The Leukemia-Associated Fusion Protein MN1-TEL Blocks TEL-Specific Recognition Sequences

The leukemia-associated fusion protein MN1-TEL combines the transcription-activating domains of MN1 with the DNA-binding domain of the transcriptional repressor TEL. Quantitative photobleaching experiments revealed that ~20% of GFP-tagged MN1 and TEL is transiently immobilised, likely due to indirect or direct DNA binding, since transcription inhibition abolished immobilisation. Interestingly, ~50% of the MN1-TEL fusion protein was immobile with much longer binding times than unfused MN1 and TEL. MN1-TEL immobilisation was not observed when the TEL DNA-binding domain was disrupted, suggesting that MN1-TEL stably occupies TEL recognition sequences, preventing binding of factors required for proper transcription regulation, which may contribute to leukemogenesis

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Stromelysin promoter is inhibited by TEL and stimulated by MN1-TEL.

<p>Expression and reporter plasmids, 100 ng and 250 ng respectively, were transiently transfected in NIH-3T3 cells. Expression of TEL results in inhibition of stomelysin promoter, whereas MN1-TEL stimulates. The non-DNA-binding mutant of MN1-TEL failed to stimulate. Equal amounts of TEL and MN1-TEL show intermediate promoter activity, indicative of competition of the proteins for the same binding sites.</p

FRAP curves and simulation data.

<p>A. GFP-MN1 FRAP curves show the fast recovery of MN1 to levels similar as GFP. In the presence of ATRA the recovery of GFP-MN1 is slightly slower. Alpha-amanitin releases the MN1 protein. Pie diagrams containing simulation data support FRAP curve data. B. TEL-GFP recovered much slower and leveled off to 70% of pre-bleach values. Simulation data calculated the presence of both a long-term bound fraction (20%; 59 s) and a short-term bound fraction (57%; 0.63 s). Alpha amanitin released the TEL-GFP protein as shown by FRAP curve and simulation data. C. GFP-MN1-TEL was largely immobile. A non DNA-binding mutant (DBDm) of GFP-MN1-TEL was mobile and diffused similarly to GFP-MN1. Alpha-amanitin treatment only partly released MN1-TEL. Simulation data calculated that 10% of the protein remains immobile for a long period (235 s).</p

Testing functionality and expression of GFP-tagged MN1, TEL and MN1-TEL.

<p>A. GFP-MN1 stimulates transcription from the MSV promoter as efficiently as MN1, both in the presence or absence of 1 uM ATRA (24 h incubation) as tested in with a luciferase assay. Western blot using anti-GFP antibody showing GFP-MN1 protein expression in stably transfected NIH 3T3 cells. GFP-MN1 located to the nucleus, where it was homogeneously distributed, with notable exception of the nucleoli, where no expression could be detected (left photograph). This expression pattern did not change upon alpha-amanitin treatment (right photograph). B. TEL and TEL-GFP are equally able to inhibit gene expression from the MSV promoter. Western blot of a cell lysate of the TEL-GFP-expressing NIH3T3 cell line is stained with anti-GFP and the two isoforms (translation starts at methionine 1 and 43) of TEL are visible. TEL-GFP is mostly nuclear in NIH3T3 cell lines (left photograph). TEL-GFP-DBDm is located in the cytoplasm (middle photograph). Expression pattern of TEL-GFP upon treatment with 50 ug/ml alpha-amanitin for 3 h (right photograph). Both MN1-TEL and GFP-MN1-TEL can weakly stimulate transcription from the MSV promoter, while the DNA-binding mutant is a weak repressor. Western blot with anti-GFP antibody of a lysate of cells expressing GFP-MN1-TEL and GFP-MN1-TEL-DBDm. GFP-MN1-TEL is nuclear, with a tendency to form aggregates upon higher expression levels (upper two photographs). GFP-MN1-TEL-DBDm is both nuclear and cytoplasmic (left lower photograph). Expression pattern of GFP-MN1-TEL did not change upon treatment with 50 ug/ml alpha-amanitin for 3 h (right lower photograph).</p

Rotavirus Vaccine Safety and Effectiveness in Infants With High-Risk Medical Conditions.

OBJECTIVES: Rotavirus vaccination has 87% to 100% effectiveness against severe rotavirus acute gastroenteritis (AGE) in healthy infants in high-income countries. Little is known whether infants with medical risk conditions (MRCs) are equally protected and if the vaccine is equally well tolerated. We conducted a quasi-experimental prospective multicenter before-after cohort study to assess the vaccine effectiveness (VE) and safety profile of the human rotavirus vaccine (HRV) among MRC infants that required prolonged or frequent postnatal care. METHODS: The Netherlands has no national rotavirus immunization program, but HRV was implemented in routine care for MRC infants in 13 Dutch hospitals. Participants in the before and after cohort, HRV unvaccinated and vaccinated, respectively, were followed for occurrence of (rotavirus) AGE. VE of at least 1 dose was estimated by using time-to-event analysis for severe rotavirus AGE. Vaccine-related serious adverse event (AEs) after HRV were retrieved systematically from medical charts. Solicited AEs after vaccinations were prospectively collected and compared between vaccination time points with or without HRV. RESULTS: In total, 1482 high-risk infants with MRC were enrolled, including 631 in the before and 851 in the after cohorts; 1302 infants were premature (88.3%), 447 were small for gestational age (30.2%), and 251 had at least 1 congenital disorder (17.0%). VE against severe rotavirus AGE was 30% (95% confidence interval [CI]: -36% to 65%). Overall, the observed number of rotavirus hospitalizations was low and not significantly different between the cohorts (2 and 2, respectively). The rate of vaccine-related serious AE was 0.24 per 100 vaccine doses. The adjusted risk ratio for any AE after HRV vaccination compared with other routine vaccinations was 1.09 (95% CI: 1.05 to 1.12) for concomitant administration and 0.91 (95% CI: 0.81 to 0.99) for single HRV administration. Gastrointestinal AEs were 10% more frequent after HRV. CONCLUSIONS: In contrast to previous findings among healthy term infants, in routine use, HRV offered limited protection to vulnerable medical risk infants. HRV is generally well tolerated in this group in single administration, but when coadministered with routine vaccines, it is associated with higher risk of (mostly gastrointestinal) AE. Our study highlights the importance of studying vaccine performance in subgroups of medically vulnerable infants