Computational approaches for circRNAs prediction and in silico characterization.

Circular RNAs (circRNAs) are single-stranded and covalently closed non-coding RNA molecules originated from RNA splicing. Their functions include regulatory potential over other RNA species, such as microRNAs, messenger RNAs and RNA binding proteins. For circRNA identification, several algorithms are available and can be classified in two major types: pseudo-reference-based and split-alignment-based approaches. In general, the data generated from circRNA transcriptome initiatives is deposited on public specific databases, which provide a large amount of information on different species and functional annotations. In this review, we describe the main computational resources for the identification and characterization of circRNAs, covering the algorithms and predictive tools to evaluate its potential role in a particular transcriptomics project, including the public repositories containing relevant data and information for circRNAs, recapitulating their characteristics, reliability and amount of data reported.

Correction: Vicencio et al. Transcriptional Signatures and Network-Based Approaches Identified Master Regulators Transcription Factors Involved in Experimental Periodontitis Pathogenesis. Int. J. Mol. Sci. 2023, 24, 14835.

In the original publication [...].

Differences in Bacterial Small RNAs in Stool Samples from Hypercholesterolemic and Normocholesterolemic Subjects.

Cholesterol metabolism is important at the physiological level as well as in several diseases, with small RNA being an element to consider in terms of its epigenetic control. Thus, the aim of this study was to identify differences between bacterial small RNAs present at the gut level in hypercholesterolemic and normocholesterolemic individuals. Twenty stool samples were collected from hypercholesterolemic and normocholesterolemic subjects. RNA extraction and small RNA sequencing were performed, followed by bioinformatics analyses with BrumiR, Bowtie 2, BLASTn, DESeq2, and IntaRNA, after the filtering of the reads with fastp. In addition, the prediction of secondary structures was obtained with RNAfold WebServer. Most of the small RNAs were of bacterial origin and presented a greater number of readings in normocholesterolemic participants. The upregulation of small RNA ID 2909606 associated with Coprococcus eutactus (family Lachnospiraceae) was presented in hypercholesterolemic subjects. In addition, a positive correlation was established between small RNA ID 2149569 from the species Blautia wexlerae and hypercholesterolemic subjects. Other bacterial and archaeal small RNAs that interacted with the LDL receptor (LDLR) were identified. For these sequences, the prediction of secondary structures was also obtained. There were significant differences in bacterial small RNAs associated with cholesterol metabolism in hypercholesterolemic and normocholesterolemic participants.

Transcriptional Signatures and Network-Based Approaches Identified Master Regulators Transcription Factors Involved in Experimental Periodontitis Pathogenesis.

Periodontitis is a chronic inflammatory disease characterized by the progressive and irreversible destruction of the periodontium. Its aetiopathogenesis lies in the constant challenge of the dysbiotic biofilm, which triggers a deregulated immune response responsible for the disease phenotype. Although the molecular mechanisms underlying periodontitis have been extensively studied, the regulatory mechanisms at the transcriptional level remain unclear. To generate transcriptomic data, we performed RNA shotgun sequencing of the oral mucosa of periodontitis-affected mice. Since genes are not expressed in isolation during pathological processes, we disclose here the complete repertoire of differentially expressed genes (DEG) and co-expressed modules to build Gene Regulatory Networks (GRNs) and identify the Master Transcriptional Regulators of periodontitis. The transcriptional changes revealed 366 protein-coding genes and 42 non-coding genes differentially expressed and enriched in the immune response. Furthermore, we found 13 co-expression modules with different representation degrees and gene expression levels. Our GRN comprises genes from 12 gene clusters, 166 nodes, of which 33 encode Transcription Factors, and 201 connections. Finally, using these strategies, 26 master regulators of periodontitis were identified. In conclusion, combining the transcriptomic analyses with the regulatory network construction represents a powerful and efficient strategy for identifying potential periodontitis-therapeutic targets.

Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group A Streptococcus.

Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIP-seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP-seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.

Mitochondrial E3 ubiquitin ligase 1 (MUL1) as a novel therapeutic target for diseases associated with mitochondrial dysfunction.

Mitochondrial E3 ubiquitin ligase 1 (MUL1) is a mitochondrial outer membrane-anchored protein-containing transmembrane domain in its N- and C-terminal regions, where both are exposed to the cytosol. Interestingly the C-terminal region has a RING finger domain responsible for its E3 ligase activity, as ubiquitin or in SUMOylation, interacting with proteins related to mitochondrial fusion and fission, cell survival, and tumor suppressor process, such as Akt. Therefore, MUL1 is involved in various cellular processes, such as mitochondrial dynamics, inter-organelle communication, proliferation, mitophagy, immune response, inflammation and cell apoptosis. MUL1 is expressed at a higher basal level in the heart, immune system organs, and blood. Here, we discuss the role of MUL1 in mitochondrial dynamics and its function in various pathological models, both in vitro and in vivo. In this context, we describe the role of MUL1 in: (1) the inflammatory response, by regulating NF-κB activity; (2) cancer, by promoting cell death and regulating exonuclear function of proteins, such as p53; (3) neurological diseases, by maintaining communication with other organelles and interacting with proteins to eliminate damaged organelles and; (4) cardiovascular diseases, by maintaining mitochondrial fusion/fission homeostasis. In this review, we summarize the latest advances in the physiological and pathological functions of MUL1. We also describe the different substrates of MUL1, acting as a positive or negative regulator in various pathologies associated with mitochondrial dysfunction. In conclusion, MUL1 could be a potential key target for the development of therapies that focus on ensuring the functionality of the mitochondrial network and, furthermore, the quality control of intracellular components by synchronously modulating the activity of different cellular mechanisms involved in the aforementioned pathologies. This, in turn, will guide the development of targeted therapies.

Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response.

Mesenchymal stem cells (MSCs) have fueled ample translation for the treatment of immune-mediated diseases. They exert immunoregulatory and tissue-restoring effects. MSC-mediated transfer of mitochondria (MitoT) has been demonstrated to rescue target organs from tissue damage, yet the mechanism remains to be fully resolved. Therefore, we explored the effect of MitoT on lymphoid cells. Here, we describe dose-dependent MitoT from mitochondria-labeled MSCs mainly to CD4+ T cells, rather than CD8+ T cells or CD19+ B cells. Artificial transfer of isolated MSC-derived mitochondria increases the expression of mRNA transcripts involved in T-cell activation and T regulatory cell differentiation including FOXP3, IL2RA, CTLA4, and TGFß1, leading to an increase in a highly suppressive CD25+ FoxP3+ population. In a GVHD mouse model, transplantation of MitoT-induced human T cells leads to significant improvement in survival and reduction in tissue damage and organ T CD4+ , CD8+ , and IFN-γ+ expressing cell infiltration. These findings point to a unique CD4+ T-cell reprogramming mechanism with pre-clinical proof-of-concept data that pave the way for the exploration of organelle-based therapies in immune diseases.

Insights into agar and secondary metabolite pathways from the genome of the red alga Gracilaria domingensis (Rhodophyta, Gracilariales).

Gracilariales is a clade of florideophycean red macroalgae known for being the main source of agar. We present a de novo genome assembly and annotation of Gracilaria domingensis, an agarophyte alga with flattened thallus widely distributed along Central and South American Atlantic intertidal zones. In addition to structural analysis, an organizational comparison was done with other Rhodophyta genomes. The nuclear genome has 78 Mbp, with 11,437 predicted coding genes, 4,075 of which did not have hits in sequence databases. We also predicted 1,567 noncoding RNAs, distributed in 14 classes. The plastid and mitochondrion genome structures were also obtained. Genes related to agar synthesis were identified. Genes for type II galactose sulfurylases could not be found. Genes related to ascorbate synthesis were found. These results suggest an intricate connection of cell wall polysaccharide synthesis and the redox systems through the use of L-galactose in Rhodophyta. The genome of G. domingensis should be valuable to phycological and aquacultural research, as it is the first tropical and Western Atlantic red macroalgal genome to be sequenced.

Long noncoding RNAs are involved in multiple immunological pathways in response to vaccination.

Understanding the mechanisms of vaccine-elicited protection contributes to the development of new vaccines. The emerging field of systems vaccinology provides detailed information on host responses to vaccination and has been successfully applied to study the molecular mechanisms of several vaccines. Long noncoding RNAs (lncRNAs) are crucially involved in multiple biological processes, but their role in vaccine-induced immunity has not been explored. We performed an analysis of over 2,000 blood transcriptome samples from 17 vaccine cohorts to identify lncRNAs potentially involved with antibody responses to influenza and yellow fever vaccines. We have created an online database where all results from this analysis can be accessed easily. We found that lncRNAs participate in distinct immunological pathways related to vaccine-elicited responses. Among them, we showed that the expression of lncRNA FAM30A was high in B cells and correlates with the expression of immunoglobulin genes located in its genomic vicinity. We also identified altered expression of these lncRNAs in RNA-sequencing (RNA-seq) data from a cohort of children following immunization with intranasal live attenuated influenza vaccine, suggesting a common role across several diverse vaccines. Taken together, these findings provide evidence that lncRNAs have a significant impact on immune responses induced by vaccination.

Epigenetic Reader BRD4 (Bromodomain-Containing Protein 4) Governs Nucleus-Encoded Mitochondrial Transcriptome to Regulate Cardiac Function.

BACKGROUND: BET (bromodomain and extraterminal) epigenetic reader proteins, in particular BRD4 (bromodomain-containing protein 4), have emerged as potential therapeutic targets in a number of pathological conditions, including cancer and cardiovascular disease. Small-molecule BET protein inhibitors such as JQ1 have demonstrated efficacy in reversing cardiac hypertrophy and heart failure in preclinical models. Yet, genetic studies elucidating the biology of BET proteins in the heart have not been conducted to validate pharmacological findings and to unveil potential pharmacological side effects. METHODS: By engineering a cardiomyocyte-specific BRD4 knockout mouse, we investigated the role of BRD4 in cardiac pathophysiology. We performed functional, transcriptomic, and mitochondrial analyses to evaluate BRD4 function in developing and mature hearts. RESULTS: Unlike pharmacological inhibition, loss of BRD4 protein triggered progressive declines in myocardial function, culminating in dilated cardiomyopathy. Transcriptome analysis of BRD4 knockout mouse heart tissue identified early and specific disruption of genes essential to mitochondrial energy production and homeostasis. Functional analysis of isolated mitochondria from these hearts confirmed that BRD4 ablation triggered significant changes in mitochondrial electron transport chain protein expression and activity. Computational analysis identified candidate transcription factors participating in the BRD4-regulated transcriptome. In particular, estrogen-related receptor α, a key nuclear receptor in metabolic gene regulation, was enriched in promoters of BRD4-regulated mitochondrial genes. CONCLUSIONS: In aggregate, we describe a previously unrecognized role for BRD4 in regulating cardiomyocyte mitochondrial homeostasis, observing that its function is indispensable to the maintenance of normal cardiac function.

OUTBREAK: a user-friendly georeferencing online tool for disease surveillance.

The current COVID-19 pandemic has already claimed more than 3.7 million victims and it will cause more deaths in the coming months. Tools that track the number and locations of cases are critical for surveillance and help in making policy decisions for controlling the outbreak. However, the current surveillance web-based dashboards run on proprietary platforms, which are often expensive and require specific computational knowledge. We developed a user-friendly web tool, named OUTBREAK, that facilitates epidemic surveillance by showing in an animated graph the timeline and geolocations of cases of an outbreak. It permits even non-specialist users to input data most conveniently and track outbreaks in real-time. We applied our tool to visualize the SARS 2003, MERS, and COVID19 epidemics, and provided them as examples on the website. Through the zoom feature, it is also possible to visualize cases at city and even neighborhood levels. We made the tool freely available at https://outbreak.sysbio.tools/ . OUTBREAK has the potential to guide and help health authorities to intervene and minimize the effects of outbreaks.

A regulatory RNA is involved in RNA duplex formation and biofilm regulation in Sulfolobus acidocaldarius.

Non-coding RNAs (ncRNA) are involved in essential biological processes in all three domains of life. The regulatory potential of ncRNAs in Archaea is, however, not fully explored. In this study, RNA-seq analyses identified a set of 29 ncRNA transcripts in the hyperthermophilic archaeon Sulfolobus acidocaldarius that were differentially expressed in response to biofilm formation. The most abundant ncRNA of this set was found to be resistant to RNase R treatment (RNase R resistant RNA, RrrR(+)) due to duplex formation with a reverse complementary RNA (RrrR(-)). The deletion of the RrrR(+) gene resulted in significantly impaired biofilm formation, while its overproduction increased biofilm yield. RrrR(+) was found to act as an antisense RNA against the mRNA of a hypothetical membrane protein. The RrrR(+) transcript was shown to be stabilized by the presence of the RrrR(-) strand in S. acidocaldarius cell extracts. The accumulation of these RrrR duplexes correlates with an apparent absence of dsRNA degrading RNase III domains in archaeal proteins.

A Guide to Scientific Crowdfunding.

Crowdfunding represents an attractive new option for funding research projects, especially for students and early-career scientists or in the absence of governmental aid in some countries. The number of successful science-related crowdfunding campaigns is growing, which demonstrates the public's willingness to support and participate in scientific projects. Putting together a crowdfunding campaign is not trivial, however, so here is a guide to help you make yours a success.

Prediction of MicroRNAs in the Epstein-Barr Virus Reveals Potential Targets for the Viral Self-Regulation.

Studies involving miRNAs have opened discussions about their broad participation in viral infections. Regarding the Human gammaherpesvirus 4 or Epstein-Barr virus (EBV), miRNAs are important regulators of viral and cellular gene expression during the infectious process, promoting viral persistence and, in some cases, oncogenic processes. We identified 55 miRNAs of EBV type 2 and inferred the viral mRNA target to self-regulate. This data indicate that gene self-repression is an important strategy for maintenance of the viral latent phase. In addition, a protein network was constructed to establish essential proteins in the self-regulation process. We found ten proteins that work as hubs, highlighting BTRF1 and BSRF1 as the most important proteins in the network. These results open a new way to understand the infection by EBV type 2, where viral genes can be targeted for avoiding oncogenic processes, as well as new therapies to suppress and combat the persistent viral infection.

StructRNAfinder: an automated pipeline and web server for RNA families prediction.

BACKGROUND: The function of many noncoding RNAs (ncRNAs) depend upon their secondary structures. Over the last decades, several methodologies have been developed to predict such structures or to use them to functionally annotate RNAs into RNA families. However, to fully perform this analysis, researchers should utilize multiple tools, which require the constant parsing and processing of several intermediate files. This makes the large-scale prediction and annotation of RNAs a daunting task even to researchers with good computational or bioinformatics skills. RESULTS: We present an automated pipeline named StructRNAfinder that predicts and annotates RNA families in transcript or genome sequences. This single tool not only displays the sequence/structural consensus alignments for each RNA family, according to Rfam database but also provides a taxonomic overview for each assigned functional RNA. Moreover, we implemented a user-friendly web service that allows researchers to upload their own nucleotide sequences in order to perform the whole analysis. Finally, we provided a stand-alone version of StructRNAfinder to be used in large-scale projects. The tool was developed under GNU General Public License (GPLv3) and is freely available at http://structrnafinder.integrativebioinformatics.me . CONCLUSIONS: The main advantage of StructRNAfinder relies on the large-scale processing and integrating the data obtained by each tool and database employed along the workflow, of which several files are generated and displayed in user-friendly reports, useful for downstream analyses and data exploration.

CEMiTool: a Bioconductor package for performing comprehensive modular co-expression analyses.

BACKGROUND: The analysis of modular gene co-expression networks is a well-established method commonly used for discovering the systems-level functionality of genes. In addition, these studies provide a basis for the discovery of clinically relevant molecular pathways underlying different diseases and conditions. RESULTS: In this paper, we present a fast and easy-to-use Bioconductor package named CEMiTool that unifies the discovery and the analysis of co-expression modules. Using the same real datasets, we demonstrate that CEMiTool outperforms existing tools, and provides unique results in a user-friendly html report with high quality graphs. Among its features, our tool evaluates whether modules contain genes that are over-represented by specific pathways or that are altered in a specific sample group, as well as it integrates transcriptomic data with interactome information, identifying the potential hubs on each network. We successfully applied CEMiTool to over 1000 transcriptome datasets, and to a new RNA-seq dataset of patients infected with Leishmania, revealing novel insights of the disease's physiopathology. CONCLUSION: The CEMiTool R package provides users with an easy-to-use method to automatically implement gene co-expression network analyses, obtain key information about the discovered gene modules using additional downstream analyses and retrieve publication-ready results via a high-quality interactive report.

Expression analysis and in silico characterization of intronic long noncoding RNAs in renal cell carcinoma: emerging functional associations.

BACKGROUND: Intronic and intergenic long noncoding RNAs (lncRNAs) are emerging gene expression regulators. The molecular pathogenesis of renal cell carcinoma (RCC) is still poorly understood, and in particular, limited studies are available for intronic lncRNAs expressed in RCC. METHODS: Microarray experiments were performed with custom-designed arrays enriched with probes for lncRNAs mapping to intronic genomic regions. Samples from 18 primary RCC tumors and 11 nontumor adjacent matched tissues were analyzed. Meta-analyses were performed with microarray expression data from three additional human tissues (normal liver, prostate tumor and kidney nontumor samples), and with large-scale public data for epigenetic regulatory marks and for evolutionarily conserved sequences. RESULTS: A signature of 29 intronic lncRNAs differentially expressed between RCC and nontumor samples was obtained (false discovery rate (FDR) < 5%). A signature of 26 intronic lncRNAs significantly correlated with the RCC five-year patient survival outcome was identified (FDR < 5%, p-value ≤ 0.01). We identified 4303 intronic antisense lncRNAs expressed in RCC, of which 22% were significantly (p < 0.05) cis correlated with the expression of the mRNA in the same locus across RCC and three other human tissues. Gene Ontology (GO) analysis of those loci pointed to 'regulation of biological processes' as the main enriched category. A module map analysis of the protein-coding genes significantly (p < 0.05) trans correlated with the 20% most abundant lncRNAs, identified 51 enriched GO terms (p < 0.05). We determined that 60% of the expressed lncRNAs are evolutionarily conserved. At the genomic loci containing the intronic RCC-expressed lncRNAs, a strong association (p < 0.001) was found between their transcription start sites and genomic marks such as CpG islands, RNA Pol II binding and histones methylation and acetylation. CONCLUSION: Intronic antisense lncRNAs are widely expressed in RCC tumors. Some of them are significantly altered in RCC in comparison with nontumor samples. The majority of these lncRNAs is evolutionarily conserved and possibly modulated by epigenetic modifications. Our data suggest that these RCC lncRNAs may contribute to the complex network of regulatory RNAs playing a role in renal cell malignant transformation.

Effects of Trimetazidine on Right Ventricular Function and Ventricular Remodeling in Patients with Pulmonary Artery Hypertension: A Randomised Controlled Trial.

BACKGROUND: Pulmonary artery hypertension (PAH) is a chronic and progressive disease. Although current therapy has improved the disease prognosis, PAH has a poor survival rate. The key feature leading to disease progression and death is right ventricular (RV) failure. METHODS AND RESULTS: We assessed the role of trimetazidine, a fatty acid beta-oxidation (FAO) inhibitor, in right ventricular function, remodeling, and functional class in PAH patients, with a placebo-controlled double-blind, case-crossover trial. Twenty-seven PAH subjects were enrolled, randomized, and assigned to trimetazidine or placebo for three months and then reallocated to the other study arm. The primary endpoint was RV morphology and function change after three months of treatment. Secondary endpoints were the change in exercise capacity assessed by a 6 min walk test after three months of treatment and the change in pro-BNP and Galectin-3 plasma levels after three months. Trimetazidine use was safe and well-tolerated. After three months of treatment, patients in the trimetazidine group showed a small but significant reduction of RV diastolic area, and a substantial increase in the 6 min walk distance (418 vs. 438 mt, p = 0.023), without significant changes in biomarkers. CONCLUSIONS: A short course of trimetazidine is safe and well-tolerated on PAH patients, and it is associated with significant increases in the 6MWT and minor but significant improvement in RV remodeling. The therapeutic potential of this drug should be evaluated in larger clinical trials.

Administration of Secretome Derived from Human Mesenchymal Stem Cells Induces Hepatoprotective Effects in Models of Idiosyncratic Drug-Induced Liver Injury Caused by Amiodarone or Tamoxifen.

Drug-induced liver injury (DILI) is one of the leading causes of acute liver injury. While many factors may contribute to the susceptibility to DILI, obese patients with hepatic steatosis are particularly prone to suffer DILI. The secretome derived from mesenchymal stem cell has been shown to have hepatoprotective effects in diverse in vitro and in vivo models. In this study, we evaluate whether MSC secretome could improve DILI mediated by amiodarone (AMI) or tamoxifen (TMX). Hepatic HepG2 and HepaRG cells were incubated with AMI or TMX, alone or with the secretome of MSCs obtained from human adipose tissue. These studies demonstrate that coincubation of AMI or TMX with MSC secretome increases cell viability, prevents the activation of apoptosis pathways, and stimulates the expression of priming phase genes, leading to higher proliferation rates. As proof of concept, in a C57BL/6 mouse model of hepatic steatosis and chronic exposure to AMI, the MSC secretome was administered endovenously. In this study, liver injury was significantly attenuated, with a decrease in cell infiltration and stimulation of the regenerative response. The present results indicate that MSC secretome administration has the potential to be an adjunctive cell-free therapy to prevent liver failure derived from DILI caused by TMX or AMI.