Genome-scale deconvolution of RNA structure ensembles.

RNA structure heterogeneity is a major challenge when querying RNA structures with chemical probing. We introduce DRACO, an algorithm for the deconvolution of coexisting RNA conformations from mutational profiling experiments. Analysis of the SARS-CoV-2 genome using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and DRACO, identifies multiple regions that fold into two mutually exclusive conformations, including a conserved structural switch in the 3' untranslated region. This work may open the way to dissecting the heterogeneity of the RNA structurome.

Factor VIII promotes angiogenesis and vessel stability regulating extracellular matrix proteins.

Not available.

The acetyltransferase p300 is recruited in trans to multiple enhancer sites by lncSmad7.

The histone acetyltransferase p300 (also known as KAT3B) is a general transcriptional coactivator that introduces the H3K27ac mark on enhancers triggering their activation and gene transcription. Genome-wide screenings demonstrated that a large fraction of long non-coding RNAs (lncRNAs) plays a role in cellular processes and organ development although the underlying molecular mechanisms remain largely unclear (1,2). We found 122 lncRNAs that interacts directly with p300. In depth analysis of one of these, lncSmad7, is required to maintain ESC self-renewal and it interacts to the C-terminal domain of p300. lncSmad7 also contains predicted RNA-DNA Hoogsteen forming base pairing. Combined Chromatin Isolation by RNA precipitation followed by sequencing (ChIRP-seq) together with CRISPR/Cas9 mutagenesis of the target sites demonstrate that lncSmad7 binds and recruits p300 to enhancers in trans, to trigger enhancer acetylation and transcriptional activation of its target genes. Thus, these results unveil a new mechanism by which p300 is recruited to the genome.

Intragenic DNA methylation prevents spurious transcription initiation.

In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cancer.

The importance of ventilatory thresholds to define aerobic exercise intensity in cardiac patients and healthy subjects.

BACKGROUND: Although structured exercise training is strongly recommended in cardiac patients, uncertainties exist about the methods for determining exercise intensity (EI) and their correspondence with effective EI obtained by ventilatory thresholds. We aimed to determine the first (VT1 ) and second ventilatory thresholds (VT2 ) in cardiac patients, sedentary subjects, and athletes comparing VT1 and VT2 with EI defined by recommendations. METHODS: We prospectively enrolled 350 subjects (mean age: 50.7±12.9 years; 167 cardiac patients, 150 healthy sedentary subjects, and 33 competitive endurance athletes). Each subject underwent ECG, echocardiography, and cardiopulmonary exercise testing. The percentages of peak VO2 , peak heart rate (HR), and HR reserve were obtained at VT1 and VT2 and compared with the EI definition proposed by the recommendations. RESULTS: VO2 at VT1 corresponded to high rather than moderate EI in 67.1% and 79.6% of cardiac patients, applying the definition of moderate exercise by the previous recommendations and the 2020 guidelines, respectively. Most cardiac patients had VO2  values at VT2 corresponding to very-high rather than high EI (59.9% and 50.3%, by previous recommendations and 2020 guidelines, respectively). A better correspondence between ventilatory thresholds and recommended EI domains was observed in healthy subjects and athletes (90% and 93.9%, respectively). CONCLUSIONS: EI definition based on percentages of peak HR and peak VO2  may misclassify the effective EI, and the discrepancy between the individually determined and the recommended EI is particularly relevant in cardiac patients. A ventilatory threshold-based rather than a range-based approach is advisable to define an appropriate level of EI.

Bicuspid aortic valve and sports: From the echocardiographic evaluation to the eligibility for sports competition.

Bicuspid aortic valve (BAV) is the most common congenital heart defect in adults. Although a BAV may remain without clinical consequences for a lifetime, it can deteriorate in aortic valve stenosis and regurgitation and aortic dilatation. Unfortunately, the impact of regular training on patients with BAV and its natural course is not fully understood, although preliminary evidence suggests that the progression of valvular disease occurs primarily in an independent manner from sports practice. The current review aims to report how to perform a comprehensive echocardiographic examination in athletes with BAV and analyze the current literature on the influence of sports practice and how it impacts the aortic valve in athletes with BAV. The article also summarizes the current recommendations on sports eligibility and disqualification for competitive athletes with BAV.

Genome-Wide Analysis of Smad7-Mediated Transcription in Mouse Embryonic Stem Cells.

Smad7 has been identified as a negative regulator of the transforming growth factor TGF-ß pathway by direct interaction with the TGF-ß type I receptor (TßR-I). Although Smad7 has also been shown to play TGF-ß unrelated functions in the cytoplasm and in the nucleus, a comprehensive analysis of its nuclear function has not yet been performed. Here, we show that in ESCs Smad7 is mainly nuclear and acts as a general transcription factor regulating several genes unrelated to the TGF-ß pathway. Loss of Smad7 results in the downregulation of several key stemness master regulators, including Pou5f1 and Zfp42, and in the upregulation of developmental genes, with consequent loss of the stem phenotype. Integrative analysis of genome-wide mapping data for Smad7 and ESC self-renewal and pluripotency transcriptional regulators revealed that Smad7 co-occupies promoters of highly expressed key stemness regulators genes, by binding to a specific consensus response element NCGGAAMM. Altogether, our data establishes Smad7 as a new, integral component of the regulatory circuitry that controls ESC identity.

The acute impact of an ultramarathon on right heart: A 12-lead ECG study.

BACKGROUND: Some concerns exist about possible detrimental effects on cardiac function of ultra-endurance competitions. The aim of this study was to evaluate the acute effects of an ultramarathon by comparing pre- and post-race 12-lead ECG features. METHODS: A total of 301 competitive athletes (mean age: 48 ± 9 years) running a 50-km ultramarathon were analyzed. Twelve-lead ECG was collected the day before the race and immediately at the finish line. According to the Italian law, athletes could have participated only after undergoing pre-participation screening that ruled out the presence of an underlying heart disease. RESULTS: After the race a significant increase in P-wave voltage (P < .001) and P-wave duration (P < .001) was found as compared to pre-race data with a higher percentage of athletes fulfilling the ECG criteria for right atrial enlargement (RAE; from 3% to 17%, P < .001). The presence of RAE post-race significantly correlated with age, hours of training/week, and years of training and inversely with time at the finish line and the final position in the ranking. T-wave and R-wave amplitude (P < .001) and QTc-interval duration (P < .001) significantly increased after the race. No significant differences in terms of supraventricular or ventricular arrhythmias were found. CONCLUSIONS: A sizeable proportion of athletes running a 50-km ultramarathon demonstrated post-race ECG signs of right heart overload but no arrhythmias. This finding supports the hypothesis that ultra-endurance races may induce transient right heart overload.

Normal and abnormal QT interval duration and its changes in preadolescents and adolescents practicing sport.

AIMS: Twelve-lead electrocardiogram (ECG) is an established tool in the evaluation of athletes, providing information about life-threatening cardiovascular diseases, such as long QT syndrome. However, the interpretation of ECG is sometimes challenging in children, particularly for the repolarization phase. The aim of this prospective, longitudinal study was to determinate the distribution of QT interval in children practicing sport and to evaluate changes in QT duration overtime. METHODS AND RESULTS: A population of 1473 preadolescents practising sport (12.0 ± 1.8 years, 7-15 years) was analysed. Each athlete was evaluated at baseline, mid-term, and end of the study (mean follow-up: 3 ± 1 years). QT interval was corrected with Bazett (B) and Fridericia (F) formulae. At baseline QT interval corrected with the Bazett formula (QTcB) was 412 ± 25 ms and QT interval corrected with the Fridericia formula (QTcF) 387 ± 21 ms, with no changes during follow-up. Ten children (0.68%) had an abnormal QTc. In those with QTcB and QTcF ≥480 ms, QTc duration persisted abnormal during the follow-up and they were disqualified. Conversely, children with 460 ms < (QTcB) <480 ms had a normal QTc interval at the end of the study. These children had also a normal QTcF. Mean difference in the calculation of QT between the two formulae was 25 ± 11 ms (P < 0.0001). For resting heart rate (HR) ≥82 b.p.m., QTcF was independent from HR contrary to QTcB. CONCLUSION: Normal QTc interval does not change over time in preadolescents. A minority of them has a QTc ≥480 ms; in these subjects, QTc interval remains prolonged. The use of Bazett and Fridericia correction formulae is not interchangeable and the Fridericia correction should be preferred in preadolescents with a resting HR ≥82 b.p.m.

In vivo probing of nascent RNA structures reveals principles of cotranscriptional folding.

Defining the in vivo folding pathway of cellular RNAs is essential to understand how they reach their final native conformation. We here introduce a novel method, named Structural Probing of Elongating Transcripts (SPET-seq), that permits single-base resolution analysis of transcription intermediates' secondary structures on a transcriptome-wide scale, enabling base-resolution analysis of the RNA folding events. Our results suggest that cotranscriptional RNA folding in vivo is a mixture of cooperative folding events, in which local RNA secondary structure elements are formed as they get transcribed, and non-cooperative events, in which 5'-halves of long-range helices get sequestered into transient non-native interactions until their 3' counterparts have been transcribed. Together our work provides the first transcriptome-scale overview of RNA cotranscriptional folding in a living organism.

High-throughput single-base resolution mapping of RNA 2΄-O-methylated residues.

Functional characterization of the transcriptome requires tools for the systematic investigation of RNA post-transcriptional modifications. 2΄-O-methylation (2΄-OMe) of the ribose moiety is one of the most abundant post-transcriptional modifications of RNA, although its systematic analysis is difficult due to the lack of reliable high-throughput mapping methods. We describe here a novel high-throughput approach, named 2OMe-seq, that enables fast and accurate mapping at single-base resolution, and relative quantitation, of 2΄-OMe modified residues. We compare our method to other state-of-art approaches, and show that it achieves higher sensitivity and specificity. By applying 2OMe-seq to HeLa cells, we show that it is able to recover the majority of the annotated 2΄-OMe sites on ribosomal RNA. By performing knockdown of the Fibrillarin methyltransferase in mouse embryonic stem cells (ESCs) we show the ability of 2OMe-seq to capture 2΄-O-Methylation level variations. Moreover, using 2OMe-seq data we here report the discovery of 12 previously unannotated 2΄-OMe sites across 18S and 28S rRNAs, 11 of which are conserved in both human and mouse cells, and assigned the respective snoRNAs for all sites. Our approach expands the repertoire of methods for transcriptome-wide mapping of RNA post-transcriptional modifications, and promises to provide novel insights into the role of this modification.

The long intergenic non-coding RNA CCR492 functions as a let-7 competitive endogenous RNA to regulate c-Myc expression.

In mammals the cell-cycle progression through the G1 phase is a tightly regulated process mediated by the transcriptional activation of early genes in response to mitogenic stimuli, whose dysregulation often leads to tumorigenesis. We here report the discovery by RNA-seq of cell-cycle regulated (CCR) long intergenic non-coding RNAs (lincRNAs), potentially involved in the control of the cell-cycle progression. We identified 10 novel lincRNAs expressed in response to serum treatment in mouse embryonic fibroblasts (MEFs) and in BALB/c fibroblasts, comparably to early genes. By loss-of-function experiments we found that lincRNA CCR492 is required for G1/S progression, localizes in the cell cytoplasm and contains 4 let-7 microRNA recognition elements (MREs). Mechanistically, CCR492 functions as a competing endogenous RNA (ceRNA) to antagonize the function of let-7 microRNAs, leading to the de-repression of c-Myc. Moreover, we show that ectopic expression of CCR492 along with a constitutively active H-Ras promotes cell transformation. Thus, we identified a new lincRNA expressed as an early gene in mammalian cells to regulate the cell-cycle progression by upregulating c-Myc expression.

RNA structure framework: automated transcriptome-wide reconstruction of RNA secondary structures from high-throughput structure probing data.

SUMMARY: The rapidly increasing number of discovered non-coding RNAs makes the understanding of their structure a key feature toward a deeper comprehension of gene expression regulation. Various enzymatic- and chemically- based approaches have been recently developed to allow whole-genome studies of RNA secondary structures. Several methods have been recently presented that allow high-throughput RNA structure probing (CIRS-seq, Structure-seq, SHAPE-seq, PARS, etc.) and unbiased structural inference of residues within RNAs in their native conformation. We here present an analysis toolkit, named RNA Structure Framework (RSF), which allows fast and fully-automated analysis of high-throughput structure probing data, from data pre-processing to whole-transcriptome RNA structure inference. AVAILABILITY AND IMPLEMENTATION: RSF is written in Perl and is freely available under the GPLv3 license from http://rsf.hugef-research.org. CONTACT: salvatore.oliviero@hugef-torino.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

TET1 is controlled by pluripotency-associated factors in ESCs and downmodulated by PRC2 in differentiated cells and tissues.

Ten-eleven translocation (Tet) genes encode for a family of hydroxymethylase enzymes involved in regulating DNA methylation dynamics. Tet1 is highly expressed in mouse embryonic stem cells (ESCs) where it plays a critical role the pluripotency maintenance. Tet1 is also involved in cell reprogramming events and in cancer progression. Although the functional role of Tet1 has been largely studied, its regulation is poorly understood. Here we show that Tet1 gene is regulated, both in mouse and human ESCs, by the stemness specific factors Oct3/4, Nanog and by Myc. Thus Tet1 is integrated in the pluripotency transcriptional network of ESCs. We found that Tet1 is switched off by cell proliferation in adult cells and tissues with a consequent genome-wide reduction of 5hmC, which is more evident in hypermethylated regions and promoters. Tet1 downmodulation is mediated by the Polycomb repressive complex 2 (PRC2) through H3K27me3 histone mark deposition. This study expands the knowledge about Tet1 involvement in stemness circuits in ESCs and provides evidence for a transcriptional relationship between Tet1 and PRC2 in adult proliferating cells improving our understanding of the crosstalk between the epigenetic events mediated by these factors.

Snai1 promotes ESC exit from the pluripotency by direct repression of self-renewal genes.

Although much is known about the pluripotency self-renewal circuitry, the molecular events that lead embryonic stem cells (ESCs) exit from pluripotency and begin differentiation are largely unknown. We found that the zinc finger transcription factor Snai1, involved in gastrulation and epithelial-mesenchymal transition, is already expressed in the inner cell mass of the preimplantation blastocysts. In ESCs, Snai1 does not respond to TGFß or BMP4 signaling but it is induced by retinoic acid treatment, which induces the binding, on the Snai1 promoter, of the retinoid receptors RARγ and RXRα, the dissociation of the Polycomb repressor complex 2 which results in the decrease of H3K27me3, and the increase of histone H3K4me3. Snai1 mediates the repression of pluripotency genes by binding directly to the promoters of Nanog, Nr5a2, Tcl1, c-Kit, and Tcfcp2l1. The transient activation of Snai1 in embryoid bodies induces the expression of the markers of all three germ layers. These results suggest that Snai1 is a key factor that triggers ESCs exit from the pluripotency state and initiate their differentiation processes.

First clinical evaluation of an atrial haemodynamic sensor lead for automatic optimization of cardiac resynchronization therapy.

AIMS: One option to improve cardiac resynchronization therapy (CRT) responder rates lies in the optimization of pacing intervals. A haemodynamic sensor embedded in the SonRtip atrial lead measures cardiac contractility and provides a systematic automatic atrioventricular and interventricular delays optimization. This multi-centre study evaluated the safety and performance of the lead, up to 1 year. METHODS AND RESULTS: A total of 99 patients were implanted with the system composed of the lead and a CRT-Defibrillator device. Patients were followed at 1, 3, 6, and 12 months post-implant. The primary safety objective was to demonstrate that the atrial lead complication free rate was superior to 90% at 3-months follow-up visit. A lead handling questionnaire was filled by implanting investigators. Lead electrical performances and the performance of the system to compute AV and VV delays were evaluated at each study visit over 1 year. The complication free rate at 3 months post-implant was 99.0% [95%CI 94.5-100.0%], P < 0.001. Electrical performances of the lead were adequate whatever the atrial lead position and remained stable over the study period. The optimization algorithm was able to compute AV and VV delays in 97% of patients, during >75% of the weeks. CONCLUSION: The atrial lead is safe to implant and shows stable electrical performance over time. It therefore offers a promising tool for automatic CRT optimization to further improve responder rates to CRT.

Three-dimensional imaging and photostimulation by remote-focusing and holographic light patterning.

Access to three-dimensional structures in the brain is fundamental to probe signal processing at multiple levels, from integration of synaptic inputs to network activity mapping. Here, we present an optical method for independent three-dimensional photoactivation and imaging by combination of digital holography with remote-focusing. We experimentally demonstrate compensation of spherical aberration for out-of-focus imaging in a range of at least 300 µm, as well as scanless imaging along oblique planes. We apply this method to perform functional imaging along tilted dendrites of hippocampal pyramidal neurons in brain slices, after photostimulation by multiple spots glutamate uncaging. By bringing extended portions of tilted dendrites simultaneously in-focus, we monitor the spatial extent of dendritic calcium signals, showing a shift from a widespread to a spatially confined response upon blockage of voltage-gated Na(+) channels.

Involvement of N4BP2L1, PLEKHA4, and BEGAIN genes in breast cancer and muscle cell development.

Patients with breast cancer show altered expression of genes within the pectoralis major skeletal muscle cells of the breast. Through analyses of The Cancer Genome Atlas (TCGA)-breast cancer (BRCA), we identified three previously uncharacterized putative novel tumor suppressor genes expressed in normal muscle cells, whose expression was downregulated in breast tumors. We found that NEDD4 binding protein 2-like 1 (N4BP2L1), pleckstrin homology domain-containing family A member 4 (PLEKHA4), and brain-enriched guanylate kinase-associated protein (BEGAIN) that are normally highly expressed in breast myoepithelial cells and smooth muscle cells were significantly downregulated in breast tumor tissues of a cohort of 50 patients with this cancer. Our data revealed that the low expression of PLEKHA4 in patients with menopause below 50 years correlated with a higher risk of breast cancer. Moreover, we identified N4BP2L1 and BEGAIN as potential biomarkers of HER2-positive breast cancer. Furthermore, low BEGAIN expression in breast cancer patients with blood fat, heart problems, and diabetes correlated with a higher risk of this cancer. In addition, protein and RNA expression analysis of TCGA-BRCA revealed N4BP2L1 as a promising diagnostic protein biomarker in breast cancer. In addition, the in silico data of scRNA-seq showed high expression of these genes in several cell types of normal breast tissue, including breast myoepithelial cells and smooth muscle cells. Thus, our results suggest their possible tumor-suppressive function in breast cancer and muscle development.

Gut microbiota depletion delays somatic peripheral nerve development and impairs neuromuscular junction maturation.

Gut microbiota is responsible for essential functions in human health. Several communication axes between gut microbiota and other organs via neural, endocrine, and immune pathways have been described, and perturbation of gut microbiota composition has been implicated in the onset and progression of an emerging number of diseases. Here, we analyzed peripheral nerves, dorsal root ganglia (DRG), and skeletal muscles of neonatal and young adult mice with the following gut microbiota status: a) germ-free (GF), b) gnotobiotic, selectively colonized with 12 specific gut bacterial strains (Oligo-Mouse-Microbiota, OMM12), or c) natural complex gut microbiota (CGM). Stereological and morphometric analyses revealed that the absence of gut microbiota impairs the development of somatic median nerves, resulting in smaller diameter and hypermyelinated axons, as well as in smaller unmyelinated fibers. Accordingly, DRG and sciatic nerve transcriptomic analyses highlighted a panel of differentially expressed developmental and myelination genes. Interestingly, the type III isoform of Neuregulin1 (NRG1), known to be a neuronal signal essential for Schwann cell myelination, was overexpressed in young adult GF mice, with consequent overexpression of the transcription factor Early Growth Response 2 (Egr2), a fundamental gene expressed by Schwann cells at the onset of myelination. Finally, GF status resulted in histologically atrophic skeletal muscles, impaired formation of neuromuscular junctions, and deregulated expression of related genes. In conclusion, we demonstrate for the first time a gut microbiota regulatory impact on proper development of the somatic peripheral nervous system and its functional connection to skeletal muscles, thus suggesting the existence of a novel 'Gut Microbiota-Peripheral Nervous System-axis.'