The identification and functional annotation of RNA structures conserved in vertebrates

Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization, and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for conserved RNA structures (CRSs), leveraging structure-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ∼516,000 human genomic regions containing CRSs. We find that a substantial fraction of human–mouse CRS regions (1) colocalize consistently with binding sites of the same RNA binding proteins (RBPs) or (2) are transcribed in corresponding tissues. Additionally, a CaptureSeq experiment revealed expression of many of our CRS regions in human fetal brain, including 662 novel ones. For selected human and mouse candidate pairs, qRT-PCR and in vitro RNA structure probing supported both shared expression and shared structure despite low abundance and low sequence identity. About 30,000 CRS regions are located near coding or long noncoding RNA genes or within enhancers. Structured (CRS overlapping) enhancer RNAs and extended 3′ ends have significantly increased expression levels over their nonstructured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality.</jats:p

Goal directed fluid removal with furosemide versus placebo in intensive care patients with fluid overload:A trial protocol for a randomised, blinded trial (GODIF trial)

Funding Information: SW has received a grant from Merchant Jakob Ehrenreich and wife Grete Ehrenreich's Foundation to production of trial drug for the GODIF trial. AP has received research funding from the Novo Nordisk Foundation, Health Insurance Denmark (Sygeforsikringen Danmark), Fresenius Kabi, Denmark, and Pfizer, Denmark. MO has received research funding from Fresenius Medical Care, Baxter and Biomerieux. MHB has received research funding for the GODIF trial from Novo Nordisk Foundation, Jakob Madsen's and wife Olga Madsen's Foundation, Svend Andersen's Foundation, and Health Insurance Denmark (Sygeforsikringen Danmark). No authors received any financial gain. All other authors declared no conflicts of interest. Publisher Copyright: © 2022 The Authors. Acta Anaesthesiologica Scandinavica published by John Wiley & Sons Ltd on behalf of Acta Anaesthesiologica Scandinavica Foundation.Background: Fluid overload is a risk factor for mortality in intensive care unit (ICU) patients. Administration of loop diuretics is the predominant treatment of fluid overload, but evidence for its benefit is very uncertain when assessed in a systematic review of randomised clinical trials. The GODIF trial will assess the benefits and harms of goal directed fluid removal with furosemide versus placebo in ICU patients with fluid overload. Methods: An investigator-initiated, international, randomised, stratified, blinded, parallel-group trial allocating 1000 adult ICU patients with fluid overload to infusion of furosemide versus placebo. The goal is to achieve a neutral fluid balance. The primary outcome is days alive and out of hospital 90 days after randomisation. Secondary outcomes are all-cause mortality at day 90 and 1-year after randomisation; days alive at day 90 without life support; number of participants with one or more serious adverse events or reactions; health-related quality of life and cognitive function at 1-year follow-up. A sample size of 1000 participants is required to detect an improvement of 8% in days alive and out of hospital 90 days after randomisation with a power of 90% and a risk of type 1 error of 5%. The conclusion of the trial will be based on the point estimate and 95% confidence interval; dichotomisation will not be used. ClinicalTrials.gov identifier: NCT04180397. Perspective: The GODIF trial will provide important evidence of possible benefits and harms of fluid removal with furosemide in adult ICU patients with fluid overload.Peer reviewe

Cross-species high-resolution transcriptome profiling suggests biomarkers and therapeutic targets for ulcerative colitis

Background: Ulcerative colitis (UC) is a disorder with unknown etiology, and animal models play an essential role in studying its molecular pathophysiology. Here, we aim to identify common conserved pathological UC-related gene expression signatures between humans and mice that can be used as treatment targets and/or biomarker candidates.Methods: To identify differentially regulated protein-coding genes and non-coding RNAs, we sequenced total RNA from the colon and blood of the most widely used dextran sodium sulfate Ulcerative colitis mouse. By combining this with public human Ulcerative colitis data, we investigated conserved gene expression signatures and pathways/biological processes through which these genes may contribute to disease development/progression.Results: Cross-species integration of human and mouse Ulcerative colitis data resulted in the identification of 1442 genes that were significantly differentially regulated in the same direction in the colon and 157 in blood. Of these, 51 genes showed consistent differential regulation in the colon and blood. Less known genes with importance in disease pathogenesis, including SPI1, FPR2, TYROBP, CKAP4, MCEMP1, ADGRG3, SLC11A1, and SELPLG, were identified through network centrality ranking and validated in independent human and mouse cohorts.Conclusion: The identified Ulcerative colitis conserved transcriptional signatures aid in the disease phenotyping and future treatment decisions, drug discovery, and clinical trial design

Furosemide versus placebo for fluid overload in intensive care patients—The randomised GODIF trial second version : Statistical analysis plan

Publisher Copyright: © 2023 The Authors. Acta Anaesthesiologica Scandinavica published by John Wiley & Sons Ltd on behalf of Acta Anaesthesiologica Scandinavica Foundation.Background: Fluid overload is associated with increased mortality in intensive care unit (ICU) patients. The GODIF trial aims to assess the benefits and harms of fluid removal with furosemide versus placebo in stable adult patients with moderate to severe fluid overload in the ICU. This article describes the detailed statistical analysis plan for the primary results of the second version of the GODIF trial. Methods: The GODIF trial is an international, multi-centre, randomised, stratified, blinded, parallel-group, pragmatic clinical trial, allocating 1000 adult ICU patients with moderate to severe fluid overload 1:1 to furosemide versus placebo. The primary outcome is days alive and out of hospital within 90 days post-randomisation. With a power of 90% and an alpha level of 5%, we may reject or detect an improvement of 8%. The primary analyses of all outcomes will be performed in the intention-to-treat population. For the primary outcome, the Kryger Jensen and Lange method will be used to compare the two treatment groups adjusted for stratification variables supplemented with sensitivity analyses in the per-protocol population and with further adjustments for prognostic variables. Secondary outcomes will be analysed with multiple linear regressions, logistic regressions or the Kryger Jensen and Lange method as suitable with adjustment for stratification variables. Conclusion: The GODIF trial data will increase the certainty about the effects of fluid removal using furosemide in adult ICU patients with fluid overload. Trial Registrations: EudraCT identifier: 2019-004292-40 and ClinicalTrials.org: NCT04180397.Peer reviewe

Deciphering the Regulation of the Mannitol Operon Paves the Way for Efficient Production of Mannitol in <i>Lactococcus lactis</i>

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positive bacteria, mtlF and mtlD, encoding the enzyme IIA component (EIIA(mtl)) of the mannitol phosphotransferase system (PTS) and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA, encoding the enzyme IIBC component (EIIBC(mtl)) of the mannitol PTS, mtlR, encoding a transcriptional activator, and mtlF, as well as a separately expressed mtlD gene. The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive element (cre) genes. The presence of carbon catabolite repression was demonstrated and was shown to be relieved in stationary-phase cells. The transcriptional activator MtlR (mtlR), in some Gram-positive bacteria, is repressed by phosphorylation by EIIA(mtl), and when we knocked out mtlF, we indeed observed enhanced expression from the two promoters, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary-phase cells as biocatalysts, we attained 10.1 g/liter mannitol with a 55% yield, which, to the best of our knowledge, is the highest titer ever reported for L. lactis. Summing up, the results of our study should be useful for improving the mannitol-producing capacity of this important industrial organism. IMPORTANCE Lactococcus lactis is the most studied species of the lactic acid bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis to produce mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated and demonstrate how this impacts mannitol production capability. We harnessed this information and managed to establish efficient mannitol production without introducing foreign genes

Ecological Implications of Fish Removal: Insights from Gut-Content Analysis of roach (<i>Rutilus rutilus</i>) and European perch (<i>Perca fluviatilis</i>) in a Eutrophic Shallow Lake

Large reductions in fish biomass are common both as a method of managing lake ecosystems by fish removals (biomanipulation), and as naturally occurring fish kills. To further understand how fish reductions change feeding patterns of fish we studied the diets of small-medium sized roach (Rutilus rutilus) and European perch (Perca fluviatilis) on a monthly basis using gut-content analysis during an 18-month period before and following a whole lake fish removal in a eutrophic shallow lake. Further, we performed in-depth analyses of zoobenthos communities of the profundal and littoral zones, as well as analyzing the zooplankton community in the littoral and pelagic part of the lake to estimate abundance and biomass of potential diet items. We found that, in general, there was a trend towards increased zoobenthivory in both species and among all sized fish after the fish removal, regardless of prior diet preference. Reduced piscivory among larger perch (&gt;150 mm), and reduced zooplanktivory among smaller perch and roach