Long non-coding RNAs: definitions, functions, challenges and recommendations.

Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease.

The status of the human gene catalogue.

Scientists have been trying to identify every gene in the human genome since the initial draft was published in 2001. In the years since, much progress has been made in identifying protein-coding genes, currently estimated to number fewer than 20,000, with an ever-expanding number of distinct protein-coding isoforms. Here we review the status of the human gene catalogue and the efforts to complete it in recent years. Beside the ongoing annotation of protein-coding genes, their isoforms and pseudogenes, the invention of high-throughput RNA sequencing and other technological breakthroughs have led to a rapid growth in the number of reported non-coding RNA genes. For most of these non-coding RNAs, the functional relevance is currently unclear; we look at recent advances that offer paths forward to identifying their functions and towards eventually completing the human gene catalogue. Finally, we examine the need for a universal annotation standard that includes all medically significant genes and maintains their relationships with different reference genomes for the use of the human gene catalogue in clinical settings.

Congenital Zika virus infection impacts on male mouse offspring's reproductive biology.

In brief: Congenital ZIKV infection promotes alarming effects on male offspring's reproductive biology. This study showed the presence of the ZIKV antigen in the testis parenchyma, decreased testosterone levels, and sperm abnormalities in male offspring born to infected mothers. Abstract: Infection with ZIKV during pregnancy is associated with fetal developmental problems. Although neurological issues are being explored more in experimental studies, limited research has focused on the reproductive health consequences for offspring born to infected mothers. In this context, this study aimed to assess the impact of ZIKV infection during pregnancy on the testes and sperm of adult male offspring. Female mice were intraperitoneally inoculated with a Brazil strain of ZIKV during the 5.5th day of embryonic gestation. The offspring were evaluated 12 weeks after birth to analyze cellular and molecular changes in the testes and sperm. A novel approach combining variable-angle spectroscopic ellipsometry and machine learning modeling was also introduced for sperm sample analysis. The study revealed the presence of ZIKV protein in the testis parenchyma of adult male offspring born to infected mothers. It was shown that the testes exhibited altered steroidogenesis and inflammatory mediators, in addition to significant issues with spermiogenesis that resulted in sperm with DNA fragmentation, head defects, and protamination failure. Additionally, sperm dielectric properties and artificial intelligence showed potential for rapid identification and classification of sperm samples from infected mice. These findings provide crucial insights into the reproductive risks for men born from ZIKV-infected pregnant women.

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.

Interaction of Sox2 with RNA binding proteins in mouse embryonic stem cells.

Sox2 is a master transcriptional regulator of embryonic development. In this study, we determined the protein interactome of Sox2 in the chromatin and nucleoplasm of mouse embryonic stem (mES) cells. Apart from canonical interactions with pluripotency-regulating transcription factors, we identified interactions with several chromatin modulators, including members of the heterochromatin protein 1 (HP1) family, suggesting a role for Sox2 in chromatin-mediated transcriptional repression. Sox2 was also found to interact with RNA binding proteins (RBPs), including proteins involved in RNA processing. RNA immunoprecipitation followed by sequencing revealed that Sox2 associates with different messenger RNAs, as well as small nucleolar RNA Snord34 and the non-coding RNA 7SK. 7SK has been shown to regulate transcription at gene regulatory regions, which could suggest a functional interaction with Sox2 for chromatin recruitment. Nevertheless, we found no evidence of Sox2 modulating recruitment of 7SK to chromatin when examining 7SK chromatin occupancy by Chromatin Isolation by RNA Purification (ChIRP) in Sox2 depleted mES cells. In addition, knockdown of 7SK in mES cells did not lead to any change in Sox2 occupancy at 7SK-regulated genes. Thus, our results show that Sox2 extensively interacts with RBPs, and suggest that Sox2 and 7SK co-exist in a ribonucleoprotein complex whose function is not to regulate chromatin recruitment, but could rather regulate other processes in the nucleoplasm.

Non-adiabatic effects in the H3+ spectrum.

The effect of non-adiabatic coupling on the computed rovibrational energy levels amounts to about 2 cm-1 for H3+ and must be included in high-accuracy calculations. Different strategies to obtain the corresponding energy shifts are reviewed in the article. A promising way is to introduce effective vibrational reduced masses that depend on the nuclear configuration. A new empirical method that uses the stockholder atoms-in-molecules approach to this effect is presented and applied to H3+. Furthermore, a highly accurate potential energy surface for the D3+ isotopologue, which includes relativistic and leading quantum electrodynamic terms, is constructed and used to analyse the observed rovibrational frequencies for this molecule. Accurate band origins are obtained that improve existing data. This article is part of a discussion meeting issue 'Advances in hydrogen molecular ions: H3+, H5+ and beyond'.

Core-valence stockholder AIM analysis and its connection to nonadiabatic effects in small molecules.

A previous theory of separation of motions of core and valence fractions of electrons in a molecule [J. R. Mohallem et al., Chem. Phys. Lett. 501, 575 (2011)] is invoked as basis for the useful concept of Atoms-in-Molecules (AIM) in the stockholder scheme. The output is a new tool for the analysis of the chemical bond that identifies core and valence electron density fractions (core-valence stockholder AIM (CVSAIM)). One-electron effective potentials for each atom are developed, which allow the identification of the parts of the AIM which move along with the nuclei (cores). This procedure results in a general method for obtaining effective masses that yields accurate non-adiabatic corrections to vibrational energies, necessary to attain cm-1 accuracy in molecular spectroscopy. The clear-cut determination of the core masses is exemplified for either homonuclear (H2+, H2) or heteronuclear (HeH+, LiH) molecules. The connection of CVSAIM with independent physically meaningful quantities can resume the question of whether they are observable or not.

Cryptic Species in Proechimys goeldii (Rodentia, Echimyidae)? A Case of Molecular and Chromosomal Differentiation in Allopatric Populations.

The spiny rats of the genus Proechimys have a wide distribution in the Amazon, covering all areas of endemism of this region. We analyzed the karyotype and cytochrome b (Cyt b) sequences in Proechimys goeldii from 6 localities representing 3 interfluves of the eastern Amazon. A clear separation of P. goeldii into 2 monophyletic clades was observed, both chromosomally and based on Cyt b sequences: cytotype A (2n = 26x2640;/27x2642;, NF = 42) for samples from the Tapajos-Xingu interfluve and cytotype B (2n = 24x2640;/25x2642;, NF = 42) for samples from the Xingu-Tocantins interfluve and east of the Tocantins River. The karyotypes differ in a pericentric inversion and a centric fusion/fission and an average nucleotide divergence of 6.1%, suggesting cryptic species. Meiotic analysis confirmed the presence of a XX/XY1Y2 multiple sex chromosome determination system for both karyotypes. The karyotypes also vary from the literature (2n = 24, NF = 42, XX/XY). The autosome translocated to the X chromosome is different both in size and morphology to P. cf. longicaudatus, which also has a multiple sex chromosome determination system (2n = 14x2640;/15x2640;x2642;/16x2640;/17x2642;, NF = 14). The Xingu River is a barrier that separates populations of P. goeldii, thus maintaining their allopatric nature and providing an explanation for the molecular and cytogenetic patterns observed for the Xingu River but not the Tocantins River.

Algorithms for predicting COVID outcome using ready-to-use laboratorial and clinical data.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging crisis affecting the public health system. The clinical features of COVID-19 can range from an asymptomatic state to acute respiratory syndrome and multiple organ dysfunction. Although some hematological and biochemical parameters are altered during moderate and severe COVID-19, there is still a lack of tools to combine these parameters to predict the clinical outcome of a patient with COVID-19. Thus, this study aimed at employing hematological and biochemical parameters of patients diagnosed with COVID-19 in order to build machine learning algorithms for predicting COVID mortality or survival. Patients included in the study had a diagnosis of SARS-CoV-2 infection confirmed by RT-PCR and biochemical and hematological measurements were performed in three different time points upon hospital admission. Among the parameters evaluated, the ones that stand out the most are the important features of the T1 time point (urea, lymphocytes, glucose, basophils and age), which could be possible biomarkers for the severity of COVID-19 patients. This study shows that urea is the parameter that best classifies patient severity and rises over time, making it a crucial analyte to be used in machine learning algorithms to predict patient outcome. In this study optimal and medically interpretable machine learning algorithms for outcome prediction are presented for each time point. It was found that urea is the most paramount variable for outcome prediction over all three time points. However, the order of importance of other variables changes for each time point, demonstrating the importance of a dynamic approach for an effective patient's outcome prediction. All in all, the use of machine learning algorithms can be a defining tool for laboratory monitoring and clinical outcome prediction, which may bring benefits to public health in future pandemics with newly emerging and reemerging SARS-CoV-2 variants of concern.

Pinstripe: a suite of programs for integrating transcriptomic and proteomic datasets identifies novel proteins and improves differentiation of protein-coding and non-coding genes.

MOTIVATION: Comparing transcriptomic data with proteomic data to identify protein-coding sequences is a long-standing challenge in molecular biology, one that is exacerbated by the increasing size of high-throughput datasets. To address this challenge, and thereby to improve the quality of genome annotation and understanding of genome biology, we have developed an integrated suite of programs, called Pinstripe. We demonstrate its application, utility and discovery power using transcriptomic and proteomic data from publicly available datasets. RESULTS: To demonstrate the efficacy of Pinstripe for large-scale analysis, we applied Pinstripe's reverse peptide mapping pipeline to a transcript library including de novo assembled transcriptomes from the human Illumina Body Atlas (IBA2) and GENCODE v10 gene annotations, and the EBI Proteomics Identifications Database (PRIDE) peptide database. This analysis identified 736 canonical open reading frames (ORFs) supported by three or more PRIDE peptide fragments that are positioned outside any known coding DNA sequence (CDS). Because of the unfiltered nature of the PRIDE database and high probability of false discovery, we further refined this list using independent evidence for translation, including the presence of a Kozak sequence or functional domains, synonymous/non-synonymous substitution ratios and ORF length. Using this integrative approach, we observed evidence of translation from a previously unknown let7e primary transcript, the archetypical lncRNA H19, and a homolog of RD3. Reciprocally, by exclusion of transcripts with mapped peptides or significant ORFs (>80 codon), we identify 32 187 loci with RNAs longer than 2000 nt that are unlikely to encode proteins. AVAILABILITY AND IMPLEMENTATION: Pinstripe (pinstripe.matticklab.com) is freely available as source code or a Mono binary. Pinstripe is written in C# and runs under the Mono framework on Linux or Mac OS X, and both under Mono and .Net under Windows. CONTACT: m.dinger@garvan.org.au or j.mattick@garvan.org.au SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Proechimys (Rodentia, Echimyidae): characterization and taxonomic considerations of a form with a very low diploid number and a multiple sex chromosome system.

BACKGROUND: Proechimys is the most diverse genus in family Echimyidae, comprising 25 species (two of which are polytypic) and 39 taxa. Despite the numerous forms of this rodent and their abundance in nature, there are many taxonomic problems due to phenotypic similarities within the genus and high intraspecific variation. Extensive karyotypic variation has been noted, however, with diploid numbers (2n) ranging from 14 to 62 chromosomes. Some heteromorphism can be found, and 57 different karyotypes have been described to date. RESULTS: In the present work, we describe a cytotype with a very low 2n. Specimens of Proechimys cf. longicaudatus were collected from two different places in northern Mato Grosso state, Brazil (12°54″S, 52°22″W and 9°51'17″S, 58°14'53″W). The females and males had 16 and 17 chromosomes, respectively; all chromosomes were acrocentric, with the exception of the X chromosome, which was bi-armed. The sex chromosome system was found to be XY1Y2, originating from a Robertsonian rearrangement involving the X and a large acrocentric autosome. Females had two Neo-X chromosomes, and males had one Neo-X and two Y chromosomes. NOR staining was found in the interstitial region of one autosomal pair. CONCLUSIONS: Comparison of this karyotype with those described in the literature revealed that Proechimys with similar karyotypes had previously been collected from nearby localities. We therefore suggest that this Proechimys belongs to a different taxon, and is either a new species or one that requires reassessment.

lncRNAdb: a reference database for long noncoding RNAs.

Large numbers of long RNAs with little or no protein-coding potential [long noncoding RNAs (lncRNAs)] are being identified in eukaryotes. In parallel, increasing data describing the expression profiles, molecular features and functions of individual lncRNAs in a variety of systems are accumulating. To enable the systematic compilation and updating of this information, we have developed a database (lncRNAdb) containing a comprehensive list of lncRNAs that have been shown to have, or to be associated with, biological functions in eukaryotes, as well as messenger RNAs that have regulatory roles. Each entry contains referenced information about the RNA, including sequences, structural information, genomic context, expression, subcellular localization, conservation, functional evidence and other relevant information. lncRNAdb can be searched by querying published RNA names and aliases, sequences, species and associated protein-coding genes, as well as terms contained in the annotations, such as the tissues in which the transcripts are expressed and associated diseases. In addition, lncRNAdb is linked to the UCSC Genome Browser for visualization and Noncoding RNA Expression Database (NRED) for expression information from a variety of sources. lncRNAdb provides a platform for the ongoing collation of the literature pertaining to lncRNAs and their association with other genomic elements. lncRNAdb can be accessed at: http://www.lncrnadb.org/.

Early variation of inflammatory indexes refines prognostic prediction in patients with hepatocellular carcinoma under systemic treatment.

Prognostic markers in advanced hepatocellular carcinoma (HCC) are relevant for clinical decisions. Variations in inflammatory indexes, such as neutrophil-to-lymphocyte ratio (NLR) or platelet-to-lymphocyte ratio (PLR), may correlate with outcomes. In the present study, it was aimed to assess the prognostic role of inflammation indexes in patients with HCC and the evolutionary behavior of these variables within the first month of treatment in a cohort of patients treated with sorafenib from 2009-2021. Subgroups were divided based on the median of each variable ('low' or 'high)'. Survival was estimated using the Kaplan-Meier method. Hazard Ratio (HR) with 95% confidence interval (CI) were estimated using Cox regression models. A total of 373 patients were included, most Child-Pugh-A (83.1%) and BCLC-C (74%). Child-Pugh-A (P=0.011), performance status 0 (P<0.001), no ascites (P<0.001) and NLR<2.6 (P<0.001) were independently associated with improved survival. Baseline PLR was not correlated with survival (P=0.137). Patients who maintained low NLR at baseline and at 1 month (reference subgroup) had improved survival (18.6 months, 95% CI:15.4-22.0) compared with the subgroup that maintained high NLR at baseline and at 1 month (4.2 months, 95% CI:3.6-5.9), with HR: 3.80 (95% CI: 2.89-4.96). The subgroup with low NLR at baseline and high NLR at 1 month had a worse prognosis compared with the reference group (HR:1.4, 95% CI: 1.1-2.0), whereas the subgroup with high NLR at baseline and low at 1 month had similar outcome (HR:1.2, 95% CI: 0.8-1.6). It was concluded that evolutionary variation of NLR has a prognostic role in HCC patients under systemic therapy. This finding suggested that systemic inflammation and early modulation of the immune environment during treatment may correlate with outcomes.

The status of the human gene catalogue.

Scientists have been trying to identify all of the genes in the human genome since the initial draft of the genome was published in 2001. Over the intervening years, much progress has been made in identifying protein-coding genes, and the estimated number has shrunk to fewer than 20,000, although the number of distinct protein-coding isoforms has expanded dramatically. The invention of high-throughput RNA sequencing and other technological breakthroughs have led to an explosion in the number of reported non-coding RNA genes, although most of them do not yet have any known function. A combination of recent advances offers a path forward to identifying these functions and towards eventually completing the human gene catalogue. However, much work remains to be done before we have a universal annotation standard that includes all medically significant genes, maintains their relationships with different reference genomes, and describes clinically relevant genetic variants.

New, fast, and precise method of COVID-19 detection in nasopharyngeal and tracheal aspirate samples combining optical spectroscopy and machine learning.

Fast, precise, and low-cost diagnostic testing to identify persons infected with SARS-CoV-2 virus is pivotal to control the global pandemic of COVID-19 that began in late 2019. The gold standard method of diagnostic recommended is the RT-qPCR test. However, this method is not universally available, and is time-consuming and requires specialized personnel, as well as sophisticated laboratories. Currently, machine learning is a useful predictive tool for biomedical applications, being able to classify data from diverse nature. Relying on the artificial intelligence learning process, spectroscopic data from nasopharyngeal swab and tracheal aspirate samples can be used to leverage characteristic patterns and nuances in healthy and infected body fluids, which allows to identify infection regardless of symptoms or any other clinical or laboratorial tests. Hence, when new measurements are performed on samples of unknown status and the corresponding data is submitted to such an algorithm, it will be possible to predict whether the source individual is infected or not. This work presents a new methodology for rapid and precise label-free diagnosing of SARS-CoV-2 infection in clinical samples, which combines spectroscopic data acquisition and analysis via artificial intelligence algorithms. Our results show an accuracy of 85% for detection of SARS-CoV-2 in nasopharyngeal swab samples collected from asymptomatic patients or with mild symptoms, as well as an accuracy of 97% in tracheal aspirate samples collected from critically ill COVID-19 patients under mechanical ventilation. Moreover, the acquisition and processing of the information is fast, simple, and cheaper than traditional approaches, suggesting this methodology as a promising tool for biomedical diagnosis vis-à-vis the emerging and re-emerging viral SARS-CoV-2 variant threats in the future.

The evolution of knowledge on genes associated with human diseases.

Thousands of biomedical scientific articles, including those describing genes associated with human diseases, are published every week. Computational methods such as text mining and machine learning algorithms are now able to automatically detect these associations. In this study, we used a cognitive computing text-mining application to construct a knowledge network comprising 3,723 genes and 99 diseases. We then tracked the yearly changes on these networks to analyze how our knowledge has evolved in the past 30 years. Our systems approach helped to unravel the molecular bases of diseases and detect shared mechanisms between clinically distinct diseases. It also revealed that multi-purpose therapeutic drugs target genes that are commonly associated with several psychiatric, inflammatory, or infectious disorders. By navigating this knowledge tsunami, we were able to extract relevant biological information and insights about human diseases.

Doege-Potter syndrome associated to metastatic solitary fibrous tumor.

Solitary fibrous tumor (SFT) is a rare fibroblastic mesenchymal neoplasm with an estimated annual incidence of 0.35 per 100,000 individuals. Doege-Potter syndrome is a paraneoplastic syndrome related to solitary fibrous tumor clinically characterized by hypoglycemia, occurring in less than 5% of cases. Herein, we report a case of metastatic SFT associated with recurrent severe hypoglycemia. A 43-year-old male with a noncontributory medical history presented with a painless and progressive growing mass in the right thigh. The histological evaluation rendered the diagnosis of SFT, and tumor resection was performed. One year after the operation, on the oncological follow-up, he was admitted to the emergency unit, manifesting an early-morning seizure associated with a severe hypoglycemia. The laboratory findings of non-islet cell tumor hypoglycemia (NICTH) in the background of a relapsed metastatic solitary fibrous tumor were consistent with the diagnosis of Doege-Potter syndrome. Hepatic embolization associated with oral glucocorticoid was an efficient palliative treatment to control the hypoglycemic crisis and allow hospital discharge.

Complex architecture and regulated expression of the Sox2ot locus during vertebrate development.

The Sox2 gene is a key regulator of pluripotency embedded within an intron of a long noncoding RNA (ncRNA), termed Sox2 overlapping transcript (Sox2ot), which is transcribed in the same orientation. However, this ncRNA remains uncharacterized. Here we show that Sox2ot has multiple transcription start sites associated with genomic features that indicate regulated expression, including highly conserved elements (HCEs) and chromatin marks characteristic of gene promoters. To identify biological processes in which Sox2ot may be involved, we analyzed its expression in several developmental systems, compared to expression of Sox2. We show that Sox2ot is a stable transcript expressed in mouse embryonic stem cells, which, like Sox2, is down-regulated upon induction of embryoid body (EB) differentiation. However, in contrast to Sox2, Sox2ot is up-regulated during EB mesoderm-lineage differentiation. In adult mouse, Sox2ot isoforms were detected in tissues where Sox2 is expressed, as well as in different tissues, supporting independent regulation of expression of the ncRNA. Sox2dot, an isoform of Sox2ot transcribed from a distal HCE located >500 kb upstream of Sox2, was detected exclusively in the mouse brain, with enrichment in regions of adult neurogenesis. In addition, Sox2ot isoforms are transcribed from HCEs upstream of Sox2 in other vertebrates, including in several regions of the human brain. We also show that Sox2ot is dynamically regulated during chicken and zebrafish embryogenesis, consistently associated with central nervous system structures. These observations provide insight into the structure and regulation of the Sox2ot gene, and suggest conserved roles for Sox2ot orthologs during vertebrate development.

RNA regulation of epigenetic processes.

There is increasing evidence that dynamic changes to chromatin, chromosomes and nuclear architecture are regulated by RNA signalling. Although the precise molecular mechanisms are not well understood, they appear to involve the differential recruitment of a hierarchy of generic chromatin modifying complexes and DNA methyltransferases to specific loci by RNAs during differentiation and development. A significant fraction of the genome-wide transcription of non-protein coding RNAs may be involved in this process, comprising a previously hidden layer of intermediary genetic information that underpins developmental ontogeny and the differences between species, ecotypes and individuals. It is also evident that RNA editing is a primary means by which hardwired genetic information in animals can be altered by environmental signals, especially in the brain, indicating a dynamic RNA-mediated interplay between the transcriptome, the environment and the epigenome. Moreover, RNA-directed regulatory processes may also transfer epigenetic information not only within cells but also between cells and organ systems, as well as across generations.