates tubular/cyst cell proliferation, the metabolic profile, mitochondrial function, and the redox state. Together, these results suggest that a short fragment of PC1 is sufficient to suppress cystic phenotype and open the door to the exploration of gene therapy strategies for ADPKD.

Achieving Histologic Normalization in Ulcerative Colitis Is Associated With a Reduced Risk of Subsequent Dysplasia.

BACKGROUND: Complete histologic normalization is associated with improved clinical outcomes in ulcerative colitis (UC). However, it is currently unknown what effect achieving histologic normalization has on the development of dysplasia. METHODS: We performed a retrospective analysis of 495 patients with a confirmed diagnosis of UC from a tertiary center. Patients were categorized according to the best histologic assessment they had during their disease course: histologic normalization, histologic quiescence, or persistent histologic activity. We assessed dysplasia rates in these patient groups after achieving histologic normalization or histologic quiescence, or 8 years after UC diagnosis in those with persistent histologic activity. Kaplan-Meier graphs and Cox regression analyses were performed to estimate this effect. RESULTS: The incidence rate of dysplasia development after achieving histologic normalization was statistically significantly less when compared with the incidence rate after achieving histologic quiescence (P = 0.001) and in those with persistent histologic activity 8 years after UC diagnosis (P = 0.033). In multivariate analysis, at any point throughout UC duration, dysplasia development was statistically lower in those with histologic normalization (adjusted hazard ratio [aHR], 0.32; 95% confidence interval [CI], 0.13-0.81) but not in those with histologic quiescence (aHR, 0.52; 95% CI, 0.25-1.10), compared with those with persistent histologic inflammation. When assessing the time after achieving histologic normalization, histologic quiescence, or 8 years post UC diagnosis in those with persistent histologic activity, we found that patients with histologic normalization had a subsequent decreased risk of developing dysplasia (aHR, 0.09; 95% CI, 0.01-0.72), compared with patients without normalization. CONCLUSIONS: Histologic normalization is associated with a decreased risk in patients with UC of developing subsequent dysplasia, compared with patients without histologic normalization. These findings have implications for surveillance intervals.